JP2004524023A - Specific human antibodies for selective cancer therapy - Google Patents

Abstract

A Fe molecule, a constituent thereof, any fragment thereof, or a constituent of a fragment, having enhanced binding properties to selectively and / or specifically bind to a target cell in a manner that favors other cells. A peptide or polypeptide, wherein the binding selectivity or specificity is primarily determined by the first hypervariable region, and wherein the Fv is a scFv or dsFv, optionally having one or more tags. Is aimed at. Enhanced binding is substantially exposed on or in a target comprising one cell in a manner that favors other cells on or in which binding sites are substantially unavailable and / or not expressed. Directed and / or overexpressed binding site. The invention is further directed to methods for isolating such peptides and polypeptides from phage display libraries, and nucleic acid molecules encoding them. The present invention provides pharmaceutical compositions and kits for the diagnosis and treatment of diseases, particularly cancer, most particularly acute myeloid leukemia, comprising the peptide or polypeptide.

Description

【０１１５】
［１２９.］ リーダー配列は、破線で下線が入れられている。Ｖ_H 領域は太字のアミノ酸配列によってコードされる。この特異的クローンは、生殖細胞系列Ｖ_H３−ＤＰ３２から誘導されている。しかしながら各クローンの生殖細胞系統は、その特定の由来により左右される（以下参照）。囲み内のアミノ酸配列は、このライブラリに由来する全てのクローンの中の超可変領域であるＶ_H−ＣＤＲ３配列についてコードする。Ｖ_H及びＶ_L 領域と合流するスペーサ領域は、イタリック体で示されたアミノ酸によってコードされた柔軟なポリペプチドである。最後にＶ_L 領域が提示されている。全てのクローン内の融合Ｖ_L フラグメントは、生殖細胞系統ＩＧＬＶ３ＳＩの単一の突然変異を受けていないＶ遺伝子から誘導され、ここでは、波線により下線が付されたｃ−ｍｙｃタグが後に続いている。
【０１１６】
［１３０.］ Ｖ_Hフクグメントのレパートリ（４９の生殖細胞系統からの）がまず第１に、ライブラリ供給業者により免疫化されていないヒトの末梢血リンパ球の再配置されたＶ−遺伝子からＲＣＲによって生成された（「実験未使用レパートリ」と呼ぶ）。Ｖ_H−配列の起源（生殖細胞系統）を、以下のウェブサイトの１つを用いて相同性試験（Blast探索）によって同定することができる。
【０１１７】
［１３１.］ 複数の方法のうちの１つにおいて、抗体の結合特性を最適化することができる。もとのリード化合物に関するより高い結合親和性を得るために抗体を最適化する１つの方法は、リード化合物内のアミノ酸残基を置換してより高い可変性を導入するか又は配列を拡大することに基づいている。例えば、もとのＶ_L領域全体を、異なる抗体亜型からのＶ_L領域で置換することができる。
【０１１８】
［１３２.］ 結合親和性を最適化する付加的な方法は、ファージミド表示突然変異誘発ライブラリを構築することにある。ファージミド表示突然変異誘発ライブラリにおいては、Ｖ_H及びＶ_LＣＤＲ３内のコア配列の各アミノ酸が好ましくは当該技術分野において既知の保存的要領でその他のあらゆるアミノ酸により独立して置換されるような形で、オリゴヌクレオチドが合成される。本発明は、表示された抗体フラグメント及びファージビリオンから抽出可能な可溶性抗体フラグメントが同じ生物活性をもつ、ファージ上で表示された一組の特異的抗体ｓｃＦｖを提供している。
【０１１９】
［１３３.］ 本書で用いられるファージ表示ライブラリは、免疫化されていないヒトの末梢血リンパ球から構築されたものであり、Ｆｖペプチドは、標的細胞の表面上の以前に特徴づけされていない未精製抗原に対して選択された。本書で使用する以前に特徴づけされていない未精製の抗原という語は、現行の作業に先立つ生化学的又は分子手段によって、同定、特徴づけ、単離又は精製されておらず、かつ観察された単離された抗体フラグメントに対する選択的及び／又は特異的結合によって当該作業の中で観察されている又は予測されている細胞の表面上で提示されたリガンドを意味する。
【０１２０】
［１３４.］ 本発明のｓｃＦｖは、標的細胞に対する増強された結合を表示する。増強された結合は、特異的表面マーカーに向けられている。特異的表面マーカーというのは、細胞膜内で隔絶され循環する認識分子にアクセス可能な分子である。表面マーカーの存在は、本書に記述されているバイオパンニング技術を介したファージ表示技術の開発を可能にした。本発明においては、さまざまな細胞型を特徴づけしかつ区別するためならびにそのさまざまな形態でのＦｖｓのための結合部位として役立つために、特異的表面マーカーが利用される。その特徴的表面マーカーに従って、さまざまな造血細胞型を区別することができ、類似の形で、病的又は癌細胞は、それらの型及び期に独特である表面マーカーを表示する。
【０１２１】
［１３５.］ ｓｃＦｖクローンの選択は、２つの異なるバイオパンニング戦略により達成可能である：すなわち、
１．標的細胞として病的又は癌細胞を使用することによる直接的選択、及び
【０１２２】
２．第２状態にある第１の細胞の結合部位が、実質的に露呈又は表示されている未知のリガンドを含むことになる、第２の例えば活性化、励起、修飾、変更又は妨害を受けた状態にある第１の例えば正常な細胞を使用することになる、段階的選択。免疫交差反応性により、結果として得られたクローンは、その後のバイオパンニング又は選択段階の後に第２の細胞上に新規の及び未知のリガンドに対し選択的及び／又は特異的に結合することができる。さらなる任意の増幅及びその後の精製に続いて、ターゲティング分子を第２の細胞上の未知のリガンドに選択的及び／又は特異的な精製済み認識分子の認識部位から構築することができる。
【０１２３】
［１３６.］ 本発明の１実施形態においては、第１の細胞は正常細胞であり得、第１の状態は未活性化状態であり、第２の状態は、活性化、励起、修飾、変更又は妨害を受けた状態であり得る。段階的選択における第２の細胞は、ヒト細胞であり得る。本発明のもう１つの実施形態では、段階的選択における第２の細胞は、病的細胞である。より好ましい実施形態においては、段階的選択における第２の細胞は、制限的な意味なく癌腫、肉腫、白血病、線腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫といった癌腫である。より好ましい実施形態においては、第２の細胞は白血病細胞である。最も好ましい実施形態においては、第２の細胞は、ＡＭＬ細胞である。
【０１２４】
［１３７.］ 本発明のより好ましい実施形態は、第２の細胞のリガンドに対するその選択的及び／又は特異的結合が主として第１の超可変領域により決定されているペプチド又はポリペプチドを提供する。さらに一層好ましい実施形態においては、第１の超可変領域は、配列番号８−２４から成るグループの中から選択されたアミノ酸配列をもつＣＤＲ３領域である。
【０１２５】
［１３８.］ 本発明のもう１つの実施形態は、本発明のペプチド又はポリペプチドにより結合された第２の細胞のリガンドを提供している。さらなる実施形態は、本発明のペプチド又はポリペプチドにより結合されたリガンドを認識し結合するあらゆる分子を提供している。
【０１２６】
［１３９.］ 癌細胞に対する結合の増強は、正常細胞内での発現に比べた癌細胞内でのリガンドの過剰発現及び／又は結合部位の露呈に起因する可能性が最も高い。本書で使用されているように、リガンドの過剰発現という語は、特定の細胞型及び／又は特定の細胞サイクル期において通常サイレント化している遺伝子又はその産物の発現、又はこの特定の細胞型についての正常な非悪性条件下で基底レベルで発現される遺伝子の増大した発現として定義づけされる。
【０１２７】
［１４０.］ 本発明のさらに好ましい実施形態においては、バイオパンニング手順の標的細胞は、細胞懸濁液の中に含有されている。造血細胞は懸濁状態で得られ、血球懸濁液とファージライブラリを混合しその後いくつかの緩衝液で洗浄することによってバイオパンニングを実施することができる。ファージは、ヒト細胞から抽出され、増幅され、表示された抗体フラグメント配列が決定される。
【０１２８】
［１４１.］ 本発明のさらに一層好ましい実施形態においては、血球懸濁液は白血病細胞を含む。最も好ましい実施形態においては、血球懸濁液は、ＡＭＬ細胞を含む。対象の発明のもう１つの実施形態では、標的細胞は、単離された器官又はその一部から誘導される。
【０１２９】
［１４２.］ 対象の発明のもう１つの実施形態においては、標的細胞又は第２の細胞は、１つの細胞系統から誘導されている。細胞系統は、Ｆｖクローンの結合特性を容易に決定できるように培養され操作され得る。さらに、細胞系統は、診断キットの開発においても有用であり得る。
【０１３０】
［１４３.］ 好ましい実施形態においては、細胞系統は、制限的意味なく、ジャーカット、Molt-４、ＨＳ−６０２、Ｕ９３７、ＴＦ−１、ＴＨＰ−１、ＫＧ−１、ＭＬ−２及びＨＵＴ−７８細胞系統といったような造血細胞系統である。
【０１３１】
［１４４.］ 本発明の好ましい実施形態では、ＣＤＲ３領域は、８４のカセット（配列番号３０−１１３）のうちのいずれか１つの中に又はその上に構築、挿入、カップリング又は融合される。より好ましい実施形態では、ＣＤＲ３領域は、４９のカセット（配列番号：３０−３２、３５、３７−３９、４１、４３、４５、４６、４８、５１、５４、５７、５９−６８、７０、７１、７６−８５、８７、８９−９２、９４、９７、９９、１０３、１０６、１１２及び１１３のうちの１つの中又は上に構築、挿入、カップリング、又は融合される。最も好ましい実施形態においては、ＣＤＲ３は、配列番号６１のカセット又はそれと少なくとも９０％の配列類似性をもつ上述の配列のいずれかのＣ末端に構築、挿入、カップリング又は融合されている。
【０１３２】
［１４５.］ １つの実施形態においては、カセットのアミノ酸配列は明示的に固定されており、一方、置換、挿入又は付着された配列は高度に可変的であり得る。カセットは、各々が最終構成体にとって重大な機能を包含する複数のドメインで構成され得る。本発明の特定の実施形態のカセットは、Ｎ末端から枠組領域１、（ＦＲ１）、ＣＤＲ１、枠組領域２（ＦＲ２）、ＣＤＲ２及び枠組領域３（ＦＲ３）を含んで成る。
【０１３３】
［１４６.］ 本発明の１実施形態においては、カセット内で全く異なる領域を置換することが可能である。例えば、カセットのＣＤＲ２及びＣＤＲ１の超可変領域は、非保存的又は好ましくは保存的アミノ酸置換によって置換又は修飾可能である。より特定的には、配列番号３０〜１１３又はそのフラグメントから成るグループの中から選択された連続するアミノ酸のカセットのＣＤＲ２及びＣＤＲ１領域は、それぞれ配列番号１１５及び１１４によって置換され得る。さらに一層特定的には、配列番号３０−３２、３５、３７−３９、４１、４３、４５、４６、４８、５１、５４、５７、５９−６８、７０、７１、７６−８５、８７、８９−９２、９４、９７、９９、１０３、１０６、１１２及び１１３又はそれらのフラグメントから成るグループの中から選択された連続するアミノ酸のカセットのＣＤＲ２及びＣＤＲ１領域がそれぞれ配列番号１１５及び１１４によって置換され得る。
【０１３４】
［１４７.］ 発明の好ましい実施形態においては、ペプチド又はポリペプチドは、重鎖及び軽鎖を含み、各鎖は、それぞれＣＤＲ３、ＣＤＲ２、及びＣＤＲ１領域である第１、第２及び第３の超可変領域を含む。結合の選択性及び特異性は、特に１つの鎖のＣＤＲ３領域によって、可能な場合には軽鎖のＣＤＲ３領域によって又好ましくは重鎖のＣＤＲ３領域によって決定され、二次的には、軽鎖好ましくは重鎖のＣＤＲ２及びＣＤＲ１領域によって決定される。結合選択性及び特異性は同様に、第１、第２及び／又は第３の超可変領域をフランキングする上流側又は下流側領域によっても二次的に影響され得る。
【０１３５】
［１４８.］ 好ましい実施形態においては、ペプチド又はポリペプチドのＣＤＲ３領域は、配列番号８〜２４から成るグループの中から選択されたアミノ酸配列を有する。
【０１３６】
［１４９.］ より好ましくは、重鎖のＣＤＲ３領域は、配列番号８〜２４を含むグループの中から選択された１つのアミノ酸配列を有し、ＣＤＲ２は配列番号１１５と同一のアミノ酸配列をもち、ＣＤＲ１領域は配列番号１１４と同一のアミノ酸配列を有する。
【０１３７】
［１５０.］ 本発明の最も好ましい実施形態においては、ＣＤＲ３領域は、配列番号８と同一のアミノ酸配列を有する。
【０１３８】
［１５１.］ 重鎖及び軽鎖に加えて、Ｆｖは、０〜２０個のアミノ酸残基の柔軟なスペーサを含む。スペーサは、分枝鎖又は重鎖であり得る。スペーサの２つの可能な配列は、配列番号１２３及び１２４と同一である。
【０１３９】
［１５２.］ 本発明の好ましい実施形態は、配列番号８と同一のＣＤＲ３配列及び配列番号２５と同一の完全ｓｃＦｅ 配列を伴うｓｃＦｖである。
【０１４０】
［１５３.］ 本発明のもう１つの好ましい実施形態は、配列番号２０と同一のＣＤＲ３配列及び配列番号２０３と同一の完全ｓｃＦｖ 配列を伴うｓｃＦｖである。
【０１４１】
［１５４.］ 本発明の最も好ましい実施形態においては、ＣＤＲ３、ＣＤＲ２及びＣＤＲ１領域はそれぞれ配列番号８、１１５及び１１４のアミノ酸を有する。
【０１４２】
［１５５.］ 本発明の１実施形態においては、Ｆｖペプチドは、それ自体、配列番号３０−１１３を含むグループの中から選択されたアミノ酸配列を伴うカセットを含む可変重鎖のＣＤＲ１及びＣＤＲ２領域；配列番号８−２４を含むグループの中から選択されたアミノ酸配列をもつ好ましくは可変重鎖のＣＤＲ３領域；配列番号１１７のアミノ酸配列をもつＣＤＲ３領域をフランキングする上流側領域；配列番号１１６のアミノ酸配列をもつＣＤＲ３領域をフランキングする下流側領域；配列番号１２３又は１２４の０〜２０個のアミノ酸残基のスペーサ；配列番号７という配列をもつ可変軽鎖を含んで成る。
【０１４３】
［１５６.］ 同様にして、もう１つの実施形態においては、ＣＤＲ２領域にフランキングする上流側領域は、配列番号１１９というアミノ酸配列を有し、ＣＤＲ２領域にフランキングする下流側領域は配列番号１１８というアミノ酸配列を有し、ＣＤＲ１領域をフランキングする上流側領域は配列番号１２１というアミノ酸配列を有し、ＣＤＲ１領域をフランキングする下流側領域は、配列番号１２０というアミノ酸配列を有する。
【０１４４】
［１５７.］ 本発明の好ましい実施形態は、第２及び第３の超可変領域がそれぞれＣＤＲ２及びＣＤＲ１の超可変領域であり、ＣＤＲ３アミノ酸配列が配列番号８であり、ＣＤＲ２アミノ酸配列が配列番号１１５であり、ＣＤＲ１アミノ酸配列が配列番号１１４であり、ＣＤＲ３領域をフランキングする上流側領域が配列番号１１７のアミノ酸配列を有し、ＣＤＲ３領域をフランキングする下流側領域が配列番号１１６のアミノ酸配列を有し、ＣＤＲ２領域をフランキングする上流側領域が配列番号１１９のアミノ酸配列を有し、ＣＤＲ２領域をフランキングする下流側領域が配列番号１１８のアミノ酸配列を有し、ＣＤＲ１領域をフランキングする上流側領域が配列番号１２１のアミノ酸配列を有し、ＣＤＲ１ 領域をフランキングする下流側領域が配列番号１２０のアミノ酸配列を有するペプチド又はポリペプチドを提供している。
【０１４５】
［１５８.］ 本発明のもう１つの実施形態は、第１、第２及び第３の超可変領域をもつ第１の鎖及び第１、第２及び第３の超可変領域をもつ第２の鎖を含んで成るＦｖ分子において、第１鎖の超可変領域のうちの１つが、配列番号８〜２４から成るグループの中から選択された１つの配列をもち、第２鎖の超可変領域のうちの１つが、配列番号１〜６及び１２５〜２０２から成るグループの中から選択された１つの配列を有し、第１、第２及び第３の超可変領域は、それぞれＣＤＲ３、ＣＤＲ２及びＣＤＲ１領域であり、ＦｖはｓｃＦｖ又はｄｓＦｖであり、任意には単数又は複数のタグを有するＦｖ分子を提供している。
【０１４６】
［１５９.］ 本名発明のもう１つの実施形態は、（ｉ）第１鎖及び第２鎖が各々、配列番号８〜２４から成るグループの中から選択された第１の超可変領域を含んで成るか；又は（ｉｉ）第１鎖の第１の超可変領域及び第２鎖の第１の超可変領域が同一であり、配列番号８〜２４から成るグループの中から選択されているか；又は（ｉｉｉ）第１鎖の第１の超可変領域が配列番号８〜２４から成るグループの中から選択されており、かつ第２鎖の第１の超可変領域が配列番号１〜６及び１２５〜２０２から成るグループの中から選択されているか；又は（ｉｖ）第１鎖の第１の超可変領域が、配列番号１〜６及び１２５〜２０２から成るグループの中から選択され、第２鎖の第１の超可変領域が配列番号８〜２４から成るグループの中から選択されている、ペプチド又はポリペプチドを提供している。
【０１４７】
［１６０.］ もう１つの実施形態は、第１鎖の第２及び第３の超可変領域がそれぞれ配列番号１１４及び１１５である、本発明のペプチド又はポリペプチドを提供する。
【０１４８】
［１６１.］ 本書に記述され詳細に説明されているアミノ酸残基２５個以下の全てのアミノ酸配列について、これらのアミノ酸配列がその範囲内で１個又は２個のアミノ酸置換を内含することそして好ましくは置換が保存的アミノ酸置換であることが、該発明のさらなる実施形態として理解され考慮されるものとする。本書に記述され詳細に説明されているアミノ酸残基が２５個より多い全てのアミノ酸配列について、これらのアミノ酸配列がもとの配列に対し９０％以上の配列類似性をもつアミノ酸配列をその範囲内に内含することが該発明のさらなる実施形態として理解され考慮されるものとする（Altschul et al., Nucleic Acids Res., ２５、３３８９−３４０２（１９９７））。類似の又は相同なアミノ酸というのは、例えば酸性、塩基性、芳香族、サイズ、正又は負に帯電した、極性、非極性といった類似の特性を示す同一でないアミノ酸として定義づけされる。
【０１４９】
［１６２.］ アミノ酸の類似性又は相同性又は配列の類似性の百分率は、２つの異なるペプチド又はポリペプチドのアミノ酸配列を比較することによって決定される。２つの配列は、通常専用に設計されたさまざまなコンピュータプログラムの１つを使用することによって整列させられ、各位置におけるアミノ酸残基が比較される。次に、アミノ酸の同一性又は相同性が決定される。その後、アミノ酸類似性百分率を決定するためにアルゴリズムが適用される。ペプチド、ポリペプチド又はタンパク質分子の間の微妙な関係の検出に対する感度が大幅に増大したことから、一般にアミノ酸配列を比較することが好ましい。タンパク質比較には、保存的アミノ酸置換の存在を考慮に入れることができ、こうして、同一でないアミノ酸が類似の物理的及び／又は化学的特性を有する場合、１つの不整合がなお正の評点を生み出す可能性がある（Altschul et al., Nucleic Acids Rs., ２５，３３８９−３４０２（１９９７））。
【０１５０】
［１６３.］ 該発明の１実施形態においては、軽鎖及び重鎖の各々の３つの超可変領域は２つの鎖の間及び鎖内部及び／又は鎖間の３つの超可変部位の間で相互交換され得る。
【０１５１】
［１６４.］ 当業者であれば、本発明のペプチド又はポリペプチドの特異的及び／又は選択的な結合の実証には、適切な負の対照の使用が必要である、ということに気づくことだろう。１つの適切な負の対照は、本発明のペプチド又はポリペプチドとほぼ同一であるが超可変ＣＤＲ３領域内に唯一の差異があるアミノ酸配列をもつペプチド又はポリペプチドでありうる。もう１つの適切な負の対照は、本発明のペプチド又はポリペプチドと同じサイズ及び／又は全体的３次元構造をもつものの完全に無関係のアミノ酸配列を有するペプチド又はポリペプチドでありうる。もう１つの適切な負の対照は、本発明のペプチド又はポリペプチドと比べた場合に全く異なる物理的及び化学的特性をもつペプチド又はポリペプチドであり得る。本発明の展開において使用される負の対照は、配列番号２８と同一のＣＤＲ３配列をもつＮ１４、及び配列番号２９と同一のＣＤＲ３配列をもつＣ１８１と呼称される。しかしながら、その他の負の対照も同様に適切であり得る。
【０１５２】
［１６５.］ もう１つの実施形態は、本発明のＦｖペプチド又はポリペプチドをコードする核酸分子好ましくはＤＮＡ分子を提供する。
【０１５３】
［１６６.］ 本発明の好ましい実施形態においては、Ｆｖの選択的結合を最適化するため、Ｆｖに対する主要な結合選択性及び／又は特異性を付与するＣＤＲ３配列を、任意のその他の重鎖生殖細胞系列まで移動させることができる。より特定的には、これらを８４の可能な重鎖生殖細胞系列のうちの１つまで移動させることができる。これらの８４の生殖細胞系列（配列番号：３０−１１３）には、（ａ）請求対象のファージクローンが当初単離された生殖細胞系列、（ｂ）ファージ表示ライブラリ内で利用可能な４８の付加的な生殖細胞系列及び（ｃ）本書で請求されている３５個の代替的生殖細胞系列が含まれる（Tomlinson et al., J. Mol.Biol., ２２７（３）：７７６−７９８（１９９２））。ＣＤＲ３領域自体に合わせて、ＣＤＲ３領域の局所的線形又は３次元環境は、適正なＣＤＲ３結合を誘導又は推進する上で潜在的に１つの役割を果たす可能性がある。例えば本書で配列番号８〜２４、１２５として記され４９の生殖細胞系列配列（配列番号：３０〜３２、３５、３７−３９、４１、４３、４５、４６、４８、５１、５７、５９−６８、７０、７１、７６−８５、８７、８９−９２、９４、９７、９９、１０３、１０６、１１２及び１１３）のいずれかから誘導されたＣＤＲ３配列のいずれかをもつペプチドも同様に、対象の発明により包含されている。
【０１５４】
［１６７.］ 生殖細胞系列ＤＰ−３２は、本発明の複数のクローンのためのカセットである。この生殖細胞系列のＣ末端は、ファージ表示ライブラリの調製を助けるためコンセンサス配列で置換された。配列番号６１の７個のカルボキシ末端アミノ酸は、配列番号１２２の７個のアミノ酸配列により置換された。
【０１５５】
［１６８.］ 本発明のＦｖｓのＣＤＲ３領域は、ＡＭＬ細胞に特異的に結合するコア配列Ａｒｇ／Ｇｌｙ／Ｌｙｓ Ｐｈｅ Ｐｒｏを含有し得る。かかるＣＤＲ３領域の８つの例が表２に提示されている。モチーフは、各ケースにおいてＣＤＲ３領域の３つのＮ末端アミノ酸残基と一致するが、これをＣＤＲ３領域内の他の場所に位置設定することも可能である。代替的には、モチーフは、より大きな結合又はターゲティング又は認識分子の一部分のアンカー又は結合領域を構築する又は構成するために用いられるか又は単独で標的ビヒクルとして使用される結合モチーフである。
【０１５６】
［１６９.］ 本発明のさらなる実施形態においては、Ｒ₁及びＲ₂の各々が０〜１５個、好ましくは１〜９個のアミノ酸残基を含みＸがＡrg、Ｇly又はＬysのいずれかであるＲ₁−Ｘ Ｐhe Ｐro−Ｒ₂のアミノ酸配列を含んで成る、結合モチーフが提供されている。最も好ましくは、ＣＤＲ３は、Ｒ₁−ＸＰhe Ｐro−Ｒ₂のアミノ酸配列を含み、ここでＲ₁及びＲ₂が各々０〜１５個のアミノ酸残基を含み、ＸがＡrg、Ｇly、又はＬysのいずれかである。
【０１５７】
［１７０.］ 対象の発明のペプチド又はポリペプチドのもう１つの好ましい実施形態においては、ペプチドがその生物活性を維持する一方で、ペプチドのＣ末端又はＮ末端のいずれかに対して１〜１０００個のアミノ酸を付加することができる。本発明の好ましい実施形態においては、ペプチドがその生物活性を維持する一方で、ペプチド又はポリペプチドのＣ末端又はＮ末端のいずれかに対し１５０〜５００個のアミノ酸を付加することができる。本発明のもう１つの好ましい実施形態においては、ペプチド又はポリペプチドがその生物活性を維持する一方で、ペプチド又はポリペプチドのＣ末端又はＮ末端のいずれかに対し８００〜１０００個のアミノ酸を付加することができる。
【０１５８】
［１７１.］ コアアミノ酸配列を拡張するための例としては、Ｉｇのコアとしてリード化合物を用いて、フルサイズの免疫グロブリンＩｇを構築することによるものがある。フルサイズのＩｇは、細胞の細胞分解活性の活性化又は補体を介して内因性細胞分解活性を誘発することのできる免疫グロブリンクラスに属する可能性がある（例えばＩｇＧ１、ＩｇＧ２又はＩｇＧ３）。フルサイズのＩｇは、強く結合する抗体の免疫グロブリンクラスに属する可能性もある（例えばＩｇＧ４）。結合時点で、フルサイズのＩｇは、例えば免疫応答を開始するべく体の防御メカニズムのためのフラグとして作用することによって、細胞内細胞シグナリングを形質導入することによって又は標的細胞に対する損傷をひき起こすことによってといった数多くのやり方のうちの単数又は複数のやり方で作用し得る。
【０１５９】
［１７２.］ 本発明の１つの好ましい実施形態は、組換え型ポリペプチドとして発現され真核細胞系統内で産生されたＩｇ分子を提供している。本発明の好ましい１実施形態においては、ＩｇポリペプチドはＩｇＧポリペプチドであり、これは哺乳動物細胞系統内で産生される。より好ましい実施形態においては、哺乳動物細胞系統は、ＣＭＶプロモータを含んで成る。
【０１６０】
［１７３.］ 本発明の好ましい実施形態においては、ＩｇＧ分子は、軽鎖及び重鎖の両方において、ＣＤＲ３、ＣＤＲ３及びＣＤＲ１の超可変領域を含む。本発明のより好ましい実施形態においては、Ｆｖ分子は、それぞれ配列番号８、１１５及び１１４をもつＣＤＲ３、ＣＤＲ２及びＣＤＲ１領域を含む。ＣＤＲ３、ＣＤＲ２及びＣＤＲ１領域は、重鎖及び軽鎖のものであり得る。
【０１６１】
［１７４.］ 本発明のさらに好ましい実施形態は、配列番号２７と同一の配列を伴う軽鎖、配列番号２６と同一の配列を伴う重鎖又はそれらと少なくとも９０％の配列類似性をもつ重鎖及び軽鎖を有するＩｇＧ分子を提供している。本発明の最も好ましい実施形態においては、ＩｇＧの２つの重鎖は同一であり、ＩｇＧの２つの軽鎖は同一である。
【０１６２】
［１７５.］ もう１つの実施形態においては、対象の発明のペプチドは、多価のＦｖ形態へと折畳むように構築される。
【０１６３】
［１７６.］ 本発明は、ｓｃＦｖ分子を含むＹ１又はＹ１７のペプチド又はポリペプチドを提供する。本書で使用されているｓｃＦｖというのは、同じものであっても、異なるものであってもよいヒト抗体の重鎖の可変領域及びヒト抗体の軽鎖の可変領域で構成され、かつその中で重鎖の可変領域が軽鎖の可変領域に対し連結、リンク、融合又は共有結合により付着されているか又はそれと会合されている、分子として定義づけされる。
【０１６４】
［１７７.］ Ｙ１及びＹ１７ｓｃＦｖ構成体は、Ｙ１又はＹ１７の抗体の超可変ドメインの単数又は複数を包含するｓｃＦｖ分子の多量体（例えば二量体、三量体、四量体など）であり得る。全てのｓｃＦｖ由来の構成体及びフラグメントは、その他の細胞に有利な形で標的細胞に対し選択的及び／又は特異的に結合するべく増強された結合特性を保持している。結合選択性及び／又は特異性は、超可変領域により主として決定される。
【０１６５】
［１７８.］ 軽鎖及び重鎖の可変ドメイン内の超可変ループは、相補的決定領域（ＣＤＲ）と呼ばれる。重鎖及び軽鎖の各々の中にはＣＤＲ１、ＣＤＲ２及びＣＤＲ３領域が存在する。これらの領域のうちの最も可変的なものは、重鎖のＣＤＲ３領域である。ＣＤＲ３領域は、Ｉｇ分子の最も露呈された領域であるものと理解され、本書で規定されている通り、観察された選択的及び／又は特異的結合を主として担っている部位である。
【０１６６】
［１７９.］ 対象の発明のＹ１及びＹ１７ペプチドは、多価のＦｖ形態へと折畳むように構築され得る。Ｙ１及びＹ１７多量体形態が、結合親和性及び特異性及び血中半減期の増大を改善するように構築された。
【０１６７】
［１８０.］ その他の研究者により、ｓｃＦｖの多価形態が産生されてきた。１つのアプローチは２つのｓｃＦｖをリンカーとリンクさせることであった。もう１つのアプローチには、リンケージのために２つのｓｃＦｖｓ間のジスルフィド結合を使用することが関与している。二量体又は三量体Ｆｖの産生に対する最も単純なアプローチは、Holliger et al., ＰＮＡＳ，９０，６４４４−６４４８（１９９３）及びA.Kortt, et al., Protein Eng., １０，４２３−４３３（１９９７）により報告された。かかる方法の１つは、ＦＯＳ及びＪＵＮタンパク質領域の配列を付加してｓｃＦｖのＣ末端でその間にロイシンジッパーを形成することによりｓｃＦｖｓの二量体を作るように設計された。Kostelny SA et al., J Immunol. １９９２ Mar １；１４８（５）：１５４７−５３；De Kruif J et al., J Biol Chem. １９９６ Mar２９；２７１（１３）：７６３０−４。もう１つの方法は、ｓｃＦｖのＣ末端でストレプトアビジンコーディング配列を付加することによって四量体を作るように設計された。ストレプトアビジンは、４つのサブユニットで構成されており、従ってｓｃＦｖストレプトアビジンが折畳まれた時点で、４つのサブユニットが自らを収容して四量体を形成する。Kipriyanov SM et al., Hum Antibodies Hybridomas, １９９５；６（３）；９３−１０１。さらにもう１つの方法においては、二量体、三量体及び四量体を作るため、問題のタンパク質の中に遊離システインが導入される。問題のタンパク質を遊離システインに架橋するため、可変的数（２〜４）のマレイミド基を伴うペプチドベースの架橋剤が使用された。Cochran JR et al., Immunity, ２０００Mar；１２（３）：２４１−５０。
【０１６８】
［１８１.］ この系ではファージライブラリ（本書で以上に記述されている通りのもの）は、１つの抗体のＦｖ領域の１価形態へと折畳むことのできるｓｃＦｖｓを表示するように設計された。さらに、同じく本書に以上で論述されている構成体は、細菌発現のために適している。遺伝子工学処理されたｓｃＦｖｓは、隣接してコードされた１５個のアミノ酸の柔軟なペプチドスペーサによって結合された重鎖及び軽鎖の可変領域を含む。好ましいスペーサは（Ｇｌｙ₄Ｓｅｒ）₃である。このスペーサの長さは、そのアミノ酸構成体要素と共に、嵩高でないスペーサを提供し、これによりＶＨ及びＶＬ領域は、その標的に対する有効な結合を提供する機能的Ｆｖドメインへと折畳むことができる。
【０１６９】
［１８２.］ 本発明は、当該技術分野における既知のあらゆる方法によって調製されたＹ１及びＹ１７の多量体に向けられている。多量体特に二量体を形成する好ましい方法は、２つの単量体の間にジスルフィド結合を形成するべくシステイン残基を利用する。この実施形態においては、二量体は、二量体形成を容易にするべく（Ｙ１−ｃｙｓ ｓｃＦｃ又はＹ１二量体と呼ばれる）ｓｃＦｖｓのカルボン末端上にシステインを付加することによって形成される。ＤＮＡ構成体が作られ（例２Ｄ及び６Ｄ参照）トランスフェクションのために使用された後、Ｙ１二量体が産生ベクター内で発現され、in vitroで再度折畳まれた。タンパク質は、ＳＤＳ−ＰＡＧＥ、ＨＰＬＣ及びＦＡＣＳによって分析された。抗体の２リットル発酵バッチが行なわれた。E. coli菌株Ｂ２１内でＹ１〜ｃｙｓを発現させた後、再折畳みがアルギニン内で行なわれた。再折畳みの後、タンパク質は、透析され、Ｑ−セファロース及びゲルろ過（sephadex７５）により精製された。２つのピークがＳＤＳ−ＰＡＧＥ（未還元）及びゲルろ過によって検出された。ピークは別々に収集され、ＦＡＣＳにより分析された。ジャーカット細胞に対する単量体及び二量体の結合がＦＡＣＳによって検査された。二量体による結合には、同じ染色レベルについて単量体抗体の量が１００分の１しか必要とされず、これは、二量体がより大きな結合力を有することを表わしていた。二量体再折畳みのための条件が決定され、９０％を上回る二量体（mg数量）を含む材料が、その後の透析、クロマトグラフィ及びゲルろ過段階の後に産生された。精製済み二量体は、酸化条件下でゲルろ過及びＳＤＳ−ＰＡＧＥ分析によって特徴づけされた。ラジオレセプタアッセイ、ＥＬＩＳＡ及びＦＡＣＳ分析により、二量体の結合能力が確認された。
【０１７０】
［１８３.］ ＣＯＮＹ１ ｓｃＦ抗体フラグメントがＹ１ｓｃＦｖから誘導される。Ｙ１ｓｃＦｖのｍｙｃタグをコードするＤＮＡ 配列は除去され、アミノ酸リジン、アラニン、リジン（ＫＡＫ）をコードする合成オリゴヌクレオチドＤＮＡ配列により置換された。
【０１７１】
［１８４.］ Ｙ１二量体と（ＣＯＮＹ１とも呼ばれる）ｓｃＦｖ単量体の結合を比較するため、ＫＧ−１細胞についてin vitroで結合競合実験が行なわれた。さらに、これらの実験は、全ＹＩＩｇＧのｓｃＦｖＹ１単量体に対する結合を比較した。この研究を実施するために、Ｙ１ ＩｇＧ Ｙ１ ＩｇＧはビオチンで標識づけされた。この研究は、Ｙ１ＩｇＧがＩＧＹコービオチンと競合したことを明らかにした。関連性のないヒトＩｇＧは、標識づけされたＹ１ ＩｇＧと競合しなかった。Ｙ１ｓｃＦｖｓ（５μｇ及び１０μｇ）は、Ｙ１ ＩｇＧ−Biotin（５０ng）と部分的に競合した。研究は同様に、１ｎｇのＩｇＧ Ｙ１−ＦＩＴＣが、１μｇのｓｃＦｖ ＦＩＴＣと同じ程度までＫＧ−１細胞に（血清無しで）結合したが、血清が存在する場合、大部分のＹ１ ＩｇＧ結合は「遮断される」ということをも示した。これらの研究は同様に、ラジオレセプタアッセイ、ＥＬＩＳＡ又はＦＡＣＳによって分析されるように、Ｙ１二量体の結合が少なくともｓｃＦｖ単量体のものより少なくとも２０倍高いことも示した。
［１８５.］ さらにもう１つの実施形態においては、（ＹＩ−ｃｙｓ−ｋａｋ ｓｃＦｖと呼ばれる）カルボキシ末端におけるシステインに加えて、リジン−アラニン−リジンが加えられた。このｓｃＦｖ構成体のアミノ酸配列は以下に再現されている。
【０１７２】
【化２】

