JP7140743B2 - Fc結合能を有する融合タンパク質を含む細胞外小胞の使用 - Google Patents
Fc結合能を有する融合タンパク質を含む細胞外小胞の使用 Download PDFInfo
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- JP7140743B2 JP7140743B2 JP2019502253A JP2019502253A JP7140743B2 JP 7140743 B2 JP7140743 B2 JP 7140743B2 JP 2019502253 A JP2019502253 A JP 2019502253A JP 2019502253 A JP2019502253 A JP 2019502253A JP 7140743 B2 JP7140743 B2 JP 7140743B2
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Description
エキソソームポリペプチド-Fc結合ポリペプチド-Fc結合ポリペプチド
エキソソームポリペプチドドメイン-Fc結合ポリペプチド-エキソソームポリペプチドドメイン-Fc結合ポリペプチド
エキソソームポリペプチドドメイン-Fc結合ポリペプチドA-エキソソームポリペプチドドメイン-Fc結合ポリペプチドB
シンテニン-細胞質TNRF-フォールドオン三量体化ドメイン-膜貫通TNFRドメイン-Zドメイン-細胞外TNFRドメイン
シンデカン-ロイシンジッパードメイン-gp130細胞質ドメイン-gp130膜貫通ドメイン-Fc結合ポリペプチド-gp130細胞外ドメイン
- CD81-プロテインA/GCD81第2ループ(非限定的な例として、配列番号75)
- CD9-ZZドメインCD9S第2ループ(非限定的な例として、配列番号76)
- FCAR細胞外ドメイン-2XGGGgSリンカー-Lamp2b(非限定的な例として、配列番号55)
- FCAR細胞外ドメイン-4XGSリンカー-Lamp2b(非限定的な例として、配列番号54)
- FCGR1A細胞外ドメイン-2XGGGgSリンカーLamp2b(非限定的な例として、配列番号49)
- FCGR1A細胞外ドメイン-4XGSリンカー-Lamp2b(非限定的な例として、配列番号48)
- FcRN細胞外ドメイン-4XGSリンカー-Lamp2b(非限定的な例として、配列番号39)
- FcRN-2XGGGgSリンカー-Lamp2b(非限定的な例として、配列番号40)
- Gp130細胞外ドメイン-2XGGGGSリンカー-FCAR細胞外ドメイン-Gp130膜貫通ドメイン-ロイシンジッパー-N末端シンテニン(非限定的な例として、配列番号52)
- Gp130細胞外ドメイン-2XGGGGSリンカー-FCGRIA細胞外ドメイン-Gp130膜貫通ドメイン-ロイシンジッパー-N末端シンテニン(非限定的な例として、配列番号46)
- Gp130細胞外ドメイン-2XGGGGSリンカー-FcRN細胞外ドメイン-Gp130膜貫通ドメイン-ロイシンジッパー-N末端シンテニン(非限定的な例として、配列番号43)
- Gp130細胞外ドメイン-2XGGGGSリンカー-Zドメイン-Gp130膜貫通ドメイン-ロイシンジッパー-N末端シンテニン(非限定的な例として、配列番号34)
- トランスフェリン受容体-2XGGGGSリンカー-FCAR細胞外ドメイン(非限定的な例として、配列番号56)
- トランスフェリン受容体-2XGGGGSリンカー-FCGR1A細胞外ドメイン(非限定的な例として、配列番号50)
- トランスフェリン受容体-2XGGGGSリンカー-FcRN細胞外ドメイン(非限定的な例として、配列番号41)
- トランスフェリン受容体-2XGGGGSリンカー-Zドメイン(非限定的な例として、配列番号38)
- CD63-FCAR細胞外ドメインCD63第1ループおよびCD63第2ループ(非限定的な例として、配列番号53)
- CD63-FCGR1A細胞外ドメインCD63第1ループおよびCD63第2ループ(非限定的な例として、配列番号47)
- CD63-FcRN細胞外ドメインCD63第1ループおよびCD63第2ループ(非限定的な例として、配列番号44)
