WO2011132713A1 - 核内移行性を有する脂質膜構造体 - Google Patents
核内移行性を有する脂質膜構造体 Download PDFInfo
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- WO2011132713A1 WO2011132713A1 PCT/JP2011/059738 JP2011059738W WO2011132713A1 WO 2011132713 A1 WO2011132713 A1 WO 2011132713A1 JP 2011059738 W JP2011059738 W JP 2011059738W WO 2011132713 A1 WO2011132713 A1 WO 2011132713A1
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- lipid membrane
- membrane structure
- polypeptide
- lipid
- cells
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- A61K47/6905—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 the form being a colloid or an emulsion
- A61K47/6911—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 the form being a colloid or an emulsion the form being a liposome
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- 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
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- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
Definitions
- the present invention relates to a lipid membrane structure having nuclear translocation properties. More specifically, the present invention relates to a lipid membrane structure such as a liposome that can be easily transferred into the nucleus of immune cells, particularly the nucleus of dendritic cells.
- a method of encapsulating a drug in a liposome that is a lipid membrane structure has been proposed as a means of specifically transporting the drug to the affected area.
- the effectiveness of liposomes encapsulating an antitumor agent has been reported in the field of malignant tumor treatment.
- a lipid membrane structure that can be used for gene expression a multifunctional envelope-type nanostructure (MEND: Multifunctionallopeenvelope-type nano ⁇ device; hereinafter, it may be abbreviated as “MEND”.
- MEND Multifunctionallopeenvelope-type nano ⁇ device
- Drug Delivery System 22-2, 115pp.115-122, 2007, etc.
- This structure can be used as a drug delivery system for selectively delivering a gene or the like into a specific cell, and is known to be useful for, for example, tumor gene therapy.
- lipid membrane structures As a means to deliver target substances such as drugs, nucleic acids, peptides, polypeptides, and sugars to specific sites such as target organs and tumor tissues using lipid membrane structures, the surface of lipid membrane structures is a functional molecule.
- a number of methods of modifying with have been proposed.
- Lipid membrane structures encapsulating drugs such as antitumor agents reach the target cells and are taken into cells by endocytosis and become encapsulated in endosomes. The received drug is released into the cytoplasm.
- Liposomes (Biochemistry, 43, pp.5618- modified with a peptide (GALA: Biochemistry, 26, pp.2964-2972, 1987) in order to enhance drug release from liposomes incorporated into endosomes 5623, 2004) and MEND (Japanese Patent Laid-Open No. 2006-28030) have been proposed.
- a liposome whose outer surface is modified with octaarginine International Publication WO2005 / 32593; WO Journal of Controlled Release, 98, pp.317-323, 2004
- bilamellar liposomes with lipid membranes modified with nuclear translocation peptides International Publication WO2006 / 101201
- surface modification with monosaccharides such as galactose and mannose Liposomes
- Multi-lipid membrane structures (T-MEND) modified with monosaccharides showed fusion properties with lipid membranes and nuclear membranes, and it was reported that gene expression efficiency could be improved in the in-vitro test results.
- a nucleic acid encoding an antigenic protein can be introduced into the nucleus of an immune cell, in particular, a dendritic cell having an antigen-presenting action, the protein transcribed and translated from the nucleic acid in the dendritic cell is expressed on the surface of the dendritic cell.
- the living body can acquire immunity to the protein. From such a viewpoint, there is a need for a technique for efficiently delivering a nucleic acid into the nucleus of immune cells such as dendritic cells.
- the efficiency of nucleic acid introduction when introducing a nucleic acid into the nucleus of a dendritic cell using a lipid membrane structure such as MEND described above is sufficiently higher than that of other cells such as tumor cells and liver parenchymal cells. is not.
- the introduced nucleic acid Before the introduced nucleic acid is finally expressed in the nucleus, it must undergo various intracellular kinetic processes such as intracellular uptake, endosomal escape, nuclear translocation, and nuclear transcription.
- a nuclear membrane consisting of two membranes always exists intact, and this nuclear membrane is presumed to impede the ability of lipid membrane structures to translocate into the nucleus. Therefore, in order to deliver nucleic acids to the nuclei of dendritic cells using lipid membrane structures, how to break through the above-mentioned processes, particularly the barrier by the nuclear membrane composed of two membranes, is a very important issue. It becomes.
- KALA peptide A 27 amino acid residue polypeptide called KALA peptide is known, and it has been reported that it can form a complex with plasmid DNA using its own cationic charge (Biochemistry, 36, pp. 3008-3017, 1997), there is no suggestion in the above publication whether this peptide promotes nuclear translocation of lipid membrane structures.
- An object of the present invention is to provide a means for efficiently delivering a nucleic acid into the nucleus of an immune cell, particularly a dendritic cell having antigen-presenting ability. More specifically, it is an object of the present invention to provide a lipid membrane structure capable of efficiently delivering a nucleic acid into the nucleus of immune cells such as dendritic cells.
- MEND international publication WO2005 / 32593
- octaarginine polypeptide which is a functional polypeptide capable of enhancing nuclear translocation ability, and endosome escape ability.
- GALA peptide which is a functional polypeptide to be imparted
- polypeptide expression from the nucleic acid was almost I was not able to admit.
- T-MEND multi-lipid membrane structure
- the present inventors have found that when the lipid membrane of a lipid membrane structure such as MEND encapsulating nucleic acid is modified with KALA peptide, the efficiency of nucleic acid introduction into the nucleus of immune cells such as dendritic cells is remarkable. It was found to increase.
- the present invention has been completed based on the above findings.
- a lipid membrane structure for delivering a substance into the nucleus of a cell, wherein the lipid membrane is a polypeptide of the following (a) and / or (b): (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted and / or substituted and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and a lipid membrane into the nucleus of a cell
- a lipid membrane structure modified with a polypeptide having activity to promote structure migration is provided.
- the above lipid membrane structure wherein the lipid membrane structure is a liposome; the above lipid membrane structure wherein the cell is an immune cell, preferably a dendritic cell; And / or the above-mentioned lipid membrane structure wherein the polypeptide of (b) is modified with a hydrophobic group, preferably a stearyl group or a cholesteryl group, and the hydrophobic group is inserted into the lipid membrane;
- PEG PEG
- the substance to be delivered is a nucleic acid, for example, any of the above lipid membrane structures, which is a functional nucleic acid such as a nucleic acid containing a gene or siRNA; Lipid membrane structure; the lipid membrane structure described above, wherein the DNA is DNA bound to a vector DNA not containing CpG; the DNA is bound to a vector DNA not containing CpG, and the DNA is free of CpG A lipid membrane structure as described above; the lipid membrane structure as described above, wherein the substance to be delivered is a nucleic acid encoding an antigen polypeptide to be presented on the surface of immune cells, preferably on the surface of dendritic cells; Any of the lipid membrane structures described above, which is a sex envelope nanostructure (MEND); any of the lipid membrane structures described above, in which a nucleic acid and a
- MEND sex envelope nanostructure
- the lipid membrane structure described above used for introducing a nucleic acid encoding an antigen polypeptide to be presented on the surface of an immune cell, preferably a dendritic cell, into the nucleus of the cell; for immunotherapy against the antigen polypeptide
- the above lipid membrane structure for use is the above lipid membrane structure wherein the antigen polypeptide is a surface polypeptide specific for cancer cells.
- a pharmaceutical composition comprising this lipid membrane structure as an active ingredient, preferably a pharmaceutical composition comprising a nucleic acid as a substance to be delivered is also provided by the present invention.
- the present invention provides a method for delivering a nucleic acid in the nucleus of a cell, preferably in the nucleus of an immune cell, more preferably in the nucleus of a dendritic cell in the body of a mammal, including a human,
- a method comprising the step of administering to the animal the above lipid membrane structure encapsulated therein; a method of presenting an antigenic polypeptide on the surface of immune cells, preferably dendritic cells, in the body of mammals including humans.
- a method comprising the step of administering to the animal the above lipid membrane structure encapsulating a nucleic acid encoding the antigen polypeptide.
- a method for obtaining immunity to an antigen polypeptide by presenting the antigen polypeptide on the surface of an immune cell, preferably a dendritic cell, in a living body of a mammal including humans, which encodes the antigen polypeptide A method comprising the step of administering to the animal the above lipid membrane structure having nucleic acid encapsulated therein; and a cancer cell-specific surface on the surface of an immune cell, preferably a dendritic cell, in a mammal including human.
- An immunotherapy of a malignant tumor in which a surface polypeptide is presented to acquire immunity to the polypeptide comprising the step of administering to the animal the above lipid membrane structure in which a nucleic acid encoding the polypeptide is encapsulated
- a method of including is provided.
- polypeptide used for promoting the migration of a lipid membrane structure into the nucleus of a cell, preferably into the nucleus of an immune cell, more preferably into the nucleus of a dendritic cell
- the lipid membrane structure provided by the present invention can efficiently migrate into the nucleus of any cell such as immune cells including dendritic cells, and efficiently transfer substances such as nucleic acids encapsulated in the nucleus.
- the polypeptide encoded by the nucleic acid can be expressed upon release.
- a nucleic acid encoding a polypeptide is introduced into the nucleus of a dendritic cell using the lipid membrane structure of the present invention, the polypeptide transcribed and translated from the nucleic acid is presented on the surface of the dendritic cell, Can acquire immunity to the polypeptide, so that effective immunotherapy can be performed against the desired polypeptide.
- the lipid membrane structure provided by the present invention itself can exert an adjuvant action on dendritic cells and can promote the production of various cytokines.
- tumor exacerbation and proliferation can be remarkably suppressed regardless of the presence or absence of an adjuvant.
- gene expression efficiency is remarkably improved by removing the CpG sequence from the vector part in the DNA to be encapsulated and, if necessary, removing the CpG sequence from the DNA encoding the protein to be expressed. .
- FIG. 4 is a graph showing gene expression efficiencies of four-membrane cationic T-MEND and anionic T-MEND in which a lipid membrane is modified with polypeptide (a) (KALA). It is the figure which showed the result of having introduce
- FIG. 3 shows that KALA modified on the MEND surface has an adjuvant effect on dendritic cells.
- FIG. 7 is a diagram showing a step of constructing a CpGfree plasmid DNA having a new multiple cloning site as the first step of the method for constructing plasmid DNA (2) in Example 6.
- FIG. 10 is a diagram showing a step of incorporating a luciferase gene into CpGfree-NEWmcs as the second step of the method for constructing plasmid DNA (2) in Example 6.
- FIG. 9 is a diagram showing a step of removing one remaining CpG sequence as the third step of the method for constructing plasmid DNA (2) in Example 6.
