JP7311933B2 - 金ナノ粒子-アプタマー結合体をベースとする抗体伝達体およびその製造方法 - Google Patents
金ナノ粒子-アプタマー結合体をベースとする抗体伝達体およびその製造方法 Download PDFInfo
- Publication number
- JP7311933B2 JP7311933B2 JP2022518165A JP2022518165A JP7311933B2 JP 7311933 B2 JP7311933 B2 JP 7311933B2 JP 2022518165 A JP2022518165 A JP 2022518165A JP 2022518165 A JP2022518165 A JP 2022518165A JP 7311933 B2 JP7311933 B2 JP 7311933B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- aptamer
- present
- cells
- igg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/44—Antibodies bound to carriers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6025—Nucleotides
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3517—Marker; Tag
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Ceramic Engineering (AREA)
- Inorganic Chemistry (AREA)
- Nanotechnology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
前記金ナノ粒子の表面に結合するアプタマー(aptamer)と、を含む、抗体伝達体を提供する。
前記金ナノ粒子の表面に結合するアプタマー(aptamer)と、を含む、抗体伝達体を提供する。
1-1.動物細胞培養(mammalian cell culture)
HeLa(ヒト子宮頸がん)細胞は、ダルベッコ変法イーグル培地(DMEM)で培養し、A549(ヒト扁平上皮がん)細胞は、2.5mM HEPES(N-(2-ヒドロキシエチル)ピペラジン-N’-2-エタンスルホン酸)バッファーを含むRPMI-1640培地で培養した。また、Human melanoma細胞であるMalme-3M、SK-MEL-2細胞は、RPMI-1640培地で培養し、A2058細胞は、ダルベッコ変法イーグル培地で培養した。すべての培地は、10% 加熱不活性化したウシ胎児血清(Caisson,USA)および1%ペニシリン-ストレプトマイシン(Welgene,Korea)を含む。
金ナノ粒子-アプタマー結合体は、二次構造の形成を防止するために、80℃で5分間プレインキュベートさせた。その後、前記金ナノ粒子-アプタマー結合体(AuNP-AptIgG)(1nM)およびモノクローナル抗体を5mM MgCl2(pH7.2)が含まれた1x PBSで常温で10分間反応させ、以後、13,000xgで遠心分離後、上澄み液を除去した。これから製造されたAuNP-AptIgG-antibody複合体は、500mM NaClおよび1M KClが追加されたTBSTを使って洗浄した。
細胞内への抗体伝達を観察するために、リシンコーティングされた10mmカバースリップ(cover slip)上で培養された細胞に金ナノ粒子-アプタマー-抗体複合体(AuNP-AptIgG-antibody complex)を処理した後、1時間培養した後、4%パラホルムアルデヒド(paraformaldehyde)(Sigma,USA)で固定させた。次に、共焦点顕微鏡(laser scanning confocal microscopy)(Carl Zeiss ZEN 2011,Germany)を用いてFITC(490nm excitation,525nm emission)およびAlexa546(556nm excitation,573nm emission)蛍光を探知した。
細胞株を96-wellプレートに接種し、細胞が付着するように一晩中培養した。以後、前記細胞を濃度を異ならせた(0、0.88、1.65および3.3nM)AuNP-AptIgG-BRAFV600Eと培養させ、細胞生存能を測定した(Yeom,J.H.et al.Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of BAX mRNA.PLoS One 8,e75369(2013))。
モノクローナル抗体のFcドメインに結合するアプタマーを用いて抗体伝達体を製造し、その概略的な模式図は図1に示した。
(イ)DNAアプタマー前処理
本発明による抗体伝達体を製造するために、モノクローナル抗体のFcドメインを探知して、これに特異的に結合するIgG-アプタマー(IgG-aptamer)を使用した。より具体的に、前記IgG-アプタマーは、3’末端がチオール基で修飾されたアプタマーであって、5’-TAATACGACTCACTATAGCAATGGTACGGTACTTCCCCACTCACCGGGTACCTGCCGCTCCCAAAAGTGCACGCTACTTTGCTAAAAAAAAAAAA-3’(SH)(配列番号1)の塩基配列からなる。乾燥した前記DNAアプタマーを最終濃度が100μMになるように水に溶かした後、50μlのオリゴに10μlの1N DTT(dithiothreitol)を入れ、室温で15分間反応させた。所望しないチオール基分子を含むDTTを除去するために、エチルアセテート(Ethyl acetate)50μlを入れて混ぜた後、遠心分離して上澄み液を除去し、この過程を3回繰り返した。その後、エタノール沈殿法(EtOH precipitating method)を用いてアプタマーを沈殿させた。
