JP7083820B2 - 膜マーカー - Google Patents
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- JP7083820B2 JP7083820B2 JP2019517354A JP2019517354A JP7083820B2 JP 7083820 B2 JP7083820 B2 JP 7083820B2 JP 2019517354 A JP2019517354 A JP 2019517354A JP 2019517354 A JP2019517354 A JP 2019517354A JP 7083820 B2 JP7083820 B2 JP 7083820B2
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Description
-上に定義される二重特異性抗体と、
-レンチウイルスによる感染症、特にHIVまたはSIVに関連した感染症の治療における使用のための、抗レトロウイルス剤の組み合わせ、との両方、
-およびもしかすると(potentially)、細胞内リザーバを根絶する組成物を含む、組成物に関する。
本発明によって取り組まれる問題は、インビトロモデルおよび有効な抗ウイルス(抗レトロウイルス)治療中のHIV-1に感染した患者の一次細胞のエクスビボ表現型調査によって、感染した細胞の特異的マーカーを同定し、検証することである。感染した細胞の表面上の、特にCD32マーカーの特異的発現のインビトロおよびエクスビボを用いて、HIV-1に感染した患者におけるウイルスリザーバを標的とし、排除すること、したがって感染した患者においてウイルスを決定的に根絶するのを可能にする有効な治療法を提案することができる。
1.ウイルス産生およびVLP
Vpxおよびウイルス粒子を含有するVLPは、293T細胞中のDNAのリン酸カルシウム形質移入についての標準プロトコルに従って産生した。VLP-Vpxは、8μgのpSIV3+プラスミドおよび2μgのpMD2-G VSV-Gプラスミドを同時形質移入することによって産生した。形質移入後16時間で培養培地を置き換えた後、48時間後にVLPを抽出し、それらを遠心分離し、それらを0.45mmフィルター上で濾過し、それらを超遠心分離によって100倍遠心分離した。HIV-1-CMV-eGFPウイルス粒子は、5μgのpHRETプラスミド、5μgのpsPAX2パッケージングプラスミド、および2μgのpMD2-Gプラスミドを同時形質移入することによって産生した。濃縮後、p24力価ウイルスストックを、ELISAによって測定し、感染力価(MOI)を、293T細胞上の力価によって測定した。
健康なドナーからの末梢血単核細胞を、密度勾配(Ficoll)によって単離し、24ウェルプレート上でVLP-Vpxの存在下、最終300μLの完全培地(RPMI 10%SVF)中2.106細胞/ウェルの濃度で12時間培養した。次いで、HIV-1-CMV-eGFP(10倍のMOIに相当する1μg p24)を添加することによって、細胞を感染させた。対照として、VLP-Vpx、HIV-1-CMV-egFPの存在下で排他的に細胞を培養するか、または未治療のままにした。感染の3日後に、静止状態の感染した(XH+)TCD4+細胞(CD69-HLA-DR-)、HIV-1-CMV-eGFP(XH-)で排他的に治療された静止状態のTCD4+細胞、および対照(XまたはNT)を、ソーターによって単離した。ソートした細胞を、β-メルカプトエタノールを添加したRA1中に再懸濁し、全RNA抽出前に-80℃で貯蔵した。
XH+、XH-、X、およびNT分画に由来する全RNAを、GE Healthcare Illustra RNAミニキットを使用して抽出した。RNAの品質を、Agilentからの2100 Bioanalyzer上で、およびRNA Nanochipによって分析した。次いで、Illuminaライブラリーを確立した。シーケンシング前に、試料を多重化した。異なる分画について、正則化対数変換遺伝子発現カウントの主成分分析を実行した。
有効に治療されたHIV-1患者からの末梢血単核細胞(ウイルス負荷<20コピーのRNA HIV-1/mL血液)を、密度勾配(Ficoll)によって単離した。
健康なドナーおよびHIV-1患者からの末梢血のインビトロ感染に由来する細胞を、抗CD3、抗CD4、抗CD32、抗HLA-DR、および抗CD69抗体を使用してマークし、FACSによって分析した。HIV-1患者からの新鮮な細胞を、抗CD3、抗CD4、抗CD32、および抗HLA-DR抗体を使用してマークし、CD32マーカーの発現の関数としてSH800(Sony)を使用してソートするために、IgG2アイソタイプ対照をマークした(全TCD4+、TCD4+ CD32-、TCD4+ CD32低、TCD4+ CD32+)。各下位集団について、ソートした細胞の一部を、全HIV-1 DNAの定量化のために乾燥ペレット中-80℃で保持し、第2の部分を、誘導性およびウイルス増幅試験のために培養した。
HIV-1患者の血液から単離した異なる分画のDNAを、QIAamp DNAマイクロキット(Qiagen)を使用して精製した。DNA濃度を、β-グロビンqPCRによって決定した。1細胞当たりの全HIV-1 DNAのコピー数を、超高感度qPCR(Bicentric)によって決定した。
マーカー候補を同定するために、発明者らは、健康なドナーに由来する静止状態のTCD4リンパ球が初めて感染するのを可能にするインビトロモデルを開発した。実際に、これらの細胞は、先行する活性化シグナル(TCRまたはPHA/IL2による活性化)なしに、HIV-1による感染に対して許容状態ではない。これらの細胞中の制限に関与するSAMHD1タンパク質を同定する発明者らは、Vpxタンパク質(SIVmac251ウイルスによりコードされる)を含有するVLPによる治療の開発を可能にし、制限を取り除くことを可能にし、活性化シグナルを必要とすることなく、感染の配向を可能にした。健康なドナーからのPBMCが、VLP-Vpxで治療され、次いでHIV-1-CMV-GFPに感染したこのモデル(図1)を使用して、メッセンジャーRNA上でRNA-seq実験を実行するために、感染細胞(GFP+)の全RNAを抽出した。VLP-Vpxで治療されたが、感染しなかったPBMC、ならびに非感染細胞は、対照として使用した。統計的および生物情報学的分析は、静止状態のTCD4リンパ球の転写プログラムに対するHIV-1感染の影響を決定するのを可能にした。
