WO2019103151A1 - Lipid membrane structure for delivering nucleic acid to within cell - Google Patents

Lipid membrane structure for delivering nucleic acid to within cell Download PDF

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WO2019103151A1
WO2019103151A1 PCT/JP2018/043528 JP2018043528W WO2019103151A1 WO 2019103151 A1 WO2019103151 A1 WO 2019103151A1 JP 2018043528 W JP2018043528 W JP 2018043528W WO 2019103151 A1 WO2019103151 A1 WO 2019103151A1
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lipid membrane
membrane structure
kala
lipid
nucleic acid
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PCT/JP2018/043528
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French (fr)
Japanese (ja)
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英万 秋田
尚也 三浦
菜穂 舘下
浩揮 田中
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国立大学法人千葉大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a lipid membrane structure for introducing nucleic acid into cells.
  • immunotherapy which treats diseases by inducing or enhancing an immune response, is focused on the treatment of cancer.
  • it is required to induce a specific immune response to an antigenic substance by administering the antigenic substance to a living body or introducing it into isolated immune cells.
  • the antigenic substance is a polypeptide
  • it is possible to use a DNA encoding the polypeptide instead of the polypeptide itself to induce a desired immune response.
  • DNA is generally referred to as a DNA vaccine.
  • Patent Document 1 discloses a DNA-encapsulated liposome in which a lipid membrane is modified with a specific peptide called KALA peptide for delivering DNA into the nucleus of immune cells, particularly dendritic cells. It is done.
  • the KALA peptide is a short polypeptide comprising a repeating unit of the amino acid sequence KAALA (K is lysine, A is alanine, L is leucine).
  • the amino acid sequence is represented by a single letter code.
  • KALA peptide is a peptide that is known to form a complex with DNA (Non-patent Document 1), but in Patent Document 1, it is used as a functional peptide for promoting the ability of liposomes to enter the nucleus. ing.
  • Patent Document 2 discloses a DNA-encapsulated liposome that has been modified with a modified peptide that maintains the function of promoting the nuclear import of KALA peptide.
  • the liposomes disclosed in Patent Document 1 and Patent Document 2 all exemplify the form of a liposome in which a complex of DNA and a polycationic substance is enclosed by a lipid membrane.
  • endocytosis uptake of liposome into cell escape of liposome from endosome to cytoplasm, transfer of liposome into cell nucleus and release of the complex in cell nucleus, transcription of nucleic acid
  • a large number of processes are required for the function of the delivered nucleic acid to be performed, such as protein translation and further antigen presentation on the cell surface.
  • the present inventors have delivered an mRNA encoding an antigenic polypeptide into an antigen-presenting cell, and encapsulated mRNA for the purpose of enhancing the ability to induce an immune response by omitting transcription of mRNA from DNA.
  • the lipid membrane structure described in 1 was prepared and transfected into antigen-presenting cells. However, it was newly confirmed that the antigen presentation intensity of the transfected cells was almost the same as that of the cells transfected with the lipid membrane structure described in Patent Document 1 in which DNA was encapsulated as a comparative object.
  • the present invention is intended to deliver nucleic acids into cells to exert desired functions more efficiently, and in particular to deliver nucleic acids into antigen-presenting cells to further enhance the antigen presentation strength of transfected cells. , Aims to provide a new means of intracellular delivery.
  • the present inventors transfect a lipid membrane structure in which mRNA is retained on the surface of a lipid membrane into an antigen-presenting cell, compared to the case where a lipid membrane structure encapsulating DNA is introduced, compared to the case of transfected cells.
  • the inventors have found that the antigen presentation intensity can be increased, and completed the following invention.
  • the peptide chain contains at least three repeating units of KALA (however, K may be R in any one or two or more of the repeating units, and / or
  • the lipid membrane is modified by at least one of a polypeptide having 12 to 50 amino acid residues, in which one or two or more of them, A and H between K and L may be H)
  • the polypeptide contains 3 or 4 repeating units of KALA in the peptide chain (however, K may be R in any one or two or more of the repeating units, and / or Or the lipid according to (1), which is a polypeptide having 12 to 30 amino acid residues in the number of amino acid residues between which K and L may be A in any one of the repeating units.
  • Membrane structure (3) The lipid membrane structure according to (1) or (2), wherein the polypeptide is a polypeptide consisting of any of the amino acid sequences shown in the following a) to g).
  • the function required for the nucleic acid can be efficiently exhibited.
  • delivery of mRNA encoding an antigenic polypeptide into an antigen-presenting cell can enhance the antigen presentation strength in the antigen-presenting cell, and can be used in immunotherapy targeting a desired antigenic polypeptide Can produce various immune cells.
  • a lipid membrane structure modified by the KALA peptide or variant thereof in which the nucleic acid is retained on the surface is required for the desired function as compared to the lipid membrane structure in which the nucleic acid is encapsulated inside the lipid membrane.
  • the amount of nucleic acid used can be reduced, which is advantageous in reducing manufacturing costs and avoiding undesirable side effects and the like.
  • FIG. 5 is a graph showing antigen-specific CD8 + T cell induction by dendritic cells transfected with KALA peptide-modified liposomes, the surface of which holds mRNA encoding OVA.
  • FIG. 10 is a graph showing CTL activity induction when KALA peptide-modified liposomes having different amounts of OVA mRNA retained on their surface are administered subcutaneously or intravenously.
  • the present invention relates to a lipid membrane-modified lipid membrane structure for delivering nucleic acid into cells, wherein the nucleic acid is retained on the surface thereof.
  • the polypeptide used in the present invention contains at least three repeating units of KALA in the peptide chain (with the proviso that K is R in any one or more of the repeating units). And / or a polypeptide having 12 to 50 amino acid residues in which one or two or more of the repeating units may interpose K and L and A may be H).
  • the polypeptide used in the present invention comprises at least 3, for example 3 to 10, preferably 3 to 7, and more preferably 3 or 4 KAA repeat units in the peptide chain.
  • K may be R in any one or two or more of the repeating units, and / or any one or two or more of the repeating units, preferably 1 to 3 A, which is sandwiched between K and L in one piece, more preferably in one piece, may be H.
  • the number of amino acid residues of the polypeptide is 12 to 50, preferably 12 to 40, and more preferably 12 to 30.
  • a typical example of the polypeptide used in the present invention is a polypeptide consisting of the amino acid sequence shown in the following a) to g).
  • a) WEAKLAKALAKALAKHLAKALAKALKA (SEQ ID NO: 1)
  • b) WEAKLAKALAKALAK HLA KALAKALKACEA SEQ ID NO: 2
  • c) WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 3)
  • WEAKLAKALAKALAK HLA SEQ ID NO: 4
  • e) KALAKALAKALAKALA SEQ ID NO: 5
  • KALAKALAKALA SEQ ID NO: 6
  • g) WEARLARALARALARHLARALA RA (SEQ ID NO: 7)
  • KALA peptide KALA peptide is known to exhibit a so-called ⁇ -helix structure in which the repeating unit is one cycle under neutral conditions of about pH 7.4, and a random coil structure under weakly acidic conditions of about pH 5.0 There is.
  • the KALA peptide is disposed on the surface of the lipid membrane structure as disclosed in the above-mentioned Patent Document 1 (International Publication WO 2011/132713), thereby enhancing the ability of the lipid membrane structure to be transferred into the cell nucleus It has a function. It is also known that the whole peptide is positively charged by a plurality of Lys residues, and has a function of forming a complex with negatively charged DNA (International Publication WO 2011/132713).
  • b) is a peptide consisting of an amino acid sequence in which three amino acid residues are added to the C-terminus of the KALA peptide, and like the KALA peptide, it has an ⁇ -helix structure and a random coil structure depending on pH, It has a function of enhancing the ability of the membrane construct to move into the cell nucleus and the ability to form a complex with a nucleic acid.
  • c) A peptide consisting of an amino acid sequence in which KALKA at the C-terminus of KALA peptide is deleted; , E) and f) are peptides consisting of an amino acid sequence in which the repeating unit of KALA is repeated 4 or 3 respectively, g) is a peptide consisting of an amino acid sequence in which all Ks are substituted for R in the amino acid sequence .
  • c) to f) are the peptides disclosed in the above-mentioned Patent Document 2 (International Publication WO 2015/098907), and have the same function as the KALA peptide.
  • International Publication WO 2011/132713 and corresponding US Application Publication US 2013/0122054, and International Publication WO 2015/098907 are all incorporated herein by reference.
  • polypeptides may also be used in the present invention: A polypeptide comprising an amino acid sequence in which K is R in any one or more of the repeating units of KALA in the amino acid sequences shown in a) to f) above; A polypeptide comprising an amino acid sequence in which A is H which is sandwiched between K and L in any one of the repeating units of KALA in the amino acid sequence shown in the above e) or f); A polypeptide consisting of an amino acid sequence in which KHLA is KALA in the amino acid sequences shown in a) to d) above; A polypeptide consisting of an amino acid sequence in which RHLA is RALA in the amino acid sequence shown in g).
  • polypeptides in the present invention are shown in the polypeptide consisting of the amino acid sequence shown in a), the polypeptide consisting of the amino acid sequence shown in c), the polypeptide consisting of the amino acid sequence shown in e) and g) A polypeptide consisting of the amino acid sequence
  • the polypeptides used in the present invention are collectively referred to as KALA peptide variants.
  • KALA peptide variants can be prepared biologically using appropriate host vector systems, utilizing a variety of genetic recombination techniques available to one skilled in the art.
  • the KALA peptide variant may be prepared using a solid phase synthesis method or other organic chemical method utilizing peptide synthesis reaction or an automatic peptide synthesizer such as a peptide synthesizer.
  • a “lipid membrane structure modified by one or more of polypeptides” is a lipid membrane structure having one or more of KALA peptide variants on the surface of the lipid membrane, specifically, It means a lipid membrane structure existing on the surface of the lipid membrane in a state where these can be in contact with other substances, particularly when the lipid membrane structure is in the form of a liposome, on the outer surface of the lipid membrane
  • a lipid membrane structure in which all of the variants are buried in the layer of the lipid bilayer, or a lipid membrane structure in which all of the KALA peptide variants exist in a lipid membrane-closed internal space a so-called polypeptide-encapsulated lipid membrane structure It is distinguished from However, as long as the KALA peptide variant is present on the surface of the lipid membrane, it is also inside the lipid bilayer, the interior space of the lipid membrane structure closed by the lipid membrane, or the inner surface of the lipid membrane facing this interior space It is acceptable for the poly
  • the KALA peptide variant is a dehydration reaction between a stearyl group, a derivative having a cholesteryl group and any other hydrophobic group, in particular, an amino group at the N-terminus of the polypeptide and a carboxylic acid group of a fatty acid having a medium chain length or more.
  • a fatty acid derivative prepared by The derivative of KALA peptide variant is to anchor the polypeptide on the surface of lipid membrane so that the polypeptide moiety remains on the surface of lipid membrane by the hydrophobic group being buried in the lipid membrane of lipid membrane structure it can.
  • the nucleic acids used in the present invention include, in addition to DNA or RNA, their analogues or derivatives (eg, peptide nucleic acid (PNA) and phosphorothioate DNA etc.).
  • the nucleic acid may be single stranded or double stranded, and may be linear or circular. In addition, it may be a nucleotide compound such as cGAMP (a dimer of AMP and GMP) which is STING ligand.
  • the length of the nucleic acid is not particularly limited.
  • Preferred nucleic acids in the present invention are those that are desired to be delivered intracellularly, particularly in the cytoplasm, and examples include nucleic acid adjuvants such as dsRNA, cGAMP, CpG ODN, polyIC, mRNA encoding an antigenic polypeptide, And antisense nucleic acids and inhibitory nucleic acids such as siRNA can be mentioned. Two or more types of nucleic acids can be simultaneously held in the lipid membrane structure.
  • the lipid membrane structure of the first aspect is excellent in the function of delivering a nucleic acid into the cytoplasm of an immune cell, particularly an antigen-presenting cell, and the nucleic acid used for this purpose is an mRNA encoding an antigenic polypeptide Is preferable, and the mRNA and the nucleic acid adjuvant may be combined.
  • the antigenic polypeptide any polypeptide having antigenicity can be selected.
  • a lipid membrane structure “having a nucleic acid on its surface” is a lipid membrane structure having a nucleic acid on the surface of the lipid membrane, specifically a lipid membrane of a lipid membrane in which the nucleic acid can be contacted with other substances.
  • a lipid membrane structure present on the surface in particular when the lipid membrane structure is in the form of a liposome, means a lipid membrane structure present on the outer surface of the lipid membrane, and the nucleic acid is closed with the lipid membrane It is distinguished from lipid membrane structures in which the so-called nucleic acid is present in the inner space.
  • the nucleic acid may be present in the interior space closed by the lipid membrane.
  • the nucleic acid mainly exists electrostatically bound to the KALA peptide variant on the surface of the lipid membrane structure, but may be bound to the surface of the lipid membrane according to other embodiments as long as the intended function is not impaired. Good.
  • the free KALA peptide variant forms a complex with the nucleic acid through electrostatic interaction, even if this complex is introduced into cells as it is, it does not have a lipid membrane and fusion with the biomembrane to be overcome is difficult. As a result, it is considered unsuitable for delivery of nucleic acid to the cytoplasm. It has been shown that even when a complex of free KALA peptide and pDNA is actually added to dendritic cells, cytokine production is not recognized because it is not recognized by the cytoplasmic nucleic acid sensor (Miura, N. et al., Nucleic Acids Res., 2015, 43, 1317-31). On the other hand, since the lipid membrane structure of the first aspect has a membrane fusogenic lipid, it is expected that the nucleic acid can be efficiently delivered to cells and the function thereof can be exerted.
  • the form of the lipid membrane structure in the first aspect is not particularly limited, and examples thereof include liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, wormlike micelles, and amorphous layered structures. It can be mentioned.
  • the preferred form of lipid membrane structure is a liposome.
  • the lipid membrane structure may be described as an example of a liposome, but the lipid membrane structure of the first aspect is not limited to the liposome.
  • Examples of the lipid constituting the lipid membrane of the lipid membrane structure include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids.
  • Examples of phospholipids and phospholipid derivatives include phosphatidyl ethanolamine, phosphalysyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, cardiolipin, sphingomyelin, ceramide phosphoryl ethanolamine, ceramide phosphoryl glycerol, ceramide phosphoryl glycerol phosphate, 1 And 2-dimyristoyl-1,2-deoxyphosphatidyl choline, plasmalogen, dioleoylphosphatidyl ethanolamine, phosphatidic acid and the like, which may be used alone or in combination of two or more.
  • the fatty acid residue in these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms.
  • lauric acid, myristic acid, palmitic acid, stearin Examples include acyl groups derived from fatty acids such as acid, oleic acid and linoleic acid.
  • phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.
  • glycolipids examples include glyceroglycolipids (eg, sulfoxyribosyl glycerides, diglycosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides, glycosyl diglycerides), glycosphingolipids (eg, galactosylcerebroside, lactosylcerebroside, gangliosides) and the like. It can be mentioned.
  • glyceroglycolipids eg, sulfoxyribosyl glycerides, diglycosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides, glycosyl diglycerides
  • glycosphingolipids eg, galactosylcerebroside, lactosylcerebroside, gangliosides
  • sterols examples include animal-derived sterols (eg, cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterols) (eg, stigmasterol, sitosterol, campesterol, Brashcasterol), sterol derived from a microorganism (eg, thymosterol, ergosterol) and the like.
  • animal-derived sterols eg, cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol
  • plant-derived sterols eg, stigmasterol, sitosterol, campesterol, Brashcasterol
  • sterol derived from a microorganism eg, thymosterol, ergosterol
  • saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid and myristic acid.
  • a tertiary amine and a lipid having a disulfide bond can also be used as a lipid component.
  • a lipid is disclosed, for example, in International Publication WO 2013/73480, and by having a tertiary amine and a disulfide bond in the molecule, it has pH responsiveness and membrane destabilizing action under a reducing environment.
  • the lipid represented by the following formula (1) disclosed in the above-mentioned International Publication is preferably as a constituent lipid of a lipid membrane structure.
  • X a and X b are independently X 1 or X 2 ; s is 1 or 2; R 4 represents an alkyl group having 1 to 6 carbon atoms, n a and n b are independently 0 or 1, R 1a and R 1b independently represent an alkylene group having 1 to 6 carbon atoms, R 2a and R 2b independently represent an alkylene group having 1 to 6 carbon atoms, Y a and Y b independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, R 3a and R 3b independently represent a sterol residue, a fat-soluble vitamin residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms)
  • lipid represented by the formula (1) for example, a lipid such as myristic acid conjugated lipid (PalmM), a retinoic acid conjugated lipid (PalmA), and a tocopherol conjugated lipid (PalmE) as a side chain
  • PalmM myristic acid conjugated lipid
  • PalmA retinoic acid conjugated lipid
  • PalmE tocopherol conjugated lipid
  • Palm E is preferably a neutral lipid and can be preferably used in the present invention because gene expression can be achieved even in the presence of serum. Palm E is described in paragraph [0056] of International Publication WO 2013/73480. Compound B-2-5 in Table 1 of It is.
  • the disclosures of the above-mentioned International Publication and the corresponding disclosure of US application publication US2014 / 0335157 are incorporated herein by reference.
  • the amount of the above-mentioned lipid to be used is not particularly limited, but in general, it can be about 5 to 50% by weight with respect to the total lipid constituting the lipid membrane structure.
  • appropriate Palm species can be selected according to the purpose while referring to these publications.
  • Particularly preferable lipids constituting the lipid membrane of the lipid membrane structure of the first aspect include dioleoylphosphatidylethanolamine (DOPE), phosphatidic acid (PA), phosphatidylserine (PS) and cardiolipin (CL). Can.
  • DOPE dioleoylphosphatidylethanolamine
  • PA phosphatidic acid
  • PS phosphatidylserine
  • CL cardiolipin
  • lipid-like materials exhibiting intracellular environmental responsiveness described in International Publication WO 2016/121942, International Publication WO 2016/027699, and International Publication WO 2013/073480 can also be mentioned.
  • the charge amount of KALA peptide variant, and the charge ratio of KALA peptide variant to nucleic acid they constitute a lipid membrane structure capable of electrostatically binding to nucleic acid. It can adjust suitably in the range acquired.
  • the lipid membrane structure of the first aspect is a membrane stabilizer such as sterol or glycerin or a fatty acid ester thereof, an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, And one or more types of substances selected from the group consisting of membrane polypeptides and the like may be included as a component of the lipid membrane or a component to be enclosed by the lipid membrane.
  • a membrane stabilizer such as sterol or glycerin or a fatty acid ester thereof
  • an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene
  • a charged substance a charged substance
  • one or more types of substances selected from the group consisting of membrane polypeptides and the like may be included as a component of the lipid membrane or a component to be enclosed by the lipid membrane.
  • Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane; and cationic polymers etc.
  • Examples of the charged substance that can give a negative charge can be mentioned dicetyl phosphate, cholesteryl hemisuccinate, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and the like.
  • the membrane polypeptide includes, for example, a membrane-surrounding polypeptide or an integral membrane polypeptide. The compounding quantity of these substances is not specifically limited, According to the objective, it can select suitably.
  • the lipid membrane structure of the first aspect may be provided with any one or more functions such as a temperature change sensitivity function, a membrane permeability function, a gene expression function, and a pH sensitivity function. it can.
  • a temperature change sensitivity function e.g., a temperature change sensitivity function
  • a membrane permeability function e.g., a membrane permeability function
  • a gene expression function e.g., a pH sensitivity function
  • a pH sensitivity function e.g., pH sensitivity function
  • the retention property of the lipid membrane structure containing a nucleic acid containing a gene, etc. in blood is improved, and the capture rate by reticular endothelial tissue such as liver and spleen is decreased.
  • endocytosis in target cells it is possible to efficiently escape lipid membrane structures from endosomes.
  • thermo change sensitive lipid derivative capable of imparting a temperature change sensitivity function
  • dipalmitoyl phosphatidyl choline and the like can be mentioned.
  • pH sensitive lipid derivative capable of imparting a pH sensitive function for example, dioleoylphosphatidylethanolamine etc. can be mentioned.
  • the lipid membrane structure of the first aspect is glycophorin, ganglioside GM1, phosphatidylinositol or other lipid derivative or polyalkylene glycol or other hydrophilic polymer capable of enhancing its retention in blood, GALA promoting endosomal prolapse ability Peptides, MPC polymers that enhance biocompatibility, antibodies that can specifically bind to receptors or antigens on the cell surface of interest, polypeptides containing multiple consecutive arginine residues, structural disruption of lipid membranes You may have INF7 grade
  • lipid A monophosphoryl lipid A (MPLA), Endocine (Eurocine)
  • a lipid adjuvant such as glucopyranosyl lipid adjuvant, saccharides, peptides, low molecular weight compounds, metal compounds and the like
  • MPLA monophosphoryl lipid A
  • Eurocine Endocine
  • a lipid adjuvant such as glucopyranosyl lipid adjuvant, saccharides, peptides, low molecular weight compounds, metal compounds and the like
  • a particularly preferred lipid adjuvant according to the invention is MPLA.
  • the content of the lipid adjuvant in the lipid membrane structure may be about 0.0003 to 3% by weight of the total lipid, preferably 0.001 to 1% by weight, and more preferably 0.003 to 0.3% by weight of the total lipid.
  • a liposome which is a preferred form of lipid membrane structure
  • a solution in which a lipid component constituting a lipid membrane is dissolved in an organic solvent such as chloroform is dried to form a lipid mixture, and then an aqueous solvent is added to the dried lipid mixture to homogenize or super
  • Liposomes in which an aqueous solvent is sealed are prepared by performing sonication, high-pressure injection or the like to emulsify.
  • Liposomes can be produced. Liposomes may be prepared by known methods other than hydration, such as reverse phase evaporation.
