Pharmaceutical composition for treating or preventing malignant tumor

Abstract

To provide a pharmaceutical composition for treating or preventing malignant tumor.SOLUTION: Pharmaceutical composition for treating or preventing malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing extracellular vesicles for treating or preventing malignant tumors, and a pharmaceutical composition for treating or preventing malignant tumors, comprising the extracellular vesicles. More specifically, the present disclosure relates to cellular control of malignant tumors using extracellular vesicles derived from mesenchymal stem cells, and a technique for treating or preventing malignant tumors. [Background technology]

[0002] Mesenchymal stem cells (MSCs, multipotent mesenchymal stromal cells, or stromal cells; hereafter referred to as MSCs) can be isolated from various tissues in the body, including bone marrow, adipose tissue, dental pulp, umbilical cord, and umbilical cord blood. When cultured and expanded MSCs are administered to the body, they home to diseased tissues and differentiate into numerous different cell types. They are known to perform immunomodulatory, anti-inflammatory, and tissue repair functions through phagocytosis by antigen-presenting cells and paracrine effects on nearby cells. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides pharmaceutical compositions that utilize such properties of MSCs to treat or prevent malignant tumors.

[0004] Thus, the present disclosure provides: (Item 1) A pharmaceutical composition for treating or preventing malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). (Item 2) The composition described in the above item, wherein the EV is tetraspanin-positive. (Item 3) The composition of any one of the preceding items, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9. (Item 4) The composition of any one of the preceding items, wherein the EV contains ganglioside GM1. (Item 5) The composition of any one of the preceding items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item 6) The composition of any one of the preceding items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item 7) The composition of any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item 7a) The composition according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item 8) The composition of any one of the preceding items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item 9) The composition according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item 10) The composition according to any one of the preceding items, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item 11) The composition according to any one of the preceding items, wherein the EV is a mixture containing exosomes as a main component. (Item 12) The composition of any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item 13) The composition according to any one of the preceding items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item 14) The composition according to any one of the preceding items, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item 15) The composition according to any one of the preceding items for inducing cell death in the malignant tumor. (Item A1) A method for producing extracellular vesicles (EVs) for treating or preventing malignant tumors, comprising: Providing human tissue-derived mesenchymal stem cells (MSCs); Culturing the MSCs; a step of subjecting the culture solution containing the cultured MSCs to at least one ultracentrifugation process to obtain a concentrated solution containing extracellular vesicles (EVs); A method comprising: (Item A2) The method according to the preceding item, wherein the ultracentrifugation treatment is carried out at least twice. (Item A3) The method according to any one of the preceding items, wherein the ultracentrifugation is a centrifugation process at at least about 100,000 x g or more. (Item A4) The method according to any one of the preceding items, wherein the human tissue comprises at least one selected from the group consisting of bone marrow, umbilical cord blood, adipose tissue, fetal appendages, dental pulp, and periodontal ligament. (Item A5) The method according to any one of the preceding items, wherein the fetal appendages include at least one selected from the group consisting of placenta, fetal membrane, umbilical cord, and amniotic fluid. (Item A6) The method according to any one of the preceding items, wherein the EVs are tetraspanin-positive. (Item A7) The method according to any one of the preceding items, wherein the tetraspanins include at least one selected from the group consisting of CD63, CD81, and CD9. (Item A8) The method according to any one of the preceding items, wherein the EV contains ganglioside GM1. (Item A9) The method according to any one of the preceding items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item A10) The method according to any one of the preceding items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item A11) The method according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item A11a) The method according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item A12) The method according to any one of the preceding items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item A13) The method according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item A14) The method according to any one of the preceding items, wherein the EVs comprise at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item A15) The method according to claim A14, wherein the EVs are a mixture mainly composed of exosomes. (Item A16) The method according to any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item A17) The method according to any one of the preceding items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item A18) The method according to any one of the preceding items, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item A19) The method according to any one of the preceding items for inducing cell death in the malignant tumor. (Item A20) A pharmaceutical composition for treating or preventing malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs), wherein the EVs are produced by the method described in any one of the preceding items. (Item B1) A composition for inhibiting the proliferation of intracellular molecules in malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). (Item B2) The composition described in the above item, wherein the EV is tetraspanin-positive. (Item B3) The composition of any one of the preceding items, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9. (Item B4) The composition of any one of the preceding items, wherein the EV contains ganglioside GM1. (Item B5) The composition of any one of the preceding items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item B6) The composition of any one of the preceding items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item B7) The composition of any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item B7a) The composition according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item B8) The composition of any one of the preceding items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item B9) The composition according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item B10) The composition according to any one of the preceding items, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item B11) The composition according to any one of the preceding items, wherein the EV is a mixture containing exosomes as a main component. (Item B12) The composition of any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item B13) The composition according to any one of the preceding items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item B14) The composition according to any one of the preceding items, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item B15) The composition according to any one of the preceding items for inducing cell death in the malignant tumor. (Item C1) An inhibitor or suppressor of Notch signaling in malignant tumor cells, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). (Item C2) The inhibitor or suppressor described in the above item, wherein the EV is tetraspanin-positive. (Item C3) The inhibitor or suppressor according to any one of the preceding items, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9. (Item C4) The inhibitor or suppressor according to any one of the preceding items, wherein the EV contains ganglioside GM1. (Item C5) The inhibitor or suppressor described in any one of the above items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item C6) The inhibitor or suppressor described in any one of the above items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item C7) The inhibitor or suppressor according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item C7a) The inhibitor or suppressor according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item C8) The inhibitor or suppressor according to any one of the above items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item C9) The inhibitor or suppressor according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item C10) The inhibitor or suppressor described in any one of the above items, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item C11) The inhibitor or suppressor according to any one of the preceding items, wherein the EV is a mixture containing exosomes as a main component. (Item C12) The inhibitor or suppressor according to any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item C13) The inhibitor or suppressor according to any one of the above items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item C14) The inhibitor or suppressor described in any one of the above items, wherein the hematopoietic malignancy includes at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item C15) An inhibitor or suppressor described in any one of the above items for inducing cell death in the malignant tumor. (Item D1) A composition for suppressing the expression of components of the γ-secretase complex in malignant tumor cells, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). (Item D2) The composition described in the above item, wherein the component molecule comprises at least one selected from the group consisting of presenilin 1, nicastrin, PEN-2, and APH-1. (Item D3) The composition of any one of the preceding items, wherein the EV is tetraspanin-positive. (Item D4) The composition of any one of the preceding items, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9. (Item D5) The composition of any one of the preceding items, wherein the EV contains ganglioside GM1. (Item D6) The composition of any one of the preceding items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item D7) The composition of any one of the preceding items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item D8) The composition of any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item D8a) The composition according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item D9) The composition of any one of the preceding items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item D10) The composition according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item D11) The composition according to any one of the preceding items, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item D12) The composition according to any one of the preceding items, wherein the EV is a mixture containing exosomes as a main component. (Item D13) The composition of any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item D14) The composition according to any one of the preceding items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item D15) The composition according to any one of the preceding items, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item D16) The composition according to any one of the preceding items for inducing cell death in the malignant tumor. (Item E1) 1. A method for treating or preventing a malignant tumor in a patient, the method comprising administering to the patient a composition comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). (Item E2) The method according to the above items, further comprising the step of administering at least one selected from the group consisting of chemotherapy, radiation therapy, remission induction therapy, post-remission therapy, salvage therapy, and pre-transplantation therapy. (Item E3) The method of any one of the preceding items, wherein the chemotherapy comprises administration of cyclophosphamide, vincristine, doxorubicin, dexamethasone, cytarabine, methotrexate, nelarabine, or any combination thereof. (Item E4) The method according to any one of the preceding items, wherein the EVs are tetraspanin-positive. (Item E5) The method according to any one of the preceding items, wherein the tetraspanins include at least one selected from the group consisting of CD63, CD81, and CD9. (Item E6) The method according to any one of the preceding items, wherein the EV contains ganglioside GM1. (Item E7) The method according to any one of the preceding items, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule. (Item E8) The method according to any one of the preceding items, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. (Item E9) The method according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin. (Item E9a) The method according to any one of the preceding items, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.). (Item E10) The method according to any one of the preceding items, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix. (Item E11) The method according to any one of the preceding items, wherein the shape of the EV is approximately disc-shaped. (Item E12) The method according to any one of the preceding items, wherein the EVs comprise at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof. (Item E13) The method according to any one of the preceding items, wherein the EV is a mixture containing exosomes as a main component. (Item E14) The method according to any one of the preceding items, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling. (Item E15) The method according to any one of the preceding items, wherein the malignant tumor is a hematopoietic malignant tumor. (Item E16) The method according to any one of the preceding items, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma. (Item E17) The method according to any one of the preceding items for inducing cell death in the malignant tumor.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] Note that features and significant actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings. [Effects of the Invention]

