WO2016167367A1 - Targeting amphiphilic nanocarrier and method for producing same - Google Patents

Targeting amphiphilic nanocarrier and method for producing same Download PDF

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WO2016167367A1
WO2016167367A1 PCT/JP2016/062184 JP2016062184W WO2016167367A1 WO 2016167367 A1 WO2016167367 A1 WO 2016167367A1 JP 2016062184 W JP2016062184 W JP 2016062184W WO 2016167367 A1 WO2016167367 A1 WO 2016167367A1
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amphiphilic
sctab
nanocarrier
membrane
liposomes
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PCT/JP2016/062184
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French (fr)
Japanese (ja)
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一成 秋吉
満 安藤
理紗子 三浦
晋一 澤田
洋 珠玖
泰 赤堀
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国立大学法人京都大学
国立大学法人三重大学
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Priority to JP2017512607A priority Critical patent/JP6763545B2/en
Publication of WO2016167367A1 publication Critical patent/WO2016167367A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a targeted amphiphilic nanocarrier and a method for producing the same.
  • Protein and peptide modifications to these drug carriers include [1] construction of genes that express the targeted polypeptide, [2] process of purifying ligands from genetically modified organisms that express these genes of interest, [3] purification It requires a multi-step preparation process, such as the process of chemically binding a substance with a hydrophobic substance, etc., and the process of [4] mixing a hydrophobically targeted polypeptide with a liposome. Since it is complicated, it has a problem that the yield of the target-oriented drug carrier is low.
  • Patent Documents 1 to 5 relate to a preparation method in which a targeting polypeptide and a hydrophobic compound are modified to a drug carrier via a linker
  • Patent Document 6 is a biotin-modified targeting polypeptide modified with avidin.
  • Targeted drug carriers are prepared by utilizing the specific binding affinity of avidin and biotin for lipids.
  • Patent Documents 7 to 10 disclose methods of using immunoliposomes, which are target-directed drug carriers, as contrast agents and therapeutic agents.
  • An object of the present invention is to provide a production method capable of easily obtaining an amphiphilic nanocarrier capable of binding to a target.
  • Another object of the present invention is to provide an amphiphilic nanocarrier that can bind to a target.
  • the present inventor has designed a fusion protein in which a membrane-binding domain is fused to a targeting polypeptide, and uses a cell-free protein expression system, so that spontaneous expression of the targeting polypeptide and spontaneous chaperone-like activity of the lipid occur.
  • a method for preparing a target-directed drug carrier is provided in one step.
  • the membrane protein is incorporated into the lipid membrane by the chaperone-like activity of the lipid simultaneously with the expression of the membrane protein.
  • the targeting polypeptide can be incorporated into a lipid carrier in a single step.
  • the present invention relates to the following amphiphilic nanocarrier and a method for producing the same.
  • Item 1. Targeted amphiphilic nanocarriers that bind a fusion protein of a targeting polypeptide and a membrane-binding domain.
  • Item 2. Item 2. The targeted amphiphilic nanocarrier according to Item 1, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody, or an antigen-binding fragment thereof.
  • Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR Item 2.
  • the targeted amphiphilic nanocarrier according to Item 1 which is an antigen-binding fragment selected from the group consisting of: Item 4.
  • Item 4. The targeted amphiphilic nanocarrier according to Item 3, wherein the targeted polypeptide is a single-chain antibody fragment (scFv).
  • Item 5. The targeted amphiphilic nanocarrier according to any one of Items 1 to 4, wherein the amphiphilic nanocarrier is a liposome.
  • Item 6. Item 6.
  • Item 7 In the presence of an amphiphilic nanocarrier, a fusion protein of a targeting polypeptide and a membrane-binding domain is synthesized by a cell-free protein synthesis system, and the targeting amphipathic nanoparticle to which the fusion protein is bound, Carrier manufacturing method.
  • Item 8. Item 8. The method for producing a targeted amphiphilic nanocarrier according to Item 7, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody or an antigen-binding fragment thereof.
  • Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR Item 8.
  • Item 10 The method for producing a targeted amphiphilic nanocarrier according to Item 9, wherein the targeted polypeptide is a single-chain antibody fragment (scFv).
  • Item 11 Item 11.
  • the preparation operation is a single step, the time required for the preparation is greatly reduced, and the yield of the target-oriented drug carrier is high.
  • fusion proteins can be designed in the same way regardless of hydrophobic or hydrophilic targeting polypeptides, the range of applications is wide and it is easy to impart multiple types of target directivity.
  • genetic information is already known, it can be developed as a tailor-made medicine since a target-oriented drug carrier can be prepared in a short time.
  • A Fusion protein (antiEGFR scTab) expression vector (pDNA),
  • B AntiEGFR scTab-incorporated liposome preparation and purification method conceptual diagram (A) Western blot analysis of each fraction after fusion protein synthesis in a cell-free protein synthesis system in the presence of liposomes, W is before purification.
  • AntiEGFR scTab expression vector pDNA
  • pDNA AntiEGFR scTab expression vector
  • Western blot analysis of each fraction after expression of various scTabs in a cell-free protein synthesis system
  • Binding affinity of various scTab-presenting liposomes for EGFR Flow cytometric analysis after incubation of various scTab presenting liposomes with HeLa cells
  • Plasma profile after scTab-presenting liposomes administered into the tail vein of mice Disappearance from tumor after intratumoral administration of scTab-presented liposomes in solid tumor model mice
  • the targeted amphiphilic nanocarrier is composed of a fusion protein of a targeting polypeptide and a membrane-binding domain and an amphiphilic nanocarrier.
  • the target of the targeting polypeptide includes cells, particularly cells related to diseases such as cancer cells.
  • the targeting polypeptide examples include peptide hormones, receptor ligands, antibodies or antigen-binding fragments thereof, and antibodies and antigen-binding fragments thereof are preferably exemplified.
  • the antibody may be any of IgG, IgM, IgA, IgD, IgE, etc., an antibody derived from a mammal such as human, monkey, mouse, rat, goat, rabbit, cow, pig, dog, cat, humanized antibody, etc. Preferred examples include human antibodies and humanized antibodies.
  • an antibody composed of one kind of protein such as a Bactrian camel antibody, a human dromedary antibody, or a llama antibody is also preferable.
  • a membrane-binding domain may be bound to the light chain or heavy chain, preferably the heavy chain.
  • Antibody antigen-binding fragments include Fab, Fab ′, F (ab ′) 2 , single-chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv), CDR ScFv is preferable.
  • Membrane binding domains include CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27, CD28, CD29, CD30, CD40 , CD44, CD45, CDw52, CD56, CD58, CD69, CD72, TNF ⁇ R, RGF ⁇ R, TSHR, VEGFR / VPFR, FGFR, EGFR, PTHrPR, PDGFR, EPO-R, GCSF-R Connexins, and the transmembrane domains of CD28 and PDGFR are preferred.
  • the fusion protein of the targeting polypeptide of the present invention and the membrane-binding domain may have a leader sequence.
  • amphiphilic nanocarriers examples include liposomes, exosomes, bicelles, nanodisks, polymer micelles, polymer liposomes, and nanogels, with liposomes being preferred.
  • an amphiphilic nanocarrier may be produced first and added to a cell-free protein synthesis system (in vitro protein synthesis system) of a fusion protein, or fused with an amphiphilic nanocarrier synthesis system.
  • a cell-free protein synthesis system for proteins may coexist and both synthesis systems may proceed simultaneously.
  • the cell-free protein synthesis system means a system for synthesizing a protein by coupling a transcription reaction and a translation reaction in one tube. For example, in an Eppendorf tube, mix the cell extract, substrates (nucleotides and amino acids) necessary for protein synthesis, buffers and salts, DNA encoding the fusion protein (fusion protein gene), and RNA polymerase at the appropriate temperature. When heated, the transcription / translation reaction occurs and the fusion protein is synthesized.
  • Escherichia coli extract, E. coli reconstructed cell-free protein synthesis system PURE ⁇ ⁇ ⁇ system
  • rabbit reticulocyte wheat germ cell extract
  • insect cell extract can be used as the cell extract.
  • a ribosome synthesis system dissolves lipids constituting liposomes in an organic solvent such as diethyl ether, isopropyl ether or chloroform, and then evaporates and removes the organic solvent under reduced pressure.
  • an organic solvent such as diethyl ether, isopropyl ether or chloroform
  • a system that synthesizes liposomes by adding an aqueous solution containing an active ingredient encapsulated inside the liposomes after mixing into a thin film and mixing at a temperature slightly higher than the phase transition temperature can be mentioned.
  • the membrane-binding domain of the fusion protein is incorporated into the lipid membrane of the amphiphilic nanocarrier, and the antibody or antigen-binding fragment thereof, etc.
  • the targeted polypeptide will be presented towards the outside of the amphiphilic nanocarrier.
  • the targeting polypeptide is fused to the membrane-binding domain at a position (the N-terminal side or the C-terminal side of the membrane-binding domain) that faces outward when the membrane-binding domain is incorporated into the liposome.
  • amphiphilic nanocarrier used in the present invention is known, it can be produced according to a known method or a commercially available product can be used.
  • An active ingredient such as a physiologically active substance such as a nucleic acid, protein, or drug can be contained in the amphiphilic nanocarrier used in the present invention.
  • amphiphilic nanocarrier other than liposome can be used in the same manner.
  • the liposome may be either a multilamellar liposome or a single membrane liposome.
  • the size of the liposome is about 40 nm to 100 ⁇ m, preferably about 50 nm to 50 ⁇ m, particularly about 60 nm to 10 ⁇ m.
  • As the liposome either a normal nanometer size liposome or a giant liposome may be used.
  • the size of the giant liposome is usually about 1 to 100 micrometers.
  • Ordinary nanometer-sized liposomes have a size of about 40 nm to 300 nm, preferably about 50 nm to 200 nm, particularly about 60 nm to 150 nm.
  • the size (particle diameter) of the liposome can be adjusted by passing it through a filter having a small pore diameter using an extruder.
  • Liposomes can be produced by any conventionally known method such as sonication, reverse phase evaporation, freeze-thaw, lipid lysis, or spray drying.
  • Examples of the constituent components of liposomes include phospholipids and cholesterols.
  • Phospholipids include phosphatidylethanolamines such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE); DPPC), phosphatidylcholines such as distearoylphosphatidylcholine (DSPC); phosphatidylserines such as dipalmitoylphosphatidylserine (DPPS); dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA) Phosphatidic acids such as dipalmitoyl phosphatidyl Examples include phosphatidylinositols such as nositol (DPPI) and distearoylphosphatidylinositol (DSPI), and natural phospholipids such as egg yolk lecithin, soybean lecithin
  • a phospholipid having a polyethylene glycol (PEG) chain for example, DSPE PEG, mPEG (methoxyPEG) -DSPE (molecular weight of PEG, 750, 1000, 2000, 5000,10000,20000, 30000, 40000) be able to.
  • PEG polyethylene glycol
  • Cholesterols include cholesterol (Chol), 3 ⁇ - [N- (dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoylcholesterol (TC-Chol), or Cholesterol PEG, mPEG Cholesterol having a polyethylene glycol (PEG) chain such as (methoxy PEG)-Cholesterol (molecular weight of PEG, 1000, 2000, 5000,10000,20000, 30000, 40000).
  • PEG polyethylene glycol
  • these lipids can be used alone or in combination.
  • the liposome can include at least one cationic lipid.
  • Cationic lipids include DC-6-14 (O, O'-ditetradecanoyl-N- ( ⁇ -trimethylammonioacetyl) diethanolamine chloride), DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N , N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy-3-trimethylammonio propane), DC-Chol (3 ⁇ -N- (N ', N',-dimethyl-aminoethane) -carbamol cholesterol), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum tri
  • Phospholipids or cholesterol derivatives modified with PEG can also be used as constituents of the liposome.
  • Phospholipids and cholesterol derivatives modified with PEG can construct so-called stealth liposomes in which the PEG chain covers the liposome surface and is not attacked by the immune system and becomes long-term blood-retaining.
  • the drug encapsulated inside the amphiphilic nanocarrier may be any drug and is not particularly limited.
  • a preferred drug is an antitumor agent.
  • Antitumor agents include hormonal therapeutic agents (eg, phosfestol, diethylstilbestrol, chlorotrianiserin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone.
  • hormonal therapeutic agents eg, phosfestol, diethylstilbestrol, chlorotrianiserin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone.
  • Mepartricin, raloxifene, olmeloxifen, levormeloxifen, antiestrogens eg, tamoxifen citrate, toremifene citrate, etc.
  • pill preparations mepithiostane, testrolactone, aminoglutethimide, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.), droloxifene, epithiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (eg, fadrozole hydrochloride, anastrozo) , Letrozole, exemestane, borozole, formestane, etc.), antiandrogens (eg, flutamide, bicalutamide, nilutamide, etc.), 5 ⁇ -reductase inhibitors (eg, finasteride, e
  • the types of cancer that are targeted by anti-tumor agents are colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, stomach cancer, biliary tract cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer.
  • Cervical cancer, endometrial cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor, etc. preferably colorectal cancer, stomach cancer , Head and neck cancer, lung cancer, breast cancer, pancreatic cancer, biliary tract cancer, liver cancer.
  • physiologically active substances such as drugs, nucleic acids and proteins may be used alone or in combination of two or more.