  • the liposome which is a preferred form of the first aspect may be either a unilamellar liposome or a multilamellar liposome, but a unilamellar liposome is preferred.
  • a derivative of KALA peptide variant is dissolved in an organic solvent such as chloroform together with a lipid component constituting a lipid membrane to form a lipid mixture containing a derivative of KALA peptide variant.
  • the liposome which is a preferable form of the first aspect may be produced by preparing a liposome by adding an aqueous solvent or the like and further adding a nucleic acid.
  • the addition amount of the derivative of KALA peptide variant is 1 to 50 mol%, preferably 5 to 30 mol%, relative to the total lipid amount of the lipid membrane structure.
  • the addition amount of the nucleic acid is not particularly limited, but may be appropriately adjusted in the range of 1 to 20% by weight with respect to 1 part by weight of the lipid membrane structure.
  • the composition of the aqueous solvent (dispersion medium) enclosed in the inside is not particularly limited as long as the lipid membrane structure can be stably dispersed.
  • buffers such as phosphate buffer, citrate buffer and phosphate buffered saline, physiological saline, medium for cell culture and the like can be mentioned.
  • These aqueous solvents (dispersion media) may further contain monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, polyhydric alcohols (aqueous solutions) and the like.
  • drugs, proteins and other physiologically active substances may be contained.
  • the size of the liposome which is a preferred embodiment of the first aspect, can be made uniform by extracting the lipid membrane inclusion body or the liposome after production through a membrane filter under high pressure.
  • the size is not particularly limited, but the average particle size is about 50 nm to 5 ⁇ m, preferably about 50 nm to 400 nm, more preferably about 50 nm to 300 nm, and still more preferably about 100 nm to 250 nm. Good.
  • a liposome which is a preferable form of the first aspect there may be mentioned a unilamellar liposome or multilamellar liposome which is a form dispersed in an aqueous solvent or a form obtained by freeze-drying or spray-drying an aqueous dispersion.
  • Liposomes in the form of an aqueous dispersion or in the form of a dry product can be prepared according to methods common to those skilled in the art.
  • the preferred form of the lipid membrane structure is a unilamellar liposome in the form of an aqueous dispersion or in the form of a dry product.
  • the type of cells targeted for nucleic acid delivery by the lipid membrane structure of the first aspect is not particularly limited, and appropriate cells can be targeted depending on the function or delivery purpose expected for the nucleic acid.
  • Preferred examples of the cells include immune cells.
  • immune cells antigen-presenting cells such as macrophages, dendritic cells and B cells are particularly preferable, and dendritic cells are particularly preferable.
  • an antigen-presenting cell is translated from mRNA by delivering an mRNA encoding an antigenic polypeptide into the cytoplasm of an antigen-presenting cell such as dendritic cell using the lipid membrane structure of the first aspect Since the polypeptide is presented on the surface of the antigen-presenting cell, and the living body can acquire immunity against the polypeptide, an effective immunotherapy targeting the desired antigenic polypeptide can be performed.
  • the present invention provides, as another embodiment, a method of producing transfected immune cells for immunotherapy in vitro, which comprises the step of incubating the immune cells and the lipid membrane structure according to the first aspect described above.
  • Particularly preferred immune cells are antigen-presenting cells such as macrophages, dendritic cells and B cells, and dendritic cells are most preferred.
  • the antigenic protein Transfected antigen-presenting cells having antigen-presenting ability with respect to Furthermore, instead of the above lipid membrane structure, a lipid membrane structure carrying both an mRNA encoding an antigenic polypeptide and a nucleic acid adjuvant or a lipid membrane structure carrying a mRNA encoding an antigenic polypeptide and a nucleic acid
  • a lipid membrane structure carrying a mRNA encoding an antigenic polypeptide and a nucleic acid By using a mixture with a lipid membrane structure retaining an adjuvant, it is also possible to produce antigen-presenting cells with further enhanced immunity inducing ability.
  • the transfected immune cells are administered to a subject in need of immunotherapy, it is preferable to use immune cells collected from the subject.
  • the transfected immune cells produced according to this embodiment are specific for the antigenic protein in subjects requiring immunotherapy targeting the antigenic polypeptide, preferably humans, particularly cancer patients. Can induce an immune response, and can be used in immunotherapy, particularly in cancer immunotherapy. Therefore, the present invention requires the steps of incubating the immune cells and the lipid membrane structure of the first aspect described above to transfect the immune cells, and immunotherapy the effective amount of the transfected immune cells. Immunotherapy comprising the step of administering to a subject is also provided as yet another embodiment.
  • the delivery of the nucleic acid to cells, preferably immune cells, using the lipid membrane structure of the first aspect can also be carried out by directly administering the lipid membrane structure to a living body.
  • the present invention thus provides, as a further aspect, a pharmaceutical composition comprising the lipid membrane structure of the first aspect as an active ingredient.
  • the lipid membrane structure used in the pharmaceutical composition of the above aspect has a means for improving the biocompatibility and retention in blood of lipid membrane fusion described in the above-mentioned International Publications WO2011 / 132713 and WO2015 / 098907. It is preferable to adopt.
  • the pharmaceutical composition of the above aspect can be used according to a dosage form, usage, dose and the like generally known as a liposome drug or preparation.
  • the pharmaceutical composition can be used in the form of parenteral preparations such as injections, drips and the like, and as carriers that can be used for such parenteral preparations, for example, physiological saline, glucose, An aqueous carrier such as an isotonic solution containing D-sorbitol and the like can be mentioned.
  • the pharmaceutical composition of the above aspect may further contain ingredients such as pharmaceutically acceptable buffers, stabilizers, preservatives and other additives.
  • Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can, according to the form of the preparation, for example, from the ingredients described in the 17th Revised Japanese Pharmacopoeia and other specifications within the scope of ordinary practice Therefore, they can be selected appropriately and used.
  • the pharmaceutical composition of the above aspect comprising as an active ingredient the lipid membrane structure of the first aspect carrying an mRNA encoding an antigenic protein as an active ingredient is a subject requiring immunotherapy targeting the antigenic polypeptide.
  • an immune response specific to the antigenic protein can be induced against immune cells in the body, and immunotherapy, particularly cancer immunotherapy are preferably used.
  • immunotherapy comprising administering an effective amount of the pharmaceutical composition of the above aspect to a subject in need thereof is also provided as a further aspect of the present invention.
  • the administration method of the pharmaceutical composition is not particularly limited, but in the case of a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, subcutaneous administration and the like can be mentioned.
  • intravascular administration preferably intravenous administration
  • intraperitoneal administration preferably intraperitoneal administration
  • enteral administration preferably subcutaneous administration and the like
  • subcutaneous administration preferably intracutaneous administration
  • the pharmaceutical composition is administered to a living body by intravenous administration.
  • KALA peptide-modified liposomes containing nucleic acid on the surface (final lipid concentration: 0.183 mM, final nucleic acid concentration: 0.013 mg / mL)
  • a nucleic acid / lipid ratio of 4 ⁇ g nucleic acid / 55 nmol lipid hereinafter referred to as pDNA KALA-Lipoplex or mRNA KALA-Lipoplex) was produced.
  • KALA peptide-modified liposomes (final lipid concentration: 0.275 mM, final nucleic acid concentration: 0.020 mg / mL; hereinafter referred to as pDNA KALA-MEND or mRNA KALA-MEND etc.) encapsulating the nucleic acid inside were produced. .
  • mRNA DOTAP-Lipoplex A comparative cationic liposome (final lipid concentration: 0.183 mM, final mRNA concentration: 0.013 mg / mL; hereinafter referred to as mRNA DOTAP-Lipoplex) was prepared.
  • Table 1 shows the results of measurement of particle size distribution and zeta potential of liposomes produced using mRNA encoding OVA (1239 nucleotides, the method of preparation is described in Example 3 (2)) as nucleic acid in (1) and (2). Shown in. No significant difference was observed between the two liposomes in any of particle diameter, zeta potential and polydispersity index (Pdl).
  • the femur and tibia are isolated from C57BL / 6J mice or Balb / c mice (6- to 8-week-old females) euthanized by cervical dislocation and disinfected with 70% ethanol, and then 20 mL of PBS (-) in a sterile petri dish Dipped in The bone was cut at both ends and bone marrow cells were pushed into a petri dish using 20 mL of complete medium using a 1 mL syringe fitted with a 26G needle (Terumo). The cell suspension was collected through a 40 ⁇ m cell strainer (Falcon) into a 50 mL conical tube and centrifuged at 500 g for 5 minutes at 4 ° C.
  • Falcon cell strainer
  • the supernatant was removed, and the remaining cell pellet was suspended in 1 mL of ACK Lysing buffer (Lonza) and incubated at room temperature for 5 minutes to destroy erythrocytes. 9 mL of complete medium was added, mixed, and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed, and the remaining cell pellet was suspended in 10 mL of complete medium and centrifuged at 500 g for 5 minutes at 4 ° C. After repeating the same operation, the cells were suspended in 10 mL of complete medium, and then inoculated into a 100 mm culture dish and incubated at 37 ° C. under 5% CO 2 .
  • non-adherent cells were recovered by lightly washing the bottom of the dish with the supernatant. After cell counting, suspend in 10 ng / mL mGM-CSF (R & D systems) -containing complete medium (complete medium for BMDC) to 1.0 ⁇ 10 6 cells / mL, and make 1 mL / well in a 24-well plate. Sowed. After removing suspended cells to leave clumps of cells 2 and 4 days after seeding, 1 mL of complete medium for BMDC was added to each well. Six days after the seeding, floating cells and weakly adherent cells in each well were collected by pipetting and used for experiments as immature dendritic cells (Bone Marrow-derived Dendritic Cells, BMDC).
  • BMDC immature dendritic cells
  • Luciferase-encoding pDNA a DNA fragment encoding Luciferase was inserted into pCpGfree-MCS (invivogen), prepared from E. coli using Endofree Plasmid Giga Kit) or mRNA ( The pDNA having a sequence encoding Luciferase downstream of the T7 promoter was prepared by in vitro transcription using mMESSAGE mMACHINETM T7 ULTRA Transcription Kit (Thermofischer Scientific) (1924 nucleotides) as an example.
  • mMESSAGE mMACHINETM T7 ULTRA Transcription Kit Thermofischer Scientific (1924 nucleotides
  • the amount of BMDC derived from C57BL / 6J mice and each of the above-mentioned liposomes is 1.5 mL so that the amount of 4.0 ⁇ 10 5 cells of BMDC / pDNA or mRNA is equivalent to 0.4 ⁇ g of each liposome / 500 ⁇ L of serum-free BMDC complete medium
  • 1000 ⁇ L of complete medium for BMDC was collected in a 1.5 mL tube, centrifuged at 4 ° C., 500 g for 5 minutes, and the supernatant was removed.
  • the supernatant remaining in each well was collected in a corresponding 1.5 mL tube, and then the wells were washed with 1000 ⁇ L PBS ( ⁇ ) and collected in a corresponding 1.5 mL tube.
  • PBS ⁇
  • 75 ⁇ l each of a 5-fold dilution of Reporter Lysis Buffer (Promega) with DDW was added to the wells.
  • the supernatant was removed after centrifuging a 1.5 mL tube containing the supernatant at 500 g for 5 minutes at 4 ° C.
  • the resulting cell pellet was suspended in 40 ⁇ L of Lysis buffer in the corresponding well, and returned to the original well. Incubated at -80 ° C for more than 30 minutes.
  • Lysis buffer in the well was collected in a 1.5 mL tube and centrifuged at 20000 g at 4 ° C. for 2 minutes. The supernatant was aliquoted into another 1.5 mL tube.
  • the amount of luminescence (RLU) when 20 ⁇ L of the supernatant was mixed with 50 ⁇ L of Luciferase assay system (Promega) was quantified by GLOMAX (Promega).
  • the amount of protein (mg) in 20 ⁇ L of the supernatant was quantified using TaKaRa BCA Protein Assay Kit (Takara Bio). The obtained luminescence amount (RLU) was divided by the amount of protein (mg) to calculate Luciferase activity (RLU / mg protein).
  • BMDC transfected with mRNA KALA-Lipoplex exhibited the highest luciferase activity when the same amount of pDNA or mRNA was used.
  • coli or mRNA prepared by in vitro transcription of pDNA having a sequence encoding OVA downstream of T7 promoter with mMESSAGE mMACHINETM T7 ULTRA Transcription Kit (Thermofischer Scientific)) as a nucleic acid Using the method described in Example 1, pDNA KALA-Lipoplex, mRNA KALA-Lipoplex, pDNA KALA-MEND, mRNA KALA-MEND, and mRNA DOTAP-Lipoplex were respectively prepared. In addition, mRNA R8-Lipoplex was prepared by replacing STR-KALA with the same amount of STR-octaarginine (R8) in Example 1 (1).
  • BMDCs from C57BL / 6J mice and each of the above-mentioned liposomes in an amount of 2.0 ⁇ 10 6 cells of BMDC / pDNA or mRNA equivalent to 0.25 to 1.0 ⁇ g of each liposome / 1000 ⁇ L of complete medium for serum-free BMDCs The mixture was mixed in a 1.5 mL tube, seeded on a 12 well plate and incubated for 2 hours. After adding 1000 ⁇ L of complete medium for BMDC and further incubating for 21 hours, cells were collected by pipetting, centrifuged at 4 ° C., 500 g for 5 minutes, and the supernatant was removed. The cells were washed twice with 1000 ⁇ L of complete medium.
  • BMADC transfected with mRNA KALA-Lipoplex exhibits the highest antigen presentation strength, equivalent to 1 ⁇ g of pDNA KALA-Lipoplex
  • the antigen-presenting intensity of BMADC transfected with KALA-Lipoplex is higher than that of BMDC transfected with. It was confirmed that it would rise depending on the amount of
  • Example 4 Evaluation of immunoinducing ability of BMDC transfected with KALA-Lipoplex (1) Immunization of BMDC to which an antigen gene has been introduced by KALA-Lipoplex BMDC derived from C57BL / 6J mouse and mRNA KALA- prepared in Example 3 Each liposome of Lipoplex, mRNA KALA-MEND and mRNA DOTAP-Lipoplex, in the amount of BMDC / mRNA of 2.0 ⁇ 10 6 cells, is equivalent to 0.025 to 2.0 ⁇ g of each liposome / 1000 ⁇ L of complete medium for serum-free BMDC The mixture was mixed in a 1.5 mL tube, seeded on a 12 well plate and incubated for 2 hours.
  • Immunization was carried out by administering 20 ⁇ L each of the BMDC or PBS subcutaneously to the soles of C57BL / 6J mice (6- to 8-week-old females). A second immunization was performed similarly, one week after the first immunization.
  • a complete medium containing 1000 ⁇ L of 2.5 ⁇ L / mL Goldi Plug (BD), 1 ⁇ g / mL OVA 257-264 was added to the wells and suspended by pipetting.
  • Splenocytes were OVA stimulated by incubation at 37 ° C., 5% CO 2 for 6 hours.
  • the cells were recovered by pipetting from the wells and washed twice with 5 mL of ice cold complete medium.
  • the cells were suspended in 5 mL of ice cold complete medium, and the cell concentration was measured.
  • Intracellular staining was performed by suspending the cells with 50 ⁇ L of 1.2 ⁇ g / mL PE anti-mouse IFN ⁇ and incubating at 4 ° C. for 30 minutes. The cells were washed twice with 500 ⁇ L of TF Perm / Wash buffer (BD), suspended with 500 ⁇ L of FACS buffer, analyzed by a flow cytometer, and the percentage of interferon ⁇ (IFN ⁇ ) producing cells in CD3 + CD8 + cells was measured.
  • BD TF Perm / Wash buffer
  • FACS buffer FACS buffer
  • Erythrocytes were destroyed by suspending with 1 mL of ACK Lysing buffer per spleen and incubating for 5 minutes at room temperature. Five volumes of complete medium was added, mixed, and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed and the remaining cell pellet was washed with 10 mL of complete medium. After suspending in 30 mL of complete medium, the cell concentration was measured. The spleen cells were divided into two groups (peptide-treated group and non-treated group) through a 40 ⁇ m cell strainer. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed and suspended in complete medium to 1.0 ⁇ 10 7 cells / mL.
  • peptide-treated group 2 mM OVA 257-264 peptide was quickly suspended by adding 1/400 of the medium volume.
  • the peptide-treated and non-treated groups were incubated at 37 ° C., 5% CO 2 for 1 hour. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed and washed twice with 10 mL of complete medium. Washed with 10 mL PBS (-).
  • the peptide-treated group was suspended with 1 ⁇ M CFSE / PBS and the non-treated group with 0.1 ⁇ M CFSE / PBS so as to be 3.0 ⁇ 10 7 cells / mL, and incubated in a 37 ° C.
  • spleens were excised from the mice and added to complete medium.
  • the spleen cells in the inside of the spleen were put out in complete medium, and the cell mass was loosened by pipetting, and then collected through a nylon mesh into a 1.5 mL tube. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed.
  • the red blood cells were disrupted by suspending with 1 mL of ACK Lysing buffer and incubating for 5 minutes at room temperature. The supernatant was removed after dilution with 9 mL of FACS buffer and centrifugation at 500 g for 5 minutes at 4 ° C. After washing the cells with 5 mL of FACS buffer, the cells were suspended with 5 mL of FACS buffer.
  • the CTL activity was calculated from the ratio of peptide-treated group to non-treated group using a flow cytometer.
  • BMDC transfected with mRNA KALA-Lipoplex can induce antigen-specific cytotoxic activity in vivo even with an amount of 1/10 or less of the amount of mRNA encapsulated in mRNA KALA-MEND.
  • Example 5 Evaluation of Membrane Consistency of KALA-Lipoplex Blood collected from ICR mice (4 to 6 weeks old male) in the presence of heparin was mixed with ice cold saline to make 10 mL. After centrifugation at 400 g for 10 minutes at 4 ° C., the supernatant was removed. The same operation was repeated three times to obtain a red blood cell suspension. Triton-X100 / PBS (Triton-X100 final concentration: 0.02% (w / v)) and an appropriate amount of erythrocyte suspension are mixed to make 250 ⁇ L, 200 ⁇ L is added to 96 well plate after vortexing, and it is at 540 nm Absorbance was measured.
  • Triton-X100 / PBS Triton-X100 final concentration: 0.02% (w / v)
  • the amount of red blood cell suspension at which the absorbance at 540 nm was 1.0 was calculated.
  • the amount of erythrocyte suspension, and mRNA KALA-Lipoplex, mRNA KALA-MEND, mRNA DOTAP-Lipoplex, and mRNA R8-Lipoplex (final lipid equivalent: 2.5, 5.0, 10 ⁇ M), and 20 mM prepared in Example 3
  • Malic acid / PBS (pH 5.5, 6.5, 7.4) was mixed to make 250 ⁇ L. The mixture was incubated at 37 ° C. and 1600 rpm for 30 minutes with stirring.
  • mRNA KALA-Lipoplex showed particularly high hemolytic activity, suggesting that mRNA KALA-Lipoplex may be excellent in escape efficiency due to its high biomembrane fusion ability in endolysosome under acidic conditions.
  • Example 6 Evaluation of mRNA Release Ability of KALA-Lipoplex Suspension Containing mRNA KALA-Lipoplex, mRNA KALA-MEND or mRNA DOTAP-Lipoplex, or 0.1 ⁇ g of OVA mRNA prepared in Example 3 in an amount corresponding to 0.1 ⁇ g mRNA 10 ⁇ L of the solution was mixed with 8 ⁇ L of 0.031 to 10 mg / mL Poly- ( ⁇ , ⁇ ) -DL-aspartic acid sodium salt mol wt 2,000-11,000 (Sigma), 2 ⁇ L of 1.45 M NaCl, and incubated at 37 ° C. for 30 minutes.
  • Example 7 Antitumor effect of KALA-Lipoplex having mRNA encoding NY-ESO-1 on the surface (1) Preparation of KALA-Lipoplex loaded with NY-ESO-1 mRNA Cancer antigen NY-ESO downstream of T7 promoter 1.
  • a thin film of a lipid mixture prepared by mixing 6.25 ⁇ L of MPLA / mL and 45 ⁇ L of chloroform in a test tube to retain NYESO1 mRNA on the surface, a lipid membrane A KALA peptide-modified liposome (NYESO1 / MPLA KALA-Lipoplex; final concentration of lipid: 0.183 mM, final concentration of nucleic acid: 0.01 mg / mL, nucleic acid lipid ratio 3 ⁇ g nucleic acid / 55 nmol lipid) was prepared.
  • the particle diameter, zeta potential and polydispersity index (Pdl) of NYESO1 mRNA KALA-Lipoplex, mRNA + CpG-ODN, mRNA + pDNA, mRNA + tpRNA and NYESO1 / MPLA KALA-Lipoplex are shown in Table 2.
  • Example 7 (4) Confirmation of Adjuvant Effect by MPLA
  • the same amount of Luciferase mRNA is retained on the surface instead of NYESO1 mRNA, and the content of MPLA contained in the lipid membrane is 0 KALA-Lipoplex (Luc / MPLA KALA Lipoplex) was prepared, each adjusted to 0.0015 ⁇ g, 0.0059 ⁇ g, 0.023 ⁇ g or 0.094 ⁇ g.
  • Transfection was carried out using the immature dendritic cells (BMDC) derived from Balb / c mice induced in Example 2 (1) and Luc / MPLA KALA Lipoplex using the same procedure as in Example 2 (2). Luciferase activity in BMDC was measured.
  • the amount of interleukin-6 (IL-6) production of BMDC was measured using Mouse IL-6 Quantikine ELISA Kit (R & D systems). The results are shown in FIG.
