JP4334271B2 - Antibody sequence creation device, antibody sequence creation method, program, and recording medium - Google Patents

Description

ここで、ΔＥは存在率であり、ｉは抗体断片配列情報の番号（ｉは１〜ｎ−ｆ＋１の範囲の整数である（ｎは抗体配列情報の長さ、ｆは抗体断片配列情報の長さである。）。）であり、δｅｘｉｓｔ（ｊ）は変異前に対する変異後のｊ番目の抗体断片配列情報に一致する他の生物種に関するアミノ酸断片配列情報の存在率であり、ｓｉｍ（ｓ'，ｓ）は変異前の抗体断片配列情報「ｓ」と変異後のアミノ酸断片配列情報「ｓ'」との抗体活性維持関数（本実施形態においては「１」を設定）であり、ｅｘｉｓｔ（ｓ'，ｊ）およびｅｘｉｓｔ（ｓ，ｊ）は変異後のｊ番目の抗体断片配列情報に一致する他の生物種に関するアミノ酸断片配列情報の存在率および変異前のｊ番目の抗体断片配列情報に一致する他の生物種に関するアミノ酸断片配列情報の存在率である。
【０２５３】
なお、ｅｘｉｓｔ（ｓ'，ｊ）およびｅｘｉｓｔ（ｓ，ｊ）は、例えば、抗体断片配列情報「ｓ'」（「ｓ」）に一致する他の生物種に関するアミノ酸断片配列情報が存在するときは「１」を、存在しないときは「０」を与えてもよい。
【０２５４】
つまり、ΔＥは、変異前の抗体断片配列情報「ｓ」を一致する他の生物種に関するアミノ酸断片配列情報に変異させたことによる、当該変異された他の生物種に関するアミノ酸断片配列情報および当該変異の影響を受けた抗体断片配列情報「ｓ'」に一致する他の生物種に関するアミノ酸断片配列情報の存在率の変化を意味する。
【０２５５】
具体的には、ΔＥが負である場合は変異前に比べて変異後の抗体配列情報に含まれる他の生物種に関するアミノ酸断片配列情報が減少したこと、つまり、変異前に比べて変異後の抗体配列情報は他の生物種に対する抗原性が高まったことを意味する。
【０２５６】
一方、ΔＥが正である場合は、変異前に比べて変異後の抗体配列情報に含まれる他の生物種に関するアミノ酸断片配列情報が増加したこと、つまり、変異前に比べて変異後の抗体配列情報は他の生物種に対する抗原性が低下することを意味する。
【０２５７】
また、上述した抗体活性維持関数ｓｉｍ（ｓ'，ｓ）は、例えば、本実施形態においては「１」を設定したが、それ以外の定数値でもよく、また、以下の抗体活性維持関数の決定方法に基づいて定義してもよい。
【０２５８】
ここで、抗体活性維持関数ｓｉｍ（ｓ'，ｓ）の決定の方法の３つの例について説明する。
【０２５９】
（１）まず、ｓ'とｓの間におけるアミノ酸残基の違いの数に基づいて抗体活性維持関数を決定してもよい。
【０２６０】
具体的には、例えば、ｓ'（一致する他の生物種に関するアミノ酸断片配列情報あり）が「ＡＣＤＥＦＧＨＩＫ」であり、ｓ（一致する他の生物種に関するアミノ酸断片配列情報なし）が「ＹＣＤＥＦＧＨＩＫ」である場合、２つの断片配列は「Ａ」と「Ｙ」が１つ異なっているので、「ｓｉｍ（ｓ'，ｓ）＝１」となる。
【０２６１】
（２）また、ｓ'とｓの間のアミノ酸の差において、構造が異なりそうと予測される変異（予め、断片構造予測手法などを用いて情報として取得する）であればｓｉｍ（ｓ'，ｓ）の値は小さくとり、そうでなければ大きくとってもよい。
【０２６２】
具体的には、例えば、ｓ'（一致する他の生物種に関するアミノ酸断片配列情報あり）が「ＡＣＤＥＦＧＨＩＫ」であり、ｓ（一致する他の生物種に関するアミノ酸断片配列情報なし）が「ＧＣＤＥＦＧＨＩＫ」である場合において、「Ａ」と「Ｇ」の１つが異なっているが、予め取得されている情報である、例えば、「Ａ」から「Ｇ」への構造変化寄与率が「ｄｉｆｆ（Ａ，Ｇ）＝０．５」である場合、ｓｉｍ（ｓ'，ｓ）は例えばｄｉｆｆの逆数「１／０．５」、つまり、「ｓｉｍ（ｓ'，ｓ）＝１／０．５」となる。
