T cell activation / proliferation method

Abstract

The invention relates to a method for activating / proliferating T cells. Specifically, the present invention provides: a method for activation and / or proliferation of T cells, the method comprising a step of contacting a cell population containing T cells with a nucleic acid delivery vector to which at least one T cell activation ligand is added on the surface; a method for delivering nucleic acids into T cells, the method comprises a step of contacting a cell population containing T cells with (a) a nucleic acid delivery vector to which at least one T cell activation ligand is added on the surface and which contains a nucleic acid therein, or (b) at least one T cell activation ligand and a nucleic acid delivery vector to which no T cell activation ligand is added on the surface and which contains a nucleic acid therein; and a method for producing a drug containing T cells.

Description

[0001] This application is a divisional application of the application with international application date October 17, 2019, international application number PCT / JP2019 / 040937, which entered the Chinese national phase on April 15, 2021, application number 201980068068.4, and invention name “T cell activation / proliferation method”. Technical Field

[0002] The present invention relates to a nucleic acid delivery vector with a T cell activation ligand added to its surface, a method for activating and / or proliferating T cells using the nucleic acid delivery vector, a method for delivering nucleic acids into T cells, etc. The present invention also relates to a method for activating and / or proliferating T cells and a method for delivering nucleic acids into T cells, each of which characteristically comprises contacting the T cell activation ligand and the nucleic acid delivery vector with the T cells at the same time. Background Art

[0003] The research and development of cancer immunotherapy using CAR-T cells or TCR-T cells with genes of T-cell receptors (TCR) of chimeric antigen receptors (CAR) or T-cell receptors (TCR) 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 generally include using viral vectors such as lentiviral vectors to introduce CAR genes into T cells collected from patients in vitro to produce CAR-T cells, and the CAR-T cells are administered to the patients. However, this method has the problem of increasing production costs due to the cost of cell culture and viral vector preparation, because it requires multiple steps in a long period of time, such as activation / proliferation of T cells, preparation of viral vectors, gene transfer to T cells, etc.

[0004] As a method 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, which utilizes aggregates containing plasmid DNA encoding CAR and cationic polymer nanoparticles, wherein the nanoparticles are coated with a non-cationic polymer coupled to an anti-CD3 antibody fragment (Patent Document 1, Non-Patent Document 1), or utilizes a nanocarrier containing mesoporous silica encapsulating CAR-encoding DNA in the pores and coated with lipids modified with anti-CD3 antibodies on the surface (Patent Document 2).

[0005] In addition, a technique for delivering the siRNA of interest to target cells by encapsulating the siRNA in a "lipid nanoparticle (LNP)" has been reported, wherein the LNP has no internal pore structure and is composed of a cationic lipid, a non-cationic auxiliary lipid, and a ligand for delivery to the target cell. For example, it has been reported that siRNA directed to CD45 is transfected into T cells in vitro or in vivo by using an anti-CD4 antibody fragment as a targeting ligand (Patent Document 3, Non-Patent Document 2).

[0006] Furthermore, Patent Document 4 describes a cationic lipid for introducing active ingredients such as nucleic acids and the like into various cells, tissues and organs including T cells.

[0007] On the other hand, as a method for activating / proliferating T cells, a method for activating and / or proliferating T cells using beads on which anti-CD3 / CD28 antibodies are immobilized or nano-sized matrix beads has been reported (Patent Documents 5 and 6).

[0008] However, a technique in which the steps of activating / proliferating T cells and the step of introducing genes into T cells can be simultaneously performed in one chamber has not been reported so far.

[0009] [Reference list]

[0010] [Patent Document]

[0011] Patent Document 1: US2017 / 0296676

[0012] Patent Document 2: US2016 / 0145348

[0013] Patent Document 3: WO 2016 / 189532

[0014] Patent Document 4: WO 2016 / 021683

[0015] Patent Document 5: US 6,352,694

[0016] Patent Document 6: US2014 / 0087462

[0017] [Non-patent literature]

[0018] Non-patent document 1: Nature Nanotechnology 12, 813-820 (2017)

[0019] Non-patent document 2: ACS Nano, 2015, 9(7), 6706-6716 Summary of the invention

[0020] [Technical issues]

[0021] The purpose of the present invention is to shorten and simplify the production process of agents for immune cell therapy such as CAR-T therapy, to provide agents for immune cell therapy at low production costs in a short time, and to provide a safer production process for agents for immune cell therapy that eliminates the potential risk of carcinogenicity caused by viral vectors.

[0022] [Technical solution]

[0023] The present inventors have conducted intensive research in an attempt to achieve the above-mentioned purpose, and have successfully performed the steps of activating / proliferating T cells and introducing genes into T cells simultaneously in one chamber by using a nucleic acid delivery vector with a T cell activation ligand added to the surface. In addition, the present inventors surprisingly found that by simply bringing the T cell activation ligand and the nucleic acid delivery vector into contact with the T cells at the same time, the activation / proliferation of T cells and the introduction of nucleic acids into T cells can be efficiently achieved, and the present invention has been completed.

[0024] Therefore, the present invention provides the following items.

[0025] [1] A method for activating / proliferating T cells, comprising the step of contacting a cell population containing T cells with a nucleic acid delivery vector having at least one T cell activating ligand added to its surface.

[0026] [2] The method according to [1], wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[0027] [3] The method according to [1] or [2], wherein the nucleic acid delivery vector has two or more T cell activating ligands added to its surface.

[0028] [4] The method according to any one of [1] to [3], wherein the nucleic acid delivery vector is a lipid nanoparticle or a liposome.

[0029] [5] The method according to any one of [1] to [4], wherein the nucleic acid delivery vector contains a nucleic acid that inhibits the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

[0030] [6] The method according to any one of [1] to [5], wherein the nucleic acid delivery vector comprises a nucleic acid encoding CAR or TCR.

[0031] [7] The method according to any one of [1] to [6], wherein the method is performed ex vivo.

[0032] [8] A method for delivering a nucleic acid into T cells, the method comprising the step of contacting a cell population containing T cells with a nucleic acid delivery carrier having at least one T cell activating ligand added to its surface and containing a nucleic acid inside.

[0033] [9] The method described in [8], wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

[0034]

[10] The method of [8] or [9], wherein the nucleic acid delivery vector has two or more T cell activating ligands added to its surface.

[0035]

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

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

[0036]

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

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

[0037]

[13] The method described in any one of [8] to

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

[0038]

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

[13] , wherein the method is performed ex vivo.

[0039]

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

[0040]

[16] The method described in

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

[0041]

[17] The method of

[15] or

[16] , wherein the cell is contacted with two or more T cell activating ligands.

[0042]

[18] The method according to any one of

[15] to

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

[0043]

[19] The method according to any one of

[15] to

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

[0044]

[20] The method described in any one of

[15] to

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

[0045]

[21] The method according to any one of

[15] to

[20] , wherein the method is performed ex vivo.

[0046]

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

[0047]

[23] The method described in

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

[0048]

[24] The method of

[22] or

[23] , wherein the nucleic acid delivery vector has two or more T cell activating ligands added to its surface.

[0049]

[25] The method according to any one of

[22] to

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

[0050]

[26] The method according to any one of

[22] to

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

[0051]

[27] The method described in any one of

[22] to

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

[0052]

[28] The method according to any one of

[22] to

[27] , wherein the method is performed ex vivo.

[0053]

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

[0054]

[30] The method described in

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

[0055]

[31] The method of

[29] or

[30] , wherein the cell is contacted with two or more T cell activating ligands.

[0056]

[32] The method according to any one of

[29] to

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

[0057]

[33] The method according to any one of

[29] to

[32] , wherein the nucleic acid delivery vector comprises a nucleic acid that inhibits the expression of a T cell activation inhibitory factor and / or a nucleic acid encoding a T cell activation promoting factor.

[0058]

[34] The method described in any one of

[29] to

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

[0059]

[35] The method of any one of

[29] to

[34] , wherein the method is performed ex vivo.

[0060]

[36] A nucleic acid delivery vector having at least one T cell activating ligand added to its surface.

[0061]

[37] The nucleic acid delivery vector described in

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

[0062]

[38] The nucleic acid delivery vector of

[36] or

[37] , wherein the nucleic acid delivery vector has two or more T cell activating ligands added to its surface.

[0063]

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

[36] to

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

[0064]

[40] The nucleic acid delivery vector 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 promoting factor.

[0065]

[41] The nucleic acid delivery vector of any one of

[36] to

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

[0066]

[42] A drug comprising the nucleic acid delivery vector described in any one of

[36] to

[41] .

[0067]

[43] A T cell into which a nucleic acid has been delivered by the method described in any one of

[15] to

[21] .

[0068]

[44] A medicine comprising the T cell described in

[43] .

[0069]

[45] A cell culture comprising a cell population containing T cells, at least one T cell activating ligand, a nucleic acid delivery vector to which the T cell activating ligand is not added, and a culture medium.

[0070]

[46] A composition for delivering a nucleic acid into a T cell, comprising at least one T cell activating ligand and a nucleic acid delivery vector to which the T cell activating ligand is not added.

[0071]

[47] A kit for delivering a nucleic acid into a T cell, comprising at least one T cell activating ligand and a nucleic acid delivery vector to which the T cell activating ligand is not added.

[0072]

[48] ​​A T cell into which a nucleic acid has been delivered by the method described in any one of [8] to

[14] .

[0073]

[49] A medicine comprising the T cell described in

[48] .

[0074]

[50] A cell culture comprising a cell population containing T cells, a nucleic acid delivery vector having at least one T cell activating ligand added to its surface and containing a nucleic acid inside, and a culture medium.

[0075]

[51] A composition for delivering a nucleic acid into a T cell, comprising a nucleic acid delivery vector having at least one T cell activating ligand added to its surface and containing a nucleic acid inside.

[0076]

[52] A kit for delivering a nucleic acid into a T cell, comprising a nucleic acid delivery vector having at least one T cell activating ligand added to its surface and containing a nucleic acid inside.

[0077] [Beneficial Effects]

[0078] According to the present invention, the steps of activating / proliferating T cells and introducing genes into T cells can be performed simultaneously in one chamber without using a viral vector. As a result, a drug for immune cell therapy can be provided in a short time at a low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Shown is a comparison of the efficiency of gene transfer into T cells via lipid nanoparticles with anti-CD3 antibodies added to their surfaces and containing various cationic lipids (compounds 7, 11, 12, 21, 31, and 35).

[0080] Figure 2 Shown is a comparison of the efficiency of gene transfer into T cells via lipid nanoparticles with anti-CD3 antibody and / or anti-CD28 antibody added to their surfaces.

[0081] Figure 3It is shown that (I) gene transfer into T cells and (II) activation of T cells are achieved simultaneously by lipid nanoparticles with anti-CD3 antibodies and anti-CD28 antibodies added to the surface. In (I) and (II), the upper numerical value shows the concentration (μg / ml) of the encapsulated mRNA, and the lower numerical value shows the concentration (μg / ml) of the antibody. For comparison, (III) shows the efficiency of T cell activation by beads with conventionally known anti-CD3 antibodies and anti-CD28 antibodies added to the surface.

[0082] Figure 4 It was shown that luc mRNA was efficiently introduced into human peripheral blood CD3-positive total T cells by co-addition of activation stimulants and lipid nanoparticles.

[0083] Figure 5 Shown are the survival and proliferation rates of T cells transfected with luc mRNA via lipid nanoparticles.

[0084] Figure 6 It is shown that luc mRNA is efficiently introduced into human CD4 / CD8-positive T cells by co-addition of activation stimulants and lipid nanoparticles (left panel), and the survival and proliferation rates of T cells are maintained at high levels (right panel). DETAILED DESCRIPTION OF THE INVENTION

[0086] 1. Nucleic acid delivery vector of the present invention

[0087] The present invention provides a nucleic acid delivery vector having at least one T cell activating ligand added to its surface (hereinafter also referred to as "the nucleic acid delivery vector of the present invention").

[0088] As used herein, "nucleic acid delivery vector" means a vector capable of carrying a nucleic acid and delivering the nucleic acid into a cell. "Capable of delivering a nucleic acid into a cell" means that the carried nucleic acid can be delivered at least into the cytoplasm of a cell.

[0089] 1-1. Nucleic acid delivery vector

[0090] The nucleic acid delivery vector used in the present invention is not particularly limited in its structure, component molecules and nucleic acid delivery form, as long as it can carry nucleic acid as described above and can deliver the nucleic acid into cells. The representative drug delivery system (DDS) of nucleic acid is, for example, a complex formed on the basis of electrostatic interactions between them and nucleic acids using positively charged cationic liposomes, cationic polymers, etc. as carriers. The complex is combined with a negatively charged cell membrane and then incorporated into the cell by adsorptive endocytosis.

