JP3681666B2 - Method for enriching mutant proteins with modified binding properties - Google Patents

Description

【００９１】
これらＭabに対するhＧＨ上のエピトープはマッピングされており〔Cunninghamら（上記）〕、８種のＭabのうちの７種に対する結合はhＧＨが適切に折り畳まれることを必要とする。すべてのＭabのＩＣ₅₀値は、Ｍab ５および６を除いて野生型hＧＨと等価であった。Ｍab ５および６の両方は、遺伝子III融合タンパク質中でブロックされるhＧＨのカルボキシ末端の近くに結合決定基を有していることが知られている。天然および変性hＧＨの両方と反応するＭab１の相対ＩＣ₅₀値は、立体配座的に敏感なＭab ２〜５、７および８と比較して変わることがない。即ち、Ｍab１は、hＧＨ−遺伝子III融合体の濃度に対するhＧＨ標準の濃度を適合させる際のあらゆる過誤の良好な内部対照として働く。
【００９２】
実施例 VI 受容体親和性ビーズに対する結合の豊富化
これまでの研究者〔Parmley, Smith（上記）；Smith（上記）；Cwirlaら（上記）；およびDevlinら（上記）〕は、ストレプトアビジン被覆したポリスチレン製ペトリ皿または微量滴定プレートで選別することによってファージを分画していた。しかし、クロマトグラフィー系は、異なる結合親和性を有する突然変異タンパク質を表示するファージミド粒子のさらに効率的な分画を可能にする。我々は、非多孔性のオキシランビーズ（Sigma）を選択してクロマトグラフィー樹脂中のファージミド粒子の捕捉を回避した。さらに、これらビーズは小さい粒子サイズ（１μm）を有しており、量に対する表面積の比が最大になる。遊離のシステイノ残基を含有するhＧＨ受容体の細胞外ドメイン（hＧＨbp）〔Fuhら（上記）〕はこれらビーズに効率的に結合し、ファージミド粒子はウシ血清アルブミンにのみ結合させたビーズに対して極めて低い非特異的な結合を示した（表II）。
【００９３】

【００９４】
代表的な豊富化実験（表II）において、１部のhＧＨファージミドを＞３,０００部のＭ１３ＫＯ７ファージと混合した。１サイクルの結合と溶離の後、１０⁶のファージが回収され、ファージミドのＭ１３ＫＯ７ファージに対する比は２：１であった。即ち、１回の結合選択工程によって、＞５０００倍の豊富化が得られた。残存するファージミドを除去するための遊離hＧＨまたは酸処理による追加の溶離によって、さらに高い豊富化が得られた。この豊富化は、被覆ポリスチレンプレートからのバッチ溶離を用いてSmithおよびその共同研究者が得た豊富化に匹敵した〔Smith,G.P.（上記）およびParmley, Smith（上記）〕。しかし、さらに少ない容量がビーズに対して用いられている（２００μl vs ６ml）。ＢＳＡにのみ結合させたビーズを用いたときには、Ｍ１３ＫＯ７を凌ぐhＧＨファージミドの豊富化はほとんど存在しなかった。対照ビーズに対して観察されたわずかな豊富化（ｐH２.１溶離に対して〜１０倍；表２）は、ビーズに結合するＢＳＡ中に存在するウシ成長ホルモン結合タンパク質の微量の不純物に由来するものであろう。それにもかかわらず、これらのデータは、hＧＨファージの豊富化がビーズ上のhＧＨbpの存在に依存することを示し、結合がhＧＨとhＧＨbpの間の特異的な相互作用によって起こることを示唆する。
【００９５】
我々は、融合ファージミド上のhＧＨの比較的弱い結合変異体を凌ぐ野生型hＧＨの豊富化を評価して、豊富化の特異性をさらに調べ、精製ホルモンに対する結合親和性の減少を融合ファージミド選別後の豊富化因子と関連させた。Ａrg６４がＡlaで置換されたhＧＨ突然変異体（Ｒ６４Ａ）を用いて融合ファージミドを構築した。このＲ６４Ａ変異ホルモンは、hＧＨと比べて受容体結合親和性が約２０倍減少している〔それぞれ、Ｋd値が７.１nMおよび０.３４nM；Cunningham, Wells（上記）〕。Ｒ６４Ａ hＧＨ−遺伝子III融合ファージミドの力価は、野生型hＧＨファージミドの力価と同等であった。１ラウンドの結合と溶離の後（表III）、野生型hＧＨファージミドは、２種類のファージミドとＭ１３ＫＯ７の混合物から、ファージミドＲ６４Ａに対しては８倍およびＭ１３ＫＯ７ヘルパーファージに対しては〜１０⁴で豊富化された。
【００９６】

【００９７】
ファージミド粒子上に野生型遺伝子IIIおよび遺伝子III融合タンパク質の混合物を表示させることにより、遺伝子IIIに対する融合体として大きな適切に折り畳まれたタンパク質を表示するビリオンを組立てそして増殖させることができる。遺伝子III融合タンパク質のコピー数を有効に制御して、ファージミドプールにおいて十分に高いレベルになお維持されている「キレート作用」を避け、大きいエピトープライブラリー（＞１０¹⁰）の選別をすることができる。我々は、hＧＨ（２２kＤのタンパク質）を天然の折り畳まれた形態で表示させうることを示した。遊離のhＧＨで溶離し、受容体親和性ビーズ上で行なった結合選択は、低い受容体結合親和性を有することが示された突然変異hＧＨファージミド以上に野生型hＧＨファージミドを効率的に豊富化した。即ち、結合定数がナノモル範囲で低下しているファージミド粒子を選別することができる。
タンパク質−タンパク質および抗体−抗原の相互作用は、不連続エピトープによって支配されている〔Janin,J.ら, J.Mol.Biol. 204: 155-164 (1988); Argos,P., Prot.Eng. 2: 101-113 (1988); Barlow,D.J.ら, Nature 322: 747-748 (1987); およびDavies,D.R.ら, J.Biol.Chem. 263: 10541-10544 (1988)〕。即ち、結合に直接関与している残基は３次構造において近接しているが、結合に関与していない残基によって隔てられている。本発明により提供されるスクリーニング系は、さらに好都合にタンパク質−受容体の相互作用を分析し、新規かつ高親和性の結合性を有するタンパク質中の不連続エピトープを単離することを可能にするものである。
【００９８】
実施例 VII hＧＨコドン１７２、１７４、１７６、１７８においてランダム化したライブラリーからのhＧＨ突然変異体の選択
鋳型の構築
hＧＨ−遺伝子III融合タンパク質の突然変異体は、Kunkelら〔Meth.Enzymol. 154: 367-382 (1987)〕の方法を用いて構築した。鋳型ＤＮＡは、Ｍ１３-Ｋ０７ファージをヘルパーとして加えて、プラスミドpＳ０１３２（Ｍ１３遺伝子IIIのカルボキシ末端側半分に融合させた天然hＧＨ遺伝子をアルカリホスファターゼプロモーターの支配下に含有する）をＣＪ２３６細胞中で増殖させることによって調製した。１本鎖のウラシル含有ＤＮＡを突然変異誘発用に調製して、(1)hＧＨ結合タンパク質（hＧＨbp）に対する結合を大きく減少させるhＧＨ中の突然変異；および(2)親のバックグラウンドファージを検定し、それに対して選択するために使用しうる唯一の制限部位（ＫpnＩ）を導入した。Ｔ７ ＤＮＡポリメラーゼおよび以下のオリゴデオキシヌクレオチド：

を用いてオリゴヌクレオチド指向性の突然変異誘発を行なった。このオリゴは、hＧＨ中に突然変異（Ｒ１７８Ｇ、Ｉ１７９Ｔ）と共にＫpnＩ部位（上に示す）を導入する。突然変異誘発からのクローンをＫpnＩ消化によってスクリーニングし、ジデオキシＤＮＡ配列決定によって確認した。ランダム突然変異誘発の鋳型として使用されるこの得られた構築物をｐHＯ４１５と命名した。
【００９９】
h ＧＨのヘリックス４中のランダム突然変異誘発
ここでもKunkelの方法を用いて、コドン１７２、１７４、１７６、１７８をhＧＨ中のランダム突然変異誘発の標的とした。上記のようにｐHＯ４１５から１本鎖鋳型を調製し、以下のオリゴ：

のプールを用いて突然変異誘発を行なった。上に示したように、このオリゴプールはコドン１７９を野生型（Ｉle）に復帰させ、ｐH０４１５の唯一のＫpnＩ部位を破壊し、そして１７２、１７４、１７６および１７８位にランダムコドン（ＮＮＳ；ここで、ＮはＡ、Ｇ、ＣまたはＴであり、ＳはＧまたはＣである）を導入する。上記の配列に対してこのコドン選択を用いて、付加的なＫpnＩ部位を創製することはできない。このＮＮＳ同義性配列の選択により、４つの部位に３２種の可能なコドン（１つの「停止」コドンおよび少なくとも１つの各アミノ酸のためのコドン）が得られ、合計（３２）⁴＝１,０４８,５７６の可能なヌクレオチド配列（この１２％は少なくとも１つの停止コドンを含有する）、または（２０）⁴＝１６０,０００の可能なポリペプチド配列と３４,４８１の未成熟で終止した配列（即ち、少なくとも１つの停止コドンを含有する配列）が得られる。
【０１００】
最初のライブラリーの増殖
突然変異誘発産物をフェノール：クロロホルム（５０：５０）で２回抽出し、後の電気穿孔工程を面倒にする塩の添加を避けるために過剰のキャリアーtＲＮＡを用いてエタノール沈澱させた。約５０ng（１５fモル）のＤＮＡを、０.２cmキュベット中の全容量４５μlにおいて、単一パルスの電圧設定２.４９kＶ（時間定数＝４.７m秒）でＷＪＭ１０１細胞（２.８ｘ１０¹⁰細胞／ml）に電気穿孔導入した。
【０１０１】
この細胞を振盪しながら３７℃で１時間回復させ、次いで２ＹＴ培地（２５ml）、１００μg/mlカルベニシリン、およびＭ１３-Ｋ０７（感染の多重度＝１０００）と混合した。この培養物からの連続希釈物をカルベニシリン含有培地にプレーティングすることにより、８.２ｘ１０⁶の電気形質転換体が得られたことがわかった。２３℃で１０分間の後、培養物を振盪しながら３７℃で一晩（１５時間）インキュベートした。
【０１０２】
一晩インキュベートの後、細胞をペレット化し、pＬＩＢ１と命名した２本鎖ＤＮＡ（dsＤＮＡ）をアルカリ溶解法によって調製した。この上清をもう一度遠心してすべての残存細胞を除去し、ファージプールφ１と命名したファージをＰＥＧ沈澱させ、ＳＴＥ緩衝液〔１０mMトリス（ｐH７.６）、１mM ＥＤＴＡ、５０mM ＮaＣl〕(１ml）に再懸濁した。ファージ力価は、hＧＨ-g３p遺伝子III融合（hＧＨ-g³）プラスミドを含有する組換えファージミドについてはコロニー形成単位（ＣＦＵ）として、そしてＭ１３-Ｋ０７ヘルパーファージについてはプラーク形成単位（ＰＦＵ）として測定した。
【０１０３】
固定化 h ＧＨ bp を用いる結合選択
１．結合：ファージプールφ１（６ｘ１０⁹ＣＦＵ、６ｘ１０⁷ＰＦＵ）の一部を緩衝液Ａ〔リン酸緩衝食塩水、０.５％ＢＳＡ、０.０５％ツイーン２０、０.０１％チメロサール〕で４.５倍に希釈し、１.５mlのシラン処理したポリプロピレン試験管において、Ｓer２３７Ｃys突然変異を含むhＧＨbp（３５０fモル）と結合させたオキシラン-ポリアクリルアミドビーズの懸濁液（５μl）と混合した。対照として、等量のファージを、ＢＳＡだけで被覆したビーズと別の試験管で混合した。このファージを、ゆっくり回転（約７rpm）させながら室温（２３℃）で３時間インキュベートすることによって、ビーズと結合させた。以下の工程は、２００μlの一定容量を用い、室温で行なった。
【０１０４】
２．洗浄：ビーズを１５秒間回転させ、上清を除去した（上清１）。非特異的に結合したファージ／ファージミドを除去するため、このビーズを緩衝液Ａに再懸濁することにより２回洗浄し、次いでペレット化した。最後の洗浄は、緩衝液Ａ中で２時間、このビーズを回転させることにより行なった。
【０１０５】
３．hＧＨ溶離：ビーズに弱く結合したファージ／ファージミドを、hＧＨによる段階溶離によって除去した。最初の工程において、ビーズを２nM hＧＨ含有の緩衝液Ａを用いて回転させた。１７時間後に、このビーズをペレット化し、２０nM hＧＨ含有の緩衝液Ａに再懸濁し、３時間回転させ、次いでペレット化した。最後のhＧＨ洗浄において、このビーズを２００nM hＧＨ含有の緩衝液Ａに懸濁させ、３時間回転させ、次いでペレット化した。
【０１０６】
４．グリシン溶離：最も強固に結合したファージミド（即ち、hＧＨ洗浄後になお結合しているファージミド）を除去するため、ビーズをグリシン緩衝液〔１Mグリシン、ＨＣlでｐH２.０〕に懸濁させ、２時間回転させ、ペレット化した。この上清（分画「Ｇ」；２００μl）を、１Mトリス塩基（３０μl）の添加によって中和した。
【０１０７】
hＧＨbp-ビーズから溶出した分画Ｇ（１ｘ１０⁶ＣＦＵ、５ｘ１０⁴ＰＦＵ）は、実質的にＫ０７ヘルパーファージ以上にファージミドに富むことはなかった。我々は、これは、組換えファージミドの増殖中にパッケージされたＫ０７ファージがhＧＨ-g３p融合体を表示するということに起因するものと考えている。
【０１０８】
しかし、ＢＳＡ被覆された対照ビーズから溶出した分画Ｇと比較すると、hＧＨbp-ビーズは１４倍高いＣＦＵを与えた。これは、非特異的に結合したファージミド以上の、強固に結合したhＧＨを表示するファージミドの豊富化を反映するものである。
【０１０９】
５．増殖：hＧＨbp-ビーズから溶出した分画Ｇの一部（４.３ｘ１０⁵ＣＦＵ）を用いて対数増殖期のＷＪＭ１０１細胞を感染させた。形質導入は、分画Ｇ（１００μl）をＷＪＭ１０１細胞（１ml）と混合し、３７℃で２０分間インキュベートし、次いでＫ０７（感染の多重度＝１０００）を加えることによって行なった。培養物（２５mlの２ＹＴとカルベニシリン）を上記のように増殖させ、ファージの第２プール（ライブラリー１Ｇ、第１のグリシン溶離用）を上記のように調製した。
【０１１０】
ライブラリー１Ｇからのファージ（図３）を、上記のように、hＧＨbpビーズへの結合について選択した。hＧＨbpビーズから溶出した分画Ｇは、この選択においてＢＳＡビーズから溶出した分画Ｇに比べて３０倍高いＣＦＵを含んでいた。もう一度、分画Ｇの一部をＷＪＭ１０１細胞中で増殖させ、ライブラリー１Ｇ²（グリシン溶離によってこのライブラリーを２回選択したことを示す）を得た。また、２本鎖ＤＮＡ（pＬＩＢ １Ｇ²）をこの培養物から調製した。
ds ＤＮＡのＫ pn Ｉ検定および制限選択
【０１１１】
バックグラウンド（ＫpnＩ⁺）鋳型の量を減少させるため、pＬＩＢ １Ｇ²の一部（約０.５μg）をＫpnＩで消化し、ＷＪＭ１０１細胞中に電気穿孔導入した。これらの細胞を、最初のライブラリーについて記載したようにＫ０７（感染の多重度＝１００）の存在下で増殖させ、新しいファージプールpＬＩＢ３を調製した（図３）。
【０１１２】
さらに、最初のライブラリー（pＬＩＢ１）由来のdsＤＮＡの一部（約０.５μg）をＫpnＩで消化し、ＷＪＭ１０１細胞中に直接電気穿孔導入した。形質転換体を上記のように回復させ、Ｍ１３-Ｋ０７に感染させ、一晩増殖させてファージライブラリー２と命名した新しいファージライブラリーを得た（図３）。
【０１１３】
連続ラウンドの選択
(1)過剰（ＣＦＵの１０倍）の精製Ｋ０７ファージ（hＧＨを表示しない）をビーズ結合カクテル中に添加した；および(2)hＧＨ段階溶離を緩衝液Ａ単独の短い洗浄に換えたことを除き、上記のようにしてpＬＩＢ２およびpＬＩＢ３の両方に由来するファージミドに対して連続ラウンドでファージミドの結合、溶離および増殖を行なった。また、一部においてはＸＬ１-Ｂlue細胞をファージミドの増殖に用いた。
【０１１４】
ＫpnＩによるdsＤＮＡの追加の消化を、最終ラウンドのビーズ結合選択の前にpＬＩＢ ２Ｇ³およびpＬＩＢ ３Ｇ⁵に対して実施した（図３）。
【０１１５】
選択したファージミドのＤＮＡ配列決定
ＬＩＢ ４Ｇ⁴由来の４種の独立して単離したクローンおよびＬＩＢ ５Ｇ⁶由来の４種の独立して単離したクローンを、ジデオキシ配列決定によって配列決定した。これら８種クローンのすべてが、同一のＤＮＡ配列：

を有していた。即ち、これらのすべてがhＧＨの同一突然変異体（Ｅ１７４Ｓ、Ｆ１７６Ｙ）をコードしている。これらクローン中の残基１７２は野生型と同様にＬysである。また、１７２に対して選択されたコドンも野生型hＧＨと同一である。このことは、ＡＡＧが同義「ＮＮＳ」コドンのセットから可能な唯一のリジン-コドンであるので驚くには当たらない。さらに、残基１７８-Ａrgも野生型と同一であるが、ここではライブラリーから選択されたコドンはＡＡＧであって野生型hＧＨで見い出されるＣＧＣではなかった（後者コドンも「ＮＮＳ」コドンのセットを用いて可能である）。
【０１１６】
Ｋ０７感染の多重度
Ｋ０７感染の感染多重度は、組換えファージミドの増殖において重要なパラメーターである。Ｋ０７の感染多重度は、ファージミドで形質転換またはトランスフェクションされた実質的にすべての細胞が新しいファージミド粒子をパッケージしうることを確保するに十分に高いものでなければならない。さらに、それぞれの細胞中の野生型遺伝子IIIの濃度は高く維持されて、複数のhＧＨ−遺伝子III融合分子がそれぞれのファージミド粒子上に表示される可能性を減少させ、それによって結合におけるキレート作用を減少させるものであるべきである。しかし、Ｋ０７の感染多重度が高すぎるときには、Ｋ０７のパッケージングは組換えファージミドのパッケージングと競合するであろう。我々は、１〜１０％のバックグラウンドＫ０７ファージしか持たない許容しうるファージミド収量が、Ｋ０７の感染多重度が１００であるときに得られることを見い出した。
【０１１７】

【０１１８】
ファージプールを先に示したように標識した（図３）。感染多重度（moi）はファージミドの増殖の際のＫ０７感染の多重度（ＰＦＵ／細胞）を意味する。ＰＦＵを越えるＣＦＵの豊富化は、精製したＫ０７を結合工程に加えたときに示される。ＢＳＡビーズから溶出したＣＦＵを越える、hＧＨbpビーズから溶出されるＣＦＵの比が示されている。プール中に残存するＫpnＩ含有鋳型（即ち、ｐH０４１５）の分画は、dsＤＮＡをＫpnＩおよびＥcoＲＩで消化し、生成物を１％アガロースゲルで移動させ、陰性の臭化エチジウム染色ＤＮＡをレーザー-スキャニングすることによって測定した。
【０１１９】
ホルモン h ＧＨ（Ｅ１７４Ｓ、Ｆ１７６Ｙ）の受容体結合の親和性
単一のクローンが２種類の異なる選択経路（図３）から単離されたという事実は、二重突然変異体（Ｅ１７４Ｓ、Ｆ１７６Ｙ）がhＧＨbpに強く結合することを示唆した。hＧＨbpに対するこのhＧＨ突然変異体の親和性を測定するために、我々はこのhＧＨ突然変異体を、プラスミドpＢ０７２０を用いる部位指向性突然変異誘発によって構築した。このプラスミドは、鋳型としての野生型hＧＨ遺伝子、ならびにコドン１７４および１７６を変化させる次のオリゴヌクレオチド：

を含有している。得られた構築物ｐH０４５８Ｂを、突然変異ホルモンの発現のために大腸菌株１６Ｃ９に導入した。hＧＨbpに対するhＧＨ（Ｅ１７４Ｓ、Ｆ１７６Ｙ）と¹²⁵Ｉ-hＧＨの競合結合のスカッチャード分析により、この（Ｅ１７４Ｓ、Ｆ１７６Ｙ）突然変異体が野生型hＧＨの結合親和性よりも少なくとも５.０倍強固な結合親和性を有していることがわかった。
【０１２０】
実施例 VIII ホルモン−ファージのヘリックス４ランダムカセットライブラリーからのhＧＨ変異体の選択
ヒト成長ホルモン変異体を、図９に記載のファージミドを用いる本発明の方法によって調製した。
【０１２１】
脱 - 融合性のホルモン−ファージベクターの構築
我々は、(1)ファージ上でhＧＨ部分の表示をさらに有利にするように、hＧＨと遺伝子III部分の間の結合を改良する；(2)実質的に「一価の表示」を得るために融合タンパク質の発現を限定する；(3)出発ベクターに対する制限ヌクレアーゼの選択を可能にする；(4)出発ベクターからの融合タンパク質の発現を排除する；および(5)あるhＧＨ−遺伝子III融合突然変異体からの対応する遊離ホルモンの容易な発現を達成する；という目的をもって、カセット突然変異誘発〔Wellsら, Gene 34: 315-323 (1985)〕およびhＧＨ−遺伝子III融合タンパク質の発現のためのベクターを設計した。
【０１２２】
pＢＲ３２２およびf１の複製起点を含有し、大腸菌phoＡプロモーター〔Bassら, Proteins 8: 309-314 (1990)〕の支配下にhＧＨ−遺伝子III融合タンパク質（hＧＨ残基１〜１９１、これに１個のＧly残基が続き、遺伝子IIIのＰro-１９８に融合している）を発現する、pＳ０１３２のオリゴヌクレオチド指向性の突然変異誘発〔Kunkelら, Methods Enzymol. 154: 367-382 (1987)〕によってプラスミドpＳ０６４３を構築した（図９）。オリゴヌクレオチド：
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3'
を用いて突然変異誘発を行った〔このオリゴヌクレオチドは、hＧＨのＰhe-１９１に続いてＸbaＩ部位（下線部）およびアンバー停止コドン（ＴＡＧ）を導入する〕。得られた構築物pＳ０６４３においては、遺伝子IIIの一部が削除され、２つのサイレント突然変異誘発（下線部）が生じ、hＧＨと遺伝子IIIの間に次の連結点が生成する：
--- hＧＨ ---------> 遺伝子III ----->
187 188 189 190 191 am* 249 250 251 252 253 254
Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly
GGC AGC TGT GGA TTC TAG AGT GGC GGT GGC TCT GGT
これは、融合タンパク質の全体サイズをpＳ０１３２中の４０１残基からpＳ０６４３中の３５０残基まで短縮する。hＧＨに対するモノクローナル抗体を用いた実験により、ファージ粒子に組立てられる新しい融合タンパク質のhＧＨ部分は以前の長い融合体よりも接近しやすいことがわかった。
【０１２３】
ホルモンを表示するファージを増殖させるため、pＳ０６４３および誘導体をＪＭ１０１またはＸＬ１-Ｂlueなどの大腸菌のアンバー-サプレッサー株〔Bullockら, BioTechniques 5: 376-379 (1987)〕中で増殖させることができる。上に示したのは、supＥサプレッサー株中で起こるアンバーコドンにおけるＧluの置換である。また、他のアミノ酸による抑制が、周知かつ一般に入手可能な種々の大腸菌株において可能である。
【０１２４】
融合体の遺伝子III部分を含まないhＧＨ（または、突然変異体）を発現させるために、pＳ０６４３および誘導体を単に１６Ｃ９などの非サプレッサー株において増殖させることができる。この場合にはアンバーコドン（ＴＡＧ）は翻訳の終止を導き、独立したＤＮＡ構築を必要とすることなく遊離のホルモンを与える。
【０１２５】
カセット突然変異誘発のための部位を創製するため、pＳ０６４３をオリゴヌクレオチド：
(1)5'-CGG-ACT-GGG-CAG-ATA-TTC-AAG-CAG-ACC-3'
〔これは、pＳ０６４３の唯一のＢglII部位を破壊する〕；
(2)5'-CTC-AAG-AAC-TAC-GGG-TTA-CCC-TGA-CTG-CTT-CAG-GAA-GG-3'〔これは、唯一のＢstＥII部位、１塩基のフレームシフト、および非アンバー停止コドン（ＴＧＡ）を挿入する〕；および
(3)5'-CGC-ATC-GTG-CAG-TGC-AGA-TCT-GTG-GAG-GGC-3'
〔これは、新規なＢglII部位を導入する〕
で突然変異させて出発ベクターｐH０５０９を得た。ＴＧＡ停止コドンと共にフレームシフトを加えることにより、出発ベクターから遺伝子III融合体が産生され得ないことが確保される。ＢstＥII−ＢglIIセグメントをｐH０５０９から切出し、所望のコドンのところで突然変異させたＤＮＡカセットで置換した。また、hＧＨ中の他の位置におけるカセット突然変異誘発のための他の制限部位も、このホルモン−ファージベクター中に導入した。
【０１２６】
h ＧＨのヘリックス４内のカセット突然変異誘発
hＧＨのコドン１７２、１７４、１７６および１７８をランダム突然変異誘発の標的とした。この理由は、これらすべてがhＧＨの表面またはその近くに存在し、受容体結合に有意に寄与し〔CunninghamおよびWells, Science 244: 1081-1085 (1989)〕；これらすべてが十分に定義された構造内に存在し、ヘリックス４の同じ側の２「ターン」を占め；そして、これらのそれぞれがhＧＨの既知の進化変異体の中の少なくとも１つのアミノ酸で置換されているためである。
【０１２７】
我々は、それぞれの標的残基にＮＮＳ（Ｎ＝Ａ／Ｇ／Ｃ／Ｔ；Ｓ＝Ｇ／Ｃ）の置換を選択した。このＮＮＳ同義性配列の選択により、４つの部位に３２種の可能なコドン（少なくとも１つの各アミノ酸のためのコドン）が得られ、合計（３２）⁴＝１,０４８,５７６の可能なヌクレオチド配列、または（２０）⁴＝１６０,０００の可能なポリペプチド配列が得られる。唯一の停止コドンであるアンバー（ＴＡＧ）がこのコドン選択により可能であり、このコドンは大腸菌のsupＥ株においてＧlu１として抑制することができる。
【０１２８】
コドン１７２、１７４、１７６および１７８にＮＮＳを有する２種類の同義性（縮重）オリゴヌクレオチドを合成し、ホスホリル化し、アニールさせて突然変異カセットを構築した：
5'-GT-TAC-TCT-ACT-GCT-TTC-AGG-AAG-GAC-ATG-GAC-NNS-GTC-NNS-ACA-NNS-CTG-NNS-ATC-GTG-CAG-TGC-A-3'
および
5'-GA-TCT-GCA-CTG-CAC-GAT-SNN-CAG-SNN-TGT-SNN-GAC-SNN-GTC-CAT-GTC-CTT-CCT-GAA-GCA-GTA-GA-3' 。
ＢstＥIIに続いてＢglIIでｐH０５０９を消化することによってベクターを調製した。生成物を１％アガロースゲル上で移動させ、大きいフラグメントを切り出し、フェノール抽出し、エタノール沈澱させた。このフラグメントをウシ腸ホスファターゼ（Boehringer）で処理し、次いでフェノール：クロロホルム抽出し、エタノール沈澱させ、突然変異用カセットとの連結用に再懸濁した。
【０１２９】
ＸＬ１ - Ｂ lue 細胞における最初のライブラリーの増殖
連結の後、反応生成物をＢstＥIIでもう一度消化し、次いでフェノール：クロロホルム抽出し、エタノール沈澱させ、水に再懸濁した〔ＢstＥII認識部位（ＧＧＴＮＡＣＣ）が、コドン１７２の３位にＧおよび１７４にＡＣＣ（Ｔhr）コドンを含むカセット内に創製される。しかし、この工程でのＢstＥIIによる処理は、あらゆる可能な突然変異カセットに対して選択しないはずである。この理由は、実質的にすべてのカセットがヘテロ２本鎖であり、この酵素によって切断され得ないためである〕。約１５０ng（４５fモル）のＤＮＡを、０.２cmキュベット中のＸＬ１-Ｂlue細胞（０.０４５ml中に１.８ｘ１０⁹細胞）に、単一パルスの電圧設定２.４９kＶ（時間定数＝４.７m秒）で電気穿孔導入した。
【０１３０】
この細胞を振盪しながらＳ.Ｏ.Ｃ.培地において３７℃で１時間回復させ、次いで２ＹＴ培地（２５ml）、１００mg/mlカルベニシリン、およびＭ１３-Ｋ０７（moi＝１００）と混合した。２３℃で１０分間の後、培養物を振盪しながら３７℃で一晩（１５時間）インキュベートした。この培養物からの連続希釈物をカルベニシリン含有培地にプレーティングすることにより、３.９ｘ１０⁷の電気形質転換体が得られたことがわかった。
【０１３１】
一晩インキュベートの後、細胞をペレット化し、ｐH０５２９Ｅ（最初のライブラリー）と命名した２本鎖ＤＮＡ（dsＤＮＡ）をアルカリ溶解法によって調製した。この上清をもう一度遠心してすべての残存細胞を除去し、ファージプールφＨ０５２９Ｅ（最初のファージライブラリー）と命名したファージをＰＥＧ沈澱させ、ＳＴＥ緩衝液〔１０mMトリス（ｐH７.６）、１mM ＥＤＴＡ、５０mM ＮaＣl〕(１ml）に再懸濁した。ファージ力価は、hＧＨ-g３pを含有する組換えファージミドについてコロニー形成単位（ＣＦＵ）として測定した。出発ライブラリーから約４.５ｘ１０¹³ＣＦＵが得られた。
【０１３２】
出発ライブラリーの同義性（縮重）
電気形質転換体のプールからの５８のクローンを、ＢstＥII−ＢglIIカセットの領域において配列決定した。これらの中で、１７％が出発ベクターに対応しており、１７％が少なくとも１つのフレームシフトを含有しており、そして７％が４つの標的コドンの外側に非サイレント（非終止）突然変異を含有していた。我々は、４１％のクローンが上記基準のいずれかによる欠点を有しており、２.０ｘ１０⁷の当初形質転換体の合計機能的プールが残るものと結論した。それでもなお、この数は可能性あるＤＮＡ配列数をほぼ２０倍越えている。従って、我々は出発ライブラリーにおいて示されるすべての可能性ある配列を有することを確信している。
【０１３３】
我々は、選択されなかったファージの配列を調べて突然変異誘発におけるコドンの偏向度を評価した（表Ｖ）。これらの結果により、一部のコドン（および、アミノ酸）がランダムな予想に比べて多いかまたは少ないことが示されるが、このライブラリーは極めて多様性が高く、大量の「兄弟」縮重の証拠は存在しないことがわかった（表VI）。
【０１３４】
表Ｖ．選択されなかったホルモンファージのコドン分布（１８８コドンあたり）クローンを出発ライブラリー（ｐH０５２９Ｅ）から配列決定した。疑似突然変異および／またはフレームシフトを含有するクローンからのコドンを含むすべてのコドンを表にした。＊：アンバー停止コドン（ＴＡＧ）はＸＬ１-Ｂlue細胞においてはＧluとして抑制される。下線を引いたコドンは、５０％またはそれ以上に多い／少ないことが示された。