【０１７３】
［１８６.］ ＹＩ−ｃｙｓ−ｋａｋは、細菌中のλ−ｐＬベクター内で産生された。４２℃まで温度を上昇させることよってλ−ｐＬベクター内の発現が誘発された。誘発された培養から封入体が得られ、望まれない可溶性タンパク質を除去するべく水溶液により半精製された。この封入体はグアニジンの中で可溶化され、ＤＴＴにより還元され、アルギニン／ｏｘ−グルタチオンに基づく溶液中でin vitroで再度折畳まれた。再折畳み後、タンパク質は透析され、尿素／リン酸緩衝液を含む緩衝液に対して接線方向流でのろ過により濃縮された。タンパク質は、ＳＰ−カラム中のイオンクロマトグラフィにより再精製され濃縮された。
【０１７４】
［１８７.］ ＣＯＮＹ１ ｓｃＦｖ中ならびにＹ１−ｃｙｓ−ｋａｋ ｓｃＦｖ中でより高レベルの発現を得るために、我々は、ｓｃＦｖ構成体のＮ末端配列の位置２に、アラニン残基についてコードするアミノ酸を導入した。この新たに修飾された構成体で、４倍の発現レベルが得られた。
【０１７５】
［１８８.］ 血小板から誘導された抗原ＧＰＩｂ（グリコカリシン）について、単量体（ＣＯＮＹｓｃＦｖ−ＹＩ−ｋａｋとしても知られている）と二量体ＹＩ−ｃｙｓ ｋａｋ（システイン二量体）の間の結合の差を確認するべく、ＥＬＩＳＡ検定が実施された。ＧＰＩｂに対する結合を検出するために、ポリクローナル抗１本鎖抗体及び／又は新規のポリクローナル抗−Ｖ_L（ウサギ由来）及び抗ウサギＨＲＰが使用された。二量体は、単量体より２〜１００倍高い活性を示した。例えば、０．８ ＯＤ単位を達成するために、わずか０．１mg／mlの二量体に比べて、１２．８mg／mlの単量体が使用された。図１２を参照のこと。
【０１７６】
［１８９.］ 二量体は、ＳＤＳ−ｐａｇｅ電気泳動、ゲルろ過クロマトグラフィ、ＥＬＩＳＡ、ラジオレセプタ結合及びＦＡＣＳにより特徴づけされた。二量体の見かけの親和性は、結合活性力に起因して、単量体よりも高いものであった。この効果は、グリコカリシンに対するＥＬＩＳＡ、ＫＧ−１細胞に対するＦＡＣＳ及びラジオレセプタアッセイ内での競合により確認された。
【０１７７】
［１９０.］ Superdex７５ゲルろ過カラムからの再折畳み及び精製後の二量体のプロファイルを描くため、ＨＰＬＣが実施された。図１０では、Ｙ１−ｃｙｓ−ｋａｋ（二量体）は、左側で最初のピーク（〜１０．８分）であり、その後のピークは単量体（〜１２分）である。同じカラム上でランさせたタンパク質サイズマーカーに従うと、二量体は約５２ｋＤａで、単量体は２６ｋＤａである。二量体と単量体の間のバランスは、再折畳み条件（酸化された作用物質の濃度及び再折畳み緩衝液中のタンパク質の濃度）を変動させることによって変更できる。二量体及び単量体は、super-dex７５カラム内のクロマトグラフィにより分離された。
【０１７８】
［１９１.］ 図１１では、二量体及び単量体の混合集団を伴うゲルが示されている。還元された形態では、２つの単量体間の還元に起因して、単量体が見られ、未還元形態では（ゲルろ過実験の場合のように）２つの集団、すなわち約３０ｋＤａの単量体画分と約６０ｋＤａの二量体が見られる。
【０１７９】
［１９２.］ さらに、ＫＧ−１細胞に対するＦＡＣＳ結合分析は、２又は３段階結合検定が実施された時点で、二量体が単量体よりもさらに感応性が高いということを示した。ＦＩＴＣによって直接標識づけされた二量体は、単量体よりもわずかに利点を示した（材料使用量が約１０分の１少ない）。二量体を競合物質として用いたＫＧ−１細胞上のラジオレセプタアッセイは、二量体が単量体よりも３０倍高い効率をもつことを示した。
【０１８０】
［１９３.］ スペーサの長さを変動させることは、二量体、三量体及び四量体を形成するさらにもう１つの好ましい方法である（当該技術分野において往々にしてそれぞれ、diabodies, triabodies及びtetrabodiesと呼ばれる）。ｓｃＦｖの２つの可変鎖をつなぐスペーサが一般的に＜？＞まで短縮される条件の下で、二量体が形成される。この短縮されたスペーサは、同じ分子からの２つの可変鎖が１つの機能的Ｆｖドメイン内に折畳むのを防ぐ。その代り、該ドメインはもう１つの分子の相補的ドメインと強制的に対合されて２つの結合ドメインを作り出す。好ましい方法では、diabody構築のために、わずか５個のアミノ酸（Ｇｌｙ₄Ｓｅｒ）のスペーサが使用された。この二量体は、２つの同一のｓｃＦｖｓからか又は２つの異なるｓｃＦｖｓ集団から形成され得、親ｓｃＦｖ(ｓ)の選択的及び／又は特異的な増強された結合活性を保持しかつ／又は増大した結合強度又は親和性を示すことができる。
【０１８１】
［１９４.］ 類似の要領で、ｓｃＦｖの２つの可変鎖をつなぐスペーサが一般的に５個未満のアミノ酸残基まで短縮され、同じ分子からの２つの可変鎖が１つの機能的Ｆｖドメインへと折畳むのを防ぐ条件下で、triabodiesが形成される。その代り、３つの分離したｓｃＦｖ分子が会合して三量体を形成する。好ましい方法においては、この柔軟なスペーサを完全に除去することによって、triabodies が得られた。triabodyは３つの同一のｓｃＦｖｓからか又は２つ又は３つの異なるｓｃＦｖｓ集団から形成され得、親ｓｃＦｖ(ｓ)の選択的及び／又は特異的な増強された結合活性を保持しかつ／又は増大した結合強度又は親和性を示すことができる。
【０１８２】
［１９５.］ 類似の要領で、ｓｃＦｖの２つの可変鎖をつなぐスペーサが一般的に５個未満のアミノ酸残基まで短縮され、同じ分子からの２つの可変鎖が１つの機能的Ｆｖドメインへと折畳むのを防ぐ条件下で、tetrabodiesが形成される。その代り、４つの分離したｓｃＦｖ分子が会合して四量体を形成する。tetrabodyは４つの同一のｓｃＦｖｓからか又は異なるｓｃＦｖｓ集団からの１〜４個の個々の単位から形成され得、親ｓｃＦｖ(ｓ)の選択的及び／又は特異的な増強された結合活性を保持しかつ／又は増大した結合強度又は親和性を示すはずである。
【０１８３】
［１９６.］ スペーサの長さが一般にアミノ酸残基５個未満である条件下でtriabodies又はtetrabodiesのいずれが形成されるかは、混合物内の特定のｓｃＦｖ(ｓ)のアミノ酸配列及び反応条件により左右される。
【０１８４】
［１９７.］ 好ましい方法においては、ビオチン／ストレプトアビジン会合を介して四量体が形成される。（本書でＹ１−biotag又はＹ１−Ｂと呼ばれている）ビオチンで酵素的に標識づけされる能力をもつ新規の発酵構成体が作り出された。ＢirＡ酵素のための基質である配列が、Ｙ１Ｃ−末端で付加された。ＢirＡ酵素は、この配列内でリジン残基に対しビオチンを付加する。Ｙ１−biotagはE.coli中でクローニングされ、発現された。封入体材料は単離され、再度折畳まれた。折畳まれたタンパク質の純度は９５％を上回り、１−Ｌ培養（小規模で最適化されていない条件）から１００mgを上回る純度が得られた。この形態の分子量は、ＨＰＬＣ、ＳＤＳ−ＰＡＧＥ及び質量分光法に従って、ｓｃＦｖのものと類似していることがわかった。Ｙ１−biotagは、ＦＡＣＳ分析のために最も一貫性ある試薬であることが発見された。しかしながら、ＫＧ−１細胞に結合するＹ１−biotagが血清の存在下で検査された場合、血清不在下での匹敵する結合のために高い濃度（１０倍以上）が必要とされる。それでも、この構成体は、分子の結合部位が無傷の状態にとどまっている特異的ビオチニル化という利点を提供した。さらに、各々の分子は、１つのビオチンのみで標識づけされ、各分子はカルボキシ末端上で１つのビオチンを受け取る。
【０１８５】
［１９８.］ 標識づけを所望の場所で１つのビオチン／分子に制限することで、ストレプトアビジンでの四量体の産生が可能となった。四量体は、ストレプトアビジン−ＰＥでＹ１−Ｂをインキュベートすることによって形成された。
【０１８６】
［１９９.］ ＦＡＣＳ分析では、Ｙ１−biotag及びストレプトアビジン−ＰＥにより作られた四量体が、血清不在下でＹ１ｓｃＦｖ 単量体より１００〜１０００倍高い感応性をもつことを示した。ストレプトアビジン−ＰＥでのＹ１−biotag四量体は、Ｙ１−反応性細胞系統（ＫＧ−１）の１つに特異的に結合すると思われる。バックグラウンド結合からのこの反応の格差は非常に高く、低量のレセプタを検出するための高い感応性を提供した。Ｙ１−ＳＡＶ四量体での正常な全血のＦＡＣＳ評価は、反応性の高い集団が全く存在しないことを示した。単球及び顆粒球は、低い程度で陽性であった。例えばＫＧ−１細胞の場合のような陽性結果が存在した細胞系統では、四量体は少なくとも１００倍反応性が高かった。
【０１８７】
［２００.］ 次に、四量体は、細胞標本と共にインキュベートされた。低用量のＹ１四量体（５ng）が、細胞系統（ＫＧ−１）に良く結合し、その他のＹ１抗体形態で以前に観察されたものよりも１０〜２０倍高い応答を提供する。様々な用量の四量体で陰性の細胞系統を検査した場合、わずかな反応が見られた。
【０１８８】
［２０１.］ 本発明の１実施形態は、
第１及び第２の細胞上の未知の免疫交差反応性結合部位に結合するターゲティング分子を同定するための方法において、（ａ）第１の状態ではなく第２の状態において、１つの未知のリガンドを含む結合部位を実質的に露呈又は表示する第１の標的細胞に対して行われ、かくして認識分子の第１の集団を生成する単数又は複数回のバイオパンニング段階；（ｂ）第２の認識分子集団を生成するべく第１の細胞の未知のリガンドに対する免疫交差反応性をもつ１つの未知のリガンドを含む結合部位を表示する第２の細胞に対して実施される、段階（ａ）の認識分子の結果として得られたストックから開始した後続するバイオパンニング及び／又は選択段階を実施する段階；（ｃ）段階（ｂ）の認識分子の第２の集団の増幅及び精製段階；及び（ｄ）第２の細胞上の未知のリガンドについて選択的かつ／又は特異的であるターゲティング分子を含んで成るペプチド又はポリペプチドを段階（ｃ）の精製済み認識分子の認識部位から構築する段階、を含んで成る、ターゲティング分子の同定方法を提供する。
【０１８９】
［２０２.］ １つの好ましい実施形態は、第１の細胞が正常な細胞であり、第１の状態が活性化されていない状態であり、第２の状態が、活性化、励起、修飾、変更又は妨害を受けた状態であるようになっている。より好ましい実施形態では、第２の細胞は病的細胞である。さらにより好ましい実施形態では、病的細胞は癌細胞である。この癌細胞は、癌腫、肉腫、白血病、腺腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫であり得るがこれらに制限されるわけではない。さらに一層好ましい実施形態では、細胞は白血病細胞である。最も好ましい実施形態では、白血病細胞は、ＡＭＬ細胞である。
【０１９０】
［２０３.］ 医薬品の製造における、任意には薬学的作用物質と会合された、又はそれに付着、カップリング、組合せ、リンク又は融合された状態にある、ペプチド又はポリペプチドの使用を提供している。好ましい１実施形態では、医薬品は、病的細胞に対抗する活性をもつ。より好ましい実施形態においては、活性は、癌細胞に対するものである。癌細胞は、癌腫、肉腫、白血病、腺腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫であり得るが、これらに制限されるわけではない。さらに一層好ましい実施形態においては、癌細胞は白血病細胞である。最も好ましい実施形態においては、白血病細胞はＡＭＬ細胞である。
【０１９１】
［２０４.］ 本発明の１実施形態は、異なる単量体ｓｃＦｖｓの混合物及び／又は、異なるｓｃＦｖｓから構築されたdiabodies又はtriabodies又はtetrabodiesの混合物を含んで成る薬学組成物を提供している。
【０１９２】
［２０５.］ さらにもう１つの実施形態は、医薬品の製造における、任意には薬学的作用物質と会合された、又はそれに付着、カップリング、組合せ、リンク又は融合させられた状態にある、本発明のペプチド又はポリペプチドの使用を提供している。該医薬品は、病的細胞に対抗する活性、より特定的には癌細胞に対抗する活性を有することができる。癌細胞は、癌腫、肉腫、白血病、線腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫であり得るが、これらに制限されるわけではない。より好ましい実施形態においては、医薬品は白血病細胞に対し活性をもつ。最も好ましい実施形態においては、医薬品は、ＡＭＬ細胞に対して活性をもつ。前記細胞に対する該医薬品の活性は、癌様成長の遅延、あらゆる成長の完全な予防又は癌様細胞の死滅をひき起こす可能性がある。
【０１９３】
［２０６.］ 本発明の１実施形態においては、医薬品又は薬学組成物の活性は、細胞成長を阻害することによる。
【０１９４】
［２０７.］ 本発明のペプチド又はポリペプチドは、癌細胞好ましくは白血病細胞そして最も好ましくはＡＭＬ細胞の成長を阻害する上で使用するための組成物、好ましくは薬学組成物を調製する目的で使用することができる。本発明の１実施形態においては、ペプチド又はポリペプチドは、癌細胞の成長の阻害において使用するための組成物を調製する目的で使用でき、この組成物は、癌細胞に選択的及び／又は特異的な薬学的リガンドをもつ少なくとも１つの化合物を含んで成る。
【０１９５】
［２０８.］ 対象の発明のペプチド又はポリペプチドは、単独で患者に対して投与することができ、そうでなければ、薬学的有効量の薬学的作用物質、薬学的に有効な担体そして任意にはアジュバントと会合された、又はこれに接合、リンク又は融合された状態で医薬品又は薬学組成物を含むものとして投与可能である。かかる薬学組成物は、タンパク質、希釈剤及び酸化防止剤を内含していてよい（Osol et al. (eds.), Remington’s Pharmaceutical Sciences（第１６版）、Mack Publishing Company（１９８０）参照）。
【０１９６】
［２０９.］ もう１つの実施形態においては、薬学的作用物質は、ペプチド結合によって本発明のペプチド又はポリペプチドにリンクされている抗体又はそのフラグメントである。
【０１９７】
［２１０.］ 好ましい１実施形態においては、毒素は、例えば、ゲロニン、シュードモナス外毒素（ＰＥ）、ＰＥ４０、ＰＥ３８、ジフテリア毒素、リシン又はそれらの修飾又は誘導体である。
【０１９８】
［２１１.］ 好ましい実施形態においては、使用される放射性同位元素は、位置特定及び／又は療法用に用いることのできるガンマ放出源、陽電子放出源及びＸ線放出源及び療法用に用いることのできるベータ放出源及びアルファ放出源を内含する。
【０１９９】
［２１２.］ 対象の発明の特定の実施形態においては、治療用放射性同位元素は、¹¹¹インジウム、¹¹³インジウム、^99mレニウム、¹⁰⁵レニウム、¹⁰¹レニウム、^99mテクネチウム、^121mテルリウム、^122mテルリウム、^125mテルリウム、¹⁶⁵ツリウム、¹⁶⁷ツリウム、¹⁶⁸ツリウム、¹²³イオジン、¹²⁶イオジン、¹³¹イオジン、¹³³イオジン、^81mクリプトン、³³キセノン、⁹⁹イットリウム、²¹³ビスマス、⁷⁷ブロミン、¹⁸フルオリン、⁹⁵ルテニウム、⁹⁷ルテニウム、¹⁰³ルテニウム、¹⁰⁵ルテニウム、¹⁰⁷水銀、²⁰³水銀、⁶⁷ガリウム及び⁶⁸ガリウムを含むグループの中から選択される。
【０２００】
［２１３.］ 対象の発明のもう１つの特定の実施形態においては、抗癌剤は、ドキソルビシン、アドリアマイシン、ｃｉｓ−プラチナ、トキソール、カリケアマイシン、ビンクリスチン、シタラビン（Ａｒａ−Ｃ）、シクロホスファミド、プレドニゾン、ダウノルビシン、イダルビシン、フルダラビン、クロラムブシル、インターフェロンアルファ、ヒドロキシウレア、テモゾロマイド、サリドマイド、ブレオマイシン及びそれらの誘導体を含むグループの中から選択される。
【０２０１】
［２１４.］ 本発明の１実施形態は、一定量の本発明のペプチド又はポリペプチドと癌細胞を接触させる段階を含む癌細胞の成長の阻害方法を提供している。好ましい実施形態においては、癌細胞は、癌腫、肉腫、白血病、腺腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫であり得るが、これらに制限されるわけではない。より好ましい実施形態においては、癌細胞は白血病細胞である。最も好ましい実施形態では、白血病細胞は、ＡＭＬ細胞である。本発明の１実施形態は、患者のin vivo及びex vivo処理を可能にする。本発明のより特定的な実施形態は、異常な基幹細胞を除去するべく自己由来骨髄のex vivoパージングを可能にする。
【０２０２】
［２１５.］ 本発明のさらに特定的な実施形態においては、白血病患者の血液を、抗癌剤に接合された本発明のペプチド又はポリペプチドを含む系を通してex vivoで循環させることができる。結合した細胞及び未結合の抗癌剤の除去後、血球を患者の体内に再度導入させることができる。代替的には、白血病患者の血液を、固相に付着させられた本発明のペプチド又はポリペプチドを含む系を通してex vivoで循環させることが可能である。系内を通過し固相に付着した本発明のペプチド又はポリペプチドに結合しなかった細胞は、患者の体内に再導入させることができる。
【０２０３】
［２１６.］ 本発明のもう１つの好ましい実施形態においては、ペプチド又はポリペプチドは、移植に先立ち異常な基幹細胞を除去するべく懸濁状態のex vivo自己由来骨髄用に利用される。異常基幹細胞のパージングは、本発明のペプチド又はポリペプチド（すなわちターゲティング分子）、その構成体、フラグメント、構成体のフラグメント又はそのフラグメントの構成体が結合されている固体支持体（例えば磁気ビーズ及び親和性カラム、ただしこれらに制限されるわけではない）上に懸濁液を走行させることによって実施可能である。かくしてex vivoでパージされた骨髄はこのとき、自己由来の骨髄移植のために使用できる。この好ましい実施形態は、白血病患者の骨髄から放出された基幹細胞に結合するものの健康なドナーの骨髄から放出された基幹細胞には結合しないファージミドクローン（Ｙ１）の本発明における同定に基づいている。類似の要領で、Ｙ１ファージミドクローンは、ＦＡＣＳ分析により異常であることが見極められた芽細胞ならびに白血病細胞に結合する。
【０２０４】
［２１７.］ 本書では、芽細胞は、哺乳動物生体内の全ての循環細胞にとっての前駆体である一次細胞として定義づけされる。それらの始原特性のため、芽細胞は、成人生体の体内には有意な量で循環しているところが発見されていない。外因性刺激無しでの循環する芽細胞の存在は、例えば造血系の悪性腫瘍を表わすものであり、その後のその消滅は、悪性疾患の緩解を表わしている可能性がある。
【０２０５】
［２１８.］ 本発明のもう１つの実施形態においては、薬学組成物は予防のために用いられる。
【０２０６】
［２１９.］ 好ましい実施形態においては、本発明の２つ以上のペプチド又はポリペプチドが組合わされて１つの混合物を形成する。
【０２０７】
［２２０.］ 本書で使用されている混合物は、単一の調製物の中に含まれる異なる種の２つ以上の分子又は粒子として定義づけされる。分子の異なる種は、共有化学結合も非共有化学結合も形成しない。
【０２０８】
［２２１.］ 対象の発明の１実施形態においては、対象の発明のペプチド又はポリペプチドは、薬学的作用物質にリンク、融合又は接合されている。
【０２０９】
［２２２.］ 対象の発明のもう１つの実施形態においては、ペプチドと薬学的作用物質の間のリンクは、直接的リンクである。本書で使用されているように、２つ以上の隣接する分子の間の直接的リンクが、分子内の元素又は元素群の間の化学的結合を介して得られる。化学的結合は、例えばイオン結合、共有結合、疎水性結合、親水性結合、静電結合又は水素結合であり得る。結合は、アミン、カルボキシ、アミド、ヒドロキシル、ペプチド及びジスルフィドを含むグループの中から選択され得るが、これらに制限されるわけではない。直接的リンクは好ましくはプロテアーゼ耐性結合でありうる。
【０２１０】
［２２３.］ さらにもう１つの実施形態においては、ペプチドと薬学的作用物質の間のリンクは、リンカー化合物により作用される。本明細書及びクレーム中で使用されているリンカー化合物は、２つ以上の部分を互いにつなぐ化合物として定義づけされる。リンカーは、直鎖又は有枝鎖リンカーでありうる。有枝鎖リンカーは、２重分岐、３重分岐又は４重以上の有枝化合物から成り得る。リンカー化合物は、ジカルボン酸、マレミドヒドラジド、ＰＤＰＨ、カルボン酸ヒドラジド、及び小型ペプチドであり得るが、これらに制限されるわけではない。その他のリンカー化合物の例としては、コハク酸、グルタル酸、及びアジピン酸といったジカルボン酸；Ｎ−〔ε−マレイミドカプロン酸〕ヒドラジド、４−〔Ｎ−マレイミドメチル〕ジクロヘキサン−１−カルボキシヒドラジド及びＮ−〔Ｋ−マレイミドウンデカン酸〕ヒドラジド〕といったようなマレイミドヒドラジド；サルファヒドリル反応性タンパク質に接合された（３−〔２−ピリジルジチオ〕プロピオニルヒドラジド）といったＰＤＰＨリンカー；２〜５個の炭素から選択されたカルボン酸ヒドラジド；及び例えば抗癌医薬品ドクソルビシンの遊離糖とｓｃＦｖの間の小型ペプチドリンカを用いた直接的カップリングが含まれる。小型ペプチドには、ＡＵ１、ＡＵ５、ＢＴａｇ、ｃ−ｍｙｃ、ＦＬＡＧ、Gｌｕ−Ｇｌｕ、ＨＡ、Ｈｉｓ６、ＨＳＶ、ＨＴＴＰＨＨ、ＩＲＳ、ＫＴ３、プロテインＣ、Ｓ・Ｔａｇ（商標）、Ｔ７、Ｖ５、ＶＳＶ−Ｇ、及びＫＡＫ Ｔａｇが含まれるが、これらに制限されるわけではない。
【０２１１】
［２２４.］ 対象の発明のペプチド又はポリペプチドの既知のあらゆる投与方法、例えば、静脈内、筋内、皮下、局所的、気管内、鞘内、腹腔内、リンパ内、鼻腔内、舌下、経口、直腸内、膣内、吸入式、口腔内、皮内、経皮又は胸腔内投与を利用することができる。
【０２１２】
［２２５.］ 静脈内投与のためには、製剤は好ましくは、患者に投与される量が所望の組成物の約０．１mg〜約１０００mgという有効量となるような形で調製されることになる。より好ましくは、投与される量は所望の組成物約１mg〜約５００mgの範囲内にあるものとする。本発明の組成物は、広い服用量範囲全体にわたり有効であり、治療すべき疾病についての詳細、患者の体内でのペプチド又はポリペプチドベースの薬学組成物の半減期、薬学的作用物質及び薬学組成物の物理的及び化学的特性、薬学組成物の投与様式、治療又は診断を受けるべき患者についての詳細ならびに治療にあたる医師が重要とみなしたその他のパラメータといった要因により左右される。
【０２１３】
［２２６.］ 経口投与用の薬学組成物は、錠剤、液体、エマルジョン、懸濁液、シロップ、丸薬、キャプレット又はカプセルの形をしていてよい。薬学組成物は同様に、装置に入れて投与することもできる。
【０２１４】
［２２７.］ 局所施用のための薬学組成物は、クリーム、軟こう、ローション、貼付剤、溶液、懸濁液又はゲルの形をとることができる。
【０２１５】
［２２８.］ さらに、薬学組成物は、固体、液体又は持続放出性製剤用に調製可能である。
【０２１６】
［２２９.］ 本発明に従って生成される抗体フラグメントを含む組成物は、従来の薬学的に受容可能な希釈剤又は担体を含んでいてよい。錠剤、丸薬、キャプレット及びカプセルは、ラクトース、デンプン及びステアリン酸マグネシウムといったような従来の賦形剤を内含し得る。座薬は、ろう及びグリセロールといった賦形剤を内含することができる。注射可能な溶液は、食塩水といったような無菌の発熱物質無しの媒質を含み、緩衝剤、安定剤又は防腐剤を内含する。従来の腸溶性コーティングも使用することができる。
【０２１７】
［２３０.］ 対象の発明は同様に、当該技術分野において既知の合成手段により抗体フラグメントを産生する方法も包含する。
【０２１８】
［２３１.］ 本発明の１実施形態は、腫瘍の診断用位置特定及び／又は画像化において使用するための画像化剤に付着、カップリング、組合せ、リンク又は融合された本発明の少なくとも１つのペプチド又はポリペプチドを含んで成る薬学組成物を含む。
【０２１９】
［２３２.］ 本発明のさらにもう１つの実施形態は、標示用マーカー分子に結合された本発明のペプチドを含む画像化剤を含んで成る、治療の前、途中又は後の治療の有効性をin vitroで分析するための診断用キットを提供している。本発明はさらに、癌、より特定的には腫瘍の診断用位置特定及び／又は画像化のために画像化剤を使用する方法において、
ａ）細胞を組成物と接触させる段階；
ｂ）細胞に結合した放射能を測定する段階；
ｃ）腫瘍を視覚化する段階、
を含んで成る方法を提供している。
【０２２０】
［２３３.］ 本発明の好ましい実施形態においては、キットの画像化剤は、螢光染料であり、このキットは、癌、より特定的には血液関連の癌、例えば白血病、リンパ腫及び骨髄腫の治療の有効性の分析を提供する。例えば診断時点、治療中、緩解中及び再発中といった疾病の各期における染色強度及び画像化剤により染色された細胞の百分率を決定するために、ＦＡＣＳ分析が用いられる。
【０２２１】
［２３４.］ 本発明はさらに、有効量の画像化剤、本発明のペプチド及び生理学的に受容可能な担体を含む組成物を提供する。
【０２２２】
［２３５.］ 好ましい実施形態においては、標示マーカー分子は、放射性同位元素、Ｘ線に対し不透明な元素、常磁性イオン又は螢光分子などを含む（ただしこれらに制限されるわけではない）、当該技術分野において既知の任意のマーカーである。
【０２２３】
［２３６.］ 対象の発明の特定的実施形態においては、標示用放射性同位元素は、¹¹¹インジウム、¹¹³インジウム、^99mレニウム、¹⁰⁵レニウム、¹⁰¹レニウム、^99mテクネチウム、^121mテルリウム、^122mテルリウム、^125mテルリウム、¹⁶⁵ツリウム、¹⁶⁷ツリウム、¹⁶⁸ツリウム、¹²³イオジン、¹²⁶イオジン、¹³¹イオジン、¹³³イオジン、^81mクリプトン、³³キセノン、⁹⁰イットリウム、²¹³ビスマス、⁷⁷ブロミン、¹⁸フルオリン、⁹⁵ルテニウム、⁹⁷ルテニウム、¹⁰³ルテニウム、¹⁰⁵ルテニウム、¹⁰⁷水銀、²⁰³水銀、⁶⁷ガリウム及び⁶⁸ガリウムであり得るが、これらに制限されるわけではない。
【０２２４】
［２３７.］ もう１つの好ましい実施形態に従うと、標示用マーカー分子は、螢光マーカー分子である。より好ましい実施形態に従うと、螢光マーカー分子は、フルオレセイン、フィコエリスリン又はローダミン又はその修飾又は接合体である。
【０２２５】
［２３８.］ 対象の発明は同様に、有効量の本発明の画像化剤、それにリンクされた薬学的作用物質及び生理学的に受容可能な担体を含む組成物をも考慮している。
【０２２６】
［２３９.］ 本発明は同様に、画像化剤が器官及び細胞に結合するような条件下で本発明の画像化剤と画像化すべき器官又は細胞を接触させ、結合した画像化剤を画像化させかくして該器官又は細胞を画像化させることが関与する、器官又は細胞を画像化させる方法をも提供する。
【０２２７】
［２４０.］ 対象の発明は、さらに、組成物が器官に結合するような条件下で本発明の組成物と治療すべき器官を接触させかくして該器官を治療することが関与する、in vivoで１つの器官を治療する方法を提供している。
【０２２８】
［２４１.］ 本発明の好ましい実施形態においては、例えばＦＡＣＳ分析によって細胞を監視し画像化することにより、全血中の悪性細胞、より特定的には白血病細胞を、ターゲティングするために、ペプチド又はポリペプチドを利用してもよい。正常な細胞との関係における腫瘍細胞についてより高い評点（例えば４倍高い）を受けた標本は、治療の対象となる。
【０２２９】
［２４２.］ 本発明は、癌を治療するのに有効な量の本発明のペプチド又はポリペプチドを患者に投与することを含む、癌を患う患者の治療を提供する。好ましい実施形態においては、癌は、癌腫、肉腫、白血病、腺腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫から成るグループの中から選択されている。より好ましい実施形態においては、癌は白血病であり、最も好ましい実施形態においては、白血病はＡＭＬである。
【０２３０】
［２４３.］ 最も好ましい実施形態では、本発明のペプチド又はポリペプチドは、特異的又は選択的にＡＭＬ細胞に結合する。本発明は、本発明のペプチド又はポリペプチドに結合されたＡＭＬ細胞上に提示されたリガンド、さらには、このリガンドに結合するペプチド又はポリペプチドを提供する。
【０２３１】
［２４４.］ 対象の発明の新規の抗体フラグメント又はその対応するペプチドミメティックは、さまざまな疾病及び身体条件を治療するための組成物又は医薬品の製造において使用される。
【０２３２】
［２４５.］ 対象の本発明は、ａ）標的細胞上で直接的にバイオパンニング手順によってか又は第１の状態ではなく第２の状態の第１の標的細胞上で間接的にバイオパンニング手順により又その後第２の標的細胞上で直接的にバイオパンニング手順によって一次認識部位を含む単数又は複数のターゲティング分子を単離し選択して、単数又は複数の前記ターゲティング分子を産生する段階；
ｂ）単数又は複数のターゲティング分子の増幅、精製及び同定段階；及び
ｃ）単数又は複数のターゲティング分子又はその認識部位からターゲティング作用物質を構築する段階、
を含んで成り、該ターゲティング作用物質がペプチド、ポリペプチド、抗体又は抗体フラグメント又はその多量体であり得る、ターゲティング作用物質の産生方法を提供する。
【０２３３】
［２４６.］ ターゲティング作用物質はさらに、薬学的作用物質にカップリング、付着、組合せ、リンク、融合されているか又はこの薬学的作用物質と会合された状態となるように構築され得る。
【０２３４】
［２４７.］ 本発明の好ましい実施形態においては、ターゲティング作用物質は、抗疾病又は抗癌剤である。
【０２３５】
［２４８.］ 本発明のもう１つの好ましい実施形態においては、薬学的作用物質は、放射性同位元素、毒素、オリゴヌクレオチド、組換え型タンパク質、抗体フラグメント、及び抗癌剤を含むグループの中から選択されている。該放射性同位元素は、¹¹¹インジウム、¹¹³インジウム、^99mレニウム、¹⁰⁵レニウム、¹⁰¹レニウム、^99mテクネチウム、^121mテルリウム、^122mテルリウム、^125mテルリウム、¹⁶⁵ツリウム、¹⁶⁷ツリウム、¹⁶⁸ツリウム、¹²³イオジン、¹²⁶イオジン、¹³¹イオジン、¹³³イオジン、^81mクリプトン、³³キセノン、⁹⁰イットリウム、²¹³ビスマス、⁷⁷ブロミン、¹⁸フルオリン、⁹⁵ルテニウム、⁹⁷ルテニウム、¹⁰³ルテニウム、¹⁰⁵ルテニウム、¹⁰⁷水銀、²⁰³水銀、⁶⁷ガリウム及び⁶⁸ガリウムを含むグループの中から選択されていてよい。
【０２３６】
［２４９.］ さらにもう１つの実施形態では、毒素は、ゲロニン、シュードモナス外毒素（ＰＥ）、ＰＥ４０、ＰＥ３８、ジフテリア、リシン及びそれらの修飾及び誘導体を含むグループの中から選択されている。
【０２３７】
［２５０.］ 本発明のさらにもう１つの実施形態においては、抗癌剤は、ドキソルビシン、モルホリノ−ドキソルビシン（ＭＤＯＸ）、アドリアマイシン、ｃｉｓ−プラチナ、トキソール、カリケアマイシン、ビンクリスチン、シタラビン（Ａｒａ−Ｃ）、シクロホスファミド、プレドニゾン、ダウノルビシン、イダルビシン、フルダラビン、クロラムブシル、インターフェロンアルファ、ヒドロキシウレア、テモゾロマイド、サリドマイド、ブレオマイシン及びそれらの誘導体から成るグループの中から選択されている。
【０２３８】
［２５１.］ 対象の発明は、（ａ）血液から誘導された細胞と共に、ファージ表示ライブラリをインキュベートすることが関与するバイオパンニング；（ｂ）未結合ファージを除去するための洗浄；（ｃ）血球からの結合したファージの溶出；（ｄ）結果として得られたファージの増幅；及び（ｅ）ペプチドを同定するための、結合したファージの表示されたペプチド配列の決定、により抗体フラグメントを同定するための方法を提供している。
【０２３９】
［２５２.］ 対象の発明は、
Ａ−Ｘ−Ｂ
という構造式又は構造をもつペプチド又はポリペプチドであって、式中Ｘが３〜３０個のアミノ酸の超可変ＣＤＲ３領域であり； Ａ及びＢが各々長さ１〜１０００アミノ酸のアミノ酸鎖であり得、ここでＡがアミノ末端、Ｂがカルボキシ末端である、ペプチド又はポリペプチドを提供している。
【０２４０】
［２５３.］ 本発明の好ましい実施形態においては、Ａは、１５０〜２５０個のアミノ酸残基であり、Ｂは、３５０〜５００個のアミノ酸残基である。
【０２４１】
［２５４.］ もう１つの好ましい実施形態においては、ペプチドのＣＤＲ３領域は、５〜１３個のアミノ酸残基である。
【０２４２】
［２５５.］ もう１つの好ましい実施形態においては、上述の構造式内のＸは、配列番号８〜２４から成るグループの中から選択されたアミノ酸配列である。
【０２４３】
［２５６.］ 本発明のもう１つの実施形態においては、ペプチド又はポリペプチドは、より大きい又は完全な抗体又は多量体の一部分である。
【０２４４】
［２５７.］ さらにもう１つの実施形態においては、二量体分子は２つのペプチド又はポリペプチドを含み、うち１つは本発明のペプチド又はポリペプチドである。該二量体分子は、本発明の同一の２つのペプチド又はポリペプチドを含んでいてよい。
【０２４５】
［２５８.］ 本発明の好ましい実施形態においては、Ｘは前記二量体分子内で、配列番号８〜２４から成るグループの中から選択されたアミノ酸配列である。
【０２４６】
［２５９.］ もう１つの実施形態は、本発明のペプチド又はポリペプチド又は二量体分子をコードする核酸分子を提供する。
【０２４７】
［２６０.］ 本発明は、医薬品の製造における、任意には薬学的作用物質と会合された又はそれに付着、組合せ、リンク又は融合された状態のペプチド又はポリペプチドの使用を提供する。
【０２４８】
［２６１.］ 本発明はさらに、病的細胞、より特定的には癌細胞に対抗する活性を持つ医薬品の製造におけるペプチド又はポリペプチドの使用を提供する。癌細胞は、癌腫、肉腫、白血病、腺腫、リンパ腫、骨髄腫、芽球腫、精上皮腫及び黒色腫を含むグループの中から選択され得る。より特定的には、癌細胞は、白血病細胞であり得、最も特定的には、白血病細胞は、ＡＭＬ細胞であり得る。
【０２４９】
［２６２.］ 本発明で定義づけされ以下の例中で論述されているような交換可能な系は、分子をさらに操作又は再構築する必要なく構成体の内部での再度画定された可変領域の交換又は置換を可能にするように設計された核酸構成体である。かかる系は、所望の核酸分子の急速かつ便利な調製を可能にする。
【実施例】
【０２５０】
実施例
［２６３.］ 以下の例は、本発明を理解する上で一助となるように記されているが、いかなる形であれその範囲を制限するようには意図されておらず、又そのようにみなされるべきものではない。特定の試薬及び反応条件が記述されているが、本発明の範囲により包含されるべく意図された修正を加えることが可能である。従って以下の例は、本発明をさらに例示する目的で提供されている。
【０２５１】
例１：
［２６４.］ １．細胞、細菌菌株、ｓｃＦｖファージ表示ライブラリ、細胞膜の調製及びバイオパンニング手順のためのタンパク質精製。
【０２５２】
［２６５.］ １．１ 白血病細胞の調製。血液標本を白血病患者から得た。フィコールクッション上でその他の細胞から単核細胞（一次細胞）を分離した（Iso-prep, Robbins Scientific Corp., Sunnyvale, CA, USA）。２５分間、１１０×ｇで遠心分離を実施した。界面にある細胞を収集し、ＰＢＳ中で２度洗浄した。その後、ＲＰＭＩ＋１０％のウシ胎児血清（ＦＣＳ）の中で細胞を懸濁させ、計数した。長時間の貯蔵のためには、１０％のＦＣＳと１０％のＤＭＳＯをリンパ球に添加し、それを次に、−７０℃で凍結した。
【０２５３】
［２６６.］ １．２ 定着した血小板の調製。血液バンクから入手した血小板濃縮物を３７℃で１時間インキュベートした。等体積の２．０％のパラホルムアルデヒドを添加し、血小板を４０℃で１８時間定着させた。低温食塩水で血小板を２回洗浄し（２５００×ｇで１０分間遠心分離）、食塩水中で０．０１％のＨＥＰＥＳ中で再懸濁させ、顕微鏡を用いて計数した。
【０２５４】
［２６７.］ 血漿フォン・ウィルブランド因子及びリストセチンに対する血小板の感応性を確認した。血漿フォン・ウィルブランド因子（ｖＷＦ；１８μｇ／ml）及びリストセチン（０．６mg／ＭＩ）を定着した血小板に添加し、血小板凝集を誘発し、クロノログ・ルミ・血小板凝集計により監視した。
【０２５５】
［２６８.］ １．３ 細菌菌株−ＴＧ−１及びＨＢ２１５１：０．５〜０．９のＡ₆₀₀まで細胞を成長させることにより（対数増殖細胞）、感染のために新鮮な細菌培養を調製した。ファージ伝播のためにE. coli ＴＧ−１細胞を用い、ｓｃＦｖタンパク質の産生のためには、E.coli ＨＢ２１５１細胞を使用した。
【０２５６】
［２６９.］ １．４ ｓｃＦｖ表示ファージライブラリ供給源。ｓｃＦｖライブラリ（Nissim et al., ＥＭＢＯ J., １３，６９２−６９８（１９９４））は、ＭＲＣの合意を得て A. Nissim博士により提供された。ライブラリは当初、中で柔軟なポリペプチドによってＶ_H及びＶ_LのドメインがリンクされたｓＦｖ フラグメントを表示するファージミドライブラリとして構築された。ファージミドライブラリ内で表示されたｓｃＦｖｓは、ファージの微量外皮タンパク質ｐＩＩＩのＮ末端に融合され、これはその後ｐＨＥＮ１ベクターへとサブクローニングされた（Nissim et al., ＥＭＢＯ J., １３，６９２−６９８（１９９４））。抗体フラグメントのレパートリがまず第１に、免疫化されていないヒトの末梢血リンパ球の再配列されたＶ−遺伝子からＲＣＲにより生成された（実験未使用のレパートリと呼ばれる）。レパートリを多様化するため、４９のクローニングされた一連のヒトＶ_H遺伝子セグメント内に、残基４〜１２個の重鎖ＣＤＲ３長をコードする無作為ヌクレオチド配列を導入した。全てのクローン内の融合されたＶ_L フラグメントは、生殖細胞系列 ＩＧＬＶ３Ｓ１の突然変異を受けていない単一のＶ遺伝子から誘導され、約１０⁸クローンの単一ポットライブラリを作り出す。
【０２５７】
［２７０.］ １．５ ＡＭＬ細胞からの膜調製。１０⁸個の洗浄された細胞を含有するペレットに対し１mlの低温溶解溶液（０．３Ｍのスクロース、５ｍＭのＥＤＴＡ、１ｍＭのＰＭＳＦ）を添加し、その後４℃で１１０００×ｇで２０分間スピンさせた。上清流体を廃棄し、ペレットをＴＥ（１０ｍＭのトリス、１ｍＭのＥＤＴＡ、１ｍＭのＰＭＳＦ中で再懸濁させ、上述のとおりスピンさせた。最終的ペレットを０．４のＡ₂₈₀で６mlのＰＢＳ中で再懸濁させ、２時間３７℃で、３本のMaxisorbimmuno-管（ＮＵＮＣ）をコーティングするために使用した。コーティングの後、管をＰＢＳで３回洗い流し、次に２時間ＲＴでＭＰＢＳ（ＰＢＳ中の２％の脱脂粉乳）で遮断させた。バイオパンニングの前に、管をさらに３回ＰＢＳで洗い流した。
【０２５８】
例２：
［２７１.］ ２．ファージミド粒子の操作 re：バイオパンニング手順。
【０２５９】
［２７２.］ ２．１ ファージミドの選択及び増幅：特定の関心の的であるエピトープを発現したファージミドを、次の４段階のバイオパンニング手順によりライブラリから選択した：
ａ） 標的に対するファージミド粒子の結合、
より特定的には、洗浄した標的細胞又は細胞膜に対するファージミド粒子の結合、
ｂ） 未結合ファージミド粒子の除去、より特定的には、大規模洗浄による除去、
ｃ） 結合したファージミド粒子の溶出、
ｄ） 溶出したファージミド粒子の伝播及び増幅、より特定的には、E.coli内で伝播及び増幅。
【０２６０】
［２７３.］ ２．２ クローンの同定： ４段階のバイオパンニング手順を一般的に３〜５回反復した。選択されたファージミドクローンを個々に伝播させ、さらに次の作業により特徴づけした：
ａ） ＤＮＡ配列決定、
ｂ） 複数の細胞型に対するファージの結合のex-vivoでの比較、
ｃ） 可溶性ｓｃＦｖを産生するためのE. coli HB２１５１の感染。
【０２６１】
［２７４.］ ２．３ 配列分析：ファージミド粒子内の〜８００ｂｐのコードされたｓｃＦｖ ＤＮＡを、上流側プライマ＃２０３７４３（５′−ＧＡＡＡＴＡＣＣＴＡＴ標的細胞ＣＴＡＣＧＧ）及び下流側プライマ＃１８１３９０（５′−ＴＧＡＡＴＴＴＴＣＴＧＴＡＴＧＡＧＧ）を用いたＰＣＲにより増幅した。ＡＢＩ ＰＲＩＳＭBig Dye終結サイクル配列決定キット及び上述のプライマを用いて自動ＡＢＩ ＰＲＩＳＭＤＮＡシーケンサ（３１０ Genetic Analyzer, Perkin Elmer）により両端部からＤＮＡフラグメントを完全に配列決定した。重鎖と軽鎖の間の柔軟なポリペプチド接合部領域にある２つの付加的なプライマ＃１９１１８１（５％−ＣＧＡＴＣＣＧＣＣＡＣＣＧＣＣＡＧＡＧ）及びその相補的プライマ＃１９１３４４（５′−ＣＴＣＴＧＧＣＧＧＴＧＧＣＧＧＡＴＣＧ）を配列決定のために使用した。
【０２６２】
例３：
［２７５.］ ３．バイオパンニングプロトコル
【０２６３】
［２７６.］ ３．１ 基本的バイオパンニングプロトコル：バイオパンニング手順は、上述のファージ表示技術の一部分である。当該研究作業では、３つのバイオパンニングプロトコルが開発され使用された。
ａ）プロトコルＡＭ（ＡＭＬ細胞膜パンニング／細菌溶出及びそれに続く全ＡＭＬ細胞パンニング／トリプシン溶出）
ｂ）プロトコルＹＰＲ（定着したヒト血小板パンニング／酸溶出）。
ｃ）プロトコルＹＰＮＲ（定着したヒト血小板パンニング／酸溶出）。
【０２６４】
［２７７.］ これらのプロトコルは、以下で詳述されている：
【０２６５】
［２７８.］ ３．１．１ プロトコルＡＭ
【０２６６】
［２７９.］ ３．１．１．１ 予備洗浄：−７０℃で貯蔵された患者由来の２×１０⁷個の凍結したＡＭＬ細胞を含有する１mlのアリコートを３７℃で急速解凍し、１０mlの低温２％ＰＢＳ−ミルク（ＭＰＢＳ）の中に直ちに希釈させた。細胞を室温（ＲＴ）で１２０×ｇでスピンさせ、２回洗浄し、ＭＰＢＳ内で再懸濁させ、血球計で計数した。細胞膜を、第１．５で記述されている通りに調製した。
【０２６７】
［２８０.］ ３．１．１．２ 選択は、もとのNissimライブラリからの１０¹²個のファージミドを含有する２mlのＭＰＢＳの添加により、固定化したＡＭＬ細胞膜上で実施された。３０分間管をゆっくりと撹拌し、次に撹拌することなくさらに９０分間インキュベートし、両方の段階共室温（ＲＴ）で行なった。ＡＭＬ細胞膜上での３ラウンドのパンニングの後、１ラウンドのパンニングを全ＡＭＬ細胞上で実施した。
【０２６８】
［２８１.］ ３．１．１．３ 洗浄：余剰の未結合ファージミドを除去するため、管の中味を傾瀉させ、管を１０回ＰＢＳ、０．１％ Tweenで洗浄し、ＰＢＳのみで１０回洗浄した。
【０２６９】
［２８２.］ ３．１．１．４ 溶出：管に直接、対数増殖するE.coli ＴＧ−１細胞（２ml）を添加し、３０分間３７℃でゆっくり撹拌しながらインキュベートした。上述のように、滴定のためアリコートを平板固定し、残りの体積を増幅のために平板固定した。
【０２７０】
［２８３.］ ３．１．１．５ 増幅：大きな平板からのコロニーをかき取り、プールした。〜０．５のＡ₆₀₀まで液体培養中でアンピシリン耐性 E.coli ＴＧ−１細胞のアリコート（〜１０⁷）を成長させ、次にヘルパーファージ（ＶＳＣ−Ｍ１３、Stratagene）で感染させて、大きな増幅されたファージミドストックを生成した。ファージミドを、ＰＥＧ沈殿手順（１８ａ）によりレスキューした。上述の増幅したＴ１６ＭＩストックを後続するパンニングラウンドのために使用した。以前に増幅したストックの１０¹¹個のファージミドを用いて、さらに２ラウンド、選択手順をくり返した。固定化した膜上の３回目のパンニング手順の増幅ストックをＴ１６Ｍ３と呼称した。
【０２７１】
［２８４.］ ３．１．１．６ 全細胞に対する再パンニング：３回目の膜パンニングの増幅ストック、Ｔ１６Ｍ３を用いて、無傷のＡＭＬ細胞をパンニングした。２×１０⁷個の細胞及び１０¹⁰個のファージミドコロニー形成単位（ＣＦＵ）（Nissimライブラリ）及び１０¹³個の野生型バクテリオファージＭ１３を含有する０．５mlのＭＰＢＳ最終体積の中で、４℃で２時間ゆっくりと撹拌しながら選択を実施した。結合したファージミドを、５０μｌのトリプシン：ＥＤＴＡ（０．２５％：０．２５％）を用いて洗浄済み細胞ペレットから溶出し、次に、５０μｌのＦＣＳを添加することにより中和した。滴定及び増幅のため、１mlのE.coli ＴＧ−Ｉ培養（Ａ₆₀₀＝０．５）を使用した。増幅した最終ストックをＴ１６Ｍ３．１と呼称した。
【０２７２】
［２８５.］ ３．１．２ プロトコルＹＰＲ
【０２７３】
［２８６.］ ３．１．２．１ 選択：１mlのＰＢＳ／ＨＥＰＥＳ／１％ＢＳＡ緩衝液中で１０¹¹のファージミド（Nissimライブラリ）で１０⁸個の定着したヒト血小板をパンニングすることよりクローン選択を達成した。回転により標本を混合しながらＲＴで１時間、結合を行なわせた。
【０２７４】
［２８７.］ ３．１．２．２ 細胞洗浄：低速遠心分離（３５００×ｇ）により５回血小板を洗浄し、上述のように再懸濁させた。
【０２７５】
［２８８.］ ３．１．２．３ 溶出：酸溶出技術により、定着した血小板から第１ラウンドの結合したファージミドを溶出させた。
【０２７６】
［２８９.］ ２００μｌの０．１Ｍのグリシン（ｐＨ２．２）を用いてＲＴで１０分間血小板をインキュベートした。０．５Ｍのトリス−ＨＣｌ、ｐＨ８．０での中和及び遠心分離の後、２００μｌのトリプシン−ＥＤＴＡ（０．２５％／０．０５）を添加し、５０μｌのＦＣＳの添加により中和することによって、残りの血小板結合ファージを溶出した。細胞を遠心分離によって除去し、酸及びトリプシンの両方の溶出プロトコルからの溶出済みファージを含有する上清流体を収集し、それぞれＹＰＲ（ａ）−１及びＹＰＲ（ｔ）−１ストックと呼称した。これらのストックを次に、３７℃で３０分間１mlの対数増殖中のＴＧ−１細胞を添加することで増幅させた。滴定のためアリコートを平板固定し、残った感染したE.coli 細胞を２ｘＴＶ／ＡＭＰ１５cm平板上で固定させた。平板を３０℃で一晩インキュベートした。各々のパンニングラウンド後の排出量を、滴定プレート上のコロニーの計数により決定した。
【０２７７】
［２９０.］ ３．１．２．４ 増殖： クローンを第３．１．１．５節で記述した通りに増幅させた。酸及びトリプシン溶出プロトコルからの、Ｒ１（ａ）及びＲ１（ｔ）ストックと呼ばれる〜１０¹²ファージミド／mlの増幅されたストックをそれぞれに組合わせ、その後のパンニングのラウンドで使用した。
【０２７８】
［２９１.］ ３．１．２．５ 第２及び第３のパンニングラウンドを、以下の修正を加えたＹＰＲ手順の第１のパンニングラウンドについて記述した通りに実施した。すなわち（ｉ）第２のパンニングについては、１０¹²のＲ１（ｔ）と組合わせて１０¹²のＲ１（ａ）を使用し、（ｉｉ）グリシン（ｐＨ２．２）のみで溶出を実施した。第２ラウンドの増幅した溶出液をＲ２と呼称した。（ｉｉｉ）第３のバイオパンニングラウンドについては、１０¹²のＲ２を使用し、第２ラウンドにおいて溶出を行なった。第３ラウンドの増幅ストックはＲ３と呼称された。
【０２７９】
［２９２.］ ３．１．３ ＹＰＮＲプロトコル
【０２８０】
［２９３.］ ３．１．３．１ バイオパンニングと洗浄を、基本的にＹＰＲプロトコルに記述されている通りに実施した。ただし、このプロトコルにおいては、（ｉ）溶出は、グリシン（ｐＨ２．２）での３回のパンニングラウンドの各々の後に実施し、第１回のパンニングと増幅の後には、増幅無しの２回の後続パンニングラウンドが続いた。第１、第２及び第３ラウンドは、それぞれＹＰＮＲ１、ＹＰＮＲ２、及びＹＰＮＲ３と呼称した。
【０２８１】
［２９４.］ ３．２ 負の対照ｓｃＦｖクローンの選択
【０２８２】
［２９５.］ ３．２．１ Ｎ１４ＣＤＲ３配列：全ての結合実験について、実験未使用のライブラリ（選択前）から単一のクローンを抜き取った。このクローンから、Ｎ１４と呼ばれる可溶性ｓｃＦｖとファージストックを調製した。配列分析から、それがＶ_H４−ＤＰ６５遺伝子系統群に属することがわかる。Ｎ１４ ＣＤＲ３と呼称された、このクローンによりコードされたｌｌ−merＶ_H−ＣＤＲ３の配列は、以下の通りである（配列番号２８）。
Ｐｈｅ Ｌｅｕ Ｔｈｒ Ｔｙｒ Ａｓｎ Ｓｅｒ Ｔｙｒ Ｇｌｕ Ｖａｌ Ｐｒｏ Ｔｈｒ
【０２８３】
［２９６.］ ３．２．２ Ｃ１８１ＣＤＲ３配列：結合分析実験の中で、付加的な負のクローンＣ１８１を使用した。（組換え型Ｂ型肝炎ウイルス［ＨＢＶ］粒子に対する反応性をもつ）クローンＣ１８１は、Ｖ_H３−ＤＰ３５系統群に属し、Ｃ１８１ ＣＤＲ３と呼称されたこのクローンによりコードされた９−merＶ_H−ＣＤＲ３の配列は、以下の通りである（配列番号２９）：
【０２８４】
Ｔｈｒ Ａｓｎ Ｔｒｐ Ｔｙｒ Ｌｅｕ Ａｒｇ Ｐｒｏ Ｌｅｕ Ａｓｎ
【０２８５】
例４：
［２９７.］ ４．ｓｃＦｖクローンの産生、精製、標識付け及び特徴づけ
【０２８６】
［２９８.］ ４．１ 可溶性ｓｃＦｖの産生： もとのファージミドライブラリを構築するために使用されるベクターであるｐＨＥＮ１を、ｓｃＦｖ遺伝子及びｐＩＩ１遺伝子の接合部においてコードされたアンバー停止コドンを用いて設計した。従って、選択されたクローンのベクターが、ファージミド感染によって、非サプレッサ菌株であるE.coli ＨＢ２１５１内に導入された時点で、この系は、細菌周辺質内への可溶性ｓｃＦｖの産生及び分泌を可能にする（Harrison et al., Methods in Enzymology, ２６７，８３−１０９（１９９６））。このときｓｃＦｖは、培養液から容易に回収可能である。可溶性ｓｃＦｖｓは、ＩＰＴＧで誘発されるｌａｃＺプロモータの制御下で産生される（Gilbert and Muller-Hill, ＰＮＡＳ（ＵＳ），５８，２４１５（１９６７））。
【０２８７】
［２９９.］ アンバー突然変異の上流側でベクターの中に、ｃ−ｍｙｃタグをコードする配列（アミノ酸１０個−Ｇｌｕ Ｇｌｎ Ｌｙｓ Ｌｅｕ Ｉｌｅ Ｓｅｒ Ｇｌｕ Ｇｌｕ Ａｓｐ Ｌｅｕ；配列番号１２３）が含まれている。発現されたｓｃＦｖのＣ末端は、（欧州細胞培養収集機関（ＥＣＡＣＣペプチド又はポリペプチド）９Ｅ１０−ハイブリドーマから誘導された）マウス抗−mycタグ抗体を用いて検出可能であるｃ−mycタグを担持しているはずである。
【０２８８】
［３００.］ ４．２ プロテインＡビーズアフィニティカラム上でのｓｃＦｖの精製； 選択されたクローン及び対照クローンＣ１８１のｓｃＦｖｓは全てＶ_H３系統群に属し、プロテインＡアフィニティカラム上での精製を可能にする。各クローンの誘発された培養からの周辺質画分（１００−２５０ml）を調製し、プロテインＡセファロースビーズと共にインキュベートした。結合したｓｃＦｖｓを酸性溶液（０．１Ｍのグリシン、ｐＨ３．０）によってカラムから回収し、その後トリスｐＨ８．０で溶出液を中和した。回収したタンパク質の濃度をＡ₂₈₀測定により決定し、その後、透析によってか又はＧ−２５セファロースカラム上でＰＢＳ緩衝液の交換を行なった。
【０２８９】
［３０１.］ ４．３ セファクリルＳ−２００カラム上でのＮ１４−ｓｃＦｖの精製：負のクローンＮ１４のｓｃＦｖは、Ｖ_H４ 遺伝子系統群に属し、従って、プロテイン−Ａのアフィニティカラム上で精製不可能である。ｓｃＦｖ−Ｎ１４の精製のためには、６０％の硫酸アンモニウムにより、２００mlの誘発培養の周辺質画分を沈殿させた。２mlの０．１×ＰＢＳ、５ｍＭのＥＤＴＡ、５ｍＭのＰＭＳＦ中でペレットを再懸濁させ、現行の緩衝液（０．１×ＰＢＳ、５ｍＭのＥＤＴＡ）で予め平衡化させたセファエリルＳ−２００カラム（１．５×９０cm）上に投入した。タンパク質を分画し、ＳＤＳ−ＰＡＧＥ及びウェスタン分析によって検出されるようなＮ１４−ｓｃＦｖを含有する画分をプールし、凍結乾燥させ１／１０体積のＨ₂Ｏ中で懸濁させた。このときＦＡＣＳ分析実験中で負の対照としてＮ１４−ｓｃＦｖ（標識づけされていないもの及びＦＩＴＣで標識づけされたもの）を用いた。
【０２９０】
［３０２.］ ４．４ ＦＩＴＣでの精製済みｓｃＦｖｓの標識づけ：各々の調製物からの精製されたｓｃＦｖ約１ミリグラムをＰＢＳ中で再懸濁させ、メーカーの指示事項に従って、フルオロ−タグＦＩＴＣ接合市販キット（Sigma カタログ番号ＦＩＦＣ−１）を用いて、ＦＩＦＣにカップリングさせた。
【０２９１】
［３０３.］ ４．５ 精製され標識づけされたｓｃＦｖの品質分析
【０２９２】
［３０４.］ ４．５．１ 精製及びＦＩＦＣ標識づけの後、各調製物（標識づけされたものとされないもの）のプロファイルをＳＤＳ−ＰＡＧＥ、ウェスタンブロット法、Superdex-７５カラム（Ａ２８０及びＡ４９５）を用いたＨＰＬＣ及び螢光光度法によって分析した。該分析は、各ｓｃＦｖ分子に対し約２個のＦＩＴＣ分子が接合されている状態でＮ１４ｓｃＦｖについて８０％の純度及びＶ_H３クローンについて９０％の純度を示した（２：１のＦ／Ｐ比）。
【０２９３】
［３０５.］ ４．５．２ ｓｃＦｖ特異性の保持を確認するため、ＦＩＴＣ標識づけの後の結合活性を査定した。
【０２９４】
［３０６.］ ４．６ ファージミドクローンの生化学的特徴づけ：構造を評価しさまざまなｓｃＦｖ調製物（例えば例８参照）の純度を査定するために、ＳＤＳ−ＰＡＧＥ、質量分析法（Ｙ１及びＹ１７ｓｃＦｖｓのみについて）及びＨＰＬＣを含む複数のタイプの分析を使用した。ｓｃＦｖを同定するためにはウェスタン分析及びＥＩＡを使用し、ｓｃＦｖ結合を特徴づけするためにＦＡＣＳを使用した。
【０２９５】
例５：
［３０７.］ ５．結合検定
【０２９６】
［３０８.］ 細胞に対する選択されたクローンの結合は、ファージミドレベルと可溶性ｓｃＦｖレベルの２つのレベルで評価された。
【０２９７】
［３０９.］ ５．１ ファージミドレベルでの結合
【０２９８】
［３１０.］ この目的で、選択されたクローンの各々から個別にファージミドストックを調製した。
【０２９９】
［３１１.］ ５．１．１ コロニー試験：１組の実験で、感染したE.coliにアンピシリン耐性を付与するバイオパンニングプロトコルから誘導された１０⁹の特異的ファージミドと、アンピシリン耐性をもたず「ブロッカ」として役立つ１０¹¹の野生型Ｍ１３ファージの混合物を、細胞型パネルから選択した１０⁵個の細胞と共にインキュベートした。インキュベーションと洗浄の後、結合したファージをトリプシンで溶出し、アリコートを用いてE.coliＴＧ−１を感染させた。E.coliを次に２×ＴＹ／ＡＭＰ平板上に固定し、３０℃で一晩インキュベートした。各クローンについて得たコロニーの数を計算し比較した。結果は、ファージミドの結合親和性及び特異性の１つの尺度を提供している。
【０３００】
［３１２.］ ５．１．２ 白色／青色コロニー試験：各実験が内部対照を内含しているこの試験においては、特定のファージミドを、ｐＧＥＭ７と呼ばれるもう１つの対象ファージミド（Promega Corp., Madison, Wisconsin,ＵＳＡ）と、上記第５．１．１節と同じ比率すなわち１／１００の比で混合した。このｐＧＥＭ７ファージミドは、アンピシリン耐性を有する； ただし、これは、そのＰＩＩＩ遺伝子のＮ末端でいかなる組換え型ポリペプチドも発現しない。ＩｍＭ Ｘ−galを含有するアンピシリン平板上でのＴＧ−１感染及びインキュベーションの後、コロニーを計数した。ｐＧＥＭ７を含有する得られたコロニーは青色であるが、一方特異的ファージミドから得られたコロニーは、白色である。各々の試験官について（同じ平板上で成長させられた）白色／青色コロニーの入出力比から誘導された冨化係数を次に計算した。
【０３０１】
［３１３.］ ５．１．３ ファージミドのＥＩＡ
【０３０２】
［３１４.］ ５．１．３．１ 選択された細胞に対するファージミドの結合：２４ウェル平板の表面上にアセトン：メタノール（１：１）を用いて約５×１０⁵の選択された細胞を定着させた。結合試験は１０⁹のファージミドを必要とした。１時間３７℃で結合を実施し、その後ＰＢＳ／Tween（０．０５％）で大規模に洗浄を行なった。ＰＢＳでの大規模な洗浄の後、平板をウサギ抗−Ｍ１３、抗−ウサギＩｇＧ−ＨＲＰ及び基質と共にインキュベートした。生成された色の強度は、Ａ４０５でＥＬＩＳＡ平板読取り装置により読取られ、結合されたファージミドのレベルに正比例していた。
【０３０３】
［３１５.］ 定着した血小板に対するファージミドの結合： ポリスチレンマイクロタイター−平板に１０⁸個の定着した血小板をコーティングし、４℃で一晩インキュベートした。結合を評価するために、約１０¹⁰のファージミドを使用した。平板の洗浄及びインキュベーション及び結合レベルの決定は、上記５．１．３．１で記述された通りに行なわれた。
【０３０４】
［３１６.］ ５．１．４ ヒト成長ホルモン（ｈＧＨ）、フィブリノーゲン、フィブロネクチン、ＢＳＡ、ＳＭ（脱脂粉乳）及びグリコカリシン（ＧＰＩｂのタンパク質分解フラグメント）から成るグループの中から選択された特異的タンパク質に対する結合検定を実施した。以下の要領で結合を検定した。２μｇ／ウェルで、試験対象のタンパク質の１つでポリスチレンマイクロタイター平板ウェルをコーティングした。４℃で一晩のインキュベーションの間、コーティングを進めさせた。結合をテストするため、約１０¹⁰個のファージミドを添加した。ＰＢＳでの大規模な洗浄の後、平板をウサギ抗Ｍ１３、抗−ウサギＢＲＰ、及び基質と共にインキュベートした。生成された色の強度により結合レベルを測定した。Ａ４０５で光学密度を測定した。各標本を全く同一の条件で検定し、平均を計算した。
【０３０５】
［３１７.］ ５．２ ｓｃＦｖレベルでの結合試験：ＨＢ２１５１の周辺質内で生成されたｓｃＦｖｓの結合を、ＥＩＡ及びＦＡＣＳ分析という２つの異なる検定によって複数の細胞型において比較した。
【０３０６】
［３１８.］ ５．２．１ 可溶性ｓｃＦｖのＥＩＡ：５〜１０μｇの合計タンパク質と共に約５×１０⁵のＡＭＬ細胞をインキュベートした。結合を１時間４℃で行ない、その後マウス抗−ｍｙｃ抗体、抗マウスＨＲＰ、及び基質を用いてＥＩＡを行なった。各段階の後、余剰の未結合抗体を、ＰＢＳでの３回の細胞洗浄によって除去した。生成された色の強度をＥＬＩＳＡ平板読取り装置（Ｏ.Ｄ.₄₀₅）により読みとる。上述の通り、色の強度は、結合レベルに正比例している。
【０３０７】
［３１９.］ ５．２．２ 細胞のＦＡＣＳ分析
【０３０８】
［３２０.］ 「３段階染色」手順により染色させる細胞の分析： 選択されたクローンの特異性をテストし確認するためにＦＡＣＳ分析を実施した。最初に、まずは粗製抽出物又は精製された標識づけされていないｓｃＦｖ、次にマウス抗ｍｙｃ抗体、そして最後にＦＩＴＣ又はＰＥで接合された抗マウス抗体を用いて、「３段階染色」手順を確立した。
【０３０９】
［３２１.］ ＦＡＣＳ分析には、フィコールで精製されＰＢＳ＋１％ＢＳＡ中で再懸濁された５〜８×１０⁵個の細胞が必要である。４℃で１時間結合を行なった。各段階の後、細胞を洗浄し、ＰＢＳ＋１％のＢＳＡ中で再懸濁させた。最終染色段階の後、ＰＢＳ、１％のＢＳＡ、２％のホルムアルデヒド中での再懸濁により、細胞を定着させ、次にＦＡＣＳ（Becton. Dickinson）により読取った。
【０３１０】
［３２２.］ ５．２．２．２ 一回の染色段階での、ＦＩＴＣで標識づけされたｓｃＦｖでの細胞の染色： ＰＢＳ＋１％のＢＳＡ中で５〜８×１０⁵個のフィコール精製された細胞と共にＦＩＴＣで標識づけされたｓｃＦｖをインキュベートした。結合を４℃で一時間実施した。その後、細胞を、上述の第５．２．２．１節と同じように洗浄し固定し、ＦＡＣＳにより読取った。
【０３１１】
例６： パンニング及び配列決定の結果
［３２３.］ ６．１ ＡＭプロトコルの結果
【０３１２】
［３２４.］ ６．１．１ ＡＭプロトコルについてのパンニング結果：パンニングのために用いられた推定上のプラスミド数（入力）及びＡＭプロトコルにおいて溶出された推定上の結合したファージミド数（出力）は、下表（表１）中に要約されている：
【０３１３】
【表１】