[0007] This disclosure provides extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) that can suppress the proliferation of malignant tumor cells. Administration of these EVs to mammals, including humans, is expected to have a significant therapeutic effect. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the inhibitory effect of EVs derived from bone marrow-isolated MSCs on T-ALL cell proliferation. (a) (b) (c) shows the growth curves of the T-ALL cell lines CuTLL1, Jurkat, and Molt4, respectively. [Figure 2] Figure 2 shows the inhibitory effect of EVs derived from MSCs isolated from umbilical cord blood on the proliferation of T-ALL cells. (a) (b) (c) shows the growth curves of the T-ALL cell lines CuTLL1, Jurkat, and Molt4, respectively. [Figure 3]Figure 3 is a bar graph showing the effect of EVs on inducing T-ALL cell death (apoptosis). (a) (b) (c) shows the expression of apoptosis-related molecular markers in the T-ALL cell lines CuTLL1, Jurkat, and Molt4, respectively. [Figure 4] Figure 4 is a bar graph showing the expression levels of Notch1 and gamma secretase components in EV-treated T-ALL cell lines (CuTLL1, Jurkat, and Molt4). (a) and (b) show the expression of NICD (Notch intracellular domain) and Notch1, respectively, and (c) shows the expression of the three components of gamma secretase (Presenilin 1, Nicastrin, and APH1). [Figure 5] FIG. 5 is a flow diagram illustrating the steps of a method according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] As used herein, "about" means ±10% of the preceding numerical value.

[0012] As used herein, "mesenchymal stem cells" (MSCs) refer to somatic stem cells derived from mesodermal tissue (mesenchyme), ectodermal tissue (neuroepithelial cells and neural crest cells), pluripotent stem cells (such as iPS cells), and the like. MSCs refer to cells that have the ability to differentiate into mesenchymal cells (such as bone cells, cardiomyocytes, chondrocytes, tenocytes, and adipocytes) and can proliferate while maintaining this ability. As used herein, mesenchymal stem cells refer to the same cells as stromal cells, and no particular distinction is made between the two. Mesenchymal stem cells are known to exist in bone marrow, synovium, periosteum, adipose tissue, umbilical cord, umbilical cord blood, placenta, dental pulp, muscle tissue, and the like, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells, among others.

[0013] Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and low-molecular-weight compounds. Molecules characteristic of mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers), such as CD73, CD90, CD105, and CD166. Negative markers not expressed in mesenchymal stem cells include, but are not limited to, CD19, CD34, CD45, HLA-DR, CD11b, and CD14. CD stands for Clusters of Differentiation, and HLA-DR stands for Human Leukocyte Antigen-D-Related. These positive and negative markers can be used to identify mesenchymal stem cells. These markers can be detected using immunological methods, but detection can also be performed by quantifying the mRNA levels of each molecule.

[0014] As used herein, "extracellular vesicles" (EVs) are vesicles released from cells, encompassing both nucleated and non-nucleated cell-derived particles. Examples of EVs include exosomes, microvesicles (MVs), apoptotic bodies, and mixtures thereof. Bacterial membrane vesicles secreted by bacteria are also known as EVs. While apoptotic bodies are released from apoptotic cells, exosomes and microvesicles, although of different origins and sizes, have been reported to be released from healthy and tumor cells.

[0015] Extracellular vesicles (EVs) are small, lipid bilayer-enclosed granular vesicles with diameters of approximately 20-1000 nm that are secreted by most cells. Inside EVs, information specific to the cell from which they were secreted, such as nucleic acids such as microRNA (miRNA) and proteins, is highly preserved. EVs are known to function as intercellular signaling tools involved in communication between nearby and distant cells in many cell types, and are also secreted by cells involved in various diseases such as cancer.

[0016] As used herein, "tetraspanin" refers to a protein superfamily with 33 members in mammals. The main feature of tetraspanins is their four transmembrane domains, TM1-TM4. Tetraspanins also contain small and large extracellular protein loops (SEL and LEL), also known as small and large extracellular loops (SL and LEL). The LEL contains a highly conserved CCG amino acid motif. Furthermore, an intracellular protein loop (SIL) is contained within the small intracellular loop. The N- and C-termini are localized intracellularly. Tetraspanins organize into tetraspanin-enriched microdomains (TEMs) using protein-protein interactions (PPIs). Tetraspanins can interact specifically within membranes, which contributes to their transport and anchoring on the membrane. Examples of tetraspanins include CD63, CD9, CD82, CD81, CD151, and CD53.

[0017] As used herein, "ganglioside" refers to an acidic glycosphingolipid that forms lipid rafts in the outer leaflet of the plasma membrane, primarily in neurons of the central nervous system. Gangliosides are known to be involved in cell proliferation, differentiation, adhesion, signal transduction, cell-cell interactions, tumorigenesis, and metastasis. Ganglioside accumulation is also known to be involved in various diseases. "Ganglioside GM1" functions as a transport receptor in the blood-brain barrier and is the major acylate glycolipid constituting the cell membrane of neurons.

[0018] As used herein, "endosome" refers to a general term for an organelle that controls the sorting, degradation, and recycling of various substances taken into cells by endocytosis. Endosomes are broadly classified into early endosomes, late endosomes, and recycling endosomes based on their morphological and functional characteristics. "Late endosomes" fuse with lysosomes to degrade the contents taken into the cell. "Late endosome-associated factors" are broadly interpreted to refer to any substance related to the function or action of such late endosomes, such as Tsg101 and Alix.

