WO2024019441A1 - Cell derived vesicle and liposome fusion hybrid nanoparticles and use thereof - Google Patents

Abstract

The present invention relates to the fusion technology of cell-derived vesicles and liposomes, and hybrid nanoparticles fabricated by the fusion technology, wherein the hybrid nanoparticles can exhibit both a specific tissue-targeting function and a drug delivery function. Specifically, the present inventors have discovered optimal conditions for effectively fusing cell derived vesicles and liposomes. Under the conditions, it is possible to fuse active substance-expressing cell-derived vesicles to drug-encapsulated liposomes to produce hybrid nanoparticles possessing the functions of both of them, that is, dual-functional hybrid nanoparticles. Thus, the present invention allows the fabrication of a wide range of hybrid nanoparticles depending on the purpose. In particular, hybrid nanoparticles fabricated by fusing cell-derived vesicles containing tumor antigen-specific chimeric antigen receptors to liposomes carrying photosensitizers can effectively target cancer cells through the chimeric antigen receptors and can destroy cancer cells by generating reactive oxygen species in response to light stimulation by the photosensitizers. Therefore, the hybrid nanoparticles according to the present invention can effectively target the intended tissues and deliver drugs through the combination of various cell-derived vesicles and liposomes and are expected to be a promising therapeutic platform that can be utilized as drug carriers and therapeutic agents in various fields.

Description

Cell-derived vesicle and liposome fusion hybrid nanoparticles and uses thereof

The present invention relates to hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, their use, and their manufacturing method.

The present invention claims priority based on Korean Patent Application No. 10-2022-0088450 filed on July 18, 2022, and all contents disclosed in the specification and drawings of the relevant applications are incorporated in this application.

Extracellular vesicles are nano-scale membrane structures that mediate intercellular communication by transferring cellular substances such as proteins between cells. The endoplasmic reticulum can not only preserve the cell membrane and cytoplasmic intrinsic active substances, but also can carry new substances, thereby exerting various biological activities. In particular, if a protein with target tissue-specific binding ability is expressed on the surface of extracellular vesicles, the targeting ability of the target site in vivo can be maximized, so it is expected to be a next-generation drug delivery vehicle.

However, there are limitations to commercialization of natural endoplasmic reticulum because the amount secreted from cells is extremely small and considerable effort is required to collect and concentrate. Therefore, artificial cell derived vesicles (CDV), which can be easily obtained in large quantities through mass extrusion, are utilized. Cell-derived vesicles are used as analogs of natural extracellular vesicles such as exosomes and ectosomes in various research and industrial fields. Crude-CDV can be prepared by continuously extruding cells through increasingly smaller micron-sized pores or by repeatedly passing cells through a depth filter. Afterwards, large quantities of CDV can be easily obtained by separating and purifying impurities from Crude-CDV. Since the membrane proteins and lipids of CDV are derived from the plasma membranes or organelle membranes of CDV parent cells, the physiologically active molecules (proteins, lipids, sugars, etc.) expressed by the cells are stored in the membrane or inside the membrane. Includes. In addition, cell-derived vesicles have the advantage of maximizing therapeutic efficacy because they can additionally encapsulate or combine pharmaceuticals with various characteristics in addition to the function of active substances derived from cells. However, to date, there are limitations in effectively encapsulating drugs in cell-derived endoplasmic reticulum, and research for this purpose is being actively conducted.

Meanwhile, liposomes are self-assembled bilayer structures made of lipids and are amphipathic particles that have both a hydrophobic part and a hydrophilic part. Liposomes have the advantage of excellent biocompatibility, simple manufacturing method, and ability to transport both water-soluble and fat-soluble drugs. In addition, liposomes can be manufactured by changing the surface properties in various ways, so they can be used in various fields such as cosmetics, adjuvants, and drug delivery. However, liposomes have the potential for non-specific drug delivery and may cause unexpected side effects. Therefore, there is a need to develop new drug carriers that can improve the target targeting ability of liposomes.

To solve the above problems, the present inventors developed a technology that can effectively fuse cell-derived endoplasmic reticulum and drug-encapsulated liposomes, and the hybrid nanoparticles produced through this process target specific tissues through proteins expressed in cell-derived endoplasmic reticulum. It was confirmed that targeting is possible and that the drug contained in liposomes can be delivered to target cells to exert physiological activity.

Therefore, the purpose of the present invention is to provide a method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.

Another object of the present invention is to provide hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating various diseases, comprising the hybrid nanoparticles as an active ingredient.

Another object of the present invention is to provide a drug delivery system containing the hybrid nanoparticles as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

The present invention provides a method for producing hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused, comprising the step of mixing cell-derived vesicles (CDV) and liposomes to induce their fusion. .

In one embodiment of the present invention, the fusion may be performed in 10 to 50 (v/v)% C ₁ to C ₅ alcohol solvent, but is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle and the liposome may be mixed at a ratio of 1 to 30:1 (cell-derived vesicle:liposome), but the mixture is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle may be obtained by extruding a sample containing cells into micropores, but is not limited thereto.

In another embodiment of the present invention, the diameter of the micropores may be 0.1 to 20 μm, but is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle may satisfy one or more characteristics selected from the group consisting of, but is not limited to:

(a) The diameter of cell-derived vesicles is 100 to 300 nm; and

(b) The surface charge of cell-derived vesicles is -50 to -10 mV.

In another embodiment of the present invention, the cell-derived vesicle may include a tumor antigen-specific protein, but is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle may include a chimeric antigen receptor, but is not limited thereto.

In another embodiment of the present invention, the liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), It may include, but is not limited to, one or more selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

In another embodiment of the present invention, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is used in a molar ratio of 1 to 20:1. ratio %), but is not limited to this.

In another embodiment of the present invention, the liposome may have a surface charge of 0 to 100 mV, but is not limited thereto.

In another embodiment of the present invention, the liposome may contain an active ingredient, but is not limited thereto.

Additionally, the present invention provides hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused.

In one embodiment of the present invention, the cell-derived vesicle may be manufactured by a manufacturing method including the step of extruding a sample containing cells into micropores, but is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle may include a chimeric antigen receptor on its surface, but is not limited thereto.

In another embodiment of the present invention, the cell-derived vesicle contains a chimeric antigen receptor on the surface, and the liposome may be loaded with an active ingredient, but is not limited thereto.

In another embodiment of the present invention, the hybrid nanoparticle may include the chimeric antigen receptor on its surface and the active ingredient between its lipid bilayers or in its internal compartment, but is not limited thereto.

In another embodiment of the present invention, the chimeric antigen receptor may be a tumor antigen-specific chimeric antigen receptor, but is not limited thereto.

In another embodiment of the present invention, the liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), It may include, but is not limited to, one or more selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). .

In another embodiment of the present invention, the active ingredients include peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, and viruses. , quantum dots, fluorochromes, and toxins, but is not limited thereto.

In another embodiment of the present invention, the active ingredients may be photosensitizers, but are not limited thereto.

In another embodiment of the present invention, the hybrid nanoparticle may satisfy one or more characteristics selected from the group consisting of, but is not limited to:

(a) the diameter of the hybrid nanoparticles is 100 to 300 nm; and

(b) The surface charge of the hybrid nanoparticle is -30 to +20 mV.

In another embodiment of the present invention, the hybrid nanoparticles may be manufactured by the manufacturing method of claim 1, but are not limited thereto.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent. do.

In addition, the present invention provides the prevention of cancer, comprising the step of administering a hybrid nanoparticle in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused to an individual in need thereof. Or provide a treatment method.

In addition, the present invention provides a use of hybrid nanoparticles in the prevention or treatment of cancer in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused.

In addition, the present invention provides the use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused for the production of a drug for treating cancer.

In one embodiment of the present invention, the hybrid nanoparticle may include the tumor antigen-specific chimeric antigen receptor on its surface and the anticancer agent between its lipid bilayers or in its internal compartment, but is not limited thereto.

In another embodiment of the present invention, the anticancer agent may be one or more selected from the group consisting of a chemical anticancer agent, a targeted anticancer agent, an immunological anticancer agent, and a photosensitizer, but is not limited thereto.

In another embodiment of the present invention, the anticancer agent is a photosensitizer, and the hybrid nanoparticle may generate active oxygen in response to light stimulation, but is not limited thereto.

In another embodiment of the present invention, the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, and intraocular melanoma. Tumor, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

Additionally, the present invention provides a kit for preventing or treating cancer, including the pharmaceutical composition.

In addition, the present invention provides a drug delivery system comprising, as an active ingredient, a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome carrying an anticancer agent.

In addition, the present invention includes the step of administering hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and liposomes loaded with a drug (e.g., an anticancer agent) are fused to an individual in need thereof. Provides a drug delivery method.

In addition, the present invention provides a drug delivery use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a drug-loaded liposome are fused together.

In addition, the present invention provides the use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a drug-loaded liposome are fused for the production of a drug delivery system.

