JP2004201547A - Mast cell death inducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicinal use using MITE. <P>SOLUTION: A mast cell death inducer or mast cell-related disease-preventing or treating agent contains a fused protein comprising PTD and MITF mutant as an active ingredient. In the fused protein comprising HisTag, PTD and MITF mutant, the MITF mutant is the mi mutant of MITF, wh mutant, HLH fragment or type A N-terminal region (1 to 305) fragment, and PTD is a peptide originated from TAT. DNA encodes the fused protein. The method for producing the fused protein uses a genetic engineering means. The fused protein is transferred into a mast cell to exhibit an action for inhibiting the activity of endogenous MITF, an action for inhibiting the existence of the mast cell from a precursor cell, an action for inducing mature cell into cell death (apoptosis), and the like. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００１７】
また、これらの変異体の構造類似体であっても、当該変異体と実質的に同程度のＭＩＴＦ阻害活性を有するものも本発明の範疇に含まれる。例えば、上述のアミノ酸配列において、１または複数個のアミノ酸を置換、欠失、挿入または付加したものであって、当該変異体と実質的に同程度の活性を有するものであってもよい。
【００１８】
Ｂ．ＰＴＤ
本発明で用いられるＰＴＤは、細胞内に取り込まれる（移行する）性質を有するものであれば、特に限定されるものではなく、公知のものを利用できる。具体的には、前記の文献（非特許文献６）中の１６４頁表２に列挙された各種オリゴペプチド、特許文献３、特許文献４、特許文献５で開示されたＰＴＤ用の各種オリゴペプチドなどが例示される。より好ましいＰＴＤは、ＴＡＴ由来ペプチド（アミノ酸配列はＹＧＲＫＫＲＲＱＲＲＲ、配列番号７、好ましい塩基配列は同８で各々示される）である。ＰＴＤはＭＩＴＦ変異体のＮ末側、Ｃ末側のいずれに結合していてもよい。また、結合は直接でもよく、架橋剤（リンカー）を介して間接的なものであってもよい。リンカーとしてはグリシン残基などが例示される。
【００１９】
Ｃ．その他の構成要素（ドメイン）
本発明の融合蛋白質は、精製用アフィニティクロマトにおけるリガンドに対して親和性を有するものを結合していてもよい。当該ドメインは特に限定されるものではなく、公知のものを利用することができる。このような関係にあるものとしては、抗原とその抗体、受容体とそのリガンド、Ｎｉ−ＮＴＡ（ニトリロ三酢酸）とＨｉｓＴａｇ、アビジン（またはストレプトアビジン）とビオチン、などが例示される。より好ましいのは、ＨｉｓＴａｇ（アミノ酸配列はＭＧＧＳＨＨＨＨＨＨ、配列番号９、好ましい塩基配列は同１０で各々示される）である。本ドメインは融合蛋白質のＮ末側、Ｃ末側のいずれに結合していてもよい。また、結合は直接でもよく、架橋剤（リンカー）を介して間接的なものであってもよい。リンカーとしてはグリシン残基などが例示される。
【００２０】
Ｄ．融合蛋白質の調製
本発明の融合蛋白質の調製方法としては、▲１▼融合蛋白質全体を、化学的合成手法を用いて合成する方法、▲２▼上記の各構成要素（ドメイン）を各々別個に調製した後に、化学的な反応手段を用いて結合する方法、▲３▼各構成要素をコードする遺伝子を連結した上で遺伝子工学的手法を用いて一気に融合蛋白質として発現させる方法などが例示される。▲２▼の場合、各構成要素の調製方法としては、化学的合成方法、細胞培養法、遺伝子工学的な手法を用いる方法などが挙げられる。ＰＴＤはＨＩＶ抗原からの切断・単離によっても調製することもできる。
【００２１】
例えば、融合蛋白質を遺伝子工学手法により調製する場合で説明する。
１）融合蛋白質をコードするＤＮＡを調製する。当該調製は常法により行われる。まず、ＭＩＴＦ変異体をコードする遺伝子を調製する。当該遺伝子の調製は、適当な細胞からｍＲＮＡを抽出し、逆転写酵素とＤＮＡポリメラーゼを用いてｃＤＮＡを合成し、ポリメラーゼ連鎖反応（ＰＣＲ）法で増幅することにより得ることができる。具体的には、ｍＲＮＡの抽出は、市販のｍＲＮＡ抽出用キットなどを用いて行い、逆転写、ｃＤＮＡ合成および増幅は、市販のｃＤＮＡ増幅キットなどを用いた５’−ＲＡＣＥ法（非特許文献８）または適当なプライマーを用いた逆転写ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）法などにより行うことができる。また、適当な細胞からゲノムＤＮＡを抽出し、当該遺伝子をＰＣＲで増幅して得ることもできる。また、公知の報告例、例えば、上述の非特許文献１、特許文献２で開示されたＭＩＴＦをコードする遺伝子を利用することもできる。例えば、本発明のＨＬＨ断片はＭＩＴＦのＭタイプから、ＡタイプのＮ末領域（１−３０５）断片はＭＩＴＦのＡタイプ（ＭＩＴＦ Ａ／ｐＣＤＮＡ３）から、各々調製することかできる。
【００２２】
次いで、ＰＴＤをコードする遺伝子を上記と同様の方法により調製し、ＭＩＴＦ変異体をコードする遺伝子と連結する。ＨｉｓＴａｇについても同様である。また、ＨｉｓＴａｇを担持する市販のプラスミド（ｐＴｒｃＨｉｓＢ、インビトロジェン社）を用いて、ＰＴＤ−ＭＩＴＦ変異体からなる融合蛋白質をコードするＤＮＡを挿入することにより、本発明の融合蛋白質をコードするＤＮＡを調製することができる。
【００２３】
２）ＤＮＡを適当なベクターに組込んで発現用ベクターを調製する。当該ベクターは特定のプロモーターの制御下に融合蛋白質を発現するために用いる。その組込みは常法により行われる。
【００２４】
上記得られた目的のＤＮＡを精製し、ベクターＤＮＡに導入して、宿主・ベクター系を構築することができる。宿主・ベクター系は一般に宿主細胞とコンパチブルな種に由来するレプリコンと、当該宿主を組合せて使用する。ベクターＤＮＡは、複製起点、プロモーター、制御配列（エンハンサー）、シグナル配列、リボソーム結合部位、ＲＮＡスプライス配列、ポリＡ付加部位、転写終結配列（ターミネーター）などを有する。また、形質転換細胞中で表現型の選択が可能となるマーカーの配列を有していてもよい。ベクターＤＮＡとしては、例えば、染色体、エピソーム由来のベクター、例えば細菌プラスミド由来、バクテリオファージ由来のベクター、またはバキュロウイルス、パポバウイルス、ＳＶ４０、などのウイルス由来のベクター、あるいはコスミドおよびファージミドなどが挙げられる。また、目的により発現ベクターやクローニングベクターなどを用いることができる。
【００２５】
プロモーターは公知のものが挙げられ、発現のための宿主に合わせて選択することができる。例えば、大腸菌を宿主とする場合は、ｔｒｐプロモーター、ｌａｃプロモーター、ｔｒｃプロモーター（ｔｒｐプロモーターの−３５領域とｌａｃプロモーターの−１０領域を連結した合成プロモーター）、Ｔ７プロモーターなどのプロモーターが例示される。また、当該発現ベクターはａｍｐ ｒなどのマーカー遺伝子を担持していてもよい。
【００２６】
ベクターＤＮＡに本発明に係るＤＮＡを組み込む方法は、自体公知の方法を適用し得る。例えば、適当な制限酵素を選択し、処理して目的のＤＮＡを特定部位で切断し、次いで同様に処理したベクターＤＮＡと混合し、リガーゼによって再結合する方法が用いられる。あるいは、目的のＤＮＡに適当なリンカーをライゲーションし、これを目的に適したベクターのマルチクローニングサイトへ挿入することによっても、所望の組換えベクターが得られる。また、宿主に導入するベクターＤＮＡとして発現ベクターを使用すれば、目的とする蛋白質の製造が可能である。
【００２７】
３）発現用ベクターを宿主に導入して形質転換体を調製する。形質転換は常法により行われる。宿主としては、大腸菌、枯草菌、酵母、動物細胞などを用いることができる。好ましくは大腸菌である。また、栄養要求性株、抗生物質感受性株を宿主とすることもできる。
【００２８】
形質転換体を調製する方法としては、プラスミドを直接宿主細胞内に導入する方法、プラスミドを染色体上に組込む方法などが挙げられる。前者としては、プロトプラストポリエチレングリコール法、エレクトロポレーション法などが例示される。後者としては、宿主染色体中に存在する遺伝子の一部のＤＮＡ配列をプラスミドに含有させて、その相同な配列部分を利用して、プラスミドまたはその線状断片を相同組換えにより宿主染色体上に導入することができる。
【００２９】
４）形質転換体を培養し、融合蛋白質を産生する。培養は常法により宿主に応じて適用な培地、培養条件（温度、時間など）を用いて行われる。大腸菌を用いる場合の培養条件としては通常、１５〜４３℃（好ましくは３０〜３７℃）程度で、１〜１００時間程度行う。また、必要に応じて通気や攪拌を加えることもできる。培養形式としては、回分培養、半回分培養（フェッド・バッチ培養）、連続培養のいずれであってもよい。
【００３０】
５）産生された融合蛋白質を精製する。宿主として大腸菌を用いた場合はまず菌体を超音波粉砕等の処理により当該蛋白を可溶化する。産生された融合蛋白質の精製は自体公知の手法により行うことができる（特許文献６など）。例えば、Ｎｉカラムを用いる方法、陰イオン交換体処理、透析処理などが例示される。また、本発明の融合蛋白質は変性剤（カオトロピック剤）処理後に変性剤を除去して得られたものを用いることが好ましい。変性剤としては尿素、塩酸グアニジン、チオシアン酸塩などが例示される。変性剤処理時の変性剤の添加条件としては、濃度１〜１０Ｍ程度が例示される。具体的には融合蛋白質と変性剤を接触させて処理した後に、変性剤の共存下にＮｉカラムを用いて処理を行い、さらに陰イオン交換体または透析処理により変性剤を除去する操作を行って、当該融合蛋白質を精製する。
【００３１】
Ｅ．調製された融合蛋白質の性状
本発明の融合蛋白質は、細胞内、特にマスト細胞内に移行する性質を有するものである。好ましくは、ＨｉｓＴａｇ、ＰＴＤおよびＭＩＴＦ変異体から構成される。このものは１０〜１００キロダルトン（ｋＤａ）程度の分子量を有する。好適には、Ｎ末側からみて、ＨｉｓＴａｇ、ＰＴＤ、ＭＩＴＦ変異体の順番に並んだものである。具体的な配列としては、表２に示すとおりである。
【００３２】
【表２】

