Cell-derived vesicles engineered by ankyrin and uses thereof

Abstract

According to the present invention, four types of anchor proteins, which conform to the inherent characteristics of CDV and can mediate the stable introduction of bioactive molecules, are explored as modified cell-derived vesicles (Cells-derived vesicles) that can be used as drug delivery carriers, thereby completing the present invention. It is confirmed that the anchor protein is used as a membrane protein specifically and abundantly existing in CDV, and the CDV containing the anchor protein can more stably load bioactive molecules. For example, the result of a comparative experiment using a fluorescent protein has been confirmed that the CDV into which the anchor protein is introduced has more efficiently loaded with the fluorescent protein than the CDV without the anchor protein. Moreover, it has been confirmed that when a cancer cell-targeting antibody is loaded in the modified CDV of the present invention, not only the cancer cell targeting ability is increased, but also the modified CDV is more effectively absorbed by cancer cells. That is, the CDV according to the present invention can stably load a plurality of bioactive molecules and deliver the bioactive molecules to a desired target as BioDrone modified by an anchor protein, and is expected to be used in drug delivery and treatment platforms for a plurality of drugs.

Description

Technical Field

[0001] The present invention relates to cell-derived vesicles transformed by anchoring proteins and drug delivery applications thereof.

[0002] The present invention claims priority to Korean Patent Application No. 10-2022-0086418, filed on Jul. 13, 2022, and No. 10-2023-0090542, filed on Jul. 12, 2023, which are hereby incorporated by reference into this application in their entirety. Background Art

[0003] Most drugs will spread to the position or tissue without lesions in the body to induce side effects. In order to minimize such side effects, the active targeting (active targeting) research using drug delivery system (DDS) is being actively studied. Drug delivery carriers are medical technologies that protect non-targeted sites and deliver drugs only to targeted sites by suppressing the unwanted distribution of drugs. In order to minimize side effects by regulating the absorption, distribution and discharge of nucleic acids, proteins or other low molecules, the therapeutic effect is enhanced at the site of action of the target and multiple drug delivery technologies are utilized. Effective drug delivery carriers can save the cost and time for new drug development, not only the development of new drugs, but also the development of drug delivery systems in countries such as the United States and Japan since the 1980s.

[0004] So far, liposomes (liposome), viruses, recombinant proteins, cationic polymers and various nano substances are used as carriers for drug delivery. However, cationic polymers and liposomes based thereon have the problem of being unsuitable for clinical use because of their high cytotoxicity. And, although attempts are made to make nucleic acids directly and stably pass through cell membranes by chemically deforming nucleic acid molecules, such methods are too large in terms of cost and time consumption, and require complicated procedures, and are therefore also not suitable for clinical application. And, although various drug delivery systems based on nanoparticles such as graphene quantum dots, magnetic particles, and metal nanoparticles have been developed, the cytotoxicity of the above-mentioned particles is high, and there is a structure that is unfavorable for the biopolymers such as nucleic acid molecules to be introduced into cells, and there is a problem of low efficiency of intracellular delivery. Therefore, there is a need for the development of drug delivery carriers that can not only effectively deliver various biological substances including nucleic acids to cells, but also are suitable for organisms and have low risk of side effects.

[0005] Extracellular vesicles are nano-scale membrane structures that deliver cellular substances such as proteins between cells to mediate intercellular communication. Extracellular vesicles (EVs) that are affinity to organisms and have intercellular signaling functions have attracted much attention as drug delivery systems (DDS). The endoplasmic reticulum can not only preserve the active substances inherent in the cell membrane and cytoplasm, but also carry new substances, thereby exerting a variety of biological activities. In particular, if proteins with targeted tissue-specific binding ability can be expressed on the surface of extracellular vesicles, the targeting ability of the target site in the organism can be maximized, so that it can be expected to be a new generation of drug delivery carriers.

[0006] However, there are very few natural endoplasmic reticulum isolated from cells, and considerable effort is required for collection and concentration, so there are limitations in commercialization. Therefore, artificial cell-derived vesicles (CDVs) that can be easily obtained in large quantities by batch extrusion are used. Cell-derived vesicles are different from other vesicles such as exosomes or extracellular vesicles, and are used as analogs of natural extracellular vesicles such as exosomes and extranuclear granules in various research and industrial fields. Then, a large amount of CDV can be easily obtained by separating impurities and purifying from crude CDV. The membrane proteins and lipids of CDV are derived from the plasma membranes or organelle membranes of CDV mother cells, so the membranes or the inside of the membranes contain physiologically active molecules (proteins, lipids, sugars, etc.) expressed by cells. In addition, while the functions of active substances derived from cells, cell-derived vesicles can also be enclosed in or even combined with drugs with various characteristics, which has the advantage of maximizing the therapeutic efficacy. In order to develop cell-derived vesicles as drug delivery systems, a technology that can efficiently introduce targeting ligands and therapeutic drugs is required. Summary of the invention

[0007] Technical issues

[0008] As a result of multiple studies conducted by the present invention to solve the above problems, anchor membrane proteins (also called anchoring proteins) that meet the inherent characteristics of cell-derived vesicles (CDVs) and can mediate the stable loading of bioactive molecules are discovered, thereby completing the present invention. The CDV of the present invention is a modified CDV that overexpresses the anchoring protein, and various molecules such as therapeutic drugs or targeting ligands can be stably loaded through the overexpressed anchoring protein, thereby effectively delivering the molecules to the target.

[0009] Therefore, an object of the present invention is to provide cell-derived vesicles overexpressing ankyrin.

[0010] Another object of the present invention is to provide cells (or cell lines) for producing the cell-derived vesicles.

[0011] Another object of the present invention is to provide a method for preparing the cell-derived vesicles.

[0012] Another object of the present invention is to provide a composition for drug delivery comprising the cell-derived vesicles as an active ingredient.

[0013] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and a person skilled in the art in the technical field to which the present invention belongs can clearly understand other problems not mentioned through the following description.

[0014] Technical Solution

[0015] The present invention provides a cell-derived vesicle as a cell-derived vesicle (CDVs) overexpressing an anchoring protein, wherein the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

[0016] In one example of the present invention, the anchoring protein can be inserted into the membrane of the cell-derived vesicle.

[0017] In another example of the present invention, the cell-derived vesicles may be derived from cells overexpressing the anchoring protein, but is not limited thereto.

[0018] In another example of the present invention, the cell-derived vesicles can be obtained by squeezing the cells, but is not limited thereto.

[0019] In another embodiment of the present invention, the cells may be one or more selected from the group consisting of stem cells, immune cells, blood cells, embryonic cells, fat cells and embryonic kidney cells, but are not limited thereto.

[0020] In another example of the present invention, the anchoring protein may be present at a higher level in the cell-derived vesicles than in the cells from which the cell-derived vesicles originate or the exosomes produced from the cells, but is not limited thereto.

[0021] In another example of the present invention, the anchor protein may be glycosylated or not glycosylated, but is not limited thereto.

[0022] In another example of the present invention, the anchoring protein can be combined with a biologically active molecule, but is not limited thereto.

[0023] In another example of the present invention, the bioactive molecule may be located outside or inside the membrane of the cell-derived vesicle, but is not limited thereto.

[0024] In another example of the present invention, the bioactive molecule can be one or more selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, viruses, semi-synthetic drugs, quantum dots, fluorochromes and toxins, but is not limited thereto.

[0025] In another example of the present invention, the protein can be one or more selected from the group consisting of antibodies, antibody fragments, growth factors, enzymes, nucleases, transcription factors, antigenic peptides, hormones, transport proteins, immunoglobulins, structural proteins, motor function proteins, signaling proteins, connecting proteins, viral proteins, natural proteins, recombinant proteins, protein complexes, fluorescent proteins, therapeutic proteins, chemically modified proteins and prions, but is not limited thereto.

[0026] In another example of the present invention, the antibody may be one or more selected from the group consisting of full-length antibodies, Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, miniantibodies, bispecific antibodies and nanoantibodies, but is not limited thereto.

[0027] In another example of the present invention, the bioactive molecules are targeting ligands, and the cell-derived vesicles can bind to cells expressing the targets of the targeting ligands, but are not limited thereto.

[0028] Furthermore, the present invention provides a cell for producing the cell-derived vesicle.

[0029] In addition, the present invention provides a method for preparing cell-derived vesicles, which comprises: step (S1), introducing a recombinant vector containing an anchoring protein encoding gene into cells; and step (S2), extruding the cells introduced with the recombinant vector to obtain cell-derived vesicles, wherein the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

[0030] In one embodiment of the present invention, the recombinant vector may be introduced into the cell via at least one selected from the group consisting of lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and vaccinia virus, but is not limited thereto.

[0031] In another example of the present invention, the cells may be infected with viruses at a multiplicity of infection (MOI) of 1 to 30 MOI, but the present invention is not limited thereto.

[0032] In another example of the present invention, the recombinant vector may further include a gene encoding a bioactive molecule, and the bioactive molecule is expressed in a state of being bound to the anchor protein, but is not limited thereto. That is, the bioactive molecule and the anchor protein may be expressed in the form of a fusion protein.

[0033] Furthermore, the present invention provides a cell for producing cell-derived vesicles, characterized in that an exogenous anchoring protein encoding gene is introduced, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

[0034] Furthermore, the present invention provides cell-derived vesicles obtained by squeezing the cells.

[0035] Furthermore, the present invention provides a composition for drug delivery (or a drug delivery carrier), comprising, as an effective ingredient, cell-derived vesicles overexpressing an anchoring protein, wherein the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1, and LAMP2.

[0036] Furthermore, the present invention provides the use of the cell-derived vesicles overexpressing the anchoring protein for drug delivery.

[0037] In addition, the present invention provides the use of the cell-derived vesicles overexpressing the anchoring protein for preparing a composition for drug delivery (or a drug delivery carrier).

[0038] Furthermore, the present invention provides a method for delivering a drug to an individual, cell, tissue, and / or organ, comprising the step of administering the cell-derived vesicles overexpressing the anchoring protein to the individual, cell, tissue, and / or organ in need thereof.

[0039] Furthermore, the present invention provides a pharmaceutical composition, as a pharmaceutical composition for preventing or treating a disease, comprising a cell-derived vesicle overexpressing an anchoring protein as an active ingredient, wherein the cell-derived vesicle is loaded with a drug for preventing or treating the disease, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2. In one example of the present invention, the disease may be cancer, and the drug may be an anticancer agent. In other examples of the present invention, the disease may be a brain disease, and the drug may be a therapeutic agent for brain disease.

[0040] Furthermore, the present invention provides the use of the cell-derived vesicles overexpressing the anchoring protein for preventing or treating diseases, wherein the cell-derived vesicles are loaded with a drug for preventing or treating the myopathy.

[0041] Furthermore, the present invention provides the use of the cell-derived vesicles overexpressing the anchoring protein for preparing a medicament for preventing or treating a disease.

[0042] Furthermore, the present invention provides a method for preventing or treating a disease, comprising the step of administering to an individual in need thereof a cell-derived vesicle overexpressing ankyrin, wherein the cell-derived vesicle is loaded with a pharmaceutical agent for preventing or treating the disease.

[0043] In one example of the present invention, the drug may be one or more selected from the group consisting of antibodies or fragments thereof, therapeutic proteins and therapeutic peptides, but is not limited thereto.

[0044] In another example of the present invention, the drug can be bound to the anchor protein of the cell-derived vesicle or loaded inside or in the membrane of the cell-derived vesicle, but is not limited thereto.

[0045] In another example of the present invention, the cell-derived vesicle further comprises a targeting ligand, and the targeting ligand can be bound to the anchor protein and located outside the membrane of the cell-derived vesicle, but is not limited thereto.

[0046] In another example of the present invention, the cell-derived vesicles can bind to cells expressing the target of the targeting ligand, but is not limited thereto.

[0047] In another example of the present invention, the anticancer agent can be bound to an anchoring protein, but is not limited thereto.