【０１３５】
表 VI．オープン読み枠を有する選択されなかった
（ｐH０５２９Ｅ）クローン
表示、例えばＴＷＧＳは、hＧＨ突然変異体１７２Ｔ／１７４Ｗ／１７６Ｇ／１７８Ｓを示す。ＸＬ１-Ｂlue細胞においてＧluとして翻訳されたアンバー（ＴＡＧ）コドンはεで示す。

【０１３６】
固定化 h ＧＨ bp および h ＰＲＬ bp の調製
野生型hＧＨbp〔Fuhら, J.Biol.Chem. 265: 3111-3115 (1990)〕を用いたことを除き、文献〔Bassら, Proteins 8: 309-314 (1990)〕に記載のようにして固定化hＧＨbp（「hＧＨbp-ビーズ」）を調製した。〔¹²⁵Ｉ〕hＧＨとの競合結合実験により、１μlのビーズ懸濁液あたり５８fモルの機能的hＧＨbpが結合することがわかった。
【０１３７】
プロラクチン受容体〔Cunninghamら, Science 250: 1709-1712 (1990)〕の２１１残基の細胞外ドメインを用いて、上記のように固定化hＰＲＬbp（「hＰＲＬbp-ビーズ」）を調製した。５０μMの亜鉛の存在下での〔¹²⁵Ｉ〕hＧＨとの競合結合実験により、１μlのビーズ懸濁液あたり２.１fモルの機能的hＰＲＬbpが結合することがわかった。
【０１３８】
「ブランクビーズ」は、オキシラン-アクリルアミドビーズを０.６Ｍエタノールアミン（ｐH９.２）により４℃で１５時間処理することによって調製した。
【０１３９】
固定化 h ＧＨ bp および h ＰＲＬ bp を用いる結合選択
ビーズに対するホルモン−ファージの結合を、次の緩衝液のいずれかにおいて実施した：hＧＨbpおよびブランクビーズを用いる選択に対しては緩衝液Ａ〔ＰＢＳ、０.５％ ＢＳＡ、０.０５％ツイーン２０、０.０１％チメロサール〕；亜鉛の存在下（＋Ｚn²⁺）にhＰＲＬbpを用いる選択に対しては緩衝液Ｂ〔５０mMトリス（ｐH７.５）、１０mM ＭgＣl₂、０.５％ ＢＳＡ、０.０５％ツイーン２０、１００mM ＺnＣl₂〕；または、亜鉛の非存在下（＋ＥＤＴＡ）にhＰＲＬbpを用いる選択に対しては緩衝液Ｃ〔ＰＢＳ、０.５％ ＢＳＡ、０.０５％ツイーン２０、０.０１％チメロサール、１０mM ＥＤＴＡ〕。結合選択は、次の経路のそれぞれに従って実施した：(1)ブランクビーズに対する結合、(2)hＧＨbp-ビーズに対する結合、(3)hＰＲＬbp-ビーズに対する結合（＋Ｚn²⁺）、(4)hＰＲＬbp-ビーズに対する結合（＋ＥＤＴＡ）、(5)hＧＨbpビーズによる２回の予備吸着とその後の未吸着分画のhＰＲＬbp-ビーズへの結合（「−hＧＨbp、＋hＰＲＬbp」選択）、または(6)hＰＲＬbp-ビーズによる２回の予備吸着とその後の未吸着分画のhＧＨbp-ビーズへの結合（「−hＰＲＬbp、＋hＧＨbp」選択）。後者の２つの操作は、それぞれhＰＲＬbpと結合するがhＧＨbpとは結合しない突然変異体、またはhＧＨbpと結合するがhＰＲＬbpとは結合しない突然変異体を豊富化するものと予想される。各サイクルにおけるファージの結合と溶離は、次のように実施した：
【０１４０】
１．結合：ホルモンファージの一部（通常は１０⁹〜１０¹⁰ＣＦＵ）を等量の非ホルモンファージ（pＣＡＴ）と混合し、適当な緩衝液（Ａ、ＢまたはＣ）で希釈し、１.５mlのポリプロピレン試験管において２００mlの総容量でhＧＨbp、hＰＲＬbpまたはブランクビーズの懸濁液（１０ml）と混合した。このファージを、ゆっくり回転（約７rpm）させながら室温（２３℃）で１時間インキュベートすることによってビーズと結合させた。以下の工程は、２００μlの一定容量を用い、室温で行なった。
【０１４１】
２．洗浄：ビーズを１５秒間回転させ、上清を除去した。非特異的に結合したファージ数を減少させるため、このビーズを適当な緩衝液に短時間再懸濁することにより５回洗浄し、次いでペレット化した。
【０１４２】
３．hＧＨ溶離：ビーズに弱く結合したファージを、hＧＨによる溶離によって除去した。このビーズを４００nM hＧＨ含有の適当な緩衝液と共に１５〜１７時間回転させた。この上清を「hＧＨ溶出液」およびビーズとした。このビーズを緩衝液に短時間再懸濁し、ペレット化することによって洗浄した。
【０１４３】
４．グリシン溶離：最も強固に結合したファージ（即ち、hＧＨ洗浄後になお結合しているファージ）を除去するため、ビーズをグリシン緩衝液〔緩衝液Ａに０.２Mグリシンを追加し、ＨＣlでｐH２.０にする〕に懸濁させ、１時間回転させ、ペレット化した。この上清（「グリシン溶出液」；２００μl）を、１Mトリス塩基（３０ml）の添加によって中和し、４℃で保存した。
【０１４４】
５．増殖：各組の条件のもと各組のビーズからのhＧＨ溶出液およびグリシン溶出液の一部を用いて対数増殖期のＸＬ１-Ｂlue細胞の独立した培養物を感染させた。形質導入は、ファージをＸＬ１-Ｂlue細胞（１ml）と混合し、３７℃で２０分間インキュベートし、次いでＫ０７（moi＝１００）を加えることによって行なった。培養物（２５mlの２ＹＴとカルベニシリン）を上記のように増殖させ、ファージの次のプールを上記のように調製した。
【０１４５】
ファージの結合、溶離および増殖を、上記のサイクルに従って、連続ラウンドで行なった。例えば、hＧＨbpビーズからのhＧＨ溶出液から増幅したファージをhＧＨbpビーズでもう一度選択し、hＧＨで溶離し、次いでＸＬ１-Ｂlue細胞の新鮮培養物の感染に用いた。上記の選択法のそれぞれに対して５ラウンドの選択および増殖の３ラウンドを実施した。
【０１４６】
選択したファージミドのＤＮＡ配列決定
各サイクルのhＧＨおよびグリシン溶離工程からのファージの一部をＸＬ１-Ｂlue細胞の接種に用い、これをカルベニシリンおよびテトラサイクリン含有のＬＢ培地にプレーティングして各ファージプールに由来する独立したクローンを得た。単離したコロニーから１本鎖ＤＮＡを調製し、突然変異カセット領域の配列決定を行なった。このＤＮＡ配列決定の結果を、図５、図６、図７および図８に推定アミノ酸配列としてまとめる。
【０１４７】
h ＧＨ突然変異体の発現および検定
hＧＨbpに対する選択したhＧＨ突然変異体の一部の結合親和性を測定するため、配列決定したクローン由来のＤＮＡを大腸菌株１６Ｃ９に導入した。上記のように、この株は非サプレッサー株であり、hＧＨの最後のＰhe-１９１残基の後でタンパク質の翻訳を終止させる。１本鎖ＤＮＡをこれらの形質転換に用いたが、２本鎖ＤＮＡまたは全ファージであっても、遊離ホルモンの発現用の非サプレッサー株中に容易に電気穿孔導入することができる。
【０１４８】
hＧＨの突然変異体は、hＧＨについて記載されているように、硫酸アンモニウム沈澱により浸透圧ショックを与えた細胞から調製した〔Olsonら, Nature 293: 408-411 (1981)〕。また、タンパク質濃度は、hＧＨを標準として用いて、クーマシー染色ＳＤＳ-ポリアクリルアミドゲル電気泳動ゲルのレーザー・デンシトメトリーによって測定した〔CunninghamおよびWells, Science 244: 1081-1085 (1989)〕。
【０１４９】
各突然変異体の結合親和性は、抗-受容体モノクローナル抗体（Ｍab２６３）を用い、文献〔Spencerら, J.Biol.Chem. 263: 7862-7867 (1988); Fuhら, J.Biol.Chem. 265: 3111-3115 (1990)〕記載のように¹²⁵Ｉ hＧＨを置換することによって測定した。
【０１５０】
異なる経路（図６）によって選択したhＧＨ突然変異体数の結果を表VIIに示す。これら突然変異体の多くが、hＧＨbpに対して野生型hＧＨよりも強い結合親和性を有している。最も改善された突然変異体であるＫＳＹＲは、野生型hＧＨよりも５.６倍高い結合親和性を有している。これら検定した突然変異体の中で選択された最も弱い突然変異体は、hＧＨに比べて結合親和性が約１０倍低いものでしかなかった。
【０１５１】
結合検定は、hＰＲＬbp-結合に対して選択した突然変異体について実施することもできる。
【０１５２】
表 VII. hＧＨbpに対する競合結合
それぞれの突然変異体が見い出された選択したプールを、１Ｇ（第１のグリシン選択）、３Ｇ（第３のグリシン選択）、３Ｈ（第３のhＧＨ選択）、３*（第３の選択：hＰＲＬbpに結合しなかったがhＧＨbpに結合した）と表示する。配列決定したすべてのクローンの中に現れる各突然変異体の回数を（ ）に示す。

【０１５３】
結合に及ぼす加成および非加成効果
一部の残基において、特定アミノ酸の置換が周囲残基とは独立した実質的に同じ作用を有している。例えば、１７２Ｒ／１７４ＳのバックグラウンドにおいてＦ１７６Ｙの置換は結合親和性を２.０倍減少させる（ＲＳＦＲ対ＲＳＹＲ）。同様に、１７２Ｋ／１７４ＡのバックグラウンドにおいてＦ１７６Ｙ突然変異体（ＫＡＹＲ）の結合親和性は、対応する１７６Ｆ突然変異体〔ＫＡＦＲ；CunninghamおよびWells (1989)〕よりも２.９倍弱い。
【０１５４】
他方、いくつかの選択したhＧＨ突然変異体について測定した結合定数は、残基１７２、１７４、１７６および１７８における一部のアミノ酸置換の非加成性の効果を示す。例えば、１７２Ｋ／１７６Ｙのバックグラウンドにおいて、Ｅ１７４Ｓの置換は、Ｅ１７４Ａを含有する対応する突然変異体（ＫＡＹＲ）よりも３.７倍強固にhＧＨbpと結合する突然変異体（ＫＳＹＲ）を与える結果になる。しかし、１７２Ｒ／１７６Ｙのバックグラウンドにおいて、これらＥ１７４置換の効果は逆になる。この場合、Ｅ１７４Ａ突然変異体（ＲＡＹＲ）はＥ１７４Ｓ突然変異体（ＲＳＹＲ）よりも１.５倍強固に結合する。
【０１５５】
このような近接残基における置換の結合に及ぼす非加成性効果は、いくつかの位置においてランダム化されたライブラリーから最適突然変異体を選択する手段としてのタンパク質-ファージ結合選択の用途を示すものである。詳細な構造の情報が存在せず、このような選択法がないと、最適突然変異体の発見までに多数の置換の組合せが試されることになるであろう。
【０１５６】
実施例 IX ホルモン−ファージのヘリックス１ランダムカセットライブラリーからのhＧＨ突然変異体の選択
【０１５７】
実施例VIIIに記載した方法を用いて、hＧＨbpおよび／またはhＰＲＬbpへの結合に関与しているhＧＨの別の領域、即ちヘリックス１の残基１０、１４、１８、２１をphＧＨam-g３pベクター（pＳ０６４３としても知られている；実施例VIIIを参照）におけるランダム突然変異誘発の標的とした。
【０１５８】
我々は「アンバー」hＧＨ-g３構築物（phＧＨam-g３pと呼ぶ）の使用を選択したが、この理由はそれが結合に対してより接近しやすい標的タンパク質hＧＨを作成するようであるためである。このことは、モノクローナル抗体結合の比較ＥＬＩＳＡ検定からのデータによって支持される。pＳ０１３２〔S.Bass, R.Greene, J.A.Wells, Proteins 8: 306 (1990)〕およびphＧＨam-g３の両方から生成したファージを、hＧＨのカルボキシ末端の近くに結合決定基を有することが知られている３種の抗体（Medix２、１Ｂ５.Ｇ２、および５Ｂ７.Ｃ１０）〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989); B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989); L.JinおよびJ.Wells（未公表の結果）〕、ならびにヘリックス１および３中の決定基を認識する１種の抗体（Medix１）〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989); B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)〕を用いて試験した。phＧＨam-g３からのファージミド粒子は、pＳ０１３２からのファージミド粒子よりも抗体Medix２、１Ｂ５.Ｇ２および５Ｂ７.Ｃ１０とさらに強く反応した。特に、pＳ０１３２粒子の結合は、Medix１に対する結合と比較して、Medix２および５Ｂ７.Ｃ１０の両方に対しては＞２０００倍減少し、１Ｂ５.Ｇ２に対しては＞２５倍減少した。一方、phＧＨam-g３ファージの結合は、Medix１に対する結合と比較して、Medix２、１Ｂ５.Ｇ２および５Ｂ７.Ｃ１０抗体に対してそれぞれ約１.５倍、１.２倍および２.３倍弱くなるにすぎなかった。
【０１５９】
カセット突然変異誘発によるヘリックス１ライブラリーの作成
アラニン-スキャニング突然変異誘発〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989); B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)，15, 16〕によって以前に同定されていたヘリックス１中の残基に突然変異を行なって、hＧＨおよび／またはhＰＲＬ受容体（それぞれ、hＧＨbpおよびhＰＲＬbpと呼ぶ）の細胞外ドメインの結合を変調させた。カセット突然変異誘発は実質的に文献〔J.A.Wells, M.Vasser, D.B.Powers, Gene 34: 315 (1985)〕記載のように行なった。このライブラリーは、オリゴヌクレオチド：
5'-GCC TTT GAC AGG TAC CAG GAG TTT G-3' および
5'-CCA ACT ATA CCA CTC TCG AGG TCT ATT CGA TAA C-3'
をそれぞれ用いる突然変異誘発によって唯一のＫpnＩ（hＧＨコドン２７のところ）およびＸhoＩ（hＧＨコドン６のところ）制限部位（下線部）が挿入されたphＧＨam-g３〔T.A.Kunkel, J.D.Roberts, R.A.Zakour, Methods Enzymol. 154: 367-382〕の突然変異体を用いる、４残基を一度にすべて突然変異させるカセット突然変異誘発（実施例VIIIを参照）によって作成した。また、後者のオリゴは＋１のフレームシフト（下線部の最後のＧ）を導入し、これが出発ベクターからの翻訳を終止させ、ファージミドライブラリー中の野生型のバックグラウンドを最少にした。この出発ベクターをｐH０５０８Ｂと命名した。hＧＨ残基１０、１４、１８、２１を突然変異させたヘリックス１ライブラリーは、相補性オリゴヌクレオチド：
5'-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3' および
5'-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3'
から調製したカセットを、ｐH０５０８Ｂの大きいＸhoＩ−ＫpnＩフラグメントに連結することによって作成した。このＫpnＩ部位は連結生成物の連結部において破壊され、非突然変異バックグラウンドの分析に制限酵素消化を使用することを可能にした。
【０１６０】
このライブラリーは少なくとも１０⁷の別個の形質転換体を含んでおり、このライブラリーが完全にランダム（１０⁶の異なるコドン組合せ）であるときには、平均して約１０コピーのそれぞれ可能な突然変異hＧＨ遺伝子が得られる。ＫpnＩを用いる制限分析により、ヘリックス１ライブラリー構築物の少なくとも８０％が挿入されたカセットを含有していることがわかった。
【０１６１】
このライブラリーからのhＧＨ-ファージの結合豊富化を、既述（実施例VIII）のようにオキシラン-ポリアクリルアミドビーズ（Sigma Chemical Co.）上に固定化したhＧＨbpを用いて行なった。ヘリックス１中の４つの残基（Ｆ１０、Ｍ１４、Ｈ１８およびＨ２１）が同じように突然変異され、４および６サイクル後に非野生型コンセンサスが現れた（表VIII）。ヘリックス１の疎水性面の１０位は疎水性である傾向にあり、一方、親水性面の２１および１８位は傾向的にＡsnが優勢であり、１４位には明白なコンセンサスが認められなかった（表IX）。
【０１６２】
hＧＨbpに対するこれらhＧＨ突然変異体の結合定数は、非サプレッサー大腸菌株１６Ｃ９中で遊離のホルモン変異体を発現させ、このタンパク質を精製し、そしてhＧＨbpからの標識化wt-hＧＨの競合置換により検定することによって測定した（実施例VIIIを参照）。示したように、いくつかの突然変異体がwt-hＧＨが結合するよりもhＧＨbpに強く結合する。
【０１６３】
表 VIII．hＧＨヘリックス１突然変異体の選択
ファージミド粒子上に発現されたhＧＨ変異体（残基Ｆ１０、Ｍ１４、Ｈ１８、Ｈ２１においてランダム突然変異）を、hＧＨbp-ビーズへの結合、およびhＧＨ（０.４mM）緩衝液と次いでグリシン（０.２M、ｐH２）緩衝液による溶離によって選択した（実施例VIIIを参照）。

【０１６４】
表 IX．選択したヘリックス１ライブラリーからのコンセンサス配列
観察された頻度は、表示したアミノ酸を有する配列決定した全クローンの分数である。名目頻度はＮＮＳの３２コドン縮重に基づいて計算した。最大豊富化因子は、ある残基に対する名目頻度値に依存して１１〜３２に変化する。単一のアラニン突然変異に対する〔Ｋd（Ａla突然変異体）／Ｋd（wt-hＧＨ）〕の値は、文献〔B.C.CunninghamおよびJ.A.Wells, Science 244: 1081(1989)；B.C.Cunningham, D.J.Henner, J.A.Wells, Science 247: 1461(1990)；B.C.CunninghamおよびJ.A.Wells, Proc.Natl.Acad.Sci.USA 88: 3407(1991)〕から採用した。

【０１６５】
表Ｘ．hＧＨbpに対する精製したhＧＨヘリックス１突然変異体の結合
競合結合実験は、〔¹²⁵Ｉ〕hＧＨ（野生型）、hＧＨbp（細胞外受容体ドメイン、残基１〜２３８を含む）、およびＭab２６３〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989)〕を用いて行なった。数値Ｐは、１またはそれ以上の選択ラウンド後に配列決定した全クローン中の各突然変異体の発生率を分数で示すものである。

【０１６６】
実施例Ｘ ホルモン−ファージの豊富化により予め見い出した突然変異を含むヘリックス４ランダムカセットライブラリーからのhＧＨ変異体の選択
ホルモン−ファージを用いる反復選択による改善された結合性を有する突然変異タンパク質の設計
hＧＨ進化変異体の新入の非結合相同体を用いる我々の実験から、多数の独立したアミノ酸置換を組合せることによって特定受容体との結合に関してhＧＨの累積して改善された突然変異体を得ることができると示唆される〔B.C.Cunningham, D.J.Henner, J.A.Wells, Science 247: 1461 (1990); B.C.CunninghamおよびJ.A.Wells, Proc.Natl.Acad.Sci.USA 88: 3407 (1991); H.B.Lowman, B.C.Cunningham, J.A.Wells, J.Biol.Chem. 266, 印刷中 (1991)〕。
【０１６７】
hＧＨ受容体にさらに強固に結合することがわかったヘリックス４aライブラリーから選択したヘリックス４の二重突然変異体（Ｅ１７４Ｓ／Ｆ１７６Ｙ；実施例VIIIを参照）をさらに改善する試みにおいて、ヘリックス４bライブラリーを作成した。Ｅ１７４Ｓ／Ｆ１７６Ｙ hＧＨ突然変異体をバックグラウンドの出発ホルモンとして用いて、ヘリックス４の親水性面上の１７４および１７６位の周りの残基に突然変異を行なった。
【０１６８】
カセット突然変異誘発によるヘリックス４ b ライブラリーの作成
実質的に文献〔J.A.Wells, M.Vasser, D.B.Powers, Gene 34: 315 (1985)〕記載のようにしてカセット突然変異誘発を行なった。唯一のＢstＥIIおよびＢglII制限部位が予め挿入されたphＧＨam-g３（実施例VIII）の突然変異体を用い、４残基を一度にすべて突然変異させるカセット突然変異誘発（実施例VIIIを参照）を用いて、Ｅ１７４Ｓ／Ｆ１７６Ｙバックグラウンド内で残基１６７、１７１、１７５および１７９を突然変異させたヘリックス４bライブラリーを作成した。相補性オリゴヌクレオチド：
5'-pG TTA CTC TAC TGC TTC NNS AAG GAC ATG NNS AAG GTC AGC NNS TAC CTG CGC NNS GTG CAG TGC A-3' および
5'-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC CTT SNN CAT GTC CTT SNN GAA GCA GTA GA-3'
から調製したカセットを、ベクターのＢstＥII−ＢglII部位に挿入した。このＢstＥII部位は連結したカセットにおいて削除された。このヘリックス４bライブラリー由来の１５の選択されなかったクローンを配列決定した。これらのうち、カセット挿入体を欠くものはなく、２０％がフレームシフトしており、７％が非サイレント突然変異を有していた。
【０１６９】
h ＧＨ bp 豊富化の結果
このライブラリーからのhＧＨ-ファージの結合豊富化を、既述（実施例VIII）のようにオキシラン-ポリアクリルアミドビーズ（Sigma Chemical Co.）上に固定化したhＧＨbpを用いて行なった。６サイクルの結合の後に、合理的かつ明白なコンセンサスが現れた（表XI）。興味あることに、すべての位置が極性の残基、特にＳer、ＴhrおよびＡsnを含有する傾向があった（表XII）。
【０１７０】
h ＧＨ突然変異体の検定
hＧＨbpに対するこれらhＧＨ突然変異体の一部の結合定数を、非サプレッサー大腸菌株１６Ｃ９中で遊離のホルモン変異体を発現させ、このタンパク質を精製し、そしてhＧＨbpからの標識化wt-hＧＨの競合置換により検定することによって測定した（実施例VIIIを参照）。示したように、いくつかのヘリックス４b突然変異体のhＧＨbpに対する結合親和性はwt-hＧＨのものよりも強固であった（表XIII）。
【０１７１】
h ＧＨ変異体の受容体選択性
最後に、我々は、hＰＲＬbpに結合する能力について、いくつかの一層強固なhＧＨbp結合突然変異体の結合親和性の分析を始めた。Ｅ１７４Ｓ／Ｆ１７６Ｙ突然変異体は、hＧＨに比べてhＰＲＬbpに２００倍弱く結合する。Ｅ１７４Ｔ／Ｆ１７６Ｙ／Ｒ１７８ＫおよびＲ１６７Ｎ／Ｄ１７１Ｓ／Ｅ１７４Ｓ／Ｆ１７６Ｙ／Ｉ１７９Ｔ突然変異体のそれぞれは、hＧＨに比べhＰＲＬbpに＞５００倍弱く結合する。即ち、ファージ表示法によってhＧＨの新規な受容体選択性突然変異体を得ることができる。
【０１７２】
ホルモン−ファージミド選択によって同定される特定残基の情報内容
３種のhＧＨ−ファージミドライブラリー（実施例VIII、IX、Ｘ）において突然変異させた１２残基のうち、４種が野生型残基の強い（他を排除するものではない）保存を示した（Ｋ１７２、Ｔ１７５、Ｆ１７６およびＲ１７８）。驚くべきことではないが、Ａlaに変換したときにhＧＨbpに対する結合親和性に最大の破壊（４〜６０倍）を引き起こす残基が存在した。Ａla置換が結合に対してより弱い作用（２〜７倍）を引き起こす一群の４個の他の残基（Ｆ１０、Ｍ１４、Ｄ１７１およびＩ１７９）が存在し、これらの位置はわずかの野生型コンセンサスしか示さなかった。最後に、hＰＲＬbpへの結合を促進したがhＧＨbpへの結合は促進しなかった別の４残基（Ｈ１８、Ｈ２１、Ｒ１６７およびＥ１７４）は、hＧＨbp-ビーズによる選択によって野生型hＧＨ配列に対してどのようなコンセンサスも示さなかった。事実、２つの残基（Ｅ１７４およびＨ２１）は、Ａla置換したときにhＧＨbpに対する結合親和性を２〜４倍に高める〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989)；B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)；B.C.Cunningham, D.J.Henner, J.A.Wells, Science 247: 1461 (1990)；B.C.CunninghamおよびJ.A.Wells, Proc.Natl.Acad.Sci.USA 88: 3407 (1991)〕。即ち、アラニン-スキャニング突然変異誘発データは、それぞれの位置を置換する可変性と十分合理的に関係している。事実、アラニン置換によって引き起こされる結合親和性の減少〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989)；B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)；B.C.Cunningham, D.J.Henner, J.A.Wells, Science 247: 1461 (1990)；B.C.CunninghamおよびJ.A.Wells, Proc.Natl.Acad.Sci.USA 88: 3407 (1991)〕は、３〜６ラウンドの選択後にファージミドプール中に野生型残基が見い出される割合を合理的に予言するものとなる。アラニン-スキャニングの情報は結合を変調させる側鎖を標的化するのに有用であり、ファージ選択は側鎖の最適化に、および置換のための各部位（および／または部位の組合せ）の可変性を定義するのに適している。スキャニング突然変異法〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989)；B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)〕とファージ表示の組合せは、受容体−リガンドの界面の設計およびインビトロでの分子進化の研究に対する強力な手段である。
【０１７３】
ホルモン−ファージミドライブラリーの反復豊富化の変異
hＧＨ中の組合せ突然変異が受容体に対する結合親和性に加成効果を有している場合には、結合を改良するためのホルモン−ファージミド豊富化により既知となった突然変異を、単に制限フラグメントの切断と連結または突然変異誘発によって組合わせてhＧＨの累積して最適化された突然変異体を得ることができる。
【０１７４】
一方、受容体結合を最適化するhＧＨの１つの領域における突然変異が、分子の重なり合うかまたは別の領域における突然変異と構造的または機能的に適合しないこともある。このような場合には、以下に挙げる反復豊富化法のいくつかの変法のいずれかによってホルモン−ファージミド豊富化を行なうことができる：(1)カセットまたは他の突然変異誘発法によって分子の２つ（または、恐らくはそれ以上）の領域のそれぞれにおいてランダムＤＮＡライブラリーを創製することができる：次いで、これら２つのＤＮＡライブラリーからの制限フラグメントの連結によって混合ライブラリーを創製することができる；(2)分子の１つの領域における突然変異により結合を最適化されたhＧＨ変異体を、ヘリックス４bライブラリーの例のように分子の第２の領域においてランダムに突然変異させることができる；(3)２またはそれ以上のランダムライブラリーを、ホルモン−ファージミド豊富化により、改善された結合について部分的に選択することができる：この最適化結合部位の「粗選択」の後、なお部分的な別のライブラリーを制限フラグメントの連結によって再混合して、分子の２またはそれ以上の領域において部分的に異なっている単一のライブラリーを得ることができ、次いでこのライブラリーをホルモン−ファージミド豊富化を用いて最適化結合についてさらに選択することができる。
【０１７５】
表 XI．４および６サイクルの豊富化の後にヘリックス４bライブラリーから選択したhＧＨの突然変異ファージミド
それぞれがＥ１７４Ｓ／Ｆ１７６Ｙ二重突然変異を含むhＧＨヘリックス４b突然変異体（残基１６７、１７１、１７５、１７９においてランダム突然変異）を、hＧＨbp-ビーズへの結合、およびhＧＨ（０.４mM）緩衝液と次いでグリシン（０.２M、ｐH２）緩衝液による溶離によって選択した。１つの突然変異体（＋）が疑突然変異Ｒ１７８Ｈを含んでいた。
【０１７６】

【０１７７】
表 XII．選択したライブラリーからのコンセンサス配列
観察された頻度は、表示したアミノ酸を有する配列決定した全クローンの分数である。名目頻度はＮＮＳの３２コドン縮重に基づいて計算した。最大豊富化因子は、ある残基に対する名目頻度値に依存して１１〜１６〜３２に変化する。単一のアラニン突然変異に対する〔Ｋd（Ａla突然変異体）／Ｋd（wt-hＧＨ）〕の値は、以下の文献から採用した；１７５位に対してはＴ１７５Ｓ突然変異体の値のみ〔B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells, Science 243: 1330 (1989)；B.C.CunninghamおよびJ.A.Wells, Science 244: 1081 (1989)；B.C.Cunningham, D.J.Henner, J.A.Wells, Science 247: 1461 (1990)；B.C.CunninghamおよびJ.A.Wells, Proc.Natl.Acad.Sci.USA 88: 3407 (1991)〕。
【０１７８】

【０１７９】
表 XIII．hＧＨbpに対する精製したhＧＨ突然変異体の結合
競合結合実験は、〔¹²⁵Ｉ〕hＧＨ（野生型）、hＧＨbp（細胞外受容体ドメイン、残基１〜２３８を含む）、およびＭab２６３(11)を用いて行なった。数値Ｐは、１またはそれ以上の選択ラウンド後に配列決定した全クローン中の各突然変異体の発生率を分数で示すものである。ヘリックス４b突然変異（*）はhＧＨ（Ｅ１７４Ｓ／Ｆ１７６Ｙ）のバックグラウンドにあることに注意すべきである。このヘリックス４b突然変異のリストにおいて、１６７、１７１、１７５、１７９にwt残基を有するＥ１７４Ｓ／Ｆ１７６Ｙ突然変異体（*）は下線で示す。
【０１８０】

