Transfection method

Abstract

In order to provide a novel method for safely and efficiently introducing nucleic acids, proteins or other target substances into T cells or other immune cells, the present invention provides a system for delivering target substances to immune cells comprising a combination of an ultrasound generating device and an aqueous solution or water of ultra-fine bubbles, the aqueous ultrafine bubble solution or ultrafine bubble water includes ultrafine bubbles having an average diameter of 200 nm or less and not containing phospholipids. The present invention provides, inter alia, a method for improving the delivery ability of nucleic acids or proteins to immune cells using ultrasound and the above-mentioned ultrafine bubble water, etc.

Description

This application is a divisional application of the Chinese patent application number 202080059401.8 (application date: June 25, 2020, invention name: transfection method). [Technical field]

[0001] The present invention relates to a system for delivering a target substance to immune cells, the system comprising a combination of ultrafine bubble water or an ultrafine bubble aqueous solution containing ultrafine bubbles and an ultrasonic generator; a method for increasing the delivery of nucleic acids or proteins to immune cells by using the ultrafine bubble water or aqueous solution and ultrasound; a preparation containing a combination of nucleic acids or proteins and the ultrafine bubble water or aqueous solution, for delivering the nucleic acids or proteins to immune cells by using in combination with ultrasonic irradiation; and a method for delivering nucleic acids or proteins to immune cells by contacting the immune cells with the preparation and treating the cells with ultrasound; etc. [Background Technology] Ultrasound has been mainly used as an ultrasound imaging method in the medical field. Microbubbles are making epoch-making progress in ultrasound diagnosis as ultrasound contrast agents. Recently, it is possible to use ultrasound for purposes other than diagnosis. For example, non-invasive cancer hyperthermia treatment by focusing ultrasound energy on the affected area and heating only the affected area is clinically applied to uterine fibroids and prostate cancer. In addition, research focusing on non-invasiveness and ease of spatial and temporal control is also underway to use ultrasound irradiation as a tool (sonoporation effect) for drug delivery systems (DDS) that deliver genes and drugs to target cells.

[0002] Up to now, have reported as the bubble liposome of the gene delivery system for skeletal muscle having wherein merged bubble liposome and ultrasonic technology, wherein perfluoropropane is encapsulated in the liposome modified by polyethylene glycol (PEG) (non-patent literature 1).In addition, have also reported the therapeutic drug for Duchenne muscular dystrophy, described therapeutic drug is for being combined with specific morpholino oligomer, encapsulation perfluorohydrocarbon and the bubble liposome (bubble lipid polymer complex (lipopolyplex)) of the PEG modification of the mean particle size of 50-500nm and is applied to muscle tissue or blood vessel, then the muscle tissue in vitro of ultrasonic irradiation, to realize that morpholino oligomer is efficiently introduced into muscle tissue (patent literature 1).Also have reported, gas encapsulated microbubble and ultrasound can be used for medicine and gene delivery to brain (non-patent literature 2).

[0003] It has been reported that using perfluoropropane gas as a filling gas results in smaller bubble liposomes than those using perfluorobutane gas or nitrogen gas (Non-Patent Document 3). Furthermore, it has been reported that administering a bubble lipid polymer complex, which is a combination of nanobubbles and plasmids, to ddY mice and irradiating brain tissue with ultrasound can introduce the plasmids into the vascular endothelium or extravascular area, and that the site of introduction varies depending on the gas encapsulation efficiency (Non-Patent Document 4).

[0004] However, there is a concern that the use of vesicle liposomes may lead to antigenicity problems caused by lipids. In addition, the output intensity is 1.5 to 2.5 W / cm 2 The high ultrasound exposure raises concerns about the safety of ultrasound diagnosis, leading to problems in practicality.

[0005] The inventors of the present invention have clarified that 8 Nanobubble water containing phospholipid-free nanobubbles has an excellent antibacterial effect (Patent Document 2). However, there is no report on the introduction of target substances (such as nucleic acids, proteins, etc.) into cells by combining nanobubble water with ultrasound.

[0006] The research and development of cancer immunotherapy using CAR-T cells or TCR-T cells transfected with chimeric antigen receptors (CAR) or T cell receptors (TCR) derived from cancer antigen-specific killer T cells is progressing rapidly. Current CAR-T cell therapy generally uses such a method, wherein the CAR gene is introduced into T cells collected from the patient in vitro by using viral vectors (such as lentiviral vectors, etc.) to produce CAR-T cells, and the CAR-T cells are administered to the patient, like Kymriah (trade name) and Yescarta (trade name) approved in the U.S. However, this method has the problem of high production cost due to the cost of cell culture and viral vector preparation, etc. If CAR and exogenous TCR can be selectively introduced into immune cells (such as T cells, etc.) in vivo, then in vitro preparation becomes unnecessary, and CAR- or TCR- immune cell therapy with low production cost can be provided. Similarly, even in vitro, if CAR and exogenous TCR can be selectively introduced into immune cells (such as T cells, etc.) without using viral vectors with high production costs, costs such as residual virus testing become unnecessary, and CAR- or TCR-immune cell therapy with low production costs can be provided.

[0007] However, because the number of cell divisions of immune cells (such as T cells) is small and they are floating cells, it is known that transfection efficiency is low compared with other cells. Therefore, it is necessary to have a more effective method for introducing nucleic acid etc. into immune cells. So far, there is no report on selectively introducing nucleic acid (for example, mRNA, DNA) encoding CAR or exogenous TCR into immune cells (such as T cells) by using the sonoporation effect. [Reference List] [Patent Document]

[0008] Patent Document 1: WO 2012 / 153635 Patent Document 2: WO 2015 / 182647 [Non-patent literature]

[0009] Non-patent document 1: YAKUGAKU ZASSHI 130(11)1489-1496(2010) Non-patent document 2: NATURE REVIEWS 12 161-174 (2016) Non-patent document 3: Drug Delivery 24(1)320-327(2017) Non-patent document 4: "Evaluation of pharmacokinetics in the brain byultrasound-responsive nano-bubbles in brain-directed DDS", Yuki Fuchigami et al., Nagasaki University, Abstracts of the 137th Annual Meeting of the Pharmaceutical Society of Japan (March 2017) [Summary of the invention] [Technical Issues]

[0010] An object of the present invention is to provide a novel method for safely and effectively introducing target substances such as nucleic acids and proteins into immune cells such as T cells. [Solution to the problem]

[0011] To achieve the above-mentioned objectives, the present inventors took note of the fact that the nanobubble water previously developed by the present inventors, which has excellent antibacterial effects (see Patent Document 2 mentioned above; since the International Organization for Standardization (ISO) defines bubbles smaller than 1 μm (1000 nm) as "ultrafine bubbles" (ISO 20480-1), they will be referred to as "ultrafine bubbles" rather than "nanobubbles" in this specification below) does not contain phospholipids. The average diameter of the ultrafine bubbles contained in this ultrafine bubble water is no more than 200 nm, which is smaller than conventional diameters. It is generally believed that ultrafine bubbles, when broken, cause more severe damage to cell membranes than microbubbles. However, the present inventors took the initiative to combine this ultrafine bubble water with ultrasonic irradiation and studied the introduction of nucleic acids and proteins into T cells. As a result, they unexpectedly demonstrated that ultrafine bubble water can significantly improve the efficiency of nucleic acid introduction through the sonoporation effect, and that the combination of ultrafine bubble water and ultrasound can significantly improve the introduction efficiency of proteins compared to treatment with either alone. Based on these findings, the inventors of the present invention have further studied and completed the present invention.