- FIG. 10 is a diagram showing a step of constructing a CpGfree plasmid DNA having a new multiple cloning site as the first step of the method for constructing plasmid DNA (2) in Example 6.
- FIG. 10 is a diagram showing a step of incorporating a luciferase gene into CpGfree-NEWmcs as the second step of the method for constructing plasm
- MEND surface-modified with KALA has high antitumor activity irrespective of the presence or absence of an adjuvant. It is the figure which showed the anti-tumor effect at the time of administering MEND surface-modified with KALA after tumor formation.
- Examples of the lipid constituting the lipid membrane structure of the present invention include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids.
- Examples of phospholipids and phospholipid derivatives include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1 , 2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, phosphatidic acid and the like, and these can be used alone or in combination of two or more.
- Fatty acid residues in these phospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms. Specific examples include lauric acid, myristic acid, palmitic acid, stearin Mention may be made of acyl groups derived from fatty acids such as acids, oleic acid and linoleic acid. Moreover, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.
- glycolipids examples include glyceroglycolipid (eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), sphingoglycolipid (eg, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like. Can be mentioned.
- glyceroglycolipid eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride
- sphingoglycolipid eg, galactosyl cerebroside, lactosyl cerebroside, ganglioside
- sterols examples include animal-derived sterols (e.g., cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterol) (e.g., stigmasterol, sitosterol, campesterol, Brush castrol), sterols derived from microorganisms (for example, timosterol, ergosterol) and the like.
- the saturated or unsaturated fatty acid 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.
- the form of the lipid membrane structure is not particularly limited.
- a form dispersed in an aqueous solvent a single membrane liposome, a multilamellar liposome, an O / W emulsion, a W / O / W emulsion, a spherical micelle, a string micelle Or an irregular layered structure.
- a preferred form of the lipid membrane structure of the present invention is a liposome.
- a liposome may be described as a preferred embodiment of the lipid membrane structure of the present invention, the lipid membrane structure of the present invention is not limited to liposomes.
- the lipid membrane structure of the present invention is a lipid membrane structure for delivering a substance into the nucleus of a cell, wherein the lipid membrane is a polypeptide of the following (a) and / or (b): (a) sequence A polypeptide comprising the amino acid sequence of No. 1; (b) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted and / or substituted and / or inserted in the amino acid sequence of SEQ ID No. 1. It is characterized by being modified with a polypeptide having an activity of promoting the migration of the lipid membrane structure into the nucleus of the cell.
- the polypeptide (a) represented by SEQ ID NO: 1 is a polypeptide comprising 27 amino acid residues obtained by removing 3 amino acids from the C-terminus from a known polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) comprising 30 amino acid residues. It is a peptide (hereinafter, this polypeptide may be referred to as “KALA peptide” in the present specification: chemistryBiochemistry, 36, pp.3008-3017, 1997).
- KALA peptide this polypeptide can form a complex with plasmid DNA by utilizing its own cationic charge, but suggests whether or not it promotes nuclear translocation of lipid membrane structures. There is no.
- the known KALA peptide is a functional polypeptide consisting of 30 amino acid residues called GALA peptide (Bioor. Med. Chem., 5, pp.1883-1891, 1997). Has an amino acid sequence substituted with another amino acid residue (for example, a plurality of glutamine residues are substituted with lysine residues).
- GALA peptide is a polypeptide having a function of promoting fusion of lipid membranes in response to pH and a function capable of enhancing the ability of the lipid membrane structure to escape from the endosome, and is used in the present invention. It is different from the function of KALA peptide (function of improving the nuclear translocation in cells of lipid membrane structures).
- polypeptide (b) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted and / or substituted and / or inserted in the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as “modified”)
- a polypeptide having an activity of promoting the transfer of a lipid membrane structure into the nucleus of a cell may be used.
- the activity for promoting the transfer of the lipid membrane structure into the nucleus for example, the activity for promoting the ability of the dendritic cell to transfer into the nucleus can be evaluated.
- a dendritic cell is used as a target cell, and a nucleic acid that can be expressed in its nucleus, for example, a nucleic acid that encodes a polypeptide linked downstream of a promoter operable in the nucleus of the dendritic cell is contained therein.
- a lipid membrane structure in which the lipid membrane is modified with a modified polypeptide (hereinafter referred to as ⁇ modified lipid membrane structure '') and a lipid membrane structure not modified with the modified polypeptide (hereinafter referred to as ⁇ modified lipid membrane structure '')
- the expression level of the marker polypeptide in the cell into which the modified lipid membrane structure is introduced is the expression level of the polypeptide in the cell into which the unmodified lipid membrane structure is introduced. What is necessary is just to evaluate whether it increases.
- the modified polypeptide can be used as the polypeptide (b).
- One or more of the above polypeptides (b) can be used in combination with the polypeptide (a).
- polypeptide (a) and polypeptide (b) can be biologically prepared using host cells by various gene recombination techniques available to those skilled in the art. Moreover, you may manufacture the said polypeptide by the organic-chemical method using peptide synthesis reactions, such as the solid-phase synthesis method which can be utilized for those skilled in the art. Alternatively, automatic synthesis may be performed using a peptide synthesizer.
- the means for immobilizing the polypeptide (a) and / or the polypeptide (b) to the lipid membrane of the lipid membrane structure is not particularly limited.
- the polypeptide (a) may be a hydrophobic group such as a stearyl group or a cholesteryl group.
- lipid membrane modification can be easily performed by preparing the lipid membrane structure so that the hydrophobic group is embedded in the lipid membrane of the lipid membrane structure. .
- Any hydrophobic compound residue can be used as the hydrophobic group.
- the lipid membrane modification with the polypeptide (a) and / or polypeptide (b) is applied to the inner lipid membrane in addition to the outer lipid membrane. You may go.
- the lipid membrane structure of the present invention can be used to deliver a substance into the nucleus of a cell, but the type of cell is not particularly limited, and the type of substance to be delivered and the purpose of substance delivery into the nucleus Depending on the situation, appropriate cells can be targeted.
- Preferred examples of the cell to be targeted include immune cells, and among the immune cells, antigen-presenting cells can be preferably used.
- antigen-presenting cells such as macrophages, dendritic cells, and B cells are preferred, and dendritic cells are particularly preferred.
- the lipid membrane structure can be surface-modified with an oligosaccharide compound having 3 or more sugars.
- the type of oligosaccharide compound having 3 or more sugars is not particularly limited.
- an oligosaccharide compound having about 3 to about 10 sugar units bound thereto can be used, and preferably about 3 to about 6 sugar units. Bound oligosaccharide compounds can be used.
- oligosaccharide compound for example, cellotriose (Cellotriose: ⁇ -D-glucopyranosyl- (1 ⁇ 4) - ⁇ -D-glucopyranosyl- (1 ⁇ 4) -D-glucose), chacotriose: ⁇ -L-rhamnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-rhamnopyranosyl- (1 ⁇ 4)]-D-glucose), gentianose (- ⁇ -D-fructofuranosyl ⁇ -D-glucopyranosyl- (1 6) - ⁇ -D-glucopyranoside), isomaltotriose (Isomaltotriose: ⁇ -D-glucopyranosyl- (1 6) - ⁇ -D-glucopyranosyl- (1 ⁇ 6) -D-glucose), isopanose : ⁇ -D-glucopy
- an oligosaccharide compound that is a trimer or hexamer of glucose can be used, and more preferably, an oligosaccharide compound that is a trimer or tetramer of glucose can be used.
- isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, maltohexaose, etc. can be suitably used, and among these, malto in which glucose is ⁇ 1-4 bonded. More preferred is triose, maltotetraose, maltopentaose, or maltohexaose.
- the amount of surface modification of the lipid membrane structure by the oligosaccharide compound is not particularly limited. For example, it is about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably 5 to 10 mol% with respect to the total amount of lipid. Degree.
- the method of modifying the surface of the lipid membrane structure with an oligosaccharide compound is not particularly limited.
- liposomes whose surfaces are modified with monosaccharides such as galactose and mannose are known. Therefore, the surface modification method described in this publication can be adopted.
- This method is a method in which a monosaccharide compound is bonded to a polyalkylene glycolated lipid to modify the surface of the lipid membrane structure. By this means, the surface of the lipid membrane structure can be simultaneously modified with polyalkylene glycol. preferable.
- the blood retention of liposomes can be enhanced.
- a hydrophilic polymer such as polyalkylene glycol
- polyalkylene glycol is preferable.
- polyalkylene glycol for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol and the like can be used.
- the molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.
- the surface modification of the lipid membrane structure with polyalkylene glycol can be easily performed by constructing a lipid membrane structure using, for example, a polyalkylene glycol-modified lipid as a lipid membrane constituent lipid.
- a polyalkylene glycol-modified lipid as a lipid membrane constituent lipid.
- stearyl polyethylene glycol for example, PEG45 stearate (STR-PEG45) or the like
- the surface modification of the lipid membrane structure can be performed using the polypeptide (a) and / or the polypeptide (b) modified with polyalkylene glycol.
- polyalkylene glycol modified with polyalkylene glycol.
- an appropriate phospholipid such as stearyl polyethylene glycol
- the lipid membrane modification with the polyalkylene glycol and the polypeptide (a) and / or the polypeptide (b) can be simultaneously achieved.
- surface modification with the polyalkylene glycol and the oligosaccharide compound can be simultaneously achieved by bonding the oligosaccharide compound to the polyalkylene glycol.
- the method of surface-modifying the lipid membrane structure with a polyalkylene glycol or oligosaccharide compound is not limited to the above-mentioned method.
- a lipidated compound such as stearyl polyalkylene glycol or oligosaccharide compound is used.
- surface modification can be performed by using as a constituent lipid of the lipid membrane structure.
- examples of lipid derivatives for enhancing retention in blood include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc. Can also be used.
- examples of lipid derivatives for enhancing retention in blood include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, etc.
- polyalkylene glycol dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer as well as polyalkylene glycol are used as hydrophilic polymers to enhance blood retention.
- the lipid membrane of the lipid membrane structure of the present invention may be modified with GALA.
- GALA GALA cholesterol derivative
- a lipid membrane structure surface-modified with GALA can be easily produced according to the method described in the above-mentioned publication. Can do.
- a lipid membrane structure surface-modified with GALA can be produced.
- the surface modification amount by GALA is not particularly limited, but is, for example, about 0.01 to 10 mol%, preferably about 0.1 to 4 mol%, more preferably about 1 to 3 mol%, based on the total lipid amount.