[0091] More specifically, examples of nucleic acid delivery vectors used in the present invention include, but are not limited to, lipid nanoparticles (LNP), liposomes (e.g., cationic liposomes, PEG-modified liposomes, etc.), and cationic polymers (e.g., polyethyleneimine, polylysine, polyornithine, chitosan, atelocollagen, protamine, etc.), delivery vectors in which cationic polymers are encapsulated in liposomes, etc. Alternatively, exosomes, which are a component derived from a living organism, may also be used. Lipid nanoparticles or liposomes are preferred, and lipid nanoparticles are more preferred.

[0092] 1-1-1. Lipid Nanoparticles (LNP)

[0093] In the present specification, "lipid nanoparticles (LNPs)" means particles having an average diameter of less than 1 μm and containing no macroporous structure (e.g., liposomes) or microporous structure (e.g., mesoporous materials) inside the shell of lipid aggregates containing cationic lipids and non-cationic lipids.

[0094] The components of the lipid nanoparticles are described below.

[0095] (a) Cationic lipids

[0096] In the present specification, "cationic lipid" means a lipid having a net positive charge in a low pH environment, for example, at physiological pH, in endosomes, etc. The cationic lipid used in the lipid nanoparticles used in the present invention is not particularly limited. For example, cationic lipids described in 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, US2015 / 0239834, WO2019 / 131839, etc., and the like may be mentioned.

[0097] Alternatively, mention may be made of the synthetic cationic lipids described in Dong et al. (Proc Natl Acad Sci US 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, cKK-E14) and in Love Synthetic cationic lipids described in KT et al. (Proc Natl Acad Sci US 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).

[0098] In a preferred embodiment, mention may be made of a cationic lipid represented by the following general formula and described in WO 2016 / 021683 or a salt thereof.

[0099]

[0100] in

[0101] W is of the formula -NR 1 R 2 Or -N + R 3 R 4 R 5 (Z - ),

[0102] R 1 and R 2 Each is independently C 1-4 Alkyl or hydrogen atom,

[0103] R 3 , R 4 and R 5 Each is independently C 1-4 alkyl,

[0104] Z - is an anion,

[0105] X is an optionally substituted C 1-6 Alkylene,

[0106] Y A , Y B and Y C are each independently an optionally substituted methine,

[0107] L A , L B and L C are each independently an optionally substituted methylene or a bond, and

[0108] R A1 , R A2 , R B1 , R B2 , R C1 and R C2 are each independently an optionally substituted C 4-10 alkyl.

[0109] More preferably, cationic lipids represented by the following structural formula and salts thereof may be mentioned.

[0110]

[0111]

[0112]

[0113]

[0114] Among the above-mentioned cationic lipids, more preferred are cationic lipids represented by the following structural formula and salts thereof.

[0115]

[0116] In another preferred embodiment, mention may be made of the cationic lipid represented by the following structural formula and described in WO 2019 / 131839.

[0117] A compound represented by the following formula or a salt thereof

[0118]

[0119] in

[0120] n is an integer from 2 to 5,

[0121] R is a straight chain C 1-5 Alkyl, straight chain C 7-11 Alkenyl or straight chain C 11 diene group, and

[0122] The wavy lines each independently show a cis-type or trans-type bond.

[0123] More preferably, cationic lipids represented by the following structural formula and salts thereof may be mentioned.

[0124]

[0125]

[0126]

[0127] Among the above-mentioned cationic lipids, more preferred are cationic lipids represented by the following structural formula and salts thereof.

[0128]

[0129]

[0130] In another preferred embodiment, mention may be made of a cationic lipid represented by the following general formula (III).

[0131] A compound represented by the following formula or a salt thereof,

[0132]

[0133] in

[0134] n1 is an integer from 2 to 6,

[0135] n2 is an integer from 0 to 2,

[0136] n3 is an integer from 0 to 2,

[0137] L is -C(O)O- or -NHC(O)O-,

[0138] Ra is a straight chain C 5-13 Alkyl, straight chain C 13-17 Alkenyl or straight chain C 17 Dienyl,

[0139] Rb is a straight chain C 2-9 alkyl,

[0140] Rc is a hydrogen atom or a straight chain C 2-9 alkyl,

[0141] Rd is a hydrogen atom or a straight chain C 2-9 alkyl,

[0142] Re is a straight chain C 2-9 Alkyl, and

[0143] Rf is a straight chain C 2-9 alkyl.

[0144] More preferably, cationic lipids represented by the following structural formula and salts thereof may be mentioned.

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] Among the above-mentioned cationic lipids, more preferred are cationic lipids represented by the following structural formula and salts thereof.

[0151]

[0152] The compound (III) can be produced, for example, by the following production method. Specifically, the compound (I) having the desired structure can be synthesized using suitable starting materials according to the structure of the compound (III) required in the esterification process. The salt of the compound (III) can be obtained by appropriately mixing with an inorganic base, an organic base, an organic acid, a basic or acidic amino acid.

[0153]

[0154]

[0155]

[0156] In the above structural formula, P 1 , P 2 , P 3 , P 4 , P 5 and P 6 Each is independently a protecting group, and compound (A) is of the formula: Compound (B) is of the formula: R 1 yes Compound (C) is of the formula: And R 2 yes

[0157] The starting materials or reagents used in each step of the above-mentioned production method and the obtained compound may each form a salt.

[0158] When the compound obtained in each step is a free compound, the compound can be converted into a salt of interest by a method known per se in the art. Conversely, when the compound obtained in each step is a salt, the salt can be converted into a free form or another type of salt of interest by a method known per se in the art.

[0159] The compound obtained in each step can be used in the next reaction directly or as a crude product in the form of its reaction solution. Alternatively, the compound obtained in each step can be separated and / or purified from the reaction mixture by separation methods such as concentration, crystallization, recrystallization, distillation, solvent extraction, classification or chromatography according to a conventional method.

[0160] If a starting material or reagent compound used in each step is commercially available, the commercially available product may be directly used.

[0161] In the reaction of each step, unless otherwise specified, the reaction time may vary depending on the reagent or solvent to be used and is generally 1 min to 48 hr, preferably 10 min to 8 hr.

[0162] In the reaction of each step, unless otherwise specified, the reaction temperature may vary depending on the reagent or solvent to be used and is generally -78°C to 300°C, preferably -78°C to 150°C.

[0163] In the reaction of each step, unless otherwise specified, the pressure may vary depending on the reagent or solvent used, and is generally 1 atm to 20 atm, preferably 1 atm to 3 atm.

[0164] In the reaction of each step, a microwave synthesis device such as a Biotage initiator can be used, for example. Unless otherwise specified, the reaction temperature can vary with the reagents or solvents used, and is generally room temperature to 300° C., preferably room temperature to 250° C., more preferably 50° C. to 250° C., unless otherwise specified. The reaction time can vary with the reagents or solvents used, and is generally 1 min to 48 hr, preferably 1 min to 8 hr.

[0165] In the reaction of each step, unless otherwise specified, the reagent is used in 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents relative to the substrate. In the case of using the reagent as a catalyst, the reagent is used in 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents relative to the substrate. When the reagent also serves as a reaction solvent, the reagent is used in the amount of a solvent.

[0166] In each step of the reaction, unless otherwise specified, the reaction is carried out without using a solvent or by dissolving or suspending in an appropriate solvent. Specific examples of the solvent include the following solvents.

[0167] Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butanol, 2-methoxyethanol, etc.;

[0168] Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, etc.;

[0169] Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.;

[0170] Saturated hydrocarbons: cyclohexane, hexane, heptane, etc.;

[0171] Amide: N,N-dimethylformamide, N-methylpyrrolidone, etc.;

[0172] Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.;

[0173] Nitriles: acetonitrile, etc.

[0174] Sulfoxide: dimethyl sulfoxide, etc.

[0175] Aromatic organic bases: pyridine, etc.

[0176] Anhydride: acetic anhydride, etc.

[0177] Organic acids: formic acid, acetic acid, trifluoroacetic acid, etc.;

[0178] Inorganic acid: hydrochloric acid, sulfuric acid, etc.;

[0179] Esters: ethyl acetate, isopropyl acetate, etc.;

[0180] Ketones: acetone, methyl ethyl ketone, etc.

[0181] water.

[0182] Two or more of these solvents may be used as a mixture in an appropriate ratio.

[0183] In each reaction step using a base, examples of the base that can be used are the bases listed below.

[0184] Inorganic alkali: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.;

[0185] Alkaline salts: sodium carbonate, calcium carbonate, sodium bicarbonate, etc.;

[0186] 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.

[0187] Metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, etc.;

[0188] Alkali metal hydride: sodium hydride, etc.

[0189] Metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, etc.;

[0190] Organic lithium: n-butyl lithium, sec-butyl lithium, etc.

[0191] In each reaction step using an acid or an acid catalyst, the acid or acid catalyst described below is used.

[0192] Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.;

[0193] Organic acid: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-naphthalenesulfonic acid, etc.;

[0194] Lewis acid: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous ferric chloride, etc.

[0195] Unless otherwise specified, each reaction step can be carried out according to a method known per se in the art, for example, a method described in Jikken Kagaku Koza (Encyclopedia of Experimental Chemistry), 5th edition, Vol. 13 to Vol. 19 (edited by the Chemical Society of Japan); Shin Jikken Kagaku Koza (New Encyclopedia of Experimental Chemistry), Vol. 14 to Vol. 15 (edited by the Chemical Society of Japan); Fine Organic Chemistry, 2nd edition revised edition (LF Tietze, Th. Eicher, Nankodo); Organic Name Reactions; The Reaction Mechanism and Essence, revised edition (Hideo Togo, Kodansha); Organic Syntheses Collective, Vol. I-VII (John Wiley & Sons, Inc.); Modern Organic Synthesis in the Laboratory: A Collection of Standard Experimental Procedures (Hideo Togo, Kodansha); Procedures) (Jie Jack Li, Oxford University Press); Comprehensive Heterocyclic Chemistry III, Volumes 1 to 14 (Elsevier Japan KK); Strategic Applications of Named Reactions in Organic Synthesis (translated by Kiyoshi Tomioka, Kagaku-Dojin Publishing); Comprehensive Organic Transformations (VCH Publishers, Inc.), 1989, etc.

[0196] In each step, the protection or deprotection reaction of the functional group can be carried out according to a method known per se in the art, for example, the method described in Protective Groups in Organic Synthesis, 4th edition, (Theodora W. Greene, Peter GMWuts), Wiley-Interscience, 2007; Protecting Groups, 3rd edition, (PJ Kocienski), Thieme, 2004), etc.

[0197] Examples of the protecting group for a hydroxyl group or a phenolic hydroxyl group in alcohols and the like include: an ether-type protecting group such as methoxymethyl ether, anisole, p-methoxyanisole, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether and tetrahydropyranyl ether; a carboxylate-type protecting group such as acetate; a sulfonate-type protecting group such as methanesulfonate; and a carbonate-type protecting group such as tert-butyl carbonate.

[0198] Examples of the protecting group for the carbonyl group in the aldehydes include: an acetal type protecting group such as dimethyl acetal; and a cyclic acetal type protecting group such as cyclic 1,3-dioxane.

[0199] Examples of the protecting group for the carbonyl group in 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.

[0200] Examples of the protecting group for the carboxyl group include: an ester type protecting group such as methyl ester; and an amide type protecting group such as N,N-dimethylamide.

[0201] Examples of the protecting group for the mercapto group include: an ether type protecting group such as benzyl thioether; and an ester type protecting group such as thioacetate, thiocarbonate and thiocarbamate.

[0202] Examples of protecting groups for amino groups or aromatic heterocycles such as imidazole, pyrrole or indole include: carbamate type protecting groups such as benzyl carbamate; amide type protecting groups such as acetamide; alkylamine type protecting groups such as N-triphenylmethylamine; and sulfonamide type protecting groups such as methanesulfonamide.

[0203] The protecting group can be removed using methods known per se in the art, for example, methods using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate or a trialkylsilyl halide (e.g. trimethylsilyl iodide or trimethylsilyl bromide) or reduction.

[0204] In each step utilizing the reduction reaction, examples of reducing agents that can be 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 complexes; 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. In the case of reducing a carbon-carbon double bond or triple bond, a method utilizing a catalyst such as palladium-carbon or a Lindlar catalyst can be used.