【００３３】
Ｆ．製剤化
本発明の融合蛋白質の製剤化には、自体公知の手法を用いることができる。例えば融合蛋白質に、製剤学的に許容しうる担体を添加あるいは混合すればよい。製剤化により得られた医薬組成物において、融合蛋白質の濃度として０．１〜１００μｇ／ｍＬまたは０．１〜１００ｎＭ程度が例示される。
【００３４】
Ｇ．用途
本発明のＭＩＴＦ変異体（または、それを用いた融合蛋白質）はマスト細胞内に移行することにより内在性ＭＩＴＦの活性を阻害する、前駆細胞からのマスト細胞の分化を阻害する、マスト細胞の生存を阻害する、成熟マスト細胞に細胞死（アポトーシス）を誘導する、などの作用を有する。従って、本発明の製剤は、マスト細胞が関与する各種疾患の予防・治療に有用であることが期待される。当該疾患としては、例えば、アレルギー、喘息、自己免疫疾患、肺線維症、癌、マストサイトーシス、マストサイトーマ等が挙げられる。
【００３５】
Ｈ．用法・用量
本発明の融合蛋白質の用法・用量としては、生体内において０．００１〜１０μｇ／ｍＬ程度の濃度で存在するように、投与量を選択すればよい。あるいは投与量として、１０μｇ〜５０ｍｇ程度が例示される。投与経路としては、静脈内投与、皮下投与、筋肉内投与、経皮投与、気道内投与などが例示される。
【００３６】
【実施例】
本発明をより詳細に説明するために実施例、製剤例および実験例を挙げるが、本発明はこれらにより何ら限定されるものではない。
【００３７】
実施例１
１）融合蛋白質発現プラスミドの構築
発現プラスミドは、大腸菌用発現ベクターｐＴｒｃＨｉｓＢ（インビトロジェン社、Ｎｏ．Ｖ３６０−２０）のＨｉｓＴａｇ配列の上流に存在するＮｃｏＩサイトとマルチクローニングサイト中のＨｉｎｄＩＩＩサイトの間に、精製用のＨｉｓＴａｇの下流にＰＴＤ配列が接続し、その下流にＭＩＴＦ変異体のｃＤＮＡを接続したＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦ変異体の遺伝子を挿入することにより構築した。図１を参照のこと。
【００３８】
ＭＩＴＦのｃＤＮＡへのＨｉｓＴａｇ、ＰＴＤ配列の付加はＰＣＲ法を用いて行った。まず、ＭＩＴＦ ＭタイプのｃＤＮＡ（ｐＳＵ０５４、ＭＩＴＦ−Ｍ／ｐＴ７ Ｂｌｕｅ）を鋳型にして、ＭＩＴＦおよびＢａｍＨＩサイトを含む上流部分をＰＣＲ法により増幅した。さらに４種類のプライマー（Ｍ−ｔａｔ、Ｔａｔ３、Ｔａｔ ２、Ｔａｔ １）を用いてＭＩＴＦの転写開始コドンの上流にＨｉｓＴａｇと制限酵素ＮｃｏＩの認識配列を付加するまで伸長させた後に、大腸菌用発現ベクターｐＴｒｃＨｉｓＢ（このものはｌａｃ Ｐ／ＯおよびＡｍｐ ｒを担持してなる）のＮｃｏＩサイトとＢａｍＨＩサイトの間にクローニングした。
【００３９】
各プライマーは以下の塩基配列を有する。
Ｍ−ｔａｔ（配列番号１７）： ＧＣＧＡＣＧＡＡＧＡＧＧＴＡＴＧＣＴＡＧＡＡＴＡＣＡＧＴＣＡＣＴＡＣＣ
Ｔａｔ ３（配列番号１８）： ＧＧＣＡＧＧＡＡＧＡＡＧＣＧＧＡＧＡＣＡＧＣＧＡＣＧＡＡＧＡＧＧＴＡＴＧ
Ｔａｔ ２（配列番号１９）： ＡＴＣＡＴＣＡＴＣＡＴＧＧＴＧＧＴＴＡＴＧＧＣＡＧＧＡＡＧＡＡＧＣＧＧ
Ｔａｔ １（配列番号２０）： ＴＡＡＡＣＣＡＴＧＧＧＧＧＧＴＴＣＴＣＡＴＣＡＴＣＡＴＣＡＴＣＡＴＣＡＴＧＧＴＧ
【００４０】
次いで、Ｍタイプ（ｐＳＵ０５４）、ｍｉ変異体（ｐＳＵ０６１、ＭＩＴＦ−Ｍｍｉ／ｐＴ７ Ｂｌｕｅ）、ｗｈ変異体（ｐＳＵ０６２、ＭＩＴＦ−Ｍ ｗｈ／ｐＴ７ Ｂｌｕｅ）をそれぞれＢａｍＨＩとＨｉｎｄＩＩＩ消化することにより、ＭＩＴＦｃＤＮＡの下流部分を単離した。この部分に変異部分も含まれる。単離したｃＤＮＡ断片を、上流部分をクローニングしたプラスミドのＢａｍＨＩサイトとＨｉｎｄＩＩＩサイトの間に挿入した。プラスミドｐＳＵ８１は正常型ＭＩＴＦであるＭタイプを含む融合蛋白質の遺伝子を挿入したものである。ｐＳＵ０８２はｍｉ変異体を、ｐＳＵ０８３はｗｈ変異体を含む融合蛋白質を各々挿入したものである。
【００４１】
２）融合蛋白質の発現
構築した発現プラスミドを大腸菌ＤＨ５α（東洋紡）に導入し形質転換した。５０ｍＬのＬ−Ｂｒｏｔｈ（５０μｇ／ｍＬのアンピシリンを含む）に、大腸菌を１白金耳量となるように植菌し、３７℃で１７時間培養した。さらに、１ＬのＬ−Ｂｒｏｔｈ（５０μｇ／ｍＬのアンピシリンおよび０．２％のグルコースを含む）に、培養液を１％となるように植菌した。Ａ６００の濁度が約０．１となるまで３７℃で培養した後に、ＩＰＴＧ（イソプロピルチオ−β−Ｄ−ガラクトシド）を終濃度０．４ｍＭとなるように添加した。２時間培養後に大腸菌を集菌した。
【００４２】
３）融合蛋白質の精製
大腸菌を遠心分離にて回収し、８Ｍの尿素、０．１Ｍの塩化ナトリウム、１０ｍＭのＤＴＴを含む２０ｍＭのＨＥＰＥＳ緩衝液（ｐＨ８．０、ｐＨは以下同様）中で超音波粉砕し可溶化した。得られたＬｙｓａｔｅを８Ｍの尿素、０．１Ｍの塩化ナトリウム、１０ｍＭのイミダゾール、１ｍＭのＤＴＴを含む２０ｍＭのＨＥＰＥＳ緩衝液で平衡化したＮｉ−ＮＴＡアガロース（Ｑｉａｇｅｎ社）カラムにアプライし、同緩衝液で洗浄した。カラムに結合した蛋白をＡ液として同緩衝液、Ｂ液として２００ｍＭのイミダゾールを含む同緩衝液を用いたイミダゾールの濃度直線勾配法で溶出し、ＳＤＳ−ＰＡＧＥ（Ｔｅｆｃｏ社）で解析して目的の分子量を示す画分をプールした。
【００４３】
溶出液１容量に対して、２０ｍＭのＨＥＰＥＳ緩衝液を１容量、４Ｍの尿素を含む同緩衝液を２容量、各々添加して希釈した溶液を、４Ｍの尿素、２５ｍＭの塩化ナトリウムを含む２０ｍＭのＨＥＰＥＳ緩衝液で平衡化した四級アンモニウム塩型強陰イオン交換体（商品名Ｑ−Ｓｅｐｈａｒｏｓｅ、ファルマシア社）にアプライし、同緩衝液で洗浄した。次に、２０ｍＭのＨＥＰＥＳ緩衝液で洗浄して尿素を除去した後に、１Ｍの塩化ナトリウムを含む同緩衝液でカラムに結合した蛋白を溶出した。溶出した融合蛋白質は１０％グリセロールを含むＤｕｌｂｅｃｃｏ’ｓ ＰＢＳ（等張化リン酸緩衝液）に対して透析した後に分注して−８０℃で保存した。
【００４４】
得られた最終産物をＳＤＳ−ＰＡＧＥで分析したところ、還元条件下で約５０キロダルトンの分子量を示すバンドが観察された。このバンドはウサギ抗ＭＩＴＦ抗体と反応した。なお、このウサギ抗ＭＩＴＦ抗体は、ウサギにＭＩＴＦのＣ末２０アミノ酸残基のペプチドを免疫して得られた抗血清を、同ペプチドを結合したカラムを用いてアフィニティ精製を行うことにより、同ペプチドを特異的に認識する抗体を調製したものである。
【００４５】
製剤例１
本発明の融合蛋白質、および、１０％グリセロールを含むＤｕｌｂｅｃｃｏ’ｓＰＢＳからなる組成物を調製した。
【００４６】
実験例１
１）まず、本発明の融合蛋白質が細胞内に移行しているかどうかを確認した。実施例１で調製したＭＩＴＦ変異体の融合蛋白質をＦＩＴＣ（フルオレセイン・イソチオシアネート）で常法により蛍光標識し、ＣＯＳ７細胞に添加した。添加１時間後にＦＡＣＳ（ｆｌｕｏｒｅｓｃｅｎｅ−ａｃｔｉｖａｔｅｄ ｃｅｌｌ ｓｏｒｔｅｒ） Ｃａｌｉｂｕｒ（ベクトン・ディキンソン社）を用いて、蛍光強度を測定した。その結果を表３に示す。
【００４７】
【表３】

【００４８】
表３から明らかなように強い蛍光が細胞に会合（アソシエート）していることが観察され、当該融合蛋白質が細胞内に効率よく移行していることが確認された。
【００４９】
２）本発明の融合蛋白質が細胞内に移行して内在性ＭＩＴＦの活性を阻害するか調べる目的でルシフェラーゼ分析を行った。
【００５０】
発現用プラスミドとして、正常ＭＩＴＦ発現用プラスミドｐＳＵ０６３（ＭＩＴＦ−Ｍ／ｐＣＤＮＡ３）、ｃ−ｋｉｔ遺伝子プロモーターの下流にルシフェラーゼ遺伝子を接続したプラスミドを調製するための各種ベクター、すなわち、Ｃ−ｋｉｔ−Ｒｌｕｃ発現用ベクターｐＳＵ０５３（Ｃ−ｋｉｔ／Ｒ−ｌｕｃ）、ルシフェラーゼ発現用ベクターｐＧＬ２（Ｌｌｕｃ、プロメガ社）を用いた。各プラスミドを、ＣＯＳ７細胞に導入（トランスフェクト）した。その条件は以下のとおり。プラスミドの使用量は、６ｃｍのディッシュ当たり３μｇ（ｐＳＵ０６３：ｐＳＵ０５３：ｐＧＬ２＝１：１：０．１）。プラスミドの導入にはトランスフェクションキット（Ｓｔｒａｔａｇｅｎｅ社、＃２００３８５）を用いた。
【００５１】
６ウェルプレートに１ウェル当たり１×１０^５個の細胞を植え込み、１７時間ＣＯ_２インキュベータに静置した。１０μＬのＳｏｌｕｔｉｏｎ １（前記キットに添付されている試薬。２．５Ｍ ＣａＣｌ_２からなる）に９０μＬの蒸留水と３μｇのＤＮＡを添加した後に、等量のＳｏｌｕｔｉｏｎ ２［前記キットに添付されている試薬。２×ＰＢＳ（ｐＨ６．９５）からなる］を加え、室温で２０分間静置した。混合したＤＮＡ溶液を培養液に添加した。２４時間後に上記の培地を交換し、１μｇ／ｍＬの本発明の融合蛋白質（実施例１で調製）を添加した。
【００５２】
本発明の融合蛋白質の添加４８時間後に細胞をＰＢＳ（−）で洗浄し、６穴のディシュ当たり０．５ｍＬのｐａｓｓｉｖｅ ｌｙｓｉｓ ｂｕｆｆｅｒを添加し、スクレーバーを用いて細胞を掻き採った。細胞を１．５ｍＬ容のチューブに移し、１４０００ｒｐｍ、４℃で５分間遠心した。ルシフェラーゼ活性の測定は、Ｄｕａｌ ｌｕｃｉｆｅｒａｓｅ ａｓｓａｙ ｋｉｔ（プロメガ社）を用いて行った。９６穴プレート上で２０μＬの遠心上清に、１００μＬのＬｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｒｅａｇｅｎｔ ＩＩを添加・混合し、ホタルルシフェラーゼ活性を測定した。さらに混合液に１００μＬのＳｔｏｐ＆Ｇｏ Ｒｅａｇｅｎｔを添加・混合し、ウミシイタケルシフェラーゼ（Ｒ−ｌｕｃ）活性を測定した。Ｒ−ｌｕｃの数値をｐＧＬ２で補正後、ＰＢＳ添加におけるＲ−ｌｕｃを１としたときの値に換算した。その結果を表４に示す。
【００５３】
【表４】