[0048] Effects of the Invention

[0049] The present invention has discovered four anchoring proteins that conform to the inherent characteristics of CDV and can mediate the stable introduction of bioactive molecules as modified cell-derived vesicles that can be used as drug delivery carriers, thereby completing the present invention. It is confirmed that the anchoring protein is a membrane protein that is specifically abundant in CDV, and the CDV containing the anchoring protein can load bioactive molecules more stably. For example, the results of comparative experiments using fluorescent proteins confirmed that CDVs with introduced anchoring proteins are more effectively loaded with fluorescent proteins than CDVs without anchoring proteins. In addition, it is confirmed that when cancer cell targeting antibodies are loaded in the modified CDV of the present invention, not only the cancer cell targeting ability is increased, but also it is more effectively absorbed by cancer cells. That is, the CDV of the present invention, as a BioDrone modified by anchoring proteins, can stably load a variety of bioactive molecules and deliver them to the desired target, and is expected to be used in drug delivery and treatment platforms for a variety of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1a To curate a table of candidate anchoring proteins identified as being more abundant in cell-derived vesicles than in cells or exosomes.

[0051] Figure 1b The figure is a genetic diagram of the lentiviral vector used to express the candidate anchor protein.

[0052] Figure 2a and Figure 2b After transfection of the candidate anchor protein expression vector into HEK293 cells, the transduction efficiency was confirmed by flow cytometry analysis ( Figure 2a ) and the expression level of the EGFP gene contained in the vector ( Figure 2b ) results.

[0053] Figure 3 In order to confirm the optimal transformation conditions, adherent HEK293 cells (upper) or suspended HEK293 cells (lower) were treated with viral particles expressing candidate anchoring proteins (GFP, PTGFRN, and RAB7A) at different MOIs, and the transformation efficiency was compared by detecting GFP-positive cells and GFP fluorescence intensity by flow cytometry under various conditions.

[0054] Figure 4a and Figure 4b This is the result of constructing a cell line expressing a candidate anchor protein and confirming the transformation efficiency after transforming the cells with lentivirus under optimal conditions (MOI 5). Figure 4a The results of detecting GFP fluorescence 24 hours after transformation are shown. Figure 4b The results of evaluating the transduction efficiency (left graph) and GFP intensity (right graph) by flow cytometry are shown.

[0055] Figure 5 The figure shows the results of confirming GFP-expressing cells by flow cytometry in order to screen cells expressing the fusion protein at a high level.

[0056] Figures 6a to 6d The results of confirming the overexpression of each fusion protein (candidate anchor protein) in the constructed cell line using lentivirus. After cell sorting, the transformation efficiency was confirmed by flow cytometry analysis ( Figure 6a ) and GFP intensity ( Figure 6b ) and GFP was quantified by GFP enzyme-linked immunosorbent assay (ELISA) ( Figure 6c The expression of candidate anchoring proteins was assessed by Western blotting using antibodies against each protein ( Figure 6d ).

[0057] Figures 7a to 7c After obtaining cell-derived vesicles (ankyrin-CDV) from cells expressing the candidate ankyrin, the candidate ankyrin present in the CDV is confirmed. Figure 7a The histogram obtained from the nanoparticle flow cytometer measurement results of GFP-positive CDV is shown. Figure 7b and Figure 7c The GFP signal intensity and quantitative results of CDV expressing each anchor-GFP fusion protein are shown.

[0058] Figure 8a and 8b A diagram showing CDVs containing ankyrin screened for the BioDrone platform. Figure 8a A simplified diagram showing the anchoring proteins screened in HEK2593 cells and their GFP fluorescence. Figure 8b Results of evaluation of anchoring proteins using nanoparticle flow cytometry and ELISA are shown. The number of GFP(+) particles and GFP protein per CDV is shown in the table.

[0059] Fig. 9 A flow chart showing the process of extruding ankyrin-CDV from cells using a membrane filter or a depth filter (top) and a table summarizing CDV extrusion conditions for two ankyrin-overexpressing cells (HEK-BSG and HEK-LAMP1) (bottom).

[0060] Figures 10a to 10c Results comparing the properties of BioDrone Anchor Protein-CDV according to the extrusion method are shown. Fig.10a A histogram showing GFP(+) particles, Fig.10b The number of GFP(+) particles and the GFP intensity in ankyrin-CDV squeezed through a membrane filter or a depth filter are shown. Fig.10c The results of anchor protein detection by Western blotting are shown.

[0061] Fig.11 The topological analysis results of the anchor protein by protease cleavage analysis are shown. After the BioDrone anchor protein-CDV was treated with various concentrations of proteinase K (PK), each tagged protein was detected by western blotting. The 3x Flag tag at the N-terminus and the HA tag at the C-terminus of the anchor protein were detected using anti-Flag or anti-HA conjugated with HRP, respectively.

[0062] Fig.12aThis is the gene restriction map of the recombinant vector expressing the anchor protein-trastuzumab scFv (scTTZ) fusion protein.

[0063] Figure 12b This is a diagram showing the structure of LAMP1-scTTZ fusion protein, which is a representative example of ankyrin-scTTZ fusion protein. In order to detect the fusion protein, miRFPnano3 and nanoluciferase were added to the C-terminus.

[0064] Figure 13aa to Figure 13c The method is to prepare a transformed cell line expressing the anchor protein-scTTZ fusion protein using lentivirus and then confirm the expression of the fusion protein. Figure 13aa and Figure 13ab The results of confirming the miRFPnano3 fluorescence of cells by flow cytometry 24 hours after transformation are shown. The miRFPnano3 fluorescence intensity was measured 3 weeks after the start of puromycin selection ( Fig.13b ) and luciferase activity ( Fig.13c ) to confirm that the expression rate of the fusion protein was above 90%.

[0065] Fig.14 The results of Western blot analysis of scTTZ fusion protein expressed in Expi293F are shown. The fusion protein was detected using antibodies targeting each anchor protein (BSG, ATP1B3, LAMP1, LAMP2), anti-Flag antibody, and protein L.

[0066] Fig.15a and Fig.15b The results of confirming whether the ankyrin-trastuzumab scFv (scTTZ) fusion protein binds to the target (HER2) are shown. Fig.15a The figure shows the binding of the fusion protein to FITC-labeled HER2 recombinant protein (HER2-FITC). Fig.15b The graph shows the results of confirming the relative mean fluorescence intensity (MFI) of FITC after detecting HER2-FITC bound to ankyrin-scTTZ-expressing cells by flow cytometry.

[0067] Fig.16 The figures show the results of evaluating the proportion of RFP+ vesicles in all CDV particles in order to confirm whether ssTTZ is stably introduced into CDV.

[0068] Fig.17a and Fig.17bThe results of analyzing the characteristics of LAMP1-CDV introduced with scTTZ are shown ( Fig.17a : Western blot results; Fig.17b : Analysis results of the ratio of vesicles bound to HER2-FITC relative to RFP+ vesicles).

[0069] Fig.18 The graph shows the results of measuring the binding of scTTZ-gLAMP1-CDV and scTTZ-LAMP1-CDV to HER2-FITC by flow cytometry in order to confirm the influence of the glycosylation of the anchor protein LAMP1.

[0070] Fig.19 To confirm the results of HER2 expression levels in different cancer cells.

[0071] Fig. 20 In order to confirm the binding ability of the anchor protein-CDV introduced with scTTZ to HER2-expressing cells, CT26 (HER2-negative cell line) or CT26 / hHER2 (human HER2 gene expressing cell line) was treated with gLAMP1-CDV introduced with or without scTTZ, and the degree of CDV cell binding was measured by flow cytometry.

[0072] Figures 21a to 21c The results are to compare the cell binding degree of CDV after treating HER2 high-expressing cell lines (BT-474, SK-BR-3) and HER2 negative cell lines (MDA-MB-231) with gLAMP1-CDV introduced or not with scTTZ ( Fig.21a : Histogram of the fluorescence peak shift due to binding to CFSE-labeled CDV; Figure 21b : Quantitative results of CDV binding to each cell line; Fig.21c : CDV binding extent confirmed by fluorescence measurement in HER2 high-expressing cell lines).

[0073] Fig.22a and Figure 22b The results of confirming the degree of uptake of the anchor protein-CDV introduced into scTTZ by HER2-expressing cells over time are shown ( Fig.22a ) and the fold change results of scTTZ-gLAMP1-CDV relative to gLAMP1-CDV taken up by cells ( Figure 22b ).

[0074] Fig.23 The graph shows the results of evaluating the ratio of RFP+ vesicles in all CDV particles in order to confirm whether cetuximab is stably introduced into CDV.

[0075] Fig.24 After introducing GFP, which is a fluorescent protein, into CDV with or without the anchor protein of the present invention, the results of comparing the introduction rate of GFP into CDV by fluorescence signal analysis were performed.

[0076] Best Mode for Carrying Out the Invention

[0077] The present invention has been completed by discovering four anchoring proteins that can mediate the stable introduction of bioactive molecules in accordance with the inherent characteristics of CDV as modified cell-derived vesicles that can be used as drug delivery carriers. The anchoring proteins have been confirmed to be membrane proteins that are specifically abundant in CDV, and CDV containing the anchoring proteins can load bioactive molecules more stably than ordinary CDV.

[0078] Specifically, in one embodiment of the present invention, 12 candidate anchoring membrane proteins that are more abundant in CDV than in cells or exosomes are screened by proteomic analysis of CDV, and cell lines expressing these candidate anchoring proteins are constructed and extruded to obtain CDV expressing the anchoring proteins. By comparing the expression level and distribution of anchoring proteins in each CDV, four proteins (BSG, ATP1B3, LAMP1 and LAMP2) that are most effectively introduced into CDV are selected, and it is confirmed that the selected anchoring proteins maintain the topological structure in CDV as in cells, and maintain the characteristics regardless of the CDV extrusion method. It is thus confirmed that the four proteins can be effectively used in CDV deformations such as fusion with targeting ligands or introduction of therapeutic active substances (therapeutic cargo) as membrane proteins stably present on CDV particles, thereby selecting them as anchoring proteins for modifying the CDV of the present invention (Example 1).

[0079] Thus, it was confirmed whether the CDV expressing the selected anchor protein can actually be used as a BioDrone for delivering biologically active molecules. Using the scFv site of trastuzumab, an antibody to HER2 protein, as a targeting ligand, a cell line overexpressing a fusion protein of the targeting ligand and the anchor protein was prepared and extruded to prepare a HER2-targeted CDV (HER2-anchored CDV). The CDV analysis results confirmed that the HER2-targeted scFv was stably introduced into the CDV, and that the CDV effectively targeted and bound to cancer cells expressing HER2 by loading the scFv on the surface (Example 2).

[0080] Furthermore, the results of preparing CDV loaded with GFP as a fluorescent protein confirmed that the CDV of the present invention into which the anchor protein was introduced was more effectively loaded with GFP than the CDV into which the anchor protein was not introduced, thereby confirming that when the anchor protein of the present invention is used, active ingredients or substances can be loaded in the CDV with higher efficiency (Example 3).

[0081] As described above, the CDV of the present invention is a BioDrone modified with an anchor protein, which can stably load a variety of bioactive molecules and deliver them to desired targets, and is expected to be used as a drug delivery and treatment platform for a variety of drugs.

[0082] Hereinafter, the present invention will be described in detail.

[0083] The main purpose of the present invention is to provide cell-derived vesicles overexpressing ankyrin.

[0084] In the present invention, "cell derived vesicles (CDVs)" refers to vesicles artificially prepared in nucleated cells. CDV can be generated from the cell membrane in almost all types of cells, and is characterized by having a double-layer phospholipid model morphology as a cell membrane structure. The cell-derived vesicles of the present invention are different from vesicles naturally secreted from cells, such as exosomes. Throughout this specification, the term "vesicle (vesicles or "vesicles")" refers to a vesicle that is divided into an inside and an outside by a lipid bilayer membrane composed of cell membrane components of the cell from which it is derived, has cell membrane lipids and cell membrane proteins, nucleic acids and cell components of the cell, and is smaller in size than the original cell, but is not limited to this.

