Method for proliferation of CAR-T cells, and lipid particles and kit used in the method.

Abstract

The present invention aims to provide a method for proliferating CAR-T cells that can efficiently produce CAR-T cells, as well as lipid particles and a kit for use in said method. [Solution] The method according to the embodiment is a method for proliferating CAR-T cells. This method includes: preparing a cell population containing CAR-T cells, CD209-positive cells, and lipid particles containing a first nucleic acid containing the CAR antigen gene; contacting the CD209-positive cells with the lipid particles to produce CAR antigen-presenting cells expressing the target antigen of CAR; and co-culturing the cell population and the CAR antigen-presenting cells.

Description

Technical Field

[0001] The present invention relates to a method for growing CAR-T cells, as well as lipid particles and kits used in the method.

Background Art

[0002] CAR-T cell therapy using T cells (CAR-T cells) genetically modified to produce a chimeric antigen receptor (CAR) that recognizes tumor cells has attracted attention due to its very high therapeutic effect on tumors. In this method, CAR-T cells are prepared by introducing a CAR gene into T cells collected from a patient, and then administered to the patient. The administered CAR-T cells kill the tumor cells encountered in the patient's body.

[0003] Generally, when preparing CAR-T cells, a step of stimulating the CAR-T cells is often involved to proliferate them more efficiently. For the stimulation step, for example, a method of culturing CAR-T cells together with T cell activating factors (such as interleukins) or a method of co-culturing CAR-T cells with peripheral blood mononuclear cells (PBMCs) expressing the antigen of CAR and a co-stimulatory factor as antigen-presenting cells is used.

[0004] Even with the conventional proliferation methods including the above stimulation step, the proliferation efficiency of CAR-T cells is sufficient for practical use. However, by producing CAR-T cells with higher efficiency, the manufacturing cost of preparations containing CAR-T cells can be reduced, and the therapeutic effect can be enhanced. Therefore, a method for growing CAR-T cells with higher efficiency than the conventional methods is required.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

[0006] The present invention aims to provide a method for proliferating CAR-T cells that can efficiently produce CAR-T cells, as well as lipid particles and a kit for use in said method. [Means for solving the problem]

[0007] A method for proliferating CAR-T cells according to an embodiment includes: preparing a cell population containing CAR-T cells, CD209-positive cells, and lipid particles containing a first nucleic acid including a CAR antigen gene; contacting the CD209-positive cells with the lipid particles to produce CAR antigen-presenting cells expressing the target antigen of CAR; and co-culturing the cell population and the CAR antigen-presenting cells.

[0008] The lipid particles according to this embodiment are lipid particles for producing CAR antigen-expressing cells derived from CD209-positive cells. The lipid particles are composed of a lipid membrane having a lumen, and nucleic acids containing the CAR antigen gene are encapsulated in the lumen.

[0009] A kit according to this embodiment comprises lipid particles for producing CAR antigen-expressing cells derived from CD209-positive cells. The lipid particles are composed of a lipid membrane having a lumen, and nucleic acids containing the CAR antigen gene are encapsulated in the lumen. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a cross-sectional view showing an example of lipid particles according to the first embodiment. [Figure 2] Figure 2 shows an example of a method for producing CAR antigen-expressing cells according to the first embodiment. [Figure 3] Figure 3 is a flowchart showing an example of a method for proliferating CAR-T cells according to the second embodiment. [Figure 4]Figure 4 is a schematic diagram showing an example of a method for proliferating CAR-T cells according to the second embodiment. [Figure 5] Figure 5 is a graph showing the experimental results for Example 1. [Figure 6] Figure 6 is a graph showing the experimental results for Example 2. [Figure 7] Figure 7 is a graph showing the experimental results for Example 3. [Modes for carrying out the invention]

[0011] The lipid particles, CAR-T cell proliferation method, and kit of the embodiment will be described below with reference to the drawings. Each figure is an embodiment and a schematic diagram to facilitate understanding; however, their shape, dimensions, ratios, etc. may differ from the actual product. These can be appropriately modified in accordance with the following description and known technologies.

[0012] [First Embodiment] ·Lipid particles The lipid particles of the first embodiment are used for the production of CAR antigen-expressing cells. As shown in Figure 1, the lipid particle 10 comprises, for example, a lipid membrane 1 and a first nucleic acid 2 encapsulated within the lipid membrane 1. The first nucleic acid 2 is a nucleic acid containing a gene encoding the target antigen of CAR (hereinafter referred to as the "CAR antigen gene"). More specifically, the lipid particles containing the nucleic acid with the CAR antigen gene are brought into contact with CD209-positive cells as described later, and the CAR antigen gene is introduced into the CD209-positive cells to produce CAR antigen-expressing cells.

[0013] The following provides a detailed explanation of each component.

[0014] (lipid membrane) The lipid particle 10 in this embodiment is a substantially spherical hollow body, i.e., a liposome, consisting of a lipid membrane 1 formed by the arrangement of multiple lipid molecules 1a in non-covalent bonds. The first nucleic acid 2 is encapsulated in the central lumen 1b. The lipid membrane 1 may be a lipid monolayer or a lipid bilayer. Furthermore, the lipid membrane 1 may consist of a single layer or a multilayer membrane.

[0015] The material of the lipid membrane 1 may be composed of the base lipids exemplified below, but it preferably further contains the lipid compounds exemplified below in addition to the base lipids.

[0016] As the base lipid, for example, lipids that are the main components of biological membranes can be used. The base lipid is a phospholipid or a sphingolipid, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin or cerebroside, or a combination thereof, etc.

[0017] For example, as the base lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl- 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphochlorin (DLPC), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or It is preferable to use cholesterol, or any combination thereof.

[0018] In particular, DOTAP is preferred because it is a cationic lipid, and its content can be used to adjust the acid dissociation constant of the lipid membrane 1 and, consequently, the lipid particles 10. The above base lipids readily fuse with the cell membrane, and in particular, when using diacylphosphatidylcholine and diacylphosphatidylethanolamine, the structure and particle size of the lipid particles 10 can be easily controlled, and they readily fuse with the cell membrane, making them preferred. The length of the hydrocarbon chain of the acyl group contained in the lipid is C 10 ~C 20 It is preferable that this is the case. This hydrocarbon chain may be saturated hydrocarbon groups or unsaturated hydrocarbon groups.

[0019] The base lipid may make up nearly 100% of the total lipid molecules 1a contained in the lipid membrane 1, but it is preferable that the base lipid of the lipid membrane 1 makes up about 30% to about 80% (molar ratio) of the total lipid molecules 1a.

[0020] Other lipid compounds besides the base lipid include, for example, biodegradable lipids. Specifically, biodegradable lipids represented by the formula Q-CHR2 (hereinafter referred to as "lipid compound A") can be used. (In the formula, Q is a nitrogen-containing aliphatic group that contains two or more tertiary nitrogen atoms and does not contain oxygen. R is independent of C 12 ~C 24 It is an aliphatic group, At least one R contains a linking group LR selected from the group consisting of -C(=O)-O-, -OC(=O)-, -OC(=O)-O-, -SC(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)- in its main chain or side chain.

[0021] When lipid membrane 1 contains lipid compound A, the cationic properties of the surface of lipid particles 10 can be adjusted, thereby reducing obstacles to cell delivery and increasing the nucleic acid delivery rate.

