JP2004000228A - ANTI-Fas ANTIBODY - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-Fas antibody useful as a treating agent for diseases caused by the disorder of a Fas/Fas ligand system and binding with human Fas as well as murine Fas. <P>SOLUTION: This anti-Fas antibody is a molecule having an antigen-binding domain specific to an epitope preserved among Fas's of primates and non-primates animals. <P>COPYRIGHT: (C)2004,JPO

Description

温度条件：　９４℃で２分間加熱した後、９４℃で１分、５５℃で１分、７２℃で２分の温度サイクルを３０回繰り返した後、７２℃で１０分間保温した（以下、実施例中のすべてのＰＣＲの反応温度調節は、ジーンアンプＰＣＲシステム９６００；（株）パーキンエルマー・ジャパン社製を使用した）。
【０１４６】
一方、マウスＩＬ３受容体の細胞外領域をコードするＤＮＡ断片　ＭＡＩＣは以下の条件で作製された。

温度条件：　上記に同じ。
【０１４７】
ＰＣＲ後の増幅されたＨＦＡＳ　ＤＮＡおよびＭＡＩＣ　ＤＮＡは、それぞれフェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動され、１μｇ／ｍｌの臭化エチジウムにより染色された後、紫外線照射下で検出された。検出されたそれぞれのバンド部分のゲル片を剃刀刃で切り取り、遠心管型限外濾過器セントリコン１０（アミコン社製）を装着したセントリリューター（アミコン社製）を用いてＤＮＡを電気的に溶出した。さらにこの溶出液の入ったセントリコン１０を取り出して、７５００×ｇ、約１時間の遠心分離を行うことによりＤＮＡを濃縮した。このＤＮＡをエタノール沈澱の後、２０μｌの蒸留水に溶解した。
【０１４８】
ｄ）　第二段階ＰＣＲ
ヒトＦａｓ融合タンパク質をコードするＤＮＡ断片　ＦＡＳＡＩＣは、以下の条件で作製された。

温度条件：　第一段階に同じ。
【０１４９】
ＰＣＲ後の増幅されたＦＡＳＡＩＣ　ＤＮＡは、フェノール抽出とエタノール沈澱の後、１％アガロースゲル電気泳動を行い、１μｇ／ｍｌの臭化エチジウムにより染色され、紫外線照射下で検出された。検出されたバンド部分のゲル片を剃刀刃で切り取り、このゲル片よりセントリリューターおよびセントリコンを用いてＤＮＡを溶出、濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０１５０】
ｅ）　ベクターの構築
上記ｄ）で得られたＦＡＳＡＩＣ　ＤＮＡを制限酵素ＥｃｏＲＩとＸｂａＩで消化した（ＦＡＳＡＩＣ　ＤＮＡ溶液全量を使用した）後、フェノール−クロロホルム抽出およびエタノール沈澱を行った。この沈澱を２μｌの滅菌脱イオン水に懸濁した。
【０１５１】
一方、プラスミドｐＭＥ１８Ｓ−ｍＦａｓ−ＡＩＣ　２μｇをＥｃｏＲＩおよびＸｂａＩで消化してから脱リン酸化したＤＮＡ断片を調製し、このものと上記ＥｃｏＲＩとＸｂａＩで消化したＦＡＳＡＩＣ　ＤＮＡとライゲーションキット（宝酒造（株）社製）を用いて連結した後、ハナハンの方法［Ｈａｎａｈａｎ，Ｄ．（１９８３）Ｊ．Ｍｏｌ．Ｂｉｏｌ．１６６，５５７−５８０　参照］に従って大腸菌ＤＨ５α株（ギブコ・ビーアールエル社製）の形質転換を行なった。この形質転換大腸菌株よりアルカリＳＤＳ法［Ｍａｎｉａｔｉｓ，　Ｔ．，　ｅｔ　ａｌ．　（１９８９）　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　（２ｎｄ　Ｅｄｉｔｉｏｎ），　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ，　ＮＹ参照］でプラスミドを調製し、目的のプラスミド　ｐｈＦａｓ−ＡＩＣ２を得た。
【０１５２】
このプラスミドをプラスミド大量調製キット（マキシプレップ・　ＤＮＡ精製システム：プロメガ社製）を用いて調製し、２０μｇ相当のＤＮＡをエタノール沈澱させた後、沈澱を滅菌したダルベッコＰＢＳ（−）（以下「ＰＢＳ」という；日水製薬（株）社製）　２０μｌに溶解した。
【０１５３】
ｆ）　発現
ＣＯＳ−１細胞（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　Ｎｏ．　ＣＲＬ−１６５０　）を、１０％　ウシ胎児血清（ギブコ社製）（以下「ＦＣＳ」という）を含むダルベッコ変法イーグル培地（以下「ＤＭＥＭ」という：日水製薬（株）社製）を入れた細胞培養用フラスコ（培養面積２２５ｃｍ^２；住友ベークライト（株）社製）中でセミコンフルエントになるまで３７℃、５％　炭酸ガス下で培養した後、培地を除去してから、フラスコに５ｇ／リットル　トリプシンおよび２ｇ／リットル　エチレンジアミン四酢酸二ナトリウムを含む溶液（以下「トリプシン−ＥＤＴＡ溶液」という；シグマ社製）３ｍｌを入れて３７℃で３分間保温した。フラスコから遊離した細胞をＰＢＳに懸濁して回収し、ＰＢＳで２回洗浄してから、ＰＢＳで６×１０^７細胞／ｍｌとなるように調整した。この細胞懸濁液２０μｌ（１．２×１０^６細胞）と上記で調製したプラスミド溶液２０μｌずつを混合し、電極間隔２ｍｍのチャンバー（島津製作所（株）社製）に入れ、遺伝子導入装置（ＧＴＥ−１；島津製作所（株）社製）にセットしてから６００Ｖ−３０μｓｅｃのパルスを１秒間隔で２回加えた。チャンバー内の細胞−ＤＮＡ混合液を１０％ＦＣＳを含むＤＭＥＭ　１０ｍｌに加え、細胞培養用フラスコ（培養面積７５ｃｍ^２）中で３７℃、７．５％　炭酸ガス下で２４時間培養した。次いで培養上清を除去し、無血清ＤＭＥＭで細胞を洗浄した後、無血清ＤＭＥＭ　１０ｍｌを加えて、３７℃、７．５％　炭酸ガス下でさらに２４時間培養し、培養上清を回収した。
【０１５４】
得られた培養上清を透析チューブ（排出限界分子量１２０００−１４０００；ギブコ・ビーアールエル社製）に入れ、１０ｍＭ　トリス−塩酸（ｐＨ８．０）に対して透析した後、ファルマシア社製ＦＰＬＣ装置を用いて以下に記載する条件でヒトＦａｓ融合タンパク質の粗精製を行った：
カラム：　リソースＱカラム（商標名；φ６．４×３０ｍｍ、ファルマシア社製）；
溶媒：　１０ｍＭ　トリス−塩酸（ｐＨ８．０）
流速：　５ｍｌ／分；
溶出：　塩化ナトリウム　０．１Ｍ−０．３Ｍ、３０分間の直線濃度勾配。
溶出液を５ｍｌずつ分画し、以下に記載するＥＬＩＳＡ法によりＦａｓ遺伝子発現産物の検定を行った。まず、各溶出分画　１００μｌを９６穴マイクロプレート（コースター社製）のウェルに入れ、３７℃で１時間保温した。各ウェル中の液を除去後、０．１％　ツイーン２０（Ｔｗｅｅｎ　２０）を含むＰＢＳ（ＰＢＳ−Ｔｗｅｅｎ）１００μｌ／ウェルで３回洗浄し、２％　ウシ血清アルブミン（以下「ＢＳＡ」という）を含むＰＢＳ　１００μｌ／ウェルを入れて３７℃で１時間保温した。各ウェルをＰＢＳ−Ｔｗｅｅｎ　１００μｌ／ウェルで３回洗浄した後に、抗マウスＩＬ−３受容体βサブユニットモノクローナル抗体ＨＣ（１ｍｇ／ｍｌ；医学生物学研究所（株）社製）をＰＢＳ−Ｔｗｅｅｎで１０００倍希釈した溶液　１００μｌ／ウェルを入れて３７℃で１時間保温した。さらに、各ウェルをＰＢＳ−Ｔｗｅｅｎ　１００μｌで３回洗浄した後に、ＰＢＳ−Ｔｗｅｅｎで２０００倍希釈した西洋ワサビペルオキシダーゼ標識抗マウスイムノグロブリン抗体（アマシャム社製）１００μｌ／ウェルを入れて３７℃で１時間保温してから、各ウェルをＰＢＳ−Ｔｗｅｅｎ　１００μｌで３回洗浄した。次に西洋ワサビペルオキシダーゼ基質（バイオラッド社製）１００μｌ／ウェルを入れて５分間静置してから、マイクロプレートリーダー（モデル４５０；バイオラッド社製）を用いて４１５ｎｍの吸光度を測定した。その結果、吸光度が高かった１９乃至２３番目の分画を集めて、粗精製ヒトＦａｓ融合タンパク質試料とした。
【０１５５】
実施例２　マウスの免疫およびハイブリドーマの調製
（２−１）　免疫
実施例１で得られた粗精製ヒトＦａｓ融合タンパク質溶液　１ｍｌ（全タンパク質量；　１００μｇ）に対し、２Ｎ　塩酸　２５μｌ、９％　カリミョウバン　２５０μｌ（終濃度１．１％）、２Ｎ　水酸化ナトリウム　２５μｌを添加した後、１０％（ｗ／ｖ）炭酸水素ナトリウム水溶液　約１２０μｌを添加することによりｐＨ６．５乃至７．０に調整した。室温で約３０分間放置後、Ｔ細胞の活性化のために百日咳菌死菌（和光純薬（株）社製；１．２×１０^１１菌／ｍｌ）２００μｌを添加し、これをＦａｓノックアウトマウス腹腔内に投与した。Ｆａｓノックアウトマウスは、千住らの文献に記載する方法により取得されたものを使用した［Ｓｅｎｊｕ，　Ｓ．　ｅｔ　ａｌ．　（１９９６）　Ｉｎｔｅｒｎａｔｉｏｎａｌ　Ｉｍｍｕｎｏｌｏｇｙ　８，　４２３　参照］。２週間後、粗精製ヒトＦａｓ融合タンパク質のみ（２０μｇタンパク質／マウス）を腹腔内に投与し追加免疫した。
【０１５６】
（２−２）　細胞融合
追加免疫３日後のマウスより脾臓を摘出し、これを２０ｍＭ　ＨＥＰＥＳ緩衝液（ｐＨ７．３）、３５０ｍｇ／ｍｌ　炭酸水素ナトリウム、０．０５ｍＭ　β−メルカプトエタノール、５０単位／ｍｌ　ペニシリン、５０μｇ／ｍｌ　ストレプトマイシン、３００μｇ／ｍｌ　Ｌ−グルタミン酸を含む無血清ＲＰＭＩ１６４０培地（１０．４ｇ／リットル　ＲＰＭＩ１６４０「ニッスイ」▲１▼：日水製薬（株）社製）（以下「無血清ＲＰＭＩ培地」という）１０ｍｌ中に入れ、メッシュ（セルストレイナー：ファルコン社製）上でスパーテルを用いてつぶした。メッシュを通過した細胞懸濁液を遠心して脾臓細胞を沈澱させた後、この脾臓細胞を無血清ＲＰＭＩ培地で２回洗浄してから、無血清ＲＰＭＩ培地に懸濁して細胞数を測定した。
【０１５７】
一方、１０％　ＦＣＳ（ギブコ・ビーアールエル社製）を含むＡＳＦ１０４培地（味の素（株）社製）（以下「血清入りＡＳＦ培地」という）にて、３７℃、５％　炭酸ガス存在下で細胞濃度が１×１０^８細胞／ｍｌを越えないように培養したミエローマ細胞　ＮＳ１（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　ＴＩＢ−１８　）を同様に無血清ＲＰＭＩ培地で洗浄し、無血清ＲＰＭＩ培地に懸濁して細胞数を測定した。
【０１５８】
３×１０^７個相当のＮＳ１細胞懸濁液と、３×１０^８個相当の脾臓細胞懸濁液を混合し、遠心後、上清を完全に除去した。以下の細胞融合の操作は、ペレットの入ったプラスチック遠沈管を温水を入れたビーカー中で３７℃に保温しながら実施した。このぺレットに、５０％（ｗ／ｖ）　ポリエチレングリコール　１５００（ベーリンガーマンハイム社製）１ｍｌを、ピペットの先でぺレットを撹拌しながらゆっくり添加した後、予め３７℃に加温しておいた無血清ＲＰＭＩ培地１ｍｌを２回に分けてゆっくり添加し、さらに７ｍｌの無血清ＲＰＭＩ培地を添加した。遠心後、上清を除去し、１０％　ＦＣＳを含むヒポキサンチン・アミノプテリン・チミジン培地（以下「ＨＡＴ培地」という；ベーリンガー・マンハイム社製）１０ｍｌを、ピペットの先でゆっくり撹拌しながら添加した。さらに１０％　ＦＣＳを含むＨＡＴ培地　２０ｍｌを添加した後に、細胞培養用９６穴マイクロプレートに１００μｌ／ウェルずつ分注し、３７℃、５％炭酸ガス下で培養した。７〜８日後、培地が黄色味を帯びたウエルには新しいＨＡＴ培地を１００μｌ／ウェルずつ加えた。このようにして得られた融合細胞を、以下に記載する限界希釈法によるスクリーニングに供した。
【０１５９】
（２−３）　限界希釈
４〜１０週令のＢＡＬＢ／ｃマウス雌（日本エスエルシー社より購入）より胸腺を摘出後、メッシュ（セルストレイナー；ファルコン社製）上でスパーテルを用いてつぶし、メッシュを通過した細胞を１０％　ＦＣＳを含むヒポキサンチン・チミジン培地（以下「ＨＴ培地」という；ベーリンガー・マンハイム社製）で２回洗浄した。マウス１匹分の胸腺細胞を１０％　ＦＣＳを含むＨＴ培地　３０ｍｌに懸濁したものをフィーダー細胞液とした。上記（２−２）で得られた融合細胞を含む培養液を、細胞密度に応じてフィーダー細胞液で１０乃至１００倍に希釈し、さらに融合細胞の密度が５細胞／ｍｌ、１細胞／ｍｌ、０．５細胞／ｍｌとなるように、フィーダー細胞液で段階希釈した。このようにして調製した各試料を、細胞培養用９６穴マイクロプレートに１００μｌ／ウェルずつ分注し、３７℃、５％炭酸ガス下で５日間培養した。
【０１６０】
（２−４）　選別
マウスＴリンパ腫細胞ＷＲ１９Ｌ（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　ＴＩＢ−５２　）にヒトＦａｓをコードする遺伝子を発現させたＷＲ１９Ｌ１２ａ細胞［Ｉｔｏｈ，　Ｎ．　ｅｔ．　ａｌ．　（１９９１）　Ｃｅｌｌ　６６，　２３３−２４３参照］を、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地中で、３７℃、５％炭酸ガス下で培養し増殖させてから、１×１０^７細胞／ｍｌに調整した細胞懸濁液を、Ｕ字底９６穴マイクロプレート（ヌンク社製）に５０μｌ／ウェルずつ分注し、遠心分離（９０×ｇ、４℃、１０分）した。上清を除去し、上記（２−３）で培養した融合細胞の培養上清を５０μｌ／ウェル加えて撹拌した後、氷上で１時間静置してから、遠心分離（９０×ｇ、４℃、１０分）して上清を除去した。ペレットを１００μｌ／ウェルのフローサイトメトリー用緩衝液（５％　ＦＣＳ、０．０４％（ｗ／ｖ）　アジ化ナトリウムを含むＰＢＳ）で２回洗浄した後、５００倍希釈したフルオレッセイン−５−イソチオシアネート（Ｆｌｕｏｒｅｓｃｅｉｎ−５−ｉｓｏｔｈｉｏｃｙａｎａｔｅ；以下「ＦＩＴＣ」という）標識ヤギ抗マウスＩｇＧ抗体ＩｇＧ画分（オルガノン・テクニカ社製）５０μｌを二次抗体として加え、氷上で１時間静置した。遠心分離（９０×ｇ、４℃、１０分）して上清を除去してから、ペレットをフローサイトメトリー用緩衝液１００μｌ／ウェルで２回洗浄後、３．７％　ホルマリン溶液　５０μｌを添加し、氷上で１０分静置することにより細胞を固定した。遠心分離（９０×ｇ、４℃、１０分）して上清を除去してから、再度フローサイトメトリー用緩衝液１００μｌ／ウェルで洗浄し、ペレットをフローサイトメトリー用緩衝液１００μｌ／ウェルに懸濁したものをフローサイトメトリー用試料とした。各試料中の細胞のＦＩＴＣ蛍光強度をフローサイトメーター（エピックス・エリート；コールター社製）で測定した（励起波長：４８８ｎｍ、検出波長：５３０ｎｍ）。その結果、融合細胞培養上清を添加しなかったＷＲ１９Ｌ１２ａ（ＦＩＴＣ蛍光強度約０．３）よりも明らかに高値（約１００−１０００）のＦＩＴＣ蛍光強度を示した試料に対応する融合細胞を選別した。
【０１６１】
（２−５）　クローニング
上記（２−４）で選別された細胞群について、上記（２−３）から（２−４）の一連の工程を５回繰返すことにより、ＷＲ１９Ｌ１２ａと結合するがＷＲ１９Ｌと結合しない単一な抗体を産生するハイブリドーマを数クローン得た。それらのハイブリドーマが産生する抗体について、（２−４）と同様の方法を用いて、マウスＦａｓに対する結合能を調べた。マウスＦａｓを発現する細胞としては、Ｆａｓをほとんど発現しない細胞株Ｌ５１７８Ｙ（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　Ｎｏ．　ＣＲＬ−１７２２　）にマウスＦａｓ発現ベクターを導入したＬ５１７８ＹＡ１細胞を使用した。その結果、Ｌ５１７８ＹＡ１細胞に結合し、Ｌ５１７８Ｙ細胞と結合しない抗体を産生するマウス−マウスハイブリドーマＨＦＥ７Ａを選別した。マウス−マウスハイブリドーマＨＦＥ７Ａは平成９年２月２０日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、寄託番号ＦＥＲＭ　ＢＰ−５８２８が付された。
【０１６２】
またマウス−マウスハイブリドーマＨＦＥ７Ａが産生する抗体（以下「ＨＦＥ７Ａ」という）のサブクラスを、モノクローナル抗体アイソタイピングキット（ピアース社製）を用いて調べた結果、ＩｇＧ１、κであることが判明した。
【０１６３】
実施例３　ＨＦＥ７Ａモノクローナル抗体の精製
実施例２で作出されたマウス−マウスハイブリドーマＨＦＥ７Ａ（ＦＥＲＭ　ＢＰ−５８２８）を、１０％　ＦＣＳを含むＡＳＦ培地　１リットル中で、３７℃、５％　炭酸ガス下で培養し、１×１０^６細胞／ｍｌとなるまで増殖させた。培養液を遠心分離（１０００ｒｐｍ、２分間）し、上清を捨て、沈澱した細胞を無血清ＡＳＦ培地で１回洗浄後、無血清ＡＳＦ培地１リットルに再懸濁し、３７℃、５％　炭酸ガス下で４８時間培養した。この培養液を遠心分離（１０００ｒｐｍ、２分間）し、上清を回収して透析チューブ（排除限界分子量１２０００−１４０００；ギブコ・ビーアールエル社製）に入れ、１０倍量の１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ８．０）に対して透析した。この透析チューブ内液からのＩｇＧの粗精製を、高速液体クロマトグラフィー装置（ＦＰＬＣシステム；ファルマシア社製）を用いて以下に記載する条件で行った：
カラム：　ＤＥＡＥ−セファロース　ＣＬ−６Ｂカラム（カラムサイズ１０ｍｌ；ファルマシア製）；
溶媒：　１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ８．０）
流速：　１ｍｌ／分；
溶出：　１Ｍ　塩化ナトリウムの直線濃度勾配（０−５０％、１８０分）。
溶出液を５ｍｌずつ分画し、各分画中の抗Ｆａｓ抗体価を、ヒトＦａｓ融合タンパク質を用いたＥＬＩＳＡ法により検定した。まず、実施例１で調製した粗精製ヒトＦａｓ融合タンパク質溶液をＥＬＩＳＡ用９６穴マイクロプレート中に１００μｌ／ウェル入れ、３７℃で１時間保温した後、この溶液を捨て、各ウェルをＰＢＳ−Ｔｗｅｅｎ　１００μｌ／ウェルで３回洗浄した。次に２％　ウシ血清アルブミンを含むＰＢＳ　１００μｌ／ウェルを入れて３７℃で１時間保温した。ＰＢＳ−Ｔｗｅｅｎ　１００μｌ／ウェルで３回洗浄した後に、各溶出分画１００μｌを入れて３７℃で１時間保温した。さらに、ＰＢＳ−Ｔｗｅｅｎ　１００μｌ／ウェルで３回洗浄した後に、ＰＢＳ−Ｔｗｅｅｎで２０００倍希釈した西洋ワサビペルオキシダーゼ標識抗マウスイムノグロブリン抗体（アマシャム社製）１００μｌ／ウェルを添加して３７℃で１時間反応させ、ＰＢＳ−Ｔｗｅｅｎ　１００μｌ／ウェルで３回洗浄した。次に西洋ワサビペルオキシダーゼ基質（バイオラッド社製）１００μｌ／ウェルを入れて５分間静置した後、マイクロプレートリーダーで各ウェルの４１５ｎｍの吸光度を測定した。
【０１６４】
その結果、吸光度の大きかった２１−３０番目の分画を集め、抗体アフィニティー精製用カラム（ハイトラップ・プロテインＧカラム、カラム体積　５ｍｌ；ファルマシア社製）２本に供与した。カラム内を２５ｍｌ／カラムの平衡化緩衝液（２０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０））にて洗浄した後に、１５ｍｌ／カラムの溶出緩衝液（０．１Ｍ　グリシン−塩酸（ｐＨ２．７））にて抗体を溶出した。それぞれの溶出液は、１．１２５ｍｌの１Ｍ　トリス−塩酸（ｐＨ９．０）を入れた試験管内に受け、溶出終了後ただちに遠心管型限外濾過器（セントリプレップ１０；グレースジャパン（株）製）の上部に入れて、３０００×ｇ、４℃で２時間遠心した。濾過器下部に回収された濾液を除去後、上部に１５ｍｌのＰＢＳを加えて、再び３０００×ｇ、４℃で２時間遠心する操作を５回繰り返した。ただし５回目の遠心は濾過器上部内の液量が０．５ｍｌになるまで行ない、この濾過器上部に残った液をＨＦＥ７Ａ試料とした。
【０１６５】
実施例４　ｃＤＮＡクローニング
（４−１）　ポリ（Ａ）^＋ＲＮＡの調製
実施例２で作製したマウス−マウスハイブリドーマＨＦＥ７Ａ（ＦＥＲＭ　ＢＰ−５８２８）を１０％　ＦＣＳを含むＡＳＦ培地　１リットルにて、３７℃、５％　炭酸ガス下で１×１０^６細胞／ｍｌまで増殖させた後に、細胞を遠心分離により回収し、グアニジンチオシアネート溶液（４Ｍ　グアニジンチオシアネート、１％　サルコシル、２０ｍＭ　エチレンジアミン四酢酸（ＥＤＴＡ）、２５ｍＭ　クエン酸ナトリウム（ｐＨ７．０）、１００ｍＭ　２−メルカプトエタノール、０．１％　アンチフォームＡ）で溶解し、溶液を回収した。ポリ（Ａ）^＋ＲＮＡの単離は本質的に、”Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　”［Ｍａｎｉａｔｉｓ，Ｔ．ｅｔ　ａｌ．（１９８２）　ｐｐ．１９６−１９８参照］に記載されているようにして実施した。具体的には、以下の手順で行った。
【０１６６】
回収した細胞溶解液は、各々２１Ｇの注射針を装填した１０ｍｌ容の注射筒を用いて、数回吸入排出を繰り返した。日立製作所（株）製ＲＰＳ４０−Ｔローター・バケット用のポリアロマー製の遠心管に３ｍｌの５．７Ｍ　塩化セシウム−０．１Ｍ　ＥＤＴＡ（ｐＨ７．５）を先に加えておき、その上に上記細胞溶解液を重層して遠心管を満たした。これを３００００ｒｐｍ、２０℃で１８時間遠心した後、得られたペレットを４００μｌの蒸留水に溶解し、エタノール沈澱を行った。得られたペレットを再び４００μｌの蒸留水に溶解した後、等量のクロロホルム／１−ブタノール（４：１、ｖ／ｖ）を加え撹拌し、遠心分離後水層を回収した。これを再度エタノール沈澱した後、沈澱を６００μｌの蒸留水に溶解したものを、全ＲＮＡ試料とした。
【０１６７】
このようにして得られた全ＲＮＡ　６００μｇから、オリゴ（ｄＴ）セルロースカラム・クロマトグラフィーにより、ポリ（Ａ）^＋ＲＮＡを精製した。すなわち、全ＲＮＡを吸着緩衝液（０．５Ｍ　塩化ナトリウム、２０ｍＭ　トリス−塩酸（ｐＨ７．５）、１　ｍＭ　ＥＤＴＡ、０．１％　ドデシル硫酸ナトリウム（ＳＤＳ））に溶解し、６５℃で５分間加熱した後、同溶液にて充填されたオリゴ（ｄＴ）セルロースカラム（タイプ７；ファルマシア社製）に供与し、溶出溶液（１０ｍＭ　トリス−塩酸（ｐＨ７．５）、１　ｍＭ　ＥＤＴＡ、０．０５％　ＳＤＳ）でポリ（Ａ）^＋ＲＮＡを溶出し、回収した。このような操作により１００μｇのポリ（Ａ）^＋ＲＮＡ画分を得た。
【０１６８】
（４−２）　ＨＦＥ７Ａの重鎖および軽鎖のＮ末端アミノ酸配列の決定
実施例３で精製した抗ヒトＦａｓ抗体ＨＦＥ７Ａを含む溶液の一部をＳＤＳ−ポリアクリルアミドゲル電気泳動（以下「ＳＤＳ−ＰＡＧＥ」という）した（ゲル濃度１２％、１００Ｖ定電圧、１２０分）。泳動終了後のゲルを転写緩衝液（２５ｍＭ　トリス−塩酸（ｐＨ９．５）、２０％　メタノール、０．０２％　ＳＤＳ）に浸した後、転写装置（ＫＳ−８４５１；マリソル社製）を用いて、予め転写緩衝液に浸しておいたポリビニリデンジフルオリド膜（以下「ＰＶＤＦ膜」という；ポアーサイズ　０．４５μｍ；ミリポア社製）にゲル中のタンパク質を転写した（１０Ｖ定電圧、４℃、１４時間）。転写後のＰＶＤＦ膜を洗浄緩衝液（２５ｍＭ　塩化ナトリウム、１０ｍＭ　ホウ酸ナトリウム緩衝液（ｐＨ８．０））で洗浄した後、染色液（５０％　メタノール、２０％　酢酸、０．０５％　クマシーブリリアントブルー）に５分間浸して染色してから、９０％　メタノールで脱色した。このようにしてＰＶＤＦ膜上で可視化された重鎖（移動度が小さい方のバンド）および軽鎖（移動度が大きい方のバンド）に相当するバンド部分を切りとり、脱イオン水にて洗浄した後に、気相式プロテインシークエンサー（ＰＰＳＱ−１０；島津製作所（株）社製）を用いて、自動エドマン法［Ｅｄｍａｎ，　Ｐ．，　ｅｔ　ａｌ．　（１９６７）　Ｅｕｒ．　Ｊ．　Ｂｉｏｃｈｅｍ．　１，　８０　参照］によりそれぞれのＮ末端アミノ酸配列を決定した。
【０１６９】
その結果、重鎖に相当するバンドのＮ末端アミノ酸配列は、
Ｇｌｎ−Ｘａａ−Ｇｌｎ−Ｌｅｕ−Ｇｌｎ−Ｇｌｎ−Ｐｒｏ−Ｇｌｙ−Ａｌａ−Ｇｌｕ−Ｌｅｕ　　（配列表の配列番号１６）
であり、軽鎖に相当するバンドのＮ末端アミノ酸配列は、
Ａｓｐ−Ｉｌｅ−Ｖａｌ−Ｌｅｕ−Ｔｈｒ−Ｇｌｎ−Ｓｅｒ−Ｐｒｏ−Ａｌａ−Ｓｅｒ−Ｌｅｕ−Ａｌａ−Ｖａｌ−Ｓｅｒ−Ｌｅｕ−Ｇｌｙ−Ｇｌｎ−Ａｒｇ　−Ａｌａ−Ｔｈｒ−Ｉｌｅ−Ｓｅｒ　（配列表の配列番号１７）
であった。
【０１７０】
これらのアミノ酸配列を、カバトらにより作成された抗体のアミノ酸配列データベース［Ｋａｂａｔ，　Ｅ．　Ａ．　ｅｔ　ａｌ．，　（１９９１）　ｉｎ　Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ　Ｖｏｌ．　ＩＩ，　Ｕ．Ｓ．　Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ参照］と比較したところ、ＨＦＥ７Ａの重鎖（γ１鎖）は、サブタイプ２ｂであり、軽鎖（κ鎖）は、サブタイプ３であることが判明した。そこで、これらのマウスサブタイプに属する遺伝子の５’末端側非翻訳領域の一部および３’末端側翻訳領域の最末端部分とハイブリダイズする、下記のオリゴヌクレオチドプライマーをそれぞれ合成した［前出カバトらの文献、Ｍａｔｔｉ　Ｋａｒｔｉｎｅｎ　ｅｔ　ａｌ．　（１９８８）　２５，８５９−８６５およびＨｅｉｎｒｉｃｈ，　Ｇ．　ｅｔ　ａｌ．　（１９８４）　Ｊ．　Ｅｘｐ．　Ｍｅｄ．　１５９，　４１７−４３５参照］：
５’−　ＧＡＣＣＴＣＡＣＣＡ　ＴＧＧＧＡＴＧＧＡ　−３’　（Ｈ１：配列表の配列番号１８）；
５’−　ＴＴＴＡＣＣＡＧＧＡ　ＧＡＧＴＧＧＧＡＧＡ　−３’　　（Ｈ２：配列表の配列番号１９）；
５’−　ＡＡＧＡＡＧＣＡＴＣ　ＣＴＣＴＣＡＴＣＴＡ　−３’　　（Ｌ１：配列表の配列番号２０）；
５’−　ＡＣＡＣＴＣＡＴＴＣ　ＣＴＧＴＴＧＡＡＧＣ　−３’　　（Ｌ２：配列表の配列番号２１）。
【０１７１】
（４−３）　ｃＤＮＡクローニング
マウス抗ヒトＦａｓモノクローナル抗体ＨＦＥ７Ａの重鎖および軽鎖をコードするｃＤＮＡを、逆転写酵素（ＲＴ）反応およびＰＣＲ反応を組み合わせて、上記（４−１）で調製したＨＦＥ７Ａ産生ハイブリドーマ由来のポリ（Ａ）^＋ＲＮＡ画分より任意の配列を特異的に増幅する方法（ＲＴ−ＰＣＲ）によりクローニングした。ＲＴ−ＰＣＲは、ＲＮＡ　ＰＣＲキット（ＡＭＶ）バージョン２（宝酒造（株）社製）を使用して行なった。
【０１７２】
ａ）　逆転写酵素反応
ＲＴ−ＰＣＲ用のプライマーは、上記（４−２）で合成したオリゴヌクレオチドプライマーのセット（５’末端側および３’末端側プライマー）を用い、重鎖用または軽鎖用各プライマーセットを使用したＲＴ−ＰＣＲ反応を個別に実施した。
【０１７３】

温度条件：　５５℃で３０分間、９９℃で５分間、５℃で５分間の順にインキュベートした。
【０１７４】
ｂ）　ＰＣＲ

温度条件：　９４℃で２分間加温した後、９４℃で３０秒、６０℃で３０秒、７２℃で１．５分間の温度サイクルを２８回繰り返した。
【０１７５】
反応終了後、反応液の一部をとり、１．５％　アガロースゲル電気泳動を行なった結果、重鎖用プライマーを入れた反応液では約１．４ｋｂｐのバンドが増幅されており、軽鎖用プライマーを入れた反応液では約０．７ｋｂｐのバンドが増幅されていた。
【０１７６】
これらＰＣＲで増幅されたｃＤＮＡを、ＴＡクローニングキット（インビトロジェン社製）を用いてクローニングした。まず、ＰＣＲ反応液とｐＣＲＩＩベクター（ＴＡクローニングキットに添付）５０ｎｇを、１μｌの１０倍濃度リガーゼ反応緩衝液（６ｍＭ　トリス塩酸（ｐＨ７．５）、６ｍＭ　塩化マグネシウム、５ｍＭ　塩化ナトリウム、７ｍＭ　β−メルカプトエタノール、０．１ｍＭ　ＡＴＰ、２ｍＭ　ＤＴＴ、１ｍＭ　スペルミジン、０．１ｍｇ／ｍｌ　ウシ血清アルブミン）中に溶解し、４単位のＴ４ＤＮＡリガーゼ（１μｌ）を加え、滅菌脱イオン水で全量を１０μｌとして、１４℃で１５時間保温した。次いで、０．５Ｍ　β−メルカプトエタノール　２μｌを加えたコンピテント大腸菌ＴＯＰ１０Ｆ’（ＴＡクローニングキットに添付）５０μｌに上記リガーゼ反応液２μｌを添加し、氷上で３０分間静置した後に、４２℃で３０秒間加温してから、再び氷上で５分間冷却した。このものにＳＯＣ培地（２％　トリプトン、０．５％　イーストエキストラクト、０．０５％　塩化ナトリウム、２．５ｍＭ　塩化カリウム、１ｍＭ　塩化マグネシウム、２０ｍＭ　グルコース）５００μｌを加え、３７℃で１時間振盪培養した。この培養液を１００μｇ／ｍｌのアンピシリンを含有するＬ−ブロス寒天培地（１％　トリプトン、０．５％　イーストエキストラクト、０．５％　塩化ナトリウム、０．１％　グルコース、０．６％　バクト・　アガー（ディフコ社製））プレート上に塗り広げ、３７℃にて一晩静置培養した。プレート上に現れた単一のアンピシリン耐性コロニーを選択し、このコロニーを白金耳で掻きとって、１００μｇ／ｍｌのアンピシリンを含有するＬ−ブロス培地にて培養した後に、遠心分離して回収した菌体からアルカリ法によりプラスミドＤＮＡを調製した。このようにして得られたプラスミドを、それぞれｐＣＲ−Ｈ（ＨＦＥ７Ａ重鎖をコードするｃＤＮＡを保持するプラスミド）およびｐＣＲ−Ｌ（ＨＦＥ７Ａ軽鎖をコードするｃＤＮＡを保持するプラスミド）と命名した。
【０１７７】
（４−４）　ヌクレオチド配列の解析
（４−３）で得られたプラスミドｐＣＲ−ＨおよびｐＣＲ−Ｌがそれぞれ保持する、ＨＦＡ７Ａ重鎖をコードするｃＤＮＡ（１．４ｋｂｐ）およびＨＦＥ７Ａ軽鎖をコードするｃＤＮＡ（０．７ｋｂｐ）のヌクレオチド配列を、遺伝子配列解析装置（３１０ジェネティックアナライザー；（株）パーキンエルマージャパン社製）を用いてジデオキシ法［Ｓａｎｇｅｒ，　Ｆ．，　ｅｔ　ａｌ．　（１９７７）　Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ　７４，　５４６３−５４６７参照］により決定した。
【０１７８】
その結果決定された、ＨＦＥ７Ａ重鎖および軽鎖をコードするｃＤＮＡのヌクレオチド配列を、それぞれ配列表の配列番号８および１０に示した。また、これらｃＤＮＡにコードされる、ＨＦＥ７Ａ重鎖および軽鎖の全アミノ酸配列を、それぞれ配列表の９および１１に示した。上記（４−１）で判明したＨＦＥ７Ａ重鎖のＮ末端アミノ酸配列（配列表の配列番号１６）は、配列表の配列番号９のアミノ酸番号１〜１１の配列と、不明の１残基を除き一致した。また、ＨＦＥ７Ａ軽鎖のＮ末端アミノ酸配列（配列表の配列番号１７）は、配列表の配列番号１１のアミノ酸番号１〜２２の配列と一致した。すなわち、ＨＦＥ７Ａ重鎖および軽鎖の成熟型タンパク質のＮ末端は、それぞれ配列番号９および１１のアミノ酸番号１のアミノ酸であることが明らかとなった。
【０１７９】
また、この重鎖および軽鎖のアミノ酸配列を、カバトらによる抗体のアミノ酸配列のデータベース［Ｋａｂａｔ，　Ｅ．　Ａ．，　ｅｔ　ａｌ．　（１９９１）　ｉｎ　”Ｓｅｑｕｅｎｃｅ　ｏｆ　Ｐｒｏｔｅｉｎｓｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ　Ｖｏｌ．ＩＩ”：　Ｕ．Ｓ．　Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｈｅａｌｔｈ　ａｎｄ　Ｈｕｍａｎ　Ｓｅｒｖｉｃｅｓ参照］と比較検討した結果、重鎖については、配列番号９のアミノ酸番号１から１２１が可変領域であり、アミノ酸番号１２２から４４５が定常領域であること、また軽鎖については、配列番号１１のアミノ酸番号１から１１１が可変領域であり、アミノ酸番号１１２から２１８が定常領域であることが明らかとなった。
【０１８０】
さらに、上記のごとく決定されたＨＦＥ７Ａの重鎖および軽鎖の可変領域のアミノ酸配列における、それぞれのＣＤＲの位置および配列は、カバトらの文献［前記Ｋａｂａｔ，　Ｅ．　Ａ．，　ｅｔ　ａｌ．　（１９９１）　参照］による抗体のアミノ酸配列のデータベースとの相同性を比較検討することによって決定された。該文献によれば、異なる抗体間でもサブタイプが同じであれば可変領域中のフレームワーク領域の鎖長はほぼ一定であり、またそのアミノ酸配列には共通性が認められる。一方、ＣＤＲは、それらのフレームワーク領域にはさまれて存在する固有の配列である。そこで、ＨＦＥ７Ａの重鎖および軽鎖のアミノ酸配列を同じサブタイプのものと比較検討した結果、ＨＦＥ７Ａの重鎖のＣＤＲは、配列表の配列番号９のアミノ酸番号３１〜３５（ＣＤＲＨ_１）、同アミノ酸番号５０〜６６（ＣＤＲＨ_２）および同アミノ酸番号９９〜１１０（ＣＤＲＨ_３）で示されるアミノ酸配列と決定された。またＨＦＥ７Ａの軽鎖のＣＤＲは、配列表の配列番号１１のアミノ酸番号２４〜３８（ＣＤＲＬ_１）、同アミノ酸番号５４〜６０（ＣＤＲＬ_２）および同アミノ酸番号９３〜１０１（ＣＤＲＬ_３）で示されるアミノ酸配列と決定された。
【０１８１】
実施例５　組換え抗体の調製
（５−１）　発現プラスミドの構築
実施例４でクローニングされたＨＦＥ７Ａの重鎖および軽鎖をコードするｃＤＮＡを動物細胞用発現ベクターｐＭＳ１８Ｓ［Ｈａｒａ，　Ｔ．，　ｅｔ　ａｌ．　（１９９２）　ＥＭＢＯ　Ｊ．　１１，　１８７５参照］に組み込んだ組換え発現ベクターを、以下に記載する方法で構築した。まず、プラスミドｐＣＲ−Ｈが保持するｃＤＮＡの５’末端に制限酵素ＥｃｏＲＩ認識部位を付加し、３’末端部分に制限酵素ＸｂａＩ認識部位および終止コドンを付加するためのオリゴヌクレオチドプライマー：
５’−　ＧＧＧＧＡＡＴＴＣＧ　ＡＣＣＴＣＡＣＣＡＴ　ＧＧＧＡＴＧＧＡ　−３’（Ｈ３：配列表の配列番号２２）
５’−　ＧＧＧＴＣＴＡＧＡＣ　ＴＡＴＴＴＡＣＣＡＧ　ＧＡＧＡＧＴＧＧＧＡ　ＧＡ　−３’　（Ｈ４：配列表の配列番号２３）
を合成した。また、プラスミドｐＣＲ−Ｌが保持するｃＤＮＡの５’末端に制限酵素ＥｃｏＲＩ認識部位を付加し、３’末端部分に制限酵素ＮｏｔＩ認識部位および終止コドンを付加するためのオリゴヌクレオチドプライマー：
５’−　ＧＧＧＧＡＡＴＴＣＡ　ＡＧＡＡＧＣＡＴＣＣ　ＴＣＴＣＡＴＣＴＡ　−３’　　（Ｌ３：配列表の配列番号２４）
５’−　ＧＧＧＧＣＧＧＣＣＧ　ＣＴＴＡＣＴＡＡＣＡ　ＣＴＣＡＴＴＣＣＴＧ　ＴＴＧＡＡＧＣ　−３’　（Ｌ４：配列表の配列番号２５）を合成した。この重鎖用および軽鎖用それぞれのプライマーを用い、以下の条件でＰＣＲを実施した。