【０１８１】
実施例 XI ファージミド表面におけるＦab分子の組立て
プラスミドの構築
プラスミドpＤＨ１８８は、ＨＥＲ-２受容体を認識する４Ｄ５と呼ばれるヒト化ＩgＧ抗体のＦab部分をコードしているＤＮＡを含有する。このプラスミドは大腸菌株ＳＲ１０１中に含まれ、ＡＴＣＣ〔Rockville, MD〕に寄託されている。
【０１８２】
簡単に説明すると、このプラスミドは次のようにして調製された。出発プラスミドは、上記のアルカリホスファターゼプロモーターを含有するpＳ０１３２である。ヒト成長ホルモンをコードしているＤＮＡを切り出し、プラスミドの末端を連結に適合するようにする一連の操作を行なった後、４Ｄ５をコードしているＤＮＡを挿入した。この４Ｄ５ ＤＮＡは２つの遺伝子を含有している。第１の遺伝子は、軽鎖の可変および不変領域をコードしており、その５′末端にstIIシグナル配列をコードしているＤＮＡを含有する。第２の遺伝子は４つの部分を含有し、その第１は５′末端のstIIシグナル配列をコードしているＤＮＡである。これに重鎖の可変ドメインをコードしているＤＮＡが続き、次いで重鎖不変領域の第１ドメインをコードしているＤＮＡが続き、さらにＭ１３遺伝子IIIをコードしているＤＮＡが続く。この構築物の顕著な特徴が図１０に示されている。４Ｄ５をコードしているＤＮＡの配列を図１１に示す。
【０１８３】
大腸菌の形質転換およびファージの生成
ポリエチレングリコール（ＰＥＧ）および電気穿孔の両方を用いて、ＳＲ１０１細胞にプラスミドを導入した。ＰＥＧコンピテントな細胞は、ChungおよびMiller〔Nucleic Acids Res. 16: 3580 (1988)〕の方法に従って調製し、形質転換した。電気穿孔にコンピテントな細胞は、ZabarovskyおよびWinberg〔Nucleic Acids Res. 18: 5912 (1990)〕の方法に従って調製し、次いで電気穿孔により形質転換した。ＳＯＣ培地〔Sambrookら（上記）が記載〕(１ml)に細胞を蒔いた後、振盪しながら３７℃で１時間増殖させた。この時点で、細胞濃度をＯＤ₆₀₀の光分散を用いて測定した。滴定したＫＯ７ファージストックを加えて感染多重度（ＭＯＩ）が１００となるようにし、このファージを室温で２０分間、細胞に付着させた。次いで、この混合物を２ＹＴブロス〔Sambrookら（上記）が記載〕(２５ml）中に希釈し、振盪しながら３７℃で一晩インキュベートした。翌日、５０００ｘｇで１０分間遠心することによって細胞をペレット化し、上清を集め、０.５Ｍ ＮaＣlおよび４％ＰＥＧ（最終濃度）を用いて室温で１０分間、ファージ粒子を沈澱させた。１０,０００ｘｇで１０分間遠心することによってファージ粒子をペレット化し、ＴＥＮ〔１０mMトリス（ｐH７.６）、１mM ＥＤＴＡ、および１５０mM ＮaＣl〕(１ml)に再懸濁し、４℃で保存した。
【０１８４】
抗原被覆したプレートの調製
０.１M炭酸水素ナトリウム（ｐH８.５）中のＢＳＡ（対照抗原）の０.５mg/ml溶液またはＨＥＲ−２抗原の細胞外ドメイン（ＥＤＣ）の０.１mg/ml溶液からの０.５mlづつを用いて、Falcon１２ウエル組織培養プレートのウエルを被覆した。溶液をウエルに適用した後、このプレートを揺れ台の上にて４℃で一晩インキュベートした。次いで、最初の溶液を除去し、ブロック緩衝液〔０.１M炭酸水素ナトリウム中に３０mg/ml ＢＳＡ〕(０.５ml）を適用し、そして室温で１時間インキュベートすることによって、プレートをブロックした。最後に、このブロック緩衝液を除去し、緩衝液Ａ〔ＰＢＳ、０.５％ＢＳＡ、および０.０５％ツイーン２０〕(１ml）を加え、そしてこのプレートをファージ選択に使用するまで４℃で１０日間まで保存した。
【０１８５】
ファージの選択法
約１０⁹のファージ粒子を１００倍過剰のＫＯ７ヘルパーファージおよび緩衝液Ａ（１ml）と混合した。この混合物を０.５mlづつ２つに分け、その１つをＥＣＤ被覆ウエルに適用し、他方をＢＳＡ被覆ウエルに適用した。これらのプレートを振盪させながら室温で１〜３時間インキュベートし、次いで緩衝液Ａ（１mlづつ）を用いて３０分間、３回洗浄した。プレートからのファージの溶離は次の２つの方法のいずれかにより室温で行なった：(1)０.０２５mg/mlの精製Ｍu４Ｄ５抗体（ネズミ）の最初の一晩インキュベートとそれに続いて酸溶離緩衝液〔０.２Mグリシン（ｐH２.１）、０.５％ＢＳＡ、および０.０５％ツイーン２０〕(０.４ml）による３０分間インキュベート、または(2)酸溶離緩衝液単独によるインキュベート。次いで、溶出液を１Mトリス塩基で中和し、ＴＥＮ（０.５mlづつ）を加えた。次に、これら試料を増殖させ、滴定し、４℃で保存した。
【０１８６】
ファージの増殖
溶出したファージの一部を２ＹＴブロス（０.４ml）に加え、約１０⁸の中間対数期雄性大腸菌株ＳＲ１０１と混合した。ファージを室温で２０分間、細胞に付着させ、次いで５０μg/mlカルベニシリンおよび５μg/mlテトラサイクリンを含有する２ＹＴブロス（５ml）に加えた。これら細胞を、中間対数相に到達するまで３７℃で４〜８時間増殖させた。ＯＤ₆₀₀を測定し、細胞をファージ生成用のＫＯ７ヘルパーファージに重感染させた。ファージ粒子を得た後、これらを滴定してコロニー形成単位（cfu）数を測定した。これは、あるファージストックの連続希釈液の一部を取り、中間対数期のＳＲ１０１に感染させ、５０νg／mlカルベニシリン含有のＬＢプレートにプレーティングすることにより行なった。
【０１８７】
ＲＩＡ親和性の測定
ＥＣＤ抗原に対するＦabファージおよびh４Ｄ５ Ｆabフラグメントの親和性を、競合受容体結合ＲＩＡ〔Burt,D.R., Receptor Binding in Drug Research, O'Brien,R.A.（編）, pp.3-29, Dekker, New York (1986)〕を用いて測定した。このＥＣＤ抗原は連続クロラミン-Ｔ法〔De Larco,J.E.ら, J.Cell.Physiol. 109: 143-152 (1981)〕を用いて¹²⁵Ｉで標識した。これにより、受容体１モルあたり０.４７モルのヨウ素が導入された１４μCi/μgの比活性を有する放射活性トレーサーが得られた。０.５ng（ＥＬＩＳＡによる）のＦabまたはＦabファージ、５０nCiの¹²⁵Ｉ-ＥＣＤトレーサー、およびある範囲量の未標識ＥＣＤ（６.４ng〜３２７７ng）を含有する一連の０.２ml溶液を調製し、室温で一晩インキュベートした。標識化ＥＣＤ-ＦabまたはＥＣＤ-Ｆabファージ コンプレックスを、抗ヒトＩgＧ（Fitzgerald 40-GH23）と６％ＰＥＧ８０００の添加により誘導される集合コンプレックスを形成させることによって、未結合の標識化抗原から分離した。このコンプレックスを遠心（１５,０００ｘｇで２０分間）によってペレット化し、標識化ＥＣＤの量（cpm）をガンマカウンターで測定した。解離定数（Ｋd）は、スカッチャード分析〔Scatchard,G., Ann.N.Y.Acad.Sci. 51: 660-672 (1949)〕を用いるプログラムＬＩＧＡＮＤの改良バージョン〔Munson,P.およびRothbard,D., Anal.Biochem. 107: 220-239 (1980)〕を用いて計算した。このＫd値を図１３に示す。
【０１８８】
競合細胞結合の検定
ロドゲン法を用い、¹²⁵Ｉによりネズミ４Ｄ５抗体を４０〜５０μCi/μgの比活性に標識した。一定量の標識化抗体と増加量の未標識変異Ｆabを含有する溶液を調製し、９６ウエル微量滴定皿中のほぼ全面のＳＫ-ＢＲ-３細胞培養物に加えた（標識化抗体の最終濃度は０.１nMであった）。４℃で一晩インキュベートした後、上清を除去し、細胞を洗浄し、細胞に結合した放射活性をガンマカウンターで測定した。プログラムＬＩＧＡＮＤの改良バージョン〔Munson,P.およびRothbard,D.（上記）〕を用いてデータを分析することによってＫd値を決定した。
【０１８９】
プラスミドpＤＨ１８８（ＡＴＣＣ Ｎo.６８６６３）の寄託は、特許手続上の微生物寄託の国際的承認に関するブダペスト条約の規定およびその規則（ブダペスト条約）のもとに為されている。これにより、寄託日から３０年間の生存微生物の維持が確保されている。この微生物は、ブダペスト条約の条項のもとで、およびジェネンテク，インコーポレイテッドとＡＴＣＣの間の同意のもとでＡＴＣＣから入手可能となるものであり、関連米国特許の発行またはいずれかの米国もしくは外国特許出願の公開のいずれか早い方により、だれでもこの培養物の子孫を永続的かつ制限されずに入手可能になることが保証されており、また、３５ ＵＳＣ §１２２に従って権利を付与された米国特許商標庁長官およびそれに従う規則（８８６ ＯＧ ６３８に具体的に言及している３７ ＣＦＲ §１.１４を含む）によって決定された者に該子孫が入手可能になることが保証されている。
【０１９０】
本出願の譲受人は、寄託微生物が適切な条件下で培養されたときに死滅しているか、失われているか、または破壊されているときには、通知により該培養物の生存試料と速やかに交換することに同意している。寄託培養物の入手可能性は、各国特許法に従っていずれかの政府の権限のもとに認可された権利に違反して本発明を実施するライセンスと解釈すべきではない。
【０１９１】
以上に既述した明細書は、当業者に本発明の実施を可能ならしめるに十分であると考えられる。寄託が為された態様は本発明の特定態様の独立した例示として意図されており、機能的に等価なあらゆる培養物が本発明の範囲内にあるので、本発明は寄託された微生物によってその範囲を限定されるものではない。本明細書中の物質の寄託は、本明細書中に含まれる既述が本発明のいずれかの態様（最良の態様を含む）の実施を可能にするに不適切であるということを認めたものではなく、また、特許請求の範囲をその特定の例示に限定することを意図するものでもない。実際には、本明細書中で説明および示したものに加えて本発明の種々の修飾が上記既述から当業者には明白になるものであり、これらは添付した請求の範囲内に入るものである。
【０１９２】
必然的に本発明を好ましい態様との関係で説明したが、上記明細書を読んだ後に当業者なら、本発明の思想および範囲から逸脱することなく、本明細書中に記載した対象に種々の変化、等価置換、変異を加えることができるであろう。即ち、本発明は本明細書に具体的に記載した方法以外の方法で実施することができる。従って、本願の特許証により認められる保護は添付した請求の範囲およびその等価物によってのみ限定されるものである。
【０１９３】
実施例 XII ヘリックス１およびヘリックス４ ホルモン−ファージ変異体の組合せ体からのhＧＨ変異体の選択
h ＧＨの加成変異体の作成
加成性の原理〔J.A.Wells, Biochemistry 29: 8509 (1990)〕によると、タンパク質の異なる部分の突然変異が相互に作用を及ぼしていないときであっても、それらを組合せることによって、別の分子に対する結合の遊離エネルギーにおいて加成的な変化が得られることが予想される（変化はΔΔＧ_bindingの点で加法的であるか、またはＫd＝exp〔−ΔＧ／ＲＴ〕の点で乗法的である）。即ち、結合親和性において２倍の増加を与える突然変異は、３倍の増加を引き起こす第２の突然変異と組合せたときに、出発変異体の６倍増加した親和性を有する二重突然変異を与えることが予想される。
【０１９４】
hＧＨ-ファージ選択によって得た複数の突然変異がhＧＨbp（hＧＨ−結合タンパク質；hＧＨ受容体の細胞外ドメイン）結合において累積的に良好な効果を与えるか否かを試験するため、ヘリックス１ライブラリーに由来するhＧＨの最も強固に結合する３種の変異体（実施例IX：Ｆ１０Ａ／Ｍ１４Ｗ／Ｈ１８Ｄ／Ｈ２１Ｎ、Ｆ１０Ｈ／Ｍ１４Ｇ／Ｈ１８Ｎ／Ｈ２１Ｎ、およびＦ１０Ｆ／Ｍ１４Ｓ／Ｈ１８Ｆ／Ｈ２１Ｌ）に見い出される突然変異を、ヘリックス４bライブラリーにおいて見い出される最も強固に結合する３種の変異体（実施例Ｘ：Ｒ１６７Ｎ／Ｄ１７１Ｓ／Ｔ１７５／Ｉ１７９Ｔ、Ｒ１６７Ｅ／Ｄ１７１Ｓ／Ｔ１７５／Ｉ１７９、およびＲ１６７Ｎ／Ｄ１７１Ｎ／Ｔ１７５／Ｉ１７９Ｔ）に見い出される突然変異と組合せた。
【０１９５】
１ヘリックス変異体のそれぞれからhＧＨ−ファージミド２本鎖ＤＮＡ（dsＤＮＡ）を単離し、制限酵素ＥcoＲＩおよびＢstＸＩで消化した。次いで、それぞれのヘリックス４b変異体から大きいフラグメントを単離し、それぞれのヘリックス１変異体からの小さいフラグメントと連結して表XIIIに示す新規な２ヘリックス変異体を得た。さらに、これら変異体のすべては先のhＧＨ-ファージ結合選択において得られたＥ１７４Ｓ／Ｆ１７６Ｙの突然変異を含有していた（詳細には実施例Ｘを参照）。
【０１９６】
h ＧＨの選択組合せライブラリーの作成
加成性の原理は多数の突然変異の組合せに当てはまるようであるが、一部の組合せ（例えば、Ｆ１７６Ｙを伴うＥ１７４Ｓ）は明らかに非加成性である（実施例VIIIおよびＸを参照）。例えば、ヘリックス１中に４つの突然変異とヘリックス４中に４つの突然変異を有する最も強固に結合する変異体を確実性をもって同定するためには、理想的には８残基全部を一度に突然変異させ、次いで全体的に最も強固に結合する変異体のプールを選別することになるであろう。しかし、このようなプールは、２.６ｘ１０¹⁰の異なるポリペプチドをコードしている１.１ｘ１０¹²のＤＮＡ配列（ＮＮＳコドンの縮重を用いて）からなる。全変異体（恐らく、１０¹³の形質転換体）の表示を確実にするに十分大きなランダムファージミドライブラリーを得ることは、現在の形質転換法によっては実際的ではない。
【０１９７】
我々は、第１に、連続ラウンドの突然変異誘発を用い、１つのライブラリーから最も強固に結合する変異を取り、次いで他の残基を突然変異させてさらに結合を改善することによってこの難題に取り組んだ（実施例Ｘ）。第２の方法において、我々は、加成性の原理を用いて２つの独立して選別したライブラリーからの最良の突然変異を組合わせて、改善された結合を有する複数の突然変異体を創製した（上記）。ここで我々は、ランダムまたは部分的ランダム突然変異体の組合せライブラリーを創製することによって、hＧＨ中の１０、１４、１８、２１、１６７、１７１、１７５、および１７９位における可能な突然変異の組合せをさらに探索した。ヘリックス１ライブラリーからのプールしたファージミド（独立して０、２または４サイクル選別；実施例IX）およびヘリックス４bライブラリーからのプール（独立して０、２または４サイクル選別；実施例Ｘ）を用いてhＧＨ−ファージミドの３つの異なる組合せライブラリーを作成し、この組合せ変異体プールをhＧＨbp結合について選別した。ある量の配列多様性がこれらプールのそれぞれに存在するので、得られる組合せライブラリーは手で作成しうる配列の組合せよりも多くの配列組合せを調べることができる（例えば、表XIII）。
【０１９８】
１ヘリックスライブラリーのプール（０、２または４ラウンド選択）のそれぞれからhＧＨ−ファージミドの２本鎖ＤＮＡ（dsＤＮＡ）を単離し、制限酵素ＡccＩおよびＢstＸＩで消化した。次いで、それぞれのヘリックス１変異体プールから大きいフラグメントを単離し、それぞれのヘリックス４b変異体プールからの小さいフラグメントと連結して、３つの組合せライブラリーｐH０７０７Ａ（実施例IXおよびＸに記載の未選択のヘリックス１およびヘリックス４bプール）、ｐH０７０７Ｂ（２回選択したヘリックス１プールと２回選択したヘリックス４bプール）、およびｐH０７０７Ｃ（４回選択したヘリックス１プールと４回選択したヘリックス４bプール）を得た。さらに、より少ないＤＮＡを用いて同じ連結を行ない、ｐH０７０７Ｄ、ｐH０７０７ＥおよびｐH０７０７Ｆ（それぞれ、０、２および４ラウンドの出発ライブラリーに対応する）と命名した。また、これら変異体プールのすべては、先のhＧＨ-ファージ結合選択で得られた突然変異Ｅ１７４Ｓ／Ｆ１７６Ｙを含有していた（詳細については実施例Ｘを参照）。
【０１９９】
h ＧＨ - ファージ変異体の組合せライブラリーの選別
連結生成物ｐH０７０７Ａ〜Ｆを加工処理し、記載（実施例VIII）のようにＸＬ１-Ｂlue細胞に電気導入した。コロニー形成単位（ＣＦＵ）に基づいて、それぞれのプールから得られた形質転換体の数は次のようであった：ｐH０７０７Ａから２.４ｘ１０⁶、ｐH０７０７Ｂから１.８ｘ１０⁶、ｐH０７０７Ｃから１.６ｘ１０⁶、ｐH０７０７Ｄから８ｘ１０⁵、ｐH０７０７Ｅから３ｘ１０⁵、およびｐH０７０７Ｆから４ｘ１０⁵。hＧＨ−ファージミド粒子を調製し、実施例VIIIの記載のように２〜７サイクルにわたってhＧＨbp-結合について選択した。
【０２００】
h ＧＨ−ファージミドライブラリーの迅速な選別
平衡結合親和性によって測定したときの強固に結合するタンパク質変異体についてのファージミドライブラリーの選別に加えて、受容体あるいは他の分子に対する結合のオン-速度（ｋ_on）またはオフ-速度（ｋ_off）のどちらかが変化した変異体を選別することも重要である。熱力学から、これら速度は平衡解離定数 Ｋd＝（ｋ_off／ｋ_on）に関係している。我々は、特定タンパク質の一部の変異体は結合に対して類似したＫdを有しているが、非常に異なったｋ_onおよびｋ_offを有しているものと想定している。逆に、ある変異体と別の変異体のＫdの変化は、ｋ_onに対する効果、ｋ_offに対する効果、またはその両方によるものであろう。タンパク質の薬理学的性質は、結合親和性またはｋ_onもしくはｋ_offに依存していることもある（作用の詳細な機序に依存する）。ここで我々は、より高いオン-速度を有するhＧＨ変異体を同定してｋ_onの変化の効果を調べようとした。我々は、結合時間を増加させることによって、ならびに同族リガンド（hＧＨ）による洗浄時間および／または濃縮を増加させることによって、選択をｋ_offの方に選択的に加重することができると想定している。
【０２０１】
固定化hＧＨbpに対する野生型hＧＨ−ファージミド結合の経時分析から、最終のｐH２洗浄液中に溶出されうる全hＧＨ−ファージミド粒子（全体の結合および溶離プロトコールについては実施例VIIIを参照）のうち、１０％未満が１分間のインキュベートの後に結合するが、９０％以上は１５分間のインキュベート後に結合するようである。
【０２０２】
「迅速な結合選択」のために、ｐH０７０７Ｂプール由来のファージミド粒子（ヘリックス１および４に対して独立して２回選択）を固定化hＧＨbpと１分間だけインキュベートし、次いで結合緩衝液（１ml）で６回洗浄した〔hＧＨ洗浄工程は削除し、残存するhＧＨ−ファージミド粒子はｐH２（結合緩衝液中の０.２Mグリシン）洗浄液で溶離した〕。非表示粒子に対するhＧＨ−ファージミド粒子の豊富化は、短時間の結合を用い、同族リガンド（hＧＨ）チャレンジを用いないときであっても、hＧＨ−ファージミド結合選択がランダム化プールから強固に結合する変異体を選別することを示した。
【０２０３】
h ＧＨ突然変異体の検定
これらhＧＨ突然変異体の一部のhＧＨbpに対する結合定数を、非サプレッサー大腸菌株１６Ｃ９または３４Ｂ８中で遊離のホルモン変異体を発現させ、このタンパク質を精製し、そして放射免疫沈澱検定法におけるhＧＨbpからの標識化wt-hＧＨの競合置換（実施例VIIIを参照）により検定することによって測定した。以下の表XIII-Ａにおいて、すべての変異体はセリン₁₇₄によって置換されたグルタメート₁₇₄およびチロシン₁₇₆によって置換されたフェニルアラニン₁₇₆（Ｅ１７４ＳおよびＦ１７６Ｙ）に加えて、hＧＨアミノ酸の１０、１４、１８、２１、１６７、１７１、１７５および１７９位に表中に示した追加の置換を有している。
【０２０４】

【０２０５】
以下の表XIVにおいて、hＧＨ変異体を、ファージミド結合選択法によって組合せライブラリーから選択した。表XIV中のすべてのhＧＨ変異体は２つのバックグラウンド突然変異（Ｅ１７４Ｓ／Ｆ１７６Ｙ）を含んでいる。ライブラリーｐH０７０７Ａ（パートＡ）、ｐH０７０７ＢおよびｐH０７０７Ｅ（パートＢ）またはｐH０７０７Ｃ（パートＣ）からのhＧＨ−ファージミドプールを、hＧＨbpに対する結合について２〜７サイクルで選別した。数値Ｐは、それぞれのプールから配列決定した１組のクローン中のそれぞれの変異体型の分数発生率を示す。
【０２０６】

【０２０７】
以下の表XVにおいて、hＧＨ変異体を、ファージミド結合選択法によって組合せライブラリーから選択した。表XV中のすべてのhＧＨ変異体は２つのバックグラウンド突然変異（Ｅ１７４Ｓ／Ｆ１７６Ｙ）を含んでいる。数値Ｐは、４サイクルの迅速結合選択の後に配列決定した全クローン中の特定変異体の分数発生率である。
【０２０８】

【０２０９】
以下の表XVIにおいて、hＧＨbp（１〜２３８）およびＭab５またはＭab２６３のいずれかを用いる¹²⁵Ｉ-標識化ホルモンＨ０６５０ＢＤまたは標識化hＧＨの競合置換によって結合定数を測定した。変異Ｈ０６５０ＢＤは野生型hＧＨよりも３０倍強固に結合するようである。
【０２１０】

【０２１１】
実施例 XIII プロテアーゼ基質配列を含有するhＧＨ-ファージと非基質ファージの選択豊富化
実施例Ｉに記載したように、プラスミドpＳ０１３２は、余分のグリシン残基の挿入を伴って遺伝子IIIタンパク質の残基Ｐro１９８に融合したhＧＨの遺伝子を含有している。このプラスミドを用いて、hＧＨ−遺伝子III融合産物がファージ表面に１価で表示されるhＧＨ-ファージ粒子を得ることができる（実施例IV）。この融合タンパク質は、可変性リンカー配列によって遺伝子IIIのカルボキシ末端ドメインに融合した全hＧＨタンパク質を含有している。
【０２１２】
あるタンパク質加水分解酵素の好ましい基質配列を選択するためにファージ表示法を使用することの可能性を調べるために、スブチリシンＢＰＮ'の遺伝子操作変異体を用いた〔Carter,P.ら, Proteins: Structure, function and genetics 6: 240-248 (1989)〕。この変異体（以下においてはＡ６４ＳＡＬスブチリシンと呼ぶ）は以下の突然変異：Ｓer２４Ｃys、Ｈis６４Ａla、Ｇlu１５６Ｓer、Ｇly１６９ＡlaおよびＴyr２１７Ｌeuを含んでいる。この酵素は必須の触媒残基Ｈis６４を欠いているので、その基質特異性が大きく制限されており、ある種のヒスチジン含有基質が優先的に加水分解される〔Carterら, Science 237: 394-399 (1987)〕。
【０２１３】
h ＧＨ - 基質 - ファージベクターの構築
pＳ０１３２中のリンカー領域の配列を、オリゴヌクレオチド：
5'-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3'
を用いて突然変異させ、Ａ６４ＳＡＬスブチリシンの基質配列を創製した。これにより、タンパク質配列：
Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp
がhＧＨと遺伝子IIIのカルボキシ末端ドメインの間のリンカー領域中に導入される結果が得られた（上記配列中の最初のＰhe残基はhＧＨのＰhe１９１である）。配列：
Ala-Ala-His-Tyr-Thr-Arg-Gln
はＡ６４ＳＡＬスブチリシンの良好な基質であることが既知である〔Carterら(1989)；上記〕。得られたプラスミドをpＳ０６４０と命名した。
【０２１４】
h ＧＨ - 基質 - ファージの選択豊富化
pＳ０１３２およびpＳ０６４０から導かれるファージミド粒子を実施例Ｉの記載のように作成した。最初の実験において、それぞれのファージプール（１０μlづつ）を、２０mMトリス-ＨＣl（ｐH８.６）および２.５Ｍ ＮaＣlを含有する緩衝液（１００μl）中でオキシランビーズ（実施例IIの記載のように調製；３０μl）と別々に混合した。結合と洗浄工程は実施例VIIの記載のように行なった。次いで、このビーズを５０nMのＡ６４ＳＡＬスブチリシンを含むかまたは含まない同緩衝液（４００μl）に再懸濁した。１０分間インキュベートした後、上清を集め、ファージ力価（cfu）を測定した。表XVIIは、約１０倍多い基質含有ファージミド粒子（pＳ０６４０）が、酵素の非存在下よりも酵素の存在下において溶出され、また、酵素の存在下もしくは非存在下の非基質ファージミド（pＳ０１３２）の場合よりも多く溶出されることを示している。酵素、ファージミドまたはビーズ濃度の増加はこの比率を変えることはなかった。
【０２１５】
選択豊富化法の改良
固定化ファージミドの非特異的溶離を減少させる試みにおいて、強固に結合するhＧＨ変異体を野生型hＧＨ遺伝子の代わりにpＳ０１３２およびpＳ０６４０に導入した。使用したhＧＨ変異体は実施例XIに記載した変異体（ｐH０６５０bp）であり、突然変異Ｐhe１０Ａla、Ｍet１４Ｔrp、Ｈis１８Ａsp、Ｈis２１Ａsn、Ａrg１６７Ａsn、Ａsp１７１Ｓer、Ｇlu１７４Ｓer、Ｐhe１７６ＴyrおよびＩle１７９Ｔhrを含有している。これにより、２つの新規なファージミド：pＤＭ０３９０（強固に結合するhＧＨを含有し、基質配列を含まない）およびpＤＭ０４１１（強固に結合するhＧＨおよび基質配列Ａla-Ａla-Ｈis-Ｔyr-Ｔhr-Ａrg-Ｇlnを含有する）が構築された。また、結合の洗浄および溶離のプロトコールは次のように変えた：
(i) 結合：COSTAR １２ウエル組織培養プレートを、０.５ml／ウエルの炭酸ナトリウム緩衝液（ｐH１０.０）中の２μg/ml hＧＨbpで１６時間被覆した。次いで、このプレートを、１ml／ウエルのブロック緩衝液〔０.１％w／vウシ血清アルブミンを含有するリン酸緩衝食塩水（ＰＢＳ）〕とともに２時間インキュベートし、１０mMトリス-ＨＣl（ｐH７.５）、１mM ＥＤＴＡおよび１００mM ＮaＣlを含有する検定緩衝液で洗浄した。ファージミドを再び実施例Ｉの記載のように調製し、ファージプールを上記検定緩衝液で１：４に希釈し、ウエルあたり０.５mlのファージを２時間インキュベートした。
（ii）洗浄：このプレートをＰＢＳ＋０.０５％ツイーン２０で徹底的に洗浄し、この洗浄緩衝液（１ml）とともに３０分間インキュベートした。この洗浄工程を３回繰返した。
（iii）溶離：このプレートを、２０mMトリス-ＨＣl（ｐH８.６）＋１００mM ＮaＣlからなる溶離緩衝液中で１０分間インキュベートし、次いで５００nMのＡ６４ＳＡＬスブチリシンを含むかまたは含まない上記緩衝液（０.５ml）でファージを溶離した。
【０２１６】
表XVIIは、pＳ０６４０およびpＳ０１３２について先に記載した方法と比較して、特異的に溶出した基質-ファージミド粒子の割合が劇的に増加したことを示している。この結果は、強固に結合するhＧＨ突然変異体が、野生型hＧＨに比べてhＧＨ結合タンパク質への結合に対して有意に低いオフ-速度を有していることによるものと考えられる。
【０２１７】
表 XVII．Ａ６４ＳＡＬスブチリシンによる基質-ファージミドの特異的溶離
ファージの１０倍希釈液（１０μl）を、５０μg/mlカルベニシリンを含有するＬＢ寒天プレート上のＸＬ１-Ｂlue細胞の１０μlスポット上にプレーティングすることによってコロニー形成単位（cfu）を評価した。

【０２１８】
実施例 XIV 基質配列ライブラリーの選択豊富化を用いるＡ６４ＳＡＬスブチリシンの好ましい基質の同定
実施例XIIIに記載した選択的豊富化法を用いてランダム基質配列のライブラリーから良好な基質配列を同定しようとした。
ランダム化基質カセットの挿入のためのベクターの構築
ランダム化基質カセットの導入およびその後の基質配列ライブラリーの発現に適したベクターを設計した。出発点は実施例VIIIに記載したベクターpＳ０６４３であった。オリゴヌクレオチド：
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-GCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3'
を用いて部位指向性の突然変異誘発を行ない、これによってhＧＨと遺伝子IIIの間にＡpaＩ（ＧＧＧＣＣＣ）およびＳalＩ（ＧＴＣＧＡＣ）制限部位が導入された。この新規な構築物をpＤＭ０２５３と命名した（pＤＭ０２５３の実際の配列は、
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-CCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3'
であり、下線を引いた塩基の置換は突然変異オリゴヌクレオチド中の偽錯誤によるものである）。また、pＤＭ０４１１（実施例XIII）由来のフラグメントを交換することによって、実施例中に記載した強固に結合するhＧＨ変異体を導入した。得られたライブラリーベクターをpＤＭ０４５４と命名した。
【０２１９】
ライブラリーカセットベクターの調製および突然変異カセットの挿入
ライブラリーカセットを導入するために、pＤＭ０４５４をＡpaＩ、次いでＳalＩで消化し、次に１３％ＰＥＧ８０００＋１０mM ＭgＣl₂で沈澱させ、７０％エタノールで２回洗浄し、再懸濁した。これによりベクターが効率的に沈澱するが、小さいＡpaＩ−ＳalＩフラグメントが溶液中に残る〔Paithankar,K.R.およびPrasad,K.S.N., Nucleic Acids Research 19: 1346〕。この生成物を１％アガロースゲル上を移動させ、ＡpaＩ−ＳalＩ消化したベクターを切り出し、Bandprepキット（Pharmacia）を用いて精製し、そして突然変異カセットとの連結用に再懸濁した。
【０２２０】
挿入するカセットはpＳ０６４０およびpＤＭ０４１１のリンカー領域中のＤＮＡ配列と同様の配列を含有していたが、ランダム化コドンによって置換された基質配列中のヒスチジンおよびチロシン残基のコドンを有していた。実施例VIIIに記載したようにランダム化位置のそれぞれにおいてＮＮＳ（Ｎ＝Ｇ／Ａ／Ｔ／Ｃ；Ｓ＝Ｇ／Ｃ）を置換することを選択した。突然変異カセット中に用いたオリゴヌクレオチドは、
5'-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3'（暗号鎖）および
5'-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3'（非暗号鎖）
であった。また、このカセットはＳalＩ部位を破壊し、従ってＳalＩによる消化を用いてベクター バックグラウンドを減少させることができる。これらオリゴヌクレオチドはＡpa−Ｓalカセット部位への挿入の前にホスホリル化しなかった。この理由は、続いて起こるカセット小集団のオリゴマー化が複数カセット挿入体による偽の結果を導くことがあることを恐れたためである。アニールと連結の後、反応生成物をフェノール：クロロホルム抽出し、エタノール沈澱させ、そして水に再懸濁した。最初は、バックグラウンドベクターを減少させるためのＳalＩによる消化を行なわなかった。実施例VIIIに記載したように、約２００ngをＸＬ１-Ｂlue細胞に電気穿孔導入し、ファージミドライブラリーを調製した。
【０２２１】
基質ライブラリーからの切断されやすい基質の選択
使用した選択法は、実施例XIIIにおいてpＤＭ０４１１およびpＤＭ０３９０について記載した方法と同じである。各ラウンドの選択の後、実施例VIIIにおいてhＧＨ-ファージについて記載されているように、新鮮なＸＬ１-Ｂlue細胞の培養物に形質導入し、新しいファージミドライブラリーを増殖させることによって、溶出したファージを増殖させた。選択法の進行は、溶出ファージの力価の測定および各ラウンドの選択後の個々のクローンの配列決定によってモニターした。
【０２２２】
表Ａは、１、２および３ラウンドの選択後の酵素の存在下および非存在下での溶離に対する連続ファージ力価を示すものである。特異的に溶出したファージ：非特異的に溶出したファージ（即ち、酵素を用いて溶出したファージ：酵素を用いずに溶出したファージ）の比がラウンド１からラウンド３まで劇的に増加しているのが明らかであり、このことは、良好な基質集団が各選択ラウンドにつれて増加していることを示唆する。
【０２２３】
出発ライブラリーからの１０個の単離体の配列決定は、これらのすべてが野生型pＤＭ０４６４配列からなることを示した。このことは、ＡpaＩによる消化の後にＳalＩ部位がＤＮＡフラグメントの末端に非常に近接しており、従って低効率の消化を導くことに起因している。それにもかかわらず、ライブラリー中に４００の可能な配列が存在しているにすぎず、従ってこの集団はなお特筆されるべきである。
【０２２４】
表Ｂ１およびＢ２は、ラウンド２およびラウンド３の選択後に得られた単離体の配列を示すものである。２ラウンドの選択の後に、ヒスチジン残基の高い発生が明確に存在する。これは正に予想されていたことである（実施例XIIIに記載したように、Ａ６４ＳＡＬスブチリシンは基質中にヒスチジン残基を必要とするが、この理由はそれが基質-助力の触媒機序を使用するためである）。３ラウンドの選択の後に、配列決定した１０個のクローンのそれぞれがランダム化カセット中にヒスチジンを有している。しかし、これら配列の２つはpＤＭ０４１１のものであり、出発ライブラリー中に存在していなかったものであり、従って不純物であることに注意すべきである。
【０２２５】
表Ａ．最初のファージプールおよび３ラウンドの選択豊富化からの溶出
ファージの滴定
ファージの１０倍希釈液（１０μl）を、５０μg/mlカルベニシリンを含有するＬＢ寒天プレート上のＸＬ１-Ｂlue細胞の１０μlスポット上にプレーティングすることによってコロニー形成単位（cfu）を評価した。

