Therapeutic effect enhancers that improve the effectiveness of CAR-T therapy.

Abstract

The present invention aims to provide a therapeutic effect enhancer that can sustain the effects of CAR-T cell therapy. [Solution] The therapeutic effect enhancer according to the embodiment contains a nucleic acid containing a gene encoding a target antigen of CAR and a nucleic acid containing a gene encoding a co-stimulatory factor.

Description

[Technical Field]

[0001] This invention relates to a therapeutic effect enhancer that improves the effectiveness of CAR-T therapy. [Background technology]

[0002] CAR-T cell therapy, which uses T cells genetically modified to produce chimeric antigen receptors (CARs) that recognize tumor cells (CAR-T cells), is attracting attention for its extremely high efficacy against tumors. In this method, CAR-T cells are created by introducing CAR genes corresponding to the surface antigens of tumor cells into T cells collected from the patient, and then administered to the patient. When the administered CAR-T cells encounter tumor cells that have CAR antigens on their surface in the patient's body, they kill the tumor cells.

[0003] However, it is known that the therapeutic effect of CAR-T cell therapy tends to gradually decrease from the start of treatment and does not last. This is thought to be partly due to the fact that the proportion of tumor cells that have lost CAR antigens on their cell surface gradually increases, making them evade recognition and killing by CAR-T cells.

[0004] Therefore, there is a need for methods that can sustain the effects of CAR-T cell therapy more effectively than conventional methods. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a therapeutic effect enhancer that can sustain the effects of CAR-T cell therapy. [Means for solving the problem]

[0006] The therapeutic effect enhancer for CAR-T cell therapy according to the embodiment comprises a nucleic acid encoding a CAR antigen and a nucleic acid encoding a costimulatory factor. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a schematic diagram showing the configuration of the therapeutic effect enhancer according to the first embodiment. [Figure 2] Figure 2 is a schematic diagram showing the configuration of a therapeutic effect enhancer in a modified example of the first embodiment. [Figure 3] Figure 3 is a flowchart showing the treatment method of the second embodiment. [Figure 4] Figure 4 is a graph showing the experimental results for Example 1. [Figure 5] Figure 5 is a schematic diagram that simply illustrates the experimental design of Example 2. [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. [Figure 8] Figure 8 is a schematic diagram that briefly explains the experimental design of Example 4. [Figure 9] Figure 9 is a graph showing the experimental results for Example 4. [Modes for carrying out the invention]

[0008] The following describes the therapeutic effect-enhancing agent of the embodiment and the method for enhancing CAR-T cell therapy using the therapeutic effect-enhancing agent with reference to the drawings. Each figure is an embodiment and a schematic diagram to facilitate understanding; however, the shape, dimensions, ratios, etc. may differ from the actual product. These can be appropriately modified in accordance with the following description and known technology.

[0009] [First Embodiment] • Therapeutic effect enhancers The therapeutic effect enhancer of the first embodiment is a composition used to enhance the therapeutic effect of CAR-T cell therapy, and contains a nucleic acid containing a gene encoding a target antigen of CAR (hereinafter referred to as "CAR antigen nucleic acid") and a nucleic acid containing a gene encoding a costimulatory factor (hereinafter referred to as "costimulatory factor nucleic acid").

[0010] As shown in FIG. 1, it is desirable that the therapeutic effect promoting agent 1 be provided as a composition in which lipid particles 10 are contained in a carrier 2. The carrier 2 is, for example, water, a saline solution such as physiological saline, an aqueous glycine solution, or a buffer solution such as HEPES. Further, the lipid particles 10 desirably encapsulate the CAR antigen nucleic acid 3 and the co-stimulatory factor nucleic acid 4 in their lipid membrane 5 and have directivity to tumor cells.

[0011] Hereinafter, each component will be described in detail.

[0012] (Lipid particles) The lipid particles 10 contained in the therapeutic effect promoting agent 1 of the present embodiment are substantially spherical hollow bodies composed of a lipid membrane 5 formed by non-covalent arrangement of a plurality of lipid molecules, that is, liposomes. And the CAR antigen nucleic acid 3 and the co-stimulatory factor nucleic acid 4 are encapsulated in the lumen thereof. The lipid membrane 5 may be a lipid monolayer membrane or a lipid bilayer membrane. Further, the lipid membrane 5 may be composed of a single layer of membrane or a multi-layer of membrane.

[0013] The material of the lipid membrane 5 may be composed of the base lipids exemplified below, but it is preferable to further contain the lipid compounds exemplified below in addition to the base lipids.

[0014] 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.

[0015] 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-3-dimethylammonium propane (DODAP), 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-phosphocholine (DLPC), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol, or a combination of any of these is preferably used.

[0016] Particularly, DOTAP is a cationic lipid and is preferable because the acid dissociation constant of the lipid membrane and thus the lipid particles can be adjusted by its content. The above base lipids are preferable because they easily fuse with the cell membrane, and the structure and particle size of the lipid particles can be easily controlled, and they easily fuse with the cell membrane. The length of the hydrocarbon chain of the acyl group contained in the lipid is preferably C 10 ~C 20 It may be a saturated hydrocarbon group or an unsaturated hydrocarbon group.

[0017] The base lipid may make up nearly 100% of the total lipid molecules contained in the lipid membrane 5, but it is preferable that the base lipid of the lipid membrane 5 makes up about 30% to about 80% (molar ratio) of the total lipid molecules.

[0018] 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.

