Method for activation / proliferation of t cells

Abstract

To shorten and simplify a production process of an agent for immune cell therapy such as CAR-T therapy, to provide an agent for immune cell therapy in a short period of time with a low production cost, and to provide a safer production process of an agent for immune cell therapy that eliminates a potential risk of carcinogenicity due to a virus vector.SOLUTION: The present invention provides a method for activating / proliferating T cells, including a step of contacting a cell population containing T cells with a nucleic acid delivery carrier having at least one kind of T cell activating ligand added to its surface, and a method for delivering a nucleic acid into T cells, the methods including a step of contacting a cell population containing T cells simultaneously with (a) a nucleic acid delivery carrier having at least one kind of T cell activating ligand added to its surface and containing a nucleic acid inside, or (b) at least one kind of T cell activating ligand, and a nucleic acid delivery carrier containing a nucleic acid inside and free of a T cell activating ligand added to its surface, a method for producing a medicament containing T cells and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid delivery carrier having a T cell activation ligand attached to its surface, a method for activating and / or proliferating T cells using the nucleic acid delivery carrier, a method for delivering a nucleic acid into T cells, etc. The present invention also relates to a method for activating and / or proliferating T cells and delivering a nucleic acid into T cells, which method comprises simultaneously contacting a T cell activation ligand and a nucleic acid delivery carrier with T cells.

[0002] BACKGROUND OF THE INVENTION Research and development of cancer immunotherapy using CAR-T cells or TCR-T cells transfected with chimeric antigen receptors (CARs) or T cell receptors (TCRs) derived from cancer antigen-specific killer T cells is progressing rapidly. Current CAR-T cell therapies, such as Kymriah (trade name) and Yescarta (trade name), approved in the United States, typically involve ex vivo transfection of T cells collected from the patient with a CAR gene using a viral vector such as a lentiviral vector to produce CAR-T cells, which are then administered to the patient. However, this method requires a lengthy, multi-step process, including T cell activation / expansion, viral vector preparation, and gene transduction into T cells. This poses a challenge, as it increases manufacturing costs due to the costs associated with cell culture and viral vector preparation.

[0003] As methods for introducing CAR into T cells without using a viral vector, ex vivo or in vivo transfection of CAR into T cells has been reported, using nanoparticles in which CAR-encoding plasmid DNA is aggregated with a cationic polymer and the aggregates are coated with a non-cationic polymer conjugated with an anti-CD3 antibody fragment (Patent Document 1, Non-Patent Document 1), or a nanocarrier in which mesoporous silica encapsulating CAR-encoding DNA within the pores is coated with lipids whose surface has been modified with an anti-CD3 antibody (Patent Document 2). In addition to these, a technology has been reported in which a target siRNA is encapsulated in a "lipid nanoparticle (LNP)" that does not have a pore structure and is composed of a cationic lipid, a non-cationic helper lipid, and a ligand for delivery to the target cell, and the siRNA is then delivered to the target cell. For example, it has been reported that an anti-CD45 siRNA was transfected into T cells ex vivo or in vivo using an anti-CD4 antibody fragment as a targeting ligand (Patent Document 3, Non-Patent Document 2). Furthermore, Patent Document 4 describes cationic lipids for introducing active ingredients such as nucleic acids into various cells, including T cells, tissues, or organs.

[0004] On the other hand, methods for activating and / or proliferating T cells have been reported that use beads onto which anti-CD3 / CD28 antibodies are immobilized or nano-sized matrix beads (Patent Documents 5 and 6).

[0005] However, no technology has been reported to date that allows the process of activating / proliferating T cells and the process of gene transfer into T cells to be carried out simultaneously in one pod. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 2017 / 0296676 [Patent Document 2] US 2016 / 0145348 [Patent Document 3] WO 2016 / 189532 [Patent Document 4] WO 2016 / 021683 [Patent Document 5] US 6,352,694 [Patent Document 6] US 2014 / 0087462 [Non-patent literature]

[0007] [Non-Patent Document 1] Nature Nanotechnology 12, 813-820 (2017) [Non-patent document 2] ACS Nano, 2015, 9(7), 6706-6716 Summary of the Invention [Problem to be solved by the invention]

[0008] The objective of the present invention is to shorten and simplify the manufacturing process for immune cell therapy agents such as CAR-T therapy, to provide immune cell therapy agents with low manufacturing costs in a short period of time, and to provide a safer manufacturing process for immune cell therapy agents that eliminates the potential carcinogenic risk of viral vectors. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned object, the present inventors have succeeded in simultaneously performing the process of activating / proliferating T cells and the process of gene transfer into T cells in one pod by using a nucleic acid delivery carrier with a T cell activation ligand attached to its surface. Furthermore, the present inventors unexpectedly discovered that efficient activation / proliferation of T cells and nucleic acid transfer into T cells can be achieved simply by simultaneously contacting T cells with the T cell activation ligand and the nucleic acid delivery carrier, leading to the completion of the present invention.

[0010] That is, the present invention provides the following. [1] A method for activating and / or expanding T cells, comprising the step of contacting a cell population containing T cells with a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface. [2] The method described in [1], wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28. [3] The method according to [1] or [2], wherein two or more types of T cell activating ligands are attached to the surface of the nucleic acid delivery carrier. [4] The method according to any one of [1] to [3], wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome. [5] The method described in any of [1] to [4], wherein the nucleic acid delivery carrier contains a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor. [6] The method according to any one of [1] to [5], wherein the nucleic acid delivery carrier contains a nucleic acid encoding a CAR or a TCR. [7] The method according to any one of [1] to [6], which is carried out ex vivo. [8] A method for delivering a nucleic acid into a T cell, comprising the step of contacting a cell population containing a T cell with a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface and containing a nucleic acid therein. [9] The method described in [8], wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[10] The method described in [8] or [9], wherein two or more types of T cell activating ligands are attached to the surface of the nucleic acid delivery carrier.

[11] The method according to any one of [8] to

[10] , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

[12] The method according to any one of [8] to

[11] , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoter.

[13] The method according to any one of [8] to

[12] , wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

[14] The method according to any one of [8] to

[13] , which is carried out ex vivo.

[15] A method for delivering a nucleic acid into a T cell, comprising the step of simultaneously contacting a cell population containing a T cell with at least one T cell activation ligand and a nucleic acid delivery carrier containing a nucleic acid therein and having no T cell activation ligand attached to its surface.

[16] The method according to

[15] , wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[17] The method according to

[15] or

[16] , wherein two or more T cell activation ligands are contacted.

[18] The method according to any one of

[15] to

[17] , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

[19] The method according to any one of

[15] to

[18] , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoter.

[20] The method according to any one of

[15] to

[19] , wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

[21] The method according to any one of

[15] to

[20] , which is carried out ex vivo.

[22] A method for producing a pharmaceutical comprising T cells, comprising the step of contacting a cell population containing T cells with a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface and containing a nucleic acid therein.

[23] The method according to

[22] , wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[24] The method according to

[22] or

[23] , wherein two or more types of T cell activating ligands are attached to the surface of the nucleic acid delivery carrier.

[25] The method according to any one of

[22] to

[24] , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

[26] The method according to any one of

[22] to

[25] , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

[27] The method according to any one of

[22] to

[26] , wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

[28] The method according to any one of

[22] to

[27] , which is carried out ex vivo.

[29] A method for producing a pharmaceutical comprising T cells, comprising the step of simultaneously contacting a cell population containing T cells with at least one T cell activation ligand and a nucleic acid delivery carrier containing a nucleic acid therein and having no T cell activation ligand attached to its surface.

[30] The method described in

[29] , wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[31] The method according to

[29] or

[30] , wherein two or more T cell activation ligands are contacted.

[32] The method according to any one of

[29] to

[31] , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

[33] The method according to any one of

[29] to

[32] , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoter.

[34] The method according to any one of

[29] to

[33] , wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

[35] A method according to any one of

[29] to

[34] , which is carried out ex vivo.

[36] A nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface.

[37] The nucleic acid delivery carrier described in

[36] , wherein the T cell activation ligand comprises an antibody against CD3 and / or an antibody against CD28.

[38] A nucleic acid delivery carrier described in

[36] or

[37] , wherein two or more types of T cell activation ligands are attached to the surface of the nucleic acid delivery carrier.

[39] The nucleic acid delivery carrier according to any one of

[36] to

[38] , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

[40] A nucleic acid delivery carrier according to any one of

[36] to

[39] , which contains a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoter.

[41] A nucleic acid delivery carrier according to any one of

[36] to

[40] , which contains a nucleic acid encoding a CAR or a TCR.

[42] A pharmaceutical comprising the nucleic acid delivery carrier according to any one of

[36] to

[41] .

[43] T cells into which nucleic acid has been delivered by any of the methods described in

[15] to

[21] .

[44] A pharmaceutical comprising the T cells described in

[43] .

[45] A cell culture comprising a cell population containing T cells, at least one T cell activation ligand, a nucleic acid delivery carrier to which no T cell activation ligand is attached on its surface, and a medium.

[46] A composition for delivering a nucleic acid to a T cell, comprising at least one T cell activation ligand and a nucleic acid delivery carrier to which no T cell activation ligand is attached on the surface.

[47] A kit for nucleic acid delivery to T cells, comprising at least one T cell activation ligand and a nucleic acid delivery carrier that does not have a T cell activation ligand attached to its surface.

[48] ​​T cells into which nucleic acid has been delivered by any of the methods described in [8] to

[14] .

[49] A pharmaceutical comprising the T cells described in

[48] .

[50] A cell culture comprising a cell population containing T cells, a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface and containing a nucleic acid therein, and a medium.

[51] A composition for delivering a nucleic acid to a T cell, comprising a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface and containing a nucleic acid therein.

[52] A kit for nucleic acid delivery to T cells, comprising a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface and containing a nucleic acid therein. [Effects of the Invention]

[0011] According to the present invention, the process of activating / proliferating T cells and the process of gene transfer into T cells can be carried out simultaneously in one pod without using a viral vector, making it possible to provide an immune cell therapy agent with low manufacturing costs in a short period of time. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a comparison of the efficiency of gene transfer into T cells using lipid nanoparticles containing various cationic lipids (compounds 7, 11, 12, 21, 31, and 35) with anti-CD3 antibodies attached to the surface. [Figure 2]FIG. 1 shows a comparison of the efficiency of gene transfer into T cells using lipid nanoparticles having anti-CD3 antibody and / or anti-CD28 antibody attached to their surfaces. [Figure 3] Figure 1 shows that gene transfer into T cells (I) and T cell activation (II) were simultaneously achieved using lipid nanoparticles with anti-CD3 and anti-CD28 antibodies attached to their surfaces. In (I) and (II), the upper values ​​indicate the encapsulated mRNA concentration (μg / ml), and the lower values ​​indicate the antibody concentration (μg / ml). For comparison, (III) shows the T cell activation efficiency using conventional beads with anti-CD3 and anti-CD28 antibodies attached to their surfaces. [Figure 4] FIG. 1 shows that luc mRNA is efficiently transferred into human peripheral blood CD3-positive pan T cells by co-addition of an activation stimulus and lipid nanoparticles. [Figure 5] FIG. 1 shows the survival and proliferation rates of T cells transfected with luc mRNA by lipid nanoparticles. [Figure 6] This figure shows that co-addition of an activation stimulus and lipid nanoparticles efficiently introduces luc mRNA into human CD4 / CD8-positive T cells (left), and maintains high levels of T cell survival and proliferation (right).

[0013] (Detailed Description of the Invention) 1. Nucleic acid delivery carrier of the present invention The present invention provides a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface (hereinafter also referred to as "the nucleic acid delivery carrier of the present invention"). Here, the term "nucleic acid delivery carrier" refers to a carrier that can carry a nucleic acid and deliver the nucleic acid into a cell. "Capable of delivery into a cell" means that the nucleic acid carried can be delivered at least into the cytoplasm of the cell.

[0014] 1-1. Nucleic acid delivery carrier The nucleic acid delivery carrier used in the present invention is not particularly limited in structure, constituent molecules, or nucleic acid-carrying form, as long as it can carry nucleic acids and deliver the nucleic acids into cells, as described above. Representative nucleic acid drug delivery systems (DDS) include complexes formed based on electrostatic interactions between positively charged cationic liposomes, cationic polymers, etc., and nucleic acids. After binding to the negatively charged cell membrane, the complexes are taken up into cells by adsorptive endocytosis.