[0012] That is, the present invention relates to [1] A system for delivering a target substance to immune cells, the system comprising ultrafine bubble water or an ultrafine bubble aqueous solution containing ultrafine bubbles having an average diameter of not more than 200 nm and not containing phospholipids, and a combination of an ultrasonic generator; [2] The system according to [1], wherein the ultrafine bubble aqueous solution comprises one or more components selected from one or more surfactants, a hydrophilic resin, and a buffer, wherein the one or more surfactants are selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; [3] The system according to [1] or [2], wherein the ultrafine bubble aqueous solution is composed of a nonionic surfactant and / or a hydrophilic resin; [3-1] The system according to [1] or [2], wherein the ultrafine bubble aqueous solution is composed of an ionic surfactant and / or a hydrophilic resin; [4] The system according to any one of [1] to [3], wherein the ultrafine bubbles are composed of perfluorocarbons or air; [5] The system according to any one of [1] to [4], wherein the ultrafine bubbles have an average diameter of 50 nm to 200 nm; [6] The system according to any one of [1] to [5], wherein the ultrafine bubbles have a d90 / d10 ratio of not greater than 5; [7] The system according to any one of [1] to [6], wherein the ultrafine bubbles in the ultrafine bubble water or ultrafine bubble aqueous solution have a mass of not less than 1.0×10 8 The density of bubbles / mL; [8] The system according to any one of [1] to [7], wherein the ultrasonic output intensity in the ultrasonic generator is not greater than 720 mW / cm 2 ; [9] The system according to any one of [1] to [8], wherein in the ultrasonic generator, the ultrasonic output intensity is 50-500 mW / cm 2 And the ultrasound frequency is 0.5-10MHz;

[10] The system according to any one of [1] to [9], wherein the target substance is a nucleic acid or a protein;

[11] The system of

[10] , wherein the nucleic acid encodes a chimeric antigen receptor or an exogenous T cell receptor;

[12] The system according to any one of [1] to

[11] , wherein the immune cell is a T cell;

[13] The system according to

[11] or

[12] , for delivering nucleic acid to immune cells;

[14] A method for increasing the delivery of nucleic acids or proteins to immune cells by using ultrafine bubble water or an ultrafine bubble aqueous solution containing ultrafine bubbles having an average diameter of not more than 200 nm and not containing phospholipids and ultrasound;

[15] A preparation comprising a combination of a nucleic acid or protein and ultrafine bubble water or an ultrafine bubble aqueous solution, the ultrafine bubble water or ultrafine bubble aqueous solution comprising ultrafine bubbles having an average diameter of not more than 200 nm and not containing phospholipids, for delivering the nucleic acid or protein to immune cells, wherein an effective amount of the nucleic acid or protein is delivered to the immune cells by combined use with ultrasonic irradiation;

[16] A method for delivering a nucleic acid or protein to immune cells, the method comprising contacting the cells with the preparation according to

[15] and treating them with ultrasound; wait. [Beneficial Effects of the Invention]

[0013] According to the system for delivering target substances to immune cells of the present invention, by irradiating ultrasound with an output intensity that does not adversely affect the living body, target substances (such as nucleic acids, proteins, etc.) can be non-invasively and selectively introduced into immune cells (such as T cells, etc.). Therefore, a highly safe introduction system can be provided. In the system, since there is no need to use liposomes, there is no need to add special additives (such as phospholipids), low-cost production can be achieved, and the antigenicity problem caused by phospholipids does not occur. Generally, it is believed that when broken, ultrafine bubbles damage cell membranes more severely than microbubbles and are therefore not suitable for cell transfection. Unexpectedly, according to the present invention, the use of ultrafine bubbles can significantly improve the efficiency of introducing target substances into cells compared to the case of using microbubbles. Furthermore, in the ultrafine bubble water or the ultrafine bubble aqueous solution, ultrafine bubbles having an average diameter of not more than 200 nm are stable for a long period of time. [Brief Description of the Drawings]

[0014] Figure 1 This demonstrates that combining an aqueous solution of ultrafine bubbles with ultrasound improves the efficiency of plasmid introduction into T cells. The vertical axis shows the GFP fluorescence intensity per viable cell. US (ultrasound irradiation): - (absent), + (present); bubbles (ultrafine bubbles): Cat (positive charge), An (negative charge), - (absent). Figure 2 This demonstrates that combining an aqueous solution of ultrafine bubbles with ultrasound improves the efficiency of protein introduction into T cells. The vertical axis shows the GFP fluorescence intensity per viable cell. US (ultrasound irradiation): - (absent), + (present); bubbles (ultrafine bubbles): Cat (positive charge), An (negative charge), - (absent). I. System of the Present Invention The present invention provides a system for delivering a target substance to immune cells, comprising ultrafine bubble water or an ultrafine bubble aqueous solution containing ultrafine bubbles with an average diameter of no more than 200 nm and no phospholipids, and a combination of an ultrasonic generator (hereinafter also referred to as the "system of the present invention").

[0015] In this specification, "delivering a target substance to an immune cell" means that a substance that is difficult to pass through the cell membrane (e.g., a water-soluble compound that does not diffuse passively, a compound with a large molecular weight, or a compound that does not contain a selective transporter or receptor) passes through the cell membrane and is transferred into the cell. Therefore, the system of the present invention can deliver a target substance to a cell by any mechanism, and examples of such mechanisms include, but are not limited to, temporarily opening a pore in the cell membrane.

[0016] In this specification, "immune cell" means a cell that participates in an immune response, and examples include lymphocytes such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, etc., granulocytes such as neutrophils, eosinophils, basophils, etc., monocytes, macrophages, dendritic cells, etc., and unipotent or multipotent stem cells or progenitor cells that can eventually differentiate into them (excluding embryonic and other totipotent or multipotent stem cells). "Immune cell" can be a separate specific immune cell or a heterogeneous cell population including multiple types of immune cells (such as lymphocytes), as long as it is a cell population containing any of the cells mentioned above. The lymphocytes mentioned above can be produced by collecting, for example, peripheral blood, bone marrow, and umbilical cord blood from humans or non-human mammals, or by differentiating from stem cells (such as iPS cells, etc.) by methods known per se. When the isolated immune cells (e.g., T cells) into which the target substance has been introduced by applying the present invention are used to treat a disease (such as cancer, etc.), it is preferred to collect cells from the subject to be treated itself or from a donor whose MHC type matches that of the subject to be treated. Alternatively, the lymphocytes can be cells present in a single animal.

[0017] In a preferred embodiment, the "immune cells" are isolated and purified T cells. In another preferred embodiment, the immune cells may be a cell population containing T cells (eg, T cells present in a single animal, etc.). In this specification, "T cell" means a type of white blood cell found in lymphoid organs or peripheral blood, etc., and is characterized by differentiation and maturation mainly in the thymus and expression of TCR. Examples of T cells that can be used in various embodiments of the present invention include cytotoxic T cells (CTLs) which are CD8-positive cells, helper T cells which are CD4-positive cells, regulatory T cells, effector T cells, etc., and cytotoxic T cells are preferred.

[0018] The target substance that will be delivered to immune cell by the system of the present invention is not particularly limited, as long as it is owing to being delivered to immune cell and can give the preferred physiologically active material of cell.Its example includes but is not limited to polymeric compound [for example, nucleic acid, small RNA / DNA such as siRNA (for example, FAM-siRNA, manufactured by NIPPON GENECO., LTD.), ssRNA, shRNA, miRNA, S-oligoDNA (phosphorothioate) etc.), gene (for example, plasmid DNA, mRNA etc.) etc., protein (also including peptide) (for example, antibody (for example, IgG (for example, Alexa-IgG, manufactured by Invitrogen)) etc.), polysaccharide (for example, dextran, fluorescein isothiocyanate dextran etc.) etc.], low molecular weight compound (for example, fluorescein, sodium fluorescein etc.) etc. Wherein, polymeric compound and low molecular weight compound can be preferably mentioned. They are further preferably polymeric compound. They are further preferably nucleic acid and protein. They are particularly preferably nucleic acid and antibody.

[0019] In this specification, "ultrafine bubbles" may contain a gas having a pressure higher than normal atmospheric pressure, and the interior of the ultrafine bubbles may be a vacuum. As used herein, "vacuum" means a space filled with a gas having a pressure lower than normal atmospheric pressure. In the present invention, "ultrafine bubbles containing no phospholipids" refer to ultrafine bubbles whose outer shells do not form a phospholipid bilayer structure. In the present invention, "ultrafine bubble water" refers to water containing ultrafine bubbles. In the present invention, "ultrafine bubble aqueous solution" refers to an aqueous solution containing ultrafine bubbles. The aqueous solution constituting the ultrafine bubble aqueous solution contains, for example, one or more substances selected from the following: 1) one or more surfactants selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, 2) Hydrophilic resin and 3) Buffer As a component.

[0020] Examples of the "anionic surfactant" in the present invention include sodium lauryl sulfate and the like. Examples of the “nonionic surfactant” in the present invention include glycerol fatty acid esters (e.g., glycerol monostearate, etc.), sucrose fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan monostearate, sorbitan monolaurate, etc.), polyglycerol fatty acid esters, polyoxyethylene (hydrogenated) castor oil, polyoxyethylene sorbitan fatty acid esters (e.g., sorbitan polyoxyethylene laurate (e.g., polysorbate 20, etc.), polyoxyethylene sorbitan oleate (e.g., Examples of the present invention include polyethylene glycol fatty acid esters, polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyethylene glycol (macrogol), polyoxyethylene polyoxypropylene glycol (e.g., poloxamer 407, poloxamer 235, poloxamer 188, poloxamine, etc.). Among these, polyoxyethylene sorbitan laurate (e.g., polysorbate 20), polyoxyethylene sorbitan oleate (e.g., polysorbate 80), etc. are preferred. Polysorbate 20 or polysorbate 80 are further preferred, and polysorbate 80 is particularly preferred. Examples of the "cationic surfactant" in the present invention include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, dequalinium chloride and the like. Examples of the "amphoteric surfactant" in the present invention include cocamidopropyl betaine, cocamidopropyl hydroxysulfonate, and the like. The above-mentioned surfactants may be used alone, or two or more thereof may be used in combination.

