WO2019131770A1 - 核酸含有脂質ナノ粒子及びその用途 - Google Patents
核酸含有脂質ナノ粒子及びその用途 Download PDFInfo
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Definitions
- the present invention provides a lipid nanoparticle containing a nucleic acid encoding a chimeric antigen receptor or a T cell receptor, using the lipid nanoparticles to express a chimeric antigen receptor or an exogenous T cell receptor in a target immune cell.
- the present invention relates to methods, and their pharmaceutical applications and the like.
- CAR-T cells transfected with chimeric antigen receptor (CAR) or T cell receptor (TCR) derived from cancer antigen-specific killer T cells It is progressing.
- CAR-T cell therapy like Kymriah (trade name) and Yescarta (trade name) approved in the United States, uses TIV cells obtained from patients ex vivo CAR using a viral vector such as lentiviral vector.
- a gene is introduced to produce CAR-T cells, and the CAR-T cells are administered to a patient.
- this method there is a problem that the production cost increases due to the cost of cell culture, preparation of virus vector and the like.
- CARs and exogenous TCRs can be introduced selectively in vivo by immune cells such as T cells in vivo, ex vivo preparation is not required, and it is possible to provide CAR- or TCR-immune cell therapy with low manufacturing cost. Become. In addition, if it is possible to selectively introduce CARs and exogenous TCRs such as T cells without using expensive viral vectors ex vivo, the cost for the virus survival test etc. becomes unnecessary. It is possible to provide low-cost CAR- or TCR-immune cell therapy.
- nanoparticles have been prepared by aggregating plasmid DNA encoding CAR with a cationic polymer and coating the aggregates with a non-cationic polymer conjugated with an anti-CD3 antibody fragment (Patent Document 1, Non-patent Document 1), Ex vivo or in vivo transfer of CAR to T cells using nano-carriers (Patent Document 2) coated with lipid that has been surface-modified with an anti-CD3 antibody, and mesoporous silica in which CAR encoding DNA is encapsulated in small pores Is being reported.
- the target siRNA in "lipid nanoparticles (LNP)" which has no pore structure inside, and consists of cationic lipid and noncationic helper lipid, and ligand for delivery to target cells
- LNP lipid nanoparticles
- a technology has been reported that delivers the siRNA to target cells by encapsulating
- T-cells were transfected ex vivo or in vivo with siRNA against CD45 using an anti-CD4 antibody fragment as a targeting ligand (Patent Document 3, Non-patent Document 2).
- nucleic acids eg, mRNA, DNA
- CAR CAR
- exogenous TCR TCR
- the object of the present invention is to provide a novel transfection technique capable of efficiently introducing CAR or an exogenous TCR in an immune cell selective manner such as T cell in vivo or ex vivo, thereby achieving low production cost of CAR- or It is to provide TCR-immune cell therapy.
- another object of the present invention is to provide more safe CAR- or TCR- immunocell therapy which avoids the problem of antigenicity by viral proteins.
- the present inventors have selected nucleic acids encoding CAR or exogenous TCR using LNP, selectively in vivo and ex vivo immune cells such as T cells. It succeeded in efficiently introducing, and came to complete this invention.
- Lipid nanoparticles containing the following (a) to (c): (A) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor; (B) cationic lipids; and (c) noncationic lipids. [2]
- the cationic lipid is Formula (I):
- L 1 is a C 1-22 alkylene group, a C 2-22 alkenylene group or a C 3-22 alkadienylene group
- n is an integer of 0 or 1
- R 1 is (1) Hydrogen atom
- (2) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 may be substituted with a substituent linear C 1-22 alkyl selected from alkenyl groups Group
- (3) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 C 2-22 alkenyl substituted optionally may be linear with a substituent selected from an alkenyl group
- (4) a linear C 3 optionally substituted by one or two substituents selected from a linear C 1-22 alkyl group and a linear C 2-22 alkenyl group -22 alkadienyl group
- R 2 is —CH 2 —O—CO—R 5
- R 3 is —CH 2 —O—CO—
- 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.
- Nanoparticles [7] The lipid nanoparticle according to [5], wherein the ligand is a ligand comprising an antibody against CD3 and / or an antigen binding domain of an antibody against CD28. [8] The lipid nanoparticle according to [5], wherein the ligand is a ligand comprising an antibody against CD3 and an antigen binding domain of an antibody against CD28. [9] A medicine comprising the lipid nanoparticle according to any one of [1] to [8]. [10] The medicine according to [9], which is a preventive or therapeutic drug for cancer. [11] The medicament according to [9], which gene-transfers a chimeric antigen receptor or an exogenous T cell receptor to in vivo immune cells and induces its expression.
- a chimeric antigen receptor or exogenous T for in vivo immune cells of a mammal which comprises administering the lipid nanoparticle according to any one of [1] to [8] to the mammal Methods for gene transfer and expression of cell receptors.
- a chimeric antigen receptor or exogenous T to in vivo T cells of the mammal which comprises administering the lipid nanoparticles of any of [1] to [8] to the mammal Methods for gene transfer and expression of cell receptors.
- a method for the prophylaxis or treatment of cancer in a mammal which comprises administering the lipid nanoparticle of any one of [1] to [8] to the mammal.
- [21] [ex] expressing a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticles according to any of [1] to [8] to a culture medium containing ex vivo immune cells a medicament comprising an in vivo immune cell.
- [22] [1] to [8] to express a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticle according to any of the above to a culture solution containing ex vivo T cells ex A medicament comprising an in vivo T cell.
- the medicine according to [21] or [22] which is a preventive or therapeutic drug for cancer.
- a chimeric antigen receptor or an exogenous T is added to an ex vivo immune cell, characterized by adding the lipid nanoparticle according to any of [8] to a culture solution containing the ex vivo immune cell Methods for gene transfer and expression of cell receptors.
- a chimeric antigen receptor or exogenous T to ex vivo T cells characterized in that the lipid nanoparticles according to any of [1] to [8] are added to a culture solution containing ex vivo T cells. Methods of expressing cellular receptors.
- a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticle of any one of [1] to [8] to a culture medium containing ex vivo immune cells to a mammal A method for preventing or treating cancer, which comprises administering ex vivo immune cells expressing the body.
- a chimeric antigen receptor or an exogenous T cell receptor obtained by adding the lipid nanoparticle of any one of [1] to [8] to a culture solution containing ex vivo T cells to a mammal A method for preventing or treating cancer, which comprises administering ex vivo T cells expressing the body.
- [31] Accepting a chimeric antigen obtained by adding the lipid nanoparticles according to any one of [1] to [8] to a culture solution containing ex vivo immune cells, for producing a preventive / therapeutic agent for cancer Use of ex vivo immune cells expressing body or exogenous T cell receptors.
- [32] Accepting a chimeric antigen obtained by adding the lipid nanoparticles according to any one of [1] to [8] to a culture solution containing ex vivo T cells, for producing a preventive / therapeutic agent for cancer Use of ex vivo T cells expressing body or exogenous T cell receptors.
- ex which expresses a chimeric antigen receptor or an exogenous T cell receptor, comprising the step of adding the lipid nanoparticle according to any of [1] to [8] to a culture medium containing ex vivo immune cells
- a method of producing a medicament comprising in vivo immune cells [34] [ex] expressing a chimeric antigen receptor or an exogenous T cell receptor, comprising the step of adding the lipid nanoparticles according to any of [1] to [8] to a culture solution containing ex vivo T cells
- CAR or exogenous TCR can be introduced selectively into immune cells such as T cells efficiently not only ex vivo but also in vivo, CAR- or TCR-immunization can be performed at low cost.
- Cell therapy can be provided.
- antigenicity problems caused by viral proteins can be avoided.
- FIG. 1 shows the results of CD19 CAR expression analysis by flow cytometry analysis of human primary cultured T cells transfected with hCD3 / hCD28-compound 12-pcDNA3.1-hCD19CAR.
- FIG. 2 shows CD19 CAR expression by flow cytometry analysis of human primary cultured T cells transfected with hCD3 / hCD28-compound 21-pcDNA3.1-hCD19CAR and hCD3 / hCD28-compound 35-pcDNA3.1-hCD19CAR The analysis results are shown.
- FIG. 3 shows the percent cytotoxicity of Nalm-6 and Daudi upon addition of human primary culture T cells transfected with CD19 CAR.
- Lipid Nanoparticles (LNP) of the Present Invention provides the following (a) to (c): (A) a nucleic acid encoding a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR); A lipid nanoparticle comprising (b) a cationic lipid; and (c) a noncationic lipid (hereinafter, also referred to as “the lipid nanoparticle of the present invention” or “the LNP of the present invention”).
- lipid nanoparticles (LNP) means that the inside of the molecular assembly constituted by the above (b) and (c) does not have a small pore structure (eg, mesoporous material), and the average diameter is 1 ⁇ m Means less than particles.
- a small pore structure eg, mesoporous material
- the average diameter is 1 ⁇ m Means less than particles.
- CAR Chimeric Antigen Receptor
- TCR Exogenous T Cell Receptor
- a-1 Nucleic Acids Encoding CAR CAR binds antigen of antibody linked to T cell signaling domain
- An artificially constructed hybrid protein comprising a domain (eg, scFv).
- Characteristics of CAR include the ability to convert T cell specificity and reactivity to selected targets in a non-MHC restricted manner, taking advantage of the antigen binding properties of monoclonal antibodies.
- Non-MHC restricted antigen recognition confers on T cells expressing CAR the ability to recognize the antigen independently of antigen processing, thereby bypassing the major mechanism of tumor escape.
- CAR advantageously does not dimerize with endogenous TCR ⁇ and ⁇ chains.
- the CAR used for the lipid nanoparticles of the present invention is a surface antigen (eg, cancer antigen) that target immune cells (eg, T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, etc.) should recognize It comprises an antigen binding domain of an antibody, an extracellular hinge domain, a transmembrane domain and an intracellular T cell signaling domain which can specifically recognize a peptide, a surface receptor and the like whose expression is enhanced in cancer cells.
- target immune cells eg, T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, etc.
- Examples of surface antigens which the antigen binding domain specifically recognizes include various cancers (eg, acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (eg, medulloblasts) Cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder or pleural cancer, nasal, nasal or middle ear cancer, oral cancer Cancer of the vulva, chronic myeloid cancer, colon cancer, esophagus cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (eg, head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, larynx Cancer, leukemia (eg, acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia), humoral tumor, liver cancer, lung cancer (e
- the antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment that can specifically recognize a target antigen, but in consideration of the ease of CAR production, the light chain variable region and heavy chain It is desirable that it is a single chain antibody (scFv) linked with a variable region via a linker peptide.
- the arrangement of the light chain variable region and the heavy chain variable region in the single chain antibody is not particularly limited as long as both can reconstitute a functional antigen binding domain, but generally from the N terminal side to the light chain variable region-linker peptide-heavy It can be designed in the order of chain variable regions.
- the linker peptide a known linker peptide usually used for the production of a single chain antibody can be used.
- DNAs encoding light chain variable regions and DNA encoding heavy chain variable regions prepared, for example, by cloning light chain genes and heavy chain genes from antibody-producing cells and performing PCR using them as templates, for example Alternatively, they can be chemically synthesized from sequence information of existing antibodies.
- a DNA encoding a single-chain antibody can be obtained by ligating the obtained DNA fragments and the DNA encoding the linker peptide according to an appropriate method.
- a leader sequence is further added to the N-terminal side of the antigen binding domain in order to present CAR on the surface of immune cells.
- domains derived from T cell surface molecules usually used in the art can be used as appropriate, and examples thereof include domains derived from CD8 ⁇ and CD28. It is not limited.
- an intracellular signal transduction domain for example, one having a CD3 ⁇ chain, a costimulatory transmission motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, 4-1BB, etc., between a transmembrane domain and a CD3 ⁇ ⁇ chain
- a costimulatory transmission motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, 4-1BB, etc.
- Nucleic acid sequence information encoding the extracellular hinge domain, transmembrane domain and intracellular signaling domain is well known in the art, and one of ordinary skill in the art can easily use T cells to obtain each domain based on such information. Can be obtained.
- a DNA encoding CAR is obtained by ligating the thus obtained DNA fragments encoding the antigen binding domain, extracellular hinge domain, transmembrane domain and intracellular signaling domain, respectively, according to a conventional method. be able to.
- the obtained CAR-encoding DNA may be inserted into an expression vector, preferably a plasmid vector, which contains a promoter functional in T cells as it is or after addition of an appropriate linker and / or nuclear localization signal and the like.
- Promoters functional in T cells include SR ⁇ promoter constitutive in mammalian cells, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR HSV-TK (herpes simplex virus thymidine kinase) promoter and the like can be used, but is not limited thereto.
- gene promoters such as CD3, CD4 and CD8 that express T cells specifically can also be used.
- RNA encoding CAR preferably mRNA can be prepared by transcribing into mRNA in an in vitro transcription system known per se, using an expression vector containing the DNA encoding CAR as a template.
- T cell receptor is composed of a dimer of TCR chain ( ⁇ chain, ⁇ chain), and is an antigen or the antigen-HLA.
- MHC human leukocyte type antigen
- Each TCR chain is composed of a variable region and a constant region, and there are three complementarity determining regions (CDR1, CDR2, CDR3) in the variable region.
- the TCR used in the present invention includes not only those in which the ⁇ chain and ⁇ chain of the TCR constitute a heterodimer but also those in which a homodimer is constituted. Furthermore, the TCR includes those in which part or all of the constant region has been deleted, those in which the amino acid sequence has been recombined, those in which a soluble TCR (soluble TCR) is used, and the like.
- exogenous TCR means that it is 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 identical to or different from the endogenous TCR expressed by T cells that are target cells of the lipid nanoparticles of the present invention.
- the nucleic acid encoding the TCR used in the lipid nanoparticles of the present invention is an ⁇ chain and ⁇ chain of TCR which can specifically recognize the surface antigen (eg, cancer antigen peptide etc.) to be recognized by the target T cell. It is a nucleic acid that encodes.
- the nucleic acid can be prepared by a method known per se. When the amino acid sequence or nucleic acid sequence of the TCR of interest is known, for example, chemically overlapping DNA strands or RNA strands, or partially overlapping oligo DNA short strands synthesized are used based on the sequences. By connecting using the PCR method or Gibson Assembly method, it is possible to construct a DNA encoding the full length or a part of the TCR of the present invention.
- the T cell of interest can be isolated from a cell population containing T cells expressing the TCR of interest, and a nucleic acid encoding the TCR can be obtained from the T cells.
- a cell population eg, PBMC
- a living organism eg, human
- these cell populations are stimulated in the presence of an epitope of a cell surface antigen recognized by the TCR of interest.
- Culture, and from this cell population, specificities to cells expressing the cell surface antigen and cell surface antigens such as CD8 and CD4 are used as indicators to specify cells expressing the cell surface antigen by a known method Can be selected.
- the specificity of T cells for cells expressing the surface antigen can be measured using, for example, a dextromer assay, an ELISPOT assay or a cytotoxicity assay.