【０２２６】
表Ｂ１．２ラウンドの選択豊富化の後の溶出ファージの配列
すべてのタンパク質配列はＡＡ＊＊ＴＲＱの形態にあるはずである（ここで、＊はランダム化コドンを表す）。以下の表においては、ランダム化コドンおよびアミノ酸に下線を引く。

表Ｂ２．３ラウンドの選択豊富化の後の溶出ファージの配列
すべてのタンパク質配列はＡＡ＊＊ＴＲＱの形態にあるはずである（ここで、＊はランダム化コドンを表す）。以下の表においては、ランダム化コドンおよびアミノ酸に下線を引く。

【配列表】

【図面の簡単な説明】
【図１】図１：線状ファージの表面に大きいタンパク質を表示（顕示）させるための方法および改変された受容体結合性を豊富化する方法を示すものである。hＧＨの全暗号配列をＭ１３遺伝子IIIのカルボキシ末端ドメインに融合させたプラスミドphＧＨ-Ｍ１３gIIIを構築した。この融合タンパク質の転写はlacプロモーター／オペレーター配列の支配下にあり、分泌はstIIシグナル配列によって指令されている。ファージミド粒子は「ヘルパー」ファージＭ１３ＫＯ７による感染によって得られ、hＧＨを表示する粒子はhＧＨ受容体を含む親和性マトリックスに結合させることによって豊富化することができる。野生型遺伝子III（Ｍ１３ＫＯ７ファージから導く）はファージの先端の４〜５コピーの複数矢印により図示し、融合タンパク質（ファージミドphＧＨ-Ｍ１３gIIIから導く）はhＧＨの折り畳み図形（矢印の頭部を置換）によって図示した。
【図２】図２：全ファージ粒子の免疫ブロットはhＧＨがファージと同時移動することを示す。塩化セシウム勾配で精製したファージミド粒子を二重のウエルに付し、３７５mMトリス、４０mMグリシン（ｐH９.６）緩衝液中、１％アガロースゲルで電気泳動を行なった。このゲルを、２％ＳＤＳおよび２％β−メルカプトエタノールを含む移転緩衝液〔２５mMトリス（ｐH８.３）、２００mMグリシン、２０％メタノール〕中に２時間浸漬し、次いで移転緩衝液中で６時間すすいだ。次いで、ゲル中のタンパク質をイモビロン膜（Millipore）上に電気ブロットした。１組の試料を含む膜はクーマシーブルーで染色してファージタンパク質の位置を示した（Ａ）。二重の膜をポリクローナルウサギ抗-hＧＨ抗体と反応させ、次いで西洋ワサビペルオキシダーゼ-コンジュゲート化したヤギ抗-ウサギＩgＧ抗体と反応させることによって、膜のhＧＨを免疫染色した（Ｂ）。レーン１はＭ１３ＫＯ７親ファージを含み、これはhＧＨを欠いているのでクーマシーブルー染色した膜においてのみ観察することができた。レーン２および３はホルモン ファージミド粒子の別の調製物を含んでおり、クーマシーおよびhＧＨ免疫染色の両方で観察することができた。親のＭ１３ＫＯ７ファージとホルモン ファージミド粒子の間の移動距離の差異は、内部にパッケージされたゲノムの大きさの相違を反映するものである（それぞれ、８.７kbと５.１kb）。
【図３】図３：コドン１７２、１７４、１７６および１７８のところをランダム化したhＧＨ-ファージライブラリーの選択法における各工程をまとめる図である。hＧＨ（Ｒ１７８Ｇ、Ｉ１７９Ｔ）遺伝子および唯一のＫpnＩ制限部位を含有する鋳型分子ｐH０４１５を本明細書に記載のように突然変異誘発し、大腸菌株ＷＪＭ１０１中に電気導入して最初のファージミドライブラリー（ライブラリー１）を得た。ライブラリー１の一部（約２％）を、本明細書に記載のように最初の選択ラウンドに直接用いてライブラリー１Ｇを得た。一方、２本鎖ＤＮＡ（dsＤＮＡ）をライブラリー１から調製し、制限酵素ＫpnＩで消化して鋳型のバックグラウンドを排除し、ＷＪＭ１０１中に電気導入してライブラリー２を得た。これらに続く選択ラウンド（または、ＫpnＩ消化；陰影を付けた箱）とその後のファージミドの増殖は、本明細書に記載した方法に従い、矢印で示したように行なった。ライブラリー４Ｇ⁴からの４種の独立したクローンおよびライブラリー５Ｇ⁶からの４種の独立したクローンをジデオキシ配列決定法によって配列決定した。これらクローンのすべてが、hＧＨ突然変異体（Ｇlu １７４ Ｓer、Ｐhe １７６ Ｔyr）に一致する同一ＤＮＡ配列を有していた。
【図４】図４：結晶学により決定したブタ成長ホルモンの２.８Å折り畳み図から導いたhＧＨの構造モデルを示すものである。hＧＨ−結合タンパク質への結合を強く変調させるhＧＨ中の残基の位置を陰影を付けた円内に示した。結合親和性において１０倍以上の減少（黒丸）、４〜１０倍の減少（・）、増加（○）、または２〜４倍の減少（・）を引き起こすアラニン置換が示されている。α−ヘリックス領域におけるらせん輪投影により、これらの両極性が明らかになった。黒くした、陰影を付けた、あるいは陰影を付けていない残基は、それぞれ荷電、極性、または非極性を示す。ヘリックス４において、突然変異のための最も重要な残基は親水性の面上にある。
【図５】図５：表示した経路（hＧＨ溶離、グリシン溶離、または予吸着後のグリシン溶離）に対するラウンド１および３の選択工程からの多数のクローンを配列決定した後にわかったhＧＨの１７２、１７４、１７６および１７８位のアミノ酸置換を示すものである（例えば、ＫＳＹＲという表示はhＧＨ突然変異体１７２Ｋ／１７４Ｓ／１７６Ｙ／１７８Ｒを表す）。非機能的な配列（即ち、ベクター バックグラウンド、または他の未成熟に終わった突然変異体および／またはフレームシフトした突然変異体）は「ＮＦ」として示している。非サイレントの疑似突然変異（即ち、上に挙げた組の標的残基以外）を含む機能的な配列は「＋」で示している。すべての配列決定クローンの中で１回を越えて現れたが異なるＤＮＡ配列を有するタンパク質配列は「＃」で示している。配列決定クローンの中で１回を越えて現れ、そして同じＤＮＡ配列を有するタンパク質配列は「＊」で示している。３ラウンドの選択の後に２種の異なる汚染配列が見い出されたことに注意すべきである（これらのクローンはカセット突然変異体に一致しなかったが、先に構築したホルモン ファージに一致した）。pＳ０６４３汚染体は野生型hＧＨ-ファージに一致する（hＧＨ「ＫＥＦＲ」）。第３ラウンドのグリシン選択したファージのプールに多いｐH０４５７汚染体は、先に同定したhＧＨの突然変異体「ＫＳＹＲ」に一致する。これら汚染体の増幅は、まれにしか発生しない突然変異体を選択するためのホルモン−ファージ選択法の能力を強調するものである。また、これらの配列の集中性は３種すべての経路において顕著であった（即ち、ＲまたはＫは１７２および１７８位に最も多く発生し、ＹまたはＦは１７６位に最も多く発生し、そしてＳ、Ｔ、Ａおよびその他の残基は１７４位に発生する）。
【図６】図６：亜鉛の存在下にhＰＲＬbp-ビーズ上で選択したファージからの配列を示すものである。表示は図５で説明したものと同じである。ここでは、配列の集中性は予測できないが、最もストリンジェントな（グリシン）選択条件下で疎水性配列への偏りがあるようである（Ｌ、ＷおよびＰ残基がこのプールに多く見い出される）。
【図７】図７：亜鉛の非存在下にhＰＲＬbp-ビーズ上で選択したファージからの配列を示すものである。表示は図５で説明したものと同じである。図６の配列とは対照的に、ここでの配列は比較的親水性が高いようである。hＧＨ溶離による４ラウンドの選択の後、２種類のクローン（ＡＮＨＱおよびＴＬＤＴ／１７１Ｖ）がプールに多く存在する。
【図８】図８：ブランクのビーズ上で選択したファージからの配列を示すものである。表示は図５で説明したものと同じである。グリシン溶離による３ラウンドの選択の後、同胞体は観察されず、非機能的な配列のバックグラウンド水準が保持された。
【図９】図９：ｐHＯ４１５からのファージミドfl起点（ori）の構築を示すものである。カセット突然変異誘発およびhＧＨ−遺伝子III融合タンパク質の発現のためのこのベクターを次のように構築した。pＢＲ３２２およびflの複製起点を含有し、大腸菌phoＡプロモーターの制御下にhＧＨ−遺伝子III融合タンパク質（hＧＨの残基１〜１９１に１個のＧly残基が続き、これが遺伝子IIIのＰro-１９８に融合している）を発現する、pＳ０１３２のオリゴヌクレオチド指向性の突然変異誘発によって、プラスミドpＳ０６４３を構築した。以下のオリゴヌクレオチド：
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3'
を用いて突然変異誘発を行なった。このオリゴヌクレオチドは、hＧＨのＰhe-１９１に続いてＸbaＩ部位（下線部）とアンバー終止コドン（ＴＡＧ）を導入した。
【図１０】図１０：Ａは、ＨＥＲ-２受容体に指向性のＦabヒト化抗体の軽鎖および重鎖（可変および不変ドメイン１）をコードしているＤＮＡを含有するプラスミドpＤＨ１８８挿入体を図示したものである。Ｖ_LおよびＶ_Hはそれぞれ軽鎖および重鎖の可変領域である。Ｃ_kはヒトκ軽鎖の不変領域である。ＣＨ１_G1はヒトγ１鎖の最初の不変領域である。両暗号領域は細菌性のstIIシグナル配列から始まっている。Ｂは、５Ａに示した挿入体を含有する全プラスミドpＤＨ１８８を図示するものである。このプラスミドを大腸菌ＳＲ１０１細胞に導入し、ヘルパーファージを添加した後、このプラスミドはファージ粒子にパッケージされる。これら粒子の一部はＦab-pIII融合体を表示する（ここで、pIIIはＭ１３遺伝子III ＤＮＡによってコードされているタンパク質である）。このプラスミド図中のセグメントは５Ａに示した挿入体に対応している。
【図１１Ａ】図１１Ａ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｂ】図１１Ｂ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｃ】図１１Ｃ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｄ】図１１Ｄ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｅ】図１１Ｅ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｆ】図１１Ｆ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｇ】図１１Ｇ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１１Ｈ】図１１Ｈ：ファージミド表面に発現された４Ｄ５ Ｆab分子をコードしているＤＮＡのヌクレオチド配列（配列番号２５）を示すものである。軽鎖のアミノ酸配列（配列番号２６）も、重鎖pIII融合体のアミノ酸配列（配列番号２７）と同様に示されている。
【図１２】図１２：変異Ｆabファージミドからの野生型４Ｄ５ Ｆabファージミドの豊富化を示すものである。１：１,０００の比の野生型ファージミドと変異４Ｄ５ Ｆabファージミドの混合物を、ＨＥＲ-２受容体の細胞外ドメインタンパク質で被覆したプレートで選択した。各ラウンドの選択の後、溶離したファージミドの一部を大腸菌に感染させ、プラスミドＤＮＡを調製した。次いで、このプラスミドＤＮＡをＥcoＲＶおよびＰstＩで消化し、５％ポリアクリルアミドゲルで分離し、臭化エチジウムで染色した。バンドをＵＶ光のもとで可視化した。野生型および変異プラスミドに起因するバンドは矢印で示した。第１ラウンドの選択は酸条件のもとでのみ溶離した。以後のラウンドは、酸溶離（図の左側）または実施例VIIIに記載した方法を用いるヒト化４Ｄ５抗体洗浄工程とその後の酸溶離（図の右側）のいずれかによって溶離した。３種の変異４Ｄ５ Ｆab分子を調製した。即ち、Ｈ９１Ａ（Ｖ_L鎖の９１位のアミノ酸ヒスチジンをアラニンに突然変異させた；図中の「Ａ」レーンに示す）、Ｙ４９Ａ（Ｖ_L鎖の４９位のアミノ酸チロシンをアラニンに突然変異させた；図中の「Ｂ」レーンに示す）、ならびにＹ９２Ａ（Ｖ_L鎖の９２位のアミノ酸チロシンをアラニンに突然変異させた；図中の「Ｃ」レーンに示す）である。アミノ酸位置の数え方はKabatらに従った〔「免疫学的挿入体のタンパク質の配列」, 第4版, U.S.Dept of Health and Human Services, Public Health Service, Nat'l.Institute of Health, Bethesda, MD (1987)〕。
【図１３】図１３：実験プロトコール中に記載したＲＩＡ親和性測定のスカッチャード分析が示されている。結合した標識化ＥＣＤ抗原の量がｘ軸に示されており、結合量を遊離量で割った数値がｙ軸に示されている。線の傾きはＫaを示し、計算したＫdは１／Ｋaである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the production and systematic selection of novel binding proteins with altered binding properties to target molecules. More specifically, the present invention relates to a method for producing an external polypeptide that mimics the binding activity of a natural binding partner.
[0002]
[Prior art]
A binding partner is a substance that specifically binds to each other, usually through non-covalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
[0003]
In the past, binding partners have been traditionally used by various methods, including collection from nature (eg, antibody-antigen and ligand-receptor pairing) and by accidental identification (eg, using random screening of candidate molecules). Preparation). In some cases, these two methods are combined. For example, protein or polypeptide variants (such as polypeptide fragments) containing key functional residues involved in binding have been prepared. These polypeptide fragments are then derivatized by methods similar to traditional drug development. Examples of such derivatization would include methods such as cyclization to conformally constrain polypeptide fragments to yield new candidate binding partners.
[0004]
The problem with using conventional methods is that natural ligands may not have the properties suitable for all therapeutic applications. Furthermore, polypeptide ligands may not be usable for some target substances. Also, methods for producing non-natural synthetic binding partners are often expensive and difficult and usually require complex synthetic methods to obtain each candidate. The inability to investigate the structural characteristics of the resulting candidate so that reasonable drug design methods can be applied to further optimize the candidate molecule further hinders these methods. Yes.
[0005]
In an attempt to overcome these problems, Geysen [Immun.Today 6: 364-369 (1985)] and Geysen et al.[ Mol.Immun. 23: 709-715 (1986)] proposed the use of polypeptide synthesis to provide a framework for the identification and manufacture of systematic repetitive binding partners. According to Geysen et al. (Id.), A short polypeptide such as a dipeptide is first screened for the ability to bind to the target molecule. The most active dipeptide is then selected for the next test. This test consists of linking another residue to the starting dipeptide (or by internally modifying the components of the initial starting dipeptide) and then screening this set of candidates for the desired activity. This process is repeated until a binding partner with the desired properties is identified.
[0006]
The method of Geysen et al. Is hampered by the inconvenience that the chemistry underlying the method, ie peptide synthesis, produces molecules with secondary and tertiary structures that are unclear or varied. As the number of iterations of selection increases, random interactions increase at an accelerated rate between the various substituents of the polypeptide and a true random population of interacting molecules with reproducible higher order structures cannot be obtained. For example, interactions between the side chains of amino acids (these are far apart in sequence but spatially adjacent) occur arbitrarily. In addition, sequences that do not promote conformationally stable secondary structure can give complex peptide-side chain interactions that may prevent side chain interactions of certain amino acids with the target molecule. Such complex interactions are facilitated by the flexibility of the polypeptide candidate polyamide backbone. Candidates can also exist in multiple conformations, making it difficult to identify conformers that interact with or bind to targets with the greatest affinity or specificity, and rational drug design Make it difficult.
[0007]
The last problem when using Geysen's iterative polypeptide method is that there is currently no practical method for producing, screening and analyzing highly diverse variants. With 20 natural amino acids, the total number of all combinations of hexapeptides that must be synthesized is 64,000,000. Even if such highly diversified peptides are produced, it is possible to rapidly screen a mixture of such highly diversified peptides and select peptides having high affinity for the target molecule. There is no way. Currently, each “attachment” peptide must be recovered in sufficient quantity to effect protein sequencing.
[0008]
Biological selection and screening have been chosen as an alternative to overcome many of the problems inherent in Geysen's method. Biological selection and screening is a powerful tool for exploring protein function and isolating mutant proteins with the desired properties [Shortle, Protein Engineering, Oxender and Fox, Ed., ARLiss, Inc., NY , pp. 103-108 (1988); and Bowie et al., Science 247: 1306-1310 (1990)]. However, the resulting selection or screening can only be applied to one or a few related proteins.
[0009]
More recently, Smith and his collaborators [Smith, Science 228: 1315-1317 (1985); and Parmley and Smith, Gene 73: 305-318 (1985)] have introduced short gene fragments into fd phage ("fusions" It has been shown that small protein fragments (10-50 amino acids) can be efficiently “displayed” on the surface of linear phages by insertion into gene III of “phage”). The gene III minor coat protein (approximately 5 copies at one end of the virion) is important for proper phage assembly and infection by attachment to E. coli pilus [Rasched et al., Microbiol. Rev. 50: 401 -427 (1986)]. Recently, "fusion phage" have been described as exogenous proteins [Devlin et al., Science 249: 404-406 (1990)] or antibodies [Scott et al., Science 249: 386-390 (1990); and Cwirla et al., Proc. Natl. Acad. .USA 87: 6378-6382 (1990)] has been found useful for displaying short mutant peptide sequences to identify peptides that can react.
[0010]
However, there are several important limitations when using such “fusion phage” to identify modified peptides or proteins with new or enhanced binding properties. First, large inserts probably disrupt the function of gene III, thus destroying the assembly and infectivity of the phage, so that only when displaying proteins with less than 100, preferably less than 50 amino acid residues, a fusion phage Have been shown to be useful [Parmley et al.,Gene 73: 305-318 (1988)]. Secondly, conventional methods cannot select peptides from a library that has the greatest binding affinity for the target molecule. For example, after a random peptide library has been thoroughly screened with anti-β endorphin monoclonal antibodies, Cwirla and co-workers have identified medium affinity peptides from high-affinity peptides (Kd ~ 0.4 μM) fused to phage. (Kd-10 μM) could not be separated. Furthermore, extremely high affinity (Kd˜7nMThe parental β-endorphin peptide sequence with) was not selected from the epitope library.
[0011]
Ladner [WO 90/02802] discloses a method for selecting novel binding proteins displayed on the outer surface of viral particles and cells (heterologous proteins have up to 164 amino acid residues). Is good). This method is intended to isolate and amplify the displayed protein to design a new group of binding proteins with the desired affinity for the target molecule. More specifically, Ladner displays a protein having a “first protein binding domain” ranging from 46 residues (clambin) to 164 residues (T4 lysozyme) fused to an M13 gene III coat protein. "Fusion phage" is disclosed. Ladner teaches the use of these proteins “not larger than necessary” because it is relatively easy to arrange restriction sites into relatively small amino acid sequences, and is a 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI). Is preferred. Small fusion proteins such as BPTI are preferred when the target is a protein or macromolecule, while relatively large fusion proteins such as T4 lysozyme are preferred for small target molecules such as steroids, which are small for such large proteins This is because it has a tear and a groove that can be fitted with a molecule. This preferred protein BPTI is named Smith et al. Or de la Cruz et al. [J. Biol. Chem. 263: 4318-4322 (1988)] present at the site of gene III or at one of its ends together with a second synthetic copy of gene III to present a “partial” unmodified gene III protein It has been proposed to do so. Ladner does not address the problem of successfully selecting high affinity peptides from random peptide libraries, which hinders prior art biological selection and screening methods.
[0012]
Human growth hormone (hGH) is greatly involved in the regulation of normal human growth and development. This 22,000 dalton pituitary hormone exhibits numerous biological effects including, inter alia, linear growth (body formation), lactation, macrophage activation, insulin-like and diabetic effects [Chawla, RK, Ann. Rev, Med. 34: 519 (1983); Edwards, CK et al., Science 239: 769 (1988); Thomer, MO et al., J. Clin. Invest. 81: 745 (1988)]. Growth hormone deficiency in children leads to dwarfism, which has been successfully treated by external administration of hGH for over 10 years. hGH is a member of a group of homologous hormones including placental lactogen, prolactin, and other genetic and species variants or growth hormones [Nicoll, C.S. et al., Endocrine Reviews 7: 169 (1986)]. hGH is unique among these and exhibits broad species specificity, cloned somatic receptors [Leung, DW et al., Nature 330: 537 (1987)] or prolactin receptors [Boutin, JM et al., Ce 53]. : 69 (1988)]. The cloned hGH gene is secreted in E. coli [Chang, CN et al., Gene 55: 189 (1987)], and its DNA and amino acid sequences have been reported [Goeddel et al., Nature 281: 544. (1979); Gray et al., Gene 39: 247 (1985)]. The three-dimensional structure of hGH cannot be used. However, the three-dimensional folding pattern of porcine growth hormone (pGH) has been reported with normal resolution and accuracy [Abdel-Meguid, S.S. et al., Proc. Natl. Acad. Sci. USA 84: 6434 (1987)]. Human growth hormone receptor and antibody epitopes have been identified by homolog-scanning mutagenesis [Cunningham et al., Science 243: 1330 (1989)]. The structure of a novel amino-terminal methionyl bovine growth hormone containing the spliced sequence of human growth hormone including histidine 18 and histidine 21 has been shown [US Patent No. 4,880,910].
[0013]
Human growth hormone (hGH) causes a variety of physiological and metabolic effects in various animal models, including linear skeletal growth, lactation, macrophage activation, insulin-like and diabetic effects [RKChawla et al., Annu. Rev. Med. 34: 519 (1983); OGPIsaksson et al., Annu. Rev. Physiol. 47: 483 (1985); CKEdwards et al., Science 239: 769 (1988); MOThorner and MLVance, J. Clin. Invest. 82: 745 (1988); JPHughes and HGFriesen, Ann. Rev. Physiol. 47: 469 (1985)]. These biological effects are guided by the interaction between hGH and specific cell receptors.
[0014]
[Problems to be solved by the invention]
Accordingly, it is an object of the present invention to provide a quick and efficient method for the systematic production of candidate binding substances.
Another object of the present invention is to produce a conformationally stable candidate binding substance displayed on the surface of phagemid particles.
Another object of the present invention is to produce a candidate binding substance consisting of a fusion protein of a phage coat protein and a heterologous polypeptide [wherein the polypeptide has a length of 100 amino acids or more and is composed of one subunit. And may be displayed on phagemid particles (the polypeptide is encoded in the genome of this phagemid)).
Yet another object of the present invention is to provide or display all peptidyl moieties that may participate in non-covalent binding interactions and to provide these moieties in a sterically restricted manner. It provides a method for producing and selecting a binding material with sufficient flexibility.
[0015]
[Means for Solving the Problems]
The above purpose is
(a) a replicable expression vector containing a first gene encoding a polypeptide and a second gene encoding at least a portion of a natural or wild-type phage coat protein, wherein the first and The second gene is heterologous and the transcriptional regulatory elements are operably linked to the first and second genes, thus resulting in a gene fusion encoding the fusion protein);
(b) mutating at one or more selected positions within the first gene of the vector to generate a group of related plasmids;
(c) transforming a suitable host cell with the plasmid;
(d) infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein;
(e) Under conditions suitable for the formation of recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, only a small amount of phagemid particles can transfer more than one copy of the fusion protein to the surface of the particles. Culturing the transformed and infected host cells, adjusting the conditions as indicated;
(f) contacting the phagemid particle with a target molecule to bind at least a portion of the phagemid particle to the target molecule; and
(g) separating bound phagemid particles from unbound particles;
This was achieved by providing a method for selecting novel binding polypeptides consisting of: Preferably, the method further comprises the step of transforming a suitable host cell with a recombinant phagemid particle that binds to the target molecule and repeating steps (d)-(g) one or more times.
[0016]
Furthermore, a method of selecting a novel binding protein consisting of more than one subunit is achieved by selecting a novel binding peptide as follows:
A replicable expression vector containing transcriptional regulatory elements operably linked to DNA encoding the desired protein comprising one or more subunits, wherein at least one of the subunits is encoded The DNA being fused is fused to DNA encoding at least a portion of the phage coat protein;
Mutating DNA encoding the desired protein at one or more selected positions to generate a group of related vectors;
Transforming a suitable host cell with the vector;
Infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein;
-Only a small amount of phagemid particles will display more than one copy of the fusion protein on the surface of the particle under conditions suitable for the formation of recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host. Culturing the transformed and infected host cells, adjusting the conditions as follows;
Contacting the phagemid particle with a target molecule to bind at least a portion of the phagemid particle to the target molecule; and
• separating bound phagemid particles from unbound particles;
Consists of.
[0017]
In the method of the present invention, the plasmid is under strict control of transcriptional regulatory elements and cultured such that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 1%. It is preferred that the conditions are adjusted.
[0018]
It is also preferred that the amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% of the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably, this amount is less than 20%.
[0019]
Usually, in the methods of the invention, the expression vector will further contain a secretory signal sequence fused to the DNA encoding the respective subunit of the polypeptide and the transcriptional regulatory element will be a promoter system. Preferred promoter systems are LacZ, λ_PL, TAC, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
[0020]
The first gene will also usually encode a mammalian protein. Preferred proteins will be selected from: human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A chain, insulin B chain, proinsulin, relaxin A chain, relaxin B chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and leutinizing hormone (LH), glycoprotein hormone receptor, calcitonin, glucagon, factor VIII, antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hematopoietic growth hormone, tumor necrosis factor α and β, enkephalinase , Human serum Albumi , Mueller inhibitor, mouse gonadotropin related peptide, microbial protein such as β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, hormone or growth factor receptor; integrin, thrombopoietin, protein A or D, rheumatoid factor Nerve growth factors such as NGF-β, platelet growth factor, transforming growth factor (TGF) such as TGF-α and TGF-β, insulin-like growth factors I and II, insulin-like growth factor binding protein, CD-4, DNase, latency related peptide, erythropoietin, osteoinductive factor, interferon such as interferon α, β and γ, colony stimulating factor (CSF) such as M-CSF, GM-CSF and G-CSF, interleukin (IL), such as IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope protein, such as GP120, GP140, atrial natriuretic peptide A, B or C, immunoglobulins, and fragments of any of the above proteins.
[0021]
Preferably, the first gene encodes a polypeptide of one or more subunits comprising about 100 or more amino acid residues, and a plurality of rigid secondary molecules displaying a plurality of amino acids that can interact with the target. It will be folded to form a structure. Preferably, the first gene will be mutated at a codon corresponding only to an amino acid capable of interacting with the target so that the integrity of the rigid secondary structure is preserved.
[0022]
Typically, the methods of the invention will use helper phage selected from M13KO7, M13R408, M13-VCS and PhiX174. A preferred helper phage is M13KO7 and a preferred coat protein is the gene III coat protein of M13 phage. A preferred host is E. coli, which is a protease-deficient strain of E. coli. A novel hGH variant selected by the method of the present invention was detected. A phagemid expression vector containing a repressible stop codon functionally placed between the polypeptide and the nucleic acid encoding the phage coat protein was constructed.
[0023]
The following discussion will be best understood by referring to FIG. In its simplest form, the method of the present invention comprises a novel binding polypeptide (such as a protein ligand) having a desired, usually high affinity for a target molecule from a library of structurally related binding polypeptides. It consists of a method to choose. A library of structurally related polypeptides fused to a phage coat protein is prepared by mutagenesis and preferably contains a single copy of each related polypeptide containing the DNA encoding the polypeptide. Display (display) on the surface of the phagemid particle. These phagemid particles are then contacted with the target molecule to separate the particles with the highest affinity for the target from the low affinity particles. The high affinity binder is then amplified by infection of the bacterial host and the competitive binding process is repeated. This process is repeated until a polypeptide with the desired affinity is obtained.
[0024]
The novel binding polypeptides or ligands produced by the methods of the present invention are useful as diagnostic or therapeutic agents (eg, agonists or antagonists) that are themselves used in the treatment of biological organisms. It is also possible to proceed with rational drug design using structural analysis of the selected polypeptide.
[0025]
As used herein, “binding polypeptide” means any polypeptide that binds to a target molecule with selective affinity. Preferably, the polypeptide is a protein, most preferably a protein containing about 100 or more amino acid residues. Usually the polypeptide will be a hormone or antibody or a fragment thereof.
[0026]
“High affinity” as used herein is <10 under physiological conditions.^-FiveM, preferably <10^-7It means the affinity constant (Kd) of M.
[0027]
As used herein, a “target molecule” is any molecule for which it is desired to produce a ligand, not necessarily a protein. However, the target is preferably a protein, and most preferably the target is a receptor such as a hormone receptor.
[0028]
As used herein, “humanized antibody” refers to an antibody in which the complementarity determining regions (CDRs) of a mouse or other non-human antibody are bound to the framework of a human antibody. Human antibody framework means all human antibodies except CDRs.
[0029]
I.Selection of polypeptides for display on the surface of phage
The first step in the method of the invention is to select a polypeptide having a rigid secondary structure that is exposed on the surface of the polypeptide for display on the surface of the phage.
[0030]
As used herein, “polypeptide” means any molecule that can be expressed by a specific DNA sequence. The polypeptides of the present invention consist of more than one subunit, each subunit being encoded by a separate DNA sequence.
[0031]
As used herein, “hard secondary structure” is, for example, α-helix, 3_TenBy any helix, pi helix, parallel and antiparallel β-sheet, and any polypeptide segment that exhibits a regular repeating structure found in reverse turns and the like. Certain “disordered” structures that lack recognizable geometrical order also form domains or “patches” of amino acid residues that can interact with the target, and the overall shape of the structure is It is included in the definition of a rigid secondary structure unless destroyed by amino acid substitution. Certain disordered structures are thought to be a combination of reverse turns. The geometry of these rigid secondary structures is well defined by the φ and ψ twist angles around the α-carbon of the peptide “backbone”.
[0032]
What is needed to expose the secondary structure to the polypeptide surface is to provide a domain or “patch” of amino acid residues that can be exposed to and bound to the target molecule. This is basically an amino acid residue that is replaced by mutagenesis, and a “library” of structurally related (mutant) binding polypeptides displayed on the surface of the phage by this mutagenesis. From this, a novel polypeptide ligand is selected. Usually, mutagenesis or substitution of amino acid residues towards the interior of the polypeptide is avoided, thereby preserving the overall structure of a rigid secondary structure. Some substitutions of amino acids in rigid internal regions, particularly with hydrophobic amino acid residues, may be tolerated because these conservative substitutions are not considered to distort the overall structure of the polypeptide. .
[0033]
A repeated cycle of “polypeptide” selection is used to select higher affinity binding by phagemid selection of multiple amino acid changes selected by multiple selection cycles. Following the first round of phagemid selection (which involves selection of amino acids in the ligand polypeptide or a first region is involved), additional rounds of phagemid selection are performed in other regions or amino acids of the ligand polypeptide. This phagemid selection cycle is repeated until the desired affinity of the ligand polypeptide is achieved. To illustrate this method, the hGH phagemid selection of Example VIII was cycled. In the first cycle, mutations were made at amino acids 172, 174, 176 and 178 of hGH and phagemids were selected. In the second cycle, amino acids 167, 171, 175 and 179 of hGH were phagemid selected. In the third cycle, amino acids 10, 14, 18 and 21 of hGH were phagemid selected. Optimal amino acid changes from the previous cycle can be introduced into the polypeptide prior to selection for the next cycle. For example, amino acid substitutions 174 (serine) and 176 (tyrosine) of hGH were introduced into hGH prior to phagemid selection of amino acids 167, 171, 175 and 179 of hGH.
[0034]
From the above, it will be appreciated that the amino acid residues that form the binding domain of a polypeptide may be present on different subunits of the polypeptide, rather than being sequentially linked. That is, the binding domain follows a specific secondary structure at the binding site, not a primary structure. Thus, mutations may be introduced into codons that encode amino acids within a specific secondary structure at the site from the inside to the outside of the polypeptide so that they have the potential to interact with the target. It is normal. For illustration purposes, the positions of residues in hGH known to strongly modulate binding to hGH-binding proteins are shown in FIG. 2 [Cunningham et al.,Science 247: 1461-1465 (1990)]. That is, representative sites suitable for mutagenesis include residues 172, 174, 176 and 178 of helix 4 as well as residue 64 in the “disordered” secondary structure.
[0035]
A polypeptide selected as a ligand for a target need not normally bind to the target. That is, for example, a glycoprotein hormone such as TSH can be selected as a ligand for the FSH receptor, and a library of mutant TSH molecules is used in the method of the present invention to obtain novel drug candidates.
[0036]
That is, the present invention contemplates any polypeptide that binds to the target molecule and includes antibodies. Preferred polypeptides are those that have pharmaceutical uses. Further preferred polypeptides include: growth hormone (including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone); parathyroid hormone; thyroid stimulating hormone; thyroxine; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; reutizing hormone; glucagon; factor VIII; antibody; pulmonary surfactant; plasminogen activator [eg urokinase or human tissue type plasminogen Activator (t-PA)]; Bombesin; Factor IX; Thrombin; Hematopoietic growth factor; Tumor necrosis factor alpha and beta; Enkephalinase; Serum albumin (eg, human serum albumin); Muller inhibitor; Relaxin A chain; Relaxin B chain; prorelaxin; ma Sugonadotropin-related peptide; microbial protein (eg, β-lactamase); tissue factor protein; inhibin; activin; vascular endothelial growth factor; hormone or growth factor receptor; integrin; thrombopoietin; protein A or D; rheumatoid factor; Factors (eg, NGF-β); platelet-derived growth factors; fibroblast growth factors (eg, aFGF and bFGF); epidermal growth factors; transforming growth factors (TGF) (eg, TGF-α and TGF-β) Insulin-like growth factor I and II; insulin-like growth factor binding protein; CD-4; DNase; latency-related peptide; erythropoietin; osteoinductive factor; interferon (eg, interferon α, β and γ); CSF) For example, M-CSF, GM-CSF and G-CSF); Interleukin (IL) (eg, IL-1, IL-2, IL-3, IL-4); Superoxide dismutase; Decay promoting factor; Atrial Natriuretic peptides A, B or C; viral antigens (eg part of the HIV envelope); immunoglobulins; and fragments of any of the above polypeptides. In addition, substitutions, insertions or deletions can be made at one or more predetermined amino acid residues on the polypeptide to obtain, for example, a product with improved biological properties. Also included are fragments of these polypeptides, particularly biologically active fragments. Further preferred polypeptides of the present invention are human growth hormone, atrial natriuretic peptides A, B and C, endotoxin, subtilisin, trypsin, and other serine proteases.
[0037]
Further preferred polypeptide hormones are any amino acid sequences produced in the first cell, specifically for receptors on the same cell type (autocrine hormone) or a second cell type (non-autocrine). A polypeptide hormone that can be defined as an amino acid sequence that binds and causes a physiological response characteristic of receptor-bearing cells. Such polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and pituitary gland polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle stimulating hormone, thyrotropin, chorionic gonadotropin, Corticotropin, α or β-melanocyte stimulating hormone, β-lipotropin, γ-lipotropin and endorphins; hypothalamic release inhibiting hormones such as corticotropin releasing factor, growth hormone releasing inhibitory hormone, growth hormone releasing factor; and atrial natriuretic peptide A , Other polypeptide hormones such as B and C are included.
[0038]
II.Obtaining the first gene (gene 1) encoding the desired polypeptide
A gene encoding the desired polypeptide (ie, a polypeptide having a rigid secondary structure) can be obtained by methods known in the art [in general, Sambrook et al.,Molecular Biology: A laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)]. If the sequence of this gene is known, the DNA encoding this gene may be chemically synthesized [Merrfield,J.Am.Chem.Soc. 85: 2149 (1963)]. If the sequence of this gene is unknown, or if this gene has not been previously isolated, from a cDNA library (prepared from RNA from the appropriate tissue in which the desired gene is expressed) Alternatively, it can be cloned from a suitable genomic DNA library. The gene is then isolated using a suitable probe. Suitable probes for cDNA libraries include monoclonal or polyclonal antibodies (but when cDNA library expression libraries), oligonucleotides, and complementary or homologous cDNAs or fragments thereof. Probes that can be used to isolate a desired gene from a genomic DNA library include cDNA encoding the same or similar genes or fragments thereof, homologous genomic DNA or DNA fragments, and oligonucleotides It is. Screening of the cDNA or genomic library with the selected probe is performed using standard methods described in Chapters 10-12 of Sambrook et al. (Supra).
[0039]
Another method for isolating the gene encoding the desired protein is to use the polymerase chain reaction (PCR) described in section 14 of Sambrook et al. (Supra). This method requires the use of oligonucleotides that hybridize to the desired gene. That is, to obtain an oligonucleotide, at least a portion of the DNA sequence of this gene must be known.
After the gene is isolated, it can be inserted into a suitable vector (preferably a plasmid) for amplification as generally described by Sambrook et al. (Supra).
[0040]
III.Construction of replicable expression vector
Although several types of vectors are available and can be used in the practice of the present invention, plasmid vectors are the preferred vectors for use in the present invention. This is because these vectors can be constructed relatively easily and can be easily amplified. In general, plasmid vectors contain a variety of components including promoters, signal sequences, phenotypic selection genes, origins of replication, and other necessary components, as is known to those skilled in the art.
[0041]
The most commonly used promoters in prokaryotic vectors include the lacZ promoter system, alkaline phosphatase phoA promoter, bacteriophage λP_LPromoters (temperature-sensitive promoters), tac promoters (hybrid trp-lac promoters regulated by lac repressor), tryptophan promoters, and bacteriophage T7 promoters. See Section 17 of Sambrook et al. (Supra) for a general description of promoters. Although the most commonly used promoters exist, other suitable microbial promoters can be used as well.
[0042]
Preferred promoters for the practice of the present invention are promoters that can be tightly regulated so that expression of the fusion gene can be controlled. The problem that remained unrecognized in the prior art is believed to be that the display of multiple copies of the fusion protein on the surface of the phagemid particle led to multiple point binding of the phagemid and the target. This action, called “chelation”, selects the wrong “high affinity” polypeptide when multiple copies of the fusion protein are displayed in close proximity to each other on the phagemid particle and the target is “chelated”. It is thought that it gives the result. When multipoint binding occurs, the effective or apparent Kd will be as high as the product of the individual Kd for each copy of the displayed fusion protein. This action may be the reason why Cwirla and his collaborators (above) were unable to separate medium affinity peptides from relatively high affinity peptides.
[0043]
By tightly controlling the expression of the fusion protein so that only a small amount (ie, less than about 1%) of the phagemid particles contain multiple copies of the fusion protein, “chelation” is overcome and high affinity It has been found that an appropriate selection of polypeptides is possible. That is, depending on the promoter, host culture conditions are adjusted to maximize the number of phagemid particles containing a single copy of the fusion protein and to minimize the number of phagemid particles containing multiple copies of the fusion protein.
[0044]
Preferred promoters used in the practice of the present invention are the lacZ promoter and the phoA promoter. This lacZ promoter is regulated by the lac repressor protein laci, so that transcription of the fusion gene can be controlled by manipulation of the amount of lac repressor protein. For illustration purposes, the phagemid containing this lacZ promoter is grown in a cell line containing a copy of the laci repressor gene, which is a repressor of the lacZ promoter. Examples of cell lines containing the lac i gene include JM101 and XL1-Blue. Alternatively, host cells can be co-transfected with a plasmid containing both the repressor laci and lacZ promoters. Sometimes both of the above methods are used simultaneously. That is, phagemid particles containing the lacZ promoter are propagated in a cell line containing the lac i gene and this cell line is co-transfected with a plasmid containing both the lacZ and lac i genes. Usually, when a gene is to be expressed in the above-described transfected host, an inducer such as isopropylthiogalactoside (IPTG) is added. However, in the present invention, this step is omitted, (a) minimizing the expression of the gene III fusion protein and minimizing the copy number (ie the number of gene III fusions relative to the number of phagemids), and (b ) Prevent inferior or improper packaging of phagemids caused by inducers such as IPTG, even at low concentrations. Usually, when no inducer is added, the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins / number of phagemid particles). The most preferred promoter used in the practice of the present invention is phoA. This promoter is thought to be regulated by the amount of inorganic phosphate in the cell, and this phosphate down regulates the activity of the promoter. That is, the activity of the promoter can be increased by decreasing the phosphate of the cell. The desired result is achieved by growing the cells in a phosphate rich medium such as 2YT or LB and controlling the expression of the gene III fusion.
