Method for producing nanovesicles derived from the outer membrane of Gram-negative bacteria and use thereof

Abstract

The present invention relates to a method for producing Gram-negative bacterial outer membrane-derived nanovesicles and their use. The present invention also provides a method for treating or diagnosing cancer using Gram-negative bacterial outer membrane-derived nanovesicles. Furthermore, the present invention provides a method for loading a disease treatment or diagnostic substance onto Gram-negative bacterial outer membrane-derived nanovesicles and delivering it to target cells or tissues. In addition, a method for preventing and treating various diseases such as cancer and bacterial / viral infections by co-administering Gram-negative bacterial outer membrane-derived nanovesicles with an antigen or administering Gram-negative bacterial outer membrane-derived nanovesicles expressing the antigen is provided.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0065067, filed on May 27, 2022, and the entire specification of the above is a reference for this application.

[0002] The present invention relates to a method for producing Gram-negative bacterial outer membrane-derived nanovesicles and their use. More specifically, it provides a method for producing Gram-negative bacterial outer membrane-derived nanovesicles and a method for reducing side effects in vivo and in vitro, enhancing efficacy, and delivering to target cells and tissues using the Gram-negative bacterial outer membrane-derived nanovesicles, and relates to pharmaceutical compositions and methods for enhancing stability and efficacy as therapeutic agents, drug delivery, and / or vaccine carriers for various diseases including cancer, and methods for treating or preventing various diseases such as cancer and bacterial / viral infections.

Background Art

[0003] All cells, including Gram-negative bacteria, are known to naturally secrete extracellular vesicles. The extracellular vesicles secreted from the Gram-negative bacteria are also known as outer membrane vesicles (OMV). The outer membrane vesicles are 20 - 200 nm in size and have substances with various biological activities such as proteins, lipids, genetic materials, lipopolysaccharides (LPS), metabolites, etc. The outer membrane vesicles perform various functions such as contributing to the delivery of proteins or genetic materials, the removal of competing bacteria and the promotion of bacterial survival, delivering toxins to host cells, and regulating the immune response.

[0004] According to recent research findings, outer membrane vesicles naturally secreted by various Gram-negative bacteria contain various substances that can activate the immune system. Since they are non-living entities with the bacteria themselves removed, they are safer than the bacteria themselves and not only have a direct therapeutic effect on various diseases such as cancer, but are also used or being developed clinically as drug carriers for treating these diseases or as vaccine carriers for preventing or treating various diseases such as cancer, COVID-19, and meningitis.

[0005] When outer membrane vesicles naturally secreted by Gram-negative bacteria are injected via the vein of cancer-developed mice, it is known to induce an anti-cancer immune response that effectively kills cancer tissue without significant side effects.

[0006] Furthermore, due to the characteristic of nanosize, outer membrane vesicles naturally secreted by Gram-negative bacteria can specifically accumulate in cancer tissue through enhanced permeability and retention (EPR) effects. Therefore, after loading drugs for treating or diagnosing cancer, they can be used as drug carriers.

[0007] On the other hand, when outer membrane vesicles naturally secreted by Gram-negative bacteria are injected into the abdominal cavity of mice three times at one-week intervals, and then the Gram-negative bacteria are injected into the abdominal cavity of the mice one week after the last injection, it is known that the mice can be prevented from dying due to the bacteria. The outer membrane vesicles are presumed to prevent death caused by bacteria through a mechanism that induces Th1 and Th17 immune responses. In fact, outer membrane vesicles are used or being developed clinically as vaccine carriers for preventing or treating various diseases such as cancer, COVID-19, and meningitis.

[0008] In addition, various therapeutic agents are loaded to enhance the therapeutic and preventive effects of outer membrane vesicles, or bacteria-derived extracellular vesicles are used in which various proteins such as cytokines, growth factors, and antibodies (antibodies that target target cells or target tissues, neutralize the activity of antigens, or perform various functions) are expressed as fusion proteins on outer membrane proteins. Various attempts are being made.

[0009] However, outer membrane vesicles naturally secreted by Gram-negative bacteria are not suitable for mass production because the amount is very small, the purification process is complex and difficult, and the yield is low. In addition, they contain intracellular substances such as periplasmic-derived proteins, inner membrane-derived proteins, cytoplasmic-derived proteins, excessive genetic materials, and various metabolites that are unnecessary for the delivery of disease treatment and diagnostic substances and vaccines. Since these substances may reduce the efficiency of drug loading or cause side effects when administered in the body, it is necessary to develop a method for removing them.

[0010] To overcome the above problems, outer membrane-derived nanovesicles (OMNV) of Gram-negative bacteria have been artificially produced and developed as therapeutic agents, drug carriers, and / or vaccine carriers for various diseases including cancer.

[0011] Generally, outer membrane-derived nanovesicles are produced by treating Gram-negative bacteria with ethylenediaminetetraacetic acid (EDTA) and lysozyme, disrupting them by sonication, etc., separating the outer membrane and inner membrane using high-speed centrifugation, treating them with a detergent such as sodium lauoryl sarcosinate (Sarkosyl) to remove the inner membrane, separating only the outer membrane components using high-speed centrifugation, and then using a method such as sonication or extrusion.

[0012] However, the above method for producing outer membrane-derived nanovesicles has a complicated process and many limitations in mass production for clinical use due to problems such as the need to use multiple substances such as lysozyme. Therefore, it is necessary to develop a method to overcome these limitations.

Summary of the Invention

Problems to be Solved by the Invention

[0013] As a result of research to solve the above-mentioned conventional problems, the present inventors found that when Sarkosyl is treated with Gram-negative bacteria, the inner membrane is removed, and only the outer membrane from which the inner membrane and intracellular substances are removed can be extracted without ultra-high-speed centrifugation. The present invention was completed by finding a method for producing nanovesicles using the extracted outer membrane.

[0014] In addition, the present invention aims to provide a method for treating or diagnosing cancer using Gram-negative bacteria outer membrane-derived nanovesicles, and a method for loading and delivering disease treatment or diagnostic substances to the outside, inside, or outer membrane of the outer membrane-derived nanovesicles. In addition, by administering Gram-negative bacteria outer membrane-derived nanovesicles expressing fusion proteins of outer membrane proteins and various proteins such as cytokines, growth factors, and antibodies to target cells or tissues, enhancing the therapeutic effect, reducing side effects, and further enhancing the delivery to target cells or tissues and the therapeutic effect and reducing side effects, the present invention aims to provide a method for preventing and treating various diseases and enhancing the therapeutic effect and reducing side effects. In addition, the present invention aims to provide a method for preventing and treating various diseases by co-administering Gram-negative bacteria outer membrane-derived nanovesicles with an antigen or administering Gram-negative bacteria outer membrane-derived nanovesicles expressing the antigen.

[0015] However, the technical problems to be achieved by the present invention are not limited to the problems described above, and other problems not described will be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0016] To achieve the above object of the present invention, the present invention provides a method for producing nanovesicles derived from the outer membrane of Gram-negative bacteria, the method comprising the following steps: (a) removing the inner membrane from Gram-negative bacteria to obtain the outer membrane; and (b) producing nanovesicles in the suspension containing the outer membrane.

[0017] To achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating diseases, comprising nanovesicles derived from the outer membrane of Gram-negative bacteria, wherein the nanovesicles derived from the outer membrane of Gram-negative bacteria have therapeutic activity.

[0018] In addition, to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating diseases, consisting of nanovesicles derived from the outer membrane of Gram-negative bacteria, wherein the nanovesicles derived from the outer membrane of Gram-negative bacteria have therapeutic activity.

[0019] In addition, to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating diseases, essentially consisting of nanovesicles derived from the outer membrane of Gram-negative bacteria, wherein the nanovesicles derived from the outer membrane of Gram-negative bacteria have therapeutic activity.

[0020] To achieve another object of the present invention, the present invention provides a vaccine composition for preventing or treating diseases, comprising nanovesicles derived from the outer membrane of Gram-negative bacteria.

[0021] To achieve another object of the present invention, the present invention provides the use of the nanovesicles derived from the outer membrane of Gram-negative bacteria according to the present invention for manufacturing a pharmaceutical composition for treating diseases.

[0022] To achieve another object of the present invention, the present invention provides a method for treating a disease, comprising administering an effective amount of a pharmaceutical composition comprising the nanovesicles derived from the outer membrane of Gram-negative bacteria according to the present invention to an individual in need thereof.

[0023] The present invention will be described in detail below. The present invention provides a method for producing nanovesicles derived from the outer membrane of Gram-negative bacteria, the method comprising the following steps: (a) removing the inner membrane from Gram-negative bacteria to obtain the outer membrane; and (b) producing nanovesicles in a suspension containing the outer membrane.

[0024] In the present invention, the "Gram-negative bacteria" means a group of bacteria that cannot maintain the color of the crystal violet reagent when distinguishing bacteria by Gram staining. Gram-negative bacteria have a cell wall composed of an outer membrane, a periplasmic space containing proteins such as lipoproteins, a peptidoglycan layer, and an inner membrane (or cytoplasmic membrane). Among these, the outer membrane is composed of an asymmetric lipid bilayer, with the inner leaf of the lipid layer being composed of phospholipids and the outer leaf being composed of lipopolysaccharides. In addition, the outer membrane houses outer membrane proteins such as OmpA, OmpE, OmpC, LptD, and BamA.

[0025] In one embodiment of the present invention, the Gram-negative bacterium can be a bacterium of a genus selected from the group consisting of the genera Escherichia, Helicobacter, Hemophilus, Neisseria, Cyanobacterium, Klebsiella, Acetobacter, Acinetobacter, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Thiobacter, Borrelia, Burkholderia, Serratia, and Treponema, but is not limited thereto. In a preferred embodiment of the present invention, the Gram-negative bacterium can be a bacterium belonging to the genus Escherichia, Salmonella, or Pseudomonas.

[0026] The above-mentioned "nanoendosome" of the present invention is demarcated inside and outside by a lipid bilayer membrane composed of the cell membrane components of the originating bacterium, contains the bacterial cell membrane lipids and cell membrane proteins, has the same topology, but genetic materials such as nucleic acids and intracellular substances such as intracellular proteins are removed, meaning that it is smaller in size than the original cell, but is not limited thereto.

[0027] In the present invention, the nanovesicles are not particularly limited as long as their diameters are at the nanometer level. For example, the diameters are about 20 - 290 nm, 20 - 280 nm, 20 - 270 nm, 20 - 260 nm, 20 - 250 nm, 20 - 240 nm, 20 - 230 nm, 20 - 220 nm, 20 - 210 nm, 20 - 200 nm, 20 - 190 nm, 20 - 180 nm, 20 - 170 nm, 20 - 160 nm, 20 - 150 nm, 20 - 140 nm, 20 - 130 nm, 20 - 120 nm, 20 - 110 nm, 20 - 100 nm, 20 - 90 nm, 20 - 80 nm, 20 - 70 nm, 20 - 60 nm, 20 - 50 nm, 20 - 40 nm, 20 - 30 nm, 30 - 300 nm, 30 - 290 nm, 30 - 280 nm, 30 - 270 nm, 30 - 260 nm, 30 - 250 nm, 30 - 240 nm, 30 - 230 nm, 30 - 220 nm, 30 - 210 nm, 30 - 200 nm, 30 - 190 nm, 30 - 180 nm, 30 - 170 nm, 30 - 160 nm, 30 - 150 nm, 30 - 140 nm, 30 - 130 nm, 30 - 120 nm, 30 - 110 nm, 30 - 100 nm, 30 - 90 nm, 30 - 80 nm, 30 - 70 nm, 30 - 60 nm, 30 - 50 nm, 30 - 40 nm, 40 - 300 nm, 40 - 290 nm, 40 - 280 nm, 40 - 270 nm, 40 - 260 nm, 40 - 250 nm, 40 - 240 nm, 40 - 230 nm, 40 - 220 nm, 40 - 210 nm, 40 - 200 nm, 40 - 190 nm, 40 - 180 nm, 40 - 170 nm, 40 - 160 nm, 40 - 150 nm, 50 - 300 nm, 50 - 290 nm, 50 - 280 nm, 50 - 270 nm, 50 - 260 nm, 50 - 250 nm, 50 - 240 nm, 50 - 230 nm, 50 - 220 nm, 50 - 210 nm, 50 - 200 nm, 50 - 190 nm, 50 - 180 nm, 50 - 170 nm, 50 - 160 nm, 50 - 150nm, 50 - 140 nm, 50 - 130 nm, 50 - 120 nm, 50 - 110 nm, 50 - 100 nm, 50 - 90 nm, 50 - 80 nm, 50 - 70 nm, 50 - 60 nm, 60 - 300 nm, 60 - 290 nm, 60 - 280 nm, 60 - 270 nm, 60 - 260 nm, 60 - 250 nm, 60 - 240 nm, 60 - 230 nm, 60 - 220 nm, 60 - 210 nm, 60 - 200 nm, 60 - 190 nm, 60 - 180 nm, 60 - 170 nm, 60 - 160 nm, 60 - 150 nm, 60 - 140 nm, 60 - 130 nm, 60 - 120 nm, 60 - 110 nm, 60 - 100 nm, 60 - 90 nm, 60 - 80 nm, 60 - 70 nm, 70 - 300 nm, 70 - 290 nm, 70 - 280 nm, 70 - 270 nm, 70 - 260 nm, 70 - 250 nm, 70 - 240 nm, 70 - 230 nm, 70 - 220 nm, 70 - 210 nm, 70 - 200 nm, 70 - 190 nm, 70 - 180 nm, 70 - 170 nm, 70 - 160 nm, 70 - 150 nm, 70 - 140 nm, 70 - 130 nm, 70 - 120 nm, 70 - 110 nm, 70 - 100 nm, 70 - 90 nm, 70 - 80 nm, 80 - 300 nm, 80 - 290 nm, 80 - 280 nm, 80 - 270 nm, 80 - 260 nm, 80 - 250 nm, 80 - 240 nm, 80 - 230 nm, 80 - 220 nm, 80 - 210 nm, 80 - 200 nm, 80 - 190 nm, 80 - 180 nm, 80 - 170 nm, 80 - 160 nm, 80 - 150 nm, 80 - 140 nm, 80 - 130 nm, 80 - 120 nm, 80 - 110 nm, 80 - 100 nm, 80 - 90 nm, 90 - 300 nm, 90 - 290 nm, 90 - 280 nm, 90 - 270 nm, 90 - 260 nm, 90 - 250 nm, 90 - 240 nm, 90 - 230 nm, 90 - 220 nm, 90 - 210 nm, 90 - 200 nm, 90 - 190 nm, 90 - 180 nm, 90 - 170 nm, 90 - 160 nm, 90 - 150 nm, 90 - 140 nm, 90 - 130 nm, 90 - 120 nm, 90 - 110 nm, 90 - 100 nm, 100 - 300nm, 110 - 290 n m, 120 - 280 nm, 130 - 270 nm, 140 - 260 nm, 150 - 250 nm, 160 - 240 nm, 170 - 230 nm, 180 - 220 nm, or 190 - 210 nm.

