Novel recombinant plasma membrane-based endoplasmic reticulum for cancer therapy.

Abstract

To provide a recombinant plasma membrane-based endoplasmic reticulum for safe anti-cancer therapy that can actively and efficiently kill cancer cells even without anti-cancer drugs, while not functioning under conditions other than the cancer microenvironment. [Solution] The present invention relates to a recombinant plasma membrane-based endoplasmic reticulum, and more specifically, to a recombinant plasma membrane-based endoplasmic reticulum containing in its membrane a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced with arginine, and a pharmaceutical composition for cancer treatment containing the recombinant plasma membrane-based endoplasmic reticulum.

Description

[Technical field]

[0001] The present invention relates to a novel recombinant plasma membrane-based vesicle, more particularly to a novel recombinant plasma membrane-based vesicle for cancer therapy. [Background technology]

[0002] Cancer refers to a group of diseases associated with abnormal cell growth that has the potential to infiltrate and metastasize to other parts of the body. As of 2015, there are more than 90 million cancer patients worldwide, and about 14 million new cancer patients are diagnosed each year. Cancer accounts for 15.7% of human deaths, and the most common cancers in men are lung cancer, prostate cancer, colon cancer, and stomach cancer, and in women are breast cancer, colon cancer, lung cancer, and cervical cancer.

[0003] Various therapeutic approaches have been attempted to treat cancer, including chemotherapy using various anticancer drugs, radiation therapy using radiation, and antibody therapy targeting specific biological molecules related to cancer. However, the anticancer drugs used in chemotherapy and radiation therapy have serious side effects because they also affect normal cells, and cancer cells often acquire resistance to the anticancer drugs, resulting in treatment failure or recurrence.

[0004] Recently, anti-cancer immunotherapy utilizing one's own immune system has shown surprising results in clinical trials, but due to the complexity of cancer, it has a limiting effect, showing effectiveness in an average of less than 30% of patients. This is because cancer cells are recognized as "self" by one's own immune cells, and therefore it is important to have immune cells recognize cancer cells as "non-self" to induce phagocytosis of cancer cells and further amplified immune responses.

[0005] Exosomes are cell-derived vesicles present in all biological fluids, including blood, urine, and cell culture media, and are also called extracellular vesicles or microvesicles. The size of exosomes is known to be 30-100 nm, and they are secreted from cells when multivesicular bodies fuse with the cell membrane, secreted directly through the cell membrane, or budding directly from the cell membrane. Exosomes are known to play important roles in various processes such as coagulation, intercellular signaling, and metabolic waste management. Exosomes have important advantages as drug delivery vehicles compared to liposomes and polymeric nanoparticles in that they have a composition similar to that of human cells themselves and are non-immunogenic (Ha et al., Acta Pharm. Sin. B. 6(4):287-296, 2016). In relation to this, there have been various attempts to use exosomes to deliver anticancer drugs such as doxorubicin to tumor tissues (Tian et al., Biomaterials. 35: 2383-2390, 2014), deliver paclitaxel and doxorubicin to the brain through the blood-brain barrier (Yang et al., Pharm. Res. 32: 2003-2014, 2015), deliver catalase through the blood-brain barrier to treat Parkinson's disease (Haney et al., J. Control Release. 207: 18-30, 2015), and deliver siRNA specific to certain genes to treat cancer (Shtam et al., Cell Commun. Signal. 11: 88, 2013).

[0006] Extracellular vesicles refer to particulate structures in which various biomolecules, such as proteins with various functions, nucleic acid molecules such as RNA, or lipids, which are released or secreted from cells into the extracellular environment, are encapsulated in the same lipid bilayer cell membrane as the cell membrane from which they originated. Extracellular vesicles refer to plasma membrane-based endoplasmic reticulum with an average diameter of 100 nm to 1 μm, which is larger than exosomes, which usually have a size of 30 to 100 nm.

[0007] A cell-derived nanovesicle is a nano-sized vesicle surrounded by a plasma membrane, which is a nano-sized cell membrane component, formed by artificial methods such as extrusion of cells through a microfluidic channel or multi-stage filtration, and is distinct from exosomes and extracellular vesicles that are naturally produced and secreted by cells.

[0008] Meanwhile, VSV-G (vesicular stomatitis virus glycoprotein) is the only viral glycoprotein present in the virion membrane of vesicular stomatitis virus, and acts as a protein for attachment and fusion of the virus to target cells. The VSV-G protein is a transmembrane protein containing two N-linked glycans, and in the absence of other viral proteins, it can initiate membrane fusion in a low pH-dependent manner. The VSV-G protein is used as a direct gene transfer carrier to form a complex with nucleic acid molecules such as DNA, or has been effectively used in gene therapy by producing more stable and high-titer pseudotyped murine leukemia virus (MLV)-based retrovirus and lentivirus-based vectors. However, recently, the possibility of using it to transfer various proteins other than genes to target cells has been proposed (Mangeot et al., Mol. Ther. 19(9):1656-1666, 2011).

[0009] As a result of research into the structure and function of the VSV-G protein, it has been found that the histidines at the 60th, 162nd, and 407th amino acid residues form a cluster in the mature protein from which the signal sequence has been removed, and act as a pH sensor (Roche et al., Science, 313:187-191, 2006; Roche et al., Science, 315:843-848, 2007). Recently, it has been reported that when the histidine at the 162nd amino acid is mutated to arginine (H162R), membrane fusion is induced at pH 6.8, which is the physiological pH surrounding cancer cells, and the death of cancer cells is promoted when neural stem cells expressing the VSV-G mutant (H162R) are administered (Zhu et al., Mol. Ther. 21(8):1492-1497, 2013).

[0010] However, the methods disclosed in the above-mentioned prior art are difficult to apply to actual clinical practice because of the difficulty in obtaining supply and demand for neural stem cells and the fact that the cancer cell killing effect is not very large. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention is intended to solve various problems including the above problems, and aims to provide a recombinant plasma membrane-based endoplasmic reticulum for safe anti-cancer therapy that does not function under conditions other than the cancer microenvironment while actively and efficiently killing cancer cells without anti-cancer drugs. In particular, the present invention aims to provide a recombinant plasma membrane-based endoplasmic reticulum that "xenogenizes" cancer cells so that they are recognized as "enemies" by immune cells. However, these problems are merely examples and do not limit the scope of the present invention. [Means for solving the problem]

[0012] According to one aspect of the present invention, there is provided a recombinant plasma membrane-based vesicle in which a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced by arginine is introduced into the membrane.

[0013] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising the recombinant plasma membrane-based vesicle as an active ingredient.

[0014] According to another aspect of the present invention, there is provided a pharmaceutical composition for cancer treatment comprising as an active ingredient a recombinant plasma membrane-based endoplasmic reticulum having a virus-derived membrane fusogenic membrane protein introduced into the membrane.

[0015] According to another aspect of the present invention, there is provided use of a recombinant plasma membrane-based endoplasmic reticulum having a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced by arginine, or a recombinant plasma membrane-based endoplasmic reticulum having a virus-derived membrane fusogenic membrane protein in which the VSV-G mutant protein is introduced into the membrane, in the production of a cancer therapeutic agent.

[0016] According to another aspect of the present invention, there is provided a method for treating cancer in an individual, comprising administering to said individual the recombinant plasma membrane-based vesicle or any one or more of the pharmaceutical compositions described above. Effect of the Invention