[0205] In each step utilizing an oxidation reaction, examples of the oxidizing agent that can be used include: peroxyacids such as meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, and tert-butyl hydroperoxide; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; chlorites such as sodium chlorite; periodates such as sodium periodate; hypervalent iodine reagents such as iodosobenzene; manganese reagents such as manganese dioxide and potassium permanganate; lead reagents such as lead tetraacetate; chromium reagents such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), and Jones reagent; halogenated compounds such as N-bromosuccinimide (NBS); oxygen; ozone; sulfur trioxide-pyridine complex; osmium tetroxide; selenium dioxide; and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).

[0206] In each step of the free radical cyclization reaction, examples of free radical initiators that can be used include: azo compounds such as azobisisobutyronitrile (AIBN); water-soluble free radical initiators such as 4-4'-azobis-4-cyanovaleric acid (ACPA); triethylboron in the presence of air or oxygen; and benzoyl peroxide. Examples of free radical initiators used include tributyltinane, tris(trimethylsilyl)silane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, and samarium iodide.

[0207] In each step utilizing the Wittig reaction, examples of the Wittig reagent that can be used include alkylene phosphanes. The alkylene phosphanes can be prepared by a method known per se in the art, such as a reaction between a phosphonium salt and a strong base.

[0208] In each step utilizing the Horner-Emmons reaction, examples of the reagent that can be used include phosphonoacetic acid esters such as methyl dimethylphosphonoacetate and ethyl diethylphosphonoacetate, and bases such as alkali metal hydrides and organic lithiums.

[0209] In each step utilizing Friedel-Crafts reaction, examples of reagents that can be used include Lewis acid and acid chloride or alkylating agents (e.g., alkyl halides, alcohols, and olefins). Alternatively, an organic or inorganic acid can be used in place of Lewis acid, and an acid anhydride such as acetic anhydride can be used in place of acid chloride.

[0210] In each step utilizing the aromatic nucleophilic substitution reaction, a nucleophile (eg, amine or imidazole) and a base (eg, basic salt or organic base) can be used as reagents.

[0211] In each step utilizing a nucleophilic addition reaction using a carbanion, a nucleophilic 1,4-addition reaction using a carbanion (Michael addition reaction), or a nucleophilic substitution reaction using a carbanion, examples of the base that can be used to generate a carbanion include organic lithium reagents, metal alkoxides, inorganic bases, and organic bases.

[0212] In each step utilizing the Grignard reaction, examples of the Grignard reagent include aryl magnesium halides such as phenylmagnesium bromide, and alkylmagnesium halides such as methylmagnesium bromide, isopropylmagnesium bromide. The Grignard reagent can be prepared by a method known per se in the art, such as a reaction between an alkyl halide or an aryl halide and magnesium metal in ether or tetrahydrofuran as a solvent.

[0213] In each step of the Knoevenagel condensation reaction, an active methylene compound having two electron withdrawing groups on the side (such as malonic acid, diethyl malonate or malononitrile) and a base (such as an organic base, a metal alkoxide or an inorganic base) can be used as reagents.

[0214] In each step utilizing the Vilsmeier-Haack reaction, phosphorus oxychloride and an amide derivative (eg, N,N-dimethylformamide) can be used as reagents.

[0215] In each step of the azidation reaction using alcohol, alkyl halide or sulfonate, examples of the azidation reagent that can be used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide and sodium azide. In the case of azidation of alcohols, for example, a method using diphenylphosphoryl azide and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), a method using trimethylsilyl azide and Lewis acid, etc. can be used.

[0216] In each step of the reductive amination reaction, examples of the reducing agent that can be used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen and formic acid. When the substrate is an amine compound, examples of the carbonyl compound that can be used include polyoxymethylene and aldehydes such as acetaldehyde and ketones such as cyclohexanone. When the substrate is a carbonyl compound, examples of the amine that can be used include primary amines such as ammonia and methylamine and secondary amines such as dimethylamine.

[0217] In each step utilizing the Mitsunobu reaction, azodicarboxylic acid esters such as diethyl azodicarboxylate (DEAD) and diisopropyl azodicarboxylate (DIAD) and triphenylphosphine can be used as reagents.

[0218] In each step utilizing the esterification, amidation or ureidation reaction, examples of the reagent that can be used include acyl halides such as acyl chloride or acyl bromide, and activated carboxylic acids such as anhydrides, active esters or sulfates. Examples of activating agents for carboxylic acids include: carbodiimide condensation agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD); triazine condensation agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hydrate (DMT-MM); carbonate condensation agents such as 1,1-carbonyldiimidazole (CDI); diphenylphosphoryl azide (DPPA); benzothiazol-1-yloxy-tris(dimethylamino)phosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent); thionyl chloride; short-chain alkyl haloformate such as ethyl chloroformate; O-(7-azabenzothiazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); sulfuric acid; and combinations thereof. In the case of using a carbodiimide condensing agent, it may be beneficial to add an additive such as 1-hydroxybenzothiazole (HOBt), N-hydroxysuccinimide (HOSu), or dimethylaminopyridine (DMAP) to the reaction.

[0219] In each step utilizing the coupling reaction, examples of metal catalysts that can be used include palladium compounds such as palladium acetate (II), tetrakis (triphenylphosphine) palladium (0), dichlorobis (triphenylphosphine) palladium (II), dichlorobis (triethylphosphine) palladium (II), tris (dibenzylideneacetone) dipalladium (0), 1,1'-bis (diphenylphosphino) ferrocene palladium chloride (II) and palladium acetate (II); nickel compounds such as tetrakis (triphenylphosphine) nickel (0); rhodium compounds such as tris (triphenylphosphine) rhodium chloride (III); cobalt compounds; copper compounds such as copper oxide and copper (I) iodide; and platinum compounds. It may also be beneficial to add a base to the reaction. Examples of such bases include inorganic bases and basic salts.

[0220] In each step of the thiocarbonylation reaction, phosphorus pentasulfide is generally used as the thiocarbonylation reagent. A reagent having a 1,3,2,4-dithiodiphosphatane-2,4-disulfide structure such as 2,4-bis(4-methoxyphenyl-1,3,2,4-dithiodiphosphatane-2,4-disulfide (Lawesson's reagent) may be used instead of phosphorus pentasulfide.

[0221] In each step utilizing the Wohl-Ziegler reaction, examples of halogenating agents that can be used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine and sulfuryl chloride. The reaction can be accelerated by further adding a free radical initiator such as heat, light, benzoyl peroxide or azobisisobutyronitrile.

[0222] In each step of the halogenation reaction using a hydroxyl group, examples of halogenating agents that can be used include acyl halides of hydrohalic acids or inorganic acids; examples include hydrochloric acid, thionyl chloride and phosphorus oxychloride for chlorination, and 48% hydrobromic acid for bromination. In addition, a method for obtaining an alkyl halide from alcohols by the action of triphenylphosphine and carbon tetrachloride or carbon tetrabromide, etc. can also be used. Alternatively, a method for synthesizing an alkyl halide by a 2-step reaction including converting an alcohol into a sulfonic acid ester and subsequent reaction with lithium bromide, lithium chloride or sodium iodide can also be used.

[0223] In each step utilizing the Arbuzov reaction, examples of the reagent that can be used include alkyl halides such as ethyl bromoacetate and phosphites such as triethyl phosphite and triisopropyl phosphite.

[0224] In each step utilizing the sulfone-esterification reaction, examples of the sulfonylating agent to be used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride and p-toluenesulfonic anhydride, and trifluoromethanesulfonic anhydride.

[0225] In each step utilizing the hydrolysis reaction, an acid or a base may be used as a reagent. In the case of performing an acid hydrolysis reaction of a tert-butyl ester, a reagent such as formic acid, triethylsilane, etc. may be added to reductively capture the by-product tert-butyl cation.

[0226] In each step utilizing the dehydration reaction, examples of the dehydrating agent that can be used include sulfuric acid, phosphorus pentoxide, phosphorus oxychloride, N,N'-dicyclohexylcarbodiimide, aluminum oxide, and polyphosphoric acid.

[0227] The salt of the compound represented by each of the above structural formulas is preferably a pharmaceutically acceptable salt. Examples thereof include salts with inorganic bases (e.g., alkali metal salts such as sodium salts, potassium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; aluminum salts; ammonium salts), salts with organic bases (e.g., salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris (hydroxymethyl) methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N, 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) or salts with acidic amino acids (salts with aspartic acid, glutamic acid).

[0228] The ratio (mol %) of the cationic lipid to the total lipid present in the lipid nanoparticles is, for example, about 10% to about 80%, preferably about 20% to about 70%, more preferably about 40% to about 60%; however, the ratio is not limited thereto.

[0229] Only one kind of the above-mentioned cationic lipids may also be used, or two or more kinds thereof may be used in combination. When a plurality of cationic lipids are used, the ratio of all cationic lipids is preferably as described above.

[0230] (b) Non-cationic lipids

[0231] In the present specification, "non-cationic lipid" means a lipid other than the cationic lipid, and is a lipid that does not have a net positive charge at a selected pH, such as physiological pH, etc. Examples of the non-cationic lipid used in the lipid nanoparticles of the present invention include phospholipids, steroids, PEG lipids, etc.

[0232] In order to enhance the delivery of nucleic acid to, for example, T cells, there is no particular limitation on phospholipids, as long as they stably maintain nucleic acid and do not inhibit fusion with cell membranes (plasma membranes and organelle membranes). For example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dilinolenoylphosphatidylcholine, etc. may be mentioned.

[0233] Preferred phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), more preferably DOPC, DPPC, POPC and DOPE.

[0234] The ratio (mol %) of phospholipids to total lipids present in the lipid nanoparticles may be, for example, about 0% to about 90%, preferably about 5% to about 30%, and more preferably about 8% to about 15%.

[0235] Only one kind of the above-mentioned phospholipids may be used, or two or more kinds thereof may be used in combination. When a plurality of phospholipids are used, the ratio of all the phospholipids is preferably as described above.

[0236] As steroids, mention may be made of 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, preferably cholesterol.

[0237] The ratio (mol %) of steroid to total lipid present in the lipid nanoparticles may be, for example, about 10% to about 60%, preferably about 12% to about 58%, and more preferably about 20% to about 55%.

[0238] Only one kind of the above-mentioned steroids may be used, or two or more kinds thereof may be used in combination. When a plurality of steroids are used, the ratio of all steroids is preferably as described above.

[0239] In this specification, "PEG lipid" means any complex of polyethylene glycol (PEG) and lipid. PEG lipid is not particularly limited, as long as it has the effect of inhibiting the aggregation of lipid nanoparticles. For example, it can be mentioned that PEG (PEG-DAA) coupled with dialkyloxypropyl, PEG (PEG-DAG) coupled with diacylglycerol (SUNBRIGHT GM-020 (NOF CORPORATION)), PEG (PEG-PE) coupled with phospholipids such as phosphatidylethanolamine, PEG (PEG-Cer) coupled with ceramide, PEG (PEG-cholesterol) coupled with cholesterol or its derivative or mixture, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglyceride (PEG-C-DOMG), 1-[8'-(1,2-dimyristoyl-3-propoxy)-formamide-3',6-dioxaoctyl]carbamoyl-ω-methyl-poly (ethylene glycol) (2KPEG-DMG) etc. Preferred PEG lipids include PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer and mixtures thereof, more preferably PEG-DAA conjugates selected from PEG-didecyloxypropyl conjugates, PEG-dilauryloxypropyl conjugates, PEG-dimyristyloxypropyl conjugates, PEG-dipalmityloxypropyl conjugates, PEG-distearyloxypropyl conjugates and mixtures thereof.

[0240] In addition to the methoxy group, a maleimide group, an N-hydroxysuccinimide group, etc. for binding to a T cell activating ligand can also be used as the free end of PEG. For example, SUNBRIGHT DSPE-0201MA (NOF) can be used as a PEG lipid having a functional group that binds to a T cell activating ligand (sometimes referred to as a "terminal reactive PEG lipid" in this specification).

[0241] The ratio (mol %) of PEG lipid to total lipid present in the lipid nanoparticles of the present invention may be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, and more preferably about 0.7% to about 2%.

[0242] The ratio (mol %) of the terminal reactive PEG lipid in the above-mentioned total PEG lipid may be, for example, about 10% to about 100%, preferably about 30% to about 100%, and more preferably about 40% to about 100%.

[0243] Only one of the above-mentioned PEG lipids may be used, or two or more thereof may be used in combination. When a plurality of PEG lipids are used, the ratio of all PEG lipids is preferably as described above.