[0028] In the present invention, the "nanovesicles derived from the outer membrane of Gram - negative bacteria" means nanovesicles artificially produced using the outer membrane of Gram - negative bacteria according to a method including steps (a) and (b).

[0029] In the method for producing the nanovesicles according to the present invention, step (a) is a step of removing the inner membrane from Gram - negative bacteria to obtain the outer membrane.

[0030] In one embodiment of the present invention, step (a) can be carried out by a method of treating Gram - negative bacteria with an anionic surfactant. The above - mentioned "anionic surfactant" is a substance having the property that anions generated by dissociation in water adsorb on the surface of the aqueous solution and reduce the surface tension. Non - limiting examples of the anionic surfactant include sodium dodecyl sulfate (SDS), Sarkosyl, lithium dodecyl sulfate, sodium 1 - octane sulfonic acid, etc., but are not limited thereto. In a preferred embodiment of the present invention, the anionic surfactant can be Sarkosyl.

[0031] In one embodiment of the present invention, in step (a), the anionic surfactant can be treated at a final concentration of 0.1 - 5% (w / v) in a culture solution or suspension containing Gram - negative bacteria, preferably at a final concentration of 0.1 - 4% (w / v), more preferably at a final concentration of 0.1 - 3% (w / v), even more preferably at a final concentration of 0.1 - 2% (w / v), and most preferably at a final concentration of 0.5 - 2% (w / v).

[0032] In the present invention, the "culture solution" means that a gram-negative bacterium is contained in a medium containing nutrient components that allow the gram-negative bacterium to survive and grow, and the "suspension" means that after separating only the bacteria from the culture solution containing the gram-negative bacterium by means such as centrifugation, the separated gram-negative bacterium is suspended in a medium or buffer. In a preferred embodiment of the present invention, the "culture solution" and the "suspension" can be interpreted interchangeably.

[0033] In one embodiment of the present invention, in the step (a), the anionic surfactant can be treated at a temperature of 4 to 60 °C for 5 to 120 minutes, preferably at a temperature of 4 to 60 °C for 5 to 60 minutes, more preferably at a temperature of 4 to 60 °C for 10 to 60 minutes, even more preferably at a temperature of 4 to 60 °C for 10 to 50 minutes, and most preferably at a temperature of 4 to 60 °C for 20 to 40 minutes, but is not limited thereto.

[0034] In one embodiment of the present invention, in the step (a) above, the anionic surfactant can be treated once or repeatedly. In the step (a) above, when the anionic surfactant is treated repeatedly, after treating the anionic surfactant in the culture solution or suspension containing the gram-negative bacterium for a certain period of time, the residual anionic surfactant contained in the supernatant is removed through means such as centrifugation, and after suspending the gram-negative bacterium in a medium or buffer, the process of re-treating the anionic surfactant can be repeated. In the present invention, the above "repeatedly" can mean 2 to 10 times, preferably 2 to 5 times, more preferably 2 to 4 times, and most preferably 2 to 3 times. In the step (a) above, when the anionic surfactant is treated repeatedly, the treatment conditions (i.e., treatment concentration, treatment time, treatment temperature, etc.) of the surfactant treated each time may be the same or different.

[0035] In the step (a), after treating Gram-negative bacteria with an anionic surfactant, a process for distinguishing the outer membrane from the inner membrane, cytoplasmic components, etc. can be further carried out. For example, after the step (a), any method for separating and purifying the outer membrane in the art, such as centrifugation, filtration, dialysis, size exclusion chromatography, Capto Core chromatography, tangential flow filtration, etc., can be further carried out, but it is not limited thereto.

[0036] In the present invention, the step (b) is a process of applying energy to the outer membrane of the Gram-negative bacteria separated in the step (a) to form nanovesicles. In one embodiment of the present invention, the step (b) is to apply a culture solution or suspension containing the outer membrane derived from Gram-negative bacteria to any one or more means selected from the group consisting of microfluidics, sonication, extrusion, treatment with an alkaline solution, treatment with a homogenizer or disperser, and nitrogen cavitation, but it is not limited thereto.

[0037] In one embodiment of the present invention, a process of purifying, separating, and collecting nanovesicles derived from the outer membrane of Gram-negative bacteria contained in the culture solution or suspension after the step (b) can be further carried out. For the purification or separation, methods known in the art can be applied without limitation. For example, it can be separated or purified by a method selected from the group consisting of ultra-high-speed centrifugation, density gradient ultra-high-speed centrifugation, ultrafiltration method, size exclusion chromatography, ion exchange chromatography, Capto Core chromatography, immunoaffinity separation method, microfluidic technology separation method, aqueous two-phase system, polymer-based precipitation method, ultrasonic decomposition method, and extrusion method, but it is not limited thereto.

[0038] According to the above method of the present invention, it is possible to provide an increased yield compared to the prior art for separating naturally secreted outer membrane vesicles. For example, based on the same volume of bacterial culture, outer membrane-derived nanovesicles can be obtained at a yield that is at least about 2 to 1000 times higher on a particle number basis than naturally secreted bacterial outer membrane vesicles. For example, based on the same volume of bacterial culture, outer membrane-derived nanovesicles can be obtained at a level that is at least about 2 to 100 times higher on a total protein basis than naturally secreted bacterial outer membrane vesicles. For example, based on the same volume of bacterial culture, outer membrane-derived nanovesicles with a purity (total particle number relative to total protein amount) that is at least about 2 to 100 times higher than that of naturally secreted bacterial outer membrane vesicles can be obtained. Also, the outer membrane-derived nanovesicles produced by the present invention have a very low possibility of containing non-outer membrane-derived components such as peripheral cytoplasm, inner membrane, and cytoplasm.

[0039] In one embodiment of the present invention, the outer membrane-derived nanovesicles produced by the above method can be characterized by lacking at least one or more non-outer membrane-derived components among the components of the bacteria from which they are derived. The above "non-outer membrane-derived components" include peptidoglycan, surrounding cytoplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes.

[0040] In one embodiment of the present invention, the outer membrane-derived nanovesicles produced by the above method can be characterized by lacking at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 100% of the content of at least one or more non-outer membrane-derived components compared to the outer membrane vesicles naturally secreted by the bacteria from which they are derived.

[0041] In one embodiment of the present invention, the Gram-negative bacteria can be characterized by being transformed.

[0042] In the present invention, the above-mentioned "transformation" means the modification of the genotype of bacteria by the introduction of exogenous polynucleotides, and regardless of the method used for the transformation, it means that the exogenous polynucleotides have been introduced into the bacteria. The exogenous polynucleotides introduced into the bacteria can be integrated into and maintained in the bacterial genome or maintained without being integrated, and the present invention includes both. The above exogenous polynucleotides can be inserted into a vector and used for the transformation of the bacteria.

[0043] In the present invention, the above-mentioned "vector" is utilized for the replication or expression of the exogenous polynucleotides and generally includes one or more of a signal sequence, an origin of replication, one or more marker genes, enhancers, promoters, and transcription termination sequences. A plasmid, which is a type of vector, means a linear or circular double-stranded DNA molecule to which an outer polynucleotide fragment can bind. Other forms of vectors are viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), where additional DNA fragments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within the bacteria (e.g., bacterial vectors including bacterial origin and episomal mammalian vectors) into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) are integrated into the interior of the bacterial genome by introduction into the bacteria and are thereby replicated together with the bacterial genome.

[0044] In the present invention, the vector can be understood to have the same meaning as an "expression vector", which is a form of an expressible vector for the polynucleotide. One polynucleotide sequence is "operably linked" to the regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence. The regulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of the nucleic acid to which it is operably linked. The regulatory sequence can exert its influence, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or the nucleic acid). The regulatory sequence includes a promoter, an enhancer, and other expression regulatory elements.

[0045] The vector can be introduced into the interior of bacteria and the bacteria can be transformed by known methods in the art, such as, but not limited to, transient transfection, microinjection, transduction, fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and known methods for introducing exogenous polynucleotides into bacteria.

[0046] In the present invention, "expression" means that a protein or polypeptide is produced in bacteria.

[0047] In the present invention, the "exogenous protein" means a protein or peptide that is not intrinsically expressed in the bacterium and is expressed in the cytoplasm and / or cell membrane of the bacterium by artificial transformation. The exogenous protein can be induced to be expressed in the bacterium by transforming the bacterium with a vector into which a polynucleotide encoding the exogenous protein has been inserted.

[0048] In a preferred embodiment of the present invention, the exogenous protein may include, but is not limited to, one or more selected from the group consisting of an antigen protein / or antigen peptide that is expressed on the outer membrane of the bacterium or can be exposed outside the outer membrane, an antigen protein or antigen peptide that can be exposed inside the outer membrane, an immunopotentiating protein, an immunosuppressive protein, a cell adhesion molecule, an antibody, a target-inducing protein, a cell membrane fusion protein, a cytokine, an enzyme, a growth factor, an extracellular domain of a membrane receptor, a marker protein, fragments thereof, and fusion proteins thereof.

[0049] In one embodiment of the present invention, the Gram-negative bacterium can be characterized in that the expression of an endogenous protein is reduced or knocked out. The "endogenous protein" refers to a protein that is specific to the Gram-negative bacterium or is naturally expressed within the Gram-negative bacterium.

[0050] In the present invention, the endogenous protein whose expression may be decreased or absent may include other endogenous proteins whose expression has not decreased, and / or non-essential ones that functionally overlap with secreted proteins (e.g., with respect to bacterial growth and / or viability, e.g., in a biomanufacturing environment). In one embodiment, the endogenous protein whose expression may be decreased or absent in the present invention may be a protein involved in non-essential or secretion-related functions. In one embodiment, the endogenous protein whose expression may be decreased or absent may be a protein related to immune function, disease resistance, bacterial communication, and / or secretion. In a preferred embodiment, the endogenous protein whose expression may be decreased or absent may be a bacterial toxicity-related protein.

[0051] In some embodiments, decreasing the expression of an endogenous protein in bacteria includes removing, e.g., deleting, a copy of the gene encoding the endogenous protein. In some embodiments, decreasing the expression of a bacterial endogenous protein includes removing, e.g., deleting, all (e.g., both) copies of the gene encoding the endogenous protein from the bacterial genome. In some embodiments, decreasing the expression of a bacterial endogenous protein includes removing, e.g., deleting, a regulatory nucleic acid sequence, such as a promoter or enhancer, operably linked to the gene encoding the endogenous protein. In some embodiments, decreasing the expression of a bacterial endogenous protein includes removing, e.g., deleting, all (e.g., both) copies of a regulatory nucleic acid sequence, such as a promoter or enhancer, operably linked to the gene encoding the endogenous protein.

[0052] Removal of the expression of the endogenous protein in the present invention, for example, deletion, involves introducing a deletion, substitution, or insertion mutation into the gene encoding the endogenous protein or into a regulatory nucleic acid sequence operably linked to the gene encoding the endogenous protein, such as a promoter or enhancer. For example, the mutation can reduce the expression of the endogenous protein.