[0022] As lipid compound A, it is preferable to use lipids having structures represented by the following formulas (1-01) and (1-02) because they offer superior nucleic acid introduction efficiency. In the following description, the lipid of formula (1-01) will be referred to as FFT10, and the lipid of formula (1-02) will be referred to as FFT20.

[0023] [ka]

[0024] [ka]

[0025] When using lipid membrane 1 containing lipid compound A as described above, it is possible to increase the amount of nucleic acid encapsulated and improve the nucleic acid delivery rate. Furthermore, it is possible to reduce cell death in the cells to which the nucleic acid has been introduced. In particular, the use of FFT10 and FFT20 is preferred because they provide particularly excellent nucleic acid encapsulation and nucleic acid delivery efficiency.

[0026] The lipid membrane 1 preferably further contains lipids that suppress aggregation by adjusting the surface charge of the lipid particles 10. Such lipids are, for example, PEG-modified lipids, particularly polyethylene glycol dimyristoyl glycerol (DMG-PEG), maleimide-DMG-PEG, polyamide oligomers derived from omega-amino(oligoethylene glycol) alkanoic acid monomers (U.S. Patent No. 6,320,017), and monosialogangliosides, and are preferably present in an amount of about 1% to about 5% (molar ratio) relative to the total lipids of the lipid membrane 1.

[0027] Other lipids that may be included in the lipid membrane 1 include, for example, relatively low-toxicity lipids to adjust toxicity, lipids having functional groups that bind ligands to lipid particles 10, and lipid sterols (e.g., cholesterol) to suppress leakage of encapsulated substances. It is particularly preferable that cholesterol is included in the lipid membrane 1.

[0028] Furthermore, it is preferable that the lipid membrane 1 of the lipid particle 10 consists of 50% or more cationic lipids. For example, if the lipid membrane 1 is composed of FFT10 and FFT20, DOPE and / or DOTAP, cholesterol, and DMG-PEG and / or maleimide-DMG-PEG, it is preferable that FFT10, FFT20, and DOTAP make up 50% or more of the total lipid composition (molar ratio).

[0029] Furthermore, it is preferable that the lipid membrane 1 contains FFT10 and FFT20 in equal proportions of each other in terms of lipid composition (molar ratio), and that the respective proportions are 10% to 30%. Specifically, the proportions of FFT10 and FFT20 are preferably 10%, 15%, 20%, 25%, or 30%.

[0030] Furthermore, in the lipid composition (molar ratio) of the lipid membrane, it is preferable that DOTAP be 10% or more and less than or equal to the respective proportions of FFT10 and FFT20. Specifically, when the respective proportions of FFT10 and FFT20 are 10%, the proportion of DOTAP should be 10%, and when the respective proportions of FFT10 and FFT20 are 20%, the proportion of DOTAP should be 10%, 15%, or 20%.

[0031] As described above, the lipid membrane 1 of the lipid particle 10 is preferred when it contains the lipid compounds FFT10 and FFT20, DOPE and / or DOTAP, cholesterol, and DMG-PEG or maleimide-DMG-PEG, because it exhibits particularly excellent nucleic acid encapsulation and nucleic acid delivery efficiency.

[0032] In particular, if lipid membrane 1 has the lipid composition (A) or (B) shown in Table 1 below, the efficiency of nucleic acid introduction into CD209-positive cells, as described later, can be further enhanced.

[0033] [Table 1]

[0034] (nucleic acid) Nucleic acid 12 is, for example, a single-stranded or double-stranded cyclic, linear, or branched nucleic acid. Nucleic acid 12 is, for example, DNA, RNA, PNA, or any of these derivatives. A derivative is DNA, RNA, or PNA with a nucleotide analog inserted, or DNA, RNA, or PNA with any of its ends modified by a label or functional group.

[0035] If the first nucleic acid 2 is RNA, it is preferable that it is modified to be resistant to degradation. For example, such modification can be any known modification that prevents the RNA from being degraded by RNase or the like. Such modifications include, for example, the use / introduction of modified / artificial nucleotides to RNA, the use / addition of non-natural sequences, or the addition of natural / non-natural CAP structures.

[0036] Modified nucleotides include, for example, pseudouridine, 5-methylcytidine, 1-methylpseudridine, 5-methoxyuridine, and 1-methyladenosine. Artificial nucleotides include, for example, BNA (Bridged nucleicacid), LNA (Locked nucleicacid), or PNA (Peptide nucleicacid).

[0037] Non-natural sequences are, for example, artificially created base sequences that do not exist in nature, such as random base sequences or hybrid sequences of natural / non-natural amino acids and nucleic acids. It is preferable to add non-natural sequences to the ends of RNA, for example.

[0038] Examples of natural CAP structures include CAP0 (m7GpppN) and CAP1 (m7GpppNm). Examples of unnatural CAP structures include ARCA (Anti-ReverseCapAnalog) or LNA-guanosine. Unnatural CAP structures are preferably attached to the 5' end of RNA, for example.

[0039] The first nucleic acid 2 is a nucleic acid containing a CAR antigen gene. In this disclosure, a CAR antigen is a protein, glycan, or glycolipid that can be expressed on the surface of CD209-positive cells, as described later, so as to bind to a CAR expressed in immune cells, for example. A gene encoding a protein generally known as a CAR antigen may be used as the CAR antigen gene, or a modified protein having a similar function, a fusion protein with additional domains, or a gene encoding a novel protein having a similar function may be used.

[0040] Herein, an example of a CAR in this disclosure will be described in detail. A CAR includes, for example, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0041] (a) Extracellular domain The extracellular domain is located on the outer surface of the T cell membrane and is a domain that specifically binds to the target of the CAR. For example, the extracellular domain includes an antigen-binding fragment of an anti-target monoclonal antibody, such as an scFv fragment. As the monoclonal antibody, for example, antibodies from rodents (mouse, rat, rabbit, etc.), human antibodies or humanized antibodies, or their antigen-binding sites can be used. The scFv fragment is a structure in which the light chain variable region (VL) and heavy chain variable region (VH) of an immunoglobulin are linked via a linker. As the linker, for example, a peptide linker consisting of a peptide in which amino acids are linked in a linear chain can be used. The peptide linker is, for example, a linker composed of glycine and serine (e.g., a GGS linker or GS linker). For example, a linker with 5 to 25 amino acid residues can be used. Alternatively, if the tumor-specific antigen that is the target of the CAR is a receptor, the extracellular domain may use a ligand that binds to that receptor.

[0042] The type and composition of the extracellular domain are selected depending on the type of CAR target. In this embodiment, the CAR target is the CAR antigen described above.

[0043] (b) Transmembrane domain The transmembrane domain is a domain located in the T cell membrane between the extracellular domain and the intracellular signaling domain. Transmembrane domains such as CD28, CD3ε, CD8α, CD3, CD4, or 4-1BB can be used. Alternatively, transmembrane domains composed of artificially constructed polypeptides can also be used.

[0044] (c) intracellular signaling domain The intracellular signaling domain is located on the inner side of the T cell membrane and transmits the signal necessary for T cell activation when the extracellular domain binds to a target antigen. The intracellular signaling domain includes, for example, a domain (first domain) for transmitting signals via the TCR complex. CD3ζ or FcεRIγ can be used as the first domain. CD3ζ is preferably used.