- the cell population containing the T cells may be, for example, a living organism having many cells expressing cell surface antigens recognized by the TCR of interest (eg, patients with diseases such as cancer, or an epitope of the antigen or a pulse of the epitope) (T cell-containing population in contact with dendritic cells).
- the nucleic acid of the present invention can be obtained by extracting a DNA from the isolated T cell by a conventional method, amplifying the TCR gene based on the nucleic acid sequence of the constant region of TCR, using the DNA as a template, and cloning.
- RNA is extracted from cells by a conventional method to synthesize cDNA, which is used as a template and an antisense primer complementary to a nucleic acid encoding each of the constant regions of TCR ⁇ chain and ⁇ chain to obtain 5'-RACE ( It can also be prepared by performing Rapid amplification of cDNA ends.
- 5'-RACE may be performed by a known method, and can be performed using a commercially available kit such as, for example, SMART PCR cDNA Synthesis Kit (manufactured by Clontech).
- the DNA encoding each of the ⁇ chain and ⁇ chain of the obtained TCR can be inserted into an appropriate expression vector in the same manner as the DNA encoding the above-mentioned CAR.
- the DNA encoding the ⁇ chain and the DNA encoding the ⁇ chain may be inserted into the same vector or into separate vectors. When inserted into the same vector, the expression vector may express both strands polycistronically or monocistronically.
- RNA encoding each chain of TCR preferably mRNA can be prepared, for example, using the expression vector as a template and in the same manner as the RNA encoding CAR.
- cationic lipid means a lipid that has a net positive charge at a selected pH, such as physiological pH.
- the cationic lipid used for the lipid nanoparticles of the present invention is not particularly limited, and, for example, WO 2015/011633, WO 2016/021683, WO 2011/153491, 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, etc., and the like It can be mentioned.
- cationic lipids represented by the following structural formula described in WO 2015/011633 can be mentioned.
- cationic lipids represented by the following structural formula.
- cationic lipids represented by the following structural formula described in WO 2016/021683 can be mentioned.
- W has the formula -NR 1 R 2 or formula -N + R 3 R 4 R 5 - and, (Z) R 1 and R 2 are each independently a C 1-4 alkyl group or a hydrogen atom, R 3 , R 4 and R 5 are each independently a C 1-4 alkyl group, Z - is an anion, X is a C 1-6 alkylene group which may be substituted, Y A , Y B and Y C are each independently an optionally substituted methine group, L A , L B and L C independently represent an optionally substituted methylene group or a bond, R A1 , R A2 , R B1 , R B2 , R C1 and R C2 each independently represent a C 4-10 alkyl group which may be substituted. ] Or a salt thereof.
- cationic lipids represented by the following structural formula can be mentioned.
- cationic lipids represented by the following structural formula.
- Another preferred embodiment includes a cationic lipid represented by the following formula (II) (hereinafter, also referred to as “compound (II)”).
- n is an integer of 2 to 5
- R represents a linear C 1-5 alkyl group, a linear C 7-11 alkenyl group or a linear C 11 alkadienyl group
- Each wavy line indicates a cis- or trans-type bond independently.
- cationic lipids represented by the following structural formula can be mentioned.
- cationic lipids represented by the following structural formula.
- Compound (II) can be produced, for example, by the following process.
- both a compound in which both of the wavy lines are in a cis form and a compound in which one or both of the wavy lines are in a trans form are produced by the same production methods as described below. be able to.
- the salt of compound (II) can be obtained by appropriate mixing with an inorganic base, an organic base, an organic acid, a basic or acidic amino acid.
- the raw materials and reagents used in each step of the above-mentioned production method, and the obtained compounds may each form a salt.
- the compound obtained in each step When the compound obtained in each step is a free compound, it can be converted to a target salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted to a free form or another type of desired salt by known methods.
- the compound obtained in each step may be used as the reaction solution or as a crude product and then used in the next reaction, or the compound obtained in each step may be concentrated from the reaction mixture according to a conventional method It can be isolated and / or purified by separation means such as crystallization, recrystallization, distillation, solvent extraction, fractionation, chromatography and the like.
- the reaction time may vary depending on the reagent and solvent to be used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 10 minutes to 8 hours.
- the reaction temperature may vary depending on the reagent and solvent to be used, but unless otherwise specified, it is usually -78 ° C to 300 ° C, preferably -78 ° C to 150 ° C.
- the pressure may differ depending on the reagent and solvent to be used, but unless otherwise specified, it is usually 1 to 20 atm, preferably 1 to 3 atm.
- a microwave synthesizer such as Biotage's Initiator may be used.
- the reaction temperature may vary depending on the reagent and solvent to be used, but unless otherwise specified, it is usually room temperature to 300 ° C., preferably room temperature to 250 ° C., more preferably 50 ° C. to 250 ° C.
- the reaction time may vary depending on the reagent and solvent used, but unless otherwise specified, it is usually 1 minute to 48 hours, preferably 1 minute to 8 hours.
- the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, relative to the substrate, unless otherwise specified.
- the reagent is used as a catalyst, the reagent is used in 0.001 equivalent to 1 equivalent, preferably 0.01 equivalent to 0.2 equivalent, relative to the substrate.
- the reagent also serves as the reaction solvent, the amount of the solvent is used.
- 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 and the like
- Halogenated hydrocarbons dichloromethane, carbon tetrachloride etc .
- Nitriles acetonitrile, etc .
- Sulfoxides dimethyl sulfoxide and the like
- Aromatic organic bases pyridine and the like
- Acid anhydrides acetic anhydride etc .
- Inorganic bases sodium hydroxide, potassium hydroxide, magnesium hydroxide etc .
- Basic salts sodium carbonate, calcium carbonate, sodium hydrogen carbonate 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 .
- Organic lithiums n-butyllithium, sec-butyllithium and the like.
- 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 acid boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous iron chloride and the like.
- protection or deprotection reaction of a functional group is carried out according to a known method, for example, Wiley-Interscience 2007, “Protective Groups in Organic Synthesis, 4th Ed.” (Theodora W. Greene, Peter G. M. Wuts). It is carried out according to the method described in Thieme company 2004 "Protecting Groups 3rdEd.” (P. J. Kocienski) and the like.
- protecting groups for hydroxyl group and phenolic hydroxyl group such as alcohol include, for example, ether type such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, tetrahydropyranyl ether and the like Protective groups; carboxylic acid ester type protective groups such as acetic acid ester; sulfonic acid ester type protective groups such as methane sulfonic acid ester; carbonate type protective groups such as t-butyl carbonate and the like.
- Examples of the protecting group for the carbonyl group of aldehyde include an acetal type protecting group such as dimethyl acetal; and a cyclic acetal type protecting group such as cyclic 1,3-dioxane.
- Examples of the protective group for the carbonyl group of ketone include ketal type protective groups such as dimethyl ketal; cyclic ketal type protective groups such as cyclic 1,3-dioxane; oxime type protective groups such as O-methyloxime; And hydrazone type protective groups such as dimethyl hydrazone.
- protecting groups for carboxyl groups include ester-type protecting groups such as methyl ester; and amide-type protecting groups such as N, N-dimethylamide.
- thiol protecting group examples include ether-type protecting groups such as benzyl thioether; and ester-type protecting groups such as thioacetic acid ester, thiocarbonate and thiocarbamate.
- the removal of the protecting group can be carried out by known methods, for example, acid, base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halide (eg, trimethylsilyl iodide) , Trimethylsilyl bromide), a reduction method, or the like.
- the reducing agent used is lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), sodium borohydride
- Metal hydrides such as triacetoxyborohydride and tetramethylammonium hydride; boranes such as borane-tetrahydrofuran complex; Raney nickel; Raney cobalt; hydrogen; formic acid and the like.
- Raney nickel or Raney cobalt can be used in the presence of hydrogen or formic acid.
- a catalyst such as palladium-carbon or Lindlar catalyst.
- examples of the oxidizing agent used include m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, peracids such as t-butyl hydroperoxide, etc .; tetrabutyl ammonium perchlorate, etc.
- MCPBA m-chloroperbenzoic acid
- hydrogen peroxide hydrogen peroxide
- peracids such as t-butyl hydroperoxide, etc .
- tetrabutyl ammonium perchlorate etc.
- Perchlorates such as sodium chlorate; chlorites such as sodium chlorite; periodic acids such as sodium periodate; high-valent iodine reagents such as iodosylbenzene; manganese dioxide Reagents having manganese such as potassium manganate; Leads such as lead tetraacetate; pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), reagents having chromium such as Jones reagent; N-bromosuccinimide (NBS) Halogen compounds such as; oxygen; ozone; sulfur trioxide / pyridine complex; male tetraoxide Um; dioxide Zeren; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and the like.
- PCC pyridinium chlorochromate
- PDC pyridinium dichromate
- NBS N-bromosuccinimide
- radical initiator When performing radical cyclization reaction in each process, as a radical initiator used, azo compounds such as azobisisobutyronitrile (AIBN); 4-4′-azobis-4-cyanopentanoic acid (ACPA) Water soluble radical initiators; triethyl boron in the presence of air or oxygen; benzoyl peroxide and the like. Further, as a radical reaction agent to be used, tributylstannane, tristrimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, samarium iodide and the like can be mentioned.
- AIBN azobisisobutyronitrile
- ACPA 4-4′-azobis-4-cyanopentanoic acid
- tributylstannane tristrimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, samarium iod
- Examples of the Wittig reagent used include alkylidene phosphoranes and the like.
- Alkylidene phosphoranes can be prepared by known methods, for example, by reacting a phosphonium salt with a strong base.
- phosphonoacetic acid esters such as methyl dimethylphosphonoacetate and ethyl diethylphosphonoacetate
- bases such as alkali metal hydrides and organic lithiums It can be mentioned.
- examples of reagents used include Lewis acids and acid chlorides or alkylating agents (eg, halogenated alkyls, alcohols, olefins, etc.).
- Lewis acids eg, halogenated alkyls, alcohols, olefins, etc.
- an organic acid or inorganic acid can be used, and instead of the acid chloride, an acid anhydride such as acetic anhydride can be used.
- a nucleophile eg, amines, imidazole etc.
- a base eg, basic salts, organic bases etc.
- nucleophilic addition reaction When performing nucleophilic addition reaction with carbanion, nucleophilic 1,4-addition reaction with carbanion (Michael addition reaction), or nucleophilic substitution reaction with carbanion in each step, a base used to generate carbanion And organic lithiums, metal alkoxides, inorganic bases, organic bases and the like.
- examples of the Grignard reagent include aryl magnesium halides such as phenyl magnesium bromide; and alkyl magnesium halides such as methyl magnesium bromide and isopropyl magnesium bromide.
- 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.
- active methylene compounds eg, malonic acid, diethyl malonate, malononitrile etc.
- bases eg, organic bases, etc. sandwiched between two electron withdrawing groups Metal alkoxides, inorganic bases
- examples of the azidation agent to be used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide, sodium azide and the like.
- DPPA diphenylphosphoryl azide
- examples of azidation agent to be used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide, sodium azide and the like.
- DPPA diphenylphosphoryl azide
- DBU 1,8-diazabicyclo [5,4,0] undec-7-ene
- the reducing agent used includes sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, formic acid and the like.
- the substrate is an amine compound
- examples of the carbonyl compound used include paraformaldehyde as well as aldehydes such as acetaldehyde and ketones such as cyclohexanone.
- the amines to be used include ammonia, primary amines such as methylamine; secondary amines such as dimethylamine, and the like.
- azodicarboxylic acid esters eg, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.
- triphenylphosphine eg, triphenylphosphine
- acyl chloride such as acid chloride and acid bromide
- acid anhydride active ester
- sulfuric acid ester And activated carboxylic acids.
- Carbodiimide-based condensing agents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD) as a carboxylic acid activating agent; 4- (4,6-dimethoxy-1,3,5-) Triazine-based condensing agents such as triazin-2-yl) -4-methylmorpholinium chloride-n-hydrate (DMT-MM); Carbonate-based condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenyl Phosphorus azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukayama reagent); thionyl chloride; haloformic acid such as ethyl chloroformate Lower alkyl; O- (7-azabenzotriazol-1-yl)
- additives such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP) and the like may be further added to the reaction.
- HOBt 1-hydroxybenzotriazole
- HOSu N-hydroxysuccinimide
- DMAP dimethylaminopyridine
- the metal catalyst used is palladium (II) acetate, tetrakis (triphenylphosphine) palladium (0), dichlorobis (triphenylphosphine) palladium (II), dichlorobis (triethyl) Phosphine compounds such as phosphine) palladium (II), tris (dibenzylideneacetone) dipalladium (0), 1,1'-bis (diphenyl phosphino) ferrocene palladium (II) chloride, palladium (II) acetate etc; Nickel compounds such as phenyl phosphine) nickel (0); rhodium compounds such as tris (triphenyl phosphine) rhodium (III) chloride; cobalt compounds; copper compounds such as copper oxide and copper (I) iodide; platinum compounds etc. Be Furthermore, a base may be added to the reaction,
- diphosphorus pentasulfide is used as the thiocarbonylating agent, but in addition to diphosphorus pentasulfide, 2,4-bis (4-methoxyphenyl) Reagents with 1,3,2,4-dithiadiphosphetan-2,4-disulfide structure such as 1,3), 2,4-dithiadiphosphetan-2,4-disulfide (Lowesson's reagent) May be used.
- N-iodosuccinimide N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, sulfuryl chloride and the like
- NBS N-bromosuccinimide
- NCS N-chlorosuccinimide
- the reaction can be accelerated by adding a radical initiator such as heat, light, benzoyl peroxide, azobisisobutyronitrile or the like to the reaction.
- an acid halide of a hydrohalic acid and an inorganic acid specifically, in chlorination, hydrochloric acid, thionyl chloride, oxy
- a method of obtaining a halogenated alkyl from an alcohol by the action of triphenylphosphine and carbon tetrachloride or carbon tetrabromide may be used.
- a method may be used in which a halogenated alkyl is synthesized through a two-step reaction in which an alcohol is converted to a sulfonic acid ester and then reacted with lithium bromide, lithium chloride or sodium iodide.
- examples of the reagent to be used include alkyl halides such as ethyl bromoacetate; and phosphites such as triethyl phosphite and tri (isopropyl) phosphite.
- a sulfone esterification reaction is carried out in each step, as a sulfonating agent to be used, methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic acid anhydride, p-toluenesulfonic acid anhydride, trifluoromethanesulfonic acid anhydride Things etc.
- an acid or a base is used as a reagent.
- formic acid, triethylsilane or the like may be added to reductively trap the by-produced t-butyl cation.
- examples of the dehydrating agent used include sulfuric acid, phosphorus pentoxide, phosphorus oxychloride, N, N'-dicyclohexylcarbodiimide, alumina, polyphosphoric acid and the like.
- a cationic lipid represented by the following formula (III) (hereinafter, also referred to as “compound (III)”) can be mentioned.
