JP4469467B2 - Cell growth inhibitor - Google Patents

Description

【００８９】
表中の値は
［励起光３４０ｎｍによる５００ｎｍの蛍光強度］／［励起光３８０ｎｍによる５００ｎｍの蛍光強度］の変化量を表す（高い値ほど強い反応を表す）。４回の測定の平均値±ＳＤ。
【００９０】
実施例２：マウスの免疫および脾臓細胞からのファージミドライブラリーの作製ファージミドライブラリーの作製はリコンビナント・ファージアンチボディー・システム（ファルマシア社製）の方法に準じた。
抗体遺伝子取得のための免疫は以下のように行った。Ｓｆ９昆虫細胞２×１０⁷ 細胞を細胞培養フラスコに播種して３０分間静置の後、前述のｐｃ△１−４９ＴＲ−３ウイルスをＭＯＩ＝２で感染させ、２７℃で４０時間培養した。この感染細胞を回収してＰＢＳ（０．９％ＮａＣｌを含む２０ｍＭリン酸ナトリウム緩衝液）で２回洗浄後、０．２Ｍアジ化ナトリウムを含むＰＢＳ４０ｍｌに懸濁した。この細胞懸濁液を室温で３０分間放置したのち５０μＬを分取して等量の０．２％トレパンブルー染色液と混合し、光学顕微鏡で細胞を観察したところ、すべての細胞が青く染まったことから、回収した感染細胞が１００％死細胞となったと判断した。この死細胞化した感染昆虫細胞をＰＢＳで２回洗浄した後、約３ｍｌのＰＢＳに懸濁した。得られた死細胞化感染昆虫細胞は約６×１０⁷細胞であった。この細胞３ｍｌに不活化百日咳死菌（シグマ社）を３０μＬ添加した後、ＢＡＬＢ／ｃ系マウスに各１ｍｌずつ腹腔内投与した。その後２週間おきに４回、同様の方法で免疫を行った後、最後の免疫実施から４日後にマウスを屠殺して脾臓の摘出を行った。
【００９１】
△１−４９ＴＲ発現昆虫細胞を免疫したマウスから脾臓を摘出し、ｍＲＮＡを抽出した。これを鋳型としてＨｏｎｇ Ｚｈｏｕらの報告（Nucleic Acid Reserch, 1994, vol. 22, No.5, 888-889）のＰＣＲプライマーを使用して抗体のＨ鎖、Ｌ鎖の遺伝子をそれぞれ別々に増幅した。得られたＤＮＡをリンカーを用いて連結し、アガロースゲル電気泳動により約７５０ｂｐのＤＮＡ断片を分離、回収した。このＤＮＡを制限酵素ＮｏｔＩとＳｆｉＩで消化し、再びアガロースゲル電気泳動により、約７５０ｂｐのＤＮＡ断片を分離、回収した。こうして得られたＤＮＡフラグメントを抗Ｄ１−４９ＴＲ発現昆虫細胞ｓｃＦｖ／ＮｏｔＩ−ＳｆｉＩと名付けた。
【００９２】
アマーシャムファルマシアバイオテク社のｐＣＡＮＴＡＢ５ベクターを制限酵素ＮｏｔＩとＳｆｉＩで消化し、アガロースゲル電気泳動によりＤＮＡ断片を分離、回収した。このＤＮＡ断片にヒスチジンＴａｇアダプター（配列番号１５）をＴＡＫＡＲＡのＬｉｇａｔｉｏｎ ｋｉｔ ＶｅｒII（ＴＭ）でライゲーションし、大腸菌ＤＨ１０Ｂ株に導入した。この遺伝子ベクター導入大腸菌を１００μｇ／ｍＬのアンピシリンを含むＬＢ−アガロース固形培地に播種し、クローンを取得した。そのうちの２０クローンについてプラスミドＤＮＡをＱＩＡＧＥＮ社プラスミド抽出キットによって取得し、Ｅ−ｔａｇプライマーとＰＥ ａｐｐｌｉｅｄ Ｂｉｏｓｙｔｅｍｓ社ＡＢＩ ＰＲＩＳＭ（ＴＭ） Ｄｙｅ ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ ＦＳ Ｒｅａｄｙ Ｒｅａｃｔｉｏｎ Ｋｉｔおよび同社ＡＢＩ Ｍｏｄｅｌ３７３Ａ ＤＮＡシークェンサーを用いて塩基配列を解析することで、ヒスチジンＴａｇアダプターが正しい方向にクローニングされたベクターを選択し、これをｐＣＡＮＴＡＢ６ＨＥベクターと名づけた。さらにこのｐＣＡＮＴＡＢ６ＨＥベクターを制限酵素ＮｏｔＩおよびＳｆｉＩで切断したベクターをｐＣＡＮＴＡＢ６ＨＥ／ＮｏｔＩ−ＳｆｉＩと名づけた。
【００９３】
抗Ｄ１−４９ＴＲ発現昆虫細胞ｓｃＦｖ／ＮｏｔＩ−ＳｆｉＩとｐＣＡＮＴＡＢ６ＨＥ／ＮｏｔＩ−ＳｆｉＩをライゲーションキット（ＴＡＫＡＲＡ社製）を用いてライゲーション反応を行った。このライゲーション溶液とコンピテントセルＴＧ１−ｔｒを用いて形質転換を行った。
【００９４】
アンピシリン（最終濃度１００μｇ／ｍｌ）を含む２×ＹＴ（トリプトン １６ｇ、イーストエクストラクト１０ｇ、ＮａＣｌ ５ｇ／Ｌ）寒天プレートに形質転換した液をまき、３７℃で一晩インキュベートした。プレート上に生じたコロニーをかきとって２×ＹＴ培地に懸濁し、これをファージミドライブラリーとした（ライブラリーのサイズは約１．３×１０⁸ｐｆｕ）。作製したファージミドライブラリーはグリセロールを最終２５％程度になるように加えてマイナス８０℃に保存した。
【００９５】
実施例３：△１−４９ＴＲ発現昆虫細胞を用いるパンニング
実施例２で得られたファージミドライブラリー１００μｌをアンピシリン（最終濃度１００μｇ／ｍｌ）、グルコース（２％）を含む２×ＹＴ培地５０ｍｌに加え、６００ｎｍのＯＤが０．６になるまで３７℃で培養した。ヘルパーファージＭ１３ＫＯ７（５×１０¹⁰ｐｆｕ／ｍｌ）を５５μｌ加え、１時間感染させた。
【００９６】
遠心により菌体を回収し、アンピシリン（最終濃度１００μｇ／ｍｌ）、カナマイシン（最終濃度５０μｇ／ｍｌ）を含む２×ＹＴ培地に懸濁し、３７℃で一晩振とう培養した。培養液を遠心後、上清を回収し、５分の１量の２０％ポリエチレングリコール、２．５Ｍ ＮａＣｌ溶液を加えてファージを沈殿回収した。回収したファージは滅菌水に懸濁した。
【００９７】
次に２％スキムミルクを含む溶液中でファージとＡｃＭＮＰＶ感染昆虫細胞を混合し、室温で３０分間反応させた。遠心により細胞に結合したファージを除き、上澄みを再度ＡｃＭＮＰＶ感染昆虫細胞と混合した。この操作を１０回繰り返すことによりＡｃＭＮＰＶ感染昆虫細胞に結合するファージを除去した。
【００９８】
続いてそのファージ溶液と△１−４９ＴＲ発現昆虫細胞を混合し、室温で１時間反応させた。遠心により結合しなかったファージ（上澄み）を除き、さらに５回の洗い操作によって非特異的な結合を排除した。その後０．１Ｍ ＨＣｌで細胞からファージをはがして回収し、等量の１Ｍ Ｔｒｉｓ−ＨＣｌで中和した。こうして回収したファージを大腸菌ＴＧ１−ｔｒに１時間感染させ、アンピシリン（最終濃度１００μｇ／ｍｌ）を含む２×ＹＴ寒天プレート上で一晩培養することによりコロニーを作製し、回収したものを△１−４９ＴＲ発現昆虫細胞１回パンニングファージミドライブラリーとした。
【００９９】
ここまでのファージ調製、細胞に結合するファージの回収という操作を４回繰り返すことによって、△１−４９ＴＲ発現昆虫細胞に結合するファージミドを濃縮した。各パンニングステップにおいて溶出されてきたファージ数は１回目から順に、８×１０⁷ｐｆｕ、１×１０⁸ｐｆｕ、１×１０⁹ｐｆｕ、６×１０¹⁰ｐｆｕである。
【０１００】
実施例４：精製ｓｃＦｖの調製
［標準的なｓｃＦｖ産生株培養法およびペリプラズム画分取得方法］
（１）ｓｃＦｖ産生大腸菌株を下記前培養液３０ｍｌに植菌して３０℃、１６０回転／分で終夜培養を行う。（前培養液組成：２×ＹＴ培地、１００μｇ／ｍｌアンピシリン、０．５％グルコース含有）
（２）終夜培養液３０ｍｌを本培養液３００ｍｌに加える。（本培養液組成：２×ＹＴ培地、１００μｇ／ｍｌアンピシリン、０．４Ｍショ糖含有）
（３）３０℃、１６０回転／分で１時間培養後、最終濃度１ｍＭになるようにＩＰＴＧ（イソプロピルチオガラクトシド）を加え、ｓｃＦｖの産生を誘導する。さらに、３０℃、１６０回転／分で６時間培養する。
（４）培養液を５００ｍｌの遠心管に移し、４℃、５５００回転／分で２０分間遠心する。
（５）沈殿物を回収し、１．８ｍｌの１０×ＨＥＰＥＳ緩衝液（１ｍＭ ＡＰＭＳＦ含有、４℃）に懸濁する。
（６）４０ｍｌの遠心管に移し、氷上で１５分間静置する。（５分おきにボルテックスで攪拌）
（７）４℃、８０００回転／分で１０分間遠心する。
（８）上清を回収し４℃に保存する。
（９）沈殿物を１６．２ｍｌ注射用水（１ｍＭ ＡＰＭＳＦ含有、４℃）に懸濁する。
（１０）氷上で２０分間静置する。（５分おきにボルテックスで攪拌）
（１１）４℃、１２０００回転／分で２０分間遠心する。
（１２）上清を回収し、（８）で保存してあった上清と合わせて「ペリプラズム画分」とする。
（１３）４℃に保存または−２０℃で凍結保存する。
【０１０１】
［標準的なｓｃＦｖ精製方法］
（１）ペリプラズム画分（３０ｍｌ）の約１／１０量の３ｍｌのＮｉ−ＮＴＡアガロース（アガロース担体の量として１／２０量）を測りとる。
（２）室温で６００回転／分で３分間遠心する。
（３）上清を捨て、１．５ｍｌの脱イオン水を加え、攪拌する。
（４）真空ポンプで吸引し、１０−２０分間脱気を行う。
（５）３０ｍｌボリュームのカラムにＮｉ−ＮＴＡアガロースを装填する。
（６）１時間室温で静置する。
（７）１５ｍｌ緩衝液１（ＨＥＰＥＳ−Ｔｙｒｏｄｅ、１５０ｍＭ ＮａＣｌ、２０ｍＭ イミダゾール、ｐＨ８）でカラムを平衡化する。
（８）３０ｍｌのペリプラズム画分を４℃で０．５ｍｉｎ／ｍｌの滴下速度で２回アプライする。以下の操作はすべて４℃で行う。
（９）１５ｍｌ緩衝液２（ＨＥＰＥＳ−Ｔｙｒｏｄｅ、１５０ｍＭ ＮａＣｌ、１０ｍＭ イミダゾール、１ｍＭ ＡＰＭＳＦ、ｐＨ８）で洗浄する。
（１０）１．５ｍｌ緩衝液３（ＨＥＰＥＳ−Ｔｙｒｏｄｅ、６５０ｍＭ ＮａＣｌ、２５０ｍＭ イミダゾール、ｐＨ８）で２回ｓｃＦｖを溶出する。
（１１）Ｍｉｌｌｉｃｕｐ ＬＧＣ（ミリポア社製）で濃縮を行う。
（１２）１０００倍量のＨＥＰＥＳ−Ｔｙｒｏｄｅ緩衝液で透析を２回行う。
（１３）精製されたｓｃＦｖを回収し、４℃または−２０℃で凍結保存する。
【０１０２】
実施例５：リコンビナントＴＲ発現昆虫細胞結合性の評価
実施例１で作製した△１−４９ＴＲ発現昆虫細胞で４回パンニングしたファージを大腸菌ＨＢ２１５１に感染させ、３７℃で一晩インキュベートし、コロニーを形成させた。
【０１０３】
こうして得られたファージミドがトランスフォームされた大腸菌ＨＢ２１５１株をアンピシリン（最終濃度１００μｇ／ｍｌ）、グルコース（最終濃度０．５％）を含む２×ＹＴ培地中、３０℃で一晩振とう培養した。培養液を１０μｌとり、１００μｌのアンピシリン（最終濃度１００μｇ／ｍｌ）を含む培地に懸濁し、３０℃で１時間振とう培養した。ここで最終濃度１ｍＭになるようにＩＰＴＧを加え、さらに６時間振とう培養を続けた後、遠心によりペレットを回収した。これに１０×Ｈｅｐｅｓ−Ｔｙｒｏｄｅ緩衝液を加えて菌体を懸濁した後、４℃で１５分静置した。最後に蒸留水を加えて１×Ｈｅｐｅｓ−Ｔｙｒｏｄｅ緩衝液（１０ｍＭ ＨＥＰＥＳ、１２９ｍＭ ＮａＣｌ、８．９ｍＭ ＮａＨＣＯ₃、２．８ｍＭ ＫＣｌ、０．８ｍＭ ＫＨ₂ＰＯ₄、５．６ｍＭ Ｄ−グルコース、０．８ｍＭ ＭｇＣｌ₂／Ｌ）の状態に戻したものをペリプラズム画分として回収した。これを結合評価用のｓｃＦｖサンプルとした。
【０１０４】
△１−４９ＴＲ発現昆虫細胞に対する結合性の評価は以下のように行った。
まず９６ウエルマルチスクリーンプレート（ミリポア社製）を３％ウシ血清アルブミンでコーティングしたところに、△１−４９ＴＲ発現昆虫細胞とＡｃＭＮＰＶ感染昆虫細胞をそれぞれ抗原として加え、調製したｓｃＦｖを混合し、１時間室温で反応させ、ｓｃＦｖのＣ末端側に存在するＥ ｔａｇ部分を認識するＨＲＰ／抗Ｅ ｔａｇ抗体コンジュゲートで検出した。こうして５４０個のコロニーを評価して△１−４９ＴＲ発現昆虫細胞に結合し、ＡｃＭＮＰＶ感染昆虫細胞には結合しないｓｃＦｖを３４個選択した。
【０１０５】
実施例６：ヒト巨核芽球系細胞を用いるＣａ流入阻害評価
実施例４において作製したｓｃＦｖのトロンビン惹起カルシウム流入阻害能を、以下に示す方法によって評価した。すなわち、巨核球細胞系の株化細胞をＨＥＰＥＳ−ｔｙｒｏｄｅ緩衝液に懸濁し、最終濃度１μＭのＦｕｒａ２−ＡＭ（和光純薬社製）を３７℃で５０分間インキュベートしたのち、ＨＥＰＥＳ−ｔｙｒｏｄｅ緩衝液で２回洗浄し、細胞密度が３．３３×１０⁶ｃｅｌｌｓ／ｍｌになるように調製した。次にその細胞懸濁液３００μｌに実施例４において作製したｓｃＦｖ溶液を２００μｌ混合し、室温で３０分間インキュベートしたのち、最終濃度が１ｍＭになるようにＣａＣｌ₂を添加し、さらに室温で５分間インキュベートした。そこにヒト血漿由来トロンビン（シグマ社製）を最終濃度が０．４Ｕ／ｍｌになるように添加し、添加３０秒後の細胞内カルシウム流入量をＣＡＦ−１００（日本分光社製）を用いて測定した。その例として、４種類のクローン（Ｎ０２１、Ｎ０２２、Ｎ０２３、Ｎ０２５）について、ｓｃＦｖ溶液のかわりにＨＥＰＥＳ−ｔｙｒｏｄｅ緩衝液とインキュベートした場合のカルシウム流入量を１００としたときの相対値および阻害率を算出した（表２参照）。
【０１０６】
【表２】

【０１０７】
実施例７：血小板凝集阻害評価
健常人より０．３８％クエン酸加血として採血し、９００回転／分で１５分遠心することにより血小板を豊富に含む血漿を取得した。その血漿をセファロース２Ｂを用いＨＥＰＡＳ−Ｔｙｒｏｄｅ緩衝液にて展開することにより血小板を精製し、洗浄血小板浮遊液（＞２×１０⁸／ｍｌ）を調製した。得られた血小板浮遊液２１０μｌにｓｃＦｖ溶液９０μｌを混合し、２０分間静置した。そのうち２８５μｌをキュッベットに分取し、アグリゴメーターＰＡＭ８Ｃ（メバニクス株式会社）を用いて攪拌しつつ、３７℃にて２分間で安定化した。その血小板浮遊液の濁度を測定しつつ、７．５μｌのＣａＣｌ₂ 水溶液（最終濃度１．５ｍＭ）を添加した。さらに７．５μｌのヒトトロンビン（最終濃度０．１〜０．２Ｕ／ｍｌ）を添加して血小板の凝集を惹起し、その濁度の変化から血小板の凝集度を１１分間測定することによってｓｃＦｖによる血小板凝集阻害を評価した。トロンビンで惹起された最大凝集度を１００％としたときのｓｃＦｖの阻害活性の例を表３に示した。
【０１０８】
【表３】