[0017] According to one embodiment of the present invention as described above, the present invention can effectively treat cancer without relying on a complicated mechanism such as gene transfer. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 is a schematic diagram illustrating the mechanism of action of recombinant exosomes containing mutant VSV-G H162R (hereinafter abbreviated as 'mVSV-G') according to one embodiment of the present invention, which induces anti-cancer immune effects in the cancer cell microenvironment (pH 6.8). [Figure 2A] 1 is a plasmid map showing a schematic structure of a plasmid DNA for producing recombinant exosomes according to one embodiment of the present invention. [Figure 2B] 1 shows the amino acid sequences showing the mutation at position 162 of wild-type VSV-G and mutant VSV-G proteins for producing recombinant exosomes according to one embodiment of the present invention, and the nucleic acid sequences of polynucleotides encoding the same. [Diagram 3] FIG. 1 is a flow chart showing a process for producing recombinant exosomes containing mVSV-G according to one embodiment of the present invention. [Figure 4] 1 shows the results of Western blot analysis of recombinant exosomes according to one embodiment of the present invention and cell extracts transfected with an mVSV-G gene construct to produce the recombinant exosomes. [Figure 5A] 1 is a photograph taken by a transmission electron microscope of recombinant exosomes containing mVSV-G produced according to one embodiment of the present invention. [Figure 5B] 1 is a histogram showing the results of dynamic light scattering analysis of the particle size of the recombinant exosomes. [Figure 6A] This is a graph showing the results of confirming the degree of membrane fusion with cancer cells due to pH change when three types of cancer cells (4T1-Luc, EL4-Ova, and CT26.CL25) were treated with mVSVG-Exo according to one embodiment of the present invention (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 6B] FIG. 1 shows photographs taken under a fluorescent microscope after staining with anti-VSV-G antibody and anti-Cadherin antibody (green) to examine whether or not membrane fusion with cancer cells occurs due to pH change when 4T1-Luc cells are treated with mVSVG-Exo according to one embodiment of the present invention. [Figure 6C]This is a photograph showing the results of Western blot analysis of the presence or absence of expression of LDLR (low density lipoprotein receptor), which is known as a receptor for VSV-G, on the surface of various cells, including three types of cancer cells (4T1-Luc, EL4-Ova, and CT26.CL25). [Figure 7A] This is a histogram showing the results of flow cytometry to observe whether mVSV-G-Exo according to one embodiment of the present invention promotes fusion between cancer cells after being fused to the surface of three types of cancer cells (4T1-Luc, EL4-Ova, and CT26.CL25) by changing the pH. [Figure 7B] 7B is a graph showing the results of quantitatively measuring the results of FIG. 7A. [Figure 7C] This is a graph showing the results of a cell viability analysis to determine whether mVSV-G-Exo according to one embodiment of the present invention directly induces the death of cancer cells (4T1-Luc, EL4-Ova, and CT26.CL25) (*: P<0.05; **: P<0.01). [Figure 8A] 1 is a graph quantified results of a fluorescence microscopy analysis of the effects of recombinant exosomes containing mVSV-G according to one embodiment of the present invention and control exosomes (Con-Exo) on phagocytosis by macrophages and dendritic cells against 4T1-Luc breast cancer cells. [Figure 8B] 1 is a graph quantified results of a fluorescence microscopy analysis of the effects of recombinant exosomes containing mVSV-G according to one embodiment of the present invention and control exosomes (Con-Exo) on phagocytosis by macrophages and dendritic cells against EL4-Ova lymphoma cells. [Figure 8C] FIG. 1 is a graph quantified from fluorescence microscopy analysis of the effects of recombinant exosomes containing mVSV-G according to one embodiment of the present invention and control exosomes (Con-Exo) on phagocytosis by macrophages and dendritic cells against CT46.CL25 colon cancer cells (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 9]FIG. 1 is a graph showing the changes in relative expression ratios of CD40 (left) and CD86 (right) in bone marrow-derived dendritic cells after treating the bone marrow-derived dendritic cells with recombinant exosomes containing mVSV-G according to one embodiment of the present invention (mVSVG-Exo) and control exosomes (Con-Exo) to investigate whether VSVG can act as a TLR4 agonist to activate the function of dendritic cells (*: P<0.05; **: P<0.01). [Figure 10A] FIG. 1 is a graph comparing the size of cancer cells over time in a 4T1-Luc breast cancer tumor model animal (Balb / c, 7-week-old female mouse) administered recombinant exosomes (200 μg) containing mVSV-G according to one embodiment of the present invention (squares are control group, circles are control exosomes not containing mVSV-G, triangles are recombinant exosomes containing mVSV-G according to one embodiment of the present invention). [Figure 10B] FIG. 10B is a graph showing the results of measuring the weight of the cancer tissue excised from the experimental animals sacrificed 16 days after the injection of the cancer cells in FIG. 10A. [Figure 10C] This is a graph showing the changes in body weight of animals used in the experiment (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 11A] FIG. 1 is a graph comparing the size of cancer cells over time in an EL4-Ova lymphoma tumor model animal (C57BL / 6, 7-week-old female mouse) administered recombinant exosomes (200 μg) containing mVSV-G according to one embodiment of the present invention (squares are control group, circles are control exosomes not containing mVSV-G, triangles are recombinant exosomes containing mVSV-G according to one embodiment of the present invention). [Figure 11B] FIG. 11B is a graph showing the results of measuring the weight of the cancer tissue excised from the experimental animals sacrificed 16 days after the injection of the cancer cells in FIG. 11A. [Figure 11C] This is a graph showing the changes in body weight of animals used in the experiment (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 12]Recombinant exosomes containing mVSV-G according to one embodiment of the present invention (mVSVG-Exo), recombinant exosomes containing wild-type VSV-G (wtVSVG-Exo), control exosomes (Con-Exo), and PBS as a control were administered into the cancer tissue of a tumor model animal (EL4-Ova-injected C57BL / 6 mouse), the excised cancer tissue was disaggregated into single cells, stained with an anti-VSV-G antibody, and then subjected to flow cytometry (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 13A] 1 shows the results of administering recombinant exosomes containing mVSV-G (mVSVG-Exo) according to one embodiment of the present invention, recombinant exosomes containing wild-type VSV-G (wtVSVG-Exo), control exosomes (Con-Exo), and PBS as a control into the cancer tissue of a tumor model animal (EL4-Ova-injected C57BL / 6 mouse), dissociating the excised tumor-draining lymph nodes into single cells, and then performing flow cytometry using dendritic cell markers anti-CD11c antibody and anti-H-2kb Ova antibody. [Figure 13B] 1 is a graph showing the results of flow cytometry using anti-CD11c antibody and anti-CD40 antibody after dissociating the excised tumor-draining lymph node into single cells. [Figure 13C] This is a graph showing the results of flow cytometry using anti-CD11c antibody and anti-CD86 antibody after dissociating the excised tumor-draining lymph node into single cells (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 13D] FIG. 1 is a series of photographs showing the results of analyzing the degree of infiltration of CD8 T cells in tumor tissues using a fluorescent microscope after staining tumor tissue slices with an anti-CD8 antibody. [Figure 13E] FIG. 13D is a graph showing the quantification results of the degree of infiltration of CD8 T cells into cancer tissues (***: P<0.001). [Figure 14A]FIG. 13 is a graph showing the results of administering recombinant exosomes containing mVSV-G (mVSVG-Exo) according to one embodiment of the present invention, recombinant exosomes containing wild-type VSV-G (wtVSVG-Exo), control exosomes (Con-Exo), and PBS as a control into cancer tissue of a tumor model animal (EL4-Ova-injected C57BL / 6 mouse), isolating CD11c-positive dendritic cells and F4 / 80-positive macrophages from the excised tumor tissue, and co-culturing them with OT-1 CD8 T cells isolated from the spleen of an OT-1 transformed mouse at a ratio of 1:5, and then analyzing the INF-γ expression level in the culture medium by ELISA. [Figure 14B] The graph shows the results of ELISA analysis of the INF-γ expression level in the culture medium after treating single-celled spleen cells from spleen tissue removed from the experimental animals with PBS as a control group and 10 μg / ml of ovalbumin, a cancer-specific antigen, for 24 hours (*: P<0.05; **: P<0.01; ***: P<0.001). [Figure 15A] FIG. 1 shows a graph (left) showing the results of measuring the volume of tumor tissue over time after administration of recombinant exosomes (mVSVG-Exo) containing mVSV-G according to one embodiment of the present invention, control exosomes (Con-Exo), and PBS as a control into the tumor tissue of nude mice in which cancer was induced by subcutaneous injection of EL4-Ova cancer cells, and a graph (right) showing the results of measuring the weight of the tumor tissue excised after the mice were sacrificed 17 days after cancer cell injection, showing the results of an experiment using low-dose administration (100 μg twice). [Figure 15B] FIG. 1 shows a graph (left) showing the results of measuring the volume of tumor tissue over time after administration of recombinant exosomes (mVSVG-Exo) containing mVSV-G according to one embodiment of the present invention, control exosomes (Con-Exo), and PBS as a control into the tumor tissue of nude mice in which cancer was induced by subcutaneous injection of EL4-Ova cancer cells, and a graph (right) showing the results of measuring the weight of the tumor tissue excised after the mice were sacrificed 17 days after cancer cell injection. The graph shows the results of an experiment using a high dose (100 μg four times) administration using the same model. [Figure 15C]The graph (left) shows the results of measuring the volume of tumor tissue over time after administration of recombinant exosomes (mVSVG-Exo) containing mVSV-G according to one embodiment of the present invention into the tumor tissue of C57BL / 6 mice in which cancer was induced by subcutaneous injection of EL4-Ova cancer cells, intraperitoneally at 3-day intervals starting one day before injection of anti-CD8 antibody into cancer cells. The graph (right) shows the results of measuring the weight of tumor tissue excised from mice sacrificed on day 18 after cancer cell injection. Recombinant IgG was used as the control antibody (***: P<0.001). [Figure 15D] FIG. 1 shows a graph (left) showing the results of measuring the volume of tumor tissue over time after administration of recombinant exosomes (mVSVG-Exo) containing mVSV-G according to one embodiment of the present invention, control exosomes (Con-Exo), and PBS as a control into the tumor tissue of BATF3 KO mice (CD103 and CD8 deficient) in which cancer was induced by subcutaneous injection of EL4-Ova cancer cells, and a graph (right) showing the results of measuring the weight of tumor tissue excised after sacrifice of the mice 17 days after cancer cell injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Definitions of terms: The term "fusogenic membrane protein" as used herein refers to a viral membrane protein that plays a key role in inducing attachment to a host cell and membrane fusion in order for a virus to penetrate the host cell. A representative example is the envelope glycoprotein (VSV-G) derived from vesicular stomatitis virus.

[0020] The term "vesicular stomatitis virus envelope glycoprotein (VSV-G protein)" as used herein is the only viral glycoprotein present in the virion membrane of vesicular stomatitis virus and acts as a viral attachment and fusion protein to target cells. The VSV-G protein is a transmembrane protein containing two N-linked glycans, and in the absence of other viral proteins, it can initiate membrane fusion in a low pH-dependent manner. The VSV-G protein has been used as a direct gene transfer carrier to form a complex with nucleic acid molecules such as DNA, or has been effectively used in gene therapy by producing more stable and high-titer pseudotyped murine leukemia virus (MLV)-based retrovirus and lentivirus-based vectors. However, recently, its potential use for transferring various proteins other than genes to target cells has been suggested (Mangeot et al., Mol. Ther. 19(9):1656-1666, 2011).

