JP2004509621A - Ligand / receptor-specific converters that redirect antibodies to receptors on pathogens - Google Patents

Abstract

The present invention relates generally to compositions and methods for preventing and treating pathogens such as bacteria, yeast, parasites, fungi, viruses, and human diseases, including but not limited to cancer. In particular, the embodiments described herein relate to the manufacture and use of ligand / receptor specific converters that redirect existing antibodies in a subject to receptors present on a pathogen.

Description

【００４２】
抗原性ドメイン
病原体または毒素は多数の異なるエピトープを提示し得るため、リガンド／受容体特異性変換体に用いられ得る抗原性ドメインの多様性も非常に広い。すなわち、リガンド／受容体特異性変換体中に組入れられ得る抗原性ドメインとしては、細菌、真菌、植物、糸状菌、ウイルス、癌細胞、および毒素のエピトープが挙げられる。所望の抗原性ドメインは、天然獲得免疫または予防接種により、被験体中にすでに存在する病原体のエピトープを含む。例えば小児期疾患を引き起こす病原体のエピトープは、抗原性ドメインとして用いられ得る。
【００４３】
いくつかの実施形態は、別個に被験体に投与される抗体と相互作用する抗原性ドメインを有する。例えば特異性変換体の抗原性ドメインと相互作用する抗体は、特異性変換体と同時投与され得る。さらに、リガンド／受容体特異性変換体と相互作用する抗体は、被験体中には通常は存在し得ないが、被験体は生物学的物質（例えば血清、血液、または組織）の導入により抗体を獲得し得る。例えば輸血を受ける被験体は多数の抗体を獲得し、そのうちのいくつかはリガンド／受容体特異性変換体の抗原性ドメインと相互作用し得る。リガンド／受容体特異性変換体に用いるためのいくつかの好ましい抗原性ドメインは、ウイルスエピトープ、例えば単純ヘルペスウイルス、Ｂ型肝炎ウイルス、ＴＴウイルス、およびポリオウイルス（これらに限定されない）を含む。
【００４４】
いくつかの実施形態では、抗原性ドメインが、「高力価抗体」により認識される病原体または毒素のエピトープを含むのも好ましい。病原体または毒素のエピトープが高力価抗体により認識されるか否かを確定するためのアプローチは以下で提示する。リガンド／受容体特異性変換体の抗原性ドメイン中に含入され得る病原体のエピトープとしては、スウェーデン特許第９９０１６０１−６号、米国特許第５，８６９，２３２号、Ｍｏｌ． Ｉｍｍｕｎｏｌ． ２８：７１９−７２６（１９９１）、およびＪ． Ｍｅｄ． Ｖｉｒｏｌ． ３３：２４８−２５２（１９９１）に開示されたペプチド配列上のエピトープが挙げられる。表２は、いくつかの好ましい抗原性ドメインのアミノ酸配列を提示する。
【００４５】
表２よりも後のセクションでは、リガンド／受容体特異性変換体の調製をより詳細に説明する。
【００４６】
【表２】

【００４７】
細菌、寄生生物、真菌、糸状菌、ウイルス、および癌細胞上の受容体と相互作用するリガンド／受容体特異性変換体の製造方法
いくつかの実施形態では、特異性ドメインおよび抗原性ドメインは別個に製造され、その後一緒に連結され（例えばリンカーにより、または共通担体分子との会合により）、また他の実施形態では、特異性ドメインおよび抗原性ドメインは同一分子の一部として製造される。例えば表１に列挙された特異性ドメインのいずれかが、表２の抗原性ドメインのいずれかと連結される。特異性および抗原性ドメインは別個に製造され、リンカーまたは担体分子により連結されて一緒にされ得る（例えばビオチニル化特異性ドメイン、ストレプトアビジン、およびビオチニル化抗原性ドメインを含む複合体）が、リガンド／受容体特異性変換体は融合タンパク質として作製されるのが好ましい。したがって、好ましい実施形態は、表２中のいずれかの抗原性ドメインに連結された表１中のいずれかの特異性ドメインを有する融合タンパク質を含む。
【００４８】
リガンド／受容体特異性変換体は、タンパク質工学処理、タンパク質化学、有機化学、および分子生物学の慣用的方法にしたがって作製され得る。さらに、いくつかの企業は、注文作製ペプチドを製造するが、リガンド／受容体特異性変換体は、このような会社に所望のリガンド／受容体特異性変換体の配列を提供し、特定の仕様書にしたがって作用物質を製造するそれらのサービスを用いることにより得てもよい（例えばＢａｃｈｅｍ ＡＧ， Ｓｗｉｔｚｅｒｌａｎｄ）。好ましくはリガンド／受容体特異性変換体は、当業界で既知の技法、例えばＭｅｒｒｉｆｉｅｌｄ ｅｔ ａｌ．， Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ． ８５：２１４９（１９６４）， Ｈｏｕｇｈｔｅｎ ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ８２：５１：３２（１９８５）， Ｓｔｅｗａｒｔ ａｎｄ Ｙｏｕｎｇ（Ｓｏｌｉｄ ｐｈａｓｅ ｐｅｐｔｉｄｅ ｓｙｎｔｈｅｓｉｓ， Ｐｉｅｒｃｅ Ｃｈｅｍ Ｃｏ．， Ｒｏｃｋｆｏｒｄ， ＩＬ（１９８４）およびＣｒｅｉｇｈｔｏｎ， １９８３， Ｐｒｏｔｅｉｎｓ： Ｓｔｒｕｃｔｕｒｅｓ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｐｒｉｎｃｉｐｌｅｓ， Ｗ．Ｈ． Ｆｒｅｅｍａｎ ＆ Ｃｏ．， Ｎ．Ｙ．により記述された技法を用いて、化学合成方法（例えば固相ペプチド合成）により調製される。
【００４９】
一アプローチによれば、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ４３０Ａペプチド合成機（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， Ｆｏｓｔｅｒ Ｃｉｔｙ， ＣＡ）を用いて、固相ペプチド合成が実施される。各合成は、ｐ−メチルベンジルヒドリルアミン固相支持体樹脂（Ｐｅｐｔｉｄｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ， Ｌｏｕｉｓｖｉｌｌｅ， ＫＹ）を使用し、酸加水分解によりペプチドが固体支持体から切り離されると、カルボキシル末端アミドを産生する。使用前に、カルボキシル末端アミドは除去することができ、高速液体クロマトグラフィー（例えばＰｅｐＳ−１５　Ｃ１８カラム（Ｐｈａｒｍａｃｉａ， Ｕｐｐｓａｌａ， Ｓｗｅｄｅｎ）を用いた逆相高速液体クロマトグラフィー（ＲＰ−ＨＰＬＣ））によりリガンド／受容体特異性変換体が精製され、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ４７３Ａペプチド合成機でシーケンシングされる。代替的合成アプローチは、自動ペプチド合成機（Ｓｙｒｏ， Ｍｕｌｔｉｓｙｎｔｅｃｈ， Ｔｕｂｉｎｇｅｎ， Ｇｅｒｍａｎｙ）および９−フルオレニルメトキシカルボニル（ｆｍｏｃ）保護化アミノ酸（Ｍｉｌｌｉｇｅｎ， Ｂｅｄｆｏｒｄ， ＭＡ）を用いる。
【００５０】
リガンド／受容体特異性変換体は化学的に合成され得るが、一方、当業界で既知の技法を用いた組換えＤＮＡ技術によりこれらのポリペプチドを産生する方が効率的であり得る。このような方法を用いて、リガンド／受容体特異性変換体をコードするヌクレオチド配列ならびに適切な転写および翻訳制御シグナルを含む発現ベクターを構築し得る。これらの方法としては、例えばｉｎ　ｖｉｔｒｏ組換えＤＮＡ技法、合成技法、およびｉｎ　ｖｉｖｏ遺伝子組換えが挙げられる。あるいは、リガンド／受容体特異性変換体をコードし得るＲＮＡは、例えば合成機を用いて化学的に合成され得る。例えばＯｌｉｇｏｎｕｃｌｅｏｔｉｄｅ Ｓｙｎｔｈｅｓｉｓ， １９８４， Ｇａｉｔ， Ｍ． Ｊ． ｅｄ．， ＩＲＬ Ｐｒｅｓｓ， Ｏｘｆｏｒｄに記載された技法を参照されたい。
【００５１】
種々の宿主発現ベクター系が、リガンド／受容体特異性変換体を発現するために利用され得る。リガンド／受容体特異性変換体は、可溶性分子である場合には培養から、すなわちペプチドまたはポリペプチドが分泌されない場合には宿主細胞から、およびペプチドまたはポリペプチドが細胞から分泌される場合には培地から得られる。しかしながら、発現系は、膜に結合したリガンド／受容体特異性変換体を発現するように工学処理された宿主細胞も含む。このような発現系からのリガンド／受容体特異性変換体の精製または濃縮は、適切な洗剤および脂質ミセルならびに当業者に既知の方法を用いて成し遂げられ得る。
【００５２】
用いられ得る発現系としては、微生物、例えばリガンド／受容体特異性変換体をコードするヌクレオチド配列を含む組換えバクテリオファージＤＮＡ、プラスミドＤＮＡまたはコスミドＤＮＡ発現ベクターにより形質転換された細菌（例えば大腸菌または枯草菌）など；リガンド／受容体特異性変換体をコードするヌクレオチド配列を含む組換え酵母発現ベクターにより形質転換された酵母（例えばサッカロミセス属、ピキア属）；リガンド／受容体特異性変換体をコードする核酸を含む組換えウイルス発現ベクター（バキュロウイルス属）に感染した昆虫細胞系；あるいはリガンド／受容体特異性変換体をコードする核酸を含有する組換え発現構築物を保有する哺乳類細胞系（例えばＣＯＳ、ＣＨＯ、ＢＨＫ、２９３、３Ｔ３）が挙げられるが、これらに限定されない。
【００５３】
細菌系では、多数の発現ベクターは、リガンド／受容体特異性変換体に関して意図された用途に応じて適宜に選択され得る。例えば、多くの量が必要とされる（例えばリガンド／受容体特異性変換体の製剤組成物の生成のために）場合、容易に精製され得る高レベルの融合タンパク質産物の発現を指図するベクターが望ましい。このようなベクターとしては、大腸菌発現ベクターｐＵＲ２７８（Ｒｕｔｈｅｒ ｅｔ ａｌ．， ＥＭＢＯ Ｊ．， ２：１７９１（１９８３））（この場合、融合タンパク質が産生されるように、リガンド／受容体特異性変換体コード配列は、ｌａｃＺコード領域を有する枠内でベクターに別々に連結され得る）；ｐＩＮベクター（Ｉｎｏｕｙｅ ＆ Ｉｎｏｕｙｅ， Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．， １３：３１０１−３１０９（１９８５）； Ｖａｎ Ｈｅｅｋｅ ＆ Ｓｃｈｕｓｔｅｒ， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， ２６４：５５０３−５５０９（１９８９））等が挙げられるが、これらに限定されない。ｐＧＥＸベクターは、グルタチオンＳ−トランスフェラーゼ（ＧＳＴ）とともに、融合タンパク質のような外来ポリペプチドを発現するためにも用いられ得る。概して、このような融合タンパク質は可溶性であり、グルタチオン−アガロースビーズへの吸着とその後の遊離グルタチオンの存在下での溶離により、溶解細胞から精製され得る。クローニングされた標的遺伝子産物がＧＳＴ部分から放出されるように、ＰＧＥＸベクターは、トロンビンまたはＸａ因子プロテアーゼ切断部位を含むよう設計される。
【００５４】
昆虫系では、Ａｕｔｏｇｒａｐｈａ ｃａｌｉｆｏｒｎｉｃａ核多角体病ウイルス（ＡｃＮＰＶ）が外来遺伝子を発現するためのベクターとして用いられる。ウイルスはＳｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａ細胞中で成長する。リガンド／受容体特異性変換体遺伝子コード配列は、ウイルスの非必須領域（例えばポリヘドリン遺伝子）中に別々にクローニングされ、ＡｃＮＰＶプロモーター（例えばポリヘドリンプロモーター）の制御下に置かれる。リガンド／受容体特異性変換体遺伝子コード配列を適切に挿入すると、ポリヘドリン遺伝子の不活性化および非閉塞性組換えウイルス（すなわちポリヘドリン遺伝子によりコードされるタンパク質様被膜を欠くウイルス）の産生を生じる。これらの組換えウイルスは次に、挿入遺伝子を発現するためのＳｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａ細胞を感染させるために用いられる。（例えばＳｍｉｔｈ ｅｔ ａｌ．， Ｊ． Ｖｉｒｏｌ． ４６：５８４（１９８３）および米国特許第４，２１５，０５１号（Ｓｍｉｔｈ）参照）。
【００５５】
哺乳類宿主細胞では、多数のウイルスベースの発現系が利用され得る。アデノウイルスが発現ベクターとして用いられる場合、リガンド／受容体特異性変換体をコードする核酸配列は、アデノウイルス転写／翻訳制御複合体、例えば後期プロモーターおよびトリパルタイトリーダー配列に連結され得る。このキメラ遺伝子は次に、ｉｎ　ｖｉｔｒｏまたはｉｎ　ｖｉｖｏ組換えによりアデノウイルスゲノム中に挿入され得る。ウイルスゲノムの非必須領域（例えば領域Ｅ１またはＥ３）中に挿入すると、感染宿主中で生育可能であり、かつリガンド／受容体特異性変換体遺伝子産物を発現し得る組換えウイルスを得ることができる。（例えばＬｏｇａｎ ＆ Ｓｈｅｎｋ， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ８１：３６５５−３６５９（１９８４）参照）。特定開始シグナルも、挿入リガンド／受容体特異性変換体ヌクレオチド配列を効率的に翻訳するために必要とされ得る（例えばＡＴＧ開始コドンおよび隣接配列）。ほとんどの場合、おそらくはＡＴＧ開始コドンを含めた外因性翻訳制御シグナルが提供されるべきである。さらに、開始コドンは、全挿入物を確実に翻訳するために、所望のコード配列のリーディングフレームと同調して存在する必要がある。これらの外因性翻訳制御シグナルおよび開始コドンは、天然および合成の両方の、種々の起点を有し得る。発現効率は、適切な転写エンハンサー要素、転写ターミネーター等を組み込むことによっても高めることができる。（Ｂｉｔｔｎｅｒ ｅｔ ａｌ．， Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌ．， １５３：５１６−５４４（１９８７）参照）。
【００５６】
さらに、挿入配列の発現を調整する、あるいは所望の特定方式で遺伝子産物を修飾し、プロセシングする宿主細胞株が選択され得る。タンパク質産物の、このような修飾（例えばグリコシル化）およびプロセシング（例えば切断）は、いくつかの実施形態において重要であり得る。異なる宿主細胞は、タンパク質および遺伝子産物の翻訳後のプロセシングおよび修飾に関する特徴的および特異的メカニズムを有する。適切な細胞株または宿主系を選択することにより、発現された外来タンパク質を確実に、的確な修飾およびプロセシングをし得る。この目的のために、遺伝子産物の一次転写体の適正なプロセシング、グリコシル化、およびリン酸化のための細胞機構を保有する真核生物宿主細胞が用いられ得る。このような哺乳類宿主細胞としては、ＣＨＯ、ＶＥＲＯ、ＢＨＫ、ＨｅＬａ、ＣＯＳ、ＭＤＣＫ、２９３、３Ｔ３、およびＷＩ３８が挙げられるが、これらに限定されない。
【００５７】
組換えタンパク質を長期間、高収率に産生するためには、安定発現させることが好ましい。例えば、工学処理により上記リガンド／受容体特異性変換体を安定的に発現する細胞株が得られる。複製のウイルス起点を含む発現ベクターを用いるというよりむしろ、宿主細胞は適切な発現制御要素（例えばプロモーター、エンハンサー配列、転写ターミネーター、ポリアデニル化部位等）および選択可能マーカーにより制御されるＤＮＡを用いて形質転換され得る。外来ＤＮＡの導入後、工学処理細胞は濃縮培地中で１〜２日間成長させることができ、その後、選択培地に切り替えられる。組換えプラスミド中の選択可能マーカーは、選択に対する耐性を付与し、かつ細胞がそれらの染色体中にプラスミドを安定的に組み込み、細胞が成長して細胞増殖巣を形成し、これは次に細胞株中でクローニングされ、拡張される。この方法は、リガンド／受容体特異性変換体を発現する細胞株を工学処理するために適宜に用いられる。
【００５８】
多数の選択系が用いられ、例としては単純ヘルペスウイルスチミジンキナーゼ（Ｗｉｇｌｅｒ， ｅｔ ａｌ．， Ｃｅｌｌ １１：２２３（１９７７））、ヒポキサンチン−グアニンホスホリボシルトランスフェラーゼ（Ｓｚｙｂａｌｓｋａ ＆ Ｓｚｙｂａｌｓｋｉ， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ４８：２０２６（１９６２））、およびアデニンホスホリボシルトランスフェラーゼ（Ｌｏｗｙ， ｅｔ ａｌ．， Ｃｅｌｌ ２２：８１７（１９８０））遺伝子が挙げられるが、これらに限定されない。これらはそれぞれ、ｔｋ．ｓｕｐ．−、ｈｇｐｒｔ．ｓｕｐ．−またはａｐｒｔ．ｓｕｐ．−細胞中で用いられ得る。さらに、抗代謝産物耐性が以下の遺伝子に関する選択の基礎として用いられ得る：メトトレキセートに対する耐性を付与するｄｈｆｒ（Ｗｉｇｌｅｒ， ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ７７：３５６７（１９８０）； Ｏ’Ｈａｒｅ， ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ７８： １５２７ （１９８１））；ミコフェノール酸に対する耐性を付与するｇｐｔ（Ｍｕｌｌｉｇａｎ ＆ Ｂｅｒｇ， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ７８：２０７２（１９８１））；アミノグリコシドＧ−４１８に対する耐性を付与するｎｅｏ（Ｃｏｌｂｅｒｒｅ−Ｇａｒａｐｉｎ， ｅｔ ａｌ．， Ｊ． Ｍｏｌ． Ｂｉｏｌ． １５０：１（１９８１））；およびハイグロマイシンに対する耐性を付与するｈｙｇｒｏ（Ｓａｎｔｅｒｒｅ， ｅｔ ａｌ．， Ｇｅｎｅ ３０：１４７（１９８４））。
【００５９】
以下のセクションでは、リガンド／受容体特異性変換体の特徴付けアッセイをより詳細に説明する。
【００６０】
リガンド／受容体特異性変換体の特徴付けアッセイ
好ましくは、リガンド／受容体特異性変換体は、受容体と相互作用する能力、および／または被験体中に存在し得る抗体と相互作用する能力に関して分析される。「特徴付けアッセイ」という用語は、リガンド／受容体特異性変換体に関してなされるアッセイ、実験または分析を指し、これは受容体（例えば細菌、ウイルス、糸状菌または真菌中に存在する表面受容体）または抗体（例えば病原体上に見出されるエピトープを認識する抗体）と相互作用する能力、あるいは病原体の増殖に対するリガンド／受容体特異性変換体の能力を評価する。「特徴付けアッセイ」という用語に含まれるのは、結合試験（例えば酵素イムノアッセイ（ＥＩＡ）、酵素結合イムノアッセイ（ＥＬＩＳＡ）、競合的結合アッセイ、コンピューター生成結合アッセイ、支持体結合試験、ならびに１および２ハイブリッド系）、および感染性試験（例えばウイルス感染、増殖および宿主細胞への接着の低減）である。
【００６１】
好ましい結合アッセイは、多重作用物質を用いる。多重作用物質の一形態は、支持体上にあるリガンド／受容体特異性変換体またはそれらの断片を含む組成物に関する。別の形態の多重作用物質は、支持体上にある受容体、または支持体にある抗体であってリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体を含む組成物を包含する。「支持体」は、このような分子を連結または固定するために用いられる担体、タンパク質、樹脂、細胞膜または任意の高分子構造であり得る。固体支持体としては、反応トレーのウエルの壁、試験管、ポリスチレンビーズ、磁気ビーズ、ニトロセルロースストリップ、膜、微小粒子（例えばラテックス粒子）、動物細胞、デュラサイト（登録商標）、人工細胞等が挙げられるが、これらに限定されない。リガンド／受容体特異性変換体は、例えば支持体上のヒドロキシ、カルボキシまたはアミノ基および反応基を介した共有結合により、無機支持体、例えば酸化ケイ素物質（例えばシリカゲル、ゼオライト、珪藻土またはアミノ化ガラス）とも連結され得る。
【００６２】
いくつかの多重作用物質では、高分子支持体は、疎水性非共有的相互作用によりリガンド／受容体特異性変換体の一部、受容体またはリガンドと相互作用する疎水性表面を有する。いくつかの場合には、支持体の疎水性表面は、ポリマー（例えばプラスチック）、または疎水性基が連結されていた任意のその他のポリマー（例えばポリスチレン、ポリエチレンまたはポリビニル）である。さらに、リガンド／受容体特異性変換体、受容体、またはリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体は、支持体、例えばタンパク質およびオリゴ／多糖（例えばセルロース、デンプン、グリコーゲン、キトサンまたはアミノ化セファロース）と共有的に結合され得る。これらの後者の多重作用物質では、分子上の反応基、例えばヒドロキシ基またはアミノ基は、担体上の反応基と連結し、共有結合をさせるために用いられる。付加的多重作用物質は、リガンド／受容体特異性変換体、受容体、またはリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体と結合するように化学的に活性化されているその他の反応基を有する支持体を含む。例えば臭化シアン活性化マトリックス、エポキシ活性化マトリックス、チオおよびチオプロピルゲル、ニトロフェニルクロロホルメートおよびＮ−ヒドロキシスクシンイミドクロロホルメート結合、あるいはオキシランアクリル支持体が用いられ得る（Ｓｉｇｍａ）。さらに、いくつかの実施形態では、リポソームまたは脂質二重層（天然または合成）を支持体として用いることが意図され、リガンド／受容体特異性変換体、受容体、またはリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体は膜表面に結合され得るか、あるいはリポソーム工学処理における技法により、膜中に組入れられ得る。一アプローチによれば、リポソーム多重支持体は、表面上に曝露されるリガンド／受容体特異性変換体、受容体、またはリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体を含む。
【００６３】
より柔軟性を高め、支持体により発生する任意の立体障害を克服するために、リガンド／受容体特異性変換体、受容体、またはリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体、および支持体との間に適切な長さのリンカー（例えば、λファージの柔軟性領域に類似するよう工学処理された「λリンカー」）を挿入することも意図される。最適結合を可能にするリンカーの適切な長さの決定は、本明細書中に詳述された特徴付けアッセイにおいて、種々のリンカーとの結合分子をスクリーニングすることにより見出され得る。
【００６４】
リガンド／受容体特異性変換体を特徴づけするためのいくつかのアプローチは、上記多重体を用いる。例えば、支持体に結合したリガンド／受容体特異性変換体は、「遊離した」接着受容体と接触され、会合が直接的（例えば標識接着受容体を用いることにより）または間接的に（例えば接着受容体に対する標識リガンドを用いることにより）確定され得る。したがって、受容体と支持体結合候補リガンド／受容体特異性変換体との会合によって、候補リガンド／受容体特異性変換体が真正リガンド／受容体特異性変換体として同定される。あるいは支持体結合接着受容体は、「遊離」リガンド／受容体特異性変換体と接触されて、会合したリガンド／受容体特異性変換体の量が直接的に（例えば標識リガンド／受容体特異性変換体を用いることにより）または間接的に（例えばリガンド／受容体特異性変換体の抗原性ドメインに対する標識抗体を用いることにより）確定され得る。同様に、支持体上にあるリガンド／受容体特異性変換体の抗原性ドメインに特異的な抗体、および標識リガンド／受容体特異性変換体（または二次検出試薬、例えば標識受容体またはリガンド／受容体特異性変換体に対する抗体）を用いることにより、リガンド／受容体特異性変換体の抗原性ドメインと結合する抗体の能力が確定され得る。
【００６５】
いくつかの特徴付けアッセイは、標的受容体および再指向された抗体と相互作用するリガンド／受容体特異性変換体の能力を評価するが、一方、他の特徴付けアッセイは、リガンド／受容体特異性変換体が標的受容体および再指向抗体の両方と結合し得るか否かを確定するように設計される。概して、特徴付けアッセイは、以下のように分類され得る：（１）ｉｎ　ｖｉｔｒｏ特徴付けアッセイ、（２）細胞特徴付けアッセイ、および（３）ｉｎ　ｖｉｖｏ特徴付けアッセイ。
【００６６】
各種類の特徴付けアッセイの考察を、以下のセクションで提示する。
【００６７】
ｉｎ　ｖｉｔｒｏ特徴付けアッセイ
リガンド／受容体特異性変換体が特定の受容体と結合するか否か、および被験体中にある抗体がリガンド／受容体特異性変換体と結合し得るか否かを確定するために用いられ得る、多数の種類のｉｎ　ｖｉｔｒｏアッセイが存在する。もっとも簡単には、受容体が支持体（例えばペトリ皿）に結合され、リガンド／受容体特異性変換体と受容体との会合が、上記のように直接または間接的にモニタリングされる。同様に、リガンド／受容体特異性変換体の抗原性ドメインに対する一次抗体（例えば被験体中に見出される抗体）が支持体に結合され、リガンド／受容体特異性変換体と一次抗体との会合が直接的に（例えば標識リガンド／受容体特異性変換体を用いて）または間接的に（例えば標識受容体、または一次抗体により認識されるエピトープと競合しないリガンド／受容体特異性変換体上のエピトープと相互作用する標識二次抗体を用いて）確定され得る。
【００６８】
別のアプローチでは、受容体が支持体に結合され、リガンド／受容体特異性変換体が受容体に結合され、一次抗体がリガンド／受容体特異性変換体に結合されるサンドイッチ型アッセイを含む。標識一次抗体が用いられる場合、受容体／特異性変換体／一次抗体の複合体の存在は、直接的に確定され得る。受容体／特異性変換体／一次抗体の複合体の存在は、例えば一次抗体とリガンド／受容体特異性変換体との結合を妨げないエピトープで一次抗体と反応する標識二次抗体を用いることにより、間接的に確定することもできる。いくつかの場合には、無標識二次抗体と反応させて標識三次抗体を用い、したがって受容体／特異性変換体／一次抗体／二次抗体／標識三次抗体の複合体を形成するのが望ましい。
【００６９】
以下の実施例は、フィブリノーゲンのＣ末端ドメイン由来の特異性ドメインが凝集因子（Ｃｌｆ）とフィブリノーゲンとの結合を抑制するか否かを確定するために実施された特徴付けアッセイを説明する。
【００７０】
実施例１
この実施例では、フィブリノーゲン（Ｆｉｂ）のＣ末端ドメインに対応するいくつかのペプチドが、凝集因子（Ｃｌｆ）とフィブリノーゲンとの結合を遮断する能力に関して分析した（表３参照）。ｆｍｏｃ　ｃｈｅｍｉｓｔｒｙ（Ｓｙｒｏ， ＭｕｌｔｉＳｙｎＴｅｃｈ， Ｇｅｒｍａｎｙ）を用いたペプチド合成における標準技法を用いて、これらのペプチドを製造した。好ましくは、逆相ＨＰＬＣによりペプチドを精製する。次に競合酵素イムノアッセイを実施して、ペプチドがＣｌｆおよびフィブリノーゲン間の相互作用を遮断することができるか否かを確定した。これらの実験の結果を表３に示す。Ｃｌｆおよびフィブリノーゲン間の相互作用を抑制することが見出されたフィブリノーゲン由来の最小ペプチドは、ＨＬＧＧＡＫＱＡＧＤ（配列番号１２４）であった。アラニンおよびリシンを用いてこの最小ペプチドの最初の２個のアミノ酸を置換すると、ペプチドがＣｌｆおよびフィブリノーゲン間の相互作用を遮断する能力に有意な作用を及ぼした（例えばペプチドＡＬＧＧＡＫＱＡＧＤ（配列番号１２３）はＣｌｆ／フィブリノーゲン相互作用を遮断できなかった）。
【００７１】
【表３】