[0019] When the lipid membrane 5 contains lipid compound A, the cationic properties of the surface of the lipid particles 10 can be adjusted, thereby reducing obstacles during cell introduction and increasing the nucleic acid introduction rate. Furthermore, using the lipid membrane 5 containing lipid compound A can increase the amount of nucleic acid contained within it, thus further increasing the nucleic acid introduction rate.

[0020] In particular, it is preferable to use a lipid having the structure represented by the following formulas (1-01) and / or (1-02) as lipid compound A, as this provides better efficiency in introducing nucleic acids into tumor cells. That is, it is preferable to include the lipids of the following formulas (1-01) and / or (1-02) in the lipid membrane, as this allows the lipid particles to be given a directivity to tumor cells. 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.

[0021] [ka]

[0022] [ka]

[0023] The lipid membrane 5 preferably further contains lipids that suppress aggregation by adjusting the surface charge of the lipid particles 10 (hereinafter referred to as aggregation-reducing lipids). Such lipids are preferably, 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.

[0024] Other lipids may include structure-forming lipids in the lipid membrane 5. Structure-forming lipids include, for example, relatively low-toxicity lipids for adjusting toxicity, lipids having functional groups for attaching ligands to lipid particles 10, and lipid sterols (e.g., cholesterol) for suppressing leakage of encapsulated substances. In particular, it is preferable that cholesterol is included in the lipid membrane 5 as a structure-forming lipid.

[0025] As described above, it is preferable that the lipid membrane 5 of the lipid particle 10 is composed of at least one lipid compound from the group consisting of cationic lipids, neutral lipids, aggregation-reducing lipids, and structure-forming lipids, in addition to FFT10 and / or FFT20, because this results in particularly excellent nucleic acid encapsulation and nucleic acid introduction efficiency.

[0026] (nucleic acid) CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4 are, for example, single-stranded or double-stranded circular, linear, or branched nucleic acids. CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4 are, for example, DNA, RNA, PNA, or derivatives thereof. Derivatives include DNA, RNA, or PNA with a nucleotide analog inserted, or DNA, RNA, or PNA with any of its ends labeled or modified with a functional group. CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4 may be in any of the following forms: mRNA, plasmid, or polynucleotide.

[0027] If the CAR antigen nucleic acid 3 and / or the costimulatory factor nucleic acid 4 are RNA, it is preferable that they be modified to be resistant to degradation. For example, such modifications may be any known modifications that prevent 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.

[0028] 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).

[0029] 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.

[0030] 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.

[0031] CAR antigen nucleic acid 3 is a nucleic acid containing a gene encoding a CAR target antigen, as described above. In this disclosure, a CAR target antigen is a protein, glycan, or glycolipid expressed on the surface of tumor cells that binds to CAR-T cells. 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.

[0032] 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.

[0033] (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 antigen specific to the tumor cell that is the target of the CAR is a receptor, the extracellular domain may use a ligand that binds to that receptor.

[0034] 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.

[0035] (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.

[0036] (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.

[0037] 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.

[0038] 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.

[0039] (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.

[0040] 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.

[0041] 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. CAR antigens should be antigens that are targeted by CARs (i.e., match the type of CAR of interest) and are generally known as substances that cannot be detected in normal cells. Furthermore, tumor-associated antigens or tumor-specific antigens may be antigens present in the extracellular matrix, or proteins containing mutations identified by genomic analysis and / or tumor-suggestive expression studies.

[0042] 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.

[0043] 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.

[0044] Costimulatory factor nucleic acid 4 is a nucleic acid containing the gene encoding the costimulatory factor, as described above. In this disclosure, a "costimulatory factor" is a protein that is naturally expressed on the membrane of antigen-presenting cells such as dendritic cells, and transmits an auxiliary signal for activation to T cells by binding to and interacting with costimulatory receptors on T cells. Costimulatory factors are also generally referred to as "costimulatory molecules," "co-stimulatory factors," or "co-stimulatory molecules."

[0045] In this disclosure, the costimulatory factor nucleic acid 4 can be a gene encoding a protein generally known as a costimulatory factor. Specifically, costimulatory factor nucleic acid 4 may be a nucleic acid encoding a costimulatory factor selected from the group consisting of 4-1-BBL, CD80, CD86, ICOSL, and OX40L. Alternatively, 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.

[0046] (Nucleic acid condensed peptide) Nucleic acid condensation peptides are peptides 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. For example, it is preferable to use cationic peptides as such peptides. Cationic peptides can condense nucleic acids by, for example, filling the gaps in the helical shape of anionic nucleic acids and reducing those gaps.

[0047] A preferred nucleic acid condensed peptide is, for example, a peptide containing 45% or more cationic amino acids. A more preferred nucleic acid condensed peptide 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.

[0048] The above nucleic acid condensed peptide preferably has the following amino acid sequence. RQRQRYYRQRQRGGRRRRRR (Sequence ID 1) RQRQRGGRRRRRR (Sequence ID 2)

[0049] Such nucleic acid condensing peptides allow for efficient condensation of nucleic acids using cationic arginine, while also weakening the anionic properties of the nucleic acids, thus enabling efficient encapsulation of nucleic acids within lipid particles 10. Furthermore, because nucleic acid condensing peptides efficiently dissociate nucleic acids within cells, it is possible to efficiently express CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4 introduced into tumor cells.

[0050] Alternatively, a nucleic acid condensed peptide has RRRRRR (a third amino acid sequence) at one end and RRRRRR (a fourth 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 third amino acid sequence, the fourth amino acid sequence, and the intermediate sequences.

[0051] Such nucleic acid condensed peptides preferably have the following amino acid sequence. RRRRRRYYRQRQRGGRRRRRR(Sequence ID 3)

[0052] Such nucleic acid condensing peptides have strong cationic properties at both ends and high binding affinity to nucleic acids. Therefore, they 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.