[0021] Examples of the "hydrophilic resin" in the present invention include acrylic resins (e.g., polyacrylamide, polyacrylic acid, polymethyl methacrylate), vinyl resins (e.g., polyvinyl pyrrolidone, poly(vinyl alcohol) (PVA), polyvinyl ethyl ether), and polysaccharides (e.g., tragacanth gum, caraya gum, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hyaluronic acid, agarose, and curdlan). Among these, poly(vinyl alcohol) and hydroxypropyl cellulose are preferred. More preferred is poly(vinyl alcohol). The above-mentioned hydrophilic resins may be used alone, or two or more thereof may be used in combination.

[0022] Examples of the "buffer" in the present invention include acidic buffers (e.g., acetate buffer, citrate buffer, diluted Mclvaine buffer), neutral buffers (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, and phosphate-buffered saline (PBS)). As the buffer, diluted Mclvaine buffer is preferred.

[0023] The aqueous solution constituting the "ultrafine bubble aqueous solution" of the present invention is preferably an aqueous solution composed of: 1) one or more surfactants selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; and / or 2) a hydrophilic resin. Among these, for example, aqueous solutions composed of nonionic surfactants and / or hydrophilic resins are preferred. Furthermore, aqueous solutions composed of nonionic surfactants are also preferred. Further preferred are aqueous solutions composed of 1) one or two selected from polysorbate 80 and polysorbate 20 and / or 2) poly(vinyl alcohol). Further preferred are aqueous solutions composed of polysorbate 80 and / or poly(vinyl alcohol). Aqueous solutions composed of polysorbate 80 are particularly preferred. In another preferred embodiment, as the aqueous solution constituting the "ultrafine bubble aqueous solution", for example, an aqueous solution composed of an ionic (anionic or cationic) surfactant and / or a hydrophilic resin can be mentioned.

[0024] Examples of the "gas" constituting the ultrafine bubbles in the present invention include, but are not limited to, one or a mixture of two or more selected from perfluorocarbons (e.g., perfluoropropane (C3F8), perfluorobutane, etc.), air, nitrogen, ozone, oxygen, argon, carbon dioxide, and helium. Among them, perfluorocarbons (e.g., perfluoropropane, perfluorobutane, etc.), air, nitrogen, ozone, oxygen, and argon are preferred. Perfluorocarbons (e.g., perfluoropropane, perfluorobutane, etc.) and air are more preferred. When air is used, ultrafine bubbles can be easily produced at low cost. Air is further preferred.

[0025] The "ultrafine bubble aqueous solution" in the present invention is preferably an ultrafine bubble aqueous solution composed of: (A) an aqueous solution composed of a nonionic surfactant and / or a hydrophilic resin; and (B) ultrafine bubbles composed of one or more gases selected from perfluorocarbons, air, and the like. More preferably, it is an ultrafine bubble aqueous solution composed of: (A) an aqueous solution composed of 1) one or both of polysorbate 80 and polysorbate 20 and / or 2) poly(vinyl alcohol); and (B) ultrafine bubbles composed of one or more gases selected from perfluorocarbons and air. Even more preferably, it is an ultrafine bubble aqueous solution composed of: (A) an aqueous solution composed of polysorbate 80 and / or poly(vinyl alcohol); and (B) ultrafine bubbles composed of perfluorocarbons and / or air. Particularly preferred is an ultrafine bubble aqueous solution composed of: polysorbate 80 and ultrafine bubbles composed of air.

[0026] The "ultrafine bubbles" of the present invention do not contain amphiphilic phospholipids (such as liposomes), and thus can provide a safer preparation that does not exhibit antigenicity.

[0027] The "ultrafine bubbles" in the present invention have an average diameter of about not more than 200 nm, preferably 10 nm to 200 nm, more preferably 50 nm to 200 nm, and even more preferably 100 nm to 180 nm. The "average diameter" in this specification means the particle size (mode diameter) corresponding to the most frequent distribution value (maximum value of number %). In this specification, "ultrafine bubble water" or "ultrafine bubble aqueous solution" refers to water or an aqueous solution in which gas particles (ultrafine bubbles) having a diameter of 1000 nm or less are stably present. The ultrafine bubble water or ultrafine bubble aqueous solution of the present invention (hereinafter also referred to as "ultrafine bubble water, etc. of the present invention") typically contains ultrafine bubbles with an average diameter of approximately 200 nm or less.

[0028] In the present invention, ultrafine bubbles ideally have a uniform size. For example, when the ultrafine bubble diameters corresponding to the cumulative 10% and cumulative 90% from the smaller diameter side of the distribution based on the number of ultrafine bubbles are d10 and d90, respectively, the "d90 / d10 ratio" is preferably no greater than 5, and more preferably no greater than 4.5.

[0029] In the present invention, the number of ultrafine bubbles contained in ultrafine bubble water or the like refers to the number of ultrafine bubbles present in 1 mL of ultrafine bubble water or ultrafine bubble aqueous solution, and is sometimes referred to as "ultrafine bubble density" in this specification. The number of ultrafine bubbles contained in ultrafine bubble water or the like in the present invention is not particularly limited. The lower limit of "ultrafine bubble density" is, for example, not less than 1.0×10 8 bubbles / mL, preferably not less than 2.0×10 8bubbles / mL, more preferably not less than 2.5×10 8 The upper limit of “ultrafine bubble density” is, for example, not more than 2.0×10 9 bubbles / mL, preferably no more than 1.0×10 9 The "ultrafine bubble density" in the present invention is, for example, 1.0×10 8 -2.0×10 9 bubbles / mL, preferably 2.0×10 8 -1.0×10 9 bubbles / mL, more preferably 2.5×10 8 -1.0×10 9 bubbles / mL.

[0030] The ultrafine bubble diameter (including the ultrafine bubble average diameter, the same below), the distribution based on the number of ultrafine bubbles (including the d90 / d10 ratio, the same below), and the number of ultrafine bubbles can be measured by a method using laser beam scattering based on Brownian motion (e.g., NanoSight Ltd, LM20, LM10, etc.), a method based on resistance change (e.g., Beckman Coulter, Multisizer 4, etc.), a method based on laser diffraction scattering (e.g., Shimadzu Corporation, SALD-7100H, etc.), a method using Mie scattering (e.g., NIPPON DENSHOKU INDUSTRIES CO., LTD., NP-500T, etc.), etc. The ultrafine bubble diameter and the distribution based on the number of ultrafine bubbles used in the present invention are those measured by a tracking method (tracking method) of laser beam scattering using NanoSight (instrument name LM10) manufactured by NanoSight Ltd. or according to the method. The ultrafine bubble diameter, the distribution based on the number of ultrafine bubbles, and the number of ultrafine bubbles can generally be measured immediately after the ultrafine bubble water, etc. is produced, or after long-term storage. The ultrafine bubble diameter, the distribution based on the number of ultrafine bubbles, and the number of ultrafine bubbles of the ultrafine bubble water, etc. in the present invention are stably maintained over an extremely long period of time (e.g., about 6 months to 2 years), and therefore, the ultrafine bubble diameter, the distribution based on the number of ultrafine bubbles, and the number of ultrafine bubbles can be measured immediately before use after the ultrafine bubble water is sealed and stored for a certain period of time after production.

[0031] In this specification, the "water" containing ultrafine bubbles (and the "water" used as the "aqueous solution" containing ultrafine bubbles) is not particularly limited, and for example, tap water, deionized water, distilled water, sterile distilled water, purified water for injection, ultrapure water, etc. can be used. For injection use, sterile distilled water, purified water for injection, etc. are preferred.