[0019] As used herein, the term "adhesion molecule" refers to a molecule that mediates the mutual proximity of two or more cells (cell adhesion) or the adhesion between a cell and a substrate. Adhesion molecules are generally divided into molecules involved in cell-to-cell adhesion (cell-cell adhesion) and molecules involved in the adhesion between a cell and an extracellular matrix (cell-substrate adhesion). Examples of adhesion molecules include integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.), tetraspanins, and ICAM-1, and these adhesion molecules are known to play an important role in receptor-mediated endocytosis of extracellular vesicles. As used herein, the term "adhesion molecule" includes molecules other than proteins as long as they mediate cell adhesion.

[0020] As used herein, the term "malignant tumor" refers to a tumor in which the morphology or arrangement of tumor cells differs from that of the normal cells from which they originate, and which exhibits invasive or metastatic properties. A malignant tumor is a malignant lesion that is not self-limited in its growth but has the ability to invade adjacent tissues and metastasize to distant tissues. Examples of malignant tumors include, but are not limited to, hematopoietic malignancies, neurogenic tumors including brain tumors, the following cancers classified as carcinomas such as squamous cell carcinoma and adenocarcinoma (head and neck cancer, skin cancer, esophageal cancer, thyroid cancer, gastric cancer, lung cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, liver cancer, prostate cancer, uterine cancer, ovarian cancer, breast cancer, kidney cancer, bladder cancer, colon cancer, etc.), melanoma, bone and soft tissue tumors, as well as lymphoma, leukemia, and myeloma.

[0021] As used herein, "hematopoietic malignancies" refers to malignant tumors in the hematopoietic organs, also referred to as hematologic malignancies. Examples of hematopoietic malignancies include leukemia, malignant lymphoma, and multiple myeloma. Leukemia, depending on its course and the origin of the hematologic malignant tumor cells, includes T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia, chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL). In the hematopoietic malignancies of the present disclosure, lymphoid malignancies are preferred, more preferably T-cell acute lymphoblastic leukemia (T-ALL) or T-cell acute lymphoblastic leukemia, and even more preferably T-cell acute lymphoblastic leukemia (T-ALL).

[0022] As used herein, "Notch signaling" refers to an evolutionarily conserved pathway in multicellular organisms that determines cell fate during development and maintains adult tissue homeostasis. The Notch pathway mediates cell-cell contact signal transduction. Within this pathway, both signal-sending and signal-receiving cells are affected by ligand-receptor crosstalk, thereby controlling a series of cell fate decisions in the development of the nervous, cardiac, immune, and endocrine systems. Notch receptors are single-pass transmembrane proteins consisting of a functional extracellular domain (NECD), a transmembrane domain (TM), and an intracellular domain (NICD). There are four classes of Notch receptors, 1 to 4. Notch receptors undergo processing, such as cleavage (S1 cleavage) and glycosylation, in the endoplasmic reticulum or Golgi apparatus of signal-receiving cells, resulting in Ca2+-stabilized heterodimers consisting of a membrane-inserted TM-NICD and a noncovalently bound NECD. The processed Notch receptor is transported to the plasma membrane by endosomes, where it becomes capable of binding to its ligand through regulation by Deltex and inhibition by NUMB.

[0023] Delta-like members (DLL1, DLL3, and DLL4) and members of the Jagged family (JAG1 and JAG2) function as ligands for Notch signaling receptors. Upon ligand binding, the NECD is cleaved from the TM-NICD domain by TACE (TNF-α ADAM metalloproteinase-converting enzyme) (S2 cleavage). The NECD remains bound to the ligand and undergoes endocytosis and recycling via Mib ubiquitination in the signal-sending cell. In the signal-receiving cell, γ-secretase releases NICD from the TM (S3 cleavage). This release allows NICD to translocate to the nucleus, where it associates with the CSL (CBF1 / Su(H) / Lag-1) transcription factor complex and induces the activation of Notch canonical target genes, including Myc, p21, and the HES family.

[0024] It has been reported that inhibition of Notch signaling inhibits the self-renewal and maintenance of stemness of cancer stem cells. It is also known that hyperactivating mutations of Notch signaling lead to T-cell acute lymphoblastic leukemia, and that hypoactivating mutations of Notch signaling lead to basal cell carcinoma, a type of skin cancer. Thus, depending on the tissue in which it acts, Notch signaling has dual properties, acting as both an oncogene and a tumor suppressor gene. It is also known that activation of Notch signaling is important for the self-renewal and maintenance of undifferentiated potential of hematopoietic stem cells in blood cells.

[0025] As used herein, "tumor associated with Notch signaling" refers to any tumor associated with Notch signaling, including, but not limited to, T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL). T-ALL / T-LBL (T-cell acute lymphoblastic leukemia / T-lymphoblastic lymphoma) is a disease in which precursor T cells become tumors (derived from precursor T cells). The origin of the malignant tumor cells is the same, and it is called T-ALL when the cells grow primarily in the bone marrow, and T-LBL when they grow primarily in the lymph nodes. In the latest WHO classification (2018), T-ALL / T-LBL is named T-lymphoblastic leukemia / lymphoma, but they are essentially the same disease. Therefore, as used herein, T-ALL / T-LBL may be any name given to a disease in which precursor T cells become tumors.

[0026] As used herein, "treatment" refers broadly to either preventive and / or therapeutic treatment, and in a narrower sense to alleviating, attenuating, or improving at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, for example, suppressing the onset of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or arresting the symptoms of a disease or condition, for the purpose of improving (curing) the pathological condition. As used herein, "treatment" refers to alleviating, attenuating, or improving at least one symptom of a disease or condition, for the purpose of improving (curing) the pathological condition.

[0027] As used herein, "prevention" refers to preventing the development of clinical symptoms of a disease state in a subject who may be exposed to or susceptible to the disease state but who has not yet experienced or exhibited symptoms of the disease state.

[0028] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0029] In one aspect of the present disclosure, a pharmaceutical composition for treating or preventing malignant tumors is provided, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). In one embodiment, the technology of the present disclosure allows for the separation and purification of EVs from bone marrow- or umbilical cord blood-derived MSCs by ultracentrifugation without the use of polymers. The resulting EVs can then be used to inhibit the proliferation of malignant tumor cells. When EVs are separated and purified from bone marrow- or umbilical cord blood-derived MSCs by ultracentrifugation without the use of polymers, the EVs exhibit the structural characteristic of the so-called exosome fraction, a discoidal shape of approximately 100 nm. Furthermore, the EVs exhibit functional characteristics, such as immunoregulatory and tissue regeneration effects, which are more systemic. Therefore, unlike EVs prepared by conventional polymer-based methods, the EVs exhibit improved therapeutic or preventive effects.

[0030] Thus, in one aspect of the present disclosure, there is provided a method for producing extracellular vesicles (EVs) for treating or preventing malignant tumors, the method comprising the steps of providing human tissue-derived mesenchymal stem cells (MSCs), culturing the MSCs, and subjecting the culture medium containing the cultured MSCs to at least one ultracentrifugation process to obtain a concentrated solution containing extracellular vesicles (EVs).

[0031] In recent years, it has become clear that the therapeutic effects of cell therapy are mediated by EVs derived from cells, and their effectiveness has been demonstrated in models of refractory immune disorders. For example, Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445 reported that EVs isolated from human bone marrow MSC cultures using polymer precipitation and centrifugation improved the immunological and pathological conditions and survival rates of acute graft-versus-host disease. However, this method uses commercially available EV isolation kits containing potentially immunogenic polymers, which may pose a problem when applied to human therapy.