【０２６３】
また、具体的には、例えば、ｓ'（一致する他の生物種に関するアミノ酸断片配列情報あり）が「ＡＣＤＥＦＧＨＩＫ」であり、ｓ（一致する他の生物種に関するアミノ酸断片配列情報なし）が「ＡＣＤＥＦＷＨＩＫ」である場合において、「Ｇ」と「Ｗ」の１つが異なっているが、予め取得されている情報である、例えば、「Ｇ」から「Ｗ」への構造変化寄与率が「ｄｉｆｆ（Ｇ，Ｗ）＝３．０」である場合、「ｓｉｍ（ｓ'，ｓ）＝１／３．０」となる。
【０２６４】
（３）さらに、ｓ'とｓの間のアミノ酸の差において、アフィニティに大きく寄与すると予測される変異（予め、情報として取得する）であれば、ｓｉｍ（ｓ'，ｓ）の値は大きくとり、そうでなければ小さくとってもよい。
【０２６５】
これにて、抗体の変異を決定する計算方法の説明を終了する。
【０２６６】
［実施例］
つぎに、本発明の方法をマウス抗体配列のヒト化に適用して、本発明の方法が正しい結果を導く可能性があるかを、以下の実施例を用いて検証する。
【０２６７】
まず、本実施例において、当該ヒト化の状態の算出・表示は、以下の手順（以下、ヒト化状態算出・表示手順と記す。）にて行った。
【０２６８】
１．抗体配列情報作成装置１００は、取得部１０２ａの処理により、マウス抗体配列情報を取得し、抗体配列情報ファイル１０６ａの所定の領域に格納する。
【０２６９】
２．抗体配列情報作成装置１００は、スライド分割部１０２ｉの処理により、取得されたマウス抗体配列情報を先頭から１アミノ酸残基ずつスライドしながら予め定められた「９」の長さのマウス抗体断片配列情報に分割し、抗体断片配列情報ファイル１０６ｂの所定の領域に格納する。
【０２７０】
３．抗体配列情報作成装置１００は、一致検索部１０２ｃの処理により、分割した各マウス抗体断片配列情報を問い合わせ（ｑｕｅｒｙ）配列として、non-reduntant sequence data で 種が human である外部のデータベース（以下、「ｎｒ．Ｈｕｍａｎ」という。）、または、「Ｈｕｍａｎ，Ｉｇ ＴｃＲ」データベースにアクセスし、一致するヒト断片配列情報を、ＢＬＡＳＴを用いて検索する。
【０２７１】
４．抗体配列情報作成装置１００は、制御部１０２の処理により、当該一致検索部１０２ｃの処理の結果、存在率として、検索された場合には「１」を検索されなかった場合には「０」を付加する。
【０２７２】
５．抗体配列情報作成装置１００は、制御部１０２の処理により、マウス抗体配列情報のアミノ酸残基ごとに、以下の数式２により、ヒト断片配列情報の存在率を算出する。
【数２】

ここで、Ｅ（ｉ）は残基番号ｉ（ｉは１〜ｎ（ｎはマウス抗体配列情報の長さである。）の値をとる整数である。）に関係するアミノ酸残基におけるヒト断片配列情報の存在率であり、ｆは予め定められたマウス抗体断片配列情報の長さ（本実施例においては「９」である。）であり、ｅｘｉｓｔ（ｓ，ｊ）はｊ番目のアミノ酸残基を先頭に持つマウス抗体断片配列情報に一致するヒト断片配列情報の存在率（本実施例においては存在する場合は「１」を、存在しない場合は「０」を与える。）である。
【０２７３】
つまり、Ｅ（ｉ）は、ｉ番目のアミノ酸残基を含む各マウス抗体断片配列情報（数はｆに一致する。）に一致するヒト断片配列情報の存在率を意味する。なお、取り得る値は、０〜ｆ（本実施例においては、「９」）である。
【０２７４】
６．抗体配列情報作成装置１００は、制御部１０２の処理により、算出された残基ごとのヒト断片存在率を出力装置１１４にグラフ表示する。
【０２７５】
これにて、ヒト化状態算出・表示手順の説明を終了する。なお、以下に説明するグラフは、当該ヒト化状態算出・表示手順に従って表示されたものである。
【０２７６】
［実施例１：従来技術で用いられた、および、ヒト化されたマウスハイブリドーマ細胞「CRA4」由来のマウスモノクローナル抗体(以下「CRA4」と呼ぶ)のヒト化状態］