[0015] More specifically, examples of nucleic acid delivery carriers used in the present invention include, but are not limited to, lipid nanoparticles (LNPs), liposomes (e.g., cationic liposomes, PEG-modified liposomes, etc.), cationic polymers (e.g., polyethyleneimine, polylysine, polyornithine, chitosan, atelocollagen, protamine, etc.), and liposomes encapsulating cationic polymers. Alternatively, exosomes, which are biologically derived components, can also be used. Preferably, the carrier is a lipid nanoparticle or liposome, more preferably a lipid nanoparticle.

[0016] 1-1-1. Lipid nanoparticles (LNPs) As used herein, the term "lipid nanoparticle (LNP)" refers to a particle with an average diameter of less than 1 μm that does not have a large pore structure (e.g., liposome) or a small pore structure (e.g., mesoporous material) inside the outer shell of a lipid assembly containing cationic lipids and non-cationic lipids. The components of lipid nanoparticles are described below.

[0017] (a) Cationic lipids As used herein, the term "cationic lipid" refers to a lipid that has a net positive charge in a low pH environment such as physiological pH or in an endosome. The cationic lipid used in the lipid nanoparticles of the present invention is not particularly limited, and examples thereof include, but are not limited to, WO 2016 / 021683, WO 2015 / 011633, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US Examples of the cationic lipids include those described in WO2015 / 0239834, WO2019 / 131839, etc. Alternatively, Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr 15;111(15):5753) (e.g., K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, cKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K-E12, cWK-E12, PK500-O12, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12, cIK-E12, cKK-E10, cKK-E14, and cKK-E16, preferably cKK-E12 and cKK-E14), Love KT et al. (Proc Natl Acad Sci U S A. 2010 May 25;107(21):9915) (e.g., C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96 and C12-200, preferably C14-110, C16-96 and C12-200).

[0018] In a preferred embodiment, examples include cationic lipids represented by the following general formula described in WO 2016 / 021683:

[0019] [ka]

[0020] [In the formula, W is a compound of the formula -NR 1 R 2 or formula-N + R 3 R 4 R 5 (Z - )of, R 1 and R 2 are each independently 1-4 an alkyl group or a hydrogen atom, R 3 , R 4 and R 5 are each independently 1-4 The alkyl group, Z - represents the anion, X is an optionally substituted C 1-6 The alkylene group, Y A , Y B and Y C each independently represents an optionally substituted methine group, L A , L B and L C each independently represents an optionally substituted methylene group or a bond, R A1 , R A2 , R B1 , R B2 , R C1 and R C2 each independently represents an optionally substituted C 4-10 represents an alkyl group.] A compound represented by the formula (I) or a salt thereof.

[0021] More preferred examples include cationic lipids represented by the following structural formula:

[0022] [ka]

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] [ka]

[0031] [ka]

[0032] and their salts.

[0033] Of the above cationic lipids, cationic lipids represented by the following structural formula are more preferred.

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] and their salts.

[0038] In another preferred embodiment, examples include cationic lipids represented by the following general formula described in WO2019 / 131839.

[0039] [ka]

[0040] [In the formula, n is an integer from 2 to 5, R is a linear C 1-5 Alkyl group, linear C 7-11 Alkenyl group or linear C 11 The alkadienyl group, The wavy lines each independently represent cis or trans bonds. show.] A compound represented by the formula (I) or a salt thereof.

[0041] More preferred examples include cationic lipids represented by the following structural formula:

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] [ka]

[0050] [ka]

[0051] [ka]

[0052] [ka]

[0053] and their salts.

[0054] Of the above cationic lipids, cationic lipids represented by the following structural formula are more preferred.

[0055] [ka]

[0056] [ka]

[0057] [ka]

[0058] and their salts.

[0059] In another preferred embodiment, the cationic lipid is represented by the following general formula (III):

[0060] [ka]

[0061] [In the formula, n1 is an integer between 2 and 6, n2 is an integer between 0 and 2, n3 is an integer between 0 and 2, L is -C(O)O- or -NHC(O)O-; Ra is a linear C 5-13 Alkyl group, linear C 13-17 Alkenyl group or linear C 17 The alkadienyl group, Rb is a linear C 2-9 The alkyl group, Rc is a hydrogen atom or a linear C 2-9 The alkyl group, Rd is a hydrogen atom or a linear C 2-9 The alkyl group, Re is a linear C 2-9 The alkyl group, Rf is a linear C 2-9 represents an alkyl group.] A compound represented by the formula (I) or a salt thereof.

[0062] More preferred examples include cationic lipids represented by the following structural formula:

[0063] [ka]

[0064] [ka]

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] [ka]

[0076] [ka]

[0077] [ka]

[0078] [ka]

[0079] [ka]

[0080] [ka]

[0081] and their salts.

[0082] Of the above cationic lipids, cationic lipids represented by the following structural formula are more preferred.

[0083] [ka]

[0084] [ka]

[0085] and their salts.

[0086] Compound (III) can be produced, for example, by the following method. In particular, during esterification, compound (I) of the desired structure can be synthesized by using appropriate raw materials depending on the structure of the target compound (III). Furthermore, salts of compound (III) can be obtained by appropriately mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.

[0087] [ka]

[0088] [ka]

[0089] [ka]

[0090] [ka]

[0091] The raw materials and reagents used in each step of the above-mentioned production methods, as well as the resulting compounds, may each form a salt.

[0092] When the compound obtained in each step is a free compound, it can be converted into the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted into the free compound or another desired type of salt by a known method.

[0093] The compound obtained in each step can be used in the next reaction either as a reaction solution or as a crude product, or the compound obtained in each step can be isolated and / or purified from the reaction mixture by a separation means such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractional distillation, chromatography, etc., according to a conventional method.

[0094] When the raw materials and reagent compounds for each step are commercially available, the commercially available products can be used as they are.

[0095] In the reaction of each step, the reaction time may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 10 minutes to 8 hours.

[0096] In the reactions of each step, the reaction temperature may vary depending on the reagents and solvents used, but is usually from -78°C to 300°C, preferably from -78°C to 150°C, unless otherwise specified.

[0097] In the reaction of each step, the pressure may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 atm to 20 atm, preferably 1 atm to 3 atm.

[0098] In the reactions of each step, a microwave synthesis apparatus such as Biotage's Initiator may be used. The reaction temperature may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually room temperature to 300°C, preferably room temperature to 250°C, and more preferably 50°C to 250°C. The reaction time may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 1 minute to 8 hours.

[0099] In the reactions of each step, unless otherwise specified, the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, relative to the substrate. When a reagent is used as a catalyst, the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents, relative to the substrate. When a reagent also serves as a reaction solvent, the reagent is used in the amount of the solvent.

[0100] Unless otherwise specified, the reactions in each step are carried out without solvent or by dissolving or suspending the compound in a suitable solvent. Specific examples of the solvent include the following:

[0101] Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butyl alcohol, 2-methoxyethanol, etc.; Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, etc.; Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.; Saturated hydrocarbons: cyclohexane, hexane, heptane, etc.; Amides: N,N-dimethylformamide, N-methylpyrrolidone, etc.; Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.; Nitriles: acetonitrile, etc.; Sulfoxides: dimethyl sulfoxide, etc.; Aromatic organic bases: pyridine, etc.; Acid anhydrides: acetic anhydride, etc.; Organic acids: formic acid, acetic acid, trifluoroacetic acid, etc.; Inorganic acids: hydrochloric acid, sulfuric acid, etc.; Esters: ethyl acetate, acetic acid isopropyl ester, etc.; Ketones: acetone, methyl ethyl ketone, etc.; water. The above solvents may be used by mixing two or more kinds in an appropriate ratio.

[0102] When a base is used in the reaction of each step, for example, the bases shown below are used.

[0103] Inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.; Basic salts: sodium carbonate, calcium carbonate, sodium bicarbonate, etc.; Organic bases: triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, imidazole, piperidine, etc.; Metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, etc.; Alkali metal hydrides: sodium hydride, etc.; Metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, etc.; Organolithium compounds: n-butyllithium, sec-butyllithium, etc.

[0104] When an acid or an acid catalyst is used in the reaction of each step, for example, the following acids or acid catalysts are used.

[0105] Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.; Organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.; Lewis acids: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous iron chloride, etc.

[0106] Unless otherwise specified, the reactions in each step are carried out according to known methods, for example, those described in "Experimental Chemistry Lectures, 5th Edition, Vol. 13-19 (edited by the Chemical Society of Japan); "New Experimental Chemistry Lectures, Vol. 14-15 (edited by the Chemical Society of Japan); "Precise Organic Chemistry, Revised 2nd Edition" (L.F. Tietze, Th. Eicher, Nankodo); "Revised Organic Named Reactions: Their Mechanisms and Key Points" (by Hideo Togo, Kodansha); "ORGANIC SYNTHESES Collective Volumes I-VII" (John Wiley & Sons Inc.); "Modern Organic Synthesis in the Laboratory: A Collection of Standard Experimental Procedures" (by Jie Jack Li, Oxford University Press); "Comprehensive Heterocyclic Chemistry III, Vol. 1-14" (Elsevier Japan Co., Ltd.); "Strategies for Organic Synthesis Learned from Named Reactions" (translated and supervised by Kiyoshi Tomioka, published by Kagaku Dojin); and "Comprehensive Organic Transformations" (VCH Publishers). This is done in accordance with the method described in the 1989 publication of the Japanese Society of Cardiology (Scientific Name: JIS K 1989).

[0107] In each step, the protection or deprotection reaction of a functional group is carried out in accordance with a known method, for example, the method described in "Protective Groups in Organic Synthesis, 4th Ed." (Theodora W. Greene, Peter G.M. Wuts), published by Wiley-Interscience, 2007; "Protecting Groups, 3rd Ed." (P.J. Kocienski), published by Thieme, 2004; etc.

[0108] Examples of the protecting group for the hydroxyl group of an alcohol or the like or a phenolic hydroxyl group include ether-type protecting groups such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylic acid ester-type protecting groups such as acetate ester; sulfonic acid ester-type protecting groups such as methanesulfonate ester; and carbonate-type protecting groups such as t-butyl carbonate.

[0109] Examples of the protecting group for the carbonyl group of an aldehyde include acetal-type protecting groups such as dimethyl acetal; and cyclic acetal-type protecting groups such as cyclic 1,3-dioxane.

[0110] Examples of protecting groups for the carbonyl group of ketones include ketal-type protecting groups such as dimethyl ketal; cyclic ketal-type protecting groups such as cyclic 1,3-dioxane; oxime-type protecting groups such as O-methyloxime; and hydrazone-type protecting groups such as N,N-dimethylhydrazone.

[0111] Examples of the carboxyl-protecting group include ester-type protecting groups such as methyl ester; and amide-type protecting groups such as N,N-dimethylamide.

[0112] Examples of the thiol-protecting group include ether-type protecting groups such as benzylthioether; and ester-type protecting groups such as thioacetate, thiocarbonate, and thiocarbamate.

[0113] Examples of protecting groups for amino groups and aromatic heterocycles such as imidazole, pyrrole, and indole include carbamate-type protecting groups such as benzylcarbamate; amide-type protecting groups such as acetamide; alkylamine-type protecting groups such as N-triphenylmethylamine; and sulfonamide-type protecting groups such as methanesulfonamide.

[0114] The protecting group can be removed by known methods, such as a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, or a trialkylsilyl halide (e.g., trimethylsilyl iodide, trimethylsilyl bromide), or a reduction method.

[0115] In each step, when a reduction reaction is carried out, examples of the reducing agent used include metal hydrides such as lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), sodium borohydride, and tetramethylammonium triacetoxyborohydride; boranes such as borane tetrahydrofuran complex; Raney nickel; Raney cobalt; hydrogen; and formic acid. For example, Raney nickel or Raney cobalt can be used in the presence of hydrogen or formic acid. When reducing a carbon-carbon double bond or triple bond, a catalyst such as palladium-carbon or Lindlar's catalyst can be used.