[0045] Furthermore, the intracellular signaling domain may further include a domain (second domain) for transmitting co-stimulatory signals. As the second domain, for example, the intracellular domain of a co-stimulatory molecule such as CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, or ICOS can be used. Preferably, CD28 or 4-1BB is used.

[0046] The first and second domains may each contain one of the elements listed above, or they may be configured by linking multiple identical or different elements in a tandem manner. The order in which the first and second domains are linked is not particularly limited, but it is preferable to place the second domain on the transmembrane domain side. The first and second domains may be linked directly, or a linker may be interposed between them. As the linker, for example, a peptide linker consisting of a peptide in which 2 to 15 amino acids are linked in a linear chain can be used.

[0047] (d) Other elements CAR may also contain other elements. These other elements may include, for example, a leader sequence (signal peptide) that promotes CAR secretion, such as the leader sequence of the GM-CSF receptor. A spacer domain may also be placed between the extracellular domain and the transmembrane domain. The spacer domain can promote the binding of the CAR to the target. For example, an Fc fragment of human IgG (e.g., human IgG1, human IgG4) can be used as the spacer domain. Alternatively, a portion of the extracellular domain of CD28 or CD8α can be used as the spacer domain. Furthermore, a spacer domain may also be placed between the transmembrane domain and the intracellular signaling domain.

[0048] A CAR gene is, for example, a combination of genes corresponding to each of the above domains in an appropriate order. The CAR gene sequence may include the full length of any of the above CAR genes, or it may include only a portion of them.

[0049] In this disclosure, CAR antigens are typically antigens associated with tumor cells or antigens specific to tumors. Antigens associated with tumor cells are those that are significantly or prominently expressed (i.e., specifically or selectively expressed) in tumor cells, surrounding tissue cells, or organisms containing tumor cells, compared to non-tumor cells. A CAR antigen may be any antigen that is a target of a CAR (i.e., matches the type of CAR of interest) and is generally known as a tumor-associated antigen or tumor-specific antigen. Tumor-associated antigens or tumor-specific antigens may also be antigens present in the extracellular matrix, or proteins containing mutations identified by genomic analysis and / or tumor-indicative expression studies.

[0050] If the above tumor is, for example, a B-cell lymphoma, multiple myeloma, retinoblastoma, pulmonary vesicle tumor, or central nervous system tumor, the CAR antigen may be, for example, CD19 antigen, CD20 antigen, GD2 antigen, CD22 antigen, CD30 antigen, CD33 antigen, CD44 variant 7 / 8 antigen, CEA antigen, Her2 / neu antigen, MUC1 antigen, MUC4 antigen, MUC6 antigen, IL-13 receptor-alpha2, immunoglobulin light chain, PSMA antigen, or VEGF receptor 2. Alternatively, if the above tumor is a myeloid tumor such as a leukemia stem cell, leukemia progenitor cell, or leukemia cell, the CAR antigen may be GM-CSF, which is a ligand for the GM-CSF (granulocyte-monocyte colony-stimulating factor) receptor. Alternatively, in the case of neuroblastoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, astrocytoma, Ewing's sarcoma, glioblastoma, retinoblastoma, rhabdomyosarcoma, non-small cell lung cancer, prostate cancer, and urothelial carcinoma, the CAR antigens may be FAM150A, FAM150B, and fragments of ALK that bind to the extracellular ligand-binding domain.

[0051] In addition, depending on the type of tumor being targeted, CAR antigens include EphB4, EGFR variant 3, EphA2, EphB2, EGFR, GD2, Glypican-3, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, κ light chain, CD38, CD44, CD44 variant 6, CD70, CD116, CD123, CD138, CD171, CEA, CSPG4, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD3, and HLA-AI MAGE. A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis Y, MCSP, Mesoserine, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGR receptor 2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, fibronectin, tenascin, ALK (anaplastic lymphoma kinase), or antigens present in the extracellular matrix such as CEA in the necrotic region of a tumor may also be used. Furthermore, the full-length or partial base sequence of a combination of multiple of these genes may also be used.

[0052] (Nucleic acid condensed peptide) Nucleic acid condensation peptide 3 is a peptide that can reduce the volume occupied by nucleic acids by condensing more nucleic acids, and can efficiently encapsulate a large amount of nucleic acids within lipid particles 10. It is preferable to use a cationic peptide as such a peptide. Cationic peptides can condense nucleic acids by, for example, filling the gaps in the helical shape of anionic nucleic acids and shrinking those gaps.

[0053] A preferred nucleic acid condensed peptide 3 is, for example, a peptide containing 45% or more cationic amino acids. A more preferred nucleic acid condensed peptide 3 has RRRRRR (first amino acid sequence) at one end and the sequence RQRQR (second amino acid sequence) at the other end. It also contains zero or one or more intermediate sequences consisting of RRRRRR or RQRQR between the two amino acid sequences. Furthermore, it contains two or more neutral amino acids between two adjacent sequences among the first amino acid sequence, the second amino acid sequence, and the intermediate sequence. The neutral amino acids are, for example, G or Y.

[0054] The nucleic acid condensed peptide 3 described above preferably has the following amino acid sequence. RQRQRYYRQRQRGGRRRRRR (Sequence ID 1) RQRQRGGRRRRRR (Sequence ID 2)

[0055] This nucleic acid condensation peptide 3 efficiently condenses nucleic acids using cationic arginine and weakens the anionic properties of the nucleic acids, thus enabling efficient encapsulation of nucleic acids within lipid particles 1. Furthermore, because nucleic acid condensation peptide 3 efficiently dissociates nucleic acids in cells, it is possible to efficiently express the first nucleic acid introduced into cells within CD209-positive cells.

[0056] Alternatively, nucleic acid condensed peptide 3 has RRRRRR (third amino acid sequence) at one end and RRRRRR (fourth amino acid sequence) at the other end. It contains zero or one or more intermediate sequences consisting of RRRRRR or RQRQR between the two amino acid sequences. In addition, it contains two or more neutral amino acids between two adjacent sequences among the third amino acid sequence, the fourth amino acid sequence, and the intermediate sequence.

[0057] Such nucleic acid condensed peptide 3 preferably has the following amino acid sequence. RRRRRRYYRQRQRGGRRRRRR(Sequence ID 3)

[0058] Such nucleic acid condensing peptide 3 has strong cationic properties at both ends and high binding affinity to nucleic acids. Therefore, it can condense nucleic acids more efficiently and encapsulate more nucleic acids within the lipid particles 10. As a result, the amount of nucleic acids remaining outside the lipid particles 10 is reduced, thereby preventing aggregation of the lipid particles 10 with each other, making it easier for the lipid particles 10 to be taken up into cells.

[0059] Furthermore, nucleic acid condensed peptide 3 having the following amino acid sequence can also be used in combination with any of the above nucleic acid condensed peptide 3s. GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(M9)(Sequence No. 4)

[0060] This peptide can further condense the nucleic acid aggregates condensed with the nucleic acid condensation peptide 3. Therefore, it is possible to obtain lipid particles with even smaller particle sizes. The smaller the particle size, the easier it is for the lipid particles to be taken up by CD209-positive cells, making it possible to introduce nucleic acids into the genome of CD209-positive cells more efficiently. For example, the first nucleic acid 2 can be condensed by stirring and mixing it with the nucleic acid condensation peptide 3 before encapsulating it in lipid particles 10.

