JP4334931B2 - Novel antibody enzyme production method and novel antibody enzyme - Google Patents

Description

【０１６８】
表１に示したように、◎、○、及び●に該当する株が１６株（全体の３４．０％）見出された。クラスがκ鎖であるマウス由来のＬ鎖では４７株中の１２株（２５．５％）が、上記◎あるいは○に当てはまった。
【０１６９】
表１に示す全４７株のgermlineに関する分類結果から、触媒三つ組残基構造を構築するものを抽出したものを表２に示す。
【０１７０】
【表２】

【０１７１】
上記表２に示したように、タイプ▲１▼（または▲１▼＊）の触媒三つ組残基構造を構成している抗体には共通性があり、１９株の内の９株がbb1で、３株がcr1であった。また、先に述べたi41SL1-2-Lは、germlineがbd2であった。
【０１７２】
次に、germlineのbb1、cr1、bd2に共通している特徴を調べた。
【０１７３】
【表３】

【０１７４】
上記表３に示したように、これらのgermlineはどれも超可変領域１（ＣＤＲ１）が１６個のアミノ酸残基で構成されていた。ＣＤＲ配列は、抗体が抗原を強く認識するために大きく変異を起こす箇所である（これが超可変領域と呼ばれる由縁である）。
【０１７５】
そこで、Asp1、Ser27A、His93の配列とgermlineとの関係について更に解析を進めた。
【０１７６】
図１１（ａ）（ｂ）（ｃ）にgermlineが、チーベ（Thiebe）らの分類で、bb1である１ＤＢＡ株について行った分子モデリングの結果を示した。ＦＲをワイヤーで、ＣＤＲをリボンで示した。Asp１はＦＲ１、Ser27AはＣＤＲ１、His93はＣＤＲ３とそれぞれ一次配列の中の異なる領域に存在している。ところが図１１（ｂ）（ｃ）に示すように、これら３つのアミノ酸残基は、ちょうどＣＤＲ３ループのHis93が存在する先端付近に位置し、His93と同じ高さ、すなわち水平方向で見た時のほぼ同一平面上に位置していることがわかる。
【０１７７】
これらは超可変領域のループが集中している空間の先端部分に当たるので、立体障害なしに抗原を取り込むことができると考えられる位置である。また、図１１（ａ）に示すように、ＣＤＲ１を構成するアミノ酸が１６個の場合は、ＣＤＲ１のループは、ちょうどこの付近で緩やかに波をうつ構造をとり、これより先端部分（２７Ｂから３１番の８アミノ酸残基）は、他のループより突出している。このため、上記抗体酵素は、立体障害なしに抗原ペプチドを取り込むことができる位置に触媒三つ組残基構造を有することとなり、抗原ペプチドをスムーズに分解することができると考えられる。そして、この立体構造上の特徴は上記表２で示した１０株の抗体Ｌ鎖および上記i41SL1-2-Lと共通していた。
【０１７８】
次に、Thiebeらのデータ（Thiebe R, Schanble KF et al, Eur J Immunol, 29(7), 2082-2081(1999)）のgermlineの中でAsp1、Ser27A、His93の出現頻度について調べてみると、１番にアスパラギン酸残基を使用するのは全９３種類中で５８種類あり、全体の６２．３％であった（アスパラギン酸残基は１番のみならず、可変領域全体に広く分布しているアミノ酸残基である）。セリン（Ser）残基の２７ＡはＣＤＲ１内にあるが、２７Ａ付近に存在するSerは触媒三つ組残基構造を構成することができる。
【０１７９】
こうしたSerを入れると有効なSerを含むgermlineが９２種類あった。つまり、Asp１とＣＤＲ１部分のSerが立体構造上近い位置に配置するgermlineは多数存在することになる。これに対し、Hisは出現率が低く９３番目に存在するgermlineはわずか７種類（bb1、cr1、bl1、cs1、bd2、bj2、8-16）のみであった。しかもその内の６種類（8-16以外のもの）は、全て触媒三つ組残基構造を構成でき、ＣＤＲ１が１６アミノ酸残基からから構成されているbb1等のgermlineである。つまり、表３で示したbb1、cr1、bl1、cs1、bd2、bj2の６種類は、抗原を分解する為に、理想的な位置にアミノ酸残基を配置しているgermlineといえる。
【０１８０】
次に、これらのgermlineから産生された抗体が抗原分解活性を有するか否かを実験的に調べた。本発明者らはこれまでに百数十種類のモノクローナル抗体を作製しており、その中の３４株についてgermline（抗体軽鎖（マウス型κ鎖））を決定した。その中で、実験に供したもののデータをまとめて表４に示す。
【０１８１】
【表４】

【０１８２】
なお、表１、表２、表４においては、タイプ◎：Asp1、Ser27A、His93により触媒三つ組残基構造を構成するもの、タイプ○：Asp1、Ser26、His93により触媒三つ組残基構造を構成するもの、タイプ●：「Asp1、Ser27AorSer26、His93」以外の組み合わせ（例えば、His90やHis91を使用）で触媒三つ組残基を構成するもの、タイプ＊：germlineから変異したことで触媒三つ組残基構造を構築できなくなったもの、タイプ×：germlineから変異したアミノ酸を含めて触媒三つ組残基構造を構築したものとする。
【０１８３】
先に議論したように超可変領域の先端部分でHisを持つ触媒三つ組残基構造の構築が可能なgermline由来の株は２１株見つかった。また、２株（No.16、18）でHisを持つ触媒三つ組残基構造を構築できなかった。
【０１８４】
即ち、触媒三つ組残基構造を持つ抗体サブユニット（軽鎖、重鎖）の多くにおいてペプチダーゼ活性、及び／又はプロテアーゼ活性を認めたことから、これらの抗体についてgermlineを調べてみると、ＣＤＲ１が１６個のアミノ酸残基で構成されていること、そしてカバット（KABAT）の分類でヒスチジン残基を９３番目（又はその近傍）に持つという共通点があった（タイプ▲４▼）。これは、ある特定のgermlineで既に酵素活性を持つ抗体Ｌ鎖が用意されていることを示唆する重要な知見である。
【０１８５】
また、触媒三つ組残基構造が存在する抗体の中でこれまでに酵素作用に関する実験を進めてきた株は41S-2（germline：hf24／すでに公表済）およびi41SL1-2（bd2）であるが、これ以外の株についても更に実験を進めており、HpU-2抗体の重鎖、HpU-9抗体の軽鎖、HpU-18抗体の軽鎖、さらにi41-7抗体の軽鎖および重鎖が、ペプチダーゼ活性、プロテアーゼ活性を有することが明らかとなった。
【０１８６】
また、i41-7抗体は、ＣＤＲ−１が１１個の19-25のgermlineに属していた。そこで、ＣＤＲ−１が１１個のgermline全てを検索したところ、19-25以外に、19-14、19-17、19-23、12-41（これらは表３にボックスで表示）に、触媒三つ組残基構造が認められた。さらに詳細に調べてみると、ＣＤＲ−１が１１個のアミノ酸残基で構成されていること、そしてカバット（KABAT）の分類で、ヒスチジン残基を９１番目（又は５５番目）に持つという共通点があった（タイプ▲４▼＊）。
【０１８７】
以上の知見をまとめると次のことがいえる。▲１▼天然型抗体酵素には大きく分けて２つのタイプが存在すると考えられる。１つは、既にgermlineの段階で触媒三つ組残基構造の構築に有利な配列を持つタイプで、チーベ（Thiebe）らの分類で、少なくともbb1、cr1、bl1、cs1、bd2、bj2、hf24、19-25、19-14、19-17、19-23、12-41、21-12の１３通りがこれに当たる。hf24の場合には、germlineに存在するHis91の他に、突然変異によりHis53が表れ、これが触媒三つ組残基構造を構築した可能性もある。▲２▼触媒三つ組残基構造の構築に有利な配列を持つgermlineはThiebeらの分類では僅か１３種類（全体で９３種類であるから１３．９％）である。
【０１８８】
しかし、ＰＤＢによる検索では約２０％、本発明者らが所有しているモノクローナル抗体では約３０％の抗体から触媒三つ組残基構造となり得るものが見つかっており、予想以上の高い確率で上記のgermlineがリクルートされている。
【０１８９】
さらに、結論として、このように、抗体の塩基配列から立体構造を予測し、表３に示すボックスで示されるgermlineを持つ抗体を選択すれば軽鎖について相当の高い確率で効率的に抗体酵素を取得する事が可能である（結論▲１▼）。
【０１９０】
また、重鎖については軽鎖の考え方を当てはめると,これまでに41S-2抗体、i41SL1-2抗体、i41-7抗体の重鎖についてはHis、Asp、Serの触媒三つ組残基構造を構成し、酵素活性を認めた。41S-2抗体、i41SL1-2抗体、i41-7抗体の重鎖のFamilyは、それぞれ、VH10、VH1、VH1である。一方、7C4抗体、HpU-2抗体の重鎖にはHis、Asp、Serによる触媒三つ組残基構造はなく、Glu、Asp、Serで三つ組残基を構成し、酵素活性を認めた。7C4抗体、HpU-2抗体の重鎖のFamilyはそれぞれ、VH5、VH1である。特に、この両者（7C4抗体、HpU-2抗体の重鎖）はすべて同じ組み合わせ（Ser84、Glu85、Asp86）で触媒三つ組残基構造を構成していた。つまり重鎖についてはVH1、VH5、VH10が効率的に抗体酵素として活性を発揮できるFamilyである（結論▲２▼）。
【０１９１】
また、本来触媒三つ組残基構造を有していない抗体でさえ、遺伝子工学的にgermline又はFamilyに相当する触媒三つ組残基構造を導入すれば、これも高い効率で抗体酵素を取得する事が可能である事が本発明からいえる（結論▲３▼）。
さらに、図３７に示すように、ＰＤＢデータの抗体軽鎖（κ型）４７種類のうち、１８種類がHisを持つ触媒三つ組残基構造を有していた（３８．３％）。また、図３８に示すように、本発明者らの所有する抗体軽鎖（κ型）の３７種類のデータのうち、２０種類がHisを持つ触媒三つ組残基構造を有していた（５４．０％）。これらを合計すると、立体構造から見出される抗体軽鎖のうちで、酵素活性を持つ抗体軽鎖の検出率は45.2%となる。これが、抗体軽鎖を検出できる一般的な確率である。
【０１９２】
ところが、上記のようにgermlineを特定すれば酵素活性を持つと考えられるHisを持つ触媒三つ組残基構造を有する抗体は88.1%（PDBデータ；16/19＝84.2%（図３７参照）、本発明者らの所有する株に関するデータ；21/23＝91.3%（図３８参照）、計；（16+21）/（19+23）＝37/42＝88.1％）とその確率は100%に近づく。なお、100%とならないのは本来触媒三つ組残基構造をもつものが、抗体の成熟過程で突然変異を起こてHisを失い、Hisを持つ触媒三つ組残基構造を失うものがあるからである。ＰＤＢデータの抗体で１種類、本発明者らが所有する抗体で２種類が変異していた。つまり、上記のgermlineに特定すれば、ほぼ完全に酵素活性を持つ抗体を取得する事ができる（結論▲４▼）。
【０１９３】
〔実施例４：軽鎖、重鎖の調製〕
精製抗体に対して限外濾過を繰り返すことによって、低分子のプロテアーゼインヒビターを除去した。これに続き、０．２Ｍ b-mercaptoethanolによる還元反応（１５℃、３ｈｒ）と０．３Ｍ iodoacetamideによるアルキル化反応（１５℃、１５ｍｉｎ）を行い、重鎖と軽鎖とに分離した。これを限外濾過濃縮後、Protein-Pak 300カラム（Millipore Corporation Waters Chromatography Division）を用いてＨＰＬＣ精製した。移動相には６Ｍ guanidine 溶液（ｐＨ６．５）を使用し、流速は０．１５ｍＬ/ｍｉｎとした。
【０１９４】
分取した重鎖及び軽鎖の溶液は、６Ｍ guanidine溶液で希釈調製後、ＰＢＳ溶液（１３７ｍＭ ＮａＣｌ、２．７ｍＭ ＫＣｌ、１．５ｍＭ ＫＨ₂ＰＯ₄、８．０ｍＭ Ｎａ₂ＨＰＯ₄、ｐＨ７．３）に対して透析してrefolding（再構成）した後に、実験条件に合わせてバッファー交換した。
【０１９５】
〔実施例５：抗体酵素の活性測定〕
触媒三つ組残基構造と酵素活性との関係を明らかにする目的で、この構造を持つと推定されたi41SL1-2抗体軽鎖（i41SL1-2-L、germline bd2由来）および重鎖（i41SL1-2-H）、i41-7抗体軽鎖（i41-7-L、germline 19-25由来）および重鎖（i41-7-H）についてペプチド基質に対する分解反応を実施した。
【０１９６】
具体的には、i41SL1-2抗体の重鎖、軽鎖のそれぞれに対して、配列番号９に示すＣＤＲ１抗原ペプチド（RSSKSLLYSNGNTYLY）を反応させた。
【０１９７】
反応条件は、５％ ＤＭＳＯ、９ｍＭ ＨＥＰＥＳ、７．５ｍＭ リン酸バッファー（ｐＨ７．１）、２５℃のもとで行った。i41SL1-2-Lは０．４μM、i41SL1-2-Hは０．２μM、抗原ペプチドは５０μMの濃度で実験を行った。
【０１９８】
その結果は、図６に示す。（●）はi41SL1-2-Lと抗原ペプチドとを反応させたもの、（○）は抗原ペプチドだけを反応させたもの、（▲）はi41SL1-2-Hと抗原ペプチドとを反応させたものを示す。図６（ａ）（ｂ）に示すように、重鎖、軽鎖ともに反応時間の経過と共に、抗原ペプチドの濃度が低下し、最終的には検出できなくなった。そこで、時間の経過に伴って新たにＨＰＬＣクロマトグラム上で検出したピーク部分を分取して質量分析したところ、抗原ペプチドの１番目のアルギニン残基と２番目のセリン残基との間が切断されたＣ末端側の断片であることがわかった。この事からi41SL12-L、及びi41SL12-Hは、抗原ペプチドを分解することが明らかとなった。
【０１９９】
また、i41-7抗体の軽鎖、重鎖それぞれに対して、配列番号１０、１１、１２に示す３種類の合成ペプチドを反応させた。i41-7抗体は、タンパク質を抗原として得たもので、現在までのところエピトープを決定していない。本来はエピトープに相当するペプチドを基質として酵素活性を検討するべきところであるが、我々がこれまでに得た抗体酵素はタンパク質には高い特異性を示すけれども、短いペプチドに対する特異性は低い。そこで、本実験では分子量数百Ｄａから２．３ｋＤａの３種類の合成ペプチドを基質として使用した。
【０２００】
反応条件は、１５ｍＭ リン酸バッファー（ｐＨ６．５）、２５℃のもとで行った。配列番号１０、１１、１２に示す合成ペプチドはそれぞれ１２０μＭ、i41-7-Lは０．８μM、i41-7-Hは０．４μMの濃度で用いた。
【０２０１】
結果を図１０（ａ）（ｂ）に示す。（●）は配列番号１０に示すペプチドＴＰ４１−１とi41-7-L又はi41-7-Hとを、（○）は配列番号１０に示すペプチドＴＰ４１−１のみを、（▲）は配列番号１１に示すペプチドとi41-7-L又はi41-7-Hとを、（△）は配列番号１１に示すペプチドのみを、（■）は配列番号１２に示すペプチドとi41-7-L又はi41-7-Hとを、（□）は配列番号１２に示すペプチドのみを、反応させたものを示す。
【０２０２】
図１０（ａ）（ｂ）に示すように、i41-7-L、及びi41-7-Hは、これらのペプチドを全て分解した。
【０２０３】
また、i41-7抗体の軽鎖（i41-7-L）による抗原タンパク質の分解反応をSDS-PAGEにより解析した。基質として用いたのは、抗体41S-2の軽鎖（41S-2-L）である。なお、この基質として用いた41S-2-Lは、refoldingが不完全で抗体酵素としての活性がないものである。
【０２０４】
分解反応は、上記と同様の条件で行い、41S-2-Lは２μＭの濃度で用い、i41-7抗体の軽鎖は０．８μＭの濃度で用いた。結果を図１０（ｃ）に示す。レーンＭは、タンパク質分子量マーカーを、レーン１〜５はそれぞれ、反応開始から０、２、４、６、８日目におけるi41-7-Lと41S-2-Lとの反応液を電気泳動した結果を示す。25.0ｋDaの位置のバンドが抗原タンパク質41S-2-Lを示しており、22.5ｋDaの位置のバンドが41S-2-Lの分解産物を示している。なお、コントロールとして、i41-7抗体の軽鎖のみを反応させた場合と、41S-2-Lのみを反応させた場合の結果を示す。このSDS-PAGEの結果に基づき、i41-7-Lによる41S-2-Lの分解反応をグラフ化したものも併せて示す。
【０２０５】
この図１０（ｃ）のSDS-PAGEの結果及びグラフから、25.0ｋDaの位置のバンドが分解され、22.5ｋDaの位置のバンドが生じているのがわかる。また、コントロールから基質の41S-2-Lのみの反応では、25.0ｋDaの位置のバンドは分解されていない。このことから、i41-7抗体の軽鎖は、抗原タンパク質41S-2-Lを分解することが示された。
【０２０６】
次に、i41-7完全抗体、軽鎖（i41-7-L）、重鎖（i41-7-H）、及びＭＡ−２抗体のポリペプチド分解能を調べた。i41-7完全抗体とは、重鎖と軽鎖とからなる完全な抗体である。ＭＡ−２抗体（germline：aq4）は、メタンフェタミンに対するモノクローナル抗体であって、立体構造中に触媒三つ組残基構造を持たない抗体である。
【０２０７】
反応条件は、上記実験と同一で行い、基質として、配列番号１０に示すポリペプチドＴＰ４１−１（図１２中では「ＴＰ」と記載している）を１２０μＭの濃度で用いた。i41-7完全抗体、i41-7-L、ＭＡ−２抗体は０．８μＭの濃度で、i41-7-Hは０．４μＭの濃度で用いた。
【０２０８】
結果を図１２に示す。（●）はi41-7-LとＴＰ４１−１とを、（□）はi41-7-HとＴＰ４１−１とを、（△）はi41-7完全抗体とＴＰ４１−１とを、（■）はＭＡ−２抗体とＴＰ４１−１とを、（○）はコントロールとしてＴＰ４１−１のみを反応させた。
【０２０９】
図１２に示すように、i41-7抗体の重鎖、軽鎖はともに、反応開始後しばらくの間、ほとんどペプチドを分解しない誘導期を示し、その後、ペプチドを分解する活性期を示すことがわかった。さらに、結果は図示しないが、ペプチドが完全分解された後、再びペプチドを添加すると、i41-7抗体の重鎖、軽鎖はともに、誘導期なしにすぐにペプチドを分解した。この結果から、i41-7抗体の重鎖、軽鎖はともに、誘導期から活性期へと移る際に、コンフォメーション変化するものと考えられる。また、一度コンフォメーション変化すれば、その後も活性型として維持されると考えられる。
【０２１０】
また、重鎖と軽鎖とが揃った完全なi41-7抗体は、ポリペプチドを分解しなかった。これは、i41-7完全抗体は、Ｓ−Ｓ結合や非共有結合等により、軽鎖と重鎖とが強く結合しているため、ポリペプチドと接触しても活性を持つ形態にコンフォメーション変化できないためと考えられる。
【０２１１】
また、立体構造中に触媒三つ組残基構造を有さないＭＡ−２抗体は、重鎖、軽鎖ともに、ポリペプチドを分解しなかった。また、結果は図示しないが、同じく触媒三つ組残基構造を有しないＭＡ−１５抗体軽鎖（germline：ba9）、７Ｃ４抗体軽鎖（germline：af4）、HpU-9抗体重鎖、及びHpU-18抗体重鎖もポリペプチド及び抗原タンパク質を分解しなかった。
【０２１２】
この結果から、立体構造中に触媒三つ組残基構造を有する抗体酵素は、ポリペプチドや抗原タンパク質を切断又は／分解する活性を有することが示された。
【０２１３】
〔実施例６：i41SL1-2-Lおよびi41SL1-2-Hの抗原ペプチドに対する分解活反応の速度論的解析〕
i41SL1-2-Lおよびi41SL1-2-HのＣＤＲ１抗原ペプチドに対する分解反応のプロファイルを詳細に追跡すると、抗原ペプチドとの反応初期においてペプチドの濃度がほとんど変化しない（ペプチドがほとんど分解されない）「誘導期」が認められた（この長さは実験条件によって異なる）。ところが、一度ペプチドを分解して活性状態にある抗体溶液に再度抗原ペプチドを添加すると、今度は誘導期が認められなくなった。そこで、抗原ペプチド再添加反応における分解初速度を測定し、Michaelis-Menten式による解析を行った。
【０２１４】
その結果、図７（ａ）（ｂ）に示すように、軽鎖、重鎖ともにHanes-Woolfプロットにおいて高い相関を示した。従って、これらはMichaelis-Menten式に従う酵素反応であると言える。ここで得た動力学的パラメータをトリプシン（trypsin）（異なる合成ペプチドを基質として求めた値）と比較して表５に示した。
【０２１５】
【表５】