[0053] Furthermore, nucleic acid condensed peptides having the following amino acid sequences can also be used in combination with any of the above nucleic acid condensed peptides. GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(M9)(Sequence No. 4)

[0054] This peptide can further condense nucleic acid aggregates condensed with the above-mentioned nucleic acid condensing peptide. 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 tumor cells, making it possible to introduce nucleic acids into the genome of tumor cells more efficiently. For example, before encapsulating them in lipid particles 10, the CAR antigen nucleic acid 3 and the costimulatory factor nucleic acid 4 can be condensed by stirring and mixing them with the nucleic acid condensing peptide.

[0055] While the use of nucleic acid condensed peptides is preferable due to the effects described above, it may not be necessary to use them depending on the type of nucleic acid used or the cell culture conditions.

[0056] The lipid particles 10 may contain further components in addition to the CAR antigen nucleic acid 3, the costimulatory factor nucleic acid 4, and the nucleic acid condensed peptide. 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.

[0057] Lipid particles 10 can be produced, 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 produced by adding an aqueous buffer containing the components to be encapsulated, such as CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4, to a mixture obtained by impregnating the lipid membrane 5 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 produced by adding ethanol obtained by impregnating the lipid membrane 5 material to an aqueous buffer containing the components to be encapsulated, such as CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4, 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.

[0058] 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.

[0059] The therapeutic effect enhancer may further contain a substance that improves the storage stability of lipid particles, i.e., a storage stabilizer. Storage stabilizers include pH adjusters, buffering agents, tonicity adjusters, and cryoprotective agents. Specifically, the storage stabilizer for lipid particles is not limited to albumin, lipoproteins, apolipoproteins, glycoproteins such as globulin, etc. Alternatively, it may be a pharmaceutically acceptable agent that brings the pharmaceutical composition closer to a physiological state, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Alternatively, it may be a lipophilic free radical quencher such as α-tocopherol that suppresses damage by free radicals. Furthermore, it may be a lipid protective agent such as a water-soluble chelator such as ferrioxamine that suppresses lipid peroxidation damage and improves storage stability. The storage stabilizer is preferably added to a solution containing the lipid particles 10 after the formation of the lipid particles 10.

[0060] The therapeutic effect enhancer may be sterilized by conventional methods. The therapeutic effect enhancer may also be supplied as a liquid or as a dry powder. If in powder form, it can be used by dissolving it in a suitable liquid, for example.

[0061] 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.

[0062] In a further embodiment, the therapeutic effect enhancer 1 may be provided as a composition containing two types of lipid particles on a carrier: lipid particles 10a containing CAR antigen nucleic acid 3 and lipid particles 10b containing costimulatory factor nucleic acid 4 (see Figure 2). In this case, the lipid particles 10a and 10b are configured to exhibit targeting of tumor cells by containing, for example, lipid compound A as the lipid constituting the respective lipid membranes 5a and 5b, and in particular by containing FFT10 and / or FFT20. The two types of lipid particles 10a and lipid particles 10b may be composed of the same lipid composition or different lipid compositions, provided that they satisfy the preferred conditions for lipid composition described in the first embodiment. That is, the lipid compositions of lipid membranes 5a and 5b may be the same or different. Such a therapeutic effect enhancer 1 can be prepared by separately preparing the lipid particles 10a and lipid particles 10b and mixing the carriers containing each of the lipid particles.

[0063] [Second Embodiment] • Procedures to enhance the therapeutic effect of CAR-T cell therapy The method of the second embodiment is a procedure to enhance the therapeutic effect of CAR-T cell therapy on the target of treatment, and uses the therapeutic effect-enhancing agent of the first embodiment. An example of the method of the second embodiment will be described below with reference to Figure 3.

[0064] The method of the second embodiment includes a preparation step (S1) of preparing a therapeutic effect enhancer containing lipid particles encapsulating CAR antigen nucleic acid and costimulatory factor nucleic acid, and CAR-T cells; a gene transfer step (S2) of administering the therapeutic effect enhancer to a subject; and a treatment step (S3) of administering CAR-T cells to a subject.

[0065] Here, "tumor cells" in this disclosure refer to cells that constitute an epithelial tumor, a non-epithelial tumor, or a malignant tumor consisting of both epithelial and non-epithelial components, and are generally also called "cancer cells." Tumor cells include all stages of the disease, for example, a state in which the malignant tumor remains within the organ of origin, a state in which the malignant tumor has spread to surrounding tissues, a state in which the malignant tumor has metastasized to lymph nodes, and a state in which the malignant tumor has metastasized to distant organs. CAR-T cells are designed to express CARs that bind to the surface antigens of these tumor cells.

[0066] The therapeutic effect enhancer prepared in preparation step (S1) is, for example, a composition containing lipid particles 10 encapsulating CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4. Alternatively, the therapeutic effect enhancer may be a composition containing two types of lipid particles, 10a encapsulating CAR antigen nucleic acid 3 and 10b encapsulating costimulatory factor nucleic acid 4, on a carrier.

[0067] In the gene transfer step (S2), the therapeutic effect enhancer prepared in the preparation step (S1) is administered to the target, thereby enabling contact between the therapeutic effect enhancer and tumor cells. When the target is a living organism, cells other than tumor cells are present, but the therapeutic effect enhancer in this embodiment contains lipid particles that are directional to tumor cells, and therefore has little effect on normal cells other than tumor cells.