  • Example 8 Evaluation of Antigen-Specific Cytotoxic Activity of KALA-Lipoplex Holding OVA mRNA on the Surface 8 ⁇ g of the mRNA content of KALA-Lipoplex, the mRNA holding OVA mRNA on the surface KALA-Lipoplex (OVA mRNA KALA-Lipoplex 8-12, final concentration of lipid: 0.183 mM, final concentration of nucleic acid: 0.013 with 55 nmol lipid, 10 ⁇ g / 55 nmol lipid or 12 ⁇ g / 55 nmol lipid-supported OVA mRNA on the surface mg / mL, 0.017 mg / mL, 0.020 mg / mL)) were produced.
  • the particle size, zeta potential and polydispersity index (Pdl) of OVA mRNA KALA-Lipoplex 8-12 are shown in Table 3.
  • Intravenous administration iv, dose 1 ⁇ g mRNA / mouse
  • subcutaneous administration sc, dose 2 ⁇ g mRNA / mouse of OVA mRNA KALA-Lipoplex 8-12 in place of BMDC in the in vivo CTL assay of Example 4
  • CTL activity Antigen specific cytotoxic activity
  • the CTL activity of the control group to which PBS was administered was less than 10%, whereas the increase of CTL activity was confirmed in all of the groups to which OVA mRNA KALA-Lipoplex 8 to 12 was administered, and in particular OVA mRNA KALA-Lipoplex 8 A marked increase in CTL activity was observed in the group that received iv intravenous administration.

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Abstract

[Problem] The purpose of the present invention is to provide a novel intracellular delivery means for delivering a nucleic acid to within a cell and thereby causing a desired function to be exhibited more efficiently, namely, for delivering a nucleic acid to within an antigen-presenting cell and thereby increasing the antigen presentation strength of a transfected cell. [Solution] Provided is a lipid membrane structure that is for delivering a nucleic acid to within a cell, in which a lipid membrane is modified by at least one polypeptide having 12-50 amino acid residues including at least three K-A-L-A repeating units within the peptide chain thereof (wherein K may be R in one or more of the repeating units, and/or the A sandwiched between K and L may be H in one or more of the repeating units), and wherein a nucleic acid is held on the surface of the lipid membrane structure.

Description

核酸を細胞内に送達するための脂質膜構造体Lipid membrane structure for delivering nucleic acid into cells
　本発明は、核酸を細胞内に導入するための脂質膜構造体に関する。 The present invention relates to a lipid membrane structure for introducing nucleic acid into cells.
　近年、免疫応答を誘導又は増強することで疾患を治療する免疫療法が、がんの治療を中心に注目されている。免疫療法の主な態様において、抗原性物質を生体に投与したり又は分離された免疫細胞に導入したりすることによる、抗原性物質に対する特異的免疫応答を誘導することが求められる。抗原性物質がポリペプチドである場合、ポリペプチドそのものに代えて、ポリペプチドをコードするDNAを利用して所望の免疫応答を誘導することも可能である。このようなDNAは、一般にDNAワクチンと呼ばれる。 BACKGROUND ART In recent years, immunotherapy, which treats diseases by inducing or enhancing an immune response, is focused on the treatment of cancer. In the main aspect of immunotherapy, it is required to induce a specific immune response to an antigenic substance by administering the antigenic substance to a living body or introducing it into isolated immune cells. When the antigenic substance is a polypeptide, it is possible to use a DNA encoding the polypeptide instead of the polypeptide itself to induce a desired immune response. Such DNA is generally referred to as a DNA vaccine.
　DNAワクチンを用いた免疫療法では、DNAからのポリペプチドの発現効率を高めることが重要である。DNAからmRNAへの転写は細胞核内で行われることから、DNAを細胞核内に効率よく送達するための脂質膜構造体、特にDNAを封入したリポソームの利用が提唱されている。例えば、特許文献１には、免疫細胞、特に樹状細胞の核内にDNAを送達するための、脂質膜がKALAペプチドと称される特定のペプチドで修飾された、DNAを封入したリポソームが開示されている。 In immunotherapy using a DNA vaccine, it is important to enhance the expression efficiency of a polypeptide from DNA. Since transcription from DNA to mRNA is performed in the cell nucleus, it has been proposed to use a lipid membrane structure, in particular a liposome encapsulating DNA, for efficiently delivering the DNA into the cell nucleus. For example, Patent Document 1 discloses a DNA-encapsulated liposome in which a lipid membrane is modified with a specific peptide called KALA peptide for delivering DNA into the nucleus of immune cells, particularly dendritic cells. It is done.
　KALAペプチドは、K-A-L-A（Kはリジン、Aはアラニン、Lはロイシン）というアミノ酸配列の繰り返し単位を含む短鎖のポリペプチドである。なお本願では、配列表を除き、アミノ酸配列は一文字表記で表す。KALAペプチドは、DNAとコンプレックスを形成することが知られているペプチドである（非特許文献１）が、特許文献１においては、リポソームの核内移行能を促進するための機能性ペプチドとして利用されている。また、特許文献２には、KALAペプチドの核内移行促進機能を維持した改変ペプチドで修飾された、DNAを封入したリポソームが開示されている。 The KALA peptide is a short polypeptide comprising a repeating unit of the amino acid sequence KAALA (K is lysine, A is alanine, L is leucine). In the present application, except for the sequence listing, the amino acid sequence is represented by a single letter code. KALA peptide is a peptide that is known to form a complex with DNA (Non-patent Document 1), but in Patent Document 1, it is used as a functional peptide for promoting the ability of liposomes to enter the nucleus. ing. Patent Document 2 discloses a DNA-encapsulated liposome that has been modified with a modified peptide that maintains the function of promoting the nuclear import of KALA peptide.
　特許文献１及び特許文献２に開示されているリポソームはいずれも、DNAとポリカチオン性物質との複合体を脂質膜で封入したリポソームの形態を例示している。リポソームを用いたトランスフェクションでは、エンドサイトーシスによる細胞内へのリポソームの取り込み、エンドソームから細胞質へのリポソームの脱出、細胞核内へのリポソームの移行と細胞核内での前記複合体の放出、核酸の転写、タンパク質の翻訳、さらに細胞表面への抗原提示等、送達された核酸の機能の発揮までに多数のプロセスが要求される。 The liposomes disclosed in Patent Document 1 and Patent Document 2 all exemplify the form of a liposome in which a complex of DNA and a polycationic substance is enclosed by a lipid membrane. In the case of transfection using liposome, endocytosis uptake of liposome into cell, escape of liposome from endosome to cytoplasm, transfer of liposome into cell nucleus and release of the complex in cell nucleus, transcription of nucleic acid A large number of processes are required for the function of the delivered nucleic acid to be performed, such as protein translation and further antigen presentation on the cell surface.
国際公開WO2011/132713International Publication WO2011 / 132713 国際公開WO2015/098907International Publication WO2015 / 098907
　本発明者らは、抗原性ポリペプチドをコードするmRNAを抗原提示細胞内に送達し、DNAからmRNAの転写を省略することで免疫応答誘導能を高めることを目的として、mRNAを封入した特許文献１に記載の脂質膜構造体を調製し、これを抗原提示細胞にトランスフェクションした。しかしながら、トランスフェクションされた細胞の抗原提示強度は、比較対象としてDNAを封入した特許文献１に記載の脂質膜構造体によってトランスフェクションされた細胞のそれと殆ど変わらないことが、新たに確認された。 The present inventors have delivered an mRNA encoding an antigenic polypeptide into an antigen-presenting cell, and encapsulated mRNA for the purpose of enhancing the ability to induce an immune response by omitting transcription of mRNA from DNA. The lipid membrane structure described in 1 was prepared and transfected into antigen-presenting cells. However, it was newly confirmed that the antigen presentation intensity of the transfected cells was almost the same as that of the cells transfected with the lipid membrane structure described in Patent Document 1 in which DNA was encapsulated as a comparative object.
　本発明は、核酸を細胞内に送達させて所望の機能をより効率的に発揮させるための、特に抗原提示細胞内に核酸を送達させてトランスフェクションされた細胞の抗原提示強度をより高めるための、新たな細胞内送達手段を提供することを目的とする。 The present invention is intended to deliver nucleic acids into cells to exert desired functions more efficiently, and in particular to deliver nucleic acids into antigen-presenting cells to further enhance the antigen presentation strength of transfected cells. , Aims to provide a new means of intracellular delivery.
　本発明者らは、脂質膜の表面にmRNAを保持した脂質膜構造体を抗原提示細胞にトランスフェクションすることで、DNAを封入した脂質膜構造体を導入したときと比較してトランスフェクション細胞の抗原提示強度を高めることができることを見出し、以下の発明を完成させた。 The present inventors transfect a lipid membrane structure in which mRNA is retained on the surface of a lipid membrane into an antigen-presenting cell, compared to the case where a lipid membrane structure encapsulating DNA is introduced, compared to the case of transfected cells. The inventors have found that the antigen presentation intensity can be increased, and completed the following invention.
（１）ペプチド鎖中にK-A-L-Aの繰り返し単位を少なくとも3個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個若しくは2個以上においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～50個のポリペプチドの1種以上により脂質膜が修飾された、細胞内に核酸を送達するための脂質膜構造体であって、核酸をその表面に保持した、前記脂質膜構造体。
（２）ポリペプチドが、ペプチド鎖中にK-A-L-Aの繰り返し単位を3又は4個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～30個のポリペプチドである、（１）に記載の脂質膜構造体。
（３）ポリペプチドが下記ａ）～ｇ）に示されるアミノ酸配列のいずれかからなるポリペプチドである、（１）又は（２）に記載の脂質膜構造体。
　　ａ）WEAKLAKALAKALAKHLAKALAKALKA（配列番号１）
　　ｂ）WEAKLAKALAKALAKHLAKALAKALKACEA（配列番号２）
　　ｃ）WEAKLAKALAKALAKHLAKALA（配列番号３）
　　ｄ）WEAKLAKALAKALAKHLA（配列番号４）
　　ｅ）KALAKALAKALAKALA（配列番号５）
　　ｆ）KALAKALAKALA（配列番号６）
　　ｇ）WEARLARALARALARHLARALARALRA（配列番号７）
（４）脂質膜構造体がリポソームである、（１）～（３）のいずれか一項に記載の脂質膜構造体。
（５）ポリペプチドが疎水性基で修飾されており、前記疎水性基が脂質膜に挿入されてなる、（１）～（４）のいずれか一項に記載の脂質膜構造体。
（６）核酸が核酸アジュバント及び／又は抗原性ポリペプチドをコードするmRNAである、（１）～（５）のいずれか一項に記載の脂質膜構造体。
（７）脂質アジュバントをさらに含む、（１）～（６）のいずれか一項に記載の脂質膜構造体。
（８）免疫細胞の細胞質に核酸を送達するための、（１）～（７）のいずれか一項に記載の脂質膜構造体。
（９）免疫細胞と（１）～（８）のいずれか一項に記載の脂質膜構造体とをインキュベーションする工程を含む、免疫療法に用いるためのトランスフェクションされた免疫細胞をインビトロで製造する方法。 (1) The peptide chain contains at least three repeating units of KALA (however, K may be R in any one or two or more of the repeating units, and / or The lipid membrane is modified by at least one of a polypeptide having 12 to 50 amino acid residues, in which one or two or more of them, A and H between K and L may be H) A lipid membrane structure for delivering a nucleic acid into a cell, wherein the nucleic acid is retained on the surface thereof.
(2) The polypeptide contains 3 or 4 repeating units of KALA in the peptide chain (however, K may be R in any one or two or more of the repeating units, and / or Or the lipid according to (1), which is a polypeptide having 12 to 30 amino acid residues in the number of amino acid residues between which K and L may be A in any one of the repeating units. Membrane structure.
(3) The lipid membrane structure according to (1) or (2), wherein the polypeptide is a polypeptide consisting of any of the amino acid sequences shown in the following a) to g).
a) WEAKLAKALAKALAKHLAKALAKALKA (SEQ ID NO: 1)
b) WEAKLAKALAKALAK HLA KALAKALKACEA (SEQ ID NO: 2)
c) WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 3)
d) WEAKLAKALAKALAK HLA (SEQ ID NO: 4)
e) KALAKALAKALAKALA (SEQ ID NO: 5)
f) KALAKALAKALA (SEQ ID NO: 6)
g) WEARLARALARALARHLARALA RA (SEQ ID NO: 7)
(4) The lipid membrane structure according to any one of (1) to (3), wherein the lipid membrane structure is a liposome.
(5) The lipid membrane structure according to any one of (1) to (4), wherein the polypeptide is modified with a hydrophobic group, and the hydrophobic group is inserted into a lipid membrane.
(6) The lipid membrane structure according to any one of (1) to (5), wherein the nucleic acid is mRNA encoding a nucleic acid adjuvant and / or an antigenic polypeptide.
(7) The lipid membrane structure according to any one of (1) to (6), further comprising a lipid adjuvant.
(8) The lipid membrane structure according to any one of (1) to (7) for delivering a nucleic acid to the cytoplasm of an immune cell.
(9) In vitro production of transfected immune cells for use in immunotherapy, comprising the step of incubating the immune cells and the lipid membrane structure according to any one of (1) to (8) Method.
　本発明によると、KALAペプチド又はそのバリアントにより修飾された脂質膜構造体を用いて細胞内に核酸を送達することで、核酸に求められる機能を効率的に発揮させることができる。例えば、抗原性ポリペプチドをコードするmRNAを抗原提示細胞内に送達することで、当該抗原提示細胞における抗原提示強度を高めることができ、所望の抗原性ポリペプチドを標的とした免疫療法において利用可能な免疫細胞を製造することができるようになる。また、核酸をその表面に保持したKALAペプチド又はそのバリアントにより修飾された脂質膜構造体は、脂質膜の内側に核酸を封入した脂質膜構造体と比較して、所望の機能の発揮に必要とされる核酸の使用量を減らすことができ、製造コストの低減及び望ましくない副作用等の回避等において有利である。 According to the present invention, by delivering a nucleic acid into cells using a lipid membrane structure modified with KALA peptide or a variant thereof, the function required for the nucleic acid can be efficiently exhibited. For example, delivery of mRNA encoding an antigenic polypeptide into an antigen-presenting cell can enhance the antigen presentation strength in the antigen-presenting cell, and can be used in immunotherapy targeting a desired antigenic polypeptide Can produce various immune cells. In addition, a lipid membrane structure modified by the KALA peptide or variant thereof in which the nucleic acid is retained on the surface is required for the desired function as compared to the lipid membrane structure in which the nucleic acid is encapsulated inside the lipid membrane. The amount of nucleic acid used can be reduced, which is advantageous in reducing manufacturing costs and avoiding undesirable side effects and the like.
ルシフェラーゼをコードするプラスミドDNA（pDNA）又はmRNAをその表面に保持したKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞におけるルシフェラーゼ活性を示すグラフである。It is a graph which shows the luciferase activity in the dendritic cell transfected by the KALA peptide modified liposome which hold | maintained the plasmid DNA (pDNA) or mRNA which encoded the luciferase on the surface. OVAをコードするpDNA又はmRNAをその表面に保持したKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞における抗原提示量を示すグラフである。It is a graph which shows the antigen presentation amount in the dendritic cell transfected with the KALA peptide modified liposome which hold | maintains pDNA or mRNA which encoded OVA on the surface. OVAをコードするmRNAをその表面に保持したKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞による抗原特異的CD8+T細胞誘導を示すグラフである。FIG. 5 is a graph showing antigen-specific CD8 + T cell induction by dendritic cells transfected with KALA peptide-modified liposomes, the surface of which holds mRNA encoding OVA. OVAをコードするmRNAをその表面に保持したKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞によるCTL活性誘導を示すグラフである。It is a graph which shows CTL activity induction by dendritic cells transfected with KALA peptide modified liposome which retained the mRNA which codes OVA on the surface. OVAをコードするmRNAをその表面に保持したKALAペプチド修飾リポソームの膜融合性を示すグラフである。It is a graph which shows the fusogenicity of the KALA peptide modified liposome which hold | maintained the mRNA which codes OVA on the surface. OVAをコードするmRNAをその表面に保持したKALAペプチド修飾リポソームのmRNA放出を示すグラフである。It is a graph which shows the mRNA release of the KALA peptide modified liposome which hold | maintained the mRNA which codes OVA on the surface. がん抗原NY-ESO-1をコードするmRNAをその表面に保持したKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞による、腫瘍細胞CT26/NY-ESO-1が皮下移植されたマウスにおける腫瘍増殖の抑制を示すグラフである。Tumor growth in mice subcutaneously implanted with tumor cell CT26 / NY-ESO-1 by dendritic cells transfected with KALA peptide-modified liposomes bearing on the surface the mRNA encoding the cancer antigen NY-ESO-1 Is a graph showing suppression of ルシフェラーゼをコードするmRNAをその表面に保持し、脂質アジュバントであるモノホスホリルリピドＡ（MPLA）を脂質膜に含むKALAペプチド修飾リポソームでトランスフェクションされた樹状細胞における、ルシフェラーゼ活性及びインターロイキン-6の産生量を示すグラフである。Luciferase activity and interleukin-6 were obtained in dendritic cells transfected with KALA peptide-modified liposomes containing the luciferase-encoding mRNA on its surface and containing lipid adjuvant monophosphoryl lipid A (MPLA) in the lipid membrane It is a graph which shows the amount of production. 異なる量のOVA mRNAをその表面に保持したKALAペプチド修飾リポソームを皮下投与又は静脈投与したときの、CTL活性誘導を示すグラフである。FIG. 10 is a graph showing CTL activity induction when KALA peptide-modified liposomes having different amounts of OVA mRNA retained on their surface are administered subcutaneously or intravenously.
　本発明の第１の態様は、ペプチド鎖中にK-A-L-Aの繰り返し単位を少なくとも3個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個若しくは2個以上においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～50個のポリペプチドの1種以上により脂質膜が修飾された、細胞内に核酸を送達するための脂質膜構造体であって、核酸をその表面に保持した、前記脂質膜構造体に関する。 In the first aspect of the present invention, at least three repeating units of KALA are contained in the peptide chain (with the proviso that K is R in any one or two or more of the repeating units, and And / or one or more of a polypeptide having 12 to 50 amino acid residues in which one or two or more of the repeating units in which A and H between K and L may be H) The present invention relates to a lipid membrane-modified lipid membrane structure for delivering nucleic acid into cells, wherein the nucleic acid is retained on the surface thereof.
　本発明において使用されるポリペプチドは、ペプチド鎖中にK-A-L-Aの繰り返し単位を少なくとも3個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個若しくは2個以上においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～50個のポリペプチドである。 The polypeptide used in the present invention contains at least three repeating units of KALA in the peptide chain (with the proviso that K is R in any one or more of the repeating units). And / or a polypeptide having 12 to 50 amino acid residues in which one or two or more of the repeating units may interpose K and L and A may be H).
　本発明において使用されるポリペプチドは、ペプチド鎖中にK-A-L-Aの繰り返し単位を少なくとも3個、例えば3～10個、好ましくは3～7個、より好ましくは3又は4個含む。また、該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個若しくは2個以上、好ましくは1～3個、より好ましくは1個においてKとLに挟まれるAがHとなっていてもよい。また、ポリペプチドのアミノ酸残基数は12～50個、好ましくは12～40個、より好ましくは12～30個である。 The polypeptide used in the present invention comprises at least 3, for example 3 to 10, preferably 3 to 7, and more preferably 3 or 4 KAA repeat units in the peptide chain. In addition, K may be R in any one or two or more of the repeating units, and / or any one or two or more of the repeating units, preferably 1 to 3 A, which is sandwiched between K and L in one piece, more preferably in one piece, may be H. In addition, the number of amino acid residues of the polypeptide is 12 to 50, preferably 12 to 40, and more preferably 12 to 30.
　本発明において使用されるポリペプチドの典型例は、下記ａ）～ｇ）に示されるアミノ酸配列からなるポリペプチドである。
　　ａ）WEAKLAKALAKALAKHLAKALAKALKA（配列番号１）
　　ｂ）WEAKLAKALAKALAKHLAKALAKALKACEA（配列番号２）
　　ｃ）WEAKLAKALAKALAKHLAKALA（配列番号３）
　　ｄ）WEAKLAKALAKALAKHLA（配列番号４）
　　ｅ）KALAKALAKALAKALA（配列番号５）
　　ｆ）KALAKALAKALA（配列番号６）
　　ｇ）WEARLARALARALARHLARALARALRA（配列番号７） A typical example of the polypeptide used in the present invention is a polypeptide consisting of the amino acid sequence shown in the following a) to g).
a) WEAKLAKALAKALAKHLAKALAKALKA (SEQ ID NO: 1)
b) WEAKLAKALAKALAK HLA KALAKALKACEA (SEQ ID NO: 2)
c) WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 3)
d) WEAKLAKALAKALAK HLA (SEQ ID NO: 4)
e) KALAKALAKALAKALA (SEQ ID NO: 5)
f) KALAKALAKALA (SEQ ID NO: 6)
g) WEARLARALARALARHLARALA RA (SEQ ID NO: 7)
　ａ）は、前出の非特許文献１（Wyman, T. B. et al., Biochemistry, 1997, 36, 3008-17；この文献は、その全てが参照により本明細書に組み込まれる）に開示されたKALAペプチドである。KALAペプチドは、pH7.4前後の中性条件下では前記繰り返し単位を１周期とするいわゆるαヘリックス構造を、またpH5.0前後の弱酸性条件下ではランダムコイル構造をそれぞれ呈することが知られている。KALAペプチドは、前出の特許文献１（国際公開WO2011/132713）に開示されるように、脂質膜構造体の表面に配置されることで、脂質膜構造体の細胞核内への移行能を高める機能を有する。また、複数のLys残基によってペプチド全体が正に荷電しており、負に荷電しているDNAとコンプレックスを形成する機能を有することも知られている（国際公開WO2011/132713）。 a) is disclosed in the aforementioned non-patent document 1 (Wyman, TB et al., Biochemistry, 1997, 36, 3008-17; this document is incorporated herein by reference in its entirety). KALA peptide. KALA peptide is known to exhibit a so-called α-helix structure in which the repeating unit is one cycle under neutral conditions of about pH 7.4, and a random coil structure under weakly acidic conditions of about pH 5.0 There is. The KALA peptide is disposed on the surface of the lipid membrane structure as disclosed in the above-mentioned Patent Document 1 (International Publication WO 2011/132713), thereby enhancing the ability of the lipid membrane structure to be transferred into the cell nucleus It has a function. It is also known that the whole peptide is positively charged by a plurality of Lys residues, and has a function of forming a complex with negatively charged DNA (International Publication WO 2011/132713).