[0244] 1-1-2. Liposomes

[0245] As another preferred nucleic acid delivery carrier used in the present invention, liposomes can be mentioned. As liposomes, those conventional liposomes for nucleic acid to DDS of cells can be used in the same manner. For example, liposomes prepared by mixing various cationic lipids (such as DOTMA, DOTAP, DDAB, DMRIE, etc.) developed as transfection reagents with neutral lipids (such as DOPE, cholesterol, etc.) of the fusion membrane released from endosomes are widely used. Liposomes in which functional molecules such as PEG, pH responsive membrane fusion peptides, membrane penetration promoting peptides, etc. are added to the liposome surface can also be used.

[0246] 1-2. T cell activation ligands

[0247] In the nucleic acid delivery vector of the present invention, a T cell activating ligand is added to the surface of the nucleic acid delivery vector.

[0248] The T cell activation ligand used in the present invention is not particularly limited, as long as it is a molecule that interacts with the surface molecules of T cells to promote the activation and / or proliferation of T cells. For example, it can be mentioned that there is a surface molecule CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, CD40L, etc. that specifically binds to CD3 coupled to TCR and is responsible for the signal transduction by TCR and is known as a co-stimulatory factor for T cell activation, and transduces activation / proliferation signals and auxiliary signals in T cells or antigen presenting cells. Such molecules can be physiological ligands (or receptors) of the above-mentioned T cell surface molecules, or can be non-physiological ligands (or receptors) with agonist activity. As non-physiological ligands, it is preferably mentioned that agonist antibodies.

[0249] More preferably, the T cell activating ligand used in the present invention comprises an antibody against CD3 and / or an antibody against CD28. The antibody against CD3 and the antibody against CD28 each specifically bind to CD3 and CD28 expressed on the target T cells to be induced to activate and / or proliferate (for example, when the target T cells are derived from humans, the antibody against CD3 and the antibody against CD28 are ideally anti-human CD3 antibodies and anti-human CD28 antibodies, respectively), and may be a complete antibody or a fragment thereof (for example, Fab, F(ab') 2 , Fab', scFv, Fv, reduced antibodies (rIgG), dsFv, sFv, diabodies, triabodies, etc.), as long as they have the ability to stimulate these surface molecules of T cells and transduce signals in T cells. There is no particular limitation on the subclass of the antibody, but IgG antibodies are preferred.

[0250] When an agonist antibody such as an antibody against CD3 or an antibody against CD28 is used as the T cell activating ligand, commercially available anti-CD3 antibodies, anti-CD28 antibodies, etc. can also be used as long as it is a complete antibody molecule, or the antibody can also be isolated from the culture medium of the cells producing the antibody. On the other hand, when the ligand is any of the above antibody fragments, by treating the complete antibody with a reducing agent (e.g., 2-mercaptoethanol, dithiothreitol) or a peptidase (e.g., papain, pepsin, ficin), or by isolating the nucleic acid encoding the fragment of the anti-CD3 antibody, anti-CD28 antibody, etc. in the same manner as described later in obtaining the nucleic acid to be encapsulated in the nucleic acid delivery vector, the antibody fragment can be produced recombinantly using it.

[0251] As the T cell activating ligand, only one may be used, or two or more may be used in combination. Preferably, two or more are combined. When two or more T cell activating ligands are used in combination, at least one is preferably an antibody against CD3 or an antibody against CD28, more preferably an antibody against CD3. Particularly preferably, both antibodies against CD3 and antibodies against CD28 may be used as T cell activating ligands.

[0252] When at least an antibody against CD3 and an antibody against CD28 are used in combination as the T cell activating ligand, the molar ratio of the two added to the surface of the nucleic acid delivery carrier of the present invention is 1:4-4:1, preferably 1:2-2:1.

[0253] When two or more T cell activating ligands are used in combination, the T cell activating ligands can be added to the surface of the nucleic acid delivery vector separately, or they can be compounded and added to the surface of the nucleic acid delivery vector, as long as the T cell activation activity of each is maintained. For example, when the two T cell activating ligands are antibodies (e.g., antibodies against CD3 and antibodies against CD28), they can be provided as bispecific antibodies known per se.

[0254] In the nucleic acid delivery vector of the present invention, the T cell activation ligand can be attached to the shell in any way, as long as it is present on the surface of the nucleic acid delivery vector. For example, when the lipid nanoparticles containing the terminal reactive PEG lipids as non-cationic lipids are used as nucleic acid delivery vectors, the ligand can be added to the end of PEG. For example, the lipid nanoparticles labeled with ligand (antibody) can be prepared by reacting the sulfhydryl group of the antibody with the PEG lipid (e.g. SUNBRIGHT DSPE-0200MA) having a maleimide group introduced into the end. Even when the liposomes modified with PEG are used as nucleic acid delivery vectors, the T cell activation ligand can also be added to the surface of the liposomes in the same manner.

[0255] 1-3. Nucleic Acid Contained in the Nucleic Acid Delivery Vector of the Present Invention

[0256] The nucleic acid delivery vector of the present invention that takes the form of not containing nucleic acid can also be used for inducing activation and / or proliferation of T cells.In a preferred embodiment, activation and / or proliferation of T cells and the delivery of nucleic acid in T cells can use encapsulated nucleic acid to carry out simultaneously in one step.Therefore, in a preferred embodiment, the nucleic acid delivery vector of the present invention also contains nucleic acid to be delivered in the T cell.

[0257] When the nucleic acid delivery vector of the present invention contains a nucleic acid inside, the nucleic acid is not particularly limited as long as the nucleic acid itself or its transcript or translation product has a function of changing the T cell to a desired state in the T cell.

[0258] 1-3-1. Nucleic acid that inhibits the expression of T cell activation inhibitory factor

[0259] In a preferred embodiment, the nucleic acid delivery vector of the present invention contains a nucleic acid that suppresses the expression of a T cell activation inhibitory factor internally. The T cell activation inhibitory factor as the target is not particularly limited, as long as it suppresses the activation of T cells. For example, it can be mentioned that negative signals are transmitted to the activation and / or proliferation of T cells after receiving stimulation from antigen presenting cells or tumor cells. Cell surface molecules such as immune checkpoint factors (such as CTLA-4, PD-1, TIM-3, LAG-3, TGIT, BTLA, VISTA (PD-1H), etc.), CD160, Cbl-b, endogenous TCR, etc.

[0260] The nucleic acid that inhibits the expression of T cell activation inhibitory factor may act at any level selected from the transcription level of the gene encoding the factor, the post-transcriptional regulation level, the protein translation level, the post-translational modification level, etc. Therefore, examples of the nucleic acid that inhibits the expression of 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 processing from the initial transcript into mRNA, nucleic acids that inhibit translation from mRNA into protein (e.g., antisense nucleic acids, miRNA), or nucleic acids that degrade mRNA (e.g., siRNA, ribozymes, miRNA), etc. Although it is preferred to use substances that act at any of these levels, substances that complementarily bind to mRNA to inhibit translation into protein or degrade mRNA are preferred. As the nucleic acid, mention may be made of

[0261] (a) a nucleic acid having RNAi activity against mRNA encoding the factor or a precursor thereof,

[0262] (b) an antisense nucleic acid directed against mRNA encoding the factor,

[0263] (c) a ribozyme nucleic acid directed against mRNA encoding the factor,

[0264] etc.

[0265] The nucleotide sequence (cDNA) of mRNA encoding each T cell activation inhibitory factor is known, and the sequence information can be obtained, for example, from public databases (eg, NCBI, EMBL, DDBJ, etc.).

[0266] (a) Nucleic acid having RNAi activity against mRNA encoding T cell activation inhibitory factor or its precursor

[0267] As a nucleic acid having RNAi activity against mRNA encoding T cell activation inhibitor, it can be mentioned that it is a double-stranded RNA consisting of an oligoRNA complementary to the target mRNA and its complementary strand, i.e., siRNA. The siRNA can be designed, for example, based on the cDNA sequence information of the target gene according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). As a short hairpin RNA (shRNA) precursor of siRNA, it can be designed by appropriately selecting a linker sequence (e.g., about 5-25 bases) capable of forming a ring structure, and connecting the sense strand and antisense strand of the siRNA through the linker sequence.

[0268] The siRNA and / or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such websites include, but are not limited to, siDESIGN Center provided by Dharmacon (http: / / dharmacon.horizondiscovery.com / jp / design-center / ?rdr=true&LangType=1041&pageid=17179928204), siRNA Target Finder provided by GenScript (https: / / www.genscript.com / tools / sirna-target-finder), etc.

[0269] In this specification, microRNA (miRNA) targeting mRNA encoding T cell activation inhibitor is also defined as being included in the nucleic acid having RNAi activity against the mRNA. For miRNA, the primary microRNA (pri-miRNA) as the primary transcript is first transcribed from the gene encoding the miRNA, and then processed by Drosha into a precursor microRNA (pre-miRNA) with a characteristic hairpin structure of about 70 bases in length, which is transported from the nucleus to the cytoplasm and further processed by Dicer to become a mature miRNA, which is taken up by RISC and acts on the target mRNA. Therefore, pre-miRNA or pri-miRNA, preferably pre-miRNA, can also be used as a precursor of miRNA.

[0270] miRNAs can be searched 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 / ) published by the Whitehead Institute in the United States, DIANA-micro-T-CDS (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=microT_CDS / index) published by the Alexander Fleming Center for Biomedical Sciences in Greece, etc. Alternatively, miRNAs targeting mRNA encoding T cell activation inhibitors can also be searched using TarBase (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8 / index), which is a database of miRNAs that have been experimentally proven to act on the target mRNA, and is published by the University of Thessaly, Pasteur Institute, etc.

[0271] The nucleotide molecules constituting siRNA and / or shRNA or miRNA and / or pre-miRNA may be natural RNA or DNA. In order to improve stability (chemical stability and / or stability against enzymes) and specific activity (affinity for RNA), various chemical modifications known per se may be included.

[0272] siRNA can be prepared by a method comprising the following steps: using a DNA / RNA automatic synthesizer to synthesize the sense strand and antisense strand of the target sequence on the mRNA, respectively, denaturing at about 90 to 95° C. for about 1 minute in a suitable annealing buffer, and annealing at about 30 to 70° C. for about 1 to 8 hours. In addition, siRNA can also be prepared by synthesizing shRNA as a precursor of siRNA and cleaving the shRNA with dicer. MiRNA and pre-miRNA can be synthesized by a DNA / RNA automatic synthesizer based on their sequence information.

[0273] In this specification, the nucleic acid designed to produce siRNA or miRNA for mRNA encoding T cell activation inhibitor in vivo is also defined as being included in the nucleic acid with RNAi activity for the mRNA. Examples of such nucleic acids include expression vectors constructed to express the above-mentioned shRNA or siRNA or miRNA or pre-miRNA, etc. As a promoter, a polII promoter (e.g., CMV immediate early promoter) can be used. It is a common practice to use a polIII promoter to ensure accurate transcription of short RNA. Examples of polIII promoters include mouse and human U6-snRNA promoters, human H1-RNase PRNA promoters, human valine-tRNA promoters, etc. In addition, sequences in which there are 4 or more continuous Ts are used as transcription termination signals. The expression cassettes of miRNA and pre-miRNA can also be produced in the same manner as in shRNA.

[0274] (b) Antisense nucleic acid against mRNA encoding T cell activation inhibitory factor

[0275] The antisense nucleic acid for the mRNA encoding the T cell activation inhibitor is a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof, which has the function of inhibiting protein synthesis by specifically binding to the target mRNA to form a stable duplex. The antisense nucleic acid can be DNA or RNA or DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous RNase H, thereby undergoing selective degradation of the target RNA. There is no particular restriction on the length of the target region of the antisense nucleic acid, as long as the hybridization of the antisense nucleic acid ultimately inhibits translation into protein, and can be the entire sequence or partial sequence of the mRNA encoding the protein. Short antisense nucleic acids may be about 10 bases, and long antisense nucleic acids can be the entire sequence of mRNA or initial transcripts. In addition, the antisense nucleic acid can be a nucleic acid that inhibits translation into protein by hybridizing with the target mRNA or initial transcript, and it can also be a nucleic acid (antigene) that can be combined with these genes as double-stranded DNA to form a triplex and inhibit transcription into RNA.

[0276] The nucleotide molecules constituting the antisense nucleic acid may also be modified in the same manner as in the case of the above-mentioned siRNA and the like so as to improve stability, specific activity and the like.

[0277] The antisense oligonucleotide can be prepared as follows: the target sequence of mRNA or primary transcript is determined based on the cDNA sequence or genomic DNA sequence of the target gene, and its complementary sequence is synthesized using a commercially available DNA / RNA automatic synthesizer.

[0278] In this specification, nucleic acids designed to produce antisense RNA against mRNA encoding T cell activation inhibitor in vivo are also defined as being included in antisense nucleic acids against the mRNA. Such nucleic acids can be exemplified by expression vectors constructed to express the above antisense RNA, etc. As a promoter, a polII promoter or a polIII promoter can be appropriately selected and used according to the length of the antisense RNA to be transcribed.