【０２１６】
上記表５に示すように、軽鎖の触媒効率（kcat/Km）はトリプシンと同等、重鎖もトリプシンの約1/10という高い値を示した事から、本抗体酵素の重鎖、軽鎖ともに、天然の酵素に匹敵する高い酵素活性を持つといえる。ところが、各パラメータを見ると基質に対する親和性（Km）については、本抗体酵素の重鎖、軽鎖がトリプシンよりも強いのに対して、回転数（kcat）については、重鎖、軽鎖ともにトリプシンの約1/200の値を示し、両者の性質に違いが認められた。この結果は、i41SL1-2-Lやi41SL1-2-Hが基質に対する高い親和性を有するという抗体としての性質を残しながら、ポリペプチドを完全に分解するという酵素活性を発揮していることを示している。
【０２１７】
〔実施例７：ＨｐＵ−２０およびＵＡ−１５のＨ鎖およびＬ鎖の調製〕
上述の実施例１に記載の手順に従って、ＨｐＵ−２０、ＵＡ−１５という２種の抗ＨＰウレアーゼマウスモノクローナル抗体を作製し、上述の実施例２と同様の方法で、モノクローナル抗体のアミノ酸配列および塩基配列が決定され、三次元立体構造解析が実施された。
【０２１８】
その結果、推定されたＨｐＵ−２０（Ｌ鎖）の可変領域の立体構造を図１３に、ＨｐＵ−２０（Ｈ鎖）の可変領域の立体構造を図１４に、ＵＡ−１５（Ｈ鎖およびＬ鎖）の可変領域の立体構造を図２２に模式的に示す。これらの立体構造予測から、ＨｐＵ−２０のＬ鎖およびＨ鎖、ＵＡ−１５のＬ鎖については、触媒三つ組残基様構造が認められた。一方、ＵＡ−１５（Ｈ鎖）には、触媒三つ組残基様構造は認められなかった。
【０２１９】
さらに、上述の実施例４と同様の方法で、ＨｐＵ−２０およびＵＡ−１５のＨ鎖およびＬ鎖が調製された。
【０２２０】
〔実施例８：抗体酵素ＨｐＵ−２０Ｌ鎖およびＨ鎖を用いた酵素活性試験１〕ＨｐＵ−２０−ＬおよびＨｐＵ−２０−ＨのTP41-1ペプチド（配列番号３１）に対する酵素活性試験を実施した。この分解反応は、以下のような材料、条件、手順で実施された。
【０２２１】
（材料）
▲１▼：HpU-20 mAb（in15mM PB pH6.5、精製・透析終了後、濾過滅菌されたもの。）
▲２▼：HpU-20抗体
▲２▼−１：Lot.1［HpU-20-L］( in15mM PB pH6.5、濃度94.4μg/ml)
▲２▼−２：Lot.1［HpU-20-H］( in15mM PB pH6.5、濃度70.8μg/ml)
▲２▼−３：Lot.2［HpU-20-L］( in15mM PB pH6.5、濃度96.7μg/ml)
▲２▼−４：Lot.2［HpU-20-H］( in15mM PB pH6.5、濃度103.3μg/ml)
▲３▼：精製された TP41-1 ペプチド
（反応液）
▲１▼：HpU-20-L：0.8μM(20μg/ml)
▲２▼：HpU-20-H：0.4μM(20μg/ml)
▲３▼：HpU-20 mAb：0.8μM(120μg/ml)
▲４▼：TP41-1 ペプチド：120μM(284μg/ml)
（反応条件）
反応温度：25℃
小試験管、マイクロチップ、15mM PB(pH6.5)：滅菌処理
実験操作：クリーンベンチ内
（方法）
（１）：TP41-1を15mM PB pH6.5で1.7mg/mlに調製し、濾過滅菌する。
（２）：次に、（１）の1.7mgTP41-1を15mM PB pH6.5で568μg/mlに調製する。
（３）：20抗体のH、L鎖を15mM PB pH6.5でそれぞれ40μg/mlに調製する。
（４）：（２）と（３）を1：1の割合で混合する。
（５）：HPLCでペプチドの経時変化を追跡する。
【０２２２】
この実験の結果については、Lot.1を図１７に、Lot.２を図１８に示す。図１７、図１８のグラフでは、横軸に反応時間（時間）を、縦軸に基質であるTP41ペプチドの濃度（μＭ）を示す。
【０２２３】
図１７、１８のグラフに示すように、調製したLot.1およびLot.2において、若干の反応性の違いはあるものの、ともに再現性よくTP41ペプチドを完全に分解した。これらの結果から、ＨｐＵ−２０−ＬおよびＨｐＵ−２０−Ｈは、TP41-1ペプチドの分解反応において酵素活性を有することが確認された。
〔実施例９：ＨｐＵ−２０Ｌ鎖およびＨ鎖を用いた酵素活性試験２〕
続いて、活性化したＨｐＵ−２０Ｌ鎖およびＨ鎖を用いて、タンパク質分解実験を以下のような材料、反応液の組成・種類、反応条件、方法で実施した。
【０２２４】
（材料）
▲１▼：活性化した（一度TP41ペプチドを完全に分解した）Lot.2［HpU-20-L］(in15mM PB pH6.5、02.10.15分離、 02.10.16精製、02.10.21 PBS透析終了、02.10.21〜22 PBにbuffer交換。4.2ヶ月保存、濃度96.7μg/ml)
▲２▼：ＨＰ菌ウレアーゼ（M.W=522600）02.07.01精製、濃度1.84mg/ml
▲３▼：BSA SIGUMA A-6003 Lot51K7600
▲４▼：HSA WAKO 019-10503 LotKSH6886
（反応液組成）
▲１▼：HpU-20-L：0.4μM(20μg／ml)
▲２▼：HP菌ウレアーゼ：52nM(28μｇ／ml)
▲３▼：BSA：0.4μM(28μg／ml)
▲４▼：HAS：0.4μM(28μg／ml)
（反応条件）
反応温度：25℃
小試験管、マイクロチップ、15mM PB(pH6.5)：滅菌処理
実験操作：クリーンベンチ内
（方法）
（１）HSA、BSAを15mM PB pH6.5で2.2mg/mlに調製し、濾過滅菌する。
（２）次に、（１）の1.7mg/ml TP41-1を15mM PB pH6.5で55μg/mlに調製する。
（３）1.84mg/ml HP菌ウレアーゼを15mM PB pH6.5で55μg/mlに調製する。
（４）活性化したHpU-20-Lと（２）、（３）を1：1の割合で混合する。
（５）SDS-PAGEで抗体及び、各タンパクの変化を追跡する
（反応液の種類）
▲１▼：0.4μM HpU-20-L+0.4μM BSA：320μl
▲２▼：0.4μM HpU-20-L+0.4μM HSA：320μl
▲３▼：0.4μM HpU-20-L+52nM HP菌ウレアーゼ：320μl
▲４▼：0.4μM HpU-20-Lのみ：320μl
▲５▼：0.4μM BSAのみ：320μl
▲６▼：0.4μM HSAのみ：320μl
▲７▼：52nM HPウレアーゼのみ：320μl
本実験の結果を図１９〜図２１に示す。図１９〜図２１は、反応開始１時間後の試料のＳＤＳ−ＰＡＧＥの結果を示す図である。なお、各レーンにおける試料については、Ｍはマーカーであり、▲１▼〜▲７▼は、上記の各反応液である。
【０２２５】
図１９〜図２１に示す結果から、約１時間の反応でＨｐＵ−２０−ＬはＢＳＡを全く分解しないことがわかる。ＨＳＡに対しては２８．５ｋＤａに薄いバンドが見られるがコントロールのＨＳＡはほとんど変化しておらず非常に弱い分解しか起こしていないことがわかる。これに対し、ＨＰ菌ウレアーゼに対しては、約１時間の反応で、５０ｋＤａに強いバンドが、２６ｋＤａに弱いバンドが観察された。また、βサブユニットのバンドはコントロールに比べ、26.7%減少していた。αサブユニットのバンドについては反応前後でほとんど変化していなかった。この結果から、ＨｐＵ−２０−Ｌは、ＨＰ菌ウレアーゼのβサブユニットを特異的に分解することが明らかとなった。
【０２２６】
〔実施例１０：ＵＡ−１５Ｌ鎖およびＨ鎖を用いた酵素活性試験〕
ＵＡ−１５−ＬおよびＵＡ−１５−Ｈを用いて、TP41-1ペプチド（配列番号３１）に対する酵素活性試験を実施した。この分解反応は、以下のような反応液を用いて、以下のような反応条件で実施された。
【０２２７】
（反応液）
▲１▼：Lot.1［UA-15-L］：0.8μM(20μg/ml)
▲２▼：Lot.1［UA-15-H］：0.4μM(20μg/ml)
▲３▼：Lot.2［UA-15-L］：0.8μM(20μg/ml)
▲４▼：Lot.2［HpU-20-H］：0.4μM(20μg/ml)
▲５▼：TP41-1ペプチド：120μM（284μg/ml）
（反応条件）
反応温度：25℃
小試験管、マイクロチップ、15mM PB(pH6.5)：滅菌処理
実験操作：クリーンベンチ内
この実験の結果については、Lot.1を図２３に、Lot.２を図２４に示す。図２３、図２４のグラフでは、横軸に反応時間（時間）を、縦軸に基質であるTP41ペプチドの濃度（μＭ）を示す。図２３および図２４に示すグラフからも分かるように、ＵＡ−２０のＬ鎖についてはTP41-1ペプチドを分解する酵素活性が確認されたがＵＡ−２０のＨ鎖については、酵素活性は確認されなかった。
【０２２８】
続いて、反応液にLot.2におけるUA-15-Lの反応液に、ペプチド基質（TP41-1）を再添加して、１５℃、２５℃、３７℃の各温度でペプチドの分解実験を実施した。
【０２２９】
結果を図２５に示す。この結果から、反応温度が２５℃の場合が、UA-15-Lの酵素活性が最も高いことが明らかとなった。
【０２３０】
さらに、上記の反応条件で、反応液にLot.1［UA-15-L］を用いて、一度TP41-1ペプチドを完全に分解した後、この溶液にTP41-1ペプチドを、５μM、１０μM、１５μM、２０μM、３０μM、６５μM、８０μMの各濃度で添加して、ＵＡ−１５−Ｌによる速度論的解析を行った。その結果を図２６および表６に示す。
【０２３１】
【表６】

【０２３２】
〔実施例１１：ＥＣＬ２Ｂ−４のＨ鎖およびＬ鎖の調製〕
上述の実施例１に記載の手順に従って、ＥＣＬ２Ｂ−４という抗ＨＰウレアーゼマウスモノクローナル抗体を作製し、上述の実施例２と同様の方法で、モノクローナル抗体のアミノ酸配列および塩基配列が決定され、三次元立体構造解析が実施された。
【０２３３】
その結果配列が決定された、ＥＣＬ２Ｂ−４軽鎖のアミノ酸配列、および、それをコードするｃＤＮＡの塩基配列を図２９に示し、ＥＣＬ２Ｂ−４重鎖のアミノ酸配列、および、それをコードするｃＤＮＡの塩基配列を図３０に示す。なお、図２９、図３０では、可変領域（ＣＤＲ−１〜ＣＤＲ−３）に下線を付して示す。
【０２３４】
次に、配列決定された各アミノ酸配列を基にして、分子モデリングが行われた。図２７には、分子モデリングによって推定されたＥＣＬ２Ｂ−４−Ｌの可変領域の３次元構造を示す。また、図２８には、分子モデリングによって推定されたＥＣＬ２Ｂ−４−Ｈの可変領域の３次元構造を示す。
【０２３５】
〔実施例１２：ＥＣＬ２Ｂ−４抗体の酵素活性試験〕
続いて、ＥＣＬ２Ｂ−４−Ｌ、ＥＣＬ２Ｂ−４−Ｈの酵素活性を確認するためのペプチド分解試験が以下のようにして行われた。
【０２３６】
先ず、ＥＣＬ２Ｂペプチド（配列番号２７）の分解反応が、２５℃の１５ｍＭリン酸バッファー（ｐＨ６．５）中で実施された。反応をモニタリングするために、２０μｌの反応液がカラム温度４０℃、定組成条件下のＲＰ−ＨＰＬＣ（Jasco製）に注入された。
【０２３７】
この分解反応の反応液として、以下の▲１▼〜▲５▼が作製された。
▲１▼ECL2B-4(L)0.8μM+ECL2B ペプチド：50μM：800μl作製
▲２▼ECL2B-4(H)0.4μM+ ECL2B ペプチド：50μM：800μl作製
▲３▼ECL2B ペプチド：50μM：500μl作製
▲４▼ECL2B-4(L)：0.8μM：300μl作製
▲５▼ECL2B-4(H)：0.4μM：300μl作製
その結果を図３１〜図３３に示す。図３１のグラフでは、横軸に反応時間（時間）を、縦軸に基質であるECL2Bペプチドの濃度（μＭ）を示す。また、図３２（ａ）〜（ｃ）には、ＨＰＬＣを使用してモニタリングされた結果を示す。また、図３３には、この酵素分解実験における速度論的解析結果を示す。図３３（ａ）は、基質（ＥＣＬ２Ｂペプチド）濃度と分解速度との関係を示すグラフであり、図３３（ｂ）は、Hanes-Woolfプロットの結果を示すグラフである。
【０２３８】
そして、上記Hanes-Woolfプロットを用いて算出したＶmax、Ｋｍ、kcat、kcat/Km値（平均プロット）を以下の表７に示す。
【０２３９】
【表７】

【０２４０】
これらの結果から、ＥＣＬ２Ｂ−４−ＬおよびＥＣＬ２Ｂ−４−Ｈは、ECL2Bペプチドの分解反応における酵素活性を有することが確認された。それゆえ、ECL2B-4-LおよびECL2B-4-Hは、CCR-5の細胞外領域を構成するペプチドを狙って、完全に分解することのできる抗体酵素であることが確認された。
【０２４１】
続いて、ＥＣＬ２Ｂ−４−ＬおよびＥＣＬ２Ｂ−４−ＨのTP41-1ペプチド（配列番号３１）に対する酵素活性試験を実施した。この分解反応は、２５℃の１５ｍＭ リン酸バッファー（ｐＨ６．５）中で実施された。反応をモニタリングするために、２０μｌの反応液がカラム温度４０℃、定組成条件下のＲＰ−ＨＰＬＣ（Jasco製）に注入された。
【０２４２】
この分解反応の反応液として、以下の▲１▼〜▲５▼が作製された。
▲１▼ECL2B-4(L)0.8μM+TP41-1 ペプチド：120μM：800μl作製
▲２▼ECL2B-4(H)0.4μM+TP41-1 ペプチド：120μM：800μl作製
▲３▼TP41-1 ペプチド：120μM：500μl作製
▲４▼ECL2B-4(L)：0.8μM：300μl作製
▲５▼ECL2B-4(H)：0.4μM：300μl作製
その結果を図３４〜図３６に示す。図３４のグラフでは、横軸に反応時間（時間）を、縦軸に基質であるECL2Bペプチドの濃度（μＭ）を示す。また、図３５（ａ）〜（ｃ）には、ＨＰＬＣを使用してモニタリングされた結果を示す。また、図３６には、この酵素分解実験における速度論的解析結果を示す。図３６（ａ）は、基質（TP41-1ペプチド）濃度と分解速度との関係を示すグラフであり、図３６（ｂ）は、Hanes-Woolfプロットの結果を示すグラフである。
【０２４３】
そして、上記Hanes-Woolfプロットを用いて算出したＶmax、Ｋｍ、kcat、kcat/Km値（平均プロット）を以下の表８に示す。
【０２４４】
【表８】

【０２４５】
これらの結果から、ＥＣＬ２Ｂ−４−ＬおよびＥＣＬ２Ｂ−４−Ｈは、TP41-1ペプチドの分解反応においても低い酵素活性を有することが確認された。また、表７と表８とを比較すれば分かるように、ECL2B-4-Lは、本来の抗原であるECL2Bペプチドの方をTP41ペプチドよりも速く分解していることが実験によって示され、ECL2B-4-Lが確かにECL2Bに対する抗体酵素であることが証明された。
【０２４６】
【発明の効果】
以上のように、本発明に係る抗体酵素又は遺伝子等を用いることにより、種々の感染症や癌等の病気の治療、診断に利用可能である。また、目的とする抗体酵素の取得により、新しい臨床診断法の開発にも繋がる。
【０２４７】
さらに、本発明に係る抗体酵素又は遺伝子等を用いることにより、新しいバイオマテリアルとして新型のバイオセンサにも応用可能であり、病気の診断、環境測定などの検査等にも利用可能である。
【０２４８】
また、本発明に係る抗体酵素又は遺伝子等を用いることにより食品工業、化学工業における効果的な合成手段などにも展開できると考えられる。
【０２４９】
また、本発明の抗体酵素の生産方法によれば、免疫学的に作製される抗体の中から、選択的に、ポリペプチドや抗原タンパク質を切断又は分解する酵素活性を有する抗体酵素を効率的に取得することができるという効果を奏する。
【０２５０】
また、本発明の抗体酵素の生産方法によれば、従来公知の遺伝子工学的な手法を用いて、容易に、ポリペプチドや抗原タンパク質を切断又は分解する酵素活性を有する抗体酵素を効率的に作製することができるという効果を奏する。
【０２５１】
【配列表】