【００５４】
なお、表中のｍｉはＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｍｉ変異体からなる融合蛋白質、ｗｈはＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｗｈ変異体からなる融合蛋白質、を各々示す。
【００５５】
ＣＯＳ細胞が発現するルシフェラーゼ活性も本発明の融合蛋白質存在下で阻害され、当該融合蛋白質が細胞内に移行して内在性ＭＩＴＦを阻害することが示唆された。
【００５６】
実験例２
本発明の融合蛋白質がＳＣＦで誘導されるマスト細胞の分化系にどのように影響するかを確認した。
【００５７】
正常マウスから骨髄細胞を調製し、その２×１０^６個／穴を、１０％のＦＣＳ、０．１ｍＭの非必須アミノ酸、１ｍＭのピルビン酸ナトリウム、２ｍＭのグルタミン、１００Ｕ／ｍＬのペニシリン、１００μｇ／ｍＬのストレプトマイシン、５０μＭの２−メルカプトエタノール、２μｇ／ｍＬの本発明の融合蛋白質（ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｗｈ変異体）を含むＲＰＭＩ１６４０中で骨髄細胞因子（ＳＣＦ、ＩＢＬ社）５０〜１００ｎｇ／ｍＬの存在下に２４穴プレートにて培養した。週に一度培地を半量交換し、その際にＳＣＦも添加した。３７℃で２１日間培養した後に、マスト細胞数、ｃ−ｋｉｔおよびＩｇＥ受容体の発現量を測定した。
【００５８】
マスト細胞数の測定はトルイジン青染色法によった。トルイジン青染色は非特許文献９に従い、ｐＨ２．７の染色液を用いて行った。
【００５９】
ｃ−ｋｉｔの発現量の測定はＦＡＣＳを用いて行った。細胞を、０．１％のＮａＮ_３、０．１％のＢＳＡを含むＰＢＳ１００μＬに懸濁し、５〜１０μｇ／ｍＬのＲ−ＰＥ（Ｒ−ｐｈｙｃｏｅｒｙｔｈｒｉｎ）標識抗マウスｃ−ｋｉｔ抗体（ＰｈａｒＭｉｎｇｅｎ社）あるいはＲ−ＰＥ標識ラットＩｇＧ２Ｂ，ｋ ｉｓｏｔｙｐｅ ｓｔａｎｄａｒｄ（ＰｈａｒＭｉｎｇｅｎ社）と氷中で１時間インキュベーションした。洗浄後に１μｇ／ｍＬのヨウ化ピリジウムを添加して、ＦＡＣＳ Ｃａｌｉｂｕｒ（前記）で解析した。ＩｇＥ受容体の発現量の測定は、５μｇ／ｍＬのＦＩＴＣ標識マウスＩｇＥを用いて同様にＦＩＴＣによる解析を行った。マスト細胞数に関する結果を表５に示す。
【００６０】
【表５】

【００６１】
本発明の融合蛋白質の添加群では、培養２１日目でマスト細胞数が未添加群に比べて１０分の１以下に減少していた。また、ｃ−ｋｉｔの発現に関する培養２１日目の結果を表６に示す。
【００６２】
【表６】

【００６３】
本発明の融合蛋白質の添加群では、培養２１日目でｃ−ｋｉｔの発現量が未添加群に比べて１０分の１以下に減少していた。また、ＩｇＥ受容体の発現量は変化がなかった（実験データは示さず）。このように本発明の融合蛋白質はマスト細胞前駆細胞のＳＣＦ受容体ｃ−ｋｉｔの発現を特異的に阻害し、その結果、マスト細胞の分化を強く阻害することが確認された。
【００６４】
実験例３
本発明の融合蛋白質の、成熟マスト細胞への影響を確認する目的で、線維芽細胞との共培養系でのマスト細胞生存への影響を確認した。
【００６５】
正常マウスから脾細胞を調製し、１０％のＦＣＳ、０．１ｍＭの非必須アミノ酸、１ｍＭのピルビン酸ナトリウム、２ｍＭのグルタミン、１００Ｕ／ｍＬのペニシリン、１００μｇ／ｍＬのストレプトマイシン、５０μＭの２−メルカプトエタノールを含むＲＰＭＩ１６４０中でＩＬ−３（ＧＥＮＺＹＭＥ社）またはＷＥＨＩ−３細胞ｃｏｎｄｉｔｉｏｎｅｄ培地（ＷＥＨＩ−３ＣＭ）存在下に３週間以上培養して、脾臓由来培養マスト細胞（ＳＭＣ）を得た。６穴プレートに線維芽細胞のＮＩＨ−３Ｔ３（理研セルバンク）をコンフルエントまで培養し、当該ＳＭＣをＩＬ−３およびＷＥＨＩ−３ＣＭを含まず、かつ、１μｇ／ｍＬの本発明の融合蛋白質を含む、上記の培地に懸濁し、３×１０^５個で播種した。２〜３日ごとに新鮮培地に交換した。３７℃で１５日間培養した後に、マスト細胞数、ヒスタミン量およびキマーゼ活性を測定した。各種実験系を表７に示す。
【００６６】
【表７】

【００６７】
ヒスタミンの定量はＲＩＡ法（栄研化学）によった。顆粒中のキマーゼ活性の測定は特異的な合成基質であるＮ−サクシニル−Ａｌａ−Ａｌａ−Ｐｒｏ−Ｐｈｅ−ｐＮＡ（シグマ社）を用いて行った。結果を表８に示す。
【００６８】
【表８】

【００６９】
線維芽細胞と正常マスト細胞を共培養するとマスト細胞は長期間生存し続けた。また、ＭＩＴＦに異常を持つ人為的に作成したｍｉマスト細胞（ｍｉ／ｍｉＳＭＣ、天然には存在しない）は線維芽細胞との共培養においてアポトーシスにより２週間以内に死滅した。正常マスト細胞と線維芽細胞の共培養に本発明の融合蛋白質（ｍｉ変異体型、ｗｈ変異体型）を添加すると、マスト細胞の生存は阻害され、ｍｉマスト細胞と同様に２週間後にはほとんどが死滅した。マスト細胞数の低下は、ヒスタミン量の低下およびキマーゼ活性の低下によっても確認された。この結果、本発明の融合蛋白質は、成熟マスト細胞にアポトーシスを誘導することが示唆された。
【００７０】
実験例４
本発明の融合蛋白質の添加濃度における影響を調べた。ＭＣ／９マスト細胞株（ＡＴＣＣより入手）をＦＩＴＣ標識したＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｗｈ変異体からなる融合蛋白質の存在下または非存在下に、ＦＡＣＳ染色緩衝液（０．１％ウシ血清アルブミンおよび０．１％アジ化ナトリウムを含むＤｕｌｂｅｃｃｏ’ｓ ＰＢＳ）中で氷上１時間インキュベートした。洗浄後にヨウ化ピリジウムを添加して死細胞をゲートアウトし、ＦＡＣＳ Ｃａｌｉｂｕｒ（前記）で解析した。結果を表９に示す。
【００７１】
【表９】

【００７２】
表９の結果から、本発明の融合蛋白質の添加濃度に依存して、当該融合蛋白質が細胞内により高濃度で移行していることが判明した。
【００７３】
実施例２： ＨｉｓＴａｇ−ＰＴＤ−ＨＬＨ
１）発現ベクターの構築
発現ベクター作成の鋳型としては正常型のＭＩＴＦのＭタイプ（ｐＳＵ０５４）を用いた。ＨＬＨ断片はＭタイプのＮ末１９６番目から２８５番目のアミノ酸までの領域をＰＣＲ法により増幅した。増幅断片のＮ末側には、プライマー（Ｔａｔ１、Ｔａｔ２、Ｔａｔ３およびＨＬＨ Ｆ）を用い、ＨｉｓＴａｇ、ＰＴＤと制限酵素ＮｃｏＩの認識配列を付加した。またＣ末側には制限酵素ＸｂａＩの認識配列を付加した。増幅断片をＮｃｏＩ＋ＸｂａＩで消化し、実施例１で作成したｐＳＵ０８１のＮｃｏＩ−ＸｂａＩ領域と置換した。当該増幅断片をＴＡクローニングし塩基配列が正しいことを確認した。構築したベクターｐＳＵ０８５はｌａｃプロモーター、ＨｉｓＴａｇ−ＰＴＤ−ＨＬＨ領域の融合蛋白質のＤＮＡ、ａｍｐ ｒから構成される（図２）。
【００７４】
プライマーＨＬＨ Ｆは以下の塩基配列を有する。
ＨＬＨ Ｆ（配列番号２５）：ＧＣＧＡＣＧＡＡＧＡＧＧＴＡＴＧＴＴＧＧＣＴＡＡＡＧＡＧＡＧＧ
２）実施例１に準じて融合蛋白質を産生・精製した。
【００７５】
実施例３： ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのＡタイプＮ末領域
１）発現ベクターの構築
ｐＳＵ０６４（ＭＩＴＦのＡタイプ／ｐＣＤＮＡ３、特許文献２を参照のこと）を鋳型に、２種類のプライマー（ｐＴＤ３−ＭＩＴＦａ、ＭＩＴＦＲ−Ｎ）を用いてＰＣＲを行い、Ｎ末領域（Ｎ末１番目から３０５番目までのアミノ酸）を増幅した。ｐＴＤ３−ＭＩＴＦａの５’末端にはＳａｃＩＩサイトを、ＭＩＴＦＲ−Ｎの５’末端にはＥｃｏＲＩサイトを付加した。増幅断片をＳａｃＩＩ＋ＥｃｏＲＩで消化し、ｐＳＵ０９３（ｐＴｒｃＨｉｓを基本骨格とするＰＴＤカセット発現ベクター）の同サイト間に挿入した（図３）。構築した発現ベクターｐＳＵ０８７は、ＨｉｓＴａｇ−ＰＴＤ−ＡタイプのＮ末領域の順にＤＮＡ断片がつながり、ｌａｃプロモーターの制御下に融合蛋白質が発現する。
【００７６】
各プライマーは以下の塩基配列を有する。
ｐＴＤ３−ＭＩＴＦａ（配列番号２６）： ＣＧＣＣＧＣＧＧＡＡＴＧＣＡＧＴＣＣＧＡＡＴＣＧＧＧＡＡＴＣ
ＭＩＴＦＲ−Ｎ（配列番号２７）： ＧＡＡＴＴＣＡＣＴＡＴＧＣＴＣＴＴＧＣＴＴＣＡＧＡＣＴＣＴＧＴＧＧＧＧ
【００７７】
２）実施例１に準じて融合蛋白質を産生した後、これを精製する際に、Ｎｉ−アガロースの代わりにＴＡＬＯＮ（商品名、ＣＬＯＮＴＥＣＨ社）を用いて行った。精製物は１０％グリセロール／ＰＢＳ溶液として調製した。
【００７８】
実施例４： ｐＥＴ１４ｂを用いた発現ベクターの構築
ｐＳＵ０８３（ｗｈ系）のＮｃｏＩ−ＮｄｅＩ断片とｐＥＴ１４ｂ（Ｔ７プロモーター、ＨｉｓＴａｇ、ａｍｐ ｒを連結してなる。Ｔａｋａｒａ）の同領域を置換して発現ベクターｐＳＵ１２１を構築した（図４）。このｐＳＵ１２１はＴ７プロモーターの下流に、ＨｉｓＴａｇ、ＰＴＤ配列、ＭＩＴＦのｗｈタイプがつながり、大腸菌の菌体内において、Ｔ７プロモーターの制御下に融合蛋白質（ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｗｈタイプ）を発現する。
【００７９】
実施例１に従い、大腸菌をフラスコで培養し、融合蛋白質（ｗｈ系）を産生した。Ｎｉ−ＮＴＡアガロースを用いた精製を実施例１に準じて行った後に、Ｓｌｉｄｅ−Ａ−Ｄｉａｌｙｚｅｒ（Ｐｉｅｒｃｅ）を用いて透析し脱尿素処理を行った。溶媒としては２９０ｍＭソルビトール、１０μＭ ＥＤＴＡ、１ｍＭトリス緩衝液（ｐＨ８）を用いた。培養液１Ｌ当たり約１０ｍｇの融合蛋白質が精製された。
【００８０】
実施例５： 精製工程（ＨＬＨ）
融合蛋白質（ＨＬＨ系、実施例２により調製）をＱ−セファロースカラムから１Ｍ ＮａＣｌ／２０ｍＭ ＨＥＰＥＳ（ｐＨ８）で溶出し、回収した画分を、使用前にＰＤ−１０カラム（ファルマシア）を用いてＰＢＳに置換した。
【００８１】
実施例６： 高密度培養（ｗｈ系）
発現ベクターｐＳＵ０８３を大腸菌ＨＢ１０１株に導入し、形質転換体を調製した。ＥＢＭ００１００１０培地（アンピシリン添加）を用いてジャーファーメンターで３７℃１６時間培養し、融合蛋白質を産生した。精製は実施例１に準じて行った。
【００８２】
実施例７： 高密度培養（ＨＬＨ）
ｐＳＵ０８５を用いて実施例６に準じて融合蛋白質を産生した。精製を実施例１に準じて行うに際し以下の点を変更した。Ｎｉ−ＮＴＡアガロース処理時に８Ｍ尿素の代わりに６Ｍ塩酸グアニジンを用いたこと、陰イオン交換体処理の代わりに分子量３０００カット膜を用いて濃縮した後に、０．５Ｍ ＮａＣｌ／２０ｍＭ ＨＥＰＥＳ（ｐＨ８）で平衡化したＰＤ−１０カラム（ファルマシア）で脱塩したこと。０．５Ｍ ＮａＣｌ／２０ｍＭ ＨＥＰＥＳ（ｐＨ７．４）に対して透析し分注して−８０℃で保存した。インビボで使用する際にはＮａＣｌ濃度を０．１５Ｍとなるように希釈して用いた。
【００８３】
製剤例２
融合蛋白質、１０％グリセロール、ＰＢＳからなる組成物を調製した。
【００８４】
製剤例３
融合蛋白質、０．１５〜１Ｍ ＮａＣｌ、２０ｍＭ ＨＥＰＥＳ（ｐＨ７．４〜８）溶液として調製した。
【００８５】
実験例５（精製結果）
各種融合蛋白質（実施例１〜３により調製）を大腸菌菌体より精製し、その分子量、収量（４Ｌフラスコ培養当たり）を表１０に示した。なお、融合蛋白質（ＡタイプのＮ末領域）の分子量は３７ｋＤａであった。
【００８６】
【表１０】