- GALA includes deletion and substitution of one or several amino acids in the amino acid sequence of the peptide in addition to the peptide specified by SEQ ID NO: 1 in the sequence listing of JP-A-2006-28030. And / or a modified peptide consisting of an added amino acid sequence and having substantially the same properties as GALA (for example, the ability to fuse lipid membranes under acidic conditions).
- GALA herein should not be construed as limiting in any way.
- the entire disclosure of JP-A-2006-28030 is included as a disclosure of the present specification by reference.
- the surface of the lipid membrane structure of the present invention can be modified with an MPC polymer.
- the MPC polymer is an MPC polymer obtained by polymerizing 2-methacryloyloxyethyl phosphorylcholine (MPC). Since this polymer has a molecular structure similar to that of a biological membrane, interaction with biological components such as polypeptides and blood cells is extremely small, and it has been shown to have excellent biocompatibility.
- MPC polymer includes both a homopolymer of MPC and a copolymer of MPC and other polymerization components.
- MPC polymers can be easily obtained from commercially available polymers.
- MPC homopolymer (CAS: 67881-99-6) as a registered trademark “LIPIDURE” from NOF Corporation; copolymer of MPC and butyl methacrylate (CAS: 125275-25-4); MPC, methacrylic Ternary copolymer of sodium acid and butyl methacrylate; binary copolymer of MPC and 2-hydroxy-3- (meth) acryloyloxypropyltrimethylammonium chloride; phospholipid polymer (LIPIDURE-S), etc. Can also be used in the present invention.
- the type of MPC polymer used in the present invention is not particularly limited, but for example, a copolymer of MPC and a methacrylic acid ester such as butyl methacrylate, particularly a block copolymer can be preferably used.
- the production method of this copolymer is described in detail in Japanese Patent No. 2890316, and those skilled in the art can easily produce a desired copolymer by referring to this patent publication. The entire disclosure of this patent publication is incorporated herein by reference.
- MPC copolymers can be preferably used.
- a copolymer of MPC and butyl methacrylate (BMA) for example, a copolymer having a molar ratio of MPC and BMA of 5: 5 (PMB50) or a copolymer having a molar ratio of MPC and BMA of 3: 7 (PMB30) is known.
- PMB50 a copolymer having a molar ratio of MPC and BMA of 5: 5
- PMB30 copolymer having a molar ratio of MPC and BMA of 3: 7
- PMB50 can be particularly preferably used.
- the degree of polymerization and molecular weight of the MPC polymer are not particularly limited. For example, from the viewpoint of maintaining water solubility, a polymer having an average molecular weight (weight average molecular weight) of about 5,000 to 300,000, preferably about 10,000 to 100,000 can be used.
- the method of modifying the lipid membrane structure with the MPC polymer is not particularly limited.
- the MPC polymer may be added to an aqueous dispersion of a lipid membrane structure such as a liposome and allowed to stand at room temperature for several minutes to several hours.
- the amount of the MPC polymer added to the aqueous dispersion is not particularly limited, but depending on the amount of the MPC polymer to be modified, for example, in the range of 0.01 to 1% by mass with respect to the total lipid amount of the lipid membrane structure, preferably May be added in an amount of about 0.1 to 10% by mass, more preferably about 0.1 to 3% by mass.
- the MPC polymer is rapidly incorporated into the lipid component of the lipid membrane structure, and a lipid membrane structure whose surface is modified with the MPC polymer can be prepared.
- the amount of surface modification by the MPC polymer is not particularly limited, but is, for example, in the range of about 0.1 to 5% by mass with respect to the total lipid amount of the lipid membrane structure.
- the lipid membrane structure of the present invention comprises a sterol or a membrane stabilizer such as glycerin or a fatty acid ester thereof, an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, and a membrane.
- an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene
- a charged substance and a membrane.
- One or two or more substances selected from the group consisting of polypeptides and the like may be included.
- the charged substance imparting positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammoniumpropane; or cationic polymers.
- Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.
- Examples of the membrane polypeptide include a membrane superficial polypeptide or an integral membrane polypeptide. The compounding amount of these substances is not particularly limited, and can be appropriately selected according to the purpose.
- the lipid membrane structure of the present invention can be provided with any one function or two or more functions such as a temperature change sensitivity function, a membrane permeation function, a gene expression function, and a pH sensitivity function.
- Examples of the temperature change sensitive lipid derivative capable of imparting a temperature change sensitive function include dipalmitoyl phosphatidylcholine and the like.
- Examples of the pH-sensitive lipid derivative that can impart a pH-sensitive function include dioleoylphosphatidylethanolamine.
- the lipid membrane structure of the present invention can be modified with a substance such as an antibody capable of specifically binding to a cell surface receptor or antigen, thereby improving the substance delivery efficiency into the cell nucleus. can do.
- a substance such as an antibody capable of specifically binding to a cell surface receptor or antigen
- a lipid derivative capable of reacting with a mercapto group in a monoclonal antibody or a fragment thereof such as poly (ethylene glycol) ) - ⁇ -distearoylphosphatidylethanolamine- ⁇ -maleimide, ⁇ - [N- (1,2-distearoyl-sn-glycero-3-phosphoryl-ethyl) carbamyl) - ⁇ - ⁇ 3- [2- ( By including a lipid derivative having a maleimide structure such as 2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) ethanecarboxamido] propyl ⁇ -poly (oxy-1,2-ethanedyl)
- the monoclonal antibody can be bound to the membrane surface of the lipid membrane structure.
- the surface of the lipid membrane structure of the present invention may be modified with a polypeptide containing a plurality of continuous arginine residues (hereinafter referred to as “polyarginine”).
- the polyarginine is preferably a polypeptide containing 4 to 20 consecutive arginine residues, more preferably a polypeptide consisting of only 4 to 20 consecutive arginine residues, particularly preferably octaarginine. Can do.
- Modification of the lipid membrane structure surface with polyarginine can be easily performed by using, for example, a lipid-modified polyarginine such as stearylated octaarginine as a constituent lipid of the lipid membrane structure according to the method described in the above publication. Can be done.
- a lipid-modified polyarginine such as stearylated octaarginine as a constituent lipid of the lipid membrane structure according to the method described in the above publication. Can be done.
- the surface of the lipid membrane structure of the present invention may be modified with INF7.
- INF7 is a glutamic acid-rich peptide obtained by modifying a peptide (1-23) derived from influenza HA polypeptide (HA2) .When mixed with liposomes, the lipid structure collapses and the encapsulated substance is easily released. (Biochemistry, 46, pp.13490-13504, 2007) and a delivery system in which INF7 is conjugated to polyethylene glycol tetraacrylate (PEG-TA) has also been proposed (The Journal Gene Medicine, 10, pp .1134-1149, 2008). Those skilled in the art can easily use INF7 in the present invention by referring to these publications.
- the term “INF7” includes one or several amino acids in the amino acid sequence of the above peptide in addition to the peptides specified by the sequences described in Table 1 of Biochemistry, 46, pp.13490-13504, 2007.
- a modified peptide consisting of an amino acid sequence in which is deleted, substituted and / or added and having substantially the same properties as INF7 is also included.
- the term “INF7” herein should not be construed as limiting in any way. The disclosures of the above publications and the disclosures of all documents cited in this publication are incorporated herein by reference.
- the method of modifying the lipid membrane structure with INF7 is not particularly limited, but in general, by constructing a lipid membrane structure using lipid-modified INF7 in which a lipid compound and INF7 are covalently bound as a lipid membrane-constituting lipid, A lipid membrane structure surface-modified with INF7 can be easily produced.
- lipid-modified INF for example, stearylated INF7 can be used, and this compound is described in Futaki, S. et al., Biocongug. Chem., 12 (6), pp.1005-1011, 2001. It can be easily manufactured according to the method.
- the amount of surface modification by INF7 is not particularly limited, but is generally in the range of 1 to 5 mol% with respect to the total lipid content of the lipid membrane structure, preferably about 3 to 5 mol% with respect to the total lipid content. It is.
- MEND Envelope-type nanostructures with multi-functionality
- MEND has a structure in which a core is a complex of a nucleic acid such as plasmid DNA and a cationic polymer such as protamine, and the core is enclosed in a lipid envelope membrane in the form of a liposome.
- MEND lipid envelope membranes can be equipped with peptides to adjust pH responsiveness and membrane permeability as needed, and the outer surface of lipid envelope membranes can be modified with alkylene glycols such as polyethylene glycol. it can.
- MEND Inside the lipid envelope of MEND, condensed DNA and cationic polymer are encapsulated, and designed to achieve efficient gene expression.
- MEND that can be suitably used in the present invention, a MEND in which a complex of plasmid DNA incorporating a desired gene and protamine is encapsulated inside and the outer surface of the lipid envelope is modified with oligosaccharide-conjugated PEG is preferable.
- the modification with oligosaccharide-linked PEG preferably uses stearyl polyethylene glycol to which the above-mentioned polypeptide (a) and / or polypeptide (b) is bound as a constituent lipid component.
- reviews such as DrugDDelivery System, 22-2, pp.115-122, 2007 can be referred to.
- the disclosures of the above publications and the disclosures of all documents cited in this review are hereby incorporated by reference.
- the form of the lipid membrane structure is not particularly limited, and examples thereof include a form dispersed in an aqueous solvent (for example, water, physiological saline, phosphate buffered physiological saline, etc.) and a form obtained by lyophilizing this aqueous dispersion. It is done.
- an aqueous solvent for example, water, physiological saline, phosphate buffered physiological saline, etc.
- the method for producing the lipid membrane structure is not particularly limited, and any method available to those skilled in the art can be employed.
- all the lipid components are dissolved in an organic solvent such as chloroform, and after forming a lipid film by drying under reduced pressure with an evaporator or spray drying with a spray dryer, the above mixture is dried with an aqueous solvent.
- an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier.
- it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc.
- extrusion may be performed under high pressure using a membrane filter having a uniform pore size.
- the size of the lipid membrane structure in the dispersed state is not particularly limited.
- the particle diameter is about 50 to 5 ⁇ m, preferably about 50 to 400 nm, and about 50 to 300 nm. Is preferable, and about 150 to 250 nm is more preferable.
- the particle diameter can be measured, for example, by the DLS (dynamic light scattering) method.
- the composition of the aqueous solvent is not particularly limited, and examples thereof include a buffer solution such as a phosphate buffer solution, a citrate buffer solution, and a phosphate buffered saline solution, a physiological saline solution, a medium for cell culture, and the like. Can do.
- a buffer solution such as a phosphate buffer solution, a citrate buffer solution, and a phosphate buffered saline solution, a physiological saline solution, a medium for cell culture, and the like.