【０１０９】
実施例８：滑膜細胞増殖阻害評価
実施例４に示したように、Ｎｉ−ＮＴＡカラムにて精製したｓｃＦｖについて、さらにエンドトキシンの除去を行った。Ｎｉ−ＮＴＡカラム溶出物１６０ｍｌを限外濾過膜を用いて９０ｍｌに濃縮したのち、φ０．２２μｍのフィルターで濾過した後、セファクリルＳ−１００を用いたゲル濾過クロマトグラフィーを行い、ｓｃＦｖが溶出する画分を回収した。それを再度Ｎｉ−ＮＴＡカラムにロードして６０ｍｌの溶出画分を得、それを限外濾過膜を用いて１０ｍｌに濃縮した。それを２ＬのＨＥＰＥＳ−ｔｙｒｏｄｅ緩衝液に対して２回透析した後、カニ外殻キトサン多孔性粒子を用いたアフィニティーカラムにロードし、フロースルー画分を回収し、φ０．２２μｍのフィルターで濾過した。各精製工程におけるエンドトキシン量、蛋白質量を表４に示した。
【０１１０】
【表４】

【０１１１】
次に、慢性関節リウマチ患者の滑膜組織から外植片法により初代培養したのち数代継代した滑膜細胞を、９６穴のマイクロタイタープレートにて１ウェル当たり５×１０³細胞になるように播き込み、１０％のＦＢＳを含むＤＭＥＭ培地中、３７℃、５％ＣＯ₂の条件下で３日間培養した。上清を除去し、無血清のＤＭＥＭ培地で洗浄した後に０．１％のＦＢＳを含むＤＭＥＭ培地を添加し、さらに４８時間血清飢餓条件下で培養した。そこにエンドトキシンを除去したｓｃＦｖを添加し、３０分間インキュベートした後、ヒト血漿由来トロンビン（シグマ社製）を最終濃度が１０Ｕ／ｍｌになるように添加した。添加２４時間後にチミジンのアナログであるＢｒｄＵを最終濃度１ｍＭになるように添加し、添加４８時間後に蛋白固相液により細胞核内に取り込まれたＢｒｄＵを固相化し、酵素標識された抗ＢｒｄＵ抗体を用いて検出した。トロンビンで惹起されたＢｒｄＵ取り込み量を１００％としたときのｓｃＦｖの阻害活性の例を表５に示した。
【０１１２】
【表５】

【０１１３】
実施例９：ｓｃＦｖのエピトープの決定
以下に示す９４種類の１５アミノ酸をＣ末端が固相側になるように９４本のピン上に固相合成した。該ペプチドはすべてＰＡＲ１型ヒトトロンビンレセプターの細胞外領域に包含されるペプチドである。
【０１１４】
ヒトトロンビンレセプターのアミノ末端側の１番目から１５番目のアミノ酸配列［配列番号１６］、同じく２番目から１６番目のアミノ酸配列［配列番号１７］、同じく３番目から１７番目のアミノ酸配列［配列番号１８］、同じく４番目から１８番目のアミノ酸配列［配列番号１９］、同じく５番目から１９番目のアミノ酸配列［配列番号２０］、同じく６番目から２０番目のアミノ酸配列［配列番号２１］、同じく７番目から２１番目のアミノ酸配列［配列番号２２］、同じく８番目から２２番目のアミノ酸配列［配列番号２３］、同じく９番目から２３番目のアミノ酸配列［配列番号２４］、同じく１０番目から２４番目のアミノ酸配列［配列番号２５］、同じく１１番目から２５番目のアミノ酸配列［配列番号２６］、同じく１２番目から２６番目のアミノ酸配列［配列番号２７］、同じく１３番目から２７番目のアミノ酸配列［配列番号２８］、同じく１４番目から２８番目のアミノ酸配列［配列番号２９］、同じく１５番目から２９番目のアミノ酸配列［配列番号３０］、同じく１６番目から３０番目のアミノ酸配列［配列番号３１］、同じく１７番目から３１番目のアミノ酸配列［配列番号３２］、同じく１８番目から３２番目のアミノ酸配列［配列番号３３］、同じく１９番目から３３番目のアミノ酸配列［配列番号３４］、同じく２０番目から３４番目のアミノ酸配列［配列番号３５］、同じく２１番目から３５番目のアミノ酸配列［配列番号３６］、同じく２２番目から３６番目のアミノ酸配列［配列番号３７］、同じく２３番目から３７番目のアミノ酸配列［配列番号３８］、同じく２４番目から３８番目のアミノ酸配列［配列番号３９］、同じく２５番目から３９番目のアミノ酸配列［配列番号４０］、同じく２６番目から４０番目のアミノ酸配列［配列番号４１］、同じく２７番目から４１番目のアミノ酸配列［配列番号４２］、同じく４１番目から５５番目のアミノ酸配列［配列番号４３］、同じく４２番目から５６番目のアミノ酸配列［配列番号４４］、同じく４３番目から５７番目のアミノ酸配列［配列番号４５］、同じく４４番目から５８番目のアミノ酸配列［配列番号４６］、同じく４５番目から５９番目のアミノ酸配列［配列番号４７］、同じく４６番目から６０番目のアミノ酸配列［配列番号４８］、同じく４７番目から６１番目のアミノ酸配列［配列番号４９］、同じく４８番目から６２番目のアミノ酸配列［配列番号５０］、同じく４９番目から６３番目のアミノ酸配列［配列番号５１］、同じく５０番目から６４番目のアミノ酸配列［配列番号５２］、同じく５１番目から６５番目のアミノ酸配列［配列番号５３］、同じく５２番目から６６番目のアミノ酸配列［配列番号５４］、同じく５３番目から６７番目のアミノ酸配列［配列番号５５］、同じく５４番目から６８番目のアミノ酸配列［配列番号５６］、同じく５５番目から６９番目のアミノ酸配列［配列番号５７］、同じく５６番目から７０番目のアミノ酸配列［配列番号５８］、同じく５７番目から７１番目のアミノ酸配列［配列番号５９］、同じく５８番目から７２番目のアミノ酸配列［配列番号６０］、同じく５９番目から７３番目のアミノ酸配列［配列番号６１］、同じく６０番目から７４番目のアミノ酸配列［配列番号６２］、同じく６１番目から７５番目のアミノ酸配列［配列番号６３］、同じく６２番目から７６番目のアミノ酸配列［配列番号６４］、同じく６３番目から７７番目のアミノ酸配列［配列番号６５］、同じく６４番目から７８番目のアミノ酸配列［配列番号６６］、同じく６５番目から７９番目のアミノ酸配列［配列番号６７］、同じく６６番目から８０番目のアミノ酸配列［配列番号６８］、同じく６７番目から８１番目のアミノ酸配列［配列番号６９］、同じく６８番目から８２番目のアミノ酸配列［配列番号７０］、同じく６９番目から８３番目のアミノ酸配列［配列番号７１］、同じく７０番目から８４番目のアミノ酸配列［配列番号７２］、同じく７１番目から８５番目のアミノ酸配列［配列番号７３］、同じく７２番目から８６番目のアミノ酸配列［配列番号７４］、同じく７３番目から８７番目のアミノ酸配列［配列番号７５］、同じく７４番目から８８番目のアミノ酸配列［配列番号７６］、同じく７５番目から８９番目のアミノ酸配列［配列番号７７］、同じく７６番目から９０番目のアミノ酸配列［配列番号７８］、同じく７７番目から９１番目のアミノ酸配列［配列番号７９］、同じく７８番目から９２番目のアミノ酸配列［配列番号８０］、同じく７９番目から９３番目のアミノ酸配列［配列番号８１］、同じく８０番目から９４番目のアミノ酸配列［配列番号８２］、同じく８１番目から９５番目のアミノ酸配列［配列番号８３］、同じく８２番目から９６番目のアミノ酸配列［配列番号８４］、同じく８３番目から９７番目のアミノ酸配列［配列番号８５］、同じく８４番目から９８番目のアミノ酸配列［配列番号８６］、同じく８５番目から９９番目のアミノ酸配列［配列番号８７］、同じく８６番目から１００番目のアミノ酸配列［配列番号８８］、同じく８７番目から１０１番目のアミノ酸配列［配列番号８９］、同じく８８番目から１０２番目のアミノ酸配列［配列番号９０］、同じく８９番目から１０３番目のアミノ酸配列［配列番号９１］、同じく９０番目から１０４番目のアミノ酸配列［配列番号９２］、同じく１５９番目から１７３番目のアミノ酸配列［配列番号９３］、同じく１６０番目から１７４番目のアミノ酸配列［配列番号９４］、同じく２３５番目から２４９番目のアミノ酸配列［配列番号９５］。同じく２３６番目から２５０番目のアミノ酸配列［配列番号９６］、同じく２３７番目から２５１番目のアミノ酸配列［配列番号９７］、同じく２３８番目から２５２番目のアミノ酸配列［配列番号９８］、同じく２３９番目から２５３番目のアミノ酸配列［配列番号９９］、同じく２４０番目から２５４番目のアミノ酸配列［配列番号１００］、同じく２４１番目から２５５番目のアミノ酸配列［配列番号１０１］、同じく２４２番目から２５６番目のアミノ酸配列［配列番号１０２］、同じく２４３番目から２５７番目のアミノ酸配列［配列番号１０３］、同じく２４４番目から２５８番目のアミノ酸配列［配列番号１０４］、同じく２４５番目から２５９番目のアミノ酸配列［配列番号１０５］、同じく２４６番目から２６０番目のアミノ酸配列［配列番号１０６］、同じく２４７番目から２６１番目のアミノ酸配列［配列番号１０７］、同じく２４８番目から２６２番目のアミノ酸配列［配列番号１０８］、同じく３２９番目から３４６番目のアミノ酸配列［配列番号１０９］
【０１１５】
上記９４種類のペプチドが固相化されたピンをプレコートバッファー（２％ ＢＳＡ、０．１％ Ｔｗｅｅｎ２０、０．１％アジ化ナトリウム０．０１Ｍ ＰＢＳ）に室温で６０分間浸したのちに、洗浄バッファー（０．０１Ｍ ＰＢＳ）で室温で１０分間洗浄した。次に２０μｇ／ｍｌのｓｃＦｖ溶液（１％ＢＳＡ、０．１％スキムミルク含有）に４℃で終夜反応させたのちに洗浄バッファーで４回洗浄した。次にｓｃＦｖのｃ−ｍｙｃタグを特異的に認識する抗体である９Ｅ１０にホースラディッシュペルオキシダーゼをコンジュゲートしたものを１μｇ／ｍｌ（１％ ＢＳＡ、０．１％スキムミルク、０．０１Ｍ ＰＢＳ含有）の濃度で室温で６０分間反応させた後、洗浄バッファーで４回洗浄した。次に発色剤溶液（ＡＢＴＳ）にピンを１０分間浸漬し、波長４０５ｎｍにおける発色剤溶液の吸光度を測定した。その結果、滑膜細胞増殖を阻害するｓｃＦｖクローンＮ０２２およびＮ０２５は、以下のアミノ酸配列に対して特異的に結合した。すなわち、ＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の４１番目から５５番目のアミノ酸配列［配列番号４３］、同じく４２番目から５６番目のアミノ酸配列［配列番号４４］、同じく４３番目から５７番目のアミノ酸配列［配列番号４５］、同じく４４番目から５８番目のアミノ酸配列［配列番号４６］、同じく４５番目から５９番目のアミノ酸配列［配列番号４７］、同じく４６番目から６０番目のアミノ酸配列［配列番号４８］、同じく４７番目から６１番目のアミノ酸配列［配列番号４９］、同じく４８番目から６２番目のアミノ酸配列［配列番号５０］、同じく４９番目から６３番目のアミノ酸配列［配列番号５１］、同じく５０番目から６４番目のアミノ酸配列［配列番号５２］、同じく５１番目から６５番目のアミノ酸配列［配列番号５３］である。
【０１１６】
このことから、トロンビンで惹起した滑膜細胞増殖を阻害するｓｃＦｖクローンＮ０２２およびＮ０２５のエピトープは、ＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の５２番目から５６番目のアミノ酸配列を認識することが示された。
【０１１７】
また滑膜細胞増殖を阻害しないｓｃＦｖクローンＮ０１８は、以下のアミノ酸配列に対して特異的に結合した。すなわち、ヒトトロンビンレセプターのアミノ末端側の４１番目から５５番目のアミノ酸配列［配列番号４３］、同じく４２番目から５６番目のアミノ酸配列［配列番号４４］、同じく４３番目から５７番目のアミノ酸配列［配列番号４５］、同じく４４番目から５８番目のアミノ酸配列［配列番号４６］、同じく４５番目から５９番目のアミノ酸配列［配列番号４７］、同じく４６番目から６０番目のアミノ酸配列［配列番号４８］、同じく４７番目から６１番目のアミノ酸配列［配列番号４９］、同じく４８番目から６２番目のアミノ酸配列［配列番号５０］、同じく４９番目から６３番目のアミノ酸配列［配列番号５１］、同じく５０番目から６４番目のアミノ酸配列［配列番号５２］、同じく５１番目から６５番目のアミノ酸配列［配列番号５３］である。
【０１１８】
このことから、トロンビンで惹起した滑膜細胞増殖を阻害するｓｃＦｖのエピトープは、ヒトトロンビンレセプターのアミノ末端側の５１番目から５６番目のアミノ酸配列であることが示された。
【０１１９】
実施例１０：ｓｃＦｖのＫｉｎｅｔｉｃｓ解析
滑膜細胞増殖阻害能を有するｓｃＦｖのエピトープに対するＫｉｎｅｔｉｃｓ特異性の検証を表面プライズモン共鳴センサーＢＩＡＣＯＲＥ１０００（ＢＩＡＣＯＲＥ社）を用いて行った。
【０１２０】
以下に示す２１アミノ酸を合成した（株式会社バイオロジカ）。このペプチドはＰＡＲ１型ヒトトロンビンレセプターの細胞外領域に包含され、活膜細胞増殖阻害能を有するｓｃＦｖのエピトープを含むペプチドである。
ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列［配列番号１１０］
Ａｓｎ Ｐｒｏ Ａｓｎ Ａｓｐ Ｌｙｓ Ｔｙｒ Ｇｌｕ Ｐｒｏ Ｐｈｅ Ｔｒｐ Ｇｌｕ Ａｓｐ Ｇｌｕ Ｇｌｕ Ｌｙｓ Ａｓｎ Ｇｌｕ Ｓｅｒ Ｇｌｙ Ｌｅｕ Ｔｈｒ
【０１２１】
ＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列［配列番号１１０］のペプチドに対してＫＬＨ複合体を作成した。ＰＩＥＲＣＥ社より市販されている「Ｉｍｊｅｃｔ Ｉｍｍｕｎｏｇｅｎ ＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ Ｋｉｔ Ｗｉｔｈ ＫＬＨ ａｎｄ ＢＳＡ」を用い、まずＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列ペプチド１ｍｇをＫｉｔのＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ緩衝液０．２５ｍｌに溶解し（ペプチド４ｍｇ／ｍｌ）、ＫＬＨ溶液（１０ｍｇ／ｍｌ）０．１ｍｌを加えた。さらにＥＤＣ（１０ｍｇ／ｍｌ）２５μｌをペプチド、ＫＬＨ混合液に攪拌しながらゆっくり滴下し、引き続き３０分間室温にて攪拌した。その後、ＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ緩衝液３５０μｌを添加し、反応を停止させた。その後、ＰＢＳ緩衝液ｐＨ７．４、３Ｌに対して２回透析し、ＫＬＨ複合体ペプチドを得た。
【０１２２】
ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列のＫＬＨ複合体ペプチドまたリファレンスとしてＫＬＨをＣＭ５センサーチップ（ＢＩＡＣＯＲＥ社）に固定化した。ＢＩＡＣＯＲＥ社で市販している「アミンカップリングキット」を使用し、業者添付のプログラムに準じて行なった。ＢＩＡＣＯＲＥ Ｃｏｎｔｒｏｌ Ｓｏｆｔｗａｒｅ Ｖｅｒ．１．２を用いてＣＭ５センサーチップに同社市販のＨＢＳ−ＥＰ緩衝液（０．０１Ｍ ＨＥＰＥＳ、０．１５Ｍ ＮａＣｌ、３ｍＭ ＥＤＴＡ、０．００５％ボリソルベート２０（ｖ／ｖ））を流速５μｌ／分で流した。さらにアミンカップリングキットのＥＤＣ／ＮＨＳの１：１混合液を７分間（３５μｌ）反応させ、センサーチップ表面上のカルボキシメチルデキストランを活性化させた。１００μｇ／ｍｌのＫＬＨ ＣｏｎｊｕｇａｔｅｄペプチドまたはＫＬＨを７分間（３５μｌ）反応させることによりセンサーチップ上に結合させ、さらにアミンカップリングキット中のエタノールアミンを７分間（３５μｌ）反応させて残余の活性型ＮＨＳ基をブロッキングした。
【０１２３】
上記センサーチップ上に固定化されたＫＬＨをリファレンスとし、ペプチドに対するｓｃＦｖの結合解離曲線を測定した。ＨＢＳ−ＥＰ緩衝液にてｓｃＦｖを５０μｇ／ｍｌ、２５μｇ／ｍｌ、１２．５μｇ／ｍｌ、６．２５μｇ／ｍｌに調製し、各ｓｃＦｖ溶液について、流速２０μｌ／分で３分間センサーチップに反応させた。その後、ＨＢＳ−ＥＰ緩衝液を流速２０μｌ／分で５分間流し、センサーチップを１０ｍＭ ＨＣｌで２０μｌ／分で１分間洗浄することを繰り返した。一連の操作におけるセンサーチップ表面の質量変化を測定することによってペプチドに対する各ｓｃＦｖの結合解離曲線を得た。得られた結合解離曲線と各ｓｃＦｖの濃度から、ＢＩＡｅｖａｌｕａｔｉｏｎ ｓｏｆｔｗａｒｅ ｖｅｒ．２．１を用いて同社添付のプログラムに準じ、各ｓｃＦｖの結合解離定数を算出した。その結果、各ｓｃＦｖの結合解離定数（ＫＤ値）は滑膜細胞増殖阻害活性をもつｓｃＦｖでＫＤ値：約５×１０^-7Ｍ、滑膜細胞増殖阻害活性をもたないｓｃＦｖでＫＤ値：約１×１０^-7Ｍであった（表６参照）。
【０１２４】
【表６】