[0085] The cell-derived vesicles of the present invention have a size of micrometer level. For example, the diameter of the CDV may be less than 0.2 μm. More specifically, the diameter of the CDV may be 10 nm to 100 nm, 20 nm to 100 nm, 50 nm to 100 nm, 50 nm to 300 nm, 50 nm to 400 nm, 50 nm to 500 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 180 nm, 50 nm to 160 nm, 50 nm to 150 nm, 50 nm to 100 nm, 100 nm to 300 nm, 100 nm to 250 nm, 100 nm to 200 nm, 100 nm to 180 nm, 120 nm to 300 nm, 150 nm to 300 nm, or 130 nm to 170 nm, but is not limited thereto.

[0086] The CDV of the present invention may have a size ranging from nanometers to micrometers. For example, the diameter of the CDV may be 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 50 nm to 150 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, 100 nm to 200 nm, 100 nm to 150 nm, 130 nm to 500 nm, 130 nm to 400 nm, 130 nm to 300 nm, or 130 nm to 200 nm, but is not limited thereto.

[0087] Furthermore, the surface charge of the CDV of the present invention may be a positive charge. For example, the Zeta potential of the CDV may be -20mV to +50mV, -20mV to +30mV, -20mV to +20mV, -20mV to +10mV, -20mV to +0mV, -20mV to -5mV, -20mV to -10mV, -15mV to -5mV, -15mV to -10mV, -13mV to -10mV, -12mV to -10mV, +10mV to +50mV, +20mV to +50mV, +30mV to +50mV, or +40mV to +50mV, but is not limited thereto.

[0088] The CDV of the present invention can be prepared using a method selected from the group consisting of extruding a suspension containing nucleated cells, ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical decomposition, and physical stimulation treatment by applying force to cells from the outside, but is not limited thereto. For example, the CDV of the present invention can be obtained by extruding a suspension containing cells using an extruder. The extrusion force of the extruder applied to prepare the CDV can be 5psi to 200psi, 10psi to 150psi, 10psi to 100psi, 10psi to 50psi, 10psi to 40psi, or 50psi to 100psi.

[0089] Furthermore, the cell-derived vesicles may be subjected to a step of removing the cell nucleus before the step of extruding the sample containing the cells into the micropores. The cell nucleus may be removed by centrifugation.

[0090] Furthermore, the vesicles of the present invention can be obtained by squeezing a sample containing cells into a micropore, preferably, the micropores of size from large to small can be used to squeeze the cells in sequence to obtain the vesicles. The diameter of the micropores can be 0.01 μm to 100 μm, 0.01 μm to 80 μm, 0.01 μm to 60 μm, 0.01 μm to 40 μm, 0.01 μm to 20 μm, 0.01 μm to 15 μm, 0.01 μm to 10 μm, 0.01 μm to 7 μm, 0.01 μm to 3 μm, 0.01 μm to 1 μm, 0.01 μm to 0.5 μm, 0.01 μm to 0.1 μm, 0.1 μm to 20 μm or 0.1 μm to 0.5 μm, but is not limited thereto. For example, the sequential extrusion can be achieved by using filters with pore diameters of 5 μm to 20 μm, 2 μm to 7 μm, 0.7 μm to 3 μm, and 0.1 μm to 0.5 μm, respectively. The size of the micropores can be appropriately adjusted according to the type of cells used. The CDV obtained by the process can also be subjected to an additional purification process.

[0091] Furthermore, the vesicles of the present invention can also be prepared by a preparation method comprising the step of extruding a sample containing cells through a depth filter. The retention rate of the depth filter can be 0.1 μm to 1 μm, 0.1 μm to 0.9 μm, 0.1 μm to 0.8 μm, 0.1 μm to 0.7 μm, 0.1 μm to 0.65 μm, 0.2 μm to 1 μm, 0.4 μm to 1 μm, 0.5 μm to 1 μm, 0.6 μm to 1 μm, 0.2 μm to 0.8 μm, 0.4 μm to 0.8 μm, 0.4 μm to 0.7 μm, 0.5 μm to 0.7 μm or 0.6 μm to 0.7 μm, but is not limited thereto. The extrusion force of the deep filter can be 5psi to 200psi, 10psi to 150psi, 10psi to 100psi, 10psi to 50psi, 10psi to 40psi or 50psi to 100psi, but is not limited thereto. In one example of the present invention, in order to squeeze the cells, the cell-derived vesicles of the present invention can be obtained by applying pressure to squeeze after placing a suspension containing cells into a device equipped with a deep filter. The pressure can be the pressure of nitrogen (N2). For example, in the case of preparing CDV by passing cells through a deep filter, the CDV can be squeezed by applying a nitrogen pressure of 0.1bar to 5bar, 0.1bar to 4bar, 0.1bar to 3bar, 0.1bar to 2.5bar, 0.3bar to 3bar, 0.1bar to 2.5bar or 1bar to 2.5bar, but is not limited thereto. The CDV obtained by the process can also undergo an additional purification process.

[0092] The purification process is used to remove other substances other than the CDV of the present invention (vesicles or other contaminants such as proteins, nucleic acids, etc. that are smaller in size than the CDV of the present invention) and obtain only the target CDV. Preferably, it can be achieved by adding 1 to 5 times, 1 to 4 times or 1 to 3 times the volume of the buffer solution relative to the total volume of the mixed solution of cell-derived vesicles and liposomes.

[0093] Specifically, the CDV generated by the extrusion step can be purified by passing it through a tangential flow filtration (TFF) process. In this case, preferably, the membrane used has a cutoff of 800 kDa, 750 kDa, 700 kDa, 650 kDa, 600 kDa, 500 kDa, 450 kDa, 400 kDa, 350 kDa or 300 kDa or more, but is not limited thereto. In the tangential flow filtration process, a concentration step of concentrating the concentration of the CDV suspension by more than 5 times and a step of replacing the buffer are performed. The CDV can be concentrated and impurities can be removed by the process.

[0094] After CDV is obtained through the tangential flow filtration process, a size exclusion chromatography process may be performed for additional purification. In this case, preferably, a column with a recovery range of about 35 nm to 350 nm or 70 nm to 1000 nm is used, and CDV is equivalent to particles of larger size, and CDV can be obtained by collecting fractions eluted through the column. Through the above purification process, vesicles smaller than CDV and other impurities such as proteins can be removed and high-purity uniform nano-sized cell-derived vesicles can be obtained.

[0095] In the present invention, "cell-containing sample" may be a sample containing nucleated cells or transformed cells thereof, and is a concept including culture fluid, suspension, dilution, etc. of the cells. The concept of cells includes, without limitation, cells capable of preparing vesicles.

[0096] Specifically, the "cells" of the present invention can use cells that can be separated from cell-derived vesicles without restriction, including all cells isolated from natural biological individuals. Preferably, the cell is a nucleated cell. In addition, the cell can be derived from any type of animal or plant, including humans and non-human mammals. Therefore, the type of cells from which the cell-derived vesicles of the present invention can be obtained is not particularly limited. For example, the cell can be a stem cell, an immune cell, a blood cell, a (human) embryonic cell, a fat cell and / or an embryonic kidney cell. For example, the cell-derived vesicle can be obtained from stem cells, kidney cells, embryonic kidney cells, nuclear cells (HEK293), cancer cells, acinar cells, myoepithelial cells, erythrocytes, immune cells, monocytes, dendritic cells, natural killer cells, T cells, B cells, macrophages, endothelial cells, epidermal cells, nerve cells, glial cells, astrocytes, muscle cells and platelets. Furthermore, the cells of the present invention may be a variety of immune cells, tumor cells, stem cells, acinar cells, myoepithelial cells or platelets. In one example of the present invention, the stem cells may be one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells. In other examples of the present invention, the stem cells may be mesenchymal stem cells derived from fat cells or mesenchymal stem cells derived from umbilical cord, but are not limited thereto.

[0097] In the present invention, "anchor protein" refers to a membrane protein located in the membrane of CDV, which mediates CDV loading of bioactive molecules. The anchor protein is located in the CDV membrane and loads the molecule in CDV by binding to the bioactive molecule. The bioactive molecule can be combined with the anchor protein after preparing the CDV expressing the anchor protein, or expressed in the cell in the form of binding to the anchor protein (i.e., fusion protein) to be loaded in CDV. The anchor protein of the present invention is derived from the mother cell of CDV, and can be a membrane protein of the plasma membrane of the cell, or can be a membrane protein derived from organelles such as lysosomes. The anchor protein of the present invention, as a transmembrane protein, can be a structure comprising an extracellular domain, a transmembrane domain, and an intracellular domain. Therefore, the anchor protein of the present invention exists in a state of being inserted into (or penetrating) the membrane of CDV, and the extracellular domain located outside the membrane can bind to other bioactive molecules. In addition, the intracellular domain located inside the membrane may be combined with other marker proteins (fluorescent proteins, labels, etc.) for detecting the anchoring protein. In this specification, CDV containing anchoring proteins may be referred to as anchoring protein-CDV, BioDrone CDV, etc. In the present invention, "CDV overexpressing anchoring proteins" refers to CDVs that contain excessive anchoring proteins through genetic engineering, and refers to CDVs that contain anchoring proteins at a higher level than the original CDVs naturally derived from the same mother cells. The anchoring protein can be directly overexpressed in CDV, but preferably, CDV can also be transformed with anchoring proteins by delivering excess anchoring proteins from mother cells transformed by anchoring proteins to CDV. In the present invention, transformation refers to the introduction and expression of exogenous genes, or the introduction of exogenous proteins.

[0098] The anchoring protein of the present invention is a protein specific to CDV. For example, the anchoring protein may be characterized in that it is present in CDV at a particularly high level compared to cells, other cell-derived organelles, or cell-derived endoplasmic reticulum. Specifically, the anchoring protein may be characterized in that it is present in CDV at a higher level than the mother cell from which CDV originates or the exosomes or extracellular vesicles produced by the mother cell. In addition, the anchoring protein may be a natural protein that exists naturally in the mother cell and is delivered to CDV, or an exogenous protein expressed from an exogenous gene introduced into the mother cell and delivered to CDV. In addition, the anchoring protein introduced into CDV may be glycosylated or not glycosylated.

[0099] Preferably, the anchoring protein can be selected from Basigin (BSG), ATP1B3 (ATPase Na + / K + Basigin is a transmembrane glycoprotein of the plasma membrane that can bind to a variety of ligands including cyclophilin protein. General information about Basigin can be confirmed in NCBI, a gene database, with Gene ID 682. ATP1B3 is Na+ / K+transporting subunit beta 3, LAMP1 (lysosomal-associated membrane glycoprotein 1) and LAMP2 (lysosomal-associated membrane glycoprotein 2). + / K + and H + / K + A downstream class of ATPase beta chain proteins, it is a membrane protein that properly maintains the electrochemical distribution of sodium (Na) and potassium (K) ions across the membrane, and is essential for regulating osmotic pressure, transporting organic / inorganic molecules, and electrical excitation of nerves. General information about ATP1B3 can be found in NCBI as Gene ID 483. LAMP1 and LAMP2 are glycoproteins present in the membrane of lysosomes, and are involved in lysosomal biosynthesis, autophagy, cholesterol homeostasis, etc. General information about LAMP1 and LAMP2 can be found in NCBI as Gene ID 3916 and Gene ID 3920, respectively.

[0100] The anchoring protein ATP1B3 of the present invention may include the amino acid sequence of SEQ ID NO: 1 or be formed by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Alternatively, the ATP1B3 protein may be encoded by a nucleic acid molecule including the nucleotide sequence of SEQ ID NO: 6 or be encoded by a nucleic acid molecule formed by the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

[0101] The anchor protein BSG of the present invention may include the amino acid sequence of SEQ ID NO: 2 or be formed by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. Alternatively, the BSG protein may be encoded by a nucleic acid molecule including the nucleotide sequence of SEQ ID NO: 7 or be encoded by a nucleic acid molecule formed by the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

[0102] The anchoring protein LAMP1 of the present invention may include the amino acid sequence of SEQ ID NO: 3 or be formed by the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. Alternatively, the LAMP1 protein may be encoded by a nucleic acid molecule including the nucleotide sequence of SEQ ID NO: 8 or be encoded by a nucleic acid molecule formed by the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

[0103] The anchoring protein LAMP2 of the present invention may include the amino acid sequence of SEQ ID NO: 4 or be formed by the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. Alternatively, the LAMP2 protein may be encoded by a nucleic acid molecule including the nucleotide sequence of SEQ ID NO: 9 or be encoded by a nucleic acid molecule formed by the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.

