WO2025115008A1

WO2025115008A1 - Extracellular vesicles derived from stimulated car-t cells

Info

Publication number: WO2025115008A1
Application number: PCT/IL2024/051119
Authority: WO
Inventors: Anat Aharon; Anat GLOBERSON LEVIN
Original assignee: Yeda Research and Development Co Ltd; Ichilov Tech Ltd
Current assignee: Yeda Research and Development Co Ltd; Ichilov Tech Ltd
Priority date: 2023-11-28
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-28

Abstract

The present invention provides extracellular vesicles (EVs) derived from T-cells expressing chimeric antigen receptors (CAR) stimulated with an antigen to which the CAR binds specifically, pharmaceutical compositions comprising these vesicles as well as their use in treating cancer. In particular, the present invention exemplifies EVs derived from stimulated T-cells expressing CAR that bind specifically to EGFR, CD276 or CD19, a pharmaceutical composition comprising these EVs and their use in treating cancer overexpressing EGFR, CD276 or CD 19, such as ovarian cancer and breast cancer.

Description

EXTRACELLULAR VESICLES DERIVED FROM STIMULATED CAR-T CELLS

FIELD OF THE INVENTION

The present invention relates to extracellular vesicles derived from stimulated T-cells expressing certain specific chimeric antigen receptors, pharmaceutical compositions comprising same and their use in treating cancer.

BACKGROUND OF THE INVENTION

CAR T cells are engineered T cells expressing a chimeric antigen receptor (CAR) that recognizes a specific tumor-associated antigen (TAA) which may distinguish cancer cells from healthy ones. CAR T cells are used as immunotherapy for several different oncologic diseases, especially for leukemias and lymphomas in the past few years. Several methods are used to transduce or transfect T cells with CAR ex-vivo. Transduction may be based on retro or lenti viral transduction using retronectin or polibrene and other known transduction enhancers. These methods can include the use of viral vectors or other methods to introduce the DNA or RNA. As a result, the transfected T cell contains a genomic sequence for the specific protein and presents or expresses the receptor. Upon recognition of the TAA, the CAR T cell is stimulated and can efficiently kill its target cells.

As a potent therapeutic modality, there are several adverse events that may be associated with the CAR T cell therapy, such as cytokine release syndrome (CRS) and life-threatening cytokine storm. The side effects associated with CRS may include hypotension, hypoxia, high grade fever and neurological disturbances. Another significant challenge is the low penetration of the CAR T to the solid tumor niche and overcoming the tumor-microenvironment CAR T suppression, so that this treatment can be applied to treat solid tumors and not only hematologic malignancies.

Extracellular vesicles (EVs) are membrane vesicles secreted by different types of cells including blood cells. EVs can be divided into three subpopulations: (I) exosomes have a size of 30-100 nm and up to 150 nm in diameter and are derived from endosomal compartments; (II) microvesicles have a size of about 100 nm, or 150 nm up to 1pm in diameter and are released from the cell surface via "vesiculation"; and (III) apoptotic bodies have a size of 1-5 pm in diameter and are released from apoptotic cells. EVs are present in the blood circulation under normal physiological conditions, and their levels are increased in a variety of diseases such as diabetes and related vascular complications, cardiovascular disease, hematologic malignancies as well as in solid tumors such as breast cancer. Tang et al., (Oncotarget 2015; 6(42): 44179-90) discussed in general terms different approaches for use of cellular and exosomal platforms for the treatment of cancer. WO 2019/128952 describes methods for preparing an immune cell exosomes carrying CAR obtained by isolation, and uses thereof. WO 2020/212985 and Aharon et al. (HUMAN GENE THERAPY, 2021, VOLUME 32, NUMBERS 19-20) describe inter alia EVs derived from activated T-cells expressing CAR that bind specifically to HER2 cancer antigen, pharmaceutical compositions comprising these EVs and their use in treating a cancer overexpressing HER2, such as ovarian cancer and breast cancer.

Lung cancer is the leading cause of cancer morbidity and mortality worldwide. NonSmall Cell Lung Cancer (NSCLC) is the most common subtype of lung cancer, comprising up to 85% of all cases. The B7-H3, also known as CD276 is an immune checkpoint molecule in the epithelial mesenchymal transition (EMT) pathway, overexpressed in tumor tissues, including Non-small cell lung cancer (NSCLC), and brain cancers such as glioblastoma while showing limited expression in normal tissues, becoming an attractive and promising target for cancer immunotherapy. In addition, about 19% NSCLC patients harbor the epidermal growth factor receptor (EGFR) mutation, 29% express Kirsten rat sarcoma (KRAS); 3% Anaplastic lymphoma kinase (ALK); 3% Human epidermal growth factor 2 (HER2) 1% c-ROS oncogene 1 (ROS1); 1% Rearranged during transfection (RET); Neurotrophic receptor tyrosine kinase (NTRK); Neuregulin-1 (NRG1). Other targets for CAR T therapy in lung cancer include: Mesothelin (MSLN), Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1), Mucin-1 (MUC1), Prostate Stem Cell Antigen (PSCA), lung-specific X (LUNX), variant domain 6 of CD44 gene, melanoma-associated antigen-Al (MAGE-A1), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), glypican-3 (GPC3). Targets for Small Cell Lung Cancer (SCLC) may also include CD56-and Delta-like ligand 3 (DLL-3).

About 30% of advanced lung cancer develop brain metastases (BM) and their prognosis as well as quality of life are generally poor. BM are frequent in advanced EGFR-mutated or ALK-rearranged NSCLCs, with an estimated >45% of patients with CNS involvement.

EGFR is one of most prominent oncogenes in glioblastoma, an aggressive brain tumor. It is overexpressed in approximately 60% of tumors, and more than 40% exhibit EGFR gene amplification. A particular deletion mutation referred to as EGFRvIII or delta-EGFR is found in 25% of tumors. Other optional CAR T targets for glioblastoma may also include interleukin (IL), 13Ra2 (IL-13Ra2) and ephrin-A2 (Her2).

EVs have been suggested to contain several elements of the parent cell from which they are derived, including proteins, DNA fragment, micro RNA, and mRNA. Upon release, EVs can interact with target cells via a receptor mediated mechanism, or they can directly fuse with the plasma membrane of target cells, thus releasing their content into the recipient cell. Alternatively, EVs can be internalized via endocytosis and release their content into the cytosol of target cells.

There is an unmet need for development of additional approaches for safe and efficient treatment of cancer.

SUMMARY OF THE INVENTION

The present invention is based on the observation that a population of isolated extracellular vesicles (EVs) which are derived from T-cells expressing a chimeric antigen receptor (CAR) following activation and stimulation by exposure to antigen to which the CAR binds specifically, provided outstanding anti-cancer effects. It was found that a population of EVs comprising medium to large size EVs (150-1000 nm) had an improved apoptotic activity in comparison to a population comprising mainly exosomes (small EVs), which had weak to moderate activity. Therefore, it was shown that a population of EVs obtained from stimulated CAR T cells and comprising EVs having a particle diameter size of from 30 to 1000 nm and comprising at least 25% of EVs having a particle diameter size of from 150 to 1000 nm had a profound cytotoxic effect. Moreover, it was shown that stimulation of CAR T cells with an inert carrier coated with the tumor-associated antigen to which the CAR binds specifically increases significantly the purity, homogeneity and potency of the obtained EVs.

Accordingly, provided herein are EV-based compositions and methods, providing improved anti-cancer therapy compared to known treatments.

According to one aspect, the present invention provides a population of isolated stimulated extracellular vesicles (EVs) derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm and wherein the CAR is selected from anti-EGFR CAR, anti-CD276 CAR and anti-CD19 CAR. In some examples, at least 30% of the EVs have a particle size of above 150 nm. In some examples, at least 35% of the EVs have a particle size of from 150 to 1000 nm. In some examples, at least 38% of the EVs have a particle size of from 150 to 1000 nm. In some examples, at least 40% of the EVs have a particle size of from 150 to 1000 nm. In some examples, at least 45% of the EVs have a particle size of above 150 nm. In some examples, from 25 to 80% of the EVs have a particle size of from 150 to 1000 nm and from 20 to 75% of EVs have a particle size diameter of from 30 to 150nm. In some examples, a mean size of the EVs is at least 140 nm or at least 160 nm or from 140 to 250 nm. In some examples, the EVs present the chimeric antigen receptor (CAR) of the stimulated CAR T-cells.

In some examples, the CAR is anti-EGFR. According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated anti- EGFR CAR T-cells, wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm. In some examples, at least 35% or at least 40% of the EVs have a particle size of from 150 to 1000 nm. In some examples, the EVs are derived from anti-EGFR CAR T-cells stimulated by a carrier presenting EGFR or a fragment thereof to which the CAR binds specifically. In some examples, the carrier is selected from cells expressing EGFR and beads presenting EGFR or a fragment thereof to which the CAR binds specifically. In some examples, the anti-EGFR CAR comprises the antigen binding domain of cetuximab. In some examples, the anti-EGFR CAR comprises the antigen binding domain of an antibody disclosed in US11045543B2. In some examples, the cells are selected from lung cancer cells, anal cancers cells glioblastoma cells and epithelial tumors of the head and neck cells. In some examples, the anti-EGFR CAR comprises the amino acid sequences SEQ ID NOs: 11 and 12. In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 13.

In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 14.

In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 15.

In some examples, the anti-EGFR CAR comprises the amino acid sequences SEQ ID NOs: 16 and 17. In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 17. In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 19. In some examples, the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 20. According to some embodiments, from 30 to 90% or from 40 to 80% of the EVs comprises the anti-EGFR CAR.

In some examples, the CAR is anti-CD276. According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated anti-CD276 CAR T-cells, wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm. In some examples, at least 35% or at least 40% of the EVs have a particle size of from 150 to 1000 nm. In some examples, the anti-EGFR CAR comprises the antigen binding domain of 8H9 humanized hybridoma. In some examples, the anti-EGFR CAR comprises the antigen binding domain of Enoblituzumab antibody. In some examples, the EVs are derived from anti-CD276 CAR T-cells stimulated by a carrier presenting CD276 or a fragment thereof to which the CAR binds specifically. In some examples, the carrier is selected from cells expressing CD276 and beads presenting CD276 or a fragment thereof to which the CAR binds specifically. In some examples, the cells are selected from lung cancer cells, anal cancers cells glioblastoma cells and epithelial tumors of the head and neck cells. In some examples, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NOs: 1 and 2. In some examples, the anti-CD276CAR comprises the amino acid sequence SEQ ID NO: 3. In some examples, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 4. In some examples, the anti-CD276CAR comprises the amino acid sequence SEQ ID NO: 5. In some examples, the anti-CD276CAR comprises the amino acid sequences SEQ ID NOs: 6 and

7. In some examples, the anti-CD276CAR comprises the amino acid sequence SEQ ID NO:

8. In some examples, the anti-CD276CAR comprises the amino acid sequence SEQ ID NO:

9. In some examples, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO:

10. According to some embodiments, from 30 to 90% or from 40 to 80% of the EVs comprise the anti-CD276 CAR.

In some examples, the EVs are derived from anti-CD19 CAR T-cells stimulated by a carrier presenting CD 19 or a fragment thereof to which the CAR binds specifically. According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated anti-CD19 CAR T-cells, wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm. In some examples, at least 38% or at least 40% of the EVs have a particle size of from 150 to 1000 nm. In some examples, the EVs are derived from anti-CD19 CAR T-cells stimulated by a carrier presenting CD 19 or a fragment thereof to which the CAR binds specifically. In some examples, the carrier is selected from cells expressing CD 19 and beads presenting CD 19 or a fragment thereof to which the CAR binds specifically. In some examples, the cells are selected from lung cancer cells, anal cancers cells glioblastoma cells and epithelial tumors of the head and neck cells. In some examples, the anti-CD19 CAR comprises the amino acid sequence SEQ ID NOs: 21 and 22. In some examples, the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 23. In some examples, the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 24. According to some embodiments, from 30 to 90% or from 40 to 80% of the EVs comprise the anti-CD19 CAR.

According to any one of the above examples, the EVs are cytotoxic EVs. According to some examples, the EVs may further comprise an anticancer agent or be devoid of an exogenous anti-cancer agent.

According to another aspect, the present invention provides a pharmaceutical composition comprising the isolated activated EVs as described in any one of the above examples, and a pharmaceutically acceptable carrier. In some examples, the EVs are sole anti- cancer agent in the pharmaceutical composition. In other examples, the pharmaceutical composition further comprises an additional anti-cancer agent. In some examples, the pharmaceutical composition is formulated as a formulation for injection.

The isolated activated EVs of the present invention and the pharmaceutical composition comprising the isolated activated EVs are for use in treating cancer, wherein the cancer cells present the antigen to which the CAR binds specifically. According to some examples the isolated activated EVs are derived from stimulated anti-EGFR CAR T cells and the cancer is selected from glioblastoma, lung adenocarcinoma, glioblastoma multiforme (GBM), diffuse large B-cell lymphoma (DLBC), skin cutaneous melanoma (SKCM), brain lower grade glioma (LGG), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), adrenocortical carcinoma (ACC), uterine corpus endometrial carcinoma (UCEC), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), ovarian serous cystadenocarcinoma (OV), cervical squamous cell carcinomaand endocervical adenocarcinoma (CESC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), Pancreatic adenocarcinoma (PAAD), testicular germ cell tumors (TGCT), prostate adenocarcinoma (PRAD), breast invasive carcinoma (BRCA), and bladder urothelial carcinoma (BLCA). According to some examples, the isolated activated EVs are derived from stimulated anti-EGFR CAR T cells and the cancer is selected from lung cancer, anal cancers glioblastoma, epithelial tumors and epithelial tumors of the head and neck cells.

According to some examples, the isolated activated EVs are derived from stimulated anti-CD276 CAR T cells and the cancer is selected from the group consisting of lung cancer, cancer stem cells, epithelial tumor, tumors of the head and neck cells, and glioblastoma, bladder cancer, breast cancer, cervix cancer, colorectal cancer, esophageal cancer, renal cancer, hepatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, biliary cancer, oral squamous cell carcinoma, intrauterine membranous cancer, squamous cell carcinoma, gastric cancer, glioma, glioblastoma, melanoma, and adrenal cancer.

According to some examples, the isolated activated EVs are derived from stimulated anti-CD19 CAR T cells and the cancer is selected from the group consisting B cell lymphoma, (relapsed or refractory), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), either relapsed or refractory large B cell lymphoma (LB CL) and multiple myeloma.

According to some examples, the use comprises co-administration of an additional anticancer agent. According to yet 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 isolated activated EVs of any one of the above examples, wherein the activated EVs are derived from stimulated anti-EGFR CAR T cells and the cancer is selected from lung cancer, anal cancers glioblastoma, epithelial tumors, epithelial tumors of the head and neck cells, or the EVs are derived from stimulated anti-CD276 CAR T cells and the cancer is selected from glioma, prostate cancer, endometrial cancer, skin cancers, lung cancer, cancer stem cells, epithelial tumors, epithelial tumors of the head and neck cells, glioblastoma, bladder cancer, pancreatic cancer, cervical cancer, breast cancer, intrahepatic cholangiocarcinoma, colorectal cancer, ovarian cancer, glioma, melanoma, liver cancer, prostatic cancer, oral squamous cell carcinoma, kidney cancer, gastric cancer, and adrenocortical carcinoma.

According to yet another aspect, the present invention provides a method for preparation of the isolated activated and stimulated extracellular vesicles derived from stimulated CAR T- cells, the method comprises: (Step 1) incubating CAR T-cells with a tumor-associated antigen to which the CAR binds specifically in a cell medium under conditions enabling T cell stimulation; (Step 2) separating the stimulated CAR T-cells from the cell medium; and (step 3) isolating the derived activated extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm and wherein the tumor-associated antigen is selected from EGFR and CD276. In some examples, the resulting isolated activated EVs are as described in any one of the above examples.

In some examples, the isolation of the EVs at step (3) comprises centrifugation at from 8,000xg to 30,000xg for from 0.5 to 4 hours. In some examples, the isolation of the EVs at step (3) comprises centrifugation at from 8,000xg to 30,000xg for from more than 0.5 to 4 hours. In some examples, the isolation of EVs in step (3) is performed by centrifugation at from 8,000xg to 20,000xg for from about (e.g. more than) 0.5 to 3 hours. In some examples, the isolation of EVs in step (3) is performed by centrifugation at from 15,000xg to 25,000xg for from 0.5 to 1.5 hours. In some examples, the method is devoid of centrifugation at a force above 50,000xg or above 30,000xg.

In some examples, the incubation at step (1) comprises incubation for from 6 to 96 hours. In some examples, the incubation at step (1) comprises incubating CAR T-cells with cells or carrier coated with the tumor-associated antigen to which the CAR binds specifically.

In some examples, step (2) comprises step (2ii) comprising centrifuging the medium of the previous step for from 10 to 60 min at from 1000g to 3000g and separating the pellet from the medium. In other examples, the method further comprises step (2i) before step (2ii), wherein step (2i) comprises centrifuging the medium with stimulated T-cells from step (1) for 5 to 60 min at from 200g to 600g and separating the pellet from the medium.

In some examples, of the method, the CAR T-cells are anti-EGFR CAR T-cells and the tumor-associated antigen is EGFR. In some examples, the anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NOs: 4, 5, 9 and 10.

In some examples, the method comprises incubating anti-EGFR CAR T-cells with (i) cancer cells presenting EGFR or with beads coated with EGFR or a fragment thereof to which anti-EGFR CAR binds specifically for from 16 to 96 or from 24 to 84 hours and isolating the derived activated extracellular vesicles.

In some examples of the method, the CAR T-cells are anti-CD276 CAR T-cells and the tumor-associated antigen is CD276. In some examples, the anti-CD276 CAR comprises an amino acid sequence selected from SEQ ID NOs: 14, 15, 19 and 20. In some examples, the method comprises incubating anti-CD276 CAR T-cells with (i) cancer cells presenting CD276 or with beads coated with CD276 or a fragment thereof to which anti-CD276 CAR binds specifically, for from 16 to 96 or from 24 to 84 hours and isolating the derived activated extracellular vesicles.

In some examples of the method, the CAR T-cells are anti-CD19 CAR T-cells and the tumor-associated antigen is CD19. In some examples, the anti-CD19 CAR comprises an amino acid sequence SEQ ID NOs: 24. In some examples, the method comprises incubating antiCD 19 CAR T-cells with (i) cancer cells presenting CD 19 or with beads coated with CD 19 or a fragment thereof to which anti-CD19 CAR binds specifically, for from 16 to 96 or from 24 to 84 hours and isolating the derived activated extracellular vesicles.

In some examples, the method further comprises washing the obtained EVs and/or freezing the EVs.

According to another aspect, the present invention provides a population of isolated stimulated extracellular vesicles prepared by a method according to any one of the above examples, wherein at least 25% of the isolated EVs have a particle diameter size of from 150 nm to 1000 nm.

According to another aspect, the present invention provides a method for the preparation of the isolated stimulated extracellular vesicles derived from stimulated CAR T-cells, the method comprises: (1) incubating CAR T-cells with a carrier coated with a tumor-associated antigen or a fragment thereof to which the CAR binds specifically in a cell medium under conditions enabling T cell stimulation; (2) separating the CAR T-cells from the cell medium; and (3) isolating the derived activated extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm.

According to some embodiments, instead of a carrier coated with the TAA or fragments thereof isolated cell membrane fragments comprising the TAA are used. Thus, in some embodiments, the present invention provides a method for preparation of the isolated stimulated extracellular vesicles derived from stimulated CAR T-cells, the method comprises: (1) incubating CAR T-cells with cell membrane fragments comprising a tumor-associated antigen or a fragment thereof to which the CAR binds specifically in a cell medium under conditions enabling T cell stimulation; (2) separating the CAR T-cells from the cell medium; and (3) isolating the derived activated extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm.

In some examples, the isolation of the EVs at step (3) comprises centrifugation at from 8,000xg to 30,000xg for from 0.5 to 4 hours or from above 0.5 hour to 4 hour. In some examples, the isolation is performed by centrifugation at from 8,000xg to 20,000xg from 0.5 to 3 hours or from above 0.5 to 3 hours. In some examples, the isolation is performed by centrifugation at from 15,000xg to 25,000xg for from 0.5 to 1.5 hours. In some examples, the method is devoid of centrifugation at a force above 50,000xg or above 30,000xg.

In some examples, the carrier is coated with a tumor-associated antigen or a fragment thereof comprises beads coated with the tumor-associated antigen or a fragment thereof. In some examples, the tumor-associated antigen is selected from HER2 (ErBb2), EGFR, CD276, CD19, CD38, CD24, MUC1, Mesothelin, PSCA, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, HER2, ROR1, CD70, and CD138. In some examples, wherein the tumor-associated antigen is selected from HER2, EGFR, and CD276.

In some examples, the incubation at step (1) comprises incubation for from 6 to 96 hours.

According to some embodiments, the method comprises multiple cycles of steps (1), (2) and (3). Therefore, according to some embodiments, after isolation of the EVs, the method comprises further stimulation of the CAR T cells with the carrier coated with a tumor- associated antigen or a fragment thereof to which the CAR binds specifically, i.e., repeating, step (1), separating the stimulated CAR T-cells from the cell medium (step 2); and isolating the derived activated extracellular vesicles (step 3). The conditions of the steps (1), (2) and (3) are as described above. According to some embodiments, the cycle is repeated 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. In some examples, the method further comprises adding the carrier coated with a tumor- associated antigen or a fragment thereof to which the CAR binds specifically to CAR T-cells obtained in step (2) and repeating steps (1), (2) and (3). In some examples, this step may be repeated 1, 2 or 3 times. Therefore, in some embodiments, 1, 2, or 3 additional stimulation is performed. In some examples, the step of stimulation may be repeated 4, 5, 6, 7, 8, 9 or 10 times.

In some examples, step (2) comprises step (2ii) comprising centrifuging the medium of the previous step for from 10 to 60 min at from 1000g to 3000g and separating the pellet from medium. In some examples, the method further comprises step (2i) before step (2ii), wherein step (2i) comprises centrifuging the medium with stimulated T-cells from step (1) for 5 to 60 min at from 200g to 600g and separating the pellet from the medium. In some examples of the method, the CAR T-cells are anti-EGFR CAR T-cells and the tumor-associated antigen is EGFR. In some examples, the anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NOs: 4, 5, 9 and 10. In other examples of the method, the CAR T-cells are anti- CD276 CAR T-cells and the tumor-associated antigen is CD276. In some examples of the method, the anti-CD276 CAR comprises an amino acid sequence selected from SEQ ID NOs: 14, 15, 19 and 20. In some examples of the method, the CAR T-cells are anti-CD19 CAR T- cells and the tumor-associated antigen is CD19. In some examples of the method, the anti- CD276 CAR comprises an amino acid sequence is SEQ ID NOs: 24. In further examples of the method the CAR T-cells are anti-HER2 (N29) CAR T-cells and the tumor-associated antigen is HER2. In some embodiments wherein the N29 CAR comprises the amino acid sequence SEQ ID NO: 3.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows N29 GFP CAR transduction levels (percent of CAR-T cells) in activated human PBLs isolated from healthy donors, detected by flow cytometry. The activated PBLs were transduced retrovirally and stained with specific anti-CAR antibody (aN29, dark gray) or Green Fluorescence Protein (GFP, black) expression (reporter gene for CAR) percent of CAR- T cells, transduced cells (UT) served as a control (light gray).

Fig. 2A-2F shows characteristics of beads conjugated with HER2 and labeled with anti HIS- FITC and with anti HER2- APC antibodies. Beads characteristics before conjugation with the HER2 protein (Fig. 2A-2C): beads size (Fig. 2A) and florescent intensity: APC (Fig. 2B) and FITC (Fig. 2C). One month post conjugation with HER2 protein (Fig. 2D-2F): beads size (Fig. 2D) and florescent intensity: APC (Fig. 2E) and FITC (Fig. 2F). Stable conjugation demonstrated and 98% of the spheroid beads found to be conjugated with HER2 protein after month.

Fig. 3 depicts Interferon y (IFN-y) levels secreted by N29 GFP CAR T cells stimulated with HER2 coated beads (Beads), or with ovary cancer cells expressing HER2+ (SKOV). Interferon y (IFN-y) levels in pg/ml were measured by ELISA.

Fig. 4 shows the expression of the GFP marker in EVs isolated by Method 1 N29 GFP CART cells, measured by florescent laser by flow cytometry.

Fig. 5 shows a Fluorescence-activated cell sorting (FACS) plot of Megamix calibration HER2 coated beads

Fig. 6 shows a size distribution of EVs obtained from non-labeled anti-HER2 CAR T cells stimulated with HER2 coated beads, measured by FACS.

Fig. 7 shows a size distribution of EVs obtained from non-labeled SKOV HER2+ cells, measured by FACS.

Fig. 8 shows a size distribution of EVs obtained from non-labeled untransduced (UT) cells incubated with HER2 coated beads, measured by FACS.

Fig. 9 shows a size distribution of EVs obtained from Calcein AM (green dye) labeled anti- HER2 CAR T cells stimulated with HER2+ cancer cells, measured by FACS.

Fig. 10 shows a size distribution of EVs obtained from Calcein AM (green dye) labeled anti- HER2 CAR T cells stimulated with HER2 coated beads, measured by FACS.

Fig. 11 shows size distribution of EVs obtained from Calcein AM (green dye) labeled UT cells incubated with HER2+ cancer cells, measured by FACS.

Fig. 12 shows size distribution of EVs obtained from Calcein AM (green dye) labeled UT cells stimulated with HER2 coated beads, measured by FACS.

Figs. 13-15 show the results of a Nanoparticle-Tracking Analysis (NTA; NanoSight) size analysis of EVs derived anti-HER2 CAR T cells, after 1, 2, 3 constitutive stimulations using same CAR T cells and HER2 coated beads. Fig. 13 - EVs size distribution analysis after the first (I), Fig. 14 - after the second (II), and Fig. 15 - after the third (III) stimulation, respectively.

Fig. 16 shows the mean EVs size of EVs obtained from HER2 CAR T cells after each stimulation 1, 2 and 3 (as described for Figs. 26-28), first stimulation (Stim I), second stimulation (Stim II), and third stimulation (Stim III). Fig. 17 shows the percent of large EVs (>150nm) obtained from HER2 CAR T cells calculated for each stimulation (as described for Figs. 26-28), first stimulation (Stim I), second stimulation (Stim II), and third stimulation (Stim III).

Fig. 18 shows the percent of large EVs (>150nm, gray) versus small (<150nm, black) obtained from HER2 CAR T cells calculated for each stimulation (as described for Figs. 26-28), first stimulation (Stim I), second stimulation (Stim II), and third stimulation (Stim III).

Fig. 19 shows the cytokine profile and content of EVs derived from anti-HER2 CAR T cells stimulated after each of two stimulation cycles. Ninety micrograms of protein from lysate of 1X10⁶ anti-HER2 CAR T cells stimulated twice with HER2 coated beads and EVs derived therefrom (isolated from the medium of 5X 10⁶ cells) were analyzed after each stimulation for their cytokine profile. Protein levels of EVs were calculated as a ratio of proteins levels in the parental cells (EVs /CAR T protein cargo). EVs /CAR T protein cargo after the first stimulation denoted HER2 beads stimulation 1 (light gray), EVs /CAR T protein cargo after the second stimulation denoted HER2 beads stimulation 2 (dark gray).

Fig. 20 shows Granzyme B content of isolated EVs derived from un transduced (UT) cells and anti-HER2 CAR T cells with or without stimulation using HER2 coated beads. Granzyme B content was analyzed and quantified by Western blot, and normalized to actin. EVs derived from untransduced (UT) cells, EVs derived from untransduced cells stimulated with HER2 coated beads (UT +stim on beads), EVs derived from anti-HER2 CAR T cells (HER2 CAR T), and EVs derived from anti-HER2 CAR T cells stimulated on HER2 beads (HER2 CAR T +stim on beads).

Figs. 21A-21I show magnified microscopic images of mCherry-SKBR cells (HER2+ adenocarcinoma cells expressing mCherry fluorescent protein) incubated with UT or anti- HER2 CAR T cells or with EVs derived therefrom (50ug per well) after each of a three stimulation cycles I, II, III. Fig. 21A, mCherry-SKBR, target cells incubated with UT cells served as a control for the effector cells, at a E:T ratio of 2: 1. Fig. 21B, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation I. Fig. 21C, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation II. Fig. 21D, mCherry- SKBR, target cells incubated with UT cells derived EVs after stimulation III. Fig. 21E, untreated (no incubation with effector cells or EVs) mCherry-SKBR cells served as control. 21F, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells, effector cells, at a E:T ratio of 2:1. Fig. 21G, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation I. Fig. 21H, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation II. Fig. 211, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation III.