【図面の簡単な説明】
【図１】（ａ）は本発明における実施の一形態に係る抗体酵素生産方法の一例を示す工程図であり、（ｂ）は本発明におけるその他の実施の一形態に係る抗体酵素生産方法の一例を示す工程図である。
【図２】本発明の実施の一形態に係る抗体酵素生産方法において、抗体構造解析工程を実施するシステムの構成の一例を示すブロック図である。
【図３】図２に示す抗体構造解析工程の処理手順を示すフローチャートである。
【図４】（ａ）は抗体i41SL1-2の重鎖の可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図であり、（ｂ）は抗体i41SL1-2の軽鎖の可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図である。
【図５】（ａ）は抗体i41SL1-2の軽鎖の可変領域の三次元立体構造を示す図であり、（ｂ）は抗体i41SL1-2の重鎖の可変領域の三次元立体構造を示す図である。
【図６】（ａ）は抗体i41SL1-2の軽鎖のポリペプチド分解反応の結果を示す図であり、（ｂ）は抗体i41SL1-2の重鎖のポリペプチド分解反応の結果を示す図である。
【図７】（ａ）は抗体i41SL1-2の軽鎖のポリペプチド分解反応の速度論的解析の結果を示す図であり、（ｂ）は抗体i41SL1-2の重鎖のポリペプチド分解反応の速度論的解析の結果を示す図である。
【図８】（ａ）は抗体i41-7の重鎖の可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図であり、（ｂ）は抗体i41-7の軽鎖の可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図である。
【図９】（ａ）は抗体i41-7の軽鎖の可変領域の三次元立体構造を示す図であり、（ｂ）は抗体i41-7の軽鎖の可変領域の三次元立体構造を別の角度から見た図であり、（ｃ）は抗体i41-7の重鎖の可変領域の三次元立体構造を示す図である。
【図１０】（ａ）は抗体i41-7の軽鎖のポリペプチド分解反応の結果を示す図であり、（ｂ）は抗体i41-7の重鎖のポリペプチド分解反応の結果を示す図であり、（ｃ）は、抗体i41-7の軽鎖による抗原タンパク質（活性のない41S-2-L）分解反応の結果を示す電気泳動図とグラフである。
【図１１】（ａ）は１ＤＢＡ株の重鎖と軽鎖との三次元立体構造を示す図であり、（ｂ）（ｃ）はその一部分を拡大した図である。
【図１２】 i41-7完全抗体、抗体i41-7の重鎖、軽鎖、及びＭＡ−２抗体のポリペプチド分解反応の結果を示す図である。
【図１３】ＨｐＵ−２０Ｌ鎖の可変領域の立体構造を示す模式図である。
【図１４】ＨｐＵ−２０Ｈ鎖の可変領域の立体構造を示す模式図である。
【図１５】ＨｐＵ−２０Ｌ鎖の可変領域のアミノ酸配列、及びそれをコードする遺伝子の塩基配列を示す図である。
【図１６】ＨｐＵ−２０Ｈ鎖の可変領域のアミノ酸配列、及びそれをコードする遺伝子の塩基配列を示す図である。
【図１７】ＨｐＵ−２０−ＬおよびＨｐＵ−２０−Ｈを用いてTP41-1ペプチド（図中では、ＴＰと記す）の分解実験を行った結果（Lot.１）を示す図である。
【図１８】ＨｐＵ−２０−ＬおよびＨｐＵ−２０−Ｈを用いてTP41-1ペプチド（図中では、ＴＰと記す）の分解実験を行った結果（Lot.２）を示す図である。
【図１９】ＨｐＵ−２０Ｌ鎖を用いたタンパク質分解実験において、反応開始１時間後の試料についてＳＤＳ−ＰＡＧＥを行った結果を示す図である。
【図２０】ＨｐＵ−２０Ｌ鎖を用いたタンパク質分解実験において、反応開始１時間後の試料についてＳＤＳ−ＰＡＧＥを行った結果を示す図である。
【図２１】ＨｐＵ−２０Ｌ鎖を用いたタンパク質分解実験において、反応開始１時間後の試料についてＳＤＳ−ＰＡＧＥを行った結果を示す図である。
【図２２】軽鎖と重鎖からなるＵＡ−１５の可変領域の立体構造モデリング（分子モデリング）を行った結果、推定された立体構造を示す図である。なお、本図では、軽鎖上にのみ触媒三つ組残基様構造が現れていることを示している。
【図２３】ＵＡ−１５−Ｌ（図中では、Ｌと示す）およびＵＡ−１５−Ｈ（図中では、Ｈと示す）を用いてTP41-1ペプチドの分解実験を行った結果（Lot.１）を示す図である。
【図２４】ＵＡ−１５−Ｌ（図中では、Ｌと示す）およびＵＡ−１５−Ｈ（図中では、Ｈと示す）を用いてTP41-1ペプチドの分解実験を行った結果（Lot.２）を示す図である。
【図２５】ＵＡ−１５−Ｌ（図中では、Ｌと示す）を用いて、１５℃、２５℃、３７℃の各温度でTP41-1ペプチド（図中では、ＴＰと記す）の分解実験を行った結果を示す図である。なお、この分解実験は、図２３に結果を示す実験を行った後に、TP41-1ペプチドを再度加え、反応温度の違いを見たものである。
【図２６】ＵＡ−１５−Ｌを用いたTP41-1ペプチド分解反応の速度論的解析の結果を示す図である。
【図２７】ＥＣＬ２Ｂ−４−Ｌの可変領域の立体構造を示す模式図である。
【図２８】ＥＣＬ２Ｂ−４−Ｈの可変領域の立体構造を示す模式図である。
【図２９】ＥＣＬ２Ｂ−４−Ｌの可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図である。
【図３０】ＥＣＬ２Ｂ−４−Ｈの可変領域のアミノ酸配列（一次構造）とそれをコードする塩基配列を示す図である。
【図３１】ＥＣＬ２Ｂ−４−Ｌ（図中では、Ｌと示す）およびＥＣＬ２Ｂ−４−Ｈ（図中では、Ｈと示す）を用いてECL2Bペプチドの分解実験を行った結果を示す図である。
【図３２】（ａ）は、ＥＣＬ２Ｂ−４−Ｌを用いたECL2Bペプチドの分解実験において、ＨＰＬＣを使用してECL2Bの濃度をモニタリングした結果を示す図である。（ｂ）は、ＥＣＬ２Ｂ−４−Ｈを用いたECL2Bペプチドの分解実験において、ＨＰＬＣを使用してECL2Bの濃度をモニタリングした結果を示す図である。（ｃ）は、ECL2Bペプチドの分解実験のコントロールとして、ＨＰＬＣを使用してECL2Bの濃度をモニタリングした結果を示す図である。
【図３３】酵素分解実験における速度論的解析結果を示す図である。（ａ）は、基質（ＥＣＬ２Ｂペプチド）濃度と分解速度との関係を示す図であり、（ｂ）は、Hanes-Woolfプロットの結果を示す図である。
【図３４】ＥＣＬ２Ｂ−４−Ｌ（図中では、Ｌと示す）およびＥＣＬ２Ｂ−４−Ｈ（図中では、Ｈと示す）を用いてTP41-1（図中では、ＴＰと示す）ペプチドの分解実験を行った結果を示す図である。
【図３５】（ａ）は、ＥＣＬ２Ｂ−４−Ｌを用いたTP41-1ペプチドの分解実験において、ＨＰＬＣを使用してTP41-1の濃度をモニタリングした結果を示す図である。（ｂ）は、ＥＣＬ２Ｂ−４−Ｈを用いたTP41-1ペプチドの分解実験において、ＨＰＬＣを使用してTP41-1の濃度をモニタリングした結果を示す図である。（ｃ）は、TP41-1ペプチドの分解実験のコントロールとして、ＨＰＬＣを使用してTP41-1の濃度をモニタリングした結果を示す図である。
【図３６】酵素分解実験における速度論的解析結果を示す図である。（ａ）は、基質（TP41-1ペプチド）濃度と分解速度との関係を示す図であり、（ｂ）は、Hanes-Woolfプロットの結果を示す図である。
【図３７】触媒三つ組残基様構造とgermline geneに関して、ＰＤＢデータを使って解析を行った結果を示す図である。
【図３８】触媒三つ組残基様構造とgermline geneに関して、本発明者らが所有する株について解析を行った結果を示す図である。
【符号の説明】
１０ 抗体構造解析システム
１１ 入力部
１２ 表示部
１３ 印刷部
１４ 記憶部
１５ 制御部
１６ 通信部
６０ 通信回線
６１ パーソナルコンピューター（ＰＣ）
６２ サーバー
１５１ 立体構造予測部
１５２ 触媒三つ組残基構造確認部[0001]
BACKGROUND OF THE INVENTION
INDUSTRIAL APPLICABILITY The present invention can be applied to a novel antibody enzyme production method for producing an antibody enzyme having both high molecular recognition ability and enzyme activity of an antibody with high efficiency, an antibody enzyme produced by this production method, and this production method. The present invention relates to an antibody enzyme having a sterically specific structure.
[0002]
[Prior art]
In recent years, various reports have been made on antibodies having enzyme-like activity, that is, antibody enzymes. For example, S. Paul et al. Have reported that autoantibodies isolated and purified from autoimmune disease patients have the activity of degrading VIP (neuropeptide: Vasoactive Intestinal Peptide) (Non-patent Document 1). In addition, Gabibov et al. Have reported that antibodies that enzymatically degrade DNA exist in autoantibodies isolated from patients with autoimmune diseases such as SLE (Systemic lupus erythematosus) and immune cell proliferative diseases (non-patented). Document 2 and Non-Patent Document 3).
[0003]
The present inventors have also obtained a monoclonal antibody whose antigen is a constant region of the outer membrane protein gp41 of AIDS virus (HIV) that causes AIDS (acquired immune deficiency syndrome), and details of the function of this monoclonal antibody. Was analyzed. As a result, it has been reported that the light chain region of this monoclonal antibody has very high activity that specifically degrades the outer membrane protein gp41 of HIV (Non-patent Document 4).
[0004]
Thus, since an antibody enzyme has both high molecular recognition ability and enzyme activity of an antibody, it is expected to be applied in many fields such as medical, chemical and food industries.
[0005]
Until now, as a method for producing and obtaining such an antibody enzyme, an animal or the like is immunized with a peptide or protein in a ground state, and the enzyme activity of all the obtained antibodies is measured to obtain the desired enzyme activity. Only the method of selecting and obtaining only the antibody having the above has been known.
[0006]
[Non-Patent Document 1]
Paul, S., et al., Science, 244: 1158, 1989.
[0007]
[Non-Patent Document 2]
Shuster, AM, et al., Science, 256: 665, 1992.,
[0008]
[Non-Patent Document 3]
Gabibov, AG, et al., Appl. Biochem. Biotech., 47: 293, 1994.
[0009]
[Non-Patent Document 4]
Hifumi, E., Okamoto, Y., Uda, T., J. Biosci. Bioeng.
88 (3), 323-327 (1999)
[0010]
[Problems to be solved by the invention]
However, the conventional methods for preparing and obtaining antibody enzymes as described above require a lot of labor and time, and the probability that an antibody enzyme can be found even if the enzyme activity of all antibodies is measured. Was less than 10%, and there was a problem that it was very inefficient.
[0011]
Thus, a method for efficiently obtaining or producing an antibody enzyme has not yet been found. For this reason, it is highly expected that an antibody enzyme having an excellent function, an efficient antibody enzyme acquisition method and a production method will be developed and used for further research and industry.
[0012]
The present invention has been made in view of the above-mentioned problems, and the object thereof is obtained by a method for efficiently producing an antibody enzyme having both high molecular recognition ability and enzyme activity of an antibody, and this production method. It is an object of the present invention to provide a novel antibody enzyme and an antibody enzyme having a sterically specific structure applicable to this production method.
[0013]
[Means for Solving the Problems]
As a result of intensive studies in view of the above problems, the present inventors have analyzed in detail the structure and function of an antibody having an enzymatic activity that cleaves and / or degrades a polypeptide or an antigen protein. A catalytic triad residue is also present in the three-dimensional structure, and it has been uniquely found that this catalytic triad residue and polypeptide degrading activity are related, and the present invention has been completed.
[0014]
In order to solve the above problems, an antibody enzyme production method according to the present invention includes a three-dimensional structure prediction step for predicting a three-dimensional structure of an antibody from an amino acid sequence, and a serine residue in the predicted three-dimensional structure of the antibody. An antibody structure analysis step of performing a catalytic triad residue structure confirmation step for confirming the presence of a catalytic triad residue structure in which an aspartic acid residue and a histidine residue or a glutamic acid residue are close in structure. It is characterized by including.
[0015]
In the catalyst triad residue structure confirmation step, it is preferable to use one of the following indicators in order to confirm the catalyst triad residue structure.
Indicator of type (1) or (1) *: having the same structure as the catalytic triad residue structure common to known antibody enzymes (type (1)), or the above known catalytic triad residue structure Among them, it has a structure presumed that a catalytic triad residue structure can be constituted by using amino acid residues at positions different by one residue (type {circle around (1)}).
Index of type (2): Serine residue, aspartic acid residue, and histidine residue or glutamic acid residue are present within the range of 3 to 20% in the three-dimensional structure.
Indicator of type {circle around (3)}: Use as an indicator that the antibody has a germ cell genotype derived from a known antibody enzyme.
Index of type (4): The hypervariable region 1 (CDR1) is composed of 16 amino acid residues, and the histidine residue at the 93rd position in the KABAT classification is used as an index.
Index of type (4) *: The hypervariable region 1 (CDR1) is composed of 11 amino acid residues, and has an histidine residue at the 91st or 55th in the Kabat classification. Use.
[0016]
The antibody enzyme production method preferably includes an antibody production step in which an antibody is produced by a hybridoma obtained by fusing mouse spleen lymphocytes immunized with an antigen and mouse myeloma cells. As the antigen, a hapten-conjugated protein obtained by binding a substance serving as an antigen determinant to a carrier protein can be used, but it is not particularly limited. Needless to say, the protein itself can also be used as an immunogen.
[0017]
Furthermore, the antibody enzyme production method includes a catalytic triple residue structure introduction step of introducing a catalytic triple residue structure into an antibody by genetic engineering techniques using the three-dimensional structure information obtained after the antibody structure analysis step. May be.
[0018]
The catalyst triad residue structure was newly found by the present inventors as a feature of an antibody enzyme having an activity of cleaving and / or degrading a polypeptide or an antigen protein. Therefore, according to the above method, since the antibody is screened using the catalytic triad residue structure specific to the antibody enzyme, the antibody enzyme can be produced more efficiently than before.
[0019]
The antibody enzyme according to the present invention has a catalytic triad residue structure in which a serine residue, an aspartic acid residue, a histidine residue or a glutamic acid residue are present in close proximity in the three-dimensional structure. Examples of the antibody enzyme include i41SL1-2 antibody, i41-7 antibody heavy chain, light chain, and the like, as shown in Examples below. It has been experimentally confirmed that the heavy and light chains of the i41SL1-2 antibody and the i41-7 antibody exhibit enzyme activity while having the property of an antibody having high affinity for the substrate.
[0020]
The antibody enzyme according to the present invention may also be an antibody enzyme in which the hypervariable region 1 (CDR1) is composed of 16 amino acid residues and has the 93rd histidine in the Kabat classification. Alternatively, it may be an antibody enzyme in which hypervariable region 1 (CDR1) is composed of 11 amino acid residues and has histidine at the 91st or 55th position in the KABAT classification. In addition, the mouse germ cell genotype is classified as Thiebe et al., Bb1, bl1, bd2, cr1, cs1, bj2, hf24, 12-41, 19-14, 19-17, 19-23, It may be produced by a gene selected from 19-25 and 21-12.
[0021]
As shown in the examples described later, as a result of analyzing a gene of a mouse-derived antibody enzyme having an activity of cleaving and / or degrading a polypeptide or an antigen protein, many of the antibody enzymes are derived from Thie et al. According to the mouse germ cell gene selected by classification from bb1, bl1, bd2, cr1, cs1, bj2, hf24, 12-41, 19-14, 19-17, 19-23, 19-25, 21-12 It has been experimentally confirmed that it has been produced. Therefore, mouse germ cell genes selected from the above bb1, bl1, bd2, cr1, cs1, bj2, hf24, 12-41, 19-14, 19-17, 19-23, 19-25, 21-12 Is basically an antibody enzyme having a catalytic triad residue structure in its three-dimensional structure, and has an activity of cleaving and / or degrading a polypeptide or an antigen protein. It is.
[0022]
The antibody enzyme according to the present invention may be an antibody heavy chain or a light chain.
[0023]
More specifically, the antibody enzyme according to the present invention includes an amino acid sequence represented by any one of SEQ ID NOs: 1, 3, 5, and 7, or SEQ ID NOs: 1, 3, 5, and 7. Among the amino acid sequences shown in any one, mention may be made of those comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added.
[0024]
The present invention also includes a gene encoding the above antibody enzyme. Furthermore, in the present invention, a computer program that allows a computer to execute an antibody structure analysis step in the above antibody enzyme production method and a computer program that records a computer program that causes a computer to execute the above antibody structure analysis step can be read. Recording media are also included.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.
[0026]
The present invention proposes an antibody enzyme production method that not only enables efficient acquisition of a natural antibody enzyme, but also can produce an antibody enzyme using a genetic engineering technique. An example of a novel and useful antibody enzyme obtained by this production method is provided. Therefore, in the following, the antibody enzyme production method according to the present invention will be described, and then an example of the obtained antibody enzyme and its gene, the function of the antibody enzyme, its utilization method, etc. will be described.
[0027]
(1) Antibody enzyme and catalytic triad structure
As a result of detailed analysis of the characteristics of the properties and structures of several types of antibody enzymes having the activity of cleaving and / or degrading polypeptides and antigen proteins, the present inventors cleaved polypeptides and antigen proteins. It was revealed for the first time that any antibody enzyme having an activity of degrading has a catalytic triad residue structure in its three-dimensional structure.
[0028]
Specifically, having a catalytic triad residue structure in its three-dimensional structure means that a serine residue, an aspartic acid residue, a histidine residue or a glutamic acid residue is three-dimensional in the three-dimensional structure of an antibody or antibody fragment. More specifically, it means that the distance between serine residue, aspartic acid residue, histidine residue or glutamic acid residue is within a range of at least 3 to 20 mm. Preferably, it may be within the range of 3 to 10 mm. This is because the catalytic triad residue structure and the substrate (polypeptide or antigen protein) are sufficient if the distance between the functional groups constituting the catalytic triad residue structure is in the range of 3 to 20 mm, particularly 3 to 10 mm. This is because it is considered that can react.
[0029]
In the antibody enzyme according to the present invention, the site where the catalytic triad residue structure is present is not particularly limited. The antibody is composed of a heavy chain (H chain) and a light chain (L chain). The light chain is composed of a variable region (VR) and a constant region (CR), and the variable region has a hypervariable region (CDR). As shown in the examples described later, the catalytic triad residue structure is mainly present in the light chain, and although it is not as heavy as the light chain in the heavy chain, it is clear that there is a catalytic triad residue structure. It has become. In addition, it has been experimentally revealed that an antibody having a catalytic triad residue structure in a three-dimensional structure has an enzyme activity that cleaves and / or degrades a polypeptide or an antigen protein.
[0030]
Therefore, in the antibody enzyme production method according to the present invention, it is only necessary to determine whether or not the antibody has a catalytic triad residue structure.
[0031]
(2) Overview of antibody enzyme production method
The antibody enzyme production method according to the present invention includes at least a three-dimensional structure prediction step for predicting a three-dimensional structure of an antibody from an amino acid sequence, and whether or not the catalyst triad residue structure is present in the predicted three-dimensional structure of an antibody. It is only necessary to include an antibody structure analysis step for performing a catalyst triad residue structure confirmation step for determining whether or not. As is clear from the findings of (1) above, if the three-dimensional structure of the antibody contains a catalytic triad residue structure, the possibility that the antibody is an antibody enzyme increases. Therefore, this allows efficient screening of antibody enzymes.
[0032]
More specifically, an example of the antibody enzyme production method according to the present invention includes an antibody production step, an amino acid sequence determination step, an antibody structure analysis step, and an antibody enzyme activity confirmation step, as shown in FIG. Just go out.
[0033]
(2-1) Antibody production process
The antibody production step is not particularly limited, and it is sufficient that a monoclonal antibody is produced by a hybridoma obtained by fusing mouse spleen lymphocytes immunized with an antigen and mouse myeloma cells. Alternatively, an antibody obtained from the library using the phage display method may be used.
[0034]
Specifically, antibodies can be produced by using a desired antigen, a fragment containing an antigenic determinant (epitope) thereof, a derivative thereof, an analog thereof, or a cell expressing them as an immunogen. . These include normal immunization or hybridoma method (Kohler, G. and Milstein, C., Nature 256,495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor, Immunology Today 4, 72 (1983) ) And the EBV-hybridoma method (Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 77-96 (1985)).
[0035]
Here, the antigen is not particularly limited as long as it is a polypeptide, protein, or polysaccharide.
[0036]
Specifically, if the antigen is a hapten, the antibody cannot be produced because it does not have the ability to induce antibody production or the like, but the carrier is made of a biopolymer such as a protein derived from a different species. Antibody production can be induced by obtaining an antigenic protein and immunizing with the protein. The carrier is not particularly limited, and various proteins conventionally known in this field such as ovalbumin, γ globulin, and hemocyanin can be suitably used.
[0037]
Here, the “polypeptide” includes a short peptide in which several amino acids are bonded, and a protein. Polypeptides include those naturally occurring and those synthesized by chemical and biological methods. The term “polysaccharide” as used herein refers to a combination of a plurality of so-called monosaccharides such as glucose, maltose, acetylglucosamine, and glucuronic acid, and the types of monosaccharides constituting the “polysaccharide” are conventional. It may be a known one, and is not particularly limited.
[0038]
(2-2) Amino acid sequence determination step
The amino acid sequence determination step is not particularly limited as long as the amino acid sequence can be determined from the antibody obtained in the antibody production step. Specifically, the amino acid sequence may be determined by directly reading the amino acid sequence from the obtained antibody, or the amino acid sequence may be deduced from the base sequence of the antibody gene. The amino acid sequence may be determined using the Edman method. The base sequence may be determined using a conventionally known sequencer, and the amino acid sequence may be estimated using a conventionally known software or the like, and is not particularly limited.
[0039]
(2-3) Antibody structure analysis process
The antibody structure analysis step is not particularly limited as long as it includes the three-dimensional structure prediction step and the catalyst triad residue structure confirmation step as described above. This makes it possible to efficiently confirm whether or not the desired antibody contains a catalytic triad residue structure.
[0040]
The three-dimensional structure prediction step is not particularly limited as long as the three-dimensional structure can be predicted from the amino acid sequence data obtained in the amino acid sequence determination step. Specifically, it is only necessary to be able to predict secondary structures and tertiary structures in proteins. That is, in the present invention, it is only necessary to clarify the tertiary structure of an IgG class antibody.
[0041]
Examples of the quaternary structure in an antibody include a structure in which a plurality of subunits (IgG class antibodies) having a tertiary structure are associated to form an oligomer, such as the IgA and IgM classes. In the present invention, there is no need to make a prediction because it is not particularly necessary in the confirmation stage of the catalyst triad residue structure. However, this is not limited to the case where the analysis result including the result of the quaternary structure can be effectively used for the production of the antibody enzyme, and the quaternary structure of the antibody may be predicted at the three-dimensional structure prediction stage.
[0042]
When performing the above-mentioned three-dimensional structure prediction step, conventionally known software for higher order structure analysis may be used, and the specific method is not particularly limited.
[0043]
The catalyst triad residue structure confirmation step is not particularly limited as long as it can be determined whether or not the catalyst triad residue structure described in (1) exists in the three-dimensional structure of the antibody. . Therefore, in the determination of the catalyst triad residue structure, the presence or absence of the above structure may be determined directly or indirectly.
[0044]
Examples of direct indicators used for the determination of the catalyst triad residue structure include the following indicators. In addition, as a preliminary step to determine using each of the following indices, the presence or absence of the above-mentioned catalytic triad residue structure by a simpler method of examining whether or not a histidine residue is present in the amino acid sequence of an antibody The determination may be made. This is because many antibodies usually contain less histidine residues than serine residues.
[0045]
The first index is an amino acid residue having the same structure as a catalytic triad residue structure common to known antibody enzymes, or an amino acid residue at a position different from that of the known catalytic triad residue structure by one residue. Is used as an index, and the former is referred to as type (1) and the latter as type (1) *.
[0046]
The second indicator is that in the antibody heavy chain (H chain) or antibody light chain (L chain) alone, the steric structure has a serine residue, aspartic acid residue, histidine residue or A case where each functional group with a glutamic acid residue is present is used as an index, and this is referred to as an index of type (2).
[0047]
Next, an indirect index used for determining the catalyst triad residue structure in the catalyst triad residue structure confirmation step is not particularly limited, but in the present embodiment, the germ cell type gene ( An index using the analysis result of germline gene) can be preferably used.
[0048]
Antibody light chains (L chains) are classified into two classes, κ and λ. In the case of mice, about 95% are said to be κ chains. In addition, mouse κ chain has the advantage that germline gene analysis is progressing and information on this is easily available. Here, as will be described in the examples described later, by analyzing germline, important findings suggesting that an antibody L chain having enzyme activity has already been prepared for a specific germline have been found. (See Example 3 and Table 3). Therefore, if a germline from which a known antibody enzyme is derived is examined in advance and an antibody having this germline is selected, the antibody enzyme can be efficiently obtained with high probability for the light chain. This is the index of type (3).
[0049]
Specific examples of germlines that can be used for the light chain include bb1, cr1, bl1, cs1, bd2, bj2, or 19-25 according to the classification of Thiebe et al. In the present embodiment and examples, germline is represented by Thiebe et al.
[0050]
For heavy chains (H chains), if the light chain concept is applied, antibody antibodies with VH1, VH5, and VH10 Family can be selected to efficiently obtain antibody enzymes with a considerable probability. Is possible. In addition, there are those that mutate during the maturation process of the antibody and lose the catalytic triad residue structure.Except for these, when specified in the above germline or family, antibodies with almost completely enzymatic activity are included. Can be acquired.
[0051]
In addition, a structurally characteristic structure obtained from a known antibody enzyme can be used as an index. Specifically, as will be described later in the Examples, the hypervariable region 1 (CDR1) is composed of 16 amino acid residues, and has 93th histidine residue in the Kabat classification. This characteristic is remarkable for antibody enzymes. Therefore, this can be used as an indicator of type (4). Furthermore, the hypervariable region 1 (CDR1) is composed of 11 amino acid residues, and is characterized by having a histidine residue at the 91st or 55th position in the Kabat classification (KABAT). This can be used as an indicator of type (4) *. In the present embodiment and examples, the amino acid sequence of an antibody is indicated by the classification of Kabat.
[0052]
In addition, since more reliable indicators are the above type (1) / (1) * or type (2), it is preferable to use these indicators in order to increase the certainty at the time of selection. The indicators of types (4) and (4) * may be used supplementarily. However, instead of using the indicators of types (1), (1) *, and (2), only indicators of types (3), (4), and (4) * can be used alone or in combination.
[0053]
As a means for performing the antibody structure analysis step including the catalyst triad residue structure confirmation step, in the present embodiment, the antibody structure analysis system described in (3) described later can be cited. It is not limited to.
[0054]
Further, when the three-dimensional structure of an antibody is predicted directly from the nucleotide sequence data of a gene by an antibody structure analysis system, the amino acid sequence determination step may be included as one step of the antibody structure analysis step. That is, as described in the amino acid sequence determination step, the amino acid sequence determination step for estimating the amino acid sequence from the base sequence of the antibody gene may be performed as one step of the antibody structure analysis step.
