Fas-associated factor 1 (FAF1)-encapsulating exosomes and their use as anti-cancer agents

Abstract

The present invention relates to exosomes encapsulating fas-associated factor 1 (FAF1) protein and their use in cancer treatment. The present inventors isolated exosomes encapsulating FAF1 protein from HEK293 cells in which FAF1, a known cancer-suppressing protein, is overexpressed, and administered the exosomes encapsulating FAF1 protein to tumor model mice in which pancreatic cancer cells (MIA PaCa-2), lung cancer cells (A549), colon cancer cells (HCT116), liver cancer cells (Hep3B), breast cancer cells (MDA-MB-231), kidney cancer cells (Caki-1) and cervical cancer cells (HeLa cells) were injected intratumorally into nude mice. As a result, the tumor growth suppression effect was more pronounced than that of the control group not treated with FAF1, and the FAF1-encapsulating exosomes of the present invention were found to be useful as a therapeutic agent for various cancers.

Description

[Technical Field]

[0001] Technical Field The present invention relates to exosomes containing FAS-associated factor 1 (FAF1) and their use as anticancer agents. [Background technology]

[0002] Background technology Tumors are the product of uncontrolled excessive proliferation of abnormal cells. When such tumors exhibit destructive growth, invasiveness, and metastasis, they are classified as malignant tumors, or cancer. Cancer-related deaths are the leading cause of death in Korea and one of the most important causes of death in developed countries. Until now, surgical treatments were developed and used until the 1950s, followed by radiation therapy in the 1960s and anticancer drug therapy in the 1970s. In the 1980s, rapid advances in basic science, particularly in immunology and molecular biology, led to the emergence of immunotherapy and gene therapy. Although considerable research is currently being conducted, these therapies have yet to achieve breakthroughs.

[0003] Fas (also known as the Fas receptor, CD95, Apo1, or TNFRSF6) is the most potent death receptor, and Fas and Fas ligand (Fas L) play a key role in apoptosis. When three Fas ligands bind to three Fas molecules, an adaptor protein called the Fas-associated death domain (FADD) binds to the Fas death domain (DD). The death effector domain (DED) of FADD then binds to the death effector domain (DED) of inactive caspase-8 (also known as FLICE or MACH), activating caspase-8. Together, Fas, FADD, and caspase-8 form the Fas death-inducing signaling complex (Fas-DISC), which activates effector caspases that ultimately cause cell death.

[0004] Fas-associated factor 1 (FAF1) is a protein involved in the Fas pathway and has been found in mice (Chu et al., 1995; Becker et al., 1997), quail (Frohlich et al., 1998), and humans (Ryu et al., 1999). They share high amino acid sequence homology: mouse FAF1 consists of 649 amino acids, quail FAF1 consists of 648 amino acids, and human FAF1 consists of 650 amino acids.

[0005] Previous studies on FAF1 have revealed that it has the ability to induce apoptosis, and that the minimal region for induction is the 181-381 amino acid region of FAF1. FAF1 is a member of the Fas-DISC, and the DISC is formed by the interaction of the DEDs of caspase-8 and FADD with the 181-381 amino acid region of FAF1, which is structurally similar to the DED (Ryu and Kim, 2003).

[0006] Decreased FAF1 expression has been reported in various cancers, including lung cancer, colon cancer, liver cancer, prostate cancer, brain tumors, and breast cancer (Feng et al., 2017). The FAF1 protein has attracted attention as a tumor suppressor. FAF1 is known to suppress tumor growth through NF-kB inhibition and tumor metastasis through TGF-β signaling (Park et al., 2004; Park et al., 2007). Furthermore, FAF1 is known to inhibit tumor growth by arresting tumor cell cycle in the G2 / M phase through Aurora A inhibition (Jang et al., 2008). Korean Patent Publication No. 10-2004-0101707 discloses that fragments of specific regions of FAF1 have the ability to inhibit angiogenesis, tubulogenesis, and cell proliferation, and can be used as tumor suppressors.

[0007] Extracellular vesicles were first discovered 50 years ago. Since then, they have been found to be present in every biological fluid tested, and cell lines are known to release various types of vesicles in vitro. Among extracellular vesicles, exosomes are small vesicles consisting of a phospholipid bilayer, measuring 50–200 nm in size. They are produced intracellularly and secreted extracellularly. They play an important role in intercellular communication by delivering a specific repertoire of nucleic acids (DNA and RNA), proteins, and lipids that are important for maintaining homeostasis. For example, exosomes are involved in fundamental physiological processes such as neurotransmission, antigen presentation, immune responses, organ development, and reproductive function, as well as several pathological conditions, including cancer progression, cardiovascular disease, inflammation, and prion infection.

[0008] Exosomes were first discovered during the final stage of red blood cell maturation, a process that releases and removes intracellular proteins, leaving only hemoglobin inside the red blood cell. Electron microscopy studies have shown that these exosomes are not directly separated from the plasma membrane, but are derived from specific intracellular compartments called multivesicular bodies (MVBs) and released and secreted into the extracellular space. When multivesicular bodies fuse with the plasma membrane, these vesicles are released into the extracellular environment, and are now called exosomes.

[0009] Exosomes are known to be produced and secreted by various immune cells, including B lymphocytes, T lymphocytes, dendritic cells, megakaryocytes, and macrophages, as well as stem cells and tumor cells. Exosome production has been reported to be either dependent or independent of the endosomal sorting complexes required for transport (ESCRTs), but the exact mechanism remains difficult to determine.

[0010] Related art includes Korean Patent Publication No. 2004-0015508, which discloses a method for introducing a gene for a specific antigen into a cell line, stably expressing the protein of the introduced gene within the cell line, and releasing it into the extracellular space via exosomes, as well as a method for using the exosomes as a vaccine. Furthermore, Korean Patent Publication No. 10-2053065, which relates to a pH-sensitive exosome composition using hyaluronic acid and doxorubicin, discloses a method for preparing pH-sensitive exosomes using a polymeric substance chemically bound to doxorubicin, hyaluronic acid, and 3-diethylaminopropylamine, and the cancer cell killing effect of the exosomes. Furthermore, Korean Patent Publication No. 10-2018-0078173 relates to novel exosome-based anticancer agents, and discloses recombinant exosomes displaying phagocytosis-promoting proteins, such as receptor tyrosine kinase and SIRP, on their surface, and recombinant exosomes containing anticancer proteins, such as asparaginase, protein toxins, cancer antigen-specific antibodies or fragments thereof, tumor suppressor genes, or antiangiogenic factors. Korean Patent Application No. 10-2019-0059724 also discloses a method for screening candidate therapeutic agents for neurodegenerative diseases after confirming that FAF1 is encapsulated in exosomes and secreted into the extracellular space, but no research results on cancer cells have been reported.