[0032] The example of " aqueous solution " containing ultrafine bubbles in this specification includes the component containing one or more surfactants (the surfactant is selected from anionic surfactant, nonionic surfactant, cationic surfactant and amphoteric surfactant) selected from one or more mentioned above, hydrophilic resin and buffer and further contains water of any additive commonly used in the field of pharmaceutical preparations. The example of " additive " includes electrolyte, excipient, lubricant, adhesive, disintegrant, solubilizer, suspending agent, dispersant, isotonic agent, soothing agent (soothing agent), preservative, antioxidant, colorant, sweetener, pH regulator, stabilizer, acidulant, flavoring, fluidizing agent etc. Preferred pharmacologically acceptable additive. More preferably use one or more additives selected from suspending agent, stabilizer, dispersant, isotonic agent etc. Two or more of the above-mentioned additives may be used in mixture at an appropriate ratio. These additives (including one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, hydrophilic resins and buffers) can also be directly dissolved in water to prepare an ultrafine bubble aqueous solution, as long as they do not affect the generation, stability, etc. of ultrafine bubbles; or ultrafine bubbles are generated in water without additives to obtain ultrafine bubble water, and the additives are dissolved when used to obtain an ultrafine bubble aqueous solution. As the aqueous solution, any one of an uncharged ultrafine bubble aqueous solution, a positively charged ultrafine bubble aqueous solution, and a negatively charged ultrafine bubble aqueous solution can be used. They can be properly selected according to the type of target immune cells, their surrounding microenvironment, or the type of target substance. In the method of the present invention for delivering a target substance to immune cells by using an ultrafine bubble aqueous solution and ultrasound, in some cases, for example, when the target substance is a polymer compound (e.g., nucleic acid, protein, etc.), the ultrafine bubble aqueous solution is preferably a negatively charged ultrafine bubble aqueous solution. In another embodiment, in the method of the present invention for delivering a target substance to immune cells by using an ultrafine bubble aqueous solution and ultrasound, in some cases, when the target substance is a polymer compound, the ultrafine bubble aqueous solution is preferably a positively charged ultrafine bubble aqueous solution. The charge of the ultrafine bubble aqueous solution can be appropriately adjusted, for example, by adjusting the pH of the buffer solution used. For example, to positively charge the ultrafine bubble aqueous solution, the pH of the ultrafine bubble aqueous solution is preferably 1-4. On the other hand, to negatively charge the ultrafine bubble aqueous solution, the pH of the ultrafine bubble aqueous solution is preferably 7-14.

[0033] Methods for producing ultrafine bubble water are broadly categorized into the following: methods involving the simultaneous generation of microbubbles (gas particles with a diameter of approximately 1-60 μm) and ultrafine bubbles in water, followed by flotation separation of the microbubbles to leave only the ultrafine bubbles; and methods involving direct generation of ultrafine bubbles, with the former currently being the mainstream. The former method includes high-speed cyclonic flow, in which gas is fragmented by high-speed cyclonic flow to produce a large number of microbubbles, which are then separated by flotation to leave the ultrafine bubbles in the water; and pressurized dissolution, in which gas is pressurized to supersaturation and dissolved, the solution is rapidly depressurized to generate microbubbles and ultrafine bubbles, which are then separated by flotation to leave the ultrafine bubbles in the water. The methods for producing ultrafine bubble water solutions are the same as those mentioned above.

[0034] The pressure dissolution type is preferably used as a method for producing the ultrafine bubble water or ultrafine bubble aqueous solution in the present invention. For example, the following steps 1) to 3) can be mentioned: 1) In a pressurized container pressurized to approximately 0.2 to 0.5 MPa by a pressure pump, a gas is forcibly dissolved in a liquid containing (i) one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, (ii) a hydrophilic resin, and / or (iii) a buffer solution; 2) A flash evaporation operation is performed in water through a nozzle to release the depressurized and supersaturated gas into the wastewater as microbubbles or ultrafine bubbles, thereby producing a mixture of microbubble water and ultrafine bubble water; 3) Aeration is stopped, and the mixture is allowed to stand to allow the microbubbles to naturally separate by floating. Thus, clear ultrafine bubble water is produced, in which only ultrafine bubbles remain.

[0035] Examples of the ultrafine bubble generator for generating the ultrafine bubble water or ultrafine bubble aqueous solution in the present invention include a pressure dissolution type device (e.g., nanoGALF manufactured by IDEC) TM , OM4-MD5-045 manufactured by AURA TEC CO., LTD., a microbubble generator manufactured by Nikuni Corporation, etc.), a high-speed cyclone type device (for example, YJ manufactured by Bi-clean, a microbubble generator manufactured by AQUA AIR, MICROBLADE manufactured by ROYAL ELECTRIC CO., LTD., etc.), etc. As the ultrafine bubble generator, a pressurized dissolution type device (for example, nanoGALF manufactured by IDEC) is preferred. TM ).

[0036] In the present invention, one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, hydrophilic resins and / or buffer solutions are used to produce ultrafine bubble water or ultrafine bubble aqueous solution, thereby increasing the number of ultrafine bubbles in the ultrafine bubble water or ultrafine bubble aqueous solution mentioned above.

[0037] The content of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, hydrophilic resins and / or buffer solutions in water used in the present invention is not particularly limited. The upper limit is preferably not more than 50% (W / V), more preferably not more than 20% (W / V), and further preferably not more than 10% (W / V). The lower limit is preferably not less than 0.01% (W / V), more preferably not less than 0.05% (W / V), and further preferably not less than 0.1% (W / V). As used herein, (W / V) means g / mL. When two or more surfactants, hydrophilic resins, and buffer solutions are used in combination, their total amount is the content in water.

[0038] The ultra-fine bubble water of the present invention produced as mentioned above is tightly sealed in a vial or ampoule and stored. It is preferably stored in a shaded area. The storage temperature is preferably not higher than room temperature, and more preferably not higher than 10°C.

[0039] The ultrafine bubble water or ultrafine bubble aqueous solution of the present invention is preferably produced in the presence of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, a hydrophilic resin, and / or a buffer solution. Therefore, the number of ultrafine bubbles in the ultrafine bubble water can be maintained at not less than 1.0×10 8 The bubbles / mL last for a period of time required to maintain the effects of the ultrafine bubble water etc. in the present invention (for example, the effect of perforating the cell membranes of immune cells when ultrafine bubble water etc. is used in combination with ultrasonic treatment, the effect of increasing the delivery of target substances to immune cells by using ultrafine bubble water etc. and ultrasound, etc.) (for example, when used as a matrix for a target substance that needs to be delivered through a cell membrane etc., for its effective period (for example, not less than 3 months, more preferably not less than 6 months, and further preferably not less than 1 year)). The ultrafine bubble water of the present invention can be sterilized by heating, and even after heating sterilization, the number of ultrafine bubbles can be maintained at not less than 1.0×10 8 bubbles / mL.

[0040] When the number of ultrafine bubbles in the ultrafine bubble water of the present invention is not less than 1.0×10 8When the content of bubbles / mL is less than 100,000, the dosage form of the present invention is 0.1447kJ / mL, and the dosage form is 0.1 ...

[0041] The ultrafine bubble water, etc. of the present invention is preferably produced in the presence of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants (preferably polysorbate 80 and / or polysorbate 20, more preferably polysorbate 80), and the ultrafine bubbles in the produced ultrafine bubble water, etc. have a smaller average diameter and more uniform size (i.e., a smaller d90 / d10 ratio). Therefore, the system of the present invention achieves a safer effect of delivering target substances to immune cells.

[0042] As the "ultrasonic generator" used in the system of the present invention, any one can be used as long as it can generate ultrasonic waves that meet the conditions for delivering the target substance to immune cells when used in combination with the ultrafine bubble water of the present invention. For example, any device commonly used for ultrasonic diagnosis in clinical settings, commercially available ultrasonic gene transfer devices (e.g., Sonitron GTS (manufactured by Nepa Gene Co., Ltd.), etc.) can be appropriately used.

[0043] The conditions of the "ultrasonic generator" in the present invention include, for example, the ultrasonic output intensity (the amount of ultrasonic waves passing through a unit area (cm2) perpendicular to the direction of travel of the sound wave per unit time) and the ultrasonic output intensity (the amount of ultrasonic waves passing through a unit area (cm2) perpendicular to the direction of travel of the sound wave per unit time). 2 ) of the acoustic energy) is not less than 10 mW, preferably not less than 30 mW, more preferably not less than 50 mW. There is no particular upper limit on the output intensity. In particular, when the system of the present invention is intended to be transfected into immune cells of mammals (including humans) in vivo, it is preferred that the range that does not adversely affect the animals (e.g., cytotoxicity) be used as the upper limit. For example, the third-party certification standard of the revised Pharmaceutical Affairs Law (2005) added "not more than 720 mW / cm 2 " requirement (same as the upper limit of Track 3 of the U.S. FDA), and controls ultrasonic diagnostic apparatuses in Japan so as not to exceed the upper limit. Therefore, the upper limit of ultrasonic output intensity in the system of the present invention is preferably 720 mW / cm 2 The ultrasonic output intensity is preferably 50-720 mW / cm 2 , further preferably 50-500mW / cm 2 The ultrasound output intensity used for conventional gene transfer is significantly higher than the standards mentioned above for ultrasound diagnostic applications (e.g., 1.5-2.5 W / cm2 ), so there is a high safety risk. In the system of the present invention, regardless of the molecular weight of the target substance, it can be detected with a small output intensity (preferably 50-500mW / cm 2 ) effectively delivers target substances to immune cells, and it is a very safe delivery system.

[0044] As a condition of the "ultrasonic generator" in the present invention, the ultrasonic frequency is not particularly limited and can be appropriately selected, for example, within the range of 0.5-10 MHz. The frequency currently widely used is about 1 MHz. However, since higher frequencies are believed to have less adverse effects on the body, the frequency can be appropriately selected within the range of 1 to 5 MHz, more preferably 1 to 3 MHz.