  • the nucleic acid is not particularly limited and may be any DNA, RNA, chimeric nucleic acid of DNA and RNA, DNA / RNA hybrid, or the like.
  • the nucleic acid can be any one of 1 to 3 strands, but is preferably single strand or double strand.
  • Nucleic acids may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (eg, commercially available peptide nucleic acids (PNA), etc.) or other oligomers containing special linkages (However, the oligomer contains a nucleotide having a configuration allowing base pairing or base attachment as found in DNA or RNA).
  • PNA commercially available peptide nucleic acids
  • Intramolecular nucleotide modifications such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphoro Dithioate, etc.), for example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) and sugars (eg, monosaccharides, etc.), side chain groups, intercalating compounds (Eg, acridine, psoralen, etc.), chelating Containing materials (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, or having modified bonds (eg, ⁇ -anomeric nucleic acids)
  • uncharged bonds eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • the siRNA is a nucleotide sequence homologous to the nucleotide sequence of the target gene mRNA or the initial transcript or a partial sequence thereof (preferably within the coding region) (including an intron in the case of the initial transcript) and a complementary sequence thereof. This is a single-stranded oligo RNA.
  • the length of a portion homologous to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length. Is not particularly limited as long as it can cause
  • the total length of siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases in length), but is not particularly limited as long as it can cause RNA interference. .
  • the relationship between the target nucleotide sequence and the sequence homologous to that contained in the siRNA may be 100% identical or may have base mutations (at least 70%, preferably 80%, more preferably 90%, most preferably within 95% identity range).
  • SiRNA may have an additional base at the 5 'or 3' end of 5 bases or less, preferably 2 bases, which does not form a base pair.
  • the additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA.
  • additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', tttt-3 Examples of the sequence include ', uuuuuu-3', but are not limited thereto.
  • the siRNA may be directed to any target gene.
  • the nucleic acid introduction agent of the present invention when used as a prophylactic / therapeutic agent for diseases, siRNA encapsulated in exosomes has an enhanced expression of the target disease.
  • the target is a gene involved in exacerbation, and more specifically, the antisense nucleic acid for the gene is a clinically advanced or preclinical stage gene or a newly known gene And the like.
  • SiRNA may be used alone or in combination of two or more.
  • proteins include enzymes, receptors, antibodies, antigens, interferons, and interleukins.
  • Example 1 Preparation of vector for fusion protein expression
  • Anti-EGFR single chain antibody gene was extracted from antibody gene library using phage display method.
  • FIG. 1A In designing the fusion protein, the amino acid sequence of the anti-EGFR antibody is selected as the targeting polypeptide, and the N-terminal membrane-binding domain is present in CD28 TMD used for chimeric antigen receptors and in biological membranes.
  • a known platelet-derived growth factor receptor (PDGFR) transmembrane domain (PDGFR TMD) was selected.
  • LS human immunoglobulin G leader sequence
  • scTab liposomes For purification and isolation of scTab liposomes, 25% w / v of the reaction solution after synthesis of cell-free protein and 1 mL of a solution mixed so that the density medium iodixanol (product name: OptiPrep TM ) concentration is 42% w / v. v 3 mL of iodixanol was laminated. Subsequently, 0.5 mL of 100 mM HEPES buffer solution was layered, and ultracentrifugation was performed at 197,000 ⁇ g, 4 ° C. for 2 hours. The scTab liposome was isolated by fraction-collecting the ultracentrifugated solution as fraction 1-6 from the liquid surface.
  • antiEGFR scTab-PDGFR or antiEGFR scTab-CD28 that does not contain LS at the amino terminus has a band derived from each fusion protein in the supernatant fraction, but its abundance was small compared to scTab with LS. It was also found that it is difficult to be incorporated into DOPC liposomes.
  • the hydrophobic LS is expressed immediately after the start of protein translation, allowing the amino terminus of the membrane-bound domain and the lipid of the liposome to interact from the beginning of translation, and the fusion protein is captured in the vicinity of the liposome.
  • the integration efficiency is considered to have improved.
  • the synthesis of a protein having a signal domain is performed in the rough endoplasmic reticulum.
  • the rough endoplasmic reticulum has ribosomes in close proximity to the membrane, and proteins synthesized by the ribosome are delivered to the endoplasmic reticulum through translation of the hydrophobic signal domain in concert with various factors simultaneously with translation. It is.
  • the amount of DOPC liposome to be added is changed, and cell-free protein synthesis is performed using the LS-antiEGFR scTab-CD28 expression plasmid.
  • the embedded evaluation was performed as described above (FIG. 2C). As a result, an increase in incorporation efficiency dependent on the lipid concentration was observed, and it was revealed that most of the expressed fusion protein was incorporated into the liposome at a lipid concentration of 10 mM or higher final concentration.
  • LS-antiEGFR scTab-PDGFR, LS-antiEGFR scTab-CD28 have binding activity specific to target protein (EGFR)
  • LS-antiEGFR scTab-PDGFR and LS-antiEGFR scTab-CD28-presenting DOPC liposomes were each immobilized on a plate and confirmed by ELISA (FIG. 3A).
  • LS-antiEGFR scTab-CD28-presenting DOPC liposomes can bind more EGFR than LS-antiEGFR scTab-PDGFR-presenting DOPC liposomes.
  • the strength of the binding affinity between the antibody and the antigen is defined by the amino acid sequence of the single-chain antibody variable region (scFv) used, and by its steric structure.
  • scFv single-chain antibody variable region
  • LS-antiEGFR scTab-CD28-presenting DOPC liposomes having stronger EGFR binding activity were used.
  • ELISA was performed using human EGFR (hEGFR), mouse EGFR (mEGFR), and human serum albumin (HSA) (FIG. 3B).
  • HSA human serum albumin
  • LS-antiEGFR scTab-CD28 was incorporated using a DOPC liposome having a particle size of about 160 nm, and protein quantification and phospholipid quantification were performed. As a result, it was revealed that about 30 molecules of LS-antiEGFRTscTab-CD28 were incorporated per liposome particle. Since scFv production is carried out by genetic engineering, it has the advantages of low molecular weight and easy modification.
  • the scFv antibody molecule has only one antigen-binding site for the ability to bind to an antigen, the affinity for the antigen is low, and modifications such as multivalent use have been devised.
  • the LS-antiEGFR scTab-presenting liposomes prepared by the method of the present invention have succeeded in multivalentization because many scTabs are incorporated in the liposomes, and overcome the above-mentioned problems.
  • Example 2 (1) Preparation of vector for expression of fusion protein
  • LS-antiEGFRscTab-CD28 (# 2) is used as a design template and labeled.
  • AntiEGFR scFv was selected as the targeting polypeptide
  • CD28 TMD and LS were selected as the N-terminal domain of the targeting polypeptide.
  • a fusion protein CD28 TMD in the C-terminal domain of the targeting polypeptide and constructed a vector to express them.
  • a fusion protein expression vector having no membrane-binding domain was also constructed (FIG. 7). The composition and number of each fusion protein are shown in Table 2, and hereinafter, the fusion protein is described by number.
  • Example 1 (2) the incorporation of # 1 scTab, # 2 scTab, # PD1 scTab, # PD2 scTab into DOPC liposomes revealed in Example 1 (2) is a membrane-bound domain regardless of the target polypeptide. It became clear that it was decided only by the presence or absence of. In addition, although # 3 scTab, which has only a membrane-binding domain in LS, is very slight, incorporation into DOPC liposomes was observed by addition of DOPC liposomes.
  • Example 3 EGFR binding affinity of target amphiphilic nanocarrier and binding affinity to EGFR expressing cells
  • scTab-presenting DOPC liposomes were immobilized on a microplate and bound to EGFR as an antigen. Affinity was evaluated ( Figure 9). In the examination, 50 ⁇ L of 33 nM scTab was added in an amount of protein per well and immobilized.
  • Example 1 (4) using # PD2 scTab, # 2 scTab, # 6 scTab and # 10 scTab expression plasmids that showed both good integration into the liposome membrane and binding affinity to EGFR.
  • liposomes DOPC / DMPE-RhoB / antiEGFR scTab
  • DOPC / DMPE-RhoB presenting scTab to liposomes fluorescently labeled with rhodamine B
  • the binding affinity with HeLa cells showed the same tendency as the above-mentioned ELISA results, and # PD2 scTab-presenting liposomes, # 2 scTab-presenting liposomes, and # 6 scTab-presenting liposomes showed excellent binding affinity.
  • # 2 scTab-incorporated DOPC / DMPE-RhoB liposome was administered intratumorally to solid tumor model mice prepared by transplanting HeLa cells subcutaneously in the back, and the disappearance of amphiphilic nanocarriers from the tumor tissue was evaluated.
  • the disappearance of the amphiphilic nanocarrier from the tumor was faster in the mouse administered with the # 2 scTab-presented DOPC / DMPE-RhoB liposome compared to the mouse administered with the DOPC / DMPE-RhoB liposome at the initial stage of intratumoral administration. there were.
  • Tailor-made medical care suitable for individual patients can be provided. Further, by using a fluorescent substance or a radioisotope as a drug carrier, it is possible to diagnose the expression of a contrast agent or a cell membrane protein. It is possible to produce a dialysis membrane or a filtration membrane that can remove specific molecules.

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Abstract

Provided is a method for producing an amphiphilic nanocarrier to which a fused protein consisting of a targeting polypeptide and a membrane-binding domain is bound, said method being characterized by comprising synthesizing the aforesaid fused protein in a cell-free protein synthesis system in the presence of the amphiphilic nanocarrier.

Description

標的化両親媒性ナノキャリア及びその製造方法Targeted amphiphilic nanocarrier and method for producing the same
　本発明は、標的化両親媒性ナノキャリア及びその製造方法に関する。 The present invention relates to a targeted amphiphilic nanocarrier and a method for producing the same.
　癌をはじめとした難治性疾患治療において薬物療法の急速な発展に伴い、微量で非常に優れた治療効果を示す医薬品が数多く開発されてきた。こうした医薬品は、治療効果が得られる反面、重篤な有害事象を発現するものが多いためその利用は制限されているのが現状である。そのことから、治療薬としての適用には治療効果を減弱することなく有害事象の発現を低下させる工夫が必要であり、こうした問題に対し、従来からリポソームをはじめとした薬物キャリアに治療薬を封入することでより安全性を確保した治療薬の開発が行われてきた。しかしながら、利用されている薬物キャリア自体は、疾患部位に対する標的特異性は乏しく、標的特異性の向上を目的に抗体や特定分子に結合特異性をもつタンパク質、ペプチドを修飾することで標的指向性を付与し、薬物動態を制御しようと試みられている。 Along with the rapid development of pharmacotherapy in the treatment of intractable diseases such as cancer, a large number of pharmaceuticals having a very excellent therapeutic effect have been developed. While these drugs have a therapeutic effect, the use of such drugs is limited because many drugs exhibit serious adverse events. For this reason, it is necessary to devise measures to reduce the occurrence of adverse events without diminishing the therapeutic effect for application as a therapeutic drug. To deal with these problems, therapeutic drugs have been conventionally encapsulated in drug carriers including liposomes. Therefore, the development of therapeutic drugs that ensure safety has been carried out. However, the drug carrier itself used has poor target specificity for the disease site, and the target directivity is improved by modifying proteins and peptides that have binding specificity to antibodies and specific molecules for the purpose of improving target specificity. Attempts have been made to give and control pharmacokinetics.
　こうした薬物キャリアへのタンパク質、ペプチド修飾は、[1]標的化ポリペプチドを発現する遺伝子の構築、[2]これら目的の遺伝子を発現する遺伝子組み換え生物からのリガンドを精製する過程、[3]精製物を疎水性物質などと化学的に結合させる過程、[4]疎水化された標的化ポリペプチドをリポソームなどと混合する過程を経る、といった多段階に渡る調製過程が必要であり、各操作が煩雑であることから標的指向型薬物キャリアの収率も低いといった問題を抱えている。 Protein and peptide modifications to these drug carriers include [1] construction of genes that express the targeted polypeptide, [2] process of purifying ligands from genetically modified organisms that express these genes of interest, [3] purification It requires a multi-step preparation process, such as the process of chemically binding a substance with a hydrophobic substance, etc., and the process of [4] mixing a hydrophobically targeted polypeptide with a liposome. Since it is complicated, it has a problem that the yield of the target-oriented drug carrier is low.
　特許文献１～５は、標的化ポリペプチドと疎水性化合物とをリンカーを介して薬物キャリアに修飾する調製法に関するものであり、また、特許文献６はアビジン修飾した標的化ポリペプチドとビオチン修飾した脂質をアビジン・ビオチンの特異的な結合親和性を利用することで標的指向型薬物キャリアを調製するものである。また、特許文献７～１０は、標的指向型薬物キャリアであるイムノリポソームの造影剤、治療薬としての利用方法を開示する。 Patent Documents 1 to 5 relate to a preparation method in which a targeting polypeptide and a hydrophobic compound are modified to a drug carrier via a linker, and Patent Document 6 is a biotin-modified targeting polypeptide modified with avidin. Targeted drug carriers are prepared by utilizing the specific binding affinity of avidin and biotin for lipids. Patent Documents 7 to 10 disclose methods of using immunoliposomes, which are target-directed drug carriers, as contrast agents and therapeutic agents.