【０３１４】
［３２５.］ 各々の連続するパンニングで得られた収量（出力）の冨化に留意されたい。さらに、ＡＭＬ全細胞をパンニングするのにＴ１６Ｍ３が使用された場合いかなる出力低下もなく、このことは結合したファージミドが外部細胞表面上の構成要素に特異的である可能性があること又は、この特異的系が比較的多くの非特異的結合ファージミドを含有し得ることを示唆している。
【０３１５】
［３２６.］ ６．１．２ ＡＭプロトコルについてのクローン配列結果： クローンはＴ１６Ｍ１、Ｔ１６Ｍ２及びＴ１６Ｍ３出力ストックから取り上げられ配列決定されたものの、以下に示す結果は、主としてＴ１６Ｍ３．１出力ストックから誘導されたクローンのものである（ＡＭＬ無傷細胞パンニング）。クローンＡＭ１０、ＡＭ１１及びＡＭ１２は、Ｔ１６Ｍ３ストック内で同定されたが、その後の出力においては同定されなかった。
【０３１６】
［３２７.］ Ｖ_H−ＣＤＲ３内で表示されたアミノ酸配列及びテスト対象クローンの出力におけるその頻度は、表２にまとめられている。
【０３１７】
表２．Ｔ１６Ｍ３及びＴ１６Ｍ３．１出力からの、ＡＭバイオパンニングプロトコルの後の選択されたクローン。
【０３１８】
【表２】

【０３１９】
［３２８.］ Ａrg/Ｇly Ｐhe Ｐroのアミノ酸配列は、表２中に提示された１０個の単離済みクローンのうちの７個に存在し、その中の１つのモチーフを表わしている。さらに、同定されたモチーフが、各ケースにおいてＣＤＲ３領域のＮ末端の３つのアミノ酸を表わすという点に留意されたい。従ってこのモチーフは、さらに大きいペプチド又はポリペプチド又はＦｖ分子の一部分としての又はＣＤＲ３領域の１つ又は両方の端部のいずれかを超えて延びるその他のアミノ酸残基と組合わせた形の又は単独の有効なアンカー又は結合部位である。
【０３２０】
［３２９.］ コア配列 Ａrg/Ｇly Ｐhe Ｐro に基づいて、ＡＭＬ細胞に結合するための高い親和性をもつその他のＣＤＲ３領域を構築することができる。これらは、Ａrg/Ｇly Ｐhe Ｐroコア配列を維持する一方で、添加、欠失又は突然変異により上述の５−１２−mersのいずれかを変動させることによって構築することができる。
【０３２１】
［３３０.］ 本発明のＣＤＲ３領域は、アミノ酸配列 Ｒ１−Ａrg/Ｇly Ｐhe Ｐro−Ｒ２を有し、ここでＲ１は０〜１５個のアミノ酸、好ましくは０〜９個、最も好ましくは０〜１個のアミノ酸を含み、Ｒ２は、１〜１５個、最も好ましくは１〜９個のアミノ酸を含む。Ｒ１及びＲ２は、ＡＭＬ細胞に対するＡrg/Ｇly Ｐhe Ｐro 配列の特異的結合に不利な影響を及ぼさないアミノ酸配列である。
【０３２２】
［３３１.］ 上述のクローンの軽鎖のＣＤＲ３領域は同一であり、配列番号１２５の中で列挙されている。
【０３２３】
［３３２.］ ６．２ ＹＰＲ及びＹＰＮＲプロトコルの結果
【０３２４】
［３３３.］ ６．２．１ ＹＰＲ及びＹＰＮＲプロトコルについてのパンニング結果：
パンニングのために用いられた推定上のプラスミド数（入力）及び溶出された推定上の結合したファージミド数（出力）は、下表（表３、４）中に要約されている：
【０３２５】
【表３】

【０３２６】
［３３４.］ 表３は、トリプシン溶出が、第１ラウンドにおける酸溶出と比べて、４倍大きい出力を生み出すことを実証している。
【０３２７】
［３３５.］ 増幅段階無しのＹＰＮＲプロトコルに従った再パンニングは、ファージミド感染及び細菌感染を優先的に増幅させる可能性を最小限にした。結果として得られる出力は、表４に描かれている。
【０３２８】
【表４】

【０３２９】
［３３６.］ 予想通り、表４に示された結果は、各々のパンニングラウンド後のファージ収量の減少を示している。このプロトコルは、非特異的ファージの増幅に起因する偏向を防ぐために使用された。
【０３３０】
［３３７.］ ６．２．２ ＹＰＲ及びＹＰＮＲプロトコルについてのクローン配列結果： 両方のプロトコルからの第３のパンニングからの複数のクローンを、配列決定のために選択した。表５に提示したアミノ酸配列は、重鎖（Ｖ_H−ＣＤＲ３）のＣＤＲ３領域のものである。生殖細胞系列及びＲ３出力に配列が現われた頻度も同じくこの表に示されている。
【０３３１】
表５：Ｒ３出力での、ＹＰＲバイオパンニングプロトコルの後の選択されたＹ系列クローン
【０３３２】
【表５】

【０３３３】
［３３８.］ ＹＰＮＲプロトコルからの単離されたクローンの大部分がＹ１でもあった。
【０３３４】
［３３９.］ 以上のクローンの軽鎖のＣＤＲ３領域は同一であり、配列番号１２５中に列挙されている。
【０３３５】
例７：
［３４０.］ ７．結合評価結果
【０３３６】
［３４１.］ ７．１ ＡＭＬ細胞（ＡＭクローン系列）に対する選択されたファージミドクローンの結合： クローンＡＭ７を除いて、テスト対象細胞に対するいかなる優先的な結合も検出されなかった。ファージミドとしてか又は精製されたｓｃＦｖとしての、全ての標的細胞に対するクローンＡＭ７の有意な、ただし非選択的な結合が観察された。結果は、ＡＭクローン系列についてのいかなる冨化も立証していない。
【０３３７】
［３４２.］ ７．２ Ｙクローン系列の結合
【０３３８】
［３４３.］ ７．２．１ ファージミド結合−定着された血小板を用いたＥＩＡ：２つの異なるプロトコルを用いた３回のパンニングラウンドの後、定着した血小板に対する結合についてＥＩＡによりファージクローンをテストした。選択されたクローンの各々からファージミドストックを調製し、これらのクローンを２セットのＥＩＡにおいてテストした。各標本を全く同一の条件で検定し、平均を計算した。結果は、図１にまとめられており、９個のＹ系列クローンのうちの６個が陽性ＥＩＡ反応を示すということを表わしている。最高の結合度は、クローンＹ１、Ｙ１６、Ｙ１７及びＹ−２７と結びつけられた。負の対照としてファージストックＭ１３（野生型バクテリオファージ）及びＥ６（ＣＬＬ白血病細胞について選択されたもの）を使用した。優性クローン、ファージＹ１は、定着した血小板に対する最高の結合を示し、Ｙ１７と共に、Ｍ１３又はＥ６ファージクローンよりもはるかに高い結合を示した。
【０３３９】
例８：
［３４４.］ ８．ｓｃＦｖｓの詳細な特徴づけ及びクローン結合
【０３４０】
［３４５.］８．１ ｓｃＦｖの構造と同定：Ｙ−Ｉの未変性構造を、Superdex７５カラムを用いたＨＰＬＣ分析によって及び質量分析法によって査定した。前者の方法の結果は、調製中の単量体、二量体及び四量体の存在を表わしている。質量分析法は、２６．５ｋＤの予想上の分子量を同定するのに充分な感度をもち、ｃ−mycタグが分割された場合、２４ｋＤの分子量が得られた。
【０３４１】
［３４６.］ しかしながら、上述の核酸配列及び質量分析法の結果に従うと予想分子量が２６．５ｋＤであるにもかかわらず、ＳＤＳ−ＰＡＧＥの結果は、無傷で未分割の分子が３０ｋＤというみかけの分子量を有するということを表わしている。ｃ−myc−特異的抗体を用いたウェスタン分析は、ＳＤＳ−ＰＡＧＥの３ｋＤの結果を確認し、ｃ−mycタグが無傷の分子の端部に存在するという言外の意味を裏づけた。２つの手順の結果の間の不一致は、方法の精度レベルならびに、テスト対象タンパク質の見かけの分子量を改変し得るＳＤＳ−ＰＡＧＥの現行の条件に起因している。
【０３４２】
［３４７.］ ８．２ 白血病細胞に対する血小板選択されたクローンの結合： 序の部分で記した通り、未成熟の造血細胞上で血小板細胞表面マーカーを発現することが可能である。血小板選択されたクローンの結合をＦＡＣＳ分析によってテストした。全血を染色しその後ＲＢＣ溶解を行なった後、又はIso-prep-（フィコールクッション）で精製された単核細胞について、ＦＡＣＳ分析を実施した。各クローンからｓｃＦｖｓを調製し、プロテインＡ上で精製し、ＦＩＴＣで標識づけした（第４．１節〜４．４節に記述されている通り）。非サプレッサE.coli菌株ＨＢ２１５）内での無傷のｓｃＦｖの産生を可能にするため、グルタミン酸（ＧＡＧ）についてコードするようＤＮＡ部位特異的突然変異誘発によりＹ−２７クローンのＶ_H−ＣＤＲ３内に発見されたアンバーコドン（ＴＡＧ）を突然変異させた。かかる研究のための標的細胞は、さまざまな白血病患者の新鮮な血液標本から単離された細胞である。標本は、イスラエルにある３つの医療センタから入手した。
【０３４３】
［３４８.］ クローンＹ１及びＹ１７は、テスト対象の白血病細胞に対する優先的な結合を示したが、一方その他の全てのＹ系列クローンは、バックグラウンドレベルでの結合しか示さなかった。表６は、さまざまな白血病細胞に対するＦＩＴＣで標識づけされたＹ−Ｉ及びＹ−１７の結合を示している。
【０３４４】
【表６】

【０３４５】
［３４９.］ 表６に分数の形で提示されている結果は、その細胞が各々のテスト対象抗体と陽性反応するものとしてＦＡＣＳ分析によって同定された患者の部分を表わしている。分子は、陽性患者の数を表わし、分母密度は、一定の与えられたｓｃＦｖ／細胞型組合せについてテストされた合計患者数を表わす。Ｙ−１７は全てのテスト対象細胞に対し強く結合した。この結合はかくして、細胞選択的でないものとみなされた。しかしながら、Ｙ１結合は、白血病細胞特に急性期にある白血病細胞の複数の試料についてきわめて選択的であることが発見された。Ｙ１−ｓｃＦｖ結合を、以下で記述するようにさらに分析した。
【０３４６】
［３５０.］ ３つのＡＭＬ標本に対するＹＩ結合の代表的結果は、図３に示されている。各々のケースにおいて、細胞集団の大部分が負の対照ｓｃＦｖでの染色により得られたバックグラウンド螢光のものよりもはるかに高い強度で螢光を発する。これらの結果は、各々の患者についてＹ１が合計細胞集団の異なる画分に結合するということを表わしている。各グラフ中の右側Ｙ−Ｉピークは、集団内の最少数のＹ１結合細胞を表わすものと考えられ、このピークより下の合計細胞の割合は、各標本中のＹＩ結合細胞の最小の割合を表わしている確率が最も高い。
【０３４７】
［３５１.］ ８．３ 正常な血球に対するＹ−Ｉの結合： 異なる血球型に応じて、フィコール精製された正常な血球に対するＹ１結合を分析した。正常なリンパ球に対する結合は全く検出されなかったが、Ｙ１は、被験者２８名のうち９名からのフィコール精製された単球、被験者８名のうち５名からの血小板、そして被験者４名のうち１名からの赤血球（ＲＢＣ）に対し結合した。ただし、単球調製物の全て及び好中球調製物の多くにおいてＣＤ１４−特異的抗体が細胞に結合した。この分析の要約が表７に示されている。
【０３４８】
【表７】

【０３４９】
［３５２.］ これらの結合結果は、各々のテスト対象抗体と陽性反応するものとしてＦＡＣＳ分析によって同定された正常な血液標本の部分を表わす。定着した血小板上で選択されたものの、ＦＩＴＣ−Ｙ１ ｓｃＦｖが血小板に対し相対的に低い結合親和性を示す、という点に留意されたい。
【０３５０】
［３５３.］ 図４は、フィコール精製された血小板（４ａ）及び単球ゲートがけされた細胞（４ｂ）に対するＹＩの結合を実証している。単球細胞集団上のシフトは、血小板上で観察されたものよりも大きく、計算上の平均螢光は、負の対照よりもそれぞれ３０倍及び５０倍大きい。この観察事実は、フィコール精製された単球に対し数多く接着する血小板の特性に起因する確率が最も高い。その後の実験により、全血標本で検定された場合、テスト対象の正常な単球、顆粒球、血小板又はＲＢＣのいずれにおいてもいかなるＹ１結合も観察されないということが示された。同様にして、血小板富有血漿（ＰＲＰ）から誘導された場合、血小板に対するＹ１結合が全く見られなかった。同じ結合条件下で（全血中で、ＦＡＣＳ溶解溶液でのＲＢＣ溶解［Becton Dickenson］を伴う）、Ｙ１は、Ficoll精製の後に得られるものと類似の形で白血病細胞に結合した。従って我々は、天然の条件下では、血小板又は単球上のＹ−Ｉエピトープが隠されていると結論づけることができる。フィコール精製手順の間、エピトープは露呈されて、Ｙ１により認識されるべくアクセス可能となっており、一方白血球細胞については、エピトープは、精製条件及び非精製条件の両方の下で露呈されている。
【０３５１】
［３５４.］ リンパ系列及び骨髄系列の正常な造血始原細胞に加えて、臍帯血中の造血基幹細胞（ＣＤ３４＋細胞）に対するＹ１結合をテストした。図５は、臍帯血ＣＤ３４＋基幹細胞に対するＦＩＴＣ−標識づけされたｓｃＦｖクローンの結合結果を示している。図５ａは、ＦＩＴＣ標識づけされた負の対照ｓｃＦｖに対するＣＤ３４＋ゲート済み細胞の結合の結果を示し、図５ｂは、ＦＩＴＣ標識づけされたｓｃＦｖクローンＹ１に対するＣＤ３４＋ゲート済み細胞の結合についての同じ分析を示している。図５ｃは、５ｂの場合と同じくＦＩＴＣ標識づけされたｓｃＦｖクローンＹ−Ｉ標本のＦＳＣ及びＳＳＣドットプロット分析を示している。この分析の結果は、前方散乱の差を（ＦＳＣ）を細胞サイズの標示として、臍帯血から誘導された２つのＣＤ３４＋基幹細胞亜集団の存在を表わしていた。Ｙ１は、２つの集団の小さい方のサイズの細胞に結合する。図５ｂ及び図５ｃ内の囲まれた部域は、クローンＹ１ｓｃＦｖを結合させるＣＤ３４＋細胞の亜集団の輪郭を描いている。さらなる分析により、小さい方のサイズの細胞が、細胞集団内に存在する死滅した細胞であり、Ｙ１結合が、Ｙ１により認識された細胞内リガンドの存在を表わしている可能性がある、ということがわかった。
【０３５２】
［３５５.］ ＧＭ−ＣＳＦで前処理された健康なドナーの末梢血球についても実験を行なった（ＧＭ−ＣＳＦ処理は、血流内への基幹細胞放出を動員する）。図５に示されたものと類似の結果が得られた。
【０３５３】
［３５６.］ ８．４ ＡＭＬ細胞上のさまざまな細胞マーカーに比べたＹ１ｓｃＦｖの結合特異性： ＡＭＬ患者からのフィコール精製された末梢細胞及び骨髄細胞のＹ１染色を、その他の一連の抗体によるこれらの細胞の染色と比較した。１４名の患者から得た標本についてのかかるＦＡＣＳ分析の結果が、表８にまとめられている。Ｙ１を内含するテスト対象マーカーの全てについて、さまざまな個体からの調製物内で、染色された細胞の頻度に有意な可変性が存在する、という点に留意されたい。さまざまなマーカーの結合とＹ１の結合の間の相関関係の欠如は、Ｙ１がその他のテスト対象マーカーにより結合されるリガンドのいずれにも結合せず、Ｙ−Ｉリガンドがテスト対象細胞表面マーカーのいずれをも構成しないということを示唆している。
【０３５４】
【表８】

【０３５５】
［３５７.］ 結果は、各々の個別の抗体と反応する可能性があるものとしてＦＡＣＳ分析によって同定された。一定の与えられた患者のフィコール精製された標本中の細胞の百分率として表現されている。
【０３５６】
［３５８.］ 結合検出のために必要とされるＹ１の濃度（〜１μｇ／５×１０⁵）に照らして、結果は、Ｙ１ｓｃＦｖがＡＭＬ細胞上の特異的リガンドに対して比較的高い結合親和性を有することを表わしている。
【０３５７】
［３５９.］ ＡＭＬ細胞に対するＹ１の結合を示す表８に提示された結果に加えて、我々は以上で（表６）、Ｙ１が、Ｂ−ＡＬＬ細胞を含めたテスト対象のその他の大部分のタイプの白血病細胞にも結合できるものの、これらのその他の白血病細胞試料についての標本サイズには制限があったということを示した。図６は、２人の患者から得た前Ｂ−ＡＬＬ細胞に対するＹ１ｓｃＦｖ結合のＦＡＣＳ分析を提示している。市販のＰＥ標識づけされたＣＤ１９（正常な末梢Ｂ細胞についてのマーカー：図６ａ、図６ｃ）又はＰＥ標識づけされたＣＤ３４（基幹細胞についてのマーカー：図６ｄ）のいずれかを用いた２重染色手順を、ＦＩＴＣ標識づけされた負の対照ｓｃＦｖ又はＦＩＴＣ標識づけされたＹ１ｓｃＦｖと共に利用した。図６ｂは、２重の負の対照である。負の対照染色パターンとの関係における、ＦＩＴＣ標識づけされた標本（ｓｃＦｖクローンＹ１）により結合された細胞の螢光強度（Ｘ軸）が提示されている（６ｅ及び６ｆ）。図６の結果は、テスト対象の２つの標本の各々の内部の白血病前Ｂ−ＡＬＬ細胞の大部分が、Ｙ−Ｉ結合に起因してＹ１ 細胞染色について陽性であることを実証している。
【０３５８】
［３６０.］ ８．５ 細胞系統に対するＹ１−ｓｃＦｖの結合： 悪性造血系列から誘導された複数の細胞系統を、Ｙ１により認識されるその能力についてスクリーニングした。ＦＡＣＳ分析は、Ｙ１がテスト対象細胞の多くに結合することを示している（表９）。１つのヒトＢ細胞系統と１つのマウス骨髄細胞系統のみがテストされたという点に留意されたい。重要なことに、この結合は、対数増殖中の細胞に限られていた。静止期にある細胞は一般にＹ１に結合せず、このことはすなわち、Ｙ１リガンド発現が細胞のライフサイクル中規制されているということを表わしていた。さらに、結合強度は、反応する細胞の間で異なっている。この観察事実は、異なる細胞において、リガンドの親和性又は発現レベルに差があるということを暗に意味している。
【０３５９】
【表９】

【０３６０】
［３６１.］ ８６ ＤＴＴの存在下で精製されたＹ１の結合：Ｙ１クローンがひとたび選択された時点で、ｓｃＦｖを産生するためのプロセスがさらに開発された。Ｙ１バッチのＦＩＬＣ分析の結果は、タンパク質が多量体化でき、主として単量体及び四量体を形成し、これら２つの形態の間の比率が調製物毎に異なるということを示した。均質な材料を得るためには、プロテインＡセファロースカラム上での親和性精製中に５ｍＭのＤＴＴを添加し、その後ＰＢＳ緩衝液の交換によりこれを除去した。実際、ＤＴＴ処理後、大部分（＞９０％）の材料が単量体画分内で発見された。（ＤＴＴの存在下で精製されＨＰＬＣ上で分析された）Ｙ１の単量体形態の結合とＹ１形態の混合物の結合の間には、有意な差が全く見い出されなかった。
【０３６１】
［３６２.］ ８．７ Ｙ１は白血病細胞に対する特異的クローンである： Ｙ１カセットは、Ｙ_H−ＤＰ３２生殖細胞系列に属する。同じ生殖細胞系列に由来する複数のその他のクローンを単離し、これについては例６で詳述されている。これらのクローンにはＹ１７、Ｙ−２７及びＹ−４４が含まれている。これらのクローン全ての一次配列（すなわち生殖細胞系列カセット）は、そのＣＤＲ３領域のみにおいて異なっている。しかしながら、Ｙ１のみが白血病細胞に対する選択性を示す。これらのクローンのＣＤ３配列は、表１０に要約されており、クローンの結合プロファイルは表１１に要約されている。
【０３６２】
【表１０】

【０３６３】
【表１１】

【０３６４】
［３６３.］ 表１０及び１１は、一次配列が、Ｖ_H−ＣＤＲ３領域を除いて４つのクローン間で同一であるものの、結合プロファイルはクローン毎に著しく異なっている、ということを示している。この観察事実は、Ｖ_H−ＣＤＲ３領域の配列が、抗原に対する結合部位の特異性において重要な役割を果たすという概念を強化している。ここで、ＣＤＲ３配列の長さも、それが置かれている特異的生殖細胞系列カセットも、結合特異性の主要な決定因子であると思われない、という点に留意されたい。Ｙ１７及びＹ−２７は各々、Ｙ１と同様６−merＣＤＲ３を含み、３つのクローン全ての重鎖が同一の生殖細胞系列から誘導されている。Ｙ１７及びＹ−２７の場合、造血細胞に対する選択的結合は、実証されなかった。
【０３６５】
例９
［３６４.］ ９．１ triabodiesの構築： もとのＹ１をコードするベクターｐＨＥＮ−Ｙ１を、Ｖ_L及びＶ_H領域の両方についてＲＣＲを用いて個別に増幅させた。Ｖ_LＰＣＲ反応のためには、センスオリゴヌクレオチド５′−ＡＡＣＴＣＧＡＧＴＧＡＧＣＴＧＡＣＡＣＡＧＧＡＣＣＣＴ、及びアンチセンスオリゴヌクレオチド５′−ＴＴＴＧＴＣＧＡＣＴＣＡＴＴＴＣＴＴＴＴＴＴＧＣＧＧＣＣＧＣＡＣＣを用いた。〜３５０ｂｐの予想されたサイズのｃＤＮＡ産物を精製し、配列決定し、ＸhoＩ及びＮotＩ制限酸素で消化させた。
【０３６６】
［３６５.］ （センスオリゴヌクレオチド５′−ＡＴＧＡＡＡＴＡＣＣＴＡＴＴＧＣＣＴＡＣＧＧ及びアンチセンスオリゴヌクレオチド５′−ＡＡＣＴＣＧＡＧＡＣＧＧＴＧＡＣＣＡＧＧＧＴＡＣＣを用いて）Ｖ_H領域を増幅するために、同じ手順を利用した。Ｖ_HＰＣＲ産物をＮcoＩ及びＸhoＩ制限酸素で消化させた。ＮcoＩ−ＮotＩで予め消化されたｐＨＥＮベクター内への３重連結手順利用した。最終ベクターをｐＴria−Ｙ１と呼称した。
【０３６７】
［３６６.］ E.coli形質転換の後、ＤＮＡ配列決定、タンパク質発現、及び細菌の周辺質空間からの抽出を含めたさらなる分析のため、複数のクローンを取上げた。還元条件下でのＳＤＳ−ＰＡＧＥ及びウェスタンブロット分析を実施して、Ｙ１ triabodiesのサイズを確認した。
【０３６８】
［３６７.］ ９．２ diabodiesの構築
【０３６９】
［３６８.］ 以上のものからのpTria−Ｙ１ベクターをＸhoＩ制限酵素で線形化し、合成の相補的２本鎖オリゴヌクレオチド（５′−ＴＣＧＡＧＡＧＧＴＧＧＡＧＧＣＧＧＴ及び５′ＴＣＧＡＡＣＣＧＣＣＴＣＣＡＣＣＴＣを予めアニールし、Ｙ１−重鎖とＹ１−軽鎖間でＸhoＩ部位内に連結させた。この新しいベクターを、ｐＤia−Ｙ１と呼称した。Triabodiesについて記述した通りに、ＤＮＡ配列及びタンパク質発現を確認した。
【０３７０】
［３６９.］ ９．３ diabodies及びtriabodiesの発現及び精製
【０３７１】
［３７０.］ E.coli中の発現は、基本的に、ｓｃＦｖ−Ｙ１について上述した通りであった。ただし、形質転換されたE.coli細胞の周辺質からのＹ１ diabodies及びtriabodiesの精製は異なっていた。ｓｃＦｖ Ｙ１単量体形態を、プロテインＡセファロースビーズの親和性カラム上で精製することができた。しかしながらＹ１の単量体形態をこの手順により精製しても、効果は無かった。従って、細菌から抽出された周辺質タンパク質を６０％の硫酸アンモニウムで一晩沈殿させ、Ｈ₂Ｏ中に再懸濁させ、０．１×ＰＢＳで予め平衡化したセファクリル−２００（Pharmacia）サイズ排除カラム上に装入した。画分を収集し、ＨＰＬＣで分析し、二量体又は三量体のいずれかの形態を含む別々の画分を、ＦＩＴＣ標識づけ及びＦＡＣＳ分析のために収集した。
【０３７２】
［３７１.］ ９．４ 細胞に対するＹ１ diabodies及び triabodiesの結合
【０３７３】
［３７２.］ 「３段階染色手順」を用いてジャーカット細胞に対しＦＡＣＳ分析を実施した。最初に、粗製抽出物又は精製済みの標識づけされていないｓｃＦｖが染色され、次にマウス抗−ｍｙｓ抗体、そして最後にＦＩＴＣ−又はＰＥ−接合された抗マウス抗体が染色される。ＦＡＣＳ分析は、フィコール精製されＰＢＳ＋１％ＢＳＡ中で再懸濁された５〜８×１０⁵細胞を必要とする。結合は、４℃で１時間実施された。各段階の後、細胞を洗浄し、ＰＢＳ＋１％ ＢＳＡ中で再懸濁させた。最終的染色段階の後、ＰＢＳ、１％のＢＳＡ、２％のホルムアルデヒド中で再懸濁させることにより細胞を定着させ、その後ＦＡＣＳにより読取った（Becton-Dickinson）。
【０３７４】
［３７３.］ Ｙ１−ｓｃＦｖの結合を、diabodies及び triabodiesのものと比較した。この分析（図７）中、３つの形態全ての結合プロファイルは非常に類似しており、これは、分子中の上述の修飾が、リガンドに対するＹ１の見かけの結合親和性を改変、隠匿又は破壊しなかったことを表している。
【０３７５】
［３７４.］ ９．５ Ｙ１−ｃｙｓ−ｋａｋ（システイン二量体）の産生
【０３７６】
［３７５.］ λｐＬ−ｙｌ−ｃｙｓ−ｋａｋ細菌培養１リットルを２〜３時間４２℃で誘発させた。この培養を３０分間５０００ＲＰＭで遠心分離した。１８０mlのＴＥ中でペレットを再懸濁させた（５０ｍＭのトリス−ＨＣｌ、ｐＨ７．４、２０ｍＭのＥＤＴＡ）。８mlのリゾチーム（５mg／mlのストックからのもの）を添加し、１時間インキュベートした。５ＭのＮａＣｌ及び２５mlの２５％トリトンを添加し、さらに１時間インキュベートした。この混合物を４℃で６０分間１３０００ＲＰＭで遠心分離に付した。上清を廃棄した。テッシュマイザ（又はホモジナイザ）を用いてＴＥ中にペレットを再懸濁させた。このプロセスを、封入体（ペレット）の色が灰色／明褐色となるまで、３〜４回くり返した。封入体を、６Ｍのグアニジン−ＨＣｌ、０．１ＭのトリスｐＨ７．４、２ｍＭのＥＤＴＡ中で可溶化させた（１０mlの可溶化緩衝液中の１．５グラムの封入体は〜１０mg／mlの可溶性タンパク質を提供した）。これを少なくとも４時間インキュベートした。タンパク質濃度を測定し、１０mg／mlの濃度にした。ＤＴＥを６５ｍＭの最終濃度まで添加し、室温で一晩インキュベートした。０．５Ｍのアルギニン、０．１ＭのトリスｐＨ８、２ｍＭのＥＤＴＡ、０．９ｍＭのＧＳＳＧを含有する溶液に対し１０mlのタンパク質を希釈させる（滴下による）ことにより再折畳みを開始した。再折畳み用溶液を４８時間〜１０℃でインキュベートした。タンパク質を含有する再折畳み用溶液を、２５ｍＭのリン酸緩衝液ｐＨ６、１００ｍＭの尿素を含む緩衝液中で透析し、５００mlsに濃縮した。濃縮／透析済み溶液をＳＰ−セファロースカラムに結合させ、ＮａＣｌ勾配により（最高１Ｍまで）溶出した。
【０３７７】
［３７６.］ ９．６ ＫＧ−１細胞でのラジオレセプタ結合検定（ＲＲＡ）を用いた、ＣＯＮＹ１及びＹ１−ＩｇＧとの比較におけるＳ−ＳＹ１−二量体の親和性研究
【０３７８】
［３７７.］ 検定系には、ＹＩ−ＩｇＧ構成体上のクロラミンＴ又はＣＯＮＹ１（Ｙ１ｓｃＦｖ）構成体上のBolton-Hunter試薬を用いた¹²⁵Ｉでのヨウ化によって調製された放射性リガンドの使用が関与していた。検定用管には、０．２mlあたり５×１０⁶個のＫＧ−１細胞及びＰＢＳｔ ０．１％ ＢＳＡ、ｐＨ７．４中の変動する量の標識づけされていない競合物質を伴う標識づけされたトレーサが入っていた。４℃で撹拌しながら１時間インキュベートした後、細胞を低温緩衝液で徹底的に洗浄し、放射性計数のため取り上げた。
【０３７９】
［３７８.］ 標識づけされたＹ１−ＩｇＧを用いたＲＲＡ研究では、管１本あたり２ngの¹²⁵Ｉ−Ｙ１−ＩｇＧを使用し、３つの分子の各々で競合を実施した。結果は図８中に記述されている。この図に提示された結果は、Ｓ−ＳＹ１二量体の親和性がＣＯＮＹ１の親和性より３０倍高かったということを実証している。この実験におけるＹ１−ＩｇＧの親和性のおおまかな見積りは２×１０^-8Ｍである。従って、二量体の対応する親和性は４×１０^-8Ｍである。
【０３８０】
［３７９.］ 標識づけされたＣＯＮＹ１を用いた第２のＲＲＡでは、１００ng／本の¹²⁵Ｉ−Ｙ１−ＩｇＧを使用し、３つの分子の各々で競合を実施した。結果は図９中に記されている。この図は、Ｓ−Ｓ二量体の親和性がＣＯＮＹ１の親和性より２０倍高かったということを実証している。この実験におけるＣＯＮＹ１の親和性のおおまかな見積りは１０^-6Ｍである。従って、二量体の対応する親和性は５×１０^-8Ｍである。
【０３８１】
［３８０.］ ９．７ ＧＣ（グリコカリシン）に対するＥＬＩＳＡ
【０３８２】
［３８１.］ １００μｌの精製されたグリコカリシンを４℃で一晩、９６フラットウェル付MaxiSorp平板内でインキュベートした。平板をＰＢＳＴ（ＰＢＳ＋０．０５％のTween）で３回、次に２００mlのＰＢＳＴ−ミルク（ＰＢＳＴ＋２％の脱脂乳）で、室温で１時間洗浄した。平板をＰＢＳＴで洗浄し、室温で１時間異なる濃度でＰＢＳＴ−ミルク中に単量体又は二量体（１００μｌ）を添加した。その後、平板を洗浄し、抗−Ｖ_Lポリクローナル（Ｙ１から誘導されたＶ_Lを伴う免疫化されたウサギから誘導されたもの）（ＰＢＳＴ−ミルク中で１：１００に希釈）を１時間添加した。平板を洗浄し、さらに１時間抗ウサギＨＲＰを添加した。平板を５回洗浄し、さらに１時間抗ウサギＨＲＰを添加した。平板を５回洗浄し、１００μｌのＴＭＢ基質を約１５分間添加し、次に１００μｌの０．５Ｈ₂ＳＯ₄を添加して反応を停止した。ＥＬＩＳＡ読取り装置内で４５０ｎｍで平板の光学密度を測定した。
【０３８３】
［３８２.］ ９．８ 原核生物（E.coli）系内で発現された組換え型グリコカリシン（ＧＣ）とのＹ１の反応性。
【０３８４】
［３８３.］ ヒト血小板ＧＰ１ｂ−グリコカルシン（ＧＣ、アミノ酸１〜アミノ酸４９３）のＮ末端可溶性部分をコードするＤＮＡフラグメントを、ＩＰＴＧ誘発可能な原核生物ベクターカセットへとクローニングした。新しく構築したプラスミドを包含するE.coli（ＢＬ２１、ＤＥ３）細胞を３７℃でO.D. ０．７〜０．８まで成長させ、次に、誘発のためのＩＰＴＧの存在について３時間３７℃で成長させた。天然の半精製されたヒト血小板由来のＧＣか又は、誘発された及び誘発されていない細胞から誘導されたE.coli 細胞リゼイト（合計タンパク質含有量）のいずれかが装入されたＳＤＳ−ポリアクリルアミドゲルを分析した。ｓｃＦｖ Ｙ１−ビオチニル化されたポリクローナルウサギ抗ヒトＧＣ抗体、市販のマウス抗ヒトＣＤ４２モノクローナル抗体（ＳＺ２ Immunotech, ＰＭ６４０ Serotee, ＨＩＰ１ Pharmigen, ＡＮ５１ ＤＡＫＯ）及び、ｇｐＩｂａのＮ末端に対するポリクローナル抗体（Ｓｃ−７０７１、Santa Cruz）を用いて、ウェスタンブロット分析を実施した。２つのポリクローナル抗体は、組換え型細菌由来ＧＣならびに天然のヒト血小板由来ＧＣの両方を認識した。ｓｃＦｖＹ１及び市販の抗体は、天然のヒト由来ＧＣのみを認識し、細菌由来の組換え型血小板ＧＣを認識しなかった。
【０３８５】
［３８４.］ グリコシル化及び硫酸化といったような翻訳後修飾は、ＧＣに対するｓｃＦｖ及び市販の抗体の結合にとって不可欠である。原核生物（E.coli）系には、グリコシル化及び硫酸化といったような翻訳後修飾メカニズムが欠如している。
【０３８６】
［３８５.］ ９．９ Ｙ１の四量体の作製
【０３８７】
［３８６.］ 以下の配列、ＬＮＤＩＦＥＡＱＫＩＥＷＨＥを、ＰＣＲ及びＩＰＴＧ誘発可能発現系へのクローニングによりＹ１のＣ末端で添加した。クローンをＹ１−biotagと命名した。この配列は、遊離ビオチンの不在下で、酵素がビオチンをリシン（Ｋ）残基に共有結合により連結させることのできる、酵素BirＡのための基質である（抗原特異的Ｔリンパ球の表現型分析。Science, １９９６．１０月４；２７４（５２８４）：９４−６，Altman JD et al）。この構成体は、ＢＬ２１細菌細胞中で封入体として産生された。前述の通りに再折畳みを行なった。封入体をグアニジン−ＤＴＥ内で可溶化させた。再折畳みは、アルギニン−トリス−ＥＤＴＡを含有する緩衝液中での希釈によって行なった。透析及び濃縮を実施し、その後HiTrapＱイオン交換精製を行なった。
【０３８８】
［３８７.］ BirＡ酵素（Avidityより購入）及びビオチンと共に、プロバイダによる推奨通りに、精製済みＹ１−biotag ｓｃＦｖをインキュベートした。ＨＡＢＡ試験（分子１個あたりのビオチンの量を推定するもの）により、ビオチニル化されたＹ１−biotagを分析し、１分子あたり約０．８を上回るビオチン分子が存在することが実証された。
【０３８９】
［３８８.］ ビオチニル化したＹ１−biotagをストレプトアビジン−ＰＥ（Phycoerytrin）と共にインキュベートして複合体を形成させ、これを、ＫＧ−１細胞（Ｙ１について陽性）を用いてＦＡＣＳ実験中で使用した。ストレプトアビジンは、最高４つのビオチニル化されたＹ−１−biotag分子を結合させることができる。結合の感応性は、結合力の増加に起因して、少なくとも１０倍増大した。
【０３９０】
［３８９.］ Ｙ１−biotagの配列は、以下の通りである：
【０３９１】
【化３】