The present invention relates to a fusion technology of cell-derived vesicles and liposomes and to hybrid nanoparticles manufactured through the fusion technology, wherein the hybrid nanoparticles can exert both targeting functions and drug delivery functions to target tissues. Specifically, the present inventors discovered the optimal conditions to effectively fuse cell-derived vesicles and liposomes, and through this, cell-derived vesicles expressing active ingredients and drug-encapsulated liposomes were fused to possess all of their functions. Since hybrid nanoparticles, that is, dual-functional hybrid nanoparticles, can be manufactured, a wide range of types of hybrid nanoparticles can be manufactured depending on the purpose. In particular, hybrid nanoparticles prepared by fusing a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with a photosensitizer effectively target cancer cells through the chimeric antigen receptor, and the photosensitizer It can kill cancer cells by generating active oxygen in response to light stimulation. Therefore, the hybrid nanoparticle according to the present invention can effectively target target tissues and deliver drugs through the combination of various cell-derived vesicles and liposomes, and is expected to be a promising therapeutic platform that can be used as a drug delivery vehicle and therapeutic agent in various fields. It is expected.

Figure 1a shows a schematic diagram of the structure of a chimeric antigen receptor (CAR) and a schematic diagram of HEK93T cells expressing CAR according to an embodiment.

Figure 1b shows the results of confirming the CAR expression level of HEK293 cells transfected with CAR (EGFR-CAR) plasmid according to an example by flow cytometry.

Figure 1c shows the results of Western blot confirmation of the expression of CD3zeta, a component of CAR, to confirm CAR expression in HEK293 cells transfected with EGFR-CAR plasmid.

Figure 2a is a diagram showing the production process of cell-derived endoplasmic reticulum (CDV) derived from HEK293 cells expressing EGFR-CAR.

Figure 2b shows the results of measuring the surface charge of control CDV and EGFR-CAR expressing CDV through nanoparticle tracking analysis.

Figure 2c shows the results of measuring the size of control CDV and EGFR-CAR expressing CDV.

Figure 2d shows the results of confirming the expression of the EGFR-CAR component (CD3zeta) and endoplasmic reticulum marker protein (CD63) of control CDV or EGFR-CAR expressing CDV using Western blot.

Figure 2e shows the results of confirming the cytotoxicity of each CDV by measuring the cell survival rate after treating MDA-MB-231 cells with control CDV or EGFR-CAR expressing CDV, respectively.

Figures 2f and 2g show fluorescence images of MDA-MB-231 cells treated with control or EGFR-CAR expressing CDV (1 × 10 ⁷ particles) for 4 hours to confirm the cellular uptake rate of CDV (Figure 2f; scale Bar = 100 μm) and fluorescence level measurement results (Figure 2g).

Figure 3a is a schematic diagram showing FRET analysis to evaluate fusion between liposomes. FRET liposomes fused with unlabeled control liposomes have a reduced surface density of fluorophores.

Figure 3b shows the results of measuring the surface zeta potential of control liposomes and FRET liposomes through nanoparticle tracking analysis.

Figure 3c shows the results of measuring the sizes of control liposomes and FRET liposomes.

Figure 3d shows the results of confirming the fusion rate by measuring the degree of FRET after inducing fusion by reacting control liposomes and FRET liposomes for 1 hour (Excitation/Emission wavelength: 465/520 nm).

Figure 3e shows the results of confirming the fusion rate by measuring the degree of FRET after inducing fusion by reacting FRET liposomes and HEK293 CDV for 1 hour (Excitation/Emission wavelength: 465/520 nm).

Figure 4a shows the results of measuring the sizes of liposomes with different lipid compositions through nanoparticle tracking analysis.

Figure 4b shows the results of measuring the surface zeta potential of liposomes with different lipid compositions.

Figure 4c shows the results of confirming the fusion rate by inducing fusion by reacting liposomes with different lipid compositions and HEK293 CDV for 1 hour (Excitation/Emission wavelength: 465/520 nm).

Figure 4d is a transmission electron microscope image of liposome B fused with HEK293 CDV.

Figure 4e shows the results of confirming the presence or absence of cytotoxicity after treating fibroblast or breast cancer cell lines with liposome B fused with HEK293 CDV at various concentrations.

Figure 5a is a diagram showing the structure of a liposome loaded with a photosensitizer according to one embodiment.

Figure 5b shows the results of measuring the surface zeta potential of liposomes loaded with photosensitizer through nanoparticle tracking analysis.

Figure 5c shows the results of measuring the size of liposomes loaded with photosensitizer.

Figure 5d is a TEM image of a liposome loaded with a photosensitizer.

Figure 5e shows the results of measuring the amount of active oxygen over time after irradiating a laser to a liposome loaded with a photosensitizer.

Figure 5f shows the results of measuring the degree of cancer cell death depending on whether or not laser irradiation was performed after treating breast cancer cell lines with liposomes loaded with a photosensitizer at various concentrations.

Figure 6a shows the results of measuring the surface zeta potential of a dual-functional hybrid nanoparticle fused with a photosensitizer-loaded liposome and an EGFR-CAR expressing CDV.

Figure 6b shows the results of measuring the size of the dual-functional hybrid nanoparticles.

Figure 6c shows nanoparticles in which control liposomes and control CDV were fused to a breast cancer cell line; Alternatively, the results of measuring cell viability after treatment with control liposomes and EGFR-CDV fused nanoparticles are shown (scale bar = 100 μm).

Figures 6d and 6e show nanoparticles in which control liposomes and control CDV were fused to breast cancer cell lines; Alternatively, the control liposome and EGFR-CDV fused nanoparticles were treated for 4 hours each, and then the hybrid nanoparticles absorbed into the cells were obtained through fluorescence imaging (Figure 6d), and the detected fluorescence levels were compared. (Figure 6e).

Figure 6f shows the results of confirming the cancer cell death rate depending on whether or not laser irradiation was performed after treating the breast cancer cell line with dual-functional hybrid nanoparticles.

Figure 7a shows an experimental schedule for evaluating the function of dual-functional hybrid nanoparticles in a tumor animal model.

Figure 7b shows the results of measuring the levels of liver and kidney toxicity markers in the peripheral blood of a tumor animal model intravenously administered hybrid nanoparticles to evaluate the biocompatibility of the dual-functional hybrid nanoparticles.

Figure 7c shows the results of confirming the distribution of nanoparticles in a tumor animal model in which fluorescently labeled hybrid nanoparticles were administered intravenously to evaluate the tumor-targeting ability of the dual-functional hybrid nanoparticles.

Figure 7d shows the results of administering hybrid nanoparticles to a tumor animal model to evaluate the cancer cell killing ability of dual-functional hybrid nanoparticles and then confirming the degree of tumor growth with or without laser irradiation.

Figure 8 is a schematic diagram showing the manufacturing process, structure, and mechanism of action of the hybrid nanoparticle according to the present invention.

The present invention relates to a fusion technology of cell-derived vesicles and liposomes and hybrid nanoparticles produced through the same. The present inventors have discovered optimal conditions for effective fusion of cell-derived vesicles and liposomes, and the It was confirmed that the hybrid nanoparticle can exert a dual function by maintaining the functions of both cell-derived vesicles and liposomes.

In particular, according to the fusion technology according to the present invention, a cell vesicle carrying a targeting substance such as a chimeric antigen receptor and a liposome carrying a pharmacological/physiological active ingredient can be fused, so a hybrid is produced by fusing the two. Nanoparticles can effectively exert both target tissue targeting and drug delivery functions. Therefore, through the fusion technology according to the present invention, hybrid nanoparticles with appropriate active ingredients can be manufactured according to the purpose, so a wide range of hybrid nanoparticles can be manufactured and used for the treatment of various diseases.

Therefore, the present invention provides a method for producing hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused, which includes the step of mixing cell-derived vesicles (CDV) and liposomes to induce their fusion. The purpose is to provide

Preferably, the cell-derived vesicles according to the present invention are manufactured by a method including the step of extruding a sample containing cells into micropores, and are distinguished from natural extracellular vesicles produced by cells. . Preferably, the step of extruding into the micropores may be performed by sequentially extruding cells using micropores with large micropores and small micropores.

In the present invention, “Cell derived vesicles (CDV)” refers to vesicles artificially produced from nucleated cells. CDV can be produced by being released from the cell membrane in almost all types of cells, and is characterized by having a double phospholipid membrane form, which is the structure of the cell membrane. The cell-derived vesicles of the present invention are distinct from naturally secreted vesicles, such as exosomes. Throughout this specification, the term “vesicles” is distinguished between the inside and the outside by a lipid bilayer composed of the cell membrane components of the cell from which it is derived, and has the cell membrane lipids, membrane proteins, nucleic acids, and cell components, etc. It means that the size is smaller than the original cell, but is not limited to this.

The cell-derived vesicle according to the present invention has a size at the micrometer level. For example, the diameter of the CDV may be less than 0.2 μm. More specifically, the diameter of the CDV is 50 to 300 nm, 50 to 250 nm, 50 to 200 nm, 50 to 180 nm, 50 to 160 nm, 50 to 150 nm, 50 to 100 nm, 100 to 300 nm, 100 nm. It may be, but is limited to, 250 nm, 100 to 200 nm, 100 to 180 nm, 120 to 300 nm, 150 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 180 nm, or 130 to 170 nm. It doesn't work.

Additionally, the cell-derived vesicle according to the present invention may have a negative surface charge (<0 mV). That is, the surface charge of the cell-derived vesicles is -100 to -5 mV, -80 to -5 mV, -50 to -5 mV, -40 to -5 mV, -30 to -5 mV, -50 to -10. It may be mV, -50 to -15 mV, -50 to -20 mV, -40 to -20 mV, -35 to -20 mV, or -30 to -20 mV, but is not limited thereto.