- n1 is an integer of 2 to 6
- n2 is an integer of 0 to 2
- n3 is an integer of 0 to 2
- L is -C (O) O- or -NHC (O) O-
- Ra represents a linear C 5-13 alkyl group, a linear C 13-17 alkenyl group or a linear C 17 alkadienyl group
- R b is a linear C 2-9 alkyl group
- Rc is a hydrogen atom or a linear C 2-9 alkyl group
- Rd is a hydrogen atom or a linear C 2-9 alkyl group
- Re is a linear C 2-9 alkyl group
- R f represents a linear C 2-9 alkyl group.
- cationic lipids represented by the following structural formula can be mentioned.
- cationic lipids represented by the following structural formula.
- Compound (III) can be produced, for example, by the following process.
- a salt of compound (III) can be obtained by appropriate mixing with an inorganic base, an organic base, an organic acid, a basic or acidic amino acid.
- the raw materials and reagents used in the reaction of each step in the above production method, and the reaction conditions may be the same as those described above in the production method of compound (II).
- cationic lipids represented by the following structural formula described in WO 2011/153493 can be mentioned.
- cationic lipids represented by the following structural formula.
- Another embodiment includes cationic lipids represented by the following structural formula described in WO 2013/126803.
- cationic lipids represented by the following structural formula.
- cationic lipids K-E12, H-A12, Y synthesized according to the following scheme described in Dong et al. (Proc Natl Acad Sci USA: 2014 Apr. 15; 111 (15): 5753).
- cKK-E12 and cKK-E14 are more preferable.
- cationic lipids C14-98, C18-96, and the like synthesized by the following scheme described in Love KT et al. (Proc Natl Acad Sci USA (2010 May 25; 107 (21): 9915). C14-113, C14-120, C14-120, C14-110, C16-96, C12-200.
- C14-110, C16-96 and C12-200 are more preferable.
- a cationic lipid represented by the following formula (I) (hereinafter also referred to as “compound (I)”) can be mentioned.
- L 1 is a C 1-22 alkylene group, a C 2-22 alkenylene group or a C 3-22 alkadienylene group
- n is an integer of 0 or 1
- R 1 is (1) Hydrogen atom
- (2) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 may be substituted with a substituent linear C 1-22 alkyl selected from alkenyl groups Group
- (3) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 C 2-22 alkenyl substituted optionally may be linear with a substituent selected from an alkenyl group
- (4) a linear C 3 optionally substituted by one or two substituents selected from a linear C 1-22 alkyl group and a linear C 2-22 alkenyl group -22 alkadienyl group
- R 2 is —CH 2 —O—CO—R 5
- R 3 is —CH 2 —O—CO—
- L 1 is a C 1-22 alkylene group, a C 2-22 alkenylene group or a C 3-22 alkadienylene group.
- L 1 is preferably a C 1-22 alkylene group.
- L 1 is more preferably a C 1-12 alkylene group.
- L 1 is more preferably a C 1-6 alkylene group.
- n is an integer of 0 or 1. n is preferably an integer of 1.
- R 1 is (1) Hydrogen atom, (2) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 may be substituted with a substituent linear C 1-22 alkyl selected from alkenyl groups Group, (3) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 C 2-22 alkenyl substituted optionally may be linear with a substituent selected from an alkenyl group Or (4) A linear C 3-22 alkadienyl which may be substituted by one or two substituents selected from a linear C 1-22 alkyl group and a linear C 2-22 alkenyl group It is a group.
- R 1 is preferably (1) Hydrogen atom, (2) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 6-12 alkyl group) with optionally substituted linear C 1-22 alkyl group ( Preferably, a linear C 6-12 alkyl group) or (3) one or two straight-chain C 2-22 alkenyl group (preferably straight-chain C 6-12 alkenyl group) with optionally substituted linear C 2-22 alkenyl group ( Preferred is a linear C 6-12 alkenyl group).
- R 1 is particularly preferably a hydrogen atom.
- R 2 is —CH 2 —O—CO—R 5 , —CH 2 —CO—O—R 5 or —R 5 .
- R 2 is preferably —CH 2 —O—CO—R 5 or —R 5 .
- R 2 is more preferably —CH 2 —O—CO—R 5 .
- R 3 is —CH 2 —O—CO—R 6 , —CH 2 —CO—O—R 6 or —R 6 .
- R 3 is preferably —CH 2 —O—CO—R 6 or —R 6 .
- R 3 is more preferably —CH 2 —O—CO—R 6 .
- R 4 is a hydrogen atom, —CH 2 —O—CO—R 7 , —CH 2 —CO—O—R 7 or —R 7 .
- R 4 is preferably a hydrogen atom or —CH 2 —O—CO—R 7 .
- R 4 is more preferably —CH 2 —O—CO—R 7 .
- R 5 , R 6 and R 7 are each independently (1) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 may be substituted with a substituent linear C 1-22 alkyl selected from alkenyl groups Group, (2) linear C 1-22 alkyl group and a linear C 2-22 1 or 2 C 2-22 alkenyl substituted optionally may be linear with a substituent selected from an alkenyl group Or (3) a linear C 3 which may be substituted by one or two substituents selected from a linear C 1-22 alkyl group and a linear C 2-22 alkenyl group -22 an alkadienyl group.
- R 5 , R 6 and R 7 are each independently preferably: (1) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 1-10 alkyl group) with optionally substituted linear C 1-22 alkyl group ( Preferably a linear C 4-18 alkyl group), (2) linear C 2-22 alkenyl group (preferably linear C 4-18 alkenyl group) or (3) linear C 3-22 alkadienyl group (preferably linear C 4-18 alkadienyl group).
- R 5 , R 6 and R 7 are each independently, more preferably (1) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 1-10 alkyl group) with optionally substituted linear C 1-22 alkyl group ( It is preferably a linear C 4-18 alkyl group) or (2) a linear C 2-22 alkenyl group (preferably a linear C 4-18 alkenyl group).
- R 8 and R 9 are each independently a C 1-6 alkyl group.
- R 8 and R 9 are each independently a C 1-3 alkyl group (preferably methyl).
- compound (I) is L 1 is a C 1-22 alkylene group (preferably a C 1-12 alkylene group, more preferably a C 1-6 alkylene group), n is an integer of 1,
- R 1 is (1) Hydrogen atom, (2) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 6-12 alkyl group) with optionally substituted linear C 1-22 alkyl group ( Preferably, a linear C 6-12 alkyl group) or (3) one or two straight-chain C 2-22 alkenyl group (preferably straight-chain C 6-12 alkenyl group) with optionally substituted linear C 2-22 alkenyl group ( Preferably a linear C 6-12 alkenyl group),
- R 2 is —CH 2 —O—CO—R 5 or —R 5
- R 3 is —CH 2 —O—CO—R 6 or —R 6
- R 4 is a hydrogen atom or -CH 2 -O-CO-R 7 , R 5
- compound (I) is represented by the above formula (I)
- L 1 is a C 1-12 alkylene group (preferably a C 1-6 alkylene group)
- n is an integer of 1
- R 1 is a hydrogen atom
- R 2 is -CH 2 -O-CO-R 5
- R 3 is -CH 2 -O-CO-R 6
- R 4 is -CH 2 -O-CO-R 7
- R 5 , R 6 and R 7 are each independently (1) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 1-10 alkyl group) with optionally substituted linear C 1-22 alkyl group ( Preferably a linear C 4-18 alkyl group), (2) linear C 2-22 alkenyl group (preferably linear C 4-18 alkenyl group) or (3) linear C 3-22 alkadienyl group (preferably linear C 4-18 alkadienyl group), and R 8 and R 9 are each independently a C 1-6 alkyl group (preferably a C 1-3 alky
- compound (I) is represented by the above formula (I)
- L 1 is a C 1-6 alkylene group
- n is an integer of 1
- R 1 is a hydrogen atom
- R 2 is -CH 2 -O-CO-R 5
- R 3 is -CH 2 -O-CO-R 6
- R 4 is -CH 2 -O-CO-R 7
- R 5 , R 6 and R 7 are each independently (1) one or two straight-chain C 1-22 alkyl group (preferably straight-chain C 1-10 alkyl group) with optionally substituted linear C 1-22 alkyl group ( Preferably, a linear C 4-18 alkyl group) or (2) a linear C 2-22 alkenyl group (preferably, a linear C 4-18 alkenyl group)
- R 8 and R 9 are each independently a C 1-3 alkyl group (preferably methyl), It is a compound.
- salts of the compounds represented by the above structural formulas are preferable, for example, salts with inorganic bases (eg, alkali metal salts such as sodium salts, potassium salts, etc .; calcium salts, Alkaline earth metal salts such as magnesium salts; aluminum salts, ammonium salts), salts with organic bases (eg, dimethylamine, diethylamine, pyridine, picoline, ethanolamine, diethanolamine, diethanolamine, ⁇ romethamine [ ⁇ Squirrel (hydroxymethyl) methylamine], ter t-butylamine, cyclohexylamine, benzylamine, salts with N, N-dibenzylethylenediamine), salts with inorganic acids (eg hydrofluoric acid, hydrochloric acid , Hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, salts with phosphoric acid), salts with organic acids (formic acid Acetic acid, tri
- the ratio (molar%) of the cationic lipid to the total lipid present in the lipid nanoparticles of the present invention is, for example, about 10% to about 80%, preferably about 20% to about 70%, more preferably about 40 % To about 60%, but not limited thereto.
- the above cationic lipids may be used alone or in combination of two or more. In the case of using a plurality of cationic lipids, it is preferable that the ratio of the cationic lipids as a whole be as described above.
- Noncationic lipid means a lipid other than a cationic lipid and is a lipid having no net positive charge at a selected pH such as physiological pH. is there.
- non-cationic lipids used in the lipid nanoparticles of the present invention include phospholipids, steroids, PEG lipids and the like.
- the nucleic acid is stably retained and fused with a cell membrane (plasma membrane and organelle membrane) so as to enhance delivery of the nucleic acid encoding CAR or exogenous TCR into target immune cells.
- a cell membrane plasma membrane and organelle membrane
- phosphatidylcholine phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine , Distearoylphosphatidyl choline, dilinolenoyl phosphatidyl choline, and the like.
- the salt for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoy
- Preferred phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidyl glycerol (DOPG), palmitoyl oleoyl phosphatidyl glycerol (POPG), dipalmitoyl phosphatidyl glycerol (DPPG), dioleoyl-phosphatidyl ethanolamine (DOPE), palmitoyl oleoyl phosphatidyl choline (POPC), palmitoyl oleoyl-phosphatidyl ethanolamine (POPE), and dioleoylphosphatidyl ethanolamine 4- (N-maleimidomethyl) -cyclohexane- 1-carboxylate (DOPE-mal) can be mentioned, more preferably DOPC, DPPC, POPC and DO
- the proportion (mol%) of phospholipid to total lipids present in the lipid nanoparticles of the present invention is, for example, about 0% to about 90%, preferably about 5% to about 30%, more preferably about 8% It can be up 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 above-mentioned ratio is obtained as a whole of the phospholipids.
- cholesterol As steroids, cholesterol, 5 ⁇ -cholestanol, 5 ⁇ -coprostanol, cholesteryl- (2′-hydroxy) -ethyl ether, cholesteryl- (4′-hydroxy) -butyl ether, 6-ketocholestanol, 5 ⁇ -cholestane, Examples include cholestenone, 5 ⁇ -cholestanone, 5 ⁇ -cholestanone and cholesteryl decanoate, preferably cholesterol.
- the ratio (mol%) of steroids to the total lipid present in the lipid nanoparticles of the present invention is, for example, about 10% to about 60%, preferably about 12% to about 58%, more preferably It may be about 20% to about 55%.
- the steroids described above may be used alone or in combination of two or more. When using several steroids, it is preferable to become said ratio as whole steroids.
- PEG lipid refers to any complex of polyethylene glycol (PEG) and a lipid.
- the PEG lipid is not particularly limited as long as it has an effect capable of suppressing aggregation of the lipid nanoparticles of the present invention, for example, PEG (PEG-DAA) conjugated with dialkyloxypropyl, conjugated with diacylglycerol PEG (PEG-DAG) (eg SUNBRIGHT GM-020 (Nippon Oil)), PEG conjugated with phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-Cer), cholesterol PEG conjugated with (PEG-cholesterol) or derivatives thereof, or a mixture thereof, mPEG 2000-1,2-di-O-alkyl-sn 3-carbomoyl glyceride (PEG-C-DOMG), 1- [8 ' And-(1,2-dimyristoyl-3-propan
- Preferred PEG lipids include PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer, and mixtures thereof, and more preferably PEG-didecyloxypropyl conjugate, PEG-dilauryloxypropyl conjugate A PEG-DAA conjugate selected from the group consisting of a gate, a PEG-dimyristyloxypropyl conjugate, a PEG-dipalmityloxypropyl conjugate, a PEG-distearyloxypropyl conjugate, and mixtures thereof.
- a free end of PEG in addition to a methoxy group, a maleimide group, an N-hydroxysuccinimidyl group, etc.
- PEG lipid having a functional group for binding a T cell targeting ligand (which may be referred to as “terminal reactive PEG lipid” in the present specification), for example, SUNBRIGHT DSPE-020MA, SUNBRIGHT DSPE-020MA (NOF Corporation) Can be used.
- the ratio (mol%) of PEG lipid to the total lipid present in the lipid nanoparticles of the present invention is, for example, about 0% to about 20%, preferably about 0.1% to about 5%, more preferably about It may be 0.7% to about 2%.
- the ratio (mol%) of the terminal reactive PEG lipid to the total PEG lipid mentioned above is, for example, about 10% to about 100%, preferably about 20% to about 100%, more preferably about 30% to about 100% It can be.
- the above PEG lipids may be used alone or in combination of two or more. In the case of using a plurality of PEG lipids, it is preferable to have the above ratio as the whole PEG lipid.
- the lipid nanoparticles of the present invention are immune cells, in particular T cells that are responsible for cell-mediated immunity among acquired immunity, NK cells that are responsible for innate immunity, monocytes, macrophages, dendritic cells, etc., and T that have the properties of NK cells. It is used to transduce and express CAR or exogenous TCR into NKT cells that are cells.
- the lipid nanoparticles of the present invention can further contain ligands that can target the lipid nanoparticles to immune cells, in particular T cells, in order to be efficiently delivered to target immune cells, particularly in vivo.
- (D) Ligand that can target lipid nanoparticles to T cells As a ligand that can target the lipid nanoparticles of the present invention to T cells, it specifically recognizes surface molecules that are specifically or highly expressed in T cells There is no particular limitation, as long as it can contain one or more antigen binding domains of an antibody against CD3, CD4, CD8 or CD28, and a further preferred example is an anti-CD3 antibody and / or Included are those comprising the antigen binding domain of an anti-CD28 antibody.
- preferred examples for delivery to T cells particularly in vivo include those containing only the antigen binding domain of anti-CD3 antibody.
- the term "antigen binding domain” is synonymous with the antigen binding domain that constitutes the above-mentioned CAR, but there is a limitation because CAR needs to be prepared as a nucleic acid encoding it, usually a single chain antibody is used
- the antigen binding domain as a T cell targeting ligand is contained in the form of a protein in the lipid nanoparticles of the present invention, so that not only single-chain antibodies but also complete antibody molecules, eg, Fab, F ( Any other antibody fragment such as ab ′) 2 , Fab ′, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, etc. can also be preferably used.