- aqueous solvents can stably disperse lipid membrane structures, but also glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharides, lactose, sucrose, cellobiose, trehalose.
- Disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, sugars such as erythritol, xylitol, sorbitol, mannitol, maltitol (aqueous solutions), glycerin, diglycerin, poly Glycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol -Alkyl ether, 1,3-polyhydric alcohol (aqueous solution), such as butylene glycol and the like may be added.
- aqueous solution such as butylene glycol and the like
- the lipid membrane structure dispersed in the aqueous solvent In order to stably store the lipid membrane structure dispersed in the aqueous solvent for a long period of time, it is desirable to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation suppression. From the viewpoint of chemical stability of the lipid, it is desirable to set the pH of the aqueous solvent from weakly acidic to neutral (about pH 3.0 to 8.0) and / or to remove dissolved oxygen by nitrogen bubbling or the like. .
- aqueous dispersion of the obtained lipid membrane structure is freeze-dried or spray-dried, for example, glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharide, lactose, sucrose, cellobiose, trehalose Stability can be improved by using disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, sugars such as erythritol, xylitol, sorbitol, mannitol, and maltitol (aqueous solutions) There is a case.
- disaccharides such as maltose, trisaccharides such as raffinose and merezinose
- polysaccharides such as cyclodextrin
- sugars such as erythritol, xylitol,
- a polyhydric alcohol aqueous solution
- aqueous solution such as diethylene glycol monoalkyl ether or 1,3-butylene glycol
- a substance to be delivered into the nucleus of a cell of a target tissue or organ can be encapsulated.
- the type of substance to be encapsulated is not particularly limited, but in addition to any active pharmaceutical ingredients such as antitumor agents, anti-inflammatory agents, antibacterial agents, antiviral agents, sugars, peptides, nucleic acids, low molecular weight compounds, metals Any substance such as a compound can be encapsulated.
- the nucleic acid include a nucleic acid containing a gene, and more specifically, for example, a gene incorporated in a plasmid, but are not limited to this specific embodiment. Needless to say, any gene can be used as the gene.
- the case where a nucleic acid is encapsulated will be specifically described below, but the scope of the present invention is not limited to this specific embodiment.
- a nucleic acid can be preferably encapsulated.
- the nucleic acid includes DNA or RNA, and analogs or derivatives thereof (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.).
- the nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.
- the nucleic acid may contain a gene.
- the gene may be any of oligonucleotide, DNA, or RNA.
- a gene for introduction in vitro such as transformation
- nucleic acids examples include genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes and cytokines, as well as nucleic acids having a function of regulating gene expression, such as siRNAs. Functional nucleic acids including RNA and the like can also be used, and these are also included in the term nucleic acid in the present specification.
- nucleic acid should be interpreted in the broadest sense, and should not be interpreted in a limited way in any sense.
- the gene DNA to be expressed can be bound to the vector DNA and encapsulated in the lipid membrane structure, but in order to achieve higher gene expression efficiency, the vector It is preferred that the DNA does not contain a CpG sequence, and in addition it may be more preferred that the gene DNA to be expressed does not contain a CpG sequence. It is preferable that an enhancer and / or a promoter is bound to the vector.
- a compound having a nucleic acid introduction function can also be added.
- examples of such compounds include O, O′-N-didodecanoyl-N- ( ⁇ -trimethylammonioacetyl) -diethanolamine chloride, O, O′-N-ditetradecanoyl-N- ( ⁇ -trimethyl).
- Ammonioacetyl) -diethanolamine chloride O, O'-N-dihexadecanoyl-N- ( ⁇ -trimethylammonioacetyl) -diethanolamine chloride, O, O'-N-dioctadecenoyl-N- ( ⁇ -trimethylammonioacetyl) -diethanolamine chloride, O, O ', O' '-tridecanoyl-N- ( ⁇ -trimethylammoniodecanoyl) aminomethane bromide and N- [ ⁇ -trimethylammonioacetyl] -didodecyl- D-glutamate, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl) -N, N-dimethyl-1-propaneammonium trifluoroacetate, 1 , 2-Dimy
- a lipid membrane structure encapsulating a nucleic acid can be used as a carrier for delivering the nucleic acid into the nucleus of a cell of a target tissue or organ.
- a nucleic acid containing a desired gene as the nucleic acid and use the above MEND.
- MEND a nucleic acid containing a desired gene
- by administering a lipid membrane structure encapsulating a nucleic acid containing a gene, preferably MEND, to mammals including humans the desired gene is delivered into the nucleus of the cells of the target tissue or organ and efficiently expressed.
- the administration method is not particularly limited, but parenteral administration is preferable, and intravenous administration is more preferable.
- a medicine in the form of a pharmaceutical composition can be prepared and administered together with an appropriate formulation additive.
- nucleic acid When a nucleic acid is transferred into the nucleus of an immune cell, preferably a dendritic cell, using the lipid membrane structure of the present invention, a substance such as a nucleic acid encapsulated in the nucleus is efficiently released and encoded by the nucleic acid. Polypeptides can be expressed.
- a nucleic acid encoding a polypeptide is introduced into the nucleus of a dendritic cell using the lipid membrane structure of the present invention, the polypeptide transcribed and translated from the nucleic acid is presented on the surface of the dendritic cell, Can acquire immunity to the polypeptide, so that effective immunotherapy can be performed against the desired polypeptide.
- This aspect is a particularly preferable aspect in the present invention.
- the lipid membrane structure provided by the present invention itself can exert an adjuvant action on dendritic cells and can promote the production of various cytokines. Therefore, immunotherapy can be performed very efficiently by using the lipid membrane structure of the present invention.
- immunotherapy can be performed very efficiently by using the lipid membrane structure of the present invention.
- Example 1 (a) Preparation of encapsulated nucleic acid (condensate with protamine) A plasmid encoding luciferase (pDNA, manufactured by BD Biosciences Clontech) was dissolved in 10 mM HEPES buffer to a concentration of 0.1 mg / mL. A pDNA solution was added dropwise to 150 ⁇ L of a 0.1 mg / mL (10 mM HEPES buffer) Protamine (Protamine) solution while stirring little by little to prepare a compact body of protamine and pDNA (N / P ratio is 2.2).
- pDNA plasmid encoding luciferase
- lipid membrane To obtain a lipid membrane. To this lipid membrane, the compacted body (a) was added so that the total lipid concentration was 0.55 mM, left standing at room temperature for 10 minutes for hydration, and then sonicated for 1 minute with a sonicator. Stearylated octaarginine (STR-R8) (2 mg / mL aqueous solution) synthesized by the method described in JP-A-2003-343857 was added in an amount of 5 mol% of the total lipid, and allowed to stand at room temperature for 10 minutes.
- STR-8 Stearylated octaarginine
- the solution was dissolved in a buffer (converted to 1 mM amine concentration), and 100 ⁇ L of the pDNA solution was added dropwise to 150 ⁇ L of the resulting solution while stirring in small amounts to prepare a polyrotaxane / pDNA compact (N / P ratio is 5.0).
- the compact (c) was added to this lipid membrane so that the total lipid concentration was 0.55 mM, left to stand for 10 min at room temperature to be hydrated, and then sonicated with a sonicator for 1 minute.
- a stearylated octaarginine (STR-R8) 2 mg / mL aqueous solution synthesized by the method described in Japanese Patent Application Laid-Open No. 2003-343857 was added to 10 mol% of the total lipid, and allowed to stand at room temperature for 10 minutes. It was harmonized.
- the collected cell lysate was centrifuged (4 ° C., 15,000 rpm, 5 minutes) and centrifuged, and 45 ⁇ L of the supernatant was recovered. Luciferase activity measurement (RLU / mL) was measured using the obtained supernatant. Furthermore, protein quantification (mg / mL) was performed by the BCA method, luciferase activity per unit polypeptide amount (RLU / mg protein) was calculated, and luciferase expression levels were compared. The results are shown in FIG.
- CL cardiolipin
- T-MEND four-membrane MEND
- 2 mg / mL STR-R8 was further added to SUV2 Cationic T-MEND having KALA and R8 in both inner and outer membranes was prepared by adding 10 mol% of the total lipid amount and incubating at room temperature for about 30 minutes.
- Example 3 (a) Evaluation of antigen presentation amount Plasmid DNA encoding egg white antigen (Ovalbumin), an antigen, was introduced into dendritic cells using various vectors, and the dendritic cells were collected 24 hours after transfection, in later washing 96 well plate 1 ⁇ 10 4 cells / 100 ⁇ L of dendritic cells and 2 ⁇ 10 5 cells / 100 ⁇ L of B3Z the (MHC-I) cells were seeded and cultured for 16 hours both. After washing the cells with PBS, CPRG buffer (5 mM CPRG (Roche Diagnotics), 0.125% NP-40 (Igepal CA-630, SIGMA), 9 mM MgCl 2 ) 100 ⁇ L) was added and incubated at 37 ° C. for 4 hours. Thereafter, the absorbance at 595 nm was measured using a Benchmark Plus microplate spectrophotometer (Bio-Rad). The absorbance of only untreated dendritic cells was normalized as 1. The results are shown in Figure 3.
- the lipid membrane structure of the present invention can introduce a gene into the nucleus of a dendritic cell very efficiently, and can present an antigen protein expressed from the introduced gene on the surface of the dendritic cell. Indicated.
- Example 4 Gene expression analysis of immune-related genes using microarray analysis
- a pDNA solution and a Protamine solution were each diluted to 0.1 mg / mL with 10 mM HEPES buffer.
- STR-R8 After adding the compaction body solution to the lipid membrane so that the total lipid concentration is 0.55 mM, STR-R8 alone is 5 mol% of the total lipid amount in R8-MEND, STR-KALA in R8 / KALA-MEND, STR-R8 was added to 5 mol% of the total lipid, left to stand for 10 min at room temperature to hydrate, and then sonicated with a sonicator for 1 min.
- RNA preparation Two days before transfection, JAWS-II cells were seeded on a 6-well plate at 4 ⁇ 10 5 cells / well. After washing the cells with PBS (-) 500 ⁇ L, add MEND solution diluted to 2 ⁇ g / mL (equivalent to plasmid DNA concentration) with ⁇ MEM (no serum, no antibiotics) to 1 mL cells, and add 5% at 37 ° C. It was left in a CO 2 incubator. 1, 3, and 6 hours after transfection, the cells were washed with 500 ⁇ L of PBS ( ⁇ ), and TRIzol Reagent (Invitrogen) was added to make a total of 1 mL for each sample.