[0104] On the other hand, the polypeptide (or nucleic acid molecule) represented by a specific sequence in this specification may include not only related sequences, but also other biological equivalents. That is, if the variation with biological equivalent activity of the polypeptide (nucleic acid molecule) is considered, the polypeptide (or nucleic acid molecule) of one embodiment is also interpreted as including a sequence showing essential identity (substantial identity) with the sequence shown in the sequence number. Specifically, the polypeptide (nucleic acid molecule) comprising the amino acid sequence (nucleotide sequence) represented by the specific sequence number is not limited to the related amino acid sequence (nucleotide sequence), and the variant of the amino acid sequence (nucleotide sequence) is also included in the scope of the present invention. The polypeptide molecule (nucleic acid molecule) formed by the amino acid sequence (nucleotide sequence) represented by the specific sequence number of the present invention is a concept of an equivalent of the polypeptide molecule (nucleic acid molecule) constituting it, for example, a part of the amino acid sequence (nucleotide sequence) of the polypeptide molecule (nucleic acid molecule) is deformed by deletion (deletion), substitution (substitution) or insertion (insertion), but can play a variant (variants) with the same function as the related polypeptide (nucleic acid molecule). Specifically, the polypeptide (nucleic acid molecule) disclosed in the present invention may include an amino acid sequence (nucleotide sequence) having a sequence homology of more than 70%, more preferably more than 80%, more preferably more than 90%, and most preferably more than 95% with the amino acid sequence represented by the specific sequence number. For example, it may include a polypeptide (nucleic acid molecule) having a sequence homology of 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%. The "% sequence homology" of the relevant polypeptide (nucleic acid molecule) is determined by comparing the comparison region of the two sequences optimally aligned, and a portion of the polypeptide sequence (nucleotide sequence) in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (excluding additions or deletions) in which the two sequences are optimally aligned.

[0105] The anchoring protein can bind to a biologically active molecule, and thereby the biologically active molecule can be loaded into the CDV.

[0106] In this specification, "bioactive molecules" is a general term for substances with biological or pharmaceutical activity, such as substances that target specific proteins (e.g., antibodies or ligands, etc.), substances that penetrate into cells (cytoplasm or nucleus) and are related to physiological activity regulation, substances that can express pharmacological effects, or substances that play a role in transporting to cells, and refers to substances with biological activity in various parts of the body such as cells, tissues, intercellular matrix, blood, etc. The bioactive molecules of the present invention are characterized in that they are loaded in CDV by binding to anchor proteins and still maintain their inherent biological activity. For example, the bioactive molecule can be a targeting ligand or a therapeutic substance. In the present invention, a targeting ligand refers to a component that can specifically recognize and target a specific substance (e.g., an antigen). Preferably, a targeting ligand can induce a CDV loaded with the ligand to bind to a cell expressing the specific antigen, etc. by targeting a specific antigen. In the present invention, a therapeutic substance refers to a substance that has a preventive, ameliorative and / or therapeutic effect on a disease. The type of the substance is not limited.

[0107] In the present invention, the bioactive molecule is loaded in the CDV by binding to the anchor protein, in which case the bioactive molecule may be located outside the membrane of the CDV (i.e., outside the CDV), inside the membrane of the CDV (i.e., inside the lipid bilayer), or inside the CDV. That is, the bioactive molecule may be located outside or inside the membrane of the CDV, preferably, the bioactive molecule may be located outside the membrane of the CDV by binding to the extracellular domain of the anchor protein.

[0108] The bioactive molecule can be selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, viruses, semi-synthetic substances, quantum dots, fluorescent pigments, toxins and complexes thereof. Preferably, the bioactive molecule of the present invention is a protein, glycoprotein or peptide.

[0109] In the present invention, the protein can be one or more selected from the group consisting of antibodies, antibody fragments, growth factors, enzymes, nucleases, transcription factors, antigenic peptides, hormones, transport proteins, immunoglobulins, structural proteins, motor function proteins, signaling proteins, connecting proteins, viral proteins, natural proteins, recombinant proteins, protein complexes, fluorescent proteins, therapeutic proteins, chemically modified proteins and prions, but is not limited thereto.

[0110] Non-limiting examples of the nucleic acid include DNA, RNA, ASO (antisense oligonucleotide), microRNA (miRNA), small interfering RNA (siRNA), aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino, etc.

[0111] Non-limiting examples of the compound include therapeutic drugs, toxic compounds, chemical compounds, etc. The "drug" is a broad concept including substances used to alleviate, prevent or diagnose diseases, wounds or specific symptoms. That is, the cell-permeable peptide of the present invention can be used as a delivery body for drugs for preventing or treating diseases.

[0112] In addition, the bioactive molecules of the present invention may include cholesterol, chemotherapeutics, vitamins, co-factors, 2,5-A chimeras, allozymes, aptamers, molecules that can regulate the pharmacokinetics and / or pharmacodynamics of polymers such as polyamines, polyamides, polyethylene glycols and polyethers, etc.

[0113] In one example of the present invention, the bioactive molecule is an antibody or a fragment thereof. In the present invention, "antibody" refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen (epitope), including polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. In addition, the term may include forms produced by genetic engineering such as chimeric antibodies (e.g., humanized mouse antibodies) and dual-binding antibodies (e.g., bispecific antibodies). The antibody comprises a variable region (VH: heavy chain variable region; VL: light chain variable region) of a heavy chain and / or a light chain in the composition. The variable region, as a primary structure, includes a portion that forms an antigen-binding site of an antibody molecule, and the antibody of the present invention may be composed of a fragment comprising a portion of the variable region. A linker may be provided between the light chain and heavy chain variable regions of the antibody or its antigen-binding fragment. The "linker" refers to a polypeptide that connects the light chain variable region and the heavy chain variable region to each other without damaging their original antigen-binding properties.

[0114] In the present invention, the antibody may be one or more selected from the group consisting of full-length antibodies, Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, miniantibodies, bispecific antibodies and nanoantibodies, but is not limited thereto.

[0115] In the present invention, the "fragments" of antibodies refer to (functional) fragments that retain the antigen binding function of antibodies, preferably, antigen binding fragments of the antibodies. The fragments are used not only to include scFv, (scFv)2, Fab, Fab' and F(ab')2, but also to include miniantibodies, bispecific antibodies, nanoantibodies or fragments thereof. The definition of the fragments is well known in the technical field to which the present invention belongs.

[0116] More specifically, a "single-chain Fv" or "scFv" antibody fragment refers to a protein in which the variable regions of the light chain and heavy chain of an antibody are connected using a linker formed by a peptide sequence connected by about 15 amino acids. These domains exist in a single polypeptide chain. The Fv polypeptide can contain a polypeptide linker between the VH domain and the VL domain in order to allow the scFv to bind to the antigen to form the desired structure. The term "Fv" fragment used in this specification is an antibody fragment containing a complete antibody recognition and binding site. Such a region can be formed by forming a dimer that is firmly covalently linked to one heavy chain variable region and one light chain variable region, for example, through scFv.

[0117] The "nanobody" is an antibody variable region composed only of heavy chains, also known as a VHH domain. Compared with conventional antibodies, nanobodies have a smaller size and a more stable structure, which not only improves tissue permeability and antigen accessibility, but also has the advantage of minimizing immune side effects caused by Fc.

[0118] Antibodies show antigen specificity based on sequence changes in the variable region. The variable region of the antigen binding site is divided into a structural region with less variability (framework region: FR) and a complementary determining region (CDR) with greater variability. Both the heavy chain and the light chain have three CDR sites divided into CDR1, CDR2 and CDR3 and four FR sites. The complementary determining region is a site in the variable region of an antibody that confers binding specificity to an antigen. Typically, the CDRs of each chain are named CDR1, CDR2, and CDR3 in order from the N-terminus, and are identified by the chain provided with a specific CDR.

[0119] The CDV of the present invention may contain two or more bioactive molecules. Each bioactive molecule may be of the same type or different types. For example, the CDV of the present invention may contain a therapeutic drug as a bioactive molecule together with a targeting ligand that targets cells, tissues or organs. Alternatively, the CDV of the present invention may contain a marker protein for marking the CDV while containing a targeting ligand or a therapeutic drug as a bioactive molecule. Each bioactive molecule may be in a form that is combined with an anchoring protein, or in a form that is respectively combined with different anchoring proteins, or in a form in which the first bioactive molecule is combined with the anchoring protein and the second bioactive molecule is loaded on the membrane or inside of the CDV. In the case of a form in which two or more bioactive molecules are combined with an anchoring protein, the bioactive molecules may be in a form in which they are sequentially combined with one end of the anchoring protein, or in a form in which they are respectively combined with both ends of the anchoring protein.

[0120] A person skilled in the art of the present invention can select appropriate bioactive molecules according to the target, and load them into CDV through the anchor protein of the present invention. For example, the bioactive molecule can be a targeting ligand that targets specific tissues, organs, or cells. Therefore, a CDV loaded with a targeting ligand can specifically recognize the target of the ligand. For example, in the case where the targeting ligand is an antibody or a fragment thereof, a CDV loaded with the ligand can recognize and bind to cells expressing the target (antigen) of the ligand. For example, in the case of a CDV loaded with an antibody or a fragment thereof targeting brain cells or endothelial cells of the blood-brain barrier (BBB), the antibody or fragment can be moved to the brain tissue, and a therapeutic agent for brain disease can also be loaded to deliver the therapeutic agent specifically to the brain.

[0121] Furthermore, the bioactive molecule may be a tumor antigen-specific antibody or a fragment thereof targeting cancer cells. For example, the bioactive molecule may be a targeted anticancer agent. The tumor antigen includes not only tumor-specific antigens expressed only in cancer cells, but also all tumor-associated antigens that can be expressed in normal cells but are particularly expressed at a high frequency in cancer cells or are more active in cancer cells. For example, the tumor antigen may be a surface protein that is expressed only in cancer cells or expressed at a higher frequency in cancer cells. As one embodiment of the present invention, the inventors used a fragment (scFv) of trastuzumab targeting HER2 as a bioactive molecule to prepare a HER2-specific CDV loaded with the fragment, and confirmed that the CDV specifically binds to cancer cells expressing HER2.

[0122] The complex of the biologically active molecule and the anchoring protein can be connected by physical binding, chemical binding, covalent bond, non-shared binding, peptide bond or self-assembly, or connected (bound) by a medium (eg, a linker) into an integrated or fused form.

[0123] In one example of the present invention, the bioactive molecule and the anchoring protein can be a complex generated by expressing them in a fusion state. For example, if a gene encoding the bioactive molecule and a gene encoding the anchoring protein are inserted into a vector, and the vector is used to transform an organism to express the genes inserted into the vector, the bioactive molecule and the anchoring protein are expressed as a fusion protein. Furthermore, when expressed as a fusion protein, any linker may be included between the bioactive molecule and the anchoring protein.

[0124] Furthermore, the present invention provides cells (or cell lines) for producing the cell-derived vesicles of the present invention. The description of the cells has been mentioned above and is omitted here.

[0125] Specifically, the cell is characterized in that an exogenous anchoring protein encoding gene is introduced, and the anchoring protein can be one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2. That is, the cell for producing cell-derived vesicles of the present invention can be a cell that overexpresses the anchoring protein.

[0126] The anchor protein encoding gene can be introduced into cells by inserting a recombinant vector. Therefore, the present invention can provide a recombinant vector into which the gene encoding the anchor protein is inserted as a recombinant vector for preparing the CDV of the present invention. In addition, the present invention can provide a cell into which the recombinant vector is introduced. The recombinant vector can also contain a bioactive molecule encoding gene together with the anchor protein encoding gene. Preferably, the bioactive molecule can be expressed in a state of being bound to the anchor protein. For example, the anchor protein encoding gene and the bioactive molecule encoding gene can be expressed from the recombinant vector in the form of a fusion protein.