温度条件：　９４℃で２分間加温した後、９４℃で３０秒、６０℃で３０秒、７５℃で１．５分のサイクルを２８回繰り返した。
【０１８２】
上記ＰＣＲにより増幅されたＤＮＡを、制限酵素ＥｃｏＲＩとＸｂａＩ（重鎖）あるいはＥｃｏＲＩとＮｏｔＩ（軽鎖）で消化した後に、同じく制限酵素ＥｃｏＲＩとＸｂａＩ（重鎖用）あるいはＥｃｏＲＩとＮｏｔＩ（軽鎖用）で消化し脱リン酸化した動物細胞用発現プラスミドベクター　ｐＭＥ１８Ｓ［Ｈａｒａ，　Ｔ．，　ｅｔ　ａｌ．　（１９９２）　ＥＭＢＯ　Ｊ．　１１，　１８７５参照］と混合し、１０倍濃度リガーゼ反応緩衝液（６ｍＭ　トリス塩酸（ｐＨ７．５）、６ｍＭ　塩化マグネシウム、５ｍＭ　塩化ナトリウム、７ｍＭ　β−メルカプトエタノール、０．１ｍＭ　ＡＴＰ、２ｍＭ　ＤＴＴ、１ｍＭ　スペルミジン、０．１ｍｇ／ｍｌ　ウシ血清アルブミン）中で、４単位のＴ４ＤＮＡリガーゼを加え、１４℃で１５時間保温した。このリガーゼ反応液２μｌをコンピテント大腸菌ＪＭ１０９株（宝酒造（株）社製）５０μｌに加え、氷上で３０分間放置した後に、４２℃で３０秒、氷上で５分間静置した。このものにＳＯＣ培地（２％　トリプトン、０．５％　イーストエキストラクト、０．０５％　塩化ナトリウム、２．５ｍＭ　塩化カリウム、１ｍＭ塩化マグネシウム、２０ｍＭ　グルコース）５００μｌを加え、１時間振とう培養した。以下、実施例４（４−３）に記載した方法で形質転換大腸菌株を単離し、該菌株からプラスミドＤＮＡを調製した。このようにして得られたプラスミドを、プラスミドｐＭＥ−Ｈ（ＨＦＥ７Ａ重鎖をコードするｃＤＮＡを保持する発現プラスミドベクター）およびプラスミドｐＭＥ−Ｌ（ＨＦＥ７Ａ軽鎖をコードするｃＤＮＡを保持する発現プラスミドベクター）と命名した。これらのプラスミドを保持する形質転換大腸菌株Ｅ．ｃｏｌｉ　ｐＭＥ−ＨおよびＥ．ｃｏｌｉ　ｐＭＥ−Ｌは、平成９（１９９７）年３月１２日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、それぞれ寄託番号ＦＥＲＭ　ＢＰ−５８６８およびＦＥＲＭ　ＢＰ−５８６７が付された。
【０１８３】
（５−２）　ＣＯＳ−７細胞での発現
上記（５−１）で得られたプラスミドｐＭＥ−ＨおよびｐＭＥ−ＬのＣＯＳ−７細胞への形質転換は、遺伝子導入装置（ＥＣＭ６００：ビーティーエックス社製）を用いて、電気穿孔法により行なった。まず、ＣＯＳ−７細胞（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　Ｎｏ．　ＣＲＬ−１６５１　）を１０％　ＦＣＳを含むＤＭＥＭを入れた細胞培養用フラスコ（培養面積２２５ｃｍ^２；住友ベークライト（株）社製）中でセミコンフルエントになるまで培養した後、培地を除去してから、３ｍｌのトリプシン−ＥＤＴＡ溶液（シグマ社製）を入れて３７℃で３分間保温した。遊離した細胞を回収し、ＰＢＳで２回洗浄してから、ＰＢＳで５×１０^６細胞／ｍｌとなるように調整した。また、プラスミド大量調製キット（マキシプレップ・ＤＮＡ精製システム：プロメガ社製）を用いて調製したプラスミドｐＭＥ−ＨおよびｐＭＥ−Ｌ各２０μｇをエタノール沈澱した後、それぞれ滅菌したＰＢＳ２０μｌに溶解した。なお、２種類のプラスミドでコ・トランスフェクションを行なった場合は、各プラスミドは２０μｇずつ使用した。上記のように調製した細胞懸濁液　２０μｌ（１．２×１０^６細胞）とプラスミド溶液２０μｌとを混合し、電極間隔２ｍｍのチャンバー（ビーティーエックス社製）に入れ、遺伝子導入装置にセットして、１５０Ｖ、９００μＦの設定で１０ミリ秒のパルスを１回加えた。チャンバー内の細胞−ＤＮＡ混合液を１０％　ＦＣＳを含むＤＭＥＭ　４０ｍｌに加え、細胞培養用プラスチックシャーレ中で３７℃、５％炭酸ガス下で２４時間培養した。次いで培養上清液を除去し、無血清ＤＭＥＭ培地で細胞を洗浄した後、無血清ＤＭＥＭ　４０ｍｌを加えて３７℃、５％炭酸ガス下でさらに７２時間培養し、培養上清を回収した。
【０１８４】
上記の方法により、以下に記載するプラスミドまたはプラスミドの組み合わせでそれぞれＣＯＳ−７細胞を形質転換して、形質転換体の培養上清を回収した：
［Ａ］　ｐＭＥ−Ｈのみ；
［Ｂ］　ｐＭＥ−Ｌのみ；
［Ｃ］　ｐＭＥ−ＨおよびｐＭＥ−Ｌのコ・トランスフェクション。
【０１８５】
（５−３）　形質転換体培養上清中の抗Ｆａｓ抗体の検出
上記（５−２）で調製された形質転換ＣＯＳ−７細胞培養上清中に抗Ｆａｓ抗体が産生されているか否かを、実施例３に記載したヒトＦａｓ融合タンパク質を抗原とするＥＬＩＳＡ法により調べた。その結果、ｐＭＥ−ＨおよびｐＭＥ−Ｌでコ・トランスフェクションした場合（上記［Ｃ］）のみ、培養液上清にヒトＦａｓ融合タンパク質と反応する抗体が産生されていることが確かめられた。
【０１８６】
実施例６　エピトープ限定
（６−１）　ＥＬＩＳＡ
以下に記載するペプチドを、自動ペプチド合成機（モデル４３０Ａ；（株）パーキンエルマージャパン・アプライドバイオシステムズ事業部）を使用して、Ｆｍｏｃ固相合成法［Ｃａｒｐｉｎｏ，　Ｌ．　Ａ．　ａｎｄ　Ｈａｎ，　Ｇ．Ｙ．　（１９７０）　Ｊ．　Ａｍｅｒ．　Ｃｈｅｍ．　Ｓｏｃ．　９２，　５７４８−５７４９　参照］により合成した：
Ａｒｇ−Ｌｅｕ−Ｓｅｒ−Ｓｅｒ−Ｌｙｓ−Ｓｅｒ−Ｖａｌ−Ａｓｎ−Ａｌａ−Ｇｌｎ−Ｖａｌ−Ｔｈｒ−Ａｓｐ−Ｉｌｅ−Ａｓｎ−Ｓｅｒ−Ｌｙｓ−Ｇｌｙ　−Ｌｅｕ　（Ｐ１：配列表の配列番号２６）；
Ｖａｌ−Ｔｈｒ−Ａｓｐ−Ｉｌｅ−Ａｓｎ−Ｓｅｒ−Ｌｙｓ−Ｇｌｙ−Ｌｅｕ−Ｇｌｕ−Ｌｅｕ−Ａｒｇ−Ｌｙｓ−Ｔｈｒ−Ｖａｌ−Ｔｈｒ−Ｔｈｒ−Ｖａｌ　−Ｇｌｕ　（Ｐ２：配列表の配列番号２７）；
Ｇｌｕ−Ｌｅｕ−Ａｒｇ−Ｌｙｓ−Ｔｈｒ−Ｖａｌ−Ｔｈｒ−Ｔｈｒ−Ｖａｌ−Ｇｌｕ−Ｔｈｒ−Ｇｌｎ−Ａｓｎ−Ｌｅｕ−Ｇｌｕ−Ｇｌｙ−Ｌｅｕ−Ｈｉｓ　−Ｈｉｓ−Ａｓｐ　（Ｐ３：配列表の配列番号２８）；
Ｔｈｒ−Ｇｌｎ−Ａｓｎ−Ｌｅｕ−Ｇｌｕ−Ｇｌｙ−Ｌｅｕ−Ｈｉｓ−Ｈｉｓ−Ａｓｐ−Ｇｌｙ−Ｇｌｎ−Ｐｈｅ−Ｃｙｓ−Ｈｉｓ−Ｌｙｓ−Ｐｒｏ−Ｃｙｓ　−Ｐｒｏ−Ｐｒｏ　（Ｐ４：配列表の配列番号２９）；
Ｇｌｙ−Ｇｌｎ−Ｐｈｅ−Ｃｙｓ−Ｈｉｓ−Ｌｙｓ−Ｐｒｏ−Ｃｙｓ−Ｐｒｏ−Ｐｒｏ−Ｇｌｙ−Ｇｌｕ−Ａｒｇ−Ｌｙｓ−Ａｌａ−Ａｒｇ−Ａｓｐ−Ｃｙｓ　−Ｔｈｒ−Ｖａｌ　（Ｐ５：配列表の配列番号３０）；
Ｇｌｙ−Ｇｌｕ−Ａｒｇ−Ｌｙｓ−Ａｌａ−Ａｒｇ−Ａｓｐ−Ｃｙｓ−Ｔｈｒ−Ｖａｌ−Ａｓｎ−Ｇｌｙ−Ａｓｐ−Ｇｌｕ−Ｐｒｏ−Ａｓｐ−Ｃｙｓ−Ｖａｌ　−Ｐｒｏ−Ｃｙｓ−Ｇｌｎ　（Ｐ６：配列表の配列番号３１）；
Ａｓｎ−Ｇｌｙ−Ａｓｐ−Ｇｌｕ−Ｐｒｏ−Ａｓｐ−Ｃｙｓ−Ｖａｌ−Ｐｒｏ−Ｃｙｓ−Ｇｌｎ−Ｇｌｕ−Ｇｌｙ−Ｌｙｓ−Ｇｌｕ−Ｔｙｒ−Ｔｈｒ−Ａｓｐ　−Ｌｙｓ−Ａｌａ　（Ｐ７：配列表の配列番号３２）；
Ｇｌｕ−Ｇｌｙ−Ｌｙｓ−Ｇｌｕ−Ｔｙｒ−Ｔｈｒ−Ａｓｐ−Ｌｙｓ−Ａｌａ−Ｈｉｓ−Ｐｈｅ−Ｓｅｒ−Ｓｅｒ−Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ａｒｇ−Ｃｙｓ　−Ａｒｇ　（Ｐ８：配列表の配列番号３３）；
Ｈｉｓ−Ｐｈｅ−Ｓｅｒ−Ｓｅｒ−Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ａｒｇ−Ｃｙｓ−Ａｒｇ−Ｌｅｕ−Ｃｙｓ−Ａｓｐ−Ｇｌｕ−Ｇｌｙ−Ｈｉｓ−Ｇｌｙ−Ｌｅｕ　−Ｇｌｕ−Ｖａｌ　（Ｐ９：配列表の配列番号３４）；
Ｌｅｕ−Ｃｙｓ−Ａｓｐ−Ｇｌｕ−Ｇｌｙ−Ｈｉｓ−Ｇｌｙ−Ｌｅｕ−Ｇｌｕ−Ｖａｌ−Ｇｌｕ−Ｉｌｅ−Ａｓｎ−Ｃｙｓ−Ｔｈｒ−Ａｒｇ−Ｔｈｒ−Ｇｌｎ　−Ａｓｎ−Ｔｈｒ　（Ｐ１０：配列表の配列番号３５）；
Ｇｌｕ−Ｉｌｅ−Ａｓｎ−Ｃｙｓ−Ｔｈｒ−Ａｒｇ−Ｔｈｒ−Ｇｌｎ−Ａｓｎ−Ｔｈｒ−Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ｃｙｓ−Ｌｙｓ−Ｐｒｏ−Ａｓｎ−Ｐｈｅ　−Ｐｈｅ−Ｃｙｓ　（Ｐ１１：配列表の配列番号３６）；
Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ｃｙｓ−Ｌｙｓ−Ｐｒｏ−Ａｓｎ−Ｐｈｅ−Ｐｈｅ−Ｃｙｓ−Ａｓｎ−Ｓｅｒ−Ｔｈｒ−Ｖａｌ−Ｃｙｓ−Ｇｌｕ−Ｈｉｓ−Ｃｙｓ　−Ａｓｐ−Ｐｒｏ　（Ｐ１２：配列表の配列番号３７）；
Ａｓｎ−Ｓｅｒ−Ｔｈｒ−Ｖａｌ−Ｃｙｓ−Ｇｌｕ−Ｈｉｓ−Ｃｙｓ−Ａｓｐ−Ｐｒｏ−Ｃｙｓ−Ｔｈｒ−Ｌｙｓ−Ｃｙｓ−Ｇｌｕ−Ｈｉｓ−Ｇｌｙ−Ｉｌｅ　−Ｉｌｅ−Ｌｙｓ　（Ｐ１３：配列表の配列番号３８）；
Ｃｙｓ−Ｔｈｒ−Ｌｙｓ−Ｃｙｓ−Ｇｌｕ−Ｈｉｓ−Ｇｌｙ−Ｉｌｅ−Ｉｌｅ−Ｌｙｓ−Ｇｌｕ−Ｃｙｓ−Ｔｈｒ−Ｌｅｕ−Ｔｈｒ−Ｓｅｒ−Ａｓｎ−Ｔｈｒ　−Ｌｙｓ−Ｃｙｓ　（Ｐ１４：配列表の配列番号３９）；
Ｇｌｕ−Ｃｙｓ−Ｔｈｒ−Ｌｅｕ−Ｔｈｒ−Ｓｅｒ−Ａｓｎ−Ｔｈｒ−Ｌｙｓ−Ｃｙｓ−Ｌｙｓ−Ｇｌｕ−Ｇｌｕ−Ｇｌｙ−Ｓｅｒ−Ａｒｇ−Ｓｅｒ−Ａｓｎ　　（Ｐ１５：配列表の配列番号４０）；
Ｓｅｒ−Ｓｅｒ−Ｇｌｙ−Ｌｙｓ−Ｔｙｒ−Ｇｌｕ−Ｇｌｙ−Ｇｌｙ−Ａｓｎ−Ｉｌｅ−Ｔｙｒ−Ｔｈｒ−Ｌｙｓ−Ｌｙｓ−Ｇｌｕ−Ａｌａ−Ｐｈｅ−Ａｓｎ　−Ｖａｌ−Ｇｌｕ　（Ｐ１６：配列表の配列番号４１）。
【０１８７】
Ｐ１乃至Ｐ１５は、ヒトＦａｓ細胞外領域の１−１５７番目までのアミノ酸配列の部分配列であり、それぞれ９乃至１１残基のアミノ酸配列が他のペプチドと重複している。Ｐ１６は陰性対照用のペプチドであり、ヒトＦａｓとの間に相同性はない。
【０１８８】
Ｐ１乃至Ｐ１６は、それぞれ４８μｌのジメチルスルホキシド（以下「ＤＭＳＯ」という）で完全に溶解した後、１ｍＭ　２−メルカプトエタノールを含むＰＢＳを加えて全量が０．８ｍｌとなるように調整し、ペプチド溶液とした。
【０１８９】
Ｃ末端にカルボキシル基を付加したヒトＦａｓ分子細胞外領域に対応するペプチドを、１０ｍＭ　２−メルカプトエタノールを含む０．０５Ｍ　炭酸−重炭酸緩衝液（ｐＨ９．６）で５０μｇ／ｍｌの濃度に希釈し、ＥＬＩＳＡ用９６穴プレート（ヌンク社製）に５０μｌ／ウェルずつ入れた。このプレートを４℃で一晩静置することにより、ウェル内面にペプチドを吸着させた。各ウェル内の液を捨て、ウェルをＰＢＳ−Ｔｗｅｅｎで４回洗浄した。１％（ｗ／ｖ）ウシ血清アルブミン（シグマ社製Ａ３８０３）を含むＰＢＳを１００μｌ／ウェルずつ分注し、３７℃で１時間保温した。各ウェルをＰＢＳ−Ｔｗｅｅｎで４回洗浄後、ＰＢＳで５μｇ／ｍｌに調製したＨＦＥ７Ａ、およびＣＨ１１を各ウェルに５０μｌ添加した。３７℃で１時間保温した後、各ウェルをＰＢＳ−Ｔｗｅｅｎで４回洗浄した。西洋ワサビペルオキシダーゼ標識ヤギ抗マウスイムノグロブリン抗体（アマシャム社製）をＰＢＳで１０００倍希釈したものを、５０μｌ／ウェルずつ分注し、３７℃で１時間保温した後、各ウェルをＰＢＳ−Ｔｗｅｅｎで４回洗浄した。西洋ワサビペルオキシダーゼ基質（バイオラッド社製）を各ウェルに１００μｌ／ウェルずつ分注し、室温で１５分間静置した後、マイクロプレートリーダー（コロナ社製）で各ウェルの４１５ｎｍの吸光度を測定した。なお、陽性対照としては、実施例１で調製したヒトＦａｓ融合タンパク質を合成ペプチドの代わりに用いた。
【０１９０】
その結果、上記合成ペプチドのうちＰ１１を吸着させたウェルのみが高い吸光度を示したことから、ＨＦＥ７ＡはＰ１１に含まれるアミノ酸配列と特異的に結合することが判明した（図３）。
【０１９１】
（６−２）　合成ペプチドと細胞表面のヒトＦａｓとの競合によるエピトープの限定（競合アッセイ法）
まず、以下に記載するペプチドを合成した：
Ｈｉｓ−Ｇｌｙ−Ｌｅｕ−Ｇｌｕ−Ｖａｌ−Ｇｌｕ−Ｉｌｅ−Ａｓｎ−Ｃｙｓ−Ｔｈｒ　　（Ｐ９５：配列表の配列番号４２）；
Ｇｌｕ−Ｉｌｅ−Ａｓｎ−Ｃｙｓ−Ｔｈｒ−Ａｒｇ−Ｔｈｒ−Ｇｌｎ−Ａｓｎ−Ｔｈｒ　　（Ｐ１００：配列表の配列番号４３）；
Ａｒｇ−Ｔｈｒ−Ｇｌｎ−Ａｓｎ−Ｔｈｒ−Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ｃｙｓ−Ｌｙｓ　　（Ｐ１０５：配列表の配列番号１）；
Ｌｙｓ−Ｃｙｓ−Ａｒｇ−Ｃｙｓ−Ｌｙｓ−Ｐｒｏ−Ａｓｎ−Ｐｈｅ−Ｐｈｅ−Ｃｙｓ　　（Ｐ１１０：配列表の配列番号４４）；
Ｐｒｏ−Ａｓｎ−Ｐｈｅ−Ｐｈｅ−Ｃｙｓ−Ａｓｎ−Ｓｅｒ−Ｔｈｒ−Ｖａｌ−Ｃｙｓ−Ｇｌｕ−Ｈｉｓ−Ｃｙｓ−Ａｓｐ　　（Ｐ１１５Ｌ：配列表の配列番号４５）；
Ｇｌｙ−Ｌｙｓ−Ｉｌｅ−Ａｌａ−Ｓｅｒ−Ｃｙｓ−Ｌｅｕ−Ａｓｎ−Ａｓｐ−Ａｓｎ　　（Ｄ３５５−３６４：配列表の配列番号４６）。
【０１９２】
Ｐ９５、Ｐ１００、Ｐ１０５およびＰ１１０は、ヒトＦａｓの細胞外領域中においてＰ１１に対応するアミノ酸配列の近傍（ヒトＦａｓ細胞外領域の９５−１２８番目に相当）の、１０残基からなる部分配列であり、それぞれ５残基ずつ他のペプチドとアミノ酸配列が重複する。Ｐ１１５Ｌは、Ｐ１１０と５残基重複する１０残基のアミノ酸配列：
Ｐｒｏ−Ａｓｎ−Ｐｈｅ−Ｐｈｅ−Ｃｙｓ−Ａｓｎ−Ｓｅｒ−Ｔｈｒ−Ｖａｌ−Ｃｙｓ　　（Ｐ１１５：配列表の配列番号４５のアミノ酸番号１〜１０）
の溶解度の低さを予測して、Ｃ末端側４残基分を伸長したものである。Ｄ３５５−３６４はヒトＦａｓと相同性のないペプチドで、陰性対照として使用した。Ｐ１１５Ｌ以外の上記各ペプチドはそれぞれ１６μｌのＤＭＳＯに溶解後、１ｍＭ２−メルカプトエタノールを含むＰＢＳを加えて全量が０．８ｍｌとなるように調製し、ペプチド溶液とした。Ｐ１１５Ｌは４８μｌのＤＭＳＯに溶解後、１ｍＭ　２−メルカプトエタノールを含むＰＢＳを加えて全量が０．８ｍｌとなるように調製した。
【０１９３】
ＨＦＥ７Ａ　０．２５μｇと、上記ペプチド溶液（ペプチド２００μｇ相当）のそれぞれとをマイクロ試験管中で混合し、１ｍＭ　２−メルカプトエタノールを含むＰＢＳで全量１００μｌとした。これを３７℃、１０−２０ｒｐｍで撹拌しながら２時間保温した後、ＦＣＳを最終濃度が５％となるように添加し、ペプチド−抗体混合液とした。
【０１９４】
ＷＲ１９Ｌ１２ａ細胞を、実施例２に記載した方法で増殖させてから遠心して回収し、無血清ＲＰＭＩ培地を用いて細胞密度が１×１０^７細胞／ｍｌとなるように調整した。この細胞浮遊液をＵ字底９６穴プレートに１００μｌ／ウェルずつ分注し、マイクロプレート用スウィングローターを使用して、４℃、１０００ｒｐｍ、３分間の遠心分離を行い、上清を除去した。ペレットに各ペプチド−抗体混合液を１００μｌ添加し、１〜２回ピペッティングした。、４℃で３０分間静置した後、遠心し上清を除去した。ペレットをフローサイトメトリー用緩衝液で３回洗浄してから、フローサイトメトリー用緩衝液で２５０倍に希釈したＦＩＴＣ標識ヤギ抗マウスＩｇＧ抗体（カッペル社製）を５０μｌ／ウェルずつ加え、１〜２回ピペッティングした。プレートを遮光して４℃で３０分間静置した後、遠心し上清を除去した。ペレットをフローサイトメトリー用緩衝液で３回洗浄し、組織固定用１０％中性緩衝ホルマリン液（和光純薬工業（株）社製）をＰＢＳで１０倍希釈したものを５０μｌ／ウェルずつ加え、１〜２回ピペッティングしてからプレートを遮光して４℃で１２時間以上静置して細胞を固定した。フローサイトメトリー用緩衝液　１００μｌ／ウェルを加えて懸濁後、遠心し上清を除去した。ペレットをフローサイトメトリー用緩衝液で３回洗浄した後、５００μｌ／ウェルのフローサイトメトリー用緩衝液に懸濁したものをフローサイトメーター（サイトエース−１５０；日本分光（株）社製）で解析し（励起波長：４８８ｎｍ、検出波長：５３０ｎｍ）、細胞１個あたりのＦＩＴＣ蛍光強度の平均値を算出した。さらに、ペプチド−抗体混合液を加えなかった場合の値を０％、Ｄ３５５−３６４の値を１００％として各試料の蛍光強度平均値を算出した。
【０１９５】
その結果、Ｐ１０５はＨＦＥ７ＡとＷＲ１９Ｌ１２ａ細胞との結合を強力に阻害し、Ｐ１０５のアミノ酸配列とそれぞれ半分づつ重複するＰ１００およびＰ１１０は、ＨＦＥ７ＡとＷＲ１９Ｌ１２ａ細胞との結合をそれぞれ５０％および６０％程度阻害することが判明した。Ｐ１０５のアミノ酸配列と全く重複しないＰ９５およびＰ１１５Ｌでは結合阻害はみられなかった（図４）。以上の結果から、Ｐ１０５はＨＦＥ７ＡとヒトＦａｓの結合を阻害するのに十分なアミノ酸配列であり、ＨＦＥ７Ａのエピトープは、Ｐ１０５に相当するアミノ酸配列中に含まれることが示された。なお、Ｐ１０５に相当する部分は、ヒトＦａｓとマウスＦａｓとの間でアミノ酸配列が保存されている領域である。
【０１９６】
実施例７　ＨＦＥ７ＡのサルＦａｓに対する結合能
チンパンジー（三和化学研究所熊本霊長類パーク）の末梢血４０ｍｌ、ニホンザルの末梢血２０ｍｌ、カニクイザルの末梢血２０ｍｌまたはマーモセットの末梢血３ｍｌを用いて、以下に記載する試験を行った。まず、ヘパリン（ノボヘパリン：ノボ社製）を加えた末梢血を等量のフィコール・パック溶液（比重１．０７７、ただしカニクイザル用は比重１．０７２となるように調整した：ファルマシア社製）に静かに積層し、１７００ｒｐｍで３０分間遠心して、末梢血単核球画分を回収した。この末梢血単核球画分をハンクス平衡塩溶液で２回洗浄し、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で１×１０^６細胞／ｍｌとなるように懸濁した。この懸濁液にフィトヘマグルチニン−Ｐ（シグマ社製）を終濃度　５μｇ／ｍｌとなるように加えて、３７℃、５％炭酸ガス下で２４時間培養した後、遠心して細胞を回収・洗浄してから、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で再懸濁した。この懸濁液にインターロイキン２（アマシャム社製）を終濃度　１０単位／ｍｌとなるように加え、３７℃、５％炭酸ガス下で７２時間培養することにより、活性化リンパ球を得た。
【０１９７】
次に、この活性化リンパ球　１×１０^６個を試験管に入れ、２０μｇ／ｍｌのＨＦＥ７Ａ５０μｌもしくはＰＢＳ　５０μｌに懸濁し、氷上で１時間静置してから、５００μｌのＰＢＳで３回洗浄した。この細胞を２０μｇ／ｍｌのＦＩＴＣ標識抗マウスＩｇＧ抗体（バイオリソース社製）５０μｌに懸濁し、氷上で３０分間静置した後、５００μｌのＰＢＳで３回洗浄した。この細胞を５００μｌのＰＢＳで再懸濁したものを試料として、フローサイトメーター（サイトエース：日本分光　（株）　社製）を用いて蛍光強度を測定した。蛍光強度による細胞数の度数分布を取得し、全細胞数中に占める染色細胞数の割合を計算した。その結果、ＨＦＥ７Ａを加えなかった試料では、上記のいずれの種においても染色細胞が３％未満であったのに対し、ＨＦＥ７Ａを加えた試料では、少なくとも１７％、最大で８２％の細胞が染色されていた。従って、ＨＦＥ７Ａはヒトを含む広い範囲の霊長類Ｆａｓと結合することが示された。
【０１９８】
実施例８　マウス生体内のＴ細胞に対するＨＦＥ７Ａのアポトーシス誘導活性
６週令の雌のＣ３Ｈ／ＨｅＪマウス（日本クレア社より購入）に対して、０．０５ｍｇまたは０．１ｍｇ／個体のＨＦＥ７Ａモノクローナル抗体（いずれも５００μｌのＰＢＳに溶解したもの）、あるいは５００μｌのＰＢＳのみを腹腔内投与し（一群あたり３匹）、投与４２時間後にマウスをエーテル麻酔し、胸腺を摘出した。この摘出した胸腺を１０％　ＦＣＳを含むＲＰＭＩ培地で洗浄した後に、メッシュ（セルストレイナー；ファルコン社製）上でスパーテルを用いてつぶし、メッシュを通過した細胞を１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で２回洗浄した。細胞数を測定した後、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で１×１０^６細胞／５０μｌとなるように懸濁した胸腺細胞を、Ｕ字底９６穴マイクロプレート（ヌンク社製）に５０μｌ／ウェルずつ分注し、遠心分離（９０×ｇ、４℃、１０分）した。
【０１９９】
上清を除去し、各ウェルに以下の（ａ）または（ｂ）の蛍光標識抗体溶液を加えた：
（ａ）０．５ｍｇ／ｍｌのＦＩＴＣ標識抗マウスＣＤ９５（Ｆａｓ）抗体（Ｊｏ２：ファーミンジェン社製）１０μｌ／ウェルおよび０．５ｍｇ／ｍｌのフィコエリトリン（以下「ＰＥ」という）標識抗マウスＣＤ９０抗体（Ｔｈｙ−１．２：セダレーン社製）１０μｌ／ウェル（なお、ＣＤ９０はＴ細胞に特異的に発現する表面抗原である）；
（ｂ）０．５ｍｇ／ｍｌのＦＩＴＣ標識抗マウスＣＤ４抗体（Ｌ３Ｔ４：ファーミンジェン社製）１０μｌ／ウェルおよび０．２ｍｇ／ｍｌのＰＥ標識抗マウスＣＤ８抗体（Ｌｙ−２：ファーミンジェン社製）１０μｌ／ウェル。
【０２００】
プレートを振盪してウェル内を撹拌し、氷上で１時間静置した後、遠心分離（９０×ｇ、４℃、１０分）した。上清を除去し、フローサイトメトリー用緩衝液１００μｌ／ウェルで２回洗浄した後、３．７％　ホルマリン溶液　５０μｌ／ウェルを加え、氷上で１０分静置することにより細胞を固定した。遠心分離（９０×ｇ、４℃、１０分）して上清を除去し、沈澱した細胞を再度フローサイトメトリー用緩衝液　１００μｌ／ウェルで洗浄後、フローサイトメトリー用緩衝液　１００μｌ／ウェルに懸濁した。このようにして得られた各ウェル中の細胞懸濁液を試料とし、フローサイトメーター（エピックス・エリート：コールター社製）を用いて、１×１０^４細胞／試料の蛍光を、以下に記載する条件で測定した：
励起波長：　４８８ｎｍ；
検出波長：　５３０ｎｍ（ＦＩＴＣ）、６００ｎｍ（ＰＥ）。
【０２０１】
この測定データから、各試料の細胞集団のＦＩＴＣおよびＰＥの蛍光分布を作成し、上記（ａ）の蛍光標識抗体を加えた試料については、Ｆａｓ陽性でかつＣＤ９０陽性の細胞（以下「Ｆａｓ^＋ＣＤ９０^＋」という）数の全細胞数中に占める割合を算出した。同様に、上記（ｂ）の蛍光標識抗体を加えた試料については、ＣＤ４陽性ＣＤ８陽性細胞（以下「ＣＤ４^＋ＣＤ８^＋」という）またはＣＤ４陽性ＣＤ８陰性細胞（以下「ＣＤ４^＋ＣＤ８⁻」という）の数の全細胞数中に占める割合を算出した。
【０２０２】
その結果を表１に示す。
【０２０３】
【表１】

（数値は％）
ＨＦＥ７Ａ投与群マウスの胸腺細胞では、ＰＢＳのみ投与した群と比較して、Ｆａｓを発現しているＴ細胞（Ｆａｓ^＋ＣＤ９０^＋）の割合が、どの投与量の場合でも顕著に減少していた。また、Ｆａｓの発現が顕著であることが知られているＣＤ４^＋ＣＤ８^＋細胞群およびＣＤ４^＋ＣＤ８⁻細胞群も、ＰＢＳのみ投与した群と比較してＨＦＥ７Ａ投与により顕著にその数が減少していた。
【０２０４】
以上の結果より、抗Ｆａｓモノクローナル抗体ＨＦＥ７Ａは、生体内でＦａｓ分子を発現しているＴ細胞に対するアポトーシス誘導活性を有することが示された。
【０２０５】
実施例９　自己免疫疾患モデルに対するＨＦＥ７Ａの効果
Ｆａｓリガンド遺伝子の突然変異体であり、全身性エリテマトーデス様自己免疫疾患のモデルマウスであるＭＲＬｇｌｄ　／ｇｌｄ　マウスを用いて、抗Ｆａｓモノクローナル抗体ＨＦＥ７Ａ投与の自己免疫疾患症状に対する効果を以下に記載する方法に従って検討した。
【０２０６】
１８週令のＭＲＬｇｌｄ　／ｇｌｄ　マウス（日本エスエルシー（株）社より購入）の腹腔内に、実施例３で調製したＨＦＥ７Ａ　０．２ｍｇまたは０．５ｍｇ／個体（５００μｌのＰＢＳに溶解したもの）、もしくは５００μｌ　ＰＢＳをそれぞれ単回投与した。
【０２０７】
自己免疫疾患症状により生じる各被検マウスの手首および足首の腫れを観察し、腫れの程度を評価して、各群の経時的な変化を調べた［米原伸　（１９９４）　日経サイエンス　別冊１１０　、６６−７７　参照］。その結果、ＨＦＥ７Ａ投与により、手足首の腫れの程度が著明に減少していることが明らかとなった。
【０２０８】
また、これらの被検マウスから胸腺を摘出し、上記試験例１に記載した方法により胸腺でＦａｓを発現しているＴ細胞の割合を調べた結果、試験例１と同様にＨＦＥ７Ａ投与により胸腺中のＦａｓ発現Ｔ細胞数は著しく減少していた。
【０２０９】
実施例１０　肝毒性試験
ＢＡＬＢ／ｃマウスに以下のいずれかのものを腹腔内投与した：
ｉ）　ＨＦＥ７Ａ　０．２ｍｇ（５００μｌのＰＢＳに溶解）；
ｉｉ）　ＨＦＥ７Ａ　０．５ｍｇ（５００μｌのＰＢＳに溶解）；
ｉｉｉ）　Ｊｏ２（ファーミンジェン社製）０．１ｍｇ（５００μｌのＰＢＳに溶解）；
ｉｖ）　５００μｌのＰＢＳのみ。
【０２１０】
上記のうち、Ｊｏ２はアポトーシス誘導活性を有する公知の抗マウスＦａｓ抗体である。投与８時間後、２４時間後および７２時間後のマウス（Ｊｏ２投与マウスのみ投与３時間後のマウス）から軽エーテル麻酔下で後大静脈より全採血し、各試料血液中のグルタミン酸−オキサロ酢酸トランスアミナーゼ（以下「ＧＯＴ」という）値およびグルタミン酸−ピルビン酸トランスアミナーゼ（以下「ＧＰＴ」という）値を、自動分析装置（７２５０型；日立製作所（株）社製）および同装置用試薬（トランスアミナーゼ−ＨＲＩＩ；和光純薬（株）社製）を用いて測定した。その結果、Ｊｏ２投与群では３時間後にＧＯＴ値およびＧＰＴ値が急上昇したのに対し、ＨＦＥ７Ａを投与した群のＧＯＴ値およびＧＰＴ値は、ＰＢＳのみ投与した群同様ほとんど変化しなかった（図５）。以上の結果より、ＨＦＥ７Ａは肝臓障害を誘発しないことが示された。
【０２１１】
実施例１１　劇症肝炎モデルに対する効果
抗マウスＦａｓ抗体Ｊｏ２をマウス腹腔内に投与すると、マウスは数時間の内に劇症肝炎を発症して死亡することが知られている［Ｏｇａｓａｗａｒａ，　Ｊ．，　ｅｔ　ａｌ．　（１９９３）　Ｎａｔｕｒｅ　３６４，　８０６参照］。そこで、このＪｏ２による肝障害に対するＨＦＥ７Ａ効果を検討するため、Ｊｏ２と同時もしくは後からＨＦＥ７Ａを投与した場合のマウスの生存率を調べた。６週齢のＢＡＬＢ／ｃマウス雌（一群３匹：日本エスエルシー（株）より購入）の腹腔内に、下記の条件で抗体を投与し、経時観察を行なった：
（Ａ）　Ｊｏ２　０．１ｍｇ／０．５ｍｌ；
（Ｂ）　Ｊｏ２　０．０１ｍｇ／０．５ｍｌ；
（Ｃ）　Ｊｏ２　０．１ｍｇとＨＦＥ７Ａ　０．５ｍｇ／０．５ｍｌ（同時投与）；
（Ｄ）　Ｊｏ２　０．１ｍｇとＨＦＥ７Ａ　０．０５ｍｇ／０．５ｍｌ（同時投与）；
（Ｅ）　Ｊｏ２　０．０１ｍｇ／０．２ｍｌ投与後、
２０分後にＨＦＥ７Ａ　０．１ｍｇ／０．２ｍｌを投与。
【０２１２】
その結果を図６に示した。Ｊｏ２の単独投与では、０．１ｍｇ／個体および０．０１ｍｇ／個体いずれの場合（（Ａ）および（Ｂ））も９時間以内にすべてのマウスが死亡した。しかしながら、Ｊｏ２投与時にＨＦＥ７Ａを同時投与（０．５ｍｇ／個体、０．０５ｍｇ／個体）した場合（（Ｃ）および（Ｄ））、マウスは投与数週間経過後も異常を示さなかったことから、ＨＦＥ７Ａ投与により劇症肝炎の発症を阻止できることが判明した。さらに、Ｊｏ２投与２０分後にＨＦＥ７Ａを投与した場合でも、マウスは正常であり外的症状も何ら認められなかったことから、肝臓等でのＦａｓ／Ｆａｓリガンド系を介した正常組織の障害に由来する種々の疾病に対して、ＨＦＥ７Ａが予防および治療効果を有することが示された。
【０２１３】
実施例１２　慢性関節リウマチに対する効果
１）　コラーゲン誘導関節炎に対する発症予防効果
ＢＡＬＢ／ｃマウスの雌とＤＢＡ／１Ｊマウスの雄との交配で得られたＦ１マウスであるＣＤ１Ｆ１マウス（６週齢の雌、日本チャールスリバー（株）より購入）を１週間馴化させた。文献に記載の方法［Ｐｈａｄｋｅ，　Ｋ．　（１９８５）　Ｉｍｍｕｎｏｐｈａｒｍａｃｏｌ．　１０，　５１−６０参照］に準じて、ウシＩＩ型コラーゲン　０．３％溶液（５０ｍＭ　酢酸溶液、コラーゲン技術研修会製）を５０ｍＭ　酢酸により０．２％（２ｍｇ／ｍｌ）に希釈し、等用量の完全フロイントアジュバント（ディフコ社製）を加えて乳化後、ツベルクリン針を装着したプラスチック製１ｍｌ注射筒を用い、静注用保定器に保定したマウスの尾根部皮内に１００μｌ（ウシＩＩ型コラーゲンとして１００μｇ相当）投与した。初回感作の１週間後に上記と同じ条件で追加免疫した。追加免疫時に１００μｇのＨＦＥ７Ａまたは対照用のマウスＩｇＧを腹腔内投与した（各群６匹）。初回感作後５週目から四肢の腫れが認められるようになった。肉眼的に四肢の関節を観察し、文献記載の方法［Ｗｏｏｄ，　Ｆ．　Ｄ．，ｅｔ　ａｌ．　（１９６９）　Ｉｎｔ．　Ａｒｃｈ．　Ａｌｌｅｒｇｙ　Ａｐｐｌ．　Ｉｍｍｕｎｏｌ．　３５，　４５６−４６７参照］に準じてその腫れの程度をスコア化した。スコアは、各四肢について以下の基準で判定した：
０：症状なし；
１：四肢の指などの小関節が一本のみ腫脹発赤；
２：小関節二本以上あるいは手首や足首などの比較的大きな関節が腫脹発赤；
３：一本の手や足全体が腫脹発赤。
【０２１４】
すなわち、四肢とも最大に腫れた場合の１個体のスコアは１２となる。また、四肢の合計スコアが１以上の個体を「発症マウス」とした。
【０２１５】
結果を図７に示した。非特異的なマウスＩｇＧを投与した対照群では初回感作後７週目までに全てのマウスが発症したのに対し、ＨＦＥ７Ａ投与群のうち半数のマウスは８週目までに全く発症しなかった（図７Ａ）。また、ＨＦＥ７Ａ投与群では対照群と比較して平均スコアが低かった（図７Ｂ）。
【０２１６】
２）　リウマチ患者由来滑膜細胞に対するアポトーシス誘導能
慢性関節リウマチ患者由来の滑膜細胞の生存率に与えるＨＦＥ７Ａの影響を、ミトコンドリアの還元能を指標とする以下に記載の方法により検討した。まずヒト慢性関節リウマチ患者患部より採取した滑膜組織を１０％　ウシ胎児血清（サミット社製）を含むダルベッコ改変イーグル培地（ギブコ社製）中でハサミで細切し、脂肪を除去後、終濃度５μｇ／ｍｌとなるようにコラゲナーゼ（シグマ社製）を添加し、９０分間３７℃でインキュベートした。１０％ウシ胎児血清を含むダルベッコ改変イーグル培地で３７℃、５％　炭酸ガスの条件下培養したものを、リウマチ患者由来滑膜細胞とした。
【０２１７】
かくして得られたリウマチ患者由来滑膜細胞をトリプシン処理により単細胞化した後１０％　ウシ胎児血清を含むダルベッコ改変イーグル培地で１×１０^５細胞／ｍｌとなるように懸濁してから、９６穴プレートに２×１０^４細胞／２００μｌ／ウェルとなるように播種し、３７℃、５％　炭酸ガス条件で６日間培養した。培養上清を除去後、ハンクス緩衝液（ギブコ社製）で３回洗浄し、ＨＦＥ７Ａを１０ｎｇ／ｍｌ乃至１０００ｎｇ／ｍｌ（１０倍希釈系列）および１０％ウシ胎児血清を含むダルベッコ改変イーグル培地　２００μｌを添加した。３７℃、５％炭酸ガス条件で２０時間培養後、１ｍｇ／ｍｌ　ＸＴＴ（２，３−ビス［２−メトキシ−４−ニトロ−５−スルホフェニル］−２Ｈ−テトラゾリウム−５−カルボキサニリド　イナーソルト：シグマ社製）、２５μＭ　ＰＭＳ（フェナジンメトサルフェート：シグマ社製）を５０μｌ（終濃度：２５０μｇ／ｍｌ　ＸＴＴ；５μＭ　ＰＭＳ）を添加してさらに４時間培養後、各ウェルの４５０ｎｍの吸光度を測定した。
【０２１８】
各ウェルの細胞の生存率を、下記式により算出した：
生存率（％）＝１００×（ａ−ｂ）／（ｃ−ｂ）
（式中、ａは被検ウェルの測定値、ｂは無細胞ウェルの測定値、ｃは抗体無添加のウェルの測定値をそれぞれ表す。）
その結果を表２に示した。ＨＦＥ７Ａは濃度依存的にリウマチ患者由来滑膜細胞の生存を阻害した。
【０２１９】
【表２】

実施例１３　ＨＦＥ７Ａヒト化抗体の設計
（１）ＨＦＥ７Ａの可変領域の分子モデリング
ＨＦＥ７Ａの可変領域の分子モデリングは、一般にホモロジーモデリングとして知られる手法［Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ　２０３，　１２１−１５３　（１９９１）　参照］を用いて行なった。すなわち、プロテイン・データ・バンク（Ｐｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｎｋ　：Ｃｈｅｍｉｓｔｒｙ　Ｄｅｐａｒｔｍｅｎｔ，　Ｂｕｉｌｄｉｎｇ　５５５，　Ｂｒｒｏｋｈｅａｖｅｎ　Ｎａｔｉｏｎａｌ　Ｌａｂｏｒａｔｏｒｙ，　Ｐ．Ｏ．　Ｂｏｘ　５０００，　Ｕｐｔｏｎ，　ＮＹ　１１９７３−５０００，　Ｕ．Ｓ．Ａ．参照：以下「ＰＤＢ」という）に登録されている、Ｘ線結晶構造解析が行なわれたヒト免疫グロブリンの可変領域の一次配列を、ＨＦＥ７Ａのフレームワーク部分と比較し、相同性の最も高いフレームワークの三次元構造として、軽鎖については１ＧＧＩ、重鎖として２ＨＦＬを選択した。両者を組合せて、フレームワーク部分の三次元構造（以下「フレームワークモデル」という）を構築した。
【０２２０】
チョッチアらの分類に従うと、ＨＦＥ７ＡのＣＤＲのうち、ＣＤＲＬ_２はカノニカルクラス１、ＣＤＲＬ_３はカノニカルクラス１、ＣＤＲＨ_１はカノニカルクラス１に分類された。ＣＤＲＬ_１、ＣＤＲＨ_２、ＣＤＲＨ_３は、特定のカノニカルクラスに対応しなかった。ＣＤＲＬ_２、ＣＤＲＬ_３およびＣＤＲＨ_１のＣＤＲループは、各カノニカルクラス固有のコンホメーションに固定し、フレームワークモデルに組み込んだ。ＣＤＲＬ_１はソロントンらの分類［Ｊ．　Ｍｏｌ．　Ｂｉｏｌ．　２６３，　８００−８１５　（１９９６）　参照］に従いクラスター１５Ｂのコンホメーションを用いた。ＣＤＲＨ_２およびＣＤＲＨ_３については、相同性の高い配列のコンホメーションをＰＤＢより検索し、エネルギー計算を併用することによって、最も確からしいＣＤＲループのコンホメーションを構築し、フレームワークモデルに組み込んだ。最終的には、エネルギー的に不利な原子間の接触をなくすようにエネルギー計算を行ない、ＨＦＥ７Ａの分子モデルを得た。上述の操作は市販の一般的な分子モデリングシステムを用いて行い得るが、本発明では、ＡｂＭ（オックスフォード・モレキュラー・リミテッド社製）を用いた。
【０２２１】
得られた分子モデルについては、ソフトウエアのプロチェック（ＰＲＯＣＨＥＣＫ：Ｊ．Ａｐｐｌ．　Ｃｒｙｓｔ．　（１９９３）　２６，　２８３−２９１参照）を用いて、構造の精度を評価した後、各残基の表面露出度を計算し、原子間接触の有無を検索した。
【０２２２】
（２）アクセプターの選択
ＨＦＥ７Ａの軽鎖、重鎖のサブグループは、ヒト抗体の各サブグループのコンセンサス配列との比較において、軽鎖ではサブグループκＩＶと７９％、重鎖ではサブグループＩと７９％の同一性を示した。しかし、この組合せを持つヒト抗体は存在しなかった。軽鎖、重鎖が同一の抗体に由来し、かつ、いずれも７０％以上の同一性を示すヒト抗体として、軽鎖がサブグループκＩＩＩ、重鎖がサブグループＩである８Ｅ１０’ＣＬを選択した。
【０２２３】
（３）アクセプターに移植するドナー残基の選択
ソフトウエアのカメレオン（オックスフォード・モレキュラー・リミテッド社製）を用いて、ＨＦＥ７Ａの軽鎖、重鎖それぞれとアクセプターのアミノ酸配列を整列し、先に述べたａ）からｄ）の要件に従い、以下の実施例に示すヒト化可変領域配列をデザインし、抗ヒトＦａｓヒト化抗体をコードするＤＮＡのヌクレオチド配列を含む組換えベクターとして、以下のプラスミドを構築した。
【０２２４】
実施例１４　ヒト化軽鎖をコードするＤＮＡの調製
（１）ヒト軽鎖（κ鎖）の全長をコードするｃＤＮＡのクローニング
マウス抗ヒトＦａｓ抗体ＨＦＥ７Ａの軽鎖アミノ酸配列をヒト化するに先立ち、定常領域を含むヒトイムノグロブリン（以下「Ｉｇ」という）軽鎖ｃＤＮＡのクローニングを行なった。
【０２２５】
１）プライマーの合成
ヒト軽鎖をコードするｃＤＮＡの分離は、ＰＣＲにより行なった。ＰＣＲを行なうにあたり、以下に記載する２種のプライマーを合成した：
５’−　ＧＣＧＡＡＴＴＣＴＧ　ＣＣＴＴＧＡＣＴＧＡ　ＴＣＡＧＡＧＴＴＴＣ　ＣＴＣＡ　−３’　（ＨＶＫＩＩ５−４：配列表の配列番号４７）；
５’−　ＧＣＴＣＴＡＧＡＴＧ　ＡＧＧＴＧＡＡＡＧＡ　ＴＧＡＧＣＴＧＧＡＧ　ＧＡ　−３’　（ＨＫＣＬ３−３：配列表の配列番号４８）。
【０２２６】
２）ヒトＩｇ軽鎖ｃＤＮＡを含むプラスミドの構築
ヒトＩｇ軽鎖の全長を含むｃＤＮＡは、ＰＣＲ法により作製され、プラスミド中に挿入された後、大腸菌でクローニングされた。
【０２２７】
ヒトＩｇ軽鎖の全長をコードするＨＬ−ＤＮＡ断片は、以下の条件で作製された。