つぎに、特開平７−１６５７９９号公報および特開平９−１９１８８６号公報（以下、従来技術と記す。）で用いられた、および、ヒト化されたＦｃＲマウス抗体「ＣＲＡ４」のヒト化状態について、図１２から図１５を参照して説明する。なお、一致検索におけるデータベースは「ｎｒ．Ｈｕｍａｎ」である。
【０２７７】
図１２は、本実施例における従来技術で用いられた「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。また、図１３は、本実施例における従来技術で用いられた「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。また、図１４は、本実施例における従来技術でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。さらに、図１５は、本実施例における従来技術でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。
【０２７８】
ここで、図１２を一例にグラフに表示されている事項について説明する。なお、以下に示すグラフにおいても同様である。
【０２７９】
まず、図１２において、横軸は残基番号であり、縦軸はアミノ酸残基ごとのヒト断片の存在率である。また、黒色で示す部分は一致するヒト断片配列情報が存在することを示す部分であり、灰色で示す部分は存在しなかったことを示す部分である。また、グラフの上部に示す横棒は、ＣＤＲの範囲を示すものである。
【０２８０】
図１２および図１３に示すように、ヒト化前のＣＲＡ４は、黒色で示す部分の面積が小さい、つまり、ヒト断片の存在率が少ない状態である。
【０２８１】
また、図１２から図１５に示すように、グラフの形を見るとマウス抗体とヒト化抗体では明らかに特徴の違いが見られる。例えば、うまくヒト化されている抗体では、ＣＤＲの部分を除いて、アミノ酸残基ごとのヒト断片の存在率はほぼ最大値（＝断片配列の長さ「９」）に保たれている。そして、ヒト化された抗体では、ＣＤＲに関わる部分は、ＣＤＲ中心から「Ｖ字型」を描いているのが特徴である。
【０２８２】
これにて、実施例１の説明を終了する。
【０２８３】
［実施例２：本発明でヒト化された抗体のヒト化状態（その１）］
つぎに、本発明でヒト化された抗体のヒト化状態について、図１６から図２１を参照して説明する。
【０２８４】
（２−１．ＣＲＡ４のヒト化状態（その１））
まず、従来技術で用いられたＣＲＡ４のヒト化状態について、図１６から図１８を参照して説明する。なお、本発明の方法によるＣＲＡ４のヒト化において、一致検索部１０２ｃおよび類似検索部１０２ｄは、「nr.Human」に対して検索を行った。また、抗体活性維持率決定部１０２ｆは、すべての類似するヒト酸断片配列情報に対して変異を行った。さらに、変異決定部１０２ｇは、変異前に対して変異後の抗体配列維持率が減少した場合、変異を終了した。
【０２８５】
図１６は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。また、図１７は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。さらに、図１８は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のアミノ酸配列の一例を示す図である。
【０２８６】
ここで、図１８の上部に示すアミノ酸配列は、Ｌ鎖およびＨ鎖における従来技術で用いられたＣＲＡ４（図１８に示す「mouse」）および本発明の方法でヒト化されたＣＲＡ４（図１８に示す「humanized(new)」）のアミノ酸配列である。
【０２８７】
また、図１８の下部に示すアミノ酸配列は、Ｌ鎖およびＨ鎖における従来技術でヒト化されたＣＲＡ４（図１８に示す「「humanized(con)」）および本発明の方法でヒト化されたＣＲＡ４（図１８に示す「humanized(new)」）のアミノ酸配列である。