[0053] Removal of a copy of a gene encoding an endogenous protein or a regulatory element operably linked to the gene, for example, deletion, can be achieved by any gene editing system known in the art. Exemplary gene editing systems include clustered regularly interspaced short palindromic repeats (CRISPR), zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). In some embodiments, removal of a copy of a gene encoding an endogenous protein or a regulatory element operably linked to the gene, for example, deletion, involves the use of a CRISPR-Cas9 molecule, such as a Cas9 molecule, in combination with an RNA specific to the gene encoding the endogenous protein or a regulatory nucleic acid sequence operably linked to the gene encoding the endogenous protein, such as a promoter or enhancer (e.g., gRNA, sgRNA, and / or trans-activating crRNA).

[0054] In some embodiments, reducing the expression of an endogenous bacterial protein includes reducing the expression of an mRNA transcript encoding the endogenous protein, such as a deletion in the bacteria. In some embodiments, reducing the expression of an mRNA transcript encoding an endogenous protein, such as a deletion, includes, for example, the use of siRNA that can bind to the mRNA, such as an endogenous protein or a nucleic acid encoding a regulatory nucleic acid sequence operably linked thereto, such as a promoter or enhancer. Those skilled in the art will know tools and techniques for designing and delivering siRNA to bacteria. These tools and / or techniques include, but are not limited to, those taught by the Dharmacon Horizon siDesign tool, InvivoGen siRNA Wizard, GenScript siRNA Construct service, IDT Custom Dicer-Substrate siRNA, Sigma-Aldrich siRNA Design service, or www.rnaiweb.com / RNAi / siRNA_Design / .

[0055] In one embodiment of the present invention, the transformed bacteria may be transformed to be less toxic, may be transformed to target specific cells or tissues, may be transformed to fuse with the cell membrane of the target cell, may be transformed to express a disease treatment and / or diagnostic substance, and may be transformed such that suppression of the specific substance and expression of the specific substance occur simultaneously, but is not limited thereto.

[0056] In a preferred embodiment of the present invention, the Gram-negative bacterium can be characterized in that it is transformed so as to have reduced toxicity. For example, it may be a bacterium transformed so as to reduce the toxicity of lipopolysaccharide that mediates the host's inflammatory response, or a bacterium in which the expression of an endotoxin-producing gene is decreased or deleted. Specifically, it may be a bacterium transformed to have any one or more genotypes selected from the group consisting of ΔmsbB, ΔkdsA, ΔkdsB, ΔlpxB, ΔkdtA, ΔlpxC, ΔlpxD, Δssc, ΔlpxA, and ΔhtrB, but is not limited thereto.

[0057] In one embodiment of the present invention, the Gram-negative bacterium may be transformed by substance treatment or gene introduction, or may be transformed two or more times.

[0058] In one embodiment of the present invention, the membrane of the Gram-negative bacterium-derived outer membrane nanovesicle may further contain components other than the outer membrane of the bacterium.

[0059] Examples of the components other than the outer membrane include a target inducer, a cell membrane fusogen, cyclodextrin, polyethylene glycol, hyaluronic acid, and the like. In addition, the components other than the outer membrane can be added by various methods, including chemical modification of the outer membrane.

[0060] For example, the membrane components of the Gram-negative bacterium outer membrane-derived nanovesicles are modified by a chemical method using a thiol group (-SH) or an amino group (-NH2), or proteins, polyethylene glycol, or hyaluronic acid are chemically bonded to the Gram-negative bacterium outer membrane-derived nanovesicles, so that the membrane components of the Gram-negative bacterium outer membrane-derived nanovesicles can be chemically modified. For this purpose, in the method for producing Gram-negative bacterium outer membrane-derived nanovesicles of the present invention, a step of chemically modifying the membrane components of the Gram-negative bacterium outer membrane-derived nanovesicles may be further included after the step (b).

[0061] The present invention also provides Gram-negative bacterium outer membrane-derived nanovesicles produced according to the above method.

[0062] In one embodiment of the present invention, the Gram-negative bacterium outer membrane-derived nanovesicles according to the present invention can be characterized in that at least one or more non-outer membrane-derived components among the components of the bacterium from which they are derived are lacking. The above "non-outer membrane-derived components" include peptidoglycan, surrounding cytoplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins, and ribosomes.

[0063] In one embodiment of the present invention, the Gram-negative bacterium outer membrane-derived nanovesicles according to the present invention can be characterized in that the content of at least one or more non-outer membrane-derived components is at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 100% lacking, as compared with the outer membrane vesicles naturally secreted by the bacterium from which they are derived.

[0064] In one embodiment of the present invention, the Gram-negative bacterium outer membrane-derived nanovesicles according to the present invention can be characterized in that they are treated according to the following means so that side effects are reduced when administered in vivo.

[0065] (1) Gram-negative bacteria outer membrane-derived nanovesicles can be produced using genetically transformed bacteria with reduced toxicity. For example, Gram-negative bacteria outer membrane-derived nanovesicles can be produced using bacteria transformed to reduce the toxicity of lipopolysaccharides that mediate the host's inflammatory response.

[0066] (2) The toxicity of bacteria can be reduced using drugs that inhibit the activity of endotoxins. An example of such a drug is polymyxin B. The drug may be administered in combination with Gram-negative bacteria outer membrane-derived nanovesicles, or Gram-negative bacteria outer membrane-derived nanovesicles may be produced from bacteria treated with the drug during culture.

[0067] (3) Side effects can be reduced using drugs with anti-inflammatory and / or anticoagulant effects. The drug includes aspirin. When Gram-negative bacteria outer membrane-derived nanovesicles and aspirin are administered in combination, side effects such as inflammatory reactions and blood coagulation reactions caused by Gram-negative bacteria outer membrane-derived nanovesicles can be prevented. Also, outer membrane-derived nanovesicles can be produced from bacteria treated with the drug during culture.

[0068] (4) The membrane components of the Gram-negative bacteria outer membrane-derived nanovesicles can be modified by chemical methods and used. For example, the membrane components of the Gram-negative bacteria outer membrane-derived nanovesicles are modified by chemical methods using thiol groups or amino groups, or proteins, polyethylene glycol, or hyaluronic acid is chemically bonded to the Gram-negative bacteria outer membrane-derived nanovesicles, whereby the membrane components of the Gram-negative bacteria outer membrane-derived nanovesicles can be chemically modified and used.

[0069] (5) Using sterilized Gram-negative bacteria outer membrane-derived nanovesicles can prevent infection by live bacteria. For example, sterilized Gram-negative bacteria outer membrane-derived nanovesicles can be obtained by sterilization using ultraviolet rays and gamma rays or removal of bacteria using filtration.

[0070] The method for reducing the side effects of the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention is not limited to the above examples, and each method can be used alone or in combination.

[0071] In one embodiment of the present invention, the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention can be characterized in that they themselves exhibit a therapeutic activity against diseases.

[0072] Therefore, the present invention provides a pharmaceutical composition for treating diseases, which contains the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention.

[0073] In addition, the present invention provides a pharmaceutical composition for treating diseases, which consists of the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention.

[0074] The present invention provides a pharmaceutical composition for treating diseases, which consists essentially of the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention.

[0075] In the present invention, the "disease" can include various diseases including cancer. In one embodiment of the present invention, the cancer may be selected from the group consisting of thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colorectal cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer and cervical cancer, but is not limited thereto. In another embodiment of the present invention, the disease may be selected from the group consisting of hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, arteriosclerosis, obesity, peptic ulcer, Alzheimer's disease, emphysema, skin diseases, skin infections, respiratory infections, urogenital infections, osteoarthritis, central nervous system infections and sepsis, but is not limited thereto.

[0076] In one embodiment of the present invention, the composition can be characterized in that it further contains a substance that enhances the therapeutic effect or reduces the side effects.

[0077] The substances that enhance the therapeutic effect or reduce side effects are selected from the group consisting of, but not limited to, anti-cancer agents, immunostimulants, STING agonists, anti-inflammatory agents, endotoxin inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles, and nanoparticles.

[0078] The above-mentioned "anticancer agent" blocks the DNA replication, transcription, and translation processes of cancer cells. The types of anticancer agents that can be used as the therapeutic substance of the present invention are not particularly limited. The anticancer agent can be selected under the general principles considered when selecting an anticancer agent, such as the type of cancer cells, the absorption rate of the anticancer agent (treatment period and anticancer agent administration route), the location of the tumor, and the size of the tumor.The anti-cancer agents that can be used in the present invention include DNA crosslinking agents such as cyclophosphamide, chlormethine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anti-cancer antibiotics such as dactinomycin (actinomycin D), doxorubicin (adriamycin), epirubicin, idarubicin, mitoxantrone, plicamycin, daunorubicin, mitomycin C, and bleomycin; plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, and irinotecan; and antibodies such as herceptin and rituximab, but are not limited thereto.

[0079] The "anti-inflammatory agent" is selected from the group consisting of, but not limited to, dexamethasone, indomethacin, ibuprofen, clobetasol propionate, diflorasone diacetate, halobetasol propionate, amcinonide, fluocinonide, mometasone furoate, desoximetasone, diclofenac, piroxicam, etc.

[0080] The "peptides" and "proteins" can include, without limitation, growth factors such as VEGF, EGF, etc., cytokines such as IL-1, IFN-gamma, IL-10, various antibody therapeutics, various peptides or proteins, and various proteins and peptides that can suppress cancer growth, metastasis, and inflammatory reactions in addition to DNase.

[0081] The "toxin" is a general term for substances derived from various organisms that can exhibit toxicity when absorbed into the body and can induce cell death through the toxin. In the present invention, the types of the above toxins are not particularly limited.

[0082] The "nucleic acid" is selected from the group consisting of, but not limited to, DNA, RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino.

[0083] The "nanoparticle" is selected from the group consisting of, but not limited to, iron oxide, gold, carbon nanotubes, and magnetic beads.

[0084] In one embodiment of the present invention, the substance that enhances the therapeutic effect or reduces side effects can be characterized in that it is loaded in nanovesicles derived from the outer membrane of Gram-negative bacteria.

[0085] In the present invention, the term "loaded" means, but is not limited to, exposing the required substance on the surface of the nanovesicle membrane derived from the outer membrane of Gram-negative bacteria or encapsulating it inside.

[0086] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier in addition to the active ingredient. That is, saline, sterilized water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components can be mixed and used, and other ordinary additives such as antioxidants and buffers can be further contained as necessary. Furthermore, a diluent, a dispersant, a surfactant, a binder, and / or a lubricant can be additionally added to formulate into injection preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each component using a method disclosed by an appropriate method in the art. The pharmaceutical composition of the present invention is not particularly limited in formulation, but is preferably formulated as an injection or an inhalant.

[0087] The administration method of the pharmaceutical composition of the present invention is not particularly limited, but it can be administered parenterally or orally by desired methods such as intravenous, subcutaneous, intraperitoneal, intranasal, inhalation or topical application. The dosage varies depending on factors such as the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate and disease severity. The daily dosage means the amount of the therapeutic substance of the present invention sufficient for the treatment of the disease state alleviated by administration to an individual in need of treatment. The effective amount of the therapeutic substance depends on the specific compound, disease state and its severity, and the individual in need of treatment, and this can be usually determined by those skilled in the art. As a non-limiting example, the dosage of the composition according to the present invention administered to the human body can vary depending on the patient's age, weight, gender, dosage form, health status and degree of disease. Based on an adult patient weighing 70 kg, it is generally 0.1 - 1000 mg / day, preferably 1 - 500 mg / day, and it can also be administered in divided doses once to several times a day at regular time intervals.

[0088] In one embodiment of the present invention, the pharmaceutical composition may further contain a drug that suppresses the toxicity of the nanovesicles derived from the outer membrane of Gram-negative bacteria. Also, the drug can be loaded onto the nanovesicles derived from the outer membrane of Gram-negative bacteria. The drug includes a drug that inhibits the toxicity of endotoxin, and an example thereof is polymyxin B.

[0089] The present invention also provides a vaccine composition for preventing or treating diseases, which contains the nanovesicles derived from the outer membrane of Gram-negative bacteria according to the present invention. The above-mentioned "vaccine" means artificially injecting an antigen into the human body to activate the immune system of the human body in order to prevent or treat diseases.

[0090] According to one embodiment of the present invention, since the nanovesicles derived from the outer membrane of Gram-negative bacteria according to the present invention were confirmed to have extremely excellent activity of inducing innate immunity by themselves, they can be used as an immunopotentiator (that is, a vaccine adjuvant, an adjuvant) that is administered together with an antigen to increase the in vivo immune response.

[0091] The "immune enhancer", "vaccine adjuvant", or "adjuvant" means a pharmaceutical or immunological preparation administered for the purpose of improving the immune response of a vaccine, that is, increasing the in vivo immune response to an antigen.

[0092] In one embodiment of the present invention, the vaccine composition can be characterized by further comprising an antigen.