上記組成の反応液を、再蒸留水にて最終容量１００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２２８】
このようにして作製されたＨＬ−ＤＮＡ断片は、真核生物用ＴＡクローニングキット（インビトロジェン社製）を用い、メーカー指定のプロトコールに従って、プラスミドｐＣＲ３ＤＮＡに挿入し、同キットに予め添付されているコンピテント大腸菌ＴＯＰ１０Ｆ’株に導入した。これにより、ヒトＩｇ軽鎖のｃＤＮＡであるＨＬ−ＤＮＡ断片を保持するプラスミドｐＨＬ１５−２７を得た。
【０２２９】
（２）　ＨＦＥ７Ａのヒト化抗体軽鎖発現ベクターの構築
１）ヒト化ＨＦＥ７Ａ−軽鎖発現ベクタープラスミドの構築
マウス抗ヒトＦａｓ抗体ＨＦＥ７Ａの軽鎖アミノ酸配列をヒト化するにあたり、ＨＦＥ７Ａ軽鎖アミノ酸配列のＮ末端側から４７番目のアミノ酸（プロリン）と４９番目のアミノ酸（リジン）（以下「領域Ｉ」という）をそれぞれヒト軽鎖（κ鎖）で保存されているアラニンおよびアルギニンに置き換えるとともに、８０番目のアミノ酸（ヒスチジン）、８１番目のアミノ酸（プロリン）、８２番目のアミノ酸（バリン）、８４番目のアミノ酸（グルタミン酸）、８５番目のアミノ酸（グルタミン酸）、８７番目のアミノ酸（アラニン）および８９番目のアミノ酸（スレオニン）（以下「領域ＩＩ」という）をそれぞれヒト軽鎖（κ鎖）で保存されているセリン、アルギニン、ロイシン、プロリン、アラニン、フェニルアラニン、バリンに置き換えたものを「ＨＨタイプ」とした。
【０２３０】
また、領域Ｉをヒトのアミノ酸残基に置き換え、領域ＩＩをマウスのアミノ酸残基のままとしたものを「ＨＭタイプ」とした。
【０２３１】
さらに、領域Ｉ、領域ＩＩともマウスのアミノ酸残基のままとしたものを「ＭＭタイプ」とした。
【０２３２】
以下、この３種類のヒト化抗ヒトＦａｓ抗体ＨＦＥ７Ａ−軽鎖アミノ酸配列を有する発現プラスミドをそれぞれ構築した。
【０２３３】
２）ヒト化ＨＦＥ７Ａの軽鎖可変および定常領域作製用プライマーの合成
各ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ−軽鎖の可変領域とヒトＩｇ軽鎖（κ鎖）の定常領域を融合した、ＨＨタイプポリペプチド鎖（配列表の配列番号５０）をコードするＤＮＡ（配列表の配列番号４９）、ＨＭタイプポリペプチド鎖（配列表の配列番号５２）をコードするＤＮＡ（配列表の配列番号５１）およびＭＭタイプポリペプチド鎖（配列表の配列番号５４）をコードするＤＮＡ（配列表の配列番号５３）の合成は、それぞれＰＣＲ法を組み合わせて行なった。
【０２３４】
ＰＣＲを行うにあたり、以下に記載する１３種のオリゴヌクレオチドプライマーを合成した：
５’−　ＣＣＣＡＡＧＣＴＴＡ　ＡＧＡＡＧＣＡＴＣＣ　ＴＣＴＣＡＴＣＴＡＧ　ＴＴＣＴ　−３’（７ＡＬ１Ｐ：配列表の配列番号５５）；
５’−　ＧＡＧＡＧＧＧＴＧＧ　ＣＣＣＴＣＴＣＣＣＣ　ＴＧＧＡＧＡＣＡＧＡ　ＧＡＣＡＡＡＧＴＡＣ　ＣＴＧＧ　−３’（７ＡＬ１Ｎ：配列表の配列番号５６）；
５’−　ＣＣＡＧＧＴＡＣＴＴ　ＴＧＴＣＴＣＴＧＴＣ　ＴＣＣＡＧＧＧＧＡＧ　ＡＧＧＧＣＣＡＣＣＣ　ＴＣＴＣ　−３’（７ＡＬ２Ｐ：配列表の配列番号５７）；
５’−　ＧＡＴＴＣＧＡＧＡＴ　ＴＧＧＡＴＧＣＡＧＣ　ＡＴＡＧＡＴＧＡＧＧ　ＡＧＴＣＴＧＧＧＴＧ　ＣＣＴＧ　−３’（７ＡＬ２Ｎ：配列表の配列番号５８）；
５’−　ＧＣＴＧＣＡＴＣＣＡ　ＡＴＣＴＣＧＡＡＴＣ　ＴＧＧＧＡＴＣＣＣＡ　ＧＡＣＡＧＧＴＴＴＡ　ＧＴＧＧＣ　−３’　（７ＡＬ３ＰＡ：配列表の配列番号５９）；
５’−　ＡＡＡＡＴＣＣＧＣＣ　ＧＧＣＴＣＣＡＧＡＣ　ＧＡＧＡＧＡＴＧＧＴ　ＧＡＧＧＧＴＧＡＡＧ　ＴＣＴＧＴＣＣＣＡＧ　ＡＣ　−３’　（７ＡＬ３Ｎ：配列表の配列番号６０）；
５’−　ＣＴＣＧＴＣＴＧＧＡ　ＧＣＣＧＧＣＧＧＡＴ　ＴＴＴＧＣＡＧＴＣＴ　ＡＴＴＡＣＴＧＴＣＡ　ＧＣＡＡＡＧＴＡＡＴ　ＧＡＧＧＡＴＣＣ　−３’　（７ＡＬ４Ｐ：配列表の配列番号６１）；
５’−　ＴＧＡＡＧＡＣＡＧＡ　ＴＧＧＴＧＣＡＧＣＣ　ＡＣＡＧＴＣＣＧＴＴ　ＴＧＡＴＴＴＣＣＡＧ　ＣＣＴＧＧＴＧＣＣＴ　ＴＧＡＣＣ　−３’（７ＡＬ４Ｎ：配列表の配列番号６２）；
５’−　ＧＧＴＣＡＡＧＧＣＡ　ＣＣＡＧＧＣＴＧＧＡ　ＡＡＴＣＡＡＡＣＧＧ　ＡＣＴＧＴＧＧＣＴＧ　ＣＡＣＣＡＴＣＴＧＴ　ＣＴＴＣＡ　−３’（７ＡＬＣＰ：配列表の配列番号６３）；
５’−　ＣＣＣＧＡＡＴＴＣＴ　ＴＡＣＴＡＡＣＡＣＴ　ＣＴＣＣＣＣＴＧＴＴ　ＧＡＡＧＣＴＣＴＴＴ　ＧＴＧＡＣ　−３’　（７ＡＬＣＮ：配列表の配列番号６４）；
５’−　ＴＣＴＧＴＣＣＣＡＧ　ＡＣＣＣＡＣＴＧＣＣ　ＡＣＴＡＡＡＣＣＴＧ　ＴＣＴＧＧＧＡＴＣＣ　ＣＡＧＡＴＴＣＧＡＧ　ＡＴＴＧＧ　−３’（Ｍ７ＡＬ２Ｎ：配列表の配列番号６５）；
５’−　ＧＴＴＴＡＧＴＧＧＣ　ＡＧＴＧＧＧＴＣＴＧ　ＧＧＡＣＡＧＡＣＴＴ　ＣＡＣＣＴＣＴＡＣＣ　ＡＴＣＣＡＴＣＣＴＧ　ＴＧＧＡＧ　−３’（Ｍ７ＡＬ３ＰＡ：配列表の配列番号６６）；
５’−　ＡＴＧＧＴＧＣＡＧＣ　ＣＡＣＡＧＴＣＣＧＴ　ＴＴＧＡＴＴＴＣＣＡ　ＧＣＣＴＧＧＴＧＣＣ　ＴＴＧＡＣＣＧＡＡＣ　ＧＴＣＣＧ　−３’（７ＡＬ４ＮＡ：配列表の配列番号６７）。
３）プラスミドｐ７ＡＬ−ＨＨの構築（ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖発現プラスミド）
配列表の配列番号５０に示すアミノ酸をコードする配列表の配列番号４９に示すＶＨＨ−ＤＮＡ断片は、三段階のＰＣＲを実施することにより作製され、プラスミドベクター中に挿入された後、大腸菌でクローニングされた。
【０２３５】
ａ）第一段階ＰＣＲ
ＶＨＨ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図８に示した。
【０２３６】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列およびＦＲＬ_１領域の一部をコードするＬ７Ａ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ１Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ１Ｎ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ（ストラタジーン社製）　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２３７】
ＦＲＬ_１の一部、ＣＤＲＬ_１、ＦＲＬ_２およびＣＤＲＬ_２領域の一部をコードするＬ７Ａ２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ２Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ２Ｎ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２３８】
ＣＤＲＬ_２およびＦＲＬ_３の一部をコードするＬ７Ａ３−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ３ＰＡ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ３Ｎ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２３９】
ＦＲＬ_３の一部、ＣＤＲＬ_３、ＦＲＬ_４および定常領域の一部をコードするＬ７Ａ４−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ４Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ４Ｎ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２４０】
ＦＲＬ_４の一部および定常領域をコードし、３’末端にＥｃｏＲＩ制限酵素切断部位を付加したＬ７Ａ５−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＬ１５−２７　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬＣＰ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬＣＮ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２４１】
ＰＣＲ後の各生成物２００μｌに、等量のフェノール・クロロホルム（５０％水飽和フェノール、４８％　クロロホルム、２％　イソアミルアルコール）を加え、１分間、激しく攪拌した。その後、１００００×ｇの遠心分離を行い、水層を回収した。得られた水層に、等量のクロロホルム・イソアミルアルコール（９６％　クロロホルム、４％　イソアミルアルコール）を加え、再び１分間激しく攪拌し、１００００×ｇの遠心分離により、水層を回収した（以後、この一連の操作を「フェノール抽出」という）。
【０２４２】
回収された上清に、１／１０倍量の３Ｍ　酢酸ナトリウム（ｐＨ５．２）と、２．５倍量のエタノールを加え、ドライアイスにより凍結後、１００００×ｇの遠心分離を行ない、ＤＮＡを沈澱として回収した（以後、この一連の操作を「エタノール沈澱」という）。
【０２４３】
得られたＤＮＡ沈澱物は、真空乾燥後、再蒸留水に溶解し、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＬ７Ａ１−ＤＮＡ、Ｌ７Ａ２−ＤＮＡ、Ｌ７Ａ３−ＤＮＡ、Ｌ７Ａ４−ＤＮＡおよびＬ７Ａ５−ＤＮＡに相当するＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２４４】
ｂ）第二段階ＰＣＲ
ＶＨＨ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図９に示した。
【０２４５】
前出Ｌ７Ａ１−ＤＮＡ断片とＬ７Ａ２−ＤＮＡ断片を融合したＬ７Ａ１．２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＬ７Ａ１−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＬ７Ａ２−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー７ＡＬ１Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ２Ｎ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２４６】
前出Ｌ７Ａ４−ＤＮＡ断片とＬ７Ａ５−ＤＮＡ断片を融合したＬ７Ａ４．５−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＬ７Ａ４−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＬ７Ａ５−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー７ＡＬ４Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬＣＮ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２４７】
ＰＣＲ後の増幅されたＬ７Ａ１．２−ＤＮＡおよびＬ７Ａ４．５−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出した各融合ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２４８】
ｃ）第三段階ＰＣＲ
ＶＨＨ−ＤＮＡ作製のための第三段階ＰＣＲの概要を図１０に示した。
【０２４９】
前出Ｌ７Ａ１．２−ＤＮＡ断片とＬ７Ａ４．５−ＤＮＡ断片およびＬ７Ａ３−ＤＮＡを融合したＶＨＨ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第二段階ＰＣＲで作製したＬ７Ａ１．２−ＤＮＡ溶液　　　１０μｌ
第二段階ＰＣＲで作製したＬ７Ａ４．５−ＤＮＡ溶液　　　１０μｌ
第一段階ＰＣＲで作製したＬ７Ａ３−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー７ＡＬ１Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬＣＮ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２５０】
ＰＣＲ後の増幅されたＶＨＨ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＶＨＨ−ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００ｘｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２５１】
ＶＨＨ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要は図１１に示した。
【０２５２】
得られたＶＨＨ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩと制限酵素ＥｃｏＲＩにより消化した。
【０２５３】
クローニング用プラスミドｐＨＳＧ３９９　ＤＮＡ（宝酒造（株）社製）１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、アルカリフォスファターゼ（ウシ腸由来：以下「ＣＩＰ」という）で脱リン酸化した。このようにして得られた脱リン酸化プラスミドｐＨＳＧ３９９　ＤＮＡと、予め制限酵素で消化したＶＨＨ−ＤＮＡ断片をＤＮＡライゲーションキット　バージョン２．０（宝酒造（株）社製）を用い、メーカー指定のプロトコールに従って連結した。
【０２５４】
連結したＤＮＡは、エタノール沈澱で回収し、５μｌの再蒸留水に溶解し、Ｅ．ｃｏｌｉ　ＪＭ１０９エレクトロ−セル（宝酒造（株）社製）と混合した。混合物を、ジーンパルサー／Ｅ．ｃｏｌｉパルサーキュベット　０．１ｃｍ（バイオラッド社製）に移し、メーカー指定のプロトコールに従い、ジーンパルサーＩＩ（バイオラッド社製）にて大腸菌ＪＭ１０９株に導入した（以下、この一連の操作を「形質転換」という）。形質転換操作を終えた菌体は、最終濃度１ｍＭのＩＰＴＧ（イソプロピルチオ−β−Ｄ−ガラクトシド、宝酒造（株）社製）、０．１％のＸ−Ｇａｌ（５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド、宝酒造（株）社製）および５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地［バクトトリプトン（ディフコ社製）　１０ｇ、バクトイーストエキストラクト（ディフコ社製）　５ｇ、塩化ナトリウム　１０ｇ、バクトアガー（ディフコ社製）　１５ｇ。蒸留水に溶解し、１リットルとする］上に塗布し、３７℃で一晩培養し、大腸菌形質転換体を得た。
【０２５５】
得られた白色を呈する形質転換体は、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地［バクトトリプトン（ディフコ社製）　１０ｇ、バクトイーストエキストラクト（ディフコ社製）　５ｇ、塩化ナトリウム　１０ｇ。蒸留水に溶解し、１リットルとする］２ｍｌ中で、３７℃にて一晩培養し、得られた培養物からアルカリ・ＳＤＳ法［Ｓａｍｂｒｏｏｋ，Ｊ．　ｅｔ　ａｌ．，　（１９８９）　ｉｎ　”Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　Ｓｅｃｏｎｄ　ｅｄｉｔｉｏｎ．”　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ．］に従ってプラスミドＤＮＡを抽出した。
【０２５６】
抽出したプラスミドＤＮＡを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化した後、１％　アガロースゲル電気泳動解析により、ＶＨＨ−ＤＮＡ断片を保持するクローンを選択した。
【０２５７】
以上の操作により、ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片を保持するプラスミドｐＨＳＧＨＨ７を得た。なお、プラスミドｐＨＳＧＨＨ７を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐＨＳＧＨＨ７　ＳＡＮＫ　７３４９７は、平成９年８月２２日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６０７３が付された。
【０２５８】
ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖ポリペプチド（配列表の配列番号５０）をコードするＤＮＡ（配列表の配列番号４９）を保持する発現ベクタープラスミドｐ７ＡＬ−ＨＨは、前出プラスミドｐＨＳＧＨＨ７を用いて作製した。
【０２５９】
哺乳動物細胞用発現プラスミドベクターｐＥＥ．１２．１（ロンザ社製）ＤＮＡ　１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＥＥ．１２．１　ＤＮＡ　１００ｎｇと、ＨｉｎｄＩＩＩおよびＥｃｏＲＩで消化したｐＨＳＧＨＨ７　ＤＮＡ断片１０μｇとをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。
【０２６０】
得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法に従ってプラスミドＤＮＡを抽出した。抽出したプラスミドＤＮＡをＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、１％　アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片が、サイトメガロウイルス（以下ＣＭＶという）プロモーター下流に正方向で挿入されたプラスミドｐ７ＡＬ−ＨＨを得た。
【０２６１】
４）プラスミドｐ７ＡＬ−ＨＭの構築（ヒト化ＨＭタイプＨＦＥ７Ａ−軽鎖発現プラスミド）
配列表の配列番号５２に示すアミノ酸をコードする配列表の配列番号５１に示すＶＨＭ−ＤＮＡ断片は、二段階のＰＣＲを実施することにより作製され、プラスミドベクター中に挿入された後、大腸菌でクローニングされた。
【０２６２】
ａ）第一段階ＰＣＲ
ＶＨＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図１２に示した。
【０２６３】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列およびＦＲＬ_１の一部をコードするＬ７Ａ１−ＤＮＡ断片、ＦＲＬ_１の一部、ＣＤＲＬ_１、ＦＲＬ_２およびＣＤＲＬ_２の一部をコードするＬ７Ａ２−ＤＮＡ断片、およびＦＲＬ_４の一部および定常領域をコードし、３’末端にＥｃｏＲＩ制限酵素切断部位を付加したＬ７Ａ５−ＤＮＡ断片は、ＶＨＨ−ＤＮＡ断片作製で得られたものを用いた［”（２）プラスミドｐ７ＡＬ−ＨＨの構築（ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖発現プラスミド）”および”ａ）第一段階ＰＣＲ”参照］。
【０２６４】
ＣＤＲＬ_２、ＦＲＬ_３、ＣＤＲＬ_３、ＦＲＬ_４および定常領域の一部をコードするＭＬ７Ａ３−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ３ＰＡ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬ４ＮＡ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２６５】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＭＬ７Ａ３−ＤＮＡに相当するＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００ｘｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２６６】
ｂ）第二段階ＰＣＲ
ＶＨＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図１３に示した。
【０２６７】
前出Ｌ７Ａ１．２−ＤＮＡ断片とＭＬ７Ａ３−ＤＮＡ断片および前出Ｌ７Ａ５−ＤＮＡ断片を融合したＶＨＭ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第二段階ＰＣＲで作製したＬ７Ａ１．２−ＤＮＡ溶液　　　１０μｌ
第一段階ＰＣＲで作製したＭＬ７Ａ３−ＤＮＡ溶液　　　　１０μｌ
第一段階ＰＣＲで作製したＬ７Ａ５−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー７ＡＬ１Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー７ＡＬＣＮ　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌ　とし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２６８】
ＰＣＲ後の増幅されたＶＨＭ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＶＨＭ−ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２６９】
ＶＨＭ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要は図１４に示した。
【０２７０】
得られたＶＨＭ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化した。
【０２７１】
クローニング用プラスミドｐＨＳＧ３９９　ＤＮＡ（宝酒造（株）社製）１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＨＳＧ３９９　ＤＮＡと、ＨｉｎｄＩＩＩおよびＥｃｏＲＩで消化したＶＨＭ−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。得られた白色を呈する形質転換体は、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。抽出したプラスミドＤＮＡをＨｉｎｄＩＩＩとＥｃｏＲＩで消化した後、１％　アガロースゲル電気泳動解析を行って、ＶＨＭ−ＤＮＡ断片を保持するクローンを選択した。
【０２７２】
以上の操作により、ヒト化ＨＭタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片を保持するプラスミドｐＨＳＧＨＭ１７を得た。なお、プラスミドｐＨＳＧＨＭ１７を保持する形質転換大腸菌Ｅ．ｃｏｌｉ　ｐＨＳＧＨＭ１７　ＳＡＮＫ　７３５９７は、平成９年８月２２日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６０７２が付された。
【０２７３】
配列表の配列番号５２に例示したヒト化ＨＭタイプＨＦＥ７Ａ−軽鎖ポリペプチドをコードする配列表の配列番号５１に例示したＤＮＡを保持する発現ベクタープラスミドｐ７ＡＬ−ＨＭは、前出プラスミドｐＨＳＧＨＭ１７を用いて作製した。
【０２７４】
哺乳動物細胞用発現プラスミドベクターｐＥＥ．１２．１　ＤＮＡ　１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＥＥ．１２．１　ＤＮＡ　１００ｎｇと、ＨｉｎｄＩＩＩとＥｃｏＲＩで消化したｐＨＳＧＨＭ１７−ＤＮＡ断片１０μｇとをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＥｃｏＲＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＭタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片が、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐ７ＡＬ−ＨＭを得た。
【０２７５】
５）プラスミドｐ７ＡＬ−ＭＭの構築（ヒト化ＭＭタイプＨＦＥ７Ａ−軽鎖発現プラスミド）
配列表の配列番号５４に示すアミノ酸をコードする、配列表の配列番号５３に示すＶＭＭ−ＤＮＡ断片は、三段階のＰＣＲを実施することにより作製され、プラスミドベクター中に挿入された後、大腸菌でクローニングされた。
【０２７６】
ａ）第一段階ＰＣＲ
ＶＭＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図１５に示した。
【０２７７】
５’末端にＨｉｎｄＩＩＩ切断部位を付加した、分泌シグナル配列およびＦＲＬ_１の一部をコードするＬ７Ａ１−ＤＮＡ断片、ならびに、ＦＲＬ_４の一部および定常領域をコードし、３’末端にＥｃｏＲＩ切断部位を付加したＬ７Ａ５−ＤＮＡ断片は、ＶＨＨ−ＤＮＡ断片作製で得られたものを用いた［”（２）プラスミドｐ７ＡＬ−ＨＨの構築（ヒト化ＨＨタイプＨＦＥ７Ａ−軽鎖発現プラスミド）”および”ａ）第一段階ＰＣＲ”参照］。
【０２７８】
ＦＲＬ_１の一部、ＣＤＲＬ_１、ＦＲＬ_２、ＣＤＲＬ_２およびＦＲＬ_３の一部をコードするＭＬ７Ａ２Ｍ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー７ＡＬ２Ｐ　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマーＭ７ＡＬ２Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２７９】
ＦＲＬ_３の一部、ＣＤＲＬ_３、ＦＲＬ_４および定常領域の一部をコードするＭＬ７Ａ３Ｍ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｌ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　Ｍ７ＡＬ３ＰＡ　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＬ４ＮＡ　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２８０】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＭＬ７Ａ２Ｍ−ＤＮＡおよびＭＬ７Ａ３Ｍ−ＤＮＡに相当するＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２８１】
ｂ）第二段階ＰＣＲ
ＶＭＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図１６に示した。
前出Ｌ７Ａ１−ＤＮＡ断片とＭＬ７Ａ２Ｍ−ＤＮＡ断片を融合したＭＬ７Ａ１．２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＬ７Ａ１−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＭＬ７Ａ２Ｍ−ＤＮＡ溶液　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　Ｍ７ＡＬ２Ｎ　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２８２】
ＰＣＲ後の増幅されたＭＬ７Ａ１．２−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出した融合ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２８３】
ｃ）第三段階ＰＣＲ
ＶＭＭ−ＤＮＡ作製のための第三段階ＰＣＲの概要を図１７に示した。
【０２８４】
前出ＭＬ７Ａ１．２−ＤＮＡ断片とＭＬ７Ａ３Ｍ−ＤＮＡ断片およびＬ７Ａ５−ＤＮＡ断片を融合したＶＭＭ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第二段階ＰＣＲで作製したＭＬ７Ａ１．２−ＤＮＡ溶液　　１０μｌ
第一段階ＰＣＲで作製したＭＬ７Ａ３Ｍ−ＤＮＡ溶液　　　１０μｌ
第一段階ＰＣＲで作製したＬ７Ａ５−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＬＣＮ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌ　とし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０２８５】
ＰＣＲ後の増幅されたＶＭＭ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＶＭＭ−ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０２８６】
ＶＭＭ−ＤＮＡ断片を保持するプラスミド作製方法の概要は図１８に示した。
【０２８７】
得られたＶＭＭ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化した。
【０２８８】
クローニング用プラスミドｐＨＳＧ３９９（宝酒造（株）社製）ＤＮＡ　１μｇをＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＨＳＧ３９９　ＤＮＡと、ＥｃｏＲＩとＨｉｎｄＩＩＩで消化したＶＭＭ−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。得られた白色を呈する形質転換体は、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。抽出したプラスミドＤＮＡをＨｉｎｄＩＩＩとＥｃｏＲＩで消化した後、１％　アガロースゲル電気泳動解析により、ＶＭＭ−ＤＮＡ断片を保持するクローンを選択した。
【０２８９】
以上の操作により、ヒト化ＭＭタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片を保持するプラスミドｐＨＳＧＭＭ６を得た。なお、プラスミドｐＨＳＧＭＭ６を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐＨＳＧＭＭ６　ＳＡＮＫ　７３６９７は、平成９年８月２２日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６０７１が付された。
【０２９０】
配列表の配列番号５４に例示したヒト化ＭＭタイプＨＦＥ７Ａ−軽鎖ポリペプチドをコードする配列表の配列番号５３に例示したＤＮＡを保持する発現ベクタープラスミドｐ７ＡＬ−ＭＭは、前出プラスミドｐＨＳＧＭＭ６を用いて作製した。
【０２９１】
哺乳動物細胞用発現プラスミドベクターｐＥＥ．１２．１　ＤＮＡ　１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＥＥ．１２．１　ＤＮＡ　１００ｎｇと、ＨｉｎｄＩＩＩとＥｃｏＲＩで消化したｐＨＳＧＭＭ６　ＤＮＡ断片１０μｇとをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＥｃｏＲＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＦＥ７Ａ−軽鎖ＭＭタイプの可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片が、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐ７ＡＬ−ＭＭを得た。
【０２９２】
６）ヌクレオチド配列の確認
プラスミドｐ７ＡＬ−ＨＨ、ｐ７ＡＬ−ＨＭおよびｐ７ＡＬ−ＭＭの挿入ＤＮＡが、目的とするヌクレオチドの配列を保持していることを確認するため、ヌクレオチド配列の決定を行なった。ヌクレオチドの配列を決定するにあたり作製したオリゴヌクレオチドプライマーは以下に記載する通りであった：
５’−　ＣＣＣＡＡＧＣＴＴＡ　ＡＧＡＡＧＣＡＴＣＣ　−３’　　（ＳＰ１：配列表の配列番号６８）；
５’−　ＡＴＣＴＡＴＧＣＴＧ　ＣＡＴＣＣＡＡＴＣＴ　−３’　　（ＳＰ２：配列表の配列番号６９）；
５’−　ＧＴＴＧＴＧＴＧＣＣ　ＴＧＣＴＧＡＡＴＡＡ　−３’　　（ＳＰ３：配列表の配列番号７０）；
５’−　ＣＣＣＧＡＡＴＴＣＴ　ＴＡＣＴＡＡＣＡＣＴ　−３’　　（ＳＰ４：配列表の配列番号７１）；
５’−　ＴＴＡＴＴＣＡＧＣＡ　ＧＧＣＡＣＡＣＡＡＣ　−３’　　（ＳＰ５：配列表の配列番号７２）；
５’−　ＡＧＡＴＴＧＧＡＴＧ　ＣＡＧＣＡＴＡＧＡＴ　−３’　　（ＳＰ６：配列表の配列番号７３）。
【０２９３】
各プライマーが結合する位置を、図１９に示した。ヌクレオチド配列の決定は、アルカリ・ＳＤＳ法と塩化セシウム法［いずれもＳａｍｂｒｏｏｋ，　Ｊ．　ｅｔ　ａｌ．　（１９８９　）　ｉｎ　”Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ．　Ｓｅｃｏｎｄ　ｅｄｉｔｉｏｎ．”　Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ．参照］により精製した各プラスミドＤＮＡを鋳型とし、ジデオキシヌクレオチド鎖終結法［Ｓａｎｇｅｒ，　Ｆ．　Ｓ．　ｅｔ　ａｌ．（１９７７）　Ｐｒｏ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ．　７４，　５４６３参照］で行なった。すなわち、３μｇの精製したプラスミドＤＮＡを１３μｌの再蒸留水に溶解し、２μｌの２ｍＭ　ＥＤＴＡと２μｌの２Ｎ　ＮａＯＨを加え、室温で５分間静置した。その後、４μｌの１０Ｍ　酢酸アンモニウム溶液と１００μｌのエタノールを加え、撹袢の後ドライアイス上で１０分間静置した。１５０００×ｇの遠心分離の後、得られた沈澱を８０％　エタノールで洗浄後、真空乾燥した。真空乾燥したＤＮＡを７μｌの再蒸留水に溶解し、ヌクレオチド配列決定反応に用いた。ヌクレオチド配列決定反応は７−デアザ−シークエナーゼ・バージョン２．０・キット（ｄＣＴＰ用：アマシャム社製）を用いた。前出プラスミド溶液７μｌに、予め合成したプライマー１ｐｍｏｌと、１μｌの反応緩衝液（キットに予め添付されている緩衝液）を加え、６５℃で２分間保温した。その後、室温まで徐冷しプライマーとアニーリングさせ、［α−^３２Ｐ］ｄＣＴＰ（アマシャム社製）で標識した。反応産物は、１×ＴＢＥ（１００ｍＭ　トリス、１００ｍＭ　ホウ酸、１ｍＭ　ＥＤＴＡ、ｐＨ８．３）緩衝液中、８Ｍ尿素を含む５％　ポリアクリルアミドゲル電気泳動を行なった。ゲルを乾燥した後、オートラジオグラフィーを行いヌクレオチド配列を解読した（以後、ヌクレオチド配列の決定は、上記方法により行なった）。
【０２９４】
その結果、ｐ７ＡＬ−ＨＨは、配列表の配列番号５０に示したポリペプチドをコードする配列表の配列番号４９に示すヌクレオチド配列を、ｐ７ＡＬ−ＨＭは、配列表の配列番号５２に示したポリペプチドをコードする配列表の配列番号５１に示すヌクレオチド配列を、ｐ７ＡＬ−ＭＭは、配列表の配列番号５４に示したポリペプチドをコードする配列表の配列番号５３に示すヌクレオチド配列をそれぞれ保持していることが確認された。
【０２９５】
実施例１５　ＨＦＥ７Ａのヒト化抗体重鎖発現ベクターの構築
（１）ヒト化ＨＦＥ７Ａの重鎖可変領域ＤＮＡを保持するプラスミドの構築
１）ヒト化重鎖可変領域作製用プライマーの合成
ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ−重鎖の可変領域と、ＩｇＧ−ＣＨ１領域のＮ末端側５残基のアミノ酸を含むポリペプチド鎖（配列表の配列番号７５）をコードするＤＮＡ（配列表の配列番号７４）の合成は、ＰＣＲ法を組み合わせて行なった。
【０２９６】
ＰＣＲを行なうにあたり、以下に記載する８種のプライマーを作製した：
５’−　ＧＧＧＡＡＧＣＴＴＧ　ＧＣＴＴＧＡＣＣＴＣ　ＡＣＣＡＴＧＧＧＡＴ　ＧＧＡＧＣＴＧＴＡＴ　−３’　（７ＡＨ１Ｐ：配列表の配列番号７６）；
５’−　ＴＧＡＡＧＣＣＣＣＡ　ＧＧＣＴＴＣＴＴＧＡ　ＣＣＴＣＡＧＣＣＣＣ　ＡＧＡＣＴＧＣＡＣＣ　ＡＧＴＴＧＧＡＣ　−３’（７ＡＨ１ＮＮＥＷ：配列表の配列番号７７）；
５’−　ＴＣＣＡＣＴＣＡＡＧ　ＣＣＴＣＴＧＴＣＣＡ　ＧＧＧＧＣＣＴＧＴＴ　ＴＴＡＣＣＣ　−３’　（７ＡＨ２Ｎ：配列表の配列番号７８）；
５’−　ＧＴＣＴＧＧＧＧＣＴ　ＧＡＧＧＴＣＡＡＧＡ　ＡＧＣＣＴＧＧＧＧＣ　ＴＴＣＡＧＴＧＡＡＧ　ＧＴＧＴＣＣＴＧＣＡ　ＡＧ　−３’（７ＡＨ２ＰＮＥＷ：配列表の配列番号７９）；
５’−　ＣＡＧＧＣＣＣＣＴＧ　ＧＡＣＡＧＡＧＧＣＴ　ＴＧＡＧＴＧＧＡＴＧ　ＧＧＡＧＡＧＡＴＴ　−３’（７ＡＨ３Ｐ：配列表の配列番号８０）；
５’−　ＴＣＡＧＡＴＣＴＣＡ　ＧＧＣＴＧＣＴＧＡＧ　ＣＴＣＣＡＴＧＴＡＧ　ＧＣＴＧＴＧＣＴＡＧ　ＣＧＧＡＴＧＴＧＴＣ　−３’（７ＡＨ３Ｎ：配列表の配列番号８１）；
５’−　ＴＧＧＡＧＣＴＣＡＧ　ＣＡＧＣＣＴＧＡＧＡ　ＴＣＴＧＡＧＧＡＣＡ　ＣＧＧＣＧＧＴＣＴＡ　ＴＴＡＣ　−３’（７ＡＨ４Ｐ：配列表の配列番号８２）；
５’−　ＧＡＴＧＧＧＣＣＣＴ　ＴＧＧＴＧＧＡＧＧＣ　ＴＧＡＧＧＡＧＡＣＧ　ＧＴＧＡＣＣＡＧＧＧ　ＴＣＣＣＴＴＣＧＣＣ　ＣＣＡＧＴ　−３’（７ＡＨ４Ｎ：配列表の配列番号８３）。
【０２９７】
２）プラスミドｐＢＬ７Ａ２７の構築
配列表の配列番号７５に示すアミノ酸をコードする配列表の配列番号７４に示すＶＤ−ＤＮＡ断片は、三段階のＰＣＲを実施することにより作製され、プラスミドへ挿入された後、大腸菌でクローニングされた。
【０２９８】
ａ）第一段階ＰＣＲ
ＶＤ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図２０に示した。
【０２９９】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列およびＦＲＨ_１のＮ末端側をコードするＨ７Ａ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｈ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＨ１Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ１ＮＮＥＷ　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３００】
ＦＲＨ_１の一部、ＣＤＲＨ_２およびＦＲＨ_２の一部をコードするＨ７Ａ２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｈ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＨ２Ｎ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ２ＰＮＥＷ　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３０１】
ＦＲＨ_２の一部、ＣＤＲＨ_２およびＦＲＨ_３の一部をコードするＨ７Ａ３−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｈ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＨ３Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ３Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３０２】
ＦＲＨ_３の一部、ＣＤＲＨ_３、ＦＲＨ_４およびＣＨ１領域のＮ末端側５アミノ酸残基をコードするＨ７Ａ４−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＭＥ−Ｈ　ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＨ４Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ４Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３０３】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１　μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＨ７Ａ１−ＤＮＡ、Ｈ７Ａ２−ＤＮＡ、Ｈ７Ａ３−ＤＮＡおよびＨ７Ａ４−ＤＮＡに相当するＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３０４】
ｂ）第二段階ＰＣＲ
ＶＤ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図２１に示した。
【０３０５】
前出Ｈ７Ａ１−ＤＮＡ断片とＨ７Ａ２−ＤＮＡ断片を融合したＨ７Ａ１．２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＨ７Ａ１−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＨ７Ａ２−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＨ１Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ２Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３０６】
前出Ｈ７Ａ３−ＤＮＡ断片とＨ７Ａ４−ＤＮＡ断片を融合したＨ７Ａ３．４−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＨ７Ａ３−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＨ７Ａ４−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＨ３Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ４Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３０７】
ＰＣＲ後の増幅されたＨ７Ａ１．２−ＤＮＡおよびＨ７Ａ３．４−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出した各融合ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３０８】
ｃ）第三段階ＰＣＲ
ＶＤ−ＤＮＡ作製のための第三段階ＰＣＲの概要を図２２に示した。
【０３０９】
前出Ｈ７Ａ１．２−ＤＮＡ断片とＨ７Ａ３．４−ＤＮＡ断片を融合したＶＤ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第二段階ＰＣＲで作製したＨ７Ａ１．２−ＤＮＡ溶液　　　１０μｌ
第二段階ＰＣＲで作製したＨ７Ａ３．４−ＤＮＡ溶液　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＨ１Ｐ　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＨ４Ｎ　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３１０】
ＰＣＲ後の増幅されたＶＤ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＶＤ−ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３１１】
ＶＤ−ＤＮＡ断片を保持するプラスミド作製方法の概要は図２３に示した。
【０３１２】
得られたＶＤ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩとＡｐａＩで消化した。
【０３１３】
プラスミドベクター　ｐＢＬＵＥＳＣＲＩＰＴ−ＩＩ　ＳＫ＋　ＤＮＡ（ストラタジーン社製）１μｇをＨｉｎｄＩＩＩとＡｐａＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＢＬＵＥＳＣＲＩＰＴ−ＩＩ　ＳＫ＋　ＤＮＡと、ＨｉｎｄＩＩＩとＡｐａＩで消化したＶＤ−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ（イソプロピルチオ−β−Ｄ−ガラクトシド、宝酒造（株）社製）、０．１％のＸ−Ｇａｌ（５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−ガラクトシド、宝酒造（株）社製）および５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた白色を呈する形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＡｐａＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ＶＤ−ＤＮＡ断片が挿入されたプラスミドｐＢＬ７Ａ２７を得た。
【０３１４】
（２）ヒトＩｇＧ１定常領域ゲノムＤＮＡを保持するプラスミドの構築
１）ヒトＩｇＧ１　ゲノム５’側ＤＮＡ断片作製用プライマーの合成
ヒトＩｇＧ１ゲノム５’側ＤＮＡ断片の合成は、ＰＣＲ法を用いて作製した。ＰＣＲを行なうにあたり、以下に記載する２種類のオリゴヌクレオチドプライマーを作製した：
５’−　ＧＧＧＡＡＧＣＴＴＣ　ＣＧＣＧＧＴＣＡＣＡ　ＴＧＧＣＡＣＣＡＣＣ　ＴＣＴＣＴＴＧＣＡ　−３’（５’Ｈｉｎｄ：配列表の配列番号８４）；
５’−　ＧＣＴＣＴＧＣＡＧＡ　ＧＡＧＡＡＧＡＴＴＧ　ＧＧＡＧＴＴＡＣＴＧ　ＧＡＡＴＣ　−３’（ＩＧＧＣＰＳＴＮ：配列表の配列番号８５）。
【０３１５】
２）プラスミドｐＩＧ５’０３の構築
ＨｉｎｄＩＩＩ切断配列に続き、ヒトＩｇＧ１のＣＨ１領域とイントロンを含むゲノムＤＮＡは、ヒトゲノムＤＮＡを鋳型とし、ＰＣＲ法により目的ＤＮＡ断片を分離増幅した後に、プラスミドｐＨＳＧ３９９（宝酒造（株）社製）中に挿入され、大腸菌でクローン化された。目的とするヒトＩｇＧ１のＣＨ１領域とイントロンを含むＤＮＡ（以下「ＩＧ５’−ＤＮＡ」という）断片の作製法の概要を図２４に示した。
【０３１６】
ＩＧ５’−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
ヒトゲノミックＤＮＡ（クローンテック社製）　　　　　　　２μｇ
オリゴヌクレオチドプライマー　５’Ｈｉｎｄ　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＩＧＧＣＰＳＴＮ　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３１７】
ＰＣＲ後の増幅されたＩＧ５’−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１　μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＩＧ５’−ＤＮＡバンドを、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３１８】
得られたＩＧ５’−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩとＰｓｔＩで消化した。
【０３１９】
プラスミドｐＨＳＧ３９９　ＤＮＡ（宝酒造（株）社製）１μｇを制限酵素ＨｉｎｄＩＩＩとＰｓｔＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐＨＳＧ３９９　ＤＮＡと、ＨｉｄＩＩＩとＰｓｔＩで消化したＩＧ５’−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。得られた白色を呈する形質転換体を、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＰｓｔＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ＩＧ５’−ＤＮＡ断片が挿入されたプラスミドｐＩＧ５’０３を得た。
【０３２０】
（３）ヒトＩｇＧ１定常領域ゲノムＤＮＡを保持するプラスミドの構築
１）ヒトＩｇＧ１ゲノム３’側ＤＮＡ断片作製用プライマーの合成
ヒトＩｇＧ１ゲノム３’側ＤＮＡ断片の合成は、ＰＣＲ法を用いて作製した。ＰＣＲを行なうにあたり、以下に記載する２種類のプライマーを作製した：
５’−　ＴＣＴＣＴＧＣＡＧＡ　ＧＣＣＣＡＡＡＴＣＴ　ＴＧＴＧＡＣＡＡＡＡ　ＣＴＣＡＣ　−３’（ＩＧＧＣＰＳＴＰ：配列表の配列番号８６）；
５’−　ＧＧＧＧＡＡＴＴＣＧ　ＧＧＡＧＣＧＧＧＧＣ　ＴＴＧＣＣＧＧＣＣＧ　ＴＣＧＣＡＣＴＣＡ　−３’（Ｅｃｏ３’：配列表の配列番号８７）。
【０３２１】
２）プラスミドｐＩＧ３’０８の構築
ヒトＩｇＧ１のイントロン、ヒンジ領域、イントロン、ＣＨ２領域、イントロン、ＣＨ３領域およびＥｃｏＲＩ切断配列を保持するＤＮＡは、ヒトゲノムＤＮＡを鋳型とし、ＰＣＲ法により目的ＤＮＡ断片を分離増幅した後に、プラスミドｐＨＳＧ３９９（宝酒造（株）社製）中に挿入され、大腸菌でクローン化された。目的とするヒトＩｇＧ１のヒンジ領域、ＣＨ２領域、ＣＨ３領域および、イントロンを含むゲノムＤＮＡ（以下「ＩＧ３’−ＤＮＡ」という）断片の作製法の概要を図２５に示した。
【０３２２】
ＩＧ３’−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
ヒトゲノミックＤＮＡ（クローンテック社製）　　　　　　　２μｇ
オリゴヌクレオチドプライマー　ＩＧＧＣＰＳＴＰ　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　Ｅｃｏ３’　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３２３】
ＰＣＲ後の増幅されたＩＧ３’−ＤＮＡ断片は、フェノール抽出とエタノール沈澱の後、５％　ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出した各ＤＮＡ断片を、剃刀で切り出し、セントリコン−１０およびセントリリューターを用いてゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３２４】
得られたＩＧ３’−ＤＮＡ断片を、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＥｃｏＲＩとＰｓｔＩで消化した。
【０３２５】
プラスミドｐＨＳＧ３９９　ＤＮＡ（宝酒造（株）社製）１μｇをＥｃｏＲＩとＰｓｔＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＨＳＧ３９９ＤＮＡと、ＥｃｏＲＩおよびＰｓｔＩで消化したＩＧ３’−ＤＮＡ断片をＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。白色コロニーを選択し、ＩＧ３’−ＤＮＡ断片が挿入されたプラスミドｐＩＧ３’０８を得た。
【０３２６】
（４）ヒト化ＨＦＥ７Ａ−重鎖発現ベクタープラスミドの構築
配列表の配列番号８９に示したヒト化ＨＦＥ７Ａ−重鎖ポリペプチドをコードする、配列表の配列番号８８に示したＤＮＡを保持する発現ベクタープラスミドｐＥｇ７ＡＨ−Ｈは、前出プラスミドｐＢＬ７Ａ２７、ｐＩＧ５’０３および、ｐＩＧ３’０８を用いて作製した。作製手順の概要を図２６に示した。
【０３２７】
ヒトＩｇＧ１−重鎖のＣＨ１領域とイントロンを含むｐＩＧ５’０３プラスミドＤＮＡ　１０μｇを、制限酵素ＡｐａＩと制限酵素ＫｐｎＩで消化した。一方、前出プラスミドｐＢＬ７Ａ２７　ＤＮＡ　１　μｇを、制限酵素ＡｐａＩとＫｐｎＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＢＬ７Ａ２７　ＤＮＡ　１００ｎｇと、ＡｐａＩおよびＫｐｎＩで消化したｐＩＧ５’０３　ＤＮＡ１０μｇとをＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株でクローニングした。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＡｐａＩとＫｐｎＩ、あるいはＨｉｎｄＩＩＩとＰｓｔＩで消化し、アガロースゲル電気泳動を行うことにより目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＦＥ７ＡのＶＤ−ＤＮＡ断片とＩＧ５’−ＤＮＡ断片が接続され、挿入されたプラスミドｐＢＬ７ＡＦ１８４を得た。
【０３２８】
次に、得られたプラスミドｐＢＬ７ＡＦ１８４　ＤＮＡ　１０μｇを、制限酵素ＨｉｎｄＩＩＩとＰｓｔＩで消化した。一方、前出プラスミドｐＩＧ３’０８ＤＮＡ　１μｇを、制限酵素ＨｉｎｄＩＩＩとＰｓｔＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＩＧ３’０８　ＤＮＡ　１００ｎｇと、ＨｉｎｄＩＩＩおよびＰｓｔＩで消化したｐＢＬ７ＡＦ１８４　ＤＮＡ　１０μｇを、ＤＮＡライゲーションキットバージョン２．０（宝酒造（株）社製）を用いて連結した後、大腸菌ＪＭ１０９株でクローニングした。得られた形質転換体を、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＰｓｔＩとＨｉｎｄＩＩＩあるいはＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＦＥ７ＡのＶＤ−ＤＮＡ断片と、ヒトＩｇＧ１定常領域をコードするゲノミックＤＮＡ断片が接続され、挿入されたプラスミドｐｇＨＳＬ７Ａ６２を得た。なお、プラスミドｐｇＨＳＬ７Ａ６２を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐｇＨＳＬ７Ａ６２　ＳＡＮＫ７３３９７は、平成９年８月２２日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６０７４が付された。
【０３２９】
得られたプラスミドｐｇＨＳＬ７Ａ６２ＤＮＡ　１０μｇを、制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩにより切断した。一方、哺乳動物細胞用発現プラスミドｐＥＥ．６．１　ＤＮＡ　１μｇをＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化ｐＥＥ．６．１　ＤＮＡ　１００ｎｇと、ＨｉｎｄＩＩＩおよびＥｃｏＲＩで消化したｐｇＨＳＬ７Ａ６２　ＤＮＡ　１０μｇを、ライゲーションキットを用いて連結した後、大腸菌ＪＭ１０９株でクローニングした。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドをＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、アガロースゲル電気泳動を行って目的の挿入断片の有無を確認した。このようにして、ヒト化ＨＦＥ７ＡのＶＤ−ＤＮＡ断片と、ヒトＩｇＧ１定常領域をコードするゲノミックＤＮＡとの融合断片が、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐＥｇ７ＡＨ−Ｈを得た。
【０３３０】
（５）ヌクレオチド配列の確認
ｐＥｇ７ＡＨ−Ｈの挿入ＤＮＡが、目的とするヌクレオチドの配列を保持していることを確認するため、ヌクレオチド配列の決定を行なった。ヌクレオチドの配列を決定するにあたり作製したオリゴヌクレオチドプライマーは以下に記載する通りであった：
５’−　ＡＣＡＧＣＣＧＧＧＡ　ＡＧＧＴＧＴＧＣＡＣ　−３’　　（ＩＧ０１：配列表の配列番号９０）；
５’−　ＡＧＡＣＡＣＣＣＴＣ　ＣＣＴＣＣＣＴＧＴＧ　−３’　　（ＩＧ０２：配列表の配列番号９１）；
５’−　ＧＴＧＣＡＧＧＧＣＣ　ＴＧＧＧＴＴＡＧＧＧ　−３’　　（ＩＧ０３：配列表の配列番号９２）；
５’−　ＧＣＡＣＧＧＴＧＧＧ　ＣＡＴＧＴＧＴＧＡＧ　−３’　　（ＩＧ０４：配列表の配列番号９３）；
５’−　ＧＴＴＴＴＧＧＧＧＧ　ＧＡＡＧＡＧＧＡＡＧ　−３’　　（ＩＧ０５：配列表の配列番号９４）；
５’−　ＣＣＡＧＴＣＣＴＧＧ　ＴＧＣＡＧＧＡＣＧＧ　−３’　　（ＩＧ０６：配列表の配列番号９５）；
５’−　ＣＣＴＧＴＧＧＴＴＣ　ＴＣＧＧＧＧＣＴＧＣ　−３’　　（ＩＧ０７：配列表の配列番号９６）；
５’−　ＣＧＴＧＧＴＣＴＴＧ　ＴＡＧＴＴＧＴＴＣＴ　−３’　　（ＩＧ０８：配列表の配列番号９７）；
５’−　ＣＴＴＣＣＴＣＴＴＣ　ＣＣＣＣＣＡＡＡＡＣ　−３’　　（ＩＧＰ５：配列表の配列番号９８）；
５’−　ＣＣＧＴＣＣＴＧＣＡ　ＣＣＡＧＧＡＣＴＧＧ　−３’　　（ＩＧＰ６：配列表の配列番号９９）；
５’−　ＧＣＡＧＣＣＣＣＧＡ　ＧＡＡＣＣＡＣＡＧＧ　−３’　　（ＩＧＰ７：配列表の配列番号１００）；
５’−　ＡＧＡＡＣＡＡＣＴＡ　ＣＡＡＧＡＣＣＡＣＧ　−３’　　（ＩＧＰ８：配列表の配列番号１０１）；
５’−　ＧＣＣＴＧＡＣＡＴＣ　ＴＧＡＧＧＡＣＴＣ　−３’　（Ｈ５＋：配列表の配列番号１０２）；
５’−　ＧＡＧＴＣＣＴＣＡＧ　ＡＴＧＴＣＡＧＧＣ　−３’　（Ｈ５−：配列表の配列番号１０３）；
５’−　ＧＡＧＣＡＧＴＡＣＴ　ＣＧＴＴＧＣＴＧＣＣ　ＧＣＧＣＧＣＧＣＣＡ　ＣＣＡＧ　−３’
（ＰＥＥＦ：配列表の配列番号１０４）；
５’−　ＧＧＴＡＴＧＧＣＴＧ　ＡＴＴＡＡＴＧＡＴＣ　ＡＡＴＧ　−３’　（ＰＥＥＢ：配列表の配列番号１０５）。
【０３３１】
各プライマーが結合する位置を、図２７に示した。ヌクレオチド配列の決定は、アルカリ・ＳＤＳ法と塩化セシウム法により精製した各プラスミドＤＮＡを鋳型とし、ジデオキシヌクレオチド鎖終結法で行なった。その結果、ｐＥｇ７ＡＨ−Ｈは配列表の配列番号８９に示したポリペプチドをコードする、配列表の配列番号８８に示すヌクレオチド配列を保持していることが確認された。
【０３３２】
実施例１６　ＣＯＳ−１細胞での発現
上記で得られたヒト化ＨＦＥ７Ａ−重鎖発現プラスミドおよび各ヒト化ＨＦＥ７Ａ−軽鎖発現プラスミドのサル腎臓由来細胞株ＣＯＳ−１細胞への導入を、電極間２ｍｍのチャンバー／ＦＣＴ−１３（島津製作所（株）社製）を装着した遺伝子導入装置ＧＴＥ−１（島津製作所（株）社製）を用いて、電気穿孔法により行った。まず、ＣＯＳ−１細胞（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ　Ｎｏ．　ＣＲＬ−１６５０　）を、１０％のウシ胎児血清（以下「ＦＣＳ」という：モアゲート社製）を含む最小必須アルファー培地（以下「α（＋）ＭＥＭ」という：ギブコ・ビーアールエル社製）を入れた細胞培養用フラスコ（培養面積２２５ｃｍ^２：住友ベークライト（株）社製）中でセミコンフルエントになるまで培養した。その後、培地を除去し、３ｍｌのトリプシン−ＥＤＴＡ溶液（シグマ社製）中、３７℃、３分間処理することによりＣＯＳ−１細胞をフラスコから遊離させた。遊離した細胞は、８００ｒｐｍ、２分間の遠心分離により回収し、リン酸緩衝液［０．０２％　塩化カリウム、０．０２％　リン酸２水素カリウム、０．８％　塩化ナトリウム、１．１５％　リン酸水素２ナトリウム。以下「ＰＢＳ（−）」という；日水製薬（株）社製］で２回洗浄した。洗浄したＣＯＳ−１細胞をＰＢＳ（−）で１×１０^８細胞数／ｍｌとなるよう調製し、ＣＯＳ−１細胞懸濁液とした。