　ｂ）は、前記KALAペプチドのC末端に3アミノ酸残基が付加されたアミノ酸配列からなるペプチドであり、KALAペプチドと同様に、pHに依存してαヘリックス構造とランダムコイル構造を呈する性質、脂質膜構造体の細胞核内への移行能を高める機能、及び核酸とのコンプレックス形成能を有する。ｃ）はKALAペプチドのC末端のKALKAが欠失したアミノ酸配列からなるペプチド、ｄ）はｃ）のアミノ酸配列からなるペプチドのC末端からK-A-L-Aの繰り返し単位が１個欠失したアミノ酸配列からなるペプチド、ｅ）及びｆ）はK-A-L-Aの繰り返し単位がそれぞれ4又は3個繰り返されたアミノ酸配列からなるペプチド、ｇ）はａ）のアミノ酸配列においてKが全てRに置換されたアミノ酸配列からなるペプチドである。ｃ）～ｆ）は、前出の特許文献２（国際公開WO2015/098907）に開示されているペプチドであり、KALAペプチドと同様の機能を有する。国際公開WO2011/132713及びこれに対応する米国出願公開US2013/0122054、並びに国際公開WO2015/098907は、その全てが参照により本明細書に組み込まれる。 b) is a peptide consisting of an amino acid sequence in which three amino acid residues are added to the C-terminus of the KALA peptide, and like the KALA peptide, it has an α-helix structure and a random coil structure depending on pH, It has a function of enhancing the ability of the membrane construct to move into the cell nucleus and the ability to form a complex with a nucleic acid. c) A peptide consisting of an amino acid sequence in which KALKA at the C-terminus of KALA peptide is deleted; , E) and f) are peptides consisting of an amino acid sequence in which the repeating unit of KALA is repeated 4 or 3 respectively, g) is a peptide consisting of an amino acid sequence in which all Ks are substituted for R in the amino acid sequence . c) to f) are the peptides disclosed in the above-mentioned Patent Document 2 (International Publication WO 2015/098907), and have the same function as the KALA peptide. International Publication WO 2011/132713 and corresponding US Application Publication US 2013/0122054, and International Publication WO 2015/098907 are all incorporated herein by reference.
　本発明においては、以下のポリペプチドも使用することができる：
前記ａ）～ｆ）に示されるアミノ酸配列においてK-A-L-Aの繰り返し単位のうちのいずれか1個又は2個以上においてKがRとなっているアミノ酸配列からなるポリペプチド；
前記ｅ）又はｆ）に示されるアミノ酸配列においてK-A-L-Aの繰り返し単位のいずれか1個においてKとLに挟まれるAがHとなっているアミノ酸配列からなるポリペプチド；
前記ａ）～ｄ）に示されるアミノ酸配列においてK-H-L-AがK-A-L-Aとなっているアミノ酸配列からなるポリペプチド；
前記ｇ）に示されるアミノ酸配列においてR-H-L-AがR-A-L-Aとなっているアミノ酸配列からなるポリペプチド。 The following polypeptides may also be used in the present invention:
A polypeptide comprising an amino acid sequence in which K is R in any one or more of the repeating units of KALA in the amino acid sequences shown in a) to f) above;
A polypeptide comprising an amino acid sequence in which A is H which is sandwiched between K and L in any one of the repeating units of KALA in the amino acid sequence shown in the above e) or f);
A polypeptide consisting of an amino acid sequence in which KHLA is KALA in the amino acid sequences shown in a) to d) above;
A polypeptide consisting of an amino acid sequence in which RHLA is RALA in the amino acid sequence shown in g).
　本発明において特に好ましいポリペプチドは、前記ａ）に示されるアミノ酸配列からなるポリペプチド、ｃ）に示されるアミノ酸配列からなるポリペプチド、ｅ）に示されるアミノ酸配列からなるポリペプチド及びｇ）に示されるアミノ酸配列からなるポリペプチドである。以下、特に個別に示さない限り、本発明で使用されるポリペプチドを纏めてKALAペプチドバリアントと表す。 Particularly preferred polypeptides in the present invention are shown in the polypeptide consisting of the amino acid sequence shown in a), the polypeptide consisting of the amino acid sequence shown in c), the polypeptide consisting of the amino acid sequence shown in e) and g) A polypeptide consisting of the amino acid sequence Hereinafter, unless otherwise indicated individually, the polypeptides used in the present invention are collectively referred to as KALA peptide variants.
　KALAペプチドバリアントは、当業者に利用可能な各種の遺伝子組み換え技術を利用し、適当な宿主ベクター系を用いて生物学的に調製することができる。KALAペプチドバリアントは、固相合成法その他のペプチド合成反応を利用した有機化学的方法又はペプチドシンセサイザー等のペプチド自動合成装置を利用して調製してもよい。 KALA peptide variants can be prepared biologically using appropriate host vector systems, utilizing a variety of genetic recombination techniques available to one skilled in the art. The KALA peptide variant may be prepared using a solid phase synthesis method or other organic chemical method utilizing peptide synthesis reaction or an automatic peptide synthesizer such as a peptide synthesizer.
　本発明において「ポリペプチドの1種以上により脂質膜が修飾された」脂質膜構造体とは、KALAペプチドバリアントの１種又はそれ以上を脂質膜の表面に有する脂質膜構造体、具体的にはこれらが他の物質と接触可能な状態で脂質膜の表面に、特に脂質膜構造体がリポソームの形態であるときは脂質膜の外表面に存在している脂質膜構造体を意味し、KALAペプチドバリアントの全てが脂質二重層の層内に埋没している脂質膜構造体、又はKALAペプチドバリアントの全てが脂質膜で閉鎖された内部空間に存在する、いわゆるポリペプチドが封入された脂質膜構造体とは区別される。ただし、KALAペプチドバリアントが脂質膜の表面に存在する限り、脂質二重層の内部、脂質膜で閉鎖される脂質膜構造体の内部空間、又はこの内部空間に向いた脂質膜の内側の面にもポリペプチドが存在することは差し支えない。 In the present invention, a “lipid membrane structure modified by one or more of polypeptides” is a lipid membrane structure having one or more of KALA peptide variants on the surface of the lipid membrane, specifically, It means a lipid membrane structure existing on the surface of the lipid membrane in a state where these can be in contact with other substances, particularly when the lipid membrane structure is in the form of a liposome, on the outer surface of the lipid membrane A lipid membrane structure in which all of the variants are buried in the layer of the lipid bilayer, or a lipid membrane structure in which all of the KALA peptide variants exist in a lipid membrane-closed internal space, a so-called polypeptide-encapsulated lipid membrane structure It is distinguished from However, as long as the KALA peptide variant is present on the surface of the lipid membrane, it is also inside the lipid bilayer, the interior space of the lipid membrane structure closed by the lipid membrane, or the inner surface of the lipid membrane facing this interior space It is acceptable for the polypeptide to be present.
　KALAペプチドバリアントは、ステアリル基、コレステリル基その他の任意の疎水性基を有する誘導体、特にポリペプチドのN末端のアミノ基と中鎖以上の鎖長を有する脂肪酸のカルボン酸基との間の脱水反応によって調製される脂肪酸誘導体の形態で使用されることが好ましい。KALAペプチドバリアントの誘導体は、その疎水性基が脂質膜構造体の脂質膜に埋没することで、ポリペプチド部分が脂質膜の表面に残るようにポリペプチドを脂質膜の表面にアンカリングさせることができる。 The KALA peptide variant is a dehydration reaction between a stearyl group, a derivative having a cholesteryl group and any other hydrophobic group, in particular, an amino group at the N-terminus of the polypeptide and a carboxylic acid group of a fatty acid having a medium chain length or more. Preferably it is used in the form of a fatty acid derivative prepared by The derivative of KALA peptide variant is to anchor the polypeptide on the surface of lipid membrane so that the polypeptide moiety remains on the surface of lipid membrane by the hydrophobic group being buried in the lipid membrane of lipid membrane structure it can.
　本発明において使用される核酸は、DNA又はRNAのほか、これらの類似体又は誘導体（例えば、ペプチド核酸（PNA）やホスホロチオエートDNA等）が包含される。核酸は一本鎖又は二本鎖のいずれであってもよく、線状又は環状のいずれであってもよい。また、STING ligandであるcGAMP （AMPとGMPの二量体）等のヌクレオチド化合物であってもよい。核酸の長さは、特に限定されない。 The nucleic acids used in the present invention include, in addition to DNA or RNA, their analogues or derivatives (eg, peptide nucleic acid (PNA) and phosphorothioate DNA etc.). The nucleic acid may be single stranded or double stranded, and may be linear or circular. In addition, it may be a nucleotide compound such as cGAMP (a dimer of AMP and GMP) which is STING ligand. The length of the nucleic acid is not particularly limited.
　本発明において好ましい核酸は、細胞内、特に細胞質内に送達されることが望まれる核酸であり、例として、dsRNA、cGAMP、CpG ODN、polyIC等の核酸アジュバント、抗原性ポリペプチドをコードするmRNA、及びアンチセンス核酸、siRNA等の阻害性核酸を挙げることができる。２種以上の核酸を同時に脂質膜構造体に保持させることもできる。 Preferred nucleic acids in the present invention are those that are desired to be delivered intracellularly, particularly in the cytoplasm, and examples include nucleic acid adjuvants such as dsRNA, cGAMP, CpG ODN, polyIC, mRNA encoding an antigenic polypeptide, And antisense nucleic acids and inhibitory nucleic acids such as siRNA can be mentioned. Two or more types of nucleic acids can be simultaneously held in the lipid membrane structure.
　第１の態様の脂質膜構造体は、免疫細胞、特に抗原提示細胞の細胞質内に核酸を送達する機能に優れており、この目的で使用される核酸としては、抗原性ポリペプチドをコードするmRNAが好ましく、該mRNAと核酸アジュバントとを組み合わせてもよい。抗原性ポリペプチドは、抗原性を有する任意のポリペプチドを選択することができる。 The lipid membrane structure of the first aspect is excellent in the function of delivering a nucleic acid into the cytoplasm of an immune cell, particularly an antigen-presenting cell, and the nucleic acid used for this purpose is an mRNA encoding an antigenic polypeptide Is preferable, and the mRNA and the nucleic acid adjuvant may be combined. As the antigenic polypeptide, any polypeptide having antigenicity can be selected.
　本発明において「核酸をその表面に保持した」脂質膜構造体とは、核酸を脂質膜の表面に有する脂質膜構造体、具体的には核酸が他の物質と接触可能な状態で脂質膜の表面に存在している脂質膜構造体、特に脂質膜構造体がリポソームの形態であるときは脂質膜の外表面に存在している脂質膜構造体を意味し、核酸が脂質膜で閉鎖された内部空間に存在する、いわゆる核酸が封入された脂質膜構造体とは区別される。ただし、核酸が脂質膜の表面に存在する限り、脂質膜で閉鎖された内部空間にも核酸が存在することは差し支えない。また、核酸は主に脂質膜構造体表面のKALAペプチドバリアントと静電的に結合して存在するが、目的とする機能を損なわない限り、他の態様により脂質膜の表面に結合していてもよい。 In the present invention, a lipid membrane structure “having a nucleic acid on its surface” is a lipid membrane structure having a nucleic acid on the surface of the lipid membrane, specifically a lipid membrane of a lipid membrane in which the nucleic acid can be contacted with other substances. A lipid membrane structure present on the surface, in particular when the lipid membrane structure is in the form of a liposome, means a lipid membrane structure present on the outer surface of the lipid membrane, and the nucleic acid is closed with the lipid membrane It is distinguished from lipid membrane structures in which the so-called nucleic acid is present in the inner space. However, as long as the nucleic acid is present on the surface of the lipid membrane, the nucleic acid may be present in the interior space closed by the lipid membrane. Also, the nucleic acid mainly exists electrostatically bound to the KALA peptide variant on the surface of the lipid membrane structure, but may be bound to the surface of the lipid membrane according to other embodiments as long as the intended function is not impaired. Good.
　遊離のKALAペプチドバリアントは核酸と静電的相互作用によりコンプレックスを形成するが、このコンプレックスをそのまま細胞に導入しても、脂質膜を持たないことから突破すべき生体膜との融合が困難であり、結果として細胞質への核酸の送達に不適であると考えられる。実際に遊離のKALAペプチドとpDNAとのコンプレックスを樹状細胞に加えても、細胞質内核酸センサーに認識されずにサイトカイン産生が認められないことが示されている（Miura, N. et al., Nucleic Acids Res., 2015, 43, 1317-31）。これに対し、第１の態様の脂質膜構造体は膜融合性のある脂質を有することから、細胞内に効率的に核酸を送達し、その機能を発揮させることができるものと期待される。 Although the free KALA peptide variant forms a complex with the nucleic acid through electrostatic interaction, even if this complex is introduced into cells as it is, it does not have a lipid membrane and fusion with the biomembrane to be overcome is difficult. As a result, it is considered unsuitable for delivery of nucleic acid to the cytoplasm. It has been shown that even when a complex of free KALA peptide and pDNA is actually added to dendritic cells, cytokine production is not recognized because it is not recognized by the cytoplasmic nucleic acid sensor (Miura, N. et al., Nucleic Acids Res., 2015, 43, 1317-31). On the other hand, since the lipid membrane structure of the first aspect has a membrane fusogenic lipid, it is expected that the nucleic acid can be efficiently delivered to cells and the function thereof can be exerted.
　第１の態様における脂質膜構造体の形態は特に限定されず、例えばリポソーム、O/W型エマルション、W/O/W型エマルション、球状ミセル、ひも状ミセル、又は不定型の層状構造物等を挙げることができる。脂質膜構造体の好ましい形態はリポソームである。以下、脂質膜構造体をリポソームを例として説明することがあるが、第１の態様の脂質膜構造体はリポソームには限定されない。 The form of the lipid membrane structure in the first aspect is not particularly limited, and examples thereof include liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, wormlike micelles, and amorphous layered structures. It can be mentioned. The preferred form of lipid membrane structure is a liposome. Hereinafter, the lipid membrane structure may be described as an example of a liposome, but the lipid membrane structure of the first aspect is not limited to the liposome.
　脂質膜構造体の脂質膜を構成する脂質としては、例えば、リン脂質、糖脂質、ステロール、又は飽和若しくは不飽和の脂肪酸等が挙げられる。 Examples of the lipid constituting the lipid membrane of the lipid membrane structure include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids.
　リン脂質及びリン脂質誘導体としては、例えば、ホスファチジルエタノールアミン、ホスファリジルコリン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジルグリセロール、カルジオリピン、スフィンゴミエリン、セラミドホスホリルエタノールアミン、セラミドホスホリルグリセロール、セラミドホスホリルグリセロールホスファート、1,2-ジミリストイル-1,2-デオキシホスファチジルコリン、プラスマロゲン、ジオレオイルホスファチジルエタノールアミン、ホスファチジン酸等を挙げることができ、これらは1種又は2種以上を組み合わせて用いることができる。これらリン脂質における脂肪酸残基は特に限定されないが、例えば、炭素数12～20の飽和又は不飽和の脂肪酸残基を挙げることができ、具体的には、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸等の脂肪酸由来のアシル基を挙げることができる。また、卵黄レシチン、大豆レシチン等の天然物由来のリン脂質を用いることもできる。 Examples of phospholipids and phospholipid derivatives include phosphatidyl ethanolamine, phosphalysyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, cardiolipin, sphingomyelin, ceramide phosphoryl ethanolamine, ceramide phosphoryl glycerol, ceramide phosphoryl glycerol phosphate, 1 And 2-dimyristoyl-1,2-deoxyphosphatidyl choline, plasmalogen, dioleoylphosphatidyl ethanolamine, phosphatidic acid and the like, which may be used alone or in combination of two or more. The fatty acid residue in these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms. Specifically, lauric acid, myristic acid, palmitic acid, stearin Examples include acyl groups derived from fatty acids such as acid, oleic acid and linoleic acid. In addition, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.
　糖脂質としては、例えば、グリセロ糖脂質（例えば、スルホキシリボシルグリセリド、ジグリコシルジグリセリド、ジガラクトシルジグリセリド、ガラクトシルジグリセリド、グリコシルジグリセリド）、スフィンゴ糖脂質（例えば、ガラクトシルセレブロシド、ラクトシルセレブロシド、ガングリオシド）等が挙げられる。 Examples of glycolipids include glyceroglycolipids (eg, sulfoxyribosyl glycerides, diglycosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides, glycosyl diglycerides), glycosphingolipids (eg, galactosylcerebroside, lactosylcerebroside, gangliosides) and the like. It can be mentioned.
　ステロールとしては、例えば、動物由来のステロール（例えば、コレステロール、コレステロールコハク酸、ラノステロール、ジヒドロラノステロール、デスモステロール、ジヒドロコレステロール）、植物由来のステロール（フィトステロール）（例えば、スチグマステロール、シトステロール、カンペステロール、ブラシカステロール）、微生物由来のステロール（例えば、チモステロール、エルゴステロール）等が挙げられる。 Examples of sterols include animal-derived sterols (eg, cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterols) (eg, stigmasterol, sitosterol, campesterol, Brashcasterol), sterol derived from a microorganism (eg, thymosterol, ergosterol) and the like.
　飽和又は不飽和の脂肪酸としては、例えば、パルミチン酸、オレイン酸、ステアリン酸、アラキドン酸、ミリスチン酸等の炭素数12～20の飽和又は不飽和の脂肪酸が挙げられる。 Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid and myristic acid.
　第１の態様の脂質膜構造体では、脂質成分として第3級アミン及びジスルフィド結合を有する脂質を用いることもできる。このような脂質は、例えば国際公開WO2013/73480に開示されており、分子内に第3級アミン及びジスルフィド結合を有することによりpH応答性及び還元的環境下における膜不安定化作用を有することが知られている。上記国際公開に開示された下記式(1)で表される脂質は脂質膜構造体の構成脂質として好ましい。
Figure JPOXMLDOC01-appb-C000001
 （式中、Ｘａ及びＸｂは独立して、Ｘ１又はＸ２であり；
Figure JPOXMLDOC01-appb-C000002
 ｓは1又は2であり、
Ｒ４は炭素数1～6のアルキル基を表し、
ｎａ及びｎｂは独立して、0又は1であり、
Ｒ１ａ及びＲ１ｂは独立して、炭素数1～6のアルキレン基を表し、
Ｒ２ａ及びＲ２ｂは独立して、炭素数1～6のアルキレン基を表し、
Ｙａ及びＹｂは独立して、エステル結合、アミド結合、カーバメート結合、エーテル結合又は尿素結合を表し、
Ｒ３ａ及びＲ３ｂは独立して、ステロール残基、脂溶性ビタミン残基又は炭素数12～22の脂肪族炭化水素基を表す） In the lipid membrane structure of the first aspect, a tertiary amine and a lipid having a disulfide bond can also be used as a lipid component. Such a lipid is disclosed, for example, in International Publication WO 2013/73480, and by having a tertiary amine and a disulfide bond in the molecule, it has pH responsiveness and membrane destabilizing action under a reducing environment. Are known. The lipid represented by the following formula (1) disclosed in the above-mentioned International Publication is preferably as a constituent lipid of a lipid membrane structure.
Figure JPOXMLDOC01-appb-C000001
 (Wherein, X a and X b are independently X 1 or X 2 ;
Figure JPOXMLDOC01-appb-C000002
 s is 1 or 2;
R 4 represents an alkyl group having 1 to 6 carbon atoms,
n a and n b are independently 0 or 1,
R 1a and R 1b independently represent an alkylene group having 1 to 6 carbon atoms,
R 2a and R 2b independently represent an alkylene group having 1 to 6 carbon atoms,
Y a and Y b independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond,
R 3a and R 3b independently represent a sterol residue, a fat-soluble vitamin residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms)
　式(1)で表される脂質において、側鎖として、例えばミリスチン酸を結合した脂質（PalmM）、レチノイン酸を結合した脂（(PalmA）、及びトコフェロールを結合した脂質（PalmE）等のPalm類脂質が知られているが、これらのうち、PalmEは中性脂質であり血清存在下においても遺伝子発現を達成できることから本発明において好ましく用いることができる。PalmEは国際公開WO2013/73480の段落[0056]の表1中の化合物B-2-5
Figure JPOXMLDOC01-appb-C000003
 である。上記国際公開及びこれに対応する米国出願公開US2014/0335157の開示の全てを参照により本明細書の開示として含める。 In the lipid represented by the formula (1), for example, a lipid such as myristic acid conjugated lipid (PalmM), a retinoic acid conjugated lipid (PalmA), and a tocopherol conjugated lipid (PalmE) as a side chain Although lipids are known, among them, Palm E is preferably a neutral lipid and can be preferably used in the present invention because gene expression can be achieved even in the presence of serum. Palm E is described in paragraph [0056] of International Publication WO 2013/73480. Compound B-2-5 in Table 1 of
Figure JPOXMLDOC01-appb-C000003
 It is. The disclosures of the above-mentioned International Publication and the corresponding disclosure of US application publication US2014 / 0335157 are incorporated herein by reference.