[0045] As a condition for using the "ultrasonic generator" in the present invention, the ultrasound irradiation time is not particularly limited as long as it is sufficient to deliver the target substance to the immune cells, and varies according to the ultrasound output intensity. For example, even when the output intensity is 50 mW / cm 2 When the target substance is delivered to the immune cells, the irradiation time can be 10 seconds. The ultrasound irradiation time can be, for example, 1 to 60 seconds, preferably 1 to 30 seconds, and more preferably 1 to 20 seconds. As the conditions for using the "ultrasonic generator" in the present invention, for example, there can be mentioned (i) 50 to 720 mW / cm 2 (Preferably 50 to 500 mW / cm 2 ) an ultrasonic output intensity, (ii) an ultrasonic frequency of 0.5 to 10 MHz, and (iii) an ultrasonic irradiation time of 1 to 60 seconds. Among them, preferably (i) 50 to 500 mW / cm 2 The invention also provides a combination of (i) an ultrasonic output intensity of 1 to 5 MHz, (ii) an ultrasonic frequency of 1 to 5 MHz, and (iii) an ultrasonic irradiation time of 1 to 60 seconds.

[0046] When the system of the present invention is used for the transfection of immune cells isolated from animals or by inducing the differentiation of stem cells (such as iPS cells, etc.) known per se, it may include a step of culturing immune cells after the transfection process. Therefore, the system of the present invention may further include tools (e.g., culture vessels (e.g., dishes, flasks, etc.), culture media, culture devices (e.g., CO2 incubators, etc.)) for culturing immune cells. Since immune cells proliferate in a floating state, they are preferably coated with culture vessels of non-adhesive or low-adhesive matrices. The culturing step mentioned above also includes a subculture step. When cells are subcultured by the system of the present invention, the expression intensity of the target substance introduced into the cells can be maintained.

[0047] II. Enhanced Delivery Methods of the Invention The present invention also provides a method for increasing the delivery of nucleic acids or proteins to immune cells, the method comprising using ultrafine bubble water or an ultrafine bubble aqueous solution containing ultrafine bubbles having an average diameter of no more than 200 nm and no phospholipids and ultrasound (hereinafter also referred to as the "increasing delivery method of the present invention"). The method comprises administering the ultrafine bubble water or the like of the present invention to a subject to deliver it to the vicinity of immune cells, and irradiating the immune cells with ultrasound to deliver the nucleic acid or protein to the immune cells. As used herein, "immune cells" have the same meaning as defined for the system of the present invention mentioned above.

[0048] As the ultrafine bubble water or ultrafine bubble aqueous solution to be used in the "increased delivery method of the present invention", the ultrafine bubble water of the present invention mentioned above, etc. can be used. The ultrasonic irradiation in the "increased delivery method of the present invention" can be performed under conditions similar to those used in the ultrasonic generator in the system of the present invention mentioned above.

[0049] The nucleic acid or protein for increasing the delivery of immune cells by the increase delivery method of the present invention is not particularly limited, and can be, for example, due to being delivered to immune cells and being able to give immune cells the preferred physiologically active material.The example of nucleic acid includes but is not limited to small RNA / DNA such as siRNA (for example, FAM-siRNA, manufactured by NIPPON GENECO., LTD.), ssRNA, shRNA, miRNA, S-oligoDNA (phosphorothioate) etc.), gene (for example, plasmid DNA, mRNA etc.) etc. The example of protein includes antibody (for example, IgG (for example, Alexa-IgG, manufactured by Invitrogen Corp.) etc.), peptide etc. Wherein, preferably encoding the nucleic acid of chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) etc. is as nucleic acid, and preferably antibody etc. are as protein.

[0050] The number of nucleotides in nucleic acid and the molecular weight of protein are not particularly limited. For example, when nucleic acid is small RNA / DNA (such as siRNA, antisense oligonucleotides, etc.), it is 10mer-30mer, preferably 15mer-25mer, and when nucleic acid is a nucleic acid encoding CAR or TCR (plasmid DNA), it is 1-10kb, preferably 2-8kb. In the case of protein, it is 25kDa-900kDa, preferably 25kDa-320kDa.

[0051] In a preferred embodiment, the nucleic acid is a nucleic acid encoding a CAR or an exogenous TCR. Nucleic acids encoding CAR or exogenous TCR are described in detail below.

[0052] (a) Nucleic acid encoding CAR CAR is an artificially constructed hybrid protein that contains an antigen binding domain (e.g., scFv) connected to a T cell signaling domain of an antibody. The feature of CAR is that it can convert the specificity and reactivity of T cells to a selected target in a non-MHC restricted manner by using the antigen binding properties of its monoclonal antibody. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR does not favorably dimerize with endogenous TCR α and β chains.

[0053] The CAR used in the present invention includes an antibody that can specifically recognize surface antigens (e.g., cancer antigen peptides, surface receptors upregulated in cancer cells, etc.) recognized by target immune cells (e.g., T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, etc.); an extracellular hinge domain; a transmembrane domain and an intracellular T cell signaling domain.

[0054] Examples of surface antigens specifically recognized by the antigen binding domain include, but are not limited to, various cancers (e.g., acute lymphoblastic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colorectal cancer, esophageal cancer, cervical cancer, Fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, Surface receptors upregulated in mast cell tumors, melanoma, multiple myeloma, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer; peritoneal cancer, retinal cancer and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.), such as CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B cells Mutated antigens (BCMA), CD123, CD3, prostate-specific membrane antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2, PD-L1, CEA, chondroitin sulfate proteoglycan-4, IL-13 receptor α chain, IgGκ light chain, etc.; cancer antigen peptides (for example, peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.), etc.

[0055] The antigen-binding domains used in the present invention are not particularly limited, as long as they are antibody fragments that can specifically recognize target antigens. Considering ease of CAR production, it is desirable to use single-chain antibodies (scFv), wherein light chain variable region and heavy chain variable region are connected via a linker peptide. The arrangement of the light chain variable region and heavy chain variable region in single-chain antibodies is not particularly limited, as long as the two can reconstruct functional antigen-binding domains. They can be designed in the order of light chain variable region-linker peptide-heavy chain variable region (from the N-terminal side). As a linker peptide, a linker peptide known per se and commonly used for producing single-chain antibodies can be used. The DNA encoding light chain variable region and the DNA encoding heavy chain variable region can be prepared, for example, by cloning the light chain gene and heavy chain gene from antibody production cells, and using them as templates for PCR, or by chemically synthesizing them from the sequence information of existing antibodies. The DNA encoding single-chain antibodies can be obtained by connecting each obtained DNA fragment and the DNA encoding the linker peptide via an appropriate method. In order to present CAR on the surface of immune cells, it is preferred that the leader sequence is further added to the N-terminal side of the antigen-binding domains.

[0056] As the extracellular hinge domain and the transmembrane domain, domains derived from T cell surface molecules commonly used in the art can be appropriately used, and examples thereof include, but are not limited to, corresponding domains derived from CD8α and CD28.

[0057] Examples of intracellular signal transduction domains include, but are not limited to, those having a CD3 zeta chain, those further having a costimulatory transduction motif (such as CD28, CD134, CD137, Lck, DAP10, ICOS, 4-1BB, etc.) between the transmembrane domain and the CD3 zeta chain, those having two or more costimulatory transduction motifs, etc. Any domains commonly used in the art can be used in combination.

[0058] The information of nucleic acid sequences encoding the extracellular hinge domain, transmembrane domain and intracellular signal transduction domain is well known in the art, and a person of ordinary skill in the art can easily obtain DNA fragments encoding the corresponding domains from T cells based on the information. The DNA encoding CAR can be obtained by connecting the DNA fragments encoding the antigen binding domain, extracellular hinge domain, transmembrane domain and intracellular signal transduction domain respectively obtained in this way.

[0059] The DNA encoding CAR thus obtained can be inserted directly or after adding a suitable linker and / or nuclear transfer signal, etc. into an expression vector (preferably a plasmid vector), wherein the expression vector contains a promoter that works in T cells. As a promoter that works in T cells, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. that are constitutive in mammalian cells can be used, but are not limited to these. In addition, gene promoters specifically expressed in T cells, such as CD3, CD4, CD8, etc., can also be used.

[0060] CAR-encoding RNA (preferably mRNA) can be prepared by using an expression vector containing the above-mentioned CAR-encoding DNA as a template and transcribing it into mRNA by an in vitro transcription system known per se.

