WO2019103151A1

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

Info

Publication number: WO2019103151A1
Application number: PCT/JP2018/043528
Authority: WO
Inventors: 英万秋田; 尚也三浦; 菜穂舘下; 浩揮田中
Original assignee: 国立大学法人千葉大学
Priority date: 2017-11-27
Filing date: 2018-11-27
Publication date: 2019-05-31
Also published as: JPWO2019103151A1

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

Lipid membrane structure for delivering nucleic acid into cells

The present invention relates to a lipid membrane structure for introducing nucleic acid into cells.

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.

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.

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.

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.

International Publication WO2011 / 132713 International Publication WO2015 / 098907

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.

(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.

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.

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. 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. 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. 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. 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. 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. 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 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. 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.

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.

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. 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.

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) 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) 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.

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).

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 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.

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.

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.

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. As the antigenic polypeptide, any polypeptide having antigenicity can be selected.

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.

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.

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.

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. 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.

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.

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.

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.

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.

(Wherein, 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)

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

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. 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

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) 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.

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) 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.

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.

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).

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) 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.

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).

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.

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).

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.

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

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) 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.

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.

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) 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.

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.

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

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. .

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.

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.

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.

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) 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.

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.

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.

(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.

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.

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) 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.

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.

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.

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.

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

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.
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). .
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)
The lipid membrane structure according to any one of claims 1 to 3, wherein the lipid membrane structure is a liposome.
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.
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.
The lipid membrane structure according to any one of claims 1 to 6, further comprising a lipid adjuvant.
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.
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.