[0021] As used herein, the term "plasma membrane-based vesicle" refers to a nano-sized vesicle surrounded by a cell membrane derived from a cell, and includes exosomes produced by secretion or budding from cells, extracellular vesicles, or cell-derived nanovesicles produced by processing cells using artificial methods such as extrusion.

[0022] As used herein, the term "exosomes" refers to cell-derived vesicles present in all biological fluids, including blood, urine, and cell culture media, also called extracellular vesicles or microvesicles. Exosomes are known to be 30-100 nm in size and are secreted from cells when multivesicular bodies fuse with the cell membrane or are secreted directly through the cell membrane. Exosomes are known to play important roles in diverse processes such as coagulation, intercellular signaling, and metabolic waste management.

[0023] The term "recombinant exosome" as used herein refers to an artificially produced exosome, which is obtained by transducing a gene encoding a foreign protein into a host cell capable of producing exosomes by genetic engineering to produce a transformed host cell, culturing the transformed host cell, and then collecting the exosome from the culture medium. The recombinant exosome contains the introduced foreign protein inside or in the exosome membrane.

[0024] The term "extracellular vesicles" as used herein refers to particulate structures in which various biomolecules, such as proteins with various functions, nucleic acid molecules such as RNA, or lipids, which are released or secreted from cells into the extracellular environment, are encapsulated in a lipid bilayer cell membrane that is the same as the cell membrane from which they originate. Extracellular vesicles refer to plasma membrane-based endoplasmic reticulum with an average diameter of 100 nm to 1 μm, which is larger than exosomes, which usually have a size of 30 to 100 nm.

[0025] The term "cell-derived nanovesicle" as used herein refers to a nano-sized vesicle surrounded by a plasma membrane, which is a nano-sized cell membrane component, formed by artificial methods such as extrusion of cells through a microfluidic channel or multi-step filtration, and is distinct from exosomes and extracellular vesicles that are naturally produced and secreted by cells.

[0026] The term "immunogenic cell death" as used herein refers to a type of cell death induced by cytostatics such as anthracyclines, oxaliplatin, and bortezomib, or by radiation therapy and photodynamic therapy. The immunogenic cell death is different from general cell death, and the immunological cell death of cancer cells can induce an effective anti-cancer immune response by activating dendritic cells and thereby activating specific T cell responses. A substance that induces immunogenic cell death is called an "immunogenic cell death inducer." The immunogenic cell death and immunogenic cell death inducers are well summarized in Kroemer et al. (Annu. Rev. Immunol., 31:51-72, 2013). The above references are incorporated herein by reference in their entirety.

[0027] The term "anthracycline-type anticancer agent" as used herein refers to a series of cell cycle non-specific anticancer agents used in cancer chemotherapy, derived from the bacterium Streptomyces peucetius var. caesius. Anthracycline-type anticancer agents are used to treat a variety of cancers, including leukemia, lymphoma, breast cancer, gastric cancer, uterine cancer, ovarian cancer, bladder cancer, and lung cancer, and are one of the most effective chemotherapy anticancer agents that have been developed so far. Daunorubicin was the first anthracycline anticancer drug to be discovered, followed by doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, etc. The mechanism of action of anthracycline anticancer drugs is said to be that they are inserted between the bases of DNA / RNA chains to inhibit DNA and RNA synthesis, thereby disrupting the replication of rapidly growing cancer cells, inhibiting topoisomerase II enzyme activity to inhibit the relaxation of supercoiled DNA, thereby disrupting transcription and replication, inducing damage to DNA, proteins, and cell membranes by forming iron-mediated free oxygen radicals, and inducing DNA damage responses, and inducing histone displacement from chromatin, which deregulates the epigenome and transcriptome. Recent studies have shown that doxorubicin inhibits the CD4 + It has been reported that it increases Th1 immune responses by activating cells (Park et al., Int. Immunopharmacol. 9(13-14):1530-1539, 2009), and that when dendritic cells are administered in combination with doxorubicin, it exhibits anticancer activity by inducing immunogenic cell death in osteosarcoma (Kawano et al., Oncol. Lett. 11:2169-2175, 2016).

[0028] As used herein, the term "taxane anticancer agent" or "taxane anticancer drug" refers to diterpenoid taxane derivatives extracted from plants of the Taxus sp., which are mitotic inhibitors that act by promoting the assembly and inhibiting the disassembly of microtubules in cells. Currently, commonly used drugs include paclitaxel and docetaxel, among which paclitaxel is a taxane anticancer drug extracted from the pericarp of Taxus brevifolia and was approved by the US FDA in 1992 as a treatment for intractable ovarian cancer, and docetaxel is a taxane anticancer drug derived from Taxus baccata, which has a similar efficacy to paclitaxel and is used to treat breast cancer, non-cell lung cancer, lymphoma, bladder cancer, etc., and has a higher hydrophilicity than paclitaxel. Recently, it has been revealed that taxane anticancer drugs also have a mechanism of promoting immunogenic cell death of cancer cells by reducing the sensitivity of cancer cells to cytotoxic T lymphocytes.

[0029] The term "immune checkpoint inhibitor" as used herein refers to a type of drug that blocks certain proteins produced by certain types of immune system cells, such as T lymphocytes, and some cancer cells. These proteins suppress immune responses and prevent T lymphocytes from killing cancer cells. Therefore, if such proteins are blocked, the "brakes" of the immune system are released, allowing T lymphocytes to kill cancer cells more effectively. The well-known "immune checkpoints" to date include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. PD-1 inhibitors include Pembrolizumab (trade name: Keytruda) and Nivolumab (trade name: Opdivo), and PD-L1 inhibitors, which are the ligands of PD-1, include Atezolizumab (trade name: Tecentriq) and Avelumab (trade name: Bavencio). Meanwhile, CTLA-4 inhibitors such as ipilimumab (trade name: Yervoy), which block the CTLA-4 / B7-1 / B7-2 interaction, have been approved by the FDA. In recent years, they have shown impressive success, especially in patients with metastatic melanoma or Hodgkin lymphoma, and have shown a lot of promise in clinical trials targeting patients with other types of cancer.

[0030] Detailed description of the invention: According to one aspect of the present invention, there is provided a recombinant plasma membrane-based endoplasmic reticulum having a VSV-G mutant protein introduced into the membrane, in which the 162nd amino acid, histidine, is replaced by arginine.

[0031] The recombinant plasma membrane-based vesicles are exosomes, extracellular vesicles or cell-derived nanovesicles.

[0032] The recombinant plasma membrane-based vesicles have been isolated and purified from mammalian cells, preferably human cells, which have been transformed with a genetic construct containing a polynucleotide encoding the VSV-G mutant protein and overexpress the VSV-G mutant protein.

[0033] The recombinant plasma membrane-based vesicles are obtained from cells transformed to express the VSV-G mutant protein.

[0034] According to another aspect of the present invention, a recombinant plasma membrane-based endoplasmic reticulum is provided in which a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced by arginine is introduced into the membrane and one or more immunogenic cell death inducers are incorporated inside.

[0035] The incorporation of a drug into the recombinant plasma membrane-based vesicles can be achieved by culturing cells genetically engineered to produce recombinant plasma membrane-based vesicles in a cell culture medium in which the drug is dissolved, and the isolated recombinant plasma membrane-based vesicles can be produced by placing the isolated recombinant plasma membrane-based vesicles in a solvent in which the immunogenic cell death inducer is dissolved, and treating with ultrasound to reconstruct the plasma membrane-based vesicles (Kim et al., Nanomedicine, 12(3):655-664, 2016). Alternatively, when a drug is loaded into the plasma membrane-based vesicles, a method of simply mixing the isolated plasma membrane-based vesicles with the drug in a suitable solvent or medium and then stirring and mixing for a suitable time can be used (Sun et al., Mol. Ther. 18(9):1606-1614, 2010), or in the case of a hydrophilic drug such as a nucleic acid, electroporation can be used to incorporate the drug into the plasma membrane-based vesicles.

[0036] The immunogenic cell death inducer is an anthracycline anticancer drug, a taxane anticancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide anticancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, measles virus, bleomycine, mitoxantrone, or oxaliplatin. The cardiac glycoside is used in combination with a non-immunogenic cell death inducer, the GADD34 / PP1 inhibitor is used in combination with mitomycin, the anthracycline anticancer drug is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarbicin, or barbicin, the taxane anticancer drug is paclitaxel or docetaxel, and the anti-EGFR antibody is cetuximab.

[0037] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising the recombinant plasma membrane-based vesicle as an active ingredient.

[0038] According to another aspect of the present invention, there is provided a pharmaceutical composition for cancer treatment comprising as an active ingredient a recombinant plasma membrane-based endoplasmic reticulum having a virus-derived membrane fusogenic membrane protein introduced into the membrane.