- Fab ′ having no Fc portion can be preferably used.
- These antibody fragments can be obtained by treating a complete antibody (eg, IgG) with a reducing agent (eg, 2-mercaptoethanol, dithiothreitol) or peptidase (eg, papain, pepsin, ficin), or by genetic recombination. It can be prepared using
- the T cell targeting ligand is a complete antibody molecule, commercially available anti-CD3, CD4, CD8, CD28 antibody or the like can be used, or it can be isolated from the culture solution of cells producing the antibody.
- anti-CD3, CD4 and CD8 are obtained by the same method as obtaining a nucleic acid encoding the antigen binding domain constituting the CAR.
- a nucleic acid encoding an antigen binding domain, such as a CD28 antibody can be isolated and used to recombinantly produce the antigen binding domain.
- the T cell targeting ligand may be bound to the shell in any manner as long as it is present on the surface of the lipid nanoparticles, for example, the terminal reaction as a noncationic lipid When containing a functional PEG lipid, it can be attached to the end of PEG.
- a ligand (antibody) -labeled lipid nanoparticle (“antibody-") is produced by reacting a PEG lipid (eg, SUNBRIGHT DSPE-020MA) having a maleimide group introduced at the end with the thiol group of the reduced antibody described above. LNP can be prepared.
- 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, a ligand for targeting to those immune cells is possessed on the surface of lipid nanoparticles.
- the lipid nanoparticles can be efficiently delivered at all, they may have appropriate targeting ligands for the molecules expressed on the surface of each immune cell on the surface of the lipid nanoparticles.
- NK cells those containing an antigen binding domain of an antibody against CD16, CD56, and the like can be mentioned, but not limited thereto.
- the lipid nanoparticles of the present invention can be produced, for example, using the method described in US 9,404,127.
- the lipid nanoparticles further contain a T cell targeting ligand
- they can be produced by chemically binding the T cell targeting ligand after producing the lipid nanoparticles.
- an organic solvent solution in which the components (b) and (c) are dissolved is prepared, and lipid nano is prepared by mixing (a) with water or buffer solution. After the particles are made, they can be produced by chemically linking T cell targeting ligands.
- the mixing ratio (molar ratio) of the cationic lipid, the phospholipid, the cholesterol and the PEG lipid includes, for example, 40 to 60: 0 to 20: 0 to 50: 0 to 5, but is not limited thereto.
- the blending ratio (molar ratio) of PEG lipid to the ligand is, for example, 20: 1 to 1: It may be twenty.
- the PEG lipids may comprise end-reactive PEG in a ratio (mol%) of about 10% to about 100%.
- the mixing can be performed using a pipette or a microfluidic mixing system (for example, Asia microfluidic system (Syrris) or Nanoassemblr (Precision Nanosystems)).
- the obtained lipid particles may be subjected to purification by gel filtration, dialysis and sterile filtration.
- the concentration of all lipid components in the organic solvent solution is preferably 0.5 to 100 mg / mL.
- organic solvent examples include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N, N-dimethylformamide, dimethylsulfoxide, or a mixture thereof.
- the organic solvent may contain 0-20% water or buffer.
- a buffer an acidic buffer (for example, acetate buffer, citrate buffer), a neutral buffer (for example, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) buffer, Tris (hydroxymethyl) aminomethane (Tris) buffer, phosphate buffer, phosphate buffered saline (PBS)) can be mentioned.
- an acidic buffer for example, acetate buffer, citrate buffer
- a neutral buffer for example, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES) buffer
- the flow rate of the mixture is preferably 0.1 to 10 mL / min, and the temperature is preferably 4 to 45 ° C.
- a dispersion containing components (a) to (d) is prepared by adding a nucleic acid encoding CAR or exogenous TCR to water or buffer solution. can do.
- the nucleic acid is preferably added so that the concentration in water or buffer is 0.05 to 2.0 mg / mL.
- the lipid nanoparticles of the present invention can also be produced by mixing the lipid particle dispersion 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.
- the hydrated amount can be determined using the Quant-iT TM Ribogreen (R) (Invitrogen).
- the encapsulation ratio of the nucleic acid in the lipid nanoparticles of the present invention can be calculated based on the difference in fluorescence intensity depending on the presence or absence of a surfactant (eg, Triton-X100).
- the dispersion medium can be replaced with water or buffer by dialysis.
- the dialysis is performed at 4 ° C. to room temperature using an ultrafiltration membrane with a molecular weight cut off of 10 to 20 K. Repeated dialysis may be performed. Tangential flow filtration may be used for dialysis.
- the ratio (weight ratio) of nucleic acid to lipid in the lipid nanoparticle of the present invention obtained as described above is about 0.01 to about 0.2.
- 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 a cumulant analysis of an autocorrelation function using Zetasizer Nano ZS (Malvern Instruments).
- an immune cell collected from a living body (also referred to herein as “ex vivo immune cells”) is contacted with the lipid nanoparticles of the present invention
- a method of producing ex vivo immune cells expressing the CAR or exogenous TCR by introducing a nucleic acid encoding CAR or exogenous TCR into the T cell, and the ex vivo immune cells obtained by the method Do.
- the “immune cell” is not particularly limited as long as it is a cell having the ability to damage target cells (pathogenic cells) such as cancer cells by any action mechanism (so-called immune effector cells).
- the immune cell may be a T cell.
- T cells collected from a living body are also referred to herein as "ex vivo T cells".
- the immune cell may be a cell responsible for innate immunity such as NK cells, macrophages, dendritic cells and the like.
- Allogeneic NK cells and the like are considered not to cause GVHD, while T cells have a considerable risk of causing GVHD by allogeneic (allo) transplantation even when the HLA types are matched. Therefore, if allo-ex vivo immune cells of various HLA types are prepared, they can be used in off-the-shelf.
- CAR-NK cells see, eg, US 2016/0096892, MoI Ther. 25 (8): 1769-1781 (2017), etc.
- CAR- dendritic cells, CAR-macrophages, etc. see, eg, WO 2017/019848, eLIFE. 2018 e36688 etc.
- the present invention provides a composition for inducing the expression of CAR or exogenous TCR comprising the lipid nanoparticle of the present invention.
- the immune cells (for example, T cells) into which the lipid nanoparticles of the present invention are introduced include isolated immune cells (for example, T cells) if they are a cell population including immune cells (for example, T cells) or their precursor cells. , T cells), or heterogeneous cell populations such as lymphocytes and progenitor cells of lymphocytes including pluripotent cells.
- lymphocyte means one of the leukocyte subtypes in the vertebrate immune system, and lymphocytes include T cells, B cells, and natural killer cells (NK cells).
- isolated and purified T cells are preferred.
- T cell is a kind of white blood cells found in lymphoid organs or peripheral blood, etc., and means one class of lymphocytes characterized by being differentiated and matured mainly in the thymus and expressing TCR.
- T cells that can be used in the present invention include cytotoxic T cells (CTL) that are CD8 positive cells, helper T cells that are CD4 positive cells, regulatory T cells, effector T cells, etc.
- CTL cytotoxic T cells
- helper T cells that are CD4 positive cells
- regulatory T cells e.g., regulatory T cells, effector T cells, etc.
- cytotoxic T cells e.g., cytotoxic T cells.
- the lymphocytes can be collected from, for example, peripheral blood, bone marrow and umbilical cord blood of human or non-human mammals.
- ex vivo immune cells eg, ex vivo T cells
- the cell population is the subject or the HLA type of the subject Preferably, they are taken from a donor consistent with
- lymphocytes including pluripotent cells
- pluripotent cells for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells) ), Embryonic germ stem cells (EG cells), hematopoietic stem cells, multipotent progenitor cells (MMP) that have lost the ability to self-replicate, myelolymphoid common progenitor cells (MLP), myeloid progenitor cells (MP) Granulocyte mononuclear precursor cells (GMP), macrophage-dendritic cell precursor cells (MDP), dendritic cell precursor cells (DCP) and the like.
- Undifferentiated cells such as pluripotent cells can be differentiated into various immune cells such as T cells by a method known per se.
- the method of bringing the lipid nanoparticles of the present invention into contact with ex vivo immune cells there is no particular limitation on the method of bringing the lipid nanoparticles of the present invention into contact with ex vivo immune cells.
- the lipid nanoparticles of the present invention may be added to a medium for normal immune cells.
- calcium phosphate co-precipitation method, PEG method, electroporation method, microinjection method, lipofection method, etc. may be used in combination to increase the introduction efficiency.
- the T cell when the lipid nanoparticles of the present invention contain a nucleic acid encoding an exogenous TCR as an active ingredient, the T cell from the viewpoint of upregulation of exogenous TCR, suppression of appearance of mispair TCR, or suppression of autoreactivity.
- the expression of endogenous TCR ⁇ chain and TCR ⁇ chain that is originally expressed by may be suppressed by siRNA.
- the base sequence of the nucleic acid encoding the TCR acts to suppress the expression of the endogenous TCR ⁇ chain and TCR ⁇ chain in order to avoid the effect of the siRNA on the exogenous TCR.
- the above-mentioned base sequence can be prepared by introducing a silent mutation into the nucleic acid encoding TCR obtained from nature, or chemically synthesizing an artificially designed nucleic acid.
- part or all of the constant region of the nucleic acid encoding the exogenous TCR may be replaced with a constant region derived from a non-human animal such as a mouse.
- the drug nanoparticle comprising the lipid nanoparticle of the present invention, or ex vivo immune cells into which the lipid nanoparticles are introduced is the lipid nanoparticle of the present invention, or the ex vivo immune cells into which the lipid nanoparticles are introduced
- a medicament hereinafter abbreviated as “the medicament of the present invention” comprising (eg, ex vivo T cells).
- a medicament comprising ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced
- the immune cells eg, T cells
- the immune cells express cells that express surface antigens that the CAR or exogenous TCR specifically recognizes by expressing the CAR or exogenous TCR. It can be specifically recognized and killed (eg, induce apoptosis). Therefore, by containing, as an active ingredient, a nucleic acid encoding a CAR or an exogenous TCR that recognizes a surface molecule specifically expressed or enhanced in a disease cell such as a cancer cell as a surface antigen.
- 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 mammals (human or other mammals (eg, mouse, rat, hamster) Can be safely administered to rabbits, cats, dogs, cows, sheep, monkeys (preferably humans).
- mammals human or other mammals (eg, mouse, rat, hamster)
- rabbits, cats, dogs, cows, sheep, monkeys preferably humans.
- the pharmaceutical agent of the present invention comprising the lipid nanoparticles of the present invention comprises the lipid nanoparticles comprising a known pharmaceutically acceptable carrier (excipient, diluent, bulking agent, binder, lubricant, It is preferable to prepare a pharmaceutical composition by mixing it with a flow aid, a disintegrant, a surfactant and the like), a conventional additive and the like.
- a pharmaceutically acceptable carrier excipient, diluent, bulking agent, binder, lubricant, It is preferable to prepare a pharmaceutical composition by mixing it with a flow aid, a disintegrant, a surfactant and the like), a conventional additive and the like.
- Excipients are well known to those skilled in the art, for example, phosphate buffered saline (eg, 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), hydrochloride , Aqueous solutions containing mineral acid salts such as hydrobromide, phosphate and sulfate, saline solutions, solutions such as glycol or ethanol and acetates, propionates, malonates, benzoates and the like And salts of organic acids.
- Adjuvants such as wetting or emulsifying agents, and pH buffering agents can also be used.
- formulation adjuvants such as suspending agents, preservatives, stabilizers and dispersing agents may be used.
- the pharmaceutical composition may also be in a dry form for reconstitution with a suitable sterile liquid prior to use.
- the pharmaceutical composition is in the form to be prepared (tablets, pills, capsules, powders, granules, syrups, emulsions, suspensions for oral administration such as suspensions; injections, drops, external preparations, suppositories, etc.
- it can be orally or parenterally administered systemically or locally.
- parenteral administration intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration and the like can be employed.
- acceptable buffers, solubilizers, isotonic agents and the like can be added.
- the dosage of the medicament of the present invention comprising the lipid nanoparticles of the present invention is, for example, in the range of 0.001 mg to 10 mg as the amount of nucleic acid encoding CAR or exogenous TCR per kg body weight at a time It is administered.
- it is administered in the range of 0.001 to 50 mg for a patient weighing 60 kg.
- the above dose is an example, and the dose can be appropriately selected depending on the type and administration route of the nucleic acid to be used, the age, body weight, symptoms and the like of the subject or patient to be administered.
- the medicament of the present invention comprising the lipid nanoparticles of the present invention is a mammal (human or other mammals (eg, mouse, rat, hamster, rabbit, cat, dog, dog, cow, sheep, monkey), preferably. , Human.),
- the expression of CAR or exogenous TCR in immune cells in the animal such as T cells (also referred to herein as “in vivo immune cells”, “in vivo T cells”).
- T cells also referred to herein as “in vivo immune cells”, “in vivo T cells”.
- the immune cells are cultured and / or / or stimulated using an appropriate medium and / or stimulating molecule prior to administration to a subject.
- Stimulation may be performed.
- Stimulatory molecules include, but are not limited to, cytokines, suitable proteins, other components, and the like.
- cytokines include IL-2, IL-7, IL-12, IL-15, IFN- ⁇ and the like, and preferably, IL-2 can be used.
- the concentration of IL-2 in the culture medium is not particularly limited, and is, for example, preferably 0.01 to 1 ⁇ 10 5 U / mL, more preferably 1 to 1 ⁇ 10 4 U / mL.
- suitable proteins include, for example, CD3 ligand, CD28 ligand, and anti-IL-4 antibody.
- lymphocyte stimulating factors such as lectin can also be added.
- serum or plasma may be added to the medium. The amount added to these media is not particularly limited, and 0% to 20% by volume is exemplified, and the amount of serum or plasma to be used can be changed according to the culture stage. For example, serum or plasma concentrations can be used at progressively reduced levels.
- the serum or plasma may be either self or non-self, but from the viewpoint of safety, a self-derived one is preferable.
- a drug having as an active ingredient the ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced as a parenteral administration to a subject.
- Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal and subcutaneous methods.
- the dose is appropriately selected according to the condition of the subject, body weight, age, etc., but usually 1 ⁇ 10 6 to 1 ⁇ 10 10 per subject for a 60 kg body weight as the cell number.
- the dose is preferably 1 ⁇ 10 7 to 1 ⁇ 10 9 , more preferably 5 ⁇ 10 7 to 5 ⁇ 10 8 .
- it may be administered once or multiple times.
- the medicament of the present invention having as an active ingredient the ex vivo immune cells into which the lipid nanoparticles of the present invention have been introduced can be in a known form suitable for parenteral administration, for example, injection or infusion.
- the medicament may optionally contain a pharmaceutically acceptable excipient.
- Pharmaceutically acceptable excipients include those described above.