【００７２】
以下の実施例は、リガンド／受容体特異性変換体が、ＣｌｆＡ受容体を有する細菌と相互作用するか否かを確定するために実施した特徴付けアッセイを記載する。
【００７３】
実施例２
フィブリノーゲンγ鎖配列の種々の領域に対応する特異性ドメイン（約２０アミノ酸長）を有するリガンド／受容体特異性変換体を、ｆｍｏｃ　ｃｈｅｍｉｓｔｒｙ（Ｓｙｒｏ， ＭｕｌｔｉＳｙｎＴｅｃｈ， Ｇｅｒｍａｎｙ）を用いたペプチド合成における標準技法を用いて製造し、これらのリガンド／受容体特異性変換体が、ＣｌｆＡ受容体、およびそれらの抗原性ドメインに特異的な抗体と結合する能力に関して分析した。これらのリガンド／受容体特異性変換体の配列を表４に列挙し、配列表に提示する（配列番号６０〜１０３）。この分析に用いたリガンド／受容体特異性変換体は、単純ヘルペスウイルスｇＧ２タンパク質のエピトープを提示する抗原性ドメインを有し、単純ヘルペスウイルスｇＧ２タンパク質に対するモノクローナル抗体により認識される。２：ｇ／ｍｌヤギ血清（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌｓ， Ｓｔ． Ｌｏｕｉｓ， ＭＯ）および０．５％Ｔｗｅｅｎ２０（ＰＢＳ−ＧＴ）を含有するリン酸塩緩衝化生理食塩水（ＰＢＳ）中で、これらのリガンド／受容体特異性変換体の連続希釈を行なった。９６ウエル微小滴定プレートにて、ｐＨ９．６の５０ｍＭ炭酸ナトリウム緩衝液中、＋４℃で一夜、１０μｇ／ｍｌで受容体ＣｌｆＡを受動的に吸着させた。
【００７４】
次にプレート上で６０分間、希釈したリガンド／受容体特異性変換体をインキュベートした。プレートに一次抗体を添加し、６０分間インキュベートすることにより（単純ヘルペスウイルスｇＧ２タンパク質に対するｍＡｂの１：１０００希釈）、受容体と相互作用するリガンド／受容体特異性変換体の能力を確定した。次に、ウサギ抗マウスＩｇＧ（Ｓｉｇｍａ）二次抗体、およびペルオキシダーゼ標識ヤギ抗ウサギＩｇＧ（Ｓｉｇｍａ）三次抗体により、結合一次ｍＡｂを指標した。ジニトロフェニレン−ジアミン（Ｓｉｇｍａ）を用いたインキュベーションによりプレートを展開して、４０５ｎｍでの吸光度を分析した。
【００７５】
表４に提示されたすべてのリガンド／受容体特異性変換体（配列番号６０〜１０３）は、固定化ＣｌｆＡと評価できるほどに結合し、ＨＳＶ　ｇＧ２タンパク質に特異的なｍＡｂの結合も可能にした。接着受容体および一次抗体に対するリガンド／受容体特異性変換体の親和性を確定するための上記方法は、任意の特異性ドメインおよび任意の抗原性ドメインを含む任意の候補リガンド／受容体特異性変換体に関して実施され得るが、ただし、適切な配列および接着受容体を用いることが必要である。
【００７６】
表４以後の実施例は、リガンド／受容体特異性変換体がＣｌｆＡ受容体を有する細菌と相互作用するか否かを確定するために実施した別の特徴付けアッセイを記載する。
【００７７】
【表４】

【００７８】
実施例３
凝集因子（Ｃｌｆ）と結合する特異性ドメインおよびポリオウイルス由来のエピトープに相当する抗原性ドメインを有するリガンド／受容体特異性変換体を、ｆｍｏｃ　ｃｈｅｍｉｓｔｒｙ（Ｓｙｒｏ， ＭｕｌｔｉＳｙｎＴｅｃｈ， Ｇｅｒｍａｎｙ）を用いたペプチド合成における標準技法を用いて製造した（表５参照）。これらのリガンド／受容体特異性変換体が、ＣＬＦおよびフィブリノーゲン間の相互作用を抑制する能力に関して分析した。これらの実験では、表５に記載したリガンド／受容体特異性変換体を製造し、種々の濃度のこれらの分子について、Ｃｌｆおよびフィブリノーゲンを含む酵素競合イムノアッセイを実施した。Ｃｌｆ／Ｆｉｂ相互作用を抑制するために必要な最低ペプチド濃度である最低抑制濃度を確証した。すなわち、Ｆｉｂ／Ｃｌｆ相互作用を抑制するのに必要な濃度が低いほど、より有効な阻害剤である。さらに、イムノアッセイにおいて抗ポリオウイルス抗体により認識されるペプチドの最低試験濃度である最低固相結合ペプチド濃度を確定した。表５中に星印で示した使用ペプチドのいくつか（例えばＣＰＡＬＴＡＶＥＴＧＣＴＮＰＬＡＡＨＨＬＧＧＡＫＱＡＧ（配列番号１３５）、ＨＨＬＧＧＡＫＱＡＧ−ＡＡ−ＣＰＡＬＴＡＶＥＴＧＣＴＮＰＬ（配列番号１３７）、ＣＰＡＬＴＡＶＥＴＧＣ−ＴＮＰＬＨＨＬＧＧＡＫＱＡＧ（配列番号１３９）およびＨＨＬＧＧＡＫＱＡＧ−ＣＰＡＬＴＡＶＥＴＧＣＴＮＰＬ（配列番号１４１））を、２つの人工的に導入したシステイン残基間で環化した。これらの実験は、ＨＨＬＧＧＡＫＱＡＧ−ＡＡ−ＣＰＡＬＴＡＶＥＴＧＣＴＮＰＬ（配列番号１３７）およびＨＨＬＧＧＡＫＱＡＧ−ＣＰＡＬＴＡＶＥＴＧＣＴＮＰＬ（配列番号１４２）がＣｌｆおよびフィブリノーゲン間の相互作用を効率的に抑制し、機能的ポリオウイルスエピトープを保持することを明示した。
【００７９】
【表５】