[0127] In the present invention, "recombinant vector" refers to a vector that can express peptides or proteins encoded by different types of nucleic acids inserted into the vector, preferably, a vector prepared in a manner capable of expressing a target protein (in the present invention, progranulin protein or a fragment thereof). The "vector" refers to any medium used to introduce and / or transfer bases into a host cell in vitro, in vitro or in vivo, and can be a replicon that brings about the replication of the bound fragment by binding to other DNA fragments. "Replicon" refers to any genetic unit that functions as a self-unit of DNA replication in vivo, that is, any genetic unit that can replicate by its own regulation (e.g., plasmid, bacteriophage, cosmid, chromosome, virus, etc.).

[0128] The vector of the present invention may be a linear DNA, a plasmid DNA or a recombinant viral vector, but is not limited thereto. The vector may include, for example, a plasmid vector, a cosmid vector, a phage vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, an adeno-associated virus vector and other viral vectors.

[0129] Preferably, the recombinant vector of the present invention may include a promoter as a transcription initiation factor binding to RNA polymerase, an arbitrary operator sequence for regulating transcription, a sequence encoding an appropriate mRNA (messenger RNA) ribosome binding site, and a sequence regulating the termination of transcription and interpretation, a terminator, etc. More preferably, it may also include a polyhistidine tag (an amino acid motif consisting of at least 5 or more histidine residues), a signal peptide gene, an endoplasmic reticulum retention signal peptide, a cloning site, etc. It may also include a tag gene, a screening marker gene such as an antibiotic resistance gene for screening transformants, etc. In the recombinant vector, the polynucleotide sequence of each gene is operably linked to the promoter. The term "operably linked" used in this specification refers to the functional combination between a nucleotide expression regulatory sequence such as a promoter sequence and other nucleotide sequences, through which the regulatory sequence regulates the transcription and / or interpretation of the other nucleotide sequences.

[0130] In the present invention, the gene or the recombinant vector can be transduced or transfected (transfection) to a virus production cell, i.e., to a packaging cell line. For "transduction" or "transfection", a variety of techniques used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, for example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection, etc. The virus containing the target gene (a gene encoding an anchor protein or a gene encoding a fusion protein of a biologically active molecule and an anchor protein) of the present invention can be proliferated in the packaging cell line and released outside the cell, and the virus can be transduced (transduced; transformed) to the target cell (i.e., a cell producing the CDV of the present invention). The nucleic acid of the virus transformed into the cell can be used in the production of the target protein (anchor protein or a fusion protein of an anchor protein and a biologically active molecule) by inserting into the genome of the cell or without inserting into the genome.

[0131] The present invention can provide cells separated by introduction (transformation, transfection, transduction, etc.) of the recombinant vector of the present invention. The cell cell here means a cell used to proliferate (amplify) the recombinant vector, rather than a cell that ultimately produces CDV. That is, the cell represents a host cell that directly transduces / transforms / transfects the aforementioned nucleic acid molecule or recombinant vector. For example, in the case where the expression vector is a viral vector, the cell can be a packaging cell for generating a virus containing the viral vector. Regarding the selection of an appropriate host, a person of ordinary skill in the art to which the present invention belongs can be self-evident through the enlightenment of this application.

[0132] In addition, the present invention provides a method for preparing cell-derived vesicles, comprising: step (S1), introducing a recombinant vector containing an anchor protein encoding gene into cells; and step (S2), obtaining cell-derived vesicles by extruding the cells into which the recombinant vector has been introduced.

[0133] The step (S1) is a step of replicating the expression vector inside the cell after introducing (transforming, transducing, transfecting) the recombinant vector into an appropriate host cell, or expressing a protein from the expression vector by induction. A person skilled in the art of the present invention can select an appropriate introduction method according to the type of vector and cell. For example, in the case where the recombinant vector is a viral vector, the vector can be introduced into the cell by infecting the cell with a virus containing the recombinant vector. For example, the recombinant vector can be introduced into the cell by a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a herpes simplex virus and / or a vaccinia virus containing the recombinant vector, preferably, it can be introduced into the cell by a lentivirus. In this case, the cell can be introduced into the recombinant vector by 1MOI to 30MOI, 1MOI to 20MOI, 1MOI to 15MOI, 1MOI to 10MOI, 1MOI to 8MOI, 1MOI to 7MOI or 1MOI to 5MOI of a virus (lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and / or vaccinia virus containing the recombinant vector). When two or more viruses are used to introduce a recombinant vector into a cell, a person skilled in the art can appropriately combine the viruses within the range of 1MOI to 30MOI, 1MOI to 20MOI, 1MOI to 15MOI, 1MOI to 10MOI, 1MOI to 8MOI, 1MOI to 7MOI or 1MOI to 5MOI to introduce the recombinant vector into the cell.

[0134] Therefore, the step (S1) may include a step of expressing the target gene introduced into the cell in the cell by culturing. The culturing may be performed after the recombinant vector is introduced into the cell and after a sufficient time for the target protein to be expressed in the cell. More preferably, the culturing may be performed after the target protein is expressed in the cell and after a sufficient time for the CDV containing the target protein to be produced.

[0135] The step (S2) is a step of obtaining cell-derived vesicles from cells that overexpress the anchor protein and / or bioactive molecule of the present invention by introducing the recombinant vector. The CDV can be obtained by extruding a sample containing cells. The extrusion can be performed using a filter containing micropores or a depth filter. The process of obtaining CDV by extruding cells has been described and its description is omitted here. The CDV obtained by the step (S2) is characterized in that it contains the anchor protein (and bioactive molecule) expressed from the recombinant vector.

[0136] The CDV obtained by the step (S2) is characterized in that the level of anchoring protein is higher than that of the cell (mother cell) into which the recombinant vector is introduced or the exosomes produced therefrom. Specifically, the level of anchoring protein of the CDV obtained by the step (S2) shows a level of at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, a level of 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more and / or a level of 0.5 times, 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or more, compared to the anchoring protein of the mother cell or its exosomes.

[0137] Furthermore, the present invention provides a composition for drug delivery comprising the vesicles derived from cells overexpressing ankyrin of the present invention as an active ingredient. In the present invention, "delivery" refers to delivery into a targeted cell, tissue or organ.

[0138] In the present invention, the drug is not limited to a specific type, and can include, without limitation, drugs (proteins, peptides, lipids, carbohydrates, nucleic acids, compounds, etc.) that have preventive, ameliorative, and / or therapeutic activities for specific diseases. As a non-limiting example, the drug can be one or more selected from the group consisting of antibodies or fragments thereof, therapeutic proteins, and therapeutic peptides.

[0139] In the present invention, the drug, as the bioactive molecule of the present invention, can be delivered to the targeted cell / tissue / organ in the form of binding to the anchor protein. In this case, the drug not only has the pharmacological activity for preventing / improving / treating a specific disease, but also can have the targeting function of the target at the same time. For example, the drug is an antibody-based anticancer agent (anticancer antibody). That is, the CDV of the present invention targets cancer cells through the anticancer antibody bound to the anchor protein, so that the anticancer activity can be exerted. The specific type of the anticancer antibody is not limited, and can include anticancer antibodies known in the technical field to which the present invention belongs without limitation. As a non-limiting example, the anti-cancer antibody can be cetuximab, trastuzumab, rituximab, cixutumumab, ganitumab, dalotuzumab, figitumumab, teprotumumab, robatumumab, AVE1642, BIIB022, isiratumab or a fragment thereof.

[0140] The drug may not be bound to the anchor protein, but may be loaded separately on the membrane or inside of the CDV for delivery to the target. In this case, the CDV may also include a targeting ligand for targeting cells / tissues / organs as a biologically active molecule bound to the anchor protein. For example, in the case where the drug is an anticancer agent, the CDV may include an antibody or a fragment thereof that targets a tumor antigen as a targeting ligand, and the drug may be delivered by specifically binding the targeting ligand to cancer cells. Alternatively, in the case where the drug is a therapeutic agent for brain diseases, the CDV may include an antibody or a fragment thereof that targets brain cells, and the drug may be delivered by moving the targeting ligand to brain tissue.

[0141] Furthermore, the CDV of the present invention may have two or more bioactive molecules, which may be in the form of being respectively bound to anchor proteins. For example, the CDV of the present invention may be respectively bound to anchor proteins by simultaneously comprising a targeting ligand and a drug (e.g., a therapeutic protein, a therapeutic peptide, etc.) for targeting. Therefore, the CDV may target a target through a targeting ligand, and may exert therapeutic activity by delivering a drug to a relevant target.

[0142] The drug delivery composition can be used as a pharmaceutical composition for preventing or treating a specific disease. That is, the present invention provides a pharmaceutical composition for preventing or treating a disease. It comprises a cell-derived vesicle loaded with a drug and overexpressing an anchor protein as an effective ingredient.

[0143] For example, in the case where the bioactive molecule of the present invention has a preventive, ameliorative and / or therapeutic effect on a specific disease, the CDV loaded with the bioactive molecule can be used for the preventive, ameliorative and / or therapeutic purposes of the disease. In this case, the drug not only has therapeutic activity, but also has a targeting function. Alternatively, the CDV may also contain a targeting ligand.

[0144] In other examples of the present invention, the cell-derived vesicles may contain targeting ligands as bioactive molecules, and may also contain drugs for preventing, improving and / or treating diseases in the membrane or interior of the CDV. The targeting ligands target the CDV to cells, tissues or organs to which the drugs should be delivered, so as to prevent, improve and / or treat diseases.

[0145] The disease, as a disease related to the drug, is a myopathy that can be prevented, treated and / or improved by administering the drug. Therefore, a person skilled in the art of the present invention can select an appropriate drug to apply the present invention according to the disease to be prevented, treated and / or improved.

[0146] For example, in the case where the drug is an anticancer agent, the disease may be cancer. That is, the present invention may provide a pharmaceutical composition for preventing or treating cancer, which is a pharmaceutical composition for preventing or treating cancer comprising a cell-derived vesicle overexpressing the anchoring protein of the present invention as an effective ingredient, and the cell-derived vesicle is loaded with an anticancer agent. The anticancer agent not only has anticancer activity, but also has a function of targeting cancer cells (e.g., anticancer antibodies). The anticancer agent may be bound to the anchoring protein of the CDV membrane, or may be loaded in the CDV in a form separated from the anchoring protein (e.g., located in the membrane or interior of the CDV). For example, the anticancer agent may be exposed to the outside of the CDV in a state of being bound to the anchoring protein of the CDV membrane to target cancer cells, and exerts anticancer activity against cancer cells by inducing the binding of CDV to cancer cells. In the present invention, the specific type of cancer is not limited, but can be selected from colorectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, lymphoma, blood cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, skin cancer, skin melanoma, eye melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue granuloma, urethral cancer, gastrointestinal cancer, glioblastoma, ovarian cancer, endometrial cancer, salivary gland cancer, vulvar cancer and head and neck cancer. The specific type of the anticancer agent is not limited, and can include anticancer agents with anticancer activity known in the technical field to which the present invention belongs without limitation. For example, the anticancer agent can be a protein or peptide-based anticancer agent. Alternatively, the anticancer agent can be an antibody or a fragment thereof with anticancer activity. In one example, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a cell-derived vesicle overexpressing an anchoring protein as an effective ingredient, wherein the cell-derived vesicle is loaded with trastuzumab or a fragment thereof (scFv, etc.), wherein the trastuzumab or a fragment thereof can bind to the anchoring protein.

[0147] As another example, the disease is a brain disease, and the drug loaded in the CDV can be a therapeutic agent for the brain disease. The specific type of the brain disease is not limited, and can be selected from degenerative brain diseases, Parkinson's disease, Huntington's disease, Alzheimer's disease, mild cognitive impairment, senile dementia, amyotrophic lateral sclerosis, spinocerebellar ataxia, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia, Progressive Supranuclear Palsy, Frontotemporal Dementia, ischemic stroke, cerebral hemorrhage, cerebral infarction and stroke, etc. The CDV may comprise a targeting ligand for targeting brain tissue (eg, an antibody or fragment thereof specific for brain cells or the BBB, etc.).