- the medicament may contain saline, phosphate buffered saline (PBS), medium, etc. in order to keep the cells stable. Examples of the medium include, but are not limited to, culture media such as RPMI, AIM-V, and X-VIVO10.
- a pharmaceutically acceptable carrier eg, human serum albumin
- a preservative and the like may be added to the medicament for the purpose of stabilization.
- 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.
- Example 1 (Reducing treatment of antibody) To 111 ⁇ l of 9.21 mg / ml anti-CD3 antibody (Bio X Cell) was mixed 12.3 ⁇ l of 10 mM aqueous DTT solution. Similarly, 16.6 ⁇ l of 10 mM DTT aqueous solution was mixed with 149 ⁇ l of 6.73 mg / ml IgG2a antibody (Bio X Cell). The mixture of each antibody and DTT was mixed by vortex, and the reaction was performed at room temperature for 30 minutes. The reaction solution was fractionated by HPLC (column: TSKgel G2000SWXL 7.8 mm ⁇ 30 cm, TOSOH, mobile phase: PBS) to obtain a separated solution containing a reduced antibody.
- the fractionated solution was ultraconcentrated using Amicon 0.5 ml-10K.
- the antibody protein concentration in the concentrate and the thiol group concentration were measured by absorbance at 230 nm and fluorescent color reaction using N- (7-dimethylamino-4-methylcoumarin-3-yl) maleimide (DACM), respectively.
- the yield of the reduced anti-CD3 antibody is 176 ⁇ l
- the protein concentration is 1.75 mg / ml
- the thiol group concentration is 5.14 ⁇ M
- the yield of the reduced IgG2a antibody is 86 ⁇ l
- the protein concentration is 5.19 mg / ml
- the thiol group concentration is 45.1 ⁇ M.
- MRNA encoding CD19 target CAR with 4-1BB and CD3 ⁇ as intracellular signaling domain was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer pH 5.0 to obtain 0.2 mg / ml nucleic acid solution .
- the obtained lipid solution and nucleic acid solution were mixed at a flow rate ratio of 3 ml / min: 6 ml / min by a Nanoassemblr apparatus (Precision Nanosystems) at room temperature to obtain a dispersion liquid containing a composition.
- the resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4 ° C.
- Example 3 Boding reaction between reduced antibody and Maleimide-LNP
- the reduced antibody solution was mixed with the dispersion of maleimide-LNP so that the molar concentration of the reduced antibody to maleimide was 1/20, and allowed to stand at room temperature for 4 hours. Thereafter, it was stored at 4 ° C. until the purification step.
- Example 4 (Gel filtration purification of antibody-LNP) The reaction solution of the reduced antibody and Maleimide-LNP was loaded on a gel filtration column Sepharose CL-4B (Cat No. 17-0150-01 / GE Healthcare), and fractionation was performed using D-PBS (-) as a mobile phase. Subsequently, the protein concentration of each fraction was measured to identify a fraction containing the target antibody-LNP. Antibody-LNP was filtered through a 0.2 ⁇ m syringe filter and stored at 4 ° C.
- Example 5 (Ex vivo transfection of CD8 + T cells) Spleens are collected from C57BL / 6J mice and dispersed in ACK lysis buffer (Biosource) to obtain mouse splenocytes. The obtained mouse splenocytes were cultured in complete RPMI 1640 medium containing 1 ng / ml interleukin 7 and 2 ⁇ g / ml concavalin A for 2 days, dead cell removal and CD8 Negative Isolation Kit using Ficoll density gradient centrifugation Mouse CD8 + T cells are isolated by treatment with Stemcell Technologies).
- ACK lysis buffer Biosource
- Mouse CD8 + T cells are transfected with CAR or exogenous TCR by dispersing and culturing the obtained mouse CD8 + T cells in complete RPMI 1640 medium containing 10 ng / ml interleukin 2 and antibody-LNP.
- CD8 + T cells are isolated from purchased human primary culture T cells, and human CD8 + T cells are transfected with CAR or exogenous TCR.
- Example 6 Human chronic myelogenous leukemia cell line K562 cells in which CD19, which is an evaluation for damage, is forcibly expressed are labeled with membrane dye PKH-26 (Sigma-Aldrich), washed with RPMI medium containing 10% fetal calf serum, and the same. Disperse and culture at 1 x 10 ⁇ 5 cells / ml in culture medium. The labeled damage assessment cells are aliquoted into a 96-well plate and cultured, CAR-T cells are mixed and then cultured at 37 ° C. for 3 hours, and stained using Annexin V-Brilliant Violet 421 (BioLegend) The apoptotic cells are quantified by flow cytometry.
- Example 7 (In vivo anticancer activity evaluation test) K562-CD19 cells stably expressing luciferase are tail vein administered to 6-week-old NOD-SCID mice, and a mouse blood cancer model is created through a one-week feeding period. Subsequently, human CAR-T cells 1 ⁇ 10 6 cells obtained by transfecting a nucleic acid encoding CAR ex vivo using antibody-LNP are administered tail vein once a week for 3 weeks The reduction of cancer cells by CAR-T cells is evaluated by measurement using the in vivo luminescence imaging system IVIS (PerkinElmer).
- IVIS in vivo luminescence imaging system
- PcDNA3.1-hCD19CAR encoding the CD19 target CAR was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer pH 5.5 to obtain a 0.2 mg / ml nucleic acid solution.
- MES 2-morpholinoethanesulfonic acid
- pcDNA3.1-hCD19CAR was prepared by incorporating the CD19 IgG4 28z sequence cited from WO2013 / 126712 into a multi cloning site of pcDNA3.1 (Thermo Fisher Scientific).
- the obtained lipid solution and nucleic acid solution were mixed at a flow rate ratio of 3 ml / min: 6 ml / min by a Nanoassemblr apparatus (Precision Nanosystems) at room temperature to obtain a dispersion liquid containing a composition.
- the resulting dispersion was dialyzed against water for 1 hour at room temperature and against PBS for 48 hours at 4 ° C. using Slyde-A-Lyzer (fractional molecular weight of 20 k, Thermo Scientific). Subsequently, it was filtered using a 0.2 ⁇ m syringe filter (Iwaki) and stored at 4 ° C.
- the Maleimide-LNP dissolved by 0.5% Triton X-100, it was measured pDNA concentration using Quant-iT TM PicoGreen TM dsDNA Assay Kit (Thermo Fisher Scientific). In addition, the pDNA concentration measured without adding Triton X-100 was taken as the concentration of pDNA that was 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 preparation ratio of maleimide-PEG-lipid (DSPE-020MA). The obtained values are shown in Table 1.
- Example 9 (CD19 CAR transfection test to human primary cultured T cells using antibody-LNP) Human Pan-T cells (AccuCell human peripheral blood pan-T cells, Negative selection) were prepared in medium at 1.1 ⁇ 10 6 cells / ml and seeded at 90 ⁇ l / well in a 96-well plate.
- the medium used was X-VIVO10 (Lonza) supplemented with recombinant IL-2 (Thermo Fisher Scientific) at a concentration of 30 ng / ml.
- the CD19 CAR positive rates at 3 and 6 days after the addition of antibody-LNP were 51.9% and 41.7%, respectively, and CAR positive cells were obtained with sufficient efficiency as compared to gene transfer with viral vector.
- Figure 19 shows the results of CD19 CAR expression analysis by flow cytometry analysis of human primary cultured T cells transfected with hCD3 / hCD28-compound 21-pcDNA3.1-hCD19CAR and hCD3 / hCD28-compound 35-pcDNA3.1-hCD19CAR. 2 shows.
- the CD19 CAR positive rate 3 days after antibody-LNP addition was 5.12% and 47.0%, respectively.
- Example 10 (Evaluation of cancer cell cytotoxicity by human primary cultured T cells transfected with CD19 CAR by antibody-LNP) Cell density of 1 ⁇ 10 4 cells / 100 ⁇ l / well using human pre B cell line NALM-6 and human Burkitt's lymphoma cell line Daudi labeled as a target cell using the DELFIA cytotoxicity assay kit (Perkin Elmer) And seeded on a 96-well U bottom plate. As a medium, RPMI (phenol red free) containing 10% FBS was used.
- RPMI phenol red free
- human primary cultured T cells transfected with CD19CAR by hCD3 / hCD28-compound 12-pcDNA3.1-hCD19CAR by the method described in another paragraph (CD19 CAR positive rate 3 days after addition of antibody-LNP: 19%) Were added as dispersed in 100 ⁇ l of culture medium so that the cell number ratio to the target cell was 0-16. Three hours after mixing the Target cell and the effector cell, 20 ⁇ l of culture supernatant was recovered. 20 ⁇ l of europium solution (Eu) was added to the collected culture supernatant, and the cytotoxicity was calculated from the fluorescence intensity emitted by the complex of chelating agent TDA and Eu released from the damaged target cell. . The cytotoxicity of Nalm-6 and Daudi by the addition of human primary culture T cells transfected with CD19 CAR is shown in FIG.
- Example 11 (Preparation of reduced Fab ')
- the reduced Fab 'that binds to Mleimide-LNP can be obtained from the anti-mouse CD3 ⁇ antibody (BE0001-1, BioXCell) and the anti-mouse CD28 antibody (BE0015-1, BioXCell) from the Pierce F (ab') 2 Preparation Kit (Thermo Fisher Scientific) ) was used. From 0.5 ml of 7.86 mg / ml anti-mouse CD3 ⁇ antibody, 1 ml of 1.75 mg / ml F (ab ') 2 was obtained.
- Example 12 Combination reaction of reduced Fab 'and Maleimide-LNP
- the reduced anti-mouse CD3 ⁇ Fab 'and anti-mouse CD28 Fab' were mixed to give a 1/20 molar amount relative to the maleimide of maleimide-LNP prepared by the method described above.
- the mixture was allowed to stand at room temperature for 4 hours and then stored at 4 ° C. until purification.
- Example 13 (Gel filtration purification of reduced Fab'-LNP)
- the reaction solution of reduced Fab 'and Maleimide-LNP was loaded on gel filtration column Sepharose CL-4B (Cat No. 17-0150-01 / GE Healthcare) and fractionated using D-PBS (-) as the mobile phase . Subsequently, the protein concentration of each fraction was measured to identify a fraction containing the target Fab′-LNP.
- Fab′-LNP was filtered through a 0.2 ⁇ m syringe filter and stored at 4 ° C. The particle size of the obtained antibody-LNP was measured by Zetasizer Nano ZS (Malvern Panalytical).
- Nucleic acid concentration and the antibody protein concentrations were measured using the Quant-iT TM PicoGreen TM dsDNA Assay Kit (Thermo Fisher Scientific) and ATTO-TAG TM FQ Amine-Derivatization Kit (Thermo Fisher Scientific).
- the lipid nanoparticles of the present invention can efficiently introduce CARs or exogenous TCRs ex vivo as well as in vivo efficiently in T cells, so CAR-T or TCR-T cells with low production cost can be introduced.
- a therapy can be provided.
- viral vectors are not used, antigenicity problems caused by viral proteins can be avoided, which is extremely useful as a novel platform for cancer immunotherapy.
