Nucleic acid-containing lipid nanoparticles and their uses

Abstract

To provide a novel transfection technique capable of efficiently introducing CAR or exogenous TCR selectively into immunocytes such as T cells in vivo or ex vivo, thereby providing CAR- or TCR-immunocell therapy with low production cost.SOLUTION: The present invention provides a lipid nanoparticle containing the following (a) to (c): (a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor; (b) a cationic lipid; and (c) a non-cationic lipid. The present invention also provides a CAR- or exogenous TCR-expressing immunocyte, obtained by introducing the lipid nanoparticle into in vivo or ex vivo T cells, and an in vivo or ex vivo therapeutic approach using the immunocytes for disease such as cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lipid nanoparticles containing a nucleic acid encoding a chimeric antigen receptor or a T cell receptor, a method for expressing a chimeric antigen receptor or an exogenous T cell receptor in immune cells of a subject using the lipid nanoparticles, and medical uses thereof, etc.

[0002] BACKGROUND OF THEINVENTION Research and development of cancer immunotherapy using CAR-T cells or TCR-T cells transfected with chimeric antigen receptors (CAR) or T cell receptors (TCR) derived from cancer antigen-specific killer T cells is progressing rapidly. Current CAR-T cell therapy, such as Kymriah (trade name) and Yescarta (trade name) approved in the United States, generally involves the ex vivo transduction of CAR genes into T cells collected from a patient using a viral vector such as a lentivirus vector to produce CAR-T cells, and then administering the CAR-T cells to the patient. However, this method has the problem of high manufacturing costs due to the costs involved in cell culture and preparation of viral vectors. If CAR or exogenous TCR could be selectively introduced into immune cells such as T cells in vivo, ex vivo preparation would be unnecessary, making it possible to provide CAR- or TCR-immune cell therapy with low manufacturing costs. Furthermore, if it were possible to selectively introduce CAR or exogenous TCR into immune cells such as T cells ex vivo without using viral vectors, which have high manufacturing costs, costs associated with virus residual tests, etc. would be unnecessary, making it possible to provide CAR- or TCR-immune cell therapy with low manufacturing costs.

[0003] To date, ex vivo or in vivo transfection of CAR into T cells has been reported using nanoparticles in which plasmid DNA encoding CAR is aggregated with a cationic polymer and the aggregates are coated with a non-cationic polymer conjugated with an anti-CD3 antibody fragment (Patent Document 1, Non-Patent Document 1), and a nanocarrier in which mesoporous silica with CAR-encoding DNA encapsulated within the pores is coated with lipids whose surface is modified with an anti-CD3 antibody (Patent Document 2).

[0004] In addition to these, a technology has been reported in which the desired siRNA is encapsulated in a "lipid nanoparticle (LNP)" that does not have a pore structure inside and is composed of a cationic lipid, a non-cationic helper lipid, and a ligand for delivery to a target cell, and the siRNA is delivered to the target cell. For example, it has been reported that an siRNA against CD45 was transfected ex vivo or in vivo into T cells using an anti-CD4 antibody fragment as a targeting ligand (Patent Document 3, Non-Patent Document 2).

[0005] However, there have been no reports to date of selectively introducing nucleic acids (e.g., mRNA, DNA) encoding CAR or exogenous TCR into immune cells such as T cells using LNPs. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 2017 / 0296676 [Patent Document 2] US 2016 / 0145348 [Patent Document 3] WO 2016 / 189532 [Non-patent literature]

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

[0008] The object of the present invention is to provide a novel transfection technique capable of efficiently introducing CAR or exogenous TCR selectively into immune cells such as T cells in vivo or ex vivo, thereby providing a CAR- or TCR-immune cell therapy with low production costs. Another object of the present invention is to provide a safer CAR- or TCR-immune cell therapy that avoids the problem of antigenicity caused by viral proteins. [Means for solving the problem]

[0009] As a result of intensive research to achieve the above-mentioned object, the inventors have succeeded in using LNP to selectively and efficiently introduce nucleic acids encoding CAR or exogenous TCR into immune cells such as T cells in vivo and ex vivo, thereby completing the present invention.

[0010] That is, the present invention provides the following. [1] Lipid nanoparticles comprising the following (a) to (c): (a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor; (b) a cationic lipid; and (c) Non-cationic lipids. [2] The cationic lipid is Formula (I):

[0011] [ka] [In the formula, L 1 is C 1-22 Alkylene group, C 2-22 Alkenylene group or C 3-22 is an alkadienylene group, n is an integer of 0 or 1, R 1 teeth, (1) a hydrogen atom, (2) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (3) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (4) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 is an alkadienyl group, R 2 is -CH2-O-CO-R 5 , -CH2-CO-OR 5 or -R 5 and R 3 is -CH2-O-CO-R 6 , -CH2-CO-OR 6 or -R 6 and R 4 is a hydrogen atom, -CH2-O-CO-R 7 , -CH2-CO-OR 7 or -R 7 and R 5 , R 6 and R 7 are each independently (1) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (2) Linear C 1-22 Alkyl groups and linear C 2-22A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (3) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 is an alkadienyl group, R8 and R9 are each independently C 1-6 indicates an alkyl group] The lipid nanoparticle according to [1], wherein the lipid nanoparticle is a compound represented by the formula: [3] The lipid nanoparticle described in [1] or [2], wherein the nucleic acid is mRNA or DNA. [4] The lipid nanoparticle according to any one of [1] to [3], wherein the non-cationic lipid is a phospholipid, cholesterol and / or a PEG lipid. [5] The lipid nanoparticle according to any one of [1] to [4], wherein the lipid nanoparticle has a ligand on its surface capable of targeting T cells. [6] The lipid nanoparticle described in [5], wherein the ligand is a ligand comprising an antigen-binding domain of one or more antibodies selected from the group consisting of an antibody against CD3, an antibody against CD4, an antibody against CD8, and an antibody against CD28. [7] The lipid nanoparticle described in [5], wherein the ligand is a ligand comprising an antigen-binding domain of an antibody against CD3 and / or an antibody against CD28. [8] The lipid nanoparticle described in [5], wherein the ligand is a ligand comprising antigen-binding domains of an antibody against CD3 and an antibody against CD28. [9] A pharmaceutical comprising the lipid nanoparticles described in any one of [1] to [8].

[10] The pharmaceutical according to [9], which is a preventive or therapeutic drug for cancer.

[11] The pharmaceutical according to [9], which comprises introducing a chimeric antigen receptor or an exogenous T cell receptor into in vivo immune cells and inducing their expression.

[12] The pharmaceutical according to [9], which comprises introducing a chimeric antigen receptor or an exogenous T cell receptor into in vivo T cells and inducing their expression.

[13] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into in vivo immune cells of a mammal and expressing the same, comprising administering to the mammal the lipid nanoparticles described in any one of [1] to [8].

[14] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into and expressing the same in in vivo T cells of a mammal, the method comprising administering to the mammal the lipid nanoparticles described in any one of [1] to [8].

[15] A method for preventing or treating cancer in a mammal, comprising administering to the mammal the lipid nanoparticles described in any one of [1] to [8].

[16] A lipid nanoparticle according to any one of [1] to [8] for use in the prevention and treatment of cancer.

[17] Use of the lipid nanoparticles described in any one of [1] to [8] for producing a cancer preventive or therapeutic agent.

[18] A composition for inducing expression of a chimeric antigen receptor or an exogenous T cell receptor, comprising the lipid nanoparticles according to any one of [1] to [8].

[19] Ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells.

[20] Ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells.

[21] A pharmaceutical comprising ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor, the ex vivo immune cells being obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells.

[22] A pharmaceutical comprising ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor, the ex vivo T cells being obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells.

[23] The pharmaceutical agent according to

[21] or

[22] , which is a preventive or therapeutic agent for cancer.

[24] The pharmaceutical agent described in

[21] or

[22] , which is an apoptosis-inducing drug.

[25] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into ex vivo immune cells and expressing the same, comprising adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells.

[26] A method for expressing a chimeric antigen receptor or an exogenous T cell receptor in ex vivo T cells, comprising adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells.

[27] A method for preventing or treating cancer, comprising administering to a mammal ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor, the ex vivo immune cells being obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells.

[28] A method for preventing or treating cancer, comprising administering to a mammal ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells.

[29] Ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells, for use in the prevention and treatment of cancer.

[30] Ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells, for use in the prevention and treatment of cancer.

[31] Use of ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells, for the production of a cancer preventive or therapeutic agent.

[32] Use of ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells, for the production of a cancer preventive or therapeutic agent.

[33] A method for producing a pharmaceutical containing ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor, comprising the step of adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo immune cells.

[34] A method for producing a pharmaceutical containing ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor, comprising the step of adding the lipid nanoparticles described in any one of [1] to [8] to a culture medium containing ex vivo T cells. Effect of the Invention

[0012] According to the present invention, since CAR or exogenous TCR can be efficiently introduced into immune cells such as T cells selectively not only ex vivo but also in vivo, it is possible to provide a CAR- or TCR-immune cell therapy with low production costs. In addition, since no viral vector is used, the problem of antigenicity caused by viral proteins can be avoided. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the CD19 CAR expression analysis by flow cytometry of human primary T cells transfected with hCD3 / hCD28-compound 12-pcDNA3.1-hCD19CAR. [Diagram 2]FIG. 2 shows the CD19 CAR expression analysis by flow cytometry of human primary T cells transfected with hCD3 / hCD28-compound 21-pcDNA3.1-hCD19CAR and hCD3 / hCD28-compound 35-pcDNA3.1-hCD19CAR. [Diagram 3] FIG. 3 shows the cytotoxicity rates of Nalm-6 and Daudi upon addition of CD19 CAR-transfected human primary T cells.

[0014] Detailed Description of the Invention 1. Lipid Nanoparticles (LNPs) of the Present Invention The present invention relates to the following (a) to (c): (a) a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR); (b) a cationic lipid; and (c) Non-cationic lipids The present invention provides a lipid nanoparticle comprising the above (hereinafter also referred to as "lipid nanoparticle of the present invention" or "LNP of the present invention"). As used herein, the term "lipid nanoparticle (LNP)" refers to a particle having an average diameter of less than 1 μm that does not have a small pore structure (e.g., mesoporous material) inside the molecular assembly formed by the above (b) and (c). The components (a) to (c) of the lipid nanoparticle of the present invention will be described below.

[0015] (a) a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR) (a-1) Nucleic acid encoding CAR CAR is an artificially constructed hybrid protein that contains an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. The characteristics of CAR include the ability to utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor escape. Moreover, when expressed in T cells, CAR advantageously does not dimerize with endogenous TCR α and β chains.