[0148] When the composition of the present invention (drug delivery composition, pharmaceutical composition, etc.) is used for the purpose of treating a specific disease, the content of the cell-derived vesicles for nucleic acid molecule delivery in the composition can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the state of the patient, etc. For example, based on the total weight of the composition, it can be 0.0001 weight percent to 99.9 weight percent or weight percent or 0.001 weight percent to 50 weight percent, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0149] The composition of the present invention may also contain suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipients 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.

[0150] The composition of the present invention can be formulated into powders, granules, sustained-release capsules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, alcoholic agents, lozenges, aromatic water agents, lemonade agents, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, liquid extracts, injections, capsules, perfusion solutions, plasters, skin care agents, patches, sprays, inhalants, patches, sterile injection solutions or aerosols and other external preparations for use according to various common methods. The external preparations can have dosage forms such as creams, gels, masks, sprays, ointments, plasters, lotions, liniments, pastes or papules.

[0151] The carriers, excipients and diluents that the composition of the present invention may contain may be lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0152] In formulating the preparation, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. may be used.

[0153] The tablets, powders, granules, capsules, pills and lozenges of the present invention can use the following additives: corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, sodium carboxymethyl starch and other excipients; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, carboxymethyl cellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethyl cellulose, purified shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone and other binders; 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 anhydride, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein-treated gelatin, alginate, amylose, guar gum (Guar disintegrants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium syrup, kaolin, vaseline, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, polyethylene glycol (Macrogol), synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid and other lubricants.

[0154] Additives for the liquid preparation of the present invention may include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitan fatty acid esters (Tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamin, polyvinyl pyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like.

[0155] The syrup of the present invention may contain a solution of white sugar, other sugars or sweeteners, and may contain aromatics, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. as needed.

[0156] Pure water may be used in the emulsion of the present invention, and an emulsifier, a preservative, a stabilizer, a fragrance, etc. may be used as necessary.

[0157] The suspension of the present invention can use suspending agents such as gum arabic, gum tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like, and surfactants, preservatives, stabilizers, colorants, and fragrances can be used as needed.

[0158] The injection of the present invention may contain: distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, glucose injection, glucose + sodium chloride injection, polyethylene glycol (PEG), lactic acid intravenous injection, ethanol, propylene glycol, fixed oil-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, phenyl benzoate and other solvents; sodium benzoate, sodium salicylate, sodium acetate, urea, ethyl carbamate, monoethylacetamide, butazolidine, propylene glycol, Tweens, nicotinamide, hexamethylenetetramine, dimethylacetamide and other dissolving aids; weak acids and their salts (acetic acid and sodium acetate), weak bases and their Buffers such as salt (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gelatin; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium pyrosulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2), and ethylenediaminetetraacetic acid; antioxidants such as 0.1% sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as sodium carboxymethyl cellulose, sodium alginate, Tween 80, and aluminum monostearate.

[0159] The suppository of the present invention can use cocoa butter, lanolin, Withepsol, polyethylene glycol, glycerin gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, Lanet wax, glyceryl monostearate, Tween or Span, Imhausen, propylene glycol monostearate, glycerin, pharmaceutical grade mixed fatty acid glycerides (Adeps solidus), Buytyrum Tego-G, Cebes Pharma16, hexalidebase 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, Massa estrarium A, Massa estrarium AS, Massa estrarium B, Massa estrarium C, Massa estrarium D, Massa estrarium E, Massa estrarium I, Massa estrarium T, Massa-MF, masupol, masupol-15, Neosuppostal-N, paramound-B, supposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), suppostal (N, Es), wecoby (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and other bases.

[0160] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which are prepared by mixing the extract with one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0161] Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., which may contain a variety of excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc., in addition to the commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc.

[0162] The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to a sufficient amount for treating a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type of the patient's disease, the severity of the disease, the activity of the drug, sensitivity to the drug, the administration time, the administration route and metabolic ratio, the treatment period, the drugs used simultaneously, and other factors well known in the medical field.

[0163] The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. The key point is to obtain the maximum effect with the minimum amount without side effects after considering all the above factors, which can be easily determined by a person of ordinary skill in the art to which the present invention belongs.

[0164] The compositions of the present invention can be administered to an individual by a variety of routes. All modes of administration are envisioned, for example, administration can be by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, injection into the spinal space (intradural), sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, oral or nasal spray, dermally administration, transdermally administration, etc.

[0165] The dosage of the composition of the present invention is determined according to the disease to be treated, the route of administration, the patient's age, sex, body weight and the severity of the disease and other related factors and the type of medicine as an active ingredient. Specifically, the effective amount of the composition of the present invention can be different according to the age, sex, and body weight of the patient. Usually, 0.001 mg to 150 mg per 1 kg body weight, preferably, 0.01 mg to 100 mg is administered every day or every other day, or divided into 1 to 3 times a day for administration. However, it can be increased or decreased according to the route of administration, the severity of the disease, sex, body weight, age, etc. Therefore, no matter what method, the dosage does not limit the scope of the present invention.

[0166] The term "subject" in the present invention refers to a subject in need of disease treatment, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, pigs, sheep, and cattle.

[0167] The term "administering" in the present invention means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0168] "Prevention" in the present invention refers to all actions that inhibit or delay the onset of the target disease, "treatment" refers to all actions that change the symptoms of the target disease and its metabolic abnormalities to improvement or cure by administering the pharmaceutical composition of the present invention, and "improvement" refers to all actions that reduce the relevant parameters of the target disease, for example, reduce the degree of symptoms, by administering the composition of the present invention. DETAILED DESCRIPTION

[0169] Hereinafter, examples will be described to help understanding of the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples.

[0170] Example

[0171] Example 1. Preparation of cell-derived vesicles containing ankyrin (ankyrin-CDV)

[0172] The BioDrone platform using cell-derived vesicles is to construct an effective drug delivery system by introducing targeting ligands or active cargo into cell-derived vesicles through genetic engineering. Since the CDV used in the BioDrone platform is physically and chemically similar to extracellular vesicles (EVs), it has higher biocompatibility and lower immunogenicity compared to other nano-sized carriers, and also has greater advantages than EVs in productivity. In the deformation of effective CDV by genetic engineering, it is a very important basic step to identify the anchoring protein that is specifically stable and abundant in CDV in the development of the BioDrone platform of the present invention.

[0173] 1-1. Expression of candidate anchoring proteins using recombinant vectors

[0174] Through proteome analysis of cell-derived vesicles in previous studies, 12 membrane proteins that are more abundant in CDVs than in cells and exosomes were screened as candidate anchor proteins ( Figure 1a Fusion proteins were designed based on 12 candidate membrane proteins and 2 comparative proteins (scaffold proteins selected from Codiak Biosciences). In order to rapidly prepare cells overexpressing the proteins, lentiviral vectors were used. Cloning and viral particle preparation were performed in VectorBuilder. Cloning was performed in the third-generation lentiviral vector system provided by VectorBuilder. Figure 1b The fusion protein gene designed as shown. In order to confirm the expression of each protein and other various analyses, a 3×Flag tag was inserted at the N-terminus, and an EGFP and HA tag were inserted at the C-terminus ( Figure 1b ). The target gene (gene of interest, GOI) is designed to be located under the CMV promoter. In addition, EGFP is included for expression confirmation by fluorescence and quantitative analysis by enzyme-linked immunosorbent assay. That is, each candidate anchor protein is fused with GFP, and the candidate anchor protein is indirectly detected by GFP.

[0175] Although the expression of fusion proteins prepared using plasmids and virus particles was confirmed during the preparation process of VectorBuilder, it is hoped that the expression of fusion proteins can be verified by itself. HEK293 cells, a cell line derived from human embryonic kidney, were transformed using the provided plasmids and Lipofectamine, and protein expression was verified by analyzing fluorescence expression. As a result, the expression of fluorescent protein was confirmed in all plasmids, and the transformation efficiency showed 30% to 95% (depending on the plasmid). Figure 2a ). In addition, in the fluorescence intensity analysis, the fusion protein of BASP1, RAB7A, CNP and GNAI2 showed a fluorescence intensity of about 20,000 or more, and high expression was confirmed ( Figure 2b ).

[0176] 1-2. Preparation of cell lines expressing candidate anchoring proteins (stable cell lines)

[0177] In this example, a stable cell line stably expressing a candidate anchor protein was constructed. Before preparing a cell line by transformation, the transformation efficiency was confirmed according to the cell type (attached or suspended) and MOI. The virus particles of EGFP, PTGFRN and RAB7A fusion proteins showing a transformation efficiency of more than 50% in Example 1-1 were treated with different MOIs to attached and suspended HEK293 cells, and the transformation efficiency was compared. As a result, no significant difference with the cell type was shown, but it can be confirmed that the efficiency and expression increase according to the MOI ( Figure 3 ). A high MOI is beneficial for short-term efficiency, but there is a risk of cytotoxicity caused by the virus, so it was decided to use adherent HEK293 cells selected at MOI 5 and treated with antibiotics to eliminate untransformed cells for cell line preparation.

[0178] To prepare cell lines, virus particles treated with each fusion protein were used to confirm the transformation efficiency after 24 hours ( Figure 4a and Figure 4b ), and cell screening was started by treating with 2 μg / mL puromycin 2 days later. Cells showing resistance to antibiotics were cultured to 1×10 7 After cells are sorted, additional screening is performed using a cell sorter. Figure 5 As shown, after selectively obtaining cells with high fluorescence intensity, antibiotics were added after 3 to 5 days for cell stabilization and culture was maintained. The results of analyzing the cells screened in this way showed that the transformation efficiency was more than 90% in all cells, and it was confirmed that the screening of cells expressing fusion protein was successfully achieved ( Figure 6a ). Each cell type expressed fusion proteins to different extents. Cells expressing fusion proteins of BASP1, RAB7A, and CNP showed high fluorescence intensity, while ATP1A1 and NCSTN cells showed low fluorescence intensity ( Figure 6b ).

[0179] The results of analyzing the expression of fusion proteins using GFP enzyme-linked immunosorbent assay (Abcam, Cambridge, UK) showed a tendency different from the results of fluorescence intensity evaluation. The fusion proteins of RAB7A, BSG, BASP1, GNAI3, CNP, LAMP1 and PTGFRN showed high expression, while ITGB1, NCSTN, ATP1B3, KTN1, LAMP2, SCARB2 and ATP1A1 showed relatively low expression ( Figure 6c ). In addition, for qualitative analysis, Western blot analysis using antibodies that recognize each membrane protein showed that all membrane proteins were overexpressed, although the degree of expression varied. This confirmed that the expression of the fusion protein (candidate anchor protein) met the purpose of the study ( Figure 6d ).

[0180] 1-3. Confirmation of the characteristics of CDV isolated from cells overexpressing anchoring proteins and screening of final anchoring proteins

[0181] In this example, cell-derived vesicles were isolated from the transformed cells obtained by the example and their characteristics were confirmed. The transformed cells ensured by the example were cultured to induce the production of CDV (anchor protein-CDV), and the efficiency of the candidate anchor protein expressed in each cell to CDV delivery was confirmed. The proportion of GFP (+) vesicles was analyzed by nanoparticle flow cytometer (NanoFCM, Inc. Company, Xiamen, China), and the amount of fusion protein was quantified by GFP enzyme-linked immunosorbent assay.

[0182] As a result, it was confirmed that the ratio of fusion protein GFP(+) vesicles ranged from 3.1% to 69.5%. Figure 7a ), the amount of fusion protein has a greater deviation. The amount of LAMP1, LAMP2, BSG and ATP1B3 fusion proteins introduced into CDV is large, but the proportion of CNP and GNAI3 introduced into vesicles, which are highly expressed in cells, is low ( Figure 7b and 7c ).