[0011] Exosomes protect cargo molecules from the extracellular environment, prolong their half-life, and, if necessary, enter target cells. However, not all proteins can be packaged as cargo molecules into exosomes, and even if they are packaged, the packaged cargo molecules do not enter all cells. Furthermore, the packaging of large, high-molecular-weight proteins into exosomes is technically challenging, and engineering proteins for packaging may significantly reduce the efficacy of the packaged proteins. Although FAF1 has been reported to be packaged spontaneously into exosomes in neuronal cells, whether exosome-packaged FAF1 enters and functions normally in cells varies depending on the cell type, requiring experimental verification. Therefore, if exosome-based FAF1 delivery is confirmed to function properly in cancer cells and mass production of FAF1-packaged exosomes becomes possible, FAF1-packaged exosomes may be applicable as tumor suppressors for various cancers.

[0012] Therefore, the inventors encapsulated FAF1, which is known to have tumor-suppressing function, into exosomes and delivered them to various tumors. To determine whether exosome-encapsulated FAF1 can suppress tumors, they isolated exosomes from cells in which FAF1 was overexpressed and confirmed whether FAF1 was spontaneously encapsulated in the isolated exosomes. They then treated cancer cells with exosomes encapsulating isolated FAF1, and confirmed that colony formation was suppressed. They also confirmed that when exosomes encapsulating FAF1 protein were administered to various mouse cancer models in which cancer cells had been transplanted, tumor growth was significantly inhibited compared to other control groups, including a group treated with recombinant FAF1 protein, and thus completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0013] Disclosure of the Invention technical challenges An object of the present invention is to provide a pharmaceutical composition for treating cancer, which contains exosomes encapsulating FAF1 protein as an active ingredient.

[0014] Another object of the present invention is to provide a method for producing exosomes encapsulating FAF1 protein.

[0015] A further object of the present invention is to provide a method for screening for a cancer therapeutic agent that reduces the expression of FAF1. [Means for solving the problem]

[0016] Problem solving To achieve the above-mentioned objectives, the present invention provides a pharmaceutical composition for treating cancer, which comprises exosomes encapsulating FAF1 protein as an active ingredient.

[0017] Furthermore, the present invention provides a method for producing exosomes encapsulating FAF1 protein, comprising the steps of: 1) introducing a polynucleotide encoding the FAF1 protein into cells to obtain transformed cells; 2) culturing the transformed cells; and 3) isolating exosomes from the cultured cells.

[0018] Furthermore, the present invention provides a method for screening cancer therapeutic agents, comprising the steps of: 1) treating cancer cells with reduced FAF1 expression with a test substance; 2) measuring the level of FAF1 protein in exosomes isolated from the cells in step 1); and 3) selecting a test substance that increases the level of FAF1 protein in step 2) compared to a control group not treated with the test substance. [Effects of the Invention]

[0019] Advantageous Effects of the Invention The present invention relates to Fas-associated factor 1 (FAF1) protein-encapsulated exosomes and their use in cancer treatment. The inventors isolated FAF1 protein-encapsulated exosomes from HEK293 cells overexpressing FAF1, a protein known to have tumor-suppressing functions. They then administered these FAF1 protein-encapsulated exosomes to tumor models: pancreatic cancer cells (MIA PaCa-2), lung cancer cells (A549), colon cancer cells (HCT116), liver cancer cells (Hep3B), breast cancer cells (MDA-MB-231), kidney cancer cells (Caki-1), and cervical cancer cells (HeLa) transplanted into nude mice. The FAF1-encapsulated exosomes demonstrated significant tumor growth suppression compared with controls not administered FAF1. Therefore, the FAF1-encapsulated exosomes of the present invention may be used as a therapeutic agent for various cancers. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 shows the results of Western blotting showing the amount of FAF1 encapsulated in exosomes isolated using an ultracentrifuge from HEK293 cells overexpressing FAF1. [Figure 2] Figure 2 shows the results of Western blotting, showing the amount of FAF1 encapsulated in exosomes isolated using an ultracentrifuge from HeLa cells overexpressing FAF1. [Figure 3] Figure 3 shows the results of Western blotting showing the amount of FAF1 encapsulated in exosomes isolated from HEK293 cells overexpressing FAF1 using Exo-quick-TC™. [Figure 4] Figure 4 shows the results of Western blotting showing the amount of FAF1 encapsulated in exosomes isolated from HeLa cells overexpressing FAF1 using Exo-quick-TC™. [Figure 5]Figure 5a is a photograph showing the colony formation inhibitory effect of MIA PaCa-2 cells treated with exosomes isolated from HEK293 cells overexpressing FAF1. Figure 5b is a graph showing the number of colonies formed in MIA PaCa-2 cells treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 6] Figure 6a is a photograph showing the colony formation inhibitory effect of treating A549 cells with exosomes isolated from HEK293 cells overexpressing FAF1. Figure 6b is a graph showing the number of colonies formed when A549 cells were treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 7] Figure 7a is a photograph showing the colony formation inhibitory effect of HCT116 cells treated with exosomes isolated from HEK293 cells overexpressing FAF1. Figure 7b is a graph showing the number of colonies formed when HCT116 cells were treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 8] Figure 8a is a photograph showing the colony formation inhibitory effect of Hep3B cells treated with exosomes isolated from HEK293 cells overexpressing FAF1, and Figure 8b is a graph showing the number of colonies formed when Hep3B cells were treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 9] Figure 9a is a photograph showing the colony formation inhibitory effect of MDA-MB-231 cells treated with exosomes isolated from HEK293 cells overexpressing FAF1, and Figure 9b is a graph showing the number of colonies formed when MDA-MB-231 cells were treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 10]Figure 10a is a photograph showing the colony formation inhibitory effect of HeLa cells treated with exosomes isolated from HEK293 cells overexpressing FAF1, and Figure 10b is a graph showing the number of colonies formed when HeLa cells were treated with exosomes isolated from HEK293 cells overexpressing FAF1. [Figure 11] Figure 11a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MIA PaCa-2 cells. Figure 11b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MIA PaCa-2 cells. Figure 11c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MIA PaCa-2 cells. Figure 11d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with MIA PaCa-2 cells. Figure 11e is a photograph showing changes in cell morphology of tumor tissues in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with MIA PaCa-2 cells. [Figure 12]Figure 12a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with A549 cells. Figure 12b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with A549 cells. Figure 12c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with A549 cells. Figure 12d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with A549 cells. Figure 12e is a photograph showing changes in cell morphology of tumor tissues in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with A549 cells. [Figure 13]Figure 13a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with HCT116 cells. Figure 13b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with HCT116 cells. Figure 13c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with HCT116 cells. Figure 13d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HCT116 cells. Figure 13e is a photograph showing changes in cell morphology of tumor tissues in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HCT116 cells. [Figure 14]Figure 14a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Hep3B cells. Figure 14b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Hep3B cells. Figure 14c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Hep3B cells. Figure 14d is a graph showing tumor weights in the MOCK group, recombinant FAF1 protein group, EXO-CON group, and EXO-FAF1 group when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Hep3B cells. Figure 14e is a photograph showing changes in cell morphology of tumor tissues in the MOCK group, recombinant FAF1 protein group, EXO-CON group, and EXO-FAF1 group when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Hep3B cells. [Figure 15]Figure 15a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MDA-MB-231 cells. Figure 15b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MDA-MB-231 cells. Figure 15c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with MDA-MB-231 cells. Figure 15d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with MDA-MB-231 cells. Figure 15e is a photograph showing changes in tumor tissue cell morphology in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with MDA-MB-231 cells. [Figure 16]Figure 16a is a graph showing the time course of tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with Caki-1 cells. Figure 16b is a graph showing the tumor volume in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with Caki-1 cells. Figure 16c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after administration of exosomes isolated from HEK293 cells overexpressing FAF1 to nude mice xenografted with Caki-1 cells. Figure 16d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Caki-1 cells. Figure 16e is a photograph showing changes in cell morphology of tumor tissues in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with Caki-1 cells. [Figure 17]Figure 17a is a graph showing the change in tumor volume over time in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HeLa cells. Figure 17b is a graph showing the tumor volume immediately before tumor enucleation in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HeLa cells. Figure 17c is a photograph of tumors in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups after exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HeLa cells. Figure 17d is a graph showing tumor weights in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HeLa cells. Figure 17e is a photograph showing changes in cell morphology of tumor tissues in the MOCK, recombinant FAF1 protein, EXO-CON, and EXO-FAF1 groups when exosomes isolated from HEK293 cells overexpressing FAF1 were administered to nude mice xenografted with HeLa cells. DETAILED DESCRIPTION OF THE INVENTION