[0045]
Another useful component of the vector used in the practice of the invention is a signal sequence. Usually this sequence is located immediately 5 'to the gene encoding the fusion protein and is therefore transcribed to the amino terminus of the fusion protein. However, in certain cases, this signal sequence has been shown to be present at the other 5 'position of the gene encoding the protein to be secreted. This sequence targets the protein to which it is bound across the inner membrane of the bacterial cell. The DNA encoding this signal sequence can be obtained as a restriction endonuclease fragment from any gene encoding a protein having a signal sequence. Suitable prokaryotic signal sequences are, for example, LamB or OmpF [Wong et al.Gene 68: 193 (1983)], and can be obtained from genes encoding MalE, PhoA and other genes. Preferred prokaryotic signal sequences for the practice of the present invention are Chang et al. [Gene 55: 189 (1987)] is a heat-stable enterotoxin II (STII) signal sequence of Escherichia coli.
[0046]
Another useful component of the vectors used in the practice of the invention is a phenotypic selection gene. A normal phenotypic selection gene is a gene encoding a protein that confers antibiotic resistance to a host cell. For illustration purposes, the ampicillin resistance gene (amp) and the tetracycline resistance gene (tet) are easy to use for this purpose.
[0047]
Construction of a suitable vector containing the above components as well as the gene encoding the desired polypeptide (gene 1) is performed using standard recombinant DNA methods described by Sambrook et al. (Supra). The isolated DNA fragments that are combined to form a vector are cleaved, processed, and ligated together in a specific order and orientation to create the desired vector.
[0048]
The DNA is cleaved using an appropriate restriction enzyme or enzyme group in an appropriate buffer. Usually, about 0.2-1 μg of plasmid or DNA fragment is used with about 1-2 units of the appropriate restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the restriction enzyme manufacturer. Usually an incubation time of about 1 or 2 hours at 37 ° C is adequate, but some enzymes require higher temperatures. After incubation, enzymes and other impurities are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and DNA is recovered from the aqueous fraction by precipitation with ethanol.
[0049]
In order to ligate DNA fragments together to obtain a functional vector, the ends of these DNA fragments must be matched to each other. In some cases, these ends are compatible in situ after endonuclease digestion. However, to make them compatible for ligation, it may be necessary to first convert the sticky ends, usually produced by endonuclease digestion, to blunt ends. To smooth the ends, the DNA is at least 15 ° C. at 15 ° C. with 10 units of DNA polymerase I Klenow fragment (Klenow) in the presence of four deoxynucleotide triphosphates in a suitable buffer. Process for minutes. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
[0050]
DNA gel electrophoresis can be used to size separate and select the cleaved DNA fragments. DNA can be electrophoresed on either agarose or polyacrylamide matrices. The choice of matrix will depend on the size of the DNA fragment to be separated. After electrophoresis, by electroelution, as described in Sambrook et al. (Supra), sections 6.30-6.33, or by melting agarose and extracting DNA therefrom when low melting agarose is used as the matrix. DNA is extracted from the matrix.
[0051]
DNA fragments to be ligated together (predigested with appropriate restriction enzymes so that the ends of each fragment to be ligated fit) are placed in solution in approximately equal amounts. This solution will also contain ATP, ligase buffer and ligase, eg about 10 units of T4 DNA ligase per 0.5 μg of DNA. When ligating a DNA fragment into a vector, the vector is first linearized by cutting with the appropriate restriction endonuclease (s). The linearized vector is then treated with alkaline phosphatase or bovine intestinal phosphatase. This phosphatase treatment prevents self-ligation of the vector during the ligation process.
[0052]
After ligation, the vector having the foreign gene inserted is introduced into an appropriate host cell. Prokaryotes are the preferred host cells for the present invention. Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli X1776 (ATCC No. 31,537), E. coli XL-1 Blue (stratagene) And many other strains of E. coli (eg, HB101, NM522, NM538, NM539) and many other prokaryotic genera and species can be used as well. In addition to the E. coli strains listed above, Bacillus (for example,Bacillus Subtilis), Other enteric bacteria (e.g.,Salmonella typhimuriumOrSerratia marcesans), And variousPseudomonasAll species can also be used as hosts.
[0053]
Prokaryotic transformation is readily accomplished using the calcium chloride method described in Section 1.82 of Sambrook et al. (Supra). According to another method, electroporation [Neumann et al.,EMBO J. 1: 841 (1982)] can be used to transform these cells. Transformed cells are usually selected by growth on antibiotics such as tetracycline (tet) or ampicillin (amp) (transformed cells are identified by the presence of tet and / or amp resistance genes on the vector. It is resistant to the substance).
[0054]
After selection of transformed cells, these cells are grown in culture and then the plasmid DNA (or other vector into which the foreign gene has been inserted) is isolated. Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale DNA preparation and the large scale DNA preparation described in Sambrook et al. (Supra), sections 1.25 to 1.33. The isolated DNA can be purified by methods known in the art, such as those described in Sambrook et al. (Supra), section 1.40. The purified plasmid DNA is then analyzed by restriction mapping and / or DNA sequencing. Usually, DNA sequencing is performed by Messing et al.Nucleic Acids Res. 9: 309 (1981)] or Maxam et al. [Meth.Enzymol. 65: 499 (1980)].
[0055]
IV.Gene fusion
The present invention contemplates fusing a gene (gene 1) encoding a desired polypeptide to a second gene (gene 2) so that a fusion protein is generated during transcription. Usually, gene 2 is a phage coat protein gene, preferably the gene III coat protein of phage M13 or a fragment thereof. Fusion of genes 1 and 2 can be achieved by inserting gene 2 at a specific site on the plasmid containing gene 1 or by inserting gene 1 at a specific site on the plasmid containing gene 2. it can.
[0056]
Insertion of a gene into the plasmid requires that the plasmid be cleaved at the exact location where the gene is to be inserted. That is, a restriction endonuclease site must be present at this position (preferably the only site so that the plasmid is cleaved at only one place during restriction endonuclease digestion). The plasmid is digested, treated with phosphatase and purified as described above. The gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be achieved when the end of the plasmid matches the end of the gene to be inserted. When the plasmid is cut using restriction enzymes and the gene to be inserted is isolated (creating blunt ends or compatible cohesive ends), bacteriophage is used as described in section 1.68 of Sambrook et al. DNA can be directly ligated together using a ligase such as phage T4 DNA ligase and incubating the mixture at 16 ° C. for 1-4 hours in the presence of ATP and ligase buffer. When the ends are not compatible, they are first added to the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase (which contains four deoxyribonucleotides to fill the overhanging single stranded ends of the digested DNA. Must be smoothed by the use of triphosphate). Alternatively, these ends can be made blunt using nucleases such as nuclease S1 or mung bean nuclease, which function by retreating the overhanging single strands of DNA. The DNA is then ligated using the above ligase. In some cases, the reading frame of the coding region changes, so that the end of the gene to be inserted cannot be smoothed. Oligonucleotide linkers may be used to overcome this problem. This linker serves as a bridge connecting the plasmid and the gene to be inserted. These linkers can be prepared synthetically as double-stranded or single-stranded DNA using conventional methods. These linkers have one end that matches the end of the gene to be inserted and are first linked to this gene using the ligation method described above. The other end of the linker is designed to be compatible with the ligation plasmid. When designing the linker, care must be taken not to destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid. In some cases, it may be necessary to design the linker such that they encode a portion of an amino acid, or encode one or more amino acids.
[0057]
A DNA encoding a stop codon can be inserted between gene 1 and gene 2. Such stop codons are UAG (Amber), UAA (Ocher) and UGA (Opel) [Davis et al., Microbiology, Harper & Row, New York, pp. 237, 245-47 and 274 (1980)]. A stop codon expressed in the wild-type host cell results in the synthesis of the gene 1 protein product to which the gene 2 protein is not bound. However, growth in suppressor host cells results in the synthesis of a detectable amount of fusion protein. Such suppressor host cells contain a tRNA that has been modified to insert an amino acid at the stop codon position of the mRNA, thus yielding a detectable amount of fusion protein. Such suppressor host cells include, for example, E. coli suppressor strains [Bullock et al.,Bio techniques 5: 376-379 (1987)] and is disclosed. Any accepted method can be used to place such a stop codon in the mRNA encoding the fusion polypeptide.
[0058]
This suppressible codon can be inserted between the first gene encoding the polypeptide and the second gene encoding at least a portion of the phage coat protein. Alternatively, this suppressible stop codon can be inserted adjacent to the fusion site by substitution of the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein. Growth of phagemids containing this suppressible codon in suppressor host cells results in detectably producing a fusion polypeptide containing the polypeptide and coat protein. When this phagemid is grown in a non-suppressor host cell, the polypeptide not fused to the phage coat protein is substantially freed by termination at the inserted repressible triplet encoding UAG, UAA or UGA. Synthesized. In non-suppressor cells, the polypeptide is synthesized and secreted from the host cell by the absence of fused phage coat protein (otherwise immobilizing the polypeptide to the host cell).
[0059]
V.Modification (mutation) of gene 1 at the selected position
Gene 1 encoding the desired polypeptide can be modified at one or more selected codons. A modification is defined as a substitution, deletion or insertion of one or more codons in the gene encoding the polypeptide. This modification results in a change in the amino acid sequence of the polypeptide as compared to the unmodified or native sequence of the same polypeptide. Preferably, this modification is by replacing at least one amino acid with any other amino acid in one or more regions of the molecule. This modification can be done by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated mutagenesis and cassette mutagenesis.
[0060]
A.Oligonucleotide-mediated mutagenesis
Oligonucleotide-mediated mutagenesis is a preferred method for preparing gene 1 substitution, deletion and insertion variants. This method is described in Zoller et al.Nucleic Acids Res. 10: 6487-6504 (1987)] is well known in the art. Briefly, gene 1 is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template of a plasmid containing gene 1 of unmodified or native DNA sequence. . After hybridization, DNA polymerase is used to synthesize the complete second complementary strand of this template into which the oligonucleotide primer has been introduced and encodes the selected modification in gene 1.
[0061]
Usually, oligonucleotides with a length of at least 25 nucleotides are used. Optimal oligonucleotides will have 12-15 nucleotides that are perfectly complementary to the template on either side of the nucleotide (s) encoding the mutation. This ensures that the oligonucleotide will hybridize properly to the single stranded DNA template molecule. This oligonucleotide can be obtained by methods known in the art, such as Crea et al.Proc.Natl.Acad.Sci.USA 75: 5765 (1978)] and is easily synthesized.
[0062]
This DNA template may be a vector derived from a bacteriophage M13 vector (M13mp18 and M13mp19 vectors available from commercial sources are suitable) or Veira et al.Meth.Enzymol. 153: 3 (1987)] can be obtained only by vectors containing a single-stranded phage origin of replication. That is, the DNA to be mutated must be inserted into one of these vectors in order to create a single stranded template. The preparation of single-stranded templates is described in Sambrook et al. (Supra), sections 4.21 to 4.41.
[0063]
In order to modify the native DNA sequence, the oligonucleotide is hybridized to a single stranded template under suitable hybridization conditions. A DNA polymerase, usually the Klenow fragment of DNA polymerase I, is then added and the complementary strand of the template is synthesized using this oligonucleotide as a primer for synthesis. In this way, a heteroduplex molecule in which one strand of DNA encodes a mutant form of gene 1 and the other strand (first template) encodes the natural unmodified sequence of gene 1. Is formed. This heteroduplex molecule is then introduced into a suitable host cell, usually a prokaryote such as E. coli JM101. The cells are grown and then plated on agarose plates and screened with 32-phosphate radiolabeled oligonucleotide primers to identify bacterial colonies containing mutated DNA.
[0064]
The method described immediately above can be modified to create a heteroduplex molecule in which both strands of the plasmid contain the mutation (s). This modification is as follows. A single stranded oligonucleotide is annealed to a single stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribothymidine (dTTP), is available from a modified thio-deoxyribocytosine (Amersham) called dCTP- (aS) ). This mixture is added to the template-oligonucleotide complex. When DNA polymerase is added to this mixture, a DNA strand identical to the template is created except for the mutated base. In addition, this new DNA strand contains dCTP- (aS) instead of dCTP, which serves to protect this strand from restriction endonuclease digestion. After nicking the heteroduplex template strand with an appropriate restriction enzyme, the template strand is digested with ExoIII nuclease or another suitable nuclease beyond the region containing the site (s) to be mutated. Can do. The reaction is then stopped, leaving a molecule that is only partially single-stranded. A complete DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP and DNA ligase. This homoduplex molecule can then be introduced into a suitable host cell such as E. coli JM101 as described above.
[0065]
Mutants with more than one amino acid to be substituted can be created by any of several methods. When these amino acids are in close proximity to each other in a polypeptide chain, they can be mutated simultaneously using one oligonucleotide encoding all of the desired amino acid substitutions. However, when amino acids are present at some distance from each other (separated by about 10 or more amino acids), it is comparative to create a single oligonucleotide that encodes all of the desired changes. Difficult. Alternatively, one of two different methods can be used.
[0066]
In the first method, an independent oligonucleotide is created for each amino acid to be substituted. If these oligonucleotides are then simultaneously annealed to the single stranded template DNA, the second DNA strand synthesized from this template will encode all of the desired amino acid substitutions. This first round is the same as described for the single mutation. That is, using wild-type DNA as a template, an oligonucleotide encoding the first desired amino acid substitution (s) is annealed to this template, and then a heteroduplex DNA molecule is created. In the second round of mutagenesis, the mutated DNA obtained in the first round of mutagenesis is used as a template. That is, the template already contains one or more mutations. An oligonucleotide encoding another desired amino acid substitution (s) is then annealed to this template and the resulting DNA strand encodes a mutation from both the first and second rounds of mutagenesis. doing. The obtained DNA can be used as a template in the third round of mutagenesis.
[0067]
B.Cassette mutagenesis
This method is also a preferred method for preparing gene 1 substitution, deletion and insertion mutants. This method is described by Wells et al.Gene 34: 315 (1985)]. The starting material is a plasmid (or other vector) containing gene 1, which is the gene to be mutated. The codon in gene 1 to be mutated has been determined. There must be a unique restriction endonuclease site on either side of the determined mutation site (s). If such restriction sites do not exist, these sites can be created using the oligonucleotide-mediated mutagenesis method described above and introduced at an appropriate position in gene 1. After introducing restriction sites into the plasmid, the plasmid is cut and linearized at these sites. Double-stranded oligonucleotides encoding the DNA sequence between these restriction sites and containing the desired mutation are synthesized by conventional methods. The two strands are synthesized separately and then hybridized together by conventional methods. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends that match both ends of the linearized plasmid so that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence of gene 1.
[0068]
VI.Preparation of DNA encoding the desired protein
In another aspect, the invention contemplates the production of a desired protein variant containing one or more subunits. Usually, each subunit is encoded by a different gene. Each gene encoding each subunit can be obtained by methods known in the art (see, eg, Section II). In some cases, it may be necessary to obtain genes encoding various subunits using multiple independent methods selected from any of the methods described in Section II.
[0069]
When constructing a replicable expression vector in which the desired protein contains more than one subunit, all subunits are usually the same promoter located 5 'to the DNA encoding the subunit. Alternatively, each subunit can be regulated by an independent promoter appropriately oriented in the vector, each promoter being operably linked to the DNA to be regulated. Promoter selection is performed as described in Section III above.
[0070]
In constructing a replicable expression vector containing DNA encoding a desired protein having multiple subunits, reference should be made to FIG. 10, which illustrates the vector for purposes of illustration. The DNA encoding each subunit of the antibody fragment is shown. This figure usually shows that one of the desired protein subunits is fused to a phage coat protein such as the M13 gene III. Usually, this gene fusion will contain its own signal sequence. It can be seen that another gene encodes another subunit or group of subunits, and each subunit usually has its own signal sequence. FIG. 10 also shows that a single promoter can regulate the expression of both subunits. In contrast, each subunit may be independently regulated by a different promoter. Desired protein subunit-phage coat protein fusion constructs can be prepared as described in Section IV above.
[0071]
In constructing a group of variants of the desired multi-subunit protein, the DNA encoding each subunit in the vector may be mutated at one or more positions in each subunit. When multi-subunit antibody variants are constructed, preferred mutagenesis sites encode amino acid residues that are located in the complementarity determining regions (CDRs) of either the light chain, heavy chain, or both chains. Matches the existing codon. This CDR is usually called a hypervariable region. The method of mutating the DNA encoding each subunit of the desired protein is carried out substantially as described in Section V above.
[0072]
VII.Preparation of target molecule and binding with phagemid
Target proteins such as receptors can be isolated from natural sources or prepared by recombinant methods by methods known in the art. Illustratively, the glycoprotein hormone receptor is McFarland et al.Science 245: 494-499 (1989)], and the non-glycosylated form expressed in E. coli is Fuh et al.J. Biol. Chem. 265: 3111-3115 (1990)]. Other receptors can be prepared by conventional methods.
[0073]
Purified target protein can be used in a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate, polyacrylic acid and polymethacrylic acid copolymers, nylon, neutral and ionic carriers Can be combined. Binding of target proteins to the matrix has been described in the literature [Methods in Enzymology 44 (1976)] or by other means known in the art.
[0074]
After binding the target protein to the matrix, the immobilized target is contacted with a library of phagemid particles under conditions suitable to bind at least a portion of the phagemid particle to the immobilized target. Usually, conditions including pH, ionic strength, temperature, etc. will be similar to physiological conditions.
[0075]
Bound phagemid particles (“conjugates”) with high affinity for the immobilized target are separated from the low affinity particles (and thus those that do not bind to the target) by washing. The conjugate can be dissociated from the immobilized target by various methods. These methods include wild-type ligands, competitive dissociation using pH and / or alteration of ionic strength, and methods known in the art.
[0076]
A suitable host cell is infected with the conjugate and helper phage, and the host cell is cultured under conditions suitable for amplification of phagemid particles. The phagemid particles are then collected and the selection process is repeated one or more times until a conjugate with the desired affinity for the target molecule is selected.
[0077]
If desired, the library of phagemid particles can be sequentially contacted with more than one immobilized target to improve selectivity for a particular target. For example, ligands such as hGH often have more than one natural receptor. In the case of this hGH, both growth hormone receptor and prolactin receptor bind to the hGH ligand. It would be desirable to improve the selectivity of hGH for growth hormone receptors over prolactin receptors. This is done by first contacting the phagemid particle library with the immobilized prolactin receptor, eluting it for the prolactin receptor with low affinity (ie, lower than wild type hGH) and then this low affinity prolactin “binding”. This can be achieved by contacting the “body” or non-conjugate with an immobilized growth hormone receptor and selecting a high affinity growth hormone receptor conjugate. In this case, hGH mutants with low affinity for the prolactin receptor have therapeutic use even when the affinity for the growth hormone receptor is slightly lower than that of wild-type hGH. It will be. This same method can be used to improve the selectivity of a specific hormone or protein for a primary functional receptor over its clearance receptor.
[0078]
In another aspect of the invention, improved substrate amino acid sequences can be obtained. These may be useful in creating better “cleavage sites” for protein linkers or for better protease substrates / inhibitors. In this embodiment, a molecule that can be immobilized (eg, hGH-receptor, biotin-avidin, or a molecule that can be covalently bound to a matrix) is fused to gene III by a linker. This linker is preferably 3-10 amino acids in length and acts as a substrate for the protease. A phagemid is constructed as described above (where a random library of phagemid particles having a different amino acid sequence at the binding site is prepared by random mutagenesis in the DNA encoding the linker region). The library of phagemid particles is then immobilized on a matrix and exposed to the desired protease. Phagemid particles having a preferred or better substrate amino acid sequence in the liner region of the desired protease will be eluted and an enriched pool of phagemid particles encoding the preferred linker will first be obtained. The phagemid particles are then iterated several more times to obtain an enriched pool of particles encoding the consensus sequence (s) (see Examples XIII and XIV).
[0079]
VIII.Growth hormone variants and methods of use
The cloned gene of hGH is expressed secreted in Eschericha cola [Chang, C.N. et al.,Gene 55: 189 (1987)], and its DNA and amino acid sequences have been reported [Goeddel et al.,Nature 281: 544 (1979); Gray et al.,Gene 39: 247 (1985)]. The present invention describes a novel hGH variant obtained using the phagemid selection method. Human growth hormone variants containing substitutions at positions 10, 14, 18, 21, 167, 171, 172, 174, 175, 176, 178 and 179 have been described. Variants with relatively high binding affinity are listed in Tables VII, XIII and XIV. Amino acid nomenclature for describing these variants is described below. Growth hormone variants may be administered and formulated in the same manner as regular growth hormone. The growth hormone variants of the invention can be expressed in any recombinant system capable of expressing natural or met hGH.
[0080]
Formulations of hGH for therapeutic administration include carriers, excipients or stabilizers that are optionally physiologically acceptable to the desired degree of purification of hGH.Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)] and prepared for storage in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers (eg, phosphates, citrates, and other organic acids). Buffer); antioxidants (including ascorbic acid); low molecular weight (less than about 10 residues) polypeptide; protein (eg, serum albumin, gelatin, or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone) Amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (eg, EDTA); divalent metal ions (Eg zinc, cobalt or copper); sugar alcohols (eg mannitol or Rubitoru); counter ion forming the salt (e.g., sodium); and / or nonionic surface active agents [e.g., Tween, Pluronics or polyethylene glycol (PEG)] are included. The preparation of the present invention may further contain a pharmaceutically acceptable buffer, an amino acid, a bulking agent, and / or a nonionic surfactant. These include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents and the like. Examples of these would include trimethylamate salt (“Tris buffer”), and disodium edetate. Using the phagemid of the present invention, a large amount of hGH mutant free of phage protein can be produced. To express hGH variants that do not contain the gene III portion of the fusion, pS0643 and derivatives can simply be grown in non-suppressor strains such as 16C9. In this case, the amber codon (TAG) leads to the termination of translation and free hormone is obtained without the need for independent DNA construction. The hGH variant is secreted from the host and can be isolated from the culture medium.
[0081]
Any one of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175, and I179 is any amino acid other than the amino acid found at that position in the native hGH shown herein. Can be substituted. That is, 20 amino acids listed below, including all 1, 2, 3, 4, 5, 6, 7, or 8 of amino acids F10, M14, H18, H21, R167, D171, T175 and I179 shown here Can be substituted with any of the other 19 amino acids. In a preferred embodiment, all eight amino acids listed here are substituted with another amino acid. The most preferred 8 amino acids to be substituted are shown in Table XIV in Example XII.
Amino acid nomenclature
Ala (A)
Arg (R)
Asn (N)
Asp (D)
Cys (C)
Gln (Q)
Glu (E)
Gly (G)
His (H)
Ile (I)
Leu (L)
Lys (K)
Met (M)
Phe (F)
Pro (P)
Ser (S)
Thr (T)
Trp (W)
Tyr (Y)
Val (V)
The nomenclature for the one letter hGH variant is the first to describe the deleted hGH amino acid (eg, glutamate 179), and then the inserted amino acid (eg, serine), resulting in (E1795S).
[0082]
【Example】
Without further explanation, it is believed that one skilled in the art can fully practice and utilize the invention using the foregoing description and illustrative examples. That is, the following specific examples illustrate preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.
[0083]
Example 1  Plasmid construction and hGH-phagemid particle preparation
Plasmid phGH-M13gIII (FIG. 1) was transformed into M13KO7⁷And hGH production plasmid pBO473 [Cunningham, BC et al.,Science 243: 1330-1336 (1989)]. Synthetic oligonucleotide:
5'-AGC-TGT-GGC-TTC-GGG-CCC-TTA-GCA-TTT-AAT-GCG-GTA-3 '
Was used to introduce a unique ApaI restriction site (underlined) after the last Phe191 codon of hGH in pBO473. Oligonucleotide:
5'-TTC-ACA-AAC-GAA-GGG-CCC-CTA-ATT-AAA-GCC-AGA-3 '
Was used to introduce a unique ApaI restriction site (first underlined) and a Glu197-amber stop codon (second underlined) into the M13KO7 gene III. Oligonucleotide:
5'-CAA-TAA-TAA-CGG-GCT-AGC-CAA-AAG-AAC-TGG-3 '
To introduce a unique NheI site (underlined) after the 3 'end of the gene III coding sequence. The resulting 650 base pair (bp) ApaI-NheI fragment from doubly mutated M13KO7 gene III was cloned into the large ApaI-NheI fragment of pBO473 to create plasmid pSO132. This fused the hGH carboxy terminus (Phe 191) to the Pro 198 residue of the gene III protein, inserting the glycine residue encoded from the Apa I site, and the fusion protein was transformed into the E. coli alkaline phosphatase (phoA) promoter and the stII secretion signal Sequence [Chang, CN et al.,Gene 55: 189-196 (1987)]. The phoA promoter was replaced with the lac promoter and operator for inducible expression of the fusion protein in rich medium. A 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and the Cap binding site was oligonucleotides:
5'-CACGACAGAATTCCCGACTGGAAA-3 'and 5'-CTGTTTCTAGAGTGAAATTGTTA-3 '
[These border the desired lac sequence and introduce EcoRI and XbaI restriction sites (underlined)]
Was prepared by PCR of plasmid pUC119 using This lac fragment was gel purified and ligated to the large EcoRI-XbaI fragment of pSO132 to create plasmid phGH-M13gIII. The sequence of all processed DNA junctions was confirmed by dideoxy sequencing [Sanger, F. et al.Proc.Natl.Acad.Sci.USA 74: 5463-5467 (1977)]. R64A mutant hGH phagemid was constructed as follows. That is, Arg64 Ala substitution (R64A) [Cunningham, BC, Wells, J.A.Science 244: 1081-1085 (1989)], the NsiI-BglII mutant fragment of hGH [Cunningham et al. (Supra)] was cloned between the corresponding restriction sites in the phGH-M13gIII plasmid (FIG. 1). To replace the wild type hGH sequence. The R64A hGH phagemid particles were grown and titrated as described below for wild type hGH-phagemid.
[0084]
The plasmid was introduced into a male strain of E. coli (JM101) and selected on carbenicillin plates. Single transformants were grown for 4 hours at 37 ° C. in 2YT medium (2 ml) and infected with M13KO7 helper phage (50 μl). The infected culture was diluted to 2YT (30 ml), incubated overnight, and phagemid particles were collected by precipitation with polyethylene glycol [Vierra, J., Messing, J.,Methods in Enzymology 153: 3-11 (1987)]. Usually, the titer of phagemid particles is 2-5x10¹¹It was within the range of cfu / ml. CsCl density centrifuge [Day, L.A.,J. Mol. Biol. 39: 265-277 (1969)] to remove all fusion protein unbound to the virion.
[0085]
Example II  Immunochemical analysis of hGH on fusion phage
Rabbit polyclonal antibodies against hGH were purified using protein A and coated on microtiter plates (Nunc) in 50 mM sodium carbonate buffer (pH 10) at a concentration of 2 μg / ml at 4 ° C. for 16-20 hours. After washing in PBS containing 0.05% Tween 20, hGH or hGH-phagemid particles were washed with buffer A [50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg / ml bovine serum albumin, and 05% Tween 20] in 2.0-0.002nMSerially diluted. After 2 hours at room temperature (rt), the plate was washed thoroughly and the indicated Mab [Cunningham et al. (Above)] was added at 1 μg / ml in buffer A for 2 hours at rt. After washing, horseradish peroxidase-conjugated goat anti-mouse IgG antibody was conjugated for 1 hour at rt. After the last wash, peroxidase activity was assayed using the substrate o-phenylenediamine.
[0086]
Example III  Binding and enrichment of hGH binding protein to polyacrylamide beads
Purification of an hGH receptor containing an extra cysteine residue introduced by oxirane polyacrylamide beads (Sigma) by site-directed mutagenesis at position 237 (does not adversely affect hGH binding; J. Wells: unpublished) Extracellular domain (hGHbp) [Fuh, G. et al.,J. Biol. Chem. 265: 3111-3115 (1990)]. This hGHbp is based on the resin [¹²⁵I] Conjugated to an amount of 1.7 pmol hGHbp / mg dry oxirane beads as measured by hGH binding. All unreacted oxirane groups were then blocked with BSA and Tris. As a control for non-specific binding of phagemid particles, BSA was similarly bound to the beads. The buffer for adsorption and washing contained 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg / ml BSA and 0.02% Tween 20. The elution buffer is 200 in addition to the wash buffer.nM Contains hGH or 0.2M glycine (pH 2.1). Parental phage M13KO7 was mixed with hGH phagemid particles in a ratio of approximately 3000: 1 (initial mixture) and in either 50 μl volume at room temperature with either absorbent (5 μl each; 0.2 mg acrylamide beads) for 8-12 hours. Mixed. The beads were pelleted by centrifugation and the supernatant was carefully removed. The beads were resuspended in wash buffer (200 μl) and mixed for 4 hours at room temperature (wash 1). After the second wash (wash 2), the beads were washed 200 timesnMWas eluted with hGH for 6 to 10 hours (eluent 1, eluent 2). The final elution was performed for 4 hours using glycine buffer (pH 2.1) to remove the remaining hGH phagemid particles (eluate 3). Each fraction was diluted appropriately in 2YT medium, mixed with fresh JM101, incubated at 37 ° C. for 5 minutes, and plated on LB or LB carbenicillin plates using 2YT soft agar (3 ml).
[0087]
Example IV  Construction of hGH-phagemid particles by a mixture of gene III products
The gene III protein consists of 410 residues and is divided into two domains separated by a variable linker sequence [Armstrong, J. et al.,FEBS Lett. 135: 167-172 (1981)]. The amino-terminal domain is required for attachment to E. coli pili, whereas the carboxy-terminal domain is incorporated into the coat of the phage and is required for proper phage assembly [Crissman, JW, Smith, GP ,Virology 132: 445-455 (1984)]. The signal sequence and amino terminal domain of gene III were replaced with the stII signal and the entire hGH gene [Chang et al. (Supra)] by fusion to residue 198 in the carboxy terminal domain of gene III (FIG. 1). This hGH-gene III fusion was placed under the control of the lac promoter / operator in a plasmid (phGH-M13gIII; FIG. 1) containing the β-lactamase gene of pBR322 and the ColE1 origin of replication and the intergenic region of phage f1. This vector can be easily maintained as a small plasmid vector by selection with carbenicillin, thereby avoiding reliance on a functional gene III fusion for growth. This plasmid can also be efficiently packaged into virions (ie, phagemid particles) by infection with helper phage such as M13KO7 [Viera et al. (Supra)], thereby avoiding the problem of phage assembly. . The infectious titer of phagemid based on transduction for carbenicillin resistance in this system is 2-5 × 10¹¹Changed to colony forming units (cfu) / ml. The titer of M13KO7 helper phage in these phagemid stocks is -10^TenPlaque forming units (pfu) / ml.
[0088]
This system was used to confirm previous studies [Parmley, Smith (supra)] that homogeneous expression of large proteins fused to gene III is detrimental to phage production (data not shown). For example, induction of the lac promoter in phGH-M13gIII by the addition of IPTG resulted in low phagemid titers. Furthermore, the phagemid particles obtained by co-infection with M13KO7 containing an amber mutation in gene III have very low phagemid titers (<10^Tencfu / ml). We thought that multiple copies of the gene III fusion bound to the phagemid surface could lead to multiple point binding (“chelation”) of the fused phage to the immobilized target protein. Thus, to control the copy number of the fusion protein, E. coli JM101 (lacl containing a constitutively high level of lac repressor protein).^QThe transcription of the hGH-gene III fusion was restricted by culturing the plasmid in). E. coli JM101 cultures containing phGH-M13gIII were maximally grown and infected with M13KO7 in the absence of the lac operon inducer (IPTG) (but this system is flexible and other genes III Can be equilibrated with the co-expression of the fusion protein). From the ratio of the amount of hGH per virion (based on the hGH immunoreactive material in the phagemid purified with a CsCl gradient), approximately 10% of the phagemid particles contain one copy of the hGH gene III fusion protein. It is approximated. Therefore, the titer of the fusion phage displaying the hGH gene III fusion is about 2-5 × 10^Ten/ Ml. This number represents the titer of E. coli in the culture that led them (-10⁸-10⁹/ Ml). That is, on average all E. coli cells produce 10-100 copies of phage decorated with hGH gene III fusion protein.
[0089]
Example V  Structural integrity of hGH-gene III fusion
Immunoblot analysis of the hGH-gene III phagemid (FIG. 2) shows that hGH cross-reactive material migrates simultaneously with the phagemid particles in the agarose gel. This indicates that hGH is firmly bound to the phagemid particles. The hGH-gene III fusion protein from this phagemid particle migrates as a single immunostained band, indicating that there is little degradation of hGH when hGH binds to gene III. Since about 25% of the phagemid particles are infectious, wild type gene III protein is clearly present. This is similar to the concentration assessed by UV absorption, and the specific infectivity assessment performed on wild-type M13 phage, similarly purified (by CsCl density gradient method) [Smith, GP (above) and Parmley, Smith ( the above)〕. That is, both wild type gene III and hGH-gene III fusion proteins are displayed in the phage pool.
[0090]
It was important to confirm that the tertiary structure of the displayed hGH was maintained in order to obtain the confidence that the result of the binding selection was converted to a natural protein. The structural integrity of the indicated hGH gene III fusion proteins was assessed using a monoclonal antibody (Mab) against hGH (Table I).