[0032] However, no technology has been developed to utilize MSC-derived EVs to suppress cell proliferation in rare diseases such as acute lymphoblastic leukemia (T-ALL). For example, acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy caused by precursor T cells undergoing tumorigenesis and abnormal proliferation within the body. To date, treatments involve repeated administration of multiple anticancer drugs in combination, but the survival rate for relapsed or refractory cases remains below 30% for children and below 10% for adults. The annual incidence of new T-ALL cases in Japan is approximately 500 adults and 90 children, making it a rare disease globally. Nelarabine was the last new drug to be launched in 2007, and its efficacy rate as a single agent is approximately 40%.

[0033] In one embodiment of the present disclosure, the method of the present disclosure involves ultracentrifugation of a culture medium containing MSCs to obtain a concentrated solution containing EVs, thereby eliminating the need for the use of potentially immunogenic polymers. This makes it possible to provide a composition that can be used to treat or prevent malignant tumors, including T-ALL.

[0034] (Isolation of extracellular vesicles from mesenchymal stem cells) In one embodiment of the present disclosure, extracellular vesicles (EVs) of the present disclosure can be obtained by culturing human tissue-derived mesenchymal stem cells (MSCs) and ultracentrifuging the resulting culture medium. The flow chart of each step of the method according to one embodiment of the present disclosure is shown in Figure 5.

[0035] In one embodiment, ultracentrifugation can be performed at, for example, about 4°C. In another embodiment, the rotation time for ultracentrifugation is not particularly limited and can be, for example, about 30 to about 180 minutes. Furthermore, during each ultracentrifugation step, high-purity purification can be achieved by removing contaminants (such as proteins) using a sucrose cushion, or even higher-purity exosomes can be recovered using a density gradient method. While the conditions for ultracentrifugation are arbitrary, a swing-out rotor and low-protein-adsorption tubes are preferably used as separation tools, and the separation centrifugation conditions are 4°C, 140,000 g, 70 minutes, and two cycles. These conditions are preferred because they allow exosomes with a diameter of approximately 100 nm to be obtained at high density and with high purity.

[0036] In one embodiment of the present disclosure, mesenchymal stem cells may be a cell population containing mesenchymal stem cells, and the cell population preferably comprises mesenchymal stem cells at about 20% or more, about 30% or more, about 40% or more, more preferably about 50% or more, about 60% or more, about 70% or more, even more preferably about 80% or more, particularly preferably about 90% or more, about 95% or more, about 97% or more, and most preferably about 98% or more.

[0037] Mesenchymal stem cells are cells that have the ability to differentiate into mesenchymal cells (e.g., bone cells, cardiomyocytes, chondrocytes, tenocytes, and adipocytes) and can proliferate while maintaining this ability. Mesenchymal stem cells are known to be present in bone marrow, synovium, periosteum, adipose tissue, umbilical cord, umbilical cord blood, placenta, dental pulp, and muscle tissue, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells. Therefore, for example, bone marrow-derived mesenchymal stem cells refer to mesenchymal stem cells contained in bone marrow and may also be referred to as bone marrow-derived stromal cells. In one embodiment of the present disclosure, the origin of MSCs is not particularly limited as long as they are human tissue. However, from the viewpoints of the effects of the pharmaceutical composition for treating or preventing malignant tumors of the present disclosure and ease of availability, bone marrow-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, periodontal ligament-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and fetal appendage-derived mesenchymal stem cells are preferred. In one embodiment, fetal appendages refer to any tissue or component obtained from a fetus, including, but not limited to, the placenta, fetal membrane, umbilical cord, and amniotic fluid. Under conditions requiring homogeneity, it is desirable to use a purification method involving sorting using a combination of surface markers expressed on mesenchymal stem cells, or to use iPS cell-derived mesenchymal stem cells. Furthermore, under conditions requiring immunoregulatory properties, it may be desirable to use, but is not limited to, adipose tissue-derived mesenchymal stem cells or fetal appendage-derived mesenchymal stem cells.

[0038] In one embodiment of the present disclosure, mononuclear cells can be isolated by a method suitable for each tissue from which the MSCs are derived, and then MSCs can be separated. The culture of MSCs is not particularly limited as long as the cells can be maintained, but can be performed, for example, at a temperature of approximately 37°C, in an environment of approximately 5% CO2 and approximately 21% O2. Furthermore, in one embodiment, the timing and method of MSC passage are not particularly limited.

[0039] The MSC culture medium is not particularly limited as long as it is a medium suitable for culturing MSCs. For example, a known culture medium for obtaining MSCs from bone marrow is Thermofischer / Gibco's advanced minimum essential medium (MEM), as disclosed in Yao H, Miura Y, Yoshioka S, et al. Stem Cells. 2014;32(8):2245-2255. Furthermore, a known culture medium for obtaining MSCs from umbilical cord blood is Thermofischer / Gibco's αMEM, as disclosed in Yoshioka S, Miura Y, Iwasa M, et al. Int J Hematol. 2015;102(2):218-229 and Fujii S, Miura Y, Iwasa M, et al. J Clin Exp Hematop. 2017;57(1):1-8. In one embodiment, such a medium may contain antibiotics, supplements such as glutamine and ascorbic acid, and biological materials (e.g., animal serum). Considering that the obtained cells will be used to treat diseases in animals (including humans), it is preferable that the medium contains as few biological materials as possible (e.g., serum-free medium).

[0040] In one embodiment, the cultured MSCs are passaged an appropriate number of times, taking into consideration the state of the cells, and then treated with an enzyme such as trypsin or EDTA to detach them, followed by centrifugation to recover the MSCs. Passaging can be carried out at any time as long as the cells can be maintained, but can be carried out, for example, approximately 5 days after the MSCs are seeded in the medium. Considering that the resulting cells will be used to treat diseases in animals (including humans), it is preferable to use a product that contains as few biological materials as possible (e.g., recombinant trypsin).

[0041] In one embodiment, when culturing MSCs, a drug can be added to the medium to activate the function of MSCs. For example, when activating the function of MSCs with vitamin K2, vitamin K2 at a concentration of 1 μM or 10 μM can be added to the medium for 24 hours, as described in Fujishiro A, Iwasa M, Fujii S, et al. Int J Hematol. 2020;112(3):316-330. Other examples of functional activation using drugs, including existing pharmaceuticals, are disclosed in, for example, Iwasa M, Miura Y, Fujishiro A, et al., Int J Hematol. 2017;105(5):587-597; Sugino N, Miura Y, Yao H, et al., Biochem Biophys Res Commun. 2016;469(4):823-829; and Yao H, Miura Y, Yoshioka S, et al., Stem Cells. 2014;32(8):2245-2255, and the concept is as described in Sugino N, Ichinohe T, Takaori-Kondo A, Maekawa T, Miura Y., Inflamm Regen. 2017;37:7.

[0042] MSCs whose functions have been activated with drugs have altered expression levels and qualities of secretory proteins such as cytokines and chemokines, as well as adhesion molecules, and can have excellent therapeutic effects. For example, hematopoietic function (Fujishiro A, Iwasa M, Fujii S, et al., Int J Hematol. 2020;112(3):316-330., Sugino N, Miura Y, Yao H, et al., Biochem Biophys Res Commun. 2016;469(4):823-829., and Yao H, Miura Y, Yoshioka S, et al. al., Stem Cells. 2014;32(8):2245-2255.), tissue repair function (Fujishiro A, Iwasa M, Fujii S, et al., Int J Hematol. 2020;112(3):316-330., and Sugino N, Miura Y, Yao H, et al., Biochem Biophys Res Commun. 2016;469(4):823-829.) and anti-cancer cell effects (Iwasa M, Miura Y, Fujishiro A, et al., Int J Hematol. 2017;105(5):587-597.).