[0093] The "antigen" can be interpreted as a synonym for "immunogen" and means that it can cause the formation of antibodies and / or a cellular immune response, that is, the activation of a specific immune response, which includes proteins of pathogens, recombinant proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides or polynucleotides. In one embodiment of the present invention, the antigen may be derived from one or more infectious pathogens selected from bacteria, viruses, and fungi. For example, the antigen may be the gE (glycoprotein E) antigen of varicella zoster virus; the gE (glycoprotein E) antigen of Japanese encephalitis virus; the inactivated seasonal influenza virus antigen; the haemagglutinin antigen and neuraminidase antigen of influenza virus; the spike antigen of coronavirus; the pertussis toxin antigen, filamentous haemagglutinin antigen, and pertactin antigen of Bordetella pertussis; the antigen of human papilloma virus (HPV), the capsular polysaccharide antigens of groups A, B, C, Y, and W-135 of Helicobacter pylori; the tetanus toxoid antigen of Clostridium tetani; the diphtheria toxoid antigen of Corynebacterium diphtheriae; the type 3 capsular polysaccharide antigen of Streptococcus pneumoniae; the antigen of Mycobacterium tuberculosis; the GP-120 and GP-160 antigens of human immunodeficiency virus (HIV); the cholera toxin B subunit antigen of Vibrio cholerae; the Staphylococcal enterotoxin B antigen of Staphylococcus;Antigens of Shigella polysaccharides of Shigella, vesicular stomatitis virus glycoprotein antigen of vesicular stomatitis virus, antigens of cytomegalovirus (CMV), hepatitis A / B / C / D / G virus antigens, antigens of respiratory syncytial virus (RSV) or antigens of herpes simplex virus, including but not limited to these.;

[0094] In one embodiment of the present invention, the antigen may be a cancer antigen. For example, the cancer antigen may be an antigen derived from HPV, carcinoembryonic antigen, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), HER2 / neu, mucin-1 (MUC-1), BCR / ABL, alpha-fetoprotein (AFP), antigen derived from Epstein-Barr virus (EBV), antigen derived from HBV, antigen derived from HCV, cancer antigen-125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), cancer antigen-19-9 (CA-19-9) or mutant Ras protein, Raf protein, Src protein, Myc protein, EGFR, PDGFR, VEGFR, p53, PTEN, or HER2 / neu, including but not limited to these.

[0095] In one embodiment of the present invention, the antigen may be loaded onto the Gram-negative bacteria outer membrane-derived nanovesicles according to the present invention, or may be included in the composition together with another carrier.

[0096] The vaccine composition can be administered in combination with commonly used immunopotentiators. Such vaccine adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, Alum, MF59, virosome, AS04 (a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)), AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate80), CpG DNA, flagellin, Poly I:C, AS01 (a mixture of QS-21, MPL and liposomes), AS02 (a mixture of QS-21, MPL and an oil-in-water emulsion), immune stimulating complexes (ISCOM) and immune stimulating complex matrix, etc. Further, the vaccine composition can be administered in combination with additional drugs to enhance the effectiveness of the vaccine or reduce side effects.

[0097] In one embodiment of the present invention, the vaccine composition can be used for the prevention or treatment of infectious diseases caused by bacteria, viruses, or fungi. In the present invention, the infection is selected from the group consisting of, but not limited to, skin infection, respiratory infection, urogenital infection, bone and joint infection, central nervous system infection, and sepsis.

[0098] In one embodiment of the present invention, the vaccine composition may be for the prevention or treatment of cancer. In the present invention, the cancer is selected from the group consisting of, but not limited to, thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colorectal cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer, and cervical cancer.

[0099] In one embodiment of the present invention, the vaccine composition may be for the prevention or treatment of a disease selected from the group consisting of, but not limited to, hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, arteriosclerosis, obesity, peptic ulcer, Alzheimer's disease, chronic obstructive pulmonary disease, asthma, skin diseases, and autoimmune diseases.

[0100] The administration route of the vaccine adjuvant or the vaccine composition containing the same according to one embodiment of the present invention can be administered via any general route as long as it can reach the target tissue. Such administration routes can be transdermal, mucosal administration (e.g., oral, nasal, anal, vaginal) or parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and administration into the synovial cavity, but are not limited thereto.

[0101] The vaccine composition according to the present invention can be formulated into an appropriate form together with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include carriers for parenteral administration such as water, appropriate oils, physiological saline, aqueous glucose, and glycols, and can further contain stabilizers and preservatives. Examples of appropriate stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Examples of appropriate preservatives include benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Further, the composition according to the present invention can appropriately contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, an antioxidant, etc., if necessary depending on the administration method and formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0102] The dosage of the vaccine composition to a patient varies depending on many factors including the patient's height, body surface area, age, specific compound administered, sex, administration time and route, general health status, and other drugs administered simultaneously. The pharmaceutically active DNA can be administered in an amount of 100 ng / kg body weight to 10 mg / kg body weight, more preferably 1 to 500 μg / kg (body weight), and most preferably 5 to 50 μg / kg (body weight), but the dosage can be adjusted considering those factors.

[0103] Furthermore, the vaccine composition of the present invention is administered in a pharmaceutically effective amount.

[0104] The present invention also provides the use of the gram-negative bacteria outer membrane-derived nanovesicles according to the present invention for producing a pharmaceutical composition for treating a disease.

[0105] The present invention also provides a method for treating a disease, comprising administering to a subject in need of an effective amount of a pharmaceutical composition comprising the Gram-negative bacterial outer membrane-derived nanovesicles according to the present invention.

[0106] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined according to factors including the type and severity of the individual, age, gender, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including co-administered drugs, and other factors well known in the medical field. The vaccine composition of the present invention can be administered at a dosage of 0.1 mg / kg to 1 g / kg, more preferably at a dosage of 1 mg / kg to 500 mg / kg. On the other hand, the said dosage can be appropriately adjusted according to the age, gender, and condition of the patient.

[0107] The above-mentioned "effective amount" of the present invention refers to an amount that, when administered to an individual, exhibits an improvement, treatment, detection, diagnosis, or inhibitory or reducing effect on cancer and the disease of the present invention, or cancer and the disease of the present invention. The said "individual" can be an animal, preferably a mammal, particularly an animal including a human, and can be cells, tissues, organs, etc. derived from the animal. The said individual can be a patient in need of the effect.

[0108] The above-mentioned "treatment" of the present invention comprehensively refers to improving the symptoms caused by cancer or the said disease, which may include curing the said disease, substantially preventing it, or improving the condition. It includes, but is not limited to, reducing, curing, or preventing one or most of the symptoms resulting from the said disease.

[0109] As used herein, the term "comprising" is used in the same sense as "including" or "characterized by", and in the compositions or methods according to the present invention, it does not exclude additional components or method steps not specifically mentioned. Also, the term "consisting of" means excluding additional elements, steps, or components not otherwise described. The term "essentially consisting of" means that in the scope of a composition or method, it may include substances or steps that do not substantially affect the basic characteristics in addition to the described substances or steps.

Brief Description of the Drawings

[0110]

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Embodiments for Carrying Out the Invention

[0111] Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those having ordinary knowledge in the art that these examples are merely for illustrating the present invention and should not be construed as limiting the scope of the present invention by the examples.

[0112] Example 1. Production of Nano Vesicles Derived from the Outer Membrane of Gram-Negative Bacteria To prepare nano vesicles derived from the outer membrane of Gram-negative bacteria, Escherichia coli BL21(DE3)ΔmsbB-AmCyan was cultured in vegetable peptone-based lysogeny broth (VP-LB), and the culture solution was centrifuged at 6,000×g for 20 minutes at 4°C. The supernatant was removed to obtain bacteria. The obtained bacteria were suspended using 100 mL of Tris-based saline (TBS; 50 mM Tris-HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl) per liter of the culture solution. The suspension was centrifuged at 6,000×g for 20 minutes at 4°C, and the supernatant was removed to wash the bacterial cells.

[0113] The washed bacterial cells were rapidly frozen using liquid nitrogen and then suspended in 30 mL of Buffer A (10 mM Tris-HCl (pH 8.0), 5% sucrose). They were treated with Sarkosyl at a final concentration of 1% for 30 minutes at room temperature. The suspension was centrifuged at 10,000×g for 20 minutes at 4°C, and the supernatant was removed. Subsequently, the pellet was suspended in 30 mL of Buffer A and treated at 4°C for 10 minutes with a final Sarkosyl concentration of 0.25%. The suspension was centrifuged at 10,000×g for 20 minutes at 4°C, and the supernatant was removed. The process of suspending the pellet in 50 mL of Buffer A, centrifuging the suspension at 10,000×g for 20 minutes at 4°C, and removing the supernatant was repeated a total of 3 times to remove Sarkosyl and isolate the Gram-negative bacterial outer membrane.

[0114] After suspending the Gram-negative bacterial outer membrane in 50 mL of Buffer A, Gram-negative bacterial outer membrane-derived nanovesicles were produced using a microfluidizer processor (Microfluidics, MA) at 10,000 psi through 4 cycles. The solution that passed through the microfluidizer was centrifuged at 6,000×g for 20 minutes at 4°C, and the supernatant was taken. Only substances with a molecular weight of 500 kDa or more were concentrated using the tangential flow filtration method, and dialysis was carried out using Buffer A to obtain a sample of about 7 mL. Finally, 3 mL of 2.5 M sucrose, 3 mL of 0.8 M sucrose, and 7 mL of the sample were sequentially placed in an ultracentrifuge tube and ultracentrifuged at 200,000×g for 2 hours at 4°C to finally obtain Gram-negative bacterial outer membrane-derived nanovesicles from the layer between 2.5 M sucrose and 0.8 M sucrose.

[0115] The manufacturing method of Gram-negative bacterial outer membrane-derived nanovesicles was shown schematically as in Figure 1.

[0116] Example 2. Isolation of Outer Membrane Vesicles from Gram-Negative Bacteria The bacterium used to isolate outer membrane vesicles from Gram-negative bacteria is E. coli BL21 (DE3) ΔmsbB-AmCyan. The bacterium was cultured in VP-LB, and the culture solution was centrifuged at 6,000×g for 20 minutes at 4°C. The supernatant was filtered using a 0.45 μm pore-sized filter, and the filtrate was concentrated using a tangential flow filtration method to retain only substances with a molecular weight of 100 kDa or more. The concentrated solution was ultracentrifuged at 100,000×g for 2 hours at 4°C, and the pellet was suspended in 50% iodixanol. 2 mL of 50% iodixanol suspension, 1.5 mL of 40% iodixanol, and 1 mL of 10% iodixanol were sequentially added to the ultracentrifugation tube, and ultracentrifugation was performed at 200,000 xg for 2 hours at 4°C to separate the outer membrane vesicles from Gram-negative bacteria from the layer between 40% iodixanol and 10% iodixanol.

[0117] Example 3. Characterization of Outer Membrane Vesicles and Outer Membrane-Derived Nanovesicles from Gram-Negative Bacteria 3-1. Confirmation of the Morphology and Size of Outer Membrane Vesicles and Outer Membrane-Derived Nanovesicles To analyze the morphology of the outer membrane-derived nanovesicles produced by the method of Example 1 and the outer membrane vesicles separated by the method of Example 2, observation was carried out using a transmission electron microscope. The outer membrane vesicles and outer membrane-derived nanovesicles were adsorbed onto a glow-discharged carbon-coated copper grid for 20 minutes. The grid was washed with distilled water and stained with 1% uranyl acetate for 2 seconds, and then observed with a transmission electron microscope (JEM1011, JEOL, Japan). As a result, as shown in Fig. 2a, it was confirmed that both the outer membrane vesicles and outer membrane-derived nanovesicles consisted of a lipid bilayer and had a diameter of 50-100 nm.

[0118] In addition, to measure the sizes of outer membrane vesicles and outer membrane-derived nanovesicles, the outer membrane vesicles and outer membrane-derived nanovesicles were diluted with HEPES-buffered saline (HBS, 20 mM HEPES (pH 7.4), 150 mM NaCl) at a concentration of 1 μg / mL, then placed in a cuvette and analyzed with a dynamic light scattering particle size analyzer (Zetasizer Nano ZS, Malvern Instruments, UK). As a result, as shown in Fig. 2b, it was confirmed that the average diameters of the outer membrane vesicles and outer membrane-derived nanovesicles were 70.1 ± 11.4 nm and 74.3 ± 15.8 nm, respectively.

[0119] 3-2. Confirmation of the total number of particles and total protein amount of outer membrane vesicles and outer membrane-derived nanovesicles To measure the total number of particles of outer membrane vesicles and outer membrane-derived nanovesicles, the outer membrane vesicles and outer membrane-derived nanovesicles were diluted with HBS at a concentration of 1 μg / mL, then placed in a chamber and analyzed with a nanoparticle tracking analyzer (Nanosight LM-10, Malvern Instruments, UK). As a result, as shown in Fig. 2c, the outer membrane vesicles and outer membrane-derived nanovesicles were found to have an average of (1.33 ± 0.24) x 10 11 particles and (7.35 ± 3.54) x 10 13 particles per liter of culture medium, respectively, and it was confirmed that about 550 times more outer membrane-derived nanovesicles were obtained than outer membrane vesicles based on the total number of particles.

[0120] In addition, the total protein amounts of the outer membrane vesicles and outer membrane-derived nanovesicles were quantified by Bradford assay. As a result, as shown in Fig. 2d, it was confirmed that the outer membrane vesicles and outer membrane-derived nanovesicles obtained an average of 0.812 ± 0.001 mg and 14.200 ± 0.283 mg per liter of culture medium, respectively, and it was confirmed that about 17 times more outer membrane-derived nanovesicles were obtained than outer membrane vesicles based on the total protein amount.