【０１２５】
実施例１１：ｓｃＦｖのエピトープのアミノ酸残基特異性の比較
滑膜細胞増殖阻害能を有するｓｃＦｖのエピトープにおけるアミノ酸残基特異性の検証を、表面プライズモン共鳴センサーＢＩＡＣＯＲＥ１０００（ＢＩＡＣＯＲＥ社）を用いて行った。
【０１２６】
以下に示す５種類の２１アミノ酸を合成した（株式会社バイオロジカ）。４種のペプチドはヒトトロンビンレセプターの細胞外領域に包含されるペプチドとそのアミノ酸残基置換ペプチドである。
【０１２７】
すなわち、ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列［配列番号１１２］、同じく４７番目から６７番目のアミノ酸残基中５１番目のリジンをアラニンに置換したアミノ酸配列［配列番号１１３］、同じく４７番目から６７番目のアミノ酸残基中５５番目のフェニルアラニンをアラニンに置換したアミノ酸配列［配列番号１１４］、同じく４７番目から６７番目のアミノ酸残基中５６番目のトリプトファンをアラニンに置換したアミノ酸配列［配列番号１１５］、およびコントロールの２１アミノ酸配列［配列番号１１１］
Ａｌａ Ｐｈｅ Ｌｙｓ Ａｌａ Ａｓｐ Ａｓｐ Ｇｌｙ Ｐｒｏ Ｃｙｓ Ｌｙｓ Ａｌａ Ｉｌｅ Ｍｅｔ Ｌｙｓ Ａｒｇ Ｐｈｅ Ｐｈｅ Ｐｈｅ Ａｓｎ Ｉｌｅ Ｐｈｅ
である。
【０１２８】
ＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列［配列番号１１２］のペプチドに対してＫＬＨ複合体を作成した。ＰＩＥＲＣＥ社より市販されている「Ｉｍｊｅｃｔ Ｉｍｍｕｎｏｇｅｎ ＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ Ｋｉｔ Ｗｉｔｈ ＫＬＨ ａｎｄ ＢＳＡ」を用い、ＰＡＲ１型ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列ペプチド１ｍｇをＫｉｔのＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ緩衝液０．２５ｍｌに溶解し（ペプチド４ｍｇ／ｍｌ）、ＫＬＨ溶解液（１０ｍｇ／ｍｌ）０．１ｍｌを加えた。さらにＥＤＣ（１０ｍｇ／ｍｌ）２５μｌをペプチド、ＫＬＨ混合液に攪拌しながらゆっくり滴下し、引き続き３０分間室温にて攪拌した。その後、ＥＤＣ Ｃｏｎｊｕｇａｔｉｏｎ緩衝液３５０μｌを添加し、反応を停止させた。さらにＰＢＳ緩衝液ｐＨ７．４、３Ｌに対して２回透析し、ＫＬＨ複合体ペプチドを得た。
【０１２９】
ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列のＫＬＨ複合体ペプチド、またリファレンスとしてＫＬＨをＣＭ５センサーチップ（ＢＩＡＣＯＲＥ社）に固定化した。ＢＩＡＣＯＲＥ社で市販している「アミンカップリングキット」を使用し、業者添付のプログラムに準じて行なった。ＢＩＡＣＯＲＥ Ｃｏｎｔｒｏｌ Ｓｏｆｔｗａｒｅ Ｖｅｒ．１．２を用いてＣＭ５センサーチップに同社市販のＨＢＳ−ＥＰ緩衝液（０．０１Ｍ ＨＥＰＥＳ、０．１５Ｍ ＮａＣｌ、３ｍＭ ＥＤＴＡ、０．００５％ボリソルベート２０（ｖ／ｖ））を流速５μｌ／分で流した。さらにアミンカップリングキットのＥＤＣ／ＮＨＳの１：１混合液を７分間（３５μｌ）反応させ、センサーチップ表面上のカルボキシルデキストランを活性化させた。１００μｇ／ｍｌの ＫＬＨ複合体ペプチドまたはＫＬＨを７分間（３５μｌ）反応させることによりセンサーチップ上に結合させ、さらにアミンカップリングキット中のエタノールアミンを７分間（３５μｌ）反応させて残余のＮＨＳ基をブロッキングした。
【０１３０】
上記センサーチップ上に固定化されたＫＬＨをリファレンスとし、ペプチドに対するｓｃＦｖの結合を確認した。ｓｃＦｖをＨＢＳ−ＥＰ緩衝液にて５０μｇ／ｍｌに調製し、流速１０μｌ／分で３分間反応させた。その後ＨＢＳ−ＥＰ緩衝液を流速２０μｌ／分で５分間流し、センサーチップを１０ｍＭ ＨＣｌで２０μｌ／分で１分間洗浄することを繰り返した。一連の操作におけるセンサーチップ表面の質量変化を測定することによってペプチドに対する各ｓｃＦｖの最大結合量を得た。滑膜細胞増殖活性を有するｓｃＦｖで最大結合量約３００ＲＵ、滑膜細胞増殖活性のないｓｃＦｖで最大結合量約６００ＲＵの結合値を得た。
【０１３１】
上記線センサーチップ上に固定化されたＫＬＨをリファレンスに、ペプチドに対するｓｃＦｖ５種の各ペプチドによる競合結合阻害を測定した。ＨＢＳ−ＥＰ緩衝液にてｓｃＦｖ（終濃度５０μｇ／ｍｌ）とペプチド（終濃度５μｇ／ｍｌ）の混合液を調製し、ｓｃＦｖ−ペプチド混合液を流速１０μｌ／分で３分間反応させた。
【０１３２】
コントロールペプチドでは滑膜細胞増殖阻害活性をもつｓｃＦｖ、もたないｓｃＦｖともに結合を阻害されなかった。ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸配列ペプチドでは滑膜細胞増殖阻害活性をもつｓｃＦｖ、もたないｓｃＦｖともに結合が阻害された。
【０１３３】
活膜細胞増殖阻害活性をもたないｓｃＦｖではヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸残基中、５１番目のリジンをアラニンに置換したアミノ酸ペプチド、および４７番目から６７番目のアミノ酸残基中、５５番目のフェニルアラニンをアラニンに置換したアミノ酸ペプチドでは結合阻害が認められず、アミノ末端側の４７番目から６７番目のアミノ酸残基中、５６番目のトリプトファンをアラニンに置換したアミノ酸ペプチドで結合阻害が認められた。
【０１３４】
滑膜細胞増殖阻害活性をもつｓｃＦｖではヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸残基中、５１番目のリジンをアラニンに置換したアミノ酸ペプチドおよび４７番目から６７番目のアミノ酸残基中、５５番目のフェニルアラニンをアラニンに置換したアミノ酸ペプチドで結合阻害が認められ、ヒトトロンビンレセプターのアミノ末端側の４７番目から６７番目のアミノ酸残基中、５６番目のトリプトファンをアラニンに置換したアミノ酸ペプチドでは結合阻害が認められなかった。
【０１３５】
滑膜細胞増殖阻害活性をもつｓｃＦｖ、もたないｓｃＦｖともにエピトープとして重複したアミノ酸配列を認識しているものの、そのエピトープにおける各アミノ酸残基に対する重要度に違いがあることが確認された。
【０１３６】
トロンビン惹起滑膜細胞増殖阻害にはヒトトロンビンレセプターのアミノ酸末端から５６番目のトリプトファン残基がｓｃＦｖの重要な結合部位であることが確認された。
【０１３７】
【発明の効果】
本発明の薬剤（ポリペプチドまたは化合物）は、トロンビンレセプターを介したシグナル伝達を抑制し、細胞増殖阻害活性やＴＮＦ−α、ＩＬ−６、ＩＬ−１βなどの炎症性メディエーターの産生や放出を抑制する活性を有し、各種炎症性疾患や細胞増殖性疾患の治療薬として用いられる。
【０１３８】
【配列表】
【０１３９】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a drug capable of suppressing cell proliferation by thrombin. More specifically, the present invention relates to an agent that binds to a specific region of the PAR type 1 thrombin receptor and inhibits the function of the receptor related to cell proliferation. The present invention also relates to a novel human PAR type 1 thrombin receptor, and a DNA fragment encoding the same.
[0002]
[Prior art]
Thrombin is a serine protease that participates in the final stages of the blood coagulation cascade and plays a vital role in thrombostasis. On the other hand, when thrombin binds to thrombomodulin on vascular endothelial cells, the procoagulant function is completely lost, and conversely, the activation ability of protein C is enhanced several thousand times. Protein C activated in this manner decomposes factor V and factor VIII and exhibits an anticoagulant action. Therefore, thrombin also acts as an anticoagulant factor. Although thrombomodulin is a type of thrombin receptor, a type of receptor (receptor) that transmits a thrombin signal has recently been discovered (Vu, T. K. H. et al .: Cell, 64, 1057-1068 (1991)). The first cloned signal transduction thrombin receptor (PAR1) cDNA encodes a protein of 425 amino acid residues, and the structure of a typical G protein-coupled receptor having seven transmembrane domains. have.
[0003]
Thrombin degrades the site Arg (41) -Ser (42) on the extracellular N-terminal side of the PAR1-type thrombin receptor. When the N-terminal side of the PAR1 type thrombin receptor is liberated, the newly exposed N-terminal site works as an agonist. That is, thrombin does not act as a ligand but only serves to expose a ligand (TRAP: Thrombin Receptor Agonist Peptide) inherent in the receptor. There is a site rich in acidic amino acids adjacent to the C-terminal side of this ligand site (-Glu-Asp-Glu-Glu-). This site is highly homologous to hirudin in the saliva of leech, a natural inhibitor of thrombin, and is called the hirudin-like domain, and is thought to be the site to which thrombin binds (Ayumi of Medicine, 167, 484-487 (1993)).
[0004]
The thrombin signal activates not only platelets and vascular endothelial cells but also vascular smooth muscle cells and macrophages to migrate and proliferate. In view of the overexpression of PAR1-type thrombin receptor after atherosclerosis and PTCA, it has been considered that this receptor plays an important role in the establishment of various vascular lesions.
[0005]
An antithrombin agent, that is, a thrombin inhibitor, not only inhibits the blood coagulation cascade but also suppresses the production of activated protein C in view of the function of thrombin as described above. That is, the balance between coagulation and anticoagulation on the vascular endothelium is expected to be lost, and as a side effect, for example, bleeding is a concern.
[0006]
On the other hand, thrombin receptor inhibitors suppress only cell activation such as cell proliferation and cell migration by regulating cell surface receptors without affecting the blood coagulation system at all from the viewpoint of their mechanism of action. I can expect that. That is, unlike a thrombin inhibitor, a thrombin receptor inhibitor may be a drug with few side effects of bleeding. Furthermore, it is possible to divide the target disease. For example, a thrombin inhibitor targets thrombosis, whereas a thrombin receptor inhibitor targets cell proliferative diseases.
[0007]
Next, how the reaction between thrombin and receptors on cells is related to various diseases will be described. For example, the following reports suggest that the thrombin receptor on cells is involved in synovial cell proliferation in rheumatism, platelet aggregation in arteriosclerosis, smooth muscle cell proliferation, or macrophage migration.
[0008]
PAR1-type thrombin receptor is overexpressed in the synovial tissue of rheumatic patients (Ann. Rheum. Dis., 55: 841-843 (1996)). In addition, a large amount of activated thrombin is present in the synovial fluid of rheumatic patients, and TAT (Thrombin-Anthrombin Complex) that reflects the amount of thrombin generated in the synovial fluid is several thousand compared with the plasma value of healthy subjects. It has recently been clarified that the value is twice as high (J. of Rheumatology, 23, 1505-1511 (1996); Clin. Exp. Rheumatol., 17, 161-170 (1999)).
[0009]
Furthermore, it is suggested that synovial cells derived from rheumatic patients increase tritium thymidine incorporation by adding thrombin or TRAP to the culture medium in vitro, that is, through the action of thrombin receptor. (Clinical Immunology and Immunopathology, 76: 225-233 (1995)).
[0010]
In rheumatoid arthritis, there are many new blood vessels in the synovial tissue that has proliferated, but it has been suggested that thrombin is involved in the process of angiogenesis, such as luminal formation of vascular endothelial cells. (Am. J. Physiol., 273, c239-245, 1997).
[0011]
Furthermore, it is known that the expression of matrix metalloprotease is increased in the pathology of patients with rheumatoid arthritis. Thrombin acts on vascular endothelial cells to enhance the expression of matrix metalloproteinase-1 and -3, and it has been reported that PAR1-type thrombin receptor is involved in the expression of matrix metalloproteinase-3 (among others) ( Atheroscler. Thromb. Vasc. Biol., 17, 1731-1738 (1997)). This suggests that the thrombin receptor is involved in the synovial hyperplasia of rheumatoid arthritis and the subsequent osteochondral destruction process.
[0012]
Summarizing these reports, it is considered that thrombin generated locally in the joint causes signal transduction via a thrombin receptor on the surface of synovial tissue or synovial cells, and synovial cell proliferation is induced.
[0013]
The pathogenesis of rheumatism has been clarified from recent studies as a step where synovial cell proliferation leads to osteochondral destruction. However, looking at the effects of current anti-rheumatic drugs, anti-inflammatory drugs that relieve pain are the mainstream, and there are still no drugs that effectively inhibit and regulate synovial cell proliferation. When considering early rheumatic treatment, synoviocyte growth inhibitors can be curatives rather than symptomatic treatments.
[0014]
In addition, neointimal thickening is a major factor in the progression of vascular lesions such as atherosclerosis and restenosis after PTCA, and overexpression of PAR1 thrombin receptor is observed in the neointimal tissue. (Journal of Clinical Investigation, 90, 1614-1621 (1992)).
[0015]
On the other hand, thrombin induces platelet aggregation, smooth muscle cell proliferation, and macrophage migration in vitro, so that signal transduction occurs through thrombin receptors on the surface of infiltrated macrophages and platelets and intimal smooth muscle cells. It is strongly suggested that local inflammation is promoted and smooth muscle cell proliferation occurs.
[0016]
In addition to the above, as one of reports on the PAR1 thrombin receptor and diseases, it has been recently reported that cerebral neurons (Atrocyte) undergo apoptosis by thrombin or TRAP (The Journal of Neuroscience, 17, 5316-5326). , (1997)), suggesting a relationship between thrombin receptors and cranial neuropathy. There is also a report that the thrombin receptor is also involved in the metastasis of certain human lung cancer cells (Nature Med., 4, 909-914, 1998).
[0017]
PAR1-type thrombin receptor is considered to be related to the onset or exacerbation of various diseases, not limited to the above-mentioned rheumatism, arteriosclerosis, and cranial nerve disorder, in view of its distribution and function in the living body.
[0018]
However, some reports show that antibodies against the TRAP site of the PAR type 1 thrombin receptor can suppress the proliferation of rat vascular smooth muscle cells (Circ. Res. 82, 980-987 (1998)). There is currently no report that cell growth was suppressed with an inhibitor against the human PAR1-type thrombin receptor. It has also been reported that stimulation by the TRAP site of the PAR1-type thrombin receptor may be insufficient for cell proliferation (FEBS Lett., 15, 225-228 (1993); J Surg. Res., 68, 139-144 (1997); Circulation 95, 1870-1876 (1997)). In recent years, a plurality of receptors (PAR3, PAR4, N-PAR), which are said to react with thrombin, have been reported, and the relationship between these receptors and the proliferation action of cells has not been clarified (Nature, 386, 502-506 (1997); Nature, 394, 690-694 (1998); Biochem. J., 313, 353-368 (1996)).
[0019]
Next, the phagemid technology, that is, the phage display library will be described in detail.
Proteins expressed on the surface of phages can be handled as if they were huge proteins fused with phages, and phage clones that bind to the target substance (eg, antigen protein) can be efficiently selected. Even if a small number of phages are selected, a large amount of phages can be obtained through infection and propagation in E. coli.
[0020]
The greatest feature of the phage display vector is that the DNA sequence (genotype) encoding the protein can be recovered at the same time by selecting using the property (phenotype) of the protein displayed on the phage surface as an index. Therefore, it is easy to perform analysis at the gene level.
[0021]
In order to selectively separate phage clones having specific binding ability from the library, operations such as binding to a target protein, washing, and elution are basically performed. Furthermore, in order to concentrate phage clones that bind specifically and strongly to the target protein, it is necessary to infect Escherichia coli with the phage obtained after the elution operation, propagate the phage, and repeat the selection operation with the target protein. Such a process of binding, washing, elution, and proliferation is called panning. Usually, by repeating this operation about 5 times, phages having specific binding ability can be selectively separated from the library. .
[0022]
Examples of the binding target of the phage include not only a protein but also a cell itself expressing a target protein on its surface, a carbohydrate, a nucleic acid, a chemical, and the like.
In the elution process, treatment with acid or alkali is generally used.
[0023]
Phage display is applicable to any polypeptide in principle. For example, presenting on the phage surface as a fusion protein with the outer shell protein III in the form of a single chain Fv (single chain Fv; scFv) fragment in which the VH and VL regions of the antibody are connected by a peptide linker Can do.
[0024]
Using the cDNA derived from B lymphocytes of mice immunized with antigen (target protein or cells, etc.) as a template, VH and VL genes are amplified and cloned by PCR method to present various antigen binding sites. A phagemid library can be generated. The size of the library is about 10 to the 8th power according to a normal molecular biological technique. By using this method, it is possible to simultaneously obtain many types of single-chain antibodies having different binding characteristics for a specific antigen.
[0025]
Conventional antibodies against the thrombin receptor are obtained by synthesizing amino-terminal extracellular peptides and immunizing animals as antigens. For example, the preparation of rabbit polyclonal antibodies has been reported (Circulation., 91, 2961-2971 (1995)). In addition, it has been reported that a polyclonal antibody obtained by immunizing rabbits with the peptide SFFLRNPSEDTFFEQF containing the TRAP site of rat thrombin receptor suppressed the proliferation of rat smooth muscle cells by thrombin (Circ. Res., 82, 980-987 (1998)).
[0026]
However, in our study, depending on the rabbit polyclonal antibody obtained by immunizing the peptide SFLLRNPNDKKYEPF containing the TRAP site of the human PAR1-type thrombin receptor, cell proliferation by thrombin of human vascular smooth muscle cells and human synovial cells Could not be suppressed.
[0027]
The thrombin receptor has a ligand in the molecule, and it is considered that signal transduction occurs when the TRAP moiety exposed by cleaving the amino terminal side by thrombin interacts with a specific part in the molecule. In addition, thrombin is thought to cleave the thrombin receptor and then activate the receptor one after another away from the receptor molecule. This suggests that the thrombin receptor undergoes a conformational change in the molecule after binding to thrombin and then cleaving by thrombin (American Heart Association, 1996).
[0028]
On the other hand, the scFv obtained by the phagemid technique is capable of obtaining a large number of independent clones by creating a single library, and each clone is specific for a single antigenic determinant and is identical. In recent years, it has been used for functional analysis of ligands and receptors, antagonist or agonist molecule design, and structure-activity relationship studies because it has the advantage of being able to stably produce antibodies with specificity.
[0029]
In cell proliferation signaling of the PAR1-type thrombin receptor, it has been considered that a site other than the TRAP site may be involved, but the site has not been identified by the information so far.
[0030]
However, in order to prepare an antibody against a site involved in unknown cell growth existing on the PAR type 1 thrombin receptor, a plurality of partial peptides obtained by dividing the entire sequence of the PAR type 1 thrombin receptor were immunized and obtained. It cannot be said that the efficiency of the method for examining the cell growth inhibitory activity of each antibody is poor.
[0031]
Therefore, the present inventors expressed recombinant PAR1-type human thrombin receptor lacking a thrombin cleavage site and a TRAP site on cells, and conducted extensive research on the function and structure-activity relationship. As a result, a drug that specifically suppresses the function of thrombin receptor by immunizing a mouse with cells expressing this human thrombin receptor variant as an antigen and preparing scFv against human thrombin receptor from the constructed phage display library Could get.
[0032]
The scFv of the present invention has an activity of specifically suppressing thrombin-induced proliferation of joint synovial cells obtained from patients with rheumatoid arthritis. To date, there has been no report that thrombin receptor inhibitors including antibodies to the thrombin receptor have significantly suppressed cell growth by thrombin, and this activity was first observed in the drug of the present invention.
[0033]
For example, Japanese Patent Publication No. 6-508742 discloses a method for obtaining an antagonist including an antibody against a thrombin receptor, but does not describe a suppression of cell proliferation action by the thrombin receptor antagonist.
[0034]
Furthermore, in the course of the present invention, scFv clones that recognize specific sequences on PAR1-type thrombin receptors were studied for the suppression of cell proliferation by thrombin for scFv and antibodies that can bind to various PAR1-type thrombin receptors. Only showed significant inhibitory activity. That is, it was also clarified that only some scFv clones have cytostatic activity among the scFvs that recognize the YEPFW sequence, which has been said to be a site related to the binding of the PAR1-type thrombin receptor to thrombin. This scFv clone having cytostatic activity is particularly strong in the Ca influx reaction in cells by thrombin, the inhibitory effect on platelet aggregation, and the strength of affinity for the epitope peptide compared to the scFv clone having no such activity. Therefore, it is considered difficult to obtain scFv and antibodies having cytostatic activity by conventional techniques.
[0035]
Furthermore, as a result of intensive studies by the present inventors, an scFv clone having an activity of inhibiting cell growth by thrombin is the amino acid sequence of the amino acid sequence from the 52nd to the 56th amino acid sequence of the recognition epitope human PAR1-type thrombin receptor. By replacing the 56th tryptophan residue with an alanine residue, binding to the epitope was lost.
[0036]
On the other hand, the scFv clone having no cytostatic activity capable of binding to the same epitope retained its binding property even when the 56th tryptophan of the PAR type 1 thrombin receptor epitope was replaced with alanine. In other words, the site involved in cell growth of the PAR1-type human thrombin receptor is not defined by the conventional anion external site binding region for thrombin, and the 56th position located between the hirudin-like sequence (EDEE) located immediately downstream thereof. It was shown to be a site centered on tryptophan residues.
[0037]
From the above, the present invention includes a technique for efficiently obtaining a drug capable of suppressing cell proliferation by thrombin. That is, in the method for obtaining thrombin receptor inhibitor, which is a method for evaluating thrombin inhibition of Ca influx reaction in cells and platelet aggregation inhibition as an evaluation item, a drug capable of effectively inhibiting cell growth by thrombin is used. Obtaining specific sequence information related to cell growth of the PAR1-type thrombin receptor can effectively block the cell growth action of thrombin as shown herein. It became possible to obtain antibodies and drugs.
[0038]
An example of the agent of the present invention is scFv having a mouse antibody skeleton, but humanized antibodies can be prepared by utilizing a technique such as CDR grafting based on the gene sequence. It is also possible to modify the scFv to add an Fc part, or to form a double-chain complete molecular antibody. These are expected to have different blood half-lives, and the dosage form of the antibody can be changed depending on the target disease, administration site, and administration method.
[0039]
In addition, since the scFv of the present invention recognizes a site important for cell growth of the PAR1-type thrombin receptor, a compound capable of modifying its activity by binding to a more active antibody or PAR1-type thrombin receptor using this scFv. It can be used as a tool for sorting and obtaining.
[0040]
[Problems to be solved by the invention]
The purpose of the present invention is to verify the involvement of the PAR1 thrombin receptor in cell proliferation by obtaining various types of antibodies against the PAR1 thrombin receptor, and to identify a site strongly involved in proliferation on the PAR1 thrombin receptor. Based on the information, a drug capable of effectively suppressing cell growth by thrombin is obtained.
[0041]
[Means for Solving the Problems]
The present inventors have found a polypeptide that specifically recognizes the PAR1-type human thrombin receptor, and have reached the present invention.
[0042]
That is, the present inventors expressed various PAR1-type human thrombin receptors as proteins having a function on the insect cell surface by genetic engineering. The PAR1-type thrombin receptor variants prepared by the present inventors are the following three types.
(1) △ 1-49TR