[0021] Best Mode for Carrying Out the Invention The present invention will be described in detail below.

[0022] The present invention provides a pharmaceutical composition for treating cancer, which contains exosomes encapsulating FAF1 protein as an active ingredient.

[0023] The FAF1 protein of the present invention comprises the amino acid sequence shown in SEQ ID NO:1.

[0024] FAF1 of the present invention constitutes the Fas apoptosis-inducing signaling complex (Fas-DISC) and induces apoptosis. FAF1 is known as a protein that activates several pathways and promotes apoptosis.

[0025] FAF1 is involved in cell proliferation by suppressing the G2 / M phase of the cell cycle through JNK-dependent mitochondrial dysfunction-induced cell necrosis and by negatively regulating Aurora A. It also participates in the ubiquitin-proteasome pathway by binding to ubiquitinated proteins and valosin-containing protein (VCP), controlling protein degradation; unnecessary FAF1 is ubiquitinated via parkin and then degraded by the proteasome pathway.

[0026] Furthermore, FAF1 is involved in various biochemical processes, such as apoptosis, inflammation, cell proliferation, and protein homeostasis. FAF1 is a tumor suppressor that plays a role in tumor suppression through NF-κB inhibition and suppresses tumor metastasis through TGF-β signaling.

[0027] The exosomes of the present invention are small vesicles composed of a bilayer phospholipid membrane secreted by cells. They are known to transmit signaling between cells and form disease-specific nucleic acids and proteins, which are then released into body fluids. They play an important role in intercellular communication through the intercellular transfer of specific repertoires of nucleic acids, proteins, and lipids that are important for maintaining homeostasis. For example, exosomes are involved in fundamental physiological processes such as neurotransmission, antigen presentation, immune response, organ development, and reproductive ability, as well as several pathological diseases such as cancer progression, cardiovascular disease, inflammation, and prion infection.

[0028] Exosomes are secreted into the extracellular environment after late endosomes known as multivesicular bodies (MVBs), which contain intraluminal vesicles (ILVs), fuse with the plasma membrane. The multivesicular bodies fuse with the plasma membrane to release the ILVs, and the multivesicular bodies fuse with lysosomes to degrade their contents. The material present in exosomes does not resemble the composition of cytoplasmic material; exosomes may contain RNA and protein, if desired.

[0029] Exosome production has been reported to be either dependent or independent of the endosomal sorting complex required for transport (ESCRT), but the exact mechanism remains difficult to determine. Cells secrete proteins containing signal peptides via the endoplasmic reticulum-Golgi complex. Vesicles containing proteins containing signal peptides migrate toward the plasma membrane, fuse with it, and release the protein extracellularly. However, proteins lacking signal peptides can also be secreted via non-classical secretory pathways. When proteins lacking signal peptides are secreted, they are secreted in the absence or presence of vesicles. The exact mechanisms of non-vesicular secretory pathways are unknown, but some proteins are secreted via membrane pores and ATP-binding cassette transporters. Vesicular secretion occurs via extracellular vesicles, including exosomes, and occurs via vesicles of various sizes.

[0030] The average diameter of exosomes is 50 nm to 300 nm, but the present invention is not limited to this.

[0031] Exosomes are nanosized extracellular vesicles released by most cell types and contain small RNA molecules, lipids, and proteins. Their advantages include the ability to overcome natural barriers, unique cell targeting properties, improved permeability, persistence, and biocompatibility. Based on their inherent function of transmitting biological information, the application of exosomes as therapeutic agents has attracted considerable attention.

[0032] The cancer is a cancer in which expression of FAF1 is decreased.

[0033] The cancer includes but is not limited to cervical cancer, pancreatic cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), breast cancer, kidney cancer, colon cancer, head and neck squamous cell carcinoma, bladder cancer, prostate cancer, stomach cancer, endometrial cancer, brain tumor, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal cell carcinoma), mesothelial carcinoma, esophageal cancer, adrenal gland cancer, thyroid cancer, bone tumor, glioblastoma, mesothelioma, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, leukemia and malignant lymphoma.In a preferred embodiment, cancer is pancreatic cancer, non-small cell lung cancer, colorectal cancer, liver cancer, triple-negative breast cancer, kidney cancer or cervical cancer.

[0034] The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or mixture thereof commonly used in pharmaceutical preparations. Any pharmaceutically acceptable carrier suitable for delivering the composition to a living body may be used. Specifically, the carrier may be a compound listed in the Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, glucose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. Furthermore, typical additives such as antioxidants, buffers, and bacteriostatic agents may be added, if necessary.

[0035] When a pharmaceutical composition is formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

[0036] The pharmaceutical composition of the present invention can be formulated into oral or parenteral preparations. Oral preparations include solid and liquid preparations. Solid preparations can be tablets, pills, powders, granules, capsules, or lozenges, and can also be prepared by adding at least one excipient to the composition. The excipient can be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. In addition, solid preparations can contain lubricants such as magnesium stearate and talc. Meanwhile, liquid preparations can be suspensions, solutions, emulsions, or syrups. In this case, liquid preparations can contain excipients such as wetting agents, sweeteners, flavorings, and preservatives.