前記(イ)過程を通じて前処理して沈殿したIgG-アプタマーを水に溶かした後、これを金ナノ粒子に添加し、この際、金ナノ粒子(AuNP)として、英国BBI Life Scienceから購入したGold colloid-15nm(#EM.GC15)を使用した。
(イ)DNAアプタマー前処理
本発明による抗体伝達体を製造するために、蛍光トレーサーとして多用されるフルオレセインイソチオシアネート(FITC)と特異的に結合するFITC-アプタマー(FITC-aptamer)を使用した。より具体的に、前記FITC-アプタマーは、3’末端がチオール基で修飾されたアプタマーであって、5’-GGACGGCACCACGGTCGGATCCGTGAGTTGTGACAATTTAGCGGGTGGTATTAGAGCCTACTGCCACAGCAATAGGATCGATACAGATCTAAAAAAAAAA-3’(SH)(配列番号2)の塩基配列からなる。乾燥した前記DNAアプタマーを最終濃度が100μMになるように、水に溶かした後、50μlのオリゴに10μlの1N DTT(dithiothreitol)を入れ、室温で15分間反応させた。所望しないチオール基分子を含むDTTを除去するために、エチルアセテート(Ethyl acetate)50μlを入れて混ぜた後、遠心分離して上澄み液を除去し、この過程を3回繰り返した。その後、エタノール沈殿法(EtOH precipitating method)を用いてアプタマーを沈殿させた。
前記(イ)過程を通じて前処理して沈殿したFITC-アプタマーを前記実施例2-1の(ロ)過程と同じ方法で金ナノ粒子に結合して、抗体伝達体(AuNP-AptFITC)を製造した。
3-1.共焦点顕微鏡分析
前記実施例2-1によって製造された金ナノ粒子-アプタマー結合体を用いて前記実施例1-2の方法で金ナノ粒子-アプタマー-抗体複合体を製造した。次に、金ナノ粒子-アプタマー-抗体複合体をHeLa細胞とA549細胞に処理した後、1時間培養した。培養後、PBSで洗浄した後、4%パラホルムアルデヒド(paraformaldehyde)で固定させ、固定させたサンプルを免疫染色(immunostaining)した後、共焦点顕微鏡(confocal microscope)で観察した。
AuNP-AptIgGにモノクローナル抗体PARP1、VP6、NSD2、Aurora A、Pax5、Vimentin、Rock-1、Rock-2、RhoE、P53、Mcl-1、P50、BRAFを反応させて、金ナノ粒子-アプタマー-抗体複合体を製造した後、前記抗体がHeLa細胞およびA549細胞に伝達されるかを確認するために、二次抗体であるMouse-546(Red)で標識し、前記実施例3-1の方法で共焦点顕微鏡分析を実施した。
前記実施例2-2の方法で製造されたアプタマー-金ナノ粒子結合体(AuNP-AptFITC)とFITCで標識されたモノクローナル抗体IgG(FITC-IgG)を反応させて製造された金ナノ粒子-アプタマー-抗体複合体(AuNP-AptIgG-FITC-IgG)をヒト皮膚がん細胞株(Malme-3MおよびSK-MEL2)に処理して1時間培養した後、これを共焦点蛍光顕微鏡で観察した。この際、細胞は、40x水浸対物レンズ(water immersion objective)を使って視覚化した。
ヒト皮膚がん細胞株(A2058、SK-MEL2、およびMalme-3M)へのAuNP-AptIgG-BRAFV600Eの伝達による細胞生存能を前記実施例1-4の方法で測定した。ヒト皮膚がん細胞株の治療用ターゲット分子として使用されるBRAFV600E抗体をBRAF突然変異細胞株A2058、Malme-3MにAuNP-AptIgGを用いて伝達した結果、図4に示されたように、突然変異のない皮膚がん細胞株SK-MEL2に比べて細胞生存能が減少したことを確認できた。
Claims (8)
- 金ナノ粒子と、
前記金ナノ粒子の表面に結合するアプタマー(aptamer)と、を含む、抗体運搬体であって、
抗体のFcドメインに特異的に結合する前記アプタマーは、配列番号1の塩基配列を含むか;又は、
抗体に結合するFITCに特異的に結合する前記アプタマーは、配列番号2の塩基配列を含む、抗体運搬体。 - 前記抗体運搬体は、前記アプタマーにIgG、PARP1、VP6、NSD2、Aurora A、Pax5、Vimentin、Rock-1、Rock-2、RhoE、P53、Mcl-1、P50、およびBRAFからなる群から選択される一つ以上のモノクローナル抗体を結合して細胞内に運搬することを特徴とする請求項1に記載の抗体運搬体。
- 前記金ナノ粒子は、10~20nmの直径を有することを特徴とする請求項1に記載の抗体運搬体。
- 前記抗体運搬体は、細胞の核、細胞質、およびミトコンドリアからなる群から選択される一つ以上の内部に抗体を運搬することを特徴とする請求項1に記載の抗体運搬体。
- 金ナノ粒子にアプタマー(aptamer)を結合させる段階を含む、抗体運搬体の製造方法であって、
抗体のFcドメインに特異的に結合する前記アプタマーは、配列番号1の塩基配列を含むか;又は、
抗体に結合するFITCに特異的に結合する前記アプタマーは、配列番号2の塩基配列を含む、方法。 - 請求項1に記載の抗体運搬体と運搬しようとする抗体とが結合してなる複合体。
- 請求項6に記載の複合体を有効成分として含む、がんの予防または治療用薬学的組成物。