【０３３３】
一方、アルカリ・ＳＤＳ法と塩化セシウム密度勾配遠心分離法を用いて調製したヒト化ＨＦＥ７Ａ−重鎖発現プラスミドＤＮＡ　４μｇおよびヒト化ＨＦＥ７Ａ−軽鎖発現プラスミドＤＮＡ　４μｇを混合した後、同一チューブ内でエタノール沈澱し、ＰＢＳ（−）　２０μｌに懸濁した。得られたプラスミド懸濁液２０μｌを、先に調製したＣＯＳ−１細胞懸濁液２０μｌ（２×１０^６細胞数）と混合し、電極間隔２ｍｍのチャンバー／ＦＣＴ−１３（島津製作所社製）に移し、遺伝子導入装置ＧＴＥ−１（島津製作所（株）社製）にセットした。その後、６００Ｖ、５０μＦのパルスを、１秒間隔で２回与えることにより、目的とするプラスミドＤＮＡをＣＯＳ−１細胞内に導入した。パルスを与えた後のチャンバー内の細胞−ＤＮＡ混合液を、１０％　ＦＣＳを含むα（＋）ＭＥＭ　５ｍｌに懸濁し、細胞培養用フラスコ（培養面積２５ｃｍ^２：住友ベークライト（株）社製）に移した。５％炭酸ガス下、３７℃、７２時間培養した後に培養上清を回収し、本培養上清中に含まれる発現生成物を解析した。
【０３３４】
上記の方法により、以下に記載するプラスミドの組み合わせでそれぞれＣＯＳ−１細胞を形質転換し、培養上清を回収した：
［Ａ］：プラスミドＤＮＡを含まない条件；
［Ｂ］：ｐＥｇ７ＡＨ−Ｈおよびｐ７ＡＬ−ＭＭのコ・トランスフェクション；
［Ｃ］：ｐＥｇ７ＡＨ−Ｈおよびｐ７ＡＬ−ＨＭのコ・トランスフェクション；
［Ｄ］：ｐＥｇ７ＡＨ−Ｈおよびｐ７ＡＬ−ＨＨのコ・トランスフェクション。
【０３３５】
実施例１７　ＥＬＩＳＡ法による発現産物の定量
実施例１６で調製した培養上清中のヒト化抗体の発現の確認および発現生成物の定量検定を、抗ヒトＩｇＧに対する抗体を用いた酵素結合抗体法（以下「ＥＬＩＳＡ法」という）により行なった。すなわち、９６穴プレート（マキシソープ：ヌンク社製）の各ウエルに、最終濃度１μｇ／ｍｌで固相化緩衝液（０．０５Ｍ　炭酸水素ナトリウム、０．０２％　アジ化ナトリウム、ｐＨ９．６）に溶解したヤギ抗ヒトＩｇＧ　Ｆｃ特異的ポリクローナル抗体（カペル社製）１００μｌを加え、３７℃、２時間保温し、抗体をプレートに固相化した。その後、０．０５％のツイーン−２０（バイオラッド社製）を含むＰＢＳ（−）（以後、「ＰＢＳ−Ｔ」という）３５０μｌで４回洗浄した。洗浄の終わった各ウエルに、１０％　ＦＣＳを含むα（＋）ＭＥＭで希釈した培養上清を加え、３７℃、２時間保温した。再び、ＰＢＳ−Ｔによる洗浄の後、ＰＢＳ−Ｔで５０００倍希釈したアルカリフォスファターゼ標識ヤギ抗ヒトＩｇＧ　Ｆｃ特異的ポリクローナル抗体（カルタグ・ラボ社製）１００μｌを、各ウエルに加え、３７℃、２時間保温した。再び、ＰＢＳ−Ｔによる洗浄の後、ｐ−ニトロフェニル燐酸を１０％　ジエタノールアミン（ｐＨ９．８）で１ｍｇ／ｍｌに調製した基質溶液を加えた。３７℃、３０分間から１時間保温し、４０５ｎｍでの吸光度を測定した。なお、本実験において、培養上清中に含まれるヒト化ＨＦＥ７Ａ抗体の濃度対照としては、１０％　ＦＣＳを含むα（＋）ＭＥＭで一定濃度に希釈したヒトプラズマイムノグロブリンＧ／サブクラス１（ＩｇＧ１）（バイオピュアＡＧ社製）を用いた。
【０３３６】
その結果、実施例１６で調製した培養上清中の発現生成物のうち、［Ｂ］、［Ｃ］および［Ｄ］の条件のものは、抗ヒトＩｇＧ抗体により特異的に検出された。
【０３３７】
実施例１８　Ｆａｓに対する結合活性の測定
以下に記載する方法に従って、実施例１６で調製した形質転換細胞培養上清中のＦａｓへの結合活性のＥＬＩＳＡ法による検定を行った。まず、実施例１で調製したヒトＦａｓ融合タンパク質発現ＣＯＳ−１細胞培養上清を固相化緩衝液で５倍に希釈し、９６穴プレート（マキシソープ：ヌンク社製）の各ウエルに５０μｌづつ入れ、４℃で一晩保温することにより、ウエル表面にヒトＦａｓ融合タンパク質を吸着させた。その後、各ウエルをＰＢＳ−Ｔ　３５０μｌで４回洗浄した。洗浄後、５％　ウシ血清アルブミン（和光純薬（株）社製）を含むＰＢＳ−Ｔを、各ウエルに５０μｌづつ加え、３７℃、１時間保温することによりブロッキングし、その後再び、各ウエルをＰＢＳ−Ｔで洗浄した。実施例１６で調製した培養上清は、予め、実施例１７に記載した方法により、含有する目的発現生成物の濃度を算出し、最終的な目的生成物濃度が１００ｎｇ／ｍｌとなるように１０％　ＦＣＳを含むα（＋）ＭＥＭ培地で調製しておいた。１００ｎｇ／ｍｌの濃度に調製した各発現生成物溶液を、１０％　ＦＣＳを含むα（＋）ＭＥＭ培地で２倍希釈を繰り返し、希釈系列を作製した。作製された各発現生成物希釈系列溶液５０μｌを、各ウエルに添加し、３７℃下、２時間反応させた。その後、再びＰＢＳ−Ｔで洗浄した後、ＰＢＳ−Ｔにより１００００倍希釈したアルカリフォスファターゼ標識ヤギ抗ヒトＩｇＧ　Ｆｃ特異的ポリクローナル抗体（カルタグ・ラボ社製）５０μｌを各ウエルに分注し、３７℃下、２時間反応させた。なお、対照として用いた、マウスハイブリドーマＨＦＥ７Ａより精製したＨＦＥ７Ａ（ＩｇＧ１）の検出は、アルカリフォスファターゼ標識ヤギ抗ヒトＩｇＧ　Ｆｃ特異的ポリクローナル抗体の代わりに、ＰＢＳ−Ｔにより５，０００倍希釈したアルカリフォスファターゼ標識ヤギ抗マウスＩｇＧ＋ＩｇＡ＋ＩｇＭ（ギブコ・ビーアールエル社製）を用いた。ＰＢＳ−Ｔで洗浄したのち、５０μｌのｐ−ニトロフェニル燐酸を１０％　ジエタノールアミン（ｐＨ９．８）で１ｍｇ／ｍｌに調製した基質溶液を加え、３７℃で３０分間から１時間保温した。各ウエルの４０５ｎｍの吸光度を測定することにより、各培養上清中に含まれる発現生成物のヒトＦａｓ融合タンパク質に対する結合活性を調べた。
【０３３８】
その結果、実施例１６で調製した培養上清中の発現生成物のうち、［Ｂ］、［Ｃ］および［Ｄ］の条件のものは、ヒトＦａｓ融合タンパク質との結合性を有するものであることが確認された（図２８）。
【０３３９】
実施例１９　ＨＦＥ７ＡのＦａｓへの結合に対する競合阻害
本発明で得られるヒト化抗Ｆａｓ抗体のＦａｓに対する結合は、ヒト化設計の基礎となった抗Ｆａｓマウスモノクローナル抗体であるＨＦＥ７Ａの結合を阻害するはずである。そこで、ＨＦＥ７Ａと実施例１６で調製した発現生成物とのヒトＦａｓ融合タンパク質に対する競合阻害活性を測定した。
【０３４０】
実施例３で得られた精製ＨＦＥ７Ａモノクローナル抗体　１ｍｇを、市販のアルカリフォスファターゼ標識キット（イムノ−リンク　ＡＰ　アンド　ＡＰＬラベリング　キット：ジェノシス社製）を添付のプロトコールに従って用いることにより標識した（以下、この標識抗体を「ＡＰ−ＨＦＥ７Ａ」という）。
【０３４１】
ヒトＦａｓ融合タンパク質を含むＣＯＳ−１培養上清を、固相化緩衝液で５倍希釈し、発光検出用９６穴プレート（ルミネッセント　ソリッド　アッセイ　プレート、高結合特性：コースター社製）の各ウエルに５０μｌづつ入れ、４℃で一晩保温することにより、ウエル表面にヒトＦａｓ融合タンパク質を吸着させた。吸着後、各ウエルをＰＢＳ−Ｔ３５０μｌで４回洗浄した。洗浄後、５％　ウシ血清アルブミン（和光純薬（株）社製）を含むＰＢＳ−Ｔを、各ウエルに１００μｌづつ加え、３７℃、１時間保温することによりブロッキングし、その後再び、各ウエルをＰＢＳ−Ｔで洗浄した。実施例１６で調製した培養上清は、予め、実施例１７で示す方法により、含有する目的発現生成物の濃度を算定し、最終的な目的生成物濃度が１μｇ／ｍｌとなるように１０％　ＦＣＳを含むα（＋）ＭＥＭで調製しておいた。１μｇ／ｍｌの濃度に調製した各発現生成物溶液を、１０％　ＦＣＳを含むα（＋）ＭＥＭで二倍希釈を繰り返し、希釈系列を作製した。一方、ＡＰ−ＨＦＥ７Ａを、１０％　ＦＣＳを含むα（＋）ＭＥＭにより５０ｎｇ／ｍｌに希釈し、作製した希釈系列と等量混合した。発現生成物希釈系列とＡＰ−ＨＦＥ７Ａ混合液を、洗浄の終わった各ウエルに５０μｌづつ加え、室温で一晩静置した。その後再び、各ウエルをＰＢＳ−Ｔで洗浄し、各ウエルに１００μｌのＣＤＰ−スター緩衝液（９．５８ｍｌ　ジエタノールアミン、０．２ｇ　塩化マグネシウム、０．２５ｇ　アジ化ナトリウム、ｐＨ８．５）を加え、１０分間、室温で静置した。その後、ＣＤＰ−スター緩衝液を除去し、ＣＤＰ−スター基質（１．２ｍｌ　サファイアＩＩ（トロピックス社製）、２００μｌ　ＣＤＰ−スター（トロピックス社製）、ＣＤＰ−スター緩衝液で１２ｍｌとする）を５０μｌ／ウエル加え、さらに室温で４０分間静置した。各培養上清中に含まれる発現生成物のヒトＦａｓ融合タンパク質に対するＨＦＥ７Ａとの競合阻害活性は、発光強度をルミノスキャン（タイターテック社製）により測定することにより行なった。
【０３４２】
その結果、実施例１６で調製した発現生成物は、マウスハイブリドーマより調製したＨＦＥ７ＡのヒトＦａｓ融合タンパク質に対する結合を、特異的に阻害することが確認された（図２９）。
【０３４３】
実施例２０　アポトーシス誘導活性
マウスＴリンパ腫細胞にヒトＦａｓ分子を発現させたＷＲ１９Ｌ１２ａ細胞［Ｉｔｏｈ，　Ｎ．　ｅｔ．　ａｌ．　（１９９１）　Ｃｅｌｌ　６６，　２３３−２４３］を用いて、ヒト化抗Ｆａｓ抗体を発現させたＣＯＳ細胞培養上清のアポトーシス誘導活性を検討した。まず、１０％　ＦＣＳ（ギブコ・ビーアールエル社製）を含むＲＰＭＩ１６４０培地にて、３７℃、５％　炭酸ガス存在下で３日間培養したＷＲ１９Ｌ１２ａ細胞（１×１０^５細胞／５０μｌ）を、９６穴マイクロプレート（住友ベークライト（株）社製）の各ウエルに５０μｌずつ分注した。実施例１６で調製した培養上清は、予め実施例１７に記載した方法により、含有する目的発現生成物の濃度を算定し、最終的な目的生成物濃度が１００ｎｇ／ｍｌとなるように１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で調製しておいた。１００ｎｇ／ｍｌの濃度に調製した各発現生成物溶液を、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で二倍希釈を繰り返し、希釈系列を作製した。各発現生成物希釈液　５０μｌを、各ウエルに添加し、３７℃で１時間保温した。その後、１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で細胞を洗浄した後、各ウエルの細胞を７５μｌの１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で懸濁した。その後、各ウエルに二次抗体として、１０％ＦＣＳを含むＲＰＭＩ１６４０培地で１．２５μｇ／ｍｌに調製したヤギ抗ヒトＩｇＧ　Ｆｃ特異的ポリクローナル抗体（カペル社製）を７５μｌ加えた。３７℃下、１２時間反応させた後、１ｍｇ／ｍｌのＸＴＴ（２、３−ビス２−メトキシ−４−ニトロ−５−スルホフェニル−２Ｈ−テトラゾリウム−５−カルボキサニリド　イナーソルト：シグマ社製）を含む２５μＭのＰＭＳ（フェナジンメトサルフェート：シグマ社製）５０μｌ（終濃度２５０μｇ／ｍｌ　ＸＴＴ、５μＭ　ＰＭＳ）を添加した。３時間培養した後、各ウェルの４５０ｎｍの吸光度を測定し、ミトコンドリアの還元能を指標とし、細胞の生存率を算定した。
【０３４４】
各ウェルの細胞の生存率を、下記式により算出した：
生存率（％）＝１００×（ａ−ｂ）／（ｃ−ｂ）
（式中、ａは被検ウエルの測定値を、ｂは無細胞ウエルの測定値を、ｃは抗体無添加のウエルの測定値をそれぞれ表わす）。
【０３４５】
その結果、実施例１６で調製した発現生成物は、ヒトＦａｓ抗原を発現したＴリンパ腫細胞株に対してアポトーシスを誘導することが示された（図３０）。
【０３４６】
実施例２１　ＨＦＥ７Ａの軽鎖のヒト化発現ベクターの構築
（１）　ヒト化ＨＦＥ７Ａ−軽鎖発現ベクタープラスミドの構築
実施例１４で作成されたＨＨタイプヒト化軽鎖をよりヒトに近づけるべく、ＨＨタイプ軽鎖アミノ酸のＮ末端から１番目のアミノ酸（アスパラギン酸）、８５番目（アラニン）および１０７番目（アルギニン）をそれぞれヒト軽鎖（κ鎖）で保存されているグルタミン酸、グルタミン酸およびリジンに置き換えたものを設計し、これを「ＰＤＨＨタイプ」とした。同様に、ＨＭタイプ軽鎖アミノ酸のＮ末端から１番目のアミノ酸（アスパラギン酸）および１０７番目（アルギニン）をそれぞれヒト軽鎖で保存されているグルタミン酸およびリジンに置き換えたものを設計し、これを「ＰＤＨＭタイプ」とした。以下、この２種類のヒト化抗ヒトＦａｓ抗体軽鎖アミノ酸配列をコードするＤＮＡを保持する発現プラスミドをそれぞれ構築した。
【０３４７】
１）プライマーの合成
ＰＤＨＨタイプポリペプチド鎖（配列表の配列番号１０７）をコードするＤＮＡ（配列表の配列番号１０６）およびＰＤＨＭタイプポリペプチド鎖（配列表の配列番号１０９）をコードするＤＮＡ（配列表の配列番号１０８）の合成は、ＰＣＲ法を組み合わせて行なった。
【０３４８】
ＰＣＲを行なうにあたり、前出２種のオリゴヌクレオチドプライマー　７ＡＬ１Ｐ（配列表の配列番号５５）および７ＡＬＣＮ（配列番号６４）に加え、さらに６種の配列表のプライマー：
５’−　ＧＧＴＧＡＧＡＴＴＧ　ＴＧＣＴＣＡＣＣＣＡ　ＡＴＣＴＣＣＡＧＧ　−３’　（ＬＰＤ１Ｐ：配列番号１１０）；
５’−　ＣＣＴＧＧＡＧＡＴＴ　ＧＧＧＴＧＡＧＣＡＣ　ＡＡＴＣＴＣＡＣＣ　−３’　（ＬＰＤ１Ｎ：配列番号１１１）；
５’−　ＣＣＡＴＣＴＣＴＣＧ　ＴＣＴＧＧＡＧＣＣＧ　ＧＡＧＧＡＴＴＴＴＧ　Ｃ　−３’　（ＬＰＤ２Ｐ：配列番号１１２）；
５’−　ＧＣＡＡＡＡＴＣＣＴ　ＣＣＧＧＣＴＣＣＡＧ　ＡＣＧＡＧＡＧＡＴＧ　Ｇ　−３’　（ＬＰＤ２Ｎ：配列番号１１３）；
５’−　ＣＡＡＧＧＣＡＣＣＡ　ＡＧＣＴＧＧＡＡＡＴ　ＣＡＡＡＣＧＧＡＣＴ　Ｇ　−３’　（ＬＰＤ３Ｐ：配列番号１１４）；および
５’−　ＣＡＧＴＣＣＧＴＴＴ　ＧＡＴＴＴＣＣＡＧＣ　ＴＴＧＧＴＧＣＣＴＴ　Ｇ　−３’　（ＬＰＤ３Ｎ：配列番号１１５）を作製した。
【０３４９】
２）ＰＤＨＨタイプヒト化軽鎖発現プラスミドｐＬＰＤＨＨ７５の構築
配列表の配列番号１０７に示されるアミノ酸配列をコードするＬＰＤＨＨ−ＤＮＡ断片（配列表の配列番号１０６）は、ＰＣＲ法を３回繰り返すことにより作製され、プラスミド中に挿入された後、大腸菌でクローニングされた。
【０３５０】
ａ）第一段階ＰＣＲ
ＬＰＤＨＨ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図３１に示した。
【０３５１】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列およびＦＲＬ_１領域の一部をコードするＬＰＤ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＳＧＨＨ７ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ１Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３５２】
ＦＲＬ_１領域の一部、ＣＤＲＬ_１領域、ＦＲＬ_２領域、ＣＤＲＬ_２領域およびＦＲＬ_３領域の一部をコードするＬＰＤＨＨ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＳＧＨＨ７ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＬＰＤ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ２Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３５３】
ＦＲＬ_３領域の一部、ＣＤＲＬ_３領域およびＦＲＬ_４領域をコードするＬＰＤＨＨ２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＳＧＨＨ７ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＬＰＤ２Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ３Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３５４】
ＦＲＬ_４領域の一部および定常領域をコードし、３’末端にＥｃｏＲＩ制限酵素切断部位を付加したＬＰＤＣ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＳＧＨＨ７ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＬＰＤ３Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＬＣＮ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３５５】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＰＤ１−ＤＮＡ、ＬＰＤＨＨ１−ＤＮＡ、ＬＰＤＨＨ２−ＤＮＡおよびＬＰＤＣ−ＤＮＡに相当するＤＮＡのバンドを剃刀で切り出し、それぞれセントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３５６】
ｂ）第二段階ＰＣＲ
ＬＰＤＨＨ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図３２に示した。
【０３５７】
前出ＬＰＤ１−ＤＮＡ断片、ＬＰＤＨＨ１−ＤＮＡおよびＬＰＤＨＨ２−ＤＮＡ断片を融合したＬＰＤＨＨ１．２−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第一段階ＰＣＲで作製したＬＰＤ１−ＤＮＡ溶液　　　　　　　１０μｌ
第一段階ＰＣＲで作製したＬＰＤＨＨ１−ＤＮＡ溶液　　　　　１０μｌ
第一段階ＰＣＲで作製したＬＰＤＨＨ２−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ３Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３５８】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＰＤＨＨ１．２−ＤＮＡに相当するＤＮＡのバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
ｃ）第三段階ＰＣＲ
ＬＰＤＨＨ−ＤＮＡ作製のための第三段階ＰＣＲの概要を図３３に示した。
【０３５９】
前出ＬＰＤＨＨ１．２−ＤＮＡ断片とＬＰＤＣ−ＤＮＡ断片を融合したＬＰＤＨＨ−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
第二段階ＰＣＲで作製したＬＰＤＨＨ１．２−ＤＮＡ溶液　　　１０μｌ
第一段階ＰＣＲで作製したＬＰＤＣ−ＤＮＡ溶液　　　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＬＣＮ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３６０】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＰＤＨＨ−ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３６１】
ＬＰＤＨＨ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要を図３４に示した。
【０３６２】
得られたＬＰＤＨＨ−ＤＮＡ断片は、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩと制限酵素ＥｃｏＲＩにより消化した。
【０３６３】
クローニング用プラスミドｐＨＳＧ３９９（宝酒造社製）ＤＮＡ１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰでで脱リン酸化した。このようにして得られた脱リン酸化プラスミドｐＨＳＧ３９９ＤＮＡと、予め制限酵素で消化したＬＰＤＨＨ−ＤＮＡ断片をＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用い、メーカー指定のプロトコールに従って連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。これを３７℃で培養後、白色を呈するコロニーを単離して、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、この培養物よりアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。抽出したプラスミドＤＮＡを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化した後、１％アガロースゲル電気泳動で解析することにより、ＬＰＤＨＨ−ＤＮＡ断片を保持するクローンを選択した。
【０３６４】
以上の操作により、ＰＤＨＨタイプヒト化軽鎖の可変領域をコードするＤＮＡと、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片を保持するプラスミドｐＨＳＨＨ５を得た。なお、プラスミドｐＨＳＨＨ５を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐＨＳＨＨ５　ＳＡＮＫ　７０３９８は、平成１０年２月２６日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６２７４が付された。
【０３６５】
次に、プラスミドｐＨＳＨＨ５を用いて、以下に記載する方法によりＰＤＨＨタイプヒト化軽鎖ポリペプチドをコードするＤＮＡを保持する発現ベクタープラスミドｐＬＰＤＨＨ７５を作製した。
【０３６６】
哺乳動物細胞用発現プラスミドｐＥＥ．１２．１（ロンザ社製）ＤＮＡ１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐＥＥ．１２．１ＤＮＡ１００ｎｇと、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化したｐＨＳＨＨ５ＤＮＡ断片　１０μｇをＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。
【０３６７】
得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、アルカリ・ＳＤＳ法に従ってプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、１％アガロースゲル電気泳動により挿入断片の有無を確認した。以上の操作により、ヒト化ＰＤＨＨタイプＨＦＥ７Ａ−軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片とが、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐＬＰＤＨＨ７５を得た。
【０３６８】
（３）ＰＤＨＭタイプヒト化軽鎖発現プラスミドｐＬＰＤＨＭ３２の構築
配列表の配列番号１０９に示されるアミノ酸配列をコードするＬＰＤＨＭ−ＤＮＡ断片（配列表の配列番号１０８）は、ＰＣＲ法を３回繰り返すことにより作製され、プラスミド中に挿入された後、大腸菌でクローニングされた。
【０３６９】
ａ）第一段階ＰＣＲ
ＬＰＤＨＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要を図３５に示した。
【０３７０】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列、およびＦＲＬ_１領域の一部をコードするＬＰＤ１−ＤＮＡ断片、および、ＦＲＬ_４領域の一部と定常領域をコードし、３’末端にＥｃｏＲＩ制限酵素切断部位を付加したＬＰＤＣ−ＤＮＡ断片は、いずれも上記（２）記載のＬＰＤＨＨ−ＤＮＡ断片の作製過程で得られたものを用いた。
【０３７１】
ＣＤＲＬ_１領域の一部、ＦＲＬ_２領域、ＣＤＲＬ_２領域、ＦＲＬ_３領域、ＣＤＲＬ_３領域、およびＦＲＬ_４領域をコードするＬＰＤＨＭ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐＨＳＧＨＭ１７ＤＮＡ　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＬＰＤ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ３Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３７２】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出したＬＰＤ１−ＤＮＡ、ＬＰＤＨＭ１−ＤＮＡおよびＬＰＤＣ−ＤＮＡに相当するＤＮＡバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３７３】
ｂ）第二段階ＰＣＲ
ＬＰＤＨＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要を図３６に示した。
【０３７４】
前出ＬＰＤ１−ＤＮＡ断片とＬＰＤＨＭ１−ＤＮＡ断片とを融合したＬＰＤＨＭ１．２−ＤＮＡ断片は、以下の条件で作製された。
【０３７５】
反応液組成：
第一段階ＰＣＲで作製したＬＰＤ１−ＤＮＡ溶液　　　　　　　１０μｌ
第一段階ＰＣＲで作製したＬＰＤＨＭ１−ＤＮＡ溶液　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＬＰＤ３Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３７６】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＰＤＨＭ１．２−ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３７７】
ｃ）第三段階ＰＣＲ
ＬＰＤＨＭ−ＤＮＡ作製のための第三段階ＰＣＲの概要を図３７に示した。
【０３７８】
前出ＬＰＤＨＭ１．２−ＤＮＡ断片とＬＰＤＣ−ＤＮＡ断片とを融合したＬＰＤＨＭ−ＤＮＡ断片は、以下の条件で作製された。
【０３７９】
反応液組成：
第二段階ＰＣＲで作製したＬＰＤＨＭ１．２−ＤＮＡ溶液　　　１０μｌ
第一段階ＰＣＲで作製したＬＰＤＣ−ＤＮＡ溶液　　　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＬ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　７ＡＬＣＮ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０３８０】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３８１】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＰＤＨＭ−ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０３８２】
ＬＰＤＨＭ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要は図３８に示した。
【０３８３】
まず、上記で得られたＬＰＤＨＭ−ＤＮＡ断片を、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩと制限酵素ＥｃｏＲＩにより消化した。
【０３８４】
次にクローニング用プラスミドｐＨＳＧ３９９（宝酒造社製）ＤＮＡ１μｇを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐＨＳＧ３９９　ＤＮＡと、予めＨｉｎｄＩＩＩとＥｃｏＲＩで消化したＬＰＤＨＭ−ＤＮＡ断片とを、ＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用い、メーカー指定のプロトコールに従って連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度１ｍＭのＩＰＴＧ、０．１％のＸ−Ｇａｌおよび５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。これを３７℃で培養後、白色を呈するコロニーを単離して、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、この培養物よりアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化した後、１％アガロースゲル電気泳動で解析することにより、ＬＰＤＨＭ−ＤＮＡ断片を保持するクローンを選択した。
【０３８５】
以上の操作により、ＰＤＨＭタイプヒト化軽鎖の可変領域と、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片を保持するプラスミドｐＨＳＨＭ２を得た。なお、プラスミドｐＨＳＨＭ２を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐＨＳＨＭ２　ＳＡＮＫ　７０１９８は、平成１０年２月２６日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６２７２が付された。
【０３８６】
次に、プラスミドｐＨＳＨＭ２を用いて、以下に記載する方法によりＰＤＨＭタイプヒト化軽鎖ポリペプチドをコードするＤＮＡを保持する発現ベクタープラスミドｐＬＰＤＨＭ３２を作製した。
【０３８７】
哺乳動物細胞用発現プラスミドｐＥＥ．１２．１（ロンザ社製）ＤＮＡ　１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐＥＥ．１２．１　ＤＮＡ（１００ｎｇ）と、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化したｐＨＳＨＭ２−ＤＮＡ断片（１０μｇ）とをＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、該培養物からアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＥｃｏＲＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動により挿入断片の有無を確認した。以上の操作により、ＰＤＨＭタイプヒト化軽鎖の可変領域をコードするＤＮＡと、ヒトＩｇκ鎖定常領域をコードするＤＮＡとの融合断片が、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐＬＰＤＨＭ３２を得た。
【０３８８】
（４）ヌクレオチド配列の確認
上記（２）および（３）で作製されたプラスミドｐＬＰＤＨＨ７５およびｐＬＰＤＨＭ３２が、それぞれ目的とするヌクレオチド配列を有するＤＮＡを保持していることを確認するため、ヌクレオチド配列の決定を行なった。ここで使用したプライマーは、前出ＳＰ１（配列表の配列番号６８）、ＳＰ２（配列表の配列番号６９）、ＳＰ３（配列表の配列番号７０）、ＳＰ４（配列表の配列番号７１）、ＳＰ５（配列表の配列番号７２）およびＳＰ６（配列表の配列番号７３）であった。各プライマーが結合する位置を、図１９に示した。ヌクレオチド配列の決定は、アルカリ・ＳＤＳ法と塩化セシウム法により精製した各プラスミドＤＮＡを鋳型とし、ジデオキシヌクレオチド鎖終結法で行なった。その結果、ｐＬＰＤＨＨ７５は配列表の配列番号１０６に示されるヌクレオチド配列を、ｐＬＰＤＨＭ３２は配列表の配列番号１０８に示されるヌクレオチド配列をそれぞれ保持していることが確認された。
【０３８９】
実施例２２　ヒト化重鎖発現ベクターの構築
実施例１５で作製されたヒト化ＨＦＥ７Ａ−重鎖をよりヒトに近づけるべく、該ヒト化重鎖のアミノ酸配列のＮ末端から４４番目のアミノ酸（アルギニン）および７６番目のアミノ酸（アラニン）をそれぞれヒト重鎖で保存されているグリシンおよびトレオニンに置き換えたものを設計し、これを「ＨＶタイプ」とした。このＨＶタイプヒト化重鎖アミノ酸配列をコードするＤＮＡを保持する発現プラスミドを、以下に記載する方法により構築した。
【０３９０】
（１）プライマーの合成
ＨＶタイプヒト化重鎖ポリペプチド鎖（配列表の配列番号１１７）をコードするＤＮＡ（配列表の配列番号１１６）の合成を、ＰＣＲ法を組み合わせて行なった。
【０３９１】
ＰＣＲを行なうにあたり、前出のプライマー７ＡＨ１Ｐ（配列表の配列番号７６）に加え、さらに３種のプライマー：
５’−　ＣＡＧＧＣＣＣＣＴＧ　ＧＡＣＡＧＧＧＣＣＴ　ＴＧＡＧＴＧＧＡＴＧ　−３’　（ＨＰＤ１Ｐ：配列表の配列番号１１８）；
５’−　ＣＡＴＣＣＡＣＴＣＡ　ＡＧＧＣＣＣＴＧＴＣ　ＣＡＧＧＧＧＣＣＴＧ　−３’　（ＨＰＤ１Ｎ：配列表の配列番号１１９）；および
５’−　ＧＣＴＧＡＧＣＴＣＣ　ＡＴＧＴＡＧＧＣＴＧ　ＴＧＣＴＡＧＴＧＧＡ　ＴＧＴＧＴＣＴＡＣ　−３’　（ＨＰＤ２Ｎ：配列表の配列番号１２０）を作製した。
【０３９２】
（２）プラスミドｐｇＨＰＤＨＶ３の構築
配列表の配列番号１１７のアミノ酸番号−１９から８４に示されるアミノ酸配列をコードするＨＰＤ１．２−ＤＮＡ断片は、ＰＣＲ法を２回繰り返すことにより作製され、プラスミドへ挿入された後、大腸菌でクローニングされた。
【０３９３】
ａ）第一段階ＰＣＲ
ＨＰＤ１．２−ＤＮＡ作製のための第一段階ＰＣＲの概要を図３９に示した。
【０３９４】
５’末端にＨｉｎｄＩＩＩ制限酵素切断部位を付加した、分泌シグナル配列、ＦＲＨ_１領域、ＣＤＲＨ_１領域およびＦＲＨ_２領域の一部をコードするＨＰＤ１−ＤＮＡ断片は、以下の条件で作製された。
反応液組成：
プラスミドｐｇＨＳＬ７Ａ６２　ＤＮＡ　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　７ＡＨ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＨＰＤ１Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０３９５】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３９６】
次に、ＦＲＨ_２領域の一部、ＣＤＲＨ_３領域およびＦＲＨ_３領域の一部をコードするＨＰＤ２−ＤＮＡ断片は、以下の条件で作製された。
【０３９７】
反応液組成：
プラスミドｐｇＨＳＬ７Ａ６２　ＤＮＡ　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＨＰＤ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＨＰＤ２Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０３９８】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０３９９】
ＰＣＲ後の各生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＨＰＤ１−ＤＮＡおよびＨＰＤ２−ＤＮＡに相当するＤＮＡバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０４００】
ｂ）第二段階ＰＣＲ
ＨＰＤ１．２−ＤＮＡ作製のための第二段階ＰＣＲの概要を図４０に示した。
【０４０１】
前出ＨＰＤ１−ＤＮＡ断片とＨＰＤ２−ＤＮＡ断片とを融合したＨＰＤ１．２−ＤＮＡ断片は、以下の条件で作製された。
【０４０２】
反応液組成：
第一段階ＰＣＲで作製したＨＰＤ１−ＤＮＡ溶液　　　　　　　１０μｌ
第一段階ＰＣＲで作製したＨＰＤ２−ＤＮＡ溶液　　　　　　　１０μｌ
オリゴヌクレオチドプライマー　７ＡＨ１Ｐ　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＨＰＤ２Ｎ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０４０３】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０４０４】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出された融合ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０４０５】
ＨＰＤ１．２−ＤＮＡ断片を保持するプラスミド作製方法の概要を図４１に示した。
【０４０６】
上記で得られたＨＰＤ１．２−ＤＮＡ断片を、フェノール抽出とエタノール沈澱によりさらに精製した後、制限酵素ＨｉｎｄＩＩＩと制限酵素ＳａｃＩにより消化した。
【０４０７】
前出プラスミドｐｇＨＳＬ７Ａ６２ＤＮＡ　１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＳａｃＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐｇＨＳＬ７Ａ６２　ＤＮＡと、予めＨｉｎｄＩＩＩとＳａｃＩで消化したＨＰＤ１．２−ＤＮＡ断片をＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのクロラムフェニコールを含むＬＢ液体培地で培養し、この培養物からアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＳａｃＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って、ＨＰＤ１．２−ＤＮＡ断片を保持するクローンを選択した。
【０４０８】
以上の操作により、ＨＶタイプヒト化重鎖の可変領域をコードするＤＮＡと、ヒトＩｇＧ１定常領域をコードするゲノミックＤＮＡ断片との融合断片を保持するプラスミドｐｇＨＰＤＨＶ３を得た。なお、プラスミドｐｇＨＰＤＨＶ３を保持する形質転換大腸菌　Ｅ．ｃｏｌｉ　ｐｇＨＰＤＨＶ３　ＳＡＮＫ　７０２９８は、平成１０年２月２６日付で独立行政法人産業技術総合研究所特許生物寄託センター（工業技術院生命工学工業技術研究所）に国際寄託され、受託番号ＦＥＲＭ　ＢＰ−６２７３が付された。
【０４０９】
次に、ｐｇＨＰＤＨＶ３　ＤＮＡ　１０μｇを、制限酵素ＨｉｎｄＩＩＩと制限酵素ＥｃｏＲＩにより消化した。一方、哺乳動物細胞用発現プラスミドｐＥＥ．６．１（ロンザ社製）１μｇを、予め制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドｐＥＥ．６．１　ＤＮＡ（１００ｎｇ）と、ＨｉｎｄＩＩＩとＥｃｏＲＩで消化したｐｇＨＰＤＨＶ３　ＤＮＡ（１０μｇ）とを、ＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株でクローニングした。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、該培養物からアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出た。このプラスミドＤＮＡを制限酵素ＨｉｎｄＩＩＩとＥｃｏＲＩで消化し、アガロースゲル電気泳動を行って挿入断片の有無を確認した。以上の操作により、ＨＶタイプヒト化重鎖をコードするＤＮＡが、ＣＭＶプロモーター下流に正方向で挿入されたプラスミドｐＥｇＰＤＨＶ３−２１を得た。
【０４１０】
（３）ヌクレオチド配列の確認
上記（２）で得られたプラスミドｐＥｇＰＤＨＶ３−２１が、目的とするヌクレオチド配列を有するＤＮＡを保持していることを確認するため、ヌクレオチド配列の決定を行なった。ここで用いられたプライマーは前出ＰＥＥＦ（配列表の配列番号１０４）、ＨＰＤ１Ｐ（配列表の配列番号１１８）、ＨＰＤ１Ｎ（配列表の配列番号１１９）およびＨＰＤ２Ｎ（配列表の配列番号１２０）であった。各プライマーが結合する位置を、図４２に示した。ヌクレオチド配列の決定は、アルカリ・ＳＤＳ法と塩化セシウム法により精製したプラスミドＤＮＡを鋳型とし、ジデオキシヌクレオチド鎖終結法で行なった。その結果、ｐＥｇＰＤＨＶ３−２１は配列表の配列番号１１６に示されヌクレオチド配列を保持していることが確認された。
【０４１１】
実施例２３　ＣＯＳ−１細胞用発現ベクターの構築
実施例１４および実施例２１で作製された各種ヒト化ＨＦＥ７Ａ−軽鎖発現ベクター（ｐ７ＡＬ−ＨＨ、ｐ７ＡＬ−ＨＭ、ｐ７ＡＬ−ＭＭ、ｐＬＰＤＨＨ７５およびｐＬＰＤＨＭ３２）と、実施例１５および実施例２２で作製された各種ヒト化ＨＦＥ７Ａ−重鎖発現ベクター（ｐＥｇ７ＡＨ−ＨおよびｐＥｇＰＤＨＶ３−２１）を用いて、ＣＯＳ−１細胞を用いた遺伝子発現のために最適化した発現ベクターを以下に記載する方法により作製した。
【０４１２】
１．ＣＯＳ−１細胞用軽鎖発現ベクターの構築
各種ヒト化ＨＦＥ−７Ａ軽鎖のＣＯＳ−１細胞用発現ベクターの構築法の概要を図４３に示した。
【０４１３】
（１）プロモーター増幅用プライマー（軽鎖用）の合成
ＳＲαプロモーター領域を含むＤＮＡ断片をＰＣＲにより作製するにあたり、以下に記載する２種のオリゴヌクレオチドプライマー：
５’−　ＴＧＣＡＣＧＣＧＴＧ　ＧＣＴＧＴＧＧＡＡＴ　ＧＴＧＴＧＴＣＡＧＴ　ＴＡＧ　−３’　（ＭＬＵＡ：配列表の配列番号１２１）；および
５’−　ＴＣＣＧＡＡＧＣＴＴ　ＴＴＡＧＡＧＣＡＧＡ　ＡＧＴＡＡＣＡＣＴＴ　Ｃ　−３’　（ＨＩＮＤＢ：配列表の配列番号１２２）を作製した。
【０４１４】
（２）発現ベクター（軽鎖用）の構築
ＳＲαプロモーター領域を含み、５’末端にＭｕｌＩ制限酵素切断部位、３’末端にＨｉｎｄＩＩＩ制限酵素切断部位がそれぞれ付加されたＬＳＲα−ＤＮＡ断片は、プラスミドｐＭＥ１８Ｓを鋳型とし、以下の条件で作製された。
【０４１５】
反応液組成：
プラスミドｐＭＥ１８Ｓ　ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＭＬＵＡ　　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＨＩＮＤＢ　　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０４１６】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０４１７】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されたＬＳＲα−ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０４１８】
次に、実施例１４および実施例２１で作製されたプラスミドｐ７ＡＬ−ＨＨ、ｐ７ＡＬ−ＨＭ、ｐ７ＡＬ−ＭＭ、ｐＬＰＤＨＨ７５またはｐＬＰＤＨＭ３２　１μｇを、制限酵素ＭｕｌＩとＨｉｎｄＩＩＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドＤＮＡと、予めＭｕｌＩとＨｉｎｄＩＩＩで消化したＬＳＲα−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、該培養物よりアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＭｕｌＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って、ＬＳＲα−ＤＮＡ断片を保持するクローンを選択した。
【０４１９】
以上の操作により、各種のヒト化軽鎖をコードするＣＯＳ−１細胞用発現ベクターｐＳＲＨＨ（ＨＨタイプ、ｐ７Ａ−ＨＨ由来）、ｐＳＲＨＭ（ＨＭタイプ、ｐ７Ａ−ＨＭ由来）、ｐＳＲＭＭ（ＭＭタイプ、ｐ７Ａ−ＭＭ由来）、ｐＳＲＰＤＨＨ（ＰＤＨＨタイプ、ｐＬＰＤＨＨ７５由来）およびｐＳＲＰＤＨＭ（ＰＤＭＭタイプ、ｐＬＰＤＨＭ３２由来）を得た。
【０４２０】
２．ＣＯＳ−１細胞用重鎖発現ベクターの構築
各種ヒト化重鎖のＣＯＳ−１細胞用発現ベクターの構築法の概要を図４４に示した。
【０４２１】
（１）プロモーター増幅用プライマー（重鎖用）の合成
ＳＲαプロモーター領域を含むＤＮＡ断片をＰＣＲにより作製するにあたり、前出のプライマーＨＩＮＤＢ（配列表の配列番号１２２）に加え、さらに１種のプライマー：
５’−　ＡＡＡＧＣＧＧＣＣＧ　ＣＴＧＣＴＡＧＣＴＴ　ＧＧＣＴＧＴＧＧＡＡ　ＴＧＴＧＴＧ　−３’（ＮＯＴＡ：配列表の配列番号１２３）を作製した。
【０４２２】
（２）発現ベクター（重鎖用）の構築
ＳＲαプロモーター領域を含み、５’末端にＮｏｔＩ制限酵素切断部位、３’末端にＨｉｎｄＩＩＩ制限酵素切断部位がそれぞれ付加されたＨＳＲα−ＤＮＡ断片は、プラスミドｐＭＥ１８Ｓを鋳型とし、以下の条件で作製された。
【０４２３】
反応液組成：
プラスミドｐＭＥ１８Ｓ　ＤＮＡ　　　　　　　　　　　　　２００ｎｇ
オリゴヌクレオチドプライマー　ＮＯＴＡ　　　　　　　　８０ｐｍｏｌ
オリゴヌクレオチドプライマー　ＨＩＮＤＢ　　　　　　　８０ｐｍｏｌ
ｄＮＴＰ混合液　　　　　　　　　　　　　　　　　　　　　　２０μｌ
１０×Ｐｆｕ緩衝液　　　　　　　　　　　　　　　　　　　　２０μｌ
ＰｆｕＤＮＡポリメラーゼ　　　　　　　　　　　　　　　　　１０単位
上記組成の反応液を、再蒸留水にて最終容量２００μｌとし、ＰＣＲに供試した。
【０４２４】
ＰＣＲ反応温度条件：９４℃で２分間加熱した後、９４℃で１分間、５５℃で１分間、７２℃で２分間の温度サイクルを３０回繰り返してから、７２℃で１０分間加温した。
【０４２５】
ＰＣＲ後の生成物は、フェノール抽出とエタノール沈澱の後、５％ポリアクリルアミドゲル電気泳動により分離された。電気泳動後のアクリルアミドゲルを、１μｇ／ｍｌの濃度のエチジウムブロミドにより染色し、生成物ＤＮＡを紫外線照射下で検出した。検出されＨＳＲα−ＤＮＡに相当するバンドを剃刀で切り出し、セントリコンおよびセントリリューターを用いてアクリルアミドゲルより溶出した。溶出したＤＮＡを７５００×ｇの遠心分離により濃縮し、エタノール沈澱の後、５０μｌの蒸留水に溶解した。
【０４２６】
次に、実施例１５および実施例２２で作製されたプラスミドｐＥｇ７ＡＨ−ＨまたはｐＥｇＰＤＨＶ３−２１　１μｇを、制限酵素ＮｏｔＩとＨｉｎｄＩＩＩで消化し、ＣＩＰで脱リン酸化した。この脱リン酸化プラスミドＤＮＡと、予めＮｏｔＩとＨｉｎｄＩＩＩで消化したＨＳＲα−ＤＮＡ断片とをＤＮＡライゲーションキットバージョン２．０（宝酒造社製）を用いて連結した後、大腸菌ＪＭ１０９株を形質転換し、最終濃度５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地上に塗布した。得られた形質転換体を、５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地で培養し、該培養物からアルカリ・ＳＤＳ法によりプラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＮｏｔＩとＨｉｎｄＩＩＩで消化し、アガロースゲル電気泳動を行って、ＨＳＲα−ＤＮＡ断片を保持するクローンを選択した。
【０４２７】
以上の操作により、各種のヒト化重鎖をコードするＣＯＳ−１細胞用発現ベクターｐＳＲｇ７ＡＨ（ｐＥｇ７ＡＨ−Ｈ由来）およびｐＳＲｇＰＤＨ（ｐＥｇＰＤＨＶ３−２１由来）を得た。
【０４２８】
実施例２４　ヒト化ＨＦＥ７Ａの各サブユニットをコードする遺伝子のＣＯＳ−１細胞での発現
実施例２３で得られたＣＯＳ−１細胞用発現ベクターによるＣＯＳ−１細胞の形質転換は、実施例１６に記載した方法により行なった。まず、ＣＯＳ−１細胞を、１０％ＦＣＳを含むα（＋）ＭＥＭ（ギブコ・ビーアールエル社製）を入れた細胞培養用フラスコ（培養面積２２５ｃｍ^２：住友ベークライト（株）社製）中でセミコンフルエントになるまで培養した。その後、培地を除去し、３ｍｌのトリプシン−ＥＤＴＡ溶液（シグマ社製）中、３７℃で３分間保温することにより、ＣＯＳ−１細胞をフラスコから遊離させた。遊離した細胞を、８００ｒｐｍ、２分間の遠心分離により回収し、ＰＢＳ（−）（日水製薬（株）社製）で２回洗浄してから、ＰＢＳ（−）で１×１０^８細胞数／ｍｌとなるように調製したものを、ＣＯＳ−１細胞懸濁液とした。
【０４２９】
一方、アルカリ・ＳＤＳ法と塩化セシウム密度勾配遠心分離法を用いて調製したヒト化重鎖発現ベクター（ｐＳＲｇ７ＡＨまたはｐＳＲｇＰＤＨ）ＤＮＡ　４μｇ、およびヒト化軽鎖発現ベクター（ｐＳＲＨＨ、ｐＳＲＨＭ、ｐＳＲＭＭ、ｐＳＲＰＤＨＨまたはｐＳＲＰＤＨＭ）ＤＮＡ　４μｇを下記に記載する組み合わせで混合した後、エタノール沈澱を行って、得られた沈澱をＰＢＳ（−）２０μｌに溶解した。得られたプラスミド溶液２０μｌを、先に調製したＣＯＳ−１細胞懸濁液２０μｌ（２×１０^６細胞）と混合したものを電極間隔２ｍｍのチャンバー（ＦＣＴ−１３：島津製作所（株）社製）に入れ、遺伝子導入装置ＧＴＥ−１（島津製作所（株）社製）にセットした。その後、６００Ｖ下、５０μＦのパルスを、１秒間隔で２回与えることにより、目的とするプラスミドＤＮＡをＣＯＳ−１細胞内に導入した。パルスを与えた後のチャンバー内の細胞−ＤＮＡ混合液を、１０％ウシ胎児血清を含むα（＋）ＭＥＭ　５ｍｌに懸濁し、細胞培養用フラスコ（培養面積２５ｃｍ^２：住友ベークライト社製）に移した。これを５％ＣＯ_２下、３７℃で７２時間培養した後に培養上清を回収した。
【０４３０】
上記の方法により、下記［Ａ］−［Ｆ］の条件でそれぞれＣＯＳ−１細胞を形質転換し、培養上清を回収した。
【０４３１】
［Ａ］：プラスミドＤＮＡを含まない条件；
［Ｂ］：ｐＳＲｇＰＤＨおよびｐＳＲＰＤＨＨのコ・トランスフェクション；
［Ｃ］：ｐＳＲｇＰＤＨおよびｐＳＲＰＤＨＭのコ・トランスフェクション；
［Ｄ］：ｐＳＲｇ７ＡＨおよびｐＳＲＨＨのコ・トランスフェクション；
［Ｅ］：ｐＳＲｇ７ＡＨおよびｐＳＲＨＭのコ・トランスフェクション；
［Ｆ］：ｐＳＲｇ７ＡＨおよびｐＳＲＭＭのコ・トランスフェクション。
【０４３２】
実施例２５　Ｆａｓに対する結合能の検定
実施例２４で調製されたそれぞれの培養上清試料について、実施例１７に記載した方法により、含有する目的発現生成物の濃度を算出し、最終的な目的生成物濃度が１００ｎｇ／ｍｌとなるように１０％　ＦＣＳを含むα（＋）ＭＥＭで調製した。なお、培養上清試料中の目的生成物濃度が１００ｎｇ／ｍｌを下回った場合、得られた培養上清は、まずセントリプレップ−１０（アミコン社製）を用いて濃縮された。すなわち、培養上清５ｍｌをセントリプレップ−１０に移し、３０００×ｇの遠心分離により濃縮してから、再び実施例１７に記載した方法により目的生成物濃度を算出した。
【０４３３】
次に、１００ｎｇ／ｍｌの濃度に調製された各試料溶液を１０％　ＦＣＳを含むα（＋）ＭＥＭで２倍希釈する操作を繰り返し、希釈系列を作製した。これらの試料について、ヒトＦａｓ融合タンパク質に対する結合能を実施例１８に記載したＥＬＩＳＡ法により測定した。
【０４３４】
その結果、実施例２４で調製された培養上清試料のうち、［Ｂ］、［Ｃ］、［Ｄ］、［Ｅ］および［Ｆ］の条件のものは、ヒトＦａｓ融合タンパク質との結合能を有するものを含んでいることが確認された（図４５）。
【０４３５】
実施例２６　ＨＦＥ７ＡのＦａｓへの結合に対する競合阻害
実施例２４で調製された各培養上清試料について、予め実施例１７で示す方法により、それぞれが含有する目的発現生成物の濃度を算定し、１０％　ＦＣＳを含むα（＋）ＭＥＭで最終的な目的生成物濃度が１μｇ／ｍｌとなるように調製した。なお、培養上清試料中の目的生成物濃度が１μｇ／ｍｌを下回った場合は、その培養上清をセントリプレップ−１０を用いて濃縮してから１μｇ／ｍｌとなるように調製した。
【０４３６】
次に、この１μｇ／ｍｌの濃度に調製した試料を１０％　ＦＣＳを含むα（＋）ＭＥＭで二倍希釈する操作を繰り返し、希釈系列を作製した。一方、ＡＰ−ＨＦＥ７Ａを、１０％　ＦＣＳを含むα（＋）ＭＥＭにより５０ｎｇ／ｍｌの濃度に調整し、上記希釈系列試料と等量混合した。その後、実施例１９記載の方法により、各試料のＨＦＥ７Ａに対する競合阻害活性を測定した。
【０４３７】
その結果、実施例２４で調製された培養上清試料のうち、［Ｂ］、［Ｃ］、［Ｄ］、［Ｅ］および［Ｆ］の条件のものは、ハイブリドーマ培養物より調製したＨＦＥ７ＡのヒトＦａｓ融合タンパク質に対する結合を特異的に阻害することが確認された（図４６）。
【０４３８】
実施例２７　アポトーシス誘導活性
実施例２４で調製された培養上清試料を、実施例２２記載の方法で最終的な目的生成物濃度が１００ｎｇ／ｍｌとなるように調整した（ただし、調整用培地としては１０％　ＦＣＳを含むＲＰＭＩ１６４０培地を用いた）。この１００ｎｇ／ｍｌの試料を１０％　ＦＣＳを含むＲＰＭＩ１６４０培地で二倍希釈する操作を繰り返し、希釈系列を作製した。その後、実施例２０記載の方法により、各試料のＷＲ１９Ｌ１２ａ細胞に対する細胞傷害活性を測定した。
【０４３９】
その結果、実施例２４で調製された培養上清試料のうち、［Ｂ］、［Ｃ］、［Ｄ］、［Ｅ］および［Ｆ］の条件のものは、ハイブリドーマ培養物より調製されたＨＦＥ７Ａと同様に、ヒトＦａｓ抗原を発現したＴリンパ腫細胞株に対してアポトーシスを誘導することが示された（図４７）。
【０４４０】
製剤例
本発明の分子は、水またはそれ以外の薬理学的に許容し得る溶液に溶解した無菌性溶液または懸濁液のアンプルとして使用に供される。また、無菌粉末製剤（本発明の分子を凍結乾燥するのが好ましい）をアンプルに充填しておき、使用時に薬理学的に許容し得る溶液で希釈してもよい。
【０４４１】
【発明の効果】本発明により、Ｆａｓ／Ｆａｓリガンド系に起因する各種の疾病（自己免疫疾患（全身性エリテマトーデス、橋本病、慢性関節リウマチ、移植片対宿主病、シェーグレン症候群、悪性貧血、アジソン氏病、強皮症、グッドパスチャー症候群、クローン氏病、自己免疫性溶血性貧血、自然不妊、重症筋無力症、多発性硬化症、バセドー病、突発性血小板減少性紫斑病、インスリン依存性糖尿病）、アレルギー、アトピー、動脈硬化症、心筋炎、心筋症、糸球体腎炎、再生不良性貧血、肝炎（劇症肝炎、慢性肝炎、ウイルス性肝炎（Ｃ型肝炎、Ｂ型肝炎、Ｄ型肝炎）またはアルコール性肝炎）、後天性免疫不全症候群または臓器移植後の拒絶反応）に対する予防または治療薬として有用な、霊長類および非霊長類動物のＦａｓの間で保存されているエピトープに特異的な抗原結合領域を有する分子が提供された。
【０４４２】
【配列表フリーテキスト】
配列番号１２：Ｎ１
配列番号１３：Ｃ３Ｎ
配列番号１４：Ｎ３Ｎ
配列番号１５：ＣＴＮ２
配列番号１８：Ｈ１
配列番号１９：Ｈ２
配列番号２０：Ｌ１
配列番号２１：Ｌ２
配列番号２２：Ｈ３
配列番号２３：Ｈ４
配列番号２４：Ｌ３
配列番号２５：Ｌ４
配列番号４１：コントロールペプチド
配列番号４６：コントロールペプチド
配列番号４７：ＨＶＫＩＩ５−４
配列番号４８：ＨＫＣＬ３−３
配列番号４９：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＨタイプ軽鎖
配列番号５０：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＨタイプ軽鎖
配列番号５１：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＭタイプ軽鎖
配列番号５２：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＭタイプ軽鎖
配列番号５３：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＭＭタイプ軽鎖
配列番号５４：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＭＭタイプ軽鎖
配列番号５５：７ＡＬ１Ｐ
配列番号５６：７ＡＬ１Ｎ
配列番号５７：７ＡＬ２Ｐ
配列番号５８：７ＡＬ２Ｎ
配列番号５９：７ＡＬ３ＰＡ
配列番号６０：７ＡＬ３Ｎ
配列番号６１：７ＡＬ４Ｐ
配列番号６２：７ＡＬ４Ｎ
配列番号６３：７ＡＬＣＰ
配列番号６４：７ＡＬＣＮ
配列番号６５：Ｍ７ＡＬ２Ｎ
配列番号６６：Ｍ７ＡＬ３ＰＡ
配列番号６７：７ＡＬ４ＮＡ
配列番号６８：ＳＰ１
配列番号６９：ＳＰ２
配列番号７０：ＳＰ３
配列番号７１：ＳＰ４
配列番号７２：ＳＰ５
配列番号７３：ＳＰ６
配列番号７４：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ重鎖可変領域とＩｇＧ−ＣＨ１のＮ末端の５アミノ酸
配列番号７５：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ重鎖可変領域とＩｇＧ−ＣＨ１のＮ末端の５アミノ酸
配列番号７６：７ＡＨ１Ｐ
配列番号７７：７ＡＨ１ＮＮＥＷ
配列番号７８：７ＡＨ２Ｎ
配列番号７９：７ＡＨ２ＰＮＥＷ
配列番号８０：７ＡＨ３Ｐ
配列番号８１：７ＡＨ３Ｎ
配列番号８２：７ＡＨ４Ｐ
配列番号８３：７ＡＨ４Ｎ
配列番号８４：５’Ｈｉｎｄ
配列番号８５：ＩＧＧＣＰＳＴＮ
配列番号８６：ＩＧＧＣＰＳＴＰ
配列番号８７：Ｅｃｏ３’
配列番号８８：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ重鎖
配列番号８９：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ重鎖
配列番号９０：ＩＧ０１
配列番号９１：ＩＧ０２
配列番号９２：ＩＧ０３
配列番号９３：ＩＧ０４
配列番号９４：ＩＧ０５
配列番号９５：ＩＧ０６
配列番号９６：ＩＧ０７
配列番号９７：ＩＧ０８
配列番号９８：ＩＧＰ５
配列番号９９：ＩＧＰ６
配列番号１００：ＩＧＰ７
配列番号１０１：ＩＧＰ８
配列番号１０２：Ｈ５＋
配列番号１０３：Ｈ５−
配列番号１０４：ＰＥＥＦ
配列番号１０５：ＰＥＥＢ
配列番号１０６：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＰＤＨＨタイプ軽鎖
配列番号１０７：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＰＤＨＨタイプ軽鎖
配列番号１０８：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＰＤＨＭタイプ軽鎖
配列番号１０９：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＰＤＨＭタイプ軽鎖
配列番号１１０：ＬＰＤ１Ｐ
配列番号１１１：ＬＰＤ１Ｎ
配列番号１１２：ＬＰＤ２Ｐ
配列番号１１３：ＬＰＤ２Ｎ
配列番号１１４：ＬＰＤ３Ｐ
配列番号１１５：ＬＰＤ３Ｎ
配列番号１１６：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＶタイプ重鎖
配列番号１１７：ヒト化抗Ｆａｓ抗体ＨＦＥ７Ａ　ＨＶタイプ重鎖
配列番号１１８：ＨＰＤ１Ｐ
配列番号１１９：ＨＰＤ１Ｎ
配列番号１２０：ＨＰＤ２Ｎ
配列番号１２１：ＭＬＵＡ
配列番号１２２：ＨＩＮＤＢ
配列番号１２３：ＮＯＴＡ
【０４４３】
【配列表】