[0279] (c) Ribozyme nucleic acid against mRNA encoding T cell activation inhibitory factor

[0280] The ribozyme nucleic acid that can specifically cut the internal coding region of the mRNA encoding the T cell activation inhibitory factor can also be used as the nucleic acid that inhibits the expression of the factor. "Ribozyme" is narrowly defined as an RNA with an enzymatic activity that cuts nucleic acid, but this specification also includes DNA, as long as there is a sequence-specific enzymatic activity that cuts nucleic acid. The most widely used ribozyme nucleic acid includes self-splicing RNA, which can be found in infectious RNA such as viroids, pseudoviruses, etc., and hammerhead types or hairpin types are known. When the ribozyme is used in the form of an expression vector with a DNA encoding the ribozyme, the ribozyme can be a hybrid ribozyme that is further coupled to the sequence of the modified tRNA to promote the transport of the transcript to the cytoplasm.

[0281] (d) Nucleic acid for inhibiting the expression of T cell activation inhibitory factor by genome editing

[0282] In a preferred embodiment different from the above (a)-(c), the nucleic acid for suppressing the expression of the T cell activation inhibitor can be a nucleic acid that can inactivate (knock out) the gene encoding the factor. As such nucleic acid, it can be mentioned that the nucleic acid encoding the artificial nuclease is composed of a nucleic acid sequence recognition module (e.g., CRISPR / Cas9, ZF motif, TAL effector, etc.) that can specifically identify the partial nucleotide sequence as the target in the gene and a nuclease composition that introduces double-strand breaks (DSB) into the gene within or near the target sequence. After DSB is introduced, the gene can be knocked out by repairing the insertion or deletion mutation caused by the error by non-homologous end joining (NHEJ). Alternatively, by combining with a targeting vector in which a marker gene (e.g., a reporter gene such as a fluorescent protein gene, a selective marker gene such as a drug resistance gene, etc.) is inserted into the gene sequence, it is also possible to repair gene knockout by homologous recombination (HR). In addition, endogenous TCR genes can also be knocked in with exogenous TCR genes by HR repair.

[0283] 1-3-2. Nucleic acid encoding T cell activation promoting factor

[0284] In another preferred embodiment, the nucleic acid delivery vector of the present invention contains a nucleic acid encoding a T cell activation promoting factor internally. Interested T cell activation promoting factors include, for example, the above-mentioned T cell activation ligands are combined with T cell surface molecules (e.g., CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, CD40L, etc.) that transduce activation and / or proliferation signals in T cells.

[0285] The nucleotide sequence (cDNA) of mRNA encoding each T cell activation-promoting factor is known, and the sequence information can be obtained, for example, from public databases (eg, NCBI, EMBL, DDBJ, etc.).

[0286] The nucleic acid encoding the T cell activation promoting factor can be encapsulated in the nucleic acid delivery vector of the present invention in the form of an expression vector containing the mRNA or DNA encoding the factor. The mRNA encoding the T cell activation promoting factor can be separated by a method known per se, using RNA extracted from T cells as a template and using a probe or primer prepared on the basis of its sequence information. The mRNA obtained can be encapsulated in the nucleic acid delivery vector of the present invention as it is, or can be converted into cDNA and amplified by RT-PCR.

[0287] The obtained DNA encoding the T cell activation promoting factor can be inserted into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells as it is or after adding a suitable linker and / or a nuclear localization signal, etc. Examples of the promoter 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, the HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. In addition, gene promoters that direct specific expression in T cells, such as CD3, CD4, and CD8, can also be used.

[0288] mRNA encoding a T cell activation-promoting factor can be prepared by using an expression vector containing a DNA encoding the factor as a template and transcribing it into mRNA in an in vitro transcription system known per se.

[0289] 1-3-3. Nucleic acid encoding chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR)

[0290] As mentioned above, the nucleic acid delivery vector encapsulated with nucleic acid of the present invention can simultaneously perform activation and / or proliferation of T cells and delivery of nucleic acid to T cells in one step. Therefore, by encapsulating the nucleic acid encoding CAR or TCR in the nucleic acid delivery vector of the present invention, the step of activating / proliferating T cells and the step of transferring genes into T cells can be performed simultaneously in one compartment.

[0291] That is, in a preferred embodiment, the nucleic acid delivery vector of the present invention contains a nucleic acid encoding CAR or TCR inside.

[0292] (a) Nucleic acid encoding CAR

[0293] CAR is an artificially constructed hybrid protein containing an antigen binding domain (e.g., scFv) of an antibody coupled to a T cell signaling domain. CAR is characterized by the ability to redirect the specificity and responsiveness of T cells to selected targets in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous TCR α and β chains.

[0294] The CAR to be encapsulated in the nucleic acid delivery vector of the present invention includes an antigen binding domain of an antibody that can specifically recognize a surface antigen that the target T cells should recognize (e.g., a cancer antigen peptide, a surface receptor that shows promoted expression in cancer cells, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0295] Examples of surface antigens specifically recognized by the antigen binding domain include, but are not limited to, surface antigens specifically recognized by the antigen binding domain in 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, cervical, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colorectal cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid 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 carcinoma, Surface receptors showing promoted expression in various cancers (such as leukemia, melanoma, multiple myeloma, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.), such as CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell mutant antigen, 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, IgG κ light chain, and cancer antigen peptides (such as peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.).

[0296] The antigen binding domain used in the present invention is not particularly limited, as long as it is an antibody fragment that can specifically recognize the target antigen. Considering the ease of CAR preparation, a single-chain antibody (scFv) in which the light chain variable region and the heavy chain variable region are connected by a connecting peptide is desirable. There is no particular restriction on the configuration of the light chain variable region and the heavy chain variable region in the single-chain antibody, as long as they can reconstruct a functional antigen binding domain. They can generally be designed to take the order of light chain variable region, connecting peptide and heavy chain variable region from the N-terminal side. As the connecting peptide, a known connecting peptide commonly used in the production of single-chain antibodies can be used. For example, DNA encoding light chain variable regions and DNA encoding heavy chain variable regions can be prepared by cloning light chain genes and heavy chain genes from antibody-producing cells, respectively, and using them as templates for PCR, etc., or by chemically synthesizing them from the sequence information of existing antibodies. The DNA encoding a single-chain antibody can be obtained by connecting each obtained DNA fragment to the DNA encoding the connecting peptide by a suitable method. The N-terminal side of the antigen binding domain is preferably further added with a leader sequence to present CAR to the surface of T cells.

[0297] As the extracellular hinge domain and transmembrane domain, domains derived from T cell surface molecules commonly used in the relevant technical field can be used as appropriate. For example, they include but are not limited to domains derived from CD8α and CD28.

[0298] Examples of the intracellular signal transduction domain include, but are not limited to, an intracellular signal transduction domain having a CD3ζ chain, an intracellular signal transduction domain further having an auxiliary stimulatory motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, 4-1BB, etc. between the transmembrane domain and the CD3ζ chain, an intracellular signal transduction domain having two or more auxiliary stimulatory motifs, etc. Any domain commonly used in the relevant technical field can be used in combination.

[0299] The sequence information of nucleic acids encoding the extracellular hinge domain, the transmembrane domain and the intracellular signaling domain is well known in the relevant technical field. A person of ordinary skill in the art can easily obtain a DNA fragment encoding each domain from a T cell based on such information.

[0300] The DNA encoding CAR can be obtained by connecting the DNA fragments encoding the antigen binding domain, extracellular hinge domain, transmembrane domain and intracellular signal transduction domain obtained thereby, respectively, by conventional methods.

[0301] The obtained DNA encoding CAR can be inserted into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells as is or after adding a suitable connector and / or nuclear localization signal. Examples of promoters functional in T cells include, but are not limited to, constitutive SRα promoters, SV40 promoters, LTR promoters, CMV (cytomegalovirus) promoters, RSV (Rous sarcoma virus) promoters, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoters, etc. in mammalian cells. In addition, gene promoters that direct specific expression in T cells, such as CD3, CD4, and CD8, can also be used.

[0302] RNA encoding CAR, preferably mRNA, can be prepared by using an expression vector containing DNA encoding the above-mentioned CAR as a template and transcribing it into mRNA in an in vitro transcription system known per se.

[0303] (b) Nucleic acid encoding exogenous TCR

[0304] In this specification, "T cell receptor (TCR)" means a receptor composed of a dimer of TCR chains (α-chain, β-chain), and recognizes antigens or antigen-HLA (human leukocyte type antigen) (MHC; major histocompatibility complex) complexes and transduces stimulation signals to T cells. Each TCR chain consists of a variable region and a constant region, and the variable region contains three complementary determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only TCRs in which the α and β chains of TCRs form heterodimers, but also TCRs in which they form homodimers. In addition, the TCR includes TCRs in which some or all of the constant regions are deleted, TCRs with recombinant amino acid sequences, and soluble TCRs, etc.

[0305] "Exogenous TCR" means that it is exogenous to the T cell that is the target cell of the nucleic acid delivery vector of the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the amino acid sequence of the endogenous TCR expressed by the T cell that is the target cell of the nucleic acid delivery vector of the present invention.

[0306] The nucleic acid encoding TCR encapsulated in the nucleic acid delivery vector of the present invention is a nucleic acid encoding the α chain and β chain of TCR that can specifically recognize a surface antigen (eg, cancer antigen peptide, etc.) recognized by a target T cell.

[0307] The nucleic acid can be prepared by methods known per se. When the amino acid sequence or nucleic acid sequence of the desired TCR is known, the DNA encoding the full length or a portion of the TCR of the present invention can be constructed based on the sequence by, for example, chemically synthesizing a DNA chain or RNA chain or connecting synthetic partially overlapping oligoDNA short chains by PCR or Gibson assembly.

[0308] When the sequence of required TCR is unknown, for example, the T cell of interest is separated from the cell colony containing the T cell expressing the TCR of interest, and the nucleic acid encoding the TCR can be obtained from the T cell.Specifically, a cell colony (such as PBMC) containing T cells is collected from a living organism (such as mankind), the cell colony is cultured and stimulated simultaneously in the presence of an epitope of a cell surface antigen recognized by the TCR of interest, and the cell colony can be selected from the cell colony to specifically identify the T cell expressing the cell surface antigen by known methods and using the specificity of cells such as CD8 and CD4 as an index. The specificity of the cell expressing the cell surface antigen of T cells can be measured, for example, by dextromer assay, ELISPOT assay, cytotoxicity assay, etc. The above-mentioned cell colony containing T cells is preferably collected from, for example, an organism (such as a patient suffering from a disease such as cancer, or a colony containing T cells contacted with the epitope of the antigen or a dendritic cell pulsed with the epitope) having a large number of cells expressing the cell surface antigen recognized by the TCR of interest.

[0309] The nucleic acid of the present invention can be obtained by the following method: DNA is extracted from the above-mentioned separated T cells by conventional methods, and the DNA is used as a template to amplify and clone the TCR gene on the basis of the nucleic acid sequence of the constant region of the TCR. It can also be prepared as follows: RNA is extracted from cells by conventional methods and cDNA is synthesized, and the cDNA is used as a template, and 5'-RACE (rapid amplification of cDNA ends) is performed using antisense primers complementary to the nucleic acids encoding the constant regions of the TCR α chain and β chain, respectively. 5'-RACE can be performed by known methods, and can be performed, for example, using commercially available kits such as SMART PCR cDNA synthesis kits (manufactured by clontech). The DNA encoding the α chain and β chain of the obtained TCR can be inserted into a suitable expression vector in the same manner as the DNA encoding the above-mentioned CAR. The DNA encoding the α chain and the DNA encoding the β chain can be inserted into the same vector or a separate vector. When inserted into the same vector, the expression vector can express two chains in a polycistronic or monocistronic manner. In the former case, an intervening sequence allowing polycistronic expression, such as IRES or FMV 2A, is inserted between the DNA encoding the two strands.

[0310] In addition, RNA encoding each chain of the TCR, preferably mRNA, can be prepared in the same manner as the above-mentioned RNA encoding CAR, for example, by using the expression vector as a template.

[0311] 1-4. T cell targeting ligands

[0312] Ideally, the nucleic acid delivery vector of the present invention is used for preferably ex vivo activation and / or proliferation of T cells. It also includes an embodiment of in vivo administration to an object. In this case, further a part (hereinafter also referred to as "T cell targeting ligand") capable of targeting the nucleic acid delivery vector to T cells is added to the surface of the nucleic acid delivery vector of the present invention, thus the efficiency of the targeting T cells can be improved.