【００８０】
次のセクションでは、リガンド／受容体特異性変換体が、病原体の増殖に対する作用を有するか否かを確定するために実施され得るいくつかの細胞ベース特徴付けアッセイを記載する。
【００８１】
病原体ベースの特徴付けアッセイ
別の種類の特徴付けアッセイにおいて、病原体ベースのアプローチを用いて、病原体およびリガンド／受容体特異性変換体の抗原性ドメインに対して指向されている抗体と相互作用する、リガンド／受容体特異性変換体の能力を評価する。この分析も、被験体の身体中において、リガンド／受容体特異性変換体と、病原体およびリガンド／受容体特異性変換体の抗原性ドメインに指向されている抗体との間の相互作用をした後に、被験体から病原体を取り除く（ｐｕｒｇｅ）体液性応答および細胞性応答（例えば補体固定およびマクロファージ分解）が続くため、病原体の増殖に対するリガンド／受容体特異性変換体の能力を明示する。一般的にいうと、病原体ベースの特徴付けアッセイは、培養病原体にリガンド／受容体特異性変換体を提供し、リガンド／受容体特異性変換体と、細胞またはウイルスとの会合をモニタリングすることを含む。数種類の病原体ベースの特徴付けアッセイが用いられ得る。以下の実施例は、好ましい特徴付けアッセイのいくつかをより詳細に記載する。
【００８２】
実施例４
病原体ベース特徴付けアッセイの一型は、支持体上に配置された細菌とリガンド／受容体特異性変換体を結合することを含む。したがって、Ｃｌｆａを産生する細菌（例えば黄色ブドウ球菌または大腸菌）を、培養中で、または適切な成長培地（例えばＬＢブロス、血液ブロス、ＬＢ寒天または血液寒天）中の寒天プレート上で成長させる。次に、真空下で膜上に培養を載せる（例えばドットブロット多岐管装置を用いる）ことにより、または移動を可能にするのに十分な時間、コロニー上に膜を載せることにより、細胞を膜（例えばニトロセルロースまたはナイロン）に移す。膜に結合された細胞は次に、リガンド／受容体特異性変換体の連続希釈（例えば、２００：１　ＰＢＳの総容積中に５００ｎｇ、１：ｇ、５：ｇ、１０：ｇ、２５：ｇおよび５０：ｇのリガンド／受容体特異性変換体）を提供される。一実験では、表４または表５に列挙されたリガンド／受容体特異性変換体を用いる。次に膜上で６０分間、希釈リガンド／受容体特異性変換体をインキュベートする。その後、非結合リガンド／受容体特異性変換体を除去し、ＰＢＳで膜を洗浄する（例えば１回の洗浄あたり２ｍｌのＰＢＳ／洗浄で３回洗浄）。次に、リガンド／受容体特異性変換体の抗原性ドメインと相互作用する一次抗体（例えば単純ヘルペスウイルスｇＧ２タンパク質に対するｍＡｂ）の１：１００〜１：１０００希釈液を提供し、結合反応を６０分間起こさせる。再び、ＰＢＳ（例えば１回の洗浄あたり２ｍｌのＰＢＳで３回洗浄）で膜を洗浄して、非結合一次抗体を除去する。適切な対照としては、膜自体、リガンド／受容体特異性変換体を伴わない膜上の細菌、ならびにリガンド／受容体特異性変換体を有するが一次抗体を有さない膜上の細菌が挙げられる。
【００８３】
膜上の細菌と結合したリガンド／受容体特異性変換体の量を検出するために、二次抗体（例えばウサギ抗マウスＩｇＧ（Ｓｉｇｍａ））および三次抗体（例えばペルオキシダーゼ標識ヤギ抗ウサギＩｇＧ（Ｓｉｇｍａ））を用いる。もちろん、一次抗体と相互作用する標識二次抗体も、同様に用いられ得る。上記のように、二次抗体を膜と６０分間接触させた後、ＰＢＳを用いて膜から非結合二次抗体を洗浄する（例えば１回につき２ｍｌのＰＢＳを用いて３回洗浄）。さらに、三次抗体を膜と６０分間接触させた後、ＰＢＳを用いて非結合三次抗体を膜から洗浄する（例えば１回につきｍｌのＰＢＳを用いて３回洗浄）。膜をジニトロ−フェニレン−ジアミン（Ｓｉｇｍａ）とともにインキュベートすることにより、結合三次抗体を検出し得る。
【００８４】
別のアプローチは、固定化したリガンド／受容体特異性変換体の使用を含む。したがって一次抗体（例えば単純ヘルペスウイルスｇＧ２タンパク質に対するｍＡｂ）が、ペトリ皿に結合される。いったん一次抗体が結合されれば、リガンド／受容体特異性変換体（例えば表４または表５に提示されたリガンド／受容体特異性変換体）の種々の希釈液を被覆皿に付加する。リガンド／受容体特異性変換体を一次抗体と６０分間会合させて、さらに非結合リガンド／受容体特異性変換体を洗い落とす（例えば１洗浄あたり２ｍｌのＰＢＳで３回洗浄）。適切な対照としては、一次抗体またはリガンド／受容体特異性変換体を含まないペトリ皿が挙げられる。
【００８５】
その後、細菌（例えば大腸菌）の混濁溶液をペトリ皿に加え、細菌を固定化リガンド／受容体特異性変換体と６０分間相互作用させる。次にＰＢＳで洗浄することにより（例えば１洗浄あたり２ｍｌのＰＢＳで３回洗浄）、非結合細菌を除去する。次に成長培地（例えばＬＢブロス）をペトリ皿に加え、培養を一夜インキュベートする。あるいはＬＢ寒天をペトリ皿に加え、培養を一夜インキュベートする。リガンド／受容体特異性変換体と細菌との間の相互作用は、視覚的に観察し得る（例えば混濁成長培地であれば、分光測光分析または寒天上のコロニーの出現を用いて定量し得る）。
【００８６】
上記アプローチを一部変更することにより、ウイルスと相互作用するリガンド／受容体特異性変換体の能力を当業者は評価し得る。例えばＴ４糖タンパク質の可溶性断片は、ヒト免疫不全ウイルス（ＨＩＶ）エンベロープ糖タンパク質と相互作用することが示されている（例えば米国特許第６，０９３，５３９号参照）。抗原性ドメイン（例えば表２に列挙した抗原性ドメイン）と連結される特異性ドメインを有する融合タンパク質を合成することにより、ＨＩＶエンベロープ糖タンパク質と相互作用するＴ４糖タンパク質の断片を含む特異性ドメイン（例えば米国特許第６，０３９，５３９号で提供されたＴ４糖タンパク質配列のアミノ酸１〜４１９またはその一部分）を有するリガンド／受容体特異性変換体を製造し得る。ペプチド化学を用いてリガンド／受容体特異性変換体を製造し得るが、好ましくは抗原性ドメインに連結されたＴ４糖タンパク質の断片を有する発現構築物が製造されて、適切な細胞中にトランスフェクトされる。米国特許第６，０３９，５３９号および上記に記載された発現および精製戦略も用いられ得る。
【００８７】
いったんリガンド／受容体特異性変換体が構築されたら、フィルター結合アッセイを実施する。すなわち、ＨＩＶ接種物の連続１０倍希釈液を、一定真空下でドットブロット装置中の膜（例えばニトロセルロースまたはナイロン）に適用する。次にリガンド／受容体特異性変換体の連続１０倍希釈液を結合ＨＩＶ粒子に適用する。リガンド／受容体特異性変換体を粒子と６０分間接触させた後、真空を適用して、ＰＢＳで洗浄する（例えば１回につき２ｍｌのＰＢＳで３回洗浄）。いったん非結合リガンド／受容体特異性変換体が除去されれば、抗原性ドメインと結合する一次抗体の１０倍連続希釈液を試料に加え、結合反応を６０分間起こさせる。次に真空を適用し、非結合一次抗体をＰＢＳで洗浄する（例えば１洗浄あたり２ｍｌのＰＢＳで３回洗浄）。上記と同様に、結合一次抗体の検出を行うことができる。
【００８８】
サンドイッチ型アッセイにおいても、ウイルスと相互作用するリガンド／受容体特異性変換体の能力を評価し得る。すなわち、リガンド／受容体特異性変換体の抗原性ドメインと相互作用する一次抗体を微小滴定ウエル中で固定し、リガンド／受容体特異性変換体の連続希釈液を一次抗体に付加し、上記のように一次抗体／特異性変換体の複合体を作製する。次にＨＩＶ接種物の１０倍連続希釈液を加え、結合反応を６０分間起こさせる。ＰＢＳ中での連続洗浄により、非結合ＨＩＶ粒子を除去する。放射能標識抗ＨＩＶ抗体（例えばＨＩＶ感染に罹患しているヒトからの血清から得た抗体）を用いて、結合ＨＩＶ粒子の検出を成し遂げ得る。
【００８９】
上記実施例において、細菌およびウイルスを用いた病原体ベースのアッセイを記載したが、これらのアプローチの一部を変更して、リガンド／受容体特異性変換体と哺乳類細胞の相互作用を試験し得る。例えば、癌細胞上に存在するインテグリン受容体と相互作用するリガンド／受容体特異性変換体の能力を、以下のように確定し得る。∀_Ｖ∃_３受容体を発現する黒色腫細胞（例えばＭ２１ヒト黒色腫細胞）はフィブリノーゲンと結合し、この相互作用は、ＲＧＤを含むペプチドを投与することにより遮断し得る（例えば、Ｋａｔａｄａ ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７２：７７２０（１９９７）およびＦｅｌｄｉｎｇ−Ｈａｂｅｒｍａｎｎ ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７１：５８９２−５９００（１９９６）参照）。同様に、多くのその他の種類の癌細胞は、ＲＧＤペプチドと相互作用するインテグリンを発現する。一アプローチによれば、ＲＧＤ応答性インテグリンを発現する癌細胞（例えばＭ２１ヒト黒色腫細胞）を培養して集密にする。Ｍ２１細胞は、１０％ウシ胎児血清、２０ｍＭのＨｅｐｅｓおよび１ｍＭのピルビン酸塩を含有するＤＭＥＭ培地中で成長し得る。
【００９０】
好ましくは、２０μｇ／ｍｌの最終濃度（２ｘ１０^６細胞／ｍｌ）で３７℃において３０分間、ヒドロエチジン（Ｐｏｌｙｓｃｉｅｎｃｅ， Ｉｎｃ．， Ｗａｒｒｉｎｇｔｏｎ， ＰＡ）で細胞を染色し、次に２回洗浄して、余分の染料を除去する。ヒドロエチジンはＤＮＡ中に入り込んで（ｉｎｔｅｒｃａｌａｔｅ）、細胞の赤色蛍光標識を生じるが、細胞の接着機能を損傷することはない。染色は、細胞とのリガンド／受容体特異性変換体の結合を定量化する一方法を提供する。すなわち、ヒドロエチジン染色細胞の総数を、蛍光標識一次抗体／特異性変換体の複合体に結合した細胞の数と比較することにより、結合効率を確定することができる。
【００９１】
したがって染色細胞を、ＲＧＤ配列（例えばＧＲＧＤＳＰＨＲＧＧＰＥＥ（配列番号１０４）またはＷＳＲＧＤＷＨＲＧＧＰＥＥ（配列番号１０５））を含むリガンド／受容体特異性変換体の種々の希釈液とともにインキュベートする。６０分間のインキュベーション後、１０％ウシ胎児血清、２０ｍＭのＨｅｐｅｓおよび１ｍＭのピルビン酸塩を含有するＤＭＥＭ培地中で数回洗浄する（例えば５ｍｌの培地の３回洗浄）ことにより、非結合リガンド／受容体特異性変換体を除去する。次に、リガンド／受容体特異性変換体の抗原性ドメインと相互作用する一次抗体（例えば単純ヘルペスウイルスｇＧ２タンパク質に対するｍＡｂ）の１：１００〜１：１０００希釈液を提供し、結合反応を６０分間起こさせる。その後、培地中で数回の洗浄を実施して、あらゆる非結合一次抗体を除去する。適切な対照としては、リガンド／受容体特異性変換体を含有しない染色細胞、または一次抗体を含有しない染色細胞が挙げられる。
【００９２】
一次抗体の結合後、ヤギ抗マウスＦＩＴＣ標識抗体（１：１００希釈液）（Ｓｉｇｍａ）を付加し、結合を６０分間起こさせる。再び、数回の培地洗浄を実施して、すべての非結合二次抗体を除去する。ヒドロエチジンに関しては５４３／５９０ｎｍに、およびフルオレセインに関しては４９５／５２５ｎｍに設定したフィルターを用いてフローサイトメトリーにより分析を行なう。リガンド／受容体特異性変換体／細胞の複合体との一次抗体の結合が有意に観察されるが、これは、リガンド／受容体特異性変換体が細胞に相当の影響を及ぼすことを実証する。
【００９３】
以下の実施例は、ＲＧＤ含有リガンド／受容体特異性変換体が哺乳類細胞と効果的に結合し、これらの細胞に抗体を向け直すことを証明する特徴付けアッセイを記載する。
【００９４】
実施例５
ｆｍｏｃ　ｃｈｅｍｉｓｔｒｙ（Ｓｙｒｏ， ＭｕｌｔｉＳｙｎＴｅｃｈ， Ｇｅｒｍａｎｙ）を用いたペプチド合成における標準技法を用いて、ペプチドＲＧＤＳＡＡＴＰＰＡＹＲ（配列番号１４５）を製造した。このペプチドは、インテグリン受容体、スペーサー（ＡＡ）、ならびにＢ型肝炎ウイルスｅ抗原（ＨＢｅＡｇ）上に存在するエピトープであるモノクローナル抗体５７／８により認識されるエピトープを有する抗原性ドメインと結合する特異性ドメインを有する。
【００９５】
ネズミ黒色腫細胞（ＳＰ２／０細胞）を血清無含有培地中で洗浄し、次に５０μｇ／ｍｌの濃度で、ＲＧＤＳＡＡＴＰＰＡＹＲ（配列番号１４５）ペプチドまたはＣ型肝炎ウイルス（ＨＣＶ）ＮＳ３ドメイン由来の対照ペプチドとともにインキュベートした。次に細胞を洗浄し、５７／８抗体で細胞を標識することにより、表面結合ペプチドの量を検出した。１／５００に希釈したＦＩＴＣ標識抗マウスＩｇＧ接合体により表面結合抗体を指標し、蛍光顕微鏡により表面染色のレベルを確定した。
【００９６】
この実験は、対照ペプチドとともにインキュベートした細胞は染色を示さなかったことを明示した。対照的に、ＲＧＤＳＡＡＴＰＰＡＹＲ（配列番号１４５）ペプチドとともにインキュベートした細胞は、表面発現インテグリンの位置と一致して、有意の表面染色を示した。したがって、ＲＧＤ含有リガンド／受容体特異性変換体は、インテグリン産生哺乳類細胞と効果的に結合し、これらの分子を用いて抗体を腫瘍細胞に向けさせ、その抗体を標的化し得る。
【００９７】
次のセクションでは、動物において実施される特徴付けアッセイを記載する。
【００９８】
ｉｎ　ｖｉｖｏ特徴付けアッセイ
特徴付けアッセイは、リガンド／受容体特異性変換体をｉｎ　ｖｉｖｏで評価する実験も含む。リガンド／受容体特異性変換体が、病原性感染を抑制する能力を評価するのに適した多数の動物モデルが存在する。マウスは、維持するのが容易であり、細菌感染、ウイルス感染および癌に罹り易いために好ましい。チンパンジーも、それらがヒトと遺伝的に密接に関連しているために好ましい。
【００９９】
マウスにおけるリガンド／受容体特異性変換体の効力を評価するための一アプローチを次の実施例で提供する。
【０１００】
実施例６
細菌感染を治療するリガンド／受容体特異性変換体の能力を試験するために、以下の特徴付けアッセイを実施し得る。約８週齢、体重２５ｇの数匹の雌ＣＦ−１異系交配マウス（Ｃｈａｒｌｅｓ Ｒｉｖｅｒｓ Ｌａｂｏｒａｔｏｒｉｅｓ）に試験すべきリガンド／受容体特異性変換体の抗原性ドメインを予防接種する。好ましくは、抗原性ドメインを担体と結合させ、アジュバントとともに投与する。例えば抗原性ドメインを、担体およびアジュバントの両方として作用するカギアナカサガイヘモシアニンまたはウシ血清アルブミンと融合させるか、あるいは、フロイントのアジュバントのようなアジュバント、水酸化アルミニウム、またはリソレシチンを用いることができる。いったん抗原性ドメインに対する高力価の抗体を、例えば免疫拡散またはＥＩＡにより立証することができれば、黄色ブドウ球菌ＮＴＣＣ１０６４９の一夜培養を免疫感作マウスに腹腔内接種する。黄色ブドウ球菌に関して約１００ｘＬＤ_５０またはｌｏｇ６．６を生産するよう、接種物を調整する。
【０１０１】
リガンド／受容体特異性変換体（例えば表４中に提示されたリガンド／受容体特異性変換体）の連続希釈液を注射用として滅菌水中に処方し、感染の１時間および５時間後に皮下（ＳＣ）または経口（ＰＯ）経路で投与する。同時に各試験に関して、非処置マウスに細菌接種物のｌｏｇ希釈液を接種することにより、接種物の（ｃｈａｌｌｅｎｇｅ）ＬＤ_５０を確認する。好ましくは５種類のｌｏｇ希釈範囲の細菌接種物を各１０匹の５群に接種する（１種類のｌｏｇ希釈液につきマウス１０匹）。ＬＤ_５０の１００倍の濃度の接種物を接種した非処置マウスの全群において１００％の死亡率を生じる。死亡率に関して、７日間毎日、マウスをモニタリングする。Ａｎｔｉｍｉｃｒｏｂ． Ａｇｅｎｔｓ Ｃｈｅｍｏｔｈｅｒ． ３１：１７６８−１７７４およびＰｒｏｃ． Ｓｏｃ． Ｅｘｐ． Ｂｉｏｌ． Ｍｅｄ． １９９４， ５７， ２６１−２６４に記載されたような「生存対投与量」のプロット化曲線の対数プロビット分析により、累積死亡率から５０％のマウスを防護するための平均有効用量（ＥＤ_５０）を算定し得る。当業者が理解するように、同様のアプローチを用いてウイルス感染および癌を抑制するリガンド／受容体特異性変換体の能力を試験し得る。
【０１０２】
本明細書中に記載したリガンド／受容体特異性変換体を製剤中に処方して、病原体の増殖を抑制する作用物質を必要とする被験体に投与し得る。以下のセクションでは、病原体上の受容体と相互作用するリガンド／受容体特異性変換体を含むいくつかの製剤を記載する。
【０１０３】
病原体上の受容体と相互作用するリガンド／受容体特異性変換体を含む製剤
本明細書中に記載されたリガンド／受容体特異性変換体は、病原体による感染を治療または予防する化合物を必要とする被験体に投与するための製剤中へ混入するのに適している。これらの薬理学的活性化合物をガレノス薬学の慣用的方法にしたがって加工して、ヒトを含めた動物への投与のための医薬製剤を生産し得る。有効成分を、修飾を加えてもしくは加えずに、製剤製品中に組込み得る。さらにいくつかの経路により本発明の薬理学的活性化合物を送達する製剤または治療薬の製造は、本発明の態様である。例えば病原体上の受容体と相互作用するリガンド／受容体特異性変換体をコードする配列を有するＤＮＡ、ＲＮＡおよびウイルスベクター（これらに限定されない）を、本発明の実施形態とともに用いる。リガンド／受容体特異性変換体をコードする核酸を単独でまたは他の有効成分と組合せて投与し得る。
【０１０４】
化合物は、本明細書中に記載した薬理学的有効成分と有害な反応をしない慣用的賦形剤、すなわち非経口、経腸（例えば経口）または局所適用に適した製薬上許容可能な有機または無機担体物質との混和物中に用い得る。適切な製薬上許容可能な担体としては、水、食塩溶液、アルコール、アラビアゴム、植物油、ベンジルアルコール、ポリエチレングリコール、ゼラチン、炭水化物（例えばラクトース）、アミロースまたはデンプン、ステアリン酸マグネシウム、タルク、ケイ酸、粘性パラフィン、香油、脂肪酸モノグリセリドおよびジグリセリド、ペンタエリスリトール脂肪酸エステル、ヒドロキシメチルセルロース、ポリビニルピロリドン等が挙げられるが、これらに限定されない。用い得るさらに多数のビヒクルは、Ｒｅｍｍｉｎｇｔｏｎ’ｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅｓ， １５^ｔｈ Ｅｄｉｔｉｏｎ， Ｅａｓｔｏｎ： Ｍａｃｋ Ｐｕｂｌｉｓｈｉｎｇ Ｃｏｍｐａｎｙ， ｐａｇｅｓ １４０５−１４１２ ａｎｄ １４６１−１４８７（１９７５）およびＴｈｅ Ｎａｔｉｏｎａｌ Ｆｏｒｍｕｌａｒｙ ＸＩＶ， １４^ｔｈ Ｅｄｉｔｉｏｎ， Ｗａｓｈｉｎｇｔｏｎ， Ａｍｅｒｉｃａｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ａｓｓｏｃｉａｔｉｏｎ（１９７５）に記載されている。製剤調製物を滅菌し、所望により、リガンド／受容体特異性変換体と有害な反応をしない限り、助剤、例えば滑剤、防腐剤、安定剤、湿潤剤、乳化剤、浸透圧に作用するための塩、緩衝剤、着色剤、風味剤および／または芳香性物質等と混合し得る。
【０１０５】
リガンド／受容体特異性変換体を有する特定の製剤の有効用量および投与方法は、患者の個々の必要性および要求される治療手段または予防手段に基づいて変わり得る。このような化合物の治療効力および毒性、例えばＥＤ_５０（集団の５０％に治療的に有効な用量）は、細胞培養または実験動物における標準製薬手法により確定し得る。例えばリガンド／受容体特異性変換体の有効用量は、上記特徴付けアッセイを用いて求め得る。これらのアッセイから得られたデータは、次に、ヒトを含めた他の生物体に関して用いるための一連の投与量の処方に用いる。このような化合物の投与量は、好ましくは、毒性を伴わないＥＤ_５０を含めた一連の循環濃度内である。投与量は、リガンド／受容体特異性変換体の種類、用いられる投薬形態、生物体の感受性、ならびに投与経路によってこの範囲内で変化する。
【０１０６】
リガンド／受容体特異性変換体の通常の投与量は、投与経路によって、約１〜１００，０００μｇ、約１０ｇの総用量まで変わり得る。望ましい投与量としては、約２５０：ｇ〜１ｍｇ、約５０ｍｇ〜２００ｍｇ、および約２５０ｍｇ〜５００ｍｇが挙げられる。
【０１０７】
いくつかの実施形態では、リガンド／受容体特異性変換体の用量は、好ましくは約０．１：Ｍ〜５００ｍＭの組織濃度もしくは血液濃度またはその両方を生じる。望ましい用量は、約１〜８００μＭの組織濃度もしくは血液濃度またはその両方を生じる。好ましい用量は、約１０μＭ〜約５００：Ｍより高い組織濃度もしくは血液濃度を生じる。８００：Ｍより高い組織濃度を生じる用量は好ましくはないが、それらを用いる可能性はある。血中レベルで測定した場合に組織中の安定濃度を維持するために、リガンド／受容体特異性変換体の定常注入も提供され得る。
【０１０８】
治療される患者を考慮して、個々の医者が正確な投与量を選択する。十分なレベルの活性部分を提供しあるいは所望の作用を保持するよう、投与量および投与法を調整する。考慮され得る付加的因子としては、疾患の重症度、治療される生物体の年齢、および生物体の体重またはサイズ；食餌、投与の時間および頻度、薬剤組合せ（単数または複数）、反応感受性、ならびに治療に対する耐性／応答が挙げられる。短期作用製剤組成物は毎日またはより高い頻度で投与し、一方、長期作用製剤組成物は、２日またはそれ以上の日数毎に、週１回、あるいは２週間に１回またはそれ以下の頻度で投与する。
【０１０９】
製剤の投与経路としては、局所、経皮、非経口、胃腸、経気管支および経肺胞投与が挙げられるが、これらに限定されない。経皮投与は、リガンド／受容体特異性変換体を皮膚に浸透させ得るクリーム、リンス、ゲル等の塗布により成し遂げられる。非経口経路の投与としては、電気的または直接注入、例えば中心静脈系への直接注入、静脈内、筋内、腹腔内、皮内または皮下注入が挙げられるが、これらに限定されない。胃腸経路の投与としては、経口摂取および直腸が挙げられるが、これらに限定されない。経気管支および経肺胞経路の投与としては、口または鼻腔内を介した吸入が挙げられるが、これらに限定されない。
【０１１０】
経皮または局所投与に適した本明細書中に記載されたリガンド／受容体特異性変換体を有する組成物としては、皮膚に直接塗布されるかまたは経皮用具（「経皮パッチ」）のような保護担体中に混入される製薬上許容可能な懸濁液、油、クリームおよび軟膏が挙げられるが、これらに限定されない。適切なクリーム、軟膏などの例は、例えばＰＤＲ（Ｐｈｙｓｉｃｉａｎ’ｓ Ｄｅｓｋ Ｒｅｆｅｒｅｎｃｅ）中に見出し得る。適切な経皮用具の例は、例えば米国特許第４，８１８，５４０号（Ｃｈｉｎｅｎ等、１９８９年４月４日発行）に記載されている。
【０１１１】
非経口投与に適した薬理学的活性化合物を有する組成物としては、製薬上許容可能な滅菌等張溶液が挙げられるが、これらに限定されない。このような溶液としては、中心静脈系への注入、静脈内、筋内、腹腔内、皮内または皮下注射のための食塩水およびリン酸緩衝化食塩水が挙げられるが、これらに限定されない。
【０１１２】
経気管支および経肺胞投与に適した薬理学的活性化合物を有する組成物としては、種々の種類の吸入用エーロゾルが挙げられるがこれらに限定されない。これらの経気管支および経肺胞投与に適した用具も、実施形態である。このような用具としては、アトマイザーおよび噴霧器が挙げられるが、これらに限定されない。本明細書中に記載したリガンド／受容体特異性変換体を有する組成物を送達するために、多数の形態の現在利用可能なアトマイザーおよび噴霧器を容易に適応し得る。
【０１１３】
胃腸投与に適した薬理学的活性化合物を有する組成物としては、経口摂取のための製薬上許容可能な粉末、ピルまたは液体、ならびに直腸投与のための座薬が挙げられるが、これらに限定されない。易使用性のために、胃腸投与、特に経口投与は好ましい一実施形態である。いったんリガンド／受容体特異性変換体を含む製剤が得られたら、それは病原体感染を治療または予防する必要がある生物体に投与し得る。
【０１１４】
本発明の態様は、医療設備、例えば人工器官、移植片および器具をコーティングすることも含む。医療用具に用いるのに適したコーティングは、リガンド／受容体特異性変換体を含有するゲルまたは粉末により、あるいはリガンド／受容体特異性変換体が懸濁される高分子コーティングにより提供し得る。用具のコーティングのための適切な高分子物質は、生理学的に許容可能であり、治療上有効量のリガンド／受容体特異性変換体が拡散し得る高分子物質である。適切なポリマーとしては、ポリウレタン、ポリメタクリレート、ポリアミド、ポリエステル、ポリエチレン、ポリプロピレン、ポリスチレン、ポリテトラフルオロエチレン、ポリ塩化ビニル、酢酸セルロース、シリコーンエラストマー、コラーゲン、シルク等が挙げられるが、これらに限定されない。このようなコーティングは、例えば米国特許第４，６１２，３３７号に記載されている。
【０１１５】
以下のセクションでは、本明細書中に記載したリガンド／受容体特異性変換体を用いた疾患の治療方法および予防方法を記載する。
【０１１６】
リガンド／受容体特異性変換体を用いた疾患の治療および予防
受容体を有する病原体による感染を治療および／または予防する必要がある被験体に、リガンド／受容体特異性変換体を含む製剤を投与し得る。そのような必要がある被験体としては、病原体と接触する危険がある個体、あるいは病原体にすでに感染している個体が挙げられる。標準臨床技法または標準診断技法により、これらの個体を同定し得る。
【０１１７】
例えば一アプローチによれば、細菌感染に罹患している被験体は、病原体の増殖を抑制する作用物質を必要とする被験体として同定される。その場合、治療上有効量のリガンド／受容体特異性変換体をこの被験体に提供する。この方法に用いるリガンド／受容体特異性変換体は、細菌上に存在する受容体（例えば細胞外フィブリノーゲン結合タンパク質（Ｅｆｂ）、コラーゲン結合タンパク質、ビトロネクチン結合タンパク質、ラミニン結合タンパク質、プラスミノーゲン結合タンパク質、トロンボスポンジン結合タンパク質、凝集因子Ａ（ＣｌｆＡ）、凝集因子Ｂ（ＣｌｆＢ）、フィブロネクチン結合タンパク質、コアグラーゼおよび細胞外接着タンパク質）と相互作用する特異性ドメインを含む。リガンド／受容体特異性変換体は、病原体または毒素のエピトープ、好ましくは必要がある被験体中に存在する高力価抗体により認識されるエピトープを有する抗原性ドメインも含む。被験体にリガンド／受容体特異性変換体を提供する前に、抗原性ドメインを認識する高力価抗体の存在に関して、必要がある被験体をスクリーニングするのも望ましい。このスクリーニングは、上記のように固定化した抗原性ドメインまたはリガンド／受容体特異性変換体を用いたＥＩＡまたはＥＬＩＳＡにより成し遂げ得る。
【０１１８】
同様に、特定の病因性作用物質上に存在する受容体を認識するリガンド／受容体特異性変換体を、ウイルス感染を抑制する作用物質を必要とする被験体に投与し得る。したがって、標準臨床手法および標準診断手法により、ウイルス感染を抑制する作用物質を必要とする被験体を同定する。次に、個体に感染する種類のウイルス上に存在する受容体と相互作用する治療上有効量のリガンド／受容体特異性変換体を、必要とする被験体に提供する。上記のように、リガンド／受容体特異性変換体の投与前に、リガンド／受容体特異性変換体の抗原性ドメインと相互作用するのに十分な力価の抗体を被験体が有するか否かを確定するのが望ましい。
【０１１９】
同一静脈中において、癌細胞上に存在する受容体と相互作用するリガンド／受容体特異性変換体を、癌の増殖を抑制する作用物質を必要とする被験体に投与し得る。例えば、標準臨床手法または標準診断手法により癌の増殖を抑制する作用物質を必要とする被験体を同定する。次に、被験体に感染した癌細胞上に存在する受容体と相互作用する治療上有効量のリガンド／受容体特異性変換体を、必要とする被験体に提供する。上記のように、リガンド／受容体特異性変換体の投与前に、リガンド／受容体特異性変換体の抗原性ドメインと相互作用するのに十分な力価の抗体を被験体が有するか否かを確定するのが望ましい。
【０１２０】
本明細書中に記載したリガンド／受容体特異性変換体は、疾患の発症を防止するための予防薬としても被験体に投与し得る。予防目的で（例えば細菌感染、ウイルス感染または癌を予防するために）、事実上誰にでも、本明細書中に記載したリガンド／受容体特異性変換体を投与し得る。しかしながら、特定の疾患に罹患する危険性が高い被験体を同定し、リガンド／受容体特異性変換体を提供するのが望ましい。疾患に罹患する危険性の高い被験体としては、疾患の家族暦を有する個体、高齢者または若年者、あるいは病原体と頻繁に接触する個体（例えば健康管理従事者）が挙げられる。したがって受容体を有する病原体により感染する危険性のある被験体を同定し、次に予防上有効量のリガンド／受容体特異性変換体を提供する。
【０１２１】
本明細書中に記載したリガンド／受容体特異性変換体に関する予防的一用途は、医療用具または移植片をリガンド／受容体特異性変換体で被覆または架橋することに関する。埋め込み型医療用具は、多数の細菌種の感染の病巣となることがある。このような用具関連感染は、これらの生物体が用具の表面に接着し、定着する傾向があることにより促進される。その結果、典型的には医療用具の移植に伴う生物学的有害反応を促進する傾向が低い表面を有する医療用具を開発する重大な必要性が存在する。
【０１２２】
一アプローチによれば、リガンド／受容体特異性変換体を含有する溶液中で医療用具を被覆する。移植前に、例えばリガンド／受容体特異性変換体の溶液中に医療用具（例えば人工弁）を保存する。医療用具は、リガンド／受容体特異性変換体を有する粉末またはゲル中でも被覆し得る。細菌またはウイルス受容体と相互作用する特異性変換体を含有する粉末またはゲル中で、例えば手袋、コンドームおよび子宮内用具を被覆し得る。いったん身体中に移植されれば、これらのリガンド／受容体特異性変換体は病原体による感染に対する予防的バリアとなる。
【０１２３】
いくつかの実施形態では、リガンド／受容体特異性変換体を医療用具に固定する。上記のように、医療用具は、リガンド／受容体特異性変換体が接着し得る支持体である。固定化は、リガンド／受容体特異性変換体と医療用具との間の疎水性相互作用により起こり得るが、医療用具にリガンド／受容体特異性変換体を固定するための好ましい方法は、共有結合を含む。例えば、特異性変換体上に存在する反応基と相互作用する反応基を有する医療用具を製造し得る。
【０１２４】
一アプローチによれば、２−アミノアルコール部分を含むリガンド／受容体特異性変換体と過ヨウ素酸塩とを用いて、約４〜約９のｐＨおよび約０〜約５０℃の温度を有する水性溶液中にアルデヒド−機能交換体を生成する。次に、一級アミン部分を含む医療用具の生体物質表面と、アルデヒド−機能交換体とを組合せて、イミン部分を介して支持体表面にリガンド／受容体特異性変換体を固定する。次に、イミン部分を還元剤と反応させて、二級アミン結合を介して生体物質表面に固定化リガンド／受容体特異性変換体を形成する。医療用具に分子を架橋するためのその他のアプローチ（例えば米国特許第６０１７７４１号に記載）を一部変更して、本明細書中に記載したリガンド／受容体特異性変換体を固定することができる。
【０１２５】
実施形態および実施例を参照しながら本発明を説明してきたが、本発明の精神を逸脱しない限り、種々の修正がなされ得る、と理解されるべきである。したがって本発明は、特許請求の範囲によってのみ限定される。[0001]
[Field of the Invention]
The present invention generally relates to compositions and methods for preventing and treating pathogens such as bacteria, yeast, parasites, fungi, viruses, and human diseases, including but not limited to cancer. In particular, the embodiments described herein relate to the manufacture and use of ligand / receptor specific converters that redirect existing antibodies in a subject to receptors present on a pathogen.
[0002]
[Background of the Invention]
Infection by pathogens, such as bacteria, yeasts, parasites, fungi and viruses, and the development and metastasis of cancer poses serious health problems for all animals, including humans, livestock and pets. These health threats are exacerbated by the emergence of strains that are resistant to vaccines and / or treatments. In the past, pharmacologists have relied on traditional methods of drug discovery to obtain safe and efficacious compounds for the treatment of these diseases. Traditional drug discovery methods often involve randomly selecting and blinding potential drug candidate molecules, hoping to be able to prove that they are effective treatments for some diseases. Including. However, with the advancement of molecular biology, the focus of drug development has shifted to the identification of target molecules associated with pathogenic agents and the design of compounds that interact with these target molecules.
[0003]
One promising class of target molecules is the ability to adhere to receptors found on the surface of bacteria, yeast, parasites, fungi, viruses and cancer cells, especially host cells or host proteins (eg extracellular matrix proteins) Is a receptor that Research in this area has focused primarily on the identification of receptors and their ligands, as well as the discovery of molecules that prevent ligand-receptor interactions and thereby block adhesion to host cells or proteins.
[0004]
For example, many pathogenic bacteria (eg, Staphylococcus aureus) are adhesion receptors (eg, ClfA, Efb, and FnBPA) that can bind to host extracellular matrix proteins (eg, fibrinogen, fibronectin, and laminin). (Flock, Mol. Med. Today 5: 532-533 (1999)). Researchers have shown that by providing peptides that correspond to regions of the host extracellular matrix protein, some bacteria can be blocked from attaching to the host extracellular matrix protein (Pei et al., Infection). and Immunity 67 (9): 4525-4530 (1999)). Similarly, many viruses have receptors that interact with proteins present on the surface of the host cell (see, eg, US Pat. Nos. 5,942,606 and 5,929,220). Researchers have shown that fragments of the T4 glycoprotein (host cell protein) can interact with gp120 of the human immunodeficiency virus (HIV) and that the T4 peptide can be used to prevent or treat HIV infection. (See, for example, US Pat. No. 6,093,539). In addition, researchers have shown that many types of cancer cells express receptors that interact with host extracellular matrix proteins, and that molecules that block integrin receptors are used for tissue adhesion, metastasis, angiogenesis, and tumor growth. (E.g., U.S. Patent Nos. 6,066,648, 6,087,330, 5,846,536, 5,766,591 and 5). , 627, 263). Although these inhibitory peptides have promising therapeutic potential, there remains a need for new compositions and methods for treating and preventing infection by pathogens and other diseases.
[0005]
[Brief summary of the invention]
The invention described herein relates to the manufacture, characterization and use of novel agents that bind to receptors on a pathogen and redirect existing antibodies in a subject against the pathogen. An embodiment of the present invention provides for at least one specificity domain comprising a ligand for a receptor and at least one antigenic domain linked to said specificity domain, wherein said antigenic domain comprises a pathogen or toxin epitope. Ligand / receptor specificity converter.
[0006]
Some embodiments of the ligand / receptor specificity converter comprise at least three of a peptide selected from the group consisting of an extracellular matrix protein, a ligand for a receptor on a virus, and a ligand for a receptor on a cancer cell. It has a specificity domain that includes contiguous amino acids. In some aspects of this embodiment, for example, the peptide is an extracellular matrix protein selected from the group consisting of fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin. Preferably, the extracellular matrix protein comprises at least three amino acids of the alpha chain of fibrinogen, and in a most preferred embodiment, the ligand comprises the sequence arginine-glycine-aspartic acid (RGD).
[0007]
In another embodiment, the peptide is a ligand for a receptor on a virus selected from the group consisting of T4 glycoprotein and hepatitis B virus envelope protein. In yet another aspect of this embodiment, the peptide is a ligand for a receptor on a cancer cell selected from the group consisting of a ligand for HER-2 / neu and a ligand for an integrin receptor. A preferred embodiment has a specificity domain comprising the sequence provided by one of SEQ ID NOs: 1-42.
[0008]
The ligand / receptor specificity converters described herein interact with receptors found on pathogens. In some embodiments, the receptor is a bacterial adhesion receptor, such as an extracellular fibrinogen binding protein (Efb), a collagen binding protein, a vitronectin binding protein, a laminin binding protein, a plasminogen binding protein, a thrombospondin binding protein, It is a bacterial adhesion receptor selected from the group consisting of aggregation factor A (ClfA), aggregation factor B (ClfB), fibronectin binding protein, coagulase and extracellular adhesion protein.
[0009]
The ligand / receptor specificity converters described herein also interact with antibodies present in the subject. In some embodiments, for example, the antigenic domain comprises at least three amino acids of a peptide selected from the group consisting of a herpes simplex virus protein, a hepatitis B virus protein, a TT virus protein, and a poliovirus protein. In a preferred embodiment, the ligand / receptor specificity variant has an antigenic domain that is a herpes simplex virus protein comprising one sequence selected from the group consisting of SEQ ID NO: 53 and SEQ ID NO: 54. In other desired embodiments, the antigenic domain is a hepatitis B virus protein comprising the sequence of one of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 59.
[0010]
Some ligand / receptor specificity variants also have an antigenic domain that is a TT virus protein that includes one sequence provided by one of SEQ ID NOs: 43-47 and SEQ ID NOs: 55-58. The ligand / receptor specificity variant may also have an antigenic domain that is a poliovirus protein comprising one sequence selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 51. Preferably, the ligand / receptor specificity converter has an antigenic domain that interacts with a high titer antibody. For example, in some embodiments, the antigenic domain specifically binds to antibodies present in animal sera diluted from about 1: 100 to 1: 1000 or more. The specificity converters of SEQ ID NOs: 60 to 105 are embodiments of the present invention.
[0011]
Aspects of the invention also relate to methods for treating or preventing infection or growth of a pathogen. For example, one aspect includes a method of treating and preventing a bacterial infection. The method is performed by providing a subject with a therapeutically effective amount of a ligand / receptor specific converter, wherein the ligand / receptor specific converter interacts with the receptor on the bacterium. It contains a specificity domain with an acting ligand, and an antigenicity domain containing an epitope of a pathogen or toxin. A method of treating or preventing a viral infection is also an embodiment. Accordingly, a method of treating or preventing a viral infection is performed by providing a subject with a therapeutically effective amount of a ligand / receptor specific convert, wherein the ligand / receptor specific convert is It includes a specificity domain having a ligand that interacts with a receptor on bacteria, and an antigenicity domain that includes a pathogen or toxin epitope. Similarly, a method of treating or preventing cancer is one embodiment, which method can be practiced by providing a subject with a therapeutically effective amount of a ligand / receptor specific convertor, wherein: The ligand / receptor specificity converter comprises a specificity domain having a ligand that interacts with a receptor on a cancer cell, and an antigenic domain comprising a pathogen or toxin epitope.
[0012]
[Detailed description of the invention]
The following relates to the manufacture, characterization, and use of novel agents that bind to receptors on the pathogen and redirect antibodies present in the subject to the pathogen. The term "antigen / antibody specificity converter" refers to a molecule comprising an amino acid sequence (eg, a complementarity determining region) corresponding to the amino acid sequence of an antibody linked to the amino acid sequence to which an antibody binds (eg, a pathogen epitope). It is known in the art to refer (see, for example, Sallberg et al., Biochemical & Biophysical Research Communications, 205: 1386-90 (1994) and U.S. Patent Nos. 5,869,232 and 6,040,137). . Antigen / antibody-specific converters can redirect antibodies present in a subject to pathogens, and these converters have therapeutic and diagnostic uses (Id.).
[0013]
The embodiments described herein relate to a second generation of converters, referred to as "ligand / receptor specific converters". Unlike antigen / antibody-specific converters, ligand / receptor-specific converters do not contain sequences found in antibodies. Instead, the ligand / receptor specificity converter comprises a first domain having a ligand for the receptor and a second domain having a pathogen or toxin epitope. Thus, in the present invention, the term “ligand / receptor specificity converter” refers to a receptor linked to an “antigenic domain” having at least one epitope of a pathogen or toxin (eg pertussis toxin or cholera toxin). Refers to a conversion agent comprising a "specificity domain" having at least one ligand for "" (the "ligand" is not an antibody or a portion thereof).
[0014]
A ligand / receptor specificity converter may include multiple specificity domains and multiple antigenic domains. For example, some ligand / receptor specificity converters include multiple specificity domains and / or multiple antigenic domains. Ligand / receptor specificity converters having multispecific and / or antigenic domains are described as being "multiplexed" because the multiple specificity and / or antigenic domains are fused in tandem. Be done. Other embodiments include, in addition to the specificity and antigenic domains, sequences that facilitate purification (eg, polyhistidine tail), linkers (eg, biotin and / or avidin or streptavidin, or the flexible arm of phage 8 (8 -Linkers)), and sequences that promote the stability of the ligand / receptor specific transformant (eg, provide resistance to protease digestion) or that promote the degradation of the ligand / receptor specific transformant (eg, a protease cleavage site) or Ligand / receptor specificity converters containing modifications. The specificity and antigenic domains are preferably peptides, but some ligand / receptor specificity converters are those that comprise modified or derivatized peptides, peptidomimetics or chemicals. Have a domain.
[0015]
Since the embodiments described herein can bind to many different receptors on many different pathogens, the diversity of ligand / receptor specificity converters is enormous. Thus, the term “pathogen” as used herein is in a general sense, ie, a pathogenic agent of a disease in an animal, such as bacteria, parasites, fungi, molds, viruses, and cancer cells (limited to these). Not). Similarly, the term “receptor” is used in a general sense and refers to a molecule (eg, a peptide other than the sequence found in an antibody, or a carbohydrate, lipid, nucleic acid, or combination thereof) that interacts with a “ligand”. A peptide other than the sequence normally found in antibodies, but can be a carbohydrate, lipid or nucleic acid). A “receptor,” as used herein, does not have to undergo signal transduction, but rather has a number of molecular interactions, such as adhesion (eg, integrins) and molecular signaling (eg, growth factor receptors) (these But not limited thereto). For example, the desired specificity domain includes a ligand having a peptide sequence present in an extracellular matrix protein (eg, fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin), and some specificities. The sex domain may be a bacterial adhesion receptor (eg, extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, thrombospondin binding protein, aggregation factor A (ClfA), Ligands that interact with aggregation factor B (ClfB), fibronectin binding protein, coagulase and extracellular adhesion protein).
[0016]
In other embodiments, the specificity domain comprises a ligand having a peptide sequence that interacts with a viral receptor (eg, a fragment of the T4 glycoprotein that binds gp120 or a fragment of the preS domain that binds gp170 of the hepadnavirus family). Including. In yet other embodiments, the specificity domain is a receptor (eg, HER-2 / neu (C-erbB2)) on a cancer cell or an integrin receptor, eg, a vitronectin receptor, a laminin receptor, a fibronectin receptor, a collagen. Receptor, fibrinogen receptor, ∀ ₄ ∃ ₁ Receptor, ∀ ₆ ∃ ₁ Receptor, ∀ ₃ ∃ ₁ Receptor, ∀ ₅ ∃ ₁ Receptor and ∀ _V ∃ ₃ Includes ligands that interact with the receptor. However, preferred embodiments have a specificity domain comprising at least 8 amino acids of the alpha chain of fibrinogen and / or the sequence arginine-glycine-aspartic acid (RGD), and the most preferred embodiments are those of SEQ ID NOs: 60-105. Has a specificity domain containing one sequence selected from the group consisting of:
[0017]
The desired antigenic domain has an epitope recognized by an antibody already present in the subject. For example, many people have been immunized against childhood diseases such as, but not limited to, smallpox, measles, mumps, rubella and polio. Thus, antibodies to epitopes on these pathogens can be produced by the immunized human. Desirable antigenic domains have epitopes found on one of these pathogenic agents.
[0018]
Some embodiments have an antigenic domain that interacts with an antibody administered to a subject. For example, an antibody that interacts with the antigenic domain of the ligand / receptor specificity converter can be co-administered with the ligand / receptor specificity converter. Furthermore, antibodies that interact with the ligand / receptor specificity converter may not normally be present in the subject, but the subject will acquire the antibody by the introduction of a biological substance (eg, serum, blood or tissue) I can do it. For example, a subject receiving a transfusion obtains a number of antibodies, some of which may interact with the antigenic domain of the ligand / receptor specificity converter.
[0019]
Most of the desired antigenic domains contain an epitope recognized by a high titer antibody. By “high titer antibody” is meant an antibody that has a high affinity for an antigen (eg, an epitope on an antigenic domain). For example, in a solid-phase enzyme-linked immunosorbent assay (ELISA), a high titer antibody is assayed in a suitable dilution of serum diluted approximately in the range of 1: 100 to 1: 1000, preferably about 1: 500, Corresponds to antibodies present in serum samples that are still positive. However, preferred antigenic domains include herpes simplex virus gG2 protein, hepatitis B virus s antigen (HBsAg), hepatitis B virus e antigen (HBeAg), hepatitis B virus c antigen (HBcAg), TT virus, and poliovirus. Or having a epitope found in a combination thereof or comprising a sequence selected from the group consisting of SEQ ID NOs: 43-59.
[0020]
The ligand / receptor specificity converters described herein can be made by recombinant engineering and / or conventional techniques in peptide chemistry. In some embodiments, the specificity and antigenic domains are made separately and then linked together (eg, via a linker or by association with a common carrier molecule). In other embodiments, the specificity and antigenic domains are made as part of the same molecule. According to one approach, ligand / receptor specificity converters having a specificity domain linked to an antigenic domain are generated on a peptide synthesizer. According to another approach, a nucleic acid encoding a specificity domain fused to an antigenic domain is cloned into an expression construct, transfected into a cell, and a ligand / receptor specific transformant is isolated from the cell or cell supernatant. Purified or isolated.
[0021]
Once a ligand / receptor specificity converter has been generated, it is screened to determine its ability to interact with an antibody specific for the receptor and / or antigenic domain on the pathogen. Thus, the term "characterization assay" as used herein refers to ligand / receptor specificity that interacts with an antibody specific for a receptor and / or antigenic domain on a pathogen or cancer cell or fragment thereof. Refers to an experiment or evaluation of the ability of a converter. For example, some characterization assays evaluate the ability of a ligand / receptor specificity converter to bind to a support having a receptor on a pathogen or fragment thereof, or vice versa. The ability of the receptor to bind to a support having is evaluated. Other characterization assays assess the ability of a ligand / receptor specificity converter to bind an antibody specific for the antigenic domain of the ligand / receptor specificity converter. Still other characterization assays assess the ability of ligand / receptor specific converters to be transmitted by pathogens or cancer cell growth in cultured cell lines or diseased animals.
[0022]
The ligand / receptor specific transformants described herein can be used as active ingredients in a formulation for the treatment and prevention of pathogenic infections and cancer in animals, such as humans. Formulation embodiments can be formulated in a number of ways, and can include excipients, binders, emulsifiers, carriers, and other auxiliaries in addition to the ligand / receptor specific converter. Formulations containing ligand / receptor specificity converters can be administered by a number of routes, including topical, transdermal, parenteral, gastrointestinal, transtracheal and transalveolar administration, It is not limited to these. The ligand / receptor specificity converters can also be used as coatings for medical equipment and prostheses, and can prevent infection or spread of disease. The amount of ligand / receptor specific converter provided in a pharmaceutical, therapeutic protocol or applied to a medical device can vary depending on the intended use, patient and frequency of administration.
[0023]
Some of the disclosed methods relate to the administration of a ligand / receptor-specific variant to a subject in need of treatment and / or prevention of bacterial, fungal, viral, and cancer. According to one approach, a patient suffering from a bacterial infection is provided with a ligand / receptor specific converter comprising a specificity domain that interacts with a bacterial receptor. Similarly, a subject suffering from a viral infection is provided with a ligand / receptor-specific converter comprising a specificity domain that interacts with a viral receptor, and a subject suffering from a cancer is expressed on a cancer cell. A ligand / receptor specificity converter comprising a specificity domain that interacts with a receptor of Once the receptor / specificity-converter complex has formed, it is intended that the pathogen or cancer cell be eliminated from the body by a complement fixation reaction and / or macrophage degradation.
[0024]
A subject suffering from or at risk for a disease is identified, and then a therapeutically effective amount of a ligand / receptor-specific converter that interacts with a receptor present on the pathogenic agent Are provided for treating and preventing diseases such as bacterial, fungal, and viral infections, and cancer. Thus, a subject suffering from a bacterial, fungal, viral infection, or cancer is identified by conventional clinical and diagnostic evaluations and has a therapeutically effective amount of a ligand / receptor that interacts with a particular pathogen or cancer cell. Body specific converters are provided. The ligand / receptor-specific converters described herein can be administered to all animals at risk for disease for prophylactic purposes, but can be used for individuals at high risk (eg, infants, the elderly, and pathogens). It is desirable to administer the ligand / receptor specific converter only to those who come into close contact. As noted above, high risk individuals are identified by clinical and diagnostic techniques currently available.
[0025]
The following sections provide more description of various types of ligand / receptor specific converters that interact with receptors on bacteria, parasites, fungi, fungi, viruses, and cancer cells.
[0026]
Ligand / receptor specific converters that interact with receptors on pathogens
Ligand / receptor specificity converters that interact with receptors on pathogens have a variety of chemical structures, but generally speaking, they contain at least one region (specificity domain) that binds to the receptor. And at least one region (antigenic domain) that interacts with an antibody that is specific for an epitope of a pathogen or toxin. Although the preferred ligand / receptor specificity converter is a peptide, some embodiments include a derivatized or modified peptide or peptidomimetic structure. For example, typical peptide-based ligand / receptor specificity converters have substituents that are not normally found on peptides or are incorporated into regions that are normally found on peptides but are usually but not unusual. Can be modified to have a substituent. Thus, peptide-based ligand / receptor specificity converters can be acetylated, acylated, or aminated, and the substituents that can be included on the peptide to modify the specificity converter include: Hydrogen, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl or 5- or 6-membered aliphatic or aromatic ring, including Not limited. Thus, the term "ligand / receptor specificity converter" is a broad term that includes modified or unmodified peptide structures, as well as peptidomimetics and peptidomimetic chemical structures.
[0027]
There are numerous methods for producing peptidomimetics similar to peptide-based ligand / receptor specificity converters. All natural amino acids used for the biological production of peptides have the L configuration. Synthetic peptides can be prepared utilizing L-amino acids, D-amino acids, or various combinations of amino acids in two different configurations using conventional synthetic methods. Synthetic compounds that mimic the configuration and desired characteristics of a peptide, but do not have undesired characteristics, such as flexibility (loss of conformation) and bond breaking, are known as "peptidomimetics" (eg, Spatola, A.). F. Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, and Mar 19, 1987. Methylenethio [CH ₂ S] using bioequivalents); and Szelke et al. , In peptides: Structure and Function, Procedures of the Eighth American Peptide Symposium, (Hruby and Rich, Eds.); 579-582, Pierce Chemical Co. , Rockford, III. (1983) (This document describes methyleneamino [CH at the Leu-Val amide bond in 6-13 octapeptides derived from angiotensinogen. ₂ NH] and hydroxyethylene [CHOHCH ₂ Renin inhibitors with both bioisosteres have been described)).
[0028]
In general, the design and synthesis of peptidomimetics that are similar to ligand / receptor specificity converters will depend on the sequence of the ligand / receptor specificity converter, and the desired ligand / receptor specificity converter (eg, most simulated). Starting with the configuration data (eg, geometric data, eg, bond length and bond angle) of the peptide that is likely to be converted, and further using such data, the geometry to be designed as a peptidomimetic. Including scholarship decisions. Numerous methods and techniques for performing this step are known in the art and any of those methods and techniques may be used (eg, Farmer, PS, Drug Design, (Ariens, EJ. Ed.) Vol. 10, pp. 119-143 (Academic Press, New York, London, Toronto, Sydney and San Francisco) (1980); Farmer, et al., P. 36, p. Verber et al., In TINS, 9/85, pp. 392-396; Kaltenbron et al., In J. Med. Chem. 33: 838-845 (1990); and Spatola, AF, in C. ... Emistry and Biochemistry of Amino Acids Peptides, and Proteins, Vol 7, pp 267-357, Chapter 5, "Peptide Backbone Modifications:. A Structure-Activity Analysis of Peptides Containing Amide Bond Surrogates Conformational Constraints, and Relations" (B Weisten, ed .; Marcell Dekker: New York, pub.) (1983); Kemp, DS, "Peptidomimetics and the Template Approach to. ucleation of ∃-sheets and ∀-helices in Peptides ", Tibech, Vol. 8, see pp.249-255 (1990)). Additional teachings are provided in U.S. Patent Nos. 5,288,707, 5,552,534, 5,811,515, 5,817,626, 5,817,879, 5,821. 231 and 5,874,529. Once a peptidomimetic has been designed, it can be manufactured using conventional techniques in peptide chemistry and / or organic chemistry.
[0029]
Some embodiments include more than one specificity domain and / or more than one antigenic domain. One type of ligand / receptor specificity converter having multiple specificity domains and / or antigenic domains is to have multispecific domains and / or antigenic domains that appear in tandem on the same molecule, Called "multiplexed ligand / receptor specificity converter". For example, a multiplexing specificity domain may comprise two or more ligands that interact with one type of receptor on a pathogen, two or more ligands that interact with different types of receptors on a pathogen, and It may have two or more ligands that interact with different types of receptors on different pathogens.
[0030]
Similarly, multiplexed antigenic domains can be constructed to have multiples of the same epitope of a pathogen or different epitopes of a pathogen, which can also be multiplexed. That is, some multiplexed antigenic domains are multivalent because the same epitope is repeated. In contrast, some multiplexed antigenic domains have multiple, distinct epitopes present on the same molecule in tandem. In this regard, these antigenic domains are multiplexed, but not multivalent. Further, some multiplexed antigenic domains are constructed to have different epitopes, but the different epitopes are themselves multivalent because each type of epitope is repeated.
[0031]
Some ligand / receptor specificity converters provide, in addition to specificity and antigenic domains, other elements, such as sequences that facilitate purification, greater flexibility, and reduce steric hindrance Linkers, as well as sequences that provide greater stability to ligand / receptor specificity converters (eg, resistance to protease degradation) or that promote degradation (eg, protease recognition sites) are included. For example, ligand / receptor specificity converters can include a cleavable signal sequence that facilitates cytoplasmic export of peptides and / or cleavable sequence tags that facilitate purification on antibody columns, glutathione columns, and metal columns.
[0032]
The ligand / receptor-specific converter may include elements that increase the flexibility of the molecule to reduce steric hindrance, or attach the ligand / receptor-specific converter to a support or other molecule. These elements are generally referred to as "linkers." For example, one type of linker that can be incorporated with a ligand / receptor specificity converter is avidin or streptavidin (or their ligand-biotin). By means of biotin-avidin / streptavidin binding, multiple ligand / receptor specificity converters can be linked together (eg, via a support (eg, a resin) or directly), or with individual specificity domains and Antigenic domains can be linked. Another example of a linker that can be included in a ligand / receptor specificity converter is called an "8 linker" because it has sequences found on 8 phages. The eight preferred sequences correspond to the flexible arms of the phage. These sequences are present in the ligand / receptor specificity converter (eg, between the specificity and antigenic domains or between multiples of the specificity domain and / or between multiples of the antigenic domain). It can be included to provide more flexibility and reduce steric hindrance.
[0033]
Further, the ligand / receptor specificity converter may include a sequence that confers resistance to protease degradation, or a sequence that promotes protease degradation. For example, the incorporation of multiple cysteines in the ligand / receptor specificity converter can confer greater resistance to protease degradation. The ligand / receptor specificity converters of these embodiments are expected to be retained in the body for extended periods of time, which may be beneficial for some therapeutic applications. In contrast, ligand / receptor specificity converters may also include sequences that increase the rate of degradation because they are rapidly cleared from the body. Numerous sequences that function as recognition sites for serine, cysteine, and aspartic proteases are known and can be incorporated into ligand / receptor-specific converters.
[0034]
The following sections describe the specificity domains in more detail.
[0035]
Specificity domain
Because a vast number of ligands are known to interact with receptors on bacteria, parasites, fungi, molds, viruses, and cancer cells, the specificities that can be used for ligand / receptor specificity converters Domain types vary. For example, many types of bacteria, parasites, fungi, filamentous fungi, viruses, and cancer cells interact with extracellular matrix proteins. Thus, the desired specificity domain comprises at least one ligand having a peptide sequence present in an extracellular matrix protein. That is, the specificity domain can have a ligand having a peptide sequence found in, for example, fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin.
[0036]
Researchers have mapped regions of extracellular matrix proteins that interact with several receptors (see, eg, McDevvit et al., Eur. J. Biochem., 247: 416-424 (1997); Flock, Molecular Med. Today, 5: 532 (1999); and Pei et al., Infect. And Immun. 67: 4525 (1999)). Some receptors bind to the same region of extracellular matrix proteins, some have overlapping binding domains, and some bind to completely different regions. Preferably, the ligand constituting the specificity domain has an amino acid sequence identified to be involved in adhesion to an extracellular matrix protein. However, fragments of a known ligand for any receptor on a pathogen can be used at random to generate ligand / receptor specific converters and to generate these candidate ligand / receptor specific converters. It should be understood that screening in the characterization assays described below can also identify molecules that interact with receptors on the pathogen.
[0037]
Some specificity domains have ligands that interact with the bacterial adhesion receptor, examples of receptors include extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plus Examples include, but are not limited to, minogen binding protein, thrombospondin binding protein, aggregation factor A (ClfA), aggregation factor B (ClfB), fibronectin binding protein, coagulase, and extracellular adhesion protein. Ligands having an amino acid sequence corresponding to the C-terminal portion of the gamma chain of fibrinogen have been shown to antagonize the binding of fibrinogen to ClfA, a Staphylococcus aureus adhesion receptor. (McDevvit et al., Eur. J. Biochem., 247: 416-424 (1997)). In addition, staphylococcal organisms produce more adhesion receptors, such as Efb, which binds the α-chain of fibrinogen, ClfB, which interacts with both the ∀ and ∃ chains of fibrinogen, and Fbe, which binds the ∃ chain of fibrinogen I do. (Pei et al., Infect. And Immun. 67: 4525 (1999)). Thus, a preferred specificity domain comprises at least three amino acids of a sequence present in a molecule capable of binding the bacterial adhesion receptor (eg, fibrinogen).
[0038]
The specificity domain may also include a ligand that interacts with a viral receptor. Some viral receptors and their corresponding ligands are known, and these ligands or fragments thereof can be incorporated into ligand / receptor specific converters. For example, Tong et al. Identified a hepadnavirus receptor, a 170 kd cell surface glycoprotein that interacts with the pre-S domain of the duck hepatitis B virus envelope protein (U.S. Pat. No. 5,929,220). Have determined that the T cell surface protein CD4 (or a soluble form called T4) interacts with gp120 of HIV (US Pat. No. 6,093,539). Thus, the specificity domain that interacts with the viral receptor interacts with a region of the pre-S domain of the duck hepatitis B virus envelope protein (eg, amino acid residues 80-102 or 80-104) or gp120 of HIV. It may include regions of the T cell surface protein CD4 (or a soluble form called T4), such as the extracellular domain of CD4 / T4 or a fragment thereof. There are more ligands for the viral receptor, and these molecules or fragments thereof can be used as specificity domains.
[0039]
The specificity domain may also include a ligand that interacts with a receptor present on the cancer cell. The proto-oncogene HER-2 / neu (C-erbB2) encodes the tyrosine kinase family surface growth factor receptor p185HER2. 20-30% of breast cancer patients overexpress the gene encoding HER-2 / neu (C-erbB2) due to gene amplification. Thus, a ligand / receptor specificity converter comprising a specificity domain encoding a ligand for HER-2 / neu (C-erbB2) is a preferred embodiment. Many types of cancer cells also overexpress or differentially express integrin receptors. Many preferred embodiments include a specificity domain that interacts with the integrin receptor. Although integrins interact primarily with extracellular matrix proteins, these receptors are known to interact with other ligands, such as invasin, RGD-containing peptides (ie, arginine-glycine-aspartate) and chemicals. (See, for example, U.S. Patent Nos. 6,090,944 and 6,090,388, and Brett et al., Eur J Immunol, 23: 1608 (1993)). As ligands for integrin receptor, vitronectin receptor, laminin receptor, fibronectin receptor, collagen receptor, fibrinogen receptor, ∀ ₄ ∃ ₁ Receptor, ∀ ₆ ∃ ₁ Receptor, ∀ ₃ ∃ ₁ Receptor, ∀ ₅ ∃ ₁ Receptor and ∀ _V ∃ ₁ Molecules that interact with the receptor include, but are not limited to. Table 1 also lists some preferred specificity domains. The above specificity domains are provided for illustrative purposes only and are to be construed as limiting in any way the range of specificity domains that can be used with the embodiments described herein. Should not be.
[0040]
The next section describes the antigenic domains in more detail.
[0041]
[Table 1]