[0055]
(2-4) Antibody enzyme activity confirmation step
In the antibody enzyme activity confirmation step, as a result of the analysis in the antibody structure analysis step, for the antibody having a catalytic triad residue structure and highly likely to be an antibody enzyme, the polypeptide or antigen protein is actually cleaved and / or Or if it comes to confirm whether it has the activity which decomposes | disassembles, it will not specifically limit. Specifically, a polypeptide or antigen protein containing a structure that becomes an antigen or antigenic determinant may be reacted with an antibody to observe the activity. The reaction conditions at this time, for example, antigen concentration, antibody concentration, reaction temperature and reaction time, salt concentration (type of buffer to be used) and the like are not particularly limited.
[0056]
(2-5) Catalyst triad residue structure introduction step
As another example of the antibody enzyme production method according to the present invention, as shown in FIG. 1 (b), a catalyst triad residue structure introduction step may be included. That is, even an antibody that does not originally have a catalytic triad residue structure can be produced with high efficiency by introducing a catalytic triad residue structure by genetic engineering. Therefore, a catalytic triad residue structure introducing step may be performed after the antibody structure analyzing step.
[0057]
In the catalyst triad residue structure introduction step, it is sufficient that the catalyst triad residue structure is introduced into the antibody by genetic engineering techniques using the three-dimensional structure information obtained in the antibody structure analysis step. The specific method is not particularly limited. Specifically, a conventionally known method such as a method of creating a deletion mutant using exonuclease or site-directed mutagenesis can be preferably used.
[0058]
The catalyst triad residue structure introduction step may be performed after the antibody structure analysis step. However, after the catalyst triad residue structure introduction step, the antibody enzyme activity confirmation step is performed, and the catalyst triad residue structure is introduced by introducing the catalyst triple residue structure. It is very preferable to determine whether or not the activity of cleaving and / or degrading peptides and antigenic proteins has been imparted.
[0059]
(3) System for executing antibody structure analysis process
(3-1) Example of specific configuration of antibody structure analysis system
The means for performing the antibody structure analysis step in the present invention may be an antibody structure analysis system using a computer. For example, as shown in FIG. 2, an antibody structure including an input unit 11, a display unit 12, a printing unit 13, a storage unit 14, a control unit 15, a three-dimensional structure prediction unit 151, and a catalytic triplet residue structure confirmation unit 152 The analysis system 10 can be mentioned.
[0060]
The input unit 11 is not particularly limited as long as it can input information related to the operation of the antibody structure analysis system 10, and a conventionally known input unit such as a keyboard, a tablet, or a scanner is preferably used. be able to.
[0061]
The display unit 12 displays various information such as information related to the operation of the antibody structure analysis system 10 and selection results including the predicted three-dimensional structure of the antibody and the confirmation result of the catalytic triad residue structure. Specifically, various display devices such as a known CRT display and a liquid crystal display are preferably used, but are not particularly limited.
[0062]
The printing unit 13 records (printing / image forming) various information that can be displayed on the display unit 12 on a recording material such as PPC paper. Specifically, known image forming apparatuses such as an ink jet printer and a laser printer are preferably used, but are not particularly limited.
[0063]
The display unit 12 and the printing unit 13 can be collectively expressed as output means. That is, the display unit 12 is a unit that outputs various types of information by soft copy, and the printing unit 13 is a unit that outputs various types of information by hard copy. Therefore, the output means used in the present invention is not limited to the display unit 12 and the printing unit 13 and may include other output means.
[0064]
The storage unit 14 stores various information (control information, selection results, other information, etc.) used in the antibody structure analysis system 10. Specifically, for example, semiconductor memories such as RAM and ROM, magnetic disks such as floppy (registered trademark) disks and hard disks, optical disk systems such as CD-ROM / MO / MD / DVD, IC cards (memory cards) Conventionally, various known storage means such as a card system such as an optical card can be suitably used.
[0065]
The storage unit 14 may be integrated with the antibody structure analysis system 10 to be a single device, or may be a separate external storage device. The integrated storage unit 14 and the external storage device may be provided. For example, the integrated storage unit 14 includes a built-in hard disk, a floppy (registered trademark) disk drive, a CD-ROM drive, a DVD-ROM drive and the like incorporated in the apparatus. Examples include an attached hard disk and the above-mentioned various disk drives.
[0066]
The control unit 15 controls the operation of the antibody structure analysis system 10. Specifically, as indicated by solid arrows in FIG. 2, each means of the input unit 11, display unit 12, printing unit 13, storage unit 14, three-dimensional structure prediction unit 151, and catalyst triplet residue structure confirmation unit 152 is used. In contrast, control information is output from the control unit 15. The antibody structure analysis system 10 as a whole operates by the above-described means operating in cooperation with each other based on this control information. In addition, since instruction information for operating the antibody structure analysis system 10 can be input from the input unit 11 to the control unit 15, in FIG. 2, the solid line arrows indicating the exchange of control information are both It is suitable.
[0067]
The three-dimensional structure prediction unit 151 and the catalyst triple residue structure confirmation unit 152 collectively function as analysis means. Specifically, the amino acid sequence data input from the input unit 11 is input to the three-dimensional structure prediction unit 151 via the control unit 15 (broken line in the figure). Then, the three-dimensional structure data generated by the three-dimensional structure prediction unit 151 is output to the catalyst triplet residue structure confirmation unit 152 (broken line in the figure). The catalyst triple residue structure confirmation unit 152 confirms the presence or absence of the catalyst triple residue structure from the three-dimensional structure data, and generates final analysis data based on the confirmation result (broken line in the figure). The analysis data is not particularly limited as long as it can be output by output means such as the display unit 12 and / or the printing unit 13.
[0068]
The data used for the analysis of the three-dimensional structure in the three-dimensional structure prediction unit 151 may be at least amino acid sequence data, but may be base sequence data. In this case, the three-dimensional structure predicting unit 151 only needs to be able to predict an amino acid sequence from base sequence data and generate a three-dimensional structure based on the data.
[0069]
Specific configurations of the control unit 15, the three-dimensional structure prediction unit 151, and the catalyst triplet residue structure confirmation unit 152 are not particularly limited, and conventionally known calculation means are preferably used. Although each said means may be an independent calculating means, Preferably, the said each means is a control analysis apparatus integrated as one calculating means. Specifically, a central processing unit (CPU) of a computer is arranged, and its operation is very preferable if it is configured to be executed according to a computer program.
[0070]
Therefore, the three-dimensional structure predicting unit 151 may be configured such that the CPU executes a calculation for predicting the three-dimensional structure according to a conventionally known computer program for predicting the three-dimensional structure of a protein. The confirmation unit 152 only needs to be able to execute an operation for determining whether or not the catalyst triad residue structure exists at least according to the computer program. In addition, although not shown, the antibody structure analysis system 10 includes other analysis means, and the analysis data may include data other than the catalytic triad residue structure and the three-dimensional structure.
[0071]
Further, as shown in FIG. 2, the antibody structure analysis system 10 may include a communication unit 16 so that various types of information can be input / output via a communication network including the Internet. The communication unit 16 is connected to a communication network and can transmit and receive various types of information. In FIG. 2, the antibody structure analysis system 10, personal computer (PC) 61, and server 62 on the same premises are connected to a communication line 60 to form a bus-type LAN (local area network). A LAN is also connected to a PC 61 in another area via the Internet.
[0072]
The specific configuration of the communication unit 16 is not particularly limited, and a known LAN card, LAN board, LAN adapter, modem, or the like can be suitably used.
[0073]
As the PC 61, a known personal computer equipped with a communication means such as a modem can be suitably used, and the PC 61 is not limited to a desktop type or a notebook type. The PC 61 has a basic configuration including a display unit such as a CRT display or a liquid crystal display and an input unit such as a keyboard or a mouse. For convenience of explanation, a display unit and an input unit (not shown) provided in the PC 61 are expressed as a PC display unit / PC input unit.
[0074]
The PC 61 may be provided with hardware (for example, various input means such as a scanner and various output means such as a printer) that can be externally attached to a general personal computer.
[0075]
The specific configuration of the server 62 is not particularly limited as long as it is a computer that can provide services to the PC 61 and the antibody structure analysis system 10 that are clients constituting the LAN. Further, the server 62 may also serve as a database server or a file server.
[0076]
The specific configuration of the communication line 60 is not particularly limited, and a conventionally known general communication line can be used. Also, the type of LAN constructed using this communication line 60 is not limited to the bus type, and may be a conventionally known type such as a star type or a ring type.
[0077]
Although not shown, the LAN may include other terminals such as a shared printer. In addition, although not shown, the communication network including the LAN may include various portable terminals capable of communication.
[0078]
In the network configured as described above, for example, after the antibody structure analysis process is performed in the antibody structure analysis system 10, the selection result is simply output within the antibody structure analysis system 10 (that is, the display unit 12 or the printing unit 13). Alternatively, it can be transmitted to the PC 61 via the LAN. In the PC 61, the results obtained from the antibody structure analysis system 10 can be displayed on a PC display unit or printed by a printer, and further, the above selection results can be processed by input from the PC input unit. it can.
[0079]
That is, the communication unit 16 functions not only as a communication unit but also as an input unit of the antibody structure analysis system 10. In particular, information (amino acid sequence, etc.) related to the execution of the antibody structure analysis process is transmitted or selected from a location remote from the location where the antibody structure analysis system 10 is located using the PC 61 via the Internet. Or the like, it is possible to provide a service for providing an antibody structure analysis process to an arbitrary customer.
[0080]
Further, when the PC 61 is connected to the antibody structure analysis system 10 via a LAN, for example, if there is one antibody structure analysis system 10 in a research facility or the like, other researchers can connect an information terminal such as the PC 61 or the like. Thus, the antibody structure analysis system 10 can be shared. Therefore, the present invention can be implemented more efficiently.
[0081]
Further, when the server 62 also serves as a database server or a file server, the selection result of the antibody structure analysis process performed via the communication network may be accumulated in the server 62 via the communication network. it can. As a result, the selection result can be used more effectively.
[0082]
In addition, in the present invention, the antibody structure analysis step in the present invention can be carried out by a program on a computer, and the recording medium for recording the program is downloaded from a communication network. A medium that fluidly carries the program is also included. For example, if an antibody structure analysis process program is recorded in the recording means of the server 62, the antibody structure analysis system 10 downloads and uses the antibody structure analysis process program from the server 62 as appropriate. Also good. However, when the antibody structure analysis system 10 downloads a program from the communication network, the download program is stored in advance in the body of the antibody structure analysis system 10 or installed from another recording medium. It has become.
[0083]
Furthermore, when connected to the server 62 via a communication network like the PC 61, the PC 61 itself can be used as the antibody structure analysis system 10 by downloading the antibody structure analysis process program from the server 62. Can do.
[0084]
(3-2) An example of antibody structure analysis process executed by the antibody structure analysis system
Next, a specific operation of the antibody structure analysis system 10, that is, an example of an antibody structure analysis step in the present invention will be described based on the flowchart of FIG.
[0085]
First, as a previous step, the base sequence of the monoclonal antibody obtained by the antibody production process is determined to finally obtain the amino acid sequence. Next, as step 1 (hereinafter, step is abbreviated as S), the amino acid sequence of the variable region is input from the input unit 11 among the amino acid sequences. Next, as S <b> 2, an index for confirming the catalyst triple residue structure by the catalyst triple residue structure confirmation unit 152 is set. Examples of the index include the types {circle around (1)}, {circle around (1)}, {circle around (2)}, {circle around (3)}, {circle around (4)}, and {circle around (4)} described in (2-3) above. These indicators may be used alone or in combination.
[0086]
Next, as S3, the three-dimensional structure prediction unit 151 predicts the three-dimensional structure of the antibody using the amino acid sequence data of the input variable region. Next, as S4, the catalyst triplet residue structure confirmation unit 152 uses the amino acid sequence data and the index set in S2 from the three-dimensional structure data obtained by the three-dimensional structure prediction unit 151. Analyze the arrangement of amino acid residues presumed to construct a triple residue structure and the state of mutation.
[0087]
If the structure of the catalytic triad or its mutant structure is confirmed from the analysis results, the antibody is likely to be an antibody enzyme. If none of the above structures are found, The antibody is unlikely to be an antibody enzyme. Next, in S5, final analysis data is generated from the analysis result in S4 and displayed on the display unit 12.
[0088]
Thereafter, in S6, it is determined whether or not the analysis of the catalyst triad residue structure is performed again. If it is performed again (YES in the figure), the process proceeds to S7, and it is determined whether or not the analysis is performed by changing the index using the data of the same amino acid sequence. In S7, when data of the same amino acid sequence is used (YES in the figure), the process returns to S2, and when data of a new amino acid sequence is input (NO in the figure), the process returns to S1.
[0089]
On the other hand, if the analysis of the catalyst triad residue structure is not performed again in S6 (NO in the figure), the process proceeds to S8, and if there are a plurality of obtained analysis data, is the printing unit 13 printed together? Alternatively, the data is transmitted to another PC 61 through the communication interface 16.
[0090]
The antibody structure analysis system 10 in the present embodiment described above is realized by a computer by a program for causing the antibody structure analysis method including the steps S1 to S8 described above to function. Also good.
[0091]
The program may be stored in a computer-readable recording medium. Specifically, the storage unit 14 illustrated in FIG. 3, specifically, for example, a ROM itself may be a program medium, or a program reading device is provided as the storage unit 14. May be a program medium that can be read by inserting a recording medium therein. As the program medium, a known configuration exemplified as a specific example of the storage unit 14 can be suitably used.
[0092]
In any case, the stored program may be configured to be accessed and executed by the control unit 15, or the program is read, and the read program is downloaded to a program storage area (not shown). A method of executing the program may be used. It is assumed that this download program is stored in advance in the storage unit 14 or the like. Further, the content stored in the recording medium is not limited to a program, and may be data, for example.
[0093]
As described above, the present invention includes a computer program that executes the above-described antibody structure analysis step on a computer, and a machine-readable recording medium that records the computer program that causes the computer to execute the program. Therefore, since the antibody structure analysis step of the present invention is executed by a computer using a program, the computer itself can be used as the antibody structure analysis system 10. As a result, the versatility of the present invention can be enhanced and the present invention can be easily used on a communication network.
[0094]
(4) An example of the antibody enzyme of the present invention
Next, the antibody enzyme according to the present invention includes an antibody enzyme having a sterically specific structure applicable to the above-described production method, and further includes an antibody enzyme produced by the above-described production method. . In the present embodiment, an example of the antibody enzyme according to the present invention will be described in detail below.
[0095]
(4-1) Antibody enzyme according to the present embodiment
The antibody enzyme according to the present embodiment includes (a) an antibody enzyme having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5 or 7, or (b) SEQ ID NO: 1, 3, 5 or 7 An antibody enzyme having a variable region consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown, and cleaves and / or cleaves a polypeptide or antigen protein Alternatively, an antibody enzyme having an activity of degrading may be used, but in the present embodiment, (1) i41SL1- having the amino acid sequence represented by SEQ ID NO: 1 and having a variable region consisting of 117 amino acid residues 2 heavy chain of antibody (i41SL1-2-H), (2) light chain of i41SL1-2 antibody having the amino acid sequence shown in SEQ ID NO: 3 and having a variable region consisting of 114 amino acid residues (i41SL1 -2-L), (3) array I41-7 heavy chain (i41-7-H) having the amino acid sequence shown in No. 5 and having a variable region of 120 amino acid residues, (4) amino acid sequence shown in SEQ ID NO: 7 And the light chain (i41-7-L) of i41-7 antibody having a variable region consisting of 109 amino acid residues.
[0096]
The i41SL1-2 antibody is a monoclonal antibody against the synthetic peptide (shown in SEQ ID NO: 9) of the light chain hypervariable region 1 (CDR1) of antibody 41S-2 that recognizes the constant region of the outer membrane protein gp-41 of HIV virus. . The amino acid sequences of the variable regions of the heavy and light chains of the i41SL1-2 antibody and the base sequences encoding them are shown in FIGS. 4 (a) and 4 (b).
[0097]
When the three-dimensional structures of the light chain (i41SL1-2-L) and heavy chain (i41SL1-2-H) of this i41SL1-2 antibody were predicted using a computer and analyzed, FIG. 5 (a) ( As shown in b), the i41SL1-2 antibody was found to have a catalytic triad residue structure on both i41SL1-2-H and i41SL1-2-L. That is, as shown in FIGS. 4 (b) and 5 (a), i41SL1-2-L is classified into kabat (KABAT) as the first aspartic acid residue (D1) and the 93rd histidine residue. A catalytic triad structure composed of a group (H93) and a 27A-th serine residue (S27a), or a 27D-th aspartic acid residue (D27d) and a 93-th group in the category of KABAT It is predicted to have a catalytic triad residue structure composed of the histidine residue (H93) and the 27A-th serine residue (S27a).
[0098]
As shown in FIGS. 4 (a) and 5 (b), i41SL1-2-H has a 55th aspartic acid residue (D55) and a 52nd histidine residue according to the classification of Kabat (KABAT). Catalytic triplet residue structure composed of a group (H52) and a 54th serine residue (S54), or a kabat (KABAT) classification, 100th aspartic acid (D100) and 35th histidine It is predicted to have a catalytic triad residue structure composed of a residue (H35) and a 98th serine residue (S98). The arrangement of these catalytic triad residues is near the hypervariable region in both the light and heavy chains.
[0099]
The i41-7 antibody is a monoclonal antibody against the light chain of antibody 41S-2. The amino acid sequence of the variable region of the heavy chain and light chain of the i41-7 antibody and the base sequence encoding it are shown in FIGS. 8 (a) and 8 (b).
[0100]
The three-dimensional structure of the light chain (i41-7-L) and heavy chain (i41-7-H) of this i41-7 antibody was predicted and analyzed using a computer in the same manner as the i41SL1-2 antibody. However, as shown in FIGS. 9 (a), (b) and (c), it is clear that the i41-7 antibody has a catalytic triad residue structure in both i41-7-L and i41-7-H. became. That is, as shown in FIGS. 8 (b) and 9 (a), i41-7-L is classified into kabat (KABAT), with the first aspartic acid residue (D1) and the 91st histidine residue. A catalytic triad residue structure composed of a group (H91) and a 26th serine residue (S26), or the 28th aspartic acid residue (D28) and 91st in the category of KABAT And a catalytic triad residue structure composed of the 30th (or 26th) serine residue (S30 or S26). The positions of these catalytic triad residue structures are in the vicinity of the hypervariable regions (CDRs) CDR1 and CDR3.
[0101]
In addition, as shown in FIGS. 8 (b) and 9 (b), i41-7-L is classified into Kabat (KABAT), with the 60th aspartic acid residue (D60) and the 55th histidine residue. It is predicted to have a catalytic triad residue structure composed of a group (H55) and the 52nd serine residue (S52). This is located in the vicinity of CDR2.
[0102]
As shown in FIGS. 8 (a) and 9 (c), i41-7-H is classified into Kabat (KABAT) as the 86th aspartic acid residue (D86) and the 41st histidine residue. It is predicted to have a catalytic triad residue structure composed of a group (H41) and a 40th (or 84 or 87) serine residue (S40 or S84 or S87).
[0103]
As another example of the antibody enzyme, there can be mentioned an antibody fragment of monoclonal antibody HpU-20 or UA-15 of urease of Helicobacter pylori (hereinafter referred to as HP urease).
[0104]
The antibody fragment of HpU-20 is specifically the variable region of the L chain of the HpU-20 antibody and the variable region of the H chain of the HpU-20 antibody. These two antibody enzymes are also composed of serine, histidine (or glutamic acid), and aspartic acid as a result of estimating the three-dimensional structure of a plurality of HP urease monoclonal antibodies by molecular modeling of the amino acid sequence of the variable region. It was found as an amino acid sequence (antibody fragment) that can constitute a catalytic triad residue.
[0105]
The variable region of the L chain of the HpU-20 antibody (hereinafter referred to as HpU-20-L), and the variable region of the H chain of the HpU-20 antibody (hereinafter referred to as HpU-20-H) are those of HP urease. Acts as a degrading enzyme. This is also evident from the result of degrading the β subunit of HP urease, as shown in the examples described below. The above-mentioned degraded peptide is one of the important regions for exhibiting the HP urease enzyme activity. Therefore, by decomposing this region, it is possible to completely destroy the function of HP urease and to make it impossible for HP bacteria to survive in the highly acidic human stomach. That is, the antibody enzyme of the present invention capable of degrading the peptide can efficiently sterilize HP bacteria.
[0106]
Subsequently, the structure of the HpU-20-L will be described in detail below. HpU-20-L is a variable region of the L chain of HpU-20, which is one of the HP urease monoclonal antibodies, as described above, and has the amino acid sequence shown in SEQ ID NO: 14 as a primary structure. FIG. 15 shows an example of the amino acid sequence of HpU-20-L and the base sequence of the gene encoding it in the lower part. In addition, this base sequence is a base sequence of the gene of the HpU-20 antibody L chain cloned in the present Example. In FIG. 15, the complementarity determining sites that form a complementary three-dimensional structure with the antigen molecule (HP urease) and determine the complementarity of the antibody are underlined as CDR-1, CDR-2, CDR-3. Show.
[0107]
FIG. 13 schematically shows a three-dimensional structure estimated as a result of molecular modeling of the amino acid sequence of HpU-20-L. As shown in FIG. 13, in the amino acid sequence shown in SEQ ID NO: 14, the first aspartic acid (indicated as D1 by Kabat (KABAT) classification in FIG. 13) and the 98th histidine (in FIG. 13 by Kabat classification). H93), the 97th serine (indicated as S92 by Kabat classification in FIG. 13) or the 28th serine (indicated by S27a by Kabat classification in FIG. 13) constitutes a catalytic triad residue. It is estimated to be. The germline of HpU-20-L is cr1.
[0108]
Next, the structure of HpU-20-H will be described in detail below. HpU-20-H is a variable region of the H chain of HpU-20, which is one of the HP urease monoclonal antibodies as described above, and has the amino acid sequence shown in SEQ ID NO: 16 as a primary structure. FIG. 16 shows an example of the amino acid sequence of HpU-20-H and the base sequence of the gene encoding it in the lower part. In addition, this base sequence is a base sequence of the gene of the HpU-20 antibody H chain cloned in the present Example. In FIG. 16, the complementarity determining sites are underlined as CDR-1, CDR-2, CDR-3.