[0116] In the oxidation reactions carried out in each step, examples of oxidizing agents that can be used include peracids such as m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, and t-butyl hydroperoxide; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; chlorites such as sodium chlorite; periodates such as sodium periodate; high-valent iodine reagents such as iodosylbenzene; manganese-containing reagents such as manganese dioxide and potassium permanganate; lead compounds such as lead tetraacetate; chromium-containing reagents such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), and Jones reagent; halogen compounds such as N-bromosuccinimide (NBS); oxygen; ozone; sulfur trioxide-pyridine complex; osmium tetroxide; zylene dioxide; and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).

[0117] When a radical cyclization reaction is carried out in each step, examples of the radical initiator used include azo compounds such as azobisisobutyronitrile (AIBN), water-soluble radical initiators such as 4-4'-azobis-4-cyanopentanoic acid (ACPA), triethylboron in the presence of air or oxygen, benzoyl peroxide, etc. Examples of the radical reaction reagent used include tributylstannane, tristrimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, samarium iodide, etc.

[0118] When a Wittig reaction is carried out in each step, examples of the Wittig reagent used include alkylidenephosphoranes, etc. The alkylidenephosphoranes can be prepared by a known method, for example, by reacting a phosphonium salt with a strong base.

[0119] When Horner-Emmons reaction is carried out in each step, examples of the reagent to be used include phosphonoacetates such as methyl dimethylphosphonoacetate and ethyl diethylphosphonoacetate; and bases such as alkali metal hydrides and organolithium compounds.

[0120] When a Friedel-Crafts reaction is carried out in each step, the reagents used include a Lewis acid and an acid chloride or an alkylating agent (e.g., alkyl halides, alcohols, olefins, etc.). Alternatively, an organic acid or an inorganic acid can be used instead of the Lewis acid, and an acid anhydride such as acetic anhydride can be used instead of the acid chloride.

[0121] In each step, when an aromatic nucleophilic substitution reaction is carried out, a nucleophile (eg, amines, imidazole, etc.) and a base (eg, basic salts, organic bases, etc.) are used as reagents.

[0122] In each step, when a nucleophilic addition reaction by a carbanion, a nucleophilic 1,4-addition reaction (Michael addition reaction) by a carbanion, or a nucleophilic substitution reaction by a carbanion is carried out, examples of the base used to generate the carbanion include organolithiums, metal alkoxides, inorganic bases, and organic bases.

[0123] When a Grignard reaction is carried out in each step, examples of the Grignard reagent include aryl magnesium halides such as phenyl magnesium bromide, and alkyl magnesium halides such as methyl magnesium bromide, isopropyl magnesium bromide, etc. The Grignard reagent can be prepared by a known method, for example, by reacting an alkyl halide or aryl halide with metallic magnesium using ether or tetrahydrofuran as a solvent.

[0124] In each step, when a Knoevenagel condensation reaction is carried out, an active methylene compound sandwiched between two electron-withdrawing groups (e.g., malonic acid, diethyl malonate, malononitrile etc.) and a base (e.g., organic bases, metal alkoxides, inorganic bases) are used as reagents.

[0125] In each step, when the Vilsmeier-Haack reaction is carried out, phosphoryl chloride and an amide derivative (eg, N,N-dimethylformamide, etc.) are used as reagents.

[0126] In each step, when an azidation reaction of alcohols, alkyl halides, or sulfonate esters is carried out, examples of the azidation agent used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide, sodium azide, etc. For example, when azidating alcohols, there are methods using diphenylphosphoryl azide and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) and methods using trimethylsilyl azide and a Lewis acid.

[0127] When a reductive amination reaction is carried out in each step, examples of the reducing agent used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, formic acid, etc. When the substrate is an amine compound, examples of the carbonyl compound used include paraformaldehyde, aldehydes such as acetaldehyde, and ketones such as cyclohexanone. When the substrate is a carbonyl compound, examples of the amine used include ammonia, primary amines such as methylamine, and secondary amines such as dimethylamine.

[0128] When Mitsunobu reaction is carried out in each step, azodicarboxylic acid esters (eg, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.) and triphenylphosphine are used as reagents.

[0129] In each step, when an esterification reaction, an amidation reaction, or a urea reaction is carried out, examples of the reagent to be used include acyl halides such as acid chlorides and acid bromides; and activated carboxylic acids such as acid anhydrides, activated esters, and sulfate esters. Examples of activating agents for carboxylic acids include carbodiimide-based condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD); triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM); carbonate-based condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenylphosphoric acid azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent); thionyl chloride; lower alkyl haloformates such as ethyl chloroformate; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); sulfuric acid; or combinations thereof. When a carbodiimide-based condensing agent is used, an additive such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), or dimethylaminopyridine (DMAP) may be further added to the reaction.

[0130] In each step, when a coupling reaction is carried out, examples of the metal catalyst to be used include palladium compounds such as palladium(II) acetate, tetrakis(triphenylphosphine)palladium(0), dichlorobis(triphenylphosphine)palladium(II), dichlorobis(triethylphosphine)palladium(II), tris(dibenzylideneacetone)dipalladium(0), 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) chloride, and palladium(II) acetate; nickel compounds such as tetrakis(triphenylphosphine)nickel(0); rhodium compounds such as tris(triphenylphosphine)rhodium(III) chloride; cobalt compounds; copper compounds such as copper oxide and copper(I) iodide; and platinum compounds. A base may also be added to the reaction, and examples of such bases include inorganic bases and basic salts.

[0131] When a thiocarbonylation reaction is carried out in each step, diphosphorus pentasulfide is typically used as the thiocarbonylating agent. However, in addition to diphosphorus pentasulfide, a reagent having a 1,3,2,4-dithiadiphosphetane-2,4-disulfide structure, such as 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lowesson's reagent), may also be used.

[0132] When performing the Wohl-Ziegler reaction in each step, examples of halogenating agents that can be used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, sulfuryl chloride, etc. Furthermore, the reaction can be accelerated by adding a radical initiator such as heat, light, benzoyl peroxide, or azobisisobutyronitrile to the reaction.

[0133] When a halogenation reaction of a hydroxy group is carried out in each step, the halogenating agent used may be a hydrohalic acid or an acid halide of an inorganic acid, specifically, for chlorination, hydrochloric acid, thionyl chloride, phosphorus oxychloride, etc., or for bromination, 48% hydrobromic acid, etc. Alternatively, a method may be used in which an alkyl halide is obtained from an alcohol by the reaction of triphenylphosphine with carbon tetrachloride or carbon tetrabromide, etc. Alternatively, a method may be used in which an alkyl halide is synthesized via a two-step reaction, in which the alcohol is converted to a sulfonic acid ester and then reacted with lithium bromide, lithium chloride, or sodium iodide.

[0134] When Arbuzov reaction is carried out in each step, examples of the reagent to be used include alkyl halides such as ethyl bromoacetate; and phosphites such as triethyl phosphite and tri(isopropyl) phosphite.

[0135] When a sulfone esterification reaction is carried out in each step, examples of the sulfonating agent to be used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, and trifluoromethanesulfonic anhydride.

[0136] In each step, when a hydrolysis reaction is performed, an acid or base is used as a reagent. When performing acid hydrolysis of t-butyl esters, formic acid or triethylsilane may be added to reductively trap the by-product t-butyl cation.

[0137] When a dehydration reaction is carried out in each step, examples of the dehydrating agent to be used include sulfuric acid, diphosphorus pentoxide, phosphorus oxychloride, N,N'-dicyclohexylcarbodiimide, alumina, polyphosphoric acid, and the like.

[0138] The salts of the compounds represented by the structural formulas above are preferably pharmacologically acceptable salts, and examples thereof include salts with inorganic bases (e.g., alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, ammonium salts), salts with organic bases (e.g., trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tritromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N, salts with N-dibenzylethylenediamine), salts with inorganic acids (e.g., salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid), salts with organic acids (salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), salts with basic amino acids (salts with arginine, lysine, ornithine), and salts with acidic amino acids (salts with aspartic acid, glutamic acid).

[0139] The ratio (mol %) of cationic lipids to the total lipids present in the lipid nanoparticles is, for example, but not limited to, about 10% to about 80%, preferably about 20% to about 70%, and more preferably about 40% to about 60%. The above cationic lipids may be used alone or in combination of two or more. When a plurality of cationic lipids are used, it is preferable that the cationic lipids as a whole have the above ratio.

[0140] (b) Non-cationic lipids As used herein, the term "non-cationic lipid" refers to a lipid other than a cationic lipid, which does not have a net positive charge at a selected pH, such as physiological pH. Examples of non-cationic lipids used in the lipid nanoparticles of the present invention include phospholipids, steroids, and PEG-lipids.

[0141] The phospholipid is not particularly limited as long as it stably retains nucleic acid and does not inhibit fusion with cell membranes (plasma membrane and organelle membrane) in order to enhance delivery of nucleic acid into T cells, and examples thereof include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinolenoylphosphatidylcholine.

[0142] Preferred phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), and more preferably DOPC, DPPC, POPC, and DOPE.

[0143] The ratio (mol %) of phospholipids to all lipids present in the lipid nanoparticles can be, for example, about 0% to about 90%, preferably about 5% to about 30%, and more preferably about 8% to about 15%. The above phospholipids may be used alone or in combination of two or more. When a plurality of phospholipids are used, it is preferable that the phospholipids as a whole have the above ratio.

[0144] Examples of steroids include cholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, with cholesterol being preferred.

[0145] If present, the ratio (mol %) of steroids to the total lipids present in the lipid nanoparticles can be, for example, about 10% to about 60%, preferably about 12% to about 58%, and more preferably about 20% to about 55%. The above steroids may be used alone or in combination of two or more. When a plurality of steroids are used, it is preferable that the steroids as a whole have the above ratio.

[0146] As used herein, "PEG lipid" refers to any conjugate of polyethylene glycol (PEG) and a lipid. The PEG lipid is not particularly limited as long as it has the effect of suppressing the aggregation of lipid nanoparticles, and examples thereof include PEG conjugated with dialkyloxypropyl (PEG-DAA), PEG conjugated with diacylglycerol (PEG-DAG) (e.g., SUNBRIGHT GM-020 (NOF Corp.)), PEG conjugated with phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-Cer), PEG conjugated with cholesterol (PEG-cholesterol), or derivatives thereof, or mixtures thereof, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), 1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6-dioxaotanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG), and the like. Preferred PEG lipids include PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer, and mixtures thereof, and more preferably PEG-DAA conjugates selected from the group consisting of PEG-didecyloxypropyl conjugates, PEG-dilauryloxypropyl conjugates, PEG-dimyristyloxypropyl conjugates, PEG-dipalmityloxypropyl conjugates, PEG-distearyloxypropyl conjugates, and mixtures thereof. In addition to a methoxy group, a maleimide group or an N-hydroxysuccinimidyl group can be used as the free end of PEG for binding to a T cell activating ligand. As a PEG lipid having a functional group for binding to a T cell activating ligand (sometimes referred to herein as a "terminally reactive PEG lipid"), for example, SUNBRIGHT DSPE-020MA (NOF Corp.) can be used.

[0147] The ratio (mol %) of PEG lipids to the total lipids present in the lipid nanoparticles of the present invention can be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, and more preferably about 0.7% to about 2%. The ratio (mol %) of the terminally reactive PEG lipid to the total PEG lipids can be, for example, about 10% to about 100%, preferably about 30% to about 100%, and more preferably about 40% to about 100%. The above PEG lipids may be used alone or in combination of two or more. When multiple PEG lipids are used, it is preferable that the PEG lipids as a whole have the above ratio.

[0148] 1-1-2. Liposomes Another preferred nucleic acid delivery carrier for use in the present invention is a liposome. Liposomes conventionally used in DDS for nucleic acid delivery to cells can be used in the same way. For example, liposomes prepared by mixing various cationic lipids (e.g., DOTMA, DOTAP, DDAB, DMRIE, etc.) developed as transfection reagents with membrane-fusogenic neutral lipids (e.g., DOPE, cholesterol, etc.) that promote release from endosomes are widely used. Liposomes with functional molecules such as PEG, pH-responsive membrane-fusogenic peptides, or membrane-permeation-promoting peptides attached to their surfaces can also be used.

