JP2021521817A - 新規な融合タンパク質およびこれを含む癌の予防または治療用薬学的組成物 - Google Patents
新規な融合タンパク質およびこれを含む癌の予防または治療用薬学的組成物 Download PDFInfo
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Abstract
【選択図】図15
Description
制御性T細胞(Treg)でのみLrig−1タンパク質が発現するかを確認するために、T細胞の亜型(サブセット)であるTh0、Th1、Th2、Th17およびiTregを用意した。前記iTregは、自然的に分離したnTregとは異なり、下記の組成を含む培地で分化を人工的に誘導した細胞を意味する。
制御性T細胞の表面タンパク質であるLrig−1タンパク質の細胞外ドメインを含む融合タンパク質を作製するために、Lrig−1タンパク質の細胞外ドメインの3次元立体構造を予測した。
前記塩基配列の予測は、Lrig−1タンパク質の構造をベースとする抗原決定基予測ソフトウェアであるElliproサーバ(http://tools.iedb.org/ellipro/)を用いた。前記Ellipro検索エンジンは、現存する抗原決定基を予測するアルゴリズムの中で最も信頼度が高いと知られた検索エンジンに相当してこれを用いた。
Lrig−1タンパク質が制御性T細胞に特異的なバイオマーカーとして作用できるかを検証した。
Lrig−1 mRNAから発現したLrig−1タンパク質が制御性T細胞でのみ特異的に発現するかを確認した。
Lrig−1タンパク質が細胞治療のターゲットになるためには、制御性T細胞の表面で発現してこそさらに効果的にターゲット治療が可能なため、Lrig−1タンパク質が表面で発現するか否かを確認した。
1.発現ベクターの作製
本発明による融合タンパク質を製造するために、下記表26のそれぞれの融合タンパク質をコードする各核酸配列を合成した。核酸配列の5’末端と3’末端にそれぞれNheIとEcoRI制限酵素配列を添加し、5’末端の制限酵素配列の後にコザック配列(Kozak’s sequence)(GCCACC)と、タンパク質翻訳のための開始コドンと発現したタンパク質を細胞外に分泌させるマウスIgGカッパ軽鎖シグナルペプチド(ATGGAAACCGATACTCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCTCTACCGGG)を挿入した。以後、下記表26のそれぞれの融合タンパク質をコードする核酸配列で、ヒト由来Lrig−1タンパク質の細胞外ドメイン;選択的にリンカー;ヒンジ領域;ヒトIgG1由来の重鎖不変領域2(CH2)および重鎖不変領域3(CH3);をコード化する核酸配列後には終結コドンを挿入した。NheIとEcoRIの2つの制限酵素配列を用いて本発明の融合タンパク質をコードする核酸配列をpcDNA3.1(+)発現ベクターにクローニングした。
293F細胞にポリエチレンイミンを用いて前記1.で作製した発現ベクターを形質転換させた後、37℃および8%CO2条件下で6日間培養した。培養上澄液をろ過した後、プロテインAレジンに流した後、1X PBSで洗浄した。pH3.5の0.1Mグリシン溶液を用いて溶出した後、得られた溶液にpH9.0の1M TRIS溶液を添加してpH中和した。以後、溶液を透析してPBS溶液状態にした後、濃縮して使用した。
前記製造例5で作製された本発明による配列番号21で表される融合タンパク質(配列番号25で表される核酸配列を用いた作製)がLrig−1タンパク質に対するリガンドと結合して、前記リガンドと制御性T細胞との間の相互作用を阻害することにより、最終的に制御性T細胞のエフェクターT細胞に対する増殖抑制能を減少させるかを確認するために、下記の実験を行った。具体的には、制御性T細胞とエフェクターT細胞を共培養する条件で前記製造例5の融合タンパク質を添加して、制御性T細胞のエフェクターT細胞に対する増殖抑制能の変化を確認した。その結果は図11に示した。
前記実施例6から明らかなように、前記製造例5で作製された本発明による配列番号21で表される融合タンパク質(配列番号25で表される核酸配列を用いた作製)はLrig−1リガンドを認識できることが分かるので、前記Lrig−1のリガンドが存在する免疫細胞を発掘するために、制御性T細胞のターゲットとされるナイーブT細胞から、活性化されたT細胞、Th1細胞、Th2細胞またはTh17細胞に分化を誘導させた後、これらの細胞を前記製造例5の融合タンパク質(一次抗体)および抗−ヒト−PE抗体(二次抗体)で染色した。蛍光活性細胞分類機(Fluorescence−Activated Cell Sorter;FACS)を用いてそれぞれの細胞表面で前記融合タンパク質の発現量を測定して、その結果を図12に示した。