【０３９２】
［３９０.］ 例１０： フルサイズのＹ１−ＩｇＧ１の構築
【０３９３】
［３９１.］ 全ＩｇＧ分子は、in vivoでのより長い半減期及びＡＤＣＣ又はＣＤＣ（補体依存性細胞毒性：Tomlinson, Current Opinions of Immunology, ５，８３−８９（１９９３））により媒介されるものといったようなin vivoでの細胞応答を誘発する潜在性を含め、Ｆｖ形態に比べていくつかの利点を有している。以下で記述する分子クローニングアプローチにより、我々は、Ｙ１Ｆｖ領域をフルサイズのＩｇＧＩ分子へと変換した。以下の順序でｃＤＮＡのフラグメントをつなぐことによってＹ１ＩｇＧＩ構築を達成した。
【０３９４】
［３９２.］ １０．１ 哺乳動物の発現系のための相容性あるリーダー配列： 完全ＩｇＧ分子に必要とされる要素の適切な挿入を可能にするように、交換可能な系を設計した。推定上のリーダー配列をコードする以下の相補的２本鎖オリゴヌクレオチドを合成し、アニールし、哺乳動物発現ベクターのＸhoＩ部位内に連結した（ＳＲα５プロモータの下で）。
【０３９５】
【化４】

【０３９６】
開始ＡＴＧコドンの上流側に、２つのKozak要素を含み入れた。さらに、可変的領域のサブクローニングを可能にするためＸhoＩ部位とリーダー配列の推定上の分割部位の間に内部ＥcoＲＶ部位を導入した。この修飾済みベクターをｐＢＪ−３と命名した。
【０３９７】
［３９３.］ １０．２ リーダー及び定常軽量領域コーディング配列の間にＶ１ｓｃＦｖ ｃＤＮＡ配列から誘導されたＶ_Lコーディング配列を挿入した。同様にして、リーダー及び定常重量領域コーディング配列の間にはＹ１ ｓｃＦｖ ｃＤＮＡ配列を挿入した。これは、個別にＹ_L及びＶ_H領域を得るため、もとのＹ１についてコードするベクターｐＨＥＮ−Ｙ１のＰＣＲ増幅により達成された。
【０３９８】
［３９４.］ １０．３ オリゴヌクレオチド
Ｖ_LＰＣＲ反応のためには、５′−ＴＴＴＧＡＴＡＴＣＣＡＧＣＴＧＧＴＧＧＡＧＴＣＴＧＧＧＧＧＡ（センス）及び５′−ＧＣＴＧＡＣＣＴＡＧＧＡＣＧＧＣＡＧＣＴＴＧＧＴ（アンチセンス）を使用した。〜３５０ｂｐの予想サイズのｃＤＮＡ産物を精製し、配列決定し、ＥcoＲＶ及びＡrtＩＩ制限酵素で消化した。それぞれセンス及びアンチセンスオリゴヌクレオチド ５′−ＧＧＧＡＴＡＴＣＣＡＧＣＴＧ（Ｃ／Ｇ）（Ａ／Ｔ）ＧＧＡＧＴＣＧＧＧＣ及び５′−ＧＧＡＣＴＣＧＡＧＡＣＧＧＴＧＡＣＣＡＧＧＧＴＡＣＣＴＴＧを用いて、同じ手順を利用してＶ_HｃＤＮＡ領域を増幅し精製した。
【０３９９】
［３９５.］ １０．４ 定常領域： ＩｇＧ１ｃＤＮＡについて誘導された定常λ３（ＣＬ−λ３）領域及び定常重量領域 ＣＨ１〜ＣＨ３を、以下のとおりに個々に合成した：
【０４００】
［３９６.］ １０．４．１ 定常ＣＬ−λ３領域については、センス５′−ＣＣＧＴＣＣＴＡＧＧＴＣＡＧＣＣＣＡＡＧＧＣＴＧＣ及びアンチセンス５′−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＧＡＡＣＡＴＴＣＴＧＴＡＧＧＧＧＣＣＡＣＴＧＴオリゴヌクレオチドと組合わせて通常の末梢Ｂ細胞（ＣＤ１９＋細胞）のプールから抽出したｍＲＮＡについてＲＴ−ＰＣＲを実施した。予想されたサイズ（〜４００ｂｐ）のＰＣＲ産物を精製し、配列決定し、ＡvrＩＩ及びＮotＩ制限酵素で消化させた。
【０４０１】
［３９７.］ １０．４．２ 定常ＩｇＧＩ領域（γ鎖）については、ＢＴＧで不死化させたヒトＢ細胞クローン（ＣＭＶ―クローン＃４０）を、ＰＣＲ増幅のために選択した。このクローンは、ヒトＣＭＶに対するＩｇＧＩを分泌することが示され、同様に、in vitro検定においてＡＤＣＣ応答を誘発することも示された。ＣＨ１〜ＣＨ３ ｃＤＮＡについては、オリゴヌクレオチド５′−ＣＣＧＣＴＣＧＡＧＴＧＣ（Ｔ／Ｃ）ＴＣＣＡＣＣＡＡＧＧＧＣＣＣＡＴＣ（Ｇ／Ｃ）ＧＴＣＴＴＣ（センス）及び５′−ＴＴＴＧＣＧＧＣＣＧＣＴＣＡＴＴＴＡＣＣＣ（Ａ／Ｇ）ＧＡＧＡＣＡＧＧＧＡＧＡＧＧＣＴ（アンチセンス）を合成し、ＰＣＲ増幅のために使用した。ＣＬ ｃＤＮＡコーディング配列について記述したとおりに、予想されたサイズ（〜１５００ｂｐ）のＰＣＲ産物を精製し、配列決定し、ＡvrＩＩ及びＮotＩ制限酵素で消化させた。
【０４０２】
［３９８.］ １０．５ 最終発現ベクターについては、ＥcoＲＶ−ＮotＩ予備消化済みベクター、ＥcoＲＶ−ＡvrＩＩ可変ｃＤＮＡ及びＡvrＩＩ−ＮotＩ定常領域を用いて３重連結手順を実施した。重鎖及び軽鎖発現のための最終ベクターをそれぞれＹ−Ｉ−ＨＣ及びＹ１−ＬＣと呼称した。
【０４０３】
［３９９.］ １０．６ ピューロマイシン耐性遺伝子（ＰＡＣ）に基づく２重選択を可能にするべくＹ１−ＬＣに基づいて付加的ベクター、ｐＢＪ−Ｙ１−ＬＰを構築した。このベクター内で、Ｙ１−ＬＣプラスミドのネオマイシン耐性遺伝子は、（ｐＭＣＣ−ＺＰベクターからの）ＰＡＣ遺伝子についてコードする〜１６００ｂｐのフラグメントで置き換えられた。
【０４０４】
［４００.］ １０．７ Ｙ−１−ＩｇＧ−ＨＣ及びＹ１−ＩｇＧ−ＬＣの両方の読取り枠（ＯＲＦ）とそれらのコードされたアミノ酸配列が、以下で提示されている：
【０４０５】
［４０１.］ １０．７．１ Ｙ１−ＩｇＧ−ＨＣ（Ｖ_H Ｃ_H１ Ｃ_H２ Ｃ_H３）のＯＲＦ
【０４０６】
【化５】

【０４０７】
［４０２.］ １０．７．２ Ｙ１−ＩｇＧ−ＬＣ（Ｖ_LＣ_L）のＯＲＦ
【０４０８】
【化６】

【０４０９】
［４０３.］ リーダー配列には下線が施されている。Ｖ_H及びＶ_L領域は各々、太字で示したアミノ酸配列によりコードされ、その後にはＩｇＧ１（重鎖について）、又はλ３（軽鎖について）定常領域配列が続いている。
【０４１０】
［４０４.］ １０．８ ＣＨＯ細胞中のＹ１重鎖及び軽鎖の発現。
重鎖又は軽鎖を発現する安定した細胞のトランスフェクション及び選択のために、個別にベクターＶ１−ＨＣ及びＹ１−ＬＣを使用した。Ｇ４１８についての選択及び細胞成長の後、上清中の分泌されたタンパク質を以下に記述する通りに捕捉ＥＩＡ検定及びウェスタンブロット分析によりＩｇＧＩ発現について分析した。
【０４１１】
［４０５.］ １０．８．１ 捕捉ＥＩＡ検定： ９６ウェル付き平板のウェルにマウス抗ヒトＩｇＧＩＦｃ（Sigma）を予めコーティングした。上述のものからの上清をウェルに添加し、ビオチニル化されたヤギ抗γ鎖特異的抗体（Sigma），ストレプトアビジン−ＨＲＰ及び基質について重鎖ＩｇＧ１の存在を検出した。ＥＬＩＳＡ平板読取り装置が、Ａ₄₀₅での顕色を監視した。
【０４１２】
［４０６.］ １０．８．２ ウェスタンブロット分析： 上述の細胞についての上清を１２．５％のＳＤＳ−ＰＡＧＥ上で走らせた。各鎖の発現を、（ａ）重鎖検出のためにはヤギ抗ヒトＩｇＧ−ＨＲＰ（Ｈ＋Ｌ；Sigma Cat ＃８６６７）で、又（ｂ）軽鎖検出のためにはビオチニル化されたヤギ−抗ヒトλ３鎖（Southern Biotechnology Association, （at＃２０７０−０８）で検出した。
両方の鎖の発現を、上述の検定によって確認し、フルサイズのＹ１−ＩｇＧ１を得るために同時トランスフェクションを実施した。
【０４１３】
［４０７.］ １０．９ Ｙ１−ＩｇＧの発現及び精製
【０４１４】
［４０８.］ １０．９．１ 細胞培養及びトランスフェクション： ５％ＣＯ₂の雰囲気中で３７℃で１０％のウシ胎児血清及び４０μｇ／mlのゲンタマイシンを伴うＦ−１２培地中でＣＨＯ細胞を培養した。トランスフェクションの一日前に、０．８×１０⁶個の細胞を９０mmの皿上に播種した。Fu Gene（Roche）トランスフェクション試薬技術により、１０μｇの軽鎖及び重鎖ＤＮＡで培養を同時トランスフェクションした。非選択培地中で２日間成長させた後、細胞を、５５０μｇ／mlのネオマイシン及び３μｇ／mlのピューロマイシンを含有するＦ−１２培地中で１０〜１２日間培養した。細胞をトリプシン処理し、Costarの９６ウェル平板内で１ウェルにつき０．５個の細胞の限界希釈によりクローニングした。個々のコロニーを採取し、６ウェル皿の中で成長させ、フラスコに移した。
【０４１５】
［４０９.］ １０．９．２ 重鎖及び軽鎖分泌の決定： トランスフェクションを受けたＣＨＯ細胞の上清の中に分泌された抗体の濃度を決定するためにサンドイッチＥＬＩＳＡ検定を使用した。抗体の濃度を決定するためには、以下の試薬を用いた： コーティングされた抗体としてのモノクローナル抗ヒトＩｇＧ１（Ｆｃ）（Sigma）、検出要素としてのヤギ抗ヒトＩｇＧ（γ−鎖特異的）ビオチン接合体（Sigma）及び、標準としての純粋ヒトＩｇＧ１ラムダ（Sigma）。このＥＬＩＳＡ検定に基づくと産生速度は３〜４μｇ／mlの間で変動した。
【０４１６】
［４１０.］ １０．９．３ 細胞からのＭＡｂ産生及び精製： ネオマイシン及びピューロマイシンで捕捉した１０％のウシ胎児血清を伴うＦ−１２培地の中で１びんにつき１〜２×１０⁸個の細胞という最終濃度まで、ローラーボトル内で細胞を生成させた。産生のためには、細胞を同じ培地内で、ただし２％のウシ胎児血清を伴って、さらに２日間培養した。分泌された抗体をプロテインＧ−セファロースカラム（Pharmacia）上で精製した。結合は、２０ｍＭのリン酸ナトリウム緩衝液、ｐＨ７．０で行なわれ、溶出は０．１Ｍのグリシン緩衝液ｐＨ２．５〜３．０で実施された。精製された抗体の数量は、ＵＶ吸光度により決定された。純度はＳＤＳ−ＰＡＧＥにより分析した。非変性条件下で、完全ＩｇＧ抗体は、１６０ｋＤというその予想上の分子量を有している。変性ゲル内では、重鎖及び軽鎖の両方共、それぞれ５５及び２８ｋＤという予想上の分子サイズを有する。
【０４１７】
［４１１.］ フルサイズＹ１−ＩｇＧ分子の結合：ｓｃＦｖ−Ｙ１分子の結合レベルに比べたＹ１−ＩｇＧの結合レベルを決定するために、結合実験を実施した。５nｇのＹ１−ＩｇＧをＲＡＪＩ細胞（負の対照、図７ａ）及びジャーカット細胞（Ｙ１陽性細胞、図７ｂ）の両方と反応させる２段階染色手順を利用した。検出のためには、ＰＥ標識づけしたヤギ抗−ヒトＩｇＧを使用した。同様にして、ジャーカット細胞（図７ｃ）と１μｇのｓｃＦｖ−Ｙ１を反応させ、検出のためにＰＥ標識づけされたウサギ抗−ｓｃＦｖを使用した。結果は、Ｙ１−ＩｇＧ及びｓｃＦｖ−Ｙ１の両方がジャーカット細胞に結合し、Ｙ１−ＩｇＧのものに類似した検出レベルを得るために約１０³倍多いｓｃＦｖ−Ｙ１分子が必要とされるということを示している。
【０４１８】
表の詳細な説明
［４１２.］ 表１： プロトコルＡＭから誘導されたパンニングの結果。パンニングに用いられたファージミドの推定上の数（入力）及び溶出された結合済みファージミドの推定上の数（出力）がＡＭ バイオパンニングプロトコルの４つの連続的段階についてまとめられている。各々の出力結果についての細胞供給源及び溶出培地が、各々の別々のストックを区別するのに用いた用語と共に列挙されている。
【０４１９】
［４１３.］ 表２： ＡＭバイオパンニングプロトコルの後の選択されたクローン。ＣＤＲ３領域（Ｖ_H−ＣＤＲ３サイズ）内のアミノ酸残基の数及び単離された異なるクローンタイプについてのＣＤＲ３アミノ酸配列が要約されている。さらに、２つのＡＭバイオパンニング出力つまりＴ１６Ｍ３及びＴ１６Ｍ３．１出力における各々のクローンタイプの頻度が提示されている。
【０４２０】
［４１４.］ 表３： ＹＰＲプロトコルから誘導されたパンニングの結果： パンニングに用いられたファージミドの推定上の数（入力）及び溶出された結合済みファージミドの推定上の数（出力）がまとめられている。各々の出力結果についての溶出培地が、各々個別のストックを区別するのに用いた用語と共に列挙されている。
【０４２１】
［４１５.］ 表４： ＹＰＮＲプロトコルから誘導されたパンニングの結果。パンニングに用いられたファージミドの推定上の数（入力）及び溶出された結合済みファージミドの推定上の数（出力）がＹＰＮＲバイオパンニングプロトコルの３つの連続的段階についてまとめられている。各々の出力結果についての溶出培地が、各々個別のストックを区別するのに用いた用語と共に列挙されている。
【０４２２】
［４１６.］ 表５： Ｒ３出力での、ＹＰＲバイオパンニングプロトコルの後の選択されたＹ系列クローン。Ｒ３出力ストックの中で複数の異なるクローンを同定した。同定されたクローンのＶ_H−ＣＤＲ３領域を含むアミノ酸残基の数及びアミノ酸配列、ならびに生殖細胞系列が詳述されている。
【０４２３】
［４１７.］ 表６： 白血病細胞についてのＹ１結合特異性。ＦＡＣＳ分析によって決定されるような７つの異なる白血病細胞型の各々を主として含有する細胞混合物と各々反応させられた３つの異なるｓｃＦｖクローンの結合実験の結果が提示されている。結果は、各々のテスト対象抗体とその細胞が陽性反応するものとしてＦＡＣＳ分析によって同定された患者の部分を表わす。分子は、陽性患者の数を表わし、分母は一定の与えられたｓｃＦｖ／白血病細胞型組合せについてテストされた患者の合計数を表わす。
【０４２４】
［４１８.］ 表７： フィコール精製された正常な血球に対するｓｃＦｖ結合のＦＡＣＳ分析。３つのｓｃＦｖクローンが、フィコール精製された５つの異なる正常な血球型に対する結合について各々分析される。これらの結合結果は、ＦＡＣＳ分析により同定された正常血液標本の部分を、各々のテスト対象抗体と陽性反応するものとして表わしている。
【０４２５】
［４１９.］ 表８： さまざまな細胞マーカーに対する抗体の結合とＹ１ｓｃＦｖ結合の比較。Ｙ１及びその他の一連の抗体による染色のＦＡＣＳ分析結果が表わされている。ＡＮＥ患者からのフィコール精製された末梢及び骨髄細胞を調製し、ＡＭＬ細胞上のさまざまな細胞マーカーに比べたＹ１ｓｃＦｖの結合特異性が研究された。結果は、ＦＡＣＳ分析により各Ｆｖと陽性反応するものとして同定された、一定の与えられた患者のフィコール精製された標本中の細胞の百分率として表現されている。比較のため次の４つのその他の抗体が走らせた： （１）ＣＤ１３−顆粒球及び単球についてのマーカー；（２）ＣＤ１４−単球及び好中球についてのマーカー；（３）ＣＤ３３−正常な骨髄性細胞及び白血病骨髄性細胞についてのマーカー及び（４）ＣＤ３４−基幹細胞についてのマーカー。
【０４２６】
［４２０.］ 表９： 造血細胞系統に対するＹ１の結合。ヒト白血病細胞系統の３つの異なるカテゴリ及び１つのマウス細胞系統に対するＹ１ｓｃＦｖの結合を決定するために、ＦＡＣＳ分析が実施された。Ｙ１が陽性結合した（反応性）又はしなかった（非反応性）細胞系統が列挙されている。
【０４２７】
［４２１.］ 表１０： Ｖ_H３−ＤＰ３２で単離されたクローンのＣＤＲ３配列。異なるバイオパンニング及び選択手順に従って、ＤＰ３２ 生殖細胞系列に基づく複数のクローンが単離された。クローンＹ１、Ｙ１７、Ｙ−２７及びＹ−４４が血小板上のバイオパンニング選択（ＹＰＲ及びＹＰＮＲプロトコル）の間に同定された。これらのクローンの各々のＶ_H−ＣＤＲ３領域の配列が提示されている。
【０４２８】
［４２２.］ 表１１： Ｖ_H３−ＤＰ３２で単離されたクローンの結合プロファイル。複数の造血細胞に対するＤＰ３２由来のクローンの結合特異性は、ＦＡＣＳ分析によりテストされた。
【０４２９】
［４２３.］ 本発明について、特定の例、材料及びデータを基準にして記述してきた。当業者であればわかるように、本発明のさまざまな態様を使用又は調製するための代替的手段が利用可能である。かかる代替的手段は、以下のクレームによって定義される通りの本発明の意図及び本質の範囲内に内含されるものとすべきである。
【０４３０】
［３９.］ 本発明についてここで、制限的な意味のない単なる例として、以下で記述する添付図面を参考にしながらさらに詳しく説明する。なお図面中、
【図面の簡単な説明】
【０４３１】
【図１】［４０.］ 図１は、ＥＩＡ検定により決定される通り、定着された血小板に結合するファージクローンを提示している。データは４０５ｎｍでの吸光度の一関数として提示されている。
【図２】［４１.］ 図２ａ、２ｂ及び２ｃは、ＦＡＣＳ分析によって決定されるような、３人の個別のＡＭＬ患者から得た単核細胞標本の結合を提示する。２つのＦＩＴＣ標識付けされた試験対象の標本（対照ｓｃＦｖ及びｓｃＦｖクローンＹ１）によって結合された細胞の螢光強度が提示されている。
【図３】［４２.］ 図３は、ＦＡＣＳ分析により決定される通りのフィコール精製された単球（３ｂ）及び血小板（３ａ）に対するＹ−Ｉの結合を提示している。２つのＦＩＴＣ標識付けされた試験対象標本（対照ｓｃＦｖ及びｓｃＦｖクローンＹ１）によって結合された細胞の螢光強度が提示されている。
【図４】［４３.］ 図４は、臍帯血ＣＤ３４＋基幹細胞に対するＦＩＴＣ標識づけされたｓｃＦｖクローンＹ１の結合を提示する。ＦＬ３−Ｈチャンネル内のＣＤ３４＋ゲートをかけた細胞を、ＦＬＩ−Ｈ内で、ＦＩＴＣ標識づけされた負の対照ｓｃＦｖ（図４ａ）又は、ＦＩＴＩ標識づけされたｓｃＦｖクローンＹ１（図４ｂ）に対するその結合について分析した。図４ｃは、４ｂと同じＦＩＴＩ標識づけされたｓｃＦｖクローンＹ１標本のＦＳＣ及びＳＳＣドットプロット分析を提示している。図４ｂ及び４ｃ内の円で囲まれた部域は、ｓｃＦｖクローンＹ１を結合させるＣＤ３４＋細胞の亜集団の輪郭を描いている。
【図５】［４４.］ 図５： 前−Ｂ−ＡＬＬ細胞を有する２人の患者から得た標本のＦＡＣＳ分析が提示されている： うち１つの標本は子供からのもの（５ａ、５ｃ、５ｅ）であり、もう１つは大人からのものである（５ｂ、５ｄ、５ｆ）。ＦＩＴＩ標識づけされた負の対照ｓｃＦｖ（５ａ、５ｂ）又はＦＩＴＩ標識づけされたＹ−ＩｓｃＦｖ（５ｃ、５ｄ）と合わせて、市販のＰＥ標識づけされたＣＤ１９（正常な末梢Ｂ細胞についてのマーカー；図５ａ、５ｃ）又はＰＥ標識づけされたＣＤ３４（基幹細胞のためのマーカー；図５ｄ）を利用した。図５ｂは、２重の負の対照である。負の対照染色パターンとの関係におけるＦＩＴＩ標識づけされた標本（ｓｃＦｖクローンＹ１）により結合された細胞の螢光強度（ｘ軸）が提示されている（５ｅ及び５ｆ）。
【図６】［４５.］ 図６：この図は、ジャーカット細胞を用いて実施された結合比較研究の結果を提供している。負の対照と合わせて、ＦＩＴＩ標識づけされたＹ−ＩｓｃＦｖ単量体、diabodies及びtriabodiesのジャーカット細胞に対する結合のＦＡＣＳ分析が提示されている。
【図７】［４６.］ 図７：この図は、ＩｇＧ−Ｙ−Ｉ及びｓｃＦｖ−Ｙ１の結合を比較する研究の結果を提供している。フルサイズのＩｇＧ−Ｙ１の結合とｓｃＦｖ−ＹＩ形態の結合を比較するために、２重染色手順を利用した。ＲＡＪＩ細胞（ＹＩ負細胞；図７ａ）及びジャーカット細胞（Ｙ１正細胞；図７ｂ）に対するＦＡＣＳ分析のために、５ナノグラムのＩｇＧ−ＹＩを使用した。検出のためには、ＰＥ標識づけしたヤギ抗ヒトＩｇＧを使用した。ｓｃＦｖ−ＹＩ−Ｉの結合のためには、〜１μｇ（２００倍）を使用し、その後続いて、ＰＥ標識づけされたウサギ抗−ｓｃＦｖ抗体での染色及びＦＡＣＳ分析を行なった（図７ｃ）。
【図８】［４７.］ 図８： この図は、ＹＩ 二量体、ＹＩｓｃＦｖ（ＣＯＮＹ１）及びＹＩＩｇＧの間の結合比較を示す。
【図９】［４８.］ 図９： この図は、Ｙ１スルフィド架橋二量体とＹ１ｓｃＦｖ（ＣＯＮＹ１）の間の結合比較を示す。
【図１０】［４９.］ 図１０： この図は、Ｙ１−ｃｙｓ−ｋａｋのSuperdex７５プロファイルのグラフである。
【図１１】［５０.］ 図１１： この図は、還元及び非還元性条件下での単量体に比較した二量体のサイズを明示している。
【図１２】［５１.］ 図１２： この図は、ＥＬＩＳＡ検定の結果を提供している。
【図１３】［５２.］ 図１３： この図は、抗−ＧＰＩｂα抗体のエピトープのチャートである。
【図１４】［５３.］ 図１４： この図は、アミノ酸の配列番号である。【Technical field】
[0001]
Field of the invention
[1.] The present invention relates to the field of tissue targeting and identification using phage display technology for peptides and polypeptides that specifically bind to target cells. Such peptides and polypeptides are in the Fv molecule, its constituents, any fragments thereof, or the constituents of the fragments. More specifically, the peptides and polypeptides may have anti-cancer activity and / or are associated or conjugated to an anti-cancer agent, especially for blood-related cancers.
[Background Art]
[0002]
Background of the Invention
2. Tissue-selective targeting of therapeutic agents is an emerging field in the pharmaceutical industry. New cancer treatments based on targeting have been designed to increase the specificity and potential of the treatment while reducing toxicity and thus increasing overall efficacy. Attempts to target toxins, radionucleotides and chemotherapeutic conjugates to tumors have utilized mouse monoclonal antibodies (MAbs') against tumor-associated antigens. In addition, differentiating antigens such as CD19, CD20, CD22 and CD25 have been developed and utilized as cancer-specific targets in treating hematopoietic malignancies. Although extensively studied, this approach has some limitations. One limiting condition is the difficulty in isolating suitable monoclonal antibodies that display selective binding. A second limiting condition is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation. The third limiting condition is that the immune response to the mouse antibody (human anti-mouse antibody) often results in shorter serum half-life, preventing repetitive treatment and thus reducing the therapeutic value of the antibody. -HAMA response) in the patient. This last restriction has stimulated interest in discovering human antibodies while engineering chimeric or humanized monoclonal antibodies from mice.
[0003]
[3.] There are many factors that affect the therapeutic efficacy of monoclonal antibodies (Mabs) for treating cancer. These factors include the specificity of expression of the antigen on the tumor cells, expression levels, antigenic heterogeneity and accessibility to the tumor mass. Leukemias and lymphomas are generally more responsive to treatment with antibodies than solid tumors, such as carcinomas. MAbs bind rapidly to leukemia and lymphoma cells in the bloodstream and readily penetrate malignant cells in lymphoid tissues, thus making lymphoid tumors an excellent candidate for MAb-based therapies. An ideal system would require identifying MAbs that recognize markers on the cell surface of stem cells that produce malignant progeny cells.
[0004]
[4.] To select random single-chain Fvs (scFvs) that bind to isolated and predetermined target proteins such as antibodies, hormones and receptors to aid in Mabs expression / production Phage libraries have been used. In addition, the use of antibody display libraries in general, and especially phage scFv libraries, facilitates an alternative means of finding a unique, yet unidentified, molecule to target unrecognized and undetermined cell surface portions.
[0005]
[5.] Leukemia, lymphoma and myeloma are cancers derived from bone marrow and lymphoid tissues and are involved in the uncontrolled growth of cells. Acute lymphoblastic leukemia ("ALL") is a heterogeneous disease defined by specific clinical and immunological properties. Like other forms of ALL, the definitive cause of most cases of B-cell ALL ("B-ALL") is unknown, but in many cases, the disease causes single cells to become abnormally and continuously It is the result of an acquired genetic modification in the DNA in the cells grown.
[0006]
[6.] AML is a group of neoplastic heterogeneous origins with progenitor cells that generate terminally differentiated cells of the myeloid lineage (erythrocytes, granulocytes, monocytes and platelets) under normal conditions. As with other forms of neoplasia, AML exhibits one or more types of early myeloid differentiation. Associated with acquired genetic alterations that result in the replacement of normally differentiated myeloid cells with relatively undifferentiated blasts. AML generally progresses in the bone marrow and, to a lesser extent, in secondary hematopoietic organs. AML mainly affects adults and peaks in incidence at the age of 15 to 40, but it has also been found that older adults are affected as well as children. Nearly all AML patients require treatment immediately after diagnosis to reach clinical remission without any evidence of abnormal circulating levels of undifferentiated blasts.
[0007]
[7.] Various monoclonal antibodies that induce cytolytic activity on tumor cells have been developed. A humanized version of the monoclonal antibody MuMAb4D5 directed against the extracellular domain of P185 (growth factor receptor (HER2)) has been approved by the FDA and is being used to treat human breast cancer (US Pat. 821,337 and 5,720,954). After binding, the antibody has the ability to inhibit HER2 growth factor receptor dependent tumor cell growth. In addition, a chimeric antibody against CD20 that caused a rapid depletion of peripheral B cells, including those associated with lymphoma, was recently approved by the FDA (US Pat. No. 5,843,439). Binding of this antibody to target cells results in complement dependent lysis. This product has recently been approved and is currently used to treat low-grade B-cell non-Hodgkin's lymphoma.
[0008]
[8.] Several other humanized and chimeric antibodies are under development or in clinical trials. In addition, humanized Igs that specifically react with the CD33 antigen expressed on both normal myeloid cells as well as most types of myeloid leukemia cells have been identified by the anticancer drug carikemycin, CMA-676 (Sievers et al. ., Blood, 90 (10 Suppl. 1 Part 1), 504A (1997)). This conjugate, known as the drug Mylotarg, was recently approved (Caron et al., Cancer Supplement, 73, 1049-1056 (1994)). In view of its cytolytic activity, an additional anti-CD33 antibody (HuM195) currently in clinical trials was identified as a gelonin toxin (McGraw et al., Cancer Immunol, Immunother, 39, 367-374 (1994)) and radioactive Isotope¹³¹I (Caron et al., Blood 83, 1760-1768 (1994)),⁹⁰Y (Jurci et al., Blood, 92, (10 Suppl, Part 1-2), 613A (1998)) and²¹³Conjugated to multiple cytotoxic agents, including Bi (Sgouras et al., J. Nucl, Med., 38 (5 Suppl.) 231P (1997).
[0009]
[9.] Chimeric antibodies against the leukemia antigen CD-45 (cHuLym3) are in preclinical stages for the treatment of human leukemia and lymphoma as conditioning for bone marrow transplantation (Sun et al, Cancer [immunol. Immunother., 48, 595-602 (2002)). In in vitro assays, specific cytolysis was observed in ADCC (antibody-dependent cell-mediated cytotoxicity) assays. (Henkart, Immunity, 1, 343.346 (1994); Squier and Cohen, Current Opin. Immunol., 6, 447-452 (1994)).
[0010]
[10.] These preliminary results seem promising, but they have the following limitations: The final product contains non-human sequences and results in a troublesome immune response to non-human materials such as HAMA. This HAMA response prevents repetitive treatment, resulting in a shorter serum half-life for the product. Furthermore, the above-described methods only allow the isolation of a single antibody species, and only allow the isolation of antibodies against known purified antigens. Moreover, these methods are not selective as long as they allow the isolation of antibodies to cell surface markers present on normal as well as malignant cells.
[0011]
[11.] Therefore, a method that overcomes the above-mentioned limiting conditions is desired. Further, such methods ideally allow for the identification of target ligands or markers on one or more cancer cells that are involved, for example, in mediating the metastasis of the cancer cells. Further, such a method would also allow for the production of antibodies against such targets. Phage display technology would provide such a capability.
[0012]
[12.] The use of phage display technology has allowed the isolation of scFvs containing fully human sequences. For example, recently a fully human antibody against the human TGFb2 receptor based on scFv clones derived from phage display technology was developed. This scFv converted to fully human IgG4 with the ability to compete with TGFb2 binding (Thompson et al., J. Immunol Methods, 227, 17-29 (1999)) has strong antiproliferative activity. This technique known to those skilled in the art is more particularly described in the following publications: Smith, Science, 228, 1315 (1985); Scott et al, Science, 249, 386-390 (1990). Cwirla et al, PNAS, 87, 6378-6382 (1990); Devlin et al, Science, 249, 404-406 (1990); Griffiths et al., EMBO J., 13 (14), 3245-3260 (1994). ); Bass et al., Proteins, 8, 309-314 (1990); McCafferty et al., Nature, 348, 552-554 (1990); Nissim et al., EMBO J., 13, 692-698 (1994). U.S. Patent Nos. 5,427,908, 5,432,018, 5,223,409 and 5,403,484; lib.
[0013]
[13.] Using this phage display display technology, the inventors of the present invention identified cell markers present on cells in a pathological or malignant state. Accordingly, the goal of the present invention is to identify peptides and polypeptides that recognize cell markers that are substantially exposed or overexpressed, especially on or in cells in a pathological or malignant state.
[0014]
[14.] A further goal of the present invention is to use and scale up phage display technology to help identify such peptides and polypeptides.
[0015]
[15.] A further goal of the present invention is to identify such peptides and polypeptides by immunocross-reactivity.
[0016]
[16.] Yet another goal of the present invention is that such peptides and polypeptides be completely human.
[0017]
[17.] Yet another goal of the present invention is that such peptides and polypeptides be isolated against antigens that may not necessarily be immunogenic.
[0018]
[18.] Yet another goal of the present invention is to provide peptides or polypeptides that prevent, delay or treat cancers, especially blood-related cancers, including leukemias or lymphomas.
[0019]
[19.] Yet another goal of the present invention is to use such peptides and polypeptides, associated or coupled, or alone with anti-cancer agents and / or diagnostic labels or markers to produce cancerous cells. Is to be able to target locally.
[0020]
[20.] Yet another goal of the present invention is to provide a method for producing a targeting agent for a desired ligand.
[0021]
[21.] Yet another goal of the present invention is to provide a specific motif that provides recognition of a cell marker that is overexpressed in a malignant condition and that can be used in targeting or diagnostic labels or markers construction for anticancer agents. Is to identify.
[0022]
[22.] Yet another goal of the present invention is to provide a composition comprising an effective amount of such a peptide, polypeptide or motif associated with or coupled to an anti-cancer agent or a diagnostic label or marker. It is in.
[0023]
[23.] It has been demonstrated that scFvs penetrate into tissues and are cleared of blood more rapidly than full-sized antibodies due to their relatively small size. Adams, GP, et al., Br. J. Cancer 77, 1405-1412 (1988); Hudson, PJ, Curr. Opin. Immunol. 11 (5), 548-557 (1999); Wu, AM, et al. Tumor Targeting, 4, 47 (1999). Thus, scFvs are often used in diagnostics involving radiolabels, such as tumor imaging, to allow the radiolabels to be cleared from the body more quickly. Recently, scFc multimers targeting many cancers have undergone preliminary clinical evaluation for in vivo stability and efficacy. Adams, GP, et al., Br. J. Cancer 77, 1405-1412 (1988); Wu, A. M., et al., Tumor Targeting 4, 47 (1999).
[0024]
[24.] The single-chain Fv (scFv) fragment binds to the heavy chain (V_H) And light chain (V_L) Variable domain. The linker is inserted into a functional Fv domain that allows the scFv to recognize and bind its target with similar or increased affinity of the parent antibody (V_H) And (V_L3.) The domain is long enough to fold. Commonly used linkers contain glycine and serine residues to provide flexibility and protease resistance.
[0025]
[25.] Typically, the scFv monomer has its V_HV at the C-terminus of the domain_LDesigned to be anchored to the N-terminal residue of the polypeptide by a polypeptide linker. Optionally, the opposite orientation is used: ie, V_LThe C-terminus of the domain is_HAt the N-terminal residue. Power, B, et al., J. Immun. Meth. 242, 193-204 (2000). Polypeptide linkers are typically around 12 amino acids in length. When the linker is reduced to about 3-12 amino acids, the scFvs cannot fold into a functional Fv domain and instead associate with a second scFvs to form a diabody. Furthermore, reducing the length of the linker to less than three amino acids forces scFv association into trimers or tetramers, depending on the length, composition, and orientation of the Fv domain. B.E. Powers, P. J. Hudson, J. Immun. Meth. 242 (2000) 193-194.
[0026]
[26.] Recently, multivalent antibody fragments such as scFv dimers, trimers and tetramers often provide higher apparent affinities compared to the binding of the parent antibody to the target. Have been discovered. This higher affinity offers a number of advantages including ideal pharmacokinetics for tumor targeting applications.
[0027]
[27.] Thus, greater binding affinity of these multivalent forms is desirable in diagnostic and therapeutic regimens. For example, scFv can be utilized as a blocking agent to bind a target receptor and thus block the binding of a "natural" ligand. In such cases, it is desirable to obtain a high affinity association between the scFv and the receptor, so as to reduce the probability of dissociation that may allow for undesired binding of the natural ligand to the target. In addition, this high affinity is particularly important when the target receptor is involved in adhesion and rolling, or when the target receptor is on cells that are present in areas of high purity flow, such as platelets. .
[0028]
[28.] Accordingly, an object of the present invention is a polyvalent form of Y1 and Y17 scFv. These polyvalent forms include, but are not limited to, dimers, trimers and tetramers, sometimes referred to herein as diabodies, triabodies and tetrabodies, respectively.
DISCLOSURE OF THE INVENTION
[0029]
Summary of the Invention
[29.] The present invention relates to the identification of peptides and polypeptides that bind selectively and / or specifically to target cells, especially against blood-related cancer cells, their construction, alone or singularly. Or, provided in association with, or combined with, conjugated or fused with a plurality of pharmaceutical agents.
[0030]
[30.] One embodiment of the present invention is directed to Fe molecules, constructs thereof, that have enhanced binding properties to selectively and / or specifically bind to target cells in a manner that favors other cells. , Any fragment or peptide or polypeptide comprising a fragment construct, the binding selectivity or specificity is determined primarily by the first hypervariable region, and the Fv is a single-chain Fv (“scFv”). ) Or disulfide Fv ("dsFv"), optionally having one or more tags.
[0031]
[31.] In another embodiment of the present invention, a target comprising one cell in a form advantageous to other cells in which a binding site is substantially unavailable and / or not expressed therein or therein. A Fv molecule, a construct thereof, having enhanced binding properties to selectively and / or specifically bind to a substantially exposed and / or overexpressed binding site on or in Wherein the binding selectivity or specificity is determined primarily by the first hypervariable region, wherein the Fv is a scFv or dsFv, optionally a singular or Peptides or polypeptides having multiple tags are provided.
[0032]
[32.] In a further embodiment of the present invention, an Fv molecule, construct thereof, having enhanced binding properties to selectively and / or specifically bind to a target cell in favor of other cells. Wherein the Fv molecule comprises a first chain having first, second, and third hypervariable regions and a first, second, and third hypervariable region. Comprising a second chain having a third hypervariable region, wherein one of the first chain hypervariable regions has a sequence selected from the group consisting of SEQ ID NOs: 8-24; One of the second-chain hypervariable regions has a sequence selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, and the first, second, and third hypervariable regions Are CDR3, CDR2 and A CDR1 region, Fv is an scFv or dsFv, optionally having one or more tags, peptide or polypeptide are provided.
[0033]
[33.] In a further embodiment of the present invention,
(A) the first strand and the second strand each comprise a first hypervariable region selected from the group consisting of SEQ ID NOs: 8-24; or
(B) the first hypervariable regions of the first and second chains are identical and are selected from the group consisting of SEQ ID NOs: 8-24, or
(C) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs: 8-24, and the first hypervariable region of the second chain is SEQ ID NOs: 1-6 and 125 Selected from the group consisting of ~ 202, or
(D) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, and the first hypervariable region of the second chain is Selected from the group consisting of:
[0034]
[34.] In a further embodiment of the present invention, the method binds to an unknown ligand on a first cell having a first and second state and the binding is effective in a second state, but the first is effective. Condition is substantially ineffective, and binds specifically or selectively to a ligand on a second cell due to immune cross-reactivity, wherein the Fv is a scFv or dsFv, and optionally one or more tags. There is provided a peptide or polypeptide comprising an Fv molecule, a construct thereof, or a fragment or construct of a fragment thereof, comprising:
[0035]
[35.] In a further embodiment of the present invention, there is provided a method for identifying a targeting molecule that binds to an unknown immune cross-reactive binding site on first and second cells, comprising:
(A) a first target cell that substantially exposes or displays a binding site containing one unknown ligand in a second state, but not in the first state, to generate a first population of recognition molecules; One or more biopannings performed on it;
(B) performed on a second cell displaying a binding site containing one unknown ligand with immunological cross-reactivity to the unknown ligand of the first cell to generate a second population of recognition molecules. A subsequent biopanning and / or selection step starting from the resulting stock of the recognition molecule of step (a);
(C) amplifying and purifying the second population of recognition molecules of step (b); and
(D) constructing a peptide or polypeptide comprising a targeting molecule that is selective and / or specific for an unknown ligand on a second cell from the recognition site of the purified recognition molecule of step (c);
There is provided a method for identifying a targeting molecule, comprising:
[0036]
[36.] In still another embodiment of the present invention, R₁-XPhe Pro-R_TwoComprising the amino acid sequence of₁And R_TwoEach comprise from 0 to 15 amino acid residues, and wherein X is Arg, Gly, or Lys.
[0037]
[37.] In still another embodiment of the present invention,
a) directly by a biopanning procedure on the target cells or indirectly by a biopanning procedure on a first target cell in a second state instead of the first state and then directly on a second target cell. Isolating and selecting one or more targeting molecules comprising a primary recognition site by a biopanning procedure to produce one or more of said targeting molecules;
b) amplifying, purifying and identifying one or more targeting molecules;
c) constructing a targeting agent from one or more targeting molecules;
And wherein the targeting agent is a peptide, polypeptide, antibody or antibody fragment or a multimer thereof.
[0038]
[38.] In another embodiment of the present invention, there is provided a peptide or polypeptide having the structural formula or structure of AXB, wherein X is a hypervariable CDR3 region of 3 to 30 amino acids. A and B may each be an amino acid chain of 1-1000 amino acids in length, wherein a peptide or polypeptide is provided wherein A is the amino terminus and B is the carboxy terminus.
BEST MODE FOR CARRYING OUT THE INVENTION
[0039]
DETAILED DESCRIPTION OF THE INVENTION
[54.] As used herein, the term specificity is defined as the recognition and subsequent binding of a target ligand by one or more domains within a peptide or polypeptide of the invention.
[0040]
[55.] Selectivity, as used herein, refers to the selection and binding of one cell type or state from a mixture of cell types or states that may be specific for the targeting molecule. Is defined as the ability of the targeting molecule to
[0041]
[56.] Conservative amino acid substitutions are defined as changes in the amino acid composition by changing one or two amino acids of a peptide, polypeptide or protein or fragment thereof. Substitutions generally have similar properties (e.g., charge, IEF, affinity, avidity, conformation, solubility) or activity so that the substitution does not substantially alter the properties (eg, charge, IEF, affinity, avidity, conformation, solubility) of the peptide, polypeptide or protein. For example, amino acids having acidic, basic, aromatic, size, positive or negative charge, and non-polarity). Standard substitutions that can be made for such conservative amino acid substitutions can be between groups of amino acids as follows:
(I) glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);
(Ii) aspartic acid (D) and glutamic acid (E)
(Iii) Alanine (A), serine (S) and threonine (T)
(Iv) histidine (H), lysine (K) and arginine (R),
(V) Asparagine (N) and glutamine (Q)
(Vi) Phenylalanine (F), tyrosine (Y) and tryptophan (W).
[0042]
[57.] Conservative amino acid substitutions flank in and in other parts of the molecule, such as the hypervariable regions and variable heavy chain cassettes that are primarily responsible for the selective and / or specific binding properties of the molecule. Can be done. Additionally or alternatively, the modification may reconstitute the molecule to form a full-sized antibody, diabodies (dimers), triabodies (trimers) and / or tetrabodies (tetramers) or This can be achieved by forming minibodies or microbodies.
[0043]
[58.] As used in the specification and claims herein, the Fvs can be the same or different and in which the variable region of the heavy chain is linked to the variable region of the light chain. , Linked, fused or covalently attached to or associated with a variable region of a heavy chain of a human antibody and a variable region of a light chain of a human antibody.
[0044]
[59.] A fragment of an Fv molecule is defined as any molecule smaller than the original Fv that still retains the selective and / or specific binding properties of the original Fv. Examples of such fragments include (1) a minibody containing a fragment of only the heavy chain of Fv, (2) a microbody containing a small fractional unit of the antibody heavy chain variable region (PCT Application No. PCT / IL99 / 00581), ( 3) Similar bodies containing fragments of the light chain and (4) similar bodies containing the functional units of the light chain variable region, but are not limited thereto.
[0045]
[60.] An anti-cancer agent is an agent that has anti-cancer activity, that is, any activity that inhibits the growth or differentiation of cancerous or immature precancerous cells, or any activity that inhibits metastasis of cancerous cells. In the present invention, the anticancer agent is an agent having an antiangiogenic activity for preventing, inhibiting, delaying or interrupting angiogenesis of tumor tissue, and also inhibiting, delaying or interrupting adhesion of cancerous and precancerous cells. It is also an active substance with anti-adhesion activity.
[0046]
[61.] Inhibition of cancer cell growth is defined herein as (i) preventing cancerous or metastatic growth, (ii) slowing cancerous or metastatic growth, (iii) keeping cells intact and alive. Complete prevention of the cancer cell growth or metastasis process while remaining in a state of death or (iv) death of the cancer cell. More particularly, inhibition of cancerous growth is particularly applicable to blood-related cancers such as AML, multiple melanoma or chronic lymphocytic leukemia.
[0047]
[62.] Phagemid is defined as a phage particle that carries plasmid DNA. Because it carries plasmid DNA, phagemid particles do not have enough space to accommodate the complete complement of the phagemid genome. Components missing in the phage genome are essential information for packaging phage particles. Thus, propagation of the phage requires culturing the desired phage particles in conjunction with a helper phage strain that complements the missing packaging information.
[0048]
[63.] A cassette as defined in the present invention applied to a polypeptide serves as a framework, considered as a single unit, and a given sequence of consecutive amino acids so manipulated. Means Amino acids can be substituted, inserted, removed or attached at one or both ends. Similarly, a stretch of amino acids can be substituted, inserted, removed or attached at one or both ends.
[0049]
[64.] As used herein, an immunoglobulin (Ig) molecule is defined as any one of five classes: IgG, IgA, IgD, IgE or IgM. The IgG class includes a plurality of subclasses including, without limitation, IgG1, IgG2, IgG3 and IgG4.
[0050]
[65.] Pharmaceutical composition means a formulation comprising the peptide or polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient or diluent thereof.
[0051]
[66.] Pharmaceutical agents refer to the prophylactic use of mammals, including, but not limited to, humans, cows, horses, pigs, mice, dogs, cats or any other warm-blooded animals. An agent is useful in therapy or diagnosis. The pharmaceutical agent is selected from the group comprising radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments and anti-cancer agents. Examples of such pharmaceutical agents include antiviral agents including acyclovir, ganciclovir and zidovudine; thrombotic / restenotic agents including cilostazol, dalteparin sodium, reviparin sodium and aspirin; zaltoprofen, pranoprofen, droxicam, acetyl Anti-inflammatory agents including salicylic acid 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtormetin, celecoxib, indomethacin, rofecoxib and nimesulide; Anti-autoimmune agents, including limaprost, chlorchromene, and anti-adhesion preventive agents including hyaluronic acid; But it is not limited.
[0052]
[67.] An anti-leukemia agent is an agent having anti-leukemia activity. For example, anti-leukemia agents include agents that inhibit or disrupt the growth of leukemia or premature leukemia cells, agents that kill leukemia or preleukemia (cells), leukemia or preleukemia against other anti-leukemia agents. Agents that increase cell sensitivity and agents that inhibit leukemic cell metastasis are included. According to the invention, the anti-leukemic agent can also be an agent with anti-angiogenic activity that prevents, inhibits, delays or interrupts tumor angiogenesis.
[0053]
[68.] As used herein, the term "affinity" is a measure of the strength of association (association constant) between a receptor (eg, one binding site on an antibody) and a ligand (eg, an antigenic determinant). The total strength of the non-covalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope. Low affinity antibodies bind weakly to the antigen and tend to dissociate easily, while high affinity antibodies bind the antigen more tightly and stay longer in the bound state. The term "binding power" is different from affinity because it reflects the valency of an antigen-antibody interaction.
[0054]
[69.] Specificity of antibody-antigen interaction: Although the antigen-antibody reaction is specific, in some cases, antibodies elicited by one antigen cross-react with another unrelated antigen Can be. Such cross-reactivity occurs when two different antigens share a homologous or similar epitope or its anchor region, or an antibody specific for one epitope binds to an irrelevant epitope with similar chemical properties. Happens if you do.
[0055]
[70.] Blast cells are cells at an immature stage of cell development that are distinguished by a higher cytoplasmic to nuclear ratio than resting cells.
[0056]
[71.] Platelets are disk-like cytoplasmic fragments of megakaryocytes that are dropped into the bone marrow sinus and then circulate in the peripheral bloodstream. Platelets have multiple physiological functions, including a major role in coagulation. Platelets have granules in the central part, with bright protoplasm around, but no clear nuclei.
[0057]
[72.] The term "epitope" is used herein to mean an antigenic determinant or antigenic site that interacts with an antibody, an antibody fragment, an antibody conjugate or a binding fragment thereof, or a complex that includes a T cell receptor. Have been. The term epitope is used interchangeably herein with the terms ligand, domain and binding region.
[0058]
[73.] A given cell can express on its surface a protein with a binding site (or epitope) for a given antibody, but the binding site is the first Can exist in cryptic form in cells in a state that can be referred to as phase (phase I) (eg, sterically hindered or blocked or lacks the features required for binding by antibodies) ). Stage I can be, for example, a normal, healthy, non-morbid condition. If the epitope is present in a covert form, it will not be recognized by any given antibody. That is, there is no binding of the antibody to this epitope or certain given cells in Phase I. However, epitopes may be revealed, for example, by the underlying modification itself, or by being unblocked because nearby or associated molecules have been modified or the region has undergone a conformational change. Can be done. Examples of modifications include changes in folding, changes in post-translational modifications, changes in phospholipidation, changes in sulfation, changes in glycosylation, and the like. Such modifications can occur when a cell enters a different state, which can be termed the second (phase II). Examples of the second state (s) include activation, proliferation, transformation, malignant states. Once modified, the epitope can then be exposed and the antibody can bind.
[0059]
[74.] As used herein, the term "Fab fragment" is a monovalent antigen binding fragment of an immunoglobulin. Fab fragments are comprised of a light chain and a portion of a heavy chain.
[0060]
[75.] Polyclonal antibodies are the product of an immune response and are formed by a number of different B-lymphocytes. Monoclonal antibodies are derived from single cells.
[0061]
[76.] As used herein, cohesion refers to the process by which suspended bacteria, cells, discs or other similarly sized particles are allowed to adhere and form a mass. This process is similar to precipitation, but the particles are larger and are in suspension rather than in solution.
[0062]
[77.] The term aggregation refers to the aggregation of platelets and thrombin and collagen induced in vitro as part of a sequential mechanism leading to the formation of a thrombus or thrombus.
[0063]
[78.] Gene expression patterns can be studied, for example, by analyzing the amount of gene product produced under various conditions at various times in various tissues. A gene is considered “overexpressed” if the amount of the gene product is higher than that found in a normal control, eg, a non-pathological control.
[0064]
[79.] A promoter is a region on DNA where RNA polymerase binds and initiates transcription.
[0065]
[80.] Antibodies or immunoglobulins are protein molecules that bind to an antigen. They are composed of units of four polypeptide chains (two heavy and two light chains) linked together by disulfide bonds. Each chain has a variable region spanning the constant region. These can be divided into five classes based on their heavy chain components: IgG, IgM, IgA, IgD and IgE. They are produced by B lymphocytes and recognize specific foreign antigenic determinants and facilitate the clearance of that antigen.
[0066]
[81.] Antibodies can be produced and used in a number of forms, including antibody conjugates. As used herein, the term “antibody complex (s)” means a complex of one or more antibodies with another antibody or one or more antibody fragments, or a complex of two or more antibody fragments. Have been used to be.
[0067]
[82.] F (ab ') 2 fragments are divalent antigen-binding fragments of immunoglobulin obtained by pepsin digestion. It contains both light chains and a portion of both heavy chains.
[0068]
[83.] Fc fragments are the non-antigen binding portions of immunoglobulins. It contains the carboxy-terminal portion of the heavy chain and a binding site for the Fc receptor.
[0069]
[84.] The Fd fragment is the variable region and first constant region of the heavy chain of an immunoglobulin.
[0070]
[85.] Contaminating proteins are proteins that have not been specifically selected for a specimen and may be present in the specimen.
[0071]
[86.] A peptidomimetic is a small molecule, peptide, polypeptide, lipid, polysaccharide or conjugate that has the same functional effect or activity as another entity, such as an antibody.
[0072]
[87.] A plasmid vector designed to contain an origin of replication from filamentous phage such as m13 of fd.
[0073]
[88.] There is a wide array of diseases involving pathological, altered or otherwise modified cells that express cell-specific and / or disease-specific ligands on their surface. These ligands can be utilized to effect recognition, selection, diagnosis and treatment of a particular disease through the recognition, selection, diagnosis and treatment of each individual cell. The invention provides a peptide or polypeptide comprising an Fv molecule, a constituent thereof, a fragment thereof, a constituent of the fragment or a fragment of a constituent, all having enhanced binding properties. These binding properties allow a peptide or polypeptide molecule to selectively and / or specifically bind to a target cell in a manner that is advantageous to other cells, with binding specificity and / or selectivity being primarily It is determined by the first hypervariable region. Fv can be scFv or dsFv.
[0074]
[89.] The Fv molecules described above can be used to target diseased cells. Pathological cells can be, for example, cancer cells. Examples of cancer types that can be diagnosed and / or treated by specific targeting include, without limitation, carcinoma, sarcoma, leukemia, tumor, lymphoma, myeloma, blastoma, seminomas and melanoma. included. Leukemia, lymphoma and myeloma are cancers derived from bone marrow and lymphoid tissues and are involved in the uncontrolled growth of cells.
[0075]
[90.] New approaches for diagnosing and treating diseases, especially cancer, have recently been developed. Among them are tumor targeting approaches using targeting molecules that can be selected and produced in various ways. One approach to identifying potential targeting molecules is phage display. Phage display refers to the expression of a peptide, polypeptide, antibody or protein on the surface of a filamentous bacteriophage by fusion to a phage coat protein, with the DNA encoding the displayed protein resident within the phage virion. This is a technology generated and selected by display. The scFv produced by phage display technology consists of the VDs of each of the antibody heavy and light chains linked by a flexible amino acid polypeptide spacer (Nissim et al., EMBOJ., 13, 692-698 (1994)). .
[0076]
[91.] Phage display libraries (also called phage peptide / antibody libraries, phage libraries or peptide / antibody libraries) contain large phage populations (generally 10⁸-10⁹), Each phage particle displaying a different peptide or polypeptide sequence. These peptides or polypeptides can be configured to have variable length. The indicated peptide or polypeptide can be derived from a human antibody heavy or light chain, but need not be so limited.
[0077]
[92.] In the present invention, scFv antibody libraries produced by phage display technology were used to acquire and produce targeting molecules. Flow cytometry, particularly a fluorescence activated cell sorter ("FACS"), was used to identify and isolate specific phage clones whose peptides or polypeptides recognized target cells. Phage-expressed scFv antibody fragments can be the subject of in vitro screening, enrichment and selection of high affinity clones (US Pat. No. 5,821,337; US Pat. No. 5,720,954). Thus, this type of library provides a powerful tool for generating new tools for research and clinical applications, and has numerous advantages over traditional approaches (Caron et al., Cancer Supplement, 73, 1049-1056 (1994)). The library contains a high potential for diversity of antibody molecules (Nissim et al., EMBO J., 692-698 (1994)). In this case, stable human cDNA can be used as a continuous source of material for antibody production (US Pat. No. 5,843,439). Molecular recognition and selection are not affected by the in vivo immunogenicity of the target protein candidate.
[0078]
[93.] While affinity selection of phage-displayed antibodies provides a useful method for enriching antigen-reactive scFvs from large libraries, it involves the isolation of single clones and characterization of soluble scFvs. A number of steps are required. The scFv itself can be modified to improve its affinity and / or avidity by making conservative amino acid substitutions or by producing a fragment of the scFv or a construct of this fragment.
[0079]
[94.] The scFvs of the present invention specific for different human cells and tissues may be used to form drug peptide compositions, fusions or conjugates with anti-disease and / or anti-cancer activity, and / or for diagnostic purposes Can be associated, combined, fused or linked with various pharmaceutical agents and / or radioisotopes in a pharmaceutically effective amount, optionally with a pharmaceutically effective carrier.
[0080]
[95.] Phage clones are selected and identified through a multi-step procedure known as biopanning. Biopanning is performed by incubating a phage display protein ligand variant (phage display library) with the target, removing unbound phage by a washing technique, and specifically eluting the bound phage. The eluted phage optionally undergoes further cycles of binding and optional amplification to enrich the pool of specific sequences in a manner that favors the phage clone as carrying the antibody fragment displaying the highest binding to the target. Amplified before being taken up. After multiple rounds, individual phage clones are characterized and the sequence of the peptide represented by the clone is determined by sequencing the corresponding DNA of the phage virion.
[0081]
[96.] The scFv obtained in this manner is also called a lead compound. A lead compound is defined as a compound whose final format comprises a core peptide or polypeptide. The lead compound can be modified and / or expanded, but must retain the core peptide or polypeptide or some conservatively modified form thereof. Modifications by amino acid substitutions can be made, for example, at the N-terminus, carboxy-terminus of Fv or in any of the CDR regions or upstream or downstream thereof. Modifications also include, but are not limited to, coupling to fusion proteins, drugs or toxins, building multimers, and extending to whole antibody molecules. One preferred category of lead compounds as provided in this patent is the scFv obtained as the end product of a biopanning procedure.
[0082]
[97.] Embodiments of the present invention provide at least one non-natural modification of a peptide or polypeptide of the present invention. Non-natural modifications can make the peptide or polypeptide more immunogenic or stable. Non-natural modifications include, but are not limited to, peptoid modifications, semipeptoid modifications, cyclic peptide modifications, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications.
[0083]
[98.] Selection of antigen-specific phage antibodies relied heavily on biopanning against a single immobilized antigen. When whole cells were used as targets, the selection was limited. In the present invention, whole cells are used to select specific antibodies that recognize leukemia cell surface determinants, where the specific receptor was not previously known or characterized. This method does not allow for easy adjustment of antigen enrichment or removal of unwanted dominant antibody reactivity. Furthermore, unlike those with higher affinity clones, phage can be enriched for those displaying multiple copies of the scFv. Nevertheless, this approach has become an invaluable tool for isolating novel human antibody molecules because of its advantages.
[0084]
[99.] One embodiment of the present invention relates to binding an unknown ligand on a first cell having a first and second state, wherein the binding is effective in the second state, but in the first state. Is substantially ineffective and binds specifically or selectively to a ligand on a second cell due to immune cross-reactivity, wherein the Fv is a scFv or dsFv, and optionally one or more tags. A peptide or polypeptide comprising an Fv molecule, a construct thereof, any fragment or construct of a fragment thereof.
[0085]
[100.] A further embodiment provides a peptide or polypeptide of the invention wherein the selective and / or specific binding of the peptide or polypeptide to a second cellular ligand is determined primarily by the first hypervariable region. providing.
[0086]
[101.] Yet another embodiment, is a peptide or polypeptide of the invention, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. Is provided.
[0087]
[102.] Yet another embodiment is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24, wherein the binding selectivity or specificity is secondary to the second. By one or more of the upstream region and / or the downstream region flanking the hypervariable region and / or the third hypervariable region and / or the first, second and third hypervariable regions. Provided are the peptides or polypeptides of the invention that are each affected.
[0088]
[103.] A further embodiment provides a second cell ligand bound by a peptide or polypeptide of the present invention. Such a two-cell selection protocol was based on the following: megakaryocytes are large multinucleated cells derived from hematopoietic stem cells in the bone marrow. Platelets break apart from the megakaryocyte cytoplasm and enter the peripheral blood. In vitro, a wide range of cytokines directly affects stem cells. For example, thrombopoietin increases platelet counts by directly increasing the differentiation of stem cells into megakaryocytes. Thus, these cells express multiple cell surface markers that are also found among premature cells.
[0089]
[104.] Malignant blood cells (leukemias and lymphomas) are characterized as immature cells that express cell surface proteins normally found in partially differentiated hematopoietic progenitor cells. Thus, platelets are an attractive source for the identification of premature cell surface markers expressed on diseased or malignant blood cells. In one protocol discussed below, specific cells carrying an unknown ligand were used for the initial biopanning step, without limitation. Subsequent clonal selection was performed with the desired target cells whose targeted cell surface markers are unknown, such as AML cells without limitation. In this way, phage clones obtained by biopanning on platelets can provide a tool for recognizing and binding ligands on the diseased or malignant blood cell in question.
[0090]
[105.] Targets as described above include cells derived from the isolated tissue. The isolated tissue can be a pathological tissue, more specifically, a cancerous tissue. The cancerous tissue can be from any form of malignancy, including, without limitation, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma.
[0091]
[106.] In addition to the biopanning methods described above, another approach is based on the isolation of peptides or polypeptides that bind ligands on cells as determined by direct panning on the ligands. is there.
[0092]
[107.] The present invention provides a peptide or polypeptide comprising an Fv molecule, a construct thereof, any fragment or construct of a fragment thereof. Constructs can be multimers (eg, diabodies, triabodies, tetrabodies) or full-size Ig molecules, and fragments can be minibodies or microbodies. All derived constructs and fragments retain enhanced binding properties to bind S / C to target cells in favor of other cells. Binding selectivity and / or specificity is determined primarily by the first hypervariable region, wherein the Fv is a scFv or dsFv, and optionally has one or more tags.
[0093]
[108.]
In one embodiment of the invention, the tag is inserted or attached to an Fv peptide or polypeptide, which aids in its preparation and identification and diagnosis. The tag can then be removed from the molecule. Tags can be, but are not limited to, the following tags: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPTHH, IRS, KT3, protein C, S. Tag ™, T7, V5, VSV-G (Jarvik and Telmer, Ann. Rev. Gen., 32, 601-618 (1998)) and KAK (lysine-alanine-lysine). The tag is preferably c-myc or KAK.
[0094]
[109.] The two variable chains of the Fv molecule of the present invention may be linked or linked to each other by a spacer having a length of 0 to 20 amino acid residues. The spacer may be branched or unbranched. Preferably, the linker is from 0 to 15 amino acid residues, and most preferably, the linker is (Gly) to produce a single-chain Fv ("scFv")._FourSer)_ThreeIt is. scFv can be obtained from a phage display library.
[0095]
[110.] The Fv molecule itself is composed of a first chain and a second chain, each chain comprising first, second and third hypervariable regions. The hypervariable loops within the light and heavy chain VDs are called complementary determining regions (CDRs). There are CDR1, CDR2 and CDR3 regions in each of the heavy and light chains. These regions are believed to form an antigen binding site and can be specifically modified to produce enhanced binding activity. Essentially the most variable of these regions is the CDR3 region of the heavy chain. The CDR3 region is understood to be the most exposed region of the Ig molecule and is primarily responsible for the observed selective and / or specific binding properties, as shown and provided herein. Things.
[0096]
[111.] One embodiment of the present invention relates to a method for targeting a target comprising a cell in a manner that is advantageous to other cells in which binding sites are substantially unavailable and / or not expressed. Or an Fv molecule, construct thereof, or any of the above, having enhanced binding properties to selectively and / or specifically bind to substantially exposed and / or overexpressed binding sites therein. In a peptide or polypeptide comprising a fragment or a construct of a fragment, the binding selectivity or specificity is determined primarily by the first hypervariable region, wherein the Fv is a scFv or dsFv, and optionally one or more. A peptide or polypeptide having a tag is provided.
[0097]
[112.] A further embodiment of the present invention provides a peptide or polypeptide wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. ing.
[0098]
[113.] In yet another embodiment, the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 24, wherein the binding selectivity or specificity is Secondarily, one or more of the downstream regions flanking the second and / or third hypervariable regions and / or the first, second and third hypervariable regions And / or affected by one or more of the upstream regions, wherein the second and third hypervariable regions are CDR2 and CDR1 regions, respectively.
[0099]
[114.] One embodiment of the present invention provides a peptide or polypeptide that binds to a target cell that is an activated, excited, modified, altered, disrupted, or pathological cell. In a further embodiment of the present invention, the target cells are cancer cells. The target cells can be selected without limitation from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma cells. In a preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, the leukemia cells are AML cells.
[0100]
[115.] The peptides or polypeptides of the present invention may also retain one or more of the hypervariable regions of the heavy and / or light chains and retain Fv with selective and / or specific binding properties. Any construct or modified construct. Constructs or modified constructs include scFvs, dsFvs, multimers of scFvs such as dimers, trimers, tetramers (also called diabody, triabody, tetrabody), and whole antibodies, and whole antibodies. All other multimers that are obtained and that include one or more of the hypervariable domains of the antibody are included. A peptide or polypeptide of the present invention is also a fragment of any construct or modified construct that has some or all of the binding properties of the original construct.
[0101]
[116.] The peptides or polypeptides of the invention are also constituents of fragments having some or all of the selective and / or specific binding properties of the original constituents. The Fvs described herein can selectively and / or specifically bind to a target cell and be associated or conjugated to an anti-cancer or anti-disease agent.
[0102]
[117.] The peptides, polypeptides, fragments thereof, constructs and fragments of the constructs of the Fv molecules of the present invention can be prepared in either prokaryotic or eukaryotic expression systems. In one embodiment of the invention, the eukaryotic expression system is a mammalian system, and after purification, the peptide or polypeptide produced in the mammalian expression system substantially eliminates mammalian contaminants. Not included. By eukaryotic cell system as defined in the present invention is meant an expression system for producing a peptide or polypeptide by a genetic engineering method, wherein the host cell is a eukaryotic cell. In another embodiment of the invention, a prokaryotic cell line for the production of a peptide or polypeptide of the invention uses E. coli as a host for an expression vector. After purification, the peptide or polypeptide produced in the E. coli system is substantially free of E. coli contaminating proteins. Use of a prokaryotic expression system may result in the addition of a methionine residue to the N-terminus of some or all of the sequences provided herein. Removal of the N-terminal methionine residue after production of the peptide or polypeptide to allow complete expression of the peptide or polypeptide can be performed without limitation, such as using Aeromonas aminopeptidase under appropriate conditions. Such methods can be performed by methods generally known in the art (US Pat. No. 5,763,215).
[0103]
[118.] The present invention provides for the production of scFvs based on the Fv peptides of the present invention. Promoters incorporated into vectors used for cloning and amplifying scFvs in prokaryotic cells can be selected from a wide variety of options. A promoter is a DNA sequence located upstream of a structural gene and capable of controlling gene expression. Promoters are found in their natural state in the chromosome (s) of the organism and can also be engineered into prokaryotic or eukaryotic expression vectors. Promoters engineered into specific loci on the desired DNA fragment provide for fine-tuned and precisely regulated expression of the gene in question. In the present invention, multiple promoters were used in the construct containing the gene encoding for the selected Fv. Promoters include, without limitation, the following: deo, P1-P2, osmB, λP_L, Β-lac-U5, SRα5 and CMV early promoter. Deo, upon introduction into an appropriate E. coli host, directs the host to the desired naturally occurring polypeptide or polypeptide analog thereof under the control of the constitutive E. coli-derived deoxyribonucleotide promoter deoP1-P2. Is a double-stranded DNA plasmid that confers the ability to effect the expression of DNA that encodes A more detailed description is provided in US Pat. No. 5,795,776 (Fisher, Aug. 18, 1998) and US Pat. No. 5,945,304 (Fisher, Aug. 31, 1999). ing.
[0104]
[119.] Expression of the E. coli osmB promoter is regulated by osmotic pressure. Vectors carrying this promoter can be used to produce high levels of a wide variety of combinatorial eukaryotic and prokaryotic polypeptides under the control of the osmB promoter in an E. coli host. A more detailed description is provided in US Pat. No. 5,795,776 (Fisher, Aug. 18, 1998) and US Pat. No. 5,945,304 (Fisher, Aug. 31, 1999). I have.
[0105]
[120.] λP_LIs the thermolabile inhibitor cI⁸⁵⁷Is a heat-induced lambda bacteriophage promoter regulated by. For a more detailed description, see Hendrix et al. Random II, Cold Spring Harbor Laboratory (1983).
[0106]
[121.] β-lac-U5 is a lacZ promoter (Gilbert and Muller-Hill, PNAS (US), 58, 2415 (1967).
[0107]
[122.] SRα5 is a mammalian cDNA expression system consisting of the simian virus 40 (SV40) early promoter and the RU5 segment of the human T-cell leukemia virus type 1 long terminal repeat. This expression system is one or two orders of magnitude more active than the SV40 early promoter in a wide range of cell types (Takebe et al., Molecular and Cell Biology, 8,466-472 (1998)).
[0108]
[123.] The human cytomegalovirus promoter, known as the CMV intermediate / early enhancer / promoter, is most preferably used in the present invention to enhance the expression of the construct of the cloned DNA insert in mammalian cells. You. The CMV promoter is described in Schmidt, E.V. et al., (1990) Mol Cell Biol., 10, 4406, and are U.S. Patent Nos. 5,168,062 and 5,385,839.
[0109]
[124.] In a preferred embodiment of the present invention, the promoter for inducing the phagemid system in prokaryotic cells is deo, osmB, λP_L, Β-lac-U5 and the CMV promoter. In a more preferred embodiment of the invention, the β-lac-U5 promoter was used for induction of the phagemid system in E. coli. In a most preferred embodiment, a CMV promoter is used.
[0110]
[125.] In one embodiment of the invention, the subject peptide or polypeptide comprises: (a) present only in the encoded sequence and absent in the mature protein. (B) a heavy chain variable region of approximately 135-135 amino acids, including the first hypervariable region of 4-12 amino acids to be modified; (c) truncated or deleted A spacer region of the following 20 amino acids; (d) the variable region of the light chain which also undergoes the specific modifications described herein, followed by; (e) optionally present in the last injectable product Not follow-up tag array. A spacer, typically about 15 amino acids in length in the scFv, allows the two variable chains (heavy and light) to fold into a functional Fv domain. A functional Fv domain retains selective and / or specific enhanced binding activity.
[0111]
[126.] In another embodiment, (d) above is followed by a tag sequence or label that can be used for conjugation, diagnostic and / or identification purposes. In this embodiment, the tag is designed to link between a peptide or polypeptide of the invention and an agent for treating or diagnosing a target cell.
[0112]
[127.] The spacer region of the scFv may be linear or branched and generally has the structural formula (Gly_FourSer)_nConsisting of glycine and serine residues in multiples of and generally 0 to 20 amino acids in total, preferably 0 to 15 amino acids in length, and is linear. Various multimers can be obtained by appropriately changing the spacer length. In embodiments of the invention, the length of the spacer is from 0 to 5 amino acids. In another embodiment, the length of the spacer is (less than 3 amino acids, as detailed below).
[0113]
[128.] Hereinafter, examples of the amino acid sequence of the scFv molecule of the present invention are shown.
[0114]
Embedded image