Receptor in which amino acids 1 to 49 at the amino terminus of PAR type 1 human thrombin receptor are deleted: a receptor in which a peptide moiety that is cleaved by thrombin or released by thrombin is deleted. It has a thrombin binding site or hirudin-like domain. The DNA sequence and amino acid sequence of Δ1-49TR are shown in SEQ ID NO: 1.
(2) △ 1-80TR
PAR1-type human thrombin receptor lacking amino acids 1 to 80 of the amino terminus: a receptor that does not contain a TRAP moiety, free peptide, or hirudin-like domain The DNA sequence and amino acid sequence of Δ1-80TR are shown in SEQ ID NO: 2.
(3) Full TR
Naturally occurring PAR type 1 human thrombin receptor: Each functional site is present in the molecule. The DNA sequence and amino acid sequence of Full TR are shown in SEQ ID NO: 3.
[0043]
The present inventors immunized mice with insect cells expressing each of the receptors (1) to (3) as antigens, prepared antibodies from the obtained mouse antisera, and examined the profiles of the respective antibodies. . As a result, an antagonistic activity profile was observed in the antibody obtained by immunization using Δ1-49TR-expressing insect cells as an antigen.
[0044]
Therefore, the present inventors prepared a phagemid library from spleen cells of mice immunized with Δ1-49TR-expressing cells as an antigen, and specifically recognized human thrombin receptor by adsorption / desorption, ie, panning method, from the library. The scFv to be bound was selected and obtained.
[0045]
As a result of further studies, the present inventors have found that some scFvs that recognize the PAR1-type human thrombin receptor have the following characteristics. That is, it binds to the PAR1-type thrombin receptor on the surface of human cells and has an activity of suppressing calcium influx through the thrombin receptor by thrombin, and further binds to the PAR1-type thrombin receptor on human platelets, It has been shown to have an activity of inhibiting receptor-mediated platelet aggregation, and further to have an activity of binding to thrombin receptor of human synovial cells to inhibit synovial cell proliferation via thrombin receptor by thrombin. It was.
[0046]
Furthermore, the present inventors determined the amino acid sequence of the binding site of the obtained antibody. As a method for analyzing the epitope, ELISA using a partially synthetic peptide of PAR1 type human thrombin receptor and ELISA using various thrombin receptor-expressing cells were performed. In addition, a multi-pin ELISA was carried out by solid-phase synthesis of a total of 94 kinds of peptides having a length of 15 amino acids per pin so as to include all extracellular regions of the PAR type 1 human thrombin receptor.
[0047]
Polyclonal antibodies against the TRAP moiety, which is the endogenous ligand of the thrombin receptor, or polyclonal antibodies against the hirudin-like domain, which is the thrombin binding site, bind to each functional site with multiple epitopes, and thus affect the function of the thrombin receptor. There is a possibility to give. However, it has not been clear so far which epitopes are effective as antagonists. Thus, as a result of intensive studies by the present inventors, the amino acids 51 to 55 of the amino terminal side of the PAR1-type human thrombin receptor included in the hirudin-like domain are important for the function of thrombin receptor including growth induction. It has been found that suppressing the function derived from the portion is effective as the receptor inhibitor.
[0048]
The amino acid sequence of this epitope is considered to be an important site in the activation and functional expression of the thrombin receptor. For example, the binding of an antibody to this site inhibits the interaction between thrombin and the thrombin receptor, and thrombin binds to the receptor but inhibits the interaction of TRAP to the extracellular loop of the receptor. Various possibilities are conceivable.
[0049]
It was also found that there is a site involved in growth among the 51st to 55th sequences on the amino-terminal side of this human PAR1-type thrombin receptor. That is, the present inventors have obtained a plurality of scFvs that bind to similar sites and show affinity for similar epitopes, but only some of the scFvs can inhibit thrombin cell proliferation. It was. ScFv, which can suppress cell growth by thrombin, suppresses cell growth by thrombin, whereas substitution of the 55th tryptophan residue from the amino terminal side of the PAR1 thrombin receptor with alanine loses its binding to the sequence. In the non-scFv, the binding to the epitope with the same substitution was maintained. That is, the 55th tryptophan residue from the amino terminus of the receptor is important for the binding of scFv capable of inhibiting cell growth by thrombin to the PAR1-type thrombin receptor, and this receptor has an effect on cell growth. The sequence around the 55th tryptophan residue from the amino terminus of the receptor was shown to play an important role.
[0050]
The present inventors have discovered a new fact as a result of a detailed study in the course of research for producing the above thrombin receptor and variants. That is, it has been found that there are two different base sequences from the conventionally known gene encoding PAR type 1 human thrombin receptor. The present inventors have confirmed that the gene is expressed on the cell surface in a form having a function as a PAR1-type thrombin receptor. That is, thrombin and TRAP (synthetic peptide of endogenous ligand moiety: H₂N-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH) showed intracellular signal transduction. In addition, binding of the rabbit polyclonal antibody to the TRAP moiety was confirmed by flow cytometry. Also from this point, it has been clarified that the gene sequence newly found by the present inventors is functional.
[0051]
The previously reported PAR type 1 thrombin receptor was cloned from the responsiveness to thrombin stimulation by first transfecting the megakaryocyte cell line Dami cell cDNA into Xenopus oocytes. In contrast, the present inventors cloned a gene encoding the PAR1 thrombin receptor from cDNA libraries of normal tissues derived from human aorta and placenta. If the reported cDNA is derived from a Dami cell, which is a kind of cancer cell, it may be due to a gene mutation or gene polymorphism.
[0052]
Further, from the above, it can be seen that the PAR1-type thrombin receptor found by the present inventors is different in amino acid sequence from that reported in two places. Therefore, the agent of the present invention recognizes and binds a novel receptor.
[0053]
That is, the present invention includes the following inventions.
1) A polypeptide or compound capable of suppressing cell growth by thrombin.
2) A polypeptide or compound that binds to a specific region involved in cell growth in the structure of the PAR1-type human thrombin receptor.
3) Peptide sequence X₁-Glu-Pro-X₂-Trp-X_ThreeSaid polypeptide or compound which binds to. Where X₁Is any amino acid or peptide sequence and X₂Is any amino acid, X_ThreeRepresents any amino acid or peptide sequence.
4) Amino acid sequence of amino acids 52 to 56 on the amino terminal side of the PAR1 type human thrombin receptor: X_Four-Tyr-Glu-Pro-Phe-Trp-X_FiveA polypeptide or compound that binds to the moiety. Where X_FourAnd X_FiveRepresents any amino acid or peptide sequence.
5) The polypeptide as a PAR1-type thrombin receptor antagonist which is a single-chain Fv or a fragment thereof.
6) The polypeptide as a PAR1 thrombin receptor antagonist which is an antibody or a fragment thereof.
7) The polypeptide as a PAR1-type thrombin receptor inhibitor, which inhibits a signal transduction system involved in cell growth via a thrombin receptor.
8) A modified PAR1-type thrombin receptor gene or a fragment thereof that can be used to obtain the polypeptide, and a cell that expresses the gene.
9) A method for obtaining the polypeptide or compound capable of suppressing cell growth by thrombin.
[0054]
DETAILED DESCRIPTION OF THE INVENTION
A method for producing scFv in the present invention will be described in detail.
A. Preparation of phagemid library
Δ1-49TR-expressing insect cells were made dead by sodium azide, mixed with dead cells (manufactured by Sigma) and suspended in physiological saline. After confirming that the cell morphology was similar to that of living cells, the cells were administered to the peritoneal cavity of mice. This immunization was performed 5 times at 2-week intervals, and blood was collected from the heart 4 days after the final immunization, and at the same time, spleen cells were removed.
[0055]
When preparing cells of the antigen, sodium azide can be brought into contact with the cells to prevent internalization of the receptor into the cells. Maintaining the number of receptor proteins present on the cell surface constantly to prevent internalization is necessary for sufficient immunization.
[0056]
In general, it is considered that a relatively small amount of immunization is required for a purified and purified antigen protein. For example, in the case of a monoclonal antibody, the dose per mouse may be several μg to several tens of μg.
[0057]
However, when a receptor on the cell surface is used as an antigen, various proteins exist on the cell surface. In order to raise the antibody titer of the immunized animal against the receptor, the number of receptors is required to be somewhat larger than the contaminating proteins on the cell surface. The present inventors specifically recognize a functional site reflecting the structure of the receptor by preventing internalization of the receptor into the cell with sodium azide and immunizing the animal with the thrombin receptor-expressing insect cell as an antigen. A phagemid antibody was obtained.
[0058]
The preparation of the phagemid antibody was carried out according to the protocol attached to the manufacturer using “Recombinant Page Antibody System” commercially available from Amersham Pharmacia Biotech. The pCANTAB5 vector included in the kit was designed such that a His6 sequence and an E-tag sequence were added when the cloned gene was expressed by inserting the base sequence ggccgcacatcatcatcataccacggg into the NotI site of this vector (pCANTAB5-HE). ). MRNA was obtained from the spleen of a mouse immunized with the above thrombin receptor-expressing insect cells, and an antibody cDNA was prepared. From the cDNA, immunoglobulin heavy chain and light chain antigen binding sites (VH and VL, respectively) genes were obtained using PCR primers included in the kit. The VL and VH gene fragments are ligated as a single DNA using a linker gene included in the kit, and further recognize the 5 ′ side of VH, recognize the primer containing the restriction enzyme SfiI recognition site and the 3 ′ side of VL. Then, PCR was performed using a primer containing a restriction enzyme NotI recognition site, and this VH-VL linked gene was amplified and obtained. This amplified DNA product was treated with restriction enzymes SfiI and NotI, inserted into the SfiI-NotI cloning site of the pCANTAB5-HE vector, and introduced into E. coli TG1. By adding the helper phage M13K07 to this gene-transferred Escherichia coli, an antibody protein was expressed and a phagemid library containing the antibody gene was prepared.
[0059]
B. Panning
The phagemid library prepared as described above contains many things other than the phagemid antibody against the target PAR1-type thrombin receptor. For example, since the antibody gene used here immunizes the cell itself in which the thrombin receptor is expressed by the baculovirus method, there may be many types of phagemid antibodies against the cell components and virus components. Therefore, the target phagemid antibody was concentrated by the panning method. That is, by reacting this phagemid antibody with an insect cell infected with a natural baculovirus AcMNPV, the phagemid antibody other than the target was adsorbed and removed, and then an insect cell expressing thrombin receptor (Δ1-49TR) By reacting and obtaining only the bound phagemid antibody, the phagemid antibody binding to the thrombin receptor was concentrated. This concentrated phagemid library was infected with E. coli HB2151, and each colony grown on an ampicillin-containing agar medium was obtained to obtain an E. coli clone (scFv-producing E. coli strain) capable of producing a soluble scFv.
[0060]
C. characterization
The scFv-producing strain was cultured and the periplasm fraction was obtained by a standard method, and purified by a standard method. In vitro methods for evaluating the function inhibition activity of PAR type 1 thrombin receptor include calcium influx inhibition evaluation using human megakaryoblastic cells, aggregation inhibition evaluation using human platelets, and synovial cells derived from patients with rheumatoid arthritis. The growth inhibition evaluation used was performed. In addition, epitopes on the PAR1-type thrombin receptor were determined for scFv in which the thrombin receptor function-inhibiting activity was recognized and scFv in which the activity was not observed in the above evaluation, and then the recognition peptide sequence was analyzed in more detail.
[0061]
The following describes standard scFv production, periplasmic fraction acquisition, and purification.
The scFv-producing Escherichia coli strain was cultured overnight in 2 × YT medium, and the overnight culture solution and the main culture solution were mixed at a ratio of 1:10. After incubation at 30 ° C. and 160 rpm for 1 hour, IPTG (isopropylthiogalactoside) was added at a final concentration of 1 mM and cultured for 6 hours to induce scFv production. In the scFv production system used by the present inventors, scFv is known to be produced and accumulated in the periplasmic fraction of E. coli. Therefore, after centrifuging the culture solution and suspending the bacterial cell precipitate in a 10-fold concentration of HEPES buffer, the precipitate obtained by centrifugation again is suspended in ultrapure water, and osmotic stress is applied. The supernatant was collected by centrifugation again to obtain a periplasmic fraction.
[0062]
The scFv prepared by the present inventors has a poly-His region in its amino acid sequence, and can be purified by affinity column chromatography using a Ni-NTA column utilizing the metal ion binding property of this region. The obtained periplasmic fraction was loaded onto a Ni-NTA agarose column, and after several washings, eluted with Hepes-tyrod buffer containing imidazole. Further, the eluate was concentrated using an ultrafiltration membrane and then dialyzed against Hepes-tyrode buffer to obtain purified scFv.
[0063]
Next, the scFv produced by each of the scFv-producing Escherichia coli strains obtained in steps A and B was analyzed for the thrombin receptor binding property. Purified scFv was reacted with thrombin receptor-expressing insect cells, and unreacted scFv was washed away. Next, a horseradish peroxidase (HRP) -labeled anti-E-Tag antibody that reacts with the C-terminus of scFv was bound, and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), a chromogenic substrate for HRP. The binding property was analyzed by a color development method using diammonium salt (ABTS). In addition, the binding property was similarly analyzed in cells infected with wild-type baculovirus AcMNPV, and scFv-producing Escherichia coli strains that bind to thrombin receptor-expressing insect cells but not to AcMNPV-infected insect cells were selected. The function inhibiting activity of the thrombin receptor was evaluated for the scFv produced by the selected Escherichia coli strain.
[0064]
The method for evaluating the function inhibitory activity of the thrombin receptor is described below. Expression of thrombin receptor is recognized in human megakaryoblastic cell lines, and it is known that thrombin increases intracellular calcium concentration. Thus, the increase in intracellular calcium concentration induced by thrombin was measured, and it was examined whether the scFv obtained by the above method has a function inhibitory activity. That is, fluorescent dye (Fura2-AM) was incorporated into human megakaryoblastic cell lines, incubated with scFv for 30 minutes at room temperature, human thrombin was added, and intracellular calcium influx 30 seconds after addition was measured. Then, the calcium influx inhibition activity of scFv was examined. As a result, multiple types of scFv clones that suppress the increase in intracellular calcium concentration caused by thrombin were obtained.
[0065]
As described above, it is known that thrombin receptor is expressed on platelets and aggregation is induced by thrombin. Thus, the effect of scFv on platelet aggregation induced by thrombin was examined. Platelets were purified from normal human blood and washed platelet suspensions were prepared. The scFv obtained by the above method was added to the obtained platelet suspension and allowed to stand at room temperature for 20 minutes. Further stabilization at 37 ° C for 2 minutes, CaCl₂(Final concentration 1.5 mM) was added. Next, human thrombin (final concentration 0.1-0.2 u / ml) was added to induce platelet aggregation, and the inhibition of platelet aggregation by scFv was evaluated from the change in turbidity. As a result, several types of scFv clones that suppress platelet aggregation were obtained.
[0066]
In addition, since the present inventors have found that synovial cells derived from rheumatoid arthritis patients have LPS (lipopolysaccharide) sensitivity and are proliferated by LPS, gel filtration and crab shell chitosan porous particles The endotoxin derived from Escherichia coli contained in the scFv sample purified and obtained as described above was removed in combination with the affinity adsorption removal method using
[0067]
As described above, the thrombin receptor is expressed in synovial cells, and it is known that cell proliferation is induced by thrombin stimulation. Then, it was investigated whether scFv from which endotoxin was removed by the above method had an inhibitory activity against the synovial cell proliferation ability induced by thrombin. That is, synovial cells derived from rheumatoid arthritis patients were serum-starved, incubated with scFv at 37 ° C. for 30 minutes, and then proliferated with human thrombin. The ability to synthesize DNA was measured by measuring thymidine analog (BrdU) incorporation from 24 to 48 hours after addition of thrombin, and the cell growth inhibitory activity of scFv was examined. As a result, several types of scFv clones that suppress synovial cell proliferation were obtained.
[0068]
Epitope mapping analysis was performed to determine the epitope of the scFv clone that inhibits the above synovial cell proliferation. That is, 94 peptides were solid-phase-synthesized on a pin so as to include all extracellular regions of human thrombin receptor with a length of 15 amino acids per peptide, and the synovial cell proliferation obtained by the above method was increased. The binding of inhibiting scFv was examined. As a result, it was found that the amino terminal amino acids 51 to 55 included in the hirudin-like domain of the PAR type 1 human thrombin receptor are epitopes of the synovial cell growth inhibitory scFv. Similarly, scFv clones that did not show synovial cell proliferation were also analyzed. As a result, scFv clones that recognize amino acids 50 to 55 on the amino terminal side of the PAR type 1 human thrombin receptor were found. It was.
[0069]
Furthermore, the scFv clone which recognizes the 51st to 55th amino acids from the amino terminal side of this PAR1 type human thrombin receptor and suppresses synovial proliferation, and the 50th to 55th amino acids from the amino terminal side of the PAR1 type human thrombin receptor A synthetic peptide corresponding to the 47th to 67th amino acids from the amino terminus of the PAR1-type human thrombin receptor containing the epitope was prepared for the scFv clone that recognizes the above and does not suppress synovial proliferation. Keyhole rimpet hemocyanin (KLH) ). This was immobilized on a sensor chip of a surface plasmon resonance measuring apparatus, and the affinity for each peptide was measured. There was no difference in affinity between scFv clones having synoviocyte growth inhibitory activity and scFv clones having no growth inhibitory activity.
[0070]
In addition, the scFv clone that recognizes amino acids 51 to 55 from the amino terminal side of the PAR1 type human thrombin receptor and suppresses synovial proliferation, and the 50th to 55th amino acids from the amino terminal side of the PAR1 type human thrombin receptor are shown below. The scFv clone that recognizes the amino acid of and does not suppress synovial proliferation was analyzed for the binding to a peptide in which a part of the amino acid sequence of the epitope was substituted. A synthetic peptide corresponding to amino acids 47 to 67 from the amino terminus of the PAR type 1 human thrombin receptor is conjugated to KLH and immobilized on a sensor chip of a surface plasmon resonance measuring apparatus, and this synovial cell proliferation inhibitory activity And the peptide corresponding to the 47th to 67th amino acids in which the 55th tryptophan from the amino terminus of the PAR1-type human thrombin receptor was replaced with alanine were analyzed, and immobilized on the sensor chip. Binding to the peptide was not inhibited. However, when the same analysis was performed on the scFv clone having no synovial cell proliferation inhibitory activity, the binding to the peptide immobilized on the sensor chip was strongly inhibited. This indicates that the substitution of the epitope peptide 55th tryptophan with alanine has lost the binding of the clone having synoviocyte growth inhibitory activity to the epitope, and thus the amino terminal of this PAR1-type human thrombin receptor. No. 55 to tryptophan are involved in thrombin cell proliferation activity.
[0071]
The drug (polypeptide or low molecular weight compound) containing the scFv or fragment thereof as an active ingredient according to one aspect of the present invention is brought into contact with a thrombus mainly composed of inflammatory cells, proliferated tissue, or platelets, Inhibition of signal transduction through the thrombin receptor, inhibition of cell growth, production and release of inflammatory mediators such as TNF-α, IL-6, and IL-1β, or thrombus formation by inhibiting platelet aggregation Can be used as a therapeutic agent for various inflammatory diseases, cell proliferative diseases, and thrombotic diseases.