[0042]
Antigenic domain
Since pathogens or toxins can present a number of different epitopes, the diversity of antigenic domains that can be used in ligand / receptor specificity converters is also very wide. That is, antigenic domains that can be incorporated into ligand / receptor specificity converters include epitopes on bacteria, fungi, plants, molds, viruses, cancer cells, and toxins. The desired antigenic domain comprises an epitope of a pathogen that is already present in the subject by natural acquired immunization or vaccination. For example, epitopes of pathogens that cause childhood disease can be used as antigenic domains.
[0043]
Some embodiments have an antigenic domain that interacts with an antibody that is separately administered to a subject. For example, an antibody that interacts with the antigenic domain of a specificity variant can be co-administered with the specificity variant. In addition, antibodies that interact with the ligand / receptor specificity converter may not normally be present in a subject, but the subject may be exposed to a biological substance (eg, serum, blood, or tissue) by introducing the antibody. Can be obtained. For example, a subject receiving a transfusion obtains a number of antibodies, some of which may interact with the antigenic domain of the ligand / receptor specificity converter. Some preferred antigenic domains for use in ligand / receptor specificity converters include viral epitopes such as, but not limited to, herpes simplex virus, hepatitis B virus, TT virus, and poliovirus.
[0044]
In some embodiments, it is also preferred that the antigenic domain comprises a pathogen or toxin epitope recognized by a "high titer antibody". An approach to determine whether an epitope of a pathogen or toxin is recognized by a high titer antibody is presented below. Pathogen epitopes that can be included in the antigenic domain of the ligand / receptor specificity converter include Swedish Patent No. 9901601-6, US Pat. No. 5,869,232, Mol. Immunol. 28: 719-726 (1991); Med. Virol. 33: 248-252 (1991). Table 2 provides the amino acid sequences of some preferred antigenic domains.
[0045]
The sections after Table 2 describe the preparation of ligand / receptor specificity converters in more detail.
[0046]
[Table 2]