　上記の脂質の使用量は特に限定されないが、一般的には脂質膜構造体を構成する全脂質に対して5～50重量％程度の量とすることができる。また、これらのPalm類については応用例が知られているので（Immunity, 21(4), pp.527-538, 2004; Int. Immunol., 21(4), pp.361-377, 2009; Blood, 115(10), pp.1958-1968, 2010)、これらの刊行物を参照しつつ適宜のPalm類を目的に応じて選択することができる。 The amount of the above-mentioned lipid to be used is not particularly limited, but in general, it can be about 5 to 50% by weight with respect to the total lipid constituting the lipid membrane structure. Moreover, since application examples are known for these Palms (Immunity, 21 (4), pp. 527-538, 2004; Int. Immunol., 21 (4), pp. 361-377, 2009; Blood, 115 (10), pp. 1958-1968, 2010), appropriate Palm species can be selected according to the purpose while referring to these publications.
　第１の態様の脂質膜構造体の脂質膜を構成する特に好ましい脂質としては、ジオレオイルホスファチジルエタノールアミン（DOPE）、ホスファチジン酸（PA）、ホスファチジルセリン（PS）、カルジオリピン（CL）を挙げることができる。また、国際公開WO2016/121942、国際公開WO2016/027699、国際公開WO2013/073480に記載された細胞内環境応答性を示す脂質様材料も挙げることが出来る。それらの混合量は、KALAペプチドバリアントの添加量、KALAペプチドバリアントの電荷量、KALAペプチドバリアントと核酸の電荷比に応じて、核酸と静電的に結合することができる脂質膜構造体を構成し得る範囲で適宜調整することができる。 Particularly preferable lipids constituting the lipid membrane of the lipid membrane structure of the first aspect include dioleoylphosphatidylethanolamine (DOPE), phosphatidic acid (PA), phosphatidylserine (PS) and cardiolipin (CL). Can. In addition, lipid-like materials exhibiting intracellular environmental responsiveness described in International Publication WO 2016/121942, International Publication WO 2016/027699, and International Publication WO 2013/073480 can also be mentioned. Depending on the amount of KALA peptide variant added, the charge amount of KALA peptide variant, and the charge ratio of KALA peptide variant to nucleic acid, they constitute a lipid membrane structure capable of electrostatically binding to nucleic acid. It can adjust suitably in the range acquired.
　第１の態様の脂質膜構造体は、ステロール、又はグリセリン若しくはその脂肪酸エステル等の膜安定化剤、トコフェロール、没食子酸プロピル、パルミチン酸アスコルビル、又はブチル化ヒドロキシトルエン等の抗酸化剤、荷電物質、及び膜ポリペプチド等からなる群から選ばれる1種又は2種以上の物質を、脂質膜の構成成分又は脂質膜で封入される成分として有していてもよい。 The lipid membrane structure of the first aspect is a membrane stabilizer such as sterol or glycerin or a fatty acid ester thereof, an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, And one or more types of substances selected from the group consisting of membrane polypeptides and the like may be included as a component of the lipid membrane or a component to be enclosed by the lipid membrane.
　正電荷を付与する荷電物質としては、例えば、ステアリルアミン、オレイルアミン等の飽和又は不飽和脂肪族アミン；ジオレオイルトリメチルアンモニウムプロパン等の飽和又は不飽和カチオン性合成脂質；あるいはカチオン性ポリマー等を挙げることができ、負電荷を付与する荷電物質としては、例えば、ジセチルホスフェート、コレステリルヘミスクシネート、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジン酸等を挙げることができる。膜ポリペプチドとしては、例えば、膜表在性ポリペプチド、又は膜内在性ポリペプチド等が挙げられる。これらの物質の配合量は特に限定されず、目的に応じて適宜選択することができる。 Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane; and cationic polymers etc. Examples of the charged substance that can give a negative charge can be mentioned dicetyl phosphate, cholesteryl hemisuccinate, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and the like. The membrane polypeptide includes, for example, a membrane-surrounding polypeptide or an integral membrane polypeptide. The compounding quantity of these substances is not specifically limited, According to the objective, it can select suitably.
　また、第１の態様の脂質膜構造体には、例えば、温度変化感受性機能、膜透過機能、遺伝子発現機能、及びpH感受性機能等のいずれか1つ又は2つ以上の機能を付与することができる。これらの機能を適宜付加することにより、例えば遺伝子を含む核酸等を内包する脂質膜構造体の血液中での滞留性を向上させ、肝臓や脾臓等の細網内皮系組織による捕捉率を低下させるとともに、標的細胞におけるエンドサイトーシスの後にエンドソームから効率的に脂質膜構造体を脱出させることが可能になる。 In addition, the lipid membrane structure of the first aspect may be provided with any one or more functions such as a temperature change sensitivity function, a membrane permeability function, a gene expression function, and a pH sensitivity function. it can. By appropriately adding these functions, for example, the retention property of the lipid membrane structure containing a nucleic acid containing a gene, etc. in blood is improved, and the capture rate by reticular endothelial tissue such as liver and spleen is decreased. In addition, after endocytosis in target cells, it is possible to efficiently escape lipid membrane structures from endosomes.
　温度変化感受性機能を付与することができる温度変化感受性脂質誘導体としては、例えば、ジパルミトイルホスファチジルコリン等を挙げることができる。また、pH感受性機能を付与することができるpH感受性脂質誘導体としては、例えば、ジオレオイルホスファチジルエタノールアミン等を挙げることができる。 As a temperature change sensitive lipid derivative capable of imparting a temperature change sensitivity function, for example, dipalmitoyl phosphatidyl choline and the like can be mentioned. Moreover, as a pH sensitive lipid derivative capable of imparting a pH sensitive function, for example, dioleoylphosphatidylethanolamine etc. can be mentioned.
　第１の態様の脂質膜構造体は、その血中滞留性を高めることができるグリコフォリン、ガングリオシドGM1、ホスファチジルイノシトールその他の脂質誘導体又はポリアルキレングリコールその他の親水性ポリマー、エンドソーム脱出能を促進するGALAペプチド、生体適合性を高めるMPCポリマー、対象となる細胞表面の受容体や抗原に対して特異的に結合可能な抗体、連続した複数個のアルギニン残基を含むポリペプチド、脂質膜の構造破壊を促進するINF7等を、その表面に有していてもよい。 The lipid membrane structure of the first aspect is glycophorin, ganglioside GM1, phosphatidylinositol or other lipid derivative or polyalkylene glycol or other hydrophilic polymer capable of enhancing its retention in blood, GALA promoting endosomal prolapse ability Peptides, MPC polymers that enhance biocompatibility, antibodies that can specifically bind to receptors or antigens on the cell surface of interest, polypeptides containing multiple consecutive arginine residues, structural disruption of lipid membranes You may have INF7 grade | etc., To accelerate | stimulate on the surface.
　また、使用目的に応じて、抗腫瘍剤、抗炎症剤、抗菌剤、抗ウイルス剤等の任意の医薬の有効成分のほか、例えばリピドＡ、モノホスホリルリピドＡ（MPLA）、Endocine（Eurocine社）、グルコピラノシル脂質アジュバントといった脂質アジュバントその他のアジュバント、糖類、ペプチド類、低分子化合物、金属化合物等を脂質膜構成成分として、又は脂質膜内に封入される成分として有していてもよい。本発明において特に好ましい脂質アジュバントはMPLAである。脂質膜構造体における脂質アジュバントの含有量は、総脂質量のおよそ0.0003～3重量％であればよく、好ましくは総脂質量の0.001～1重量％、より好ましくは0.003～0.3重量％である。 In addition to the active ingredient of any medicine such as antitumor agent, anti-inflammatory agent, antibacterial agent, antiviral agent according to the purpose of use, for example, lipid A, monophosphoryl lipid A (MPLA), Endocine (Eurocine) And a lipid adjuvant such as glucopyranosyl lipid adjuvant, saccharides, peptides, low molecular weight compounds, metal compounds and the like may be contained as a lipid membrane component or as a component to be enclosed in a lipid membrane. A particularly preferred lipid adjuvant according to the invention is MPLA. The content of the lipid adjuvant in the lipid membrane structure may be about 0.0003 to 3% by weight of the total lipid, preferably 0.001 to 1% by weight, and more preferably 0.003 to 0.3% by weight of the total lipid.
　脂質膜構造体の好ましい形態であるリポソームの製造は、当業者に一般的な任意の方法を基づいて製造することができる。例えば水和法による場合、脂質膜を構成する脂質成分をクロロホルム等の有機溶媒に溶解した溶液を乾燥させて脂質混合物を形成した後、水系溶媒を乾燥した脂質混合物に添加して、ホモジナイズ、超音波処理又は高圧噴射等を行って乳化する等で、水系溶媒を封入したリポソームを調製する。次に、リポソームに前述のKALAペプチドバリアントの誘導体を添加してインキュベートし、リポソームの表面にKALAペプチドバリアントの誘導体をアンカリングさせた後、核酸をさらに加えることで、第１の態様の好ましい形態であるリポソームを製造することができる。リポソームは、水和法以外の公知の方法、例えば逆相蒸発法等によって調製してもよい。第１の態様の好ましい形態であるリポソームは、一枚膜リポソーム又は多重膜リポソームのいずれでもよいが、一枚膜リポソームが好ましい。 The preparation of a liposome, which is a preferred form of lipid membrane structure, can be prepared based on any method common to those skilled in the art. For example, in the case of the hydration method, a solution in which a lipid component constituting a lipid membrane is dissolved in an organic solvent such as chloroform is dried to form a lipid mixture, and then an aqueous solvent is added to the dried lipid mixture to homogenize or super Liposomes in which an aqueous solvent is sealed are prepared by performing sonication, high-pressure injection or the like to emulsify. Next, the derivative of the aforementioned KALA peptide variant is added to the liposome and incubated, and after the derivative of the KALA peptide variant is anchored on the surface of the liposome, the nucleic acid is further added, thereby obtaining the preferred embodiment of the first aspect Certain liposomes can be produced. Liposomes may be prepared by known methods other than hydration, such as reverse phase evaporation. The liposome which is a preferred form of the first aspect may be either a unilamellar liposome or a multilamellar liposome, but a unilamellar liposome is preferred.
　また、上記の水和法に基づいた方法において、KALAペプチドバリアントの誘導体を脂質膜を構成する脂質成分とともにクロロホルム等の有機溶媒に溶解し、KALAペプチドバリアントの誘導体を含む脂質混合物を形成してから、水系溶媒を添加する等してリポソームを調製し、核酸をさらに加えることで第１の態様の好ましい形態であるリポソームを製造してもよい。 In the method based on the above-mentioned hydration method, a derivative of KALA peptide variant is dissolved in an organic solvent such as chloroform together with a lipid component constituting a lipid membrane to form a lipid mixture containing a derivative of KALA peptide variant. The liposome which is a preferable form of the first aspect may be produced by preparing a liposome by adding an aqueous solvent or the like and further adding a nucleic acid.
　第１の態様の脂質膜構造体の製造において、KALAペプチドバリアントの誘導体の添加量は、脂質膜構造体の総脂質量に対して、1～50モル%、好ましくは5～30モル%であることが好ましい。また、核酸の添加量は特に制限は無いが、脂質膜構造体1重量部に対して、1～20%重量部の範囲で適宜調節すればよい。 In the production of the lipid membrane structure of the first aspect, the addition amount of the derivative of KALA peptide variant is 1 to 50 mol%, preferably 5 to 30 mol%, relative to the total lipid amount of the lipid membrane structure. Is preferred. Further, the addition amount of the nucleic acid is not particularly limited, but may be appropriately adjusted in the range of 1 to 20% by weight with respect to 1 part by weight of the lipid membrane structure.
　第１の態様の脂質膜構造体において、その内部に封入される水系溶媒（分散媒）は、脂質膜構造体を安定に分散させることができるものであれば、その組成は特に限定されず、例えば、リン酸緩衝液、クエン酸緩衝液、リン酸緩衝生理食塩液等の緩衝液、生理食塩水、細胞培養用の培地等を挙げることができる。これら水系溶媒（分散媒）は、さらに単糖類、二糖類、三糖類、多糖類、糖アルコール、多価アルコール（水溶液）等を含んでいてもよい。また必要に応じて、薬物、タンパク質その他の生理活性物質を含んでいてもよい。 In the lipid membrane structure of the first aspect, the composition of the aqueous solvent (dispersion medium) enclosed in the inside is not particularly limited as long as the lipid membrane structure can be stably dispersed. For example, buffers such as phosphate buffer, citrate buffer and phosphate buffered saline, physiological saline, medium for cell culture and the like can be mentioned. These aqueous solvents (dispersion media) may further contain monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, polyhydric alcohols (aqueous solutions) and the like. Also, if necessary, drugs, proteins and other physiologically active substances may be contained.
　第１の態様の好ましい形態であるリポソームは、脂質膜封入体又は製造後のリポソームを高圧下でメンブランフィルターに通してイクストルージョンすることで、その大きさを揃えることもできる。大きさは特に限定されないが、その平均粒子径は50 nm～5μm程度、好ましくは50 nmから400 nm程度、より好ましくは50 nmから300 nm程度、さらに好ましくは100 nmから250 nm程度であればよい。 The size of the liposome, which is a preferred embodiment of the first aspect, can be made uniform by extracting the lipid membrane inclusion body or the liposome after production through a membrane filter under high pressure. The size is not particularly limited, but the average particle size is about 50 nm to 5 μm, preferably about 50 nm to 400 nm, more preferably about 50 nm to 300 nm, and still more preferably about 100 nm to 250 nm. Good.
　第１の態様の好ましい形態であるリポソームとしては、水系溶媒に分散した形態又は水性分散物を凍結乾燥又は噴霧乾燥した形態である一枚膜リポソーム又は多重層リポソーム等を挙げることができる。水性分散物の形態又は乾燥物の形態にあるリポソームは、当業者に一般的な方法によって調製することができる。脂質膜構造体の好ましい形態は、水性分散物の形態又は乾燥物の形態にある一枚膜リポソームである。 As a liposome which is a preferable form of the first aspect, there may be mentioned a unilamellar liposome or multilamellar liposome which is a form dispersed in an aqueous solvent or a form obtained by freeze-drying or spray-drying an aqueous dispersion. Liposomes in the form of an aqueous dispersion or in the form of a dry product can be prepared according to methods common to those skilled in the art. The preferred form of the lipid membrane structure is a unilamellar liposome in the form of an aqueous dispersion or in the form of a dry product.
　第１の態様の脂質膜構造体によって核酸を送達する対象となる細胞の種類は特に限定されず、核酸に期待される機能又は送達目的等に応じて、適宜の細胞を標的とすることができる。細胞として、好ましくは免疫細胞を挙げることができ、免疫細胞のなかでもマクロファージ、樹状細胞、B細胞等の抗原提示細胞が特に好ましく、特に樹状細胞が最も好ましい。 The type of cells targeted for nucleic acid delivery by the lipid membrane structure of the first aspect is not particularly limited, and appropriate cells can be targeted depending on the function or delivery purpose expected for the nucleic acid. . Preferred examples of the cells include immune cells. Among immune cells, antigen-presenting cells such as macrophages, dendritic cells and B cells are particularly preferable, and dendritic cells are particularly preferable.
　例えば、第１の態様の脂質膜構造体を用いて、樹状細胞等の抗原提示細胞の細胞質内に抗原性ポリペプチドをコードするmRNAを送達することで、抗原提示細胞はmRNAから翻訳されたポリペプチドを抗原提示細胞の表面に提示し、生体は当該ポリペプチドに対する免疫を獲得することができるので、所望の抗原性ポリペプチドを標的とした有効な免疫療法を行うことができるようになる。 For example, an antigen-presenting cell is translated from mRNA by delivering an mRNA encoding an antigenic polypeptide into the cytoplasm of an antigen-presenting cell such as dendritic cell using the lipid membrane structure of the first aspect Since the polypeptide is presented on the surface of the antigen-presenting cell, and the living body can acquire immunity against the polypeptide, an effective immunotherapy targeting the desired antigenic polypeptide can be performed.
　本発明は、免疫細胞と前述の第１の態様である脂質膜構造体とをインキュベーションする工程を含む、免疫療法用のトランスフェクションされた免疫細胞をインビトロで製造する方法を、別の態様として提供する。免疫細胞としては、マクロファージ、樹状細胞、B細胞等の抗原提示細胞が特に好ましく、特に樹状細胞が最も好ましい。 The present invention provides, as another embodiment, a method of producing transfected immune cells for immunotherapy in vitro, which comprises the step of incubating the immune cells and the lipid membrane structure according to the first aspect described above. Do. Particularly preferred immune cells are antigen-presenting cells such as macrophages, dendritic cells and B cells, and dendritic cells are most preferred.
　例えば、抗原性タンパク質をコードするmRNAを保持した第１の態様の脂質膜構造体と生体から分離した免疫細胞、好ましくは抗原提示細胞とを適当な培地中でインキュベーションすることで、当該抗原性タンパク質に関する抗原提示能を有するトランスフェクションされた抗原提示細胞を製造することができる。さらには、上記脂質膜構造体に代えて、抗原性ポリペプチドをコードするmRNA及び核酸アジュバントの両方を保持した脂質膜構造体又は抗原性ポリペプチドをコードするmRNAを保持した脂質膜構造体と核酸アジュバントを保持した脂質膜構造体との混合物を用いることで、免疫誘導能がさらに高められた抗原提示細胞を製造することもできる。トランスフェクションされた免疫細胞を免疫療法を必要とする対象に投与する場合、免疫細胞は当該対象から採取したものを用いることが好ましい。 For example, by incubating the lipid membrane structure of the first aspect carrying an mRNA encoding an antigenic protein and an immune cell separated from a living body, preferably an antigen presenting cell, in an appropriate medium, the antigenic protein Transfected antigen-presenting cells having antigen-presenting ability with respect to Furthermore, instead of the above lipid membrane structure, a lipid membrane structure carrying both an mRNA encoding an antigenic polypeptide and a nucleic acid adjuvant or a lipid membrane structure carrying a mRNA encoding an antigenic polypeptide and a nucleic acid By using a mixture with a lipid membrane structure retaining an adjuvant, it is also possible to produce antigen-presenting cells with further enhanced immunity inducing ability. When the transfected immune cells are administered to a subject in need of immunotherapy, it is preferable to use immune cells collected from the subject.
　本態様により製造されるトランスフェクションされた免疫細胞は、当該抗原性ポリペプチドを標的とした免疫療法を必要とする対象、好ましくはヒト、特にがん患者に対して、当該抗原性タンパク質に特異的な免疫応答を誘導することができ、免疫療法、特にがんの免疫療法において使用することができる。したがって本発明は、免疫細胞と前述の第１の態様である脂質膜構造体とをインキュベーションして免疫細胞をトランスフェクションする工程、及びトランスフェクションされた免疫細胞の有効量を免疫療法を必要とする対象に投与する工程を含む免疫療法もさらなる別の態様として提供するものである。 The transfected immune cells produced according to this embodiment are specific for the antigenic protein in subjects requiring immunotherapy targeting the antigenic polypeptide, preferably humans, particularly cancer patients. Can induce an immune response, and can be used in immunotherapy, particularly in cancer immunotherapy. Therefore, the present invention requires the steps of incubating the immune cells and the lipid membrane structure of the first aspect described above to transfect the immune cells, and immunotherapy the effective amount of the transfected immune cells. Immunotherapy comprising the step of administering to a subject is also provided as yet another embodiment.
　また、第１の態様の脂質膜構造体を用いた細胞、好ましくは免疫細胞への核酸の送達は、生体に脂質膜構造体を直接投与して行うこともできる。本発明は、したがって、第１の態様の脂質膜構造体を有効成分として含む医薬組成物をさらなる態様として提供する。上記態様の医薬組成物において用いられる脂質膜構造体は、前出の国際公開WO2011/132713及びWO2015/098907に記載された、脂質膜融合体の生体適合性や血中滞留性を向上させる手段を採用することが好ましい。 The delivery of the nucleic acid to cells, preferably immune cells, using the lipid membrane structure of the first aspect can also be carried out by directly administering the lipid membrane structure to a living body. The present invention thus provides, as a further aspect, a pharmaceutical composition comprising the lipid membrane structure of the first aspect as an active ingredient. The lipid membrane structure used in the pharmaceutical composition of the above aspect has a means for improving the biocompatibility and retention in blood of lipid membrane fusion described in the above-mentioned International Publications WO2011 / 132713 and WO2015 / 098907. It is preferable to adopt.
　上記態様の医薬組成物は、リポソーム医薬又は製剤として一般に知られている剤形、用法、用量等に準じて利用することができる。例えば、医薬組成物は、注射剤、点滴剤等の非経口製剤の形態で用いられることができ、このような非経口製剤に用いることができる担体としては、例えば、生理食塩水や、ブドウ糖、Ｄ－ソルビトール等を含む等張液といった水性担体が挙げられる。 The pharmaceutical composition of the above aspect can be used according to a dosage form, usage, dose and the like generally known as a liposome drug or preparation. For example, the pharmaceutical composition can be used in the form of parenteral preparations such as injections, drips and the like, and as carriers that can be used for such parenteral preparations, for example, physiological saline, glucose, An aqueous carrier such as an isotonic solution containing D-sorbitol and the like can be mentioned.
　上記態様の医薬組成物はさらに、薬学的に許容される緩衝剤、安定剤、保存剤その他の添加剤といった成分を含み得る。薬学的に許容される成分は当業者において周知であり、当業者が通常の実施能力の範囲内で、例えば第十七改訂日本薬局方その他の規格書に記載された成分から製剤の形態に応じて適宜選択して使用することができる。 The pharmaceutical composition of the above aspect may further contain ingredients such as pharmaceutically acceptable buffers, stabilizers, preservatives and other additives. Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can, according to the form of the preparation, for example, from the ingredients described in the 17th Revised Japanese Pharmacopoeia and other specifications within the scope of ordinary practice Therefore, they can be selected appropriately and used.