[0072]
For example, the thrombin receptor inhibitor of the present invention can be used as a preparation for intravenous injection. In that case, the scFv or a fragment thereof may be contained in a wide range, and the dosage varies depending on various conditions. These can be dissolved or dispersed in an aqueous medium usually used for intravenous injection. In addition to injections, they are used as external preparations or suppositories. Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Other compositions for parenteral administration contain one or more active substances and are formulated by conventional methods, such as topical solutions, ointments, coatings, suppositories for rectal administration and Includes pessaries.
[0073]
【Example】
Example 1:Preparation of various recombinant TR expressing cells
[Acquisition of human thrombin receptor cDNA]
CDNA was synthesized from 1 μg of human aorta-derived mRNA (purchased from Clontech) using a cDNA synthesis kit Synthesis Kit (Amersham Pharmacia Biotech). The cDNA portion of the thrombin receptor was PCR amplified with primers TR1 [SEQ ID NO: 4] and TR4 [SEQ ID NO: 5] using 1/100 of this cDNA. As a result of agarose gel electrophoresis of the PCR reaction solution, a band estimated to be a thrombin receptor cDNA fragment was observed at about 1.3 kb. This DNA fragment was extracted from the gel and purified using a DNA purification kit (QIAEXII manufactured by QIAGEN). The purified DNA fragment was inserted into the pCRII pCR2.1 (TM) vector using a TA cloning kit (Invitrogen), and plasmid purification (using a plasmid extraction kit from QIAGEN) was performed on several clones. Using these purified plasmids, DNA sequence was determined by Hitachi DNA fluorescence automatic sequencer. Six types of primers used for base sequence determination are shown in [SEQ ID NO: 6-11]. As a result, it was revealed that this DNA fragment encodes the thrombin receptor. The entire base sequence of the determined human aorta-derived thrombin receptor is shown in [SEQ ID NO: 3]. Similarly, the inventors also determined the cDNA sequence derived from human placenta, which was consistent with that derived from human aorta. The thrombin receptor cDNA sequences derived from human aorta and human placenta were compared with the thrombin receptor cDNA sequence (accession No. M62424) registered in DNA Data Base Japan (DDBJ). From the 5 'end of the portion encoding the receptor protein, the 711st base is C from G, the 712st base is from G to C, the 1091st base is from C to G, and the 1092nd base is G. From C to C, it was found that the cDNA was replaced from the display in the database. The thrombin receptor gene in the genomic DNA derived from human placenta was also analyzed for the nucleotide sequence of this site, and it was confirmed that the sequence was the same as the sequence obtained by the present inventors. The pCRII plasmid into which this human thrombin receptor cDNA was inserted was named pCR-TR14.
[0074]
[Preparation of transfer vector containing thrombin receptor cDNA]
The pCR-TR14 was cleaved with restriction enzymes EcoRI and BamHI and subjected to agarose gel electrophoresis. Then, an approximately 1.3 kb fragment containing thrombin receptor cDNA was extracted from the gel and purified using a DNA purification kit (QIAEXII manufactured by QIAGEN). On the other hand, baculovirus transfer vector pVL1393 (Invitrogen) [Technique, Vol. 2, No. 4, pp.173-188, 1990] was cleaved with restriction enzymes EcoRI and BamHI, and ligated with the above DNA fragment. Escherichia coli JM109 strain was transformed with this reaction solution, and plasmids were extracted from several transformant colonies to obtain a plasmid into which an about 1.3 kb fragment had been inserted. This was named pAc-TR14. This thrombin receptor cDNA-containing transfer vector was purified (using a plasmid extraction kit manufactured by QIAGEN) and used to produce a recombinant baculovirus for thrombin receptor expression.
[0075]
[Preparation of recombinant baculovirus for thrombin receptor expression]
For the production of recombinant baculovirus, a BaculoGold ™ kit manufactured by Fermigen was used. Sf9 insect cell 1 × 10⁶Cells were seeded in 30 mm dishes and allowed to adhere for 30 minutes at room temperature. On the other hand, 0.25 μg of BaculoGold (TM) DNA and 2 μg of pAc-TR14 plasmid DNA were mixed and allowed to stand at room temperature for 5 minutes, and then 0.5 ml transfection buffer B (25 mM HEPES pH 7.1, 125 mM CaCl₂, 140 mM NaCl) was added and mixed well. Next, the medium of the seeded Sf9 cells was extracted, and 0.5 ml of transfection buffer A (Grace medium, containing 10% bovine serum) was added. Here, cotransfection was performed by adding dropwise the transfection buffer B and the DNA solution little by little. Four hours after incubation at 27 ° C., the medium was replaced with fresh Grace medium (containing 10% bovine serum). After 5 days, the culture supernatant containing the recombinant virus was recovered, and 10-fold, 100-fold, and 1000-fold dilutions were prepared, and Sf9 cells (1 × 10⁶Cells / dish) were infected for 1 hour at room temperature. The supernatant was extracted, 2 ml of 1% SeaPlaque agarose-containing grace medium (42 ° C.) was added dropwise, and the mixture was allowed to stand at room temperature for about 10 minutes until the agarose solidified. Then, it culture | cultivated at 27 degreeC for 3 days. On day 3, 1 ml / dish of neutral red / PBS (1 g / l) was added. When virus plaques were observed on the 4th day, 6 plaques were confirmed in the dish infected with the 100 culture dilution. Several single virus plaques with agarose were extracted with a Pasteur pipette and mixed with Grace's medium to spread the virus into the medium. Sf9 cells (25 cm) obtained by monolayer culture of the virus solution derived from this single plaque²Flask culture). This was cultured at 27 ° C. for 4-5 days to recover the virus solution. 1 ml of this virus solution was placed in a 1.5 ml Eppendorf tube and centrifuged at 4 ° C. and 15000 rpm for 30 minutes to precipitate virus particles. The precipitate was suspended in 200 μl of TE (10 mM Tris, 0.1 mM EDTA) buffer, 50 μl of Lysis buffer (10% SDS, 1 mM EDTA) was added, and incubated at 60 ° C. for 20 minutes. Viral DNA in the reaction solution was recovered by phenol / chloroform extraction and ethanol precipitation. Using 1/5 of the collected viral DNA, PCR amplification was performed with primers BacF [SEQ ID NO: 12] and TR-S2 [SEQ ID NO: 7], and it was confirmed that the thrombin receptor cDNA was contained in the DNA. This recombinant baculovirus for thrombin receptor expression was named AcTRF. The virus solution was appropriately diluted and plaque assay was performed to check the titer. Further, by infecting Sf9 cells with a virus solution at MOI (Multiplicity of Infection) = 0.25, 1 to 5 × 10⁷A virus solution with a high concentration was obtained. The virus solution was stored frozen at 4 ° C and -80 ° C.
[0076]
[Construction of modified thrombin receptor cDNA]
The endogenous ligand peptide portion (TRAP) of the structure of the thrombin receptor was deleted, the thrombin binding site was retained, and the modified thrombin receptor Δ1-49TR was retained, and both the endogenous ligand peptide site and the thrombin binding site were deleted. The modified thrombin receptor Δ1-80TR cDNA was constructed and expressed in a baculoyl-insect cell system. Δ1-49TR cDNA is the site of bases 79 to 147 in the gene sequence of full-length thrombin receptor cDNA (pCR-TR14), and Δ1-80TR cDNA is the site of bases 79 to 240 of the full-length thrombin receptor. It was made to be deficient. These genes are located in the signal peptide site (amino acid residues 1 to 26) that is removed when the thrombin receptor is expressed in the cell, and in the case of Δ1-49TR when the signal is removed in the full-length thrombin receptor. It is designed to be a thrombin receptor in which 33 amino acid residues from the amino terminal site and an amino acid sequence in which 64 amino acid residues are deleted in Δ1-80TR are added. The nucleotide sequences of the Δ1-49TR and Δ1-80 cDNAs and the amino acid sequences encoded by them are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
[0077]
[Construction of Δ1-49TR cDNA]
The procedure for preparing Δ1-49 cDNA by deleting the site of base numbers 79 to 147 from full-length thrombin receptor cDNA (pCR-TR14) is shown below.
(1) A Δ1-49-5 ′ (190 bp) fragment, which is a gene fragment containing a site corresponding to the base sequence 1-185 of Δ1-49 cDNA, was prepared from pCR-TR14.
▲ 1 ▼ -1
Primer homologous to the pCRII vector (INVITROGEN) site of pCR-TR14 M13REV22 primer [SEQ ID NO: 10], and the site corresponding to base numbers 59 to 78 and the site corresponding to base numbers 148 to 157 of the full-length thrombin receptor cDNA PCR reaction was carried out using TRΔ27-49R primer [SEQ ID NO: 13] which is a primer complementary to. The composition of the reaction solution was pCR-TR14 DNA 200 ng / ml, M13REV22 primer 300 nM, TRΔ29-49 primer 300 nM, DNA polymerization mix {20 mM dNTPs mix} (Amersham Pharmacia-Biotech) 10 μl / ml and × 10 Ex^TM  High-Fidelity PCR system buffer (Boehringer Mannheim) 100 μl / ml, 3.5 U / ml Expand^TM  High-fidelity PCR system enzyme mix (Boehringer Mannheim) 10 μl / ml was used, and the reaction was performed at a reaction scale of 50 μl. Reaction conditions are GeneAmp system^R  This is a temperature cycle in which a reaction is performed at 94 ° C for 2 minutes using PCR system 9600 (Perkin Elmer), followed by 10 cycles of 94 ° C for 15 seconds → 59 ° C for 30 seconds → 72 ° C for 1 minute, followed by 72 ° C for 7 minutes. It was. The sample after the reaction is 4% NUSIEVE^R  GTG^R  Using an AGAROSE (FMC BioProducts) -TAE (40 mM Tris-acetate pH 7.6, 1 mM EDTA) buffer gel, electrophoresis was performed at 100 V for 1 hour, and a gel corresponding to a DNA chain length of 224 bp was cut out. The excised gel was extracted and purified by Hot-Phenol method to obtain about 200 ng of DNA.
[0078]
▲ 1 ▼ -2
In order to obtain a gene fragment corresponding to base numbers 148 to 367 of full-length thrombin receptor cDNA, it comprises a portion corresponding to base numbers 69 to 78 of full-length thrombin receptor cDNA and a portion corresponding to base numbers 148 to 169. PCR was performed using TRΔ27-49F primer [SEQ ID NO: 14] and TRS1 primer [SEQ ID NO: 6] consisting of a sequence complementary to base numbers 350 to 367 of the full-length thrombin receptor cDNA. The reaction solution was prepared in the same manner except that the M13REV22 primer and TRΔ27-49R primer in the reaction composition shown in (1) -1 were replaced with TRΔ27-49F primer and TRS1 primer. The reaction conditions were the same as in (1) -1. The resulting PCR reaction was 4% NUSUEVE.^TM  GTG^TM  Electrophoresis was performed at 100 V for 1 hour using an AGAROSE-TAE buffer gel. A gel corresponding to a 220 bp DNA chain length was cut out, and DNA was extracted and purified by the Hot-Phenol method. The obtained DNA fragment is about 240 ng.
[0079]
▲ 1 ▼ -3
Since it is designed so that a 20 bp complementary portion is generated between the TRΔ27-49R primer and the TRΔ27-49F primer, the 224 bp DNA fragment obtained in (1) -1 and (1) The 220 bp DNA fragment obtained in step-2 was ligated by the assembly PCR method. First, 80 ng of the 220 bp DNA fragment obtained in (1) -2 and 96 ng of the 220 bp DNA fragment obtained in (1) -2 were mixed. The reaction solution composition was DNA primer mix 10 μl / ml, × 10 Expand^TM  High-Fidelity PCR system buffer 100 μl / ml, 3.5 U / ml Expand^TM  High-Fidelity PCR system enzyme mix was 7.5 μl / ml and was performed on a 40 μl reaction scale. The reaction is GeneAmp^RAfter a reaction at 94 ° C. for 2 minutes in the PCR system, a cycle of 94 ° C. for 15 seconds → 59 ° C. for 30 seconds → 72 ° C. for 1 minute and 30 seconds was performed in a temperature cycle program in which the reaction was performed at 72 ° C. for 7 minutes. Next, 40 μl of the reaction product obtained by the reaction was added to 150 ng / ml of M13REV22 primer, 150 ng / ml of TRS1 primer, 10 μl / ml of DNA polymerization mix, × 10 Expand^TM  High-Fidelity PCR system buffer 100 μl / ml, 3.5 U Expand^TM  High-fidelity PCR system enzyme mix Each reagent was added to 6 μl / ml, adjusted to a total volume of 400 μl with distilled water, and 50 μl was dispensed into 5 reaction tubes to carry out the PCR reaction. The reaction was performed by GeneAmp PCR system 9600 at 94 ° C. for 2 minutes, followed by 10 cycles of 94 ° C. for 15 seconds → 59 ° C. for 30 seconds → 72 ° C. for 1 minute 30 seconds, and then 94 ° C. for 15 seconds → 59 ° C. for 30 seconds → 10 cycles of 72 ° C. for 1 minute and 30 seconds plus 20 seconds extension for each cycle were performed, and finally a temperature cycle program was performed in which the reaction was performed at 72 ° C. for 7 minutes. After recovering DNA from the obtained PCR reaction product by ethanol precipitation, CHROMASPIN-100 DEPC-H₂Purified with O column (CLONTECH). This was cleaved with restriction enzyme BamHI (Takara Shuzo) 30 U, and then restriction enzyme BsmI (New England BioLabs) 30 U. 4% NUSIEVE is the product of restriction enzyme treatment.^TM  GTG^TM  Electrophoresis was performed at 50 V for 1 hour using an AGAROSE-TAE buffer gel. A gel corresponding to the 190 bp DNA chain length was cut out, and DNA was extracted and purified by the Hot-Phenol method. As a result, about 400 ng of DNA corresponding to the Δ1-49-5 ′ (190 bp) DNA fragment was obtained.
[0080]
(2) Since the base numbers 255 to 1278 of the full-length thrombin receptor cDNA and the base numbers 186 to 1209 of the Δ1-49TR cDNA have the same sequence, the base number of the full-length thrombin receptor cDNA portion contained in pCR-TR14 A portion of 1 to 244 is excised and replaced with Δ1-49-5 ′ (190 bp), which is a DNA fragment containing base numbers 1 to 185 of Δ1-49TR cDNA, to obtain base number 1 of Δ1-49 cDNA. The part of ˜1209 was constructed.
[0081]
▲ 2 ▼ -1
2.3 μg of pCR-TR14DNA was cleaved with restriction enzyme BamHI 30U followed by restriction enzyme BsmI 30U, which was 0.75% SEAPLAQUE^TMGTG^TM  Electrophoresis was performed at 50 V for 1 hour on AGAROSE (FMC Bio Products) -TAE buffer gel. The gel containing the DNA fragment corresponding to 4.1 kbp was cut out, extracted from the gel by the Hot-Phenol method, and purified to obtain about 640 ng of DNA fragment.
[0082]
▲ 2 ▼ -2
80 ng of the Δ1-49-5 ′ (190 bp) fragment obtained in (1) and 64 ng of the pCR-TR14 4.1 kbp fragment obtained in (2) -1 were obtained from UNI-Amp kit (CLONTECH). Using a ligation reagent (T4 ligase 37.5 U / ml), the reaction was carried out at 20 ° C. for 20 hours at 4 ° C. for binding. The obtained binding DNA was transferred to E. coli DH-10B strain (GIBCO BRL), BRL Cell^R  E. coli pulser^RThe gene was introduced using (GIBCO BRL). Escherichia coli into which the gene was introduced was inoculated on an LB agar medium containing 100 μg / ml of ampicillin and cultured overnight at 37 ° C., and a colony that was visually observed was used as a recombinant clone. These were subjected to PCR using the primers TR1 primer and TRS1 primer corresponding to base numbers 1-20 of Δ1-49TR cDNA, and a vector containing Δ1-49 cTR DNA in which an amplification product of 300 bp was observed by electrophoresis was introduced. When the presence or absence of the clone was analyzed, it was confirmed that the target DNA was introduced into 17 clones out of 84 recombinant clones. Among these clones, one clone pCR-Δ1-49TR clone e-01 was subjected to DNA base sequence analysis. For details pCRII^RSequence primer for vector site M13REV22 primer [SEQ ID NO: 10], M13FOR24 primer [SEQ ID NO: 11], Δ1-49 TR cDNA sequence primer TR1, TRS1, TRS2 [SEQ ID NO: 4, 6, and 7], TRS3 [SEQ ID NO: 8] ], TRS4 [SEQ ID NO: 9] and ABI PRISM^TM  Dye Terminator Cycle Sequencing Ready Reaction Reaction with AmplTaq^R  Using DNA polymerase FS (Perkin Elmer), the protocol attached to the manufacturer was followed. As an analysis apparatus, ABI 373A DNA Sequencer (Applied Biosystems, currently Perkin Elmer) was used. As a result, the DNA base sequence corresponding to the Δ1-49TR cDNA of pCR-Δ1-49TR clone e-1 was completely identical with the DNA base sequence shown in SEQ ID NO: 1, and therefore the target Δ1- It was confirmed that 49TR cDNA was correctly constructed. E. coli containing this pCR-Δ1-49TR clone e-01 was transformed into CIRCLEGROW containing 100 μg / ml of ampicillin.^RIncubate overnight at 37 ° C. in the medium and transfer the contained plasmid to QIAGEN^R  Extraction and purification with PLASMID MIDIKIT (QIAGEN) gave 34 μg of pCR-Δ1-49TR clone e-01 plasmid.
[0083]
[Preparation of Δ1-49TR cDNA-containing transfer vector]
A baculovirus transfer vector containing Δ1-49TR cDNA was prepared in the same manner as the transfer vector pAC-TR14 of full-length thrombin receptor cDNA. Briefly, a 1.2 kbp Δ1-49TR cDNA portion of pCR-Δ1-49TR clone e-01 was excised with the restriction enzymes BamHI and EcoRI, and the DNA fragment was treated with the restriction enzymes BamHI and EcoRI in the pVL1393 vector. Subcloned into PCR analysis using BacF primer and TRS2 primer was performed on the obtained 84 clones of recombinant clones, and it was confirmed that Δ1-49TR cDNA was cloned in 24 clones. Escherichia coli containing one of these clones, pAc-Δ1-49TR clone e01E01 plasmid, was cultured and purified to obtain about 390 μg of plasmid.
[0084]
[Preparation of recombinant baculovirus for expressing Δ1-49TR modified thrombin receptor]
A recombinant baculovirus containing Δ1-49TR cDNA was prepared in the same manner as the preparation of recombinant baculovirus pAC-TRF for expression of full-length thrombin receptor using 2 μg of pAc-Δ1-49TR clone e01E01 recombinant transfer vector. . Among recombinant virus clones, clone pAC-Δ1-49TR-3, which was confirmed to have Δ1-49TR cDNA incorporated by PCR with BacF and TR-S2 primers, was used as a set for Δ1-49TR modified TR expression. A replacement baculovirus was used. By infecting Sf9 insect cells with MOI = 0.25, 3 × 10⁷A high concentration virus solution of pfu / ml was obtained.
[0085]
[Expression of thrombin receptor in insect cells by recombinant baculovirus]
Recombinant baculovirus AcTRF for thrombin receptor expression obtained as described above was prepared in a size of 150 cm.²1.8 × 10 seeded in flask⁷Of Sf9 cells were infected at MOI = 0.6-1.0, and statically cultured at 27 ° C. for 40-48 hours. After culturing, the infected cells were collected and confirmed to have thrombin receptor expression and function by Western blotting and calcium influx experiments.
[0086]
[Western blotting]
Collected infected cells (150cm²About 6 x 10 flasks⁷) Was washed twice with PBS. All subsequent experiments were performed at 4 ° C. Next, it is suspended in 5 ml of a buffer for homogenization (containing 20 mM TrisHCl (pH 8.5), 5 mM EDTA, COMPLETE (TM) (manufactured by Boehringer)), and 30 strokes at 2500 rpm with a Teflon homogenizer. Homogenization was performed. This homogenate was placed in a 15 ml centrifuge tube and centrifuged at 2500 rpm (about 1000 g) for 10 minutes, and the supernatant was collected. The collected supernatant was subjected to ultracentrifugation at 50000 rpm for 1 hour to precipitate the crude membrane fraction. The precipitate was suspended in 1 ml of homogenizing buffer, dispensed in 50 μl aliquots, and stored frozen at −80 ° C. As a result of quantification using Nano Orange (TM) (manufactured by Boehringer), the protein concentration was 14 mg / ml. The crude membrane fraction thus obtained was subjected to SDS polyacrylamide gel electrophoresis using 10% gel at 25 μg-50 μg / lane, and transferred to a nitrocellulose membrane. The membrane was blocked with 3% gelatin / TBS for 1 hour at room temperature, washed 3 times with TBS-0.05% Tween and once with TBS. Thereafter, the reaction was carried out overnight at room temperature in a 1% gelatin / TBS solution containing 10-20 μg / ml of anti-TRAP rabbit polyclonal antibody obtained by immunizing rabbits with TRAP, which is the endogenous ligand region of thrombin receptor. The next day, the cells were washed three times with TBS-0.05% Tween and once with TBS, and reacted with an anti-rabbit IgG goat polyclonal HRP-labeled antibody (approximately 1 μg / ml, 1% gelatin / TBS solution) at room temperature for 1 hour. Next, it was washed 3 times with TBS-0.05% Tween and twice with TBS, and developed with Konica Immunostain HRP for 10 minutes at room temperature to detect the band. As a result, specific bands of about 45 kD and about 85 kD were observed in insect cells infected with the recombinant baculovirus for thrombin receptor expression, confirming that the thrombin receptor was expressed on the insect cell membrane. .
[0087]
[Calcium inflow experiment]
Collected infected cells (approximately 3 × 10⁷) Was washed once with HEPES-Tyrode buffer (-Ca), suspended in 10 ml of HEPES-Tyrode buffer (containing 1 μg / ml Fura2-AM), and Fura2-AM was loaded at 27 ° C. for 30 minutes. The cells were collected by centrifugation at 800 rpm for 5 minutes at room temperature, washed twice with HEPES-Tyrode buffer (-Ca), and resuspended in 2 ml of HEPES-Tyrode buffer (-Ca). 2 μl of 1M CaCl in this cell suspension₂Was added to a final concentration of 1 mM and incubated for 5 minutes at room temperature, protected from light. 490 μl of the Fura2-AM-loaded infected cells prepared as described above was put in a cuvette and measured with an intracellular calcium measuring apparatus (JASCO CAF-100 type). When [fluorescence intensity of 500 nm by excitation light of 340 nm] / [fluorescence intensity of 500 nm by excitation light of 380 nm] (reflecting intracellular calcium concentration) is stabilized at about 0.02, 10 μl of 10 unit / ml thrombin or 1.25 mM TRAP is added to the cuvette. Was added to stimulate and intracellular calcium concentration was measured over time. The inflow of calcium is detected by increasing the value of [fluorescence intensity of 500 nm by excitation light 340 nm] / [fluorescence intensity of 500 nm by excitation light 380 nm]. As a result, calcium influx was specifically observed in insect cells infected with the recombinant baculovirus for thrombin receptor expression (see Table 1). From this result, it was proved that a thrombin receptor having a function was expressed in insect cells.
[0088]
[Table 1]