[0039] In the composition, the viral fusogenic membrane protein is vesicular stomatitis virus VSV-G protein, gibbon ape leukemia virus GALV.fus, influenza virus hemagglutinin, respiratory cytoplasmic virus F protein, human immunodeficiency virus gp120 or gp41, flavivirus E protein, alphavirus E1 protein, baculovirus gp64, hepatitis C virus gp31 or gp70, measles virus H protein or F protein, or Ebola virus gp1 or gp2, and the VSV-G protein is a wild-type VSV-G protein or a pH-sensitive mutant VSV-G protein in which the 162nd amino acid, histidine, is replaced with arginine.

[0040] The composition may further comprise one or more anti-cancer compounds.

[0041] The anti-cancer compound is an immunogenic cell death inducer or an immune checkpoint inhibitor, and the immunogenic cell death inducer is an anthracycline anti-cancer drug, a taxane anti-cancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide anti-cancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone, or oxaliplatin. The cardiac glycoside is used in combination with a non-immunogenic cell death inducer, the GADD34 / PP1 inhibitor is used in combination with mitomycin, the anthracycline anti-cancer drug is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or barbicin, the taxane anti-cancer drug is paclitaxel or docetaxel, and the anti-EGFR antibody is cetuximab. On the other hand, the immune checkpoint inhibitor is a PD-1 / PD-L1 interaction inhibitor or a CTLA-4 / B7-1 / B7-2 interaction inhibitor, the PD-1 / PD-L1 interaction inhibitor is an antibody targeting PD-1 or PD-L1, or a functional fragment of the antibody or a single-chain-based antibody analogue, the CTLA-4 / B7-1 / B7-2 interaction inhibitor is an antibody targeting CTLA-4, B7-1 or B7-2, or a functional fragment of the antibody or a single-chain-based antibody analogue, the PD-1 or PD-L1 targeting antibody is Pembrolizumab, Nivolumab, Atezolizumab or Avelumab, and the CTLA-4 / B7-1 / B7-2 interaction inhibitor is Ipilimumab. Whereas the functional fragment of the antibody is Fab, F(ab')2, or Fab', the single chain based antibody analogue is an scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR), nanobody or camelid antibody heavy chain fragment (VLR). H H).

[0042] Meanwhile, in the composition, the anticancer compound is encapsulated inside the plasma membrane-based endoplasmic reticulum, and may be simply mixed and formulated, or may be packaged separately and mixed immediately before use, or may be administered simultaneously or at different times. However, when the immune checkpoint inhibitor is an antibody, a functional fragment of an antibody, or a single-chain-based antibody analog, it is encapsulated inside the exosome, but can function by simply being administered in combination with the recombinant plasma membrane-based endoplasmic reticulum derived from the cell of the present invention, or by being presented on the membrane surface of the recombinant plasma membrane-based endoplasmic reticulum. When the antibody, the functional fragment of the antibody, or the antibody analogue based on a single chain is presented on the membrane surface of a recombinant plasma membrane-based vesicle, the recombinant plasma membrane-based vesicle on which the immune checkpoint inhibitor according to one embodiment of the present invention is presented on the membrane surface can be produced by transducing a gene encoding the antibody into a host cell using a genetic recombination technique in the same manner as the virus-derived membrane fusion protein used in the present invention, expressing the gene so as to be presented on the membrane surface of the host cell, and then secreting or budding from the host cell, or by artificially processing the cell to obtain a nano-sized vesicle. In this case, the antibody, the functional fragment of the antibody, or the antibody analogue based on a single chain is genetically recombined to have a separate membrane translocation domain or anchoring domain for presentation on the cell membrane surface. Alternatively, instead of genetically recombining the antibody to have the membrane translocation domain or membrane anchoring domain, it is also possible to use IgM or IgD, which is an antibody that is not secreted but is in a membrane-bound form from the beginning.

[0043] In the case of the composition, the recombinant plasma membrane-based vesicle and the immunogenic cell death inducer may be provided in the form of a premixed composition, packaged separately and mixed and administered immediately before use, or may be administered separately at regular intervals.

[0044] The pharmaceutical composition of the present invention may include a pharma- ceutically acceptable carrier. The composition including a pharma- ceutically acceptable carrier may be in various oral or parenteral dosage forms, but is preferably in a parenteral dosage form. When formulated, it is prepared using a diluent or excipient such as a typical filler, extender, binder, wetting agent, disintegrant, surfactant, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0045] The pharmaceutical composition may have any one of the dosage forms selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.

[0046] The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it may be administered by various routes such as intravenous injection, intranasal inhalation, intramuscular administration, intraperitoneal administration, and transdermal absorption.

[0047] The compositions of the present invention are administered in a pharma- ceutically effective amount.

[0048] In the present invention, the term "pharmacologically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level may be determined based on factors including the type and severity of the individual, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention is administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 to 500 mg / kg. Meanwhile, the dosage may be appropriately adjusted depending on the age, sex, and condition of the patient.

[0049] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, or may be administered in combination with other anticancer drugs, and may be administered sequentially or simultaneously with conventional anticancer drugs. It may be administered singly or multiple times. It is important to administer an amount that provides maximum efficacy with minimal side effects while taking into consideration all of the above factors, and this can be easily determined by those skilled in the art.

[0050] According to another aspect of the present invention, there is provided use of a recombinant plasma membrane-based endoplasmic reticulum having a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced by arginine, or a recombinant plasma membrane-based endoplasmic reticulum having a virus-derived membrane fusogenic membrane protein in which the VSV-G mutant protein is introduced into the membrane, in the production of a cancer therapeutic agent.

[0051] According to another aspect of the present invention, there is provided a method for treating cancer in an individual, comprising administering to said individual the recombinant plasma membrane-based vesicle or any one or more of the pharmaceutical compositions described above.

[0052] In the cancer treatment method, the individual is a human or non-human mammal.

[0053] The present inventors hypothesized that when cancer cells are treated by introducing a low pH fusogenic mutant (H162R) of the VSV-G protein, a membrane fusogenic membrane protein, onto the surface of exosomes, membrane fusion is not induced under typical physiological conditions (pH 7.4) but is induced under conditions similar to the microenvironment of cancer tissues (pH 6.8), and that such membrane fusion can kill cancer cells, and that since the VSV-G H162R mutant protein itself is a pathogen associated molecular pattern (PAMP), it enhances the phagocytosis of phagocytes and the cross-prime ability of dendritic cells (see Figure 1), and therefore produced recombinant exosomes that display the VSV-G H162R mutant protein on their surface (see Figures 2A to 5B). In fact, it was found that the recombinant exosomes prepared as described above selectively induce membrane fusion of cancer cells at pH 6.8 in in vitro experiments (see Figures 6A to 7B), but do not induce cancer cell death in cancer cells by themselves (see Figure 7C). In addition, the recombinant exosomes (mVSVG-Exo) according to one embodiment of the present invention promote phagocytosis of various cancer cells (4T1-Luc, EL4-Ova, and CT26.CL25) by macrophages and dendritic cells (see Figures 8A to 8C), and it was confirmed that this is a phenomenon caused by VSVG acting as a TLR4 agonist (see Figure 9). In fact, when administered to tumor model animals, it was confirmed that the growth of cancer cells was significantly suppressed without side effects such as weight loss (see Figures 10A to 11C), and it was confirmed by flow cytometry that VSV-G protein was significantly expressed on the cell surface of tumor tissues excised from the experimental animals (see Figure 12). The above results suggest that the recombinant exosomes of the present invention are multifunctional anticancer agents that exhibit anticancer activity through multiple mechanisms.

[0054] In addition, the present inventors performed various analyses to determine the anti-cancer mechanism of the recombinant plasma membrane-based endoplasmic reticulum of the present invention, and as a result, it was confirmed that the recombinant exosome according to one embodiment of the present invention not only increased the cancer-specific antigen expression of dendritic cells (see FIG. 13A) and promoted the maturation of dendritic cells (see FIG. 13B to FIG. 13C), but also increased the infiltration of CD8 T cells into tumor tissues (see FIG. 13D and FIG. 13E). In addition, the recombinant exosome according to one embodiment of the present invention significantly improved the cross-reducing ability of dendritic cells in cancer tissues (see FIG. 14A), and it was confirmed that such cancer-specific immune ability is induced by cancer-specific antigens, and thus has immune memory ability (see FIG. 14B).

[0055] Furthermore, the present inventors conducted animal experiments using nude mice lacking T cell immunity to confirm whether the recombinant exosome according to one embodiment of the present invention is dependent on T cell immunity. As a result, it was confirmed that the anti-cancer effect disappeared (FIG. 15A), and that the anti-cancer effect was observed when the drug dosage was doubled (FIG. 15B). However, in an animal experiment using BATF3 knockout mice lacking CD103 and CD8 dendritic cells, which play an important role in T cell immunity, no anti-cancer activity was observed even in the recombinant exosome administration group of the present invention, and it was confirmed that the recombinant exosome according to one embodiment of the present invention exhibits anti-cancer activity through dendritic cells (FIG. 15D). In order to investigate the role of CD8 T cells in the anti-cancer effect of the recombinant exosome according to one embodiment of the present invention, an animal experiment using wild-type mice in which CD8 was neutralized using an anti-CD8 antibody was conducted. It was confirmed that the anti-cancer activity of the recombinant exosome according to one embodiment of the present invention was eliminated when the anti-CD8 antibody was administered to remove CD8 T cells (FIG. 15C). These results indicate that the recombinant exosome according to one embodiment of the present invention not only enhances the innate immune response against cancer cells by inducing the maturation of dendritic cells in an individual in the tumor microenvironment, but also enhances the adaptive immune response against cancer antigens by promoting the infiltration of CD8 T cells into tumor tissues, thereby exhibiting anti-cancer activity.