[0037] Parenteral preparations include injections, suppositories, inhalation powders, spray aerosols, powders, creams, etc. Injections may contain a sterile aqueous solution, a non-aqueous solvent, a suspending agent, an emulsifier, etc. In this case, the non-aqueous solvent or suspending agent may be propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, or an injectable ester such as ethyl oleate.

[0038] The pharmaceutical composition of the present invention can be administered orally or parenterally according to desired method.Parenteral administration includes, but is not limited to, intrabronchial inhalation, intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular, intrapleural or intratumoral injection.In a preferred embodiment, the pharmaceutical composition of the present invention can be administered by intratumoral injection.

[0039] The pharmaceutical composition can be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the administration time, the administration route, the treatment period, and other concurrently used drugs. However, to achieve the desired effect, the amount of the active ingredient contained in the pharmaceutical composition of the present invention may be 0.0001 to 1,000 mg / kg, particularly 0.001 to 500 mg / kg. Administration can be once or several times daily.

[0040] The pharmaceutical compositions of the present invention can be administered alone or in combination with other therapeutic agents. When administered in combination, administration can be sequential or simultaneous.

[0041] Furthermore, the present invention provides a method for producing exosomes encapsulating FAF1 protein, comprising: 1) introducing a polynucleotide encoding the FAF1 protein into a cell to obtain a transformed cell; 2) culturing the transformed cells; and 3) isolating exosomes from the cells The present invention provides a method comprising:

[0042] The cells in step 1) are any one or more cells selected from the group consisting of B lymphocytes, T lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, tumor cells, and HEK293 cells, but are not limited thereto. In a preferred embodiment, the cells are HEK293 cells or HeLa cells.

[0043] Transduction was performed at 5 x 10 6 ~7×10 6 The treatment can be carried out by treating 6.5 × 10 cells with 5 to 10 μg of 3×Flag-FAF1 plasmid and culturing the cells, preferably 6.5 × 10 6 Treat the cells with 8 μg of 3×Flag-FAF1 plasmid and culture the cells.

[0044] Exosomes can be isolated by methods commonly used in the art for isolating exosomes. Size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, tangential flow filtration, exosome precipitation, whole exosome isolation kits, immunosorbent capture, affinity methods such as affinity capture, affinity purification, immunoassays, microfluidic separation, or a combination thereof can be performed to isolate exosomes.

[0045] According to a specific embodiment of the present invention, the culture medium is collected and centrifuged at 300 × g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes to separate the supernatant, which is then filtered through a 0.2 μm filter and centrifuged at 150,000 × g for 70 minutes using an ultracentrifuge. The supernatant is removed, the pellet is washed with PBS, and the mixture is centrifuged again at 150,000 × g for 70 minutes to remove the supernatant and isolate the exosomes remaining in the lower layer.

[0046] Isolated exosomes can be stored at -20°C and -80°C.

[0047] The term "transformation" as used herein refers to a molecular biological technique in which a DNA fragment or a plasmid carrying a foreign gene different from that of the original cell is introduced into the cell and binds to the DNA present in the original cell, thereby changing the genetic properties of the cell.

[0048] FAF1-encapsulated exosomes contain FAF1 protein and function as a carrier that transports the FAF1 protein to cancer cells or tissues.

[0049] FAF1 protein is secreted extracellularly via exosomes.

[0050] The extracellularly secreted FAF1 protein can induce apoptosis in other cells.

[0051] Apoptosis is a type of programmed cell death that can occur in multicellular organisms. It involves changes in cell morphology and intracellular biochemical changes that lead to cell death. This process culminates in cell swelling and cracking, alterations in the cell membrane, chromatin condensation and chromosome cleavage, and engulfment by phagocytes.

[0052] In contrast to necrosis, which is cell death caused by acute cell injury, apoptosis is not harmful to the organism but rather benefits its life cycle. The formation of fingers and toes during differentiation of the human embryo is a prime example of apoptosis. Furthermore, apoptosis is an important mechanism in cell replacement, tissue remodeling, and removal of damaged cells.

[0053] Apoptosis occurs in two ways: one is to remove unnecessary parts during the process of development and differentiation, and the other is to protect other cells when a cell is severely damaged and may become cancerous. However, cells that have problems during the apoptosis process become cancer cells, and these cancer cells do not die but instead continue to grow and proliferate by dividing repeatedly.

[0054] The term "culture" as used herein refers to a method for growing cells or microorganisms under appropriate artificially controlled environmental conditions. In the present invention, the method for culturing a transformant can be performed using a method widely known in the art.

[0055] The medium means a known medium used for culturing animal cells, and can be selected from the group of commercially available serum-free media, protein-free media and chemically defined media.

[0056] Furthermore, the present invention provides a method for screening a cancer therapeutic agent, comprising the steps of: 1) treating cancer cells with reduced FAF1 expression with a test substance; 2) measuring the level of FAF1 protein in exosomes isolated from the cells of step 1); and 3) selecting a test substance that increases the level of FAF1 protein in step 2) compared to a control group not treated with the test substance; The present invention provides a method comprising:

[0057] In this method, the cells in step 1) are not particularly limited, but in a preferred aspect, they are MIA PaCa-2 cells, A549 cells, HCT 116 cells, Hep3B cells, MDA-MB-231 cells, Caki-1 cells, or HeLa cells.

[0058] In this method, the test substance in step 1) refers to a substance that increases the expression of FAF1, and may be any one selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts, but is not limited to these.

[0059] As a marker for selecting candidate substances for use in the present invention, the level of FAF1 protein can be measured using methods known in the art. For example, antibodies that specifically detect proteins are commercially available and can be used to detect proteins using Western blotting, co-immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay, etc. By using such antibodies, the expression level of a particular protein can be specifically confirmed, but the present invention is not limited thereto.

[0060] If the expression level of a specific protein is increased compared to a control group not administered the test substance, the substance can be selected as a candidate for a cancer therapeutic drug.

[0061] First, we confirmed that overexpressing FAF1 in HEK 293 and HeLa cells increases the amount of FAF1 in exosomes, and that FAF1 can be secreted via exosomes (see Figures 1 to 4). FAF1, a pro-apoptotic factor, is a protein that inhibits tumorigenesis. Therefore, to confirm whether exosomes isolated from HEK 293 cells overexpressing FAF1 have an inhibitory effect on tumorigenesis, we performed tumorigenesis inhibition tests using MIA PaCa-2, A549, HCT 116, Hep3B, MDA-MB-231, or HeLa cells. We confirmed that the number of colonies formed when treated with exosomes isolated from HEK 293 cells overexpressing FAF1 was lower than when treated with exosomes isolated from HEK 293 cells not overexpressing FAF1 (see Figures 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, and 10b). Furthermore, exosomes isolated from FAF1-overexpressing HEK 293 cells were injected intratumorally into nude mice bearing xenografts of MIA PaCa-2, A549, HCT 116, Hep3B, MDA-MB-231, Caki-1, or HeLa cells. FAF1-encapsulated exosomes reduced tumor volume and weight, and FAF1 treatment induced cell necrosis (see Figures 11a-11e, 12a-12e, 13a-13e, 14a-14e, 15a-15e, 16a-16e, and 17a-17e). These results suggest that encapsulated FAF1 is secreted into the extracellular space via exosomes, is properly delivered to target cancer cells, and effectively inhibits tumor growth. Therefore, FAF1-encapsulated exosomes can be used as a cancer therapeutic agent.