[0061] (b) Nucleic acid encoding exogenous TCR In this specification, "T cell receptor (TCR)" means a receptor that is composed of a dimer of TCR chains (α chain, β chain), recognizes antigens or antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complexes, and transmits stimulation signals to T cells. Each TCR chain is composed of a variable region and a constant region, and the variable region has three complementary determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only those in which the α chain and β chain of TCR form heterodimers, but also those in which they form homodimers. In addition, TCR includes those lacking part or all of the constant region, those with recombinant amino acid sequences, those with soluble TCR, etc. "Exogenous TCR" means that it is exogenous to the T cell that is the target cell in the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR expressed by the T cell that is the target cell in the present invention.

[0062] The nucleic acid encoding the TCR used in the present invention is a nucleic acid encoding the α chain and β chain of TCR that can specifically recognize a surface antigen (eg, cancer antigen peptide, etc.) to be recognized by target T cells. The nucleic acid can be prepared by methods known per se. When the amino acid sequence or nucleic acid sequence of the target TCR is known, for example, DNA or RNA chains can be chemically synthesized based on the sequence, or DNA encoding the full length or portion of the TCR of the present invention can be constructed by ligating partially overlapping short oligoDNA chains using PCR or Gibson assembly methods.

[0063] When the sequence of target TCR is unknown, for example, from the cell colony separation target T cell containing the T cell expressing target TCR, and the nucleic acid encoding TCR can be obtained from this T cell.Specifically, from living body (for example, people) collect the cell colony (for example, PBMC) containing T cell, and cultivate it under the stimulation in the presence of the epi-position of the cell surface antigen identified by target TCR, and can be by using the specificity of the cell expressing cell surface antigen and using cell surface antigen (such as CD8, CD4 etc.) as indicator by known method from this cell colony, select the T cell of the cell of specific identification expression cell surface antigen.T cell can use for example Dextramer to measure, ELISPOT measures, cytotoxicity assay etc. to measure the specificity of the cell expressing surface antigen.Preferably from the living body (for example, patient with disease (such as cancer etc.) or the dendritic cell containing T cell colony or with epitope pulse) of the cell of the cell surface antigen with many expressions identified by target TCR for example, with antigen epitope contact) collect the cell colony containing T cell mentioned above.

[0064] The nucleic acid of the present invention is obtained in the following manner: DNA is extracted from the T cells of the separation mentioned above by a conventional method, and by using DNA as a template, the nucleic acid sequence based on the TCR constant region is amplified and the TCR gene is cloned. In addition, it can also be prepared in the following manner: RNA is extracted from cells by a conventional method, cDNA is synthesized, and 5'-RACE (cDNA end rapid amplification) is performed by using cDNA as a template and using antisense primers complementary to the nucleic acid encoding TCR α chains and β chain constant regions. 5'-RACE can be performed by known methods, and can be performed, for example, by using a commercially available kit (such as SMART PCR cDNA synthesis kit (manufactured by Clontech)). The DNA of the α chains and β chains of the obtained coding TCR can be inserted into a suitable expression vector in the same manner as the DNA encoding CAR mentioned above. The DNA encoding α chains and the DNA encoding β chains can be inserted into the same vector or a separate vector. When they are inserted into the same vector, the expression vector can express two chains polycistron or monocistron. In the former case, an intervening sequence allowing polycistronic expression, such as IRES or FMV 2A, is inserted between the DNA encoding the two chains. In addition, RNA (preferably mRNA) encoding each chain of TCR can be prepared, for example, using an expression vector as a template and in the same manner as the above-mentioned CAR-encoding RNA.

[0065] III. Formulations of the Invention The present invention also provides a preparation comprising a combination of nucleic acid or protein and ultrafine bubble water or an ultrafine bubble aqueous solution, wherein the ultrafine bubble water or ultrafine bubble aqueous solution contains ultrafine bubbles having an average diameter of no greater than 200 nm and does not contain phospholipids, for delivering nucleic acid or protein to immune cells, wherein an effective amount (an amount in which the immune cells exert the desired effect) of the nucleic acid or protein is delivered to the immune cells by combining with ultrasound irradiation (hereinafter also referred to as the "preparation of the present invention"). As used herein, "immune cell" has the same meaning as defined for the system of the present invention mentioned above.

[0066] In the preparation of the present invention, the nucleic acid or protein may be any of the nucleic acids or proteins exemplified in the "Method of Increasing Delivery of the Present Invention" mentioned above. As the ultrafine bubble water or ultrafine bubble aqueous solution in the preparation of the present invention, the ultrafine bubble water or ultrafine bubble aqueous solution described in the "System of the Present Invention" mentioned above can be used.

[0067] In the formulation of the present invention, nucleic acids or proteins can be mixed with the ultrafine bubble water of the present invention and administered to the subject as a single preparation, or they can be formulated separately and administered at the same time or at different times via the same or different routes, as long as they can coexist adjacent to immune cells at the same time.

[0068] When the nucleic acid or protein is prepared separately from the ultrafine bubble water or the like of the present invention, the nucleic acid or protein can be mixed with a pharmaceutically acceptable carrier in an amount that can be tolerated by humans or other mammals. Examples of pharmaceutically acceptable carriers include pH regulators such as monosodium phosphate, dipotassium phosphate, disodium phosphate, monopotassium phosphate, sodium hydroxide, hydrochloric acid, etc.; antibiotics such as kanamycin sulfate, erythromycin lactobionate, penicillin G potassium, etc.; stabilizers such as lactose, potassium glutamate, D-sorbitol, glycine, human serum albumin, etc.; colorants such as phenol red, etc.; isotonic agents such as sodium chloride, potassium chloride, etc.; etc.

[0069] The content of nucleic acid or protein in the preparation of the present invention is not particularly limited, as long as the cells can be given preferred characteristics (such as desired physiological activity, etc.) when they are delivered to immune cells by ultrasonic irradiation. For example, when the preparation of the present invention contains a nucleic acid encoding CAR or exogenous TCR as a nucleic acid and is administered to a mammal (including a human), the amount of nucleic acid encoding CAR or exogenous TCR administered once is in the range of 0.001mg-10mg / 1kg body weight. For example, when administered to a human patient, it is administered to a patient weighing 60kg in the range of 0.001-50mg. The dosage mentioned above is an example, and the dosage can be appropriately selected according to the type of nucleic acid to be used, the route of administration, the age, weight, symptoms, etc. of the subject or patient to be administered. On the other hand, the amount of ultrafine bubble water, etc. is not particularly limited, as long as it is sufficient to deliver nucleic acid or protein to immune cells by ultrasonic irradiation. For example, a certain amount of ultrafine bubbles is allowed to be delivered so that the ultrafine bubble density near the immune cells is 1×10 8 -10×10 8 bubbles / mL, preferably 2×10 8 -5×10 8 bubbles / mL.

[0070] IV. Delivery Methods of the Invention The present invention also provides a method for delivering nucleic acid or protein to immune cells, the method comprising contacting the cells with the formulation of the present invention mentioned above, and treating the cells with ultrasound. As used herein, the meaning of "immune cell" is the same as that defined for the system of the present invention mentioned above.

[0071] The target to which the “increased delivery method of the present invention” or the “delivery method of the present invention” is applied is not particularly limited, as long as it is an immune cell collected from a living body (also referred to as “ex vivo immune cell” in this specification) or an animal tissue containing it or an immune cell in the body (also referred to as “in vivo immune cell” in this specification). For example, humans and other mammals (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.), immune cells derived from them or tissues containing them, etc. can be mentioned. There is no particular limitation on the means for administering the ultrafine bubble water, etc. in the present invention to the subject, as long as it is an administration route that can deliver ultrafine bubbles to the vicinity of the immune cells. Below, the case of delivering a nucleic acid encoding CAR or exogenous TCR as the nucleic acid is used as a specific example, and the in vitro immune cells and the in vivo immune cells are described separately.

[0072] (a) Delivery of nucleic acids encoding CAR or exogenous TCR to immune cells ex vivo According to the delivery method of the present invention, the in vitro immune cells expressing CAR or exogenous TCR can be produced in the following manner: by combining the ultrafine bubble water etc. in the present invention and ultrasound, the nucleic acid encoding CAR or exogenous TCR is delivered to the in vitro immune cells. Therefore, the present invention also provides the in vitro immune cells obtained by the method described. " Immune cell " herein is not particularly limited, as long as it is defined in the system of the present invention mentioned above in those with the ability to destroy target cells (pathogenic cells) (such as cancer cells etc.) by a certain mechanism of action (so-called immune effector cells). Its example includes the T cells responsible for cellular immunity in acquired immunity, the NK cells, monocytes, macrophages, dendritic cells etc. responsible for natural immunity, and NKT cells etc. as the T cells with NK cell characteristics. In a preferred embodiment, immune cells can be T cells. The T cells collected from living bodies are also referred to as "in vitro T cells" in this manual. On the other hand, in another preferred embodiment, immune cells can be cells responsible for natural immunity, such as NK cells, macrophages, dendritic cells etc. Even when T cells have the same MHC type, there is still a considerable risk of GVHD caused by allogeneic (allo) transplantation, but alloNK cells, etc. are not considered to cause GVHD. Therefore, when preparing allogeneic ex vivo immune cells of various MHC types, they can be used ready-made. For CAR-NK cells, for example, CAR-dendritic cells are described in US2016 / 0096892, Mol Ther.25(8):1769-1781(2017), etc., and CAR-macrophages, etc. are described in, for example, WO 2017 / 019848, eLIFE.2018e36688, etc.