HIV-1患者の血液から単離された細胞下位集団を、IL2(50IU/mL)の存在下、PHAまたは抗CD3、抗CD28、および抗CD2ビーズ(Miltenyi)などの賦活剤の不在下または存在下で培養した。TCD4+およびTCD4+ CD32-分画を、24ウェルプレート上で1mLの完全培地当たり106細胞の濃度で培養し、p24 ELISA試験のために、上清を2日毎に回収した。
この実施例では、発明者らは、患者に由来し、CD32中で枯渇したTCD4細胞の集団がどのように、活性化後にHIVウイルスを常に再活性化し得るかを理解しようとした。
研究を継続するために、発明者らは、異なるマーカー候補が、ウイルスリザーバであるとして同定された細胞中で同時発現したかどうかを最後に試験した。
CD32に対して配向される本発明の抗体は、当業者に既知の様々な技法、特に下記のものによって産生され得る。
Claims (10)
- 哺乳動物免疫不全ウイルスに感染した細胞の細胞内リザーバの検出のための方法であって、前記方法が、インビトロまたはエクスビボで、前記哺乳動物免疫不全ウイルスに感染し、および前記哺乳動物免疫不全ウイルスに対する特定の抗レトロウイルス療法で治療した哺乳動物の細胞を、表面上にCD89分化マーカーを発現するリンパ球細胞の検出を可能にする薬剤と接触させるステップを含む、方法。
- 表面上にCD89分化マーカーを発現するリンパ球細胞の検出を可能にする前記薬剤が、抗CD89抗体である、請求項1に記載の方法。
- 前記哺乳動物免疫不全ウイルスが、ヒト免疫不全ウイルス(HIV)、サル免疫不全ウイルス(SIV)、またはネコ免疫不全ウイルス(FIV)である、請求項1または請求項2に記載の方法。
- 表面上に前記CD89分化マーカーを発現する前記リンパ球細胞が、TCD4リンパ球細胞である、請求項1~3のいずれか一項に記載の方法。
- 哺乳動物免疫不全ウイルスに感染した細胞(以下「リザーバ細胞」という。)の細胞内リザーバを検出するためのキットであって、前記リザーバ細胞の表面上のCD89分化マーカーを検出する少なくとも1種の薬剤と、前記リザーバ細胞のゲノム中の前記哺乳動物免疫不全ウイルスのDNAの存在を判定することを可能にする少なくとも1つの組成物とを含む、キット。
- 前記リザーバ細胞のゲノム中の前記哺乳動物免疫不全ウイルスのDNAの存在を判定することを可能にする前記組成物が、前記ウイルスのゲノムの配列の特定のオリゴヌクレオチドを含む、請求項5に記載のキット。
- 哺乳動物免疫不全に対する抗ウイルス療法剤を使用した治療の中止に続く哺乳動物免疫不全の再発に関する予後の診断をインビトロで行うための方法であって、前記方法が、CD89分化マーカーを表面上に発現し、かつ前記哺乳動物免疫不全の原因であるウイルスのDNAをゲノム中に有するリンパ球細胞を定量化するステップを含む、方法。
- 哺乳動物免疫不全に対する抗ウイルス療法剤を使用した治療の中止に続く哺乳動物免疫不全の完全寛解に関する診断をインビトロで行うための方法であって、前記方法が、哺乳動物免疫不全ウイルスのDNAをゲノム中に有し、かつ表面上に少なくともCD89分化マーカーを発現するリンパ球細胞の不在を検出するステップを含む、方法。
- 哺乳動物免疫不全ウイルスに感染した細胞の細胞内リザーバの検出のための方法であって、前記方法が、インビトロまたはエクスビボで、前記哺乳動物免疫不全ウイルスに感染し、ならびに特に前記哺乳動物免疫不全ウイルスに対する特定の抗レトロウイルス療法および/または前記細胞内リザーバを根絶することを目的とした療法で治療した哺乳動物の細胞を、表面上にCD89分化マーカーを発現するリンパ球細胞の検出を可能にする薬剤と接触させるステップを含む、方法。
- 哺乳動物免疫不全ウイルスに感染した哺乳動物細胞の細胞内リザーバの根絶を目的とした治療の有効性の評価のための方法であって、前記方法が、インビトロまたはエクスビボで、表面上にCD89分化マーカーを発現するリンパ球細胞の存在、量、または不在の検出を可能にする薬剤を使用して、前記細胞内リザーバを検出するステップを含む、方法。
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WO2018060978A3 (fr) | 2018-05-17 |
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US11041858B2 (en) | 2021-06-22 |
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JP7083819B2 (ja) | 2022-06-13 |
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EP3519822A2 (fr) | 2019-08-07 |
EP3519823A1 (fr) | 2019-08-07 |
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US11125751B2 (en) | 2021-09-21 |
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CA3037697C (fr) | 2023-11-14 |
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