[0062] The present invention will now be described in more detail with reference to the following examples.

[0063] However, the following examples are for the purpose of illustrating the present invention, and the present invention is not limited thereto. [Example]

[0064] Example 1: Isolation of exosomes from HEK 293 cells overexpressing FAF1 <1-1>Cell culture HEK 293 cells, a human embryonic kidney-derived cell line, were maintained in Dulbecco's modified Eagle's medium (DMEM, WelGENE, Korea) containing antibiotics, 10% fetal bovine serum (FBS, Atlas Biologicals, USA), and antibiotic-antimycoplasma agent (penicillin / streptomycin, GIBCO BRL, USA) at 5% CO2 and 37°C, with subculture every two days.

[0065] <1-2> Intracellular transduction To overexpress FAF1 in cells, 3x Flag-tagged FAF1 plasmid (8 μg) was added to 6.5 × 10 6 was introduced into the cells. Specifically, cells were seeded onto 150 mm culture dishes and cultured in DMEM containing 10% FBS at 5% CO2 and 37°C for 24 hours. 3xFlag-tagged FAF1 was prepared using a previously published method (Yu et al., 2016). A total of 180 μL of DNA:BioT (Bioland Scientific, USA) mixture was prepared using DMEM without FBS or antibiotics at a ratio of 1:1.5 and incubated at room temperature for 5 minutes. The mixture was added to the cells cultured for 24 hours and incubated at 5% CO2 and 37°C for 24 hours.

[0066] <1-3> Exosome isolation <1-3-1>Exo-quick-TC (商標) Isolation of exosomes using Exo-quick-TC (商標) Exosomes were isolated from cells transfected with 3x Flag-tagged FAF1 plasmid using a system called a ELISA kit (System Biosciences, USA). Specifically, 24 hours after transfection, the cells were treated with DMEM (System Biosciences, USA) supplemented with 10% exosome-depleted FBS and cultured for 48 hours. The culture medium was collected and centrifuged at 3,000 rpm for 15 minutes to remove cell debris from the supernatant. The collected medium (10 mL) was treated with 2 mL of Exo-quick buffer, shaken, and mixed thoroughly to achieve a medium:Exo-quick buffer ratio of 5:1. After overnight culture at 4°C, the medium was centrifuged twice at 1,500 g for 30 minutes to obtain exosomes.

[0067] <1-3-2> Isolation of exosomes using an ultracentrifuge Exosomes were isolated from cells transfected with the 3x Flag-tagged FAF1 plasmid using an ultracentrifuge (optima XE-100, BECKMAN Coulter, USA). Specifically, 24 hours after transfection, cells were treated with DMEM without FBS or antibiotics and cultured for another 24 hours. The culture medium was collected and centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes to separate the supernatant, which was then filtered through a 0.2 μm syringe filter (BioFACT, Korea).

[0068] To isolate exosomes for in vitro and in vivo experiments, samples were centrifuged at 150,000 g for 70 minutes in a BECKMAN Coulter optima XE-100 ultracentrifuge using a 45Ti rotor. The supernatant was discarded and the pellet was washed with PBS. The washed PBS solution was centrifuged again at 150,000 g for 70 minutes. The supernatant was then discarded and the remaining exosomes were collected in EP tubes using PBS.

[0069] The isolated exosomes were stored at -20°C and -80°C and thawed at 4°C before use.

[0070] Example 2: Isolation of exosomes from HeLa cells overexpressing FAF1 <2-1>Cell culture HeLa cells derived from a cervical cancer patient were maintained in DMEM (WelGENE, Korea) containing antibiotics, 10% FBS (Atlas Biologicals, USA), and antibiotic-antimycoplasma agent (penicillin / streptomycin, GIBCO BRL, USA) at 5% CO2 and 37°C, with subculture every two days.

[0071] <2-2> Intracellular transduction To overexpress FAF1 in HeLa cells, 3x Flag-tag-FAF1 plasmid (8 μg) was added to 6.5 × 10 6 was introduced into the cells. The specific experimental method was the same as in Example 1-2.

[0072] <2-3> Exosome isolation Exo-quick-TC (商標) Alternatively, exosomes were isolated from HeLa cells transfected with 3x Flag-tagged FAF1 plasmid using an ultracentrifuge. The specific experimental method was the same as in Example 1-3.

[0073] Example 3: Cultivation of cancer cell lines MIA PaCa-2 (pancreatic cancer cells), Hep3B (liver cancer cells), MDA-MB-231 (triple-negative breast cancer cells), Caki-1 (renal cancer cells), and HeLa (cervical cancer cells) were cultured in DMEM, 10% FBS, and 1% antibiotic-antimycoplasma agent (penicillin / streptomycin, GIBCO BRL, USA) at 37°C with 5% CO2 in an incubator and subcultured every 3 days. A549 (non-small cell lung cancer cells) and HCT116 (colon cancer cells) were cultured in RPMI 1640 medium with 10% FBS and 1% antibiotic-antimycoplasma agent at 37°C with 5% CO2 and subcultured every 3 days.

[0074] <Experimental Example 1> Confirmation of an increase in FAF1 in exosomes when FAF1 is overexpressed in HEK 293 cells and HeLa cells To investigate whether FAF1 is incorporated into exosomes in large amounts when FAF1 is overexpressed in HEK 293 cells and HeLa cells transduced with the 3x Flag tag-FAF10 plasmid (8 μg) of Example 1, ultracentrifugation and Exo-quick-TC (商標) The expression level of FAF1 in exosomes isolated using the method was confirmed by Western blotting.

[0075] Specifically, for Western blotting, 50 μL of mammalian lysis buffer was added to the pellet after removing the supernatant and allowed to lyse for 30 minutes on ice. An 8-10% acrylamide gel was prepared using a Western blot kit (Amersham Biosciences, UK). The lysed pellet was mixed with 5 μL of SDS sample buffer and boiled for 3 minutes. A predetermined volume was then loaded onto the gel, and proteins in the pellet were separated by size at 80-100 V. The acrylamide gel containing the separated proteins was placed on top of a nitrocellulose membrane, and a 200 mA current was applied for 2 hours to transfer the proteins to the nitrocellulose membrane. The membrane was then blocked with nonfat dry milk, and the FAF1 antibody was then coupled to the membrane, followed by a secondary antibody. The amount of FAF1 protein was confirmed using a Western blot detection kit (AbFrontier, Korea) and a ChemiDoc-It Imaging System (UVP, USA).