Fig. 22 shows a graphic presentation of red color intensity derived from the mCherry fluorescent protein as a marker for proliferation. mCherry-SKBR target cells incubated with UT cells at a E:T ratio of 2:1, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation I, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation II, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation III, untreated (no incubation with effector cells or EVs) mCherry-SKBR cells served as control., mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells, effector cells, at a E:T ratio of 2:1, mCherry-SKBR, target cells incubated with anti- HER2CAR T cells derived EVs after stimulation I, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation II, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation III.

Fig. 23 shows SKBR cells apoptosis levels following incubation with anti-HER2 CAR T cells or EVs thereof obtained after each one of three stimulations with HER2 coated beads and compared to untransduced cells (UT). Caspase 3/7 activity was calculated as the ratio of total green color intensity to cell coverage. SKBR cells incubated with anti-HER2 CAR T cells, with anti-HER2 CAR T EVs obtained from stimulation I, stimulation II, with UT Cells, and with UT EVs obtained from stimulation I, stimulation II, and Stimulation III.

Figs. 24-25 show flow cytometry analysis of anti-CD276 CAR and anti-EGFR CAR expression following transduction into PG13 cells. Plasmids encoding the anti-CD276 CAR and anti-EGFR CAR constructs linked to FLAG epitope were integrated into PG-13 viral producing packaging cells were sorted using an anti-FLAG PE conjugated antibody using flow cytometry (BD FACS Aria™ III Cell Sorter. Fig. 24, flow cytometry analysis of PG 13 cells transduced with anti-CD276 CAR. Fig. 25, flow cytometry analysis of PG 13 cells transduced with anti-EGFR CAR.

Fig. 26 shows the flow cytometry SSC:FSC gating strategy, for PBLs transduced with anti- CD276 or anti-EGFR CAR, each linked to a FLAG tag.

Fig. 27 shows the flow cytometry and transduction percentage of anti-CD276 CAR to PBLs compared to control unstained samples based on gate 1 (as detailed in Fig. 38) and detected by anti-FLAG PE antibody.

Fig. 28 shows the flow cytometry and transduction percentage of anti-EGFR CAR to PBLs compared to control unstained samples based on gate 1 (as detailed in Fig. 38) and detected by anti-FLAG PE antibody. Fig. 29 shows transduction statistics obtained from different blood donors (N=6), data are expressed as mean ± SD from separate experiments.

Fig. 30A shows a FACS analysis of EGFR expression levels on lung cancer cell lines, A549, H1975, and HCC827. Unstained A549, H1975, and HCC827 cells (US A549, US H1975, and US HCC827) were used as background control, isotype control of A549, H1975, and HCC827 cells (isotype A549, isotype H1975, and isotype HCC827), the cells stained with anti-EGFR antibody (A549, H1975, and HCC827).

Fig. 30B shows a FACS analysis of CD276 expression levels on lung cancer cell lines, A549, H1975, and HCC827. Unstained A549, H1975, and HCC827 cells (US A549, US H1975, and US HCC827) were used as background control, isotype control of A549, H1975, and HCC827 cells (isotype A549, isotype H1975, and isotype HCC827), the cells stained with anti-CD276 antibody (A549, H1975, and HCC827).

Fig. 31A shows a FACS analysis of EGFR expression levels on glioblastoma cell lines, U251. Unstained U251 cells (US) were used as background control, isotype control (isotype), the cells stained with anti-EGFR antibody (stained).

Fig. 31B shows a FACS analysis of CD276 expression levels on glioblastoma cell lines, U251. Unstained U251 cells (US) were used as background control, isotype control (isotype), the cells stained with anti-EGFR antibody (stained).

Figs. 32-35 show the cytotoxic effect (% of killing) of anti-EGFR and anti-CD276 CAR T cells (Effector cells) on different target cells at different Effector: Target cells ratios. anti- EGFR CAR T cells (gray line with squares) and Anti-CD276 CAR T cells (Black line with circles), untransduced cells served as a control (UT, dashed line with triangles). Data are expressed as mean ± SD of 4-5 samples from separate experiments (N) and analyzed by two- way ANOVA analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 32 - anti-EGFR and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line HCC827.

Fig. 33 - anti-EGFR and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line Hl 975. Fig. 34 - anti-EGFR and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line A549. Fig. 35 - anti-EGFR and anti-CD276 CAR T cytotoxic effect on glioblastoma U251 cell line.

Fig. 36 shows ELISA results for IFN-y levels (pg/mL) secreted by anti-EGFR CAR T cells (light gray, N=4) and anti-CD276 CAR T cells (dark gray, N=4), stimulated with HCC827, H1975 and A549 cells. Untransduced cells served as a control (UT, black, N=3). Data are expressed as mean ± SD of 3-4 samples from separate experiments (N) and analyzed by two- way ANOVA analysis. *p < 0.05, **p < 0.01.

Figs. 37-40 show size distribution (diameter in nm) measured by Nanoparticle-tracking analysis (NTA) of anti-EGFR CAR T and anti-CD276 CAR T EVs, CAR T cells derived from two different healthy donors or UT cells, the CAR T (effector cells) and UT (control) cells were stimulated with lung cancer cells (Target cells) at a 2:1 Effector :Target (E:T) ratio for 24h-72h. Following the stimulation EVs were isolated according to method 1 and their size was evaluated by nanoparticle-tracking analysis (NTA; NanoSight, version 3.1). Fig. 37. shows size distribution analysis of anti-EGFR CAR T EVs of donor 1. Fig. 38. shows size distribution analysis of anti-CD276 CAR T EVs of donor 1 Fig. 39. shows size distribution analysis of anti-EGFR CAR T EVs of donor 2 Fig. 40. Shows size distribution analysis of anti-CD276 CAR T EVs of donor 2 are.

Fig. 41 shows the percentages of small (<150nm, light gray) and large (>150nm, dark grey) EVs of anti-EGFR and anti-CD276 from donor 1 and donor 2 as described for Figs 37-40.

Figs. 42A-42M show the cytotoxic effect of CAR T cells and their respective EVs towards mcherry-HCC827 lung cancer cell line. Anti-EGFR and anti-CD276 CAR T cells and EVs derived therefrom were added to mCherry-HCC827 cells (5000 cell/well) 6 hrs after seeding on 96-well plates. The effect of the EVs was compared to anti-EGFR or anti-CD267 CAR T cells, or UT (untransduced) cells that were added to the target cells at a ratio of 2: 1 (E:T) or to untreated target cells. Cells were documented by IncuCyte Systems for Live-Cell Imaging every 4 h. Fig. 42A, mCherry-HCC827 with anti-EGFR EVs from donor 1. Fig. 42B, mCherry-HCC827 with anti-EGFR EVs from donor 2. Fig. 42C, mCherry-HCC827 with anti- CD276 EVs from donor 1. Fig. 42D, mCherry-HCC827 with anti-CD276 EVs from donor 2. Fig. 42E, mCherry-HCC827 with UT EVs. Fig. 42F, mCherry-HCC827 with lung cancer EVs Fig. 42G, mCherry-HCC827 with no EVs (untreated). Fig. 42H, mCherry-HCC827 with anti- EGFR CAR T cells from donor 1. Fig. 421, mCherry-HCC827 with anti-EGFR CAR T cells from donor 2. Fig. 42J, mCherry-HCC827 with anti-CD276 CAR T cells from donor 1. Fig. 42K, mCherry-HCC827 with anti-CD276 CAR T cells from donor 2. Fig. 42L, mCherry- HCC827 with UT cells. Fig. 42M, untreated mCherry-HCC827.

Fig. 43 shows mCherry-HCC827 target cells apoptosis (or proliferation) induced by Anti- EGFR and anti-CD276 CAR T cells and EVs derived therefrom compared to the controls. Apoptosis (or proliferation) was monitored over the period of 40-100 hrs by detection of the red color intensity of the mCherry protein, for each of the cultures as described for Figs.42A- 42M. mCherry-HCC827 cells with: anti-EGFR EVs from donor 1 (grey line with squares); anti-EGFR EVs from donor 2 (grey line with circles); anti-CD276 EVs from donor 1 (grey line with rhombus); anti-CD276 EVs from donor 2 (light grey line with triangles); UT EVs (grey line with upside down triangles); UT cells from donor 1 (light grey line with upside down triangles); UT cells from donor 2 (black line with upside down triangles); anti-EGFR CAR T cells from donor 1 (black line with squares); anti-EGFR CAR T cells from donor 2 (black line with circles); anti-CD276 CAR T cells from donor 1 (black line with triangle); with anti-CD276 CAR T cells from donor 2 (black line with rhombus); and with Hl 975 lung cancer EVs (light grey line with stars).

Fig. 44 shows mCherry- A549 target cells apoptosis (or proliferation) induced by Anti-EGFR and anti-CD276 CAR T cells and EVs derived therefrom compared to the controls, basically as described for Fig. 56. mCherry-A549 calls with: anti-EGFR EVs from donor 1; anti-EGFR EVs from donor 2; with anti-CD276 EVs from donor 1; with anti-CD276 EVs from donor 2; with UT EVs; with UT cells from donor 1 (light grey line with upside down triangles); with UT cells from donor 2; with anti-EGFR CAR T cells from donor 1; with anti-EGFR CAR T cells from donor 2; with anti-CD276 CAR T cells from donor 1; with anti-CD276 CAR T cells from donor 2; and with Hl 975 lung cancer EVs.

Fig. 45 shows the percentage of the killing effects of EV's and CAR-T cells stimulated (from donor 2) with HCC827 cells on mCherryHCC827 (as described for Figs. 37-4; determined by methylene blue killing assay after 96hrs of exposure. Killing (%) of mCherryHCC827 incubated with: anti-EGFR EVs (EVs EGFR, light grey), anti-CD276 EVs (EVs CD276, dark grey), untransduced cells derived EVs (EVs UT, black round corners rectangle), EVs derived from lung cancer (EVs lung cancer, white), anti-EGFR CAR T cells (CAR T EGFR, light grey with wavy lines), anti-CD276 CAR T cells (CAR TCD276, dark grey with wavy lines), untransduced cells (UT black rectangle).

Figs 46A-46E show apoptosis levels of U251 glioblastoma target cells induced by CAR T EVs were evaluated by caspase-3/7 green dye activity assay (Sartorius) and documented by IncuCyte Systems for Live-Cell Imaging. anti-EGFR or anti-CD276 CAR T EVs (50ug/200ul medium) obtained from two donors, were added to U251 glioblastoma cells (5000cell/well) To that end, Glioblastoma U251 cells were seeded in 96 well plate (5000cell/well) and compared to non-treated cells. Green caspase 3/7 activity reagent was added at time 0 and the accumulation of the green caspase 3/7 dye activity (apoptotic cells, dots) was measured as object integrated intensity (GCU lm2/image) over 4 days after 96h exposure to CAR EVs anti- EGFR or anti-CD276 compared to non-treated cells. Fig. 46A, U251 glioblastoma target cells incubated with anti-EGFR CAR T EV s from donor 1. Fig. 46B, U251 glioblastoma target cells incubated with anti-CD276 CAR T EVs from donor 1. Fig. 46C, U251 glioblastoma target cells incubated with anti-EGFR CAR T EVs from donor 2. Fig. 46D, U251 glioblastoma target cells incubated with anti-CD276 CAR T EVs from donor 2. Fig. 46E, untreated U251 glioblastoma target cells.

Fig. 47 shows apoptosis levels (intensity of green caspase 3/7 activity) over time in U251 lung cancer target cells following incubation with U251 glioblastoma target cells incubated with: anti-EGFR and anti-CD276 CAR T EVs derived from two healthy donors. U251 glioblastoma target cells incubated with: anti-EGFR CAR T EVs from donor 1 (line with circles); anti- CD276 CAR T EVs from donor 1 (line with squares); anti-EGFR CAR T EVs from donor 2 (line with triangles); anti-CD276 CAR T EVs from donor 2 (line with rhombus); untreated U251 glioblastoma target cells (line with stars).

Fig. 48 shows the percentage of the killing of U251 glioblastoma cancer cells induced by CAR T EVs anti-EGFR and anti CD276 compared to untreated cells. The percent of killing of U251 glioblastoma target cells incubated with anti-EGFR CAR T EVs (EGFR, n=2, black) and with anti-CD276 CAR T EVs (CD276, n=2, grey). Data are expressed as mean ± SD.

Figs. 49A-49F show effect of anti-EGFR and anti-CD276 CAR-T cells (Fig 49A-49C) or CAR-EVs derived therefrom (Fig 49D-49F) on cancer cell lines: HCC827 (Figs 49A and 49D), A549 (Figs 49B and 49E) and H1975 (Figs 49C and 49F) by Methylene blue killing assay.

Fig. 50A-50D show the results of Scale-up production of activated EVs from anti-HER2-CAR T cells stimulated with beads coated with HER2 protein. Fig. 50 shows N29 CAR T EVs killing effects on SKOV cells after 96 hours of co-culture.

Fig. 51A-51D shows the characteristics of beads either conjugated (Fig. 51C and 51D) with CD19 or not (Fig. 51A and Fig. 51B).

Fig. 52 shows the size of EVs isolated from CD 19 CAR T cells or UT cells that were stimulated with beads conjugated with biotinylated CD 19 antigen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a population of EVs, wherein the EVs are derived from stimulated T-cells expressing a chimeric antigen receptor (CAR), wherein at least 25% of the EVs have size of 150 nm or more.

It was surprisingly found that preparation of EVs that were derived from T-cells expressing N29 CAR specific to HER2, (denoted as N29 CAR T) and activated with ovarian cancer cells presenting HER2, and wherein the population of the EVs comprises more than 25% of EVs having size between 150 nm to 1000 nm, exhibited an outstanding cytotoxic effect towards ovarian and breast HER2 positive cancer cells. Similar results were shown for EVs obtained from stimulated anti-CD19, anti-EGFR and anti-CD276 CAR T cells. As exemplified in the examples, EVs from anti-EGFR and anti-CD276 CAR T cells provided a significant cytotoxic effect against cancer cells such as lung cancer and brain cancer. It was further shown that the effect was not lost upon freezing the EVs. Thus, the EVs may be frozen and thawed before use, which ease their 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 CD 19, related to hematology cancer or cancer cells which are HER2 negative). Moreover, the preparation of EVs from specifically activated N29 CAR T cells comprising 20 % or less EVs having the size of above 150 nm had a much weaker effect. It is clear, however, that the population of the EVs comprise also EVs having a size of below 150 nm. In other words, the population of EVs of the present invention is a mixture of microvesicles and exosomes wherein the fraction of microvesicles is 25% or more. Typically, the population of the EVs of the present invention comprises about 25 to 80 % of EVs having a size of above 150 nm (e.g., 150-1000 nm) and the rest (e.g. 20-75% of the EVs) have a size of below 150 nm, more specifically 30-150 nm. It was further found that EVs obtained from the prestimulated CAR T cells were distinguishable from EVs of control preparations (i.e. EVs from unstimulated or un-transduced T cells) by their physical properties such as size distribution and presence of certain antigen markers.

Yet another important advancement of the present application relates to a method of stimulation of CAR T cells and more specifically obtaining a population of highly unified and active EVs. Without being bound to any particular theory it was estimated that one of the drawbacks of obtaining EVs from CAR T cells stimulated by target cells presenting the tumor- associated antigen to which the CAR binds specifically is that these target cells secrete EVs by themselves. Eventually, the population of EVs obtained contains EVs secreted by CAR T cells and EVs secreted by target cells, more specifically, by cancer cells. In this way the concentration of active EVs is reduced and the EVs obtained from cancer cells may have a negative effect. Therefore, the developed method in which CAR T cells are activated by a carrier, such as inert beads, presenting/coated with the antigen to which the CAR binds specifically allows obtaining a much more enriched and homogenous population of EVs in which more than 85% of the EVs include CAR. Considering that the obtained EVs has a much lower non-specific immune reaction, this method allowed also obtain an off-the-shelf product. Therefore, according to some embodiments, the present invention provides off-the-shelf activated EVs.

According to one aspect, the present invention provides a population of isolated activated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 22% of the EVs have particle diameter size of above 150 nm. According to some embodiments, the present invention provides isolated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 22% of the EVs have a size of above 150 nm. According to other embodiments, the present invention provides a population of isolated activated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have size of above 150 nm and the EVs has a size of from 30 nm to 1000 nm. In particular, embodiments of the invention are directed to isolated activated EVs derived from CAR T-cells activated by a CAR-mediated stimulation prior to EV isolation.

The terms "extracellular vesicles" and “EVs” are used herein interchangeably and refer to a cell-derived vesicles comprising a membrane that encloses an internal space. Generally, extracellular vesicles range in diameter from 30 nm to 1000 nm, and may comprise various cargo molecules either within the internal space, displayed on the external surface of the extracellular vesicle, and/or spanning the membrane. Said cargo molecules may comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. The term extracellular vesicles comprises also the terms “exosome” and “microvesicles”. The terms “exosomes” and “nanovesicle” are used herein interchangeably and refer to small EVs having a size of between 30 to 100 nm or between 30 to 150 nm in diameter. The term “microvesicles” as used herein refers to EVs having a size of between 100 to 1000 nm in diameter or between 150 nm to 1000 nm. Generally, the EVs may comprise at least a part of the molecular contents of the cells from which they are originated, e.g. lipids, fatty acids, polypeptides, polynucleotides, proteins and/or saccharides. According to the teaching of the present invention, at least 25% of the EVs have size of above 150 nm. Alternatively, at least 25% of the EVs have a size of 150 nm or more. According to some embodiments, at least 25% of the EVs have a size of from 150 nm to 1000 nm. According to any one of the aspects and embodiments of the present invention, the activated EVs of the present invention have the size of from 30 to 1000 nm.

The extracellular vesicles of the present invention are mostly spherical and the terms "size", "particle size", and "particle diameter size" used herein interchangeably refer to the diameter of the extracellular vesicles or to the longer diameter of the extracellular vesicles. When referring to a population the particle size refers to an average particle size. Any known method for measurement of particle size may be used to determine the size of the EVs of the present invention. A non-limiting example is nanoparticle-tracking analysis (NTA).

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

According to some embodiments, at least 10% or at least 15% of the isolated activated EV have a size between 150 to 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 of more 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 of above 150 nm. According to another embodiment, at least 42 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 45 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 50 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 55 % of the activated EVs have a size of above 150 nm. According to some embodiments, at least 60 % of the activated EVs have a size of above 150 nm. According to some embodiments, at least 65 % of the activated EVs have a size of above 150 nm. According to some embodiments, at least 70 % of the activated EVs have a size of above 150 nm. According to some embodiments, from 25 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, from 25 to 35% of the activated EVs have a size of above 150 nm. According to certain embodiments, from 25 to 45% of the activated EVs have a size of above 150. According to some embodiments, from 30 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 35 to 65% of the activated EVs have a particle diameter size of above 150 nm. According to another embodiment, from 35 to 45% of the activated EVs have a size of above 150 nm. According to yet another embodiment, from 32 to 65% of the activated EVs have a size of above 150 nm. According to some embodiments, the terms "above 150 nm" and "150 nm or more" when referring to EVs have the meaning of "from about 150 nm to about 1000 nm". Therefore, according to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated T- cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm. It is clear that the rest of the EVs have an average size of below 150 nm and more specifically that the EVs have a size of from about 30 to about 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 25% to 80% of EVs having an average particle size diameter of from 150 to 1000 nm and from 20 to 75% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 35% to 65% of EVs having an average particle size diameter of from 150 to 1000 nm and from 35 to 65% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 40% to 60% of EVs having an average particle size diameter of from 150 to 1000 nm and from 40 to 60% of EVs having an average particle size diameter of from 30 to 150 nm. According to any one of the aspects and embodiments of the present invention, the activated EVs of the present invention derived/obtained from from stimulated CAR T-cells have the size of from 30 to 1000 nm.

The term "X nm or more" encompasses also the term "more than X nm" and may be replaced by it in any one of the embodiments of the present invention.

According to some embodiments, at least 0.2%, or at least 0.5% or at least 0.8% of the isolated activated EV have the size above 300 nm, e.g., between 300 to 600 nm. According to some embodiments, from about 0.3 to about 3% of the isolated activated EV have the size between 300 to 500 nm.

According to some embodiments, the mean size of the activated EVs is 130 nm or more, as measured by nanop article-tracking analysis (NTA). According to other embodiments, the mean size of the activated EVs is 132 nm or more. According to other embodiments, the mean size of the activated EVs is 135 nm or more. According to other embodiments, the mean size of the activated EVs is 137 nm or more. According to other embodiments, the mean size of the activated EVs is 140 nm or more. According to other embodiments, the mean size of the activated EVs above 140 nm. According to other embodiments, the mean size of the activated EVs is 142 nm or more. According to other embodiments, the mean size of the activated EVs is 145 nm or more. According to other embodiments, the mean size of the activated EVs is 147 nm or more. According to other embodiments, the mean size of the activated EVs is 150 nm or more. According to other embodiments, the mean size of the activated EVs is 152 nm or more. According to other embodiments, the mean size of the activated EVs is 155 nm or more. According to other embodiments, the mean size of the activated EVs is 160 nm or more. According to other embodiments, the mean size of the activated EVs above 160 nm. According to other embodiments, the mean size of the activated EVs is 162 nm or more. According to other embodiments, the mean size of the activated EVs is 165 nm or more. According to other embodiments, the mean size of the activated EVs is above 170 nm. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs have the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm.

According to some embodiments, the present invention provides isolated stimulated EVs derived from pre- stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 nm or more. According to other embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have the mean size of 140 nm or more. According to some embodiments, at least 25% of the EVs have a size of above 150 nm and the EVs have a mean 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 a population of isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 147 nm or more. According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 170 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean 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, 162 nm or more or 170 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm, and the EVs have a mean size of 150 nm or more. According to some embodiments, at least 29% of the EVs have a diameter size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, at least 30% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 32% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 35% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean 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, 162 nm or more or 170 nm or more. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size above 170 nm. According to some embodiments, at least 40% of the EVs have a size of above 150 nm and the EVs have a mean size of 170 nm or more. According to some embodiments, at least 43% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 46% of the EVs have a size of above 150 nm and the EVs have a mean 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, at least 46% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 50% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more or of 150 nm or more or of 155 nm or more. According to some embodiments, at least 60% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more or of 155 nm or more or of 160 nm or more. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs has the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm.

According to some embodiments, at least 0.5%, at least 1%, at least 1.5%, at least 2% or at least 3 % have a size of above 300 nm.

According to some embodiments, the ratio between EVs having the particle size of above 150 nm (i.e. from 150 to 1000 nm) and EVs having the particle size of below 150 nm (i.e. from 30 to 150 nm) is from 2:8 to 8:8. According to some embodiments, the ratio between EVs having the particle size of above 150 nm and EV having the particle size of below 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 having the particle size of above 150 nm and EV having the particle size of below 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 of above 150 nm, the EVs have a mean size of 132 nm or more, 135 nm or more, 137 nm or more, 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 and the ratio between EVs having the size of above 150 nm and EV having the size of below 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 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 have size above 155 nm, or above 160 nm.

The terms “derived from” and “originated from” are used herein interchangeably and refer to extracellular vesicles that are produced within, by, or from, a specified cell, cell type, or population of cells such as T-cells, and in particular, from CAR T cells.

As used herein, the terms "parent cell", "producer cell" and “original cell” include any cell from which the extracellular vesicle is derived and isolated. The terms also encompass a cell that shares a protein, lipid, sugar, or nucleic acid component of the extracellular vesicle. For example, a "parent cell" or "producer cell" includes a cell that serves as a source for the extracellular vesicle membrane. The term “original CAR T-cells” subsequently refers to CAR T-cells from which the EVs are derived.

The terms "purify," "purified," "purifying", "isolate", "isolated," and "isolating" are used herein interchangeably and refer to the state of a population (e.g., a plurality of known or unknown amount and/or concentration) of extracellular vesicles, that have undergone one or more processes of purification/isolation, e.g., a selection of the desired extracellular vesicles, or alternatively a removal or reduction of residual biological products and/or removal of undesirable extracellular vesicles, e.g. removing EVs having a particular size. According to one embodiment, the ratio of EVs to residual parent 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 initial material. According to some embodiments, the ratio is weight ratio. In some advantageous embodiments, the term “isolated” have the meaning of substantially cell-free or cell-free, and may be substituted by it.

The terms "chimeric antigen receptor" and "CAR" are used herein interchangeably 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 cells.

The extracellular portion or domain of a CAR comprises an antigen binding domain and optionally a spacer or hinge region. The antigen binding domain of the CAR targets and specifically binds to an antigen of interest, e.g. a tumor-associated antigen (TAA). The targeting regions may comprise full length heavy chain, Fab fragments, or single chain variable fragment (scFvs). The antigen binding domain can be derived from the same species or from a different species than the one in which the CAR will be used. In one embodiment, the antigen binding domain is a scFv.

The term “antigen binding portion”, “antigen binding region” and ’’antigen binding domain” are used herein interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi- specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb, which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen binding portion” of an antibody. In certain embodiments of the invention, scFv molecules are incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.

Each one of the VH and VL domains comprises 3 complementarity-determining regions (CDRs). As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain (HC) and the light chain (LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901- 917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or Hl, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).

The extracellular spacer or hinge region of a CAR is located between the antigen binding domain and a transmembrane domain. Extracellular spacer domains may include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof.

The term "transmembrane domain" refers to the region of the CAR, which crosses or bridges the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein, an artificial hydrophobic sequence or a combination thereof.

The term “intracellular domain” refers to the intracellular part of the CAR comprising an activation domain capable of activating T cells and optionally additional co-stimulatory domain(s). The intracellular domain may be an intracellular domain of a T cell receptor (e.g. the zeta chain associated with the T cell receptor complex) and/or may comprise stimulatory domains of other receptor (e.g., TNFR superfamily members) or a portion thereof, such as an intracellular activation domain (e.g., an immunoreceptor tyrosine-based activation motif (IT AM) -containing T cell activating motif), an intracellular costimulatory domain, or both. According to some embodiments, the costimulatory domain is selected from a costimulatory domain of CD28, 4-1BB, 0X40, iCOS, CD27, CD80 and CD70. According to one embodiment, the costimulatory domain is a costimulatory domain of CD28. According to one another, the costimulatory domain a costimulatory domain of 4- IBB.

The terms “binds specifically” or “specific for” with respect to an antigen-binding domain of a CAR or of an antibody, or of a fragment thereof refers to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. The term encompasses that the antigen-binding domain binds to the antibody-recognizing portion of its antigen (epitope) with high affinity, and does not bind to other unrelated epitopes with high affinity.

The CAR may be specific in some embodiments to a tumor-associated antigen. The terms “tumor-associated antigen” and “TAA” are used herein interchangeably and refer to any antigen, which is found in significantly higher concentrations in or on tumor cells than on normal cells. According to any one of the above embodiments, the CAR of the CAR T- cells specifically binds to a tumor-associated antigen (TAA). Any CAR that binds to a TAA may be used according to the teaching of the present invention. According to one embodiment, the TAA is HER2. According to another embodiment, the CAR of the CAR T-cell specifically binds to a tumor-associated antigen selected from CD19, CD38 and CD24. Other non-limiting examples of CARs that may be used are CAR against an antigen selected from MUC1, Mesothelin, PSCA, EGFR, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, HER2, R0R1, CD70, CD38, CD138, CD24, and CD19. According to one embodiment, the TAA is CD276. According to one embodiment, the TAA is EGFR. According to one embodiment, the TAA is CD 19.

The term “T cell” as used herein refers to a lymphocyte that expresses T-cell receptors and participates in a variety of cell-mediated immune reactions, as well known in art. T cells may include CD4⁺ T-cells, CD8⁺ T-cells and natural killer T-cells. The term encompasses genetically modified T-cells, e.g. transduced with a nucleic acid such as DNA or RNA, optionally using a vector. The term “CAR T cell” refers to a T-cell expressing a CAR. In certain embodiments, the invention relates to CAR T cells comprising a population of CD8⁺ T-cells.