[0073] The in vitro immune cells of the nucleic acid encoding CAR or exogenous TCR introduced by the preparation of the present invention can be the specific immune cells (for example, T cells, NK cells, monocytes, macrophages, dendritic cells, etc., NKT cells or their stem / ancestral cells) separated and purified or include various types of immune cells (such as lymphocytes) heterogeneous cell colonies, as long as it is containing the effector immune cells mentioned above or can eventually be differentiated into their single or multipotent stem cells or ancestral cells (excluding embryos and other all-powerful or multipotent stem cells) cell colonies. The example of the T cell separated and purified includes cytotoxic T cells (CTL) as CD8 positive cells, helper T cells as CD4 positive cells, regulatory T cells, effector T cells, and preferably cytotoxic T cells.

[0074] The lymphocytes mentioned above can be collected from, for example, peripheral blood, bone marrow, and umbilical cord blood of humans or non-human mammals, or produced from stem cells (such as iPS cells, etc.). When ex vivo immune cells (eg, ex vivo T cells) to which nucleic acids encoding CAR or exogenous TCR have been introduced by the preparation of the present invention are used to treat diseases (such as cancer, etc.), it is preferred to collect cell populations from the subject to be treated itself or a donor whose MHC type matches that of the subject to be treated.

[0075] Examples of unipotent or multipotent stem cells or their progenitor cells that can ultimately differentiate into immune cells include hematopoietic stem cells, myeloid-lymphoid progenitor (MLP) cells, myeloid progenitor (MP) cells, granulocyte-monocyte progenitor (GMP) cells, macrophage-dendritic cell progenitor (MDP) cells, dendritic cell precursor (DCP) cells, etc. These stem / progenitor cells can be differentiated into various immune cells, such as E cells, by methods known per se.

[0076] The method for contacting the preparation of the present invention with isolated immune cells is not particularly limited, and for example, the preparation of the present invention can be added to a universal culture medium for immune cells. Since immune cells proliferate in a floating state without adhering to a culture container, the pre-culture of isolated immune cells and contact with the preparation of the present invention are preferably carried out in a culture container coated with a non-adhesive or low-adhesive matrix. By using the ultrasonic generator and irradiation conditions described with respect to the system of the present invention, isolated immune cells can be subjected to ultrasonic irradiation.

[0077] When the preparation of the present invention particularly contains the nucleic acid encoding exogenous TCR as an active ingredient, from the perspective of increasing the expression of exogenous TCR, suppressing the occurrence of mismatched TCR or suppressing self-reactivity, siRNA can suppress the expression of endogenous TCR α chain and TCR β chain originally expressed by T cells. When the nucleic acid mentioned above is applied to the method, in order to avoid the influence of siRNA on exogenous TCR, the base sequence of the nucleic acid encoding TCR is preferably different from the base sequence corresponding to RNA, and the siRNA that suppresses the expression of endogenous TCR α chain and TCR β chain acts on the RNA (codon conversion sequence). These methods are described in, for example, WO 2008 / 153029. The base sequence mentioned above can be produced by introducing silent mutations into the nucleic acid encoding TCR obtained from nature, or by chemical synthesis of artificially designed nucleic acids. Alternatively, in order to avoid mismatching with endogenous TCR chains, some or all of the constant regions of the nucleic acid encoding exogenous TCR can be replaced with the constant regions of animals (e.g., mice) other than humans.

[0078] (b) Delivery of nucleic acids encoding CAR or exogenous TCR to immune cells in vivo According to the delivery method of the present invention, the preparation of the present invention is administered to a mammal (human or other mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, cattle, sheep, monkey), preferably human), and the tissue or organ containing immune cells (e.g., spleen, thymus, etc.) is exposed to ultrasound, thereby introducing the nucleic acid into the immune cells in the animal body, such as T cells (also referred to as "in vivo T cells" in this specification), thereby inducing the expression of CAR or exogenous TCR. In vivo immune cells specifically recognize cancer cells expressing surface antigens targeted by CAR or exogenous TCR, and kill diseased cells, thereby showing a preventive or therapeutic effect on the disease.

[0079] The preparation of the present invention in the form of, for example, an injection can deliver nucleic acids and ultrafine bubble water to the vicinity of immune cells in the body by subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, drip injection, intracerebral injection, intracerebrospinal fluid injection, etc. In addition, ultrasound irradiation can be performed by replacing the target area with a tissue or organ containing immune cells in the body through conventional ultrasound diagnosis procedures.

[0080] When the “increased delivery method of the present invention” or the “delivery method of the present invention” is applied to ex vivo immune cells (i.e., immune cells or stem cells (such as iPS cells, etc.) isolated from animals) using a method known per se, a step of culturing the immune cells after the transfection treatment can be included. The culturing step can be carried out in a culture medium commonly used for maintaining terminally differentiated immune cells or inducing more undifferentiated stem / progenitor cells to differentiate into terminally differentiated immune cells using culture containers known per se (e.g., dishes, flasks, etc.) and culture devices (e.g., CO2 incubators, etc.). Since immune cells proliferate in a floating state without adhering to the culture container, the culture of ex vivo immune cells is preferably carried out in a culture container coated with a non-adhesive or low-adhesive matrix. In addition, the culturing step mentioned above also includes a subculture step. When cells are subcultured by the “increased delivery method of the present invention” or the “delivery method of the present invention”, the expression intensity of the target substance introduced into the cells can be maintained.

[0081] V. Agents containing isolated immune cells into which target substances have been introduced by the present invention The in vitro immune cells that have been introduced into the target substance using the system of the present invention or by the delivery method of the present invention provide the desired effect (for example, obtain new physiological activity) through the action of the target substance. Therefore, it can be directly or by mixing with known pharmaceutically acceptable carriers (including excipients, diluents, fillers, adhesives, lubricants, glidants, disintegrants, surfactants, etc.), commonly used additives, etc. and formulated into pharmaceutical compositions. Excipients are well known to those skilled in the art, and adjuvants (such as wetting agents or emulsifiers, etc.) and pH buffers can also be used. In addition, formulation adjuvants (such as suspending agents, preservatives, stabilizers, dispersants, etc.) can also be used. The pharmaceutical composition mentioned above can be in dry form and will be reconstituted with a suitable sterile liquid before use. The pharmaceutical composition can be administered orally or parenterally, systemically or locally, depending on the form of the preparation (oral medicaments, such as tablets, pills, capsules, powders, granules, syrups, emulsions, suspensions, etc.; parenteral medicaments, such as injections, drip infusions, external preparations, suppositories, etc.) etc. In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, etc. are possible. In addition, when used in the form of an injection, an acceptable buffer, solubilizer, isotonic agent, etc. may also be added.

[0082] For example, when the target substance is a nucleic acid encoding CAR or exogenous TCR, the in vitro immune cells into which the target substance has been introduced can specifically recognize cells expressing surface antigens specifically recognized by CAR or exogenous TCR and kill them (e.g., inducing apoptosis). Therefore, when containing nucleic acid encoding CAR or exogenous TCR (which recognizes surface molecules specifically expressed in diseased cells (such as cancer cells, etc.) or whose expression is enhanced in the cells) as an active ingredient, nucleic acid has been introduced and in vitro immune cells expressing CAR or exogenous TCR can be used to prevent or treat diseases (such as cancer), and can be safely administered to humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys), preferably humans. In a preferred embodiment, the medicament containing the in vitro immune cells introduced into the target substance by the present invention can be a preventive drug or a therapeutic drug for cancer. The cancer used as the application target of the medicament is not particularly limited. Examples include, but are not limited to, acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colorectal cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphoblastic leukemia, The present invention relates to a group of cancers that are classified as follows: 1. Cancer of the nasopharyngeal, ovarian, and pancreatic; 2. Cancer of the retina, bladder, and esophagus; 3. Cancer of the larynx, pharynx, prostate, rectum, kidney, skin, small intestine, soft tissue, solid tumors; 4. Cancer of the ovary, ovary, and ureter; 5. Cancer of the urethra, bladder, and esophagus; 6. Cancer of the ovary, ovary, and esophagus; 7. Cancer of the urethra, bladder, and esophagus; 8. Cancer of the urethra, bladder, and esophagus; 9. Cancer of the urethra, bladder, and esophagus; 10. Cancer of the urethra, bladder, and esophagus; 11. Cancer of the urethra, bladder, and esophagus; 12. Cancer of the urethra, bladder, and esophagus; 13. Cancer of the urethra, bladder, and esophagus; 14. Cancer of the urethra, bladder, and esophagus; 15. Cancer of the urethra, bladder, and esophagus; 16. Cancer of the urethra, bladder, and esophagus; 17. Cancer of the urethra, bladder, and esophagus; 18. Cancer of the urethra, bladder, and esophagus; 19. Cancer of the urethra, bladder, and esophagus; 20. Cancer of the urethra, bladder, and esophagus; 21. Cancer of the urethra, bladder, and esophagus; 22. Cancer of the urethra