[0068] The route of administration of CAR-T cells to a living organism 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 method of administration may be local. In that case, for example, they may be directly injected into the target tissue, organ, or system. The administration schedule should be selected considering the gender, age, weight, or condition of the subject (patient). For example, it may be a single dose, or multiple doses in succession or at regular intervals.

[0069] During the gene transfer process (S2), when lipid particles 10 come into contact with tumor cells, the lipid particles 10 are taken up into the tumor cells by endocytosis or membrane fusion between the lipid particles and the tumor cell membrane. Next, CAR antigen nucleic acid 3 and costimulatory factor nucleic acid 4 are released into the tumor cells from the lipid particles 10 taken up into the tumor cells, thereby introducing the CAR antigen nucleic acid and costimulatory factor nucleic acid into the tumor cells. The introduced CAR antigen nucleic acid expresses the CAR antigen on the surface of the tumor cells, thus restoring the targetability of tumor cells that have lost their surface antigens. In addition, the introduced costimulatory factor expresses the costimulatory factor on the surface of the tumor cells.

[0070] To allow the introduced CAR antigen nucleic acid and costimulatory factor nucleic acid to be expressed, a period of time may be allowed between the gene transfer step (S2) and the treatment step (S3) described below. For example, several days to several weeks may be allowed between the administration of the treatment effect enhancer and the next treatment step (S3).

[0071] In the treatment step (S3), after the gene transfer step (S2), the CAR-T cells prepared in the preparation step (S1) are administered to the target cells. This brings the CAR-T cells into contact with tumor cells that have expressed CAR antigens and costimulatory factors through the gene transfer step (S2).

[0072] In the treatment process (S3), CAR-T cells recognize CAR antigens expressed on the surface of tumor cells and kill them. Furthermore, since CAR-T cells also express costimulatory factors on the surface of the recognized tumor cells, their tumor cell-killing ability is enhanced by activation through interaction.

[0073] Here, CAR-T cells can be any cell population created by a general method. As long as they contain T cells, CAR-T cells can be created using various cell populations. For example, the cell population containing T cells may be peripheral blood mononuclear cells (PBMCs) collected from peripheral blood. Alternatively, CAR-T cells may be created so that 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 express CAR. It is more preferable that the cell population containing T cells is a cell population collected from the subject to whom the CAR-T cells 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.

[0074] The introduction of CAR genes into T cells may be carried out using any tool or method commonly known for gene transfer into cells. Furthermore, while it is not necessary to integrate the CAR gene into the T cell genome, as long as CAR expression is achieved, integration into the genome is more preferable. Examples of gene transfer tools and methods for T cells include polycations, calcium phosphate, lipofection, liposomes, electroporation, microinjection, particle guns, retro / lentiviral vectors, and adenoviral vectors. Additionally, tools utilizing transposon vectors, homologous recombination, recombinase systems, or exogenous gene integration tools such as ZFNs and CRISPR / Cas9 genome editing tools may be used in combination.

[0075] In the method of the second embodiment, the same gene introduction step as (S2) may be repeated multiple times after the treatment step (S3), or the same treatment step as (S3) may be repeated multiple times simultaneously.

[0076] Conventionally, a method has been known that uses viruses to introduce only the CAR antigen gene into tumor cells. However, with this method, administering the virus to the body leads to the acquisition of immunity to the virus, making multiple administrations impossible and forcing a single administration. Therefore, continuous treatment was difficult with conventional methods using viruses. On the other hand, the method of this embodiment does not use viruses, so multiple administrations are possible, and even if the surface antigen of tumor cells disappears again during the course of treatment, the targetability of the tumor cells can be restored.

[0077] The method of the second embodiment allows for highly efficient gene introduction into tumor cells by using a therapeutic effect enhancer, and consequently, highly efficient killing of tumor cells. Although cells other than tumor cells exist in the living body, the therapeutic effect enhancer of this embodiment contains lipid particles that are directional to tumor cells, and is superior in that it has little effect on normal cells other than tumor cells.

[0078] In a further embodiment, the timing of administering the therapeutic effect enhancer may be after the start of CAR-T cell therapy. That is, the CAR-T cell therapy of a further embodiment may include a preparation step (S1) of preparing a therapeutic effect enhancer containing lipid particles encapsulating CAR antigen nucleic acids and costimulatory factor nucleic acids, and CAR-T cells; a treatment step (S2') of administering CAR-T cells to a subject; and a gene transfer step (S3') of administering the therapeutic effect enhancer to the subject after S2.

[0079] [example] Below, we will describe an example of using a therapeutic effect-enhancing agent prepared according to the embodiment, using the method of the embodiment. However, the embodiments of the present invention are not limited to the following example.

[0080] Example 1. Confirmation of tumor cell killing effect and persistence of therapeutic effect enhancers through cell testing. To confirm the tumor cell-killing effect and its persistence by the therapeutic effect-enhancing agent, the following cell tests A to C were performed.

[0081] Cell study A is a comparative example to confirm the tumor cell-killing effect of CAR-T cells. Ephrin receptor B4 (EphB4) CAR-T cells were used. The CAR antigen was the extracellular domain of the EphB4 protein. Specifically, in cell study A, CAR-T cells were brought into contact with tumor cells and cultured, and then further subcultured to bring the tumor cells and CAR-T cells into contact again.

[0082] Cell study B is a comparative example designed to confirm the tumor cell-killing effect of CAR-T cells after adding a therapeutic effect enhancer that does not contain a costimulatory factor. Specifically, tumor cells into which the gene was introduced using liposomes containing mRNA encoding the CAR antigen (extracellular domain of the EphB4 protein) were brought into contact with CAR-T cells and cultured. After further subculturing, the tumor cells and CAR-T cells were brought into contact again under the same conditions.