[0043] In one embodiment, the MSCs recovered by centrifugation can be used as therapeutic MSCs for treatment as they are, or after washing with a phosphate buffer solution or the like as necessary. When storing, they can be stored in a storage container using a storage solution.

[0044] In one embodiment, when EVs are isolated from a culture medium containing cultured MSCs, MSCs can be cultured in the culture medium described above, then replaced with a serum-free medium (serum-free medium) and further cultured. The resulting medium (conditioned medium) can be subjected to coarse centrifugation, microfiltration, and then ultracentrifugation one or more times to isolate an EV fraction enriched in exosomes. In one embodiment, density gradient centrifugation can be performed using sucrose, without affecting the therapeutic effect of the resulting EVs.

[0045] In one embodiment of the present disclosure, ultracentrifugation is preferably performed at least two, at least three, at least four, or at least five times. For example, the ultracentrifugation is not particularly limited as long as it can produce the EVs of the present disclosure, but can be, for example, centrifugation at at least about 100,000 x g or more, about 110,000 x g or more, about 120,000 x g or more, about 130,000 x g or more, about 140,000 x g or more, about 150,000 x g or more, about 160,000 x g or more, about 170,000 x g or more, about 180,000 x g or more, about 190,000 x g or more, or about 200,000 x g or more.

[0046] In one embodiment of the present disclosure, when ultracentrifugation is performed multiple times, the supernatant can be removed and the pellet can be washed with phosphate-buffered saline after each treatment, and the centrifugal force (× g) can be changed. Furthermore, high-purity purification can be achieved by removing contaminants (such as proteins) using a sucrose cushion, and even higher-purity exosomes can be recovered using a density gradient method.

[0047] In one embodiment, the culture dishes, microfilters, and centrifugal devices used for EV isolation are not particularly limited as long as they are suitable for EV isolation. In one embodiment, chips and tubes are preferably made of low-adsorption materials.

[0048] In one embodiment, the EV fraction separated as described above contains particles exhibiting a roughly discoid shape. The size of the EVs is not particularly limited, but includes particles with sizes of approximately 80, 90, 100, 110, 120, 130, 140, and 150 nm.

[0049] In one embodiment of the present disclosure, the isolated EVs may be tetraspanin-positive. Tetraspanin molecules are markers for exosomes and may include, for example, CD63, CD81, and CD9.

[0050] In one embodiment of the present disclosure, the isolated EVs may also contain ganglioside GM1.

[0051] In one embodiment of the present disclosure, the isolated EVs may also contain various microRNAs, cytokines, chemokines, late endosome-associated factors, and adhesion molecules. In this case, late endosome-associated factors may include, for example, Tsg101 and Alix. In one embodiment, cytokines may include, but are not limited to, IL-13, PAI-1, IL-10, TGF-β, or HLA-G.

[0052] In one embodiment of the present disclosure, the adhesion molecule may be any molecule that mediates the proximity of two or more cells to each other (cell adhesion) or the adhesion between a substrate and a cell, and examples thereof include integrins (e.g., β1 integrin, β3 integrin, αV integrin, etc.), tetraspanins, ICAM-1, etc.

[0053] In one embodiment of the present disclosure, EVs isolated by the method of the present disclosure can be obtained as exosomes, microvesicles, apoptotic bodies, and / or a mixture thereof, and preferably may be a mixture primarily composed of exosomes.

[0054] In one embodiment of the present disclosure, microRNAs comprise a group of approximately 700 molecules, including let-7a, that constitute intracellular signals capable of regulating the expression of various genes in target cells. In one embodiment, microRNAs include, but are not limited to, miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246. In one embodiment, KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes pathway analysis) of messenger RNAs commonly targeted by 10 microRNAs (e.g., miR-125a-3p) highly expressed in EVs derived from bone marrow MSCs revealed high expression of microRNAs that suppress GVHD (KEGG map number 05332) and T cell receptor signaling (KEGG map number 04660).

[0055] In one embodiment of the present disclosure, a composition comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) of the present disclosure can be used to treat or prevent malignant tumors, including, but not limited to, hematopoietic malignancies, neurogenic tumors including brain tumors, cancers classified as carcinomas such as squamous cell carcinoma and adenocarcinoma (head and neck cancer, skin cancer, esophageal cancer, thyroid cancer, gastric cancer, lung cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, liver cancer, prostate cancer, uterine cancer, ovarian cancer, breast cancer, kidney cancer, bladder cancer, and colorectal cancer), melanoma, bone and soft tissue tumors, lymphoma, leukemia, and myeloma. Examples of hematopoietic malignant tumors include leukemia, malignant lymphoma, and multiple myeloma. Leukemia, depending on its progression and the origin of the hematologic malignant tumor cells, includes T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), T-cell acute lymphocytic leukemia, chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0056] In one embodiment of the present disclosure, a composition comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) of the present disclosure can be used to treat or prevent tumors associated with Notch signaling. Without being bound by theory, the composition of the present disclosure can treat tumors associated with Notch signaling by regulating the S3 / S4 cleavage by gamma secretase, which is involved in Notch protein processing. Specifically, molecules such as microRNAs, proteins, and lipids contained in EVs act individually or in combination to suppress the activation of Notch protein by, for example, reducing the expression of the gamma secretase catalytic subunit presenilin. As a result, the expression level of NICD is reduced, which can reduce the expression of molecules associated with tumor growth, such as c-myc.

[0057] In one embodiment of the present disclosure, a composition containing EVs obtained by the method of the present disclosure can induce cell death in malignant tumors. Without being bound by theory, the composition of the present disclosure is believed to induce apoptosis by EVs enhancing the expression of pro-apoptotic molecules such as Bcl-2 associated X protein (BAX) and Bid, while suppressing the expression of anti-apoptotic molecules such as Bcl-2. Indeed, in the present disclosure, apoptotic cells positive for annexin V (AV) and propidium iodide (PI) were successfully detected.

[0058] In one embodiment, EVs derived from bone marrow MSCs obtained as described above can dose-dependently suppress the proliferation of T-ALL cell lines CuTLL1, Jurkat, and Molt4, as shown in the Examples herein.

[0059] In one embodiment, EVs derived from umbilical cord blood MSCs obtained as described above can dose-dependently suppress the proliferation of T-ALL cell lines CuTLL1, Jurkat, and Molt4, as shown in the Examples herein.

[0060] In one embodiment, EVs derived from bone marrow MSCs obtained as described above can induce apoptosis in T-ALL cell lines CuTLL1, Jurkat, and Molt4, as shown in the Examples herein.

[0061] In one embodiment, EVs derived from bone marrow MSCs obtained as described above can reduce NICD expression in T-ALL cell lines CuTLL1, Jurkat, and Molt4, as shown in the Examples herein.

[0062] In one embodiment, EVs derived from bone marrow MSCs obtained as described above can reduce the expression of gamma secretase components in T-ALL cell lines CuTLL1, Jurkat, and Molt4, as shown in the Examples herein.