[0016] The CAR used in the lipid nanoparticles of the present invention comprises an antigen-binding domain of an antibody capable of specifically recognizing a surface antigen (e.g., a cancer antigen peptide, a surface receptor whose expression is enhanced in cancer cells, etc.) to be recognized by a target immune cell (e.g., a T cell, a NK cell, a NKT cell, a monocyte, a macrophage, a dendritic cell, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0017] Examples of surface antigens that the antigen-binding domain specifically recognizes include various cancers (e.g., acute lymphocytic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous ... leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.) surface receptors with increased expression, such as CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell Examples of antigens include, but are not limited to, cancer antigen peptides (e.g., peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.), and the like.

[0018] The antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment capable of specifically recognizing a target antigen, but considering the ease of CAR production, it is preferable to use a single-chain antibody (scFv) in which a light chain variable region and a heavy chain variable region are linked via a linker peptide. The arrangement of the light chain variable region and the heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstitute a functional antigen-binding domain, but can usually be designed in the order of light chain variable region-linker peptide-heavy chain variable region from the N-terminus. As the linker peptide, a publicly known linker peptide that is usually used in the production of single-chain antibodies can be used. The DNA encoding the light chain variable region and the DNA encoding the heavy chain variable region can be prepared, for example, by cloning the light chain gene and the heavy chain gene from an antibody-producing cell, respectively, and performing PCR using them as a template, or can be chemically synthesized from the sequence information of an existing antibody. The DNA encoding the single-chain antibody can be obtained by ligating each of the obtained DNA fragments and the DNA encoding the linker peptide by an appropriate method. It is preferable that a leader sequence is further added to the N-terminus of the antigen-binding domain in order to present the CAR on the surface of immune cells.

[0019] As the extracellular hinge domain and transmembrane domain, any domain derived from a T cell surface molecule commonly used in the art can be used as appropriate, including, but not limited to, domains derived from CD8α and CD28.

[0020] Examples of intracellular signaling domains include those having a CD3ζ chain, those having an additional costimulatory signaling motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, or 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more costimulatory signaling motifs, but are not limited to these, and any domain commonly used in the art can be used in combination.

[0021] Nucleic acid sequence information encoding the extracellular hinge domain, transmembrane domain, and intracellular signaling domain is well known in the art, and a person skilled in the art can easily obtain DNA fragments encoding each domain from T cells based on that information. The DNA fragments thus obtained, each encoding the antigen-binding domain, the extracellular hinge domain, the transmembrane domain, and the intracellular signaling domain, can be ligated by standard methods to obtain DNA encoding a CAR.

[0022] The obtained DNA encoding CAR can be inserted into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells, either as is or after adding an appropriate linker and / or nuclear localization signal, etc. Examples of promoters functional in T cells include, but are not limited to, constitutive SRα promoters in mammalian cells, SV40 promoters, LTR promoters, CMV (cytomegalovirus) promoters, RSV (Rous sarcoma virus) promoters, MoMuLV (Moloney murine leukemia virus) LTRs, and HSV-TK (herpes simplex virus thymidine kinase) promoters. Gene promoters such as CD3, CD4, and CD8 that are specifically expressed in T cells can also be used.

[0023] RNA encoding a CAR, preferably mRNA, can be prepared by transcribing an expression vector containing DNA encoding the CAR as a template into mRNA in an in vitro transcription system known per se.

[0024] (a-2) Nucleic acid encoding an exogenous TCR As used herein, the term "T cell receptor (TCR)" refers to a receptor that is composed of a dimer of TCR chains (α chain, β chain) and recognizes an antigen or the antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complex to transmit a stimulatory signal to a T cell. Each TCR chain is composed of a variable region and a constant region, and the variable region has three complementarity determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only TCRs in which the α chain and β chain of TCR constitute a heterodimer, but also TCRs in which the α chain and β chain constitute a homodimer. Furthermore, the TCR also includes TCRs in which a part or all of the constant region is deleted, TCRs in which the amino acid sequence is modified, and TCRs in which the constant region is soluble. In addition, "exogenous TCR" means exogenous to T cells, which are target cells of the lipid nanoparticles of the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR expressed by the T cells, which are target cells of the lipid nanoparticles of the present invention.

[0025] The nucleic acid encoding the TCR used in the lipid nanoparticles of the present invention is a nucleic acid encoding the α and β chains of a TCR that can specifically recognize the surface antigen (e.g., a cancer antigen peptide, etc.) that the target T cell should recognize. The nucleic acid can be prepared by a method known per se. When the amino acid sequence or nucleic acid sequence of the target TCR is known, it is possible to construct a DNA encoding the full length or a part of the TCR of the present invention based on the sequence, for example, by chemically synthesizing a DNA strand or an RNA strand, or by connecting synthesized short oligo-DNA strands that overlap each other using the PCR method or the Gibson Assembly method.

[0026] When the sequence of the TCR of interest is not known, for example, the T cells of interest can be isolated from a cell population containing T cells expressing the TCR of interest, and a nucleic acid encoding the TCR can be obtained from the T cells. Specifically, a cell population (e.g., PBMC) containing T cells is collected from a living body (e.g., human), and the cell population is cultured while being stimulated in the presence of an epitope of a cell surface antigen recognized by the TCR of interest. From the cell population, T cells that specifically recognize cells expressing the cell surface antigen can be selected by a known method using the specificity for cells expressing the cell surface antigen and cell surface antigens such as CD8 and CD4 as indicators. The specificity of T cells for cells expressing the surface antigen can be measured, for example, using a dextromer assay, an ELISPOT assay, or a cytotoxicity assay. The cell population containing the T cells is preferably collected, for example, from a living body having a large number of cells expressing a cell surface antigen recognized by the target TCR (e.g., a patient with a disease such as cancer, or a T cell-containing population contacted with an epitope of the antigen or dendritic cells pulsed with the epitope).

[0027] The nucleic acid of the present invention can be obtained by extracting DNA from the isolated T cells by a conventional method, amplifying the TCR gene based on the nucleic acid sequence of the constant region of the TCR using the DNA as a template, and cloning it. Alternatively, RNA can be extracted from the cells by a conventional method, synthesizing cDNA, and using the cDNA as a template to perform 5'-RACE (Rapid amplification of cDNA ends) using antisense primers complementary to the nucleic acids encoding the constant regions of the TCR α chain and β chain, respectively. 5'-RACE may be performed by a known method, for example, using a commercially available kit such as SMART PCR cDNA Synthesis Kit (manufactured by Clontech). The obtained DNAs encoding the α chain and β chain of TCR can be inserted into an appropriate expression vector, similar to the DNA encoding the CAR. The DNA encoding the α chain and the DNA encoding the β chain may be inserted into the same vector or into separate vectors. When inserted into the same vector, the expression vector may express both chains polycistronically or monocistronically. In the former case, an intervening sequence allowing polycistronic expression, such as an IRES or FMV 2A, is inserted between the DNA encoding both strands. Furthermore, RNA, preferably mRNA, encoding each chain of the TCR can be prepared in the same manner as the RNA encoding the CAR, for example, using the expression vector as a template.

[0028] (b) Cationic lipids As used herein, "cationic lipid" refers to a lipid that carries a net positive charge at a selected pH, such as physiological pH. The cationic lipid used in the lipid nanoparticles of the present invention is not particularly limited, and examples thereof include cationic lipids described in WO 2015 / 011633, WO 2016 / 021683, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US 2015 / 0239834, and the like.

[0029] Preferred examples of the cationic lipid include those represented by the following structural formula described in WO 2015 / 011633.

[0030] [ka]

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] and their salts.

[0039] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0040] [ka]

[0041] and their salts.

[0042] Preferred examples of the cationic lipid include those represented by the following structural formula described in WO 2016 / 021683.

[0043] [ka]

[0044] [In the formula, W is a group of formula -NR 1 R 2 or formula -N + R 3 R 4 R 5 (Z - )of, R 1 and R 2 are each independently 1-4 An alkyl group or a hydrogen atom, R 3 , R 4 and R 5 are each independently 1-4 The alkyl group, Z -represents an anion, X is an optionally substituted C 1-6 The alkylene group, Y A , Y B and Y C each independently represents an optionally substituted methine group, L A , L B and L C each independently represents an optionally substituted methylene group or a bond, R A1 , R A2 , R B1 , R B2 , R C1 and R C2 each independently represents an optionally substituted C 4-10 represents an alkyl group. A compound represented by the formula:

[0045] More preferred examples include cationic lipids represented by the following structural formula:

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] [ka]

[0050] [ka]

[0051] [ka]

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] and their salts.

[0057] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] and their salts.

[0062] In another preferred embodiment, the cationic lipid represented by the following formula (II) (hereinafter, also referred to as "compound (II)") is mentioned.

[0063] [ka]

[0064] [In the formula, n is an integer from 2 to 5, R is a linear C 1-5 Alkyl group, linear C 7-11 Alkenyl group or linear C 11 The alkadienyl group is The wavy lines each independently represent cis or trans bonds. show.] A compound represented by the formula:

[0065] More preferred examples include cationic lipids represented by the following structural formula:

[0066] [ka]

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] [ka]

[0076] [ka]

[0077] and their salts.

[0078] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0079] [ka]

[0080] [ka]

[0081] [ka]

[0082] and their salts.

[0083] Compound (II) can be produced, for example, by the following production method. Compound (II) in which both wavy lines are cis bonds and compounds in which one or both wavy lines are trans bonds can be produced by the same production method as the production method shown below. In particular, in the esterification, it is possible to synthesize compound (II) of the desired structure by using an appropriate raw material according to the structure of the target compound (II). In addition, a salt of compound (II) can be obtained by appropriate mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.

[0084] [ka]

[0085] The raw materials and reagents used in each step of the above-mentioned production methods, as well as the resulting compounds, may each form a salt.

[0086] When the compound obtained in each step is a free compound, it can be converted to the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted to the free form or another type of desired salt by a known method.

[0087] The compound obtained in each step can be used in the next reaction either as a reaction solution or as a crude product, or the compound obtained in each step can be isolated and / or purified from the reaction mixture by a separation means such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractional distillation, chromatography, etc. in accordance with a conventional method.

[0088] When the raw materials and reagent compounds for each step are commercially available, the commercially available products can be used as they are.

[0089] In the reaction of each step, the reaction time may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 10 minutes to 8 hours.

[0090] In the reactions of each step, the reaction temperature may vary depending on the reagents and solvents used, but is usually from -78°C to 300°C, preferably from -78°C to 150°C, unless otherwise specified.