[0149] 1-2.T cell activating ligands The nucleic acid delivery carrier of the present invention comprises the above-mentioned nucleic acid delivery carrier having a T cell activating ligand attached to its surface. The T cell activating ligand used in the present invention is not particularly limited as long as it interacts with a T cell surface molecule and promotes T cell activation and / or proliferation. Examples include molecules that specifically bind to CD3, which couples with TCR and mediates TCR-mediated signal transduction, and to surface molecules known as costimulators for T cell activation, such as CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, and CD40L, and transduce activation / proliferation signals or co-signals into T cells or antigen-presenting cells. Such molecules may be physiological ligands (or receptors) for the above T cell surface molecules, or non-physiological ligands (or receptors) with agonistic activity. A preferred example of a non-physiological ligand is an agonist antibody.

[0150] More preferably, the T cell activating ligand used in the present invention includes an antibody against CD3 and / or an antibody against CD28. The antibody against CD3 and the antibody against CD28 may be a complete antibody or a fragment thereof (e.g., Fab, F(ab')2, Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc.), as long as they specifically bind to CD3 and CD28 expressed on target T cells, respectively, and induce activation and / or proliferation (for example, when the target T cells are of human origin, the antibody against CD3 and the antibody against CD28 are preferably anti-human CD3 and anti-human CD28 antibodies, respectively), and have the ability to stimulate these T cell surface molecules and transduce a signal into the T cells. Furthermore, there are no particular limitations on the subclass of the antibody, but IgG antibodies are preferred.

[0151] When an agonist antibody such as an antibody against CD3 or CD28 is used as the T cell activating ligand, a commercially available anti-CD3 antibody, anti-CD28 antibody, etc. can be used as long as it is a complete antibody molecule, or it can be isolated from the culture medium of cells producing the antibody. On the other hand, when the ligand is a fragment of any of the above antibodies, nucleic acids encoding a fragment of an anti-CD3 antibody, anti-CD28 antibody, etc. can be isolated by treating the complete antibody with a reducing agent (e.g., 2-mercaptoethanol, dithiothreitol) or peptidase (e.g., papain, pepsin, ficin) or by a method similar to that used to obtain nucleic acids encapsulated in nucleic acid delivery carriers described below, and then used to recombinantly produce the antibody fragment.

[0152] Although a single T cell activating ligand may be used alone or in combination of two or more, a combination of two or more is preferred. When two or more T cell activating ligands are used in combination, at least one of them is preferably an antibody against CD3 or an antibody against CD28, more preferably an antibody against CD3. Particularly preferably, both an antibody against CD3 and an antibody against CD28 can be used as the T cell activating ligand.

[0153] When at least an antibody against CD3 and an antibody against CD28 are used in combination as T cell activating ligands, the molar ratio of the two antibodies attached to the surface of the nucleic acid delivery carrier of the present invention is 1:4 to 4:1, preferably 1:2 to 2:1.

[0154] When two or more T cell activating ligands are used in combination, the T cell activating ligands may be attached separately to the surface of the nucleic acid delivery carrier, or a complex of these may be attached to the surface of the nucleic acid delivery carrier, as long as the T cell activating activity of each is maintained. For example, when the two T cell activating ligands are antibodies (e.g., an antibody against CD3 and an antibody against CD28), they can be provided as a known bispecific antibody.

[0155] In the nucleic acid delivery carrier of the present invention, the T cell activating ligand may be bound to the outer shell in any manner as long as it is present on the surface of the nucleic acid delivery carrier. For example, when lipid nanoparticles containing terminally reactive PEG lipids as non-cationic lipids are used as nucleic acid delivery carriers, the T cell activating ligand can be attached to the end of the PEG. For example, lipid nanoparticles labeled with a ligand (antibody) can be prepared by reacting a PEG lipid (e.g., SUNBRIGHT DSPE-020MA) with a maleimide group at its end with the thiol group of the reduced antibody. When PEG-modified liposomes are used as nucleic acid delivery carriers, the T cell activating ligand can be attached to the liposome surface in a similar manner.

[0156] 1-3. Nucleic acids contained in the nucleic acid delivery carrier of the present invention Although the nucleic acid delivery carrier of the present invention can be used to induce activation and / or proliferation of T cells in a form that does not contain nucleic acid, in a preferred embodiment, by encapsulating a nucleic acid, activation and / or proliferation of T cells and delivery of the nucleic acid into T cells can be performed simultaneously in a single step. Thus, in a preferred embodiment, the nucleic acid delivery carrier of the present invention further contains a nucleic acid for delivery into T cells.

[0157] When the nucleic acid delivery carrier of the present invention contains a nucleic acid therein, the nucleic acid is not particularly limited as long as the nucleic acid itself or its transcription product or translation product has the function of changing the T cell to a desired state within the T cell.

[0158] 1-3-1. Nucleic acid that suppresses the expression of T cell activation inhibitory factor In a preferred embodiment, the nucleic acid delivery carrier of the present invention contains a nucleic acid that suppresses the expression of a T cell activation inhibitory factor. The target T cell activation inhibitory factor is not particularly limited as long as it suppresses T cell activation, and examples include immune checkpoint factors (e.g., CTLA-4, PD-1, TIM-3, LAG-3, TGIT, BTLA, VISTA (PD-1H), etc.), which are cell surface molecules that receive stimulation from antigen-presenting cells or tumor cells and transmit negative signals against the activation and / or proliferation of T cells, CD160, Cbl-b, endogenous TCR, etc.

[0159] Nucleic acids that suppress the expression of a T cell activation inhibitory factor may act at any stage, such as the transcription level of the gene encoding the factor, the level of post-transcriptional regulation, the level of translation into protein, or the level of post-translational modification. Therefore, examples of nucleic acids that suppress the expression of a T cell activation inhibitory factor include nucleic acids that inhibit the transcription of the gene encoding the factor (e.g., antigene), nucleic acids that inhibit the processing of initial transcription products into mRNA, and nucleic acids that inhibit the translation of mRNA into protein (e.g., antisense nucleic acids, miRNA) or degrade mRNA (e.g., siRNA, ribozymes, miRNA). Although substances that act at any stage can be used, substances that bind complementarily to mRNA to inhibit translation into protein or degrade mRNA are preferred. Examples of such nucleic acids include: (a) a nucleic acid or a precursor thereof having RNAi activity against mRNA encoding the factor; (b) an antisense nucleic acid to the mRNA encoding the factor, and (c) a ribozyme nucleic acid for the mRNA encoding the factor etc. The nucleotide sequences of mRNA (cDNA) encoding each T cell activation inhibitor are known, and sequence information can be obtained from public databases (eg, NCBI, EMBL, DDBJ, etc.).

[0160] (a) a nucleic acid or a precursor thereof having RNAi activity against mRNA encoding a T cell activation inhibitory factor Examples of nucleic acids that have RNAi activity against mRNA encoding a T cell activation inhibitor include double-stranded RNAs (siRNAs) consisting of an oligoRNA complementary to the target mRNA and its complementary strand. siRNAs can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). Furthermore, short hairpin RNAs (shRNAs), which are precursors of siRNAs, can be designed by appropriately selecting any linker sequence (e.g., approximately 5-25 bases) capable of forming a loop structure and linking the sense and antisense strands of the siRNA via the linker sequence.

[0161] siRNA and / or shRNA sequences can be searched for using search software provided free of charge on various websites, including, but not limited to, the siDESIGN Center provided by Dharmacon (http: / / dharmacon.horizondiscovery.com / jp / design-center / ?rdr=true&LangType=1041&pageid=17179928204) and the siRNA Target Finder provided by GenScript (https: / / www.genscript.com / tools / sirna-target-finder).

[0162] As used herein, microRNAs (miRNAs) targeting mRNAs encoding T cell activation inhibitors are also defined as nucleic acids with RNAi activity against the mRNA. miRNAs are first transcribed from the genes encoding them as primary transcripts (primary-microRNAs, or pri-miRNAs). These miRNAs are then processed by Drosha into precursor-microRNAs (pre-miRNAs) with a characteristic hairpin structure (approximately 70 bases in length). These precursors are then transported from the nucleus to the cytoplasm, where they are further processed by Dicer to become mature miRNAs, which are then incorporated into RISC and act on target mRNAs. Therefore, pre-miRNAs and pri-miRNAs, preferably pre-miRNAs, can be used as precursors of miRNAs.

[0163] miRNAs can be searched for using target prediction software provided free of charge on various websites. Examples of such websites include, but are not limited to, TargetScan (http: / / www.targetscan.org / vert_72 / ) from the Whitehead Institute in the United States and DIANA-micro-T-CDS (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=microT_CDS / index) from the Alexander Fleming Center for Biomedical Sciences in Greece. Alternatively, TarBase (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8 / index), a database of miRNAs experimentally proven to act on target mRNAs, provided by the University of Cesarea and the Institut Pasteur, can be used to search for miRNAs that target mRNAs encoding T cell activation inhibitors.

[0164] The nucleotide molecules constituting siRNA and / or shRNA, or miRNA and / or pre-miRNA may be natural RNA or DNA, but may contain various chemical modifications known per se to improve stability (chemical and / or enzymatic) or specific activity (affinity with RNA).

[0165] siRNA can be prepared by synthesizing the sense and antisense strands of a target sequence on mRNA using an automated DNA / RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then annealing them at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, siRNA can be prepared by synthesizing shRNA, which serves as a precursor to siRNA, and cleaving it using a dicer. miRNA and pre-miRNA can be synthesized using an automated DNA / RNA synthesizer based on their sequence information.

[0166] As used herein, nucleic acids designed to generate siRNA or miRNA against mRNA encoding a T cell activation inhibitor in vivo are also defined as nucleic acids having RNAi activity against the mRNA. Examples of such nucleic acids include expression vectors constructed to express the above-mentioned shRNA or siRNA, or miRNA or pre-miRNA. While Pol II promoters (e.g., CMV immediate early promoters) can also be used as promoters, Pol III promoters are generally used to ensure accurate transcription of short RNAs. Pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters. Furthermore, a sequence of four or more consecutive Ts is used as a transcription termination signal. Expression cassettes for miRNA and pre-miRNA can also be prepared in the same manner as for shRNA.

[0167] (b) an antisense nucleic acid against mRNA encoding a T cell activation inhibitor; Antisense nucleic acids against mRNA encoding a T cell activation inhibitory factor are nucleic acids containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a portion thereof, and function to inhibit protein synthesis by binding to the target mRNA and forming a specific and stable duplex. Antisense nucleic acids may be DNA or RNA, or may be DNA / RNA chimeras. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H, causing selective degradation of the target RNA. The target region of an antisense nucleic acid is not particularly limited in length, as long as hybridization with the antisense nucleic acid results in inhibition of protein translation. It may be the entire sequence or a partial sequence of the mRNA encoding the protein, as short as about 10 bases, or as long as the entire sequence of the mRNA or initial transcription product. Furthermore, antisense nucleic acids not only hybridize with target mRNA or initial transcription products to inhibit protein translation, but may also bind to these double-stranded DNA genes to form a triplex and inhibit transcription into RNA (antigene).

[0168] The nucleotide molecules constituting the antisense nucleic acid may also be modified in the same manner as in the above-mentioned siRNA, etc., in order to improve stability, specific activity, etc. Antisense oligonucleotides can be prepared by determining the target sequence of mRNA or the initial transcription product based on the cDNA sequence or genomic DNA sequence of the target gene, and synthesizing a sequence complementary to this using a commercially available automated DNA / RNA synthesizer.

[0169] As used herein, nucleic acids designed to generate antisense RNA against mRNA encoding a T cell activation inhibitory factor in vivo are also defined as being included in the antisense nucleic acid against the mRNA. Examples of such nucleic acids include expression vectors constructed to express the above-mentioned antisense RNA. Depending on the length of the antisense RNA to be transcribed, either a Pol II promoter or a Pol III promoter can be appropriately selected and used as the promoter.

[0170] (c) a ribozyme nucleic acid for mRNA encoding a T cell activation inhibitory factor; Ribozyme nucleic acids capable of specifically cleaving mRNA encoding a T cell activation inhibitory factor within the coding region can also be used as nucleic acids that inhibit the expression of the factor. In a narrow sense, the term "ribozyme" refers to RNA with enzymatic activity that cleaves nucleic acids. However, in this specification, the term is used to encompass DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and are known to be of the hammerhead and hairpin types. When a ribozyme is used in the form of an expression vector containing DNA encoding it, it can also be used as a hybrid ribozyme by further linking a modified tRNA sequence to promote the translocation of the transcription product to the cytoplasm.