[0063] In one embodiment, the selective cell growth suppression of T-ALL by EVs derived from bone marrow MSCs obtained as described above is due to specific binding to T-ALL cells and the transfer of EV contents to T-ALL cells.

[0064] In one embodiment, EVs derived from bone marrow MSCs obtained as described above not only reduce the expression of the gamma secretase catalytic subunit presenilin in the T-ALL cell lines CuTLL1, Jurkat, and Molt4, but also reduce the expression of nicastrin and APH1, as shown in the Examples herein. Therefore, a composition containing the EVs of the present invention can exert a cell proliferation inhibitory effect via a molecular mechanism different from that of existing gamma secretase inhibitors.

[0065] Other features used in this system of the present disclosure may include features for any embodiment described elsewhere herein.

[0066] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and can be found in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology," Wiley, and annual updates; Sninsky, JJ et al. (1999); "PCR Applications: Protocols for Functional Genomics," Academic Press; and "Experimental Methods for Gene Transfer and Expression Analysis," a special edition of Experimental Medicine, Yodosha, 1997, all of which are incorporated herein by reference in their entirety.

[0067] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other examples include Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0068] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0069] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0070] Example 1: Isolation of EVs from MSCs In this example, a representative method for isolating EVs from MSCs is demonstrated. Two hundred thousand MSCs were seeded into a T-75 culture flask (treated) and cultured in 10 mL of advanced MEM medium (containing 5% bovine serum, 100 μM ascorbic acid, 2 mM glutamine, 100 units of penicillin, and 100 μM streptomycin) at 37°C in a humidified atmosphere with 5% CO2. After 7 days, the medium was replaced with 10 mL of serum-free medium (containing 100 μM ascorbic acid, 2 mM glutamine, 100 units of penicillin, and 100 μM streptomycin). After 24 hours, the conditioned medium was collected in a centrifuge tube and centrifuged at 2,000 g for 30 minutes. The supernatant was then passed through a 0.2 μm filter to remove large contaminants. The purified conditioned medium was then ultracentrifuged at 140,000 g for 70 minutes at 4°C. The same procedure was repeated, the supernatant was removed, and the pellet was resuspended in Dulbecco's phosphate-buffered saline. EVs derived from 100,000 MSCs were defined as one EV unit.

[0071] Example 2: Inhibitory effect of EVs derived from bone marrow MSCs on T-ALL cell proliferation 1 x 10 CuTLL1 leukemia cells in a cell culture dish 5The cells were suspended in 1 mL of culture medium at a cell density of 10 μL / mL. Subsequently, the cells were divided into three groups: one group (dashed line), one group (dotted line), and one group (solid line) that received 10 μL (= 1 EV unit) of EV solution derived from bone marrow MSCs obtained in Example 1, respectively. The number of cells in each group was measured over time. The groups receiving EV solution once (dashed line) and three times (dotted line) showed significant suppression of CuTLL1 leukemia cell proliferation (Figure 1a). Similar experiments were performed on Jurkat leukemia cells (Figure 1b) and Molt4 leukemia cells (Figure 1c). Similar to the results for CuTLL1 leukemia cells, the groups receiving EV solution once (dashed line) and three times (dotted line) showed significant suppression of leukemia cell proliferation compared to the group receiving no EV solution (solid line).

[0072] Example 3: Inhibitory effect of EVs derived from umbilical cord blood MSCs on T-ALL cell proliferation 1 x 10 CuTLL1 leukemia cells in a cell culture dish 5 Cells were suspended in 1 mL of culture medium at a cell density of 1 / mL. Subsequently, 10 μL (=1 EV unit) of EV solution derived from bone marrow MSCs (dotted line), umbilical cord blood MSCs (lot #1) (dashed line / circle plot), and umbilical cord blood MSCs (lot #2) (dashed line / triangle plot) obtained in Example 1 was added three times, and cell numbers were counted over time. Leukemia cell proliferation was significantly suppressed in the groups treated with EV solution (red, blue, and green lines) compared to the group without EV solution (solid line) (Figure 2a). Similar experiments were performed with Jurkat leukemia cells (Figure 2b) and Molt4 leukemia cells (Figure 2c). Similar results were obtained with CuTLL1 leukemia cells. The groups treated with EV solution (dotted line, dashed line / circle plot, dashed line / triangle plot) showed significantly suppressed leukemia cell proliferation compared to the group without EV solution (solid line). Data are shown as mean ± standard deviation. Statistical significance was indicated by *, P<0.05; **, P<0.01.

[0073] Example 4: Effect of EVs on T-ALL cell death (apoptosis) induction 1 x 10 CuTLL1 leukemia cells in a cell culture dish 5The cells were suspended in 1 mL of culture medium at a cell density of 10 μL / mL. Three groups were then created: one group (gray bar), one group (white bar), and one group (black bar) with 10 μL (=1 EV unit) of EV solution derived from bone marrow MSCs obtained in Example 1 added once (gray bar), three groups (white bar), and one group without addition (black bar) (Figure 3a). On day 3, these cells were double-stained with annexin V (AV) and propidium iodide (PI), and the staining rate was measured using flow cytometry. The groups receiving EV solution once (gray bar) and three times (white bar) showed a significant increase in the proportion of CuTLL1 leukemia cells in the early apoptotic phase (AV positive, PI negative) and the late apoptotic phase (AV positive, PI positive) (far right). Similar experiments were performed on Jurkat leukemia cells (Figure 3b) and Molt4 leukemia cells (Figure 3c). Similar to the results for CuTLL1 leukemia cells, the percentage of leukemia cells in the early apoptotic phase (AV-positive, PI-negative) (middle panels in Figures 3b and 3c, respectively) and the late apoptotic phase (AV-positive, PI-positive) (far right panels in Figures 3b and 3c, respectively) was significantly increased in the groups treated with EV solution once (gray bars) or three times (white bars) compared to the group without EV solution (black bars). Data are shown as mean ± standard deviation. Statistical significance is indicated by *, P<0.05; **, P<0.01.

[0074] Example 5: Expression of Notch1 molecules and gamma secretase components in EV-treated T-ALL cell lines 1 x 10 CuTLL1 leukemia cells, Jurkat leukemia cells, and Molt4 leukemia cells were cultured in a cell culture dish. 5The cells were suspended in 1 mL of culture medium at a cell density of 10 μL / mL. Subsequently, two groups were created: one group (+) with 10 μL (= 1 EV unit) of EV solution derived from bone marrow MSCs obtained in Example 1 (EV solution), and the other group (-) without EV solution. The amount of NICD expressed in these cells was measured by Western blotting. In CuTLL1 and Jurkat leukemia cells, NICD expression was significantly reduced in the EV solution-treated group (Figure 4a). Figure 4b shows the expression of Notch1 molecules, and Figure 4c shows the expression of presenilin 1, nicastrin, and APH1 molecules, as measured in the same manner as in Figure 4a. In CuTLL1 and Jurkat leukemia cells, the EV solution-treated group (+) showed significantly reduced expression of presenilin 1, nicastrin, and APH1 molecules compared to the EV solution-treated group (-). Data are presented as mean ± standard deviation. Statistical significance was indicated by *, P<0.05; **, P<0.01.

[0075] Example 6: Analysis of physical characteristics of MSC-derived EVs To confirm that MSC-derived EVs are positive for tetraspanin molecules, immunoblotting was performed to confirm the expression of the proteins. This can be done in the same manner as in Figure S1B of Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445.