[0313] There is no particular limitation on the T cell targeting ligand, as long as it can specifically recognize surface molecules that are specific or highly expressed in T cells. Preferably, it includes a ligand containing an antigen binding domain of an antibody to CD3, CD4 or CD8, more preferably an anti-CD3 antibody. Here, "antigen binding domain" is synonymous with the antigen binding domain constituting the above-mentioned CAR. However, since CAR needs to be prepared as a nucleic acid encoding it, limitations arise, and single-chain antibodies are generally used in many cases. Since the antigen binding domain as a T cell targeting ligand is contained in the lipid nanoparticle of the present invention in a protein state, not only single-chain antibodies, but also any other antibody fragments such as complete antibody molecules, Fab, F (ab')2 , Fab', Fv, reduced antibodies (rIgG), dsFv, sFv, diabodies, triabodies, etc., can also be preferably used. These antibody fragments can be prepared by treating intact antibodies (such as IgG) with a reducing agent (such as 2-mercaptoethanol, dithiothreitol) or a peptidase (such as papain, pepsin, ficin) or by using genetic recombination operations.

[0314] When 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 separated from the culture of cells producing the antibody. On the other hand, when the ligand is any one of the above-mentioned antigen binding domains (antibody fragments), the nucleic acid encoding the antigen binding domains, such as anti-CD3, CD4, CD8 antibodies, etc., is separated in the same manner as the nucleic acid encoding the antigen binding domains constituting the CAR, and the antigen binding domains can be produced using its recombination.

[0315] 2. The nucleic acid delivery vector of the present invention Production

[0316] As a representative example of the nucleic acid delivery vector of the present invention, the production example of the nucleic acid delivery vector of the present invention using lipid nanoparticles as carriers (hereinafter also referred to as "lipid nanoparticles of the present invention") is explained below. Even when other carriers such as liposomes, etc. are used, the nucleic acid delivery vector of the present invention can be obtained in the same manner by making appropriate changes according to the carrier used.

[0317] The lipid nanoparticles of the present invention can be produced, for example, by forming lipid nanoparticles according to the method described in US9,404,127 and chemically binding T cell activation ligands. Alternatively, as described in, for example, WO 2016 / 021683, an organic solvent solution dissolving cationic lipids and non-cationic lipids is prepared, the organic solvent solution is mixed with water or a buffer solution in which nucleic acids to be encapsulated in the lipid nanoparticles are dissolved to prepare lipid nanoparticles, and chemically binds T cell activation ligands (and when the lipid nanoparticles of the present invention are used in vivo, necessary T cell targeting ligands), thereby producing the lipid nanoparticles. The mixing ratio (molar ratio) of cationic lipids, phospholipids, cholesterol and PEG lipids is, for example, 40 to 60: 0 to 20: 0 to 50: 0 to 5, but the ratio is not limited thereto. When PEG lipids are blended as non-cationic lipids and T cell activation ligands are added to the ends of PEG, the mixing ratio (molar ratio) of the PEG lipids and the ligands can be, for example, 20: 1 to 1: 20. The above-mentioned PEG lipid can contain terminal reactive PEG with a ratio (mol%) of about 10% to about 100%. The above-mentioned mixing can be carried out using a pipette, a microfluid mixing system (e.g., Asia microfluidic system (Syrris)). The lipid particles obtained can be purified by gel filtration, dialysis or sterile filtration.

[0318] The concentration of the total lipid component in the organic solvent solution is preferably 0.5 to 100 mg / mL.

[0319] As the organic solvent, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide or a mixture thereof can be cited. The organic solvent can contain 0 to 20% of water or a buffer solution. As the buffer solution, an acidic buffer solution (e.g., acetate buffer solution, citrate buffer solution) or a neutral buffer solution (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPE) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, phosphate buffer solution, phosphate buffered saline (PBS) can be cited.

[0320] When a microfluidic mixing system is used for mixing, it is preferred to mix 1 volume part of an organic solvent solution with 1 to 5 volume parts of water or a buffer solution. In addition, in the system, the flow rate of the mixture (mixed solution of an organic solvent solution and water or a buffer solution) is preferably 0.1 to 10 mL / min, and the temperature is preferably 15 to 45° C.

[0321] When producing lipid particle dispersion as described above, the dispersion containing cationic lipid, non-cationic lipid, nucleic acid and T cell activating ligand can be produced by adding nucleic acid to be encapsulated in lipid nanoparticles to water or buffer. The addition of the nucleic acid preferably takes the mode that its concentration in water or buffer solution is 0.05 to 2.0 mg / mL.

[0322] Furthermore, lipid nanoparticles can also be produced by mixing a lipid particle dispersion with a nucleic acid by a method known per se.

[0323] In the lipid nanoparticles of the present invention, the content of nucleic acid is preferably 1-20 wt%. The content can be measured using Quant-iT TM (Invitrogen). In the lipid nanoparticles of the present invention, the encapsulation ratio of nucleic acids can be calculated based on the difference in fluorescence intensity with or without the addition of a surfactant (eg, Triton-X100).

[0324] The dispersion medium can be replaced by dialysis with water or a buffer solution. For dialysis, an ultrafiltration membrane with a molecular weight cutoff of 10 to 20K is used at 4°C to room temperature. Dialysis can be performed repeatedly. For dialysis, tangential flow filtration can be used.

[0325] 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.

[0326] The average particle size of the lipid nanoparticles of the present invention is preferably 10 to 200 nm. The average particle size of the lipid particles can be calculated by cumulant analysis of the autocorrelation function using, for example, a Zetasizer Nano ZS (Malvern Instruments).

[0327] 3. Methods for activating / proliferating T cells using T cell activating ligands and nucleic acid delivery vectors, and methods for delivering nucleic acids to T cells Method for producing a drug containing T cells

[0328] The present invention also provides a method for activating and / or proliferating T cells using the nucleic acid delivery vector of the present invention obtained as described above (hereinafter also referred to as "activation / proliferation method of the present invention"). The method includes the step of contacting a cell colony containing T cells with the nucleic acid delivery vector of the present invention. When used in this article, the T cell can be a T cell collected from a living organism (also referred to as "ex vivo T cell" in this specification) or a T cell in a living organism (also referred to as "in vivo T cell" in this specification), preferably an ex vivo T cell.

[0329] In a different case, when the nucleic acid delivery vector of the present invention contains nucleic acid inside, the activation / proliferation method of the present invention can deliver the nucleic acid to T cells at the same time. Therefore, the present invention also provides a method for delivering nucleic acid to T cells, which includes the step of contacting a cell colony containing T cells with a nucleic acid delivery vector of the present invention. In another embodiment, the present invention provides a method for delivering nucleic acid to T cells, which includes contacting a cell colony containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery vectors to which a T cell activation ligand is not added on the surface (hereinafter, the above two embodiments are also collectively referred to as "nucleic acid delivery method of the present invention"). In this specification, when a free T cell activation ligand not bound to a nucleic acid delivery vector is used, the ligand can be used alone or as a complex in which the ligand is bound to a carrier (e.g., Dynabeads (R) (Thermo Fisher Scientific company)). As such a complex, commercially available products (e.g., TransAct (Milteny Biotech company), Dynabeads human T-cell activator CD3 / CD28 (ThermoFisher Scientific company)) can also be used.

[0330] In another different case, the T cells activated and / or proliferated using the nucleic acid delivery vector of the present invention or the T cells to which the nucleic acid is delivered can be used as a medicament for immune cell therapy. Therefore, the present invention also provides a method for producing a medicine containing T cells, comprising the step of contacting a cell colony containing T cells with a nucleic acid delivery vector of the present invention. In another embodiment, the present invention provides a method for producing a medicine containing T cells, comprising the step of contacting a cell colony containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery vectors to which a T cell activation ligand is not added on the surface.

[0331] The cell colony containing T cells that will come into contact with the nucleic acid delivery vector of the present invention or T cell activation ligand and the nucleic acid delivery vector to which T cell activation ligand is not added on the surface can be a separated T cell, or for example, a heterogeneous cell colony such as a progenitor cell of a lymphocyte, including lymphocytes and pluripotent cells, as long as it is a cell colony containing T cells or its progenitor cell. In the present invention, "lymphocyte" refers to one of the subtypes of leukocytes in the vertebrate immune system. Examples of lymphocytes include T cells, B cells and natural killer cells (NK cells), preferably isolated and purified T cells. In the present invention, "T cells" are a type of leukocytes found in lymphoid organs, peripheral blood, etc., and refer to a class of lymphocytes characterized by differentiation and maturation and expression of TCR mainly in the thymus. Examples of T cells that can be used in the present invention include cytotoxic T cells (CTL) as CD8 positive cells, helper T cells as CD4 positive cells, regulatory T cells and effector T cells, and preferably cytotoxic T cells.

[0332] The above-mentioned lymphocytes can be collected from peripheral blood, bone marrow and umbilical cord blood of humans or non-human mammals, for example. When the ex vivo T cells after contacting with the nucleic acid delivery vector of the present invention are used to treat diseases such as cancer, the cell population is preferably collected from the person to be treated or a donor whose HLA type matches the object to be treated.

[0333] The cell colony containing T cells that will come into contact with the nucleic acid delivery vector of the present invention or the T cell activation ligand and the nucleic acid delivery vector to which the T cell activation ligand is not added on the surface can be a cell colony of T cells obtained by differentiation induction from the progenitor cells of lymphocytes containing pluripotent cells. The examples of lymphocyte progenitor cells including pluripotent cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), embryonic germ cells (EG cells), hematopoietic stem cells, multipotent progenitor cells that have lost their self-renewal potential (specialized progenitor cells: MMP), common myeloid-lymphoid progenitor cells (MLP), myeloid progenitor cells (MP), granulocyte monocyte progenitor cells (GMP), macrophage-dendritic cell progenitor cells (MDP), dendritic cell progenitor cells (DCP), etc. Undifferentiated cells such as pluripotent cells can be differentiated into T cells by methods known per se.

[0334] There is no particular limitation on the method for contacting the nucleic acid delivery vector of the present invention or T cell activation ligand and the nucleic acid delivery vector to which T cell activation ligand is not added on the surface with in vitro T cells, and the nucleic acid delivery vector of the present invention or T cell activation ligand and the nucleic acid delivery vector to which T cell activation ligand is not added on the surface can be added to a typical culture medium for T cells, for example. Therefore, on the other hand, the present invention also provides a cell culture comprising a cell colony containing T cells, a nucleic acid delivery vector of the present invention or at least one T cell activation ligand and a nucleic acid delivery vector to which T cell activation ligand is not added on the surface, and a culture medium.

[0335] In the nucleic acid delivery method of the present invention, in order to improve the efficiency of nucleic acid delivery, for example, calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method, etc. can be used in combination.

[0336] In the nucleic acid delivery method of the present invention, when the nucleic acid delivery vector of the present invention or the nucleic acid delivery vector of the surface without adding T cell activation ligand contains the nucleic acid encoding exogenous TCR in particular inside, from the point of view of improving the expression of exogenous TCR, suppressing the occurrence of mismatched TCR or suppressing self-reactivity, the expression of endogenous TCR α chain and TCR β chain inherently expressed by T cells can be suppressed by siRNA. When the above-mentioned nucleic acid is applied to the method, in order to avoid the influence of siRNA on exogenous TCR, the base sequence of the nucleic acid encoding the TCR is preferably a sequence (sequence of codon conversion type) different from the base sequence of the RNA acted on by the siRNA corresponding to the expression of the endogenous TCR α and TCR β chain. Such methods are described in, for example, WO2008 / 153029. The above-mentioned base sequence can be produced by introducing silent mutations or chemically synthesized artificially designed nucleic acids in the nucleic acid encoding TCR obtained naturally. Alternatively, in order to avoid mismatching with endogenous TCR chains, part or all of the constant regions of the nucleic acid encoding the exogenous TCR can be replaced with constant regions of animals such as mice derived from outside humans.

[0337] Alternatively, as mentioned above, genome editing techniques can be used to knock out endogenous TCR genes.