【０２８８】
また、図１８において、「ｄｉｆ」の行に付されている「＊」は、アミノ酸が異なっているアミノ酸残基であることを示す記号であり、「ｃｄｒ」の行に付されている「Ｌ」〜「Ｊ」は、ＣＤＲ部位に含まれるアミノ酸残基であることを示す記号である。
【０２８９】
図１６から図１８に示すように、Ｌ鎖においてはＣＤＲ部位以外ほぼヒト化されているが、Ｈ鎖においては１つのＣＤＲ部位がさらにヒト化されていることが理解できる。この結果は、図１４および図１５に示す従来技術でヒト化された「ＣＲＡ４」の結果と同じである。
【０２９０】
図１６および図１７と、図１４および図１５とに示す存在率において、全体の存在率の値はそれほど変わらないが、それぞれの抗体配列を比較する（図１８参照。）と違いが大きいことがわかる。
【０２９１】
そして、従来技術でヒト化されたＣＲＡ４のＨ鎖においては、アミノ酸残基の違いが大きく、１８個もの変異の差があった。また、変異の絶対数にも違いがあり、従来技術でヒト化されたＣＲＡ４の変異数は、Ｌ鎖で１９個、Ｈ鎖で２２個であった (図18を参照)。
【０２９２】
一方、本発明の方法でヒト化したＣＲＡ４の変異数は、Ｌ鎖で１３個、Ｈ鎖で１３個であり、ほぼ半分となった。変異を多く入れると、一般に抗体活性が低下する可能性が大きくなることを考慮すると、本発明の手法が抗体活性の低下を避ける可能性が高くなると思われる。
【０２９３】
（２−２．ＣＲＡ４のヒト化状態（その２））
つぎに、従来技術で用いられたＣＲＡ４のヒト化状態について、図１９から図２１を参照して説明する。なお、本発明の方法によるＣＲＡ４のヒト化において、一致検索部１０２ｃおよび類似検索部１０２ｄは、外部データベース「ｎｒ．Ｈｕｍａｎ」に対して検索を行った。また、抗体活性維持率決定部１０２ｆは、最も類似度の高いヒト酸断片配列情報に対して変異を行った。さらに、変異決定部１０２ｇは、変異前に対して変異後の抗体活性維持率が減少しても変異を終了しなかった。
【０２９４】
図１９は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の別の一例を示す図である。また、図２０は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の別の一例を示す図である。さらに、図２１は、本実施例における本発明の方法でヒト化された「ＣＲＡ４」のアミノ酸配列の別の一例を示す図である。
【０２９５】
図１９から図２１に示すように、図１６から図１８の結果同様、Ｌ鎖においてはＣＤＲ部位以外ほぼヒト化されており、Ｈ鎖においては１つのＣＤＲ部位がヒト化されていることが理解できる。
【０２９６】
また、図１９および図２０と図１４および図１５とに示す存在率において、全体の存在率の値はそれほど変わらないが、それぞれの配列を比較する（図２１参照。）と違いが大きいことがわかる。
【０２９７】
そして、従来技術でヒト化されたＣＲＡ４のＨ鎖においては、アミノ酸残基の違いが大きく、１９個もの変異の差があった。また、変異の絶対数にも違いがあり、従来技術でヒト化されたＣＲＡ４の変異数は、Ｌ鎖で１９個、Ｈ鎖で２２個であった。
【０２９８】
一方、本発明の方法でヒト化したＣＲＡ４の変異数は、Ｌ鎖で１３個、Ｈ鎖で１３個であり、ほぼ半分となった。変異を多く入れると、一般に抗体活性が低下する可能性が大きくなることを考慮すると、本発明の手法が抗体活性の低下を避ける可能性が高くなると思われる。
【０２９９】
これにて、実施例２の説明を終了する。
【０３００】
［実施例１および実施例２に対する考察］
マウス抗体(図12、13参照)においては、ヒト断片の存在率は少ない。この蛋白質がヒト中でランダムに断片化されたとすると、その断片がヒト由来である可能性は極めて低いと考えられる(つまり、抗原性があるといえる)。
【０３０１】