[0121] In addition, to confirm the purity of outer membrane vesicles and outer membrane-derived nanovesicles, the total number of particles relative to the total protein amount was calculated. As a result, as shown in Fig. 2e, the outer membrane vesicles and outer membrane-derived nanovesicles were (0.164 ± 0.024)×10 9 particles / μg and (5.177 ± 0.220)×10 9 particles / μg, respectively, and it was confirmed that the outer membrane-derived nanovesicles had a purity approximately 32 times higher than that of the outer membrane vesicles.

[0122] 3-3. Confirmation of protein distribution and presence of outer membrane proteins in outer membrane vesicles and outer membrane-derived nanovesicles First, to prepare whole cell lysates, the E. coli BL21 (DE3) ΔmsbB-AmCyan culture was centrifuged at 6,000×g for 20 minutes at 4°C. The supernatant was removed, and then the pellet was treated with RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF) and suspended. The suspension was then sonicated to obtain whole cell lysates.

[0123] To confirm the protein distribution of whole cell lysates, outer membrane vesicles, and outer membrane-derived nanovesicles, 5 μg of whole cell lysates, outer membrane vesicles, and outer membrane-derived nanovesicles were electrophoresed under non-reducing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE) and stained with Coomassie Brilliant Blue. As a result, as shown in Fig. 2f, unlike whole cell lysates and outer membrane vesicles in which protein bands of various sizes were confirmed, only a limited number of protein bands were confirmed in outer membrane-derived nanovesicles.

[0124] In addition, to confirm the presence of outer membrane proteins in whole cell lysates, outer membrane vesicles, and outer membrane-derived nanovesicles, 1 μg of whole cell lysates, outer membrane vesicles, and outer membrane-derived nanovesicles was electrophoresed under non-reducing conditions and transferred to a polyvinylidene fluoride (PVDF) membrane for semi-dry transfer. Subsequently, the membrane was treated with a 3% non-fat milk / TBS-T (Tween 20 0.05%) solution for 1 hour of blocking. Then, a primary antibody that specifically binds to the OmpA protein was treated on the membrane and reacted at room temperature for 2 hours. After washing the membrane 3 times with TBS-T (Tween 20 0.05%), a secondary antibody conjugated with peroxidase was treated and reacted at room temperature for 1 hour. After washing the membrane 3 times again with TBS-T (Tween 20 0.05%), the expression of the OmpA protein was confirmed using an enhanced chemiluminescence (ECL) substrate. As a result, as shown in Figure 2g, it was confirmed that OmpA was more highly expressed in outer membrane vesicles and outer membrane-derived nanovesicles compared to whole cell lysates.

[0125] 3-4. Measurement of AmCyan fluorescence values and total nucleic acid amounts in whole cell lysates and outer membrane-derived nanovesicles To confirm the cytoplasmic proteins in whole cell lysates and outer membrane-derived nanovesicles, the relative amount of the cytoplasmic fluorescent protein AmCyan was confirmed. Using a spectrophotometer, the fluorescence value was measured at an excitation wavelength of 457 nm and an emission wavelength of 491 nm. As a result, as shown in Figure 2h, it was confirmed that there was almost no cytoplasmic fluorescent protein in outer membrane-derived nanovesicles compared to whole cell lysates.

[0126] To confirm the total amount of nucleic acids in the whole cell lysates and the outer membrane-derived nanovesicles, we measured the absorbance at wavelength=260 nm using a microspectrophotometer. As a result, as shown in Figure 2i, it was confirmed that there was almost no nucleic acid in the outer membrane-derived nanovesicles compared to the whole cell lysates.

[0127] Taking all of the above results into consideration, we have established a method for producing and purifying highly pure nanovesicles derived from the outer membrane of Gram-negative bacteria, which have a production yield several tens to several hundreds of times higher than that of outer membrane vesicles, and in which the bacterial inner membrane is destroyed and unnecessary substances such as cytoplasmic proteins and nucleic acids are efficiently removed.

[0128] Example 4. Confirmation of the innate immunity induction function of Gram-negative bacterial outer membrane vesicles and outer membrane-derived nanovesicles In order to confirm the innate immunity induction function of the outer membrane vesicles prepared by the method of Example 1 and the outer membrane vesicles isolated by the method of Example 2, the outer membrane vesicles and the outer membrane-derived nanovesicles were incubated with HEK-Blue. TM hTLR2 and HEK-Blue TM The reaction level was confirmed by treating hTLR4, and the cytokine secretion was confirmed by treating mouse macrophages, RAW264.7.

[0129] HEK-Blue TM hTLR2 and HEK-Blue TM To confirm the degree of response of hTLR4 to outer membrane vesicles and outer membrane-derived nanovesicles, each cell (2 × 10 5(Cells / well) were allowed to adhere to the well plate for 24 hours. Then, after treating each cell with outer membrane vesicles and outer membrane-derived nanovesicles at various concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 ng / mL) for 18 hours, conditioned medium was obtained. The obtained conditioned medium was reacted with a chemiluminescence substrate to measure RLU, and RLU was corrected (normalized) using the RLU when outer membrane vesicles and outer membrane-derived nanovesicles were not treated.

[0130] To confirm the reactivity of RAW264.7 to outer membrane vesicles and outer membrane-derived nanovesicles, RAW264.7 (5×10 4 (Cells / well) were allowed to adhere to the well plate for 24 hours. Then, after treating RAW264.7 with outer membrane vesicles and outer membrane-derived nanovesicles at various concentrations (0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 ng / mL) for 24 hours, conditioned medium was obtained. mTNF-α and mIL-6 in the obtained conditioned medium were quantified by ELISA.

[0131] As a result, as shown in FIGS. 3a - d, outer membrane vesicles and outer membrane-derived nanovesicles were able to induce innate immunity in a concentration-dependent manner, and at the same concentration, it was confirmed that outer membrane vesicles induced more innate immunity than outer membrane-derived nanovesicles. These results suggest that outer membrane-derived nanovesicles have an innate immunity-inducing function while having fewer side effects compared to outer membrane vesicles.

[0132] Example 5. Characterization of Outer Membrane Nanovesicles Produced from Various Types of Gram-Negative Bacteria 5-1. Confirmation of the Morphology and Size of Outer Membrane-Derived Nanovesicles Produced from Various Types of Gram-Negative Bacteria Referring to the method of Example 1 above, E. coli BL21 (DE3) wild-type, Pseudomonas aeruginosa PAO1 wild-type, Salmonella enterica wild-type, and S. enterica ΔmsbB were cultured to produce outer membrane-derived nanovesicles. To analyze the morphology and size of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria, observation and analysis were carried out using a transmission electron microscope and a dynamic light scattering analyzer with reference to Example 3-1 above. As a result, as shown in Fig. 4a, it was confirmed that all outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria consisted of a lipid bilayer. Also, as shown in Fig. 4b, it was confirmed that the average diameters of outer membrane-derived nanovesicles of E. coli BL21 (DE3) wild-type, Pseudomonas aeruginosa PAO1 wild-type, Salmonella enterica wild-type, and S. enterica ΔmsbB were 125.5 ± 40.2 nm, 124.0 ± 39.3 nm, 144.6 ± 48.9 nm, and 154.8 ± 32.1 nm, respectively.

[0133] 5-2. Confirmation of the total number of particles and total protein amount of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria To confirm the total number of particles and total protein amount of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria, analysis and quantification were carried out using a nanoparticle tracking analyzer and Bradford assay with reference to Example 3-2 above. As a result, as shown in Fig. 4c, outer membrane-derived nanovesicles of E. coli BL21 (DE3) wild-type, P. aeruginosa PAO1 wild-type, S. enterica wild-type, and S. enterica ΔmsbB had an average of (2.10 ± 0.22) x 10 13 particles, (2.56 ± 0.09) x 10 13 particles, (3.52 ± 0.23 x 10 13 particles, (3.36 ± 0.59) x 10 13It was confirmed that particles were obtained. Also, as shown in Fig. 4d, it was confirmed that for E. coli BL21 (DE3) wild-type, P. aeruginosa PAO1 wild-type, S. enterica wild-type, and S. enterica ΔmsbB outer membrane-derived nanovesicles, 12.733 ± 1.803 mg, 11.043 ± 1.866 mg, 24.151 ± 3.077 mg, and 25.479 ± 3.619 mg were obtained per liter of culture medium, respectively.

[0134] In addition, to confirm the purity of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria, the total number of particles relative to the total protein amount was calculated. As a result, as shown in Fig. 4e, for E. coli BL21 (DE3) wild-type, P. aeruginosa PAO1 wild-type, S. enterica wild-type, and S. enterica ΔmsbB outer membrane-derived nanovesicles, they were (1.657 ± 0.112)×10 9 particles / μg, (2.353 ± 0.349)×109 particles / μg, (1.467 ± 0.140)×10 9 particles / μg, and (1.320 ± 0.193)×109 particles / μg, respectively, indicating their purity.

[0135] Example 6. Confirmation of the innate immune induction function of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria The innate immune induction function of outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria, prepared by the method of Example 5 above, was confirmed by the method of Example 4 above. As a result, as shown in Figs. 5a - d, it was confirmed that outer membrane-derived nanovesicles produced from various types of Gram-negative bacteria can induce innate immunity in a concentration-dependent manner.

[0136] Example 7. Production of outer membrane-derived nanovesicles from Gram-negative bacteria containing polyethylene glycol An attempt was made to produce outer membrane-derived nanovesicles containing polyethylene glycol by including polyethylene glycol on the surface of nanovesicles derived from the outer membrane of Gram-negative bacteria produced by the method of Example 1 above.

[0137] The nanovesicles derived from the outer membrane of Gram-negative bacteria produced by the method of Example 1 above were diluted to 1 mg / mL using HEPES-buffered saline (20 mM HEPES (pH 7.4), 150 mM NaCl), and then mixed so that the final concentrations of cholesterol-PEG and cholesterol-PEG-biotin were 20 μg / mL and 2 μg / mL, respectively. Subsequently, the mixture was reacted at 4°C for 30 minutes, and then PEGylated outer membrane-derived nanovesicles were obtained by size exclusion chromatography ( PEGylation OMNV).

[0138] PEGylation To confirm the PEGylation of OMNV, OMNV at various concentrations (0, 100, 300, 1000 ng / mL) or PEGylation OMNV was adhered at room temperature for 2 hours. Then, it was blocked with 1% BSA / PBS at room temperature for 1 hour and treated with streptavidin conjugated with peroxidase (streptavidin-HRP) at room temperature for 30 minutes. Finally, luminescence was measured using a multimode plate reader with BM chemiluminescence substrate to confirm the presence or absence of PEGylation.

[0139] As a result, as shown in Figure 6, PEGylation in the case of OMNV, it was found that cholesterol-polyethylene glycol-biotin bound to the outer membrane-derived nanovesicles and showed a high RLU. On the other hand, in the case of OMNV, it was confirmed that a low RLU was shown.

[0140] Example 8. Drug delivery using outer membrane-derived nanovesicles of Gram-negative bacteria 8-1. Anticancer drug delivery using outer membrane-derived nanovesicles from Gram-negative bacteria - 1: Outer membrane-derived nanovesicles loaded with an anticancer drug during the manufacturing process To confirm whether daunorubicn (DNR), an anticancer drug, can be delivered using the outer membrane-derived nanovesicles produced by the method of Example 1 above, outer membrane-derived nanovesicles loaded with daunorubicn were produced according to the method of Example 1. At the stage where the outer membrane of Gram-negative bacteria was separated by treating with Sarkosyl, 500 μg / mL of daunorubicn was added. Then, serial extrusion was performed to prepare outer membrane-derived nanovesicles loaded with daunorubicn (OMNV DNR ).

[0141] OMNV DNR To confirm whether daunorubicn can be delivered to target cells using OMNV DNR , Alexa 488 NHS Ester was reacted with OMNV DNR , and only the fluorescently labeled OMNV Alexa 488 OMNV DNR was separated by size exclusion chromatography ( Alexa 488 OMNV DNR ). RAW264.7 was cultured on gelatin-treated glass, and Alexa 488 OMNV DNR was treated at 1 μg / mL for 24 hours, then the nuclei of the cells were stained with Hoechst and observed under a fluorescence microscope. Furthermore, Alexa 488 OMNV DNR was treated with RAW264.7 at multiple concentrations (0, 10, 100, 1000 ng / mL), and flow cytometry was performed after 24 hours.

[0142] As a result, as shown in FIGS. 7a - b, it was confirmed that OMNV DNR delivered daunorubicn to the target cells.

[0143] 8-2. Anticancer drug delivery using outer membrane-derived nanovesicles from Gram-negative bacteria - 2: Outer membrane-derived nanovesicles loaded with an anticancer drug by various methods after manufacturing After manufacturing outer membrane-derived nanovesicles by the method of Example 1 above, in order to confirm whether daunorubicin can be delivered to target cells even when daunorubicin is loaded by various methods, after manufacturing outer membrane-derived nanovesicles by the method of Example 1 above, daunorubicin was loaded by incubation, sonication, and freezing-thawing methods.