[0183] The amounts and introduction ratios of PTGFRN and BASP1 from Codiak Biosciences selected for comparison in CDV were confirmed to be lower than those of other membrane proteins. This is presumed to be because CDV and extracellular vesicles have different production mechanisms and therefore show different introduction states.

[0184] Based on the above analysis results, it was confirmed that in order to transform CDV through the transformation of mother cells, membrane proteins specific to CDV were required, and therefore ATP1B3, BSG, LAMP1, and LAMP2 were selected as anchor proteins for the BioDrone platform.

[0185] 1-4. Confirmation of the final anchor protein for the BioDrone platform

[0186] The final four BioDrone Anchor proteins (BioDrone Anchor) screened by characterization of candidate anchor proteins of HEK-CDV were ATP1B3 and BSG, which are plasma membrane-derived proteins, and LAMP1 and LAMP2, which are known to be lysosome membrane-derived proteins.

[0187] After multiple passages, it was confirmed that each selected anchor protein was stably expressed in HEK293 cells. The schematic diagram of each anchor protein-CDV based on the original topology is shown in Figure 8a Furthermore, based on the ratio of GFP(+) particles as the result of nanoparticle flow cytometric analysis and the GFP quantitative value as the result of ELISA analysis, the number of GFP molecules (i.e., the number of molecules of indirect anchoring protein) present in each GFP(+)CDV particle was theoretically calculated ( Figure 8b ). For example, 152 and 122 GFP molecules were confirmed per BSG-CDV and LAMP1-CDV, respectively.

[0188] 1-5. Comparison of properties of anchor protein-CDV according to CDV extrusion method

[0189] There are many methods for extruding cell-derived vesicles used in the BioDrone platform, but in the test for the selected anchor protein, a membrane filter (NanoSizer MINI Liposome Extruder) was used on a small scale. This does not pose a risk in rapidly testing multiple candidate anchor proteins, but it is necessary to efficiently produce CDV in large quantities when developing and utilizing the BioDrone platform. Therefore, in order to derive the most appropriate CDV production method, it is necessary to confirm whether the characteristics of the CDV change according to the extrusion method. CDVs were extruded from two cell lines (HEK-BSG and HEK-LAMP1) that overexpressed the selected anchor proteins using either a membrane filter (ES-50) or a depth filter ( Fig. 9 ), to determine whether the properties of cell-derived vesicles vary with the extrusion method.

[0190] The distribution of GFP(+) particles was analyzed at the single particle level using CDV extruded using different methods. Fig.10a and Fig.10b ), and the abundance of anchoring proteins was compared by Western blotting ( Fig.10c ). As a result, it was confirmed that the anchor protein of the present invention showed a similar tendency regardless of the extrusion method and was present in each CDV. That is, there may be some difference in the anchor protein introduction rate, etc. depending on the type of anchor protein, but it was confirmed that there was no difference depending on the extrusion method.

[0191] 1-6. Analysis of the topological structure of the anchor protein introduced into CDV

[0192] In this example, the topology of anchor proteins was analyzed. To this end, anchor protein-CDV was treated with proteinase K to remove the extra-part of the protein present in the CDV membrane, and then the topology of each protein was confirmed by Western blotting.

[0193] When designing a plasmid construct, different tags (Flag tag and HA tag, respectively) were introduced into the N- or C-terminus for use in topological analysis of anchored proteins. The overall experiment outline is that after inducing the digestion of the extra-vesicular part of the membrane protein by treating with proteinase K, the degree of detection of the anchored protein was confirmed using antibodies against different tags introduced into the N- or C-terminus. If the part outside the vesicle corresponding to the anchored protein is the N-terminus, the degree of anchored protein detected by anti-Flag decreases with the treatment of proteinase K, and if it is the C-terminus, the degree of anchored protein detected by anti-HA decreases. It can be confirmed that after treating BSG-CDV and LAMP1-CDV with proteinase K, if the protein is detected using anti-HA, the degree of detection of proteins equivalent to the original size decreases, and after decomposing the proteins outside the vesicle, the degree of detection of small-sized proteins remaining inside increases. That is, it can be confirmed that BSG and LAMP1 still maintain their original topological structures after CDV introduction ( Fig.11 ) These results indicate that the anchoring protein of the present invention does not change its characteristics even when introduced into CDV.

[0194] In conclusion, four anchoring proteins ATP1B3, BSG, AMP1 and LAMP2 were screened out through the embodiments for modifying CDV, and it was confirmed that the anchoring proteins are stably present on CDV particles and can be introduced into CDV by fusion with targeting ligands or by introducing therapeutic cargo, thereby being used in a variety of ways in the effective and efficient modification of CDV for drug delivery.

[0195] Example 2. Development of HER2-targeted anchor protein-CDV

[0196] In order to confirm that the anchor protein-CDV of the present invention can be used as a drug delivery carrier, the targeting ligand or active substance (active cargo) is expressed in the form of a fusion protein with the anchor protein to introduce CDV, and then whether the CDV targets the target cell, binds to the cell, and is absorbed by the cell. Thus, in this embodiment, a CDV targeting HER2 (human epidermal growth factor receptor 2) is prepared and its effect is confirmed. In order to give CDV targeting directivity, the scFv (single-chain variable fragment) site of trastuzumab, which is a HER2 protein-specific antibody, is used as a targeting ligand. After constructing a cell line overexpressing trastuzumab's scFv (scTTZ) and obtaining an anchor protein-CDV imported with a HER2-targeted scFv therefrom, the tumor targeting ability of the anchor protein-CDV is evaluated in a HER2-positive tumor model.

[0197] 2-1. Preparation of trastuzumab overexpressing cell lines using lentiviral-based transformation

[0198] The targeting ligand is introduced into CDV using the four membrane proteins selected as BioDrone anchor proteins through the aforementioned examples. To this end, a cell line expressing the scFv (scTTZ) of trastuzumab, which is a HER2 antibody, is first prepared. The four selected membrane proteins (BSG, ATP1B3, LAMP1, and LAMP2) are more abundant in CDV than in cells or exosomes, and therefore, it is expected that the targeting ligand can be introduced into CDV more stably than when the anchor protein is used to overexpress the targeting ligand. In order to prepare a cell line using lentiviral transduction, a vector construct capable of expressing a fusion of the anchor protein and the trastuzumab scFv (scTTZ) is designed and lentiviral particles are prepared ( Fig.12a and Figure 12b ).

[0199] A total of 9 cell lines, including a control group, were prepared by transformation using the prepared lentiviral particles. 24 hours after transformation, it was confirmed that miRFPnano3 fluorescence could be observed under a fluorescence microscope and that the results of flow cytometry analysis showed that approximately 30% to 95% of the cells were fluorescent ( Figure 13aa and Figure 13ab The transformed cells were cultured for about a week, and the viability was about 90% at 5.0×10 5When the concentration of cells / mL reached 30mL, cells expressing scTTZ and anchoring proteins were selected by treating them with 2μg / mL of puromycin. After observing the cell status for about 3 weeks, the puromycin concentration was adjusted from 2μg / mL to 5μg / mL, and more than 90% expression was confirmed in all cell lines.

[0200] Furthermore, for the selected cells, the results of comparing the fluorescence intensity of miRFPnano3 and the luciferase activity (luciferase activity) confirmed that the fluorescence intensity and luciferase activity between cells showed a similar trend ( Fig.13b and Fig.13c That is, the expression levels of scTTZ and anchoring proteins were indirectly evaluated by fluorescence intensity analysis, and it was confirmed that both miRFPnano3 and luciferase fused to the C-terminus were stably expressed.

[0201] Then, the expression of anchor protein, scTTZ and Flag tag was analyzed by Western blotting ( Fig.14 ). As with the previous results, although the expression level of each cell type was different, when the protein size and band thickness were confirmed, it was confirmed that all proteins were well expressed. When the results of miRFPnano3 and luciferase activity were considered at the same time, among the anchoring proteins, LAMP1 and LAMP2 were particularly confirmed to express the scTTZ fusion protein more stably.

[0202] In order to confirm whether the scTTZ expressed in each cell binds to the target protein, the binding to recombinant human HER2 labeled with FITC was confirmed ( Fig.15a ). When cells without scTTZ but with only anchoring proteins were used as a control group, it was confirmed that all four anchoring proteins increased the FTIC fluorescence signal due to HER2 bound to scTTZ ( Fig.15b ). In particular, it was confirmed that although LAMP1 and LAMP2 are lysosomal membrane proteins, they are also expressed in large quantities in the plasma membrane when overexpressed, and therefore bind to a large amount of HER2 even without permeabilization of cells.

[0203] 2-2. Preparation of anchor protein-CDV introduced with trastuzumab (scFv)

[0204] For cell lines expressing scTTZ and anchoring proteins, CDV was produced by extrusion using ES-50. CDV can be purified by SEC and concentrated by Amicon to obtain a final volume of 5.0×10 10 -1.5×10 11The particle concentration and volume (yield) of CDV at different production steps are shown in Table 1.

[0205] Table 1

[0206]

[0207] The ratio of miRFPnano3 positive particles in CDV was confirmed by analyzing the extruded CDV using nanoparticle flow cytometry. Although the ratio of fluorescence varied from 4% to 38% depending on the anchor protein, it was confirmed that all anchor protein-CDV expressed fluorescence. In particular, it was confirmed that the introduction rate of ssTTZ was high when LAMP1 was used as the anchor protein, and the introduction rate of ssTTZ into CDV was even higher when glycosylated LAMP1 (gLAMP1) was used ( Fig.16 ). Therefore, subsequent experiments were performed using LAMP1-CDV and gLAMP1-CDV as representative examples.

[0208] The expression of the fusion protein of CDV with scTTZ was confirmed by Western blotting using LAMP1 as the anchor protein ( Fig.17a ), and the binding of recombinant HER2 (FITC-HER2) was confirmed using nanoparticle flow cytometry ( Fig.17b ). A thick band of LAMP1 can also be observed in CDV, and when confirmed using protein L, a relatively thicker band can be confirmed in CDV using glycosylated LAMP1 as an anchor protein. In the results of nanoparticle flow cytometry analysis, CDV using glycosylated LAMP1 showed a 5% higher expression of miRFPnano3 (using PC5 fluorescence) compared to AMP1. When HER2 labeled with FITC was bound to CDV, only 0.5% of CDV in the gLAMP1-CDV as a control group showed fluorescence, which is considered to be non-specific binding. In contrast, FITC fluorescence was confirmed in 44.5% and 35.5% of CDV in scTTZ-gLAMP1 and LAMP1-CDV, respectively, indicating that when considering the proportion of miRFPnano3-positive CDV, most of the CDV with scTTZ effectively binds to HER2.

[0209] In order to confirm the difference of scTTZ-CDV with or without LAMP1 glycosylation, CDV was attached to aldehyde / sulfate latex beads, and FITC-HER2 protein was reacted at various concentrations to compare the EC50 values ​​( Fig.18 ). First, it was confirmed that both CDVs bind to HER2 in a concentration-dependent manner. Based on this, the EC50 results were calculated to be 0.94 μg / mL for gLAMP1-CDV and 0.88 μg / mL for LAMP1-CDV. When considering the proportion of scTTZ in each CDV, it was believed that the difference in EC50 was not large, and no effect of glycosylation was observed.

[0210] 2-3. In vitro evaluation of trastuzumab (scFv)-introduced anchor protein-CDV

[0211] (1) Analysis of target cell binding

[0212] Before evaluating the binding between scTTZ-CDV and HER2-expressing cells, the HER2 expression levels in three breast cancer cells, BT-474, SK-BR-3, and MDA-MB-231, as well as CT26 and CT26 / hHER2 cells were confirmed. BT-474 and SK-BR-3 cells are known to be HER2-high-expressing cell lines, but their doubling time is 2-3 days, and they are difficult to use to prepare tumor animal models due to their slow proliferation rate. As an alternative, CT26 / hHER2 prepared by expressing human HER2 in mouse-derived cells was used. Fig.19 As shown, it was confirmed that HER2 was not expressed in MDA-MB-231 and CT26 cells, which are HER2-negative cell lines. On the contrary, it was confirmed that more than 99% of cells in BT-474 and SK-BR-3, which are HER2-positive cell lines, expressed HER2, and more than 90% of cells in CT26 / hHER2 expressed hHER2. In addition, it was confirmed that hHER2 was expressed at a level about 2 times higher in BT-474 and SK-BR-3 than in CT26 / hHER2.

