JP2021531020A - Icam−1アプタマー、その診断および治療用途 - Google Patents
Icam−1アプタマー、その診断および治療用途 Download PDFInfo
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Abstract
Description
アプタマー
最近、アプタマーと呼ばれる機能的なオリゴヌクレオチドベースの生体分子が、抗体の有望な代替物として大きな関心を集めている。アプタマー選択の技術は、病気の診断と治療に応用できるため科学界において注目されている。
細胞間接着分子(ICAM)は、免疫グロブリンスーパージーンファミリーの構造的に関連する膜貫通糖タンパク質であり、白血球に存在するβ2インテグリン分子のリガンドである(Almenar−Queralt A. et al., Am. J. Pathol., 1995, 147(5), 1278−1288; Hubbard AK et al, Free Radic. Biol. Med., 2000, 28, 1379−1386)。同定された5つのICAMのうち、ICAM−1が最もよく研究されている(Koning et al., Endothelium, 2002, 9, 161−171; Muro et al., Curr. Pharm. Des., 2005, 11, 2383−2401)。ICAM−1(細胞間接着分子1)は、ヒトにおいてICAM1遺伝子によってコードされるタンパク質である。この遺伝子は、通常、内皮細胞および免疫系の細胞上に発現する細胞表面をコードする。ICAM−1は、CD11a/CD18型またはCD11b/CD18型のインテグリンに結合し、受容体としてライノウイルスに利用される。
本発明は、ICAM−1分子に高い親和性で結合することができるRNAアプタマーの同定に基づく。特に、本発明のアプタマーは、それが生理学的に存在する部位、すなわち、ICAM−1が発現する細胞表面上で標的を特異的に認識するという課題を解決し、in vivoでそれらの使用に適していることを実証する。実際、本明細書に記載のアプタマーは、表面でICAM−1を過剰発現している細胞に直接結合できることが見出された。
UCAUGAUACGUUGCGUGAGCAACUGCGGCGCUAAAGUUUGGUUGACUAGUACAUG(配列番号1)はICAM−1に対して最も高い結合親和性を示した。
GGGAAGAGAAGGACAUAUGAUCAUGAUACGUUGCGUGAGCAACUGCGGCGCUAAAGUUUGGUUGACUAGUACAUGACCACUUGA (配列番号2)
は、同様にICAM−1に結合することができることが示された。
本発明のアプタマーは、例えば、ヌクレアーゼに対する耐性を高めるため、薬物動態を調節するため、または診断部分または治療部分とコンジュゲートさせるために修飾することができる。
配列番号1は、アプタマー10.T(55nt)の5’からの配列を示している。
5’ UCAUGAUACGUUGCGUGAGCAACUGCGGCGCUAAAGUUUGGUUGACUAG
UACAUG 3’
配列番号2は、アプタマー12c−10(84nt)の5’からの配列を示している。
5’ GGGAAGAGAAGGACAUAUGAUCAUGAUACGUUGCGUGAGCAACUGCGGCG
CUAAAGUUUGGUUGACUAGUACAUGACCACU UGA 3’
装置
フローサイトメトリーデータの取得は、BD AccuriTM C6フローサイトメーター(BD Bioscience)を使用して行った。RT−qPCRは、StepOneTM PlusリアルタイムPCRシステム(Applied Biosystems)により行った。ゲルの可視化は、Gel Doc EZ System(Bio−Rad)を用いて行った。ELONAデータは、MultiskanTM FCマイクロプレート光度計(ThermoFisher Scientific)によって取得した。
ICAM−1 細胞間接着分子−1
SELEX Systematic Evolution of Ligands by Exponential enrichment
RNA リボ核酸
DNA デオキシリボ核酸
WT 野生型
nt ヌクレオチド
HSA ヒト血清アルブミン
KD 解離定数
HMEC−1 ヒト乳腺上皮細胞−1
TNFalpha 腫瘍壊死因子アルファ
COS7 SV40遺伝物質を有するCV−1(サル)を起源とする細胞
Rt−qPCR リアルタイムポリメラーゼ連鎖反応
ICAM−1に対するアプタマーを特異的に選択するため、細胞表面に存在する他のタンパク質について異なる2つの細胞株(陽性/陰性)の代わりに、同じ細胞株(過剰発現した標的あり/なし)を用いて、cell−SELEX法に従い行った。
短いアプタマー10.T(配列番号1)が、元の分子である12C−10(配列番号2)の活性部位を含み、COS7−ICAM−1に対する高い結合と親和性を保存しているかを試験する目的で、長いアプタマーのスクリーニングに使用したのと同じ条件を維持して結合アッセイを行った。