In the present invention, the “cell” can be used without limitation as long as it is a cell capable of separating cell-derived vesicles, and includes all cells isolated from natural organisms. Preferably, the cells are nucleated cells. Additionally, the cells may be of plant origin or any type of animal, including human and non-human mammals. Therefore, there is no particular limitation on the type of cells from which the cell-derived vesicle according to the present invention can be obtained, but for example, stem cells, kidney cells, embryonic kidney cells, cancer cells, acinar cells, myoepithelial cells, red blood cells, immune cells, etc. The cell-derived vesicles can be obtained from cells, monocytes, dendritic cells, natural killer cells, T cells, B cells, macrophages, endothelial cells, epidermal cells, neurons, glial cells, astrocytes, muscle cells, and platelets. You can. It may be various types of immune cells, tumor cells, stem cells, acinar cells, myoepithelial cells, or platelets. Preferably, the stem cells include mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, and salivary gland stem cells. It may be any one or more selected from the group consisting of, and preferably may be adipocyte-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells, but is not limited thereto.

In the present invention, the vesicle is used to extrude a suspension containing nucleated cells, ultrasonic disintegration, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical disintegration, and physical stimulation by externally applying force to the cells. It can be manufactured using a method selected from the group consisting of treatments, but is not limited thereto. In the present invention, the vesicle was manufactured by extruding a suspension containing nucleated cells using an extruder. The extrusion force of the extruder applied to produce the nucleated cell-derived vesicle of the present invention may be 5 to 200 psi, 10 to 150 psi, 10 to 100 psi, 10 to 50 psi, 10 to 40 psi, or 50 to 100 psi. there is.

In addition, the vesicle according to the present invention can be obtained by extruding a sample containing cells into micropores. Preferably, the cells are sequentially extruded using those with micropores from large to small. It can be obtained by doing. The diameter of the micropores is 0.01 to 100 μm, 0.01 to 80 μm, 0.01 to 60 μm, 0.01 to 40 μm, 0.01 to 20 μm, 0.01 to 15 μm, 0.01 to 10 μm, 0.01 to 7 μm, 0.01 to 3 μm. , 0.01 to 1 μm, 0.01 to 0.5 μm, 0.01 to 0.1 μm, or 0.1 to 0.5 μm, but is not limited thereto. For example, the sequential extrusion may be performed using filters with pore diameters of 5 to 20 μm, 2 to 7 μm, 0.7 to 3 μm, and 0.1 to 0.5 μm. The size of the micropores can be appropriately adjusted depending on the type of cell used. CDV obtained through the above process may undergo additional purification.

Additionally, cell-derived vesicles may be prepared by removing the nucleus of the cell before extruding the cell-containing sample into the micropore. The cell nuclei can be removed through centrifugation.

Meanwhile, the vesicle according to the present invention can also be manufactured through a manufacturing method that includes the step of passing a sample containing cells through a depth filter and extruding it. The extrusion force of the depth filter may be 5 to 200 psi, 10 to 150 psi, 10 to 100 psi, 10 to 50 psi, 10 to 40 psi, or 50 to 100 psi, but is not limited thereto. CDV obtained through the above process may undergo additional purification.

The purification process is intended to remove other substances (vesicles smaller than the CDV of the present invention or other contaminants) in addition to the CDV of the present invention and obtain only the desired CDV, preferably by mixing cell-derived vesicles and liposomes. This can be done by adding 1 to 5 times, 1 to 4 times, or 1 to 3 times the volume of buffer solution compared to the total volume of the solution.

Specifically, CDV produced through the extrusion step is purified through a tangential flow filtration (TFF) process. The membrane used at this time preferably has a cut off of 500 kDa, 450 kDa, 400 kDa, 350 kDa, or 300 kDa or more, and a concentration step and buffer exchange step to concentrate the concentration of the CDV suspension to 5 times or more. is performed. Through the above process, CDV is concentrated and impurities are removed.

After obtaining CDV through the tangential flow filtration process, size exclusion chromatography may be further performed. At this time, it is preferable to use a column with a recovery range of about 35 to 350 nm or 70 to 1,000 nm. Since CDV corresponds to relatively large particles, CDV can be obtained by collecting the fraction eluted through the column. there is. Through this purification process, impurities such as vesicles and other proteins smaller than CDV can be removed.

In the present invention, the “sample containing cells” may be a sample containing nucleated cells or transformed cells thereof, and includes without limitation any cell capable of producing vesicles.

In the present invention, the filter used in the extrusion step can be used without limitation as long as it contains micropores and has a membrane structure capable of filtering samples, but is preferably a polycarbonate membrane filter. .

In addition, the cell-derived vesicle according to the present invention can be manufactured using a cell extruder including a support, an intermediate filter, and a membrane filter. A detailed description of the extruder can be found in Republic of Korea Patent Publication No. 10-2134906.

In one embodiment of the present invention, the production method may include the step of obtaining cell-derived vesicles from cells transformed or transfected with a recombinant vector. The cell-derived vesicle obtained at this time contains the expression product of the gene contained in the recombinant vector.

The recombinant vector is preferably a recombinant vector containing the gene to be expressed in the autologous cell-derived vesicle in which the present invention is to be practiced. Since the cell-derived vesicle according to the present invention is derived from the plasma membrane or organelle of the mother cell and may contain physiologically active molecules expressed by the mother cell, the mother cell is transformed/transfected with the desired gene, and then transformed/transfected. Vesicles derived from cells expressing the above genes can be obtained from infected cells.

There is no limitation to the type of the gene, and a person skilled in the art can introduce an appropriate gene into a cell according to the purpose and obtain a cell-derived vesicle containing the expression product of the gene therefrom. The types of expression products include proteins, peptides, glycoproteins, and nucleic acids. Preferably, the expression product is a protein targeting a specific tissue (i.e., a protein with tissue-specific binding ability). More preferably, the expression product is a protein targeting tumor tissue and/or cancer cells. The cancer cell and/or tumor tissue specific protein is not limited to a specific type and includes all ligands, receptors, signaling proteins, and antibodies or fragments thereof. In one embodiment of the present invention, the gene is a gene encoding a chimeric antigen receptor targeting tumor tissue or cancer cells.

In the present invention, “chimeric antigen receptor (CAR)” refers to synthetic receptors capable of targeting specific antigens. CAR according to the present invention is preferably a polypeptide comprising an antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain (cytoplasmic domain).

In the present invention, “antigen-binding domain” refers to a protein or polypeptide domain that can specifically recognize and bind to a target antigen. In the present invention, “antigen” refers to a polypeptide, compound, or substance that can specifically bind to humoral immune mediators such as antibodies or cellular immune mediators such as T cell receptors. . Preferably, the antigen may be a cancer cell-specific protein (i.e., expressed only in cancer cells, or particularly expressed or highly active in cancer cells), and more preferably, it may be a cancer cell-specific surface protein. That is, the antigen is a tumor antigen.

The tumor antigens are included without limitation as long as they are specifically expressed in cancer cells or have a particularly high expression level in cancer cells, and are not limited to specific types, but include EGFR, CD19, TAG72, IL13Rα2 (Interleukin 13 receptor alpha-2 subunit), CD52, CD33, CD20, TSLPR, CD22, CD30, GD3, CD171, NCAM (Neural cell adhesion molecule), FBP (Folate binding protein), Le(Y) (Lewis-Y antigen), PSCA (Prostate stem cell antigen), PSMA ( Prostate-specific membrane antigen), CEA (Carcinoembryonic antigen), HER2 (Human epidermal growth factor receptor 2), Mesothelin, CD44v6 (Hyaluronate receptor variant 6), B7-H3, Glypican-3, ROR1 (receptor tyrosine kinase like orphan receptor 1) ), Survivin, FOLR1 (folate receptor), WT1 (Wilm's tumor antigen), and VEGFR2 (Vascular endothelial growth factor 2). However, a person skilled in the art can select an appropriate tumor antigen known in the art according to the type of cancer to be treated and apply it to the present invention. Preferably, the chimeric antigen receptor according to the present invention is a chimeric antigen receptor having a breast cancer tumor antigen-specific antigen binding domain, and more preferably, is a chimeric antigen receptor having an EGFR-specific antigen binding domain. For example, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 6 or may include an antigen-binding domain consisting of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto. In addition, the EGFR-specific antigen binding domain may include the base sequence of SEQ ID NO: 1 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, “hinge region” refers to a region located between the antigen-binding domain and the transmembrane domain and serves as a flexible linker. The hinge region ensures that the antigen-binding domain is properly positioned when the antigen-binding domain binds to the antigen, thereby forming a stable bond with the antigen. The hinge region is not limited to a specific type, but may preferably be the CD8 hinge region. Preferably, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 7 or a hinge region consisting of the amino acid sequence of SEQ ID NO: 7, but is not limited thereto. Additionally, the hinge region may include the base sequence of SEQ ID NO: 2 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, “transmembrane domain (TM)” refers to any polypeptide or oligopeptide that functions to connect extracellular and intracellular signaling domains across the cell membrane. The transmembrane domain is not limited to a specific type, but is preferably a CD28 transmembrane domain. Preferably, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 8 or may include a transmembrane domain consisting of the amino acid sequence of SEQ ID NO: 8, but is not limited thereto. Additionally, the transmembrane domain may include the base sequence of SEQ ID NO: 3 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 3, but is not limited thereto.