[0056] Moreover, the present inventors have experimentally confirmed that recombinant exosomes containing wild-type VSV-G protein, not pH-sensitive VSV-G protein, in their membranes also exhibit anti-cancer activity by inducing T cell-specific immune responses, although the effect is somewhat reduced. Even if a virus-derived membrane fusogenic membrane protein is not presented on the surface of cancer cells through fusion of cancer cells with recombinant exosomes in the cancer microenvironment, the VSV-G protein itself can act as a TLR4 agonist, and it has been determined that the T cell-specific immune response induced by recombinant exosomes containing wild-type VSV-G protein in their membranes is an effect induced by activating phagocytes through binding to the TLR4 receptor of phagocytes. Therefore, it can be expected that membrane fusogenic membrane proteins derived from other viruses other than VSV-G protein can also exhibit similar effects to the wtVSVG exosomes or mVSVG exosomes used in the present invention. In fact, it has been reported that viral membrane fusion proteins, such as the GALV.fus protein derived from gibbon ape leukemia virus, enhance the anticancer effect of oncolytic herpes simplex virus (Fu et al., Mol. Ther. 7(6):748-754, 2003).

[0057] The above results were achieved without the use of other anticancer drugs, and are expected to show strong synergistic effects when administered in combination with other immunogenic cell death inducers such as doxorubicin. Therefore, the recombinant exosome according to one embodiment of the present invention is expected to be very useful in developing new anticancer therapeutic agents that exhibit strong anticancer effects while minimizing the side effects of conventional anticancer drugs.

[0058] Furthermore, in one embodiment of the present invention, even if an anticancer effect is confirmed using recombinant exosomes, cell-derived plasma membrane-based vesicles such as extracellular vesicles and cell-derived nanovesicles, which have a structure similar to that of recombinant exosomes, are also expected to exhibit the same function as the recombinant exosomes according to one embodiment of the present invention when they are produced from the transformed host cells by a conventional method, i.e., by genetic recombination, after transforming the host cells so that they contain a virus-derived membrane fusogenic membrane protein in their membranes. EXAMPLES

[0059] The present invention will be described in more detail with reference to the following examples and experimental examples. However, the present invention is not limited to the following examples and experimental examples, and may be embodied in various different forms. The following examples are provided to fully disclose the present invention and to fully convey the scope of the invention to those skilled in the art.

[0060] Example 1: Production of the VSV-G H162R construct In order to prepare recombinant exosomes containing the VSV-G H162R mutant protein, the present inventors first prepared a gene construct encoding the H162R mutant protein in which the histidine, which is the amino acid residue at position 162 of VSV-G (position 178 based on the protein disclosed in GenBank No. CAC47944), is replaced with arginine. Specifically, for this purpose, a plasmid DNA (pCMV-VSV-G Envelope Vector, RV-110, Cell Biolabs, hereinafter abbreviated as 'VSV-G construct') containing a polynucleotide (SEQ ID NO: 2) encoding the wild-type VSV-G protein (SEQ ID NO: 1) was used as a template to generate a gene construct encoding the H162R mutant protein, which is the amino acid residue at position 162 of VSV-G (position 178 based on the protein disclosed in GenBank No. CAC47944) in which the histidine is replaced with arginine. [ka] and the reverse primer set forth in SEQ ID NO:4 [ka] Site-directed mutagenesis was performed using (the bold code corresponds to the mutated amino acid, arginine).

[0061] Example 2: Production of recombinant exosomes containing VSV-G H162R The present inventors transfected HEK293T cells with the pCMV-VSV-G H162R plasmid vector (hereinafter, abbreviated as 'VSV-G H162R construct', Fig. 2A and Fig. 2B) containing the gene (SEQ ID NO: 6) encoding the VSV-G H162R protein (SEQ ID NO: 5) prepared in Example 1, and then cultured for 48 hours. The cell culture medium was then collected and centrifuged sequentially at 300g for 10 minutes, 2,000g for 10 minutes, and 10,000g for 30 minutes, filtered using a 0.2 μm filter, and then ultrafiltered again at 150,000g for 3 hours to collect the pellet (Fig. 3).

[0062] Next, in order to confirm whether the VSV-G H162R mutant protein is contained in the exosomes, a portion of the collected exosomes was disrupted and then subjected to Western blot analysis using an anti-VSV-G antibody (Abcam, ab50549) and an anti-Alix antibody (exosome marker, Santacruz, sc99010) (FIG. 4). As a result, as shown in FIG. 4, the VSV-G H162R mutant protein was detected in both the transformed HEK293T cells and the recombinant exosomes obtained therefrom. FIG. 4 also shows the results of Western blot analysis for the exosome markers Alix, CD63, and TSG 101. This means that the VSV-G H162R mutant protein is normally contained in the exosomes derived from the cells transformed to express the VSV-G H162R mutant protein according to one embodiment of the present invention.

[0063] Next, the inventors photographed the collected recombinant exosomes using a transmission electron microscope (FIG. 5A), while analyzing the particle size of the recombinant exosomes using a dynamic light scattering (DLS) analyzer (Malvern Zetasizer Nano ZS, UK) (FIG. 5B). As a result, as shown in FIG. 5B, it was confirmed that both the recombinant exosomes (mVSVG-Exo) produced according to one embodiment of the present invention and the control exosomes (Con-Exo) had a very narrow size spectrum of about 80 nm.

[0064] Comparative Example: Production of wtVSVG Exosomes As a comparative example, the inventors prepared a genetic construct to express wild-type VSV-G protein in which the 162nd amino acid is not mutated, and then prepared recombinant exosomes (wtVSVG-Exo) in which the wild-type VSV-G protein is present in the membrane using the method of Example 2.

[0065] Example 3: Preparation of doxorubicin-encapsulated recombinant exosomes To load the recombinant exosomes prepared in Example 2 with doxorubicin (DOX), an immunogenic cell death inducer, purified exosomes (~10 11 Exosomes) are first mixed with DOX in 1 mL PBS. The DOX-recombinant exosome mixture is then sonicated using a Model 505 Sonic Dismembrator with a 0.25 inch tip at 20% amplitude, 30 seconds on / off and 2 minutes cooling between cycles for a total of 6 revolutions. After sonication, the Exo-DOX solution is incubated at 37°C for 60 minutes to recover the exosome membranes. Excess free drug is removed by size exclusion chromatography using a NAP-10 Sephadex G25 column (GE Healthcare, Buckinghamshire, UK).

[0066] Experimental Example 1: Analysis of whether VSV-G H162R fuses with cancer cells in vitro The present inventors attempted to confirm whether the recombinant exosomes prepared in Example 2 actually fuse with cancer cells under in vitro conditions.

[0067] To this end, specifically, the present inventors used 5 × 10 cells each of the 4T1-Luc mouse breast cancer cell line, the EL4-Ova lymphoma cell line, and the CT26.CL25 colon cancer cell line. 5 The cells were placed in 1 ml of fusion buffer (1.8 mM NAH2PO4, 8.4 mM Na2HPO4, 10 mM HEPES, 10 mM MES, 2.5 mM NaCl, pH adjusted to 7.4, 6.8 or 5.5 with hydrochloric acid) and fused with 50 μg mVSVG-Exo or wtVSVG-Exo (exosomes made using pCMV-wild-type VSVG plasmid vector) at 37 ° C for 10 minutes, washed with culture medium, and stabilized in culture medium at 37 ° C for 1 hour. At this time, the pH of the fusion buffer was divided into groups of pH 7.4, pH 6.8, or pH 5.5. Next, the degree of fusion of mVSVG-Exo or wtVSVG-Exo to the cancer cell surface was evaluated by anti-VSVG antibody staining using a flow cytometer. As a result of the experiment, it was confirmed that wtVSVG-Exo can fuse to the surface of the cancer cell membrane at only pH 5.5, i.e., cancer cell membrane editing is possible for all cancer cells, whereas mVSVG can fuse to the surface of the cancer cell membrane not only at pH 5.5 but also at 6.8, which is the pH of the tumor microenvironment (Figure 6A). In addition, 3 × 10 4T1-Luc cells were cultured in a 4-well chamber under the same conditions one day before. 4 The cells were seeded at 100x the pH of the fusion buffer and treated with mVSVG-Exo under the same conditions as above. The pH of the fusion buffer was divided into groups of 7.4 and 6.8. The cells were then stained with anti-VSVG antibody (red) and anti-Cadherin antibody (green), which can stain cell membranes, and photographed using a confocal fluorescence microscope. As a result, it was confirmed that mVSVG-Exo was unable to fuse with the surface of the cancer cell membrane at pH 7.4, but was able to fuse with the surface at pH 6.8 (Figure 6B).