[0047]
Methods for producing ligand / receptor specific converters that interact with receptors on bacteria, parasites, fungi, molds, viruses, and cancer cells
In some embodiments, the specificity and antigenic domains are manufactured separately and then linked together (eg, by a linker or by association with a common carrier molecule), and in other embodiments, the specificity domain And the antigenic domain are produced as part of the same molecule. For example, any of the specificity domains listed in Table 1 are linked to any of the antigenic domains in Table 2. The specificity and antigenic domains can be manufactured separately and linked together by a linker or carrier molecule (eg, a complex that includes a biotinylated specificity domain, streptavidin, and a biotinylated antigenic domain), but the ligand / Preferably, the receptor specificity converter is produced as a fusion protein. Thus, a preferred embodiment includes a fusion protein having any of the specificity domains in Table 1 linked to any of the antigenic domains in Table 2.
[0048]
Ligand / receptor specificity converters can be made according to conventional methods of protein engineering, protein chemistry, organic chemistry, and molecular biology. In addition, some companies produce tailor-made peptides, but ligand / receptor specificity converters provide such companies with the desired ligand / receptor specificity converter sequences and specific specifications. It may be obtained by using those services which produce the active substance according to the written description (eg Bachem AG, Switzerland). Preferably, the ligand / receptor specificity converter is prepared using techniques known in the art, for example, Merrifield et al. , J. et al. Am. Chem. Soc. 85: 2149 (1964), Houghten et al. , Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill. (1984) and Creighton, 1983, Pharmaceuticals, Inc., USA). , NY, prepared by chemical synthesis methods (eg, solid phase peptide synthesis).
[0049]
According to one approach, solid phase peptide synthesis is performed using an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA). Each synthesis uses p-methylbenzylhydrylamine solid support resin (Peptide International, Louisville, KY) and produces a carboxyl-terminal amide when the peptide is cleaved from the solid support by acid hydrolysis. Prior to use, the carboxyl-terminal amide can be removed and the ligand / resolved by high-performance liquid chromatography (eg, reversed-phase high-performance liquid chromatography (RP-HPLC) using a PepS-15 C18 column (Pharmacia, Uppsala, Sweden)). Receptor specificity transformants are purified and sequenced on an Applied Biosystems 473A peptide synthesizer. An alternative synthetic approach uses an automated peptide synthesizer (Syro, Multisyntech, Tubingen, Germany) and 9-fluorenylmethoxycarbonyl (fmoc) protected amino acid (Milligen, Bedford, MA).
[0050]
Ligand / receptor specificity converters can be synthesized chemically, while it may be more efficient to produce these polypeptides by recombinant DNA technology using techniques known in the art. Using such methods, one can construct expression vectors that contain a nucleotide sequence that encodes a ligand / receptor specificity converter and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Alternatively, RNA that can encode a ligand / receptor specificity converter can be chemically synthesized, for example, using a synthesizer. See, eg, Oligonucleotide Synthesis, 1984, Gait, M.A. J. ed. , IRL Press, Oxford.
[0051]
A variety of host expression vector systems can be utilized to express the ligand / receptor specificity converter. The ligand / receptor specificity transformant can be obtained from a culture if it is a soluble molecule, ie, from a host cell if the peptide or polypeptide is not secreted, and from the medium if the peptide or polypeptide is secreted from the cell. Obtained from However, expression systems also include host cells engineered to express the membrane-bound ligand / receptor specificity converter. Purification or enrichment of the ligand / receptor specific transformant from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods known to those skilled in the art.
[0052]
Expression systems that can be used include microorganisms, such as bacteria (eg, E. coli or Bacillus subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing a nucleotide sequence encoding a ligand / receptor specificity converter. A yeast transformed with a recombinant yeast expression vector containing a nucleotide sequence encoding a ligand / receptor specific transformant (eg, Saccharomyces, Pichia); encoding a ligand / receptor specific transformant An insect cell line infected with a recombinant viral expression vector containing the nucleic acid (Baculovirus); or a mammalian cell line harboring a recombinant expression construct containing a nucleic acid encoding a ligand / receptor specific transformant (eg, COS, CHO, BHK, 293, 3T3). But, but it is not limited to these.
[0053]
In bacterial systems, a number of expression vectors may be selected depending on the use intended for the ligand / receptor specificity converter. For example, if large quantities are required (eg, for the production of pharmaceutical compositions of ligand / receptor specific transformants), vectors that direct the expression of high levels of fusion protein products that can be readily purified may be used. desirable. Such vectors include the E. coli expression vector pUR278 (Ruther et al., EMBO J., 2: 1791 (1983)) (in this case, a ligand / receptor-specific converter code such that a fusion protein is produced). The sequences can be separately ligated into the vector in frame with the lacZ coding region); the pIN vector (Inouye & Inouye, Nucleic Acids Res., 13: 3101-3109 (1985); Van Heke & Schuster, J. Biol. Chem., 264: 5503-5509 (1989)) and the like, but are not limited thereto. The pGEX vector can be used with glutathione S-transferase (GST) to express foreign polypeptides, such as fusion proteins. Generally, such fusion proteins are soluble and can be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product is released from the GST moiety.
[0054]
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The ligand / receptor specificity converter gene coding sequence is cloned separately into a nonessential region of the virus (eg, polyhedrin gene) and placed under the control of an AcNPV promoter (eg, polyhedrin promoter). Proper insertion of the ligand / receptor specificity converter gene coding sequence results in inactivation of the polyhedrin gene and production of a non-occlusive recombinant virus (ie, a virus lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells for expressing the inserted gene. (See, for example, Smith et al., J. Virol. 46: 584 (1983) and U.S. Patent No. 4,215,051 (Smith)).
[0055]
In mammalian host cells, a number of viral-based expression systems may be utilized. If an adenovirus is used as an expression vector, the nucleic acid sequence encoding the ligand / receptor specific convertor may be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, region E1 or E3) can result in a recombinant virus capable of growing in an infected host and expressing a ligand / receptor-specific converter gene product. . (See, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 3655-3659 (1984)). Specific initiation signals may also be required to efficiently translate the inserted ligand / receptor specificity converter nucleotide sequence (eg, ATG start codon and adjacent sequences). In most cases, exogenous translational control signals, possibly including the ATG start codon, should be provided. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can have a variety of origins, both natural and synthetic. Expression efficiency can also be increased by incorporating appropriate transcription enhancer elements, transcription terminators, and the like. (See Bittner et al., Methods in Enzymol., 153: 516-544 (1987)).
[0056]
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important in some embodiments. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. By selecting the appropriate cell line or host system, the foreign protein expressed can be reliably modified and processed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
[0057]
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, a cell line stably expressing the above ligand / receptor specific transformant can be obtained by engineering. Rather than using an expression vector containing a viral origin of replication, the host cell is transfected with appropriate expression control elements (eg, promoters, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and DNA controlled by a selectable marker. Can be converted. Following the introduction of the foreign DNA, the engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes, allowing the cells to grow and form cell foci, which in turn Cloned and expanded in This method is suitably used to engineer cell lines that express the ligand / receptor specificity transformant.
[0058]
Numerous selection systems are used, for example, herpes simplex virus thymidine kinase (Wigler, et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. USA 48: 2026 (1962)), and the adenine phosphoribosyltransferase (Lowy, et al., Cell 22: 817 (1980)) gene. These are respectively tk. sup. -, Hgprt. sup. -Or apprt. sup. -Can be used in cells. In addition, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr (Wigler, et al., Proc. Natl. Acad. Sci. USA 77: 3567 (1980), which confers resistance to methotrexate); Natl. Acad. Sci. USA 78: 1527 (1981); gpt that confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072). (1981)); neo (Colberre-Garapin, et al., J. Mol. Biol. 150: 1 (1981)) which confers resistance to aminoglycoside G-418; and hygromycin. Hygro (Santerre, et al., Gene 30: 147 (1984)).
[0059]
The following section describes the ligand / receptor specificity converter characterization assay in more detail.
[0060]
Ligand / Receptor Specificity Transformer Characterization Assay
Preferably, the ligand / receptor specificity converter is analyzed for its ability to interact with the receptor and / or to interact with antibodies that may be present in the subject. The term "characterization assay" refers to an assay, experiment or analysis performed on a ligand / receptor specificity converter, which is a receptor (eg, a surface receptor present in bacteria, viruses, molds or fungi). Alternatively, the ability to interact with an antibody (eg, an antibody that recognizes an epitope found on the pathogen), or the ability of the ligand / receptor specific transformant to propagate the pathogen is assessed. Included within the term “characterization assay” are binding tests (eg, enzyme immunoassays (EIA), enzyme linked immunoassays (ELISAs), competitive binding assays, computer generated binding assays, support binding tests, and one and two hybrids. System), and infectivity tests (eg, reduced viral infection, growth and adhesion to host cells).
[0061]
Preferred binding assays use multiple agents. One form of a multi-agent relates to a composition comprising a ligand / receptor specificity converter or a fragment thereof on a support. Another form of a multi-agent comprises a receptor on a support, or a composition comprising an antibody on a support, the antibody being specific for the antigenic domain of a ligand / receptor specificity converter. . A "support" can be a carrier, protein, resin, cell membrane or any polymeric structure used to link or immobilize such molecules. Examples of the solid support include reaction tray well walls, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles (eg, latex particles), animal cells, Durasite (registered trademark), artificial cells, and the like. But not limited thereto. Ligand / receptor specificity converters may be used, for example, by covalent attachment via a hydroxy, carboxy or amino group and a reactive group on the support, to an inorganic support such as a silicon oxide material such as silica gel, zeolite, diatomaceous earth or aminated glass. ).
[0062]
For some multi-agents, the polymeric support has a hydrophobic surface that interacts with a portion of the ligand / receptor specific transformant, receptor or ligand by hydrophobic non-covalent interactions. In some cases, the hydrophobic surface of the support is a polymer (eg, a plastic) or any other polymer to which hydrophobic groups have been attached (eg, polystyrene, polyethylene, or polyvinyl). In addition, antibodies specific for the ligand / receptor specific transformant, the receptor, or the antigenic domain of the ligand / receptor specific transformant can be used to support, eg, proteins and oligo / polysaccharides (eg, cellulose, starch, glycogen , Chitosan or aminated sepharose). In these latter multiple agents, a reactive group on the molecule, such as a hydroxy or amino group, is used to link and form a covalent bond with the reactive group on the carrier. The additional multi-agent is chemically activated to bind to an antibody specific for the ligand / receptor specific converter, the receptor, or the antigenic domain of the ligand / receptor specific converter. Includes a support having other reactive groups. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxysuccinimide chloroformate linkages, or oxirane acrylic supports can be used (Sigma). Further, in some embodiments, it is contemplated that liposomes or lipid bilayers (natural or synthetic) will be used as supports, and ligand / receptor specific converters, receptors, or ligand / receptor specific converters. Antibodies specific for the antigenic domain of can be bound to the membrane surface or can be incorporated into the membrane by techniques in liposome engineering. According to one approach, the liposome multi-support comprises an antibody specific for a ligand / receptor specific converter, a receptor, or an antigenic domain of the ligand / receptor specific converter, which is exposed on the surface. .
[0063]
To increase flexibility and overcome any steric hindrance generated by the support, specificity for the antigenic domain of the ligand / receptor-specific converter, the receptor, or the ligand / receptor-specific converter It is also contemplated to insert a linker of appropriate length between the antibody and the support (eg, a “λ linker” engineered to resemble the flexible region of λ phage). Determination of the appropriate length of a linker that allows for optimal binding can be found by screening binding molecules with various linkers in the characterization assays detailed herein.
[0064]
Some approaches for characterizing ligand / receptor specificity converters use the above multiplex. For example, a ligand / receptor specific converter bound to a support may be contacted with a "free" adhesion receptor and the association may be direct (eg, by using a labeled adhesion receptor) or indirect (eg, by adhesion). (By using a labeled ligand for the receptor). Thus, the association of the receptor with the support binding candidate ligand / receptor specificity converter identifies the candidate ligand / receptor specificity converter as a true ligand / receptor specificity converter. Alternatively, the support-bound adhesion receptor is contacted with a “free” ligand / receptor specificity converter to directly control the amount of associated ligand / receptor specificity converter (eg, labeled ligand / receptor specificity). Transformants) or indirectly (eg, by using a labeled antibody against the antigenic domain of a ligand / receptor specific transformant). Similarly, an antibody specific for the antigenic domain of the ligand / receptor specific converter on the support, and a labeled ligand / receptor specific converter (or a secondary detection reagent such as a labeled receptor or ligand / By using an antibody to the receptor-specific transformant), the ability of the antibody to bind to the antigenic domain of the ligand / receptor-specific transformant can be determined.
[0065]
Some characterization assays assess the ability of ligand / receptor specificity converters to interact with the target receptor and the redirected antibody, while other characterization assays assess ligand / receptor specificity. It is designed to determine whether a sex converter can bind to both the target receptor and the redirecting antibody. In general, characterization assays can be categorized as follows: (1) in vitro characterization assays, (2) cell characterization assays, and (3) in vivo characterization assays.
[0066]
Discussion of each type of characterization assay is provided in the following sections.
[0067]
In vitro characterization assay
Used to determine whether a ligand / receptor specific converter binds to a particular receptor and whether an antibody in a subject can bind to a ligand / receptor specific converter. There are many types of in vitro assays that can be obtained. Most simply, the receptor is bound to a support (eg, a Petri dish) and the association of the ligand / receptor specificity converter with the receptor is monitored directly or indirectly as described above. Similarly, a primary antibody (eg, an antibody found in a subject) to the antigenic domain of the ligand / receptor specific transformant is bound to the support and the association of the ligand / receptor specific transformant with the primary antibody is established. Epitopes on the ligand / receptor specific converter that do not compete directly (eg, with the labeled ligand / receptor specific converter) or indirectly (eg, on the labeled receptor, or epitope recognized by the primary antibody) (Using a labeled secondary antibody that interacts with).
[0068]
Another approach involves a sandwich-type assay in which the receptor is bound to a support, the ligand / receptor specific converter is bound to the receptor, and the primary antibody is bound to the ligand / receptor specific converter. If a labeled primary antibody is used, the presence of the receptor / specificity converter / primary antibody complex can be determined directly. The presence of the receptor / specificity converter / primary antibody complex can be determined, for example, by using a labeled secondary antibody that reacts with the primary antibody with an epitope that does not interfere with binding of the primary antibody to the ligand / receptor specificity converter. , Can also be determined indirectly. In some cases, it is desirable to react with an unlabeled secondary antibody to use a labeled tertiary antibody, thus forming a receptor / specificity converter / primary antibody / secondary antibody / labeled tertiary antibody complex. .
[0069]
The following example describes a characterization assay performed to determine whether a specificity domain from the C-terminal domain of fibrinogen inhibits the binding of aggregation factor (Clf) to fibrinogen.
[0070]
Example 1
In this example, several peptides corresponding to the C-terminal domain of fibrinogen (Fib) were analyzed for their ability to block the binding of aggregation factor (Clf) to fibrinogen (see Table 3). These peptides were prepared using standard techniques in peptide synthesis using fmoc chemistry (Syro, MultiSynTech, Germany). Preferably, the peptide is purified by reverse phase HPLC. A competitive enzyme immunoassay was then performed to determine if the peptide could block the interaction between Clf and fibrinogen. Table 3 shows the results of these experiments. The smallest fibrinogen-derived peptide found to suppress the interaction between Clf and fibrinogen was HLGGAQQAGD (SEQ ID NO: 124). Substitution of the first two amino acids of this minimal peptide with alanine and lysine had a significant effect on the ability of the peptide to block the interaction between Clf and fibrinogen (eg, peptide ALGGAQQAGD (SEQ ID NO: 123) The Clf / fibrinogen interaction could not be blocked).
[0071]
[Table 3]