アプタマー10.Tの結合について別の実験でさらに調べた。5’末端をビオチン化したアプタマー10.Tについてプルダウンアッセイを行い、その配列が、標的であるICAM−1に結合することを確認した。COS7細胞をICAM−1 cDNAで48時間一過性トランスフェクトして回収し、リゼート300μgを、1μMのビオチン化アプタマー10.T、およびネガティブコントロールとして使用するICAM−1−非関連2’F−RNA配列とインキュベートした。複合体をストレプトアビジンアガロースビーズと連続してインキュベートした。数回の洗浄と変性の後、図7に示すように、サンプルをイムノブロッティングで分析した。
アプタマー10.Tを、ヒト血清中での安定性について試験した。87%ヒト血清中で37℃にてインキュベートした。サンプルを種々の時間(T0、1、2、4、8、12、24、48、72時間)で採取し、プロテイナーゼKと37℃で1時間インキュベートして血清タンパク質を分解し、変性ゲルにロードした。結果は、アプタマー10.Tの半減期が4〜8時間であることを示した。安定性の結果を図6に示す。
イメージング用途における10.Tアプタマー使用の可能性を実証するため、C12−アミノリンカー(5’−C12−NH2)を挿入した後、RNA配列の5’末端を市販の色素Alexa Fluor488に結合させた。アミノリンカーは、塩基的触媒作用によりC12脂肪族ジアミンとの縮合により5’末端リン酸に挿入した。得られたフリーのNH2部分を、市販のAlexa Fluor 488−NHSエステルと反応させて共有結合によりアミド結合を形成した。Alexa Fluor 488−NHSエステルを高純度の無水ジメチルホルムアミド(DMF)またはジメチルスルホキシド(DMSO)に溶解し、0.1〜0.2M炭酸水素ナトリウム緩衝液(pH 8.3)中で室温にて反応させた。精製を、PAGE、続いてHPLCにより実施した。
標的に対する親和性を調べるため、アプタマー10.Tを、酵母tRNA 200μg/mLで前処理した後、濃度を上げながら(10−50−100−250−500−1000nM)COS7−ICAM−1細胞上で37℃にて15分間インキュベートした。同じ実験を、ネガティブコントロールとして非関連アプタマーA10(配列はLupold S.E. et al., Cancer Res. 2002, 62, 4029−4033に開示)を用いて行った。結合をRT−qPCRによって評価した。図8に示されるようにKd値は375.7±151.7nMであった。
ヒト血清アルブミン(HSA)のKd値を計算するため、ELONAアッセイを行った。ビオチン化アプタマー10.Tを、HSA 25nMで事前にコーティングしたまたはコーティングされていない96ウェルマイクロタイター高結合プレート上で、濃度を上げながら(1−10−100−1000nM)インキュベートした。Kd値は計算できず、10.Tは1000nM以下ではHSAと反応しないことを示した。各実験では、ビオチン化ポリクローナル抗体抗HSAをポジティブコントロールとして使用した。この実験の結果を図11に示す。
Claims (15)
- 配列番号1のRNA配列を含む、細胞間接着分子−1(ICAM−1)に結合するアプタマー。
- 最大100ヌクレオチドの長さを有することを特徴とする、請求項1に記載のアプタマー。
- 500nM〜50nMの解離定数でICAM−1に結合することができる、請求項1または2に記載のアプタマー。
- 配列番号2のRNA配列を含む、請求項1〜3のいずれかに記載のアプタマー。
- すべてのピリミジン残基が2’−フルオロピリミジンに修飾されている、請求項1〜4のいずれかに記載のアプタマー。
- 少なくとも1つの化学修飾を含むようさらなる修飾がされており、該修飾が、糖の位置での化学的置換;リン酸の位置での化学置換;および核酸の塩基の位置での化学置換、から選択される、請求項5に記載のアプタマー。
- 修飾が、修飾ヌクレオチドの組み込み、化合物とのコンジュゲーション、およびレポーター部分による標識からなる群から選択される、請求項6に記載のアプタマー。
- レポーター部分が、フルオロフォア部分、磁性または常磁性部分、放射性標識部分、親和性標識、X線部分、超音波イメージング部分、光音響イメージング部分およびナノ粒子ベース部分からなる群から選択される、請求項7に記載のアプタマー。
- ICAM1関連の病状、障害、機能不全、状態または疾患の診断、治療または視覚化における使用のための、請求項1〜8のいずれかに記載のアプタマー。
- ICAM−1関連の病状、障害、機能不全、状態または疾患が、炎症または炎症関連疾患である、請求項9に記載の使用のためのアプタマー。
- 前記診断または視覚化が、ICAM−1を発現する体組織または器官系のイメージングを含んでなる、請求項9に記載の使用のためのアプタマー。
- 前記組織および器官系が、それぞれ内皮および血管である、請求項11に記載の使用のためのアプタマー。
- 薬学的に許容される担体および賦形剤と共に、請求項1〜8のいずれかに記載のアプタマーを含有する、診断、治療またはイメージング組成物。
- 器官または組織のイメージングに適している、請求項13に記載の組成物。
- イメージングが、磁気共鳴画像法、陽電子放出断層撮影法(PET)、コンピュータ断層撮影法(CT)、超音波、光音響画像法(PAI)、近赤外蛍光(NIRF)、または単一光子放出コンピュータ断層撮影(SPECT)に基づく、請求項14に記載の組成物。
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