In the present invention, “intracellular signaling domain (cytoplasmic domain) (or may also be referred to as endodomain (ED))” refers to a polypeptide or oligopeptide inside the cell membrane. In the present invention, the intracellular signaling domain may include one or more intracellular co-stimulatory domains. The “stimulatory signal domain” and “co-stimulatory domain” are any polypeptide or oligopeptide that transmits signals that cause activation or inhibition of biological processes within the cell. means. The intracellular signaling domain is not limited to a specific type, but may preferably consist of a CD28 signaling domain and/or a CD3zeta domain. Preferably, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10, or may include a signaling domain consisting of the amino acid sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10. , but is not limited to this. In addition, the signaling domain may include the base sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5, or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 4 and/or SEQ ID NO: 5, but is not limited thereto. .

In one embodiment of the present invention, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 12 or may be composed of the amino acid sequence of SEQ ID NO: 12, but is not limited thereto. Additionally, the chimeric antigen receptor may include the base sequence of SEQ ID NO: 11 or may be encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 11, but is not limited thereto.

In the present invention, a polypeptide (polynucleotide) consisting of an amino acid sequence (nucleic acid sequence) represented by a specific sequence number is not limited to the corresponding amino acid sequence (nucleic acid sequence), and variants of the amino acid sequence (nucleic acid sequence) are within the scope of the present invention. included within. The polypeptide molecule (polynucleotide molecule) of the amino acid sequence (nucleic acid sequence) of the present invention is a functional equivalent of the polypeptide molecule (polynucleotide molecule) constituting it, for example, some amino acid sequences (nucleic acid sequences) of the polypeptide molecule are deleted ( It is a concept that includes variants that have been modified by deletion, substitution, or insertion, but can have the same functional effect as the corresponding polypeptide (polynucleotide). Specifically, the polypeptide (polynucleotide) disclosed in the present invention contains at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 90% of the amino acid sequence (nucleic acid sequence) represented by a specific sequence number. In other words, it may include an amino acid sequence (nucleic acid sequence) having more than 95% sequence homology. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology. It includes polypeptides (polynucleotides) having . The “% sequence homology” for a polypeptide (polynucleotide) is determined by comparing a comparison region with two optimally aligned sequences, and the portion of the polypeptide sequence (polynucleotide sequence) in the comparison region is consistent with the optimal alignment of the two sequences. may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions).

In the production method according to the present invention, the cells are cells that express tumor antigen-specific proteins, and the cell-derived vesicles obtained therefrom may contain the same tumor antigen-specific proteins as those expressed in the cells. Preferably, the tumor antigen-specific protein is expressed on the surface of the cell-derived vesicle. Therefore, hybrid nanoparticles obtained by fusing a liposome and a cell-derived vesicle containing a tumor antigen-specific protein contain the same tumor antigen-specific protein as that contained in the cell-derived vesicle. Preferably, the tumor antigen-specific protein is a chimeric antigen receptor specific for a tumor antigen.

In the present invention, “liposomes” refers to a structure composed of a lipid membrane surrounding an aqueous internal compartment. The liposome membrane is mainly composed of phospholipids and their derivatives. When the phospholipids and their derivatives are dispersed in an aqueous solution, a single layer or lipid bilayers are spontaneously formed in the endoplasmic reticulum. Liposomes can carry water-soluble active ingredients in an aqueous internal space and can also carry hydrophobic active ingredients in a lipid bilayer, so they can be used as carriers for various drugs.

The liposome according to the present invention preferably contains 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1, It may include one or more selected from the group consisting of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

Preferably, the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is 1 to 20:1, 1 to 15:1, 1 It may be included in a molar ratio (molar ratio %) of 1 to 10:1, 1 to 8:1, 1 to 5:1, 1 to 4:1, or 2 to 5:1. In particular, the liposome containing DOPC and DOTAP is characterized by a positive charge on the surface.

In addition, the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS): 1,2-dioleoyl-sn-glycero -3-phosphocholine (DOPC) is 1 to 10:1 to 10:1, 1 to 8:1 to 10:1, 1 to 10:1 to 8:1, 1 to 5:1 to 10:1, 1 to 10:1. It may be included in a molar ratio (molar ratio %) of 5:1 to 5:1, or 1 to 3:1 to 3:1.

In addition, the 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is 1: 1 to 20, 1: 1 It may be included in a molar ratio (molar ratio %) of 1 to 15, 1:1 to 10, 1:1 to 8, or 1:1 to 5.

Preferably, the liposome according to the present invention may have a positive surface charge (>0 mV). That is, the liposome has a surface charge of 0 to 100 mV, 0 to 80 mV, 0 to 60 mV, 0 to 50 mV, 0 to 45 mV, 10 to 100 mV, 10 to 50 mV, 20 to 60 mV, and 20 to 50 mV. It may be 50 mV, 30 to 50 mV, or 30 to 40 mV, but is not limited thereto. Through specific examples, the present inventors confirmed that effective fusion occurred when cell-derived vesicles with a negative charge on the surface were mixed with liposomes with a positive charge on the surface.

In addition, the diameter of the liposome according to the present invention is 100 to 500 nm, 100 to 400 nm, 100 to 350 nm, 100 to 320 nm, 100 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 190 nm, It may be 200 to 500 nm, 200 to 400 nm, 200 to 350 nm, 250 to 500 nm, 250 to 400 nm, 250 to 350 nm, 280 to 400 nm, 280 to 350 nm, or 280 to 320 nm. It is not limited.

In one embodiment of the present invention, the liposome may be prepared through a production method comprising the following steps:

(S1) dissolving lipids in an organic solvent;

(S2) preparing a liposome membrane by evaporating the organic solvent; and

(S3) Hydrating the liposome membrane in an aqueous solution.

In one embodiment, the organic solvent may be one or more selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether, but is not limited thereto.

In one embodiment, the aqueous solution may be phosphate-buffered saline, but is not limited thereto.

Preferably, the method for producing liposomes may further include (S4) treating the hydrated liposome membrane with ultrasound.

Preferably, the liposome according to the present invention may contain an active ingredient. The active ingredient is not limited to a specific type, and any one skilled in the art can be applied as long as it exerts the desired effect and can be loaded into liposomes. Non-limiting examples include peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, and quantum dots. , fluorochrome, and toxin, but is not limited thereto. The active ingredient may be supported inside the liposome or between lipid bilayers. Therefore, the hybrid nanoparticle obtained by fusing the liposome carrying the active ingredient and the cell-derived vesicle may contain the same active ingredient as that carried in the liposome. Preferably, the active ingredient is supported between the lipid bilayers or in the internal compartment of the hybrid nanoparticle.

In the method for producing hybrid nanoparticles according to the present invention, fusion of cell-derived vesicles and liposomes is characterized in that 10 to 50 (v/v)% C ₁ to C ₅ alcohol solvent is used. Preferably, the alcohol solvent is a C ₁ to C ₅ , C ₁ to C ₃ , or C ₁ to C ₂ alcohol solvent. Most preferably, the alcohol solvent is ethanol. Preferably, the concentration of the alcohol (ethanol) is 10 to 50 (v/v)%, 10 to 45 (v/v)%, 10 to 40 (v/v)%, 10 to 35 (v) compared to the total mixed solution. /v)%, 20 to 50 (v/v)%, 20 to 40 (v/v)%, 20 to 35 (v/v)%, 25 to 50 (v/v)%, 25 to 40 (v) /v)%, 25 to 35 (v/v)%, or 27 to 32 (v/v)%, but is not limited thereto. Specifically, the fusion can be achieved by adding alcohol to a mixed solution of cell-derived vesicles and liposomes to a final concentration of 10 to 50 (v/v)%. The fusion occurs 10 minutes to 180 minutes, 10 minutes to 150 minutes, 10 minutes to 120 minutes, 10 minutes to 100 minutes, 10 minutes to 80 minutes, and 10 minutes to 65 minutes after adding alcohol to the mixed solution of cell-derived vesicles and liposomes. minutes, 20 minutes to 100 minutes, 30 minutes to 100 minutes, 40 minutes to 100 minutes, 50 minutes to 100 minutes, or 50 minutes to 80 minutes, but is not limited thereto. Through specific examples, the present inventors have found that fusion of liposomes and cell-derived vesicles occurs more effectively when using alcohol rather than using sonication or electroporation, especially at a final concentration of 10 to 50 (v/v)% compared to the total reaction solution. It was confirmed that the most effective fusion occurred under alcohol conditions. In other words, the fusion technology of liposomes and cell-derived vesicles according to the present invention is more effective in fusing liposomes and cell-derived vesicles compared to the electroporation or sonication technology used for the fusion of liposomes or vesicles. It is characterized by the ability to fuse. In addition, the fusion technology according to the present invention can more easily produce hybrid nanoparticles in which liposomes and cell-derived vesicles are fused using an alcohol solvent. In particular, it does not require special substances such as other catalysts and can be produced using only an alcohol solvent. Since liposomes and cell-derived vesicles can be effectively fused, it is economically and time-effective as it does not require impurity removal processes.