[0091] In the reaction of each step, the pressure may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 atm to 20 atm, preferably 1 atm to 3 atm.

[0092] In the reactions of each step, a microwave synthesis device such as Biotage Initiator may be used. The reaction temperature may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually room temperature to 300° C., preferably room temperature to 250° C., and more preferably 50° C. to 250° C. The reaction time may vary depending on the reagents and solvent used, but unless otherwise specified, it is usually 1 minute to 48 hours, and preferably 1 minute to 8 hours.

[0093] In the reaction of each step, unless otherwise specified, the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, relative to the substrate. When the reagent is used as a catalyst, the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents, relative to the substrate. When the reagent also serves as a reaction solvent, the reagent is used in an amount equivalent to the solvent.

[0094] Unless otherwise specified, the reactions in each step are carried out without a solvent or by dissolving or suspending the compound in a suitable solvent. Specific examples of the solvent include the following.

[0095] Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butyl alcohol, 2-methoxyethanol, etc.; Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, etc.; Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.; Saturated hydrocarbons: cyclohexane, hexane, heptane, etc.; Amides: N,N-dimethylformamide, N-methylpyrrolidone, etc.; Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.; Nitriles: acetonitrile, etc.; Sulfoxides: dimethyl sulfoxide, etc.; Aromatic organic bases: pyridine, etc.; Acid anhydrides: acetic anhydride, etc.; Organic acids: formic acid, acetic acid, trifluoroacetic acid, etc.; Inorganic acids: hydrochloric acid, sulfuric acid, etc.; Esters: ethyl acetate, isopropyl acetate, etc.; Ketones: acetone, methyl ethyl ketone, etc.; water. The above solvents may be used as a mixture of two or more kinds in an appropriate ratio.

[0096] When a base is used in the reaction of each step, for example, the base shown below is used.

[0097] Inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.; Basic salts: sodium carbonate, calcium carbonate, sodium bicarbonate, etc.; Organic bases: triethylamine, diethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, imidazole, piperidine, etc.; Metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, etc.; Alkali metal hydrides: sodium hydride, etc.; Metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, etc.; Organolithium compounds: n-butyllithium, sec-butyllithium, etc.

[0098] When an acid or an acid catalyst is used in the reaction of each step, for example, the following acids and acid catalysts are used.

[0099] Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.; Organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.; Lewis acids: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous ferric chloride, etc.

[0100] Unless otherwise specified, the reactions in each step are carried out by known methods, for example, in Experimental Chemistry Lectures, 5th Edition, Vol. 13-19 (edited by the Chemical Society of Japan); New Experimental Chemistry Lectures, Vol. 14-15 (edited by the Chemical Society of Japan); Precision Organic Chemistry, Revised 2nd Edition (LF Tietze, Th. Eicher, Nankodo); Revised Organic Named Reactions: Their Mechanisms and Key Points (by Hideo Togo, Kodansha); ORGANIC SYNTHESES Collective Volumes I-VII (John Wiley & Sons Inc); Modern Organic Synthesis in the Laboratory A Collection of Standard Experimental Procedures (by Jie Jack Li, published by OXFORD UNIVERSITY); Comprehensive Heterocyclic Chemistry III, Vol. 1-14 (Elsevier Japan Co., Ltd.); Organic Synthesis Strategies Learned from Named Reactions (translated and supervised by Kiyoshi Tomioka, published by Kagaku Dojin); Comprehensive Organic Transformations (VCH Publishers The method is carried out in accordance with the method described in the 1989 issue of the National Institute of Standards and Technology (NIS).

[0101] In each step, the protection or deprotection reaction of a functional group is carried out in accordance with a known method, for example, the method described in "Protective Groups in Organic Synthesis, 4th Ed." (Theodora W. Greene, Peter GM Wuts), published by Wiley-Interscience in 2007; "Protecting Groups 3rd Ed." (PJ Kocienski), published by Thieme in 2004.

[0102] Examples of the protecting group for the hydroxyl group of an alcohol or the like or a phenolic hydroxyl group include ether-type protecting groups such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylate-type protecting groups such as acetate; sulfonate-type protecting groups such as methanesulfonate; and carbonate-type protecting groups such as t-butyl carbonate.

[0103] Examples of the protecting group for the carbonyl group of an aldehyde include acetal-type protecting groups such as dimethyl acetal; and cyclic acetal-type protecting groups such as cyclic 1,3-dioxane.

[0104] Examples of protecting groups for the carbonyl group of a ketone 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.

[0105] Examples of the carboxyl-protecting group include ester-type protecting groups such as methyl ester; and amide-type protecting groups such as N,N-dimethylamide.

[0106] Examples of the thiol-protecting group include ether-type protecting groups such as benzylthioether; and ester-type protecting groups such as thioacetate, thiocarbonate, and thiocarbamate.

[0107] Examples of the protecting group for an amino group or an aromatic heterocycle such as imidazole, pyrrole, or indole include carbamate-type protecting groups such as benzylcarbamate; amide-type protecting groups such as acetamide; alkylamine-type protecting groups such as N-triphenylmethylamine; and sulfonamide-type protecting groups such as methanesulfonamide.

[0108] The protecting group can be removed by a known method, for example, a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, a trialkylsilyl halide (e.g., trimethylsilyl iodide, trimethylsilyl bromide), or a reduction method.

[0109] In each step, when a reduction reaction is carried out, examples of the reducing agent used include metal hydrides such as lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), sodium borohydride, and tetramethylammonium triacetoxyborohydride; boranes such as borane tetrahydrofuran complex; Raney nickel; Raney cobalt; hydrogen; and formic acid. For example, Raney nickel or Raney cobalt can be used in the presence of hydrogen or formic acid. When a carbon-carbon double bond or triple bond is reduced, a catalyst such as palladium-carbon or Lindlar catalyst can be used.

[0110] In each step, when an oxidation reaction is carried out, examples of the oxidizing agent used include peracids such as m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, and t-butyl hydroperoxide; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; chlorites such as sodium chlorite; periodates such as sodium periodate; high-valent iodine reagents such as iodosylbenzene; manganese-containing reagents such as manganese dioxide and potassium permanganate; lead compounds such as lead tetraacetate; chromium-containing reagents such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), and Jones reagent; halogen compounds such as N-bromosuccinimide (NBS); oxygen; ozone; sulfur trioxide-pyridine complex; osmium tetroxide; zerene dioxide; and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).

[0111] In each step, when a radical cyclization reaction is carried out, examples of the radical initiator used include azo compounds such as azobisisobutyronitrile (AIBN), water-soluble radical initiators such as 4-4'-azobis-4-cyanopentanoic acid (ACPA), triethylboron in the presence of air or oxygen, benzoyl peroxide, etc. Examples of the radical reaction reagent used include tributylstannane, tristrimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, samarium iodide, etc.

[0112] When a Wittig reaction is carried out in each step, examples of the Wittig reagent used include alkylidene phosphoranes, etc. The alkylidene phosphoranes can be prepared by a known method, for example, by reacting a phosphonium salt with a strong base.

[0113] When the Horner-Emmons reaction is carried out in each step, examples of the reagent to be used include phosphonoacetates such as methyl dimethylphosphonoacetate, ethyl diethylphosphonoacetate and the like; and bases such as alkali metal hydrides, organolithium compounds and the like.

[0114] In each step, when the Friedel-Crafts reaction is carried out, the reagents used include a Lewis acid and an acid chloride or an alkylating agent (e.g., alkyl halides, alcohols, olefins, etc.). Alternatively, an organic acid or an inorganic acid can be used instead of the Lewis acid, and an acid anhydride such as acetic anhydride can be used instead of the acid chloride.

[0115] In each step, when an aromatic nucleophilic substitution reaction is carried out, a nucleophile (eg, amines, imidazole, etc.) and a base (eg, basic salts, organic bases, etc.) are used as reagents.

[0116] In each step, when a nucleophilic addition reaction by a carbanion, a nucleophilic 1,4-addition reaction (Michael addition reaction) by a carbanion, or a nucleophilic substitution reaction by a carbanion is performed, examples of the base used to generate the carbanion include organolithiums, metal alkoxides, inorganic bases, and organic bases.

[0117] When a Grignard reaction is carried out in each step, examples of the Grignard reagent include aryl magnesium halides such as phenyl magnesium bromide, etc., and alkyl magnesium halides such as methyl magnesium bromide, isopropyl magnesium bromide, etc. The Grignard reagent can be prepared by a known method, for example, by reacting an alkyl halide or aryl halide with metallic magnesium using ether or tetrahydrofuran as a solvent.

[0118] In each step, when a Knoevenagel condensation reaction is carried out, an active methylene compound sandwiched between two electron-withdrawing groups (e.g., malonic acid, diethyl malonate, malononitrile, etc.) and a base (e.g., organic bases, metal alkoxides, inorganic bases) are used as reagents.

[0119] In each step, when the Vilsmeier-Haack reaction is carried out, phosphoryl chloride and an amide derivative (eg, N,N-dimethylformamide, etc.) are used as reagents.

[0120] In each step, when an azidation reaction of alcohols, alkyl halides, or sulfonate esters is carried out, examples of the azidation agent used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide, sodium azide, etc. For example, when azidating alcohols, there are a method using diphenylphosphoryl azide and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) and a method using trimethylsilyl azide and a Lewis acid.

[0121] In each step, when a reductive amination reaction is carried out, examples of the reducing agent used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, formic acid, etc. When the substrate is an amine compound, examples of the carbonyl compound used include paraformaldehyde, aldehydes such as acetaldehyde, and ketones such as cyclohexanone. When the substrate is a carbonyl compound, examples of the amines used include ammonia, primary amines such as methylamine, and secondary amines such as dimethylamine.

[0122] In each step, when Mitsunobu reaction is carried out, an azodicarboxylate (eg, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.) and triphenylphosphine are used as reagents.

[0123] In the case where an esterification reaction, an amidation reaction, or a urea reaction is carried out in each step, examples of the reagent to be used include acyl halides such as acid chlorides and acid bromides; and activated carboxylic acids such as acid anhydrides, active esters, and sulfates. Examples of activating agents for carboxylic acids include carbodiimide-based condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD); triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM); carbonate-based condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenylphosphoric acid azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent); thionyl chloride; lower alkyl haloformates such as ethyl chloroformate; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); sulfuric acid; or combinations thereof. When a carbodiimide-based condensing agent is used, an additive such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), or dimethylaminopyridine (DMAP) may be further added to the reaction.