As described above, the activated EVs are considered to comprise at least a part of the molecular contents of the parental cells. According to some embodiments, the activated EVs of the present invention comprise the chimeric antigen receptor (CAR) of the original CAR T- cell (in at least a subset of the EVs population as 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 comprise the chimeric antigen receptor (CAR) of the original CAR T-cell. According to one embodiment, the CAR is presented on the outer membrane of the EVs. According to some embodiments, at least 10% of the isolated activated EVs of the present invention comprise the chimeric antigen receptor. According to some embodiments, at least 15% of the isolated activated EVs of the present invention comprise the chimeric antigen receptor. According to some embodiments, at least 18% of the isolated activated EVs of the present invention comprise the chimeric antigen receptor. According to some embodiments, at least 20% of the isolated activated EVs of the present invention comprise the chimeric antigen receptor. According to some embodiments, at least 3%, at least 5%, at least 10% of the isolated activated EVs of the present invention present the chimeric antigen receptor. According to some embodiments, at least 15% of the isolated activated EVs of the present invention present the chimeric antigen receptor. According to some embodiments, at least 25% or at least 30% of the isolated activated EVs of the present invention comprise the chimeric antigen receptor. According to some embodiments, from 10% to 90% of EVs present the CAR. According to some embodiments, from 15% to 85%, from 20% to 80%, from 25% to 75%, from 30% to 60%, from 20% to 60%, from 20% to 50%, from 15% to 45%, from 15% to 40% of the EVs present the CAR. According to some embodiments, the CAR is anti-HER2 CAR. According to one embodiment, the CAR is N29 CAR.

The terms "HER2" and “human HER2” are used herein interchangeably and refer to the protein known as human epidermal growth factor receptor 2, receptor tyro sine-protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (HER2) and has an extension number EC 2.7.10.1. The terms “anti Her2” or “aHer2” refers to an antigen binding domain of a CAR or of an antibody that binds specifically to human Her2. According to some embodiments, HER2 protein has an amino acid sequence SEQ ID NO: 85. According to some embodiments, the term HER2 protein encompasses also a fragment of the HER2 protein to which the CAR binds specifically. According to some embodiments, the fragment is an extracellular fragment, i.e., exposed to extracellular matrix. According to some embodiments, the extracellular fragment may refer to a complete extracellular fragment or to some section/domain thereof. According to some embodiments, the fragment comprises the amino acid sequence SEQ ID NO: 84.

For the simplicity of understanding of the invention, the reference to all SEQ ID numbers are provided in Sequence Table at the end of the detailed description.

According to some embodiments, the EVs of the present invention are originated from CAR T cell, wherein the CAR binds specifically to HER2 (anti-HER2 CAR). According to one embodiment, the anti-HER2 CAR comprises 3 complementarity determining regions (CDRs) of a light variable chain having amino acid sequence SEQ ID NO: 55 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 56. According to one embodiment, the CAR that binds specifically to HER2 is N29 CAR, as known in the art, e.g., as described in Globerson-Levin A, et al., Molecular therapy, 2014; 22(5); 1029-38. In general, N29 is a monoclonal antibody binding specifically human HER2 receptor, and N29 CAR comprises a scFv of said N29 antibody as an antigen binding domain. According to some embodiments, the CAR T-cells express N29 CAR (N29 CAR T-cells). According to one embodiment, the activated EVs are derived from activated N29 CAR T-cells. According to some embodiments, the N29 CAR has amino acid sequence SEQ ID NO: 57. According to other embodiments, the N29 CAR is encoded by DNA sequence SEQ ID NO: 58. According to some embodiments, the EVs of the present invention are originated from stimulated CAR T cell, wherein the CAR binds specifically to EGFR (anti-EGFR CAR). According to one embodiment, the CAR that binds specifically to EGFR comprises an ABD of the Cetuximab CAR. According to one embodiment, the anti-EGFR CAR comprises the antigen binding domain of an antibody disclosed in US11045543B2. According to one embodiment, the anti-EGFR CAR comprises 3 complementarity determining regions (CDRs) of a light variable chain having amino acid sequence SEQ ID NO: 11 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 12. According to one embodiment, the anti-EGFR CAR comprises 3 complementarity determining regions (CDRs) of a light variable chain having amino acid sequence SEQ ID NO: 16 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 17. According to one embodiment, the activated EVs are derived from stimulated anti-EGFR CAR T-cells. According to some embodiments, the anti-EGFR comprises a VL domain comprising the amino acid sequence SEQ ID NO: 11 and a VL domain comprising the amino acid sequence SEQ ID NO: 12. According to some embodiments, the anti-EGFR comprises an antigen binding domain comprising the amino acid sequence SEQ ID NO 13. According to some embodiments, the anti-EGFR comprises the amino acid sequence SEQ ID NO: 14. According to some embodiments, the anti-EGFR comprises the amino acid sequence SEQ ID NO: 15.

According to some embodiments, the anti-EGFR comprises a VL domain comprising the amino acid sequence SEQ ID NO: 16 and a VL domain comprising the amino acid sequence SEQ ID NO: 17. According to some embodiments, the anti-EGFR comprises an antigen binding domain comprising the amino acid sequence SEQ ID NO 18. According to some embodiments, the anti-EGFR comprises the amino acid sequence SEQ ID NO: 19. According to some embodiments, the anti-EGFR comprises the amino acid sequence SEQ ID NO: 20.

According to some embodiments, the EVs of the present invention are originated from stimulated CAR T cells, wherein the CAR binds specifically to CD276 (anti-CD276 CAR). According to one embodiment, the CAR that binds specifically to CD276 comprises an ABD of the 8H9 humanized hybridoma antibody. According to one embodiment, the anti-CD276 CAR comprises the antigen-binding domain of Enoblituzumab.

According to one embodiment, the anti-CD276CAR comprises 3 complementarity determining regions (CDRs) of a light variable chain having amino acid sequence SEQ ID NO: 1 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 2. According to some embodiments, the anti-CD276 CAR comprises a VL domain comprising the amino acid sequence SEQ ID NO: 1 and a VL domain comprising the amino acid sequence SEQ ID NO: 2. According to some embodiments, the anti-CD276 CAR comprises an antigen binding domain comprising the amino acid sequence SEQ ID NO 3. According to some embodiments, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 4. According to some embodiments, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 5.

According to one embodiment, the anti-CD276CAR comprises 3 complementarity determining regions (CDRs) of a light variable chain having amino acid sequence SEQ ID NO: 5 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 6. According to some embodiments, the anti-CD276 CAR comprises a VL domain comprising the amino acid sequence SEQ ID NO: 6 and a VH domain comprising the amino acid sequence SEQ ID NO: 7. According to some embodiments, the anti-CD276 CAR comprises an antigen binding domain comprising the amino acid sequence SEQ ID NO 8. According to some embodiments, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 9. According to some embodiments, the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 10.

According to some embodiments, the EVs of the present invention are originated from simulated CAR T cells, wherein the CAR binds specifically to CD 19 (anti-CD19 CAR). According to one embodiment, the anti-CD19 CAR comprises 3 CDRs of a light variable chain having amino acid sequence SEQ ID NO: 21 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 22. According to some embodiments, the anti-CD19 CAR comprises a VL domain comprising the amino acid sequence SEQ ID NO: 21 and a VL domain comprising the amino acid sequence SEQ ID NO: 22. According to some embodiments, the anti-CD19 CAR comprises an antigen binding domain comprising the amino acid sequence SEQ ID NO 23. According to some embodiments, the anti-CD19 comprises the amino acid sequence SEQ ID NO: 24.

According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the EVs have a particle size diameter of from 150 to 1000 nm and wherein the CAR is selected from anti-EGFR CAR, anti-CD276 CAR and anti-CD19 CAR. According to some embodiments, at least 30% of the EVs have a particle size diameter of from about 150 to 1000 nm. According to some embodiments, at least 35% of the EVs have a particle size diameter of from about 150 to 1000 nm. According to some embodiments, the present invention provides a population of isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 30%, at least 35% or at least 45% of the EVs have a particle size diameter of from about 150 to 1000 nm and the rest have a particle size diameter of from 30 to about 150 and wherein the CAR is selected from anti-EGFR CAR, anti-CD276 CAR and anti-CD19 CAR. According to any one of the above embodiments, the CAR T-cells are stimulated CAR T-cells. The terms “pre- stimulated”, “pre-activated”, "stimulated" and "activated" with respect to CAR T-cells are used herein interchangeably and refer to CAR T-cells that have been incubated with and therefore stimulated with a tumor-associated antigen to which the CAR binds specifically. Furthermore, the term refers to a state of the T-cells provided with a CAR-mediated stimulation prior to EVs isolation. Such stimulated CAR T cells may also be denoted as "specifically stimulated CAR T cell" and the EVs obtained from the stimulated CAR T cells are denoted as activated EVs, although the term activated with respect to EVs may be omitted in some of the embodiments. The stimulation is effected (performed) by incubation of CAR T cells with a specific tumor-associated antigen for a period of time sufficient to activate the T- cells, as known in the art. According to some embodiments, stimulation is performed by any known method. In some examples, stimulation is performed by incubating the CAR T cells with cells expressing or overexpressing the TAA to which CAR binds specifically. In some examples, stimulation is performed by incubating the CAR T cells with a carrier coated with (i.e. bound to) with TAA to which the CAR binds specifically. In some examples, stimulation is performed by incubating the CAR T cells with a carrier coated with a fragment of a TAA, wherein the CAR binds specifically to said fragment. Non-limiting examples of such a carrier are beads, support, petty dishes, a column or any other possible carrier. The beads may have any form, e.g. spherical or elliptic, and size. According to some embodiments, the beads are spherical and have a size in the range of several micron to several hundreds of microns. According to some embodiments, the beads are spherical and have a size of from 1 to 500 pm. According to some embodiments, the beads are spherical and have a size of from 5 to 300 pm, from 10 to 200 pm, from 15 to 100 pm, from 20 to 50 pm or from 10 to 100 pm or from 10 to 50 pm or about 35 pm. According to some embodiments, stimulation is performed by incubating the CAR T cells with fragments of cell membrane presenting the TAA to which the CAR binds specifically. The term "fragment of cell membrane" refers to any bilayer comprising the TAA. In some embodiments, the fragment of cell membrane is obtained by lysing cells comprising the TAA or rupturing cell membrane of cells comprising the TAA and subsequently purifying the fragments. This can be done by any known method. Stimulation of CAR T cells with cell membrane fragment may be used as an alternative to stimulation with cells or beads. Stimulation of CAR T cells with cell membrane fragment have the same advantages as stimulation with beads and in some cases may have additional advantages.

According to some embodiments, the incubation is performed for from 3 to 96, from 6 to 72, or from 12 to 48 hours, e.g. for 24 hours. According to some embodiments, the incubation is performed for from 48 to 96 or from 60 to 84 hours. Stimulation can for example be associated with induced cytokine production, elevation levels of IL-2, IL-5, IL-8, IL- 12, IL-17, IL-21, MCP-1 (CCL2), MIP-la (CCL3), MIP-lp (CCL4), RANTES (CCL5), MIG (CXCL9), IP10 (CXCL10), fractalkine (CX3CL1), G-CSF, GM-CSF, Flt-3L, IL-IRa, and/or TNFa, elevated expression of receptors such as CD25 (the IL-2 receptor) and CD71 (the transferrin receptor), elevated expression of co-stimulatory molecules such as CD26, CD27, CD28, CD30, CD154 or CD40L, and CD134, and detectable effector functions. Stimulation can, for example, be associated with elevated level of Granzyme in EVs. With respect to T- cells, “activation” may have also the meaning of the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation.

The terms “stimulated” and “activated” with respect to EVs mean that the EVs have been obtained from stimulated CAR T-cells as described above. Activated EVs as described herein typically manifest improved properties (e.g. anti-cancer properties characteristic of their parent CAR T-cells) compared to native, non-activated EVs (EVs obtained from nonactivated CAR T-cells). As discussed above, the activated EVs may include the content or the partial content of their parent CAR T-cells. Thus, in some embodiments, the EVs comprise or express elevated levels of cytokines and/or receptors as in their parent-activated CAR T-cells. Without wishing to be bound by a specific theory or mechanism of action, activated EVs may be distinguished from non-activated EVs by the presence of surface markers and/or intracellular markers. For example, without limitation, activated EVs may contain or express increased levels of T cell activation markers, e.g. CD25 and/or CD137 (41-BB), CD3, CD38 and HLADR. In some embodiments, the EVs present an anti-EGFR CAR. In some embodiments, the EVs present anti-CD276 CAR. In some embodiments, the EVs present an anti-HER2 CAR. In some embodiments, the EVs present anti-CD19 CAR. According to some embodiments, the CARs are as described hereinabove.

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

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

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

According to some embodiments, at least 15 % or at least 20 % of the EVs of the present invention express HLADR antigen and at least 8% of the EVs of the present invention express CD38 antigen, on their outer membrane.

According to the teaching of the present invention, activated EVs are those obtained from CAR T-cells stimulated by incubation with their corresponding TAA, i.e., a TAA to which the CAR binds specifically and consequently stimulates the T-cells. According to one embodiment, the CAR-T cells were incubated with TAA from 6 to 48 or from 12 to 36 hours. According to one embodiment, the CAR-T cells were incubated with TAA from 48 to 96 or from 60 to 84 hours. In other embodiments, the T cells were incubated with a CAR-mediated stimulation no more than 24 hours prior to EV collection, e.g. up to 18, 12 or 6 hours prior to EVs isolation. According to certain exemplary embodiments, the T cells have been stimulated by cells (e.g. tumor cells or antigen-presenting cells) presenting the TAA. According to certain exemplary embodiments, the T cells are stimulated by TAA expressed or presented by an entity such as liposomes or TAA attached to a surface of an entity such as a plate. According to certain exemplary embodiments, the T cells are stimulated by a surface presenting the TAA to which the CAR binds specifically. According to one embodiment, the TAA is HER2 and the CAR is N29 CAR. According to another embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with ovarian cancer cells expressing HER2. According to one embodiment, the ovarian cancer cells are SKOV cells. According to another embodiment, the CAR is N29 CAR and the CAR T-cells are incubated with HER2 positive breast cancer cells. According to another embodiment, the anti-HER2 CAR is N29 CAR and the CAR T-cells are incubated with HER2 antigen. According to another embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with HER2 such as recombinant protein conjugated to the beads protein. According to another embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with a fragment of HER2 protein to which N29 CAR binds specifically. According to some embodiments, the EVs are derived from stimulated N29 CAR T-cells. According to one embodiment, the EVs are derived from N29 CAR T-cells activated with HER2 positive ovarian cancer cells. According to one embodiment, the EVs are derived from N29 CAR T-cells stimulated with SKOV cells. According to one embodiment, the EVs are derived from N29 CAR T-cells activated with HER2 breast cancer cells. According to one embodiment, the EVs are derived from N29 CAR T-cells stimulated with primary HER2 positive cancer cells. According to one embodiment, the EVs are derived from N29 CAR T- cells stimulated with beads bound and presenting HER2 protein. According to one embodiment, the EVs are derived from N29 CAR T-cells stimulated with beads bound and presenting a fragment of HER2 protein to which N29 CAR binds specifically. According to some embodiments, the HER2 comprises the amino acid sequence SEQ ID NO: 84 or 85. According to one embodiment, the primary HER2 positive cancer cells are cells obtained from a subject suffering from said 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. According to some embodiments, the N29 comprises the amino acid sequence SEQ ID NO: 103.

According to one embodiment, the TAA is EGFR and the CAR is anti-EGFR CAR as described herein above, e.g. having an ABD of Cetuximab or of an antibody disclosed in US11045543B2. According to another embodiment, the CAR is anti-EGFR and the CAR T- cells were incubated with lung adenocarcinoma cells expressing EGFR. Non-limiting examples of such lung adenocarcinoma cells are A549, H1975, and HCC827 According to one embodiment, the anti-EGFR CAR T-cells were incubated with glioblastoma cells expressing EGFR. Non-limiting example of such glioblastoma cells is U251. According to another embodiment, the CAR is anti-EGFR CAR and the CAR T-cells were incubated with EGFR protein. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83. According to some embodiments, the term EGFR protein encompasses also a fragment of the EGFR protein to which the anti-EGFR CAR binds specifically. According to some embodiments, the fragment of EGFR is an extracellular fragment, i.e. exposed to extracellular matrix. According to some embodiments, the extracellular fragment may refer to a complete extracellular fragment or to some section/domain thereof. According to another embodiment, the CAR is anti-EGFR CAR and the CAR T-cells were incubated with a fragment of EGFR protein to which EGFR CAR binds specifically.

According to some embodiments, the EVs are derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises 3 CDRs of a light variable chain having amino acid sequence SEQ ID NO: 11 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 12. According to some embodiments, the EVs are derived from stimulated anti- EGFR CAR T-cells, wherein the CAR comprises the amino acid sequences SEQ ID NOs: 11 and 12, or comprising the ABD comprising the amino acid sequence SEQ ID NO: 13, or wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 14 and 15. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with adenocarcinoma cells. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with glioblastoma cells. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with beads coated with and presenting EGFR protein of a fragment thereof. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with beads coated with and presenting a fragment of EGFR protein to which the anti-EGFR CAR binds specifically. According to some embodiments, the fragment of EGFR protein is an extracellular fragment of the EGFR protein comprising the amino acid sequence SEQ ID NO: 83. 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.

According to some embodiments, the EVs are derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises 3 CDRs of a light variable chain having amino acid sequence SEQ ID NO: 16 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 17. According to some embodiments, the EVs are derived from stimulated anti- EGFR CAR T-cells, wherein the CAR comprises the amino acid sequences SEQ ID NOs: 16 and 17, or comprising the ABD comprising the amino acid sequence SEQ ID NO: 18, or wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 19 and 20. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with adenocarcinoma cells. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with glioblastoma cells. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with beads coated with and presenting EGFR protein. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83. According to one embodiment, the EVs are derived from the anti-EGFR CAR T-cells stimulated with beads coated with and presenting a fragment of EGFR protein to which the anti-EGFR CAR binds specifically. According to some embodiments, the fragment of EGFR protein is an extracellular fragment of the EGFR protein comprising the amino acid sequence SEQ ID NO: 83. 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-cell.

According to one embodiment, the TAA is CD276 and the CAR is anti-CD276 CAR as defined above. According to another embodiment, the CAR is anti-CD276 and the CAR T- cells were incubated with lung adenocarcinoma cells expressing CD276. Non-limiting examples of such lung adenocarcinoma cells are A549, H1975, and HCC827 According to one embodiment, the anti-CD276 CAR T-cells were incubated with glioblastoma cells expressing CD276. Non-limiting example of such glioblastoma cells is U251. According to another embodiment, the CAR is anti-CD276 CAR and the CAR T-cells were incubated with CD276 protein. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82. According to some embodiments, the term CD276 protein encompasses also a fragment of the CD276 protein to which the anti-CD276 CAR binds specifically. According to some embodiments, the fragment is an extracellular fragment, i.e. exposed to extracellular matrix. According to some embodiments, the extracellular fragment may refer to a complete extracellular fragment or to some section/domain thereof. According to another embodiment, the CAR is anti-CD276 CAR and the CAR T-cells were incubated with a fragment of CD276 protein to which anti-CD276 CAR binds specifically.

According to some embodiments, the EVs are derived from stimulated anti-CD276 CAR T-cells, wherein the CAR comprises 3 CDRs of a light variable chain having the amino acid sequence SEQ ID NO: 1 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 2. According to some embodiments, the EVs are derived from stimulated anti- CD276 CAR T-cells, wherein the CAR comprises the amino acid sequences SEQ ID NOs: 1 and 2, or comprising an ABD comprising the amino acid sequence SEQ ID NO: 3, or wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 4 and 5. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with adenocarcinoma cells. According to one embodiment, the EVs are derived from the anti- CD276 CAR T-cells stimulated with glioblastoma cells. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with beads coated with and presenting CD276 protein. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with beads coated with and presenting a fragment of CD276 protein to which the anti-CD276 CAR binds specifically. According to some embodiments, the fragment of CD276 protein is an extracellular fragment of the CD276 protein comprising the amino acid sequence SEQ ID NO: 82. 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-cell.

According to some embodiments, the EVs are derived from stimulated anti-CD276 CAR T-cells, wherein the CAR comprises 3 CDRs of a light variable chain having amino acid sequence SEQ ID NO: 6 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 7. According to some embodiments, the EVs are derived from stimulated anti- CD276 CAR T-cells, wherein the CAR comprises the amino acid sequences SEQ ID NOs: 6 and 7, or comprising the ABD comprising the amino acid sequence SEQ ID NO: 8, or wherein the CAR comprises an amino acid sequence selected from SEQ ID NOs: 9 and 10. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with adenocarcinoma cells. According to one embodiment, the EVs are derived from the anti- CD276 CAR T-cells stimulated with glioblastoma cells. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with beads coated with and presenting CD276 protein. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82. According to one embodiment, the EVs are derived from the anti-CD276 CAR T-cells stimulated with beads coated with and presenting a fragment of CD276 protein to which the anti-CD276 CAR binds specifically. According to some embodiments, the fragment of CD276 protein is an extracellular fragment of the CD276 protein comprising the amino acid sequence SEQ ID NO: 82. 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-cell.

According to one embodiment, the TAA is CD 19 and the CAR is anti-CD19 CAR as defined above. According to another embodiment, the CAR is anti-CD19 and the CAR T-cells were incubated with B cell lymphomas cells expressing CD19. According to one embodiment, the anti-CD19 CAR T-cells were incubated with acute lymphoblastic leukemia cells expressing CD19. According to one embodiment, the anti-CD19 CAR T-cells were incubated with chronic lymphocytic leukemia cells expressing CD19. According to another embodiment, the CAR is anti-CD19 CAR and the CAR T-cells were incubated with CD 19 protein. According to some embodiments, the CD 19 protein comprises the amino acid sequence SEQ ID NO: 80. According to some embodiments, the term CD19 protein encompasses also a fragment of the CD 19 protein to which the anti-CD19 CAR binds specifically. According to some embodiments, the fragments is an extracellular fragment, i.e. exposed to extracellular matrix. According to some embodiments, the extracellular fragment may refer to a complete extracellular fragment or to some section/domain thereof. According to another embodiment, the CAR is anti-CD19 CAR and the CAR T-cells were incubated with a fragment of CD 19 protein to which anti-CD19 CAR binds specifically. According to some embodiments, the fragment of CD19 comprises the amino acid sequence SEQ ID NO: 81. According to some embodiments, the B cell lymphoma cells and large B cell lymphoma (LBCL) are either cells of relapsed or refractory lymphoma.

According to some embodiments, the EVs are derived from stimulated anti-CD19 CAR T-cells, wherein the CAR comprises 3 CDRs of a light variable chain having amino acid sequence SEQ ID NO: 21 and 3 CDRs of a heavy variable chain having amino acid sequence SEQ ID NO: 22. According to some embodiments, the EVs are derived from stimulated anti- CD19 CAR T-cells, wherein the CAR comprises the amino acid sequences SEQ ID NOs: 21 and 22, or comprising the ABD comprising the amino acid sequence SEQ ID NO: 23, or wherein the CAR comprises an amino acid sequence of SEQ ID NOs: 24. According to one embodiment, the EVs are derived from the anti-CD19 CAR T-cells stimulated with adenocarcinoma cells. According to one embodiment, the EVs are derived from the anti-CD19 CAR T-cells stimulated with cells selected from B cell lymphomas cells, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). According to one embodiment, the EVs are derived from the anti-CD19 CAR T-cells stimulated with beads coated with and presenting CD 19 protein. According to some embodiments, the CD 19 protein comprises the amino acid sequence SEQ ID NO: 80. According to one embodiment, the EVs are derived from the anti-CD19 CAR T-cells stimulated with beads coated with and presenting a fragment of CD 19 protein to which the anti-CD19 CAR binds specifically. According to some embodiments, the fragment of CD19 comprises the amino acid sequence SEQ ID NO: 81. 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-cell.

According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the EVs obtained from stimulated CAR T cells stimulated of the present invention comprise the CAR. According to some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of the activated EVs of the present invention comprise the CAR. According to some embodiments, from 20 to 90%, from 30 to 85% or, from 40 to 90% of the resulting activated EVs of the present invention comprise the CAR. According to some embodiments, the present invention provides EVs, wherein the EVs are derived from activated N29 CAR T-cells, wherein at least 20% of the EVs have size of above 150 nm. According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells as described above, wherein at least 22% or at least 25%of the EVs have a size of from 150 nm to 1000 nm.

According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T-cells, stimulated anti-CD19 CAR T-cells or stimulated anti-CD276 CAR T-cells wherein at least 25% of the EVs have size of above 150 nm. According to some embodiments, at least 27% 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 of 150 nm or more. 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 of above 150 nm. According to another embodiment, at least 42 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 45 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 50 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 55 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 60 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 65% or at least 70 % of the activated EVs have a size of above 150 nm. According to some embodiments, from 22 to 70% or from 25 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, from 25 to 35% of the activated EVs have a size of above 150 nm. According to certain embodiments, from 25 to 45% of the activated EVs have a size of above 150. According to some embodiments, from 30 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 35 to 65% of the activated EVs have a size of above 150 nm. According to other embodiments, the mean size of the activated EVs is 135 nm or more. According to other embodiments, the mean size of the activated EVs is 140 nm or more. According to some embodiments, the mean size of the activated EVs is 145 nm or more. According to certain embodiments, the mean size of the activated EVs is 150 nm or more. According to one embodiment, the mean size of the activated EVs is 155 nm or more, 160 nm or more, or 165 nm or more. According to one embodiment, the mean size of the activated EVs is above 160 nm or above 170 nm. According to any one of the above embodiments, the EVs has a size up to 1000 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti- CD276 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 diameter size of above 150 nm and the EVs have the mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more, 167 nm or more and 170 nm 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 of above 150 nm and the EVs have the mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, stimulated anti- EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells, wherein at least 29% or at least 30% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 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 of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs have the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti- CD276 CAR T cells wherein at least 22% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, stimulated anti- EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated N29 CAR T-cells, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 35% of the EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti- CD276 CAR T cells have a particle size of above 150 nm and the EVs have a mean size of more than 150 nm or 155 nm or more or 160 nm or more. According to some embodiments, at least 40% of the EVs derived from activated N29 CAR T-cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 45% or at least 60% of the EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 55% of the EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti- CD276 CAR T cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 60% of the EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells have a size of above 150 nm and the EVs have a mean 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 of above 150 nm, the EVs have a mean size of 132 nm or more, 135 nm or more, 137 nm or more, 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 and the ratio between EVs having the size of above 150 nm and EV having the size of below 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 stimulated EVs of the present invention comprise the N29 CAR, anti-EGFR CAR, anti-CD19 CAR or anti- CD276 CAR. According to some embodiments, at least 15% of the EVs of the EVs derived from stimulated N29 CAR T-cells, stimulated anti-EGFR CAR T cells, stimulated anti-CD19 CAR T cells or stimulated anti-CD276 CAR T cells express CD3 antigen on their outer membrane. According to some embodiments, at least 20% of the EVs of the EVs derived from activated N29 CAR T-cells express HLADR antigen on their outer membrane. According to some embodiments, at least 8% of the EVs of the EVs derived from activated N29 CAR T- cells express CD38 antigen on their outer membrane. According to some embodiments, at least 20 % of the EVs of the present invention express CD3 antigen on their outer membrane, at least 20 % of the EVs of the present invention express HLADR antigen on their outer membrane and at least 10% of the EVs of the present invention express CD38 antigen on their outer membrane. According to some embodiments, the N29 CAR T are activated by incubation with HER2 positive cancer cells. According to some embodiments, the 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 stimulated extracellular vesicles derived from N29 CAR-T cells incubated from 12 to 96 hours with ovarian cancer cells expressing HER2, wherein the EVs are isolated within 24 hours post incubation. According to another embodiment, the N29 CAR-T cells were incubated from 12 to 36 hours with breast cancer cells expressing HER2, wherein the EVs are isolated within 24 hours post incubation. According to some embodiments, the EVs were isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after the incubation. According to other embodiments, the EVs are isolated within 1, 2, 3, 4, 5, 6 or 7 days after the incubation. For example, T cells may be incubated at a ratio of T cells to target cells/beads coated with HER2 of 1:1, 1.5:1 to 3:1, e.g. 2:1. According to one embodiment, the T cells are incubated with target cells at a ratio of T cells to target cells of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1. According to one embodiment, the T cells are incubated with target cells or beads coated with HER2 protein or a fragment thereof at a ratio of T cells to target cells/bead of from 3:1 to 1:3, 2:1 to 1:2 or about 1:1. According to some embodiment, the beads are coated with HER2 protein or fragments thereof, e.g. comprising the amino acid sequences SEQ ID NO: 85and 84, respectively. According to some embodiments, at least 25% or at least 30% of the EVs obtained from stimulated CAR T cells present invention comprise the N29 CAR. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the EVs obtained from stimulated CAR T cells of the present invention comprise the N29 CAR. According to some embodiments, at least 55% of the activated EVs obtained from stimulated CAR T cells of the present invention comprise the N29 CAR. According to some embodiments, at least 60% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 65% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 70% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 75% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 80% of the activated EVs obtained from stimulated CAR T cells of the present invention comprise the N29 CAR. According to some embodiments, at least 85% of the activated EVs of the present invention comprise the N29 CAR.