[0068] Meanwhile, LDLR (low-density lipoprotein receptor) is the most well-known cell membrane receptor to which VSVG binds to fuse with the cell membrane. Thus, the present inventors investigated the LDLR expression level by Western blot analysis using an anti-LDLR antibody for various cells, including cancer cells, to which recombinant exosomes according to one embodiment of the present invention are fused. As a result, as shown in FIG. 6C, it was confirmed that LDLR is not expressed in normal cells such as bone marrow-derived macrophages, bone marrow-derived dendritic cells, and spleen cells, but is expressed in cancer cells such as 4T1-Luc, EL4-Ova, and CT26.CL25, suggesting that the mVSVG-Exo produced targets and fuses only to cancer cells (FIG. 6C).

[0069] Experimental example 2: Whether recombinant exosomes promote fusion between cancer cells Next, the present inventors attempted to confirm whether mVSVG-Exo promotes fusion between cancer cells after it is fused to the surface of the cancer cells. To this end, 1 × 10 5 The cells were stained with 1 μM deep red or 1 μM green CMFDA, treated with mVSVG-Exo as described in Experimental Example 1, and fusion was performed under conditions of pH 7.4 or pH 6.8. The cells were then seeded on a 35-well culture dish, and after 24 hours, the degree of cancer cell fusion (Green CMFDA Signal, Deep red signal double positive cells) was examined by flow cytometry. As a result, as shown in Figures 7A and 7B, in the case of 4T1-Luc and CT26.CL25, it was confirmed that cell-cell fusion was significantly promoted by mVSVG-Exo at pH 6.8 rather than pH 7.4, and in the case of EL4-Ova, fusion tended to increase, even if it was not significant.

[0070] Next, the inventors investigated whether mVSVG-Exo itself induces cancer cell death. To this end, specifically, breast cancer cell line 4T1-Luc, lymphoma cell line EL4-Ova, and colon cancer cell line CT26.CL25 were fused by treating them with mVSVG-Exo, control exosomes (Con-Exo), or buffer (treatment with fusion buffer only without exosomes) using pH 7.4 or pH 6.8 fusion buffer as described in Experimental Example 1. Next, 5 × 10 3 The cells were seeded in a 96-well plate with normal medium, and 24 hours later, cell viability was measured by CCK assay. As a result, as shown in Figure 7C, there was no difference in cell viability in all experimental groups, suggesting that mVSVG-Exo does not directly induce cancer cell death.

[0071] Experimental Example 3: Analysis of phagocytosis against cancer cells The present inventors performed a phagocytosis assay to determine whether the fusogenic exosomes according to one embodiment of the present invention promote phagocytosis of cancer cells by macrophages and dendritic cells.

[0072] Specifically, the present inventors used breast cancer cell line 4T1-Luc, lymphoma cell line EL4-Ova, colon cancer cell line CT26.CL25 5×10 5The cells were fused using mVSVG-Exo, control exosomes (Con-Exo), or buffer (treated with fusion buffer only without exosomes) at pH 7.4 or pH 6.8 in the same manner as described in Experimental Example 1. After the cancer cell membrane editing, the cancer cells were stained with pH rodo SE at 120 ng / ml. Bone marrow derived macrophages and bone marrow derived dendritic cells stained with green CMFDA 1 μM were co-cultured with cancer cells stained with pH rodo SE at a ratio of 1:2 for 2 hours. Next, the extent to which the macrophages and dendritic cells phagocytosed the cancer cells was confirmed using a fluorescent microscope. As a result of the experiment, as shown in Figures 8A to 8C, it was confirmed that the phagocytosis of cancer cells by phagocytes (bone marrow derived macrophages and bone marrow derived dendritic cells) was increased only in the mVSVG-Exo treatment group in which the cancer cell membrane was edited at pH 6.8, unlike the other groups. Furthermore, when cancer cell membrane editing was performed using mVSVG-Exo at pH 6.8 and then preblocking was performed using an anti-VSVG antibody (indicated as "pre" on the graph), the enhanced phagocytosis disappeared, demonstrating that the phagocytosis-enhancing effect of the recombinant exosome according to one embodiment of the present invention is dependent on mVSVG.

[0073] This suggests that the fusogenic exosomes of the present invention exhibit anti-cancer activity based on multiple functions, not only by promoting the fusion of cancer cells but also by promoting phagocytosis by macrophages and dendritic cells against cancer cells, thereby exerting an anti-cancer effect.

[0074] Experimental Example 4: Investigating the mechanism by which recombinant exosomes promote the maturation of dendritic cells VSV-G protein is known to be a TLR4 agonist. Therefore, the present inventors investigated whether the recombinant exosome according to one embodiment of the present invention promotes the maturation of dendritic cells through the TLR4 pathway. To this end, 1×10 bone marrow-derived dendritic cells were cultured in a 6-well culture dish. 61000g were seeded on the cells and treated with 500ng of mVSVG-Exo or 500ng of Con-Exo for 24 hours. The cells were then separated and centrifuged, stained with anti-CD40 and anti-CD86 antibodies, which reflect the maturation of dendritic cells, and the maturation of dendritic cells was evaluated using a flow cytometer. As a result of the experiment, as shown in Figure 9, it was confirmed that mVSVG-Exo increased the expression levels of CD40 and CD86 in dendritic cells, which means that mVSVG-Exo can promote the maturation of dendritic cells.

[0075] Experimental Example 5: Analysis of in vivo anticancer effects Based on the results of Experimental Examples 1 to 4, the present inventors investigated whether the recombinant exosome according to one embodiment of the present invention can suppress the growth of cancer cells under in vivo conditions.

[0076] Specifically, the present inventors administered 100 μg of the recombinant exosomes (mVSVG-Exo) obtained in Example 2 or the control exosomes (Con-Exo) to 1×10 mouse 4T1-Luc breast cancer cells. 6 The mice were subcutaneously inoculated on the back (day 0) and 7-week-old Balb / c wild-type mice (female, n=21) were injected with cancer cells into the tumors on days 5 and 6. The control group was administered only PBS. The size of the cancer tissues and the weights of the experimental animals were then confirmed at 3-day intervals until day 16 after the cancer cell inoculation (Figures 10A and 10C). After 16 days from the cancer cell inoculation, all the experimental animals were sacrificed, and the cancer tissues were excised and weighed (Figure 10B).

[0077] The present inventors also inoculated 1×10 EL4-Ova cancer cells into the left dorsal flank of C57BL / 6 wild-type mice. 6The mice were subcutaneously inoculated with 100 μg of wtVSVG-Exo, 100 μg of mVSVG-Exo, 100 μg of Con-Exo, or PBS was administered intratumorally on days 6 and 7 after cancer cell inoculation. The size of the tumor and the weight of the experimental animals were measured at 3-day intervals (FIGS. 11A and 11C), and on day 18 after cancer cell injection, the mice were sacrificed and the tumor tissues were removed and weighed (FIG. 11B).

[0078] As shown in Figures 10A to 11C, the results of the experiment showed that the comparison groups, wtVSVG-Exo-administered group and mVSVG-Exo-administered group, showed significant anti-cancer effects compared to the control group, and in particular, the mVSVG-Exo-administered group showed the best anti-cancer effect compared to the other groups.

[0079] Experimental Example 6: Correlation between anticancer activity and introduced VSV-G The present inventors investigated the expression level of mVSV-G protein on the surface of tumor cells to confirm whether the in vivo anticancer effect of the recombinant exosome according to one embodiment of the present invention shown in Experimental Example 5 is due to the action of the mVSV-G protein introduced into the exosome. To this end, specifically, 1×10 EL4-Ova cancer cells were injected into the left back of C57BL / 6 wild-type mice. 6 After the cancer cell injection, the tumor size was 100 mm 3 When the tumor volume reached 100μg, 100μg of wtVSVG-Exo, 100μg of mVSVG-Exo, 100μg of Con-Exo, or PBS was administered intratumorally by injection. Two hours after administration, the cancer tissue was excised, isolated into single cells, and subjected to flow cytometry with staining using anti-VSVG antibody (Figure 12). As a result of the experiment, as shown in Figure 12, it was confirmed that VSVG was expressed on the surface of the cancer cell membrane only in the mVSVG-Exo group.

[0080] The above results demonstrate that the in vivo anti-cancer activity of the recombinant exosome according to one embodiment of the present invention is caused by the VSVG protein present in the exosome membrane being transferred to the cancer cell membrane upon fusion of the recombinant exosome with cancer cells in the tumor microenvironment.

[0081] Experimental Example 7: Study of the mechanism of anti-cancer action of recombinant exosomes 7-1: Investigation of effects on dendritic cells The present inventors investigated whether the recombinant exosome according to one embodiment of the present invention exhibits anti-cancer activity by activating the function of dendritic cells. To this end, 1×10 EL4-Ova cancer cells were injected into the left back of C57BL / 6 wild-type mice. 6 100μg of wtVSVG-Exo, 100μg of mVSVG-Exo, 100μg of Con-Exo, or PBS was administered intratumorally on the 6th and 7th days after the cancer cell injection (the day of cancer cell injection is considered as day 0). On the 18th day after the cancer cell injection, the mice were sacrificed and the cancer tissue and tumor-draining lymph nodes were excised. Next, to analyze the degree of cancer antigen expression by dendritic cells, the single-celled tumor-draining lymph nodes were stained with anti-CD11c antibody, a dendritic cell marker, and anti-H-2kb Ova antibody, which can measure the cancer-specific antigen ovalbumin loaded on MHC-1, and then analyzed by flow cytometer (Figure 13A). As a result, as shown in Figure 13A, the mVSVG-Exo-administered group showed the highest degree of cancer-specific antigen expression by dendritic cells. In addition, to analyze the maturation level of dendritic cells, single-celled tumor-draining lymph nodes were stained with anti-CD11c antibody, a dendritic cell marker, and anti-CD40 and anti-CD86 antibodies capable of evaluating the maturation level of dendritic cells, and then analyzed by a flow cytometer (FIGS. 13B and 13C). As a result of the experiment, it was confirmed that both the wtVSVG-Exo and mVSVG-Exo groups were able to promote the maturation of dendritic cells. Therefore, it was found that the effect of the recombinant exosome according to one embodiment of the present invention in promoting the function of dendritic cells is due to the function of VSVG itself, unrelated to the fusion of the recombinant exosome to cancer cells.