[0061] While it is preferable to use nucleic acid condensed peptide 3 for the effects described above, it may not be necessary to use nucleic acid condensed peptide 3 depending on the type of nucleic acid used or the cell culture conditions.

[0062] The lipid particles 10 may contain further components in addition to the first nucleic acid 2 and nucleic acid condensed peptide 3. For example, they may contain compounds that regulate intracellular nucleic acid expression, such as retinoic acid, cyclic adenosine monophosphate (cAMP), or ascorbic acid. Alternatively, they may contain, for example, peptides, polypeptides, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, other cell surface receptors and their ligands.

[0063] Lipid particles 10 can be prepared, for example, using known methods used when encapsulating small molecules in lipid particles. These known methods include, for example, the Bangam method, organic solvent extraction method, ethanol injection method, surfactant removal method, or freeze-thaw method. If the organic solvent extraction method is selected, for example, lipid particles 10 can be prepared by adding an aqueous buffer containing the components to be encapsulated, such as the first nucleic acid 2, to a mixture obtained by impregnating the lipid membrane 1 material in an organic solvent such as alcohol, stirring, and suspending the mixture. If the ethanol injection method is selected, for example, lipid particles 10 can be prepared by adding ethanol obtained by impregnating the lipid membrane 1 material to an aqueous buffer containing the components to be encapsulated, such as the first nucleic acid 2, stirring, and suspending the mixture. The ratio of nucleic acids encapsulated in the lipid particles 10 can be easily adjusted by changing the ratio of the two in the aqueous buffer. The amount of nucleic acid encapsulated can be confirmed, for example, using a commercially available DNA and RNA quantification kit.

[0064] The average particle size of the lipid particles 10 is, for example, about 50 nm to about 300 nm, preferably about 50 nm to about 200 nm. For example, the particle size can be reduced by sonication. It is also possible to adjust the size of the lipid particles 10 by passing them through a polycarbonate film or a ceramic film. The average particle size of the lipid particles 10 can be measured, for example, by a zetasizer using dynamic light scattering.

[0065] The lipid particles of the embodiments described above may be provided as a composition contained in a suitable carrier. The carrier may be, for example, water, saline solution such as physiological saline, aqueous glycine solution, or a buffer such as HEPES.

[0066] The composition containing lipid particles may further contain substances that improve storage stability. These substances are not limited to but include, for example, albumin, lipoproteins, apolipoproteins, glycoproteins such as globulins, etc. Specifically, substances that improve storage stability include pH adjusters, buffering agents, tonicity adjusters, etc. Alternatively, they may be pharmaceutically acceptable agents that bring the pharmaceutical composition closer to a physiological state, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Or, they may be lipophilic free radical quenchers such as α-tocopherol that suppress damage by free radicals. Furthermore, they may be lipid protectants such as water-soluble chelators like ferrioxamine that suppress lipid peroxidation damage and improve storage stability. The substances that improve storage stability are preferably added to the solution containing the lipid particles 10 after the formation of the lipid particles 10.

[0067] The composition containing lipid particles may be sterilized by conventional methods. The composition may also be supplied as a liquid or as a dried powder. The powdered composition can be used, for example, by dissolving it in a suitable liquid.

[0068] The concentration of lipid particles 10 in the above composition is not limited, but is preferably 0.01 to 30% by mass, more preferably 0.05 to 10% by mass. The concentration can be appropriately selected depending on the purpose.

[0069] • Method for generating CAR antigen-expressing cells The method for producing CAR antigen-expressing cells according to the first embodiment includes a preparation step (S1) of preparing CD209-positive cells and lipid particles as described above, and a gene introduction step (S2) of bringing the prepared CD209-positive cells and lipid particles into contact.

[0070] Here, CD209-positive cells in this disclosure are cells on which CD209 is present on the cell surface (more specifically, cells that express CD209, a type C lectin molecule, as a type II transmembrane pattern recognition receptor), such as dendritic cells and macrophages, but dendritic cells are preferred as CD209-positive cells in this disclosure. Dendritic cells include immature dendritic cells found in peripheral tissues and mature dendritic cells found in lymphoid tissues, but immature dendritic cells are more preferred as CD209-positive cells in this disclosure.

[0071] The CD209-positive cells in this embodiment may be collected from a cell population taken from a subject to whom the CAR-T cells ultimately obtained by the proliferation method of the second embodiment will be administered, i.e., from an autologous cell population. Alternatively, the CD209-positive cells may be collected from an allogeneic cell population or a commercially available cell population, or they may be cells differentiated from hematopoietic stem cells or cells differentiated from iPS cells.

[0072] The gene transfer step (S2) can be carried out, for example, by mixing a culture medium containing CD209-positive cells prepared in the preparation step (S1) with a solution containing lipid particles. In other words, the production method of the first embodiment can be carried out by a simple operation of bringing the lipid particles of the first embodiment, whose lipid membrane exhibits the lipid composition described above, into contact with CD209-positive cells. The production method of the first embodiment allows for highly efficient gene transfer into CD209-positive cells, and consequently, highly efficient production of CAR antigen-expressing cells.

[0073] In the method for producing CAR antigen-expressing cells of the first embodiment, the preparation step (S1) and the gene transfer step (S2) may be performed sequentially, but other steps for producing CAR antigen-expressing cells may be included in between. For example, a pre-culture step may be included between the preparation step (S1) and the gene transfer step (S2) to enhance the activity of CD209-positive cells by culturing them in advance. Also, a post-culture step may be included after the gene transfer step (S2) in which CD209-positive cells that have come into contact with lipid particles are cultured. Although the pre-culture step of CD209-positive cells as a step after the preparation step (S1) and the post-culture step of CD209-positive cells as a step after the gene transfer step (S2) have been described, the pre-culture step may be interpreted as including the preparation step (S1), or the post-culture step may be interpreted as including the gene transfer step (S2).

[0074] [Second Embodiment] • Methods for proliferating CAR-T cells The lipid particles described in the first embodiment can also be used in a method for proliferating CAR-T cells, including the production of CAR antigen-presenting cells, as a further application. An example of a method for proliferating CAR-T cells using the lipid particles of the first embodiment will be described below with reference to the drawings.

[0075] The method for proliferating CAR-T cells in the second embodiment, as shown in Figure 3, includes a preparation step (S21) for preparing lipid particles of the first embodiment, CAR-T cells, and CD209-positive cells; a preparation step (S22) for producing CAR antigen-presenting cells by introducing the CAR antigen gene into CD209-positive cells by contacting them with the lipid particles of the first embodiment; and a culture step (S23) for co-culturing CAR-T cells and CAR antigen-presenting cells. Each step will be described in detail below with reference to Figures 3 and 4.

[0076] The CAR-T cells prepared in preparation step (S21) may be produced in preparation step (S21). For example, preparation step (S21) may include a step of preparing a cell population 301 containing T cells and producing CAR-T cells 401 from this cell population 301.

[0077] T cells include CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ-T cells, or their precursor cells. Various cell populations can be used as long as they contain such T cells. For example, the cell population 301 containing T cells may be peripheral blood mononuclear cells (PBMCs) collected from peripheral blood. It is more preferable that the cell population 301 containing T cells is a cell population collected from a subject to whom the CAR-T cells ultimately obtained by the proliferation method of the second embodiment will be administered (i.e., an autologous cell population). However, allogeneic cell populations or commercially available cell populations can also be used. Furthermore, a cell population that has been processed to separate unwanted cells from the collected or obtained cell population may be used.