[0338] 4. Nucleic acid delivery containing at least one T cell activating ligand in combination and without T cell activating ligand added to the surface Vector-based nucleic acid delivery system

[0339] As mentioned above, nucleic acid can be delivered to T cells by contacting a cell colony containing T cells with at least one T cell activation ligand and any of the above-mentioned nucleic acid delivery vectors to which T cell activation ligands are not added to the surface at the same time. Therefore, the present invention also provides a nucleic acid delivery system, which contains at least one T cell activation ligand combined and a nucleic acid delivery vector to which T cell activation ligands are not added to the surface. In the nucleic acid delivery system, the T cell activation ligand and the nucleic acid delivery vector to which T cell activation ligands are not added to the surface can be provided as a composition containing both or in the form of a kit containing both as separate components. In addition to the above two components, the nucleic acid delivery kit may further contain, for example, a culture medium used in contacting a cell colony containing T cells with these components, etc., although not limited thereto. 5. An isolated T cell containing a T cell activation ligand and a nucleic acid delivery vector activated and / or proliferated or delivered with a nucleic acid. Cell-derived drugs

[0340] The present invention also provides ex vivo T cells activated / proliferated by the activation / proliferation method of the present invention, ex vivo T cells (including cell cultures containing a culture medium) carrying nucleic acids delivered by the nucleic acid delivery method of the present invention, and drugs containing the same.

[0341] The in vitro T cells activated and / or proliferated by the activation / proliferation method of the present invention specifically recognize cells expressing surface antigens specifically recognized by the TCR expressed in the T cells and kill them (e.g., induce apoptosis). In addition, the nucleic acid delivery method of the present invention is used to deliver the nucleic acid encoding CAR or exogenous TCR to the in vitro T cells expressing the CAR or exogenous TCR, specifically recognizing the cells expressing surface antigens specifically recognized by the CAR or exogenous TCR, and can kill them (e.g., induce apoptosis). Therefore, the T cells expressing TCRs that are recognized as surface antigen-specific expression or surface molecules that show enhanced expression in diseased cells such as cancer cells are activated and / or proliferated in vitro T cells and the in vitro T cells that are introduced with nucleic acids encoding CAR or exogenous TCRs that recognize the surface molecules 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, cattle, sheep, monkeys), preferably humans).

[0342] In the medicine containing in vitro T cells after contacting with the nucleic acid delivery vector of the present invention or T cell activation ligand and the nucleic acid delivery vector without adding T cell activation ligand on the surface as the active ingredient, the T cells can be cultured using a suitable culture medium before being administered to the object. In addition, the activation and / or proliferation of T cells can also be maintained or expanded by adding stimulating molecules to the culture medium. In addition, serum or plasma can be added to the culture medium. Although there is no particular restriction on the amount added to these culture media, it can be mentioned that 0 volume %-20 volume %. In addition, the amount of serum or plasma used can be changed according to the culture stage. For example, serum or plasma concentration can be progressively reduced. The source of serum or plasma can be autologous or non-autologous, and autologous is preferred from the safety aspect.

[0343] The drug containing the in vitro T cells as active ingredients after contacting the nucleic acid delivery vector of the present invention or the T cell activation ligand and the nucleic acid delivery vector without T cell activation ligand added to the surface is preferably used by parenteral administration to the subject. Examples of methods for parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal and subcutaneous administration, etc. Although the dosage is appropriately selected according to the condition, weight, age, etc. of the subject, the administration of the drug to a subject weighing 60 kg is generally such that the number of cells per dose is 1×10 6 -1×10 10 cells, preferably 1×10 7 -1×10 9 cells, more preferably 5×10 7 -5×10 8 Cells. The medicine can be administered once or in multiple separate parts. The medicine containing the in vitro T cells after contact with the nucleic acid delivery vector of the present invention as an active ingredient can be formulated into a known form suitable for parenteral administration, such as a medicament for injection or infusion. The medicine of the present invention may appropriately contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient includes those described above. The medicine may contain saline, phosphate buffered saline (PBS), culture medium, etc., to stably maintain the cells. Examples of the culture medium include, but are not limited to, culture medium such as RPMI, AIM-V, X-VIVO10, etc. For stabilization purposes, the medicine may be supplemented with a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc.

[0344] The drug containing the in vitro T cells after contacting with the nucleic acid delivery vector of the present invention or T cell activation ligand and the nucleic acid delivery vector to which the T cell activation ligand is not added on the surface as the active ingredient can be a preventive or therapeutic drug for cancer. There is no particular limitation on the cancer used as the target of the drug of the present invention. Examples include, but are not limited to, acute lymphocytic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anus, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervix, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (such as non-small cell lung cancer), lymphoma (such as 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 cancer, greater omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.

[0345] 6. Drugs containing the nucleic acid delivery vector of the present invention

[0346] The nucleic acid delivery vector of the present invention can be administered to mammals such as humans in vivo, and induce activation and / or proliferation of T cells in living organisms. In addition, the nucleic acid delivery vector of the present invention containing nucleic acids encoding CAR or exogenous TCR is administered to mammals such as humans in vivo, and the nucleic acid is delivered and expressed in T cells in living organisms, and then the T cells can be given the ability to specifically recognize cells (such as cancer cells) expressing surface antigens (such as cancer antigens) specifically recognized by CAR or exogenous TCR and kill them (such as inducing apoptosis). Therefore, the present invention also provides a medicine containing the nucleic acid delivery vector of the present invention.

[0347] The medicine containing the nucleic acid delivery vector of the present invention is preferably prepared into a pharmaceutical composition by mixing the nucleic acid delivery vector with a known pharmaceutically acceptable carrier (including excipients, diluents, extenders, adhesives, lubricants, glidants, disintegrants, surfactants, etc.) and conventional additives, etc. The excipient is well known to those of ordinary skill in the art, and includes, for example, phosphate buffered saline (for example, 0.01M phosphate, 0.138M NaCl, 0.0027M KCl, pH 7.4), aqueous solutions containing inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc., saline solutions, glycol solutions, ethanol, etc., and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. In addition, adjuvants such as wetting agents or emulsifiers and pH buffers can also be used. In addition, preparation adjuvants such as suspending agents, preservatives, stabilizers and dispersants can also be used. Alternatively, the above-mentioned pharmaceutical composition can be taken in a dry form, which is reconstituted with a suitable sterile liquid before use. Depending on the form in which it is prepared (oral preparations such as tablets, pills, capsules, powders, granules, syrups, emulsions, suspensions, etc.; parenteral preparations such as injections, drips, external preparations, suppositories, etc.), the pharmaceutical composition can be administered orally or parenterally, systemically or topically. For parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, etc. can be used. When used in an injectable form, acceptable buffers, solubilizers, isotonic agents, etc. can also be added.

[0348] The dosage of the drug of the present invention containing the nucleic acid delivery vector of the present invention is, as the amount of nucleic acid encoding CAR or exogenous TCR per 1 kg body weight per dose, in the range of, for example, 0.001 mg to 10 mg. For example, when administered to a human patient, for a patient weighing 60 kg, the dosage is in the range of 0.0001 to 50 mg. The above dosages are examples, and the dosages can be appropriately selected according to the type of nucleic acid used, the route of administration, the age, weight, symptoms, etc. of the subject or patient to be administered.

[0349] By administering to a mammal (e.g., a human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cattle, sheep, monkey), preferably a human), a drug containing the nucleic acid delivery vector of the present invention can induce the expression of CAR or exogenous TCR in T cells (in vivo T cells) in the animal. The in vivo T cells kill diseased cells such as cancer cells that express surface antigens targeted by CAR or exogenous TCR, thereby confirming the preventive or therapeutic effect against the disease.

[0350] The drug containing the nucleic acid delivery vector of the present invention can be a preventive or therapeutic drug for cancer. There is no particular limitation on the cancer used as the target of the drug of the present invention. Examples include, but are not limited to, acute lymphocytic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anus, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervix, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic myeloid cancer, colorectal cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic Leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (such as non-small cell lung cancer), lymphoma (such as 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 cancer, greater omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.

[0351] The present invention is explained in more detail below with reference to examples, which are merely illustrative and do not limit the present invention. [Example]

[0352] 1. Antibody reduction treatment

[0353] 9.21mg / ml anti-CD3 antibody and anti-CD28 antibody (Bio X Cell company) solution (111μl each) were mixed with 10mM DTT aqueous solution (12.3μl). Each mixture of antibody and DTT was mixed by vortexing to react at room temperature for 30min. The reaction mixture was fractionated by HPLC (column: TSKgel G2000SWXL 7.8mm×30cm, TOSOH, mobile phase: PBS) to give a fraction solution containing reduced antibody. The fraction solution was concentrated by ultrafiltration using Amicon 0.5ml-10K. The concentrations of antibody protein and thiol groups in the concentrate were measured by the absorbance at 230nm and the fluorescent colorimetric reaction using N-(7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM).

[0354] 2. Preparation of Maleimide-lipid Nanoparticles

[0355] A lipid mixture containing compound 7, 11, 12, 21, 31 or 35 as a 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, 10% water to give a 7.0 mg / ml lipid solution. On the other hand, luciferase mRNA (TriLink company) was dissolved in 2-morpholineethanesulfonic acid (MES) buffer (pH 5.0) to give a 0.2 mg / ml mRNA solution. The obtained lipid solution and mRNA solution were mixed at room temperature by a Nanoassemblr device (PrecisionNanosystems) at a flow rate ratio of 3 ml / min: 6 ml / min to give a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hr using Slyde-A-Lyzer (20k molecular weight fraction, Thermo Scientific) and against PBS at 4° C. for 48 hr. Subsequently, the dialyzate was filtered through a 0.2 μm syringe filter (Iwaki) and stored at 4° C.

[0356] 3. Binding reaction of reduced antibody to maleimide-lipid nanoparticles

[0357] The maleimide-lipid nanoparticle dispersion was mixed with the reduced antibody solution so that the molar concentration of the reduced antibody was 1 / 20 of the molar concentration of the maleimide and allowed to stand at room temperature for 4 hrs. The mixture was then stored at 4°C until the purification step.

[0358] 4. Gel Filtration Purification of Antibody-Lipid Nanoparticles

[0359] The reaction mixture of reduced antibody and maleimide-lipid nanoparticles was loaded onto a gel filtration column Sepharose CL-4B (Catalog No. 17-0150-01 / GE Healthcare) and fractionated using D-PBS (-) as a mobile phase. Subsequently, the protein concentration of each fraction was measured to identify the fraction containing the antibody-lipid nanoparticles of interest, thereby obtaining an antibody-lipid nanoparticle stock solution. The antibody-lipid nanoparticles were filtered through a 0.2 μm syringe filter and stored at 4°C.

[0360] 5. Particle Size Measurement of Antibody-Lipid Nanoparticles

[0361] To 1 μl of the antibody-lipid nanoparticle stock solution, 99 μl of phosphate buffered saline (137 mM NaCl, 7.99 mM Na 2 HPO 4, 2.7 mM KCl, 1.47 mM KH 2 PO 4 , pH 7.4). The obtained dispersion was subjected to dynamic light scattering measurement using Zetasizer Nano ZS (Malverninstruments), and the cumulant analysis of the autocorrelation function was performed to calculate the average particle size Z and the polydispersity index (PDI).

[0362] 6. Zeta Potential Measurement of Antibody-Lipid Nanoparticles

[0363] To 50 μl of the antibody-lipid nanoparticle stock solution, 700 μl of HEPES buffer (10 mM HEPES-NaOH, pH 7.3) was added. The resulting dispersion was subjected to electrophoretic light scattering measurement using a Zetasizer Nano ZS (Malvern instruments) to measure the zeta potential.

[0364] 7. Measurement of mRNA Encapsulation Efficiency and Concentration of Antibody-Lipid Nanoparticles

[0365] The antibody-lipid nanoparticle stock solution was diluted with TE buffer to adjust the mRNA concentration to about 4 μg / ml. The naked mRNA was diluted with TE buffer to 4 μg / ml as the mRNA concentration standard solution. The diluted antibody-lipid nanoparticle and naked mRNA concentration standard solution (60 μl each) were mixed with 60 μl TE buffer or TE buffer containing 2% Triton-X100, respectively. The mixture was allowed to stand at room temperature for 5 min and mixed with 120 μl Quant-iT TM RiboGreen (registered trademark) was mixed, and the mixture was allowed to stand for 5 min. The fluorescence intensity of the mixture was measured using an Envision microplate reader (Perkin-Elmer company). The mRNA encapsulation efficiency and mRNA concentration were calculated by the following formula.

[0366] mRNA encapsulation efficiency % = (1-F TE / F Triton )×100

[0367] mRNA concentration = (F Triton -b)×d / m

[0368] (where F TE The RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer is shown. Triton(a) shows the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer containing 2% Triton-X100, (b) and (m) show the y-axis intercept and slope obtained from the calibration curve of the concentration standard siRNA, and (d) is the dilution rate of lipid nanoparticles.

[0369] A list of the analysis results of the prepared antibody-lipid nanoparticles is shown in the table below.