- 請求項6に記載の複合体の、がんの予防または治療に用いられる薬剤の生産のための使用。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020190066491A KR102336120B1 (ko) | 2019-06-05 | 2019-06-05 | 금 나노입자-앱타머 결합체를 기반으로 하는 항체 전달체 및 이의 제조 방법 |
KR10-2019-0066491 | 2019-06-05 | ||
PCT/KR2019/011838 WO2020246660A1 (ko) | 2019-06-05 | 2019-09-11 | 금 나노입자-앱타머 결합체를 기반으로 하는 항체 전달체 및 이의 제조 방법 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2022536214A JP2022536214A (ja) | 2022-08-12 |
JP7311933B2 true JP7311933B2 (ja) | 2023-07-20 |
Family
ID=73652453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2022518165A Active JP7311933B2 (ja) | 2019-06-05 | 2019-09-11 | 金ナノ粒子-アプタマー結合体をベースとする抗体伝達体およびその製造方法 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20210100915A1 (ja) |
EP (1) | EP3821907B1 (ja) |
JP (1) | JP7311933B2 (ja) |
KR (1) | KR102336120B1 (ja) |
CN (1) | CN112334154B (ja) |
CA (1) | CA3139371A1 (ja) |
WO (1) | WO2020246660A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113252658B (zh) * | 2021-06-28 | 2021-11-09 | 中国农业大学 | 一种基于金纳米识别适配体冗余碱基的方法 |
KR20230167291A (ko) | 2022-05-30 | 2023-12-08 | 주식회사 엔이에스바이오테크놀러지 | 금나노입자-앱타머 결합체를 기반으로 하는 항균 단백질 전달체 및 이의 제조 방법 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009014404A2 (en) | 2007-07-25 | 2009-01-29 | Korea Research Institute Of Bioscience And Biotechnology | Multifunctional protein simultaneously delivering antibodies and nanoparticles |
US20140350230A1 (en) | 2011-10-17 | 2014-11-27 | Research & Business Foundation Sungkyunkwan University | Multi-functional nucleic acid-based anti-cancer drug capable of targeting and therapy, method for preparing same and anti-cancer composition comprising same |
EP2995300A1 (en) | 2013-02-13 | 2016-03-16 | Chung-Ang University Industry-Academic Cooperation Foundation | Protein transduction domain based on gold nanoparticle-aptamer conjugate and method for producing same |
KR101613020B1 (ko) | 2014-10-20 | 2016-04-18 | 서강대학교산학협력단 | 표적항원 검출용 복합체 및 이의 제조방법 |
JP2018516854A (ja) | 2015-04-07 | 2018-06-28 | メモリアル スローン ケタリング キャンサー センター | ナノ粒子イムノコンジュゲート |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101430987B1 (ko) * | 2011-07-26 | 2014-08-21 | (주) 더바이오 | 항체 결합 나노 입자의 클러스터가 결합된 자성 입자의 제조 방법, 이에 의하여 제조된 항체 결합 나노 입자의 클러스터가 결합된 자성 입자, 및 이를 이용하여 제조된 세포 독성 t 세포 확대 배양을 위한 자성 입자 어레이 |
KR102006426B1 (ko) * | 2016-04-29 | 2019-08-02 | 주식회사 파마웍스 | 금 나노입자의 레일리 산란 현상 및 비색적 분석법을 이용한 알츠하이머 진단용 바이오센서 및 상기 바이오센서를 이용한 다중검지법 |
KR101966505B1 (ko) * | 2016-12-05 | 2019-04-05 | 중앙대학교 산학협력단 | 항균용 나노 구조체 및 이의 용도 |