- CD63-ZドメインCD63第1ループおよびCD63第2ループ(非限定的な例として、配列番号35)
- TNFR細胞外ドメイン-2XGGGGSリンカー-FCAR細胞外ドメイン-TNFR膜貫通ドメイン-フォールドオン-N末端シンテニン(非限定的な例として、配列番号51)
- TNFR細胞外ドメイン-2XGGGGSリンカー-FCGRIA細胞外ドメイン-TNFR膜貫通ドメイン-フォールドオン-N末端シンテニン(非限定的な例として、配列番号45)
- TNFR細胞外ドメイン-2XGGGGSリンカー-FcRN細胞外ドメイン-TNFR膜貫通ドメイン-フォールドオン-N末端シンテニン(非限定的な例として、配列番号42)
TNFR細胞外ドメイン-2XGGGGSリンカー-Zドメイン-TNFR膜貫通ドメイン-フォールドオン-N末端シンテニン(非限定的な例として、配列番号33)
- Zドメイン-2XGGGgSリンカー-Lamp2b(非限定的な例として、配列番号37)
- Zドメイン-4XGSリンカー-Lamp2b(非限定的な例として、配列番号36)
- トランスフェリン受容体-プロテインAG (非限定的な例として、配列番号72に作動可能に融合した、非限定的な例として、配列番号10)
limomab aritox)、またはそれらの任意の組み合わせの任意の1つ以上であり得る。重要なことに、本発明は、その標的が細胞内および/または細胞外のいずれかにかかわらず、任意の抗体の送達を提供する。本発明のEVの抗体を内在化する能力は、モノクローナル抗体開発における段階変化を表し、抗原を標的とすることができ、標的細胞の細胞内小器官全体にわたって標的とする。重要なことに、本発明による抗体およびFc含有タンパク質は、検出および画像化の目的のために、例えば、フルオロフォア、MRI剤、PET剤、放射性物質、酵素、ナノ粒子、金属、有機および無機化合物等を用いて、有利にも標識され得る。
実験部分
材料および方法
・細胞培養およびトランスフェクション
HEK293T細胞は、典型的には、ATCCに推奨されるように、15cmディッシュに播種され(ディッシュ当たり9×106細胞)、血清含有DMEM中に一晩残した。次の日に、細胞を、細胞に直接添加したリポプレックス化DNAで一過性にトランスフェクトされた。手短に、DNAとポリエチレンイミン(PEI)を個別にOptiMEM中で5分間インキュベートし、その後に、20分間、室温で、共に混合した。リポプレックス化DNAと細胞を、6時間共インキュベートし、続いて、培養上清をOptiMEMに48時間交換した。ディッシュ、フラスコ、および他の細胞培養容器中で評価された他の細胞および細胞株は、骨髄由来間葉系間質細胞(BM-MSC)およびワルトン膠様質由来MSC(WJ-MSC)、羊膜細胞、線維芽細胞、様々な内皮細胞および上皮細胞、ならびに様々な免疫細胞および細胞株を含んだ。
・アッセイおよび分析
典型的にはTFFおよびLC、特にビーズ溶出液LC等の濾過の組み合わせである様々な方法を用いてEVは単離および精製された。典型的には、EV含有媒体は、回収され、300g5分間の低速度回転に供され、続いて、2000g10分間の回転により、より大きな粒子および細胞残屑を除去した。上清を0.22μmシリンジフィルターによって濾過し、種々の精製ステップに供した。大容量は、Vivaflow 50R tangential flow(TFF)装置(Sartorius)を用いて、100kDaカットオフフィルターによって、またはKR2i TFF system(SpectrumLabs)を用いて、100または300kDaカットオフ中空糸フィルターによって、透析濾過され、およそ20mlに濃縮した。予め濃縮された媒体は、続けて、AKTAprime plusまたはAKTA Pure 25クロマトグラフィーシステム(GE Healthcare Life Sciences)に接続されたビーズ溶出液カラム(HiScreen or HiTrap Capto Core 700 column, GE Healthcare Life Sciences)にロードされた。カラム平衡化のための流速設定、試料ロード、および適切なカラム清浄化の手順は、製造者の説明書に従って選択された。試料は、UV吸収クロマトグラムに従って回収され、Amicon Ultra-15 10kDa分子量カットオフスピンフィルター(Millipore)を用いて、100μlの最終容量に濃縮され、さらなる下流の分析のために-80℃で保存された。タンパク質およびRNAの溶出プロファイルを評価するために、媒体は濃縮され、100kDaおよび300kDa中空糸フィルターを用いるKR2i TFF systemによって透析濾過され、Tricorn 10/300 Sepharose 4 Fast Flow (S4FF)カラム(GE Healthcare Life Sciences)で試料分析した。