　例えば、抗原性タンパク質をコードするmRNAを保持した第１の態様の脂質膜構造体を有効成分として含む上記態様の医薬組成物は、当該抗原性ポリペプチドを標的とした免疫療法を必要とする対象、好ましくはヒト、特にがん患者に投与されることで、その体内の免疫細胞に対して当該抗原性タンパク質に特異的な免疫応答を誘導することができ、免疫療法、特にがんの免疫療法において好適に使用される。したがって、有効量の上記態様の医薬組成物を、免疫療法を必要とする対象に投与する工程を含む免疫療法も、本発明のさらなる別の態様として提供される。 For example, the pharmaceutical composition of the above aspect comprising as an active ingredient the lipid membrane structure of the first aspect carrying an mRNA encoding an antigenic protein as an active ingredient is a subject requiring immunotherapy targeting the antigenic polypeptide. Preferably, when administered to a human, particularly a cancer patient, an immune response specific to the antigenic protein can be induced against immune cells in the body, and immunotherapy, particularly cancer immunotherapy Are preferably used. Accordingly, immunotherapy comprising administering an effective amount of the pharmaceutical composition of the above aspect to a subject in need thereof is also provided as a further aspect of the present invention.
　医薬組成物の投与方法は、特に制限されないが、非経口製剤である場合は、例えば血管内投与（好ましくは静脈内投与）、腹腔内投与、腸管内投与、皮下投与等を挙げることができる。好ましい実施形態の一つにおいて、医薬組成物は、静脈内投与により生体に投与される。 The administration method of the pharmaceutical composition is not particularly limited, but in the case of a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, subcutaneous administration and the like can be mentioned. In one of the preferred embodiments, the pharmaceutical composition is administered to a living body by intravenous administration.
　以下の実施例によって本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。遺伝子導入能評価、免疫誘導能評価等のリポソームの活性評価の方法は、国際公開WO2011/132713及びWO2015/098907に記載されたものを参照することができる。 The present invention will be described in more detail by the following examples, but the present invention is not limited thereto. For methods for evaluating the activity of liposomes, such as gene transfer ability evaluation and immune induction ability evaluation, those described in International Publications WO 2011/132713 and WO 2015/098907 can be referred to.
実施例１　リポソームの調製
（１）核酸をその表面に保持したKALAペプチド修飾リポソームの調製
　2mM DOPE（エタノール溶液）45μL、2mM PA（クロロホルム：エタノール＝1：1） 10μL及びクロロホルム45μLを試験管内で混合し、DOPE：PA＝9：2（クロロホルム：エタノール＝1：1）とした後、真空デシケーターで溶媒を留去して、脂質混合物の薄膜を形成した。 Example 1 Preparation of Liposome (1) Preparation of KALA Peptide Modified Liposome Holding Nucleic Acid on Its Surface 45 μL of 2 mM DOPE (ethanol solution), 10 μL of 2 mM PA (chloroform: ethanol = 1: 1) and 45 μL of chloroform are mixed in a test tube After DOPE: PA = 9: 2 (chloroform: ethanol = 1: 1), the solvent was distilled off with a vacuum desiccator to form a thin film of a lipid mixture.
　200μLの10mM HEPES緩衝液（pH7.4）を脂質混合物に添加し、室温で15分静置してから浴槽型ソニケーターで30秒間超音波処理することで、脂質濃度に換算して0.55mM相当のリポソーム懸濁液を調製した。100μLのリポソーム懸濁液と100μLのHEPES緩衝液（pH7.4）を混合し、ボルテックスミキサーで攪拌しながら、8.25μLの1mM STR-KALAペプチド（配列番号１に示されるアミノ酸配列からなるKALAペプチドのN末端がステアリル化されたもの）を素早く添加して、脂質膜表面にKALAペプチドを配置させた。さらにボルテックスミキサーで攪拌しながら100μLの40μg/mLのpDNA又はmRNA溶液を滴下することで、核酸をその表面に保持したKALAペプチド修飾リポソーム（脂質終濃度：0.183mM、核酸終濃度：0.013mg/mL、核酸脂質比　4μg 核酸／55 nmol 脂質；以下、pDNA KALA-Lipoplex又はmRNA KALA-Lipoplexと表す）を製造した。 Add 200 μL of 10 mM HEPES buffer (pH 7.4) to the lipid mixture, allow to stand at room temperature for 15 minutes, and then sonicate for 30 seconds with a bath-type sonicator, equivalent to 0.55 mM in terms of lipid concentration A liposome suspension was prepared. A mixture of 100 μL of liposome suspension and 100 μL of HEPES buffer (pH 7.4) and stirring with a vortex mixer, 8.25 μL of 1 mM STR-KALA peptide (a KALA peptide consisting of the amino acid sequence shown in SEQ ID NO: 1) N-terminally stearylated) was rapidly added to place the KALA peptide on the lipid membrane surface. Furthermore, by adding 100 μL of a 40 μg / mL pDNA or mRNA solution dropwise while stirring with a vortex mixer, KALA peptide-modified liposomes containing nucleic acid on the surface (final lipid concentration: 0.183 mM, final nucleic acid concentration: 0.013 mg / mL) A nucleic acid / lipid ratio of 4 μg nucleic acid / 55 nmol lipid; hereinafter referred to as pDNA KALA-Lipoplex or mRNA KALA-Lipoplex) was produced.
（２）核酸を封入したKALAペプチド修飾リポソームの調製
　2mM DOPE（エタノール溶液） 42.8μL、2mM PA（クロロホルム：エタノール＝1：1） 12.2μL、クロロホルム 42.8μLを試験管内で混合し、DOPE：PA＝7：2（クロロホルム：エタノール＝1：1）とした後、真空デシケーターで溶媒を留去して、脂質混合物の薄膜を形成した。 (2) Preparation of nucleic acid-encapsulated KALA peptide-modified liposome: 42.8 μL of 2 mM DOPE (ethanol solution), 12.2 μL of 2 mM PA (chloroform: ethanol = 1: 1), 42.8 μL chloroform mixed in a test tube, DOPE: PA = After setting it to 7: 2 (chloroform: ethanol = 1: 1), the solvent was distilled off with a vacuum desiccator to form a thin film of a lipid mixture.
　0.12mg/mL 硫酸プロタミン溶液（10mM HEPES緩衝液、pH7.4）100μLを1.5mLチューブへ加え、ボルテックスミキサーで攪拌しながら、0.15mg/mLのpDNA又はmRNA溶液（10mM HEPES緩衝液、pH7.4）100μLをそれぞれ滴下して、プロタミンとpDNAとからなるコア粒子及びプロタミンとmRNAとからなるコア粒子の各懸濁液を調製した。前記脂質混合物に各コア粒子懸濁液200μLを添加し、室温で10分静置してから浴槽型ソニケーターで30秒間超音波処理することで、脂質濃度に換算して0.55mM相当のリポソーム懸濁液を調製した。100μLのリポソーム懸濁液と100μLのHEPES緩衝液（pH7.4）とを混合し、ボルテックスミキサーで攪拌しながら、5.5μLの1mM STR-KALAペプチドを素早く添加して脂質膜表面にKALAペプチドを配置させることで、核酸をその内部に封入したKALAペプチド修飾リポソーム（脂質終濃度：0.275mM、核酸終濃度：0.020mg/mL；以下、pDNA KALA-MEND又はmRNA KALA-MEND等と表す）を製造した。 Add 100 μL of 0.12 mg / mL protamine sulfate solution (10 mM HEPES buffer, pH 7.4) to a 1.5 mL tube, and while stirring with a vortex mixer, 0.15 mg / mL pDNA or mRNA solution (10 mM HEPES buffer, pH 7.4) 100 μL was added dropwise to prepare each suspension of core particles consisting of protamine and pDNA and core particles consisting of protamine and mRNA. Add 200 μL of each core particle suspension to the lipid mixture, allow to stand for 10 minutes at room temperature, and then sonicate for 30 seconds with a bath-type sonicator to obtain a liposome suspension equivalent to 0.55 mM in terms of lipid concentration The solution was prepared. Mix 100 μL of liposomal suspension with 100 μL of HEPES buffer (pH 7.4) and quickly add 5.5 μL of 1 mM STR-KALA peptide to place the KALA peptide on the lipid membrane surface while stirring with a vortex mixer As a result, KALA peptide-modified liposomes (final lipid concentration: 0.275 mM, final nucleic acid concentration: 0.020 mg / mL; hereinafter referred to as pDNA KALA-MEND or mRNA KALA-MEND etc.) encapsulating the nucleic acid inside were produced. .
（３）比較用カチオン性リポソームの調製
　4mM DOTAP（エタノール溶液） 13.8μL、2mM DOPE（エタノール溶液） 27.5μL、クロロホルム 41.3μLを試験管内で混合し、DOTAP：DOPE＝1：1（クロロホルム：エタノール＝1：1）とした後、真空デシケーターで溶媒を留去して、脂質混合物の薄膜を形成した。 (3) Preparation of Comparative Cationic Liposome 13.8 μL of 4 mM DOTAP (ethanol solution), 27.5 μL of 2 mM DOPE (ethanol solution), 41.3 μL of chloroform are mixed in a test tube, and DOTAP: DOPE = 1: 1 (chloroform: ethanol = After 1: 1), the solvent was distilled off with a vacuum desiccator to form a thin film of the lipid mixture.
　200μLの10mM HEPES緩衝液（pH7.4）を脂質混合物に添加し、室温で15分静置してから浴槽型ソニケーターで30秒間超音波処理することで、脂質濃度に換算して0.55mM相当のリポソーム懸濁液を調製した。100μLのリポソーム懸濁液と100μLのHEPES緩衝液（pH7.4）を混合し、ボルテックスミキサーで攪拌しながら、100μLの40μg/mLのmRNA溶液を滴下することで、DOTAPを構成脂質とし、KALAペプチドを有しない比較用カチオン性リポソーム（脂質終濃度：0.183mM、mRNA終濃度：0.013mg/mL；以下、mRNA DOTAP-Lipoplexと表す）を製造した。 Add 200 μL of 10 mM HEPES buffer (pH 7.4) to the lipid mixture, allow to stand at room temperature for 15 minutes, and then sonicate for 30 seconds with a bath-type sonicator, equivalent to 0.55 mM in terms of lipid concentration A liposome suspension was prepared. Mix 100 μL of liposome suspension and 100 μL of HEPES buffer solution (pH 7.4), and add 100 μL of 40 μg / mL mRNA solution dropwise while stirring with a vortex mixer to make DOTAP a component lipid, and use KALA peptide A comparative cationic liposome (final lipid concentration: 0.183 mM, final mRNA concentration: 0.013 mg / mL; hereinafter referred to as mRNA DOTAP-Lipoplex) was prepared.
　（１）及び（２）においてOVAをコードするmRNA（1239ヌクレオチド、調製方法は実施例３（２）に記載）を核酸として用いて製造したリポソームの粒度分布及びゼータ電位を測定した結果を表１に示す。粒子径、ζ（ゼータ）電位及び多分散指数（Pdl）のいずれについても両リポソーム間に大きな差は認められなかった。
Figure JPOXMLDOC01-appb-T000004
 Table 1 shows the results of measurement of particle size distribution and zeta potential of liposomes produced using mRNA encoding OVA (1239 nucleotides, the method of preparation is described in Example 3 (2)) as nucleic acid in (1) and (2). Shown in. No significant difference was observed between the two liposomes in any of particle diameter, zeta potential and polydispersity index (Pdl).
Figure JPOXMLDOC01-appb-T000004
実施例２　KALA-Lipoplexの樹状細胞に対する遺伝子導入能評価
（１）マウス骨髄由来樹状細胞の誘導
　500mLのRPMI-1640（Sigma-aldrich）培地に対し、5mLの1M HEPES（ナカライテスク）、5mLの100mM Sodium Pyruvate（ナカライテスク）、500μLの2-Mercaptethanol（Gibco）、5mLの10000μg/mL Penicillin+10000U/mL Streptomycin（ナカライテスク）、50mLの非働化FCSを添加して完全培地を調製した。頸椎脱臼により安楽死させたC57BL/6Jマウス又はBalb/cマウス（6～8週齢メス）から大腿骨・脛骨を摘出し、70%エタノールで消毒後、滅菌シャーレ内で20mLのPBS（-）に浸した。骨の両端を切断し、26G注射針（テルモ）を装着した1mLシリンジを用いて、20mLの完全培地を用いて骨髄細胞をシャーレ中に押し出した。細胞懸濁液を40μmセルストレイナー（Falcon）を通して、50mLコニカルチューブへ回収し、4℃、500g、5分で遠心した。上清を除き、残った細胞ペレットを1mLのACK Lysing buffer（Lonza）で懸濁し、室温、5分インキュベートすることで、赤血球を破壊した。9mLの完全培地を加え、混合した後、4℃、500g、5分で遠心した。上清を除き、残った細胞ペレットを10mLの完全培地で懸濁し、4℃、500g、5分で遠心した。同様の操作をもう一度行った後、10mLの完全培地で懸濁後、100mm培養ディッシュへ播種し、37℃、5%CO2下でインキュベートした。4時間以上経過した後、上清でディッシュ底を軽く洗浄することで、非接着細胞を回収した。セルカウントの後、1.0×106cells/mLとなるように、10ng/mL mGM-CSF（R&D systems）含有完全培地（BMDC用完全培地）で懸濁し、24well plateへ1mL/wellとなるように播種した。播種から2日後及び4日後に細胞の凝集塊を残して浮遊細胞を除去した後、1mLのBMDC用完全培地を各wellに添加した。播種から6日後に、各wellの浮遊細胞および弱接着細胞をピペッティングにより回収し、未成熟樹状細胞（Bone Marrow-derived Dendritic Cell、BMDC）として実験に用いた。 Example 2 Evaluation of Gene Transfer Ability of KALA-Lipoplex to Dendritic Cells (1) Induction of Dendritic Cells Derived from Mouse Bone Marrow 5 mL of 1 M HEPES (Nacalai Tesque), 5 mL to 500 mL of RPMI-1640 (Sigma-aldrich) medium Complete medium was prepared by adding 100 mM Sodium Pyruvate (Nacalai Tesque), 500 μL of 2-Mercapethanol (Gibco), 5 mL of 10000 μg / mL Penicillin + 10000 U / mL Streptomycin (Nacalai Tesque), 50 mL of inactive FCS. The femur and tibia are isolated from C57BL / 6J mice or Balb / c mice (6- to 8-week-old females) euthanized by cervical dislocation and disinfected with 70% ethanol, and then 20 mL of PBS (-) in a sterile petri dish Dipped in The bone was cut at both ends and bone marrow cells were pushed into a petri dish using 20 mL of complete medium using a 1 mL syringe fitted with a 26G needle (Terumo). The cell suspension was collected through a 40 μm cell strainer (Falcon) into a 50 mL conical tube and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed, and the remaining cell pellet was suspended in 1 mL of ACK Lysing buffer (Lonza) and incubated at room temperature for 5 minutes to destroy erythrocytes. 9 mL of complete medium was added, mixed, and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed, and the remaining cell pellet was suspended in 10 mL of complete medium and centrifuged at 500 g for 5 minutes at 4 ° C. After repeating the same operation, the cells were suspended in 10 mL of complete medium, and then inoculated into a 100 mm culture dish and incubated at 37 ° C. under 5% CO 2 . After 4 hours or more, non-adherent cells were recovered by lightly washing the bottom of the dish with the supernatant. After cell counting, suspend in 10 ng / mL mGM-CSF (R & D systems) -containing complete medium (complete medium for BMDC) to 1.0 × 10 6 cells / mL, and make 1 mL / well in a 24-well plate. Sowed. After removing suspended cells to leave clumps of cells 2 and 4 days after seeding, 1 mL of complete medium for BMDC was added to each well. Six days after the seeding, floating cells and weakly adherent cells in each well were collected by pipetting and used for experiments as immature dendritic cells (Bone Marrow-derived Dendritic Cells, BMDC).
（２）レポーター遺伝子を用いた遺伝子導入能評価
　LuciferaseをコードするpDNA（pCpGfree-MCS (invivogen)にLuciferaseをコードするDNA断片を挿入し、Endofree Plasmid Giga Kitを用いて大腸菌より調製した）又はmRNA（T7 promoterの下流にLuciferaseをコードする配列を有するpDNAを、mMESSAGE mMACHINE（商標） T7 ULTRA Transcription Kit (Thermofischer Scientific)によりin vitro転写する事で調製した（1924ヌクレオチド））を核酸として用いて、実施例１に示した方法により、4種類のリポソームmRNA KALA- Lipoplex、pDNA KALA-MEND、mRNA KALA-MEND、及びmRNA DOTAP-Lipoplexを調製した。 (2) Evaluation of gene transferability using a reporter gene Luciferase-encoding pDNA (a DNA fragment encoding Luciferase was inserted into pCpGfree-MCS (invivogen), prepared from E. coli using Endofree Plasmid Giga Kit) or mRNA ( The pDNA having a sequence encoding Luciferase downstream of the T7 promoter was prepared by in vitro transcription using mMESSAGE mMACHINETM T7 ULTRA Transcription Kit (Thermofischer Scientific) (1924 nucleotides) as an example. Four liposomal mRNAs KALA-Lipoplex, pDNA KALA-MEND, mRNA KALA-MEND, and mRNA DOTAP-Lipoplex were prepared by the method shown in 1.
　C57BL/6Jマウス由来のBMDCと上記の各リポソームとを、4.0×105cellsのBMDC/pDNA又はmRNA量で0.4μg相当の各リポソーム/500μLの血清不含BMDC用完全培地となるように1.5mLチューブ内で混合し、これを24 well plateに播種して、3時間インキュベートした。1000μLのBMDC用完全培地を各wellに添加して更に3時間インキュベートした後に、上清1000μLを1.5mLチューブに回収し、4℃、500g、5分で遠心後、上清を除いた。各wellに残った上清を対応する1.5mLチューブに回収後、wellを1000μLのPBS（-）で洗浄し、対応する1.5mLチューブに回収した。wellにはReporter Lysis Buffer（Promega）をDDWで5倍希釈したものを75μLずつ添加した。上清を入れた1.5mLチューブを4℃、500g、5分で遠心後、上清を除いた。生じた細胞ペレットを、対応するwell内のLysis buffer 40μLで懸濁し、元のwellに戻した。-80℃で30分以上インキュベートした。室温で融解し、セルスクレイパーで細胞をはがした後、well内のLysis bufferを1.5mLチューブに回収し、4℃、20000g、2分で遠心した。上清を別の1.5mLチューブに分注した。上清20μLをLuciferase assay system（Promega）50μLと混合した際の発光量（RLU）をGLOMAX（Promega）により定量した。上清20μL中のタンパク質量（mg）をTaKaRa BCA Protein Assay Kit（タカラバイオ）を用いて定量した。得られた発光量（RLU）をタンパク質量（mg）で除することで、Luciferase activity （RLU/mg protein）として算出した。 The amount of BMDC derived from C57BL / 6J mice and each of the above-mentioned liposomes is 1.5 mL so that the amount of 4.0 × 10 5 cells of BMDC / pDNA or mRNA is equivalent to 0.4 μg of each liposome / 500 μL of serum-free BMDC complete medium After mixing in a tube, this was seeded in a 24 well plate and incubated for 3 hours. After adding 1000 μL of complete medium for BMDC to each well and incubating for additional 3 hours, 1000 μL of supernatant was collected in a 1.5 mL tube, centrifuged at 4 ° C., 500 g for 5 minutes, and the supernatant was removed. The supernatant remaining in each well was collected in a corresponding 1.5 mL tube, and then the wells were washed with 1000 μL PBS (−) and collected in a corresponding 1.5 mL tube. To the wells, 75 μl each of a 5-fold dilution of Reporter Lysis Buffer (Promega) with DDW was added. The supernatant was removed after centrifuging a 1.5 mL tube containing the supernatant at 500 g for 5 minutes at 4 ° C. The resulting cell pellet was suspended in 40 μL of Lysis buffer in the corresponding well, and returned to the original well. Incubated at -80 ° C for more than 30 minutes. After thawing at room temperature and removing cells with a cell scraper, Lysis buffer in the well was collected in a 1.5 mL tube and centrifuged at 20000 g at 4 ° C. for 2 minutes. The supernatant was aliquoted into another 1.5 mL tube. The amount of luminescence (RLU) when 20 μL of the supernatant was mixed with 50 μL of Luciferase assay system (Promega) was quantified by GLOMAX (Promega). The amount of protein (mg) in 20 μL of the supernatant was quantified using TaKaRa BCA Protein Assay Kit (Takara Bio). The obtained luminescence amount (RLU) was divided by the amount of protein (mg) to calculate Luciferase activity (RLU / mg protein).
　図１に示されるように、同量のpDNA又はmRNAを使用した場合において、mRNA KALA- LipoplexでトランスフェクションされたBMDCが最も高いルシフェラーゼ活性を示すことが確認された。 As shown in FIG. 1, it was confirmed that BMDC transfected with mRNA KALA-Lipoplex exhibited the highest luciferase activity when the same amount of pDNA or mRNA was used.