[0076] When exosomes were isolated using ultracentrifugation, FAF1 expression was slightly increased in exosomes isolated from FAF1-overexpressing HEK 293 cells compared to exosomes isolated from cells not overexpressing FAF1 (Figure 1). In the case of HeLa cells, FAF1 expression was almost absent in exosomes isolated from cells not overexpressing FAF1 or from cells overexpressing FAF1 (Figure 2).

[0077] Exo-quick-TC (商標)When exosomes were isolated using this method, the expression level of FAF1 in exosomes isolated from HEK 293 cells overexpressing FAF1 was significantly increased (Figure 3). In HeLa cells, FAF1 was not detected in exosomes isolated from FAF1-overexpressing cells, but the amount of FAF1 was confirmed to be increased by the tag protein Flag (Figure 4).

[0078] Exo-quick-TC (商標) The exosome isolation method using an ultracentrifuge is simpler and has the advantage of saving time compared to the method using an ultracentrifuge, but has the disadvantage of contamination occurring during the isolation process. Furthermore, since the method using an ultracentrifuge can isolate exosomes with higher purity, the inventors isolated exosomes using an ultracentrifuge in the following experimental example to confirm the effect of highly pure exosomes.

[0079] <Experimental Example 2> Confirmation of the in vitro antitumor effect of exosomes isolated from FAF1-overexpressing HEK 293 cells Exosomes isolated from FAF1-overexpressing HEK 293 cells were evaluated for their ability to inhibit colony formation in pancreatic cancer, non-small cell lung cancer, liver cancer, and cervical cancer cell lines.

[0080] Specifically, HEK 293 cells transfected with 3x Flag-tagged FAF1 (0 μg or 8 μg) were cultured for 24 hours. To promote exosome secretion, the culture medium was then collected and exosomes were isolated using an ultracentrifuge (optima XE-100, BECKMAN Coulter). The isolated exosomes were quantified using a spectrophotometer (MECASYS, Korea). To examine the in vitro effect of FAF1-encapsulated exosomes on tumor colonization in MIA PaCa-2, A549, HCT116, Hep3B, MDA-MB-231, or HeLa cell lines, MIA PaCa-2, Hep3B, or HeLa cells were seeded at 100 cells per well in a 6-well plate, while A549, HCT116, and MDA-MB-231 cells were seeded at 200 cells per well. After 24 hours, cells were treated with exosomes without FAF1 (exosome control: EXO-CON) or exosomes with FAF1 (EXO-FAF1). For MIA PaCa-2, exosomes were measured using a spectrophotometer (MECASYS, Korea). Cells were treated with 6 μg of exosomes and cultured for 12 days. For A549, exosomes were measured using NTA. Cells were cultured at 3 x 10 8 exosomes were treated with 3.16x10 cells and then cultured for 10 days; in the case of HCT116, exosomes were measured by NTA, and cells were cultured at 3.16x10 cells. 9 The cells were treated with 12 μg of exosomes and cultured for 10 days. In the case of Hep3B, exosomes were measured using a spectrophotometer (MECASYS, Korea). The cells were treated with 12 μg of exosomes and cultured for 7 days. 9 HeLa cells were treated with 1.65x10 exosomes and cultured for 12 days. In the case of HeLa cells, exosomes were measured by NTA, and 1.65x10 cells were cultured for 12 days. 10 The cells were treated with exosomes at the respective concentrations and cultured for 10 days. The medium was replaced with fresh medium every three days. After the medium was replaced, the cells were treated with exosomes at the respective concentrations. After the experiment was completed, the cells were washed with PBS, treated with 1 mL of methanol for 20 minutes, and then treated with 1 mL of crystal violet solution (SIGMA-ALDRICH, USA) for 5 minutes to count the number of stained colonies.

[0081] <2-1> Confirmation of colony formation suppression ability in pancreatic cancer To confirm the anti-tumor effect of FAF1-encapsulated exosomes in pancreatic cancer by inhibiting colony formation, MIA PaCa-2 cells, a pancreatic cancer cell line, were treated with FAF1-encapsulated exosomes and confirmed.

[0082] As a result, as shown in Figures 5a and 5b, the group treated with EXO-FAF1 had the lowest number of colonies. As can be seen from the figures, EXO-FAF1 reduced colony formation by 24.2±7.27% compared to EXO-CON and 26.2±7.08% compared to MOCK.

[0083] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in pancreatic cancer.

[0084] <2-2> Confirmation of colony formation inhibitory activity in non-small cell lung cancer To verify the antitumor effect of FAF1-encapsulated exosomes in non-small cell lung cancer, A549 cells, a non-small cell lung cancer cell line, were treated with FAF1-encapsulated exosomes and their ability to inhibit colony formation was confirmed.

[0085] As shown in Figures 6a and 6b, the group treated with EXO-FAF1 had the lowest number of colonies. EXO-FAF1 reduced colony formation by 46.9 ± 12.88% compared to EXO-CON and by 42.7 ± 13.91% compared to MOCK.

[0086] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in non-small cell lung cancer.

[0087] <2-3> Confirmation of colony formation inhibitory activity in colon cancer To verify the antitumor effect of FAF1-encapsulated exosomes in colon cancer, HCT116 cells, a colon cancer cell line, were treated with FAF1-encapsulated exosomes and their ability to inhibit colony formation was confirmed.

[0088] As shown in Figures 7a and 7b, the EXO-FAF1 treatment group showed the lowest number of colonies. EXO-FAF1 reduced colony formation by 23.0 ± 9.07% compared to EXO-CON and by 30.9 ± 8.14% compared to MOCK.

[0089] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in colorectal cancer.

[0090] <2-4> Confirmation of colony formation inhibitory activity in liver cancer To verify the antitumor effect of FAF1-encapsulated exosomes in liver cancer, Hep3B cells, a liver cancer cell line, were treated with FAF1-encapsulated exosomes and their ability to inhibit colony formation was confirmed.

[0091] As shown in Figures 8a and 8b, the group treated with EXO-FAF1 had the lowest number of colonies. EXO-FAF1 reduced colony formation by 50.6 ± 10.21% compared to EXO-CON and 46.1 ± 11.15% compared to MOCK.

[0092] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in liver cancer.

[0093] <2-5> Confirmation of colony formation inhibition in triple-negative breast cancer To verify the colony formation-suppressing ability of FAF1-encapsulated exosomes in the antitumor effect of triple-negative breast cancer, MDB-MB-231 cells, a triple-negative breast cancer cell line, were treated with FAF1-encapsulated exosomes and the effect was confirmed.

[0094] As a result, as shown in Figures 9a and 9b, the group treated with EXO-FAF1 had the lowest number of colonies. Numerical results confirmed that EXO-FAF1 reduced colony formation by 43.8 ± 10.40% compared to EXO-CON and 59.3 ± 7.59% compared to MOCK.

[0095] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in triple-negative breast cancer.