[0370] [Table 1]

[0371]

[0372] 8. Transfection of mRNA into primary human T cells using lipid nanoparticles conjugated to anti-CD3 antibodies

[0373] Human peripheral blood CD3 positive total T cells (Precision Bioservices) were cultured at 1×10 5 The cells were seeded at a density of 10 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 as the culture medium. Subsequently, lipid nanoparticles bound to anti-CD3 antibodies encapsulating luciferase mRNA (TriLink) were added to the culture medium so that the concentration of luciferase mRNA in the culture medium was 1 μg / ml, and the mixture was incubated at 5% CO 2 The cells were placed in an incubator at 37°C for 72 hr. The luciferase expressed in the T cells was measured using the Bright-Glo Luciferase Assay System Kit (Promega). The relative luciferase luminescence intensity of the T cells supplemented with each anti-CD3 antibody-lipid nanoparticle is shown in Figure 1 middle.

[0374] 9. Transfection of mRNA into human primary cells using lipid nanoparticles conjugated to anti-CD3 and / or anti-CD28 antibodies In cultured T cells

[0375] Human peripheral blood CD3 positive total T cells (Precision Bioservices) were cultured at 1×10 5The cells were seeded at a cell density of 10 cells / well on 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 as the culture medium. Subsequently, anti-CD3 antibody-bound lipid nanoparticles, anti-CD28 antibody-bound lipid nanoparticles, a mixture of anti-CD3 antibody-bound lipid nanoparticles and anti-CD28 antibody-bound lipid nanoparticles, and lipid nanoparticles bound to a mixture of anti-CD3 antibody and anti-CD28 antibody were added to the culture medium so that the concentration of luciferase mRNA in the culture medium was 1 μg / ml, and the mixture was incubated at 5% CO 2 The cells were placed in an incubator at 37°C for 72 hr. The luciferase expressed in the T cells was measured using the Bright-Glo Luciferase Assay System Kit (Promega). The relative luciferase luminescence intensity of the T cells supplemented with each anti-CD3 antibody-lipid nanoparticle is shown in Figure 2 middle.

[0376] 10. Transfection of mRNA into primary human T cells using lipid nanoparticles conjugated to anti-CD3 / anti-CD28 antibodies Stimulation of T cell activation

[0377] Human peripheral blood CD3 positive total T cells (Precision Bioservices) were cultured at 1×10 5 Cells were seeded at a density of 10 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 as the culture medium. Subsequently, lipid nanoparticles encapsulating luciferase mRNA (TriLink) and bound to anti-CD3 antibody and anti-CD28 antibody were added to the culture medium so that the concentration of luciferase mRNA in the culture medium was 0.3 or 1 μg / ml, and the mixture was incubated at 5% CO 2 The cells were placed in an incubator at 37°C for 72 hours. As control samples for T cell activation stimulation, T cells supplemented with TransAct (Miltenyi Biotec company) and Dynabeads (Thermo Fisher Scientific company) were also prepared. The luciferase expressed in T cells was measured using the Bright-Glo Luciferase Assay System Kit (Promega). The number of viable T cells was measured using the CellTiter-Glo Kit (Promega). The expression level of luciferase is shown in Figure 3(I), and the number of surviving T cells is shown in Figure 3 (II) and (III).

[0378] 11. Preparation of Lipid Nanoparticles Encapsulating Luc mRNA

[0379] 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, 10% water to give a lipid solution of 8.1 mg / ml. On the other hand, luciferase mRNA (Luc mRNA) (TriLink company) was dissolved in 2-morpholineethanesulfonic acid (MES) buffer (pH 5.0) to give a 0.18 mg / ml mRNA solution. The obtained lipid solution and mRNA solution were mixed at room temperature by a Nanoassemblr device (Precision Nanosystems) at a flow rate ratio of 3 ml / min: 6 ml / min to give a dispersion of lipid nanoparticles encapsulating Luc mRNA. The obtained dispersion was dialyzed against water at room temperature for 1 hr using a Slyde-A-Lyzer (molecular weight fraction of 20k, Thermo Scientific) and dialyzed against PBS at 4°C for 48 hr. Subsequently, the dialysate was filtered through a 0.2 μm syringe filter (Iwaki) and stored at 4°C.

[0380] 12. Particle Size Measurement of Lipid Nanoparticles Encapsulating Luc mRNA

[0381] To 1 μl of the lipid nanoparticle stock solution encapsulating Luc mRNA, 99 μl of phosphate buffered saline (137 mM NaCl, 7.99 mM Na 2 HPO 4 , 2.7 mM KCl, 1.47 mM KH 2 PO 4 , pH 7.4). The obtained dispersion was subjected to dynamic light scattering measurement using Zetasizer Nano ZS (Malvern instruments), and the cumulant analysis of the autocorrelation function was performed to calculate the average particle size Z and the polydispersity index.

[0382] 13. Zeta Potential Measurement of Lipid Nanoparticles Encapsulating Luc mRNA

[0383] To 50 μl of the lipid nanoparticle stock solution encapsulating Luc mRNA was added 700 μl of HEPES buffer (10 mM HEPES-NaOH, pH 7.3). The resulting dispersion was subjected to electrophoretic light scattering measurement using a Zetasizer Nano ZS (Malvern instruments) to measure the zeta potential.

[0384] 14. Measurement of mRNA Encapsulation Efficiency and Concentration in Lipid Nanoparticles Encapsulating Luc mRNA

[0385] The stock solution of nanoparticles encapsulating Luc mRNA was diluted with TE buffer to adjust the mRNA concentration to about 4 μg / ml. The naked mRNA was diluted with TE buffer to 4 μg / ml as an mRNA concentration standard solution. The diluted nanoparticles encapsulating Luc mRNA and the naked mRNA concentration standard solution (60 μl each) were mixed with 60 μl TE buffer or TE buffer containing 2% Triton-X100, respectively. The mixture was allowed to stand at room temperature for 5 min and mixed with 120 μl Quant-iT TM RiboGreen (registered trademark) was mixed, and the mixture was allowed to stand for 5 min. The fluorescence intensity of the mixture was measured using an Envision microplate reader (Perkin-Elmer company). The mRNA encapsulation efficiency and mRNA concentration were calculated by the following formula.

[0386] mRNA encapsulation efficiency % = (1-F TE / F Triton )×100

[0387] mRNA concentration = (F Triton -b)×d / m

[0388] (where F TE The RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer is shown. Triton (a) shows the RiboGreen fluorescence intensity of lipid nanoparticles mixed with TE buffer containing 2% Triton-X100, (b) and (m) show the y-axis intercept and slope obtained from the calibration curve of the concentration standard siRNA, and (d) is the dilution rate of lipid nanoparticles.

[0389] The analysis results of lipid nanoparticles encapsulating Luc mRNA are shown in Table 2.

[0390] [Table 2]

[0391]

[0392]

[0393] 15. Efficient transfection of mRNA into human peripheral blood CD3 by co-addition of activation stimulants and lipid nanoparticles Positive total T cells

[0394] Human peripheral blood CD3 positive total T cells (Precision Bioservices) were cultured at 1×10 5 The cells were seeded at a cell density of 10 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) and supplemented with TransAct (Milteny Biotech) or Dynabeads human T-cell activator CD3 / CD28 (Thermo Fisher Scientific) for stimulating T cell activation according to the manufacturer's recommended protocol was used as the culture medium. Subsequently, lipid nanoparticle compound 35-luc mRNA encapsulated with luciferase mRNA (TriLink) was added to the culture medium so that the concentration of luciferase mRNA in the culture medium was 0.1, 0.3 or 1 μg / ml, and the mixture was incubated at 5% CO 2 The cells were placed in an incubator at 37°C for 72 hours. The luciferase expressed in the T cells was measured using the Bright-Glo Luciferase Assay System Kit (Promega). The survival rate and proliferation rate of the T cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega KK). The results are shown in Figure 4 and 5 It has been shown that the addition of lipid nanoparticles encapsulating Luc mRNA to T cells under activation stimulation greatly enhances transfection activity ( Figure 4 In addition, the survival and proliferation rates of T cells were maintained at high levels ( Figure 5 ).

[0395] 16. Efficient transfection of luc mRNA into human CD4 / CD8 positive T cells

[0396] The human peripheral blood leukocyte fraction Leukopak (HemaCare) was washed with the LOVO cell processing system (Fresenius), and CD4 and CD8 positive cells were collected using the cell processing system CliniMACS (Milteny Biotec). The obtained CD4 / CD8 positive cells were concentrated at 1×10 5Cells were seeded at a cell density of 10 cells / well on a flat-bottom 96-well plate (Corning). For cell culture, a medium supplemented with 26 mL OPTmizer CTS T-cell expansion supplement, 20 mL CTS Immune SR, 10 mL 200 mM L-glutamine (all from ThermoFischer Scientific), 10 mL 10 mg / ml streptomycin sulfate (MEIJI company) and 4.2 ng / ml MACS GMP recombinant human IL-2 (Milteny Biotec) was used per 1 L OPTmizer CTS T-cell expansion basal medium. In addition, as a T cell activation stimulator, TransAct (Milteny Biotech) was added according to the manufacturer's recommended protocol. Subsequently, lipid nanoparticle compound 35-luc mRNA encapsulated with luciferase mRNA (TriLink) was added to the culture medium so that the concentration of luciferase mRNA in the culture medium was 1, 3 or 10 μg / ml, and the mixture was incubated at 5% CO 2 The cells were placed in an incubator at 37°C for 72 hours. The luciferase expressed in the T cells was measured using the Bright-Glo Luciferase Assay System Kit (Promega). The survival rate and proliferation rate of the T cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega KK). The results are shown in Figure 6 It has been shown that adding lipid nanoparticles encapsulating Luc mRNA to T cells under activation stimulation greatly increased transfection activity. In addition, the survival and proliferation rates of T cells were maintained at high levels.

[0397] [Industrial Applicability]

[0398] Using the nucleic acid delivery vector of the present invention or the nucleic acid delivery method of the present invention, the step of activating / proliferating T cells and the step of introducing genes into T cells can be performed simultaneously in one chamber. As a result, a medicament for immune cell therapy can be provided at a low production cost in a short time, and since immune cell therapy can be provided at a lower cost, the present invention is extremely useful.

[0399] This application is based on patent application No. 2018-197069 filed on October 18, 2018 and 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 delivering nucleic acid into T cells, the method comprising the step of simultaneously contacting a cell population containing T cells that has not been activated with at least one T cell activating ligand and a nucleic acid delivery vector containing nucleic acid, wherein the nucleic acid delivery vector has no T cell activating ligand added to its surface, wherein the nucleic acid delivery vector is a lipid nanoparticle, and wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

2. The method of claim 1, wherein contact is made with two or more T cell activating ligands. 3 . The method according to claim 1 , wherein the nucleic acid comprises a nucleic acid that inhibits the expression of a T cell activation inhibitory factor and / or a nucleic acid that encodes a T cell activation promoting factor.

4. The method of claim 1, wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

5. The method of claim 1, wherein the method is performed ex vivo.

6. A method for producing a drug containing T cells, the method comprising the step of simultaneously contacting a cell population containing T cells that has not been activated with at least one T cell activating ligand and a nucleic acid delivery vector containing nucleic acid, wherein the nucleic acid delivery vector has no T cell activating ligand added to its surface, wherein the nucleic acid delivery vector is a lipid nanoparticle, and wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

7. The method of claim 6, wherein contact is performed with two or more T cell activating ligands.

8. The method according to claim 6, wherein the nucleic acid delivery vector comprises a nucleic acid that inhibits the expression of a T cell activation inhibitory factor and / or a nucleic acid encoding a T cell activation promoting factor.

9. The method of claim 6, wherein the nucleic acid comprises a nucleic acid encoding a CAR or a TCR.

10. The method of claim 6, wherein the method is performed ex vivo.

11. A T cell, wherein a nucleic acid has been delivered into the T cell by the method according to claim 1.

12. A medicament comprising the T cell according to claim 11.

13. A cell culture comprising a cell population containing T cells, at least one T cell activating ligand, a nucleic acid delivery vector to which no T cell activating ligand is added on the surface, and a culture medium, wherein the nucleic acid delivery vector is a lipid nanoparticle, wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

14. A composition for delivering nucleic acid to T cells, comprising at least one T cell activating ligand and a nucleic acid delivery vector to which no T cell activating ligand is added on the surface, wherein the nucleic acid delivery vector is a lipid nanoparticle, wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

15. A kit for delivering nucleic acid into T cells, comprising at least one T cell activating ligand and a nucleic acid delivery vector to which the T cell activating ligand is not added, wherein the nucleic acid delivery vector is a lipid nanoparticle, wherein the T cell activating ligand comprises an antibody against CD3 and / or an antibody against CD28.

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