実施例３　KALA-LipoplexでトランスフェクションされたBMDCの抗原提示強度評価
　オボアルブミン（OVA）をコードするpDNA（pCpGfree-MCS (invivogen)にOVAをコードするDNA断片を挿入し、Endofree Plasmid Giga Kitを用いて大腸菌より調製した）又はmRNA（T7 promoterの下流にOVAをコードする配列を有するpDNAを、mMESSAGE mMACHINE（商標） T7 ULTRA Transcription Kit (Thermofischer Scientific)によりin vitro転写する事で調製した）を核酸として用いて、実施例１に示した方法により、pDNA KALA- Lipoplex、mRNA KALA- Lipoplex、pDNA KALA-MEND、mRNA KALA-MEND、及びmRNA DOTAP-Lipoplexをそれぞれ調製した。また、実施例１（１）においてSTR-KALAを同量のSTR-オクタアルギニン（R8）に代えてmRNA R8- Lipoplexを調製した。 Example 3 Evaluation of Antigen Presenting Strength of BMDC Transfected with KALA-Lipoplex A DNA fragment encoding OVA is inserted into pDNA (pCpGfree-MCS (invivogen)) encoding ovalbumin (OVA), using Endofree Plasmid Giga Kit Prepared from E. coli or mRNA (prepared by in vitro transcription of pDNA having a sequence encoding OVA downstream of T7 promoter with mMESSAGE mMACHINETM T7 ULTRA Transcription Kit (Thermofischer Scientific)) as a nucleic acid Using the method described in Example 1, pDNA KALA-Lipoplex, mRNA KALA-Lipoplex, pDNA KALA-MEND, mRNA KALA-MEND, and mRNA DOTAP-Lipoplex were respectively prepared. In addition, mRNA R8-Lipoplex was prepared by replacing STR-KALA with the same amount of STR-octaarginine (R8) in Example 1 (1).
　C57BL/6Jマウス由来のBMDCと上記の各リポソームとを、2.0×106cellsのBMDC/pDNA又はmRNA量で0.25～1.0μg相当の各リポソーム/1000μLの血清不含BMDC用完全培地となるように1.5mLチューブ内で混合し、これを12 well plateに播種して、2時間インキュベートした。1000μLのBMDC用完全培地を添加して更に21時間インキュベートした後に、細胞をピペッティングにより回収し、4℃、500g、5分で遠心後、上清を除いた。1000μLの完全培地で2回細胞を洗浄した。400μLの完全培地で細胞を懸濁後、細胞濃度を測定した。2.0×105cellsのBMDCと1.0×105cellsのB3Z細胞（OVA257-264を特異的に認識するT細胞レセプターを発現するT細胞ハイブリドーマ。OVA257-264を提示する抗原提示細胞との相互作用により活性化されるとβ-Galを発現する）を200μLの完全培地中で混合し、96well plateに播種後、37℃、5%CO2下で16時間インキュベートした。各wellの上清を1.5mLチューブへ回収し、200μLのPBS（-）で洗浄し、対応する1.5mLチューブに回収した。4℃、500g、5分で遠心後、上清を除いた。各wellに50μLのCPRG bufferを加えると同時に、1.5mLチューブ中の細胞ペレットを50μLのCPRG bufferで懸濁し、対応するwellに加えた。37℃で4時間インキュベート後、各wellの595nmにおける吸光度を測定した。吸光度が1を超える場合は、CPRG diluentを用いて希釈し測定した。 BMDCs from C57BL / 6J mice and each of the above-mentioned liposomes in an amount of 2.0 × 10 6 cells of BMDC / pDNA or mRNA equivalent to 0.25 to 1.0 μg of each liposome / 1000 μL of complete medium for serum-free BMDCs The mixture was mixed in a 1.5 mL tube, seeded on a 12 well plate and incubated for 2 hours. After adding 1000 μL of complete medium for BMDC and further incubating for 21 hours, cells were collected by pipetting, centrifuged at 4 ° C., 500 g for 5 minutes, and the supernatant was removed. The cells were washed twice with 1000 μL of complete medium. After suspending the cells in 400 μL of complete medium, the cell concentration was measured. Mutual and 2.0 × 10 5 cells of BMDC and 1.0 × 10 5 cells antigen presenting cells presenting T cell hybridoma .ova 257-264 expressing T cells specifically recognizing receptors B3Z cells (OVA 257-264 of When activated by action, β-Gal was expressed in 200 μL of complete medium, mixed in a 96-well plate, and incubated at 37 ° C. under 5% CO 2 for 16 hours. The supernatant of each well was collected into a 1.5 mL tube, washed with 200 μL PBS (−), and collected into a corresponding 1.5 mL tube. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed. At the same time as 50 μL of CPRG buffer was added to each well, the cell pellet in a 1.5 mL tube was suspended with 50 μL of CPRG buffer and added to the corresponding wells. After 4 hours of incubation at 37 ° C., the absorbance at 595 nm of each well was measured. When the absorbance was above 1, it was diluted and measured using CPRG diluent.
　図２に示されるように、同量例えば1.0μgのpDNA又はmRNAを使用した場合において、mRNA KALA- LipoplexでトランスフェクションされたBMDCが最も高い抗原提示強度を示すこと、1μg相当のpDNA KALA-LipoplexでトランスフェクションされたBMDCよりも0.25μg相当のmRNA KALA-LipoplexでトランスフェクションされたBMDCの方が高い抗原提示強度を示すこと、mRNA KALA-LipoplexでトランスフェクションされたBMDCの抗原提示強度は、mRNAの量に依存して上昇すること等が確認された。 As shown in FIG. 2, when the same amount, for example, 1.0 μg of pDNA or mRNA is used, BMADC transfected with mRNA KALA-Lipoplex exhibits the highest antigen presentation strength, equivalent to 1 μg of pDNA KALA-Lipoplex The antigen-presenting intensity of BMADC transfected with KALA-Lipoplex is higher than that of BMDC transfected with. It was confirmed that it would rise depending on the amount of
実施例４　KALA-LipoplexでトランスフェクションされたBMDCの免疫誘導能評価
（１）KALA-Lipoplexにより抗原遺伝子を導入されたBMDCの免疫
　C57BL/6Jマウス由来のBMDCと実施例３で調製したmRNA KALA- Lipoplex、mRNA KALA-MEND及びmRNA DOTAP-Lipoplexの各リポソームとを、2.0×106cellsのBMDC/ mRNA量で0.025～2.0μg相当の各リポソーム/1000μLの血清不含BMDC用完全培地となるように1.5mLチューブ内で混合し、これを12well plateへ播種して2時間インキュベートした。1000μLの完全培地を添加して更に4時間インキュベートした後に、細胞をピペッティングにより回収し、4℃、500g、5分で遠心後、上清を除いた。1000μLのPBS（-）で2回細胞を洗浄した。400μLのPBSで細胞を懸濁後、細胞濃度を測定した。4℃、500g、5分で遠心後、上清を除いた後、125×105cells/mLとなるように細胞をPBS（-）で懸濁し、氷中へ移して、トランスフェクションされた3種のBMDCを用意した。このBMDC又はPBSをC57BL/6Jマウス（6～8週齢メス）の両足裏皮下に20μLずつ投与することで免疫処置を行った。1回目の免疫の1週間後に2回目の免疫処置を同様に行った。 Example 4 Evaluation of immunoinducing ability of BMDC transfected with KALA-Lipoplex (1) Immunization of BMDC to which an antigen gene has been introduced by KALA-Lipoplex BMDC derived from C57BL / 6J mouse and mRNA KALA- prepared in Example 3 Each liposome of Lipoplex, mRNA KALA-MEND and mRNA DOTAP-Lipoplex, in the amount of BMDC / mRNA of 2.0 × 10 6 cells, is equivalent to 0.025 to 2.0 μg of each liposome / 1000 μL of complete medium for serum-free BMDC The mixture was mixed in a 1.5 mL tube, seeded on a 12 well plate and incubated for 2 hours. After adding 1000 μL of complete medium and incubating for additional 4 hours, cells were collected by pipetting, centrifuged at 4 ° C., 500 g for 5 minutes, and the supernatant was removed. The cells were washed twice with 1000 μL PBS (−). After suspending the cells in 400 μL of PBS, the cell concentration was measured. After centrifugation at 500 g for 5 minutes at 4 ° C, after removing the supernatant, cells were suspended with PBS (-) to 125 × 10 5 cells / mL, transferred to ice, and transfected 3 A species of BMDC was prepared. Immunization was carried out by administering 20 μL each of the BMDC or PBS subcutaneously to the soles of C57BL / 6J mice (6- to 8-week-old females). A second immunization was performed similarly, one week after the first immunization.
（２）細胞内IFNγ染色による抗原特異的CD8+T細胞の誘導能評価
　2回目の免疫から1週間後に、各マウスより脾臓を摘出し、完全培地中に加えた。脾臓内部の脾臓細胞を完全培地中に出し、ピペッティングにより細胞塊をほぐした後、40μmセルストレイナーに通して50mLコニカルチューブへ回収した。4℃、500g、5分で遠心後、上清を除いた。1mLのACK Lysing bufferにより懸濁し、室温、5分インキュベートすることで、赤血球を破壊した。9mLの完全培地を加え、混合した後、4℃、500g、5分で遠心した。上清を除き、残った細胞ペレットを5mLの完全培地で2回洗浄した。5mLの完全培地で懸濁後、細胞濃度を測定した。15×106cellsの脾臓細胞を1500μLの1μg/mL OVA257-264含有完全培地で懸濁し、6well plateに播種した後、37℃、5%CO2下で1時間インキュベートした。1000μLの2.5μL/mL Goldi Plug（BD）、1μg/mL OVA257-264含有完全培地をwellに加え、ピペッティングにより懸濁した。37℃、5%CO2下で6時間インキュベートすることで脾臓細胞をOVA刺激した。wellからピペッティングにより細胞を回収し、5mLの氷冷完全培地で2回洗浄した。5mLの氷冷完全培地に懸濁し、細胞濃度を測定した。 (2) Evaluation of Inducibility of Antigen-Specific CD8 + T Cells by Intracellular IFNγ Staining One week after the second immunization, spleen was removed from each mouse and added to complete medium. The spleen cells inside the spleen were put out in complete medium, and the cell mass was loosened by pipetting, and then passed through a 40 μm cell strainer and collected into a 50 mL conical tube. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed. The red blood cells were disrupted by suspending with 1 mL of ACK Lysing buffer and incubating for 5 minutes at room temperature. 9 mL of complete medium was added, mixed, and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed and the remaining cell pellet was washed twice with 5 mL of complete medium. After suspending in 5 mL of complete medium, cell concentration was measured. 15 × 10 6 cells of spleen cells were suspended in 1500 μL of complete medium containing 1 μg / mL OVA 257-264 , seeded in a 6-well plate, and then incubated at 37 ° C. in 5% CO 2 for 1 hour. A complete medium containing 1000 μL of 2.5 μL / mL Goldi Plug (BD), 1 μg / mL OVA 257-264 was added to the wells and suspended by pipetting. Splenocytes were OVA stimulated by incubation at 37 ° C., 5% CO 2 for 6 hours. The cells were recovered by pipetting from the wells and washed twice with 5 mL of ice cold complete medium. The cells were suspended in 5 mL of ice cold complete medium, and the cell concentration was measured.
　1.0×106cellsのOVAペプチド処理脾臓細胞を4℃、500g、5分で遠心後、上清を除き、1mLのFACS buffer（2.5% BSA、0.5% NaN3）で2回洗浄した。50μLの10μg/mL anti-mouse CD16/32 antibodyで細胞を懸濁し、4℃、10分インキュベートすることでブロッキングを行った。50μLのAPC anti-mouse CD3（4μg/mL）、FITC anti-mouse CD8a（10μg/mL）を各細胞に添加し、タッピングにより混和後、4℃、30分インキュベートすることで細胞表面分子の染色を行った。1mLのFACS bufferで2回洗浄した。250μLのTF Fix/Perm Buffer（BD）で細胞を懸濁し、ボルテックスミキサーで3秒間攪拌後、4℃、45分インキュベートすることで、膜透過処理及び固定処理を行った。500μLのTF Perm/Wash buffer（BD）で2回洗浄した。50μLの1.2μg/mL PE anti-mouse IFNγ で細胞を懸濁し、4℃、30分インキュベートすることで、細胞内染色を行った。500μLのTF Perm/Wash buffer（BD）で2回洗浄し、500μLのFACS bufferで懸濁し、フローサイトメーターにより解析し、CD3+CD8+細胞におけるインターフェロンγ（IFNγ）産生細胞の割合を測定した。 After centrifuging 1.0 × 10 6 cells of OVA peptide-treated spleen cells at 500 ° C. for 5 minutes at 4 ° C., the supernatant was removed and washed twice with 1 mL of FACS buffer (2.5% BSA, 0.5% NaN 3 ). The cells were suspended in 50 μL of 10 μg / mL anti-mouse CD16 / 32 antibody and blocking was performed by incubating at 4 ° C. for 10 minutes. Add 50 μL of APC anti-mouse CD3 (4 μg / mL) and FITC anti-mouse CD8a (10 μg / mL) to each cell, mix by tapping, and incubate cell surface molecules by incubating at 4 ° C for 30 minutes. went. Washed twice with 1 mL of FACS buffer. The cells were suspended with 250 μL of TF Fix / Perm Buffer (BD), stirred for 3 seconds with a vortex mixer, and then incubated at 4 ° C. for 45 minutes to perform membrane permeabilization and fixation. Washed twice with 500 μL of TF Perm / Wash buffer (BD). Intracellular staining was performed by suspending the cells with 50 μL of 1.2 μg / mL PE anti-mouse IFNγ and incubating at 4 ° C. for 30 minutes. The cells were washed twice with 500 μL of TF Perm / Wash buffer (BD), suspended with 500 μL of FACS buffer, analyzed by a flow cytometer, and the percentage of interferon γ (IFN γ) producing cells in CD3 + CD8 + cells was measured.
　図３に示されるように、mRNA KALA-Lipoplexを投与したマウスにおいて、最も効率的に抗原特異的CD8+T細胞が誘導されることが確認された。 As shown in FIG. 3, it was confirmed that antigen-specific CD8 + T cells were most efficiently induced in mice administered mRNA KALA-Lipoplex.
（３）in vivo CTLアッセイによる抗原特異的細胞傷害性活性評価
　上記（１）と同様にC57BL/6Jマウスに3種類のBMDCのいずれかを１回免疫し、免疫から1週間後に以下の方法で調製した標的細胞を投与した。まずC57BL/6Jマウスより脾臓を摘出し、完全培地中に加えた。脾臓内部の脾臓細胞を完全培地中に出し、ピペッティングにより細胞塊をほぐした後、40μmセルストレイナーに通して50mLコニカルチューブへ回収した。4℃、500g、5分で遠心後、上清を除いた。脾臓1つあたり1mLのACK Lysing bufferにより懸濁し、室温、5分インキュベートすることで、赤血球を破壊した。5倍量の完全培地を加え、混合した後、4℃、500g、5分で遠心した。上清を除き、残った細胞ペレットを10mLの完全培地で洗浄した。30mLの完全培地で懸濁後、細胞濃度を測定した。40μmのセルストレイナーを通して脾臓細胞を2群に分けた（ペプチド処理群、及び非処理群）。4℃、500g、5分で遠心後、上清を除き、1.0×107cells/mLとなるように完全培地で懸濁した。ペプチド処理群に、2mM OVA257-264ペプチドを培地量の1/400量を添加し素早く懸濁した。ペプチド処理群、及び非処理群を37℃、5%CO2下で1時間インキュベートした。4℃、500g、5分で遠心後、上清を除き、10mLの完全培地で2回洗浄した。10mLのPBS（-）で洗浄した。3.0×107cells/mLとなるように、ペプチド処理群を1μM CFSE/PBSで、非処理群を0.1μM CFSE/PBSで懸濁し、遮光しながら37℃の水浴で10分間インキュベートした。10mLの完全培地を添加し、4℃、500g、5分で遠心後、上清を除去後、10mLの完全培地、及び10mLのPBS（-）で洗浄した。10mLのPBS（-）で細胞を懸濁し、細胞濃度を測定した。5.0×107cells/mLとなるようにPBS（-）で懸濁し、ペプチド処理群と非処理群を100μLずつ混合したものを標的細胞として、免疫マウスの尾静脈より投与した。 (3) Evaluation of antigen-specific cytotoxic activity by in vivo CTL assay As described in (1) above, C57BL / 6J mice are immunized once with any of three types of BMDC, and one week after the immunization, the following method is used. The prepared target cells were administered. First, spleens were isolated from C57BL / 6J mice and added to complete medium. The spleen cells inside the spleen were put out in complete medium, and the cell mass was loosened by pipetting, and then passed through a 40 μm cell strainer and collected into a 50 mL conical tube. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed. Erythrocytes were destroyed by suspending with 1 mL of ACK Lysing buffer per spleen and incubating for 5 minutes at room temperature. Five volumes of complete medium was added, mixed, and centrifuged at 500 g for 5 minutes at 4 ° C. The supernatant was removed and the remaining cell pellet was washed with 10 mL of complete medium. After suspending in 30 mL of complete medium, the cell concentration was measured. The spleen cells were divided into two groups (peptide-treated group and non-treated group) through a 40 μm cell strainer. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed and suspended in complete medium to 1.0 × 10 7 cells / mL. In the peptide-treated group, 2 mM OVA 257-264 peptide was quickly suspended by adding 1/400 of the medium volume. The peptide-treated and non-treated groups were incubated at 37 ° C., 5% CO 2 for 1 hour. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed and washed twice with 10 mL of complete medium. Washed with 10 mL PBS (-). The peptide-treated group was suspended with 1 μM CFSE / PBS and the non-treated group with 0.1 μM CFSE / PBS so as to be 3.0 × 10 7 cells / mL, and incubated in a 37 ° C. water bath for 10 minutes while blocking light. 10 mL of complete medium was added, and after centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed and washed with 10 mL of complete medium and 10 mL of PBS (−). The cells were suspended in 10 mL PBS (-) and the cell concentration was measured. The cells were suspended in PBS (−) to be 5.0 × 10 7 cells / mL, and 100 μL each of the peptide-treated group and the non-treated group were mixed and administered as target cells from the tail vein of immunized mice.
　標的細胞投与の20時間後、マウスより脾臓を摘出し、完全培地中に加えた。脾臓内部の脾臓細胞を完全培地中に出し、ピペッティングにより細胞塊をほぐした後、ナイロンメッシュを通して1.5mLチューブへ回収した。4℃、500g、5分で遠心後、上清を除いた。1mLのACK Lysing bufferにより懸濁し、室温、5分インキュベートすることで、赤血球を破壊した。9mLのFACS bufferで希釈し、4℃、500g、5分で遠心後、上清を除いた。5mLのFACS bufferで細胞を洗浄後、5mLのFACS bufferで細胞を懸濁した。フローサイトメーターを用いて、ペプチド処理群と非処理群の比から、CTL活性を算出した。 Twenty hours after target cell administration, spleens were excised from the mice and added to complete medium. The spleen cells in the inside of the spleen were put out in complete medium, and the cell mass was loosened by pipetting, and then collected through a nylon mesh into a 1.5 mL tube. After centrifugation at 500 g for 5 minutes at 4 ° C., the supernatant was removed. The red blood cells were disrupted by suspending with 1 mL of ACK Lysing buffer and incubating for 5 minutes at room temperature. The supernatant was removed after dilution with 9 mL of FACS buffer and centrifugation at 500 g for 5 minutes at 4 ° C. After washing the cells with 5 mL of FACS buffer, the cells were suspended with 5 mL of FACS buffer. The CTL activity was calculated from the ratio of peptide-treated group to non-treated group using a flow cytometer.
　図４に示されるように、いずれのリポソームでトランスフェクションされたBMDCも、mRNAの量に依存した抗原特異的CTL活性を示した。特にmRNA KALA-LipoplexでトランスフェクションされたBMDCは、mRNA KALA-MENDに封入されたmRNA量の1/10以下の量でも、生体において抗原特異的な細胞傷害活性を誘導することができることが確認された。 As shown in FIG. 4, all liposome-transfected BMDCs showed antigen-specific CTL activity dependent on the amount of mRNA. In particular, it has been confirmed that BMDC transfected with mRNA KALA-Lipoplex can induce antigen-specific cytotoxic activity in vivo even with an amount of 1/10 or less of the amount of mRNA encapsulated in mRNA KALA-MEND. The
実施例５　KALA-Lipoplexの膜融合性評価
　ICRマウス（4～6週齢オス）よりヘパリン存在下で採取した血液を、氷冷生理食塩水と混合して10mLとした。4℃、400g、10分で遠心後、上清を除いた。同様の操作を3回繰り返し、赤血球懸濁液とした。Triton-X100/PBS（Triton-X100終濃度:0.02%（w/v））と適当量の赤血球懸濁液を混合して250μLとし、ボルテックスミキサーで攪拌後、200μLを96well plateへ加え、540nmにおける吸光度を測定した。540nmにおける吸光度が1.0となる赤血球懸濁液量を算出した。その量の赤血球懸濁液と、実施例３で調製したmRNA KALA- Lipoplex、mRNA KALA-MEND、mRNA DOTAP-Lipoplex、及びmRNA R8- Lipoplex（脂質換算終濃度:2.5、5.0、10μM）、及び20mM リンゴ酸/PBS（pH5.5、6.5、7.4）を混合し、250μLとした。37℃、1600rpmの条件で30分間攪拌しながらインキュベートした。4℃、400g、5分で遠心後、200μLの上清を透明96well plateへ加え、540nmの吸光度を測定した。0.02%（w/v）Triron-X100中での吸光度を100%Lysis時の値として、緩衝液中での吸光度を0%Lysisの値として、それぞれのリポソームの溶血活性（%Lysis）を算出した。 Example 5 Evaluation of Membrane Consistency of KALA-Lipoplex Blood collected from ICR mice (4 to 6 weeks old male) in the presence of heparin was mixed with ice cold saline to make 10 mL. After centrifugation at 400 g for 10 minutes at 4 ° C., the supernatant was removed. The same operation was repeated three times to obtain a red blood cell suspension. Triton-X100 / PBS (Triton-X100 final concentration: 0.02% (w / v)) and an appropriate amount of erythrocyte suspension are mixed to make 250 μL, 200 μL is added to 96 well plate after vortexing, and it is at 540 nm Absorbance was measured. The amount of red blood cell suspension at which the absorbance at 540 nm was 1.0 was calculated. The amount of erythrocyte suspension, and mRNA KALA-Lipoplex, mRNA KALA-MEND, mRNA DOTAP-Lipoplex, and mRNA R8-Lipoplex (final lipid equivalent: 2.5, 5.0, 10 μM), and 20 mM prepared in Example 3 Malic acid / PBS (pH 5.5, 6.5, 7.4) was mixed to make 250 μL. The mixture was incubated at 37 ° C. and 1600 rpm for 30 minutes with stirring. After centrifugation at 4 ° C., 400 g for 5 minutes, 200 μL of the supernatant was added to a transparent 96 well plate, and the absorbance at 540 nm was measured. The hemolytic activity (% Lysis) of each liposome was calculated using the absorbance in 0.02% (w / v) Triron-X 100 as the value at 100% Lysis and the absorbance in the buffer as the 0% Lysis value. .