[0144] First, to load daunorubicin into outer membrane-derived nanovesicles by incubation, outer membrane-derived nanovesicles (1 mg / mL) and daunorubicin (500 μg / mL) were mixed and incubated at 37 °C for 6 hours. Also, to load daunorubicin into outer membrane-derived nanovesicles by sonication, outer membrane-derived nanovesicles (1 mg / mL) and daunorubicin (500 μg / mL) were mixed, and sonication was repeated 10 times at an amplitude of 50%, a cycle of 0.5, and 4 °C. Finally, to load daunorubicin into outer membrane-derived nanovesicles by freezing-thawing, outer membrane-derived nanovesicles (1 mg / mL) and daunorubicin (500 μg / mL) were mixed, frozen in liquid nitrogen for 3 minutes, and then thawed at 37 °C for 3 minutes, repeating 10 times. Size exclusion chromatography was performed to separate only the outer membrane-derived nanovesicles loaded with daunorubicin in the mixture that had undergone the incubation, sonication, and freezing-thawing processes. Finally, after manufacturing outer membrane-derived nanovesicles, the outer membrane-derived nanovesicles loaded with daunorubicin by incubation, sonication, and freezing-thawing methods were designated as OMNV DNR-Inc 、OMNV DNR-Son 、OMNV DNR-FT respectively.

[0145] OMNV DNR-Inc 、OMNV DNR-Son 、OMNV DNR-FT To confirm whether daunorubicin can be delivered to target cells using OMNV DNR-Inc 、OMNV DNR-Son, OMNV DNR-FT were each reacted with Alexa 488 NHS ester, and only the fluorescently labeled ones were separated ( Alexa 488 OMNV DNR-In , Alexa 488 OMNV DNR-Son , Alexa 488 OMNV DNR-FT ). As a result of treating these with RAW264.7 and observing them by flow cytometry and fluorescence microscopy in Example 8-1 above, as shown in FIGS. 8a-b, 9a-b, and 10a-b, it was confirmed that OMNV DNR-Inc , OMNV DNR-Son , and OMNV DNR-FT delivered daunorubicin to the target cells.

[0146] 8-3. Delivery of anti-inflammatory drugs using outer membrane-derived nanovesicles from Gram-negative bacteria To confirm whether dexamethasone (DEX), an anti-inflammatory agent, can be delivered using the outer membrane-derived nanovesicles produced by the method of Example 1 above, outer membrane-derived nanovesicles loaded with dexamethasone were produced according to the method of Example 1. At the stage of separating the outer membrane of Gram-negative bacteria by treating with Sarkosyl, 500 μg / mL of dexamethasone was added and extrusion was performed to produce outer membrane-derived nanovesicles loaded with dexamethasone (OMNV DEX ).

[0147] OMNV DEX was used to confirm whether it can induce an anti-inflammatory response. RAW264.7 (5×10 4 cells / well) was adhered to a well plate for 24 hours. Then, RAW264.7 was treated with various concentrations (10, 30, 100, 300, 1000 ng / mL) of OMNV DEX and the OMNV produced in Example 1 for 18 hours, and then conditioned medium was obtained. The mIL-6 in the obtained conditioned medium was quantified by ELISA.

[0148] As a result, as shown in FIG. 11, OMNV DEXIt was confirmed that it can suppress IL-6 secretion in macrophages in a concentration-dependent manner.

[0149] Example 9. Protein Delivery Using Outer Membrane-Derived Nanovesicles from Gram-Negative Bacteria To confirm that proteins can be delivered using the outer membrane-derived nanovesicles produced by the method of Example 1 above, outer membrane-derived nanovesicles loaded with GFP were produced according to the method of Example 1. GFP was added at 500 μg / mL at the stage of treating with Sarkosyl to separate the outer membrane of Gram-negative bacteria, and serial extrusion was performed to produce outer membrane-derived nanovesicles loaded with GFP (OMNV GFP ).

[0150] OMNV GFP To confirm that GFP can be delivered to target cells using OMNV GFP OMNV was reacted with Alexa 594 NHS Ester, and only the fluorescently labeled OMNV GFP was separated by size exclusion chromatography ([[]] Alexa 594 OMNV GFP ). RAW264.7 was cultured on gelatin-treated glass, Alexa 594 OMNV GFP After treating with 1 μg / mL for 24 hours, the nuclei of the cells were stained with Hoechst and observed under a fluorescence microscope. Furthermore, Alexa 594 OMNV GFP OMNV was treated with RAW264.7 at various concentrations (0, 10, 100, 1000 ng / mL), and flow cytometry was performed after 24 hours.

[0151] As a result, as shown in FIGS. 12a - b, it was confirmed that OMNV GFP delivered GFP to target cells.

[0152] From the above, it was confirmed that various drugs, proteins, etc. can be delivered to target cells using outer membrane-derived nanovesicles.

[0153] Example 10. Confirmation of Side Effects after Injection of Outer Membrane-Derived Nanovesicles from Gram-Negative Bacteria To confirm the side effects after injection of outer membrane-derived nanovesicles produced by the method of Example 1 above, C57BL / 6 (male, 6 weeks old) mice were injected intraperitoneally with 50 μg / head of outer membrane-derived nanovesicles, and then serum was separated from the blood of the mice 24 hours later. Subsequently, blood biochemical analysis was performed using an automatic chemistry analyzer (BS-390, Mindray, China).

[0154] As a result, as shown in Figure 13, the mice injected with outer membrane-derived nanovesicles showed no significant difference from the control group in most items, and it was confirmed that only the C-reactive protein concentration increased significantly slightly.

[0155] Taking the above results together, outer membrane-derived nanovesicles from Gram-negative bacteria have almost no side effects, and cancer treatment and diagnosis methods, delivery methods of substances for disease treatment and diagnosis, and vaccine delivery methods for disease prevention and treatment can be very usefully utilized in the development of treatments and vaccines for various diseases including cancer and infectious diseases.

[0156] Example 11. Confirmation of in vivo distribution after injection of outer membrane-derived nanovesicles from Gram-negative bacteria To confirm the in vivo distribution after injection of outer membrane-derived nanovesicles produced by the method of Example 1 above, outer membrane-derived nanovesicles fluorescently labeled with Cy7 ( Cy7 OMNV) were prepared, and free Cy7 was removed by size exclusion chromatography. Then Cy7 the whole body distribution was confirmed by time using an in vivo imaging system (IVIS) device by injecting OMNV into the peritoneal cavity of mice. In addition, major organs (thymus, spleen, heart, liver, lung, kidney, skin at the injection site, small intestine) 24 hours after injection were separated and the in-organ distribution was confirmed with an IVIS device. As a control group Cy7 Dye Control was used.

[0157] As a result, as shown in Figure 14a, Cy7Dye Control could not be confirmed in systemic distribution. However, as shown in Fig. 14b, Cy7 it was confirmed that OMNV spread around the abdominal cavity over time. Also, as shown in Figs. 14c and 14d, Cy7 Dye Control could not be confirmed in organ distribution. However, Cy7 it was confirmed that OMNV had strong signals in the liver and small intestine, and when the signals were corrected by organ weight, strong signals were found in the thymus, spleen, heart, and liver.

[0158] Example 12. Confirmation of the anti-cancer efficacy of outer membrane-derived nanovesicles from Gram-negative bacteria To confirm the anti-cancer efficacy of the outer membrane-derived nanovesicles produced by the method of Example 1 above, mouse bladder cancer cell line MB49 was injected subcutaneously into C57BL / 6 (male, 7-week-old) mice at 1x10 6 cells / head. Then, over time, the size of the cancer tissue was measured using Vernier calipers (size = 0.5 × width × length × length). When the size of the cancer tissue reached about 100 mm 3 the outer membrane-derived nanovesicles were injected three times at 3-day intervals (when injected once, 0, 1, or 10 μg / head), and the size of the cancer tissue was measured over time.

[0159] As a result, as shown in Figs. 15a - b, it was found that the outer membrane-derived nanovesicles exhibited anti-cancer efficacy.

[0160] Example 13. Confirmation of the interaction between outer membrane-derived nanovesicles from Gram-negative bacteria and dendritic cells 13-1. Confirmation of the effect of outer membrane-derived nanovesicles from Gram-negative bacteria on the survival rate and cytokine secretion of dendritic cells To confirm the effect of the outer membrane-derived nanovesicles produced by the method of Example 1 above on the survival rate and cytokine secretion of dendritic cells, bone marrow-derived dendritic cells (BMDCs) were isolated and treated with outer membrane nanovesicles, and the survival rate and cytokine secretion of dendritic cells were confirmed.

[0161] The femurs and tibias of C57BL / 6 (male, 4-week-old) mice were separated, DMEM (2.5% FBS) was injected into the bones to isolate bone marrow cells, and the cells were cultured using RPMI1640 (10% FBS, 50 μM 2-mercaptoethanol, 20 ng / mL rmGM-CSF) to isolate bone marrow-derived dendritic cells. The isolated bone marrow-derived dendritic cells (1x10 5 cells / well) were allowed to adhere to a well plate for 24 hours, and then the bone marrow-derived dendritic cells were treated with outer membrane nanovesicles at various concentrations (0, 1, 3, 10, 30, 100, 300, 1000 ng / mL) for 24 hours. Cell viability was confirmed by MTT assay, and mTNF-α, mIL-6, and mIL-12p40 were quantified by ELISA in the obtained conditioned medium.

[0162] As a result, as shown in Figure 16a, the outer membrane-derived nanovesicles did not affect the viability of dendritic cells, and as shown in Figures 16b, 16c, and 16d, it was confirmed that they could induce the secretion of mTNF-α, mIL-6, and mIL-12p40 in a concentration-dependent manner.

[0163] 13-2. Confirmation of uptake of outer membrane-derived nanovesicles from Gram-negative bacteria by dendritic cells To confirm that dendritic cells efficiently uptake the outer membrane-derived nanovesicles produced by the method of Example 1 above, outer membrane-derived nanovesicles ([[]] Alexa 594 OMNV) labeled with Alexa 594 were prepared, and free Alexa 594 was removed by size exclusion chromatography. Then Alexa 594 OMNV was treated with dendritic cells, and the uptake by dendritic cells was confirmed by fluorescence microscopy and flow cytometry. As a control group Alexa 594 Dye Control was used.

[0164] As a result, as shown in Figures 17a - b, dendritic cells Alexa 594 uptake OMNV in a concentration-dependent manner, Alexa 594 and it was confirmed that Dye Control was not taken up.

[0165] Example 14. Vaccination using outer membrane-derived nanovesicles from Gram-negative bacteria and antigens 14-1. Confirmation of the ability of outer membrane-derived nanovesicles to induce short-term immunity To confirm the short-term immune induction ability of the outer membrane-derived nanovesicles produced by the method of Example 1 above, the antigen (GFP or SARS-CoV-2 S protein) and the immunopotentiator (Alum) or CuSO4 were mixed with the outer membrane-derived nanovesicles and injected into the peritoneal cavity of C57BL / 6 (male, 4 weeks old) three times at one-week intervals. The time of the first injection was set as day 0, and blood was taken on days 7, 14, and 21, and serum was separated. The experimental procedure was as shown in Fig. 18a.

[0166] The antigen (GFP or SARS-CoV-2 S protein) was treated overnight at room temperature in a 96-well black plate (100 ng / well) and washed three times with PBS-T (Tween 20 0.05%). Then it was blocked with 1% BSA / PBS at room temperature for 1 hour and washed three times again with PBS-T (Tween 20 0.05%). The mouse serum was diluted with 1% BSA / PBS and treated at room temperature for 2 hours, and washed three times again with PBS-T (Tween 20 0.05%). Then, the secondary antibody (anti-mouse IgG-HRP) conjugated with peroxidase was diluted with 1% BSA / PBS and treated at room temperature for 1 hour, and washed three times again with PBS-T (Tween 20 0.05%). Finally, luminescence was measured using a multimode plate reader with BM chemiluminescence substrate to confirm the antigen-specific IgG titer.

[0167] As a result, as shown in Figs. 18b - 18e, it was confirmed that when the antigen, the immunopotentiator (Alum) or CuSO4 and the outer membrane-derived nanovesicles were injected together, not only the control group but also a higher antigen-specific IgG titer was shown compared to the case where only the antigen and the immunopotentiator (Alum) or CuSO4 were injected. Thus, it was confirmed that when the outer membrane-derived nanovesicles were administered in combination with the antigen and the immunopotentiator (Alum) or CuSO4, short-term immunity could be induced more effectively.

[0168] 14-2. Confirmation of the long-term immune induction ability of outer membrane-derived nanovesicles To confirm the long-term immune induction ability of outer membrane-derived nanovesicles, an antigen (GFP or SARS-CoV-2 S protein) and an immunopotentiator (Alum) or CuSO4 were mixed with outer membrane-derived nanovesicles and injected into the peritoneal cavity of C57BL / 6 (male, 4 weeks old) three times at one-week intervals. The first injection was on day 0, and blood was taken on days 21 and 49, and serum was separated. The experimental procedure was as shown in Fig. 19a.