[0089]
The values in the table are
The amount of change of [fluorescence intensity of 500 nm by excitation light of 340 nm] / [fluorescence intensity of 500 nm by excitation light of 380 nm] is represented (a higher value represents a stronger reaction). Mean value SD of 4 measurements.
[0090]
Example 2:Immunization of mice and preparation of phagemid library from spleen cellsThe preparation of the phagemid library was in accordance with the method of the recombinant phage antibody system (Pharmacia).
Immunization for antibody gene acquisition was performed as follows. Sf9 insect cell 2 × 10⁷The cells were seeded in a cell culture flask and allowed to stand for 30 minutes, and then infected with the aforementioned pcΔ1-49TR-3 virus at MOI = 2 and cultured at 27 ° C. for 40 hours. The infected cells were collected, washed twice with PBS (20 mM sodium phosphate buffer containing 0.9% NaCl), and suspended in 40 ml of PBS containing 0.2 M sodium azide. The cell suspension was allowed to stand at room temperature for 30 minutes, and 50 μL was collected and mixed with an equal amount of 0.2% trepan blue staining solution. When the cells were observed with an optical microscope, all cells were stained blue. From this, it was judged that the collected infected cells were 100% dead cells. The dead insect cells were washed twice with PBS and then suspended in about 3 ml of PBS. The resulting dead cellized insect cells are approximately 6 × 10⁷It was a cell. 30 μL of inactivated pertussis bacteria (Sigma) was added to 3 ml of the cells, and then 1 ml each was intraperitoneally administered to BALB / c mice. Thereafter, immunization was carried out 4 times every 2 weeks in the same manner, and after 4 days from the last immunization, the mice were sacrificed and the spleen was removed.
[0091]
The spleen was excised from mice immunized with Δ1-49TR expressing insect cells, and mRNA was extracted. Using this as a template, the antibody H chain and L chain genes were amplified separately using PCR primers of Hong Zhou et al. (Nucleic Acid Reserch, 1994, vol. 22, No. 5, 888-889). . The obtained DNA was ligated using a linker, and a DNA fragment of about 750 bp was separated and recovered by agarose gel electrophoresis. This DNA was digested with restriction enzymes NotI and SfiI, and a DNA fragment of about 750 bp was again separated and collected by agarose gel electrophoresis. The thus obtained DNA fragment was named anti-D1-49TR expressing insect cell scFv / NotI-SfiI.
[0092]
The pCANTAB5 vector of Amersham Pharmacia Biotech was digested with restriction enzymes NotI and SfiI, and DNA fragments were separated and collected by agarose gel electrophoresis. A histidine Tag adapter (SEQ ID NO: 15) was ligated to this DNA fragment using TAKARA's Ligation kit VerII (TM) and introduced into E. coli DH10B. This gene vector-introduced Escherichia coli was inoculated on an LB-agarose solid medium containing 100 μg / mL ampicillin to obtain a clone. Plasmid DNA of 20 clones of them was obtained by QIAGEN plasmid extraction kit, and EBI tag primer and PE applied Biosystems ABI PRISM (TM) Dye terminator Cycle Sequencing FS Ready Reaction Kit and ABI DNA sequence 37A By analyzing the above, a vector in which the histidine Tag adapter was cloned in the correct direction was selected and named pCANTAB6HE vector. Further, a vector obtained by cleaving this pCANTAB6HE vector with restriction enzymes NotI and SfiI was named pCANTAB6HE / NotI-SfiI.
[0093]
Ligation reaction of anti-D1-49TR expressing insect cells scFv / NotI-SfiI and pCANTAB6HE / NotI-SfiI was performed using a ligation kit (manufactured by TAKARA). Transformation was performed using this ligation solution and competent cells TG1-tr.
[0094]
The transformed solution was plated on 2 × YT (tryptone 16 g, yeast extract 10 g, NaCl 5 g / L) agar plate containing ampicillin (final concentration 100 μg / ml), and incubated at 37 ° C. overnight. The colonies formed on the plate were scraped and suspended in 2 × YT medium to obtain a phagemid library (the size of the library was about 1.3 × 10 × 10).⁸pfu). The prepared phagemid library was added at a final concentration of about 25% and stored at −80 ° C.
[0095]
Example 3:Panning using Δ1-49TR expressing insect cells
100 μl of the phagemid library obtained in Example 2 was added to 50 ml of 2 × YT medium containing ampicillin (final concentration 100 μg / ml) and glucose (2%), and cultured at 37 ° C. until the OD at 600 nm reached 0.6. did. Helper phage M13KO7 (5 × 10^Ten55 μl of pfu / ml) was added and infected for 1 hour.
[0096]
The cells were collected by centrifugation, suspended in 2 × YT medium containing ampicillin (final concentration 100 μg / ml) and kanamycin (final concentration 50 μg / ml), and cultured with shaking at 37 ° C. overnight. After centrifuging the culture solution, the supernatant was collected, and a 1/5 volume of 20% polyethylene glycol and 2.5 M NaCl solution was added to precipitate and collect the phage. The recovered phage was suspended in sterilized water.
[0097]
Next, phage and AcMNPV-infected insect cells were mixed in a solution containing 2% skim milk and allowed to react at room temperature for 30 minutes. Phage bound to the cells were removed by centrifugation and the supernatant was again mixed with AcMNPV infected insect cells. This operation was repeated 10 times to remove phages that bound to AcMNPV-infected insect cells.
[0098]
Subsequently, the phage solution and Δ1-49TR-expressing insect cells were mixed and reacted at room temperature for 1 hour. Phage that did not bind by centrifugation (supernatant) was removed, and non-specific binding was eliminated by 5 additional washing operations. Thereafter, phages were removed from the cells with 0.1 M HCl and recovered, and neutralized with an equal amount of 1 M Tris-HCl. The collected phages were infected with E. coli TG1-tr for 1 hour, and cultured on 2 × YT agar plates containing ampicillin (final concentration 100 μg / ml) overnight to produce colonies. 49TR-expressing insect cells were used as a single panning phagemid library.
[0099]
Phagemids that bind to Δ1-49TR-expressing insect cells were concentrated by repeating the operations of phage preparation and recovery of phages that bind to cells four times. The number of phage eluted in each panning step was 8 × 10 in order from the first.⁷pfu, 1 × 10⁸pfu, 1 × 10⁹pfu, 6 × 10^Tenpfu.
[0100]
Example 4:Preparation of purified scFv
[Standard scFv producing strain culture method and periplasm fraction acquisition method]
(1) The scFv-producing Escherichia coli strain is inoculated into 30 ml of the following preculture solution and cultured overnight at 30 ° C. and 160 rpm. (Preculture composition: 2 × YT medium, 100 μg / ml ampicillin, containing 0.5% glucose)
(2) Add 30 ml of the overnight culture to 300 ml of the main culture. (Main culture solution composition: 2 × YT medium, 100 μg / ml ampicillin, 0.4 M sucrose contained)
(3) After culturing at 30 ° C. and 160 rpm for 1 hour, IPTG (isopropylthiogalactoside) is added to a final concentration of 1 mM to induce scFv production. Furthermore, it culture | cultivates for 6 hours at 30 degreeC and 160 rotation / min.
(4) The culture solution is transferred to a 500 ml centrifuge tube and centrifuged at 4 ° C. and 5500 rpm for 20 minutes.
(5) The precipitate is collected and suspended in 1.8 ml of 10 × HEPES buffer (containing 1 mM APMSF, 4 ° C.).
(6) Transfer to a 40 ml centrifuge tube and leave on ice for 15 minutes. (Vortex every 5 minutes)
(7) Centrifuge at 4 ° C. and 8000 rpm for 10 minutes.
(8) Collect the supernatant and store at 4 ° C.
(9) The precipitate is suspended in 16.2 ml of water for injection (containing 1 mM APMSF, 4 ° C.).
(10) Let stand on ice for 20 minutes. (Vortex every 5 minutes)
(11) Centrifuge at 4 ° C. and 12,000 rpm for 20 minutes.
(12) Collect the supernatant and combine it with the supernatant stored in (8) to make the “periplasm fraction”.
(13) Store at 4 ° C. or store frozen at −20 ° C.
[0101]
[Standard scFv purification method]
(1) About 3/10 Ni-NTA agarose (1/20 amount as the amount of agarose carrier) of about 1/10 amount of periplasm fraction (30 ml) is measured.
(2) Centrifuge at 600 rpm for 3 minutes at room temperature.
(3) Discard the supernatant, add 1.5 ml of deionized water, and stir.
(4) Suction with a vacuum pump and degas for 10-20 minutes.
(5) Load a 30 ml volume column with Ni-NTA agarose.
(6) Let stand at room temperature for 1 hour.
(7) Equilibrate the column with 15 ml buffer 1 (HEPES-Tyrode, 150 mM NaCl, 20 mM imidazole, pH 8).
(8) Apply 30 ml of periplasm fraction twice at 4 ° C. at a dropping rate of 0.5 min / ml. All the following operations are performed at 4 ° C.
(9) Wash with 15 ml buffer 2 (HEPES-Tyrode, 150 mM NaCl, 10 mM imidazole, 1 mM APMSF, pH 8).
(10) Elute scFv twice with 1.5 ml buffer 3 (HEPES-Tyrode, 650 mM NaCl, 250 mM imidazole, pH 8).
(11) Concentrate with Millicup LGC (Millipore).
(12) Dialyze twice with 1000 volumes of HEPES-Tyrode buffer.
(13) The purified scFv is recovered and stored frozen at 4 ° C or -20 ° C.
[0102]
Example 5:Evaluation of recombinant TR-expressing insect cells
The phage panned four times with the Δ1-49TR-expressing insect cells prepared in Example 1 were infected with E. coli HB2151, and incubated overnight at 37 ° C. to form colonies.
[0103]
The thus obtained phagemid-transformed Escherichia coli HB2151 strain was cultured overnight at 30 ° C. in 2 × YT medium containing ampicillin (final concentration 100 μg / ml) and glucose (final concentration 0.5%). 10 μl of the culture solution was taken, suspended in a medium containing 100 μl of ampicillin (final concentration 100 μg / ml), and cultured with shaking at 30 ° C. for 1 hour. Here, IPTG was added to a final concentration of 1 mM, and shaking culture was further continued for 6 hours, and then the pellet was collected by centrifugation. To this was added 10 × Hepes-Tyrode buffer to suspend the cells, and the mixture was allowed to stand at 4 ° C. for 15 minutes. Finally, distilled water was added and 1 × Hepes-Tyrode buffer (10 mM HEPES, 129 mM NaCl, 8.9 mM NaHCO 3) was added._Three2.8 mM KCl, 0.8 mM KH₂PO_Four5.6 mM D-glucose, 0.8 mM MgCl₂/ L) was recovered as a periplasmic fraction. This was used as a scFv sample for binding evaluation.
[0104]
Evaluation of the binding to Δ1-49TR expressing insect cells was performed as follows.
First, a 96-well multiscreen plate (Millipore) was coated with 3% bovine serum albumin, Δ1-49TR-expressing insect cells and AcMNPV-infected insect cells were added as antigens, and the prepared scFv was mixed for 1 hour. The mixture was reacted at room temperature and detected with an HRP / anti-E tag antibody conjugate that recognizes the E tag portion present on the C-terminal side of scFv. Thus, 540 colonies were evaluated and 34 scFvs that bound to Δ1-49TR-expressing insect cells and did not bind to AcMNPV-infected insect cells were selected.
[0105]
Example 6:Ca influx inhibition evaluation using human megakaryocyte cells
The ability of the scFv prepared in Example 4 to inhibit thrombin-induced calcium influx was evaluated by the method shown below. That is, a megakaryocyte cell line is suspended in HEPES-tyrode buffer, and Fura2-AM (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 1 μM is incubated at 37 ° C. for 50 minutes. Washed twice and cell density 3.33 × 10⁶It was prepared to be cells / ml. Next, 200 μl of the scFv solution prepared in Example 4 was mixed with 300 μl of the cell suspension, incubated at room temperature for 30 minutes, and CaCl was adjusted to a final concentration of 1 mM.₂And further incubated at room temperature for 5 minutes. Human plasma-derived thrombin (manufactured by Sigma) was added so that the final concentration was 0.4 U / ml, and the amount of intracellular calcium influx 30 seconds after the addition was determined using CAF-100 (manufactured by JASCO Corporation). It was measured. As an example, relative values and inhibition rates are calculated for four clones (N021, N022, N023, N025) when the calcium influx is 100 when incubated with HEPES-tyrode buffer instead of scFv solution. (See Table 2).
[0106]
[Table 2]