【００８７】
実験例６（インビトロ阻害）
実験例３に準じて実験した。１０ｎＭの融合蛋白質を１７日間作用させた後にマスト細胞数、ヒスタミン、トリプターゼ、キマーゼを測定した。結果を表１１に示す。
【００８８】
【表１１】

【００８９】
表中の数値は平均±標準偏差を示す。また＊＊は溶媒群との間に、ｓｔｕｄｅｎｔ’ｓ ｔｅｓｔによりｐ＜０．０５で有意差があることを示す。また＊は同じくｐ＜０．０１で有意差があることを示す。以下同様。
【００９０】
実験例７（同上）
実験例３に準じて実験した。２０ｎＭの融合蛋白質を１５日間作用させた後にマスト細胞比率を測定した。結果を表１２に示す。本発明の融合蛋白質はマスト細胞の生存を抑制した。
【００９１】
【表１２】

【００９２】
実験例８（同上）
１）実験例２に準じて実験を行った。マウス骨髄細胞（２×１０^６／ウェル）をＳＣＦの存在下に融合蛋白質を添加して２８日間培養したときのマスト細胞数を求めた。例数は３とした。結果を表１３に示す。
【００９３】
【表１３】

【００９４】
２）実験は１）に準じて行った。融合蛋白質の添加濃度は２．５ｎＭとした。ヒスタミン濃度、キマーゼ活性を求めた。結果を表１４に示す。
【００９５】
【表１４】

【００９６】
３）実験は１）と同様に行った。融合蛋白質の添加濃度は２．５〜２０ｎＭとした。細胞数とキマーゼ活性を求めた。例数は２とした。結果を表１５に示す。
【００９７】
【表１５】

【００９８】
実験例９（ヒトマスト細胞への作用）
１）ヒトＣＤ３４陽性骨髄細胞（ＢｉｏＷｈｉｔｔａｋｅｒ社）２×１０^４／ウェルを、ＳＣＦ（１００ｎｇ／ｍｌ）、ＩＬ−６（１００ｎｇ／ｍｌ）、ＩＬ−１０（１０ｎｇ／ｍｌ）、融合蛋白質（ＨＬＨ系）を添加した培養液を週に一度、半量交換しながら９週間培養し、細胞数、回収した細胞についてｌｙｓａｔｅ中のキマーゼ活性、ヒスタミン含量を測定した。培養液は５％ＦＣＳを含むＭｅｄｉａＩ（免疫生物研究所）を用いた。例数は３とした。その結果、本発明の融合蛋白質はヒトマスト細胞分化を阻害した（表１６）。
【００９９】
【表１６】

【０１００】
２）ヒトＣＤ３４陽性骨髄細胞を１）に準じてサイトカインの存在下に８週間培養し、途中まで分化した未成熟なマスト細胞を調製した。細胞を一旦回収、遠心洗浄し、新しくプレートに培養液中で７×１０^５／ウェルずつ播種し、融合蛋白質（ＨＬＨ系を１０または５０ｎＭ添加）を添加した同培養液（前記のサイトカインを含む）を用いて培養を継続した。週に一度培養液を半量交換した。培養２週間後に細胞数、キマーゼ活性、ヒスタミン含量を測定した。また当該細胞をＰＥ標識抗ｃ−ｋｉｔ抗体（あるいはＰＥ標識対照抗体）で染色し、ＦＡＣＳで解析した。その結果、本発明の融合蛋白質は未成熟マスト細胞に作用してｃ−ｋｉｔ発現を阻害し、ヒトマスト細胞の成熟を阻害した（表１７）。
【０１０１】
【表１７】

【０１０２】
３）ヒトＣＤ３４陽性骨髄細胞を１）に準じてサイトカインの存在下に１２週間培養し、分化完了した成熟なマスト細胞を調製した。細胞を一旦回収、遠心洗浄し、新しくプレートに培養液中で２．４×１０^４／ウェルずつ播種し、融合蛋白質（ＨＬＨ系を２、１０または５０ｎＭ添加）を添加した同培養液（前記のサイトカインを含む）を用いて培養を継続した。週に一度培養液を半量交換した。培養３週間後に細胞数、ヒスタミン含量を測定した。その結果、本発明の融合蛋白質は濃度依存的にヒスタミン含量を低下させ、成熟ヒト骨髄培養マスト細胞の機能を抑制した（表１８）。
【０１０３】
【表１８】

【０１０４】
実験例１０（インビボ阻害）
１）Ｃ５７ＢＬ／６マウスに融合蛋白質（ｗｈ系）６０μｇを２００μＬのＰＢＳ中で週３回４週間まで腹腔内投与した。最終投与の翌日に腹腔細胞を回収し、以下の項目を測定した。例数は４とした。投与４週間の結果を表１９に示す。
【０１０５】
総細胞数： コールターカウンターまたは血球計数装置により測定した。
マスト細胞： スメア標本をトルイジンブルーで染色し、陽性の比率を算出した。
ヒスタミン： 腹腔細胞懸濁液にトリトンＸ１００を添加してＬｙｓａｔｅを調製し、ＬｙｓａｔｅについてＥＬＩＳＡキットにより測定した。
ｃ−ｋｉｔおよびＩｇＥ受容体両陽性細胞： 回収した腹腔細胞をＰＥ標識抗ｃ−ｋｉｔ抗体およびマウスＩｇＥ／ビオチン標識抗ＩｇＥ抗体／ＡＰＣ標識ストレプトアビジンで二重染色しＦＡＣＳで解析した。
【０１０６】
【表１９】

【０１０７】
２）１０または５０μｇの融合蛋白質（ｗｈ系、いずれも２００μＬベヒクル中で）をマウス腹腔内に２週間投与した。例数は１０とした。１）と同様にして各項目を測定した。結果を表２０に示す。
【０１０８】
【表２０】

【０１０９】
３）１０μｇの融合蛋白質（ＨＬＨ系）を３５０μＬベヒクル中でマウス腹腔内に１３日間投与した。マスト細胞数、ヒスタミン含量の測定は１）に準じて行った。結果を表２１に示す。
【０１１０】
【表２１】

【０１１１】
本発明の融合蛋白質（ｗｈ系、ＨＬＨ系）はいずれの実験系においてもインビボでマスト細胞数を減少させた。
【０１１２】
【発明の効果】
本発明によれば、ＭＩＴＦ変異体を用いて、マスト細胞の細胞死を誘発することができる。よって、マスト細胞が関与する各種疾患の予防・治療に有用な薬剤を臨床の場に提供することができる。
【０１１３】
【配列表】
配列表配列番号１：ＭＩＴＦのｍｉ変異体のアミノ酸配列
配列表配列番号２：その塩基配列
配列表配列番号３：ＭＩＴＦのｗｈ変異体のアミノ酸配列
配列表配列番号４：その塩基配列
配列表配列番号５：ｂＨＬＨ−Ｚｉｐ断片のアミノ酸配列
配列表配列番号６：その塩基配列
配列表配列番号７：ＴＡＴ由来ペプチドのアミノ酸配列
配列表配列番号８：その塩基配列
配列表配列番号９：ＨｉｓＴａｇのアミノ酸配列
配列表配列番号１０：その塩基配列
配列表配列番号１１：ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｍｉ変異体からなる融合蛋白質のアミノ酸配列
配列表配列番号１２：その塩基配列
配列表配列番号１３：ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｗｈ変異体からなる融合蛋白質のアミノ酸配列
配列表配列番号１４：その塩基配列
配列表配列番号１５：ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのｂＨＬＨ−Ｚｉｐ断片からなる融合蛋白質のアミノ酸配列
配列表配列番号１６：その塩基配列
配列表配列番号１７：プライマーＭ−Ｔａｔの塩基配列
配列表配列番号１８：プライマーＴａｔ ３の塩基配列
配列表配列番号１９：プライマーＴａｔ ２の塩基配列
配列表配列番号２０：プライマーＴａｔ １の塩基配列
配列表配列番号２１：ＭＩＴＦのＡタイプＮ末領域（１−３０５）のアミノ酸配列
配列表配列番号２２：その塩基配列
配列表配列番号２３：ＨｉｓＴａｇ−ＰＴＤ−ＭＩＴＦのＡタイプＮ末領域（１−３０５）からなる融合蛋白質のアミノ酸配列
配列表配列番号２４：その塩基配列
配列表配列番号２５：プライマーＨＬＨ Ｆ
配列表配列番号２６：プライマーｐＴＤ３−ＭＩＴＦａ
配列表配列番号２７：プライマーＭＩＴＦＲ−Ｎ
【０１１４】