WO2009/020094WO2009 / 020094 WO2009/020093WO2009 / 020093 WO2009/008489WO2009 / 008489 特開2011-115167JP2011-115167 特表2008-536944Special table 2008-536944 特表2009-512696Special table 2009-512696 特表2013-534814Special table 2013-534814 特表2010-507361Special table 2010-507361 特表2001-527534Special table 2001-527534 特表2000-510836Special table 2000-510836
　本発明は、標的に結合することができる両親媒性ナノキャリアを容易に得ることができる製造方法を提供することを目的とする。 An object of the present invention is to provide a production method capable of easily obtaining an amphiphilic nanocarrier capable of binding to a target.
　また、本発明は、標的に結合することができる両親媒性ナノキャリアを提供することを目的とする。 Another object of the present invention is to provide an amphiphilic nanocarrier that can bind to a target.
　本発明者は、標的化ポリペプチドに膜結合性ドメインを融合した融合タンパク質を設計し、無細胞タンパク質発現システムを用いることで、標的化ポリペプチドの発現と同時に脂質のシャペロン様活性による自発的な脂質膜への組込みを利用することで、一段階で簡便に標的指向型薬物キャリアの調製法を提供する。 The present inventor has designed a fusion protein in which a membrane-binding domain is fused to a targeting polypeptide, and uses a cell-free protein expression system, so that spontaneous expression of the targeting polypeptide and spontaneous chaperone-like activity of the lipid occur. By utilizing incorporation into a lipid membrane, a method for preparing a target-directed drug carrier is provided in one step.
　脂質膜存在下、無細胞タンパク質合成システムを用いることで膜タンパク質の発現と同時に、脂質のシャペロン様活性によって膜タンパク質が脂質膜に組込まれることを利用する。標的化ポリペプチドと膜結合ドメインを融合した融合タンパク質発現カセットを設計・構築し、無細胞タンパク質合成システムを利用することで、一段階の操作で脂質キャリアに標的化ポリペプチドを組込むことができる。 Using the cell-free protein synthesis system in the presence of a lipid membrane, the membrane protein is incorporated into the lipid membrane by the chaperone-like activity of the lipid simultaneously with the expression of the membrane protein. By designing and constructing a fusion protein expression cassette in which a targeting polypeptide and a membrane-binding domain are fused and utilizing a cell-free protein synthesis system, the targeting polypeptide can be incorporated into a lipid carrier in a single step.
　本発明は、以下の両親媒性ナノキャリア及びその製造方法に関する。
項１．　標的化ポリペプチドと膜結合性ドメインの融合タンパク質を結合した標的化両親媒性ナノキャリア。
項２．　標的化ポリペプチドがペプチドホルモン、受容体のリガンド、抗体又はその抗原結合性フラグメントである、項１に記載の標的化両親媒性ナノキャリア。
項３．　標的化ポリペプチドが、Fab、Fab'、F(ab')2、一本鎖抗体断片(scFv)、二量体化V領域(Diabody)、ジスルフィド安定化V領域(dsFv)又はCDRを含むペプチドからなる群から選ばれる抗原結合性フラグメントである、項１に記載の標的化両親媒性ナノキャリア。
項４．　標的化ポリペプチドが一本鎖抗体断片(scFv)である、項３に記載の標的化両親媒性ナノキャリア。
項５．　両親媒性ナノキャリアがリポソームである、項１～４のいずれか１項に記載の標的化両親媒性ナノキャリア。
項６．　融合タンパク質が、膜結合性ドメイン－scFv－膜結合性ドメイン(２つの膜結合性ドメインは、同一であっても異なっていてもよい)の構造を有する、項１～５のいずれか１項に記載の標的化両親媒性ナノキャリア。
項７．　両親媒性ナノキャリアの存在下で、標的化ポリペプチドと膜結合性ドメインの融合タンパク質を無細胞タンパク質合成系で合成させることを特徴とする、前記融合タンパク質が結合された標的化両親媒性ナノキャリアの製造方法。
項８．　標的化ポリペプチドがペプチドホルモン、受容体のリガンド、抗体又はその抗原結合性フラグメントである、項７に記載の標的化両親媒性ナノキャリアの製造方法
項９．　標的化ポリペプチドが、Fab、Fab'、F(ab')2、一本鎖抗体断片(scFv)、二量体化V領域(Diabody)、ジスルフィド安定化V領域(dsFv)又はCDRを含むペプチドからなる群から選ばれる抗原結合性フラグメントである、項７に記載の標的化両親媒性ナノキャリアの製造方法。
項１０．　標的化ポリペプチドが一本鎖抗体断片(scFv)である、項９に記載の標的化両親媒性ナノキャリアの製造方法。
項１１．　両親媒性ナノキャリアがリポソームである、項７～１０のいずれか１項に記載の標的化両親媒性ナノキャリアの製造方法。 The present invention relates to the following amphiphilic nanocarrier and a method for producing the same.
Item 1. Targeted amphiphilic nanocarriers that bind a fusion protein of a targeting polypeptide and a membrane-binding domain.
Item 2. Item 2. The targeted amphiphilic nanocarrier according to Item 1, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody, or an antigen-binding fragment thereof.
Item 3. Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR Item 2. The targeted amphiphilic nanocarrier according to Item 1, which is an antigen-binding fragment selected from the group consisting of:
Item 4. Item 4. The targeted amphiphilic nanocarrier according to Item 3, wherein the targeted polypeptide is a single-chain antibody fragment (scFv).
Item 5. Item 5. The targeted amphiphilic nanocarrier according to any one of Items 1 to 4, wherein the amphiphilic nanocarrier is a liposome.
Item 6. Item 6. The fusion protein according to any one of Items 1 to 5, wherein the fusion protein has a structure of a membrane-binding domain-scFv-membrane-binding domain (the two membrane-binding domains may be the same or different). The targeted amphiphilic nanocarrier described.
Item 7. In the presence of an amphiphilic nanocarrier, a fusion protein of a targeting polypeptide and a membrane-binding domain is synthesized by a cell-free protein synthesis system, and the targeting amphipathic nanoparticle to which the fusion protein is bound, Carrier manufacturing method.
Item 8. Item 8. The method for producing a targeted amphiphilic nanocarrier according to Item 7, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody or an antigen-binding fragment thereof. Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR Item 8. The method for producing a targeted amphiphilic nanocarrier according to Item 7, which is an antigen-binding fragment selected from the group consisting of:
Item 10. Item 10. The method for producing a targeted amphiphilic nanocarrier according to Item 9, wherein the targeted polypeptide is a single-chain antibody fragment (scFv).
Item 11. Item 11. The method for producing a targeted amphiphilic nanocarrier according to any one of Items 7 to 10, wherein the amphiphilic nanocarrier is a liposome.
　調製操作が一段階であることから、調製に要する時間も大幅に短縮され、標的指向型薬物キャリアの収率も高い。加えて、疎水性、親水性標的化ポリペプチドを問わず融合タンパク質の設計は同様に行えることから、その応用の幅は広く、複数種の標的指向性の付与も容易である。また、遺伝子情報さえ既知であれば、短時間で標的指向型薬物キャリアの調製が可能であることからテーラーメイド医薬品としても発展可能である。 Since the preparation operation is a single step, the time required for the preparation is greatly reduced, and the yield of the target-oriented drug carrier is high. In addition, since fusion proteins can be designed in the same way regardless of hydrophobic or hydrophilic targeting polypeptides, the range of applications is wide and it is easy to impart multiple types of target directivity. In addition, if genetic information is already known, it can be developed as a tailor-made medicine since a target-oriented drug carrier can be prepared in a short time.
(A)融合タンパク質(antiEGFR scTab)発現ベクター（pDNA）、(B)antiEGFR scTab組込みリポソームの調製法、および精製法の概念図(A) Fusion protein (antiEGFR scTab) expression vector (pDNA), (B) AntiEGFR scTab-incorporated liposome preparation and purification method conceptual diagram (A) リポソーム存在下、無細胞タンパク質合成系における融合タンパク質 合成後の各フラクションのウェスタンブロット分析、Wは精製前(whole)である。(B)リポソーム非存在下、無細胞タンパク質合成系における融合タンパク質合成後の各フラクションのウェスタンブロット分析 (C) 無細胞タンパク質合成系における脂質量とscTabのリポソームへの組込み相関(A) Western blot analysis of each fraction after fusion protein synthesis in a cell-free protein synthesis system in the presence of liposomes, W is before purification. (B) Western blot analysis of each fraction after fusion protein synthesis in a cell-free protein synthesis system in the absence of liposomes (C) Correlation of lipid content and scTab incorporation into liposomes in a cell-free protein synthesis system (A)antiEGFR scTab提示リポソームのEGFRに対する結合親和性（B）antiEGFR scTab提示リポソームのhEGFR、mEGFR、HSAに対する結合親和性(A) Binding affinity of antiEGFR scTab-presenting liposomes to EGFR (B) Binding affinity of antiEGFR scTab-presenting liposomes to hEGFR, mEGFR, and HSA antiEGFR scTab提示リポソームとEGFR発現細胞とのインキュベーション後のフローサイトメトリー分析Flow cytometric analysis after incubation of antiEGFR scTab presenting liposomes and EGFR expressing cells antiEGFR scTab提示リポソームとHeLa細胞とのインキュベーション後の共焦点レーザー顕微鏡像Confocal laser microscope image after incubation of antiEGFR scTab-presenting liposomes and HeLa cells HeLa細胞, MCF7細胞, Jurkat E6細胞のEGFR発現レベルを示す図。The figure which shows the EGFR expression level of a HeLa cell, MCF7 cell, and Jurkat E6 cell. 膜結合ドメインを変化させたantiEGFR scTab発現用ベクター（pDNA）AntiEGFR scTab expression vector (pDNA) with altered membrane binding domain 種々のscTabを無細胞タンパク質合成系で発現させた後の各フラクションのウェスタンブロット分析Western blot analysis of each fraction after expression of various scTabs in a cell-free protein synthesis system 種々のscTab提示リポソームのEGFRに対する結合親和性Binding affinity of various scTab-presenting liposomes for EGFR 種々のscTab提示リポソームとHeLa細胞とのインキュベーション後のフローサイトメトリー分析Flow cytometric analysis after incubation of various scTab presenting liposomes with HeLa cells scTab提示リポソームをマウス尾静脈内投与した後の血漿中プロファイルPlasma profile after scTab-presenting liposomes administered into the tail vein of mice scTab提示リポソームを固形腫瘍モデルマウスの腫瘍内投与した後の腫瘍からの消失Disappearance from tumor after intratumoral administration of scTab-presented liposomes in solid tumor model mice
　本明細書において、標的化両親媒性ナノキャリアは標的化ポリペプチドと膜結合性ドメインとの融合タンパク質および両親媒性ナノキャリアで構成される。 In the present specification, the targeted amphiphilic nanocarrier is composed of a fusion protein of a targeting polypeptide and a membrane-binding domain and an amphiphilic nanocarrier.
　標的化ポリペプチドの標的としては、細胞が挙げられ、特にがん細胞などの疾患に関連する細胞が挙げられる。 The target of the targeting polypeptide includes cells, particularly cells related to diseases such as cancer cells.
　標的化ポリペプチドとしては、ペプチドホルモン、受容体のリガンド、抗体又はその抗原結合性フラグメントが挙げられ、抗体又はその抗原結合性フラグメントが好ましく例示される。抗体としては、IgG、IgM、IgA、IgD、IgEなどのいずれでもよく、ヒト、サル、マウス、ラット、ヤギ、ウサギ、ウシ、ブタ、イヌ、ネコなどの哺乳動物由来の抗体、ヒト化抗体などが挙げられ、ヒト抗体、ヒト化抗体が好ましく例示される。また、フタコブラクダ抗体、ヒトコブラクダ抗体、ラマ抗体などの1種類のタンパク質で構成される抗体も好ましい。抗体が軽鎖と重鎖を有する場合、軽鎖又は重鎖、好ましくは重鎖に膜結合性ドメインを結合させればよい。 Examples of the targeting polypeptide include peptide hormones, receptor ligands, antibodies or antigen-binding fragments thereof, and antibodies and antigen-binding fragments thereof are preferably exemplified. The antibody may be any of IgG, IgM, IgA, IgD, IgE, etc., an antibody derived from a mammal such as human, monkey, mouse, rat, goat, rabbit, cow, pig, dog, cat, humanized antibody, etc. Preferred examples include human antibodies and humanized antibodies. In addition, an antibody composed of one kind of protein such as a Bactrian camel antibody, a human dromedary antibody, or a llama antibody is also preferable. When the antibody has a light chain and a heavy chain, a membrane-binding domain may be bound to the light chain or heavy chain, preferably the heavy chain.
　抗体の抗原結合性フラグメントとしては、Fab、Fab'、F(ab')2、一本鎖抗体断片(scFv)、二量体化V領域(Diabody)、ジスルフィド安定化V領域(dsFv)、CDRを含むペプチドなどが挙げられ、scFvが好ましい。 Antibody antigen-binding fragments include Fab, Fab ′, F (ab ′) 2 , single-chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv), CDR ScFv is preferable.