[0171] (d) Nucleic acid that suppresses the expression of T cell activation inhibitors using genome editing In a preferred embodiment other than those described in (a) to (c) above, the nucleic acid that suppresses the expression of a T cell activation inhibitory factor can be a nucleic acid capable of inactivating (knockout) the gene encoding the factor. Examples of such nucleic acids include nucleic acids encoding artificial nucleases consisting of a nucleic acid sequence recognition module (e.g., CRISPR / Cas9, ZF motif, TAL effector, etc.) that can specifically recognize a partial nucleotide sequence in the gene as a target, and a nuclease that introduces a double-strand break (DSB) into the gene within or near the target sequence. After DSB introduction, the gene can be knocked out by insertion or deletion mutation due to a non-homologous end joining (NHEJ) repair error. Alternatively, gene knockout can be achieved by homologous recombination (HR) repair by combining the gene with a targeting vector containing a marker gene (e.g., a reporter gene such as a fluorescent protein gene, or a selectable marker gene such as a drug resistance gene) inserted into the gene sequence. Alternatively, an endogenous TCR gene can be knocked in using an exogenous TCR gene via HR repair.

[0172] 1-3-2. Nucleic acid encoding a T cell activation promoter In another preferred embodiment, the nucleic acid delivery vehicle of the present invention contains a nucleic acid encoding a T cell activation-promoting factor. Examples of the target T cell activation-promoting factor include T cell surface molecules (e.g., CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, CD40L, etc.) to which the above-mentioned T cell activation ligands bind and transmit activation and / or proliferation signals into T cells. The nucleotide sequences of mRNA (cDNA) encoding each T cell activation-promoting factor are known, and sequence information can be obtained from public databases (e.g., NCBI, EMBL, DDBJ, etc.).

[0173] The nucleic acid encoding the T cell activation-promoting factor can be encapsulated in the nucleic acid delivery carrier of the present invention in the form of mRNA or an expression vector containing DNA encoding the factor. The mRNA encoding the T cell activation-promoting factor can be isolated by a method known per se, using RNA extracted from T cells as a template and probes or primers prepared based on the sequence information. The resulting mRNA can be encapsulated directly in the nucleic acid delivery carrier of the present invention, or can be converted to cDNA by RT-PCR and amplified.

[0174] The resulting DNA encoding the T cell activation-promoting factor can be inserted directly or after the addition of an appropriate linker and / or nuclear localization signal into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells. Examples of promoters functional in T cells include, but are not limited to, the constitutive SRα promoter in mammalian cells, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney murine leukemia virus) LTR, and the HSV-TK (herpes simplex virus thymidine kinase) promoter. Gene promoters that are specifically expressed in T cells, such as CD3, CD4, and CD8, can also be used.

[0175] mRNA encoding a T cell activation-promoting factor can be prepared by using an expression vector containing DNA encoding the factor as a template and transcribing it into mRNA in a known in vitro transcription system.

[0176] 1-3-3. Nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR) As described above, the nucleic acid delivery carrier of the present invention encapsulates a nucleic acid, thereby enabling activation and / or proliferation of T cells and delivery of the nucleic acid into T cells simultaneously in a single step. Therefore, by encapsulating a nucleic acid encoding a CAR or TCR in the nucleic acid delivery carrier of the present invention, the step of activating / proliferating T cells and the step of introducing a gene into T cells can be performed simultaneously in one pod.

[0177] That is, in a preferred embodiment, the nucleic acid delivery carrier of the present invention contains a nucleic acid encoding a CAR or TCR.

[0178] (a) a nucleic acid encoding a CAR CARs are artificially constructed hybrid proteins containing an antibody antigen-binding domain (e.g., scFv) linked to a T cell signaling domain. Characteristics of CARs include their ability to utilize the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous TCR α and β chains.

[0179] The CAR encapsulated in the nucleic acid delivery carrier of the present invention comprises an antigen-binding domain of an antibody that can specifically recognize a surface antigen to be recognized by target T cells (e.g., a cancer antigen peptide, a surface receptor whose expression is increased in cancer cells, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0180] Examples of surface antigens specifically recognized by the antigen-binding domain include various cancers (e.g., acute lymphocytic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulva cancer, chronic myeloid cancer, colon 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), and surface receptors whose expression is upregulated in tumors such as: myeloid leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.), e.g., CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell Examples of antigens include, but are not limited to, mutation antigens, 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, and cancer antigen peptides (e.g., peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.).

[0181] The antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment capable of specifically recognizing a target antigen. However, considering the ease of CAR production, a single-chain antibody (scFv) in which a light chain variable region and a heavy chain variable region are linked via a linker peptide is desirable. The arrangement of the light chain variable region and heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstitute a functional antigen-binding domain. Typically, the arrangement is designed in the following order from the N-terminus: light chain variable region-linker peptide-heavy chain variable region. As the linker peptide, a publicly known linker peptide commonly used in the production of single-chain antibodies can be used. DNA encoding the light chain variable region and DNA encoding the heavy chain variable region can be prepared, for example, by cloning the light chain gene and heavy chain gene, respectively, from antibody-producing cells and performing PCR using them as templates, or by chemical synthesis using the sequence information of an existing antibody. DNA encoding a single-chain antibody can be obtained by ligating the resulting DNA fragments with DNA encoding the linker peptide using an appropriate method. It is preferable that a leader sequence be further added to the N-terminus of the antigen-binding domain in order to present the CAR on the surface of T cells.

[0182] As the extracellular hinge domain and transmembrane domain, domains derived from T cell surface molecules commonly used in the art can be used as appropriate, including, but not limited to, domains derived from CD8α and CD28.

[0183] Examples of intracellular signaling domains include those having a CD3ζ chain, those having an additional costimulatory motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, or 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more costimulatory motifs, but are not limited to these, and any combination of domains commonly used in the art can be used.

[0184] Nucleic acid sequence information encoding the extracellular hinge domain, transmembrane domain, and intracellular signaling domain is well known in the art, and a person skilled in the art can easily obtain DNA fragments encoding each domain from T cells based on this information. The DNA fragments thus obtained, each encoding the antigen-binding domain, extracellular hinge domain, transmembrane domain, and intracellular signaling domain, can be ligated using standard methods to obtain DNA encoding a CAR.

[0185] The resulting DNA encoding the CAR can be inserted directly or after adding an appropriate linker and / or nuclear localization signal, etc., into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells. Examples of promoters functional in T cells include, but are not limited to, the constitutive SRα promoter in mammalian cells, the SV40 promoter, the LTR promoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney murine leukemia virus) LTR, and the HSV-TK (herpes simplex virus thymidine kinase) promoter. Gene promoters such as CD3, CD4, and CD8, which are expressed specifically in T cells, can also be used.

[0186] RNA encoding a CAR, preferably mRNA, can be prepared by using an expression vector containing DNA encoding the CAR as a template and transcribing it into mRNA in a known in vitro transcription system.

[0187] (b) a nucleic acid encoding an exogenous TCR As used herein, the term "T cell receptor (TCR)" refers to a receptor composed of a dimer of TCR chains (α chain, β chain), which recognizes an antigen or the antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complex and transmits a stimulatory signal to a T cell. Each TCR chain is composed of a variable region and a constant region, and the variable region contains three complementarity-determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only TCRs in which the α chain and β chain of the TCR form a heterodimer, but also TCRs in which the chain forms a homodimer. Furthermore, the TCR also includes TCRs in which the constant region is partially or completely deleted, TCRs with recombinant amino acid sequences, soluble TCRs, and the like. The term "exogenous TCR" means that the exogenous TCR is exogenous to the T cells that are the target cells of the nucleic acid delivery carrier of 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 cells that are the target cells of the nucleic acid delivery carrier of the present invention.

[0188] The nucleic acid encoding the TCR encapsulated in the nucleic acid delivery carrier of the present invention is a nucleic acid encoding the α chain and β chain of a TCR that can specifically recognize the surface antigen (e.g., a cancer antigen peptide, etc.) that the target T cell should recognize. The nucleic acid can be prepared by a method known per se. When the amino acid sequence or nucleic acid sequence of the target TCR is known, a DNA encoding the full length or a part of the TCR of the present invention can be constructed based on the sequence, for example, by chemically synthesizing a DNA strand or an RNA strand, or by connecting synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson Assembly.

[0189] When the sequence of a TCR of interest is not known, for example, the T cell of interest can be isolated from a cell population containing T cells expressing the TCR of interest, and a nucleic acid encoding the TCR can be obtained from the T cell. Specifically, a cell population containing T cells (e.g., PBMCs) is collected from a living organism (e.g., a human), and the cell population is cultured while being stimulated in the presence of an epitope of a cell surface antigen recognized by the TCR of interest. From this cell population, T cells that specifically recognize cells expressing the cell surface antigen can be selected by known methods using the specificity for cells expressing the cell surface antigen and cell surface antigens such as CD8 and CD4 as indicators. The specificity of T cells for cells expressing the surface antigen can be measured using, for example, a dextromer assay, an ELISPOT assay, or a cytotoxicity assay. The cell population containing the T cells is preferably collected, for example, from a living organism having a large number of cells expressing a cell surface antigen recognized by the target TCR (e.g., a patient with a disease such as cancer, or a T cell-containing population that has been contacted with an epitope of the antigen or dendritic cells pulsed with the epitope).

[0190] The nucleic acid of the present invention can be obtained by extracting DNA from the isolated T cells using a conventional method, and using the DNA as a template to amplify and clone the TCR gene based on the nucleic acid sequence of the TCR constant region. Alternatively, RNA can be extracted from the cells using a conventional method to synthesize cDNA, and then using the cDNA as a template to perform 5'-RACE (Rapid Amplification of cDNA Ends) using antisense primers complementary to the nucleic acids encoding the TCR α-chain and β-chain constant regions, respectively. 5'-RACE can be performed by known methods, for example, using a commercially available kit such as the SMART PCR cDNA Synthesis Kit (Clontech). The resulting DNAs encoding the TCR α-chain and β-chain can be inserted into an appropriate expression vector, similar to the DNA encoding the CAR. The DNA encoding the α-chain and the DNA encoding the β-chain can be inserted into the same vector or separate vectors. When inserted into the same vector, the expression vector may express both chains polycistronically or monocistronically. In the former case, an intervening sequence that allows polycistronic expression, such as an IRES or FMV 2A, is inserted between the DNA encoding both strands. Furthermore, RNA, preferably mRNA, encoding each chain of the TCR can be prepared in the same manner as the RNA encoding the CAR, for example, using the expression vector as a template.

[0191] 1-4.T cell targeting ligand The nucleic acid delivery carrier of the present invention is preferably used to activate and / or proliferate T cells ex vivo, but also includes use in the form of in vivo administration to a subject. In this case, the targeting efficiency of the nucleic acid delivery carrier to T cells can be increased by further attaching to the surface of the nucleic acid delivery carrier of the present invention a ligand that can target the nucleic acid delivery carrier to T cells (hereinafter also referred to as a "T cell targeting ligand").

[0192] The T cell targeting ligand is not particularly limited as long as it can specifically recognize a surface molecule that is specifically or highly expressed on T cells. Preferably, it contains the antigen-binding domain of an antibody against CD3, CD4, or CD8, with an anti-CD3 antibody being a more preferred example. Here, the term "antigen-binding domain" is synonymous with the antigen-binding domain constituting the CAR. However, since CARs are prepared as nucleic acids encoding them, there are limitations. Single-chain antibodies are typically used. However, since the antigen-binding domain serving as the T cell targeting ligand is contained in the lipid nanoparticles of the present invention in the form of a protein, not only single-chain antibodies but also any other antibody fragments, such as whole antibody molecules, Fab, F(ab'), Fab', Fv, reduced antibody (rIgG), dsFv, sFv, diabodies, and triabodies, can be preferably used. These antibody fragments can be prepared by treating whole antibodies (e.g., IgG) with reducing agents (e.g., 2-mercaptoethanol, dithiothreitol) or peptidases (e.g., papain, pepsin, ficin), or by genetic recombination techniques.

[0193] If the T cell-targeting ligand is a complete antibody molecule, commercially available anti-CD3, CD4, CD8 antibodies, etc. can be used, or the ligand can be isolated from the culture medium of cells producing the antibody. On the other hand, if the ligand is any of the above-mentioned antigen-binding domains (antibody fragments), nucleic acids encoding the antigen-binding domain of anti-CD3, CD4, CD8 antibodies, etc. can be isolated by methods similar to those used to obtain nucleic acids encoding the antigen-binding domains constituting the CAR, and the antigen-binding domains can be recombinantly produced using the isolated nucleic acids.