- PBS PBS
- TRIzol Reagent Invitrogen
- DEPC diethylpyrocarbonate
- RNA labeling Labeled RNA for DNA microarray experiments was prepared using the Quick Amp Labeling Kit, one-color (Agilent) according to the attached protocol. CDNA was synthesized from 500 ng of total RNA by reverse transcription. Next, cRNA labeled with cyanine3 (Cy3) -CTP was prepared from the cDNA by in vitro transcription reaction. In order to remove unreacted Cy3, the labeled cRNA was purified using RNeasy Mini Kit. About the obtained Cy3 labeled cRNA solution, the concentration and the amount of Cy3 uptake were calculated using Nano Drop, and the quality was confirmed.
- Cy3 cyanine3
- the efficiency of Cy3 incorporation (pmol / ⁇ g) of the purified labeled cRNA probe was calculated from the cRNA concentration (ng / ⁇ L) and the amount of Cy3 incorporation (pmol / ⁇ L).
- the calculation method is shown below.
- the cRNA concentration (ng / ⁇ L) was calculated by the following formula.
- cRNA concentration (ng / ⁇ L) OD 260 ⁇ 10 * ⁇ 40 ( ⁇ g / mL) * The optical path length of NanoDrop is 1 mm.
- the amount of Cy3 incorporation (pmol / ⁇ L) was calculated using the following formula.
- Cy3 uptake (pmol / ⁇ L) (OD 550 ⁇ 10 * ⁇ 1000) / (150 mM -1 cm -1 ) * The optical path length of NanoDrop is 1 mm.
- DNA microarray Hybridization of labeled cRNA to mouse oligo DNA microarray (4x44K) was performed using Gene Expression Hybridization Kit according to the attached protocol. Labeled cRNA was randomly cleaved and fragmented, and a hybridization solution was prepared using 1.65 ⁇ g of RNA. Samples were applied to microarray slides, hybridized at 65 ° C. for 17 hours, and then washed with Expression Wash Buffer containing 10% Triton X-102.
- the microarray slide was scanned using an Agilent DNA Microarray Scanner (Agilent Technologies). A 532 nm laser was used to measure Cy3. The created TIFF image was read with Feature Extraction Software, and each spot information (gene profile) was saved in text file format. The acquired expression profile was imported into GeneSpringGX11 and analyzed.
- Example 5 CTL activity of protein-encapsulated KALA-modified liposomes (1) Preparation of antigen-encapsulated R8-modified liposome and KALA-modified liposome A lipid thin film consisting of EPC / Chol / CHEMS (molar ratio 70:20:10) was prepared in a glass test tube, and OVA (5 mg / mL, 10 mM HB ) was added to a lipid concentration of 10 mM and hydrated at room temperature for 15 minutes. After hydration, the mixture was vortexed, transferred to a 15 mL conical tube, and freeze-thawed 5 times.
- EPC / Chol / CHEMS molar ratio 70:20:10
- the particle size of R8-Lip was 189.5 nm, PDI was 0.095, and the ⁇ potential was 47.8 mV.
- the particle size of KALA-Lip was 188.4 nm, PDI was 0.317, and the ⁇ potential was 33.7 mV.
- mice Female, 7-9 weeks old were subcutaneously administered with R8-Lip or KALA-Lip containing 50 ⁇ g of OVA (26G needle) .
- target cells prepared by the following method were administered. The spleen was removed from naive mice that had undergone cervical dislocation, cells were loosened in a petri dish containing 3-5 mL of RPMI1640 medium, collected using a 2.5 mL syringe, and transferred to a 50 mL conical tube through a nylon mesh.
- the supernatant was removed, suspended in 1 mL of ACK Lysing buffer (Lonza, Walkersville, MD), and incubated at room temperature for 5 minutes for hemolysis. After adding 9 mL of RPMI1640 medium, the mixture was centrifuged, further washed with 10 mL of medium, suspended in 20 mL, and transferred to two 50 mL conical tubes through nylon mesh. The cells were counted and centrifuged, and resuspended in the medium (10 7 cells / mL).
- OVA 257-264 peptide (1 mM, final concentration 5 ⁇ M) was added to one of the two, and the cell suspension was incubated at 37 ° C. under 5% CO 2 for 60 minutes. After washing with 10 mL of medium and 10 mL of PBS, 5 ⁇ M CFSE (Molecular probe) (CFSE High ) is used for cells that are pulsed with OVA 257-264 peptide, and 0.5 ⁇ M CFSE (CFES Low ) is used for cells that are not pulsed. The suspension was suspended in PBS containing 3 ⁇ 10 7 cells / mL and incubated at 37 ° C. for 10 minutes.
- the spleen was removed, the spleen was hemolyzed by the above method, washed with 10 mL of RPMI1640 medium and 10 mL of PBS, and suspended in 5 mL of FACS buffer.
- the prepared cell suspension was transferred to a FACS tube through a nylon mesh, and the number of CFSE positive cells was measured with a flow cytometer. 7,500 cells were analyzed for cells stained at a low concentration (CFSE low ).
- CTL activity was calculated by comparing the number of cells in CFSE High and CFSE Low . The error between experiments was corrected by the CFSE High / CFSE Low ratio in naive mice.
- the results are shown in FIG.
- the vertical axis shows CTL activity, and shows the percentage of cells killed among cells targeted by CTL (a group of cells pulsed with OVA 257-264 peptide). Since the target cell (CFSE High ) presents an OVA peptide to MHC class I, the cell killing effect in this evaluation is due to OVA-specific CTL.
- R8-Lip has been shown to be a carrier capable of inducing specific MHC class I presentation and anti-tumor activity in vivo (Nakamura, T. et al. Mol. Ther., 16, pp.1507- 1514, 2008). As shown by the above results, a dramatic improvement in CTL activity was observed by modifying KALA instead of R8 on the liposome surface. Both R8-Lip and KALA-Lip have no adjuvant, but the CTL activity of KALA-Lip is comparable to the CTL activity when R8-Lip is equipped with a CpG sequence-containing oligonucleotide known as an adjuvant. there were. This result supports the result of Example 4 that KALA has a high adjuvant function from the viewpoint of function.
- Example 6 Enhancement of KALA-MEND gene expression activity by vector modification of the introduced plasmid
- a Preparation of various plasmid DNAs
- the following four types of plasmid DNAs (1) Plasmid DNA containing a CpG sequence in the backbone of the plasmid DNA and also having a CpG sequence from the start codon to the stop codon of the luciferase sequence that is a marker gene (PcDNA3.1-Luc (+); 425 total CpG sequences), (2) The plasmid DNA backbone and the luciferase sequence between the start codon and the stop codon do not contain CpG sequences (pCpGfree-Luc (0); CpG sequence total 0), (3) The plasmid DNA backbone contains CpG sequence, and the luciferase sequence start codon to stop codon do not contain CpG sequence (pcDNA3.1-Luc (0); (4) Total of 332 CpG sequences), and (4) The plasmid DNA backbone does
- the expression vector pcDNA3.1 purchased from Invitrogen was used for the construction of the expression plasmid DNA containing the CpG sequence in the backbone (above (1) and (3)).
- an expression vector pCpGfree-mcs purchased from Invivogen was used for the construction of an expression plasmid DNA ((2) and (4) above) consisting of a backbone not containing a CpG sequence.
- plasmid DNA (1) a DNA fragment containing luciferase containing a CpG sequence was obtained by cleaving Promega's pGL3-basic vector restriction enzyme Hind III / Xba I. This fragment was introduced into the HindIII / XbaI site of pcDNA3.1 vector (pcDNA3.1-Luc (+)).
- double-stranded DNA having a complementary end at the restriction enzyme cleavage site after treating the multicloning site of pCpGfree-mcs (BglII-KpnI-EcoO109I-NcoI-NheI) with BglII / NheI
- BglII-PvuII-NcoI-XbaI-NheI was created (pCpGfree-NEWmcs) (FIG. 6).
- the plasmid DNA prepared by this method has only one CpG sequence after the 12th position counted from the codon of the luciferase gene (FIG. 7).
- the remaining CpG sequence is excised from pCpGfree-Luc (+1) by DraIII / NheI treatment to cut out the sequence part containing the stop codon and CpG sequence, has a terminal complementary to this restriction enzyme cleavage site, and is mutated to the CpG sequence part. It was removed by inserting the oligonucleotide added with. At this time, the EcoRI site existing immediately below the stop codon was replaced with the EcoRV site (pCpGfree-Luc (0)) (FIG. 8).
- plasmid DNA (3) insert the CpG-free luciferase gene excised from BglII / EcoRV from pCpGfree-Luc (0) created in the construction of plasmid DNA (2) into the BamHI / RV site of pcDNA3.1. (PcDNA3.1-Luc (0)).
- luciferase code obtained by PmeI / XbaI treatment from pcDNA3.1-Luc (+) prepared in the construction of plasmid DNA (1) against the PvuII / XbaI cleavage site of pCpGfree-NEWmcs It was created by inserting the gene (pCpGfree-Luc (+)).
- ACK Lysing Buffer 1 mL was added, mixed, and allowed to stand at room temperature for 3 to 5 minutes. After adding 10 mL of the medium, the supernatant was removed by centrifugation, and further washed twice with 10 mL of the medium. Next, the cells were suspended in 10 mL of medium, added to a 10 cm cell culture dish (FALCON), and cultured at 37 ° C. under 5% CO 2 for 4 hours or more. Lightly pipetted to collect only floating cells in a 50 mL conical tube, centrifuged, removed the supernatant, suspended in 10 mL of medium and counted.
- FALCON 10 cm cell culture dish
- GM-CSF final concentration 10 ng / mL
- seed 1 mL each in a 24-well plate (Corning, NY), 37 ° C, 5% Cultivation was performed for 2 days under CO 2 conditions. After 2 days and 4 days, cell clumps were left and floating cells were removed, and then 1 mL of fresh GM-CSF-containing RPMI1640 medium was added. Suspended and weakly adherent cells 6-8 days after the start of culture in the presence of GM-CSF were used as immature dendritic cells in the experiment. According to the evaluation using a CD11c antibody (PE anti-mouse CD11c, Clone: N418, BioLegend), the purity was 85 to 90%.
- CD11c antibody PE anti-mouse CD11c, Clone: N418, BioLegend
- STR-R8 Stearylated R8
- the floating cell group was collected together with the medium, centrifuged (4 ° C., 500 g, 5 minutes), and collected by removing the supernatant. Add the Reporter Lysis Buffer (1x) to the cells remaining in each well and the previously collected floating cells and mix them together to make a total of 75 ⁇ L, and then pipet at -80 ° C. Frozen. After thawing the sample frozen at ⁇ 80 ° C., the cells were removed using a cell scraper and collected in an Eppendorf tube. The collected cell lysate was centrifuged at 15,000 rpm, 4 ° C. for 2 minutes, and 50 ⁇ L of the supernatant was recovered.