[0115]
[129.] The leader sequence is underlined with a dashed line. V_H  The region is encoded by the bold amino acid sequence. This specific clone has a germline V_HIt is derived from 3-DP32. However, the germline of each clone depends on its particular origin (see below). The amino acid sequence in the box indicates the hypervariable region, V, in all clones derived from this library._H-Code for the CDR3 sequence. V_HAnd V_L The spacer region, which merges with the region, is a flexible polypeptide encoded by the amino acids shown in italics. Finally V_L The area is presented. Fusion V in all clones_L The fragment is derived from a single unmutated V gene of the germline IGLV3SI, followed by a wavy underlined c-myc tag.
[0116]
[130.] V_HThe fragment repertoire (from 49 germline lines) was first generated by RCR from rearranged V-genes of human peripheral blood lymphocytes that had not been immunized by a library supplier (see “Experimental Unused repertoire "). V_H-The origin of the sequence (germline) can be identified by a homology test (Blast search) using one of the following websites:
[0117]
[131.] In one of a number of ways, the binding properties of the antibody can be optimized. One way to optimize antibodies for higher binding affinity for the original lead compound is to replace amino acid residues in the lead compound to introduce more variability or extend the sequence Based on For example, the original V_LThe entire region is made up of V from different antibody subtypes._LCan be replaced by a region.
[0118]
[132.] An additional method of optimizing binding affinity is to construct a phagemid-displayed mutagenesis library. In the phagemid display mutagenesis library, V_HAnd V_LOligonucleotides are synthesized such that each amino acid of the core sequence within CDR3 is independently replaced, preferably by any other amino acid, in a conservative manner known in the art. The present invention provides a set of specific antibody scFvs displayed on phage, wherein the displayed antibody fragments and the soluble antibody fragments extractable from phage virions have the same biological activity.
[0119]
[133.] The phage display library used in this document was constructed from unimmunized human peripheral blood lymphocytes, and Fv peptides were previously uncharacterized on the surface of target cells. Selected for purified antigen. As used herein, the term unpurified antigen that has not been identified, characterized, isolated or purified by biochemical or molecular means prior to current work, and has not been observed A ligand displayed on the surface of a cell that has been observed or predicted in the work by selective and / or specific binding to the isolated antibody fragment.
[0120]
[134.] The scFvs of the invention display enhanced binding to target cells. Enhanced binding is directed to specific surface markers. Specific surface markers are molecules that are accessible to circulating and circulating recognition molecules within the cell membrane. The presence of surface markers has enabled the development of phage display technology via the biopanning technology described herein. In the present invention, specific surface markers are utilized to characterize and differentiate various cell types as well as serve as binding sites for Fvs in their various forms. According to its characteristic surface markers, various hematopoietic cell types can be distinguished, and in a similar manner, pathological or cancer cells display surface markers that are unique to their type and stage.
[0121]
[135.] Selection of scFv clones can be achieved by two different biopanning strategies:
1. Direct selection by using pathological or cancer cells as target cells, and
[0122]
2. A second, eg, activated, excited, modified, altered, or disrupted state in which the binding site of the first cell in the second state will comprise an unknown ligand that is substantially exposed or displayed. Stepwise selection, which will use the first, for example, normal cells. Due to immune cross-reactivity, the resulting clones can selectively and / or specifically bind to new and unknown ligands on second cells after a subsequent biopanning or selection step. . Following any further amplification and subsequent purification, a targeting molecule can be constructed from the recognition site of the purified recognition molecule selective and / or specific for the unknown ligand on the second cell.
[0123]
[136.] In one embodiment of the present invention, the first cell can be a normal cell, the first state is an unactivated state, and the second state is activation, excitation, modification, alteration. Or it may be in a disturbed state. The second cell in the stepwise selection can be a human cell. In another embodiment of the invention, the second cell in the stepwise selection is a pathological cell. In a more preferred embodiment, the second cell in the stepwise selection is a carcinoma, such as carcinoma, sarcoma, leukemia, fibroid, lymphoma, myeloma, blastoma, seminomas and melanoma without limitation. . In a more preferred embodiment, the second cell is a leukemia cell. In a most preferred embodiment, the second cell is an AML cell.
[0124]
[137.] A more preferred embodiment of the present invention provides a peptide or polypeptide whose selective and / or specific binding to a ligand of a second cell is determined primarily by the first hypervariable region. In an even more preferred embodiment, the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24.
[0125]
[138.] Another embodiment of the present invention provides a second cell ligand bound by a peptide or polypeptide of the present invention. A further embodiment provides any molecule that recognizes and binds a ligand bound by a peptide or polypeptide of the present invention.
[0126]
[139.] Enhanced binding to cancer cells is most likely due to overexpression of ligand in cancer cells and / or exposure of binding sites as compared to expression in normal cells. As used herein, the term ligand overexpression refers to the expression of a gene or product thereof that is normally silent during a particular cell type and / or during a particular cell cycle, or with respect to this particular cell type. Defined as increased expression of a gene that is expressed at basal levels under normal, non-malignant conditions.
[0127]
[140.] In a further preferred embodiment of the invention, the target cells of the biopanning procedure are contained in a cell suspension. Hematopoietic cells are obtained in suspension and biopanning can be performed by mixing the hemocyte suspension with the phage library and then washing with several buffers. Phage are extracted from human cells, amplified, and the displayed antibody fragment sequence is determined.
[0128]
[141.] In an even more preferred embodiment of the present invention, the blood cell suspension comprises leukemic cells. In a most preferred embodiment, the blood cell suspension comprises AML cells. In another embodiment of the subject invention, the target cells are derived from an isolated organ or part thereof.
[0129]
[142.] In another embodiment of the subject invention, the target cell or second cell is derived from one cell lineage. Cell lines can be cultured and manipulated so that the binding characteristics of the Fv clone can be readily determined. In addition, cell lines may be useful in developing diagnostic kits.
[0130]
[143.] In a preferred embodiment, the cell line is, without limitation, Jurkat, Molt-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2 and HUT-. Hematopoietic cell line such as the 78 cell line.
[0131]
[144.] In a preferred embodiment of the invention, the CDR3 region is constructed, inserted, coupled or fused in or on any one of the 84 cassettes (SEQ ID NOS: 30-113). In a more preferred embodiment, the CDR3 region comprises 49 cassettes (SEQ ID NOS: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71). , 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113. In a most preferred embodiment, Has CDR3 constructed, inserted, coupled or fused at the C-terminus of the cassette of SEQ ID NO: 61 or any of the above sequences having at least 90% sequence similarity thereto.
[0132]
[145.] In one embodiment, the amino acid sequence of the cassette is explicitly fixed, while the substituted, inserted or attached sequences can be highly variable. A cassette may be composed of multiple domains, each containing a function critical to the final construct. The cassette of certain embodiments of the invention comprises, from the N-terminus, framework region 1, (FR1), CDR1, framework region 2 (FR2), CDR2 and framework region 3 (FR3).
[0133]
[146.] In one embodiment of the present invention, it is possible to replace completely different regions in the cassette. For example, the CDR2 and CDR1 hypervariable regions of the cassette can be substituted or modified by non-conservative or, preferably, conservative amino acid substitutions. More specifically, the CDR2 and CDR1 regions of the cassette of contiguous amino acids selected from the group consisting of SEQ ID NOs: 30-113 or fragments thereof may be replaced by SEQ ID NOs: 115 and 114, respectively. Even more particularly, SEQ ID NOs: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89. The CDR2 and CDR1 regions of a cassette of contiguous amino acids selected from the group consisting of -92, 94, 97, 99, 103, 106, 112 and 113 or fragments thereof may be replaced by SEQ ID NOs: 115 and 114, respectively. .
[0134]
[147.] In a preferred embodiment of the invention, the peptide or polypeptide comprises a heavy chain and a light chain, wherein each chain is a CDR3, CDR2 and CDR1 region, respectively, of the first, second and third super-regions. Including the variable region. The selectivity and specificity of the binding is determined in particular by the CDR3 region of one chain, if possible by the CDR3 region of the light chain and preferably by the CDR3 region of the heavy chain, and secondarily by the light chain. Is determined by the CDR2 and CDR1 regions of the heavy chain. Binding selectivity and specificity can also be influenced secondarily by upstream or downstream regions flanking the first, second and / or third hypervariable regions.
[0135]
[148.] In a preferred embodiment, the CDR3 region of the peptide or polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24.
[0136]
[149.] More preferably, the CDR3 region of the heavy chain has one amino acid sequence selected from the group including SEQ ID NOs: 8 to 24, and CDR2 has the same amino acid sequence as SEQ ID NO: 115, The CDR1 region has the same amino acid sequence as SEQ ID NO: 114.
[0137]
[150.] In a most preferred embodiment of the present invention, the CDR3 region has the same amino acid sequence as SEQ ID NO: 8.
[0138]
[151.] In addition to the heavy and light chains, Fv contains a flexible spacer of 0-20 amino acid residues. Spacers can be branched or heavy chains. The two possible sequences of the spacer are identical to SEQ ID NO: 123 and 124.
[0139]
[152.] A preferred embodiment of the present invention is a scFv with a CDR3 sequence identical to SEQ ID NO: 8 and a complete scFe sequence identical to SEQ ID NO: 25.
[0140]
[153.] Another preferred embodiment of the present invention is a scFv with a CDR3 sequence identical to SEQ ID NO: 20 and a complete scFv sequence identical to SEQ ID NO: 203.
[0141]
[154.] In a most preferred embodiment of the present invention, the CDR3, CDR2 and CDR1 regions have the amino acids of SEQ ID NOs: 8, 115 and 114, respectively.
[0142]
[155.] In one embodiment of the present invention, the Fv peptide itself comprises a CDR1 and CDR2 region of a variable heavy chain comprising a cassette with an amino acid sequence selected from the group comprising SEQ ID NOs: 30-113; Preferably a CDR3 region of a variable heavy chain having an amino acid sequence selected from the group comprising SEQ ID NOs: 8-24; an upstream region flanking a CDR3 region having an amino acid sequence of SEQ ID NO: 117; an amino acid of SEQ ID NO: 116 A downstream region flanking a CDR3 region having a sequence; a spacer of 0 to 20 amino acid residues of SEQ ID NO: 123 or 124; and a variable light chain having a sequence of SEQ ID NO: 7.
[0143]
[156.] Similarly, in another embodiment, the upstream region flanking the CDR2 region has an amino acid sequence of SEQ ID NO: 119, and the downstream region flanking the CDR2 region is SEQ ID NO: 118. The upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121, and the downstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 120.
[0144]
[157.] In a preferred embodiment of the present invention, the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively, the CDR3 amino acid sequence is SEQ ID NO: 8, and the CDR2 amino acid sequence is SEQ ID NO: 115. The CDR1 amino acid sequence is SEQ ID NO: 114, the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117, and the downstream region flanking the CDR3 region is the amino acid sequence of SEQ ID NO: 116. The upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119, the downstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 118, and the upstream region flanking the CDR1 region. The flanking region has the amino acid sequence of SEQ ID NO: 121, and flanks the CDR1 region. Side region is provided a peptide or polypeptide having the amino acid sequence of SEQ ID NO: 120.
[0145]
[158.] Another embodiment of the present invention is directed to a first chain having first, second and third hypervariable regions and a second chain having first, second and third hypervariable regions. In a Fv molecule comprising a chain, one of the hypervariable regions of the first chain has a sequence selected from the group consisting of SEQ ID NOs: 8-24 and the hypervariable region of the second chain. One of which has one sequence selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, wherein the first, second and third hypervariable regions are CDR3, CDR2 and CDR1, respectively. Region, wherein the Fv is a scFv or dsFv, optionally providing an Fv molecule with one or more tags.
[0146]
[159.] Another embodiment of the real name invention comprises (i) a first hypervariable region wherein the first strand and the second strand each are selected from the group consisting of SEQ ID NOs: 8-24. Or (ii) the first hypervariable region of the first chain and the first hypervariable region of the second chain are identical and are selected from the group consisting of SEQ ID NOs: 8-24; (Iii) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs: 8-24, and the first hypervariable region of the second chain is SEQ ID NOs: 1-6 and 125-125; Or (iv) the first hypervariable region of the first strand is selected from the group consisting of SEQ ID NOs: 1-6 and 125-202; The first hypervariable region is selected from the group consisting of SEQ ID NOS: 8 to 24. Is provides a peptide or polypeptide.
[0147]
[160.] Another embodiment provides a peptide or polypeptide of the invention, wherein the second and third hypervariable regions of the first chain are SEQ ID NOs: 114 and 115, respectively.
[0148]
[161.] For all amino acid sequences of up to and including 25 amino acid residues described and described in detail herein, these amino acid sequences include within their scope one or two amino acid substitutions; Preferably, it is to be understood and considered that the substitution is a conservative amino acid substitution as a further embodiment of the invention. For all amino acid sequences having more than 25 amino acid residues described and described in this document, amino acid sequences having 90% or more sequence similarity with the original sequence within the range are included. Is included and understood as further embodiments of the invention (Altschul et al., Nucleic Acids Res., 25, 3389-3402 (1997)). Similar or homologous amino acids are defined as non-identical amino acids that exhibit similar properties, eg, acidic, basic, aromatic, size, positive or negatively charged, polar, non-polar.
[0149]
[162.] The percentage of amino acid similarity or homology or sequence similarity is determined by comparing the amino acid sequences of two different peptides or polypeptides. The two sequences are usually aligned by using one of a variety of specially designed computer programs, and the amino acid residues at each position are compared. Next, the amino acid identity or homology is determined. Thereafter, an algorithm is applied to determine the percent amino acid similarity. It is generally preferable to compare amino acid sequences because of the greatly increased sensitivity to detecting subtle relationships between peptide, polypeptide or protein molecules. Protein comparisons can take into account the presence of conservative amino acid substitutions, so that if non-identical amino acids have similar physical and / or chemical properties, one mismatch still produces a positive score There is a possibility (Altschul et al., Nucleic Acids Rs., 25, 3389-3402 (1997)).
[0150]
[163.] In one embodiment of the invention, the three hypervariable regions of each of the light and heavy chains are inter-related between the two chains and between the three hypervariable sites within and / or between the chains. Can be exchanged.
[0151]
[164.] One skilled in the art will be aware that demonstrating specific and / or selective binding of a peptide or polypeptide of the present invention requires the use of appropriate negative controls. Would. One suitable negative control may be a peptide or polypeptide having an amino acid sequence that is substantially identical to a peptide or polypeptide of the present invention, but has only one difference in the hypervariable CDR3 region. Another suitable negative control may be a peptide or polypeptide having the same size and / or overall three-dimensional structure as the peptide or polypeptide of the invention, but having a completely unrelated amino acid sequence. Another suitable negative control can be a peptide or polypeptide that has completely different physical and chemical properties when compared to a peptide or polypeptide of the present invention. The negative controls used in the development of the present invention are designated N14, which has the same CDR3 sequence as SEQ ID NO: 28, and C181, which has the same CDR3 sequence as SEQ ID NO: 29. However, other negative controls may be appropriate as well.
[0152]
[165.] Another embodiment provides a nucleic acid molecule, preferably a DNA molecule, encoding an Fv peptide or polypeptide of the present invention.
[0153]
[166.] In a preferred embodiment of the present invention, the CDR3 sequence that confers primary binding selectivity and / or specificity to Fv is replaced with any other heavy chain gene to optimize the selective binding of Fv. It can be moved to a cell lineage. More specifically, they can be moved to one of the 84 possible heavy chain germline. These 84 germline (SEQ ID NOs: 30-113) include (a) the germline from which the claimed phage clone was originally isolated, and (b) the 48 additional available phage display libraries. Germline and (c) the 35 alternative germline claimed herein (Tomlinson et al., J. Mol. Biol., 227 (3): 776-798 (1992)). ). In line with the CDR3 region itself, the local linear or three-dimensional environment of the CDR3 region may potentially play a role in inducing or driving proper CDR3 binding. For example, 49 germline sequences (SEQ ID NOs: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 57, 59-68) designated herein as SEQ ID NOs: 8-24, 125. , 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113), as well as peptides having any of the CDR3 sequences derived from the subject. Covered by the invention.
[0154]
[167.] Germline DP-32 is a cassette for multiple clones of the present invention. The germline C-terminus was replaced with a consensus sequence to aid in the preparation of a phage display library. The seven carboxy terminal amino acids of SEQ ID NO: 61 have been replaced by the seven amino acid sequences of SEQ ID NO: 122.
[0155]
[168.] The CDR3 region of the Fvs of the present invention may contain a core sequence Arg / Gly / Lys Phe Pro that specifically binds to AML cells. Eight examples of such CDR3 regions are provided in Table 2. The motif corresponds in each case to the three N-terminal amino acid residues of the CDR3 region, but it is also possible to position it elsewhere in the CDR3 region. Alternatively, the motif is a binding motif that is used to construct or configure an anchor or binding region for a larger binding or targeting or recognition molecule portion, or used alone as a target vehicle.
[0156]
[169.] In a further embodiment of the present invention, R₁And R_TwoHas 0 to 15, preferably 1 to 9 amino acid residues and X is Arg, Gly or Lys.₁-X Phe Pro-R_TwoA binding motif comprising the amino acid sequence of Most preferably, CDR3 is R₁-XPhe Pro-R_TwoComprising the amino acid sequence of₁And R_TwoContains 0 to 15 amino acid residues, and X is any of Arg, Gly, or Lys.
[0157]
[170.] In another preferred embodiment of the peptide or polypeptide of the subject invention, the peptide retains its biological activity while 1-1000 relative to either the C-terminus or the N-terminus of the peptide Amino acids can be added. In a preferred embodiment of the invention, the peptide retains its biological activity while adding 150 to 500 amino acids to either the C-terminus or the N-terminus of the peptide or polypeptide. In another preferred embodiment of the invention, the peptide or polypeptide retains its biological activity while adding 800-1000 amino acids to either the C-terminus or the N-terminus of the peptide or polypeptide. be able to.
[0158]
[171.] An example of extending the core amino acid sequence is by constructing a full-sized immunoglobulin Ig using a lead compound as the Ig core. Full-sized Igs may belong to the immunoglobulin class capable of inducing endogenous cytolytic activity via activation of the cytolytic activity of the cell or complement (eg IgG1, IgG2 or IgG3). Full-sized Igs may also belong to the immunoglobulin class of strongly binding antibodies (eg, IgG4). At the time of binding, the full-size Ig may transduce intracellular cell signaling or cause damage to target cells, for example by acting as a flag for the body's defense mechanisms to mount an immune response. It may work in one or more of a number of ways, such as by.
[0159]
[172.] One preferred embodiment of the present invention provides Ig molecules expressed as recombinant polypeptides and produced in eukaryotic cell lines. In one preferred embodiment of the invention, the Ig polypeptide is an IgG polypeptide, which is produced in a mammalian cell line. In a more preferred embodiment, the mammalian cell line comprises a CMV promoter.
[0160]
[173.] In a preferred embodiment of the invention, the IgG molecule comprises the hypervariable regions of CDR3, CDR3 and CDR1 in both the light and heavy chains. In a more preferred embodiment of the invention, the Fv molecule comprises CDR3, CDR2 and CDR1 regions having SEQ ID NOs: 8, 115 and 114, respectively. The CDR3, CDR2 and CDR1 regions can be of heavy and light chains.
[0161]
[174.] A further preferred embodiment of the present invention relates to a light chain having the same sequence as SEQ ID NO: 27, a heavy chain having the same sequence as SEQ ID NO: 26 or a heavy chain having at least 90% sequence similarity thereto. And an IgG molecule having a light chain. In a most preferred embodiment of the invention, the two heavy chains of the IgG are identical and the two light chains of the IgG are identical.
[0162]
[175.] In another embodiment, the subject peptides of the invention are constructed to fold into a multivalent Fv form.
[0163]
[176.] The present invention provides a peptide or polypeptide of Y1 or Y17 comprising a scFv molecule. As used herein, a scFv is composed of the variable region of the heavy chain of a human antibody and the variable region of the light chain of a human antibody, which may be the same or different, and The variable region of the heavy chain is defined as a molecule that is attached to, or associated with, the variable region of the light chain by a link, link, fusion, or covalent bond.
[0164]
[177.] The Y1 and Y17 scFv constructs can be multimers (eg, dimers, trimers, tetramers, etc.) of scFv molecules that include one or more of the hypervariable domains of the Y1 or Y17 antibodies. . All scFv-derived constructs and fragments retain enhanced binding properties to selectively and / or specifically bind to target cells in a manner that favors other cells. Binding selectivity and / or specificity is determined primarily by the hypervariable region.
[0165]
[178.] The hypervariable loops within the light and heavy chain variable domains are called complementary determining regions (CDRs). There are CDR1, CDR2 and CDR3 regions in each of the heavy and light chains. The most variable of these regions is the CDR3 region of the heavy chain. The CDR3 region is understood to be the most exposed region of the Ig molecule and is the site responsible primarily for the observed selective and / or specific binding as defined herein.
[0166]
[179.] The Y1 and Y17 peptides of the subject invention can be constructed to fold into multivalent Fv forms. Y1 and Y17 multimeric forms have been constructed to improve binding affinity and specificity and increased blood half-life.
[0167]
[180.] Multivalent forms of scFv have been produced by other researchers. One approach was to link the two scFvs with a linker. Another approach involves using a disulfide bond between the two scFvs for linkage. The simplest approach to the production of dimeric or trimeric Fv is described by Holliger et al., PNAS, 90, 6444-6448 (1993) and A. Kortt, et al., Protein Eng., 10, 423-433. (1997). One such method was designed to create a dimer of scFvs by adding the sequence of the FOS and JUN protein regions and forming a leucine zipper between them at the C-terminus of the scFv. Kostelny SA et al., J Immunol. 1992 Mar 1; 148 (5): 1547-53; De Kruif J et al., J Biol Chem. 1996 Mar 29; 271 (13): 7630-4. Another method was designed to create a tetramer by adding a streptavidin coding sequence at the C-terminus of the scFv. Streptavidin is composed of four subunits, so when the scFv streptavidin folds, the four subunits contain themselves to form a tetramer. Kipriyanov SM et al., Hum Antibodies Hybridomas, 1995; 6 (3); 93-101. In yet another method, free cysteine is introduced into the protein of interest to make dimers, trimers and tetramers. To crosslink the protein of interest to free cysteine, a peptide-based crosslinker with a variable number (2-4) of maleimide groups was used. Cochran JR et al., Immunity, 2000 Mar; 12 (3): 241-50.
[0168]
[181.] In this system, a phage library (as described herein above) was designed to display scFvs that can fold into a monovalent form of the Fv region of one antibody. . Further, the constructs also discussed herein above are suitable for bacterial expression. Genetically engineered scFvs contain heavy and light chain variable regions joined by a flexible, encoded 15 amino acid peptide spacer. A preferred spacer is (Gly_FourSer)_ThreeIt is. The length of this spacer, together with its amino acid building blocks, provides a non-bulky spacer, which allows the VH and VL regions to fold into a functional Fv domain that provides efficient binding to its target.
[0169]
[182.] The present invention is directed to multimers of Y1 and Y17 prepared by any method known in the art. A preferred method of forming multimers, especially dimers, utilizes cysteine residues to form a disulfide bond between the two monomers. In this embodiment, the dimer is formed by adding cysteine on the carboxy terminus of the scFvs (called Y1-cys scFc or Y1 dimer) to facilitate dimer formation. After the DNA construct was made (see Examples 2D and 6D) and used for transfection, the Y1 dimer was expressed in the production vector and refolded in vitro. Proteins were analyzed by SDS-PAGE, HPLC and FACS. A two liter fermentation batch of the antibody was performed. After expression of Y1-cys in E. coli strain B21, refolding was performed in arginine. After refolding, the protein was dialyzed and purified by Q-Sepharose and gel filtration (sephadex 75). Two peaks were detected by SDS-PAGE (unreduced) and gel filtration. Peaks were collected separately and analyzed by FACS. Monomer and dimer binding to Jurkat cells was examined by FACS. Binding by the dimer required only one hundredth of the amount of monomeric antibody for the same staining level, indicating that the dimer had greater avidity. The conditions for dimer refolding were determined and material containing greater than 90% dimer (mg quantity) was produced after the subsequent dialysis, chromatography and gel filtration steps. The purified dimer was characterized by gel filtration and SDS-PAGE analysis under oxidizing conditions. Radioreceptor assay, ELISA and FACS analysis confirmed the binding capacity of the dimer.
[0170]
[183.] A CONY1 scF antibody fragment is derived from Y1 scFv. The DNA sequence encoding the myc tag of Y1scFv was removed and replaced with a synthetic oligonucleotide DNA sequence encoding the amino acids lysine, alanine, lysine (KAK).
[0171]
[184.] To compare the binding of the Y1 dimer to the scFv monomer (also called CONY1), binding competition experiments were performed on KG-1 cells in vitro. In addition, these experiments compared the binding of total YIIgG to the scFvY1 monomer. To perform this study, Y1 IgG Y1 IgG was labeled with biotin. This study revealed that Y1 IgG competed with IGY cobiotin. Unrelated human IgG did not compete with the labeled Y1 IgG. Y1 scFvs (5 μg and 10 μg) partially competed with Y1 IgG-Biotin (50 ng). Studies also showed that 1 ng of IgG Y1-FITC bound to KG-1 cells to the same extent as 1 μg of scFv FITC (without serum), but in the presence of serum, most Y1 IgG binding was “blocked”. Will be done. " These studies also showed that binding of the Y1 dimer was at least 20 times higher than that of the scFv monomer, as analyzed by radioreceptor assay, ELISA or FACS.
[185.] In yet another embodiment, lysine-alanine-lysine was added in addition to the cysteine at the carboxy terminus (termed YI-cys-kak scFv). The amino acid sequence of this scFv construct is reproduced below.
[0172]
Embedded image