一方、ヒト化抗体(既知ヒト化抗体(図14,15参照)、実施例１(図16、17参照)、実施例２(図19, 20参照))においては、ヒト断片の存在率は一般に大きく、CDR(とその近辺)を除き、あるアミノ酸残基が関わる全てのヒト断片から非ヒト由来断片が存在しない状態となる。この状態下においては、CDR(とその近辺)以外の部位におけるランダムな断片化によって、ほぼヒト由来断片が得られると言えるだろう(つまり、抗原性は低い)。
【０３０２】
変異による抗原性低下と活性維持を両立させる、という課題はヒト化抗体作成時の主要な問題点である。一般に、抗原性低下と活性維持はトレードオフの関係にある。すなわち、ヒト化への変異により抗原性は低くなるが、変異が多くなってくると、活性を低下確率の増大が予想される。そこで例えば、活性低下の一つの指標例として、マウス→ヒト化抗体への変異の数を考えることができる。ここでは、変異の数が多くなる＝活性低下の可能性が高くなる、と考える。
【０３０３】
本発明によるヒト化手法(図16, 17, 19, 20)と既存手法によるヒト化(図14,15)を比較してみよう。ヒト化グラフとしてはあまり違いが認められないにも関わらず、ヒト化された配列は異なっていることが分かる(図18、19参照)。
【０３０４】
配列の変異の違いを見ると、本発明におけるヒト化手法の方が従来手法と比較して変異の数が少ないということがわかる。ヒト化としての状況は同じだが変異の数が少ない、ということは、抗原性は低いが活性は維持されている可能性が高い、ということを意味する。つまりこの指標で見る限り、従来手法と比較して抗原性と活性維持が共に(特に活性維持に関して)改善されていることがわかる。
【０３０５】
本発明では変異による抗体活性の維持をさらに深く考慮するため、例えば、抗体活性を維持するヒト断片配列があれば当該ヒト断片配列に変異させる、あるいは、抗体活性に影響のあるヒト断片配列には変異させない、などの仕組みを導入している。
【０３０６】
具体的には、本発明においては、例えば、抗体活性の維持と相関のある配列距離、つまり、抗体活性の維持という観点から見た配列間距離（例えば、配列の類似度など）などを定義することができる。
【０３０７】
また、本発明においては、抗体活性維持関数の類似度を、ＢＬＡＳＴなどで用いられる例えば、ＰＡＭ３０などの行列を用いて定義してよく、さらに、当該行列を活性維持用に作りかえて定義してもよい。
【０３０８】
具体的には、本発明において、上述した抗体活性維持関数「ｓｉｍ（ｓ'，ｓ）」などを決定してもよい。
【０３０９】
また、本発明において、例えば、電荷情報やエネルギーやアフィニティなどのアミノ酸の特徴に関する指標情報、断片配列と構造情報との相関情報、構造モデリングなどの詳細な構造情報などを利用して、より抗体活性を維持できる抗体活性維持関数などを決定してもよい。
【０３１０】
つぎに、CDRの抗原性について考察してみる。既知ヒト化抗体、実施例1、実施例2におけるヒト化抗体のCDR(とその近辺)でのグラフは、共に V 字型を描くことが分かる(図14,15,16,17,19,20)。これは、CDR付近の断片データが「nr.Human」上に存在していないことを示している。
【０３１１】
既知ヒト化抗体においてもCDRが「nr.Human」上に存在しないことに関して、1.CDRは抗原性を免除される傾向にあるので、この部分に関してのヒト化は考慮する必要がない、2.CDRは germline 配列が rearrange された遺伝子によって得られた配列であり、従って普通の nr データベース上には存在しない可能性がある。よってCDR の抗原性に関しては、CDR専用の「ヒトCDR断片データ」を「nr.Human」の代わりに用いてもよい、という二つの解釈が存在する。
【０３１２】
従来手法は基本的に1.の立場をとっていると考えられるが、本発明の枠組みは、CDRは変異させないという従来手法とは異なり、「ヒトCDR断片データ」を用いることによりCDR部位もヒト化することが可能である。すなわち我々の手法においては、CDR を変異させることも可能である。