【図面の簡単な説明】
【図１】ｐｈＦａｓ−ＡＩＣ２の構築図。
【図２】ｐＭＥ−ＨおよびｐＭＥ−Ｌの構築図。
【図３】ＨＦＥ７Ａのエピトープ限定のためのＥＬＩＳＡの結果を表す図。
【図４】ＨＦＥ７Ａのエピトープ限定のための競合アッセイの結果を表す図。
【図５】ＨＦＥ７Ａの毒性試験の結果を表す図。
【図６】劇症肝炎モデルを用いた試験の結果を表す図。
【図７】コラーゲン誘導関節炎の発症予防試験の結果を表す図。
【図８】ＶＨＨ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図９】ＶＨＨ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図１０】ＶＨＨ−ＤＮＡ作製のための第三段階ＰＣＲの概要。
【図１１】ＶＨＨ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要。
【図１２】ＶＨＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図１３】ＶＨＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図１４】ＶＨＭ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要。
【図１５】ＶＭＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図１６】ＶＭＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図１７】ＶＭＭ−ＤＮＡ作製のための第三段階ＰＣＲの概要。
【図１８】ＶＭＭ−ＤＮＡ断片を保持するプラスミド作製方法の概要。
【図１９】ヒト化軽鎖シークエンシングプライマーの結合位置を示す図。
【図２０】ＶＤ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図２１】ＶＤ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図２２】ＶＤ−ＤＮＡ作製のための第三段階ＰＣＲの概要。
【図２３】ＶＤ−ＤＮＡ断片を保持するプラスミド作製方法の概要。
【図２４】ヒトＩｇＧ１のＣＨ１領域とイントロンを含むＤＮＡ（ＩＧ５’−ＤＮＡ）断片の作製法の概要。
【図２５】ヒトＩｇＧ１のヒンジ領域、ＣＨ２領域、ＣＨ３領域およびイントロンを含むゲノムＤＮＡ（ＩＧ３’−ＤＮＡ）断片の作製法の概要。
【図２６】発現ベクタープラスミドｐＥｇ７ＡＨ−Ｈの作製手順の概要。
【図２７】ヒト化重鎖シークエンシングプライマーの結合位置を示す図。
【図２８】ヒト化抗Ｆａｓ抗体のヒトＦａｓ融合タンパク質に対する結合活性を示す図。
【図２９】ヒトＦａｓ融合タンパク質に対するＨＦＥ７Ａとヒト化抗Ｆａｓ抗体との競合阻害を示す図。
【図３０】ヒト化ＨＦＥ７ＡのＷＲ１９Ｌ１２ａに対する細胞障害活性を示す図。
【図３１】ＬＰＤＨＨ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図３２】ＬＰＤＨＨ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図３３】ＬＰＤＨＨ−ＤＮＡ作製のための第三段階ＰＣＲの概要。
【図３４】ＬＰＤＨＨ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要。
【図３５】ＬＰＤＨＭ−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図３６】ＬＰＤＨＭ−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図３７】ＬＰＤＨＭ−ＤＮＡ作製のための第三段階ＰＣＲの概要。
【図３８】ＬＰＤＨＭ−ＤＮＡ断片を保持する発現プラスミド作製方法の概要。
【図３９】ＨＰＤ１．２−ＤＮＡ作製のための第一段階ＰＣＲの概要。
【図４０】ＨＰＤ１．２−ＤＮＡ作製のための第二段階ＰＣＲの概要。
【図４１】ＨＰＤ１．２−ＤＮＡ断片を保持するプラスミド作製方法の概要。
【図４２】ＨＶタイプヒト化重鎖をコードするＤＮＡのシークエンシングプライマー結合位置を示す図。
【図４３】各種ヒト化軽鎖をＣＯＳ−１細胞で発現させるために最適化した発現ベクターの構築図。
【図４４】各種ヒト化重鎖をＣＯＳ−１細胞で発現させるために最適化した発現ベクターの構築図。
【図４５】各種形質転換ＣＯＳ−１細胞培養上清中のヒトＦａｓ融合タンパク質に対する結合活性を示す図。
【図４６】各種形質転換ＣＯＳ−１細胞培養上清中の、ヒトＦａｓ融合タンパク質とＨＦＥ７Ａの結合に対する競合阻害活性を示す図。
【図４７】各種形質転換ＣＯＳ−１細胞培養上清中のＷＲ１９Ｌ１２ａに対する細胞障害活性を示す図。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing and selecting an anti-Fas antibody that recognizes a Fas antigen that is a cell membrane molecule involved in apoptosis.
[0002]
[Prior art]
In vivo, physiological cell death that occurs due to normal cell replacement is called apoptosis, and is distinguished from necrosis (necrosis), which is a pathological cell death (for example, see Non-Patent Document 1). So-called programmed cell death is the death of certain types of cells that have been programmed to die in vivo in advance, and apoptosis is one of them. In apoptosis, phenomena such as cell surface curvature, nuclear chromatin condensation, and chromosomal DNA fragmentation are characteristically observed.
[0003]
Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize self antigens. In fact, it is known that in the process of maturation of T lymphocytes, 95% or more of cells such as cells that react with self die in the thymus (for example, see Non-Patent Document 2). It is considered that the onset of so-called autoimmune diseases is caused by the generation of autoreactive lymphocytes due to the failure of apoptosis in lymphocyte differentiation (for example, see Non-Patent Document 3).
[0004]
Such molecules involved in apoptosis include Fas (for example, see Non-Patent Document 4), tumor necrosis factor receptor (Tumor Necrosis Factor Receptor) (for example, see Non-Patent Document 5), and CD40 (for example, see Non-Patent Document 5). And non-patent document 6) and perforin / granzyme A (for example, see non-patent document 7). Fas is a transmembrane protein present on the cell surface. When a protein called Fas ligand binds to its extracellular region, it induces apoptosis in the cell. If the Fas / Fas ligand system is abnormal, cells that adversely affect homeostasis, which should be eliminated by apoptosis, remain without being eliminated, or apoptosis occurs in cells essential for homeostasis. Various obstacles occur by being induced. In the present invention, they are collectively referred to as “disease caused by abnormal Fas / Fas ligand system”.
[0005]
The following common mechanism can be found in the onset or progression of disease caused by abnormal Fas / Fas ligand system. That is, abnormal cells that express Fas but are not removed (hereinafter referred to as “abnormal cells”) attack other normal tissues or cells or abnormally proliferate, causing damage to normal tissues or cells. , Each cause the symptoms. In some cases, these disorders are caused or exacerbated by normal tissues or cells stimulated by abnormal cells that express Fas and undergo apoptosis. Specifically, examples of the diseases caused by abnormalities in the Fas / Fas ligand system include those described below.
[0006]
Autoimmune diseases: Various human autoimmune diseases (Hashimoto's disease, systemic lupus erythematosus, Sjogren's syndrome, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolysis Many associations have been reported between Fas / Fas ligand system and primary anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis).
[0007]
As a genetic abnormality of the Fas / Fas ligand system, in mice, lpr (lymphoproliferation) mice having abnormalities in Fas, lpr ^cg (Lpr gene complementing gld gene) mice and gld (generized lymphoproliferative disease) having abnormalities in Fas ligand are known, and it is evident that all exhibit various autoimmune disease symptoms accompanied by specific systemic lymphadenopathy. ing. MRLlpr mice, which are spontaneous model mice of human systemic lupus erythematosus, produce autoantibodies with marked lymphadenopathy and develop nephritis due to immune complexes. Since this mouse has an abnormality in the Fas gene, immune tolerance to self-antigens due to apoptosis through Fas does not occur in the periphery, and as a result, activated self-reactive T cells are accumulated continuously. It is presumed to exhibit the above-mentioned disease state (for example, see Non-Patent Document 8). Lymphadenopathy, hypergammaglobulinemia, CD4 in humans ⁻ CD8 ⁻ It has been reported that autoimmune disease-like cases including two pediatric cases showing remarkable expansion of T cells (for example, see Non-Patent Document 9) are caused by Fas gene abnormality (for example, , Non-Patent Documents 10 and 11), and Autoimmune Lymphoproliferative Syndrome (ALPS). Therefore, not only in mice but also in humans, the apoptosis-inducing system via Fas is greatly involved in establishing and maintaining immune tolerance to self-antigens, and it is considered that various autoimmune diseases are induced by the abnormalities.
[0008]
In addition, since the majority of T cells involved in tissue destruction by infiltrating the affected area of rheumatoid arthritis patients express Fas, it is known that rheumatoid arthritis also has an aspect as an autoimmune disease. (For example, see Non-Patent Document 12).
[0009]
Insulin-dependent diabetes mellitus is often caused by the destruction of pancreatic beta cells by self-reactive T cells, resulting in an absolute deficiency of insulin secretion. Is important.
[0010]
Furthermore, in graft-versus-host disease after bone marrow transplantation, Fas expression is increased in the target organ to be damaged, and the degree of the increased expression correlates with the degree of target organ damage (for example, see Non-Patent Document 13). ). Therefore, in preventing or treating this disease, it is necessary to prevent apoptosis of cells of the target organ and reduce the number of cells that attack the target organ.
[0011]
Allergic diseases: Inflammatory cells involved in allergic diseases are usually activated and infiltrate the lesions, apoptosis is suppressed, cell life is prolonged, and the localization of the inflammatory cells increases, and Demonstrate function. In an experimental model of eosinophilic airway inflammation in mice, administration of an anti-Fas antibody with apoptosis-inducing activity via the airway may eliminate submucosal eosinophil infiltration seen after allergen inhalation. It is known (for example, see Non-Patent Document 14). Therefore, it is possible to reduce symptoms by inducing apoptosis in inflammatory cells.
[0012]
Rheumatoid arthritis: Apart from the aforementioned autoimmune disease aspects of rheumatoid arthritis, it is known that synovial cells that are abnormally proliferating in the affected area express Fas (for example, see Non-Patent Document 15). .). When the synovial cells derived from the affected area are stimulated with an anti-Fas antibody having an apoptosis-inducing activity, apoptosis is induced (for example, see Non-Patent Document 16). That is, in the affected part of a patient with rheumatoid arthritis, the Fas / Fas ligand system is not functioning normally, and autoreactive T cells and synovial cells that abnormally proliferate are not removed while expressing Fas. I have.
[0013]
Atherosclerosis: The final image of cell death at the center of atherosclerotic lesions is necrosis (necrosis), and it has been reported that apoptosis is involved in the progression and regression processes (see, for example, Non-Patent Document 17). Electron microscopic findings of atherosclerotic lesions show an apoptotic image of smooth muscle cells with nucleus enrichment (see, for example, Non-Patent Document 18). It has also been reported that foam cells, which are a type of macrophage that gather in the intimal layer of the artery and phagocytose fat in early lesions of atherosclerosis, express Fas and induce apoptosis by an anti-Fas antibody having apoptosis-inducing activity. (For example, see Non-Patent Document 19). Arteriosclerotic lesions often involve lymphocyte infiltration, suggesting that Fas ligand of T cells and Fas of macrophages may control arteriosclerosis (for example, see Non-Patent Document 20).
[0014]
Myocarditis / cardiomyopathy: Fas / Fas ligand system may be involved in the pathogenesis of ischemic heart disease, viral heart disease, autoimmune heart disease such as dilated cardiomyopathy and chronic cardiomyopathy Is high. Myocarditis is inflammation of the heart muscle that is thought to be mainly caused by a virus such as Coxsackie virus, and typically develops from cold pain-like symptoms, followed by chest pain, arrhythmia, heart failure, shock, and the like. Cardiomyopathy is defined as "cardiomyopathy of unknown cause", and its cause is also considered to be viral infection. In an examination of a myocarditis model in a mouse with heart failure, apoptotic cells (nucleus enrichment, fragmentation) are observed in the mouse heart after virus inoculation. Increased expression of Fas was also observed in the mouse heart, suggesting that apoptosis was induced by Fas ligand derived from infiltrating inflammatory cells, mainly lymphocytes (for example, see Non-Patent Document 21). It is known that cultured rat cardiomyocytes undergo apoptosis due to ischemia, and at the same time, mRNA encoding Fas increases in the cells (for example, see Non-Patent Document 22).
[0015]
Renal disease: In many chronic kidney diseases, the remodeling of glomerular tissue leads to the accumulation of extracellular matrix in the glomerulus, which promotes glomerular sclerosis, abolition of the filtration function, and the pathology of chronic renal failure. To present. In the advanced glomerular sclerosis model, typical apoptotic images are observed in the hardened area at the electron microscope level, and an increase in glomerular apoptosis is observed in accordance with the decrease in the number of glomerular cells as the hardening progresses (For example, see Non-Patent Document 23). Also, in acute glomerulonephritis, it is known that nephritis tends to recover by reducing apoptotic mesangial cells by apoptosis (for example, see Non-Patent Documents 24 and 25). Furthermore, it has been reported that cells expressing Fas in glomeruli are significantly increased in purpura nephritis, lupus nephritis and the like (for example, see Non-Patent Document 26).
[0016]
Aplastic anemia: Fas expression is more remarkable on the surface of hematopoietic progenitor cells in patients with aplastic anemia than in normal individuals, suggesting that Fas is involved in the reduction of hematopoietic stem cells. (For example, see Non-Patent Document 27).
[0017]
Hepatitis: In fulminant hepatitis, it is known that apoptosis is induced in many hepatocytes, and when the anti-Fas antibody Jo2 is intraperitoneally administered to mice, hepatic cell death similar to fulminant hepatitis is observed. Therefore, it is considered that apoptosis of hepatocytes via Fas is involved in the onset thereof (for example, see Non-Patent Documents 28 and 29). In the immunohistological examination, enhancement of Fas expression was observed in the hepatocytoplasm of the hepatocyte necrosis part of the chronic hepatitis lesion and in the liver cell membrane of the liver disease lesion such as fatty liver (for example, Non-patent Document 30). And 31).
[0018]
In addition, Fas is expressed in the lesions of chronic persistent hepatitis C and chronic active hepatitis, all of which have hepatocytes stained sporadically and are accompanied by infiltrating lymphocytes that appear to be cytotoxic T cells around. (For example, see Non-Patent Document 32). Cytotoxic T cells have the function of inducing apoptosis in infected cells by Fas ligand on the cell surface, but also induce apoptosis in nearby normal cells. This phenomenon called neighbor injury is caused by the fact that many cells in the body express Fas when the cells themselves or nearby cells are infected.
[0019]
In addition, in the case where RNA derived from hepatitis C virus disappears by administration of interferon for chronic hepatitis C, the expression of Fas in liver tissue is significantly reduced.
[0020]
Hepatocytes from patients with acute hepatic failure have increased cell surface Fas, and the cells undergo apoptosis with an anti-Fas antibody having apoptosis-inducing activity. Furthermore, in alcoholic hepatitis, the Fas ligand is also expressed in the hepatocytes themselves inside the pseudolobule (for example, see Non-Patent Document 33). In situ hybridization studies of Fas ligand gene expression showed that expression was found in liver infiltrating lymphocytes in patients with acute liver failure and in hepatocytes themselves inside pseudolobules in cases of alcoholic cirrhosis. Apoptosis is thought to be induced by a different mechanism in cirrhosis, but in any case, it is speculated that the Fas / Fas ligand system is abnormal. It is also known that in a hepatitis model mouse, liver damage is inhibited by administration of a substance having a property of inhibiting the binding of Fas ligand to Fas (for example, see Non-Patent Document 34).
[0021]
Acquired immunodeficiency syndrome: A cause of immunodeficiency in a patient infected with a human immunodeficiency virus (hereinafter referred to as "HIV") is death of a very large number of immune cells not infected with HIV by apoptosis. Helper T cells are killed by contact with HIV, but since growth factors derived from helper T cells are essential for suppressing apoptosis of cytotoxic T cells, when helper T cells disappear, cytotoxic T cells undergo apoptosis. Wake up. Fas expression in peripheral blood lymphocytes of HIV-infected persons is correlated with the progress of the disease state (for example, see Non-Patent Documents 35 and 36). In non-infected persons, Fas-positive peripheral blood lymphocytes are stimulated by Fas. Apoptosis is not easily caused by apoptosis, but apoptosis is induced in Fas via short term in peripheral blood lymphocytes of an infected person (for example, see Non-Patent Document 37). The apoptosis of immune cells in E. coli is also thought to be due to abnormalities in the Fas / Fas ligand system.
[0022]
Rejection after organ transplantation: Rejection after organ transplantation is similar to an autoimmune disease, except that the transplanted organ targeted by cytotoxic T cells is derived from the donor. By suppressing the function of cytotoxic T cells, improvement of symptoms can be expected.
[0023]
For each of these diseases, abnormal cells (eg, autoreactive T cells in autoimmune diseases, foam cells in atherosclerosis, mesangial cells in acute glomerulonephritis, infected cells in viral infections, synovial cells in rheumatoid arthritis Protecting normal tissues or cells is an effective therapeutic measure, but a drug that only induces apoptosis through Fas can cause damage to normal tissues even if abnormal cells can be removed. A drug that is highly active and only inhibits Fas-mediated apoptosis can protect normal cells but cannot remove abnormal cells. In fact, anti-mouse Fas monoclonal antibody Jo2 having apoptosis-inducing activity causes fulminant hepatitis in mice (for example, see Non-Patent Document 38). Until now, an anti-Fas antibody that has been confirmed to have no apoptosis when administered to mice while having apoptosis-inducing activity has not been known.
[0024]
Immunoglobulin G (hereinafter simply referred to as "IgG") is a light polypeptide chain having a molecular weight of about 23,000 (hereinafter referred to as "light chain") and a heavy polypeptide chain having a molecular weight of about 50,000 (hereinafter referred to as "heavy chain"). It consists of two each. Both heavy and light chains have a repeating structure of a region in which the amino acid sequence is conserved, consisting of about 110 residues, and these constitute a basic unit (hereinafter, referred to as “domain”) of the three-dimensional structure of IgG. The heavy and light chains are composed of four consecutive and two domains, respectively. In both heavy and light chains, the amino-terminal domain has a greater variation in amino acid sequence between antibody molecules than other domains, and this domain is a variable domain (hereinafter referred to as “V domain”). Called. At the amino terminus of IgG, heavy and light chain V domains associate complementarily to form a variable region. In contrast, the remaining domains form a constant region as a whole. The constant region has a sequence characteristic of each animal species.For example, since the constant region of mouse IgG is different from the constant region of human IgG, mouse IgG is recognized as a foreign substance by the human immune system. As a result, a human anti-mouse antibody (Human Anti Mouse Antibody: hereinafter referred to as “HAMA”) response occurs (for example, see Non-Patent Document 39). Therefore, mouse antibodies cannot be repeatedly administered to humans. In order to administer such an antibody to humans, it is necessary to modify the antibody molecule so as not to cause a HAMA response while maintaining the specificity of the antibody.
[0025]
According to the result of X-ray crystal structure analysis, such a domain generally has a long cylindrical structure in which two antiparallel β sheets each having three to five β chains are overlapped. In the variable region, three loops are assembled for each of the heavy and light chain V domains to form an antigen binding site. Each of these loops is called a complementarity determining region (hereinafter, referred to as “CDR”), and the amino acid sequence is most remarkably mutated. The portion of the variable region other than the CDR generally has a role of maintaining the structure of the CDR, and is called a “framework”. Kabat et al. Collected a large number of primary sequences of heavy and light chain variable regions and prepared a table in which each primary sequence was classified into CDRs and frameworks based on the conservation of the sequences (for example, Non-Patent Document 40). reference.). In addition, each framework was classified into a plurality of subgroups having a common feature in amino acid sequence. In addition, it has been found that a corresponding framework exists between human and mouse.
[0026]
From the study on the structural characteristics of IgG, the following method for producing a humanized antibody was devised.
[0027]
In the early stage of research, a chimeric antibody was proposed in which the variable region of a mouse-derived antibody was conjugated to a human-derived constant region (for example, see Non-Patent Document 41). However, such chimeric antibodies can still induce a HAMA response, especially when administered for long periods of time, since they contain many non-human amino acid residues (see, for example, Non-Patent Document 42).
[0028]
As a method for further reducing the amino acid residues derived from non-human mammals that may express a HAMA response to humans, a method has been proposed in which only the CDR portion is incorporated into a human-derived antibody (for example, Non-Patent Documents) 43)), but grafting with CDR alone was generally not sufficient to retain immunoglobulin activity against the antigen.
[0029]
On the other hand, in 1987, using data from X-ray crystal structure analysis,
(I) In the amino acid sequence of the CDR, there are a site that directly binds to the antigen and a site that maintains the structure of the CDR itself. The three-dimensional structure that the CDR can take has a plurality of typical patterns (canonical structure). Be classified as
(Ii) It has been found that the class of the canonical structure is determined not only by CDR but also by the type of amino acid at a specific position in the framework portion (for example, see Non-Patent Document 44).
[0030]
Based on this finding, it has been suggested that when using the CDR grafting method, it is necessary to graft amino acid residues of some frameworks to human antibodies in addition to CDR sequences (for example, see Patent Document 1).
[0031]
In general, an antibody derived from a non-human mammal having a CDR to be transplanted is defined as a “donor”, and a human antibody to which the CDR is transplanted is defined as an “acceptor”. The present invention also complies with this definition.
[0032]
A point to be considered when performing the CDR transplantation method is to preserve the CDR structure as much as possible and to maintain the activity of the immunoglobulin molecule. To achieve this goal:
(I) Which subgroup should the acceptor select?
(Ii) Which amino acid residue should be selected from the donor framework
It is necessary to pay attention to the following two points.
[0033]
Queen et al. Have proposed a design method in which the amino acid residue of the donor framework is grafted together with the CDR sequence into an acceptor when the amino acid residue satisfies at least one of the following criteria (for example, see Patent Document 1):
(A) Amino acids in the acceptor framework region are rare at that position and the corresponding amino acids of the donor are common at said position of the acceptor.
(B) the amino acid is in the immediate vicinity of one of the CDRs;
(C) It is predicted that the amino acid has a side chain atom within about 3% of the CDR in a three-dimensional immunoglobulin model and is capable of interacting with an antigen or with the CDR of a humanized antibody.
[0034]
However, there is no example of successfully obtaining an IgG-type humanized anti-Fas antibody having an apoptosis-inducing activity. Further, the anti-Fas monoclonal antibody of the present invention is a monoclonal antibody having novel characteristics not known hitherto. Therefore, no humanized version of the monoclonal antibody is known.
[0035]
[Patent Document 1]
Japanese Unexamined Patent Publication No. Hei 4-502408
[Non-patent document 1]
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[Non-patent document 2]
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[Non-Patent Document 3]
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[Non-patent document 4]
"The Journal of Experimental Medicine", 1989, vol. 169, p. 1747-1756
[Non-Patent Document 5]
"Cell", 1990, Vol. 61, p. 351-359
[Non-Patent Document 6]
"Nature", 1993, vol. 364, p. 645-648
[Non-Patent Document 7]
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[Non-Patent Document 8]
Nature, 1995, vol. 373, p. 385-
[Non-Patent Document 9]
"Journal of Clinical Investigation", 1992, Vol. 90, p. 334-
[Non-Patent Document 10]
"Cell", 1995, Vol. 81, p. 935-
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[Non-Patent Document 12]
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[Non-Patent Document 18]
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[Non-Patent Document 19]
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[Non-Patent Document 20]
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[Non-Patent Document 22]
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[Non-Patent Document 23]
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[Non-Patent Document 24]
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[Non-Patent Document 25]
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[Non-Patent Document 26]
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[Non-Patent Document 27]
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[Non-Patent Document 28]
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[Non-Patent Document 29]
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[Non-Patent Document 30]
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[Non-Patent Document 31]
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[Non-Patent Document 33]
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[Non-Patent Document 34]
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[Non-Patent Document 36]
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[Non-Patent Document 37]
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[Non-Patent Document 38]
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[Non-Patent Document 39]
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[Non-Patent Document 40]
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[Non-Patent Document 41]
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[Non-Patent Document 43]
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[Non-Patent Document 44]
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[0036]
[Problems to be solved by the invention]
A first object of the present invention is to bind to Fas on the surface of such abnormal cells to induce apoptosis, and to induce apoptosis in normal cells by binding Fas ligand to Fas. Another object of the present invention is to provide a novel anti-Fas antibody having a function of preventing apoptosis, or a molecule similar thereto.
[0037]
Several monoclonal antibodies specifically binding to human Fas, including those having apoptosis-inducing activity, are known, but none of them has been found to bind to mouse Fas. Similarly, some monoclonal antibodies that bind to mouse Fas are known, but none of them bind to human Fas. That is, in evaluating the efficacy of an existing anti-human Fas monoclonal antibody as a medicine, the above-mentioned disease state model mice cannot be used. A second object of the present invention is to provide an anti-Fas antibody that binds to both human Fas and mouse Fas, or a molecule similar thereto.
[0038]
A third object of the present invention is to provide a prophylactic or therapeutic agent for the above-mentioned various diseases, comprising the above-mentioned anti-Fas antibody or a molecule similar thereto as an active ingredient.
[0039]
[Means for Solving the Problems]
The present invention
(1) a molecule having an antigen-binding region specific to an epitope conserved between primate and non-primate Fas,
(2) The molecule according to (1), wherein the primate is human.
(3) The molecule according to (1) or (2), wherein the non-primate animal is a rodent.
(4) the molecule of (3), wherein the rodent is a mouse;
(5) a molecule having an antigen-binding region specific to an epitope conserved among mammalian Fas,
(6) a monoclonal antibody produced by mouse-mouse hybridoma HFE7A (FERM BP-5828),
(7) a molecule characterized by having all the same CDRs as the six CDRs derived from the anti-human Fas monoclonal antibody produced by the mouse-mouse hybridoma HFE7A (FERM BP-5828);
(8) a molecule comprising an antigen-binding region specific to an epitope recognized by a monoclonal antibody produced by a mouse-mouse hybridoma HFE7A (FERM BP-5828),
(9) The molecule according to any one of (1) to (5), (7) or (8), which is an antibody;
(10) a heavy chain polypeptide comprising an amino acid sequence represented by the following general formula (I):
-FRH ₁ -CDRH ₁ -FRH ₂ -CDRH ₂ -FRH ₃ -CDRH ₃ -FRH ₄ -(I)
(Where, FRH ₁ Represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids,
CDRH ₁ Represents an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing;
FRH ₂ Represents an arbitrary amino acid sequence consisting of 14 amino acids,
CDRH ₂ Represents an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing;
FRH ₃ Represents an arbitrary amino acid sequence consisting of 32 amino acids,
CDRH ₃ Represents an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing;
FRH ₄ Represents an arbitrary amino acid sequence consisting of 11 amino acids,
Each amino acid is connected by a peptide bond.)
And a light chain polypeptide comprising an amino acid sequence represented by the following general formula (II):
-FRL ₁ -CDRL ₁ -FRL ₂ -CDRL ₂ -FRL ₃ -CDRL ₃ -FRL ₄ -(II)
(Where FRL ₁ Represents an arbitrary amino acid sequence consisting of 23 amino acids,
CDRL ₁ Represents an amino acid sequence represented by SEQ ID NO: 5 in the sequence listing,
FRL ₂ Represents an arbitrary amino acid sequence consisting of 15 amino acids,
CDRL ₂ Represents an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing,
FRL ₃ Represents an arbitrary amino acid sequence consisting of 32 amino acids,
CDRL ₃ Represents an amino acid sequence represented by SEQ ID NO: 7 in the sequence listing;
FRL ₄ Represents an arbitrary amino acid sequence consisting of 10 amino acids,
Each amino acid is connected by a peptide bond.)
A molecule characterized by having
(11) At least one of (1) to (5) or (7) to (10), which has at least one of the following biological properties a) to f): One of the molecules listed:
a) having apoptosis-inducing activity on T cells expressing Fas;
b) has an effect of improving autoimmune disease symptoms in MRLgld / gld mice;
c) does not induce liver damage;
d) has a therapeutic or preventive effect against fulminant hepatitis caused by anti-mouse Fas monoclonal antibody Jo2;
e) has an effect of preventing the onset of collagen-induced arthritis;
f) has an apoptosis-inducing activity on synovial cells derived from a patient with rheumatoid arthritis;
(12) The molecule according to (11), which has all of the biological properties according to (a) to (f).
(13) The molecule according to any one of (10) to (12), which is an antibody,
(14) The molecule according to (9) or (13), which is humanized.
(15) the molecule of (9) or (13), which is an immunoglobulin G-type antibody;
(16) the antibody of (6), which is humanized;
(17) (1) to (5) or (7) to (15), wherein apoptosis can be induced in abnormal cells expressing Fas and apoptosis of normal cells can be prevented. The molecule according to any one of the above,
(18) The molecule according to any one of (1) to (5), (7) to (13), (15) or (17), or the antibody according to (6), wherein the antibody is a Fas / Fas ligand. A method for evaluating the effect of a drug on a disease state affected by an interaction, comprising administering the molecule or the antibody to a model animal of the disease state, and then administering the molecule or the antibody to a change in the disease state. Comparing between a treated animal and a non-treated animal,
(19) Epitopes conserved between primate and non-primate Fas in at least one human immunoglobulin light chain or fragment thereof and at least one human immunoglobulin heavy chain or fragment thereof, respectively. It has an antigen-binding region specific for an epitope that is conserved between primate and non-primate Fas, which can be obtained from an antibody that specifically binds by grafting the respective CDR of the antibody. A humanized antibody,
(20) Similarity of the variable region between human immunoglobulin light chain and human immunoglobulin heavy chain with antibodies that specifically bind to epitopes conserved between primate and non-primate Fas, respectively. (19) The humanized antibody according to (19), wherein the antibody having the highest potential is selected.
(21) from an antibody that specifically binds to an epitope conserved between primate and non-primate Fas, the antigen recognition site of the antibody in the heavy and / or light chain framework regions of the antibody (19) or (20), wherein an amino acid important for maintaining the structure of (19) or (20) is transplanted into a human immunoglobulin light chain or a fragment thereof and / or a human immunoglobulin heavy chain or a fragment thereof. ) Described humanized antibody,
(22) It binds to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, characterized by (1) to (5), (7) to (15), (17) or (19) (21) the molecule or the antibody according to any one of the above,
(23) The amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence listing, the amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence listing, and the amino acid sequence represented by amino acid number 1 of SEQ ID NO: 54 of the sequence listing 218, a light sequence comprising any one of the amino acid sequences of amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing or the amino acid sequences of amino acid numbers 1 to 218 of SEQ ID NO: 109 in the sequence listing. The molecule or antibody according to any one of (1) to (5), (7) to (15), (17) or (19) to (22), which has a chain polypeptide;
(24) A heavy chain polypeptide comprising any one of the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 117 of the sequence listing. The molecule or the antibody according to any one of (1) to (5), (7) to (15), (17) or (19) to (23),
(25) a light chain polypeptide comprising the amino acid sequence of amino acids 1 to 218 of SEQ ID NO: 50 in the sequence listing, and a heavy chain comprising the amino acid sequence of amino acids 1 to 451 of SEQ ID NO: 89 of the sequence listing The molecule or antibody according to (23) or (24), which is composed of a polypeptide;
(26) a light chain polypeptide comprising the amino acid sequence of amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing, and a heavy chain comprising the amino acid sequence of amino acid numbers 1 to 451 of SEQ ID NO: 117 of the sequence listing The molecule or antibody according to (23) or (24), which is composed of a polypeptide;
(27) Any one of the polypeptides in the molecule or antibody according to any one of (1) to (5), (7) to (15), (17) or (19) to (26) DNA encoding
(28) a DNA encoding the light chain polypeptide or the heavy chain polypeptide of the antibody according to (6) or (16),
(29) a DNA encoding a polypeptide comprising an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 11 in the sequence listing;
(30) a DNA encoding a polypeptide comprising an amino acid sequence represented by amino acid numbers 1 to 445 of SEQ ID NO: 9 in the sequence listing,
(31) an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence listing, an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence listing, and an amino acid sequence represented by amino acid number 1 of SEQ ID NO: 54 of the sequence listing 218, any one of the amino acid sequences represented by amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing or the amino acid sequences represented by amino acid numbers 1 to 218 of SEQ ID NO: 109 in the sequence listing. A DNA encoding a polypeptide consisting of
(32) a polypeptide comprising any one of the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 89 of the Sequence Listing or the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 117 of Sequence Listing DNA encoding
(33) The DNA according to (29), which comprises the nucleotide sequence shown by nucleotide numbers 61 to 393 of SEQ ID NO: 10 in the sequence listing.
(34) The DNA according to (29), which comprises the nucleotide sequence of nucleotides 61 to 714 of SEQ ID NO: 10 in the sequence listing.
(35) the DNA according to (30), which comprises the nucleotide sequence of nucleotide numbers 58 to 420 of SEQ ID NO: 8 in the sequence listing;
(36) the DNA according to (30), which comprises the nucleotide sequence of nucleotides 58 to 1392 of SEQ ID NO: 8 in the sequence listing;
(37) a nucleotide sequence represented by nucleotide numbers 100 to 753 of SEQ ID NO: 49 in the Sequence Listing, a nucleotide sequence represented by nucleotide numbers 100 to 753 of SEQ ID NO: 51 in the Sequence Listing, and a nucleotide sequence represented by nucleotide number 100 of SEQ ID NO: 53 of the Sequence Listing (31) comprising the nucleotide sequence shown in SEQ ID NO: 753, the nucleotide sequence shown in nucleotide numbers 100 to 753 of SEQ ID NO: 106 in the sequence listing, or the nucleotide sequence shown in nucleotide sequence 100 to 753 of SEQ ID NO: 108 in the sequence listing. ) Described DNA,
(38) The DNA according to (32), comprising the nucleotide sequence represented by nucleotide numbers 84 to 2042 of SEQ ID NO: 88 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 78 to 2036 of SEQ ID NO: 116 in the sequence listing. ,
(39) a recombinant DNA vector comprising the DNA according to any one of (27) to (38),
(40) a recombinant DNA vector comprising the DNA of any one of (29), (31), (33), (34) or (37),
(41) a recombinant DNA vector comprising the DNA of any one of (30), (32), (35), (36) or (38),
(42) a host cell transformed with the recombinant DNA vector according to (39),
(43) a host cell transformed with the recombinant DNA vector according to (40) and / or (41),
(44) a host cell transformed with the recombinant DNA vector according to (40) or (41),
(45) the host cell of any one of (41) to (43), wherein the host cell is derived from a mammal;
(46) Transformed E. coli. coli pME-L (FERM BP-5867),
(47) Transformed E. coli coli pME-H (FERM BP-5868),
(48) Transformed E. coli. coli pHSGMM6 SANK 73697 (FERM BP-6071),
(49) Transformed E. coli. coli pHSGHM17 SANK 73597 (FERM BP-6072),
(50) Transformed E. coli coli pHSGHH7 SANK 73497 (FERM BP-6073),
(51) Transformed E. coli. coli pHSHM2 SANK 70198 (FERM BP-6272),
(52) Transformed E. coli coli pHSHH5 SANK 70398 (FERM BP-6274),
(53) Transformed E. coli. coli pgHSL7A62 SANK73397 (FERM BP-6074),
(54) Transformed E. coli coli pgHPDHV3 SANK70298 (FERM BP-6273),
(55) A method for producing an anti-Fas antibody, comprising culturing the host cell according to (44) or (45), and then recovering the anti-Fas antibody from the culture.
(56) mouse-mouse hybridoma HFE7A (FERM BP-5828),
(57) The antibody according to (6), wherein the mouse-mouse hybridoma HFE7A (FERM BP-5828) is cultured under conditions capable of producing immunoglobulin, and then the immunoglobulin is recovered from the culture. Production method,
(58) The molecule according to any one of (1) to (5), (7) to (15), (17) or (19) to (26), or the molecule according to (6) or (16). An agent for preventing or treating a disease caused by an abnormality in a Fas / Fas ligand system, comprising an antibody as an active ingredient;
(59) Diseases caused by abnormal Fas / Fas ligand system are autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus), allergy, atopy, artery Sclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, B, D) or alcoholic hepatitis) The prophylactic or therapeutic agent according to (58), which is selected from the group consisting of acquired immunodeficiency syndrome and rejection after organ transplantation.
(60) Diseases caused by abnormal Fas / Fas ligand system include autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture syndrome, Crohn's disease, autoimmune hemolytic anemia, spontaneous infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus) The prophylactic or therapeutic agent according to (58) or (59),
(61) the preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is allergy;
(62) the preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is rheumatoid arthritis;
(63) The preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is atherosclerosis.
(64) the prophylactic or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is myocarditis or cardiomyopathy;
(65) the prophylactic or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is glomerulonephritis;
(66) The preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is aplastic anemia.
(67) The disease caused by abnormality of the Fas / Fas ligand system is hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, B, D) or alcoholic hepatitis). The prophylactic or therapeutic agent according to (58) or (59),
(68) the preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is acquired immunodeficiency syndrome;
(69) The preventive or therapeutic agent according to (58 or 59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is rejection after organ transplantation.
[0040]
In order to obtain a mouse monoclonal antibody that binds to both human and mouse Fas using a portion where the amino acid sequence is conserved between human and mouse as an epitope, a normal mouse such as a BALB / c mouse is converted to human Fas. When immunized, cells that produce antibodies that also bind to mouse Fas are eliminated in the thymus as autoantigen-reactive antibody-producing cells. It is presumed that the Fas-Fas ligand system is involved in the elimination of self-reactive T cells in the thymus [Shin Yonehara (1994) Nikkei Science Supplement 110, 66-77]. When a mouse deficient in the Fas-Fas ligand system such as a mouse in which the gene cannot be expressed (hereinafter referred to as "Fas knockout mouse") is used as an immunized animal, an antibody that binds to mouse Fas can also be obtained. . However, the immunized animal used in the present invention is not limited to the Fas knockout mouse. For example, even if other experimental animals such as a rat and a rabbit are deficient in the Fas-Fas ligand system, the immunized animal of the present invention Can be used.
[0041]
The present inventors have produced a hybridoma that produces a novel anti-Fas monoclonal antibody that binds to human and mouse Fas by immunizing a Fas knockout mouse with human Fas and fusing spleen cells of the mouse with mouse myeloma cells. Then, the monoclonal antibody was purified from the culture supernatant. This novel anti-Fas monoclonal antibody induced apoptosis in T cells of mouse and non-human primates expressing Fas. That is, according to the present invention, at least between Fas of a primate including human and rodent Fas can be commonly recognized by the antibody of the present invention, and apoptosis can be induced by binding of the antibody of the present invention. It was revealed that various epitopes exist.
[0042]
In addition, this novel anti-Fas monoclonal antibody had the effect of reducing the symptoms of autoimmune disease model mice. Even more surprisingly, it has been found that this novel anti-Fas monoclonal antibody does not have the side effect of inducing hepatic injury, which has been known among conventional anti-Fas antibodies having apoptosis-inducing activity. In addition, the present inventors cloned the genes encoding the heavy and light chains of the novel anti-Fas monoclonal antibody from the above hybridomas, elucidated the nucleotide sequences thereof, and analyzed the nucleotide sequences encoded by those genes. The amino acid sequences of the CDRs in the amino acid sequences of the chain and light chain were determined. The present inventors have also prepared expression vectors comprising the genes encoding the heavy and light chains, respectively, and used them in the culture supernatant obtained by co-transfection of cultured animal cells with these vectors. Produced a recombinant anti-Fas antibody that reacts with Fas. The thus-obtained anti-Fas antibody and its recombinant have an activity of protecting the liver from fulminant hepatitis caused by Fas-mediated apoptosis, and are also effective for the prevention and treatment of rheumatoid arthritis, respectively. Therefore, the antibody has an activity of inducing apoptosis through Fas in abnormal cells and an activity of inhibiting apoptosis of normal cells through Fas in abnormal cells, and is effective in preventing or preventing diseases caused by abnormal Fas / Fas ligand system. It was found to be effective for treatment. The present inventors produced a recombinant antibody having no immunogenicity against humans while retaining the activity as an anti-Fas antibody by grafting the CDR amino acid sequence of the mouse-derived anti-Fas monoclonal antibody to a human antibody. And completed the present invention.
[0043]
When a monoclonal antibody commonly recognizing a homologous protein derived from a heterologous animal binds to a protein antigen, the amino acid sequence is conserved between the heterologous animals around the binding site (epitope) on the antigen side, that is, amino acid sequence homology Although it is considered that the homology is often kept high, the epitope in the Fas molecule to which the antibody of the present invention binds is not limited to the region where the homology in the primary structure is maintained. That is, the "epitope conserved between the primate and non-primate Fas" in the present invention is that homology in higher-order structure is maintained between heterologous animals, and consequently common to a single monoclonal antibody. And regions that can be recognized as described above.
[0044]
In the present invention, the “antigen-binding region” refers to an intramolecular region having the same function as an antigen-binding region in an antibody molecule. This antigen binding region is not limited to those derived from a particular antibody or combination of antibodies. Furthermore, the antigen-binding region and the binding region for an antigen in a specific antibody molecule need only be similar in that they can bind to the antigen. Is not a requirement of the antigen binding region. Therefore, for example, even when the antigen-binding region is designed based on the CDRs of a known antibody and transplanted into a human antibody, the resulting antigen-binding region is not necessarily similar to the antigen-binding region of the known antibody. However, from the viewpoint of maintaining the specific binding ability to the antigen, the similarity between the two is preferably high.
[0045]
The “molecule characterized by having an antigen-binding region” in the present invention is preferably an antibody, but is not limited thereto, as long as the antigen-binding region binds to an epitope in a Fas molecule. Thus, for example, the antigen binding region may be added to the support of an affinity purification column. However, hereinafter, unless otherwise stated, "molecules of the invention" will generally refer to antibodies.
[0046]
When the molecule of the present invention is an antibody, any of immunoglobulin G (IgG), A (IgA), E (IgE) and M (IgM) can be suitably used as the antibody. However, usually IgG is more preferred.
[0047]
In the present invention, as described above, by transplanting at least all of the amino acid sequences of CDRs from a non-human animal-derived antibody to a human antibody, immunogenicity to humans can be reduced while maintaining the function as an antibody. The artificially modified antibodies are referred to as “humanized antibodies”.
[0048]
In the present invention, "FR" refers to a framework region. For example, FRH ₁ Refers to the most N-terminal framework region in the heavy chain subunit variable region, FRL ₄ Refers to the fourth framework region from the N-terminus in the light chain subunit variable region. Similarly, for example, CDRH ₁ Refers to the CDR located at the most N-terminus in the heavy chain subunit variable region; ₃ Refers to the third CDR from the N-terminus in the light chain subunit variable region.
[0049]
BEST MODE FOR CARRYING OUT THE INVENTION
The anti-Fas monoclonal antibody of the present invention can be obtained, for example, by immunizing a Fas knockout mouse with human Fas, and culturing a hybridoma obtained by fusing spleen cells of the mouse with mouse myeloma cells.
[0050]
In the production of a monoclonal antibody, at least the following working steps are required. That is,
(A) purification of a biopolymer used as an antigen,
(B) after immunizing the animal by injecting the antigen, collecting blood and assaying the antibody titer to determine the timing of splenectomy, and then preparing antibody-producing cells;
(C) preparation of myeloma cells (hereinafter referred to as “myeloma”);
(D) cell fusion between antibody-producing cells and myeloma,
(E) selecting a hybridoma group producing the desired antibody,
(F) division into single cell clones (cloning),
(G) optionally, culturing hybridomas to produce monoclonal antibodies in large quantities, or breeding hybridoma-transplanted animals,
(H) Examination of the physiological activity of the monoclonal antibody thus produced and its recognition specificity, or examination of its properties as a labeling reagent.
[0051]
Hereinafter, a method for preparing an anti-Fas monoclonal antibody will be described in detail along the above steps, but the method for preparing the antibody is not limited thereto. For example, antibody-producing cells other than spleen cells and myeloma can also be used.
[0052]
(A) Purification of antigen
Examples of the antigen include an extracellular region of human Fas and an extracellular region of mouse interleukin 3 receptor (hereinafter referred to as “IL3R”) [Nishimura, Y .; et al. (1995) Immunol. 154, 4395-4403], a recombinant protein obtained by introducing the phFas-AIC2 expression vector into a monkey-derived cell line COS-1, expressing it, and partially purifying it (hereinafter referred to as “recombinant human Fas”). ) Is valid. phFas-AIC2 can be prepared by incorporating a DNA encoding a fusion protein of human Fas and mouse IL3R into an expression vector pME18S for animal cells. However, the DNA, vector, host and the like encoding Fas are not limited to these.
[0053]
Specifically, a transformant obtained by transforming COS-1 cells with phFas-AIC2 was cultured, and a fusion protein of human Fas and mouse IL3R produced in the culture supernatant was subjected to a resource Q column. Recombinant Fas can be partially purified by performing ion exchange chromatography using (trade name; manufactured by Pharmacia).
[0054]
Purified Fas itself present on the cell membrane of a human cell line can also be used as the antigen. In addition, the primary structure of Fas is known [Itoh, N .; , Et al. (1991) Cell 66, 233-243], a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is chemically synthesized by a method well known to those skilled in the art, and this is used as an antigen. You can also.
[0055]
(B) Preparation of antibody-producing cells
The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or alum, and the animal is immunized as an immunogen. Fas knockout mice are most preferably used as experimental animals, and such mice are described in Senju, S. et al. , Et al (1996) International Immunology 8, 423].
[0056]
The method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.
[0057]
Immunization can be performed once or multiple times at appropriate intervals (preferably at one to five week intervals). Thereafter, the antibody titer to the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operations can be enhanced by using the animal whose antibody titer is sufficiently high as a source of antibody-producing cells. Generally, it is preferable to use antibody-producing cells derived from an animal 3 to 5 days after the final immunization for subsequent cell fusion.
[0058]
The method for measuring the antibody titer used herein includes radioisotope immunoassay (hereinafter referred to as "RIA"), enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA"), fluorescent antibody method, passive hemagglutination Although various known techniques such as the method can be mentioned, the RIA method or the ELISA method is more preferable from the viewpoints of detection sensitivity, speed, accuracy, and possibility of automation of the operation.
[0059]
The measurement of the antibody titer in the present invention can be performed, for example, by the following procedure according to the ELISA method. First, purified or partially purified Fas is adsorbed on a solid surface such as a 96-well plate for ELISA, and a solid surface on which no antigen is adsorbed is a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as “BSA”). ), And after washing the surface, it is brought into contact with a serially diluted sample (for example, mouse serum) as a first antibody, and the anti-Fas antibody in the sample is bound to the antigen. Further, an antibody against a mouse antibody that is enzyme-labeled as a second antibody is added to bind to the mouse antibody, a substrate of the enzyme is added after washing, and a change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.
[0060]
(C) Myeloma preparation process
As the myeloma, a cell line generally obtained from a mouse, for example, an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma strain P3X63Ag8U. 1 (P3-U1) [Yelton, D .; E. FIG. et al. Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1) [Kohler, G. et al. et al. European J. et al. Immunology, 6, 511-519 (1976)], Sp2 / O-Ag14 (SP-2) [Shulman, M .; et al. Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, J. et al. F. et al. J. Immunology, 123, 1548-1550 (1979)], P3X63Ag8 (X63) [Horibata, K. et al. and Harris, A. W. Nature, 256, 495-497 (1975)]. These cell lines are prepared by adding 8-azaguanine to an appropriate medium, for example, 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”). Added medium], Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium; hereinafter referred to as "DMEM"). Was subcultured 3 to 4 days before cell fusion in a normal medium [eg, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Inc.)]. ⁷ Keep the above cell number.
[0061]
(D) Cell fusion
Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any part of an individual, such as spleen, lymph node, peripheral blood, or a combination thereof as appropriate. , But splenocytes are most commonly used.
[0062]
After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.
[0063]
The spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 1640) or a phosphate buffered saline (hereinafter, referred to as “PBS”), and the ratio of spleen cells to myeloma is 5: 1 to 10: 1. Mix to the extent necessary and centrifuge. After removing the supernatant and thoroughly loosening the precipitated cell group, 1 ml of a serum-free medium containing 50% (w / v) polyethylene glycol (molecular weight: 1,000 to 4,000) is added dropwise with stirring. Thereafter, 10 ml of serum-free medium is slowly added, followed by centrifugation. The supernatant was discarded again, and the precipitated cells were placed in a normal medium (hereinafter referred to as "HAT") containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as "HAT") solution and mouse interleukin-2 (hereinafter referred to as "IL-2"). Medium)), dispensed into each well of a culture plate (hereinafter, referred to as “plate”), and cultured at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide. HAT medium is supplemented as needed on the way.
[0064]
(E) Selection of hybridoma group
When the myeloma cell is an 8-azaguanine resistant strain, that is, a hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient strain, the unfused myeloma cell and a fusion cell of myeloma cells are: Cannot survive in HAT containing medium. On the other hand, a fusion cell of antibody-producing cells or a hybridoma of antibody-producing cells and myeloma cells can survive, but a fusion cell of antibody-producing cells has a long life. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma of the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.
[0065]
With respect to the hybridomas that have grown in a colony, the medium is replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the anti-Fas antibody titer is measured by, for example, an ELISA method. However, when the above fusion protein is used as an antigen for ELISA, it is necessary to remove the clone so as not to select a clone producing an antibody that specifically binds to the extracellular region of the mouse IL3 receptor. . The presence or absence of such a clone can be confirmed, for example, by ELISA using the mouse IL3 receptor or its extracellular region as an antigen.
[0066]
The method using an 8-azaguanine-resistant cell line has been described above, but other cell lines can also be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.
(F) Cloning
By measuring the antibody titer in the same manner as described in the step (b), the hybridoma that has been found to produce a specific antibody is transferred to another plate and cloned. The cloning method includes a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator. And a cell sorter for separating one cell using a cell sorter. The limiting dilution method is simple and often used.
[0067]
For the wells in which the antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those having a stable antibody titer are selected as anti-Fas monoclonal antibody-producing hybridoma strains. Furthermore, by selecting a hybridoma that produces an antibody that binds to mouse Fas by the same method, an anti-Fas monoclonal antibody-producing cell is obtained. Examples of mouse Fas used at this time include, for example, an expression plasmid vector pME18S-mFas-AIC [Nishimura, Y. et al., For a DNA encoding a fusion protein of the extracellular region of mouse Fas and the extracellular region of mouse IL3 receptor. et al. (1995) Immunol. 154, 4395-4403] into cultured animal cells and expressed, purified mouse Fas, or cells expressing mouse Fas on the cell surface.
[0068]
In addition, mouse-mouse hybridoma HFE7A, which is an anti-Fas monoclonal antibody-producing cell suitable as a base antibody for preparing the humanized anti-Fas antibody of the present invention, was obtained from the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST). International Depositary with the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology on February 19, 1997, and assigned the accession number FERM BP-5828. Therefore, for example, when preparing an antibody using mouse-mouse hybridoma HFE7A, the antibody can be prepared from step (g) described below, omitting step (f).
[0069]
(G) Preparation of monoclonal antibody by hybridoma culture
The hybridoma that has completed cloning is cultured by changing the medium from an HT medium to a normal medium. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The anti-Fas monoclonal antibody containing the prophylactic or therapeutic agent of the present invention as an active ingredient can be obtained by purifying the supernatant in this large-scale culture using a method known to those skilled in the art such as gel filtration. In addition, by growing the hybridoma in the abdominal cavity of a mouse of the same strain (for example, BALB / c as described above) or a Nu / Nu mouse, the anti-Fas containing the preventive or therapeutic agent of the present invention as an active ingredient can be obtained. Ascites containing a large amount of monoclonal antibodies can be obtained.
[0070]
As a simple purification method, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit; manufactured by Pharmacia) or the like can also be used.
[0071]
The monoclonal antibody thus obtained has high antigen specificity for human and mouse Fas.
[0072]
(H) Monoclonal antibody assay
The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, examples of the identification method include the Ouchterlony method, the ELISA method, and the RIA method. The Otterlony method is simple but requires a concentration operation when the concentration of the monoclonal antibody is low. On the other hand, when the ELISA method or the RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbed solid phase as it is, and the antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. Isotypes and subclasses can be identified. As a simpler method, a commercially available kit for identification (for example, Mouse Typer Kit; manufactured by Bio-Rad) can be used.
[0073]
Furthermore, protein quantification can be performed by the Foreign Lowry method and a method of calculating from the absorbance at 280 nm [1.4 (OD280) = 1 mg / ml immunoglobulin].
[0074]
Identification of the recognition epitope of the monoclonal antibody can be performed as follows. First, various partial structures of the molecule recognized by the monoclonal antibody are prepared. In preparing the partial structure, a method for preparing various partial peptides of the molecule using a known oligopeptide synthesis technique, a DNA sequence encoding the desired partial peptide using a gene recombination technique into a suitable expression plasmid There are methods of integration, production inside and outside the host such as E. coli, etc., but for the above purpose, it is common to use both in combination. For example, a series of polypeptides which are sequentially shortened by an appropriate length from the C-terminus or N-terminus of an antigen protein are produced using a gene recombination technique well known to those skilled in the art, and the reactivity of the monoclonal antibody against them is examined. , Determine a rough recognition site.
[0075]
After that, the oligopeptide of the corresponding portion, or a variant of the peptide, etc., are further variously synthesized using oligopeptide synthesis techniques well known to those skilled in the art, and the prophylactic or therapeutic agent of the present invention is contained as an active ingredient. Epitopes are defined by examining the binding of the monoclonal antibodies to those peptides or by examining the peptide's competitive inhibitory activity on the binding of the monoclonal antibodies to the antigen. As a simple method for obtaining various kinds of oligopeptides, commercially available kits (for example, a SPOTs kit (manufactured by Genosis Biotechnology), a series of multipin peptide synthesis kits using a multipin synthesis method (manufactured by Chiron), etc.) Can also be used.
[0076]
Next, a DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody of the present invention is prepared by preparing mRNA from a hybridoma cell producing the above-mentioned anti-Fas monoclonal antibody, and converting the mRNA into cDNA with reverse transcriptase. Can be obtained by isolating DNAs encoding the heavy or light chains of the antibody, respectively.
[0077]
For the extraction of mRNA, a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method and the like can be employed, but a guanidine thiocyanate / cesium chloride method is preferred. To prepare mRNA from cells, first, total RNA is prepared, and poly (A) such as oligo (dT) cellulose or oligo (dT) latex beads is prepared from the total RNA. ⁺ It can be carried out by a method of purifying using a carrier for RNA purification or a method of directly purifying from a cell lysate using the carrier. As a method for preparing total RNA, alkaline sucrose density gradient centrifugation [Dougherty, W. et al. G. FIG. and Hiebert, E.A. (1980) Violology 101, 466-474], a guanidine thiocyanate / phenol method, a guanidine thiocyanate / trifluorocesium method, a phenol / SDS method, etc., but a method using guanidine thiocyanate and cesium chloride [Chirgwin, J .; M. , Et al. (1979) Biochemistry 18, 5294-5299].
[0078]
Poly (A) obtained as described above ⁺ After using RNA as a template to synthesize a single-stranded cDNA by a reverse transcriptase reaction, a double-stranded cDNA can be synthesized from the single-stranded cDNA. This method includes the S1 nuclease method [Estratidis, A. et al. , Et al. (1976) Cel 17, 279-288], and the Gubler-Hoffman method [Gubler, U.S.A. and Hoffman, B .; J. (1983) Gene 25, 263-269], and the Okayama-Berg method [Okayama, H .; and Berg, P.M. (1982) Mol. Cell. Biol. 2, 161-170], and in the present invention, the polymerase chain reaction (hereinafter referred to as “PCR”) using single-stranded cDNA as a template [Saiki, R .; K. , Et al. (1988) Science 239,
487-49], that is, the so-called RT-PCR method is preferable.
[0079]
The thus obtained double-stranded cDNA is incorporated into a cloning vector, the resulting recombinant vector is introduced into a microorganism such as Escherichia coli, and transformed, and a transformant is selected using tetracycline resistance or ampicillin resistance as an index. can do. Transformation of E. coli is performed by the Hanahan method [Hanahan, D .; (1983) Mol. Biol. 166, 557-580], that is, by adding the recombinant DNA vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, it is necessary to have the above-mentioned drug resistance gene. It is also possible to use cloning vectors other than plasmids, for example, lambda phage.
[0080]
As a method for selecting a strain having cDNA encoding each subunit of the objective anti-human Fas antibody from the transformants obtained as described above, for example, the following various methods can be adopted. When the target cDNA is specifically amplified by the RT-PCR method, these operations can be omitted.
[0081]
(1) Method using polymerase chain reaction
When the whole or a part of the amino acid sequence of the target protein has been elucidated, oligonucleotide primers of sense strand and antisense strand corresponding to the part of the amino acid sequence are synthesized, and these are combined to perform polymerase chain reaction [Saiki, R. K. , Et al. (1988) Science 239, 487-49] to amplify a DNA fragment encoding the desired anti-human Fas antibody heavy or light chain subunit. As the template DNA used here, for example, a cDNA synthesized by reverse transcriptase reaction from mRNA of a hybridoma (FERM BP-5828) producing an anti-human Fas monoclonal antibody HFE7A can be used.
[0082]
The DNA fragment thus prepared can be directly incorporated into a plasmid vector using a commercially available kit or the like. ³² P, ³⁵ A target clone can also be selected by labeling with S or biotin or the like, and performing colony hybridization or plaque hybridization using this as a probe.
[0083]
For example, the monoclonal antibody HFE7A suitable as the anti-Fas monoclonal antibody of the present invention is an immunoglobulin G1 (hereinafter referred to as “IgG1”) molecule, which is composed of a heavy chain (γ1 chain) and a light chain (κ chain) subunit. Complex. As a method for examining the partial amino acid sequence of each of these subunits, each subunit is isolated using a well-known method such as electrophoresis or column chromatography, and then an automatic protein sequencer (for example, manufactured by Shimadzu Corporation) A method of analyzing the N-terminal amino acid sequence of each subunit using PPSQ-10) or the like is preferable.
[0084]
A method for collecting cDNA encoding each subunit of the anti-human Fas monoclonal antibody protein from the target transformant obtained as described above is a known method [Maniatis, T. et al. , Et al. (1982) in "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory, NY. Reference]. For example, it can be carried out by separating a fraction corresponding to vector DNA from cells and cutting out a DNA region encoding a subunit of interest from the plasmid DNA.
[0085]
(2) Screening method using synthetic oligonucleotide probe
When the whole or part of the amino acid sequence of the target protein has been elucidated (the sequence may be that of any region of the target protein as long as it is a specific sequence of a plurality of contiguous sequences), the oligo corresponding to the amino acid sequence Nucleotides are synthesized (in this case, any of a nucleotide sequence deduced with reference to codon usage or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences can be used. This can be reduced by using a probe ( ³² P, ³⁵ The DNA of the transformant is hybridized with a denatured and fixed nitrocellulose filter, and the obtained positive strain is selected.
[0086]
The determination of the sequence of the DNA thus obtained can be carried out, for example, by the Maxam-Gilbert chemical modification method [Maxam, A .; M. and Gilbert, W.C. (1980) in "Methods in Enzymology" 65, 499-576] and the dideoxynucleotide chain termination method [Messing, J. et al. and Vieira, J. et al. (1982) Gene 19, 269-276].
[0087]
In recent years, an automatic base sequence determination system using a fluorescent dye has become widespread (for example, a sequence robot “CATALYST 800” manufactured by PerkinElmer Japan and a model 373A DNA sequencer).
[0088]
By using such a system, the DNA nucleotide sequencing operation can be performed efficiently and safely. Determining the entire amino acid sequence of the heavy and light chains of the monoclonal antibody of the present invention from the nucleotide sequence of the DNA of the present invention and the N-terminal amino acid sequence data of the heavy and light chains thus determined. Can be.
[0089]
The heavy and light chains of an immunoglobulin each consist of a variable region and a constant region, and the variable region further comprises a complementarity-determining region (hereinafter referred to as “CDR”; both heavy and light chains have three positions) and the same. Consists of adjacent framework regions (4 each for heavy and light chains).
[0090]
Among them, the amino acid sequence of the constant region is common between antibodies having the same immunoglobulin subclass regardless of the type of antigen. On the other hand, although the amino acid sequences of the variable regions, and especially the CDRs, are unique to each antibody, studies comparing the amino acid sequence data of many antibodies show that the CDR positions and the length of the framework sequences are the same as each other. It is known that the antibody subunits belonging to the group are almost similar [Kabat, E. et al. A. , Et al. (1991) in "Sequence of Proteins of Immunological Interest Vol. II": U.S. Pat. S. See Department of Health and Human Services]. Therefore, by comparing each amino acid sequence of the heavy chain and light chain of an antibody such as the anti-Fas monoclonal antibody HFE7A with known amino acid sequence data, the positions of CDRs, framework regions and constant regions in each amino acid sequence can be determined. Can be determined. In addition, FRH ₁ That is, the chain length of the framework region closest to the N-terminal side of the heavy chain may be usually shorter than (30 amino acids). For example, as the same subtype as HFE7A of mouse IgG1, the framework region has a minimum of 18 amino acids. Is known [Kabat et al., Supra. reference]. From this, in the antibody of the present invention, the chain length of the N-terminal framework region of the heavy chain is set to be 18 amino acids or more and 30 amino acids or less as long as the function as an anti-Fas antibody is not impaired, It is. The amino acid numbers of the immunoglobulin heavy chains in the present invention are FRH unless otherwise specified. ₁ Is 30 amino acids.
[0091]
In addition, a peptide having the same amino acid sequence as the CDR of the light chain or heavy chain determined as described above, or a peptide having a continuous partial amino acid sequence therein, is subjected to an artificial modification operation to make the CDR By approaching the three-dimensional structure formed in the Fas antibody molecule, it is possible to impart binding activity to Fas alone [see, for example, US Pat. No. 5,331,573]. Therefore, a peptide having the same amino acid sequence as that of the CDR or a continuous partial amino acid sequence therein is also encompassed in the molecule of the present invention, and is thus protected against a disease caused by an abnormality in the Fas-Fas ligand system. Or it can be used for treatment.
[0092]
In preparing a variant in which any one or more amino acids in the amino acid sequence have been deleted, a cassette mutation method [Toshimitsu Kishimoto, "Neochemical Chemistry Laboratory Course 2, Nucleic Acid III, Recombinant DNA Technology" 242- 251].
[0093]
Such various DNAs are obtained, for example, by the phosphite triester method [Hunkapiller, M .; , Et al. (1984), Nature 310, 105-111] and the like. The codon for the desired amino acid is known per se, and its selection may be arbitrarily determined. For example, the codon for the desired amino acid may be determined according to a conventional method in consideration of the frequency of codon usage of the host used. These nucleotide sequence codons can be partially modified by site-directed mutagenesis using a primer consisting of a synthetic oligonucleotide encoding the desired modification (cytospecific mutagenesis) [Mark, D. et al. F. , Et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666] and the like.
[0094]
Whether or not a certain DNA hybridizes with DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody of the present invention can be determined, for example, by the random primer method [Feinberg, A. et al. P. and Vogelstein, B.W. (1983) Anal. biochem. 132, 6-13] and the nick translation method [Maniatis, T .; , Et al. (1982) in "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory, NY. [Α- ³² It can be determined by conducting an experiment as described below using a probe DNA labeled with [P] dCTP or the like.
[0095]
First, the DNA to be examined is adsorbed on, for example, a nitrocellulose membrane or a nylon membrane, and if necessary, treated with alkali denaturation or the like, and then solidified by heating or ultraviolet rays. This membrane was combined with a prehybridization solution containing 6 × SSC (1 × SSC is 0.15 M sodium chloride, 0.015 M trisodium citrate solution), 5% Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS). After soaking and incubating at 55 ° C. for 4 hours or more, the previously prepared probe was added to the same prehybridization solution at a final specific activity of 1 × 10 5 ⁶ Add to a concentration of cpm / ml and keep at 60 ° C overnight. Thereafter, the operation of washing the film with 6 × SSC at room temperature for 5 minutes is repeated several times, and further washed with 2 × SSC for 20 minutes, followed by autoradiography.
[0096]
Using the method as described above, hybridize from any cDNA library or genomic library with DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody which is the basis of the humanized anti-Fas antibody of the present invention. Can be isolated [Maniatis, T .; , Et al. (1982) in "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory, NY. reference].
[0097]
By incorporating each DNA obtained as described above into an expression vector, it is possible to introduce the DNA into prokaryotic or eukaryotic host cells and express the respective genes in those host cells.
[0098]
Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. In order to express a gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing a replication origin and a promoter sequence such as lac UV5. . The vector preferably has a sequence capable of imparting selectivity based on the phenotypic trait in the transformed cell. For example, Escherichia coli includes E. coli. The JM109 strain derived from E. coli K12 strain and the like are often used, and as the vector, pBR322 and pUC-based plasmids are generally often used, but are not limited thereto, and any known various strains and vectors can be used. In Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) PL promoter, polypeptide chain Examples include, but are not limited to, the elongation factor Tu (tufB) promoter.
[0099]
As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 [Ohmura, K. et al. , Et al. (1984) J.C. Biochem. 95, 87-93] and the like, but is not limited thereto. As the promoter, a regulatory sequence of the Bacillus subtilis α-amylase gene is often used, and if necessary, a DNA sequence encoding the signal peptide sequence of α-amylase can be ligated to allow extracellular secretion. .
[0100]
Eukaryotic host cells include cells such as vertebrates and yeasts. Examples of vertebrate cells include COS-1 [Gluzman, Y. (1981) Cell 23, 175-182]. As yeast, baker's yeast (Saccharomyces cerevisiae), alcohol-assimilating yeast (Pichia pastoris), fission yeast (Schizossaccharomyces pombe), and the like are used. Cells such as those exemplified herein are commonly used as host cells, but are not limited thereto.
[0101]
As a vertebrate cell expression vector, those having a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence, and the like which are usually located upstream of the gene to be expressed can be used. It may have a replication origin. Examples of such expression vectors include pSV2dhfr with the SV40 early promoter [Subramani, S .; , Et al. (1981) Mol. Cell. Bio. 1, 854-864], etc., but is not limited thereto.
[0102]
Examples of yeast expression vectors include, for example, the promoter of alcohol dehydrogenase gene [Bennetzen, J. et al. L. and Hall, B .; D. (1982) Biol. Chem. 257, 3018-3025], and a promoter of a galactose metabolism enzyme gene (such as gal10) [Ichikawa, K. et al. , Et al. (1993) Biosci. Biotech. Biochem. 57, 1686-1690], etc., and if necessary, by linking a DNA sequence encoding the signal peptide sequence of the yeast gene, extracellular secretion becomes possible, but is not limited thereto. .
[0103]
For example, when COS-1 is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS-1, and has a transcription promoter, a transcription termination signal, and an RNA splice. Expression vectors with sites can be used. The expression vector is prepared by the DEAE-dextran method [Luthman, H .; and Magnusson, G .; (1983) Nucleic Acids Res. 11, 1295-1308], a calcium phosphate-DNA coprecipitation method [Graham, F. et al. L. and van der Eb, A. J. (1973) Virology 52, 456-457] and electroporation [Neumann, E. et al. , Et al. (1982) EMBO J .; 1, 841-845], and the like, whereby desired transformed cells can be obtained.
[0104]
In particular, by co-transfecting COS-1 with an expression vector comprising DNA encoding the heavy chain and an expression vector comprising DNA encoding the same light chain, these DNAs can be simultaneously obtained. A transformant producing the recombinant anti-Fas antibody can be obtained. However, the method for producing the recombinant anti-Fas antibody is not limited to the above. For example, a single expression vector capable of retaining both DNAs encoding each of a heavy chain and a light chain and expressing them at the same time And a method of transforming COS-1 cells with the vector can also be used.
[0105]
The desired transformant obtained above can be cultured according to a method well known to those skilled in the art, and the culture produces a recombinant anti-Fas antibody inside or outside the transformant cells. As the medium used for the culture, various ones commonly used depending on the host cell used can be appropriately selected. For example, in the case of COS-1, the medium may be RPMI1640 medium or Dulbecco's modified Eagle medium (DMEM). If necessary, a serum component such as fetal calf serum (FCS) may be used.
[0106]
The culture temperature at the time of culturing the transformant may be any temperature as long as it does not significantly lower the protein synthesis ability in the cell, but the culture is preferably performed at 32 to 42 ° C, most preferably at 37 ° C. . If necessary, the cells can be cultured in air containing 1 to 10% (v / v) carbon dioxide.
[0107]
The E. coli strain E. coli transformed with a recombinant DNA vector that expresses the DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody of the present invention in animal cells. coli pME-H and E. coli. coli pME-L has been internationally deposited with the National Institute of Advanced Industrial Science and Technology (AIST) on March 12, 1997, by the National Institute of Advanced Industrial Science and Technology (AIST), under the accession number FERMBP-. 5868 and FERM BP-5867. Therefore, cultured animal cells such as COS-1 are transformed with the recombinant DNA vector isolated from the deposited strain, and the transformed cells are cultured to produce a recombinant anti-Fas antibody in the culture. be able to.
[0108]
The fraction containing the anti-Fas antibody protein, which is produced intracellularly or extracellularly in the transformant as described above, can be separated by various known protein separation procedures utilizing the physical properties, chemical properties, and the like of the protein. -Can be purified. Specific examples of such a method include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various types of chromatography, dialysis methods, combinations thereof, and the like.
[0109]
In order to humanize an anti-Fas mouse monoclonal antibody, it is necessary to design the amino acid sequence of the variable region such that the entire determined CDR sequence and a part of the amino acid residues of the FR sequence are transplanted into a human antibody. This design follows the following method.
[0110]
Conventionally, when designing humanization, the guideline for selecting the acceptor subgroup is:
{Circle around (1)} Using a natural combination of a heavy chain and a light chain of a known human antibody having a natural amino acid sequence in its entirety,
(2) The combination as a subgroup to which the heavy and light chains belong is preserved, but the heavy and light chains are derived from different human antibodies, and have the same identity as the amino acid sequences of the donor heavy and light chains. Using a high amino acid sequence or a consensus sequence,
Either is selected. In the present invention, the above guidelines can be followed.
(3) The heavy chain and light chain FRs having the highest identity to the donor FRs are selected from a library of human antibody primary sequences without considering the combination of the subgroups.
It is also possible to adopt the method described above. By these selection methods, the identity of the amino acid in the FR portion between the donor and the acceptor can be at least 70% or more. By employing this method, the number of amino acid residues to be transplanted from the donor can be reduced, and the induction of the HAMA response can be reduced.
[0111]
In addition, the operation of predicting the tertiary structure from the primary sequence of the antibody molecule (hereinafter, this operation is referred to as “molecular modeling”) has a limitation in its prediction accuracy, and amino acid residues that rarely appear in the subgroup to which the donor belongs Role cannot be fully specified. According to the method of Queen et al., It is generally difficult to determine which amino acid residue, donor or acceptor, should be selected at such a position. According to the selection method of (3), the chance of making such a judgment can be significantly reduced.
[0112]
The present inventors have further improved this method of humanization by providing novel methods for identifying amino acids from the donor FRs that are important for maintaining the structure and function of the donor CDRs. I have.
[0113]
After the human acceptor molecule of each of the light chain and the heavy chain is selected, a method of selecting an amino acid residue to be transplanted from the FR of the donor is as described below.
[0114]
When the amino acid sequences of the donor and the acceptor are aligned, and the amino acid residues are different at the corresponding positions of the FRs, it is necessary to determine which residue to select. Must be selected so as not to impair the three-dimensional structure of the CDRs.
[0115]
Queen et al., In the above-mentioned PCT Publication No. 4-502408, proposed a method of grafting an amino acid residue on an FR to an acceptor together with a CDR sequence when the amino acid residue meets at least one of the following requirements:
1) Amino acids in the human FR region of the acceptor are rare at that position and the corresponding amino acids of the donor are common at said position of the acceptor;
2) the amino acid is in the immediate vicinity of one of the CDRs;
3) It is expected that the amino acid has a side chain atom within about 3% of the CDR in a three-dimensional immunoglobulin model and is capable of interacting with an antigen or with the CDR of a humanized antibody.
[0116]
Since the residue represented by 2) often exhibits the property of 3), the requirement of 2) is deleted in the present invention, and two additional requirements are provided. That is, according to the present invention, the amino acid residues on the donor FR to be transplanted with the CDR are as follows:
a) whether the amino acid in the acceptor FR is rare at that position and the corresponding amino acid of the donor is common at that position;
b) whether the amino acid is expected to interact in a three-dimensional structural model with a constituent amino acid atom of a CDR and an antigen or a CDR loop to be transplanted;
c) whether the position is a canonical class determining residue;
d) whether the position constitutes the interface between the heavy and light chains,
In this case, the amino acid residue is grafted from the donor FR.
[0117]
According to the requirement a), amino acids found at a frequency of 90% or more at the relevant position for antibodies of the same subclass are defined as “ordinary” and amino acids found at a frequency of less than 10% as “rare” in accordance with the Kabat table described above. I do.
[0118]
In the requirement (c), whether or not “the position is a residue determining a canonical class” can be uniquely determined according to the above-mentioned table of Choccia.
[0119]
For the requirements of b) and d), molecular modeling of the antibody variable region is required in advance. As the software for molecular modeling, any commercially available software can be adopted, but AbM (manufactured by Oxford Molecular Limited) can be preferably used.
[0120]
Since there is a certain limit in the prediction accuracy of molecular modeling, in the present invention, by referring to the experimental results of X-ray crystallography of the variable regions of various antibodies, it is possible to confirm the certainty of the structural prediction obtained from molecular modeling. A distinction is made between two stages.
[0121]
In the present invention, in the three-dimensional structure of the variable region constructed by molecular modeling software such as AbM, the distance between two atoms is larger than the value obtained by adding 0.5 ° to the sum of the van der Waals radii. When the length was short, it was estimated that the two atoms were in contact with Van der Waals. When the distance between polar atoms such as amide nitrogen and carbonyl oxygen in the main chain and side chain is shorter than the average hydrogen bond distance of 2.9 ° plus 0.5 °, it is estimated that hydrogen bonds exist between them. Further, when the distance between the atoms having opposite electric charges is shorter than 2.85 ° + 0.5 °, it is estimated that an ion pair is formed between them.
[0122]
On the other hand, from the experimental results of X-ray crystal structure analysis of the variable regions of various antibodies, regardless of the subgroup, the positions on the FR where the contact with the CDR is frequently found are 1, 2, 3 in the light chain. At positions 4, 4, 5, 23, 35, 36, 46, 48, 49, 58, 69, 71, 88, and 2, 4, 27, 28, 29, 30, 36, 38, 46, The positions of positions 47, 48, 49, 66, 67, 69, 71, 73, 78, 92, 93, 94, 103 are specified (all numbers are amino acid numbers defined in the above-mentioned Kabat et al. Document). (The same applies hereinafter.) Applying the same criteria as in molecular modeling, the amino acid residues at these positions are found in contact with the amino acid residues of the CDR in two thirds of the known antibody variable region. Based on these findings, b) "the amino acid is expected to interact with an antigen or a CDR loop to be transplanted in the three-dimensional structural model of the amino acid in the three-dimensional structural model" means the following requirements. .
[0123]
In molecular modeling, the position of the FR where the possibility of contact between the FR and the CDR is predicted matches one of the positions where the contact between the FR and the CDR is frequently detected experimentally by X-ray crystallography. In this case, transplantation of donor amino acid residues is preferred. Otherwise, this requirement b) is not taken into account.
[0124]
d) “The position constitutes the contact surface between the heavy chain and the light chain” means the following requirements. From the results of X-ray crystallographic analysis of the variable regions of various antibodies, the amino acid residues at positions 36, 38, 43, 44, 46, 49, 87, 98 in the light chain, and 37, 39 in the heavy chain , 45, 47, 91, 103, and 104 are frequently found to make heavy chain-light chain contact. In molecular modeling, the possibility of heavy chain-light chain contact is foreseen, and if the position matches any of the positions described above, transplantation of donor amino acid residues is preferred. Otherwise, this requirement d) is not taken into account.
[0125]
DNAs encoding the variable regions of the heavy and light chains of the humanized anti-Fas antibody of the present invention can be produced by the method described below.
[0126]
For example, a plurality of polynucleotide fragments of 60 to 70 nucleotides consisting of a partial nucleotide sequence of the DNA are chemically synthesized so as to be alternate on the sense side and the antisense side, and then each polynucleotide fragment is annealed to obtain a DNA ligase. To obtain DNA having DNAs encoding the variable regions of the heavy and light chains of the desired humanized anti-Fas antibody.
[0127]
As another method, a DNA encoding the entire amino acid sequence of the acceptor variable region is separated from human lymphocytes, and the CDR-encoding region is subjected to nucleotide substitution by a method well known to those skilled in the art to introduce a restriction enzyme cleavage sequence. I do. After cleavage of the region with the corresponding restriction enzyme, a nucleotide sequence encoding the CDR of the donor is synthesized and ligated with DNA ligase to encode the variable regions of the desired humanized anti-Fas antibody heavy and light chains. DNA can be obtained.
[0128]
Further, in the present invention, the overlap extension PCR method described below (Holton et al., Gene, 77 , 61-68, (1989)) to obtain DNAs encoding the variable regions of the heavy and light chains of the desired humanized anti-Fas antibody.
[0129]
That is, two kinds of DNAs respectively encoding two kinds of amino acid sequences desired to be connected are referred to as (A) and (B) for convenience. (A) a 20 to 40 nucleotide sense primer that anneals to the 5 ′ side (hereinafter, this primer is referred to as (C)) and (B) a 20 to 40 nucleotide antisense primer that anneals to the 3 ′ side (Hereinafter, this primer is referred to as (D)). Furthermore, a chimeric sense primer (hereinafter, this primer is referred to as (E)) in which 20 to 30 nucleotides on the 3 ′ side of (A) and 20 to 30 nucleotides on the 5 ′ side of (B) are linked, and A complementary antisense primer (hereinafter, this primer is referred to as (F)) is synthesized. By using an appropriate vector DNA containing (A) as a substrate and performing PCR using sense primer (C) and chimeric antisense primer (F), (B) is added to the 3 ′ end of (A). DNA having 20 to 30 nucleotides added at the 5 'end can be obtained (this newly obtained DNA is referred to as (G)). Similarly, by using an appropriate vector DNA containing (B) as a substrate and performing PCR using an antisense primer (D) and a chimeric sense primer (E), (A) is added to the 5 ′ end of (B). ) Can be obtained (hereinafter, newly obtained DNA is referred to as (H)). (G) and (H) thus obtained have complementary nucleotide sequences at 40 to 60 nucleotides on the 3 ′ side of (G) and 40 to 60 nucleotides on the 5 ′ side of (H). . When PCR is performed by mixing the amplified (G) and (H), (G) and (H) become single-stranded in the first denaturation reaction, and most of the DNA is recovered in the subsequent annealing reaction. However, some DNAs form a heteroDNA duplex which anneals with the complementary nucleotide sequence region. In the subsequent extension reaction, the protruding single-stranded portion is repaired, and a chimeric DNA in which (A) and (B) are linked (hereinafter, this DNA is referred to as (I)) can be obtained. Further, by using this (I) as a substrate and performing PCR using a sense primer (C) and an antisense primer (D), (I) can be amplified. In the present invention, the DNA encoding the CDR regions of the heavy and light chains of the anti-human Fas mouse monoclonal antibody, the DNA encoding the FR region of human immunoglobulin IgG, and the DNA encoding the secretion signal of human immunoglobulin IgG Can be carried out as (A) and (B) on a case-by-case basis, respectively.
[0130]
The codon for the desired amino acid is known per se, and its selection may be arbitrarily determined. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used. These nucleotide sequence codons can be partially modified by site-specific mutagenesis using a primer consisting of a synthetic oligonucleotide encoding the desired modification (Mark, DF, et al.) According to a conventional method. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666). Therefore, when each primer is chemically synthesized, by designing each primer in advance to introduce a point mutation, it is possible to obtain DNAs encoding the variable regions of the heavy and light chains of the desired anti-Fas antibody. it can.
[0131]
By incorporating each of the thus obtained DNAs of the present invention into an expression vector, prokaryotic or eukaryotic host cells can be transformed. Furthermore, each gene can be expressed in each host cell by introducing an appropriate promoter and a sequence related to expression into these vectors.
[0132]
In addition, three types of transformants, each of which contains DNA encoding the variable region of the light chain of the humanized anti-Fas antibody of the present invention, coli pHSGMM6 SANK 73697 strain; coli pHSGHM17 SANK 73597 strain and E. coli pHSGHM17 SANK 73597 strain. E. coli pHSGHH7 SANK 73497 strain, and a transformant into which DNA encoding the variable region of the heavy chain of the humanized anti-Fas antibody of the present invention has been incorporated. The E. coli pgHSL7A62 SANK73397 strain was internationally deposited on August 22, 1997 at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology). BP-6071, FERM BP-6072 and FERM BP-6073, and FERM BP-6074. In addition, two transformants incorporating DNA encoding the variable region of the light chain of the humanized anti-Fas antibody of the present invention, coli pHSHM2 SANK 70198 strain and E. coli pHSHM2 SANK 70198 strain. E. coli pHSHH5 SANK 70398 strain, and a transformant into which DNA encoding the variable region of the heavy chain of the humanized anti-Fas antibody of the present invention has been incorporated. E. coli pgHPDHV3 SANK 70298 strain was internationally deposited on February 26, 1998 at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology). FERM BP-6272 and FERM BP-6274, and FERM BP-6273 are attached. Therefore, it is possible to obtain a DNA encoding each subunit of the humanized anti-Fas antibody protein by, for example, isolating a plasmid from the deposited strain or performing PCR using the extract of the deposited strain as a template. it can.
[0133]
According to the above method, a recombinant anti-Fas antibody can be easily produced with high yield and high purity.
[0134]
As a method for confirming that the recombinant anti-Fas antibody thus produced specifically binds to Fas, for example, the same ELISA method as in the case of evaluating the antibody titer at the time of mouse immunization is suitable.
[0135]
The molecules of the invention (not limited to antibodies) have the following functional properties a) to f), each of which can be confirmed, for example, by the method described in each item:
a) Apoptosis-inducing activity on Fas-expressing T cells: The thymus is excised from the mouse to which the molecule of the present invention has been administered, and the obtained thymocytes are used as anti-mouse Fas antibodies and antibodies that specifically recognize T cells. Apoptosis-inducing activity on Fas-expressing T cells by measuring the percentage of cells bound by both antibodies by flow cytometry;
b) Reduction of autoimmune disease symptoms in MRLgld / gld mice: MRLgld / gld mice in which a gene encoding a Fas ligand has a mutation and exhibit autoimmune disease-like symptoms [Shin Yonehara (1994) Nikkei Science Supplement 110 , 66-77] to determine whether the intraperitoneal administration of the molecule of the present invention reduces swelling of the limbs, which is an autoimmune disease-like symptom of the mouse;
c) Does not induce liver damage: Peripheral blood is collected from BALB / c mice to which the molecule of the present invention has been administered, and the glutamic acid-oxaloacetate transaminase (hereinafter referred to as “GOT”) level and glutamic acid-pyruvate transaminase (hereinafter referred to as “GOT”). The "GPT" value is measured using an automatic analyzer (for example, Model 7250; manufactured by Hitachi, Ltd.) and a reagent for the device (for example, transaminase-HRII; manufactured by Wako Pure Chemical Industries, Ltd.). When administered to a living body, the molecule of the present invention does not increase blood GOT and GPT levels, which are indicators of liver damage;
d) Therapeutic or preventive effect against fulminant hepatitis: In the experimental system in which anti-mouse Fas monoclonal antibody Jo2 is administered to mice to induce fulminant hepatitis, when the molecule of the present invention is administered simultaneously with or after Jo2 administration Investigate the effect of
e) Effect of preventing the onset of collagen-induced arthritis: Examining the effect of the administration of the molecule of the present invention on a rheumatoid arthritis model induced by administering an emulsion comprising collagen and complete Freund's adjuvant to mice;
f) Apoptosis-inducing activity on rheumatoid arthritis patient-derived synovial cells: Synovial cells collected from the affected area of rheumatoid arthritis patients are cultured, and the survival rate of the cells when the molecule of the present invention is contained in a medium is examined. .
[0136]
The molecule of the present invention does not induce apoptosis in the activities described in a) and f) above, that is, the activity of inducing apoptosis in abnormal cells, and the activities described in c) and d) in above, that is, does not induce apoptosis in normal cells. It is a substance having novel properties and having an activity of inhibiting apoptosis, and is useful as a component to be contained in a preventive or therapeutic agent for a disease caused by abnormal Fas / Fas ligand system.
[0137]
In addition, the fact that the molecule of the present invention has apoptosis-inducing activity means that cells (for example, human lymphocyte cell line HPB-ALL (Morikawa, S., et al. (1978) Int. J) Cancer 21, 166-170) or Jurkat (American Type Culture No. TIB-152) or the like, and the viability thereof is measured by an MTT assay (Green, LM, et al. (1984) J. Immunological). Methods 70, 257-268) and the like.
[0138]
Molecules of the present invention, particularly anti-Fas antibodies whose immunogenicity for humans is almost the same as human antibodies, can be used for diseases caused by the Fas / Fas ligand system (autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis) , Graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, vasedo Disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus), allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral) Effective against hepatitis (hepatitis C, hepatitis B, hepatitis D) or alcoholic hepatitis), acquired immunodeficiency syndrome or rejection after organ transplantation It can be used as a prophylactic or therapeutic agent to. Such a prophylactic or therapeutic agent can be administered in various forms, such as oral administration by tablets, capsules, granules, powders, syrups, etc., or injections, drops, suppositories. And parenteral administration.
[0139]
The dose varies depending on symptoms, age, body weight, etc., but in the case of oral administration, it is usually about 0.1 mg to 1000 mg per day for an adult, and these are administered once or divided into several times. be able to. In the case of parenteral administration, a single dose of 0.1 mg to 1000 mg can be administered by subcutaneous injection, intramuscular injection or intravenous injection.
[0140]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
[0141]
Example 1 Preparation of antigen
To obtain soluble human Fas that does not contain the transmembrane region, the extracellular region of human Fas and the mouse interleukin 3 (IL3) receptor [Gorman, D .; M. et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 5459-5463], a plasmid vector for expression of a fusion protein with the extracellular region (hereinafter referred to as "human Fas fusion protein") was prepared. DNA encoding the human Fas fusion protein was prepared by the PCR method.
[0142]
a) Mold
In performing the PCR, the template used was an expression plasmid vector pME18S-mFas-AIC [Nishimura, Y. et al.] For a DNA encoding a fusion protein of the extracellular region of mouse Fas and the extracellular region of mouse IL3 receptor. et al. (1995) Immunol. 154, 4395-4403] and the plasmid vector pCEV4 carrying the cDNA encoding human Fas [Itoh, N. et al. , Et al. (1991) Cell 66, 233-243].
[0143]
b) Primer
In performing the PCR, the following oligonucleotide primers were synthesized:
5′-GGGGAATTCC AGTACGGAGGT TGGGGAAGCT CTTT-3 ′ (N1: SEQ ID NO: 12 in Sequence Listing),
5′-GTTTCTTCTG CCCTGTTCAC CAAGTTAGAT CTGGA-3 ′ (C3N: SEQ ID NO: 13 in Sequence Listing),
5′-TCCAGATCTA ACTTGGTGAC AGAGGCAGAA GAAAC-3 ′ (N3N: SEQ ID NO: 14 in Sequence Listing),
5'-CCCTCTAGAC GCGTCACGTG GGCATCAC-3 '(CTN2: SEQ ID NO: 15 in Sequence Listing).
[0144]
Oligonucleotides such as primers for PCR were all synthesized using an automatic DNA synthesizer (Model 380B; manufactured by PerkinElmer Japan Applied Biosystems Division) according to the manual [Matteuucci, M .; D. and Caruthers, M .; H. (1981) Am. Chem. Soc. 103, 3185-3191]. After completion of the synthesis, each oligonucleotide was cleaved from the support and deprotected, and the resulting solution was freeze-dried to form a powder. This powder was dissolved in distilled water and stored frozen at -20 ° C until use.
[0145]
c) First stage PCR
DNA fragment HFAS encoding the extracellular region of human Fas was prepared under the following conditions. In performing the PCR, an LA PCR kit (Takara Shuzo Co., Ltd.) was used.

Temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle was repeated 30 times at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes, and then the temperature was kept at 72 ° C. for 10 minutes (hereinafter referred to as “Execution”). The reaction temperature of all PCRs in the examples was controlled by GeneAmp PCR System 9600; manufactured by PerkinElmer Japan.
[0146]
On the other hand, a DNA fragment MAIC encoding the extracellular region of mouse IL3 receptor was prepared under the following conditions.

Temperature conditions: Same as above.
[0147]
The amplified HFAS DNA and MAIC DNA after PCR were subjected to 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation, stained with 1 μg / ml ethidium bromide, and detected under ultraviolet irradiation. Was. A gel piece of each detected band portion was cut off with a razor blade, and DNA was electrically eluted using a centriluter (manufactured by Amicon) equipped with a centrifugal tube ultrafilter Centricon 10 (manufactured by Amicon). . Further, the Centricon 10 containing the eluate was taken out, and the DNA was concentrated by centrifugation at 7,500 × g for about 1 hour. This DNA was dissolved in 20 μl of distilled water after ethanol precipitation.
[0148]
d) Second stage PCR
DNA fragment FASAIC encoding human Fas fusion protein was prepared under the following conditions.

Temperature conditions: Same as the first step.
[0149]
The amplified FASAIC DNA after PCR was subjected to 1% agarose gel electrophoresis after phenol extraction and ethanol precipitation, stained with 1 μg / ml ethidium bromide, and detected under ultraviolet irradiation. The gel piece of the detected band portion was cut off with a razor blade, DNA was eluted from the gel piece using a centrilutor and a centricon, concentrated, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0150]
e) Vector construction
The FASAIC DNA obtained in d) above was digested with the restriction enzymes EcoRI and XbaI (using the entire amount of the FASAIC DNA solution), followed by phenol-chloroform extraction and ethanol precipitation. This precipitate was suspended in 2 μl of sterile deionized water.
[0151]
Separately, 2 μg of plasmid pME18S-mFas-AIC was digested with EcoRI and XbaI, and then a dephosphorylated DNA fragment was prepared. This fragment was combined with FASAIC DNA digested with EcoRI and XbaI and a ligation kit (Takara Shuzo Co., Ltd.) ), Followed by the method of Hanahan [Hanahan, D .; (1983) J. Amer. Mol. Biol. 166,557-580], and Escherichia coli DH5α strain (manufactured by Gibco BRL) was transformed. An alkaline SDS method [Maniatis, T .; , Et al. (1989) in Molecular Cloning: See A Laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory, NY] to obtain the desired plasmid phFas-AIC2.
[0152]
This plasmid was prepared using a plasmid large-scale preparation kit (Maxiprep DNA purification system: manufactured by Promega), and 20 μg of DNA was precipitated with ethanol, and the precipitate was sterilized with Dulbecco's PBS (-) (hereinafter referred to as “PBS”). Called Nissui Pharmaceutical Co., Ltd.).
[0153]
f) expression
COS-1 cells (American Type Culture Collection No. CRL-1650) were prepared using Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") containing 10% fetal calf serum (manufactured by Gibco) (hereinafter referred to as "FCS"). Cell culture flask (culture area 225 cm) containing Pharmaceutical Co., Ltd.) ² , 5% CO2 in a flask containing 5 g / L trypsin and 2 g / L ethylenediaminetetraacetic acid diacetate. 3 ml of a solution containing sodium (hereinafter, referred to as “trypsin-EDTA solution”; manufactured by Sigma) was added, and the mixture was kept at 37 ° C. for 3 minutes. The cells released from the flask were suspended in PBS and collected, washed twice with PBS, and then washed with PBS at 6 × 10 5 ⁷ Adjusted to cells / ml. 20 μl of this cell suspension (1.2 × 10 ⁶ Cells) and 20 μl of the above-prepared plasmid solution were mixed, placed in a chamber (manufactured by Shimadzu Corporation) with an electrode spacing of 2 mm, and set in a gene transfer apparatus (GTE-1; manufactured by Shimadzu Corporation). After that, a pulse of 600 V-30 μsec was applied twice at 1 second intervals. The cell-DNA mixture in the chamber was added to 10 ml of DMEM containing 10% FCS, and a flask for cell culture (culture area 75 cm) was added. ² ) At 37 ° C under 7.5% carbon dioxide for 24 hours. Next, after removing the culture supernatant and washing the cells with serum-free DMEM, 10 ml of serum-free DMEM was added, and the cells were further cultured at 37 ° C. and 7.5% carbon dioxide for 24 hours to collect the culture supernatant.
[0154]
The obtained culture supernatant was placed in a dialysis tube (discharge limit molecular weight 12,000-14000; manufactured by Gibco BRL), dialyzed against 10 mM Tris-hydrochloric acid (pH 8.0), and then subjected to a Pharmacia FPLC apparatus. The crude purification of human Fas fusion protein was performed using the conditions described below:
Column: Resource Q column (trade name; φ6.4 × 30 mm, manufactured by Pharmacia);
Solvent: 10 mM Tris-HCl (pH 8.0)
Flow rate: 5 ml / min;
Elution: Sodium chloride 0.1M-0.3M, 30 minutes linear gradient.
The eluate was fractionated in 5 ml portions, and the Fas gene expression product was assayed by the ELISA method described below. First, 100 μl of each eluted fraction was placed in a well of a 96-well microplate (manufactured by Coaster) and kept at 37 ° C. for 1 hour. After removing the liquid in each well, the wells were washed three times with 100 μl / well of PBS containing 0.1% Tween 20 (Tween 20) and containing 2% bovine serum albumin (hereinafter referred to as “BSA”). 100 μl / well of PBS was added and incubated at 37 ° C. for 1 hour. After washing each well three times with 100 μl / well of PBS-Tween, the anti-mouse IL-3 receptor β subunit monoclonal antibody HC (1 mg / ml; manufactured by Medical Biology Laboratories) was washed with PBS-Tween. 100 μl / well of a 1000-fold diluted solution was added and incubated at 37 ° C. for 1 hour. Further, after each well was washed three times with 100 μl of PBS-Tween, 100 μl / well of horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 2000-fold with PBS-Tween was added, and incubated at 37 ° C. for 1 hour. Then, each well was washed three times with 100 μl of PBS-Tween. Next, 100 μl / well of a horseradish peroxidase substrate (manufactured by Bio-Rad) was added and allowed to stand for 5 minutes, and the absorbance at 415 nm was measured using a microplate reader (Model 450; manufactured by Bio-Rad). As a result, the 19th to 23rd fractions having high absorbance were collected and used as a crude purified human Fas fusion protein sample.
[0155]
Example 2 Immunization of mice and preparation of hybridomas
(2-1) Immunity
To 1 ml of the partially purified human Fas fusion protein solution obtained in Example 1 (total protein amount: 100 μg), 25 μl of 2N hydrochloric acid, 250 μl of 9% potassium alum (1.1% final concentration), and 25 μl of 2N sodium hydroxide were added. After that, the pH was adjusted to 6.5 to 7.0 by adding about 120 μl of a 10% (w / v) aqueous sodium hydrogen carbonate solution. After standing at room temperature for about 30 minutes, pertussis killed bacteria (manufactured by Wako Pure Chemical Industries, Ltd .; 1.2 × 10 ¹¹ (Bacteria / ml) 200 μl was added and administered intraperitoneally to Fas knockout mice. As the Fas knockout mouse, a mouse obtained by the method described in Senju et al. Was used [Senju, S. et al. et al. (1996) International Immunology 8, 423]. Two weeks later, only partially purified human Fas fusion protein (20 μg protein / mouse) was intraperitoneally administered for booster immunization.
[0156]
(2-2) Cell fusion
The spleen was removed from the mouse 3 days after the booster immunization, and the spleen was extracted with 20 mM HEPES buffer (pH 7.3), 350 mg / ml sodium bicarbonate, 0.05 mM β-mercaptoethanol, 50 units / ml penicillin, 50 μg / ml streptomycin, Serum-free RPMI1640 medium containing 300 μg / ml L-glutamic acid (10.4 g / liter RPMI1640 “Nissui” (1): manufactured by Nissui Pharmaceutical Co., Ltd.) (hereinafter referred to as “serum-free RPMI medium”) It was crushed using a spatula on a mesh (Cell Strainer: Falcon). After the cell suspension passed through the mesh was centrifuged to precipitate spleen cells, the spleen cells were washed twice with serum-free RPMI medium, and then suspended in serum-free RPMI medium to measure the number of cells.
[0157]
On the other hand, cells were cultured in an ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FCS (manufactured by Gibco BRL) (hereinafter referred to as “ASF medium containing serum”) at 37 ° C. in the presence of 5% carbon dioxide gas. Concentration is 1 × 10 ⁸ Myeloma cells NS1 (American Type Culture Collection TIB-18) cultured not to exceed cells / ml were similarly washed with serum-free RPMI medium and suspended in serum-free RPMI medium to measure the number of cells.
[0158]
3 × 10 ⁷ NS1 cell suspension equivalent to 3 × 10 ⁸ An equivalent number of spleen cell suspensions were mixed, centrifuged, and the supernatant was completely removed. The following cell fusion operation was performed while keeping the plastic centrifuge tube containing the pellet in a beaker containing warm water at 37 ° C. 1 ml of 50% (w / v) polyethylene glycol 1500 (manufactured by Boehringer Mannheim) is slowly added to the pellet while stirring the pellet with a pipette tip. 1 ml of serum RPMI medium was slowly added in two portions, and 7 ml of serum-free RPMI medium was further added. After centrifugation, the supernatant was removed, and 10 ml of a hypoxanthine / aminopterin / thymidine medium (hereinafter referred to as “HAT medium”; Boehringer Mannheim) containing 10% FCS was added with gentle stirring at the tip of a pipette. After adding 20 ml of HAT medium containing 10% FCS, 100 μl / well was dispensed into a 96-well microplate for cell culture and cultured at 37 ° C. under 5% carbon dioxide gas. Seven to eight days later, 100 μl / well of fresh HAT medium was added to the wells with yellowish medium. The thus obtained fusion cells were subjected to screening by the limiting dilution method described below.
[0159]
(2-3) Limit dilution
After extracting the thymus from a BALB / c mouse female (purchased from Japan SLC) 4 to 10 weeks old, it was crushed using a spatula on a mesh (Cell Strainer; Falcon), and the cells that passed through the mesh were reduced to 10%. The plate was washed twice with a hypoxanthine / thymidine medium containing FCS (hereinafter referred to as "HT medium"; manufactured by Boehringer Mannheim). Thymic cells for one mouse were suspended in 30 ml of HT medium containing 10% FCS to obtain a feeder cell solution. The culture solution containing the fused cells obtained in the above (2-2) is diluted 10 to 100 times with a feeder cell solution depending on the cell density, and the density of the fused cells is further reduced to 5 cells / ml and 1 cell / ml. Was serially diluted with a feeder cell solution to a concentration of 0.5 cells / ml. Each sample thus prepared was dispensed into a 96-well microplate for cell culture at 100 μl / well and cultured at 37 ° C. under 5% carbon dioxide for 5 days.
[0160]
(2-4) Sorting
WR19L12a cells obtained by expressing a gene encoding human Fas in mouse T lymphoma cells WR19L (American Type Culture Collection TIB-52) [Itoh, N .; et. al. (1991) Cell 66, 233-243] was cultured and grown in RPMI1640 medium containing 10% FCS at 37 ° C. under 5% CO 2, and then 1 × 10 5 ⁷ The cell suspension adjusted to cells / ml was dispensed at 50 μl / well into a U-bottom 96-well microplate (manufactured by Nunc) and centrifuged (90 × g, 4 ° C., 10 minutes). The supernatant was removed, the culture supernatant of the fused cells cultured in the above (2-3) was added at 50 μl / well, and the mixture was stirred, left on ice for 1 hour, and then centrifuged (90 × g, 4 ° C.). , 10 minutes) and the supernatant was removed. The pellet was washed twice with 100 μl / well of a flow cytometry buffer (PBS containing 5% FCS, 0.04% (w / v) sodium azide) and then diluted 500-fold with fluorescein-5- 50 μl of isothiocyanate (Fluorescein-5-isothiocyanate; hereinafter referred to as “FITC”) labeled goat anti-mouse IgG antibody IgG fraction (manufactured by Organon Technica) was added as a secondary antibody, and the mixture was allowed to stand on ice for 1 hour. After removing the supernatant by centrifugation (90 × g, 4 ° C., 10 minutes), the pellet was washed twice with 100 μl / well of a flow cytometry buffer, and 50 μl of a 3.7% formalin solution was added. The cells were fixed by standing on ice for 10 minutes. The supernatant was removed by centrifugation (90 × g, 4 ° C., 10 minutes), washed again with 100 μl / well of flow cytometry buffer, and the pellet was suspended in 100 μl / well of flow cytometry buffer. The cloudy one was used as a sample for flow cytometry. The FITC fluorescence intensity of the cells in each sample was measured with a flow cytometer (Epics Elite; manufactured by Coulter) (excitation wavelength: 488 nm, detection wavelength: 530 nm). As a result, a fused cell corresponding to a sample showing a FITC fluorescence intensity clearly higher (about 100-1000) than WR19L12a (FITC fluorescence intensity about 0.3) to which no fusion cell culture supernatant was added was selected. .
[0161]
(2-5) Cloning
A single antibody that binds to WR19L12a but does not bind to WR19L is obtained by repeating the series of steps (2-3) to (2-4) five times for the cell group selected in (2-4). A few clones of hybridomas producing were obtained. For the antibodies produced by those hybridomas, the binding ability to mouse Fas was examined using the same method as in (2-4). As the cells expressing mouse Fas, L5178YA1 cells obtained by introducing a mouse Fas expression vector into a cell line L5178Y (American Type Culture Collection No. CRL-1722) which hardly expresses Fas were used. As a result, mouse-mouse hybridoma HFE7A that produces an antibody that binds to L5178YA1 cells and does not bind to L5178Y cells was selected. The mouse-mouse hybridoma HFE7A was internationally deposited on February 20, 1997, with the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary (National Institute of Bioscience and Biotechnology), under the deposit number FERM BP-5828. Was done.
[0162]
The subclass of the antibody (hereinafter, referred to as "HFE7A") produced by the mouse-mouse hybridoma HFE7A was examined using a monoclonal antibody isotyping kit (Pierce), and as a result, IgG1 and κ were found.
[0163]
Example 3 Purification of HFE7A monoclonal antibody
The mouse-mouse hybridoma HFE7A (FERM BP-5828) produced in Example 2 was cultured in 1 liter of ASF medium containing 10% FCS at 37 ° C. under 5% carbon dioxide gas, and 1 × 10 6 ⁶ Cells were grown to cells / ml. The culture was centrifuged (1000 rpm, 2 minutes), the supernatant was discarded, and the precipitated cells were washed once with a serum-free ASF medium, resuspended in 1 liter of a serum-free ASF medium, and incubated at 37 ° C., 5% CO 2 gas. The cells were cultured under the following conditions for 48 hours. This culture solution is centrifuged (1000 rpm, 2 minutes), and the supernatant is collected and put into a dialysis tube (exclusion limit molecular weight 12,000 to 14000; manufactured by Gibco BRL), and 10 times the volume of 10 mM sodium phosphate buffer. The solution (pH 8.0) was dialyzed. The crude purification of IgG from the solution in the dialysis tube was performed using a high-performance liquid chromatography apparatus (FPLC system; manufactured by Pharmacia) under the conditions described below:
Column: DEAE-Sepharose CL-6B column (column size: 10 ml; manufactured by Pharmacia);
Solvent: 10 mM sodium phosphate buffer (pH 8.0)
Flow rate: 1 ml / min;
Elution: linear concentration gradient of 1 M sodium chloride (0-50%, 180 min).
The eluate was fractionated in 5 ml portions, and the anti-Fas antibody titer in each fraction was assayed by ELISA using a human Fas fusion protein. First, 100 μl / well of the roughly purified human Fas fusion protein solution prepared in Example 1 was placed in a 96-well microplate for ELISA, and incubated at 37 ° C. for 1 hour. Then, this solution was discarded, and each well was placed in 100 μl of PBS-Tween. / Well 3 times. Next, 100 μl / well of PBS containing 2% bovine serum albumin was added, and the mixture was incubated at 37 ° C. for 1 hour. After washing three times with 100 μl / well of PBS-Tween, 100 μl of each eluted fraction was added and incubated at 37 ° C. for 1 hour. Further, after washing three times with 100 μl / well of PBS-Tween, 100 μl / well of horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 2000-fold with PBS-Tween was added, and reacted at 37 ° C. for 1 hour. And washed three times with 100 μl / well of PBS-Tween. Next, 100 μl / well of a horseradish peroxidase substrate (manufactured by Bio-Rad) was added and allowed to stand for 5 minutes, and then the absorbance at 415 nm of each well was measured with a microplate reader.
[0164]
As a result, the 21st to 30th fractions having a large absorbance were collected and supplied to two antibody affinity purification columns (Hitrap Protein G column, column volume 5 ml; manufactured by Pharmacia). After washing the inside of the column with 25 ml / column equilibration buffer (20 mM sodium phosphate buffer (pH 7.0)), 15 ml / column elution buffer (0.1 M glycine-hydrochloric acid (pH 2.7)) Eluted the antibody. Each eluate was placed in a test tube containing 1.125 ml of 1M Tris-hydrochloric acid (pH 9.0), and immediately after completion of elution, a centrifugal tube type ultrafilter (Centreprep 10; manufactured by Grace Japan Co., Ltd.) And centrifuged at 3000 × g at 4 ° C. for 2 hours. After removing the filtrate collected at the lower part of the filter, 15 ml of PBS was added to the upper part, and the operation of centrifuging again at 3000 × g and 4 ° C. for 2 hours was repeated five times. However, the fifth centrifugation was performed until the amount of liquid in the upper part of the filter became 0.5 ml, and the liquid remaining on the upper part of the filter was used as an HFE7A sample.
[0165]
Example 4 cDNA cloning
(4-1) Poly (A) ⁺ Preparation of RNA
The mouse-mouse hybridoma HFE7A (FERM BP-5828) prepared in Example 2 was added to 1 liter of an ASF medium containing 10% FCS at 37 ° C. under 5% carbon dioxide gas. ⁶ After growing to cells / ml, cells were harvested by centrifugation and guanidine thiocyanate solution (4M guanidine thiocyanate, 1% sarkosyl, 20 mM ethylenediaminetetraacetic acid (EDTA), 25 mM sodium citrate (pH 7.0), 100 mM 2- The solution was dissolved with mercaptoethanol, 0.1% antifoam A) and the solution was recovered. Poly (A) ⁺ Isolation of RNA is essentially as described in "Molecular Cloning A Laboratory Manual" [Maniatis, T .; et al. (1982) pp. 196-198]. Specifically, the following procedure was performed.
[0166]
The collected cell lysate was repeatedly inhaled and discharged several times using a 10-ml syringe equipped with a 21-G injection needle. 3 ml of 5.7 M cesium chloride-0.1 M EDTA (pH 7.5) was previously added to a polyallomer centrifuge tube for an RPS40-T rotor bucket manufactured by Hitachi, Ltd., and the cell lysate was added thereto. The solution was overlaid to fill the centrifuge tube. This was centrifuged at 30,000 rpm at 20 ° C. for 18 hours, and the obtained pellet was dissolved in 400 μl of distilled water, followed by ethanol precipitation. After dissolving the obtained pellet again in 400 μl of distilled water, an equal amount of chloroform / 1-butanol (4: 1, v / v) was added and stirred, and the aqueous layer was collected after centrifugation. This was again precipitated with ethanol, and the precipitate was dissolved in 600 μl of distilled water to obtain a total RNA sample.
[0167]
Poly (A) was obtained from 600 μg of the total RNA thus obtained by oligo (dT) cellulose column chromatography. ⁺ RNA was purified. That is, total RNA is dissolved in an adsorption buffer (0.5 M sodium chloride, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS)) and heated at 65 ° C. for 5 minutes. After that, the solution was applied to an oligo (dT) cellulose column (type 7; manufactured by Pharmacia) packed with the same solution, and an elution solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05% SDS) was added. ) With poly (A) ⁺ RNA was eluted and collected. By such an operation, 100 μg of poly (A) ⁺ An RNA fraction was obtained.
[0168]
(4-2) Determination of N-terminal amino acid sequences of heavy and light chains of HFE7A
A part of the solution containing the anti-human Fas antibody HFE7A purified in Example 3 was subjected to SDS-polyacrylamide gel electrophoresis (hereinafter referred to as “SDS-PAGE”) (gel concentration: 12%, constant voltage of 100 V, 120 minutes). After the electrophoresis, the gel was immersed in a transfer buffer (25 mM Tris-HCl (pH 9.5), 20% methanol, 0.02% SDS), and then transferred using a transfer device (KS-8451; manufactured by Marisol). The protein in the gel was transferred to a polyvinylidene difluoride membrane (hereinafter referred to as “PVDF membrane”; pore size 0.45 μm; manufactured by Millipore) previously immersed in a transfer buffer (10 V constant voltage, 4 ° C., 14 hours). . After the transfer, the PVDF membrane is washed with a washing buffer (25 mM sodium chloride, 10 mM sodium borate buffer (pH 8.0)) and then stained (50% methanol, 20% acetic acid, 0.05% Coomassie brilliant blue). For 5 minutes, and then destained with 90% methanol. In this manner, the bands corresponding to the heavy chain (the band with the lower mobility) and the light chain (the band with the higher mobility) visualized on the PVDF membrane were cut out, and washed with deionized water. , A gas phase protein sequencer (PPSQ-10; manufactured by Shimadzu Corporation) using an automated Edman method [Edman, P. et al. , Et al. (1967) Eur. J. Biochem. 1, 80] was determined for each N-terminal amino acid sequence.
[0169]
As a result, the N-terminal amino acid sequence of the band corresponding to the heavy chain
Gln-Xaa-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu (SEQ ID NO: 16 in Sequence Listing)
And the N-terminal amino acid sequence of the band corresponding to the light chain is
Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Val-Ser-Leu-Gly-Gln-Arg-Ala-Thr-Ile-Ser (SEQ ID NO: 17 in Sequence Listing) )
Met.
[0170]
These amino acid sequences are stored in an antibody amino acid sequence database [Kabat, E. et al. A. et al. , (1991) in Sequences of Proteins of Immunological Interest Vol. II, U.S. S. Comparison with Department of Health and Human Services], it was found that the heavy chain (γ1 chain) of HFE7A is subtype 2b and the light chain (κ chain) is subtype 3. Therefore, the following oligonucleotide primers were synthesized which hybridize with a part of the 5'-terminal untranslated region and the most terminal part of the 3'-terminal translated region of the genes belonging to these mouse subtypes, respectively. Et al., Matti Kartinen et al. (1988) 25, 859-865 and Heinrich, G. et al. et al. (1984) J.C. Exp. Med. 159, 417-435]:
5′-GACCTCACCA TGGGATGGA-3 ′ (H1: SEQ ID NO: 18 in Sequence Listing);
5′-TTTACAGGAGAGTGGGAGA-3 ′ (H2: SEQ ID NO: 19 in Sequence Listing);
5′-AAGAAGCATC CTCTCATCTA-3 ′ (L1: SEQ ID NO: 20 in Sequence Listing);
5′-ACACTCATTC CGTTTGAAGC-3 ′ (L2: SEQ ID NO: 21 in Sequence Listing).
[0171]
(4-3) cDNA cloning
The cDNA encoding the heavy and light chains of the mouse anti-human Fas monoclonal antibody HFE7A was combined with a reverse transcriptase (RT) reaction and a PCR reaction to obtain a poly (A) derived from the HFE7A-producing hybridoma prepared in (4-1) above. ) ⁺ Cloning was performed by a method (RT-PCR) for specifically amplifying any sequence from the RNA fraction. RT-PCR was performed using RNA PCR kit (AMV) version 2 (manufactured by Takara Shuzo Co., Ltd.).
[0172]
a) Reverse transcriptase reaction
As the primer for RT-PCR, a set of oligonucleotide primers synthesized in the above (4-2) (5′-end and 3′-end primers) was used, and each primer set for heavy chain or light chain was used. RT-PCR reactions were performed individually.
[0173]

Temperature conditions: Incubation was performed at 55 ° C. for 30 minutes, at 99 ° C. for 5 minutes, and at 5 ° C. for 5 minutes.
[0174]
b) PCR

Temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1.5 minutes was repeated 28 times.
[0175]
After the completion of the reaction, a part of the reaction solution was taken and subjected to 1.5% agarose gel electrophoresis. As a result, in the reaction solution containing the heavy chain primer, a band of about 1.4 kbp was amplified. In the reaction solution containing the primer, a band of about 0.7 kbp was amplified.
[0176]
The cDNA amplified by these PCRs was cloned using a TA cloning kit (Invitrogen). First, 50 ng of the PCR reaction solution and pCRII vector (attached to the TA cloning kit) were added to 1 μl of a 10-fold concentration ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol). , 0.1 mM ATP, 2 mM DTT, 1 mM spermidine, 0.1 mg / ml bovine serum albumin), add 4 units of T4 DNA ligase (1 μl), make up to 10 μl with sterile deionized water, at 14 ° C. It was kept warm for 15 hours. Next, 2 μl of the ligase reaction solution was added to 50 μl of competent Escherichia coli TOP10F ′ (attached to the TA cloning kit) to which 2 μl of 0.5 M β-mercaptoethanol was added, and the mixture was allowed to stand on ice for 30 minutes, and then at 42 ° C. for 30 seconds. After warming, it was cooled again on ice for 5 minutes. To this, 500 μl of an SOC medium (2% tryptone, 0.5% yeast extract, 0.05% sodium chloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, 20 mM glucose) was added, followed by shaking culture at 37 ° C. for 1 hour. . This culture was used as an L-broth agar medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 0.6% Bacto agar) containing 100 μg / ml ampicillin. (Manufactured by Difco)) The cells were spread on a plate and cultured at 37 ° C. overnight. A single ampicillin-resistant colony that appeared on the plate was selected, this colony was scraped off with a platinum loop, cultured in an L-broth medium containing 100 μg / ml ampicillin, and then collected by centrifugation. Plasmid DNA was prepared from the body by the alkaline method. The plasmids thus obtained were named pCR-H (plasmid holding the cDNA encoding the HFE7A heavy chain) and pCR-L (plasmid holding the cDNA encoding the HFE7A light chain), respectively.
[0177]
(4-4) Analysis of nucleotide sequence
Nucleotide sequences of cDNA encoding HFA7A heavy chain (1.4 kbp) and cDNA encoding HFE7A light chain (0.7 kbp), which are respectively retained by plasmids pCR-H and pCR-L obtained in (4-3). Using a gene sequence analyzer (310 Genetic Analyzer; manufactured by PerkinElmer Japan Co., Ltd.) [Sanger, F. et al. , Et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467].
[0178]
The nucleotide sequences of the cDNAs encoding the HFE7A heavy and light chains determined as a result are shown in SEQ ID NOs: 8 and 10 in the sequence listing, respectively. The entire amino acid sequences of the HFE7A heavy and light chains encoded by these cDNAs are shown in Sequence Listings 9 and 11, respectively. The N-terminal amino acid sequence of the HFE7A heavy chain found in (4-1) above (SEQ ID NO: 16 in the sequence listing) is the same as the sequence of amino acids 1 to 11 in SEQ ID NO: 9 except for one unknown residue. Matched. The N-terminal amino acid sequence of the HFE7A light chain (SEQ ID NO: 17 in the sequence listing) was identical to the sequence of amino acids 1 to 22 of SEQ ID NO: 11 in the sequence listing. That is, it was revealed that the N-terminus of the mature HFE7A heavy and light chain proteins was the amino acid of amino acid number 1 of SEQ ID NOS: 9 and 11, respectively.
[0179]
In addition, the amino acid sequences of the heavy and light chains can be obtained from the database of antibody amino acid sequences by Kabat et al. [Kabat, E. et al. A. , Et al. (1991) in "Sequence of Proteins of Immunological Interest Vol. II": U.S. Pat. S. As a result of a comparison with heavy chain, amino acid numbers 1 to 121 of SEQ ID NO: 9 are a variable region, amino acid numbers 122 to 445 are a constant region, and a light chain Revealed that amino acid numbers 1 to 111 of SEQ ID NO: 11 are the variable region, and amino acid numbers 112 to 218 are the constant region.
[0180]
Further, in the amino acid sequences of the variable regions of the heavy and light chains of HFE7A determined as described above, the positions and sequences of the respective CDRs are described in Kabat et al. A. , Et al. (1991)] and the homology of the amino acid sequence of the antibody to a database was determined. According to the literature, the chain length of the framework region in the variable region is almost constant if the subtype is the same between different antibodies, and the amino acid sequences have commonality. On the other hand, CDRs are unique sequences that are sandwiched between these framework regions. Then, as a result of comparing and examining the amino acid sequences of the heavy chain and light chain of HFE7A with those of the same subtype, the CDRs of the heavy chain of HFE7A were found to have the amino acid numbers 31 to 35 (CDRH) of SEQ ID NO: 9 in the sequence listing. ₁ ), Amino acids 50-66 (CDRH) ₂ ) And amino acids 99-110 (CDRH) ₃ ) Was determined. Further, the CDRs of the light chain of HFE7A are as follows: amino acids 24 to 38 of SEQ ID NO: 11 in the sequence listing (CDRL ₁ ), Amino acids 54-60 (CDRL) ₂ ) And amino acids 93-101 (CDRL) ₃ ) Was determined.
[0181]
Example 5 Preparation of Recombinant Antibody
(5-1) Construction of expression plasmid
The cDNA encoding the heavy and light chains of HFE7A cloned in Example 4 was converted into an animal cell expression vector pMS18S [Hara, T .; , Et al. (1992) EMBO J .; 11, 1875] was constructed by the method described below. First, an oligonucleotide primer for adding a restriction enzyme EcoRI recognition site to the 5 ′ end of the cDNA held by the plasmid pCR-H and adding a restriction enzyme XbaI recognition site and a stop codon to the 3 ′ end:
5'-GGGGAATTCG ACTCACCATGGGATGGA-3 '(H3: SEQ ID NO: 22 in Sequence Listing)
5′-GGGTCTAGAC TATTTACAG GAGAGTGGGA GA-3 ′ (H4: SEQ ID NO: 23 in Sequence Listing)
Was synthesized. Oligonucleotide primers for adding a restriction enzyme EcoRI recognition site to the 5 ′ end of the cDNA held by the plasmid pCR-L and adding a restriction enzyme NotI recognition site and a stop codon to the 3 ′ end:
5′-GGGGAATTCA AGAAGCATCC TCTCATCTA-3 ′ (L3: SEQ ID NO: 24 in Sequence Listing)
5′-GGGGCGGCCG CCTACTAACA CTCATTCTCTG TTGAAGC-3 ′ (L4: SEQ ID NO: 25 in Sequence Listing) was synthesized. Using each of the primers for the heavy and light chains, PCR was performed under the following conditions.

Temperature conditions: After heating at 94 ° C for 2 minutes, a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 75 ° C for 1.5 minutes was repeated 28 times.
[0182]
After the DNA amplified by the above PCR is digested with restriction enzymes EcoRI and XbaI (heavy chain) or EcoRI and NotI (light chain), the same restriction enzymes EcoRI and XbaI (for heavy chain) or EcoRI and NotI (for light chain) are used. ) Digested and dephosphorylated expression plasmid vector for animal cells pME18S [Hara, T .; , Et al. (1992) EMBO J .; 11, 1875] and a 10-fold ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine) , 0.1 mg / ml bovine serum albumin), 4 units of T4 DNA ligase was added, and the mixture was incubated at 14 ° C for 15 hours. 2 μl of this ligase reaction solution was added to 50 μl of competent Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.), allowed to stand on ice for 30 minutes, and then allowed to stand at 42 ° C. for 30 seconds and on ice for 5 minutes. To this, 500 μl of an SOC medium (2% tryptone, 0.5% yeast extract, 0.05% sodium chloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, 20 mM glucose) was added, followed by shaking culture for 1 hour. Hereinafter, a transformed E. coli strain was isolated by the method described in Example 4 (4-3), and a plasmid DNA was prepared from the strain. The plasmids thus obtained were combined with plasmids pME-H (an expression plasmid vector carrying the cDNA encoding the HFE7A heavy chain) and plasmid pME-L (an expression plasmid vector carrying the cDNA encoding the HFE7A light chain). Named. Transformed E. coli strains carrying these plasmids coli pME-H and E. coli. coli pME-L has been internationally deposited on March 12, 1997 at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) with deposit numbers FERM BP. -5868 and FERM BP-5867.
[0183]
(5-2) Expression in COS-7 cells
Transformation of the plasmids pME-H and pME-L obtained in the above (5-1) into COS-7 cells was performed by electroporation using a gene transfer device (ECM600: manufactured by Beatty X). . First, COS-7 cells (American Type Culture Collection No. CRL-1651) were placed in a cell culture flask (culture area 225 cm) containing DMEM containing 10% FCS. ² ; Sumitomo Bakelite Co., Ltd.) until the cells became semi-confluent. After removing the medium, 3 ml of a trypsin-EDTA solution (Sigma) was added and incubated at 37 ° C. for 3 minutes. The released cells are collected, washed twice with PBS, and then washed with 5 × 10 5 with PBS. ⁶ Adjusted to cells / ml. In addition, 20 μg of each of plasmids pME-H and pME-L prepared using a plasmid large-scale preparation kit (Maxiprep / DNA Purification System: manufactured by Promega) was ethanol-precipitated, and then dissolved in 20 μl of sterilized PBS. When co-transfection was performed with two types of plasmids, 20 μg of each plasmid was used. 20 μl of the cell suspension prepared as described above (1.2 × 10 ⁶ The cells were mixed with 20 μl of the plasmid solution, placed in a chamber (manufactured by Beatty X Corp.) with an electrode spacing of 2 mm, set in a gene transfer apparatus, and a pulse of 10 msec was applied once at a setting of 150 V and 900 μF. The cell-DNA mixture in the chamber was added to 40 ml of DMEM containing 10% FCS, and cultured in a plastic dish for cell culture at 37 ° C. under 5% carbon dioxide for 24 hours. Next, the culture supernatant was removed, and the cells were washed with a serum-free DMEM medium. Then, 40 ml of serum-free DMEM was added, and the cells were further cultured at 37 ° C. under 5% carbon dioxide for 72 hours to collect the culture supernatant.
[0184]
COS-7 cells were each transformed with the plasmids or combinations of plasmids described below, and the culture supernatants of the transformants were collected by the above method:
[A] pME-H only;
[B] pME-L only;
[C] Co-transfection of pME-H and pME-L.
[0185]
(5-3) Detection of anti-Fas antibody in culture supernatant of transformant
Whether the anti-Fas antibody was produced in the culture supernatant of the transformed COS-7 cell prepared in the above (5-2) was determined by the ELISA method using the human Fas fusion protein as an antigen described in Example 3 as an antigen. Examined. As a result, it was confirmed that only in the case of co-transfection with pME-H and pME-L ([C] above), an antibody that reacts with the human Fas fusion protein was produced in the culture supernatant.
[0186]
Example 6 Restriction of epitope
(6-1) ELISA
The peptide described below was synthesized using an automatic peptide synthesizer (Model 430A; PerkinElmer Japan Applied Biosystems Division) using the Fmoc solid-phase synthesis method [Carpino, L. et al. A. and Han, G .; Y. (1970) J.I. Amer. Chem. Soc. 92, 5748-5549].
Arg-Leu-Ser-Ser-Lys-Ser-Val-Asn-Ala-Gln-Val-Thr-Asp-Ile-Asn-Ser-Lys-Gly-Leu (P1: SEQ ID NO: 26 in Sequence Listing);
Val-Thr-Asp-Ile-Asn-Ser-Lys-Gly-Leu-Glu-Leu-Arg-Lys-Thr-Val-Thr-Thr-Val-Glu (P2: SEQ ID NO: 27 in Sequence Listing);
Glu-Leu-Arg-Lys-Thr-Val-Thr-Thr-Val-Glu-Thr-Gln-Asn-Leu-Glu-Gly-Leu-His-His-Asp (P3: SEQ ID NO: 28 in Sequence Listing);
Thr-Gln-Asn-Leu-Glu-Gly-Leu-His-His-Asp-Gly-Gln-Phe-Cys-His-Lys-Pro-Cys-Pro-Pro (P4: SEQ ID NO: 29 in Sequence Listing);
Gly-Gln-Phe-Cys-His-Lys-Pro-Cys-Pro-Pro-Gly-Glu-Arg-Lys-Ala-Arg-Asp-Cys-Thr-Val (P5: SEQ ID NO: 30 in Sequence Listing);
Gly-Glu-Arg-Lys-Ala-Arg-Asp-Cys-Thr-Val-Asn-Gly-Asp-Glu-Pro-Asp-Cys-Val-Pro-Cys-Gln (P6: SEQ ID NO: 31 in Sequence Listing) );
Asn-Gly-Asp-Glu-Pro-Asp-Cys-Val-Pro-Cys-Gln-Glu-Gly-Lys-Glu-Tyr-Thr-Asp-Lys-Ala (P7: SEQ ID NO: 32 in Sequence Listing);
Glu-Gly-Lys-Glu-Tyr-Thr-Asp-Lys-Ala-His-Phe-Ser-Ser-Lys-Cys-Arg-Arg-Cys-Arg (P8: SEQ ID NO: 33 in Sequence Listing);
His-Phe-Ser-Ser-Lys-Cys-Arg-Arg-Cys-Arg-Leu-Cys-Asp-Glu-Gly-His-Gly-Leu-Glu-Val (P9: SEQ ID NO: 34 in Sequence Listing);
Leu-Cys-Asp-Glu-Gly-His-Gly-Leu-Glu-Val-Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr (P10: SEQ ID NO: 35 in Sequence Listing);
Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr-Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys (P11: SEQ ID NO: 36 in Sequence Listing);
Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys-Glu-His-Cys-Asp-Pro (P12: SEQ ID NO: 37 in Sequence Listing);
Asn-Ser-Thr-Val-Cys-Glu-His-Cys-Asp-Pro-Cys-Thr-Lys-Cys-Glu-His-Gly-Ile-Ile-Lys (P13: SEQ ID NO: 38 in Sequence Listing);
Cys-Thr-Lys-Cys-Glu-His-Gly-Ile-Ile-Lys-Glu-Cys-Thr-Leu-Thr-Ser-Asn-Thr-Lys-Cys (P14: SEQ ID NO: 39 in Sequence Listing);
Glu-Cys-Thr-Leu-Thr-Ser-Asn-Thr-Lys-Cys-Lys-Glu-Glu-Gly-Ser-Arg-Ser-Asn (P15: SEQ ID NO: 40 in Sequence Listing);
Ser-Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile-Tyr-Thr-Lys-Lys-Glu-Ala-Phe-Asn-Val-Glu (P16: SEQ ID NO: 41 in Sequence Listing).
[0187]
P1 to P15 are partial sequences of the amino acid sequence from position 1 to position 157 of the extracellular region of human Fas, and the amino acid sequence of 9 to 11 residues overlaps with other peptides. P16 is a peptide for negative control and has no homology with human Fas.
[0188]
Each of P1 to P16 was completely dissolved in 48 μl of dimethyl sulfoxide (hereinafter referred to as “DMSO”), and then adjusted to a total volume of 0.8 ml by adding PBS containing 1 mM 2-mercaptoethanol. did.
[0189]
A peptide corresponding to the extracellular region of the human Fas molecule having a carboxyl group added to the C-terminus was diluted to a concentration of 50 μg / ml with a 0.05 M carbonate-bicarbonate buffer (pH 9.6) containing 10 mM 2-mercaptoethanol. 50 μl / well was placed in a 96-well ELISA plate (manufactured by Nunc). The plate was allowed to stand at 4 ° C. overnight to adsorb the peptide on the inner surface of the well. The solution in each well was discarded, and the well was washed four times with PBS-Tween. PBS containing 1% (w / v) bovine serum albumin (A3803 manufactured by Sigma) was dispensed at 100 μl / well and incubated at 37 ° C. for 1 hour. After washing each well four times with PBS-Tween, 50 μl of HFE7A and CH11 adjusted to 5 μg / ml with PBS were added to each well. After incubating at 37 ° C. for 1 hour, each well was washed four times with PBS-Tween. A horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 1000-fold with PBS was dispensed at 50 μl / well, incubated at 37 ° C. for 1 hour, and then each well was washed with PBS-Tween for 4 hours. Washed twice. A horseradish peroxidase substrate (manufactured by Bio-Rad) was dispensed at 100 μl / well into each well, and allowed to stand at room temperature for 15 minutes. Then, the absorbance at 415 nm of each well was measured with a microplate reader (manufactured by Corona). As a positive control, the human Fas fusion protein prepared in Example 1 was used instead of the synthetic peptide.
[0190]
As a result, only the wells to which P11 was adsorbed among the synthetic peptides showed high absorbance, indicating that HFE7A specifically bound to the amino acid sequence contained in P11 (FIG. 3).
[0191]
(6-2) Restriction of epitope by competition between synthetic peptide and human Fas on cell surface (competition assay method)
First, the peptides described below were synthesized:
His-Gly-Leu-Glu-Val-Glu-Ile-Asn-Cys-Thr (P95: SEQ ID NO: 42 in Sequence Listing);
Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr (P100: SEQ ID NO: 43 in Sequence Listing);
Arg-Thr-Gln-Asn-Thr-Lys-Cys-Arg-Cys-Lys (P105: SEQ ID NO: 1 in Sequence Listing);
Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys (P110: SEQ ID NO: 44 in Sequence Listing);
Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys-Glu-His-Cys-Asp (P115L: SEQ ID NO: 45 in Sequence Listing);
Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-Asp-Asn (D355-364: SEQ ID NO: 46 in Sequence Listing).
[0192]
P95, P100, P105 and P110 are partial sequences consisting of 10 residues near the amino acid sequence corresponding to P11 in the extracellular region of human Fas (corresponding to positions 95 to 128 of the extracellular region of human Fas). The amino acid sequence overlaps with other peptides by 5 residues each. P115L has a 10 residue amino acid sequence that overlaps 5 residues with P110:
Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys (P115: amino acids 1 to 10 of SEQ ID NO: 45 in the sequence listing)
Is expected to have low solubility, and is extended by 4 residues on the C-terminal side. D355-364 is a peptide with no homology to human Fas and was used as a negative control. After dissolving each of the above peptides except P115L in 16 μl of DMSO, PBS containing 1 mM 2-mercaptoethanol was added to adjust the total volume to 0.8 ml to prepare a peptide solution. P115L was dissolved in 48 μl of DMSO, and then adjusted to a total volume of 0.8 ml by adding PBS containing 1 mM 2-mercaptoethanol.
[0193]
0.25 μg of HFE7A and each of the above peptide solutions (equivalent to 200 μg of peptide) were mixed in a micro test tube, and the total volume was adjusted to 100 μl with PBS containing 1 mM 2-mercaptoethanol. This was kept at 37 ° C. for 2 hours while stirring at 10 to 20 rpm, and then FCS was added to a final concentration of 5% to prepare a peptide-antibody mixed solution.
[0194]
WR19L12a cells were grown by the method described in Example 2, collected by centrifugation, and grown to a cell density of 1 × 10 4 using serum-free RPMI medium. ⁷ Adjusted to cells / ml. The cell suspension was dispensed at 100 μl / well into a U-shaped bottom 96-well plate, and centrifuged at 4 ° C. and 1000 rpm for 3 minutes using a microplate swing rotor to remove the supernatant. 100 μl of each peptide-antibody mixture was added to the pellet, and pipetting was performed once or twice. After standing at 4 ° C. for 30 minutes, the mixture was centrifuged to remove the supernatant. The pellet was washed three times with the flow cytometry buffer, and then 50 μl / well of a FITC-labeled goat anti-mouse IgG antibody (manufactured by Kappel) diluted 250-fold with the flow cytometry buffer was added. Pipette once. The plate was allowed to stand at 4 ° C. for 30 minutes in the dark, and then centrifuged to remove the supernatant. The pellet was washed three times with a flow cytometry buffer, and a 10% neutral buffered formalin solution for tissue fixation (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 10-fold with PBS was added at 50 μl / well. After pipetting once or twice, the plate was allowed to stand at 4 ° C. for 12 hours or more while shielding the light from light to fix the cells. After 100 μl / well of a buffer for flow cytometry was added and suspended, the mixture was centrifuged and the supernatant was removed. After the pellet was washed three times with a flow cytometry buffer, the suspension suspended in 500 μl / well of a flow cytometry buffer was analyzed with a flow cytometer (Cytoace-150; manufactured by JASCO Corporation). (Excitation wavelength: 488 nm, detection wavelength: 530 nm), and the average value of the FITC fluorescence intensity per cell was calculated. Furthermore, the value when no peptide-antibody mixture was added was 0%, and the value of D355-364 was 100%, and the average fluorescence intensity of each sample was calculated.
[0195]
As a result, P105 strongly inhibited the binding between HFE7A and WR19L12a cells, and P100 and P110, which overlap the amino acid sequence of P105 by half each, inhibited the binding between HFE7A and WR19L12a cells by about 50% and 60%, respectively. It has been found. No binding inhibition was observed for P95 and P115L, which do not completely overlap the amino acid sequence of P105 (FIG. 4). From the above results, it was shown that P105 has an amino acid sequence sufficient to inhibit the binding of HFE7A to human Fas, and that the epitope of HFE7A is included in the amino acid sequence corresponding to P105. The portion corresponding to P105 is a region where the amino acid sequence is conserved between human Fas and mouse Fas.
[0196]
Example 7 Binding ability of HFE7A to monkey Fas
The tests described below were performed using 40 ml of peripheral blood from chimpanzees (Sanwa Chemical Laboratory Kumamoto Primate Park), 20 ml of peripheral blood of Japanese macaques, 20 ml of peripheral blood of cynomolgus monkeys, or 3 ml of peripheral blood of marmoset. First, peripheral blood to which heparin (Novoheparin: manufactured by Novo) was added was quietly added to an equal volume of Ficoll-Pak solution (specific gravity: 1.077; for cynomolgus monkeys, the specific gravity was adjusted to 1.072: manufactured by Pharmacia). And centrifuged at 1700 rpm for 30 minutes to collect a peripheral blood mononuclear cell fraction. The peripheral blood mononuclear cell fraction was washed twice with Hanks' balanced salt solution, and then washed with RPMI1640 medium containing 10% FCS at 1 × 10 6. ⁶ The cells were suspended to give cells / ml. Phytohemagglutinin-P (manufactured by Sigma) was added to this suspension to a final concentration of 5 μg / ml, and the mixture was cultured at 37 ° C. for 24 hours under 5% carbon dioxide gas, and then centrifuged to collect and wash the cells. Thereafter, the cells were resuspended in RPMI1640 medium containing 10% FCS. Activated lymphocytes were obtained by adding interleukin 2 (manufactured by Amersham) to this suspension to a final concentration of 10 units / ml and culturing at 37 ° C. under 5% carbon dioxide for 72 hours.
[0197]
Next, this activated lymphocyte 1 × 10 ⁶ The cells were placed in a test tube, suspended in 50 μl of 20 μg / ml HFE7A or 50 μl of PBS, left on ice for 1 hour, and then washed three times with 500 μl of PBS. The cells were suspended in 50 μl of a 20 μg / ml FITC-labeled anti-mouse IgG antibody (manufactured by BioResource), allowed to stand on ice for 30 minutes, and then washed three times with 500 μl of PBS. Using the cells resuspended in 500 μl of PBS as a sample, the fluorescence intensity was measured using a flow cytometer (Cytoace: manufactured by JASCO Corporation). The frequency distribution of the number of cells based on the fluorescence intensity was obtained, and the ratio of the number of stained cells to the total number of cells was calculated. As a result, in the sample without HFE7A, less than 3% of the cells were stained in any of the above-mentioned species, whereas in the sample with HFE7A, at least 17% and at most 82% of the cells were stained. It had been. Thus, HFE7A was shown to bind to a wide range of primates, including humans.
[0198]
Example 8 Apoptosis-inducing activity of HFE7A on T cells in vivo in mice
For a 6-week-old female C3H / HeJ mouse (purchased from CLEA Japan), 0.05 mg or 0.1 mg / individual HFE7A monoclonal antibody (both dissolved in 500 μl of PBS) or 500 μl of PBS Was administered intraperitoneally (3 mice per group), and 42 hours after administration, the mice were anesthetized with ether and the thymus was removed. The extracted thymus was washed with RPMI medium containing 10% FCS, and then crushed using a spatula on a mesh (Cell Strainer; manufactured by Falcon), and the cells that passed through the mesh were twice washed with RPMI1640 medium containing 10% FCS. Washed. After measuring the number of cells, 1 × 10 6 was added to RPMI1640 medium containing 10% FCS. ⁶ Thymocytes suspended at 50 μl / cell were dispensed at 50 μl / well into a U-bottom 96-well microplate (manufactured by Nunc) and centrifuged (90 × g, 4 ° C., 10 minutes).
[0199]
The supernatant was removed and the following fluorescently labeled antibody solution (a) or (b) was added to each well:
(A) 0.5 mg / ml FITC-labeled anti-mouse CD95 (Fas) antibody (Jo2: manufactured by Pharmingen) 10 μl / well and 0.5 mg / ml phycoerythrin (hereinafter referred to as “PE”)-labeled anti-mouse CD90 antibody (Thy-1.2: manufactured by Cedarane) 10 μl / well (CD90 is a surface antigen specifically expressed on T cells);
(B) 10 μl / well of 0.5 mg / ml FITC-labeled anti-mouse CD4 antibody (L3T4: manufactured by Pharmingen) and 0.2 mg / ml of PE-labeled anti-mouse CD8 antibody (Ly-2: manufactured by Pharmingen) ) 10 μl / well.
[0200]
The plate was shaken to stir the well, left on ice for 1 hour, and then centrifuged (90 × g, 4 ° C., 10 minutes). After removing the supernatant and washing twice with 100 μl / well of a buffer for flow cytometry, 50 μl / well of a 3.7% formalin solution was added, and the cells were fixed by allowing to stand on ice for 10 minutes. The supernatant was removed by centrifugation (90 × g, 4 ° C., 10 minutes), and the precipitated cells were washed again with 100 μl / well of flow cytometry buffer and suspended in 100 μl / well of flow cytometry buffer. It became cloudy. Using the cell suspension in each well thus obtained as a sample, 1 × 10 4 was measured using a flow cytometer (Epix Elite: manufactured by Coulter). ⁴ The fluorescence of the cells / sample was measured under the conditions described below:
Excitation wavelength: 488 nm;
Detection wavelength: 530 nm (FITC), 600 nm (PE).
[0201]
From the measurement data, the fluorescence distribution of FITC and PE of the cell population of each sample was prepared. For the sample to which the fluorescently labeled antibody of (a) was added, Fas-positive and CD90-positive cells (hereinafter referred to as “Fas ⁺ CD90 ⁺ ) Of the total cell number was calculated. Similarly, for the sample to which the fluorescent-labeled antibody of (b) above was added, CD4 + CD8 + cells (hereinafter “CD4 ⁺ CD8 ⁺ ") Or CD4-positive CD8-negative cells (hereinafter" CD4 ⁺ CD8 ⁻ ) In the total number of cells.
[0202]
Table 1 shows the results.
[0203]
[Table 1]