[0173]
[186.] YI-cys-kak was produced in a λ-pL vector in bacteria. Increasing the temperature to 42 ° C induced expression in the λ-pL vector. Inclusion bodies were obtained from the induced cultures and semi-purified with an aqueous solution to remove unwanted soluble proteins. This inclusion body was solubilized in guanidine, reduced by DTT, and refolded in vitro in an arginine / ox-glutathione-based solution. After refolding, the protein was dialyzed and concentrated by filtration in a tangential flow against a buffer containing a urea / phosphate buffer. The protein was repurified and concentrated by ion chromatography in a SP-column.
[0174]
[187.] To obtain higher levels of expression in the CONY1 scFv as well as in the Y1-cys-kak scFv, we introduced an amino acid encoding an alanine residue at position 2 of the N-terminal sequence of the scFv construct. did. A 4-fold expression level was obtained with this newly modified construct.
[0175]
[188.] Binding between the monomer (also known as CONYscFv-YI-kak) and the dimer YI-cys kak (cysteine dimer) for the platelet-derived antigen GPIb (glycocalysin) An ELISA test was performed to confirm the difference between the two. To detect binding to GPIb, polyclonal anti-single-chain antibodies and / or novel polyclonal anti-V_L(From rabbit) and anti-rabbit HRP were used. The dimer showed 2 to 100 times higher activity than the monomer. For example, to achieve 0.8 OD units, 12.8 mg / ml monomer was used as compared to only 0.1 mg / ml dimer. See FIG.
[0176]
[189.] Dimers were characterized by SDS-page electrophoresis, gel filtration chromatography, ELISA, radioreceptor binding and FACS. The apparent affinity of the dimer was higher than the monomer due to avidity. This effect was confirmed by ELISA for glycocalysin, FACS for KG-1 cells and competition in the radioreceptor assay.
[0177]
[190.] HPLC was performed to profile the dimer after refolding and purification from a Superdex 75 gel filtration column. In FIG. 10, Y1-cys-kak (dimer) is the first peak on the left (〜10.8 min), and the subsequent peak is the monomer (〜12 min). According to a protein size marker run on the same column, the dimer is about 52 kDa and the monomer is 26 kDa. The balance between dimer and monomer can be altered by varying the refolding conditions (the concentration of oxidized agent and the concentration of protein in the refolding buffer). Dimers and monomers were separated by chromatography in a super-dex 75 column.
[0178]
[191.] FIG. 11 shows a gel with a mixed population of dimers and monomers. In the reduced form, monomers are seen due to the reduction between the two monomers, and in the unreduced form, as in two populations (as in the gel filtration experiment), a single monomer of about 30 kDa. A dimer of about 60 kDa is seen with the body fraction.
[0179]
[192.] In addition, FACS binding analysis on KG-1 cells indicated that the dimer was more sensitive than the monomer when a two- or three-step binding assay was performed. The dimer directly labeled by FITC showed a slight advantage over the monomer (approximately 10 times less material used). Radioreceptor assays on KG-1 cells using the dimer as a competitor showed that the dimer was 30 times more efficient than the monomer.
[0180]
[193.] Varying the length of the spacer is yet another preferred method of forming dimers, trimers and tetramers (often in the art, diabodies, triabodies and called tetrabodies). The spacer connecting the two variable chains of the scFv is generally <? Under conditions reduced to>, a dimer is formed. This shortened spacer prevents two variable chains from the same molecule from folding into one functional Fv domain. Instead, the domain is forced to pair with the complementary domain of another molecule, creating two binding domains. In a preferred method, only 5 amino acids (Gly_FourSer) spacers were used. The dimer can be formed from two identical scFvs or from two different scFvs populations, retaining and / or increasing the selective and / or specific enhanced avidity of the parent scFv (s) The binding strength or affinity.
[0181]
[194.] In a similar manner, the spacer connecting the two variable chains of the scFv is generally shortened to less than 5 amino acid residues, and the two variable chains from the same molecule are combined into one functional Fv domain. Under conditions that prevent folding, triabodies are formed. Instead, three separate scFv molecules associate to form a trimer. In a preferred method, triabodies were obtained by completely removing the flexible spacer. Triabodies can be formed from three identical scFvs or from two or three different scFvs populations, retaining and / or increasing the selective and / or specific enhanced avidity of the parent scFv (s) It can indicate binding strength or affinity.
[0182]
[195.] In a similar manner, the spacer connecting the two variable chains of the scFv is generally shortened to less than 5 amino acid residues, and the two variable chains from the same molecule are combined into one functional Fv domain. Tetrabodies are formed under conditions that prevent folding. Instead, four separate scFv molecules associate to form a tetramer. A tetrabody can be formed from four identical scFvs or from one to four individual units from a different scFvs population, retaining the selective and / or specific enhanced binding activity of the parent scFv (s). And / or should exhibit increased binding strength or affinity.
[0183]
[196.] Whether triabodies or tetrabodies are formed under the condition that the length of the spacer is generally less than 5 amino acid residues depends on the amino acid sequence of the specific scFv (s) in the mixture and the reaction conditions. Is done.
[0184]
[197.] In a preferred method, tetramers are formed via biotin / streptavidin association. A new fermentation construct has been created that has the ability to be enzymatically labeled with biotin (referred to herein as Y1-biotag or Y1-B). A sequence that was a substrate for the BirA enzyme was added at the Y1C-terminus. The BirA enzyme adds biotin to lysine residues within this sequence. Y1-biotag was cloned and expressed in E. coli. The inclusion body material was isolated and refolded. The purity of the folded protein was greater than 95% and the 1-L cultures (small, non-optimized conditions) yielded greater than 100 mg purity. The molecular weight of this form was found to be similar to that of the scFv according to HPLC, SDS-PAGE and mass spectroscopy. Y1-biotag was found to be the most consistent reagent for FACS analysis. However, when Y1-biotag binding to KG-1 cells is tested in the presence of serum, higher concentrations (> 10-fold) are required for comparable binding in the absence of serum. Nevertheless, this construct provided the advantage of specific biotinylation, in which the binding site of the molecule remained intact. Further, each molecule is labeled with only one biotin, and each molecule receives one biotin on the carboxy terminus.
[0185]
[198.] Restricting labeling to one biotin / molecule where desired allowed the production of tetramers with streptavidin. Tetramers were formed by incubating Y1-B with streptavidin-PE.
[0186]
[199.] FACS analysis showed that the tetramer made with Y1-biotag and streptavidin-PE was 100-1000 times more sensitive than the Y1 scFv monomer in the absence of serum. The Y1-biotag tetramer with streptavidin-PE appears to specifically bind to one of the Y1-reactive cell lines (KG-1). The disparity of this reaction from background binding was very high, providing high sensitivity for detecting low levels of receptor. FACS evaluation of normal whole blood with the Y1-SAV tetramer indicated that there was no highly responsive population. Monocytes and granulocytes were to a lesser extent positive. In cell lines where a positive result was present, such as in the case of KG-1 cells, the tetramer was at least 100-fold more reactive.
[0187]
[200.] Next, the tetramer was incubated with the cell preparation. Low doses of Y1 tetramer (5 ng) bind well to the cell line (KG-1) and provide a 10-20 fold higher response than previously observed with other Y1 antibody forms. When negative cell lines were tested with various doses of tetramer, a slight response was seen.
[0188]
[201.] One embodiment of the present invention is:
A method for identifying a targeting molecule that binds to an unknown immune cross-reactive binding site on first and second cells, comprising: (a) one unknown ligand in a second state instead of the first state; One or more biopanning steps performed on a first target cell substantially exposing or displaying a binding site comprising, thus producing a first population of recognition molecules; (b) a second recognition The recognition of step (a), which is performed on a second cell displaying a binding site containing one unknown ligand having immunological cross-reactivity to the unknown ligand of the first cell to generate a molecular population. Performing a subsequent biopanning and / or selection step starting from the resulting stock of molecules; (c) amplifying and purifying the second population of recognition molecules of step (b); and ( And b.) Constructing a peptide or polypeptide comprising a targeting molecule that is selective and / or specific for the unknown ligand on the second cell from the recognition site of the purified recognition molecule of step (c). And a method for identifying a targeting molecule.
[0189]
[202.] One preferred embodiment is that the first cell is a normal cell, the first state is a non-activated state, and the second state is activation, excitation, modification, alteration. Or, it is in a state of being obstructed. In a more preferred embodiment, the second cell is a pathological cell. In an even more preferred embodiment, the diseased cell is a cancer cell. The cancer cells can be, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminomas and melanoma. In an even more preferred embodiment, the cells are leukemia cells. In a most preferred embodiment, the leukemia cells are AML cells.
[0190]
[203.] Provides the use of a peptide or polypeptide in the manufacture of a medicament, optionally associated with, or attached to, coupled to, associated with, linked to or fused to a pharmaceutical agent. . In one preferred embodiment, the medicament has activity against diseased cells. In a more preferred embodiment, the activity is against cancer cells. The cancer cells can be, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. In an even more preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, the leukemia cells are AML cells.
[0191]
[204.] One embodiment of the present invention provides a pharmaceutical composition comprising a mixture of different monomeric scFvs and / or a mixture of diabodies or triabodies or tetrabodies constructed from different scFvs.
[0192]
[205.] Yet another embodiment of the present invention is a method of manufacturing a medicament, optionally associated with, or attached to, coupled to, associated with, linked to or fused to a pharmaceutical agent. Or the use of a peptide or polypeptide of the invention. The medicament may have activity against pathological cells, more specifically against cancer cells. The cancer cells can be, but are not limited to, carcinoma, sarcoma, leukemia, fibroids, lymphoma, myeloma, blastoma, seminomas and melanoma. In a more preferred embodiment, the medicament has activity against leukemia cells. In a most preferred embodiment, the medicament has activity on AML cells. The activity of the medicament on the cells may delay cancerous growth, completely prevent any growth or cause the death of cancerous cells.
[0193]
[206.] In one embodiment of the present invention, the activity of the medicament or pharmaceutical composition is by inhibiting cell growth.
[0194]
[207.] The peptide or polypeptide of the present invention is used for preparing a composition, preferably a pharmaceutical composition, for use in inhibiting the growth of cancer cells, preferably leukemia cells and most preferably AML cells. can do. In one embodiment of the invention, the peptide or polypeptide can be used for preparing a composition for use in inhibiting the growth of cancer cells, wherein the composition is selective and / or specific for cancer cells. At least one compound having a specific pharmaceutical ligand.
[0195]
[208.] The peptide or polypeptide of the subject invention can be administered to a patient alone, or otherwise a pharmaceutically effective amount of a pharmaceutical agent, a pharmaceutically effective carrier and optionally Can be administered in association with, or conjugated, linked to, or fused to an adjuvant, including a medicament or pharmaceutical composition. Such pharmaceutical compositions may include proteins, diluents and antioxidants (see Osol et al. (Eds.), Remington's Pharmaceutical Sciences (16th edition), Mack Publishing Company (1980)).
[0196]
[209.] In another embodiment, the pharmaceutical agent is an antibody or fragment thereof linked to a peptide or polypeptide of the invention by a peptide bond.
[0197]
[210.] In one preferred embodiment, the toxin is, for example, gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or a modification or derivative thereof.
[0198]
[211.] In a preferred embodiment, the radioisotopes used can be used for gamma, positron and X-ray sources and therapeutics that can be used for localization and / or therapy. Includes beta and alpha sources.
[0199]
[212.] In certain embodiments of the subject invention, the therapeutic radioisotope is¹¹¹indium,¹¹³indium,^99mrhenium,¹⁰⁵rhenium,¹⁰¹rhenium,^99mtechnetium,^121mTellurium,^122mTellurium,^125mTellurium,¹⁶⁵thulium,¹⁶⁷thulium,¹⁶⁸thulium,^{one two Three}Iodine,¹²⁶Iodine,¹³¹Iodine,¹³³Iodine,^81mkrypton,³³xenon,⁹⁹yttrium,²¹³Bismuth,⁷⁷Bromin,¹⁸Fluorin,⁹⁵ruthenium,⁹⁷ruthenium,¹⁰³ruthenium,¹⁰⁵ruthenium,¹⁰⁷mercury,²⁰³mercury,⁶⁷Gallium and⁶⁸Selected from the group containing gallium.
[0200]
[213.] In another particular embodiment of the subject invention, the anticancer agent is doxorubicin, adriamycin, cis-platinum, toxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone. , Daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, bleomycin and derivatives thereof.
[0201]
[214.] One embodiment of the present invention provides a method for inhibiting the growth of cancer cells, comprising contacting the cancer cells with a fixed amount of a peptide or polypeptide of the present invention. In a preferred embodiment, the cancer cells can be, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. In a more preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, the leukemia cells are AML cells. One embodiment of the present invention allows for in vivo and ex vivo treatment of a patient. A more specific embodiment of the present invention allows for ex vivo purging of autologous bone marrow to remove abnormal stem cells.
[0202]
[215.] In a more specific embodiment of the invention, the blood of a leukemia patient can be circulated ex vivo through a system comprising a peptide or polypeptide of the invention conjugated to an anticancer agent. After removal of bound cells and unbound anticancer agent, blood cells can be reintroduced into the patient. Alternatively, the blood of a leukemia patient can be circulated ex vivo through a system comprising a peptide or polypeptide of the invention attached to a solid phase. Cells that have not passed through the system and have not bound to the peptide or polypeptide of the invention attached to the solid phase can be reintroduced into the patient's body.
[0203]
[216.] In another preferred embodiment of the present invention, the peptide or polypeptide is utilized for ex vivo autologous bone marrow in suspension to remove abnormal basal cells prior to transplantation. Purging of aberrant stem cells can be accomplished by binding a peptide or polypeptide of the invention (ie, a targeting molecule), a constituent, a fragment, a fragment of a constituent, or a constituent of a fragment thereof, to a solid support (eg, magnetic beads and affinity beads). This can be done by running the suspension over an acidic column, but not limited to. The bone marrow thus purged ex vivo can then be used for autologous bone marrow transplantation. This preferred embodiment is based on the present invention's identification of a phagemid clone (Y1) that binds to stem cells released from the bone marrow of a leukemia patient but does not bind to stem cells released from the bone marrow of a healthy donor. In a similar manner, the Y1 phagemid clone binds to blasts and leukemia cells determined to be abnormal by FACS analysis.
[0204]
[217.] In this document, blast cells are defined as primary cells that are precursors to all circulating cells in a mammalian organism. Due to their primordial properties, blasts have not been found to circulate in significant amounts in adult humans. The presence of circulating blasts without exogenous stimuli, for example, is indicative of a malignant tumor of the hematopoietic system, and its subsequent disappearance may indicate remission of the malignant disease.
[0205]
[218.] In another embodiment of the present invention, the pharmaceutical composition is used for prophylaxis.
[0206]
[219.] In a preferred embodiment, two or more peptides or polypeptides of the invention are combined to form a mixture.
[0207]
[220.] As used herein, a mixture is defined as two or more molecules or particles of different species contained in a single preparation. Different species of the molecule do not form covalent or non-covalent chemical bonds.
[0208]
[221.] In one embodiment of the subject invention, the peptide or polypeptide of the subject invention is linked, fused or conjugated to a pharmaceutical agent.
[0209]
[222.] In another embodiment of the subject invention, the link between the peptide and the pharmaceutical agent is a direct link. As used herein, a direct link between two or more adjacent molecules is obtained via a chemical bond between elements or groups of elements within the molecule. The chemical bond can be, for example, an ionic, covalent, hydrophobic, hydrophilic, electrostatic or hydrogen bond. The bond may be selected from, but not limited to, the group comprising amine, carboxy, amide, hydroxyl, peptide and disulfide. The direct link may preferably be a protease resistant bond.
[0210]
[223.] In yet another embodiment, the link between the peptide and the pharmaceutical agent is acted upon by a linker compound. As used herein and in the claims, a linker compound is defined as a compound that joins two or more moieties together. The linker can be a linear or branched linker. Branched linkers may consist of double-branched, triple-branched or quadruple or more branched compounds. Linker compounds can be, but are not limited to, dicarboxylic acids, maleimide hydrazides, PDPH, carboxylic acid hydrazides, and small peptides. Examples of other linker compounds include dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid; N- [ε-maleimidocaproic acid] hydrazide, 4- [N-maleimidomethyl] diclohexane-1-carboxyhydrazide and N Maleimide hydrazide, such as-[K-maleimidoundecanoic acid] hydrazide; PDPH linker, such as (3- [2-pyridyldithio] propionyl hydrazide) conjugated to sulfahydryl-reactive protein; selected from 2-5 carbons Carboxylic acid hydrazides; and, for example, direct coupling between the free sugar of the anticancer drug doxorubicin and the scFv using a small peptide linker. Small peptides include AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, protein C, S • Tag ™, T7, V5, VSV-G. , And KAK Tag, but are not limited to these.
[0211]
[224.] Any known method of administering the peptide or polypeptide of the subject invention, eg, intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, intranasal, sublingual, Oral, rectal, vaginal, inhalation, buccal, intradermal, transdermal or intrathoracic administration can be utilized.
[0212]
[225.] For intravenous administration, the formulation is preferably prepared in such a manner that the amount administered to the patient will be an effective amount of about 0.1 mg to about 1000 mg of the desired composition. Become. More preferably, the amount administered will be in the range of about 1 mg to about 500 mg of the desired composition. The compositions of the present invention are effective over a wide dosage range and provide details about the disease to be treated, the half-life of the peptide or polypeptide-based pharmaceutical composition in the patient's body, the pharmaceutical agent and the pharmaceutical composition. It depends on factors such as the physical and chemical properties of the product, the mode of administration of the pharmaceutical composition, the details of the patient to be treated or diagnosed, and other parameters that the treating physician deems important.
[0213]
[226.] Pharmaceutical compositions for oral administration may be in the form of tablets, liquids, emulsions, suspensions, syrups, pills, caplets, or capsules. The pharmaceutical composition can also be administered in a device.
[0214]
[227.] Pharmaceutical compositions for topical application may be in the form of creams, ointments, lotions, patches, solutions, suspensions or gels.
[0215]
[228.] Additionally, pharmaceutical compositions can be prepared for solid, liquid or sustained release formulations.
[0216]
[229.] The compositions comprising the antibody fragments produced according to the present invention may include a conventional pharmaceutically acceptable diluent or carrier. Tablets, pills, caplets, and capsules may contain conventional excipients such as lactose, starch and magnesium stearate. Suppositories may include excipients such as waxes and glycerol. Injectable solutions include sterile pyrogen-free media such as saline and include buffers, stabilizers or preservatives. Conventional enteric coatings can also be used.
[0217]
[230.] The subject invention also encompasses methods for producing antibody fragments by synthetic means known in the art.
[0218]
[231.] One embodiment of the present invention relates to at least one of the present invention attached, coupled, combined, linked or fused to an imaging agent for use in diagnostic localization and / or imaging of tumors. Pharmaceutical compositions comprising the peptides or polypeptides.
[0219]
[232.] Yet another embodiment of the present invention relates to a method for determining the efficacy of a treatment before, during or after a treatment comprising an imaging agent comprising a peptide of the invention conjugated to a marker marker molecule. We provide diagnostic kits for in vitro analysis. The present invention further provides a method of using an imaging agent for diagnostic localization and / or imaging of cancer, more particularly, tumor,
a) contacting the cells with the composition;
b) measuring the radioactivity bound to the cells;
c) visualizing the tumor;
A method comprising:
[0220]
[233.] In a preferred embodiment of the invention, the imaging agent of the kit is a fluorescent dye, and the kit is used for the treatment of cancer, more particularly blood-related cancers such as leukemia, lymphoma and myeloma. Provides an analysis of the effectiveness of treatment. FACS analysis is used to determine the intensity of staining and the percentage of cells stained by the imaging agent at each stage of the disease, for example, at the time of diagnosis, during treatment, during remission and during relapse.
[0221]
[234.] The present invention further provides compositions comprising an effective amount of an imaging agent, a peptide of the present invention, and a physiologically acceptable carrier.
[0222]
[235.] In a preferred embodiment, the marker marker molecules include, but are not limited to, radioisotopes, X-ray opaque elements, paramagnetic ions or fluorescent molecules. Any marker known in the art.
[0223]
[236.] In certain embodiments of the subject invention, the indicating radioisotope is:¹¹¹indium,¹¹³indium,^99mrhenium,¹⁰⁵rhenium,¹⁰¹rhenium,^99mtechnetium,^121mTellurium,^122mTellurium,^125mTellurium,¹⁶⁵thulium,¹⁶⁷thulium,¹⁶⁸thulium,^{one two Three}Iodine,¹²⁶Iodine,¹³¹Iodine,¹³³Iodine,^81mkrypton,³³xenon,⁹⁰yttrium,²¹³Bismuth,⁷⁷Bromin,¹⁸Fluorin,⁹⁵ruthenium,⁹⁷ruthenium,¹⁰³ruthenium,¹⁰⁵ruthenium,¹⁰⁷mercury,²⁰³mercury,⁶⁷Gallium and⁶⁸It can be, but is not limited to, gallium.
[0224]
[237.] According to another preferred embodiment, the marker marker molecule is a fluorescent marker molecule. According to a more preferred embodiment, the fluorescent marker molecule is fluorescein, phycoerythrin or rhodamine or a modification or conjugate thereof.
[0225]
[238.] The subject invention also contemplates compositions comprising an effective amount of an imaging agent of the present invention, a pharmaceutical agent linked thereto, and a physiologically acceptable carrier.
[0226]
[239.] The present invention also provides for contacting an imaging agent of the invention with an organ or cell to be imaged under conditions such that the imaging agent binds to organs and cells and imaging the bound imaging agent. Also provided is a method of imaging an organ or cell, which involves imaging the organ or cell.
[0227]
[240.] The subject invention further involves in vivo treating the organ to be treated with the composition of the invention under conditions such that the composition binds to the organ, in vivo. A method for treating one organ is provided.
[0228]
[241.] In a preferred embodiment of the present invention, a peptide or a peptide for targeting malignant cells in whole blood, more specifically leukemia cells, for example by monitoring and imaging cells by FACS analysis. Polypeptides may be utilized. Specimens that receive a higher score (eg, 4 times higher) for tumor cells in relation to normal cells are eligible for treatment.
[0229]
[242.] The invention provides for the treatment of a patient suffering from cancer, comprising administering to the patient an amount of a peptide or polypeptide of the invention effective to treat cancer. In a preferred embodiment, the cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a more preferred embodiment, the cancer is leukemia, and in a most preferred embodiment, the leukemia is AML.
[0230]
[243.] In a most preferred embodiment, the peptides or polypeptides of the invention specifically or selectively bind to AML cells. The present invention provides ligands presented on AML cells bound to the peptides or polypeptides of the invention, as well as peptides or polypeptides that bind to this ligand.
[0231]
[244.] The novel antibody fragments of the subject invention or their corresponding peptidomimetics are used in the manufacture of compositions or medicaments for treating various diseases and conditions.
[0232]
[245.] The subject invention may comprise a) a biopanning procedure directly on the target cells or a biopanning procedure indirectly on the first target cell in the second state instead of the first state. Also subsequently isolating and selecting one or more targeting molecules comprising the primary recognition site by a biopanning procedure directly on the second target cell to produce one or more said targeting molecules;
b) amplifying, purifying and identifying one or more targeting molecules;
c) constructing a targeting agent from one or more targeting molecules or recognition sites thereof;
And wherein the targeting agent can be a peptide, polypeptide, antibody or antibody fragment or a multimer thereof.
[0233]
[246.] The targeting agent may be further coupled, attached, combined, linked, fused to, or otherwise associated with the pharmaceutical agent.
[0234]
[247.] In a preferred embodiment of the present invention, the targeting agent is an anti-disease or anti-cancer agent.
[0235]
[248.] In another preferred embodiment of the invention, the pharmaceutical agent is selected from the group comprising radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anti-cancer agents. I have. The radioisotope is¹¹¹indium,¹¹³indium,^99mrhenium,¹⁰⁵rhenium,¹⁰¹rhenium,^99mtechnetium,^121mTellurium,^122mTellurium,^125mTellurium,¹⁶⁵thulium,¹⁶⁷thulium,¹⁶⁸thulium,^{one two Three}Iodine,¹²⁶Iodine,¹³¹Iodine,¹³³Iodine,^81mkrypton,³³xenon,⁹⁰yttrium,²¹³Bismuth,⁷⁷Bromin,¹⁸Fluorin,⁹⁵ruthenium,⁹⁷ruthenium,¹⁰³ruthenium,¹⁰⁵ruthenium,¹⁰⁷mercury,²⁰³mercury,⁶⁷Gallium and⁶⁸It may be selected from a group containing gallium.
[0236]
[249.] In yet another embodiment, the toxin is selected from the group comprising gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria, ricin, and modifications and derivatives thereof.
[0237]
[250.] In yet another embodiment of the present invention, the anti-cancer agent is doxorubicin, morpholino-doxorubicin (MDOX), adriamycin, cis-platinum, toxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclo It is selected from the group consisting of phosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, bleomycin and derivatives thereof.
[0238]
[251.] The subject invention provides (a) biopanning that involves incubating a phage display library with cells derived from blood; (b) washing to remove unbound phage; (c) blood cells. (D) amplification of the resulting phage; and (e) determination of the displayed peptide sequence of the bound phage to identify the peptide, to identify antibody fragments. Offers a way.
[0239]
[252.] The subject invention is:
A-X-B
Wherein X is a hypervariable CDR3 region of 3 to 30 amino acids; A and B may each be an amino acid chain of 1 to 1000 amino acids in length. Wherein A is the amino terminus and B is the carboxy terminus.
[0240]
[253.] In a preferred embodiment of the present invention, A is 150-250 amino acid residues and B is 350-500 amino acid residues.
[0241]
[254.] In another preferred embodiment, the CDR3 region of the peptide is between 5 and 13 amino acid residues.
[0242]
[255.] In another preferred embodiment, X in the above structural formula is an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24.
[0243]
[256.] In another embodiment of the invention, the peptide or polypeptide is part of a larger or complete antibody or multimer.
[0244]
[257.] In yet another embodiment, the dimeric molecule comprises two peptides or polypeptides, one of which is a peptide or polypeptide of the present invention. The dimer molecule may comprise two identical peptides or polypeptides of the invention.
[0245]
[258.] In a preferred embodiment of the present invention, X is an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 24 in the dimer molecule.
[0246]
[259.] Another embodiment provides a nucleic acid molecule encoding a peptide or polypeptide or dimer molecule of the invention.
[0247]
[260.] The present invention provides the use of a peptide or polypeptide in the manufacture of a medicament, optionally in association with, or attached to, associated with, linked to or fused to a pharmaceutical agent.
[0248]
[261.] The present invention further provides the use of a peptide or polypeptide in the manufacture of a medicament having activity against pathological cells, more particularly cancer cells. The cancer cells can be selected from a group including carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. More specifically, the cancer cells can be leukemia cells, and most particularly, the leukemia cells can be AML cells.
[0249]
[262.] Interchangeable systems as defined in the present invention and as discussed in the examples below provide for the redefined variable region within the construct without the need to further manipulate or reconstruct the molecule. Nucleic acid constructs designed to allow for exchange or substitution. Such a system allows for rapid and convenient preparation of the desired nucleic acid molecule.
【Example】
[0250]
Example
[263.] The following examples are set forth to assist in understanding the invention, but are not and are not intended to limit its scope in any way. Not something to be done. Although specific reagents and reaction conditions have been described, it is possible to make modifications intended to be encompassed by the scope of the present invention. Accordingly, the following examples are provided to further illustrate the present invention.
[0251]
Example 1:
[264.] 1. Preparation of cells, bacterial strains, scFv phage display libraries, cell membrane preparation and protein purification for biopanning procedures.
[0252]
[265.] 1.1 Preparation of leukemia cells. Blood specimens were obtained from leukemia patients. Mononuclear cells (primary cells) were separated from other cells on a Ficoll cushion (Iso-prep, Robbins Scientific Corp., Sunnyvale, CA, USA). Centrifugation was performed at 110 × g for 25 minutes. Cells at the interface were collected and washed twice in PBS. The cells were then suspended in RPMI + 10% fetal calf serum (FCS) and counted. For prolonged storage, 10% FCS and 10% DMSO were added to the lymphocytes, which were then frozen at -70 ° C.
[0253]
[266.] 1.2 Preparation of fixed platelets. Platelet concentrate obtained from the blood bank was incubated at 37 ° C. for 1 hour. An equal volume of 2.0% paraformaldehyde was added and the platelets were fixed at 40 ° C. for 18 hours. Platelets were washed twice with cold saline (centrifugation at 2500 × g for 10 minutes), resuspended in 0.01% HEPES in saline and counted using a microscope.
[0254]
[267.] The sensitivity of platelets to plasma von Willebrand factor and ristocetin was confirmed. Plasma von Willebrand factor (vWF; 18 μg / ml) and ristocetin (0.6 mg / MI) were added to the fixed platelets to induce platelet aggregation and monitored by a chronolog-Lumi platelet aggregometer.
[0255]
[268.] 1.3 Bacterial strains-TG-1 and HB2151: A of 0.5-0.9.₆₀₀Fresh bacterial cultures were prepared for infection by growing cells up to (exponentially growing cells). E. coli TG-1 cells were used for phage propagation and E. coli HB2151 cells were used for production of scFv proteins.
[0256]
[269.] 1.4 scFv display phage library source. The scFv library (Nissim et al., EMBO J., 13, 692-698 (1994)) was provided by Dr. A. Nissim with MRC agreement. The library initially had V_HAnd V_LWas constructed as a phagemid library displaying the sFv fragments to which the domains were linked. The scFvs displayed in the phagemid library were fused to the N-terminus of the phage trace coat protein pIII, which was subsequently subcloned into the pHEN1 vector (Nissim et al., EMBO J., 13, 692-698 (1994). )). A repertoire of antibody fragments was first generated by RCR from rearranged V-genes of non-immunized human peripheral blood lymphocytes (referred to as the naive repertoire). To diversify the repertoire, a series of 49 cloned human V_HWithin the gene segment, a random nucleotide sequence encoding a heavy chain CDR3 length of 4-12 residues was introduced. Fused V in all clones_L The fragment was derived from a single unmutated V gene of the germline IGLV3S1, and⁸Create a single pot library of clones.
[0257]
[270.] 1.5 Membrane preparation from AML cells. 10⁸1 ml of cold lysing solution (0.3 M sucrose, 5 mM EDTA, 1 mM PMSF) was added to the pellet containing the washed cells and then spun at 11000 xg for 20 minutes at 4 ° C. The supernatant fluid was discarded and the pellet was resuspended in TE (10 mM Tris, 1 mM EDTA, 1 mM PMSF and spun as above. The final pellet was 0.4 A₂₈₀And resuspended in 6 ml of PBS for 2 hours at 37 ° C. and used to coat three Maxisorbimmuno-tubes (NUNC). After coating, the tubes were rinsed three times with PBS and then blocked with MPBS (2% non-fat dry milk in PBS) at RT for 2 hours. Prior to biopanning, the tubes were rinsed three more times with PBS.
[0258]
Example 2:
[271.] 2. Manipulation of phagemid particles re: Biopanning procedure.
[0259]
[272.] 2.1 Selection and amplification of phagemids: Phagemids that expressed epitopes of particular interest were selected from the library by the following four-step biopanning procedure:
a) binding of phagemid particles to the target,
More specifically, binding of phagemid particles to washed target cells or cell membranes,
b) removal of unbound phagemid particles, more particularly removal by extensive washing;
c) elution of the bound phagemid particles,
d) Propagation and amplification of eluted phagemid particles, more particularly in E. coli.
[0260]
[273.] 2.2 Identification of clones: The four-stage biopanning procedure was generally repeated 3-5 times. Selected phagemid clones were propagated individually and further characterized by the following work:
a) DNA sequencing,
b) Ex-vivo comparison of phage binding to multiple cell types
c) Infection of E. coli HB2151 to produce soluble scFv.
[0261]
[274.] 2.3 Sequence analysis: 〜800 bp of encoded scFv DNA in phagemid particles was ligated with upstream primer # 203743 (5′-GAAATAACCCTAT target cell CTACGG) and downstream primer # 181390 (5′-TGAATTTTCTGTATGAGG). Amplified by PCR using DNA fragments were completely sequenced from both ends using an ABI PRISM Big Dye termination cycle sequencing kit and an automated ABI PRISM DNA sequencer (310 Genetic Analyzer, Perkin Elmer) using the primers described above. Two additional primers # 191181 (5% -CGATCCGCCCACCGCCAGAG) and its complementary primer # 191344 (5'-CTCTGGCGGGTGGCGGATCG) in the flexible polypeptide junction region between the heavy and light chains were sequenced. used.
[0262]
Example 3:
[275.] 3. Biopanning protocol
[0263]
[276.] 3.1 Basic Biopanning Protocol: The biopanning procedure is part of the phage display technique described above. In this work, three biopanning protocols were developed and used.
a) Protocol AM (AML cell membrane panning / bacteria elution followed by whole AML cell panning / trypsin elution)
b) Protocol YPR (fixed human platelet panning / acid elution).
c) Protocol YPNR (fixed human platelet panning / acid elution).
[0264]
[277.] These protocols are detailed below:
[0265]
[278.] 3.1.1 Protocol AM
[0266]
[279.] 3.1.1.1 Prewash: 2 x 10 from patient stored at -70 ° C.⁷A 1 ml aliquot containing the frozen AML cells was quickly thawed at 37 ° C. and immediately diluted in 10 ml of cold 2% PBS-milk (MPBS). Cells were spun at 120 × g at room temperature (RT), washed twice, resuspended in MPBS and counted on a hemocytometer. Cell membranes were prepared as described in Section 1.5.
[0267]
[280.] 3.1.1.2 Selection is to select 10 from the original Nissim library.¹²This was performed on immobilized AML cell membranes by the addition of 2 ml of MPBS containing one phagemid. Tubes were gently agitated for 30 minutes, then incubated for an additional 90 minutes without agitation, and both steps were performed at room temperature (RT). After three rounds of panning on AML cell membrane, one round of panning was performed on all AML cells.
[0268]
[281.] 3.1.1.3 Washing: To remove excess unbound phagemid, decant the tube and wash the tube 10 times with PBS, 0.1% Tween and 10 times with PBS only. Washed.
[0269]
[282.] 3.1.1.4 Elution: Exponentially growing E. coli TG-1 cells (2 ml) were added directly to the tube and incubated for 30 minutes at 37 ° C. with gentle agitation. Aliquots were plated for titration and the remaining volumes were plated for amplification as described above.
[0270]
[283.] 3.1.1.5 Amplification: Colonies from large plates were scraped and pooled. ~ 0.5 A₆₀₀Aliquots of ampicillin-resistant E. coli TG-1 cells in liquid culture until⁷) Was grown and then infected with helper phage (VSC-M13, Stratagene) to generate large amplified phagemid stocks. Phagemid was rescued by the PEG precipitation procedure (18a). The amplified T16MI stock described above was used for a subsequent panning round. 10 of the previously amplified stock¹¹The selection procedure was repeated two more rounds with phagemids. The amplified stock of the third panning procedure on the immobilized membrane was designated T16M3.
[0271]
[284.] 3.1.1.6 Re-panning on whole cells: Intact AML cells were panned using the T16M3 amplification stock from the third membrane panning. 2 × 10⁷Cells and 10^TenPhagemid colony forming units (CFU) (Nissim library) and 10¹³Selection was carried out in a final volume of 0.5 ml MPBS containing the individual wild-type bacteriophage M13 at 4 ° C. with gentle stirring for 2 hours. Bound phagemid was eluted from the washed cell pellet with 50 μl trypsin: EDTA (0.25%: 0.25%) and then neutralized by adding 50 μl FCS. For titration and amplification, 1 ml of E. coli TG-I culture (A₆₀₀= 0.5) was used. The final stock that was amplified was designated T16M3.1.
[0272]
[285.] 3.1.2 Protocol YPR
[0273]
[286.] 3.1.2.1 Selection: 10 in 1 ml PBS / HEPES / 1% BSA buffer¹¹10 phagemids (Nissim library)⁸Clone selection was achieved by panning of established human platelets. Binding was performed for 1 hour at RT while mixing the samples by rotation.
[0274]
[287.] 3.1.2.2 Cell Wash: Platelets were washed 5 times by low speed centrifugation (3500 xg) and resuspended as described above.
[0275]
[288.] 3.1.2.3 Elution: The first round of bound phagemid was eluted from the established platelets by acid elution technique.
[0276]
[289.] Platelets were incubated for 10 minutes at RT with 200 μl of 0.1 M glycine, pH 2.2. After neutralization and centrifugation with 0.5 M Tris-HCl, pH 8.0, add 200 μl trypsin-EDTA (0.25% / 0.05) and neutralize by adding 50 μl FCS. Eluted the remaining platelet-binding phage. Cells were removed by centrifugation and supernatant fluid containing eluted phage from both acid and trypsin elution protocols was collected and designated as YPR (a) -1 and YPR (t) -1 stocks, respectively. These stocks were then amplified by adding 1 ml of exponentially growing TG-1 cells for 30 minutes at 37 ° C. Aliquots were plated for titration, and the remaining infected E. coli cells were fixed on 2xTV / AMP 15 cm plates. Plates were incubated overnight at 30 ° C. The output after each panning round was determined by counting the colonies on the titration plate.
[0277]
[290.] 3.1.2.4 Growth: Clones were amplified as described in section 3.1.1.5. Called R1 (a) and R1 (t) stocks from acid and trypsin elution protocols-10¹²The phagemid / ml amplified stock was combined in each case and used in subsequent rounds of panning.
[0278]
[291.] 3.1.2.5 The second and third panning rounds were performed as described for the first panning round of the YPR procedure with the following modifications. That is, for (i) the second panning, 10¹²10 in combination with R1 (t)¹²(1) Elution was performed only with glycine (pH 2.2). The second round amplified eluate was designated as R2. (Iii) For the third biopanning round, 10¹²Elution was performed in the second round using R2. The third round of amplified stock was designated R3.
[0279]
[292.] 3.1.3 YPNR protocol
[0280]
[293.] 3.1.3.1 Biopanning and washing were performed essentially as described in the YPR protocol. However, in this protocol, (i) elution was performed after each of the three panning rounds with glycine (pH 2.2), and after the first panning and amplification, two rounds without amplification were performed. A subsequent panning round followed. The first, second, and third rounds were designated as YPNR1, YPNR2, and YPNR3, respectively.
[0281]
[294.] 3.2 Selection of negative control scFv clones
[0282]
[295.] 3.2.1 N14 CDR3 sequence: For all binding experiments, a single clone was picked from an unexperimented library (before selection). From this clone, a soluble scFv designated N14 and a phage stock were prepared. From sequence analysis, it is V_HIt can be seen that the gene belongs to the 4-DP65 gene family. The 11-merV encoded by this clone, designated N14 CDR3_H-The sequence of CDR3 is as follows (SEQ ID NO: 28):
Phe Leu Thr Tyr Asn Ser Tyr Glu Val Pro Thr
[0283]
[296.] 3.2.2 C181 CDR3 sequence: In a binding assay experiment, an additional negative clone C181 was used. Clone C181 (reactive with recombinant hepatitis B virus [HBV] particles)_HThe 9-merV encoded by this clone, which belongs to the 3-DP35 family and was designated C181 CDR3_H-The sequence of CDR3 is as follows (SEQ ID NO: 29):
[0284]
Thr Asn Trp Tyr Leu Arg Pro Leu Asn
[0285]
Example 4:
[297.] 4. Production, purification, labeling and characterization of scFv clones
[0286]
[298.] 4.1 Production of soluble scFv: pHEN1, a vector used to construct the original phagemid library, was designed using an amber stop codon encoded at the junction of the scFv and pII1 genes. did. Thus, when the vector of the selected clone was introduced into the non-suppressor strain E. coli HB2151 by phagemid infection, this system enabled the production and secretion of soluble scFv into the bacterial periplasm. (Harrison et al., Methods in Enzymology, 267, 83-109 (1996)). At this time, scFv can be easily recovered from the culture solution. Soluble scFvs are produced under the control of the lacZ promoter induced by IPTG (Gilbert and Muller-Hill, PNAS (US), 58, 2415 (1967)).
[0287]
[299.] A vector encoding a c-myc tag (10 amino acids—Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu; SEQ ID NO: 123) is contained in the vector upstream of the amber mutation. The C-terminus of the expressed scFv carries a c-myc tag that can be detected using a mouse anti-myc tag antibody (derived from the European Cell Culture Collection (ECACC peptide or polypeptide) 9E10-hybridoma). Should be.
[0288]
[300.] 4.2 Purification of scFv on protein A bead affinity column; all scFvs of selected and control clone C181 were V_HIt belongs to three families and allows for purification on a protein A affinity column. Periplasmic fractions (100-250 ml) from the induced culture of each clone were prepared and incubated with protein A Sepharose beads. The bound scFvs were recovered from the column with an acidic solution (0.1 M glycine, pH 3.0), after which the eluate was neutralized with Tris pH 8.0. The concentration of the recovered protein is A₂₈₀Determined by measurement, followed by a PBS buffer exchange either by dialysis or on a G-25 Sepharose column.
[0289]
[301.] 4.3 Purification of N14-scFv on Sephacryl S-200 column: The scFv of the negative clone N14 was V_HIt belongs to the 4 gene family and therefore cannot be purified on protein-A affinity columns. For purification of scFv-N14, 200 ml of the periplasmic fraction of the induced culture was precipitated with 60% ammonium sulfate. Sephaeryl S-200 column resuspended in 2 ml 0.1 × PBS, 5 mM EDTA, 5 mM PMSF and pre-equilibrated with current buffer (0.1 × PBS, 5 mM EDTA) (1.5 × 90 cm). Proteins were fractionated and fractions containing N14-scFv as detected by SDS-PAGE and Western analysis were pooled, lyophilized and 1/10 volume H_TwoSuspended in O. At this time, N14-scFv (unlabeled and labeled with FITC) was used as a negative control in the FACS analysis experiment.
[0290]
[302.] 4.4 Labeling of purified scFvs with FITC: Approximately 1 milligram of purified scFv from each preparation was resuspended in PBS and fluoro-tagged FITC conjugated according to the manufacturer's instructions. Coupling to FIFC was performed using a commercial kit (Sigma Catalog No. FIFC-1).
[0291]
[303.] 4.5 Quality analysis of purified and labeled scFv
[0292]
[304.] 4.5.1 After purification and FIFC labeling, the profile of each preparation (labeled and unlabeled) was analyzed by SDS-PAGE, Western blot, Superdex-75 columns (A280 and A495). Was analyzed by HPLC using HPLC and fluorometry. The analysis shows 80% purity and V for N14 scFv with about 2 FITC molecules conjugated to each scFv molecule._HThree clones showed 90% purity (2: 1 F / P ratio).
[0293]
[305.] 4.5.2 To confirm retention of scFv specificity, the binding activity after FITC labeling was assessed.
[0294]
[306.] 4.6 Biochemical characterization of phagemid clones: SDS-PAGE, mass spectrometry (Y1 and Y17 scFvs) to assess structure and assess the purity of various scFv preparations (see eg, Example 8). Only) and HPLC. Western analysis and EIA were used to identify the scFv, and FACS was used to characterize the scFv binding.
[0295]
Example 5:
[307.] 5. Combination test
[0296]
[308.] Binding of selected clones to cells was assessed at two levels: phagemid levels and soluble scFv levels.
[0297]
[309.] 5.1 Binding at the phagemid level
[0298]
[310.] For this purpose, phagemid stocks were prepared individually from each of the selected clones.
[0299]
[311.] 5.1.1 Colony test: one set of experiments derived from a biopanning protocol that confers ampicillin resistance to infected E. coli.⁹Specific phagemid and serves as a "blocker" without ampicillin resistance.¹¹Of the wild-type M13 phage were selected from a cell-type panel.^FiveIncubated with cells. After incubation and washing, the bound phage was eluted with trypsin and an aliquot was used to infect E. coli TG-1. E. coli was then fixed on 2 × TY / AMP plates and incubated at 30 ° C. overnight. The number of colonies obtained for each clone was calculated and compared. The results provide one measure of phagemid binding affinity and specificity.
[0300]
[312.] 5.1.2 White / blue colony test: In this test, where each experiment includes an internal control, a particular phagemid was replaced with another target phagemid called pGEM7 (Promega Corp., Madison , Wisconsin, USA) at the same ratio as in section 5.1.1 above, that is, 1/100. This pGEM7 phagemid is ampicillin resistant; however, it does not express any recombinant polypeptide at the N-terminus of its PIII gene. After TG-1 infection and incubation on ampicillin plates containing ImM X-gal, colonies were counted. The resulting colonies containing pGEM7 are blue, while the colonies obtained from specific phagemids are white. The enrichment factor derived from the input / output ratio of white / blue colonies (grown on the same plate) for each tester was then calculated.
[0301]
[313.] 5.1.3 EIA of phagemid
[0302]
[314.] 5.1.3.1 Binding of phagemid to selected cells: about 5 x 10 using acetone: methanol (1: 1) on the surface of a 24-well plate.^FiveOf the selected cells were established. Binding test is 10⁹Of phagemids. Binding was performed for 1 hour at 37 ° C., followed by extensive washing with PBS / Tween (0.05%). After extensive washing with PBS, the plates were incubated with rabbit anti-M13, anti-rabbit IgG-HRP and substrate. The intensity of the color generated was read by an ELISA plate reader at A405 and was directly proportional to the level of bound phagemid.
[0303]
[315.] Phagemid binding to established platelets: polystyrene microtiter-10 plates⁸Individual fixed platelets were coated and incubated at 4 ° C. overnight. Approximately 10 to assess binding^TenPhagemid was used. Washing and incubation of plates and determination of binding levels were performed as described in 5.1.3.1 above.
[0304]
[316.] 5.1.4 Binding to a specific protein selected from the group consisting of human growth hormone (hGH), fibrinogen, fibronectin, BSA, SM (skim milk powder) and glycocalysin (proteolytic fragment of GPIb) An assay was performed. Binding was tested as follows. At 2 μg / well, polystyrene microtiter plate wells were coated with one of the proteins to be tested. The coating was allowed to proceed during the overnight incubation at 4 ° C. About 10 to test the join^TenPhagemids were added. After extensive washing with PBS, plates were incubated with rabbit anti-M13, anti-rabbit BRP, and substrate. The binding level was determined by the intensity of the color produced. The optical density was measured at A405. Each sample was tested under exactly the same conditions, and the average was calculated.
[0305]
[317.] 5.2 Binding test at scFv level: The binding of scFvs generated in the periplasm of HB2151 was compared in multiple cell types by two different assays, EIA and FACS analysis.
[0306]
[318.] 5.2.1 EIA of soluble scFv: about 5 × 10 with 5-10 μg total protein^FiveAML cells were incubated. Binding was performed for 1 hour at 4 ° C., followed by EIA using mouse anti-myc antibody, anti-mouse HRP, and substrate. After each step, excess unbound antibody was removed by three cell washes with PBS. The intensity of the color produced was measured using an ELISA plate reader (OD).₄₀₅). As mentioned above, color intensity is directly proportional to the coupling level.
[0307]
[319.] 5.2.2 FACS analysis of cells
[0308]
[320.] Analysis of cells stained by the "three-step staining" procedure: FACS analysis was performed to test and confirm the specificity of the selected clones. A "three-step staining" procedure was established, first using a crude extract or purified unlabeled scFv, then a mouse anti-myc antibody, and finally an anti-mouse antibody conjugated with FITC or PE. did.
[0309]
[321.] For FACS analysis, 5-8 × 10 5 purified by Ficoll and resuspended in PBS + 1% BSA.^FiveIndividual cells are required. Binding was performed at 4 ° C. for 1 hour. After each step, cells were washed and resuspended in PBS + 1% BSA. After the final staining step, cells were fixed by resuspension in PBS, 1% BSA, 2% formaldehyde, and then read by FACS (Becton. Dickinson).
[0310]
[322.] 5.2.2.2 Staining of cells with FITC-labeled scFv in one staining step: 5-8 x 10 in PBS + 1% BSA^FiveThe FITC-labeled scFv was incubated with the ficoll purified cells. Coupling was performed at 4 ° C. for 1 hour. The cells were then washed and fixed as described in section 5.2.2.1 above, and read by FACS.
[0311]
Example 6:  Panning and sequencing results
[323.] 6.1 Result of AM Protocol
[0312]
[324.] 6.1.1 Panning results for the AM protocol: The estimated number of plasmids used for panning (input) and the number of bound phagemids eluted in the AM protocol (output) are: The following table (Table 1) summarizes:
[0313]
[Table 1]