[0194] 2. Production of the nucleic acid delivery carrier of the present invention Below, we will explain an example of the production of the nucleic acid delivery carrier of the present invention (hereinafter also referred to as "lipid nanoparticles of the present invention") when lipid nanoparticles are used as the carrier, as a representative example of the nucleic acid delivery carrier of the present invention. However, even when other carriers such as liposomes are used, the nucleic acid delivery carrier of the present invention can be obtained in the same way by making appropriate changes depending on the carrier used.

[0195] The lipid nanoparticles of the present invention can be produced by preparing lipid nanoparticles using, for example, the method described in US Pat. No. 9,404,127 and then chemically conjugating a T cell activating ligand to the lipid nanoparticles. Alternatively, as described in WO 2016 / 021683, lipid nanoparticles can be produced by preparing an organic solvent solution containing cationic lipids and non-cationic lipids, mixing the solution with water or a buffer solution containing nucleic acid to be encapsulated in the lipid nanoparticles, and then chemically conjugating a T cell activating ligand (and optionally a T cell targeting ligand when the lipid nanoparticles of the present invention are used in vivo). Examples of the molar ratio of cationic lipid, phospholipid, cholesterol, and PEG lipid include, but are not limited to, 40:60:0 to 20:0 to 50:0 to 5. Furthermore, when PEG lipid is used as the non-cationic lipid and a T cell activating ligand is attached to the end of the PEG, the molar ratio of the PEG lipid to the ligand can be, for example, 20:1 to 1:20. The PEG-lipid may contain about 10% to about 100% terminal reactive PEG (mol %). The mixing can be performed using a pipette or a microfluidic mixing system (e.g., Asia microfluidic system (Syrris)). The resulting lipid particles may be purified by gel filtration or subjected to dialysis and sterile filtration. The concentration of all lipid components in the organic solvent solution is preferably 0.5 to 100 mg / mL.

[0196] Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and mixtures thereof. The organic solvent may contain 0 to 20% water or a buffer solution. Examples of buffer solutions include acidic buffer solutions (e.g., acetate buffer solutions, citrate buffer solutions) and neutral buffer solutions (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solutions, tris(hydroxymethyl)aminomethane (Tris) buffer solutions, phosphate buffer solutions, and phosphate-buffered saline (PBS)).

[0197] When mixing is performed using a microfluidic mixing system, it is preferable to mix 1 volume part of the organic solvent solution with 1 to 5 volume parts of water or buffer solution. In this system, the flow rate of the mixture (mixture of the organic solvent solution and water or buffer solution) is preferably 0.1 to 10 mL / min, and the temperature is preferably 15 to 45°C.

[0198] When preparing a lipid particle dispersion as described above, a nucleic acid to be encapsulated in lipid nanoparticles can be added to water or a buffer solution to prepare a dispersion containing cationic lipids, non-cationic lipids, nucleic acids, and T cell activating ligands. The nucleic acid is preferably added to water or a buffer solution so that its concentration in water or a buffer solution is 0.05 to 2.0 mg / mL. Alternatively, lipid nanoparticles can be produced by mixing a lipid particle dispersion with the nucleic acid by a method known per se. The content of the nucleic acid in the lipid nanoparticles of the present invention is preferably 1 to 20% by weight. TM Measurement can be performed using Ribogreen (registered trademark) (Invitrogen). The encapsulation rate of the nucleic acid in the lipid nanoparticles of the present invention can be calculated based on the difference in fluorescence intensity between the presence and absence of a surfactant (e.g., Triton-X100).

[0199] The dispersion medium can be replaced with water or a buffer solution by dialysis. Dialysis is carried out at 4°C to room temperature using an ultrafiltration membrane with a molecular weight cutoff of 10 to 20K. Dialysis may be repeated. Tangential flow filtration may also be used for dialysis.

[0200] The ratio (weight ratio) of nucleic acid to lipid in the lipid nanoparticles of the present invention obtained as described above is about 0.01 to about 0.2.

[0201] The average particle size of the lipid nanoparticles of the present invention is preferably 10 to 200 nm, and can be calculated by cumulant analysis of the autocorrelation function using, for example, a Zetasizer Nano ZS (Malvern Instruments).

[0202] 3. Method for activating / proliferating T cells using T cell activating ligands and nucleic acid delivery carriers, method for delivering nucleic acids into T cells, and method for producing T cell-containing medicines The present invention also provides a method for activating and / or expanding T cells using the nucleic acid delivery carrier of the present invention obtained as described above (hereinafter also referred to as the "activation / expansion method of the present invention"). The method comprises the step of contacting a cell population containing T cells with the nucleic acid delivery carrier of the present invention. Here, the T cells may be T cells collected from a living body (also referred to herein as "ex vivo T cells") or T cells within a living body (also referred to herein as "in vivo T cells"), with ex vivo T cells being preferred.

[0203] In another aspect, when a nucleic acid delivery carrier of the present invention contains a nucleic acid, the nucleic acid can be simultaneously delivered into T cells by implementing the activation / proliferation method of the present invention. Accordingly, the present invention also provides a method for delivering a nucleic acid into T cells, comprising the step of contacting a cell population containing T cells with a nucleic acid delivery carrier of the present invention. In another embodiment, the present invention also provides a method for delivering a nucleic acid into T cells, comprising the step of simultaneously contacting a cell population containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery carriers without a T cell activation ligand attached to its surface (hereinafter, both of the above embodiments are collectively referred to as the "nucleic acid delivery method of the present invention"). When a free T cell activation ligand not bound to a nucleic acid delivery carrier is used herein, the ligand may be used alone or in a complex in which the ligand is bound to a carrier (e.g., Dynabeads® (Thermo Fisher Scientific)). Alternatively, commercially available products (eg, TransAct (Milteny Biotech), Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher Scientific)) may be used as such complexes.

[0204] In yet another aspect, T cells activated and / or expanded using the nucleic acid delivery carrier of the present invention, or T cells to which a nucleic acid has been delivered, can be used as an immune cell therapy agent. Accordingly, the present invention also provides a method for producing a pharmaceutical containing T cells, comprising the step of contacting a cell population containing T cells with the nucleic acid delivery carrier of the present invention. In another embodiment, the present invention also provides a method for producing a pharmaceutical containing T cells, comprising the step of simultaneously contacting a cell population containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery carriers to which no T cell activation ligand has been attached to the surface.

[0205] The cell population containing T cells to be contacted with the nucleic acid delivery carrier of the present invention, or with a T cell activation ligand or a nucleic acid delivery carrier without a T cell activation ligand attached to its surface, may be any cell population containing T cells or their precursor cells, and may be isolated T cells or a heterogeneous cell population, such as lymphocytes or lymphocyte precursor cells including pluripotent cells. In the present invention, "lymphocyte" refers to one of the subtypes of white blood cells in the immune system of vertebrates, including T cells, B cells, and natural killer cells (NK cells). Isolated and purified T cells are preferred. In the present invention, "T cell" refers to a type of white blood cell found in lymphoid organs or peripheral blood, and refers to a classification of lymphocytes characterized by differentiation and maturation mainly in the thymus and expression of TCR. Examples of T cells that can be used in the present invention include CD8-positive cytotoxic T cells (CTLs), CD4-positive helper T cells, regulatory T cells, and effector T cells, with cytotoxic T cells being preferred.

[0206] The lymphocytes can be collected from, for example, peripheral blood, bone marrow, or umbilical cord blood of a human or non-human mammal. When ex vivo T cells contacted with the nucleic acid delivery carrier of the present invention are used to treat a disease such as cancer, the cell population is preferably collected from the subject or a donor whose HLA type matches that of the subject.

[0207] The cell population containing T cells to be contacted with the nucleic acid delivery carrier of the present invention, or with a T cell activation ligand or a nucleic acid delivery carrier without a T cell activation ligand attached to its surface, may be a cell population in which lymphocyte precursor cells, including pluripotent cells, have been induced to differentiate into T cells. Examples of lymphocyte precursor cells, including pluripotent cells, include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), hematopoietic stem cells, multipotent progenitor cells that have lost self-renewal ability (MMPs), common myelo-lymphoid progenitors (MLPs), myeloid precursor cells (MPs), granulocyte-monocyte precursor cells (GMPs), macrophage-dendritic cell precursor cells (MDPs), and dendritic cell precursor cells (DCPs). Undifferentiated cells, such as pluripotent cells, can be differentiated into T cells by methods known per se.

[0208] There are no particular limitations on the method for contacting the nucleic acid delivery carrier of the present invention, or a T cell activation ligand and a nucleic acid delivery carrier without a T cell activation ligand attached to its surface, with ex vivo T cells, but for example, the nucleic acid delivery carrier of the present invention, or a T cell activation ligand and a nucleic acid delivery carrier without a T cell activation ligand attached to its surface, may be added to a normal T cell culture medium. Thus, in another aspect, the present invention also provides a cell culture comprising a cell population containing T cells, the nucleic acid delivery carrier of the present invention, or at least one T cell activation ligand and a nucleic acid delivery carrier without a T cell activation ligand attached to its surface, and a culture medium. In the nucleic acid delivery method of the present invention, for example, calcium phosphate coprecipitation, PEG, electroporation, microinjection, lipofection, etc. may be used in combination to increase the efficiency of nucleic acid delivery.

[0209] In the nucleic acid delivery method of the present invention, when the nucleic acid delivery carrier of the present invention or a nucleic acid delivery carrier without a T cell activation ligand attached to its surface contains a nucleic acid encoding an exogenous TCR, the expression of the endogenous TCRα and TCRβ chains naturally expressed by the T cell may be suppressed by siRNA, from the viewpoint of increasing the expression of the exogenous TCR, suppressing the appearance of mispaired TCRs, or suppressing non-autoreactivity. When applying the nucleic acid to the method, it is preferable that the nucleotide sequence of the nucleic acid encoding the TCR be a sequence different from the nucleotide sequence corresponding to the RNA on which the siRNA acting to suppress the expression of the endogenous TCRα and TCRβ chains acts (a codon-altered sequence), in order to avoid the effect of the siRNA on the exogenous TCR. These methods are described, for example, in WO 2008 / 153029. The nucleotide sequence can be prepared by introducing silent mutations into a nucleic acid encoding a naturally occurring TCR or by chemically synthesizing an artificially designed nucleic acid. Alternatively, to avoid mispairing with endogenous TCR chains, part or all of the constant region of the nucleic acid encoding the exogenous TCR may be replaced with a constant region derived from an animal other than human, such as a mouse. Alternatively, as described above, endogenous TCR genes can be knocked out using genome editing techniques.

[0210] 4. A nucleic acid delivery system comprising a combination of at least one T cell activation ligand and a nucleic acid delivery carrier on the surface of which no T cell activation ligand is attached. As described above, nucleic acids can be delivered into T cells by simultaneously contacting a cell population containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery carriers to which no T cell activation ligand is attached on its surface. Accordingly, the present invention also provides a nucleic acid delivery system comprising a combination of at least one T cell activation ligand and a nucleic acid delivery carrier to which no T cell activation ligand is attached on its surface. In this nucleic acid delivery system, the T cell activation ligand and the nucleic acid delivery carrier to which no T cell activation ligand is attached on its surface may be provided as a composition comprising both, or may be provided as separate components in the form of a kit. In addition to the above-mentioned components, the nucleic acid delivery kit may further include, for example, a medium to be used when contacting the cell population containing T cells with these components, but is not limited to this.

[0211] 5. A pharmaceutical comprising ex vivo T cells activated and / or expanded by a T cell activating ligand and a nucleic acid delivery carrier, or to which a nucleic acid has been delivered. The present invention also provides ex vivo T cells activated and / or expanded by the activation / expansion method of the present invention, or ex vivo T cells to which a nucleic acid has been delivered by the nucleic acid delivery method of the present invention (including a cell culture further comprising a medium), and pharmaceuticals containing them.