Additionally, in the method for producing hybrid nanoparticles according to the present invention, cell-derived vesicles and liposomes can be mixed at a number ratio of 1 to 30:1 (cell-derived vesicles: liposomes). Specifically, the cell-derived vesicle:liposome is 1 to 30:1, 1 to 25:1, 1 to 20:1, 1 to 15:1, 1 to 10:1, 5 to 30:1, 5 to 20. : 1, 5 to 15 : 1, 5 to 10 : 1, 7 to 10 : 1, or 8 to 10 : 1, but is not limited thereto.

In the present invention, 'effective fusion' means that a hybrid nanoparticle obtained by fusion of a cell-derived vesicle and a liposome is manufactured into one structure containing both the cell-derived vesicle-derived component and the liposome-derived component. do.

Additionally, the present invention provides hybrid nanoparticles in which cell-derived vesicles and liposomes are fused.

Specific descriptions of the cell-derived vesicle and the liposome are as described above.

The hybrid nanoparticle is a fusion of cell-derived vesicles and liposomes, and is characterized by containing both components derived from each of the cell-derived vesicles and liposomes. For example, the membrane of the hybrid nanoparticle may be composed of the membrane components of the cell-derived vesicle and the liposome. Preferably, the membrane of the hybrid nanoparticle is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1, It may include one or more selected from the group consisting of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), preferably 1,2- It may include dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

In particular, hybrid nanoparticles prepared by fusing cell-derived vesicles expressing a specific protein and liposomes loaded with a specific active ingredient contain both the protein and the active ingredient, and thus can exert all of their activities and functions. (i.e., dual-functional hybrid nanoparticles). Preferably, the protein derived from the cell-derived vesicle is located on the surface of the hybrid nanoparticle, and the active ingredient derived from the liposome is trapped between the lipid bilayers of the hybrid nanoparticle or stored in an internal compartment inside the lipid bilayer. It may be possible, but it is not limited to this.

In one embodiment of the present invention, the hybrid nanoparticle can be manufactured by fusing a cell-derived vesicle containing a chimeric antigen receptor and a drug-loaded liposome. Therefore, the hybrid nanoparticle can be produced by fusing the chimeric antigen receptor with a liposome loaded with the drug. and all of the above drugs. Preferably, in the hybrid nanoparticle, the chimeric antigen receptor is located on the surface of the nanoparticle, and the drug can be captured within the lipid bilayer of the nanoparticle or encapsulated in an internal compartment inside the lipid bilayer.

Preferably, the chimeric antigen receptor may be a tumor antigen-specific chimeric antigen receptor that specifically binds to the tumor antigen. Therefore, hybrid nanoparticles obtained by fusing a cell-derived vesicle containing the tumor antigen-specific chimeric antigen receptor with a liposome also contain the tumor antigen-specific chimeric antigen receptor, thereby killing cancer cells and tumor tissues. It can be targeted.

Hybrid nanoparticles targeting cancer cells through the chimeric antigen receptor can be introduced into target cells and deliver drugs (derived from liposomes) loaded on the nanoparticles to the corresponding cells. At this time, the drug is preferably an anticancer agent.

The diameter of the hybrid nanoparticle according to the present invention is 100 to 300 nm, 100 to 250 nm, 100 to 200 nm, 100 to 180 nm, 100 to 150 nm, 130 to 300 nm, 130 to 250 nm, 130 to 200 nm, It may be 130 to 180 nm, 150 to 300 nm, 150 to 250 nm, 150 to 200 nm, or 150 to 180 nm, but is not limited thereto.

In addition, the hybrid nanoparticle according to the present invention has a surface charge of -30 to +20 mV, -25 to +20 mV, -20 to +20 mV, -15 to +20 mV, -30 to +10 mV, -25 mV. to +10 mV, -20 to +10 mV, -15 to +10 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -15 to 0 mV, -30 to -5 mV, It may be -25 to -5 mV, -20 to -5 mV, or -15 to -5 mV, but is not limited thereto.

Preferably, the hybrid nanoparticles may be manufactured using the method for producing hybrid nanoparticles according to the present invention.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent. do.

Specific descriptions of the cell-derived vesicles, liposomes, and hybrid nanoparticles prepared by fusing them are as described above.

The hybrid nanoparticle may contain a tumor antigen-specific chimeric antigen receptor derived from the cell-derived vesicle on the surface, and an anticancer agent derived from the liposome between lipid bilayers or in an internal compartment. Therefore, the hybrid nanoparticle can target cancer cells/tumor tissues through the chimeric antigen receptor and deliver the anticancer agent to the cancer cells/tumor tissues.

The anticancer agent may be one or more selected from the group consisting of chemical anticancer agents, targeted anticancer agents, immune anticancer agents, and photosensitizers, but is not limited thereto, as long as it exerts an anticancer effect and can be carried in liposomes or hybrid nanoparticles of the present invention. May be included without limitation. For example, the anticancer drugs include doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, floxuridine, and vinorelbine. , gemcitabine, leuprolide, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozocin, valubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, It may be one or more selected from the group consisting of doxyfluridine, vinblastine, mitomycin, prednisone, afinitor, and mitoxantrone, but is not limited thereto.

Preferably, the anticancer agent is a photosensitizer. In the present invention, “photosensitizer” refers to a drug that exhibits specific activity in response to light stimulation. Preferably, the photosensitizer refers to a substance that generates reactive oxygen by chemically reacting with light stimulation and oxygen. Therefore, hybrid nanoparticles containing the photosensitizer generate active oxygen only under light stimulation, so they do not exert cytotoxicity in the absence of light stimulation, but when exposed to light stimulation, they generate active oxygen and cause cell death. . In other words, hybrid nanoparticles containing a photosensitizer selectively exert an anticancer effect, thereby significantly lowering the risk of side effects due to non-specific anticancer activity. In the present invention, the photosensitizer may be any agent that can generate active oxygen in response to photostimulation, but is preferably porphyrin, temoporfi (THPC), or verteporphine. (Verteporfin), Rostaporfin, Talaporfin sodium, Padeliporfin, Chlorin e6, Zinc Pthalocyanine, Pheophorbide a , and sulftalanzinc (Suftalanzinc), etc.

Additionally, the photosensitizer may generate active oxygen in response to light stimulation with a wavelength of 500 to 800 (nm), but is not limited thereto. More specifically, the photosensitizer may have a wavelength of 500 to 800, 500 to 750, 500 to 700, 500 to 680, 600 to 800, 600 to 750, 600 to 700, or 600 to 680 (nm).

Additionally, the pharmaceutical composition according to the present invention may be administered sequentially or simultaneously with the photostimulation treatment, but is not limited thereto.

In the present invention, “cancer” includes both solid cancer and blood cancer. In one embodiment of the present invention, the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, and intraocular melanoma. , uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma , urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and It may be one or more selected from the group consisting of pituitary adenoma, but is not limited thereto. Preferably, the cancer may be characterized as a cancer expressing EGFR. Here, the chimeric antigen receptor contained in the hybrid nanoparticle is characterized as being a chimeric antigen receptor specific for an antigen that is specifically expressed in the target cancer or is expressed at a particularly high level in the cancer.

The content of the hybrid nanoparticles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight, based on the total weight of the composition. It may be %, but is not limited to this. The content ratio is a value based on the dry amount with the solvent removed.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, paste preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease. Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and body weight, and is generally administered at 0.001 to 150 mg, preferably 0.01 to 100 mg, per kg of body weight every day or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

Additionally, the present invention provides a kit for preventing or treating cancer comprising the pharmaceutical composition. In addition to the composition containing the hybrid nanoparticles, the kit according to the present invention may include other components, compositions, solutions, devices, etc. commonly required for the prevention or treatment of cancer. For example, the kit may further include cell-derived vesicles, liposomes, etc. for producing the hybrid nanoparticles, and may further include instructions containing information related to the hybrid nanoparticles (e.g., manufacturing method, etc.). there is.

In addition, the present invention provides a drug delivery system comprising, as an active ingredient, a hybrid nanoparticle fused with a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome carrying an anticancer agent.

The drug delivery system is characterized in that it delivers the anticancer agent to target cells. Preferably, the target cell of the drug delivery system is a cancer cell or tumor tissue, and more preferably, a cancer cell or tumor tissue that expresses a tumor antigen to which the chimeric antigen receptor contained in the hybrid nanoparticle specifically binds.

The terms used in the present invention are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person working in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

Throughout the specification of the present invention, when a part is said to “include” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary. The terms "about", "substantially", etc. used throughout the specification of the present invention are used to mean at or close to that value when manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention Precise or absolute figures are used to aid understanding and to prevent unscrupulous infringers from taking unfair advantage of the disclosure.

Throughout the specification of the present invention, the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of constituent elements.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

experiment material

The materials used in the following examples and experimental examples are as follows:

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2-1,3 -benzoxadiazol-4-yl)(NBD-PS), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(Rhod-PE), and mini-extruder was purchased from Avanti Polar Lipids; n-Dodecyl βULTROL grade) was purchased from Merck; DiR and temoporfin (mTHPC) were purchased from Sigma-Aldrich; The EGFR-CAR plasmid was constructed with anti-EGFR single-chain variable fragment, CD8 hinge domain, CD28 transmembrane domain, CD28 endodomain, and CD3zeta domain.