[0083] Cell test C is an example to confirm the tumor cell-killing effect of CAR-T cells after adding the therapeutic effect-enhancing agent of the first embodiment. Specifically, CAR-T cells were brought into contact with tumor cells into which genes had been introduced using liposomes containing mRNA encoding CAR antigen and costimulatory factors (CD80 and 4-1BBL), and then cultured. After further subculturing, the tumor cells and CAR-T cells were brought into contact again under the same conditions.

[0084] • Preparation of tumor cells Alveolar rhabdomyosarcoma cell line Rh30 suspended in Dulbecco's modified Eagle (DMEM) medium supplemented with 10% fetal bovine serum was divided into 1 × 10⁻¹⁶ cells. 5 Cells were seeded in a 4-well culture plate and prepared by incubating in an incubator at 37°C with a 5% CO2 atmosphere.

[0085] • Preparation of CAR-T cells Human peripheral blood mononuclear cells (PBMCs) were subjected to a procedure to introduce the CAR gene, and a cell population containing CAR-T cells was created. The human peripheral blood mononuclear cells (PBMCs) were cultured in ALyS705 (containing IL7: 10 ng / mL and IL15: 5 ng / mL) with 5% artificial serum (Institute of Cell Science) added, in an incubator at 37°C under a 5% CO2 atmosphere. CAR-T cells were produced using the piggyBAC method with moth-derived DNA transferase. Specifically, plasmid DNA for CAR expression and plasmid DNA for piggyBAC expression were introduced into PBMCs by electroporation.

[0086] • Preparation of lipid particles For cell study B, mRNA encoding the extracellular domain of the CAR antigen EphB4 was used as the nucleic acid to be encapsulated in the lipid particles. For cell study C, mRNA encoding the extracellular domain of the CAR antigen EphB4, as well as mRNA encoding the costimulatory factors CD80 and 4-1BBL, were used. The solutions containing each mRNA were mixed with ethanol-soluble lipid solutions with the lipid composition described in Table 1 below. These solutions were then mixed with 10 mM HEPES (pH 7.3), washed by centrifugal ultrafiltration, and concentrated to obtain the respective solutions containing the lipid particles used in cell studies B and C.

[0087] [Table 1]

[0088] • Gene transfer into tumor cells using lipid particles In cell studies B and C, the respective lipid particles were brought into contact with tumor cells to introduce genes into the tumor cells. Specifically, the respective lipid particle-containing solutions prepared as described above were added to DMEM medium containing 10% fetal bovine serum containing tumor cells Rh30, which was prepared as described above, and genes were introduced. After mixing with the lipid particle-containing solutions, the cells were cultured for one day to induce gene expression.

[0089] • Measurement of the killing effect of CAR-T cells on tumor cells In cell study A, after the preparation of Rh30 tumor cells and EphB4 CAR-T cells, a culture medium containing both was mixed, and the survival rate of tumor cells, i.e., the killing rate by EphB4 CAR-T cells, was measured after 5 days. In cell studies B and C, after the expression of the transgene in Rh30 tumor cells and the preparation of EphB4 CAR-T cells were completed, a culture medium containing both was mixed, and the survival rate of tumor cells, i.e., the killing rate by EphB4 CAR-T cells, was measured after 5 days. The CAR-T cell killing rate (%) of tumor cells was calculated by dividing the number of tumor cells in each condition by the number of tumor cells in the negative control (number of tumor cells cultured without the addition of CAR-T cells), and then multiplying this value by 100. Furthermore, in cell studies A, B, and C, after the killing rate measurement, the surviving CAR-T cells were brought into contact with tumor cells to which the gene had been newly introduced by contacting lipid particles, and the killing rate by CAR-T cells was measured.

[0090] ·result The experimental results for Example 1 are shown in Figure 4. In Figure 4, the values ​​on the vertical axis represent the mortality rate, "1st" on the horizontal axis indicates the result of the first mortality rate measurement, and "2nd" on the horizontal axis indicates the result of the second mortality rate measurement after subculturing.

[0091] As shown in Figure 4, in cell study A, a control study, the first-time killing rate was approximately 60%, but the second-time killing rate was very low. This confirmed that tumor cells Rh30 were evading the immune system. In cell study B, a killing rate of over 90% was observed in the first trial, but the second-time killing rate was approximately 70%, indicating a decrease in the killing effect of EphB4 CAR-T cells on tumor cells Rh30.

[0092] On the other hand, in cell test C, the killing rate was over 90% in the first trial, and approximately 90% in the second trial, confirming the sustained killing effect of EphB4 CAR-T cells even against Rh30 tumor cells that can evade the immune system. Furthermore, it was confirmed that EphB4 CAR-T cells acquire a sustained killing effect on tumor cells by being activated upon contact with Rh30 tumor cells expressing CAR antigens and costimulatory factors. These results indicate that the expression of costimulatory factors CD80 and 4-1BBL is effective in sustaining the killing effect of CAR-T cells against tumor cells.

[0093] Example 2. Confirmation of the tumor cell-killing effect of therapeutic effect enhancers through animal testing. To confirm the tumor cell-killing effect of the presence or absence of a co-stimulatory factor, the following animal studies A to D were conducted. First, the experimental designs of animal studies A to D will be briefly explained with reference to Figure 5.

[0094] As shown in Figure 5(a), animal study A is a comparative example to confirm the proliferation rate of untreated tumor cells. Specifically, tumor cells 6, which originally express small amounts of CAR antigen as untreated tumor cells, were transplanted into mice, and their proliferation rate was measured over time.