[0091]
These epitopes on hGH for Mabs have been mapped [Cunningham et al. (Supra)] and binding to 7 of the 8 Mabs requires hGH to be properly folded. All Mab ICs₅₀The values were equivalent to wild type hGH except for Mabs 5 and 6. Both Mabs 5 and 6 are known to have binding determinants near the carboxy terminus of hGH that is blocked in the gene III fusion protein. Relative IC of Mab1 reacting with both natural and modified hGH₅₀The values remain unchanged compared to conformationally sensitive Mabs 2-5, 7 and 8. That is, Mabl serves as a good internal control for any errors in adapting the concentration of hGH standards to the concentration of hGH-gene III fusion.
[0092]
Example VI  Enrichment of binding to receptor affinity beads
Previous investigators [Parmley, Smith (above); Smith (above); Cwirla et al. (Above); and Devlin et al. (Above]) have selected by sorting on streptavidin-coated polystyrene Petri dishes or microtiter plates. Phage was fractionated. However, the chromatographic system allows for a more efficient fractionation of phagemid particles displaying muteins with different binding affinities. We selected non-porous oxirane beads (Sigma) to avoid capture of phagemid particles in the chromatography resin. In addition, these beads have a small particle size (1 μm), maximizing the ratio of surface area to quantity. The extracellular domain (hGHbp) of the hGH receptor containing free cysteino residues [Fuh et al. (Supra)] binds efficiently to these beads and the phagemid particles bind to bovine serum albumin only. It showed very low non-specific binding (Table II).
[0093]

[0094]
In a representative enrichment experiment (Table II), 1 part of hGH phagemid was mixed with> 3,000 parts of M13KO7 phage. After one cycle of binding and elution, 10⁶Of phage were recovered and the ratio of phagemid to M13KO7 phage was 2: 1. That is,> 5000 times enrichment was obtained by one binding selection step. Further elution was obtained by additional elution with free hGH or acid treatment to remove residual phagemids. This enrichment was comparable to the enrichment obtained by Smith and co-workers using batch elution from coated polystyrene plates [Smith, G.P. (above) and Parmley, Smith (above)]. However, a smaller volume is used for the beads (200 μl vs 6 ml). When using beads conjugated only to BSA, there was little enrichment of hGH phagemid over M13KO7. The slight enrichment observed for the control beads (-10-fold to pH 2.1 elution; Table 2) is due to trace impurities of bovine growth hormone binding protein present in BSA binding to the beads. It will be a thing. Nevertheless, these data indicate that hGH phage enrichment is dependent on the presence of hGHbp on the beads, suggesting that binding occurs by a specific interaction between hGH and hGHbp.
[0095]
We evaluated the enrichment of wild-type hGH over a relatively weak binding mutant of hGH on the fusion phagemid to further investigate the specificity of the enrichment and to determine the reduction of binding affinity for purified hormone after selection of the fusion phagemid. Associated with the enrichment factor. A fusion phagemid was constructed using an hGH mutant (R64A) in which Arg64 was replaced with Ala. This R64A mutant hormone has about 20-fold decrease in receptor binding affinity compared to hGH [Kd values of 7.1 nM and 0.34 nM, respectively, Cunningham, Wells (above)]. The titer of R64A hGH-gene III fusion phagemid was comparable to that of wild type hGH phagemid. After one round of binding and elution (Table III), the wild-type hGH phagemid was derived from a mixture of two phagemids and M13KO7, 8 fold for phagemid R64A and -10 for M13KO7 helper phage.^FourEnriched with.
[0096]

[0097]
By displaying a mixture of wild type gene III and gene III fusion proteins on phagemid particles, virions that display large properly folded proteins as fusions to gene III can be assembled and propagated. Effectively control the copy number of the gene III fusion protein to avoid “chelation” that is still maintained at a sufficiently high level in the phagemid pool, avoiding large epitope libraries (> 10^Ten) Can be selected. We have shown that hGH (22 kD protein) can be displayed in its native folded form. Binding selections eluted with free hGH and performed on receptor affinity beads efficiently enriched wild-type hGH phagemids over mutant hGH phagemids that were shown to have low receptor binding affinity. . That is, it is possible to select phagemid particles whose binding constant is reduced in the nanomolar range.
Protein-protein and antibody-antigen interactions are governed by discontinuous epitopes [Janin, J. et al.,J. Mol. Biol. 204: 155-164 (1988); Argos, P.,Prot.Eng. 2: 101-113 (1988); Barlow, D.J., et al.,Nature 322: 747-748 (1987); and Davies, D.R., et al.,J. Biol. Chem. 263: 10541-10544 (1988)]. That is, residues that are directly involved in binding are close in tertiary structure but are separated by residues that are not involved in binding. The screening system provided by the present invention more advantageously allows for the analysis of protein-receptor interactions and the isolation of discontinuous epitopes in proteins with novel and high affinity binding properties. It is.
[0098]
Example VII  Selection of hGH mutants from libraries randomized at hGH codons 172, 174, 176, 178
Template construction
Mutants of the hGH-gene III fusion protein are described by Kunkel et al.Meth.Enzymol. 154: 367-382 (1987)]. Template DNA is propagated in CJ236 cells with plasmid pS0132 (containing the native hGH gene fused to the carboxy-terminal half of M13 gene III under the control of the alkaline phosphatase promoter) with M13-K07 phage as a helper Prepared. Single-stranded uracil-containing DNA is prepared for mutagenesis and (1) mutations in hGH that greatly reduce binding to hGH binding protein (hGHbp); and (2) parental background phage is assayed. Introduced a unique restriction site (KpnI) that could be used to select against it. T7 DNA polymerase and the following oligodeoxynucleotides:

Was used for oligonucleotide-directed mutagenesis. This oligo introduces a KpnI site (shown above) with mutations (R178G, I179T) in hGH. Clones from the mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. This resulting construct used as a template for random mutagenesis was named pHO415.
[0099]
h Random mutagenesis in helix 4 of GH
Again, using Kunkel's method, codons 172, 174, 176, 178 were targeted for random mutagenesis in hGH. A single-stranded template is prepared from pHO415 as described above and the following oligos:

Mutagenesis was performed using a pool of As indicated above, this oligopool reverted codon 179 to wild type (Ile), destroyed the unique KpnI site of pH0415, and random codons (NNS; where 172, 174, 176 and 178; , N is A, G, C or T and S is G or C). Using this codon selection for the above sequence, no additional KpnI site can be created. This selection of NNS synonymous sequences resulted in 32 possible codons at 4 sites (one “stop” codon and at least one codon for each amino acid) for a total of (32)^Four= 1,048,576 possible nucleotide sequences (12% of which contain at least one stop codon), or (20)^Four= 160,000 possible polypeptide sequences and 34,481 immature terminated sequences (ie sequences containing at least one stop codon) are obtained.
[0100]
First library propagation
The mutagenized product was extracted twice with phenol: chloroform (50:50) and ethanol precipitated with excess carrier tRNA to avoid the addition of salts that complicate later electroporation steps. About 50 ng (15 fmol) of DNA in a total volume of 45 μl in a 0.2 cm cuvette with a single pulse voltage setting of 2.49 kV (time constant = 4.7 msec) (2.8 × 10 4 cells)^TenCells / ml) were electroporated.
[0101]
The cells were allowed to recover for 1 hour at 37 ° C. with shaking and then mixed with 2YT medium (25 ml), 100 μg / ml carbenicillin, and M13-K07 (multiplicity of infection = 1000). By plating serial dilutions from this culture onto carbenicillin-containing medium, 8.2 × 10 8⁶It was found that an electrotransformant was obtained. After 10 minutes at 23 ° C., the cultures were incubated overnight (15 hours) at 37 ° C. with shaking.
[0102]
After overnight incubation, the cells were pelleted and a double stranded DNA (dsDNA) designated pLIB1 was prepared by the alkaline lysis method. The supernatant was centrifuged once more to remove all remaining cells, and the phage designated phage pool φ1 was PEG precipitated and re-suspended in STE buffer [10 mM Tris (pH 7.6), 1 mM EDTA, 50 mM NaCl] (1 ml). Suspended. The phage titer is determined by hGH-g3p gene III fusion (hGH-g^Three) Recombinant phagemids containing plasmids were measured as colony forming units (CFU) and for M13-K07 helper phage as plaque forming units (PFU).
[0103]
Immobilization h GH bp Join selection with
1. Binding: Phage pool φ1 (6 × 10⁹CFU, 6x10⁷A portion of PFU) was diluted 4.5 times with buffer A (phosphate buffered saline, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal) and treated with 1.5 ml of silane. Were mixed with a suspension (5 μl) of oxirane-polyacrylamide beads conjugated with hGHbp (350 fmol) containing the Ser237Cys mutation. As a control, an equal volume of phage was mixed in a separate tube with beads coated with BSA alone. The phage was bound to the beads by incubating at room temperature (23 ° C.) for 3 hours with slow rotation (about 7 rpm). The following steps were performed at room temperature using a constant volume of 200 μl.
[0104]
2. Washing: The beads were spun for 15 seconds and the supernatant was removed (Supernatant 1). The beads were washed twice by resuspending in buffer A to remove non-specifically bound phage / phagemid and then pelleted. The final wash was performed by spinning the beads in buffer A for 2 hours.
[0105]
3. hGH elution: Phage / phagemids that were weakly bound to the beads were removed by step elution with hGH. In the first step, the beads were spun using buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted, resuspended in buffer A containing 20 nM hGH, rotated for 3 hours, and then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH, spun for 3 hours and then pelleted.
[0106]
4). Glycine elution: To remove the most tightly bound phagemid (ie, phagemid still bound after hGH wash), suspend the beads in glycine buffer [1M glycine, pH 2.0 with HCl] and rotate for 2 hours. And pelletized. The supernatant (fraction “G”; 200 μl) was neutralized by addition of 1M Tris base (30 μl).
[0107]
Fraction G eluted from hGHbp-beads (1 × 10⁶CFU, 5x10^FourPFU) was not substantially richer in phagemid than K07 helper phage. We believe this is due to the fact that the K07 phage packaged during propagation of the recombinant phagemid displays the hGH-g3p fusion.
[0108]
However, hGHbp-beads gave 14 times higher CFU when compared to fraction G eluted from BSA-coated control beads. This reflects the enrichment of phagemids that display tightly bound hGH over non-specifically bound phagemids.
[0109]
5. Proliferation: A fraction of fraction G eluted from hGHbp-beads (4.3 x 10^FiveCFU) was used to infect WJM101 cells in logarithmic growth phase. Transduction was performed by mixing fraction G (100 μl) with WJM101 cells (1 ml), incubating at 37 ° C. for 20 minutes, and then adding K07 (multiplicity of infection = 1000). Cultures (25 ml of 2YT and carbenicillin) were grown as described above and a second pool of phage (Library 1G, for first glycine elution) was prepared as described above.
[0110]
Phages from library 1G (FIG. 3) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained 30 times higher CFU than fraction G eluted from BSA beads in this selection. Once again, a portion of fraction G was grown in WJM101 cells and library 1G²(Indicating that this library was selected twice by glycine elution). In addition, double-stranded DNA (pLIB 1G²) Was prepared from this culture.
ds DNA K pn I test and restriction selection
[0111]
Background (KpnI⁺) PLIB 1G to reduce the amount of template²A part (about 0.5 μg) of the lysate was digested with KpnI and electroporated into WJM101 cells. These cells were grown in the presence of K07 (multiplicity of infection = 100) as described for the initial library and a new phage pool pLIB3 was prepared (FIG. 3).
[0112]
Furthermore, a part of dsDNA (about 0.5 μg) derived from the first library (pLIB1) was digested with KpnI and directly electroporated into WJM101 cells. Transformants were recovered as described above, infected with M13-K07, and grown overnight to obtain a new phage library designated phage library 2 (FIG. 3).
[0113]
Select consecutive rounds
(1) An excess (10 times CFU) of purified K07 phage (not displaying hGH) was added into the bead-bound cocktail; and (2) except that the hGH step elution was replaced with a short wash of buffer A alone. The phagemids from both pLIB2 and pLIB3 were bound, eluted and propagated in successive rounds as described above. In some cases, XL1-Blue cells were used for phagemid growth.
[0114]
Additional digestion of dsDNA with KpnI was performed before pLIB 2G prior to the final round of bead binding selection.^ThreeAnd pLIB 3G^Five(Fig. 3).
[0115]
DNA sequencing of selected phagemids
LIB 4G^Four4 independently isolated clones from LIB and 5G⁶Four independently isolated clones from were sequenced by dideoxy sequencing. All of these 8 clones have the same DNA sequence:

Had. That is, all of these encode the same mutant of hGH (E174S, F176Y). Residue 172 in these clones is Lys as in the wild type. The codon selected for 172 is also the same as wild type hGH. This is not surprising since AAG is the only lysine-codon possible from the set of synonymous “NNS” codons. Furthermore, residue 178-Arg is also identical to the wild type, but here the codon selected from the library was AAG and not the CGC found in wild type hGH (the latter codon was also a set of “NNS” codons). Is possible).
[0116]
Multiplicity of K07 infection
The multiplicity of infection of K07 infection is an important parameter in the growth of recombinant phagemid. The multiplicity of infection of K07 must be high enough to ensure that virtually all cells transformed or transfected with phagemid can package new phagemid particles. Furthermore, the concentration of wild type gene III in each cell is kept high, reducing the likelihood that multiple hGH-gene III fusion molecules will be displayed on each phagemid particle, thereby chelating upon binding. Should be reduced. However, when K07 multiplicity of infection is too high, K07 packaging will compete with recombinant phagemid packaging. We have found that an acceptable phagemid yield with only 1-10% background K07 phage is obtained when the multiplicity of infection of K07 is 100.
[0117]

[0118]
The phage pool was labeled as indicated previously (Figure 3). The multiplicity of infection (moi) means the multiplicity of K07 infection (PFU / cell) during the growth of phagemid. Enrichment of CFU over PFU is shown when purified K07 is added to the binding step. The ratio of CFU eluted from hGHbp beads over CFU eluted from BSA beads is shown. The fraction of the KpnI containing template (ie pH0415) remaining in the pool digests dsDNA with KpnI and EcoRI, migrates the product on a 1% agarose gel, and laser-scans negative ethidium bromide stained DNA. Was measured.
[0119]
hormone h Receptor binding affinity of GH (E174S, F176Y)
The fact that a single clone was isolated from two different selection pathways (FIG. 3) suggested that the double mutant (E174S, F176Y) binds strongly to hGHbp. To determine the affinity of this hGH mutant for hGHbp, we constructed this hGH mutant by site-directed mutagenesis using the plasmid pB0720. This plasmid contains the wild type hGH gene as a template and the following oligonucleotides that change codons 174 and 176:

Contains. The resulting construct pH0458B was introduced into E. coli strain 16C9 for expression of mutant hormones. hGH (E174S, F176Y) against hGHbp¹²⁵Scatchard analysis of competitive binding of I-hGH revealed that this (E174S, F176Y) mutant had a binding affinity that was at least 5.0 times stronger than that of wild-type hGH. .
[0120]
Example VIII  Selection of hGH variants from a hormone-phage helix 4 random cassette library
Human growth hormone mutants were prepared by the method of the present invention using the phagemid described in FIG.
[0121]
Prolapse - Construction of a fusogenic hormone-phage vector
We (1) improve the binding between hGH and the gene III part to further favor the display of the hGH part on phage; (2) to obtain a substantially “monovalent display” Limit expression of the fusion protein; (3) allow selection of restriction nucleases relative to the starting vector; (4) eliminate expression of the fusion protein from the starting vector; and (5) certain hGH-gene III fusion mutations. Cassette mutagenesis [Wells et al., With the goal of achieving easy expression of the corresponding free hormone from the body;Gene 34: 315-323 (1985)] and vectors for the expression of hGH-gene III fusion proteins were designed.
[0122]
Contains the origin of replication of pBR322 and f1, and contains the E. coli phoA promoter [Bass et al.,Proteins 8: 309-314 (1990)], hGH-gene III fusion protein (hGH residues 1-191, followed by one Gly residue, fused to Pro-198 of gene III) PS0132 oligonucleotide-directed mutagenesis [Kunkel et al.,Methods Enzymol. 154: 367-382 (1987)] to construct plasmid pS0643 (FIG. 9). Oligonucleotide:
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3 '
[This oligonucleotide introduces the Phe-191 of hGH followed by an XbaI site (underlined) and an amber stop codon (TAG)). In the resulting construct pS0643, part of gene III is deleted, two silent mutagenesis (underlined) occurs, and the following junction is generated between hGH and gene III:
--- hGH ---------> Gene III ----->
187 188 189 190 191 am * 249 250 251 252 253 254
Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly
GGC AGC TGT GGA TTC TAG AGT GGC GGT GGC TCT GGT
This shortens the overall size of the fusion protein from 401 residues in pS0132 to 350 residues in pS0643. Experiments with monoclonal antibodies against hGH have shown that the hGH portion of the new fusion protein assembled into phage particles is more accessible than previous long fusions.
[0123]
In order to grow phage displaying hormones, pS0643 and derivatives were transformed into E. coli amber-suppressor strains such as JM101 or XL1-Blue [Bullock et al.,BioTechniques 5: 376-379 (1987)]. Shown above is the Glu substitution at the amber codon that occurs in the supE suppressor strain. In addition, suppression by other amino acids is possible in various well-known and generally available E. coli strains.
[0124]
To express hGH (or mutants) that do not contain the gene III portion of the fusion, pS0643 and derivatives can simply be grown in non-suppressor strains such as 16C9. In this case, the amber codon (TAG) leads to termination of translation and gives the free hormone without the need for independent DNA construction.
[0125]
To create a site for cassette mutagenesis, pS0643 is an oligonucleotide:
(1) 5'-CGG-ACT-GGG-CAG-ATA-TTC-AAG-CAG-ACC-3 '
[This destroys the unique BglII site of pS0643];
(2) 5'-CTC-AAG-AAC-TAC-GGG-TTA-CCC-TGA-CTG-CTT-CAG-GAA-GG-3 ′ [this inserts a unique BstEII site, a one base frameshift, and a non-amber stop codon (TGA)]; and
(3) 5'-CGC-ATC-GTG-CAG-TGC-AGA-TCT-GTG-GAG-GGC-3 '
[This introduces a new BglII site]
To obtain the starting vector pH0509. Adding a frameshift with the TGA stop codon ensures that no gene III fusion can be produced from the starting vector. The BstEII-BglII segment was excised from pH 0509 and replaced with a mutated DNA cassette at the desired codon. Other restriction sites for cassette mutagenesis at other positions in hGH were also introduced into this hormone-phage vector.
[0126]
h Cassette mutagenesis in helix 4 of GH
hGH codons 172, 174, 176 and 178 were targeted for random mutagenesis. This is because all of these are present at or near the surface of hGH and contribute significantly to receptor binding [Cunningham and Wells,Science 244: 1081-1085 (1989)]; all of these are present in a well-defined structure and occupy two “turns” on the same side of helix 4; and each of these is a known evolutionary variant of hGH This is because it is substituted with at least one amino acid.
[0127]
We chose NNS (N = A / G / C / T; S = G / C) substitution for each target residue. This selection of NNS synonymous sequences resulted in 32 possible codons (codons for at least one amino acid) at four sites, for a total of (32)^Four= 1,048,576 possible nucleotide sequences, or (20)^Four= 160,000 possible polypeptide sequences are obtained. The only stop codon, amber (TAG), is possible by this codon selection and this codon can be suppressed as Glu1 in the supE strain of E. coli.
[0128]
Two synonymous (degenerate) oligonucleotides with NNS at codons 172, 174, 176 and 178 were synthesized, phosphorylated and annealed to construct a mutation cassette:
5'-GT-TAC-TCT-ACT-GCT-TTC-AGG-AAG-GAC-ATG-GAC-NNS-GTC-NNS-ACA-NNS-CTG-NNS-ATC-GTG-CAG-TGC-A-3 '
and
5'-GA-TCT-GCA-CTG-CAC-GAT-SNN-CAG-SNN-TGT-SNN-GAC-SNN-GTC-CAT-GTC-CTT-CCT-GAA-GCA-GTA-GA-3 '.
The vector was prepared by digesting pH0509 with BstEII followed by BglII. The product was run on a 1% agarose gel, the large fragment was excised, phenol extracted and ethanol precipitated. This fragment was treated with bovine intestinal phosphatase (Boehringer), then phenol: chloroform extracted, ethanol precipitated and resuspended for ligation with the mutagenesis cassette.
[0129]
XL1 - B lue Growth of the first library in cells
After ligation, the reaction product was digested once more with BstEII, then phenol: chloroform extracted, ethanol precipitated, and resuspended in water [BstEII recognition site (GGTNACC) is at position 3 of codon 172GAnd to 174ACCCreated in a cassette containing the (Thr) codon. However, treatment with BstEII in this step should not select for any possible mutation cassette. This is because virtually all cassettes are heteroduplex and cannot be cleaved by this enzyme]. Approximately 150 ng (45 fmol) of DNA was transferred to XL1-Blue cells (1.8 x 10 in 0.045 ml) in a 0.2 cm cuvette.⁹The cells were electroporated with a single pulse voltage setting of 2.49 kV (time constant = 4.7 msec).
[0130]
The cells were allowed to recover for 1 hour at 37 ° C. in SOC medium with shaking and then mixed with 2YT medium (25 ml), 100 mg / ml carbenicillin, and M13-K07 (moi = 100). After 10 minutes at 23 ° C., the cultures were incubated overnight (15 hours) at 37 ° C. with shaking. By plating serial dilutions from this culture onto carbenicillin-containing medium, 3.9 × 10⁷It was found that an electrotransformant was obtained.
[0131]
After overnight incubation, the cells were pelleted and a double stranded DNA (dsDNA) designated pH0529E (first library) was prepared by the alkaline lysis method. The supernatant was centrifuged once more to remove all remaining cells, and a phage designated phage pool φH0529E (first phage library) was PEG precipitated and STE buffer [10 mM Tris (pH 7.6), 1 mM EDTA, 50 mM. Resuspended in NaCl (1 ml). Phage titers were measured as colony forming units (CFU) for recombinant phagemids containing hGH-g3p. Approximately 4.5x10 from departure library¹³CFU was obtained.
[0132]
Synonymity of starting library (degenerate)
58 clones from the pool of electrotransformants were sequenced in the region of the BstEII-BglII cassette. Of these, 17% correspond to the starting vector, 17% contain at least one frameshift, and 7% have non-silent (non-stopping) mutations outside the four target codons. Contained. We have 41% clones have the disadvantages according to any of the above criteria, and 2.0 × 10⁷It was concluded that a total functional pool of the original transformants would remain. Nevertheless, this number is almost 20 times greater than the number of possible DNA sequences. We are therefore confident that we have all possible sequences shown in the starting library.
[0133]
We evaluated the codon bias in mutagenesis by examining the sequences of unselected phages (Table V). These results indicate that some codons (and amino acids) are more or less than random predictions, but this library is highly diverse and evidences a large amount of “sibling” degeneracy Was found to be absent (Table VI).
[0134]
Table V. Unselected hormone phage codon distribution (per 188 codon) clones were sequenced from the starting library (pH 0529E). All codons were tabulated, including codons from clones containing pseudomutations and / or frameshifts. *: The amber stop codon (TAG) is suppressed as Glu in XL1-Blue cells. The underlined codons were shown to be more / less than 50% or more.