[0107]
Example 7:Evaluation of platelet aggregation inhibition
Blood was collected as a 0.38% citrated blood from a healthy person and centrifuged at 900 rpm for 15 minutes to obtain plasma rich in platelets. Platelets were purified by developing the plasma with Sepharose 2B in HEPAS-Tyrode buffer, and washed platelet suspension (> 2 × 10⁸/ Ml) was prepared. 90 μl of scFv solution was mixed with 210 μl of the obtained platelet suspension and allowed to stand for 20 minutes. Of these, 285 μl was dispensed into a cuvette and stabilized at 37 ° C. for 2 minutes while stirring with an aggregometer PAM8C (Mebanics Corp.). While measuring the turbidity of the platelet suspension, 7.5 μl CaCl₂Aqueous solution (final concentration 1.5 mM) was added. In addition, 7.5 μl of human thrombin (final concentration 0.1-0.2 U / ml) was added to induce platelet aggregation, and the degree of platelet aggregation was measured for 11 minutes from the change in turbidity. Platelet aggregation inhibition was evaluated. Table 3 shows examples of scFv inhibitory activity when the maximum aggregation degree induced by thrombin is 100%.
[0108]
[Table 3]

[0109]
Example 8:Synovial cell growth inhibition evaluation
As shown in Example 4, the endotoxin was further removed from the scFv purified with the Ni-NTA column. Fractions from which 160 ml of Ni-NTA column eluate was concentrated to 90 ml using an ultrafiltration membrane, filtered through a 0.22 μm filter, and then subjected to gel filtration chromatography using Sephacryl S-100 to elute scFv Minutes were collected. It was loaded again onto a Ni-NTA column to obtain 60 ml elution fraction, which was concentrated to 10 ml using an ultrafiltration membrane. It was dialyzed twice against 2 L of HEPES-tyrode buffer, then loaded onto an affinity column using crab shell chitosan porous particles, and the flow-through fraction was collected and filtered through a 0.22 μm filter. . Table 4 shows the amounts of endotoxin and protein in each purification step.
[0110]
[Table 4]