[0076] MSC-derived EVs were also observed using a transmission electron microscope to confirm their disc-shaped morphology. This can be done in the same manner as in Figure S1C of Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445. The size of MSC-derived EVs was also confirmed using nanoparticle tracking assay (NTA) or resistive nanopulse spectroscopy (TRPS). Analysis can also be performed using the qNano® (IZON) system, which is based on the TRPS method, in the same manner as in Figure S1D of Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445.

[0077] Protein concentration can also be measured using the Bradford assay, for example. This can be done in the same way as the method described in the EV preparation section on page 434 of Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445. Microarrays and next-generation sequencing, commonly used for comprehensive gene expression analysis, and proteomics, commonly used for comprehensive protein expression analysis, can be applied to identify EV internal components. Cytokine array analysis is an example of a simple method for semi-comprehensively identifying the expression of specific proteins, including adhesion molecules (Figure S11 in Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445). Figure 4 in the same publication shows an example of analysis using miRNA microarrays. The expression of genes or proteins obtained by comprehensive or semi-comprehensive analysis can be confirmed using quantitative PCR or immunoblotting, respectively.

[0078] By measuring particle size using nanoparticle tracking assays (NTA) or resistive nanopulse pulse (TRPS), it is possible to analyze the particle distribution of exosomes, microvesicles, and apoptotic bodies, or small EVs (≦200 nm) and large EVs (>200 nm). Analysis can also be performed using the qNano® (IZON Corporation), which is based on the TRPS method, as shown in Figure S1D in Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445.

[0079] Example 7: Other therapeutic targets: examples other than T-ALL Examples of the use of the composition of the present disclosure for diseases other than T-ALL will be described below. Patients with NOTCH gene amplification or activating mutations in the NOTCH gene, for example, 1 × 10 for colorectal cancer patients for the former and 1 × 10 for breast cancer patients for the latter. 5 A composition containing EVs secreted by individual MSCs (herein referred to as 1 EV dose) is administered at a concentration of 1 EV / mL, for example, 1 to 3 times, and tumor regression is confirmed by imaging tests such as colonoscopy or PET-CT scans, or blood tumor marker tests such as CEA. The dosage and frequency of administration are determined appropriately depending on the type of disease and patient characteristics.

[0080] The target diseases for treatment include patients with malignant tumors (not limited to T-ALL and T-LBL, and not limited to hematopoietic tumors but also solid tumors) who have been found to have abnormalities, including mutations in the NOTCH gene and genes controlled by the NOTCH gene, as determined by comprehensive cancer genome profiling tests such as Foundation One (registered trademark) CDx, and can also be treated as personalized medicine.

[0081] Furthermore, it can also be applied as personalized medicine for non-malignant tumor diseases (e.g., Alzheimer's disease) in which abnormalities including mutations in the NOTCH gene and genes controlled by the NOTCH gene have been observed.

[0082] Example 8: Further Demonstration of Graft-Versus-Host Disease (GVHD) and Tumor Regression An example of the use of the composition of the present disclosure in GVHD patients will be described. For patients with GVHD who are refractory to initial steroid therapy, EVs are administered in combination with steroids at a concentration of 1 EV / mL one to three times. The dosage and frequency of administration are determined appropriately depending on the type of disease and patient characteristics. While the timing of EV administration is not limited to a specific day after transplantation, it is expected to be more effective early in the course of GVHD. The therapeutic effect is confirmed by clinical findings (e.g., regression of skin rash, reduction in watery stool frequency), laboratory test values ​​(e.g., improvement in hyperbilirubinemia), and histopathological findings (e.g., suppression of intestinal epithelial cell loss, reduction in inflammatory cell infiltration around the bile duct). The immune regulatory function of EVs is confirmed by an increase in peripheral blood regulatory T cells and a shift from activated T cells to naive T cells. Fujii S, Miura Y, Fujishiro A, et al. Stem Cells. 2018;36(3):434-445.

[0083] Example 9: Relationship between apoptosis and gamma-secretase complex components The ability of the composition of the present disclosure to induce cell death (apoptosis) in T-ALL cells and suppress the expression of gamma-secretase complex components is predicted by, for example, identifying target gene groups using bioinformatics such as miRDB and KEGG pathway analysis based on miRNA information obtained by comprehensive analysis using microarray or next-generation sequencers, and then confirming the expression of individual molecules using quantitative PCR or immunoblotting.

[0084] For comprehensive analysis of molecular groups, general omics analysis can be used, in addition to microarray methods and next-generation sequencers.

[0085] Example 10: Combination with additional ingredients Chemotherapy regimens for use in combination with the composition or EVs of the present disclosure include the following common treatment regimens for T-ALL / LBL: The composition containing the EVs of the present disclosure can be combined with chemotherapy that appropriately combines cyclophosphamide, vincristine, doxorubicin, and dexamethasone (e.g., Hyper CVAD therapy), chemotherapy that appropriately combines cytarabine and methotrexate (e.g., MA therapy), nelarabine, radiation, etc., and can be used for remission induction therapy, post-remission therapy (maintenance therapy, intensive therapy), salvage therapy for relapsed or refractory cases, and pre-hematopoietic cell transplantation therapy. The timing of administration of the composition containing EVs disclosed herein may be before, during, or after these treatment regimens. Because antitumor effects are expected to be greater when the tumor cell burden is low, EVs are preferably administered alone or in combination with multidrug chemotherapy as post-remission treatment (maintenance treatment, intensification treatment). EVs are administered one to three times at a concentration of 1 EV / mL. The dosage and frequency of administration are determined appropriately depending on the type of disease and patient characteristics.

[0086] The EV-containing composition of the present disclosure is used in combination with radiation irradiation, for example, when lymph nodes are invaded by tumor cells due to T-LBL, causing proliferation and swelling. In this case, it is desirable to administer EVs after completion of radiation therapy (2 Gy once daily x 15 to 20 times = 30 Gy to 40 Gy in total). EVs are administered at a concentration of 1 EV / mL, one to three times. The dosage and frequency of administration are determined appropriately depending on the type of disease and the characteristics of the patient.

[0087] Example 11: Analysis of the Use of Notch Signal Inhibitors Tissues from patients with Notch signaling-related cancers are obtained from bone marrow or peripheral blood in the case of T-ALL, and from lymph nodes in the case of T-LBL. For patients with other Notch signaling-related cancers, tissues are obtained from resected specimens of the affected cancer. In either case, the obtained tissue is isolated and cultured in vitro or in three-dimensional culture, or xenografted (in vivo) into immunodeficient mice (e.g., nude mice), and exposed to EVs at a concentration of 1 EV / mL. NICD expression levels in cancer cells are measured using immunoblotting to confirm their use as Notch signaling inhibitors.

[0088] (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0089] According to the present disclosure, extracellular vesicles (EVs) obtained from mesenchymal stem cells (MSCs) can suppress the proliferation of malignant tumor cells. When a composition containing such EVs is administered to mammals, including humans, it can exert a high therapeutic effect, and therefore, it is expected to be applied in the medical field.