実施例
実施例1Fc結合ポリペプチドを含むEV(Fc-結合EV)へのIgGの結合
実施例2:抗HER2抗体の送達のためのFCGR1A Lamp2B EV
実施例3:Fc結合EVを介する抗体の細胞内取り込みおよび送達
Claims (9)
- 研究ツールとして、診断ツールとして、画像化ツールとして、生物学的基準材料(RM)として、実験対照として、および/または実験標準としての使用のための、少なくとも1つのFc結合ポリペプチドを含む細胞外小胞(EV)を含む組成物であって、
前記少なくとも1つのFc結合ポリペプチドが少なくとも1つのエキソソームポリペプチドと共に融合タンパク質の一部を形成し、前記少なくとも1つのFc結合ポリペプチドが、前記EVの外部表面上に提示される、組成物。 - 前記使用が、フローサイトメトリー、イメージングフローサイトメトリー、ナノ粒子トラッキング解析、ナノイメージング、共焦点顕微鏡、蛍光顕微鏡、動的光散乱、電子顕微鏡、発光に基づく検出、蛍光相関分光法、蛍光共鳴エネルギー移動(FRET)に基づく技術、磁気、蛍光、および/または浮力に基づく標識に基づく陽性もしくは陰性の選択技術、粒子の密度、電荷、もしくはサイズに基づく陽性もしくは陰性の選択技術、または任意のそれらの組み合わせにおけるものである、請求項1に記載の組成物。
- 前記Fc結合ポリペプチドがFc含有タンパク質のFcドメインに結合している、請求項1または2に記載の組成物。
- Fc含有タンパク質が抗体である、請求項3に記載の組成物。
- Fc含有タンパク質が少なくとも1つのフルオロフォア、少なくとも1つの放射性標識、少なくとも1つのPETリガンド、少なくとも1つのMRI剤、ナノ粒子、無機化合物、または任意の他の検出部分に結合している、請求項3または4に記載の組成物。
- 前記使用が、フローサイトメトリー、ナノ粒子トラッキング解析、および/または動的光散乱のための生物学的RMとしてのものである、請求項1~5のいずれか1項に記載の組成物。
- 前記RMが、補償、較正、標準化、または検証のために使用される、請求項6に記載の組成物。
- 前記EV自体が、少なくとも1つの検出部分を含む、請求項1~7のいずれか1項に記載の組成物。
- 前記少なくとも1つの検出部分が、少なくとも1つのフルオロフォア、少なくとも1つの染料、少なくとも1つの蛍光ポリペプチド、少なくとも1つの放射性標識、少なくとも1つのPET剤、少なくとも1つのMRI剤、無機化合物、有機化合物、または任意の他の検出部分を含む群から選択される、請求項8に記載の組成物。
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JP2007295929A (ja) | 2006-04-05 | 2007-11-15 | Geno Membrane:Kk | イヌBsep遺伝子 |
WO2015058148A1 (en) | 2013-10-17 | 2015-04-23 | Children's Hospital Los Angeles | Antibody dependent exosome therapy |
Non-Patent Citations (1)
Title |
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CHEN Chien-Sheng et al.,Protein G-liposomal nanovesicles as universal reagents for immunoassay,Talanta,2015年,Vol.67,pp.205-211 |
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