CN106932591B (zh) * | 2017-01-17 | 2019-05-24 | 华中师范大学 | 一种基于适配体检测细胞因子的非标记电化学生物传感器及细胞因子实时检测方法 |
KR101873840B1 (ko) | 2017-12-05 | 2018-07-04 | 한림대학교 산학협력단 | 금나노 입자가 담지된 대식세포와 연속광파 방식의 확산광 분광법을 이용한 폐질환 영상 진단 방법 |
KR102254421B1 (ko) | 2017-12-05 | 2021-05-20 | 성화정 | 패션후르츠 과육과 씨의 혼합 추출물을 유효성분으로 함유하는 혈전증의 예방 또는 치료용 약학적 조성물 및 건강 기능 식품 |
CN108309995A (zh) * | 2018-01-16 | 2018-07-24 | 华中师范大学 | 一种基于化学信号诱导的刺激-响应磁性纳米粒子的药物释放体系 |
-
2019
- 2019-06-05 KR KR1020190066491A patent/KR102336120B1/ko active IP Right Grant
- 2019-09-11 CA CA3139371A patent/CA3139371A1/en active Pending
- 2019-09-11 CN CN201980028325.1A patent/CN112334154B/zh active Active
- 2019-09-11 WO PCT/KR2019/011838 patent/WO2020246660A1/ko unknown
- 2019-09-11 EP EP19923712.4A patent/EP3821907B1/en active Active
- 2019-09-11 JP JP2022518165A patent/JP7311933B2/ja active Active
-
2020
- 2020-10-27 US US17/081,356 patent/US20210100915A1/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009014404A2 (en) | 2007-07-25 | 2009-01-29 | Korea Research Institute Of Bioscience And Biotechnology | Multifunctional protein simultaneously delivering antibodies and nanoparticles |
US20140350230A1 (en) | 2011-10-17 | 2014-11-27 | Research & Business Foundation Sungkyunkwan University | Multi-functional nucleic acid-based anti-cancer drug capable of targeting and therapy, method for preparing same and anti-cancer composition comprising same |
EP2995300A1 (en) | 2013-02-13 | 2016-03-16 | Chung-Ang University Industry-Academic Cooperation Foundation | Protein transduction domain based on gold nanoparticle-aptamer conjugate and method for producing same |
KR101613020B1 (ko) | 2014-10-20 | 2016-04-18 | 서강대학교산학협력단 | 표적항원 검출용 복합체 및 이의 제조방법 |
JP2018516854A (ja) | 2015-04-07 | 2018-06-28 | メモリアル スローン ケタリング キャンサー センター | ナノ粒子イムノコンジュゲート |
Non-Patent Citations (1)
Title |
---|
Ha Minh Hiep et al.,"RNA aptamer-based optical nanostructured sensor for highly sensitive and label-free detection of antigen-antibody reactions",Analytical and Bioanalytical Chemistry,2010年,Vol.396,p.2575-2581 |
Also Published As
Publication number | Publication date |
---|---|
CN112334154B (zh) | 2024-07-12 |
EP3821907A1 (en) | 2021-05-19 |
EP3821907A4 (en) | 2022-03-23 |
EP3821907C0 (en) | 2023-10-25 |
EP3821907B1 (en) | 2023-10-25 |
CN112334154A (zh) | 2021-02-05 |
JP2022536214A (ja) | 2022-08-12 |
WO2020246660A1 (ko) | 2020-12-10 |
CA3139371A1 (en) | 2020-12-10 |
KR102336120B1 (ko) | 2021-12-08 |
US20210100915A1 (en) | 2021-04-08 |