[0124] In each step, when a coupling reaction is carried out, examples of the metal catalyst 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)ferrocenepalladium (II) chloride, and palladium acetate (II); nickel compounds such as tetrakis(triphenylphosphine)nickel (0); rhodium compounds such as tris(triphenylphosphine)rhodium (III) chloride; cobalt compounds; copper compounds such as copper oxide and copper iodide (I); and platinum compounds. A base may also be added to the reaction, and examples of such bases include inorganic bases and basic salts.

[0125] When a thiocarbonylation reaction is carried out in each step, diphosphorus pentasulfide is typically used as the thiocarbonylating agent. In addition to diphosphorus pentasulfide, a reagent having a 1,3,2,4-dithiadiphosphetane-2,4-disulfide structure, such as 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lowesson's reagent), may also be used.

[0126] In each step, when the Wohl-Ziegler reaction is carried out, examples of the halogenating agent used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, sulfuryl chloride, etc. Furthermore, the reaction can be accelerated by adding a radical initiator such as heat, light, benzoyl peroxide, or azobisisobutyronitrile to the reaction.

[0127] In each step, when a halogenation reaction of a hydroxy group is carried out, examples of the halogenating agent used include hydrohalic acid and an acid halide of an inorganic acid, specifically, for chlorination, hydrochloric acid, thionyl chloride, phosphorus oxychloride, etc., and for bromination, 48% hydrobromic acid, etc. may be mentioned. In addition, a method of obtaining an alkyl halide from an alcohol by the reaction of triphenylphosphine with carbon tetrachloride or carbon tetrabromide, etc. may be used. Alternatively, a method of synthesizing an alkyl halide through a two-step reaction in which an alcohol is converted into a sulfonic acid ester and then reacted with lithium bromide, lithium chloride, or sodium iodide may be used.

[0128] When Arbuzov reaction is carried out in each step, examples of the reagent used include alkyl halides such as ethyl bromoacetate and the like; and phosphites such as triethyl phosphite, tri(isopropyl) phosphite and the like.

[0129] When a sulfone esterification reaction is carried out in each step, examples of the sulfonating agent used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride, and the like.

[0130] In each step, when hydrolysis is performed, an acid or base is used as a reagent. When acid hydrolysis of t-butyl ester is performed, formic acid or triethylsilane may be added to reductively trap the by-product t-butyl cation.

[0131] When a dehydration reaction is carried out in each step, examples of the dehydrating agent to be used include sulfuric acid, diphosphorus pentoxide, phosphorus oxychloride, N,N'-dicyclohexylcarbodiimide, alumina, polyphosphoric acid and the like.

[0132] In another preferred embodiment, the cationic lipid represented by the following formula (III) (hereinafter, also referred to as "compound (III)") is mentioned.

[0133] [ka]

[0134] [In the formula, n1 is an integer from 2 to 6, n2 is an integer from 0 to 2, n3 is an integer between 0 and 2, L is -C(O)O- or -NHC(O)O-; Ra is a linear C 5-13 Alkyl group, linear C 13-17 Alkenyl group or linear C 17 The alkadienyl group is Rb is a linear C 2-9 The alkyl group, Rc is a hydrogen atom or a linear C 2-9 The alkyl group, Rd is a hydrogen atom or a linear C 2-9 The alkyl group, Re is a linear C 2-9 The alkyl group, Rf is linear C 2-9 represents an alkyl group. A compound represented by the formula:

[0135] More preferred examples include cationic lipids represented by the following structural formula:

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140]

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[0141]

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[0142]

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[0143]

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[0144]

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[0145]

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[0146]

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[0147]

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[0148]

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[0149]

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[0150] [ka]

[0151] [ka]

[0152] [ka]

[0153] [ka]

[0154] and their salts.

[0155] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0156] [ka]

[0157] [ka]

[0158] and their salts.

[0159] Compound (III) can be produced, for example, by the following process. In particular, during esterification, it is possible to synthesize compound (I) of a desired structure by using an appropriate raw material according to the structure of the target compound (III). In addition, a salt of compound (III) can be obtained by appropriately mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.

[0160] [ka]

[0161] [ka]

[0162] [ka]

[0163] [ka]

[0164] The raw materials and reagents used in the reactions in each step in the above-mentioned production method, as well as the reaction conditions, may be the same as those described above in the production method of compound (II).

[0165] In another embodiment, the cationic lipid may be represented by the following structural formula described in WO 2011 / 153493.

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] [ka]

[0171]

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[0172]

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[0173]

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[0174]

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[0175]

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[0176]

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[0177]

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[0178]

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[0179]

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[0180]

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[0181]

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[0182]

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[0183]

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[0184]

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[0185]

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[0186]

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[0187]

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[0188]

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[0189]

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[0190]

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[0191]

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[0192]

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[0193]

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[0194]

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[0195]

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[0196]

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[0197]

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[0198]

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[0199]

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[0200]

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[0201]

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[0202]

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[0203]

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[0204]

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[0205]

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[0206]

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[0207]

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[0208]

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[0209]

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[0210]

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[0211]

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[0212]

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[0213]

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[0214] [ka]

[0215] [ka]

[0216] [ka]

[0217] [ka]

[0218] [ka]

[0219] and their salts.

[0220] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0221] [ka]

[0222] and their salts.

[0223] In another embodiment, the cationic lipid may be represented by the following structural formula described in WO 2013 / 126803.

[0224] [ka]

[0225] [ka]

[0226] [ka]

[0227] [ka]

[0228] [ka]

[0229] and their salts.

[0230] Of the above cationic lipids, more preferred are cationic lipids represented by the following structural formula:

[0231] [ka]

[0232] and its salts.

[0233] In another embodiment, the method of Dong et al. (Proc Natl Acad Sci US A. 2014 Apr 2014) 15;111(15):5753), cationic lipids 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 are synthesized according to the following scheme.

[0234] [ka]

[0235] Among the above cationic lipids, more preferred ones include cKK-E12 and cKK-E14.

[0236] In another embodiment, the cationic lipids C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96, and C12-200 are synthesized according to the following scheme described in Love KT et al. (Proc Natl Acad Sci US A. 2010 May 25; 107(21):9915).

[0237] [ka]

[0238] Among the above cationic lipids, more preferred ones include C14-110, C16-96 and C12-200.

[0239] In a particularly preferred embodiment, the cationic lipid represented by the following formula (I) (hereinafter, also referred to as "compound (I)") is mentioned.

[0240] [ka]

[0241] [In the formula, L 1 is C 1-22 Alkylene group, C 2-22 Alkenylene group or C 3-22 is an alkadienylene group, n is an integer of 0 or 1, R 1 teeth, (1) a hydrogen atom, (2) Linear C 1-22 Alkyl groups and linear C 2-22A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (3) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (4) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 is an alkadienyl group, R 2 is -CH2-O-CO-R 5 , -CH2-CO-OR 5 or -R 5 and R 3 is -CH2-O-CO-R 6 , -CH2-CO-OR 6 or -R 6 and R 4 is a hydrogen atom, -CH2-O-CO-R 7 , -CH2-CO-OR 7 or -R 7 and R 5 , R 6 and R 7 are each independently (1) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (2) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (3) Linear C 1-22 Alkyl groups and linear C 2-22A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 is an alkadienyl group, R8 and R9 are each independently C 1-6 indicates an alkyl group] Or its salt.

[0242] L 1 is C 1-22 Alkylene group, C 2-22 Alkenylene group or C 3-22 It is an alkadienylene group. L 1 is preferably C 1-22 It is an alkylene group. L 1 More preferably, C 1-12 It is an alkylene group. L 1 More preferably, C 1-6 It is an alkylene group.

[0243] n is an integer of 0 or 1. n is preferably an integer equal to 1.

[0244] R 1 teeth, (1) a hydrogen atom, (2) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (3) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (4) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 It is an alkadienyl group. R 1is preferably (1) a hydrogen atom, (2) One or two linear C 1-22 Alkyl groups (preferably linear C 6-12 A straight-chain C 1-22 Alkyl groups (preferably linear C 6-12 alkyl group), or (3) One or two linear C 2-22 Alkenyl groups (preferably linear C 6-12 Alkenyl group) 2-22 Alkenyl groups (preferably linear C 6-12 alkenyl group). R 1 is particularly preferably a hydrogen atom.

[0245] R 2 is -CH2-O-CO-R 5 , -CH2-CO-OR 5 or -R 5 It is. R 2 is preferably -CH2-O-CO-R 5 or -R 5 It is. R 2 More preferably, —CH—O—CO—R 5 It is.

[0246] R 3 is -CH2-O-CO-R 6 , -CH2-CO-OR 6 or -R 6 It is. R 3 is preferably -CH2-O-CO-R 6 or -R 6 It is. R 3 More preferably, —CH—O—CO—R 6 It is.

[0247] R 4 is a hydrogen atom, -CH2-O-CO-R 7, -CH2-CO-OR 7 or -R 7 It is. R 4 is preferably a hydrogen atom or -CH2-O-CO-R 7 It is. R 4 More preferably, —CH—O—CO—R 7 It is.

[0248] R 5 , R 6 and R 7 are each independently (1) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 1-22 Alkyl groups, (2) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 2-22 an alkenyl group, or (3) Linear C 1-22 Alkyl groups and linear C 2-22 A straight-chain C group optionally substituted with one or two substituents selected from an alkenyl group. 3-22 It is an alkadienyl group. R 5 , R 6 and R 7 are each independently preferably (1) One or two linear C 1-22 Alkyl groups (preferably linear C 1-10 A straight-chain C 1-22 Alkyl groups (preferably linear C 4-18 alkyl group), (2) Linear C 2-22 Alkenyl groups (preferably linear C 4-18 alkenyl group), or (3) Linear C 3-22 Alkadienyl groups (preferably linear C 4-18alkadienyl group). R 5 , R 6 and R 7 are each independently more preferably (1) One or two linear C 1-22 Alkyl groups (preferably linear C 1-10 A straight-chain C 1-22 Alkyl groups (preferably linear C 4-18 alkyl group), or (2) Linear C 2-22 Alkenyl groups (preferably linear C 4-18 alkenyl group).

[0249] R8 and R9 are each independently C 1-6 It is an alkyl group. R8 and R9 are each independently C 1-3 It is an alkyl group (preferably methyl).

[0250] Preferably, compound (I) is represented by the above formula (I): L 1 But, C 1-22 An alkylene group (preferably C 1-12 Alkylene group, more preferably C 1-6 alkylene group), n is an integer equal to 1; R 1 but, (1) a hydrogen atom, (2) One or two linear C 1-22 Alkyl groups (preferably linear C 6-12 A straight-chain C 1-22 Alkyl groups (preferably linear C 6-12 alkyl group), or (3) One or two linear C 2-22 Alkenyl groups (preferably linear C 6-12 Alkenyl group) 2-22 Alkenyl groups (preferably linear C 6-12 alkenyl group), R 2 But -CH2-O-CO-R 5 or -R 5 and R 3 But -CH2-O-CO-R 6 or -R 6 and R 4 is a hydrogen atom or -CH2-O-CO-R 7 and R 5 , R 6 and R 7 However, each independently, (1) One or two linear C 1-22 Alkyl groups (preferably linear C 1-10 A straight-chain C 1-22 Alkyl groups (preferably linear C 4-18 alkyl group), (2) Linear C 2-22 Alkenyl groups (preferably linear C 4-18 alkenyl group), or (3) Linear C 3-22 Alkadienyl groups (preferably linear C 4-18 alkadienyl group), and R8 and R9 are each independently C 1-6 Alkyl group (preferably C 1-3 alkyl group, particularly preferably methyl); It is a compound.

[0251] More preferably, compound (I) is represented by the above formula (I): L 1 But, C 1-12 An alkylene group (preferably C 1-6 alkylene group), n is an integer equal to 1; R 1 is a hydrogen atom, R 2 But -CH2-O-CO-R 5 and R 3 But -CH2-O-CO-R6 and R 4 But -CH2-O-CO-R 7 and R 5 , R 6 and R 7 However, each independently, (1) One or two linear C 1-22 Alkyl groups (preferably linear C 1-10 A straight-chain C 1-22 Alkyl groups (preferably linear C 4-18 alkyl group), (2) Linear C 2-22 Alkenyl groups (preferably linear C 4-18 alkenyl group), or (3) Linear C 3-22 Alkadienyl groups (preferably linear C 4-18 alkadienyl group), and R8 and R9 are each independently C 1-6 Alkyl group (preferably C 1-3 alkyl group, particularly preferably methyl); It is a compound.

[0252] More preferably, compound (I) is represented by the above formula (I): L 1 But, C 1-6 is an alkylene group, n is an integer equal to 1; R 1 is a hydrogen atom, R 2 But -CH2-O-CO-R 5 and R 3 But -CH2-O-CO-R 6 and R 4 But -CH2-O-CO-R 7 and R 5 , R 6 and R 7 However, each independently, (1) One or two linear C1-22 Alkyl groups (preferably linear C 1-10 A straight-chain C 1-22 Alkyl groups (preferably linear C 4-18 alkyl group), or (2) Linear C 2-22 Alkenyl groups (preferably linear C 4-18 alkenyl group) and R8 and R9 are each independently C 1-3 is an alkyl group (preferably methyl); It is a compound.

[0253] As the salt of the compound represented by each of the structural formulas above, a pharmacologically acceptable salt is preferable, and examples thereof include salts with inorganic bases (e.g., alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt, ammonium salt), salts with organic bases (e.g., trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tritromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N, salts with N-dibenzylethylenediamine), salts with inorganic acids (e.g., salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid), salts with organic acids (salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), salts with basic amino acids (salts with arginine, lysine, ornithine) or salts with acidic amino acids (salts with aspartic acid, glutamic acid).

[0254] The ratio (mol %) of cationic lipids to the total lipids present in the lipid nanoparticles of the present invention is, for example, about 10% to about 80%, preferably about 20% to about 70%, and more preferably about 40% to about 60%, but is not limited thereto. The above cationic lipids may be used alone or in combination of two or more. When a plurality of cationic lipids are used, it is preferable that the cationic lipids as a whole have the above ratio.

[0255] (c) Non-cationic lipids As used herein, "non-cationic lipid" refers to a lipid other than a cationic lipid, which does not have a net positive charge at a selected pH, such as physiological pH. Non-cationic lipids used in the lipid nanoparticles of the present invention include, for example, phospholipids, steroids, PEG lipids, etc.

[0256] The phospholipid is not particularly limited as long as it stably retains the nucleic acid and does not inhibit fusion with the cell membrane (plasma membrane and organelle membrane) in order to enhance the delivery of the nucleic acid encoding a CAR or an exogenous TCR into the target immune cell, and examples thereof include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinolenoylphosphatidylcholine.

[0257] Preferred phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), more preferably DOPC, DPPC, POPC, and DOPE.

[0258] The ratio (mol %) of phospholipids to all lipids present in the lipid nanoparticles of the present invention can be, for example, about 0% to about 90%, preferably about 5% to about 30%, and more preferably about 8% to about 15%. The above phospholipids may be used alone or in combination of two or more. When a plurality of phospholipids are used, it is preferable that the phospholipids as a whole have the above ratio.

[0259] Steroids include cholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, preferably cholesterol.

[0260] The ratio (mol %) of steroids to the total lipids present in the lipid nanoparticles of the present invention, if present, can be, for example, about 10% to about 60%, preferably about 12% to about 58%, and more preferably about 20% to about 55%. The above steroids may be used alone or in combination of two or more. When a plurality of steroids are used, it is preferable that the steroids as a whole have the above ratio.

[0261] As used herein, "PEG lipid" refers to any conjugate of polyethylene glycol (PEG) and a lipid. The PEG lipid is not particularly limited as long as it has the effect of suppressing aggregation of the lipid nanoparticles of the present invention, and examples thereof include PEG bound to dialkyloxypropyl (PEG-DAA), PEG bound to diacylglycerol (PEG-DAG) (e.g., SUNBRIGHT GM-020 (NOF Corp.)), PEG bound to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-Cer), PEG conjugated with cholesterol (PEG-cholesterol) or derivatives thereof, or mixtures thereof, mPEG2000-1,2-di-O-alkyl-sn3-carbomoyl glyceride (PEG-C-DOMG), 1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6-dioxaotanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG), and the like. Preferred PEG lipids include PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer, and mixtures thereof, more preferably PEG-DAA conjugates selected from the group consisting of PEG-didecyloxypropyl conjugates, PEG-dilauryloxypropyl conjugates, PEG-dimyristyloxypropyl conjugates, PEG-dipalmityloxypropyl conjugates, PEG-distearyloxypropyl conjugates, and mixtures thereof. In addition to a methoxy group, a maleimide group or an N-hydroxysuccinimidyl group can be used as the free end of PEG for binding a T cell targeting ligand, which will be described later. As a PEG lipid having a functional group for binding a T cell targeting ligand (sometimes referred to as a "terminally reactive PEG lipid" in this specification), for example, SUNBRIGHT DSPE-020MA or SUNBRIGHT DSPE-020MA (NOF Corp.) can be used.

[0262] The ratio (mol %) of PEG lipid to the total lipids present in the lipid nanoparticles of the present invention can be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, and more preferably about 0.7% to about 2%. The ratio (mol %) of the terminally reactive PEG lipid to the total PEG lipid may be, for example, about 10% to about 100%, preferably about 20% to about 100%, and more preferably about 30% to about 100%. The above PEG lipids may be used alone or in combination of two or more. When a plurality of PEG lipids are used, it is preferable that the PEG lipids as a whole have the above ratio.

[0263] The lipid nanoparticles of the present invention are used to introduce and express CAR or exogenous TCR into immune cells, particularly T cells responsible for cellular immunity among acquired immunity, NK cells, monocytes, macrophages, dendritic cells, etc., responsible for innate immunity, and NKT cells, which are T cells having the properties of NK cells. Therefore, the lipid nanoparticles of the present invention can further contain a ligand capable of targeting the lipid nanoparticles to immune cells, particularly T cells, in order to be efficiently delivered to target immune cells, particularly in vivo.

[0264] (d) Ligands capable of targeting lipid nanoparticles to T cells The ligand capable of targeting the lipid nanoparticles of the present invention to T cells is not particularly limited as long as it can specifically recognize a surface molecule that is specifically or highly expressed in T cells, but preferably contains one or more antigen-binding domains of antibodies against CD3, CD4, CD8 or CD28, and more preferably contains an antigen-binding domain of an anti-CD3 antibody and / or an anti-CD28 antibody. In addition, a preferred example, particularly for in vivo delivery to T cells, is one that contains only the antigen-binding domain of an anti-CD3 antibody. Here, the "antigen-binding domain" is synonymous with the antigen-binding domain constituting the CAR, but since CAR needs to be prepared as a nucleic acid that encodes it, there are restrictions, and single-chain antibodies are usually used in many cases, but since the antigen-binding domain as a T cell targeting ligand is contained in the lipid nanoparticles of the present invention in the form of a protein, not only single-chain antibodies but also any other antibody fragments such as, for example, complete antibody molecules, Fab, F(ab')2, Fab', Fv, reduced antibodies (rIgG), dsFv, sFv, diabodies, triabodies, etc. can be preferably used. In particular, for delivery to target immune cells in vivo, Fab' having no Fc portion can be preferably used. These antibody fragments can be prepared by treating a complete antibody (e.g., IgG) with a reducing agent (e.g., 2-mercaptoethanol, dithiothreitol) or a peptidase (e.g., papain, pepsin, ficin), or by genetic recombination techniques.

[0265] If the T cell targeting ligand is a complete antibody molecule, commercially available anti-CD3, CD4, CD8, CD28 antibodies, etc. can be used, or the ligand can be isolated from the culture medium of cells producing the antibody. On the other hand, if the ligand is any of the above antigen-binding domains (antibody fragments), a nucleic acid encoding the antigen-binding domain of an anti-CD3, CD4, CD8, CD28 antibody, etc. can be isolated by a method similar to that for obtaining a nucleic acid encoding the antigen-binding domain constituting the CAR, and the antigen-binding domain can be recombinantly produced using the isolated nucleic acid.

[0266] In the lipid nanoparticle of the present invention, the T cell targeting ligand may be bound to the outer shell in any manner as long as it is present on the surface of the lipid nanoparticle, but for example, when the non-cationic lipid contains a terminal reactive PEG lipid, it can be added to the end of PEG.For example, the lipid nanoparticle labeled with a ligand (antibody) (sometimes referred to as "antibody-LNP") can be prepared by reacting the PEG lipid (e.g., SUNBRIGHT DSPE-020MA) with a maleimide group at the end with the thiol group of the above-mentioned reduced antibody.

[0267] When the lipid nanoparticles of the present invention are used for gene transfer to immune cells other than T cells, such as NK cells and dendritic cells, the lipid nanoparticles can be delivered efficiently even if they do not have a ligand for targeting those immune cells on the surface of the lipid nanoparticles, but the lipid nanoparticles may have a suitable targeting ligand for a molecule expressed on the surface of each immune cell. For example, in the case of NK cells, those containing the antigen-binding domain of antibodies against CD16 and CD56, etc., are included, but are not limited thereto.