- Luciferase activity measurement (RLU / mL) was measured using the obtained supernatant. Furthermore, protein quantification (mg / mL) by BCA method was performed, and luciferase activity (RLU / mg protein) per unit protein amount was calculated.
- FIG. The left figure shows the gene expression activity of R8-MEND and KALA-MEND in BMDC when (1) pcDNA3.1-Luc (+) and (4) pCpGfree-Luc (0) prepared in (a) is used.
- Both R8-MEND and KALA-MEND showed a high gene expression activity promoting effect when pCpGfree-Luc (0) was used.
- the gene expression level (10 7) that enables antigen presentation based on the results of Examples 2 and 3 using JAWS II. RUL / mg protein level) was achieved.
- Example 7 Cancer prevention vaccine effect (a) Preparation of CpG-free OVA expression plasmid DNA An antigen (OVA) gene sequence excluding all CpG sequences was designed (1161 bp DNA sequence shown in SEQ ID NO: 2 in the sequence listing), and custom synthesis was performed (TAKARA). This sequence was subcloned into the NcoI / NheI site among the multiple cloning sites of pCpGfree-NEWmcs (pCpGfree-OVA (0)).
- OVA antigen gene sequence excluding all CpG sequences was designed (1161 bp DNA sequence shown in SEQ ID NO: 2 in the sequence listing), and custom synthesis was performed (TAKARA). This sequence was subcloned into the NcoI / NheI site among the multiple cloning sites of pCpGfree-NEWmcs (pCpGfree-OVA (0)).
- RPMI 1640 (with serum and antibiotics) 500 ⁇ L was added to each well, and after 21 hours, cells were collected. The floating cells and the adherent cells peeled off by pipetting in PBS were collected and collected by centrifugation (4 ° C., 500 g, 5 minutes).
- Tumor volume tumor volume (mm 3 ) was calculated by the formula of major axis ⁇ minor axis ⁇ minor axis ⁇ 0.52.
- Example 8 Tumor growth inhibitory effect (a) Gene transfer of antigen (OVA) -expressing plasmid DNA into BMDC Dendritic cells isolated and differentiated by the method described in Example 6 (b) so that they become 4 ⁇ 10 5 cells / well in a 24-well plate Sowing.
- KALA-MEND prepared using CpG-free OVA expression plasmid DNA (pCpGfree-OVA (0)) was prepared according to the method described in Example 6 (c). MEND was diluted with RPMI 1640 (no serum, no antibiotics) so that the amount of pDNA was 0.8 ⁇ g / well, and prepared to 500 ⁇ L.
- a MEND solution was applied to each well and incubated in a 37 ° C., 5% CO 2 environment. After 3 hours, 500 ⁇ L of RPMI 1640 (with serum and antibiotics) was added to each well, and the cells were collected after 21 hours. The floating cells and the adherent cells peeled off by pipetting in PBS were collected and collected by centrifugation (4 ° C., 500 g, 5 minutes).
- KALA-MEND can provide an antitumor effect even if administered after the tumor has formed.
- the lipid membrane structure provided by the present invention can efficiently migrate into the nucleus of any cell such as immune cells including dendritic cells, and efficiently transfer substances such as nucleic acids encapsulated in the nucleus. Since it can be released to express the polypeptide encoded by the nucleic acid, and it can also exert an efficient adjuvant effect, effective immunotherapy can be performed on the desired polypeptide. There is a feature.
Abstract
Description
(a)配列番号1に記載のアミノ酸配列からなるポリペプチド;
(b)配列番号1に記載のアミノ酸配列において1個又は数個のアミノ酸が欠失及び/又は置換及び/又は挿入されたアミノ酸配列からなるポリペプチドであって、細胞の核内への脂質膜構造体の移行を促進する活性を有するポリペプチド
で修飾された脂質膜構造体が提供される。
(a)配列番号1に記載のアミノ酸配列からなるポリペプチド;
(b)配列番号1に記載のアミノ酸配列において1個又は数個のアミノ酸が欠失及び/又は置換及び/又は挿入されたアミノ酸配列からなるポリペプチド
が提供される。
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、ホスファチジン酸など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、リノール酸など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、大豆レシチンなどの天然物由来のリン脂質を用いることもできる。
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、ミリスチン酸などの炭素数12~20の飽和又は不飽和の脂肪酸が挙げられる。
例1
(a)封入する核酸の調製(プロタミンによる凝縮体)
ルシフェラーゼをコードしているプラスミド(pDNA、BD Biosciences Clontech社製)を10 mM HEPES緩衝液に0.1 mg/mLとなる様に溶解した。0.1 mg/mL(10 mM HEPES緩衝液)の プロタミン(Protamine)溶液150μLにpDNA溶液を100μL少量ずつ攪拌しながら滴下して、プロタミンとpDNAのコンパクション体を調製した(N/P比は2.2)。
1 mM DOPE 112.5μL及び1 mM CHEMS 25μLをDOPE:CHEMS=9:2となるようにガラス試験管に加えた。さらに、Biochemistry, 43, pp.5618-5628, 2004に記載の方法に従って合成された1mg/mL Chol-GALA、及び特開2003-343857号公報に記載された方法に準じて合成した1mg/mLステアリル化KALA(STR-KALA)を総脂質濃度の1~8%分の修飾量となるように加え、さらに全量が200μLとなるようにクロロホルムを加えた後、デシケーターで減圧乾燥して溶媒を留去させて脂質膜を得た。この脂質膜に、総脂質濃度が0.55 mMとなるように上記(a)のコンパクション体を添加し、室温で10分間静置して水和させた後、ソニケーターで1分間超音波処理した。特開2003-343857号公報に記載の方法で合成したステアリル化オクタアルギニン(STR-R8)(2 mg/mL水溶液を総脂質の 5 mol%分添加し、室温で10 分間静置した。
ルシフェラーゼをコードしているプラスミド(pDNA、BD Biosciences Clontech社製)を10 mM HEPES緩衝液に0.1 mg/mLとなる様に溶解した。J. Control. Release, 131, pp.137-144, 2008に記載の方法に従って合成されたポリロタキサン(平均ポリエチレングリコール鎖長4,000、シクロデキストリン貫通数29、1鎖内平均カチオン数46)を10 mM HEPES緩衝液により溶解し(1 mM アミン濃度換算)、得られた溶液150μLにpDNA溶液を100μL少量ずつ攪拌しながら滴下して、ポリロタキサンとpDNAのコンパクション体を調製した(N/P比は5.0)。
1 mM DOPE 107 μL及び1 mM ホスファチジン酸(PA) 30.5μLをDOPE:PA=7:2となるようにガラス試験管に加えた。特開2003-343857号公報に記載された方法に準じて合成した1mg/mLのSTR-KALAを総脂質濃度の1.5~5%分の修飾量(8.25μL~27.5μL)となるように加え、さらに全量が200μLとなるようにクロロホルムを加えた後、デシケーターで減圧乾燥して溶媒を留去させて脂質膜を得た。この脂質膜に、総脂質濃度が0.55 mMとなるように上記(c)のコンパクション体を添加し、室温で10 min静置し水和させた後、ソニケーターで1分間超音波処理した。特開2003-343857号公報に記載の方法で合成したステアリル化オクタアルギニン(STR-R8)2 mg/mL水溶液を総脂質の 10 mol%となるように添加し、室温で10 分間静置し水和させた。
JAWS II細胞を8×104cells/ウェルとなるように24 ウェルプレートに播種し、αMEM培地で24 時間培養した。培養後の細胞をPBS 500 μLで洗浄した後、上記(b)及び(d)で作製したリポソームとコントロールリポソーム(ステアリル化KALA及びChol-GALA未修飾)をpDNA量が0.4μg/wellとなる様にαMEM に添加した後、各ウェルに加えて37 ℃、5% CO2で3時間インキュベーションした。インキュベーション後に細胞をPBS 500 μLで洗浄した後、αMEM 1 mLを各ウェルに添加し、37℃、5% CO2の条件下でさらに21時間インキュベーションして形質転換細胞を作製した。