前記製造例5で作製された本発明による配列番号21で表される融合タンパク質(配列番号25で表される核酸配列を用いた作製)の固形癌に対する治療効果を確認するために、図13に示すように、B16F10黒色腫細胞(メラノーマ細胞)をマウスなどに3×105細胞の量で皮下注射した後、4日、8日、12日目に前記製造例5の融合タンパク質を200ugの量で腹腔内注射した。前記メラノーマ細胞の移植後、時間の経過による腫瘍の体積変化を測定して、その結果を図14に示した。
前記製造例34で作製された本発明による配列番号78で表される融合タンパク質(配列番号82で表される核酸配列を用いた作製)の固形癌に対する治療効果を確認するために、CT−26大膓癌細胞をマウスなどに3×105細胞の量で皮下注射した後、4日、8日、12日目に前記製造例34の融合タンパク質を200ugの量で腹腔内注射した。前記大膓癌細胞の移植後、時間の経過による腫瘍の体積変化を測定して、その結果を図15に示した。
Claims (21)
- Lrig−1(leucine−rich and immunoglobulin−like domains1)タンパク質の細胞外ドメインおよび免疫グロブリンFc領域を含む融合タンパク質。
- 前記Lrig−1タンパク質の細胞外ドメインは、配列番号1または3で表される、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、CH1、CH2、CH3およびCH4ドメインからなる群より選択される1つ〜4つのドメインを含む、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、IgG、IgA、IgD、IgEまたはIgM由来Fc領域、重鎖不変領域2(CH2)、重鎖不変領域3(CH3)、ヒンジ、その断片、これらの組み合わせ、またはこれらの組み合わせを含むハイブリッドFcである、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、IgG、IgA、IgD、IgEまたはIgM由来のCH2およびCH3ドメインを含む、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、IgG1、IgG2、IgG3またはIgG4由来のCH2およびCH3ドメインを含む、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、ヒンジ領域を含む、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、IgG、IgA、IgM、IgD、IgE、またはアバタセプト(Abatacept)由来のヒンジ領域を含む、請求項7に記載の融合タンパク質。
- 前記ヒンジ領域は、IgG1、IgG2、IgG3、IgG4、IgDまたはアバタセプト(Abatacept)由来のヒンジ領域を含む、請求項7に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、配列番号5または6で表されるCH2およびCH3を含む、請求項1に記載の融合タンパク質。
- 前記免疫グロブリンFc領域は、配列番号7〜10のいずれか1つで表されるヒンジ領域を含む、請求項1に記載の融合タンパク質。
- 前記Lrig−1タンパク質の細胞外ドメインは、前記免疫グロブリンFc領域のN−末端またはC−末端に直接連結される、請求項1に記載の融合タンパク質。
- 前記Lrig−1タンパク質の細胞外ドメインは、リンカーを介して前記免疫グロブリンFc領域のN−末端またはC−末端に連結される、請求項1に記載の融合タンパク質。
- 前記リンカーは、配列番号11で表されるペプチドリンカー;および配列番号12で表されるペプチドリンカー;の少なくとも1つである、請求項13に記載の融合タンパク質。
- 請求項1〜14のいずれか1項に記載の融合タンパク質をコードする核酸分子。
- 請求項15に記載の核酸分子が挿入された発現ベクター。
- 請求項16に記載の発現ベクターが形質感染した宿主細胞株。
- 請求項1〜14のいずれか1項に記載の融合タンパク質を有効成分として含む癌の予防または治療用薬学組成物。
- 前記癌は、固形癌である、請求項18に記載の薬学組成物。
- 前記癌は、胃癌、肝臓癌、膠細胞腫、卵巣癌、大膓癌、頭頚部癌、膀胱癌、腎細胞癌、乳癌、転移癌、前立腺癌、膵臓癌、黒色腫または肺癌である、請求項18に記載の薬学組成物。
- 請求項1〜14のいずれか1項に記載の融合タンパク質を個体に投与するステップを含む癌の予防または治療方法。
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