【図面の簡単な説明】
【図１】本発明の融合蛋白質（ｍｉ系、ｗｈ系）および当該融合蛋白質を発現するプラスミドｐＳＵ０８２（ｍｉ系／ｐＴｒｃＨｉｓＢ）、ｐＳＵ０８３（ｗｈ系／ｐＴｒｃＨｉｓＢ）の構造を概略的に示したものである。
【図２】本発明の融合蛋白質（ＨＬＨ系）および当該融合蛋白質を発現するプラスミドｐＳＵ０８５（ＨＬＨ系／ｐＴｒｃＨｉｓＢ）の構造を概略的に示したものである。
【図３】本発明の融合蛋白質（ＡタイプＮ末領域）を発現するプラスミドｐＳＵ０８７（ＡタイプＮ末領域／ｐＳＵ０９３）の構築手順を概略的に示したものである。
【図４】本発明の融合蛋白質（ｗｈ系）を発現するプラスミドｐＳＵ１２１（ｗｈ系／ｐＥＴ１４）の構築手順を概略的に示したものである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a pharmaceutical use using the MITF mutant. More specifically, the present invention relates to a pharmaceutical use of a fusion protein comprising a mutant MITF.
[0002]
[Prior art]
MITF (abbreviation of microphthalmia-associated transcription factor) is a kind of transcription control factor existing in a living body, and is a protein having a c-kit gene expression regulating ability specific to mast cells.
[0003]
MITF is a known substance (Non-Patent Document 1), but the first thing found was a gene encoding MITF. That is, the gene was isolated as a causal gene in mi / mi mice. In the mi / mi mouse, normal MITF is not expressed because of a mutation in the MITF gene, that is, a deletion of one amino acid in the transcription activation region of the MITF gene. The mi / mi mouse is a mutant mouse having ocular hypoplasia, melanocyte deficiency, mast cell loss, and osteopetrosis as main symptoms, and tissues such as melanocytes, mast cells, retinal pigment epithelial cells, and osteoclasts. Presents abnormal differentiation. The abnormal differentiation of these cells in mi / mi mice is caused by the fact that normal MITF is not expressed and gene transcription is not activated by MITF.
[0004]
Analysis of the expression tissue distribution by Northern blot using normal mouse tissues or cultured cell lines showed that MITF mRNA was expressed in heart, melanocytes, and mast cells. In recent years, it has been reported that MITF includes isoforms having different sequences at the 5 ′ end of cDNA, that is, melanocyte type (M type), heart type (H type) and A type (Non-Patent Document 2). . The MITF gene is composed of 10 (M type) or 11 (A, H type) exons, and exon 2 and subsequent ones are almost common in all three types. The M type is further classified into two types depending on whether an exon 5b consisting of 18 bases encoding 6 amino acids is added or not. The types of A and H completely match after exon 1B, and the exon 1 at the 5 'end is different. The A, H type and M type are common after exon 2, but the M type does not have exon 1B upstream of exon 2 and is linked to specific exon 1. In addition, it is clear from the genome sequence that each type of promoter is different.
[0005]
The MITF protein is presumed to be composed of a nuclear localization region, a transcription activation region, a DNA binding region, a dimer formation region, and an activation region of MITF itself. It is common. The MITF protein is a transcription control factor having bHLH-Zip (basic-helix-loop-helix / leucine zipper) at the center of its structure, forms a dimer, binds to DNA, and transcribes the target gene. Activate. It has been reported that there is a large difference between the A-type and H-type transcription activity, and it is presumed that exon 1 having a different gene sequence between both types has a function of controlling the transcription activity.
[0006]
In addition, it has been reported that MITF protein acts as a transcriptional regulator in melanocytes, and is involved in melanocyte proliferation / differentiation, melanin synthesis, and the like.
[0007]
It has been reported that also in mast cells, the MITF protein acts as a transcriptional regulator that regulates c-kit gene expression (a transcription factor that activates the c-kit promoter) (Non-Patent Document 3). The c-kit gene is expressed in hematopoietic progenitor cells, mast cells, pigment cells, and germ cells, and the proliferation and differentiation of these cells are controlled by the action of Sl factor. In mast cells, the MITF protein is thought to be involved in maintaining the mast cell survival by controlling c-kit gene expression.
[0008]
It has long been reported that mast cells are involved in allergic diseases (Non-Patent Document 4). In addition, diseases involving mast cells in addition to allergic diseases include autoimmune diseases, pulmonary fibrosis, cancer, mast cytosis, and mast cytomas.
[0009]
On the other hand, PTD (abbreviation of protein transduction domain) is a general term for domains having a function of translocating (incorporating) proteins into cells through biological membranes. For example, analysis by domain unit in the HIV antigen revealed that the TAT-derived peptide had a function of transferring the HIV antigen into normal T cells, and this was confirmed to be a factor in cell infection. (Non-Patent Document 5). Based on these findings, there are reports that various PTDs having the same function as TAT exist, and a method of fusing these PTDs and various proteins to translocate into cells (Non-patent Document 6). ).
[0010]
However, no report has been found so far on a technique for migrating MITF or mast cells into cells using PTD.
[0011]
[Patent Document 1]
WO 00/47765 pamphlet
[Patent Document 2]
WO 01/66735 pamphlet
[Patent Document 3]
JP-T-2001-513987
[Patent Document 4]
JP 2002-505077 A
[Patent Document 5]
Japanese Unexamined Patent Publication No. Hei 5-505102
[Patent Document 6]
JP-T-2002-512808
[Non-patent document 1]
Cell, 1993, 74, 395-404.
[Non-patent document 2]
Biochemistry, 1999, Vol. 71, No. 1, 61-64.
[Non-Patent Document 3]
Blood, 1996, 88: 4, 1225-1233.
[Non-patent document 4]
IgE, must cells and the allergic response (Ciba Foundation symposium) 147, John Wiley & Sons publisher, Chichester, UK. , 1989
[Non-Patent Document 5]
Cell, 1988, 55, 1179-1188.
[Non-Patent Document 6]
Current Opinion in Molecular Therapeutics 2000, 2000, Vol. 2, No. 2, pp. 162-167.
[Non-Patent Document 7]
Trends in Genetics, 1995, Vol. 11, No. 11, pp. 442-448.
[Non-Patent Document 8]
Proceeding National Academic Society USA (Proc. Natl. Acad. Sci. USA), 1988, 85, 8998-9002.
[Non-Patent Document 9]
Current Protocols in Immunology (Wiley) Section 7.25.2 [Current Protocols in Immunology (Wiley) Section]
[0012]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel pharmaceutical use using a MITF mutant.
[0013]
[Means for Solving the Problems]
The present inventors have conducted studies in consideration of the above circumstances, and as a result, have found that a combination of a MITF mutant and a PTD can induce cell death of mast cells, thereby completing the present invention.
[0014]
That is, the present invention relates to an agent for inducing cell death of mast cells containing a fusion protein composed of PTD and MITF mutant as an active ingredient; and a mast cell containing a fusion protein composed of PTD and MITF mutant as an active ingredient. A fusion protein comprising HisTag, PTD and MITF mutant, wherein the MITF mutant is a mi mutant, a wh mutant, an HLH fragment or an A-type N-terminal region of MITF (1-305) A fragment, wherein the PTD is a TAT-derived peptide; the fusion protein; a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier; and a method for producing the fusion protein using genetic engineering techniques. The production method comprising the step of producing a fusion protein by the method; and a DNA encoding the fusion protein. It is. The following details will be described.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
A. MITF mutant
The MITF mutant used in the present invention is not particularly limited as long as it is a mutant of natural (wild-type) MITF and has MITF inhibitory activity. Specifically, mi mutants of MITF, wh mutants of MITF, and other MITF mutants, for example, splicing variants (any of A-form, H-form, M-form, and N-form) or a portion of MITF Structures having MITF inhibitory activity, for example, a bHLH-Zip (M-form N-terminal 196 to 285) fragment, an N-terminal fragment containing that portion, an A-form N-terminal region (N-terminal 1 (Th to 305 th) fragments are exemplified. Specific examples include the MITF mutants disclosed in Non-Patent Document 7, Patent Document 1, Patent Document 2, and the like. Particularly preferred mutants are mi mutant, wh mutant, bHLH-Zip fragment (hereinafter, HLH fragment), and N-terminal region (1-305) fragment of Form A. Table 1 shows the relationship between these amino acid sequences and base sequences.
[0016]
[Table 1]

[0017]
In addition, even structural analogs of these mutants having substantially the same MITF inhibitory activity as the mutants are included in the scope of the present invention. For example, in the above amino acid sequence, one or more amino acids may be substituted, deleted, inserted or added, and may have substantially the same activity as the mutant.
[0018]
B. PTD
The PTD used in the present invention is not particularly limited as long as it has a property of being taken up (transferred) into cells, and a known one can be used. Specifically, various oligopeptides listed in Table 2 on page 164 in the aforementioned literature (Non-Patent Document 6), various oligopeptides for PTD disclosed in Patent Documents 3, Patent Documents 4 and 5, and the like. Is exemplified. A more preferred PTD is a TAT-derived peptide (the amino acid sequence is YGRKKRRQRRR, SEQ ID NO: 7, and the preferred base sequence is represented by SEQ ID NO: 8). The PTD may be bound to either the N-terminal or C-terminal of the MITF mutant. Further, the bond may be direct or indirect via a cross-linking agent (linker). Examples of the linker include a glycine residue.
[0019]
C. Other components (domains)
The fusion protein of the present invention may bind to a protein having affinity for a ligand in affinity chromatography for purification. The domain is not particularly limited, and a known domain can be used. Examples of such a relationship include an antigen and its antibody, a receptor and its ligand, Ni-NTA (nitrilotriacetic acid) and HisTag, and avidin (or streptavidin) and biotin. More preferred is HisTag (the amino acid sequence is MGGGSHHHHHH, SEQ ID NO: 9, and the preferred nucleotide sequence is SEQ ID NO: 10). This domain may be linked to either the N-terminal or C-terminal of the fusion protein. Further, the bond may be direct or indirect via a cross-linking agent (linker). Examples of the linker include a glycine residue.
[0020]
D. Preparation of fusion protein
The method for preparing the fusion protein of the present invention includes: (1) a method of synthesizing the entire fusion protein using a chemical synthesis technique; and (2) a method of preparing each of the above-mentioned constituent elements (domains) separately, And (3) a method in which genes encoding the respective constituent elements are linked and then expressed as a fusion protein at once using a genetic engineering technique. In the case of (2), examples of a method for preparing each component include a chemical synthesis method, a cell culture method, a method using a genetic engineering technique, and the like. PTD can also be prepared by cleavage and isolation from HIV antigen.
[0021]
For example, a case where a fusion protein is prepared by a genetic engineering technique will be described.
1) Prepare a DNA encoding the fusion protein. The preparation is performed by a conventional method. First, a gene encoding the MITF mutant is prepared. The gene can be prepared by extracting mRNA from appropriate cells, synthesizing cDNA using reverse transcriptase and DNA polymerase, and amplifying the cDNA by polymerase chain reaction (PCR). Specifically, mRNA is extracted using a commercially available kit for extracting mRNA, and reverse transcription, cDNA synthesis and amplification are performed using a 5′-RACE method using a commercially available cDNA amplification kit (Non-patent Document 8). ) Or reverse transcription-polymerase chain reaction (RT-PCR) using appropriate primers. Alternatively, it can be obtained by extracting genomic DNA from an appropriate cell and amplifying the gene by PCR. In addition, known report examples, for example, the gene encoding MITF disclosed in Non-patent Document 1 and Patent Document 2 described above can also be used. For example, the HLH fragment of the present invention can be prepared from the MITF M type, and the A type N-terminal region (1-305) fragment can be prepared from the MITF A type (MITF A / pCDNA3).
[0022]
Next, a gene encoding PTD is prepared in the same manner as described above, and ligated to a gene encoding the MITF mutant. The same applies to HisTag. Further, a DNA encoding the fusion protein of the present invention is prepared by inserting a DNA encoding the fusion protein comprising the PTD-MITF mutant using a commercially available plasmid (pTrcHisB, Invitrogen) carrying HisTag. be able to.
[0023]
2) An expression vector is prepared by incorporating DNA into an appropriate vector. The vector is used to express the fusion protein under the control of a specific promoter. The incorporation is performed by a conventional method.
[0024]
The obtained target DNA can be purified and introduced into a vector DNA to construct a host-vector system. The host-vector system generally uses the host in combination with a replicon derived from a species compatible with the host cell. The vector DNA has an origin of replication, a promoter, a control sequence (enhancer), a signal sequence, a ribosome binding site, an RNA splice sequence, a polyA addition site, a transcription termination sequence (terminator), and the like. It may also have a marker sequence that allows phenotype selection in transformed cells. Examples of the vector DNA include a chromosome, an episome-derived vector, for example, a bacterial plasmid-derived, bacteriophage-derived vector, or a virus-derived vector such as baculovirus, papovavirus, SV40, or a cosmid or phagemid. In addition, an expression vector, a cloning vector, or the like can be used depending on the purpose.
[0025]
Promoters include known promoters, which can be selected according to the host for expression. For example, when Escherichia coli is used as a host, examples thereof include promoters such as a trp promoter, a lac promoter, a trc promoter (a synthetic promoter in which the −35 region of the trp promoter and the −10 region of the lac promoter are linked), and a T7 promoter. Further, the expression vector may carry a marker gene such as amp r.
[0026]
As a method for incorporating the DNA according to the present invention into the vector DNA, a method known per se can be applied. For example, a method is used in which an appropriate restriction enzyme is selected and treated to cut the target DNA at a specific site, then mixed with a similarly treated vector DNA, and religated with ligase. Alternatively, a desired recombinant vector can be obtained by ligating an appropriate linker to the target DNA, and inserting this into a multicloning site of a vector suitable for the target. In addition, if an expression vector is used as a vector DNA to be introduced into a host, a target protein can be produced.
[0027]
3) A transformant is prepared by introducing an expression vector into a host. Transformation is performed by a conventional method. As a host, Escherichia coli, Bacillus subtilis, yeast, animal cells and the like can be used. Preferably it is Escherichia coli. In addition, auxotrophic strains and antibiotic-sensitive strains can be used as hosts.
[0028]
Examples of a method for preparing a transformant include a method of directly introducing a plasmid into a host cell and a method of integrating a plasmid into a chromosome. Examples of the former include a protoplast polyethylene glycol method and an electroporation method. In the latter case, the DNA sequence of a part of the gene present in the host chromosome is contained in the plasmid, and the homologous sequence portion is used to introduce the plasmid or its linear fragment into the host chromosome by homologous recombination. can do.
[0029]
4) Culture the transformant to produce a fusion protein. The cultivation is performed by a conventional method using a medium and culture conditions (temperature, time, etc.) applicable to the host. Culture conditions when Escherichia coli is used are usually at about 15 to 43 ° C (preferably 30 to 37 ° C) for about 1 to 100 hours. Moreover, ventilation and stirring can be added as needed. The culture format may be any of batch culture, semi-batch culture (fed-batch culture), and continuous culture.
[0030]
5) Purify the produced fusion protein. When Escherichia coli is used as a host, the protein is first solubilized by treatment such as ultrasonic grinding. Purification of the produced fusion protein can be performed by a method known per se (Patent Document 6, etc.). For example, a method using a Ni column, anion exchanger treatment, dialysis treatment and the like are exemplified. Further, it is preferable to use the fusion protein of the present invention obtained by removing the denaturant after treatment with the denaturant (chaotropic agent). Examples of the denaturing agent include urea, guanidine hydrochloride, and thiocyanate. The concentration of the denaturant during the treatment with the denaturant is, for example, about 1 to 10 M in concentration. Specifically, after treating by contacting the fusion protein with a denaturing agent, a treatment is performed using a Ni column in the presence of the denaturing agent, and further, an operation of removing the denaturing agent by anion exchanger or dialysis treatment is performed. Then, the fusion protein is purified.
[0031]
E. FIG. Properties of the prepared fusion protein
The fusion protein of the present invention has a property of translocating into cells, particularly mast cells. Preferably, it is composed of HisTag, PTD and MITF variants. It has a molecular weight of the order of 10 to 100 kilodaltons (kDa). Preferably, when viewed from the N-terminal side, HisTag, PTD, and MITF mutant are arranged in this order. The specific arrangement is as shown in Table 2.
[0032]
[Table 2]