上記(e)の形質転換細胞をPBS 500μLで洗浄し、そのPBSを1.5 mLサンプルチューブに回収し、遠心(4℃、2,000 rpm, 2分間)して上清を除去した。各ウェルにReporter Lysis Buffer(Promega社)75μLを添加し、-80℃のリーザーに移して凍結した。凍結させたサンプルを解凍させた後、セルスクレーパーを用いて細胞をはがし、1.5 mLサンプルチューブに回収した。回収した細胞溶解液を遠心(4℃、15,000 rpm、5分間)して遠心分離し、その上清45μLを回収した。得られた上清を用いてルシフェラーゼ活性測定(RLU/mL)を測定した。さらにBCA法によるタンパク定量(mg/mL)を行い、単位ポリペプチド量当たりのルシフェラーゼ活性(RLU/ mg protein)を算出し、ルシフェラーゼ発現量を比較した。結果を図1に示す。
(a)正電荷コアT-MENDの調製
3.3 mM カルジオリピン(CL) 84 μL及び10 mM DOPE 27.5 μLをCL:DOPE=1:1となるようにガラス試験管に加えた。1 mg/mLのSTR-KALAを総脂質濃度の0.5%、1%、2%、及び5%の修飾量となるようにそれぞれ加え、さらに全量が200μLとなるようにクロロホルムを加えた後、デシケーターで減圧乾燥して溶媒を留去させて脂質膜を得た。この脂質膜に総脂質濃度が0.55 mMとなるように10 mM HEPES溶液を1mL加え、水和した脂質膜をバスタイプソニケーターを用いてガラス試験管から剥離させた後、プローブ型ソニケーターを用いて10分間超音波処理した。超音波処理後、遠心分離(15,000 rpm、20℃、5分間)して上清を回収する操作を3回行い、STR-KALA濃度の異なる4種類のリポソーム(SUV1-1~5)を回収した。
ルシフェラーゼをコードしているプラスミド(pDNA、BD Biosciences Clontech社製)を10 mM HEPES緩衝液に0.1 mg/mLとなるように溶解した。0.1 mg/mL pDNA 60μLにアミン濃度換算で1 mMのポリロタキサン溶液(平均ポリエチレングリコール鎖長4,000、シクロデキストリン貫通数29、1鎖内平均カチオン数46) 90μLを少量ずつ攪拌しながら滴下して、ポリロタキサンとpDNAのコンパクション体を調製した(N/P比は0.5)。
例1(e)に記載の方法に従って、上記(a)及び(b)でそれぞれ調製した2種のT-MENDを用いて形質転換細胞を作製し、さらに例1(f)に記載の方法に従って、各細胞におけるルシフェラーゼの発現量を測定した。その結果、カチオン性T-MENDにおいて、KALAの修飾密度依存的に封入された遺伝子発現の上昇が認められた(図2)。また、アニオン性T-MENDに対しても、内膜と外膜をKALAで修飾することで遺伝子発現が10倍以上に上昇した。特にアニオン性T-MENDは市販の遺伝子導入試薬であるLipofectamine PLUSよりも高い遺伝子発現を示した。
(a)抗原提示量の評価
種々のベクターを用いて、抗原である卵白抗原(Ovalbumin)をコードするプラスミドDNAを樹状細胞に導入し、トランスフェクションから24時間後に樹状細胞を回収し、培地で洗浄後96 well plateに1×104 cells/100μLの樹状細胞と2×105 cells/100μLのB3Z(MHC-I)細胞を播種し、16時間共培養した。その後PBSで細胞を洗い、CPRG buffer(5 mM CPRG(Roche Diagnotics), 0.125% NP-40(Igepal CA-630, SIGMA), 9 mM MgCl2となるように二回蒸留水に溶解し、1 mLずつ分注して-20℃で遮光保存したもの)100μLを添加して37℃で4時間インキュベートした。その後、Benchmark Plus microplate spectrophotometer(Bio-Rad)を用いて595 nmの吸光度を測定した。未処理の樹状細胞のみの吸光度を1として規格化した。結果を図3に示す。
(a) R8-MEND及びR8/KALA-MENDの調製
ベクターのコアとして、pDNA溶液、Protamine溶液をそれぞれ、10 mM HEPES bufferで0.1 mg/mLに希釈した。0.1 mg/mL Protamine 150 μLに対し、0.1 mg/mL pDNA 100 μLを攪拌しながら少量ずつ滴下してProtamineとpDNAのコンパクション体を調製した(N/P比=2.2)。脂質膜は、DOPE:CHEMS=9:2となるように、1 mM DOPE 112.5 μL、1 mM CHEMS 25 μLをガラス試験管に加え、全量が200 μLとなるようにCHCl3を加えた後、デシケーターで減圧乾燥し、溶媒を留去して脂質膜を得た。脂質膜に総脂質濃度が0.55 mMとなるようにコンパクション体溶液を添加した後、R8-MENDにおいてはSTR-R8単独を総脂質量の5mol%、、R8/KALA-MENDにおいてはSTR-KALA、STR-R8を総脂質の5 mol%となるようにそれぞれ添加し、室温で10 min静置し水和させた後、ソニケーターで1 min超音波処理した。
トランスフェクション 2日前にJAWS-II細胞を4×105 cells/wellとなるように6 well plateに播種した。細胞をPBS(-)500 μLでwashした後、αMEM(血清なし、抗生物質なし)によって2 μg/mL(プラスミドDNA濃度相当)に希釈したMEND溶液を1 mL細胞に加え、37℃で5% CO2インキュベーター内に静置した。トランスフェクションしてから1、3、及び6時間後に細胞をPBS(-) 500 μLで洗浄し、サンプルごとに合計1 mLとなるようにTRIzol Reagent(invitrogen社)を加えた。Cell scraperで細胞をはがし、エッペンドルフチューブに回収した。1分間 ボルテックスにより混合して-80℃で保存した。mRNAの精製に際し、各サンプルを室温で解凍した後、クロロホルムを200 μL加え、15 秒間 ボルテックスにより混合した。室温で3分間インキュベーションした後、4℃環境下で12,000 rpmで15 min遠心した。上清を他のエッペンドルフチューブに移し、イソプロパノールを500 μL加えた後に室温で10 minインキュベーションした。4℃環境下で12,000 rpm、10 min遠心した後、上清を除き、pelletに75%エタノールを1 mLを加えてボルテックスにより混合し、遠心した(4℃、12,000 rpm、4分間)。上清を除去し、ジエチルピロカーボネート(DEPC)処理水を25 μL加え、10 min インキュベーションし、Nano Drop(Thermo Scientific社)を用い、260 nmにおける吸光度よりRNA量を定量した。
DNAマイクロアレイ実験用のラベル化RNAをQuick Amp Labeling Kit、one-color(Agilent)を用いて添付プロトコルに従い調製した。Total RNA 500 ngから逆転写反応によりcDNAを合成した。次に、cDNAからin vitro転写反応によってCyanine3(Cy3)-CTPでラベルしたcRNAを調製した。未反応のCy3を除去するために、ラベル化cRNAをRNeasy Mini Kitを用いて精製した。得られたCy3ラベル化cRNA溶液について、Nano Dropを用いて濃度とCy3取り込み量を算出し、品質を確認した。
(1)以下の式で、cRNA濃度(ng/μL)を算出した。
cRNA濃度(ng/μL)=OD260×10※×40(μg/mL)
※ NanoDropの光路長は1 mm
(2)以下の式で、Cy3の取り込み量(pmol/μL)を算出した。
Cy3取り込み量(pmol/μL)=(OD550×10※×1000)/(150 mM-1cm-1)
※ NanoDropの光路長は1 mm
(3)以下の式で、Cy3の取り込み効率(pmol/μg)を算出した。
Cy3取り込み効率(pmol/μg)= Cy3取り込み量(pmol/μL)/ cRNA濃度(ng/μL)
全Cy3ラベル化cRNAサンプルに関して、cRNAの収量が1.65 μg以上かつCy3の取り込み効率が9 pmol/μg以上であることを確認した。
マウスオリゴDNAマイクロアレイ(4x44K)へのラベル化cRNAのハイブリダイゼーションは、Gene Expression Hybridization Kitを用いて添付プロトコルに従い実験を行った。ラベル化cRNAをランダムに切断してフラグメント化し、1.65 μgのRNAを使用してハイブリダイゼーション溶液を調製した。マイクロアレイスライドにサンプルをアプライし、65℃、17時間ハイブリダイゼーションを行った後、10% Triton X-102を含むExpression Wash Bufferでスライドを洗浄した。
(1)コントロールスポットの除去
解析ソフトGeneSpring GX11にデータを取り込むと、アレイの四隅を認識させるスポットなどを含むコントロールスポットまで検出するため、これらのスポットの除去を行った。
(2)フラグの除去
スポットの検出時に、形が乱れている、蛍光が飽和している、などシグナルの状況に応じてPresent(P)、Marginal(M)、Absent(A)の3段階のフラグが評価される。(P、M、Aの順に信頼度が低下する)。全サンプル中少なくとも1サンプルでPresentの評価が下された遺伝子を抽出した。
(3)低発現遺伝子の除去
発現レベルの低い遺伝子は、同一サンプル同士の発現レベルもばらつくため、除去を行った。シグナル強度の絶対値であるRaw値が全サンプル中少なくとも1サンプルで、100以上の遺伝子を抽出した。以上のQuality controlを行った後、21995遺伝子を対象として解析を進めた。
(1)抗原封入R8修飾リポソーム、KALA修飾リポソームの調整
ガラス試験管にEPC/Chol/CHEMS(モル比70:20:10)からなる脂質薄膜を調製し、OVA(5 mg/mL, 10 mM HB)を脂質濃度が10 mMとなるように添加後、室温で15分間水和した。水和後、ボルテックスにより混合し15 mLコニカルチューブに移し、凍結融解を5回行った。400 nmのメンブレンフィルターでエクストリュージョンし、超遠心(43,000 rpm, 4℃, 30 min)して上清を除去後、ペレットをHBG 200 μLで静かにすすぎ、適量のHBGに再懸濁した。下記の項にある手順に従い、OVA濃度及び脂質濃度を定量した。実験に使用する前にSTR-R8(10 mg/mL)又はSTR-KALA(10 mg/mL)を脂質モル濃度の7.5%添加し、室温で30分間以上インキュベートすることで、R8-Lip及びKALA-Lipを調製した。R8-Lipの粒子径は189.5 nm、PDIは0.095、ζ電位は47.8 mVであり、KALA-Lipの粒子径は188.4 nm、PDIは0.317、ζ電位は33.7 mVであった。
C57BL/6マウス(雌、7-9週齢)に50 μgのOVAを内封したR8-LipもしくはKALA-Lipを皮下投与した(26G針)。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。頚椎脱臼したnaiveマウスより脾臓を摘出し、RPMI1640培地3~5 mLの入ったシャーレ中で細胞をほぐし2.5 mLシリンジを用いて回収後、ナイロンメッシュを通して50 mLコニカルチューブに移した。遠心(1600~1700 rpm、4℃、5分)後、上清を除去し、ACK Lysing buffer(Lonza, Walkersville, MD) 1 mLに懸濁して室温で5 分間インキュベートし溶血した。RPMI1640培地9 mLを添加後遠心し、さらに培地 10 mLで洗浄後、20 mLに懸濁しナイロンメッシュを通して50 mLコニカルチューブ2本に移した。細胞を計数後遠心し、培地に再懸濁した(107 cells/mL)。2本のうち1本にOVA257-264ペプチド(1 mM、終濃度 5 μM)を添加し、この細胞懸濁液を37℃、5% CO2条件下で60分間インキュベートした。培地10 mL及びPBS 10 mLで洗浄後、OVA257-264ペプチドによってパルスした細胞群は5 μM CFSE(Molecular probe) (CFSEHigh) により、パルスしていない細胞群は0.5 μM CFSE (CFESLow)を含むPBSに3×107 cells/mLとなるよう懸濁し、37℃で10分間インキュベーションした。RPMI1640培地10 mLで2回、PBS 10 mLで2回洗浄後、最終的にPBSに懸濁した(5×107 cells/mL)。投与直前に異なる濃度で染色した2種の細胞を等量混合し、免疫したマウスに尾静脈より投与した(1×107 cells/200 μL/mouse、26G針)。