[0169] The separated serum was measured for antigen-specific IgG titer as shown in Example 14-1 above. As a result, as shown in Figs. 19b - 19e, it was confirmed that not only the case where the antigen, immunopotentiator (Alum) or CuSO4 outer membrane-derived nanovesicles were injected together but also the case where only the antigen and immunopotentiator (Alum) or CuSO4 were injected showed a higher antigen-specific IgG titer than the control group. Thus, it was confirmed that co-administration of outer membrane-derived nanovesicles with an antigen and an immunopotentiator (Alum) or CuSO4 can more effectively induce long-term antigen-specific IgG titers.

[0170] Also, to confirm the effect of outer membrane-derived nanovesicles on long-term T cell activation, an antigen (GFP or SARS-CoV-2 S protein) and an immunopotentiator (Alum) or CuSO4 outer membrane-derived nanovesicles were mixed and injected into the peritoneal cavity of C57BL / 6 (male, 4 weeks old) three times at one-week intervals. The first injection was on day 0, and the spleen was separated from the mouse on day 49. Only T cells were separated from the separated spleen and treated with the antigen for 72 hours to obtain a conditioned medium. The mIFN-γ in the obtained conditioned medium was quantified by ELISA. The experimental procedure was as shown in Fig. 20a.

[0171] As a result, as shown in FIGS. 20b-20e, it was confirmed that not only the control group but also the case where the antigen, immunopotentiator (Alum) or outer membrane-derived nanovesicles were injected together showed higher antigen-specific mIFN-γ secretion than the case where only the antigen and immunopotentiator (Alum) or CuSO4 were injected. Thus, it was confirmed that when outer membrane-derived nanovesicles are administered in combination with an antigen and an immunopotentiator, a long-term antigen-specific Th1 response can be induced more effectively.

[0172] Overall, it was confirmed that when outer membrane-derived nanovesicles are administered in combination with an antigen and an immunopotentiator (Alum) or CuSO4, long-term immunity can be induced more effectively.

[0173] 14-3. Confirmation of the ability to induce immunity by the injection route of outer membrane-derived nanovesicles To confirm the ability to induce immunity by the injection route of outer membrane-derived nanovesicles, an antigen (H5N1 hemagglutinin), an immunopotentiator (Alum), and outer membrane-derived nanovesicles were mixed and injected into the peritoneal cavity or thigh muscle of C57BL / 6 (male, 4 weeks old) three times at one-week intervals. The time point of the first injection was designated as day0, and blood was collected on days 21 and 45, and serum was separated. The experimental procedure was as shown in FIG. 21a.

[0174] The separated serum was measured for antigen-specific IgG titer as shown in Example 14-1 above. As a result, as shown in FIGS. 21b-21e, both injection routes showed higher antigen-specific IgG titers when the antigen, immunopotentiator, and outer membrane-derived nanovesicles were injected together than not only the control group but also the case where only the antigen and immunopotentiator were injected. Thus, it was confirmed that when outer membrane-derived nanovesicles are administered in combination with an antigen and an immunopotentiator by peritoneal or intramuscular injection, a long-term antigen-specific IgG titer can be induced more effectively.

[0175] To summarize the above results, nanovesicles derived from the outer membrane of gram-negative bacteria have almost no side effects and can be very useful in the development of cancer treatment and diagnosis methods, delivery methods for disease treatment and diagnostic substances, and delivery methods for vaccines for disease prevention and treatment, as well as in the development of treatments and vaccines for various diseases, including cancer and infectious diseases.

[0176] In summary, the above results suggest that nanovesicles derived from the outer membrane of gram-negative bacteria can be used as a next-generation vaccine delivery vehicle to effectively induce the formation of neutralizing antibodies in the short and long term, and can be used as a vaccine to prevent the development of cancers such as viral infections and cervical cancer caused by viral infections, as well as bacterial infections.

[0177] Example 15. Preparation of Gram-negative bacterial outer membrane derived nanovesicles expressing outer membrane protein display system-based antigens To prepare gram-negative bacteria outer membrane-derived nanovesicles expressing an antigen based on the outer membrane protein display system, as shown in FIG. 22a, the signal sequences of the outer membrane protein (OmpA or LolB), the antigen (S1-RBD (SARS-CoV-2 Spike protein 1 receptor-binding domain) or H5N1 hemagglutinin), and His6-tag were inserted into the multiple cloning site of the pHCE vector, and E. coli BL21(DE3)ΔmsbB was transformed to overexpress the antigen in the gram-negative bacteria outer membrane. The transformed gram-negative bacteria were used to prepare outer membrane-derived nanovesicles expressing the antigen by the method of Example 1 above ( S1 RBD- OmpA OMNV, S1 RBD- LolB OMNV, H5N1-LolB OMNV).

[0178] To analyze the characteristics of the outer membrane-derived nanovesicles expressing the antigen, the size, yield, and purity of the outer membrane-derived nanovesicles expressing the antigen were measured by the methods of Examples 3-1 and 3-2 above. The results are as shown in Fig. 22b. On the other hand, to confirm the presence of the antigen in the outer membrane-derived nanovesicles, Western blotting was performed using a His6-tag specific primary antibody by the method of Example 3-3 above. As a result, as shown in Figs. 22c, 22d, and 22e, a His6-tag specific band was confirmed, and it was confirmed that the antigen was present in the outer membrane-derived nanovesicles.

[0179] Example 16. Confirmation of the ability of Gram-negative bacteria outer membrane-derived nanovesicles expressing an antigen based on an outer membrane protein display system to induce dendritic cell cytokine secretion The Gram-negative bacteria outer membrane-derived nanovesicles expressing an antigen based on an outer membrane protein display system produced by the method of Example 15 above were treated with bone marrow-derived dendritic cells by the method of Example 13-1 above to obtain a conditioned medium. mTNF-α, mIL-6, and mIL-12p40 were quantified by ELISA in the obtained conditioned medium.

[0180] As a result, as shown in Figs. 23a-f, it was confirmed that the outer membrane-derived nanovesicles expressing an antigen based on an outer membrane protein display system could induce the secretion of mTNF-α, mIL-6, and mIL-12p40 in a concentration-dependent manner in dendritic cells.

[0181] Example 17. Vaccination using Gram-negative bacteria outer membrane-derived nanovesicles expressing an antigen based on an outer membrane protein display system 17-1. Intraperitoneal administration model To confirm the short-term and long-term immune induction abilities of Gram-negative bacterium outer membrane-derived nanovesicles expressing an outer membrane protein display system-based antigen produced by the method of Example 15, Gram-negative bacterium outer membrane-derived nanovesicles expressing the antigen were injected into the abdominal cavity of C57BL / 6 (male, 4 weeks old) three times at 5- to 6-day intervals. The first injection was on day 0, and blood was collected on days 5, 11, and 17 to separate serum. The experimental procedure is as shown in Fig. 24a.

[0182] Next, antigen-specific IgG titers were confirmed according to the method of Example 14-1. As a result, as shown in Figs. 24b and 24c, it was confirmed that when Gram-negative bacterium outer membrane-derived nanovesicles expressing the antigen were injected, higher antigen-specific IgG titers were shown compared to the control group.

[0183] Also, according to the method of Example 14-2, the serum and spleen of the mice were separated on day 49, and the long-term immune induction ability of Gram-negative bacterium outer membrane-derived nanovesicles expressing an outer membrane protein display system-based antigen was confirmed. As a result, as shown in Figs. 24d and 24e, it was confirmed that when Gram-negative bacterium outer membrane-derived nanovesicles expressing the antigen were injected, higher antigen-specific IgG titers and mIFN-γ secretion were shown compared to the control group.

[0184] 17-2. Intramuscular administration model To confirm the short-term and long-term immune induction abilities of Gram-negative bacterium outer membrane-derived nanovesicles expressing the antigen, Gram-negative bacterium outer membrane-derived nanovesicles expressing the antigen were injected into the thigh muscle of C57BL / 6 (male, 4 weeks old) three times at 5- to 6-day intervals. The first injection was on day 0, and blood was collected on days 5, 11, and 17 to separate serum. The experimental procedure is as shown in Fig. 25a.

[0185] Next, the antigen-specific IgG titer was measured according to the method of Example 14-1 above, and as a result, it was confirmed that the antigen-expressing Gram-negative bacteria outer membrane-derived nanovesicles injected showed a higher antigen-specific IgG titer than the control group, as shown in Figures 25b and 25c.

[0186] Example 18. Confirmation of safety of nanovesicles derived from the outer membrane of Gram-negative bacteria expressing antigens based on the outer membrane protein display system In order to confirm the safety of the nanovesicles derived from the outer membrane of Gram-negative bacteria expressing an antigen based on the outer membrane protein display system prepared by the method of Example 15, the nanovesicles derived from the outer membrane of Gram-negative bacteria expressing the antigen were injected intraperitoneally into mice (0, 10, 50, 100 μg / head, single injection). The survival and weight of the mice were observed up to 48 hours after injection. As a result, as shown in Figures 26a-f, no significant difference was observed between the control group and the group injected with the nanovesicles derived from the outer membrane of Gram-negative bacteria expressing the antigen.

[0187] Taking the results of Examples 15, 16, 17, and 18 together, in order to enhance the therapeutic and preventive efficacy and vaccine delivery efficacy of outer membrane vesicles using an outer membrane protein display system utilizing outer membrane proteins of gram-negative bacteria such as OmpA, LolB, OmpF, ClyA, etc., the gram-negative bacterial outer membrane-derived nanovesicles composed of fusion proteins expressed alone or in combination with various proteins such as various peptides, cytokines, growth factors, antibodies, antigen peptides, and antigen proteins have almost no side effects and can be very useful in cancer treatment and diagnosis methods, delivery methods for disease treatment and diagnosis substances, and vaccine delivery methods for disease prevention and treatment, as well as in the development of treatments and vaccines for various diseases including cancer and infectious diseases.

[0188] Example 19. Method for loading proteins into Gram-negative bacterial outer membrane derived nanovesicles To develop a method for loading therapeutic substances, diagnostic substances, immune-enhancing substances, or vaccine antigens onto Gram-negative bacteria outer membrane-derived nanovesicles, according to the schematic diagram of Figure 27a, Gram-negative bacteria outer membrane-derived nanovesicles were mixed with GFP as a model substance. After that, GFP was loaded using electroporation, sonication, and freeze-thawing methods for the control group. Then, the amount of loaded GFP was analyzed by size exclusion chromatography (using the standard curve made with purified GFP). As shown in Figure 27b, it was confirmed that about 8.4 ng, 14.8 ng, 20.4 ng, and 24.4 ng of GFP were loaded onto 1 μg of Gram-negative bacteria outer membrane-derived nanovesicles (OMNV) by the control group, electroporation, sonication, and freeze-thawing methods, respectively.

[0189] In addition, to enhance the efficiency of loading therapeutic substances, diagnostic substances, immune-enhancing substances, or vaccine antigens onto Gram-negative bacteria outer membrane-derived nanovesicles, methods such as treating the Sarkosyl-treated outer membrane or purified Gram-negative bacteria outer membrane-derived nanovesicles with a well-known alkaline solution to open the membrane, loading the therapeutic substance, diagnostic substance, immune-enhancing substance, or vaccine antigen, and then re-sealing can also be used.

[0190] Example 20. Production of Gram-negative bacteria outer membrane-derived nanovesicles by various methods In order to produce outer membrane-derived nanovesicles from Gram-negative bacteria without using ultra-high speed centrifugation, after culturing E. coli BL21(DE3)ΔmsbB-AmCyan in VP-LB using the method of Example 1 above, the outer membrane of Gram-negative bacteria was separated using Sarkosyl. Then, outer membrane-derived nanovesicles from Gram-negative bacteria were produced using a microfluidic processor, and centrifugation, tangential flow filtration, and dialysis were carried out to obtain a sample of about 7 mL. Finally, outer membrane-derived nanovesicles from Gram-negative bacteria were finally obtained from the fraction obtained through Capto Core 700 (OMNV(UC-free)). On the other hand, using E. coli BL21(DE3)ΔmsbB, outer membrane-derived nanovesicles were also produced by the method of Example 1 (OMNV(UC)).

[0191] Example 21. Comparison of characteristics by the method for producing outer membrane nanovesicles of Gram-negative bacteria 21-1. Confirmation of morphology and size by the method for producing outer membrane-derived nanovesicles To analyze the morphology and size of OMNV(UC) and OMNV(UC-free) produced by the method of Example 20 above, observation and analysis were carried out using a transmission electron microscope and a dynamic light scattering analyzer with reference to Example 3-1 above. As a result, as shown in Fig. 28a, it was confirmed that both OMNV(UC) and OMNV(UC-Free) consist of a lipid bilayer. Also, as shown in Fig. 28b, it was confirmed that the average diameters of OMNV(UC) and OMNV(UC-free) are 74.3±15.8 nm and 105.1±10.7 nm, respectively.