[0212] Ex vivo T cells activated and / or expanded by the activation / expansion method of the present invention can specifically recognize and kill (e.g., induce apoptosis of) cells expressing a surface antigen specifically recognized by the TCR expressed by the T cells. Furthermore, ex vivo T cells to which a nucleic acid encoding a CAR or exogenous TCR has been delivered by the nucleic acid delivery method of the present invention express the CAR or exogenous TCR, and thereby can specifically recognize and kill (e.g., induce apoptosis of) cells expressing the surface antigen specifically recognized by the CAR or exogenous TCR. Therefore, ex vivo T cells, which are activated and / or proliferated T cells that express a TCR that recognizes a surface molecule that is specifically expressed or whose expression is enhanced in diseased cells such as cancer cells as a surface antigen, or ex vivo T cells into which a nucleic acid encoding a CAR or exogenous TCR that recognizes the surface molecule has been delivered, can be used for the prevention or treatment of diseases such as cancer, and can be safely administered to mammals (humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys; preferably humans)).

[0213] In pharmaceuticals containing as an active ingredient the nucleic acid delivery carrier of the present invention, or ex vivo T cells contacted with a T cell activation ligand or a nucleic acid delivery carrier not having a T cell activation ligand attached to its surface, the T cells may be cultured in an appropriate medium before administration to a subject. Stimulatory molecules may also be added to the medium to maintain and / or amplify the activation and / or proliferation of T cells. Furthermore, serum or plasma may be added to the medium. The amount of these substances added to the medium is not particularly limited, but examples include 0% to 20% by volume. The amount of serum or plasma used can be varied depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources, but autologous sources are preferred from a safety perspective.

[0214] Pharmaceuticals containing as active ingredients the nucleic acid delivery carrier of the present invention, or ex vivo T cells contacted with a T cell activating ligand or a nucleic acid delivery carrier not having a T cell activating ligand attached to its surface, are preferably administered parenterally to a subject. Examples of parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is selected appropriately depending on the condition, weight, age, etc. of the subject, but typically, a single dose of 1 x 10 cells is administered to a subject weighing 60 kg. 6 ~1×10 10 Preferably 1 x 10 7 ~1×10 9 5×10 7 ~5×10 8 The ex vivo T cells are administered in a single dose or multiple doses. Pharmaceuticals containing ex vivo T cells contacted with the nucleic acid delivery carrier of the present invention as active ingredients can be prepared in known forms suitable for parenteral administration, such as injections or infusions. The pharmaceuticals may contain pharmacologically acceptable excipients, as appropriate. Examples of pharmacologically acceptable excipients include those described above. The pharmaceuticals may contain physiological saline, phosphate-buffered saline (PBS), culture media, etc., to stably maintain the cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, and X-VIVO10. Pharmaceutically acceptable carriers (e.g., human serum albumin), preservatives, etc., may also be added to the pharmaceuticals for stabilization purposes.

[0215] A medicament containing, as an active ingredient, the nucleic acid delivery carrier of the present invention, or a T cell activation ligand and ex vivo T cells contacted with a nucleic acid delivery carrier not having a T cell activation ligand attached to its surface, can be a cancer prevention or treatment drug. The cancers to which the medicament of the present invention is applicable are not particularly limited, and examples thereof include acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, chronic myeloid cancer, colon 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), and the like. , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, etc., but are not limited thereto.

[0216] 6. Medicine containing the nucleic acid delivery carrier of the present invention The nucleic acid delivery carriers of the present invention can induce the activation and / or proliferation of T cells in vivo by administering them to mammals such as humans. Furthermore, the nucleic acid delivery carriers of the present invention, which contain a nucleic acid encoding a CAR or an exogenous TCR, can be administered in vivo to mammals such as humans to deliver and express the nucleic acid in T cells in vivo, thereby endowing the T cells with the ability to specifically recognize and kill (e.g., induce apoptosis of) cells (e.g., cancer cells) that express a surface antigen (e.g., a cancer antigen) specifically recognized by the CAR or exogenous TCR. Therefore, the present invention also provides a pharmaceutical comprising the nucleic acid delivery carrier of the present invention.

[0217] Pharmaceuticals containing the nucleic acid delivery carriers of the present invention are preferably prepared as pharmaceutical compositions by mixing the nucleic acid delivery carriers with known pharmaceutically acceptable carriers (including excipients, diluents, bulking agents, binders, lubricants, flow aids, disintegrants, surfactants, etc.) and conventional additives. Excipients are well known to those skilled in the art, and include, for example, phosphate-buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, physiological saline, glycol or ethanol solutions, and salts of organic acids such as acetate, propionate, malonate, and benzoate. In addition, auxiliary agents such as wetting agents or emulsifiers, and pH buffers may also be used. Furthermore, formulation auxiliary agents such as suspending agents, preservatives, stabilizers, and dispersing agents may also be used. The pharmaceutical compositions may also be in a dry form for reconstitution with an appropriate sterile liquid prior to use. The pharmaceutical composition can be administered systemically or locally, orally or parenterally, depending on the form it is prepared in (oral preparations such as tablets, pills, capsules, powders, granules, syrups, emulsions, and suspensions; parenteral preparations such as injections, infusions, topical preparations, and suppositories). Parenteral administration can include intravenous, intradermal, subcutaneous, rectal, and transdermal administration. When used in the form of an injection, acceptable buffers, solubilizers, isotonicity agents, and the like can also be added.

[0218] The dosage of a pharmaceutical containing a nucleic acid delivery carrier of the present invention is, for example, 0.001 mg to 10 mg of nucleic acid encoding a CAR or exogenous TCR per kg of body weight per administration. For example, when administered to a human patient, the dosage is 0.001 to 50 mg for a patient weighing 60 kg. The dosages listed above are merely examples, and can be appropriately selected depending on the type of nucleic acid used, the route of administration, and the age, body weight, and symptoms of the recipient or patient.

[0219] When administered to a mammal (human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey); preferably, human), a pharmaceutical comprising the nucleic acid delivery carrier of the present invention can induce the expression of a CAR or exogenous TCR in T cells within the animal (in vivo T cells), and the in vivo T cells kill diseased cells such as cancer cells that express the surface antigen targeted by the CAR or exogenous TCR, thereby exhibiting a preventive or therapeutic effect against the disease.

[0220] A medicament comprising the nucleic acid delivery carrier of the present invention can be a preventive or therapeutic drug for cancer. The cancers to which the medicament of the present invention is applicable are not particularly limited, and examples thereof include acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, chronic myeloid cancer, colon 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), and the like. , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.

[0221] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and the present invention is not limited to these examples. [Example]

[0222] 1. Antibody reduction treatment 111 μl each of 9.21 mg / ml anti-CD3 antibody and anti-CD28 antibody (Bio X Cell) solutions was mixed with 12.3 μl of 10 mM DTT solution. The antibody and DTT mixture was vortexed and incubated at room temperature for 30 minutes. The reaction mixture was fractionated by HPLC (column: TSKgel G2000SWXL 7.8 mm x 30 cm, Tosoh Corporation, mobile phase: PBS) to obtain a fraction containing the reduced antibody. The fraction was then ultraconcentrated using an Amicon 0.5 ml-10K column. The antibody protein concentration and thiol group concentration in the concentrate were measured by absorbance at 230 nm and a fluorescent color reaction using N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), respectively.

[0223] 2. Preparation of Maleimide-lipid Nanoparticles A lipid mixture containing compounds 7, 11, 12, 21, 31, or 35 as the cationic lipid (cationic lipid:DPPC:Cholesterol:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA = 60:10.6:28:1.4:1 molar ratio) was dissolved in 90% EtOH and 10% water to obtain a 7.0 mg / ml lipid solution. Luciferase mRNA (TriLink) was dissolved in 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) to obtain a 0.2 mg / ml mRNA solution. The resulting lipid and mRNA solutions were mixed at room temperature using a NanoAssemblr device (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hour using a Slyde-A-Lyzer (20k molecular weight cutoff, Thermo Scientific) and against PBS for 48 hours at 4°C, then filtered using a 0.2 μm syringe filter (Iwaki) and stored at 4°C.

[0224] 3. Conjugation reaction of reduced antibody and maleimide-lipid nanoparticles The maleimide-lipid nanoparticle dispersion was mixed with the reduced antibody solution so that the molar concentration of the reduced antibody relative to the maleimide was 1 / 20, and the mixture was left to stand at room temperature for 4 hours. It was then stored at 4°C until the purification step.

[0225] 4. Gel Filtration Purification of Antibody-lipid Nanoparticles The reaction mixture of reduced antibody and maleimide-lipid nanoparticles was loaded onto a gel filtration column, Sepharose CL-4B (Cat. No. 17-0150-01 / GE Healthcare), and fractionated using D-PBS(-) as the mobile phase. The protein concentration of each fraction was then measured to identify the fraction containing the desired antibody-lipid nanoparticles, yielding the antibody-lipid nanoparticle stock solution. The antibody-lipid nanoparticles were filtered through a 0.2 μm syringe filter and stored at 4°C.

[0226] 5. Particle Size Measurement of Antibody-lipid Nanoparticles 99 μl of phosphate-buffered saline (137 mM NaCl, 7.99 mM NaHPO, 2.7 mM KCl, 1.47 mM KHPO, pH 7.4) was added to 1 μl of the antibody-lipid nanoparticle stock solution. The resulting dispersion was subjected to dynamic light scattering measurements using a Zetasizer Nano ZS (Malvern Instruments). Cumulant analysis of the autocorrelation function was performed to determine the Z-average particle size and polydispersity index (PDI).

[0227] 6. Zeta Potential Measurement of Antibody-lipid Nanoparticles 700 μl of HEPES buffer (10 mM HEPES-NaOH, pH 7.3) was added to 50 μl of the antibody-lipid nanoparticle stock solution, and the resulting dispersion was subjected to electrophoretic light scattering measurement using a Zetasizer Nano ZS (Malvern Instruments) to measure the ζ potential.

[0228] 7. Measurement of mRNA Encapsulation Rate and Concentration in Antibody-Lipid Nanoparticles The antibody-lipid nanoparticle stock solution was diluted with TE buffer to adjust the mRNA concentration to approximately 4 μg / ml. Naked mRNA was also diluted with TE buffer to 4 μg / ml as a standard solution for mRNA concentration. 60 μl of the diluted antibody-lipid nanoparticles and naked mRNA standard solution were mixed with 60 μl of TE buffer or TE buffer containing 2% Triton-X100. After allowing to stand at room temperature for 5 minutes, 120 μl of Quant-iT TM RiboGreen (registered trademark) was mixed in and the mixture was allowed to stand for another 5 minutes. The fluorescence intensity of the mixture was measured using an Envision microplate reader (Perkin-Elmer). The mRNA encapsulation rate and mRNA concentration were calculated using the following formulas.

[0229] % mRNA inclusion rate = (1-F TE / F Triton ) x 100 mRNA concentration = (F Triton -b) × d / m (However, F TE indicates the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer, and F Triton indicates the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer containing 2% Triton-X100. b and m indicate the y-intercept and slope obtained from the calibration curve of the concentration-standard siRNA. d indicates the dilution rate of the lipid nanoparticles.

[0230] The analytical results of the prepared antibody-lipid nanoparticles are listed in the table below.

[0231] [Table 1]

[0232] 8. mRNA transfection into human primary T cells using anti-CD3 antibody-conjugated lipid nanoparticles Human peripheral blood CD3-positive pan-T cells (Precision Bioservices) were added at 1 × 10 5Cells were seeded into round-bottom 96-well plates (Corning) at a cell density of 1000 cells / well. The serum-free hematopoietic cell medium, X-VIVO10 (Lonza), supplemented with 30 ng / ml recombinant IL-2 (Thermo Fisher Scientific) was used. Anti-CD3 antibody-conjugated lipid nanoparticles encapsulating luciferase mRNA (TriLink) were then added to the medium at a luciferase mRNA concentration of 1 μg / ml and incubated in a 5% CO2 incubator at 37°C for 72 hours. Luciferase expression in T cells was measured using the Bright-Glo Luciferase Assay System kit (Promega). The relative luciferase luminescence intensity of T cells treated with each anti-CD3 antibody-lipid nanoparticle is shown in Figure 1.