[0096] <2-6> Confirmation of colony formation inhibitory activity in cervical cancer To confirm the colony formation-suppressing ability of FAF1-encapsulated exosomes in the antitumor effect of cervical cancer, HeLa cells, a cervical cancer cell line, were treated with FAF1-encapsulated exosomes and confirmed.

[0097] As a result, as shown in Figures 10a and 10b, the group treated with EXO-FAF1 had the lowest number of colonies. Numerical results confirmed that EXO-FAF1 reduced colony formation by 38.4±6.45% compared to EXO-CON and 38.0±6.49% compared to MOCK.

[0098] These results suggest that FAF1-encapsulated exosomes suppress tumorigenesis in cervical cancer.

[0099] <Experimental Example 3> Confirmation of the in vivo antitumor effect of exosomes isolated from FAF1-overexpressing HEK 293 cells in a tumor xenograft animal model To investigate the in vivo tumor-suppressive effects of exosomes isolated from FAF1-overexpressing HEK293 cells, MIA PaCa-2, A549, HCT 116, Hep3B, MDA-MB-231, Caki-1, and HeLa cell lines were mixed with 100 μL of PBS and 100 μL of Matrigel (Corning Life Science, USA) and xenografted into the dorsal region of 8-week-old male BALB / cSLC nu / nu mice (Central Lab. Animal Inc., Korea) through a 26G syringe. Tumor volume was measured as a function of the long axis x short axis. 2 The calculation was based on a 0.5 × 0.5 standard deviation. Tumor volumes were 80–140 mm 3 When tumor volume reached 1000 mg / kg / day, mice were intratumorally injected with PBS, recombinant FAF1 (AngioLab, Korea), EXO-CON, and EXO-FAF1 every other day for a total of four injections. Tumor volumes were measured for the MIA PaCa-2 cell line up to day 43, the A549 cell line up to day 48, the HCT116 cell line up to day 30, the Hep3B cell line up to day 35, the MDA-MB-231 cell line up to day 40, the Caki-1 cell line up to day 46, and the HeLa cell line up to day 41. On the final day, mice were euthanized, and tumors were enucleated, weighed, and examined by H&E staining.

[0100] Tissues extracted for H&E staining were preserved in formalin solution. After 24 hours, they were dehydrated in 70% ethanol for 20 minutes, followed by two 20-minute cycles in 95% ethanol, and finally two 20-minute cycles in 100% ethanol. They were then incubated in xylene and paraffin for 3 hours each using a tissue processor (Leica, Germany). For tissue sectioning, the tissues were fixed in paraffin and cut into 5-μm sections using a microtome. The sections were then placed on glass slides. Before staining, the tissues were incubated for 20 minutes at 60°C on a slide warmer. Next, the tissues were incubated three times in xylene for 3 minutes and 30 seconds each. This was followed by two cycles of 100% ethanol, followed by one cycle of 95%, 80%, and 70% ethanol, each for 3 minutes and 30 seconds. The tissues were then washed in running water for 3 minutes. Next, the slides were incubated in hematoxylin for 5 minutes, followed by a 10-minute wash, followed by a 4-second wash in hydrochloric acid, a 10-second wash in 1% ammonia, and a 1-minute 10-second wash in eosin. The slides were then placed in 70% ethanol, 85% ethanol, and then incubated twice in 100% ethanol for 1 minute 30 seconds. After the ethanol wash, the slides were incubated in a 1:1 mixture of 100% ethanol and xylene for 1 minute 30 seconds. Finally, the slides were incubated in xylene for 3 minutes, followed by another 4 minutes in fresh xylene. Shandon (商標) The slides were covered with a glass cover using Synthetic Mountant (Thermo Scientific, USA). After staining, the slides were observed under a microscope.

[0101] <3-1>Efficacy evaluation in pancreatic cancer xenograft animal models MIA PaCa-2 cells, a pancreatic cancer cell line, were xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 11a and 11b, tumor volume in the FAF1-encapsulated exosome-treated group was reduced by 60.46 ± 13.33% compared to the PBS-treated group, 55.40 ± 15.04% compared to the EXO-CON-treated group, and 59.71 ± 13.58% compared to the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 11c and 11d, tumor weight in the FAF1-encapsulated exosome-treated group was reduced by 52.55 ± 12.13% compared to the PBS-treated group, 46.74 ± 13.61% compared to the EXO-CON-treated group, and 45.28 ± 13.98% compared to the recombinant FAF1 (AngioLab, Korea)-treated group.

[0102] Furthermore, the cell morphology of the tumor tissue was observed by H&E staining. As shown in Figure 11e, the cytoplasmic size of the tumor tissue in the HEK Exo-FAF1-treated group was smaller than that in the mock-treated, recombinant FAF1-treated, and HEK Exo-CON-treated groups, confirming that HEK Exo-FAF1 treatment induced cell necrosis.

[0103] These results indicate that exosome-mediated FAF1 suppresses tumorigenesis in vivo.

[0104] <3-2>Efficacy evaluation in non-small cell lung cancer xenograft model animals A549 cells, a non-small cell lung cancer cell line, were xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 12a and 12b, tumor volume was reduced by 75.45 ± 9.89% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 72.77 ± 10.97% in the EXO-CON-treated group, and 75.14 ± 10.01% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 12c and 12d, tumor weight was reduced by 59.07 ± 13.88% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 61.31 ± 13.12% in the EXO-CON-treated group, and 63.19 ± 12.48% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within tumors in the EXO-FAF1-treated group, as shown in Figure 12e.

[0105] <3-3>Efficacy evaluation in colon cancer xenograft model animals The colon cancer cell line HCT 116 was xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 13a and 13b, tumor volume was reduced by 66.87 ± 7.62% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 47.87 ± 12.00% in the EXO-CON-treated group, and 56.93 ± 9.91% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 13c and 13d, tumor weight was reduced by 71.88 ± 5.66% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 40.12 ± 12.05% in the EXO-CON-treated group, and 60.09 ± 8.03% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within the tumors in the EXO-FAF1-treated group, as shown in Figure 13e.

[0106] <3-4>Efficacy evaluation in liver cancer xenograft model animals The liver cancer cell line Hep3B was xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 14a and 14b, tumor volume was reduced by 76.16 ± 11.61% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 70.74 ± 14.25% in the EXO-CON-treated group, and 62.44 ± 18.29% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 14c and 14d, tumor weight was reduced by 73.48 ± 33.22% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 66.62 ± 42.16% in the EXO-CON-treated group, and 44.44 ± 70.87% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within the tumors in the EXO-FAF1-treated group, as shown in Figure 14e.