[0109]
FIG. 14 schematically shows a predicted three-dimensional structure as a result of molecular modeling of the amino acid sequence of HpU-20-H. As shown in FIG. 14, in the amino acid sequence shown in SEQ ID NO: 16, the 90th aspartic acid (indicated as D86 by the classification of Kabat (KABAT) in FIG. 14), the 89th glutamic acid (in FIG. 14, by the classification of Kabat) E85) and the 88th serine (indicated as S84 by Kabat classification in FIG. 14) are presumed to constitute a catalytic triad residue.
[0110]
The above-mentioned antibody fragment of UA-15 is specifically the variable region of the L chain of the UA-15 antibody. As in the case of HpU-20-L and the like described above, this antibody enzyme was estimated from its three-dimensional structure by molecular modeling of the amino acid sequence of its variable region from among a plurality of HP urease monoclonal antibodies. It was found as an amino acid sequence (antibody fragment) capable of constituting a catalytic triad residue consisting of histidine and aspartic acid.
[0111]
The variable region of the L chain of the UA-15 antibody (hereinafter referred to as UA-15-L) acts as a degrading enzyme for HP urease. Then, by decomposing HP urease, it is possible to completely destroy the function of HP urease and to make it impossible for HP bacteria to survive in the highly acidic human stomach. That is, the antibody enzyme of the present invention capable of degrading the peptide can efficiently sterilize HP bacteria.
[0112]
Next, the structure of the UA-15-L will be described in detail below. UA-15-L is a variable region of the L chain of UA-15, one of the HP urease monoclonal antibodies, as described above, and has the amino acid sequence shown in SEQ ID NO: 18 as a primary structure.
[0113]
FIG. 22 schematically shows a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of UA-15 composed of a light chain and a heavy chain. In FIG. 22, the light chain is indicated by L and the heavy chain is indicated by H. As shown in FIG. 22, in the amino acid sequence shown in SEQ ID NO: 18, the first aspartic acid (referred to as Asp1 by the classification of Kabat (KABAT) in FIG. 22), the 28th serine (in FIG. 22, It is presumed that the 94th histidine (referred to as His90 in FIG. 22 according to the classification of Kabat) constitutes a catalytic triad residue according to the classification of Kabat.
[0114]
The variable region of the heavy chain of UA-15 has the amino acid sequence shown in SEQ ID NO: 20 as a primary structure, but the structure predicted to be a catalytic triad residue in this heavy chain of UA-15 is Since it does not exist, it has no activity as an antibody enzyme. The base sequence of the gene encoding the variable region of the heavy chain of UA-15 is also described as SEQ ID NO: 21.
[0115]
Still another example of the antibody enzyme is an antibody fragment of the monoclonal antibody ECL2B-4 of the chemokine receptor CCR-5. More specifically, the antibody fragment of ECL2B-4 includes a light chain variable region of ECL2B-4 (ie, ECL2B-4-L) and a heavy chain variable region of ECL2B-4 (ie, ECL2B). -4-H).
[0116]
These two antibody enzymes are obtained from a monoclonal antibody obtained by using a peptide constituting the extracellular region of the chemokine receptor CCR-5 as an immunogen. The two antibody enzymes are composed of catalytic triad residues consisting of serine, histidine (or glutamic acid), and aspartic acid as a result of estimating the three-dimensional structure by molecular modeling of the amino acid sequence of the variable region of the monoclonal antibody. It was searched as a possible amino acid sequence (antibody fragment).
[0117]
The antibody enzymes ECL2B-4-L and ECL2B-4-H also degrade the extracellular region peptide (peptide consisting of the amino acid sequence shown in SEQ ID NO: 27) of the chemokine receptor CCR-5, as shown in Examples below. It acts as a degrading enzyme for the chemokine receptor CCR-5.
[0118]
That is, the above-mentioned two antibody enzymes can completely degrade this CCR-5 and lose its function. Therefore, it is considered that the above antibody enzyme can be effectively used as an anti-HIV drug that prevents HIV infection or suppresses the progression of AIDS symptoms.
[0119]
Next, the structure of the ECL2B-4-L will be described in detail below. ECL2B-4-L is a variable region of the light chain of monoclonal antibody ECL2B-2 that uses the extracellular region peptide of CCR-5 as an immunogen, and has the amino acid sequence shown in SEQ ID NO: 23 as a primary structure.
[0120]
FIG. 27 schematically shows a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of ECL2B-4-L. As shown in FIG. 27, in the amino acid sequence shown in SEQ ID NO: 23, the first aspartic acid (in FIG. 27, indicated as D1 by the classification of Kabat (KABAT)), the 28th serine (in FIG. 27, It is presumed that the 31st histidine (indicated as H27d in FIG. 27 by the Kabat classification) constitutes a catalytic triad residue, according to the Kabat classification. In place of the 31st histidine, the 98th histidine (in FIG. 27, indicated as H93 by Kabat classification) may be used. In place of the 31st serine, the 28th (referred to as S27a in FIG. 27), 32nd (referred to as S27e in FIG. 27), and 97th (referred to as S92 in FIG. 27). Either of them may be used.
[0121]
Next, the structure of the ECL2B-4-H will be described in detail below. ECL2B-4-H is the variable region of the heavy chain of monoclonal antibody ECL2B-4 that uses the extracellular region peptide of CCR-5 as an immunogen, and has the amino acid sequence shown in SEQ ID NO: 25 as the primary structure.
[0122]
FIG. 28 schematically shows a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of ECL2B-4-H. As shown in FIG. 28, in the amino acid sequence shown in SEQ ID NO: 25, the 92nd aspartic acid (in FIG. 28, it is written as D86 according to the classification of Kabat (KABAT)), the 65th serine (in FIG. 28, It is presumed that the 48th glutamic acid (denoted as E46 according to the Kabat classification in FIG. 28) constitutes a catalytic triad residue according to the Kabat classification. Instead of the 92nd aspartic acid, it may be the 64th aspartic acid (denoted as D59 in FIG. 28 by Kabat classification). Further, instead of the 65th serine, the 90th serine (referred to as S84 in FIG. 28) may be used. In place of the 48th glutamic acid, the 91st glutamic acid (referred to as E85 in FIG. 28) may be used.
[0123]
(4-2) Gene according to the present embodiment
The gene according to the present embodiment is (a) a gene encoding an antibody enzyme having a variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, 3, 5 or 7, or (b) SEQ ID NO: 1, 3, 5 Or an antibody enzyme having a variable region consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in 7; Any gene may be used as long as it encodes an antibody enzyme having an activity of cleaving and / or degrading. In the present embodiment, (1) a gene having a variable region consisting of a base sequence (cDNA) represented by SEQ ID NO: 2 (i41SL1 -2-H gene), (2) gene having a variable region consisting of the base sequence (cDNA) shown in SEQ ID NO: 4 (i41SL1-2-L gene), (3) base sequence shown in SEQ ID NO: 6 a gene having a variable region consisting of (cDNA) (i41-7-H gene) and (4) a gene having a variable region consisting of the base sequence (cDNA) shown in SEQ ID NO: 8 (i41-7-L gene) Illustrated. These genes are derived from mice (Mus musculus).
[0124]
Examples of other genes according to the present embodiment include genes having the nucleotide sequences shown in SEQ ID NOs: 15 and 17. The gene consisting of the base sequence shown in SEQ ID NO: 15 is one of the base sequences of the gene (cDNA) encoding the variable region of the L chain of HpU-20, and the gene consisting of the base sequence shown in SEQ ID NO: 17 is It is one of the base sequences of a gene (cDNA) encoding the variable region of HpU-20 H chain. However, the gene according to the present invention is not limited to this, and may be various genes encoding antibody enzymes having the amino acid sequences shown in SEQ ID NOs: 14 and 16, and further genes encoding variants thereof. .
[0125]
As another gene according to the present embodiment, a gene consisting of the base sequence shown in SEQ ID NO: 19 can also be exemplified. The gene consisting of the base sequence shown in SEQ ID NO: 19 is one of the base sequences of the gene (cDNA) encoding the variable region of the UA-15 L chain. However, the gene according to the present invention is not limited thereto, and may be various genes encoding an antibody enzyme having the amino acid sequence shown in SEQ ID NO: 19, and further a gene encoding a variant thereof.
[0126]
Examples of other genes according to the present embodiment include those consisting of the base sequence shown in SEQ ID NO: 24 and those consisting of the base sequence shown in SEQ ID NO: 26. The gene consisting of the base sequence shown in SEQ ID NO: 24 is one of the base sequences of the gene encoding the variable region of the L chain of ECL2B-4, and the gene consisting of the base sequence shown in SEQ ID NO: 26 is that of ECL2B-4 It is one of the base sequences of genes encoding the variable region of the H chain.
[0127]
The “gene” of the present invention includes RNA and DNA. RNA includes mRNA, and DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA serving as a sense strand or an anti-coding strand serving as an antisense strand (the antisense strand may be used as a probe or an antisense strand). Available as a drug). Furthermore, the “gene” of the present invention includes sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences) in addition to the sequence encoding the protein (a) or (b). It may be a thing.
[0128]
(4-3) Functions of antibody enzymes i41SL1-2 and i41-7
The above-mentioned i41SL1-2 antibody and i41-7 antibody are, as shown in Examples described later, heavy chain (i41SL1-2-H, i41-7-H), light chain (i41SL1-2-L, i41-7- Both L) were experimentally confirmed to have the activity of cleaving and / or degrading polypeptides and antigenic proteins (see FIGS. 6 (a) (b) and 10 (a) (b) (c)). Moreover, although a result is not shown, when protein HSA is made to react with said i41SL1-2-H, i41-7-H, i41SL1-2-L, and i41-7-L as control, degradation of HSA is Did not happen. From this, it was shown that the above-mentioned i41SL1-2 antibody and i41-7 antibody have high substrate specificity for both heavy chain and light chain.
[0129]
In particular, as shown in FIGS. 10 (a) and 10 (b), both the heavy and light chains of the i41-7 antibody have peptidase activity and are short peptides, regardless of specificity. It became clear that the reaction rate was different, but it was cleaved or decomposed. In addition, the reaction rate of the short peptide was larger than that of the long peptide. This is not a problem of specificity, and it is considered that short peptides have a small molecular weight and are easy to access the binding site of the i41-7 antibody.
[0130]
In FIG. 10C, i41-7-L decomposes 41S-2-L (25 kDa), which is an antigenic protein, and a 22.5 kDa band appears. Moreover, since i41-7-L did not decompose unrelated proteins such as BSA, it can be said that i41-7-L specifically decomposed antigen proteins.
[0131]
That is, i41SL1-2-H, i41-7-H, i41SL1-2-L, and i41-7-L are specifically cleaved or degraded when the substrate is a protein, and the substrate is a polypeptide ( (A polypeptide of about 30 mer or more) is cleaved / degraded to a certain degree, and when the substrate is a shorter peptide (about 30 mer or less), it is cleaved // decomposed rather non-specifically. Conceivable.
[0132]
Therefore, the above i41SL1-2-H, i41-7-H, i41SL1-2-L, and i41-7-L are antibody enzymes that have both excellent substrate recognition ability of antibodies and substrate conversion ability of enzymes. It became clear that there was.
[0133]
The antibody MA-2 (monoclonal antibody against methamphetamine) that does not have a catalytic triad residue structure in the three-dimensional structure does not show any antigen degradation reaction (see FIG. 12). As will be described later, the heavy chains of HpU-9 and HpU-18 antibodies do not have His and cannot have a catalytic triad residue-like structure. Both heavy chains did not show any peptidase activity.
[0134]
Therefore, it has been shown that an antibody enzyme having a catalytic triad residue structure in the three-dimensional structure has an activity of cleaving and / or degrading a polypeptide or an antigen protein.
[0135]
Moreover, as shown in the Example mentioned later, the kinetic analysis of the degradation reaction with respect to the antigen peptide of the light chain of the said i41SL1-2 antibody and a heavy chain was conducted (refer FIG. 7 (a) (b), Table 5). . As a result, the i41SL1-2 antibody showed a light chain catalytic efficiency (kcat / Km) equivalent to that of trypsin, and a heavy chain as high as about 1/10 of that of trypsin. Therefore, it can be said that the i41SL1-2 antibody has a high enzyme activity comparable to that of natural proteolytic enzymes in both light and heavy chains. Furthermore, it is clear that i41SL1-2-L and i41SL1-2-H exhibit enzyme activity of degrading polypeptides and antigen proteins while retaining the properties of antibodies that have high affinity for substrates. It became.
[0136]
Therefore, it has been experimentally shown that the antibody enzyme according to the present invention has both activity equivalent to that of natural proteolytic enzyme and high affinity and specificity for a specific substrate of the antibody.
[0137]
(4-4) Production method of antibody enzyme according to the present embodiment
The antibody enzyme according to the present embodiment can be produced by the above-described antibody enzyme production method according to the present invention. In particular, in the present embodiment, the enzyme enzyme is analyzed by a three-dimensional structure analysis by germline analysis results or molecular modeling. Producing antibodies with activity.
[0138]
Specifically, among mouse germlines, the enzyme activity was confirmed in light chains derived from bd2, 19-25, and hf24 according to the classification of Thie et al. (Especially bd2 and 19-25 were derived from germline). A catalytic triad structure is constructed with amino acid residues.In hf24, histidine residues are caused by mutations). In addition to this, the antibody light chain derived from cr1 (HpU18) and cs1 (HpU9) (both of which are presumed to construct a catalytic triad structure with amino acid residues derived from germline) are also directed against peptides. Degradation activity is recognized. HpU-2-H (J558.h) has no histidine residue and has a catalytic triad residue structure composed of Asp86, Glu85, and Ser84 (or Ser87), and has the ability to degrade polypeptides and antigenic proteins. Indicated. This is an important finding that suggests that many antibodies with enzyme activity exist in nature and that germline is an indicator for finding this type of antibody. Details will be described in an embodiment described later.
[0139]
(4-5) Antibody enzyme according to the present invention and method for using the gene
The antibody enzyme, gene, and the like according to the present invention are novel antibody enzymes that can be produced based on the knowledge that they have a catalytic triad residue structure in a three-dimensional structure that has not been conventionally known. Therefore, it has the following usefulness.
[0140]
By using the present invention, for example, an amino acid sequence of an antibody prepared by a conventionally known immunological technique (for example, a base sequence is determined using a conventionally known genetic engineering technique, and an amino acid sequence deduced therefrom) ), The three-dimensional structure of the antibody is estimated by a computer, and the polypeptide and the antigen protein are cleaved and / or decomposed by examining whether the three-dimensional structure has a structure capable of forming a catalytic triad residue structure. It is possible to predict an antibody enzyme having activity and obtain it efficiently.
[0141]
Further, by using the present invention, for example, by using a conventionally known genetic engineering technique, by introducing a structure capable of forming a catalytic triad residue structure into a three-dimensional structure, the polypeptide and the antigen protein are cleaved and / or It is also possible to artificially produce an antibody enzyme having an activity of degrading.
[0142]
In addition, conventionally known genetic methods are not particularly limited. For example, site-directed mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), PCR, etc. are used. 1 or more in the nucleotide sequence of the gene obtained as described above by using a known mutant protein production method such as a method for introducing mutation into the nucleotide sequence or a mutant strain production method by inserting a transposon. Can be made by modifying such that the bases are replaced, deleted, inserted, and / or added. Moreover, you may utilize a commercially available kit for preparation of a mutein.
[0143]
The antibody enzyme obtained and prepared by the method as described above has both the substrate recognition ability with high antibody specificity and the enzyme activity. For this reason, for example, for infectious diseases caused by bacteria and viruses entering the living body, the antibody enzymes specific to the bacteria and viruses that cause them are obtained or prepared, and the antibody enzymes are diagnosed. It can be used for treatment. In addition, for example, cancer can be diagnosed and treated using the antibody enzyme by obtaining or preparing an antibody enzyme that specifically recognizes a polypeptide that is specifically expressed in cancer cells. Is possible.
[0144]
In addition to the above-described use as pharmaceuticals, there is a possibility that it will lead to the development of innovative drugs that overcome intractable diseases and drug resistance in the future for new drugs that have never existed before.
[0145]
In addition, RIA (Radioimmunoassay) and ELISA (Enzyme-Linked Immunosorbent Assay) are currently widely used for immunological diagnosis. The target antibody enzyme is obtained and the antibody enzyme is used. This will lead to the development of new clinical diagnostic methods.
[0146]
Furthermore, antibody enzymes that combine the high molecular recognition ability of antibodies and the substrate conversion ability of enzymes can be applied to new biosensors as new biomaterials, and can also be used for diagnostics, environmental measurements, and other tests. Is possible.
[0147]
In addition, since the antibody enzyme according to the present invention has almost the same activity as that of a natural enzyme, its catalytic activity (enzyme activity) is used to make a reaction accelerator such as a catalyst used in the food industry and the chemical industry. Can also be developed.
[0148]
The present invention also includes a gene encoding the above-described antibody enzyme or a mutant gene thereof. The gene according to the present invention or a mutant gene thereof can be introduced into various host cells to express the antibody enzyme according to the present invention or a mutant protein thereof. The introduced gene or mutant gene of the present invention may exist as a vector in the host cell, or may be included as “external” DNA or “additional” DNA in the genomic DNA of the host cell. As used herein, “external” DNA refers to DNA that is not naturally present in the host cell genome but has been inserted into the host cell genome as a result of artificial manipulation. The “added” DNA means a DNA that is naturally present in the genome of a specific host cell, but is additionally inserted into the genome of the host cell as a result of artificial manipulation.
[0149]
The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell may be appropriately selected. That is, according to the type of the host cell, a promoter sequence may be appropriately selected in order to reliably express the gene, and this and the gene according to the present invention incorporated into various plasmids may be used as an expression vector. Furthermore, the expression vector may contain not only a promoter sequence but also a terminator sequence.
[0150]
In addition, various markers may be used to confirm whether or not the above gene or mutant gene has been introduced into the host cell, and whether or not the gene or mutant gene is reliably expressed in the host cell. For example, a gene deleted in the host cell is used as a marker, and a plasmid or the like containing this marker is introduced into the host cell together with an expression vector containing the gene according to the present invention. Thereby, the introduction of the gene according to the present invention can be confirmed from the expression of the marker gene.
[0151]
The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe), Xenopus laevis oocytes, cultured cells of various mammals, or insects Although cultured cells etc. can be mentioned, it is not specifically limited.
[0152]
A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. .
[0153]
As described above, the antibody enzyme and its gene according to the present invention can be applied not only to diagnosis and treatment of various infectious diseases and cancers but also to various reagents for research.
[0154]
A conventionally known method can be applied to the pharmaceutical preparation of the gene or antibody enzyme according to the present invention, and it is not particularly limited. For example, in the case of a research reagent, the gene according to the present invention may be converted into a transformation kit, or the antibody enzyme according to the present invention may be mass-produced in a heterologous expression system and purified.
[0155]
In addition, each antibody enzyme shown in FIG. 4 (a), FIG. 4 (b), FIG. 8 (a), FIG. 8 (b), FIG. 15, FIG. 16, FIG. Since the amino acid number in the amino acid sequence is a number according to Kabat's classification, it is different from the amino acid number shown in each corresponding SEQ ID NO.
[0156]
【Example】
In the following Examples, the results of examining the physical properties of the antibody enzymes according to the present invention: i41SL1-2-H, i41-7-H, i41SL1-2-L, and i41-7-L will be described in order. .
[0157]
[Example 1: Production of antibody]
The i41SL1-2 antibody and i41-7 antibody were performed according to the following method.
[0158]
Specifically, first, the synthetic polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 or the light chain of the antibody 41S-2 shown in SEQ ID NO: 13 was respectively converted to m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). ) Or glutaraldehyde was bound to a carrier protein and used as an antigen protein for immunization. KLH (Keyhole Limpet Hemocyanin) was used as a carrier protein. Next, Balb / c mice were immunized with the antigen protein.
[0159]
Subsequently, the spleen was removed from the immunized mouse to prepare spleen cells. These spleen cells and myeloma cells derived from mouse bone marrow separately prepared were fused with polyethylene glycol and then subjected to HAT selection. And the antibody production positive cell group was selected by the screening by ELISA (ELISA) method, those cell groups were cloned, and the antibody production hybridoma was obtained. The hybridoma was transplanted into the abdominal cavity of the mouse, and ascites was collected to obtain a monoclonal antibody. As the i41-7 antibody, one that reacted well with the 41S-2 antibody light chain was selected at the screening stage. The prepared antibody was purified by affinity chromatography.
[0160]
The purity of the purified antibody was confirmed by SDS-PAGE.
[0161]
[Example 2: Three-dimensional structure analysis of monoclonal antibody]
First, the base sequences of various monoclonal antibodies were determined. Specifically, after extracting mRNA from various antibody-producing cells, cDNA was synthesized by RT-PCR. The antibody gene is amplified by PCR using this as a template, and incorporated into pGEM-T vectors (pGEM-T (registered trademark) and p-GEM-T (registered trademark) Easy Vector System, Promega Corporation, USA). Subcloned into E. coli JM109. After liquid culture, the plasmid in which the target gene was incorporated was purified and the nucleotide sequence was determined.
[0162]
Next, as reference data, the amino acid sequence of the monoclonal antibody variable region deduced from the base sequence (35 strains) and the amino acid sequence of the antibody registered in the Protein Data Bank (PDB) (Research Collaboratory for Structural Bioinformatics) (56 Used). For these, first, the three-dimensional structure of the antibody was constructed with the software AbM (Oxford molecular Ltd., UK), and then the energy was minimized with Discover (Molecular Simulations Inc., USA).
[0163]
[Example 3: Analysis of germline gene]
Among antibodies subjected to three-dimensional conformational analysis, Vκ germline was searched using Ig BLAST (National Center for Biotechnology Information) for antibodies derived from mice and whose L chain class was k-type. In addition to estimating the germline from which each antibody was derived, we analyzed in detail the amino acid residue arrangement and mutation status from germline for the amino acid residues presumed to construct a catalytic triad residue structure. . Specifically, it was performed as follows.
[0164]
Molecular modeling was carried out by the method described in Example 2 above for 56 monoclonal antibodies randomly extracted from Protein Data Bank (PDB) and 34 strains randomly extracted from the monoclonal antibodies owned by the present inventors. The three-dimensional structure of the variable region was analyzed. In the present invention, molecular modeling was performed using the PDB sequence data, and the three-dimensional structure of the variable region was analyzed. This is because all antibody data can be equally evaluated by analyzing the three-dimensional structure using the same software. That is, it is based on the idea that evaluation is not performed using uneven data, such as evaluation using X-ray crystal structure analysis data in some cases and evaluation using molecular modeling data in other cases.
Of the 56 strains randomly extracted from PDB, 52 strains were derived from mice and the remaining 4 strains were derived from humans. Antibody light chains are classified into two classes, κ and λ. In the case of mice, about 95% are said to be κ chains. In addition, mouse κ chain has the advantage that germline gene analysis is progressing and information on this is easily available. Therefore, mouse κ chain (47 strains) was analyzed in more detail.
[0165]
From these, strains related to the catalytic triad residue structure were selected and classified as follows. First, those comprising a catalytic triad residue structure by Asp1, Ser27A, and His93 common to i41SL1-2-L described above were classified as type ◎. And what constitutes a catalytic triad structure by Asp1, Ser26, His93 is classified as type ○, and a catalytic triad residue is combined with a combination other than “Asp1, Ser27AorSer26, His93” (for example, using His90 or His91). The constituents are categorized as type ●, and those that can no longer construct catalytic triad residues due to mutation from germline are classified as type x, and the catalytic triad residual structure including amino acids mutated from germline is constructed. Were classified as type *.
[0166]
First, Table 1 shows the classification results for germline of all 47 strains.
[0167]
[Table 1]