[0135]
table VI. Not selected with open reading frame
(PH0529E) clone
Indications, eg TWGS, indicate hGH mutant 172T / 174W / 176G / 178S. The amber (TAG) codon translated as Glu in XL1-Blue cells is indicated by ε.

[0136]
Immobilization h GH bp and h PRL bp Preparation of
Wild-type hGHbp [Fuh et al.,J. Biol. Chem. 265: 3111-3115 (1990)] (Bass et al.,Proteins 8: 309-314 (1990)] to prepare immobilized hGHbp (“hGHbp-beads”). [¹²⁵I] Competitive binding experiments with hGH showed that 58 fmol of functional hGHbp was bound per μl of bead suspension.
[0137]
Prolactin receptor (Cunningham et al.,Science 250: 1709-1712 (1990)], an immobilized hPRLbp ("hPRLbp-bead") was prepared as described above. In the presence of 50 μM zinc¹²⁵I] Competitive binding experiments with hGH revealed that 2.1 fmol of functional hPRLbp was bound per μl of bead suspension.
[0138]
“Blank beads” were prepared by treating oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) at 4 ° C. for 15 hours.
[0139]
Immobilization h GH bp and h PRL bp Join selection with
Hormone-phage binding to the beads was performed in one of the following buffers: Buffer A [PBS, 0.5% BSA, 0.05% Tween 20, for selection using hGHbp and blank beads. 0.01% thimerosal]; in the presence of zinc (+ Zn²⁺) For selection using hPRLbp buffer B [50 mM Tris (pH 7.5), 10 mM MgCl₂0.5% BSA, 0.05% Tween 20, 100 mM ZnCl₂Or buffer C [PBS, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal, 10 mM EDTA] for selection using hPRLbp in the absence of zinc (+ EDTA). Binding selection was performed according to each of the following pathways: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+ Zn).²⁺), (4) binding to hPRLbp-beads (+ EDTA), (5) two pre-adsorptions with hGHbp beads followed by binding of unadsorbed fraction to hPRLbp-beads ("-hGHbp, + hPRLbp" selection), or (6) Two pre-adsorptions with hPRLbp-beads and subsequent binding of unadsorbed fractions to hGHbp-beads (selection of "-hPRLbp, + hGHbp"). The latter two manipulations are expected to enrich for mutants that bind to hPRLbp but not hGHbp, respectively, or mutants that bind hGHbp but not hPRLbp. Phage binding and elution in each cycle was performed as follows:
[0140]
1. Binding: part of a hormone phage (usually 10⁹-10^TenCFU) is mixed with an equal volume of non-hormone phage (pCAT), diluted with the appropriate buffer (A, B or C) and hGHbp, hPRLbp or blank beads in a total volume of 200 ml in a 1.5 ml polypropylene tube. And a suspension (10 ml). The phage was bound to the beads by incubating at room temperature (23 ° C.) for 1 hour with slow rotation (about 7 rpm). The following steps were performed at room temperature using a constant volume of 200 μl.
[0141]
2. Washing: The beads were spun for 15 seconds and the supernatant was removed. To reduce the number of non-specifically bound phage, the beads were washed 5 times by briefly resuspending in an appropriate buffer and then pelleted.
[0142]
3. hGH elution: Phage that weakly bound to the beads were removed by elution with hGH. The beads were spun for 15-17 hours with an appropriate buffer containing 400 nM hGH. This supernatant was used as “hGH eluate” and beads. The beads were washed by resuspending briefly in buffer and pelleting.
[0143]
4). Glycine elution: To remove the most tightly bound phage (ie, phage still bound after hGH washing), the beads were added to glycine buffer (0.2 M glycine in buffer A and pH 2.0 with HCl. And then rotated for 1 hour to pelletize. The supernatant (“Glycine eluate”; 200 μl) was neutralized by the addition of 1M Tris base (30 ml) and stored at 4 ° C.
[0144]
5. Proliferation: Under each set of conditions, an independent culture of XL1-Blue cells in logarithmic growth was infected with a portion of hGH eluate and glycine eluate from each set of beads. Transduction was performed by mixing the phage with XL1-Blue cells (1 ml), incubating at 37 ° C. for 20 minutes, and then adding K07 (moi = 100). The culture (25 ml of 2YT and carbenicillin) was grown as above and the next pool of phage was prepared as above.
[0145]
Phage binding, elution and growth were performed in successive rounds according to the cycle described above. For example, phage amplified from hGH eluate from hGHbp beads were again selected with hGHbp beads, eluted with hGH, and then used to infect fresh cultures of XL1-Blue cells. Five rounds of selection and three rounds of growth were performed for each of the above selection methods.
[0146]
DNA sequencing of selected phagemids
A portion of the phage from each cycle of hGH and glycine elution steps was used for inoculation of XL1-Blue cells and was plated on LB medium containing carbenicillin and tetracycline to obtain independent clones from each phage pool. . Single-stranded DNA was prepared from the isolated colonies and the mutation cassette region was sequenced. The results of this DNA sequencing are summarized as deduced amino acid sequences in FIG. 5, FIG. 6, FIG. 7 and FIG.
[0147]
h GH mutant expression and assay
In order to determine the binding affinity of some of the selected hGH mutants for hGHbp, DNA from sequenced clones was introduced into E. coli strain 16C9. As mentioned above, this strain is a non-suppressor strain and terminates protein translation after the last Phe-191 residue of hGH. Although single-stranded DNA was used for these transformations, even double-stranded DNA or whole phage can be easily electroporated into non-suppressor strains for free hormone expression.
[0148]
hGH mutants were prepared from cells subjected to osmotic shock by ammonium sulfate precipitation as described for hGH [Olson et al.,Nature 293: 408-411 (1981)]. The protein concentration was measured by laser densitometry of Coomassie-stained SDS-polyacrylamide gel electrophoresis gel using hGH as a standard [Cunningham and Wells,Science 244: 1081-1085 (1989)].
[0149]
The binding affinity of each mutant was determined by using an anti-receptor monoclonal antibody (Mab263) and the literature [Spencer et al.,J. Biol. Chem. 263: 7862-7867 (1988); Fuh et al.,J. Biol. Chem. 265: 3111-3115 (1990)]¹²⁵Measured by replacing I hGH.
[0150]
The results for the number of hGH mutants selected by different routes (Figure 6) are shown in Table VII. Many of these mutants have a stronger binding affinity for hGHbp than wild type hGH. The most improved mutant, KSYR, has a binding affinity that is 5.6 times higher than wild type hGH. The weakest mutant selected among these tested mutants had only about 10-fold lower binding affinity compared to hGH.
[0151]
Binding assays can also be performed on mutants selected for hPRLbp-binding.
[0152]
table VII. Competitive binding to hGHbp
The selected pool in which each mutant was found was selected as 1G (first glycine selection), 3G (third glycine selection), 3H (third hGH selection), 3 * (third selection: hPRLbp Is bound to hGHbp). The number of each mutant appearing in all sequenced clones is shown in ().

[0153]
Additive and non-additive effects on bonding.
In some residues, certain amino acid substitutions have substantially the same effect independent of surrounding residues. For example, substitution of F176Y in the background of 172R / 174S reduces binding affinity by a factor of 2.0 (RSFR vs. RSYR). Similarly, in the background of 172K / 174A, the binding affinity of the F176Y mutant (KAYR) is 2.9 times weaker than the corresponding 176F mutant [KAFR; Cunningham and Wells (1989)].
[0154]
On the other hand, the binding constants measured for some selected hGH mutants show the non-additive effect of some amino acid substitutions at residues 172, 174, 176 and 178. For example, in the background of 172K / 176Y, substitution of E174S results in a mutant that binds hGHbp (KSYR) 3.7 times more strongly than the corresponding mutant containing E174A (KAYR). . However, in the 172R / 176Y background, the effects of these E174 substitutions are reversed. In this case, the E174A mutant (RAYR) binds 1.5 times more tightly than the E174S mutant (RSYR).
[0155]
This non-additive effect on the binding of substitutions at adjacent residues demonstrates the use of protein-phage binding selection as a means of selecting optimal mutants from libraries randomized at several positions Is. Without detailed structural information and without such a selection method, many substitution combinations would be tried before finding the optimal mutant.
[0156]
Example IX  Selection of hGH mutants from hormone-phage helix 1 random cassette library
[0157]
Using the method described in Example VIII, another region of hGH involved in binding to hGHbp and / or hPRLbp, ie, residues 10, 14, 18, 21 of helix 1, was transferred to the phGHam-g3p vector (pS0643). Also known as; see Example VIII) as a target for random mutagenesis.
[0158]
We chose to use the “Amber” hGH-g3 construct (referred to as phGHam-g3p) because it appears to create a target protein hGH that is more accessible for binding. This is supported by data from a comparative ELISA assay for monoclonal antibody binding. pS0132 [S. Bass, R. Greene, J. A. Wells,Proteins 8: 306 (1990)] and phages generated from both phGHam-g3 were expressed by three antibodies known to have binding determinants near the carboxy terminus of hGH (Medix2, 1B5.G2, and 5B7). C10) [BCCunningham, P. Jhurani, P. Ng, JAWells,Science 243: 1330 (1989); B.C.Cunningham and J.A.Wells,Science 244: 1081 (1989); L. Jin and J. Wells (unpublished results)], and one antibody (Medix1) that recognizes determinants in helices 1 and 3 [BCCunningham, P. Jhurani, P .Ng, JAWells,Science 243: 1330 (1989); B.C.Cunningham and J.A.Wells,Science 244: 1081 (1989)]. Phagemid particles from phGHam-g3 reacted more strongly with antibodies Medix2, 1B5.G2 and 5B7.C10 than phagemid particles from pS0132. In particular, the binding of pS0132 particles was reduced> 2000 times for both Medix2 and 5B7.C10 and> 25 times for 1B5.G2 compared to binding to Medix1. On the other hand, the binding of phGHam-g3 phage is about 1.5 times, 1.2 times, and 2.3 times weaker than that of Medix1, compared to Medix2, 1B5.G2 and 5B7.C10 antibodies, respectively It wasn't too much.
[0159]
Creation of helix 1 library by cassette mutagenesis
Alanine-scanning mutagenesis (B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells,Science 243: 1330 (1989); B.C.Cunningham and J.A.Wells,Science 244: 1081 (1989), 15, 16] by mutating residues in helix 1 previously identified by hGH and / or hPRL receptors (referred to as hGHbp and hPRLbp, respectively) Modulated domain binding. Cassette mutagenesis is substantially described in the literature [J.A.Wells, M.Vasser, D.B.Powers,Gene 34: 315 (1985)]. This library contains oligonucleotides:
5'-GCC TTT GAC AGG TAC CAG GAG TTT G-3 'and
5'-CCA ACT ATA CCA CTC TCG AGG TCT ATT CGA TAA C-3 '
PhGHam-g3 [T.A.Methods Enzymol. 154: 367-382] was made by cassette mutagenesis (see Example VIII), which mutates all four residues at once. The latter oligo also introduced a +1 frame shift (G at the end of the underline), which terminated translation from the starting vector and minimized wild-type background in the phagemid library. This starting vector was named pH0508B. A helix 1 library mutated at hGH residues 10, 14, 18, 21 is a complementary oligonucleotide:
5'-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3 'and
5'-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3 '
Was prepared by ligation to a large XhoI-KpnI fragment of pH 0508B. This KpnI site was destroyed at the junction of the ligation product, allowing restriction enzyme digestion to be used for analysis of non-mutated background.
[0160]
This library is at least 10⁷Separate transformants, and this library is completely random (10⁶On average, about 10 copies of each possible mutant hGH gene is obtained. Restriction analysis using KpnI showed that at least 80% of the helix 1 library construct contained the inserted cassette.
[0161]
Enrichment of hGH-phage from this library was performed using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as previously described (Example VIII). Four residues in Helix 1 (F10, M14, H18 and H21) were mutated in the same way and a non-wild type consensus appeared after 4 and 6 cycles (Table VIII). Position 10 of the hydrophobic surface of helix 1 tends to be hydrophobic, while positions 21 and 18 of the hydrophilic surface tend to be predominantly Asn, with no obvious consensus at position 14. (Table IX).
[0162]
The binding constant of these hGH mutants to hGHbp is expressed by expressing a free hormonal variant in non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labeled wt-hGH from hGHbp. (See Example VIII). As shown, some mutants bind to hGHbp more strongly than wt-hGH binds.
[0163]
table VIII. Selection of hGH helix 1 mutant
HGH variants expressed on phagemid particles (random mutations at residues F10, M14, H18, H21) were bound to hGHbp-beads and hGH (0.4 mM) buffer followed by glycine (0.2 M , PH 2) by elution with buffer (see Example VIII).

[0164]
table IX. Consensus sequence from selected helix 1 library
The observed frequency is the fraction of all clones sequenced with the indicated amino acid. Nominal frequency was calculated based on NNS 32 codon degeneracy. The maximum enrichment factor varies from 11 to 32 depending on the nominal frequency value for a residue. The value of [Kd (Ala mutant) / Kd (wt-hGH)] for a single alanine mutation is given in the literature [B. C. Cunningham and J. A. Wells,Science 244: 1081 (1989); B.C.Cunningham, D.J.Henner, J.A.Wells,Science 247: 1461 (1990); B.C. Cunningham and J.A. Wells,Proc.Natl.Acad.Sci.USA 88: 3407 (1991)].

[0165]
Table X. Binding of purified hGH helix 1 mutant to hGHbp
Competition binding experiments¹²⁵I] hGH (wild type), hGHbp (extracellular receptor domain, including residues 1-238), and Mab263 [B.C. Cunningham, P. Jhurani, P.Ng, J.A. Wells,Science 243: 1330 (1989)]. The numerical value P represents the fractional occurrence of each mutant in all clones sequenced after one or more selection rounds.

[0166]
Example X  Selection of hGH variants from a helix 4 random cassette library containing mutations previously found by hormone-phage enrichment
Design of muteins with improved binding by iterative selection using hormone-phages
From our experiments using new unbound homologues of hGH evolutionary variants, we obtain cumulative and improved mutants of hGH for binding to specific receptors by combining multiple independent amino acid substitutions [BCCunningham, DJHenner, JAWells,Science 247: 1461 (1990); B.C.Cunningham and J.A.Wells,Proc.Natl.Acad.Sci.USA 88: 3407 (1991); H.B.Lowman, B.C.Cunningham, J.A.Wells,J. Biol. Chem. 266, printing (1991)].
[0167]
In an attempt to further improve a double mutant of helix 4 (E174S / F176Y; see Example VIII) selected from the helix 4a library found to bind more tightly to the hGH receptor, the helix 4b library It was created. Mutations were made at residues around positions 174 and 176 on the hydrophilic face of helix 4 using the E174S / F176Y hGH mutant as the background starting hormone.
[0168]
Helix 4 by cassette mutagenesis b Creating a library
Substantially the literature [J.A.Wells, M.Vasser, D.B.Powers,Gene 34: 315 (1985)] cassette mutagenesis was performed as described. Using cassette mutagenesis (see Example VIII) using a mutant of phGHam-g3 (Example VIII) pre-inserted with only BstEII and BglII restriction sites, mutating all four residues at once A helix 4b library was created in which residues 167, 171, 175 and 179 were mutated in the E174S / F176Y background. Complementary oligonucleotide:
5'-pG TTA CTC TAC TGC TTC NNS AAG GAC ATG NNS AAG GTC AGC NNS TAC CTG CGC NNS GTG CAG TGC A-3 'and
5'-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC CTT SNN CAT GTC CTT SNN GAA GCA GTA GA-3 '
The cassette prepared from was inserted into the BstEII-BglII site of the vector. This BstEII site was deleted in the linked cassette. Fifteen unselected clones from this helix 4b library were sequenced. Of these, none lacked the cassette insert, 20% were frameshifted and 7% had non-silent mutations.
[0169]
h GH bp Results of enrichment
Enrichment of hGH-phage from this library was performed using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as previously described (Example VIII). After 6 cycles of binding, a reasonable and clear consensus appeared (Table XI). Interestingly, all positions tended to contain polar residues, especially Ser, Thr and Asn (Table XII).
[0170]
h GH mutant assay
The binding constants of some of these hGH mutants to hGHbp were expressed by expressing a free hormonal variant in non-suppressor E. coli strain 16C9, purifying the protein, and competitive displacement of labeled wt-hGH from hGHbp. Measured by assay (see Example VIII). As shown, the binding affinity for hGHbp of some helix 4b mutants was stronger than that of wt-hGH (Table XIII).
[0171]
h Receptor selectivity of GH mutant
Finally, we began analyzing the binding affinity of several more robust hGHbp binding mutants for their ability to bind hPRLbp. The E174S / F176Y mutant binds 200 times weakly to hPRLbp compared to hGH. Each of the E174T / F176Y / R178K and R167N / D171S / E174S / F176Y / I179T mutants binds> 500-fold weakly to hPRLbp compared to hGH. That is, a novel receptor-selective mutant of hGH can be obtained by the phage display method.
[0172]
Information content of specific residues identified by hormone-phagemid selection
Of the 12 residues mutated in the 3 hGH-phagemid libraries (Examples VIII, IX, X), 4 showed strong (but not exclusive) conservation of wild-type residues (K172, T175, F176 and R178). Not surprisingly, there were residues that caused the greatest disruption (4-60 fold) in binding affinity for hGHbp when converted to Ala. There is a group of four other residues (F10, M14, D171 and I179) that cause the Ala substitution to have a weaker effect on binding (2-7 fold), and these positions have only a few wild-type consensus Not shown. Finally, another four residues (H18, H21, R167 and E174) that promoted binding to hPRLbp but not hGHbp were selected against the wild-type hGH sequence by selection with hGHbp-beads. There was no such consensus. In fact, two residues (E174 and H21) increase the binding affinity for hGHbp by 2-4 fold when Ala substituted [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells,Science 243: 1330 (1989); B.C. Cunningham and J.A. Wells,Science 244: 1081 (1989); B.C.Cunningham, D.J.Henner, J.A.Wells,Science 247: 1461 (1990); B.C. Cunningham and J.A. Wells,Proc.Natl.Acad.Sci.USA 88: 3407 (1991)]. That is, alanine-scanning mutagenesis data is reasonably related to the variability that replaces each position. In fact, the reduction in binding affinity caused by alanine substitution (B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells,Science 243: 1330 (1989); B.C. Cunningham and J.A. Wells,Science 244: 1081 (1989); B.C.Cunningham, D.J.Henner, J.A.Wells,Science 247: 1461 (1990); B.C. Cunningham and J.A. Wells,Proc.Natl.Acad.Sci.USA 88: 3407 (1991)] reasonably predicts the proportion of wild-type residues found in the phagemid pool after 3-6 rounds of selection. Alanine-scanning information is useful for targeting side chains that modulate binding, and phage selection can be used for side chain optimization and variability at each site (and / or combination of sites) for substitution. It is suitable for defining. Scanning mutation method (B.C.Cunningham, P.Jhurani, P.Ng, J.A.Wells,Science 243: 1330 (1989); B.C. Cunningham and J.A. Wells,Science 244: 1081 (1989)] and phage display are powerful tools for designing receptor-ligand interfaces and for studying molecular evolution in vitro.
[0173]
Mutations in iterative enrichment of hormone-phagemid libraries
If the combination mutation in hGH has an additive effect on the binding affinity for the receptor, the mutation known by hormone-phagemid enrichment to improve the binding is simply Combined by cleavage and ligation or mutagenesis, a cumulative optimized mutant of hGH can be obtained.
[0174]
On the other hand, mutations in one region of hGH that optimize receptor binding may be structurally or functionally incompatible with mutations in overlapping or other regions of the molecule. In such cases, hormone-phagemid enrichment can be performed by any of several variations of the iterative enrichment method listed below: (1) 2 molecules of the molecule by cassette or other mutagenesis methods. A random DNA library can be created in each of one (or perhaps more) regions: a mixed library can then be created by ligation of restriction fragments from these two DNA libraries; 2) hGH variants that are optimized for binding by mutation in one region of the molecule can be randomly mutated in the second region of the molecule, as in the example of a helix 4b library; (3) Two or more random libraries can be linked to improved binding by hormone-phagemid enrichment. TePartialAfter “crude selection” of this optimized binding site, yet another partial library can be remixed by ligation of restriction fragments and partially in two or more regions of the molecule Can be obtained, and this library can then be further selected for optimized binding using hormone-phagemid enrichment.
[0175]
table XI. Mutated phagemid of hGH selected from helix 4b library after 4 and 6 cycles of enrichment
HGH helix 4b mutants, each containing an E174S / F176Y double mutation (random mutations at residues 167, 171, 175, 179), binding to hGHbp-beads, and hGH (0.4 mM) buffer And then elution with glycine (0.2 M, pH 2) buffer. One mutant (+) contained a suspicious mutation R178H.
[0176]

[0177]
table XII. Consensus sequence from the selected library
The observed frequency is the fraction of all clones sequenced with the indicated amino acid. Nominal frequency was calculated based on NNS 32 codon degeneracy. The maximum enrichment factor varies from 11 to 16 to 32 depending on the nominal frequency value for a residue. The value of [Kd (Ala mutant) / Kd (wt-hGH)] for a single alanine mutation was taken from the following literature; only the value of the T175S mutant for position 175 [BCCunningham , P.Jhurani, P.Ng, JAWells,Science 243: 1330 (1989); B.C. Cunningham and J.A. Wells,Science 244: 1081 (1989); B.C.Cunningham, D.J.Henner, J.A.Wells,Science 247: 1461 (1990); B.C. Cunningham and J.A. Wells,Proc.Natl.Acad.Sci.USA 88: 3407 (1991)].
[0178]

[0179]
table XIII. Binding of purified hGH mutant to hGHbp
Competition binding experiments¹²⁵I] Performed with hGH (wild type), hGHbp (extracellular receptor domain, including residues 1-238), and Mab263 (11). The numerical value P represents the fractional occurrence of each mutant in all clones sequenced after one or more selection rounds. Note that the helix 4b mutation (*) is in the background of hGH (E174S / F176Y). In this list of helix 4b mutations, the E174S / F176Y mutant (*) with wt residues at 167, 171, 175, 179 is underlined.
[0180]