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Abstract
Description
キメラ抗原受容体(CAR)又はがん抗原特異的キラーT細胞由来のT細胞受容体(TCR)を遺伝子導入したCAR-T細胞又はTCR-T細胞によるがん免疫療法の研究・開発が急速に進展している。現在のCAR-T細胞療法は、米国で承認されたKymriah(商品名)やYescarta(商品名)のように、患者から採取したT細胞にレンチウイルスベクター等のウイルスベクターを用いてex vivoでCAR遺伝子を導入してCAR-T細胞を製造し、当該CAR-T細胞を患者に投与するという方法が一般的である。しかし、この方法では、細胞培養やウイルスベクター等の調製にかかるコストにより製造原価が高くなるという課題がある。in vivoでT細胞等の免疫細胞選択的にCARや外因性TCRを導入することができれば、ex vivoでの調製が不要となり、製造原価の低いCAR-もしくはTCR-免疫細胞療法の提供が可能となる。またex vivoでも、製造原価の高いウイルスベクターを用いずにT細胞等の免疫細胞選択的にCARや外因性TCRを導入することができれば、ウイルス残存試験等にかかるコストが不要になることにより、製造原価の低いCAR-もしくはTCR-免疫細胞療法の提供が可能となる。
[1]以下の(a)~(c)を含む脂質ナノ粒子:
(a)キメラ抗原受容体または外因性T細胞受容体をコードする核酸;
(b)カチオン性脂質;及び
(c)非カチオン性脂質。
[2]前記カチオン性脂質が、
式(I):
[式中、
L1は、C1-22アルキレン基、C2-22アルケニレン基またはC3-22アルカジエニレン基であり、
nは、0または1の整数であり、
R1は、
(1)水素原子、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(4)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R2は、-CH2-O-CO-R5、-CH2-CO-O-R5または-R5であり、
R3は、-CH2-O-CO-R6、-CH2-CO-O-R6または-R6であり、
R4は、水素原子、-CH2-O-CO-R7、-CH2-CO-O-R7または-R7であり、
R5、R6およびR7は、それぞれ独立して、
(1)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R8およびR9は、それぞれ独立して、C1-6アルキル基を示す]
で表される化合物またはその塩である、[1]に記載の脂質ナノ粒子。
[3]前記核酸がmRNA又はDNAである、[1]または[2]に記載の脂質ナノ粒子。
[4]前記非カチオン性脂質が、リン脂質、コレステロール及び/又はPEG脂質である、[1]~[3]のいずれかに記載の脂質ナノ粒子。
[5]前記脂質ナノ粒子がT細胞へ標的化し得るリガンドを表面に有する、[1]~[4]のいずれかに記載の脂質ナノ粒子。
[6]前記リガンドが、CD3に対する抗体、CD4に対する抗体、CD8に対する抗体およびCD28に対する抗体からなる群より選択される1以上の抗体の抗原結合ドメインを含むリガンドである、[5]に記載の脂質ナノ粒子。
[7]前記リガンドが、CD3に対する抗体および/またはCD28に対する抗体の抗原結合ドメインを含むリガンドである、[5]に記載の脂質ナノ粒子。
[8]前記リガンドが、CD3に対する抗体およびCD28に対する抗体の抗原結合ドメインを含むリガンドである、[5]に記載の脂質ナノ粒子。
[9][1]~[8]のいずれかに記載の脂質ナノ粒子を含有してなる医薬。
[10]癌の予防又は治療薬である、[9]に記載の医薬。
[11]in vivo免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、その発現を誘導する、[9]に記載の医薬。
[12]in vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、その発現を誘導する、[9]に記載の医薬。
[13]哺乳動物に対し、[1]~[8]のいずれかに記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物のin vivo免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
[14]哺乳動物に対し、[1]~[8]のいずれかに記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物のin vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
[15]哺乳動物に対し、[1]~[8]のいずれかに記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物における癌の予防又は治療方法。
[16]癌の予防・治療に使用するための、[1]~[8]のいずれかに記載の脂質ナノ粒子。
[17]癌の予防・治療剤を製造するための、[1]~[8]のいずれかに記載の脂質ナノ粒子の使用。
[18][1]~[8]のいずれかに記載の脂質ナノ粒子を含有してなるキメラ抗原受容体または外因性T細胞受容体発現誘導用組成物。
[19][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞。
[20][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞。
[21][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞、を含有してなる医薬。
[22][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞、を含有してなる医薬。
[23]癌の予防又は治療薬である、[21]または[22]に記載の医薬。
[24]アポトーシス誘導薬である、[21]または[22]に記載の医薬。
[25][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加することを特徴とする、ex vivo 免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
[26][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加することを特徴とする、ex vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を発現させる方法。
[27]哺乳動物に対し、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞を投与することを特徴とする、癌の予防又は治療方法。
[28]哺乳動物に対し、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞を投与することを特徴とする、癌の予防又は治療方法。
[29]癌の予防・治療に使用するための、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞。
[30]癌の予防・治療に使用するための、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞。
[31]癌の予防・治療剤を製造するための、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞の使用。
[32]癌の予防・治療剤を製造するための、[1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞の使用。
[33][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加する工程を含む、キメラ抗原受容体または外因性T細胞受容体を発現するex vivo免疫細胞を含有してなる医薬の製造方法。
[34][1]~[8]のいずれかに記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加する工程を含む、キメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞を含有してなる医薬の製造方法。
1.本発明の脂質ナノ粒子(LNP)
本発明は、以下の(a)~(c):
(a)キメラ抗原受容体(CAR)または外因性T細胞受容体(TCR)をコードする核酸;
(b)カチオン性脂質;及び
(c)非カチオン性脂質
を含む脂質ナノ粒子(以下、「本発明の脂質ナノ粒子」、「本発明のLNP」ともいう)を提供する。
本明細書において「脂質ナノ粒子(LNP)」とは、上記(b)及び(c)により構成される分子集合体の内部に、小孔構造(例、メソポーラス材料)を有しない、平均径1μm未満の粒子を意味する。
以下、本発明の脂質ナノ粒子の構成要素(a)~(c)について説明する。
(a-1)CARをコードする核酸
CARは、T細胞シグナル伝達ドメインに連結された抗体の抗原結合ドメイン(例、scFv)を含む、人工的に構築されたハイブリッドタンパク質である。CARの特徴には、モノクローナル抗体の抗原結合特性を利用して、非MHC拘束的様式でT細胞の特異性及び反応性を、選択された標的に対して転換する能力が挙げられる。非MHC拘束的抗原認識は、CARを発現するT細胞に、抗原プロセシングと無関係に抗原を認識する能力を与え、それにより、腫瘍エスケープの主要な機構を迂回する。さらに、T細胞中で発現されると、CARは、有利なことに、内在性TCR α鎖及びβ鎖と二量体化しない。
そのようにして得られた、抗原結合ドメイン、細胞外ヒンジドメイン、膜貫通ドメイン及び細胞内シグナル伝達ドメインをそれぞれコードするDNA断片を、常法により連結することにより、CARをコードするDNAを取得することができる。
本明細書において、「T細胞受容体(TCR)」とは、TCR鎖(α鎖、β鎖)のダイマーから構成され、抗原又は該抗原-HLA(ヒト白血球型抗原)(MHC;主要組織適合遺伝子複合体)複合体を認識してT細胞へ刺激シグナルを伝達する受容体を意味する。それぞれのTCR鎖は可変領域と定常領域から構成され、可変領域には、3つの相補性決定領域(CDR1、CDR2、CDR3)が存在する。また、本発明で使用されるTCRには、TCRのα鎖とβ鎖がヘテロダイマーを構成しているものだけでなく、ホモダイマーを構成しているものも包含される。さらに、該TCRには、定常領域の一部若しくは全部を欠損したものや、アミノ酸配列を組み換えたもの、可溶性TCR(soluble TCR)としたものなども包含される。
尚、「外因性TCR」とは、本発明の脂質ナノ粒子の標的細胞であるT細胞にとって外因性であることを意味する。外因性TCRのアミノ酸配列は、本発明の脂質ナノ粒子の標的細胞であるT細胞が発現する内因性TCRと同一であっても、異なっていてもよい。
該核酸は自体公知の方法により調製することができる。目的のTCRのアミノ酸配列または核酸配列が公知である場合には、該配列に基づき、例えば、化学的にDNA鎖やRNA鎖を合成するか、もしくは合成した一部オーバーラップするオリゴDNA短鎖を、PCR法やGibson Assembly法を利用して接続することにより、本発明のTCRの全長又は一部をコードするDNAを構築することが可能である。
また、TCRの各鎖をコードするRNA、好ましくはmRNAは、例えば、該発現ベクターを鋳型として、前記CARをコードするRNAと同様にして、調製することができる。
本明細書において、「カチオン性脂質」とは、生理学的pHなどの選択したpHにおいて、正味の正電荷を持つ脂質を意味する。本発明の脂質ナノ粒子に使用されるカチオン性脂質は特に限定されないが、例えば、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、等に記載のカチオン性脂質などが挙げられる。
Wは、式-NR1R2又は式-N+R3R4R5(Z-)を、
R1及びR2は、それぞれ独立して、C1-4アルキル基又は水素原子を、
R3、R4及びR5は、それぞれ独立して、C1-4アルキル基を、
Z-は、陰イオンを、
Xは、置換されていてもよいC1-6アルキレン基を、
YA、YB及びYCは、それぞれ独立して、置換されていてもよいメチン基を、
LA、LB及びLCは、それぞれ独立して、置換されていてもよいメチレン基又は結合手を、
RA1、RA2、RB1、RB2、RC1及びRC2は、それぞれ独立して、置換されていてもよいC4-10アルキル基を示す。]
で表される化合物又はその塩。
nは、2~5の整数を、
Rは、直鎖状C1-5アルキル基、直鎖状C7-11アルケニル基又は直鎖状C11アルカジエニル基を、
波線は、それぞれ独立して、シス型又はトランス型の結合を
示す。]
で表される化合物又はその塩。
エーテル類:ジエチルエーテル、ジイソプロピルエーテル、ジフェニルエーテル、テトラヒドロフラン、1,2-ジメトキシエタンなど;
芳香族炭化水素類:クロロベンゼン、トルエン、キシレンなど;
飽和炭化水素類:シクロヘキサン、ヘキサン、ヘプタンなど;
アミド類:N,N-ジメチルホルムアミド、N-メチルピロリドンなど;
ハロゲン化炭化水素類:ジクロロメタン、四塩化炭素など;
ニトリル類:アセトニトリルなど;
スルホキシド類:ジメチルスルホキシドなど;
芳香族有機塩基類:ピリジンなど;
酸無水物類:無水酢酸など;
有機酸類:ギ酸、酢酸、トリフルオロ酢酸など;
無機酸類:塩酸、硫酸など;
エステル類:酢酸エチル、酢酸イソプロピルエステルなど;
ケトン類:アセトン、メチルエチルケトンなど;
水。
上記溶媒は、二種以上を適宜の割合で混合して用いてもよい。
塩基性塩類:炭酸ナトリウム、炭酸カルシウム、炭酸水素ナトリウムなど;
有機塩基類:トリエチルアミン、ジエチルアミン、ピリジン、4-ジメチルアミノピリジン、N,N-ジメチルアニリン、1,4-ジアザビシクロ[2.2.2]オクタン、1,8-ジアザビシクロ[5.4.0]-7-ウンデセン、イミダゾール、ピペリジンなど;
金属アルコキシド類:ナトリウムエトキシド、カリウムtert-ブトキシド、ナトリウムtert-ブトキシドなど;
アルカリ金属水素化物類:水素化ナトリウムなど;
金属アミド類:ナトリウムアミド、リチウムジイソプロピルアミド、リチウムヘキサメチルジシラジドなど;
有機リチウム類:n-ブチルリチウム、sec-ブチルリチウムなど。
有機酸類:酢酸、トリフルオロ酢酸、クエン酸、p-トルエンスルホン酸、10-カンファースルホン酸など;
ルイス酸:三フッ化ホウ素ジエチルエーテル錯体、ヨウ化亜鉛、無水塩化アルミニウム、無水塩化亜鉛、無水塩化鉄など。
n1は、2~6の整数を、
n2は、0~2の整数を、
n3は、0~2の整数を、
Lは、-C(O)O-又は-NHC(O)O-を、
Raは、直鎖状C5-13アルキル基、直鎖状C13-17アルケニル基又は直鎖状C17アルカジエニル基を、
Rbは、直鎖状C2-9アルキル基を、
Rcは、水素原子又は直鎖状C2-9アルキル基を、
Rdは、水素原子又は直鎖状C2-9アルキル基を、
Reは、直鎖状C2-9アルキル基を、
Rfは、直鎖状C2-9アルキル基を示す。]
で表される化合物又はその塩。
L1は、C1-22アルキレン基、C2-22アルケニレン基またはC3-22アルカジエニレン基であり、
nは、0または1の整数であり、
R1は、
(1)水素原子、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(4)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R2は、-CH2-O-CO-R5、-CH2-CO-O-R5または-R5であり、
R3は、-CH2-O-CO-R6、-CH2-CO-O-R6または-R6であり、
R4は、水素原子、-CH2-O-CO-R7、-CH2-CO-O-R7または-R7であり、
R5、R6およびR7は、それぞれ独立して、
(1)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R8およびR9は、それぞれ独立して、C1-6アルキル基を示す]
又はその塩。
L1は、好ましくは、C1-22アルキレン基である。
L1は、より好ましくは、C1-12アルキレン基である。
L1は、さらに好ましくは、C1-6アルキレン基である。
nは、好ましくは、1の整数である。
(1)水素原子、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または、
(4)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基である。
R1は、好ましくは、
(1)水素原子、
(2)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC6-12アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC6-12アルキル基)、または、
(3)1つまたは2つの直鎖状のC2-22アルケニル基(好ましくは直鎖状のC6-12アルケニル基)で置換されていてもよい直鎖状のC2-22アルケニル基(好ましくは直鎖状のC6-12アルケニル基)である。
R1は、特に好ましくは、水素原子である。
R2は、好ましくは、-CH2-O-CO-R5または-R5である。
R2は、より好ましくは、-CH2-O-CO-R5である。
R3は、好ましくは、-CH2-O-CO-R6または-R6である。
R3は、より好ましくは、-CH2-O-CO-R6である。
R4は、好ましくは、水素原子または-CH2-O-CO-R7である。
R4は、より好ましくは、-CH2-O-CO-R7である。
(1)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基である。
R5、R6およびR7は、それぞれ独立して、好ましくは、
(1)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC1-10アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC4-18アルキル基)、
(2)直鎖状のC2-22アルケニル基(好ましくは直鎖状のC4-18アルケニル基)、または
(3)直鎖状のC3-22アルカジエニル基(好ましくは直鎖状のC4-18アルカジエニル基)である。
R5、R6およびR7は、それぞれ独立して、より好ましくは、
(1)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC1-10アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC4-18アルキル基)、または
(2)直鎖状のC2-22アルケニル基(好ましくは直鎖状のC4-18アルケニル基)である。
R8およびR9は、それぞれ独立して、C1-3アルキル基(好ましくはメチル)である。
L1が、C1-22アルキレン基(好ましくはC1-12アルキレン基、より好ましくはC1-6アルキレン基)であり、
nが、1の整数であり、
R1が、
(1)水素原子、
(2)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC6-12アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC6-12アルキル基)、または、