[0168]
As shown in Table 1, 16 strains (34.0% of the total) were found that corresponded to ◎, ○, and ●. In the L chain derived from a mouse whose class is κ chain, 12 out of 47 strains (25.5%) corresponded to the above ◎ or ○.
[0169]
Table 2 shows the results of the classification of the germline of all 47 strains shown in Table 1 and the ones that construct the catalytic triad residue structure.
[0170]
[Table 2]

[0171]
As shown in Table 2 above, there is a commonality among the antibodies constituting the catalytic triad residue structure of type (1) (or (1) *), and 9 out of 19 strains are bb1, Three strains were cr1. In addition, i41SL1-2-L mentioned above had germline bd2.
[0172]
Next, the characteristics common to germline bb1, cr1, and bd2 were examined.
[0173]
[Table 3]

[0174]
As shown in Table 3 above, each of these germlines had hypervariable region 1 (CDR1) composed of 16 amino acid residues. The CDR sequence is a place where the antibody undergoes a large mutation because it strongly recognizes the antigen (this is the reason called hypervariable region).
[0175]
Therefore, we further analyzed the relationship between Asp1, Ser27A, and His93 sequences and germline.
[0176]
FIGS. 11 (a), (b), and (c) show the results of molecular modeling performed by germline on the 1DBA strain, which is bb1, according to the classification of Thiebe et al. FR is indicated by a wire and CDR is indicated by a ribbon. Asp1 exists in FR1, Ser27A exists in CDR1, and His93 exists in CDR3 in different regions in the primary sequence. However, as shown in FIGS. 11 (b) and 11 (c), these three amino acid residues are located in the vicinity of the tip of the CDR3 loop where His93 is present and at the same height as His93, that is, when viewed in the horizontal direction. It can be seen that they are located on substantially the same plane.
[0177]
Since these hit the tip of the space where the loops of the hypervariable region are concentrated, it is considered that the antigen can be taken in without steric hindrance. Further, as shown in FIG. 11 (a), when the number of amino acids constituting CDR1 is 16, the CDR1 loop has a wave-like structure that is gently waved in the vicinity of this, and the tip portion (from 27B to 31B) The number 8 amino acid residue) protrudes from the other loops. For this reason, the antibody enzyme has a catalytic triad residue structure at a position where the antigenic peptide can be taken in without steric hindrance, and it is considered that the antigenic peptide can be decomposed smoothly. These three-dimensional structural features were common to the 10 antibody L chains and i41SL1-2-L shown in Table 2 above.
[0178]
Next, in the germline of the data of Thiebe et al. (Thiebe R, Schanble KF et al, Eur J Immunol, 29 (7), 2082-2081 (1999)), the frequency of Asp1, Ser27A and His93 was examined. There were 58 types of aspartic acid residues used in No. 1 in 58 types, which was 62.3% of the total (aspartic acid residues were widely distributed not only in No. 1 but also in the entire variable region. Amino acid residues). Although serine (Ser) residue 27A is in CDR1, Ser present in the vicinity of 27A can constitute a catalytic triad residue structure.
[0179]
There were 92 types of germlines containing effective Ser when such Ser was included. In other words, there are a large number of germlines in which Ser in the Asp1 and CDR1 portions are arranged at positions close to each other in the three-dimensional structure. On the other hand, His had a low appearance rate and there were only 7 types of germlines (bb1, cr1, bl1, cs1, bd2, bj2, 8-16). Moreover, six of them (other than 8-16) are germlines such as bb1 that can form a catalytic triad residue structure, and CDR1 is composed of 16 amino acid residues. In other words, the six types bb1, cr1, bl1, cs1, bd2, and bj2 shown in Table 3 can be said to be germlines in which amino acid residues are arranged at ideal positions in order to decompose the antigen.
[0180]
Next, it was experimentally investigated whether or not the antibodies produced from these germlines have antigenolytic activity. The present inventors have produced hundreds of types of monoclonal antibodies so far, and determined germline (antibody light chain (mouse κ chain)) for 34 strains. Among them, the data of those subjected to the experiment are shown together in Table 4.
[0181]
[Table 4]