(Value is%)
In the thymocytes of the mice to which HFE7A was administered, the T cells expressing Fas (Fas ⁺ CD90 ⁺ ) Was significantly reduced at all doses. CD4, which is known to have remarkable Fas expression, ⁺ CD8 ⁺ Cell group and CD4 ⁺ CD8 ⁻ The number of the cell group was also significantly reduced by the administration of HFE7A as compared with the group to which only PBS was administered.
[0204]
The above results indicate that the anti-Fas monoclonal antibody HFE7A has apoptosis-inducing activity on T cells expressing Fas molecules in vivo.
[0205]
Example 9 Effect of HFE7A on autoimmune disease model
Using the MRLgld / gld mouse, which is a mutant of the Fas ligand gene and a model mouse for systemic lupus erythematosus-like autoimmune disease, the effect of the administration of the anti-Fas monoclonal antibody HFE7A on the symptoms of autoimmune disease is described below. investigated.
[0206]
Intraperitoneally 18-week-old MRL gld / gld mice (purchased from Japan SLC, Inc.), 0.2 mg or 0.5 mg of HFE7A prepared in Example 3 (dissolved in 500 μl of PBS), Alternatively, 500 μl of PBS was each administered once.
[0207]
Swelling of the wrist and ankle of each test mouse caused by autoimmune disease symptoms was observed, the degree of swelling was evaluated, and the change over time in each group was examined [Shin Yonehara (1994) Nikkei Science Supplement 110, 66] -77]. As a result, it became clear that the degree of swelling of the wrist and ankle was significantly reduced by HFE7A administration.
[0208]
In addition, the thymus was excised from these test mice, and the percentage of T cells expressing Fas in the thymus was examined by the method described in Test Example 1 above. The number of T cells expressing Fas significantly decreased.
[0209]
Example 10 Hepatotoxicity test
BALB / c mice were administered intraperitoneally with any of the following:
i) 0.2 mg of HFE7A (dissolved in 500 μl of PBS);
ii) HFE7A 0.5 mg (dissolved in 500 μl PBS);
iii) Jo2 (Pharmingen) 0.1 mg (dissolved in 500 μl of PBS);
iv) 500 μl of PBS only.
[0210]
Of the above, Jo2 is a known anti-mouse Fas antibody having apoptosis-inducing activity. Eight hours, 24 hours and 72 hours after administration, whole blood was collected from the posterior vena cava under light ether anesthesia from mice at 8 hours, 72 hours after administration, and glutamate-oxaloacetate transaminase in each sample blood. (Hereinafter referred to as “GOT”) value and glutamate-pyruvate transaminase (hereinafter referred to as “GPT”) value were measured by an automatic analyzer (Model 7250; manufactured by Hitachi, Ltd.) and a reagent for the same (Transaminase-HRII; The measurement was carried out using Kojun Pharmaceutical Co., Ltd.). As a result, the GOT value and the GPT value increased sharply after 3 hours in the Jo2-administered group, whereas the GOT value and the GPT value in the HFE7A-administered group hardly changed as in the group administered only with PBS (FIG. 5). . The above results indicated that HFE7A did not induce liver damage.
[0211]
Example 11 Effect on fulminant hepatitis model
It is known that when the anti-mouse Fas antibody Jo2 is administered intraperitoneally to mice, the mice develop fulminant hepatitis within a few hours and die [Ogasawara, J. et al. , Et al. (1993) Nature 364, 806]. Therefore, in order to examine the effect of HFE7A on liver injury caused by Jo2, the survival rate of mice when HFE7A was administered simultaneously with or after Jo2 was examined. Antibodies were administered intraperitoneally to 6-week-old BALB / c mouse females (3 mice per group: purchased from Japan SLC, Inc.) under the following conditions and observed over time:
(A) Jo2 0.1 mg / 0.5 ml;
(B) Jo2 0.01 mg / 0.5 ml;
(C) Jo2 0.1 mg and HFE7A 0.5 mg / 0.5 ml (simultaneous administration);
(D) Jo2 0.1 mg and HFE7A 0.05 mg / 0.5 ml (simultaneous administration);
(E) After administration of 0.01 mg / 0.2 ml of Jo2,
20 minutes later, HFE7A was administered at 0.1 mg / 0.2 ml.
[0212]
FIG. 6 shows the result. In the case of single administration of Jo2, all mice died within 9 hours in both cases of 0.1 mg / individual and 0.01 mg / individual ((A) and (B)). However, when HFE7A was co-administered (0.5 mg / individual, 0.05 mg / individual) during the administration of Jo2 ((C) and (D)), the mice did not show any abnormality even after several weeks of administration. It was found that the administration of HFE7A can prevent the onset of fulminant hepatitis. Furthermore, even when HFE7A was administered 20 minutes after the administration of Jo2, the mouse was normal and no external symptoms were observed. Therefore, it was derived from the damage of normal tissues via the Fas / Fas ligand system in the liver and the like. HFE7A has been shown to have prophylactic and therapeutic effects on various diseases.
[0213]
Example 12 Effect on Rheumatoid Arthritis
1) Preventive effects on collagen-induced arthritis
A CD1F1 mouse (a 6-week-old female, purchased from Charles River Japan Co., Ltd.), an F1 mouse obtained by mating a female BALB / c mouse with a male DBA / 1J mouse, was acclimated for 1 week. The method described in the literature [Phadke, K .; (1985) Immunopharmacol. 10, 51-60], a 0.3% solution of bovine type II collagen (50 mM acetic acid solution, manufactured by Collagen Technology Workshop) was diluted to 50% acetic acid to 0.2% (2 mg / ml), and the Of Freund's complete Freund's adjuvant (manufactured by Difco) and emulsified. Then, using a plastic 1 ml syringe equipped with a tuberculin needle, 100 μl (as bovine type II collagen) was injected into the base of the ridge of the mouse held in an intravenous incubator. (Equivalent to 100 μg). One week after the first sensitization, booster immunization was performed under the same conditions as above. At the time of booster, 100 μg of HFE7A or mouse IgG for control was intraperitoneally administered (6 mice in each group). From 5 weeks after the first sensitization, swelling of the extremities began to be observed. The joints of the extremities are observed macroscopically, and the method described in the literature [Wood, F. et al. D. , Et al. (1969) Int. Arch. Allergy Appl. Immunol. 35, 456-467], and the degree of swelling was scored. The score was determined for each limb according to the following criteria:
0: No symptoms;
1: Only one small joint such as a finger of a limb swells and reddens;
2: Swelling and redness of two or more small joints or relatively large joints such as wrists and ankles;
3: One hand or foot is swollen and red.
[0214]
That is, the score of one individual when all the limbs are swollen to the maximum is 12. An individual having a total limb score of 1 or more was defined as a “mouse onset”.
[0215]
The results are shown in FIG. In the control group to which non-specific mouse IgG was administered, all mice developed by 7 weeks after the first sensitization, whereas half of the mice in the HFE7A administration group did not develop any disease by 8 weeks. (FIG. 7A). The average score was lower in the HFE7A-administered group than in the control group (FIG. 7B).
[0216]
2) Apoptosis-inducing ability for synovial cells from rheumatic patients
The effect of HFE7A on the survival rate of synovial cells derived from patients with rheumatoid arthritis was examined by the method described below using mitochondrial reduction ability as an index. First, synovial tissue collected from the affected part of a human rheumatoid arthritis patient was minced with scissors in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Sumit), and fat was removed. Collagenase (manufactured by Sigma) was added at 5 μg / ml, and the mixture was incubated at 37 ° C. for 90 minutes. Cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum under conditions of 37 ° C. and 5% carbon dioxide gas was used as rheumatoid patient-derived synovial cells.
[0219]
The thus-obtained synovial cells derived from a rheumatic patient were converted into single cells by trypsin treatment and then 1 × 10 5 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. ⁵ After suspending to 2 cells / ml, 2 × 10 ⁴ Cells were seeded at 200 μl / well and cultured at 37 ° C. under 5% carbon dioxide for 6 days. After removing the culture supernatant, the cells were washed three times with Hanks buffer (manufactured by Gibco), and 200 μl of Dulbecco's modified Eagle's medium containing 10 ng / ml to 1000 ng / ml of HFE7A (10-fold dilution series) and 10% fetal bovine serum was added. Was added. After culturing at 37 ° C. and 5% carbon dioxide for 20 hours, 1 mg / ml XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide inner salt: Sigma Corporation 50 μl (final concentration: 250 μg / ml XTT; 5 μM PMS) and 25 μM PMS (phenazine methosulfate: Sigma) were added, and the mixture was further cultured for 4 hours, and the absorbance at 450 nm of each well was measured.
[0218]
The cell viability in each well was calculated by the following formula:
Survival rate (%) = 100 × (ab) / (c−b)
(In the formula, a represents a measured value of a test well, b represents a measured value of a cell-free well, and c represents a measured value of a well in which no antibody is added.)
The results are shown in Table 2. HFE7A inhibited the survival of rheumatoid patient-derived synovial cells in a concentration-dependent manner.
[0219]
[Table 2]

Example 13 Design of HFE7A Humanized Antibody
(1) Molecular modeling of the variable region of HFE7A
Molecular modeling of the variable region of HFE7A was performed using a method generally known as homology modeling [see Methods in Enzymology 203, 121-153 (1991)]. That is, a protein data bank (Chemistry Department, Building 555, Brokhaven National Laboratory, PO Box 5000, Upton, NY, 1993, NY 1197, NY 1173-1000). The primary sequence of the variable region of the human immunoglobulin subjected to X-ray crystal structure analysis registered with the HFE7A framework portion is compared with the framework portion of HFE7A, and the three-dimensional structure of the framework having the highest homology is identified as the light chain. Was selected as 1GGI and 2HFL as the heavy chain. By combining both, a three-dimensional structure of the framework part (hereinafter referred to as “framework model”) was constructed.
[0220]
According to the classification of Cottia et al., Among CDRs of HFE7A, CDRL ₂ Is Canonical Class 1, CDRL ₃ Is Canonical Class 1, CDRH ₁ Was classified into canonical class 1. CDRL ₁ , CDRH ₂ , CDRH ₃ Did not correspond to a particular canonical class. CDRL ₂ , CDRL ₃ And CDRH ₁ CDR loops were fixed to a conformation specific to each canonical class and incorporated into the framework model. CDRL ₁ Is the classification of Sorongton et al [J. Mol. Biol. 263, 800-815 (1996)]. CDRH ₂ And CDRH ₃ For, the conformation of a highly homologous sequence was searched from the PDB, and the most probable CDR loop conformation was constructed by using energy calculation in combination, and was incorporated into the framework model. Finally, an energy calculation was performed so as to eliminate the contact between atoms that are disadvantageous in terms of energy, and a molecular model of HFE7A was obtained. The above operation can be performed using a commercially available general molecular modeling system. In the present invention, AbM (manufactured by Oxford Molecular Limited) was used.
[0221]
For the obtained molecular model, the accuracy of the structure was evaluated using software procheck (PROCHECK: see J. Appl. Cryst. (1993) 26, 283-291), and then the surface exposure of each residue was performed. The degree was calculated and the presence or absence of interatomic contact was searched.
[0222]
(2) Acceptor selection
The light chain and heavy chain subgroups of HFE7A show 79% identity with the subgroup κIV in the light chain and 79% with subgroup I in the heavy chain in comparison with the consensus sequence of each subgroup of human antibodies. Was. However, no human antibody with this combination was present. As a human antibody in which the light chain and the heavy chain are derived from the same antibody, and both show 70% or more identity, 8E10'CL whose light chain is subgroup κIII and whose heavy chain is subgroup I was selected. .
[0223]
(3) Selection of donor residue to be transplanted to acceptor
Using a software chameleon (manufactured by Oxford Molecular Limited), the amino acid sequences of the light chain and heavy chain of HFE7A and the acceptor were aligned, and the following procedures were performed in accordance with the requirements of a) to d) described above. The humanized variable region sequences shown in the examples were designed, and the following plasmids were constructed as recombinant vectors containing the nucleotide sequence of the DNA encoding the anti-human Fas humanized antibody.
[0224]
Example 14 Preparation of DNA Encoding Humanized Light Chain
(1) Cloning of cDNA encoding full length human light chain (κ chain)
Prior to humanizing the light chain amino acid sequence of mouse anti-human Fas antibody HFE7A, human immunoglobulin (hereinafter referred to as “Ig”) light chain cDNA containing a constant region was cloned.
[0225]
1) Synthesis of primer
The cDNA encoding the human light chain was separated by PCR. In carrying out the PCR, the following two primers were synthesized:
5′-GCGAATTCTG CCTGACTACTA TCAGAGTTTC CTCA-3 ′ (HVKII5-4: SEQ ID NO: 47 in Sequence Listing);
5'-GCTCTAGATAG AGGTGAAAGA TGAGCTGGAG GA-3 '(HKCL3-3: SEQ ID NO: 48 in Sequence Listing).
[0226]
2) Construction of plasmid containing human Ig light chain cDNA
A cDNA containing the full length human Ig light chain was prepared by PCR, inserted into a plasmid, and cloned in E. coli.
[0227]
An HL-DNA fragment encoding the full length human Ig light chain was prepared under the following conditions.