[0268] 2. Production of lipid nanoparticles of the present invention The lipid nanoparticles of the present invention can be produced, for example, by using the method described in US9,404,127. When the lipid nanoparticles further contain a T cell targeting ligand, they can be produced by chemically binding the T cell targeting ligand after the lipid nanoparticles are produced. Alternatively, as described in WO2016 / 021683, for example, an organic solvent solution in which the above components (b) and (c) are dissolved is prepared, and the lipid nanoparticles are produced by mixing with water or a buffer solution in which (a) is dissolved, and then the T cell targeting ligand is chemically bound to the lipid nanoparticles. Examples of the mixture ratio (molar ratio) of the cationic lipid, phospholipid, cholesterol, and PEG lipid include, but are not limited to, 40 to 60: 0 to 20: 0 to 50: 0 to 5. In addition, when a PEG lipid is mixed as a non-cationic lipid and a T cell targeting ligand is added to the end of PEG, the mixture ratio (molar ratio) of the PEG lipid and the ligand can be, for example, 20: 1 to 1: 20. The PEG lipid may contain about 10% to about 100% terminal reactive PEG as a ratio (mol%). The mixing may be performed using a pipette or a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassembblr (Precision Nanosystems)). The obtained lipid particles may be purified by gel filtration, or subjected to dialysis and sterile filtration. The concentration of all lipid components in the organic solvent solution is preferably 0.5 to 100 mg / mL.

[0269] Examples of the organic solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and mixtures thereof. The organic solvent may contain 0 to 20% water or a buffer solution. Examples of the buffer solution include acidic buffer solutions (e.g., acetate buffer solutions, citrate buffer solutions) and neutral buffer solutions (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solutions, tris(hydroxymethyl)aminomethane (Tris) buffer solutions, phosphate buffer solutions, and phosphate buffered saline (PBS)).

[0270] When mixing is performed using a microfluidic mixing system, it is preferable to mix 1 to 5 parts by volume of water or buffer solution with 1 part by volume of organic solvent solution. In the system, the flow rate of the mixture (mixture of organic solvent solution and water or buffer solution) is preferably 0.1 to 10 mL / min, and the temperature is preferably 4 to 45°C.

[0271] When preparing a lipid particle dispersion as described above, a nucleic acid encoding CAR or exogenous TCR may be added to water or a buffer solution to prepare a dispersion containing components (a) to (d). The nucleic acid is preferably added so that the concentration in water or a buffer solution is 0.05 to 2.0 mg / mL. The lipid nanoparticles of the present invention can also be produced by mixing a lipid particle dispersion liquid and the nucleic acid by a method known per se. The content of the nucleic acid in the lipid nanoparticles of the present invention is preferably 1 to 20% by weight. TM It can be measured using Ribogreen (registered trademark) (Invitrogen). The encapsulation rate of the nucleic acid in the lipid nanoparticles of the present invention can be calculated based on the difference in fluorescence intensity between the presence and absence of the addition of a surfactant (e.g., Triton-X100).

[0272] The dispersion medium can be replaced with water or a buffer solution by dialysis. Dialysis is carried out at 4°C to room temperature using an ultrafiltration membrane with a molecular weight cutoff of 10 to 20K. Dialysis may be repeated. Tangential flow filtration may also be used for dialysis.

[0273] In the lipid nanoparticles of the present invention obtained as described above, the ratio (weight ratio) of nucleic acid to lipid is about 0.01 to about 0.2.

[0274] 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, for example, by performing cumulant analysis of the autocorrelation function using a Zetasizer Nano ZS (Malvern Instruments).

[0275] 3. Ex vivo immune cells transfected with lipid nanoparticles of the present invention The present invention provides a method for producing ex vivo immune cells expressing a CAR or an exogenous TCR by contacting the lipid nanoparticles of the present invention with immune cells collected from a living body (also referred to as "ex vivo immune cells" in this specification) and introducing a nucleic acid encoding a CAR or an exogenous TCR into the T cells, and an ex vivo immune cell obtained by the method. Here, the "immune cells" are not particularly limited as long as they are cells (so-called immune effector cells) that have the ability to damage target cells (pathogenic cells) such as cancer cells by some mechanism of action, and examples of such cells include T cells that are responsible for cellular immunity in acquired immunity, NK cells, monocytes, macrophages, dendritic cells, etc. that are responsible for natural immunity, and NKT cells, which are T cells having the properties of NK cells. In a preferred embodiment, the immune cells may be T cells. T cells collected from a living body are also referred to as "ex vivo T cells" in this specification. On the other hand, in another preferred embodiment, the immune cells may be cells that are responsible for natural immunity, such as NK cells, macrophages, and dendritic cells. Although T cells have a considerable risk of causing GVHD through allogeneic (allo) transplantation even when the HLA type is matched, allogeneic NK cells and the like are thought not to cause GVHD. Therefore, if allogeneic ex vivo immune cells of various HLA types are prepared, they can be used off-the-shelf. CAR-NK cells are described, for example, in US2016 / 0096892, Mol Ther. 25(8): 1769-1781 (2017), and CAR-dendritic cells, CAR-macrophages, and the like are described, for example, in WO2017 / 019848, eLIFE. 2018 e36688, and the like. In another aspect, the present invention provides a composition for inducing expression of a CAR or exogenous TCR, comprising the lipid nanoparticle of the present invention.

[0276] The immune cells (e.g., T cells) to which the lipid nanoparticles of the present invention are introduced may be isolated specific immune cells (e.g., T cells) or heterogeneous cell populations such as lymphocytes and lymphocyte precursors including pluripotent cells, so long as they are immune cells (e.g., T cells) or cell populations containing precursors thereof. In the present invention, "lymphocytes" refers to one of the subtypes of white blood cells in the immune system of vertebrates, and examples of lymphocytes include T cells, B cells, and natural killer cells (NK cells). Preferably, the immune cells are isolated and purified T cells. In the present invention, "T cells" refers to a type of white blood cell found in lymphoid organs or peripheral blood, and is a classification of lymphocytes characterized by being differentiated and matured mainly in the thymus and expressing TCR. Examples of T cells that can be used in the present invention include cytotoxic T cells (CTLs) that are CD8 positive cells, helper T cells, regulatory T cells, and effector T cells that are CD4 positive cells, and preferably cytotoxic T cells.

[0277] The lymphocytes can be collected from, for example, peripheral blood, bone marrow, and umbilical cord blood of humans or non-human mammals. When ex vivo immune cells (e.g., ex vivo T cells) to which the lipid nanoparticles of the present invention have been introduced are used for the treatment of diseases such as cancer, the cell population is preferably collected from the subject of treatment or a donor whose HLA type matches that of the subject of treatment.

[0278] Examples of lymphocyte precursor cells including pluripotent cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), hematopoietic stem cells, multipotent progenitors (MMPs) that have lost the ability to self-replicate, myelo-lymphoid common progenitors (MLPs), myeloid precursor cells (MPs), granulocyte mononuclear precursor cells (GMPs), macrophage-dendritic cell precursor cells (MDPs), dendritic cell precursor cells (DCPs), etc. Undifferentiated cells such as pluripotent cells can be differentiated into various immune cells, for example, T cells, by a method known per se.

[0279] There is no particular limitation on the method for contacting the lipid nanoparticles of the present invention with ex vivo immune cells, but for example, the lipid nanoparticles of the present invention may be added to a normal immune cell culture medium. Alternatively, in order to increase the introduction efficiency, for example, calcium phosphate coprecipitation, PEG, electroporation, microinjection, lipofection, etc. may be used in combination.

[0280] When the lipid nanoparticles of the present invention contain a nucleic acid encoding an exogenous TCR as an active ingredient, the expression of the endogenous TCRα chain and TCRβ chain that the T cell naturally expresses may be suppressed by siRNA from the viewpoint of increasing the expression of the exogenous TCR, suppressing the appearance of mispaired TCR, or suppressing autoreactivity. When the nucleic acid is applied to the method, it is preferable that the base sequence of the nucleic acid encoding the TCR is a sequence (codon-converted sequence) different from the base sequence corresponding to the RNA on which the siRNA that suppresses the expression of the endogenous TCRα chain and TCRβ chain acts in order to avoid the effect of the siRNA on the exogenous TCR. These methods are described, for example, in International Publication No. 2008 / 153029. The base sequence can be prepared by introducing silent mutations into a nucleic acid encoding a TCR obtained from nature or by chemically synthesizing an artificially designed nucleic acid. Alternatively, in order to avoid mispairing with the endogenous TCR chain, a part or all of the constant region of the nucleic acid encoding the exogenous TCR may be replaced with a constant region derived from an animal other than human, for example, a mouse.

[0281] 4. A pharmaceutical comprising the lipid nanoparticles of the present invention or ex vivo immune cells into which the lipid nanoparticles have been introduced The present invention provides a pharmaceutical comprising the lipid nanoparticles of the present invention, or ex vivo immune cells (e.g., ex vivo T cells) into which the lipid nanoparticles have been introduced (hereinafter abbreviated as "the pharmaceutical of the present invention"). (4-1. Pharmaceuticals Comprising Ex Vivo Immune Cells Introduced with Lipid Nanoparticles of the Present Invention) Immune cells (e.g., T cells) into which the lipid nanoparticles of the present invention have been introduced can express CAR or exogenous TCR, thereby specifically recognizing cells expressing a surface antigen specifically recognized by the CAR or exogenous TCR, and killing (e.g., inducing apoptosis) the cells. Therefore, by containing, as an active ingredient, a nucleic acid encoding a CAR or exogenous TCR that recognizes a surface molecule that is specifically expressed or whose expression is enhanced in diseased cells such as cancer cells as a surface antigen, the ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced can be used for the prevention or treatment of diseases such as cancer, and can be safely administered to mammals (humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, preferably humans).

[0282] (4-2. Medicine containing the lipid nanoparticles of the present invention) The pharmaceutical composition of the present invention containing the lipid nanoparticles of the present invention is preferably prepared by mixing the lipid nanoparticles with known pharma- ceutically acceptable carriers (including excipients, diluents, bulking agents, binders, lubricants, flow aids, disintegrants, surfactants, etc.) or conventional additives. Excipients are well known to those skilled in the art, and include, for example, phosphate-buffered saline (e.g., 0.01M phosphate, 0.138M NaCl, 0.0027M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, physiological saline, glycol or ethanol solutions, and salts of organic acids such as acetate, propionate, malonate, and benzoate. In addition, auxiliary agents such as wetting agents or emulsifiers, and pH buffers can also be used. In addition, formulation auxiliary agents such as suspending agents, preservatives, stabilizers, and dispersants may also be used. The pharmaceutical composition may also be in a dry form for reconstitution with an appropriate sterile liquid before use. The pharmaceutical composition can be administered orally or parenterally, systemically or locally, depending on the preparation form (oral preparations such as tablets, pills, capsules, powders, granules, syrups, emulsions, and suspensions; parenteral preparations such as injections, drips, external preparations, and suppositories). When administered parenterally, it can be administered intravenously, intradermally, subcutaneously, rectally, transdermally, etc. When used in the form of an injection, an acceptable buffer, solubilizer, isotonicity agent, etc. can also be added.