According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated anti-EGFR CART-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, wherein at least 25% of the EVs have size of from 150 nm to 1000 nm. According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated anti-EGFR CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, wherein at least 25% of the EVs have size of above 150 nm. According to some embodiments, at least 27% 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 of 150 nm or more. 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 of above 150 nm. According to another embodiment, at least 42 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 45 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 50 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 55 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 60 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 62 or at least 65 % of the activated EVs have a size of above 150 nm. According to some embodiments, from 22 to 70% or from 25 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, from 25 to 35% of the activated EVs have a size of above 150 nm. According to certain embodiments, from 25 to 45% of the activated EVs have a size of above 150. According to some embodiments, from 30 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 35 to 65% of the activated EVs have a size of above 150 nm. According to one embodiment, from 50 to 75% of the activated EVs have a size of above 150 nm. According to one embodiment, from 50 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 55 to 75% of the activated EVs have a size of above 150 nm. According to one embodiment, from 60 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, the mean size of the activated EVs is 135 nm or more. According to other embodiments, the mean size of the activated EVs is 140 nm or more. According to some embodiments, the mean size of the activated EVs is 145 nm or more. According to certain embodiments, the mean size of the activated EVs is 150 nm or more. According to one embodiment, the mean size of the activated EVs is 155 nm or more, 160 nm or more, or 165 nm or more. According to one embodiment, the mean size of the activated EVs is above 160 nm or above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 and 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 diameter size of above 150 nm and the EVs have the mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more, 167 nm or more and 170 nm or more. According to some embodiments, the present invention provides isolated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 and wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 29% or at least 30% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 35% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs have the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 22% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-EGFR CAR T-cells, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 35% of the EVs derived from stimulated anti-EGFR CAR T-cells have a particle size of above 150 nm and the EVs have a mean size of more than 150 nm or 155 nm or more or 160 nm or more wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 40% of the EVs derived from stimulated anti-EGFR CAR T-cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 45% or at least 60% of the EVs derived from the stimulated anti-EGFR CAR T-cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 25% to 80% of EVs having an average particle size diameter of from 150 to 1000 nm and from 20 to 75% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 35% to 65% of EVs having an average particle size diameter of from 150 to 1000 nm and from 35 to 65% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 40% to 60% of EVs having an average particle size diameter of from 150 to 1000 nm and from 40 to 60% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, at least 55% of the EVs derived from stimulated the anti-EGFR CAR T-cells have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 60% of the EVs derived from stimulated anti-EGFR CAR T-cells have a size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 25% of the EVs have a size of above 150 nm, the EVs have a mean size of 132 nm or more, 135 nm or more, 137 nm or more, 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 and the ratio between EVs having the size of above 150 nm and EV having the size of below 150 nm is from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, 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 the anti-EGFR CAR. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 55% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 60% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 65% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 70% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 75% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 80% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 85% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 90% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, at least 95% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to some embodiments, from 50% to 95, from 55 to 90%, from 60 to 85% or from 65 to 80% of the activated EVs of the present invention comprise the anti-EGFR CAR. According to another embodiment, the CAR is anti-EGFR and the CAR T-cells were incubated with lung adenocarcinoma cells expressing EGFR. Non-limiting examples of such lung adenocarcinoma cells are A549, Hl 975, and HCC827 According to one embodiment, the anti-EGFR CAR T- cells were incubated with glioblastoma cells expressing EGFR. Non-limiting examples of such glioblastoma cells is U251. According to another embodiment, the CAR is anti-EGFR CAR and the CAR T-cells were incubated with EGFR protein. According to another embodiment, the CAR is anti-EGFR CAR and the CAR T-cells were incubated with a fragment of EGFR protein to which EGFR CAR binds specifically. According to one embodiment, the EVs are derived from anti-EGFR CAR T-cells stimulated with beads coated with and presenting EGFR protein, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to one embodiment, the EVs are derived from anti-EGFR CAR T- cells stimulated with beads coated with and presenting a fragment of EGFR protein to which the anti-EGFR CAR binds specifically, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some exemplary embodiments, the present invention provides isolated stimulated extracellular vesicles derived from stimulated anti-EGFR CAR-T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 and incubated from 12 to 96 hours with cancer cells or beads presenting EGFR or a fragment thereof as described above, wherein the EVs are isolated within 24 hours post incubation. According to another embodiment, the stimulated anti-EGFR CAR-T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 and were incubated from 48 to 84 hours as described above and the EVs are isolated within 24 hours post incubation. According to some embodiments, the EVs were isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after the incubation. According to other embodiments, the EVs are isolated within 1, 2, 3, 4, 5, 6 or 7 days after the incubation. For example, T cells may be incubated at a ratio of T cells to target cells or beads coated with target protein of 3:1 to 1:3, or 1.5:1 to 3:1, e.g. 2:1. According to one embodiment, the T cells are incubated with target cells or beads coated with the target protein at a ratio of T cells to target cells of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1. According to some embodiments, the stimulated anti-EGFR CAR T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 were stimulated 2 or 3 cycles with the beads. According to some embodiments, the beads are coated with EGFR protein having the amino acid sequence SEQ ID NO: 500 or with an extracellular fragment of the protein.

According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated anti-CD276 CART -cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 22% of the EVs have size of above 150 nm. According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have size of above 150 nm. According to some embodiments, at least 27% 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 of 150 nm or more. 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 of above 150 nm. According to another embodiment, at least 42 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 45 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 50 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 55 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 60 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 65% or at least 70 % of the activated EVs have a size of above 150 nm. According to some embodiments, from 22 to 70% or from 25 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, from 25 to 35% of the activated EVs have a size of above 150 nm. According to certain embodiments, from 25 to 45% of the activated EVs have a size of above 150. According to some embodiments, from 30 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 35 to 65% of the activated EVs have a size of above 150 nm. According to other embodiments, the mean size of the activated EVs is 135 nm or more. According to other embodiments, the mean size of the activated EVs is 140 nm or more. According to some embodiments, the mean size of the activated EVs is 145 nm or more. According to certain embodiments, the mean size of the activated EVs is 150 nm or more. According to one embodiment, the mean size of the activated EVs is 155 nm or more, 160 nm or more, or 165 nm or more. According to one embodiment, the mean size of the activated EVs is above 160 nm or above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, 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 diameter size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more, 167 nm or more and 170 nm or more. According to some embodiments, the present invention provides isolated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 29% or at least 30% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 35% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs has the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 22% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 35% of the EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 have a particle size of above 150 nm and the EVs have a mean size of more than 150 nm or 155 nm or more or 160 nm or more. According to some embodiments, at least 40% of the EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 45% or at least 60% of the EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 25% to 80% of EVs having an average particle size diameter of from 150 to 1000 nm and from 20 to 75% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 35% to 65% of EVs having an average particle size diameter of from 150 to 1000 nm and from 35 to 65% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 40% to 60% of EVs having an average particle size diameter of from 150 to 1000 nm and from 40 to 60% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, at least 55% of the EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and lOhave a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 60% of the EVs derived from stimulated anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 have a size of above 150 nm and the EVs have a mean 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 of above 150 nm, the EVs have a mean size of 132 nm or more, 135 nm or more, 137 nm or more, 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 and the ratio between EVs having the size of above 150 nm and EV having the size of below 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 the anti-CD276 CAR. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the activated

EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 55% of the activated EVs of the present invention comprise the anti-

CD276 CAR. According to some embodiments, at least 60% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 65% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 70% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 75% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 80% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 85% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 90% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, at least 95% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to some embodiments, from 50% to 95, from 55 to 90%, from 60 to 85% or from 65 to 80% of the activated EVs of the present invention comprise the anti-CD276 CAR. According to another embodiment, the CAR is anti-CD276 and the CAR T-cells were incubated with lung adenocarcinoma cells expressing CD276. Non-limiting examples of such lung adenocarcinoma cells are A549, Hl 975, and HCC827 According to one embodiment, the anti- CD276 CAR T-cells were incubated with glioblastoma cells expressing CD276. Non-limiting examples of such glioblastoma cells is U251. According to another embodiment, the CAR is anti-CD276 CAR and the CAR T-cells were incubated with CD276 protein. According to another embodiment, the CAR is anti-CD276 CAR and the CAR T-cells were incubated with a fragment of CD276 protein to which CD276 CAR binds specifically. According to one embodiment, the EVs are derived from anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 stimulated with beads coated with and presenting CD276 protein. According to one embodiment, the EVs are derived from anti-CD276 CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 stimulated with beads coated with and presenting a fragment of CD276 protein to which the anti-CD276 CAR binds specifically According to some exemplary embodiments, the present invention provides isolated stimulated extracellular vesicles derived from stimulated anti-CD276 CAR-T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 incubated from 12 to 96 hours with cancer cells or beads presenting CD276 or a fragment thereof as described above, wherein the EVs are isolated within 24 hours post incubation. According to another embodiment, the stimulated anti-CD276 CAR-T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 were incubated from 24 to 96, from 36 to 86, from 48 to 86, from 60 to 86 hours or about 72 hours as described above and the EVs are isolated within 24 hours post incubation. According to some embodiments, the EVs were isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after the incubation. According to other embodiments, the EVs are isolated within 1, 2, 3, 4, 5, 6 or 7 days after the incubation. For example, T cells may be incubated at a ratio of T cells to target cells or to beads coated with the target protein of about 1:1, from 1.5:1 to 3:1, e.g. 2:1, from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, about 2:3, or about 1:1. According to one embodiment, the T cells are incubated with target cells or beads coated with the target protein at a ratio of T cells to target cells/bead of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1. According to some embodiments, the stimulated anti-CD276 CAR T cells were stimulated 2 or 3 cycles with the beads. According to some embodiments, the stimulated anti-CD276 CAR T cells were stimulated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles with the beads. According to some embodiments, the CD276 protein to which the anti-CD276 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 82. According to some embodiments, the fragment of the CD276 protein is an extracellular fragment.

According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated anti-CD19 CART-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 22% of the EVs have size of above 150 nm. According to some embodiments, the present invention provides EVs, wherein the EVs are derived from stimulated the anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have size of above 150 nm. According to some embodiments, at least 27% 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 of 150 nm or more. 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 of above 150 nm. According to another embodiment, at least 42 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 45 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 50 % of the activated EVs have a size of above 150 nm. According to another embodiment, at least 55 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 60 % of the activated EVs have a size of above 150 nm. According to yet another embodiment, at least 65 or at least 70 % of the activated EVs have a size of above 150 nm. According to some embodiments, from 22 to 70% or from 25 to 70% of the activated EVs have a size of above 150 nm. According to other embodiments, from 25 to 35% of the activated EVs have a size of above 150 nm. According to certain embodiments, from 25 to 45% of the activated EVs have a size of above 150. According to some embodiments, from 30 to 70% of the activated EVs have a size of above 150 nm. According to one embodiment, from 35 to 65% of the activated EVs have a size of above 150 nm. According to other embodiments, a mean size of the activated EVs is 135 nm or more. According to other embodiments, a mean size of the activated EVs is 140 nm or more. According to some embodiments, a mean size of the activated EVs is 145 nm or more. According to certain embodiments, a mean size of the activated EVs is 150 nm or more. According to one embodiment, a mean size of the activated EVs is 155 nm or more, 160 nm or more, or 165 nm or more. According to one embodiment, a mean size of the activated EVs is above 160 nm or above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, 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 diameter size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more, 167 nm or more and 170 nm or more. According to some embodiments, the present invention provides isolated EVs derived from stimulated the anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 29% or at least 30% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 35% of the EVs have a size of above 150 nm and the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and above 170 nm. According to some embodiments, the terms "above X nm" and "X nm or more" when referring to EVs has the meaning of from about X nm to about 1000 nm. According to some embodiments, the rest of the EVs have an average size of below 150 nm and more specifically from about 30 to about 150 nm. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 22% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 135 or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 155 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 160 nm or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 165 nm or more. According to some embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have a size of above 150 nm and the EVs have a mean size of 147 nm or more. According to some embodiments, at least 35% of the EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 have a particle size of above 150 nm and the EVs have a mean size of more than 150 nm or 155 nm or more or 160 nm or more. According to some embodiments, at least 40% of the EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 45% or at least 60% of the EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 25% to 80% of EVs having an average particle size diameter of from 150 to 1000 nm and from 20 to 75% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 35% to 65% of EVs having an average particle size diameter of from 150 to 1000 nm and from 35 to 65% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 40% to 60% of EVs having an average particle size diameter of from 150 to 1000 nm and from 40 to 60% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, at least 55% of the EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24have a particle size of above 150 nm and the EVs have a mean size of 155 nm or more or 160 nm or more or 165 nm or more. According to some embodiments, at least 60% of the EVs derived from stimulated anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 have a size of above 150 nm and the EVs have a mean 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 of above 150 nm, the EVs have a mean size of 132 nm or more, 135 nm or more, 137 nm or more, 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 and the ratio between EVs having the size of above 150 nm and EV having the size of below 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 the anti-CD19 CAR. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 55% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 60% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 65% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 70% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 75% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 80% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 85% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 90% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, at least 95% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to some embodiments, from 50% to 95, from 55 to 90%, from 60 to 85% or from 65 to 80% of the activated EVs of the present invention comprise the anti-CD19 CAR. According to another embodiment, the CAR is anti-CD19 and the CAR T-cells were incubated with lung adenocarcinoma cells expressing CD19. Non-limiting examples of such lung adenocarcinoma cells are A549, Hl 975, and HCC827 According to one embodiment, the anti-CD19 CAR T- cells were incubated with glioblastoma cells expressing CD19. Non-limiting example of such glioblastoma cells is U251. According to another embodiment, the CAR is anti-CD19 CAR and the CAR T-cells were incubated with CD 19 protein. According to another embodiment, the CAR is anti-CD19 CAR and the CAR T-cells were incubated with a fragment of CD 19 protein to which CD 19 CAR binds specifically. According to one embodiment, the EVs are derived from anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 stimulated with beads coated with and presenting CD19 protein. According to one embodiment, the EVs are derived from anti-CD19 CAR T-cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 stimulated with beads coated with and presenting a fragment of CD 19 protein to which the anti-CD19 CAR binds specifically According to some exemplary embodiments, the present invention provides isolated stimulated extracellular vesicles derived from stimulated anti-CD19 CAR-T cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 incubated from 12 to 96 hours with cancer cells or beads presenting CD 19 or a fragment thereof as described above, wherein the EVs are isolated within 24 hours post incubation. According to another embodiment, the stimulated anti-CD19 CAR-T cells comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 were incubated from 24 to 96, from 36 to 86, from 48 to 86, from 60 to 86 hours or about 72 hours as described above and the EVs are isolated within 24 hours post incubation. According to some embodiments, the EVs were isolated within 30, 36, 42, 48, 60, 72, 84, 96 hours after the incubation. According to other embodiments, the EVs are isolated within 1, 2, 3, 4, 5, 6 or 7 days after the incubation. For example, T cells may be incubated at a ratio of T cells to target cells or to beads coated with the target protein of about 1:1, from 1.5:1 to 3:1, e.g. 2:1, from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, about 2:3, or about 1:1. According to one embodiment, the T cells are incubated with target cells or beads coated with the target protein at a ratio of T cells to target cells/bead of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1. According to some embodiments, the stimulated anti-CD19 CAR T cells were stimulated 2 or 3 or 4-10 cycles with the beads. According to some embodiments, the CD19 protein to which the anti- CD19 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 80. According to some embodiments, the fragment of the CD 19 protein to which the anti-CD19 CAR binds specifically is an extracellular domain and optionally comprises the amino acid sequence SEQ ID NO: 81. According to some embodiments, the present invention provides an off-the-shelf product comprising activated EVs according to any one of the above embodiments.

According to any one of the above embodiments, the activated EVs are cytotoxic EVs, i.e. exhibiting target- specific (e.g. tumor-directed) cytotoxicity. According to one embodiment, the activated EVs exhibit cytotoxic activity toward cancer cells. According to a further embodiment, the activated EVs of the present invention exhibit cytotoxic activity specifically toward cancer cells (e.g. toward those exhibiting or expressing the TAA to which the CAR of the parent cells is directed). According to some embodiments, the cytotoxic activity is apoptosis.

The isolated activated EVs of the invention have been unexpectedly found to exert outstanding anti-tumor effects even in the absence of an exogenously added anti-cancer agent or pay load. Thus, in some embodiments, the invention relates to isolated activated EVs of the invention devoid of any exogenous anti-cancer agent.

According to another embodiment, the activated EVs further comprise an anticancer agent. The terms “anti-cancer”, “anti-neoplastic” and “anti-tumor” when referred to a compound, an agent, moiety or a composition are used herein interchangeably and refer to a compound, drug, antagonist, inhibitor, or modulator having anticancer properties or the ability to inhibit or prevent the growth, function or proliferation of and/or causing destruction of cells, and in particular tumor cells. According to some embodiments, the anti-cancer agent is selected from chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, an anti-cancer agent is a chemotherapeutic. The term "exogenous anti-cancer agent" as used herein refers to anti-cancer agent that was loaded into the EVs after their isolation from the T-cells.

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

Each and every embodiment related to isolated activated EVs as described in any one of the above aspects applies herein as well.

According to any aspect or embodiment of the present invention the term "at least X% of EVs having size above 150 nm" may be replaced by the term "on average X% or more of EVs have size above 150 nm". Thus, according to some embodiments, the present invention provides isolated stimulated EVs derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein on average 22% or more of the EVs have a particle size diameter of above 150 nm. According to some embodiments, on average 25% or more, 30% or more, 35% or more, or 40% or more of the EVs have a particle size diameter of above 150 nm. In some embodiments, the upper limit of the size of the EVs of the present invention is 1000 nm.

The term “pharmaceutical composition” as used herein refers to a composition comprising a therapeutic agent (such as activated and isolated EVs of the present invention) formulated together with one or more pharmaceutically acceptable carriers.

The term “therapeutically effective amount” of EVs is an amount of EVs that, when administered to a subject will have the intended therapeutic effect. The therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of the cognitive impairment, and the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

Formulation of pharmaceutical compositions may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluidextracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, spirits, capsules, creams, troches, 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.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. Solid carriers or excipients such as, for example, lactose, starch or talcum or liquid carriers such as, for example, water, fatty oils or liquid paraffins.

Other carriers or excipients which may be used include, but are not limited to, materials derived from animal or vegetable proteins, such as the gelatins, dextrins and soy, wheat and psyllium seed proteins; gums such as acacia, guar, agar, and xanthan; polysaccharides; alginates; carboxymethylcelluloses; carrageenans; dextrans; pectins; synthetic polymers such as polyvinylpyrrolidone; polypeptide/protein or polysaccharide complexes such as gelatinacacia complexes; sugars such as mannitol, dextrose, galactose and trehalose; cyclic sugars such as cyclodextrin; inorganic salts such as sodium phosphate, sodium chloride and aluminium silicates; and amino acids having from 2 to 12 carbon atoms and derivatives thereof 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.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application typically include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol (or other synthetic solvents), antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, phosphates), and agents that adjust tonicity (e.g., sodium chloride, dextrose). The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, for example. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose glass or plastic vials.

Pharmaceutical compositions adapted for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which can contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Such compositions can also comprise water, alcohols, polyols, glycerine and vegetable oils, for example. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Such compositions preferably comprise a therapeutically effective amount of a compound of the invention and/or other therapeutic agent(s), together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities and compositions that do not produce an adverse, allergic, or other untoward reactions when administered to an animal, or human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by a government drug regulatory agency, e.g., the United States Food and Drug Administration (FDA) Office of Biologies standards.

The terms “enrich”, "enriched" or "enriching" are used interchangeably and refer to a composition comprising higher content and/or concentration of extracellular vesicles than the initial composition. In some embodiments, the composition or the pharmaceutical composition of the present invention are enriched compositions, i.e. has the amount and/or concentration of extracellular vesicles higher that the initial amount and/or concentration obtained upon purification of the EVs. In some embodiments, the concentration of activated EVs is 1.1, 1.5, 2, 3, 5, 10, 50, 100, 500 or 1000 times and above higher compared to the starting material.

According to one embodiment, the pharmaceutical composition comprises activated and isolated EVs derived from stimulated CAR T-cells of the present invention. According to one embodiment, the CAR T-cells were stimulated by incubation with the TAA to which the CAR binds specifically. According to one embodiment, the CAR T-cells were incubated with the TAA from 1 to 96 hour. According to some embodiments, the CAR T-cells were incubated with TAA from 8 to 48 or from 12 to 36 hour. According to one embodiment, the TAA is HER2 and the CAR is N29 CAR. According to another embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with ovarian cancer cells presenting HER2. According to another embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with beads coated with HER2 protein to which N29 CAR binds specifically or with a fragment of HER2. According to a further embodiment, the CAR is N29 CAR and the CAR T-cells were incubated with breast cancer cells presenting HER2. According to one embodiment, the ovarian cancer cells are SKOV cells. According to a further embodiment, the CAR is N29 CAR and the CAR T-cells are N29 CAR T-cells incubated with primary HER2 positive cells.

According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated T-cells expressing an anti-HER2 CAR, and a pharmaceutically acceptable carrier, wherein at least 22 or at least 25% of the EVs have size of above 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated N29 CAR T-cells, and a pharmaceutically acceptable carrier, wherein at least 25% of the EVs have size of above 150 nm.

According to one embodiment, present invenit provides a pharmaceutical composition, wherein the TAA is EGFR and the CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to another embodiment, the CAR is an anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 and the CAR T-cells were incubated with cancer cells presenting EGFR such as lung adenocarcinoma cells or glioblastoma cells. According to a further embodiment, the CAR is an anti-EGFR CAR comprising an amino acid sequence selected from and the CAR T-cells were incubated with inert beads covered/bound to EGFR or a fragment thereof to anti-EGFR CAR binds specifically. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83. According to some embodiments, the term EGFR protein encompasses also a fragment of the EGFR protein to which the anti-EGFR CAR binds specifically. According to some embodiments, the fragment is an extracellular fragment, i.e. exposed to extracellular matrix.

According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated T-cells expressing an anti-EGFR CAR, and a pharmaceutically acceptable carrier, wherein at least from 25 to 80% of the EVs have size of from 150 nm to 1000 nm and the rest have the size of from about 30 to about 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated anti-EGFR CAR T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, and a pharmaceutically acceptable carrier, wherein from about 25% to about 80% of the EVs have size of from about 150 nm to about 1000 nm. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83.

According to one embodiment, the TAA is CD276 and the CAR is stimulated anti- CD276 CAR. According to another embodiment, the CAR is an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 and the CAR T-cells were incubated with cancer cells presenting CD276 such as lung adenocarcinoma cells or glioblastoma cells. According to a further embodiment, is an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 and the CAR T-cells were incubated with inert bead covered/bound to CD276 or a fragment thereof to which anti-CD276 CAR binds specifically. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82.

According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated T-cells expressing an anti-HER2 CAR, and a pharmaceutically acceptable carrier, wherein from 25 to 80% of the EVs have size of above 150 nm and the rest have the size of from about 30 to about 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated anti-CD276 CAR T cells wherein the anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, and a pharmaceutically acceptable carrier, wherein from about 25% to about 80% of the EVs have size of from about 150 nm to about 1000 nm.

According to one embodiment, the TAA is CD 19 and the CAR is stimulated anti-CD19 CAR. According to another embodiment, the CAR is an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 and the CAR T-cells were incubated with cancer cells presenting CD 19 such as B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). According to a further embodiment, the anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 and the CAR T-cells were incubated with inert bead covered/bound to CD 19 or a fragment thereof to which anti-CD19 CAR binds specifically. According to some embodiments, the CD19 protein to which the anti-CD19 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 80. According to some embodiments, the fragment of CD 19 protein to which the anti-CD19 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 81. According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated T-cells expressing an anti-CD19 CAR, and a pharmaceutically acceptable carrier, wherein at least from 25 to 80% of the EVs have size of from 150 nm to 1000 nm and the rest (remaining EVs) have the size of from about 30 to about 150 nm. According to some embodiments, the present invention provides a pharmaceutical composition comprising the isolated activated EVs derived from stimulated anti-CD19 CAR T cells comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24, and a pharmaceutically acceptable carrier, wherein from about 25% to about 80% of the EVs have size of from about 150 nm to about 1000 nm.

According to some embodiments, the T-cells are selected from CD4+ T-cells, CD8+ T- cells and a combination thereof. According to one embodiment, the CAR T-cells are stimulated with corresponding TAA or cells expressing said TAA.

According to some embodiments, the pharmaceutical composition is devoid of any additional anti-tumor agents. In other embodiments, the invention relates to pharmaceutical compositions comprising the isolated activated EVs of the invention as a sole anti-cancer agent.

The terms “substantially devoid”, “essentially devoid”, “devoid”, “does not include” and “does not comprise” may be used interchangeably and when referring to a composition that does not include, contain or comprise a particular component, e.g. said composition comprises less than 0.1 wt%, less than 0.01 wt%, or less than 0.001 wt% of the component. In some embodiments, the term devoid contemplates a composition comprising traces of the devoid component such as traces of a component used in the purification process. When referring to process, the term have the meaning of excluding a particular step.

According to other embodiments, the composition further comprises an additional anticancer agent. According to some embodiments, the anti-cancer agent is selected from chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, an anti-cancer agent is a chemotherapeutic agent. In other embodiments, the additional anti-cancer agent is CAR T-cells, wherein the CAR of said T- cells differs from the CAR of the CAR T-cells from which the EVs are originated.

According to some embodiments, the pharmaceutical composition is a cell-free composition. According to any one of the above embodiments, the pharmaceutical composition of the present invention is for use in treating cancer.

The term “treating cancer” as used herein should be understood to e.g. encompass treatment resulting in a decrease in tumor size; a decrease in rate of tumor growth; stasis of tumor size; a decrease in the number of metastasis; a decrease in the number of additional metastasis; a decrease in invasiveness of the cancer; a decrease in the rate of progression of the tumor from one stage to the next; inhibition of tumor growth in a tissue of a mammal having a malignant cancer; control of establishment of metastases; inhibition of tumor metastases formation; regression of established tumors as well as decrease in the angiogenesis induced by the cancer, inhibition of growth and proliferation of cancer cells and so forth. The term “treating cancer” as used herein should also be understood to encompass prophylaxis such as prevention as cancer reoccurs after previous treatment (including surgical removal) and prevention of cancer in an individual prone (genetically, due to life style, chronic inflammation and so forth) to develop cancer. As used herein, “prevention of cancer” is thus to be understood to include prevention of metastases, for example after surgical procedures or after chemotherapy.

As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. According to one embodiment, cancer is a solid tumor. According to one embodiment, cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma, stomach, mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarian carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and/or liver metastases.

According to some embodiments, the cancer is cancer which cells present the antigen to which the CAR binds specifically. According to some embodiments, the cancer present HER2 antigen. According to other embodiments, the cancer cells present CD19. According to other embodiments, the cancer cells present CD276. According to other embodiments, the cancer cells present EGFR. According to yet another embodiment, the cancer present CD38 antigen.

According to some embodiments, the cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma, stomach, liver, pancreatic and brain cancers and hematology malignancies. In one embodiment, the pharmaceutical composition comprising isolated activated EVs derived from activated anti-HER2 CAR T-cells is for use in treating HER2 positive cancer.

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

The terms "HER2 positive" and "HER2+" are used herein interchangeably and refer to cells overexpressing HER2 antigen.