[0233] 9. mRNA transfection into human primary T cells using lipid nanoparticles conjugated with anti-CD3 and / or anti-CD28 antibodies Human peripheral blood CD3-positive pan-T cells (Precision Bioservices) were added at 1 × 10 5 Cells were seeded at a cell density of 1000 cells / well in a round-bottom 96-well plate (Corning). Serum-free hematopoietic cell culture medium X-VIVO10 (Lonza) supplemented with 30 ng / ml recombinant IL-2 (Thermo Fisher Scientific) was used. Subsequently, anti-CD3 antibody-conjugated lipid nanoparticles, anti-CD28 antibody-conjugated lipid nanoparticles, a mixture of anti-CD3 and anti-CD28 antibody-conjugated lipid nanoparticles, and a mixture of anti-CD3 and anti-CD28 antibody-conjugated lipid nanoparticles containing luciferase mRNA (TriLink) were added to the culture medium at a luciferase mRNA concentration of 1 μg / ml and incubated in a 5% CO2 incubator at 37°C for 72 hours. Luciferase expression in T cells was measured using the Bright-Glo Luciferase Assay System kit (Promega). The relative luciferase luminescence intensity of T cells treated with each antibody-lipid nanoparticle is shown in Figure 2.

[0234] 10. mRNA transfection into human primary T cells and stimulation of T cell activation using anti-CD3 / anti-CD28 antibody-conjugated lipid nanoparticles Human peripheral blood CD3-positive pan-T cells (Precision Bioservices) were added at 1 × 10 5 Cells were seeded into round-bottom 96-well plates (Corning) at a cell density of 1000 cells / well. The culture medium used was serum-free hematopoietic cell culture medium X-VIVO10 (Lonza) supplemented with 30 ng / ml recombinant IL-2 (Thermo Fisher Scientific). Subsequently, lipid nanoparticles conjugated with anti-CD3 and anti-CD28 antibodies encapsulating luciferase mRNA (TriLink) were added to the culture medium at luciferase mRNA concentrations of 0.3 and 1 μg / ml, respectively, and the culture was incubated at 37°C in a 5% CO2 incubator for 72 hours. As a control sample for T cell activation stimulation, T cells supplemented with TransAct (Miltenyi Biotec) and Dynabeads (Thermo Fisher Scientific) were also prepared. Luciferase expression in T cells was measured using the Bright-Glo Luciferase Assay System kit (Promega). The number of viable T cells was measured using a CellTiter-Glo kit (Promega). The expression level of luciferase is shown in Figure 3(I), and the number of viable T cells is shown in Figures 3(II) and (III).

[0235] 11. Preparation of Luc mRNA-encapsulated lipid nanoparticles A lipid mixture containing Compound 35 as a cationic lipid (Compound 35:DPPC:Cholesterol:SUNBRIGHT GM-020:SUNBRIGHT = 60:10.6:28:1.4 molar ratio) was dissolved in 90% EtOH and 10% water to obtain an 8.1 mg / ml lipid solution. Luciferase mRNA (Luc mRNA) (TriLink) was dissolved in 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) to obtain a 0.18 mg / ml mRNA solution. The resulting lipid and mRNA solutions were mixed at room temperature using a NanoAssemblr device (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion of Luc mRNA-encapsulated lipid nanoparticles. The resulting dispersion was dialyzed against water at room temperature for 1 hour using a Slyde-A-Lyzer (20k molecular weight cutoff, Thermo Scientific) and against PBS for 48 hours at 4°C, then filtered using a 0.2 μm syringe filter (Iwaki) and stored at 4°C.

[0236] 12. Particle size measurement of Luc mRNA-encapsulated lipid nanoparticles 99 μl of phosphate-buffered saline (137 mM NaCl, 7.99 mM NaHPO, 2.7 mM KCl, 1.47 mM KHPO, pH 7.4) was added to 1 μl of the Luc mRNA-encapsulating lipid nanoparticle stock solution. The resulting dispersion was subjected to dynamic light scattering measurements using a Zetasizer Nano ZS (Malvern Instruments). The Z-average particle size and polydispersity index were determined by cumulant analysis of the autocorrelation function.

[0237] 13. Zeta potential measurement of Luc mRNA-encapsulated lipid nanoparticles 50 μL of the Luc mRNA-encapsulating lipid nanoparticle stock solution was mixed with 700 μL of HEPES buffer (10 mM HEPES-NaOH, pH 7.3), and the resulting dispersion was subjected to electrophoretic light scattering measurements using a Zetasizer Nano ZS (Malvern Instruments) to measure the ζ potential.

[0238] 14. Measurement of mRNA encapsulation rate and concentration of Luc mRNA-encapsulated lipid nanoparticles The stock solution of Luc mRNA-encapsulated lipid nanoparticles was diluted with TE buffer to adjust the mRNA concentration to approximately 4 μg / ml. Naked mRNA was also diluted to 4 μg / ml with TE buffer to prepare a standard mRNA concentration solution. 60 μl of the diluted Luc mRNA-encapsulated lipid nanoparticles and the standard naked mRNA concentration solution were mixed with 60 μl of TE buffer or TE buffer containing 2% Triton-X100. After allowing to stand at room temperature for 5 minutes, 120 μl of Quant-iT™ RiboGreen® was added and allowed to stand for another 5 minutes. The fluorescence intensity of the mixture was measured using an Envision microplate reader (Perkin-Elmer). The mRNA encapsulation rate and mRNA concentration were calculated using the following formula:

[0239] % mRNA inclusion rate = (1-F TE / F Triton ) x 100 mRNA concentration = (F Triton -b) × d / m (However, F TE indicates the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer, and F Triton indicates the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer containing 2% Triton-X100. b and m indicate the y-intercept and slope obtained from the calibration curve of the concentration-standard siRNA. d indicates the dilution rate of the lipid nanoparticles.

[0240] The analytical results of the Luc mRNA-encapsulating lipid nanoparticles are shown in Table 2.

[0241] [Table 2]

[0242] 15. Highly efficient luc mRNA transfection into human peripheral blood CD3+ pan T cells by co-addition of activation stimuli and lipid nanoparticles Human peripheral blood CD3-positive pan-T cells (Precision Bioservices) were added at 1 × 10 5 Cells were seeded at a density of 1000 cells / well in a round-bottom 96-well plate (Corning). Serum-free hematopoietic cell culture medium X-VIVO10 (Lonza) was used, supplemented with 30 ng / ml recombinant IL-2 (Thermo Fisher Scientific) and either TransAct (Milteny Biotech) or Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher Scientific), which stimulate T cells, according to the manufacturer's recommended protocol. Subsequently, lipid nanoparticle compound 35-luc mRNA encapsulating luciferase mRNA (TriLink) was added to the culture medium at luciferase mRNA concentrations of 0.1, 0.3, or 1 μg / ml. The cultures were incubated at 37°C in a 5% CO2 incubator for 72 hours. Luciferase expression in T cells was measured using the Bright-Glo Luciferase Assay System kit (Promega). The survival and proliferation rates of T cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). The results are shown in Figures 4 and 5. The addition of lipid nanoparticles encapsulating Luc mRNA to T cells under stimuli dramatically improved transfection activity (Figure 4). Furthermore, the survival and proliferation rates of T cells were maintained at high levels (Figure 5).

[0243] 16. Highly efficient luc mRNA transfection into human CD4 / CD8+ T cells by co-addition of activation stimuli and lipid nanoparticles Human peripheral blood leukocyte fraction Leukopak (HemaCare) was washed using the LOVO Cell Processing System (Fresenius), and CD4 and CD8 positive cells were collected using the cell processing system CliniMACS (Milteny Biotec). The resulting CD4 / CD8 positive cells were 1 × 10 5 Cells were seeded into flat-bottom 96-well plates (Corning) at a cell density of 1000 cells / well. For cell culture, the medium used consisted of 26 mL of OPTmizer CTS T-cell Expansion Supplement, 20 mL of CTS Immune SR, 10 mL of L-Glutamine 200 mM (ThermoFischer Scientific), 10 mL of Streptomycin Sulfate 10 mg / mL (MEIJI), and 4.2 ng / mL of MACS GMP Recombinant Human IL-2 (Milteny Biotech) per 1 L of OPTmizer CTS T-Cell Expansion Basal Medium. TransAct (Milteny Biotech) was added as a T cell activation stimulant according to the manufacturer's recommended protocol. Next, lipid nanoparticle compound 35-luc mRNA encapsulating luciferase mRNA (TriLink) was added to the culture medium at luciferase mRNA concentrations of 1, 3, or 10 μg / ml and incubated in a 5% CO2 incubator at 37°C for 72 hours. Luciferase expression in T cells was measured using the Bright-Glo Luciferase Assay System kit (Promega). T cell viability and proliferation were measured using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). The results are shown in Figure 6. The addition of lipid nanoparticles encapsulating Luc mRNA to activated T cells dramatically improved transfection activity. Furthermore, T cell viability and proliferation were maintained at high levels. [Industrial Applicability]

[0244] By using the nucleic acid delivery carrier of the present invention or the nucleic acid delivery method of the present invention, the process of activating / proliferating T cells and the process of gene transfer into T cells can be carried out simultaneously in one pod, making it possible to produce immune cell therapy agents with low manufacturing costs in a short period of time, which is extremely useful in that it allows immune cell therapy to be provided at a lower cost.

[0245] This application is based on Japanese Patent Application No. 2018-197069 filed on October 18, 2018, and Japanese Patent Application No. 2019-124629 filed on July 3, 2019, the contents of which are incorporated herein by reference in their entirety.

Claims

1. A method for activating and / or expanding T cells, the method comprising a step of contacting a cell population containing T cells with a nucleic acid delivery carrier having at least one T cell activation ligand attached to its surface.

2. The method of claim 1 , wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

3. The method according to claim 1, wherein two or more types of T cell activation ligands are attached to the surface of the nucleic acid delivery carrier.

4. The method of claim 1 , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

5. The method according to claim 1, wherein the nucleic acid delivery carrier contains a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

6. The method of claim 1, wherein the nucleic acid delivery carrier contains a nucleic acid encoding a CAR or a TCR.

7. The method of claim 1, which is carried out ex vivo.

8. A method for delivering a nucleic acid into a T cell, comprising the step of contacting a cell population containing a T cell with a nucleic acid delivery carrier having at least one type of T cell activation ligand attached to its surface and containing a nucleic acid therein, wherein the nucleic acid does not include a nucleic acid encoding a CAR or TCR.

9. The method of claim 8, wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

10. The method according to claim 8, wherein two or more types of T cell activation ligands are attached to the surface of the nucleic acid delivery carrier.

11. The method of claim 8 , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

12. The method according to claim 8 , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

13. The method of claim 8, which is carried out ex vivo.

14. A method for delivering a nucleic acid into a T cell, comprising the steps of simultaneously contacting a cell population containing a T cell with at least one type of T cell activation ligand and a nucleic acid delivery carrier containing a nucleic acid therein and having no T cell activation ligand attached to its surface.

15. The method of claim 14, wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

16. The method of claim 14, wherein two or more T cell activation ligands are contacted.

17. The method of claim 14, wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

18. The method according to claim 14 , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

19. The method of claim 14, wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

20. The method of claim 14, which is carried out ex vivo.

21. A method for producing a pharmaceutical comprising T cells, comprising the step of simultaneously contacting a cell population containing T cells with at least one type of T cell activation ligand, and a nucleic acid delivery carrier containing a nucleic acid therein and not having a T cell activation ligand attached to its surface.

22. The method of claim 21 , wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

23. The method of claim 21 , wherein two or more T cell activating ligands are contacted.

24. The method of claim 21 , wherein the nucleic acid delivery carrier is a lipid nanoparticle or a liposome.

25. The method according to claim 21 , wherein the nucleic acid comprises a nucleic acid that suppresses the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

26. 22. The method of claim 21 , wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

27. 22. The method of claim 21, which is carried out ex vivo.

28. A T cell into which a nucleic acid has been delivered by the method of claim 14.

29. A pharmaceutical comprising the T cell described in claim 28.

30. A cell culture comprising a cell population including T cells, at least one T cell activation ligand, a nucleic acid delivery carrier to which no T cell activation ligand is attached on its surface, and a medium.

31. A composition for delivering a nucleic acid to a T cell, comprising at least one T cell activation ligand and a nucleic acid delivery carrier to which no T cell activation ligand is attached to the surface.

32. A kit for delivering a nucleic acid to a T cell, comprising at least one T cell activation ligand and a nucleic acid delivery carrier to which no T cell activation ligand is attached to its surface.

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