[0033]
F. Formulation
For preparation of the fusion protein of the present invention, a method known per se can be used. For example, a pharmaceutically acceptable carrier may be added to or mixed with the fusion protein. In the pharmaceutical composition obtained by the formulation, the concentration of the fusion protein is, for example, about 0.1 to 100 μg / mL or about 0.1 to 100 nM.
[0034]
G. FIG. Use
The mutant MITF of the present invention (or a fusion protein using the same) inhibits the activity of endogenous MITF by migrating into mast cells, inhibits differentiation of mast cells from precursor cells, and survives mast cells. And has the effect of inducing cell death (apoptosis) in mature mast cells. Therefore, the preparation of the present invention is expected to be useful for prevention and treatment of various diseases involving mast cells. Examples of the disease include allergy, asthma, autoimmune disease, pulmonary fibrosis, cancer, mast cytosis, mast cytoma and the like.
[0035]
H. Usage and dosage
The dose and dosage of the fusion protein of the present invention may be selected so that the fusion protein is present in the living body at a concentration of about 0.001 to 10 μg / mL. Alternatively, the dose is, for example, about 10 μg to 50 mg. Examples of the administration route include intravenous administration, subcutaneous administration, intramuscular administration, transdermal administration, and intratracheal administration.
[0036]
【Example】
The present invention will be described in more detail with reference to Examples, Formulation Examples and Experimental Examples, which by no means limit the present invention.
[0037]
Example 1
1) Construction of fusion protein expression plasmid
The expression plasmid is a PTD sequence downstream of the HisTag for purification, between the NcoI site upstream of the HisTag sequence of the Escherichia coli expression vector pTrcHisB (Invitrogen, No. V360-20) and the HindIII site in the multiple cloning site. Was constructed, and a HisTag-PTD-MITF mutant gene to which a MITF mutant cDNA was connected was inserted downstream thereof. See FIG.
[0038]
The addition of the HisTag and PTD sequences to the MITF cDNA was performed using the PCR method. First, using the MITF M type cDNA (pSU054, MITF-M / pT7 Blue) as a template, the upstream portion containing the MITF and BamHI sites was amplified by PCR. Furthermore, the expression vector for Escherichia coli was extended using four types of primers (M-tat, Tat3, Tat2, and Tat1) until the recognition sequence of HisTag and the restriction enzyme NcoI was added upstream of the transcription initiation codon of MITF. It was cloned between the NcoI and BamHI sites of pTrcHisB, which carries lac P / O and Ampr.
[0039]
Each primer has the following nucleotide sequence.
M-tat (SEQ ID NO: 17): GCGACGAAGAGGTATGCTTAGAATACATCTCACTACC
Tat 3 (SEQ ID NO: 18): GGCAGGGAAGAAGCGGAGACAGCGACGAAGAGGTATG
Tat 2 (SEQ ID NO: 19): ATCATCATCATGGGTGGTTATGGCAGGAAGAAGCGG
Tat 1 (SEQ ID NO: 20): TAAACCCATGGGGGGTTCTCATCATCATCATCATCATCATGGTG
[0040]
Next, the M type (pSU054), the mi mutant (pSU061, MITF-Mmi / pT7 Blue), and the wh mutant (pSU062, MITF-M wh / pT7 Blue) were digested with BamHI and HindIII, respectively, to thereby obtain the downstream portion of the MITF cDNA. Was isolated. This part also includes a mutated part. The isolated cDNA fragment was inserted between the BamHI site and the HindIII site of the plasmid in which the upstream portion was cloned. Plasmid pSU81 is one into which the gene of the fusion protein containing M type, which is normal MITF, has been inserted. pSU082 was obtained by inserting a mi mutant, and pSU083 was obtained by inserting a fusion protein containing a wh mutant.
[0041]
2) Expression of fusion protein
The constructed expression plasmid was introduced into E. coli DH5α (Toyobo) for transformation. Escherichia coli was inoculated into 50 mL of L-Broth (containing 50 μg / mL of ampicillin) so as to make one loopful of E. coli, and cultured at 37 ° C. for 17 hours. Further, 1 L of L-Broth (containing 50 μg / mL of ampicillin and 0.2% of glucose) was inoculated with the culture solution to 1%. After culturing at 37 ° C. until the turbidity of A600 became about 0.1, IPTG (isopropylthio-β-D-galactoside) was added to a final concentration of 0.4 mM. After culturing for 2 hours, Escherichia coli was collected.
[0042]
3) Purification of fusion protein
Escherichia coli was recovered by centrifugation, and ultrasonically crushed and solubilized in a 20 mM HEPES buffer solution (pH 8.0, pH is hereinafter the same) containing 8 M urea, 0.1 M sodium chloride and 10 mM DTT. The obtained Lysate was applied to a Ni-NTA agarose (Qiagen) column equilibrated with a 20 mM HEPES buffer containing 8 M urea, 0.1 M sodium chloride, 10 mM imidazole, and 1 mM DTT, and the same buffer was used. And washed. The protein bound to the column was eluted by the same buffer solution as solution A and imidazole concentration linear gradient using the same buffer solution containing 200 mM imidazole as solution B, and analyzed by SDS-PAGE (Tefco) to analyze the objective protein. Fractions showing molecular weight were pooled.
[0043]
For 1 volume of the eluate, 1 volume of 20 mM HEPES buffer and 2 volumes of the same buffer containing 4 M urea were added and diluted, and the resulting solution was diluted with 20 mM HEPES buffer containing 4 M urea and 25 mM sodium chloride. The solution was applied to a quaternary ammonium salt type strong anion exchanger (trade name: Q-Sepharose, Pharmacia) equilibrated with a HEPES buffer, and washed with the same buffer. Next, after washing with 20 mM HEPES buffer to remove urea, the protein bound to the column was eluted with the same buffer containing 1 M sodium chloride. The eluted fusion protein was dialyzed against Dulbecco's PBS (isotonic phosphate buffer) containing 10% glycerol, and then aliquoted and stored at -80 ° C.
[0044]
When the obtained final product was analyzed by SDS-PAGE, a band having a molecular weight of about 50 kDa was observed under reducing conditions. This band reacted with rabbit anti-MITF antibody. The rabbit anti-MITF antibody was obtained by immunizing a rabbit with a peptide having 20 amino acid residues at the C-terminus of MITF and subjecting the serum to affinity purification using a column to which the peptide was bound. An antibody that specifically recognizes was prepared.
[0045]
Formulation Example 1
A composition consisting of the fusion protein of the present invention and Dulbecco's PBS containing 10% glycerol was prepared.
[0046]
Experimental example 1
1) First, it was confirmed whether or not the fusion protein of the present invention was transferred into cells. The MITF mutant fusion protein prepared in Example 1 was fluorescently labeled with FITC (fluorescein isothiocyanate) by a conventional method, and added to COS7 cells. One hour after the addition, the fluorescence intensity was measured using FACS (fluorescene-activated cell sorter) Calibur (Becton Dickinson). Table 3 shows the results.
[0047]
[Table 3]