[0314]
[325.] Note the enrichment of the yield (output) obtained with each successive panning. In addition, there was no loss of power when T16M3 was used to pan AML whole cells, indicating that bound phagemids may be specific for components on the outer cell surface, or Suggest that the target system may contain a relatively large number of non-specific binding phagemids.
[0315]
[326.] 6.1.2 Clone sequence results for AM protocol: Although clones were picked and sequenced from the T16M1, T16M2 and T16M3 output stocks, the results shown below were derived primarily from the T16M3.1 output stock. Clone (AML intact cell panning). Clones AM10, AM11 and AM12 were identified in the T16M3 stock but not in subsequent output.
[0316]
[327.] V_H-The amino acid sequence displayed in CDR3 and its frequency in the output of the clone under test are summarized in Table 2.
[0317]
Table 2. Selected clones after AM biopanning protocol from T16M3 and T16M3.1 outputs.
[0318]
[Table 2]

[0319]
[328.] The amino acid sequence of Arg / Gly Phe Pro is present in seven of the ten isolated clones presented in Table 2 and represents one of the motifs. Furthermore, it should be noted that the motifs identified represent in each case the N-terminal 3 amino acids of the CDR3 region. Thus, this motif may be in the form of a single peptide or polypeptide or Fv molecule or in combination with other amino acid residues extending beyond either one or both ends of the CDR3 region, or alone. An effective anchor or binding site.
[0320]
[329.] Based on the core sequence Arg / GlyPhePro, other CDR3 regions with high affinity for binding to AML cells can be constructed. These can be constructed by altering any of the above 5-12-mers by additions, deletions or mutations while maintaining the Arg / Gly Phe Pro core sequence.
[0321]
[330.] The CDR3 region of the present invention has the amino acid sequence R1-Arg / GlyPhePro-R2, where R1 is 0-15 amino acids, preferably 0-9, most preferably 0-1. R2 comprises 1 to 15, most preferably 1 to 9 amino acids. R1 and R2 are amino acid sequences that do not adversely affect the specific binding of the Arg / GlyPhePro sequence to AML cells.
[0322]
[331.] The CDR3 regions of the light chains of the above clones are identical and are listed in SEQ ID NO: 125.
[0323]
[332.] 6.2 Results of YPR and YPNR protocols
[0324]
[333.] 6.2.1 Panning results for YPR and YPNR protocols:
The putative plasmid numbers used for panning (input) and the putative bound phagemid numbers eluted (output) are summarized in the following tables (Tables 3, 4):
[0325]
[Table 3]

[0326]
[334.] Table 3 demonstrates that trypsin elution produces 4 times greater output than acid elution in the first round.
[0327]
[335.] Re-panning according to the YPNR protocol without the amplification step minimized the potential for preferential amplification of phagemid and bacterial infections. The resulting output is depicted in Table 4.
[0328]
[Table 4]

[0329]
[336.] As expected, the results shown in Table 4 indicate a decrease in phage yield after each panning round. This protocol was used to prevent bias due to amplification of non-specific phage.
[0330]
[337.] 6.2.2 Clone sequencing results for YPR and YPNR protocols: Multiple clones from a third panning from both protocols were selected for sequencing. The amino acid sequence presented in Table 5 contains the heavy chain (V_H-CDR3). The frequency of sequence appearance in the germline and R3 output is also shown in this table.
[0331]
Table 5: Selected Y-series clones after YPR biopanning protocol at R3 output
[0332]
[Table 5]

[0333]
[338.] Most of the clones isolated from the YPNR protocol were also Y1.
[0334]
[339.] The CDR3 regions of the light chains of the above clones are identical and are listed in SEQ ID NO: 125.
[0335]
Example 7:
[340.] 7. Combined evaluation result
[0336]
[341.] 7.1 Binding of selected phagemid clones to AML cells (AM clone lineage): Except for clone AM7, no preferential binding to test cells was detected. Significant, but non-selective binding of clone AM7 to all target cells, either as phagemids or as purified scFv, was observed. The results do not demonstrate any enrichment for the AM clone line.
[0337]
[342.] 7.2 Linking of Y clone series
[0338]
[343.] 7.2.1 Phagemid binding-EIA with fixed platelets: After three panning rounds with two different protocols, phage clones were tested by EIA for binding to fixed platelets. Phagemid stocks were prepared from each of the selected clones and these clones were tested in two sets of EIAs. Each sample was tested under exactly the same conditions, and the average was calculated. The results are summarized in FIG. 1 and show that 6 out of 9 Y-series clones show a positive EIA response. The highest binding was associated with clones Y1, Y16, Y17 and Y-27. Phage stocks M13 (wild-type bacteriophage) and E6 (selected for CLL leukemia cells) were used as negative controls. The dominant clone, phage Y1, showed the highest binding to settled platelets, with Y17, much higher binding than the M13 or E6 phage clones.
[0339]
Example 8:
[344.] 8. Detailed characterization and clone binding of scFvs
[0340]
[345.] 8.1 Structure and identification of scFv: The native structure of YI was assessed by HPLC analysis using a Superdex 75 column and by mass spectrometry. The results of the former method indicate the presence of monomers, dimers and tetramers during preparation. Mass spectrometry was sensitive enough to identify an expected molecular weight of 26.5 kD, and when the c-myc tag was resolved, a molecular weight of 24 kD was obtained.
[0341]
[346.] However, despite the predicted molecular weight of 26.5 kD according to the nucleic acid sequence and mass spectrometry results described above, the SDS-PAGE results show an apparent molecular weight of 30 kD for intact and unresolved molecules. Has been represented. Western analysis using a c-myc-specific antibody confirmed the 3 kD result of SDS-PAGE, confirming the implicit significance that the c-myc tag is present at the end of the intact molecule. The discrepancy between the results of the two procedures is due to the accuracy level of the method as well as the current conditions of SDS-PAGE, which can alter the apparent molecular weight of the protein under test.
[0342]
[347.] 8.2 Binding of platelet-selected clones to leukemia cells: As noted in the introduction, it is possible to express platelet cell surface markers on immature hematopoietic cells. The binding of platelet selected clones was tested by FACS analysis. FACS analysis was performed after staining whole blood followed by RBC lysis or on mononuclear cells purified by Iso-prep- (Ficoll cushion). ScFvs were prepared from each clone, purified on protein A, and labeled with FITC (as described in Sections 4.1-4.4). To allow the production of intact scFv in the non-suppressor E. coli strain HB215), the DNA site-directed mutagenesis of the Y-27 clone was performed by DNA site-directed mutagenesis to encode for glutamic acid (GAG)._H-Amber codon (TAG) found in CDR3 was mutated. Target cells for such studies are cells isolated from fresh blood specimens of various leukemia patients. Specimens were obtained from three medical centers in Israel.
[0343]
[348.] Clones Y1 and Y17 showed preferential binding to the leukemia cells tested, while all other Y lineage clones showed only background level binding. Table 6 shows the binding of FITC-labeled YI and Y-17 to various leukemia cells.
[0344]
[Table 6]

[0345]
[349.] The results presented in fractions in Table 6 represent the portion of the patient whose cells were identified by FACS analysis as positive for each antibody tested. The numerator represents the number of positive patients and the denominator density represents the total number of patients tested for a given scFv / cell type combination. Y-17 bound strongly to all test cells. This binding was thus considered non-cell selective. However, Y1 binding was found to be highly selective for multiple samples of leukemic cells, especially those in the acute phase. Y1-scFv binding was further analyzed as described below.
[0346]
[350.] Representative results of YI binding on three AML specimens are shown in FIG. In each case, the majority of the cell population fluoresces at a much higher intensity than that of the background fluorescence obtained by staining with the negative control scFv. These results indicate that for each patient, Y1 binds to a different fraction of the total cell population. The right YI peak in each graph is considered to represent the least number of Y1-binding cells in the population, and the percentage of total cells below this peak represents the minimum percentage of YI-binding cells in each sample. The probability of showing is the highest.
[0347]
[351.] 8.3 Y-I binding to normal blood cells: Y1 binding to ficoll-purified normal blood cells was analyzed according to different blood cell types. No binding to normal lymphocytes was detected, but Y1 was ficoll-purified monocytes from 9 of 28 subjects, platelets from 5 of 8 subjects, and of 4 subjects Bound to erythrocytes (RBC) from one person. However, CD14-specific antibodies bound to cells in all monocyte preparations and many of the neutrophil preparations. A summary of this analysis is shown in Table 7.
[0348]
[Table 7]

[0349]
[352.] These binding results represent the portion of the normal blood sample identified by FACS analysis as positively reactive with each antibody of interest. FITC-Y1 scFv selected on fixed platelets, but relatively low binding to plateletsAffinityIt should be noted that
[0350]
[353.] FIG. 4 demonstrates the binding of YI to ficoll-purified platelets (4a) and monocyte-gated cells (4b). The shift on the monocyte cell population is greater than that observed on platelets, and the calculated mean fluorescence is 30 and 50 times greater than the negative control, respectively. This observation is most likely due to the property of platelets that adhere to ficoll-purified monocytes in large numbers. Subsequent experiments showed that no Y1 binding was observed in any of the normal monocytes, granulocytes, platelets or RBCs tested when assayed on whole blood specimens. Similarly, no Y1 binding to platelets was seen when derived from platelet-rich plasma (PRP). Under the same binding conditions (with whole blood RBC lysis with FACS lysis solution [Becton Dickenson]), Y1 bound to leukemic cells in a manner similar to that obtained after Ficoll purification. Thus, we can conclude that under natural conditions, the Y-I epitope on platelets or monocytes is masked. During the Ficoll purification procedure, the epitope is exposed and accessible for recognition by Y1, while for leukocytes, the epitope is exposed under both purified and non-purified conditions.
[0351]
[354.] In addition to normal hematopoietic progenitor cells of the lymphoid and myeloid lineages, Y1 binding to hematopoietic stem cells (CD34 + cells) in cord blood was tested. FIG. 5 shows the binding results of FITC-labeled scFv clones to cord blood CD34 + stem cells. FIG. 5a shows the results of binding of CD34 + gated cells to FITC-labeled negative control scFv, and FIG. 5b shows the same analysis for binding of CD34 + gated cells to FITC-labeled scFv clone Y1. ing. FIG. 5c shows FSC and SSC dot plot analysis of the FITC-labeled scFv clone Y-I specimen as in 5b. The results of this analysis indicated the presence of two subpopulations of CD34 + stem cells derived from cord blood using the difference in forward scatter (FSC) as an indication of cell size. Y1 binds to the smaller size cells of the two populations. The boxed areas in FIGS. 5b and 5c outline the subpopulation of CD34 + cells that bind clone Y1 scFv. Further analysis indicates that the smaller sized cells are dead cells present in the cell population, and that Y1 binding may be indicative of the presence of an intracellular ligand recognized by Y1. all right.
[0352]
[355.] Experiments were also performed on peripheral blood cells of healthy donors pre-treated with GM-CSF (GM-CSF treatment mobilizes stem cell release into the bloodstream). Results similar to those shown in FIG. 5 were obtained.
[0353]
[356.] 8.4 Binding specificity of Y1 scFv compared to various cell markers on AML cells: Y1 staining of Ficoll purified peripheral and bone marrow cells from AML patients, Compared to cell staining. The results of such FACS analysis on specimens from 14 patients are summarized in Table 8. Note that for all of the markers under test, including Y1, there is significant variability in the frequency of stained cells in preparations from different individuals. The lack of correlation between the binding of various markers and the binding of Y1 indicates that Y1 does not bind to any of the ligands bound by the other test markers, and that the Y-I ligand does not bind to any of the cell surface markers to be tested. Does not even constitute
[0354]
[Table 8]

[0355]
[357.] The results were identified by FACS analysis as likely to react with each individual antibody. Expressed as the percentage of cells in a Ficoll-purified specimen of a given patient.
[0356]
[358.] The concentration of Y1 required for binding detection (（1 μg / 5 × 10^FiveIn light of ()), the results show that Y1 scFv has a relatively high binding affinity for specific ligands on AML cells.
[0357]
[359.] In addition to the results presented in Table 8 showing the binding of Y1 to AML cells, we now (Table 6) show that Y1 is the primary target of most other tested subjects, including B-ALL cells. Although they could also bind to leukemia cell types, they indicated that sample size was limited for these other leukemia cell samples. FIG. 6 presents a FACS analysis of Y1 scFv binding to pre-B-ALL cells obtained from two patients. Double staining with either commercially available PE-labeled CD19 (marker for normal peripheral B cells: FIGS. 6a, 6c) or PE-labeled CD34 (marker for stem cells: FIG. 6d). The procedure was utilized with a FITC-labeled negative control scFv or a FITC-labeled Y1 scFv. FIG. 6b is a double negative control. The fluorescence intensity (X-axis) of the cells bound by the FITC-labeled specimen (scFv clone Y1) in relation to the negative control staining pattern is presented (6e and 6f). The results in FIG. 6 demonstrate that the majority of pre-leukemic B-ALL cells inside each of the two specimens tested are positive for Y1 cell staining due to YI binding.
[0358]
[360.] 8.5 Binding of Y1-scFv to cell lines: Multiple cell lines derived from a malignant hematopoietic lineage were screened for their ability to be recognized by Y1. FACS analysis indicates that Y1 binds to many of the cells tested (Table 9). Note that only one human B cell line and one mouse bone marrow cell line were tested. Importantly, this binding was restricted to exponentially growing cells. Cells in the quiescent phase generally did not bind to Y1, indicating that Y1 ligand expression was regulated during the life cycle of the cell. Furthermore, the binding strength varies between the responding cells. This observation implies that there is a difference in ligand affinity or expression level in different cells.
[0359]
[Table 9]

[0360]
[361.] Binding of purified Y1 in the presence of 86 DTT: Once the Y1 clone was selected, a process for producing scFv was further developed. The results of the FIC analysis of the Y1 batch showed that the protein was able to multimerize, mainly forming monomers and tetramers, and the ratio between these two forms varied from preparation to preparation. To obtain a homogeneous material, 5 mM DTT was added during affinity purification on a Protein A Sepharose column, which was then removed by changing the PBS buffer. In fact, after DTT treatment, the majority (> 90%) of the material was found in the monomer fraction. No significant difference was found between the binding of the monomeric form of Y1 and the binding of the mixture of Y1 forms (purified in the presence of DTT and analyzed on HPLC).
[0361]
[362.] 8.7 Y1 is a specific clone for leukemia cells: The Y1 cassette contains Y_H-Belongs to the DP32 germline. Several other clones from the same germline were isolated and are described in detail in Example 6. These clones include Y17, Y-27 and Y-44. The primary sequence of all of these clones (ie, the germline cassette) differs only in its CDR3 region. However, only Y1 shows selectivity for leukemia cells. The CD3 sequences of these clones are summarized in Table 10, and the binding profiles of the clones are summarized in Table 11.
[0362]
[Table 10]

[0363]
[Table 11]

[0364]
[363.] Tables 10 and 11 show that the primary sequence is V_H-Although identical among the four clones except for the CDR3 region, this indicates that the binding profiles are significantly different from clone to clone. This observation indicates that V_H-Reinforces the notion that the sequence of the CDR3 region plays an important role in the specificity of the binding site for an antigen. It should be noted here that neither the length of the CDR3 sequence, nor the specific germline cassette in which it is located, appears to be a major determinant of binding specificity. Y17 and Y-27 each contain a 6-mer CDR3, similar to Y1, in which the heavy chains of all three clones are derived from the same germline. In the case of Y17 and Y-27, no selective binding to hematopoietic cells was demonstrated.
[0365]
Example 9
[364.] 9.1 Construction of triabodies: The vector pHEN-Y1 encoding the original Y1 was_LAnd V_HBoth regions were separately amplified using RCR. V_LFor the PCR reaction, the sense oligonucleotide 5'-AACTCGAGGTGAGCTGACACAGGACCCT and the antisense oligonucleotide 5'-TTTGTCCGACTCATTCTTTTTTTGCGGCGCACC were used. The expected size cDNA product of ３５０350 bp was purified, sequenced and digested with XhoI and NotI restriction oxygen.
[0366]
[365.] V (using the sense oligonucleotide 5'-ATGAAATACCATTGCCTACGG and the antisense oligonucleotide 5'-AACTCGGACGGGTGACCAGGGTACC)_HThe same procedure was used to amplify the region. V_HPCR products were digested with NcoI and XhoI restriction oxygen. A triple ligation procedure into the pHEN vector previously digested with NcoI-NotI was utilized. The final vector was called pTria-Y1.
[0367]
[366.] Following E. coli transformation, multiple clones were picked for further analysis, including DNA sequencing, protein expression, and extraction from bacterial periplasmic space. SDS-PAGE and Western blot analysis under reducing conditions were performed to confirm the size of Y1 triabodies.
[0368]
[367.] 9.2 Construction of diabodies
[0369]
[368.] The pTria-Y1 vector from above was linearized with XhoI restriction enzyme, and the synthetic complementary double stranded oligonucleotides (5′-TCGAGAGGTGGAGGCGGT and 5′TCGAACCGCCTCCACCTCC were pre-annealed, and Y1-heavy chain and Y1 -Ligation within the XhoI site between the light chains, this new vector was called pDia-Y1 DNA sequence and protein expression were confirmed as described for Triabodies.
[0370]
[369.] 9.3 Expression and purification of diabodies and triabodies
[0371]
[370.] Expression in E. coli was essentially as described above for scFv-Y1. However, the purification of Y1 diabodies and triabodies from the periplasm of transformed E. coli cells was different. The scFv Y1 monomer form could be purified on an affinity column of Protein A Sepharose beads. However, purification of the monomeric form of Y1 by this procedure had no effect. Therefore, the periplasmic proteins extracted from the bacteria were precipitated overnight with 60% ammonium sulfate,_TwoResuspended in O and loaded onto Sephacryl-200 (Pharmacia) size exclusion column pre-equilibrated with 0.1 × PBS. Fractions were collected and analyzed by HPLC, and separate fractions containing either dimer or trimer forms were collected for FITC labeling and FACS analysis.
[0372]
[371.] 9.4 Binding of Y1 diabodies and triabodies to cells
[0373]
[372.] FACS analysis was performed on Jurkat cells using the "three-step staining procedure". First, the crude extract or purified unlabeled scFv is stained, then the mouse anti-mys antibody, and finally the FITC- or PE-conjugated anti-mouse antibody. FACS analysis was performed on 5-8 × 10 5 ficoll purified and resuspended in PBS + 1% BSA.^FiveRequires cells. Coupling was performed at 4 ° C. for 1 hour. After each step, cells were washed and resuspended in PBS + 1% BSA. After the final staining step, cells were fixed by resuspension in PBS, 1% BSA, 2% formaldehyde, then read by FACS (Becton-Dickinson).
[0374]
[373.] Binding of Y1-scFv was compared to that of diabodies and triabodies. In this analysis (FIG. 7), the binding profiles of all three forms were very similar, as the above-described modifications in the molecule altered, concealed or destroyed the apparent binding affinity of Y1 for the ligand. It means that there was no.
[0375]
[374.] 9.5 Production of Y1-cys-kak (cysteine dimer)
[0376]
[375.] One liter of λpL-yl-cys-kak bacterial culture was induced for 2-3 hours at 42 ° C. The culture was centrifuged at 5000 RPM for 30 minutes. The pellet was resuspended in 180 ml TE (50 mM Tris-HCl, pH 7.4, 20 mM EDTA). 8 ml of lysozyme (from a 5 mg / ml stock) was added and incubated for 1 hour. 5M NaCl and 25 ml of 25% Triton were added and incubated for another hour. This mixture was centrifuged at 13000 RPM for 60 minutes at 4 ° C. The supernatant was discarded. The pellet was resuspended in TE using a tissuemizer (or homogenizer). This process was repeated 3-4 times until the color of the inclusions (pellets) was gray / light brown. Inclusion bodies were solubilized in 6 M guanidine-HCl, 0.1 M Tris pH 7.4, 2 mM EDTA (1.5 grams of inclusion bodies in 10 ml of solubilization buffer were -10 mg / ml Provided soluble protein). This was incubated for at least 4 hours. The protein concentration was measured and brought to a concentration of 10 mg / ml. DTE was added to a final concentration of 65 mM and incubated overnight at room temperature. Refolding was initiated by diluting (by dropping) 10 ml of protein into a solution containing 0.5 M arginine, 0.1 M Tris pH 8, 2 mM EDTA, 0.9 mM GSSG. The refolding solution was incubated for 48 hours at ℃ 10 ° C. The refolding solution containing the protein was dialyzed in a buffer containing 25 mM phosphate buffer pH 6, 100 mM urea and concentrated to 500 mls. The concentrated / dialysed solution was bound to a SP-Sepharose column and eluted with a NaCl gradient (up to 1M).
[0377]
[376.] 9.6 Affinity study of S-SY1-dimer in comparison with CONY1 and Y1-IgG using radioreceptor binding assay (RRA) in KG-1 cells
[0378]
[377.] Chloramine T on YI-IgG construct or Bolton-Hunter reagent on CONY1 (Y1 scFv) construct was used for the assay system.¹²⁵I involved the use of radioligands prepared by iodination with I. In the test tube, 5 x 10 per 0.2 ml⁶KG-1 cells and a labeled tracer with varying amounts of unlabeled competitor in PBSt 0.1% BSA, pH 7.4. After incubation for 1 hour at 4 ° C. with shaking, the cells were washed thoroughly with cold buffer and picked up for radioactivity counting.
[0379]
[378.] In RRA studies using labeled Y1-IgG, 2 ng / tube was used.¹²⁵Competition was performed with each of the three molecules using I-Y1-IgG. The results are described in FIG. The results presented in this figure demonstrate that the affinity of the S-SY1 dimer was 30 times higher than that of CONY1. A rough estimate of the affinity of Y1-IgG in this experiment was 2 × 10^-8M. Therefore, the corresponding affinity of the dimer is 4 × 10^-8M.
[0380]
[379.] In a second RRA with labeled CONY1, 100 ng / line¹²⁵Competition was performed with each of the three molecules using I-Y1-IgG. The results are shown in FIG. This figure demonstrates that the affinity of the SS dimer was 20 times higher than that of CONY1. A rough estimate of the affinity of CONY1 in this experiment was 10^-6M. Therefore, the corresponding affinity of the dimer is 5 × 10^-8M.
[0381]
[380.] 9.7 ELISA for GC (Glycocalicin)
[0382]
[381.] 100 μl of purified glycocalicin was incubated overnight at 4 ° C in MaxiSorp plates with 96 flat wells. Plates were washed three times with PBST (PBS + 0.05% Tween) and then with 200 ml PBST-milk (PBST + 2% skim milk) for 1 hour at room temperature. Plates were washed with PBST and monomer or dimer (100 μl) was added in PBST-milk at different concentrations for 1 hour at room temperature. Thereafter, the plate is washed, and the anti-V_LPolyclonal (V derived from Y1_L(Derived from immunized rabbits) (diluted 1: 100 in PBST-milk) for 1 h. Plates were washed and anti-rabbit HRP was added for an additional hour. Plates were washed 5 times and anti-rabbit HRP was added for an additional hour. The plate is washed 5 times, 100 μl of TMB substrate is added for about 15 minutes, then 100 μl of 0.5H_TwoSO_FourWas added to stop the reaction. The optical density of the plates was measured at 450 nm in an ELISA reader.
[0383]
[382.] 9.8 Reactivity of Y1 with recombinant glycocalicin (GC) expressed in prokaryotic (E. coli) systems.
[0384]
[383.] A DNA fragment encoding the N-terminal soluble portion of human platelet GP1b-glycocalcin (GC, amino acids 1 to 493) was cloned into an IPTG-inducible prokaryotic vector cassette. E. coli (BL21, DE3) cells containing the newly constructed plasmid are grown at 37 ° C. to an OD of 0.7-0.8, then grown for 3 hours at 37 ° C. for the presence of IPTG for induction. Was. SDS-polyacrylamide loaded with either GC from native semi-purified human platelets or E. coli cell lysates (total protein content) derived from induced and non-induced cells The gel was analyzed. scFv Y1-biotinylated polyclonal rabbit anti-human GC antibody, commercially available mouse anti-human CD42 monoclonal antibody (SZ2 Immunotech, PM640 Serotee, HIP1 Pharmigen, AN51 DAKO) and polyclonal antibody against the N-terminus of gpIba (Sc-7071, Santa Western blot analysis was performed using Cruz). The two polyclonal antibodies recognized both recombinant bacterial GC and native human platelet GC. scFvY1 and a commercially available antibody recognized only natural human-derived GC, and did not recognize bacterial-derived recombinant platelet GC.
[0385]
[384.] Post-translational modifications such as glycosylation and sulfation are essential for the binding of scFv and commercially available antibodies to GC. Prokaryotic (E. coli) systems lack post-translational modification mechanisms such as glycosylation and sulfation.
[0386]
[385.] 9.9 Preparation of tetramer of Y1
[0387]
[386.] The following sequence, LNDIFEAQKIEWHE, was added at the C-terminus of Y1 by PCR and cloning into an IPTG inducible expression system. The clone was named Y1-biotag. This sequence is a substrate for the enzyme BirA, which allows the enzyme to covalently link biotin to a lysine (K) residue in the absence of free biotin (phenotypic analysis of antigen-specific T lymphocytes). Science, Oct. 4, 1996; 274 (5284): 94-6, Altman JD et al). This construct was produced as inclusion bodies in BL21 bacterial cells. Refolding was performed as described above. Inclusion bodies were solubilized in guanidine-DTE. Refolding was performed by dilution in a buffer containing arginine-Tris-EDTA. Dialysis and concentration were performed, followed by HiTrapQ ion exchange purification.
[0388]
[387.] Purified Y1-biotag scFv was incubated with BirA enzyme (purchased from Avidity) and biotin as recommended by the provider. The HABA test (estimating the amount of biotin per molecule) analyzed the biotinylated Y1-biotag and demonstrated that there were more than about 0.8 biotin molecules per molecule.
[0389]
[388.] Biotinylated Y1-biotag was incubated with streptavidin-PE (Phycoerytrin) to form a complex, which was used in FACS experiments using KG-1 cells (positive for Y1). Streptavidin can bind up to four biotinylated Y-1-biotag molecules. The binding sensitivity was increased at least 10-fold due to the increased binding force.
[0390]
[389.] The sequence of Y1-biotag is as follows:
[0391]
Embedded image

[0392]
[390.] Example 10: Construction of full size Y1-IgG1
[0393]
[391.] All IgG molecules are those mediated by longer half-life in vivo and ADCC or CDC (complement dependent cytotoxicity: Tomlinson, Current Opinions of Immunology, 5, 83-89 (1993)). Have several advantages over the Fv form, including the potential to elicit cellular responses in vivo, such as By the molecular cloning approach described below, we converted the Y1Fv region into a full-sized IgGI molecule. Y1IgGI construction was achieved by joining the cDNA fragments in the following order.
[0394]
[392.] 10.1 Compatible leader sequences for mammalian expression systems: Exchangeable systems were designed to allow for the proper insertion of the elements required for a complete IgG molecule. The following complementary double-stranded oligonucleotide encoding the putative leader sequence was synthesized, annealed, and ligated into the XhoI site of the mammalian expression vector (under the SRα5 promoter).
[0395]
Embedded image

[0396]
Two Kozak elements were included upstream of the start ATG codon. In addition, an internal EcoRV site was introduced between the XhoI site and the putative split site of the leader sequence to allow subcloning of the variable region. This modified vector was named pBJ-3.
[0397]
[393.] 10.2 V derived from the V1 scFv cDNA sequence between the leader and constant light region coding sequences._LThe coding sequence was inserted. Similarly, a Y1 scFv cDNA sequence was inserted between the leader and the constant weight region coding sequence. This is individually Y_LAnd V_HTo obtain the region, this was achieved by PCR amplification of the vector pHEN-Y1 coding for the original Y1.
[0398]
[394.] 10.3 Oligonucleotides
V_LFor the PCR reaction, 5'-TTTGATATCCAGCTGGTGGAGTCTGGGGGA (sense) and 5'-GCTGACCTAGGACGGCAGCTTGGT (antisense) were used. The expected size cDNA product of ３５０350 bp was purified, sequenced and digested with EcoRV and ArtII restriction enzymes. Using the same procedure with the sense and antisense oligonucleotides 5'-GGGATATCCAGCTG (C / G) (A / T) GGAGTCGGGGC and 5'-GGACTCGAGACGGTGACCAGGGTACCTTG, respectively,_HThe cDNA region was amplified and purified.
[0399]
[395.] 10.4 Constant region: The constant λ3 (CL-λ3) region and constant weight region CH1-CH3 derived for the IgG1 cDNA were synthesized individually as follows:
[0400]
[396.] 10.4.1 For the constant CL- [lambda] 3 region, RNA from pools of normal peripheral B cells (CD19 + cells) extracted in combination with oligonucleotides of the sense 5'-CCGTCCTAGGTCAGCCCAAGGCTGC and the antisense 5'-TTTGCGGCCGCTCATGAACATTCTGTAGGGGGCCACTGT was used. Was subjected to RT-PCR. PCR products of the expected size (（400 bp) were purified, sequenced and digested with AvrII and NotI restriction enzymes.
[0401]
[397.] 10.4.2 For the constant IgGI region (γ chain), a BTG immortalized human B cell clone (CMV-clone # 40) was selected for PCR amplification. This clone was shown to secrete IgGI against human CMV and was also shown to elicit an ADCC response in an in vitro assay. For CH1 to CH3 cDNA, oligonucleotides 5'-CCGCTCGAGTGC (T / C) TCCACCAAGGCCCCATC (G / C) GTCTTC (sense) and 5'-TTTGCGGCCGCTCATTTACCC (A / G) GAGACAGGGAGAGGCT (antisense) are synthesized and PCR is performed. Used for. PCR products of the expected size (（1500 bp) were purified, sequenced, and digested with AvrII and NotI restriction enzymes as described for the CL cDNA coding sequence.
[0402]
[398.] 10.5 For the final expression vector, a triple ligation procedure was performed using EcoRV-NotI predigested vector, EcoRV-AvrII variable cDNA and AvrII-NotI constant region. The final vectors for heavy and light chain expression were designated YI-HC and Y1-LC, respectively.
[0403]
[399.] 10.6 An additional vector, pBJ-Y1-LP, was constructed based on Y1-LC to allow double selection based on the puromycin resistance gene (PAC). In this vector, the neomycin resistance gene of the Y1-LC plasmid was replaced with a １1600 bp fragment encoding for the PAC gene (from the pMCC-ZP vector).
[0404]
[400.] 10.7 The open reading frames (ORFs) of both Y-1-IgG-HC and Y1-IgG-LC and their encoded amino acid sequences are presented below:
[0405]
[401.] 10.7.1 Y1-IgG-HC (V_H C_H1 C_H2 C_H3) ORF
[0406]
Embedded image

[0407]
[402.] 10.7.2 Y1-IgG-LC (V_LC_L) ORF
[0408]
Embedded image

[0409]
[403.] The leader sequence is underlined. V_HAnd V_LEach region is encoded by an amino acid sequence shown in bold, followed by an IgG1 (for heavy chain) or λ3 (for light chain) constant region sequence.
[0410]
[404.] 10.8 Expression of Y1 heavy and light chains in CHO cells.
Vectors V1-HC and Y1-LC were used separately for transfection and selection of stable cells expressing heavy or light chains. After selection for G418 and cell growth, secreted proteins in the supernatant were analyzed for IgGI expression by capture EIA assay and Western blot analysis as described below.
[0411]
[405.] 10.8.1 Capture EIA assay: The wells of a 96-well plate were pre-coated with mouse anti-human IgGIFc (Sigma). Supernatants from above were added to the wells to detect the presence of heavy chain IgG1 for biotinylated goat anti-γ chain specific antibody (Sigma), streptavidin-HRP and substrate. If the ELISA plate reader is A₄₀₅Was monitored for color development.
[0412]
[406.] 10.8.2 Western blot analysis: Supernatants for the above cells were run on 12.5% SDS-PAGE. Expression of each chain was determined by (a) goat anti-human IgG-HRP (H + L; Sigma Cat # 8667) for heavy chain detection and (b) biotinylated goat-antibody for light chain detection. Human λ3 chain (Southern Biotechnology Association, (at # 2070-08)) was detected.
Expression of both chains was confirmed by the assay described above, and co-transfection was performed to obtain full-sized Y1-IgG1.
[0413]
[407.] 10.9 Expression and Purification of Y1-IgG
[0414]
[408.] 10.9.1 Cell culture and transfection: 5% CO_TwoCHO cells were cultured in F-12 medium with 10% fetal calf serum and 40 μg / ml gentamicin at 37 ° C. in an atmosphere. One day before transfection, 0.8 × 10⁶Individual cells were seeded on a 90 mm dish. Cultures were co-transfected with 10 μg of light and heavy chain DNA by Fu Gene (Roche) transfection reagent technology. After two days of growth in non-selective medium, cells were cultured for 10-12 days in F-12 medium containing 550 μg / ml neomycin and 3 μg / ml puromycin. Cells were trypsinized and cloned in Costar 96-well plates by limiting dilution of 0.5 cells per well. Individual colonies were picked, grown in 6-well dishes, and transferred to flasks.
[0415]
[409.] 10.9.2 Determination of heavy and light chain secretion: A sandwich ELISA assay was used to determine the concentration of antibody secreted into the supernatant of transfected CHO cells. The following reagents were used to determine antibody concentration: monoclonal anti-human IgG1 (Fc) (Sigma) as coated antibody, goat anti-human IgG (γ-chain specific) biotin as detection element Conjugate (Sigma) and pure human IgG1 lambda as standard (Sigma). Based on this ELISA assay, production rates varied between 3-4 μg / ml.
[0416]
[410.] 10.9.3 MAb production and purification from cells: 1-2 x 10 per bottle in F-12 medium with 10% fetal bovine serum captured with neomycin and puromycin.⁸Cells were generated in roller bottles to a final concentration of individual cells. For production, cells were cultured in the same medium but with 2% fetal calf serum for an additional 2 days. The secreted antibody was purified on a protein G-Sepharose column (Pharmacia). Binding was performed with 20 mM sodium phosphate buffer, pH 7.0, and elution was performed with 0.1 M glycine buffer, pH 2.5-3.0. The quantity of purified antibody was determined by UV absorbance. Purity was analyzed by SDS-PAGE. Under non-denaturing conditions, a complete IgG antibody has its predicted molecular weight of 160 kD. In denaturing gels, both the heavy and light chains have expected molecular sizes of 55 and 28 kD, respectively.
[0417]
[411.] Full-size Y1-IgG molecule binding: Binding experiments were performed to determine the binding level of Y1-IgG relative to the binding level of scFv-Y1 molecule. A two-step staining procedure was used in which 5 ng of Y1-IgG was reacted with both RAJI cells (negative control, FIG. 7a) and Jurkat cells (Y1 positive cells, FIG. 7b). For detection, PE-labeled goat anti-human IgG was used. Similarly, Jurkat cells (FIG. 7c) were reacted with 1 μg of scFv-Y1, and PE-labeled rabbit anti-scFv was used for detection. The results indicate that both Y1-IgG and scFv-Y1 bind to Jurkat cells and require about 10 to obtain a level of detection similar to that of Y1-IgG.^ThreeThis indicates that twice as many scFv-Y1 molecules are required.
[0418]
Detailed description of the table
[412.]Table 1: Panning result derived from protocol AM. The putative number of phagemids used for panning (input) and the putative number of eluted bound phagemids (output) are summarized for four consecutive steps of the AM biopanning protocol. The cell source and elution media for each output are listed, along with the terms used to distinguish each separate stock.
[0419]
[413.]Table 2: Selected clones after AM biopanning protocol. CDR3 region (V_H-CDR3 size) and the CDR3 amino acid sequences for the different clone types isolated are summarized. In addition, the frequencies of each clonal type in two AM biopanning outputs, T16M3 and T16M3.1 outputs, are presented.
[0420]
[414.]Table 3: Panning results derived from YPR protocol: The estimated number of phagemids used for panning (input) and the estimated number of eluted bound phagemids (output) are summarized. The elution media for each output is listed along with the terms used to distinguish each individual stock.
[0421]
[415.]Table 4: Panning results derived from YPNR protocol. The putative number of phagemids used for panning (input) and the putative number of eluted bound phagemids (output) are summarized for three consecutive steps of the YPNR biopanning protocol. The elution media for each output is listed along with the terms used to distinguish each individual stock.
[0422]
[416.]Table 5: Selected Y-series clones after YPR biopanning protocol at R3 output. Several different clones were identified in the R3 output stock. V of the clone identified_H-The number and amino acid sequence of the amino acid residues including the CDR3 region, and the germline are detailed.
[0423]
[417.]Table 6: Y1 binding specificity for leukemia cells. The results of binding experiments of three different scFv clones each reacted with a cell mixture predominantly containing each of the seven different leukemia cell types as determined by FACS analysis are presented. The results represent the portion of the patient identified by FACS analysis as positive for each test antibody and its cells. The numerator represents the number of positive patients and the denominator represents the total number of patients tested for a given scFv / leukemia cell type combination.
[0424]
[418.]Table 7: FACS analysis of scFv binding to Ficoll purified normal blood cells. Three scFv clones are each analyzed for binding to five different ficoll purified normal blood cell types. These binding results represent the portion of the normal blood sample identified by FACS analysis as positively reactive with each of the antibodies to be tested.
[0425]
[419.]Table 8: Comparison of antibody binding and Y1 scFv binding to various cell markers. The results of FACS analysis of staining with Y1 and other series of antibodies are shown. Ficoll purified peripheral and bone marrow cells from ANE patients were prepared and the binding specificity of Y1scFv compared to various cell markers on AML cells was studied. Results are expressed as the percentage of cells in Ficoll purified specimens of a given patient, identified as positively reactive with each Fv by FACS analysis. Four other antibodies were run for comparison: (1) markers for CD13-granulocytes and monocytes; (2) markers for CD14-monocytes and neutrophils; (3) CD33-normal Markers for myeloid cells and leukemic myeloid cells and (4) markers for CD34- stem cells.
[0426]
[420.]Table 9: Y1 binding to hematopoietic cell lineage. FACS analysis was performed to determine the binding of Y1 scFv to three different categories of human leukemia cell lines and one mouse cell line. Cell lines positively bound (reactive) or not (reactive) by Y1 are listed.
[0427]
[421.]Table 10: V_H3- CDR3 sequence of clone isolated with DP32. According to different biopanning and selection procedures, multiple clones based on the DP32 germline were isolated. Clones Y1, Y17, Y-27 and Y-44 were identified during biopanning selection on platelets (YPR and YPNR protocols). The V of each of these clones_H-The sequence of the CDR3 region is presented.
[0428]
[422.]Table 11: V_HBinding profile of clones isolated with 3-DP32. The binding specificity of DP32-derived clones to multiple hematopoietic cells was tested by FACS analysis.
[0429]
[423.] The invention has been described with reference to specific examples, materials and data. As those skilled in the art will appreciate, alternative means for using or preparing the various aspects of the present invention are available. Such alternatives should be included within the spirit and scope of the invention as defined by the following claims.
[0430]
[39.] The present invention will now be described in more detail, by way of non-limiting example only, with reference to the accompanying drawings described below. In the drawing,
[Brief description of the drawings]
[0431]
FIG. 1 presents phage clones that bind to established platelets, as determined by EIA assay. Data is presented as a function of absorbance at 405 nm.
[41.] Figures 2a, 2b and 2c present the binding of mononuclear cell specimens from three individual AML patients as determined by FACS analysis. The fluorescence intensity of the cells bound by the two FITC-labeled specimens of the test subject (control scFv and scFv clone Y1) is presented.
[42.] FIG. 3 presents the binding of YI to Ficoll purified monocytes (3b) and platelets (3a) as determined by FACS analysis. The fluorescence intensity of the cells bound by the two FITC-labeled test specimens (control scFv and scFv clone Y1) is presented.
[43.] FIG. 4 presents the binding of FITC-labeled scFv clone Y1 to cord blood CD34 + stem cells. The CD34 + gated cells in the FL3-H channel were ligated in FILI-H to FITC-labeled negative control scFv (FIG. 4a) or FITI-labeled scFv clone Y1 (FIG. 4b). Was analyzed. FIG. 4c presents an FSC and SSC dot plot analysis of the same FITI-labeled scFv clone Y1 specimen as 4b. The circled areas in FIGS. 4b and 4c outline the subpopulation of CD34 + cells that bind scFv clone Y1.
[44.] FIG. 5: FACS analysis of specimens obtained from two patients with pre-B-ALL cells is presented: one specimen from a child (5a, 5c, 5e) and the other is from an adult (5b, 5d, 5f). Combined with FITI-labeled negative control scFv (5a, 5b) or FITI-labeled Y-IscFv (5c, 5d), commercially available PE-labeled CD19 (marker for normal peripheral B cells; FIGS. 5a, 5c) or PE-labeled CD34 (a marker for stem cells; FIG. 5d) was utilized. FIG. 5b is a double negative control. The fluorescence intensity (x-axis) of the bound cells by the FITI-labeled specimen (scFv clone Y1) in relation to the negative control staining pattern is presented (5e and 5f).
[45.] FIG. 6: This figure provides the results of a comparative binding study performed using Jurkat cells. FACS analysis of the binding of FITI-labeled Y-IscFv monomers, diabodies and triabodies to Jurkat cells, together with a negative control, is presented.
[46.] FIG. 7: This figure provides the results of studies comparing the binding of IgG-YI and scFv-Y1. To compare the binding of full size IgG-Y1 to that of the scFv-YI form, a double staining procedure was utilized. For FACS analysis on RAJI cells (YI negative cells; FIG. 7a) and Jurkat cells (Y1 positive cells; FIG. 7b), 5 nanograms of IgG-YI were used. For detection, PE-labeled goat anti-human IgG was used. For binding of scFv-YI-I, １1 μg (200 ×) was used, followed by staining with PE-labeled rabbit anti-scFv antibody and FACS analysis (FIG. 7c).
[47.] FIG. 8: This figure shows the binding comparison between YI dimer, YIscFv (CONY1) and YIIgG.
[48.] FIG. 9: This figure shows the binding comparison between Y1 sulfide bridged dimer and Y1 scFv (CONY1).
[49.] FIG. 10: This figure is a graph of the Superdex75 profile of Y1-cys-kak.
[50.] FIG. 11: This figure demonstrates the size of the dimer compared to the monomer under reducing and non-reducing conditions.
[51.] FIG. 12: This figure provides the results of the ELISA assay.
[52.] FIG. 13: This figure is a chart of the epitopes of anti-GPIbα antibodies.
[53.] FIG. 14: This figure is the SEQ ID NO of the amino acid.