[0107] <3-5>Efficacy evaluation in triple-negative breast cancer xenograft model animals The triple-negative breast cancer cell line MDA-MB-231 was xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 15a and 15b, tumor volume in the FAF1-encapsulated exosome-treated group was reduced by 80.38 ± 8.34% compared to the PBS-treated group, 68.32 ± 13.46% compared to the EXO-CON-treated group, and 67.22 ± 13.93% compared to the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 15c and 15d, tumor weight in the FAF1-encapsulated exosome-treated group was reduced by 77.03 ± 11.83% compared to the PBS-treated group, 69.63 ± 15.66% compared to the EXO-CON-treated group, and 62.50 ± 19.33% compared to the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within tumors in the EXO-FAF1-treated group, as shown in Figure 15e.

[0108] <3-6>Efficacy evaluation in renal cancer xenograft model animals The Caki-1 cell line, a renal cancer cell line, was xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 16a and 16b, tumor volume was reduced by 54.23 ± 12.40% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 43.05 ± 15.21% in the EXO-CON-treated group, and 53.56 ± 12.60% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 16c and 16d, tumor weight was reduced by 42.11 ± 16.22% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group and 31.25 ± 19.26% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within the tumors in the EXO-FAF1-treated group, as shown in Figure 16e.

[0109] <3-7>Efficacy evaluation in cervical cancer xenograft model animals HeLa cells, a cervical cancer cell line, were xenografted into immunodeficient nude mice to determine whether FAF1-encapsulated exosomes have an inhibitory effect on tumor formation. As shown in Figures 17a and 17b, tumor volume was reduced by 66±11.33% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 56.4±14.52% in the EXO-CON-treated group, and 57.1±14.30% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, as shown in Figures 17c and 17d, tumor weight was reduced by 60±17.59% in the FAF1-encapsulated exosome-treated group compared to the PBS-treated group, 48.6±21.39% in the EXO-CON-treated group, and 70.7±12.17% in the recombinant FAF1 (AngioLab, Korea)-treated group. Furthermore, necrosis was observed within the tumors in the EXO-FAF1-treated group, as shown in Figure 17e. Further aspects of the present invention are described below: [Section 1] A pharmaceutical composition for treating cancer, comprising exosomes encapsulating Fas-associated factor 1 (FAF1) protein as an active ingredient. [Section 2] Item 1. The pharmaceutical composition of Item 1, wherein the FAF1 protein comprises the amino acid sequence set forth in SEQ ID NO:1. [Section 3] Item 1. The pharmaceutical composition according to Item 1, wherein the exosomes have a diameter of 50 to 200 nm. [Section 4] Item 1. The pharmaceutical composition according to Item 1, wherein the FAF1 protein encapsulated in the exosome suppresses tumorigenesis in other cells. [Section 5] Item 1. The pharmaceutical composition according to Item 1, wherein the cancer is cervical cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, head and neck squamous cell carcinoma, bladder cancer, prostate cancer, stomach cancer, endometrial cancer, brain tumor, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, endometrial mesothelial cancer, esophageal cancer, adrenal cancer, thyroid cancer, bone tumor, glioblastoma, mesothelioma, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, leukemia, or malignant lymphoma. [Section 6] Item 1. The pharmaceutical composition according to Item 1, wherein FAF1 expression is decreased in cancer. [Section 7] Item 1. The pharmaceutical composition according to Item 1, wherein the exosomes of the composition deliver FAF1 to target tumor cells. [Section 8] Item 10. The pharmaceutical composition according to Item 1, wherein the composition is administered orally, by intrabronchial inhalation, or by intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular, intrapleural or intratumoral injection. [Section 9] Item 1. The pharmaceutical composition according to item 1, wherein the composition is administered alone or simultaneously or sequentially with other therapeutic agents. [Section 10] Item 3. The pharmaceutical composition according to Item 2, wherein FAF1 of SEQ ID NO: 1 is encapsulated in exosomes. [Section 11] A method for producing exosomes encapsulating FAF1 protein, comprising: 1) introducing a polynucleotide encoding the FAF1 protein into a cell to obtain a transformed cell; 2) culturing the transformed cells; and 3) isolating exosomes from the cells A method comprising: [Section 12] Item 12. The method according to Item 11, wherein the cells in step 1) are any one or more cells selected from the group consisting of B lymphocytes, T lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, tumor cells, and human embryonic kidney 293 (HEK293) cells. [Section 13] A method for screening a cancer therapeutic agent, comprising: 1) treating cancer cells with reduced FAF1 expression with a test substance; 2) measuring the level of FAF1 protein in exosomes isolated from the cells of step 1); and 3) selecting a test substance that increases the level of FAF1 protein in step 2) compared to a control group not treated with the test substance; A method comprising: [Section 14] Item 14. The method of item 13, wherein the cells are MIA PaCa-2 cells, MDA-MB-231 cells, HCT 116 cells, Hep3B cells, Caki-1 cells, A549 cells, or HeLa cells. [Section 15] A method for treating cancer, comprising administering exosomes encapsulating FAF1 protein to an individual with reduced FAF1 expression. [Section 16] Use of FAF1 protein-encapsulating exosomes in the manufacture of cancer therapeutic drugs.

Claims

1. A pharmaceutical composition for treating cancer, comprising as an active ingredient an exosome encapsulating Fas-associated factor 1 (FAF1) protein, wherein the cancer is selected from the group consisting of cervical cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, head and neck squamous cell carcinoma, bladder cancer, prostate cancer, gastric cancer, endometrial cancer, brain tumor, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, endometrial mesothelial cancer, esophageal cancer, adrenal cancer, thyroid cancer, bone tumor, glioblastoma, mesothelioma, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, leukemia, and malignant lymphoma.

2. 2. The pharmaceutical composition of claim 1, wherein the FAF1 protein comprises the amino acid sequence set forth in SEQ ID NO:

1.

3. 2. The pharmaceutical composition of claim 1, wherein the exosomes have a diameter of 50 to 200 nm.

4. The pharmaceutical composition according to claim 1, wherein the FAF1 protein encapsulated in the exosome suppresses tumorigenesis in other cells.

5. The pharmaceutical composition of claim 1, wherein expression of FAF1 is decreased in cancer.

6. The pharmaceutical composition of claim 1 , wherein the exosomes of the composition deliver FAF1 to target tumor cells.

7. 10. The pharmaceutical composition of claim 1, wherein the composition is administered orally, by intrabronchial inhalation, or by intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular, intrapleural, or intratumoral injection.

8. 10. The pharmaceutical composition of claim 1, wherein the composition is administered alone or simultaneously and sequentially with other therapeutic agents.

9. The pharmaceutical composition according to claim 2, wherein FAF1 of SEQ ID NO: 1 is encapsulated in an exosome.

10. Use of FAF1 protein-encapsulating exosomes in the manufacture of a cancer therapeutic agent, wherein the cancer is cervical cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, head and neck squamous cell carcinoma, bladder cancer, prostate cancer, gastric cancer, endometrial cancer, brain tumor, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, endometrial mesothelial cancer, esophageal cancer, adrenal cancer, thyroid cancer, bone tumor, glioblastoma, mesothelioma, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, leukemia, or malignant lymphoma.

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