[0048]
As is clear from Table 3, it was observed that strong fluorescence was associated with the cell (associate), and it was confirmed that the fusion protein was efficiently transferred into the cell.
[0049]
2) Luciferase analysis was performed to determine whether the fusion protein of the present invention migrated into cells and inhibited the activity of endogenous MITF.
[0050]
As an expression plasmid, a normal MITF expression plasmid pSU063 (MITF-M / pCDNA3), various vectors for preparing a plasmid in which a luciferase gene is connected downstream of the c-kit gene promoter, that is, for expression of C-kit-Rluc The vector pSU053 (C-kit / R-luc) and the luciferase expression vector pGL2 (Lluc, Promega) were used. Each plasmid was introduced (transfected) into COS7 cells. The conditions are as follows. The amount of plasmid used was 3 μg per dish of 6 cm (pSU063: pSU053: pGL2 = 1: 1: 0.1). For the introduction of the plasmid, a transfection kit (Stratagene, # 200385) was used.
[0051]
1 × 10 per well in a 6-well plate ⁵ Cells for 17 hours ₂ Placed in the incubator. 10 μL of Solution 1 (reagent attached to the kit. 2.5 M CaCl 2 ₂ ), 90 μL of distilled water and 3 μg of DNA were added thereto, and an equal amount of Solution 2 [reagent attached to the kit. Consisting of 2 × PBS (pH 6.95)] and allowed to stand at room temperature for 20 minutes. The mixed DNA solution was added to the culture. After 24 hours, the above medium was replaced, and 1 μg / mL of the fusion protein of the present invention (prepared in Example 1) was added.
[0052]
48 hours after the addition of the fusion protein of the present invention, the cells were washed with PBS (-), 0.5 mL of passive lysis buffer was added per 6-well dish, and the cells were scraped off using a scraper. The cells were transferred to a 1.5 mL tube and centrifuged at 14000 rpm at 4 ° C. for 5 minutes. The measurement of luciferase activity was performed using Dual Luciferase assay kit (Promega). 100 μL of Luciferase Assay Reagent II was added to and mixed with 20 μL of the centrifuged supernatant on a 96-well plate, and firefly luciferase activity was measured. Further, 100 μL of Stop & Go Reagent was added to and mixed with the mixture, and the Renilla luciferase (R-luc) activity was measured. After correcting the value of R-luc with pGL2, the value was converted to a value when R-luc in PBS addition was set to 1. Table 4 shows the results.
[0053]
[Table 4]

[0054]
In the table, mi represents a fusion protein comprising a HisTag-PTD-MITF mi mutant, and wh represents a fusion protein comprising a HisTag-PTD-MITF wh mutant.
[0055]
The luciferase activity expressed by COS cells was also inhibited in the presence of the fusion protein of the present invention, suggesting that the fusion protein migrates into cells and inhibits endogenous MITF.
[0056]
Experimental example 2
It was confirmed how the fusion protein of the present invention affects the differentiation system of mast cells induced by SCF.
[0057]
Bone marrow cells were prepared from normal mice, and 2 × 10 ⁶ Cells / well were prepared with 10% FCS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM 2-mercaptoethanol, 2 μg / mL Was cultured in a 24-well plate in RPMI1640 containing the fusion protein of the present invention (HisTag-PTD-MITF wh mutant) in the presence of 50 to 100 ng / mL of bone marrow cell factor (SCF, IBL). Half the medium was changed once a week, at which time SCF was also added. After culturing at 37 ° C for 21 days, the number of mast cells, c-kit and the expression level of IgE receptor were measured.
[0058]
The number of mast cells was measured by the toluidine blue staining method. Toluidine blue staining was performed using a staining solution of pH 2.7 according to Non-Patent Document 9.
[0059]
The expression level of c-kit was measured using FACS. Cells are grown with 0.1% NaN ₃ Suspended in 100 μL of PBS containing 0.1% BSA, and 5 to 10 μg / mL of R-PE (R-phycoerythrin) -labeled anti-mouse c-kit antibody (PharMingen) or R-PE-labeled rat IgG2B, kisotype standard. (PharMingen) for 1 hour in ice. After washing, 1 μg / mL of pyridium iodide was added and analyzed by FACS Calibur (described above). The measurement of the expression level of the IgE receptor was similarly performed by FITC analysis using 5 μg / mL FITC-labeled mouse IgE. Table 5 shows the results regarding the number of mast cells.
[0060]
[Table 5]

[0061]
In the group to which the fusion protein of the present invention was added, the number of mast cells was reduced to one-tenth or less on the 21st day of culture as compared to the group without addition. Table 6 shows the results of the expression of c-kit on day 21 of culture.
[0062]
[Table 6]

[0063]
In the group to which the fusion protein of the present invention was added, the expression level of c-kit was reduced to one-tenth or less on day 21 of the culture as compared to the group without addition. In addition, the expression level of the IgE receptor did not change (experimental data not shown). Thus, it was confirmed that the fusion protein of the present invention specifically inhibited the expression of the SCF receptor c-kit in mast cell precursor cells, and as a result, strongly inhibited mast cell differentiation.
[0064]
Experimental example 3
In order to confirm the effect of the fusion protein of the present invention on mature mast cells, the effect on mast cell survival in a co-culture system with fibroblasts was confirmed.
[0065]
Splenocytes were prepared from normal mice, 10% FCS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM 2-mercaptoethanol Was cultured for more than 3 weeks in RPMI1640 containing IL-3 (GENZYME) or WEHI-3 cell-conditioned medium (WEHI-3CM) to obtain spleen-derived cultured mast cells (SMC). The fibroblast NIH-3T3 (RIKEN cell bank) is cultured to confluence in a 6-well plate, and the SMC is free from IL-3 and WEHI-3CM and contains 1 μg / mL of the fusion protein of the present invention. 3 × 10 ⁵ Individually sown. The medium was replaced with a fresh medium every 2-3 days. After culturing at 37 ° C. for 15 days, the number of mast cells, the amount of histamine and the chymase activity were measured. Table 7 shows various experimental systems.
[0066]
[Table 7]

[0067]
Histamine was quantified by the RIA method (Eiken Chemical). The measurement of chymase activity in the granules was performed using N-succinyl-Ala-Ala-Pro-Phe-pNA (Sigma) which is a specific synthetic substrate. Table 8 shows the results.
[0068]
[Table 8]

[0069]
When fibroblasts and normal mast cells were co-cultured, the mast cells survived for a long time. In addition, artificially created mimast cells (mi / miSMC, not naturally occurring) having an abnormality in MITF died within 2 weeks by apoptosis in co-culture with fibroblasts. When the fusion protein (mi mutant type, wh mutant type) of the present invention is added to the co-culture of normal mast cells and fibroblasts, the survival of mast cells is inhibited, and almost two weeks after mimast cells, the mast cells die. did. The decrease in mast cell number was also confirmed by a decrease in the amount of histamine and a decrease in chymase activity. As a result, it was suggested that the fusion protein of the present invention induces apoptosis in mature mast cells.
[0070]
Experimental example 4
The influence of the addition concentration of the fusion protein of the present invention was examined. The MC / 9 mast cell line (obtained from ATCC) was prepared in the presence or absence of a fusion protein consisting of a wh mutant of FITC-labeled HisTag-PTD-MITF in a FACS staining buffer (0.1% bovine serum albumin and Incubated for 1 hour on ice in Dulbecco's PBS containing 0.1% sodium azide. After washing, pyridium iodide was added to gate out dead cells and analyzed by FACS Calibur (supra). Table 9 shows the results.
[0071]
[Table 9]

[0072]
From the results in Table 9, it was found that the fusion protein migrated into the cell at a higher concentration depending on the added concentration of the fusion protein of the present invention.
[0073]
Example 2: HisTag-PTD-HLH
1) Construction of expression vector
As a template for preparing an expression vector, a normal MITF M type (pSU054) was used. The HLH fragment amplified the region from the 196th to 285th amino acids of the N-terminal of the M type by PCR. On the N-terminal side of the amplified fragment, HisTag, PTD and a recognition sequence for restriction enzyme NcoI were added using primers (Tat1, Tat2, Tat3 and HLHF). Further, a recognition sequence for a restriction enzyme XbaI was added to the C-terminal side. The amplified fragment was digested with NcoI + XbaI and replaced with the NcoI-XbaI region of pSU081 created in Example 1. The amplified fragment was subjected to TA cloning to confirm that the nucleotide sequence was correct. The constructed vector pSU085 is composed of lac promoter, DNA of fusion protein of HisTag-PTD-HLH region, and ampr (FIG. 2).
[0074]
The primer HLHF has the following nucleotide sequence.
HLHF (SEQ ID NO: 25): GCGACGAAGAGGTATGTTGGCTAAGAGAGG
2) A fusion protein was produced and purified according to Example 1.
[0075]
Example 3: A type N-terminal region of HisTag-PTD-MITF
1) Construction of expression vector
Using pSU064 (A type of MITF / pCDNA3; see Patent Document 2) as a template and two types of primers (pTD3-MITFa, MITFR-N), PCR was performed, and the N-terminal region (from the N-terminal first). 305 amino acids). A SacII site was added to the 5 ′ end of pTD3-MITFa, and an EcoRI site was added to the 5 ′ end of MITFR-N. The amplified fragment was digested with SacII + EcoRI, and inserted between the same sites of pSU093 (a PTD cassette expression vector having pTrcHis as a basic skeleton) (FIG. 3). In the constructed expression vector pSU087, DNA fragments are connected in the order of HisTag-PTD-A type N-terminal region, and the fusion protein is expressed under the control of the lac promoter.
[0076]
Each primer has the following nucleotide sequence.
pTD3-MITFa (SEQ ID NO: 26): CCGCGCGGAATGCAGTCCCGAATCGGGAATC
MITFR-N (SEQ ID NO: 27): GAATTCACTATGCTCTGTCTCAGACTCTGTGGGGGG
[0077]
2) After producing a fusion protein according to Example 1, purification was performed using TALON (trade name, CLONTECH) instead of Ni-agarose. The purified product was prepared as a 10% glycerol / PBS solution.
[0078]
Example 4: Construction of expression vector using pET14b
An expression vector pSU121 was constructed by substituting the NcoI-NdeI fragment of pSU083 (wh strain) with the same region of pET14b (T7 promoter, HisTag, ampr; Takara) (FIG. 4). This pSU121 is linked with HisTag, PTD sequence and MIH wh type downstream of the T7 promoter, and expresses a fusion protein (HisTag-PTD-MITF wh type) under the control of the T7 promoter in E. coli cells.
[0079]
According to Example 1, Escherichia coli was cultured in a flask to produce a fusion protein (wh system). After purification using Ni-NTA agarose was performed in accordance with Example 1, dialysis was performed using a Slide-A-Dializer (Pierce) and deurea treatment was performed. As a solvent, 290 mM sorbitol, 10 μM EDTA, and 1 mM Tris buffer (pH 8) were used. About 10 mg of the fusion protein was purified per liter of the culture solution.
[0080]
Example 5: Purification step (HLH)
The fusion protein (HLH system, prepared according to Example 2) was eluted from the Q-Sepharose column with 1 M NaCl / 20 mM HEPES (pH 8), and the collected fraction was purified using a PD-10 column (Pharmacia) before use. Was replaced by
[0081]
Example 6: High density culture (wh system)
The expression vector pSU083 was introduced into E. coli HB101 to prepare a transformant. The cells were cultured in a jar fermenter at 37 ° C. for 16 hours using EBM0010010 medium (with ampicillin) to produce a fusion protein. Purification was performed according to Example 1.
[0082]
Example 7: High density culture (HLH)
A fusion protein was produced according to Example 6 using pSU085. When performing purification according to Example 1, the following points were changed. 6M guanidine hydrochloride was used instead of 8M urea at the time of Ni-NTA agarose treatment, and after concentrating using a molecular weight cut-off membrane instead of anion exchanger treatment, equilibrated with 0.5M NaCl / 20 mM HEPES (pH 8). Desalted with a modified PD-10 column (Pharmacia). It was dialyzed against 0.5 M NaCl / 20 mM HEPES (pH 7.4), aliquoted, and stored at -80 ° C. When used in vivo, it was used after diluting the NaCl concentration to 0.15M.
[0083]
Formulation Example 2
A composition comprising the fusion protein, 10% glycerol and PBS was prepared.
[0084]
Formulation Example 3
It was prepared as a fusion protein, 0.15 to 1 M NaCl, 20 mM HEPES (pH 7.4 to 8) solution.
[0085]
Experimental example 5 (result of purification)
Various fusion proteins (prepared according to Examples 1 to 3) were purified from E. coli cells, and their molecular weights and yields (per 4 L flask culture) are shown in Table 10. The molecular weight of the fusion protein (A-type N-terminal region) was 37 kDa.
[0086]
[Table 10]