　膜結合性ドメインとしては、CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD11a、CD11b、CD11c、CD13、CD14、CD18、CD19、CD20、CD22、CD23、CD27、CD28、CD29、CD30、CD40、CD44、CD45、CDw52、CD56、CD58、CD69、CD72、TNFαR、RGFβR、TSHR、VEGFR/VPFR、FGFR、EGFR、PTHrPR、PDGFR、EPO-R、GCSF-Rなどの膜タンパク質に存在する膜貫通ドメイン、コネキシンが挙げられ、CD28、PDGFRの膜貫通ドメインが好ましい。 Membrane binding domains include CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CD20, CD22, CD23, CD27, CD28, CD29, CD30, CD40 , CD44, CD45, CDw52, CD56, CD58, CD69, CD72, TNFαR, RGFβR, TSHR, VEGFR / VPFR, FGFR, EGFR, PTHrPR, PDGFR, EPO-R, GCSF-R Connexins, and the transmembrane domains of CD28 and PDGFR are preferred.
　本発明の標的化ポリペプチドと膜結合性ドメインとの融合タンパク質は、リーダー配列を有していてもよい。 The fusion protein of the targeting polypeptide of the present invention and the membrane-binding domain may have a leader sequence.
　両親媒性ナノキャリアとしては、リポソーム、エキソソーム、バイセル、ナノディスク、高分子ミセル　高分子リポソーム、ナノゲルなどが挙げられ、リポソームが好ましい。 Examples of amphiphilic nanocarriers include liposomes, exosomes, bicelles, nanodisks, polymer micelles, polymer liposomes, and nanogels, with liposomes being preferred.
  本発明の製造方法では、両親媒性ナノキャリアを先に製造し、融合タンパク質の無細胞タンパク質合成系（インビトロでのタンパク質合成系）に加えてもよく、両親媒性ナノキャリアの合成系と融合タンパク質の無細胞タンパク質合成系を共存させて、両方の合成系を同時に進行させてもよい。 In the production method of the present invention, an amphiphilic nanocarrier may be produced first and added to a cell-free protein synthesis system (in vitro protein synthesis system) of a fusion protein, or fused with an amphiphilic nanocarrier synthesis system. A cell-free protein synthesis system for proteins may coexist and both synthesis systems may proceed simultaneously.
　ここで、無細胞タンパク質合成系とは、転写反応と翻訳反応とを一つのチューブ内で共役させ、タンパク質を合成する系を意味する。例えば、エッペンドルフチューブなどで、細胞抽出液、タンパク質合成に必要な基質類（ヌクレオチドやアミノ酸）、緩衝液や塩類、融合タンパク質をコードするＤＮＡ（融合タンパク質遺伝子）及びＲＮＡポリメラーゼを混合し、適切な温度に加温すると、転写・翻訳反応がおこり、融合タンパク質が合成される。細胞抽出液としては、大腸菌抽出液、大腸菌再構築型無細胞タンパク質合成系（PURE system）ウサギ網状赤血球、コムギ胚芽の細胞抽出液、昆虫細胞抽出液を使用することができる。また、両親媒性ナノキャリアの合成系として、例えばリボソーム合成系は、リポソームを構成する脂質をジエチルエーテル、イソプロピルエーテル又はクロロホルムなどの有機溶媒に溶解した後、有機溶媒を減圧下で蒸発除去して薄膜とした後に、リポソーム内部に封入する有効成分を含む水溶液を加えて、相転移温度よりやや高め温度で混合することでリポソームを合成する系が挙げられる。 Here, the cell-free protein synthesis system means a system for synthesizing a protein by coupling a transcription reaction and a translation reaction in one tube. For example, in an Eppendorf tube, mix the cell extract, substrates (nucleotides and amino acids) necessary for protein synthesis, buffers and salts, DNA encoding the fusion protein (fusion protein gene), and RNA polymerase at the appropriate temperature. When heated, the transcription / translation reaction occurs and the fusion protein is synthesized. As the cell extract, Escherichia coli extract, E. coli reconstructed cell-free protein synthesis system (PURE ウ サ ギ system) rabbit reticulocyte, wheat germ cell extract, and insect cell extract can be used. In addition, as a synthesis system for amphiphilic nanocarriers, for example, a ribosome synthesis system dissolves lipids constituting liposomes in an organic solvent such as diethyl ether, isopropyl ether or chloroform, and then evaporates and removes the organic solvent under reduced pressure. A system that synthesizes liposomes by adding an aqueous solution containing an active ingredient encapsulated inside the liposomes after mixing into a thin film and mixing at a temperature slightly higher than the phase transition temperature can be mentioned.
　融合タンパク質がインビトロで合成された時点でリポソームなどの両親媒性ナノキャリアが存在すると、両親媒性ナノキャリアの脂質膜に融合タンパク質の膜結合性ドメインが組込まれ、抗体又はその抗原結合性フラグメントなどの標的化ポリペプチドが両親媒性ナノキャリアの外側に向けて提示されることになる。標的化ポリペプチドは、膜結合性ドメインがリポソームに組込まれたときに外側を向くような位置（膜結合性ドメインの N 末端側もしくはC末端側）で膜結合性ドメインに融合される。 When an amphiphilic nanocarrier such as a liposome is present when the fusion protein is synthesized in vitro, the membrane-binding domain of the fusion protein is incorporated into the lipid membrane of the amphiphilic nanocarrier, and the antibody or antigen-binding fragment thereof, etc. Of the targeted polypeptide will be presented towards the outside of the amphiphilic nanocarrier. The targeting polypeptide is fused to the membrane-binding domain at a position (the N-terminal side or the C-terminal side of the membrane-binding domain) that faces outward when the membrane-binding domain is incorporated into the liposome.
　本発明で使用する両親媒性ナノキャリアは公知であるので、公知の方法に従い製造されるか、市販品を使用することができる。 Since the amphiphilic nanocarrier used in the present invention is known, it can be produced according to a known method or a commercially available product can be used.
  本発明で使用する両親媒性ナノキャリアの内部に、核酸、タンパク質、薬物などの生理活性物質などの有効成分を含有させることができる。 有効 An active ingredient such as a physiologically active substance such as a nucleic acid, protein, or drug can be contained in the amphiphilic nanocarrier used in the present invention.
　以下に、両親媒性ナノキャリアとしてリポソームを例にとり説明するが、リポソーム以外の両親媒性ナノキャリアも同様に使用できる。 Hereinafter, explanation will be given by taking liposome as an example of amphiphilic nanocarrier, but amphiphilic nanocarrier other than liposome can be used in the same manner.
　リポソームは、多重層リポソーム、一枚膜リポソームのいずれであってもよい。リポソームの大きさは４０ｎｍ～１００μｍ程度、好ましくは５０ｎｍ～５０μｍ程度、特に６０ｎｍ～１０μｍ程度である。リポソームは、通常のナノメートルサイズのリポソームとジャイアントリポソームのいずれを使用してもよい。ジャイアントリポソームのサイズは通常１～１００マイクロメーター程度である。通常のナノメートルサイズのリポソームの大きさは、４０ｎｍ～３００ｎｍ程度、好ましくは５０ｎｍ～２００ｎｍ程度、特に６０ｎｍ～１５０ｎｍ程度である。リポソームのサイズ(粒子径)は、エクストルーダーを用いて、孔径の小さいフィルターを通過させることによって調節可能である。 The liposome may be either a multilamellar liposome or a single membrane liposome. The size of the liposome is about 40 nm to 100 μm, preferably about 50 nm to 50 μm, particularly about 60 nm to 10 μm. As the liposome, either a normal nanometer size liposome or a giant liposome may be used. The size of the giant liposome is usually about 1 to 100 micrometers. Ordinary nanometer-sized liposomes have a size of about 40 nm to 300 nm, preferably about 50 nm to 200 nm, particularly about 60 nm to 150 nm. The size (particle diameter) of the liposome can be adjusted by passing it through a filter having a small pore diameter using an extruder.
　リポソームは超音波処理法、逆相蒸発法、凍結融解法、脂質溶解法、噴霧乾燥法などの従来公知の任意の方法により製造することができる。 Liposomes can be produced by any conventionally known method such as sonication, reverse phase evaporation, freeze-thaw, lipid lysis, or spray drying.
　リポソームの構成成分としては、リン脂質、コレステロール類などが挙げられる。 Examples of the constituent components of liposomes include phospholipids and cholesterols.
　リン脂質としては、ジパルミトイルホスファチジルエタノールアミン（ＤＰＰＥ）、ジオレオイルホスファチジルコリン（ＤＯＰＣ）、ジミリストイルホスファチジルエタノールアミン（ＤＭＰＥ）、ジステアロイルホスファチジルエタノールアミン（ＤＳＰＥ）などのホスファチジルエタノールアミン類；ジパルミトイルホスファチジルコリン（ＤＰＰＣ）、ジステアロイルホスファチジルコリン（ＤＳＰＣ）などのホスファチジルコリン類；ジパルミトイルホスファチジルセリン（ＤＰＰＳ）、ジステアロイルホスファチジルセリン（ＤＳＰＳ）などのホスファチジルセリン類；ジパルミトイルホスファチジン酸（ＤＰＰＡ）、ジステアロイルホスファチジン酸（ＤＳＰＡ）などのホスファチジン酸類、ジパルミトイルホスファチジルイノシトール（ＤＰＰＩ）、ジステアロイルホスファチジルイノシトール（ＤＳＰＩ）などのホスファチジルイノシトール類などが挙げられ、卵黄レシチン、大豆レシチン、リゾレシチン等の天然リン脂質、あるいはこれらを常法によって水素添加したものを使用することができる。 Phospholipids include phosphatidylethanolamines such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE); DPPC), phosphatidylcholines such as distearoylphosphatidylcholine (DSPC); phosphatidylserines such as dipalmitoylphosphatidylserine (DPPS); dipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA) Phosphatidic acids such as dipalmitoyl phosphatidyl Examples include phosphatidylinositols such as nositol (DPPI) and distearoylphosphatidylinositol (DSPI), and natural phospholipids such as egg yolk lecithin, soybean lecithin, and lysolecithin, or those obtained by hydrogenating these in a conventional manner may be used. it can.
　リン脂質として、ポリエチレングリコール(PEG)鎖を有するリン脂質、例えばDSPE PEG, mPEG（メトキシPEG）-DSPE（PEGの分子量、750, 1000, 2000, 5000,10000,20000, 30000, 40000）を使用することができる。 As the phospholipid, use is made of a phospholipid having a polyethylene glycol (PEG) chain, for example, DSPE PEG, mPEG (methoxyPEG) -DSPE (molecular weight of PEG, 750, 1000, 2000, 5000,10000,20000, 30000, 40000) be able to.
　コレステロール類としては、コレステロール（Ｃｈｏｌ）、３β－[Ｎ－(ジメチルアミノエタン)カルバモイル]コレステロール（ＤＣ－Ｃｈｏｌ）、Ｎ－（トリメチルアンモニオエチル）カルバモイルコレステロール（ＴＣ－Ｃｈｏｌ）、或いはCholesterol PEG, mPEG（メトキシPEG）- Cholesterol（PEGの分子量、1000, 2000, 5000,10000,20000, 30000, 40000）などのポリエチレングリコール(PEG)鎖を有するコレステロールが挙げられる。 Cholesterols include cholesterol (Chol), 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoylcholesterol (TC-Chol), or Cholesterol PEG, mPEG Cholesterol having a polyethylene glycol (PEG) chain such as (methoxy PEG)-Cholesterol (molecular weight of PEG, 1000, 2000, 5000,10000,20000, 30000, 40000).
　リポソームの構成成分は、これらの脂質を単独であるいは組み合わせて使用することができる。 As a component of the liposome, these lipids can be used alone or in combination.
　リポソームには、少なくとも１種のカチオン性脂質を包含させることができる。 The liposome can include at least one cationic lipid.
　カチオン性脂質としては、DC-6-14（O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride）、DODAC(dioctadecyldimethylammonium chloride）、DOTMA（N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium）、DDAB（didodecylammonium bromide）、ＤＯＴＡＰ（1,2-dioleoyloxy-3-trimethylammonio propane）、DC-Chol（3β-N-(N',N',-dimethyl-aminoethane)-carbamol cholesterol）、DMRIE（1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium）、DOSPA（2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminum trifluoroacetate）等が挙げられる。 Cationic lipids include DC-6-14 (O, O'-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride), DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N , N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy-3-trimethylammonio propane), DC-Chol (3β-N- (N ', N',-dimethyl-aminoethane) -carbamol cholesterol), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate) .
　リポソームの構成成分として、DSPE-PG10G、DSPE-PEG350やPEG-コレステロールのようにPEGなどで修飾されたリン脂質やコレステロール誘導体を使用することもできる。PEGで修飾されたリン脂質やコレステロール誘導体は、PEG鎖がリポソーム表面を覆うことで免疫系により攻撃されなくなり長期血中滞留性となる、いわゆるステルスーリポソームを構築することができる。 Phospholipids or cholesterol derivatives modified with PEG, such as DSPE-PG10G, DSPE-PEG350, and PEG-cholesterol, can also be used as constituents of the liposome. Phospholipids and cholesterol derivatives modified with PEG can construct so-called stealth liposomes in which the PEG chain covers the liposome surface and is not attacked by the immune system and becomes long-term blood-retaining.