[0083] As the dosage of the medicament containing the isolated immune cells into which the target substance has been introduced by the present invention, for example, a nucleic acid encoding CAR or exogenous TCR is administered in a one-time amount in the range of 0.001 mg-10 mg / 1 kg body weight. For example, for administration to human patients, 0.001-50 mg is administered to a patient weighing 60 kg. The dosage mentioned above is an example, and the dosage can be appropriately selected according to the type of nucleic acid to be used, the route of administration, the age, weight, symptoms of the subject or patient to be administered, etc. The agent containing the isolated immune cells into which the target substance has been introduced by the present invention is preferably administered parenterally to the subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration methods. The dose is appropriately selected according to the subject's condition, weight, age, etc. Generally, it is administered so that the number of cells per administration to a subject weighing 60 kg is generally 1×10 6 -1×10 10 cells, preferably 1×10 7 -1×10 9 cells, more preferably 5×10 7 -5×10 8 Furthermore, it can be administered once or divided into multiple administrations.

[0084] The present invention is further described below by referring to Examples and Examples; however, the present invention is not limited thereto in any sense. Unless otherwise specified, in the following Reference Examples and Examples, "%" indicates weight / volume %. [Example]

[0085] Reference Example 1: Preparation of ultrafine bubble water Polysorbate 80 (2 g) (0.1% polysorbate 80) was dissolved in water for injection (2 L) or diluted Mclivaine buffer (pH 3.0, 2 L) and an ultrafine bubble generator (nanoGALF manufactured by IDEC) was used with the following settings: TM FZ1N-02) was used to prepare an aqueous solution of ultrafine bubbles. When an acidic buffer solution (diluted Mclivaine buffer solution: pH 3.0) was used, positively charged ultrafine bubbles were generated, while when water for injection was used, negatively charged ultrafine bubbles were generated. Gas used for preparation: Air Bubble water flow rate: about 4.0L / min Dissolution pressure: 300KPa±5%(sd) The prepared ultrafine bubble aqueous solution was subjected to high-pressure steam sterilization for 30 minutes at 121° C. to 124° C. using an autoclave as appropriate. After sterilization, the ultrafine bubble average diameter, ultrafine bubble density, and d90 / d10 ratio were measured by a tracking method utilizing laser beam scattering using LM10 (NanoSight Ltd.).

[0086] The results are shown below. Average diameter of ultrafine bubbles: 120nm±16nm Ultrafine bubble density: 4×10 8 bubbles / mL d90 / d10 ratio: 3.3

[0087] Example 1 Introduction of genes into T cells Jurkat E6.1 (3×10 4 Cells (100 cells, 0.5 mL) were seeded into 48 wells. After removing the culture medium, 0.5 mL of ultrafine bubble / RPMI culture medium containing 5 μg of pGFP was added. Ultrafine bubble / RPMI culture medium prepared as follows was used as the transfection medium: 1 L of the ultrafine bubble aqueous solution prepared in Reference Example 1 was added to RPMI culture medium powder, and 2 g / L NaHCO3 and 10% FBS were added.

[0088] After adding the culture medium containing pGFP, an ultrasonic generator (NEPAGENE) was used at a frequency of 1 MHz and 0.5 W / cm 2The output intensity of 500 μg / mL is carried out ultrasonic irradiation and continues 10s.By cell culture 2h, then remove transfection medium, and cell is cultivated 48h in RPMI culture medium (culture medium).After this, by cell collection, the cell lysis solution with 0.15mL is cracked completely, and the fluorescence intensity of GFP is measured with fluorescence spectrophotometer.Also the number of surviving cells is measured, it is converted into the fluorescence intensity of each surviving cell, and compare. The results are Figure 1 Shown in.

[0089] Example 2 Introduction of proteins into T cells Jurkat E6.1 (3×10 4 Cells, 0.5 mL) were seeded in 48 wells. After removing the culture medium, 0.5 mL of ultrafine bubble / RPMI culture medium containing 5 μg of IgG-FITC was added (4 mL of ultrafine bubble / RPMI culture medium was added to 40 μL of IgG-FITC). Ultrafine bubble / RPMI culture medium prepared as follows was used as a transfection medium: 1 L of the ultrafine bubble aqueous solution prepared in Reference Example 1 was added to RPMI culture medium powder, and 2 g / L NaHCO3 and 10% FBS were added.

[0090] After adding the culture medium containing IgG-FITC, an ultrasonic generator (NEPAGENE) was used at a frequency of 1 MHz and 0.5 W / cm 2 The output intensity of IgG-FITC was used to carry out ultrasonic irradiation for 10s. Cells were cultured for 2h, then the transfection medium was removed, and the cells were cultured for 48h in RPMI culture medium (culture medium). The fluorescence intensity of IgG-FITC was measured by fluorescence spectrophotometer. The number of surviving cells was also measured, converted into the fluorescence intensity of each surviving cell, and compared. The results are Figure 2 Shown in. [Industrial Applicability]

[0091] The system of the present invention can effectively deliver target substances (such as nucleic acids, proteins, etc.) to immune cells through low-output ultrasound irradiation, and thus can provide a highly safe system for delivering drugs to immune cells. Compared with the use of conventional microbubbles, the use of ultrafine bubbles can significantly improve the efficiency of introducing target substances into cells. In addition, since the system does not require the use of liposomes, it can be manufactured at low cost and is highly safe. In summary, the system of the present invention is particularly useful as a novel DDS that enters immune cells.

[0092] This application is based on patent application No. 2019-119164 filed in Japan (filing date: June 26, 2019), the contents of which are incorporated herein in their entirety.

Claims

1. A system for delivering nucleic acids or proteins to immune cells, the system comprising a combination of a negatively charged ultrafine bubble aqueous solution containing ultrafine bubbles having an average diameter of no greater than 200 nm and containing no phospholipids, and an ultrasonic generator, wherein the negatively charged ultrafine bubble aqueous solution comprises one or more surfactants selected from anionic surfactants and nonionic surfactants, and wherein the ultrasonic output intensity in the ultrasonic generator is no greater than 50-500 mW / cm 2 .

2. The system of claim 1, wherein the ultrafine bubbles are composed of perfluorocarbons or air.

3. The system of claim 1, wherein the ultrafine bubbles have an average diameter of 50 nm to 200 nm.

4. The system of claim 1, wherein the ultrafine bubbles have a d90 / d10 ratio of no greater than 5.

5. The system according to claim 1, wherein the ultrafine bubbles in the ultrafine bubble water or ultrafine bubble aqueous solution have a density of not less than 1.0×10 8 The density of bubbles / mL.

6. The system according to claim 1, wherein in the ultrasonic generator, the ultrasonic output intensity is 50-500 mW / cm 2 And the ultrasonic frequency is 0.5-10 MHz.

7. The system of claim 1, wherein the nucleic acid encodes a chimeric antigen receptor or an exogenous T cell receptor.

8. The system of claim 1, wherein the immune cells are T cells.

9. The system of claim 1, for delivering nucleic acids to immune cells.

10. A method for increasing the delivery of nucleic acids or proteins to immune cells, the method comprising using a negatively charged ultrafine bubble aqueous solution comprising ultrafine bubbles having an average diameter of not more than 200 nm and not containing phospholipids and ultrasound, wherein the negatively charged ultrafine bubble aqueous solution comprises one or more surfactants selected from anionic surfactants and nonionic surfactants, wherein the ultrasound output intensity in the ultrasound generator is not more than 50-500 mW / cm 2 .

11. A preparation comprising a combination of a nucleic acid or protein and a negatively charged ultrafine bubble aqueous solution, the ultrafine bubble aqueous solution comprising ultrafine bubbles having an average diameter of no greater than 200 nm and being free of phospholipids, for delivering the nucleic acid or protein to immune cells, wherein an effective amount of the nucleic acid or protein is delivered to the immune cells by combination with ultrasound irradiation, wherein the negatively charged ultrafine bubble aqueous solution comprises one or more surfactants selected from anionic surfactants and nonionic surfactants, and wherein the ultrasound output intensity in the ultrasound generator is no greater than 50-500 mW / cm 2 .

12. An in vitro method for delivering nucleic acids or proteins to immune cells, said method comprising contacting said cells with the preparation according to claim 11 and treating them with ultrasound.

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