【０３１３】
上記1.の立場で考えるか、2.の立場で考えるかによってCDRの取り扱いが変わるが、どちらの方向でヒト化を進めるのがよいかは、この部位が抗原性にどの程度深く関与しているかに依存する。
【０３１４】
もし、CDR部位が抗原性にあまり深く関与していないのであれば、本発明において、例えばヒト化においてCDRをあまり考慮しなくて良い(上記1.の立場)。つまり、本発明においては、抗体配列情報をCDRおよびフレームワーク領域に分割し、当該フレームワーク領域のみをヒト化の対象とすることで、より変異の少ないヒト化抗体を作成してもよい。
【０３１５】
一方、CDRが抗原性に深く関与しているのであれば、本発明において、例えば、まず上記「ヒトCDR断片データ」のようなCDRの断片データベースを作成してもよい。なお、このデータベースを参照することにより、抗原性の排除をさらに効果的に行うことができる。
【０３１６】
抗体活性の維持に関しては、CDRは抗原と直接結合する部位であることが多く、この部位の変異が直接の失活原因となり得るため、CDR部位のヒト化には慎重な取り扱いを要する。そこで、本発明においては、上記抗体活性維持関数の他に、より活性に敏感な抗体活性維持関数を導入してもよく、また、例えば立体構造モデリング手法等を陽に取り入れてもよい。
【０３１７】
［実施例３：本発明でヒト化された抗体のヒト化状態（その２）］
つぎに、本発明の方法でヒト化された抗組織因子抗体のヒト化状態について、図２２から図２８を参照して説明する。なお、例とした抗体配列は、マウス抗体としてはPDBコード「1ahw」の配列、既知ヒト化抗体としては「1jps」の配列である。
【０３１８】
図２２は、本実施例において用いられたマウス抗体「１ａｈｗ」のＬ鎖のヒト化状態の一例を示す図である。また、図２３は、本実施例において用いられたマウス抗体「１ａｈｗ」のＨ鎖のヒト化状態の一例を示す図である。
【０３１９】
また、図２４は、本実施例において用いられた既知ヒト化マウス抗体「１ｊｐｓ」のＬ鎖のヒト化状態の一例を示す図である。また、図２５は、本実施例において用いられた既知ヒト化抗体「１ｊｐｓ」のＨ鎖のヒト化状態の一例を示す図である。
【０３２０】
また、図２６は、本実施例において本発明の方法で作成されたヒト化抗体「humanized(new) 1ahw」のＬ鎖のヒト化状態の一例を示す図である。また、図２７は、本実施例において本発明の方法で作成されたヒト化抗体「humanized(new) 1ahw」のＨ鎖のヒト化状態の一例を示す図である。
【０３２１】
さらに、図２８は、本実施例において本発明の方法で作成されたヒト化抗体「humanized(new) 1ahw」のアミノ酸配列の一例を示す図である。
【０３２２】
図２２から図２５を見る限りは、既知ヒト化抗体「1jps」も効果的にヒト化がされているように見えるが、図２４から図２７を見ると、本発明の方法で作成されたヒト化抗体「humanized(new) 1ahw」(図26,図27を参照)のほうがＣＤＲ付近の断片配列においてさらにヒト化されていることが理解できる。このことから、本発明の方法により、抗体活性が高く維持され、かつ、抗原性が効果的に排除されたヒト化抗体を作成できることが理解できる。
【０３２３】
これにて、実施例３の説明を終了する。
【０３２４】
［他の実施の形態］
さて、これまで本発明の実施の形態について説明したが、本発明は、上述した実施の形態以外にも、上記特許請求の範囲に記載した技術的思想の範囲内において種々の異なる実施の形態にて実施されてよいものである。
【０３２５】
例えば、抗体配列作成装置１００がスタンドアローンの形態で処理を行う場合を一例に説明したが、抗体配列作成装置１００とは別筐体で構成されるクライアント端末からの要求に応じて処理を行い、その処理結果を当該クライアント端末に返却するように構成してもよい。