[0283] The dosage of the pharmaceutical of the present invention containing the lipid nanoparticle of the present invention is, for example, 0.001 mg to 10 mg of nucleic acid encoding CAR or exogenous TCR per kg of body weight per administration. For example, when administered to a human patient, the dosage is 0.001 to 50 mg for a patient weighing 60 kg. The dosages mentioned above are examples, and can be appropriately selected depending on the type of nucleic acid used, the administration route, and the age, body weight, symptoms, etc. of the subject or patient.

[0284] The pharmaceutical of the present invention, which comprises the lipid nanoparticles of the present invention, can be administered to a mammal (human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey); preferably, human) to induce the expression of CAR or exogenous TCR in immune cells within the animal, such as T cells (also referred to herein as "in vivo immune cells" or "in vivo T cells"). The in vivo immune cells specifically recognize cancer cells, etc., that express the surface antigen targeted by the CAR or exogenous TCR, and kill the diseased cells, thereby exhibiting a preventive or therapeutic effect against the disease.

[0285] In the case of a medicine containing ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced as an active ingredient, the immune cells may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before being administered to a subject. Examples of stimulatory molecules include, but are not limited to, cytokines, appropriate proteins, and other components. For example, in the case of T cells, examples of cytokines include IL-2, IL-7, IL-12, IL-15, IFN-γ, etc., and preferably IL-2 can be used. The concentration of IL-2 in the medium is not particularly limited, but is preferably, for example, 0.01 to 1 × 10 5 U / mL, more preferably 1 to 1 × 10 4 U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, lymphocyte stimulating factors such as lectin can also be added. Furthermore, serum or plasma may be added to the medium. The amount of these to be added to the medium is not particularly limited, but is exemplified as 0% by volume to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be reduced stepwise. The serum or plasma may be derived from either autologous or non-autologous sources, but from the viewpoint of safety, it is preferable to use autologous sources.

[0286] The medicine containing the ex vivo immune cells introduced with lipid nanoparticles of the present invention as an active ingredient is preferably administered parenterally to a subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but usually, the number of cells is usually 1 x 10 6 ~1×10 10 Preferably 1×10 7 ~1×10 9 10, more preferably 5×10 7 ~5×10 8 The drug is administered so that the number of cells is one. The drug may be administered once or multiple times. The drug of the present invention, which contains the ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced as an active ingredient, may be in a known form suitable for parenteral administration, such as an injection or infusion. The drug may contain a pharmacologically acceptable excipient as appropriate. Examples of pharmacologically acceptable excipients include those described above. The drug may contain physiological saline, phosphate buffered saline (PBS), culture medium, etc., in order to stably maintain the cells. Examples of culture medium include, but are not limited to, RPMI, AIM-V, X-VIVO10, etc. In addition, the drug may contain a pharmacologic acceptable carrier (e.g., human serum albumin), a preservative, etc. for the purpose of stabilization.

[0287] The medicament of the present invention may be a preventive or therapeutic drug for cancer. The cancer to which the medicament of the present invention is applied is not particularly limited, and examples thereof include acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nose, nasal cavity, or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, etc., are not limited thereto.

[0288] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and the present invention is not limited to these. EXAMPLES

[0289] Example 1 (Antibody reduction treatment) 111 μl of 9.21 mg / ml anti-CD3 antibody (Bio X Cell) was mixed with 12.3 μl of 10 mM DTT solution. Similarly, 149 μl of 6.73 mg / ml IgG2a antibody (Bio X Cell) was mixed with 16.6 μl of 10 mM DTT solution. The mixture of each antibody and DTT was mixed using a vortex and reacted at room temperature for 30 minutes. The reaction solution was fractionated by HPLC (column: TSKgel G2000SWXL 7.8 mm x 30 cm, TOSOH, mobile phase: PBS) to obtain a fraction containing reduced antibody. The fraction was ultraconcentrated using an Amicon 0.5 ml-10K. The antibody protein concentration and thiol group concentration in the concentrate were measured by absorbance at 230 nm and fluorescence color reaction using N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), respectively. The yield of the reduced anti-CD3 antibody was 176 μl, the protein concentration was 1.75 mg / ml, and the thiol group concentration was 5.14 μM, and the yield of the reduced IgG2a antibody was 86 μl, the protein concentration was 5.19 mg / ml, and the thiol group concentration was 45.1 μM.

[0290] Example 2 (Preparation of Maleimide-LNP) A lipid mixture (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 obtain a 7.0 mg / ml lipid solution. As the cationic lipid, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate (compound 7) described in WO2016 / 021683 and N,N,N-trimethyl-5-oxo-5-(3-((3-pentyloctanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propoxy)pentan-1-aminium iodide (compound 8) were mixed at a molar ratio of 59.1:0.9 and used. The mRNA encoding the CD19-targeted CAR having 4-1BB and CD3ζ as the intracellular signaling domain was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer pH 5.0 to obtain a 0.2 mg / ml nucleic acid solution. The obtained lipid solution and nucleic acid solution were mixed at room temperature with a Nanoassemblyr device (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 48 hours using a Slyde-A-Lyzer (20k molecular weight cutoff, Thermo Scientific). Then, it was filtered using a 0.2 μm syringe filter (Iwaki) and stored at 4°C.

[0291] Example 3 (Binding reaction between reduced antibody and Maleimide-LNP) The maleimide-LNP dispersion was mixed with the reduced antibody solution so that the molar concentration of the reduced antibody against maleimide was 1 / 20, and the mixture was left to stand at room temperature for 4 hours.The mixture was then stored at 4°C until the purification step.

[0292] Example 4 (Gel filtration purification of antibody-LNP) The reaction solution of the reduced antibody and Maleimide-LNP was loaded onto a gel filtration column Sepharose CL-4B (Cat No. 17-0150-01 / GE Healthcare) and fractionated using D-PBS(-) as the mobile phase. The protein concentration of each fraction was then measured to identify the fraction containing the antibody-LNP of interest. The antibody-LNP was filtered through a 0.2 μm syringe filter and stored at 4°C.

[0293] Example 5 (Ex vivo transfection into CD8+ T cells) Spleens were collected from C57BL / 6J mice and dispersed in ACK lysing buffer (Biosource) to obtain mouse splenocytes. The obtained mouse splenocytes were cultured for 2 days in complete RPMI 1640 medium containing 1 ng / ml interleukin 7 and 2 μg / ml concavalin A, and mouse CD8+ T cells were isolated by removing dead cells using Ficoll density gradient centrifugation and processing with a CD8 Negative Isolation Kit (Stemcell Technologies). The obtained mouse CD8+ T cells were dispersed and cultured in complete RPMI 1640 medium containing 10 ng / ml interleukin 2 and antibody-LNP to transfect mouse CD8+ T cells with CAR or exogenous TCR. Similarly, CD8+ T cells are isolated from purchased human primary cultured T cells, and the human CD8+ T cells are transfected with CAR or exogenous TCR.

[0294] Example 6 (In vitro cytotoxicity evaluation of CAR-T cells) The CD19-expressing human chronic myeloid leukemia cell line K562 cells, which serve as the cytotoxicity evaluation cells, are labeled with membrane dye PKH-26 (Sigma-Aldrich), washed with RPMI medium containing 10% fetal calf serum, and dispersed and cultured at 1 x 10^5 cells / ml in the same medium. The labeled cytotoxicity evaluation cells are dispensed into a 96-well plate and cultured, and after mixing with CAR-T cells, they are cultured at 37°C for 3 hours. The apoptotic cells are quantified by staining with Annexin V-Brilliant Violet 421 (BioLegend) and performing flow cytometry.

[0295] Example 7 (In vivo anticancer activity evaluation test) K562-CD19 cells stably expressing luciferase were administered to 6-week-old NOD-SCID mice via the tail vein, and a mouse blood cancer model was created after a one-week rearing period. Human CAR-T cells (1x10^6 cells) were then transfected ex vivo with CAR-encoding nucleic acid using antibody-LNP, and administered once a week for three weeks via the tail vein, and the reduction of cancer cells by CAR-T cells was evaluated by measurement using an in vivo bioluminescence imaging system IVIS (PerkinElmer).

[0296] Example 8 (Preparation of Maleimide-LNP using cationic lipids) The lipid mixture (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% 25 mM acetate buffer pH 4.0 to obtain a 10 mg / ml lipid solution. The cationic lipids used were 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-tetradec-9-enoate (compound 12), 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2-((dodecanoyloxy)methyl)propane-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate (compound 21), and 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate (compound 35). pcDNA3.1-hCD19CAR encoding CD19-targeted CAR was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer pH 5.5 to obtain a 0.2 mg / ml nucleic acid solution. pcDNA3.1-hCD19CAR was prepared by incorporating the CD19 IgG4 28z sequence cited in WO2013 / 126712 into the multi cloning site of pcDNA3.1 (Thermo Fisher Scientific). The obtained lipid solution and nucleic acid solution were mixed at room temperature with a Nanoassemblr device (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 48 hours using a Slyde-A-Lyzer (20k molecular weight cutoff, Thermo Scientific). Then, the solution was filtered using a 0.2 μm syringe filter (Iwaki) and stored at 4°C.

[0297] (Measurement of nucleic acid concentration of maleimide-LNP and calculation of estimated maleimide concentration) Maleimide-LNP was dissolved in 0.5% Triton X-100 and analyzed by Quant-iT TM PicoGreen TM The pDNA concentration was measured using a dsDNA Assay Kit (Thermo Fisher Scientific). The pDNA concentration measured without the addition of Triton X-100 was taken as the concentration of pDNA not encapsulated in LNP, and the pDNA encapsulation rate in LNP was calculated. The estimated maleimide concentration was calculated by multiplying the measured pDNA concentration by the maleimide-PEG-lipid (DSPE-020MA) loading ratio. The obtained values ​​are shown in Table 1.