Claims

1. A pharmaceutical composition for treating or preventing malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs).

2. The composition of claim 1 , wherein the EV is tetraspanin-positive.

3. The composition of claim 2 , wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9.

4. The composition according to any one of claims 1 to 3, wherein the EV contains ganglioside GM1.

5. The composition according to any one of claims 1 to 4, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule.

6. The composition of claim 5, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246.

7. The composition according to claim 5 , wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (β1 integrin, β3 integrin, αV integrin, etc.).

8. The composition of claim 5, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix.

9. The composition according to any one of claims 1 to 8, wherein the EV has a substantially discoid shape.

10. The composition according to any one of claims 1 to 9, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof.

11. The composition according to claim 10, wherein the EV is a mixture containing exosomes as a main component.

12. The composition according to any one of claims 1 to 11, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling.

13. The composition of any one of claims 1 to 12, wherein the malignant tumor is a hematopoietic malignant tumor.

14. 14. The composition of claim 13, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

15. The composition according to any one of claims 1 to 14, for inducing cell death in said malignant tumor.

16. A method for producing extracellular vesicles (EVs) for treating or preventing malignant tumors, comprising: Providing human tissue-derived mesenchymal stem cells (MSCs); Culturing the MSCs; a step of subjecting the culture solution containing the cultured MSCs to at least one ultracentrifugation treatment to obtain a concentrated solution containing extracellular vesicles (EVs); A method comprising:

17. 17. The method of claim 16, wherein the ultracentrifugation process is performed at least twice.

18. The method according to claim 16 or 17, wherein the ultracentrifugation is a centrifugation at at least about 100,000 x g or more.

19. The method of any one of claims 16 to 18, wherein the human tissue comprises at least one selected from the group consisting of bone marrow, umbilical cord blood, adipose tissue, fetal appendages, dental pulp, and periodontal ligament.

20. The method of any one of claims 16 to 19, wherein the fetal appendage comprises at least one selected from the group consisting of a placenta, a fetal membrane, an umbilical cord, and amniotic fluid.

21. The method of any one of claims 16 to 20, wherein the EV is tetraspanin-positive.

22. 22. The method of claim 21, wherein the tetraspanins comprise at least one selected from the group consisting of CD63, CD81, and CD9.

23. The method according to any one of claims 16 to 22, wherein the EV contains ganglioside GM1.

24. The method according to any one of claims 16 to 23, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule.

25. 25. The method of claim 24, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246.

26. The method of claim 24, wherein the adhesion molecule comprises at least one selected from the group consisting of integrins (β1 integrin, β3 integrin, αV integrin, etc.).

27. The method of claim 24, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix.

28. The method according to any one of claims 16 to 27, wherein the EV has a substantially disc-like shape.

29. The method according to any one of claims 16 to 28, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof.

30. The method according to claim 29, wherein the EV is the mixture mainly composed of exosomes.

31. The method of any one of claims 16 to 30, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling.

32. The method of any one of claims 16 to 31, wherein the malignant tumor is a hematopoietic malignant tumor.

33. 33. The method of claim 32, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

34. The method according to any one of claims 16 to 33, for inducing cell death in the malignant tumor.

35. A pharmaceutical composition for treating or preventing malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs), wherein the EVs are produced by the method according to any one of claims 16 to 34.

36. A composition for inhibiting the proliferation of intracellular molecules in malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs).

37. 37. The composition of claim 36, wherein the EV is tetraspanin positive.

38. 38. The composition of claim 37, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9.

39. The composition according to any one of claims 36 to 38, wherein the EV contains ganglioside GM1.

40. The composition of any one of claims 36 to 39, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule.

41. 41. The composition of claim 40, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246.

42. 41. The composition of claim 40, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin.

43. The composition of claim 40, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix.

44. The composition according to any one of claims 36 to 43, wherein the EV has a substantially discoid shape.

45. The composition according to any one of claims 36 to 44, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof.

46. The composition according to claim 45, wherein the EV is a mixture mainly composed of exosomes.

47. The composition of any one of claims 36 to 46, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling.

48. The composition of any one of claims 36 to 47, wherein the malignant tumor is a hematopoietic malignant tumor.

49. 49. The composition of claim 48, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

50. The composition according to any one of claims 36 to 49, for inducing cell death in the malignant tumor.

51. An agent for inhibiting or suppressing Notch signaling in cells of malignant tumors, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs).

52. The inhibitor or suppressor of claim 51, wherein the EV is tetraspanin positive.

53. The inhibitor or suppressor of claim 52, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9.

54. The inhibitor or suppressor according to any one of claims 51 to 53, wherein the EV contains ganglioside GM1.

55. The inhibitor or suppressor according to any one of claims 51 to 54, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule.

56. The inhibitor or suppressor of claim 55, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246.

57. The inhibitor or suppressor of claim 55, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin.

58. The inhibitor or suppressor described in claim 55, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix.

59. The inhibitor or suppressor according to any one of claims 51 to 58, wherein the shape of the EV is approximately disc-shaped.

60. The inhibitor or suppressor according to any one of claims 51 to 59, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof.

61. The inhibitor or suppressor described in claim 60, wherein the EV is a mixture mainly composed of exosomes.

62. The inhibitor or suppressor according to any one of claims 51 to 61, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling.

63. The inhibitor or suppressor according to any one of claims 51 to 62, wherein the malignant tumor is a hematopoietic malignant tumor.

64. The inhibitor or suppressor of claim 63, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

65. The inhibitor or suppressor according to any one of claims 51 to 64, for inducing cell death in the malignant tumor.

66. A composition for suppressing the expression of a component molecule of the γ-secretase complex in malignant tumor cells, comprising extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs).

67. 67. The composition of claim 66, wherein the component molecule comprises at least one selected from the group consisting of presenilin 1, nicastrin, PEN-2, and APH-1.

68. 68. The composition of claim 66 or 67, wherein the EV is tetraspanin positive.

69. 69. The composition of claim 68, wherein the tetraspanin comprises at least one selected from the group consisting of CD63, CD81, and CD9.

70. The composition according to any one of claims 66 to 69, wherein the EV contains ganglioside GM1.

71. The composition of any one of claims 66 to 70, wherein the EV contains at least one selected from the group consisting of microRNA, cytokine, chemokine, late endosome-associated factor, and adhesion molecule.

72. 72. The composition of claim 71, wherein the microRNA comprises at least one selected from the group consisting of miR-125a-3p, miR-21, miR-223, miR-145, miR-4732, and miR-1246.

73. 72. The composition of claim 71, wherein the adhesion molecule comprises at least one selected from the group consisting of β1 integrin, β3 integrin, and αV integrin.

74. The composition of claim 71, wherein the late endosome-associated factor comprises at least one selected from the group consisting of Tsg101 and Alix.

75. The composition of any one of claims 66 to 74, wherein the EV has a substantially discoid shape.

76. The composition according to any one of claims 66 to 75, wherein the EV comprises at least one selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and mixtures thereof.

77. The composition of claim 76, wherein the EV is a mixture mainly composed of exosomes.

78. The composition of any one of claims 66 to 77, wherein the malignant tumor comprises a tumor whose growth is associated with Notch signaling.

79. 79. The composition of any one of claims 66 to 78, wherein the malignant tumor is a hematopoietic malignant tumor.

80. 80. The composition of claim 79, wherein the hematopoietic malignancy comprises at least one selected from the group consisting of T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

81. The composition according to any one of claims 66 to 80, for inducing cell death in the malignant tumor.

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