[0078] Furthermore, it is preferable to culture the cell population 301, which includes T cells, in an appropriate medium and under appropriate culture conditions before generating CAR-T cells. Culturing the cells enhances their activity, allowing for more efficient subsequent introduction of the CAR gene.

[0079] Next, as shown in Figure 4, CAR-T cells 401 are created by introducing the CAR gene into T cells and expressing CAR. The introduction of the CAR gene into T cells can be carried out using any tool or method that is generally known as a gene transfer tool or method for cells. Furthermore, as long as CAR is expressed, it is not necessary to integrate the CAR gene into the T cell genome or not, but it is more preferable to integrate the CAR gene into the genome. As tools or methods for gene transfer into cells, for example, polycations, calcium phosphate, lipofection, liposomes, electroporation, microinjection, particle guns, retro / lentiviral vectors, adenovirus vectors, etc. may be used. In addition, tools utilizing transposon vectors, homologous recombination, recombinase systems, or exogenous gene integration tools such as genome editing tools such as ZFN and CRISPR / Cas9 may be used in combination.

[0080] The following describes the procedure for preparing CAR-T cells when using a nucleic acid delivery reagent 20 containing the nucleic acid of the CAR gene as a tool for introducing the CAR gene into T cells. This nucleic acid delivery reagent 20 may be, for example, lipid particles containing the nucleic acid of the CAR gene, or a viral vector containing the nucleic acid of the CAR gene.

[0081] First, a nucleic acid delivery reagent 20 containing the nucleic acid of the CAR gene is brought into contact with a cell population 301 containing T cells. This contact introduces the CAR gene into the T cells. This contact can be carried out, for example, by adding the nucleic acid delivery reagent to a suspension of the cell population 301, by adding beads coated with the nucleic acid delivery reagent 20 to a suspension of the cell population 301, or by adding a suspension of the cell population 301 to a container whose surface is coated with the nucleic acid delivery reagent 20. Subsequently, CAR-T cells 401 can be produced by culturing the cell population 301 that has been in contact with the nucleic acid delivery reagent 20.

[0082] The nucleic acid contained in the nucleic acid introduction reagent 20 may include additional nucleic acids in addition to the nucleic acid containing the CAR gene. Such nucleic acids are, for example, DNA or RNA that have the function of catalyzing the integration of foreign DNA into the genome, or the function of modifying DNA such as DNA methylation, demethylation, repair, and / or binding. For example, these nucleic acids may be DNA or RNA that encodes a protein having the above-mentioned modification activity. By including these nucleic acids, it is possible to add the above-mentioned modifications to the CAR gene sequence introduced into the genome or the surrounding sequence, for example, and to further modify the function of T cells.

[0083] It has been explained that the proliferation method of this embodiment may include a step of producing CAR-T cells from the cell population 301 as a separate step prior to the preparation step (S21), but the step of producing CAR-T cells from the cell population 301 may also be interpreted as being part of the preparation step (S21).

[0084] Next, the preparation step (S22) will be described. In the preparation step (S22), as shown in Figures 3 and 4, the lipid particles 10 are brought into contact with the CD209-positive cells 101. Contact can be carried out, for example, by adding a solution or composition containing the lipid particles 10 to a cell suspension in which the CD209-positive cells 101 are suspended. Alternatively, contact may be carried out by adding a solution or composition containing the lipid particles 10 to a container or the like on which the CD209-positive cells 101 are attached to or fixed to the surface. Alternatively, contact may be carried out by adding a cell suspension in which the CD209-positive cells 101 are suspended to a container or the like on which the lipid particles 10 are attached to or fixed to the surface.

[0085] Lipid particles 10 are taken up by CD209-positive cells 101 through endocytosis caused by contact with the lipid particles 10, or by membrane fusion between the lipid particles and the cell membrane of CD209-positive cells 101. The first nucleic acid 22 is released from the lipid particles 10 taken up into the CD209-positive cells 101, thereby introducing the first nucleic acid 22 into the CD209-positive cells 101. The introduced first nucleic acid 22 expresses the CAR antigen 23 within the CD209-positive cells 101, thereby creating CAR antigen-expressing cells 201.

[0086] Next, in the co-culture step (S23), the CAR-T cells 401 prepared in the preparation step (S21) and the CAR antigen-expressing cells 201 prepared in the preparation step (S22) are co-cultured. For example, co-culture can be performed by adding the CAR-T cells 401 and the CAR antigen-expressing cells 201 to a container 501 containing the basic culture medium 502. The basic culture medium 502 used for co-culture should be a medium suitable for culturing both the CAR-T cells 401 and the CAR antigen-presenting cells 201. The culture conditions should preferably be suitable for the survival and proliferation of both the CAR-T cells 401 and the CAR antigen-presenting cells 201.

[0087] In the co-culture step (S23), the CAR antigen-expressing cells 201 may be directly or indirectly attached to or fixed to one surface of the solid phase. The solid phase is, for example, a container 501 made of metal, resin, gel, or fiber, but it does not necessarily have to be in the shape of a container; it may be a plate or a sheet, etc. When such a solid phase is used in a method for growing CAR-T cells, the co-culture step (S23) can be easily carried out by adding a cell suspension containing CAR-T cells 401 to the surface of the solid phase to which the CAR antigen-expressing cells 201 are attached or fixed. Furthermore, in the subsequent recovery step, the grown CAR-T cells 401 can be easily recovered by replacing the basic medium 502, leaving the CAR antigen-expressing cells 201 fixed to the solid phase.

[0088] In the co-culture step (S23), CAR-T cells 401 are activated when CAR antigen-expressing cells 201 present the antigen. Therefore, in the method of this embodiment, CAR antigen-expressing cells are also referred to as "CAR antigen-presenting cells." As shown in the examples described later, using cells derived from CD209-positive cells as CAR antigen-expressing cells results in higher CAR-T cell proliferation efficiency than using CAR antigen-presenting cells derived from other cells or cell groups (e.g., PBMCs).

[0089] In this embodiment, CAR antigen-presenting cells 201 are CD209-positive cells expressing CAR antigens. Such CD209-positive cells generally play an important role in the innate immune detection of pathogens and the subsequent activation of adaptive immunity in living organisms. More specifically, CD209-positive cells in living organisms (e.g., dendritic cells and macrophages) take in and degrade pathogens through phagocytosis and present target antigens to T cells, thereby initiating an adaptive immune response and inducing T cell activity and differentiation, as well as regulatory T cell differentiation. Furthermore, CD209-positive cells in living organisms also secrete cytokines, interferons, or growth factors that enhance and regulate the immune response. Therefore, by using CD209-positive cells as CAR antigen-presenting cells in co-culture, CAR-T cells can be proliferated more efficiently than when using other cells or cell groups (e.g., PBMCs). This is thought to be because CD209-positive cells, which are the body's natural antigen-presenting cells, are more likely to stimulate CAR-T cells.