[0182]
In Tables 1, 2, and 4, type ◎: Asp1, Ser27A, and His93 constitute the catalytic triad residue structure, Type ○: Asp1, Ser26, His93 constitutes the catalytic triad residue structure , Type ●: Composing the catalytic triad with a combination other than “Asp1, Ser27AorSer26, His93” (for example, using His90 or His91), Type *: Catalytic triad structure can be constructed by mutating from germline It is assumed that the residue structure of the catalyst triad including the amino acid mutated from type x: germline is constructed.
[0183]
As discussed above, 21 germline-derived strains were found that can construct catalytic triad residues with His at the tip of the hypervariable region. In addition, a catalytic triad residue structure with His could not be constructed in two strains (No. 16, 18).
[0184]
That is, since peptidase activity and / or protease activity was observed in many antibody subunits (light chain, heavy chain) having a catalytic triad residue structure, when germline was examined for these antibodies, CDR1 was 16 It has a common feature that it is composed of amino acid residues, and has a histidine residue at the 93rd position (or its vicinity) in the classification of KABAT (type (4)). This is an important finding that suggests that an antibody L chain having an enzymatic activity has already been prepared for a specific germline.
[0185]
Among the antibodies with catalytic triad residues, the strains that have so far conducted experiments on enzyme action are 41S-2 (germline: hf24 / already published) and i41SL1-2 (bd2). Further strains are also being experimented with, the HpU-2 antibody heavy chain, the HpU-9 antibody light chain, the HpU-18 antibody light chain, and the i41-7 antibody light chain and heavy chain, It was revealed that it has peptidase activity and protease activity.
[0186]
In addition, the i41-7 antibody belonged to 19-25 germline with 11 CDR-1. Therefore, when all the 11 germlines with CDR-1 were searched, in addition to 19-25, 19-14, 19-17, 19-23, 12-41 (these are indicated by boxes in Table 3) A triple residue structure was observed. When examined in more detail, CDR-1 is composed of 11 amino acid residues, and in common with Kabat (KABAT), it has a histidine residue at the 91st (or 55th) position. (Type ▲ 4 ▼ *).
[0187]
In summary, the following can be said. (1) It is considered that there are two types of natural antibody enzymes. One is a type that already has a favorable sequence for the construction of a catalytic triad structure at the germline stage. According to the classification of Thie et al., At least bb1, cr1, bl1, cs1, bd2, bj2, hf24, 19 -25, 19-14, 19-17, 19-23, 12-41, and 21-12 correspond to this. In the case of hf24, in addition to His91 present in germline, His53 was revealed by mutation, which may have constituted a catalytic triad residue structure. (2) The germline having a sequence advantageous for the construction of the catalytic triad residue structure is only 13 types (13.9% because 93 types in total) according to the classification of Thiebe et al.
[0188]
However, about 20% of the searches by PDB and about 30% of the monoclonal antibodies owned by the present inventors have been found to be a catalytic triad residue structure, and the above-mentioned germline has a higher probability than expected. Has been recruited.
[0189]
Furthermore, as a conclusion, if a three-dimensional structure is predicted from the base sequence of the antibody and an antibody having the germline shown in the box shown in Table 3 is selected, the antibody enzyme can be efficiently and efficiently obtained for the light chain. It is possible to obtain (conclusion (1)).
[0190]
If the concept of light chain is applied to the heavy chain, the 41S-2 antibody, i41SL1-2 antibody, and i41-7 antibody heavy chain have so far formed a catalytic triad structure of His, Asp, and Ser. Enzyme activity was observed. The heavy chain families of the 41S-2 antibody, the i41SL1-2 antibody, and the i41-7 antibody are VH10, VH1, and VH1, respectively. On the other hand, the heavy chain of the 7C4 antibody and the HpU-2 antibody did not have a catalytic triad residue structure with His, Asp, and Ser, and Glu, Asp, and Ser comprised a triplet residue, and enzyme activity was observed. The families of the heavy chains of the 7C4 antibody and the HpU-2 antibody are VH5 and VH1, respectively. In particular, both of these (7C4 antibody, HpU-2 antibody heavy chain) all constituted the catalytic triad residue structure in the same combination (Ser84, Glu85, Asp86). In other words, regarding the heavy chain, VH1, VH5, VH10 is a family that can efficiently exhibit activity as an antibody enzyme (conclusion (2)).
[0191]
In addition, even an antibody that does not originally have a catalytic triad residue structure can be obtained with high efficiency by introducing a catalytic triad residue structure corresponding to germline or Family by genetic engineering. This can be said from the present invention (conclusion (3)).
Furthermore, as shown in FIG. 37, 18 types of 47 antibody light chains (κ type) in the PDB data had a catalytic triad residue structure having His (38.3%). Further, as shown in FIG. 38, among 37 types of data of the antibody light chain (κ type) possessed by the present inventors, 20 types had a catalytic triad residue structure having His (54. 0%). When these are added together, the detection rate of antibody light chains having enzyme activity among the antibody light chains found from the three-dimensional structure is 45.2%. This is a general probability that an antibody light chain can be detected.
[0192]
However, if a germline is specified as described above, 88.1% of the antibody having a catalytic triad residue structure having His, which is considered to have enzyme activity (PDB data; 16/19 = 84.2% (see FIG. 37)), the present invention Data on the stocks owned by them; 21/23 = 91.3% (see Fig. 38), total; (16 + 21) / (19 + 23) = 37/42 = 88.1%) and the probability approaches 100% . The reason why it does not become 100% is that those having a catalytic triad residue structure inherently have a mutation in the maturation process of the antibody and lose His, and some lose the catalytic triad residue structure having His. One type of antibody in the PDB data and two types in the antibody owned by the present inventors were mutated. In other words, if the above germline is specified, an antibody having almost complete enzyme activity can be obtained (Conclusion (4)).
[0193]
[Example 4: Preparation of light chain and heavy chain]
Low molecular weight protease inhibitors were removed by repeated ultrafiltration on the purified antibody. This was followed by a reduction reaction with 0.2 M b-mercaptoethanol (15 ° C., 3 hr) and an alkylation reaction with 0.3 M iodoacetamide (15 ° C., 15 min) to separate heavy and light chains. This was concentrated by ultrafiltration, and then purified by HPLC using a Protein-Pak 300 column (Millipore Corporation Waters Chromatography Division). A 6M guanidine solution (pH 6.5) was used as the mobile phase, and the flow rate was 0.15 mL / min.
[0194]
The separated heavy chain and light chain solutions were diluted with 6M guanidine solution and then PBS solution (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH). ₂ PO _Four 8.0 mM Na ₂ HPO _Four After dialysis against pH 7.3) and refolding, the buffer was exchanged according to the experimental conditions.
[0195]
[Example 5: Measurement of antibody enzyme activity]
In order to clarify the relationship between the catalytic triad residue structure and enzyme activity, the i41SL1-2 antibody light chain (derived from i41SL1-2-L, germline bd2) and heavy chain (i41SL1-2), which are presumed to have this structure. -H), i41-7 antibody light chain (from i41-7-L, germline 19-25) and heavy chain (i41-7-H) were subjected to a degradation reaction against a peptide substrate.
[0196]
Specifically, the CDR1 antigen peptide (RSSKSLLYSNGNTYLY) shown in SEQ ID NO: 9 was reacted with each of the heavy chain and light chain of the i41SL1-2 antibody.
[0197]
The reaction conditions were 5% DMSO, 9 mM HEPES, 7.5 mM phosphate buffer (pH 7.1), 25 ° C. Experiments were performed at a concentration of 0.4 μM for i41SL1-2-L, 0.2 μM for i41SL1-2-H, and 50 μM for the antigenic peptide.
[0198]
The result is shown in FIG. (●) is the reaction of i41SL1-2-L and antigenic peptide, (○) is the reaction of only antigenic peptide, (▲) is the reaction of i41SL1-2-H and antigenic peptide Indicates. As shown in FIGS. 6 (a) and 6 (b), the concentration of the antigen peptide decreased with the passage of the reaction time for both the heavy chain and the light chain, and finally became undetectable. Therefore, when the peak portion newly detected on the HPLC chromatogram was collected and analyzed by mass spectrometry with the passage of time, the portion between the first arginine residue and the second serine residue of the antigen peptide was cleaved. It was found to be a C-terminal fragment. From this, it became clear that i41SL12-L and i41SL12-H degrade the antigenic peptide.
[0199]
In addition, three types of synthetic peptides shown in SEQ ID NOs: 10, 11, and 12 were reacted with the light chain and heavy chain of the i41-7 antibody, respectively. The i41-7 antibody is obtained by using a protein as an antigen, and the epitope has not been determined so far. Originally, the enzyme activity should be examined using a peptide corresponding to the epitope as a substrate, but the antibody enzyme that we have obtained so far shows high specificity for the protein, but the specificity for short peptides is low. Therefore, in this experiment, three kinds of synthetic peptides having a molecular weight of several hundred Da to 2.3 kDa were used as substrates.
[0200]
The reaction conditions were 15 mM phosphate buffer (pH 6.5) and 25 ° C. The synthetic peptides shown in SEQ ID NOs: 10, 11, and 12 were used at a concentration of 120 μM, i41-7-L at 0.8 μM, and i41-7-H at a concentration of 0.4 μM, respectively.
[0201]
The results are shown in FIGS. 10 (a) and 10 (b). (●) shows peptide TP41-1 and i41-7-L or i41-7-H shown in SEQ ID NO: 10, (◯) shows only peptide TP41-1 shown in SEQ ID NO: 10, and (▲) shows SEQ ID NO: 11 and i41-7-L or i41-7-H, (Δ) shows only the peptide shown in SEQ ID NO: 11, and (■) shows the peptide shown in SEQ ID NO: 12 and i41-7-L or i41. -7-H, (□) shows the reaction of only the peptide shown in SEQ ID NO: 12.
[0202]
As shown in FIGS. 10 (a) and 10 (b), i41-7-L and i41-7-H all degraded these peptides.
[0203]
In addition, the degradation reaction of the antigen protein by the light chain (i41-7-L) of the i41-7 antibody was analyzed by SDS-PAGE. The light chain (41S-2-L) of antibody 41S-2 was used as the substrate. Note that 41S-2-L used as this substrate is incompletely refolding and has no activity as an antibody enzyme.
[0204]
The degradation reaction was performed under the same conditions as described above. 41S-2-L was used at a concentration of 2 μM, and the light chain of the i41-7 antibody was used at a concentration of 0.8 μM. The results are shown in FIG. Lane M is the protein molecular weight marker, and lanes 1 to 5 are each electrophoresed with the reaction solution of i41-7-L and 41S-2-L at 0, 2, 4, 6, and 8 days from the start of the reaction. Results are shown. The band at the position of 25.0 kDa indicates the antigen protein 41S-2-L, and the band at the position of 22.5 kDa indicates the degradation product of 41S-2-L. As a control, the results when only the light chain of the i41-7 antibody is reacted and when only 41S-2-L is reacted are shown. Based on this SDS-PAGE result, a graph showing the degradation reaction of 41S-2-L by i41-7-L is also shown.
[0205]
From the result of SDS-PAGE and the graph of FIG. 10C, it can be seen that the band at the position of 25.0 kDa is decomposed and the band at the position of 22.5 kDa is generated. In addition, in the reaction of the substrate 41S-2-L alone from the control, the band at the 25.0 kDa position is not decomposed. This showed that the light chain of the i41-7 antibody degrades the antigen protein 41S-2-L.
[0206]
Next, the polypeptide resolution of the i41-7 complete antibody, light chain (i41-7-L), heavy chain (i41-7-H), and MA-2 antibody was examined. The i41-7 complete antibody is a complete antibody consisting of a heavy chain and a light chain. The MA-2 antibody (germline: aq4) is a monoclonal antibody against methamphetamine and does not have a catalytic triad residue structure in the three-dimensional structure.
[0207]
The reaction conditions were the same as in the above experiment, and the polypeptide TP41-1 (denoted as “TP” in FIG. 12) shown in SEQ ID NO: 10 was used as a substrate at a concentration of 120 μM. The i41-7 complete antibody, i41-7-L, and MA-2 antibody were used at a concentration of 0.8 μM, and i41-7-H was used at a concentration of 0.4 μM.
[0208]
The results are shown in FIG. (●) shows i41-7-L and TP41-1, (□) shows i41-7-H and TP41-1, (△) shows i41-7 complete antibody and TP41-1, (■ ) Reacted with MA-2 antibody and TP41-1, and (◯) reacted only with TP41-1 as a control.
[0209]
As shown in FIG. 12, both the heavy chain and the light chain of the i41-7 antibody show an induction period in which the peptide is hardly degraded for a while after the start of the reaction, and then show an active period in which the peptide is degraded. It was. Further, although the results are not shown, when the peptide was added again after the peptide was completely degraded, both the heavy and light chains of the i41-7 antibody immediately degraded the peptide without the induction period. From this result, it is considered that both the heavy chain and the light chain of the i41-7 antibody undergo a conformational change when moving from the induction phase to the active phase. In addition, once the conformation changes, it is considered that the active form is maintained thereafter.
[0210]
In addition, the complete i41-7 antibody in which the heavy chain and light chain are aligned did not degrade the polypeptide. This is because the i41-7 complete antibody has a light chain and a heavy chain that are strongly bound by SS bond or non-covalent bond, etc., so the conformation changes to a form that is active even when contacting with the polypeptide. It is thought that it is impossible.
[0211]
In addition, the MA-2 antibody that does not have a catalytic triad residue structure in the three-dimensional structure did not degrade the polypeptide in either the heavy chain or the light chain. Although the results are not shown, MA-15 antibody light chain (germline: ba9), 7C4 antibody light chain (germline: af4), HpU-9 antibody heavy chain, and HpU-18, which also have no catalytic triad residue structure. The antibody heavy chain also did not degrade the polypeptide or antigenic protein.
[0212]
From this result, it was shown that an antibody enzyme having a catalytic triad residue structure in the three-dimensional structure has an activity of cleaving or decomposing a polypeptide or an antigen protein.
[0213]
[Example 6: Kinetic analysis of i41SL1-2-L and i41SL1-2-H degradation activity for antigenic peptides]
When the degradation profile of the i41SL1-2-L and i41SL1-2-H against the CDR1 antigen peptide is traced in detail, the peptide concentration hardly changes in the initial reaction with the antigen peptide (the peptide is hardly degraded). (This length depends on the experimental conditions). However, once the peptide was decomposed and the antigenic peptide was added again to the active antibody solution, the induction period was no longer recognized. Therefore, the initial degradation rate in the antigen peptide re-addition reaction was measured and analyzed using the Michaelis-Menten equation.
[0214]
As a result, as shown in FIGS. 7A and 7B, both the light chain and the heavy chain showed high correlation in the Hanes-Woolf plot. Therefore, it can be said that these are enzyme reactions according to the Michaelis-Menten equation. The kinetic parameters obtained here are shown in Table 5 in comparison with trypsin (value obtained using different synthetic peptides as substrates).
[0215]
[Table 5]