Claims (259)

A Fe molecule, a constituent thereof, any fragment thereof, or a fragment thereof, having enhanced binding properties to selectively and / or specifically bind to the target cell in a manner that is advantageous to a cell other than the target cell. A peptide or polypeptide comprising a construct, wherein the binding selectivity or specificity is primarily determined by the first hypervariable region, and wherein the Fv is a scFv or dsFv, optionally having one or more tags. Or a polypeptide. The peptide or polypeptide according to claim 1, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. The first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24, and the second hypervariable region has a secondary binding selectivity or specificity. The peptide according to claim 1 , wherein the peptide is affected by one or more of the three hypervariable regions and / or the upstream or downstream regions flanking the first, second and third hypervariable regions. Or a polypeptide. 3. The peptide or polypeptide of claim 2 , wherein the first hypervariable region is a scFv having SEQ ID NO: 25, which is the same CDR3 region as SEQ ID NO: 8. 2. The peptide or polypeptide of claim 1 , wherein the scFv molecule comprises a linear or branched spacer of no more than 20 amino acid residues. The peptide or polypeptide according to claim 5 , wherein the spacer is SEQ ID NO: 123 or SEQ ID NO: 124. 2. The peptide or polypeptide according to claim 1 , wherein the target cell is an abnormal, pathological cell that has been activated, excited, modified, altered, interfered with. The peptide or polypeptide according to claim 7 , wherein the diseased cell is a cancer cell. 8. The peptide or polypeptide of claim 7 , wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma cells. The peptide or polypeptide according to claim 9 , wherein the cell is a leukemia or myeloma cell. 10. The peptide or polypeptide according to claim 9 , wherein the leukemia or myeloma cells are B cell malignancies. The peptide or polypeptide according to claim 10 , wherein the leukemia cell is acute myeloid leukemia. SEQ ID NO: 8 further comprising a cassette of contiguous amino acids having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-113 or having at least 90% amino acid similarity thereto. 3. The peptide or polypeptide of claim 2 , wherein the CDR3 region having an amino acid sequence selected from the group consisting of ~ 24 provides one framework into which is assembled, inserted, attached, attached, combined or fused. . The cassette has SEQ ID NOS: 30-32, 33, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94. 14. The peptide or polypeptide of claim 13 , having an amino acid sequence selected from or having at least 90% amino acid similarity thereto. 14. The peptide or polypeptide of claim 13 , wherein the cassette has the amino acid sequence of SEQ ID NO: 61 or has at least 90% amino acid similarity thereto. 16. The peptide or polypeptide of claim 15 , wherein the cassette has the amino acid sequence of SEQ ID NO: 61 or has at least 90% amino acid similarity thereto. 16. The peptide or polypeptide of claim 15 , wherein the seven carboxy terminal amino acid residues of SEQ ID NO: 61 have been replaced by the seven amino acid residues of SEQ ID NO: 122. 4. The peptide or polypeptide of claim 3 , wherein the second and third hypervariable regions are CDR2 and CDR1 hypervariable regions, respectively. 3. The peptide or polypeptide according to claim 2 , wherein the CDR3 region has the amino acid sequence of SEQ ID NO: 8. 19. The peptide or polypeptide of claim 18 , wherein the CDR2 and CDR1 regions have the amino acid sequences of SEQ ID NO: 115 and SEQ ID NO: 114, respectively. 4. The peptide of claim 3 , wherein the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively, wherein the CDR3, CDR2 and CDR1 regions have the amino acid sequences of SEQ ID NOs: 8, 115 and 114, respectively. Polypeptide. The peptide or polypeptide according to claim 3 , wherein the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117, and the downstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 116. The second hypervariable region is a CDR2 hypervariable region, the upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119, and the downstream region flanking the CDR2 region is the amino acid sequence of SEQ ID NO: 118. The peptide or polypeptide according to claim 3 , having the formula: The third hypervariable region is a CDR1 hypervariable region, the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121, and the downstream region flanking the CDR1 region is the amino acid sequence of SEQ ID NO: 120. The peptide or polypeptide according to claim 3 , having the formula: 19. The peptide or polypeptide according to claim 18 , wherein the CDR2 and CDR1 regions of the cassette of contiguous amino acids selected from the group consisting of SEQ ID NOs: 30-113 or fragments thereof are replaced by SEQ ID NOs: 115 and 114, respectively. peptide. SEQ ID NOs: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99,103,106,112 and 113 or CDR2 and CDR1 regions of contiguous amino acids of the cassette selected from the group consisting of fragments are replaced by SEQ ID NO: 115 and 114, according to claim 18 A peptide or polypeptide of (A) the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively.
(B) the amino acid sequence of CDR3 is SEQ ID NO: 8,
(C) the amino acid sequence of CDR2 is SEQ ID NO: 115,
(D) the amino acid sequence of CDR1 is SEQ ID NO: 114,
(E) the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117,
(F) the downstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 116,
(G) the upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119,
(H) the downstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 118,
(I) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121,
(J) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 120,
A peptide or polypeptide according to claim 3 . The peptide or polypeptide according to claim 1 , wherein the Fv is a scFv obtainable from a phage display library. A phage display library has been constructed from unimmunized human peripheral blood lymphocytes, and the scFv peptides have been selected against previously uncharacterized crude antigen on the surface of target cells. A peptide or polypeptide according to claim 28 . 29. The peptide of claim 28 , wherein biopanning comprises binding the phage to a target, removing unbound phage, eluting the bound phage, and propagating and amplifying the eluted phage. Or a method for selecting or identifying a polypeptide. An Fv molecule, its constituents, having enhanced binding properties to selectively and / or specifically bind to substantially exposed and / or overexpressed binding sites on or in target cells, In a peptide or polypeptide comprising any of the fragments or constructs of the fragments, binding to the target cell may occur in other cells in which the binding site is substantially unavailable and / or not expressed in or within. Peptides or polypeptides which advantageously occur, wherein the binding selectivity or specificity is mainly determined by the first hypervariable region, wherein the Fv is a scFv or dsFv, and wherein the Fv optionally has one or more tags . 32. The peptide or polypeptide of claim 31 , wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. The first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 24, wherein the binding selectivity or specificity is secondarily increased; Affected by the third hypervariable region and / or one or more upstream or downstream regions flanking the first, second and third hypervariable regions, wherein the second and third hypervariable regions are 32. The peptide or polypeptide of claim 31 , which is a CDR2 and CDR1 region, respectively. Fv molecules, their constituents, any fragments or constituents of fragments, having enhanced binding properties to selectively and / or specifically bind to target cells in favor of other cells. Wherein the Fv molecule comprises a first chain having first, second, and third hypervariable regions and a second chain having first, second, and third hypervariable regions. And wherein one of the first chain hypervariable regions has a sequence selected from the group consisting of SEQ ID NOs: 8-24, and one of the second chain hypervariable regions comprises Having one sequence selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, wherein the first, second and third hypervariable regions are CDR3, CDR2 and CDR1 regions, respectively; Is scFv or dsF In it, Fv is optionally has one or more tags, peptide or polypeptide. (A) the first strand and the second strand each comprise a first hypervariable region selected from the group consisting of SEQ ID NOs: 8-24; or (b) the first strand of the first strand. The hypervariable region and the first hypervariable region of the second chain are identical and are selected from the group consisting of SEQ ID NOs: 8-24; or (c) the first hypervariable region of the first chain is Is selected from the group consisting of SEQ ID NOs: 8-24 and the first hypervariable region of the second strand is selected from the group consisting of SEQ ID NOs: 1-6 and 125-202;
(D) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs: 1-6 and 125-202; Selected from the group consisting of
A peptide or polypeptide according to claim 34 . 35. The peptide or polypeptide of claim 34 , wherein the second and third hypervariable regions of the first chain are SEQ ID NOS: 114 and 115, respectively. (A) binding to an unknown ligand on a first cell having a first and second state, wherein the binding is effective in the second state, but is substantially ineffective in the first state; and (B) due to immune cross-reactivity, specifically or selectively binds to a ligand on a second cell, wherein the Fv is a scFv or dsFv, and the Fv optionally has one or more tags ,
A peptide or polypeptide comprising an Fv molecule, its constituents, any fragment or constituent of the fragment. 38. The peptide or polypeptide according to claim 37 , wherein the first cell is a normal cell. 38. The peptide or polypeptide of claim 37 , wherein the first state is an unactivated state and the second state is an activated, excited, modified, altered, or interfered state. 38. The peptide or polypeptide of claim 37 , wherein the second cell is a pathological cell. 42. The peptide or polypeptide according to claim 40 , wherein the diseased cell is a cancer cell. 42. The peptide or polypeptide of claim 40 , wherein the pathological cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. . 43. The peptide or polypeptide according to claim 42 , wherein the pathological cell is a leukemia cell. 44. The peptide or polypeptide of claim 43 , wherein the leukemia cell is an acute myeloid leukemia cell. 38. The peptide or polypeptide of claim 37 , wherein the selective and / or specific binding of the peptide or polypeptide to a second cell ligand is determined primarily by the first hypervariable region. 46. The peptide or polypeptide of claim 45 , wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. The first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 24, wherein the binding selectivity or specificity is secondarily increased; 47. The method according to claim 46 , wherein the third hypervariable region and / or the first, second and third hypervariable regions are flanked by one or more of the upstream region or the downstream region, respectively. A peptide or polypeptide according to claim 1. A ligand expressed by a second cell and capable of being bound by the peptide or polypeptide of claim 37 . A molecule that recognizes and binds the ligand of claim 48 . A nucleic acid molecule encoding a peptide or polypeptide according to any one of claims 1, 31, 34 or 37. 51. The nucleic acid molecule according to claim 50 , wherein the nucleic acid is DNA. 38. The peptide or polypeptide of claim 37 , wherein the first and second states of the first cell are the same, and wherein the first cell is derived from one cell line. Cell lines, Jurkat, are selected from the group consisting of Molt-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2 and HUT-78, claim 52 4. The peptide or polypeptide according to item 1. A method for identifying a targeting molecule that binds to an unknown immune cross-reactive binding site on first and second cells, comprising:
(A) performing one or more biopannings on a first target cell that substantially exposes or displays a binding site containing at least one unknown ligand in a second state instead of the first state; Thus producing a first population of recognition molecules;
(B) performing on a second cell displaying a binding site comprising at least one unknown ligand having immunological cross-reactivity to the unknown ligand of the first cell to generate a second population of recognition molecules. Performing a subsequent biopanning and / or selection step starting from the first population of recognition molecules of step (a);
(C) amplifying and purifying the second population of recognition molecules of step (b); and (d) a targeting molecule that is selective and / or specific for an unknown ligand on the second cell. Constructing a peptide or polypeptide from the recognition site of the purified recognition molecule of step (c);
A method for identifying a targeting molecule, comprising: A first cell is a normal cell, a state in which the first state is not activated, the second state, activating, exciting, modifications, and states modified or interference claim 54. The method according to 54 . 55. The method of claim 54 , wherein the second cell is a pathological cell. 57. The method of claim 56 , wherein the pathological cells are cancer cells. 57. The method of claim 56 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 59. The method of claim 58 , wherein the cells are leukemia cells. 60. The method of claim 59 , wherein the leukemia cells are acute myeloid leukemia cells. 38. The peptide or polypeptide according to claim 1 or 37 , in the manufacture of a medicament, optionally associated or attached, coupled, combined, linked or fused to a pharmaceutical agent. use. 62. Use according to claim 61 , wherein the medicament has activity against pathological cells. 63. The use according to claim 62 , wherein the pathological cells are cancer cells. 63. The use according to claim 62 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma cells. 65. The use according to claim 64 , wherein the cell is a leukemia cell. 66. The use according to claim 65, wherein the leukemia cells are acute myeloid leukemia cells. 38. The peptide or polymorph according to claim 1 or 37 , for use as a medicament, optionally associated with or attached, coupled, combined, linked or fused to a pharmaceutical agent. peptide. 68. The peptide or polypeptide of claim 67 , wherein the medicament has activity against diseased cells. 69. The peptide or polypeptide of claim 68 , wherein the diseased cell is a cancer cell. 69. The peptide or polypeptide of claim 68 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 71. The peptide or polypeptide of claim 70 , wherein the cell is a leukemia cell. 72. The peptide or polypeptide of claim 71, wherein the leukemia cells are acute myeloid leukemia cells. 38. Use of a peptide or polypeptide according to claim 1 or claim 37 for the preparation of a composition for use in inhibiting the growth of pathological or cancer cells. 74. Use of a peptide or polypeptide according to claim 73 , wherein the cell is a leukemia cell. 75. Use of a peptide or polypeptide according to claim 74 , wherein the leukemia cell is an acute myeloid leukemia cell. A composition for use in inhibiting the growth of cancer cells, the composition comprising at least one compound having a pharmaceutical ligand selective and / or specific for cancer cells. 38. Use of a peptide or polypeptide according to claim 1 or claim 37 . Claim 1 or Claim 1 wherein the pharmaceutically effective amount of a pharmaceutical agent, optionally associated with, attached to, coupled to, associated with, linked to or fused with a pharmaceutically effective carrier. 38. A composition comprising at least one peptide according to 37 . 78. The composition of claim 77 , wherein the peptide or polypeptide and the pharmaceutical agent are linked via a linker compound. 79. The composition of claim 78 , wherein the linker compound is selected from the group consisting of dicarboxylic acids, maleimide hydrazides, PDPH, carboxylic acid hydrazides, and small peptides. The small peptides are AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, protein C, S.Tag ™, T7, V5, VSV-G and 80. The composition of claim 79 , wherein the composition is selected from the group consisting of KAK-Tag. Tags are AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S • Tag ™, T7, V5, VSV-G, and 38. The peptide or polypeptide according to any one of claims 1, 31, 34 and 37 , wherein the peptide or polypeptide is selected from the group consisting of KAK-Tag. 78. The composition of claim 77 , wherein the pharmaceutical agent is selected from the group consisting of radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anti-cancer agents. Radioisotope, indium, ¹¹¹ Indium, ¹¹³ Indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ^99m technetium, ^121m tellurium, ^122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ Thulium, ¹⁶⁸ Thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, groups of ⁶⁷ gallium and ⁶⁸ gallium 83. The composition of claim 82 , wherein the composition is selected from: 83. The composition of claim 82 , wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifications and derivatives thereof. Anticancer agents include doxorubicin, morpholino-doxorubicin (MDOX), adriamycin, cis-platinum, toxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha 83. The composition of claim 82 , wherein the composition is selected from the group consisting of: hydroxyurea, temozolomide, thalidomide, bleomycin, and derivatives thereof. 38. A method of inhibiting cell growth comprising contacting a cell with a quantity of the peptide or polypeptide of claim 1 or 37 . 87. The method of claim 86 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 91. The method of claim 87 , wherein the cell is a leukemia cell. 89. The method of claim 88 , wherein the leukemia cells are acute myeloid leukemia cells. 38. At least one peptide according to claim 1 or 37 attached, coupled, coupled, linked or fused to an imaging agent for use in diagnostic localization and imaging of a tumor. Pharmaceutical composition. 38. A method for treating a patient suffering from a disease or cancer, comprising administering to the patient a peptide or polypeptide according to claim 1 or 37 in an amount effective to treat the disease or cancer. 92. The method of claim 91 , wherein the disease or cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. 93. The method of claim 92 , wherein the disease is leukemia. 94. The method of claim 93 , wherein the leukemia is acute myeloid leukemia. 38. The peptide or polypeptide according to claim 1 or claim 37 , wherein the Fv specifically or selectively binds to acute myeloid leukemia (AML) cells. A ligand displayed on AML cells bound by the peptide or polypeptide of claim 95 . A peptide or polypeptide that binds to the ligand of claim 96 . 38. For in vitro analysis of the efficacy of a treatment before, during or after treatment comprising a peptide or polypeptide of claim 1 or claim 37 attached, coupled, combined, linked or fused to a marker. Diagnostic kit. The kit according to claim 98 , wherein the marker marker molecule is a fluorescent marker. 100. The kit of claim 99 , wherein the fluorescent marker is selected from the group consisting of fluorescein, rhodamine, phycoerythrin, and modifications and conjugates thereof. The kit according to claim 98 , which is used for diagnosis of a disease or cancer. 38. The peptide or polypeptide according to claim 1 or claim 37 , wherein the construct is an Ig polypeptide. 103. The method for producing a peptide or polypeptide of claim 102 , wherein the Ig polypeptide is expressed as a recombinant polypeptide and is produced in a eukaryotic cell system. 104. The method of claim 103 , wherein the eukaryotic system is a mammalian cell line. 103. The peptide or polypeptide of claim 102 , wherein said Ig polypeptide is an IgG polypeptide. 106. The peptide or polypeptide of claim 105 , wherein the IgG polypeptide comprises CDR3, CDR2, and CDR1 regions having SEQ ID NOs: 8, 115, and 114, respectively. 107. The IgG polypeptide of claim 106 , wherein the CDR3, CDR2 and CDR1 regions are heavy chain regions. 107. The IgG polypeptide of claim 106 , wherein the CDR3, CDR2 and CDR1 regions are light chain regions. 103. The IgG polypeptide of claim 102 , wherein the IgG has a heavy chain comprising SEQ ID NO: 26 and a light chain comprising SEQ ID NO: 27 or a chain having at least 90% amino acid similarity thereto. 38. The method for producing a peptide or polypeptide of claim 1 or claim 37 , wherein the peptide or polypeptide is produced in a prokaryotic or eukaryotic cell system. 112. The method of claim 110 , wherein the prokaryotic system comprises E. coli comprising an expression vector and the eukaryotic system is a mammalian cell line. Expression vectors prokaryotic systems, osmB, deo, β-lac -U5, λP L, SRα5, and comprising a promoter selected from the group consisting of CMV, The method of claim 111. A binding motif comprising the amino acid sequence of R ₁ -X Phe Pro-R _{2 wherein} each of R ₁ and R ₂ comprises 0 to 15 amino acid residues and X is any of Arg, Gly or Lys. A peptide or polypeptide comprising CDR3 comprises the amino acid sequence of R ₁ -XPhe Pro-R ₂ , wherein R ₁ and R ₂ each comprise from 0 to 15 amino acid residues, and X is any of Arg, Gly, or Lys; A peptide or polypeptide according to any one of claims 2, 34 or 46 . 38. The peptide or polypeptide of claim 1 or claim 37 , wherein the peptide or polypeptide includes at least one non-naturally occurring modification. 115. The peptide or polypeptide of claim 115 , wherein said non-naturally occurring modification renders the peptide or polypeptide more immunogenic or more stable. Wherein the at least one modification, peptide modifications, semipeptoids modified, cyclic peptide modification, N- terminus modification, C terminus modification, peptide bond modification, are selected from the group consisting of backbone modifications, and residue modification, claim 116. The peptide or polypeptide according to 116 . 68. The peptide or polypeptide of any one of claims 1, 31, 34, 37 or 67 for ex vivo purging of autologous bone marrow to remove abnormal cells. a) directly by a biopanning procedure on the target cells or indirectly by a biopanning procedure on a first target cell in a second state instead of the first state and then directly on a second target cell. Isolating and selecting one or more targeting molecules comprising a primary recognition site by a biopanning procedure to produce one or more of said targeting molecules;
b) amplifying, purifying and identifying one or more targeting molecules; and c) constructing a targeting agent from the one or more targeting molecules.
Otherwise, the targeting agent can be a peptide, polypeptide, antibody or antibody fragment or a multimer thereof. 120. The method of claim 119 , wherein the targeting agent is coupled, attached, combined, linked, fused, or associated with the pharmaceutical agent. 121. The method of claims 119 and 120 , wherein the targeting agent is an anti-disease or anti-cancer agent. 121. The method of claim 120 , wherein the pharmaceutical agent is selected from the group consisting of radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anti-cancer agents. Radioisotope, ¹¹¹ Indium, ¹¹³ Indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ^99m technetium, ^121m tellurium, ^122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ Thulium, ¹⁶⁸ Thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ Iodine, ^{81 m} krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorin, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium 123. The method of claim 122 , wherein the method has been selected. 130. The method of claim 122 , wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria, lysine, and modifications and derivatives thereof. The anticancer agent is selected from the group consisting of vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, bleomycin and derivatives thereof. 123. The method of claim 122 , wherein A-X-B
Wherein X is a hypervariable CDR3 region of 3 to 30 amino acids; A and B may each be an amino acid chain of 1 to 1000 amino acids in length. Wherein A is the amino terminus and B is the carboxy terminus. 127. The peptide of claim 126 , wherein A is 150-250 amino acid residues and B is 350-500 amino acid residues. 127. The peptide of claim 126, wherein the CDR3 region is between 5 and 13 amino acid residues. 127. The peptide or polypeptide of claim 126 , wherein X is an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. 130. The peptide or polypeptide of claim 127 , which is part of a larger or intact antibody or multimer. 127. A dimeric molecule comprising two peptides or polypeptides, one of which is the peptide or polypeptide of claim 126 . 127. A dimeric molecule comprising two peptides or polypeptides of claim 126 that are identical. 133. The dimer molecule according to claim 131 or 132 , wherein X is an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24. 130. A nucleic acid molecule encoding the peptide or polypeptide of claim 126 or the dimer molecule of claim 130 . 105. The method of claim 104 , wherein the mammalian cell line comprises the SRα5 promoter. 105. The method of claim 104 , wherein the mammalian cell line comprises a CMV promoter. A peptide or polypeptide substantially as described herein. A Fe molecule, a constituent thereof, any fragment thereof, or a constituent of a fragment, having enhanced binding properties to selectively and / or specifically bind to a target cell in a manner that favors other cells. In a peptide or polypeptide comprising, the binding selectivity or specificity is determined primarily by the first hypervariable region, wherein the first hypervariable region is an amino acid selected from the group consisting of SEQ ID NO: 8 or 20 A peptide or polypeptide which is a CDR3 region comprising a sequence, wherein the Fv is a scFv or dsFv, optionally having one or more tags. An upstream or downstream region whose binding selectivity or specificity flanks the second hypervariable region, the third hypervariable region and / or the first, second and third hypervariable regions 139. The peptide or polypeptide of claim 138 , wherein said peptide or polypeptide is affected by one or more of the following. 139. The peptide or polypeptide of claim 138 , wherein the first hypervariable region is a scFv having SEQ ID NO: 25, which is the same CDR3 region as SEQ ID NO: 8. 139. The peptide or polypeptide of claim 138 , wherein the first hypervariable region is a scFv having SEQ ID NO: 203, which is a CDR3 region identical to SEQ ID NO: 20. 139. The peptide or polypeptide of claim 138 , wherein the scFv molecule comprises a linear or branched spacer of no more than 20 amino acid residues. 144. The peptide or polypeptide of claim 142 , wherein the spacer is SEQ ID NO: 123 or SEQ ID NO: 124. 139. The peptide or polypeptide of claim 138 , wherein the target cell is an abnormal, pathological, activated, excited, modified, altered, disrupted cell. 153. The peptide or polypeptide of claim 144 , wherein said diseased cell is a cancer cell. 153. The peptide or polypeptide of claim 144 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 147. The peptide or polypeptide of claim 146 , wherein the cell is a leukemia or myeloma cell. 147. The peptide or polypeptide of claim 146 , wherein the leukemia or myeloma cell is a B cell malignancy. 147. The peptide or polypeptide of claim 147 , wherein the leukemia cell is acute myeloid leukemia. SEQ ID NO: 8 further comprising a cassette of contiguous amino acids having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-113 or having at least 90% amino acid similarity thereto. 139. The peptide or polypeptide of claim 138 , wherein the CDR3 region having an amino acid sequence selected from the group consisting of -20 provides one framework into which the CDR3 region is constructed, inserted, attached, attached, combined, or fused. . The cassette has SEQ ID NOS: 30-32, 33, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94. 150. The peptide or polypeptide of claim 150 , having an amino acid sequence selected from or having at least 90% amino acid similarity thereto. 160. The peptide or polypeptide of claim 150 , wherein the cassette has the amino acid sequence of SEQ ID NO: 61 or has at least 90% amino acid similarity thereto. 153. The peptide or polypeptide of claim 152 , wherein the cassette has the amino acid sequence of SEQ ID NO: 61 or has at least 90% amino acid similarity thereto. 153. The peptide or polypeptide of claim 152 , wherein the seven carboxy terminal amino acid residues of SEQ ID NO: 122 have been replaced by the seven amino acid residues of SEQ ID NO: 122. 139. The peptide or polypeptide of claim 138 , wherein the second and third hypervariable regions are CDR2 and CDR1, respectively. 139. The peptide or polypeptide of claim 138 , wherein the CDR3 region has the amino acid sequence of SEQ ID NO: 8. 139. The peptide or polypeptide of claim 138 , wherein the CDR3 region has the amino acid sequence of SEQ ID NO: 20. 160. The peptide or polypeptide of claim 155 , wherein the CDR2 and CDR1 regions have the amino acid sequences of SEQ ID NO: 115 and SEQ ID NO: 114, respectively. 141. The peptide or claim of claim 140 , wherein the second and third hypervariable regions are CDR2 and CDR1 hypervariable regions, respectively, and wherein the CDR3, CDR2 and CDR1 regions have the amino acid sequences of SEQ ID NOs: 8, 115 and 114, respectively. Polypeptide. 141. The peptide of claim 140 , wherein the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively, wherein the CDR3, CDR2 and CDR1 regions have the amino acid sequences of SEQ ID NOs: 20, 115 and 114, respectively. Polypeptide. 140. The peptide or polypeptide of claim 139 , wherein the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117, and the downstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 116. The second hypervariable region is a CDR2 hypervariable region, the upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119, and the downstream region flanking the CDR2 region is the amino acid sequence of SEQ ID NO: 118. 140. The peptide or polypeptide according to claim 139 , comprising: The third hypervariable region is a CDR1 hypervariable region, the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121, and the downstream region flanking the CDR1 region is the amino acid sequence of SEQ ID NO: 120. 140. The peptide or polypeptide according to claim 139 , comprising: 155. The peptide or polypeptide of claim 155 , wherein the CDR2 and CDR1 regions of the contiguous amino acid cassette selected from the group consisting of SEQ ID NOs: 30-113 or fragments thereof are replaced by SEQ ID NOs: 115 and 114, respectively. peptide. SEQ ID NOs: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99,103,106,112 and 113 or CDR2 and CDR1 regions of contiguous amino acids of the cassette selected from the group consisting of fragments are replaced by SEQ ID NO: 115 and 114, claim 155 A peptide or polypeptide of (A) the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively.
(B) the amino acid sequence of CDR3 is SEQ ID NO: 8,
(C) the amino acid sequence of CDR2 is SEQ ID NO: 115,
(D) the amino acid sequence of CDR1 is SEQ ID NO: 114,
(E) the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117,
(F) the downstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 116,
(G) the upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119,
(H) the downstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 118,
(I) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121,
(J) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 120,
139. A peptide or polypeptide according to claim 139 . (A) the second and third hypervariable regions are the hypervariable regions of CDR2 and CDR1, respectively.
(B) the amino acid sequence of CDR3 is SEQ ID NO: 20,
(C) the amino acid sequence of CDR2 is SEQ ID NO: 115,
(D) the amino acid sequence of CDR1 is SEQ ID NO: 114,
(E) the upstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 117,
(F) the downstream region flanking the CDR3 region has the amino acid sequence of SEQ ID NO: 116,
(G) the upstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 119,
(H) the downstream region flanking the CDR2 region has the amino acid sequence of SEQ ID NO: 118,
(I) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 121,
(J) the upstream region flanking the CDR1 region has the amino acid sequence of SEQ ID NO: 120,
139. A peptide or polypeptide according to claim 139 . 139. The peptide or polypeptide of claim 138 , wherein the Fv is a scFv obtainable from a phage display library. A phage display library has been constructed from unimmunized human peripheral blood lymphocytes, and the scFv peptides have been selected against previously uncharacterized crude antigen on the surface of target cells. 170. A peptide or polypeptide according to claim 165 . 170. The peptide of claim 165 , wherein biopanning comprises binding phage to the target, removing unbound phage, eluting the bound phage, and propagating and amplifying the eluted phage. Or a method for selecting or identifying a polypeptide. An Fv molecule, its constituents, having enhanced binding properties to selectively and / or specifically bind to substantially exposed and / or overexpressed binding sites on or in target cells, In a peptide or polypeptide comprising any of the fragments or constructs of the fragments, binding to the target cell may occur in other cells in which the binding site is substantially unavailable and / or not expressed in or within. Advantageously occurring, the binding selectivity or specificity is mainly determined by the first hypervariable region, wherein the first hypervariable region is a CDR3 region consisting of SEQ ID NO: 8 or 20, wherein Fv is scFv or A peptide or polypeptide which is a dsFv, wherein the Fv optionally has one or more tags. One or more upstream flanking the second hypervariable region, the third hypervariable region and / or the first, second and third hypervariable regions with a secondary binding selectivity or specificity 172. The peptide or polypeptide of claim 171 , wherein the peptide or polypeptide is affected by a region or a downstream region, and wherein the second and third hypervariable regions are CDR2 and CDR1 regions, respectively. Fv molecules, their constituents, any fragments or constituents of fragments, having enhanced binding properties to selectively and / or specifically bind to target cells in favor of other cells. Wherein the Fv molecule comprises a first chain having first, second, and third hypervariable regions and a second chain having first, second, and third hypervariable regions. And wherein one of the first chain hypervariable regions comprises the sequence of SEQ ID NO: 8 or 20, and one of the second chain hypervariable regions comprises the sequence of SEQ ID NOs: 1-6 and 125-202. The first, second, and third hypervariable regions are CDR3, CDR2, and CDR1 regions, respectively, wherein Fv is scFv or dsFv, and Fv is optional. Singular or plural Having a tag, a peptide or polypeptide. (A) the first hypervariable region of the first chain and the first hypervariable region of the second chain are identical and are selected from the group consisting of SEQ ID NOs: 8 or 20; or (b) Wherein the first hypervariable region of one chain is selected from the group consisting of SEQ ID NOs: 8 or 20, and the first hypervariable region of the second chain consists of SEQ ID NOs: 1-6 and 125-202 Is selected from; or
(D) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, and the first hypervariable region of the second chain is selected from the SEQ ID NOs: 8 or 20; Selected from the group consisting of
173. A peptide or polypeptide according to claim 173 . 173. The peptide or polypeptide of claim 173 , wherein the second and third hypervariable regions of the first chain are SEQ ID NOS: 114 and 115, respectively. (A) binding to an unknown ligand on a first cell having a first and second state, wherein the binding is effective in the second state, but is substantially ineffective in the first state; and (B) binding specifically or selectively to a ligand on a second cell due to immune cross-reactivity, wherein the Fv is a scFv or a dsFv, and the Fv optionally has one or more tags; And wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 8 or 20.
A peptide or polypeptide comprising an Fv molecule, its constituents, any fragment or constituent of the fragment. 177. The peptide or polypeptide of claim 176 , wherein said first cell is a normal cell. 177. The peptide or polypeptide of claim 176 , wherein the first state is an unactivated state and the second state is an activated, excited, modified, altered, or interfered state. 177. The peptide or polypeptide of claim 176 , wherein said second cell is a pathological cell. 178. The peptide or polypeptide of claim 179 , wherein said pathological cell is a cancer cell. 179. The peptide or polypeptide of claim 179 , wherein the pathological cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. . 183. The peptide or polypeptide of claim 181 , wherein the diseased cell is a leukemia cell. 183. The peptide or polypeptide of claim 182 , wherein the leukemia cell is an acute myeloid leukemia cell. 177. The peptide or polypeptide of claim 176 , wherein the selective and / or specific binding of the peptide or polypeptide to a second cell ligand is determined primarily by the first hypervariable region. An upstream region or a downstream wherein the binding selectivity or specificity flanks the second hypervariable region, the third hypervariable region and / or the first, second and third hypervariable regions. 177. The peptide or polypeptide of claim 176 , each affected by one or more of the flanking regions. 177. A ligand expressed by a second cell and capable of being bound by the peptide or polypeptide of claim 176 . 189. A molecule that recognizes and binds the ligand of claim 186 . 178. A nucleic acid molecule encoding a peptide or polypeptide according to any one of claims 138, 171, 173 or 176. 189. The nucleic acid molecule of claim 188 , wherein the nucleic acid is DNA. 177. The peptide or polypeptide of claim 176 , wherein the first and second states of the first cell are the same, and wherein the first cell is derived from one cell line. 190. The peptide of claim 190 , wherein the cell line is selected from the group consisting of Jurkat, Molt-4, HS-602, U937, TF-1, THP-1, KG-1, and HUT-78. Or a polypeptide. 181. The peptide or polypeptide of claim 138 or 176 , in the manufacture of a medicament, optionally associated with or attached, coupled, combined, linked to or fused to a pharmaceutical agent. use. 192. Use according to claim 192, wherein the medicament has activity against diseased cells. 194. Use according to claim 193 , wherein the pathological cells are cancer cells. 194. The use of claim 193 , wherein the cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 196. The use of claim 195 , wherein the cell is a leukemia cell. 197. Use according to claim 196, wherein the leukemia cells are acute myeloid leukemia cells. 181. The peptide or poly- m according to claim 138 or 176 for use as a medicament, optionally associated with, or attached to, coupled to, associated with, linked to or fused with a pharmaceutical agent. peptide. 199. The peptide or polypeptide of claim 198 , wherein the medicament has activity against diseased cells. 200. The peptide or polypeptide of claim 199 , wherein the diseased cell is a cancer cell. 199. The peptide or polypeptide of claim 199 , wherein the cell is selected from the group consisting of a carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 220. The peptide or polypeptide of claim 201 , wherein said cell is a leukemia cell. 202. The peptide or polypeptide of claim 202, wherein the leukemia cell is an acute myeloid leukemia cell. 178. Use of a peptide or polypeptide according to claim 138 or 176 for preparing a composition for use in inhibiting the growth of pathological or cancer cells. 204. Use of a peptide or polypeptide according to claim 204 , wherein the cell is a leukemia cell. 205. Use of a peptide or polypeptide according to claim 205 , wherein the leukemia cell is an acute myeloid leukemia cell. A composition for use in inhibiting the growth of cancer cells, the composition comprising at least one compound having a pharmaceutical ligand selective and / or specific for cancer cells. 178. Use of a peptide or polypeptide according to claim 138 or 176 . 138 or claim 138 or claim 138 , wherein the pharmaceutically effective amount of the pharmaceutical agent, optionally associated with, attached to, coupled to, associated with, or fused to the pharmaceutically effective carrier. 176. A composition comprising at least one peptide according to 176 . 210. The composition of claim 208 , wherein the peptide or polypeptide and the pharmaceutical agent are linked via a linker compound. 210. The composition of claim 209 , wherein the linker compound is selected from the group consisting of dicarboxylic acids, maleimide hydrazides, PDPH, carboxylic acid hydrazides, and small peptides. Small peptides from AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S.Tag ™, T7, V5 and VSV-G 210. The composition of claim 210 , wherein the composition is selected from the group consisting of: The tag consists of AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, protein C, S • Tag ™, T7, V5 and VSV-G. 177. The peptide or polypeptide of any one of claims 138, 171, 173, and 176 , wherein the peptide or polypeptide is selected from a group. 210. The composition of claim 208 , wherein the pharmaceutical agent is selected from the group consisting of radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anti-cancer agents. Radioisotope, indium, ¹¹¹ Indium, ¹¹³ Indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ^99m technetium, ^121m tellurium, ^122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ Thulium, ¹⁶⁸ Thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, groups of ⁶⁷ gallium and ⁶⁸ gallium 213. The composition of claim 213 , wherein the composition is selected from: 213. The composition of claim 213 , wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, lysine, and modifications and derivatives thereof. The anticancer agent is doxorubicin, adriamycin, cis-platinum, toxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide 213. The composition of claim 213 , wherein the composition is selected from the group consisting of: bleomycin and derivatives thereof. 178. A method of inhibiting cell growth comprising contacting a cell with a quantity of the peptide or polypeptide of claim 138 or 176 . 218. The method of claim 217 , wherein said cells are selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 218. The method of claim 218 , wherein the cells are leukemia cells. 220. The method of claim 219 , wherein the leukemia cells are acute myeloid leukemia cells. 181. At least one peptide according to claim 138 or 176 attached, coupled, combined, linked or fused to an imaging agent for use in diagnostic localization and imaging of tumors. Pharmaceutical composition. 177. A method of treating a patient suffering from a disease comprising administering to the patient an amount of the peptide or polypeptide of claim 138 or 176 effective to treat the disease. 222. The method of claim 222 , wherein the disease is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. 223. The method of claim 223 , wherein the disease is leukemia. 224. The method of claim 224 , wherein the leukemia is acute myeloid leukemia. 177. The peptide or polypeptide of claim 138 or 176 , wherein the Fv specifically or selectively binds to acute myeloid leukemia (AML) cells. 226. A ligand displayed on AML cells bound by the peptide or polypeptide of claim 226 . 228. A peptide or polypeptide that binds to the ligand of claim 227 . 177. For in vitro analysis of therapeutic efficacy before, during or after treatment, comprising a peptide or polypeptide of claim 138 or 176 attached, coupled, combined, linked or fused to a marker. Diagnostic kit. 230. The kit of claim 229 , wherein the marker marker molecule is a fluorescent marker. 230. The kit of claim 230 , wherein the fluorescent marker is selected from the group consisting of fluorescein, rhodamine, phycoerythrin, and modifications and conjugates thereof. 230. The kit of claim 229 for use in diagnosing a disease or cancer. 178. The peptide or polypeptide of claim 139 or 176 , wherein said construct is an Ig polypeptide. 234. The method for producing a peptide or polypeptide of claim 233 , wherein the Ig polypeptide is expressed as a recombinant polypeptide and produced in a eukaryotic cell system. 234. The method of claim 234 , wherein the eukaryotic system is a mammalian cell line. 233. The peptide or polypeptide of claim 233 , wherein said Ig polypeptide is an IgG polypeptide. 237. The peptide or polypeptide of claim 236 , wherein the IgG polypeptide comprises CDR3, CDR2 and CDR1 regions having SEQ ID NOs: 8, 115 and 114, respectively. 237. The peptide or polypeptide of claim 236 , wherein the IgG polypeptide comprises CDR3, CDR2 and CDR1 regions having SEQ ID NOs: 20, 115 and 114, respectively. 237. The IgG polypeptide of claim 237 , wherein the CDR3, CDR2 and CDR1 regions are heavy chain regions. 239. The IgG polypeptide of claim 238 , wherein the CDR3, CDR2 and CDR1 regions are heavy chain regions. 237. The IgG polypeptide of claim 237 , wherein the CDR3, CDR2 and CDR1 regions are light chain regions. 239. The IgG polypeptide of claim 238 , wherein the CDR3, CDR2 and CDR1 regions are light chain regions. 233. The IgG polypeptide of claim 233 , wherein the IgG has a heavy chain comprising SEQ ID NO: 26 and a light chain comprising SEQ ID NO: 27 or a chain having at least 90% amino acid similarity thereto. 180. The method of producing a peptide or polypeptide of claim 139 or 176 , wherein the peptide or polypeptide is produced in a prokaryotic or eukaryotic cell system. 265. The method of claim 244 , wherein the prokaryotic system comprises E. coli comprising an expression vector and the eukaryotic system is a mammalian cell line. Expression vectors prokaryotic systems, osm B, deo, β- lac-U5, comprising a promoter selected from the group consisting of .lambda.P _L and CMV, The method of claim 245. CDR3 comprises the amino acid sequence of R ₁ -XPhe Pro-R ₂ , wherein R ₁ and R ₂ each comprise from 0 to 15 amino acid residues, and X is any of Arg, Gly, or Lys; 178. A peptide or polypeptide according to any one of claims 138 or 176 . 177. The peptide or polypeptide of claim 138 or 176 , wherein said peptide or polypeptide includes at least one non-naturally occurring modification. 249. The peptide or polypeptide of claim 248 , wherein said non-naturally occurring modification renders the peptide or polypeptide more immunogenic or more stable. Wherein the at least one modification, peptide modifications, semipeptoids modified, cyclic peptide modification, N- terminus modification, C terminus modification, peptide bond modification, are selected from the group consisting of backbone modifications, and residue modification, claim 249. The peptide or polypeptide according to 249 . 199. A peptide or polypeptide according to any one of claims 138, 171, 173, 176 or 198 for ex vivo purging of autologous bone marrow to remove abnormal cells. A-X-B
Wherein X is a hypervariable CDR3 region comprising SEQ ID NO: 3 or 20; and A and B may each be an amino acid chain of 1-1000 amino acids in length. Wherein A is the amino terminus and B is the carboxy terminus. 252. The peptide of claim 252 , wherein A is 150-250 amino acid residues and B is 350-500 amino acid residues. 250. The peptide or polypeptide of claim 253 , which is part of a larger or intact antibody or multimer. 258. A dimeric molecule comprising two peptides or polypeptides, one of which is the peptide or polypeptide of claim 252 . 258. A dimeric molecule comprising two peptides or polypeptides of claim 252 that are identical. 257. A nucleic acid molecule encoding the peptide or polypeptide of claim 252 or the dimer molecule of claim 254 . 235. The method of claim 235 , wherein the mammalian cell line comprises the SRα5 promoter. 235. The method of claim 235 , wherein the mammalian cell line comprises a CMV promoter.

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