[0090] Furthermore, in order to transform cells or cell populations other than CD209-positive cells into CAR antigen-presenting cells, it is necessary to introduce other genes (e.g., costimulators). On the other hand, CD209-positive cells can function as CAR antigen-presenting cells in the proliferation method of this embodiment simply by introducing the target CAR antigen gene. Therefore, the proliferation method of this embodiment is preferable because its gene introduction process can be carried out with fewer elements than conventional methods, thereby reducing costs and effort, and preventing a decrease in quality and yield due to contamination.

[0091] Furthermore, the proliferation method of this embodiment, as described in the first embodiment, allows for the creation of a group of CAR antigen-presenting cells with significantly high CAR antigen gene expression rates by using lipid particles with a specific lipid composition. By using this group of CAR antigen-presenting cells, it is possible to further activate CAR-T cells and increase the proliferation rate of CAR-T cells. In other words, by applying the method of this embodiment, which includes a co-culture step with CAR antigen-presenting cells, to a cell population containing CAR-T cells, the CAR-T cell content in the cell population can be improved.

[0092] The proliferation method of this embodiment may further include a step of recovering the obtained CAR-T cells after the culture step (S23). For example, CAR-T cells 401 in the basic medium 502 can be recovered by general processes such as pipetting and centrifugation.

[0093] In a further embodiment, a cell preparation may be made using CAR-T cells proliferated by the method of this embodiment. The cell preparation may contain CAR-T cells in a therapeutically effective amount. The cell preparation may also contain agents that protect CAR-T cells, such as dimethyl sulfoxide (DMSO) or serum albumin; agents that prevent the growth of microorganisms, such as antibiotics; and agents that activate, proliferate, or induce differentiation of cells, such as vitamins, cytokines, growth factors, or steroids.

[0094] The administration route of CAR-T cells or cell preparations obtained by the proliferation method of this embodiment is not particularly limited. For example, they may be administered by intravenous injection, intra-arterial injection, intra-portal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Alternatively, the administration method may be local. In that case, for example, they may be directly injected into the target tissue, organ, or system. The administration schedule may be selected considering the gender, age, weight, or condition of the subject (patient), and may be a single dose or a series of continuous or periodic doses.

[0095] [Third Embodiment] ·kit The kit of the third embodiment is a kit used for a method of proliferating CAR-T cells and comprises lipid particles for producing CAR antigen-presenting cells as described in the first embodiment. Furthermore, the kit of the third embodiment may also comprise a nucleic acid delivery reagent for introducing CAR genes into CAR-T cells as described in the second embodiment. However, in the kit of the third embodiment, the lipid particles of the first embodiment and the nucleic acid delivery reagent of the second embodiment are not provided mixed together (i.e., not provided as a composition), but rather provided as separate reagents in the kit.

[0096] The kit may further contain other components in the above composition or as a separate composition. These other components may include cell culture media and / or antibodies, peptides, etc., for activating T cells. It may also contain substances that improve the storage stability of lipid particles and / or nucleic acid delivery reagents, for example, any of the storage stability improving substances exemplified in the first embodiment.

[0097] The kit may comprise a solid phase having a surface to which CD209-positive cells can be directly or indirectly attached or fixed. The solid phase may be a container made of, for example, metal, resin, gel, or fiber, but does not necessarily have to be in the shape of a container; it may be a plate or sheet, etc. In the kit, lipid particles (or nucleic acid introduction reagents) may be provided attached or fixed directly or indirectly to one surface of the solid phase.

[0098] Direct attachment or immobilization can be achieved, for example, by directly coating the surface of a solid phase with lipid particles or nucleic acid delivery reagents and drying them. Indirect attachment or immobilization can be achieved, for example, by coating the solid phase with lipid particles or nucleic acid delivery reagents via a peptide, resin, or antibody and drying them.

[0099] When using a kit equipped with such a solid phase for CAR-T cell proliferation, the gene transfer process can be easily carried out by adding a cell suspension containing various cells to the surface to which the lipid particles (or nucleic acid delivery reagent) of the solid phase are attached or fixed. For example, if lipid particles are attached or fixed to the solid phase surface in a free-moving manner, the lipid particles can be released into the cell suspension by adding a cell suspension containing CD209-positive cells to the solid phase surface, allowing them to come into contact with the CD209-positive cells. Similarly, if nucleic acid delivery reagents are attached or fixed to the solid phase surface in a free-moving manner, the nucleic acid delivery reagents can be released into the cell suspension by adding a cell suspension containing CAR-T cells to the solid phase surface, allowing them to come into contact with the CAR-T cells.

[0100] [example] The following describes examples of how lipid particles, CAR antigen-presenting cells, and ultimately CAR-T cells were produced and used by the method of the embodiment. However, the embodiments of the present invention are not limited to the examples below.

[0101] Example 1. Selection of lipid composition of lipid particles suitable for gene transfer into dendritic cells. To determine the lipid composition of lipid particles suitable for gene transfer into dendritic cells, the experiment in Example 1 was performed using the following procedure.

[0102] • Preparation of lipid particles mRNA encoding GFP (green protein) was used as the nucleic acid to be encapsulated in the lipid particles. After mixing the solution containing this mRNA with ethanol-soluble lipid solutions of lipid compositions (1) to (19), the mixture was further mixed with 10 mM HEPES (pH 7.3), and then washed and concentrated by centrifugal ultrafiltration to obtain a total of 19 solutions containing lipid particles with different lipid compositions.

[0103] [Table 2]

[0104] • Preparation of dendritic cells Dendritic cells (DCs) were isolated from PBMCs (peripheral blood mononuclear cells) using the adhesion method and cultured. After thawing frozen PBMCs (Lonza), PBMCs suspended in ALyS705 (Cell Science Institute) were placed in a 96-well culture plate. 6 200 μL of (cells / mL) was added and the mixture was incubated at 37°C in a 5% CO2 atmosphere for 4 hours. After removing the culture plate from the incubator and removing the culture medium from the wells, non-adherent cells were removed by washing three times with phosphate-buffered saline (PBS). After washing, 200 μL of ALyS705 (containing IL4: 10 ng / mL and GM-CSF: 10 ng / mL) was added and cell culture was continued at 37°C in a 5% CO2 atmosphere.

[0105] • Introduction of nucleic acids into dendritic cells using lipid particles Each of the 19 solutions containing lipid particles with different lipid compositions was used to add GFP mRNA to each well of the culture plate containing dendritic cells. In other words, the solutions containing lipid particles were dispensed so that each well contained lipid particles with one of the lipid compositions (1) to (19), meaning that each well contained lipid particles with a different lipid composition. After dispensing, the culture plates were placed in an incubator at 37°C under a 5% CO2 atmosphere, and dendritic cells were cultured.

[0106] • Measurement of gene transferability in dendritic cells After 24 hours of culture, the culture plates were removed from the incubator, and the GFP fluorescence intensity and cell proliferation activity of the cells were measured. GFP fluorescence intensity was measured using a fluorescence microplate reader (Infinite F200 PRO, Tecan). After measuring fluorescence intensity, cell proliferation activity was measured using the Premix WST-1 Cell Proliferation Assay System (Takara Bio). ALiS705 containing Premix WST-1 solution was added to the wells of the culture plate, and the plate was left to stand in an incubator at 37°C with a 5% CO2 atmosphere. After 30 minutes, the culture plate was removed from the incubator, and the absorbance of the wells was measured. The value of cell proliferation activity was calculated by measuring the absorbance at 450 nm and 690 nm, and dividing the OD450 nm value by the OD690 nm value. Lipid particle evaluation was performed using the product of the fluorescence intensity value and the cell activity value as an indicator.