(3)1つまたは2つの直鎖状のC2-22アルケニル基(好ましくは直鎖状のC6-12アルケニル基)で置換されていてもよい直鎖状のC2-22アルケニル基(好ましくは直鎖状のC6-12アルケニル基)であり、
R2が、-CH2-O-CO-R5または-R5であり、
R3が、-CH2-O-CO-R6または-R6であり、
R4が、水素原子または-CH2-O-CO-R7であり、
R5、R6およびR7が、それぞれ独立して、
(1)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC1-10アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC4-18アルキル基)、
(2)直鎖状のC2-22アルケニル基(好ましくは直鎖状のC4-18アルケニル基)、または
(3)直鎖状のC3-22アルカジエニル基(好ましくは直鎖状のC4-18アルカジエニル基)であり、かつ
R8およびR9が、それぞれ独立して、C1-6アルキル基(好ましくはC1-3アルキル基、特に好ましくはメチル)である、
化合物である。
L1が、C1-12アルキレン基(好ましくはC1-6アルキレン基)であり、
nが、1の整数であり、
R1が、水素原子であり、
R2が、-CH2-O-CO-R5であり、
R3が、-CH2-O-CO-R6であり、
R4が、-CH2-O-CO-R7であり、
R5、R6およびR7が、それぞれ独立して、
(1)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC1-10アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC4-18アルキル基)、
(2)直鎖状のC2-22アルケニル基(好ましくは直鎖状のC4-18アルケニル基)、または
(3)直鎖状のC3-22アルカジエニル基(好ましくは直鎖状のC4-18アルカジエニル基)であり、かつ
R8およびR9が、それぞれ独立して、C1-6アルキル基(好ましくはC1-3アルキル基、特に好ましくはメチル)である、
化合物である。
L1が、C1-6アルキレン基であり、
nが、1の整数であり、
R1が、水素原子であり、
R2が、-CH2-O-CO-R5であり、
R3が、-CH2-O-CO-R6であり、
R4が、-CH2-O-CO-R7であり、
R5、R6およびR7が、それぞれ独立して、
(1)1つまたは2つの直鎖状のC1-22アルキル基(好ましくは直鎖状のC1-10アルキル基)で置換されていてもよい直鎖状のC1-22アルキル基(好ましくは直鎖状のC4-18アルキル基)、または
(2)直鎖状のC2-22アルケニル基(好ましくは直鎖状のC4-18アルケニル基)
であり、かつ
R8およびR9が、それぞれ独立して、C1-3アルキル基(好ましくはメチル)である、
化合物である。
上記のカチオン性脂質は1種のみを用いてもよいし、2種以上を組み合わせて用いてもよい。複数のカチオン性脂質を用いる場合、カチオン性脂質全体として前記の比率となることが好ましい。
本明細書において、「非カチオン性脂質」とは、カチオン性脂質以外の脂質を意味し、生理学的pHなどの選択したpHにおいて、正味の正電荷を持たない脂質である。本発明の脂質ナノ粒子に使用される非カチオン性脂質としては、例えば、リン脂質、ステロイド類、PEG脂質等が挙げられる。
上記のリン脂質は1種のみを用いてもよいし、2種以上を組み合わせて用いてもよい。複数のリン脂質を用いる場合、リン脂質全体として前記の比率となることが好ましい。
上記のステロイド類は1種のみを用いてもよいし、2種以上を組み合わせて用いてもよい。複数のステロイド類を用いる場合、ステロイド類全体として前記の比率となることが好ましい。
PEGの自由末端としてはメトキシ基の他に、後述するT細胞標的化リガンドを結合するためのマレイミド基やN-ヒドロキシスクシンイミジル基等を用いることができる。T細胞標的化リガンドを結合するための官能基を有するPEG脂質(本明細書中「末端反応性PEG脂質」という場合がある)としては、例えばSUNBRIGHT DSPE-020MAや、SUNBRIGHT DSPE-020MA(日油)を用いることができる。
上記の全PEG脂質に占める末端反応性PEG脂質の比率(モル%)は、例えば、約10%~約100%、好ましくは約20%~約100%、より好ましくは約30%~約100%であり得る。
上記のPEG脂質は1種のみを用いてもよいし、2種以上を組み合わせて用いてもよい。複数のPEG脂質を用いる場合、PEG脂質全体として前記の比率となることが好ましい。
本発明の脂質ナノ粒子をT細胞へ標的化し得るリガンドとしては、T細胞で特異的に又は高発現している表面分子を特異的に認識し得るものであれば、特に制限はないが、好ましくは、CD3、CD4、CD8又はCD28に対する抗体の1つ以上の抗原結合ドメインを含むものであり、さらに好ましい例としては抗CD3抗体及び/又は抗CD28抗体の抗原結合ドメインを含むものが挙げられる。また、特にin vivoでのT細胞への送達における好ましい例として、抗CD3抗体の抗原結合ドメインのみを含むものが挙げられる。ここで「抗原結合ドメイン」とは、前記CARを構成する抗原結合ドメインと同義であるが、CARはそれをコードする核酸として調製する必要があるため制約があり、通常は一本鎖抗体を用いる場合が多いが、T細胞標的化リガンドとしての抗原結合ドメインは、タンパク質の状態で本発明の脂質ナノ粒子に含有させるので、一本鎖抗体だけでなく、例えば、完全抗体分子、Fab、F(ab’)2、Fab’、Fv、還元抗体(rIgG)、dsFv、sFv、ダイアボディ、トリアボディ等の他の任意の抗体フラグメントも、好ましく使用することができる。特にin vivoでの標的免疫細胞への送達においては、Fc部分を有さないFab’を好ましく使用することができる。これらの抗体フラグメントは、完全抗体(例、IgG)を還元剤(例、2-メルカプトエタノール、ジチオスレイトール)やペプチダーゼ(例、パパイン、ペプシン、フィシン)で処理することにより、あるいは遺伝子組換え操作を用いて調製することができる。
本発明の脂質ナノ粒子は、例えば、US9,404,127に記載される方法を用いて製造することができる。脂質ナノ粒子がT細胞標的化リガンドをさらに含む場合には、脂質ナノ粒子を作製後にT細胞標的化リガンドを化学結合することにより製造することができる。あるいは、WO2016/021683に記載されるように、例えば、上記成分(b)と(c)を溶解した有機溶媒溶液を調製し、(a)を溶解した水もしくは緩衝液と混合することにより脂質ナノ粒子を作成した後に、T細胞標的化リガンドを化学結合することにより製造することができる。カチオン性脂質、リン脂質、コレステロール及びPEG脂質の混合比(モル比)としては、例えば、40~60:0~20:0~50:0~5が挙げられるが、それらに限定されない。また、非カチオン性脂質としてPEG脂質を配合し、T細胞標的化リガンドをPEGの末端に付加する場合、PEG脂質と該リガンドとの配合比(モル比)は、例えば、20:1~1:20であり得る。上記PEG脂質には末端反応性PEGを比率(モル%)として約10%~約100%で含み得る。上記混合は、ピペットや微小流体混合システム(例えば、Asia microfluidic system(Syrris)やNanoassemblr(Precision Nanosystems))を用いて行うことができる。得られた脂質粒子は、ゲルろ過による精製や、透析および滅菌ろ過に付してもよい。
有機溶媒溶液中の全脂質成分の濃度は、好ましくは0.5~100mg/mLである。
また、本発明の脂質ナノ粒子は、脂質粒子分散液と該核酸を自体公知の方法で混和することにより製造することもできる。
本発明の脂質ナノ粒子における該核酸の含量は、好ましくは、1~20重量%である。該含量は、Quant-iTTMRibogreen(登録商標)(Invitrogen)を用いて測定することができる。また、本発明の脂質ナノ粒子における該核酸の内封率は、界面活性剤(例、Triton-X100)添加の有無による蛍光強度の差をもとに算出することができる。
本発明は、生体から採取した免疫細胞(本明細書において「ex vivo 免疫細胞」ともいう)に、本発明の脂質ナノ粒子を接触させ、該T細胞内にCAR又は外因性TCRをコードする核酸を導入することにより、該CAR又は外因性TCRを発現するex vivo 免疫細胞を製造する方法、並びに該方法により得られるex vivo 免疫細胞を提供する。ここで「免疫細胞」としては、がん細胞等の標的細胞(病原細胞)を、何らかの作用機序により障害し得る能力を有する細胞(いわゆる免疫エフェクター細胞)であれば特に制限はないが、例えば、獲得免疫のうち細胞性免疫を担うT細胞や、自然免疫を担うNK細胞、単球、マクロファージ、樹状細胞等、並びにNK細胞の性質を有するT細胞であるNKT細胞などが挙げられる。好ましい一実施態様においては、免疫細胞はT細胞であり得る。生体から採取したT細胞を、本明細書において「ex vivo T細胞」ともいう。一方、別の好ましい実施態様においては、免疫細胞は、NK細胞、マクロファージ、樹状細胞等の自然免疫を担う細胞であり得る。T細胞はHLAタイプが一致する場合でも、同種異系(アロ)移植によりGVHDを引き起こすリスクが相当程度あるのに対し、アロNK細胞等はGVHDを引き起こさないと考えられている。従って、種々のHLAタイプのアロex vivo免疫細胞を準備しておけば、off-the-shelfでの使用が可能となる。CAR-NK細胞については、例えば、US2016/0096892、Mol Ther. 25(8): 1769-1781 (2017)等に、CAR-樹状細胞、CAR-マクロファージ等については、例えば、WO2017/019848、eLIFE. 2018 e36688等に記載されている。
また、別の側面では、本発明は、本発明の脂質ナノ粒子を含有してなるCARまたは外因性TCRの発現誘導用組成物を提供する。
本発明は、本発明の脂質ナノ粒子、又は該脂質ナノ粒子が導入されたex vivo 免疫細胞(例、ex vivo T細胞)を含有してなる医薬(以下「本発明の医薬」と略記する。)を提供する。
(4-1.本発明の脂質ナノ粒子が導入されたex vivo 免疫細胞を含有してなる医薬)
本発明の脂質ナノ粒子が導入された免疫細胞(例、T細胞)は、CAR又は外因性TCRを発現することにより、該CAR又は外因性TCRが特異的に認識する表面抗原を発現する細胞を特異的に認識し、これを殺傷(例、アポトーシスを誘導)することができる。従って、表面抗原として、がん細胞等の疾患細胞で特異的に発現するか、発現が亢進している表面分子を認識するCAR又は外因性TCRをコードする核酸を活性成分として含有させることにより、本発明の脂質ナノ粒子が導入されたex vivo 免疫細胞は、がん等の疾患の予防又は治療のために用いることができ、哺乳動物(ヒト又は他の哺乳動物(例:マウス、ラット、ハムスター、ウサギ、ネコ、イヌ、ウシ、ヒツジ、サル。好ましくは、ヒト)に安全に投与することができる。
本発明の脂質ナノ粒子を含有してなる本発明の医薬は、該脂質ナノ粒子に、公知の薬学的に許容される担体(賦形剤、希釈剤、増量剤、結合剤、滑沢剤、流動助剤、崩壊剤、界面活性剤等などが含まれる)や慣用の添加剤などと混合して医薬組成物として調製することが好ましい。賦形剤は、当業者にはよく知られており、例えば、リン酸緩衝生理食塩水(例えば、0.01Mリン酸塩、0.138M NaCl、0.0027M KCl、pH7.4)、塩酸塩、臭化水素酸塩、リン酸塩、硫酸塩などの鉱酸塩を含有する水溶液、生理食塩液、グリコール又はエタノールなどの溶液及び酢酸塩、プロピオン酸塩、マロン酸塩、安息香酸塩などの有機酸の塩などが挙げられる。また、湿潤剤又は乳化剤などの補助剤、及びpH緩衝剤も使用することができる。さらに、懸濁化剤、保存剤、安定化剤及び分散剤などの製剤補助剤などを用いてもよい。また、上記医薬組成物は、使用前に適切な無菌の液体により再構成するための乾燥形態であってもよい。該医薬組成物は、調製する形態(錠剤、丸剤、カプセル剤、散剤、顆粒剤、シロップ剤、乳剤、懸濁液などの経口投与剤;注射剤、点滴剤、外用剤、坐剤などの非経口投与剤)等に応じて、全身的に又は局所的に、経口投与又は非経口投与することができる。非経口投与する場合には、静脈投与、皮内投与、皮下投与、直腸投与、経皮投与すること等が可能である。また注射剤型で用いる場合には、許容される緩衝剤、溶解補助剤、等張剤等を添加することもできる。
(抗体の還元処理)
9.21 mg/mlの抗CD3抗体(Bio X Cell社)111 μlに10 mMの DTT水溶液12.3 μlを混合した。同様に、6.73 mg/mlのIgG2a抗体(Bio X Cell社)149 μlに10 mMのDTT水溶液16.6 μlを混合した。各抗体とDTTの混合液はvortexにより混合し、室温にて30分間反応を行った。反応液はHPLC(カラム:TSKgel G2000SWXL 7.8 mm×30 cm, TOSOH社, 移動相:PBS)により分画を行い、還元抗体を含む分取液を得た。分取液はAmicon 0.5 ml-10Kを用いて限外濃縮を行った。濃縮液中の抗体タンパク濃度、およびチオール基濃度はそれぞれ230 nmの吸光度およびN-(7-ジメチルアミノ-4-メチルクマリン-3-yl)マレイミド(DACM)を用いた蛍光呈色反応により測定した。なお、還元抗CD3抗体の収量は176 μl、タンパク濃度1.75 mg/ml, チオール基濃度5.14 μMであり、還元IgG2a抗体の収量は86 μl、タンパク濃度5.19 mg/ml、チオール基濃度45.1 μMであった。
(Maleimide-LNPの調製)
脂質混合物(カチオン性脂質:DPPC:Cholesterol:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1, モル比)を、90% EtOH、10% 水に溶解して、7.0 mg/mlの脂質溶液を得た。カチオン性脂質としては、WO2016/021683に記載される3-((5-(ジメチルアミノ)ペンタノイル)オキシ)-2,2-ビス(((3-ペンチルオクタノイル)オキシ)メチル)プロピル 3-ペンチルオクタノアート(化合物7)とN,N,N-トリメチル-5-オキソ-5-(3-((3-ペンチルオクタノイル)オキシ)-2,2-ビス(((3-ペンチルオクタノイル)オキシ)メチル)プロポキシ)ペンタン-1-アミニウム ヨージド(化合物8)を59.1:0.9(モル比)で混合して使用した。細胞内シグナル伝達ドメインとして4-1BBとCD3ζを有するCD19標的CARをコードするmRNAは10 mM 2-モルホリノエタンスルホン酸(MES)緩衝液pH 5.0に溶解して0.2 mg/mlの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、Nanoassemblr装置 (Precision Nanosystems社)によって、流速比 3 ml/min:6ml/minで混合し、組成物を含む分散液を得た。得られた分散液は、Slyde-A-Lyzer(20kの分画分子量、Thermo Scientific社)を用いて水に対して室温で1時間、PBSに対して4℃で48時間透析を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてろ過を行い、4℃に保存した。
(還元抗体とMaleimide-LNPの結合反応)
maleimideに対する還元抗体のモル濃度が1/20となるようにmaleimide-LNP分散液に還元抗体溶液を混合し、室温にて4時間静置した。その後、精製工程まで4℃に保管した。
(抗体-LNPのゲルろ過精製)
還元抗体とMaleimide-LNPの反応液をゲルろ過カラムSepharose CL-4B (Cat No.17-0150-01 / GE Healthcare)にロードし、D-PBS(-)を移動相として分画を行った。続いて、各フラクションのタンパク濃度を測定する事により、目的とする抗体-LNPが含まれる分画を同定した。抗体-LNPは0.2μmのシリンジフィルターによってろ過を行い、4℃に保存した。
(CD8+ T細胞へのex vivoトランスフェクション)
脾臓をC57BL/6Jマウスより採取し、ACK lysing buffer(Biosource)に分散することでマウス脾細胞を得る。得られたマウス脾細胞を1 ng/ml interleukin 7および2 μg/ml concavalin Aを含有するcomplete RPMI 1640培地で2日間の培養し、Ficoll密度勾配遠心を用いた死細胞除去とCD8 Negative Isolation Kit (Stemcell Technologies)を用いた処理により、マウスCD8+ T細胞を分離する。得られたマウスCD8+ T細胞を10 ng/ml interleukin 2および抗体-LNPを含有するcomplete RPMI 1640培地に分散して培養することにより、マウスCD8+ T細胞にCARまたは外因性TCRをトランスフェクションする。
同様にして、購入したヒト初代培養T細胞よりCD8+ T細胞を分離し、ヒトCD8+ T細胞にCARまたは外因性TCRをトランスフェクションする。
(CAR-T細胞のin vitro細胞障害性評価)
障害性評価細胞であるCD19を強制発現させたヒト慢性骨髄性白血病細胞株K562細胞をmembrane dye PKH-26 (Sigma-Aldrich)によって標識し、10% fetal calf serumを含むRPMI培地によって洗浄し、同培地に1 x 10^5 cells/mlで分散して培養する。標識された障害性評価細胞を96-well plateに分注して培養し、CAR-T細胞を混合後に37℃で3時間培養し、Annexin V-Brilliant Violet 421 (BioLegend)を用いた染色を行った上でフローサイトメトリーを行うことにより、アポトーシスを起こした細胞を定量する。
(in vivo抗がん活性評価試験)
ルシフェラーゼを安定発現するK562-CD19細胞を6週齢のNOD-SCIDマウスに尾静脈投与し、1週間の飼育期間を経る事でマウス血液がんモデルを作成する。続いて、抗体-LNPを用いてex vivoでCARをコードする核酸をトランスフェクションして得られたヒトCAR-T細胞1×10^6 cellsを1週間に一度、3週に亘って尾静脈投与し、in vivo発光イメージングシステム IVIS (PerkinElmer)を用いた測定により、CAR-T細胞によるがん細胞の減少を評価する。
(カチオン性脂質を用いたMaleimide-LNPの調製)