The reaction solution having the above composition was adjusted to a final volume of 100 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0228]
The HL-DNA fragment thus prepared was inserted into a plasmid pCR3DNA using a TA cloning kit for eukaryotes (manufactured by Invitrogen) according to the protocol specified by the manufacturer, and the competent kit previously attached to the kit was used. It was introduced into E. coli TOP10F 'strain. As a result, a plasmid pHL15-27 carrying an HL-DNA fragment which is a cDNA of a human Ig light chain was obtained.
[0229]
(2) Construction of humanized antibody light chain expression vector for HFE7A
1) Construction of humanized HFE7A-light chain expression vector plasmid
In humanizing the light chain amino acid sequence of the mouse anti-human Fas antibody HFE7A, the 47th amino acid (proline) and the 49th amino acid (lysine) from the N-terminal side of the HFE7A light chain amino acid sequence (hereinafter referred to as "region I") Are replaced with alanine and arginine, respectively, which are conserved in the human light chain (κ chain), and the amino acid at position 80 (histidine), the amino acid at position 81 (proline), the amino acid at position 82 (valine), the amino acid at position 84 ( Glutamic acid), the 85th amino acid (glutamic acid), the 87th amino acid (alanine) and the 89th amino acid (threonine) (hereinafter referred to as “region II”), each of which is conserved in a human light chain (κ chain); Place on arginine, leucine, proline, alanine, phenylalanine, valine The replacement was designated as "HH type".
[0230]
The “HM type” was obtained by replacing region I with human amino acid residues and leaving region II as mouse amino acid residues.
[0231]
Furthermore, those in which the amino acid residues of mouse in both region I and region II were left as "MM type".
[0232]
Hereinafter, expression plasmids having these three types of humanized anti-human Fas antibody HFE7A-light chain amino acid sequences were respectively constructed.
[0233]
2) Synthesis of primers for preparing light chain variable and constant regions of humanized HFE7A
DNA encoding the HH-type polypeptide chain (SEQ ID NO: 50 in the sequence listing) obtained by fusing the variable region of each humanized anti-Fas antibody HFE7A-light chain with the constant region of a human Ig light chain (κ chain) SEQ ID NO: 49), DNA encoding the HM type polypeptide chain (SEQ ID NO: 52 in the sequence listing) (SEQ ID NO: 51 in the sequence listing) and DNA encoding the MM type polypeptide chain (SEQ ID NO: 54 in the sequence listing) The synthesis of SEQ ID NO: 53) in the column list was performed by a combination of the PCR methods.
[0234]
In performing the PCR, the following thirteen oligonucleotide primers were synthesized:
5'-CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCT-3 '(7AL1P: SEQ ID NO: 55 in Sequence Listing);
5′-GAGAGGGTGG CCCTCTCCCC TGGAGACAGA GACAAAGTAC CTGG-3 ′ (7AL1N: SEQ ID NO: 56 in Sequence Listing);
5′-CCAGGTACTTTTGTCTCTGTCTCCAGGGGAGAGGGCCACCCTCTC-3 ′ (7AL2P: SEQ ID NO: 57 in Sequence Listing);
5'-GATTCGAGATTGGATGCAGCATAGATGAGAGTCTGGGTGCCTG-3 '(7AL2N: SEQ ID NO: 58 in Sequence Listing);
5'-GCTGCATCCAATCTCGAATCTGGGATCCCAGACAGGTTTAGTGGC-3 '(7AL3PA: SEQ ID NO: 59 in Sequence Listing);
5′-AAAATCCGCC GGCTCCAGAC GAGAGATGGT GAGGGTGAAG TCTGTCCCAG AC-3 ′ (7AL3N: SEQ ID NO: 60 in Sequence Listing);
5′-CTCGTCTGGA GCCGGCGGAT TTTGCAGTCT ATTACTGTCA GCAAAGTAAT GAGGATCC-3 ′ (7AL4P: SEQ ID NO: 61 in Sequence Listing);
5′-TGAAGACAGA TGGTGCAGCC ACATCCGGT TGATTTCCAG CCTGGTGCCT TGACC -3 ′ (7AL4N: SEQ ID NO: 62 in Sequence Listing);
5′-GGTCAAGGCA CCAGGCTGGA AATCAAACGG ACTGTGGCTG CACATCTGT CTTCA -3 ′ (7ALCP: SEQ ID NO: 63 in Sequence Listing);
5'-CCCCGAATTCT TACTAACACT CTCCCCTGTT GAAGCTCTTT GTGAC-3 '(7ALCN: SEQ ID NO: 64 in Sequence Listing);
5′-TCTGTCCCAG ACCCACTGCC ACTAAACCTG TCTGGGATCC CAGATTCGAG ATTGG -3 ′ (M7AL2N: SEQ ID NO: 65 in Sequence Listing);
5′-GTTTAGTGGC AGTGGGTCTG GGACAGACTTT CACCCTCTACC ATCCATCCTG TGGAG -3 ′ (M7AL3PA: SEQ ID NO: 66 in Sequence Listing);
5′-ATGGTGCAGC CACAGTCCGT TTGATTTCCA GCCTGGTGCC TTGACCGAAC GTCCG -3 ′ (7AL4NA: SEQ ID NO: 67 in Sequence Listing).
3) Construction of plasmid p7AL-HH (Humanized HH type HFE7A-light chain expression plasmid)
The VHH-DNA fragment represented by SEQ ID NO: 49 encoding the amino acid represented by SEQ ID NO: 50 in the sequence listing was prepared by performing three-step PCR, inserted into a plasmid vector, and then cloned into E. coli. Was done.
[0235]
a) First stage PCR
FIG. 8 shows an outline of the first step PCR for preparing VHH-DNA.
[0236]
Secretory signal sequence and FRL with HindIII restriction enzyme cleavage site added to 5 'end ₁ The L7A1-DNA fragment encoding a part of the region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7AL1N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase (Stratagene) 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0237]
FRL ₁ Part of the CDRL ₁ , FRL ₂ And CDRL ₂ The L7A2-DNA fragment encoding a part of the region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL2P 80pmol
Oligonucleotide primer 7AL2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0238]
CDRL ₂ And FRL ₃ L7A3-DNA fragment encoding a part of was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL3PA 80pmol
Oligonucleotide primer 7AL3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, followed by heating at 72 ° C for 10 minutes.
[0239]
FRL ₃ Part of the CDRL ₃ , FRL ₄ And an L7A4-DNA fragment encoding a part of the constant region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL4P 80pmol
Oligonucleotide primer 7AL4N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, followed by heating at 72 ° C for 10 minutes.
[0240]
FRL ₄ And an L7A5-DNA fragment which encodes a constant region and adds an EcoRI restriction enzyme cleavage site to the 3 ′ end were prepared under the following conditions.
Reaction liquid composition:
Plasmid pHL15-27 200ng
Oligonucleotide primer 7ALCP 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, followed by heating at 72 ° C for 10 minutes.
[0241]
An equal amount of phenol / chloroform (50% water-saturated phenol, 48% chloroform, 2% isoamyl alcohol) was added to 200 μl of each product after PCR, and the mixture was vigorously stirred for 1 minute. Thereafter, centrifugation at 10,000 × g was performed to collect an aqueous layer. An equal amount of chloroform / isoamyl alcohol (96% chloroform, 4% isoamyl alcohol) was added to the obtained aqueous layer, the mixture was vigorously stirred for 1 minute again, and the aqueous layer was collected by centrifugation at 10,000 × g (hereinafter referred to as “water layer”). This series of operations is referred to as "phenol extraction").
[0242]
To the recovered supernatant, 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volume of ethanol were added, frozen with dry ice, centrifuged at 10,000 × g, and the DNA was recovered. This was recovered as a precipitate (hereinafter, this series of operations is referred to as "ethanol precipitation").
[0243]
The obtained DNA precipitate was dried in vacuum, dissolved in double-distilled water, and separated by 5% polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected L7A1-DNA, L7A2-DNA, L7A3-DNA, L7A4-DNA and L7A5-DNA were cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0244]
b) Second stage PCR
FIG. 9 shows an outline of the second step PCR for preparing VHH-DNA.
[0245]
The L7A1.2-DNA fragment obtained by fusing the aforementioned L7A1-DNA fragment and L7A2-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
L7A1-DNA solution prepared by the first step PCR 10 μl
L7A2-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7AL2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0246]
The L7A4.5-DNA fragment obtained by fusing the above-mentioned L7A4-DNA fragment and L7A5-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
L7A4-DNA solution prepared by the first step PCR 10 μl
L7A5-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL4P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0247]
The amplified L7A1.2-DNA and L7A4.5-DNA fragments after PCR were separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. Each detected fusion DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0248]
c) Third stage PCR
FIG. 10 shows an outline of the third step PCR for preparing VHH-DNA.
[0249]
The VHH-DNA fragment obtained by fusing the above-mentioned L7A1.2-DNA fragment, L7A4.5-DNA fragment and L7A3-DNA was prepared under the following conditions.
Reaction liquid composition:
L7A1.2-DNA solution prepared by the second step PCR 10 μl
L7A4.5-DNA solution prepared by the second step PCR 10 μl
L7A3-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0250]
The amplified VHH-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VHH-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0251]
The outline of the method for preparing an expression plasmid that holds a VHH-DNA fragment is shown in FIG.
[0252]
The obtained VHH-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and EcoRI.
[0253]
1 μg of a cloning plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) was previously digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with alkaline phosphatase (bovine intestinal origin: hereinafter referred to as “CIP”). The dephosphorylated plasmid pHSG399 DNA thus obtained and the VHH-DNA fragment digested with restriction enzymes in advance were ligated using DNA Ligation Kit version 2.0 (Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer. did.
[0254]
The ligated DNA was recovered by ethanol precipitation, dissolved in 5 μl of double-distilled water. E. coli JM109 electro-cell (Takara Shuzo Co., Ltd.). The mixture was mixed with Gene Pulser / E. coli pulsar cuvette 0.1 cm (manufactured by Bio-Rad) and introduced into Escherichia coli JM109 strain using Gene Pulser II (manufactured by Bio-Rad) according to the protocol specified by the manufacturer (hereinafter, this series of operations is referred to as “transformation”). ). After completion of the transformation, the cells were treated with IPTG (isopropylthio-β-D-galactoside, Takara Shuzo Co., Ltd.) at a final concentration of 1 mM, and 0.1% X-Gal (5-bromo-4-chloro-). LB agar medium containing 3-indolyl-β-D-galactoside, Takara Shuzo Co., Ltd.) and 50 μg / ml chloramphenicol [Bactotryptone (Difco) 10 g, Bacto yeast extract (Difco) 5 g, 10 g of sodium chloride, 15 g of Bacto agar (manufactured by Difco). Dissolved in distilled water to make 1 liter], and cultured overnight at 37 ° C. to obtain an E. coli transformant.
[0255]
The resulting white transformant was an LB liquid medium containing 10 μg / ml chloramphenicol [10 g of bactotripton (manufactured by Difco), 5 g of bactoeast extract (manufactured by Difco), and 10 g of sodium chloride. Dissolve in distilled water to make 1 liter] in 2 ml at 37 ° C. overnight, and use the alkaline SDS method [Sambrook, J. et al. et al. , (1989) in "Molecular Cloning A Laboratory Manual Second Edition." Cold Spring Harbor Laboratory Press. ] To extract plasmid DNA.
[0256]
After the extracted plasmid DNA was digested with restriction enzymes HindIII and EcoRI, a clone holding the VHH-DNA fragment was selected by 1% agarose gel electrophoresis analysis.
[0257]
By the above operation, plasmid pHSGHH7 carrying a fusion fragment of the variable region of the humanized HH type HFE7A-light chain and the DNA encoding the human Igκ chain constant region was obtained. In addition, transformed Escherichia coli harboring plasmid pHSGHH7. coli pHSGHH7 SANK 73497 was internationally deposited on August 22, 1997, at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) with accession number FERM BP-6073. Was done.
[0258]
The expression vector plasmid p7AL-HH carrying DNA (SEQ ID NO: 49 in the sequence listing) encoding the humanized HH type HFE7A-light chain polypeptide (SEQ ID NO: 50 in the sequence listing) was prepared using the aforementioned plasmid pHSGHH7. .
[0259]
Expression plasmid vector pEE. 1 μg of 12.1 (Lonza) DNA was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pEE. 12.1 100 ng of DNA was ligated to 10 μg of pHSGHH7 DNA fragment digested with HindIII and EcoRI using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109 strain to 50 μg. / Ml of ampicillin on LB agar medium.
[0260]
The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted according to the alkaline SDS method. The extracted plasmid DNA was digested with HindIII and EcoRI, and subjected to 1% agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, the fusion fragment of the variable region of the humanized HH type HFE7A-light chain and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the cytomegalovirus (hereinafter referred to as CMV) promoter. The plasmid p7AL-HH was obtained.
[0261]
4) Construction of plasmid p7AL-HM (Humanized HM type HFE7A-light chain expression plasmid)
The VHM-DNA fragment shown in SEQ ID NO: 51 of the Sequence Listing encoding the amino acid shown in SEQ ID NO: 52 in the Sequence Listing was prepared by performing two-step PCR, inserted into a plasmid vector, and cloned into Escherichia coli. Was done.
[0262]
a) First stage PCR
FIG. 12 shows an outline of the first step PCR for preparing VHM-DNA.
[0263]
Secretory signal sequence and FRL with HindIII restriction enzyme cleavage site added to 5 'end ₁ L7A1-DNA fragment encoding a part of ₁ Part of the CDRL ₁ , FRL ₂ And CDRL ₂ L7A2-DNA fragment encoding a part of ₄ The L7A5-DNA fragment, which codes for a part of E. coli and a constant region and adds an EcoRI restriction enzyme cleavage site at the 3 ′ end, used was obtained from the preparation of the VHH-DNA fragment [“(2) Plasmid p7AL-HH” (Humanized HH type HFE7A-light chain expression plasmid) "and" a) First Step PCR "].
[0264]
CDRL ₂ , FRL ₃ , CDRL ₃ , FRL ₄ And a ML7A3-DNA fragment encoding a part of the constant region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL3PA 80pmol
Oligonucleotide primer 7AL4NA 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0265]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected DNA band corresponding to ML7A3-DNA was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0266]
b) Second stage PCR
FIG. 13 shows an outline of the second step PCR for preparing VHM-DNA.
[0267]
The VHM-DNA fragment obtained by fusing the aforementioned L7A1.2-DNA fragment with the ML7A3-DNA fragment and the aforementioned L7A5-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
L7A1.2-DNA solution prepared by the second step PCR 10 μl
10 μl of ML7A3-DNA solution prepared by the first step PCR
L7A5-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0268]
The amplified VHM-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VHM-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0269]
FIG. 14 shows an outline of a method for preparing an expression plasmid holding a VHM-DNA fragment.
[0270]
The obtained VHM-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and EcoRI.
[0271]
1 μg of a cloning plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pHSG399 DNA was ligated with a VHM-DNA fragment digested with HindIII and EcoRI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109 strain, The cells were spread on an LB agar medium containing IPTG at a final concentration of 1 mM, X-Gal at 0.1% and chloramphenicol at 50 μg / ml. The obtained white transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and plasmid DNA was extracted by an alkaline SDS method. After the extracted plasmid DNA was digested with HindIII and EcoRI, 1% agarose gel electrophoresis analysis was performed to select a clone holding the VHM-DNA fragment.
[0272]
By the above operation, a plasmid pHSGHM17 carrying a fusion fragment of the variable region of the humanized HM type HFE7A-light chain and the DNA encoding the human Igκ chain constant region was obtained. The transformed E. coli E. coli harboring plasmid pHSGHM17 was used. E. coli pHSGHM17 SANK 73597 was internationally deposited on August 22, 1997 with the National Institute of Advanced Industrial Science and Technology (AIST) at the National Institute of Advanced Industrial Science and Technology (AIST) under the accession number FERM BP-6072. Was done.
[0273]
The expression vector plasmid p7AL-HM carrying the DNA exemplified in SEQ ID NO: 51 of the Sequence Listing encoding the humanized HM type HFE7A-light chain polypeptide exemplified in SEQ ID NO: 52 of the Sequence Listing was prepared using the aforementioned plasmid pHSGHM17. Produced.
[0274]
Expression plasmid vector pEE. 12.1 1 μg of DNA was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pEE. 12.1 100 ng of DNA and 10 μg of pHSGHM17-DNA fragment digested with HindIII and EcoRI were ligated using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109 strain. The cells were spread on an LB agar medium containing 50 μg / ml of ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with EcoRI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, a plasmid p7AL-HM in which a fusion fragment of the humanized HM type HFE7A-light chain variable region and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained. .
[0275]
5) Construction of plasmid p7AL-MM (humanized MM type HFE7A-light chain expression plasmid)
The VMM-DNA fragment shown in SEQ ID NO: 53, which encodes the amino acid shown in SEQ ID NO: 54 in the sequence listing, was prepared by performing a three-step PCR, inserted into a plasmid vector, and then transformed into E. coli. Cloned.
[0276]
a) First stage PCR
FIG. 15 shows an outline of the first step PCR for preparing VMM-DNA.
[0277]
Secretory signal sequence and FRL with HindIII cleavage site added at 5 'end ₁ L7A1-DNA fragment encoding a part of ₄ The L7A5-DNA fragment, which codes for a part of E. coli and the constant region and has an EcoRI cleavage site added to the 3 ′ end, was obtained from the preparation of the VHH-DNA fragment [“(2) Construction of plasmid p7AL-HH” (Humanized HH type HFE7A-light chain expression plasmid) "and" a) First step PCR "].
[0278]
FRL ₁ Part of the CDRL ₁ , FRL ₂ , CDRL ₂ And FRL ₃ ML7A2M-DNA fragment encoding a part of was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer 7AL2P 80pmol
Oligonucleotide primer M7AL2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0279]
FRL ₃ Part of the CDRL ₃ , FRL ₄ And a ML7A3M-DNA fragment encoding a part of the constant region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-L DNA
Oligonucleotide primer M7AL3PA 80pmol
Oligonucleotide primer 7AL4NA 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0280]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected ML7A2M-DNA and ML7A3M-DNA were cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0281]
b) Second stage PCR
FIG. 16 shows an outline of the second step PCR for preparing VMM-DNA.
The ML7A1.2-DNA fragment obtained by fusing the aforementioned L7A1-DNA fragment and ML7A2M-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
L7A1-DNA solution prepared by the first step PCR 10 μl
10 μl of ML7A2M-DNA solution prepared by the first step PCR
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer M7AL2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0282]
The amplified ML7A1.2-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected fusion DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0283]
c) Third stage PCR
FIG. 17 shows an outline of the third step PCR for preparing VMM-DNA.
[0284]
The VMM-DNA fragment obtained by fusing the ML7A1.2-DNA fragment, ML7A3M-DNA fragment and L7A5-DNA fragment described above was prepared under the following conditions.
Reaction liquid composition:
10 μl of ML7A1.2-DNA solution prepared by the second step PCR
ML7A3M-DNA solution prepared by the first step PCR 10 μl
L7A5-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0285]
The amplified VMM-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VMM-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0286]
The outline of the method for preparing a plasmid that holds a VMM-DNA fragment is shown in FIG.
[0287]
The obtained VMM-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and EcoRI.
[0288]
1 μg of cloning plasmid pHSG399 (manufactured by Takara Shuzo Co., Ltd.) DNA was digested with HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pHSG399 DNA was ligated to a VMM-DNA fragment digested with EcoRI and HindIII using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109 strain. The cells were spread on an LB agar medium containing IPTG at a final concentration of 1 mM, X-Gal at 0.1% and chloramphenicol at 50 μg / ml. The obtained white transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and plasmid DNA was extracted by an alkaline SDS method. After the extracted plasmid DNA was digested with HindIII and EcoRI, a clone holding the VMM-DNA fragment was selected by 1% agarose gel electrophoresis analysis.
[0289]
By the above operation, plasmid pHSGMM6 carrying a fusion fragment of the variable region of the humanized MM type HFE7A-light chain and the DNA encoding the human Igκ chain constant region was obtained. In addition, transformed Escherichia coli carrying the plasmid pHSGMM6. coli pHSGMM6 SANK 73697 was internationally deposited on August 22, 1997, at the National Institute of Advanced Industrial Science and Technology (AIST) by the National Institute of Advanced Industrial Science and Technology (AIST), under the accession number FERM BP-6071. Was done.
[0290]
The expression vector plasmid p7AL-MM carrying the DNA exemplified in SEQ ID NO: 53 of the Sequence Listing encoding the humanized MM type HFE7A-light chain polypeptide exemplified in SEQ ID NO: 54 of the Sequence Listing was prepared by using the aforementioned plasmid pHSGMM6. Produced.
[0291]
Expression plasmid vector pEE. 12.1 1 μg of DNA was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pEE. 12.1 100 ng of DNA and 10 μg of pHSGMM6 DNA fragment digested with HindIII and EcoRI were ligated using DNA Ligation Kit Version 2.0 (Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109 strain to 50 μg. / Ml of ampicillin on LB agar medium. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with EcoRI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. In this way, a plasmid p7AL-MM in which a fusion fragment of a humanized HFE7A-light chain MM type variable region and a DNA encoding a human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained. .
[0292]
6) Confirmation of nucleotide sequence
The nucleotide sequence was determined in order to confirm that the inserted DNAs of the plasmids p7AL-HH, p7AL-HM and p7AL-MM retain the nucleotide sequence of interest. Oligonucleotide primers generated in determining the nucleotide sequence were as described below:
5'-CCCAAGCTTA AGAAGCATCC-3 '(SP1: SEQ ID NO: 68 in Sequence Listing);
5′-ATCTATGCTGCATCCAATCT-3 ′ (SP2: SEQ ID NO: 69 in Sequence Listing);
5'-GTTGTGTGCC TGCTGAATAA-3 '(SP3: SEQ ID NO: 70 in Sequence Listing);
5'-CCCGAATTCT TACTAACACT-3 '(SP4: SEQ ID NO: 71 in Sequence Listing);
5'-TTATTCAGCA GGCACACAAC-3 '(SP5: SEQ ID NO: 72 in Sequence Listing);
5'-AGATTGGGATG CAGCATAGAT-3 '(SP6: SEQ ID NO: 73 in Sequence Listing).
[0293]
The positions to which each primer binds are shown in FIG. The nucleotide sequence was determined by the alkali-SDS method and the cesium chloride method [both of Sambrook, J. et al. et al. (1989) in "Molecular Cloning, A Laboratory Manual. Second edition." Cold Spring Harbor Laboratory Press. Using the plasmid DNA purified by the above method as a template and a dideoxynucleotide chain termination method [Sanger, F. et al. S. et al. (1977) Pro. Natl. Acad. Sci. USA. 74, 5463]. That is, 3 μg of the purified plasmid DNA was dissolved in 13 μl of double distilled water, 2 μl of 2 mM EDTA and 2 μl of 2N NaOH were added, and the mixture was allowed to stand at room temperature for 5 minutes. Thereafter, 4 μl of a 10 M ammonium acetate solution and 100 μl of ethanol were added, and after stirring, the mixture was allowed to stand on dry ice for 10 minutes. After centrifugation at 15000 × g, the obtained precipitate was washed with 80% ethanol and dried under vacuum. The vacuum-dried DNA was dissolved in 7 μl of double distilled water and used for nucleotide sequencing reactions. For the nucleotide sequencing reaction, 7-deaza-sequenase version 2.0 kit (for dCTP: manufactured by Amersham) was used. To 7 μl of the above plasmid solution, 1 pmol of a primer synthesized in advance and 1 μl of a reaction buffer (a buffer supplied in advance with the kit) were added, and the mixture was incubated at 65 ° C. for 2 minutes. Thereafter, the mixture was gradually cooled to room temperature, annealed with the primer, and [α- ³² [P] dCTP (Amersham). The reaction product was subjected to 5% polyacrylamide gel electrophoresis containing 1 M of urea in 1 × TBE (100 mM Tris, 100 mM boric acid, 1 mM EDTA, pH 8.3) buffer. After drying the gel, autoradiography was performed to decode the nucleotide sequence (hereinafter, the nucleotide sequence was determined by the above method).
[0294]
As a result, p7AL-HH is the nucleotide sequence shown in SEQ ID NO: 49 of the sequence listing encoding the polypeptide shown in SEQ ID NO: 50 in the sequence listing, and p7AL-HM is the polypeptide shown in SEQ ID NO: 52 in the sequence listing P7AL-MM holds the nucleotide sequence shown in SEQ ID NO: 53 of the sequence listing encoding the polypeptide shown in SEQ ID NO: 54 in the sequence listing, respectively. It was confirmed that.
[0295]
Example 15 Construction of humanized antibody heavy chain expression vector for HFE7A
(1) Construction of plasmid holding heavy chain variable region DNA of humanized HFE7A
1) Synthesis of primer for preparing humanized heavy chain variable region
DNA encoding the variable region of the humanized anti-Fas antibody HFE7A-heavy chain and a polypeptide chain (SEQ ID NO: 75 in the sequence listing) containing the N-terminal 5 amino acids of the IgG-CH1 region (SEQ ID NO: 75 in the sequence listing) The synthesis of 74) was performed by combining PCR methods.
[0296]
In performing the PCR, the following eight primers were prepared:
5′-GGGAAGCTTG GCTTGACCTC ACCATGGGAT GGAGCTGTAT-3 ′ (7AH1P: SEQ ID NO: 76 in Sequence Listing);
5'-TGAAGCCCCA GGCTTCTTGA CCTCAGCCCC AGACTGCACC AGTTGGAC -3 '(7AH1NNEW: SEQ ID NO: 77 in Sequence Listing);
5'-TCCACTCAAG CCCTCTTCCA GGGGCCTGTT TTACCC-3 '(7AH2N: SEQ ID NO: 78 in Sequence Listing);
5′-GTCTGGGGCT GAGGTCAAGA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AG-3 ′ (7AH2PNEW: SEQ ID NO: 79 in Sequence Listing);
5'-CAGGCCCCTG GACAGAGGCT TGAGTGGATG GGAGAGATT -3 '(7AH3P: SEQ ID NO: 80 in Sequence Listing);
5'-TCAGATCTCA GGCTGCTGAG CTCCATGTAG GCTGTGCTAG CGGATGTGTC -3 '(7AH3N: SEQ ID NO: 81 in Sequence Listing);
5′-TGGAGCTCAG CAGCCTGAGA TCTGAGGACA CGGCGGTCTTA TTAC-3 ′ (7AH4P: SEQ ID NO: 82 in Sequence Listing);
5′-GATGGGCCCT TGGTGGAGGC TGAGGAGACG GTGACCAGGG TCCCTTCGCC CCAGT-3 ′ (7AH4N: SEQ ID NO: 83 in Sequence Listing).
[0297]
2) Construction of plasmid pBL7A27
The VD-DNA fragment shown in SEQ ID NO: 74 encoding the amino acid shown in SEQ ID NO: 75 in the sequence listing was prepared by performing three-step PCR, inserted into a plasmid, and cloned in E. coli. .
[0298]
a) First stage PCR
FIG. 20 shows the outline of the first step PCR for preparing VD-DNA.
[0299]
Secretion signal sequence and FRH with a HindIII restriction enzyme cleavage site added to the 5 'end ₁ The H7A1-DNA fragment encoding the N-terminal side of was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-H DNA
Oligonucleotide primer 7AH1P 80pmol
Oligonucleotide primer 7AH1NNEW 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0300]
FRH ₁ Part of the CDRH ₂ And FRH ₂ The H7A2-DNA fragment encoding a part of was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-H DNA
Oligonucleotide primer 7AH2N 80pmol
Oligonucleotide primer 7AH2PNEW 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0301]
FRH ₂ Part of the CDRH ₂ And FRH ₃ The H7A3-DNA fragment encoding a part of was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-H DNA
Oligonucleotide primer 7AH3P 80pmol
Oligonucleotide primer 7AH3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0302]
FRH ₃ Part of the CDRH ₃ , FRH ₄ And a H7A4-DNA fragment encoding the N-terminal 5 amino acid residues of the CH1 region was prepared under the following conditions.
Reaction liquid composition:
200 ng of plasmid pME-H DNA
Oligonucleotide primer 7AH4P 80pmol
Oligonucleotide primer 7AH4N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0303]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected H7A1-DNA, H7A2-DNA, H7A3-DNA and H7A4-DNA were excised with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0304]
b) Second stage PCR
FIG. 21 shows the outline of the second step PCR for preparing VD-DNA.
[0305]
The H7A1.2-DNA fragment obtained by fusing the aforementioned H7A1-DNA fragment and H7A2-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
H7A1-DNA solution prepared by the first step PCR 10 μl
H7A2-DNA solution prepared in the first step PCR 10 μl
Oligonucleotide primer 7AH1P 80pmol
Oligonucleotide primer 7AH2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0306]
The H7A3.4-DNA fragment obtained by fusing the aforementioned H7A3-DNA fragment and H7A4-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
H7A3-DNA solution prepared by the first step PCR 10 μl
H7A4-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AH3P 80pmol
Oligonucleotide primer 7AH4N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0307]
The amplified H7A1.2-DNA and H7A3.4-DNA fragments after PCR were separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. Each detected fusion DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0308]
c) Third stage PCR
FIG. 22 shows an outline of the third step PCR for preparing VD-DNA.
[0309]
The VD-DNA fragment obtained by fusing the H7A1.2-DNA fragment and the H7A3.4-DNA fragment described above was prepared under the following conditions.
Reaction liquid composition:
10 μl of H7A1.2-DNA solution prepared by the second step PCR
H7A3.4-DNA solution prepared by the second step PCR 10 μl
Oligonucleotide primer 7AH1P 80pmol
Oligonucleotide primer 7AH4N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0310]
The amplified VD-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VD-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0311]
FIG. 23 shows an outline of a method for preparing a plasmid holding a VD-DNA fragment.
[0312]
The obtained VD-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and ApaI.
[0313]
1 μg of the plasmid vector pBLUESCRIPT-II SK + DNA (manufactured by Stratagene) was digested with HindIII and ApaI, and dephosphorylated with CIP. This dephosphorylated pBLUESCRIPT-II SK + DNA was ligated to a VD-DNA fragment digested with HindIII and ApaI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), and transformed into Escherichia coli JM109. After conversion, IPTG (isopropylthio-β-D-galactoside, manufactured by Takara Shuzo Co., Ltd.) at a final concentration of 1 mM, and 0.1% X-Gal (5-bromo-4-chloro-3-indolyl-β-D) were used. -Galactoside, manufactured by Takara Shuzo Co., Ltd.) and 50 μg / ml ampicillin on an LB agar medium. The obtained white transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with ApaI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, the plasmid pBL7A27 into which the VD-DNA fragment was inserted was obtained.
[0314]
(2) Construction of plasmid holding human IgG1 constant region genomic DNA
1) Synthesis of primer for preparing DNA fragment on 5 'side of human IgG1 genome
The synthesis of the DNA fragment on the 5 'side of the human IgG1 genome was prepared using the PCR method. In performing the PCR, two types of oligonucleotide primers were prepared as described below:
5'-GGGAAGCTTC CGCGGTCACA TGGCACCACC TCTCTGTGCA -3 '(5'Hind: SEQ ID NO: 84 in Sequence Listing);
5′-GCTCTGCAGA GAGAAGATTG GGAGTTACTG GAATC-3 ′ (IGGCPSTN: SEQ ID NO: 85 in Sequence Listing).
[0315]
2) Construction of plasmid pIG5'03
The genomic DNA containing the human IgG1 CH1 region and the intron following the HindIII cleavage sequence is inserted into plasmid pHSG399 (Takara Shuzo) after separating and amplifying the target DNA fragment by PCR using human genomic DNA as a template. And cloned in E. coli. FIG. 24 shows an outline of a method for preparing a DNA fragment (hereinafter referred to as “IG5′-DNA”) containing the desired human IgG1 CH1 region and intron.
[0316]
The IG5'-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
Human genomic DNA (Clontech) 2 μg
Oligonucleotide primer 5'Hind 80pmol
Oligonucleotide primer IGGCPSTN 80 pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0317]
The amplified IG5'-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected IG5'-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0318]
The obtained IG5'-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and PstI.
[0319]
1 μg of plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) was digested with restriction enzymes HindIII and PstI, and dephosphorylated with CIP. The dephosphorylated plasmid pHSG399 DNA was ligated with an IG5'-DNA fragment digested with HidIII and PstI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), followed by transformation of Escherichia coli JM109. Then, it was spread on an LB agar medium containing 1 mM of IPTG, 0.1% of X-Gal, and 50 μg / ml of chloramphenicol. The resulting white transformant was cultured in an LB liquid medium containing 50 μg / ml chloramphenicol, and the plasmid DNA was extracted by the alkaline SDS method. This plasmid was digested with PstI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, a plasmid pIG5'03 into which the IG5'-DNA fragment was inserted was obtained.
[0320]
(3) Construction of plasmid holding human IgG1 constant region genomic DNA
1) Synthesis of primer for preparing DNA fragment on 3 'side of human IgG1 genome
The synthesis of the DNA fragment on the 3 ′ side of the human IgG1 genome was prepared using the PCR method. In performing the PCR, two types of primers were prepared as described below:
5′-TCTCTGCAGA GCCCAAATCT TGTGACAAAA CTCAC-3 ′ (IGGCPSTP: SEQ ID NO: 86 in Sequence Listing);
5′-GGGGAATTCG GGAGCGGGGCTTTGCCGGCCG TCGCACTCA -3 ′ (Eco3 ′: SEQ ID NO: 87 in Sequence Listing).
[0321]
2) Construction of plasmid pIG3'08
DNA containing human IgG1 intron, hinge region, intron, CH2 region, intron, CH3 region and EcoRI-cleaved sequence is obtained by separating and amplifying a target DNA fragment by PCR using human genomic DNA as a template, and then plasmid pHSG399 (Takara Shuzo ( And cloned in E. coli. FIG. 25 shows an outline of a method for preparing a genomic DNA (hereinafter, referred to as “IG3′-DNA”) fragment containing the desired human IgG1 hinge region, CH2 region, CH3 region, and intron.
[0322]
The IG3'-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
Human genomic DNA (Clontech) 2 μg
Oligonucleotide primer IGGCPSTP 80pmol
Oligonucleotide primer Eco3 '80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR temperature conditions: After heating at 94 ° C for 2 minutes, a temperature cycle of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes was repeated 30 times, and then heating was performed at 72 ° C for 10 minutes.
[0323]
The amplified IG3'-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. Each detected DNA fragment was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0324]
The obtained IG3'-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes EcoRI and PstI.
[0325]
1 μg of plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) was digested with EcoRI and PstI, and dephosphorylated with CIP. The dephosphorylated pHSG399 DNA and the IG3'-DNA fragment digested with EcoRI and PstI were ligated using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), and Escherichia coli JM109 was transformed. The cells were spread on an LB agar medium containing 1 mM IPTG, 0.1% X-Gal and 50 μg / ml chloramphenicol. White colonies were selected to obtain a plasmid pIG3'08 into which the IG3'-DNA fragment was inserted.
[0326]
(4) Construction of humanized HFE7A-heavy chain expression vector plasmid
The expression vector plasmid pEg7AH-H carrying the DNA shown in SEQ ID NO: 88, encoding the humanized HFE7A-heavy chain polypeptide shown in SEQ ID NO: 89 in the sequence listing, was obtained from the plasmids pBL7A27 and pIG5'03 described above. And it was produced using pIG3'08. FIG. 26 shows an outline of the manufacturing procedure.
[0327]
10 μg of pIG5'03 plasmid DNA containing the human IgG1-heavy chain CH1 region and intron was digested with restriction enzymes ApaI and KpnI. On the other hand, 1 μg of the above plasmid pBL7A27 DNA was digested with restriction enzymes ApaI and KpnI, and dephosphorylated with CIP. 100 ng of this dephosphorylated pBL7A27 DNA and 10 μg of pIG5'03 DNA digested with ApaI and KpnI were ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), and then cloned into Escherichia coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with ApaI and KpnI or HindIII and PstI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, the plasmid pBL7AF184 was obtained in which the VD-DNA fragment of humanized HFE7A and the IG5'-DNA fragment were connected and inserted.
[0328]
Next, 10 μg of the obtained plasmid pBL7AF184 DNA was digested with restriction enzymes HindIII and PstI. On the other hand, 1 μg of the aforementioned plasmid pIG3′08 DNA was digested with restriction enzymes HindIII and PstI, and dephosphorylated with CIP. 100 ng of this dephosphorylated pIG3'08 DNA and 10 μg of pBL7AF184 DNA digested with HindIII and PstI were ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), and then cloned into Escherichia coli JM109 strain. . The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and the plasmid DNA was extracted by the alkaline SDS method. This plasmid was digested with PstI and HindIII or HindIII and EcoRI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, the plasmid pgHSL7A62 was obtained in which the VD-DNA fragment of humanized HFE7A was connected to the genomic DNA fragment encoding the human IgG1 constant region. In addition, transformed Escherichia coli carrying the plasmid pgHSL7A62. E. coli pgHSL7A62 SANK73397 was internationally deposited on August 22, 1997, with the National Institute of Advanced Industrial Science and Technology (AIST) at the National Institute of Advanced Industrial Science and Technology (AIST) under the accession number FERM BP-6074. Was.
[0329]
10 μg of the obtained plasmid pgHSL7A62 DNA was digested with restriction enzymes HindIII and EcoRI. On the other hand, the expression plasmid pEE. 6.1 μg of DNA was digested with HindIII and EcoRI and dephosphorylated with CIP. This dephosphorylated pEE. 6.1 ng of 100 ng of DNA and 10 μg of pgHSL7A62 DNA digested with HindIII and EcoRI were ligated using a ligation kit, and then cloned into Escherichia coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with HindIII and EcoRI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, a plasmid pEg7AH-H was obtained in which the fusion fragment of the humanized HFE7A VD-DNA fragment and the genomic DNA encoding the human IgG1 constant region was inserted in the forward direction downstream of the CMV promoter.
[0330]
(5) Confirmation of nucleotide sequence
The nucleotide sequence was determined in order to confirm that the inserted DNA of pEg7AH-H retained the nucleotide sequence of interest. Oligonucleotide primers generated in determining the nucleotide sequence were as described below:
5′-ACAGCCGGGA AGGTGTGCAC-3 ′ (IG01: SEQ ID NO: 90 in Sequence Listing);
5′-AGACACCCTC CCCTCCTGTG-3 ′ (IG02: SEQ ID NO: 91 in Sequence Listing);
5′-GTGCAGGGCC TGGGTTTAGGG-3 ′ (IG03: SEQ ID NO: 92 in Sequence Listing);
5′-GCACGGTGGG CATGTGTGAG-3 ′ (IG04: SEQ ID NO: 93 in Sequence Listing);
5′-GTTTTGGGGG GAAGAGGAAG-3 ′ (IG05: SEQ ID NO: 94 in the sequence listing);
5′-CCAGTCCTGG TGCAGGACGG-3 ′ (IG06: SEQ ID NO: 95 in Sequence Listing);
5′-CCTGTGGTTC TCGGGGCTGC-3 ′ (IG07: SEQ ID NO: 96 in Sequence Listing);
5′-CGTGGTCTTG TAGTTGTTCT-3 ′ (IG08: SEQ ID NO: 97 in Sequence Listing);
5'-CTTCCTCTTC CCCCCAAAAC-3 '(IGP5: SEQ ID NO: 98 in Sequence Listing);
5′-CCGTCCTGCA CCAGGAACTGG-3 ′ (IGP6: SEQ ID NO: 99 in Sequence Listing);
5′-GCAGCCCCGA GAACCACAGG-3 ′ (IGP7: SEQ ID NO: 100 in Sequence Listing);
5′-AGAACAACTA CAAGACCACG-3 ′ (IGP8: SEQ ID NO: 101 in Sequence Listing);
5'-GCCTGACATC TGAGGATCC-3 '(H5 +: SEQ ID NO: 102 in Sequence Listing);
5'-GAGTCCTCAG ATGTCAGGC-3 '(H5-: SEQ ID NO: 103 in Sequence Listing);
5'-GAGCAGACTCGTTGCTGCCGCCGCGGCCACCAG-3 '
(PEEF: SEQ ID NO: 104 in the sequence listing);
5'-GGTATGGCTG ATTAATGATC AATG-3 '(PEEB: SEQ ID NO: 105 in Sequence Listing).
[0331]
The position where each primer binds is shown in FIG. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using each plasmid DNA purified by the alkali-SDS method and the cesium chloride method as a template. As a result, it was confirmed that pEg7AH-H had a nucleotide sequence represented by SEQ ID NO: 88 in the sequence listing, encoding the polypeptide represented by SEQ ID: 89 in the sequence listing.
[0332]
Example 16 Expression in COS-1 cells
The humanized HFE7A-heavy chain expression plasmid and each humanized HFE7A-light chain expression plasmid obtained above were introduced into monkey kidney-derived cell line COS-1 cells by introducing a 2 mm chamber between electrodes / FCT-13 (Shimadzu Corporation). This was performed by an electroporation method using a gene transfer apparatus GTE-1 (manufactured by Shimadzu Corporation) equipped with the above-described method. First, COS-1 cells (American Type Culture Collection No. CRL-1650) were prepared using a minimum essential alpha medium (hereinafter referred to as “α (+) MEM) containing 10% fetal calf serum (hereinafter referred to as“ FCS ”: manufactured by Moregate). ": A cell culture flask (culture area 225 cm) containing Gibco BRL ² : Sumitomo Bakelite Co., Ltd.) until semi-confluent. Thereafter, the medium was removed, and the cells were treated in 3 ml of trypsin-EDTA solution (manufactured by Sigma) at 37 ° C. for 3 minutes to release COS-1 cells from the flask. The released cells were collected by centrifugation at 800 rpm for 2 minutes, and a phosphate buffer [0.02% potassium chloride, 0.02% potassium dihydrogen phosphate, 0.8% sodium chloride, 1.15% phosphoric acid] Disodium oxyhydrogen. Hereinafter, referred to as “PBS (−)”; manufactured by Nissui Pharmaceutical Co., Ltd.]. Wash the washed COS-1 cells with PBS (−) at 1 × 10 ⁸ The COS-1 cell suspension was prepared so as to have a cell number / ml.
[0333]
On the other hand, after mixing 4 μg of humanized HFE7A-heavy chain expression plasmid DNA and 4 μg of humanized HFE7A-light chain expression plasmid DNA prepared using the alkali-SDS method and the cesium chloride density gradient centrifugation method, ethanol was mixed in the same tube. The precipitate was precipitated and suspended in 20 μl of PBS (−). 20 μl of the obtained plasmid suspension was mixed with 20 μl of the previously prepared COS-1 cell suspension (2 × 10 ⁶ The cells were transferred to a chamber / FCT-13 (manufactured by Shimadzu Corporation) having an electrode spacing of 2 mm, and set in a gene transfer apparatus GTE-1 (manufactured by Shimadzu Corporation). Thereafter, the target plasmid DNA was introduced into COS-1 cells by applying a pulse of 600 V and 50 μF twice at 1 second intervals. The cell-DNA mixture in the chamber after the pulse application was suspended in 5 ml of α (+) MEM containing 10% FCS, and the suspension was cultured in a cell culture flask (culture area 25 cm). ² : Sumitomo Bakelite Co., Ltd.). After culturing at 37 ° C. for 72 hours under 5% carbon dioxide, the culture supernatant was collected and the expression products contained in the main culture supernatant were analyzed.
[0334]
COS-1 cells were each transformed with the following combinations of plasmids and the culture supernatant was collected by the above method:
[A]: conditions not containing plasmid DNA;
[B]: co-transfection of pEg7AH-H and p7AL-MM;
[C]: co-transfection of pEg7AH-H and p7AL-HM;
[D]: Co-transfection of pEg7AH-H and p7AL-HH.
[0335]
Example 17 Quantification of Expression Products by ELISA
Confirmation of expression of the humanized antibody in the culture supernatant prepared in Example 16 and quantitative assay of the expression product were performed by an enzyme-linked immunosorbent assay (hereinafter, referred to as "ELISA") using an antibody against anti-human IgG. . That is, each well of a 96-well plate (Maxi Soap: manufactured by Nunc) was added to a solid phase buffer (0.05 M sodium hydrogen carbonate, 0.02% sodium azide, pH 9.6) at a final concentration of 1 μg / ml. 100 μl of a dissolved goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Capel) was added, the mixture was incubated at 37 ° C. for 2 hours, and the antibody was immobilized on a plate. Thereafter, the plate was washed four times with 350 μl of PBS (−) containing 0.05% Tween-20 (manufactured by Bio-Rad) (hereinafter referred to as “PBS-T”). The culture supernatant diluted with α (+) MEM containing 10% FCS was added to each well after washing, and the mixture was kept at 37 ° C. for 2 hours. After washing with PBS-T again, 100 μl of an alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Caltag Lab) diluted 5000-fold with PBS-T was added to each well, and the mixture was added at 37 ° C. for 2 hours. Insulated. After washing with PBS-T again, a substrate solution prepared by adjusting p-nitrophenyl phosphoric acid to 1 mg / ml with 10% diethanolamine (pH 9.8) was added. After keeping the temperature at 37 ° C. for 30 minutes to 1 hour, the absorbance at 405 nm was measured. In this experiment, as a control for the concentration of the humanized HFE7A antibody contained in the culture supernatant, human plasma immunoglobulin G / subclass 1 (IgG1) diluted to a certain concentration with α (+) MEM containing 10% FCS was used. (Manufactured by BioPure AG) was used.
[0336]
As a result, of the expression products in the culture supernatant prepared in Example 16, those under the conditions of [B], [C] and [D] were specifically detected by the anti-human IgG antibody.
[0337]
Example 18 Measurement of binding activity to Fas
According to the method described below, the binding activity to Fas in the culture supernatant of the transformed cells prepared in Example 16 was assayed by ELISA. First, the culture supernatant of the COS-1 cell expressing the human Fas fusion protein prepared in Example 1 was diluted 5-fold with the immobilization buffer, and 50 μl was added to each well of a 96-well plate (Maxisorp: Nunc). The mixture was kept at 4 ° C. overnight, and the human Fas fusion protein was adsorbed on the well surface. Thereafter, each well was washed four times with 350 μl of PBS-T. After washing, 50 μl of PBS-T containing 5% bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and the well was blocked by incubating at 37 ° C. for 1 hour. Washed with PBS-T. The concentration of the target expression product contained in the culture supernatant prepared in Example 16 was calculated in advance by the method described in Example 17, and the final concentration of the target product was adjusted to 100 ng / ml. It was prepared in an α (+) MEM medium containing% FCS. Each expression product solution adjusted to a concentration of 100 ng / ml was repeatedly diluted 2-fold with an α (+) MEM medium containing 10% FCS to prepare a dilution series. 50 μl of the prepared dilution series of each expression product was added to each well, and reacted at 37 ° C. for 2 hours. Then, after washing again with PBS-T, 50 μl of an alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Caltag Lab) diluted 10000-fold with PBS-T was dispensed into each well, and the temperature was lowered at 37 ° C. And reacted for 2 hours. The detection of HFE7A (IgG1) purified from mouse hybridoma HFE7A, which was used as a control, was performed by alkaline phosphatase labeled 5,000-fold diluted with PBS-T instead of alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody. Goat anti-mouse IgG + IgA + IgM (manufactured by Gibco BRL) was used. After washing with PBS-T, a substrate solution prepared by adjusting 50 μl of p-nitrophenyl phosphoric acid to 1 mg / ml with 10% diethanolamine (pH 9.8) was added, and the mixture was incubated at 37 ° C. for 30 minutes to 1 hour. The binding activity of the expression product contained in each culture supernatant to the human Fas fusion protein was determined by measuring the absorbance at 405 nm of each well.
[0338]
As a result, among the expression products in the culture supernatant prepared in Example 16, those having the conditions of [B], [C], and [D] have binding properties to the human Fas fusion protein. This was confirmed (FIG. 28).
[0339]
Example 19 Competitive inhibition of HFE7A binding to Fas
The binding of the humanized anti-Fas antibody obtained in the present invention to Fas should inhibit the binding of HFE7A, the anti-Fas mouse monoclonal antibody on which the humanization design was based. Therefore, the competitive inhibitory activity of HFE7A and the expression product prepared in Example 16 on human Fas fusion protein was measured.
[0340]
1 mg of the purified HFE7A monoclonal antibody obtained in Example 3 was labeled using a commercially available alkaline phosphatase labeling kit (Immuno-Link AP and APL labeling kit: manufactured by Genosis) according to the attached protocol (hereinafter, this labeled antibody). Is referred to as “AP-HFE7A”).
[0341]
The COS-1 culture supernatant containing the human Fas fusion protein was diluted 5-fold with the immobilization buffer, and 50 μl was added to each well of a 96-well plate for luminescence detection (luminescent solid assay plate, high binding property: manufactured by Coaster). The human Fas fusion protein was adsorbed on the surface of the well by keeping it at 4 ° C. overnight. After the adsorption, each well was washed four times with 350 μl of PBS-T. After washing, 100 μl of PBS-T containing 5% bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and the well was blocked by incubating at 37 ° C. for 1 hour. Washed with PBS-T. The concentration of the target expression product contained in the culture supernatant prepared in Example 16 was previously calculated by the method described in Example 17, and 10% was adjusted so that the final target product concentration was 1 μg / ml. It had been prepared with α (+) MEM containing FCS. Each expression product solution adjusted to a concentration of 1 μg / ml was repeatedly diluted two-fold with α (+) MEM containing 10% FCS to prepare a dilution series. On the other hand, AP-HFE7A was diluted to 50 ng / ml with α (+) MEM containing 10% FCS, and mixed with the prepared dilution series in an equal amount. 50 μl of the expression product dilution series and the AP-HFE7A mixture were added to each well after washing, and the mixture was allowed to stand at room temperature overnight. Thereafter, each well was washed again with PBS-T, and 100 µl of CDP-star buffer (9.58 ml of diethanolamine, 0.2 g of magnesium chloride, 0.25 g of sodium azide, pH 8.5) was added to each well, and 10 wells were added. Let stand at room temperature for minutes. Thereafter, the CDP-star buffer was removed, and a CDP-star substrate (made up to 1.2 ml with 1.2 ml sapphire II (manufactured by Tropics), 200 μl CDP-star (manufactured by Tropics), and CDP-star buffer) was used. 50 μl / well was added, and the mixture was left still at room temperature for 40 minutes. The competitive inhibitory activity of the expression product contained in each culture supernatant with HFE7A for the human Fas fusion protein was determined by measuring the luminescence intensity using a luminoscan (manufactured by Titertec).
[0342]
As a result, it was confirmed that the expression product prepared in Example 16 specifically inhibited the binding of HFE7A prepared from a mouse hybridoma to a human Fas fusion protein (FIG. 29).
[0343]
Example 20 Apoptosis-inducing activity
WR19L12a cells expressing human Fas molecules in mouse T lymphoma cells [Itoh, N .; et. al. (1991) Cell 66, 233-243] was used to examine the apoptosis-inducing activity of a COS cell culture supernatant expressing a humanized anti-Fas antibody. First, WR19L12a cells (1 × 10 5) cultured in RPMI1640 medium containing 10% FCS (manufactured by Gibco BRL) at 37 ° C. in the presence of 5% carbon dioxide for 3 days. ⁵ (Cells / 50 μl) was dispensed at 50 μl into each well of a 96-well microplate (manufactured by Sumitomo Bakelite Co., Ltd.). The concentration of the target expression product contained in the culture supernatant prepared in Example 16 was calculated in advance by the method described in Example 17, and 10% was adjusted so that the final target product concentration was 100 ng / ml. It was prepared in RPMI 1640 medium containing FCS. Each expression product solution adjusted to a concentration of 100 ng / ml was repeatedly diluted two-fold with an RPMI1640 medium containing 10% FCS to prepare a dilution series. 50 μl of each expression product dilution was added to each well and incubated at 37 ° C. for 1 hour. After washing the cells with RPMI 1640 medium containing 10% FCS, the cells in each well were suspended in 75 μl of RPMI 1640 medium containing 10% FCS. Thereafter, 75 μl of a goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Capel) adjusted to 1.25 μg / ml in RPMI1640 medium containing 10% FCS was added to each well as a secondary antibody. After reacting at 37 ° C. for 12 hours, 1 mg / ml of XTT (2,3-bis-2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide inner salt: manufactured by Sigma) is included. 50 μl of 25 μM PMS (phenazine methosulfate: manufactured by Sigma) (final concentration 250 μg / ml XTT, 5 μM PMS) was added. After culturing for 3 hours, the absorbance at 450 nm of each well was measured, and the cell viability was calculated using the mitochondrial reducing ability as an index.
[0344]
The cell viability in each well was calculated by the following formula:
Survival rate (%) = 100 × (ab) / (c−b)
(Where a represents the measured value of the test well, b represents the measured value of the cell-free well, and c represents the measured value of the well without the antibody added).
[0345]
As a result, the expression product prepared in Example 16 was shown to induce apoptosis in a T lymphoma cell line that expressed the human Fas antigen (FIG. 30).
[0346]
Example 21 Construction of Humanized Expression Vector of Light Chain of HFE7A
(1) Construction of humanized HFE7A-light chain expression vector plasmid
In order to make the HH-type humanized light chain prepared in Example 14 more human-like, the first amino acid (aspartic acid), the 85th (alanine), and the 107th (arginine) from the N-terminus of the HH-type light chain amino acid were replaced with Glutamate, glutamic acid, and lysine which were respectively conserved in human light chains (κ chains) were designed and designated as “PDHH type”. Similarly, an HM-type light chain amino acid in which the first amino acid (aspartic acid) and the 107th amino acid (arginine) from the N-terminus were replaced with glutamic acid and lysine conserved in the human light chain, respectively, was designed. PDHM type ". Hereinafter, expression plasmids carrying DNAs encoding the light chain amino acid sequences of these two types of humanized anti-human Fas antibodies were respectively constructed.
[0347]
1) Synthesis of primer
DNA encoding the PDHH type polypeptide chain (SEQ ID NO: 107 in the Sequence Listing) (SEQ ID NO: 106 in the Sequence Listing) and DNA encoding the PDHM type polypeptide chain (SEQ ID NO: 109 in the Sequence Listing) (SEQ ID NO: 108 in the Sequence Listing) The synthesis of ()) was performed by combining the PCR method.
[0348]
In performing the PCR, in addition to the above-mentioned two oligonucleotide primers 7AL1P (SEQ ID NO: 55 in the sequence listing) and 7ALCN (SEQ ID NO: 64), six other primers in the sequence listing:
5′-GGTGGAATTG TGCTCACCCA ATCTCCAGG-3 ′ (LPD1P: SEQ ID NO: 110);
5'-CCTGGAGATT GGGTGAGCAC AATCTCACC-3 '(LPD1N: SEQ ID NO: 111);
5'-CCATCTCTCG TCTGGAGCCG GAGGATTTTG C-3 '(LPD2P: SEQ ID NO: 112);
5'-GCAAAATCCC CCGGCTCCAG ACGAGAGATG G-3 '(LPD2N: SEQ ID NO: 113);
5′-CAAGGCACCCA AGCTGGAAAT CAAACGGACT G-3 ′ (LPD3P: SEQ ID NO: 114); and
5′-CAGTCCGTTT GATTTCCAGC TTGGTGCCTTG-3 ′ (LPD3N: SEQ ID NO: 115) was prepared.
[0349]
2) Construction of PDHH type humanized light chain expression plasmid pLPDHH75
An LPDHH-DNA fragment (SEQ ID NO: 106 in the sequence listing) encoding the amino acid sequence shown in SEQ ID NO: 107 in the sequence listing was prepared by repeating the PCR method three times, inserted into a plasmid, and then cloned in E. coli. Was done.
[0350]
a) First stage PCR
FIG. 31 shows the outline of the first-step PCR for LPDHH-DNA production.
[0351]
Secretory signal sequence and FRL with HindIII restriction enzyme cleavage site added to 5 'end ₁ The LPD1-DNA fragment encoding a part of the region was prepared under the following conditions.
Reaction liquid composition:
Plasmid pHSGHH7 DNA 200ng
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer LPD1N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0352]
FRL ₁ Part of the region, CDRL ₁ Region, FRL ₂ Region, CDRL ₂ Region and FRL ₃ The LPDHH1-DNA fragment encoding a part of the region was prepared under the following conditions.
Reaction liquid composition:
Plasmid pHSGHH7 DNA 200ng
Oligonucleotide primer LPD1P 80pmol
Oligonucleotide primer LPD2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0353]
FRL ₃ Part of the region, CDRL ₃ Region and FRL ₄ The LPDHH2-DNA fragment encoding the region was prepared under the following conditions.
Reaction liquid composition:
Plasmid pHSGHH7 DNA 200ng
Oligonucleotide primer LPD2P 80pmol
Oligonucleotide primer LPD3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0354]
FRL ₄ An LPDC-DNA fragment encoding a part of the region and the constant region and having an EcoRI restriction enzyme cleavage site added to the 3 ′ end was prepared under the following conditions.
Reaction liquid composition:
Plasmid pHSGHH7 DNA 200ng
Oligonucleotide primer LPD3P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0355]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected LPD1-DNA, LPDHH1-DNA, LPDHH2-DNA and LPDC-DNA were cut out with a razor and eluted from an acrylamide gel using a centricon and a centrilutor, respectively. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0356]
b) Second stage PCR
FIG. 32 shows an outline of the second step PCR for producing LPDHH-DNA.
[0357]
The LPDHH1.2-DNA fragment obtained by fusing the above LPD1-DNA fragment, LPDHH1-DNA and LPDHH2-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
LPD1-DNA solution prepared by the first step PCR 10 μl
LPDHH1-DNA solution prepared by the first step PCR 10 μl
LPDHH2-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer LPD3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0358]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band of the detected DNA corresponding to LPDHH1.2-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
c) Third stage PCR
FIG. 33 shows an outline of the third step PCR for producing LPDHH-DNA.
[0359]
The LPDHH-DNA fragment obtained by fusing the LPDHH1.2-DNA fragment and the LPDC-DNA fragment was prepared under the following conditions.
Reaction liquid composition:
LPDHH1.2-DNA solution prepared by the second step PCR 10 μl
10 μl of LPDC-DNA solution prepared by the first step PCR
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0360]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LPDHH-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0361]
FIG. 34 shows an outline of a method for preparing an expression plasmid retaining the LPDHH-DNA fragment.
[0362]
The obtained LPDHH-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and EcoRI.
[0363]
1 μg of cloning plasmid pHSG399 (manufactured by Takara Shuzo) DNA was previously digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. The dephosphorylated plasmid pHSG399 DNA thus obtained and the LPDHH-DNA fragment previously digested with restriction enzymes were ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer. The JM109 strain was transformed and spread on LB agar medium containing 1 mM IPTG, 0.1% X-Gal and 50 μg / ml chloramphenicol. After culturing at 37 ° C., a white colony was isolated, cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and a plasmid DNA was extracted from the culture by an alkaline SDS method. The extracted plasmid DNA was digested with restriction enzymes HindIII and EcoRI, and analyzed by 1% agarose gel electrophoresis to select a clone retaining the LPDHH-DNA fragment.
[0364]
By the above operation, a plasmid pHSHH5 carrying a fusion fragment of the DNA encoding the variable region of the humanized light chain of the PDHH type and the DNA encoding the constant region of the human Igκ chain was obtained. In addition, transformed Escherichia coli carrying the plasmid pHSHH5. E. coli pHSHH5 SANK 70398 was internationally deposited on February 26, 1998 at the National Institute of Advanced Industrial Science and Technology (AIST) by the National Institute of Advanced Industrial Science and Technology (AIST) and given the accession number FERM BP-6274. Was done.
[0365]
Next, using the plasmid pHSHH5, an expression vector plasmid pLPDHH75 carrying a DNA encoding a PDHH type humanized light chain polypeptide was prepared by the method described below.
[0366]
Expression plasmid pEE. 1 μg of 12.1 (Lonza) DNA was previously digested with restriction enzymes HindIII and EcoRI and dephosphorylated with CIP. This dephosphorylated plasmid pEE. After ligation of 12.1 DNA (100 ng) and 10 μg of a pHSHH5 DNA fragment previously digested with restriction enzymes HindIII and EcoRI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), Escherichia coli JM109 was transformed, and 50 μg / ml of ampicillin was transformed. Was spread on an LB agar medium containing.
[0367]
The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted according to the alkaline SDS method. This plasmid DNA was digested with restriction enzymes HindIII and EcoRI, and the presence or absence of the inserted fragment was confirmed by 1% agarose gel electrophoresis. By the above operation, a plasmid pLPDHH75 in which the fusion fragment of the humanized PDHH type HFE7A-light chain variable region and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained.
[0368]
(3) Construction of PDHM-type humanized light chain expression plasmid pLPDHM32
An LPDHM-DNA fragment (SEQ ID NO: 108 in the sequence listing) encoding the amino acid sequence shown in SEQ ID NO: 109 in the sequence listing was prepared by repeating the PCR method three times, inserted into a plasmid, and then cloned into E. coli. Was done.
[0369]
a) First stage PCR
FIG. 35 shows an outline of the first step PCR for producing LPDHM-DNA.
[0370]
A secretory signal sequence with a HindIII restriction enzyme cleavage site added to the 5 ′ end, and FRL ₁ LPD1-DNA fragment encoding a part of the region, and FRL ₄ LPDC-DNA fragments encoding a part of the region and the constant region and having an EcoRI restriction enzyme cleavage site added to the 3 ′ end were all obtained from the LPDHH-DNA fragment production process described in (2) above. Using.
[0371]
CDRL ₁ Part of the region, FRL ₂ Region, CDRL ₂ Region, FRL ₃ Region, CDRL ₃ Region, and FRL ₄ The LPDHM1-DNA fragment encoding the region was prepared under the following conditions.
Reaction liquid composition:
Plasmid pHSGHM17 DNA 200ng
Oligonucleotide primer LPD1P 80pmol
Oligonucleotide primer LPD3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0372]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected LPD1-DNA, LPDHM1-DNA and LPDC-DNA were cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0373]
b) Second stage PCR
FIG. 36 shows an outline of the second step PCR for producing LPDHM-DNA.
[0374]
The LPDHM1.2-DNA fragment obtained by fusing the above-mentioned LPD1-DNA fragment and LPDHM1-DNA fragment was prepared under the following conditions.
[0375]
Reaction liquid composition:
LPD1-DNA solution prepared by the first step PCR 10 μl
LPDHM1-DNA solution prepared by the first step PCR 10 μl
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer LPD3N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0376]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LPDHM1.2-DNA was excised with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0377]
c) Third stage PCR
FIG. 37 shows an outline of the third step PCR for producing LPDHM-DNA.
[0378]
The LPDHM-DNA fragment obtained by fusing the above-described LPDHM1.2-DNA fragment and the LPDC-DNA fragment was prepared under the following conditions.
[0379]
Reaction liquid composition:
LPDHM1.2-DNA solution prepared by the second step PCR 10 μl
10 μl of LPDC-DNA solution prepared by the first step PCR
Oligonucleotide primer 7AL1P 80pmol
Oligonucleotide primer 7ALCN 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0380]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0381]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LPDHM-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0382]
The outline of the method for preparing an expression plasmid retaining the LPDHM-DNA fragment is shown in FIG.
[0383]
First, the LPDHM-DNA fragment obtained above was further purified by phenol extraction and ethanol precipitation, and then digested with the restriction enzymes HindIII and EcoRI.
[0384]
Next, 1 μg of cloning plasmid pHSG399 (manufactured by Takara Shuzo) was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. The dephosphorylated plasmid pHSG399 DNA was ligated with an LPDHM-DNA fragment previously digested with HindIII and EcoRI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer. Was transformed and plated on LB agar medium containing 1 mM IPTG, 0.1% X-Gal and 50 μg / ml chloramphenicol. After culturing at 37 ° C., a white colony was isolated, cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes HindIII and EcoRI, and analyzed by 1% agarose gel electrophoresis to select a clone retaining the LPDHM-DNA fragment.
[0385]
By the above operation, plasmid pHSHM2 carrying a fusion fragment of the variable region of the PDHM-type humanized light chain and the DNA encoding the human Igκ chain constant region was obtained. In addition, transformed Escherichia coli carrying the plasmid pHSHM2. E. coli pHSHM2 SANK 70198 was internationally deposited on February 26, 1998 at the National Institute of Advanced Industrial Science and Technology (AIST) by the National Institute of Advanced Industrial Science and Technology (AIST) and given the accession number FERM BP-6272. Was done.
[0386]
Next, using plasmid pHSHM2, an expression vector plasmid pLPDHM32 carrying DNA encoding a PDHM-type humanized light chain polypeptide was prepared by the method described below.
[0387]
Expression plasmid pEE. 1 μg of 12.1 (Lonza) DNA was previously digested with restriction enzymes HindIII and EcoRI and dephosphorylated with CIP. This dephosphorylated plasmid pEE. 12.1 DNA (100 ng) and pHSHM2-DNA fragment (10 μg) previously digested with restriction enzymes HindIII and EcoRI were ligated using DNA ligation kit version 2.0 (Takara Shuzo), and Escherichia coli JM109 strain was ligated. It was transformed and spread on LB agar medium containing 50 μg / ml ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes EcoRI and HindIII, and the presence or absence of the inserted fragment was confirmed by agarose gel electrophoresis. By the above operations, a plasmid pLPDHM32 in which a fusion fragment of the DNA encoding the variable region of the PDHM-type humanized light chain and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained. .
[0388]
(4) Confirmation of nucleotide sequence
The nucleotide sequence was determined in order to confirm that the plasmids pLPDHH75 and pLPDHM32 prepared in (2) and (3) above each had a DNA having the target nucleotide sequence. The primers used here were SP1 (SEQ ID NO: 68 in the Sequence Listing), SP2 (SEQ ID NO: 69 in the Sequence Listing), SP3 (SEQ ID NO: 70 in the Sequence Listing), SP4 (SEQ ID NO: 71 in the Sequence Listing), and SP5. (SEQ ID NO: 72 in the sequence listing) and SP6 (SEQ ID NO: 73 in the sequence listing). The positions to which each primer binds are shown in FIG. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using each plasmid DNA purified by the alkali-SDS method and the cesium chloride method as a template. As a result, it was confirmed that pLPDHH75 has the nucleotide sequence shown in SEQ ID NO: 106 in the sequence listing, and pLPDHM32 has the nucleotide sequence shown in SEQ ID NO: 108 in the sequence listing.
[0389]
Example 22 Construction of humanized heavy chain expression vector
In order to make the humanized HFE7A-heavy chain produced in Example 15 closer to humans, the amino acid at position 44 (arginine) and the amino acid at position 76 (alanine) from the N-terminal of the amino acid sequence of the humanized heavy chain were replaced with human, respectively. Substitutions of glycine and threonine conserved in the heavy chain were designed and designated as "HV type". An expression plasmid carrying the DNA encoding the HV type humanized heavy chain amino acid sequence was constructed by the method described below.
[0390]
(1) Synthesis of primer
DNA (SEQ ID NO: 116 in the Sequence Listing) encoding the HV type humanized heavy chain polypeptide chain (SEQ ID NO: 117 in the Sequence Listing) was synthesized by a combination of PCR methods.
[0391]
In carrying out the PCR, in addition to the above primer 7AH1P (SEQ ID NO: 76 in the sequence listing), three more primers:
5′-CAGGCCCCTG GACAGGGCCT TGAGTGGATG-3 ′ (HPD1P: SEQ ID NO: 118 in Sequence Listing);
5'-CATCCACTCA AGGCCCTGTC CAGGGGCCTG-3 '(HPD1N: SEQ ID NO: 119 in Sequence Listing); and
5′-GCTGAGCTCC ATGTAGGCTG TGCTAGTGGA TGGTTCTAC-3 ′ (HPD2N: SEQ ID NO: 120 in Sequence Listing) was prepared.
[0392]
(2) Construction of plasmid pgHPDHV3
The HPD1.2-DNA fragment encoding the amino acid sequence represented by amino acid numbers -19 to 84 of SEQ ID NO: 117 in the sequence listing was prepared by repeating the PCR method twice, inserted into a plasmid, and cloned into E. coli. Was done.
[0393]
a) First stage PCR
FIG. 39 shows the outline of the first-step PCR for preparing HPD1.2-DNA.
[0394]
Secretion signal sequence, FRH, with a HindIII restriction enzyme cleavage site added to the 5 'end ₁ Region, CDRH ₁ Region and FRH ₂ An HPD1-DNA fragment encoding a part of the region was prepared under the following conditions.
Reaction liquid composition:
Plasmid pgHSL7A62 DNA 200ng
Oligonucleotide primer 7AH1P 80pmol
Oligonucleotide primer HPD1N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0395]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0396]
Next, FRH ₂ Part of the region, CDRH ₃ Region and FRH ₃ An HPD2-DNA fragment encoding a part of the region was prepared under the following conditions.
[0397]
Reaction liquid composition:
Plasmid pgHSL7A62 DNA 200ng
Oligonucleotide primer HPD1P 80pmol
Oligonucleotide primer HPD2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0398]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0399]
Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected HPD1-DNA and HPD2-DNA were cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0400]
b) Second stage PCR
FIG. 40 shows the outline of the second step PCR for preparing HPD1.2-DNA.
[0401]
The HPD1.2-DNA fragment obtained by fusing the aforementioned HPD1-DNA fragment and HPD2-DNA fragment was prepared under the following conditions.
[0402]
Reaction liquid composition:
HPD1-DNA solution prepared by the first step PCR 10 μl
HPD2-DNA solution prepared by the first-step PCR 10 μl
Oligonucleotide primer 7AH1P 80pmol
Oligonucleotide primer HPD2N 80pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0403]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0404]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band corresponding to the detected fusion DNA was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0405]
FIG. 41 shows an outline of a method for preparing a plasmid holding the HPD1.2-DNA fragment.
[0406]
The HPD1.2-DNA fragment obtained above was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and SacI.
[0407]
1 μg of the above plasmid pgHSL7A62 DNA was previously digested with restriction enzymes HindIII and SacI, and dephosphorylated with CIP. After ligating the dephosphorylated plasmid pgHSL7A62 DNA and an HPD1.2-DNA fragment previously digested with HindIII and SacI using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), Escherichia coli JM109 strain was transformed. It was spread on an LB agar medium containing chloramphenicol at a final concentration of 50 μg / ml. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes SacI and HindIII, and subjected to agarose gel electrophoresis to select a clone retaining the HPD1.2-DNA fragment.
[0408]
By the above operation, plasmid pgHPDHV3 carrying a fusion fragment of the DNA encoding the variable region of the HV type humanized heavy chain and the genomic DNA fragment encoding the human IgG1 constant region was obtained. In addition, transformed Escherichia coli carrying the plasmid pgHPDHV3. E. coli pgHPDHV3 SANK 70298 was internationally deposited on February 26, 1998 with the National Institute of Advanced Industrial Science and Technology (AIST) at the National Institute of Advanced Industrial Science and Technology (AIST), under the accession number FERM BP-6273. Was done.
[0409]
Next, 10 μg of pgHPDHV3 DNA was digested with restriction enzymes HindIII and EcoRI. On the other hand, the expression plasmid pEE. 6.1 μg (manufactured by Lonza) was digested with restriction enzymes HindIII and EcoRI in advance and dephosphorylated with CIP. This dephosphorylated plasmid pEE. 6.1 DNA (100 ng) and pgHPDHV3 DNA (10 μg) digested with HindIII and EcoRI were ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo), and then cloned into Escherichia coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes HindIII and EcoRI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the inserted fragment. By the above operation, plasmid pEgPDHV3-21 in which the DNA encoding the HV type humanized heavy chain was inserted in the forward direction downstream of the CMV promoter was obtained.
[0410]
(3) Confirmation of nucleotide sequence
The nucleotide sequence was determined in order to confirm that the plasmid pEgPDHV3-21 obtained in the above (2) holds a DNA having the target nucleotide sequence. The primers used here were PEEF (SEQ ID NO: 104 in the Sequence Listing), HPD1P (SEQ ID NO: 118 in the Sequence Listing), HPD1N (SEQ ID NO: 119 in the Sequence Listing), and HPD2N (SEQ ID NO: 120 in the Sequence Listing). Was. The position where each primer binds is shown in FIG. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using plasmid DNA purified by the alkali-SDS method and the cesium chloride method as a template. As a result, pEgPDHV3-21 was confirmed to have the nucleotide sequence shown in SEQ ID NO: 116 in the sequence listing.
[0411]
Example 23 Construction of expression vector for COS-1 cells
Various humanized HFE7A-light chain expression vectors (p7AL-HH, p7AL-HM, p7AL-MM, pLPDHH75 and pLPDHM32) prepared in Example 14 and Example 21, and in Example 15 and Example 22. Using various humanized HFE7A-heavy chain expression vectors (pEg7AH-H and pEgPDHV3-21), expression vectors optimized for gene expression using COS-1 cells were prepared by the method described below.
[0412]
1. Construction of light chain expression vector for COS-1 cells
FIG. 43 shows an outline of a method for constructing expression vectors for various humanized HFE-7A light chains for COS-1 cells.
[0413]
(1) Synthesis of promoter amplification primer (for light chain)
In preparing a DNA fragment containing the SRα promoter region by PCR, the following two oligonucleotide primers are used:
5′-TGCACGCGTG GCTGTGGAAT GTGTGTCAGT TAG-3 ′ (MLUA: SEQ ID NO: 121 in Sequence Listing); and
5′-TCCGAAGCTT TTAGAGCAGA AGTAACACTTC-3 ′ (HINDB: SEQ ID NO: 122 in Sequence Listing) was prepared.
[0414]
(2) Construction of expression vector (for light chain)
The LSRα-DNA fragment containing the SRα promoter region and having a MulI restriction enzyme cleavage site at the 5 ′ end and a HindIII restriction enzyme cleavage site at the 3 ′ end was prepared using the plasmid pME18S as a template under the following conditions.
[0415]
Reaction liquid composition:
200 ng of plasmid pME18S DNA
Oligonucleotide primer MLUA 80 pmol
Oligonucleotide primer HINDB 80 pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0416]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0417]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LSRα-DNA was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0418]
Next, 1 μg of the plasmids p7AL-HH, p7AL-HM, p7AL-MM, pLPDHH75 or pLPDHM32 prepared in Example 14 and Example 21 were digested with restriction enzymes MulI and HindIII, and dephosphorylated with CIP. The dephosphorylated plasmid DNA was ligated with an LSRα-DNA fragment previously digested with MulI and HindIII using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.). The cells were spread on an LB agar medium containing 50 μg / ml of ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes MulI and HindIII, and agarose gel electrophoresis was performed to select a clone retaining the LSRα-DNA fragment.
[0419]
By the above operation, the expression vectors pSRHH (derived from HH type, p7A-HH), pSRHM (derived from HM type, p7A-HM), pSRMM (MM type, derived from p7A-HM) for COS-1 cells encoding various humanized light chains are obtained. MM), pSRPDHH (PDHH type, derived from pLPDHH75) and pSRPDHM (PDMM type, derived from pLPDHM32) were obtained.
[0420]
2. Construction of heavy chain expression vector for COS-1 cells
The outline of the method for constructing expression vectors for various humanized heavy chains for COS-1 cells is shown in FIG.
[0421]
(1) Synthesis of promoter amplification primer (for heavy chain)
In preparing a DNA fragment containing the SRα promoter region by PCR, in addition to the above-mentioned primer HINDB (SEQ ID NO: 122 in the sequence listing), one more primer:
5′-AAAGCGGCCG CTGCTAGCTT GGCTTGTGGAA TGTGTG-3 ′ (NOTA: SEQ ID NO: 123 in Sequence Listing) was prepared.
[0422]
(2) Construction of expression vector (for heavy chain)
An HSRα-DNA fragment containing the SRα promoter region and having a NotI restriction enzyme cleavage site at the 5 ′ end and a HindIII restriction enzyme cleavage site at the 3 ′ end was prepared using the plasmid pME18S as a template under the following conditions.
[0423]
Reaction liquid composition:
200 ng of plasmid pME18S DNA
Oligonucleotide primer NOTA 80pmol
Oligonucleotide primer HINDB 80 pmol
dNTP mixture 20μl
20 μl of 10 × Pfu buffer
Pfu DNA polymerase 10 units
The reaction solution having the above composition was adjusted to a final volume of 200 μl with double-distilled water and subjected to PCR.
[0424]
PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, followed by heating at 72 ° C. for 10 minutes.
[0425]
The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band corresponding to the detected HSRa-DNA was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.
[0426]
Next, 1 μg of the plasmid pEg7AH-H or pEgPDHV3-21 prepared in Example 15 and Example 22 was digested with restriction enzymes NotI and HindIII, and dephosphorylated with CIP. This dephosphorylated plasmid DNA was ligated with an HSRα-DNA fragment previously digested with NotI and HindIII using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.). The cells were spread on an LB agar medium containing 50 μg / ml of ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes NotI and HindIII, and subjected to agarose gel electrophoresis to select a clone retaining the HSRa-DNA fragment.
[0427]
Through the above operations, expression vectors pSRg7AH (derived from pEg7AH-H) and pSRgPDH (derived from pEgPDHV3-21) for COS-1 cells encoding various humanized heavy chains were obtained.
[0428]
Example 24 Expression of genes encoding humanized HFE7A subunits in COS-1 cells
Transformation of COS-1 cells with the expression vector for COS-1 cells obtained in Example 23 was performed by the method described in Example 16. First, COS-1 cells were placed in a cell culture flask (culture area 225 cm) containing α (+) MEM (manufactured by Gibco BRL) containing 10% FCS. ² : Sumitomo Bakelite Co., Ltd.) until semi-confluent. Thereafter, the medium was removed, and COS-1 cells were released from the flask by keeping the mixture at 37 ° C. for 3 minutes in 3 ml of a trypsin-EDTA solution (manufactured by Sigma). The released cells were collected by centrifugation at 800 rpm for 2 minutes, washed twice with PBS (−) (manufactured by Nissui Pharmaceutical Co., Ltd.), and then washed with 1 × 10 4 with PBS (−). ⁸ The COS-1 cell suspension was prepared so as to have a cell number / ml.
[0429]
On the other hand, 4 μg of a humanized heavy chain expression vector (pSRg7AH or pSRgPDH) DNA prepared using the alkali-SDS method and cesium chloride density gradient centrifugation, and a humanized light chain expression vector (pSRHH, pSRHM, pSRMM, pSRPDH or pSRPDHM) 4) DNA (4 μg) was mixed in the combination described below, followed by ethanol precipitation, and the obtained precipitate was dissolved in PBS (−) 20 μl. 20 μl of the obtained plasmid solution was added to 20 μl of the previously prepared COS-1 cell suspension (2 × 10 ⁶ The resulting mixture was placed in a chamber (FCT-13: manufactured by Shimadzu Corporation) with an electrode spacing of 2 mm, and set in a gene transfer apparatus GTE-1 (manufactured by Shimadzu Corporation). Thereafter, the target plasmid DNA was introduced into the COS-1 cells by applying a pulse of 50 μF at 600 V twice at 1 second intervals. The cell-DNA mixture in the chamber after applying the pulse was suspended in 5 ml of α (+) MEM containing 10% fetal bovine serum, and the suspension was cultured in a cell culture flask (culture area 25 cm). ² : Sumitomo Bakelite Co., Ltd.). This is 5% CO ₂ After culturing at 37 ° C. for 72 hours, the culture supernatant was recovered.
[0430]
By the above method, COS-1 cells were transformed under the following conditions [A] to [F], and the culture supernatant was recovered.
[0431]
[A]: conditions not containing plasmid DNA;
[B]: co-transfection of pSRgPDH and pSRPDHH;
[C]: co-transfection of pSRgPDH and pSRPDHM;
[D]: co-transfection of pSRg7AH and pSRHH;
[E]: co-transfection of pSRg7AH and pSRHM;
[F]: co-transfection of pSRg7AH and pSRMM.
[0432]
Example 25 Assay of binding ability to Fas
For each of the culture supernatant samples prepared in Example 24, the concentration of the target expression product contained was calculated by the method described in Example 17 so that the final target product concentration was 100 ng / ml. Prepared with α (+) MEM containing 10% FCS. When the concentration of the target product in the culture supernatant sample was lower than 100 ng / ml, the obtained culture supernatant was first concentrated using Centriprep-10 (manufactured by Amicon). That is, 5 ml of the culture supernatant was transferred to Centriprep-10, concentrated by centrifugation at 3000 × g, and the concentration of the target product was calculated again by the method described in Example 17.
[0433]
Next, the operation of diluting each sample solution adjusted to a concentration of 100 ng / ml twice with α (+) MEM containing 10% FCS was repeated to prepare a dilution series. For these samples, the binding ability to the human Fas fusion protein was measured by the ELISA method described in Example 18.
[0434]
As a result, among the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E] and [F] showed the binding ability to human Fas fusion protein. (Fig. 45).
[0435]
Example 26 Competitive inhibition of HFE7A binding to Fas
For each culture supernatant sample prepared in Example 24, the concentration of the target expression product contained therein was calculated in advance according to the method described in Example 17, and finally the α (+) MEM containing 10% FCS was used. The desired product concentration was adjusted to 1 μg / ml. When the concentration of the target product in the culture supernatant sample was lower than 1 μg / ml, the culture supernatant was concentrated using Centriprep-10 and then adjusted to 1 μg / ml.
[0436]
Next, the operation of diluting the sample prepared at a concentration of 1 μg / ml twice with α (+) MEM containing 10% FCS was repeated to prepare a dilution series. On the other hand, AP-HFE7A was adjusted to a concentration of 50 ng / ml with α (+) MEM containing 10% FCS, and mixed with the diluted series sample in an equal amount. Thereafter, the competitive inhibitory activity on HFE7A of each sample was measured by the method described in Example 19.
[0437]
As a result, among the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E], and [F] were those of HFE7A prepared from the hybridoma culture. It was confirmed that the binding to the human Fas fusion protein was specifically inhibited (FIG. 46).
[0438]
Example 27 Apoptosis-inducing activity
The culture supernatant sample prepared in Example 24 was adjusted by the method described in Example 22 so that the final target product concentration was 100 ng / ml (however, the medium for adjustment contains 10% FCS). RPMI 1640 medium was used). The operation of diluting the 100 ng / ml sample twice with RPMI1640 medium containing 10% FCS was repeated to prepare a dilution series. Thereafter, the cytotoxic activity of each sample on WR19L12a cells was measured by the method described in Example 20.
[0439]
As a result, among the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E] and [F] were HFE7A prepared from the hybridoma culture. Similarly, it was shown to induce apoptosis in a T lymphoma cell line that expressed the human Fas antigen (FIG. 47).
[0440]
Formulation example
The molecules of the invention are provided for use as ampules in sterile solutions or suspensions in water or other pharmacologically acceptable solutions. Alternatively, a sterile powder preparation (preferably freeze-dried of the molecule of the present invention) may be filled in an ampoule and diluted with a pharmacologically acceptable solution at the time of use.
[0441]
According to the present invention, various diseases caused by the Fas / Fas ligand system (autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison) Disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes) , Allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, hepatitis B, hepatitis D)) or During Fas of primate and non-primate animals useful as prophylactic or therapeutic agents against alcoholic hepatitis), acquired immunodeficiency syndrome or rejection after organ transplantation) Molecule is provided with a specific antigen binding region to an epitope being conserved.
[0442]
[Sequence List Free Text]
SEQ ID NO: 12: N1
SEQ ID NO: 13: C3N
SEQ ID NO: 14: N3N
SEQ ID NO: 15: CTN2
SEQ ID NO: 18: H1
SEQ ID NO: 19: H2
SEQ ID NO: 20: L1
SEQ ID NO: 21: L2
SEQ ID NO: 22: H3
SEQ ID NO: 23: H4
SEQ ID NO: 24: L3
SEQ ID NO: 25: L4
SEQ ID NO: 41: control peptide
SEQ ID NO: 46: control peptide
SEQ ID NO: 47: HVKII5-4
SEQ ID NO: 48: HKCL3-3
SEQ ID NO: 49: Humanized anti-Fas antibody HFE7A HH type light chain
SEQ ID NO: 50: Humanized anti-Fas antibody HFE7A HH type light chain
SEQ ID NO: 51: Humanized anti-Fas antibody HFE7A HM type light chain
SEQ ID NO: 52: Humanized anti-Fas antibody HFE7A HM type light chain
SEQ ID NO: 53: humanized anti-Fas antibody HFE7A MM type light chain
SEQ ID NO: 54: humanized anti-Fas antibody HFE7A MM type light chain
SEQ ID NO: 55: 7AL1P
SEQ ID NO: 56: 7AL1N
SEQ ID NO: 57: 7AL2P
SEQ ID NO: 58: 7AL2N
SEQ ID NO: 59: 7AL3PA
SEQ ID NO: 60: 7AL3N
SEQ ID NO: 61: 7AL4P
SEQ ID NO: 62: 7AL4N
SEQ ID NO: 63: 7ALCP
SEQ ID NO: 64: 7ALCN
SEQ ID NO: 65: M7AL2N
SEQ ID NO: 66: M7AL3PA
SEQ ID NO: 67: 7AL4NA
SEQ ID NO: 68: SP1
SEQ ID NO: 69: SP2
SEQ ID NO: 70: SP3
SEQ ID NO: 71: SP4
SEQ ID NO: 72: SP5
SEQ ID NO: 73: SP6
SEQ ID NO: 74: Humanized anti-Fas antibody HFE7A heavy chain variable region and N-terminal 5 amino acids of IgG-CH1
SEQ ID NO: 75: Humanized anti-Fas antibody HFE7A heavy chain variable region and N-terminal 5 amino acids of IgG-CH1
SEQ ID NO: 76: 7AH1P
SEQ ID NO: 77: 7AH1NNEW
SEQ ID NO: 78: 7AH2N
SEQ ID NO: 79: 7AH2PNEW
SEQ ID NO: 80: 7AH3P
SEQ ID NO: 81: 7AH3N
SEQ ID NO: 82: 7AH4P
SEQ ID NO: 83: 7AH4N
SEQ ID NO: 84: 5 ′ Hind
SEQ ID NO: 85: IGGCPSTN
SEQ ID NO: 86: IGGCPSTP
SEQ ID NO: 87: Eco3 ′
SEQ ID NO: 88: humanized anti-Fas antibody HFE7A heavy chain
SEQ ID NO: 89: humanized anti-Fas antibody HFE7A heavy chain
SEQ ID NO: 90: IG01
SEQ ID NO: 91: IG02
SEQ ID NO: 92: IG03
SEQ ID NO: 93: IG04
SEQ ID NO: 94: IG05
SEQ ID NO: 95: IG06
SEQ ID NO: 96: IG07
SEQ ID NO: 97: IG08
SEQ ID NO: 98: IGP5
SEQ ID NO: 99: IGP6
SEQ ID NO: 100: IGP7
SEQ ID NO: 101: IGP8
SEQ ID NO: 102: H5 +
SEQ ID NO: 103: H5-
SEQ ID NO: 104: PEEF
SEQ ID NO: 105: PEEB
SEQ ID NO: 106: humanized anti-Fas antibody HFE7A PDHH type light chain
SEQ ID NO: 107: humanized anti-Fas antibody HFE7A PDHH type light chain
SEQ ID NO: 108: humanized anti-Fas antibody HFE7A PDHM type light chain
SEQ ID NO: 109: humanized anti-Fas antibody HFE7A PDHM type light chain
SEQ ID NO: 110: LPD1P
SEQ ID NO: 111: LPD1N
SEQ ID NO: 112: LPD2P
SEQ ID NO: 113: LPD2N
SEQ ID NO: 114: LPD3P
SEQ ID NO: 115: LPD3N
SEQ ID NO: 116: humanized anti-Fas antibody HFE7A HV type heavy chain
SEQ ID NO: 117: humanized anti-Fas antibody HFE7A HV type heavy chain
SEQ ID NO: 118: HPD1P
SEQ ID NO: 119: HPD1N
SEQ ID NO: 120: HPD2N
SEQ ID NO: 121: MLUA
SEQ ID NO: 122: HINDB
SEQ ID NO: 123: NOTA
[0443]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a construction diagram of phFas-AIC2.
FIG. 2 is a construction diagram of pME-H and pME-L.
FIG. 3 shows the results of ELISA for defining the epitope of HFE7A.
FIG. 4 shows the results of a competition assay for defining the epitope of HFE7A.
FIG. 5 shows the results of a toxicity test of HFE7A.
FIG. 6 is a diagram showing the results of a test using a fulminant hepatitis model.
FIG. 7 is a diagram showing the results of a test for preventing the onset of collagen-induced arthritis.
FIG. 8: Overview of the first step PCR for VHH-DNA production.
FIG. 9 is an outline of the second step PCR for producing VHH-DNA.
FIG. 10: Summary of the third step PCR for VHH-DNA production.
FIG. 11 shows an outline of a method for preparing an expression plasmid having a VHH-DNA fragment.
FIG. 12: Overview of first step PCR for VHM-DNA production.
FIG. 13. Schematic of the second stage PCR for VHM-DNA production.
FIG. 14 shows an outline of a method for preparing an expression plasmid having a VHM-DNA fragment.
FIG. 15: Overview of the first step PCR for VMM-DNA production.
FIG. 16 is an outline of the second step PCR for preparing VMM-DNA.
FIG. 17: Summary of the third step PCR for VMM-DNA production.
FIG. 18 shows an outline of a method for preparing a plasmid holding a VMM-DNA fragment.
FIG. 19 is a diagram showing binding positions of humanized light chain sequencing primers.
FIG. 20. Outline of the first step PCR for VD-DNA production.
FIG. 21: Outline of the second step PCR for VD-DNA production.
FIG. 22. Summary of the third step PCR for VD-DNA production.
FIG. 23 shows an outline of a method for preparing a plasmid holding a VD-DNA fragment.
FIG. 24 shows an outline of a method for producing a DNA (IG5′-DNA) fragment containing the CH1 region and intron of human IgG1.
FIG. 25 shows an outline of a method for preparing a genomic DNA (IG3′-DNA) fragment containing the hinge region, CH2 region, CH3 region and intron of human IgG1.
FIG. 26 shows an outline of a procedure for preparing an expression vector plasmid pEg7AH-H.
FIG. 27 shows binding positions of humanized heavy chain sequencing primers.
FIG. 28 shows the binding activity of a humanized anti-Fas antibody to a human Fas fusion protein.
FIG. 29 is a view showing the competitive inhibition of HFE7A and humanized anti-Fas antibody against human Fas fusion protein.
FIG. 30 shows the cytotoxic activity of humanized HFE7A on WR19L12a.
FIG. 31. Overview of the first step PCR for LPDHH-DNA production.
FIG. 32. Overview of the second step PCR for production of LPDHH-DNA.
FIG. 33. Summary of the third step PCR for LPDHH-DNA production.
FIG. 34 shows an outline of a method for preparing an expression plasmid retaining an LPDHH-DNA fragment.
FIG. 35. Summary of first step PCR for LPDHM-DNA production.
FIG. 36. Summary of second stage PCR for LPDHM-DNA production.
FIG. 37. Summary of third step PCR for LPDHM-DNA production.
FIG. 38 shows an outline of a method for preparing an expression plasmid retaining an LPDHM-DNA fragment.
FIG. 39. Summary of first step PCR for HPD1.2-DNA production.
FIG. 40. Summary of second stage PCR for HPD1.2-DNA production.
FIG. 41 shows an outline of a method for preparing a plasmid holding an HPD1.2-DNA fragment.
FIG. 42 is a diagram showing binding positions of DNAs encoding HV type humanized heavy chains with a sequencing primer.
FIG. 43 is a construction diagram of an expression vector optimized for expressing various humanized light chains in COS-1 cells.
FIG. 44 is a construction diagram of an expression vector optimized for expressing various humanized heavy chains in COS-1 cells.
FIG. 45 shows the binding activity to human Fas fusion protein in the culture supernatant of various transformed COS-1 cells.
FIG. 46 is a graph showing the competitive inhibitory activity on the binding between human Fas fusion protein and HFE7A in the culture supernatant of various transformed COS-1 cells.
FIG. 47 is a graph showing cytotoxicity to WR19L12a in the culture supernatant of various transformed COS-1 cells.

Claims (17)

An antibody obtained by humanizing a monoclonal antibody produced by a mouse-mouse hybridoma HFE7A (FERM BP-5828), having the following features 1) to 5):
1) having an antigen-binding region specific to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
_{2) -FRH 1 -CDRH 1 -FRH 2} -CDRH 2 -FRH 3 -CDRH 3 -FRH 4 having a heavy chain polypeptide that comprises an amino acid sequence represented by the following general formula (I) - (I)
(Wherein, FRH ₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acid sequences, CDRH ₁ represents an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, and FRH ₂ represents an arbitrary amino acid sequence consisting of 14 amino acids. CDRH ₂ represents the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, FRH ₃ represents any amino acid sequence consisting of 32 amino acids, and CDRH ₃ represents the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing. represents the amino acid sequence, FRH ₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, each amino acid is linked at each peptide bond.) and the amino acid sequence represented by the following general formula (II) A light chain polypeptide comprising:
_{3) -FRL 1 -CDRL 1 -FRL 2} -CDRL 2 -FRL 3 -CDRL 3 -FRL 4 having a light chain polypeptide that comprises an amino acid sequence represented by the following general formula (II) - (II)
(Wherein, FRL ₁ represents any amino acid sequence consisting of 23 amino acid sequences, CDRL ₁ represents the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, and FRL ₂ represents any amino acid sequence consisting of 15 amino acid sequences. Represents an amino acid sequence, CDRL ₂ represents an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, FRL ₃ represents an arbitrary amino acid sequence consisting of 32 amino acids, and CDRL ₃ represents a sequence represented by SEQ ID NO: 7 in the sequence listing. Represents an amino acid sequence, and FRL ₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, and each amino acid is linked by a peptide bond.);
3) has an apoptosis-inducing activity on T cells expressing Fas;
4) Does not induce liver damage.
5) Immunoglobulin type G antibody. The antibody according to claim 1, which has a therapeutic or preventive effect on fulminant hepatitis induced by the anti-mouse Fas monoclonal antibody Jo2. The antibody according to claim 1 or 2, wherein the antibody has the biological property according to at least one of the following a) to c):
a) It has an effect of improving autoimmune disease symptoms in MRLgld / gld mice;
b) has a preventive effect on collagen-induced arthritis;
c) It has apoptosis-inducing activity on synovial cells derived from patients with rheumatoid arthritis. An antibody according to claim 1 or 2, characterized in that it has all of the biological properties described under a) to d) below:
a) It has an effect of improving autoimmune disease symptoms in MRLgld / gld mice;
b) has a therapeutic or preventive effect on fulminant hepatitis induced by anti-mouse Fas monoclonal antibody Jo2;
c) has an effect of preventing the onset of collagen-induced arthritis;
d) It has apoptosis-inducing activity on synovial cells derived from patients with rheumatoid arthritis. The antibody according to any one of claims 1 to 4, wherein apoptosis can be induced in abnormal cells expressing Fas and apoptosis of normal cells can be prevented. An antibody composed of the light chain polypeptide described in 1) and the heavy chain polypeptide described in 2) below, wherein the amino acid sequence of at least one of the light chain polypeptide and the heavy chain polypeptide is 1 Or, several amino acids have been deleted, substituted or added, comprising an amino acid sequence, and having an antigen-binding region specific to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing,
An antibody having apoptosis-inducing activity against T cells expressing Fas:
1) The amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence listing, the amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence listing, and the amino acid sequence 1 to 218 of SEQ ID NO: 54 of the sequence listing Or any one of the amino acid sequences of amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing or the amino acid sequences of amino acid numbers 1 to 218 of SEQ ID NO: 109 in the sequence listing. Light chain polypeptide;
2) a heavy-chain polypeptide consisting of any one of the amino acid sequences of amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or the amino acid sequences of amino acid numbers 1 to 451 of SEQ ID NO: 117 of the sequence listing . From a light chain polypeptide consisting of the amino acid sequence represented by amino acid Nos. 1 to 218 of SEQ ID No. 50 in the Sequence Listing and a heavy chain polypeptide consisting of the amino acid sequence represented by amino acid Nos. 1 to 451 of SEQ ID No. 89 of the Sequence Listing The antibody comprises a light chain polypeptide and / or a heavy chain polypeptide, wherein at least one of the amino acid sequences has an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and An antibody having an antigen-binding region specific to the amino acid sequence represented by No. 1 and having apoptosis-inducing activity on T cells expressing Fas. From a light chain polypeptide consisting of the amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing and a heavy chain polypeptide consisting of the amino acid sequence represented by amino acid sequence 1 to 451 of SEQ ID NO: 117 of the sequence listing The antibody comprises at least one amino acid sequence of a light chain polypeptide and a heavy chain polypeptide, wherein the amino acid sequence has an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and expresses Fas. An antibody having an apoptosis-inducing activity against a specific T cell. A DNA encoding any one of the polypeptide chains in the antibody according to any one of claims 1 to 8. A DNA encoding the light chain polypeptide or the heavy chain polypeptide of the antibody according to any one of claims 1 to 8. A recombinant DNA vector comprising the DNA according to claim 9 or 10. A host cell transformed with the recombinant DNA vector according to claim 11. 13. The host cell according to claim 12, wherein the host cell is derived from a mammal. A medicament comprising the antibody according to any one of claims 1 to 8 as an active ingredient. A method for producing a mouse-mouse hybridoma HFE7A, comprising the following steps 1) and 2):
1) immunizing an animal deficient in the Fas-Fas ligand system with Fas;
2) A step of fusing spleen cells excised from an animal immunized with Fas with myeloma cells and selecting a hybridoma that produces an antibody that binds to Fas. A method for selecting a mouse-mouse hybridoma HFE7A, comprising the following steps 1) and 2):
1) Immunizing an animal deficient in the Fas-Fas ligand system with Fas:
2) A step of fusing spleen cells excised from an animal immunized with Fas with myeloma cells and selecting a hybridoma that produces an antibody that binds to Fas. 18. The method according to claim 16 or 17, wherein the animal deficient in the Fas-Fas ligand system is a mouse in which the gene encoding Fas has been rendered incapable of expression (hereinafter, referred to as "Fas knockout mouse"). .

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