[0095] As shown in Figure 5(b), animal study B is a comparative example to confirm the proliferation rate of untreated tumor cells expressing a costimulatory factor. Specifically, tumor cells 7 expressing a costimulatory factor were created by using the therapeutic effect enhancer of the first embodiment on tumor cells 6 that originally express a small amount of CAR antigen, and these tumor cells 7 were transplanted into mice, and their proliferation rate was measured over time.

[0096] As shown in Figure 5(c), animal study C is a comparative example to confirm the killing effect of CAR-T cells 8 on untreated tumor cells. Specifically, tumor cells 6 expressing small amounts of CAR antigen were transplanted into mice, and then CAR-T cells 8 were administered to the mice, and the proliferation rate of tumor cells 6 was measured over time.

[0097] As shown in Figure 5(d), Animal Test D is an example to confirm the killing effect of CAR-T cells on tumor cells expressing a costimulatory factor using the therapeutic effect enhancer of the first embodiment. Specifically, tumor cells 6 that originally express a small amount of CAR antigen were transplanted into mice with costimulatory factor-expressing tumor cells 7, which were created using the therapeutic effect enhancer 1. Then, CAR-T cells 8 were administered to the mice, and the proliferation rate of the costimulatory factor-expressing tumor cells 7 was measured over time.

[0098] • Preparation of tumor cells The tumor cells used in animal studies A to D were all Rh30, the same as in Example 1, and these Rh30 cells expressed small amounts of CAR antigen. However, as mentioned above, in animal studies B and D, the mice were transplanted with Rh30 cells into which genes encoding costimulatory factors had been introduced, and the costimulatory factors introduced were CD80 and 4-1BBL. These tumor cells were prepared by incubating them in DMEM medium supplemented with 10% fetal bovine serum in an incubator at 37°C and a 5% CO2 atmosphere.

[0099] • Preparation of CAR-T cells The CAR-T cells used in animal studies A-D were EphB4 CAR-T cells, the same as in Example 1. Specifically, a cell population containing CAR-T cells was created by introducing the CAR gene into PBMCs. The PBMCs were cultured in ALyS705 (containing IL7: 10 ng / mL and IL15: 5 ng / mL) with 5% artificial serum (Cell Science Institute) added, in an incubator at 37°C under a 5% CO2 atmosphere. The CAR-T cells were produced using the piggyBAC method with moth-derived DNA transferase. Specifically, plasmid DNA for CAR expression and plasmid DNA for piggyBAC expression were introduced into PBMCs by electroporation.

[0100] • Tumor cell transplantation and CAR-T cell administration In animal studies A-D, the animals transplanted with each type of tumor cell were NSG® mice. Specifically, the prepared tumor cells (2 × 10⁻¹⁴) were transplanted into each animal. 6A solution containing EphB4 CAR-T cells (0.1 mL) was subcutaneously administered to mice to create tumor-bearing mice. In animal studies C and D, 0.2 mL (10 × 10) of a solution containing EphB4 CAR-T cells was administered 7 days after tumor cell transplantation. 6 The cells were administered by subcutaneous injection. In the control studies, animal studies A and B, 0.2 mL of phosphate buffer (HEPES) was administered via tail vein 7 days after tumor cell transplantation.

[0101] • Measurement of tumor cell proliferation rate During animal studies A-D, tumor cell volume was measured over time as an indicator of tumor cell proliferation. Tumor cell volume was calculated by measuring the tumor diameter over time with calipers and using the formula (short diameter × short diameter × long diameter) / 2. The tumor size of the mouse was 2000 mm. 3 At that point, all the mice were euthanized.

[0102] ·result The experimental results for Example 2 are shown in Figure 6. In Figure 6, the vertical axis represents tumor volume, and the horizontal axis represents the number of days elapsed since tumor cell transplantation. The results of Animal Experiment A confirm that the tumor volume increased over time. Furthermore, although the rearing of Animal Experiment B was discontinued on day 28 due to the death of the mice, a similar increasing trend in tumor volume to that of Animal Experiment A was observed, indicating that the expression of co-expression factors alone does not affect the proliferation rate of tumor cells.

[0103] In animal study C, the tumor volume was about half the size of that in animal study A at 32 days after tumor cell transplantation, but by 36 days it had reached the same level as in animal study A. On the other hand, although an increase in tumor volume was observed in animal study D, from 25 days after tumor cell transplantation onwards, the tumor volume was generally about 25% of that in animal studies B to D. Therefore, it became clear that expressing not only CAR antigens but also costimulatory factors resulted in a significantly higher and more effective killing effect on CAR-T cells.

[0104] Example 3. Animal study to confirm the expression of costimulatory factors in tumor cells administered with a therapeutic effect enhancer. Animal experiment E below was performed to confirm the enhanced expression of CAR antigen and the expression of costimulatory factors in tumor cells by the therapeutic effect promoting agent.

[0105] · Preparation of tumor cells The tumor cells used in animal test E were the same Rh30 as in Examples 1 and 2. Rh30 was prepared by standing in an incubator at 37 °C and 5% CO2 atmosphere using DMEM medium supplemented with 10% fetal bovine serum.

[0106] · Preparation of therapeutic effect promoting agent As the nucleic acid encapsulated in lipid particles, in animal test E, the same mRNA encoding the extracellular domain of CAR antigen EphB4 and the mRNAs encoding costimulatory factors CD80 and 4-1BBL as in cell test C of Example 1 and animal test D of Example 2 were used. After mixing the solution containing each mRNA with the ethanol-dissolved lipid solution having the lipid composition described in Table 1 above, it was further mixed with 10 mM HEPES (pH 7.3) and washed and concentrated by centrifugal ultrafiltration to obtain a solution containing lipid particles (that is, the therapeutic effect promoting agent of the first embodiment).