　両親媒性ナノキャリアの内部に封入される薬物は任意の薬物が使用され特に限定されないが、例えば、抗腫瘍剤、抗高血圧剤、抗低血圧剤、抗精神病剤、鎮痛剤、抗鬱剤、抗躁剤、抗不安剤、鎮静剤、催眠剤、抗癲癇剤、オピオイドアゴニスト、喘息治療剤、麻酔剤、抗不整脈剤、関節炎治療剤、鎮痙剤、ＡＣＥインヒビター、鬱血除去剤、抗生物質、抗狭心症剤、利尿剤、抗パーキンソン病剤、気管支拡張剤、抗利尿剤、利尿剤、抗高脂血症剤、免疫抑制剤、免疫調節剤、制吐剤、抗感染症剤、抗新生物剤、抗真菌剤、抗ウイルス剤、抗糖尿病剤、抗アレルギー剤、解熱剤、抗痛風剤、抗ヒスタミン剤、止痒剤、骨調節剤、心血管剤、コレステロール低下剤、抗マラリア剤、鎮咳剤、去痰剤、粘液溶解剤、鼻詰り用薬剤、ドパミン作動剤、消化管用薬剤、筋弛緩剤、神経筋遮断剤、副交感神経作動剤、プロスタグランジン、興奮薬、食欲抑制剤、甲状腺剤又は抗甲状腺剤、ホルモン、抗偏頭痛剤、抗肥満剤、抗炎症剤などとして作用し得るものが挙げられる。好ましい薬物は抗腫瘍剤である。抗腫瘍剤としては、ホルモン療法剤（例えば、ホスフェストロール、ジエチルスチルベストロール、クロロトリアニセリン、酢酸メドロキシプロゲステロン、酢酸メゲストロール、酢酸クロルマジノン、酢酸シプロテロン、ダナゾール、アリルエストレノール、ゲストリノン、メパルトリシン、ラロキシフェン、オルメロキフェン、レボルメロキシフェン、抗エストロゲン（例、クエン酸タモキシフェン、クエン酸トレミフェンなど）、ピル製剤、メピチオスタン、テストロラクトン、アミノグルテチイミド、ＬＨ－ＲＨアゴニスト（例、酢酸ゴセレリン、ブセレリン、リュープロレリンなど）、ドロロキシフェン、エピチオスタノール、スルホン酸エチニルエストラジオール、アロマターゼ阻害薬（例、塩酸ファドロゾール、アナストロゾール、レトロゾール、エキセメスタン、ボロゾール、フォルメスタンなど）、抗アンドロゲン（例、フルタミド、ビカルタミド、ニルタミドなど）、５α－レダクターゼ阻害薬（例、フィナステリド、エプリステリドなど）、副腎皮質ホルモン系薬剤（例、デキサメタゾン、プレドニゾロン、ベタメタゾン、トリアムシノロンなど）、アンドロゲン合成阻害薬（例、アビラテロンなど）、レチノイドおよびレチノイドの代謝を遅らせる薬剤（例、リアロゾールなど）などが挙げられ、なかでもＬＨ－ＲＨアゴニスト（例、酢酸ゴセレリン、ブセレリン、リュープロレリンなど））、アルキル化剤（例えば、ナイトロジェンマスタード、塩酸ナイトロジェンマスタード－Ｎ－オキシド、クロラムブチル、シクロフォスファミド、イホスファミド、チオテパ、カルボコン、トシル酸インプロスルファン、ブスルファン、塩酸ニムスチン、ミトブロニトール、メルファラン、ダカルバジン、ラニムスチン、リン酸エストラムスチンナトリウム、トリエチレンメラミン、カルムスチン、ロムスチン、ストレプトゾシン、ピポブロマン、エトグルシド、カルボプラチン、シスプラチン、ミボプラチン、ネダプラチン、オキサリプラチン、アルトレタミン、アンバムスチン、塩酸ジブロスピジウム、フォテムスチン、プレドニムスチン、プミテパ、リボムスチン、テモゾロミド、トレオスルファン、トロフォスファミド、ジノスタチンスチマラマー、カルボコン、アドゼレシン、システムスチン、ビゼレシン）、代謝拮抗剤（例えば、メルカプトプリン、６－メルカプトプリンリボシド、チオイノシン、メトトレキサート、エノシタビン、シタラビン、シタラビンオクフォスファート、塩酸アンシタビン、５－ＦＵ系薬剤（例、フルオロウラシル、テガフール、ＵＦＴ、ドキシフルリジン、カルモフール、ガロシタビン、エミテフールなど）、アミノプテリン、ロイコボリンカルシウム、タブロイド、ブトシン、フォリネイトカルシウム、レボフォリネイトカルシウム、クラドリビン、エミテフール、フルダラビン、ゲムシタビン、ヒドロキシカルバミド、ペントスタチン、ピリトレキシム、イドキシウリジン、ミトグアゾン、チアゾフリン、アンバムスチン）、抗癌性抗生物質（例えば、アクチノマイシンＤ、アクチノマイシンＣ、マイトマイシンＣ、クロモマイシンＡ３、塩酸ブレオマイシン、硫酸ブレオマイシン、硫酸ペプロマイシン、塩酸ダウノルビシン、塩酸ドキソルビシン、塩酸アクラルビシン、塩酸ピラルビシン、塩酸エピルビシン、ネオカルチノスタチン、ミスラマイシン、ザルコマイシン、カルチノフィリン、ミトタン、塩酸ゾルビシン、塩酸ミトキサントロン、塩酸イダルビシン）、植物由来抗癌剤（例えば、エトポシド、リン酸エトポシド、硫酸ビンブラスチン、硫酸ビンクリスチン、硫酸ビンデシン、テニポシド、パクリタキセル、ドセタクセル、ビノレルビン、カンプトテシン、塩酸イリノテカン）、免疫療法剤（ＢＲＭ）（例えば、ピシバニール、クレスチン、シゾフィラン、レンチナン、ウベニメクス、インターフェロン、インターロイキン、マクロファージコロニー刺激因子、顆粒球コロニー刺激因子、エリスロポイエチン、リンホトキシン、ＢＣＧワクチン、コリネバクテリウムパルブム、レバミゾール、ポリサッカライドＫ、プロコダゾール）、細胞増殖因子ならびにその受容体の作用を阻害する薬剤（例えば、トラスツズマブ（ハーセプチン（商標）；抗ＨＥＲ２抗体）、ＺＤ１８３９（イレッサ）、グリーベック（ＧＬＥＥＶＥＣ）などの抗体医薬）が挙げられる。抗腫瘍剤の対象となる癌の種類としては、結腸・直腸癌、肝臓癌、腎臓癌、頭頸部癌、食道癌、胃癌、胆道癌、胆のう・胆管癌、膵臓癌、肺癌、乳癌、卵巣癌、子宮頚癌、子宮体癌、膀胱癌、前立腺癌、精巣腫瘍、骨・軟部肉腫、白血病、悪性リンパ腫、多発性骨髄腫、皮膚癌、脳腫瘍等が挙げられ、好ましくは結腸・直腸癌、胃癌、頭頸部癌、肺癌、乳癌、膵臓癌、胆道癌、肝臓癌が挙げられる。 The drug encapsulated inside the amphiphilic nanocarrier may be any drug and is not particularly limited. For example, an antitumor agent, antihypertensive agent, antihypertensive agent, antipsychotic agent, analgesic agent, antidepressant, anti Acupuncture, anxiolytic, sedative, hypnotic, antidepressant, opioid agonist, asthma treatment, anesthetic, antiarrhythmic, arthritis treatment, antispasmodic, ACE inhibitor, decongestant, antibiotic, antianginal , Diuretic, antiparkinsonian, bronchodilator, diuretic, diuretic, antihyperlipidemic, immunosuppressant, immunomodulator, antiemetic, anti-infective, anti-neoplastic, Antifungal agent, antiviral agent, antidiabetic agent, antiallergic agent, antipyretic agent, antigout agent, antihistamine agent, antidiarrheal agent, bone regulator, cardiovascular agent, cholesterol lowering agent, antimalarial agent, antitussive agent, expectorant, mucus Solubilizer, nasal plug, dopamine agonist Gastrointestinal drugs, muscle relaxants, neuromuscular blockers, parasympathomimetics, prostaglandins, stimulants, appetite suppressants, thyroid or antithyroid agents, hormones, anti-migraine agents, anti-obesity agents, anti-inflammatory agents And the like that can act as such. A preferred drug is an antitumor agent. Antitumor agents include hormonal therapeutic agents (eg, phosfestol, diethylstilbestrol, chlorotrianiserin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone. , Mepartricin, raloxifene, olmeloxifen, levormeloxifen, antiestrogens (eg, tamoxifen citrate, toremifene citrate, etc.), pill preparations, mepithiostane, testrolactone, aminoglutethimide, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.), droloxifene, epithiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (eg, fadrozole hydrochloride, anastrozo) , Letrozole, exemestane, borozole, formestane, etc.), antiandrogens (eg, flutamide, bicalutamide, nilutamide, etc.), 5α-reductase inhibitors (eg, finasteride, epristeride, etc.), corticosteroids (eg, dexamethasone) , Prednisolone, betamethasone, triamcinolone, etc.), androgen synthesis inhibitors (eg, abiraterone), retinoids and drugs that slow the metabolism of retinoids (eg, riarosol), among others, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.)), alkylating agents (eg, nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, Thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, sodium estramsine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etoglucid, carboplatin, cisplatin, Miboplatin, nedaplatin, oxaliplatin, altretamine, ambermustine, dibrospium hydrochloride, fotemustine, prednimustine, pumitepa, ribomustine, temozolomide, treosulphane, trophosphamide, dinostatin timamarer, carbocon, adzelesin, systemustin, biselecin) Antimetabolites (eg mercaptopurine, 6-mercaptopurine riboside, thioino , Methotrexate, enositabine, cytarabine, cytarabine ocphosphate, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid Folinate calcium, levofolinate calcium, cladribine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim, idoxyuridine, mitoguazone, thiazofurin, ambmustine), anticancer antibiotics (eg, actinomycin D, actinomycin C, Mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromy sulfate , Daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocalcinostatin, misramicin, sarcomomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride), plant-derived anticancer agents (for example, , Etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine, camptothecin, irinotecan hydrochloride, immunotherapeutic agent (BRM) (eg, picibanil, crestin, schizophyllan, fenibran, lentinone, ubenix Interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, procodazole), agents that inhibit the action of cell growth factors and their receptors (eg, trastuzumab (Herceptin ™; anti-HER2 antibody), ZD1839 (Iressa) And antibody drugs such as Gleevec). The types of cancer that are targeted by anti-tumor agents are colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, stomach cancer, biliary tract cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer. Cervical cancer, endometrial cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor, etc., preferably colorectal cancer, stomach cancer , Head and neck cancer, lung cancer, breast cancer, pancreatic cancer, biliary tract cancer, liver cancer.
　これらの薬剤、核酸、タンパク質などの生理活性物質は、１種単独で用いてもよく、２種以上を併用してもよい。 These physiologically active substances such as drugs, nucleic acids and proteins may be used alone or in combination of two or more.
　核酸としては、特に制限はなく、ＤＮＡ、ＲＮＡ、ＤＮＡとＲＮＡのキメラ核酸、ＤＮＡ／ＲＮＡのハイブリッド等いかなるものであってもよい。また、核酸は１～３本鎖のいずれも用いることができるが、好ましくは１本鎖又は２本鎖である。核酸は、プリンまたはピリミジン塩基のＮ－グリコシドであるその他のタイプのヌクレオチド、あるいは非ヌクレオチド骨格を有するその他のオリゴマー（例えば、市販のペプチド核酸（ＰＮＡ）等）または特殊な結合を含有するその他のオリゴマー（但し、該オリゴマーはＤＮＡやＲＮＡ中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する）などであってもよい。さらには公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、１個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合（例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど）を持つもの、電荷を有する結合または硫黄含有結合（例えば、ホスホロチオエート、ホスホロジチオエートなど）を持つもの、例えば蛋白質（ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチドなど）や糖（例えば、モノサッカライドなど）などの側鎖基を有しているもの、インターカレート化合物（例えば、アクリジン、プソラレンなど）を持つもの、キレート化合物（例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など）を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの（例えば、αアノマー型の核酸など）であってもよい。好ましい核酸としては、siRNAなどのRNAが挙げられる。 The nucleic acid is not particularly limited and may be any DNA, RNA, chimeric nucleic acid of DNA and RNA, DNA / RNA hybrid, or the like. The nucleic acid can be any one of 1 to 3 strands, but is preferably single strand or double strand. Nucleic acids may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (eg, commercially available peptide nucleic acids (PNA), etc.) or other oligomers containing special linkages (However, the oligomer contains a nucleotide having a configuration allowing base pairing or base attachment as found in DNA or RNA). Furthermore, those with known modifications, such as those with labels known in the art, capped, methylated, one or more natural nucleotides substituted with analogs, Intramolecular nucleotide modifications, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphoro Dithioate, etc.), for example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) and sugars (eg, monosaccharides, etc.), side chain groups, intercalating compounds (Eg, acridine, psoralen, etc.), chelating Containing materials (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, or having modified bonds (eg, α-anomeric nucleic acids) It may be. Preferred nucleic acids include RNA such as siRNA.