[0213] Next, the binding of scTTZ-introduced CDV to CT26 / hHER2 and HER2 was confirmed. CFSE fluorescently labeled scTTZ-gLAMP1-CDV and gLAMP1-CDV as a control group were cultured with CT26 / hHER2 or control group CT26 cells, and the relative degree of binding was compared by measuring the fluorescence intensity by flow cytometry. Fig. 20As shown, in CT26 cells, which are HER2-negative cell lines, no fluorescence difference due to the two CDVs was observed regardless of the presence or absence of scTTZ. In contrast, in the case of CT26 / hHER2 cells, the histogram peak of flow cytometry analysis shifted by about 6% due to the binding of scTTZ-gLAMP1-CDV, confirming that scTTZ-gLAMP1-CDV bound to the cells.

[0214] Then, the binding of scTTZ-gLAMP1-CDV was evaluated using HER2 high-expressing cells. BT-474 and SK-BR-3 were used as HER2 high-expressing cell lines, and MDA-MB-231 cells, a HER2 negative cell line, were used as a control group. As a result, no binding difference between CDVs was observed for MDA-MB-231 cells. On the contrary, in BT-474 and SK-BR-3, a peak shift of about 7.5%-11% was confirmed in scTTZ-gLAMP1-CDV relative to the control group CDV ( Fig.21a ). The results of quantifying the fluorescence intensity of CDV bound to cells showed that approximately 1.5 times and 1.8 times more CDV was bound to SK-BR-3 and BT-474 cells, respectively ( Figure 21b ). In addition, when the binding of HER2 high-expressing cell lines to scTTZ-LAMP1-CDV was confirmed by comparing the luciferase activity fused to CDV instead of using fluorescence intensity, it was confirmed that the binding to SK-BR-3 and BT-474 cells was increased by 1.8 times and 1.7 times, respectively ( Fig.21c ). It is expected that when a full-length antibody other than scFv is used, the binding ability to the target protein as described above can be further improved. The above results indicate that the anchor protein-CDV of the present invention can stably introduce antibody-based drugs into CDV via the anchor protein, and can effectively target target cells based on the antibody.

[0215] (2) Analysis of uptake into target cells

[0216] Next, to confirm whether CDV increased its uptake into cells by ssTTZ, BT-474 cells were treated with DiO-stained gLAMP1-CDV and scTTZ-LAMP1-CDV, and the uptake into cells was confirmed. In order to confirm the difference in the amount of uptake into cells at 15 minutes, 30 minutes, 60 minutes, and 180 minutes of culturing cells, DiO fluorescence was compared by flow cytometry. In this case, the results were analyzed considering the relative fluorescence intensity of different samples ( Fig.22a). As a result, it was confirmed that compared with the control group, scTTZ-LAMP1-CDV was absorbed by cells about 2.62 times more at 15 minutes, 1.79 times more at 30 minutes, 1.65 times more at 1 hour, and 1.24 times more at 3 hours. Moreover, the largest difference was shown at 15 minutes, and the difference gradually decreased with the increase of culture time ( Figure 22b ), which can be seen as the increased uptake into cells due to the binding of scTTZ initially introduced into CDV to HER2 on the cell surface (receptor-mediated endocytosis). Over time, the uptake of CDV into cells is achieved depending on the presence or absence of scTTZ.

[0217] In summary, it was confirmed that the targeting ligand capable of targeting a specific protein can be effectively introduced into CDV using the anchor protein of the present invention as a medium, and the CDV introduced with the targeting ligand can bind to or be absorbed by cancer cells expressing the target protein, and it is believed that the anchor protein-CDV of the present invention can be used as an effective drug delivery carrier. In particular, the use of anchor protein to introduce a targeting ligand into CDV has been confirmed not only by the scFv of trastuzumab, but also by the scFv of cetuximab ( Fig.23 ), it is expected that the anchor protein-CDV of the present invention can be effectively used in the loading and delivery of various targeting ligands.

[0218] Example 3. Development of anchor protein-CDV for introduction of GFP

[0219] In order to confirm the utilization of the anchor protein-CDV of the present invention, an anchor protein-CDV into which GFP as a fluorescent protein is introduced was prepared to confirm whether the fluorescent protein is normally introduced into the CDV. Therefore, after preparing lentiviral particles by preparing a vector construct capable of expressing GFP in a form fused with BSG, one of the anchor proteins of the present invention, the lentiviral particles were used to transform HEK293 cells. Cells without anchor proteins but only overexpressing GFP were used as a control group. After CDV was squeezed out of each cell, the fluorescence of CDV was detected using a nanoparticle flow cytometer and an enzyme-linked immunosorbent assay. The results showed that in the case of CDV into which BSG-GFP was introduced, GFP fluorescence was detected in more than 75% of the CDV. On the contrary, in the case of CDV into which only GFP was introduced, GFP fluorescence was detected in only about 13% of the CDV ( Fig.24 ). The above results indicate that, when the anchor protein of the present invention is used, active ingredients or substances can be introduced into CDV more efficiently.

[0220] The above description of the present invention is only for illustration, and it can be understood by those skilled in the art that the present invention can be easily transformed into other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the above described embodiments are only for illustration and not for limitation, no matter from which aspect.

[0221] Table 2

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] *trastuzumab: trastuzumab

[0228] Industrial Applicability

[0229] The present invention has discovered four anchoring proteins that conform to the inherent characteristics of CDV and can mediate the stable introduction of bioactive molecules as modified cell-derived vesicles that can be used as drug delivery carriers, thereby completing the present invention. It is confirmed that the anchoring protein is a membrane protein that is specifically abundant in CDV, and the CDV containing the anchoring protein can load bioactive molecules more stably. For example, the results of comparative experiments using fluorescent proteins confirmed that CDVs with introduced anchoring proteins are more effectively loaded with fluorescent proteins than CDVs without anchoring proteins. In addition, it is confirmed that when cancer cell targeting antibodies are loaded in the modified CDV of the present invention, not only the cancer cell targeting ability is increased, but also it is more effectively absorbed by cancer cells. That is, the CDV of the present invention, as a BioDrone modified by anchoring proteins, can stably load a variety of bioactive molecules and deliver them to the desired target, and is expected to be used in drug delivery and treatment platforms for a variety of drugs.

Claims

1. A cell-derived vesicle overexpressing an anchoring protein, characterized in that: The anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

2. The cell-derived vesicle according to claim 1, characterized in that The anchoring protein is inserted into the membrane of the cell-derived vesicle.

3. The cell-derived vesicle according to claim 1, characterized in that The cell-derived vesicles are derived from cells overexpressing the anchoring protein.

4. The cell-derived vesicle according to claim 3, characterized in that The cell-derived vesicles are obtained by extruding the cells.

5. The cell-derived vesicle according to claim 3, characterized in that The cells are at least one selected from the group consisting of stem cells, immune cells, blood cells, embryonic cells, fat cells and embryonic kidney cells.

6. The cell-derived vesicle according to claim 1, characterized in that The anchoring protein is present at a higher level in the cell-derived vesicles than in the cells from which the cell-derived vesicles originate or in exosomes produced from the cells.

7. The cell-derived vesicle according to claim 1, characterized in that The anchoring protein binds to the biologically active molecule.

8. The cell-derived vesicle according to claim 7, characterized in that The bioactive molecule is located outside or inside the membrane of the cell-derived vesicle.

9. The cell-derived vesicle according to claim 7, characterized in that The bioactive molecule is one or more selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, viruses, semi-synthetic substances, quantum dots, fluorescent pigments and toxins.

10. The cell-derived vesicle according to claim 9, characterized in that The protein is one or more selected from the group consisting of antibodies, antibody fragments, growth factors, enzymes, nucleases, transcription factors, antigenic peptides, hormones, transport proteins, immunoglobulins, structural proteins, motor function proteins, signaling proteins, connecting proteins, viral proteins, natural proteins, recombinant proteins, protein complexes, fluorescent proteins, therapeutic proteins, chemically modified proteins and prions.

11. The cell-derived vesicle according to claim 10, characterized in that The antibody is one or more selected from the group consisting of full-length antibodies, Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, miniantibodies, bispecific antibodies and nanoantibodies.

12. The cell-derived vesicle according to claim 7, characterized in that The biologically active molecule is a targeting ligand, and the cell-derived vesicles bind to cells that express a target of the targeting ligand.

13. A method for preparing the cell-derived vesicles according to claim 1, comprising: Step (S1), introducing a recombinant vector containing an anchor protein encoding gene into a cell; as well as Step (S2), extruding the cells into which the recombinant vector has been introduced to obtain cell-derived vesicles, is characterized in that: The anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

14. The method for preparing cell-derived vesicles according to claim 13, characterized in that: The recombinant vector is introduced into the cell through at least one selected from the group consisting of lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and vaccinia virus containing the recombinant vector.

15. The method for preparing cell-derived vesicles according to claim 14, characterized in that: The cells were infected with virus at 1 to 30 MOI.

16. The method for preparing cell-derived vesicles according to claim 13, characterized in that: The recombinant vector also contains a gene encoding a biologically active molecule, and the biologically active molecule is expressed in a state of being bound to the anchor protein.

17. A cell for producing cell-derived vesicles, characterized in that: An exogenous anchor protein encoding gene is introduced, and the anchor protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

18. A cell-derived vesicle, characterized in that: Obtained by squeezing the cells according to claim 17.

19. A composition for drug delivery, characterized in that The invention relates to a vesicle derived from a cell in which an anchoring protein is overexpressed as an effective ingredient, wherein the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

20. The drug delivery composition according to claim 19, characterized in that The drug is one or more selected from the group consisting of antibodies or fragments thereof, therapeutic proteins and therapeutic peptides.

21. The drug delivery composition according to claim 19, characterized in that The drug is bound to the anchoring protein of the cell-derived vesicle or loaded in the interior or membrane of the cell-derived vesicle.

22. The drug delivery composition according to claim 19, characterized in that The cell-derived vesicle further comprises a targeting ligand, which is bound to the anchor protein and is located outside the membrane of the cell-derived vesicle.

23. The drug delivery composition according to claim 22, characterized in that The cell-derived vesicles bind to cells expressing the target of the targeting ligand.

24. A pharmaceutical composition for preventing or treating cancer, comprising vesicles derived from cells overexpressing ankyrin as an effective ingredient, characterized in that: The cell-derived vesicles are loaded with an anticancer agent, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1, and LAMP2.

25. The pharmaceutical composition for preventing or treating cancer according to claim 24, characterized in that: The anticancer agent binds to the anchoring protein.

26. Use of a cell-derived vesicle overexpressing an anchoring protein, characterized in that: The cell-derived vesicles are used for drug delivery, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

27. A use of a cell-derived vesicle overexpressing an anchoring protein, characterized in that: The cell-derived vesicles are used to prepare a composition for drug delivery, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

28. A drug delivery method, comprising the step of administering to an individual in need thereof a cell-derived vesicle overexpressing one or more anchoring proteins selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2, wherein: The cell-derived vesicles are loaded with drugs.

29. A method for preventing or treating cancer, comprising the step of administering to an individual in need thereof a cell-derived vesicle overexpressing one or more anchoring proteins selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2, wherein: The cell-derived vesicles are loaded with anti-cancer agents.

30. A use of a cell-derived vesicle overexpressing an anchoring protein, characterized in that: The cell-derived vesicle is used for preventing or treating cancer, the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2, and the cell-derived vesicle is loaded with an anticancer agent.

31. Use of a cell-derived vesicle overexpressing ankyrin, characterized in that: The cell-derived vesicle is used to prepare a medicament for preventing or treating cancer, and the anchoring protein is one or more selected from the group consisting of Basigin, ATP1B3, LAMP1 and LAMP2.

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