[0082] 7-2: Investigation into the effect of CD8 T cells on infiltration into cancer tissue The present inventors investigated the effect of the recombinant exosome according to one embodiment of the present invention on the infiltration of CD8 T cells into cancer tissues. To this end, specifically, the cancer tissues excised from the experimental animals in Experimental Example 7-1 were embedded in an OCT compound and frozen, and then frozen slices were prepared and stained with an anti-CD8 antibody, and the infiltration level of CD8 T cells into the cancer tissues was comparatively analyzed using a fluorescent microscope (FIGS. 13D and 13E). As a result, the mVSVG-Exo administration group showed the highest infiltration level of CD8 T cells. In particular, there was no difference between the wtVSVG-Exo administration group and the control group and the control exosome administration group, and it was found that the CD8 T cell infiltration-promoting activity of the recombinant exosome according to one embodiment of the present invention is not only the function of VSVG itself but also the effect of fusion with cancer cells in the tumor microenvironment.

[0083] 7-3: Analysis of cross-cropping capacity The present inventors investigated whether the recombinant exosomes according to one embodiment of the present invention have the cross-reactivity to CD8 T cells. To this end, 1×10 EL4-Ova cancer cells were injected into the left dorsal flank of C57BL / 6 wild-type mice. 6100μg of wtVSVG-Exo, 100μg of mVSVG-Exo, 100μg of Con-Exo, or PBS was administered intratumorally on the 6th and 7th days after the cancer cell injection (the day of cancer cell injection is considered as day 0). On the 18th day after the cancer cell injection, the mice were sacrificed and the cancer tissues and spleen tissues were excised. To evaluate the cross-prime ability of macrophages and dendritic cells, dendritic cells (CD11c-positive cells) and macrophages (F4 / 80-positive cells) were separated from the single-celled cancer tissues using F4 / 80 or CD11c magnetic particles. Next, OT-1 CD8 T cells were isolated from the spleen of OT-1 transgenic mice that had OT-1 CD8 T cells capable of recognizing ovalbumin loaded on MHC1 using a CD8 T cell column, and the OT-1 CD8 T cells were co-cultured with dendritic cells or macrophages isolated from cancer tissues at a ratio of 1:5 in culture medium for 3 days. After 3 days, the medium was collected and ELISA analysis was performed using anti-IFN-γ antibody for each group to analyze the expression level of INF-γ (Figure 14A). As a result, it was confirmed that the cross-reducing ability of dendritic cells isolated from cancer tissues of rats treated with mVSVG-Exo was significantly improved compared to other groups.

[0084] 7-4: Immune memory capacity survey The present inventors investigated whether the recombinant exosome according to one embodiment of the present invention can memorize cancer antigens and trigger a cancer-specific immune response in the case of recurrence of the same cancer after the completion of cancer treatment. To this end, the spleen tissues extracted from the experimental animals in Experimental Example 7-3 were separated into single cells, and then 5×10 6The cells were seeded in 12-well culture dishes with 1 ml of culture medium and treated with PBS or 10 μg / ml of egg albumin, a cancer-specific antigen, for 24 hours. The medium was then collected and the expression level of IFN-γ in the medium was analyzed by ELISA analysis using an anti-IFN-γ antibody for each group (Figure 14B). As a result, as shown in Figure 14B, a significant increase in cancer antigen-specific immune response was observed in the wtVSVG-Exo-administered group and the mVSVG-Exo-administered group, and in particular, the best cancer antigen-specific immune response was observed in the mVSVG-Exo-administered group.

[0085] 7-5: Identification of immune cells related to the anti-cancer activity of recombinant exosomes The present inventors attempted to investigate what immune cells the recombinant exosomes according to one embodiment of the present invention are dependent on. First, in order to confirm whether anticancer effects due to enhanced phagocytosis of phagocytes are observed in nude mice lacking T cell immunity, EL4-Ova cancer cells (1×10 6 10 pieces were subcutaneously injected into the left back of nude mice to induce cancer, and on the 6th day (the day of cancer cell injection is considered as day 0) and the 7th day after the cancer cell injection, 100 μg of mVSVG-Exo, 100 μg of Con-Exo, or PBS was administered by injection into the tumor. The size of the tumor was measured at 3-day intervals, and on the 18th day after the cancer cell injection, the mice were sacrificed and the cancer tissues were excised and weighed (FIG. 15A). As a result, as shown in FIG. 15A, it was confirmed that the anti-cancer effect of mVSVG-Exo shown in C57BL / 6 mice in which T cells were present disappeared in nude mice, and thus it can be seen that the anti-cancer effect of the recombinant exosome according to one embodiment of the present invention is dependent on T cell immunity. In order to confirm whether the anti-cancer effect due to the enhancement of phagocytosis of phagocytes is also shown in nude mice lacking T cell immunity, 1×10 EL4-Ova cancer cells were injected into the nude mice at 3 days after the cancer cell injection. 6The tumor was induced by subcutaneous injection of mVSVG-Exo, Con-Exo or PBS into the tumor on the 5th day (the day of tumor cell injection is considered as day 0), 6th day, 7th day and 8th day after tumor cell injection (2 times more than other experiments). The tumor size was measured at 3-day intervals, and on the 17th day after tumor cell injection, the mouse was sacrificed and the tumor tissue was excised and weighed (FIG. 15B). As a result, as shown in FIG. 15B, it was confirmed that the anti-cancer effect of mVSVG-Exo was also observed in nude mice when the existing drug dosage was doubled and the experiment was further progressed when the tumor was small. This is presumed to be because the recombinant exosome of the present invention induces an innate immune response by promoting the phagocytosis of phagocytes in addition to increasing the infiltration ability of CD8 T cells.

[0086] Next, to confirm what type of T cells the effect of mVSVG-Exo is dependent on, the inventors injected 1 × 10 EL4-Ova cancer cells into the left back of C57BL / 6 wild-type mice. 6 The tumor was induced by subcutaneous injection of mVSVG-Exo, Con-Exo, or PBS was administered intratumorally on the 6th and 7th days (the day of cancer cell injection is considered as day 0) after the cancer cell injection. Next, 150 μg of anti-CD8 neutralizing antibody was intraperitoneally administered at 3-day intervals from 1 day before the cancer cell injection to remove CD8 T cells. The size of the tumor was measured at 3-day intervals, and on the 18th day after the cancer cell injection, the mouse was sacrificed and the tumor tissue was excised and weighed (Figure 15C). As a result, as confirmed in Figure 15C, it was confirmed that the anti-cancer effect of mVSVG-Exo shown in C57BL / 6 mice with preserved T cell immunity disappeared in mice without CD8 T cells. This suggests that the anti-cancer effect of mVSVG-Exo is dependent on CD8 T cell immunity.

[0087] Finally, we injected 1 × 10 EL4-Ova cancer cells into the left back of BATF3 knockout mice, which lack CD103 and CD8 dendritic cells, which play the most important role in forming T cell immunity. 6100μg of mVSVG-Exo, 100μg of Con-Exo, or PBS was administered intratumorally on the 6th and 7th days after the cancer cell injection (the day of cancer cell injection is considered as day 0). The size of the tumor was measured at 3-day intervals, and on the 18th day after the cancer cell injection, the mouse was sacrificed and the tumor tissue was excised and weighed (FIG. 15D). As a result, as shown in FIG. 15D, it was confirmed that the anti-cancer effect of mVSVG-Exo shown in C57BL / 6 mice in which CD103 and CD8 dendritic cells were present was abolished in BATF3 knockout mice, and thus it was found that the anti-cancer effect of the recombinant exosome according to one embodiment of the present invention is dependent on CD103 and CD8 dendritic cells, and T cell immunity.

[0088] The results suggest that the recombinant exosomes containing the VSV-G H162R mutant protein according to one embodiment of the present invention promote a specific anti-cancer immune effect under the pH conditions of the microenvironment of cancer tissues. The anti-cancer effect of the recombinant exosomes containing the VSV-G H162R mutant protein was achieved without the aid of other anti-cancer drugs, and a significant synergistic effect is expected when other anti-cancer drugs, particularly immunogenic cell death inducers, are encapsulated or co-treated. In particular, when the anti-cancer drug is incorporated into the recombinant exosomes according to the embodiments of the present invention, which are membrane structures, the recombinant exosomes can deliver the incorporated anti-cancer drug to the inside of the cancer cells by specific fusion with the cancer cells, thereby minimizing side effects caused by the anti-cancer drug acting on general cells. [Industrial Applicability]

[0089] Therefore, the recombinant plasma membrane-based endoplasmic reticulum containing the recombinant exosome according to one embodiment of the present invention not only has anticancer activity by itself, but is also expected to have even stronger anticancer activity when used in combination with other anticancer drugs, and therefore can be very usefully utilized in the development of new anticancer drugs that exhibit strong effects while minimizing side effects. [Sequence List Free Text]

[0090] SEQ ID NO:1 is the amino acid sequence of the wild-type VSV-G protein.