According to other embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from anti-HER2 CAR T-cells stimulated with HER2 specific activation, is for use in treating HER2 positive ovarian cancer. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-HER2 CAR T-cells is for use in treating HER2 positive breast cancer. According to alternative embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-HER2 CAR T-cells is for use in treating HER2 positive ovarian cancer. According to some embodiments, anti-HER2 CAR is N29 CAR. According to some embodiments, the pharmaceutical composition comprising activated and isolated EVs of the present invention derived from activated N29 CAR T-cells is for use in treating breast cancer. According to a further embodiment, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated N29 CAR T-cells is for use in treating ovarian cancer. According to yet another embodiment, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated N29 CAR T-cells is for use in treating lung adenocarcinoma or stomach 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 diameter size of above 150 nm and/or the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and 170 nm 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 of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more, of 155 nm or more, of 160 nm or more, or of 165 nm or more. According to some embodiments, the ratio between EVs having the particle size of above 150 nm and EV having the particle size of below 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 20 % of the EVs of the present invention express CD3 antigen on their outer membrane and/or at least 20 % of the EVs of the present invention express HLADR antigen on their outer membrane and/or at least 10% of the EVs of the present invention express CD38 antigen on their outer membrane. 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, the N29 CAR-T cells were incubated from 12 to 36 hours with breast cancer cells expressing HER2, wherein the EVs are isolated within 24 hours post incubation. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles, derived from N29 CAR-T cells incubated from 12 to 36 hours with ovarian cancer cells expressing HER2, wherein the EVs are isolated within 24 hours post incubation. For example, T cells may be incubated at a ratio of T cells to target cells of 1.5:1 to 3:1, e.g. 2:1. According to one embodiment, the T cells are incubated with target cells at a ratio of T cells to target cells of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1.

In one embodiment, the pharmaceutical composition comprising isolated activated EVs derived from stimulated anti-EGFR CAR T-cells comprising an anti-EGFR CAR according to any one of the above embodiments is for use in treating EGFR positive cancer. In one embodiment, the pharmaceutical composition comprising isolated activated EVs derived from stimulated anti-EGFR CAR T-cells comprising an anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 is for use in treating EGFR positive cancer, i.e. cancer overexpressing EGFR. According to some embodiments, the EGFR positive cancer is selected from glioblastoma, lung adenocarcinoma, glioblastoma multiforme (GBM), diffuse large B-cell lymphoma (DLBC), skin cutaneous melanoma (SKCM), brain lower grade glioma (LGG), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), adrenocortical carcinoma (ACC), uterine corpus endometrial carcinoma (UCEC), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), ovarian serous cystadenocarcinoma (OV), cervical squamous cell carcinomaand endocervical adenocarcinoma (CESC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), Pancreatic adenocarcinoma (PAAD), testicular germ cell tumors (TGCT), prostate adenocarcinoma (PRAD), breast invasive carcinoma (BRCA), and bladder urothelial carcinoma (BLCA). nonsmall-cell lung cancer, colorectal cancer, squamous-cell carcinoma of the head and neck, and pancreatic cancer, Renal Cell Cancer, Cutaneous Squamous Cell Carcinoma in Skin (SCC), Bone Tumor. According to some embodiments, the EGFR positive cancer is selected from glioblastoma, lung adenocarcinoma, glioblastoma multiforme (GBM), diffuse large B-cell lymphoma (DLBC), skin cutaneous melanoma (SKCM), brain lower grade glioma (LGG), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), and adrenocortical carcinoma (ACC).

The terms "EGFR positive" and "EGFR+" are used herein interchangeably and refer to cells overexpressing EGFR antigen.

According to other embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from anti-EGFR CAR T-cells stimulated with EGFR specific stimulation, is for use in treating EGFR-positive lung cancer. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-EGFR CAR T-cells is for use in treating EGFR positive glioblastoma. According to some embodiments, anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, the pharmaceutical composition comprising activated and activated EVs of the present invention derived from stimulated anti-EGFR CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 is for use in treating lung cancer. According to a further embodiment, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-EGFR CAR T- cells is for use in treating glioblastoma. According to some embodiments, the pharmaceutical composition comprises isolated activated EVs derived from stimulated anti-EGFR CAR T- cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, 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 diameter size of from 150 nm to 1000 nm and/or the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and 170 nm or more. According to other embodiments, the activated EVs derived from stimulated anti-EGFR CAR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more, of 155 nm or more, of 160 nm or more, or of 165 nm or more. According to some embodiments, the ratio between EVs having the particle size of above 150 nm and EV having the particle size of below 150 nm is from 1:4 to 1:2 or about 1:4, about 1:3, about 2:3, or about 1:1. According to some embodiments, at least 40 % or at least 45% or at least 50 % or at least 55 % or at least 60 % or at least 70 % of the EVs of the present invention express anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 on their surface. According to another embodiment, the anti-EGFR CAR-T cells were incubated from 12 to 96 or from 48 to 84 hours with a carrier presenting EGFR, such as cells or beads coated with EGFR, wherein the EVs are isolated within 24 hours post incubation. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles, derived from anti-EGFR CAR-T cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 incubated from 48 to 84 hours with cancer cells expressing EGFR, wherein the EVs are isolated within 24 hours post incubation. For example, T cells may be incubated at a ratio of T cells to target cells or to beads coated with the target protein of about 1:1, from 3:1 to 1:3, from 2:1 to 1:2, from 1.5:1 to 3:1, e.g. 2:1, from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, about 2:3, or about 1:1. According to one embodiment, the T cells are incubated with target cells or beads coated with the target protein at a ratio of T cells to target cells/bead of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1.

In one embodiment, the pharmaceutical composition comprising isolated activated EVs derived from stimulated anti-CD276 CART-cells is for use in treating CD276 positive cancer.

According to some embodiments, the CD276 positive cancer is selected from glioma, prostate cancer, endometrial cancer, skin cancers, lung cancer, cancer stem cells, epithelial tumors, epithelial tumors of the head and neck cells, glioblastoma, bladder cancer, pancreatic cancer, cervical cancer, breast cancer, intrahepatic cholangiocarcinoma, colorectal cancer, ovarian cancer, glioma, melanoma, liver cancer, prostatic cancer, oral squamous cell carcinoma, kidney cancer, gastric cancer, and adrenocortical carcinoma. The terms "CD276 positive" and "CD276+" are used herein interchangeably and refer to cells overexpressing CD276 antigen. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82.

According to other embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from anti-CD276 CAR T-cells stimulated with CD276 specific activation, is for use in treating CD276-positive lung cancer. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD276 CAR T-cells is for use in treating CD276-positive glioblastoma. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD276 CAR T-cells is for use in treating CD276-positive bladder cancer. According to some embodiments, anti-CD276 CAR is anti-CD276 CAR comprising an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, the pharmaceutical composition comprising activated and isolated EVs of the present invention derived from stimulated anti-CD276 CAR T-cells comprising an anti- CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 is for use in treating lung cancer. According to a further embodiment, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-CD276 CAR T-cells comprising an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 is for use in treating glioblastoma. According to some embodiments, the pharmaceutical composition comprises isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, 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 diameter size of from 150 nm to 1000 nm and/or the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and 170 nm or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD276 CAR T-cells comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more, of 155 nm or more, of 160 nm or more, or of 165 nm or more. According to some embodiments, the ratio between EVs having a particle size of above 150 nm and EV having the particle size of below 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 40 % or at least 45% or at least 50 % or at least 55 % or at least 60 % or at least 70 % of the EVs of the present invention express anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 on their surface. According to another embodiment, the anti-CD276 CAR-T cells comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 were incubated from 12 to 96 hours with a carrier presenting CD276 such as cells or beads coated with CD276, wherein the EVs are isolated within 24 hours post incubation. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles, derived from anti-CD276 CAR-T cells comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 incubated from 48 to 84 hours with cancer cells expressing CD276, wherein the EVs are isolated within 24 hours post incubation. For example, T cells may be incubated at a ratio of T cells to target cells or to beads coated with the target protein of about 1:1, from 3:1 to 1:3, from 2:1 to 1:2, from 1.5:1 to 3:1, e.g. 2:1, from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, about 2:3, or about 1:1.. According to one embodiment, the T cells are incubated with target cells or coated beads at a ratio of T cells to target cells of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1.

In one embodiment, the pharmaceutical composition comprising isolated activated EVs derived from stimulated anti-CD19 CART -cells is for use in treating CD 19 positive cancer.

According to some embodiments, the CD 19 positive cancer is selected from B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), large B cell lymphoma (LB CL) and multiple myeloma. According to some embodiments, the B cell lymphoma cells and large B cell lymphoma (LBCL) are either cells of relapsed or refractory lymphoma.

The terms "CD19 positive" and "CD19+" are used herein interchangeably and refer to cells overexpressing CD 19 antigen. According to some embodiments, the CD 19 protein comprises the amino acid sequence SEQ ID NO: 80.

According to other embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from anti-CD19 CAR T-cells stimulated with CD 19 specific stimulation, is for use in treating CD 19-positive lung cancer. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD19 CAR T-cells is for use in treating CD 19- positive B cell lymphoma. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD19 CAR T-cells is for use in treating CD 19-positive large B cell lymphoma (LBCL). According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD19 CAR T-cells is for use in treating CD19-positive multiple myeloma. According to some embodiments, the B cell lymphoma cells and large B cell lymphoma (LBCL) are either cells of relapsed or refractory lymphoma. According to some embodiments, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from activated anti-CD19 CAR T-cells is for use in treating CD 19 positive CLL or ALL. According to some embodiments, anti-CD19 CAR is anti-CD19 CAR comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, the pharmaceutical composition comprising activated and isolated EVs of the present invention derived from stimulated anti-CD19 CAR T-cells comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 is for use in treating lung cancer. According to a further embodiment, the pharmaceutical composition comprising isolated activated EVs of the present invention derived from stimulated anti-CD19 CAR T-cells comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 is for use in treating B cell lymphoma. According to some embodiments, the pharmaceutical composition comprises isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24, 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 diameter size of from 150 nm to 1000 nm and/or the EVs have a mean size selected from 132 or more, 135 nm or more, 137 nm or more, 140 nm or more, 142 nm or more, 145 nm or more, 147 nm or more, 150 nm or more, 152 nm or more, 155 nm or more, 160 nm or more, 162 nm or more, 165 nm or more and 170 nm or more. According to other embodiments, the present invention provides isolated activated EVs derived from stimulated anti-CD19 CAR T-cells comprising the amino acid sequence SEQ ID NO: 24, wherein at least 25% of the EVs have a size of above 150 nm, and the EVs have a mean size of 140 or more. According to some embodiments, at least 29% of the EVs have a size of above 150 nm and the EVs have a mean size of 150 nm or more, of 155 nm or more, of 160 nm or more, or of 165 nm or more. According to some embodiments, the ratio between EVs having the particle size of above 150 nm and EV having the particle size of below 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 40 % or at least 45% or at least 50 % or at least 55 % or at least 60 % or at least 70 % of the EVs of the present invention express anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 on their surface. According to another embodiment, the anti-CD19 CAR-T cells comprising the amino acid sequence SEQ ID NO: 24 were incubated from 12 to 96 hours with a carrier presenting CD 19 such as cells or beads coated with CD 19, wherein the EVs are isolated within 24 hours post incubation. According to some exemplary embodiments, the present invention provides isolated activated extracellular vesicles, derived from anti-CD19 CAR-T cells comprising the amino acid sequence SEQ ID NO: 24 incubated from 48 to 84 hours with cancer cells expressing CD19, wherein the EVs are isolated within 24 hours post incubation. For example, T cells may be incubated at a ratio of T cells to target cells or to beads coated with the target protein of about 1:1, from 3:1 to 1:3, from 2:1 to 1:2, from 1.5:1 to 3:1, e.g. 2:1, from 1:4 to 1:2 or about 1:3 to about 1:1.5, about 1:2 to about 1:1.3, about 2:3, or about 1:1. According to one embodiment, the T cells are incubated with target cells or coated beads at a ratio of T cells to target cells of from 15:1 to 1:5, from 10:1 to 1:4 from 8:1 to 1:3 from 5:1 to 1:2 or from 3:1 to 1:1.

According to some embodiments, the stimulation comprises activation with primary cancer cells obtained from the subject having the cancer. According to one embodiment, the primary cancer cells are obtained from the cancer tissue of a subject having the cancer.

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

The pharmaceutical composition of the present invention may be administered by any known method.

The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including selfadministration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

According to one embodiment, the pharmaceutical composition is formulated as a solution for injection. According to another embodiment, the pharmaceutical composition is systemically administered. According to some embodiments, the pharmaceutical composition is injected, e.g. intravenously or intramuscularly injected. According to one embodiment, the pharmaceutical composition is administered locally. According to some embodiments, the pharmaceutical composition is administered intratumorally. According to another embodiment, the pharmaceutical composition is administered in a proximity to tumor.

In some embodiments, the invention relates to the pharmaceutical compositions comprising the isolated activated EVs of the invention is for use in treating cancer as a sole anti-cancer agent.

According to some embodiments, the pharmaceutical composition of the present invention is co-administered with an additional anti-cancer agent.

According to some embodiments, the anti-cancer compound is selected from chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons, and antagonistic agents targeting cytokines, cytokine receptors or antigens associated with tumor cells. In some embodiments, an anti-cancer agent is a chemotherapeutic. In other embodiments, the anti-cancer agent is CAR T-cells.

According to some embodiments, the co-administration of the pharmaceutical composition of the present invention and of additional anti-tumor compound or agent is performed in a regimen selected from a single combined composition, separate individual compositions administered substantially at the same time, and separate individual compositions administered under separate schedules and include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term “co-administration” encompasses administration of a first and second agent in a substantially simultaneous manner, such as in a single dosage form, e.g., a capsule or tablet having a fixed ratio of first and second amounts, or in multiple dosage forms for each. The agents can be administered in a sequential manner in either order. When co-administration involves the separate administration of each agent, the agents are administered sufficiently close in time to have the desired effect (e.g., complex formation).

The term “sequential manner” refers to an administration of two compounds at a different time, and optionally in different modes of administration. The agents can be administered in a sequential manner in either order.

The terms “substantially simultaneous manner” refers to administration of 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.

According to another aspect, the present invention provides a method of 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 the pharmaceutical composition of the present invention. According to one embodiment, the cancer is selected from breast cancer, ovarian cancer, lung adenocarcinoma and stomach cancer. In some embodiments, the present invention provides a method of treating cancer selected from breast cancer, ovarian cancer, lung adenocarcinoma, stomach cancer and any one of the cancer as described herein above by administering an effective amount of isolate activated EVs derived from activated CAR T-cells expressing N29 CAR, wherein at least 25% of the EVs have size of from 150 nm to 1000 nm.

Each and every embodiment related to isolated activated EVs of the present invention as described in any one of the above aspects applies herein as well.

According to another embodiment, the method further comprises co-administration of an additional anti-cancer agent. According to some embodiments, the anti-cancer agent is a chemotherapeutic agent. According to other embodiment, the anti-cancer agent is a composition comprising CAR T-cells.

According to another aspect, the present invention provides use of the EVs according to the present invention for preparation of a medicament for treating cancer.

According to a further aspect, the present invention provides a method of preparation of the isolated activated extracellular vesicles of the present invention. According to some embodiments, the present invention provides a method for preparation of the isolated stimulated extracellular vesicles derived from stimulated CAR T-cells wherein at least 22 % or at least 25 % of the EVs have size of above 150 nm, wherein the method comprises incubating the CAR T-cells with a tumor-associated antigen to which the CAR binds specifically under conditions enabling T cell stimulation, and isolating the derived activated extracellular vesicles. According to some embodiments, the present invention provides a method for preparation of the isolated stimulated extracellular vesicles derived from stimulated CAR T-cells wherein at least 25 % or from 25 to 80% of the EVs have size of from about 150 nm to about 1000 nm, wherein the method comprises incubating the CAR T-cells with a tumor-associated antigen to which the CAR binds specifically under conditions enabling T cell stimulation, and isolating the derived activated extracellular vesicles.

According to some embodiments, the method of preparation comprises (1) incubating CAR T-cell with a tumor-associated antigen to which CAR binds specifically, wherein the incubation is performed in a cell medium under conditions enabling T cell stimulation; (2) separating T-cell from the medium ; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 size above 150 nm. According to one embodiment, at least 25 % of the EVs have size of more than 150 nm. According to one embodiment, at least 25 % of the resulting EVs have size of from 150 nm to 1000 nm. According to some embodiments, the resulting population of isolated activated EVs derived from stimulated CAR T-cells comprises from 25% to 80% of EVs having an average particle size diameter of from 150 to 1000 nm and from 20 to 75% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 35% to 65% of EVs having an average particle size diameter of from 150 to 1000 nm and from 35 to 65% of EVs having an average particle size diameter of from 30 to 150 nm. According to some embodiments, the population of isolated activated EVs derived from stimulated CAR T-cells comprises from 40% to 60% of EVs having an average particle size diameter of from 150 to 1000 nm and from 40 to 60% of EVs having an average particle size diameter of from 30 to 150 nm.

According to some embodiments, separating T-cell from the cell medium of step (2) comprises separating the cell medium from cells and large cell particles. The term "large cell particles" refer to cell particles above 1 pm, such as cell debris, organelles etc. As a result, cell medium comprising EVs of the present invention is obtained. The separation may be effected in one step or in several steps. According to some embodiments, separating T-cell from the medium comprises the following steps: step (2i) comprising centrifuging the medium with stimulated T-cell for 5 to 60 min at from 200g to 600g and separating/collecting the pellet, thereby separating the pellet from the medium, and step (2ii) comprising centrifuging cell medium obtained from step (2i) (supernatant) for from 10 to 60 min at from 1000g to 3000g and separating the resulted pellet from medium. According to some embodiments, the method comprises only step (2ii). Thus, according to some embodiments, step 2 comprises centrifuging cell medium obtained from previous step (step 1) for from 10 to 60 min at from 1000g to 3000g and separating the resulting pellet from the cell medium. Cell medium obtained after centrifugation may be denoted as supernatant.

According to some embodiments, step (2ii) comprises centrifuging for from 10 to 50 min, or for 10 to 30min or for 10 to 20 min at from 1000 to 2000g. According to some embodiments, step (2i) comprises centrifuging the medium for from 5 to 15 min at from 200 to 600g or at about 400g. According to some embodiments, step (2i) comprises centrifuging the medium for from 5 to 30 min at from 200 to 600g or at about 400g. According to some embodiments, separating T-cell from the medium comprises the following steps: step (2i) comprising centrifuging the medium with stimulated T-cell for 5 to 15 min at from 200g to 600g and separating/collecting the pellet, thereby separating the pellet from the medium, and step (2ii) comprising centrifuging cell medium obtained from step (2i) for from 10 to 30 min at from 1000g to 3000g and separating the resulted pellet from medium. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg and collecting the supernatant for EVs purification.

According to some embodiments, the method comprises only step (2ii). In case step (2i) is absent, (2ii) comprises centrifuging cell medium obtained from step (1). According to other embodiments, the method may further comprise other steps before step 3, wherein the centrifugation force is not above 5,000g.

The steps (2i), (2ii) or their combination should be short enough to precipitate the cells and large cell particles, but not the EVs of the present invention. Thus, in some embodiments, a short centrifugation at step (2i) may be preferred.

Alternatively, the separation of cell medium comprising EVs of the present invention from cells and large cell particles is a continuous process performed as known in art, e.g. by microfluidic system, hollow-fiber bioreactor technology (Whitford W. et al. www.GENengnews.com 2015), nanoscale separation array (Wunsch BH. Nature nanotechnology 2016), or magnetic nanowires (Lim J. J Nanobiotechnology. 2019).

According to some embodiments, the T-cells obtained in step (2i) or in step (2i) may be recycled, i.e. used for further preparation of EVs of the present invention. Thus, the cells collected in step (2i) or in step (2i) are incubated with a tumor-associated antigen to which CAR bind specifically in a cell medium under conditions enabling T cell activation to initiate the process and then separated from the medium in step (2) as described above. The number of cycles in which CAR T-cells are used in the preparation of the EVs of the present invention is limited only by the ability of the T-cells to generate EVs of the present invention having all properties as described above.

The terms “incubating” or “incubation” are used herein interchangeably and refer to a process of contacting or exposing CAR T-cells with the desired entity, under conditions enabling T cell stimulation. According to one embodiment, the CAR T-cell are incubated with the TAA for at least 1 hour. According to another embodiment, the incubation is for at least 6, 12, 18 or 24 hours. According to another embodiment, the incubation is for from 1 to 96 hour. According to some embodiments, the incubation is for from 6 to 84, from 12 to 72, from 18 to 60, from 24 to 48 or from 30 to 32 hours. According to another embodiment, the incubation is for from 6 to 48, from 12 to 42, from 18 to 36 hours. According to another embodiment, the incubation is for from 20 to 30 hours. According to some embodiments, the incubation is for from 24 to 96 hours, from 36 to 96 hours, from 48 to 96 hours, from 48 to 84 hours, from 60 to 84 hour or about 72 hours. In other embodiments, the incubation is performed for about 24 hours. According to some embodiments, incubation comprises incubation with any carrier presenting the TAA. According to some embodiments, incubation comprises incubation with cells presenting TAA. According to other embodiments, incubation comprises incubation with inert carrier covered and/or bound with the TAA. The inert carrier may be any carrier as known in the art. In some embodiments, the carrier is a bead or plurality of beads covered with the TAA. The beads of the present invention may be of any form and suitable size According to some embodiments, the beads are spherical. According to other embodiments, the beads are ellipsoid. In other embodiments, the carrier is a polymer with multiple binding site occupied by the TAA. In some embodiments, the polymer is polysaccharide, such as branched or straight saccharide. In some embodiments, the carrier is dextran polymer backbone such as dexamer. In some embodiments, the inert carrier is cell membrane fragment comprising the TAA. According to some embodiments, stimulation is performed by incubating the CAR T cells with fragments of cell membrane presenting the TAA to which the CAR binds specifically.

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

According to other embodiment, upon purification, the ratio of EVs to cells is at least 2, 3, 4, 5, 6, 8 or 10 times or alternatively and typically at least 50, at least 100, at least 500 or at least 1000 times higher than in the initial material According to some embodiments, the purification provides EVs substantially free of cells. According to other embodiments, purification provides cell-free EVs composition. According to some embodiments, incubation of CAR T-cell with a TAA comprises incubation with a complete TAA, a part of TAA to which the CAR binds specifically (epitope or epitope-comprising portion) or with an entity that expresses said TAA such as a complete cell, an EV expressing said TAA or any carrier such as liposomes expressing said TAA. According to some embodiments, incubation comprises incubation with cells presenting the TAA.

According to some embodiments, isolating the EVs (step 3) comprises low force centrifugation of the medium comprising the EVs. According to some embodiments, the isolation comprises centrifugation at from 8,000g to 30,000 for from 0.5 to 4 hours. According to some embodiments, the isolation comprises centrifugation at from 8,000g to 30,000 for more than 0.5 to 4 hours. According to one embodiment, the isolation comprises centrifugation at from 8,000g to 30,000 for from 0.5 to 3 hours. According to another embodiment, the isolation comprises centrifugation at from 8,000g to 30,000 for from 0.5 to 2 hours. According to yet embodiment, the isolation comprises centrifugation at from 8,000g to 30,000 for from 0.5 to 1.5 hours.

According to some embodiments, step (3) is devoid of centrifugation at a force above 30,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 31,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 35,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 50,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 70,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 80,000g. According to some embodiments, step (3) is devoid of centrifugation at a force above 90,000g. According to any one of the above embodiments, the whole process is devoid of ultracentrifugation. According to some embodiments, the whole process is devoid of centrifugation at a force above 30,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 31,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 35,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 70,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 80,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 90,000g.

According to some embodiments, the isolation of the EVs comprises centrifugation at from 8,000g to 20,000 for from 0.5 to 4 hours. According to one embodiment, the isolation comprises centrifugation at from 8,000g to 20,000 for from 0.5 to 3 hours. According to one embodiment, the isolation comprises centrifugation at from 8,000g to 20,000 for from 0.5 to 2.5 hours. According to one embodiment, the isolation comprises centrifugation at from 8,000g to 20,000 for from 0.5 to 2 hours. According to one embodiment, the isolation comprises centrifugation at from 8,000g to 20,000 for from 0.5 to 1.5 hours. According to some embodiments, the isolation comprises centrifugation at from 16,000g to 22,000 for from 0.5 to 4 hours or from 0.5 to 3 hours or from 0.5 to 2 hours or for from 0.5 to 1.5 hours. According to some embodiments, the isolation comprises centrifugation at about 20,000g for 0.5 to 2.5 hours.

According to some embodiments, the isolation comprises centrifugation at from 8,000g to 15,000 for from 0.5 to 4 hours or from 0.5 to 3 hours or from 0.5 to 2.5 hours or from 0.5 to 2 hours or for from 0.5 to 1.5 hours.

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

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

According to some embodiments, the present invention provides a method for the preparation of the isolated activated extracellular vesicles derived from stimulated CAR T- cells, the method comprises: (1) incubating CAR T-cells with a carrier coated with a protein or a peptide to which the CAR binds specifically under conditions enabling T cell stimulation thereby stimulating the CAR T cells; (2) separating the CAR T-cells from the cell medium; (3) isolating the derived extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm. The method encompasses any one of the embodiments related to a method of preparation as described above and below. According to some embodiments, the method is devoid of centrifugation above 30,000g, or above 50,000g, or above 70,000g. According to some embodiments, at least 25%, at least 30%, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to some embodiments, from 20 to 90%, from 30 to 85% or, from 40 to 90% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to any one of the above embodiments, the CAR of the CAR T-cells specifically binds to a tumor-associated antigen (TAA). Any CAR that binds to a TAA may be used according to the teaching of the present invention. According to some embodiment, the TAA selected from MUC1, Mesothelin, PSCA, EGFR, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, HER2, ROR1, CD70, CD38, CD138, CD24, and CD19. According to one embodiment, the TAA is CD276. HER2. According to one embodiment, the TAA is CD276. According to one embodiment, the TAA is EGFR. According to one embodiment, the TAA is CD 19.

According to some embodiments, the CAR is anti-HER2 CAR. According to some embodiments, the CAR is N29 CAR. According to certain embodiments, the stimulation comprises stimulation of anti-HER2 CAR T cells, such as N29 CAR T-cell with ovarian cancer cells presenting HER2, such as SKOV cells. According to some embodiments, the stimulation comprises stimulation of anti-HER2 CAR T cells, such as N29 CAR T-cell with breast cancer cells presenting HER2. According to some embodiments, the stimulation comprises incubation of N29 CAR T-cell with beads coated with HER2 protein. According to some embodiments, the stimulation comprises the incubation of N29 CAR T-cell with beads coated with a fragment of HER2 protein to which N29 CAR binds specifically. According to some embodiments, the HER2 protein comprises the amino acid sequence of SEQ ID NO: 85. According to some embodiments, the fragment of HER2 protein comprises the amino acid sequence of SEQ ID NO: 84. According to some embodiments, at least 25% or at least 30% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 55% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 60% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 65% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 70% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 75% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 80% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, at least 85% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, from 20 to 90% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, from 30 to 85% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, from 40 to 90% of the resulting activated EVs of the present invention comprise the N29 CAR. According to some embodiments, N29 CAR comprises the amino acid sequence SEQ ID NO: 57.

According to some embodiments, the CAR is anti-EGFR CAR. According to some embodiments, anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to certain embodiments, the stimulation comprises stimulation of anti-EGFR CAR T cells, such as anti-EGFR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 with lung cancer cells presenting EGFR, such as A549, H1975, and HCC827. According to some embodiments, the stimulation comprises stimulation of anti-EGFR CAR T cells, such as anti-EGFR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 with glioblastoma cells presenting EGFR such as U251. According to some embodiments, the stimulation comprises incubation of anti-EGFR CAR T cells, such as anti- EGFR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 with beads coated with EGFR protein. According to some embodiments, the EGFR protein comprises the amino acid sequence SEQ ID NO: 83. According to some embodiments, the stimulation comprises the incubation of anti-EGFR CAR T cells, such as anti-EGFR T-cells comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 with beads coated with a fragment of EGFR protein to which the anti-EGFR CAR, such as anti-EGFR comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, binds specifically. According to some embodiments, the fragment is an extracellular fragment of EGFR protein comprising the amino acid sequence SEQ ID NO: 83. According to some embodiments, at least 25% or at least 30% of the activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 55% of the activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 60% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 65% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR T cells, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 70% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 75% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 80% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti- EGFR comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, at least 85% of the activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, from 30 to 90%, from 40 to 85%, from 45 to 80% of the resulting activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR, comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20.