　ｓｉＲＮＡとは、標的遺伝子のｍＲＮＡもしくは初期転写産物のヌクレオチド配列又はその部分配列（好ましくはコード領域内）（初期転写産物の場合はイントロン部分を含む）に相同なヌクレオチド配列とその相補鎖からなる二本鎖オリゴＲＮＡである。ｓｉＲＮＡに含まれる、標的ヌクレオチド配列と相同な部分の長さは、通常、約１８塩基以上、例えば約２０塩基前後（代表的には約２１～２３塩基長）の長さであるが、ＲＮＡ干渉を引き起こすことが出来る限り、特に限定されない。また、ｓｉＲＮＡの全長も、通常、約１８塩基以上、例えば約２０塩基前後（代表的には約２１～２３塩基長）の長さであるが、ＲＮＡ干渉を引き起こすことが出来る限り、特に限定されない。 The siRNA is a nucleotide sequence homologous to the nucleotide sequence of the target gene mRNA or the initial transcript or a partial sequence thereof (preferably within the coding region) (including an intron in the case of the initial transcript) and a complementary sequence thereof. This is a single-stranded oligo RNA. The length of a portion homologous to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length. Is not particularly limited as long as it can cause The total length of siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases in length), but is not particularly limited as long as it can cause RNA interference. .
　標的ヌクレオチド配列と、ｓｉＲＮＡに含まれるそれに相同な配列との関係については、１００％一致していてもよいし、塩基の変異があってもよい（少なくとも７０％、好ましくは８０％、より好ましくは９０％、最も好ましくは９５％以上の同一性の範囲内であり得る）。 The relationship between the target nucleotide sequence and the sequence homologous to that contained in the siRNA may be 100% identical or may have base mutations (at least 70%, preferably 80%, more preferably 90%, most preferably within 95% identity range).
　ｓｉＲＮＡは、５’又は３’末端に５塩基以下、好ましくは２塩基からなる、塩基対を形成しない、付加的な塩基を有していてもよい。該付加的塩基は、ＤＮＡでもＲＮＡでもよいが、ＤＮＡを用いるとｓｉＲＮＡの安定性を向上させることができる。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’等の配列が挙げられるが、これに限定されるものではない。 SiRNA may have an additional base at the 5 'or 3' end of 5 bases or less, preferably 2 bases, which does not form a base pair. The additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples of the sequence include ', uuuuu-3', but are not limited thereto.
　ｓｉＲＮＡは任意の標的遺伝子に対するものであってよいが、本発明の核酸導入剤を疾患の予防・治療剤として用いる場合には、エキソソーム中に封入されるｓｉＲＮＡは、その発現亢進が対象疾患の発症および／または増悪に関与する遺伝子を標的とするものであることが好ましく、より具体的には、その遺伝子に対するアンチセンス核酸が、臨床もしくは前臨床段階に進んでいる遺伝子や新たに知られた遺伝子を標的とするもの等が挙げられる。 The siRNA may be directed to any target gene. However, when the nucleic acid introduction agent of the present invention is used as a prophylactic / therapeutic agent for diseases, siRNA encapsulated in exosomes has an enhanced expression of the target disease. It is preferable that the target is a gene involved in exacerbation, and more specifically, the antisense nucleic acid for the gene is a clinically advanced or preclinical stage gene or a newly known gene And the like.
　ｓｉＲＮＡは、１種のみで使用してもよく、２種以上を組み合わせて使用してもよい。 SiRNA may be used alone or in combination of two or more.
　タンパク質としては、酵素、受容体、抗体、抗原、インターフェロン、インターロイキンなどが挙げられる。 Examples of proteins include enzymes, receptors, antibodies, antigens, interferons, and interleukins.
　以下、本発明を実施例を用いてより詳細に説明するが、本発明が実施例に限定されないことはいうまでもない。 Hereinafter, although the present invention will be described in more detail with reference to examples, it goes without saying that the present invention is not limited to the examples.
実施例１
（１）融合タンパク質発現のためのベクターの調製
　抗EGFR一本鎖抗体遺伝子は、抗体遺伝子ライブラリーからファージディスプレイ法を用いて抽出した。無細胞タンパク質合成システムを用いてリポソームへ直接抗体を組み込むために、標的化ポリペプチドと３種類の膜結合ドメインからなる４種類の融合タンパク質をデザインし、それらを発現するベクター（pDNA）を構築した(図１A)。融合タンパク質のデザインにあたり、標的化ポリペプチドとしては抗EGFR抗体のアミノ酸配列を選択し、N末端の膜結合性ドメインとしてはキメラ抗原受容体に用いられているCD28 TMDと生体膜に存在することが知られている血小板由来成長因子受容体(PDGFR)の膜貫通ドメイン（PDGFR TMD）を選択した。また分泌シグナルであるヒト免疫グロブリンGリーダー配列（LS）も膜結合性ドメインの1つとして選択し、併せてLSを挿入したベクターを構築した。各融合タンパク質の構成と番号を表１に示す。 Example 1
(1) Preparation of vector for fusion protein expression Anti-EGFR single chain antibody gene was extracted from antibody gene library using phage display method. In order to incorporate antibodies directly into liposomes using a cell-free protein synthesis system, we designed four types of fusion proteins consisting of targeting polypeptides and three types of membrane-binding domains, and constructed vectors (pDNA) to express them. (FIG. 1A). In designing the fusion protein, the amino acid sequence of the anti-EGFR antibody is selected as the targeting polypeptide, and the N-terminal membrane-binding domain is present in CD28 TMD used for chimeric antigen receptors and in biological membranes. A known platelet-derived growth factor receptor (PDGFR) transmembrane domain (PDGFR TMD) was selected. In addition, a human immunoglobulin G leader sequence (LS), which is a secretion signal, was selected as one of the membrane-binding domains, and a vector in which LS was inserted was constructed. Table 1 shows the composition and number of each fusion protein.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（２）融合タンパク質のリポソームへの組込みによる標的化両親媒性ナノキャリアの製造
　各antiEGFR scTab発現プラスミド、リポソーム、再構成型無細胞発現系(PURESYSTEM)を混合し、37℃、4時間インキュベートした（図1B）。無細胞タンパク質合成系は、T7 プロモータ依存的なmRNAの転写に必要なT7RNAポリメラーゼ、アミノ酸、tRNA、リボソームの混合液（キット製品でマスターミックスになっているもの）にT7 プロモータを有するscTab発現プラスミドを200ngとリポソームを添加することで最終的な反応溶液量が25μLになるように調製した溶液である。scTabリポソームの精製・単離は、無細胞タンパク質合成後の反応溶液と密度媒体iodixanol（製品名OptiPrepTM）の濃度が42% w/vになるように混合した溶液1mLの上に25% w/v iodixanol 3mLを積層した。続いて100mM HEPES緩衝溶液0.5mLを積層し、197,000×g、4℃、2時間超遠心分離を行った。超遠心分離後の溶液を液面よりフラクション１－６として分画回収することで、scTabリポソームを単離した。 (2) Production of targeted amphiphilic nanocarrier by incorporation of fusion protein into liposome Each antiEGFR scTab expression plasmid, liposome, reconstituted cell-free expression system (PURESYSTEM) were mixed and incubated at 37 ° C for 4 hours ( Figure 1B). The cell-free protein synthesis system uses a T7 promoter-containing scTab expression plasmid in the T7 RNA polymerase, amino acid, tRNA, and ribosome mixture (which is the master mix in the kit product) necessary for T7 promoter-dependent mRNA transcription. A solution prepared by adding 200 ng and liposomes so that the final reaction solution volume is 25 μL. For purification and isolation of scTab liposomes, 25% w / v of the reaction solution after synthesis of cell-free protein and 1 mL of a solution mixed so that the density medium iodixanol (product name: OptiPrep ™ ) concentration is 42% w / v. v 3 mL of iodixanol was laminated. Subsequently, 0.5 mL of 100 mM HEPES buffer solution was layered, and ultracentrifugation was performed at 197,000 × g, 4 ° C. for 2 hours. The scTab liposome was isolated by fraction-collecting the ultracentrifugated solution as fraction 1-6 from the liquid surface.
（３）融合タンパク質のリポソームへの組込み評価
　各antiEGFR scTabのリポソームへの組込み評価は、DOPCリポソーム存在下、PURESYSTEMを用いて各antiEGFR scTabの発現を行い、超遠心法で分離することで各分画を得て、western blot法にて行った。(図2A)。その結果、アミノ末端にLSを含むLS-antiEGFR scTab-PDGFR、あるいはLS-antiEGFR scTab-CD28は上清分画に各融合タンパク質由来のバンドが認められたことから、DOPCリポソームに組込まれることが明らかになった。一方、アミノ末端に LS を含まないantiEGFR scTab-PDGFR、あるいはantiEGFR scTab-CD28は上清分画に各融合タンパク質由来のバンドが認められるものの、LSを有するscTabと比べその存在量は少なかったことからもDOPCリポソームに組込まれにくいことが明らかになった。また、リポソーム存在下での製造において、上清分画に認められたLS-antiEGFR scTab-PDGFR、あるいはLS-antiEGFR scTab-CD28がDOPCリポソームのシャペロン活性による凝集抑制によるものかを検証する為に、リポソーム非存在下で無細胞タンパク質合成を行った（図2B）。その結果、リポソーム非存在下では沈殿分画にのみ各融合タンパク質が認められたことから、上清分画に存在する各融合タンパク質はリポソームに組込まれることで可溶化され、上清分画に移行したと考えられる。 (3) Evaluation of incorporation of fusion proteins into liposomes Evaluation of incorporation of each antiEGFR scTab into liposomes was performed by expressing each antiEGFR scTab using PURESYSTEM in the presence of DOPC liposomes and separating them by ultracentrifugation. And was performed by western blot method. (Figure 2A). As a result, LS-antiEGFR scTab-PDGFR containing LS at the amino terminus or LS-antiEGFR scTab-CD28 was found to be incorporated into DOPC liposomes, as bands from each fusion protein were observed in the supernatant fraction. Became. On the other hand, antiEGFR scTab-PDGFR or antiEGFR scTab-CD28 that does not contain LS at the amino terminus has a band derived from each fusion protein in the supernatant fraction, but its abundance was small compared to scTab with LS. It was also found that it is difficult to be incorporated into DOPC liposomes. In addition, in the production in the presence of liposomes, in order to verify whether LS-antiEGFR scTab-PDGFR or LS-antiEGFR scTab-CD28 found in the supernatant fraction is due to aggregation suppression by chaperone activity of DOPC liposomes, Cell-free protein synthesis was performed in the absence of liposomes (FIG. 2B). As a result, in the absence of liposomes, each fusion protein was found only in the precipitate fraction, so each fusion protein present in the supernatant fraction was solubilized by incorporation into the liposome and transferred to the supernatant fraction. It is thought that.
　以上のことから、タンパク質翻訳開始直後に疎水性のLSが発現することで、翻訳初期から膜結合性ドメインのアミノ末端とリポソームの脂質が相互作用することができ、融合タンパク質はリポソーム近傍に捕捉されたまま翻訳が進行するため、組込み効率が向上したと考えられる。細胞内において、シグナルドメインを持つタンパク質の合成は粗面小胞体で行われる。粗面小胞体には、膜に近接するようにリボソームが存在し、リボソームで合成されるタンパク質は、翻訳と同時に種々の因子と協奏的に疎水性シグナルドメインが貫通する形で小胞体に受け渡される。このことからも、今回アミノ末端に膜結合性ドメインを付加した結果は、細胞と異なり小胞体膜へ輸送する因子は存在しないが、細胞内でタンパク質が小胞体膜へ輸送される現象を一部反映する形でリボソーム膜に提示されたと考えられる。また、アミノ末端にLSを有さないantiEGFR scTab-PDGFR、antiEGFR sc Tab-CD28はLSを有するLS-antiEGFR scTab-PDGFR、LS-antiEGFR scTab-CD28それぞれと比べ融合タンパク質合成量が少ないことも明らかとなった。 Based on the above, the hydrophobic LS is expressed immediately after the start of protein translation, allowing the amino terminus of the membrane-bound domain and the lipid of the liposome to interact from the beginning of translation, and the fusion protein is captured in the vicinity of the liposome. As the translation proceeds, the integration efficiency is considered to have improved. In the cell, the synthesis of a protein having a signal domain is performed in the rough endoplasmic reticulum. The rough endoplasmic reticulum has ribosomes in close proximity to the membrane, and proteins synthesized by the ribosome are delivered to the endoplasmic reticulum through translation of the hydrophobic signal domain in concert with various factors simultaneously with translation. It is. From this fact, the result of adding a membrane-binding domain to the amino terminus is different from the cell in that there are no factors that transport to the endoplasmic reticulum membrane, but some of the phenomenon that proteins are transported to the endoplasmic reticulum membrane in the cell. It is thought that it was presented on the ribosome membrane in a reflecting manner. It is also clear that antiEGFR scTab-PDGFR, antiEGFR sc Tab-CD28 without LS at the amino terminus has less fusion protein synthesis than LS-antiEGFR scTab-PDGFR and LS-antiEGFR scTab-CD28 with LS, respectively. became.
　続いて融合タンパク質のリポソームへの組込みが脂質量依存的に変動するかを検証する為に、添加するDOPCリポソームの量を変え、LS-antiEGFR scTab-CD28発現プラスミドを用いて無細胞タンパク質合成を行った後、上述の通り組込み評価を行った（図2C）。その結果、脂質濃度依存的な組込み効率の増大が認められ、終濃度10mM以上の脂質濃度で発現融合タンパク質の殆どがリポソームに組込まれることが明らかとなった。 Subsequently, in order to verify whether the incorporation of the fusion protein into the liposome varies depending on the amount of lipid, the amount of DOPC liposome to be added is changed, and cell-free protein synthesis is performed using the LS-antiEGFR scTab-CD28 expression plasmid. After that, the embedded evaluation was performed as described above (FIG. 2C). As a result, an increase in incorporation efficiency dependent on the lipid concentration was observed, and it was revealed that most of the expressed fusion protein was incorporated into the liposome at a lipid concentration of 10 mM or higher final concentration.