[0072]
The following example describes a characterization assay performed to determine whether a ligand / receptor specific convertor interacts with a ClfA receptor bearing bacterium.
[0073]
Example 2
Ligand / receptor specificity converters having specificity domains (approximately 20 amino acids in length) corresponding to various regions of the fibrinogen γ chain sequence were prepared by standard techniques in peptide synthesis using fmoc chemistry (Syro, MultiSynTech, Germany). And analyzed these ligand / receptor specificity converters for their ability to bind to antibodies specific for the ClfA receptor and their antigenic domains. The sequences of these ligand / receptor specificity converters are listed in Table 4 and presented in the sequence listing (SEQ ID NOs: 60-103). The ligand / receptor specific transformant used in this analysis has an antigenic domain presenting an epitope of the herpes simplex virus gG2 protein and is recognized by a monoclonal antibody against the herpes simplex virus gG2 protein. 2: These ligands / receptors in phosphate buffered saline (PBS) containing g / ml goat serum (Sigma Chemicals, St. Louis, MO) and 0.5% Tween 20 (PBS-GT). Serial dilutions of the body-specific transformants were made. Receptor ClfA was passively adsorbed at 10 μg / ml in a 96-well microtiter plate at + 4 ° C. overnight in 50 mM sodium carbonate buffer at pH 9.6.
[0074]
The diluted ligand / receptor specific transformant was then incubated on the plate for 60 minutes. The ability of the ligand / receptor specific transformant to interact with the receptor was determined by adding the primary antibody to the plate and incubating for 60 minutes (1: 1000 dilution of mAb against herpes simplex virus gG2 protein). Next, the bound primary mAb was indexed with a rabbit anti-mouse IgG (Sigma) secondary antibody and a peroxidase-labeled goat anti-rabbit IgG (Sigma) tertiary antibody. Plates were developed by incubation with dinitrophenylene-diamine (Sigma) and analyzed for absorbance at 405 nm.
[0075]
All ligand / receptor specificity converters presented in Table 4 (SEQ ID NOs: 60-103) bound appreciably to immobilized ClfA, and also allowed binding of mAbs specific to the HSV gG2 protein. . The above method for determining the affinity of a ligand / receptor specificity converter for an adhesion receptor and a primary antibody comprises any candidate ligand / receptor specificity conversion comprising any specificity domain and any antigenic domain. It can be performed on the body, provided that an appropriate arrangement and adhesion receptor are used.
[0076]
The examples following Table 4 describe another characterization assay performed to determine whether a ligand / receptor specific convertor interacts with a bacterium having a ClfA receptor.
[0077]
[Table 4]