According to some embodiments, the CAR is anti-CD276 CAR. According to some embodiments, the CAR is anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to certain embodiments, the stimulation comprises stimulation of anti-CD276 CAR T cells, such as anti-CD276 T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, with lung cancer cells presenting CD276, such as A549, H1975, and HCC827. According to some embodiments, the stimulation comprises stimulation of anti-CD276 CAR T cells, such as anti- CD276 T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 with glioblastoma cells presenting CD276 such as U251. According to some embodiments, the stimulation comprises incubation of anti-CD276 CAR T cells, such as anti-CD276 T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 with beads coated with CD276 protein. According to some embodiments, the CD276 protein comprises the amino acid sequence SEQ ID NO: 82. According to some embodiments, the stimulation comprises the incubation of anti-CD276 CAR T cells, such as anti-CD276 T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, with beads coated with a fragment of CD276 protein to which the anti-CD276 CAR, such as anti-CD276 a CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, binds specifically. According to some embodiments, the fragment of CD276 protein is an extracellular fragment of the CD276 protein comprising the amino acid sequence SEQ ID NO: 82. According to some embodiments, at least 25% or at least 30% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 55% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 60% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti- CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 65% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 70% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 75% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 80% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, at least 85% of the resulting activated EVs of the present invention comprise the anti-CD276 CAR, such as anti- CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, from 30 to 90%, from 40 to 85%, from 45 to 80% of the resulting activated EVs of the present invention comprise anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10.

According to some embodiments, the CAR is anti-CD19 CAR. According to some embodiments, the CAR is anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to certain embodiments, the stimulation comprises stimulation of anti-CD19 CAR T cells, such as anti-CD19 T-cell comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 with cancer cells selected from B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). According to some embodiments, the stimulation comprises incubation of anti-CD19 CAR T cells, such as anti-CD19 T-cell comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 with beads coated with CD 19 protein. According to some embodiments, the CD 19 protein to which the anti- CD19 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 80. According to some embodiments, the stimulation comprises the incubation of anti-CD19 CAR T cells, such as anti-CD19 T-cell comprising a CAR comprising the amino acid sequence SEQ ID NO: 24 with beads coated with a fragment of CD 19 protein to which the anti-CD19 CAR, such as anti-CD19 a CAR comprising the amino acid sequence SEQ ID NO: 24, binds specifically. According to some embodiments, the fragment of the CD 19 protein to which the anti-CD19 CAR binds specifically comprises the amino acid sequence SEQ ID NO: 81. According to some embodiments, at least 25% or at least 30% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 55% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 60% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti- CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 65% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 70% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 75% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti- CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 80% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, at least 85% of the resulting activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, from 30 to 90%, from 40 to 85%, from 45 to 80% of the resulting activated EVs of the present invention comprise anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24.

According to some embodiments, the incubation is for from 9 to 48 hours. According to some embodiments, the incubation is for from 12 to 36 hours. According to some embodiments, the incubation is for from 18 to 36 hours. In some embodiments, the derived stimulated extracellular vesicles are isolated immediately or within 24 or within 48 hours after separation of the CAR T cells from the medium. According to some embodiments, the incubation is for from 24 to 108 hours. According to some embodiments, the incubation is for from 36 to 96 hours. According to some embodiments, the incubation is for from 48 to 96 hours. According to some embodiments, the incubation is for from 60 to 108 hours. According to some embodiments, the incubation is for from 60 to 96 hours. According to some embodiments, the incubation is for from 60 to 84 hours.

According to some embodiments, the method comprises washing the EVs pellet at least once, e.g. 1, 2, 3 or more times at step (4).

According to some embodiments, the method further comprises freezing the EVs at a temperature below -60°C, e.g. at -80°C or below. According to some embodiment, a cryoprotecting buffer may be used to protect the EVs during the freezing process.

According to some embodiments, the method including stimulation of the CAR T cells with beads coated with a TAA to which the CAR binds specifically further comprises yet another cycle of incubation of CAR T cells with beads coated with TAA and purification of the EVs. Steps (1), (2) and (3) may be considered as one cycle of EVs purification. Therefore, the method may comprise several cycles, e.g., 1, 2, 3, 4 or 5 cycles. In some embodiments, the method may comprise 6, 7, 8, 9, or 10 stimulation cycles. Therefore, according to some embodiments, the method comprises from 2 to 10 cycles, each cycle comprising steps (1), (2) and (3) as described in any one of the above embodiments. To clarify, in some embodiments, the CAR T cells may be bound to beads loaded with TAA and may be co-precipitated and coresuspended with the beads.

In other embodiments, the method further comprises adding an inert carrier coated with TAA to which CAR binds specifically. According to some embodiments, the adding of the beads may be performed before the first cycle only or before each one of the cycles. According to some embodiments, the amount of the added beads may vary between cycles and is adapted to maintain the desired concentration of the TAA to improve the outcome. According to some embodiments, the present invention provides a method for preparation of the isolated extracellular vesicles derived from stimulated CAR T-cells, the method comprises: (0) adding to a CAR T cell culture an inert carrier coated with TAA to which CAR binds specifically, (1) incubating CAR T-cells with the carrier under conditions enabling T cell stimulation; (2) separating the CAR T-cells from the cell medium; (3) isolating the derived activated extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm; and (4) adding an inert carrier coated with TAA to which CAR binds specifically to CAR T-cells obtained in step (2) and repeating steps (1), (2) and (3). According to some embodiments, step (4) may be repeated 2, 3, 4 or 5 times.

According to some embodiments, the present invention provides a method of preparation of activated extracellular vesicles comprising the following steps: (0) adding inert beads coated with HER2 protein or fragment thereof to a culture of N29 CAR T-cells; (1) incubating the N29 CAR T-cells with the inert beads coated with HER2 or a fragment thereof under conditions enabling T cell stimulation for from 6 to 96 hours; (2) separating the N29 CAR T-cell from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 from 25 % to 80% of the isolated EVs have particle size of from 150 nm to 1000 nm and from 20 to 75 % of the and isolated EVs have particle size of from about 30 to about 150 nm. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg. According to some embodiments, the method comprises adding inert beads coated with HER2 or a fragment thereof to a culture of N29 CAR T-cells obtained in step (2) and repeating steps (l)-(5). According to some embodiments, the whole process is devoid of centrifugation at a force above 30,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 31,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 35,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 70,000g or above 90,000g. According to some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the activated EVs of the present invention comprise the N29 CAR. According to some embodiments, the HER2 protein comprises the amino acid sequence of SEQ ID NO: 85. According to some embodiments, the fragment of HER2 protein comprises the amino acid sequence of SEQ ID NO: 84. According to some embodiments, the N29 comprises the amino acid sequence of SEQ ID NO: 57.

According to some embodiments, the present invention provides a method of preparation stimulated extracellular vesicles comprising the following steps: (0) adding inert beads coated with EGFR or a fragment thereof to a culture of anti-EGFR CAR T cells, such as anti-EGFR CAR T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20, (1) incubating the anti-EGFR CAR T cells with the inert beads coated with EGFR protein or a fragment thereof under conditions enabling T cell stimulation for from 6 to 96 hours; (2) separating the anti-EGFR CAR T cells, the anti-EGFR CAR T-cell from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 from 25 % to 80% of the isolated EVs have particle size of from 150 nm to 1000 nm and from 20 to 75 % of the and isolated EVs have particle size of from about 30 to about 150 nm. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg. According to some embodiments, the method comprises adding inert beads coated with EGFR protein or a fragment thereof to a culture of anti-EGFR CAR T cells, such as anti-EGFR CAR T-cell comprising a CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20 obtained in step (2) and repeating step (l)-(5). According to some embodiments, the whole process is devoid of centrifugation at a force above 30,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 31,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 35,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 70,000g or above 90,000g. According to some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the activated EVs of the present invention comprise anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to some embodiments, from 40% to 85%, from 45 to 80% or from 50 to 75% of the activated EVs of the present invention comprise the anti-EGFR CAR, such as anti-EGFR CAR comprising an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20. According to any one of the above embodiments, the EGFR coating the beads comprises the amino acid sequence SEQ ID NO: 82. In other embodiments, the beads are coated with a fragment of the EGFR protein is a fragment of the EGFR protein comprising the amino acid sequence SEQ ID NO: 82.

According to some embodiments, the present invention provides a method of preparation stimulated extracellular vesicles comprising the following steps: (0) adding inert beads coated with CD276 protein or fragment thereof to a culture of anti-CD276 CAR T cells, such as anti-CD276 CAR T-cell comprising an anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10; (1) incubating the anti-CD276 CAR T cells with the inert beads coated with CD276 protein or a fragment thereof under conditions enabling T cell stimulation for from 6 to 96 hours; (2) separating the anti-CD276 CAR T cells from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 from 25 % to 80% of the isolated EVs have particle size of from 150 nm to 1000 nm and from 20 to 75 % of the and isolated EVs have particle size of from about 30 to about 150 nm. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg. According to some embodiments, the method comprises adding inert beads coated with CD276 protein or a fragment thereof to a culture of anti-CD276 CAR T cells, such as anti-CD276 CAR T-cell comprising anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10 obtained in step (2) and repeating step (l)-(5). According to some embodiments, the whole process is devoid of centrifugation at a force above 30,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 31,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 35,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 70,000g or above 90,000g. According to some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10. According to some embodiments, from 40% to 85%, from 45 to 80% or from 50 to 75% of the activated EVs of the present invention comprise the anti-CD276 CAR, such as anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID Nos: 4, 5, 9 and 10. According to any one of the above embodiments, the CD276 coating the beads comprises the amino acid sequence SEQ ID NO: 82. In other embodiments, the beads are coated with a fragment of CD276 comprising the amino acid sequence SEQ ID NO: 82.

According to some embodiments, the present invention provides a method of preparation stimulated extracellular vesicles comprising the following steps: (0) adding inert beads coated with CD 19 protein or fragment thereof to a culture of anti-CD19 CAR T cells, such as anti-CD19 CAR T-cell comprising an anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24; (1) incubating the anti-CD19 CAR T cells with the inert beads coated with CD 19 protein or a fragment thereof under conditions enabling T cell stimulation for from 6 to 96 hours; (2) separating the anti-CD19 CAR T cells from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 from 25 % to 80% of the isolated EVs have particle size of from 150 nm to 1000 nm and from 20 to 75 % of the and isolated EVs have particle size of from about 30 to about 150 nm. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg. According to some embodiments, the method comprises adding inert beads coated with CD 19 protein or a fragment thereof to a culture of anti-CD19 CAR T cells, such as anti-CD19 CAR T-cell comprising anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24 obtained in step (2) and repeating step (l)-(5). According to some embodiments, the whole process is devoid of centrifugation at a force above 30,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 31,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 35,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 50,000g. According to some embodiments, the whole process is devoid of centrifugation at a force above 70,000g or above 90,000g. According to some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24. According to some embodiments, from 40% to 85%, from 45 to 80% or from 50 to 75% of the activated EVs of the present invention comprise the anti-CD19 CAR, such as anti-CD19 CAR comprising an amino acid sequence selected from SEQ ID NOs: 4, 5, 9 and 10. According to any one of the above embodiments, the CD 19 coating the beads comprises the amino acid sequence SEQ ID NO: 80. In other embodiments, the beads are coated with a fragment of CD19 comprises the amino acid sequence SEQ ID NO: 81. In some embodiments, the carrier used instead of beads is cell membrane fragment comprising CD 19 or a fragment thereof.

According to some embodiments, the method for preparation is Method 1 described in the Examples. According to some embodiments, the method for preparation is Method 5 a described in the Examples. According to some embodiments, the method for preparation is Method 3 as described in the Examples.

Typically, the T cells comprise a CD8⁺ T cell population. 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 at least CD4⁺ and CD8⁺ CAR T-cells.

According to some embodiments, the present invention provides isolated stimulated extracellular vesicles prepared by the method according to any one of the above embodiments. Thus, the present invention provides isolated stimulated extracellular vesicles prepared by the following steps: (1) incubating CAR T-cell with tumor-associated antigen to which CAR binds specifically under conditions enabling T cell stimulation, preferably for from 6 to 96 hours; (2) separating the T-cell from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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% or at least 25 % of the isolated EVs have particle size of 150 nm and more. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500xg.

According to some embodiments, the present invention provides isolated stimulated extracellular vesicles prepared by the method according to any one of the above embodiments. Thus, the present invention provides isolated stimulated extracellular vesicles prepared by the following steps: (1) incubating CAR T-cell with beads coated a tumor-associated antigen to which CAR binds specifically under conditions enabling T cell stimulation thereby obtaining stimulated CAR T cells, preferably for from 6 to 96 hours; (2) separating the T-cell from the medium comprising the EVs by centrifuging for 10 to 30 min at from 200 to 600g and collecting the pellet; and centrifuging the remained medium for from 10 to 30 min at from 1000 to 3000g and discarding/collecting the resulted pellet thereby obtaining medium comprising EVs; (3) isolating the EVs from the medium by centrifugation at from 8,000g to 30,000 for from 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 at least 25 % of the isolated EVs have particle size of 150 nm to 1000 nm. According to some embodiments, separating the T-cell from the medium comprising the EVs comprises centrifuging for 5 to 15 min at about 400g, and centrifuging the supernatant for 10 to 20 min at about 1500g. According to some embodiments, the method is devoid centrifugation above 30,000g or above 50,000g. According to some embodiments, the resulting activated EVs comprise at least 25%, at least 30%, at least 35% or at least 40%, or at least 45% or at least 50% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to some embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to some embodiments, from 20 to 90%, from 30 to 85% or, from 40 to 90% of the resulting activated EVs of the present invention comprise the CAR of the CAR T cells. According to any one of the above embodiments, the CAR of the CAR T-cells specifically binds to a tumor-associated antigen (TAA). Any CAR that binds to a TAA may be used according to the teaching of the present invention. According to some embodiment, the TAA selected from MUC1, Mesothelin, PSCA, EGFR, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, HER2, R0R1, CD70, CD38, CD138, CD24, and CD19. According to one embodiment, the TAA is HER2. According to one embodiment, the TAA is CD276. According to one embodiment, the TAA is EGFR. According to one embodiment, the TAA is CD 19.

According to some embodiments, the isolated stimulated extracellular vesicles prepared by the method of the present invention is a off-the shelf EVs.

According to another embodiment, the method further comprises a step of enrichment of a population of EVs comprising the CAR. The step of enrichment of CAR-EVs may comprise use of magnetic beads conjugated to the specific antibodies against CAR or against CD3 or by any other sorting method.

Table of sequences number

The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated 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 materially alter the basic and novel characteristics of the claimed compositions or methods.

As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10%, or +/- 5%, +/-1%, or even +/-0.1% from the specified value.

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

EXAMPLES

Materials and methods

T-cell preparation

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). PBLs were activated in non-tissue culture-treated 6-well plates, pre-coated with both purified anti-human CD3 and purified anti-human CD28 for 48 hours at 37°C. Activated lymphocytes were harvested and subjected to two consecutive retroviral transductions in RetroNectin pre-coated, non-tissue culture-treated 6-well plates supplemented with human IL- 2 (100 lU/mL). After transduction, cells were cultured in the presence of 350 lU/mL IL-2 for 24-72 hours. Transduction efficiency was monitored by flow cytometry. Activated but nontransduced (non-infected) cells were included as T cell controls (Eshhar Z, J Immunol Methods 2001,248:67-76). N29 CAR targets HER2 expressed on ovarian cancer cells and anti-CD19 CAR targets CD19 expressed on hematopoietic malignant cells.

N29 CAR has a light chain variable fragment as set forth in SEQ ID NO:21 and a heavy chain variable fragment as set forth in SEQ ID NO:22. The complete CAR N29 in encoded by DNA sequence as set forth in SEQ ID NO: 54 and has amino acid sequence as set forth in SEQ ID NO: 24.

Extracellular Vesicles samples

Throughout the below experiments, unless stated otherwise, extracellular vesicles were obtained from N29 CAR T-cells or from Non-transduced T cells each either incubated with target cells: SKOV (HER2+) or OVCAR (HER2-), or not. The cells were incubated for 24 hours. EVs were isolated from cell medium at the end of 24 hours of cell stimulation on target cells.

The following notification of the samples is used in the Examples:

Sample 1. T cells expressing N29 CAR after stimulated with SKOV (HER2+) cells are denoted as: N29 on SKOV;

Sample 2. T cells expressing N29 CAR after incubation with OVCAR (HER2-) cells are denoted as: N29 on OVCAR;

Sample 3. Non- Non-transduced T cells incubated with SKOV( HER2+) cells are denoted as: UT on SKOV;

Sample 4. Non-transduced T cells incubated with OVCAR (HER2-) cells are denoted as: UT on OVCAR;

Sample 5. Medium of N29 CART cells is denoted as: N29 on medium;

Sample 6. Medium of non-transduced T cells (without stimulation) is denoted as: UT on medium;

Sample 7. Target cell medium obtained from SKOV is denoted as: SKOV on medium; and Sample 8. Target cell medium obtained from OVCAR cells is denoted as: OVCAR on medium.

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.

EVs from each of the above samples were isolated/purified by one of Methods 1-5 described below. Extracellular vesicle (EVs) isolation and analysis

Method 1

The medium of CAR T-cells was collected and centrifuged for 5-10 min at 400g, supernatant was further centrifuged for 15-30 (about 20) min at 1500xg. Supernatant was further centrifuged for 1 h at 20,000xg (20Kxg). An EV pellet was then frozen in aliquots at -80°C.

Method 2

The supernatant obtained in Method 1 (after centrifugation at 20,000xg) was further centrifuged for 60min at 100,000xg and the pellet (20K-100Kxg pellet) was then frozen in aliquots at -80°C.

Method 3

The medium of CAR T-cells was collected and centrifuged for 5-10 min at 400g, supernatant was further centrifuged for 15-30 (about 20) min at 1500xg, then supernatant was further centrifuged for 30min at 10,000xg. The EV pellet was then frozen in aliquots at -80°C. Method 4

The supernatant from Method 3 was further centrifuged for 1 lOmin at 70,000xg and the pellet was then frozen in aliquots at -80°C.

Method 5

The medium of CAR T-cells was collected and centrifuged for 5-10 min at 400g, supernatant was further centrifuged for 15-30 (about 20) min at 1500xg, then supernatant was further centrifuged for 180min at 10,000xg, and the pellet was then frozen in aliquots at -80°C. Size assessment

EVs size and concentration were evaluated by Nanoparticle-tracking analysis (NT A) that can measured particles in the range of 50-2000 nm. NTA was performed using a NanoSight NS300 system with a CMOS camera and 532-nm laser (Malvern Instruments. Malvern, UK), each sample was measured three times. Since the EVs are mostly spherical particles, the size refers to the diameter of the EVs.

Beads having 0.7 pm size were used to set the appropriate size gate for large EVs analysis by flow cytometry. Fluorescent labeled antibodies were used to validate the expression of specific antigens.

Protein assay

In order to calculate the amount of protein in the EVs specimens and to use the same amount of protein in each well, EVs were measured by bicinchoninic acid (BCA) a colorimetric method for detection and quantitation of total proteins or by Thermo Scientific™ NanoDrop™.

Cells viability - XTT assay

In order to assess the quantity of the exposed cells and their viability, the metabolic activity of the targeted cells were analyzed. The ability of CAR T cells-EVs to reduce the tetrazolium salt XTT to orange-colored compounds of formazan was measured. The intensity of the dye is correlated to the number of viable cells and was monitored by ELISA reader. Cell lines

MDA231 HER2 positive breast cancer cells, ovarian cancer cells, (SKOV and SKOV/Luc) (the latter stably expresses the firefly luciferase gene) and pancreatic adenocarcinoma (CAP AN) are all HER2 positive cell lines and thus are a potential target for N29 CART EVs.

MDA231 HER2 negative, Ovarian cancer cells (OVCAR cells), Raji cells, B lymphocytes of Burkitt's lymphoma, which are all HER2 negative cell and are therefore nontarget cells for the EVs from N29 CAR T-cell, served as control cells.

Cytotoxic effect of EVs

The cytotoxic effects EVs on target cells were viewed and documented by light microscope and measured by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega).

At the end of exposure to EVs, target cells nuclei were stained with Hoechst 33342 staining solution (ABCAM), indicating total cells number while cells apoptosis were measured by ANNEXIN /PI kit (MEBCYTO, MBL, MA, USA) according to the manufacture instruction. The cytotoxic effects of EVs on target cells were followed and documented by:

1. Light microscope (ZOE™ Fluorescent Cell Imager, Bio-Rad), analyzed by Image J software;

2. INCUCYTE (Sartorius, Germany), a live-cell imaging and analysis. The percentage of apoptotic target cells were calculated from the number of cells labeled with PI / total cells nuclei number or PI / total cells area. In addition number of cells labeled with CASPAS 3/7 or with cytotoxic dye was measured.

Statistical analysis

GraphPad Prism 4, Bonferroni's Multiple Comparison, one way ANOVA test and nonparametric Mann Whitney t- test. Example 1. Characterization of the extracellular vesicles obtained in Method 1

Extracellular vesicles were obtained from stimulated T cells expressing N29 CAR or from un-transduced (UT) T cells using Methods 1-5. The results are described in details in WO 2020/212985 incorporated herein by reference and in Aharon et al. (HUMAN GENE THERAPY, 2021, VOLUME 32, NUMBERS 19-20)

Shortly, a population of EVs obtained by methods in which no ultracentrifugation at a force above 20,000g was used comprise a mixture of exosomes and microvesicles and showed a much profound cytotoxic effect than a population of exosomes only. EVs obtained by Method 1 were compared with EVs obtained by the same method from un-transduced T cells and with EVs from unstimulated CAR T cells (see for also Aharon et al. (HUMAN GENE THERAPY, 2021, VOLUME 32, NUMBERS 19-20). Aharon et al., shows that EVs obtained from untranduced T cells and unstimulated CAR T cells behave similarly and do not show cytotoxic effect.

Typically, studies are concentrated in obtaining and testing exosomes. However, we empirically discovered that presence of 25% or more of microvesicles, i.e. EVs having the size of 150-1000 nm significantly improved the cytotoxic effect of the population of EVs. The distribution of EVs size obtained by Methods 1-5 are presented in Table 1.

Table 2. Percent of EVs having size above 150nm

Method 1 provided the best results and therefore, most of the further examples exploited it. As can be seen, EVs obtained by Method 1 comprise more than 40% of EVs having the size of 150-1000 nm and the rest were exosomes (30-150 nm). Example 2. Characterization of CAR EVs isolated from CAR T cells stimulated on coated beads

Transduction efficacy

For the preparation of CAR T cells, retroviral transduction of T cells was performed as described in the materials and methods section. Briefly, PBLs were isolated from the blood of healthy human donors by density gradient centrifugation on Ficoll-Paque (Axis-shield, Oslo, Norway). PBLs were activated in tissue culture non-treated 6-well plates pre-coated with anti-human CD3 and anti-human CD28 for 48 h at 370C. Activated lymphocytes were harvested and subjected to two consecutive retroviral transductions in RetroNectin pre-coated, tissue culture-non-treated 6-well plates supplemented with human IL-2 (100 lU/mL). After transduction, cells were cultured in the presence of 350 lU/mL IL-2 for 24 - 72 h. To evaluate transduction efficiency, cells were stained with the specific anti-CAR antibody, anti-N29 or with anti-GFP antibody (GFP reporter gene for the N29 CAR). The percentage of positive cells was determined by flow cytometry. Results are presented in Fig. 1. Cells expressing the transduced CAR and stained with anti-N29 (aN29), activated cells not expressing the CAR were included as a T cell control (untransduced, UT), cells stained with GFP (GFP).

As can be seen in Fig. 1 the transduction rate of the N29 CAR was 55-60%. In other words, 55-60% of the cells were transduced cells that expressed the N29 CAR. The successfully transduced cells (N29 GFP CAR T cells) were isolated and further characterized.

Beads conjugation with the HER2 protein

Anti-Biotin MACSiBead Particles Beads (Miltenyi Biotec, Germany) were conjugated with a fragment of human HER2/ErB2 protein comprising the amino acid sequence set forth in SEQ ID NO: 84 (His & AVI Tag, Sino biological) according to manufacture instructions (30 pg total biotinylated protein per IxlO⁸ Anti-Biotin MACSiBead Particles). HER2 conjugated beads were labeled with anti HIS-FITC and with anti HER2- APC antibodies (BD). Beads characteristics before conjugation and one month after conjugation are presented in Fig. 2A- 2F. Before conjugation with the HER2 protein: Figs. 2A: beads size and florescent intensity: Figs. 2B - APC and Figs. 2C - FITC; and one month post conjugation Figs. 2D beads size and florescent intensity; Figs. 2E - APC and Figs. 2F - FITC. Stable conjugation demonstrated and 98% of the spheroid beads found to be conjugated with HER2 protein after month.

CAR T cells activity - Cytokine secretion

The successfully transduced cells (N29 GFP CAR T cells) were isolated and further characterized. The N29 CAR T cells' IFN-y secretion levels following a stimulation with HER2 coated beads were evaluated and compared to IFN-y secretion levels of N29 GFP CAR T cells stimulated with SKOV (HER2+) target cells. N29 CAR T cells were divided into two stimulation treatments, 2xl0⁸ cells per stimulation treatment. The cells were incubated with about 2xl0⁸ of HER2 coated beads (average beads size 3.5 pm)

The EVs were obtained using Method 1 described above. Shortly, CAR T cells were stimulated with HER2- conjugated beads in ratio of 1:1 for 24-72hrs, at 37°C or with 1:1 SKOV target cells for 24-72hrs, at 37°C. The EVs from CAR T cells stimulated with beads coated with the targeted protein were obtained as follows: cell media was collected and centrifuged at 400 g for 5-10 min and the cell pellet was discarded. The supernatant was then centrifuged for 15-30 min (about 20) at 1500 g and the cell debris was then discarded. The original supernatant was then submitted to centrifugation at 20,000 g for 1 h at 40C. The resulting EV pellets were frozen in aliquots at -80°C. The EVs from CAR T cells stimulated with target cells were obtained as described above.

Fig 3 presents IFN-y secretion levels (pg/ml) of N29 GFP CAR T cells stimulated with HER2 coated beads (Beads) or with SKOV cells (SKOV). As can be seen in Fig. 3, higher levels of IFN-y secretion were found in N29 CAR GFP T cells stimulated with HER2 coated beads compared to the same cells stimulated with target cells ovary cancer cells (SKOV) expressed HER2.

EVs derived from N29-GFP CAR T- were analyzed for the appearance of green color. The green fluorescence derived from the GFP in the EVs was measured by florescent laser by flow cytometry. The results are presented in Fig. 4. As can be seen from the figure, about 28% of EVs isolated from GFP CAR T cells were colored, in other words, contained the green GFP CAR fragment.

Example 3. Purity of EVs derived from CAR T cells stimulated with beads coated with the target protein

Without being limited to any particular theory it is estimated that target cells expressing the target protein/antigen of interest used for stimulation of CAR T cells secrete EVs just as CAR T cells. Therefore, it is estimated that when producing activated EVs from CAR T cells stimulated by target cells, the resulting EVs are a mixture of EVs originated from both the CAR-T cells and the target cells. In a previous experiment with HER2 as a target protein and with stimulation with HER2+ cells, -40% of the resulting EVs were derived from the CAR T cells (CAR T EVs). To increase EVs purity and avoid the unwanted effect of EVs derived from cancer cells, an inert system of HER2 recombinant protein coated beads with was developed, produced, calibrated and used for the stimulation of CAR T cells. Following CAR T transduction, specific anti-HER2 CAR-T or untransduced (UT) cells were stimulated with HER2 -bound beads or with HER2+ cancer cells. To differentiate EVs derived from CAR-T cells stimulated with HER2 coated beads or with HER2+ cancer cells, a portion of anti-HER2 CAR-T cells and UT cells were labeled with Calcein AM Dye (green) before cell stimulation. Next, HER2 CAR-T cells and UT cells were stimulated for 24 h with HER2 coated beads or with HER2+ cancer cells. IFN-y secretion levels by the stimulated CAR T cells were evaluated and found comparable to the secretion levels using SKOV cells as previously explained.

The EVs were isolated as described in Example 2. The isolated EVs derived from unlabeled and Calcein AM labeled CAR-T cells and UT cells media were analyzed by (Fluorescence-activated cell sorting) FACS analysis. Results are presented in Figs. 5-12. Fig. 5 depicts Megamix HER2 coated beads plot. Fig. 6 depicts EVs obtained from non-labeled anti-HER2 CAR T cells stimulated with HER2 coated beads. Fig. 7 depicts EVs obtained from non-labeled SKOV HER2+ cells. Fig. 8 depicts EVs obtained from non-labeled UT cells stimulated with HER2 coated beads.

Fig. 9 depicts EVs obtained from Calcein AM labeled anti-HER2 CAR T cells stimulated with HER2+ cancer cells. Fig. 10 depicts EVs obtained from Calcein AM labeled anti-HER2 CAR T cells stimulated with HER2 coated beads. Fig. 11 depicts EVs obtained from Calcein AM labeled UT cells stimulated with HER2+ cancer cells. Fig. 12 depicts EVs obtained from Calcein AM labeled UT cells stimulated with HER2 coated beads.