Example 1. Cell culture conditions

HEK293 and NIH3T3 cells were purchased from the Korean Cell Line Bank, and MDA-MB-231 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HEK293 and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C and 5% CO ₂ . NIH3T3 cells were cultured at 37°C and 5% CO ₂ in Roswell Park Memorial Institute Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

Example 2. Preparation of liposomes

Liposomes were prepared by thin film hydration method. Several types of lipids were combined at various molar ratios (see Tables 1 and 2), dissolved in chloroform, and liposomes were prepared in a round bottom flask. Next, the sample was placed in a rotary evaporator and used under vacuum and 60°C conditions for 15 minutes to evaporate chloroform. Next, the lipid film was treated with 1 mL of PBS for 30 minutes to hydrate, and then subjected to ultrasound at 20% amplitude (pulse mode; 3 seconds/3 seconds) for 7 minutes and 30 seconds to obtain lipid bilayer liposomes. The obtained liposomes were stored at 4°C.

Example 3. Preparation of CAR cell-derived vesicles

To prepare HEK293 cells expressing CAR (CAR-HEK293), HEK293 cells were transfected with Geneporter3000 transfection reagent (Genlantis) according to the manufacturer's protocol. Seeded HEK293 cells showed 80% confluency on the day of transfection. The plasmid DNA was diluted and mixed in GP3K diluent and the GP3K reagent was diluted in serum-free medium, and the mixed reagents were incubated at room temperature for 5 minutes to prepare lipoplexes for transfection. Complete medium was removed from the plated cells, replaced with serum-free medium, and then lipoplex was treated. After 24 hours of culture, transfected cells were collected. The obtained cells were centrifuged to remove nuclei and sonicated with a tip-sonicator at 20% ultrasound amplitude (pulse mode, 3 sec/3 sec) for 30 sec. Treated cells were successively extruded through polycarbonate membrane filters (Whatman, UK) with pore sizes of 10 μm, 5 μm, 1 μm, and 400 nm using a mini extruder (Avanti Polar Lipids, AL, USA). Cell-derived vesicles (CDV) expressing CAR were obtained. The protein concentration of the prepared CDV was quantified using the BCA protein assay kit (Thermo Fisher, Korea) according to the manufacturer's protocol.

Example 4. Characterization of CDV

The size and shape of CDV were analyzed using a transmission electron microscope (TEM, H-7600, Hitachi). CDV size, surface zeta potential, and particle number were measured using Nanoparticle Tracking Analysis (NTA, Zetaview, Particle Metrix, USA).

Example 5. Analysis of in vitro viability and cytotoxicity

To determine the effect of nanoparticles on cell viability, the Cell Counting Kit-8 (CCK-8) assay kit was used according to the manufacturer's protocol to measure viability and cytotoxicity of MDA-MB-231 and NIH3T3 cells. did. Various nanovesicles were treated in a number of 1×10 ⁷ to 1×10 ⁹ in serum-free medium in which cells were cultured for 4 hours. After processing liposomes loaded with temoporfin (photosensitizer) in serum-free medium, the medium was changed to culture medium and a laser of 12 J intensity was illuminated using a 671 nm laser source (1000MWCW400F, LaserLab, Korea). did. After 24 hours, CCK-8 reagent was applied to each well. Cell viability was calculated by measuring the absorbance of each well at 450 nm for 30 minutes with a microplate reader (Synergy H1, BioTek, Winooski, USA).

Example 6. Fluorescence resonance energy transfer (FRET)-based fusion assay

Liposomes and CDV were mixed in a black plate maintained at 37°C in a microplate reader (Synergy H1, BioTek, USA). CDV and liposomes were mixed in PBS at a liposome/CDV ratio of 1/9 (1×10 ¹¹ pieces). All vesicles were counted by NTA, and the total volume of the reaction mixture was 200 μL. Triton x-100 or ethanol was added to a final concentration of 2% or 30%, respectively. Additionally, sonication or electroporation was performed at 40 kHz or bipolar (50 voltage, 10 μs, 5 pulses (ECM830, BTX Apparatus, USA)) for 20 minutes. During the CDV-liposome fusion process, the lipid mixture was monitored by FRET. Additionally, donor fluorescent liposomes containing the same molar ratio (1.5%) of fluorescent lipids NBD-PS and Rhod-PE were prepared (see Tables 1 and 2). When NBD is excited, the fluorescence resonance energy is transferred to rhodamine in a FRET process that depends on the distance between the two fluorophores. Fusion was monitored by following NBD fluorescence intensity (excitation at 460 nm, emission at 535 nm) at 37°C. After 1 h, the fusion reaction was stopped by adding 10 μl of DDM (25 mg/mL) to solubilize all liposomes and the NBD fluorescence intensity was measured at infinite dilution: Max(NBD). The fusion curve was normalized using the following equation:

NBD fluorescence increase (%) = [NBD-Min(NBD)]/[Max(NBD)-Min(NBD)]

Here, Min(NBD) means the lowest NBD fluorescence value at all times.

Example 7. Singlet oxygen generation test

To measure the production of reactive oxygen species in liposomes loaded with temoporphine, a stock solution of singlet oxygen sensor green reagent (SOSG reagent, Thermo Fisher Scientific, USA) in methanol was diluted with distilled water to prepare a test solution. SOSG reagent was added to temoporphine-loaded liposomes, exposed to a 671 nm laser source (LaserLab, Korea), and monitored using a microplate reader at 504/525 nm wavelengths (excitation/emission).

Example 8. Evaluation of in vitro cancer targeting efficiency of CAR-CDV

To study the targeting efficiency of CDV, MDA-MB-231 cells were seeded in 24-well plates and cultured overnight before experiments. After 24 h, the cells were washed with PBS, the medium was replaced with serum-free medium, and incubated with DiR-labeled CDV for 4 h. After completion of incubation, the serum-free medium was replaced with culture medium again. After 24 hours, cells were observed using a confocal laser scanning microscope (CLSM, Fluoview FV1000, Olympus, Japan). Image analysis was performed using the Fiji analysis program. The degree of fluorescence was quantified by measuring the average value of n > 3 pictures for each sample.

Example 9. Flow cytometry

After transfection of the plasmid into HEK293 cells, cells from each group were washed with PBS and suspended in FACS buffer. Suspended cells were treated with Fc blocker (BD Bioscience, Franklin Lakes, NJ, USA) for 15 minutes at 4°C and then incubated with dye-conjugated antibodies for 1 hour at 4°C. Samples labeled with antibodies were washed three times with FACS buffer and fixed with 4% paraformaldehyde. EGFR scFv CAR expression levels in cells were analyzed using FITC-anti-Fab antibody (Jackson ImmunoResearch, West Grove, PA, USA). CAR-expressing cells were quantified by flow cytometry (BD FACS Canto II, BD Biosciences, San Jose, CA, USA), and data were analyzed with FACS Diva software (BD Biosciences, San Jose, CA, USA).

Example 10. Protein preparation and Western blot analysis

Proteins from cell lysates or CDV were separated by SDS-PAGE and then transferred to nitrocellulose membrane. Blots were blocked with 5% skim milk for 1 hour at room temperature, treated with diluted primary antibody, incubated overnight at 4°C, and then incubated with HRP-conjugated secondary antibody (Invitrogen, USA) for 1 hour at room temperature. . Staining of the membrane was developed using ECL detection reagent (Bio-rad, USA). CD3zeta (Abcam, USA) was used as a CAR structural marker. CD63 and TSG101 (Invitrogen, USA) were used as exosome markers. GAPDH was used as a control.

Example 11. Animal experiments

All in vivo experiments were performed in accordance with the approved protocol guidelines of the Animal Care Committee of the Catholic University Animal Hospital (Republic of Korea, CUK-IACUC-2021-017-01). All mice were housed under temperature and light controlled conditions (12 h light/dark cycle). Eight-week-old female BABL/c nude mice were stabilized for one week and then used in animal experiments. To prepare the mouse tumor model, MDA-MB-231 cells (1 × 10 ⁶ cells) were subcutaneously inoculated into the right flank of 9-week-old BALB/c nude mice. After 7 days, when the average tumor volume reached 100 mm ^{3 ,} mice were used for the experiment. After the mouse was completely anesthetized with isoflurane, nanovesicles were injected intravenously into the mouse. Twenty-four hours later, the tumor was irradiated twice with a laser (laser dose, 100 J/cm ² ) at 12-hour intervals using a 671 nm fiber-coupled laser system (LaserLab, Korea). Tumor size was measured with calipers, and tumor volume was calculated using the formula width × width × length × 0.5. To evaluate the evaluation of the in vivo tumor targeting specificity of CDV in a tumor mouse model, images were obtained via a fluorescently labeled organism bioimaging device (FOBI, Neo-Science, Suwon, Korea).

Example 12. Blood biochemistry analysis

To investigate the toxicity of hybrid vesicles in organs, fused vesicles were injected intravenously into Balb/c nude mice. One week later, blood samples were harvested by intracardiac blood collection. Samples were centrifuged at 3000 rpm for 20 min to separate serum from whole blood. The levels of AST, ALT, ALP, CREA, GLU, CPK, Ca, and NA/K in serum were quantified using an automated chemistry analyzer (DRI-CHEM NX500i, Fujifilm, Japan).

Example 13. Statistical analysis

Statistical analyzes were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). All data were evaluated as mean ± standard deviation (SD) or standard error of the mean (SEM). ANOVA was used to examine three or more groups, and t-test was used to examine two groups. The figure legend indicates the significance level of the p-value.