[0107] ·result The experimental results for Example 1 are shown in Figure 5. In Figure 5, the values ​​on the vertical axis represent the cumulative values ​​of GFP fluorescence intensity and cell proliferation activity, and the values ​​on the horizontal axis represent the compositions corresponding to each of the lipid compositions (1) to (19) listed in Table 2.

[0108] As shown in Figure 5, when genes are introduced using lipid particles with lipid composition (14) or (15) in Table 2, the transduction efficiency of CAR antigen genes (the abundance of CAR antigen-presenting cells) is significantly higher compared to when genes are introduced using lipid particles with other lipid compositions. In other words, it can be said that using lipid particles with lipid composition (14) or (15) in Table 2 is particularly efficient in producing CAR antigen-presenting cells. Note that lipid composition (14) is the same as lipid composition (A) in Table 1, and lipid composition (15) is the same as lipid composition (B) in Table 1.

[0109] Furthermore, the value obtained by integrating GFP fluorescence intensity and cell proliferation activity takes into account both the gene transfer efficiency for each lipid particle with a different lipid composition and the subsequent proliferation activity of CAR antigen-presenting cells. Therefore, it can be seen that the lipid particles with lipid compositions (14) or (15) in Table 2 exhibit high performance (high gene transfer efficiency and low cytotoxicity) in gene transfer to dendritic cells.

[0110] From the above results, it can be seen that when FFT10 and FFT20 are present in equal proportions of each other in terms of lipid composition (molar ratio), with a blending ratio of 10% to 30%, and when DOTAP is present in 10% or more of the lipid composition (molar ratio), and with a blending ratio less than or equal to that of FFT10 or FFT20, the gene transfer efficiency by lipid particles is high and the cytotoxicity is low, thus enabling the efficient production of dendritic cells expressing CAR antigens, which is therefore preferable.

[0111] Example 2. Comparison of CAR-T cell proliferation efficiency between the liposome-DC method and the AP method. To compare the proliferation efficiency of CAR-T cells using the proliferation method of the second embodiment (hereinafter referred to as the "liposome-DC method") with the proliferation efficiency of CAR-T cells using the conventional proliferation method (hereinafter referred to as the "AP method"), the experiment in Example 2 was carried out using the following procedure.

[0112] • Preparation of dendritic cells Dendritic cells (DCs) were isolated from peripheral blood mononuclear cells (PBMCs) using the adhesion method and cultured. After thawing frozen PBMCs (Lonza), PBMCs suspended in ALyS705 (Cell Science Institute) were placed in a 24-well culture plate. 6 Cells were added (per well) and the incubator was placed at 37°C in a 5% CO2 atmosphere for 4 hours. After removing the culture plate from the incubator and removing the culture medium from the wells, non-adherent cells were removed by washing three times with phosphate-buffered saline (PBS). After washing, ALyS705 (containing IL4: 10 ng / mL and GM-CSF: 10 ng / mL) was added, and cell culture was continued at 37°C in a 5% CO2 atmosphere.

[0113] • Preparation of PBMC Human peripheral blood mononuclear cells (PBMCs) were cultured in ALyS705() (containing IL7: 10 ng / mL and IL15: 5 ng / mL) supplemented with 5% artificial serum (Institute of Cell Science) in an incubator at 37°C under a 5% CO2 atmosphere.

[0114] • Production of CAR-T cells We introduced the CAR gene into prepared PBMCs to create a cell population containing CAR-T cells for co-culture with CAR antigen-presenting cells. Specifically, the created CAR-T cells were co-cultured with CAR antigen-presenting cells in a form that was part of a cell population that also included non-CAR-T cell populations derived from PBMCs. We created CAR-T cells (EphB4 CAR-T) that express a chimeric antibody receptor that recognizes EphB4. The CAR-T cells were created using the piggyBAC method with moth-derived DNA transferase. That is, plasmid DNA for CAR expression and plasmid DNA for piggyBAC expression were introduced into PBMCs by electroporation.

[0115] • Preparation of lipid particles Lipid particles were prepared as follows. mRNA encoding the EphB4 antigen (hereinafter referred to as "EphB4-mRNA") was used as the nucleic acid to be encapsulated in the lipid particles. After mixing the solution containing this mRNA with an ethanol-soluble lipid solution (i.e., FFT10 / FFT20 / DOPE / DOTAP / cholesterol / PEG-DMG=20 / 20 / 10 / 10 / 36 / 4 (mol%)), the mixture was further mixed with 10 mM HEPES (pH 7.3), and the solution was washed and concentrated by centrifugal ultrafiltration to obtain a solution containing lipid particles encapsulating EphB4-mRNA.

[0116] • Production of CAR antigen-presenting cells for use in liposome-DC synthesis In the liposome-DC method, CAR antigen-presenting cells for co-culture with CAR-T cells were created by introducing EphB4-mRNA as the CAR antigen gene into dendritic cells prepared as described above. Specifically, lipid particles were added to each dendritic cell to a concentration of 2.0 μg RNA / well, and the mixture was cultured in an incubator at 37°C under a 5% CO2 atmosphere.

[0117] • Preparation of CAR antigen-presenting cells for use in the AP method In the AP method, CAR antigen-presenting cells for co-culture with CAR-T cells were created by introducing EphB4-plasmid DNA as the CAR antigen gene into the PBMC prepared as described above using electroporation, and were cultured in an incubator at 37°C under a 5% CO2 atmosphere.

[0118] • Co-culture using liposome-DC method In the liposome-DC method, CAR antigen-presenting cells derived from dendritic cells prepared as described above were co-cultured with a cell population containing CAR-T cells. Specifically, 1 day after the introduction of EphB4-mRNA and the start of culture, the cell population containing CAR-T cells was added to the dendritic cell-derived CAR antigen-presenting cells, and the culture was started by placing the cells in an incubator at 37°C under a 5% CO2 atmosphere.

[0119] Co-culture using the AP method In the AP method, PBMC-derived CAR antigen-presenting cells prepared as described above were co-cultured with a cell population containing CAR-T cells. Specifically, PBMC-derived CAR antigen-presenting cells were harvested from the cell suspension three days after the introduction of EphB4-plasmid DNA and the start of culture, and cultured together with the cell population containing CAR-T cells in an incubator at 37°C and a 5% CO2 atmosphere.

[0120] • Measurement of the abundance of T cells and CAR-T cells At 14 days after the start of culture, co-culture using the liposome-DC method and co-culture using the AP method were both completed. Fluorescent immunofluorescence staining was performed for the T cell surface antigens CD3 and EphB4 CAR, and the abundance of cells expressing these antigens was measured by FACS.

[0121] ·result The experimental results for Example 2 are shown in Figure 6. Figure 6(a) shows the results of CAR-T cell proliferation using the liposome-DC method, and (b) shows the results of CAR-T cell proliferation using the AP method. Figures 6(a) and (b) are dot plots with the expression level of EphB4 CAR (i.e., the abundance of CAR-T cells in the cell population) on the vertical axis and the expression level of CD3, a T cell surface antigen (i.e., the abundance of T cells in the cell population) on the horizontal axis.