As can be seen in Fig. 5, there are two picks indicating bead size of 0.5 and 0.9 pm As can be seen in Fig. 7 the EVs also shed from the cancer cells that used for the CAR T stimulation. As can be seen in Fig. 8 EVs also shed from UT cells however to a less extent. This was made to understand the background of non- stimulated UT non labeled EVs. As can be seen in Figs. 9-10, -38% of the EVs population of anti-HER2 CAR-T EVs following stimulation on target cells were labeled in green, meaning -38% of the EVs originated specifically from the HER2 CAR-T cells (Fig. 9). In contrast, when stimulated with HER2 coated beads, 94% of the EVs population were labeled in green, meaning 94% of the EVs originated specifically from the HER2 CAR-T Cells (Fig. 10). As can be seen in Figs. 11-12, about 47% of the EVs population of UT EVs following stimulation on target cells were labeled in green, meaning -47% of the EVs originated specifically from the UT cells (Fig. 11). In contrast, when stimulated with HER2 coated beads, 90% of the EVs population were labeled in green, meaning 90% of the EVs originated specifically from the UT cells (Fig. 12).

When analyzing EVs originated from stimulation on target cells -38% of stimulated

HER2 CAR-T EVs and 47% of stimulated UT EVs were labeled in green (Figs. 9 and Fig. 11, respectively). Without being bound by any theory or mechanism of action, it is estimated that the remaining EVs in the mixture are originated from the non-labeled SKOV target cells used for the CAR-T stimulation. In comparison, 94% of EVs derived from HER2 CAR-T EVs obtained from CAR-T cells stimulated on beads and 90% of stimulated UT EVs stimulated on beads were labeled in green (Figs. 10 and Fig. 12, respectively). Using beads for stimulation increased significantly (more than 2-fold) the percent of EVs of interest in the total EVs population by at least two-fold (from -38% to 94%). The results indicate the high purity of CAR-T EVs samples. The EVs secreted from cells stimulated with beads-bound protein are originated from the CAR-T cells only and are devoid of EVs from the target cells that potentially may have adverse effect. Hence, stimulations with beads coated with a protein/antigen of interest is advantageous in achieving highly pure product (e.g., population of isolated EVs in which most of the EVs are derived from cells of interest, isolated EVs with high purity). The production of CAR T derived EVs, particularly the stimulation of the CAR T cells with specific protein/antigen coated beads is pertinent to diverse proteins and is not limited to the HER2 protein. The protein or antigen of interest may be a Tumor-associated Antigen (TAA), a Tumor Specific Antigen (TSA), or any protein or antigen of interest.

Example 4. Multiple stimulations of CAR T cells

To produce a large amount of EVs derived from the same CAR T cells, three constitutive stimulations were done using the same CAR T cells and coated beads. Supernatant were collected every 24 hour, the EVs were separated and isolated and media was re-added to produce an additional batch of harvest (each such 24-hours incubation after the EVs were isolated (or not present) in the media is referred to as one stimulation, e.g. stimulation 1/2/3). Next the CAR T cells underwent stimulation with HER2-coated beads as described in Examples 8 and the EVs were isolated as described in that example. The size and concentrations of the resulting EVs were measured by Nanoparticle-Tracking Analysis (NTA; NanoSight). The NTA results are presented in Figs. 13-15, EVs size distribution analysis after the first (I), second (II), and third (III) stimulation, respectively.

As can be seen in Figs. 13 15, the EV pellets of the three stimulations contained vesicles in a variety of size.

The mean EVs size of each stimulation was calculated for each, first stimulation (Stim I), second stimulation (Stim II), and third stimulation (Stim III), results are presented in Fig. 16. As can be seen in Fig. 16, the average size of EVs obtained from HER2 CAR T cells of the three stimulations were similar (Stimulation 1: 174.4+14.80 nm; Stimulation 2: 184.0+17.17 nm, Stimulation 3: 178.3+12.00 nm).

The percent of large (>150nm, i.e. 150-1000 nm) and small EVs (<150nm, i.e. 30-150 nm) for each one of the stimulations 1 (Stim I), 2 (Stim II), and 3 (Stim III), was analyzed, and the results are presented in Figs. 17-18. Fig. 17 depicts the average percentage of large EVs (>150nm) for each stimulation. Fig. 18 depicts the percent of large EVs (gray) Vs small EVs (black) of each stimulation.

As can be seen in Figs. 17-18, the percentage of EVs larger than 150nm found to be similar in the three stimulations (Fig. 17). The small EVs (<150nm) accounted for -50% and the large EVs (>150 nm) accounted for -50% (Stim 1: 48.04+ 5.726%; Stim 2: 51.96+ 6.516%, Stim 3: 50.91+ 9.707%) (Fig. 18). In other words, the ratio between exosomes and microvesicles was about 1:1.

Example 5. Cytokine content of EVs derived from CAR-T cells

To assess the anti-cancer and cytotoxic activity potential of the EVs derived from anti- HER2 CAR-T cells stimulated twice with HER2 coated beads, the cytokines profile and content of the EVs was evaluated after each stimulation and compare to their parental CAR-T cells. The cytokine content of stimulated CAR T cells by HER2 beads and their related EVs was measured by protein array and was calculated as median fluorescence intensity after reduction of the background. Cells and EVs were lysate (90 pg of each sample) and loaded on protein array assay. Protein levels of EVs were calculated as a ratio of proteins levels in the parental cells (EVs /CAR T protein cargo) and are summarized on the graph. Cell proteins (50 pg) were isolated from 10⁶ cells and the EV proteins (50 pg) were isolated from the medium of 5xl0⁶ cells. Results are presented in Fig. 19, EVs /CAR T protein cargo after the first stimulation denoted HER2 beads stimulation 1 (light gray), EVs /CAR T protein cargo after the second stimulation denoted HER2 beads stimulation 2 (dark gray).

As can be seen in Fig. 19, the concentrations of most cytokine in parental CAR-T cells stimulated with HER2 coated beads were equal to or higher than those measured in their related EVs. However, the levels of IL- 16 and Macrophage Colony-Stimulating Factor (M- CSF) were higher in EVs sample obtained after the first stimulation of HER2 CAR T cells with HER2-coated beads compared to their parental cells. The levels of IL-4, IL-12p70 and IL- 17 were higher in EVs sample obtained after the second stimulation of HER2 CAR T cells with HER2 coated beads compared to their parental cells. Next, the levels of Granzyme B were measured and analyzed. Granzyme B is a serine protease granzyme secreted by cytotoxic T cells and natural killers and mediates apoptosis in target cells. Isolated EVs derived from the four samples of cell cultures: untransduced cells and CAR T cells with or without stimulation using HER2 coated beads were lysed and analyzed and quantified by Western blot, and normalized to actin. Results are presented in Fig. 20 showing Granzyme B content in the EVs derived from untransduced cells (UT), EVs derived from untransduced cells incubated with HER2 coated beads (UT +stim on beads), EVs derived from anti-HER2 CAR T cells (ErbB2 CAR T), and EVs derived from anti-HER2 CAR T cells stimulated on HER2 beads (HER2 CAR T +stim on beads).

As can be seen in Fig. 20, the levels of Granzyme B (expressed as a ratio of actin) were four times higher in EVs obtained from CAR T cells after one stimulation with HER2 coated beads compared to EVs obtained from non- stimulated CAR T cells or compared to EVs isolated from UT cells with or without stimulation.

Example 6. Cytotoxic effects of EVs derived from CAR T stimulated with coated beads

The cytotoxicity of EVs derived from CAR-T cells stimulated twice (consecutive stimulations) with HER2 coated beads was evaluated. Each batch of stimulation was collected and tested separately in order to compare the cytotoxic efficiency between each stimulation. SKBR (HER2+) adenocarcinoma cells expressing mCherry red fluorescent protein (mCherry- SKBR, target cells 5000/96 plate well) were incubated with UT T cells or CAR-T cells (effector cells) at a ratio of 2:1 Effector :Target cells (E:T) or with EVs (50pg/96 plate well) obtained from UT T-cells or CAR-T cells after each stimulation I, II, III compared to untreated SKBR cells. Magnified microscopic images of the tested cultures are presented in Figs. 21A- 211. Fig. 21A shows the image of mCherry-SKBR target cells incubated with UT cells, served as a control for the effector cells, at a E:T ratio of 2:1. Fig. 21B, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation I. Fig. 21C, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation II. Fig. 21D, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation III. Fig. 21E, untreated (no incubation with effector cells or EVs) mCherry-SKBR cells served as control. 21F, mCherry- SKBR, target cells incubated with anti-HER2 CAR T cells, effector cells, at a E:T ratio of 2: 1. Fig. 21G, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation I. Fig. 21H, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation II. Fig. 211, mCherry-SKBR, target cells incubated with anti-HER2CAR T cells derived EVs after stimulation III. As can be seen in Figs. 21A-21I, in both control cultures, mCherry-SKBR incubated with UT cells or not incubated with any cells (untreated), appear to proliferate and a vast area of the well is covered. The mCherry-SKBR cells incubated with UT derived EVs from each of the stimulations I, II, and III, also appeared to proliferate and cover the majority of the well’s area. In contrast, in the cell cultures of mCherry-SKBR cell incubated with HER2 CAR T cells, or EVs obtained therefrom after each stimulation, I, II, III, there was no proliferation, and the few detected cells exhibited morphological changed typical to cell apoptosis (nonadherent apoptotic round cells).

Target cells apoptosis (or proliferation) was monitored over the period of 96 hours by detection of the red color intensity of the mCherry protein, for each of the cultures as described above. Results are presented in Fig. 22, mCherry-SKBR, target cells incubated with UT cells served as a control for the effector cells, at a E:T ratio of 2:1, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation I, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation II, mCherry-SKBR, target cells incubated with UT cells derived EVs after stimulation III, untreated (no incubation with effector cells or EVs) mCherry-SKBR cells served as control), mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells, effector cells, at a E:T ratio of 2:1, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation I, mCherry- SKBR, target cells incubated with anti-HER2CAR T cells derived EVs after stimulation II, mCherry-SKBR, target cells incubated with anti-HER2 CAR T cells derived EVs after stimulation III.

As can be seen in Fig. 22, the increase in red color intensity of mCherry labeled cells indicates high cell proliferation while a decrease indicates cell death. A decrease of red color intensity was observed in mCherry-SKBR cells incubated with anti-HER2 CAR T cells for the 45 hrs and from there on the red color intensity remained substantially the same, indicating cell death. For the first 25 hrs a moderate increase in the intensity of the red color was detected in all other cell cultures, a small decrease was observed for the next 5 hrs. However, in the mCherry-SKBR cells incubated with UT cells and EVs obtained from UT cells with three consecutive stimulations using HER2 coated beads an exponential increase in red color intensity was observed. This increase in color intensity indicates cell proliferation. In other words, the UT cells and EVs derived therefrom exhibited no cytotoxic effect. In contrast, CAR-T EVs, obtained from three consecutive stimulations using HER2 coated beads, induced cell death, the red color intensity unchanged from the 30^th hr to the 96^th hr. The CAR-T EVs from stimulation I and III results were comparable, CAR-T EVs from stimulation II exhibited a slightly higher decrease in color intensity compared to stimulation I and III. However, There was no significant change between EVs of stimulation II compared to stimulation I or III.

It is clear from the results that CAR T cells and EVs obtained from the three consecutive stimulations exerted an anti-cancer cytotoxic activity and mediated cell death of target cells.

Caspase 3/7 activity

Caspase 3/7 is a cellular marker for apoptosis it is stained green once activated, indicating apoptosis in target cells. SKBR cells (5000 cells/well in 96 plate well) were incubated with UT or anti-HER2 CAR T cells or EVs thereof (50pg/per well in 96 plate well) obtained after each one of the three stimulations with HER2 coated beads. The SKBR cells were documented by Incucyte for 92 hours in 4h intervals. Caspase 3/7 activity was calculated as the ratio of total green color intensity to cell coverage. Results are presented in Fig. 23, SKBR cells incubated with anti-HER2 CAR T cells, with anti-HER2 CAR T EVs obtained from stimulation I, stimulation II, with UT Cells, and with UT EVs obtained from stimulation I, stimulation II, and Stimulation III.

As can be seen in Fig. 23, UT T cells or UT EVs obtained from each of the three stimulation cycles showed little to no increase in caspase activity and had no effect on SKBR cells’ apoptosis. In contrast, anti-HER2 CAR T cells showed a steep increase in caspase activity at the time period between the 16^th hr to the 36^th hr which than stabilizes at high level of activity. Anti-HER2 CAR T EVs obtained from stimulation I and II showed a more moderate yet consistent increase in caspase 3/7 activity over time. It is apparent that HER2 CAR T cells and by their related EVs induce caspase activity and target cells’ apoptosis over time.

The results indicate that the CAR T EVs of the present invention obtained from CAR T cells stimulation or multiple stimulation cycles have a cytotoxic and anti-cancer activity and effect towards their corresponding target cells.

Example 7. Establishment of viral particles Producers PG13 stable cell lines for effective transduction of lymphocytes with EGER and CD276 CAR-T cells

The production of retroviral vectors was achieved through the utilization of the PG 13 packaging cell line, which is derived from NIH3T3 mouse cells. These PG13 cells are stably transfected with the Moloney murine leukemia virus gag-pol proteins and the Gibbon ape leukemia virus envelope protein. Plasmids encoding the anti-CD276 CAR (the DNA sequence encoding the CARs are as set forth in SEQ ID NOs: 35 and 40 and anti-EGFR CAR constructs (the sequence the DNA sequence encoding the CARs are set forth in SEQ ID NO: 45 and 50) including a FLAG epitope (as a marker for detection and sorting) were integrated into PG-13 viral producing packaging cells. The transduction was performed as follows:

Day 1 - Separate PG 13 packaging cell plate into several as needed (according to standard cell culture plate separation protocol) and incubate until needed. Viral medium consists of 87% DMEM (Dulbecco's Modified Eagle's Medium), 10% FCS (Fetal Calf Serum), 1% L-glutamine, 1% antibiotics mix (penicillin, streptomycin & nystatin) & 1% sodium pyruvate - all at stock concentration.

Separate frozen lymphocytes and commence activation in an activation plate and incubate for two days at 5% CO2 at 37°C.

Day 2 - Suction of all PG 13 packaging cell plates’ supernatant and adding of 5ml of medium to each plate. This is done on order to work with smaller volumes at the time of addition of the virus to the lymphocytes, so as to not change their concentration too much.

Day 3 - Add 1.5 ml/well of viral supernatant supplemented with 100 U/ml of IL-2 to an FN coated 6-well plate and incubate for 30 minutes at 37°C.

Harvest activated lymphocytes from the stimulating plate, wash with PBS and resuspend, 2.5x106 in 1.5 ml of viral supernatant + 100 U/ml IL-2. Add to the RN plate containing the viral supernatant.

After 6 h at 37°C 7.5% CO2, remove the viral supernatant gently and replace with 4 ml of RPMLFCS + 100 U/ml IL-2 medium. Incubate over night at 37°C 5% CO2.

Day 4 - Repeat day 3.

Day 5 - harvest the lymphocytes by vigorous pipetting with PBS and washing of the wells to obtain all cells. Re-suspend the cells in RPMLFCS medium with 350 U/ml of IL-2, and incubate in 37°C 5% CO2.

The stimulation of the CAR T cells was performed usually at days 7-8.

The retroviral backbone encoding each CAR was integrated into these cells, enabling constitutive production of secreted retroviral vectors. Following integration, infected PG 13 cells were sorted using an anti-FLAG PE conjugated antibody using flow cytometry (BD FACS Aria™ III Cell Sorter). Results of the Flow cytometry analysis of anti-CD276 CAR and anti-EGFR expression in infected PG13 cells are presented in Figs. 24-25, respectively. The fluorescence intensity represents the expression level of the CAR on the surface of the sorted PG13 cells. As can be seen in Figs 24-25, 95.56% of the cells expressed anti-CD276 CAR (Fig. 24), and 91.28% of the cells expressed anti-EGFR CAR (Fig. 25).

The resulting cell lines served as continuous retrovirus producers, capable of generating retroviruses carrying the anti-CD276, or anti-EGFR CAR constructs.

Example 8. T cells transduction

For the production of anti-CD276, or anti-EGFR CAR T cells the respective retroviral vectors, carrying the anti-CD276 or anti-EGFR CAR genes, were used to infect T cells, enabling the integration of CAR genes into the T cell genome. This transduction process (as detailed in T-cell preparation section) allowed the modified T cells to express CARs on their surface, equipping them with the ability to recognize and eliminate cancer cells expressing the corresponding antigens. The efficiency of transduction was assessed by flow cytometry, the transduced cells were stained with an anti-FLAG PE antibody (PE anti-DYKDDDDK Tag Antibody Rat IgG2a, X clone #L5 0.2 mg/ml, 0.125 pg per 1- 10⁶ cell) to detect the presence of the introduced CAR construct.

Fig. 26 presents the flow cytometry SSC:FSC gating strategy. Figs. 27-28 presents the flow cytometry and transduction percentage of anti-CD276 and anti-EGFR CAR, respectively, to PBLs compared to control unstained samples based on gate 1 (as presented in Fig. 26) and detected by anti-FLAG PE antibody. Fig. 29, presents transduction statistics obtained from different blood donors (N=6), data are expressed as mean ± SD from separate experiments.

As can be seen in Fig. 26, 79.6 % of the cells expressed the CAR construct were detected by the anti-FLAG antibody. It is clear from Figs 27-29 that 41.3+3.2% of the PBLs were transduced with anti-CD276 CAR, and that 36.85+9.3% of the PBLs were transduced with anti-EGFR CAR.

Example 9. EGFR and CD276 expression in lung adenocarcinoma and glioblastoma cell lines

To examine EGFR and CD276 expression on target cells for CAR, both antigens were evaluated across various lung cancer and glioblastoma cell lines. The selected lung adenocarcinoma cell lines A549, H1975, and HCC827 and glioblastoma cells U251 were stained with anti-EGFR and anti-CD276 antibodies, along with unstained respective cells and isotype controls. The fluorescence signal intensity was measured by FACS. Results for the expression levels of EGFR and CD276 on lung cancer cell lines are presented in Figs. 30A- 30B, respectively. Unstained A549, H1975, and HCC827 cells (US A549, US H1975, and US HCC827), isotype control of A549, H1975, and HCC827 cells (isotype A549, isotype H1975, and isotype HCC827), the cells stained with anti-EGFR antibodies or with anti-CD276 antibodies (A549, H1975, and HCC827). Results for the expression levels of EGFR and CD276 glioblastoma U251 cell line are presented in Figs. 31A-31B, respectively. Unstained U251 cells (US) were used as background control, isotype control (isotype), the cells stained with anti-EGFR antibody (stained).

As can be seen in Figs. 30A-30B, A549 and Hl 975 lung cell lines exhibited similar levels of EGFR expression, while HCC827 cell line demonstrated a higher signal intensity compared to the controls. However, CD276 expression on HCC827 and H1975 cell line showed similar signal intensities, while A549 cell line displayed a slightly lower signal compared to the controls. As can be seen in Figs. 44A-44B, both EGFR and CD267 expression levels on glioblastoma cell line U251 were comparable to unstained cells or to isotype control. In conclusion, FACS analysis showed that A549 (adenocarcinoma human alveolar basal epithelial cells) and lung cancer epithelial cell lines Hl 975 and HCC827 have high levels of both antigens EGFR and CD267. Glioblastoma U251 cells express high level of EGFR and moderate level of CD276

Example 10. Anti-EGFR and anti-CD276 CAR T cells cytotoxic activity against lung cancer cells and glioblastoma cells

The cytotoxicity activity and killing percentage induced by anti-EGFR CAR T cells and anti-CD276 CAR T cells against target cells were assessed by methylene blue. Anti-EGFR CAR comprised the amino acid sequence SEQ ID NO: 15 or 20. Anti-CD276 CAR comprised the amino acid sequence SEQ ID NO: 5 or 10. Three different lung adenocarcinoma cell lines (HCC827, H1975 and A549) and one cells line of glioblastoma (U251) were used as target cells. For the antitumor cytotoxicity assay of anti-CD276 or anti-EGFR CAR-T cells (Effector cells), Effector T cells were co-cultured with cells of each of the target cell lines at various Effector (E): Target (T) ratios (10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, 0.3125:1) in a total volume of 200pL for 24 hours. Figs. 32-35, present the cytotoxic effect (% of killing) of different E:T ratios of anti-EGFR CAR T cells (gray line with squares) and anti-CD276 CAR T cells (Black line with circles), compared to untransduced cells (UT, dashed line with triangles). Data are expressed as mean ± SD of 4-5 samples from separate experiments (N) and analyzed by two- way ANOVA analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared each concentration time point to un-transduced (UT) cells. Fig. 32 shows an anti-EGFR CAR T and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line HCC827. Fig. 33, anti- EGFR and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line Hl 975. Fig. 34, anti-EGFR and anti-CD276 CAR T cytotoxic effect on lung adenocarcinoma cell line A549. Fig. 35, anti-EGFR and anti-CD276 CAR T cytotoxic effect on glioblastoma U251 cell line.

As can be seen in Figs 32-34, anti-EGFR CAR T cells and anti-CD276 CAR T cells in ratio 2.5:1 and up induced significant killing effects (above 75% killing) compared to UT cells of HCC827 and H1975 lung cancer cells. The anti-EGFR CAR T cells induced killing in a ratio-dependent manner on A549 cells reaching about 70% killing at an E:T ratio of 10:1. The anti-CD276 CAR T cells induced about 25% of killing on A549 cells reaching about 70% killing from E:T ratio of 2.5:1 and above. As can be seen in Fig 35, both anti-EGFR CAR T cells and anti-CD276 CAR T cells in ratio 2.5:1 and up induced significant killing effects (above 75% killing) compared to UT cells of glioblastoma U251 cells.

Next, IFN-y secretion levels by the anti-EGFR CAR T cells and anti-CD276 CAR T cells following the co-culture/stimulation with lung cancer target cells, were evaluated by ELISA. Fig. 36 presents ELISA results for the IFN-y levels (pg/mL) secreted by anti-EGFR CAR T cells (light gray, N=4) and anti-CD276 CAR T cells (dark gray, N=4), stimulated with HCC827, H1975 and A549 cells, untransduced cells served as a control (UT, black, N=3). Data are expressed as mean ± SD of 3-4 samples from separate experiments (N) and analyzed by two-way ANOVA analysis. *p < 0.05, **p < 0.01.

As can be seen in Fig. 36, both anti-EGFR CAR T cells and anti-CD276 CAR T cells co-cultured/stimulated with HCC827 and Hl 975 lung cancer cells secreted significantly higher IFN-y compared to UT cells. A more moderate increase in IFN-y secretion by both anti- EGFR CAR T cells and anti-CD276 CAR T cells co-cultured/stimulated with A549 cells compared to UT cells.

Example 11. Anti-EGFR and anti-CD276 CAR T derived EVs production and size distribution

For the production of EVs from anti-EGFR CAR T and anti-CD276 CAR T, CAR T cells derived from two different healthy donors or UT cells were stimulated for 24h-72h with lung cancer cells at a 2:1 E:T ratio. Following the stimulation, the EVs from each CAR T cell line were isolated according to Method 1. EVs sizes and concentrations were measured by nanoparticle-tracking analysis (NTA; NanoSight, version 3.1). To this end, all samples were diluted 1:100 with filtered (0.025um filter) PBS before analysis. Results for size distribution analysis of anti-EGFR CAR T EVs of donor 1 are presented in Fig. 37. Results for size distribution analysis of anti-CD276 CAR T EVs of donor 1 are presented in Fig. 38. Results for size distribution analysis of anti-EGFR CAR T EVs of donor 2 are presented in Fig. 39. Results for size distribution analysis of anti-CD276 CAR T EVs of donor 2 are presented in Fig. 40.

The percentages of small (<150nm, (30-150 nm) light gray) and large (>150nm, (150- 1000 nm) dark grey) EVs of anti-EGFR and anti-CD276 from donor 1 and donor 2 are presented in Fig. 41.

As can be seen in Figs. 37-41, the percentage of anti-CD276 EVs larger than 150nm accounted for -60% or -70% of the EV’s from donor 1 and donor 2 respectively. The percentage of anti-EGFR EVs larger than 150nm accounted for -40% or -70% of the EV’s from donor 1 and donor 2, respectively.

Example 12. CAR-T cells and CAR T EVs cytotoxicity towards lung cancer target cells

The anti-EGFR and anti-CD267 CAR T cells and their related CAR T EVs, as described in Example 11, were further evaluated for their anti-cancer activity and cytotoxic effect towards mCherry-HCC827 and mCherry-A549 lung cancer cells. mCherry-HCC827 and mCherry-A549 cells were seeded on 96-well plates (5000 cell/well). After 6h, EVs (50ug/200ul medium) obtained from UT cells or anti-EGFR or anti-CD267 CAR T cells stimulated with H1975 lung cancer cells, or EVs from target cells H1975, were added to mCherry-HCC827 and mCherry-A549 target cells. The anti-cancer activity and cytotoxic effect of the EVs was compared to anti-EGFR or anti-CD267 CAR T cells, or UT cells that were added to the target cells at a ratio of 2:1 (E:T) or to untreated target cells. Cells were documented by IncuCyte Systems for Live-Cell Imaging every 4 h. Red fluorescent intensity of the mCherry-HCC827 and mCherry-A549 over time were calculated. Results of the cytotoxic effect towards mcherry-HCC827 are presented in Figs. 42A-56M. Fig. 42A, mCherry-HCC827 with anti-EGFR EVs from donor 1. Fig. 42B, mCherry-HCC827 with anti- EGFR EVs from donor 2. Fig. 42C, mCherry-HCC827 with anti-CD276 EVs from donor 1. Fig. 42D, mCherry-HCC827 with anti-CD276 EVs from donor 2. Fig. 54E, mCherry- HCC827 with UT EVs. Fig. 42F, mCherry-HCC827 with lung cancer EVs Fig. 54G, mCherry-HCC827 with no EVs (untreated). Fig. 42H, mCherry-HCC827 with anti-EGFR CAR T cells from donor 1. Fig. 421, mCherry-HCC827 with anti-EGFR CAR T cells from donor 2. Fig. 42J, mCherry-HCC827 with anti-CD276 CAR T cells from donor 1. Fig. 42K, mCherry-HCC827 with anti-CD276 CAR T cells from donor 2. Fig. 42L, mCherry-HCC827 with UT cells. Fig. 42M, untreated mCherry-HCC827. The results indicate that the CAR T EVs of the present invention obtained from CAR T cells with anti CD267 or anti EGFR expressing cells specifically killed and had a cytotoxic effect, anti-cancer activity towards their corresponding target lung cancer cells.

Target cells apoptosis (or proliferation) was monitored over the period of 40-100 hrs by detection of the red color intensity of the mCherry protein, for each of the cultures as described above. Results of the apoptosis/proliferation of mCherry-HCC827 are presented in Fig. 43. mCherry-HCC827 with anti-EGFR EVs from donor 1 (grey line with squares); mCherry-HCC827 with anti-EGFR EVs from donor 2 (grey line with circles); mCherry - HCC827 with anti-CD276 EVs from donor 1 (grey line with rhombus); mCherry-HCC827 with anti-CD276 EVs from donor 2 (light grey line with triangles); mCherry-HCC827 with UT EVs (grey line with upside down triangles); mCherry-HCC827 with UT cells from donor

1 (light grey line with upside down triangles); mCherry-HCC827 with UT cells from donor 2 (black line with upside down triangles); mCherry -HCC827 with anti-EGFR CAR T cells from donor 1 (black line with squares); mCherry-HCC827 with anti-EGFR CAR T cells from donor

2 (black line with circles); mCherry-HCC827 with anti-CD276 CAR T cells from donor 1 (black line with triangle); mCherry-HCC827 with anti-CD276 CAR T cells from donor 2 (black line with rhombus); and mCherry -HCC827 with Hl 975 lung cancer EVs (light grey line with stars).

As can be seen in Fig. 43, both anti-EGFR and anti-CD267 CAR T cells and EVs obtained therefrom exhibited low red color intensity, indicating that the target cells underwent cell apoptosis. The red color intensity remained low and stable throughout the HOhrs of detection. EVs derived from lung cancer cells kept low red color intensity for about 80 hrs after which the intensity increased gradually. In contrast untransduced cells and their EVs exhibited a gradual increase in the red color intensity between 40 to 60 hrs, after which a steeper increase in color intensity was observed.