[0091] SEQ ID NO:2 is the nucleic acid sequence of a polynucleotide encoding the wild-type VSV-G protein.

[0092] SEQ ID NO:3 is the nucleic acid sequence of the forward primer used to clone a polynucleotide encoding the wild-type VSV-G protein.

[0093] SEQ ID NO:4 is the nucleic acid sequence of the reverse primer used to clone the polynucleotide encoding the wild-type VSV-G protein.

[0094] SEQ ID NO:5 is the amino acid sequence of the H162R mutant VSV-G protein according to one embodiment of the present invention.

[0095] SEQ ID NO:6 is the nucleic acid sequence of a polynucleotide encoding the H162R mutant VSV-G protein.

[0096] SEQ ID NO: 7 is the amino acid sequence surrounding the histidine 162nd amino acid of the wild-type VSV-G protein.

[0097] SEQ ID NO:8 is the amino acid sequence surrounding the arginine 162nd amino acid of the H162R mutant VSV-G protein.

[0098] SEQ ID NO: 9 is the nucleic acid sequence of a polynucleotide encoding a peptide surrounding the histidine, which is the 162nd amino acid, of the wild-type VSV-G protein.

[0099] SEQ ID NO: 10 is the nucleic acid sequence of a polynucleotide encoding a peptide surrounding the arginine 162nd amino acid of the H162R mutant VSV-G protein.

[0100] The present invention has been described with reference to the embodiments and experimental examples, but these are merely illustrative, and those skilled in the art will understand that various modifications and equivalent embodiments and experimental examples are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the claims.

[0101] The present disclosure includes the following aspects. [Appendix 1] A recombinant plasma membrane-based endoplasmic reticulum in which a mutant VSV-G protein in which the 162nd amino acid, histidine, is replaced by arginine has been introduced into the membrane. [Appendix 2] 2. The recombinant plasma membrane-based vesicle of claim 1, which is an exosome, an extracellular vesicle or a cell-derived nanovesicle. [Appendix 3] The recombinant plasma membrane-based endoplasmic reticulum according to claim 1, which has been transformed with a genetic construct comprising a polynucleotide encoding the VSV-G mutant protein, and isolated and purified from a mammalian cell that overexpresses the VSV-G mutant protein. A recombinant plasma membrane-based endoplasmic reticulum in which a VSV-G mutant protein in which the 162nd amino acid, histidine, is replaced by arginine is introduced into the membrane and one or more immunogenic cell death inducers are incorporated inside. [Appendix 5] The recombinant plasma membrane-based endoplasmic reticulum according to claim 4, wherein the immunogenic cell death inducer is an anthracycline anticancer drug, a taxane anticancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide anticancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone or oxaliplatin. [Appendix 6] A pharmaceutical composition for cancer treatment comprising the recombinant plasma membrane-based vesicle according to any one of claims 1 to 5 as an active ingredient. [Appendix 7] 7. The pharmaceutical composition for treating cancer according to claim 6, further comprising one or more anti-cancer compounds. [Appendix 8] 8. The pharmaceutical composition for cancer treatment according to claim 7, wherein the anticancer compound is an immunogenic cell death inducer or an immune checkpoint inhibitor. [Appendix 9] The pharmaceutical composition for cancer treatment according to claim 8, wherein the immunogenic cell death inducer is an anthracycline anticancer drug, a taxane anticancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide anticancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone or oxaliplatin. [Appendix 10] 10. The pharmaceutical composition for cancer treatment according to claim 9, wherein the anthracycline anticancer drug is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarbicin, or barbicin. [Appendix 11] 10. The pharmaceutical composition for cancer treatment according to claim 9, wherein the taxane anticancer drug is paclitaxel or docetaxel. [Appendix 12] 10. The pharmaceutical composition for cancer treatment according to claim 9, wherein the anti-EGFR antibody is cetuximab. [Appendix 13] A pharmaceutical composition for cancer treatment comprising as an active ingredient a recombinant plasma membrane-based endoplasmic reticulum having a viral-derived membrane fusogenic membrane protein introduced into the membrane. [Appendix 14] The pharmaceutical composition for cancer treatment described in Appendix 13, wherein the plasma membrane-based vesicle is an exosome, an extracellular vesicle or a cell-derived nanovesicle. [Appendix 15] The composition for cancer treatment described in Appendix 13, wherein the viral membrane fusogenic membrane protein is vesicular stomatitis virus VSV-G protein, gibbon ape leukemia virus GALV.fus, influenza virus hemagglutinin, respiratory cytoplasmic virus F protein, human immunodeficiency virus gp120 or gp41, flavivirus E protein, alphavirus E1 protein, baculovirus gp64, hepatitis C virus gp31 or gp70, measles virus H protein or F protein, or Ebola virus gp1 or gp2. [Appendix 16] 16. The pharmaceutical composition for cancer treatment according to any one of claims 12 to 15, further comprising one or more anti-cancer compounds. [Appendix 17] 17. The pharmaceutical composition for cancer treatment according to claim 16, wherein the anticancer compound is an immunogenic cell death inducer or an immune checkpoint inhibitor. [Appendix 18] 18. The pharmaceutical composition for cancer treatment according to claim 17, wherein the immunogenic cell death inducer is an anthracycline anticancer drug, a taxane anticancer drug, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide anticancer drug, a GADD34 / PP1 inhibitor, LV-tSMAC, measles virus, bleomycin, mitoxantrone or oxaliplatin. [Appendix 19] 19. The pharmaceutical composition for cancer treatment according to claim 18, wherein the anthracycline anticancer drug is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarbicin, or barbicin. [Appendix 20] The taxane anticancer drug according to claim 18, which is paclitaxel or docetaxel. A pharmaceutical composition for treating cancer. [Appendix 21] The pharmaceutical composition for cancer treatment according to claim 18, wherein the anti-EGFR antibody is cetuximab. The pharmaceutical composition for cancer treatment described in Appendix 17, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 interaction inhibitor or a CTLA-4 / B7-1 / B7-2 interaction inhibitor. [Appendix 23] 23. The pharmaceutical composition for cancer treatment described in Appendix 22, wherein the PD-1 / PD-L1 interaction inhibitor is an antibody targeting PD-1 or PDL1, or a functional fragment of the antibody, or a single-chain-based antibody analogue. [Appendix 24] 24. The pharmaceutical composition for cancer treatment according to claim 23, wherein the antibody targeting PD-1 or PDL1 is pembrolizumab, nivolumab, atezolizumab or avelumab. [Appendix 25] 23. The pharmaceutical composition for cancer treatment described in Appendix 22, wherein the CTLA-4 / B7-1 / B7-2 interaction inhibitor is an antibody targeting CTLA-4, B7-1 or B7-2, or a functional fragment of said antibody or a single-chain-based antibody analogue. [Appendix 26] 26. The pharmaceutical composition for cancer treatment according to claim 25, wherein the CTLA-4 / B7-1 / B7-2 interaction inhibitor is ipilimumab. [Appendix 27] 26. The pharmaceutical composition for cancer treatment according to claim 23 or 25, wherein said single chain based antibody analogue is an scFv, sdAb, diabody, monobody, variable lymphocyte receptor (VLR), nanobody or camelid heavy chain fragment (VHH). [Appendix 28] 17. The pharmaceutical composition for cancer treatment according to claim 16, wherein the anticancer compound is encapsulated inside the recombinant exosome. [Appendix 29] A method for treating cancer in an individual, comprising administering to said individual a therapeutically effective amount of a pharmaceutical composition for treating cancer described in Appendix 6. [Appendix 30] A method for treating cancer in an individual, comprising administering to said individual a therapeutically effective amount of a pharmaceutical composition for treating cancer according to claim 13.

Claims

1. A pharmaceutical composition for cancer treatment comprising: a recombinant plasma membrane-based endoplasmic reticulum having a VSV-G mutant protein, in which the 178th amino acid of the VSV-G protein shown in SEQ ID NO: 1 is substituted for histidine with arginine, introduced into the membrane as an active ingredient; and a pharma- ceutical acceptable carrier, The recombinant plasma membrane-based endoplasmic reticulum is an exosome. The pharmaceutical composition for treating cancer.

2. The pharmaceutical composition for cancer treatment described in claim 1, wherein the endoplasmic reticulum is transformed with a gene construct containing a polynucleotide encoding the VSV-G mutant protein and is isolated and purified from a mammalian cell overexpressing the VSV-G mutant protein.

3. 1. Use of a pharmaceutical composition comprising a recombinant plasma membrane-based vesicle in the manufacture of a medicament for the treatment of cancer, comprising: A VSV-G mutant protein in which the 178th amino acid, histidine, of the VSV-G protein shown in SEQ ID NO:1 is replaced with arginine is introduced into the membrane, The recombinant plasma membrane-based endoplasmic reticulum is an exosome. The above uses.

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