[0216]
As shown in Table 5 above, the light chain catalytic efficiency (kcat / Km) was equivalent to that of trypsin, and the heavy chain was as high as about 1/10 of that of trypsin. Both have high enzyme activity comparable to that of natural enzymes. However, looking at each parameter, the heavy chain and light chain of this antibody enzyme are stronger than trypsin with respect to the affinity (Km) for the substrate, whereas the heavy chain and light chain are both the rotation speed (kcat). A value of about 1/200 of trypsin was shown, and a difference was observed between the properties of both. This result shows that i41SL1-2-L and i41SL1-2-H exhibit the enzymatic activity of completely degrading the polypeptide while retaining the property as an antibody that has high affinity for the substrate. ing.
[0217]
[Example 7: Preparation of H and L chains of HpU-20 and UA-15]
According to the procedure described in Example 1 above, two types of anti-HP urease mouse monoclonal antibodies HpU-20 and UA-15 were prepared, and the amino acid sequence and base of the monoclonal antibody were determined in the same manner as in Example 2 above. The sequence was determined and a 3D structure analysis was performed.
[0218]
As a result, the three-dimensional structure of the estimated variable region of HpU-20 (L chain) is shown in FIG. 13, the three-dimensional structure of the variable region of HpU-20 (H chain) is shown in FIG. 14, and UA-15 (H chain and L chain). The three-dimensional structure of the variable region of the chain) is schematically shown in FIG. From these three-dimensional structure predictions, a catalytic triad residue-like structure was observed for the L and H chains of HpU-20 and UA-15. On the other hand, no catalytic triad residue-like structure was observed in UA-15 (H chain).
[0219]
Furthermore, HpU-20 and UA-15 H and L chains were prepared in the same manner as in Example 4 above.
[0220]
[Example 8: Enzyme activity test 1 using antibody enzyme HpU-20 L chain and H chain] An enzyme activity test was conducted on TP41-1 peptide (SEQ ID NO: 31) of HpU-20-L and HpU-20-H. . This decomposition reaction was carried out with the following materials, conditions, and procedures.
[0221]
(material)
(1): HpU-20 mAb (in15 mM PB pH 6.5, filtered and sterilized after completion of purification and dialysis)
(2): HpU-20 antibody
(2) -1: Lot.1 [HpU-20-L] (in15 mM PB pH 6.5, concentration 94.4 μg / ml)
(2) -2: Lot.1 [HpU-20-H] (in15 mM PB pH 6.5, concentration 70.8 μg / ml)
(2) -3: Lot.2 [HpU-20-L] (in15 mM PB pH 6.5, concentration 96.7 μg / ml)
(2) -4: Lot.2 [HpU-20-H] (in15 mM PB pH 6.5, concentration 103.3 μg / ml)
(3): Purified TP41-1 peptide
(Reaction solution)
(1): HpU-20-L: 0.8 μM (20 μg / ml)
(2): HpU-20-H: 0.4 μM (20 μg / ml)
(3): HpU-20 mAb: 0.8 μM (120 μg / ml)
(4): TP41-1 Peptide: 120 μM (284 μg / ml)
(Reaction conditions)
Reaction temperature: 25 ° C
Small test tube, microchip, 15 mM PB (pH 6.5): Sterilization treatment
Experimental operation: Inside a clean bench
(Method)
(1): TP41-1 is adjusted to 1.7 mg / ml with 15 mM PB pH 6.5, and sterilized by filtration.
(2): Next, 1.7 mg TP41-1 of (1) is prepared to 568 μg / ml with 15 mM PB pH 6.5.
(3): The 20 antibody H and L chains are prepared to 40 μg / ml each with 15 mM PB pH6.5.
(4): (2) and (3) are mixed at a ratio of 1: 1.
(5): Follow the change of peptide with time by HPLC.
[0222]
Regarding the results of this experiment, Lot.1 is shown in FIG. 17 and Lot.2 is shown in FIG. In the graphs of FIGS. 17 and 18, the horizontal axis represents the reaction time (hours), and the vertical axis represents the concentration (μM) of the substrate TP41 peptide.
[0223]
As shown in the graphs of FIGS. 17 and 18, in the prepared Lot.1 and Lot.2, the TP41 peptide was completely decomposed with good reproducibility, although there was a slight difference in reactivity. From these results, it was confirmed that HpU-20-L and HpU-20-H have enzyme activity in the degradation reaction of TP41-1 peptide.
[Example 9: Enzyme activity test 2 using HpU-20 L chain and H chain]
Subsequently, using the activated HpU-20 L chain and H chain, a proteolysis experiment was carried out using the following materials, the composition and type of reaction solution, reaction conditions, and methods.
[0224]
(material)
(1): Activated (once the TP41 peptide has been completely degraded) Lot.2 [HpU-20-L] (in15 mM PB pH6.5, 02.10.15 separation, 02.10.16 purification, 02.10.21 PBS dialysis completed , 02.10.21-22 PB buffer exchange. 4.2 months storage, concentration 96.7μg / ml)
(2): HP bacteria urease (MW = 522600) 02.07.01 purification, concentration 1.84 mg / ml
(3): BSA SIGUMA A-6003 Lot51K7600
(4): HSA WAKO 019-10503 LotKSH6886
(Reaction solution composition)
(1): HpU-20-L: 0.4 μM (20 μg / ml)
(2): HP bacteria urease: 52 nM (28 μg / ml)
(3): BSA: 0.4 μM (28 μg / ml)
(4): HAS: 0.4 μM (28 μg / ml)
(Reaction conditions)
Reaction temperature: 25 ° C
Small test tube, microchip, 15 mM PB (pH 6.5): Sterilization treatment
Experimental operation: Inside a clean bench
(Method)
(1) Prepare HSA and BSA at 2.2 mg / ml with 15 mM PB pH 6.5, and sterilize by filtration.
(2) Next, 1.7 mg / ml TP41-1 of (1) is prepared to 55 μg / ml with 15 mM PB pH 6.5.
(3) 1.84 mg / ml HP fungal urease is prepared at 55 mM / ml with 15 mM PB pH 6.5.
(4) The activated HpU-20-L and (2) and (3) are mixed at a ratio of 1: 1.
(5) Follow changes in antibodies and proteins by SDS-PAGE
(Type of reaction solution)
(1): 0.4 μM HpU-20-L + 0.4 μM BSA: 320 μl
(2): 0.4 μM HpU-20-L + 0.4 μM HSA: 320 μl
(3): 0.4 μM HpU-20-L + 52 nM HP bacteria urease: 320 μl
(4): 0.4 μM HpU-20-L only: 320 μl
(5): 0.4 μM BSA only: 320 μl
(6): 0.4 μM HSA only: 320 μl
▲ 7 ▼: 52nM HP urease only: 320μl
The results of this experiment are shown in FIGS. FIGS. 19-21 is a figure which shows the result of SDS-PAGE of the sample 1 hour after the reaction start. For the samples in each lane, M is a marker, and (1) to (7) are the above reaction solutions.
[0225]
From the results shown in FIGS. 19 to 21, it can be seen that HpU-20-L does not decompose BSA at all in a reaction of about 1 hour. Although a thin band can be seen at 28.5 kDa for HSA, it can be seen that the control HSA hardly changed and only very weak degradation occurred. On the other hand, a strong band at 50 kDa and a weak band at 26 kDa were observed in the reaction for about 1 hour for HP bacterial urease. In addition, the β subunit band decreased by 26.7% compared to the control. The α subunit band hardly changed before and after the reaction. From this result, it was revealed that HpU-20-L specifically degrades the β subunit of HP urease.
[0226]
[Example 10: Enzyme activity test using UA-15 L chain and H chain]
Using UA-15-L and UA-15-H, an enzyme activity test against TP41-1 peptide (SEQ ID NO: 31) was performed. This decomposition reaction was carried out under the following reaction conditions using the following reaction solution.
[0227]
(Reaction solution)
(1) Lot.1 [UA-15-L]: 0.8μM (20μg / ml)
(2) Lot.1 [UA-15-H]: 0.4 μM (20 μg / ml)
(3): Lot.2 [UA-15-L]: 0.8 μM (20 μg / ml)
(4): Lot.2 [HpU-20-H]: 0.4 μM (20 μg / ml)
(5): TP41-1 peptide: 120 μM (284 μg / ml)
(Reaction conditions)
Reaction temperature: 25 ° C
Small test tube, microchip, 15 mM PB (pH 6.5): Sterilization treatment
Experimental operation: Inside a clean bench
Regarding the results of this experiment, Lot.1 is shown in FIG. 23 and Lot.2 is shown in FIG. In the graphs of FIGS. 23 and 24, the horizontal axis represents the reaction time (hours), and the vertical axis represents the concentration (μM) of the substrate TP41 peptide. As can be seen from the graphs shown in FIG. 23 and FIG. 24, the enzyme activity for decomposing TP41-1 peptide was confirmed for the UA-20 L chain, but the enzyme activity was confirmed for the UA-20 H chain. There wasn't.
[0228]
Subsequently, the peptide substrate (TP41-1) was re-added to the reaction solution of UA-15-L in Lot.2 to the reaction solution, and peptide degradation experiments were performed at 15 ° C, 25 ° C, and 37 ° C. Carried out.
[0229]
The results are shown in FIG. From this result, it was revealed that the enzyme activity of UA-15-L was highest when the reaction temperature was 25 ° C.
[0230]
Furthermore, after the TP41-1 peptide was once completely degraded using Lot.1 [UA-15-L] as a reaction solution under the above reaction conditions, the TP41-1 peptide was added to this solution at 5 μM, 10 μM, Kinetic analysis with UA-15-L was performed by adding each concentration of 15 μM, 20 μM, 30 μM, 65 μM, and 80 μM. The results are shown in FIG.
[0231]
[Table 6]

[0232]
[Example 11: Preparation of ECL2B-4 H and L chains]
According to the procedure described in Example 1 above, an anti-HP urease mouse monoclonal antibody called ECL2B-4 was prepared, and the amino acid sequence and base sequence of the monoclonal antibody were determined in the same manner as in Example 2 above, and three-dimensional Three-dimensional structure analysis was performed.
[0233]
FIG. 29 shows the amino acid sequence of the ECL2B-4 light chain and the base sequence of the cDNA encoding it, and the sequence of the amino acid sequence of the ECL2B-4 heavy chain and the cDNA encoding it. The base sequence is shown in FIG. In FIG. 29 and FIG. 30, variable regions (CDR-1 to CDR-3) are underlined.
[0234]
Next, molecular modeling was performed based on each amino acid sequence determined. FIG. 27 shows the three-dimensional structure of the variable region of ECL2B-4-L estimated by molecular modeling. FIG. 28 shows a three-dimensional structure of the variable region of ECL2B-4-H estimated by molecular modeling.
[0235]
[Example 12: Enzyme activity test of ECL2B-4 antibody]
Subsequently, a peptide degradation test for confirming the enzyme activities of ECL2B-4-L and ECL2B-4-H was performed as follows.
[0236]
First, the degradation reaction of the ECL2B peptide (SEQ ID NO: 27) was carried out in 15 mM phosphate buffer (pH 6.5) at 25 ° C. In order to monitor the reaction, 20 μl of the reaction solution was injected into RP-HPLC (manufactured by Jasco) under a constant temperature condition at a column temperature of 40 ° C.
[0237]
The following (1) to (5) were prepared as reaction solutions for this decomposition reaction.
(1) ECL2B-4 (L) 0.8μM + ECL2B Peptide: 50μM: 800μl
(2) ECL2B-4 (H) 0.4μM + ECL2B peptide: 50μM: 800μl
(3) Preparation of ECL2B peptide: 50 μM: 500 μl
(4) ECL2B-4 (L): 0.8μM: 300μl
(5) ECL2B-4 (H): 0.4 μM: 300 μl produced
The results are shown in FIGS. In the graph of FIG. 31, the horizontal axis represents the reaction time (hours), and the vertical axis represents the concentration (μM) of the substrate ECL2B peptide. Moreover, the result monitored using HPLC in FIG. 32 (a)-(c) is shown. FIG. 33 shows the kinetic analysis results in this enzymatic degradation experiment. FIG. 33 (a) is a graph showing the relationship between the substrate (ECL2B peptide) concentration and the degradation rate, and FIG. 33 (b) is a graph showing the results of the Hanes-Woolf plot.
[0238]
Table 7 below shows Vmax, Km, kcat, and kcat / Km values (average plots) calculated using the Hanes-Woolf plot.
[0239]
[Table 7]

[0240]
From these results, it was confirmed that ECL2B-4-L and ECL2B-4-H have enzyme activity in the degradation reaction of ECL2B peptide. Therefore, it was confirmed that ECL2B-4-L and ECL2B-4-H are antibody enzymes that can be completely degraded by targeting peptides constituting the extracellular region of CCR-5.
[0241]
Then, the enzyme activity test with respect to TP41-1 peptide (sequence number 31) of ECL2B-4-L and ECL2B-4-H was implemented. This degradation reaction was carried out in 15 mM phosphate buffer (pH 6.5) at 25 ° C. In order to monitor the reaction, 20 μl of the reaction solution was injected into RP-HPLC (manufactured by Jasco) under a constant temperature condition at a column temperature of 40 ° C.
[0242]
The following (1) to (5) were prepared as reaction solutions for this decomposition reaction.
(1) ECL2B-4 (L) 0.8μM + TP41-1 Peptide: 120μM: 800μl
(2) ECL2B-4 (H) 0.4μM + TP41-1 Peptide: 120μM: 800μl
(3) TP41-1 peptide: 120 μM: 500 μl
(4) ECL2B-4 (L): 0.8μM: 300μl
(5) ECL2B-4 (H): 0.4 μM: 300 μl produced
The results are shown in FIGS. In the graph of FIG. 34, the horizontal axis represents the reaction time (hours), and the vertical axis represents the concentration (μM) of the substrate ECL2B peptide. Moreover, the result monitored using HPLC in FIG. 35 (a)-(c) is shown. FIG. 36 shows the kinetic analysis results in this enzymatic degradation experiment. FIG. 36 (a) is a graph showing the relationship between the substrate (TP41-1 peptide) concentration and the degradation rate, and FIG. 36 (b) is a graph showing the results of the Hanes-Woolf plot.
[0243]
Table 8 below shows Vmax, Km, kcat, and kcat / Km values (average plots) calculated using the Hanes-Woolf plot.
[0244]
[Table 8]

[0245]
From these results, it was confirmed that ECL2B-4-L and ECL2B-4-H have low enzyme activity even in the degradation reaction of TP41-1 peptide. In addition, as can be seen from a comparison between Table 7 and Table 8, it has been experimentally shown that ECL2B-4-L degrades the original antigen ECL2B peptide faster than the TP41 peptide. It was proved that -4-L is indeed an antibody enzyme against ECL2B.
[0246]
【The invention's effect】
As described above, by using the antibody enzyme or gene according to the present invention, it can be used for treatment and diagnosis of various diseases such as infectious diseases and cancer. In addition, acquisition of the target antibody enzyme leads to the development of a new clinical diagnostic method.
[0247]
Furthermore, by using the antibody enzyme or gene according to the present invention, it can be applied to a new biosensor as a new biomaterial, and can also be used for tests such as disease diagnosis and environmental measurement.
[0248]
It is also considered that the antibody enzyme or gene according to the present invention can be used for effective synthetic means in the food industry and the chemical industry.
[0249]
In addition, according to the method for producing an antibody enzyme of the present invention, an antibody enzyme having an enzyme activity for selectively cleaving or degrading a polypeptide or an antigen protein is efficiently selected from immunologically produced antibodies. There is an effect that it can be acquired.
[0250]
Further, according to the method for producing an antibody enzyme of the present invention, an antibody enzyme having an enzyme activity for easily cleaving or degrading a polypeptide or an antigen protein can be efficiently produced using a conventionally known genetic engineering technique. There is an effect that can be done.
[0251]
[Sequence Listing]

[Brief description of the drawings]
1A is a process diagram showing an example of an antibody enzyme production method according to an embodiment of the present invention, and FIG. 1B is a diagram of an antibody enzyme production method according to another embodiment of the present invention. It is process drawing which shows an example.
FIG. 2 is a block diagram showing an example of the configuration of a system that performs an antibody structure analysis step in the antibody enzyme production method according to one embodiment of the present invention.
3 is a flowchart showing a processing procedure of an antibody structure analysis step shown in FIG.
FIG. 4 (a) is a view showing the amino acid sequence (primary structure) of the variable region of the heavy chain of antibody i41SL1-2 and the base sequence encoding it; (b) is the light chain of antibody i41SL1-2. It is a figure which shows the amino acid sequence (primary structure) of a variable region, and the base sequence which codes it.
5A is a diagram showing the three-dimensional structure of the variable region of the light chain of antibody i41SL1-2, and FIG. 5B is the diagram showing the three-dimensional structure of the variable region of the heavy chain of antibody i41SL1-2. FIG.
6A is a diagram showing the result of the polypeptide degradation reaction of the light chain of antibody i41SL1-2, and FIG. 6B is the diagram showing the result of the polypeptide degradation reaction of the heavy chain of antibody i41SL1-2. is there.
FIG. 7A is a diagram showing the results of kinetic analysis of the polypeptide degradation reaction of the light chain of antibody i41SL1-2, and FIG. 7B is the result of the polypeptide degradation reaction of the heavy chain of antibody i41SL1-2. It is a figure which shows the result of a kinetic analysis.
FIG. 8 (a) is a view showing the amino acid sequence (primary structure) of the variable region of the heavy chain of antibody i41-7 and the base sequence encoding it; (b) is the light chain of antibody i41-7. It is a figure which shows the amino acid sequence (primary structure) of a variable region, and the base sequence which codes it.
9A is a diagram showing the three-dimensional structure of the variable region of the light chain of antibody i41-7, and FIG. 9B shows the three-dimensional structure of the variable region of the light chain of antibody i41-7. (C) is a diagram showing the three-dimensional structure of the variable region of the heavy chain of antibody i41-7.
FIG. 10 (a) is a view showing the result of the polypeptide degradation reaction of the light chain of antibody i41-7, and FIG. 10 (b) is the view showing the result of the polypeptide degradation reaction of the heavy chain of antibody i41-7. (C) is an electrophoretic diagram and a graph showing the results of an antigen protein (inactive 41S-2-L) degradation reaction by the light chain of antibody i41-7.
11A is a diagram showing a three-dimensional structure of a heavy chain and a light chain of a 1DBA strain, and FIGS. 11B and 11C are enlarged views of a part thereof.
FIG. 12 shows the results of polypeptide degradation reaction of i41-7 complete antibody, heavy chain and light chain of antibody i41-7, and MA-2 antibody.
FIG. 13 is a schematic diagram showing the three-dimensional structure of the variable region of the HpU-20 L chain.
FIG. 14 is a schematic diagram showing the three-dimensional structure of the variable region of the HpU-20 heavy chain.
FIG. 15 shows the amino acid sequence of the variable region of the HpU-20 L chain and the base sequence of the gene encoding it.
FIG. 16 shows the amino acid sequence of the variable region of the HpU-20 heavy chain and the base sequence of the gene encoding it.
FIG. 17 is a diagram showing the results (Lot. 1) of a decomposition experiment of TP41-1 peptide (in the figure, indicated as TP) using HpU-20-L and HpU-20-H.
FIG. 18 is a diagram showing the result (Lot. 2) of the degradation experiment of TP41-1 peptide (in the figure, indicated as TP) using HpU-20-L and HpU-20-H.
FIG. 19 is a diagram showing the results of SDS-PAGE performed on a sample 1 hour after the start of the reaction in a protein degradation experiment using HpU-20 L chain.
FIG. 20 is a diagram showing the results of SDS-PAGE performed on a sample 1 hour after the start of the reaction in a proteolysis experiment using an HpU-20 L chain.
FIG. 21 is a diagram showing the results of SDS-PAGE performed on a sample 1 hour after the start of the reaction in a proteolysis experiment using an HpU-20 L chain.
FIG. 22 is a diagram showing a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of UA-15 consisting of a light chain and a heavy chain. In this figure, it is shown that a catalytic triad residue-like structure appears only on the light chain.
FIG. 23 shows the result of a decomposition experiment of TP41-1 peptide using UA-15-L (shown as L in the figure) and UA-15-H (shown as H in the figure) (Lot. It is a figure which shows 1).
FIG. 24 shows the results of TP41-1 peptide degradation experiments using UA-15-L (shown as L in the figure) and UA-15-H (shown as H in the figure) (Lot. It is a figure which shows 2).
FIG. 25: Decomposition experiment of TP41-1 peptide (denoted as TP in the figure) at 15 ° C., 25 ° C. and 37 ° C. using UA-15-L (denoted as L in the figure) It is a figure which shows the result of having performed. In this decomposition experiment, after performing the experiment whose result is shown in FIG. 23, the TP41-1 peptide was added again to see the difference in reaction temperature.
FIG. 26 shows the results of kinetic analysis of TP41-1 peptide degradation reaction using UA-15-L.
FIG. 27 is a schematic diagram showing a three-dimensional structure of a variable region of ECL2B-4-L.
FIG. 28 is a schematic diagram showing the three-dimensional structure of the variable region of ECL2B-4-H.
FIG. 29 shows the amino acid sequence (primary structure) of the variable region of ECL2B-4-L and the base sequence encoding it.
FIG. 30 shows the amino acid sequence (primary structure) of the variable region of ECL2B-4-H and the base sequence encoding it.
FIG. 31 shows the results of an ECL2B peptide degradation experiment using ECL2B-4-L (shown as L in the figure) and ECL2B-4-H (shown as H in the figure). .
FIG. 32 (a) shows the results of monitoring the concentration of ECL2B using HPLC in the ECL2B peptide degradation experiment using ECL2B-4-L. (B) is a figure which shows the result of having monitored the density | concentration of ECL2B using HPLC in the degradation experiment of ECL2B peptide using ECL2B-4-H. (C) is a figure which shows the result of having monitored the density | concentration of ECL2B using HPLC as control of the decomposition | disassembly experiment of ECL2B peptide.
FIG. 33 is a diagram showing a kinetic analysis result in an enzymatic degradation experiment. (A) is a figure which shows the relationship between a substrate (ECL2B peptide) density | concentration and a decomposition rate, (b) is a figure which shows the result of a Hanes-Woolf plot.
FIG. 34 shows the peptide of TP41-1 (shown as TP in the figure) using ECL2B-4-L (shown as L in the figure) and ECL2B-4-H (shown as H in the figure). It is a figure which shows the result of having conducted decomposition | disassembly experiment.
FIG. 35 (a) is a diagram showing the results of monitoring the concentration of TP41-1 using HPLC in the TP41-1 peptide degradation experiment using ECL2B-4-L. (B) is a figure which shows the result of having monitored the density | concentration of TP41-1 using HPLC in the decomposition | disassembly experiment of TP41-1 peptide using ECL2B-4-H. (C) is a figure which shows the result of having monitored the density | concentration of TP41-1 using HPLC as control of the decomposition experiment of TP41-1 peptide.
FIG. 36 is a diagram showing a kinetic analysis result in an enzyme degradation experiment. (A) is a figure which shows the relationship between a substrate (TP41-1 peptide) density | concentration and a decomposition rate, (b) is a figure which shows the result of a Hanes-Woolf plot.
FIG. 37 shows the results of analysis using PDB data for the catalytic triad residue-like structure and germline gene.
FIG. 38 shows the results of analysis of strains owned by the present inventors regarding the catalytic triad residue-like structure and germline gene.
[Explanation of symbols]
10 Antibody structure analysis system
11 Input section
12 Display section
13 Printing department
14 Storage unit
15 Control unit
16 Communication Department
60 communication line
61 Personal computer (PC)
62 servers
151 3D structure prediction unit
152 catalytic triad residue structure confirmation part

Claims (5)

An antibody enzyme, which is an antibody fragment against urease of Helicobacter pylori and acts as a peptide degrading enzyme, comprising the amino acid sequence shown in SEQ ID NO: 16. A gene encoding the antibody enzyme according to claim 1. The gene according to claim 2, comprising the base sequence shown in SEQ ID NO: 17. A vector comprising the gene according to claim 2 or 3. A host cell into which the gene according to claim 2 or 3 has been introduced.

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