[0111]
Next, synovial cells that were subcultured after several primary passages from the synovial tissue of patients with rheumatoid arthritis by the explant method were transferred to 5 × 10 5 per well in a 96-well microtiter plate.^ThreeInoculate cells to 37 ° C, 5% CO in DMEM medium containing 10% FBS.₂For 3 days. The supernatant was removed, washed with serum-free DMEM medium, DMEM medium containing 0.1% FBS was added, and the cells were further cultured under serum starvation conditions for 48 hours. The scFv from which endotoxin was removed was added thereto and incubated for 30 minutes, and then human plasma-derived thrombin (manufactured by Sigma) was added to a final concentration of 10 U / ml. 24 hours after the addition, BrdU, which is an analog of thymidine, was added to a final concentration of 1 mM, and 48 hours after the addition, BrdU incorporated into the cell nucleus was immobilized by a protein solid phase solution, and an enzyme-labeled anti-BrdU antibody was immobilized. Detected. Table 5 shows examples of scFv inhibitory activity when the amount of BrdU incorporation induced by thrombin is 100%.
[0112]
[Table 5]

[0113]
Example 9:Determination of the epitope of scFv
The following 94 types of 15 amino acids were solid-phase synthesized on 94 pins so that the C-terminal was on the solid-phase side. The peptides are all peptides included in the extracellular region of the PAR type 1 human thrombin receptor.
[0114]
1st to 15th amino acid sequence [SEQ ID NO: 16] on the amino terminal side of the human thrombin receptor, also 2nd to 16th amino acid sequence [SEQ ID NO: 17], and 3rd to 17th amino acid sequence [SEQ ID NO: 18] ], From the 4th to 18th amino acid sequence [SEQ ID NO: 19], from the 5th to 19th amino acid sequence [SEQ ID NO: 20], from the 6th to 20th amino acid sequence [SEQ ID NO: 21], also from the 7th 21st amino acid sequence [SEQ ID NO: 22], 8th to 22nd amino acid sequence [SEQ ID NO: 23], 9th to 23rd amino acid sequence [SEQ ID NO: 24], 10th to 24th amino acid Sequence [SEQ ID NO: 25], also the 11th to 25th amino acid sequence [SEQ ID NO: 26], also 12 26th amino acid sequence [SEQ ID NO: 27], 13th to 27th amino acid sequence [SEQ ID NO: 28], 14th to 28th amino acid sequence [SEQ ID NO: 29], 15th to 29th amino acid Sequence [SEQ ID NO: 30], 16th to 30th amino acid sequence [SEQ ID NO: 31], 17th to 31st amino acid sequence [SEQ ID NO: 32], 18th to 32nd amino acid sequence [SEQ ID NO: 33] ], 19th to 33rd amino acid sequence [SEQ ID NO: 34], 20th to 34th amino acid sequence [SEQ ID NO: 35], 21st to 35th amino acid sequence [SEQ ID NO: 36], 22nd To the 36th amino acid sequence [SEQ ID NO: 37], also from the 23rd to 37th amino acid sequence SEQ ID NO: 38], also from the 24th to 38th amino acid sequence [SEQ ID NO: 39], from the 25th to 39th amino acid sequence [SEQ ID NO: 40], from the 26th to 40th amino acid sequence [SEQ ID NO: 41], Similarly, the 27th to 41st amino acid sequence [SEQ ID NO: 42], the 41st to 55th amino acid sequence [SEQ ID NO: 43], the 42nd to 56th amino acid sequence [SEQ ID NO: 44], and the 43rd to 57th amino acid sequence. The amino acid sequence [SEQ ID NO: 45], the 44th to 58th amino acid sequence [SEQ ID NO: 46], the 45th to 59th amino acid sequence [SEQ ID NO: 47], the 46th to 60th amino acid sequence [ SEQ ID NO: 48], the 47th to 61st amino acid sequence [SEQ ID NO: 49], also the 48th 62nd amino acid sequence [SEQ ID NO: 50], 49th to 63rd amino acid sequence [SEQ ID NO: 51], 50th to 64th amino acid sequence [SEQ ID NO: 52], 51st to 65th amino acid sequence Amino acid sequence [SEQ ID NO: 53], 52nd to 66th amino acid sequence [SEQ ID NO: 54], 53rd to 67th amino acid sequence [SEQ ID NO: 55], 54th to 68th amino acid sequence [SEQ ID NO: 56], 55th to 69th amino acid sequence [SEQ ID NO: 57], 56th to 70th amino acid sequence [SEQ ID NO: 58], 57th to 71st amino acid sequence [SEQ ID NO: 59], 58 From the 72nd amino acid sequence [SEQ ID NO: 60], also from the 59th to 73rd amino acid sequence SEQ ID NO: 61], 60th to 74th amino acid sequence [SEQ ID NO: 62], 61st to 75th amino acid sequence [SEQ ID NO: 63], 62nd to 76th amino acid sequence [SEQ ID NO: 64], Also from the 63rd to 77th amino acid sequence [SEQ ID NO: 65], from 64th to 78th amino acid sequence [SEQ ID NO: 66], from 65th to 79th amino acid sequence [SEQ ID NO: 67], from 66th to 80th The amino acid sequence [SEQ ID NO: 68], the 67th to 81st amino acid sequence [SEQ ID NO: 69], the 68th to 82nd amino acid sequence [SEQ ID NO: 70], and the 69th to 83rd amino acid sequence [ SEQ ID NO: 71], also from the 70th to 84th amino acid sequence [SEQ ID NO: 72], also from 71 The 85th amino acid sequence [SEQ ID NO: 73], the 72nd to 86th amino acid sequence [SEQ ID NO: 74], the 73rd to 87th amino acid sequence [SEQ ID NO: 75], and the 74th to 88th amino acid sequence. Amino acid sequence [SEQ ID NO: 76], 75th to 89th amino acid sequence [SEQ ID NO: 77], 76th to 90th amino acid sequence [SEQ ID NO: 78], 77th to 91st amino acid sequence [SEQ ID NO: 79], also from the 78th to 92nd amino acid sequence [SEQ ID NO: 80], from the 79th to 93rd amino acid sequence [SEQ ID NO: 81], from the 80th to 94th amino acid sequence [SEQ ID NO: 82], also from 81 95th to 95th amino acid sequence [SEQ ID NO: 83], also the 82nd to 96th amino acid sequence [SEQ ID NO: 84], 83rd to 97th amino acid sequence [SEQ ID NO: 85], 84th to 98th amino acid sequence [SEQ ID NO: 86], 85th to 99th amino acid sequence [SEQ ID NO: 87] Also from the 86th to the 100th amino acid sequence [SEQ ID NO: 88], from the 87th to the 101st amino acid sequence [SEQ ID NO: 89], from the 88th to the 102nd amino acid sequence [SEQ ID NO: 90], also from the 89th 103rd amino acid sequence [SEQ ID NO: 91], 90th to 104th amino acid sequence [SEQ ID NO: 92], 159th to 173rd amino acid sequence [SEQ ID NO: 93], 160th to 174th amino acid sequence [SEQ ID NO: 94], similarly, amino acid sequence from position 235 to position 249 [sequence Issue 95]. Also from the 236th to 250th amino acid sequence [SEQ ID NO: 96], from 237th to 251st amino acid sequence [SEQ ID NO: 97], from 238th to 252nd amino acid sequence [SEQ ID NO: 98], from 239th to 253 The amino acid sequence [SEQ ID NO: 99], the 240th to 254th amino acid sequence [SEQ ID NO: 100], the 241st to 255th amino acid sequence [SEQ ID NO: 101], and the 242nd to 256th amino acid sequence [ SEQ ID NO: 102], amino acid sequence from 243 to 257 [SEQ ID NO: 103], amino acid from 244 to 258 [SEQ ID NO: 104], amino acid from 245 to 259 [SEQ ID NO: 105], Also from 246th to 260th a No acid sequence [SEQ ID NO: 106], amino acid sequence from 247 to 261 [SEQ ID NO: 107], amino acid sequence from 248 to 262 [SEQ ID NO: 108], amino acid sequence from 329 to 346 [sequence] Number 109]
[0115]
After immersing the pins on which 94 kinds of peptides were immobilized in precoat buffer (2% BSA, 0.1% Tween 20, 0.1% sodium azide 0.01M PBS) at room temperature for 60 minutes, washing buffer Wash with (0.01M PBS) at room temperature for 10 minutes. Next, the mixture was reacted at 20 ° C./scFv solution (containing 1% BSA and 0.1% skim milk) at 4 ° C. overnight, and then washed 4 times with a washing buffer. Next, the concentration of 1Eg / ml (containing 1% BSA, 0.1% skim milk, 0.01M PBS) of 9E10, which is an antibody specifically recognizing the cF-myc tag of scFv, conjugated with horseradish peroxidase The mixture was reacted at room temperature for 60 minutes and then washed 4 times with a washing buffer. Next, the pin was immersed in the color former solution (ABTS) for 10 minutes, and the absorbance of the color former solution at a wavelength of 405 nm was measured. As a result, scFv clones N022 and N025 that inhibit synovial cell proliferation specifically bound to the following amino acid sequences. That is, the amino acid sequence from the 41st to 55th amino acid sequence [SEQ ID NO: 43] on the amino terminal side of the PAR1 type human thrombin receptor, the 42nd to 56th amino acid sequence [SEQ ID NO: 44], and the 43rd to 57th amino acid sequence. [SEQ ID NO: 45], also the amino acid sequence from the 44th to the 58th amino acid [SEQ ID NO: 46], the amino acid sequence from the 45th to the 59th amino acid [SEQ ID NO: 47], and the amino acid sequence from the 46th to the 60th [SEQ ID NO: 48] The 47th to 61st amino acid sequence [SEQ ID NO: 49], the 48th to 62nd amino acid sequence [SEQ ID NO: 50], the 49th to 63rd amino acid sequence [SEQ ID NO: 51], and the 50th amino acid sequence. 64th amino acid sequence [SEQ ID NO: 52], also the 51st to 65th amino acids Is acid sequence [SEQ ID NO: 53].
[0116]
This indicates that the epitopes of scFv clones N022 and N025 that inhibit the synovial cell proliferation induced by thrombin recognize the amino acid sequences 52 to 56 on the amino terminal side of the PAR1 human thrombin receptor. .
[0117]
Moreover, scFv clone N018 which does not inhibit synovial cell proliferation specifically bound to the following amino acid sequence. That is, the amino acid sequence from the 41st to 55th amino acid sequence [SEQ ID NO: 43] on the amino terminal side of the human thrombin receptor, the 42nd to 56th amino acid sequence [SEQ ID NO: 44], and the 43rd to 57th amino acid sequence [sequence] No. 45], the 44th to 58th amino acid sequence [SEQ ID NO: 46], the 45th to 59th amino acid sequence [SEQ ID NO: 47], the 46th to 60th amino acid sequence [SEQ ID NO: 48], 47th to 61st amino acid sequence [SEQ ID NO: 49], 48th to 62nd amino acid sequence [SEQ ID NO: 50], 49th to 63rd amino acid sequence [SEQ ID NO: 51], 50th to 64th Amino acid sequence [SEQ ID NO: 52], and also from the 51st to 65th amino acid sequence [ Column number 53 is a.
[0118]
From this, it was shown that the epitope of scFv that inhibits synovial cell proliferation induced by thrombin is the amino acid sequence from the 51st to the 56th amino acid side of the human thrombin receptor.
[0119]
Example 10:Kinetics analysis of scFv
Kinetics specificity for the scFv epitope having the ability to inhibit synovial cell proliferation was verified using a surface plasmon resonance sensor BIACORE1000 (BIACORE).
[0120]
The following 21 amino acids were synthesized (Biologica Inc.). This peptide is included in the extracellular region of the PAR1-type human thrombin receptor and contains a scFv epitope having an ability to inhibit active cell growth.
Amino acid sequence of amino acids 47 to 67 of the human thrombin receptor [SEQ ID NO: 110]
Asn Pro Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu Glu Lys Asn Glu Ser Gly Leu Thr
[0121]
A KLH complex was prepared for a peptide having the amino acid sequence of the 47th to 67th amino acids [SEQ ID NO: 110] on the amino terminal side of the PAR1 human thrombin receptor. First, 1 mg of the 47th to 67th amino acid sequence peptide of the amino terminal side of the PAR type 1 human thrombin receptor was transferred to the EDC Conjugation Buffer 0 of Kit using the “Immun Immunogen EDC Conjugation Kit With KLH and BSA” commercially available from PIERCE. Dissolved in 25 ml (peptide 4 mg / ml) and 0.1 ml of KLH solution (10 mg / ml) was added. Further, 25 μl of EDC (10 mg / ml) was slowly added dropwise to the peptide / KLH mixture with stirring, and the mixture was subsequently stirred for 30 minutes at room temperature. Thereafter, 350 μl of EDC Conjugation Buffer was added to stop the reaction. Then, it dialyzed twice with respect to PBS buffer pH7.4, 3L, and obtained KLH complex peptide.
[0122]
The KLH complex peptide having the amino acid sequence from the 47th to the 67th amino acid sequence on the amino terminal side of the human thrombin receptor or KLH as a reference was immobilized on a CM5 sensor chip (BIACORE). The “amine coupling kit” commercially available from BIACORE was used in accordance with the program attached to the supplier. BIACORE Control Software Ver. 1.2 using CMBS sensor chip with commercially available HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (v / v)) at a flow rate of 5 μl / min. Washed away. Further, a 1: 1 mixture of EDC / NHS from the amine coupling kit was reacted for 7 minutes (35 μl) to activate carboxymethyldextran on the sensor chip surface. 100 μg / ml of KLH Conjugated Peptide or KLH was allowed to react on the sensor chip for 7 minutes (35 μl), and ethanolamine in the amine coupling kit was reacted for 7 minutes (35 μl) to react with the remaining active NHS groups. Blocked.
[0123]
Using the KLH immobilized on the sensor chip as a reference, the scFv binding dissociation curve for the peptide was measured. The scFv was adjusted to 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml with HBS-EP buffer, and each scFv solution was reacted with the sensor chip at a flow rate of 20 μl / min for 3 minutes. . Thereafter, HBS-EP buffer was flowed at a flow rate of 20 μl / min for 5 minutes, and the sensor chip was repeatedly washed with 10 mM HCl at 20 μl / min for 1 minute. The binding dissociation curve of each scFv for the peptide was obtained by measuring the mass change of the sensor chip surface in a series of operations. From the obtained binding dissociation curve and the concentration of each scFv, BIAevaluation software ver. The binding dissociation constant of each scFv was calculated using 2.1 according to the program attached to the company. As a result, the binding dissociation constant (KD value) of each scFv is scFv having synovial cell proliferation inhibitory activity, and the KD value is about 5 × 10.^-7M, scFv without synovial cell proliferation inhibitory activity, KD value: about 1 × 10^-7M (see Table 6).
[0124]
[Table 6]

[0125]
Example 11:Comparison of amino acid residue specificity of epitopes of scFv
Verification of amino acid residue specificity in the epitope of scFv having the ability to inhibit synovial cell proliferation was performed using a surface plasmon resonance sensor BIACORE1000 (BIACORE).
[0126]
Five types of 21 amino acids shown below were synthesized (Biologica Co., Ltd.). The four peptides are peptides included in the extracellular region of the human thrombin receptor and amino acid residue-substituted peptides.
[0127]
That is, the amino acid sequence from the 47th to the 67th amino acid sequence [SEQ ID NO: 112] on the amino terminal side of the human thrombin receptor, and the amino acid sequence in which the 51st lysine in the 47th to 67th amino acid residues is substituted with alanine [SEQ ID NO: 113] ], The amino acid sequence in which the 55th phenylalanine in the 47th to 67th amino acid residues is substituted with alanine [SEQ ID NO: 114], and the 56th tryptophan in the 47th to 67th amino acid residues is also substituted with alanine. Amino acid sequence [SEQ ID NO: 115] and control 21 amino acid sequence [SEQ ID NO: 111]
Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe
It is.
[0128]
A KLH complex was prepared for a peptide having the amino acid sequence from the 47th to the 67th amino acid sequence [SEQ ID NO: 112] on the amino terminal side of the PAR1-type human thrombin receptor. Using “Image Immunogen EDC Conjugation Kit With KLH and BSA” commercially available from PIERCE, 1 mg of the amino acid sequence peptide from the 47th to the 67th amino acid side of the PAR1 human thrombin receptor was added to the EDC Conjugation buffer solution of Kit. It melt | dissolved in 25 ml (peptide 4 mg / ml), and 0.1 ml of KLH solution (10 mg / ml) was added. Further, 25 μl of EDC (10 mg / ml) was slowly added dropwise to the peptide / KLH mixed solution while stirring, and then stirred for 30 minutes at room temperature. Thereafter, 350 μl of EDC Conjugation buffer was added to stop the reaction. Furthermore, it dialyzed twice against PBS buffer pH7.4, 3L, and obtained KLH complex peptide.
[0129]
A KLH complex peptide having the amino acid sequence from the 47th to the 67th amino acid sequence on the amino terminal side of the human thrombin receptor and KLH as a reference were immobilized on a CM5 sensor chip (BIACORE). The “amine coupling kit” commercially available from BIACORE was used in accordance with the program attached to the supplier. BIACORE Control Software Ver. 1.2 using CMBS sensor chip with commercially available HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (v / v)) at a flow rate of 5 μl / min. Washed away. Further, a 1: 1 mixture of EDC / NHS from the amine coupling kit was reacted for 7 minutes (35 μl) to activate carboxyl dextran on the sensor chip surface. 100 μg / ml of KLH conjugate peptide or KLH was allowed to react on the sensor chip for 7 minutes (35 μl), and ethanolamine in the amine coupling kit was reacted for 7 minutes (35 μl) to remove the remaining NHS groups. Blocked.
[0130]
Using KLH immobilized on the sensor chip as a reference, the binding of scFv to the peptide was confirmed. scFv was adjusted to 50 μg / ml with HBS-EP buffer, and reacted at a flow rate of 10 μl / min for 3 minutes. Thereafter, HBS-EP buffer was flowed at a flow rate of 20 μl / min for 5 minutes, and the sensor chip was repeatedly washed with 10 mM HCl at 20 μl / min for 1 minute. The maximum amount of each scFv binding to the peptide was obtained by measuring the mass change of the sensor chip surface in a series of operations. A scFv with synovial cell proliferating activity gave a maximum binding amount of about 300 RU, and a scFv without synovial cell proliferating activity gave a binding value of about 600 RU.
[0131]
With reference to KLH immobilized on the above-mentioned line sensor chip, competitive binding inhibition by each peptide of the scFv5 species against the peptide was measured. A mixture of scFv (final concentration 50 μg / ml) and peptide (final concentration 5 μg / ml) was prepared in HBS-EP buffer, and the scFv-peptide mixture was reacted at a flow rate of 10 μl / min for 3 minutes.
[0132]
The control peptide did not inhibit the binding of both scFv with and without synoviocyte growth inhibitory activity. In the amino acid sequence peptide from the 47th to the 67th amino acid side of the human thrombin receptor, the binding was inhibited for both scFv having and synovial cell proliferation inhibitory activity.
[0133]
In scFv having no active membrane cell growth inhibitory activity, an amino acid peptide in which the 51st lysine is substituted with alanine in the 47th to 67th amino acid residues on the amino terminal side of the human thrombin receptor, and the 47th to 67th Among amino acid residues, amino acid peptide in which 55th phenylalanine is substituted with alanine does not inhibit binding, and amino acid peptide in which 56th tryptophan is substituted with alanine in 47th to 67th amino acid residues on the amino terminal side Binding inhibition was observed.
[0134]
In scFv having synoviocyte growth inhibitory activity, among the 47 th to 67 th amino acid residues on the amino terminal side of the human thrombin receptor, the amino acid peptide in which the 51 st lysine is substituted with alanine and the 47 th to 67 th amino acid residues Among them, the amino acid peptide in which the 55th phenylalanine was substituted with alanine was found to inhibit binding, and the amino acid peptide in which the 56th tryptophan was substituted with alanine in the 47th to 67th amino acid residues on the amino terminal side of the human thrombin receptor. No binding inhibition was observed.
[0135]
Although both scFv with and without synoviocyte growth inhibitory activity recognized an overlapping amino acid sequence as an epitope, it was confirmed that there is a difference in importance for each amino acid residue in the epitope.
[0136]
For thrombin-induced synoviocyte growth inhibition, it was confirmed that the 56th tryptophan residue from the amino acid terminal of the human thrombin receptor is an important binding site for scFv.
[0137]
【The invention's effect】
The agent (polypeptide or compound) of the present invention suppresses signal transduction through the thrombin receptor and suppresses cell growth inhibitory activity and production and release of inflammatory mediators such as TNF-α, IL-6, and IL-1β. It is used as a therapeutic agent for various inflammatory diseases and cell proliferative diseases.
[0138]
[Sequence Listing]
[0139]

Claims (4)

  PAR1-type thrombin receptor partial sequence that binds to an epitope containing Tyr-Glu-Pro-Phe-Trp, or an epitope in which Phe in Tyr-Glu-Pro-Phe-Trp contained in the epitope is replaced with Ala And a single-chain Fv or a fragment thereof, or an antibody or a fragment thereof that suppresses cell proliferation by thrombin.   PAR1-type thrombin receptor partial sequence that binds to an epitope containing Tyr-Glu-Pro-Phe-Trp, or an epitope in which Phe in Tyr-Glu-Pro-Phe-Trp contained in the epitope is replaced with Ala However, a single-chain Fv or a fragment thereof that does not bind to an epitope in which Trp in Tyr-Glu-Pro-Phe-Trp contained in the epitope is substituted with Ala and suppresses cell growth by thrombin, or an antibody or Its fragments.   The single-chain Fv or fragment thereof, or the antibody or fragment thereof according to claim 1 or 2, which inhibits a signal transduction system involved in cell proliferation via a thrombin receptor. A modified PAR1-type thrombin receptor gene represented by SEQ ID NO: 1.