脂質混合物(カチオン性脂質:DPPC:Cholesterol:SUNBRIGHT GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1, モル比)を、90% EtOH、10% 25 mM酢酸バッファー pH 4.0に溶解して、10 mg/mlの脂質溶液を得た。カチオン性脂質としては、3-((5-(ジメチルアミノ)ペンタノイル)オキシ)-2,2-ビス(((9Z)-テトラデカ-9-エノイルオキシ)メチル)プロピル (9Z)-テトラデカ-9-エノアート(化合物12)と2-(((4-(ジメチルアミノ)ブタノイル)オキシ)メチル)-2-((ドデカノイルオキシ)メチル)プロパン-1,3-ジイル (9Z,9'Z)ビス-テトラデカ-9-エノアート(化合物21)、および2-(((4,5-ジブチルノナノイル)オキシ)メチル)-2-(((5-(ジメチルアミノ)ペンタノイル)オキシ)メチル)プロパン-1,3-ジイル ジデカノアート(化合物35)を使用した。CD19標的CARをコードするpcDNA3.1-hCD19CARを10 mM 2-モルホリノエタンスルホン酸(MES)緩衝液pH 5.5に溶解して0.2 mg/mlの核酸溶液を得た。なお、pcDNA3.1-hCD19CARは、WO2013/126712より引用したCD19 IgG4 28z 配列を、pcDNA3.1(Thermo Fisher Scientific)のmulti cloning siteへ組み込み作成した。得られた脂質溶液および核酸溶液を、室温で、Nanoassemblr装置 (Precision Nanosystems社)によって、流速比 3 ml/min:6ml/minで混合し、組成物を含む分散液を得た。得られた分散液は、Slyde-A-Lyzer(20kの分画分子量、Thermo Scientific社)を用いて、水に対して室温で1時間、PBSに対して4℃で48時間透析を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてろ過を行い、4℃に保存した。
Maleimide-LNPを0.5% Triton X-100によって溶解し、Quant-iTTM PicoGreenTM dsDNA Assay Kit(Thermo Fisher Scientific)を用いてpDNA濃度を測定した。また、Triton X-100を添加せずに測定したpDNA濃度をLNPに内封されなかったpDNAの濃度とし、LNPへのpDNA内封率を算出した。推定maleimide濃度は、測定されたpDNA濃度にmaleimide-PEG-lipid (DSPE-020MA)の仕込み比率を乗じる事によって算出した。得られた各値は表1に示した。
DTTを用いて還元したanti-human CD3 antibody(BE0001-2, BioXCell)およびanti-human/monkey CD28 antibody(BE0248, BioXCell)を等量ずつ混合し、maleimide-LNPのmaleimideに対して1/20モル量となるように混合した。Maleimide-LNPと還元抗体の濃度と体積は表2に示した。混合液は室温にて4時間静置した後、精製工程まで4℃に保管した。
還元抗体とMaleimide-LNPの反応液をゲルろ過カラムSepharose CL-4B (Cat No.17-0150-01 / GE Healthcare)にロードし、D-PBS(-)を移動相として分画を行った。続いて、各フラクションのタンパク濃度を測定する事により、目的とする抗体-LNPが含まれる分画を同定した。抗体-LNPは0.2μmのシリンジフィルターによってろ過を行い、4℃に保存した。得られた抗体-LNPの粒子径はZetasizer Nano ZS (Malvern Panalytical)によって測定した。核酸濃度と抗体タンパク濃度はそれぞれQuant-iTTM PicoGreenTM dsDNA Assay Kit(Thermo Fisher Scientific)およびATTO-TAGTM FQ Amine-Derivatization Kit(Thermo Fisher Scientific)を用いて測定した。各分析結果の値は表3に示した。
(抗体-LNPを用いたヒト初代培養T細胞へのCD19 CARトランスフェクション試験)
Human Pan-T Cells (AccuCell human peripheral blood pan-T cells, Negative selection) を1.1 ×106 cells/mlに培地で調製し、96-well plateに90μl/wellで播種した。培地にはrecombinant IL-2 (Thermo Fisher Scientific)を30 ng/mlの濃度で添加したX-VIVO10 (Lonza) を用いた。続いて、30μg/mlのpcDNA3.1-hCD19CAR濃度にPBSで希釈した抗体-LNP10μlを培地に添加し、37℃、5% CO2インキュベーターにて3日および6日間の培養を行った。
96-well plateにて培養したヒト初代培養T細胞は1.5 mlチューブに回収し、Recombinant human CD19 protein , Fc Chimera Active, Biotin (Abcam)を2μl添加し、氷上にて30分間静置した。続いて、200 μlの1% FBSを添加したCell Wash (BD) の添加と300×g, 5分間の遠心による洗浄を2回行った後、上清を除去して100 μlの1% FBS, Cell Washに細胞を分散した。細胞の分散液には、0.2μlのBrilliant Violet 421 Streptavidinを添加してピペッティングによる混合の後、氷上に30分間静置した。染色した細胞は200μlの1% FBS Cell Washと遠心による洗浄を3回行い、フィルターろ過後、200μlの 1% FBS Cell Washに分散してLSRFortessa(BD)によるフローサイトメトリー解析を行った。
hCD3/hCD28-化合物12-pcDNA3.1-hCD19CARによるトランスフェクションを行ったヒト初代培養T細胞の、フローサイトメトリー解析によるCD19 CAR発現解析結果を図1に示した。抗体-LNP添加後3日および6日におけるCD19 CAR陽性率はそれぞれ51.9%および41.7%であり、ウイルスベクターによる遺伝子導入と比較しても十分な効率でCAR陽性細胞が得られた。
hCD3/hCD28-化合物21-pcDNA3.1-hCD19CARおよびhCD3/hCD28-化合物35-pcDNA3.1-hCD19CARによるトランスフェクションを行ったヒト初代培養T細胞の、フローサイトメトリー解析によるCD19 CAR発現解析結果を図2に示した。抗体-LNP添加後3日おけるCD19 CAR陽性率はそれぞれ5.12%および47.0%であった。
(抗体-LNPによりCD19 CARをトランスフェクションしたヒト初代培養T細胞によるがん細胞障害性評価)
DELFIA 細胞障害性アッセイキット(Perkin Elmer)を用いて標識したヒトpre B細胞株NALM-6、およびヒトバーキットリンパ腫細胞株Daudiをtarget cellとして、1×104cell/100 μl/wellの細胞密度にて96-well U bottom plateに播種した。培地には10% FBS含有RPMI (phenol red free)を用いた。続いて、別項に記載の方法にてhCD3/hCD28-化合物12-pcDNA3.1-hCD19CAR によりCD19CARをトランスフェクションしたヒト初代培養T細胞(抗体-LNP添加後3日おけるCD19 CAR陽性率:19%)をeffector cellとして、target cellとの細胞数比が0~16となるように100μlの培地に分散して添加した。Target cellとeffector cellを混合してから3時間後、20μlの培養上清を回収した。回収した培養上清には20 μlのeuropium solution(Eu)を添加し、障害を受けたtarget cellより放出されたキレート剤TDAとEuの複合体により発せられる蛍光強度から、細胞障害率を算出した。
CD19 CARをトランスフェクションしたヒト初代培養T細胞の添加によるNalm-6およびDaudiの細胞障害率を図3に示した。
(還元Fab'の調製)
Mleimide-LNPと結合する還元Fab'は、anti-mouse CD3ε antibody(BE0001-1, BioXCell)およびanti-mouse CD28 antibody(BE0015-1, BioXCell)よりPierce F(ab')2 Preparation Kit(Thermo Fisher Scientific)を用いて調製した。7.86 mg/mlのanti-mouse CD3ε antibody 0.5 mlからは1.75 mg/mlのF(ab')2が1 ml得られた。3.86 mg/mlのanti-mouse CD28 antibody 0.5 mlからは0.97 mg/mlのF(ab')2が0.62 ml得られた。各F(ab')2には還元剤として2-アミノエタンチオールp-トルエンスルホン酸塩を40 mMの濃度で混合し、37℃にて1時間静置した。得られたFab'はZeba Spin Desalting Columns, 7K MWCO-0.5 ml (Thermo Fischer Scientific)により精製し、Maleimide-LNPとの反応まで4℃に保存した。なお、Fab'のタンパク濃度、およびチオール基濃度はそれぞれ230 nmの吸光度およびN-(7-ジメチルアミノ-4-メチルクマリン-3-yl)マレイミド(DACM)を用いた蛍光呈色反応により測定した。
(還元Fab’とMaleimide-LNPの結合反応)
還元したanti-mouse CD3εFab'およびanti-mouse CD28 Fab'は、上記の方法によって調製されたmaleimide-LNPのmaleimideに対して1/20モル量となるように混合した。混合液は室温にて4時間静置した後、精製工程まで4℃に保管した。
(還元Fab’-LNPのゲルろ過精製)
還元Fab'とMaleimide-LNPの反応液をゲルろ過カラムSepharose CL-4B (Cat No.17-0150-01 / GE Healthcare)にロードし、D-PBS(-)を移動相として分画を行った。続いて、各フラクションのタンパク濃度を測定する事により、目的とするFab'-LNPが含まれる分画を同定した。Fab'-LNPは0.2μmのシリンジフィルターによってろ過を行い、4℃に保存した。得られた抗体-LNPの粒子径はZetasizer Nano ZS (Malvern Panalytical)によって測定した。核酸濃度と抗体タンパク濃度はそれぞれQuant-iTTM PicoGreenTM dsDNA Assay Kit(Thermo Fisher Scientific)およびATTO-TAGTM FQ Amine-Derivatization Kit(Thermo Fisher Scientific)を用いて測定した。
Claims (34)
- 以下の(a)~(c)を含む脂質ナノ粒子:
(a)キメラ抗原受容体または外因性T細胞受容体をコードする核酸;
(b)カチオン性脂質;及び
(c)非カチオン性脂質。 - 前記カチオン性脂質が、
式(I):
[式中、
L1は、C1-22アルキレン基、C2-22アルケニレン基またはC3-22アルカジエニレン基であり、
nは、0または1の整数であり、
R1は、
水素原子、
直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R2は、-CH2-O-CO-R5、-CH2-CO-O-R5または-R5であり、
R3は、-CH2-O-CO-R6、-CH2-CO-O-R6または-R6であり、
R4は、水素原子、-CH2-O-CO-R7、-CH2-CO-O-R7または-R7であり、
R5、R6およびR7は、それぞれ独立して、
(1)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC1-22アルキル基、
(2)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC2-22アルケニル基、または
(3)直鎖状のC1-22アルキル基および直鎖状のC2-22アルケニル基から選ばれる1つまたは2つの置換基で置換されていてもよい直鎖状のC3-22アルカジエニル基であり、
R8およびR9は、それぞれ独立して、C1-6アルキル基を示す]
で表される化合物またはその塩である、請求項1に記載の脂質ナノ粒子。 - 前記核酸がmRNA又はDNAである、請求項1に記載の脂質ナノ粒子。
- 前記非カチオン性脂質が、リン脂質、コレステロール及び/又はPEG脂質である、請求項1に記載の脂質ナノ粒子。
- 前記脂質ナノ粒子がT細胞へ標的化し得るリガンドを表面に有する、請求項1に記載の脂質ナノ粒子。
- 前記リガンドが、CD3に対する抗体、CD4に対する抗体、CD8に対する抗体およびCD28に対する抗体からなる群より選択される1以上の抗体の抗原結合ドメインを含むリガンドである、請求項5に記載の脂質ナノ粒子。
- 前記リガンドが、CD3に対する抗体及び/又はCD28に対する抗体の抗原結合ドメインを含むリガンドである、請求項5に記載の脂質ナノ粒子。
- 前記リガンドが、CD3に対する抗体及びCD28に対する抗体の抗原結合ドメインを含むリガンドである、請求項5に記載の脂質ナノ粒子。
- 請求項1に記載の脂質ナノ粒子を含有してなる医薬。
- 癌の予防又は治療薬である、請求項9に記載の医薬。
- in vivo免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、その発現を誘導する、請求項9に記載の医薬。
- in vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、その発現を誘導する、請求項9に記載の医薬。
- 哺乳動物に対し、請求項1に記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物のin vivo免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
- 哺乳動物に対し、請求項1に記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物におけるin vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
- 哺乳動物に対し、請求項1に記載の脂質ナノ粒子を投与することを特徴とする、該哺乳動物における癌の予防又は治療方法。
- 癌の予防・治療に使用するための、請求項1に記載の脂質ナノ粒子。
- 癌の予防・治療剤を製造するための、請求項1に記載の脂質ナノ粒子の使用。
- 請求項1に記載の脂質ナノ粒子を含有してなるキメラ抗原受容体または外因性T細胞受容体発現誘導用組成物。
- 請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞。
- 請求項1に記載の脂質ナノ粒子をex vivoT細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞。
- 請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞、を含有してなる医薬。
- 請求項1に記載の脂質ナノ粒子をex vivoT細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞、を含有してなる医薬。
- 癌の予防又は治療薬である、請求項21に記載の医薬。
- アポトーシス誘導薬である、請求項21に記載の医薬。
- 請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加することを特徴とする、ex vivo 免疫細胞にキメラ抗原受容体または外因性T細胞受容体を遺伝子導入し、発現させる方法。
- 請求項1に記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加することを特徴とする、ex vivo T細胞にキメラ抗原受容体または外因性T細胞受容体を発現させる方法。
- 哺乳動物に対し、請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞を投与することを特徴とする、癌の予防又は治療方法。
- 哺乳動物に対し、請求項1に記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞を投与することを特徴とする、癌の予防又は治療方法。
- 癌の予防・治療に使用するための、請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞。
- 癌の予防・治療に使用するための、請求項1に記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞。
- 癌の予防・治療剤を製造するための、請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞の使用。
- 癌の予防・治療剤を製造するための、請求項1に記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加して得られたキメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞の使用。
- 請求項1に記載の脂質ナノ粒子をex vivo 免疫細胞を含む培養液に添加する工程を含む、キメラ抗原受容体または外因性T細胞受容体を発現するex vivo 免疫細胞を含有してなる医薬の製造方法。
- 請求項1に記載の脂質ナノ粒子をex vivo T細胞を含む培養液に添加する工程を含む、キメラ抗原受容体または外因性T細胞受容体を発現するex vivo T細胞を含有してなる医薬の製造方法。
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Publication number | Publication date |
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CN118634318A (zh) | 2024-09-13 |
KR20200104360A (ko) | 2020-09-03 |
BR112020013201A2 (pt) | 2020-12-01 |
CN111542338B (zh) | 2024-06-18 |
CN118512596A (zh) | 2024-08-20 |
IL275567A (en) | 2020-08-31 |
CN118593692A (zh) | 2024-09-06 |
TW201929868A (zh) | 2019-08-01 |
EA202091566A1 (ru) | 2020-09-18 |
CN118512584A (zh) | 2024-08-20 |
CN118512583A (zh) | 2024-08-20 |
AU2018397910A2 (en) | 2020-08-06 |
JP2023099136A (ja) | 2023-07-11 |
IL275567B2 (en) | 2024-03-01 |
EP3733211A1 (en) | 2020-11-04 |
US20210052646A1 (en) | 2021-02-25 |
SG11202006033YA (en) | 2020-07-29 |
IL275567B1 (en) | 2023-11-01 |
JPWO2019131770A1 (ja) | 2020-12-24 |
MX2020006843A (es) | 2020-09-03 |
AU2018397910A1 (en) | 2020-07-16 |
CN111542338A (zh) | 2020-08-14 |
EP3733211A4 (en) | 2021-11-24 |
CA3087147A1 (en) | 2019-07-04 |
CO2020008972A2 (es) | 2020-07-31 |
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