[0181]
Example XI  Assembly of Fab molecules on the phagemid surface
Plasmid construction
Plasmid pDH188 contains DNA encoding the Fab portion of a humanized IgG antibody called 4D5 that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR101 and has been deposited with ATCC [Rockville, MD].
[0182]
Briefly, this plasmid was prepared as follows. The starting plasmid is pS0132 containing the alkaline phosphatase promoter described above. A DNA encoding human growth hormone was excised, and a series of operations were performed so that the ends of the plasmid were adapted for ligation, and then a DNA encoding 4D5 was inserted. This 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain and contains DNA encoding the stII signal sequence at its 5 'end. The second gene contains four parts, the first of which is the DNA encoding the 5 'end stII signal sequence. This is followed by the DNA encoding the heavy chain variable domain, followed by the DNA encoding the first domain of the heavy chain constant region, followed by the DNA encoding the M13 gene III. The salient features of this construct are shown in FIG. The sequence of the DNA encoding 4D5 is shown in FIG.
[0183]
E. coli transformation and phage production
The plasmid was introduced into SR101 cells using both polyethylene glycol (PEG) and electroporation. PEG-competent cells include Chung and Miller [Nucleic Acids Res. 16: 3580 (1988)] and transformed. Cells that are competent for electroporation are Zabarovsky and Winberg.Nucleic Acids Res. 18: 5912 (1990)] and then transformed by electroporation. Cells were seeded in SOC medium (described in Sambrook et al. (Supra)) (1 ml) and grown at 37 ° C. for 1 hour with shaking. At this point, the cell concentration is OD₆₀₀Was measured using light dispersion. A titrated KO7 phage stock was added to give a multiplicity of infection (MOI) of 100 and the phages were allowed to attach to the cells for 20 minutes at room temperature. This mixture was then diluted in 2YT broth (described by Sambrook et al. (Supra)) (25 ml) and incubated overnight at 37 ° C. with shaking. The next day, the cells were pelleted by centrifugation at 5000 xg for 10 minutes, the supernatant was collected, and phage particles were precipitated with 0.5 M NaCl and 4% PEG (final concentration) for 10 minutes at room temperature. Phage particles were pelleted by centrifugation at 10,000 × g for 10 minutes, resuspended in TEN [10 mM Tris (pH 7.6), 1 mM EDTA, and 150 mM NaCl] (1 ml) and stored at 4 ° C.
[0184]
Preparation of antigen-coated plates
0.5 ml each from a 0.5 mg / ml solution of BSA (control antigen) in 0.1 M sodium bicarbonate (pH 8.5) or a 0.1 mg / ml solution of the extracellular domain (EDC) of HER-2 antigen. Was used to coat the wells of a Falcon 12 well tissue culture plate. After the solution was applied to the wells, the plate was incubated overnight at 4 ° C. on a rocking platform. The plate was then blocked by removing the initial solution, applying blocking buffer [30 mg / ml BSA in 0.1 M sodium bicarbonate] (0.5 ml) and incubating at room temperature for 1 hour. Finally, the block buffer is removed, buffer A [PBS, 0.5% BSA, and 0.05% Tween 20] (1 ml) is added, and the plate is used at 4 ° C. until used for phage selection. Stored for up to 10 days.
[0185]
Phage selection method
About 10⁹Of phage particles were mixed with 100-fold excess of KO7 helper phage and buffer A (1 ml). This mixture was divided into two 0.5 ml portions, one applied to an ECD coated well and the other applied to a BSA coated well. The plates were incubated at room temperature for 1-3 hours with shaking and then washed 3 times for 30 minutes with buffer A (1 ml each). Phage elution from the plate was performed at room temperature by either of the following two methods: (1) Initial overnight incubation of 0.025 mg / ml of purified Mu4D5 antibody (murine) followed by acid elution buffer. Incubate with [0.2 M glycine (pH 2.1), 0.5% BSA, and 0.05% Tween 20] (0.4 ml) for 30 minutes, or (2) Incubate with acid elution buffer alone. The eluate was then neutralized with 1M Tris base and TEN (0.5 ml increments) was added. These samples were then grown, titrated and stored at 4 ° C.
[0186]
Phage growth
A portion of the eluted phage is added to 2YT broth (0.4 ml) and about 10⁸Of mid log phase male E. coli strain SR101. Phages were allowed to attach to the cells for 20 minutes at room temperature and then added to 2YT broth (5 ml) containing 50 μg / ml carbenicillin and 5 μg / ml tetracycline. The cells were grown for 4-8 hours at 37 ° C. until the mid-log phase was reached. OD₆₀₀And the cells were superinfected with KO7 helper phage for phage production. After obtaining the phage particles, they were titrated to determine the number of colony forming units (cfu). This was done by taking a portion of a serial dilution of a phage stock, infecting mid-log phase SR101, and plating on LB plates containing 50 νg / ml carbenicillin.
[0187]
Measurement of RIA affinity
The affinity of Fab phage and h4D5 Fab fragment for ECD antigen was determined by competitive receptor binding RIA [Burt, D.R.,Receptor Binding in Drug Research, O'Brien, R.A. (ed.), Pp.3-29, Dekker, New York (1986)]. This ECD antigen is obtained by the continuous chloramine-T method [De Larco, J.E. et al.,J.Cell.Physiol. 109: 143-152 (1981)]¹²⁵Labeled with I. As a result, a radioactive tracer having a specific activity of 14 μCi / μg into which 0.47 mol of iodine was introduced per mol of the receptor was obtained. 0.5 ng (by ELISA) Fab or Fab phage, 50 nCi¹²⁵A series of 0.2 ml solutions containing I-ECD tracer and a range of unlabeled ECD (6.4 ng-3277 ng) were prepared and incubated overnight at room temperature. Labeled ECD-Fab or ECD-Fab phage complexes were separated from unbound labeled antigen by forming aggregate complexes induced by the addition of anti-human IgG (Fitzgerald 40-GH23) and 6% PEG8000. The complex was pelleted by centrifugation (15,000 xg for 20 minutes) and the amount of labeled ECD (cpm) was measured with a gamma counter. The dissociation constant (Kd) is determined by Scatchard analysis [Scatchard, G.,Ann.NYAcad.Sci. 51: 660-672 (1949)], an improved version of the program LIGAND [Munson, P. and Rothbard, D.,Anal.Biochem. 107: 220-239 (1980)]. This Kd value is shown in FIG.
[0188]
Competitive cell binding assay
Using the rhodogen method,¹²⁵I labeled murine 4D5 antibody with a specific activity of 40-50 μCi / μg. A solution containing a fixed amount of labeled antibody and increasing amounts of unlabeled mutant Fab was prepared and added to almost the entire SK-BR-3 cell culture in a 96-well microtiter dish (final concentration of labeled antibody). Was 0.1 nM). After overnight incubation at 4 ° C., the supernatant was removed, the cells were washed, and the radioactivity bound to the cells was measured with a gamma counter. Kd values were determined by analyzing the data using an improved version of the program LIGAND [Munson, P. and Rothbard, D. (above)].
[0189]
The deposit of the plasmid pDH188 (ATCC No. 68663) is made under the provisions of the Budapest Treaty and the rules (Budapest Treaty) concerning the international recognition of the deposit of microorganisms in the patent procedure. This ensures the maintenance of viable microorganisms for 30 years from the date of deposit. This microorganism is made available from the ATCC under the terms of the Budapest Treaty and with the consent of Genentech, Inc. and ATCC, and is issued by the relevant US patent or any US or foreign The earlier of the publication of the patent application, which guarantees that anyone of this culture's progeny will be made available permanently and unrestricted, and is entitled to the US according to 35 USC §122 It is guaranteed that the offspring will be made available to those determined by the Office of the Patent and Trademark Office and the rules that follow (including 37 CFR §1.14, which specifically refers to 886 OG 638).
[0190]
The assignee of the present application will promptly replace a living sample of the culture upon notification when the deposited microorganism is killed, lost or destroyed when cultured under appropriate conditions I agree. The availability of deposited cultures should not be construed as a license to practice the invention in violation of rights granted under the authority of any government in accordance with national patent laws.
[0191]
The foregoing specification is considered to be sufficient to enable one skilled in the art to practice the invention. The deposited embodiment is intended as an independent illustration of a particular embodiment of the present invention, and since any functionally equivalent culture is within the scope of the invention, the invention is covered by the deposited microorganism. Is not limited. Deposits of substances in this specification have acknowledged that the statements contained herein are inadequate to allow implementation of any aspect of the invention, including the best mode. It is not intended to be limiting, nor is it intended to limit the scope of the claims to that particular example. Indeed, various modifications of the invention in addition to those described and shown herein will become apparent to those skilled in the art from the foregoing description, which fall within the scope of the appended claims It is.
[0192]
Although the present invention has been described in connection with the preferred embodiments, it will be appreciated by those skilled in the art after reading the above specification that various objects may be found in the subject matter described herein without departing from the spirit and scope of the invention. Changes, equivalent substitutions and mutations could be added. That is, the present invention can be carried out by methods other than those specifically described in this specification. Accordingly, the protection granted by this patent is limited only by the appended claims and equivalents thereof.
[0193]
Example XII  Selection of hGH variants from helix 1 and helix 4 hormone-phage variant combinations
h Creation of additive mutants of GH
The principle of additivity (J.A. Wells,Biochemistry 29: 8509 (1990)], even when mutations in different parts of the protein do not interact with each other, by combining them, they are additive in the free energy of binding to another molecule. Change is expected (change is ΔΔG_bindingOr additive in terms of Kd = exp [−ΔG / RT]). That is, a mutation that gives a 2-fold increase in binding affinity results in a double mutation with a 6-fold increased affinity of the starting variant when combined with a second mutation that causes a 3-fold increase. Expected to give.
[0194]
In order to test whether multiple mutations obtained by hGH-phage selection have a cumulatively good effect on hGHbp (hGH-binding protein; extracellular domain of hGH receptor) binding, The mutations found in the three most tightly binding variants of hGH from which they were derived (Example IX: F10A / M14W / H18D / H21N, F10H / M14G / H18N / H21N, and F10F / M14S / H18F / H21L) , Found in the three tightest binding variants found in the helix 4b library (Example X: R167N / D171S / T175 / I179T, R167E / D171S / T175 / I179, and R167N / D171N / T175 / I179T) Combined with mutations.
[0195]
HGH-phagemid double stranded DNA (dsDNA) was isolated from each of the 1 helix mutants and digested with restriction enzymes EcoRI and BstXI. The large fragment was then isolated from each helix 4b variant and ligated with the small fragment from each helix 1 variant to yield the novel two helix variants shown in Table XIII. In addition, all of these mutants contained the E174S / F176Y mutation obtained in the previous hGH-phage binding selection (see Example X for details).
[0196]
h Creation of GH selective combination library
While the principle of additivity seems to apply to a number of mutation combinations, some combinations (eg, E174S with F176Y) are clearly non-additive (see Examples VIII and X). For example, to reliably identify the most tightly bound variants with 4 mutations in helix 1 and 4 mutations in helix 4, ideally all 8 residues are suddenly Mutants will then be screened for the pool of mutants that bind most tightly overall. However, such a pool is 2.6x10^Ten1.1 × 10 encoding different polypeptides¹²DNA sequence (using the degeneracy of NNS codons). All mutants (probably 10¹³It is not practical to obtain a random phagemid library large enough to ensure the display of the transformants) by current transformation methods.
[0197]
We first address this challenge by using sequential rounds of mutagenesis, taking the most tightly binding mutation from one library, and then mutating other residues to further improve binding. Worked (Example X). In the second method, we combine the best mutations from two independently selected libraries using the principle of additivity to create multiple mutants with improved binding. (Above). Here we combine possible mutations at positions 10, 14, 18, 21, 167, 171, 175, and 179 in hGH by creating a combinatorial library of random or partially random mutants. Further explored. Pooled phagemids from the helix 1 library (independently 0, 2 or 4 cycle selection; Example IX) and pools from the helix 4b library (independently 0, 2 or 4 cycle selection; Example X) Were used to generate three different combinatorial libraries of hGH-phagemids and this combinatorial mutant pool was screened for hGHbp binding. Because a certain amount of sequence diversity exists in each of these pools, the resulting combinatorial library can examine more sequence combinations than can be generated manually (eg, Table XIII).
[0198]
Double-stranded DNA (dsDNA) of hGH-phagemid was isolated from each of the 1-helix library pools (0, 2 or 4 round selections) and digested with restriction enzymes AccI and BstXI. Large fragments were then isolated from each helix 1 mutant pool and ligated with small fragments from each helix 4b mutant pool to generate three combinatorial libraries pH0707A (unselected as described in Examples IX and X). Helix 1 and helix 4b pool), pH0707B (2 times selected helix 1 pool and 2 times selected helix 4b pool), and pH0707C (4 times selected helix 1 pool and 4 times selected helix 4b pool). In addition, the same ligation was performed using less DNA and named pH0707D, pH0707E and pH0707F (corresponding to 0, 2 and 4 rounds of the starting library, respectively). All of these mutant pools also contained the mutation E174S / F176Y obtained from the previous hGH-phage binding selection (see Example X for details).
[0199]
h GH - Selection of combinatorial library of phage variants
The ligated products pH0707A-F were processed and electro-introduced into XL1-Blue cells as described (Example VIII). Based on colony forming units (CFU), the number of transformants obtained from each pool was as follows: pH 0707A to 2.4 × 10⁶, PH 0707B to 1.8x10⁶, PH 0707C to 1.6x10⁶, PH 0707D to 8x10^Five, PH 0707E to 3x10^Five, And pH 0707F to 4 × 10^Five. hGH-phagemid particles were prepared and selected for hGHbp-binding for 2-7 cycles as described in Example VIII.
[0200]
h Rapid selection of GH-phagemid library
In addition to screening phagemid libraries for protein variants that bind tightly as measured by equilibrium binding affinity, the on-rate of binding to receptors or other molecules (k_on) Or off-speed (k_offIt is also important to select mutants in which either of them has changed. From thermodynamics, these rates are the equilibrium dissociation constants Kd = (k_off/ K_on). We have found that some variants of certain proteins have similar Kd for binding, but very different k_onAnd k_offIt is assumed that Conversely, the Kd change between one mutant and another is k_onEffect on k_offThis may be due to effects on or both. The pharmacological properties of a protein are binding affinity or k_onOr k_off(Depending on the detailed mechanism of action). Here we identify hGH mutants with higher on-rates and k_onI tried to investigate the effect of changes. We increase the selection by increasing the binding time and by increasing the washing time and / or enrichment with the cognate ligand (hGH)._offIt is assumed that it can be selectively weighted towards
[0201]
From time course analysis of wild type hGH-phagemid binding to immobilized hGHbp, less than 10% of the total hGH-phagemid particles that can be eluted in the final pH2 wash (see Example VIII for overall binding and elution protocol) Appear to bind after 1 minute incubation, but over 90% appear to bind after 15 minutes incubation.
[0202]
For “rapid binding selection”, phagemid particles from the pH0707B pool (selected twice independently for helices 1 and 4) were incubated with immobilized hGHbp for only 1 minute, then with binding buffer (1 ml). Washed 6 times (the hGH wash step was deleted and the remaining hGH-phagemid particles were eluted with pH2 (0.2 M glycine in binding buffer) wash). Enrichment of hGH-phagemid particles to non-displayed particles uses short-term binding and mutations that bind hGH-phagemid binding selection tightly from the randomized pool even when cognate ligand (hGH) challenge is not used Shown to sort the body.
[0203]
h GH mutant assay
The binding constants of some of these hGH mutants to hGHbp were expressed in free suppressor mutants in non-suppressor E. coli strains 16C9 or 34B8, the protein was purified and labeled from hGHbp in a radioimmunoprecipitation assay. Measured by assaying by competitive displacement of conjugated wt-hGH (see Example VIII). In Table XIII-A below, all variants are serine₁₇₄Glutamate substituted by₁₇₄And tyrosine₁₇₆Phenylalanine substituted by₁₇₆In addition to (E174S and F176Y), the hGH amino acids have the additional substitutions shown in the table at positions 10, 14, 18, 21, 167, 171, 175 and 179.
[0204]

[0205]
In Table XIV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection method. All hGH variants in Table XIV contain two background mutations (E174S / F176Y). HGH-phagemid pools from libraries pH0707A (Part A), pH0707B and pH0707E (Part B) or pH0707C (Part C) were screened in 2-7 cycles for binding to hGHbp. The value P indicates the fractional incidence of each variant type in a set of clones sequenced from each pool.
[0206]

[0207]
In Table XV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection method. All hGH variants in Table XV contain two background mutations (E174S / F176Y). The value P is the fractional incidence of a particular variant in all clones sequenced after 4 cycles of rapid binding selection.
[0208]

[0209]
In Table XVI below, hGHbp (1-238) and either Mab5 or Mab263 are used.¹²⁵Binding constants were determined by competitive displacement of I-labeled hormone H0650BD or labeled hGH. Mutation H0650BD appears to bind 30 times more tightly than wild type hGH.
[0210]

[0211]
Example XIII  Selective enrichment of hGH-phage and non-substrate phage containing protease substrate sequences
As described in Example I, plasmid pS0132 contains the gene for hGH fused to residue Pro198 of gene III protein with the insertion of an extra glycine residue. This plasmid can be used to obtain hGH-phage particles in which the hGH-gene III fusion product is monovalently displayed on the phage surface (Example IV). This fusion protein contains the entire hGH protein fused to the carboxy-terminal domain of gene III by a variable linker sequence.
[0212]
To examine the possibility of using phage display to select a preferred substrate sequence for a protein hydrolase, a genetically engineered mutant of subtilisin BPN 'was used [Carter, P. et al.,Proteins: Structure, function and genetics 6: 240-248 (1989)]. This variant (hereinafter referred to as A64SAL subtilisin) contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly limited and certain histidine-containing substrates are preferentially hydrolyzed [Carter et al.,Science 237: 394-399 (1987)].
[0213]
h GH - Substrate - Construction of phage vector
The sequence of the linker region in pS0132 is the oligonucleotide:
 5'-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3 '
Was used to create a substrate sequence for A64SAL subtilisin. This results in a protein sequence:
Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp
Was introduced into the linker region between hGH and the carboxy-terminal domain of gene III (the first Phe residue in the sequence is hGH Phe191). Array:
Ala-Ala-His-Tyr-Thr-Arg-Gln
Is known to be a good substrate for A64SAL subtilisin [Carter et al. (1989); supra]. The resulting plasmid was named pS0640.
[0214]
h GH - Substrate - Selective enrichment of phage
Phagemid particles derived from pS0132 and pS0640 were made as described in Example I. In the first experiment, each phage pool (10 μl each) was added to oxirane beads (as described in Example II) in a buffer (100 μl) containing 20 mM Tris-HCl (pH 8.6) and 2.5 M NaCl. Preparation; 30 μl) and mixed separately. The binding and washing steps were performed as described in Example VII. The beads were then resuspended in the same buffer (400 μl) with or without 50 nM A64SAL subtilisin. After 10 minutes of incubation, the supernatant was collected and the phage titer (cfu) was measured. Table XVII shows that about 10 times more substrate-containing phagemid particles (pS0640) were eluted in the presence of enzyme than in the absence of enzyme, and for non-substrate phagemid (pS0132) in the presence or absence of enzyme. It shows that it elutes more than the case. Increasing enzyme, phagemid or bead concentration did not change this ratio.
[0215]
Improved selection enrichment method
In an attempt to reduce non-specific elution of immobilized phagemid, tightly binding hGH mutants were introduced into pS0132 and pS0640 instead of the wild type hGH gene. The hGH variant used is the variant described in Example XI (pH 0650 bp) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr. This results in two new phagemids: pDM0390 (contains tightly bound hGH, no substrate sequence) and pDM0411 (strongly bound hGH and substrate sequence Ala-Ala-His-Tyr-Thr-Arg-Gln) Containing) was constructed. Also, the binding wash and elution protocol was changed as follows:
(i) Binding: COSTAR 12 well tissue culture plates were coated with 2 μg / ml hGHbp in 0.5 ml / well sodium carbonate buffer (pH 10.0) for 16 hours. The plate was then incubated for 2 hours with 1 ml / well blocking buffer [phosphate buffered saline (PBS) containing 0.1% w / v bovine serum albumin] and 10 mM Tris-HCl (pH 7.5). ) Washed with assay buffer containing 1 mM EDTA and 100 mM NaCl. The phagemid was again prepared as described in Example I, the phage pool was diluted 1: 4 with the above assay buffer, and 0.5 ml of phage per well was incubated for 2 hours.
(Ii) Wash: The plate was washed thoroughly with PBS + 0.05% Tween 20 and incubated with this wash buffer (1 ml) for 30 minutes. This washing process was repeated three times.
(Iii) Elution: The plate is incubated for 10 minutes in elution buffer consisting of 20 mM Tris-HCl (pH 8.6) +100 mM NaCl, and then the above buffer with or without 500 nM A64SAL subtilisin (0.5 ml). ) To elute the phage.
[0216]
Table XVII shows a dramatic increase in the proportion of specifically eluted substrate-phagemid particles compared to the method previously described for pS0640 and pS0132. This result is thought to be due to the tightly binding hGH mutant having a significantly lower off-rate for binding to hGH binding protein compared to wild type hGH.
[0217]
table XVII. Specific elution of substrate-phagemid by A64SAL subtilisin
Colony forming units (cfu) were assessed by plating 10-fold dilutions of phage (10 μl) onto 10 μl spots of XL1-Blue cells on LB agar plates containing 50 μg / ml carbenicillin.

[0218]
Example XIV  Identification of preferred substrates for A64SAL subtilisin using selective enrichment of substrate sequence libraries
We tried to identify good substrate sequences from a library of random substrate sequences using the selective enrichment method described in Example XIII.
Construction of vector for insertion of randomized substrate cassette
A vector was designed that was suitable for the introduction of a randomized substrate cassette and subsequent expression of the substrate sequence library. The starting point was the vector pS0643 described in Example VIII. Oligonucleotide:
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-GCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3 '
Was used for site-directed mutagenesis, which introduced an ApaI (GGGCCC) and SalI (GTCGAC) restriction site between hGH and gene III. This new construct was named pDM0253 (the actual sequence of pDM0253 is
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-CCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3 '
And the underlined base substitution is due to pseudo-error in the mutant oligonucleotide). Also, the strongly binding hGH mutant described in the Examples was introduced by exchanging fragments derived from pDM0411 (Example XIII). The resulting library vector was named pDM0454.
[0219]
Preparation of library cassette vector and insertion of mutation cassette
To introduce the library cassette, pDM0454 was digested with ApaI and then with SalI and then 13% PEG8000 + 10 mM MgCl.₂Precipitated with, washed twice with 70% ethanol and resuspended. This effectively precipitates the vector but leaves a small ApaI-SalI fragment in solution [Paithankar, K.R. and Prasad, K.S.N.,Nucleic Acids Research 19: 1346]. The product was run on a 1% agarose gel, the ApaI-SalI digested vector was excised, purified using the Bandprep kit (Pharmacia) and resuspended for ligation with the mutation cassette.
[0220]
The cassette to be inserted contained a sequence similar to the DNA sequence in the linker region of pS0640 and pDM0411, but had histidine and tyrosine residue codons in the substrate sequence replaced by randomized codons. We chose to replace NNS (N = G / A / T / C; S = G / C) at each of the randomized positions as described in Example VIII. The oligonucleotides used in the mutation cassette are
5'-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3 '(cryptographic chain) and
5'-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3 '(unencrypted chain)
Met. This cassette also destroys the SalI site and thus digestion with SalI can be used to reduce the vector background. These oligonucleotides did not phosphorylate prior to insertion into the Apa-Sal cassette site. The reason for this is that the subsequent oligomerization of the cassette subpopulation was afraid that it could lead to false results with multiple cassette inserts. After annealing and ligation, the reaction product was phenol: chloroform extracted, ethanol precipitated and resuspended in water. Initially, digestion with SalI to reduce the background vector was not performed. As described in Example VIII, about 200 ng was electroporated into XL1-Blue cells to prepare a phagemid library.
[0221]
Selection of easily cleaved substrates from a substrate library
The selection method used is the same as described for pDM0411 and pDM0390 in Example XIII. After each round of selection, the eluted phage were transformed by transducing a culture of fresh XL1-Blue cells and growing a new phagemid library as described for hGH-phage in Example VIII. Allowed to grow. The progress of the selection method was monitored by measuring the titer of eluted phage and sequencing individual clones after each round of selection.
[0222]
Table A shows serial phage titers for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection. The ratio of specifically eluted phage: nonspecifically eluted phage (ie phage eluted with enzyme: phage eluted without enzyme) has increased dramatically from round 1 to round 3. This is evident, suggesting that the good substrate population increases with each selection round.
[0223]
Sequencing of 10 isolates from the starting library showed that all of these consisted of the wild type pDM0464 sequence. This is due to the fact that the SalI site is very close to the end of the DNA fragment after digestion with ApaI, thus leading to low efficiency digestion. Nevertheless, there are only 400 possible sequences in the library, so this population should still be noted.
[0224]
Tables B1 and B2 show the sequences of isolates obtained after round 2 and round 3 selection. After two rounds of selection, there is clearly a high incidence of histidine residues. This was exactly what was expected (as described in Example XIII, A64SAL subtilisin requires a histidine residue in the substrate because it uses a substrate-assisted catalytic mechanism. To do that). After 3 rounds of selection, each of the 10 sequenced clones has histidine in the randomized cassette. However, it should be noted that two of these sequences are of pDM0411, which were not present in the starting library and are therefore impurities.
[0225]
Table A. Elution from initial phage pool and 3 rounds of selective enrichment
Titration of phage
Colony forming units (cfu) were assessed by plating 10-fold dilutions of phage (10 μl) onto 10 μl spots of XL1-Blue cells on LB agar plates containing 50 μg / ml carbenicillin.

[0226]
Table B1. Sequence of eluted phage after two rounds of selective enrichment
All protein sequences should be in the form AA ** TRQ (where * represents a randomized codon). In the table below, randomized codons and amino acids are underlined.

Table B2. Sequence of eluted phage after 3 rounds of selective enrichment
All protein sequences should be in the form AA ** TRQ (where * represents a randomized codon). In the table below, randomized codons and amino acids are underlined.

[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a method for displaying large proteins on the surface of filamentous phages and a method for enriching modified receptor binding. Plasmid phGH-M13gIII was constructed in which the entire coding sequence of hGH was fused to the carboxy terminal domain of M13 gene III. Transcription of this fusion protein is under the control of the lac promoter / operator sequence and secretion is directed by the stII signal sequence. Phagemid particles are obtained by infection with “helper” phage M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor. The wild type gene III (derived from M13KO7 phage) is illustrated by 4-5 copies of multiple arrows at the top of the phage, and the fusion protein (derived from phagemid phGH-M13gIII) is represented by the hGH folding figure (replaces the head of the arrow) Illustrated.
FIG. 2: Immunoblot of total phage particles shows that hGH co-migrates with phage. Phagemid particles purified with a cesium chloride gradient were applied to double wells and electrophoresed on a 1% agarose gel in 375 mM Tris, 40 mM glycine (pH 9.6) buffer. The gel was soaked in transfer buffer [25 mM Tris (pH 8.3), 200 mM glycine, 20% methanol] containing 2% SDS and 2% β-mercaptoethanol for 2 hours, and then 6 hours in transfer buffer. Rinse. The protein in the gel was then electroblotted onto an immobilon membrane (Millipore). The membrane containing a set of samples was stained with Coomassie blue to indicate the location of the phage protein (A). The membrane hGH was immunostained by reacting the double membrane with a polyclonal rabbit anti-hGH antibody and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (B). Lane 1 contains the M13KO7 parental phage, which can only be observed in the Coomassie blue stained membrane because it lacks hGH. Lanes 2 and 3 contained another preparation of hormone phagemid particles and could be observed with both Coomassie and hGH immunostaining. Differences in migration distance between the parental M13KO7 phage and the hormone phagemid particles reflect differences in the size of the genome packaged inside (8.7 kb and 5.1 kb, respectively).
FIG. 3 is a diagram summarizing each step in the method for selecting an hGH-phage library in which codons 172, 174, 176 and 178 are randomized. The template molecule pH0415 containing the hGH (R178G, I179T) gene and a unique KpnI restriction site was mutagenized as described herein and electrotransferred into E. coli strain WJM101 to generate the initial phagemid library (library 1) was obtained. A portion of library 1 (about 2%) was used directly in the first round of selection as described herein to obtain library 1G. On the other hand, double-stranded DNA (dsDNA) was prepared from library 1, digested with restriction enzyme KpnI to eliminate the background of the template, and introduced into WJM101 to obtain library 2. Subsequent rounds of selection (or KpnI digestion; shaded boxes) and subsequent growth of phagemids were performed as indicated by the arrows according to the methods described herein. Library 4G^Four4 independent clones and library 5G from⁶Four independent clones from were sequenced by dideoxy sequencing. All of these clones had identical DNA sequences that matched the hGH mutant (Glu 174 Ser, Phe 176 Tyr).
FIG. 4: A structural model of hGH derived from a 2.8-fold folding diagram of porcine growth hormone determined by crystallography. The positions of the residues in hGH that strongly modulate binding to hGH-binding protein are shown in shaded circles. Shown are alanine substitutions that cause a 10-fold or greater decrease in binding affinity (black circles), a 4-10 fold decrease (•), an increase (◯), or a 2-4 fold decrease (•). Spiral ring projection in the α-helix region revealed these polarities. Residues that are blackened, shaded, or unshaded indicate charge, polarity, or nonpolarity, respectively. In helix 4, the most important residue for mutation is on the hydrophilic side.
FIG. 5: 172, 174 of hGH found after sequencing a large number of clones from round 1 and 3 selection steps for the indicated pathway (hGH elution, glycine elution, or glycine elution after pre-adsorption). The amino acid substitutions at positions 176 and 178 are indicated (for example, the designation KSYR represents the hGH mutant 172K / 174S / 176Y / 178R). Non-functional sequences (ie, vector background, or other immature mutants and / or frameshifted mutants) are indicated as “NF”. Functional sequences containing non-silent pseudomutations (ie, other than the set of target residues listed above) are indicated by “+”. Protein sequences that appear more than once in all sequencing clones but have different DNA sequences are indicated by “#”. Protein sequences that appear more than once in the sequencing clone and have the same DNA sequence are indicated by “*”. Note that two different contaminating sequences were found after three rounds of selection (these clones did not match the cassette mutant but matched the previously constructed hormone phage). The pS0643 contaminant matches the wild type hGH-phage (hGH “KEFR”). The pH0457 contaminant, abundant in the third round glycine-selected phage pool, matches the previously identified mutant “KSYR” of hGH. The amplification of these contaminants highlights the ability of the hormone-phage selection method to select rarely occurring mutants. Also, the concentration of these sequences was significant in all three pathways (ie, R or K occurs most frequently at positions 172 and 178, Y or F occurs most often at position 176, and S , T, A and other residues occur at position 174).
FIG. 6: Sequence from phage selected on hPRLbp-beads in the presence of zinc. The display is the same as described in FIG. Here, sequence concentration is unpredictable, but appears to be biased towards hydrophobic sequences under the most stringent (glycine) selection conditions (L, W and P residues are found in this pool a lot) .
FIG. 7: Sequence from phage selected on hPRLbp-beads in the absence of zinc. The display is the same as described in FIG. In contrast to the arrangement of FIG. 6, the arrangement here appears to be relatively hydrophilic. After 4 rounds of selection by hGH elution, two types of clones (ANHQ and TLDT / 171V) are present in the pool.
FIG. 8: Sequence from phage selected on blank beads. The display is the same as described in FIG. After 3 rounds of selection by glycine elution, no siblings were observed and the background level of non-functional sequence was retained.
FIG. 9: Construction of phagemid fl origin (ori) from pHO415. This vector for cassette mutagenesis and expression of hGH-gene III fusion protein was constructed as follows. Contains an origin of replication for pBR322 and fl, and under the control of the E. coli phoA promoter, an hGH-gene III fusion protein (residues 1-191 of hGH followed by one Gly residue, which is fused to gene III Pro-198 Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132. The following oligonucleotides:
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3 '
Was used for mutagenesis. This oligonucleotide introduced a Phe-191 of hGH followed by an XbaI site (underlined) and an amber stop codon (TAG).
FIG. 10: A shows plasmid pDH188 insert containing DNA encoding light and heavy chains (variable and constant domain 1) of Fab humanized antibody directed against the HER-2 receptor. It is illustrated. V_LAnd V_HAre the variable regions of the light and heavy chains, respectively. C_kIs the constant region of the human kappa light chain. CH1_G1Is the first invariant region of the human γ1 chain. Both coding regions start with the bacterial stII signal sequence. B illustrates the entire plasmid pDH188 containing the insert shown in 5A. After introducing this plasmid into E. coli SR101 cells and adding helper phage, this plasmid is packaged into phage particles. Some of these particles display Fab-pIII fusions, where pIII is a protein encoded by M13 gene III DNA. The segment in this plasmid diagram corresponds to the insert shown in 5A.
FIG. 11A shows the nucleotide sequence (SEQ ID NO: 25) of DNA encoding 4D5 Fab molecule expressed on the surface of phagemid. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11B shows the nucleotide sequence (SEQ ID NO: 25) of DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11C: shows the nucleotide sequence (SEQ ID NO: 25) of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11D shows the nucleotide sequence (SEQ ID NO: 25) of DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11E: shows the nucleotide sequence (SEQ ID NO: 25) of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11F shows the nucleotide sequence of DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11G shows the nucleotide sequence of DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 11H: shows the nucleotide sequence (SEQ ID NO: 25) of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain (SEQ ID NO: 26) is also shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27).
FIG. 12 shows enrichment of wild-type 4D5 Fab phagemid from mutant Fab phagemids. A mixture of wild type phagemid and mutant 4D5 Fab phagemid in a ratio of 1: 1,000 was selected on plates coated with the extracellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid was infected with E. coli to prepare plasmid DNA. The plasmid DNA was then digested with EcoRV and PstI, separated on a 5% polyacrylamide gel and stained with ethidium bromide. Bands were visualized under UV light. Bands attributed to wild type and mutant plasmids are indicated by arrows. The first round of selection eluted only under acid conditions. Subsequent rounds were eluted either by acid elution (left side of the figure) or a humanized 4D5 antibody wash step using the method described in Example VIII followed by acid elution (right side of the figure). Three mutant 4D5 Fab molecules were prepared. That is, H91A (V_LThe amino acid histidine at position 91 of the chain was mutated to alanine; shown in “A” lane in the figure), Y49A (V_LThe amino acid tyrosine at position 49 of the chain was mutated to alanine; shown in “B” lane in the figure), as well as Y92A (V_LThe amino acid tyrosine at position 92 of the chain was mutated to alanine; shown in “C” lane in the figure). The amino acid position was counted according to Kabat et al. ("Protein sequence of immunological insert", 4th edition, USDept of Health and Human Services, Public Health Service, Nat'l. Institute of Health, Bethesda, MD (1987)].
FIG. 13: Scatchard analysis of RIA affinity measurements as described in the experimental protocol is shown. The amount of bound labeled ECD antigen is shown on the x-axis, and the numerical value obtained by dividing the amount bound by the amount released is shown on the y-axis. The slope of the line indicates Ka, and the calculated Kd is 1 / Ka.

Claims (10)

A phagemid expression vector comprising a transcriptional regulatory element operably linked to a gene fusion encoding a fusion protein, the gene fusion comprising a first gene encoding a polypeptide and a phage coat protein A second gene encoding at least a portion of
The vector does not contain a gene encoding a mature coat protein, and
(I) does not contain the complete phage genome and / or
(Ii) transformation of host cells with the vector does not produce phage particles in the absence of helper phage and / or
(Iii) an amino acid-encoded triplet in which a DNA triplet codon encoding a repressible stop codon of mRNA is inserted between the fusion ends of the first and second genes or adjacent to the gene fusion junction A phagemid expression vector substituted for a codon,
However,
(a) the vector does not contain a gene encoding a mature coat protein;
(b) does not contain the complete phage genome,
(c) transformation of host cells with the vector does not produce phage particles in the absence of helper phage;
Only one selected from the group consisting of
(d) an amino acid-encoded triplet in which a DNA triplet codon encoding a repressible stop codon of mRNA is inserted between or adjacent to the fusion ends of the first and second genes Excludes vectors containing in combination with phagemid expression vectors substituted for codons.   The phagemid vector according to claim 1, wherein the stop codon is UAG, UAA or UGA.   The phagemid expression vector according to claim 1 or 2, wherein the phage coat protein is encoded by gene III of filamentous phage. The phagemid expression vector according to claim 3, wherein the first gene is a mammalian protein or a synthetic protein .   Protein is growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A chain, insulin B chain, proinsulin, relaxin A chain, relaxin B chain , Prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), reutizing hormone (LH), glycoprotein hormone receptor, calcitonin, glucagon, factor VIII, antibody, pulmonary surfactant, urokinase, streptokinase, Human tissue type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hematopoietic growth factor, tumor necrosis factor α and β, enkephalinase, human serum albumin, Mueller inhibitor, mouse gonadotropin related peptide, β-lactamase, tissue factor Protein, inhibin, activin, vascular endothelial growth factor, integrin receptor, thrombopoietin, proteins A and D, rheumatoid factor, nerve growth factor (NGF-β), platelet growth factor, transforming growth factor (TGF); TGF-α And TGF-β, insulin-like growth factor I and II, insulin-like growth factor binding protein, CD-4, DNase, latency-related peptide, erythropoietin, osteoinductive factor, interferon α, β and γ, colony stimulating factor (CSF) ), M-CSF, GM-CSF and G-CSF, interleukin (IL), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope protein GP120 and GP140, atrial natriuretic peptide A, And C, a phagemid expression vector according to claim 1 protein with a subunit more than one of the above proteins, immunoglobulins, and which are selected from the group consisting of fragments.   A host cell containing the phagemid expression vector according to any one of claims 1 to 5 and a helper phage having a gene encoding a coat protein. The amount or the number of phagemid particles that comprise at least a part of one of the phagemid expression vectors according to any one of claims 1 to 5 and that display two or more copies of the fusion protein on the particle surface is 20% A group of phagemid particles that are less than. A method for selecting a novel binding polypeptide comprising:
(a) constructing a family of phagemid expression vectors according to any one of claims 1 to 5 (wherein the phagemid expression vector contains a mutant first gene encoding a mutant polypeptide);
(b) transforming a suitable host cell with a phagemid expression vector;
(c) infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein sufficient to produce recombinant phagemid particles;
(d) Under the conditions suitable for formation of recombinant phagemid particles containing at least a part of one of the phagemid expression vectors according to any one of claims 1 to 5 and capable of transforming a host, Culturing transformed and infected host cells, wherein the amount or number of phagemid particles displaying more than 2 copies of the fusion protein on the particle surface is less than 20%;
(e) contacting the recombinant phagemid particle with a target molecule to bind at least a portion of the phagemid particle to the target molecule; and
(f) separating phagemid particles that bind to the target molecule from unbound particles;
A method comprising the steps.   A group of phagemid particles produced by the method of claim 8. 9. The method of claim 8, wherein the first gene encodes a polypeptide linked to a linker amino acid sequence, and the phagemid expression vector contains a mutant first gene encoding a variant linker amino acid sequence, Step (f)
(f) contacting the bound phagemid particle with a protease capable of hydrolyzing the bound amino acid sequence of at least a portion of the bound phagemid particle;
(g) isolating hydrolyzed phagemid particles; and optionally,
(h) further contacting a suitable host cell with the hydrolyzed phagemid particle and repeating steps (d)-(g);
The method of claim 8, wherein

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