[Experimental example]

Experimental Example 1. Preparation of EGFR-CAR expressing cell-derived vesicles (EGFR-CAR-CDV)

Chimeric antigen receptor (CAR) is a targeting receptor that binds to specific proteins expressed by cancer cells. In other words, because CAR gives cells the ability to target cancer cells, it is mainly expressed in T cells, natural killer cells (NK cells), or macrophages, and induces the immune cells to effectively target and kill cancer cells. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that binds to epidermal growth factor (EGF). EGFR is associated with various diseases such as solid tumors (lung cancer, colon cancer, breast cancer, etc.) and SARS-CoV-2 infection.

To prepare anti-EGFR-CAR cells, HEK293 cells were transfected with anti-EGFR-CAR (“” plasmid (Figure 1a). To confirm whether the cells express EGFR-CAR, CAR of transfected HEK293 cells As a result of confirming the expression level by flow cytometry, it was confirmed that there were a large number of cells expressing CAR compared to all cells (Figure 1b). In addition, the expression of CD3zeta, a component of CAR, was confirmed by Western blot, and it was confirmed that it was transfected with EGFR-CAR plasmid. It was confirmed that the transfected HEK293 cells expressed CD3zeta at a high level (Figure 1c). These results showed that the transfected HEK293 cells expressed anti-EGFR-CAR at a high level.

Afterwards, the transfected cells were sequentially extruded in a mini extruder through membrane filters with various pore sizes to prepare anti-EGFR-CAR expressing cell-derived vesicles (CDVs) (Figure 2A). In order to confirm the characteristics of the obtained EGFR-CDV, the surface charge and size of the control CDV and EGFR-CAR expressing CDV were first measured through nanoparticle tracking analysis (NTA). As a result, the surface charge and size of each CDV were determined. It was confirmed that there was no significant difference in size (Figures 2b and 2c). In addition, as a result of confirming the expression of EGFR-CAR marker protein (CD3zeta) and endoplasmic reticulum marker protein (CD63) in control CDV or EGFR-CAR expressing CDV using Western blot, expression of endoplasmic reticulum marker protein was confirmed in both CDVs. , EGFR-CAR CDV was confirmed to maintain a high level of expression of CD3zeta (Figure 2d). Subsequently, the cell survival rate was measured after treating MDA-MB-231 cells with control CDV or EGFR-CAR expressing CDV, respectively. As a result, it was confirmed that there was no difference in cell survival rate of each CDV treatment group compared to the untreated control group. Control CDV and EGFR-CAR expressing CDV were confirmed to have no cytotoxicity (Figure 2e). In other words, the above results show that CDV expressing anti-EGFR-CAR has an anti-EGFR-CAR structure, endoplasmic reticulum characteristics, and biocompatibility. EGFR is well known to be expressed in breast cancer cells. Therefore, in this experimental example, MDA-MB-231, a breast cancer cell line expressing EGFR, was selected to confirm the target cell targeting ability of CDV. After treating MDA-MB-231 cells with control or EGFR-CAR expressing CDV (1 × 10 ⁷ particles) for 4 hours, fluorescence images of the cells were taken and the level of fluorescence was analyzed. As a result, cells of EGFR-CAR expressing CDV were found to be It was confirmed that the absorption rate was higher than that of the control CDV (Figures 2f and 2g). The above results support that EGFR-CDV has better cancer cell-targeting properties than control CDV.

Experimental Example 2. Fusion analysis of EGFR-CAR-CDV and liposome using FRET technique

Fluorescence resonance energy transfer (FRET) is a fluorescence-based technique that monitors interactions (such as fusion) between liposomes. To develop a novel fusion method of CDV and liposomes, fusion between liposomes was first attempted. To evaluate the degree of fusion between liposomes, control liposomes and FRET liposomes were fused in various ways and NBD fluorescence was analyzed (i.e., NBD to analyze the energy transfer efficiency from to Rhod). FRET liposomes fused with unlabeled control liposomes have a reduced surface density of fluorophores, and the molar ratio composition of the two fluorescent probes (NBD-PS and Rhod-PE) and lipid is important in FRET analysis (Table 1 and Figure 3a). As a result of measuring the surface zeta potential of the control liposome and FRET liposome through nanoparticle tracking analysis, it was confirmed that there was no significant difference in the surface charge and size of the two liposomes (Figures 3b and 3c).

To induce liposome fusion, control liposomes and FRET liposomes having the lipid composition shown in Table 1 were mixed, then ethanol was added to a concentration of 30% EtOH compared to the total solution, and after 1 hour, a volume of phosphoric acid three times the total volume was added. It was purified by adding buffered saline. In order to compare liposome fusion by other methods (sonication, electroporation, etc.), the degree of FRET of liposomes induced by fusion by each method was measured, and the results showed that the group that used the method using a 30% EtOH solution or electroporation performed It was confirmed that fusion occurred as the fluorescence value of NBD increased. In particular, the group fused with 30% EtOH showed a similar fluorescence value to Triton x-100, a positive control well known to dissolve lipid membranes, and showed a higher fluorescence value than Triton x-100 1 hour after fusion ( Figure 3d). However, as a result of measuring the degree of FRET after inducing fusion of FRET liposomes and HEK293 CDV with the lipid composition shown in Table 1 in the same manner, there was no change in fluorescence value, unlike the group using Triton x-100, which was the positive control, and the negative control group. Fluorescence values similar to those of the phosphoPBS group were confirmed, showing that fusion between the FRET liposome and CDV was not triggered under any conditions except triton x-100 (FIG. 3E).

Experimental Example 3. Establishment of liposome composition capable of fusing with EGFR-CAR-CDV

As confirmed in the above examples, FRET liposomes with the compositions in Table 1 did not fuse with CDV under any conditions (FIG. 3e). Therefore, the present inventors determined that the lipid composition of liposomes affects the degree of fusion with CDV, and synthesized liposomes with various lipid compositions to confirm liposome compositions that can efficiently fuse with CDV (Table 2).

As a result of checking the characteristics of the three liposomes, the sizes of liposomes A, B, and C in that order were 163 nm, 179 nm, and 198 nm, respectively (Figure 4a). Additionally, the surface charges were -36, +40, and -36 mV, respectively (Figure 4b). As a result of comparing the fluorescence resonance energy transfer rates by fusing each FRET liposome with different lipid composition and CDV with 30% EtOH, a statistically significantly higher transfer rate was observed only in liposome B, which is a cationic liposome. It was found that it was effectively fused with CDV (Figures 4c and 4d). The above results mean that liposomes with a positive charge on the surface are suitable for fusion with CDV in 30% EtOH. Next, to evaluate the biocompatibility of liposome B, fibroblast cell line (NIH3T3) and breast cancer cell line (MDA-MB-231) were treated with liposome B with various particle numbers and the cell viability was confirmed. As a result, liposome B was not cytotoxic. It was found that (Figure 4e).

Experimental Example 4, Evaluation of breast cancer cell targeting ability of hybrid nanovesicles

Dual-functionalized nanoparticles that can target cancer and deliver cancer-specific drugs were created. Specifically, first, a hydrophobic photosensitizer (mTHPC), which generates reactive oxygen species (ROS) in response to laser, was encapsulated in cationic liposomes (Figure 5a). As a result of checking the size of the liposome encapsulated with the photosensitizer, it was confirmed that the diameter was about 307 nm (Figure 5c) and that it had a positive surface charge (32 mV) on the surface (Figure 5b). In addition, as a result of TEM imaging of the liposome encapsulated with the photosensitizer, a spherical shape was observed (Figure 5d). Next, the amount of active oxygen was measured by irradiating a laser to the mTHPC-loaded liposome. As a result, the amount of active oxygen increased as the laser irradiation time increased. The liposome loaded with the photosensitizer responded to the laser and produced active oxygen. It was confirmed that was generated normally (Figure 5e). In addition, when liposomes loaded with photosensitizer were treated with breast cancer cell lines (MDA-MB-231) and cell death was compared with and without laser irradiation, no death of cancer cells was observed in the group without laser irradiation, but in the group with laser irradiation, cell death was not observed. It was confirmed that significant cancer cell death was induced in the group (Figure 5f). The above results show that liposomes loaded with a photosensitizer can effectively induce cancer cell death by generating active oxygen upon laser irradiation.

Next, hybrid nanovesicles were prepared by fusing liposomes loaded with a photosensitizer and CDV expressing EGFR-CAR, and their properties and effects were confirmed. The hybrid nanovesicles were prepared by fusing liposomes and CDV with 30% EtOH as in Experimental Example 2. The sizes of control CDV, EGFR-CDV, liposomes fused with control CDV, and liposomes fused with EGFR-CDV were 145, 165, 132, and 173 nm, respectively, showing no significant difference in size (Figure 6b). In addition, as a result of checking the surface charges of each, the surface charge of CDV was about -25 Mv, while the surface charge of the hybrid nanoparticle fused with CDV and cationic liposome was about -10 mV, and the surface charge of CDV was reduced by cationic liposome. An increase was confirmed (Figure 6a). Subsequently, the cytotoxicity and cancer cell killing effects of the hybrid nanoparticles were confirmed. First, as a result of treating the breast cancer cell line (MDA-MB-231) with the hybrid nanoparticles, it was confirmed that they had almost no toxicity (Figure 6c). In addition, after treating breast cancer cell lines with hybrid nanoparticles, the cellular uptake rate of the nanoparticles was confirmed using CAR. As a result, it was confirmed that the cellular uptake rate of liposomes fused with EGFR-CDV was higher compared to liposomes fused with control CDV. (Figures 6d and 6e). Next, after treating breast cancer cell lines with hybrid nanoparticles, the cell death rate was checked with or without laser irradiation. As a result, cancer cell death was not induced in the group that was not irradiated with laser, but most cancer cells were killed in the group that was irradiated with laser. It was found that (Figure 6f). The above results show that the hybrid nanoparticle according to the present invention can be effectively absorbed into cancer cells by targeting cancer cells through EGFR-CAR, and can kill cancer cells by generating active oxygen in response to laser within cancer cells. In other words, the hybrid nanoparticle according to the present invention exhibits excellent cancer cell targeting ability and cancer cell killing function by effective fusion of CDV expressing cancer cell-specific CAR and liposome loaded with photosensitizer.