[0078]
Example 3
A ligand / receptor-specific converter having a specific domain that binds to an aggregation factor (Clf) and an antigenic domain corresponding to a poliovirus-derived epitope was used in peptide synthesis using fmoc chemistry (Syro, MultiSynTech, Germany). Manufactured using standard techniques (see Table 5). These ligand / receptor specificity converters were analyzed for their ability to suppress the interaction between CLF and fibrinogen. In these experiments, the ligand / receptor specific converters listed in Table 5 were prepared, and enzyme competition immunoassays containing Clf and fibrinogen were performed on various concentrations of these molecules. The lowest inhibitory concentration, the lowest peptide concentration required to suppress the Clf / Fib interaction, was established. That is, the lower the concentration required to suppress the Fib / Clf interaction, the more effective the inhibitor. In addition, the lowest solid phase bound peptide concentration, which was the lowest test concentration of the peptide recognized by the anti-poliovirus antibody in the immunoassay, was determined. Some of the peptides used indicated by an asterisk in Table 5 (eg, CPALTAVETGTCNPLAAAHLGHGAKQAG (SEQ ID NO: 135), HHLGGAKQAG-AA-CPALTAVETGTCTNPL (SEQ ID NO: 137), CPALTAVETGC-TNPLHHLGGGAQTAGHGPNGLGHTAGGHCTGLAGTAGGNCT )) Was cyclized between two artificially introduced cysteine residues. These experiments demonstrate that HHLGGAKQAG-AA-CPALTAVETGTCTNPL (SEQ ID NO: 137) and HHLGGAKQAG-CPALTAVETGTCTNPL (SEQ ID NO: 142) efficiently suppress the interaction between Clf and fibrinogen and retain a functional poliovirus epitope. did.
[0079]
[Table 5]