　図５に示されるように、いずれのリポソームもpH5.5において溶血活性を示した一方、pH6.5及びpH7.4では溶血はあまり認められなかった。なかでもmRNA KALA-Lipoplexは特に高い溶血活性を示していたことから、酸性条件であるエンドライソソームにおいてmRNA KALA-Lipoplexはその高い生体膜膜融合能により脱出効率に優れる可能性が示唆された。 As shown in FIG. 5, while all the liposomes exhibited hemolytic activity at pH 5.5, hemolysis was hardly observed at pH 6.5 and pH 7.4. Above all, mRNA KALA-Lipoplex showed particularly high hemolytic activity, suggesting that mRNA KALA-Lipoplex may be excellent in escape efficiency due to its high biomembrane fusion ability in endolysosome under acidic conditions.
実施例６　KALA-LipoplexのmRNA放出能評価
　0.1μg mRNAに相当する量の実施例３で調製したmRNA KALA- Lipoplex、mRNA KALA-MEND若しくはmRNA DOTAP-Lipoplex、又は0.1μgのOVA mRNAを含む懸濁液10μLを、0.031～10mg/mL Poly-（α,β）-DL-aspartic acid sodium salt mol wt 2,000-11,000（Sigma） 8μL、1.45M NaCl 2μLと混合し、37℃、30分間インキュベートした。6×Loading dye（TOYOBO） 4μLを混合し、10,000倍希釈Gel Red（Biotium）含有1%アガロースゲルに全量をアプライした。4℃で2時間電気泳動し、Gel Doc EZ（Bio-rad）により撮影し、各リポソームからのmRNA放出量を比較した。 Example 6 Evaluation of mRNA Release Ability of KALA-Lipoplex Suspension Containing mRNA KALA-Lipoplex, mRNA KALA-MEND or mRNA DOTAP-Lipoplex, or 0.1 μg of OVA mRNA prepared in Example 3 in an amount corresponding to 0.1 μg mRNA 10 μL of the solution was mixed with 8 μL of 0.031 to 10 mg / mL Poly- (α, β) -DL-aspartic acid sodium salt mol wt 2,000-11,000 (Sigma), 2 μL of 1.45 M NaCl, and incubated at 37 ° C. for 30 minutes. Four μL of 6 × Loading dye (TOYOBO) was mixed, and the whole amount was applied to 1% agarose gel containing 10,000-fold diluted Gel Red (Biotium). Electrophoresis was performed at 4 ° C. for 2 hours, photographed with Gel Doc EZ (Bio-rad), and the amount of mRNA released from each liposome was compared.
　図６に示されるように、mRNA KALA-Lipoplexは、mRNA KALA-MEND又はmRNA DOTAP-Lipoplexと比較してmRNAを効率的に放出することが確認された。 As shown in FIG. 6, it was confirmed that mRNA KALA-Lipoplex releases mRNA efficiently as compared to mRNA KALA-MEND or mRNA DOTAP-Lipoplex.
実施例７　NY-ESO-1をコードするmRNAを表面に有するKALA-Lipoplexの抗腫瘍効果
（１）NY-ESO-1 mRNA搭載KALA-Lipoplexの調製
　T7 promoterの下流にがん抗原NY-ESO-1をコードするcDNA（配列番号８）を有するpDNAを元に、mMESSAGE mMACHINE（商標） T7 ULTRA Transcription Kit (Thermofischer Scientific）によりin vitro転写することで調製したmRNA（UTRを含む814ヌクレオチド、NYESO1 mRNAと表す）を核酸として用いて、実施例１（１）に示した方法により、NYESO1 mRNAを表面に保持したKALA-Lipoplex（NYESO1 mRNA KALA-Lipoplex；脂質終濃度：0.183mM、核酸終濃度：0.013mg/mL、核酸脂質比　4μg 核酸／55nmol 脂質）を作製した。 Example 7 Antitumor effect of KALA-Lipoplex having mRNA encoding NY-ESO-1 on the surface (1) Preparation of KALA-Lipoplex loaded with NY-ESO-1 mRNA Cancer antigen NY-ESO downstream of T7 promoter 1. An mRNA (UTR containing 814 nucleotides, NYESO1 mRNA) prepared by in vitro transcription using mMESSAGE mMACHINETM T7 ULTRA Transcription Kit (Thermofischer Scientific) based on pDNA having a cDNA encoding SEQ ID NO: 1 (SEQ ID NO: 8) KALA-Lipoplex (NYESO1 mRNA KALA-Lipoplex; final lipid concentration: 0.183 mM, nucleic acid final concentration: 0.013 mg) using the NYESO1 mRNA on the surface by the method described in Example 1 (1) using A nucleic acid / lipid ratio of 4 μg (nucleic acid / 55 nmol lipid) was prepared.
（２）アジュバントを含むKALA-Lipoplexの調製
　CpG ODN 0.06μg：NYESO1 mRNA 3.94μg（CpG ODN/mRNA =1/64）、pDNA .（pCpGfree-NewMCS）12μg：NYESO1 mRNA 3.88μg（pDNA/mRNA = 1/32）、及びtpRNA 2.00μg：NYESO1 mRNA 2.00μg（tpRNA/mRNA = 1/1）の各混合物を核酸として用いて、実施例１（１）に示した方法により、NYESO1 mRNAと各核酸アジュバントとを表面に保持したKALA-Lipoplex（mRNA+CpG-ODN、mRNA+pDNA、mRNA+tpRNA、脂質終濃度：0.183mM、核酸終濃度：0.013mg/mL、核酸脂質比　4μg 核酸／55nmol 脂質）を作製した。 (2) Preparation of KALA-Lipoplex containing an adjuvant CpG ODN 0.06 μg: NYESO1 mRNA 3.94 μg (CpG ODN / mRNA = 1/64), pDNA. 12 (pCpGfree-NewMCS) 12 μg: NYESO1 mRNA 3.88 μg (pDNA / mRNA = 1 / 32) and 2.00 μg of tpRNA: 2.00 μg of NYESO1 mRNA (tpRNA / mRNA = 1/1) as a nucleic acid, according to the method shown in Example 1 (1), NYESO1 mRNA and each nucleic acid adjuvant Prepare KALA-Lipoplex (mRNA + CpG-ODN, mRNA + pDNA, mRNA + tpRNA, final lipid concentration: 0.183 mM, final nucleic acid concentration: 0.013 mg / mL, nucleic acid lipid ratio 4 μg nucleic acid / 55 nmol lipid) did.
　さらに、NYESO1 mRNAと、2mM DOPE（エタノール溶液）45μL、2mM PA（クロロホルム：エタノール＝1：1） 10μL、0.09 mg/mL MPLA（クロロホルム：メタノール＝75：25）をクロロホルムで16倍希釈した0.0056 mg/mL MPLA 6.25μL及びクロロホルム45μLを試験管内で混合して調製される脂質混合物の薄膜とを用いて、実施例１（１）と同様の操作を行い、NYESO1 mRNAを表面に保持し、脂質膜にMPLAを含むKALAペプチド修飾リポソーム（NYESO1/MPLA KALA-Lipoplex；脂質終濃度：0.183 mM、核酸終濃度：0.01 mg/mL、核酸脂質比　3μg 核酸／55nmol 脂質）を製造した。 Furthermore, 0.0056 mg of NYESO1 mRNA, 45 μL of 2 mM DOPE (ethanol solution), 10 μL of 2 mM PA (chloroform: ethanol = 1: 1), 0.09 mg / mL MPLA (chloroform: methanol = 75: 25) diluted with chloroform 16 times The same procedure as in Example 1 (1) is carried out using a thin film of a lipid mixture prepared by mixing 6.25 μL of MPLA / mL and 45 μL of chloroform in a test tube to retain NYESO1 mRNA on the surface, a lipid membrane A KALA peptide-modified liposome (NYESO1 / MPLA KALA-Lipoplex; final concentration of lipid: 0.183 mM, final concentration of nucleic acid: 0.01 mg / mL, nucleic acid lipid ratio 3 μg nucleic acid / 55 nmol lipid) was prepared.
　NYESO1 mRNA KALA-Lipoplex、mRNA＋CpG-ODN、mRNA＋pDNA、mRNA+tpRNA及びNYESO1/MPLA KALA-Lipoplexの粒子径、ζ電位及び多分散指数（Pdl）を表２に示す。
Figure JPOXMLDOC01-appb-T000005
 The particle diameter, zeta potential and polydispersity index (Pdl) of NYESO1 mRNA KALA-Lipoplex, mRNA + CpG-ODN, mRNA + pDNA, mRNA + tpRNA and NYESO1 / MPLA KALA-Lipoplex are shown in Table 2.
Figure JPOXMLDOC01-appb-T000005
（３）NYESO1 mRNAを表面に保持したKALA-Lipoplexの抗腫瘍効果
　実施例２（１）で誘導したBalb/cマウス由来の未成熟樹状細胞（BMDC）と上記（１）で製造したKALA-Lipoplexとを用いて、実施例４の（１）と同様の操作を行い、NYESO1 mRNA KALA-Lipoplex 及びNYESO1/MPLA KALA-LipoplexでトランスフェクションされたBMDCをそれぞれ作製した。 (3) Antitumor effect of KALA-Lipoplex having NYESO1 mRNA retained on the surface Immature dendritic cells (BMDC) derived from Balb / c mice induced in Example 2 (1) and KALA prepared in the above (1) The same procedure as in (4) of Example 4 was performed using Lipoplex to prepare BMDCs transfected with NYESO1 mRNA KALA-Lipoplex and NYESO1 / MPLA KALA-Lipoplex, respectively.
　6～8週齢のBalb/cマウス（n=5）にNY-ESO-1を発現しているCT26腫瘍細胞（1.0×106 cells）を皮下移植し（0日）、7日目に腫瘍サイズを測定した。7日目と14日目に上記BMDC（各1.0 × 106 cells、mRNA量1.0μg）をそれぞれ足裏皮下に投与して免疫処置を行い、30日目まで腫瘍サイズを経時的に測定した。結果を図７に示す。 Six to eight-week-old Balb / c mice (n = 5) were subcutaneously transplanted with CT26 tumor cells (1.0 × 10 6 cells) expressing NY-ESO-1 (day 0), and tumors were observed on day 7 The size was measured. On the 7th and 14th day, the BMDC (each 1.0 × 10 6 cells, mRNA amount: 1.0 μg) was administered subcutaneously to the sole of the foot to perform immunization, and the tumor size was measured over time until the 30th day. The results are shown in FIG.
　BMDCに代えてPBSを足裏皮下に投与したコントロールマウス群（図７のPBS）と比較して、NYESO1 mRNA KALA-Lipoplex 又はNYESO1/MPLA KALA-LipoplexでトランスフェクションされたBMDCを投与した群（図７のmRNA及びmRNA/MPLA）では移植腫瘍の増殖が抑制されることが、特にNYESO1/MPLA KALA-LipoplexでトランスフェクションされたBMDCを投与した群では腫瘍の増殖が強く抑制されることが確認された。 A group receiving BMDC transfected with NYESO1 mRNA KALA-Lipoplex or NYESO1 / MPLA KALA-Lipoplex as compared to a group of control mice (PBS in FIG. 7) administered PBS subcutaneously in place of BMDC (FIG. 7) (7) mRNA and mRNA / MPLA) were confirmed to suppress the growth of transplanted tumors, and in particular, it was confirmed that tumor growth was strongly suppressed in the group to which BNDCs transfected with NYESO1 / MPLA KALA-Lipoplex were administered. The
（４）MPLAによるアジュバント効果の確認
　本実施例７（１）の操作に準じて、NYESO1 mRNAに代えて同量のLuciferase mRNAを表面に保持し、さらに脂質膜に含まれるMPLAの含有量を0、0.0015μg、0.0059μg、0.023μg又は0.094μgにそれぞれ調節したKALA-Lipoplex（Luc/MPLA KALA Lipoplex）を製造した。実施例２（１）で誘導したBalb/cマウス由来の未成熟樹状細胞（BMDC）とLuc/MPLA KALA Lipoplexとを用いて、実施例２（２）と同様の操作を行って、トランスフェクションしたBMDCにおけるLuciferase activityを測定した。また、BMDCのinterleukin-6（IL-6）産生量を、Mouse IL-6 Quantikine ELISA Kit（R&D systems）を用いて測定した。結果を図８に示す。 (4) Confirmation of Adjuvant Effect by MPLA In accordance with the procedure of Example 7 (1), the same amount of Luciferase mRNA is retained on the surface instead of NYESO1 mRNA, and the content of MPLA contained in the lipid membrane is 0 KALA-Lipoplex (Luc / MPLA KALA Lipoplex) was prepared, each adjusted to 0.0015 μg, 0.0059 μg, 0.023 μg or 0.094 μg. Transfection was carried out using the immature dendritic cells (BMDC) derived from Balb / c mice induced in Example 2 (1) and Luc / MPLA KALA Lipoplex using the same procedure as in Example 2 (2). Luciferase activity in BMDC was measured. Moreover, the amount of interleukin-6 (IL-6) production of BMDC was measured using Mouse IL-6 Quantikine ELISA Kit (R & D systems). The results are shown in FIG.
　脂質膜中のMPLA含有量を増加させてもLuciferase activityの変化は殆ど見られなかった一方、IL-6産生量はMPLA含有量と共に増加することが確認された。 While the change in Luciferase activity was hardly observed even when the MPLA content in the lipid membrane was increased, it was confirmed that the IL-6 production amount increased with the MPLA content.
実施例８　OVA mRNAを表面に保持したKALA-Lipoplexの抗原特異的細胞傷害性活性評価
　実施例３に示される操作に準じて、OVA mRNAを表面に保持したmRNA KALA- LipoplexのmRNA含有量を8μg／55 nmol 脂質、10μg／55 nmol 脂質又は12μg／55 nmol 脂質としたOVA mRNAを表面に保持したKALA-Lipoplex（OVA mRNA KALA-Lipoplex 8～12、脂質終濃度：0.183 mM、核酸終濃度：0.013mg/mL、0.017mg/mL、0.020mg/mL））を製造した。OVA mRNA KALA-Lipoplex 8～12の粒子径、ζ電位及び多分散指数（Pdl）を表３に示す。
Figure JPOXMLDOC01-appb-T000006
 Example 8 Evaluation of Antigen-Specific Cytotoxic Activity of KALA-Lipoplex Holding OVA mRNA on the Surface According to the procedure shown in Example 3, 8 μg of the mRNA content of KALA-Lipoplex, the mRNA holding OVA mRNA on the surface KALA-Lipoplex (OVA mRNA KALA-Lipoplex 8-12, final concentration of lipid: 0.183 mM, final concentration of nucleic acid: 0.013 with 55 nmol lipid, 10 μg / 55 nmol lipid or 12 μg / 55 nmol lipid-supported OVA mRNA on the surface mg / mL, 0.017 mg / mL, 0.020 mg / mL)) were produced. The particle size, zeta potential and polydispersity index (Pdl) of OVA mRNA KALA-Lipoplex 8-12 are shown in Table 3.
Figure JPOXMLDOC01-appb-T000006
　実施例４（３）のin vivo CTLアッセイにおいてBMDCの代わりにOVA mRNA KALA-Lipoplex 8～12を静脈内投与（iv、投与量1μg mRNA／匹）又は皮下投与（sc、投与量2μg mRNA／匹）したときの抗原特異的細胞傷害性活性（CTL activity）を測定した。結果を図９に示す。 Intravenous administration (iv, dose 1 μg mRNA / mouse) or subcutaneous administration (sc, dose 2 μg mRNA / mouse of OVA mRNA KALA-Lipoplex 8-12 in place of BMDC in the in vivo CTL assay of Example 4 (3) Antigen specific cytotoxic activity (CTL activity) was measured. The results are shown in FIG.
　PBSを投与したコントロール群のCTL activityは10％未満であるのに対して、OVA mRNA KALA-Lipoplex 8～12を投与した群ではいずれもCTL activityの上昇が確認され、特にOVA mRNA KALA-Lipoplex 8を静脈投与した群においてCTL activityの顕著な上昇が確認された。 The CTL activity of the control group to which PBS was administered was less than 10%, whereas the increase of CTL activity was confirmed in all of the groups to which OVA mRNA KALA-Lipoplex 8 to 12 was administered, and in particular OVA mRNA KALA-Lipoplex 8 A marked increase in CTL activity was observed in the group that received iv intravenous administration.

Claims (9)

  1. 　ペプチド鎖中にK-A-L-Aの繰り返し単位を少なくとも3個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個若しくは2個以上においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～50個のポリペプチドの1種以上により脂質膜が修飾された、細胞内に核酸を送達するための脂質膜構造体であって、核酸をその表面に保持した、前記脂質膜構造体。 The peptide chain contains at least three repeating units of KALA (provided that K is R in any one or two or more of the repeating units and / or any of the repeating units) An intracellular cell in which a lipid membrane is modified by one or more of a polypeptide having 12 to 50 amino acid residues, wherein A and H may be sandwiched between K and L in one or two or more. A lipid membrane structure for delivering a nucleic acid to the lipid membrane structure, wherein the nucleic acid is retained on the surface thereof.
  2. 　ポリペプチドが、ペプチド鎖中にK-A-L-Aの繰り返し単位を3又は4個含む（ただし該繰り返し単位のうちのいずれか1個若しくは2個以上においてKがRとなっていてもよく、及び／又は該繰り返し単位のうちのいずれか1個においてKとLに挟まれるAがHとなっていてもよい）アミノ酸残基数が12～30個のポリペプチドである、請求項１に記載の脂質膜構造体。 The polypeptide comprises 3 or 4 repeating units of KALA in the peptide chain (however, K may be R in any one or more of the repeating units and / or the repeating unit The lipid membrane structure according to claim 1, which is a polypeptide in which the number of amino acid residues between A and H between K and L in any one of the units is 12 to 30). .
  3. 　ポリペプチドが下記ａ）～ｇ）に示されるアミノ酸配列のいずれかからなるポリペプチドである、請求項１又は２に記載の脂質膜構造体。
    　　ａ）WEAKLAKALAKALAKHLAKALAKALKA（配列番号１）
    　　ｂ）WEAKLAKALAKALAKHLAKALAKALKACEA（配列番号２）
    　　ｃ）WEAKLAKALAKALAKHLAKALA（配列番号３）
    　　ｄ）WEAKLAKALAKALAKHLA（配列番号４）
    　　ｅ）KALAKALAKALAKALA（配列番号５）
    　　ｆ）KALAKALAKALA（配列番号６）
    　　ｇ）WEARLARALARALARHLARALARALRA（配列番号７）     The lipid membrane structure according to claim 1 or 2, wherein the polypeptide is a polypeptide consisting of any of the amino acid sequences shown in the following a) to g).
    a) WEAKLAKALAKALAKHLAKALAKALKA (SEQ ID NO: 1)
    b) WEAKLAKALAKALAK HLA KALAKALKACEA (SEQ ID NO: 2)
    c) WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 3)
    d) WEAKLAKALAKALAK HLA (SEQ ID NO: 4)
    e) KALAKALAKALAKALA (SEQ ID NO: 5)
    f) KALAKALAKALA (SEQ ID NO: 6)
    g) WEARLARALARALARHLARALA RA (SEQ ID NO: 7)
  4. 　脂質膜構造体がリポソームである、請求項１～３のいずれか一項に記載の脂質膜構造体。 The lipid membrane structure according to any one of claims 1 to 3, wherein the lipid membrane structure is a liposome.
  5. 　ポリペプチドが疎水性基で修飾されており、前記疎水性基が脂質膜に挿入されてなる、請求項１～４のいずれか一項に記載の脂質膜構造体。 The lipid membrane structure according to any one of claims 1 to 4, wherein the polypeptide is modified with a hydrophobic group, and the hydrophobic group is inserted into a lipid membrane.
  6. 　核酸が核酸アジュバント及び／又は抗原性ポリペプチドをコードするmRNAである、請求項１～５のいずれか一項に記載の脂質膜構造体。 The lipid membrane structure according to any one of claims 1 to 5, wherein the nucleic acid is mRNA encoding a nucleic acid adjuvant and / or an antigenic polypeptide.
  7. 　脂質アジュバントをさらに含む、請求項１～６のいずれか一項に記載の脂質膜構造体。 The lipid membrane structure according to any one of claims 1 to 6, further comprising a lipid adjuvant.
  8. 　免疫細胞の細胞質に核酸を送達するための、請求項１～７のいずれか一項に記載の脂質膜構造体。 The lipid membrane structure according to any one of claims 1 to 7, for delivering a nucleic acid to the cytoplasm of an immune cell.
  9. 　免疫細胞と請求項１～８のいずれか一項に記載の脂質膜構造体とをインキュベーションする工程を含む、免疫療法に用いるためのトランスフェクションされた免疫細胞をインビトロで製造する方法。

          A method of producing transfected immune cells in vitro for use in immunotherapy, comprising the step of incubating the immune cells and the lipid membrane structure according to any one of claims 1 to 8.

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