（４）標的化両親媒性ナノキャリアのEGFR結合特異性
　リポソームへ組込まれた融合タンパク質（LS-antiEGFR scTab-PDGFR、LS-antiEGFR scTab-CD28）が標的タンパク質（EGFR）特異的に結合活性を有するかを検証するために、LS-antiEGFR scTab-PDGFRおよびLS-antiEGFR scTab-CD28提示DOPCリポソームをそれぞれプレートに固定化し、ELISA法にて確認した(図3A)。その結果、LS-antiEGFR scTab-CD28提示DOPCリポソームはLS-antiEGFR scTab-PDGFR提示DOPCリポソームより多くのEGFRと結合できることが明らかとなった。抗体と抗原との結合親和性の強度は用いている一本鎖抗体可変領域（scFv）のアミノ酸配列、強いては、その立体構造により規定される。膜結合性ドメインとしてPDGFRを用いた場合、疎水性が高いためリポソームへの組込みは安定されるが、EGFRとの結合親和性が低下するような抗原認識部位の立体構造が変化したと考えられる。そのことからも、単に疎水性の高い膜結合性ドメインを用いてリポソームへの組込みを安定させるだけではなく、scFvの結合親和性の低下を避けることが可能な活性と挿入のバランスに優れた疎水性ドメインを用いることが融合タンパク質提示DOPCリポソームの調製にあたり望まれた。 (4) EGFR binding specificity of targeted amphiphilic nanocarriers Fusion proteins incorporated into liposomes (LS-antiEGFR scTab-PDGFR, LS-antiEGFR scTab-CD28) have binding activity specific to target protein (EGFR) In order to verify this, LS-antiEGFR scTab-PDGFR and LS-antiEGFR scTab-CD28-presenting DOPC liposomes were each immobilized on a plate and confirmed by ELISA (FIG. 3A). As a result, it was revealed that LS-antiEGFR scTab-CD28-presenting DOPC liposomes can bind more EGFR than LS-antiEGFR scTab-PDGFR-presenting DOPC liposomes. The strength of the binding affinity between the antibody and the antigen is defined by the amino acid sequence of the single-chain antibody variable region (scFv) used, and by its steric structure. When PDGFR is used as a membrane-binding domain, incorporation into a liposome is stable because of its high hydrophobicity, but the three-dimensional structure of an antigen recognition site that reduces the binding affinity to EGFR is considered to have changed. Therefore, it is not only a stable membrane-binding domain with a highly hydrophobic membrane-binding domain, but also a hydrophobicity with a good balance between activity and insertion that can prevent a decrease in the binding affinity of scFv. The use of sex domains was desired in the preparation of fusion protein displaying DOPC liposomes.
　そこで、以降の検討ではEGFR結合活性がより強いLS-antiEGFR scTab-CD28提示DOPCリポソームを用いた。LS-antiEGFR scTab-CD28提示DOPCリポソームの結合特異性を調べるため、ヒトEGFR（hEGFR）、マウスEGFR（mEGFR）、ヒト血清アルブミン（HSA）を用いてELISAを行った（図3B）。その結果、LS-antiEGFR scTab-CD28提示リポソームはhEGFRに対して強い結合親和性を示した。抗原ではないHSAとは結合親和性が認められず、また、LS-antiEGFR scTab-CD28提示DOPCリポソームの代わりに単なるDOPCを固定化した場合ではEGFRの非特異的な吸着が認められなかった。加えて、マウスEGFRを添加した場合ヒトEGFR添加と同様LS-antiEGFR scTab-CD28提示DOPCリポソームとの結合が認められたことからLS-antiEGFR scTab-CD28はヒトとマウスのEGFRに交差性を持つことが明らかとなった。 Therefore, in the subsequent examinations, LS-antiEGFR scTab-CD28-presenting DOPC liposomes having stronger EGFR binding activity were used. To examine the binding specificity of LS-antiEGFRantiscTab-CD28-presented DOPC liposomes, ELISA was performed using human EGFR (hEGFR), mouse EGFR (mEGFR), and human serum albumin (HSA) (FIG. 3B). As a result, LS-antiEGFR scTab-CD28-presenting liposomes showed strong binding affinity for hEGFR. No binding affinity was observed with HSA that is not an antigen, and nonspecific adsorption of EGFR was not observed when mere DOPC was immobilized instead of DOPC liposomes presenting LS-antiEGFR scTab-CD28. In addition, when mouse EGFR was added, binding to LS-antiEGFRTscTab-CD28-presented DOPC liposomes was observed in the same way as human EGFR was added, so LS-antiEGFR scTab-CD28 has cross-reactivity to human and mouse EGFR. Became clear.
　続いて、リポソーム１粒子あたりどのくらいのLS-antiEGFR scTab-CD28が組込まれているか、約160nm粒子径を持つDOPCリポソームを用いてLS-antiEGFR scTab-CD28を組込み、タンパク質定量、リン脂質定量を行ったところ、リポソーム１粒子あたり約 30分子のLS-antiEGFR scTab-CD28が組込まれていることが明らかとなった。scFv作製は、遺伝子工学的に行われているため、分子量が小さく、また改変が容易であるという利点がある。しかしながら、scFv抗体1分子が持つ抗原との結合能は抗原結合部位が1つしかないために、抗原との親和性は低く、その利用には多価化するなどの改変について考案されてきた。本発明の方法で調製したLS-antiEGFR scTab提示リポソームは、多くのscTabがリポソームに組込まれていることから、多価化に成功しており、前述のような課題を克服した。 Subsequently, how much LS-antiEGFR scTab-CD28 was incorporated per liposome particle, LS-antiEGFR scTab-CD28 was incorporated using a DOPC liposome having a particle size of about 160 nm, and protein quantification and phospholipid quantification were performed. As a result, it was revealed that about 30 molecules of LS-antiEGFRTscTab-CD28 were incorporated per liposome particle. Since scFv production is carried out by genetic engineering, it has the advantages of low molecular weight and easy modification. However, since the scFv antibody molecule has only one antigen-binding site for the ability to bind to an antigen, the affinity for the antigen is low, and modifications such as multivalent use have been devised. The LS-antiEGFR scTab-presenting liposomes prepared by the method of the present invention have succeeded in multivalentization because many scTabs are incorporated in the liposomes, and overcome the above-mentioned problems.
（５）標的化両親媒性ナノキャリアのEGFR発現細胞に対する結合親和性
　ローダミンBで蛍光標識したリポソーム（DOPC/DMPE-RhoB）にLS-antiEGFR scTabを提示したリポソーム（DOPC/DMPE-RhoB/LS-antiEGFR scTab）を用いて、EGFR発現細胞とDOPC/DMPE-RhoB/ LS-antiEGFR scTab-CD28リポソームとの結合親和性に関して、図６に示すEGFR発現量が異なる細胞株を用いてフローサイトメトリー(図4)と共焦点レーザー顕微鏡観察(図5)を行った。フローサイトメトリーの結果より、各細胞のEGFR発現量に依存的なDOPC/DMPE-RhoB/LS-antiEGFR scTabリポソームの結合が認められた。このことから、 LS-antiEGFR scTab提示リポソームは細胞膜に存在するEGFRを認識して細胞と結合することが明らかとなった。 (5) Binding Affinity of Targeted Amphiphilic Nanocarriers to EGFR-expressing Cells Liposomes (DOPC / DMPE-RhoB / LS-) displaying LS-antiEGFR scTab on liposomes fluorescently labeled with rhodamine B (DOPC / DMPE-RhoB) Using antiEGFR scTab), flow cytometry (FIG. 6) using cell lines with different EGFR expression levels shown in FIG. 6 with respect to the binding affinity between EGFR-expressing cells and DOPC / DMPE-RhoB / LS-antiEGFR scTab-CD28 liposomes. 4) and confocal laser microscope observation (Fig. 5). From the results of flow cytometry, binding of DOPC / DMPE-RhoB / LS-antiEGFR scTab liposomes depending on the EGFR expression level of each cell was observed. From this, it became clear that the LS-antiEGFR scTab-presenting liposome recognizes EGFR present in the cell membrane and binds to the cell.
　また、DOPC/DMPE-RhoB/抗EGFR scFv-myc-CD28をHeLa細胞に添加した後、共焦点レーザー顕微鏡観察を行った結果、フローサイトメトリーと同様の傾向が認められた。特に、DOPC/DMPE-RhoB/LS-antiEGFR scTabリポソームを添加した細胞では、細胞膜上にローダミンB由来の強い蛍光シグナルが認められることから、LS-antiEGFR scTab提示リポソームはEGFRに強い親和性を示した。 Also, after adding DOPC / DMPE-RhoB / anti-EGFR scFv-myc-CD28 to HeLa cells, confocal laser microscope observation was performed. As a result, the same tendency as in flow cytometry was observed. In particular, in cells to which DOPC / DMPE-RhoB / LS-antiEGFR scTab liposomes were added, a strong fluorescent signal derived from rhodamine B was observed on the cell membrane, so LS-antiEGFR scTab-presenting liposomes showed a strong affinity for EGFR. .
実施例２
（１）融合タンパク質発現のためのベクターの調製
　リポソーム膜への組込み効率と結合親和性のバランスが調和した融合タンパク質の創出を目的に、LS-antiEGFRscTab-CD28(#2)を設計鋳型とし、標識化ポリペプチドにantiEGFR scFvを選択し、標的化ポリペプチドのN末端ドメインにはCD28 TMDとLSを選択した。また、標的化ポリペプチドのC末端ドメインにCD28 TMDとする融合タンパク質をデザインし、それらを発現するベクターを構築した。併せて、膜結合性ドメインを有さない融合タンパク質発現ベクターも構築した (図７)。各融合タンパク質の構成と番号を表２に示し、以降では融合タンパク質を番号で記載する。 Example 2
(1) Preparation of vector for expression of fusion protein For the purpose of creating a fusion protein that balances the efficiency of incorporation into the liposome membrane and the binding affinity, LS-antiEGFRscTab-CD28 (# 2) is used as a design template and labeled. AntiEGFR scFv was selected as the targeting polypeptide, and CD28 TMD and LS were selected as the N-terminal domain of the targeting polypeptide. In addition, we designed a fusion protein CD28 TMD in the C-terminal domain of the targeting polypeptide and constructed a vector to express them. In addition, a fusion protein expression vector having no membrane-binding domain was also constructed (FIG. 7). The composition and number of each fusion protein are shown in Table 2, and hereinafter, the fusion protein is described by number.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（２）融合タンパク質のリポソームへの組込みによる標的化両親媒性ナノキャリアの製造および組込み評価
　上記ベクターを用いて、実施例１（２）の製造法と同様な無細胞蛋白質合成システムを用いて、各融合タンパク質がリポソームに組込まれた標的指向型キャリアを製造し、リポソームへの組込みを評価した（図8）。その結果、膜結合ドメインを有さない#4 scFv, #11 scFvではDOPCリポソームの添加に依らず、発現した融合タンパク質は上清分画には存在せず、全て沈殿分画に存在した。この結果より、実施例１（２）で明らかとなった#1 scTab、 #2 scTab、 #PD1 scTab、 #PD2 scTabのDOPCリポソームへの組込みは、標的化ポリペプチドは関係なく、膜結合性ドメインの有無のみで決定することが明らかとなった。また、LSしか膜結合性ドメインを有さない#3 scTabにおいては非常に僅かであるが、DOPCリポソームの添加によってDOPCリポソームへの組込みが認められた。以上の結果は、実施例１（２）で明らかとなったLSを有する#2 scTab、#PD2 scTabの方が、LSを有さない#1 scTab、#PD1 scTabより組込み効率が良いという結果を裏付けるものであり、LSにより翻訳途中のscTabが緩やかにリポソーム膜と相互作用し、続く翻訳によりTMDが合成されscTabがリポソーム膜に強固に組込まれると考えられる。一方で、CD28 TMDを有する他のscTab（#5 scTab, #6 scTab, #9 scTab, #10 scTab）は、CD28 TMDの融合部位に依らず良好な組込み効率を示した。また、#8 scTabの発現量は非常に少なく、リポソームへの組込みを評価する量の#8 scTabを得ることが出来なかった。 (2) Production of targeted amphiphilic nanocarrier by incorporation of fusion protein into liposome and evaluation of incorporation Using the above vector, a cell-free protein synthesis system similar to the production method of Example 1 (2), Targeted carriers in which each fusion protein was incorporated into liposomes were produced, and their incorporation into liposomes was evaluated (FIG. 8). As a result, in the case of # 4 scFv and # 11 scFv having no membrane binding domain, the expressed fusion protein was not present in the supernatant fraction and was present in the precipitate fraction irrespective of the addition of DOPC liposome. From this result, the incorporation of # 1 scTab, # 2 scTab, # PD1 scTab, # PD2 scTab into DOPC liposomes revealed in Example 1 (2) is a membrane-bound domain regardless of the target polypeptide. It became clear that it was decided only by the presence or absence of. In addition, although # 3 scTab, which has only a membrane-binding domain in LS, is very slight, incorporation into DOPC liposomes was observed by addition of DOPC liposomes. The above results indicate that # 2 scTab and # PD2 scTab having LS revealed in Example 1 (2) have better integration efficiency than # 1 scTab and # PD1 scTab having no LS. This is supported by the fact that scTab during translation slowly interacts with the liposome membrane by LS, and TMD is synthesized by subsequent translation and scTab is firmly incorporated into the liposome membrane. On the other hand, other scTabs with CD28 TMD (# 5 scTab, # 6 scTab, # 9 scTab, # 10 scTab) showed good integration efficiency regardless of the fusion site of CD28 TMD. In addition, the expression amount of # 8 scTab was very small, and it was not possible to obtain # 8 scTab in an amount for evaluating the incorporation into liposomes.