[0298] [Table 1]

[0299] (Binding reaction of two types of mixed reduced antibodies and Maleimide-LNP) Equal amounts of anti-human CD3 antibody (BE0001-2, BioXCell) and anti-human / monkey CD28 antibody (BE0248, BioXCell) reduced with DTT were mixed, and the mixture was mixed at 1 / 20 molar amount relative to the maleimide in maleimide-LNP. The concentrations and volumes of maleimide-LNP and reduced antibody are shown in Table 2. The mixture was left to stand at room temperature for 4 hours and then stored at 4°C until the purification process.

[0300] [Table 2]

[0301] (Gel filtration purification of antibody-LNP) The reaction solution of the reduced antibody and Maleimide-LNP was loaded onto a gel filtration column Sepharose CL-4B (Cat No. 17-0150-01 / GE Healthcare) and fractionated using D-PBS(-) as the mobile phase. The protein concentration of each fraction was then measured to identify the fraction containing the antibody-LNP of interest. The antibody-LNP was filtered through a 0.2 μm syringe filter and stored at 4°C. The particle size of the antibody-LNP obtained was measured using a Zetasizer Nano ZS (Malvern Panalytical). The nucleic acid concentration and antibody protein concentration were measured using Quant-iT TM PicoGreen TM dsDNA Assay Kit (Thermo Fisher Scientific) and ATTO-TAG TM Measurements were performed using the FQ Amine-Derivatization Kit (Thermo Fisher Scientific). The values ​​of each analysis result are shown in Table 3.

[0302] [Table 3]

[0303] Example 9 (CD19 CAR transfection test into human primary T cells using antibody-LNP) Human Pan-T Cells (AccuCell human peripheral blood pan-T cells, Negative selection) 1.1 × 10 6The cells were prepared in medium at 30 μg / ml and seeded in a 96-well plate at 90 μl / well. X-VIVO10 (Lonza) medium supplemented with recombinant IL-2 (Thermo Fisher Scientific) at a concentration of 30 ng / ml was used. Next, 10 μl of antibody-LNP diluted with PBS to a concentration of 30 μg / ml of pcDNA3.1-hCD19CAR was added to the medium, and the cells were cultured for 3 and 6 days at 37°C in a 5% CO2 incubator.

[0304] (CD19CAR expression evaluation by flow cytometry) Human primary T cells cultured in a 96-well plate were collected in a 1.5 ml tube, and 2 μl of recombinant human CD19 protein, Fc Chimera Active, Biotin (Abcam) was added and left on ice for 30 minutes. Next, 200 μl of Cell Wash (BD) containing 1% FBS was added and washed twice by centrifugation at 300×g for 5 minutes, after which the supernatant was removed and the cells were dispersed in 100 μl of 1% FBS, Cell Wash. 0.2 μl of Brilliant Violet 421 Streptavidin was added to the cell dispersion, mixed by pipetting, and left on ice for 30 minutes. The stained cells were washed three times with 200 μl of 1% FBS Cell Wash and centrifuged, filtered, dispersed in 200 μl of 1% FBS Cell Wash, and analyzed by flow cytometry using an LSRFortessa (BD). The results of CD19 CAR expression analysis by flow cytometry of human primary cultured T cells transfected with hCD3 / hCD28-compound 12-pcDNA3.1-hCD19CAR are shown in Figure 1. The CD19 CAR positivity rates 3 and 6 days after addition of antibody-LNP were 51.9% and 41.7%, respectively, and CAR-positive cells were obtained with sufficient efficiency compared to gene transfer using a viral vector. The results of CD19 CAR expression analysis by flow cytometry of human primary cultured T cells transfected with hCD3 / hCD28-compound 21-pcDNA3.1-hCD19CAR and hCD3 / hCD28-compound 35-pcDNA3.1-hCD19CAR are shown in Figure 2. The CD19 CAR positivity rates 3 days after antibody-LNP addition were 5.12% and 47.0%, respectively.

[0305] Example 10 (Evaluation of cancer cytotoxicity by human primary cultured T cells transfected with CD19 CAR using antibody-LNP) The human pre-B cell line NALM-6 and the human Burkitt lymphoma cell line Daudi were labeled using the DELFIA Cytotoxicity Assay Kit (Perkin Elmer) at 1 × 10 4 The cells were seeded in a 96-well U-bottom plate at a cell density of 100 μl / well. RPMI (phenol red free) containing 10% FBS was used as the medium. Next, human primary cultured T cells (CD19 CAR positive rate 3 days after antibody-LNP addition: 19%) transfected with CD19CAR using hCD3 / hCD28-compound 12-pcDNA3.1-hCD19CAR by the method described in a separate section were dispersed in 100 μl of medium and added as effector cells so that the cell number ratio with the target cells was 0 to 16. 3 hours after mixing the target cells and effector cells, 20 μl of culture supernatant was collected. 20 μl of europium solution (Eu) was added to the collected culture supernatant, and the cell damage rate was calculated from the fluorescence intensity emitted by the complex of the chelating agent TDA and Eu released from the damaged target cells. The cytotoxicity rates of Nalm-6 and Daudi upon addition of CD19 CAR-transfected human primary T cells are shown in Figure 3 .

[0306] Example 11 (Preparation of reduced Fab') Reduced Fab' to be bound to Mleimide-LNP was prepared from anti-mouse CD3ε antibody (BE0001-1, BioXCell) and anti-mouse CD28 antibody (BE0015-1, BioXCell) using the Pierce F(ab')2 Preparation Kit (Thermo Fisher Scientific). 1 ml of 1.75 mg / ml F(ab')2 was obtained from 0.5 ml of 7.86 mg / ml anti-mouse CD3ε antibody. 0.62 ml of 0.97 mg / ml F(ab')2 was obtained from 0.5 ml of 3.86 mg / ml anti-mouse CD28 antibody. Each F(ab')2 was mixed with 2-aminoethanethiol p-toluenesulfonate at a concentration of 40 mM as a reducing agent and incubated at 37°C for 1 hour. The resulting Fab' was purified using Zeba Spin Desalting Columns, 7K MWCO-0.5 ml (Thermo Fischer Scientific) and stored at 4°C until reaction with Maleimide-LNP. The protein concentration and thiol group concentration of Fab' were measured by absorbance at 230 nm and a fluorescent color reaction using N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), respectively.

[0307] Example 12 (Binding reaction between reduced Fab' and Maleimide-LNP) The reduced anti-mouse CD3ε Fab' and anti-mouse CD28 Fab' were mixed at 1 / 20 molar amount to the maleimide of the maleimide-LNP prepared by the above method. The mixture was left to stand at room temperature for 4 hours and then stored at 4°C until the purification step.

[0308] Example 13 (Gel filtration purification of reduced Fab'-LNP) The reaction mixture of reduced Fab' and Maleimide-LNP was loaded onto a gel filtration column Sepharose CL-4B (Cat No. 17-0150-01 / GE Healthcare) and fractionated using D-PBS(-) as the mobile phase. The protein concentration of each fraction was then measured to identify the fraction containing the desired Fab'-LNP. The Fab'-LNP was filtered through a 0.2 μm syringe filter and stored at 4°C. The particle size of the resulting antibody-LNP was measured using a Zetasizer Nano ZS (Malvern Panalytical). The nucleic acid concentration and antibody protein concentration were measured using Quant-iT TM PicoGreen TM dsDNA Assay Kit (Thermo Fisher Scientific) and ATTO-TAG TM Measurements were performed using the FQ Amine-Derivatization Kit (Thermo Fisher Scientific). [Industrial Applicability]

[0309] The lipid nanoparticles of the present invention can efficiently and selectively introduce CAR or exogenous TCR into T cells not only ex vivo but also in vivo, and therefore can provide CAR-T or TCR-T cell therapy with low production costs. In addition, since they do not use a viral vector, they can avoid the problem of antigenicity caused by viral proteins, and are extremely useful as a new platform for cancer immunotherapy.

[0310] This application is based on patent application No. 2017-252616 filed in Japan on December 27, 2017, the contents of which are incorporated in their entirety by reference herein.

Claims

1. A lipid nanoparticle comprising the following (a) to (c), the lipid nanoparticle having a ligand on its surface capable of targeting T cells: (a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor; (b) a cationic lipid selected from the group consisting of the following compounds 12, 21, 35 and 7; and (c) non-cationic lipids. 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】

2. The lipid nanoparticle of claim 1 , wherein the nucleic acid is mRNA or DNA.

3. The lipid nanoparticle of claim 1, wherein the non-cationic lipid is a phospholipid, cholesterol and / or a PEG lipid.

4. The lipid nanoparticle of claim 1, wherein the PEG lipid is a terminally reactive PEG lipid.

5. The lipid nanoparticle of claim 1, wherein the ligand is a ligand comprising an antigen-binding domain of one or more antibodies selected from the group consisting of an antibody against CD3, an antibody against CD4, an antibody against CD8, and an antibody against CD28.

6. The lipid nanoparticle of claim 1, wherein the ligand is a ligand comprising an antigen-binding domain of an antibody against CD3 and / or an antibody against CD28.

7. The lipid nanoparticle of claim 6, wherein the ligand is a ligand comprising the antigen-binding domains of an antibody against CD3 and an antibody against CD28.

8. A pharmaceutical comprising the lipid nanoparticles according to claim 1.

9. The pharmaceutical composition according to claim 8, which is a preventive or therapeutic agent for cancer.

10. The pharmaceutical according to claim 8, which induces expression of a chimeric antigen receptor or an exogenous T cell receptor by gene transfer into in vivo immune cells.

11. The pharmaceutical according to claim 8, which induces expression of a chimeric antigen receptor or an exogenous T cell receptor by gene transfer into in vivo T cells.

12. The lipid nanoparticle of claim 1 for use in the prevention or treatment of cancer.

13. Use of the lipid nanoparticles described in claim 1 for producing a cancer prophylactic or therapeutic agent.

14. A composition for inducing expression of a chimeric antigen receptor or an exogenous T cell receptor, comprising the lipid nanoparticles described in claim 1.

15. A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into ex vivo immune cells and expressing the same, comprising adding the lipid nanoparticles described in claim 1 to a culture medium containing ex vivo immune cells.

16. A method for expressing a chimeric antigen receptor or an exogenous T cell receptor in ex vivo T cells, comprising adding the lipid nanoparticles described in claim 1 to a culture medium containing ex vivo T cells.

17. A method for producing a pharmaceutical comprising ex vivo immune cells expressing a chimeric antigen receptor or an exogenous T cell receptor, comprising the step of adding the lipid nanoparticles described in claim 1 to a culture medium containing ex vivo immune cells.

18. The method includes adding the lipid nanoparticles according to claim 1 to a culture solution containing ex vivo T cells. A method for producing a medicament comprising ex vivo T cells expressing a chimeric antigen receptor or an exogenous T cell receptor.

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