[0087]
Experimental Example 6 (in vitro inhibition)
The experiment was performed according to Experimental Example 3. After allowing the 10 nM fusion protein to act for 17 days, the number of mast cells, histamine, tryptase and chymase were measured. Table 11 shows the results.
[0088]
[Table 11]

[0089]
The numerical values in the table indicate mean ± standard deviation. In addition, ** indicates that there is a significant difference at p <0.05 from the solvent group by the student's test. * Also indicates that there is a significant difference at p <0.01. The same applies hereinafter.
[0090]
Experimental example 7 (same as above)
The experiment was performed according to Experimental Example 3. After a 20-nM fusion protein was allowed to act for 15 days, the mast cell ratio was measured. Table 12 shows the results. The fusion protein of the present invention suppressed mast cell survival.
[0091]
[Table 12]

[0092]
Experimental example 8 (same as above)
1) An experiment was performed according to Experimental Example 2. Mouse bone marrow cells (2 × 10 ⁶ / Well), the number of mast cells when the fusion protein was added in the presence of SCF and cultured for 28 days was determined. The number of cases was 3. Table 13 shows the results.
[0093]
[Table 13]

[0094]
2) The experiment was performed according to 1). The addition concentration of the fusion protein was 2.5 nM. Histamine concentration and chymase activity were determined. Table 14 shows the results.
[0095]
[Table 14]

[0096]
3) The experiment was performed in the same manner as in 1). The addition concentration of the fusion protein was 2.5 to 20 nM. Cell number and chymase activity were determined. The number of cases was 2. Table 15 shows the results.
[0097]
[Table 15]

[0098]
Experimental example 9 (action on human mast cells)
1) Human CD34-positive bone marrow cells (BioWhittaker) 2 × 10 ⁴ Per well, the medium containing SCF (100 ng / ml), IL-6 (100 ng / ml), IL-10 (10 ng / ml), and fusion protein (HLH system) was added once a week while changing the volume by 9%. After culturing for a week, the number of cells and the chymase activity and histamine content in lysate of the recovered cells were measured. As a culture solution, MediaI (Institute for Immunity and Biology) containing 5% FCS was used. The number of cases was 3. As a result, the fusion protein of the present invention inhibited human mast cell differentiation (Table 16).
[0099]
[Table 16]

[0100]
2) Human CD34-positive bone marrow cells were cultured for 8 weeks in the presence of cytokines according to 1) to prepare immature mast cells that had been partially differentiated. The cells are collected once, centrifugally washed, and freshly plated on a plate at 7 × 10 ⁵ Per well, and culturing was continued using the same culture solution (including the above-mentioned cytokine) to which the fusion protein (HLH system was added at 10 or 50 nM) was added. The culture medium was exchanged once a week by half. Two weeks after the culture, the cell number, chymase activity, and histamine content were measured. The cells were stained with a PE-labeled anti-c-kit antibody (or a PE-labeled control antibody) and analyzed by FACS. As a result, the fusion protein of the present invention acted on immature mast cells to inhibit c-kit expression and inhibit maturation of human mast cells (Table 17).
[0101]
[Table 17]

[0102]
3) Human CD34-positive bone marrow cells were cultured for 12 weeks in the presence of cytokines according to 1) to prepare mature mast cells that had completed differentiation. The cells are collected once, centrifuged and washed, and newly plated on a plate at 2.4 × 10 4 ⁴ Per well, and culturing was continued using the same culture medium (containing the above-mentioned cytokines) to which the fusion protein (2, 10 or 50 nM of the HLH system was added) was added. The culture medium was exchanged once a week by half. After 3 weeks of culture, the cell number and histamine content were measured. As a result, the fusion protein of the present invention reduced the histamine content in a concentration-dependent manner and suppressed the function of mature human bone marrow cultured mast cells (Table 18).
[0103]
[Table 18]

[0104]
Experimental Example 10 (in vivo inhibition)
1) C57BL / 6 mice were intraperitoneally administered 60 μg of the fusion protein (wh type) three times a week for 4 weeks in 200 μL of PBS. The day after the final administration, the peritoneal cells were collected, and the following items were measured. The number of cases was four. Table 19 shows the results of the administration for 4 weeks.
[0105]
Total cell number: Measured with a Coulter counter or a hemocytometer.
Mast cells: Smear specimens were stained with toluidine blue and the ratio of positive was calculated.
Histamine: Lyton was prepared by adding Triton X100 to the peritoneal cell suspension, and Lysate was measured using an ELISA kit.
Both c-kit and IgE receptor positive cells: The collected peritoneal cells were double-stained with PE-labeled anti-c-kit antibody and mouse IgE / biotin-labeled anti-IgE antibody / APC-labeled streptavidin and analyzed by FACS.
[0106]
[Table 19]

[0107]
2) 10 or 50 μg of the fusion protein (wh type, both in 200 μL vehicle) were intraperitoneally administered to the mice for 2 weeks. The number of cases was 10. Each item was measured in the same manner as in 1). The results are shown in Table 20.
[0108]
[Table 20]

[0109]
3) 10 μg of the fusion protein (HLH system) was administered intraperitoneally to the mice in a 350 μL vehicle for 13 days. The number of mast cells and histamine content were measured according to 1). The results are shown in Table 21.
[0110]
[Table 21]

[0111]
The fusion protein of the present invention (wh system, HLH system) reduced mast cell numbers in vivo in all experimental systems.
[0112]
【The invention's effect】
According to the present invention, mast cell death can be induced using the MITF mutant. Therefore, a drug useful for prevention and treatment of various diseases involving mast cells can be provided in a clinical setting.
[0113]
[Sequence list]
Sequence Listing SEQ ID NO: 1: amino acid sequence of mi variant of MITF
Sequence Listing SEQ ID NO: 2: Its base sequence
Sequence Listing SEQ ID NO: 3: Amino acid sequence of wh mutant of MITF
Sequence Listing SEQ ID NO: 4: Its base sequence
Sequence Listing SEQ ID NO: 5: amino acid sequence of bHLH-Zip fragment
Sequence Listing SEQ ID NO: 6: Its base sequence
SEQ ID NO: 7: Amino acid sequence of TAT-derived peptide
Sequence Listing SEQ ID NO: 8: Its base sequence
SEQ ID NO: 9: Amino acid sequence of HisTag
Sequence Listing SEQ ID NO: 10: Its base sequence
Sequence Listing SEQ ID NO: 11: Amino acid sequence of fusion protein consisting of mi variant of HisTag-PTD-MITF
Sequence Listing SEQ ID NO: 12: Its base sequence
SEQ ID NO: 13: Amino acid sequence of fusion protein consisting of wh mutant of HisTag-PTD-MITF
Sequence Listing SEQ ID NO: 14: Its base sequence
Sequence Listing SEQ ID NO: 15: Amino acid sequence of fusion protein consisting of bHLH-Zip fragment of HisTag-PTD-MITF
Sequence Listing SEQ ID NO: 16: Its base sequence
Sequence Listing SEQ ID NO: 17: Base sequence of primer M-Tat
SEQ ID NO: 18: Nucleotide sequence of primer Tat3
SEQ ID NO: 19: Nucleotide sequence of primer Tat2
SEQ ID NO: 20: Base sequence of primer Tat 1
SEQ ID NO: 21: Amino acid sequence of A-type N-terminal region (1-305) of MITF
Sequence Listing SEQ ID NO: 22: Its base sequence
Sequence Listing SEQ ID NO: 23: Amino acid sequence of fusion protein comprising A-type N-terminal region (1-305) of HisTag-PTD-MITF
Sequence Listing SEQ ID NO: 24: Its base sequence
Sequence Listing SEQ ID NO: 25: Primer HLHF
Sequence Listing SEQ ID NO: 26: primer pTD3-MITFa
Sequence Listing SEQ ID NO: 27: primer MITFR-N
[0114]

[Brief description of the drawings]
FIG. 1 schematically shows the structure of a fusion protein (mi system, wh system) of the present invention and plasmids pSU082 (mi system / pTrcHisB) and pSU083 (wh system / pTrcHisB) expressing the fusion protein. .
FIG. 2 schematically shows the structure of a fusion protein (HLH type) of the present invention and a plasmid pSU085 (HLH type / pTrcHisB) expressing the fusion protein.
FIG. 3 schematically shows a procedure for constructing a plasmid pSU087 (A type N-terminal region / pSU093) expressing the fusion protein (A-type N-terminal region) of the present invention.
FIG. 4 schematically shows a procedure for constructing a plasmid pSU121 (wh / pET14) expressing the fusion protein (wh) of the present invention.

Claims (25)

An agent for inducing cell death of mast cells, comprising a fusion protein comprising a PTD (protein transduction domain) and a MITF (micropharmacia-associated transscription factor) mutant as an active ingredient. 2. The cell death-inducing agent according to claim 1, wherein the fusion protein comprises HisTag, PTD and MITF mutant. The MITF mutant is a mi mutant, a wh mutant, an HLH (basic-helix-loop-helix / leucine zipper) fragment or an A-type N-terminal region (1-305) fragment of the MITF, and the PTD is a TAT-derived peptide. The cell death inducing agent according to claim 1. The cell death-inducing agent according to claim 1, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO: 11, 13, 15 or 23 in the sequence listing. The cell death-inducing agent according to claim 4, wherein one or more amino acids of the fusion protein are substituted, deleted, inserted or added, and the activities are substantially equivalent. The cell death-inducing agent according to claim 1, wherein the active ingredient is obtained by treating the fusion protein with a denaturing agent (chaotropic agent) and then removing the denaturing agent. 2. The cell death inducer according to claim 1, wherein the denaturant is urea, guanidine hydrochloride or thiocyanate. An agent for preventing or treating a disease involving mast cells, comprising a fusion protein composed of a PTD and a MITF mutant as an active ingredient. 9. The preventive or therapeutic agent according to claim 8, wherein the disease involving mast cells is allergy, asthma, autoimmune disease, pulmonary fibrosis, cancer, mast cytoma or mast cytosis. 9. The prophylactic / therapeutic agent according to claim 8, wherein the fusion protein comprises HisTag, PTD and MITF mutant. In a fusion protein composed of HisTag, PTD and MITF mutant, the MITF mutant is a mi mutant, a wh mutant, an HLH fragment or an A type N-terminal region (1-305) fragment of MITF, and the PTD is TAT. The fusion protein, which is a derived peptide. The fusion protein according to claim 11, wherein the fusion protein has an amino acid sequence represented by SEQ ID NO: 11, 13, 15, or 23 in Sequence Listing. 13. The fusion protein according to claim 12, wherein the fusion protein has one or more amino acids substituted, deleted, inserted or added, and has substantially the same activity. A pharmaceutical composition comprising the fusion protein according to claim 11 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 14, wherein the concentration of the fusion protein is 0.1 to 100 µg / ml or 0.1 to 100 nM. 12. The method for producing a fusion protein according to claim 11, comprising a step of producing the fusion protein using a genetic engineering technique. 17. The method according to claim 16, further comprising denaturant treatment, Ni column treatment, and denaturant removal. 17. The production method according to claim 16, wherein the fusion protein is produced using a host expression system derived from Escherichia coli. 17. The production method according to claim 16, wherein the denaturing agent is removed by a treatment step of an anion exchanger or dialysis. 17. The method according to claim 16, wherein the denaturing agent is urea, guanidine hydrochloride or thiocyanate. 17. The method according to claim 16, wherein the concentration of the modifier added during the treatment with the modifier is 1 to 10M. A DNA encoding the fusion protein according to claim 11. The DNA according to claim 22, wherein the fusion protein has an amino acid sequence represented by SEQ ID NOs: 11, 13, 15, or 23 in the sequence listing. 24. The DNA according to claim 23, wherein the fusion protein is obtained by substituting, deleting, inserting or adding one or more amino acids and has substantially the same activity. The DNA according to claim 22, wherein the DNA has a base sequence represented by SEQ ID NO: 12, 14, 16, or 24 in the sequence listing.

Images