JP3683278B2 - Mouse monoclonal anti-idiotype antibody 11D10 and method of use thereof - Google Patents

Description

別の改変において、推定11D10活性を有する試験フラグメントを、Ab1とAb2との間、またはAb1とHMFGとの間の結合を妨げる能力について試験する。この試験はいくつかの適用においてより感受性であり得る。何故ならば、11D10とAb1との間のより低いアフィニティー相互作用は非常に弱くて安定な結合を形成できないが、十分な濃度で存在させた場合は、別のリガンド−レセプター対の結合を妨げるのに十分であるからである。HMFGは精製された抗原またはHMFG発現細胞として提供され得る。アッセイは、Ab1またはHMFGまたはAb2いずれかを標識し、分離を容易にするために必要に応じてリガンド−レセプター対の他のメンバーを固体支持体に固定化させることによって行われ得る。試験フラグメントを標識試薬と共にインキュベートし、次いで、混合物を固定化標的または試験細胞に提示して、試験フラグメントが結合を阻害し得るかを測定する。阻害の程度は11D10活性に相関する。
競合アッセイの種々の例は実施例のセクションで後記する。11D10ポリペプチド活性を示す１つの試験は、種々の量の11D10ポリペプチドの存在下で、放射性標識Ab1(MC-10)の半精製もしくは精製HMFGへの結合を測定することである。例えば、実施例１を参照のこと。次いで、Ab1-HMFG混合物を11D10ポリペプチドで被覆したプレートに添加し、そして結合を標識Ab1単独の結合と比較する。好ましくは、この試験は標識Ab1の非飽和量で行って、少量の競合HMFGでの結合の変化を検出する。インタクトな11D10で行ったこの試験の例を実施例１に示す。別の競合アッセイにおいて、（MCF-7またはSKBR3のような）HMFGポジティブ標的細胞をコンフルエントな単層として96ウェル組織培養プレート中で増殖させる。11D10ポリペプチドの非存在下および存在下での放射性標識Ab1(MC-10)の結合を測定する。阻害の程度は、インタクトな11D10または他の11D10ポリペプチドの阻害と比較され得る。インタクトな11D10を用いるこの競合アッセイの例を実施例１に示す。11D10 scFvとインタクトな11D10との間の阻害の程度を比較するこのアッセイの別の例を実施例８に示す。
Ab1に結合しない非関連の抗イディオタイプ抗体のようなネガティブコントロールと比較する場合に阻害があれば、11D10ポリペプチドはAb1に結合すると考えられる。
上記の全てのアッセイを用いれば、標識分子を、放射性同位体（すなわち、¹²⁵I）を用いる、ならびにビチオン化分子、および酵素検出のための分子、蛍光標識および化学発光標識のような非放射性標識を用いるような種々の方法で標識し得ることが当業者に明らかである。
また、上記試験を用いて、種々の11D10ポリペプチドフラグメントの特徴を比較し得る。例えば、競合アッセイを行うことができ、ここで、第１の11D10ポリペプチドが、種々の量の第２の11D10ポリペプチドの存在下で、Ab1(MC-10)への結合に対して競合する。このような試験は、結合アフィニティーまたは他の特徴の相対的程度を示し得る。
11D10ポリペプチドを特徴付ける別の方法は、免疫応答を生じるそれらの能力を試験することである。本明細書で用いる「免疫応答」とは体液性応答、細胞性応答、またはその両方のいずれかを示す。本明細書で用いる「免疫応答を誘発する能力」は、ヒトを含むいずれかの個体に関する。
11D10ポリペプチドが体液性応答を生じる能力は、11D10ポリペプチドの投与後に11D10ポリペプチドに結合する抗体の存在を試験することによって測定し得る。この抗体（Ab3）は、11D10ポリペプチドの投与前には存在しないか、あるいは低量で存在したことが理解される。免疫原性は、好ましくは、先の抗11D10応答を伴わない個体で試験される。適切な個体の例は限定されるものではないがマウス、ウサギ、サルおよびヒトを含む。この試験のために、個体に11D10ポリペプチドを投与する。投与当たりの量および投与回数は、個体に応じて変化する。インタクトな11D10を用いる本発明者らの先の実験に基づくと、マウスでは、用量および３回の投与当たりCFAおよびIFAの存在下で、KLHにカップリングした11D10ポリペプチドの約100μgを要した。サルはほぼ２mgを要する。本発明の目的のために、ヒトに投与され得る11D10ポリペプチドの範囲は、約10μg〜10mg、好ましくは100μg〜10mg、好ましくは500μg〜８mg、より好ましくは１mg〜４mg、なおより好ましくは約２mgである。
Ab3の存在は、まず、血清をオートロガスな免疫グロブリンと共にプレインキュベートしてイソタイプおよびアロタイプ抗原決定基に対する抗体をブロックし、次いで、例えば、ELISAまたはRIAを用いて、HMFGおよび／または11D10ポリペプチドに対する結合について血清を試験することによって決定され得る。例えば、予め反応させた血清の異なる希釈をマイクロタイタープレート上に被覆した11D10（または11D10ポリペプチド）と反応させる。非関連Ab2をコントロールとして供する。洗浄の後、例えば、同種サンドイッチアッセイにおいて¹²⁵I−標識11D10を用いて、Ab3−11D10複合体を標識する。このアッセイからの結果を11D10ポリペプチドの投与前に得られた結果と比較する。マウスにおいてインタクトな11D10によって誘発されたAb3のこのような検出用アッセイのより詳細な記載を実施例１に記載する。あるいは、ヒト大腸癌腫LS174-T細胞のようなHMFGポジティブ細胞への結合が免疫フローサイトメトリーを用いて試験され得る。
HMFGに対するAb3の結合はまた、HMFGポジティブ組織サンプルでの免疫沈降もしくはそれとの免疫反応性、またはドットブロット分析によって決定され得る。ドットブロット分析の一つの方法において、HMFGの半精製抽出物をニトロセルロースフィルターに直接ブロットする。次いで、フィルターを、Ab3を含有する血清と共にインキュベートし、酵素結合抗免疫グロブリンによって反応を発現させる（実施例１）。もしAb3がHMFGに結合すれば、ポジティブなブロットが出現するはずである。組織サンプルでの試験には、イムノペルオキシダーゼアッセイが使用され得る（実施例１）。
所望ならば、11D10ポリペプチドによって誘発されたAb3はさらに特徴付けされ得る。例えば、競合アッセイが、Ab3がAb1イディオトープを共有するか否かを決定するために行われ得る。この試験では、11D10ポリペプチドで免疫化した個体由来の血清を、標識11D10ポリペプチド（またはインタクトな11D10）のAb1への結合の阻害について試験する。阻害はAb3およびAb1が少なくとも類似の結合決定基を含有することを示す。同様に、（部分的に精製した、精製した、またはHMFGポジティブ細胞の表面上にあるを問わず）HMFGに対する結合についてのAb3とAb1との競合は、固定量の標識Ab1(MC-10)を、Ab3含有血清またはAb1調製物およびHMFG（または、MCF-7またはSKBR3のようなHMFG関連細胞）の種々の希釈物と共に同時インキュベートすることによって試験され得る。これらの試験を実施例１においてインタクトな11D10について説明する。
当業者に明らかにように、上記したアッセイを使用し、Ab3は、順に11D10ポリペプチドを特徴付けるために用いられ得る。
11D10ポリペプチドを特徴付ける別の方法は、細胞傷害性である抗体を誘発するその能力を試験することによる。補体が媒介する細胞傷害性（CMC）の決定のために、SKBR3（標的）細胞（すなわち、HMFGを発現する細胞）を⁵¹Crで標識する。標識は、約10⁶細胞をほぼ200μCiのNa₂SO₄と共に37℃で60分間インキュベートし、続いて洗浄することによって達成され得る。このアッセイは、抗体を含有すると考えられる血清を添加し、そしてインキュベートすることによって行なわれる。次いで、LS174-T細胞で予め吸着されたモルモット血清（または他の補体源）を添加する。37℃における適切なインキュベーション時間の後、次いで、⁵¹Cr放出の程度を測定し、そして未オプソニン化コントロール細胞の⁵¹Cr放出の程度と比較する。⁵¹Crの放出はCMC活性と相関する。Herlynら(1981)Int.J.Cancer 27:769。
11D10ポリペプチドを特徴付ける別の方法は、ADCC応答に関与する抗HMFG抗体を誘発するその能力を試験することによる。Chereshら(1986)Cancer Research 46:5112-5118。このアッセイでは、培養ヒトMCF-7またはSKBR3細胞（すなわち、それらの表面でHMFGを発現する細胞）が⁵¹Crで標識され、標的細胞として使用される。正常ヒト末梢血単核細胞（PBMC）がエフェクター細胞として使用される。好ましくは、ADCCアッセイは、100：１のエフェクター：標的細胞比にて、熱不活化血清の存在下で４時間行われるが、他の適切な条件が用いられ得る。次いで、⁵¹Cr放出の量を測定する。
本発明の11D10ポリペプチドはまた、細胞性応答を誘発するそれらの能力によって特徴付けられ得る。本明細書で用いる「細胞性応答」とは、Ｔ細胞が関与する応答であり、そしてインビトロまたはインビボで観察され得る。
細胞性免疫応答を検出する１つの方法は、Ｔ細胞増殖活性についてアッセイすることによる。この試験では、細胞性免疫応答を、11D10ポリペプチドと共にインキュベートした末梢血単核細胞（PBM）の増殖によって測定する。11D10ポリペプチドの所要数の投与後に末梢血単核細胞を血液から単離し、そして種々の濃度の11D10ポリペプチドと共にインキュベートする。マウスを用いる場合、Ｔ細胞は脾臓から得られる。Ｔ細胞は、例えば、Ficoll^TMのようなグラジエント上の遠心分離によって富化され得る。PHAのような非特異的マイトジェンをポジティブコントロールとして供する：非関連抗イディオタイプ抗体とのインキュベーションをネガティブコントロールとして供する。好ましくは、刺激細胞は、特に組織適合性Call II抗原に関して、応答細胞とオートロガスである。PBMを適切な日数の間インキュベートさせることにより増殖させた後、［³Ｈ］チミジン取込みを測定する。多くの場合、適切な時間は５日間である。所望ならば、いずれのＴ細胞サブセットが増殖するかの決定がフローサイトメトリーを用いて行われ得る。必要に応じて、脾臓Ｔ細胞は、モノクローナル抗体RL.172（抗CD4⁺）またはmAb.168（抗CD8⁺）および補体とのインキュベーションによって、増殖アッセイ前にCD4⁺またはCD8⁺細胞いずれかを予め枯渇され得る。
細胞性免疫応答を検出する別の方法は、Ｔ細胞の細胞傷害性（CTL）活性について試験することである。この試験では、Ｔリンパ球（すなわち、富化Ｔ細胞集団）を、標準的⁵¹Cr放出アッセイにおいて標的として用いるために、（典型的には、脾臓細胞から）単離する。Kantorら(1992)J.Natl.Cancer Inst.84:1084-1091。⁵¹Cr放出アッセイの例は以下の通りである。略言すると、HMFGポジティブ腫瘍細胞（典型的には、１〜２×10⁶細胞）を標的細胞として約200μCiのNa₂ ⁵¹CrO₄(Amersham Corp.,Arlington Heights,Ill)で37℃で60分間放射性標識し、続いて、徹底的に洗浄して取り込まれなかった同位体を除去する。次いで、共に培地に再懸濁したＴ細胞および標的（１×10⁴／ウェル）を、96ウェルＵ底プレート(Costar Corp.)中、種々のエフェクター：標的比にて組み合わせる。プレートを100×ｇで５分間遠心して細胞接触を開始させ、そして５％CO₂にて37℃で４または16時間インキュベートする。インキュベーション後、上清をSupernatant Collection System(Skatron,Inc.,Sterling,VA)を用いて収集し、そして放射能をガンマカウンター(Beckman Instruments)で定量する。⁵¹Crの自然放出をエフェクターの非存在下における標的のインキュベーションによって決定し、他方、⁵¹Crの最大または全放出を0.1％Triton X-100中における標的のインキュベーションによって決定する。⁵¹Crの特異的放出パーセントは以下の式によって決定する。
特異的放出パーセント＝［（実験−自然）/（最大−自然）］×100
11D10ポリペプチドを特徴付ける別の方法は、HMFG関連腫瘍の進行を改善し、遅延させ、および／またはその程度を減少させるそれらの能力を試験することである。このような試験は炎症インジケーター、ラジオシンチグラフィー、または循環HMFGレベルの測定を含み得る（このようなアッセイは商業的に入手できる）。
11D10ポリペプチドの使用および使用する方法
11D10ポリペプチドは多数の用途を有する。11D10ポリペプチドは、個体における免疫応答、好ましくは抗HMFG応答を誘導するために用いられ得る。それらはまた、Ab3のレベルを検出およびモニターするため、またはAb3を精製するために用いられ得る。11D10ポリペプチドはまた、例えば、結腸直腸ガン、ある種の肺ガン（腺ガン）、胃ガン、膵ガン、およびある種の乳ガンのようなHMFG関連疾患の処置に有用である。
このように、本発明は、免疫応答を誘導するのに有効な量で11D10ポリペプチドを投与する工程を包含する、個体において免疫応答を誘導する方法を含む。好ましくは、個体はHMFG関連腫瘍を有する。この意味では、「有効量」とは、体液性および／または細胞性を問わず、測定可能な免疫応答を誘発するのに十分な量である。有効量は１回またはそれ以上の投与で投与され得る。
本発明はまた、生物学的サンプル中の11D10と結合する抗体（すなわち、Ab3および／またはAb1）を検出する方法を含む。これらの方法は、例えば、個体中のAb1またはAb3レベルをモニターするための臨床状況において、ならびにAb3の市販生産が望まれる産業状況において適用可能である。これらの方法は、サンプル中のAb3および／またはAb1を11D10ポリペプチドと、Ab3および／またはAb1と11D10ポリペプチドとの間の安定な複合体の形成を可能とするのに適切な条件下で接触させ、もしあれば形成された安定な複合体を検出することを要する。多数の免疫アッセイ方法が当該分野で公知であり、そして本明細書中に記載されている。さらなる例示のために、潜在的にAb3および／またはAb1を含有する試験サンプルが、典型的には（例えば、放射性同位体または酵素で）検出可能に標識した11D10ポリペプチドの所定の非限定量と混合され得る。液相アッセイにおいては、未反応試薬は、濾過またはクロマトグラフィーのような分離技術によって除去される。これらの免疫アッセイ技術において、複合体と会合した標識の量は、サンプル中に存在するAb3および／またはAb1の量と正に相関する。同様のアッセイが設計され得る。そこでは、試験サンプル中のAb3および／またはAb1が、限定量の11D10ポリペプチドへの結合について標識抗体と競合する。ここでは、標識の量は、サンプル中のAb3および／またはAb1の量と負に相関する。Ab3および／またはAb1レベルを測定するための適切なサンプルは生物学的サンプルであり、血清または血漿を含み、好ましくは血清である。他のサンプルは組織サンプルを含む。
さらに、本発明はまた、Ab3（および／またはAb1）を含有する生物学的サンプルを11D10ポリペプチドと接触させる工程、およびもしあればそれにより形成された複合体を得る工程を包含する、Ab3（またはAb1）を精製する方法を含む。典型的には、11D10ポリペプチドを、アフィニティーカラム精製のためにアフィニティーマトリックスにカップリングさせる。このような方法は当該分野では日常的であり、本明細書中に詳細に記載する必要はない。
また、本発明には、有効量の11D10ポリペプチドを投与する工程を包含する、HMFG関連腫瘍のようなHMFG関連疾患を処置する方法が含まれる。「HMFG関連腫瘍」は、HMFG、特に腫瘍細胞の表面で発現されたHMFGを含有する腫瘍であり、その例は上記されている。この意味では、処置のための有効量は、疾患状態を緩和するのに十分な量である。有効量は、１回または１回より多い投与にて与えられ得る。有効量の11D10ポリペプチドでの個体の処置は、例えば、そのように処置されない個体と比較して、疾患の進行の速度を減少させ得る。
別の実施態様において、HMFG関連疾患を有する個体においてＴ細胞応答を刺激する方法が提供される。このＴ細胞応答は、11D10ポリペプチド、特にHMFGに対して相同である11D10ポリペプチドを用いる、Ｔ細胞の増殖および／または細胞傷害性Ｔ細胞活性の促進として現れ得る。11D10ポリペプチドは、（ポリペプチドまたは11D10ポリペプチドをコードするポリヌクレオチドを含有するプラスミドのいずれかとして）直接に投与され得るか、あるいは適切な細胞のエクスビボ培養に添加され得る。11D10ポリペプチドは、例えば、所望のＴ細胞活性を刺激するのに有効な量で、単離された末梢血単核細胞に添加される。次いで、刺激されたＴ細胞が個体に再導入される。添加される11D10ポリペプチドの量は、個体の状態、従前および／または併用の処置手順、および使用される他の物質のようないくつかの要因に依存する。
本発明のポリペプチドは単独でまたは所望の活性／目的を促進する他の因子と併用して使用され得る。11D10ポリペプチドはまた、相互に種々に組み合わせて使用され得る。この意味では、「因子」とは種々の物質のいずれでもあり得る。さらに、「併用して」とは、因子がポリペプチドと同時に、その前に、またはその後に使用され得ることを意味する。因子はまた、融合タンパク質のようなポリペプチドに共有結合され得るか、またはポリペプチドに物理的に近接し得る。所望の活性は、11D10ポリペプチドの使用における所望の目的を容易にし、増強し、促進し、または調節するいずれの活性でもある。
使用され得る因子は、サイトカイン、リンホカイン、アジュバント、および薬物を含むが、これらに限定されない。因子はまた、リポソームのようなポリペプチドの送達を容易とする物質、またはポリペプチドの、特定の標的、例えば、細胞レセプターへの送達を促進する物質を含む。例えば、１つ以上の11D10ポリペプチドが、GM-CSFのようなサイトカインもまた含有する融合タンパク質として生産され得る。あるいは、１つ以上の11D10ポリペプチドは、GM-CSFのようなサイトカインと共に投与され得る。
本発明はまた、例えば、ラジオシンチグラフィーまたは放射線療法のために、標識抗HMFG抗体（Ab1）を受容した個体から標識（例えば、放射能）を除去するために11D10ポリペプチドを使用する方法を含む。本発明はまた、放射性標識抗HMFG抗体を治療用量で投与し、続いてモル過剰の11D10ポリペプチドを投与する方法を包含する。
本発明の目的のための11D10の使用は、上記で議論され、そして同様にそれらの原理が11D10ポリヌクレオチドに適用される。腫瘍部位に局在化されないいずれの抗HMFGをも位置決定し結合させるために、標識抗HMFGよりも十分にモル過剰である11D10ポリペプチドの量が選択される。投与のタイミングおよび11D10ポリペプチドの量は、放射性標識抗体の性質、使用される放射性同位体のタイプ、および個体の状態に依存する。好ましくは、11D10ポリペプチドと抗HMFG抗体とのモル比は、少なくとも約５：１、より好ましくは約25：１〜200：1である。好ましくは、11D10ポリペプチドは、個体が抗HMFG抗体を受容した５〜24時間後に投与される。
抗HMFG抗体に結合した11D10ポリペプチド（特に、MC-10）について、腫瘍細胞表面上の抗HMFGの検出は、腫瘍細胞と11D10ポリペプチドとを抗HMFG抗体への結合を可能にするのに十分な時間接触させ、そして抗HMFG抗体に結合したいずれかの11D10の存在を検出することにより達成され得る。実験的なパラメータの開発（例えば、11D10のポリペプチドの量または反応時間）は、十分に当該分野の技術内である経験的な決定である。
11D10、11D10ポリヌクレオチドおよび／または11D10ポリペプチドを含む薬学的組成物およびワクチン
本発明は、11D10、11D10ポリヌクレオチドおよび／または11D10ポリペプチドを含有する薬学的組成物およびワクチンを含む。このような薬学的組成物／ワクチンは、免疫応答を誘発するのに、および／または乳ガンのようなHMFG関連疾患の処置に有用である。薬学的組成物／ワクチンは、単独で、または化学療法または放射線治療のような他の形態の治療と併用してのいずれかで、HMFG関連疾患を緩和または改善し得る。薬学的に受容可能な賦形剤において有効量の11D10、11D10ポリヌクレオチドおよび／または11D10ポリペプチドから成るこれらの薬学的組成物は、単位投与量形態、滅菌非経口溶液または懸濁液、滅菌の非経口以外の溶液または経口溶液または懸濁液、水中油型または油中水型エマルジョンなどでのヒトおよび動物への全身投与に適する。処方または非経口および非経口でない薬物送達は当該分野で公知であり、そしてRemingtons' Pharmaceutical Sciences第18版，Mack Publishing(1990)に記載されている。
薬学的に受容可能な賦形剤は、薬理学上有効な物質の投与を容易とする比較的不活性な物質である。例えば、賦形剤は、ワクチン組成物に形態および粘稠度を与え得、あるいは希釈剤として作用し得る。適切な賦形剤は、安定化剤、湿潤剤および乳化剤、浸透圧を変化させるための塩、カプセル剤、緩衝液、および皮膚浸透促進剤を含むが、これらに限定されない。薬学的に受容可能な賦形剤の例はRemingtons' Pharmaceutical Sciences(1990)、前掲に記載されている。
１つの実施態様において、11D10ポリペプチドを含有する薬学的組成物は、例えば、個体由来の末梢血単球（PBM）のエクスビボ培養を刺激するために用いられる。次いで、PBMを個体に再導入する。薬学的組成物は、単独でまたはリンホカインのような他の生体応答改変因子と組み合わせて用いられる。
１つのタイプの薬学的組成物はワクチンである。従って、本発明はまた、有効量の11D10、11D10ポリヌクレオチド、11D10ポリペプチド、またはそれらの組合せおよび薬学的に受容可能な賦形剤を含むワクチンを含む。これらのワクチンは、特に、個体（特に、HMFG関連腫瘍のような進行性のHMFG関連疾患を有する個体）の免疫応答を誘発するために使用され得る。好ましくは、免疫応答は、ヒト乳脂肪球抗体の産生を含む。これらのワクチンはHMFG関連疾患の処置、調節、および／または緩和に特に有用である。
11D10を含むワクチンの投与は上記で議論した。
上述した11D10ポリヌクレオチドを含有するワクチンは、いわゆる「遺伝子免疫化」またはDNAワクチンのために使用され得、ここでは、抗原ポリペプチドをコードするポリヌクレオチドが、保護的免疫応答を誘発するために、宿主細胞に導入される。Tangら(1992)Nature 356:152-154。一旦細胞核に入ったら、プラスミドは環状の非複製エピソームとして持続し得、これは用量依存的かつ長期の発現に導く。Spoonerら(1995)Gene Therapy 2:173-180。ポリヌクレオチドを用いる免疫化が細胞性応答ならびに体液性応答を生じることが示されている。Spoonerら(1995);Wangら(1995)Human Gene Therapy 6:407-418。遺伝子免疫化は、感染の危険がなく免疫応答を誘発するビヒクルとしての生微生物または弱毒微生物の多くの利点を有する。
好ましくは、11D10ポリヌクレオチドは、プロモーター、エンハンサーおよびシグナル配列のような転写および翻訳のための適切な制御配列を含有するプラスミドベクターとして導入される。１またはそれ以上の11D10ポリヌクレオチドが単一クローニングベクター内で使用され得、および／または複数のベクターが使用され得る。複数の11D10ポリヌクレオチドが使用される場合、それらはベクター内にインフレームで挿入されるか、または別々のプロモーターの制御下に置かれるべきである。使用される11D10ポリヌクレオチドの長さおよび／またはタイプは変化し得、そしてワクチン投与の臨床目的、個体の状態、および個体の免疫学的プロフィールのようないくつかの要因に依存する。さらに、免疫応答を増強し、容易とし、および／または増加させる他の物質をコードするポリヌクレオチドもまた、ベクターに挿入され得る。GM-CSFのようなこのような物質の例は上述した。
例えば、１つの実施態様においては、11D10のscFvをコードするポリヌクレオチドが上記発現ベクター（プラスミド）の１つに挿入される。別の例においては、図19に示される11D10フラグメントをコードするポリヌクレオチドが、ワクチンとして投与するために発現ベクターに挿入される。別の例においては、11D10の免疫原性フラグメントをコードするポリヌクレオチドが、発現ベクターに挿入される。
11D10ポリヌクレオチドを使用する別のタイプのワクチンは、いわゆる発現ライブラリー免疫化であり、ここでは、（11D10の種々の部分をコードする）11D10ポリヌクレオチドの発現ライブラリーを用いて宿主を免疫化する。Barryら(1995)Nature 377:632-635。得られた複数部分に分かれた非感染性ワクチンは、可能な免疫原として複数のペプチドを提示するので、特に有用であることが判明し得る。複数の免疫原の提示は、それを投与する各特定の宿主（すなわち、個体）が免疫学的に有効なポリペプチドを選択し得るというさらなる利点を有し、それは個体間で変化し得る。11D10ポリペプチドの発現で用いる発現ライブラリーは包括的（すなわち、全11D10分子を集合的にコードする）であり得るか、あるいは部分的であり得る。免疫化用の発現ライブラリーは、適切なベクター系を用い、上述した一般的組換え方法によって作製される。典型的には、11D10ポリヌクレオチドは分泌を媒介するシグナル配列とインフレームに融合される。
投与すべき11D10ポリヌクレオチドの量は、投与の様式および経路（すなわち、直接的注射−対−エクスビボ培養およびトランスフェクション）、11D10ポリヌクレオチドによりコードされる11D10ポリペプチド、（免疫学的および／または疾患状態のような）個体の状態、および所望の目的のようないくつかの要因に依存する。典型的には、直接的に投与する場合、投与当たりの量は約10μg〜１mg、好ましくは25μg〜500μg、より好ましくは30μg〜250μg、さらにより好ましくは50〜100μgである。
別の実施態様において、11D10ポリヌクレオチドは、ワクチン処方のためのコードされた11D10ポリペプチドを発現できる生ウイルスまたは弱毒化ウイルスまたはウイルスベクターにおいて用いられる。例として、アデノウイルス、アデノ随伴レトロウイルス（AAV）、およびSV40が挙げられるが、これらに限定されない。好ましくは、ウイルスはワクシニアである。組換えワクシニアウイルスは、免疫原性ウイルス粒子と共に11D10ポリヌクレオチドによってコードされる11D10ポリペプチドを有効に共提示するための強力な因子を提供し得る。ワクシニアウイルスベクターの構築は上述された。一般に、組換えウイルスベクターを宿主細胞のインビボ感染を行うのに十分な量で添加する。この量は使用するウイルスのタイプ、コードされる11D10ポリペプチドの性質、個体の状態、および所望の結果に依存する。（11D10ポリペプチドまたはscFvのような11D10ポリペプチドを含有する11D10変異体をコードし得る）組換えワクシニアは、用量当たり約10⁷〜10⁸プラーク形成単位でワクチン接種のために直接使用され得る。ワクシニアは、非経口的に(例えば、皮下または筋肉内注射によって)、ならびに粘膜を介して（例えば、鼻的に）、経口的に、または吸入によって投与され得る。あるいは、ワクシニアは、ワクシニア感染細胞を介して投与され得る。この技術において、腫瘍細胞のような適切な細胞を培養中のワクシニアで感染させる。次いで、感染細胞を個体に再導入する。ワクシニアで細胞を感染させる方法およびそれらの感染細胞を再導入する方法は記載されている。例えば、Moss(1990)参照のこと。
ワクチンはまた、１以上の11D10ポリペプチドから調製され得る。11D10ポリペプチドは、上述の方法のうちのいずれかによって、特に適切な発現ベクターからの精製によって調製され得る。１つの実施態様において、ワクチンは、１以上の11D10ポリペプチドを含む。11D10ポリペプチドは、中性もしくは塩形態としてワクチンに処方され得る。薬学的に受容可能な塩は、（11D10ポリペプチドの遊離アミノ酸で形成された）酸付加塩を含み、これは、例えば、塩酸またはリン酸のような無機塩、あるいは酢酸、シュウ酸、酒石酸、マレイン酸などのような有機酸で形成される。また、遊離カルボキシル基で形成された塩はまた、例えば、水酸化ナトリウム、水酸化カリウム、水酸化アンモニウム、水酸化カルシウム、または水酸化第二鉄のような無機塩基、およびイソプロピルアミン、トリエチルアミン、２−エチルアミノエタノール、ヒスチジン、プロカインなどのような有機塩基から誘導され得る。
別の実施態様において、ｂ型肝炎表面抗原のようなウイルス粒子に融合させた11D10ポリペプチドを含有するワクチンが提供される。
活性成分として11D10ポリヌクレオチドまたはポリペプチドを含有するワクチンの調製は、当該分野における標準的な実施を含む。典型的には、このようなワクチンは注射剤として、液体溶液または懸濁液のいずれかとして調製される；注射に先立って液体中の溶液または懸濁液に適切な固体形態もまた調製され得る。ワクチンはまた乳化され得るか、あるいは11D10ポリペプチドおよび／またはポリヌクレオチドはリポソームと会合され得る。
ワクチン中の11D10、11D10ポリペプチドおよび／または11D10ポリヌクレオチドはそのまま使用され得るが、しばしば、薬学的に受容可能な賦形剤と混合される。適切な賦形剤は、例えば、水、生理食塩水、生理学的緩衝化生理食塩水、デキストロース、グリセロール、エタノール、およびそれらの組合せである。所望ならば、ワクチンはまた、湿潤剤または乳化剤、pH緩衝剤、安定化剤、および／またはアジュバントのような少量の補助物質を含有し得る。アジュバントの例は、上述した。獣医学的用途および動物における抗体の生産のために、フロイントのアジュバントの分裂促進成分が使用され得る。アジュバントの選択は、特にヒトに使用することを意図する場合は、部分的に、アジュバントの存在下におけるワクチンの安定性、投与経路、およびアジュバントの調節受容性に依存する。例えば、ミョウバンは、ヒトにおけるアジュバントとしての使用について米国食品医薬品局（FDA）によって認可されている。11D10ポリヌクレオチドを含有するワクチンを用いて免疫応答を増強させるために、カチオン性脂質中のカプセル化が使用され得る。11D10ポリペプチドの送達のために、リポソーム中へのカプセル化もまた適し得る。細胞への送達のために、ポリヌクレオチドおよび／またはポリペプチドをパッケージングするのに適したリポソームは、当該分野で公知である。
11D10ポリペプチドは、特に11D10ポリペプチドが100アミノ酸またはそれ未満のアミノ酸を含む場合、その免疫原性を増強させるために、必要に応じて化学的に処理され得る。このような処理は、例えば、グルタルアルデヒドでの架橋；キーホールリンペットヘモシアニン（KLH）または破傷風トキソイドのようなタンパク質キャリアへの連結を含み得る。
最適下の免疫応答が本発明のワクチンによって誘導されたサプレッサーＴ細胞に起因すると推定される場合、シクロホスファミド（100mg/kg体重）もまた腹腔内投与され得る。
本発明のワクチンは、典型的には、例えば、皮下、筋肉内、腹腔内、または皮内のいずれかで、注射によって非経口的に投与される。投与はまた、鼻孔内、肺内（例えば、エアロゾルによる）、経口、および静脈内であり得る。他の投与様式に適したさらなる処方は、坐薬、およびある場合には経口処方を含む。投与経路は、処置すべき個体の状態および所望の臨床的効果に依存する。
投与は、免疫応答（体液性および／または細胞性）の誘発のような、所望の測定可能なパラメーターが検出されるまで一週間毎または二週間毎のベースで開始し得る。投与は、次いで、二週間毎または一カ月毎のようなより少い頻度ベースで継続され得る。11D10を含むワクチンについて、好ましくは、投与は、最初の４回の投与は２週間毎に与えられ、続いて１カ月毎に与えれる。
ワクチンは、投与処方に適合する様式で、予防的および／または治療的に有効となるような量で投与される。投与すべき量は、処置すべき個体、抗体を合成する個体の免疫系の能力、投与経路、および所望の保護の程度に依存する。投与するために必要な活性成分の正確な量は、実施者の判断に依存し得、そして個体に特有であり得る。11D10、11D10ポリヌクレオチドおよび11D10ポリペプチドについての一般投与量範囲は上述に示した。
典型的には、ワクチンは一連の用量にて投与され、免疫応答を初回刺激するための一群の用量で開始し、続いてより間隔をあけた「維持」用量で行う。例えば、ワクチンは免疫応答を確立させるために一週間毎ベースで、続いて応答を維持するために二週間毎または一月毎の注射で投与され得る。
ワクチン中の11D10ポリペプチドおよび／または11D10ポリヌクレオチドは、単独で、他の11D10ポリペプチドおよび／またはポリヌクレオチドと組み合わせて、インタクトな11D10と組み合わせて、ならびに／あるいは所望の効果を増強し、容易とし、または調節する、リンホカインおよび薬物のような他の物質と組み合わせて与えられ得る。このような物質の例は上述した。11D10ポリペプチドは溶液中の11D10ポリペプチドの混合物を調製するか、または融合タンパク質を合成することによって組み合わせられ得る。
本発明のワクチンはまた、HMFGをコードするポリヌクレオチドまたはそのフラグメントを含有する組換えワクシニアおよび／あるいはGM-CSFのようなリンホカインをコードするポリヌクレオチドを含有する組換えワクチンと併用して投与され得る。さらに、本発明のワクチンは、確立されているかまたは実験的であるかを問わず、他の治療様式と併用して使用され得る。このような使用は、例えば、化学療法または放射線療法のような、他の治療様式の投与のみと比較して、ワクチンの投与が臨床的結果を改善する場合に示される。
11D10ワクチンの免疫原性は、Ab3のレベルを測定しおよび／または疾患状態をモニターすることによってモニターされ得る。RIAまたはELISAを用いるAb3の検出および測定ならびにＴ細胞活性（すなわち、増殖および／または細胞傷害活性）の測定は上述した。一例として、Ab3は以下のように定量され得る。マイクロタイタープレートをMC-10（Ab1）で被覆し、そして固定量の125Ｉ−標識11D10ポリペプチドと反応させる。阻害剤として精製MC-10を用いて標準阻害曲線を作成する。異なる希釈の血清を、Ab1-Ab2反応を阻害する能力について試験し、そして血清中のAb3の量を標準阻害曲線から見積もる。あるいは、Ｔ細胞応答は、上述したアッセイのうちのいずれかを用いてモニターされ得る。疾患状態は、腫瘍関連マーカー、Ｘ線、CTスキャン、および他の測定可能な臨床的発現の測定のような当該分野における標準的技術を用いてモニターされ得る。
本明細書中に記載したものに限定されない多数の代替的なワクチン組成物が免疫反応を誘導するのに効果的であり得ることが認識される。全てのこのような組成物は、それらが11D10ポリヌクレオチドまたはポリペプチドを活性成分として含むのであれば、本発明の範囲内で具体化される。
11D10、11D10ポリヌクレオチドおよび／またはポリペプチドを含むキット
本発明はまた、11D10、11D10ポリヌクレオチドおよび／またはポリペプチドを含有するキット（好ましくは、診断キット）を含む。本発明の11D10、11D10ポリヌクレオチドおよび／または11D10ポリペプチドを用いる診断手順は、診断研究所、実験研究所、実施者、または私的な個人によって行われ得る。本発明により具体化されるキットは、本明細書中に開示したもののいずれかのような抗HMFGまたは抗11D10活性についてのアッセイを行うことを可能とするものを含み、それにより、それらの活性を検出および／または定量する。本発明によって具体化されるキットはまた、例えば、エクスビボまたはインビボでトランスフェクトされた細胞における11D10ポリヌクレオチドの検出を可能とするキットを含む。これらのキットは、11D10の可変領域またはその一部をコードするポリヌクレオチドを含むポリヌクレオチドの検出または定量のために使用され得る。11D10可変領域とハイブリダイズするが（すなわち、安定なハイブリッドを形成する）、（本出願の出願時には公知の）他の可変領域のポリヌクレオチドとはハイブリダイズし得ない11D10ポリヌクレオチド（本明細書中に記載される）が、特に適切である。
例えば、生物学的サンプル中のAb3の存在が、11D10ポリペプチドを用いて試験され得る。このサンプルは、Ab3の富化のために、必要に応じて前処理され得る。
本発明のキットは、適切なパッケージングにおける11D10、11D10ポリヌクレオチドおよび／またはポリペプチドを含む。キットは、必要に応じて、手順において有用なさらなる成分を提供し得る。これらの必要に応じた成分は、以下を包含するが、これらに限定されない：緩衝液、捕獲試薬（capture reagent）、発現試薬（developing reagent）、標識、反応表面、検出手段、コントロールサンプル、使用説明書、および説明的資料。
以下の実施例を例示のために提供するが、本発明はこれらに限定されない。
実施例
実施例１．11D10抗イディオタイプ抗体の産生および特徴づけ
モノクローナル抗イディオタイプ抗体11D10を産生するハイブリドーマ細胞株を作製し、そして、以下の記載に従って同定した。所望の特異性と機能性を有する抗体を得るためには、免疫化手順およびスクリーニング手順の両方の局面が重要であった。11D10は初期に産生された多くのAb2の１つであり、そして最も望ましい特性を備えた候補として同定された。
この免疫化抗体(Ab1)は、マウス抗HMFGモノクローナル抗体MC-10（この節ではBrE-1）と言う）であった。応答動物もまたマウスであったため、生成したAb2はBrE-1のイディオタイプの特性に対するものと予想された。しかし、その中の一画分のみがBrE-1パラトープに対するものであり、さらにより小さい集団が、免疫原性でありそしてAb3を誘発し得、そしてさらにより小さな集団が腫瘍関連抗原と交差反応するAb3を誘発する。
BrE-1をオートロガスな種において十分に免疫原性とするために、キャリアKLHに結合させ、フロイントアジュバントで乳化した。これを用量の間が２週間のみの通常でないスケジュールでレシピエント動物に反復投与した。このスケジュールに従って５匹のマウスを免疫化した。約３匹のマウスで、４回目の免疫化後にのみ実質的な応答が起きた。応答動物に、５回目のBrE-1静注投与量によって追加抗原刺激を行い、脾臓細胞を単離し、そして各動物から個別にハイブリドーマを調製した。クローニングは標準的な技術に従って行った。
スクリーニング手順は、以下の四つの重要な工程を包含する：(1)BrE-1に結合する抗体についてのポジティブ選択、(2)アイソタイプまたはアロタイプ決定基を認識する抗体についてのネガティブ選択、(3)HMFGに対するBrE-1の結合を阻害する能力ついてのポジティブ選択、および(4)マウスおよびウサギの両方において、最初の腫瘍関連抗原（HMFG）に対する体液性免疫応答を誘導する能力ついてのポジティブ選択。この節の残り部分（rest）は、スクリーニング手順の概要を提供し、この手順は続く節でより詳細に示される。
BrE-1と反応するが、同じアロタイプ決定基またはアイソタイプ決定基を共有する他の標的モノクローナル抗体とは反応しないクローンを同定するための初期スクリーニングを、イムノアッセイによって行った。重要なアッセイは、BrE-1を固相に付着させ、培養上清で重層し、そして放射性ヨウ素標識BrE-1で発現させるサンドイッチRIAであった。このアッセイでは、ハイブリドーマ上清中の抗体が、機能的に二価であって、かつ捕獲BrE-1と発現するBrE-1との間をつなぎ得ることが要求される。イディオタイプ特異的であり、かつこのアッセイで強いシグナルを発した数個のクローンをさらなる研究用に選択した。
BrE-1のHMFGへの結合をブロックするためにAb2を必要とする競合アッセイにより次のスクリーニングを行った。これによると、Ab2がBrE-1のパラトープを認識したことが立証された。HMFGは、細胞表面でHMFGを発現するヒト乳房細胞腫瘍株である、MCF-7細胞の形態で提供された。このアッセイの性質上、Ab2が腫瘍細胞上でその特異的な提示様式でBrE-1と腫瘍抗原との間の相互作用をブロックすることが必要である。最低限、それ以前のスクリーニング試験を通過した候補Ab2は、BrE-1の細胞への結合を少なくとも85％阻害することが必要とされた。この最低限を実質的に超えたAb2は約３個あり、11D10がほぼ最高レベルの阻害を提供した。
最終的なスクリーニング試験は、候補Ab2をレシピエントに注入したときに、所望の特異性のAb3を誘発し得るか否かの決定であった。マウス腹水から十分な量のAb2を調製し、そしてマウスとウサギで試験した。これらの試験動物由来の血清を、最初に、免疫化に使用した同じ標識Ab2を用いるサンドイッチイムノアッセイにおいてAb3の存在についてアッセイした。次いで、ポジティブの試験結果を示す血清を、腫瘍関連抗原、すなわちHMFGに対して反応するAb3の能力についてアッセイした。HMFGの半純粋な調製物を用いてマイクロタイタープレートを被覆し、系列希釈した試検血清を重層し、そして標識抗免疫グロブリンを用いて、結合したAb3を検出した。HMFGに結合するAb3の力価により、抗HMFG誘導物質としてのその能力の反映としてAb2の「質」を規定した。
モノクローナル抗体11D10は、最高の質を有する抗イディオタイプとして出現し、そして本発明で具体化される種々の成分、組成物、および手順の基礎となる。
材料および方法
細胞：ハイブリドーマ株の生成に用いた融合パートナーは、ATCCからNo.CRL-1580として入手でき、P3X63Ag8.653と祖先が関係するマウス非分泌性ミエローマ細胞株P3-653であった。樹立ヒト細胞株を、他の箇所で記載されたように(Seonら(1984)J.Immunol.132:2089)、５％ウシ胎児血清を補充したRPMI1640中で培養した。
HMFG：脱脂HMFGを、Roberto L.Cerianiから供給を受け、そして彼のプロトコルに従って調製した（Ceraniら(1977)Proc.Natl.Acad.Sci.USA 74:582-586）。簡単には、人乳の洗浄された乳脂画分を、２容量のクロロホルムで２回抽出し、そして１容量のエーテルで２回洗浄し、次いで凍結乾燥した。タンパク質の濃度を、Lowry法により決定した。
BrE-1抗体：マウスモノクローナル抗体BrE-1（MC-10）を、Dr.R.L.Cerianiより豊富に提供を受け、そしてそれはWO 89/07268に記載されている。プリスタンで初回刺激した個々のマウスに、２〜10×10⁶個の生存細胞を腹腔内注入することによって、BrE-1ハイブリドーマの腹水を調製した。この腹水から45％飽和硫酸アンモニウム沈殿およびその後のProtein Ａ Sepharose^TM CL-4B（Eyら（1978）Immunochemistry 15:429）上のクロマトグラフィーによってIgG画分を単離した。単離されたIgGの純度を、免疫拡散法、免疫電気泳動、および高圧液体クロマトグラフィー(HPLC)分画によってチェックした。異なるアイソタイプの他の非関連のAb1およびAb2を、コントロールとして使用した。
BrE-1のF(ab')₂フラグメントの調製：標準的なペプシン消化（Parham（1983年）J.Immunol 131:2895）によりF(ab')₂フラグメントを調製した。要約すれば、BrE-1腹水由来のIgG画分を0.1Mクエン酸緩衝液（pH3.5）に対して透析し、そしてペプシン（25Tg/mg IgG）を用いて370Cで８時間消化した。切断後、3.0Mトリス緩衝液（pH8.6）でpHを7.0に調整し、そして低温で溶液をリン酸緩衝化生理食塩水(PBS)に対して透析した。消化物をSepharose^TM６カラムを用いるHPLCによって分離した。単離されたF(ab')₂の純度は免疫拡拡散法により、および標準的なELISAでの抗アイソタイプ試薬との反応により決定した。
抗体のKLHとのカップリング：Maloneyら(1985)、Hybridoma 4:191によって記載された方法に従って、BrE-1をキーホールリンペットヘモシニアン(KLH)とカップリングした。抗体ストック溶液(1mg/ml)を新たに希釈したグルタルアルデヒド溶液の存在下（最終濃度0.05％）でPBS中のKLH(1mg/ml)と混合した。混合物を室温で１時間くるくると回転させ、次いで、PBSに対して40Cで完全に透析した。
同系BALB/cマウスの免疫化：BALB/cの雌を、２ヶ月の期間にわたって４回免疫化した。最初の注入は、完全フロイントアジュバントで乳化した100TgのBrE-1を用いて腹腔内に与えた。次の２回の注入は、不完全フロイントアジュバント中のKLHとカップリングした100TgのBrE-1を皮下注入または腹腔内注入のいずれかで与えた。マウスは時々採血を行い、そしてBrE-1のF(ab')₂フラグメントおよびコントロールとして正常プールBALB/cマウス血清IgGを用いることにより、結合アッセイ中のELISAによって抗イディオタイプ活性をチェックした。融合の３日前に、PBS中のBrE-1を静脈内に注入してマウスを追加免疫した。
抗イディオタイプハイブリドーマの生成
ハイブリドーマ株の生成に用いた融合パートナーは、ATCCからNo.CRL-1580として入手でき、P3X63Ag8.653と祖先が関係するマウス非分泌性ミエローマ細胞株P3-653であった。樹立ヒト細胞株を他の箇所で記載されたように(Seonら(1984)J.Immunol.132:2089)、５％ウシ胎児血清を補充したRPMI1640中で培養した。
ハイブリドーマは、本質的に、OiおよびHerzenberg（（1980）Selected Methods of Cellular Immunology、MishellおよびShiigi編、Freeman Publs.351〜372）の方法に従って生成した。50％ポリエチレングリコール（PEG、分子量約4500）の存在下、免疫化マウス由来の脾臓細胞を、P3-653細胞と1:1から10:1の割合で混合した。次に、融合細胞を洗浄し、そして培養した。ヒポキサンチン-アミノプテリン-チミジン培地を用いてハイブリッドを選択した。
ハイブリドーマクローンを分泌する抗イディオタイプ抗体(Ab2)の初期選択
RIAによってハイブリドーマクローンの初期スクリーニングを行った。精製したBrE-1を、クロラミンT法（Hunter(1970)Proc.Soc.Exp.Biol.Med.133:989）により放射性ヨウ素標識した。BrE-1またはコントロール抗体（種々のアイソタイプおよび非関連の特異性のモノクローナル抗体、ならびにBALB/cの正常IgG）を、PVCプレート上に500ng/ウェルで被覆した。40Cで一晩インキュベートした後に、プレートを、PBS中の１％ウシ血清アルブミン（BSA）でブロックした。被覆したプレートを、ハイブリドーマ上清の系列希釈と共に４時間インキュベートし、そして約50,000cpmの¹²⁵I-BrE-1を用いて発現させた。RIAアッセイは、抗体に対するストリンジェントな特異性試験であり、そしてまた、抗体が２つのBrE-1分子の間をつなぎ得ることを必要とする。
加えて、ELISAアッセイを行い、各クローンのクラスおよびサブクラスを決定する。ELISAを、BrE-1抗体（または、コントロール）を用いて500ng/ウェルでマイクロタイタープレートを被覆することにより行った。40Cで一晩インキュベートした後に、プレートを、PBS中の１％BSAでブロックした。100TIのハイブリドーマ培養上清または20×濃縮物を、室温で４時間、ウェル中でインキュベートする。PBSで洗浄した後に、プレートを、アルカリホスファターゼ標識抗アイソタイプ試薬と共に室温で４時間または40Cで一晩さらにインキュベートし、そして基質を用いて発色させた。特定のIgGサブクラスの抗体を、プロテインAクロマトグラフィーにより容易に精製し、そしてそれは有用なエフェクター機能を有し得る。
1300の初期融合物のウェル由来の培養上清を、初めにスクリーニングした。RIAにおいてBrE-10と反応するが、コントロールの免疫グロブリンに一致するアイソタイプまたはアロタイプとは反応しない、42のAb2ハイブリドーマを得た。
所望の特性を備えるこれらのスクリーニングアッセイから多くのモノクローナルAb2分泌細胞株が出現した。その中にモノクローナル抗体11D10が存在した。
モノクローナルAb2がBrE-1イディオタイプに特異的であることの確認
Ab2のイディオタイプ特異性はAb1への直接結合により確認された。種々の精製Ab2を¹²⁵Iで標識し、そしてモノクローナル抗TAA Ab1のパネルで被覆したプレートへの結合について試験した。¹²⁵I-11D10を用いた実験の結果を図６に示す。結果は平均cpmで示される（n=3、標準偏差10％未満）。11D10は、BrE-1とほぼ独占的に結合し；試験された他のAb1のいずれとも交差反応性は実質的に無かった。
Ab2のイディオタイプ特異性をまた、Ab1およびAb2の位置を逆転することにより確認した。100ng、300ng、および1000ngの精製したAb2でプレートを被覆し、種々の標識Ab1と反応させた。¹²⁵I-BrE-1および¹²⁵I-BrE-3が含まれる。BrE-3は、別の腫瘍関連HMFGエピトープに特異的なIgG1であり、そしてコントロールとして作用する。11D10をこの方法により試験した実験の結果を表１に示し、そしてBrE-1についての特異性を確認した。

BrE-1イディオタイプに対する特異性を、競合実験においてさらに確立する。種々の標識Ab2を、Ab2s、Ab1、および他のマウスの免疫グロブリンを含む非標識競合物のパネルの異なるメンバーと混合した。次いで、Ab2を、BrE-1被膜プレートに対する結合について試験した。最も良好なAb2では、250ngの非標識のAb2またはBrE-1を競合物として使用して90％を超える阻害を観察した。実際に、他の免疫グロブリンを潜在的な競合物として使用して、10Tgまでの濃度で阻害は得られなかった。11D10が試験されたAb2であった代表的な結果を、表２に示す（平均cpm、n=3、標準偏差10％未満）。

BrE-1パラトープに対する抗イディオタイプについてのスクリーニング
Ab2がBrE-1のパラトープに対するものか否かを決定するため、Ab2を使用して放射性標識BrE-1の、ヒト乳癌腫細胞株MCF-7またはSKBR3上に発現されたHMFGへの結合について競合させた。
アッセイを行うために、標的細胞を96ウェル組織培養プレート中でコンフルエントな単層として増殖させた。試験Ab2の種々の希釈物（培養上清または精製抗体のいずれか）を標識BrE-1と混合し、次いで、マイクロタイタープレート中のコンフルエントな細胞培養物に加えた。アッセイの阻害パーセントを次式に従って計算した。

ここでR_Tはインヒビターを有する実験ウェルの平均cpmであり；R_cは平均バックグラウンドcpmであり；そしてR_MAXはいずれのインヒビターも有さない平均最大結合である。
図７は、11D10を競合抗体として用いて行った、このタイプの実験の結果を示す。250ngのAb2 11D10は、HMFG発現細胞に対する標識BrE-1の結合を90％以上阻害した（図７）。
このステージで試験された42個のモノクローナル抗体（11D10を含む）のうちの約24個は、HMFG発現細胞またはHMFG抽出物に対する標識BrE-1の結合を、プレート結合アッセイにおいて約25ngという少量で阻害した。精製された3H1（無関係の特異性のマウスモノクローナル抗体）を、コントロール競合物として使用し、そしてそれは細胞に対するBrE-1の結合を阻害しなかった。一般的に、少なくとも85％の阻害を生じるAb2は、スクリーニング工程においてこの段階を通過したと考えられた。
結合特異性の確認
最も有望なAb2について、BrE-1への結合の特異性を確認するため、HMFGを用いる確認実験を行った。
約40,000cpmの¹²⁵I-BrE-1を、HMFGの半精製調製物と、同時にインキュベートした。この抗体Ag混合物をAb2被覆プレートに添加し（500ng/ウェル）、そしてHMFGの結合阻害能力を測定した。Ab2の量は、結合し得たBrE-1の量に関して限定されず、そしてそれ故、少量の競合HMFGに対する鋭敏なインジケーターであった。
これまでに記載したスクリーニング試験において、ポジティブと判断された24個の抗体産生クローンのうち６個を使用して、Ab2の供給源としてマウスの腹水を調製した。このAb2を、標準的な技術によりProtein Aアフィニティー樹脂を用いるクロマトグラフィーによって精製した。
11D10に対するBrE-1の結合は、HMFGにより阻害される。
腫瘍特異的免疫応答を誘発し得る抗イディオタイプのスクリーニング
これらの実験の主な目的は抗HMFG免疫応答を誘発し得る抗イディオタイプを見出すことであるので、次のスクリーニング工程は、動物モデルにおいてその免疫原性を試験することであった。Ab2は免疫原性であるだけでなく、腫瘍抗原HMFGと交差反応するAb3を惹起し得る。
従って、HMFG発現細胞との競合実験で最も強い結果をもたらしたAb2を、免疫化実験で試験するために取り上げた。
試験されるべき各Ab2について、５匹のBALB/cマウスおよび２匹のニュージーランドシロウサギを免疫化した。マウスについては、Ab2をKLHに結合させた。２週間のスケジュールで、マウス一匹あたり50μgを注射し、そしてウサギ一匹あたり200μgを注射した。最初の注射液は、フロイントの完全アジュバント中で調製し、次の注射液はフロイントの不完全アジュバント中で調製した。血清を、分析のために規則正しく回収した。最初に、Ab3の力価を、抗体の捕獲および検出の際にAb2の両方を用いて標準的なサンドイッチラジオイムノアッセイで測定した。Ab2に対する抗体応答は、５回目の免疫後に実質的なレベルに達した。
次に、アッセイを、HMFG調製物でコートしたプレートで行った。血清をウェル中でインキュベートし、そして結合した抗体を酵素結合抗免疫グロブリンで検出した。このアッセイには、抗体が元の腫瘍関連抗原に結合することが必要であり、そして抗イディオタイプで免疫化することによって誘導されたAb3の少なくとも一部が腫瘍抗原特異的であることを確立する。HMFG特異的Ab3のレベルを連続希釈によって力価測定し、そして免疫化Ab2の「質」を規定した。
11D10モノクローナル抗体は、試験される候補の中で、最も高い質を有することが明らかになった。
11D10により誘発されたAb3が所望の特異性を有することの確認
11D10の治療目的が腫瘍関連抗原にタンパク質に対する免疫反応を誘発するその能力に依存するので、得られたAb3の特異性を続く多数の実験で確認した。
Ab3含有血清(抗アイソタイプおよび抗アロタイプ活性を欠く)は、標識BrE-1の11D10への結合またはその逆をほぼ完全に阻害した。このことは、Ab3抗体がAb1とイディオタイプを共有することを示す。Ab3産生動物がKLHに結合した11D10またはフロイントのアジュバント中で乳化した11D10のどちらで免疫された場合でも、類似の結果が得られた。
11D10で免疫化したマウス由来の脾細胞を、モノクローナルAb3産生細胞株を作製するために用いた。前節で記載したものと同様の競合実験により、モノクローナルAb3がBrE-1と同じ様式でHMFGに結合することが示された。
結合実験を、11D10によって誘導されたAb3が腫瘍細胞株で発現したときにHMFGに結合し得るか否かを決定するために行った。図８は、種々のAb3調製物の乳ガン細胞株SKBR3への直接結合の結果を示す。ポリクローナルマウスAb3、ポリクローナルウサギAb3、およびモノクローナルマウスAb3を、マウス免疫グロブリンでの吸着によって調製し、そしていくつかの希釈液での結合について試験した。11D10で免疫化した動物由来のポリクローナルおよびモノクローナルAb3血清の両方は、HMFGポジティブ細胞株MCF-7およびSKBr3に結合したが、抗原陰性メラノーマ細胞株M21/P6には結合しなかった。
種々の競合実験を行って、11D10によって誘導されたAb3が所望の特異性を有することを確認した。マイクロタイターウェル中のSKBR3細胞のコンフルエントな単層培養物を、競合因子としての50,000cpmの¹²⁵I-BrE-1および種々のAb3希釈物と反応させた。特異性に関連のないモノクローナル抗体(1E3)を、ネガティブコントロールとして用いた。結果を図９に示す。20μgのモノクローナルAb3は、結合を25％阻害した。1/50希釈に希釈したポリクローナルAb3血清を含むマウスまたはウサギの血清は、それぞれ38％および30％阻害した。このことは、Ab3がAb1と同一のHMFGエピトープに結合することを示した。これらの条件下で完全に結合しなかったことにより、Ab3のいくつかはAb1よりもHMFGに対してより低い親和性および親和力を有しうることが示唆される。
11D10で免疫化したマウス由来の脾細胞をＴ細胞増殖アッセイに用いた。脾細胞を半精製したHMFGの存在下で５日間培養し、次いで[³H]チミジンをパルスした。コントロールよりも11D10で免疫化した動物由来の細胞での取り込みが大きいことは、Id特異的細胞性免疫応答の存在と一致する。11D10-Alugel免疫化ウサギは、半精製したHMFG調製物に対してDTH皮膚反応をいくらか示したが、純粋CEA(ネガティブ特異性コントロール)に対しては示さなかった。HMFGはいくつかのエピトープを含みそして精製形態では入手できないので、本発明者らは、反応の特異性について確認できない。
mAb1およびmAb3のドットブロット分析
種々の希釈のHMFG抗原を、ニトロセルロースフィルターにトランスブロットし、そしてBrE-1(Ab1)およびmAb3と反応させた(図10)。レーン１〜３は、HMGFでトランスブロットし、そしてそれぞれ、BrE-1(10μg/ml)、コントロール1E3 IgG1(50μg/ml)、およびmAb3 IgG1(50μg/ml)と共にインキュベートした。反応をヤギ抗マウスIgGアルカリホスファターゼ試薬および基質を用いることによって進行させた。染色は同じであったが、Ab1でより強力であった一方でコントロール抗体はネガティブであった。
乳ガン患者での交差反応イディオタイプについての適合実験
本発明者らは、50人の無作為に選出した乳ガン患者由来の血清を研究して、そのいずれかがAb2 11D10によって認識される予め存在する適合イディオタイプを有するか否かを決定した。マイクロタイタープレートを、250ng/ウェルの11D10(Ab2)の精製F(ab')₂フラグメントでコートし、そして1:100希釈の患者の血清と共にインキュベートし、そしてヤギ抗ヒトIgG酵素標識抗体を用いて発現させた。Ab2への血清の結合を、アルカリホスファターゼ結合抗ヒトIgG(γ鎖特異性)抗体および基質を用いて検出した。
図11に示すように、これらの乳ガン患者の血清のうち少数(8/50)が、11D10と反応する抗体のレベルが上昇した。抗Id治療での選択基準は、疾患それ自身が宿主でのＢ細胞およびＴ細胞のプライミング状態を誘導するという仮定に基づいている。対応する適合しているIdを発現している患者において、Ab2刺激によって既にプライムされているこのようなＢ細胞およびＴ細胞が効果的に刺激され得るという仮説がたてられる。乳ガン患者由来の血清に適合するIdの発見により、これらがAb2 11D10での抗Id免疫治療活性化のための潜在的な候補として特に適切であり得ることが示唆される。
MX-1腫瘍保持BALB/cヌードマウスでの¹¹¹In-BrE-1のクリアランス研究
11D10が放射線免疫治療において過剰の放射標識した抗体に結合させるための第２の試薬として適切であるか否かを決定するために、本発明者らはクリアランス研究を行った。各BALB/cヌードマウスに、20μCiの2.9μgの¹¹¹In-BrE-1を注射した。24時間後、20μgの抗Id 11D10を６匹のマウスの一群に注射して、30分後に屠殺しそして別の群を40時間後に屠殺した。マウスのコントロール群は、抗Id-11D10を30分後および40時間後に注射しなかった。マウスを屠殺した後、血液および器官を取り出して放射活性レベルを測定した。結果を表３に示し、そして１gmの¹¹¹In-BrE-1±SEMあたりのパーセント用量で表した。値は、平均％SEMで表した。30分および40時間の両方で、コントロールと比較して、実験群で肝臓を除くほとんど全ての器官(特に血液、腎臓、筋肉、および肺)で放射活性の顕著なクリアランスが存在した。腫瘍の保持については30分では影がなかった；しかし、40時間後、処置群でいくらかのクリアランスが存在した。

実施例２：11D10 cDNAのクローニングおよび配列決定
他に特定しなければ、全てのクローニング技術は、本質的に、Sambrookら(1989)に記載されたものであり、そして使用した全ての試薬は製造者の指示に従った。
ポリヌクレオチド配列を、11D10産生細胞株由来のメッセンジャーRNAを単離することによって、11D10抗体について得た。各配列決定のために、総RNAを１×10⁷の11D10ハイブリドーマ細胞から単離した。メッセンジャーRNAを、オリゴチミジレート-セルロースカラムのクロマトグラフィーを２サイクル通過させることによって調製した。mRNAの収量は約100μgであった。一本鎖cDNAを、SuperScript Preamplification kit(GIBCO/BRL)を用いて合成した。
重鎖可変領域を配列決定するために、PCRを、以下のマウスγ１定常領域のアミノ酸126〜119に対応する逆方向(3')プライマー

および正方向プライマーの種々の混合物(マウス可変領域サブグループのＮ末端リーダー配列に対応する)を用いて、cDNAで行った。陽性反応をもたらした(5')正方向プライマーは：

であり、これはアミノ酸-20〜-13に対応する(Ｉ＝イノシン、Ｒ＝ＡまたはＧ、Ｙ＝ＣまたはＴ、Ｋ＝ＧまたはＴ、Ｓ＝ＣまたはＧ、Ｗ＝ＡまたはＴ)。
増幅したcDNAのフラグメントを、pT7プラスミドにサブクローニングし、そしてNovaBlueコンピテント細胞を供給者(Novagen)によって提供されたプロトコルを用いて形質転換した。組換えコロニーを色選択によって拾い、そしてプラスミドDNAをミニプレップ手順によって調製した。二本鎖プラスミドのDNA配列を、Sequenase Version 2.0キット(USB,Cleveland,Ohio)を用いて決定した。プラスミド中のDNA挿入片の配列を、プラスミドに特異的なプライマーを用いて両方向から決定した；すなわちT7プロモータープライマー(TAATACGACTCACTATAGGGG-配列番号38)およびU-19プライマー(GTTTTCCCAGTCACGACGT-配列番号39)。少なくとも８個のクローンを、配列決定のために拾った。
11D10軽鎖可変領域の配列を、同様の様式で決定した。PCRでポジティブな結果を生じた正方向および逆方向のプライマーは：

これは、それぞれリーダー配列のアミノ酸-20〜-10、およびマウスκ鎖定常領域の122〜116に対応する。
PCR増幅でのエラー率を最小にするために、pfuDNAポリメラーゼ(Stratagene,an Diego)を、全ての次の実験での増幅に用いた。この熱安定性DNAポリメラーゼでの変異頻度は、Taqポリメラーゼと比較して1/10である。
単離されたcDNAが11D10重鎖および軽鎖に対応することの確認を、単離された抗体のＮ末端のアミノ酸配列決定によって得た。50μgの精製11D10抗体をサンプルローディング緩衝液(50mM Tris-HCl(pH6.8)、１％SDS、１％グリセロール、0.1％βメルカプトエタノール)で希釈して、100℃で３分間加熱する。変性させたタンパク質を、SDS含有7.5％ポリアクリルアミドゲル(BioRad Miniprotean II Dual Slab Cell)にロードし、そして200Ｖで１時間電気泳動に供した。ゲル中のタンパク質を、Towbinら((1979)Proc.Natl.Acad.Sci.USA.78:4350-4354)に記載された手順によって、150mAで一晩、ポリビニリデンジフルオライド(PVDF)メンブレンに移した。転移緩衝液は、25mM Tris、192mMグリシン、20％(v/v)メタノールを含む。メンブレンを、50％メタノール−50％酢酸中の0.1％Coomasie Brilliant Blueに素早く浸して染色し、その後40％メタノールおよび10％酢酸を含む溶液で洗浄した。室温でメンブレンを乾燥させた後、染色された重鎖および軽鎖のバンドを、清潔なカミソリ刃で切り出した。メンブレン片上のタンパク質を、パルス標識した液体化学およびオンラインフェニル−エチオノハイダンション(ethiohydantion)アミノ酸同定を用いて、Applied Biosystem Model 477Aタンパク質シーケンサーを使用して、自動Edman分解によるＮ末端ミクロ配列決定に供した。各タンパク質を、10〜15の分解サイクルに供し、そして各サイクルからの変換された切断産物を、逆相HPLCによって分析した。
モノクローナル抗体11D10の重鎖および軽鎖可変領域の核酸配列および対応するアミノ酸配列を、それぞれ図１および図２に示す。
図１では、リーダー配列の３番目のアミノ酸(アミノ酸-18)およびフレームワーク３の25番目のアミノ酸(アミノ酸81)はエラーであることが明らかである。当業者に明らかであるように、ヌクレオチドトリプレット「GCC」によってコードされるアミノ酸はＡ、すなわちアラニン(アミノ酸-18)であり、そしてヌクレオチドトリプレット「GAA」によってコードされるアミノ酸はＥ、すなわちグルタミン酸(アミノ酸81)である。同様に、図３Ａにおいて、アミノ酸81(フレームワーク３内)は、Ｅまたはグルタミン酸である。配列番号58は、本開示と共に提出された「図１」を読むタイプされたシート上の誤植で示されるように、間違ったアミノ酸翻訳を示す。配列番号２は、正しいアミノ酸翻訳を示す。
核酸配列を、本実施例で先に記載したように、抗体産生細胞株由来のmRNAのPCR増幅によって得た。アミノ酸配列を、続いて、遺伝コードを用いてポリヌクレオチド配列を翻訳することによって得た。正確なアミノ酸は、遺伝コードのために自明である。正確なアミノ酸配列はまた、本開示の支持においてアクセス番号HB-12020でATCCに寄託された抗体産生細胞株に固有のものである。
重鎖および軽鎖ポリヌクレオチドならびにアミノ酸配列を、National Center for Biotechnology InformationでBLASTアルゴリズムを用いて、PDB,SwissProt,PIR,SPUpdate、GwnPept、およびGPUpdateデータベースから入手可能な配列と比較した。比較は、1996年１月19日に行った。
11D10軽鎖可変領域のDNA配列に最も密接に適合した10のデータベースDNA配列のなかでは、同一のものはなかった。11D10 DNA配列と約８〜27の差異が存在した(これは約６〜17アミノ酸の差異に相当する)。６番目の適合した配列(名称＞gb/M59920/MUSIQKAA3)は、マウスκVJ生殖様配列であり、そしておそらく11D10軽鎖が由来するプロトタイプ遺伝子を表す。11D10 DNA配列は、14個の位置で生殖系配列と異なるが、これは約７アミノ酸の位置の差異に相当する。
11D10重鎖可変領域のDNA配列に最も密接に適合した10のデータベースのDNA配列の中では、同一のものはなかった。10の配列のうち９が、３〜12塩基対長く、これはVDJ接合部内のスプライシングの差異に依存した。さらに、接合部の外側の11D10 DNA配列と比較して、約15〜43点の差異が存在し、これは11〜23アミノ酸の差異に相当した。
従って、11D10 DNAによってコードされるアミノ酸配列と最も密接に適合したデータベースのDNAによってコードされるアミノ酸配列との間には、少なくとも約18アミノ酸の差異が存在する。点の差異は、おそらく、抗体産生細胞株を作製するために用いる動物でのこの細胞株の発達の間に、生殖系配列の体細胞変異によって生じる。
図４は、11D10軽鎖可変領域コード配列に最も密接に適合した10のポリヌクレオチド配列を示す。図５は、11D10重鎖可変領域コード配列に最も密接に適合した10のポリヌクレオチド配列を示す。
軽鎖および重鎖可変領域アミノ酸配列を、他の既知の配列とNational Center for Biotechnology Informationで、BLASTアルゴリズムを用いて、PDB,SwissProt,PIR,SPUpdate,GenPept、およびGPUpdateデータベースから入手可能な配列と比較した。比較は、1996年１月19日に行った。
BLAST検索で見出された15の最も密接したアミノ酸配列は、表６に示す識別名を有する。

図26(A)および(B)は、BLASTサーチに見出される15の最も近い配列との11D10軽および重鎖アミノ酸配列の比較描写である。パネル(A)は、軽鎖の比較を示す。パネル(B)は、重鎖の比較を示す。11D10配列と同一の残基は、点（．）で示す。重鎖VDJ接合点の近くの整列を改善するために導入したギャップは、二重線（＝）で示す。
11D10軽鎖可変領域のものに最も密接に適合した50のデータベースのアミノ酸配列のうちで、同一のものはなかった。11D10は、15の最も近い配列とは最小７および平均約12の置換の相違で異なった。相違の集まりが、CDR1の末端の近くに起こった。他の相違は、CDR3およびフレームワークに起こった。
11D10重鎖可変領域のものに最も密接に適合した50のデータベースのアミノ酸配列のうちで、同一のものはなかった。以下は、比較から推定された主要点を要約する。
最も近い適合は、別の抗イディオタイプ（命名gp|X64805|MMAIDHCH_1）としてそのGen Bankの命名によって示される重鎖可変領域とであった。残基１と98との間（VDJ接合の前）に11の置換があり、残基98の後は７置換の相違があった。
11D10は、長さが40の重鎖配列と異なっていた。他の配列は、VDJ接合点の近くで、５残基までとより長いか、または２残基ほどより短かった。
２つの最も密接に適合した配列を除いて、残基１と98との間には、少なくとも18そして平均では約22の置換の相違があった。
種々のDおよびJ領域遺伝子は、プロトタイプV遺伝子とともに用いられるようである。50の配列のうちの１つのみが、同じD領域を用いているようであった。D領域内に点相違が存在し、そして３残基のスプライシング相違が存在した。
最も密接に適合した50の配列にとって、全部で18の挿入、欠失、および置換の相違が存在した。他の49の配列は、全部で少なくとも25および平均で約30の挿入、欠失、および置換の相違を有した。
相違は、可変領域全体に現れる。
図26(C)は、15の最も最も近い適合した配列の軽および重鎖可変領域のアミノ酸コンセンサス配列を示す（配列番号47および配列番号48）。これらは、組み立てられた軽鎖VJ遺伝子および重鎖VDJ遺伝子のプロトタイプを表す。コンセンサス配列と同一である11D10配列中の残基は、点（．）で示す。CDRは、アスタリスク（￥）で示す。また、N→C（大文字）またはC→N（小文字）の方向で、ヒト乳脂肪グロブリン（HMFG）由来のフラグメントを示す。
以下のポイントを推論し得る：
・11D10は、コンセンサス配列から少なくとも18残基離れている。これらのうちの７個は軽鎖に生じ、そして11個は重鎖に生じる。
・点相違は、11D10軽および重鎖可変領域配列全体に起こる。８個はCDR内に生じ、10個はCDR外に生じる。
・HMFGフラグメントとの11D10配列の整列は、点変異に依存しない。
可変領域フラグメント配列、特に重鎖VDJ接合点を包囲する配列または図26(C)に示される任意の点相違は、11D10の免疫学的活性を有する本発明のポリペプチドの開発において興味深い。VDJ接合点または任意の点相違の近くのコード配列は、本発明のポリヌクレオチドの開発において興味深い。
実施例３：サルにおける11D10による乳ガン特異的応答の誘導
細胞株
HMFG抗原を発現するヒト乳ガン細胞株MCF-7を、10％FCS、1％L-グルタミン、ペニシリン、およびストレプトマイシンを補充したRPMI 1640培地中で増殖させ、そして抗腫瘍応答の検出に用いた。ヒト黒色腫細胞株M21/P6（Scripps Research Institute,La Jolla,CAのRalph Reisfeid博士の好意により提供された）およびＴ細胞株MOLT-4（両方ともHMFGネガティブである）を同じ培地で増殖させ、そしてネガティブコントロールとして用いた。
抗体
分子量400,000のHMFG分子で別個のそして特異的なエピトープを識別するAb1 mAb MC-10(IgG2b、κ)を、実施例１に記載のように、抗Id mAb 11D10(IgG1-κ)の産生のために、同系のBALB/cマウスを免疫するために用いた。mAb2 3H1(IgG1-κ)は、ヒトCEA（Bhattacharya-Chatterjeeら、（1990）J.Immunol.14S:Z758-2765）に関連するマウス抗Id mAbであり、そしてコントロールとして用いた。
アジュバント
抗Idワクチンの免疫原生を増強するためには、アジュバントが必要である。水酸化アルミニウムは、米国食品医薬品局により、ヒトでのアジュバントとしての使用が認められている。それゆえ、本発明者らは、この前臨床研究において、以下に記載のように、水酸化アルミニウムで沈降させたAb2 11D10でサルを免疫化した。
抗体の調製
11D10を、実施例１に記載のように得た。mAb2 3H1(IgG1-κ)は、ヒトCEA（Bhattacharya-Chatterjeeら、（1990）J.Immunol.14S:Z758-2765)に関連するマウス抗Id mAbであり、コントロールとして用いた。
水酸化アルミニウム沈降
アジュバントを、抗Idワクチンの免疫原性を増強するために用いた。水酸化アルミニウムは、米国食品医薬品局により、ヒトでのアジュバントとしての使用が認められている。それゆえ、本発明者らは、この前臨床研究において、Ab2 11D10でサルを免疫化した。簡単に述べれば、精製したmAb抗Id(Ab2)の５mgのアリコートに、１mlの2％ Alu-Gel S(Serva Fine Biochem,Inc.,Garden City,Long Island,NY)を添加した。次いで、Dulbecco's-PBSを用いて容量を10.0mlに調整し、そして混合物をボルテックス上で室温で１時間インキュベートした。次いで混合物を、24℃にて2000rpmで10分間遠心分離した。ゲル層に結合したmAbの量は、上清中の結合していない抗体の量の分光光度測定により決定した。Alu-Gelで沈降させた抗体を、使用するまで４℃で保存した。これらの手順は、層流フード内で無菌的に行い、そして最終産物を滅菌して、抗Id 11D10 Alu-Gelとして鮮明にラベルし、そして発熱物質を含まない、滅菌ガラスバイアルに等分した。
サルの免疫化
カニクイザルを、11D10または3H1コントロールを用いて免疫化した。サルを、White Stands Research Institutes（Alamogordo,NM）に収容した。体重が３〜４kgの雄ザルと雌ザルとの対を、２mgの11D10または3H1のいずれかを用いて、４つの異なる部位で０日、14日、28日、および42日に、それぞれ皮内に免疫化した。２匹のサルだけを、経済的理由のために単回投与量で各抗Id(Ad2)について使用した。２mgの投与量を、異なる抗Idワクチンを用いた事前の前臨床試験および臨床試験に基づいて選択した。血液サンプルを、免疫前および各免疫の10日後に採取した。
毒性
サルにおけるAb3応答の誘導は、動物において明白な副作用を全く引き起こさなかった。複数回の免疫の結果として、接種部位における穏やかな局部的膨張、および炎症しか観察されなかった。サルを、物理的検察および体重測定により日常的にチェックした。異常の徴候は全く示さなかった。
水酸化アルミニウム沈降させたAb2での免疫化によって誘導された体液免疫性の発達
Ab2への特異的Ab3応答。免疫化したサル由来の血清を、抗-抗Id抗体の存在について試験した。血清を、正常マウス免疫グロブリンで予備インキュベートして、アイソタイプおよびアロタイプの決定基に対するサル抗体をブロックし、次いで、マイクロタイタープレートにコートした免疫化抗Id(11D10)との反応によって抗-抗Id(Ab3)の存在をRIAによってチェックした。無関係のAb2を、コントロールとして用いた。洗浄した後、抗原-抗体反応を、均一サンドイッチRIA中で¹²⁵I標識抗Id試薬を用いてタグした。前免疫性血清およびコントロールAb2 3H1で免疫化したサル由来の血清もまた、これらのアッセイに用いた。さらに、¹²⁵I標識モノクローナルAb2 3H1を、コントロールとして用いた。
Ab3のイディオトープ分析。ポジティブ反応を上記の方法で得る場合、これらのサル由来のAb3血清を、マイクロタイタープレートに結合させたBrE-1(Ab1)への¹²⁵I標識11D10の結合を阻害する能力またはその反対（プレートの11D10への放射標識したBrE-1の結合を阻害）についてチェックした。無関係のAb1-Ab2系を、コントロールとして用いた（Bhattacharya-Chatterjeeら、(1990);Bhattacharya-Chatterjeeら、(1987)J.Immunol.139:1354-13605）。これは、サル血清中のAb3が、BrE-1(Ab1)を有するイディオトープを共有するかどうかを示した。Ab3血清によるAb1-Ab2結合の阻害アッセイもまた、Ab3が真の抗-抗Idであるかどうかを示している。
腫瘍抗原へのAb3の結合。天然の標的抗原への直接の体液免疫応答を評価するために、サルAb3血清を、RIAにおいてHMFGを発現することが知られている細胞株との反応性について試験した。さらに、血清を、マイクロタイタープレートにコートしたHMFG抗原の可溶化半精製調製物への反応性をチェックした。抗原-抗体反応を、ELISAにおいて、¹²⁵I標識抗ヒト免疫グロブリン試薬またはアルカリホスファターゼで標識した抗ヒト免疫グロブリンを用いることにより検出した。予備免疫性血清を、コントロールとして用いた。無関係のCEAもまた、このアッセイのコントロールとして用いた。HMFG抗原に結合しているサルAb3血清のアイソタイプを、抗ヒトアイソタイプ特異的試薬を用いるELISAによって決定した。
腫瘍細胞株へのAb3の結合もまた、免疫フローサイトメトリーによってチェックした。抗原ポジティブMCF-7細胞（１×10⁶／ウェル）を、100μlのAb1(MC-10)およびAb3とともに、４℃で60分間反応させた。洗浄の後、細胞を、ヤギ抗マウスまたはヤギ抗ヒトF(ab')₂ IgG-FITC標識抗体（Tago,Burlingame,CA）のいずれかとともに、４℃で30分間インキュベートした。次いで、これを２回洗浄し、２％のパラホルムアルデヒドで固定し、そして免疫フローサイトメトリー（FACStar,Becton,Dickinson,San Jose,CA）によって分析した。抗原ネガティブMOLT-4細胞を、このアッセイのコントロールとして用いた。
過免疫化サル血清由来の抗-抗Id抗体（Ab3）の精製。20mlの過免疫血清を、Sepharose4Bに結合させた免疫化抗Id免疫グロブリン（11D10-IgG1）からなる免疫吸着カラムに通した。抗-抗Id抗体（Ab3）を、0.1Mグリシン-塩酸緩衝液（pH2.4）で溶出し、そして３MトリスでpH7.0に中和した。次いで、溶出した抗体を、Sepharose4Bに結合させた無関係のアイソタイプ-アロタイプ適合抗Id mAbからなる免疫吸着カラムに通して抗アイソタイプ反応性および抗アロタイプ反応性を除去した。通した抗体を濃縮し、そして精製Ab3として用いた。Ab3のアイソタイプを、ヒト抗アイソタイプ特異的抗原（Tago）を用いるELISAによって決定した。
Ab3のエピトープ分析。サルに生じるAb3、およびAb1(BrE-1)が、同一の抗原決定基に結合することを示すために、精製Ab3によって抗原ポジティブ腫瘍細胞株MCF-7またはHMFG抗原へのBrE-1の結合の阻害を、RIAによってチェックした（Bhattacharya-Chatterjeeらに記載される）（1990）。
HMFGを用いる精製Ab3のスロットブロット分析。ポリビニリデン二フッ化物の膜を、メタノールで５分間活性化し、0.02MPBS（pH7.0）に移した。種々の濃度のタンパク質（５μg、２μg、１μg）を、Hybrislot機器（BRL Life Technologies,Gaithersburg,MD）を用いて膜に吸着させた。次いで、膜をPBS中の２％BSAで２時間振盪しながらブロックし、続いて、20μg/mlのAb1またはAb3の溶液の５mlとともに、３時間振盪しながらインキュベーションした。次いで、膜をPBS中の１％BSAで５回洗浄し、そしてアルカリホスファターゼ標識ヤギ抗ヒト免疫グロブリンまたはヤギ抗マウス免疫グロブリン（１：100希釈）とともに90分間インキュベートし、洗浄し、ならびにBio-Radキットに供給された基質を用いて発色させた。
Id特異的増殖応答のためのアッセイ
新鮮な末梢血単核細胞を、標準Ficoll-Hypaque密度勾配遠心法によって単離し、そして５×10³細胞／ウェルを、種々の濃度の11D10およびコントロール3H1（10ng〜２μg）とともに、５％の熱失活化FCS、ペニシリン、およびストレプトマイシンを有するRPMI1640培地中でインキュベートした。非特異的マイトジェンフィトヘマグルチニン-Pを、２μg/ウェルおよび１μg/ウェルでポジティブコントロールとして用いた。細胞を５％の二酸化炭素を含む空気中で37℃で５日間インキュベートした後、[³H]チミジン（１μCi／ウェル）で20時間パルスした。データを、[³H]チミジン取り込みの平均計測値（３連のウェル）／分として表した。SDのデータは、各決定値の10％未満であった。
結果
サルにおける抗-抗Id(Ab3)応答の誘導。サル由来の血清を、４回目の免疫化の後に10日得て、サンドイッチRIAによるAb3応答およびAb1にへのAb2の結合の阻害について分析した（表４）。これらのアッセイのために、血清を、正常マウス免疫グロブリン（500μg/ml）で前処理して、抗アイソタイプ反応性および抗アロタイプ反応性をブロックした。サンドイッチRIAのために、４回目の免疫化の後に得られたAB3血清を、正常マウス免疫グロブリン（500μg/ml）を含むPBSで希釈した。血清を、アッセイの前に正常マウスIgGとともに予備インキュベートした。１：40希釈のAb3を、抗Id mAb 11D10または3H1とともにインキュベートし、マイクロタイタープレートにコートし、次いでサンドイッチアッセイにおいて¹²⁵I標識11D10または3H1（-50,000cpm）と反応させた。結果は、サンドイッチアッセイにおける結合したcpmとして表している。結果は、平均のcpm（n＝３）として示してある。データのSDは、10％未満だった、次いで、本発明者らは、サルAb3血清の半精製したHMFGへの結合を試験した。11D10で免疫化したサル由来のAb3血清（PRO723およびPRO872）は、免疫化Ab2（11D10）に特異的に結合したが、無関係のAb2（3H1）との反応性は最小であった。サルAb3はまた、Ab1への放射線標識したAb2の結合を、１：40の希釈時でさえ、それぞれ91および95％阻害した（表４）。前免疫化血清または無関係のAb2 3H1で免疫化したサルから得られた血清（PRO541およびPRO667）では、阻害はなかった。精製11D10を、プレートをコートするために用い（250ng/ウェル）、そして11D10への放射線標識したBrE-1（約50,000cpm）の結合を、種々の希釈のAb3血清の存在下での阻害について試験した。平行して行ったコントロール実験において、無関係のAb1-Ab2系（mAb 5019-3H1）をコントロールとして用いた。Ab3応答の速度論は、サルPRO723由来の血清を用いて図12に示し、これは、Ab2への放射線標識したAb1の結合の阻害を示す。同様の反応性が、サルPRO872由来の血清で見られた。これらの結果は、サルAb3血清はAb1とイディオタイプを共有していることを示している。

抗腫瘍細胞抗体応答の誘導。11D10免疫化サル血清がHMFGポジティブ乳ガン細胞に特異的に結合するかどうか決定するために、サルAb3血清の乳ガン細胞株MCF-7への結合をELISAによって試験した。種々の希釈のAb3血清を、96ウェルマイクロタイタープレートで増殖させた細胞のコンフルエントな単層に加え、そしてヤギ抗ヒトIgG（重鎖および軽鎖特異的）酵素標識抗体を用いて発達させた。吸光度（OD）を、１時間後に読みとった。サルPRO872を、Ab2 11D10で免疫化した。サルPRP667を、コントロール無関係のAb2(3H1)で免疫化した。前免疫サル血清をまた、コントロールとして用いた。サルAb3血清（.....）の黒色腫細胞株M21/P6への結合を、ELAISAによって同じプロトコルに従って試験した。結果を、405nmでの平均吸光度（n＝３）として示す。データのSDは、各アッセイについて10％未満であった。図13で示されるように、血清（種々の希釈での４回目の免疫化の後に得られる）は、MCF-7細胞と反応したが、抗原ネガティブ黒色腫細胞株M21/P6とは反応しなかった。次いで、本発明発明者らは、サルAb3血清の半精製したHMFGへの結合を試験した。プレートを、HMFG（２mg/ウェル）でコートし、そしてAb2 11D10で免疫化したサル（PRO723）ならびに同じアイソタイプおよびアロタイプの無関係のAb2 3H1で免疫化したサル（PRO667）由来のAb3血清（１：100希釈）と反応させた。前免疫性血清およびPBS-BSAをまた、コントロールとして用いた。結果は図９であり、そして、405nmでの平均吸光度（OD）として表した。平行したコントロール実験において、同一の血清を、精製CEAでコートしたプレート上でチェックした。11D10免疫化サル血清との反応性は見られなかった；得られた吸光度は、FBS-BSAコントロールで得られた物に匹敵した。結果を、405nmでの平均吸光度（n＝３）として表す。データのSDは10%未満であった。実験値とコントロール値との間の差違は、統計学的に有意であった。Ab3血清はまた、ELISAによって、マイクロタイタープレートにコートされた半精製したHMFGに特異的に結合した（図14）。前免疫サルまたは無関係のAb2(3H1)で免疫化したサル由来のコントロール血清は、感知できるほどのHMFGへの結合は示さなかった。平行した実験において、サルPRO723由来の同一のAb3を、CEAでコートしたプレートに上で比較したところ、ネガティブであった。
細胞表面HMFGとの反応性を決定するために、MCF-7細胞を、免疫フローサイトメトリーによって試験した。図10に示しているように、Ab2免疫化サル由来のAb3は、Ab1で得られる結合パターン（示していない）に類似した別個の結合（図15-A）を示した。有意な結合は、HMFGを発現しないMOLT-4細胞では得られなかった（図15-B）。
次いで、Ab3抗体を上記のように血清から精製した。精製したAb3の反応性を、ELISAによってチェックした。プレートを、HMFG（２mg/ml、100ng/ウェル）でコートし、そして室温で一晩インキュベートした。プレートを、PBS中の１％BSAでブロックし、そして11D10またはコントロールAb2(3H1)のいずれかで免疫化したサル由来の精製したAb3（20mg/ml）と反応させた。PBS-BSAをネガティブコントロールとして用いた。CEAでコートしたプレートにおいて、mAb 8019（抗CEA）をポジティブコントロールとして用いた。結果は図16であり、そして405nmでの平均吸光度（OD）（n＝３）として表す。データのSDは、10％未満であった。サルAb3は、マイクロタイタープレートにコートしたHMFGと特異的に反応したが、一方、反応性はコントロールCEAでコートしたプレートでは得られなかった。
精製したAb3のHMFGへの特異性を、スロットブロット分析によって、さらに確認した（図17）。図６において、レーン１および２を半精製したHMFGでコートし、そしてレーン３および４を精製したCEAでコートした。膜を、Ab1（レーン１）、精製したAb3（レーン２および３）、およびmAb8019中の抗CEA（レーン４）とともにインキュベートした。HMFGとの反応性は示されたが、CEAとの反応性は示されなかった。
MCF-7細胞結合へのマウスAb1およびサルAb3の競合。Ab3がAb1と同様の結合部位を有する場合、Ab3は、MCF-7細胞上でのHMFGへの結合についてAb1を競合するはずである。マイクロタイタープレート中のMCF-7細胞のコンフルエントな単層を、種々の濃度の精製したAb3および固定量の¹²⁵I標識したAb1（約50,000cpm）と反応させた。阻害の割合を算出し、そしてインヒビターに対して（mg Ab3）プロットした。固定した量の放射性標識したAb1を、種々の量の精製したAb3またはコントロールAb3調製物およびMCF-7細胞とともに同時インキュベートした（図18）。100ngの精製したAb3は、結合を60％阻害し、そして１mgの精製したAb3は80％をこえる阻害を与えたが、一方、５mgの濃度で用いられたコントロールAb3は、何らかの阻害も産生しなかった。これらの結果は、Ab2免疫サル抗体はAb1のような同一の抗原に結合することを示している；従って、Ab3調製物はAb1'特性を有する抗体分子を含む。
抗Idへの細胞応答。細胞性免疫応答を、水酸化アルミニウム沈降した抗Id抗体（11D10）および水酸化アルミニウム沈降した抗iD抗体（3H1）とともにインキュベートした抹消血単核細胞の増殖によって測定した。免疫化サル由来の末梢血リンパ球（PBL）を、インビトロでの11d10-Alugelまたは3H1-Alugelでの刺激への応答性についてアッセイした。PBLを、11D10 Alugel^TM（２μg〜100ng）および3H1-Alugel（２μg〜100ng）とともに５日間培養し、そして増殖を、[³H]チミジンの取り込みにより測定した。サルPRO723から得た末梢血単核細胞をインビトロで11D10（１mg/ml）；コントロールの無関係のAb2 3H1（１mg/ml）で刺激した。同様に、サルPRO872から得られた末梢血単核細胞を、インビトロで11D10（１mg/ml）；無関係のAb2 3H1（１mg/ml）で刺激した。刺激を、[³H]チミジンパルスの取り込みの程度によって測定した。抗原を全く含まない培養培地をまた、コントロールとして用いた。免疫化後の末梢血単核細胞を、３回目の免疫化後10日目に回収した。結果を、図19に示し、そして３連のウェルの平均cpmとして表す。データのSDは、各決定値について10%未満であった。実験値とコントロール値との間の相違は、統計学的に有意なものであった。ポジティブ増殖応答は、サルPRO872およびPRO723の両方において見られ、これはマイトジェンフィトヘマグルチン-Pへの応答にほぼ匹敵した。前免疫細胞は、抗Id抗体に対して増殖応答を示さなかったが、免疫後の細胞は、有意に応答した。また、アイソタイプ適合3H1抗Id抗体への、わずかではあるが重要な応答が存在し、これは11D10応答よりも有意に小さかった；これは、免疫グロブリン分子の非Id成分への応答を表しているようである。増殖応答を、最初３回目の注射後に記録し、そして同様の反応性を、４回目の免疫化後に得た。血液の供給が制限されていたので、本発明者らは、半精製したHMFG抗原の存在下でのＴ細胞増殖を試験できなかった。従って、リンパ球において誘導される細胞性免疫応答の抗原特異性、ならびにこれらのリンパ球の表現型を確立できなかった。
実施例４：11D10のヒトへの投与
注入あたり1、2、4、または8mgのいずれかの11D10でアルミニウム沈降された抗Id 11D10を用いる免疫
患者を、0、14、28、および42日に、皮内注入によって、11D10の４用量のうちの１つを受けるように無作為に選択した。各用量群は、３人から８人の患者を含む。水酸化アルミニウム沈降を、Herlynら(1987)Proc.Natl.Acad.Sci.USA 84.805Sの方法に従って実施する。簡潔には、ワクチンは、0.2% Alu-Gel Sを含有する水酸化アルミニウム沈降後の抗Id 11D10からなる。精製モノクローナル抗Id(Ab2)の５mgのアリコートに、２% Alu-Gel S（Serva Fine Biochem,Inc.,Garden City,Long Island,NY）の1mlを添加する。次に、容量をD-PBSで10.0mlに調整し、そして混合物を撹拌しながら、室温で１時間インキュベートする。次に、混合物を24℃にて10分間、2000rpmで遠心分離する。ゲル層に結合された抗体の量を、分光分析によって、上清中の非結合抗体量を測定することにより、決定する。Alu-Gel沈降されたAbを、使用するまで４℃にて保存する。全ての操作は、層流フード(hood)下で実施される。最終産物を滅菌し、抗Id 11D10-Alu-Gelとして明らかに標識され、発熱物質を含まない滅菌ガラスバイアルに分配する。水酸化アルミニウム沈降されたイディオトープの活性を、ELISAでのAb1 F(ab')2への結合を試験することによってモニターする。最終的に、そのロットの生物学的活性を、記載されているように（Bhattacharya-Chatterjeeら(1987)J.Immunol.139:1354;Bhattacharya-Chatterjeeら(1988)J.Immunol.141=1398）、イディオトープキャリアでの免疫後に、小動物の血清で確かめる。最終充満産物を、モルモットにおいてエンドトキシン、不妊症、および一般的な安全性について、試験する。次いで、水酸化アルミニウム沈降された11D10を、投与前に、水浴中で45℃にて30分間加熱処理する。
体液性免疫
抗抗Id抗体（Ab3）を生じる患者の体液性免疫応答を特徴づける。抗体を、以下の(a)から(e)について評価する：(a)Ab3が実際にAb1抗体を含有するかどうかを決定するための、腫瘍細胞または単離された半精製抗原への結合；(b)細胞傷害性効果のメディエーターとしてのエフェクター細胞または補体による、乳ガン細胞に対する細胞傷害性活性；(c)抗ヒトイソタイプ特異的試薬によるイソタイプの特徴づけ；(d)Ab3のイディオトープ分析（すなわち、Ab1とのイディオトープの共有）；および、(e)Ab3のエピトープ分析（すなわち、Ab1と同じエピトープへの結合）。
アルミニウム沈降されたAb2による免疫によって誘導された、体液性免疫の出現を、プロトコールに示されている種々の時点で、患者から得られた血清を試験することによって評価する。血清を、まず、全ヒト抗マウス抗体（HAMA）応答（抗イソ／アロ／および抗抗イディオタイプ抗体）について、サンドイッチRIAによって試験する。簡潔には、マイクロタイタープレートを11D10でコートし、そして患者血清の種々の希釈物とともにインキュベートする。洗浄後に、抗原-抗体反応は、均一系サンドイッチRIAにおいて、¹²⁵I-標識された抗Id 11D10を用いて標識される。11D10は、無傷の(intact)IgG1として注入されるので、患者はHAMA応答をマウントすることが期待される。
(a)Ab2に応答する特異的Ab3応答：ポジティブHAMA応答を有する、免疫された患者からの血清を、抗抗イディオタイプ抗体の存在について試験する。血清を、イソタイプおよびアロタイプ決定基に対するヒト抗体をブロックするために、正常マウス免疫グロブリンとともにプレイキュベートし、次いで、抗抗Id(Ad3)の存在について、マイクロタイタープレートにコートされた免疫抗Id(11D10)との反応を、RIAによって確認する。無関係のAb2をコントロールとして使用する。洗浄後に、抗原-抗体反応を、上記のように、均一系サンドイッチRIAにおいて¹²⁵I-標識抗Id試薬を用いて標識する。前処理、非免疫血清、および正常ドマーからの血清を、これらのアッセイにおけるコントロールとして使用する。
(b)Ab3のイディオトープ分析：ポジティブ反応が上記(a)で得られる場合、それらの処置患者からのAb3血清を、マイクロタイタープレートに結合されたMC-10(Ab1)への¹²⁵I-標識11D10結合を阻害する能力、またはその逆（プレート上の11D10への放射標識MC-10の結合の阻害）について確かめる。非関連Ab1-Ab2システムを、コントロールとして使用する。この試験は、患者血清中のAb3が、MC-10(Ab1)と、イディオトープを共有するか否かを実証する。Ab3によるAb1-Ab2結合の阻害アッセイはまた、Ab3が真の抗抗イディオタイプであるか否かを実証する。
(c)Ab3の腫瘍抗原への結合：ネガティブ標的抗原に対して指向される体液性免疫応答を評価するために、患者Ab3血清を、HMFGを発現することが知られている細胞株との反応性について、RIAにおいて試験する。さらに、血清を、マイクロタイタープレート上にコートされた、HMFG抗原の可溶化半精製調製物に対する反応性について確かめる。抗原-抗体反応を、ELISAにおいて¹²⁵I-標識抗ヒトIg試薬またはアルカリホスファターゼ標識抗ヒト-Igを使用することにより検出する。免疫前血清および非関連抗原をコントロールとして使用する。HMFG抗原へ結合するヒトAb3血清のイソタイプを、抗ヒトイソタイプ特異的試薬を使用して、ELISAにより決定する。これらの実験について、HMFGを、Laroccaら（1992）Hybridoma 11(2):191-201の方法によって、E.coliから融合タンパク質として得る。
(d)Ab3のエピトープ分析：処置患者において生成されたAb3およびAb1(MC-10)が、同じ抗原決定基に結合することを実証するために、Ab3血清による、Agポジティブ腫瘍細胞株MCF-7またはHMFG抗原に対するMC-10結合の阻害を、RIAによって確かめる。
(e)Ab3の細胞傷害性活性：患者血清中のAb3が、腫瘍細胞に特異的に結合する場合、エフェクター細胞および／または補体と共合して、これらの細胞を溶解するAb3の能力を、標準ADCC（Chereshら(1985)Proc.Natl.Acad.Sci.USA 83:515）、またはCMCアッセイ（Herlynら(1981)Int.J.Cancer 27:769）によって試験する。しかし、Ab3の細胞傷害性活性はそのイソタイプに依存し得、IgG1は、ADCCにおいて有効であり、およびIgG1、IgG2、IgG3、およびIgMは、CMCにおいて有効である。
患者の細胞性免疫応答に対する11D10免疫の効果の測定
進行性の乳ガンを有する患者の11D10-Alugelでの免疫が、T細胞（そのうちのいくつかが、その腫瘍細胞に対して細胞溶解活性を示し得る）の活性に導くかどうかを試験するために、患者のT細胞は、Ab2βによってインビボで活性化される場合、インビトロでのAb2β、抗原、または増殖を伴う自己腫瘍細胞での刺激、および潜在的なCTL誘導に応答するはずである。
(a)患者の末梢血単核細胞（PBMC）の増殖応答を、免疫抗Id 11D10、コントロールAb2、または半精製HMFG抗原の存在下で試験する。あるいは、PBMCを、照射自己腫瘍細胞（可能な場合、手術中低温保存されるか、または短時間培養で維持される）で刺激する。細胞が増殖する場合、それらは、増殖に関与する細胞のサブタイプを決定するために、フローサイトメトリーによって表現型を分類する。増殖応答は、³H-チミジンの取り込みによって測定され、そして前治療PBMC Stimulation指標に比較される。このアッセイに対して、血液サンプルを、３回および４回の免疫後、および最終治療の１ヶ月後に回収する。
(b)インビトロ細胞傷害性活性：細胞性免疫プロフィールを、自己ガン細胞（可能な場合）、または同種異系MC-10Agポジティブ腫瘍細胞に対する、T細胞の細胞傷害性活性を、⁵¹Cr放出アッセイにおいて、インビトロで試験することによって評価する。Ertleら(22)によって示唆されるように、抗Id抗体の、エフェクター相レベルに限定されたMHCではない、細胞傷害性T細胞を誘導する能力は、標的として自己腫瘍細胞を使用する困難を克服し得、さらに自己腫瘍細胞の使用を促進し得る。乳ガン患者および健常ドナーからの末梢血リンパ球調製物を、バイオビーズに不溶化するか、またはプラスチックプレート上にコートした、抗Id（10ng〜100μgの範囲）の種々の用量とともにインキュベートする。最適数のリンパ球を、Mishell-Dutton培養培地で２ml中の最適濃度のイディオトープワクチンとともに、135mmペトリ皿中で培養する。細胞を５〜６日後に採集し、記載(84)されるように、⁵¹Cr標識標的に対する、４時間または18時間の⁵¹Cr放出細胞傷害性アッセイにおけるエフェクター細胞として使用する。可能な場合はいつでも、自己腫瘍細胞（診断中または治療前に低温保存される）を、標的細胞として使用する。新鮮腫瘍細胞株を、ヌードマウスまたは細胞培養において、増殖および維持することにより、少なくとも選択された何人かの患者において、自己細胞の供給を有するこのは可能であり得る。同種異系MC-10 Agポジティブ腫瘍細胞株はまた、標的として同時に試験される。腫瘍細胞殺傷が存在する場合、特異性を特徴づけるために以下の実験を行う：
(i)リンパ球が、乳腫瘍細胞に対して細胞傷害性になる場合、それらを、抗原を発現しない多数の標的（すなわち、リンパ球、その他のガン腫細胞株）を用いる細胞傷害アッセイにおいて試験する。細胞傷害性の欠損は、標的が、おそらく乳ガン関連Agであることを示唆する。
(ii)リンパ球を、⁵¹Cr-標識腫瘍細胞を用いる細胞傷害性アッセイにおいて使用および試験される、Ab2βと同じイソタイプの非関連抗イディオタイプハイブリドーマとともにインキュベートする。細胞傷害性の欠損は、抗Idの効果が特異的であることを示す。
別の実験において、患者のリンパ球を、照射自己腫瘍細胞（可能な場合）とともにインキュベートする。細胞を５〜６日後に採集し、そして生細胞を、リンパ球分離培地で精製する。細胞傷害性を、⁵¹Cr-標識自己標的腫瘍細胞に対して測定する。
このインビトロ細胞傷害性アッセイは、前治療後に、すなわち、４回の免疫および最後の免疫の１ヶ月後に得られた血液から単離されたPBMCを利用する。
11D10の最適用量の決定
さらなる臨床試験用の最適用量を、２つの基準に従って選択する：
(a)最大Ab3応答を誘導するAb2用量を決定する。これは、阻害アッセイによって決定され得る。
(b)最大Ab3結合を誘導するAb2用量は、腫瘍に応答する（Ab1'応答）。
Ab3およびAb1'応答の定量
患者血清中の抗抗Id抗体（Ab3）の発現を、RIA阻害研究によって、以下のように定量する。簡潔には、マイクロタイタープレートをMC-10-IgG1（Ab1）でコートし、そして¹²⁵I-標識Ab2の固定化量と反応させる。標準阻害曲線を、インヒビターとして精製MC-10 IgG1を使用して作成する。次に、抗イソアロタイプ活性を涸渇された患者血清を、異なる希釈でのAb1-Ab2反応を阻害する能力について確認し、そして血清中のAb1様抗体の量を、標準阻害曲線から評価する。Ab3応答の誘導および持続期間を、種々の用量レベル間で比較し得る。多数の用量で、Ab3応答または持続期間の間に実質的な差異が存在しない場合、半精製HMFG抗原でコートされたプレートに対するELISAアッセイにより、血清中の特異的抗腫瘍応答(Ab1')の力価を比較する。
インビトロ研究。Ab1'またはAb3ポジティブな患者血清が循環する場合、それは、患者の腫瘍細胞もしくは循環腫瘍抗原に（Agが血液中に微少量分泌されたとしても）結合し得るか、またはそれらが低親和性であるかを示し得る。患者のPBMCを、腫瘍特異的抗体の誘導のために、抗原またはAb2を用いてインビトロで刺激する。これには、治療前および次いで４回免疫の１ヶ月後に回収される血液から得た末梢血単核細胞（PBMC）を、改変Mishell-Dutton培養（DeFreitasら(1985)Curr.Top Microbiol.Immunol.119:75）において、種々の濃度の11D10、または非関連Ab2、またはHMFG抗原（10μg〜110ng）とともに培養する。培養上清を採集し、そしてまず最初に、特異的ヒト免疫グロブリン産生についてELISAアッセイにより確認し、そしてラジオイムノアッセイによりAb2の不溶化調製物への結合について確認する。さらに、上清を、¹²⁵I-標識MC-10（Ab1）とAb2βとの間の反応を阻害する能力によって、イディオトープ保有分子の含有量について試験する。上清を、MC-10 Ag-ポジティブヒト乳ガン腫細胞、およびAg-ネガティブリンパ球とのそれらの反応性について、Ab1'抗体評価用のRIAまたはELISAアッセイ（感度＞1ng）によって、¹²⁵I-標識抗ヒトIg試薬を用いる結合アッセイにおいてもまた、確認する。
Ab2βの効果の特異性は、同じイソタイプの非関連Ab2とともに、PBMCをインキュベートすることによってモニターする。Ab3ポイジティブ患者のみがこのインビトロ研究に包含されるので、Ab2βで刺激されたPBMCは、Ab2β（11D10）に結合する抗体を分泌し、そしてポジティブコントロールとして作用するはずである。
Ab4応答の潜在的な誘導
ネットワーク仮説(network hypothesis)に従って、Ab2で免疫された患者はまた、Ab2の特異性を模擬し得る、Ab4応答（抗抗抗Id）を最終的に誘導し得る。この可能性を研究するために、Ab3ポジティブ患者の（抗イソおよび抗アロタイプ抗体の涸渇された）血清を、記載されてるように、ELISAまたはRIAによって、MC-10（Ab1）と反応させる。ポジティブおよびネガティブコントロールを、記載のように、Ab3アッセイに対して包含させる。このアッセイのための血清を、最終治療の３ヶ月後に得る。
実施例５：進行性HMFG関連疾患を伴う患者への11D10の投与によって誘発された免疫応答の分析
INDを、アルミニウムで沈降された抗Id 11D10を有する乳ガン患者の臨床治験用に、米国食品医薬品局から得た（BB-IND番号5745）。５人の患者を、フェーズIb試験に登録した。全患者は、以前に標準的な治療法によって治療された進行性の乳ガンを有し、彼らの腫瘍細胞は、モノクローナル抗体MC-10（BrE1）によって同定すると、乳ガン抗原HMFGに対してポジティブであった。患者を注射あたり、１mg、２mg、４mg、または８mg用量の11D10-Alugel（アルミニウム）のいずれかに無作為化した。彼らを、最低４回の注射に対して隔週に皮内注入により免疫した。治療は、患者の疾患が快方に向かうまで、月基準で継続した。
第１の患者は、これまでのところ、８mgの８回免疫を受け；第２の患者は、4mgの４回の免疫を受け；第３の患者は、２mgの４回免疫を受けた。第４および第５の患者は、それぞれ、１mgおよび４mgの用量を受け、最近研究に加えられた。
毒 性
毒性は、注入部位で軽度の紅斑(erytherma)および硬化を伴った局所反応のみを有し、最小であった。抗Id処置は、造血細胞、腎機能、または肝機能に対して、有害な影響を何も有さなかった。
抗イディオタイプに対する体液性応答
アルミニウム沈降された抗Id 11D10（実施例４に記載されたように調製された）での免疫によって誘導された体液性免疫の出現を、治療前およびワクチンでの各治療後の患者からの血清を試験することにより評価した。第１の患者３人からの超免疫血清（４回免疫後）は、免疫Ab2 11D10に対する抗イソ／アロ／抗抗イディオタイプ応答を含む、全ヒト抗マウス抗体応答の有意なレベルを示した。第１の患者からの典型的なデーターを図20に示す。次に、これらの患者からの血清を、ラジオイムノアッセイによって、プレート上で、¹²⁵I-MC-10（Ab1）のAb2 11D10への結合またはその逆（プレート上での放射標識Ab2のAb1への結合）を阻害する能力について確認した。これらの反応を、イソタイプおよびアロタイプ決定基に対するヒト抗体をブロックするために、過剰の正常マウスIgの存在下で行った。図21は、第一患者についてのデーターを示す。約40%の阻害が、血清の1:40希釈にて得られたが、一方、９%および22%のみの阻害が、同じ希釈にて、第２および第３の患者血清によって得られた。７回の免疫後に、第１の患者は、血清の1:40希釈で83%の阻害を示した。全患者３人からの免疫前血清は、阻害を示さなかった。これらの結果は、患者１が、抗抗イディオタイプ抗体（Ab3）を有意にマウントし、そして他の２人の患者は、ある程度のAb3反応性を有したことを示す。
次に、抗Id 11D10が、抗腫瘍抗原（HMFG）の特異的抗体応答を、免疫された患者において誘導し得るか否かを、調査した。これについては、４回の免疫後に得られた血清を、マイクロタイタープレート上にコートされたHMFG抗原（Dr.Cerianiから得た融合タンパク質;Laroccaら(1992)）に対して、ELISAアッセイによって試験した。結果を表５に示し、そして３連のウエルの平均として表す（S.D.＜10%）。

患者１および３からのAb3血清は、免疫前血清と比較すると、HMFG抗原への特異的結合を示した。患者２からの免疫前および免疫後血清は両方とも、HMFGへの非特異的結合を示し、これは、免疫によって増大しなかった。
抗イディオタイプに対する細胞性免疫応答
細胞性免疫応答を、アルミニウム沈降させた抗Id 11D10、およびイソ、アロタイプ適合性コントロール抗Id 3H1とともにインキュベートした、末梢血単核細胞の増殖によって測定した。ポジティブ増殖応答は、患者１のみにおいて認められたが（図22）、他の２人の患者においては認められなかった。患者１からの免疫前細胞は増殖応答を有さなかったが、一方、超免疫細胞は、抗Id 11D10に対して有意な応答を有した。コントロール抗Id 3H1に対する応答もまた存在した；この応答は、11D10応答よりも有意に弱く、マウス免疫グロブリン分子の非イディオタイプ成分に対する応答を示すようである。
結果は、抗Id 11D10が、進行性乳ガン患者において、体液性および細胞性免疫応答の両方を誘導し得ることを示唆する。
実施例６：11D10ポリペプチドフラグメントをコードする組換えワクシニアベクターの構築
プラスミド構築および組換えワクシニアウイルスの産生
一般ワクシニアベクター（rvv）の構築スキームを図18に示す。本発明者らは、ワクシニアウイルス野生型WR株のTK遺伝子の全配列（GenBank,受託番号J02425）を、国立バイオテクノロジー情報センター（NCBI）よりBLASTプログラムを用いて取得した。Aitschulら(1990)J.Mol.Biol.215:403-410。配列データから、順方向および逆方向PCRプライマー5'-CAGATGGAAGGGCCCAAC(配列番号42)および5'-GATTGATGCATATCATTACC(配列番号43)を合成した。これらはそれぞれTK配列のヌクレオチド22〜39および727〜708に相当する（Hrubyら(1983)Pro.Natl.Acad.Sci.USA 80:3411-3415)。プラスミドpGEM-7Zf(+)（Promega）へ挿入するため、Apa I部位（下線）を順方向プライマーに導入し、Nsi I部位（下線）を逆方向プライマーに導入した。ワクシニアの野生型WR株由来のDNAを単離し、TK遺伝子をPCRで増幅した。予想通りのサイズ（約700bp）のDNAフラグメントがPCRで得られた。このDNAを、低融点アガロースにおける電気泳動で分離し、GELase(Epicentre Tech.)で消化することにより精製した。Apa IおよびNsi Iでの消化後、TK DNAフラグメントをpGEM-7Zf(+)に連結した。得られたプラスミド（pGEM-TK）を標準的な形質転換法で増幅した。挿入を制限マッピングにより確認した。
プロモーター7.5Kを野生型ワクシニアウイルスから、7.5Kプロモーター配列のヌクレオチド69〜88および335〜312に相当する、順方向プライマー5'-GTTATCGATGTCGAATAGCC(配列番号44)および逆方向プライマー5'-TTGCTGCAGATTGAGTACTGTTCT（配列番号45）を使用してPCRにより増幅した。Cochranら(1985)J.Virol.54:30-37。Cla I部位（順方向）とPst I部位（逆方向）をプライマーに含ませた。増幅したDNAフラグメントをPst Iで消化した。5'末端をポリヌクレオチドキナーゼでリン酸化したより小さいオリゴヌクレオチドを有するポリヌクレオチドアダプターを合成した。半リン酸化したアダプターをPst Iで消化したPCR増幅7.5KプロモーターDNAフラグメントに連結した。産物をCla I/Eco RI消化pGEM-TKで消化した。
11D10ポリペプチドをコードするcDNA挿入物を、pVVのNco I部位とXmaI(Sma I)部位との間に挿入する。このプラスミドはまた、scFv cDNAの5'末端にV_Hのリーダー配列を含む。所望であれば、E.coliβガラクトシダーゼのcDNAを含むワクシニアコントロールプラスミドが構築され得る。
rvvの構築
rvvを、Mackettら（DNA Cloning，第２巻、D.M.Glover編、IRL Press 1985）の手順に従って、CV-1細胞を用い、ワクシニアプラスミドとワクシニアウイルスの野生型WR株との相同組換えによって構築する。βガラクトシダーゼ（コントロール）を発現する組換えウイルスクローンを、5'-ブロモデオキシウリジンと5-ブロモ-4-クロロ-3-インドイル-β-D-ガラクトシダーゼ(X-Gal)の存在下でTK 143B細胞上での増殖により選択する。青色の組換えウイルスを、パスツールピペットで拾いプラークを精製する。クローン選択の第二工程として、11D10cDNAをプローブとして使用し、抽出したDNAのサザンブロットを行う。rvvの更なる選択を、ウイルス感染したCV-1または他の任意の真核細胞の培養上清をELISAによりアッセイすることにより行う。細胞に結合した11D10ポリペプチドがrvvに存在する場合（すなわち、リーダー配列が欠失されている場合）、細胞溶解物をアッセイする。MC-10(Ab1)をプローブとして用いるウェスタンブロットもまた行う。ワクシニアウイルスによって合成された11D10ポリペプチドの生物学的活性を、上記のように細胞結合阻害によって決定する。11D10ポリヌクレオチドを含むrvvクローンを、0.1%のニュートラルレッドで染色することによって選択し、上記のようにプラークを精製する。ウイルスクローンを、標準技術を用い、高力価溶解物中に増殖させる。Mackettら(1982)Proc.Natl.Acad.Sci.USA 79:7415-7419。典型的には、最高量の11D10ポリペプチドを産生するクローンを、さらなる研究のために選択する。
組換えワクシニアウイルスによって発現された11D10ポリペプチド（外来タンパク質）のアッセイ
CV-1細胞を、10％ウシ胎児血清ならびに1ml当たり100単位のペニシリンおよび100μgのストレプトマイシンを補充したDulbeccoの改変Eagle培地(DMEM)で、25cm²フラスコあるいは６ウェルのクラスターフラスコにおいて培養する。細胞にrvvを30のMOIで接種する。ウイルスを2時間、37℃で組織培養インキュベーターにおいて吸収させ、その後、接種物を培養培地で置換し、そしてインキュベーションを継続する。示された時間のインキュベーション後、上清を取り出し、分泌された11D10ポリペプチドをアッセイする。コントロールとして、模擬感染させた細胞からの上清を使用する。11D10ポリペプチドのアッセイを、例えば実施例１および５に記載されたように、MC-10(Ab1)との結合について試験することによって、実行し得る。rvv-lacZによって産生されたβ-D-ガラクトピラノシドを、Miller（Experiments in Molecular Genetics,Cold Spring Harbor Pines 1972）に従って、p-ニトロ-β-D-ガラクトピラノシドを基質として用いてアッセイする。ウイルス感染細胞の培養上清を、アッセイの前にウイルスを不活化するためにβ-プロピオネートで処理する。Corcoranら(1988)J.Parasit.74:763。³HチミジンのNFS60細胞による取り込みを、細胞増殖の尺度として使用した。Jaffeeら(1993)Cancer Res.53:2221-2226。模擬感染CV-1細胞由来の上清の存在下での、³Hチミジン取り込みによる放射能をバックグラウンドとして差し引いた。ポジティブコントロールとしておよび生物学的活性の標準として、インタクトな11D10を使用する。あるいは、GM-CSFの標準溶液を使用し得る。
ワクシニア11D10ワクチンの試験
ワクシニア投与については、10⁴〜10⁷pfuのウイルス価をマウスに注射する。注射は皮下、筋肉内、皮内または腹腔内であり得る。免疫は毎週行う。マウスはAb3（Ab1'を含む）の測定のため各免疫の７日後に採血する。Ｔ細胞免疫性の発展についての試験を追加免疫の10日後に行う。マウスをまた、腫瘍チャレンジによって試験し得、そこで腫瘍細胞注射後の生存をモニターする。
ウイルス感染腫瘍細胞を介するワクシニア投与については、自己腫瘍細胞を、10％（vol/vol）のウシ胎児血清、２mMグルタミンおよびゲンタマイシンを含むEagle培地中で維持する。75cm²フラスコ中のコンフルエント細胞の単層に、（3×10⁸）プラーク形成単位（pfu）のrvvを接種する。37℃で２時間後、接種物をDMEMに置換し、インキュベーションをさらに24時間継続した。顕微鏡下での検査の後、細胞をスクレーパーで回収し、PBSで２回洗浄し、所望の濃度（10³〜10⁵/200μl）でPBSに再懸濁する。６〜８週齢の雌性C57BL/6マウスをHarlan Bioproducts for Science Inc.（IN）から購入する。動物の左後脇腹に細胞性ワクチンを皮下注射し、２週間後、右後脇腹にチャレンジ用に腫瘍細胞を注射する。腫瘍チャレンジ後、マウスの生存および腫瘍の存在を毎日モニターする。腫瘍が測定可能であれば、腫瘍を週毎にカリパスで二次元に測定し、体積を式（幅²×長さ）／２を使用して計算する。明白であるが、体積を正確に測定するには小さすぎる腫瘍は０体積として記録されるが、腫瘍発現はポジティブとして記録される。腫瘍体積は、120日の観察期間にわたって実際に腫瘍を発生したマウスについてのみ平均する。０値は、最終的には腫瘍を発生するが所定の時点では腫瘍がなかったマウスについて含める。
統計学的評価
統計学的評価はSigmaStatソフトウェア（Jandel,Inc.San Rafael CA USA）を用いて行う。0.05未満のP値を統計学的に有意とみなす。
実施例７：11D10融合タンパク質をコードする発現ベクターの構築
11D10の重鎖のVDJ可変領域をコードするcDNAを、プラスミドpUC911D10Vから単離し、図25に示したベクターに連結した。このベクターではマウスGM-CSFあるいはIL-2の成熟ペプチドをコードするDNAフラグメントを、CH₃エキソンの3'側に連結した。これはCH₃エキソンの最終コドンと停止コドンとの間にCla I部位、そしてその停止コドンの3'側にNotI部位を生成することによって実現された。11D10の軽鎖をコードするcDNAを、図25に示した発現プラスミドpSV184-ΔHneoに組み込んだ。pSV184ΔHneoプラスミドはShinら((1989)Meth.Enzymol.178:459-476)によって以前に記載された。
最初のトランスフェクション後、11D10の軽鎖を含むプラスミドを、Shinら(1989)の変法に従い、エレクトロポレーションにより以下のように上記細胞にトランスフェクトした。細胞を遠心分離によって増殖培地から取り出し、Iscoveの改変Dulbecco培地（IMDM,GIBCO）で２回洗浄した。細胞を4.5ｘ10⁶/mlの濃度で冷IMDM（血清なし）に再懸濁した。この懸濁液から0.9ml（4×10⁶細胞）を0.4cm BioRadキュベットに加えた。約20μgのpSV211D10V_H-サイトカインDNAを、pSV211D10V_H-サイトカインのEco RI部位の間に存在しない制限酵素（例えばPvuII）で線状化した。フェノール/クロロホルム抽出およびエタノール沈澱の後、DNAを50μl未満のIMDMに懸濁し、細胞懸濁液に加えた。細胞懸濁液-DNA混合物を氷中で10分間冷却した後、Bio-Rad Gene Pulser Transfection Apparatusにより200V、960μFの電気容量で電気パルスを印加した。10分間の氷中でのインキュベーション後、細胞を、10％ウシ胎児血清を含む96mlのIMDMに懸濁し、８つの96ウェルのプレートに、マルチチャンネルピペットにより125μl／ウェル（5×10³細胞／ウェル）で分配した。２日後、１ウェル当たり10％ウシ胎児血清および25μg／mlのマイコフェノール酸を含む125μlのIMDMを加えた。４日後、培地を各ウェルから半分除去し、125μlの1×IMDM-マイコフェノール酸培地を加えた。クローンが可視化した後（約10〜15日後）、トランスフェクトーマの検出のためのMC-10でのアッセイのために培養上清を取り出した。
表６は、構築した種々の発現プラスミドを示す。mIL-2およびmGM-CSFは、マウスのIL-2およびGM-CSFをそれぞれ意味する。V_HおよびV_Lはシグナルペプチドを含んでいた。

高産生トランスフェクトーマの検出には、細胞を限界希釈（１細胞／ウェル）後、96ウェルのプレートにプレートする。マイクロウェル中のクローンが可視化したが、まだ小さなままである場合、培養上清を、機能的な抗体の検出について、サンドイッチラジオイムノアッセイによりアッセイする。96ウェルプレートをMC-10でコートし、トランスフェクトーマからの50μlの培養上清を、コートした抗体と反応させる。各トランスフェクトーマにより産生される機能的な抗体の量は¹²⁵I-標識MC-10によるラジオイムノアッセイによって決定される。高抗体産生トランスフェクタントのさらなる評価については、選択したクローン由来の培養上清の種々の希釈物を同様にアッセイする。
重鎖GM-CSF融合物を含むプラスミドを、Oiら（1986）BioTechniques 4:214-221の技法を使用し、原形質融合によりSp2/0細胞にトランスフェクトした。高産生クローンを融合タンパク質の最初の生化学的特徴付けのために選択した。
２回のELISAを以下のように実施した。アッセイ１では、マイクロタイタープレートを標準濃度のヤギ抗ヒトκ軽鎖（Orgomon Teknika Corp.West Chester,PA）でコートし、BSAでブロックし、そして洗浄した。種々の試験構築物を発現する細胞の培養物由来の上清を、次にウェルでインキュベートした。洗浄後、ウェルにアルカリホスファターゼ結合重鎖特異的ヤギ抗ヒトIgG1（Sigma）を重層し、通常の方法で発色させた。陽性反応は、上清が、予測したタイプのヒト重鎖および軽鎖の定常領域の両方を有する免疫グロブリンを含んでいることを示した。アッセイ２を、ビオチン結合ラット抗マウスGM-CSF（Pharmingen）、続いてアビジンペルオキシダーゼ結合物（Sigma）を使用して発色させた以外は類似の様式で行った。陽性反応は、上清が、ヒト軽鎖およびGM-CSF成分（重鎖Ｃ末端に期待される）を有する免疫グロブリンを含んでいることを示した。
表７は、９クローンの上清を重鎖にカップリングした軽鎖の存在（アッセイ１）およびGM-GSF活性（アッセイ２）についてアッセイした結果を示す。結果は、軽鎖、重鎖、およびGM-CSFについて決定基を同じ分子上に含む、多くの結合タンパク質が得られたことを示す。
融合タンパク質を、Sepharose-protein Aカラムを使用するアフィニティークロマトグラフィーにより単離し、下記のように以下の抗体および標準を用い特徴づける：モノクローナルマウス抗ヒトIL-2（Genzyme,code,DNA-1）、標準天然ヒトIL-2（BRL cat.#13288-014）；モノクローナル抗マウスIL-2（UBI,cat.#05-115）、標準マウス組換えIL-2（Sigma,cat,#I 4517）；ヒトGM-CSF標準を用いたヒトGM-CSFアッセイキット（R&D System cat.DGM00）、ラット抗マウスGM-CSF（BRL,cat.#13306-014）、標準マウス組換えGM-CSF（UBI,cat.#01-167）。

精製したキメラ（すなわち融合）タンパク質を、還元条件下および非還元条件下でSDSポリアクリルアミドゲル電気泳動により分析した。分子量標準および精製した11D10をこの実験に含めた。タンパク質のバンドをクーマシーブリリアントブルー染色により染色した。MC-10（Ab1）と11D10（元のAb2）およびHMFG（名目上の抗原）との結合に対する精製融合タンパク質の効果を、阻害RIAにより、この融合タンパク質のAb1結合特異性を確立するためのAb2の特徴付けのために、予め行ったように研究した。標識Ab1とSKBR3（HMFG発現乳ガン細胞株）細胞との結合の融合タンパク質による阻害は、抗原抗体結合の特異性についてのさらなる支持を提供する。マウスIL-2の生物学的活性を、CTLL-2Ｔ細胞の懸濁液を用いる細胞増殖アッセイにより決定する。サンプルおよび標準を、完全RPMI-10培地に系列希釈し、50μlのアリコートを96ウェルプレートのウェルに分注した。CTLL-2細胞を活動対数期まで増殖させ、完全RPMI-10で洗浄して、残りのIL-2を除去する。細胞を完全RPMI-10中に1×10⁵細胞／mlで懸濁する。細胞を３群に分割し、１セットにモノクローナル抗マウスIL-2を投与し、１セットに抗ヒトIgGγ鎖を投与し、そして残りの１セットにこれらの抗体の希釈に使用する溶液を投与する。細胞懸濁液（50μl、5×10³細胞）を各ウェルに加える。細胞をCO₂インキュベーターで37℃にて24時間インキュベートする。³H-チミジンを加え、インキュベーションをさらに24時間継続し、その後細胞を回収し、そして取り込まれた放射能を測定する。抗体の有無によるウェル中のカウントの差分を、サンプルあるいは標準の正味の生物学的活性とみなす。11D10もまたこれらアッセイに含め、抗体自体がCTLL-2の細胞増殖を誘導し得るかどうかを決定する。ヒトIL-2の生物学的活性を、特異的な抗体の存在下および非存在下で、CTLL-2細胞を用いて同様に評価した。GM-CSFの生物学的活性については、類似の細胞増殖アッセイを行う。マウスおよびヒトのGM-CSFについては、NFS-60細胞（Holmesら(1985)Proc.Natl.Acad.Sci(USA)82:6687-6691）およびM-07e細胞（Genetics Institute）をそれぞれ使用する。
データ解釈、予想される結果、起こり得る問題および代替のストラテジー。還元条件下でのSDS-PAGEは、11D10および融合タンパク質の両方について25Kd付近のタンパク質バンドを示すと予測される；より高分子量のバンドは、トランスフェクトーマが重鎖と融合したサイトカイン−抗体融合タンパク質を産生したことを示す。非還元条件下の電気泳動により、テトラマーの免疫グロブリンが形成されたかどうかを決定することは可能である。その場合、融合タンパク質は一本のバンドを形成し、その分子量はサイトカイン分子の２倍に相当する量だけ高いはずである、すなわち、融合タンパク質は、サイトカインに関してはダイマーである。サイトカインに対するELISAおよび生物学的アッセイは、サイトカイン分子が発現されているかどうか、そして生物学的に活性であるかどうかを示す。標準組換えサイトカインを用いる定量アッセイは、融合タンパク質として存在するサイトカインの生物学的活性が、遊離のサイトカインと比較して、匹敵するかあるいは増強されているかどうかを示す。コントロールの抗体と比較して対応する抗体による、サイトカインの生物学的活性の阻害は、サイトカインの作用が特異的であることを示す。抗ヒトIgGγ鎖による、サイトカインの生物学的活性の阻害は、サイトカイン部分がIgとの融合分子として存在していることを示す。
実施例８：11D10scFの発現および特徴付け
11D10のためのV_H-(GGGS)₃-V_L（配列番号46）をコードするcDNA構築物を調製する。この11D10フラグメントに対するcDNAを、pET-22b(+)プラスミドベクター（Novagen,Madison,WI）に組み込み、E.coli中で発現させる。配列分析を行い、V_Lのフレームワークに結合したV_Hのカルボキシ末端を含みリーダー領域を含まないプラスミド構築物を確認する。pET-22b(+)は、６つの連続したヒスチジン残基（His₆）からなるニッケルイオン結合ドメインを含む。His₆ドメインは、ニッケルに対して高親和性を有し、これを組換え11D10scFvの精製に使用する。
細胞結合競合アッセイを、11D10scFvが、インタクトな11D10によって示される抗原模倣物を保持しているかどうかを調べるために行う。HMFGポジティブMCF-7あるいはSKBR3（50μl容量で1×10⁵細胞／ウェル）を96ウェルプレートに播く。細胞を、[¹²⁵I]MC-10（Ab1）（100,000cpm）とともに室温で2時間、漸増濃度の11D10あるいは11D10scFvフラグメントの非存在下あるいは存在下でインキュベートする。％阻害を以下の式に従い計算する。

ここで、R₇は実験ウェルの平均放射能であり、R_MAXはタンパク質が全く存在しないときの放射能であり、そしてR_Cはバックグラウンドの放射能である。
実施例９：マウスにおける組換え11D10ポリヌクレオチドワクチンの試験
組換え候補11D10ポリヌクレオチドワクチンを本明細書中に記載のように調製する。10〜15匹の雌性C57BL/6マウス（６〜８週齢）からなる２群を、Bhattacharya-Chatterjeeら(1988)により記述されるグルタルアルデヒドを使用してKLHとカップリングした50〜100μg用量の精製プラスミドで筋肉内に免疫した。
さらに、種々の投与経路（例えば、筋肉内、皮内、皮下、および腹腔内）を比較する。
Ab3（Ab1'を含む）産生を確認するために、各免疫の７日後に、上記のようにマウスから採血する。追加免疫の10日後に、Ｔ細胞増殖アッセイ用の脾臓を単離するために、各群３匹のマウスを屠殺する。
観察された任意の効果が特異的であるか否かを決定するため、非特異的な体液性あるいは細胞性免疫（注射されたポリヌクレオチドによって誘導されるサイトカイン産生のような間接メカニズムによる）との対比として、以下のコントロールを使用する：(a)11D10ポリヌクレオチドインサートを有さないプラスミド；(b)11D10ポリヌクレオチドインサートを逆（すなわちアンチセンス）方向に有するプラスミド；および(c)無関係のAb2をコードするポリヌクレオチドを含むプラスミド。
実施例１０：HMFG関連疾患が進行性の患者への11D10の投与により誘発される免疫応答のさらなる解析
実施例５に記載のようなミョウバン（BB-IND#5745）で沈澱させた抗Id 11D10を用いた乳ガン患者の臨床試験のための米国FDA第Ib相を、12人の患者を含むように拡張した。全ての患者が進行性の乳ガンに罹患しており、以前に標準的な治療法で処理されており、そして彼らの腫瘍細胞は、モノクローナル抗体MC-10（BrE1）によって規定されるように乳ガン抗原HMFGについてポジティブであった。患者を、ランダムに１回の注射につき1mg，2mg，4mg，または8mgの用量の11D10アルゲル（ミョウバン）に割り当てた。この患者を、皮内注射で１週間おきに最低４回免疫した。患者の疾患が進展するまで、治療を月単位で継続した。患者は疾患の活動について非常に綿密に監視され、そして疾患の進展あるいは死亡のために全員がこの研究からはずされた。12人の患者のこの研究の詳細を表８に示す。

毒性
毒性は、軽い紅斑および硬化を伴う、注射部位での局所的な反応のみとわずかであった。抗Id処置は、造血細胞、腎機能あるいは肝機能に対してにいかなる有害効果も有さなかった。
抗イディオタイプに対する体液性応答
ミョウバン沈澱させた抗Id 11D10（実施例４に記載されたように調製）を用いた免疫によって誘導された体液性免疫の発達を、治療前およびワクチンでの各処置後の患者の血清を試験することにより評価した。10人の患者の内の５人から得た超免疫血清（4回目の免疫後）は、Ab2 11D10での免疫に対して、抗イソ／アロ／抗抗イディオタイプ応答を含む、顕著なレベルの全ヒト抗マウス抗体応答を示した。次に、これらの患者の血清を、ラジオイムノアッセイにより¹²⁵I-MC-10(Ab1)とプレート上のAb2 11D10との結合を阻害する能力あるいはその逆（プレート上のAb1に対する放射標識Ab2の結合の阻害）について、チェックをした。これらの反応を、イソタイプおよびアロタイプの決定基に対してヒト抗体をブロックするために、過剰な正常マウスIgの存在下で行った。
図27AおよびBは、患者１（患者１のデータは実施例５でも議論されている）を含む１０人の患者のデータを示す。患者１、５、６、７および12由来の血清は、希釈率１：100でさえも有意な阻害を示した。患者１、５、６、７および12由来の免疫前血清はいかなる阻害も示さなかった。まとめると、これらの結果は、患者１、５、６、７および12は、有意な抗抗イディオタイプ抗体（Ab3）を生じたが、他の患者（２、３、８、９、および10）は、有意なAb3反応性を生じなかったことを示す。
次に、本発明者らは、免疫患者において抗Id 11D10が抗腫瘍抗原（HMFG）特異的抗体応答を誘導し得るかどうかを調べた。このためには、４回目の免疫後に血清の患者１、６および12から得た血清を、抗Id 11D10カラムでアフィニティー精製し、精製したAb3を、マイクロタイタープレート上にコートしたHMFG抗原（Ceriani博士から得た融合タンパク質；Laroccaら(1992)）に対してELISAアッセイにより試験した。
結果を図28に示す。患者#1、６、および12からの精製したAb3血清は、コントロール抗Id 3H1で処置した大腸ガン患者から得た精製したAb3と比較して、HMFG抗原に特異的な結合を示した。11D10で免疫した患者の血清における抗体（Ab1'）のアイソタイプは、ほとんどがIgGであった。
抗イディオタイプに対する細胞性免疫応答
細胞性免疫応答を、ミョウバンで沈澱させた抗Id 11D10およびイソ、アロタイプがマッチしたコントロールの抗Id 3H1とともにインキュベートした末梢血単核細胞の増殖により測定した。ポジティブの増殖応答は、患者#1、５、６、および12において見られたが、試験した他の患者では見られなかった。図29Aおよび29Bは、それぞれ患者#1および５のデータを示す。患者の免疫前細胞は増殖応答は有さないが、超免疫細胞は、抗Id 11D10に対する有意な応答を有した。コントロールの抗Id 3H1に対する応答も存在した；この応答は11D10に対する反応より弱く、マウス免疫グロブリン分子の非イディオタイプ成分に対する応答を表しているようである。
この結果は、実施例５の知見は、抗Id 11D10が、進行性の乳ガン患者（他の多くの処置も受けていた）において、体液性および細胞性の両方の免疫応答を誘導し得ることを支持することを確証する。
前述の発明は、明瞭な理解のために例示および実施例によってある程度詳細に記載されているが、特定の変更および改変を実行することは当業者に明らかである。したがって、記載および実施例は、本発明の範囲を制限すると解釈されるべきではなく、本発明の範囲は添付の請求の範囲によって正確に記載される。
部分配列表
(xi)配列：配列番号１

(2)配列番号２のための情報

(2)配列番号３のための情報

(2)配列番号４のための情報

Cross-reference to related applications
This application is filed on Dec. 20, 1995, with provisional patent application __________ (former U.S. Patent Application No. 08 / 575,762 (attorney reference number 30414-20003.00)) and Jan. 29, 1996. We claim the benefits of filed US Provisional Patent Application ______ (formerly US Patent Application No. 08 / 591,965 (Attorney Inquiry Number 30414-20003.00), all of which are incorporated herein by reference. Incorporated as.
Statement on rights to inventions made under government-sponsored research
The present invention is based on research incentives obtained from the National Institutes of Health (RO1 CA 47860, CA 56701, PO1 CA 59306, CA 57165, RO1 CA 6000, UO1 65748 and CRF incentive PO1 CA 42767). It was made with the help of The US federal government has certain rights in this invention.
Technical field
The present invention relates to monoclonal anti-idiotype antibodies. More particularly, the present invention relates to anti-idiotype antibody 11D10 that elicits an immune response against specific epitopes of human milk fat globules (HMFG), and polynucleotide and polypeptide sequences for 11D10.
Background of the Invention
Despite extensive medical research and many advances, cancer remains the second leading cause of death in the United States. In women, breast cancer is the most common cause of cancer death. Conventional treatment modalities (eg, surgery, radiation therapy, and chemotherapy) are widely used and there are many successes, while still significant failures exist, especially for solid tumors. Mortality from cancers such as breast cancer remains high, and there is a strong need for alternative modalities.
Immunotherapy of human cancer using tumor cells, or tumor-derived vaccines, has been hampered for several reasons. It is consistently difficult to obtain large amounts or purified tumor-associated antigens. These are often chemically ambiguous and difficult to purify. Furthermore, they still have the problem of immunobiological response to tumor antigens. In other words, the question is whether cancer patients can effectively increase their immune responses to their tumors. Tumor associated antigens (TAA) are often part of "self" and usually have a very poor immune response in tumor-bearing hosts due to tolerance to the antigen (eg, T cell mediated suppression) To trigger. Furthermore, cancer patients tend to be immunosuppressed and respond only to certain T cell-dependent antigens.
Immunobiologists have learned that by changing the molecular environment, weak antigens (with respect to eliciting an immune response) can be converted into strong antigens. Altering the hapten carrier activates T cell helper cells and makes the overall immune response more potent. Thus, changing carriers can also convert tolerant antigens into effective antigens. McBridge et al. (1986)Br.J.Cancer 53: 707. Often, the immunological status of cancer patients is suppressed, so that patients can only respond to specific T-dependent antigens and not to other antigenic forms. In view of these, it makes sense to introduce molecular changes to tumor-associated antigens before using them in patients as vaccines. Unfortunately, this is not achievable for most tumor antigens. Because they are not well identified and their purification is very difficult.
Lindemann ((1973)Ann.Immunol.124: 171-184) and Jerne ((1974)Ann.Immunol.125: 373-389) provides an excellent approach for converting epitope structure into idiotypic determinants expressed on the surface of antibodies. According to this network concept, immunization with a given tumor-associated antigen results in the production of an antibody against this tumor-associated antigen (referred to as Ab1), which in turn is a series of anti-idiotypic antibodies (referred to as Ab2) against Ab1. Used to make. Some of these Ab2 molecules can effectively mimic the three-dimensional structure of tumor-associated antigens identified by Ab1. These particular anti-idiotypes, called Ab2β, fit the Ab1 paratope and express an internal image of the tumor-associated antigen. Ab2β can induce a specific immune response similar to that induced by the original tumor-associated antigen and can thus be used as a surrogate tumor-associated antigen. Immunization with Ab2β can lead to the generation of anti-anti-idiotype antibodies (Ab3) that recognize the corresponding original tumor-associated antigen identified by Ab1. Because of this Ab1-like reactivity, it is also called Ab3 or Ab1 ′, indicating that Ab3 may differ from other idiotypes derived from Ab1.
A promising approach to cancer treatment is immunotherapy using anti-idiotype antibodies. In this form of treatment, antibodies that mimic the epitope of the tumor-associated protein are administered in an effort to stimulate the patient's immune system against the tumor via the tumor-associated protein. WO 91/11465 describes a method of stimulating an immune response against malignant cells or infectious agents using primate anti-idiotype antibodies in humans. However, not all anti-idiotype antibodies can be used in a treatment regime for tumors. First, antibodies raised against Ab1 are only partially limited in their reactivity to Ab1 paratopes (ie non-reactive with characteristics common to other potential antibodies of the host). Sex). Second, anti-idiotype antibodies are not necessarily immunogenic. Third, even though anti-idiotypes elicit an immune response, only a few of these immunogenic anti-idiotypes elicit an immune response against tumor antigens and other less specific Does not induce against antigen. Furthermore, since different cancers have widely varying molecular and clinical features, it is suggested that anti-idiotypic therapy should be evaluated based on tumor origin and antigen expression, as the case may be. The
Anti-Id monoclonal antibodies that are structurally similar to tumor-associated antigens are used as antigen substitutes in cancer patients. Herlyn et al. (1987)PNAS 84: 8055-8059; Mittleman et al. (1992)PNAS 89: 466-470; Chatterjee et al. (1993)Ann.NYAcad.Sci.690: 376-377. Anti-Id has been proposed to provide partial analogs of tumor-associated antigens in an immunogenic situation.
Human milk fat globules (HMFG) are milk fat globules secreted into breast milk by breast epithelial cells and consist of lipid droplets encased in a plasma membrane. Thus, HMFG is a rich source of overcoat-bound antigen. One antigenic component of HMFG is a high molecular weight, membrane-bound mucin associated with breast cancer and other cancers such as the ovary, lung, and pancreas. This mucin contains a protein having a known amino acid sequence derived from cDNA. Semi-purified HMFG is available in small quantities from several sources and can be used in plasmaological assays. Peterson et al. (1990)Hybridoma 9: 221-235. However, HMFG is very difficult to isolate and purify, and purified HMFG cannot be used for patient immunization or animal experiments. Furthermore, because some epitopes on HMFG are common with normal tissues, particularly non-penetrating glycoproteins, immunization with intact HMFG molecules can elicit potentially autoimmune responses. On the other hand, an immune response against tumor-associated epitopes is much more desirable.
A series of mouse monoclonal antibodies (mAbs) that recognize components of HMFG have been described to be associated primarily with human breast cancer and not with most normal cells. Chatterjee et al. (1993)Ann.NYAcad.Sci.690: 376-377; Ceriani et al. (1983)Somatic cell genet.9: 415-427. Among these mAbs, MC-10 (BrE-1) reacts most specifically to mucin-like proteins of breast cancer cells that are more than 80% of the high molecular weight (molecular weight 400,000) present at high density, And it is minimally expressed in some normal tissues such as lung epithelial lining and renal tubules. Ceriani et al. (1983); Ceriani et al. (1990)Antibody Immunoconjugates and Radiopharmaceuticals 3: 181-198.
Recurrent breast cancer cannot be treated with standard treatment. Therefore, new therapeutic approaches for this disease are needed. The present invention overcomes deficiencies in the prior art by providing a monoclonal anti-idiotype antibody (11D10) as an antigen (Ag) that elicits an immune response against HMFG in non-human primates. These may be useful for the treatment of anti-tumor immunity in patients with advanced HMFG-related cancers (such as breast cancer).
All publications cited herein are hereby incorporated by reference in their entirety.
Summary of the Invention
The present invention provides a mouse anti-idiotype antibody 11D10 capable of eliciting an immune response to human milk fat globules (HMFG) against high molecular weight mucins. The present invention also encompasses polypeptides comprising at least part of the variable region of anti-idiotype antibody 11D10, and polynucleotides encoding these polypeptides. The invention also encompasses pharmaceutical compositions and vaccines comprising 11D10, 11D10 polypeptides and / or 11D10 polynucleotides. The invention also includes diagnostic kits and methods using 11D10, 11D10 polypeptides and / or 11D10 polynucleotides, including methods of treating HMFG-related tumors.
Furthermore, the object of the present invention is to provide compositions and methods of use of anti-idiotype (anti-Id) monoclonal 11D10 polynucleotides and polypeptides for inducing anti-tumor immunity in patients with HMFG-related diseases such as breast cancer. It is to be.
Accordingly, in one aspect, the invention encompasses monoclonal anti-idiotype antibody 11D10 produced by hybridoma cell line ATCC Deposit No. 12020 or progeny thereof.
In another aspect, the invention includes a hybridoma cell line designated ATCC Deposit No. 12020 or progeny thereof.
In another aspect, the present invention also includes an isolated polynucleotide comprising a sequence encoding a polypeptide having the immunological activity of monoclonal anti-idiotype antibody 11D10. Here, the polypeptide comprises at least 5 consecutive amino acids of the variable region of 11D10.
In another aspect, the invention is an isolated polynucleotide composed of a region of at least 15 contiguous nucleotides, wherein the region is stable from SEQ ID NO: 5 to SEQ ID NO: 14. Provided is a polynucleotide capable of forming a stable duplex with a polynucleotide comprising the light chain variable coding sequence of SEQ ID NO: 1 under conditions that do not form a hybrid. The present invention is also an isolated polynucleotide composed of a region of at least 15 contiguous nucleotides, which region does not form a stable hybrid with SEQ ID NO: 15 to SEQ ID NO: 32. A polynucleotide capable of forming a stable duplex with a polynucleotide comprising the heavy chain variable coding sequence of SEQ ID NO: 3 under the conditions is provided.
Another aspect of the present invention is a cloning vector and expression vector comprising the polynucleotide of the present invention. Also included are host cells containing the polynucleotides of the invention.
Another aspect of the invention is a polypeptide having the immunological activity of monoclonal anti-idiotype antibody 11D10. Here, the polypeptide comprises a sequence of at least 5 consecutive amino acids from the variable region of 11D10.
In another aspect, 11D10 polypeptides are provided that comprise a region homologous to HMFG.
In another aspect, the present invention provides a fusion polypeptide comprising an 11D10 polypeptide. Polymeric 11D10 polypeptides and humanized antibodies comprising 11D10 polypeptides are also included in the invention.
In another aspect, the invention encompasses pharmaceutical compositions and vaccines comprising an effective amount of 11D10, 11D10 polypeptide and / or 11D10 polynucleotide.
In another aspect, the invention provides a method of eliciting an anti-HMFG immune response in an individual having a progressive HMFG-related disease, wherein the method comprises an effective amount of 11D10, 11D10 polynucleotide and / or 11D10 polypeptide. Administering to an individual.
Another aspect of the invention is a method for removing anti-HMFG antibody from an individual who has received anti-human milk fat globulin (HMFG) antibody, comprising the step of administering 11D10.
In another aspect, the present invention provides a method for detecting the presence of anti-HMFG antibody bound to tumor cells, wherein the method contacts the tumor cells with 11D10 for a time sufficient to bind to the anti-HMFG antibody. And a method of detecting the presence of any 11D10 bound to the anti-HMFG antibody.
In another aspect, a method for detecting an anti-HMFG immune response in an individual is provided. These methods include contacting and forming a biological sample from an individual and 11D10 under conditions that allow a stable complex to form between 11D10 and an antibody that binds to 11D10, and Detecting any stable complex.
In another aspect, a method for detecting an antibody that binds to 11D10 in a biological sample is provided. These methods include contacting a sample-derived antibody from an individual with an 11D10 or 11D10 polypeptide under conditions that allow the formation of a stable antigen-antibody complex, and the stability formed, if any. A process for detecting complex complexes.
Another aspect of the present invention is a method for alleviating human milk fat globule related disease in an individual having a progressive human milk fat globule related disease. These methods require administration of an effective amount of 11D10 to the individual.
In another aspect, the present invention also provides a detection kit or quantification kit comprising 11D10, 11D10 polypeptide or 11D10 polynucleotide in suitable packaging.
These and other objects of the present invention will become readily apparent to those skilled in the art from the following detailed description and drawings. Here, only the preferred embodiments of the present invention are shown and described by way of example only as the best mode of carrying out the invention. As will be readily appreciated, the invention can be modified by those skilled in the art without departing from the spirit and scope of the invention.
[Brief description of the drawings]
FIG. 1 shows the light chain variable region of 11D10 and the cDNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of adjacent residues. CDR and framework regions are shown. Correct translation should indicate A or alanine for amino acid-18 and E or glutamic acid for amino acid 81.
FIG. 2 shows the heavy chain variable region of 11D10 and the cDNA sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of adjacent residues.
FIG. 3 shows the amino acid sequences of the light chain variable region of 11D10 (amino acids 1 to 107 of SEQ ID NO: 2; FIG. 3A) and the heavy chain variable region (amino acids 1 to 118 of SEQ ID NO: 4; FIG. 3B). Each variable region consists of four framework regions and three CDRs. In FIG. 3A, the correct translation for amino acid 81 should indicate E or glutamic acid.
FIG. 4 shows the 10 polynucleotides most closely matched to the sequence encoding the 11D10 light chain variable region in a database survey. These sequences have the designations SEQ ID NO: 5 to SEQ ID NO: 14.
FIG. 5 shows the 10 nucleotide sequence that most closely matches the sequence encoding the 11D10 heavy chain variable region in a database survey. These sequences have the designations SEQ ID NO: 15 to SEQ ID NO: 32. The dash corresponds to the omitted region that is not homologous to sequence 3.
FIG. 6 is a bar graph showing the anti-Id specificity (IgG1, κ) of 11D10. Binding of 125I-labeled 11D10 (approximately 25,000 cpm) to various mouse monoclonal proteins and anti-HMFG (breast TAA) antibodies was determined using direct RIA. The isotypes of the monoclonal proteins are as follows: MC-10 (BrE-1, IgG2b, κ) first bar; IE3 (IgGI, κ) second bar; 4DC6 (IgG1, I), third bar BrE-3 (IgG1, κ) fourth rod; Hy-Clone IgG2b, fifth rod; Hy-Clone IgG3, k, sixth rod; IgG3, Hy-Clone IgA, seventh rod; and MOPC 104E (IgM, I), eighth bar; RWP1-1 (IgG2b, k), ninth bar.
FIG. 7 is a graph showing inhibition of binding of MC-10 (BrE-1) to MCF-7 and SKBR3 cells by purified Ab2. Black circles indicate competition for 11D10 binding to MCF-7 cells; white circles indicate binding of 11D10 to SKBR3 cells; white squares indicate binding of 3H1 to MCF-7 or SKBR3 cells.
FIG. 8 is a graph showing the binding of absorbed polyclonal mouse and rabbit Ab3 serum and mAb3 binding to the breast cancer cell line SKBR3 by ELISA. Open circles indicate 11D10-2F7 (mAb3); closed circles indicate mouse Ab3 serum; open triangles indicate rabbit Ab3 serum; open squares indicate 1E3 control.
FIG. 9 is a graph showing inhibition of binding of MC-10 (BrE-1) to SKBR3 cells by mAb3 and polyclonal mouse and rabbit Ab3 serum. Black circles indicate rabbit (Ab3) serum (# 123); white circles indicate 11D10-2F7 (mAb3); white squares indicate mouse Ab3 serum; white triangles indicate pre-immune serum (control).
FIG. 10 is a halftone copy of transblot analysis of HMFG on mAb1 and mAb3 on nitrocellulose paper. Lane 1, MC-10 (10 μg / ml); Lane 2, IE3 IgG1 (control; 50 μg / ml); Lane 3, mAb3 IgG1 (50 μg / ml).
FIG. 11 is a graph showing the expression level of 11D10 anti-Id-reactive antibody in the serum of breast cancer patients.
FIG. 12 is a graph showing inhibition of Ab1 (mAb MC-10) binding to 11D10 (Ab2) by monkey (PRO 723) Ab3 serum by RIA. Black circles indicate sera after three immunizations; white circles indicate sera after two immunizations; black squares indicate sera after one immunization; x indicates sera before immunization.
FIG. 13 is a bar graph showing the binding of monkey Ab3 serum to the breast cancer cell line MCF-7 by ELISA. Open bars indicate sera before immunization; hatched bars indicate immune sera; white bars indicate control sera. The dotted line shows the binding of monkey Ab3 serum to the melanoma cell line M21 / P6.
FIG. 14 is a bar graph showing binding of monkey Ab3 serum to semi-purified HMFG by ELISA. First bar, immune serum; second bar, preimmune serum; third bar, control serum; fourth bar, bovine serum albumin (BSA).
Figures 15A and 15B show immunoflow cytometry analysis of monkey Ab3 serum on MCF-7 cells. In FIG. 15A, tumor cells were reacted with preimmune serum and Ab3 serum from monkeys immunized with 11D10 (1: 100 dilution). In FIG. 15B, MOLT-4 cells that do not express HMFG were reacted with preimmune serum and immune monkey Ab3 serum raised against 11D10.
FIG. 16 is a bar graph showing the binding of purified monkey Ab3 to HMFG or purified CEA by ELISA. The left part of the figure shows the plate coated with HMFG; the right part of the figure shows the plate coated with CEA. In the left part of the figure, the first bar represents Ab3; the second bar represents control Ab3; the third bar represents PBS-BSA. In the right part of the figure, the first bar represents Ab3; the second bar represents PBS-BSA; the third bar represents anti-CEA.
FIG. 17 is a halftone copy of slot blot analysis with HMFG or purified CEA. Different concentrations of HMFG (lanes 1 and 2) and CEA (lanes 3 and 4) were absorbed into the polyvinylidene difluoride membrane. Membranes were incubated with Ab1 (lane 1), purified Ab3 (lanes 2 and 3) and anti-CEA mAb (control Ab1) (lane 4).
FIG. 18 is a graph showing inhibition of Ab1 binding to MCF-7 cells by purified Ab3. Open circles indicate Ab3 purified from monkeys immunized with 11D10; closed circles indicate control Ab3 purified from monkeys immunized with control 3H1.
FIG. 19 is a bar graph representing a T cell proliferation assay using monkey peripheral blood mononuclear cells (PBM). The left part of the graph shows PBM stimulated with 11D10; the right part of the graph shows PBM stimulated with 3H1. For each part half, the left box shows PBM from monkey # 872; the right part shows PBM from monkey # 723. White bars indicate pre-immune serum; black bars indicate post-immunization serum.
FIG. 20 is a graph showing the reactivity of Ab3 in patient serum (patient # 1) after 11D10 administration as measured by radioimmunoassay. White circles indicate preimmune serum; black circles indicate postimmune serum.
FIG. 21 is a graph showing the inhibition of Ab1 binding to 11D10 by patient serum (patient # 1). White circles indicate preimmune serum; black circles indicate postimmune serum.
FIG. 22 is a bar graph showing T cell proliferation by patient peripheral blood lymphocytes. For each pair of bars, white bars represent pre-immune cells; black bars represent post-immunization cells. The stimulants tested were: medium, first pair of bars; 11D10, second pair of bars; 3H1, third pair of bars; PHA, fourth pair of bars.
FIG. 23 shows the variable region of the light chain of 11D10 (amino acids 41-60, 34-56, and 85-107 of SEQ ID NO: 2) and the heavy chain (amino acids 43-62 of SEQ ID NO: 4) and the tandem repeats of HMFG ( A selected amino acid sequence comparison between SEQ ID NO: 33 and SEQ ID NO: 34) is shown. Matched amino acids are indicated by solid lines.
FIG. 24 shows a scheme for construction of pVV, a universal vaccinia vector (plasmid) for expression of 11D10 polynucleotides. White box indicates vaccinia TK gene; hatched box indicates 7.5K vaccinia promoter. Restriction sites are as follows: A, Apa I; Ns, Nsi I; C, Cla I; E, Eco RI; P, Pst I; Nc, Nco I; Sm, Sma I. (E) and (C) show potential Eco RI and Cla I sites, respectively. Three stop codons are indicated by S1, S2, and S3. VL and VR indicate the left and right parts of the vaccinia flanking sequence. TK and 7.5K were obtained by PCR using DNA from vaccinia wild-type WR strain.
FIG. 25 shows plasmids (FIG. 25A) and chimeras (FIG. 25B) suitable for the production of 11D10 fusion proteins.
FIG. 26 (AC) is a list comparing the amino acid sequence of the 11D10 variable region with the sequences of 15 immunoglobulin light and heavy chains obtained from a GenBank database search. Panel A shows the sequence closest to the mature 11D10 light chain variable region (contained in SEQ ID NO: 2); Panel B shows the sequence closest to the mature 11D10 heavy chain variable region (contained in SEQ ID NO: 4). Residues that are identical to 11D10 are indicated by dots (.); Gaps introduced to improve alignment for heavy chain VDJ binding are indicated by double lines (=). Panel C shows the light and heavy chain variable region consensus sequences (SEQ ID NO: 47 and SEQ ID NO: 48) and compares them to the sequence of 11D10. The variable region of 11D10 showed unique splicing differences for heavy chain VDJ binding, and an additional 18 point differences from the original sequence were located in both the light and heavy chains.
Figures 27A and 27B are graphs showing inhibition of Ab1 binding to 11D10 by patient serum. FIG. 27A shows data for patients # 1 (white squares), # 2 (white diamonds), # 3 (white circles), # 5 (white triangles), and # 7 (squares with a lattice pattern). FIG. 27B shows data for patients # 6 (white squares), # 8 (white diamonds), # 9 (white circles), # 11 (white triangles), and # 12 (squares with a lattice pattern). Open circles indicate sera before immunization; closed circles indicate sera after immunization.
FIG. 28 is a bar graph showing reactivity of affinity purified Ab3 from patient serum following administration of 11D10 as measured by radioimmunoassay (RIA) (patient # 5 (first pair of bars), # 6 ( Second pair of bars) and # 1 (third pair of bars)). The fourth pair of bars (“X”) shows Ab3 of an irrelevant patient. The fifth pair of bars represents MC10; the sixth pair of bars represents phosphate buffered saline (PBS). In each pair of bars, the black bar represents IgG and the white bar represents IgM. Open circles indicate sera before immunization; closed circles indicate sera after immunization.
FIGS. 29A and 29B are bar graphs showing T cell proliferation by the patient's peripheral blood lymphocytes. For each pair of bars, white bars indicate pre-immunization; black (or hatched) bars indicate cells after immunization. FIG. 29A shows data for patient # 1. FIG. 29B shows data for patient # 5. The stimuli tested were: 11D10, first pair of bars; 4DC6, second pair of bars; PHA, third pair of bars; medium, fourth pair of bars.
BEST MODE FOR CARRYING OUT THE INVENTION
We found a monoclonal anti-idiotypic antibody 11D10 that escapes immune tolerance and induces a specific immune response against different and specific epitopes of human breast fat globules (HMFG), a breast cancer-associated antigen. . The immune response elicited by 11D10 typically includes both humoral and cellular responses. Thus, 11D10 is expected to be useful in treating HMFG related diseases. The hybridoma producing 11D10 was incorporated on January 17, 1996, under the terms of the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures, American Type Culture Collection (ATCC) (12301 Parklawn Drive, Rockville, MD, USA 20852). This has been given accession number 12020. A complete description of 11D10, including its generation and characterization, can be found in Shared Patent Application No. 08 / 575,762 (US Provisional Application Number __; Attorney Inquiry Number 30414-20003.00).
Cancer patients are often immunosuppressed and tolerant to various tumor associated antigens (TAA), including HMFG. Inducing such an active immune response against TAA represents an important challenge in cancer therapy. The present invention uses a network theory approach to vaccine therapy using internal image antigens. Immunization with a given antigen results in the production of antibodies against the antigen. As used herein, “Ab1” represents an anti-tumor monoclonal antibody; “Ab2” represents an anti-idiotype monoclonal antibody: and “Ab3” represents an anti-anti-idiotype monoclonal antibody.
We have found that 11D10 is effective in inducing an immune response (humoral and / or cellular) against HMFG in all tested mammals that are not autoantigens. Importantly, we have also found that 11D10 elicits an immune response in patients with advanced HMFG-related diseases, particularly breast cancer. This is particularly important. Because many of these patients are moderately or severely compromised because of their previous treatment and / or the nature of the disease, or both, and often receive 11D10 as a last resort. This is because the. While not wishing to adhere to a particular theory, one way in which the induction of an immune response can occur is that the binding site of 11D10 can present a region that is partially similar to an epitope in HMFG ( Against other epitopes that give higher immunogenicity). An epitope of HMFG that is similar to that of 11D10 was identified by anti-HMFG mAb MC-10 (Ab1), which recognizes a different and specific epitope on HMFG, and this is syngeneic BALB / c mice Was used to immunize for the production of anti-Id mAb 11D10. These studies show that the antibodies of the invention are useful for generating an immune response and treating HMFG-related diseases such as breast cancer in individuals with advanced disease. The inventors also believe that 11D10 is effective in the treatment of HMFG-related diseases in individuals with high tumor burden. This is also useful for the detection of Ab1 and / or Ab3.
We have also found the polynucleotide sequence encoding the variable region of 11D10 and the polypeptide fragment of 11D10 encoded thereby. Thus, the present invention provides a polynucleotide sequence encoding anti-idiotype antibody 11D10 and functionally equivalent fragments thereof, polypeptide fragments of 11D10, recombinant methods for producing these 11D10 polynucleotides and polypeptides, Includes pharmaceutical and vaccine compositions comprising 11D10 polynucleotides and polypeptides, diagnostic kits comprising 11D10 polynucleotides and polypeptides, and methods of using 11D10 polypeptides and / or 11D10 polynucleotides. These polypeptides and polynucleotides are useful for the evaluation and treatment of HMFG related diseases such as breast cancer. These and other uses of the 11D10 polynucleotides and 11D10 polypeptides of the present invention are discussed in more detail below.
The entire sequence of the 11D10 light chain and heavy chain constant regions has not been determined, but is predicted to be identical or nearly identical to the sequences of other mouse immunoglobulin molecules.
For the mouse kappa light chain constant region, four genetic allotypes encoding two protein allotypes have been published by Solin et al. (1993) Immunogenetics 37; 401-407 (incorporated herein by reference). . FIG. 1 of Solin et al. Shows mouse and rat immunoglobulin kappa chain gene sequences, compares sequences within kappa chain constant regions of different strains, and highlights allotype differences. Κ chain constant region sequences for BALB / c, PL, SJL, and M. spretus are included. Other naturally occurring allotypes are possible.
A mouse γ1 heavy chain constant region DNA sequence from a newborn mouse was published by Honjo et al. (1979) Cell 18: 559-568 (incorporated herein by reference). FIG. 5 of Honjo et al. Shows the germline DNA sequence along with the encoded protein sequence. The above line shows another protein sequence obtained from mouse myeloma MOPC 21. Other naturally occurring allotypes are possible.
None of the 10 database DNA sequences that most closely matched the sequence of the 11D10 light chain variable region were identical. There are about 8-27 differences from the 11D10 DNA sequence, which correspond to about 6-17 amino acid differences. The sixth matched sequence (designated> gb / M59920 / MUSIQKAA3) is a mouse κVJ germ-like sequence and probably represents the prototypic gene from which the 11D10 light chain was derived. The 11D10 DNA sequence differs from the germline sequence at 14 positions, which corresponds to a point difference of about 7 amino acids.
None of the 10 database DNA sequences that most closely matched the sequence of the 11D10 heavy chain variable region were identical. Nine of the ten sequences were 3-12 base pairs long due to splicing differences within VDJ binding. In addition, there are about 15-43 point differences outside the junction as compared to the 11D10 DNA sequence, which corresponds to about 11-23 amino acid differences.
Thus, at least about 18 amino acid differences existed between the amino acid sequence encoded by 11D10 DNA and the sequence encoded by the most closely matched database DNA. This difference is believed to have been caused by somatic mutations in germline sequences during the development of antibody producing cells in the animals used to generate the antibodies.
The amino acid sequence of the 11D10 variable region was compared to other known immunoglobulin molecules (Example 2). Both the light and heavy chain polypeptide variable region sequences for 11D10 were unique.
None of the 50 database amino acid sequences most closely matched to the sequence of the 11D10 light chain variable region were identical. 11D10 differed by a minimum of 7 substitutions from the 15 closest sequences and an average of about 12 substitution differences, which included non-conservative substitutions across the variable region.
None of the 50 database amino acid sequences that most closely matched the sequence of the 11D10 heavy chain variable region were identical. The following summarizes the main points deduced from the comparison.
The closest match sequence had 11 substitutions between residue 1 and residue 98 (before VDJ binding) and 7 substitution differences after residue 98.
11D10 differed in length from most heavy chain sequences by 1-5 residues.
There were an average of about 30 insertion, deletion, and substitution differences between 11D10 and 50 matched sequences.
FIG. 26 panel C provides a comparison with the consensus sequence from the database sequence of the 11D10 amino acid light and heavy chain sequences. In addition to splicing differences for heavy chain VDJ binding, there are at least 18 differences between 11D10 and the consensus sequence, presumably resulting from somatic mutations during antibody maturation. Point differences occur across the light and heavy chain variable regions.
Of particular interest in developing 11D10 derivatives with 11D10 immunological activity are regions of 11D10 polynucleotide or polypeptide sequences that contain portions of heavy chain VDJ binding. Also of interest are regions that span at least one, preferably two, more preferably three or more point differences between the 11D10 amino acid sequence or the amino acid sequence encoded by SEQ ID NO: 5 to SEQ ID NO: 32.
Useful materials and processes of the invention are made possible by the provision of polynucleotides encoding 11D10 and 11D10. These sequences allow for the design of polypeptides that can be useful, for example, as vaccines for the treatment of HMFG-related diseases or as reagents for detecting the presence of Ab1 and / or Ab3. Furthermore, these sequences allow for the design of polynucleotides useful as probes or primers for detection and amplification of 11D10 target regions, as well as 11D10 polynucleotides useful as vaccines.
Definition
As used herein, the terms “11D10”, “11D10 antibody”, and “11D10 monoclonal antibody” are used interchangeably to refer to immunoglobulin produced by the 11D10 hybridoma cell line deposited with the ATCC. The The production and characterization of 11D10 is described in Example 1. 11D10 is an anti-idio, which contains an epitope that is at least partially similar to the individual and specific epitopes of human milk fat globule (HMFG), which is mainly expressed at high density by breast cancer cells. Type antibody (Ad2). Different biological functions are associated with 11D10, including but not limited to the ability to induce binding to Ab1 and / or Ab3 and immune responses (humoral and / or cellular) against HMFG. . Unless otherwise specified, the term “intact 11D10” refers to the amino acid sequence of the entire 11D10 molecule. A “fragment” of 11D10 is a part of 11D10. The definition of “11D10” includes enzymatic cleavage and / or chemical treatment of intact antibodies that contain the entire heavy and light chain variable regions of 11D10 and can be bound to MC-10 by standard immunoassays. Fragments produced (eg Fab, F (ab ')₂, And F (ab ′)) are also included.
As used herein, 11D10 “immunological activity” refers to any of the following activities: (a) ability to bind to Ab1 (MC-10); (b) 11D10 in a specific manner. Ability to inhibit the binding of MC-10 to MC-10 (Ab1) or the binding of MC-10 to HMFG; (c) a specific immune response, in particular an antibody (humoral) response and / or a T cell response, and the fectors resulting therefrom Ability to trigger functions. Antibody responses include antibody mediated functions such as antibody dependent cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC). T cell responses include helper T cell function, cytotoxic T cell function, inflammatory inducer T cells, and T cell suppression. Immunological activity is measured by radioimmunoassay (RIA), solid phase enzyme immunoassay (ELISA), complement binding, opsonization, detection of T cell proliferation, and various⁵¹It can be measured by using standard methods known in the art, such as a Cr release assay. These methods are known in the art and are particularly described herein. A compound that can elicit a specific immune response according to any of these criteria is said to be "immunogenic". “Immunogenic” refers to the ability to elicit a specific humoral and / or cellular immune response.
“Activity”, “function”, or “feature” of 11D10 are used interchangeably and refer to various properties of 11D10. Examples of 11D10 function include binding to Ab1 and / or Ab3, induction of Ab3, and / or induction of a cellular immune response (preferably an anti-HMFG response), and improvement or alleviation of HMFG-related diseases. It is not limited to.
An antibody having “identical characteristics” that are identical to another antibody has structural (ie, physical) and / or functional (ie, chemical) properties that make the antibody identical to another antibody. Means that. Similarly, a hybridoma having “identical characteristics” of the cells of the hybridoma cell line is a hybridoma having structural and / or functional properties that are identical to the hybridoma cell line being compared. For purposes of the present invention, the antibody's identifying characteristics are, but are not limited to, those features associated with 11D10 as described above; the hybridoma's identifying characteristics are those associated with a hybridoma producing 11D10. .
The “variable region” of 11D10 refers to one or a combination of the variable region of the light chain of 11D10 or the variable region of the heavy chain of 11D10.
As used herein, GM-GSF, IL-2, and other biologically active molecules include fragments and derivatives based on the respective parent molecule having the same biological or physiological function. Means that.
As used herein, a “progeny” of a hybridoma is a hybridoma derivative that is completely identical to the original (parent) cell due to mutation or other adaptation. Monoclonal antibodies are produced that may or may not be identical, but maintain the ability to avoid immune tolerance (ie, cause an immune response against HMFG).
“HMFG” is an abbreviation for human milk fat globule. HMFG is a number of proteinaceous (and therefore antigenic) components. As used herein, it includes HMFG expression as prepared by the method of Ceriani et al. ((1977) Proc. Natl. Acad. Sci. USA 74: 582-586) along with antigenically related substances. A semi-purified extract of a breast cancer cell line, which includes HMFG expressed in breast cancer cells and a more highly purified product. HMFG includes a known amino acid sequence of high molecular weight mucin (its epitope is recognized by monoclonal antibody MC-10, which is used as Ab1 in the induction of 11D10). Thus, the immunological reactivity of anti-HMFG induced by immunizing an animal with 11D10 preferably binds to a polypeptide epitope related to the epitope recognized by MC-10.
For purposes of the present invention, an “HMFG-related disease” or “HMFG-related tumor” is a disease state or tumor associated with an HMFG antigen (particularly expressed on the surface of a cell) that binds to MC-10 (Ab1). It is.
As used herein, “polynucleotide” is a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, and / or their analogs. As used herein, the terms “polynucleotide” and “nucleotide” are used interchangeably. A polynucleotide can have any three-dimensional structure and can perform any function known or unknown. The term “polynucleotide” includes double-stranded, single-stranded, and triple helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide is known to form double-stranded forms and double-stranded forms. Or each of the two predicted complementary single stranded forms.
The following are non-limiting examples of polynucleotides: gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any Isolated DNA of sequences, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Purine and pyrimidine analogs are known in the art and are aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-amino. Methyl uracil, inosine, N6-isopentenyl adenine 1-methyl adenine, 1-methyl pseudouracil, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine, 3- Examples include, but are not limited to, methylcytosine, 5-methylcytosine, pseudouracil, 5-pentynyluracil, and 2,6-diaminopurine. The use of uracil as a thymine substituent in deoxyribonucleic acid is also considered an analogous form of pyrimidine.
If present, modifications to the nucleotide structure may be applied before or after assembly of the polymer. The sequence of nucleotides can be hindered by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition include, for example, “caps”, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications (eg, modifications at uncharged bonds (eg, methylphosphonates) , Phosphotriesters, phosphoamidates, carbamates, etc.), and modifications with charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine) (ply-L-lysine)), including pendant moieties, intercalators (eg, acridine, psoralen, etc.), chelators (eg, metals, radioactive metals, boron, metal oxides, etc.) Modified, including alkylators, modified bonds (eg alpha anomers) Modified with an acid, etc.), as well as unmodified forms of the polynucleotide.
In addition, any of the hydroxyl groups normally present in sugars can be replaced with phosphonate groups, phosphate groups and protected with standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or It can be bound to a solid support. The 5 'and 3' terminal OH groups can be phosphorylated or substituted with amine or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.
Polynucleotides also include 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (eg, arabinose , Xylose or lyxose), pyranose sugars, furanose sugars, cedoheptulose, acyclic analogs, and abasic nucleoside analogs (eg, methylriboside), but generally similar to ribose or deoxyribose sugars known in the art It may contain a form.
As described above, one or more phosphodiester bonds can be replaced by another linking group. These other linking groups are those where the phosphate is P (O) S (“thioate”), P (S) S (“dithioate”), (O) NR₂("Amidate"), P (O) R, P (O) OR ', CO, or CH₂Including, but not limited to, embodiments substituted by ("formacetal"). Here, each R or R ′ is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl containing an ether (—O—) bond as necessary. (cycloalky), cycloalkenyl, or araldyl. Not all bonds of a polynucleotide need be the same.
Although conventional sugars and bases are used to apply the methods of the invention, substitution of similar forms of sugars, purines and pyrimidines may result in an end product such that another backbone structure similar to the polyamide backbone may be advantageous. Can be advantageous in designing.
A “fragment” (also referred to as a “region”) of an 11D10 polynucleotide (ie, a polynucleotide encoding 11D10) is a polynucleotide consisting of at least nine consecutive nucleotides of the variable region of 11D10 (ie, at least 11D10 variable). Code part of the region). Preferred fragments comprise a region encoding at least 5 consecutive amino acids of the variable region of 11D10. More preferably, it comprises at least 10 consecutive amino acids of the variable region, even more preferably at least 15 consecutive amino acids of the variable region.
As used herein, the term “recombinant” polynucleotide can either be (1) not associated with all or part of the polynucleotide with which it is naturally associated, or (2) it is naturally Contemplates polynucleotides that are linked to a polynucleotide other than the linked polynucleotide, or (3) are not naturally occurring, genomic, cDNA, semi-synthetic, or synthetic origin.
The terms “polypeptide”, “oligopeptide”, “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be hindered by non-amino acids. The term is also naturally or mediated (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling component). Amino acid polymers modified by This definition also includes, for example, one or more amino acid analogs (including, for example, unnatural amino acids), as well as polypeptides containing other modifications known in the art. Since the polypeptides of the present invention are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.
A polypeptide “fragment” (also referred to as a “region”) of 11D10 comprises at least 5 consecutive amino acids, more preferably at least 8 consecutive amino acids, and even more preferably at least about 10 consecutive amino acids of the sequence of 11D10. A polypeptide comprising the amino acid sequence of 11D10, wherein at least three amino acids are derived from the variable region of 11D10. For purposes of the present invention, a fragment of 11D10 may be identified and characterized by any of the following functions: (a) homology with HMFG; (b) ability to bind to Ab1 or Ab3; (c ) The ability to elicit an immune response (ie a humoral and / or cellular response), preferably an anti-HMFG immune response; (d) improvement, delay, or delay of HMFG-related tumors and / or HMFG-related disease states Ability to improve or mitigate. Item (b), (c) or (d) falls within the term “immunologically reactive”. The 11D10 fragment can have any one, more than one, or all of the functions identified above. Methods for determining these functions (a)-(d) are described below.
11D10 polypeptides that are “homologous” to or share homologies with HMFG aligned the amino acid sequences of HMFG and 11D10 polypeptides in any way, including the same or opposite directions relative to each other In some cases, it means that at least 2, preferably 3, and more preferably 4 consecutive amino acids in the polypeptide correspond to HMFG. Since functional peptide fragments can be very small for the purposes of the present invention, only a few amino acids can be matched (eg, the requirement for contiguous amino acids required for binding sites and / or antigen presentation). The number can be as few as 2-5 amino acids). An 11D10 polypeptide “containing a region of homology” to HMFG shares homology to HMFG within its amino acid sequence, as defined above.
A “fusion polypeptide” is a polypeptide comprising a region at a position in the sequence that is different from the naturally occurring position. This region can usually be present in a different protein and bound in a fusion polypeptide; alternatively, they can usually be present in the same protein but are placed in a new configuration in the fusion polypeptide.
As used herein, “immune response” refers to a humoral response, a cellular response, or both.
A “functionally equivalent fragment” of an 11D10 polypeptide or polynucleotide retains at least one property and / or function of the 11D10 polypeptide or polynucleotide. For example, the sequence can be altered by adding additional nucleotides or peptides, as is known in the art, so that the functionality of the sequence that induces immunity is not altered. Other examples are sequence deletions and / or substitutions. Alternatively, the sequence can be altered by substituting nucleotides or amino acids, or by combining additions, deletions or substitutions. As will be apparent to those skilled in the art, the functionality of a polypeptide sequence that induces immunity includes other features and / or activities of the sequence, such as binding to Ab1 and / or Ab3. Furthermore, “inducing immunity” will be apparent to those skilled in the art to include any aspect of the immune response, such as a humoral response or a cellular response. It is also clear that the functionality of a polynucleotide sequence depends in part on its intended use, and any functionality maintained on a fragment of a polynucleotide meets this definition. For example, a “functionally equivalent fragment” of an 11D10 polynucleotide can be a fragment that retains the ability to hybridize so that the desired polynucleotide can be used as a probe. Alternatively, a “functionally equivalent fragment” of an 11D10 polynucleotide is a fragment of 11D10 (a variable region of the variable region) in which the polynucleotide has a function associated with intact 11D10, and preferably a function associated with induction of anti-HMFG immunity. Can be coded). A functionally equivalent fragment of an 11D10 polypeptide or polynucleotide can have the same, enhanced, or decreased function when compared to an 11D10 polypeptide or polynucleotide. Other functions of 11D10 are listed above. Functionally equivalent fragments have at least 9 nucleotides or at least 5 amino acids, preferably have at least 15 nucleotides or at least 10 amino acids, and even more preferably have at least 25 nucleotides or at least 20 amino acids.
“Cell line” or “cell culture” refers to a higher eukaryotic cell grown or maintained in vitro. It is understood that a cell derivative may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell.
A “vector” is a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. This term refers to vectors that function primarily for insertion of nucleic acid molecules into cells, replication of vectors that function primarily for replication of nucleic acids, and expression vectors that function for transcription and / or translation of DNA or RNA. Including. Also included are vectors that provide more than one of the above functions.
A “host cell” includes an individual cell or cell culture that can be a recipient of a vector or a recipient of uptake of nucleic acid molecules and / or proteins. A host cell contains the progeny of a single host cell, and this progeny is not necessarily completely (in the genome of morphological or total DNA complementation) from the original parent cell due to natural, accidental or intentional mutation. May not be identical. Host cells include cells transfected in vivo with a polynucleotide of the invention.
An “expression vector” is defined as a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. "Expression system" usually refers to a suitable host cell containing an expression vector that can function to yield a desired expression product.
A “signal sequence” is a short amino acid sequence directed to a newly synthesized secreted protein or membrane protein through and through a cellular membrane such as the endoplasmic reticulum. The signal sequence is typically at the N-terminal portion of the polypeptide and is cleaved after the polypeptide crosses the membrane.
“Heterologous” means derived from (ie, obtained from) a genotype that differs from the rest of what is being compared. For example, the polynucleotide may be located in a plasmid or vector from a different source by genetic engineering techniques, thereby becoming a heterologous polynucleotide. A promoter that is linked to a coding sequence that is not naturally linked is a heterologous promoter.
An “isolated” or “purified” antibody, polynucleotide or polypeptide is one that is substantially free of the substance with which it is naturally associated. Substantially free means that there is at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the substance with which it naturally associates.
A “vaccine” is a pharmaceutical composition for human or animal use that is administered with the intention of conferring to a recipient a certain degree of specific immunological reactivity against a particular target or group of targets. Immunological reactivity refers to antibodies or cells that are immunologically reactive to the target (particularly B cells, plasma cells, helper T cells, and cytotoxic T lymphocytes, and their precursors), Or any combination thereof. For the purposes of the present invention, this target is the tumor associated antigen HMFG, or any tumor associated antigen bound by 11D10. Immunological reactivity may be desired for experimental purposes, for treatment of certain conditions, or for exclusion of certain substances.
A “stable double-stranded” polynucleotide (or “stable complex” formed between any two or more components in a biochemical reaction) is A duplex or mixture that lasts long enough between detection, including any additional washing steps or other operations that may occur in the meantime.
A biological “sample” encompasses a variety of sample types obtained from an individual and typically used in diagnostic procedures or assays. This definition includes solid tissue samples such as blood and other fluid samples of biological origin, biopsy specimens or tissue cultures, or cells and their progeny obtained therefrom. This definition also includes samples that have been manipulated by any method after their acquisition, such as by treatment with reagents, solubilization, or enrichment of certain components such as proteins or polynucleotides. The term “biological sample” encompasses clinical samples and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
“Treatment” as used herein is an approach for obtaining beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical outcomes, whether detectable or undetectable, may be improved symptoms, reduced degree of disease, stabilized (ie, not worsened) disease, disease, Including, but not limited to, preventing the spread (ie, metastasis) of the disease, delaying or prolonging disease progression, ameliorating or alleviating the disease state, and reducing (whether partially or wholly). “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
“Remission” of a disease is a reduction in the degree of disease state and / or undesirable clinical symptoms and / or progression compared to not administering 11D10, 11D10 polynucleotide, and / or 11D10 polypeptide. It means that the passage of time is slowed or lengthened.
An “effective amount” is an amount sufficient to produce an advantageous or desired clinical result. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of 11D10, 11D10 polynucleotide, and / or 11D10 polypeptide is an amount sufficient to induce an immune response, particularly an anti-HMFG response. With respect to treatment, an “effective amount” of an 11D10, 11D10 polynucleotide, and / or 11D10 polypeptide is an amount sufficient to alleviate, ameliorate, stabilize, reverse, prolong, or delay the progression of a HMFG-related disease state. is there. The detection and measurement of these indicators of efficacy is discussed below.
An “individual” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals and pets.
General technology
In practicing the invention, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are within the skill of the art are used. Such techniques are described in “Molecular Cloning: A Laboratory Manual”, 2nd edition (Sambrook et al., 1989); “Oligonucleotides Synthesis” (MJGait, 1984); “Animal Cell Culture” (RIFreshney, 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (DMWei and CC Blackwell); “Gene Transfer Vectors for Mammalian Cells” (JMMiller and MPCalos, 1987); “Current Protocols” in Molcecular Biology ”(edited by FM Ausubel et al., 1987);“ PCR: The Polymerase Chain Reaction ”(edited by Mullis et al., 1994); Explained.
These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and should therefore be considered when considering aspects of these inventions. Systems that are particularly useful for individual aspects are discussed below.
11D10
In one embodiment, the invention encompasses a monoclonal anti-idiotype antibody (referred to herein as “anti-Id”) produced by the hybridoma cell line ATCC No. 12020 or its progeny. The invention also encompasses a hybridoma cell line designated by ATCC No. 12020. Production and characterization is described in Example 1 and below.
In another embodiment, the invention encompasses a purified antibody having the same identifying characteristics as the antibody produced by the hybridoma cell line designated ATCC No. 12020. The invention also encompasses a hybridoma that identifies all of the cellular characteristics of the hybridoma cell line designated ATCC No. 12020.
The invention also includes 11D10 coupled to a label that can produce a detectable signal. These bound antibodies are useful, for example, for quantification or imaging of Ab1 (and / or Ab3). Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, and other antibodies. The label can be covalently attached to 11D10 or attached to 11D10 via a second reagent (eg, secondary antibody, protein A, or biotin-avidin complex). Methods for labeling antibodies are known in the art and need not be described in detail herein.
11D10 generation and selection
Selection of Ab1 that elicits anti-Id (Ad2). A series of cell type-specific mouse monoclonals ("mAbs") that recognize components of the human milk fat globule (HMFG) membrane that are associated with breast cancer but not with most normal tissues have been produced. Ceriani et al. (1983); Taylor-Papadimitriou et al. (1981)Int.J.Cancer 28: 17-21. In these mAbs, MC-10 (also called BrE1) is very limited and it is present in negligible amounts in human breast epithelial cells and is increased at least 10-fold in breast cancer cells It is specific in the sense that it reacts with high molecular weight (molecular weight 400,000) mucins. WO 8907268; EP 401247. This antibody is cytotoxic to breast cancer cells in in vitro studies. Ceriani et al. (1983); Peterson et al. (1990).
mAb MC-10 has a very limited histopathological distribution in normal tissues. MC-10 only binds to several regions of the lung and the epithelial lining of the scattered distal convoluted tubules of the kidney, normal breast and many other normal epithelia (colon , Pancreas, stomach, thyroid, bladder, liver) and other normal tissues (adrenal glands, brain, lymph nodes, myocardium, ovary, pancreas, testis) and no obvious histopathological connection. On the other hand, a high proportion of various human tumors (including breast, endometrium, lung, ovary, and pancreas) bind severely to mAb MC-10. Formalin-fixed tumors studied for MC-10 binding (positive number / total) include the following: breast cancer (carcinoma (CA)) (144/182), colon CA (3/27), duodenal CA (0 / 1), endometrial CA (7/14), kidney CA (0/11), lung CA (41/47), ovary CA (20/26), pancreas CA (9/15), prostate CA (0 / 2), salivary gland CA (0/3), stomach CA (2/7), thyroid CA (0/7), hepatocholangio CA (8/33), islet cell CA (0/2), lymphoma (0/20), melanoma (0/23), meningioma (0/5), Merkel cell CA (4/9), mesothelioma (1/11), neuroblastoma (0/2) Eosinophilic granuloma (1/1), paraganglioma (0/10), pleoadenoma (0/7). For sarcomas: unclassified (0/1), alveoli (0/1), angiosarcoma (0/1), clear cell (0/2), cystic sarcoma (0/1), epithelioid cells (5 / 12), Ewing (0/1), fibrosarcoma (0/1), leiomyoma (0/2), liposarcoma (0/1), malignant fibrohistiocytoma (0/2), synovium Mesothelioma (0/7), spindle cell CA (5/16), undifferentiated (1/9); Schwann cell tumor (0/3), seminoma (0/4), teratoma (0/3) ), Thymoma (0/8), transitional epithelial cell CA (5/10), undifferentiated CA (7/29), Warthin's tumor (0/1). Ceriani et al. (1990). We also studied hematopoietic cells for the presence of MC-10 Ag by FACS analysis in our laboratory, and those cells (including granulocytes and platelets) were negative for antigen. I found out. Positive control MCF-7 cells were well stained with mAb MC-10. Thus, 11D10 has potential for use in a wide variety of cancers where HMFG is detected.
Therefore, mAb MC-10 is selected for anti-Id production. Because it is initially expressed in high density by human breast cancer and some other tumor cells, but by immunoperoxidase staining in normal adult tissues or by flow cytometry analysis in hematopoietic cells containing granulocytes Because it defines a unique and specific epitope of high molecular weight mucins of human milk fat globules (HMFG) that cannot be found.
The breast cancer associated epitope defined by the monoclonal antibody MC-10 is a suitable target for active immunotherapy against these tumors. This Ag is expressed in more than 80% in the case of breast cancer and is present in high density in tumor tissue compared to a small number of normal tissues that contain trace amounts of this Ag. Ceriani et al. (1983); Taylor-Papadimitriou et al. (1991). Ag flows in the circulation in a very small amount. Peterson et al. (1990)Hybridoma 9: 221-235. Circulating low levels of Ag do not appear to interfere with the binding of radiolabeled anti-HMFGmAb to tumor targets in in vivo studies of advanced breast cancer patients. The limited specificity of MC-10 along with the high ability of MC-10 to bind to representative breast cancer cell lines MCF-7 and SKBR3 makes it an excellent target for Ab2 hybridoma production. We have purified MC-10 (IgG2b) for anti-idiotype production._κ) (Lot number 5319001).
Generation of monoclonal anti-idiotype hybridomas and selection of 11D10. By immunizing naïve mice with MC-10 anti-HMFG antibody, 11D10 was obtained and an anti-idiotypic response was obtained. Isogenic BALB / c mice were immunized 4 times with MC-10 (Ab1) and their spleen cells were fused with non-secreting mouse melanoma P3-653 cells. To obtain an anti-idiotype with all the desired characteristics, a large-scale screening process was used that involved four important steps: (1) Positive selection of antibodies bound to MC-10 (2) negative selection for antibodies that recognize isotype or allotype determinants; (3) positive selection for the ability to inhibit the binding of MC-10 to HMFG; (4) original tumor-associated antigen in both mice and rabbits; Positive selection of the ability to induce a humoral immune response against (HMFG).
Several Ab2 hybridomas specific to the immunized MC-10 Id were obtained and did not react with either isotype or allotype determinants. To determine if these Ab2s are directed against the paratopes of MC-10, the binding of radiolabeled MC-10 to the breast tumor bacterial strains MCF-7 and SKBr3 was determined using various amounts of Ab2 hybridoma culture supernatants. Studied in the presence of. Ab2, which can inhibit MC-10 binding to these cells, was grown and purified from ascites for further study. Different purified Ab2 were prepared as vaccines and injected into naïve mice and rabbits on a biweekly schedule. After four injections, serum samples were titrated for the presence of Ab3 that binds not only to Ab2 to be immunized but also to HMFG. Ab2, which reproducibly induces high titers of Ab3 with the desired specificity, was named 11D10. Further details of the method used to obtain 11D10 are provided in Example 1.
The immune response in animals immunized with 11D10 was further characterized. Immune sera from both mice and rabbits immunized with 11D10 compete with MC-10 for binding to the breast cancer cell lines MCF-7 or SKBr3 and inhibit the binding of radioiodine labeled MC-10 to 11D10 (FIG. 7). This indicates that mouse and rabbit Ab3 can share the idiotype with Ab1 (MC-10) and probably bind to the same epitope as Ab1.
Monoclonal Ab3 that binds to the MC-10 positive antigen is also obtained from mice immunized with 11D10. Ab3 (both polyclonal and monoclonal) reacts with HMFG Ag semi-purified by dot blot analysis and stains MCF-7 cells by immunoperoxidase method. In addition, rabbit Ab3 serum opsonized tumor cell lines MCF-7 and SKBr3 in a complement-mediated cytotoxicity (CMC) assay.
We have also discovered that administration of 11D10 to non-human primates (cynomolgus monkeys) produces an immune response (both humoral and cellular) (Example 3;Cancer Res.(1995) 55: 1525-1530). Ab3 produced in response to 11D10 was specific for HMFG (FIGS. 12-17). The antibody (Ab3) concentration was very high, and 1.32 mg of purified Ab3 was recovered from 30 ml of serum (44 μg / ml serum). This purified Ab3 was able to inhibit the binding of radiolabeled Ab1 to the HMFG positive breast cancer cell line MCF-7 by more than 60% in an amount of only 100 ng.
In addition to humoral immunity, the cellular immune response in monkeys was studied by T cell proliferation assay. Peripheral blood immune lymphocytes (PBL) from monkeys that received 11D10 were substantially proliferated when challenged with 11D10 in vitro but not with the unrelated controls Ab2, 3H1 Was described (Figure 19). This suggests Id-specific cellular proliferation.
For clinical application of anti-idiotype antibodies for immunotherapy, it is an essential condition to demonstrate the induction of specific anti-TAA antibodies in different species of animals.
More importantly, humans with HMFG-related tumors are tolerant to HMFG antigens, but we have also avoided 11D10 immune tolerance and progressive human milk fat globule-related disease It has been found to elicit an immune response in individuals with (especially breast cancer). Three patients with HMFG positive advanced breast cancer, who had failed standard treatment, were administered 11D10 (Example 5). Initial data showed that all three of these patients developed antibodies that were anti-HMFG (FIGS. 20-21); patient # 2 had non-specific binding but some Ab3 activity. Had. Furthermore, one of the patients showed a cellular immune response as evidenced by a T cell proliferation assay (FIG. 22). Upon further analysis (using affinity purified Ab3), it was found that only one of these three patients developed an antibody that was anti-HMFG (Figures 27 and 28; Example 10). . In evaluating additional patients (total 12) collected in this study (Example 10), we found that 5 out of the 10 patients tested were radiolabeled MC-10 to 11D10. It was found that anti-HMFG antibodies were developed as assessed by inhibition of (Ab1) binding. A total of 4 patients (# 1, 5, 6, and 12) showed a cellular immune response (Figure 29). A more detailed description of this study is found in Examples 5 and 10.
Preparation of 11D10
The antibody of the present invention can be obtained by several methods. 11D10 can be produced from the hybridoma ATCC number HB12020 described herein. Methods for isolating antibodies are well known in the art. For example, Harlow and Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and Sambrook et al. (1989)Molecular Cloning: A Laboratory ManualSee Cold Spring Harbor Laboratory. Antibodies can be obtained through tissue culture or from hybridomas derived from mouse ascites and can be purified using conventional techniques for antibody purification. These techniques are known in the art. For example, the cells can be cultured in a suitable medium and the spent medium can be used as a source of antibodies. If desired, matrix coated channels or beads and cell co-cultures can be included to promote the growth of antibody producing cells. In order to produce large amounts of antibody, it is usually more convenient to obtain ascites. Such methods are known in the art and generally involve injecting the hybridoma cells into an untreated immunologically histocompatible or immune tolerant mammal (especially a mouse). . Mammals are treated as needed by pre-administering an appropriate composition (eg, pristane) for the production of ascites. Preferably, 11D10 is purified from the ascites of BALB / c using recombinant protein G-agarose chromatography followed by protein-A-CL-sepharose 4B chromatography.
Alternatively, 11D10 is a sequence and information provided herein and techniques known in the art (eg, commercially available automated peptide synthesizers, such as those manufactured by Applied Biosystems, Inc (Foster City, Calif.)). Can be synthesized chemically.
The 11D10 antibody is an IgG1 mouse subclass and can be isolated by any technique appropriate for this isotype of immunoglobulin. Purification methods include salting out (eg, using ammonium sulfate), ion exchange chromatography (eg, on a cationic or anionic exchange column at neutral pH, and stepwise ionic strength gradients. Elution with increasing), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins (eg, protein A, protein G, hydroxyapatite, and anti-immunoglobulin). 11D10 can also be purified on an affinity column containing MC-10 paratope, for example in the form of purified Ab1 or Ab3.
11D10 can also be obtained by use of conventional recombinant methods such as those described in Sambrook et al. (1989). For example, using the sequences and information provided herein, a polynucleotide encoding either the heavy or light chain of 11D10 can be expressed in an appropriate expression vector (which includes regulatory sequences for transcription (eg, promoters). Can be cloned). Expression vectors are sequentially introduced into the host cell. Host cells are grown under appropriate conditions such that the polynucleotide is transcribed and translated into protein. The heavy and light chains of 11D10 can be produced separately and then joined by disulfide bond rearrangement. Alternatively, a vector with separate polynucleotides encoding each strand of 11D10, or a vector with a single polypeptide encoding both strands as separate transcripts can be transfected into a single host cell, It can then produce the entire molecule and assemble. Preferably, the host cell is a higher eukaryotic cell capable of providing a normal carbohydrate supplement of the molecule. Thus, 11D10 produced in a host cell can be purified using standard techniques in the art. Next, a polynucleotide encoding 11D10 for use in the production of 11D10 by any of these methods can be obtained sequentially from the hybridoma producing 11D10, or provided herein. It can be produced synthetically or recombinantly from a DNA sequence.
When 11D10 is administered to an individual, 11D10 is preferably at least 80% pure, more preferably at least 90% pure, even more preferably still even more preferably at least 95% pure, More preferably about 97% pure, even more preferably about 99% pure, even more preferably at least about 99.5% pure, and free of pyrogens and other contaminants. In this case, the purity percentage is calculated as the weight percent of the total protein content of the preparation.
11D10 uses and 11D10 usage.
11D10 has several uses. 11D10 can be used to elicit an immune response in individuals with advanced HMFG-related tumors, thereby treating the individual's HMFG-related tumors. Preferably, the immune response is anti-HMFG. Furthermore, 11D10 can be used to detect antibodies that bind to HMFG or 11D10. 11D10 can also be used to remove undesired excess labeled Ab1 from the circulation of patients previously treated with labeled monoclonal anti-HMFG antibody. The label can be any label attached to an antibody appropriate for its intended use, including, for example, radioisotopes, toxic sites such as toxins, and drugs. 11D10 is also useful for enhancing tumor detection in imaging.
Use of 11D10 to elicit an immune response or in treatment. The present invention encompasses a method of eliciting an immune response in an individual having a progressive HMFG-related disease (eg, HMFG-related tumor) that entails administering an effective amount of 11D10 to the individual. In this sense, an “effective amount” is an amount sufficient to elicit a humoral and / or cellular, immune response. Preferably, the immune response comprises a product of anti-HMFG.
Subjects suitable for administration of 11D10 antibody can be identified by a number of different criteria. For example, 11D10 can be administered to experimental animals to study the effects of 11D10 on the immune response or to obtain useful reagents such as antibodies or cell lines specific for anti-HMFG.
In preferred embodiments, 11D10 can be used to elicit an immune response and / or to treat and / or alleviate advanced HMFG-related diseases (eg, HMFG-related tumors). “HMFG-related tumors” are those HMFG antigens that are specifically expressed on the surface of tumor cells (eg, breast, endometrial cancer, ovary, transitional cell carcinoma and undifferentiated cancer (other examples are described above)). That is, it is a tumor containing HMFG-related antigen). As used herein, a “progressive” HMFG-related tumor means that there is detectable metastasis (ie, a detectable tumor mass at a site other than the primary site of the tumor). The mass is preferably detected by imaging techniques known in the art (eg X-ray or CT scan). An effective amount of 11D10 is administered to an individual with a progressive HMFG-related tumor for the induction, mitigation, or treatment of an immune response. Administration of an effective amount of 11D10 to individuals with advanced HMFG-related tumors may slow or slow the progression of the disease or improve the disease compared to other individuals not so treated .
In some situations involving advanced HMFG-related tumors (especially advanced breast cancer), individuals receiving 11D10 are either due to previous treatments, the disease itself, or both properties It is understood that an immunocompromised state can be moderate to severe. Thus, the time required to mount an immune response and / or the number of injections of 11D10 and / or the amount of 11D10 per administration can vary. For example, an individual may need longer time to elicit an immune response once 11D10 has been administered. In this case, it is recommended that the individual continue to be monitored for an immune response, even if no initial (ie within one month) immune response is detected. In another example, an individual may require a greater than average number of injections to elicit an immune response.
One possible indicator of the efficacy of 11D10 administration (whether an immune response and / or treatment was induced or whether 11D10 administration is indicated) is the density of HMFG on tumor cells. This density can vary widely from individual to individual and can vary over the course of 11D10 administration and / or over the course of the disease. As used herein, the “density” of HMFG may refer to either or both of: (a) of cells having HMFG on their surface per whole cell in a given biological sample. Number; (b) Amount of HMFG on each cell surface. Density (a) is calculated paying attention to the number of cells in the stained sample, or otherwise indicates the presence of HMFG divided by the total number of cells. This density is preferably greater than about 20%, more preferably greater than about 30%, more preferably greater than about 50%, even more preferably greater than about 70%, even more preferably greater than about 80%, Most preferably greater than about 90%. Accordingly, the present invention has an HMFG density greater than about 20%, preferably greater than about 30%, more preferably greater than 70%, even more preferably greater than about 80%, and most preferably greater than about 90%. Administration of 11D10 to an individual.
The density (b) is indicated, for example, by the relative intensity of cell staining (or the intensity of any measurement that indicates the presence of HMFG) in a sample from one individual relative to a sample from another individual. For this density, one skilled in the art can empirically determine the density. Density (b) is for normal tissues (ie, cells lacking HMFG, or unaffected cells), and therefore the preferred range is greater than unaffected cells when detected by immunohistochemical staining density. Is about 1.5 times higher, preferably about 3 times higher, more preferably about 10 times higher. Unaffected cells can also be from the same individual.
This is not to say that individuals with low density (eg, less than about 50%) are not shown for administration of 11D10. While not wishing to be bound by a single theory, administration of 11D10 produces a series of immune responses (eg, cytotoxic T cell responses) that produce a more general response that is effective against HMFG-related tumors. It can be triggered. However, the lower density may indicate that further treatment is desirable.
It is understood that density can also be used as an indicator of the degree of disease and response to administration of 11D10. For example, a sample taken from an individual at the start of 11D10 administration may exhibit about 80% density (ie, about 80% of cells exhibit HMFG). Samples taken from the same site after receiving 11D10 may exhibit a density of only about 50%. This indicates that HMFG expressing cells are destroyed. Similarly, if the staining intensity of a sample from an individual who has received 11D10 disappears upon receiving 11D10, this indicates that HMFG-bearing tumor cells have been destroyed.
For the purpose of eliciting or treating an immune response against individuals with advanced HMFG-related tumors, 11D10 is administered parenterally, preferably intracutaneously. Other routes of administration include, but are not limited to, intramuscular, subcutaneous, and intradermal. 11D10 can also be administered indirectly by treatment of cultured cells followed by introduction of these cultured cells into an individual.
The amount of 11D10 administered depends on several factors such as the individual's condition and the route of administration. Preferably, the dose per administration is in the range of about 0.1 mg to about 20 mg. More preferably, the dose is in the range of about 0.5 mg, more preferably in the range of about 1 mg to about 8 mg. Preferably, the dose is about 2 mg to about 8 mg. 11D10 is typically administered in 4 injections every 2 weeks, followed by monthly if necessary. The timing of subsequent injections (ie, maintenance dose) will depend, inter alia, on the symptoms and response of the individual being treated. In order to determine when maintenance (additional) administration should be given (typically about every 3 months), for example, the level of Ab3 can be monitored, preferably by the diagnostic methods described herein.
Preferably, 11D10 is administered with a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient is a relatively inert substance that facilitates administration of a pharmaceutically effective substance. For example, the excipient can impart form or consistency to the vaccine composition, or can act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for various osmotic pressures, encapsulating agents, buffering agents, and skin penetration enhancers. Examples of pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences (Alfonso R. Gennaro ed., 18th edition, 1990).
Preferably, 11D10 is used with an adjuvant that enhances the presentation of 11D10 or enhances the immune response to 11D10. Suitable adjuvants include aluminum hydroxide, alum, QS-21 (US Pat. No. 5,057,540), precursors and modified forms thereof (eg, DHEA-S, sulfonated form of DHEA) (US Pat. No. 5,407,684). And 5,077,284), β-2 microglobulin (WO 91/16924), muramyl dipeptide, muramyl tripeptide (US Pat. No. 5,171,568), and monophosphoryl lipid A (US Pat. No. 4,436,728; WO 92/16231). ) And its derivatives (eg Detox^TM), And BCG (US Pat. No. 4,726,947). Other suitable adjuvants include aluminum salts, squalene mixtures (SAF-1), muramyl peptides, saponin derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera Toxin B subunits, polyphosphazenes and derivatives, and immunostimulatory complexes (ISCOMs) as described in Takahashi et al. (1990) Nature 344: 873-875 include, but are not limited to. For use in veterinary medicine and production of antibodies in animals, a Freund's adjuvant mitogenic composition can be used.
The choice of adjuvant depends in part on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the prescribed acceptability of the adjuvant, particularly when intended for human use. For example, alum is approved by the US Food and Drug Administration (FDA) for use in the human body as an adjuvant and used in our clinical trials. 11D10 can be administered in a precipitated form; for example, alum precipitated 11D10 can be used. The preparation of aluminum hydroxide precipitate 11D10 is described in Examples 3 and 4. When QS-21 is used, preferably 100 μg is used as each dose, which is preferably administered subcutaneously within about 30 minutes after mixing with 11D10 (care should be taken to gently mix). Detox^TMIs preferably used as each dose, which is preferably administered subcutaneously within about 30 minutes after mixing with 11D10. The manufacturer may generally provide recommendations regarding quantity, volume, preparation, and route of administration.
Alternatively, 11D10 can be encapsulated in liposomes, for example. Liposomes suitable for packaging polypeptides for delivery to cells are known in the art.
11D10 can be heat treated prior to administration, and the heat treatment can be performed in the presence of an adjuvant (eg, alum). For example, 11D10 can be heated at about 40 ° C. to 60 ° C., preferably 45 ° C. to 55 ° C., for about 5 minutes to 2 hours, preferably 15 minutes to 1 hour. The heat treatment is more preferably performed at 45 ° C. for 30 minutes in a sterile vial in a water bath. The heat treatment can be performed at any time prior to administration. Preferably, the heat treatment is performed within 7 days after administration. Other heat treatment methods can be used as long as the desired activity of 11D10 is not significantly lost.
For the purpose of eliciting an immune response, 11D10 can be administered in unmodified form. It may be preferred to modify 11D10 to improve immunogenicity. Methods for improving immunogenicity include cross-linking with reagents such as glutaraldehyde or bifunctional binders, or binding to multivalent platform molecules, among others. Immunogenicity can also be improved by binding to protein carriers, particularly those containing T cell epitopes.
11D10 can be used alone or in combination with other factors that promote the desired activity / purpose. In this sense, the “factor” can be any of a variety of substances. Furthermore, “in combination with” means that the factor can be used simultaneously, before or after 11D10. The desired activity is any activity that facilitates, enhances, promotes or modulates the desired purpose when 11D10 is used. Factors that can be used include, but are not limited to, cytokines, lymphokines, adjuvants, and drugs. Agents also include substances that facilitate delivery of 11D10 (eg, liposomes) or substances that facilitate delivery of 11D10 to specific targets (eg, cell receptors). For example, 11D10 can be administered with a cytokine such as GM-CSF.
To determine the effect of administration of 11D10, the individual can be monitored for either an antibody against HMFG (humoral) or a cellular immune response, or a combination thereof.
For example, serum or plasma is obtained from an individual to determine the level of HMFG antibody (Ab3) in a biological sample. The sample is optionally enriched for immunoglobulin before the assay is performed (this is not usually required). When mouse immunoglobulin (eg, 11D10) is used as an assay reagent, the sample is preferably pretreated to remove anti-mouse immunoglobulin activity. This can be done, for example, by depletion on a mouse immunoglobulin column, or by mixing nonspecific mouse immunoglobulin into the sample and removing any immunoprecipitates that have formed.
To perform this assay, anti-HMFG that may be present in the sample is contacted with a non-limiting amount of an antigenic equivalent of HMFG. This includes isolated HMFG, nitrocellulose with HMFG attached by direct blotting or transcription from polyacrylamide gels, cells expressing HMFG (eg, MCF-7 or SKBR3 cells), membranes from such cells It can be a preparation or an immobilized tissue section containing HMFG. Alternatively, an anti-idiotype, in particular 11D10, can be used.
Once the immune complex is formed, it is separated from the HMFG analog that was not normally complexed, and the amount of complex present is determined. The complex can be separated, for example, by centrifugation of recovered cells, or immunoprecipitation, or capture by a solid phase. The amount of complex present can be measured by supplying a labeled HMFG analog directly or by incubating with a second reagent. Alternatively, a competition assay can be performed, where the sample is first incubated with an HMFG analog, and then an unrestricted amount of labeled anti-HMFG reagent is added that competes with anti-HMFG that may be present in the sample. Suitable labels include radioactive labels, enzyme labels, fluorescent labels, and chemiluminescent labels. Using a solution known to be free of anti-HMFG, a standard curve is constructed and solutions with various relative concentrations of anti-HMFG are used in place of the sample. Samples containing an unknown amount of anti-HMFG are usually assayed in parallel, and the relative amount of anti-HMFG contained therein is determined by comparison with a standard curve. Preferred assays for determining the level of anti-HMFG using the 11D10 antibody are described in further detail in the following sections.
The isotype of the anti-HMFG antibody can be determined by including an isotype specific reagent in the immunoassay either at the separation step or at the labeling step. For example, anti-human IgG can be used to isolate or detect IgG class antibodies present in clinical samples of human origin. The presence of IgG class specific anti-HMFG usually indicates a memory response. The presence of an IgM class anti-HMFG usually indicates an ongoing immune stimulation (eg, may be due to the presence of an HMFG-expressing tumor) or treatment with 11D10.
If desired, anti-HMFG antibodies detected in a biological sample can be further characterized; for example, by competing with anti-MC10 (Ab1) they are specific for the relevant epitope on HMFG. It is determined whether there is. Competition assays between Ab1 and Ab3 are described in detail in the Examples section.
An anti-HMFG antibody can also be tested to determine if it is cytotoxic. Complement-mediated cytotoxicity (CMC), for example⁵¹Determined using Cr-labeled HMFG expressing target cells (eg, MCF-7 or SKBR3). The sign is about 10⁶About 200μCi Na₂ ⁵¹CrO_FourCan be achieved by incubating at 37 ° C. for 60 minutes followed by washing. This assay is performed by incubating the antibody (or clinical sample containing the antibody) with the target cells. The opsonized cells are then washed and incubated with a complement source; for example, guinea pig serum is pre-adsorbed to remove intrinsic antibody activity. After an appropriate incubation period at 37 ° C,⁵¹Release of Cr into the medium is determined and compared to that from non-opsonized control cells.⁵¹The release of Cr into the medium is measured and compared to that from non-opsonized control cells.⁵¹Cr release correlates with CMC activity.
Another way to characterize anti-HMFG antibodies is by testing their ability to participate in ADCC responses (Cheresh et al. (1986), Cancer Res. 46: 5112). Radiolabeled HMFG expressing target cells are incubated with anti-HMFG (in the form of heat inactivated serum) and effector cells. Normal human peripheral blood mononuclear cells (PBMC) are suitable effector cells, and preferably an effector: target ratio of about 100 is used. Released after about 4 hours at 37 ° C⁵¹The proportion of Cr is determined as a measure of ADCC activity.
The cellular immune response in subjects receiving 11D10 can be quantified by performing standard functional assays for specific T cell activity.
One type of assay measures T cell proliferation. In this test, peripheral blood mononuclear cells (PBMC) are obtained from whole blood samples collected from treated subjects. For laboratory animals, spleen cells can also be used. T cells can be enriched by centrifuging with a gradient such as, for example, Ficoll (TM). The cells are then cultured in the presence of various concentrations of HMFG or (more commonly) irradiated HMFG expressing cells. Preferably, the stimulator cell is autologous with the responder cell, particularly with respect to histocompatibility class II antigen.
Another type of assay measures T cell cytotoxicity. In this study, the enriched population of T cells is⁵¹Used to degrade Cr-labeled HMFG expressing target cells (prepared as described above). Preferably, the effector cells are autologous with the target cells, particularly with respect to histocompatibility class I antigens. The population of T cells can be primed with HMFG or an appropriate cell line, if desired. The T cells are then^FourCombine with various labeled target cells in various ratios (eg, in the wells of a microtiter plate). Plates are optionally centrifuged to initiate cell contact and the cells are incubated together at 37 ° C. for 4-16 hours.⁵¹Percentage of specific release of Cr into the medium is based on comparison of labeled targets cultured alone (negative control) and targets degraded with detergents such as 0.1% Triton (TX) X-100 (positive control) , Measured.
Other suitable measures that measure the effects of 11D10 administration include clinical trials that may be appropriate to determine the progression of a suspicious type of cancer. Such tests can include inflammation indicators, mammography, and radioscintigraphy (described elsewhere in this disclosure).
Use of 11D10 to perform immunoassays. Another manner in which 11D10 can be used is to assay the presence of antibodies or other immune components that bind to 11D10 or HMFG. Such compounds can be present after therapeutic administration of 11D10 or can occur naturally due to the presence of HMFG expressing tumors in an immunocompetent host. The assay can be performed on a biological sample, usually a clinical sample.
In one embodiment of the invention, 11D10 is used to detect the presence of anti-HMFG (especially anti-11D10 idiotype) that may be present in a biological sample. Samples are suitably prepared by enriching for antibody activity as necessary prior to conducting the assay. If a biological sample is suspected of containing antibodies that are active against non-idiotype regions of 11D10 (especially anti-mouse immunoglobulin), the assay should be performed to eliminate them or avoid their detection Preferably it is done. The anti-mouse immunoglobulin antibody can be removed from the sample, for example, by precipitation with normal mouse IgG or by adsorption by mouse Ig adsorption. Binding of anti-mouse immunoglobulin antibodies (particularly antibodies specific for the Fc region) can be minimized by careful selection of assay reagents. 11D10 F (ab ')₂Or Fab fragments and other mouse immunoglobulin reagents are particularly suitable.
After the sample is properly prepared, it is mixed with the sample and excess functionally equivalent 11D10 under conditions that allow a complex to form between 11D10 and any anti-HMFG that may be present. The amount of complex was then determined and compared to the complex formed with a standard sample containing a known amount of anti-HMFG within the expected range. Complex formation can be observed by immunoprecipitation or nephrometry, but labeling (eg,¹²⁵It is generally more sensitive to use reagents labeled with radioisotopes such as I), enzymes such as peroxidase and β-galactosidase, or fluorescent dyes such as fluorescein.
Antibody assays can be performed in the liquid phase. For example, anti-HMFG can be mixed with label 11D10. Alternatively, anti-HMFG in the sample can be used to compete with labeled anti-HMFG for binding sites on 11D10. In general, bound label and unbound label are separated to quantify the percent binding. Suitable methods of separation include gel filtration chromatography and precipitation with antibodies to immunoglobulin from the species from which the sample was obtained (optionally in the presence of polyethylene glycol). Alternatively, the percentage of bound and unbound label can be determined in situ using, for example, a fluorescent / quenched label pair or an enzyme / inhibitor label pair. See, for example, U.S. Pat. No. 3,996,345 Ullma et al.).
It is usually more convenient to perform capture assays using reagents bound to a solid phase (eg, polyethylene test tubes, microtiter plate wells, or magnetic beads). In a competitive capture assay, unlabeled anti-HMFG in the sample competes with labeled anti-HMFG for binding to 11D10. 11D10 can be bound directly to a solid support or can be captured later using, for example, anti-11D10. In this assay, the amount of label associated with the solid phase is inversely related to the amount of anti-HMFG in the sample.
In a sandwich capture assay, anti-HMFG is captured by 11D10 bound to the solid phase directly or through a second reagent. After washing, anti-HMFG is detected using an appropriate species of anti-immunoglobulin, or a second 11D10 antibody to which the label binds directly or indirectly. Alternatively, anti-immunoglobulin can be bound to a solid phase and label 11D10 is used to simplify the sandwich type assay. If the anti-immunoglobulin used is isotype specific, then the class of antibody can also be determined. In this type of assay, the amount of label bound to the solid phase is positively correlated with the amount of anti-HMFG in the sample.
Other methods of measuring specific antibodies are known in the art and can be applied to measure anti-HMFG by using 11D10 as the target antigen. All such applied methods are envisaged in the present invention. Further description of specific embodiments is provided in the Examples section.
11D10 can also be used to measure the level of cellular anti-HMFG activity, particularly the anti-11D10 idiotype. In a preferred embodiment, 11D10 is used to identify anti-HMFG T cells. T cells are defined for this purpose as lymphocytes expressing T cell receptors that bind to the 11D10 idiotype. 11D10 can be labeled and contacted with a population of cells suspected of containing anti-HMFGT cells. Alternatively, unlabeled 11D10 can be mixed with cells and subsequently combined with a labeled secondary reagent such as labeled anti-mouse immunoglobulin or protein A. Suitable labels for this purpose include radioactive labels and fluorescent labels. The use of fluorescent labels also separates anti-HMFG cells from non-specific cells in a fluorescence activated cell sorter.
Use of 11D10 to remove label Ab1. The invention also encompasses methods of using 11D10 for removal of label (eg, radioactive material) from an individual that has received labeled anti-HMFG antibody (Ab1), eg, for radioscintigraphy or radiotherapy. One common problem in the use of antibody-targeted radionuclides (ie, radioimmunotherapy) is the presence of excess Ab1 in a system that limits the dose of radiolabeled antibody for treatment. Furthermore, effective imaging using radiolabeled antibodies is hampered by excessive circulating radiolabeled antibodies, which often take several days to be removed from the circulatory system and tissue. In these methods of the invention, 11D10 is administered to an individual at a specific time after administration of labeled anti-HMFG. The intent is to complex 11D10 with anti-HMFG at sites other than the tumor (eg, circulatory system and gaps), thereby promoting its clearance. As a result, the level of labeled moieties (eg, radioisotopes) in unaffected tissues was reduced and tumor images (compared to adjacent tissues) were enhanced. Similarly, if a subject is given a radionuclide for irradiation of a tumor site, it is desirable to reduce incidental exposure of unaffected tissue. Accordingly, the present invention encompasses treatment methods in which a radiolabeled anti-HMFG antibody is administered at a therapeutic dose followed by a molar excess of 11D10.
In any of these applications, the amount of 11D10 will be a molar amount sufficient to exceed the amount of labeled anti-HMFG to place and bind to any anti-HMFG that is not localized at the tumor site. Selected. The time of administration and the amount of 11D10 will depend on the nature of the radiolabeled antibody, the type of radioisotope used, and the condition of the individual. Preferably, the molar ratio of 11D10 to anti-HMFG antibody is at least about 5: 1, more preferably from about 25: 1 to 200: 1. Preferably, 11D10 is administered within 5 to 24 hours after the individual receives the anti-HMFG antibody.
Use of 11D10 to detect anti-HMFG antibody bound to tumor cells. The invention also encompasses a method of detecting the presence of anti-HMFG antibody that binds to tumor cells, the method comprising: forming tumor cells by treating with 11D10 for a sufficient amount of time to bind to anti-HMFG antibodies; Detecting all the presence of the complex. This intent allows 11D10 to be used to detect anti-HMFG pre-bound to tumor cells; or promotes binding of anti-HMFG to tumor cells by forming multivalent anti-HMFG / 11D10 immune complexes. It is to be. In the former example, 11D10 is provided with a detectable label or by means to which the label can bind. In the latter example, either anti-HMFG or 11D10 is provided with a label.
This strategy can also be used, for example, to identify HMFG antigen-bearing cells in isolated cell suspensions. Cells are incubated sequentially or simultaneously with anti-HMFG and 11D10, washed and then labeled cells are detected. Preferred labels for this embodiment include fluorescent labels such as fluorescein, rhodamine, and Texas Red. If desired, labeled cells are separated from unlabeled cells using any solid phase positive or negative immunoselection technique known in the art, for example, by separation with a fluorescence activated cell sorter or by affinity separation. obtain.
This strategy can be used, for example, for detection or imaging of a tumor in an affected subject. Anti-HMFG and 11D10 are administered to the subject (usually continuously) and are accumulated at the tumor site. Suitable labels include radiolabels (eg,¹¹¹In,¹³¹I and⁹⁹mTc). Tumors are then detected or visualized using standard methods of radioscintigraphy.
11D10 polynucleotide
The invention includes a polynucleotide encoding an anti-idiotype antibody 11D10 or a fragment of 11D10 based on the polynucleotide sequences shown in FIGS. These polynucleotides are isolated and / or produced by chemical and / or recombinant methods, or combinations of these methods. Unless otherwise stated, the term “polynucleotide” or “11D10 polynucleotide” should include all embodiments of the polynucleotide of the invention.
The 11D10 polynucleotides of the invention are useful as probes, primers, and in pharmaceutical preparations including vaccines in expression systems. Particularly useful applications of this polypeptide are discussed below.
Accordingly, the present invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide having 11D10 immunological activity, wherein the polypeptide comprises at least 5 consecutive amino acids of the 11D10 variable region. . In one embodiment, the coding polynucleotide sequence encodes a variable region derived from a light chain. In another embodiment, the coding polynucleotide sequence encodes a variable region derived from a heavy chain.
The present invention also provides the 11D10 polynucleotide shown in FIGS. In one embodiment, an isolated polynucleotide that encodes a polypeptide having an immunological activity of 11D10 is provided, wherein the polynucleotide comprises the 11D10 of SEQ ID NO: 2 (FIG. 1). Contains at least 5 consecutive amino acids of the variable light chain. In another embodiment, an isolated polynucleotide encoding a polypeptide having an immunological activity of 11D10 is provided, wherein the peptide is a variable of 11D10 as shown in SEQ ID NO: 4 (FIG. 2). Contains at least 5 consecutive amino acids of the heavy chain. In another embodiment, the (variable region) coding polypeptide sequence is shown in SEQ ID NO: 1 (FIG. 1). In another embodiment, the (variable region) encoding polynucleotide sequence is shown in SEQ ID NO: 3 (FIG. 2). The polynucleotide sequence may be similar to the sequence shown in SEQ ID NO: 1 (FIG. 1) or SEQ ID NO: 3 (FIG. 2), with minor changes designed to optimize codon usage or stability. Or may vary significantly. Given the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, designing such polynucleotides is within the ability of one skilled in the art. FIG. 1 shows the nucleotide sequence of the variable region of the light chain of 11D10 (SEQ ID NO: 1) and the derived amino acid sequence (SEQ ID NO: 2). FIG. 2 shows the nucleotide sequence (SEQ ID NO: 3) and the derived amino acid sequence (SEQ ID NO: 1) of the variable region of the heavy chain of 11D10. The nucleotide sequence of SEQ ID NO: 1 is 435 base pairs and was obtained from the clone described in Example 2. The polynucleotide sequence of SEQ ID NO: 3 is 467 base pairs and was obtained as described in Example 2.
In another embodiment, the invention provides at least about 60 contiguous nucleotides of SEQ ID NO: 1, preferably 70 contiguous nucleotides, preferably at least about 80 contiguous nucleotides, more preferably at least about 100 contiguous nucleotides. And even more preferably comprising a polynucleotide encoding a portion of the 11D10 light chain variable region comprising at least about 150 contiguous nucleotides. The present invention also includes at least about 15 contiguous nucleotides of CDR1 encoding the sequence of the 11D10 light chain variable region, preferably at least about 25 contiguous nucleotides, more preferably at least about 30 contiguous nucleotides. A polynucleotide encoding a part of the chain variable region is included. The invention also includes at least about 10 contiguous nucleotides of CDR2 or CDR3 encoding the sequence of the 11D10 light chain variable region, preferably at least about 15 contiguous nucleotides, and even more preferably at least about 20 contiguous nucleotides A polynucleotide encoding a portion of the 11D10 light chain variable region.
In another embodiment, the invention provides at least about 60 contiguous nucleotides of SEQ ID NO: 3, preferably at least about 70 contiguous nucleotides, preferably at least about 80 contiguous nucleotides, more preferably at least about 100 contiguous nucleotides. A polynucleotide encoding a portion of the 11D10 heavy chain variable region, comprising still more preferably at least about 150 contiguous nucleotides. The present invention also provides a polynucleotide encoding a portion of the 11D10 heavy chain variable region comprising at least 10 consecutive nucleotides of CDR1 encoding the sequence of the 11D10 heavy chain variable region, preferably at least about 15 consecutive nucleotides. Including. The present invention also provides at least about 15 contiguous nucleotides of CDR2 or CDR3 encoding the sequence of the 11D10 heavy chain variable region, preferably at least about 20 contiguous nucleotides, preferably at least about 25 contiguous nucleotides, more preferably Polynucleotides encoding portions of the 11D10 heavy chain variable region comprising at least about 35 contiguous nucleotides, and even more preferably at least about 50 contiguous nucleotides.
In another embodiment, the invention includes any of the aforementioned 11D10 polynucleotides, wherein said polynucleotide encodes at least 5 amino acids of a complementarity determining region (CDR). CDRs are described below.
The present invention includes modifications to the 11D10 polynucleotides described above, such as deletions, substitutions, additions or changes in properties in any nucleic acid moiety. A “modification” is any difference in the nucleotide sequence compared to the polynucleotide set forth herein that encodes an 11D10 polypeptide fragment and / or any difference in the nucleic acid portion of the polynucleotide. Such changes can be useful to facilitate cloning and expression modification of 11D10 polynucleotides. Such changes can also be useful in imparting desired properties, such as stability, to the polynucleotide. The definition of polynucleotide provided herein provides examples of these modifications.
The present invention includes 11D10 polynucleotides comprising their full length (unprocessed), processed polynucleotides, coding polynucleotides, non-coding polynucleotides, or portions thereof, of 11D10 polynucleotides. However, these polynucleotides contain a region encoding at least a part of the variable region of 11D10. Also encompassed are mRNA and cDNA sequences and fragments thereof comprising a portion of a variable region coding segment.
The invention also provides functionally equivalent variants and derivatives of 11D10 that may enhance, decrease, or do not significantly affect the properties of the polypeptide encoded thereby and functional Polynucleotides encoding for fragments equivalent to. These functionally equivalent variants, derivatives and fragments exhibit the ability to induce an immune response, preferably an anti-HMFG immune response. For example, changes in the DNA sequence that do not change the encoded amino acid sequence, as well as changes that result in conservative substitution of amino acid residues, changes that result in one or several amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs A change that results in a change that does not significantly affect the properties of the encoded polypeptide. Nucleotide substitutions that do not change the encoded amino acid residue may be useful for optimizing gene expression in different systems. Appropriate substitutions are known to those skilled in the art and are made, for example, to reflect preferred codon usage in a particular expression system. In another example, an alternatively spliced polynucleotide can give rise to a functionally equivalent fragment or variant of 11D10. Alternative processed polynucleotide sequence variants are mRNAs that differ in sequence from each other but are derived from the same genomic region, eg, 1) use of alternative promoters; 2) use of alternative polyadenylation sites; or 3) Defined as the polynucleotide sequence corresponding to the mRNA resulting from the use of alternative splice sites.
The 11D10 polynucleotides of the invention also include polynucleotides that encode other 11D10 fragments. Polynucleotides encoding 11D10 fragments are useful, for example, as probes, therapeutic agents, and as templates encoding various functional and / or binding domains of 11D10. Thus, the present invention forms stable hybrids with polynucleotides encoding the light chain or heavy chain variable region of 11D10, but does not hybridize with other immunoglobulins encoding regions known at the time of filing this application. At least 15 contiguous nucleotides, more preferably at least about 20 contiguous nucleotides, more preferably at least about 25 contiguous nucleotides, more preferably at least about 35 contiguous nucleotides, more preferably at least about 50 contiguous nucleotides Even more preferably at least about 75 contiguous nucleotides, even more preferably at least about 100 contiguous nucleotides, even more preferably at least about 200 contiguous nucleotides, still more preferably at least about 300 contiguous nucleotide regions. Including poly Including the Kureochido. In one embodiment, the region comprises a polynucleotide comprising a variable light chain encoding the sequence of SEQ ID NO: 1 under conditions where the region does not form a stable hybrid with SEQ ID NO: 5 to SEQ ID NO: 14. Heavy chains can be formed. In another embodiment, the region is stable to a polynucleotide comprising a variable heavy chain encoding the sequence of SEQ ID NO: 3 under conditions where the region does not form a stable hybrid with SEQ ID NO: 5 to SEQ ID NO: 32. Heavy chains can be formed.
In another embodiment, the 11D10 polynucleotide fragment comprises about 15, preferably 20, and even more preferably 30 bases of the sequence shown in FIG. 1 (SEQ ID NO: 1) or FIG. 2 (SEQ ID NO: 3). A fragment of this size can encode the binding site of an Ab1 or Ab3 antibody. Suitable fragments are those that specifically hybridize to 11D10 DNA or RNA so that they are effective as primers or probes. Primers are particularly useful in polymerase chain reaction (PCR).
Hybridization reactions can be performed under conditions of different “stringency”. This condition is a condition that increases the stringency of hybridization reactions well known and published in the art. See, for example, Sambrook and Maniatis. Examples of relevant conditions are (in order of increasing stringency): incubation temperatures of 25 ° C., 37 ° C., 50 ° C. and 68 ° C .; 10 × SSC, 6 × SSC, 1 × SSC, 0.1 × SSC (where SSC 0.15 M NaCl and 15 mM citrate buffer) and their equivalent concentrations using other buffer systems; 0%, 25%, 50% and 75% formamide concentrations; incubation conditions from 5 minutes to 24 hours One, two or more wash steps; 1, 2 or 15 minutes wash incubation time; and 6 × SSC, 1 × SSC, 0.1 × SSC, or deionized water wash solutions.
"T_m"Is the temperature in degrees Celsius at which 50% of the polynucleotide duplex made from the complementary strand hydrogen bonded in antiparallel by Watson-Crick base pairing dissociates into a single strand under experimental conditions. T_mCan be predicted according to the following standard formula:
T_m= 81.5 + 16.6log [Na⁺] +0.41 (% G / C) -0.61 (% F) -600 / L
Where [Na⁺] Is the cation (usually sodium ion) concentration in mol / L; (% G / C) is the number of G and C residues as a percentage of all residues in the duplex; (% F) Is the percent formamide (wt / vol) in solution; and L is the number of nucleotides in each strand of the duplex.
Useful 11D10 polynucleotides that encode fragments of 11D10 give rise to polynucleotide fragments (eg, based on SEQ ID NO: 1 in FIG. 1 or SEQ ID NO: 3 in FIG. 2) and code for the function of interest thereby Can be obtained by testing the polypeptide. Alternatively, if there is a desired 11D10 polypeptide, the polynucleotide sequence can be derived from the amino acid sequence of the 11D10 polypeptide. For example, 11D10 polypeptide binds to Ab1 and / or Ab3. Or they can be tested for their ability to elicit an immune response. Assays for these various functions are described below.
The invention also includes polynucleotides that encode 11D10 derivatives or variants, including one or more 11D10 polypeptides, such as polynucleotides encoding scFvs, polymers, fusion proteins, and chimeras. These forms of 11D10 are described below.
The invention also provides a polynucleotide covalently linked to a detectable label. Such polynucleotides are useful, for example, as probes for detecting related nucleotide sequences.
Preparation of 11D10 polynucleotide
The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR.
Methods for chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. One skilled in the art can generate the desired DNA sequence using the sequences provided herein and a commercially available DNA synthesizer.
To prepare 11D10 polynucleotides using recombinant methods, a polynucleotide containing the desired sequence can be inserted into a suitable vector, which is then introduced into a suitable host cell for replication and amplification. obtain. The polynucleotide can be inserted into the host cell by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, conjugation or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrating vector (such as a plasmid) or can be integrated into the host cell genome. The polynucleotide so amplified can be isolated from host fat by methods well known in the art. See, for example, Sambrook et al. (1989).
Alternatively, PCR allows for DNA sequence regeneration. PCR technology is well known in the art and includes U.S. Pat.Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, andPCR: The Polymerase Chain Reaction, Edited by Mullis et al., Birkauswer Press, Boston (1994).
RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. If the cells are replicated and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example as described in Sambrook et al. (1989).
When used as a vaccine, Horn et al. ((1995)Human gene therapy 6: 565-573), which produces a plasmid DNA for pharmaceutical use suitable for administration, a plasmid containing 11D10 polynucleotide is prepared.
Cloning and expression vectors containing 11D10 polynucleotides
The invention further includes various vectors having cloned 11D10 polynucleotides. These vectors can be used for the expression of recombinant polypeptides as well as a source of 11D10 polynucleotides. Cloning vectors can be used as a means of storing polynucleotides in a depository in order to obtain replicate copies of the 11D10 polynucleotides they contain, or for future recovery. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. Vectors can also be used when it is desirable to have intact cells capable of expressing the 11D10 polypeptide in an individual and thus synthesizing the polypeptide, such as in gene therapy. Suitable cloning and expression vectors include any vector known in the art, such as vectors used in bacterial expression systems, mammalian expression systems, yeast expression systems and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, Gacesa and Ramji,Vectors, John Wiley & Sons (1994).
Cloning and expression vectors typically contain a selectable marker (eg, a gene encoding a protein necessary for the survival or growth of host cells transformed with the vector), but such marker genes It can be carried on another polynucleotide sequence co-introduced into the cell. Only those host cells into which the selection gene has been introduced survive and / or grow under selective conditions. Typical selection genes are: (a) proteins that confer resistance to antibiotics or other toxin substances (eg, ampicillin, neomycin, methotrexate, etc.); (b) proteins that complement auxotrophic defects; or (c) Encodes proteins that provide important nutrients that are not available from complex media. The selection of an appropriate marker gene depends on the host cell, and appropriate genes for different hosts are known in the art. Cloning and expression vectors also typically contain a replication system that is recognized by the host.
Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. The selected cloning vector can vary according to the host cell intended for use, and useful cloning vectors generally have the ability to self-replicate and carry a single target for a particular restriction endonuclease. And / or carry a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses (eg, pUC18, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28). These cloning vectors and many other cloning vectors are available from suppliers such as BioRad, Strategene, and Invitrogen.
An expression vector is generally a replicable polynucleotide construct that contains a polynucleotide encoding the 11D10 polypeptide of interest. The polynucleotide encoding 11D10 polypeptide is operably linked to appropriate transcriptional control elements such as promoters, enhancers and terminators. For expression (ie, translation), one or more translation control elements such as a ribosome binding site, a translation initiation site, and a stop codon are also usually required. These regulatory elements (transcriptional and translational elements) can be derived from 11D10 nucleotides (ie, 11D10 genes) or they can be heterologous (ie, derived from other genes and / or other organisms). ). A polynucleotide sequence encoding a signal peptide may also be included to allow the 11D10 polypeptide to cross and / or remain in the cell membrane or to be secreted from the cell. A number of expression vectors suitable for expression in eukaryotic cells, including yeast, avian and mammalian cells, are known in the art. One example of an expression vector is pcDNA3 (Invitrogen, San Diego CA), where transcription is driven by a cytomegalovirus (CMV) early promoter / enhancer. This vector also contains multiple restriction enzyme recognition sites for insertion of the 11D10 polynucleotide of interest. Another example of an expression vector (system) is the baculovirus / insect system.
Vectors containing the polynucleotide of interest include electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other material transfection; microprojectile bombardment; lipofection; and infection ( Here, the vector can be introduced into the host cell by any number of suitable means, including infectious agents such as the vaccinia virus described below. The choice of means for introducing the vector or 11D10 polynucleotide often depends on the host cell.
Once introduced into a suitable host cell, such as E. coli or COS-7, the expression of 11D10 polypeptide can be measured using any assay described herein. For example, the presence of 11D10 polypeptide can be detected by RIA or ELISA of the culture supernatant or cell lysate (if 11D10 polypeptide is secreted).
Particularly useful expression vectors for 11D10 polynucleotides are vaccinia viruses comprising 11D10 polynucleotide sequences, which can also be used in vaccine preparation. Moss (1991)Science 252: 1662-1667. To introduce a polynucleotide sequence encoding an 11D10 polypeptide, including an 11D10 polypeptide fragment, into a vaccinia, the polynucleotide sequence of interest is first combined with flanking sequences homologous to vaccinia DNA that are not essential for replication. Insert into a plasmid containing the vaccinia virus promoter. The plasmid-containing cells are then infected with vaccinia, which leads to a low level of homologous recombination between the plasmid and the virus, and the vaccinia promoter and polynucleotide sequence encoding the 11D10 polypeptide is transferred into the vaccinia virus genome. Is done. Typically, the 11D10 polynucleotide is inserted into the viral tk (thymidine kinase) gene. Insertion at the tk site attenuates the virus more than 10,000 times compared to the wild type (Flexner et al. (1989)Vaccine 88 (Cold Spring Harbor Laboratory), 179-184). Recombinant virus is tk^-Identified by phenotype. Preferably, expression of the 11D10 polynucleotide is under the control of a vaccinia early / late promoter (7.5K), whereby the resulting 11D10 polypeptide can be expressed in infected cells throughout the viral life cycle. However, other promoters known in the art such as pH 6, synthetic promoters, SV40 promoters, or promoters derived from adenovirus can be used. Expression of 11D10 polypeptide occurs in cells infected with recombinant vaccinia or individuals immunized with live recombinant vaccinia virus. Construction of a vaccinia vector for expression of 11D10 polypeptide is provided in Example 4. Any one of several strains of vaccinia, including but not limited to WR, ALVAC and NYVAC may be used. ALVAC and NYVAC strains are used to infect avian cells.
A vaccinia vector of the invention may contain one or more polynucleotides encoding 11D10 polypeptide. A vaccinia vector also encodes a polynucleotide sequence that encodes other polypeptides that enhance, facilitate, or modulate the desired result, such as, but not limited to, lymphokines including IL-2, IL-4 and GM-CSF. Can be included. A preferred lymphokine is GM-CSF. When using GM-CSF, by means of recombination methods, eliminating AU-rich elements from the 3 ′ untranslated region of RNA transcripts and / or eliminating sequences in the 5 ′ untranslated region that may form hairpin loops Is also preferred. Also encompassed by the present invention are vaccinia vectors encoding recombinant 11D10 variants, including 11D10 polypeptides, such as scFvs, chimeras, and polymers (described below).
Host cells transformed with 11D10 polynucleotides
Another embodiment of the invention is a host cell transformed (ie, containing) an 11D10 polynucleotide and / or a vector having an 11D10 polynucleotide sequence, as described above. Both prokaryotic and eukaryotic host cells can be used. Prokaryotic hosts include bacterial cells such as E. coli and mycobacteria. Among eukaryotic hosts are yeast, insects, birds, plants and mammalian cells. Host systems are known in the art and need not be described in detail herein. One example of a mammalian host cell is NSO, which can be obtained from the European Collection of Cell Cultures (UK). For example, transfection of NSO cells with a plasmid driven by the cytomegalovirus (CMV) promoter, followed by amplification of this plasmid with glutamine synthetase provides a useful system for protein production. Cockett et al. (1990)Bio / Technology 8: 662-667.
The host cells of the invention can be used, inter alia, as a reservoir for 11D10 polynucleotides and / or as a vehicle for the production of 11D10 polynucleotides and polypeptides. They can also be used as vehicles for in vivo delivery of 11D10 polypeptides.
Uses and methods of use of 11D10 polynucleotides
The polynucleotides of the present invention have several uses. 11D10 polynucleotides are useful, for example, in expression systems for recombinant production of 11D10 or 11D10 fragments. They are also useful as hybridization probes to assay for the presence of 11D10 polynucleotide (or related) sequences in a sample using methods well known to those skilled in the art. In addition, 11D10 polynucleotides are also useful as primers to achieve amplification of the desired polynucleotide. The polynucleotides of the invention are also useful as vaccines and for gene therapy.
The 11D10 polynucleotide of the present invention can be used as a primer for amplification of a polynucleotide encoding 11D10 or a fragment thereof, as in polymerase chain reaction (PCR). PCR was described above. The conditions for carrying out the PCR reaction depend on the desired specificity, which can be adjusted depending on the primers used and the reaction conditions. Such adjustments are known in the art and need not be described in detail herein.
11D10 polynucleotides can also be used, for example, as hybridization probes for the detection of the presence of 11D10 polynucleotides in cells. For example, 11D10 polynucleotides can be used as probes to determine the presence of 11D10 polynucleotide sequences in cells used in gene therapy. For these methods, obtain an appropriate cell sample or cell-derived sample (both samples that are believed to contain an 11D10 polynucleotide sequence) and contact the 11D10 polynucleotide probe with a sample-derived polynucleotide and an 11D10 polynucleotide probe. Test for the presence of nucleotides. This method is performed to allow hybridization to occur between the 11D10 probe and the 11D10 polynucleotide of interest, and the resulting hybrid complex (if any) is detected. Such methods require procedures well known in the art such as cell culture, polynucleotide preparation, hybridization, and detection of hybrid complexes formed, if any. Using similar methods, the probes can also be used to detect vectors that are then used to produce 11D10 polypeptides, intact 11D10, or recombinant mutant forms of 11D10.
The 11D10 polypeptides of the present invention produce 11D10 polypeptides, intact 11D10, or recombinant forms of 11D10, including intact 11D10 with enhanced, equivalent or different desired properties Can be used in the expression system. These recombinant forms are made using routine methods in the art. Examples of recombinant forms of 11D10 and 11D10 polypeptides include, but are not limited to, hybrids, chimeras, single chain variants, and fusion proteins, including other components such as cytokines. A more detailed description of these 11D10 recombinant forms and 11D10 polypeptides and their production is provided below.
Another use for 11D10 polynucleotides is in vaccines and gene therapy. The general principle is to administer the polynucleotide so as to either promote or attenuate expression of the polypeptide encoded therein. Accordingly, the present invention encompasses methods of inducing an immune response and methods of treatment comprising administering to an individual an effective amount of 11D10 polynucleotide. In these methods, an 11D10 polynucleotide encoding an 11D10 polypeptide is administered to an individual either directly or via cells transfected with the 11D10 polynucleotide. Preferably, the 11D10 polynucleotide is replicated in the cell. Thus, the 11D10 polynucleotide is operably linked to a suitable promoter, such as a heterologous promoter that is endogenously active in cells of the target tissue type. Invasion of the polynucleotide into the cell is accomplished by techniques known in the art, such as through a viral expression vector such as a vaccinia or adenoviral vector, or by coupling the polynucleotide to a cationic liposome. Preferably, the 11D10 polynucleotide is in the form of a circular plasmid, preferably in a superhelical configuration. Preferably, once in the cell nucleus, the plasmid persists as a circular non-replicating epitope molecule. In vitro mutagenesis can then be performed using plasmid constructs, for example, to encode more immunogenic molecules or T cell episomes having the desired HLA motif.
To determine whether a plasmid containing the 11D10 polynucleotide can be expressed in eukaryotic cells, eukaryotic organisms such as COS-7 cells, CHO (bird origin) cells, or HeLa (human origin) cells, for example. Cells can be transfected with the plasmid. The expression resulting in 11D10 polypeptide is then measured by RIA or ELISA. Western blots can be performed using cell lysates using MC-10 (Ab1) as a probe to confirm 11D10 polypeptide associated with the cells. Alternatively, for smaller 11D10 polypeptides, expression can be detected by constructing a plasmid such that the resulting 11D10 polypeptide is recombinantly labeled, eg, using an enzyme label. In addition, characterization of the expressed 11D10 polypeptide is achieved by performing purification of the 11D10 polypeptide followed by a functional assay described herein (eg, a cell binding inhibition assay).
The invention also includes ex vivo transfection of 11D10 polynucleotides, wherein cells removed from an individual are transfected with a vector encoding 11D10 polypeptide and reintroduced into the individual. Suitable transfected cells include but are not limited to peripheral blood mononuclear cells.
The therapeutic administration of 11D10 polynucleotide is described in more detail below.
11D10 polypeptide
The invention includes 11D10 polypeptide fragments that contain at least a portion of the variable region of 11D10, and proteins comprising 11D10 fragments. A polypeptide fragment of 11D10 that can include any region or subregion of SEQ ID NO: 2 (FIG. 1) or SEQ ID NO: 4 (FIG. 2), provided that the fragment includes at least a portion of the variable region, is based on the following criteria: (A) ability to bind to Ab1 and / or Ab3; (b) ability to induce an immune response against HMFG; (c) homology to any portion of HMFG (ie, substantial sequence identity); (d ) Identified and characterized by any (one or more) ability to alleviate, ameliorate, reduce or delay HMFG-related diseases, particularly HMFG-related tumors.
Polypeptide fragments of 11D10 are used in pharmaceutical compositions and vaccines as their diagnostic tools for monitoring Ab1 and / or Ab3 levels, their use in generating antibodies that bind to HMFG, and labels It has a variety of uses, including their use in removing labeled Ab1 from individuals that have received anti-HMFG antibodies.
Unless otherwise indicated, the term “11D10 polypeptide” encompasses all embodiments of the polypeptides of the invention.
The invention includes a polypeptide having an immunological activity of 11D10, wherein the polypeptide is composed of a sequence of at least 5 consecutive amino acids from the variable region of 11D10. In one embodiment, the variable region is derived from the light chain, and more particularly is shown in SEQ ID NO: 2 (FIG. 1). In another embodiment, the variable region is derived from a heavy chain and, more particularly, is shown in SEQ ID NO: 4 (FIG. 2). In another embodiment, the five consecutive amino acid sequences are derived from complementarity determining regions (CDRs).
The amino acid sequences of SEQ ID NO: 2 (FIG. 1) and SEQ ID NO: 4 (FIG. 2) are shown in FIG. 3, which show the framework and CDR sequences of the 11D10 light and heavy chain variable regions, respectively. The framework sequence is responsible for correct β-sheet folding of the VL and VH domains and interstrand interactions that bring the domains together. Complementarity determining regions (CDRs) refer to the six hypervariable sequences (3 from VL and 3 from VH) of the variable region that are thought to form antigen binding sites together. The delinearization of these regions and the identification of the 11D10 leader sequence was based on the search and analysis of Kabat's immunology database with the BLAST program.
Another embodiment of the invention is a polypeptide fragment of 11D10 comprising a sequence selected from the group consisting of the amino acid sequences (fragments) shown in FIG. These polypeptides represent functional subregions (ie, frameworks and CDRs) of the light and heavy chain variable regions. Preferably, these 11D10 polypeptides comprise a CDR.
The present invention also provides at least 25 contiguous amino acids, preferably at least 28, more preferably at least 30 contiguous amino acids, even more preferably at least about 35 of the variable region shown in SEQ ID NO: 2 (FIG. 2). Contiguous amino acids, even more preferably at least about 50 contiguous amino acids, or at least 5 contiguous amino acids of its CDR1 or CDR2, preferably at least 7 contiguous amino acids, preferably at least 8 contiguous amino acids, more preferably A polypeptide fragment of the 11D10 light chain variable region comprising at least about 10 contiguous amino acids, or at least 7 contiguous amino acids of its CDR3, preferably at least 8 contiguous amino acids, more preferably at least 9 contiguous amino acids Including.
In another embodiment, the invention provides at least 17 contiguous amino acids, preferably at least 20 contiguous amino acids, preferably at least about 25 contiguous amino acids of the variable region shown in SEQ ID NO: 4 (FIG. 1). More preferably at least about 35 consecutive amino acids, even more preferably at least about 50 consecutive amino acids, or five consecutive amino acids of CDR1 thereof, or at least six consecutive amino acids of CDR2 or CDR3, preferably at least Polypeptide fragments of the 11D10 heavy chain variable region comprising 7 consecutive amino acids, more preferably at least about 10 consecutive amino acids.
The size of the 11D10 polypeptide fragment can vary widely since the length required to produce activity can be very small, but the maximum length is typically not detrimental to produce activity. The minimum size must be sufficient to provide the desired function. For example, a binding site on a polypeptide can be as short as about 5 amino acids in length, while other binding sites are formed by a collection of amino acids that are spatially proximal but not in adjacent sequences. . Thus, the present invention includes an 11D10 polypeptide fragment comprising a portion of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 1) or SEQ ID NO: 4 (FIG. 2), wherein the 11D10 polynucleotide is about 5 amino acids. It is long. The present invention also provides a polypeptide fragment of 11D10 comprising a portion of the amino acid sequence shown in SEQ ID NO: 2 (FIG. 1) or SEQ ID NO: 4 (FIG. 2), wherein the 11D10 polynucleotide is about 10, 15 25, 30, 50, 100, or 150 amino acids in length. The present invention also provides an 11D10 polypeptide fragment comprising a portion of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 1) or SEQ ID NO: 4 (FIG. 2), having at least about 5 amino acids and at most about 100 amino acids. To do. As will be apparent to those skilled in the art, regardless of their size, these 11D10 polypeptides can also be associated or bound to other substances or factors to facilitate the function and / or specificity of the 11D10 polypeptide. , Enhance, or regulate. Examples of such modifications will be described later.
In another embodiment, 11D10 polypeptide fragments are provided that contain regions of homology to HMFG, particularly tandemrepeats of 20 amino acids within HMFG. See, for example, Larocca et al. (1992) Hybridoma 11: 191-201. Such homologous fragments are nominally similar to the high molecular weight mucin antigen of HMFG nominally and can therefore participate in antigen presentation by mimicking HMFG (final target antigen). These 11D10 polypeptides also cooperate with class I major histocompatibility complex (MHC) antigens to participate in antigen presentation and thus cause killing of cytotoxic T cells. FIG. 23 shows an alignment between similar sequences of 11D10 and HMFG when the amino acid sequences are aligned in both directions (ie, aligned in the same and reverse directions). Examples of regions of homology to HMFG included in the present invention are as follows (SEQ ID NO: 2; amino acid numbering based on amino acids 1 to 107 in FIG. 3): (a) amino acids 51 to 52 of the light chain; Amino acids 54 to 56; amino acids 92 to 93; and (b) amino acids 57 to 58 of the heavy chain. Accordingly, the present invention also provides about amino acid 50 to about amino acid 53, about amino acid 50 to about amino acid 56, about amino acid 92 to about amino acid 93 of the sequence shown in FIG. 3-A (amino acids 1 to 107 of SEQ ID NO: 2), Or an 11D10 polypeptide comprising the amino acid sequence from about amino acid 90 to about amino acid 94, and the sequence shown in FIG. 3-B (amino acids 1-118 of SEQ ID NO: 4) from about amino acid 57 to about amino acid 58, from about amino acid 56 to about Polypeptides comprising amino acid 58, or about amino acid 53 to about amino acid 58 are included.
The present invention includes modifications to 11D10 polypeptides, including functionally equivalent fragments of 11D10 polypeptides that do not significantly affect their properties, and variants with enhanced or decreased activity. Polypeptide modifications are routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides that involve conservative substitution of amino acid residues, one or more deletions or additions of amino acids that do not significantly adversely alter functional activity, or the use of chemical analogs. . Amino acid residues that can be conservatively substituted for each other include, but are not limited to: glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine; Arginine; and phenylalanine / tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as glycosylation, acetylation and phosphorylation with different sugars. Preferably, amino acid substitutions are conservative, i.e., the substituted amino acid has chemical properties similar to those of the original amino acid. Such conservative substitutions are known in the art and examples are provided above. Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as a variable region. Changes in the variable region can alter binding affinity and / or specificity. Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution and chelation. The modification can be used for the attachment of a label for an immunoassay, for example, the attachment of a radioactive moiety for a radioimmunoassay. Modified 11D10 polypeptides can be made using procedures established in the art and screened using standard assays known in the art, some of which are described below in the Examples.
The invention also includes a fusion protein comprising one or more 11D10 polypeptides. In one embodiment, at least 10 contiguous amino acids of the variable light chain region shown in SEQ ID NO: 2 (FIG. 1) and at least 10 amino acids of the variable heavy chain region shown in SEQ ID NO: 4 (FIG. 2) A fusion polypeptide comprising is provided. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises an 11D10 light chain variable region and a heavy chain variable region. For purposes of the present invention, an 11D10 fusion protein contains one or more 11D10 polypeptides and another amino acid sequence that is not bound to it by the native molecule, eg, a heterologous sequence or a homologous sequence from another region. . Useful heterologous sequences include, but are not limited to, sequences that provide secretion from the host cell, sequences that enhance immunological reactivity, or coupling of the polypeptide to an immunoassay support or vaccine carrier. Includes a facilitating sequence. Another example is a so-called bacterial “superantigen” such as Staphylococcus enterotoxin A (SEA). Dohlsiten et al. (1994) Proc. Natl. Acad. Sci. USA 91: 8945-8949. For example, an 11D10 polypeptide can be fused to a biological response modifier. Examples of biological response modifiers include, but are not limited to, cytokines such as GM-CSF, interleukin-2 (IL-2), interleukin-4 (IL-4), and γ-interferon Or include lymphokines. Accordingly, the present invention includes 11D10 fusion polypeptides comprising GM-CSF or IL-2. FIG. 25 shows an example of a plasmid construct for fusion of the 11D10 polypeptide with a preferred lymphokine GM-CSF or IL-2. Cotransfection of this plasmid (encoding 11D10 heavy chain, as indicated) with a plasmid encoding 11D10 light chain also produces an 11D10 fusion polypeptide. Preferably, a plasmid encoding 11D10 light chain is first transfected into Sp2 / 0 or NSO cells by protoplast fusion (Shin et al. (1989) Meth. Enzym. 178: 459-476) followed by electroporation. The plasmid containing the coding sequence of the 11D10 heavy chain is transfected into the high producing clone from the first transfection. Shin et al. (1989). These procedures are described in more detail in Example 7.
Antibodies produced as a result of transfection of the plasmid (either by co-transfection or sequential transfection) (ie, antibodies comprising heavy and light chains) will form a light chain attached to the heavy chain. It can be detected by any assay that detects. Such assays are routine in the art. For example, non-reducing SDS gel electrophoresis can be used to detect the presence (indicated by molecular weight) of antibody molecules that contain both light and heavy chains. Another example of an assay that detects light chains bound to heavy chains is an ELISA as follows. Microtiter plates are coated with standard concentrations of goat anti-human kappa light chain antibody, blocked with BSA and washed. The coated plate is reacted with culture supernatants of cells expressing various test constructs. After washing, the plates are then treated with goat anti-human γ-1 antibody with alkaline phosphatase conjugate and developed in the usual manner. Absorbance is measured at 405 nm. Where the fusion antibody comprises a biologically responsive molecule such as a cytokine, the antibody can also be detected, for example, by using an assay that measures the reactivity of the cytokine. Such assays are known in the art and need not be described in detail herein. For example, GM-CSF fusion 11D10 antibody and / or 11D10 polypeptide can be detected as follows. The plate is coated with goat anti-human kappa antibody and the coated plate is reacted with the culture supernatant (if the fusion is secreted). The reacted plate is then treated with rat antibody against the mouse GM-CSF / biotin conjugate and the resulting complex is detected by measuring absorbance at 490 nm. These assays are described in more detail in Example 7.
Alternatively, the plasmid of FIG. 25 can be transfected into a heavy chain loss mutant. For example, heavy chain loss mutants can be obtained by treating 2 × 10 7 11D10 cells with fluorescein-labeled rabbit anti-mouse IgG (H chain specific, DAKO Corporation, Carpinteria, Calif.) According to the supplier's instructions. it can. Stained and unstained cell populations are analyzed with a fluorescence activated cell sorter. Unstained cells are collected in sterile tubes and placed in 96-well plates at 1 cell / well by limiting dilution. The culture supernatant is then assayed by ELISA using goat anti-mouse IgG (heavy chain specific) and goat anti-mouse κ. Clones with kappa positive and IgG negative phenotypes are subcloned at least 3 times to obtain stable 11D10 (-H) mutants. A putative heavy chain loss mutant (11D10 (-H)) clone can be isolated and the light chain variable region cDNA sequenced to confirm that the remaining light chain is the light chain of 11D10. Reverse mRNA PCR for 11D10 VH is performed with two sets of 5′- and 3′-primers used for cloning of 11D10 (−H) cDNA (Example 2). The heavy chain loss mutant should not produce a detectable DNA band. Transfection of these cells with heavy chain constructs can then be accomplished using standard methods in the art such as electroporation.
11D10 fusion polypeptides can be generated, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide region is encoded in the desired relationship. These fusion proteins can be useful for enhancing, modifying, and / or facilitating the activity of the 11D10 polypeptide.
The invention also includes 11D10 modified recombinant forms, including 11D10 polypeptides, ie, 11D10 polypeptides that contain at least a portion of the variable region of 11D10 shown in FIGS. As used herein, a “modified” or “recombinant” form of 11D10 contains an 11D10 polypeptide that differs in sequence and / or configuration from intact 11D10. The recombinant form of the 11D10 antibody included in the present invention is a hybrid antibody, wherein one pair of heavy chain and light chain is homologous to those of the first antibody, The other pair is homologous with a different second antibody. For the purposes of the present invention, one pair of light and heavy chains is derived from 11D10. Typically, each of these two pairs binds to a different epitope of HMFG. Such hybrids can also be formed using chimeric chains, as described below.
In another embodiment, an 11D10 chimera is provided in which the heavy and / or light chain is a fusion protein. Typically, the constant domain of the chain is from one particular species and / or class, and the variable domain is from a different species and / or class. For example, a “humanized” 11D10 antibody is an antibody in which the constant region is of human origin and the variable region is derived from 11D10 (ie, mouse). Also, an antibody having a humanized variable region in which the framework region is derived from a human sequence but the CDR region comprises an 11D10 amino acid sequence is exemplified in the present invention. For example, see EP0329400. Also illustrated are chimeric functional fragments. One example is a humanized Fab fragment, which contains a human hinge region, a human first constant region, a human kappa light chain or heavy chain constant region, and a variable region from 11D10. Humanized 11D10 Fab fragments are generated in order to form Fab dimers. Typically, the 11D10 fusion proteins and 11D10 chimeras of the present invention are made by preparing and expressing the polynucleotides encoding them using the recombinant methods described herein, Can also be prepared by other means known in the art including, for example, chemical synthesis.
Another example of a modified recombinant form of 11D10 included in the present invention is a modified antibody, which refers to an antibody having an altered amino acid sequence of 11D10. Using standard recombinant techniques, the 11D10 antibody can be designed to obtain the desired properties. For example, changes in the amino acid sequence can result in greater immunogenicity of the resulting 11D10 polypeptide. Changes range from changing one or more amino acids to complete redesign of a region, eg, the constant region. In general, changes in the constant region may achieve desired cellular process characteristics, such as changes in complement fixation, membrane interaction, and other effector functions. Changes in the variable region can be made to alter the binding properties. Modified / recombinant 11D10 antibodies can also be designed to facilitate specific delivery of substances (such as lymphokines) to effector cells. Other amino acid sequence modifications are described above.
The invention also includes 11D10 single chain variable region fragments (“scFv”). Single chain variable region fragments are made by linking light and / or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) Science 242: 423-426. An example of a linking peptide is (GGGGS) 3 (SEQ ID NO: 35), which bridges about 3.5 nm between the carboxy terminus of one variable region and the amino terminus of another variable region. Other sequence linkers have been designed and used. Bird et al. (1988). The linker can in turn be modified for further functions such as drug attachment or attachment to a solid support.
Accordingly, one embodiment of the present invention is that at least 10 contiguous amino acids of the light chain variable region shown in SEQ ID NO: 2 (FIG. 1) and the heavy chain variable shown in SEQ ID NO: 4 (FIG. 2). A fusion polypeptide comprising at least 10 contiguous amino acids of a region, wherein the amino acid segments are linked by a linker polypeptide of about 5-20 amino acids. In another embodiment, the fusion polypeptide (scFv) comprises a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 1) and a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 4 (FIG. 2).
Any peptide with sufficient mobility and length can be used as a linker in scFv. Usually, the linker is selected to have little to no immunogenicity. With respect to the 11D10 component of scFv, all or part of the heavy and / or light chain may be used. Typically, the entire variable region is included in the scFv. For example, a light chain variable region can be linked to a heavy chain variable region. Alternatively, part of the light chain variable region can be linked to all or part of the heavy chain variable region. For asymmetric linkers such as (GGGGS) 3 (SEQ ID NO: 35), the scFv can be assembled in either order, eg, VH-(linker)-VL or VL-(linker)-VH. Can be. However, when expressed in E. coli, there can be differences in the expression levels of these two configurations. It is also possible to construct a hybrid (or two-phase) scFv (where one component is an 11D10 polypeptide and the other component is a different polypeptide such as a T cell epitope). Is possible. (X)-(Linker)-(X)-(Linker)-(X) (where X is an 11D10 polypeptide or a combination of an 11D10 polypeptide and another polypeptide) A simple tandem scFv can also be made.
Single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, an appropriate plasmid containing a polynucleotide encoding scFv is either a eukaryotic cell such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell such as E. coli. In any suitable host cell. A polynucleotide encoding the scFv of interest can be made by routine manipulation such as ligation of the polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.
A particularly useful system for the production of 11D10 scFv is the plasmid vector pET-22b (+) (Novagen, Madison, WI) in E. coli. pET-22b (+) contains a nickel ion binding domain consisting of six consecutive histidine residues that serves as the basis for scFv purification. This example (shown in Example 7) is for illustrative purposes only and is not limiting. Another example of a vector that can be used is pcDNA3 (Invitrogen, San Diego, Calif.) As described above.
When E. coli is used for scFv production, the conditions should be such that the scFv polypeptide can ensure optimal tertiary and quaternary structure. Depending on the plasmid used (particularly the activity of the promoter) and the host cell, it may be necessary to regulate the production of scFv. For example, the use of weaker promoters or lower temperature expression may be necessary to optimize scFv production. Alternatively, scFv expression in eukaryotic cells such as yeast, insects, plants or mammals may be appropriate.
Various scFvs can be tested for binding activity, for example, by testing for direct binding to Ab1, or by using them in the competition experiments described herein. Any of the assays described below that test fragments for 11D10 activity can be used to test scFv. For example, radiolabeled Ab1 (MC-10) in the absence or presence (increasing amount) of the scFv to be tested is converted to HMFG such as MCF-7 cells.⁺React with cells. The percent inhibition observed is compared to 11D10 or another Ab2. 11D10 scFv is characterized as capable of binding if the scFv inhibits Ab1 binding to HMFG positive cells when compared to a negative control such as an unrelated anti-idiotype antibody. Alternatively, scFv can be characterized using other immunological assays described herein, such as the ability to elicit an immune response. Furthermore, scFvs can be constructed with or without immunoglobulin leader sequences (for secretion) depending on whether a secreted or cell-associated form of scFv is desired.
In another embodiment, a single chain 11D10 antibody polypeptide is provided that includes no linker or a very short non-flexible linker. These so-called “bivalent” antibodies cannot participate in intrachain interactions because of the absence of a linker (or because of the presence of very short linkers) and therefore interact with other single chains. , Form “diabodies”. For example, a bivalent 11D10 antibody polypeptide can be made using recombinant methods in either of the following configurations: VL--VH or VH--VL.
The present invention also includes polymeric forms of 11D10 polypeptides. As used herein, polymeric forms of 11D10 polypeptides contain multiple (ie, more than one) 11D10 polypeptides. In one embodiment, a linear polymer of 11D10 polypeptide is provided. These 11D10 linear polymers can be bound to a carrier. These linear polymers can include multiple copies of a single 11D10 polypeptide, or a combination of different 11D10 polypeptides, and can have tandem 11D10 polypeptides, or 11D10 polypeptides separated by other amino acid sequences. These linear polymers can be made using standard recombinant methods well known in the art. In another embodiment, an 11D10 multiple antigen peptide (MAP) is provided. MAP has a small immunologically inert core with a radially branched lysine dendrite, on which a large number of 11D10 polypeptides can be tethered (ie, covalently linked). Posnett et al. (1988) J. Biol. Chem. 263: 1719-1725; Tam (1989) Meth. Enz. 168: 7-15. The result is a large macromolecule with a high molar ratio of 11D10 polypeptide to core. MAP is a useful and effective immunogen and an antigen useful for assays such as ELISA. 11D10 MAP can be made synthetically and can be obtained commercially (Quality Controlled Biochemicals, Inc., Hopkinton, Mass.). In a typical MAP system, the core matrix consists of 8 amino acids to anchor three levels of lysine and 11D10 polypeptide. MAP can be synthesized by any method known in the art, for example, a solid phase method. For example, R.B.Merrifield (1963) J.Am.Chem.Soc.85: 2149.
In another embodiment of the invention, the immunogenicity of the 11D10 polypeptides is prepared in an expression system that is fused or assembled with the particle-forming protein such that, for example, the 11D10 polypeptides are associated with the hepatitis B surface antigen. Can be enhanced. See, for example, US Pat. No. 4,722,840. A construct in which the 11D10 polypeptide is directly linked to the particle-forming protein coding sequence results in a hybrid that is immunogenic with respect to the 11D10 polypeptide. Furthermore, all prepared vectors contain epitopes specific for HBV with varying degrees of immunogenicity, such as, for example, pre-S peptides. Therefore, particles constructed from particle-forming proteins containing 11D10 sequences are immunogenic with respect to 11D10 and HBV. These forms of 11D10 polypeptides can be made in eukaryotic cells such as yeast or mammalian cells.
In another embodiment, the 11D10 polypeptide can be bound to a carrier. If the 11D10 polypeptide takes the correct configuration to provide a binding site, but is very small enough to be immunogenic, the polypeptide can be linked to a suitable carrier. Numerous techniques for obtaining such a connection are known in the art and need not be described in detail herein. Any carrier that does not itself induce the production of antibodies that are detrimental to the host may be used. Suitable carriers are proteins; polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, etc .; polymeric amino acids such as polyglutamic acid, polylysine, etc .: amino acid copolymers; and inactive viruses Particles, or macromolecules that are typically large and slowly metabolized, such as attenuated bacteria, such as Salmonella. Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art. As will be apparent to those skilled in the art, 11D10 polypeptides, such as fusion proteins, and the above recombinant forms of 11D10 can be fused in turn with other amino acid sequences. For example, 11D10 scFv can be fused to a cytokine such as IL-2. FIG. 25 provides an example of a plasmid construct that produces such a fusion protein.
The 11D10 polypeptides of the present invention can be identified in a number of ways. For example, the light and heavy chain variable regions can be screened by preparing a series of short polypeptides that span the entire variable region amino acid sequence together. For example, it is routine to test each polypeptide for the presence of the desired property by starting with a 50-mer or 20-mer polypeptide. Screening for such polypeptides is well within the skill of those in the art. Also, protein sequences to identify potentially interesting polypeptides, for example, computer analysis of homology to HMFG, or identify putative regions associated with idiotype-anti-idiotype contacts, and then test these It is also known to implement computer algorithms based on molecular recognition theory to prepare these polypeptides containing regions.
Those skilled in the art will readily understand that the various forms and derivatives of 11D10 described in this section can be combined in various ways to produce other 11D10 polypeptides with desirable properties. For example, 11D10 polypeptides with modified residues can be included in the MAP. In another example, 11D10 scFv is fused to a cytokine such as IL-2.
Polypeptide preparation
The polypeptides of the present invention can be made by procedures known in the art. Polypeptides can be produced by proteolysis or other degradation of 11D10, by the recombinant methods described above (ie, single or fusion polypeptides), or by chemical synthesis. 11D10 polypeptides, particularly short polypeptides of up to about 50 amino acids, can be conveniently made by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, 11D10 polypeptides can be produced by an automated polypeptide synthesizer using solid phase methods.
Preferably, the polypeptide is at least partially purified from other cellular components. Preferably, the polypeptide is at least 50% pure. In this sense, purity is calculated as a weight percent of the total protein content of the preparation. More preferably, the protein is 50-70% pure. More highly purified polypeptides can also be obtained and are included in the present invention. For clinical use, the polypeptide is preferably highly purified and is at least about 80% pure, preferably at least about 90% pure, more preferably at least about 95% pure, and even more preferably at least about 99% pure. There are no pyrogens or other contaminants. Protein purification methods are known in the art and are not described in detail herein. Alternatively, if the 11D10 polypeptide is expressed in a suitable storage medium such as a plant seed, the 11D10 polypeptide need not be purified and may be administered without purification. Fiedler et al. (1995) Biotechnology 13: 1090-1093.
The 11D10 polypeptide can be obtained from intact 11D10, which can in turn be isolated from a hybridoma that produces 11D10 (ATCC HB 12020), which is co-owned US patent application Ser. No. 08 / 575,762 (tentative number). No. ______; agent case number 30414-20003.00). Techniques for isolating antibodies from hybridomas are well known in the art. See, for example, Harlow and Lane (1988). Once intact 11D10 is obtained, 11D10 polypeptides can be obtained by intact 11D10 degradation, for example, by using a proteolytic enzyme (proteinase). Examples of proteolytic enzymes include, but are not limited to, trypsin, plasmin and thrombin. Intact 11D10 can be incubated with one or more proteinases or digested sequentially. The nature and extent of proteolytic cleavage depends on the desired polypeptide length and the enzyme used. These techniques are well known in the art. Alternatively, or in addition, intact 11D10 can be treated with a disulfide reducing agent to dissociate the molecule.
11D10 polypeptides can be made by chemical synthesis using techniques known in the art.
11D10 polypeptides can also be produced by expression systems using recombinant methods. If 11D10 polynucleotides encoding 11D10 polypeptides are available, it is possible to construct expression vectors encoding intact 11D10, functionally equivalent fragments thereof, or recombinant forms of 11D10. The polynucleotide encoding the desired 11D10 polypeptide can be an expression vector suitable for any convenient host, regardless of whether it contains a signal sequence that allows secretion, whether in a fused or mature form. Can be linked. Both eukaryotic and prokaryotic host systems can be used. The polypeptide is then isolated from lysed cells or culture medium and purified to the extent necessary for its intended use. Purification or isolation of the polypeptide expressed in the host system can be accomplished by any method known in the art. For example, a cDNA encoding intact 11D10 or a fragment thereof can be operably linked to a suitable promoter, inserted into an expression vector, and transfected into a suitable host cell. The host cell is then cultured under conditions that allow transcription and translation to recover the desired polypeptide. Other segments that control transcription or translation may also be used, such as signal sequences that direct the polypeptide (ie, for secretion) to specific cellular compartments. Examples of prokaryotic host cells are known in the art and include, for example, E. coli. Examples of eukaryotic host cells are known in the art and include yeast, birds, insects, plants, and animal cells such as COS7, HeLa, CHO and other mammalian cells.
The polypeptides of the invention can also be expressed using recombinant vaccinia virus as a vector. This application is particularly useful in vaccine formulations because vaccinia virus carriers containing heterologous antigenic determinants have been shown to be successful immunogens. Expression of 11D10 polypeptides and their use in vaccinia vectors are described above and below.
Characterization of 11D10 polypeptide
The 11D10 polypeptides of the present invention can be characterized in several ways. For example, 11D10 polypeptides can be tested for the ability to bind to Ab1 and / or Ab3. Alternatively, 11D10 polypeptides can be tested for their ability to elicit an immune response, preferably an anti-HMFG response. 11D10 polypeptides can also be tested for their ability to alleviate or ameliorate HMFG-related diseases, such as HMFG-related tumors. While only one of these properties is necessary for the polypeptide to be present in order to be compatible with the present invention, it is understood that more than one of these properties can be present.
The ability of an 11D10 polypeptide to bind to Ab1 and / or Ab3 can be assessed in several ways. In one study, the binding of 11D10 polypeptide to Ab1 can be achieved directly as described in Example 1 (FIG. 1), for example, Ab1 or Ab3 coated with radiolabeled 11D10 polypeptide on a microtiter plate. Can be tested by, for example, radioimmunoassay (RIA).
In another procedure, binding to Ab1 or Ab3 is measured by a competitive immunoassay. In one modification of this procedure, the binding of labeled 11D10 polypeptide or functionally equivalent fragment to Ab1 (MC-10) can be achieved by different Ab1, other Ab2, 11D10 or analogs thereof, other 11D10 polypeptides, HMFG. Or measured in the presence of extracts containing HMFG, or other proteins. The percent inhibition is calculated by the following formula:

In another modification, test fragments with putative 11D10 activity are tested for the ability to prevent binding between Ab1 and Ab2 or between Ab1 and HMFG. This test may be more sensitive in some applications. This is because the lower affinity interaction between 11D10 and Ab1 is so weak that it cannot form a stable bond, but when present in sufficient concentration, it prevents the binding of another ligand-receptor pair. Because it is enough. HMFG can be provided as purified antigen or HMFG expressing cells. The assay can be performed by labeling either Ab1 or HMFG or Ab2 and immobilizing other members of the ligand-receptor pair to a solid support as needed to facilitate separation. The test fragment is incubated with a labeling reagent and then the mixture is presented to an immobilized target or test cell to determine whether the test fragment can inhibit binding. The degree of inhibition correlates with 11D10 activity.
Various examples of competitive assays are described below in the Examples section. One test showing 11D10 polypeptide activity is to measure the binding of radiolabeled Ab1 (MC-10) to semi-purified or purified HMFG in the presence of various amounts of 11D10 polypeptide. See, for example, Example 1. The Ab1-HMFG mixture is then added to the plate coated with 11D10 polypeptide and binding is compared to binding of labeled Ab1 alone. Preferably, this test is performed with an unsaturated amount of labeled Ab1 to detect binding changes with small amounts of competing HMFG. An example of this test performed on an intact 11D10 is shown in Example 1. In another competitive assay, HMFG positive target cells (such as MCF-7 or SKBR3) are grown as confluent monolayers in 96-well tissue culture plates. The binding of radiolabeled Ab1 (MC-10) in the absence and presence of 11D10 polypeptide is measured. The degree of inhibition can be compared to inhibition of intact 11D10 or other 11D10 polypeptides. An example of this competition assay using intact 11D10 is shown in Example 1. Another example of this assay comparing the degree of inhibition between 11D10 scFv and intact 11D10 is shown in Example 8.
If there is inhibition when compared to a negative control such as an unrelated anti-idiotype antibody that does not bind to Ab1, the 11D10 polypeptide will bind to Ab1.
Using all the above assays, the labeled molecule can be labeled with a radioisotope (ie,¹²⁵It will be apparent to those skilled in the art that labeling can be accomplished in a variety of ways using I) and using non-radioactive labels such as biotinylated molecules and molecules for enzyme detection, fluorescent and chemiluminescent labels.
The above test can also be used to compare the characteristics of various 11D10 polypeptide fragments. For example, a competition assay can be performed in which a first 11D10 polypeptide competes for binding to Ab1 (MC-10) in the presence of varying amounts of a second 11D10 polypeptide. . Such tests can indicate the relative degree of binding affinity or other characteristics.
Another way to characterize 11D10 polypeptides is to test their ability to generate an immune response. As used herein, “immune response” refers to either a humoral response, a cellular response, or both. As used herein, “ability to elicit an immune response” refers to any individual, including humans.
The ability of an 11D10 polypeptide to produce a humoral response can be measured by testing for the presence of antibodies that bind to the 11D10 polypeptide after administration of the 11D10 polypeptide. It is understood that this antibody (Ab3) was not present prior to administration of the 11D10 polypeptide or was present in low amounts. Immunogenicity is preferably tested in individuals without a prior anti-11D10 response. Examples of suitable individuals include but are not limited to mice, rabbits, monkeys and humans. For this study, an 11D10 polypeptide is administered to the individual. The amount per administration and the number of administrations vary depending on the individual. Based on our previous experiments with intact 11D10, mice required approximately 100 μg of 11D10 polypeptide coupled to KLH in the presence of CFA and IFA per dose and in 3 doses. Monkeys require approximately 2 mg. For purposes of the present invention, the range of 11D10 polypeptides that can be administered to humans is about 10 μg to 10 mg, preferably 100 μg to 10 mg, preferably 500 μg to 8 mg, more preferably 1 mg to 4 mg, even more preferably about 2 mg. It is.
The presence of Ab3 first pre-incubates sera with autologous immunoglobulin to block antibodies against isotype and allotype antigenic determinants and then binds to HMFG and / or 11D10 polypeptides using, for example, ELISA or RIA Can be determined by testing the serum. For example, different dilutions of pre-reacted serum are reacted with 11D10 (or 11D10 polypeptide) coated on a microtiter plate. Unrelated Ab2 serves as a control. After washing, for example in homogeneous sandwich assays¹²⁵I-labeled 11D10 is used to label the Ab3-11D10 complex. The results from this assay are compared to the results obtained before administration of 11D10 polypeptide. A more detailed description of such a detection assay for Ab3 induced by intact 11D10 in mice is described in Example 1. Alternatively, binding to HMFG positive cells such as human colon carcinoma LS174-T cells can be tested using immunoflow cytometry.
Ab3 binding to HMFG can also be determined by immunoprecipitation or immunoreactivity with HMFG positive tissue samples, or by dot blot analysis. In one method of dot blot analysis, a semi-purified extract of HMFG is blotted directly onto a nitrocellulose filter. The filter is then incubated with serum containing Ab3 and the reaction is expressed by enzyme-linked anti-immunoglobulin (Example 1). If Ab3 binds to HMFG, a positive blot should appear. For testing on tissue samples, an immunoperoxidase assay can be used (Example 1).
If desired, Ab3 induced by the 11D10 polypeptide can be further characterized. For example, a competition assay can be performed to determine whether Ab3 shares the Ab1 idiotope. In this test, sera from individuals immunized with 11D10 polypeptide are tested for inhibition of binding of labeled 11D10 polypeptide (or intact 11D10) to Ab1. Inhibition indicates that Ab3 and Ab1 contain at least similar binding determinants. Similarly, competition between Ab3 and Ab1 for binding to HMFG (whether partially purified, purified, or on the surface of HMFG positive cells) will result in a fixed amount of labeled Ab1 (MC-10). Can be tested by co-incubation with various dilutions of Ab3-containing serum or Ab1 preparation and HMFG (or HMFG-related cells such as MCF-7 or SKBR3). These tests are described for intact 11D10 in Example 1.
As will be apparent to those skilled in the art, using the assays described above, Ab3 can in turn be used to characterize the 11D10 polypeptide.
Another way to characterize 11D10 polypeptides is by testing their ability to elicit antibodies that are cytotoxic. For determination of complement-mediated cytotoxicity (CMC), SKBR3 (target) cells (ie, cells expressing HMFG)⁵¹Label with Cr. The sign is about 10⁶Cells approximately 200 μCi Na₂SO_FourCan be achieved by incubating at 37 ° C. for 60 minutes followed by washing. This assay is performed by adding and incubating serum suspected of containing the antibody. Guinea pig serum (or other complement source) pre-adsorbed with LS174-T cells is then added. After an appropriate incubation time at 37 ° C, then⁵¹Measure the extent of Cr release, and control of non-opsonized control cells⁵¹Compare with the extent of Cr release.⁵¹Cr release correlates with CMC activity. Herlyn et al. (1981)Int.J.Cancer 27: 769.
Another way to characterize 11D10 polypeptide is by testing its ability to elicit anti-HMFG antibodies involved in ADCC responses. Cheresh et al. (1986)Cancer research 46: 5112-5118. In this assay, cultured human MCF-7 or SKBR3 cells (ie, cells that express HMFG on their surface)⁵¹Labeled with Cr and used as target cells. Normal human peripheral blood mononuclear cells (PBMC) are used as effector cells. Preferably, the ADCC assay is performed at an effector: target cell ratio of 100: 1 for 4 hours in the presence of heat inactivated serum, although other suitable conditions can be used. Then⁵¹Measure the amount of Cr released.
The 11D10 polypeptides of the present invention can also be characterized by their ability to elicit cellular responses. As used herein, a “cellular response” is a response involving T cells and can be observed in vitro or in vivo.
One method of detecting a cellular immune response is by assaying for T cell proliferative activity. In this test, the cellular immune response is measured by proliferation of peripheral blood mononuclear cells (PBM) incubated with 11D10 polypeptide. Peripheral blood mononuclear cells are isolated from blood after administration of the required number of 11D10 polypeptides and incubated with various concentrations of 11D10 polypeptide. When using mice, T cells are obtained from the spleen. T cells are, for example, Ficoll^TMCan be enriched by centrifugation on a gradient such as A non-specific mitogen such as PHA serves as a positive control: Incubation with an unrelated anti-idiotype antibody serves as a negative control. Preferably, the stimulator cells are responder cells and autologous, particularly with respect to histocompatible Call II antigen. After growing the PBM by incubating for an appropriate number of days,^ThreeH] Thymidine incorporation is measured. In many cases, a suitable time is 5 days. If desired, a determination of which T cell subsets will grow can be made using flow cytometry. If necessary, splenic T cells can be obtained by monoclonal antibody RL.172 (anti-CD4⁺) Or mAb.168 (anti-CD8⁺) And complement incubation, CD4 prior to proliferation assay⁺Or CD8⁺Any of the cells can be pre-depleted.
Another method of detecting a cellular immune response is to test for T cell cytotoxicity (CTL) activity. In this study, T lymphocytes (ie, enriched T cell populations) are⁵¹Isolated (typically from spleen cells) for use as a target in Cr release assays. Kantor et al. (1992)J.Natl.Cancer Inst.84: 1084-1091.⁵¹An example of a Cr release assay is as follows. In short, HMFG positive tumor cells (typically 1-2 × 10⁶200μCi Na as target cell)₂ ⁵¹CrO_Four(Amersham Corp., Arlington Heights, Ill) is radiolabeled at 37 ° C. for 60 minutes, followed by extensive washing to remove unincorporated isotopes. T cells and target (1 × 10 6) both resuspended in medium^Four/ Well) are combined at various effector: target ratios in 96 well U-bottom plates (Costar Corp.). Plates are centrifuged at 100 xg for 5 minutes to initiate cell contact and 5% CO₂Incubate at 37 ° C for 4 or 16 hours. After incubation, the supernatant is collected using the Supernatant Collection System (Skatron, Inc., Sterling, VA) and radioactivity is quantified with a gamma counter (Beckman Instruments).⁵¹Spontaneous release of Cr was determined by target incubation in the absence of effector, while⁵¹Maximum or total release of Cr is determined by target incubation in 0.1% Triton X-100.⁵¹The specific release percentage of Cr is determined by the following formula:
Specific release percentage = [(experimental-natural) / (maximum-natural)] x 100
Another way to characterize 11D10 polypeptides is to test their ability to improve, retard, and / or reduce the extent of HMFG-related tumor progression. Such tests can include measurement of inflammation indicators, radioscintigraphy, or circulating HMFG levels (such assays are commercially available).
Uses and methods of use of 11D10 polypeptides
11D10 polypeptides have a number of uses. The 11D10 polypeptide can be used to induce an immune response in an individual, preferably an anti-HMFG response. They can also be used to detect and monitor Ab3 levels or to purify Ab3. 11D10 polypeptides are also useful for the treatment of HMFG-related diseases such as colorectal cancer, certain lung cancers (adenocarcinoma), gastric cancer, pancreatic cancer, and certain breast cancers.
Thus, the present invention includes a method of inducing an immune response in an individual comprising administering an 11D10 polypeptide in an amount effective to induce an immune response. Preferably, the individual has an HMFG associated tumor. In this sense, an “effective amount” is an amount sufficient to elicit a measurable immune response, whether humoral and / or cellular. An effective amount can be administered in one or more administrations.
The invention also includes a method of detecting an antibody (ie, Ab3 and / or Ab1) that binds to 11D10 in a biological sample. These methods are applicable, for example, in clinical situations for monitoring Ab1 or Ab3 levels in an individual, as well as in industrial situations where commercial production of Ab3 is desired. These methods contact Ab3 and / or Ab1 in a sample under conditions suitable to allow the formation of a stable complex between 11D10 polypeptide and Ab3 and / or Ab1 and 11D10 polypeptide. It is necessary to detect the stable complex formed, if any. A number of immunoassay methods are known in the art and are described herein. For further illustration, a test sample potentially containing Ab3 and / or Ab1 is typically a predetermined non-limiting amount of 11D10 polypeptide that is detectably labeled (eg, with a radioisotope or enzyme). Can be mixed. In liquid phase assays, unreacted reagents are removed by separation techniques such as filtration or chromatography. In these immunoassay techniques, the amount of label associated with the complex is positively correlated with the amount of Ab3 and / or Ab1 present in the sample. Similar assays can be designed. There, Ab3 and / or Ab1 in the test sample competes with the labeled antibody for binding to a limited amount of 11D10 polypeptide. Here, the amount of label is negatively correlated with the amount of Ab3 and / or Ab1 in the sample. Suitable samples for measuring Ab3 and / or Ab1 levels are biological samples, including serum or plasma, preferably serum. Other samples include tissue samples.
Furthermore, the present invention also includes contacting a biological sample containing Ab3 (and / or Ab1) with an 11D10 polypeptide, and obtaining a complex formed thereby, if any. Or a method of purifying Ab1). Typically, 11D10 polypeptide is coupled to an affinity matrix for affinity column purification. Such methods are routine in the art and need not be described in detail herein.
The invention also includes a method of treating an HMFG-related disease, such as an HMFG-related tumor, comprising administering an effective amount of 11D10 polypeptide. An “HMFG-related tumor” is a tumor containing HMFG, particularly HMFG expressed on the surface of tumor cells, examples of which are described above. In this sense, an effective amount for treatment is an amount sufficient to alleviate the disease state. An effective amount can be given in one or more doses. Treatment of an individual with an effective amount of a 11D10 polypeptide can, for example, reduce the rate of disease progression compared to an individual that is not so treated.
In another embodiment, a method is provided for stimulating a T cell response in an individual having an HMFG-related disease. This T cell response may manifest as promotion of T cell proliferation and / or cytotoxic T cell activity using 11D10 polypeptides, particularly 11D10 polypeptides that are homologous to HMFG. The 11D10 polypeptide can be administered directly (either as a polypeptide or a plasmid containing a polynucleotide encoding 11D10 polypeptide) or can be added to an ex vivo culture of appropriate cells. 11D10 polypeptide is added to the isolated peripheral blood mononuclear cells, for example, in an amount effective to stimulate the desired T cell activity. The stimulated T cells are then reintroduced into the individual. The amount of 11D10 polypeptide added will depend on several factors such as the condition of the individual, conventional and / or combination treatment procedures, and other materials used.
The polypeptides of the invention can be used alone or in combination with other factors that promote the desired activity / purpose. 11D10 polypeptides can also be used in various combinations with each other. In this sense, the “factor” can be any of a variety of substances. Furthermore, “in combination” means that the factors can be used at the same time, before, or after the polypeptide. An agent can also be covalently linked to a polypeptide, such as a fusion protein, or physically in close proximity to the polypeptide. The desired activity is any activity that facilitates, enhances, promotes or modulates the desired purpose in the use of the 11D10 polypeptide.
Factors that can be used include, but are not limited to, cytokines, lymphokines, adjuvants, and drugs. Agents also include substances that facilitate delivery of the polypeptide, such as liposomes, or substances that facilitate delivery of the polypeptide to a particular target, eg, a cellular receptor. For example, one or more 11D10 polypeptides can be produced as a fusion protein that also contains a cytokine such as GM-CSF. Alternatively, one or more 11D10 polypeptides can be administered with a cytokine such as GM-CSF.
The invention also includes methods of using 11D10 polypeptides to remove label (eg, radioactivity) from an individual that has received a labeled anti-HMFG antibody (Ab1), eg, for radioscintigraphy or radiotherapy. . The invention also encompasses a method of administering a radiolabeled anti-HMFG antibody at a therapeutic dose followed by a molar excess of 11D10 polypeptide.
The use of 11D10 for the purposes of the present invention is discussed above, and those principles apply to 11D10 polynucleotides as well. An amount of 11D10 polypeptide that is in a sufficient molar excess over labeled anti-HMFG is selected to locate and bind any anti-HMFG that is not localized at the tumor site. The timing of administration and the amount of 11D10 polypeptide will depend on the nature of the radiolabeled antibody, the type of radioisotope used, and the condition of the individual. Preferably, the molar ratio of 11D10 polypeptide to anti-HMFG antibody is at least about 5: 1, more preferably about 25: 1 to 200: 1. Preferably, the 11D10 polypeptide is administered 5-24 hours after the individual has received the anti-HMFG antibody.
For 11D10 polypeptides conjugated to anti-HMFG antibodies (especially MC-10), detection of anti-HMFG on the tumor cell surface is sufficient to allow tumor cells and 11D10 polypeptides to bind to anti-HMFG antibodies. Can be achieved by contacting for a long time and detecting the presence of any 11D10 bound to the anti-HMFG antibody. The development of experimental parameters (eg, 11D10 polypeptide amount or reaction time) is an empirical decision that is well within the skill of the art.
Pharmaceutical compositions and vaccines comprising 11D10, 11D10 polynucleotides and / or 11D10 polypeptides
The invention includes pharmaceutical compositions and vaccines containing 11D10, 11D10 polynucleotides and / or 11D10 polypeptides. Such pharmaceutical compositions / vaccines are useful for eliciting an immune response and / or for the treatment of HMFG related diseases such as breast cancer. The pharmaceutical composition / vaccine may alleviate or ameliorate HMFG-related diseases, either alone or in combination with other forms of treatment such as chemotherapy or radiation therapy. These pharmaceutical compositions consisting of an effective amount of 11D10, 11D10 polynucleotide and / or 11D10 polypeptide in a pharmaceutically acceptable excipient may comprise a unit dosage form, a sterile parenteral solution or suspension, a sterile Suitable for systemic administration to humans and animals in non-parenteral solutions or oral solutions or suspensions, oil-in-water or water-in-oil emulsions, and the like. Formulation or parenteral and non-parenteral drug delivery is known in the art and described in Remingtons' Pharmaceutical Sciences 18th edition, Mack Publishing (1990).
Pharmaceutically acceptable excipients are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, an excipient can impart form and consistency to the vaccine composition, or can act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for changing osmotic pressure, capsules, buffers, and skin penetration enhancers. Examples of pharmaceutically acceptable excipients are described in Remingtons' Pharmaceutical Sciences (1990), supra.
In one embodiment, a pharmaceutical composition containing 11D10 polypeptide is used, for example, to stimulate ex vivo culture of peripheral blood monocytes (PBM) from an individual. The PBM is then reintroduced into the individual. The pharmaceutical composition is used alone or in combination with other biological response modifiers such as lymphokines.
One type of pharmaceutical composition is a vaccine. Accordingly, the present invention also includes a vaccine comprising an effective amount of 11D10, 11D10 polynucleotide, 11D10 polypeptide, or combinations thereof and a pharmaceutically acceptable excipient. These vaccines can be used in particular to elicit an immune response in an individual, particularly an individual with a progressive HMFG-related disease such as an HMFG-related tumor. Preferably, the immune response includes the production of human milk fat globule antibodies. These vaccines are particularly useful for the treatment, modulation and / or alleviation of HMFG related diseases.
Administration of vaccines containing 11D10 was discussed above.
Vaccines containing the 11D10 polynucleotides described above can be used for so-called “gene immunization” or DNA vaccines, in which the polynucleotide encoding the antigen polypeptide is used to elicit a protective immune response. Introduced into the host cell. Tang et al. (1992) Nature 356: 152-154. Once in the cell nucleus, the plasmid can persist as a circular, non-replicating episome, leading to dose-dependent and long-term expression. Spooner et al. (1995) Gene Therapy 2: 173-180. It has been shown that immunization with polynucleotides results in a cellular response as well as a humoral response. Spooner et al. (1995); Wang et al. (1995) Human Gene Therapy 6: 407-418. Genetic immunization has many advantages of live or attenuated microorganisms as vehicles that elicit an immune response without the risk of infection.
Preferably, the 11D10 polynucleotide is introduced as a plasmid vector containing appropriate control sequences for transcription and translation such as promoters, enhancers and signal sequences. One or more 11D10 polynucleotides can be used in a single cloning vector and / or multiple vectors can be used. If multiple 11D10 polynucleotides are used, they should be inserted in-frame into the vector or placed under the control of separate promoters. The length and / or type of 11D10 polynucleotide used can vary and depend on several factors such as the clinical purpose of vaccination, the condition of the individual, and the individual's immunological profile. In addition, polynucleotides encoding other agents that enhance, facilitate and / or increase the immune response can also be inserted into the vector. Examples of such substances such as GM-CSF are described above.
For example, in one embodiment, a polynucleotide encoding 11D10 scFv is inserted into one of the above expression vectors (plasmids). In another example, a polynucleotide encoding the 11D10 fragment shown in FIG. 19 is inserted into an expression vector for administration as a vaccine. In another example, a polynucleotide encoding an immunogenic fragment of 11D10 is inserted into an expression vector.
Another type of vaccine that uses 11D10 polynucleotides is so-called expression library immunization, in which the host is immunized with an expression library of 11D10 polynucleotides (encoding various portions of 11D10). . Barry et al. (1995) Nature 377: 632-635. The resulting multi-part non-infectious vaccine can prove to be particularly useful because it presents multiple peptides as possible immunogens. The presentation of multiple immunogens has the additional advantage that each particular host (ie, an individual) that administers it can select an immunologically effective polypeptide, which can vary from individual to individual. The expression library used for expression of 11D10 polypeptides can be comprehensive (ie, collectively encode all 11D10 molecules) or can be partial. An expression library for immunization is prepared by the general recombination method described above using an appropriate vector system. Typically, 11D10 polynucleotides are fused in-frame with a signal sequence that mediates secretion.
The amount of 11D10 polynucleotide to be administered depends on the mode and route of administration (ie, direct injection vs. ex vivo culture and transfection), the 11D10 polypeptide encoded by the 11D10 polynucleotide, (immunological and / or disease It depends on the individual's condition (such as condition) and several factors such as the desired purpose. Typically, when administered directly, the amount per dose is about 10 μg to 1 mg, preferably 25 μg to 500 μg, more preferably 30 μg to 250 μg, even more preferably 50 to 100 μg.
In another embodiment, the 11D10 polynucleotide is used in a live or attenuated virus or viral vector that can express the encoded 11D10 polypeptide for vaccine formulation. Examples include, but are not limited to, adenovirus, adeno-associated retrovirus (AAV), and SV40. Preferably the virus is vaccinia. Recombinant vaccinia virus can provide a powerful factor for effectively co-presenting the 11D10 polypeptide encoded by the 11D10 polynucleotide with the immunogenic viral particle. The construction of the vaccinia virus vector has been described above. Generally, the recombinant viral vector is added in an amount sufficient to effect in vivo infection of the host cell. This amount will depend on the type of virus used, the nature of the encoded 11D10 polypeptide, the condition of the individual, and the desired result. Recombinant vaccinia (which may encode 11D10 variants containing 11D10 polypeptides or 11D10 polypeptides such as scFv) is about 10 per dose.⁷~Ten⁸It can be used directly for vaccination with plaque forming units. Vaccinia can be administered parenterally (eg, by subcutaneous or intramuscular injection), as well as through the mucosa (eg, nasally), orally, or by inhalation. Alternatively, vaccinia can be administered via vaccinia infected cells. In this technique, appropriate cells, such as tumor cells, are infected with vaccinia in culture. The infected cells are then reintroduced into the individual. Methods for infecting cells with vaccinia and methods for reintroducing those infected cells have been described. For example, see Moss (1990).
Vaccines can also be prepared from one or more 11D10 polypeptides. 11D10 polypeptides can be prepared by any of the methods described above, particularly by purification from an appropriate expression vector. In one embodiment, the vaccine comprises one or more 11D10 polypeptides. 11D10 polypeptides can be formulated into vaccines as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino acids of the 11D10 polypeptide), which include, for example, inorganic salts such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, Formed with an organic acid such as maleic acid. In addition, salts formed with free carboxyl groups also include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and isopropylamine, triethylamine, 2 -Can be derived from organic bases such as ethylaminoethanol, histidine, procaine and the like.
In another embodiment, a vaccine is provided that contains an 11D10 polypeptide fused to a viral particle such as hepatitis b surface antigen.
Preparation of vaccines containing 11D10 polynucleotides or polypeptides as active ingredients includes standard practice in the art. Typically, such vaccines are prepared as injections, either as liquid solutions or suspensions; solid forms suitable for solutions or suspensions in liquid prior to injection can also be prepared. . Vaccines can also be emulsified or 11D10 polypeptides and / or polynucleotides can be associated with liposomes.
The 11D10, 11D10 polypeptide and / or 11D10 polynucleotide in the vaccine can be used as such, but is often mixed with pharmaceutically acceptable excipients. Suitable excipients are, for example, water, saline, physiologically buffered saline, dextrose, glycerol, ethanol, and combinations thereof. If desired, the vaccine may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, and / or adjuvants. Examples of adjuvants are described above. For veterinary applications and production of antibodies in animals, Freund's adjuvant mitogenic components can be used. The choice of adjuvant depends, in part, on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the regulatory acceptability of the adjuvant, particularly when intended for human use. For example, alum is approved by the US Food and Drug Administration (FDA) for use as an adjuvant in humans. Encapsulation in cationic lipids can be used to enhance the immune response with vaccines containing 11D10 polynucleotides. For delivery of 11D10 polypeptide, encapsulation in liposomes may also be suitable. Liposomes suitable for packaging polynucleotides and / or polypeptides for delivery to cells are known in the art.
An 11D10 polypeptide can be chemically treated as necessary to enhance its immunogenicity, particularly when the 11D10 polypeptide contains 100 amino acids or less. Such treatment may include, for example, cross-linking with glutaraldehyde; linking to a protein carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxoid.
Cyclophosphamide (100 mg / kg body weight) can also be administered intraperitoneally if it is assumed that the suboptimal immune response is due to suppressor T cells induced by the vaccines of the invention.
The vaccines of the invention are typically administered parenterally by injection, eg, either subcutaneously, intramuscularly, intraperitoneally, or intradermally. Administration can also be intranasal, intrapulmonary (eg, by aerosol), oral, and intravenous. Additional formulations suitable for other modes of administration include suppositories, and in some cases oral formulations. The route of administration will depend on the condition of the individual to be treated and the desired clinical effect.
Administration can be initiated on a weekly or biweekly basis until the desired measurable parameter is detected, such as the induction of an immune response (humoral and / or cellular). Administration can then be continued on a less frequent basis, such as every two weeks or every month. For vaccines comprising 11D10, preferably administration is given every 2 weeks for the first 4 doses, followed by every month.
The vaccine is administered in an amount that is prophylactically and / or therapeutically effective in a manner compatible with the dosage formulation. The amount to be administered depends on the individual to be treated, the ability of the individual's immune system to synthesize antibodies, the route of administration, and the degree of protection desired. The exact amount of active ingredient required to administer can depend on the judgment of the practitioner and can be unique to the individual. General dosage ranges for 11D10, 11D10 polynucleotides and 11D10 polypeptides are given above.
Typically, vaccines are administered in a series of doses, starting with a group of doses to prime the immune response, followed by more spaced “maintenance” doses. For example, the vaccine can be administered on a weekly basis to establish an immune response, followed by biweekly or monthly injections to maintain the response.
An 11D10 polypeptide and / or 11D10 polynucleotide in a vaccine alone, in combination with other 11D10 polypeptides and / or polynucleotides, in combination with intact 11D10, and / or enhances and facilitates the desired effect. Or given in combination with other substances such as lymphokines and drugs that regulate. Examples of such materials are described above. 11D10 polypeptides can be combined by preparing a mixture of 11D10 polypeptides in solution or synthesizing a fusion protein.
The vaccines of the present invention can also be administered in combination with a recombinant vaccine containing a polynucleotide encoding HMFG or a recombinant vaccinia containing a fragment thereof and / or a lymphokine such as GM-CSF. . Furthermore, the vaccines of the present invention can be used in combination with other treatment modalities, whether established or experimental. Such use is indicated when administration of the vaccine improves clinical outcome compared to administration of other treatment modalities alone, such as chemotherapy or radiation therapy.
The immunogenicity of the 11D10 vaccine can be monitored by measuring the level of Ab3 and / or monitoring the disease state. The detection and measurement of Ab3 using RIA or ELISA and the measurement of T cell activity (ie proliferation and / or cytotoxic activity) were described above. As an example, Ab3 can be quantified as follows. Microtiter plates are coated with MC-10 (Ab1) and reacted with a fixed amount of 125 I-labeled 11D10 polypeptide. A standard inhibition curve is generated using purified MC-10 as an inhibitor. Different dilutions of sera are tested for the ability to inhibit the Ab1-Ab2 response and the amount of Ab3 in the serum is estimated from a standard inhibition curve. Alternatively, the T cell response can be monitored using any of the assays described above. The disease state can be monitored using standard techniques in the art such as tumor associated markers, x-rays, CT scans, and other measurable clinical expression measurements.
It will be appreciated that a number of alternative vaccine compositions, not limited to those described herein, may be effective in inducing an immune response. All such compositions are embodied within the scope of the present invention if they comprise an 11D10 polynucleotide or polypeptide as an active ingredient.
Kit comprising 11D10, 11D10 polynucleotide and / or polypeptide
The present invention also includes kits (preferably diagnostic kits) containing 11D10, 11D10 polynucleotides and / or polypeptides. Diagnostic procedures using the 11D10, 11D10 polynucleotides and / or 11D10 polypeptides of the present invention can be performed by diagnostic laboratories, experimental laboratories, practitioners, or private individuals. Kits embodied by the present invention include those that allow one to perform an assay for anti-HMFG or anti-11D10 activity, such as any of those disclosed herein, thereby reducing their activity. Detect and / or quantify. Kits embodied by the invention also include kits that allow for detection of 11D10 polynucleotides in, for example, ex vivo or in vivo transfected cells. These kits can be used for the detection or quantification of a polynucleotide comprising a polynucleotide encoding the variable region of 11D10 or a portion thereof. 11D10 polynucleotides that hybridize to 11D10 variable regions (ie, form stable hybrids) but cannot hybridize to other variable region polynucleotides (known at the time of filing this application) Are particularly suitable.
For example, the presence of Ab3 in a biological sample can be tested using the 11D10 polypeptide. This sample can be pretreated as needed for Ab3 enrichment.
The kits of the invention comprise 11D10, 11D10 polynucleotides and / or polypeptides in suitable packaging. The kit can optionally provide additional components useful in the procedure. These optional components include, but are not limited to: buffers, capture reagents, developing reagents, labels, reaction surfaces, detection means, control samples, instructions for use. And explanatory material.
The following examples are provided for illustration, but the invention is not limited thereto.
Example
Example 1. Production and characterization of 11D10 anti-idiotype antibody
A hybridoma cell line producing the monoclonal anti-idiotype antibody 11D10 was generated and identified as described below. In order to obtain antibodies with the desired specificity and functionality, aspects of both the immunization procedure and the screening procedure were important. 11D10 is one of many Ab2 produced early and was identified as a candidate with the most desirable properties.
This immunizing antibody (Ab1) was a mouse anti-HMFG monoclonal antibody MC-10 (referred to as BrE-1 in this section). Since the responding animal was also a mouse, the resulting Ab2 was expected to be directed to the idiotypic properties of BrE-1. However, only one fraction is against the BrE-1 paratope, an even smaller population is immunogenic and can induce Ab3, and an even smaller population cross-reacts with tumor-associated antigens Trigger Ab3.
In order to make BrE-1 sufficiently immunogenic in autologous species, it was bound to carrier KLH and emulsified with Freund's adjuvant. This was repeatedly administered to recipient animals on an unusual schedule with only two weeks between doses. Five mice were immunized according to this schedule. About 3 mice had a substantial response only after the fourth immunization. Responding animals were boosted with the fifth BrE-1 intravenous dose, spleen cells were isolated, and hybridomas were prepared individually from each animal. Cloning was performed according to standard techniques.
The screening procedure involves four important steps: (1) positive selection for antibodies that bind to BrE-1, (2) negative selection for antibodies that recognize isotype or allotype determinants, (3) Positive selection for the ability to inhibit the binding of BrE-1 to HMFG, and (4) positive selection for the ability to induce a humoral immune response against the first tumor associated antigen (HMFG) in both mice and rabbits. The rest of this section provides an overview of the screening procedure, which is shown in more detail in the following section.
Initial screening to identify clones that react with BrE-1 but do not react with other target monoclonal antibodies that share the same allotype or isotype determinant was performed by immunoassay. The important assay was a sandwich RIA where BrE-1 was attached to a solid phase, layered with culture supernatant, and expressed with radioiodine labeled BrE-1. This assay requires that the antibody in the hybridoma supernatant be functionally divalent and able to bridge between captured BrE-1 and expressed BrE-1. Several clones that were idiotype specific and gave strong signals in this assay were selected for further study.
The following screening was performed by a competitive assay that required Ab2 to block the binding of BrE-1 to HMFG. This proves that Ab2 recognized the BrE-1 paratope. HMFG was provided in the form of MCF-7 cells, a human breast cell tumor line that expresses HMFG on the cell surface. The nature of this assay requires that Ab2 blocks the interaction between BrE-1 and tumor antigen in a specific presentation manner on tumor cells. At a minimum, candidate Ab2 that passed previous screening tests was required to inhibit BrE-1 binding to cells by at least 85%. There were about 3 Ab2s that substantially exceeded this minimum, and 11D10 provided nearly the highest level of inhibition.
The final screening test was a determination of whether Ab3 of the desired specificity could be induced when candidate Ab2 was injected into the recipient. A sufficient amount of Ab2 was prepared from mouse ascites and tested in mice and rabbits. Sera from these test animals were first assayed for the presence of Ab3 in a sandwich immunoassay with the same labeled Ab2 used for immunization. Sera showing positive test results were then assayed for the ability of Ab3 to react against a tumor-associated antigen, ie HMFG. A micropure plate was coated with a semi-pure preparation of HMFG, overlaid with serially diluted test sera, and labeled anti-immunoglobulin was used to detect bound Ab3. The titer of Ab3 binding to HMFG defined the “quality” of Ab2 as a reflection of its ability as an anti-HMFG inducer.
Monoclonal antibody 11D10 emerges as the highest quality anti-idiotype and is the basis for the various components, compositions, and procedures embodied in the present invention.
Materials and methods
The fusion partner used to generate the cell: hybridoma line was the mouse non-secretory myeloma cell line P3-653, available from ATCC as No. CRL-1580 and related to P3X63Ag8.653 and ancestors. Established human cell lines as described elsewhere (Seon et al. (1984)J.Immunol.132: 2089) and cultured in RPMI 1640 supplemented with 5% fetal calf serum.
HMFG: Delipidated HMFG was supplied by Roberto L. Ceriani and was prepared according to his protocol (Cerani et al. (1977)Proc.Natl.Acad.Sci.USA 74: 582-586). Briefly, the washed milk fat fraction of human milk was extracted twice with 2 volumes of chloroform and washed twice with 1 volume of ether and then lyophilized. Protein concentration was determined by the Lowry method.
BrE-1 antibody: The mouse monoclonal antibody BrE-1 (MC-10) was abundantly provided by Dr. R.L. Ceriani and is described in WO 89/07268. 2-10 x 10 for individual mice primed with pristane⁶Ascites fluid of BrE-1 hybridoma was prepared by injecting a viable cell into the peritoneal cavity. From this ascites, 45% saturated ammonium sulfate precipitation followed by Protein A Sepharose^TM CL-4B (Ey et al. (1978)Immunochemistry 15: 429) The IgG fraction was isolated by chromatography above. The purity of the isolated IgG was checked by immunodiffusion, immunoelectrophoresis, and high pressure liquid chromatography (HPLC) fractionation. Other unrelated Ab1 and Ab2 of different isotypes were used as controls.
BrE-1 F (ab ')₂Fragment preparation: standard pepsin digestion (Parham, 1983)J.Immunol 131: 2895) to F (ab ')₂Fragments were prepared. In summary, the IgG fraction from BrE-1 ascites was dialyzed against 0.1 M citrate buffer (pH 3.5) and digested with pepsin (25 Tg / mg IgG) at 370 C for 8 hours. After cleavage, the pH was adjusted to 7.0 with 3.0 M Tris buffer (pH 8.6) and the solution was dialyzed against phosphate buffered saline (PBS) at low temperature. Sepharose digestion^TMSeparation by HPLC using 6 columns. Isolated F (ab ')₂The purity of was determined by immunodiffusion and reaction with anti-isotype reagents in a standard ELISA.
Coupling of antibodies with KLH: Maloney et al. (1985),Hybridoma BrE-1 was coupled with keyhole limpet hemocyanin (KLH) according to the method described by 4: 191. Antibody stock solution (1 mg / ml) was mixed with KLH (1 mg / ml) in PBS in the presence of freshly diluted glutaraldehyde solution (final concentration 0.05%). The mixture was rotated for 1 hour at room temperature and then dialyzed completely at 40 C against PBS.
Immunization of syngeneic BALB / c mice: BALB / c females were immunized 4 times over a 2 month period. The first infusion was given intraperitoneally with 100 Tg BrE-1 emulsified with complete Freund's adjuvant. The next two infusions were given either 100Tg BrE-1 coupled with KLH in incomplete Freund's adjuvant, either subcutaneously or intraperitoneally. Mice occasionally draw blood and BrE-1 F (ab ')₂Anti-idiotypic activity was checked by ELISA during the binding assay by using normal pooled BALB / c mouse serum IgG as a fragment and control. Three days prior to fusion, mice were boosted with BrE-1 in PBS injected intravenously.
Generation of anti-idiotype hybridomas
The fusion partner used to generate the hybridoma line was available from ATCC as No. CRL-1580 and was the mouse non-secretory myeloma cell line P3-653, which is related to P3X63Ag8.653 and its ancestors. Established human cell lines as described elsewhere (Seon et al. (1984)J.Immunol.132: 2089) and cultured in RPMI 1640 supplemented with 5% fetal calf serum.
Hybridomas were generated essentially according to the methods of Oi and Herzenberg ((1980) Selected Methods of Cellular Immunology, edited by Mishell and Shiigi, Freeman Publs. 351-372). Spleen cells from immunized mice were mixed with P3-653 cells at a ratio of 1: 1 to 10: 1 in the presence of 50% polyethylene glycol (PEG, molecular weight about 4500). The fused cells were then washed and cultured. Hybrids were selected using hypoxanthine-aminopterin-thymidine medium.
Initial selection of anti-idiotype antibody (Ab2) that secretes hybridoma clones
Initial screening of hybridoma clones was performed by RIA. Purified BrE-1 was chloramine T method (Hunter (1970)Proc.Soc.Exp.Biol.Med.133: 989). BrE-1 or control antibodies (monoclonal antibodies of various isotypes and unrelated specificities, as well as BALB / c normal IgG) were coated on PVC plates at 500 ng / well. After overnight incubation at 40C, the plates were blocked with 1% bovine serum albumin (BSA) in PBS. The coated plate is incubated for 4 hours with serial dilutions of the hybridoma supernatant and about 50,000 cpm¹²⁵It was expressed using I-BrE-1. The RIA assay is a stringent specificity test for an antibody and also requires that the antibody be able to bridge between two BrE-1 molecules.
In addition, an ELISA assay is performed to determine the class and subclass of each clone. ELISA was performed by coating microtiter plates at 500 ng / well with BrE-1 antibody (or control). After overnight incubation at 40C, the plates were blocked with 1% BSA in PBS. 100 TI hybridoma culture supernatant or 20 × concentrate is incubated in the wells for 4 hours at room temperature. After washing with PBS, the plates were further incubated with alkaline phosphatase labeled anti-isotype reagent for 4 hours at room temperature or overnight at 40C and developed with substrate. Certain IgG subclass antibodies are readily purified by protein A chromatography and may have useful effector functions.
Culture supernatants from 1300 early fusion wells were initially screened. Forty-two Ab2 hybridomas were obtained that reacted with BrE-10 in RIA but did not react with isotypes or allotypes consistent with control immunoglobulins.
Many monoclonal Ab2 secreting cell lines emerged from these screening assays with the desired properties. Among them was monoclonal antibody 11D10.
Confirmation that monoclonal Ab2 is specific for the BrE-1 idiotype
The idiotype specificity of Ab2 was confirmed by direct binding to Ab1. Various purified Ab2¹²⁵Tested for binding to plates labeled with I and coated with a panel of monoclonal anti-TAA Ab1.¹²⁵The result of the experiment using I-11D10 is shown in FIG. Results are expressed as mean cpm (n = 3, standard deviation less than 10%). 11D10 bound almost exclusively to BrE-1; there was virtually no cross-reactivity with any of the other Ab1 tested.
The idiotype specificity of Ab2 was also confirmed by reversing the positions of Ab1 and Ab2. Plates were coated with 100 ng, 300 ng, and 1000 ng of purified Ab2 and reacted with various labeled Ab1.¹²⁵I-BrE-1 and¹²⁵I-BrE-3 is included. BrE-3 is an IgG1 specific for another tumor associated HMFG epitope and acts as a control. The results of experiments in which 11D10 was tested by this method are shown in Table 1 and confirmed the specificity for BrE-1.

Specificity for the BrE-1 idiotype is further established in competition experiments. Various labeled Ab2 were mixed with different members of a panel of unlabeled competitors including Ab2s, Ab1, and other murine immunoglobulins. Ab2 was then tested for binding to BrE-1 coated plates. For the best Ab2, over 90% inhibition was observed using 250 ng of unlabeled Ab2 or BrE-1 as competitors. In fact, no inhibition was obtained at concentrations up to 10 Tg using other immunoglobulins as potential competitors. Representative results of 11D10 being tested Ab2 are shown in Table 2 (mean cpm, n = 3, standard deviation less than 10%).

Screening for anti-idiotype against BrE-1 paratope
Use Ab2 to compete for binding of radiolabeled BrE-1 to HMFG expressed on human breast carcinoma cell lines MCF-7 or SKBR3 to determine if Ab2 is against the BrE-1 paratope I let you.
To perform the assay, target cells were grown as confluent monolayers in 96 well tissue culture plates. Various dilutions of test Ab2 (either culture supernatant or purified antibody) were mixed with labeled BrE-1 and then added to confluent cell cultures in microtiter plates. The percent inhibition of the assay was calculated according to the following formula:

Where R_TIs the average cpm of experimental wells with inhibitors; R_cIs the average background cpm; and R_MAXIs the mean maximum binding without any inhibitor.
FIG. 7 shows the results of this type of experiment performed using 11D10 as a competing antibody. 250 ng of Ab2 11D10 inhibited the binding of labeled BrE-1 to HMFG expressing cells by 90% or more (FIG. 7).
About 24 of the 42 monoclonal antibodies tested at this stage (including 11D10) inhibit the binding of labeled BrE-1 to HMFG-expressing cells or HMFG extracts in as little as about 25 ng in a plate binding assay did. Purified 3H1 (an irrelevant specificity mouse monoclonal antibody) was used as a control competitor and it did not inhibit BrE-1 binding to cells. In general, Ab2 that produced at least 85% inhibition was considered to have passed this stage in the screening process.
Confirmation of binding specificity
For the most promising Ab2, a confirmation experiment using HMFG was performed to confirm the specificity of binding to BrE-1.
About 40,000cpm¹²⁵I-BrE-1 was incubated simultaneously with a semi-purified preparation of HMFG. This antibody Ag mixture was added to Ab2 coated plates (500 ng / well) and the ability of HMFG to inhibit binding was measured. The amount of Ab2 was not limited with respect to the amount of BrE-1 that could be bound, and was therefore a sensitive indicator for small amounts of competing HMFG.
Ascites fluid was prepared as a source of Ab2 using 6 of 24 antibody-producing clones judged positive in the screening tests described so far. This Ab2 was purified by chromatography using Protein A affinity resin by standard techniques.
The binding of BrE-1 to 11D10 is inhibited by HMFG.
Screening for anti-idiotypes that can induce tumor-specific immune responses
Since the main goal of these experiments was to find anti-idiotypes that could elicit an anti-HMFG immune response, the next screening step was to test its immunogenicity in animal models. Ab2 is not only immunogenic, but can elicit Ab3 that cross-reacts with the tumor antigen HMFG.
Therefore, Ab2 that gave the strongest results in competition experiments with HMFG expressing cells was taken up for testing in immunization experiments.
For each Ab2 to be tested, 5 BALB / c mice and 2 New Zealand white rabbits were immunized. For mice, Ab2 was conjugated to KLH. On a two week schedule, 50 μg was injected per mouse and 200 μg per rabbit. The first injection was prepared in Freund's complete adjuvant and the second injection was prepared in Freund's incomplete adjuvant. Serum was collected regularly for analysis. Initially, Ab3 titers were measured in a standard sandwich radioimmunoassay using both Ab2 during antibody capture and detection. The antibody response to Ab2 reached a substantial level after the fifth immunization.
The assay was then performed on plates coated with HMFG preparation. Serum was incubated in the wells and bound antibody was detected with enzyme-linked anti-immunoglobulin. This assay requires that the antibody binds to the original tumor-associated antigen, and establishes that at least a portion of Ab3 induced by immunizing with an anti-idiotype is tumor antigen-specific . The level of HMFG-specific Ab3 was titrated by serial dilution and the “quality” of immunized Ab2 was defined.
The 11D10 monoclonal antibody was found to have the highest quality among the candidates tested.
Confirmation that Ab3 induced by 11D10 has the desired specificity
Since the therapeutic purpose of 11D10 depends on its ability to elicit an immune response against the protein on tumor-associated antigens, the specificity of the resulting Ab3 was confirmed in a number of subsequent experiments.
Ab3-containing serum (lack of anti-isotype and anti-allotype activity) almost completely inhibited binding of labeled BrE-1 to 11D10 or vice versa. This indicates that the Ab3 antibody shares an idiotype with Ab1. Similar results were obtained when Ab3-producing animals were immunized with 11D10 conjugated to KLH or 11D10 emulsified in Freund's adjuvant.
Spleen cells from mice immunized with 11D10 were used to generate monoclonal Ab3-producing cell lines. Competition experiments similar to those described in the previous section showed that monoclonal Ab3 binds to HMFG in the same manner as BrE-1.
Binding experiments were performed to determine whether Ab3 induced by 11D10 can bind to HMFG when expressed in tumor cell lines. FIG. 8 shows the results of direct binding of various Ab3 preparations to the breast cancer cell line SKBR3. Polyclonal mouse Ab3, polyclonal rabbit Ab3, and monoclonal mouse Ab3 were prepared by adsorption with mouse immunoglobulin and tested for binding at several dilutions. Both polyclonal and monoclonal Ab3 sera from animals immunized with 11D10 bound to HMFG positive cell lines MCF-7 and SKBr3, but not to the antigen negative melanoma cell line M21 / P6.
Various competition experiments were performed to confirm that Ab3 induced by 11D10 has the desired specificity. Confluent monolayer cultures of SKBR3 cells in microtiter wells with 50,000 cpm as a competitor¹²⁵Reacted with I-BrE-1 and various Ab3 dilutions. A monoclonal antibody (1E3) not related to specificity was used as a negative control. The results are shown in FIG. 20 μg of monoclonal Ab3 inhibited binding by 25%. Mouse or rabbit serum containing polyclonal Ab3 serum diluted to 1/50 dilution inhibited by 38% and 30%, respectively. This indicated that Ab3 binds to the same HMFG epitope as Ab1. The lack of complete binding under these conditions suggests that some of Ab3 may have a lower affinity and affinity for HMFG than Ab1.
Splenocytes from mice immunized with 11D10 were used for T cell proliferation assays. Spleen cells were cultured in the presence of semi-purified HMFG for 5 days, then [^ThreeH] thymidine was pulsed. The greater uptake in cells from animals immunized with 11D10 than in the control is consistent with the presence of an Id-specific cellular immune response. 11D10-Alugel immunized rabbits showed some DTH skin reaction to the semi-purified HMFG preparation, but not to pure CEA (negative specificity control). Since HMFG contains several epitopes and is not available in purified form, we cannot confirm the specificity of the reaction.
Dot blot analysis of mAb1 and mAb3
Various dilutions of HMFG antigen were transblotted to nitrocellulose filters and reacted with BrE-1 (Ab1) and mAb3 (FIG. 10). Lanes 1-3 were transblotted with HMGF and incubated with BrE-1 (10 μg / ml), control 1E3 IgG1 (50 μg / ml), and mAb3 IgG1 (50 μg / ml), respectively. The reaction proceeded by using goat anti-mouse IgG alkaline phosphatase reagent and substrate. The staining was the same, but more intense with Ab1, while the control antibody was negative.
Conformance experiments on cross-reactive idiotypes in breast cancer patients
We studied sera from 50 randomly selected breast cancer patients and determined whether any of them had pre-existing matched idiotypes recognized by Ab2 11D10. Microtiter plates were purified from 250 ng / well of 11D10 (Ab2) F (ab ')₂The fragments were coated and incubated with a 1: 100 dilution of patient serum and expressed using goat anti-human IgG enzyme-labeled antibody. Serum binding to Ab2 was detected using alkaline phosphatase-conjugated anti-human IgG (γ chain specific) antibody and substrate.
As shown in FIG. 11, a small number (8/50) of sera from these breast cancer patients had increased levels of antibodies that react with 11D10. The selection criteria for anti-Id therapy is based on the assumption that the disease itself induces the priming state of B and T cells in the host. It is hypothesized that in patients expressing the corresponding matched Id, such B and T cells already primed by Ab2 stimulation can be effectively stimulated. The discovery of Id compatible with sera from breast cancer patients suggests that they may be particularly suitable as potential candidates for anti-Id immunotherapy activation with Ab2 11D10.
MX-1 tumor-bearing BALB / c in nude mice¹¹¹In-BrE-1 clearance study
To determine whether 11D10 is suitable as a second reagent for binding to excess radiolabeled antibody in radioimmunotherapy, we performed clearance studies. For each BALB / c nude mouse, 2.9 μg of 20 μCi¹¹¹In-BrE-1 was injected. After 24 hours, 20 μg of anti-Id 11D10 was injected into a group of 6 mice, sacrificed after 30 minutes and another group was sacrificed after 40 hours. The mouse control group was not injected with anti-Id-11D10 after 30 minutes and 40 hours. After the mice were sacrificed, blood and organs were removed and the radioactivity levels were measured. The results are shown in Table 3 and 1 gm¹¹¹Expressed as percent dose per In-BrE-1 ± SEM. Values were expressed as mean% SEM. At both 30 minutes and 40 hours, there was significant clearance of radioactivity in almost all organs (especially blood, kidney, muscle, and lung) except the liver in the experimental group compared to the control. There was no shadow at 30 minutes for tumor retention; however, there was some clearance in the treatment group after 40 hours.

Example 2: Cloning and sequencing of 11D10 cDNA
Unless otherwise specified, all cloning techniques were essentially those described in Sambrook et al. (1989) and all reagents used were in accordance with the manufacturer's instructions.
Polynucleotide sequences were obtained for the 11D10 antibody by isolating messenger RNA from the 11D10 producing cell line. 1x10 total RNA for each sequencing⁷Of 11D10 hybridoma cells. Messenger RNA was prepared by passing through two cycles of chromatography on an oligothymidylate-cellulose column. The yield of mRNA was about 100 μg. Single-stranded cDNA was synthesized using SuperScript Preamplification kit (GIBCO / BRL).
To sequence the heavy chain variable region, PCR was performed with the reverse (3 ′) primer corresponding to amino acids 126-119 of the following mouse γ1 constant region:

And various mixtures of forward primers (corresponding to the N-terminal leader sequence of the mouse variable region subgroup) were performed on the cDNA. The (5 ') forward primer that resulted in a positive reaction is:

Which corresponds to amino acids -20 to -13 (I = inosine, R = A or G, Y = C or T, K = G or T, S = C or G, W = A or T).
The amplified cDNA fragment was subcloned into pT7 plasmid and NovaBlue competent cells were transformed using the protocol provided by the supplier (Novagen). Recombinant colonies were picked by color selection and plasmid DNA was prepared by a miniprep procedure. The DNA sequence of the double stranded plasmid was determined using the Sequenase Version 2.0 kit (USB, Cleveland, Ohio). The sequence of the DNA insert in the plasmid was determined from both directions using primers specific for the plasmid; ie T7 promoter primer (TAATACGACTCACTATAGGGG-SEQ ID NO: 38) and U-19 primer (GTTTTCCCAGTCACGACGT-SEQ ID NO: 39). At least 8 clones were picked for sequencing.
The sequence of the 11D10 light chain variable region was determined in a similar manner. The forward and reverse primers that produced a positive PCR result are:

This corresponds to amino acids -20 to -10 of the leader sequence and 122 to 116 of the mouse kappa chain constant region, respectively.
To minimize the error rate in PCR amplification, pfu DNA polymerase (Stratagene, an Diego) was used for amplification in all subsequent experiments. The mutation frequency in this thermostable DNA polymerase is 1/10 compared to Taq polymerase.
Confirmation that the isolated cDNA corresponds to the 11D10 heavy and light chains was obtained by N-terminal amino acid sequencing of the isolated antibody. 50 μg of purified 11D10 antibody is diluted with sample loading buffer (50 mM Tris-HCl (pH 6.8), 1% SDS, 1% glycerol, 0.1% β mercaptoethanol) and heated at 100 ° C. for 3 minutes. The denatured protein was loaded on a 7.5% polyacrylamide gel containing SDS (BioRad Miniprotean II Dual Slab Cell) and subjected to electrophoresis at 200 V for 1 hour. Proteins in the gel were applied to polyvinylidene difluoride (PVDF) membranes at 150 mA overnight by the procedure described in Towbin et al. ((1979) Proc. Natl. Acad. Sci. USA. 78: 4350-4354). Moved. The transfer buffer contains 25 mM Tris, 192 mM glycine, 20% (v / v) methanol. Membranes were stained by rapid immersion in 0.1% Coomasie Brilliant Blue in 50% methanol-50% acetic acid and then washed with a solution containing 40% methanol and 10% acetic acid. After drying the membrane at room temperature, the stained heavy and light chain bands were cut out with a clean razor blade. Proteins on membrane strips were subjected to N-terminal microsequencing by automated Edman degradation using an Applied Biosystem Model 477A protein sequencer using pulse-labeled liquid chemistry and online phenyl-ethiohydantion amino acid identification . Each protein was subjected to 10-15 degradation cycles and the converted cleavage products from each cycle were analyzed by reverse phase HPLC.
The nucleic acid sequences and corresponding amino acid sequences of the heavy and light chain variable regions of monoclonal antibody 11D10 are shown in FIGS. 1 and 2, respectively.
In FIG. 1, it is clear that the third amino acid of the leader sequence (amino acid -18) and the 25th amino acid of framework 3 (amino acid 81) are in error. As will be apparent to those skilled in the art, the amino acid encoded by the nucleotide triplet “GCC” is A, ie, alanine (amino acid-18), and the amino acid encoded by the nucleotide triplet “GAA” is E, ie, glutamic acid (amino acid 81). Similarly, in FIG. 3A, amino acid 81 (within framework 3) is E or glutamic acid. SEQ ID NO: 58 shows an incorrect amino acid translation, as indicated by a typographical error on a typed sheet reading “FIG. 1” filed with this disclosure. SEQ ID NO: 2 shows correct amino acid translation.
Nucleic acid sequences were obtained by PCR amplification of mRNA from antibody producing cell lines as described previously in this example. The amino acid sequence was subsequently obtained by translating the polynucleotide sequence using the genetic code. The exact amino acid is self-evident because of the genetic code. The exact amino acid sequence is also unique to the antibody producing cell line deposited with the ATCC under accession number HB-12020 in support of this disclosure.
Heavy and light chain polynucleotide and amino acid sequences were compared to sequences available from the PDB, SwissProt, PIR, SPUpdate, GwnPept, and GPUpdate databases using the BLAST algorithm at the National Center for Biotechnology Information. The comparison was made on January 19, 1996.
None of the 10 database DNA sequences most closely matched to the DNA sequence of the 11D10 light chain variable region were identical. There were about 8-27 differences from the 11D10 DNA sequence (this corresponds to about 6-17 amino acid differences). The sixth matched sequence (name> gb / M59920 / MUSIQKAA3) is a mouse κVJ germ-like sequence and probably represents a prototype gene from which the 11D10 light chain is derived. The 11D10 DNA sequence differs from the germline sequence at 14 positions, which corresponds to a position difference of about 7 amino acids.
Of the 10 database DNA sequences that most closely matched the DNA sequence of the 11D10 heavy chain variable region, none were identical. Of the 10 sequences, 9 were 3-12 base pairs longer, depending on splicing differences within the VDJ junction. In addition, there were approximately 15-43 differences compared to the 11D10 DNA sequence outside the junction, which corresponded to 11-23 amino acid differences.
Thus, there is at least about 18 amino acid differences between the amino acid sequence encoded by 11D10 DNA and the amino acid sequence encoded by the most closely matched database DNA. The point difference is probably caused by somatic mutations in the germline sequence during development of this cell line in the animal used to generate the antibody producing cell line.
FIG. 4 shows the 10 polynucleotide sequences most closely matched to the 11D10 light chain variable region coding sequence. FIG. 5 shows the 10 polynucleotide sequences most closely matched to the 11D10 heavy chain variable region coding sequence.
Compare light and heavy chain variable region amino acid sequences with other known sequences and sequences available from the PDB, SwissProt, PIR, SPUpdate, GenPept, and GPUpdate databases using the BLAST algorithm at the National Center for Biotechnology Information did. The comparison was made on January 19, 1996.
The 15 closest amino acid sequences found in the BLAST search have the identifiers shown in Table 6.

Figures 26 (A) and (B) are comparative depictions of 11D10 light and heavy chain amino acid sequences with the 15 closest sequences found in the BLAST search. Panel (A) shows a light chain comparison. Panel (B) shows a heavy chain comparison. Residues that are identical to the 11D10 sequence are indicated by a dot (.). Gaps introduced to improve alignment near the heavy chain VDJ junction are indicated by double lines (=).
Of the 50 database amino acid sequences that most closely matched those of the 11D10 light chain variable region, none were identical. 11D10 differed from the 15 closest sequences with a minimum of 7 and an average of about 12 substitution differences. A collection of differences occurred near the end of CDR1. Other differences occurred in CDR3 and the framework.
Of the 50 database amino acid sequences that most closely matched those of the 11D10 heavy chain variable region, none were identical. The following summarizes the main points estimated from the comparison.
The closest match was with the heavy chain variable region indicated by its Gen Bank designation as another anti-idiotype (nomenclature gp | X64805 | MMAIDHCH_1). There were 11 substitutions between residues 1 and 98 (before the VDJ junction), and there were 7 substitution differences after residue 98.
11D10 was different from the heavy chain sequence 40 in length. Other sequences were longer, up to 5 residues, or as short as 2 residues, near the VDJ junction.
With the exception of the two most closely matched sequences, there were at least 18 substitution differences between residues 1 and 98 on average.
Various D and J region genes appear to be used with the prototype V gene. Only one of the 50 sequences appeared to use the same D region. There was a point difference within the D region and a three residue splicing difference.
There were a total of 18 insertion, deletion, and substitution differences for the 50 most closely matched sequences. The other 49 sequences had a total of at least 25 and an average of about 30 insertions, deletions, and substitution differences.
Differences appear throughout the variable region.
FIG. 26 (C) shows the amino acid consensus sequences of the 15 closest matched sequences of the light and heavy chain variable regions (SEQ ID NO: 47 and SEQ ID NO: 48). These represent assembled light chain VJ gene and heavy chain VDJ gene prototypes. Residues in the 11D10 sequence that are identical to the consensus sequence are indicated by a dot (.). CDR is indicated by an asterisk (¥). In addition, a fragment derived from human milk fat globulin (HMFG) is shown in the direction of N → C (upper case) or C → N (lower case).
The following points can be inferred:
• 11D10 is at least 18 residues away from the consensus sequence. Of these, 7 occur in the light chain and 11 occur in the heavy chain.
• Differences occur throughout the 11D10 light and heavy chain variable region sequences. 8 occur within the CDR and 10 occur outside the CDR.
• The alignment of the 11D10 sequence with the HMFG fragment is independent of point mutations.
The variable region fragment sequences, particularly those surrounding the heavy chain VDJ junction or any differences shown in FIG. 26 (C) are of interest in the development of polypeptides of the invention having 11D10 immunological activity. Coding sequences near the VDJ junction or any point differences are of interest in the development of the polynucleotides of the invention.
Example 3: Induction of breast cancer specific response by 11D10 in monkeys
Cell line
Human breast cancer cell line MCF-7 expressing HMFG antigen was grown in RPMI 1640 medium supplemented with 10% FCS, 1% L-glutamine, penicillin, and streptomycin and used to detect anti-tumor responses. Human melanoma cell line M21 / P6 (provided courtesy of Dr. Ralph Reisfeid of Scripps Research Institute, La Jolla, CA) and T cell line MOLT-4 (both are HMFG negative) are grown in the same medium, And it was used as a negative control.
antibody
Ab1 mAb MC-10 (IgG2b, κ), which distinguishes distinct and specific epitopes with a molecular weight of HMFG molecule of 400,000, is used for the production of anti-Id mAb 11D10 (IgG1-κ) as described in Example 1. Were used to immunize syngeneic BALB / c mice. mAb2 3H1 (IgG1-κ) is a human CEA (Bhattacharya-Chatterjee et al. (1990)J.Immunol.14S: Z758-2765), a mouse anti-Id mAb and used as a control.
Adjuvant
An adjuvant is required to enhance the immunogenicity of the anti-Id vaccine. Aluminum hydroxide is approved for use as an adjuvant in humans by the US Food and Drug Administration. Therefore, in this preclinical study, we immunized monkeys with Ab2 11D10 precipitated with aluminum hydroxide as described below.
Antibody preparation
11D10 was obtained as described in Example 1. mAb2 3H1 (IgG1-κ) is a human CEA (Bhattacharya-Chatterjee et al. (1990)J.Immunol.14S: Z758-2765) is a mouse anti-Id mAb used as a control.
Aluminum hydroxide precipitation
Adjuvant was used to enhance the immunogenicity of the anti-Id vaccine. Aluminum hydroxide is approved for use as an adjuvant in humans by the US Food and Drug Administration. Therefore, we immunized monkeys with Ab2 11D10 in this preclinical study. Briefly, 1 ml of 2% Alu-Gel S (Serva Fine Biochem, Inc., Garden City, Long Island, NY) was added to a 5 mg aliquot of purified mAb anti-Id (Ab2). The volume was then adjusted to 10.0 ml with Dulbecco's-PBS and the mixture was incubated on a vortex at room temperature for 1 hour. The mixture was then centrifuged at 2000 rpm for 10 minutes at 24 ° C. The amount of mAb bound to the gel layer was determined by spectrophotometric measurement of the amount of unbound antibody in the supernatant. The antibody precipitated with Alu-Gel was stored at 4 ° C. until use. These procedures were performed aseptically in a laminar flow hood and the final product was sterilized, clearly labeled as anti-Id 11D10 Alu-Gel, and aliquoted into sterile pyrogen free glass vials.
Monkey immunization
Cynomolgus monkeys were immunized with 11D10 or 3H1 controls. Monkeys were housed at White Stands Research Institutes (Alamogordo, NM). Male and female monkey pairs weighing 3-4 kg, intradermally at 0, 14, 28, and 42 days at 4 different sites using 2 mg of either 11D10 or 3H1, respectively Immunized with. Only two monkeys were used for each anti-Id (Ad2) at a single dose for economic reasons. The 2 mg dose was selected based on prior preclinical and clinical trials with different anti-Id vaccines. Blood samples were taken before immunization and 10 days after each immunization.
toxicity
Induction of Ab3 response in monkeys did not cause any obvious side effects in animals. As a result of multiple immunizations, only mild local swelling and inflammation at the site of inoculation was observed. Monkeys were routinely checked by physical examination and body weight measurement. There was no sign of abnormality.
Development of humoral immunity induced by immunization with aluminum hydroxide precipitated Ab2
Specific Ab3 response to Ab2. Sera from immunized monkeys were tested for the presence of anti-anti-Id antibodies. Serum was preincubated with normal mouse immunoglobulin to block monkey antibodies against isotype and allotype determinants and then anti-anti-Id (11D10) by reaction with immunized anti-Id (11D10) coated on microtiter plates. The presence of Ab3) was checked by RIA. Irrelevant Ab2 was used as a control. After washing, the antigen-antibody reaction is performed in a homogeneous sandwich RIA.¹²⁵Tagging with I-labeled anti-Id reagent. Preimmune sera and sera from monkeys immunized with control Ab2 3H1 were also used in these assays. further,¹²⁵I-labeled monoclonal Ab2 3H1 was used as a control.
Ab3 idiotope analysis. If a positive reaction is obtained by the above method, Ab3 sera from these monkeys are added to BrE-1 (Ab1) bound to a microtiter plate.¹²⁵We checked for the ability to inhibit the binding of I-labeled 11D10 or vice versa (inhibition of radiolabeled BrE-1 binding to 11D10 on the plate). An irrelevant Ab1-Ab2 system was used as a control (Bhattacharya-Chatterjee et al. (1990); Bhattacharya-Chatterjee et al. (1987)J.Immunol.139: 1354-13605). This indicated whether Ab3 in monkey serum shared an idiotope with BrE-1 (Ab1). An inhibition assay of Ab1-Ab2 binding by Ab3 serum also shows whether Ab3 is a true anti-anti-Id.
Binding of Ab3 to tumor antigen. To assess a direct humoral immune response to the natural target antigen, monkey Ab3 serum was tested for reactivity with cell lines known to express HMFG in RIA. In addition, the serum was checked for reactivity to a solubilized semi-purified preparation of HMFG antigen coated on a microtiter plate. Antigen-antibody reaction in ELISA¹²⁵Detection was by using anti-human immunoglobulin labeled with I-labeled anti-human immunoglobulin reagent or alkaline phosphatase. Preimmune serum was used as a control. Irrelevant CEA was also used as a control for this assay. The isotype of monkey Ab3 serum binding to HMFG antigen was determined by ELISA using anti-human isotype specific reagents.
Ab3 binding to tumor cell lines was also checked by immunoflow cytometry. Antigen positive MCF-7 cells (1x10⁶/ Well) was reacted with 100 μl of Ab1 (MC-10) and Ab3 at 4 ° C. for 60 minutes. After washing, the cells are treated with goat anti-mouse or goat anti-human F (ab ′)₂ Incubated with either IgG-FITC labeled antibody (Tago, Burlingame, CA) for 30 minutes at 4 ° C. This was then washed twice, fixed with 2% paraformaldehyde, and analyzed by immunoflow cytometry (FACStar, Becton, Dickinson, San Jose, Calif.). Antigen negative MOLT-4 cells were used as a control for this assay.
Purification of anti-anti-Id antibody (Ab3) from hyperimmunized monkey serum. 20 ml of hyperimmune serum was passed through an immunosorbent column consisting of immunized anti-Id immunoglobulin (11D10-IgG1) conjugated to Sepharose 4B. Anti-anti-Id antibody (Ab3) was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.4) and neutralized to pH 7.0 with 3 M Tris. The eluted antibody was then passed through an immunosorbent column consisting of an irrelevant isotype-allotype matched anti-Id mAb conjugated to Sepharose 4B to remove anti-isotype and anti-allotype reactivity. The passed antibody was concentrated and used as purified Ab3. The isotype of Ab3 was determined by ELISA using human anti-isotype specific antigen (Tago).
Ab3 epitope analysis. To demonstrate that Ab3 occurring in monkeys and Ab1 (BrE-1) bind to the same antigenic determinant, purified Ab3 demonstrated the binding of BrE-1 to the antigen-positive tumor cell line MCF-7 or HMFG antigen. Inhibition was checked by RIA (described in Bhattacharya-Chatterjee et al.) (1990).
Slot blot analysis of purified Ab3 using HMFG. The polyvinylidene difluoride membrane was activated with methanol for 5 minutes and transferred to 0.02 M PBS (pH 7.0). Various concentrations of protein (5 μg, 2 μg, 1 μg) were adsorbed to the membrane using a Hybrislot instrument (BRL Life Technologies, Gaithersburg, MD). The membrane was then blocked with 2% BSA in PBS for 2 hours with shaking, followed by incubation with 5 ml of a 20 μg / ml Ab1 or Ab3 solution for 3 hours with shaking. The membrane was then washed 5 times with 1% BSA in PBS and incubated for 90 minutes with alkaline phosphatase-labeled goat anti-human immunoglobulin or goat anti-mouse immunoglobulin (1: 100 dilution), washed, and Bio-Rad Color was developed using the substrate supplied in the kit.
Assays for Id-specific proliferative responses
Fresh peripheral blood mononuclear cells are isolated by standard Ficoll-Hypaque density gradient centrifugation and 5 × 10 5^ThreeCells / wells were incubated with various concentrations of 11D10 and control 3H1 (10 ng-2 μg) in RPMI 1640 medium with 5% heat-inactivated FCS, penicillin, and streptomycin. Non-specific mitogen phytohemagglutinin-P was used as a positive control at 2 μg / well and 1 μg / well. After incubating the cells for 5 days at 37 ° C. in air containing 5% carbon dioxide,^ThreeH] thymidine (1 μCi / well) was pulsed for 20 hours. Data,^ThreeIt was expressed as the average measurement of [H] thymidine incorporation (triplicate wells) / min. SD data was less than 10% of each decision.
result
Induction of anti-anti-Id (Ab3) response in monkeys. Monkey-derived sera were obtained 10 days after the fourth immunization and analyzed for inhibition of Ab3 response and Ab2 binding to Ab1 by sandwich RIA (Table 4). For these assays, sera were pretreated with normal mouse immunoglobulin (500 μg / ml) to block anti-isotype and anti-allotype reactivity. For sandwich RIA, AB3 serum obtained after the fourth immunization was diluted with PBS containing normal mouse immunoglobulin (500 μg / ml). Serum was preincubated with normal mouse IgG prior to assay. A 1:40 dilution of Ab3 was incubated with anti-Id mAb 11D10 or 3H1, coated onto a microtiter plate, and then in a sandwich assay¹²⁵Reaction with I-labeled 11D10 or 3H1 (-50,000 cpm). Results are expressed as bound cpm in the sandwich assay. Results are shown as average cpm (n = 3). The SD of the data was less than 10%, and then we tested the binding of monkey Ab3 serum to semi-purified HMFG. Ab3 sera from monkeys immunized with 11D10 (PRO723 and PRO872) specifically bound to immunized Ab2 (11D10), but had minimal reactivity with irrelevant Ab2 (3H1). Monkey Ab3 also inhibited the binding of radiolabeled Ab2 to Ab1 by 91 and 95%, respectively, even at 1:40 dilution (Table 4). There was no inhibition in preimmunized sera or sera obtained from monkeys immunized with irrelevant Ab2 3H1 (PRO541 and PRO667). Purified 11D10 was used to coat the plate (250 ng / well) and the binding of radiolabeled BrE-1 (approximately 50,000 cpm) to 11D10 was tested for inhibition in the presence of various dilutions of Ab3 serum did. In a parallel control experiment, an irrelevant Ab1-Ab2 system (mAb 5019-3H1) was used as a control. The kinetics of the Ab3 response is shown in FIG. 12 using sera from monkey PRO723, which shows inhibition of radiolabeled Ab1 binding to Ab2. Similar reactivity was seen with sera from monkey PRO872. These results indicate that monkey Ab3 serum shares an idiotype with Ab1.

Induction of anti-tumor cell antibody response. To determine whether 11D10 immunized monkey serum specifically binds to HMFG positive breast cancer cells, the binding of monkey Ab3 serum to the breast cancer cell line MCF-7 was tested by ELISA. Various dilutions of Ab3 serum were added to confluent monolayers of cells grown in 96-well microtiter plates and developed with goat anti-human IgG (heavy and light chain specific) enzyme-labeled antibodies. Absorbance (OD) was read after 1 hour. Monkey PRO872 was immunized with Ab2 11D10. Monkey PRP667 was immunized with control-independent Ab2 (3H1). Preimmune monkey serum was also used as a control. The binding of monkey Ab3 serum (.....) to the melanoma cell line M21 / P6 was tested according to the same protocol by ELISA. Results are shown as mean absorbance at 405 nm (n = 3). The SD of the data was less than 10% for each assay. As shown in FIG. 13, serum (obtained after the fourth immunization at various dilutions) reacted with MCF-7 cells but not with the antigen-negative melanoma cell line M21 / P6. It was. The inventors then tested the binding of monkey Ab3 serum to semi-purified HMFG. Plates were coated with HMFG (2 mg / well) and immunized with Ab2 11D10 (PRO723) and Ab3 serum from monkeys immunized with the same isotype and allotype of unrelated Ab2 3H1 (PRO667) (1: 100 Reaction). Preimmune serum and PBS-BSA were also used as controls. The result is FIG. 9 and expressed as mean absorbance (OD) at 405 nm. In parallel control experiments, identical sera were checked on plates coated with purified CEA. There was no reactivity with 11D10 immunized monkey serum; the absorbance obtained was comparable to that obtained with the FBS-BSA control. Results are expressed as mean absorbance at 405 nm (n = 3). Data SD was less than 10%. The difference between experimental and control values was statistically significant. Ab3 serum also specifically bound to semi-purified HMFG coated on microtiter plates by ELISA (FIG. 14). Control sera from preimmunized monkeys or monkeys immunized with irrelevant Ab2 (3H1) did not show appreciable binding to HMFG. In a parallel experiment, the same Ab3 from monkey PRO723 was negative when compared above to a CEA coated plate.
To determine reactivity with cell surface HMFG, MCF-7 cells were examined by immunoflow cytometry. As shown in FIG. 10, Ab3 from Ab2 immunized monkeys showed distinct binding (FIG. 15-A) similar to the binding pattern obtained with Ab1 (not shown). Significant binding was not obtained in MOLT-4 cells that do not express HMFG (FIG. 15-B).
Ab3 antibody was then purified from serum as described above. The reactivity of the purified Ab3 was checked by ELISA. Plates were coated with HMFG (2 mg / ml, 100 ng / well) and incubated overnight at room temperature. Plates were blocked with 1% BSA in PBS and reacted with purified Ab3 (20 mg / ml) from monkeys immunized with either 11D10 or control Ab2 (3H1). PBS-BSA was used as a negative control. In plates coated with CEA, mAb 8019 (anti-CEA) was used as a positive control. The results are in FIG. 16 and are expressed as mean absorbance (OD) at 405 nm (n = 3). The SD of the data was less than 10%. Monkey Ab3 reacted specifically with HMFG coated on microtiter plates, whereas no reactivity was obtained with plates coated with control CEA.
The specificity of purified Ab3 for HMFG was further confirmed by slot blot analysis (FIG. 17). In FIG. 6, lanes 1 and 2 were coated with semi-purified HMFG and lanes 3 and 4 were coated with purified CEA. Membranes were incubated with Ab1 (lane 1), purified Ab3 (lanes 2 and 3), and anti-CEA in mAb8019 (lane 4). Reactivity with HMFG was shown, but no reactivity with CEA.
Competition of mouse Ab1 and monkey Ab3 to MCF-7 cell binding. If Ab3 has a similar binding site as Ab1, Ab3 should compete with Ab1 for binding to HMFG on MCF-7 cells. Confluent monolayers of MCF-7 cells in microtiter plates were prepared with various concentrations of purified Ab3 and fixed amounts of¹²⁵Reaction with I-labeled Ab1 (approximately 50,000 cpm). Percentage of inhibition was calculated and plotted against inhibitor (mg Ab3). A fixed amount of radiolabeled Ab1 was co-incubated with various amounts of purified Ab3 or control Ab3 preparation and MCF-7 cells (FIG. 18). 100 ng of purified Ab3 inhibited binding by 60%, and 1 mg of purified Ab3 gave more than 80% inhibition, whereas the control Ab3 used at a concentration of 5 mg produced no inhibition. It was. These results indicate that the Ab2 immune monkey antibody binds to the same antigen, such as Ab1; therefore, the Ab3 preparation contains antibody molecules with Ab1 ′ properties.
Cell response to anti-Id. The cellular immune response was measured by proliferation of peripheral blood mononuclear cells incubated with aluminum hydroxide precipitated anti-Id antibody (11D10) and aluminum hydroxide precipitated anti-iD antibody (3H1). Peripheral blood lymphocytes (PBL) from immunized monkeys were assayed for responsiveness to stimulation with 11d10-Alugel or 3H1-Alugel in vitro. PBL, 11D10 Alugel^TM(2 μg to 100 ng) and 3H1-Alugel (2 μg to 100 ng) for 5 days and proliferation is [^ThreeMeasured by [H] thymidine incorporation. Peripheral blood mononuclear cells obtained from monkey PRO723 were stimulated in vitro with 11D10 (1 mg / ml); control irrelevant Ab2 3H1 (1 mg / ml). Similarly, peripheral blood mononuclear cells obtained from monkey PRO872 were stimulated in vitro with 11D10 (1 mg / ml); irrelevant Ab2 3H1 (1 mg / ml). Stimulus, [^ThreeIt was measured by the degree of uptake of H] thymidine pulses. Culture medium without any antigen was also used as a control. Peripheral blood mononuclear cells after immunization were collected 10 days after the third immunization. The results are shown in FIG. 19 and are expressed as the average cpm of triplicate wells. The SD of the data was less than 10% for each decision value. The difference between experimental and control values was statistically significant. A positive proliferative response was seen in both monkeys PRO872 and PRO723, which was almost comparable to the response to mitogen phytohemagglutin-P. Preimmune cells did not show a proliferative response to anti-Id antibodies, but post-immunized cells responded significantly. There was also a small but important response to the isotype-matched 3H1 anti-Id antibody, which was significantly less than the 11D10 response; this represents a response to the non-Id component of the immunoglobulin molecule It seems. Proliferative responses were recorded after the first third injection and similar reactivity was obtained after the fourth immunization. Due to limited blood supply, we were unable to test T cell proliferation in the presence of semi-purified HMFG antigen. Therefore, the antigen specificity of the cellular immune response induced in lymphocytes as well as the phenotype of these lymphocytes could not be established.
Example 4: Administration of 11D10 to humans
Immunization with anti-Id 11D10 aluminized with either 1, 2, 4, or 8 mg of 11D10 per injection
Patients were randomly selected to receive one of four doses of 11D10 by intradermal injection on days 0, 14, 28, and 42. Each dose group contains 3 to 8 patients. Aluminum hydroxide precipitation is performed according to the method of Herlyn et al. (1987) Proc. Natl. Acad. Sci. USA 84.805S. Briefly, the vaccine consists of anti-Id 11D10 after aluminum hydroxide precipitation containing 0.2% Alu-Gel S. Add 1 ml of 2% Alu-Gel S (Serva Fine Biochem, Inc., Garden City, Long Island, NY) to a 5 mg aliquot of purified monoclonal anti-Id (Ab2). The volume is then adjusted to 10.0 ml with D-PBS and the mixture is incubated for 1 hour at room temperature with stirring. The mixture is then centrifuged at 2000 rpm for 10 minutes at 24 ° C. The amount of antibody bound to the gel layer is determined by measuring the amount of unbound antibody in the supernatant by spectroscopic analysis. Alu-Gel precipitated Ab is stored at 4 ° C. until use. All operations are performed under a laminar hood. The final product is sterilized and dispensed into sterile glass vials clearly labeled as anti-Id 11D10-Alu-Gel and containing no pyrogen. The activity of aluminum hydroxide precipitated idiotope is monitored by testing binding to Ab1 F (ab ′) 2 in ELISA. Finally, the biological activity of the lot is as described (Bhattacharya-Chatterjee et al. (1987) J. Immunol. 139: 1354; Bhattacharya-Chatterjee et al. (1988) J. Immunol. 141 = 1398) Check with small animal sera after immunization with idiotope carrier. The final filled product is tested for endotoxin, infertility, and general safety in guinea pigs. The aluminum hydroxide precipitated 11D10 is then heat treated in a water bath at 45 ° C. for 30 minutes prior to administration.
Humoral immunity
Characterize the humoral immune response of patients who develop anti-anti-Id antibodies (Ab3). The antibodies are evaluated for the following (a) to (e): (a) binding to tumor cells or isolated semi-purified antigen to determine whether Ab3 actually contains Ab1 antibody; (b) Cytotoxic activity against breast cancer cells by effector cells or complement as mediators of cytotoxic effects; (c) characterization of isotypes by anti-human isotype specific reagents; (d) idiotope analysis of Ab3 (Ie, sharing an idiotope with Ab1); and (e) epitope analysis of Ab3 (ie, binding to the same epitope as Ab1).
The appearance of humoral immunity induced by immunization with aluminum precipitated Ab2 is assessed by testing sera obtained from patients at various times indicated in the protocol. Serum is first tested by sandwich RIA for total human anti-mouse antibody (HAMA) response (anti-iso / allo / and anti-anti-idiotype antibodies). Briefly, microtiter plates are coated with 11D10 and incubated with various dilutions of patient serum. After washing, the antigen-antibody reaction is performed in a homogeneous sandwich RIA¹²⁵Label with I-labeled anti-Id 11D10. Since 11D10 is injected as intact IgG1, patients are expected to mount a HAMA response.
(a) Specific Ab3 response in response to Ab2: Sera from immunized patients with a positive HAMA response are tested for the presence of anti-anti-idiotype antibodies. Serum was precubated with normal mouse immunoglobulin to block human antibodies against isotype and allotype determinants, and then immune anti-Id (coated on microtiter plates for the presence of anti-anti-Id (Ad3) ( The reaction with 11D10) is confirmed by RIA. Irrelevant Ab2 is used as a control. After washing, the antigen-antibody reaction is performed in a homogeneous sandwich RIA as described above.¹²⁵Label with I-labeled anti-Id reagent. Pretreatment, non-immune serum, and serum from normal domers are used as controls in these assays.
(b) Abio idiotope analysis: If a positive response is obtained in (a) above, Ab3 sera from those treated patients were transferred to MC-10 (Ab1) bound to a microtiter plate.¹²⁵Determine the ability to inhibit I-labeled 11D10 binding, or vice versa (inhibition of radiolabeled MC-10 binding to 11D10 on the plate). An unrelated Ab1-Ab2 system is used as a control. This study demonstrates whether Ab3 in patient serum shares an idiotope with MC-10 (Ab1). Inhibition assays of Ab1-Ab2 binding by Ab3 also demonstrate whether Ab3 is a true anti-anti-idiotype.
(c) Binding of Ab3 to tumor antigen: reaction of patient Ab3 serum with a cell line known to express HMFG to evaluate the humoral immune response directed against the negative target antigen Sex is tested in RIA. In addition, the serum is checked for reactivity against a solubilized semi-purified preparation of HMFG antigen coated on a microtiter plate. Antigen-antibody reaction in ELISA¹²⁵Detection is by using I-labeled anti-human Ig reagent or alkaline phosphatase-labeled anti-human-Ig. Pre-immune serum and unrelated antigens are used as controls. The isotype of human Ab3 serum that binds to the HMFG antigen is determined by ELISA using anti-human isotype specific reagents. For these experiments, HMFG is obtained as a fusion protein from E. coli by the method of Larocca et al. (1992) Hybridoma 11 (2): 191-201.
(d) Epitope analysis of Ab3: To demonstrate that Ab3 and Ab1 (MC-10) generated in treated patients bind to the same antigenic determinant, the Ag positive tumor cell line MCF-7 with Ab3 serum Alternatively, inhibition of MC-10 binding to HMFG antigen is confirmed by RIA.
(e) Ab3 cytotoxic activity: When Ab3 in patient sera specifically binds to tumor cells, Ab3's ability to lyse these cells by combining with effector cells and / or complement. Standard ADCC (Cheresh et al. (1985) Proc. Natl. Acad. Sci. USA 83: 515), or CMC assay (Herlyn et al. (1981) Int. J. Cancer 27: 769). However, the cytotoxic activity of Ab3 may depend on its isotype, IgG1 is effective in ADCC, and IgG1, IgG2, IgG3, and IgM are effective in CMC.
Measuring the effect of 11D10 immunity on the cellular immune response of patients
To test whether immunization with 11D10-Alugel in patients with advanced breast cancer leads to the activity of T cells, some of which may show cytolytic activity against the tumor cells When a patient's T cells are activated in vivo by Ab2β, they should respond to in vitro stimulation with Ab2β, antigen, or autologous tumor cells with proliferation, and potential CTL induction.
(a) The proliferative response of the patient's peripheral blood mononuclear cells (PBMC) is tested in the presence of immune anti-Id 11D10, control Ab2, or semi-purified HMFG antigen. Alternatively, PBMC are stimulated with irradiated autologous tumor cells (where possible, cryopreserved during surgery or maintained in short-term culture). When cells proliferate, they classify the phenotype by flow cytometry to determine the subtypes of cells involved in proliferation. Proliferative response is^ThreeMeasured by H-thymidine incorporation and compared to pre-treatment PBMC Stimulation index. For this assay, blood samples are collected after 3 and 4 immunizations and 1 month after the final treatment.
(b) In vitro cytotoxic activity: Cellular immune profile, T cell cytotoxic activity against autologous cancer cells (if possible), or allogeneic MC-10Ag positive tumor cells,⁵¹Assess by testing in vitro in a Cr release assay. As suggested by Ertle et al. (22), the ability of anti-Id antibodies to induce cytotoxic T cells, not MHC restricted to effector phase levels, overcomes the difficulty of using autologous tumor cells as targets And may further facilitate the use of autologous tumor cells. Peripheral blood lymphocyte preparations from breast cancer patients and healthy donors are incubated with various doses of anti-Id (range 10 ng to 100 μg) insolubilized in biobeads or coated on plastic plates. The optimal number of lymphocytes is cultured in 135 mm Petri dishes with optimal concentration of idiotope vaccine in 2 ml in Mishell-Dutton culture medium. Cells are harvested after 5-6 days and as described (84)⁵¹4 or 18 hours against Cr-labeled target⁵¹Used as effector cells in Cr release cytotoxicity assay. Whenever possible, autologous tumor cells (which are cryopreserved during diagnosis or prior to treatment) are used as target cells. This may be possible with the supply of autologous cells in at least some selected patients by growing and maintaining fresh tumor cell lines in nude mice or cell culture. Allogeneic MC-10 Ag positive tumor cell lines are also tested simultaneously as targets. If tumor cell killing is present, the following experiment is performed to characterize specificity:
(i) If lymphocytes become cytotoxic to breast tumor cells, they are tested in a cytotoxicity assay using multiple targets that do not express the antigen (ie, lymphocytes, other carcinoma cell lines). To do. The cytotoxic defect suggests that the target is probably breast cancer associated Ag.
(ii) lymphocytes⁵¹Incubate with an unrelated anti-idiotype hybridoma of the same isotype as Ab2β used and tested in a cytotoxicity assay using Cr-labeled tumor cells. A cytotoxic defect indicates that the effect of anti-Id is specific.
In another experiment, patient lymphocytes are incubated with irradiated autologous tumor cells (when possible). Cells are harvested after 5-6 days and viable cells are purified on lymphocyte separation medium. Cytotoxicity,⁵¹Measure against Cr-labeled self-targeted tumor cells.
This in vitro cytotoxicity assay utilizes PBMC isolated from blood obtained after pre-treatment, i.e. 4 immunizations and 1 month after the last immunization.
Determination of the optimal dose of 11D10
The optimal dose for further clinical trials is selected according to two criteria:
(a) Determine the Ab2 dose that induces the maximal Ab3 response. This can be determined by inhibition assays.
(b) The Ab2 dose that induces maximum Ab3 binding responds to the tumor (Ab1 ′ response).
Quantification of Ab3 and Ab1 'responses
The expression of anti-anti-Id antibody (Ab3) in patient serum is quantified by RIA inhibition studies as follows. Briefly, a microtiter plate is coated with MC-10-IgG1 (Ab1), and¹²⁵React with the immobilized amount of I-labeled Ab2. A standard inhibition curve is generated using purified MC-10 IgG1 as inhibitor. Next, patient sera depleted of anti-isoallotypic activity is checked for the ability to inhibit the Ab1-Ab2 response at different dilutions, and the amount of Ab1-like antibody in the sera is assessed from a standard inhibition curve. The induction and duration of the Ab3 response can be compared between various dose levels. At multiple doses, if there is no substantial difference between Ab3 response or duration, the ELISA assay against a plate coated with semi-purified HMFG antigens can boost the strength of the specific anti-tumor response (Ab1 ') in serum. Compare values.
In vitro study. If Ab1 'or Ab3 positive patient sera circulate, it can bind to the patient's tumor cells or circulating tumor antigens (even if Ag is secreted in the blood in small amounts) or they have low affinity Can indicate Patient PBMC are stimulated in vitro with antigen or Ab2 for induction of tumor-specific antibodies. Peripheral blood mononuclear cells (PBMCs) obtained from blood collected before treatment and then one month after four immunizations were prepared from modified Michel-Dutton cultures (DeFreitas et al. (1985) Curr. Top Microbiol. Immunol. 119: 75) are cultured with various concentrations of 11D10, or unrelated Ab2, or HMFG antigen (10 μg-110 ng). Culture supernatants are collected and first confirmed for specific human immunoglobulin production by ELISA assay and by radioimmunoassay for binding of Ab2 to the insolubilized preparation. Furthermore, the supernatant¹²⁵The content of idiotope-carrying molecules is tested by their ability to inhibit the reaction between I-labeled MC-10 (Ab1) and Ab2β. Supernatants were assayed for MC-10 Ag-positive human breast carcinoma cells and their reactivity with Ag-negative lymphocytes by RIA or ELISA assay (sensitivity> 1 ng) for evaluation of Ab1 ′ antibodies.¹²⁵Confirmation is also performed in binding assays using I-labeled anti-human Ig reagents.
The specificity of the Ab2β effect is monitored by incubating PBMC with an unrelated Ab2 of the same isotype. Since only Ab3 positive patients are included in this in vitro study, PBMCs stimulated with Ab2β should secrete antibodies that bind to Ab2β (11D10) and serve as a positive control.
Potential induction of Ab4 response
According to the network hypothesis, patients immunized with Ab2 can also ultimately induce an Ab4 response (anti-anti-anti-Id), which can mimic the specificity of Ab2. To study this possibility, sera from Ab3 positive patients (depleted of anti-iso and anti-allotype antibodies) are reacted with MC-10 (Ab1) by ELISA or RIA as described. Positive and negative controls are included for the Ab3 assay as described. Serum for this assay is obtained 3 months after the final treatment.
Example 5: Analysis of immune response elicited by administration of 11D10 to patients with advanced HMFG-related diseases
IND was obtained from the US Food and Drug Administration for clinical trials of breast cancer patients with anti-Id 11D10 precipitated with aluminum (BB-IND # 5745). Five patients were enrolled in the Phase Ib trial. All patients had advanced breast cancer that was previously treated with standard therapies, and their tumor cells were positive for the breast cancer antigen HMFG, as identified by monoclonal antibody MC-10 (BrE1). It was. Patients were randomized to either 1 mg, 2 mg, 4 mg, or 8 mg doses of 11D10-Alugel (aluminum) per injection. They were immunized by intradermal injection every other week for a minimum of 4 injections. Treatment continued on a monthly basis until the patient's illness improved.
The first patient so far received 8 mg 8 immunizations; the second patient received 4 mg 4 immunizations; the third patient received 2 mg 4 immunizations. The fourth and fifth patients received doses of 1 mg and 4 mg, respectively, and were recently added to the study.
Poisonous
Toxicity was minimal with only a local reaction with mild erytherma and hardening at the site of injection. Anti-Id treatment had no adverse effect on hematopoietic cells, kidney function, or liver function.
Humoral response to anti-idiotype
The appearance of humoral immunity induced by immunization with aluminum-precipitated anti-Id 11D10 (prepared as described in Example 4), and serum from patients before treatment and after each treatment with vaccine. Evaluation was made by testing. Hyperimmune sera from 3 first patients (after 4 immunizations) showed significant levels of total human anti-mouse antibody responses, including anti-iso / allo / anti-anti-idiotype responses to immune Ab2 11D10. Typical data from the first patient is shown in FIG. The sera from these patients are then collected on a plate by radioimmunoassay.¹²⁵The ability of I-MC-10 (Ab1) to bind to Ab2 11D10 or vice versa (radiolabeled Ab2 binding to Ab1 on the plate) was confirmed. These reactions were performed in the presence of excess normal mouse Ig to block human antibodies against isotype and allotype determinants. FIG. 21 shows data for the first patient. About 40% inhibition was obtained with the 1:40 dilution of serum, whereas only 9% and 22% inhibition was obtained with the second and third patient sera at the same dilution. After 7 immunizations, the first patient showed 83% inhibition at a 1:40 dilution of serum. Preimmune sera from all three patients showed no inhibition. These results indicate that patient 1 was significantly mounted with anti-anti-idiotype antibody (Ab3) and the other two patients had some Ab3 reactivity.
Next, it was investigated whether anti-Id 11D10 could induce a specific antibody response of anti-tumor antigen (HMFG) in immunized patients. For this, sera obtained after 4 immunizations were tested by ELISA assay against HMFG antigen (fusion protein obtained from Dr. Ceriani; Larocca et al. (1992)) coated on microtiter plates. . Results are shown in Table 5 and are expressed as the average of triplicate wells (S.D. <10%).

Ab3 sera from patients 1 and 3 showed specific binding to HMFG antigen when compared to preimmune sera. Both pre-immune and post-immune sera from patient 2 showed non-specific binding to HMFG, which was not increased by immunization.
Cellular immune response to anti-idiotype
The cellular immune response was measured by proliferation of peripheral blood mononuclear cells incubated with an aluminum-precipitated anti-Id 11D10 and iso, allotype compatible control anti-Id 3H1. A positive proliferative response was observed only in patient 1 (FIG. 22), but not in the other two patients. Preimmune cells from patient 1 had no proliferative response, whereas hyperimmune cells had a significant response to anti-Id 11D10. There was also a response to the control anti-Id 3H1; this response was significantly weaker than the 11D10 response and appears to indicate a response to the non-idiotypic component of the mouse immunoglobulin molecule.
The results suggest that anti-Id 11D10 can induce both humoral and cellular immune responses in patients with advanced breast cancer.
Example 6: Construction of recombinant vaccinia vector encoding 11D10 polypeptide fragment
Plasmid construction and production of recombinant vaccinia virus
FIG. 18 shows a construction scheme of a general vaccinia vector (rvv). The present inventors obtained the entire TK gene sequence (GenBank, accession number J02425) of vaccinia virus wild type WR strain from the National Center for Biotechnology Information (NCBI) using the BLAST program. Aitschul et al. (1990)J. Mol. Biol.215: 403-410. From sequence data, forward and reverse PCR primer 5'-CAGATGGAAGGGCCCAAC (SEQ ID NO: 42) and 5'-GATTGATGCATATCATTACC (SEQ ID NO: 43) was synthesized. These correspond to nucleotides 22-39 and 727-708 of the TK sequence, respectively (Hruby et al. (1983)Pro.Natl.Acad.Sci.USA 80: 3411-3415). For insertion into plasmid pGEM-7Zf (+) (Promega), an Apa I site (underlined) was introduced into the forward primer and an Nsi I site (underlined) was introduced into the reverse primer. DNA from the wild-type WR strain of vaccinia was isolated and the TK gene was amplified by PCR. A DNA fragment of the expected size (about 700 bp) was obtained by PCR. The DNA was separated by electrophoresis in low melting point agarose and purified by digestion with GELase (Epicentre Tech.). After digestion with Apa I and Nsi I, the TK DNA fragment was ligated into pGEM-7Zf (+). The resulting plasmid (pGEM-TK) was amplified by standard transformation methods. Insertion was confirmed by restriction mapping.
Forward primer 5'-GTT from the wild type vaccinia virus, promoter 7.5K, corresponding to nucleotides 69-88 and 335-312 of the 7.5K promoter sequenceATCGATGTCGAATAGCC (SEQ ID NO: 44) and reverse primer 5'-TTGCTGCAGAmplified by PCR using ATTGAGTACTGTTCT (SEQ ID NO: 45). Cochran et al. (1985)J. Virol.54: 30-37. The Cla I site (forward direction) and Pst I site (reverse direction) were included in the primer. The amplified DNA fragment was digested with PstI. A polynucleotide adapter with a smaller oligonucleotide phosphorylated at the 5 'end with polynucleotide kinase was synthesized. The half-phosphorylated adapter was ligated to a PCR amplified 7.5K promoter DNA fragment digested with PstI. The product was digested with Cla I / Eco RI digested pGEM-TK.
A cDNA insert encoding 11D10 polypeptide is inserted between the Nco I and XmaI (Sma I) sites of pVV. This plasmid also contains a V at the 5 'end of the scFv cDNA._HContains the leader sequence. If desired, a vaccinia control plasmid containing the E. coli β-galactosidase cDNA can be constructed.
rvv construction
rvv is constructed by homologous recombination between a vaccinia plasmid and a wild-type WR strain of vaccinia virus using CV-1 cells according to the procedure of Macckett et al. (DNA Cloning, Volume 2, edited by DMGlover, IRL Press 1985) . Recombinant virus clones expressing β-galactosidase (control) were TK 143B cells in the presence of 5′-bromodeoxyuridine and 5-bromo-4-chloro-3-indoyl-β-D-galactosidase (X-Gal) Select by growth above. Pick up the blue recombinant virus with a Pasteur pipette and purify the plaque. As a second step of clone selection, Southern blotting of the extracted DNA is performed using 11D10 cDNA as a probe. Further selection of rvv is performed by assaying the culture supernatant of virus infected CV-1 or any other eukaryotic cell by ELISA. If 11D10 polypeptide bound to the cell is present in rvv (ie, the leader sequence has been deleted), the cell lysate is assayed. Western blots using MC-10 (Ab1) as a probe are also performed. The biological activity of the 11D10 polypeptide synthesized by vaccinia virus is determined by cell binding inhibition as described above. Rvv clones containing 11D10 polynucleotides are selected by staining with 0.1% neutral red and plaques are purified as described above. Viral clones are grown in high titer lysates using standard techniques. Mackett et al. (1982)Proc.Natl.Acad.Sci.USA 79: 7415-7419. Typically, clones producing the highest amount of 11D10 polypeptide are selected for further study.
Assay of 11D10 polypeptide (foreign protein) expressed by recombinant vaccinia virus
CV-1 cells were grown to 25 cm in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 100 units penicillin and 100 μg streptomycin per ml.²Incubate in flasks or 6-well cluster flasks. Cells are inoculated with rvv at a MOI of 30. Virus is absorbed for 2 hours at 37 ° C. in a tissue culture incubator, after which the inoculum is replaced with culture medium and incubation is continued. After the indicated time of incubation, the supernatant is removed and the secreted 11D10 polypeptide is assayed. As a control, supernatant from mock infected cells is used. Assays for 11D10 polypeptides can be performed by testing for binding to MC-10 (Ab1), eg, as described in Examples 1 and 5. β-D-galactopyranoside produced by rvv-lacZ is assayed using p-nitro-β-D-galactopyranoside as a substrate according to Miller (Experiments in Molecular Genetics, Cold Spring Harbor Pines 1972) To do. The culture supernatant of virus-infected cells is treated with β-propionate to inactivate the virus prior to assay. Corcoran et al. (1988)J.Parasit.74: 763.^ThreeUptake of H-thymidine by NFS60 cells was used as a measure of cell proliferation. Jaffee et al. (1993)Cancer Res.53: 2221-2226. In the presence of supernatant from mock-infected CV-1 cells,^ThreeRadioactivity due to H thymidine incorporation was subtracted as background. Intact 11D10 is used as a positive control and as a standard for biological activity. Alternatively, a standard solution of GM-CSF can be used.
Vaccinia 11D10 vaccine trial
For vaccinia administration, 10^Four~Ten⁷Mice are injected with the viral titer of pfu. Injection can be subcutaneous, intramuscular, intradermal or intraperitoneal. Immunization is done weekly. Mice are bled 7 days after each immunization for measurement of Ab3 (including Ab1 ′). A test for the development of T cell immunity is performed 10 days after the boost. Mice can also be tested by tumor challenge, where survival after tumor cell injection is monitored.
For vaccinia administration via virus-infected tumor cells, autologous tumor cells are maintained in Eagle's medium containing 10% (vol / vol) fetal calf serum, 2 mM glutamine and gentamicin. 75cm²In a monolayer of confluent cells in a flask, (3 x 10⁸) Inoculate rvv of plaque forming units (pfu). After 2 hours at 37 ° C., the inoculum was replaced with DMEM and incubation continued for an additional 24 hours. After examination under the microscope, the cells are recovered with a scraper, washed twice with PBS and the desired concentration (10^Three~Ten^Five/ 200 μl) and resuspend in PBS. 6-8 week old female C57BL / 6 mice are purchased from Harlan Bioproducts for Science Inc. (IN). The cellular vaccine is injected subcutaneously into the left hind flank of the animal and two weeks later tumor cells are injected for challenge into the right hind flank. Following tumor challenge, mice are monitored daily for survival and tumor presence. If the tumor is measurable, measure the tumor two-dimensionally with a caliper weekly and calculate the volume (²X length) / 2. Obviously, tumors that are too small to accurately measure volume are recorded as 0 volume, while tumor expression is recorded as positive. Tumor volumes are averaged only for mice that actually developed tumors over a 120 day observation period. A zero value is included for mice that eventually developed a tumor but no tumor at a given time.
Statistical evaluation
Statistical evaluation is performed using SigmaStat software (Jandel, Inc. San Rafael CA USA). P values less than 0.05 are considered statistically significant.
Example 7: Construction of an expression vector encoding 11D10 fusion protein
A cDNA encoding the heavy chain VDJ variable region of 11D10 was isolated from plasmid pUC911D10V and ligated into the vector shown in FIG. In this vector, a DNA fragment encoding the mature peptide of mouse GM-CSF or IL-2_ThreeConnected to 3 'side of exon. This is CH_ThreeThis was achieved by creating a Cla I site between the final and stop codons of the exon, and a NotI site 3 'to the stop codon. The cDNA encoding the light chain of 11D10 was incorporated into the expression plasmid pSV184-ΔHneo shown in FIG. The pSV184ΔHneo plasmid is described by Shin et al. ((1989)Meth.Enzymol.178: 459-476).
After the first transfection, the plasmid containing the 11D10 light chain was transfected into the cells by electroporation according to a modification of Shin et al. (1989) as follows. Cells were removed from the growth medium by centrifugation and washed twice with Iscove's modified Dulbecco medium (IMDM, GIBCO). 4.5x10 cells⁶Resuspended in cold IMDM (no serum) at a concentration of / ml. 0.9 ml (4 x 10) from this suspension⁶Cells) was added to a 0.4 cm BioRad cuvette. About 20μg pSV211D10V_H-Cytokine DNA, pSV211D10V_H-Linearized with a restriction enzyme (eg PvuII) not present between the Eco RI sites of the cytokine. After phenol / chloroform extraction and ethanol precipitation, DNA was suspended in less than 50 μl IMDM and added to the cell suspension. The cell suspension-DNA mixture was cooled in ice for 10 minutes, and then an electric pulse was applied with a Bio-Rad Gene Pulser Transfection Apparatus at an electric capacity of 200 V and 960 μF. After incubation in ice for 10 minutes, the cells were suspended in 96 ml IMDM containing 10% fetal calf serum, and 125 μl / well (5 × 10 5) with a multichannel pipette in 8 96-well plates.^ThreeCells / well). Two days later, 125 μl IMDM containing 10% fetal calf serum and 25 μg / ml mycophenolic acid per well was added. After 4 days, half of the medium was removed from each well and 125 μl of 1 × IMDM-mycophenolic acid medium was added. After the clones were visualized (after about 10-15 days), the culture supernatant was removed for assay with MC-10 for detection of transfectoma.
Table 6 shows the various expression plasmids constructed. mIL-2 and mGM-CSF refer to mouse IL-2 and GM-CSF, respectively. V_HAnd V_LContained a signal peptide.

For detection of high production transfectomas, cells are limiting diluted (1 cell / well) and then plated into 96 well plates. If clones in the microwells are visualized but remain small, the culture supernatant is assayed by sandwich radioimmunoassay for detection of functional antibodies. A 96-well plate is coated with MC-10 and 50 μl of culture supernatant from the transfectoma is reacted with the coated antibody. The amount of functional antibody produced by each transfectoma is¹²⁵Determined by radioimmunoassay with I-labeled MC-10. For further evaluation of high antibody producing transfectants, various dilutions of culture supernatants from selected clones are similarly assayed.
A plasmid containing the heavy chain GM-CSF fusion was transfected into Sp2 / 0 cells by protoplast fusion using the technique of Oi et al. (1986) BioTechniques 4: 214-221. High producing clones were selected for initial biochemical characterization of the fusion protein.
Two ELISAs were performed as follows. In Assay 1, microtiter plates were coated with a standard concentration of goat anti-human kappa light chain (Orgomon Teknika Corp. West Chester, Pa.), Blocked with BSA, and washed. Supernatants from cultures of cells expressing the various test constructs were then incubated in the wells. After washing, the wells were overlaid with alkaline phosphatase-conjugated heavy chain specific goat anti-human IgG1 (Sigma) and developed in the usual manner. A positive reaction indicated that the supernatant contained immunoglobulins with both the human heavy and light chain constant regions of the expected type. Assay 2 was performed in a similar manner except that color was developed using biotin-conjugated rat anti-mouse GM-CSF (Pharmingen) followed by avidin peroxidase conjugate (Sigma). A positive reaction indicated that the supernatant contained an immunoglobulin with a human light chain and a GM-CSF component (expected at the heavy chain C-terminus).
Table 7 shows the results of assaying the supernatant of 9 clones for the presence of light chain coupled to heavy chain (Assay 1) and GM-GSF activity (Assay 2). The results show that many binding proteins were obtained that contained determinants on the same molecule for light chain, heavy chain, and GM-CSF.
The fusion protein is isolated by affinity chromatography using a Sepharose-protein A column and characterized using the following antibodies and standards as follows: monoclonal mouse anti-human IL-2 (Genzyme, code, DNA-1), Standard natural human IL-2 (BRL cat. # 13288-014); monoclonal anti-mouse IL-2 (UBI, cat. # 05-115), standard mouse recombinant IL-2 (Sigma, cat, #I 4517); Human GM-CSF assay kit using human GM-CSF standard (R & D System cat. DGM00), rat anti-mouse GM-CSF (BRL, cat. # 13306-014), standard mouse recombinant GM-CSF (UBI, cat . # 01-167).

Purified chimeric (ie, fusion) protein was analyzed by SDS polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Molecular weight standards and purified 11D10 were included in this experiment. Protein bands were stained by Coomassie Brilliant Blue staining. The effect of the purified fusion protein on the binding of MC-10 (Ab1) to 11D10 (original Ab2) and HMFG (the nominal antigen), and the inhibitory RIA, Ab2 to establish the Ab1 binding specificity of this fusion protein For the characterization of Inhibition of the binding between labeled Ab1 and SKBR3 (HMFG expressing breast cancer cell line) cells by a fusion protein provides further support for the specificity of antigen-antibody binding. The biological activity of mouse IL-2 is determined by a cell proliferation assay using a suspension of CTLL-2 T cells. Samples and standards were serially diluted in complete RPMI-10 medium and 50 μl aliquots were dispensed into wells of a 96 well plate. CTLL-2 cells are grown to active log phase and washed with complete RPMI-10 to remove any remaining IL-2. 1x10 cells in complete RPMI-10^FiveSuspend in cells / ml. Cells are divided into three groups, one set is administered with monoclonal anti-mouse IL-2, one set is administered with anti-human IgG gamma chain, and the other set is administered with the solution used to dilute these antibodies. . Cell suspension (50 μl, 5 × 10^ThreeCells) to each well. CO cells₂Incubate for 24 hours at 37 ° C in an incubator.^ThreeH-thymidine is added and incubation is continued for an additional 24 hours, after which the cells are harvested and the incorporated radioactivity is measured. The difference in counts in the well with and without antibody is considered the net biological activity of the sample or standard. 11D10 is also included in these assays to determine whether the antibody itself can induce cell proliferation of CTLL-2. The biological activity of human IL-2 was similarly assessed using CTLL-2 cells in the presence and absence of specific antibodies. A similar cell proliferation assay is performed for the biological activity of GM-CSF. For mouse and human GM-CSF, NFS-60 cells (Holmes et al. (1985)Proc.Natl.Acad.Sci(USA) 82: 6687-6691) and M-07e cells (Genetics Institute), respectively.
Data interpretation, expected results, possible problems and alternative strategies. SDS-PAGE under reducing conditions is expected to show a protein band around 25 Kd for both 11D10 and the fusion protein; the higher molecular weight band is a cytokine-antibody fusion with the transfectoma fused to the heavy chain Indicates that the protein was produced. It is possible to determine whether tetrameric immunoglobulins have been formed by electrophoresis under non-reducing conditions. In that case, the fusion protein will form a single band and its molecular weight should be higher by an amount corresponding to twice that of the cytokine molecule, ie the fusion protein is a dimer with respect to the cytokine. ELISA and biological assays for cytokines indicate whether the cytokine molecule is expressed and biologically active. Quantitative assays using standard recombinant cytokines indicate whether the biological activity of cytokines present as fusion proteins is comparable or enhanced compared to free cytokines. Inhibition of the biological activity of the cytokine by the corresponding antibody compared to the control antibody indicates that the action of the cytokine is specific. Inhibition of cytokine biological activity by the anti-human IgGγ chain indicates that the cytokine moiety is present as a fusion molecule with Ig.
Example 8: Expression and characterization of 11D10scF
V for 11D10_H-(GGGS)_Three-V_LA cDNA construct encoding (SEQ ID NO: 46) is prepared. The cDNA for this 11D10 fragment is incorporated into the pET-22b (+) plasmid vector (Novagen, Madison, Wis.) And expressed in E. coli. Perform sequence analysis and V_LV combined with other frameworks_HConfirm that the plasmid construct contains the carboxy terminus of and no leader region. pET-22b (+) contains 6 consecutive histidine residues (His₆A nickel ion binding domain. His₆The domain has a high affinity for nickel and is used for the purification of recombinant 11D10scFv.
A cell binding competition assay is performed to determine if 11D10scFv retains the antigen mimetic exhibited by intact 11D10. HMFG positive MCF-7 or SKBR3 (1 × 10 in 50 μl volume)^FiveCells / well) in a 96-well plate. Cells,¹²⁵I] Incubate with MC-10 (Ab1) (100,000 cpm) for 2 hours at room temperature in the absence or presence of increasing concentrations of 11D10 or 11D10scFv fragment. The% inhibition is calculated according to the following formula:

Where R₇Is the average radioactivity in the experimental well and R_MAXIs the radioactivity in the absence of any protein, and R_CIs the background radioactivity.
Example 9: Testing of recombinant 11D10 polynucleotide vaccine in mice
A recombinant candidate 11D10 polynucleotide vaccine is prepared as described herein. Two groups of 10-15 female C57BL / 6 mice (6-8 weeks old) were coupled with KLH using glutaraldehyde as described by Bhattacharya-Chatterjee et al. (1988) at 50-100 μg doses Immunized intramuscularly with the purified plasmid.
In addition, different routes of administration (eg, intramuscular, intradermal, subcutaneous, and intraperitoneal) are compared.
To confirm Ab3 (including Ab1 ′) production, blood is drawn from mice as described above 7 days after each immunization. Ten days after the boost, 3 mice in each group are sacrificed to isolate the spleen for T cell proliferation assay.
With non-specific humoral or cellular immunity (by indirect mechanisms such as cytokine production induced by injected polynucleotides) to determine if any observed effect is specific In contrast, the following controls are used: (a) a plasmid without the 11D10 polynucleotide insert; (b) a plasmid with the 11D10 polynucleotide insert in the reverse (ie, antisense) orientation; and (c) an irrelevant Ab2. A plasmid containing the encoding polynucleotide.
Example 10: Further analysis of immune response elicited by administration of 11D10 to patients with progressive HMFG-related disease
US FDA Phase Ib expanded for clinical trials of breast cancer patients with anti-Id 11D10 precipitated with alum (BB-IND # 5745) as described in Example 5 to include 12 patients did. All patients have advanced breast cancer, have been previously treated with standard therapies, and their tumor cells are breast cancer antigens as defined by monoclonal antibody MC-10 (BrE1) Positive about HMFG. Patients were randomly assigned to 11D10 Argel (alum) at doses of 1 mg, 2 mg, 4 mg, or 8 mg per injection. The patient was immunized at least 4 times every other week by intradermal injection. Treatment continued on a monthly basis until the patient's disease progressed. Patients were monitored very closely for disease activity and all were removed from the study because of disease progression or death. Details of this study for 12 patients are shown in Table 8.

toxicity
Toxicity was negligible with only a local reaction at the injection site with mild erythema and hardening. Anti-Id treatment did not have any adverse effect on hematopoietic cells, kidney function or liver function.
Humoral response to anti-idiotype
Examining patient sera before treatment and after each treatment with vaccine for the development of humoral immunity induced by immunization with alum-precipitated anti-Id 11D10 (prepared as described in Example 4) It was evaluated by. Hyperimmune sera from 5 of 10 patients (after the 4th immunization) showed significant levels of anti-iso / allo / anti-anti-idiotype responses to immunization with Ab2 11D10 All human anti-mouse antibody response was shown. The sera of these patients are then analyzed by radioimmunoassay.¹²⁵The ability to inhibit the binding of I-MC-10 (Ab1) to Ab2 11D10 on the plate or vice versa (inhibition of binding of radiolabeled Ab2 to Ab1 on the plate) was checked. These reactions were performed in the presence of excess normal mouse Ig to block human antibodies against isotype and allotype determinants.
FIGS. 27A and B show data for 10 patients including patient 1 (patient 1 data is also discussed in Example 5). Sera from patients 1, 5, 6, 7 and 12 showed significant inhibition even at a dilution of 1: 100. Preimmune sera from patients 1, 5, 6, 7 and 12 did not show any inhibition. Taken together, these results show that patients 1, 5, 6, 7 and 12 produced significant anti-anti-idiotypic antibodies (Ab3) while other patients (2, 3, 8, 9, and 10). Indicates that no significant Ab3 reactivity occurred.
Next, we investigated whether anti-Id 11D10 can induce anti-tumor antigen (HMFG) specific antibody responses in immunized patients. To this end, sera obtained from sera patients 1, 6 and 12 after the fourth immunization were affinity purified with an anti-Id 11D10 column and purified Ab3 was coated on a microtiter plate (Dr. Ceriani) From the fusion protein obtained from Larocca et al. (1992).
The results are shown in FIG. Purified Ab3 sera from patients # 1, 6, and 12 showed specific binding to HMFG antigen compared to purified Ab3 obtained from colon cancer patients treated with control anti-Id 3H1. The antibody (Ab1 ′) isotype in the serum of patients immunized with 11D10 was mostly IgG.
Cellular immune response to anti-idiotype
Cellular immune responses were measured by proliferation of peripheral blood mononuclear cells incubated with anti-Id 11D10 precipitated with alum and iso, allotype matched control anti-Id 3H1. A positive proliferative response was seen in patients # 1, 5, 6, and 12, but not in other patients tested. Figures 29A and 29B show data for patients # 1 and 5, respectively. Although the patient's pre-immune cells had no proliferative response, the hyperimmune cells had a significant response to anti-Id 11D10. There was also a response to the control anti-Id 3H1; this response was weaker than the response to 11D10 and appears to represent a response to the non-idiotypic component of the mouse immunoglobulin molecule.
This result indicates that the findings of Example 5 indicate that anti-Id 11D10 can induce both humoral and cellular immune responses in patients with advanced breast cancer (which also received many other treatments). Make sure you support.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the description and examples should not be construed as limiting the scope of the invention, which is precisely described by the appended claims.
Partial sequence table
(xi) Sequence: SEQ ID NO: 1

(2) Information for SEQ ID NO: 2

(2) Information for SEQ ID NO: 3

(2) Information for SEQ ID NO: 4

Claims (18)

A light chain comprising a variable region having the same amino acid sequence as the amino acid sequence of the variable region of SEQ ID NO: 2 and a heavy chain comprising a variable region having the same amino acid sequence as the amino acid sequence of the variable region of SEQ ID NO: 4. antibody. The antibody of claim 1, wherein the antibody is a monoclonal antibody. A hybridoma cell line indicated by ATCC No. 12020 or its progeny. A pharmaceutical composition comprising an effective amount of the antibody of claim 1 or 2 and a pharmaceutically acceptable excipient. A vaccine comprising an effective amount of the antibody of claim 1 or 2 and a pharmaceutically acceptable excipient. A composition for raising an immune response in an individual with a progressive human milk fat globule-related disease, comprising an effective amount of the antibody according to claim 1 or 2. 6. A vaccine according to claim 5 for eliciting an immune response in an individual with progressive human milk fat globule-related disease. 7. The composition of claim 6 , wherein the progressive human milk fat globule related disease is breast cancer. A composition for removing a labeled anti-HMFG antibody from an individual who has received a labeled anti-human milk fat globule (HMFG) antibody, comprising the antibody according to claim 1 or 2. A method for detecting the presence of an anti-human milk fat globule (HMFG) antibody bound to a tumor cell, sufficient to bind the tumor cell and the antibody of claim 1 or 2 to the anti-HMFG antibody. 3. A method comprising the step of contacting for a period of time and detecting the presence of the antibody of claim 1 or 2 bound to the anti-HMFG antibody. A method for detecting an anti-human milk fat globule immune response in an individual comprising:
(a) a biological sample derived from the individual and the antibody of claim 1 or 2 are stable between the antibody of claim 1 or 2 and the antibody that binds to the antibody of claim 1 or 2 Contacting under conditions that allow formation of a complex, and (b) detecting any stable complexes formed. A composition for alleviating a human milk fat globule-related disease in an individual having a progressive human milk fat globule-related disease, comprising an effective amount of the antibody according to claim 1 or 2. A kit for detecting or quantifying anti-human milk fat globule antibody,
A kit comprising the antibody of claim 1 or 2 in suitable packaging. The antibody of claim 1 or 2 comprises a detectable label, the kit of claim 1 3. A vaccine for raising an immune response in an individual with a progressive human milk fat globule-related disease, comprising an effective amount of the antibody of claim 1 or 2. The vaccine according to claim 15 , wherein the progressive human milk fat globule-related disease is breast cancer. A vaccine for removing labeled anti-HMFG antibody from an individual who has received labeled anti-human milk fat globule (HMFG) antibody, comprising the antibody according to claim 1 or 2. A vaccine for alleviating human milk fat globule-related disease in an individual having a progressive human milk fat globule-related disease, comprising an effective amount of the antibody according to claim 1 or 2.

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