[0122] The dot plots in Figures 6(a) and (b) show that the Q2 fraction contains CD3-positive (T lymphocyte) and EphB4 CAR-positive cells (CAR-expressing cells), i.e., CAR-T cells. In the AP method, the CAR-T cell content in the cell population was approximately 14%, while in the liposome-DC method, the CAR-T cell content in the cell population was approximately 35%.

[0123] These results demonstrate that the liposome-DC method is superior to the AP method as a method for proliferating CAR-T cells, and that is, the method of proliferating CAR antigen-presenting cells derived from dendritic cells is superior to the method of proliferating CAR antigen-presenting cells derived from PBMCs.

[0124] Example 3. Effect of maleimide liposomes on gene transfer into dendritic cells To verify the effect of maleimide liposomes on gene transfer into dendritic cells, the experiment in Example 3 was performed using the following procedure.

[0125] • Preparation of lipid particles Lipid particles were prepared as follows. CAR antigen gene mRNA was used as the nucleic acid to be encapsulated in the lipid particles. This mRNA-containing solution was mixed with either an ethanol-soluble lipid solution of composition (15) in Table 2, or an ethanol-soluble lipid solution of composition (15) with maleimide-PEG-DMG further added (FFT10 / FFT20 / DOPE / DOTAP / cholesterol / PEG-DMG / maleimide-PEG-DMG=20 / 20 / 10 / 10 / 36 / 2 / 2 (mol%)). The mixture was then further mixed with 10 mM HEPES (pH 7.3), followed by washing and concentration using centrifugal ultrafiltration to obtain two solutions containing lipid particles with different lipid compositions.

[0126] • Introduction of nucleic acids into dendritic cells using lipid particles Two different solutions, each containing lipid particles with different lipid compositions, were added to each well of the culture plate containing dendritic cells, so that the mRNA level of the CAR antigen gene was 2.0 μg RNA / well. Specifically, one well contained lipid particles with lipid composition (15), and the other well contained lipid particles further containing maleimide-PEG-DMG. After dispensing, the culture plate was placed in an incubator at 37°C under a 5% CO2 atmosphere to introduce nucleic acids into the dendritic cells.

[0127] • Detection of dendritic cells expressing CAR antigens Approximately one day after culturing, each culture plate was removed from the incubator, and cells expressing EphB4 were detected by fluorescent immunoassay. The percentage of dendritic cells expressing the CAR antigen gene out of the total number of dendritic cells was then calculated.

[0128] ·result The experimental results for Example 3 are shown in Figure 7. The values ​​on the vertical axis of Figure 7 represent the percentage of dendritic cells expressing the CAR antigen gene, (A) on the horizontal axis shows the case where lipid particles with lipid composition (15) described in Table 2 were used, and (B) shows the case where lipid particles further containing the maleimide-PEG-DMG described above were used.

[0129] As shown in Figure 7, when gene transfer was performed using lipid particles further containing maleimide-PEG-DMG as described above, the proportion of CAR antigen-presenting cells was higher compared to when gene transfer was performed using lipid particles with the lipid composition (15) listed in Table 2. In other words, it was shown that using lipid particles further containing maleimide-PEG-DMG in addition to FFT10, FFT20, DOPE, DOTAP, cholesterol, and PEG-DMG resulted in higher gene transfer efficiency to dendritic cells. [Explanation of Symbols]

[0130] 1,21...lipid particle, 1b...lumen, 2,22...first nucleic acid, 2a...CAR antigen gene 3...Nucleic acid condensed peptide, 4...Second nucleic acid, 4a...Transposase gene sequence, 10, 20, 21...lipid particles, 23...CAR antigen, 101...CD209 positive cells, 201…CAR antigen-presenting cells, 301…Cell population including T cells, 401…CAR-T cells

Claims

1. Lipid particles for producing CAR antigen-expressing cells derived from CD209-positive cells, The aforementioned lipid particles are composed of a lipid membrane having a lumen. Lipid particles having nucleic acids containing the CAR antigen gene encapsulated within the aforementioned lumen.

2. The lipid particle according to claim 1, wherein the lipid membrane contains 50% or more cationic lipids in terms of lipid composition (molar ratio).

3. The aforementioned lipid membrane is Formula Q-CHR ２ (In the formula, Q is a nitrogen-containing aliphatic group that contains two or more tertiary nitrogen atoms and does not contain oxygen. R is independent of C １２ ~C ２４ It is an aliphatic group, The lipid particle according to claim 2, wherein at least one R contains a lipid compound represented in its main chain or side chain by a linking group LR selected from the group consisting of -C(=O)-O-, -O-C(=O)-, -O-C(=O)-O-, -S-C(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)-.

4. The lipid particle according to claim 3, wherein the lipid compound is a lipid compound of formula (1-01) and / or a lipid compound of formula (1-02). 【Chemistry 1】 【Chemistry 2】

5. The lipid particles according to claim 4, wherein the lipid compound of formula (1-01) and the lipid compound of formula (1-02) each account for 10% to 30% of the lipid composition (molar ratio) of the lipid membrane, and are in equal proportions to each other.

6. The lipid particle according to claim 3, wherein the lipid membrane further comprises a base lipid or a lipid that suppresses aggregation of the lipid particles.

7. The lipid particles according to claim 6, wherein the base lipid is 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

8. The lipid particles according to claim 6, wherein the lipid that suppresses aggregation of the lipid particles is polyethylene glycol dimyristoyl glycerol (DMG-PEG) or maleimide DMG-PEG.

9. The lipid particle according to claim 4, wherein the lipid membrane comprises the lipid compound of formula (1-01) and the lipid compound of formula (1-02), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and polyethylene glycol dimyristoylglycerol (DMG-PEG) or maleimide DMG-PEG.

10. The lipid particle according to claim 1, wherein the CAR antigen gene is the full length or a portion of one or more genes selected from EphB4, EGFR variant 3, ALK, and EphA2.

11. The lipid particle according to claim 1, wherein the nucleic acid is DNA, RNA, PNA, or a derivative thereof.

12. The lipid particle according to claim 1, wherein the nucleic acid is a single-stranded or double-stranded, cyclic, linear, or branched nucleic acid.

13. Lipid particles according to any one of claims 1 to 12, and A substance that improves the storage stability of the aforementioned lipid particles A kit for producing CAR antigen-expressing cells, comprising at least [the specified element].

14. The kit according to claim 13, wherein the lipid particles are directly or indirectly attached to or fixed to a solid phase.

15. The kit according to claim 14, wherein the solid phase is a container, plate, or sheet made of metal, resin, gel, or fiber.

16. A method for proliferating genetically modified T cells (CAR-T cells) that express a chimeric antigen receptor (CAR), A step of preparing a cell population containing CAR-T cells, CD209-positive cells, and lipid particles according to any one of claims 1 to 12; A step of producing CAR antigen-presenting cells by contacting the CD209-positive cells with the lipid particles and introducing the nucleic acid into the CD209-positive cells; and The step of co-culturing the CAR-T cells and the CAR antigen-presenting cells. Methods that include...

17. The method according to claim 16, wherein the CD209-positive cells are dendritic cells.

18. The method according to claim 16, wherein the cell population containing CAR-T cells is produced from a cell population containing T cells.

19. The method according to claim 18, wherein the cell population including the T cells is peripheral blood mononuclear cells (PBMCs).

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