Results of the apoptosis/proliferation of mCherry-A549 are presented in Fig. 44. The figure shows the results for mCherry-A549 cells incubated with anti-EGFR EVs or anti- CD276 CAR T from donor 1 and donor 2 in comparison to incubation with UT cells or Hl 975 lung cancer EVs.

As can be seen that the activated EVs obtained from stimulated anti-EGFR CAR T cells and stimulated anti-CD276 CAR T induced a statistically significant cytotoxic effect seen by incocyte followup of mCherry expression of the target cells.

The Red fluorescent intensity were calculated over time and the percentage of killing effects of EV's and CAR-T cells stimulated with HCC827 cells were determined by methylene blue killing assay after 96hrs of exposure. Fig. 45 presents the percentage of killing of mCherry-A549/mCherry827 by anti-EGFR EVs (EVs EGFR, light grey), anti-CD276 EVs (EVs CD276, dark grey), untrnsduced cells derived EVs (EVs UT, black round corners rectangle), EVs derived from lung cancer (EVs lung cancer, white), anti-EGFR CAR T cells (CAR T EGFR, light grey with wavy lines), anti-CD276 CAR T cells (CAR TCD276, dark grey with wavy lines), untransduced cells (UT black rectangle).

As can be seen in Fig. 45, while exposure of the target cells to UT cells, UT EVs or to target cells EVs induced target cell proliferation over time, exposure of target cells to activated CAR T EVs against EGFR or against CD267 induced high percentage of target cells’ killing even higher than the parental CAR T cells. EVs from both anti-CD276 and anti-EGFR CAR T induced statistically significant high percentage of killing (>75%) compared to UT EVs (p<0.0001), and even higher than their parental cells.

Example 13. CAR-T cells and CAR T EVs cytotoxicity towards glioblastoma cells target cells

Apoptosis levels of U251 glioblastoma target cells induced by CAR T EVs were evaluate by caspase-3/7 green dye activity assay (Sartorius) and documented by IncuCyte Systems for Live-Cell Imaging. To that end, Glioblastoma U251 cells were seeded in 96 well plate (5000cell/well). Next anti-EGFR or anti-CD276 CAR T EVs (50ug/200ul medium) obtained from two donors, were added to the wells and compared to non-treated wells. Green caspase 3/7 activity reagent was added at time 0 and documented by IncuCyte after 40 hours every 4 hours. The apoptosis levels induced by CAR T EVs was compared to the apoptosis levels of untreated cells. Accumulation of the green caspase 3/7 dye activity (apoptotic cells, green dots) measured as object integrated intensity (GCU • lm2/image) over 4 days after 96h exposure to CAR EVs anti-EGFR or anti-CD276 compared to non-treated cells. Results are presented in Figs. 46A-46E. Fig. 46A, U251 glioblastoma target cells incubated with anti- EGFR CAR T EVs from donor 1. Fig. 46B, U251 glioblastoma target cells incubated with anti-CD276 CAR T EVs from donor 1. Fig. 46C, U251 glioblastoma target cells incubated with anti-EGFR CAR T EVs from donor 2. Fig. 46D, U251 glioblastoma target cells incubated with anti-CD276 CAR T EVs from donor 2. Fig. 46E, untreated U251 glioblastoma target cells.

As can be seen in Figs. 46A-46E, EVs from stimulated anti-EGFR and anti-CD276 CAR T from the two donors induced apoptosis in glioblastoma target cells. Apoptotic bodies were documented all across the glioblastoma cell culture. Next, Apoptosis levels (intensity of green caspase 3/7 activity) over time were calculated. The results are presented in Fig. 47, U251 glioblastoma target cells incubated with: anti-EGFR CAR T EVs from donor 1 (line with circles); anti-CD276 CAR T EVs from donor 1 (line with squares); anti-EGFR CAR T EVs from donor 2 (line with triangles); anti-CD276 CAR T EVs from donor 2 (line with rhombus); untreated U251 glioblastoma target cells (line with stars).

As can be seen in Fig. 47, the intensity of caspase 3/7 activity increased over time in cancer target cells treated with the CAR T EVs compared to untreated cells. The anti-EGFR and anti-CD276 CAR T EVs from both donors exerted anti-cancer and cytotoxic activity and induced significant levels of apoptosis over time in glioblastoma cancer cells.

The percentage of killing of glioblastoma cancer cells induced by EVs derived from anti EGFR CAR T and anti CD276 CAR T cells compared to untreated cells was calculated as well. Fig. 48 present the percent of killing of U251 glioblastoma target cells incubated with anti-EGFR CAR T EVs (EGFR, n=2, black) and with anti-CD276 CAR T EVs (CD276, n=2, grey). Data are expressed as mean ± SD for EVs obtained from T cells from donor 1 and 2.

As can be seen in Fig. 48A and 48B, the activated CAR T EVs induced high percentage of U251 glioblastoma target cells’ killing, anti-EGFR CAR T EVs, -77% percent killing, and anti-CD276 CAR T EVs, -64.4% percent killing.

In a further experiment, the efficacy of anti-EGFR CAR T cells, anti-CD276 CAR T cells, or EVs derived from these CAR T cells was assessed using Methylene blue killing assay of HCC827 (non-small cell lung cancer cell line), A549, and H1975 cell lines. The EVs were obtained using Method 1 as described above from CAR T cells stimulated by cancer cells. The experiment for CAR T cells was performed with different effector-to-target (E:T) ratios, compared to untreated (UT) cells. Different amount (and concentrations) of EVs were used, up to 50pg EVs per well. The results are presented in Figs. 49A-49F. Figs. 49A-49C show results for CAR T cells and Figs. 49D-49F show results for the efficacy of activated EVs of the present invention. A clear dose dependency is seen for the cell treatment by CAR T cell and by activated EVs from CAR T cells. It can be seen that EVs therapy reached 60%-80% killing of the cancer cells, while the plateau was not reached. Therefore, it is assumed that 100% killing may be reached upon increase of EVs concentration. In addition, considering that the EVs were obtained from CAR T cells that were stimulated by cancer cells comprising a TAA to which the CAR binds specifically, only 40-50% of the EVs comprise CAR, as shown in Example 3. We assume that by increasing the amount of "active" EVs, i.e. EVs comprising the CAR, either by increasing the amount the EVs, by using a system in which the stimulation of CAR T cells is done without the use of cancer cells as described above or by combining these a higher killing effect will be obtained.

Summary of the results

Summarizing all the presented results it can be clearly seen that only the population of EVs from Sample 1 (N29 co-cultured with SKOV) and purified by Methods 1, 3, and 5 provided a strong toxic (apoptotic) effect on HER2 positive cells: such as ovarian cancer cells (SKOV) cells and HER2 positive breast cancer cells (MDA231). These populations comprise a high content of large EVs, at least 25 % of the EVs have a size of 150 nm. The best results were obtained by EVs from Sample 1 purified by Method 1. At least 30 % of these EVs have a size of above 150 nm. It also can be seen that EVs obtained by Methods 2 and 4 that comprised much smaller EVs (in fact comprised mostly exosomes) did not generate a high rate of apoptosis, and definitely not at the same level as samples with high content of large EVs.

It is also clear that stimulation of CAR T cells with beads coated with a target of interest (HER2, EGFR or CD276) yielded a population of EVs with high purity of specifically derived from the CAR T cells (>90% of total EVs) which kept their anti-cancer and cytotoxic activity. Additionally, the multiple stimulation cycle with the coated beads, while collecting EVs after each cycle, allowed the re-use of the same CAR T cells, and provided populations of EVs comprising high percentages of EVs specifically derived from the CAR T cells. The EVs populations from each of the stimulation cycles comprise high content of large EVs (-50%) and provided a strong anti-cancer and cytotoxic effect on the relevant target cells (-60-75% target cells killing). It was further shown that the killing effect of EVs is dose-dependent.

Example 14. Scale-up production.

In order to increase the EVs yield we used different bioreactor systems.

For example, for scale up we used GREX cell culture platform, a methods and process optimization for large-scale CAR T expansion and thus CAR T EVs production. The process in the GREX may include activation transduction and proliferation of CAR T cells or only part of the stages. Specifically, EVs were isolated from N29 CAR T cells that were stimulated with HER2 coated beads in flask or in GREX. (The results are presented in Figs. 50A-50D. Fig. 50A - EVs protein concentration, Fig. 50B - EVs count /ml (NTA analysis), Fig. 50C - EVs mean size, Fig. 50D - percentage of EVs larger then 150nm, Fig. 50E shows N29 CAR T EVs killing effects on SKOV cells after 96 hours of co-culture).

Other technologies: Hollow fiber bioreactors (HFBRs) have increasingly been implemented for EV production. In these dynamic setups, cells are expanded on cylindrical hollow fibers, which can host 100-fold more cells than common T-flasks (M. Lu, Eur. J. Pharm. Biopharm. 2017). Alternative bioreactors 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.

Example 15. Preparation of EVs anti-CD19 CAR T cells stimulated with beads coated with CD19 protein

Beads conjugation with the CD 19 protein

Anti-Biotin MACSiBead Particles Beads (Miltenyi Biotec, Germany) were conjugated with a fragment of human CD 19 protein comprising the amino acid sequence as set forth in amino acid sequence SEQ ID NO: 81 according to manufacture instructions (30 pg total biotinylated protein per IxlO⁸ Anti-Biotin MACSiBead Particles). CD19 conjugated beads were labeled with anti-HIS-FITC and with anti CD19-APC antibodies (BD). Beads characteristics before conjugation and after conjugation are presented in Fig. 51A-51D. Conjugation demonstrated and 89% of the spheroid beads found to be conjugated with CD 19 protein.

The EVs were obtained using Method 1. Anti-CD19 CAR T Cells were stimulated with CD19- conjugated beads in ratio of 1:1 for 24-72hrs, at 37°C. The EVs from CAR T cells stimulated with beads coated with the targeted protein were obtained as follows: cell media was collected and centrifuged at 400 g for 5-10 min and the cell pellet was discarded. The supernatant was then centrifuged for 15-30 min (about 20) at 1500 g and the cell debris was then discarded. The original supernatant was then subjected to centrifugation at 20,000 g for 1 h at 40°C. The resulting EV pellets were frozen in aliquots at -80°C. The EVs from CAR T cells stimulated with target cells were obtained as described above. The size of the EVs after 1 or 2 stimulation is presented in Fig 52.

In an alternative example, EVs from anti-CD19 CAR T were obtained by stimulating anti-CD19 CAR T cells with cell membrane fragments comprising CD19 for 24-72hrs, at 37°C and further prepared as described above. Example 16. Effects of CD19 CAR T cells and CD19 CAR T EVs on NAML6 CD19⁺- GFP cells

EVs were isolated from 4 samples: 1) UT cells, 2) UT cells incubated with CD19- conjugated beads, 3) CD19 CAR T cells, 4) CD19 CAR T cells stimulated with CD19- conjugated beads.

CD 19 NAML6 - GFP target cells (peripheral blood lymphoma cell line) were co culture with UT or CD 19 CAR T cells or with EVs samples (1-4) for 24h. Target cells apoptosis was validated by Annexin V- APC.

Example 17. Preparation of EVs obtained from CAR-T cells stimulated with an antigen bound to inert beads.

T cells expressing anti-EGFR CAR are incubated for from 12 to 96 hour, and therefore, stimulated with beads coated with EGFR protein or with the peptide/fragment of the EGFR to which the CAR binds specifically. In some examples, the supernatant is collected and an additional batch of beads is added and a second batch of the supernatant is collected. This step is repeated once again in other examples. In one example, the EGFR protein has the amino acid sequence SEQ ID NO: 83.

T cells expressing anti-CD276 CAR are incubated for from 12 to 96 and therefore stimulated with beads coated with CD276 protein or with a peptide from the CD276 to which the CAR binds specifically. In some examples, the supernatant is collected and an additional batch of beads is added and a second batch of the supernatant is collected. This step is repeated once again in other examples. In one example, the CD276 protein has the amino acid sequence SEQ ID NO: 82.

T cells expressing anti-CD38 CAR are incubated for from 12 to 96 and therefore stimulated with beads coated with CD38protein or with the peptide from the CD38 to which the CAR binds specifically. In some examples, the supernatant is collected and an additional batch of beads is added and a second batch of the supernatant is collected. This step is repeated once again in other examples.

T cells expressing anti-CD138 CAR are incubated for from 12 to 96 and therefore stimulated with beads coated with CD138protein or with the peptide from the CD 138 to which the CAR binds specifically. In some examples, the supernatant is collected and an additional batch of beads was added and a second batch of the supernatant is collected. This step is repeated once again in other examples. T cells expressing anti-CD19 CAR are incubated for from 12 to 96 and therefore stimulated with beads coated with CD 19 protein or with the peptide from the CD 19 to which the CAR binds specifically, or with fragments of cell membrane expressing CD19. In some examples, the supernatant is collected and an additional batch of beads was added and a second batch of the supernatant is collected. This step is repeated once again in other examples.

In other examples, stimulation of CAR T cells is performed by incubating the CAR T cells with fragments of cell membrane expressing EGFR, CD276, CD138 or CD19.

The EVs from the above-mentioned stimulated CAR T cells are obtained as follows: cell media comprising stimulated CAR T cells is collected and centrifuged at 400 g for 5-10 min and the cell pellet was discarded (this step is relevant if no additional stimulation is made). The supernatant is then centrifuged for 15-30 min (about 20) at 1500 g and the cell debris was then discarded. The resulting supernatant is then submitted to centrifugation at 20,000 g for 1 h at 4°C and the pellet is collected. The resulting EV pellets are frozen in aliquots at -80°C.

Example 18. Preparation of EVs obtained from CAR-T cells stimulated with an antigen bound to a tissue culture plate

In order to facilitate the stimulation of CAR T-cells toward manufacturing “off the shelf’ CAR-T cell derived activated EVs, we stimulate CAR-T cells with the antigen which is coated/bound to the tissue culture plates or beads. This overcomes the need of using target cells, growing them and side effects that may be caused by remnants or residual components of these target cells. Several binding protocols are tested to enhance the accessibility of antigen to the CART in order to form the immunological synapse.

The EVs from CAR T cells stimulated with beads coated with the targeted protein were obtained as follows: cell media was collected and centrifuged at 400 g for 5-10 min and the cell pellet was discarded. The supernatant was then centrifuged for 15-30 (about 20) min at 1500 g and the cell debris was then discarded. The original supernatant was then submitted to centrifugation at 20,000 g for 1 h at 4°C. The resulting EV pellets were frozen in aliquots at - 80°C. The EVs from CAR T cells stimulated with target cells were obtained as described

Example 19. Anti-cancer efficacy of the EVs

The efficacy of the EVs of the present invention in treating cancer in vivo is tested in hematological and solid tumor models. Cancer cell lines are injected to immunodeficient mice. For solid tumor models, we inject cell lines originating from ovarian cancer (SKOV, OVCAR), intraperitoneally, or subcutaneously. Alternatively, we use a model of pancreas tumor by injecting pancreatic cancer cells (Capan) subcutaneously or orthotopically. Cell line that originated from breast cancer (MDA-MB-231) are injected subcutaneously. For the hematological cancer model, we inject intravenously a cell line originated from lymphoma.

EVs samples 1-8 are purified from activated CAR T cell culture by method 1 (20,000g, 60min) or 5 (10,000g 180min). EVs samples are labeled with a fluorescent dye such as XenoLight DiR as described before (Ohno S. Molecular Therapy 2013). The labelled EVs (4- 100 pg) are injected intravenously or intratumorally to mice bearing the transplanted tumor cells twice a week for 4 weeks. 12 and 24 hours after each injection the locations of the EVs and tumor size is monitored using an In Vivo Imaging System (IVIS). At the end of 4 weeks brain, heart, spleen, liver, lung, kidney, small intestine, colon, and tumor tissues are harvested for pathology validation.

Example 20. CAR T derived EVs function as an allogeneic "off the shelf" therapy without triggering an immunogenic response.

Immunocompetent allogeneic mice were implanted with a lymphoma tumor cell line (A20) from a given mouse strain (Balb/c). The mice were then treated using anti-CD19 CAR T cells or anti- CD19 CAR T EVs of the present invention obtained as described above. These therapies were compared for their efficacy and side effects as Graft-versus-host disease (GVHD) symptoms are monitored. One or more EV injections are needed to achieve a therapeutic effect, and that requires large-scale EV production. This can be achieved by multiple stimulations of CAR T cells in a large scale platform. We believe that CD19 CAR-T E Vs have the potential for being an "off-the-shelf" product and thus facilitate the accessibility of treatment for cancer patients.

Example 21. Efficacy on activated EVs obtained from stimulated CAR T cells in vivo

On day 0, A549 and H1975 NSCLC cell lines expressing a luciferase signal (IxlO⁶ cells per mouse) are injected intravenously into mice. Thirteen days later, bioluminescent imaging using the IVIS Lumina is conducted to confirm tumor establishment, and mice are grouped into treatment cohorts to ensure balanced groups.

On day 14, mice receive a pre-conditioning treatment consisting of either irradiation (200 rad) or an intraperitoneal (IP) injection of cyclophosphamide (200 mg/kg). Over the next 10-25 weeks, mice are monitored weekly by measuring body weight and imaged via IVIS 1- 2 times per week. The following treatments are administered to mice with established cancer, either comprising A549 or H1975 NSCLC cell lines:

1. Anti-CD276 CAR T cells

2. Anti-CD19 CAR T cells

3. Anti-EGFR CAR T cells

4. EVs derived from 1.

5. EVs derived from 2.

6. EVs derived from 3.

7. EVs derived from untranduced T cells

8. Saline

CAR-EVs (3-200 pg) are administered 1-3 times per week for 15 weeks by intravenous tail injection, intravitreal injection, or inhalation to assess therapeutic efficacy (nasal approach). During the first 5 weeks the mice are imaged using IVIS Lumina and weighed. At the end of the experiment, the mice are sacrificed and the size of the tumor is measured.

Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

1. A population of isolated activated extracellular vesicles (EV s) derived from stimulated T-cells expressing a chimeric antigen receptor (CAR T-cells), wherein at least 25% of the activated EVs have a particle size diameter of from 150 to 1000 nm and wherein the CAR is selected from anti-EGFR CAR, anti-CD276 CAR and anti-CD19 CAR.

2. The population of activated EVs according to claim 1, wherein (i) at least 40% or at least 45% of the EVs have a particle size of above 150 nm, (ii) a mean size of the EVs is at least 140 nm or at least 160 nm, (iii) the activated EVs have the size of from 30 to 1000 nm, or (iv) any combinations of (i), (ii) and (iii).

3. The population of activated EVs according to claim 1 or 2, comprising from 20 to 75% of EVs having a particle size diameter of from 30 to 150nm.

4. The population of activated EVs according to any one of claims 1 to 3, wherein the EVs present the chimeric antigen receptor (CAR) of the stimulated CAR T-cells.

5. The population of activated EVs according to any one of claims 1 to 4, wherein the CAR is anti-EGFR.

6. The population of activated EVs according to claim 5, wherein the anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20.

7. The population of activated EVs according to any one of claims 1 to 4, wherein the CAR is anti-CD276 CAR.

8. The population of activated EVs according to claim 7, wherein the anti-CD276 CAR comprising an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10.

9. The population of activated EVs according to any one of claims 1 to 4, wherein the CAR is anti-CD19 CAR.

10. The population of activated EVs according to claims 9, wherein the anti-CD19 CAR comprising the amino acid sequence SEQ ID NO: 24.

11. The population of activated EVs according to any one of claims 1 to 10, wherein the EVs are derived from CAR T-cells stimulated by a carrier presenting the tumor-associated antigen or a fragment thereof to which the CAR binds specifically.

12. The population of activated EVs according to claim 11, wherein carrier is selected from cells expressing the tumor-associated antigen and an inert carrier presenting the tumor- associated antigen or fragment thereof to which the CAR binds specifically, preferably wherein the inert carrier is beads.

13. The population of activated EVs according to any one of claims 1 to 12, wherein at least 50% of the EVs comprise the CAR of the CAR T cells.

14. The population of activated EVs according to any one of claims 1 to 13, wherein the EVs are cytotoxic EVs.

15. The population of activated EVs according to any one of claims 1 to 14, wherein the EVs further comprising an anticancer agent.

16. The activated EVs according to any one of claims 1 to 14, wherein the EVs are devoid of an exogenous anti-cancer agent.

17. A pharmaceutical composition comprising the population of isolated activated EVs according to any one of claims 1 to 16, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, comprising said population of EVs as a sole anti-cancer agent or further comprising an additional anti-cancer agent.

19. The pharmaceutical composition according to any one of claims 17 to 18, formulated as a formulation for injection.

20. The pharmaceutical composition according to any one of claims 17 to 19, for use in treating cancer, wherein the cancer cells present the antigen to which the CAR binds specifically.

21. The pharmaceutical composition for use according to claim 20, wherein the pharmaceutical composition comprises a population of activated EVs derived from stimulated anti-EGFR CAR T cells and wherein the cancer is selected from lung cancer, anal cancers glioblastoma and epithelial tumors and epithelial tumors of the head and neck, glioblastoma, lung adenocarcinoma, glioblastoma multiforme (GBM), diffuse large B-cell lymphoma (DLBC), skin cutaneous melanoma (SKCM), brain lower grade glioma (LGG), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), and carcinomanon-small-cell lung cancer, colorectal cancer,, and pancreatic cancer, renal cell cancer, cutaneous squamous cell carcinoma in skin (SCC), and bone tumor.

22. The pharmaceutical composition for use according to claim 21 , wherein the anti-EGFR CAR comprises an amino acid sequence selected from SEQ ID NO: 14, 15, 19 and 20.

23. The pharmaceutical composition for use according to claim 20, wherein the pharmaceutical composition comprises population of activated EVs derived from stimulated anti-CD276 CAR T cells and wherein the cancer is selected from glioma, prostate cancer, endometrial cancer, skin cancers, lung cancer, cancer stem cells, epithelial tumors, epithelial tumors of the head and neck cells, glioblastoma, bladder cancer, pancreatic cancer, cervical cancer, breast cancer, intrahepatic cholangiocarcinoma, colorectal cancer, ovarian cancer, melanoma, liver cancer, prostatic cancer, oral squamous cell carcinoma, kidney cancer, gastric cancer, and adrenocortical carcinoma,

24. The pharmaceutical composition for use according to claim 23, wherein the anti- CD276 CAR comprises an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10.

25. The pharmaceutical composition for use according to claim 20, wherein the pharmaceutical composition comprises a population of activated EVs derived from stimulated anti-CD19 CAR T cells and wherein the cancer is selected from B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

26. The pharmaceutical composition for use according to claim 25, wherein the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 24.

27. The pharmaceutical composition for use according to any one of claims 20 to 26, wherein (i) the pharmaceutical composition is administered systemically or intratumorally, (ii) the use comprises co-administration of an additional anti-cancer agent, or (iii) both (i) and (ii).

28. A method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of isolated activated EVs according to any one of claims 1 to 19, wherein (i) the activated EVs are derived from stimulated anti-EGFR CAR T cells and the cancer is selected from lung cancer cells, anal cancers cells glioblastoma cells and epithelial tumors and epithelial tumors of the head and neck cells. Glioblastoma, lung adenocarcinoma, glioblastoma multiforme (GBM), diffuse large B-cell lymphoma (DLBCL), skin cutaneous melanoma (SKCM), epithelial tumors and epithelial tumors of the head and neck cells, brain lower grade glioma (LGG), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), and carcinoma, (ii) the EVs are derived from stimulated anti-CD276 CAR T cells and the cancer is selected from lung cancer, cancer stem cells, epithelial tumors, epithelial tumors of the head and neck, bladder cancer, breast cancer, cervix cancer, colorectal cancer, esophageal cancer, renal cancer, hepatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, biliary cancer, oral squamous cell carcinoma, intrauterine membranous cancer, squamous cell carcinoma, gastric cancer, glioma, glioblastoma, melanoma, and adrenal cancer, or (iii) the EVs are derived from stimulated anti-CD19 CAR T cells and the cancer is selected from B cell lymphomas, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

29. A method for preparation of a population of isolated stimulated extracellular vesicles derived from stimulated CAR T-cells according to any one of claims 1 to 25, the method comprises: (1) incubating CAR T-cells with a tumor-associated antigen to which the CAR binds specifically in a cell medium under conditions enabling T cell stimulation; (2) separating the stimulated CAR T-cells from the cell medium; and (3) isolating the derived activated extracellular vesicles, thereby obtaining a population of isolated activated EVs, wherein at least 25% of the EVs have a size of from 150 nm to 1000 nm and wherein the tumor-associated antigen is selected from EGFR, CD276 and CD 19, wherein the method is devoid of centrifugation at a force above 50,000xg or above 30,000xg.

30. A method for preparation of isolated stimulated extracellular vesicles derived from stimulated CAR T-cells, the method comprising: (1) incubating CAR T-cells with an inert carrier coated with a tumor-associated antigen or a fragment thereof to which the CAR binds specifically in a cell medium under conditions enabling T cell stimulation; (2) separating the CAR T-cells from the cell medium; and (3) isolating the derived activated extracellular vesicles from the medium thereby obtaining a population of isolated activated EVs, wherein at least 25% of the activated EVs have a size of from 150 nm to 1000 nm, wherein the method is devoid of centrifugation at a force above 50,000xg or above 30,000xg.

31. The method according to claim 30, wherein the carrier coated with a tumor-associated antigen or a fragment thereof comprises beads coated with the tumor-associated antigen or a fragment thereof.

32. The method according to any one of claims 30 to 31, wherein the tumor-associated antigen is selected from HER2, EGFR, CD276, CD19, CD38, CD24, MUC1, Mesothelin, PSCA, EPCAM, CEA, PSMA, GPC3, LMP1, CD133, cMET, GD2, ROR1, CD70, HLADR, and CD138, preferably wherein the tumor-associated antigen is selected from HER2, EGFR, CD276 and CD19.

33. The method according to any one of claims 30 to 32, wherein at least 50% of the resulting EVs comprise the CAR of the CAR T cells.

34. The method according to any one of claims 30 to 33, wherein the method comprises repeating from 2 to 10 times the sequence of steps (1), (2) and (3).

35. The method according to any one of claims 30 to 34, wherein the method further comprises adding the carrier coated with a tumor-associated antigen or a fragment thereof to which the CAR binds specifically to CAR T-cells obtained prior to step (1).

36. The method according to any one of claims 29 and 32 to 35, wherein (i) the CAR T- cells are anti-EGFR CAR T-cells, the tumor-associated antigen is EGFR and the anti-EGFR CAR comprises an amino selected from SEQ ID NO: 14, 15, 19 and 20, (ii) the CAR T-cells are anti-CD276 CAR T-cells, the tumor-associated antigen is CD276 and the anti-CD276 CAR comprises an amino acid sequence selected from SEQ ID NO: 4, 5, 9 and 10, or (iii) the CAR T-cells are anti-CD19 CAR T-cells, the tumor-associated antigen is CD 19 and the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 24.

37. The method according to any one of claims 29 and 32 to 36, wherein EGFR comprises the amino acid sequence SEQ ID NO: 83, CD276 protein comprises the amino acid sequence SEQ ID NO: 82, and CD19 protein comprises the amino acid sequence SEQ ID NO: 81.

38. The method according to claims 32, wherein the CAR T-cells are anti-HER2 CAR T- cells and the tumor-associated antigen is HER2, preferably wherein anti-HER2 CAR comprises the amino acid sequence SEQ ID NO: 103 and/or HER2 protein or a fragment thereof comprises the amino acid sequences SEQ ID NO: 85 and 84, respectively.

39. The method according to any one of claims 29 to 38 wherein the isolation of the EVs at step (3) comprises centrifugation at from 8,000xg to 30,000xg for from 0.5 to 4 hours and/or wherein the activated EVs have the size of from 30 to 1000 nm.

40. The method according to any one of claims 29 to 39, wherein the incubation at step (1) comprises (i) incubation for from 6 to 96 hours and/or (ii) incubating CAR T-cells with cells or a carrier presenting the tumor-associated antigen to which the CAR binds specifically.

41. The method according to any one of claims 29 to 40, wherein step (2) comprises a step (2ii) comprising centrifuging the medium of the previous step for from 10 to 60 min at from

1000g to 3000g and separating the pellet from medium and optionally a step (2i) before step (2ii), wherein step (2i) comprises centrifuging the medium with stimulated T-cells from step (1) for 5 to 60 min at from 200g to 600g and separating the pellet from the medium.

42. A population of isolated stimulated extracellular vesicles (EVs) prepared by a method according to any one of claims 29 to 41.