Experimental Example 5. Evaluation of breast cancer tumor targeting ability of hybrid nanovesicles

Since the cancer cell targeting ability and drug delivery effect of the hybrid nanoparticle according to the present invention were confirmed in vitro through the previous example, in this example, a tumor mouse model was created to evaluate the function of the hybrid nanovesicle. Specifically, according to the animal experiment schedule shown in Figure 7a, hybrid nanovesicles fused with liposomes loaded with EGFR-CAR expressing CDV and photosensitizer were intravenously administered to a tumor mouse model, and then laser was irradiated to kill the tumor caused by the hybrid nanovesicles. The killing effect was evaluated. First, as a result of evaluating the blood toxicity of systemically injected hybrid nanoparticles, no significant changes occurred in the levels of liver and kidney toxicity markers compared to the control group, confirming that the hybrid nanoparticles according to the present invention have excellent biocompatibility. (Figure 7b). In addition, the distribution of nanovesicles in the body was confirmed through fluorescence imaging 24 hours after intravenous injection of hybrid nanovesicles stained with a fluorescent substance. As a result, hybrid nanoparticles fused with CDV expressing EGFR-CAR showed tumor growth compared to the control group. As the amount accumulated in the area was significantly high, it was confirmed that the tumor targeting ability of liposomes fused with CDV expressing cancer cell-specific CAR was superior (Figure 7c). In addition, as a result of intravenous injection of hybrid nanoparticles and then irradiation with a laser to track the tumor size depending on whether or not the laser was irradiated, the tumor size in the group irradiated with the laser was significantly reduced compared to the group not irradiated with the laser. According to the present invention, It was confirmed that the hybrid nanoparticles were effectively absorbed into cancer cells and then responded to the laser to generate active oxygen, effectively killing cancer cells and inhibiting tumor growth (Figure 7d).

Through the above experimental examples, it was confirmed that the hybrid nanoparticle according to the present invention can effectively target cancer cells through cancer cell-specific CAR and kill cancer cells by effectively delivering drugs for photodynamic therapy into cancer cells. In addition, the hybrid nanoparticles have excellent biocompatibility and do not induce cell death unless irradiated with a laser, so the risk of side effects due to non-specific drug activity can be significantly reduced. In particular, the present inventors have discovered an effective fusion method of CDV and liposomes, and various types of CDV and liposomes can be fused without being limited to the type of active ingredient (FIG. 8). Therefore, through the present invention, those skilled in the art can prepare hybrid nanoparticles with dual functions through the fusion of liposomes loaded with various drugs and CDV expressing appropriate active ingredients according to the purpose, and the nanoparticles can be used for the treatment of various drugs. It is expected that it can be used for.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Table 3 below shows sequence information of the components used in the present invention.

The present invention relates to a fusion technology of cell-derived vesicles and liposomes and to hybrid nanoparticles produced through the fusion technology, wherein the hybrid nanoparticles can exert both targeting functions and drug delivery functions to target tissues. Specifically, the present inventors discovered the optimal conditions to effectively fuse cell-derived vesicles and liposomes, and through this, cell-derived vesicles expressing active ingredients and drug-encapsulated liposomes were fused to possess all of their functions. Since hybrid nanoparticles, that is, dual-functional hybrid nanoparticles, can be manufactured, a wide range of types of hybrid nanoparticles can be manufactured depending on the purpose. In particular, hybrid nanoparticles prepared by fusing a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with a photosensitizer effectively target cancer cells through the chimeric antigen receptor, and the photosensitizer It can kill cancer cells by generating active oxygen in response to light stimulation. Therefore, the hybrid nanoparticle according to the present invention can effectively target target tissues and deliver drugs through a combination of various cell-derived vesicles and liposomes, and is expected to be a promising therapeutic platform that can be used as a drug delivery vehicle and therapeutic agent in various fields. It is expected.

Claims (31)

A method for producing hybrid nanoparticles in which cell-derived vesicles (CDV) and liposomes are fused, comprising the step of mixing cell-derived vesicles (CDV) and liposomes to induce their fusion. According to paragraph 1, A method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, characterized in that the fusion is carried out in an alcohol solvent of 10 to 50 (v/v)% of C ₁ to C ₅ . According to paragraph 1, A method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, wherein the cell-derived vesicles and the liposomes are mixed at a ratio of 1 to 30:1 (cell-derived vesicles: liposomes). According to paragraph 1, A method of producing a hybrid nanoparticle in which a cell-derived vesicle and a liposome are fused, characterized in that the cell-derived vesicle is obtained by extruding a sample containing cells into a micropore. According to clause 4, A method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, characterized in that the diameter of the micropores is 0.1 to 20 μm. According to paragraph 1, The cell-derived vesicle satisfies one or more characteristics selected from the group consisting of: A method of producing a hybrid nanoparticle in which a cell-derived vesicle and a liposome are fused: (a) The diameter of cell-derived vesicles is 100 to 300 nm; and (b) The surface charge of cell-derived vesicles is -50 to -10 mV. According to paragraph 1, A method of producing a hybrid nanoparticle in which a cell-derived vesicle and a liposome are fused, wherein the cell-derived vesicle contains a chimeric antigen receptor. According to paragraph 1, The liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero Hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, characterized in that they contain at least one selected from the group consisting of -3-phosphocholine (DOPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Manufacturing method. According to clause 8, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is characterized in that it is contained in a molar ratio (molar ratio %) of 1 to 20: 1. A method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused. According to paragraph 1, A method for producing hybrid nanoparticles in which cell-derived vesicles and liposomes are fused, wherein the liposome has a surface charge of 0 to 100 mV. According to paragraph 1, A method of producing a hybrid nanoparticle in which a cell-derived vesicle and a liposome are fused, wherein the liposome is loaded with an active ingredient. Hybrid nanoparticles fused with cell-derived vesicles (CDV) and liposomes. According to clause 12, The cell-derived vesicle is a hybrid nanoparticle, characterized in that it is manufactured by a manufacturing method comprising the step of extruding a sample containing cells into micropores. According to clause 12, The cell-derived vesicle contains a chimeric antigen receptor on its surface, and the liposome contains an active ingredient. According to clause 14, The hybrid nanoparticle is characterized in that it contains the chimeric antigen receptor on its surface and the active ingredient between its lipid bilayers or in its internal compartment. In paragraph 14. A hybrid nanoparticle, characterized in that the chimeric antigen receptor is a tumor antigen-specific chimeric antigen receptor. According to clause 12, The liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero A hybrid nanoparticle containing one or more selected from the group consisting of -3-phosphocholine (DOPC), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). According to clause 14, The active ingredients include peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorescent dyes. (fluorochrome), and a hybrid nanoparticle, characterized in that at least one selected from the group consisting of toxins. According to clause 14, Hybrid nanoparticles, characterized in that the active ingredients are photosensitizers. According to clause 12, The hybrid nanoparticle satisfies one or more characteristics selected from the group consisting of: (a) the diameter of the hybrid nanoparticles is 100 to 300 nm; and (b) The surface charge of the hybrid nanoparticle is -30 to +20 mV. According to clause 12, The hybrid nanoparticle is characterized in that it is manufactured by the manufacturing method of claim 1. A pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a hybrid nanoparticle in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused. According to clause 22, A pharmaceutical composition for the prevention or treatment of cancer, wherein the hybrid nanoparticle contains the tumor antigen-specific chimeric antigen receptor on its surface and the anticancer agent between the lipid bilayers or in an internal compartment thereof. According to clause 22, A pharmaceutical composition for the prevention or treatment of cancer, wherein the anticancer agent is at least one selected from the group consisting of a chemical anticancer agent, a targeted anticancer agent, an immunological anticancer agent, and a photosensitizer. According to clause 24, A pharmaceutical composition for the prevention or treatment of cancer, wherein the anticancer agent is a photosensitizer, and the hybrid nanoparticle generates active oxygen in response to light stimulation. According to clause 22, The above cancers include breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer One selected from the group consisting of cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition for preventing or treating cancer, characterized by the above. A kit for preventing or treating cancer, comprising the pharmaceutical composition of claim 22. A drug delivery system comprising, as an active ingredient, a hybrid nanoparticle in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused. A method for preventing or treating cancer, comprising the step of administering a hybrid nanoparticle in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused to an individual in need thereof. Use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused for the prevention or treatment of cancer. Use of hybrid nanoparticles in which a cell-derived vesicle containing a tumor antigen-specific chimeric antigen receptor and a liposome loaded with an anticancer agent are fused for the production of a drug for the prevention or treatment of cancer.

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