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WO2016167367A1 - Nanosupport amphiphile ciblant et son procédé de production - Google Patents

Nanosupport amphiphile ciblant et son procédé de production Download PDF

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Publication number
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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Japanese (ja)
Inventor
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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Mie University NUC
Kyoto University NUC
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Mie University NUC
Kyoto University NUC
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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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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

L'invention concerne un procédé de production d'un nanosupport amphiphile auquel une protéine fusionnée composée d'un polypeptide de ciblage et d'un domaine de liaison de membrane est liée, ledit procédé étant caractérisé en ce qu'il comprend la synthèse de la protéine de fusion précitée dans un système acellulaire de synthèse de protéines en présence du nanosupport amphiphile.
PCT/JP2016/062184 2015-04-15 2016-04-15 Nanosupport amphiphile ciblant et son procédé de production Ceased WO2016167367A1 (fr)

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WO2023027082A1 (fr) * 2021-08-23 2023-03-02 積水化学工業株式会社 Liposome-exosome hybride lié par un peptide, exosome lié par un peptide, composition les contenant et procédé pour leur formation
JP2025505427A (ja) * 2022-01-28 2025-02-26 リサーチ アンド ビジネス ファウンデーション サンキュンクワン ユニヴァーシティ 膜構造化タンパク質が結合している抗体を含む抗体結合脂質ナノ粒子

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Publication number Priority date Publication date Assignee Title
WO2023027082A1 (fr) * 2021-08-23 2023-03-02 積水化学工業株式会社 Liposome-exosome hybride lié par un peptide, exosome lié par un peptide, composition les contenant et procédé pour leur formation
JP2025505427A (ja) * 2022-01-28 2025-02-26 リサーチ アンド ビジネス ファウンデーション サンキュンクワン ユニヴァーシティ 膜構造化タンパク質が結合している抗体を含む抗体結合脂質ナノ粒子
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