(a)各種プラスミドDNAの調製
以下の4種類のプラスミドDNA:(1)プラスミドDNAのバックボーンにCpG配列を含み、かつマーカー遺伝子であるルシフェラーゼ配列の開始コドンからストップコドンにもCpG配列を有するプラスミドDNA(pcDNA3.1-Luc(+);CpG配列合計425個)、(2)プラスミドDNAのバックボーン及びルシフェラーゼ配列の開始コドンからストップコドンまでの間のいずれにもCpG配列を含まないもの(pCpGfree-Luc(0); CpG配列合計0個)、(3)プラスミドDNAのバックボーンにCpG配列を含み、ルシフェラーゼ配列の開始コドンからストップコドンにはCpG配列を含まないもの(pcDNA3.1-Luc (0);CpG配列合計332個)、及び(4)プラスミドDNAのバックボーンにはCpG配列を含まず、ルシフェラーゼ配列の開始コドンからストップコドンにはCpG配列を含むもの(pCpGfree-Luc(+);CpG配列合計98個)を作成した。
頚椎脱臼したC57BL/6マウス(6~8週齢)より大腿骨および頸骨を摘出し、70%エタノールで軽く消毒した後、PBSに浸した。骨の両端を切断し、培地入りの1 mLシリンジ(26G針)で骨髄細胞をRPMI1640培地中に押しだした。細胞懸濁液を40 μmのセルストレイナー(FALCON)を通して50 mLコニカルチューブに移した。遠心(450 g、4℃、5分間)後上清を除去し、タッピングして細胞を分散させた後、ACK Lysing Buffer1 mLを添加、混合し、室温で3~5分間静置した。培地10 mLを添加後、遠心して上清を除去し、さらに培地10 mLで2回洗浄した。次に、細胞を培地10 mLに懸濁し、10 cm細胞培養ディッシュ(FALCON)に添加し、37℃、5% CO2条件下で4時間以上培養した。軽くピペッティングして浮遊細胞のみを50 mLコニカルチューブに回収し、遠心、上清除去後、培地10 mLに懸濁して計数した。1×106 cells/mLとなるよう培地に懸濁し、GM-CSF(終濃度10 ng/mL)を添加後、24 well plate(Corning, NY)に1 mLずつ播種し、37℃、5% CO2条件下で2日間培養した。2日後および4日後に細胞の凝集塊を残し浮遊細胞を除去した後、新しいGM-CSF含有RPMI1640培地1 mLを添加した。GM-CSF存在下で培養開始後6~8日目の浮遊及び弱付着細胞を未成熟樹状細胞として実験に用いた。CD11c抗体(PE anti-mouse CD11c, Clone: N418, BioLegend)を用いた評価では純度は85~90%であった。
上記(a)で得られた各種プラスミドDNAを10 mM HEPES緩衝液に0.1 mg/mLとなる様に溶解した。0.1 mg/mL(10 mM HEPES緩衝液)の プロタミン(Protamine)溶液150 μLにpDNA溶液を100μL少量ずつ攪拌しながら滴下し、プロタミンとpDNAのコンパクション体を調製した(N/P比は2.2)。
上記(b)に示す方法により誘導したBMDCを24wellプレートに4×105 cells/wellとなるように播種した。調製したMENDをpDNA量が0.4 μg/well含むようにRPMI 1640(血清なし、抗生物質なし)で希釈し、500 μLとなるように調製した。各wellにMEND溶液をアプライし、37℃、5% CO2環境下でインキュベーションした。3時間後にRPMI 1640(血清、抗生物質有り) 500μLを各wellに添加し、さらに21時間後にルシフェラーゼ活性を測定した。ルシフェラーゼ活性の測定に関して、浮遊している細胞群はメディウムごと回収し、遠心(4℃、500 g、5分間)し、上清を除去することで集めた。各wellに残っている細胞、及び先に回収した浮遊細胞群にReporter Lysis Buffer(1×)を添加して両者を混合した後、合計75 μLにした後、ピペッティングを行い、-80℃で凍結した。-80℃で凍結させたサンプルを解凍させた後、セルスクレーパーを用いて細胞をはがし、エッペンドルフチューブに回収した。回収した細胞溶解液を15,000 rpm、4℃、2分間遠心分離し、その上清50 μLを回収した。得られた上清を用いてルシフェラーゼ活性測定(RLU/mL)を測定した。さらにBCA法によるタンパク定量(mg/mL)行い、単位タンパク質量当たりのルシフェラーゼ活性(RLU/ mg protein)を算出した。
(a)CpGフリーOVA発現プラスミドDNAの調製
CpG配列をすべて除いた抗原(OVA)遺伝子配列を設計し(配列表の配列番号2に示す1161bpのDNA配列)、カスタム合成を行った(TAKARA)。本配列を、pCpGfree-NEWmcsのマルチクローニングサイトのうち、NcoI/NheIサイトにサブクローニングした(pCpGfree-OVA(0))。
例6(b)に記載した方法により単離・分化された樹状細胞を24wellプレートに4×105 cells/wellとなるように播種した。CpGフリーのOVA発現プラスミドDNA(pCpGfree-OVA(0))を用いて調製されたKALA-MENDを例6(c)に記載した方法に従って調製した。MENDをpDNA量が0.4 μg/well含むようにRPMI 1640(血清なし、抗生物質なし)で希釈し、500 μLとなるように調製した。各wellにMEND溶液をアプライし、37℃、5% CO2環境下でインキュベーションした。3時間後にRPMI 1640(血清、抗生物質有り) 500μLを各wellに添加し、さらに21 時間後に細胞を回収した。浮遊している細胞及びPBS中でピペッティングすることによってはがした付着細胞をまとめ、遠心(4℃、500 g、5分間)することで回収した。
抗原(OVA)発現腫瘍細胞(E.G7-OVA細胞)を移植する14日前、7日前に、C57BL/6マウス(雌、8週齢)に(1)DCのアジュバントであるCpGのみを作用させたBMDC、(2)KALA-MENDを用いて非抗原タンパクであるルシフェラーゼを発現させたBMDC、(3)KALA-MENDを用いて抗原タンパク(OVA)を発現させたBMDC、(4)KALA-MENDを用いて抗原タンパク(OVA)を発現させ、かつCpGアジュバントにより活性化したBMDCを4×105 cells/40μL PBSになるように調製し、全量をマウスの足の裏に投与した。2回目の免疫から1週間後に8×105個のE.G7-OVA細胞を左脇腹に移植し、経時的に腫瘍体積を測定した。腫瘍体積(tumor volume (mm3))は長径×短径×短径×0.52の式により算出した。
(a)BMDCへの抗原(OVA)発現プラスミドDNAの遺伝子導入
例6(b)に記載した方法により単離・分化された樹状細胞を24wellプレートに4×105 cells/wellとなるように播種した。CpGフリーのOVA発現プラスミドDNA(pCpGfree-OVA(0))を用いて調製されたKALA-MENDを例6(c)に記載した方法に従って調製した。MENDをpDNA量が0.8 μg/well含むようにRPMI 1640(血清なし、抗生物質なし)で希釈し、500 μLとなるように調製した。各wellにMEND溶液をアプライし、37℃、5% CO2環境下でインキュベーションした。3 時間後にRPMI 1640(血清、抗生物質有り) 500μLを各wellに添加し、さらに21時間後に細胞を回収した。浮遊している細胞及びPBS中でピペッティングすることによってはがした付着細胞をまとめ、遠心(4℃、500 g、5分間)することで回収した。
8×105個の抗原(OVA)発現腫瘍細胞(E.G7-OVA細胞)をC57BL/6マウス(雌、8週齢)の左脇腹に移植して15日目及び22日目に、(1)未処理のBMDC、(2)KALA-MENDを用いて非抗原タンパクであるルシフェラーゼを発現させたBMDC、(3)KALA-MENDを用いて抗原タンパク(OVA)を発現させたBMDCを40μL PBS中に4x 105個のBMDCが入るように懸濁し、足の裏に投与した。経時的に腫瘍体積を測定し、腫瘍体積(tumor volume (mm3))は長径×短径×短径×0.52の式により算出した。
Claims (15)
- 細胞の核内に物質を送達するための脂質膜構造体であって、脂質膜が下記の(a)及び/又は(b)のポリペプチド:
(a)配列番号1に記載のアミノ酸配列からなるポリペプチド;
(b)配列番号1に記載のアミノ酸配列において1個又は数個のアミノ酸が欠失及び/又は置換及び/又は挿入されたアミノ酸配列からなるポリペプチドであって、細胞の核内への脂質膜構造体の移行を促進する活性を有するポリペプチド
で修飾された脂質膜構造体。 - 脂質膜構造体がリポソームである請求項1に記載の脂質膜構造体。
- 細胞が免疫細胞である請求項1又は2に記載の脂質膜構造体。
- 上記(a)及び/又は(b)の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た請求項1ないし3のいずれか1項に記載の脂質膜構造体。
- 連続した複数個のアルギニン残基を含むポリペプチドを表面に有する請求項1ないし4のいずれか1項に記載の脂質膜構造体。
- ポリアルキレングリコールを表面に有する請求項1ないし5のいずれか1項に記載の脂質膜構造体。
- 免疫細胞表面に提示すべき抗原ポリペプチドをコードする核酸を該細胞の核内に導入するために用いる請求項1ないし6のいずれか1項に記載の脂質膜構造体。
- 送達すべき物質が内部に封入された請求項1ないし7のいずれか1項に記載の脂質膜構造体。
- 内部に核酸及びカチオン性ポリマーが封入された請求項8に記載の脂質膜構造体。
- 送達すべき物質が免疫細胞表面に提示すべき抗原ポリペプチドをコードする核酸である請求項8又は9に記載の脂質膜構造体。
- 上記抗原ポリペプチドに対する免疫療法に用いるための請求項10に記載の脂質膜構造体。
- 該核酸がDNAであり、該DNAがCpGを含まないDNAである請求項10又は11に記載の脂質膜構造体。
- 請求項8ないし12のいずれか1項に記載の脂質膜構造体を有効成分として含む医薬組成物。
- 悪性腫瘍の予防及び/又は治療のために用いる請求項13に記載の医薬組成物。
- 細胞の核内への脂質膜構造体の移行を促進するために用いるポリペプチドであって、下記の(a)及び/又は(b)のポリペプチド:
(a)配列番号1に記載のアミノ酸配列からなるポリペプチド;
(b)配列番号1に記載のアミノ酸配列において1個又は数個のアミノ酸が欠失及び/又は置換及び/又は挿入されたアミノ酸配列からなるポリペプチド。
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Also Published As
Publication number | Publication date |
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EP2572706A4 (en) | 2014-02-26 |
JPWO2011132713A1 (ja) | 2013-07-18 |
CN103037840A (zh) | 2013-04-10 |
US20150202154A1 (en) | 2015-07-23 |
US20130122054A1 (en) | 2013-05-16 |
JP5794541B2 (ja) | 2015-10-14 |
EP2572706A1 (en) | 2013-03-27 |
US8981044B2 (en) | 2015-03-17 |
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