[0107] · Tumor cell transplantation and administration of therapeutic effect promoting agent Tumor cells were transplanted into NSG mice in the same manner as in animal tests A to D of Example 2. Specifically, the prepared tumor cells (2×10 6 cells / 0.1 mL) were subcutaneously administered to the mice to prepare tumor-bearing mice. When tumor engraftment was confirmed, 50 μL of the therapeutic effect promoting agent (200 ng mRNA / μL) was administered into the tumor. Three days after the administration of the therapeutic effect promoting agent, the tumor was excised, fixed with formalin, and then paraffin sections were prepared. For the prepared paraffin sections, immunohistochemical staining (IHC) was performed using anti-EphB4 antibody for anti-CAR antigen and anti-CD80 antibody and anti-4-1BBL antibody for anti-costimulatory factor antibody. The results of IHC for each antibody were photographed with Vectra (registered trademark) 3, and the diaminobenzidine (DAB) positive rate was analyzed with image analysis software inForm.

[0108] · Results The experimental results are shown in Figure 7. Figure 7(a) is a pie chart showing the DAB positivity rate (intensity) of EphB4, (b) is a pie chart showing the DAB positivity rate (intensity) of CD80, and (c) is a pie chart showing the DAB positivity rate (intensity) of 4-1BBL. In the pie chart, "0", "+1", "+2", and "+3" represent the magnitude of the detected intensity, with the intensity increasing in this order.

[0109] Referring to Figure 7, the DAB positivity rate for CD80 was very low, while EphB4 and 4-1BBL showed high DAB positivity rates. This result indicates that in the tumors administered with the therapeutic effect enhancer in animal study E, both the CAR antigen and co-expression factors contained in the therapeutic effect enhancer were expressed. Therefore, it was confirmed that the therapeutic effect enhancer of the first embodiment introduced and expressed the co-stimulatory factors in tumor cells.

[0110] Example 4. Animal studies to confirm the tumor cell killing effect and persistence of therapeutic effect enhancers. To confirm the tumor cell-killing effect and its persistence by the therapeutic effect-enhancing agent, the following animal studies F to H were conducted. First, the experimental designs of animal studies F to H will be briefly explained with reference to Figure 8.

[0111] As shown in Figure 8(a), Animal Study F is a comparative example to confirm the proliferation rate of untreated tumor cells. Animal Study F follows almost the same procedure as Animal Study A in Example 2, but differs in the time period from tumor cell transplantation to HEPES administration and the number of HEPES administrations.

[0112] As shown in Figure 8(a), animal study G is a comparative example to confirm the killing effect of CAR-T cells 8 on tumor cells 6 when the therapeutic effect-enhancing agent of the present invention is not used. Specifically, tumor cells 6 that originally express a small amount of CAR antigen were transplanted into mice, and then CAR-T cells 8 were administered to the mice, and the proliferation rate of the tumor cells was measured over time.

[0113] As shown in Figure 8(a), animal study H is an example to confirm the killing effect of CAR-T cells 8 on tumor cells 6 when using the therapeutic effect enhancer 1 of the first embodiment. Specifically, tumor cells 6 that originally express a small amount of CAR antigen were transplanted into mice, then the therapeutic effect enhancer 1 was administered to the mice, followed by administration of CAR-T cells 8, and then the therapeutic effect enhancer 1 was administered again at a later date, and the proliferation rate of tumor cells 6 was measured over time.

[0114] • Preparation of tumor cells The tumor cells used in animal studies F-H were Rh30, the same as in Examples 1, 2, and 3. Specifically, Rh30 cells were prepared by incubating DMEM medium with 10% fetal bovine serum in an incubator at 37°C and a 5% CO2 atmosphere.

[0115] • Preparation of CAR-T cells The CAR-T cells used in animal studies F-H were EphB4 CAR-T cells, the same as in Examples 1 and 2. Specifically, a cell population containing CAR-T cells was created by introducing the CAR gene into PBMCs. The PBMCs were cultured in ALyS705 (containing IL7: 10 ng / mL and IL15: 5 ng / mL) with 5% artificial serum (Cell Science Institute) added, in an incubator at 37°C under a 5% CO2 atmosphere. CAR-T cells were produced using the piggyBAC method with moth-derived DNA transferase. Specifically, plasmid DNA for CAR expression and plasmid DNA for piggyBAC expression were introduced into PBMCs by electroporation.

[0116] • Preparation of therapeutic effect enhancers As nucleic acids to be encapsulated in the lipid particles, animal test H used mRNA encoding the extracellular domain of the CAR antigen EphB4 and mRNA encoding the costimulators CD80 and 4-1BBL, the same as in cell test C of Example 1 and animal test D of Example 2. After mixing the solutions containing each mRNA with ethanol-soluble lipid solutions with the lipid composition described in Table 1 above, the mixture was further mixed with 10 mM HEPES (pH 7.3), washed by centrifugal ultrafiltration, and concentrated to obtain a solution containing lipid particles (i.e., the therapeutic effect enhancer of the first embodiment).