KR20200139946A (ko) | 2020-12-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Hosseini et al. | The use of nanoparticles as a promising therapeutic approach in cancer immunotherapy | |
Jiang et al. | Progress and challenges in developing aptamer-functionalized targeted drug delivery systems | |
Kang et al. | Developing an antibody-binding protein cage as a molecular recognition drug modular nanoplatform | |
JP7455510B2 (ja) | 悪性脳腫瘍における標的化粒子の浸透、分布および応答のための組成物及び方法 | |
Huang et al. | Nanomedicine–a promising therapy for hematological malignancies | |
EP2714094B1 (en) | Anti-nucleolin agent-conjugated nanoparticles | |
JP6346265B2 (ja) | Dnaインターカレーティング剤の細胞送達 | |
JP7311933B2 (ja) | 金ナノ粒子-アプタマー結合体をベースとする抗体伝達体およびその製造方法 | |
US20140275509A1 (en) | Multifunctionalized materials | |
US11666664B2 (en) | Self assembling molecules for targeted drug delivery | |
US20200370049A1 (en) | Methods and compositions for treating cancer using peptide nucleic acid-based agents | |
JP2007527881A (ja) | 薬学的に活性な物質を細胞特異的に濃縮するためのタンパク質ベースのナノ粒子形態の支持システム | |
JP7177131B2 (ja) | アポトーシスを誘導するための組成物及び方法 | |
JP2009533350A (ja) | B細胞悪性疾患を処置するための組成物および方法 | |
JP2022541650A (ja) | ペプチド核酸系の作用物質を用いて癌を治療するための方法および組成物 | |
JP7069135B2 (ja) | アルブミンベースのナノ医薬品を使用するための組成物及び方法 | |
JP2019517477A (ja) | 薬物抵抗性癌のための薬物送達ナノ粒子および治療 | |
KR101973535B1 (ko) | 그래핀 양자점 복합체 | |
Shakil et al. | Theranostic potentials of gold nanomaterials in hematological malignancies | |
JP2022541765A (ja) | 抗grp78抗体およびその使用方法 | |
US20230065416A1 (en) | Clathrin-chimetic antibody receptor constructs for immune cell activation therapy in vivo | |
Pandey et al. | Aptamer-guided Selective Delivery of Therapeutics to Breast Cancer Cells Expressing Specific Biomarkers | |
Banerjee et al. | Prodrug Conjugate Strategies in Targeted Anticancer Drug Delivery Systems | |
Gollakota | Commercial Feasibility of the Multicomponent Nanochain Therapy | |
JP2020507636A (ja) | 細胞表面チオールとの反応のためのチオール反応性基を含有する細胞内在化コンジュゲート |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20211201 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20211201 |
|
RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7426 Effective date: 20220408 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20220408 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221227 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230324 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230608 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230630 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7311933 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |