JP5913202B2 - 改善された特異性を有する新規免疫グロブリン結合タンパク質 - Google Patents
改善された特異性を有する新規免疫グロブリン結合タンパク質 Download PDFInfo
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- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
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Description
本願は、2008年8月11日に出願された米国仮特許出願61/188,549号および2009年4月16日に出願された米国仮特許出願61/212,812号(これらの各々の内容全体が、参照により、本明細書中に組み込まれる。)の優先権の利益を主張する。
本発明は、免疫グロブリンに対して改善された結合特異性を有する、修飾された免疫グロブリン結合タンパク質、例えば、ブドウ球菌プロテインA、並びにこれを製造及び使用する方法に関する。
本開示をより容易に理解できるようにするために、まず、ある種の用語を定義する。さらなる定義は、詳細な説明を通じて記載する。
SpAは、細菌スタフィロコッカス・オーレウスに由来する約42kDaのタンパク質であり、N末端に5つの直列の高度に相同な細胞外免疫グロブリン(Ig)結合ドメイン(E、D、A、B及びCと表記される。)を含有する。SpAの各細胞外ドメインは、異なるIg結合部位を有する。1つの部位はFcγ(IgのIgGクラスの定常領域)に対するものであり、他の部位は、ある種のIg分子のFab部分(抗原認識に必要なIgの部分)に対するものである。ドメインの各々がFab結合部位を含有すると報告されている。SpAの非Ig結合部分はC末端に位置しており、X領域又はXドメインと表記される。
本発明のSpA変異物は、本分野において公知のあらゆる適切な方法を用いて作製することができる。例えば、Sambrook、Fritsch及びManiatisによるMolecular Cloningという題名の研究室マニュアル中に記載されているものなどの、核酸の部位特異的突然変異誘発のための標準的な技術を例えば使用し得る。さらに、ポリメラーゼ連鎖反応(PCR)突然変異誘発を含む標準的な分子生物学技術を使用し得る。
SpA変異物の作製に続いて、免疫グロブリンのFab又はFc部分へのSpA変異物の結合特異性は、本分野における標準的な技術及び本明細書中に記載されている技術を用いて決定される。
本発明は、抗体のFc部分に対する親和性を有し、及び抗体のFab部分に対して低下した親和性を有する又は親和性を有さない少なくとも1つの免疫グロブリン結合タンパク質を含むクロマトグラフィーマトリックスを調製する方法も提供する。一実施形態において、このような方法は、(a)単離されたSpAドメイン(例えば、E、D、A、B、C又はZ)をコードする核酸配列を準備すること;(b)アラニン又はトリプトファン以外のアミノ酸によって、少なくとも29位のグリシンが置換されている変異物タンパク質をコードするように前記核酸配列を変異させること;(c)宿主細胞(例えば、適切な原核細胞又は真核細胞)中の変異物タンパク質を発現させること;(d)変異物タンパク質を宿主細胞から回収すること;及び(e)固体支持体へ変異物タンパク質を連結させることを含む。
SpA変異物を含有するベクターの構築
プロテインAの「Bドメイン」をコードする遺伝子は、DNA合成装置を用いた標準的な方法によって合成される。遺伝子の5’末端は、開始メチオニンに対するコドンを含み、及び遺伝子の3’末端に6つのヒスチジンコドンを含む。この遺伝子は、ベクターpJ56:8620中に与えられる。親ベクターpJ56は、アンピシリン、カナマイシン、クロラムフェニコール及びゲンタマイシンに対する耐性を付与する。続いて発現ベクター中に遺伝子をクローニングするための適切な制限酵素部位が、遺伝子の5’末端及び3’末端の両方に導入される。ベクターのプラスミド地図が図5に示されている。
プロテインA変異物の発現及び精製
様々なSpA変異物を発現させるために、あらゆる適切な細菌発現系を使用することができる。例えば、タンパク質は、pET11a(Novagen,Madison WI)などのpETベクターから、株BL21(DE3)(Promega,Madison WI)などのエシェリヒア・コリ(Eschericia coli)中で発現され得る。プレートから単一コロニーを拾い上げ、100μg/mLアンピシリンを含有するLB培地中において、約37℃で一晩増殖させる。100μg/mLアンピシリンを含有する新鮮なLB培地中に、一晩培養物を100倍希釈し、600nmの光学密度が0.8であるような細胞密度まで増殖させる。1mMイソプロピル−β−D−チオガラクトピラノシドの添加後、細胞をさらに2時間増殖させる。SDS−PAGE分析及びウェスタンブロッティングによって発現を確認する。典型的なプロテインAウェスタンブロットが図8に示されており、図8は、イー・コリBL21(DE3)細胞中のベクターPET11a:8620からのプロテインAの発現がニワトリIgY抗プロテインA抗体を用いて検出されることを示している。
Fc及びFab断片へのSpA変異物の結合
続いて、NiNTAアガロース樹脂(Qiagen)を用いて、Fc及びFabを結合する能力に関して、各SpA変異物を試験する。JacksonImmunoResearch(WestGrove,PA)から、Fc及びF(ab)2断片を入手する。20mMイミダゾールを含有するリン酸緩衝化生理的食塩水中に、0.11mg/mLの濃度まで各SpA変異物を希釈し、0.44mg/mLの濃度までFcを希釈し、0.92mg/mLの濃度までF(ab)2を希釈する。NiNTAアガロース樹脂を再懸濁し、樹脂スラリー約20μLを、0.5mLの微小遠心管中にピペットで分注する。続いて、樹脂を沈降させ、上清を廃棄する。20mMイミダゾールを加えたPBS約100μLで樹脂を洗浄し、室温で約15分間温置し、再度沈降させる。上清を廃棄する。約250μLの0.11mg/mLの各SpA変異物又はPBS対照を、2つ組みで、樹脂カラム上に搭載し、室温で約30分間温置する。樹脂を沈降させ、分析のために上清を保存する。毎回、20mMイミダゾールを加えたPBS100μLで、沈降物を3回洗浄し、5分間温置し、再度沈降させる。上清を廃棄する。樹脂とともに約30分間温置することによって、SpA変異物の各々又はPBS対照との結合に関して、Fc又はF(ab)2の約100μLを試験する。続いて、樹脂を沈降させ、分析のために上清を保存する。毎回、20mMイミダゾールを加えたPBS約100μLで、樹脂を再度3回洗浄し、5分間温置し、沈降させる。今回は、上清を廃棄する。20mMイミダゾールを加えたPBS約100μLで、結合された画分を溶出し、約15分間温置し、沈降させる。さらなる分析のために、上清を保存する。続いて、図9から12に図示されているように、Bio−Rad Laboratories,CAから購入した4から15%勾配のゲル上でのSDS−PAGEによって、試料を分析する。
Fab binding:Melissa A.Starovasnik,Mark P.O’connell,Wayne J.Fairbrother,And Robert F.Kelley(1999).Antibody variable region binding by Staphylococal proteinA:Thermodynamic analysis and location of the Fv binding site on E−domain.Protein Science 8,1423−1431.
Fc binding constant:Susanne Gulich,Mathias Uhlen,Sophia Hober(2000) Protein engineering of an IgG−binding domain allows milder elution conditions during affinity chromatography.Journal of Biotechnology 76,233−244.
Claims (7)
- スタフィロコッカス・オーレウスのプロテインAの1つ又はそれ以上のCドメインを含む単離された免疫グロブリン結合タンパク質であって、
前記1つ又はそれ以上の単離されたドメインは、配列番号11に記載されているアミノ酸配列、又は配列番号11に記載の配列と少なくとも90%同一性を有し且つ配列番号10に記載の配列と少なくとも90%同一性を有するアミノ酸配列を含み、
さらに前記1つ又はそれ以上のドメインは、少なくとも29位のグリシンがリジン、アルギニン及びロイシンからなる群から選択されるアミノ酸で置換されており、
前記免疫グロブリン結合タンパク質は、免疫グロブリンG(IgG)のFc部分を結合するが、29位にグリシンを含む免疫グロブリン結合タンパク質と比べて、免疫グロブリンG(IgG)のFab部分への低下した結合を示し、
前記Fab部分への低下した結合が、29位にグリシンを含む免疫グロブリン結合タンパク質と比べて、15%未満低下した結合である、
前記単離された免疫グロブリン結合タンパク質。 - 29位のグリシンがリジン残基で置換されている、請求項1に記載の免疫グロブリン結合タンパク質。
- スタフィロコッカス・オーレウスのプロテインAのカルボキシ末端領域の少なくとも一部をさらに含む、請求項1に記載の免疫グロブリン結合タンパク質。
- 請求項1に記載の免疫グロブリン結合タンパク質をコードする核酸分子。
- 請求項4に記載の核酸分子を含む宿主細胞。
- 固体支持体へ連結された、請求項1に記載の免疫グロブリン結合タンパク質を含むクロマトグラフィーマトリックス。
- (a)1つ又はそれ以上の免疫グロブリンを含む試料を準備する工程;
(b)前記1つ又はそれ以上の免疫グロブリンがマトリックスに結合するような条件下で、請求項6に記載のマトリックスと試料を接触する工程;及び
(c)適切な条件下で溶出することによって、1つ又はそれ以上の結合された免疫グロブリンを回収する工程;
を含む、試料から1つ又はそれ以上の免疫グロブリンをアフィニティー精製する方法。
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