[0117] • Tumor cell transplantation and CAR-T cell administration In animal studies F-H, tumor cells were transplanted into NSG mice, similar to animal studies A-D in Example 2. Specifically, the prepared tumor cells (2 × 10⁻¹⁴) were transplanted. 6 A solution containing HEPES or EphB4 CAR-T cells (10 × 10 mL) was administered subcutaneously to mice to create tumor-bearing mice. In comparative animal studies F and G, once the tumors had grown to a certain size, a solution containing HEPES or EphB4 CAR-T cells (10 × 10 mL) was administered subcutaneously to mice. 6 0.2 mL (cells / mL) was administered via tail vein.

[0118] • Administration of CAR-T cells and therapeutic effect enhancers In animal experiment H, 50 μL of a therapeutic effect enhancer (200 ng mRNA / μL) was administered into the tumor the day before HEPES or CAR-T cells were administered in animal experiments F and G. The following day, a solution containing EphB4 CAR-T cells (10 × 10) was administered. 6 0.2 mL (cells / mL) was administered via tail vein, and the following day, 50 μL of a therapeutic effect enhancer (200 ng mRNA / μL) was administered again into the tumor. The time course of the above animal studies F to H is shown in Figure 8(b).

[0119] • Measurement of tumor cell proliferation rate In animal studies F-H, as in animal studies A-D, the volume of tumor cells was measured over time as an indicator of tumor cell proliferation during the rearing period. The tumor cell volume was calculated by measuring the tumor diameter over time with calipers and using the formula (short diameter × short diameter × long diameter) / 2. The tumor size of the mouse was 2000 mm². 3At that point, all the mice were euthanized.

[0120] ·result The experimental results for Example 4 are shown in Figure 9. In Figure 9, the vertical axis represents tumor volume, and the horizontal axis represents the number of days elapsed since CAR-T cell administration. From the results of animal study F, it can be confirmed that the tumor volume increased over time from around 23 days after CAR-T cell administration. In animal study G, the tumor volume increased over time from around 29 to 33 days after CAR-T cell administration, and until day 43, the tumor volume was about half that of animal study F, but from day 47 onwards, a tumor volume similar to that of animal study F was observed. From these results, it can be seen that CAR-T cell administration delayed the onset of tumor cell proliferation, but ultimately the killing effect of CAR-T cells was limited.

[0121] In animal study H, tumor volume increased over time from around 43 to 47 days after CAR-T cell administration, but even at 50 days, the tumor volume was significantly smaller compared to animal studies F and G. Therefore, it was clear that the therapeutic effect-enhancing agent of this embodiment exerted a high killing effect on CAR-T cells. [Explanation of Symbols]

[0122] 1... Therapeutic effect enhancer, 10, 10a, 10b... Lipid particles, 2...carrier, 3...CAR antigen nucleic acid, 4...costimulatory factor nucleic acid, 5,5a,5b...lipid membrane, 6…Tumor cells expressing small amounts of CAR antigen, 7…Costimulatory factor-expressing tumor cells, 8…CAR-T cells

Claims

1. A therapeutic effect enhancer that promotes the therapeutic effect of CAR-T cell therapy, comprising a nucleic acid containing a gene encoding a target antigen of CAR and a nucleic acid containing a gene encoding a co-stimulatory factor.

2. The aforementioned therapeutic effect-enhancing agent contains lipid particles, The therapeutic effect enhancer according to claim 1, wherein the lipid particles encapsulate a nucleic acid containing a gene encoding the target antigen of the CAR and a nucleic acid containing a gene encoding the costimulator in a lipid membrane.

3. The therapeutic effect enhancer according to claim 2, wherein the lipid membrane comprises a lipid compound of the following formula (1-01) and / or a lipid compound represented by formula (1-02). 【Chemistry 1】 【Chemistry 2】

4. The therapeutic effect enhancer according to claim 3, wherein the lipid membrane further comprises at least one lipid compound selected from the group consisting of cationic lipids, neutral lipids, aggregation-reducing lipids, and structure-forming lipids.

5. The aforementioned therapeutic effect-enhancing agent contains two types of lipid particles, One of the lipid particles contains nucleic acids that include a gene encoding the target antigen of the CAR, The other lipid particle contains nucleic acid including the gene encoding the aforementioned costimulator. A therapeutic effect enhancer according to claim 1.

6. The aforementioned one lipid particle and the aforementioned other lipid particle are composed of a lipid membrane containing the lipid compound of formula (1-01) and / or the lipid compound represented by formula (1-02) below. The lipid composition of the lipid membrane of one of the aforementioned lipid particles and the lipid composition of the lipid membrane of the other lipid particle are either the same or different. A therapeutic effect enhancer according to claim 5. 【Transformation 3】 【Chemistry 4】

7. The therapeutic effect enhancer according to claim 6, wherein the lipid membrane of one lipid particle and the other lipid particle further comprises at least one lipid compound selected from the group consisting of cationic lipids, neutral lipids, aggregation-reducing lipids, and structure-forming lipids.

8. The therapeutic effect enhancer according to claim 1, wherein the nucleic acid containing the gene encoding the costimulator is the full length or a portion of at least one nucleic acid selected from the group consisting of 4-1BBL, CD80, CD86, ICOSL, and OX40L.

9. The therapeutic effect enhancer according to claim 8, wherein the nucleic acid containing the gene encoding the costimulator is at least one nucleic acid selected from the group consisting of DNA, mRNA, plasmids, or polynucleotides.

10. The therapeutic effect enhancer according to claim 2, comprising a storage stabilizer for the lipid particles.

11. The therapeutic effect enhancer according to claim 5, comprising a storage stabilizer for one of the lipid particles and the other lipid particle.

12. The therapeutic effect enhancer according to claim 10 or 11, wherein the storage stabilizer is a pH adjuster, a buffering agent, a tonicity adjuster, and a cryoprotectant.

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