[0080]
The next section describes some cell-based characterization assays that can be performed to determine if a ligand / receptor specific transformant has an effect on pathogen growth.
[0081]
Pathogen-based characterization assays
In another type of characterization assay, ligand / receptor specificity interacts with an antibody directed against the antigenic domain of the pathogen and ligand / receptor specificity transformant using a pathogen based approach. Evaluate the ability of the converter. This analysis is also based on the interaction between ligand / receptor specific transformants and antibodies directed against the antigenic domains of the pathogen and ligand / receptor specific transformants in the subject's body. The humoral and cellular responses (eg, complement fixation and macrophage degradation) of the pathogen from the subject follow, thus demonstrating the ability of the ligand / receptor-specific converter to propagate the pathogen. Generally speaking, a pathogen-based characterization assay provides a cultured pathogen with a ligand / receptor specific converter and monitors the association of the ligand / receptor specific converter with a cell or virus. Including. Several pathogen-based characterization assays can be used. The following examples describe some of the preferred characterization assays in more detail.
[0082]
Example 4
One type of pathogen-based characterization assay involves binding a ligand / receptor specific converter with a bacterium located on a support. Thus, bacteria producing Clfa (eg, Staphylococcus aureus or E. coli) are grown in culture or on agar plates in a suitable growth medium (eg, LB broth, blood broth, LB agar or blood agar). The cells are then membrane-coated by placing the culture on a membrane under vacuum (eg, using a dot blot manifold device) or by placing the membrane on a colony for a time sufficient to allow migration. (Eg, nitrocellulose or nylon). The cells bound to the membrane are then serially diluted in ligand / receptor specific transformants (eg, 500 ng, 1: g, 5: g, 10: g, 25: g in a total volume of 200: 1 PBS). And 50: g ligand / receptor specificity converter). In one experiment, the ligand / receptor specificity converters listed in Table 4 or Table 5 are used. The diluted ligand / receptor specific converter is then incubated on the membrane for 60 minutes. Thereafter, the unbound ligand / receptor-specific converter is removed and the membrane is washed with PBS (eg, 3 washes with 2 ml of PBS / wash per wash). Next, a 1: 100 to 1: 1000 dilution of a primary antibody (e.g., a mAb against herpes simplex virus gG2 protein) that interacts with the antigenic domain of the ligand / receptor specificity transformant is provided and the binding reaction is allowed to proceed for 60 minutes. Wake up. Again, the membrane is washed with PBS (eg, 3 washes with 2 ml of PBS per wash) to remove unbound primary antibody. Suitable controls include bacteria on the membrane itself, on the membrane without the ligand / receptor specificity converter, and on the membrane with the ligand / receptor specificity converter but without the primary antibody. .
[0083]
To detect the amount of ligand / receptor specific converter bound to the bacteria on the membrane, a secondary antibody (eg, rabbit anti-mouse IgG (Sigma)) and a tertiary antibody (eg, peroxidase-labeled goat anti-rabbit IgG (Sigma)) ) Is used. Of course, labeled secondary antibodies that interact with the primary antibody can be used as well. After contacting the secondary antibody with the membrane for 60 minutes as described above, the unbound secondary antibody is washed from the membrane with PBS (eg, three times with 2 ml of PBS each time). Further, after the tertiary antibody is brought into contact with the membrane for 60 minutes, the unbound tertiary antibody is washed from the membrane with PBS (eg, three times with ml of PBS each time). Bound tertiary antibody can be detected by incubating the membrane with dinitro-phenylene-diamine (Sigma).
[0084]
Another approach involves the use of immobilized ligand / receptor specificity converters. Thus, a primary antibody (eg, a mAb against the herpes simplex virus gG2 protein) is bound to the Petri dish. Once the primary antibody is bound, various dilutions of the ligand / receptor specific converter (eg, the ligand / receptor specific converter listed in Table 4 or Table 5) are added to the coated dish. The ligand / receptor-specific converter is associated with the primary antibody for 60 minutes to further wash off unbound ligand / receptor-specific converter (eg, 3 washes with 2 ml of PBS per wash). Suitable controls include a Petri dish containing no primary antibody or ligand / receptor specificity converter.
[0085]
Thereafter, a turbid solution of bacteria (eg, E. coli) is added to the Petri dish and the bacteria are allowed to interact with the immobilized ligand / receptor-specific converter for 60 minutes. Unbound bacteria are then removed by washing with PBS (eg, 3 washes with 2 ml of PBS per wash). The growth medium (eg, LB broth) is then added to the Petri dish and the culture is incubated overnight. Alternatively, add LB agar to the Petri dish and incubate the culture overnight. The interaction between the ligand / receptor specificity converter and the bacterium can be visually observed (eg, in turbid growth media, can be quantified using spectrophotometric analysis or the appearance of colonies on agar). .
[0086]
By modifying the above approach, one of skill in the art can evaluate the ability of the ligand / receptor specificity converter to interact with the virus. For example, soluble fragments of the T4 glycoprotein have been shown to interact with the human immunodeficiency virus (HIV) envelope glycoprotein (see, eg, US Pat. No. 6,093,539). By synthesizing a fusion protein having a specificity domain linked to an antigenicity domain (eg, the antigenic domains listed in Table 2), the specificity domain comprising a fragment of the T4 glycoprotein that interacts with the HIV envelope glycoprotein ( For example, a ligand / receptor specific transformant having amino acids 1-419 of the T4 glycoprotein sequence provided in U.S. Patent No. 6,039,539 or a portion thereof can be prepared. Although peptide chemistry can be used to produce ligand / receptor specific transformants, preferably an expression construct having a fragment of the T4 glycoprotein linked to an antigenic domain is produced and transfected into appropriate cells. You. The expression and purification strategies described in US Pat. No. 6,039,539 and above may also be used.
[0087]
Once the ligand / receptor specificity converter has been constructed, a filter binding assay is performed. That is, a serial 10-fold dilution of the HIV inoculum is applied under constant vacuum to a membrane (eg, nitrocellulose or nylon) in a dot blot apparatus. A serial 10-fold dilution of the ligand / receptor specificity converter is then applied to the bound HIV particles. After contacting the ligand / receptor specificity converter with the particles for 60 minutes, vacuum is applied and washed with PBS (eg, three times with 2 ml of PBS each time). Once the unbound ligand / receptor specific transformant has been removed, a 10-fold serial dilution of the primary antibody that binds the antigenic domain is added to the sample and the binding reaction is allowed to occur for 60 minutes. A vacuum is then applied and the unbound primary antibody is washed with PBS (eg, 3 washes with 2 ml of PBS per wash). As described above, detection of the bound primary antibody can be performed.
[0088]
In a sandwich-type assay, the ability of the ligand / receptor-specific converter to interact with the virus can also be evaluated. That is, the primary antibody that interacts with the antigenic domain of the ligand / receptor-specific converter is immobilized in the microtiter well, and a serial dilution of the ligand / receptor-specific converter is added to the primary antibody, A primary antibody / specificity converter complex is prepared as described above. A 10-fold serial dilution of the HIV inoculum is then added and the binding reaction is allowed to take place for 60 minutes. Unbound HIV particles are removed by continuous washing in PBS. Detection of bound HIV particles can be accomplished using a radiolabeled anti-HIV antibody (eg, an antibody obtained from serum from a human suffering from an HIV infection).
[0089]
Although the above examples describe pathogen-based assays using bacteria and viruses, some of these approaches can be modified to test the interaction of ligand / receptor specific transformants with mammalian cells. For example, the ability of a ligand / receptor specificity converter to interact with an integrin receptor present on a cancer cell can be determined as follows. ∀ _V ∃ ₃ Receptor expressing melanoma cells (eg, M21 human melanoma cells) bind to fibrinogen and this interaction can be blocked by administering a peptide comprising RGD (eg, Katada et al., J. Biol). Chem. 272: 7720 (1997) and Felding-Habermann et al., J. Biol. Chem. 271: 5892-5900 (1996)). Similarly, many other types of cancer cells express integrins that interact with the RGD peptide. According to one approach, cancer cells that express RGD-responsive integrins (eg, M21 human melanoma cells) are cultured to confluence. M21 cells can grow in DMEM medium containing 10% fetal calf serum, 20 mM Hepes and 1 mM pyruvate.
[0090]
Preferably, a final concentration of 20 μg / ml (2 × 10 ⁶ Cells / ml) are stained with hydroethidine (Polyscience, Inc., Warrington, PA) for 30 minutes at 37 ° C. and then washed twice to remove excess dye. Hydroethidine intercalates into the DNA, resulting in a red fluorescent label of the cell, but without impairing the cell's adhesive function. Staining provides one way to quantify the binding of ligand / receptor specificity converter to cells. That is, the binding efficiency can be determined by comparing the total number of hydroethidine-stained cells with the number of cells bound to the fluorescent-labeled primary antibody / specificity converter complex.
[0091]
Thus, the stained cells are incubated with various dilutions of a ligand / receptor-specific converter containing an RGD sequence (eg, GRGDSPHRGGPEE (SEQ ID NO: 104) or WSRGDWHRGGPEE (SEQ ID NO: 105)). Following a 60 minute incubation, the unbound ligand / receptor is washed several times in DMEM medium containing 10% fetal calf serum, 20 mM Hepes and 1 mM pyruvate (eg, three washes of 5 ml of medium). Remove body-specific transformants. Next, a 1: 100 to 1: 1000 dilution of a primary antibody (e.g., a mAb against herpes simplex virus gG2 protein) that interacts with the antigenic domain of the ligand / receptor specificity transformant is provided and the binding reaction is allowed to proceed for 60 minutes. Wake up. Thereafter, several washes in the medium are performed to remove any unbound primary antibody. Suitable controls include stained cells that do not contain the ligand / receptor specificity converter, or that do not contain the primary antibody.
[0092]
After binding of the primary antibody, a goat anti-mouse FITC-labeled antibody (1: 100 dilution) (Sigma) is added and binding is allowed to occur for 60 minutes. Again, several media washes are performed to remove any unbound secondary antibody. Analysis is performed by flow cytometry using a filter set at 543/590 nm for hydroethidine and 495/525 nm for fluorescein. Binding of the primary antibody to the ligand / receptor specific converter / cell complex is significantly observed, demonstrating that the ligand / receptor specific converter has a significant effect on cells. .
[0093]
The following examples describe characterization assays that demonstrate that RGD-containing ligand / receptor specificity converters bind effectively to mammalian cells and redirect antibodies to these cells.
[0094]
Example 5
The peptide RGDSAATPPAYR (SEQ ID NO: 145) was prepared using standard techniques for peptide synthesis using fmoc chemistry (Syro, MultiSynTech, Germany). This peptide binds to an integrin receptor, a spacer (AA), and an antigenic domain having an epitope recognized by monoclonal antibody 57/8, an epitope present on the hepatitis B virus e antigen (HBeAg). Have a domain.
[0095]
Murine melanoma cells (SP2 / 0 cells) are washed in serum-free medium and then at a concentration of 50 μg / ml, the RGDSAATPPAYR (SEQ ID NO: 145) peptide or a control peptide from the hepatitis C virus (HCV) NS3 domain. And incubated. Next, the cells were washed, and the amount of the surface-bound peptide was detected by labeling the cells with the 57/8 antibody. The surface-bound antibody was indexed with a 1/500 diluted FITC-labeled anti-mouse IgG conjugate, and the level of surface staining was determined by fluorescence microscopy.
[0096]
This experiment demonstrated that cells incubated with the control peptide showed no staining. In contrast, cells incubated with the RGDSAATPPAYR (SEQ ID NO: 145) peptide showed significant surface staining, consistent with the location of surface expressed integrins. Thus, RGD-containing ligand / receptor specificity converters can effectively bind to integrin-producing mammalian cells and use these molecules to direct and target antibodies to tumor cells.
[0097]
The next section describes the characterization assays performed on animals.
[0098]
In vivo characterization assay
Characterization assays also include experiments that evaluate ligand / receptor specificity converters in vivo. There are a number of animal models suitable for assessing the ability of a ligand / receptor specific transformant to suppress a pathogenic infection. Mice are preferred because they are easy to maintain and are susceptible to bacterial infection, viral infection and cancer. Chimpanzees are also preferred because they are genetically closely related to humans.
[0099]
One approach to assessing the efficacy of ligand / receptor specificity converters in mice is provided in the following examples.
[0100]
Example 6
To test the ability of a ligand / receptor specificity converter to treat a bacterial infection, the following characterization assay may be performed. Several female CF-1 outbred mice (Charles Rivers Laboratories), approximately 8 weeks of age and weighing 25 g, are vaccinated with the antigenic domain of the ligand / receptor specificity converter to be tested. Preferably, the antigenic domain is combined with a carrier and administered with an adjuvant. For example, the antigenic domain can be fused with K. limpet hemocyanin or bovine serum albumin, which acts as both a carrier and an adjuvant, or an adjuvant, such as Freund's adjuvant, aluminum hydroxide, or lysolecithin can be used. Once a high titer of antibody against the antigenic domain can be established, for example, by immunodiffusion or EIA, immunized mice are intraperitoneally inoculated with an overnight culture of S. aureus NTCC 10649. About 100xLD for Staphylococcus aureus ₅₀ Or adjust the inoculum to produce log 6.6.
[0101]
Serial dilutions of the ligand / receptor specific converter (eg, the ligand / receptor specific converter provided in Table 4) are formulated in sterile water for injection and subcutaneously (1 and 5 hours after infection) SC) or the oral (PO) route. For each test, challenge LD at the same time by inoculating untreated mice with a log dilution of the bacterial inoculum. ₅₀ Check. Preferably, 5 groups of bacterial inoculum in 5 log dilution ranges are inoculated into 5 groups of 10 animals each (10 mice per 1 log dilution). LD ₅₀ Results in 100% mortality in all groups of untreated mice inoculated with 100 times the concentration of the inoculum. Mice are monitored daily for mortality for 7 days. Antimicrob. Agents Chemother. 31: 1768-1774 and Proc. Soc. Exp. Biol. Med. The mean effective dose (ED) for protecting 50% of mice from cumulative mortality was determined by log probit analysis of a plot of "survival versus dose" as described in 1994, 57, 261-264. ₅₀ ) Can be calculated. As one of skill in the art will appreciate, a similar approach may be used to test the ability of ligand / receptor specific transformants to suppress viral infection and cancer.
[0102]
The ligand / receptor specific converters described herein can be formulated in a formulation and administered to a subject in need of an agent that inhibits the growth of a pathogen. The following sections describe some formulations containing ligand / receptor specificity converters that interact with receptors on pathogens.
[0103]
Formulations containing ligand / receptor specificity converters that interact with receptors on pathogens
The ligand / receptor specificity converters described herein are suitable for incorporation into a formulation for administration to a subject in need of a compound that treats or prevents infection by a pathogen. These pharmacologically active compounds can be processed according to conventional methods of galenic pharmacy to produce pharmaceutical formulations for administration to animals, including humans. The active ingredient can be incorporated into pharmaceutical products, with or without modification. The manufacture of a formulation or therapeutic that delivers a pharmacologically active compound of the present invention by several routes is an aspect of the present invention. For example, but not limited to, DNA, RNA, and viral vectors having sequences encoding ligand / receptor specificity converters that interact with receptors on pathogens are used with embodiments of the present invention. Nucleic acids encoding ligand / receptor specificity converters may be administered alone or in combination with other active ingredients.
[0104]
The compound is a conventional excipient that does not adversely react with the pharmacologically active ingredients described herein, ie, a pharmaceutically acceptable organic or organic suitable for parenteral, enteral (eg, oral) or topical application. It can be used in admixture with inorganic carrier substances. Suitable pharmaceutically acceptable carriers include water, saline solution, alcohol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrate (eg lactose), amylose or starch, magnesium stearate, talc, silicic acid, Examples include, but are not limited to, viscous paraffins, balms, fatty acid mono- and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. Further vehicles that can be used are described in Remmington's Pharmaceutical Sciences, 15 ^th Edition, Easton: Mack Publishing Company, pages 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14. ^th Edition, Washington, American Pharmaceutical Association (1975). The formulation preparation is sterilized and, if desired, does not adversely react with the ligand / receptor specificity converter, auxiliaries such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, for acting on osmotic pressure. It can be mixed with salts, buffers, colorants, flavors and / or fragrances and the like.
[0105]
The effective dosage and method of administration of a particular formulation having a ligand / receptor specificity converter can vary based on the individual needs of the patient and the required therapeutic or prophylactic measure. Therapeutic efficacy and toxicity of such compounds, such as ED ₅₀ (Therapeutically effective dose in 50% of the population) can be determined by standard pharmaceutical techniques in cell culture or experimental animals. For example, an effective dose of a ligand / receptor specificity converter can be determined using the characterization assay described above. The data obtained from these assays is then used in formulating a range of dosage for use with other organisms, including humans. The dosage of such compounds is preferably that of ED without toxicity. ₅₀ And within a series of circulating concentrations. Dosage will vary within this range depending upon the type of ligand / receptor specific converter, the dosage form employed, the sensitivity of the organism, and the route of administration.
[0106]
Typical dosages of the ligand / receptor specificity converter can vary from about 1 to 100,000 μg, up to a total dose of about 10 g, depending on the route of administration. Desirable dosages include about 250: g to 1 mg, about 50 mg to 200 mg, and about 250 mg to 500 mg.
[0107]
In some embodiments, the dose of the ligand / receptor-specific converter preferably results in a tissue or blood concentration of about 0.1: M to 500 mM or both. Desirable doses will result in a tissue or blood concentration of about 1-800 μM or both. Preferred doses result in tissue or blood concentrations greater than about 10 μM to about 500: M. Doses that result in tissue concentrations higher than 800: M are not preferred, but they may be used. A constant infusion of the ligand / receptor specific converter may also be provided to maintain a stable concentration in the tissue as measured at the blood level.
[0108]
The exact dose will be selected by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to retain the desired effect. Additional factors that may be considered include the severity of the disease, the age of the organism to be treated, and the weight or size of the organism; diet, time and frequency of administration, drug combination (s), response sensitivity, and Resistance / response to treatment. Short-acting pharmaceutical compositions are administered daily or more frequently, while long-acting pharmaceutical compositions are administered every two or more days, once a week, or once every two weeks or less. Administer.
[0109]
Routes of administration of the formulation include, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar administration. Transdermal administration is accomplished by application of a cream, rinse, gel, or the like, which allows the ligand / receptor specific converter to penetrate the skin. Parenteral routes of administration include, but are not limited to, electrical or direct injection, such as direct injection into the central venous system, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Gastrointestinal routes of administration include, but are not limited to, oral ingestion and rectum. Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation via the mouth or intranasally.
[0110]
Compositions having the ligand / receptor specific converters described herein that are suitable for transdermal or topical administration include those that are applied directly to the skin or in transdermal devices ("transdermal patches"). Such as, but not limited to, pharmaceutically acceptable suspensions, oils, creams and ointments incorporated into such protective carriers. Examples of suitable creams, ointments, and the like may be found in, for example, the PDR (Physician's Desk Reference). Examples of suitable transdermal devices are described, for example, in U.S. Patent No. 4,818,540 (Chien et al., Issued April 4, 1989).
[0111]
Compositions having a pharmacologically active compound suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for injection into the central venous system, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
[0112]
Compositions having pharmacologically active compounds suitable for transbronchial and transalveolar administration include, but are not limited to, various types of inhalable aerosols. Devices suitable for these transbronchial and transalveolar administrations are also embodiments. Such devices include, but are not limited to, atomizers and nebulizers. Numerous forms of currently available atomizers and nebulizers can be readily adapted to deliver compositions having the ligand / receptor specificity converters described herein.
[0113]
Compositions having a pharmacologically active compound suitable for gastrointestinal administration include, but are not limited to, pharmaceutically acceptable powders, pills or liquids for oral ingestion, and suppositories for rectal administration. For ease of use, gastrointestinal administration, especially oral administration, is a preferred embodiment. Once a formulation containing the ligand / receptor specificity converter is obtained, it can be administered to an organism in need of treating or preventing a pathogen infection.
[0114]
Aspects of the invention also include coating medical equipment, such as prostheses, implants, and devices. Coatings suitable for use in medical devices can be provided by gels or powders containing the ligand / receptor specific converter, or by polymeric coatings in which the ligand / receptor specific converter is suspended. Suitable polymeric materials for coating the device are those that are physiologically acceptable and capable of diffusing a therapeutically effective amount of the ligand / receptor specific converter. Suitable polymers include, but are not limited to, polyurethane, polymethacrylate, polyamide, polyester, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl chloride, cellulose acetate, silicone elastomer, collagen, silk, and the like. Such coatings are described, for example, in U.S. Pat. No. 4,612,337.
[0115]
The following sections describe methods for treating and preventing diseases using the ligand / receptor specificity converters described herein.
[0116]
Treatment and prevention of diseases using ligand / receptor specificity converters
A subject in need of treating and / or preventing infection by a receptor-bearing pathogen can be administered a formulation comprising a ligand / receptor-specific converter. Subjects in need of such include individuals who are at risk for contact with the pathogen or who are already infected with the pathogen. These individuals can be identified by standard clinical or diagnostic techniques.
[0117]
For example, according to one approach, a subject suffering from a bacterial infection is identified as a subject in need of an agent that suppresses the growth of a pathogen. In that case, a therapeutically effective amount of the ligand / receptor specificity converter is provided to the subject. The ligand / receptor-specific converter used in this method includes a receptor present on bacteria (for example, extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, It contains specific domains that interact with thrombospondin binding protein, aggregation factor A (ClfA), aggregation factor B (ClfB), fibronectin binding protein, coagulase and extracellular adhesion protein). The ligand / receptor specificity converter also comprises an antigenic domain having an epitope of a pathogen or toxin, preferably an epitope recognized by a high titer antibody present in a subject in need thereof. Prior to providing a subject with a ligand / receptor specificity converter, it may also be desirable to screen the subject in need for the presence of a high titer antibody that recognizes the antigenic domain. This screening can be accomplished by EIA or ELISA using an antigenic domain or ligand / receptor specificity converter immobilized as described above.
[0118]
Similarly, a ligand / receptor specific convertor that recognizes a receptor present on a particular pathogenic agent can be administered to a subject in need of an agent that suppresses viral infection. Thus, standard clinical and diagnostic techniques identify subjects in need of an agent that suppresses viral infection. The subject in need is then provided with a therapeutically effective amount of a ligand / receptor specific converter that interacts with a receptor present on a virus of the type that infects the individual. As described above, prior to administration of the ligand / receptor-specific converter, whether the subject has sufficient antibody titer to interact with the antigenic domain of the ligand / receptor-specific converter Is desirably determined.
[0119]
In the same vein, a ligand / receptor specific converter that interacts with a receptor present on a cancer cell can be administered to a subject in need of an agent that inhibits cancer growth. For example, a subject in need of an agent that inhibits cancer growth is identified by standard clinical or diagnostic techniques. The subject in need is then provided with a therapeutically effective amount of a ligand / receptor specific converter that interacts with a receptor present on a cancer cell infected with the subject. As described above, prior to administration of the ligand / receptor-specific converter, whether the subject has sufficient antibody titer to interact with the antigenic domain of the ligand / receptor-specific converter Is desirably determined.
[0120]
The ligand / receptor specificity converters described herein can also be administered to a subject as a prophylactic to prevent the onset of disease. For prophylactic purposes (eg, to prevent bacterial, viral, or cancer), virtually anyone can be administered a ligand / receptor specific converter described herein. However, it is desirable to identify subjects at increased risk of suffering from a particular disease and to provide ligand / receptor specificity converters. Subjects at high risk of having the disease include individuals with a family history of the disease, the elderly or young, or individuals who frequently come into contact with the pathogen (eg, health care workers). Thus, a subject at risk for infection by a receptor-bearing pathogen is identified, and then a prophylactically effective amount of a ligand / receptor specific converter is provided.
[0121]
One prophylactic use of the ligand / receptor specific converter described herein relates to coating or cross-linking a medical device or implant with the ligand / receptor specific converter. Implantable medical devices can be focal points for infection of many bacterial species. Such device-related infections are facilitated by the tendency of these organisms to adhere and settle on the surface of the device. As a result, there is a significant need to develop medical devices that have surfaces that are less prone to promoting adverse biological reactions typically associated with implanting medical devices.
[0122]
According to one approach, the medical device is coated in a solution containing the ligand / receptor specificity converter. Prior to implantation, the medical device (eg, a prosthetic valve) is stored, for example, in a solution of the ligand / receptor specificity converter. The medical device can also be coated in a powder or gel with the ligand / receptor specificity converter. For example, gloves, condoms and intrauterine devices can be coated in powders or gels containing specificity converters that interact with bacterial or viral receptors. Once implanted in the body, these ligand / receptor-specific converters provide a protective barrier to infection by pathogens.
[0123]
In some embodiments, the ligand / receptor specificity converter is immobilized on a medical device. As noted above, the medical device is a support to which the ligand / receptor specificity converter can adhere. Although immobilization can occur by hydrophobic interaction between the ligand / receptor specific converter and the medical device, a preferred method for immobilizing the ligand / receptor specific converter on the medical device is covalent bonding including. For example, a medical device having a reactive group that interacts with a reactive group present on the specificity converter can be manufactured.
[0124]
According to one approach, an aqueous solution having a pH of about 4 to about 9 and a temperature of about 0 to about 50 ° C. using a ligand / receptor specificity converter comprising a 2-amino alcohol moiety and periodate. An aldehyde-function exchanger is formed in the solution. Next, the surface of the biological material of the medical device containing the primary amine moiety is combined with the aldehyde-function exchanger, and the ligand / receptor specific converter is immobilized on the support surface via the imine moiety. Next, the imine moiety is reacted with a reducing agent to form an immobilized ligand / receptor specific converter on the surface of the biological material via a secondary amine bond. Other approaches to cross-linking molecules to medical devices (eg, as described in US Pat. No. 6,017,741) can be modified in part to immobilize the ligand / receptor specific converters described herein. .
[0125]
While the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

Claims (22)

Ligand / receptor specificity comprising at least one specificity domain comprising a ligand for a receptor and at least one antigenic domain linked to said specificity domain comprising an antigenic domain comprising a pathogen or toxin epitope Transform body. The method of claim 1, wherein the specificity domain comprises at least three consecutive amino acids of a peptide selected from the group consisting of an extracellular matrix protein, a ligand for a receptor on a virus, and a ligand for a receptor on a cancer cell. The described ligand / receptor specificity converter. The ligand / receptor specific converter according to claim 2, wherein the peptide is an extracellular matrix protein selected from the group consisting of fibrinogen, collagen, vitronectin, laminin, plasminogen, thrombospondin, and fibronectin. . 3. The ligand / receptor specific converter according to claim 2, wherein the peptide is a ligand for a receptor on a virus selected from the group consisting of T4 glycoprotein and hepatitis B virus envelope protein. 3. The ligand / receptor specific transformant of claim 2, wherein the peptide is a ligand for a receptor on a cancer cell selected from the group consisting of a ligand for HER-2 / neu and a ligand for an integrin receptor. The specificity domains are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, A sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 124. Ligand / receptor specificity converter according to claim 1. 4. The ligand / receptor specific converter according to claim 3, wherein the extracellular matrix protein comprises at least 3 amino acids of the alpha chain of fibrinogen. The ligand / receptor specificity converter of claim 1, wherein the ligand comprises a sequence of arginine-glycine-aspartic acid (RGD). 2. The ligand / receptor specificity converter of claim 1, wherein said receptor is found on a pathogen. 2. The ligand / receptor specificity converter of claim 1, wherein said receptor is a bacterial adhesion receptor. The bacterial adhesion receptor includes extracellular fibrinogen binding protein (Efb), collagen binding protein, vitronectin binding protein, laminin binding protein, plasminogen binding protein, thrombospondin binding protein, aggregation factor A (ClfA), aggregation factor B 11. The ligand / receptor specific transformant of claim 10, wherein the transformant is selected from the group consisting of (ClfB), fibronectin binding protein, coagulase, and extracellular adhesion protein. 2. The ligand / receptor of claim 1, wherein the antigenic domain comprises at least three amino acids of a peptide selected from the group consisting of a herpes simplex virus protein, a hepatitis B virus protein, a TT virus protein, and a poliovirus protein. Body-specific converter. 13. The ligand / receptor specific transformant according to claim 12, wherein the antigenic domain is a herpes simplex virus protein comprising at least one sequence selected from the group consisting of SEQ ID NO: 53 and SEQ ID NO: 54. The ligand according to claim 12, wherein the antigenic domain is a hepatitis B virus protein comprising at least one sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 59. / Receptor specificity converter. The antigenic domain is at least one selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 13. The ligand / receptor specificity converter of claim 12, which is a TT virus protein comprising two sequences. 13. The ligand / receptor specific transformant according to claim 12, wherein the antigenic domain is a poliovirus protein comprising one sequence selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 51. 2. The ligand / receptor specificity converter of claim 1, wherein said antigenic domain interacts with a high titer antibody. 18. The ligand / receptor specific transformant of claim 17, wherein the antigenic domain specifically binds to antibodies present in animal sera diluted from about 1: 100 to 1: 1000 or more. The sequence of the ligand / receptor specificity transformant is SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, Sequence No. 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, Sequence No. 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, Column number 104, SEQ ID NO: 105, SEQ ID NO: 137, and is selected from the group consisting of SEQ ID NO: 142, ligand / receptor specificity converter according to claim 1. Providing a subject with a therapeutically effective amount of a ligand / receptor specific converter comprising a specificity domain having a ligand that interacts with a receptor on a bacterium and an antigenicity domain having an epitope of a pathogen or toxin. A method of treating or preventing a bacterial infection, including. Providing a subject with a therapeutically effective amount of a ligand / receptor specific converter comprising a specificity domain having a ligand that interacts with a receptor on a virus, and an antigenicity domain having an epitope of a pathogen or toxin. A method for treating or preventing a viral infection, including. Providing a subject with a therapeutically effective amount of a ligand / receptor specific converter comprising a specific domain having a ligand that interacts with a receptor on a cancer cell and an antigenic domain having a pathogen or toxin epitope A method for treating or preventing cancer.