　良好な組込み効率を示した#PD2、#2、#5、#6、#7、#9、#10 scTabについてscTab提示DOPCリポソームの物性を評価したところ（表３）、いずれのscTabを組込んだDOPCリポソームも提示前のDOPCリポソームと比べ粒子径の変化は認められなかった。また、DOPCリポソーム１粒子あたり組込まれたscTab量は約20 － 50分子であることが明らかとなった。 # PD2, # 2, # 5, # 6, # 7, # 9, # 10 which showed good incorporation efficiency were evaluated for the physical properties of scPC-presented DOPC liposomes (Table 3). Any scTab was incorporated. However, the DOPC liposome did not change in particle size compared to the DOPC liposome before presentation. In addition, it was revealed that the amount of scTab incorporated per DOPC liposome particle was about 20 to 50 molecules.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（３）標的化両親媒性ナノキャリアのEGFR結合親和性およびEGFR発現細胞に対する結合親和性
実施例１（３）と同様にscTab提示DOPCリポソームをマイクロプレートに固定化し、抗原であるEGFRとの結合親和性を評価した（図９）。検討にあたり、各ウェルあたりタンパク質量で33nM scTabを50μL添加し、固定化した。その結果、いずれのscTab 提示DOPCリポソームを固相化した場合でもEGFRとの結合親和性が認められ、特にN末端とC末端に膜結合性ドメインを有する融合タンパク質（#2、#6、#PD2、#7scTab）提示リポソームは強い結合親和性を示した。しかしながら、scTab分子内に膜結合性ドメインが１つしかない#5 scTab、#9 scTab 提示リポソームにおいては、EGFRとの結合親和性は低いものであった。scFvは熱力学的に不安定であり、タンパク質の構造変化によりそのままでは抗原との結合親和性が容易に減弱することが知られている。以上より、scTabが結合親和性を示すには、scFvの自由度を制限することで熱力学的な安定性をもたらすことが重要であり、それを可能とすると考えられるscFvの両末端に膜結合性ドメインを有することが望まれる。 (3) EGFR binding affinity of target amphiphilic nanocarrier and binding affinity to EGFR expressing cells As in Example 1 (3), scTab-presenting DOPC liposomes were immobilized on a microplate and bound to EGFR as an antigen. Affinity was evaluated (Figure 9). In the examination, 50 μL of 33 nM scTab was added in an amount of protein per well and immobilized. As a result, even when any scTab-presented DOPC liposome was immobilized, binding affinity to EGFR was observed, especially fusion proteins with membrane-binding domains at the N-terminus and C-terminus (# 2, # 6, # PD2 , # 7scTab) presenting liposomes showed strong binding affinity. However, the # 5 scTab and # 9 scTab presenting liposomes having only one membrane-binding domain in the scTab molecule had a low binding affinity with EGFR. It is known that scFv is thermodynamically unstable, and its binding affinity with an antigen is easily reduced as it is due to a structural change of the protein. From the above, in order for scTab to exhibit binding affinity, it is important to bring about thermodynamic stability by limiting the degree of freedom of scFv, and it is thought that this is possible by membrane binding to both ends of scFv It is desirable to have a sex domain.
　続いて、リポソーム膜ヘの良好な組込みと、EGFRとの結合親和性をともに示した#PD2 scTab、#2 scTab、#6 scTab、および、#10 scTab発現プラスミドを用いて実施例１（４）と同様に、ローダミンBで蛍光標識したリポソーム（DOPC/DMPE-RhoB）にscTabを提示したリポソーム（DOPC/DMPE-RhoB/ antiEGFR scTab）を製造し、EGFR陽性細胞であるHeLa細胞との結合親和性をフローサイトメトリーによって評価した（図１０）。その結果、HeLa細胞との結合親和性は上述のELISAの結果と同様の傾向を示し、#PD2 scTab提示リポソーム、#2 scTab提示リポソーム、#6 scTab提示リポソームが優れた結合親和性を示した。 Subsequently, Example 1 (4) using # PD2 scTab, # 2 scTab, # 6 scTab and # 10 scTab expression plasmids that showed both good integration into the liposome membrane and binding affinity to EGFR. In the same manner as above, liposomes (DOPC / DMPE-RhoB / antiEGFR scTab) presenting scTab to liposomes fluorescently labeled with rhodamine B (DOPC / DMPE-RhoB) are produced and binding affinity to EGFR-positive HeLa cells Was evaluated by flow cytometry (FIG. 10). As a result, the binding affinity with HeLa cells showed the same tendency as the above-mentioned ELISA results, and # PD2 scTab-presenting liposomes, # 2 scTab-presenting liposomes, and # 6 scTab-presenting liposomes showed excellent binding affinity.
（４）標的化両親媒性ナノキャリアの体内動態
　上記#2 scTabベクターを用いて、実施例１（４）の製造法と同様な無細胞蛋白質合成システムを用いて、#2 scTabがDOPC/DMPE-RhoBリポソームに組込まれた標的化両親媒性ナノキャリアを製造し、正常マウスへ尾静脈内投与を行い血中からの消失を評価した（図１１）。その結果、静脈内投与後のリポソームの血漿中プロファイルはDOPC/DMPE-RhoBリポソーム、#2 scTab組込みDOPC/DMPE-RhoBリポソームにおいて大きな差は認められなかった。 (4) Pharmacokinetics of targeted amphipathic nanocarriers Using the cell-free protein synthesis system similar to the production method of Example 1 (4), using the # 2 scTab vector, # 2 scTab becomes DOPC / DMPE. -Targeted amphiphilic nanocarriers incorporated into -RhoB liposomes were produced and administered to normal mice via tail vein to evaluate disappearance from the blood (FIG. 11). As a result, the plasma profile of liposomes after intravenous administration was not significantly different between DOPC / DMPE-RhoB liposomes and # 2 scTab-incorporated DOPC / DMPE-RhoB liposomes.
　続いて、HeLa細胞を背部皮下に移植することで作製した固形腫瘍モデルマウスへ#2 scTab組込みDOPC/DMPE-RhoBリポソームを腫瘍内投与し、腫瘍組織からの両親媒性ナノキャリアの消失を評価した結果（図１２）、腫瘍内投与初期ではDOPC/DMPE-RhoBリポソーム投与マウスと比べ、#2 scTab提示DOPC/DMPE-RhoBリポソームを投与したマウスにおいて腫瘍からの両親媒性ナノキャリアの消失は速やかであった。しかしながら、６時間後においてDOPC/DMPE-RhoBリポソーム投与マウスの腫瘍ではリポソームの存在が認められなかったのに対し、#2 scTab提示DOPC/DMPE-RhoBリポソーム投与マウスの腫瘍ではscTab提示リポソームの存在が認められた。このことから、腫瘍組織で発現が亢進しているEGFRに対する結合親和性を有するantiEGFR scTabをリポソームへ組込むことで、antiEGFR scTabリポソームは腫瘍組織内のEGFRと結合し、より効率的に腫瘍組織に滞留したと考えられる。 Subsequently, # 2 scTab-incorporated DOPC / DMPE-RhoB liposome was administered intratumorally to solid tumor model mice prepared by transplanting HeLa cells subcutaneously in the back, and the disappearance of amphiphilic nanocarriers from the tumor tissue was evaluated. As a result (FIG. 12), the disappearance of the amphiphilic nanocarrier from the tumor was faster in the mouse administered with the # 2 scTab-presented DOPC / DMPE-RhoB liposome compared to the mouse administered with the DOPC / DMPE-RhoB liposome at the initial stage of intratumoral administration. there were. However, the presence of liposomes was not observed in the tumors of mice administered with DOPC / DMPE-RhoB liposomes after 6 hours, whereas the presence of scTab-presenting liposomes in tumors of mice administered with # 2 scTab-presented DOPC / DMPE-RhoB liposomes. Admitted. From this, by incorporating antiEGFR scTab having binding affinity for EGFR whose expression is enhanced in the tumor tissue into the liposome, the antiEGFR scTab liposome binds to the EGFR in the tumor tissue and more efficiently stays in the tumor tissue It is thought that.
患者個人個人に適したテーラーメイド医療が提供可能である。また、薬物キャリアに蛍光物質や放射性同位体を用いることで、造影剤や細胞膜タンパク質の発現を診断可能である。特定分子の除去が可能となる透析膜や濾過膜を作製することが可能である。 Tailor-made medical care suitable for individual patients can be provided. Further, by using a fluorescent substance or a radioisotope as a drug carrier, it is possible to diagnose the expression of a contrast agent or a cell membrane protein. It is possible to produce a dialysis membrane or a filtration membrane that can remove specific molecules.

Claims (11)

  1. 標的化ポリペプチドと膜結合性ドメインの融合タンパク質を結合した標的化両親媒性ナノキャリア。 Targeted amphiphilic nanocarriers that bind a fusion protein of a targeting polypeptide and a membrane-binding domain.
  2. 標的化ポリペプチドがペプチドホルモン、受容体のリガンド、抗体又はその抗原結合性フラグメントである、請求項１に記載の標的化両親媒性ナノキャリア。 2. The targeted amphiphilic nanocarrier of claim 1, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody or an antigen-binding fragment thereof.
  3. 標的化ポリペプチドが、Fab、Fab'、F(ab')2、一本鎖抗体断片(scFv)、二量体化V領域(Diabody)、ジスルフィド安定化V領域(dsFv)又はCDRを含むペプチドからなる群から選ばれる抗原結合性フラグメントである、請求項１に記載の標的化両親媒性ナノキャリア。 Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR The targeted amphiphilic nanocarrier according to claim 1, which is an antigen-binding fragment selected from the group consisting of.
  4. 標的化ポリペプチドが一本鎖抗体断片(scFv)である、請求項３に記載の標的化両親媒性ナノキャリア。 4. The targeted amphiphilic nanocarrier according to claim 3, wherein the targeting polypeptide is a single chain antibody fragment (scFv).
  5. 両親媒性ナノキャリアがリポソームである、請求項１～４のいずれか１項に記載の標的化両親媒性ナノキャリア。 The targeted amphiphilic nanocarrier according to any one of claims 1 to 4, wherein the amphiphilic nanocarrier is a liposome.
  6. 融合タンパク質が、膜結合性ドメイン－scFv－膜結合性ドメイン(２つの膜結合性ドメインは、同一であっても異なっていてもよい)の構造を有する、請求項１～５のいずれか１項に記載の標的化両親媒性ナノキャリア。 The fusion protein according to any one of claims 1 to 5, wherein the fusion protein has a structure of a membrane-binding domain-scFv-membrane-binding domain (the two membrane-binding domains may be the same or different). Targeted amphiphilic nanocarriers according to 1.
  7. 両親媒性ナノキャリアの存在下で、標的化ポリペプチドと膜結合性ドメインの融合タンパク質を無細胞タンパク質合成系で合成させることを特徴とする、前記融合タンパク質が結合された標的化両親媒性ナノキャリアの製造方法。 In the presence of an amphiphilic nanocarrier, a fusion protein of a targeting polypeptide and a membrane-binding domain is synthesized by a cell-free protein synthesis system, and the targeting amphipathic nanoparticle to which the fusion protein is bound, Carrier manufacturing method.
  8. 標的化ポリペプチドがペプチドホルモン、受容体のリガンド、抗体又はその抗原結合性フラグメントである、請求項７に記載の標的化両親媒性ナノキャリアの製造方法 The method for producing a targeted amphiphilic nanocarrier according to claim 7, wherein the targeting polypeptide is a peptide hormone, a receptor ligand, an antibody or an antigen-binding fragment thereof.
  9. 標的化ポリペプチドが、Fab、Fab'、F(ab')2、一本鎖抗体断片(scFv)、二量体化V領域(Diabody)、ジスルフィド安定化V領域(dsFv)又はCDRを含むペプチドからなる群から選ばれる抗原結合性フラグメントである、請求項７に記載の標的化両親媒性ナノキャリアの製造方法。 Targeting polypeptide is a peptide containing Fab, Fab ′, F (ab ′) 2 , single chain antibody fragment (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) or CDR The method for producing a targeted amphiphilic nanocarrier according to claim 7, which is an antigen-binding fragment selected from the group consisting of:
  10. 標的化ポリペプチドが一本鎖抗体断片(scFv)である、請求項９に記載の標的化両親媒性ナノキャリアの製造方法。 The method for producing a targeted amphiphilic nanocarrier according to claim 9, wherein the targeting polypeptide is a single-chain antibody fragment (scFv).
  11. 両親媒性ナノキャリアがリポソームである、請求項７～１０のいずれか１項に記載の標的化両親媒性ナノキャリアの製造方法。 The method for producing a targeted amphiphilic nanocarrier according to any one of claims 7 to 10, wherein the amphiphilic nanocarrier is a liposome.
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