[0192] 21-2. Confirmation of total particle number and total protein amount by the method for producing outer membrane-derived nanovesicles To confirm the total particle number and total protein amount of OMNV(UC) and OMNV(UC-free) produced by the method of Example 20 above, analysis and quantification were carried out using a nanoparticle tracking analyzer and a Bradford assay with reference to Example 3-2 above. As a result, as shown in Fig. 28c, OMNV(UC) and OMNV(UC-free) have an average of (7.35±0.35) x 10 per liter of culture medium 13particles, (7.45 ± 0.21) x 10 13 It was confirmed that particles were obtained. Also, as shown in Fig. 28d, it was confirmed that OMNV (UC) and OMNV (UC-free) were obtained at an average of 14.200 ± 0.283 mg and 4.400 ± 0.566 mg per liter of the culture medium, respectively.

[0193] Furthermore, to confirm the purity of OMNV(UC) and OMNV(UC-free), the total number of particles relative to the total protein amount was calculated. As a result, as shown in Fig. 28e, OMNV(UC) and OMNV(UC-free) were (5.353 ± 0.107) × 10 9 particles / μg and (15.183 ± 0.921) × 10 9 particles / μg, respectively, indicating their purity.

[0194] Example 22. Confirmation of innate immune induction function by the method for producing outer membrane-derived nanovesicles The innate immune induction functions of OMNV(UC) and OMNV(UC-free) produced by the method of Example 20 above were confirmed by the method of Example 4 above. As a result, as shown in Figs. 29a to d, it was confirmed that OMNV(UC) and OMNV(UC-free) can induce innate immunity in a concentration-dependent manner to almost the same extent.

[0195] Example 23. Confirmation of anti-cancer efficacy by the method for producing outer membrane-derived nanovesicles To confirm the anti-cancer efficacy of OMNV(UC) and OMNV(UC-free) produced by the method of Example 20 above, mouse bladder cancer cell line MB49 was injected subcutaneously into C57BL / 6 (male, 7 weeks old) mice at 5 x 10 5 cells / head, and then the size of the cancer tissue was measured using vernier calipers over time (size = 0.5 × width × length × length). When the size of the cancer tissue reached about 100 mm 3 OMNV (UC) and OMNV (UC-free) were injected three times at 3-day intervals (10 μg / head for one injection), and the size of the cancer tissue was measured over time.

[0196] As a result, as shown in Fig. 30, it was found that OMNV(UC) and OMNV(UC-free) exhibited anti-cancer efficacy.

[0197] Taking the above results together, for the increase in stability in vivo and in vitro including blood, the reduction of side effects, the enhancement of the efficacy of disease treatment, the enhancement of the efficacy of drug delivery systems, and the enhancement of the efficacy of vaccine delivery systems, various substances such as anti-cancer compounds, anti-inflammatory compounds, various immunopotentiators including STING Agonist, peptides, proteins, nucleic acids (mRNA, siRNA, DNA, plasmid, etc.), aptamers, toxins, and various forms of antigens, or their complexes are loaded (loading and / or display) alone or in combination inside or on the surface of outer membrane-derived nanovesicles, thereby reducing the side effects of outer membrane-derived nanovesicles from Gram-negative bacteria and effectively enhancing the stability and efficacy as a disease therapeutic agent, drug delivery, and / or vaccine delivery system.

[0198] The Gram-negative bacterium outer membrane-derived nanovesicles according to the present invention are produced by various methods and are technologies that can be utilized in various fields (Figure 31). The Gram-negative bacterium outer membrane-derived nanovesicles use various methods to perform additional 1) size modulation and homogenization, 2) reduction of side effects, increase in stability in vivo and in vitro including blood, enhancement of the effectiveness of disease treatment, improvement of the effectiveness of drug delivery, improvement of the effectiveness of vaccine delivery, various immune enhancers including anticancer compounds, anti-inflammatory compounds, STING Agonist, peptides, proteins, nucleic acids (mRNA, siRNA, DNA, plasmid, etc.), aptamers, toxins, and various forms of antigens, etc., various substances or their complexes alone or in combination are loaded (loading and / or display) into the interior or onto the surface of the outer membrane-derived nanovesicles, and compounds, peptides, proteins, fusion proteins, nucleic acids, aptamers, toxins, various forms of antigens, polymers, lipids, etc., various substances or their complexes alone or in combination required for cell or tissue targeting are loaded (loading and / or display) into the interior or onto the surface of the outer membrane-derived nanovesicles, 3) By combining the above methods, the side effects of the Gram-negative bacterium outer membrane-derived nanovesicles can be reduced, and the stability and efficacy as a disease treatment agent, drug delivery vehicle and / or vaccine delivery vehicle can be effectively enhanced.

[0199] In addition, the method for producing the Gram-negative bacterium outer membrane-derived nanovesicles of the present invention in which substances for enhancing disease treatment efficacy, drug delivery vehicle efficacy, and vaccine delivery vehicle efficacy are loaded (loading and / or display) alone or in combination can be used for therapeutic, drug delivery, vaccine, or experimental purposes in vitro or in vivo.

[0200] Furthermore, when considering the above results comprehensively, Gram-negative bacteria outer membrane-derived nanovesicles or Gram-negative bacteria outer membrane-derived nanovesicles loaded with therapeutic, diagnostic, and vaccine substances can be administered at well-known dosages by techniques well-known to those skilled in the medical or veterinary arts, taking into account factors such as the age, gender, species, breed of the individual, and the condition of the recipient animal and the route of administration. The route of administration can be transdermal, administration through mucous membranes (e.g., oral, nasal, anal, vaginal) or parenteral routes (intramuscular, subcutaneous, intravenous, intravenous, intraperitoneal, or intrasynovial). The therapeutic, diagnostic, and vaccine compositions can be administered alone or concomitantly or sequentially with other treatments or therapies. Dosage forms can include suspensions, syrups or elixirs, and formulations for oral, parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration, such as sterile suspensions or emulsions. The therapeutic, diagnostic, and vaccine compositions can be administered as sprays, mixed with food and / or water, or delivered to an appropriate carrier, diluent, or excipient, such as an additional mixture with sterile water, physiological saline, glucose, etc. The said compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffers, adjuvants, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the desired formulation and route of administration. Standard pharmaceutical textbooks such as "Remington's Pharmaceutical Sciences, 1990" can be referred to in order to manufacture appropriate formulations without undue experimentation.

Industrial Applicability

[0201] The method for producing Gram-negative bacteria outer membrane-derived nanovesicles, the cancer treatment and diagnosis methods using the same, the delivery method of substances for disease treatment and diagnosis, and the vaccine delivery method for disease prevention and treatment can be very usefully utilized in the development of treatments and vaccines for various diseases including cancer and infectious diseases, and have very high industrial applicability.

Claims

1. A method for producing outer membrane-derived nanovesicles from Gram-negative bacteria, the method comprising the following steps: (a) removing the inner membrane from Gram-negative bacteria to obtain the outer membrane; and (b) producing nanovesicles in a suspension containing the outer membrane.

2. The method according to claim 1, wherein the Gram-negative bacteria are selected from the group consisting of the genus Escherichia, the genus Helicobacter, the genus Hemophilus, the genus Neisseria, the genus Cyanobacterium, the genus Klebsiella, the genus Acetobacter, the genus Acinetobacter, the genus Enterobacter, the genus Chlamydia, the genus Vibrio, the genus Pseudomonas, the genus Salmonella, the genus Thiobacter, the genus Borrelia, the genus Burkholderia, the genus Serratia, and the genus Treponema.

3. The method according to claim 1, wherein the Gram-negative bacteria are transformed.

4. The method according to claim 3, wherein the Gram-negative bacteria are Gram-negative bacteria transformed so as to weaken the toxicity of the outer membrane-derived nanovesicles.

5. The method according to claim 3, wherein the Gram-negative bacteria are genetically engineered Gram-negative bacteria having any one or more genotypes selected from the group consisting of ΔmsbB, ΔkdsA, ΔkdsB, ΔlpxB, ΔkdtA, ΔlpxC, ΔlpxD, Δssc, ΔlpxA, and ΔhtrB.

6. The method according to claim 3, wherein the Gram-negative bacteria are Gram-negative bacteria transformed so as to express a cell membrane fusion substance.

7. The method according to claim 3, wherein the Gram-negative bacteria are Gram-negative bacteria transformed so as to be targeted to specific cells or tissues.

8. The production method according to claim 3, wherein the Gram-negative bacterium is transformed to express one or more selected from the group consisting of an antigen protein, an antigen peptide, an immunopotentiating protein, an immunosuppressive protein, a cell adhesion molecule, an antibody, a target-inducing protein, a cell membrane fusion protein, a cytokine, an enzyme, a growth factor, an extracellular domain of a membrane receptor, a marker protein, fragments thereof, and fusion proteins thereof.

9. The production method according to any one of claims 3 to 8, wherein the Gram-negative bacterium is a Gram-negative bacterium that has been transformed two or more times.

10. The production method according to claim 1, wherein the membrane of the Gram-negative bacterium-derived nanovesicle further contains components other than the outer membrane of the Gram-negative bacterium.

11. The production method according to claim 10, wherein the component other than the cell membrane of the Gram-negative bacterium is selected from the group consisting of a target-inducing substance, a cell membrane fusion substance, cyclodextrin, polyethylene glycol, and hyaluronic acid.

12. The production method according to claim 1, wherein the membrane component of the Gram-negative bacterium outer membrane-derived nanovesicle is chemically modified.

13. The production method according to claim 12, wherein the membrane component of the Gram-negative bacterium outer membrane-derived nanovesicle is chemically modified using a thiol group or an amine group, or a protein, polyethylene glycol, or hyaluronic acid is chemically bonded to the Gram-negative bacterium outer membrane-derived nanovesicle.

14. The production method according to claim 1, wherein the inner membrane of the Gram-negative bacterium is removed by treating the Gram-negative bacterium with Sarkosyl.

15. The production method according to claim 1, further comprising a step of separating the Gram-negative bacterium outer membrane-derived nanovesicle by a method selected from the group consisting of ultra-high speed centrifugation, density gradient ultra-high speed centrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, Capto Core chromatography, immunoaffinity separation, microfluidic technology separation, aqueous two-phase system, polymer-based precipitation, sonication, and extrusion after the step (b).

16. Gram-negative bacterium outer membrane-derived nanovesicles produced according to the method of claim 1.

17. A pharmaceutical composition for treating diseases, comprising gram-negative bacteria outer membrane-derived nanovesicles according to claim 16.

18. The pharmaceutical composition according to claim 17, wherein the disease is selected from the group consisting of thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colorectal cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer, and cervical cancer.

19. The pharmaceutical composition according to claim 17, wherein the disease is selected from the group consisting of hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, arteriosclerosis, obesity, peptic ulcer, Alzheimer's disease, emphysema, chronic obstructive pulmonary disease, asthma, skin diseases, and autoimmune diseases.

20. The pharmaceutical composition according to claim 17, further comprising a substance that enhances the therapeutic effect or reduces side effects.

21. The pharmaceutical composition according to claim 20, wherein the substance that enhances the therapeutic effect or reduces side effects is loaded on the gram-negative bacteria outer membrane-derived nanovesicles.

22. The pharmaceutical composition according to claim 20, wherein the substance that enhances the therapeutic effect or reduces side effects is one or more selected from the group consisting of anti-cancer agents, immune enhancers, STING agonists, anti-inflammatory agents, endotoxin inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles, and nanoparticles.

23. The pharmaceutical composition according to claim 22, wherein the nucleic acid is selected from the group consisting of DNA, RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino.

24. The pharmaceutical composition according to claim 22, wherein the nanoparticles are selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads.

25. A vaccine composition for preventing or treating diseases, comprising gram-negative bacteria outer membrane-derived nanovesicles according to claim 16.

26. The vaccine composition according to claim 25, further comprising an antigen.

27. The vaccine composition according to claim 26, wherein the antigen is loaded on the gram-negative bacteria outer membrane-derived nanovesicles.

28. The vaccine composition according to claim 26, wherein the antigen is a bacterium-derived antigen, a virus-derived antigen, a fungus-derived antigen, a cancer-derived antigen; or a mutation of Ras protein, Raf protein, Src protein, Myc protein, EGFR, PDGFR, VEGFR, p53, PTEN, or HER2 / neu.

29. The vaccine composition according to claim 25, wherein the disease is an infection caused by bacteria, viruses, or fungi.

30. The vaccine composition according to claim 29, wherein the infectious disease is selected from the group consisting of skin infectious diseases, respiratory infectious diseases, urogenital infectious diseases, bone and joint infectious diseases, central nervous system infectious diseases, and sepsis.

31. The vaccine composition according to claim 25, wherein the disease is selected from the group consisting of thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colon cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer, and cervical cancer.

32. The vaccine composition according to claim 25, wherein the disease is selected from the group consisting of hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, arteriosclerosis, obesity, peptic ulcer, Alzheimer's disease, chronic obstructive pulmonary disease, asthma, skin diseases, and autoimmune diseases.

33. The vaccine composition according to claim 25, wherein the vaccine is used for combined administration of a drug or an immunopotentiator for the purpose of increasing efficacy or reducing side effects.

34. Use of the gram-negative bacteria outer membrane-derived nanovesicles according to claim 16 for manufacturing a pharmaceutical composition for treating diseases.

35. A method for treating a disease, comprising administering an effective amount of a pharmaceutical composition comprising the gram-negative bacteria outer membrane-derived nanovesicles according to claim 16 to an individual in need thereof.

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