【０３２６】
また、実施形態において説明した各処理のうち、自動的に行なわれるものとして説明した処理の全部または一部を手動的に行うこともでき、あるいは、手動的に行なわれるものとして説明した処理の全部または一部を公知の方法で自動的に行うこともできる。
【０３２７】
この他、上記文書中や図面中で示した処理手順、制御手順、具体的名称、各種の登録データや検索条件等のパラメータを含む情報、画面例、データベース構成については、特記する場合を除いて任意に変更することができる。
【０３２８】
また、抗体配列作成装置１００に関して、図示の各構成要素は機能概念的なものであり、必ずしも物理的に図示の如く構成されていることを要しない。
例えば、抗体配列作成装置１００の各部または各装置が備える処理機能、特に制御部１０２にて行なわれる各処理機能については、その全部または任意の一部を、ＣＰＵ（Ｃｅｎｔｒａｌ Ｐｒｏｃｅｓｓｉｎｇ Ｕｎｉｔ）および当該ＣＰＵにて解釈実行されるプログラムにて実現することができ、あるいは、ワイヤードロジックによるハードウェアとして実現することも可能である。なお、プログラムは、後述する記録媒体に記録されており、必要に応じて抗体配列作成装置１００に機械的に読み取られる。
【０３２９】
すなわち、ＲＯＭまたはＨＤなどの記憶部１０６などには、ＯＳ（Ｏｐｅｒａｔｉｎｇ Ｓｙｓｔｅｍ）と協働してＣＰＵに命令を与え、各種処理を行うためのコンピュータプログラムが記録されている。このコンピュータプログラムは、ＲＡＭ等にロードされることによって実行され、ＣＰＵと協働して制御部１０２を構成する。また、このコンピュータプログラムは、抗体配列作成装置１００に対して任意のネットワーク３００を介して接続されたアプリケーションプログラムサーバに記録されてもよく、必要に応じてその全部または一部をダウンロードすることも可能である。
【０３３０】
また、本発明にかかるプログラムを、コンピュータ読み取り可能な記録媒体に格納することもできる。ここで、この「記録媒体」とは、フレキシブルディスク、光磁気ディスク、ＲＯＭ、ＥＰＲＯＭ、ＥＥＰＲＯＭ、ＣＤ−ＲＯＭ、ＭＯ、ＤＶＤ等の任意の「可搬用の物理媒体」や、各種コンピュータシステムに内蔵されるＲＯＭ、ＲＡＭ、ＨＤ等の任意の「固定用の物理媒体」、あるいは、ＬＡＮ、ＷＡＮ、インターネットに代表されるネットワークを介してプログラムを送信する場合の通信回線や搬送波のように、短期にプログラムを保持する「通信媒体」を含むものとする。
【０３３１】
また、「プログラム」とは、任意の言語や記述方法にて記述されたデータ処理方法であり、ソースコードやバイナリコード等の形式を問わない。なお、「プログラム」は必ずしも単一的に構成されるものに限られず、複数のモジュールやライブラリとして分散構成されるものや、ＯＳ（Ｏｐｅｒａｔｉｎｇ Ｓｙｓｔｅｍ）に代表される別個のプログラムと協働してその機能を達成するものをも含む。なお、実施の形態に示した各装置において記録媒体を読み取るための具体的な構成、読み取り手順、あるいは、読み取り後のインストール手順等については、周知の構成や手順を用いることができる。
【０３３２】
記憶部１０６に格納される各種のデータベース等（抗体配列情報ファイル１０６ａ〜アミノ酸断片配列情報データベース１０６ｇ）は、ＲＡＭ、ＲＯＭ等のメモリ装置、ハードディスク等の固定ディスク装置、フレキシブルディスク、光ディスク等のストレージ手段であり、各種処理やウェブサイト提供に用いる各種のプログラムやテーブルやファイルやデータベースやウェブページ用ファイル等を格納する。
【０３３３】
また、抗体配列作成装置１００は、既知のパーソナルコンピュータ、ワークステーション等の情報処理端末等の情報処理装置にプリンタやモニタやイメージスキャナ等の周辺装置を接続し、該情報処理装置に本発明の方法を実現させるソフトウェア（プログラム、データ等を含む）を実装することにより実現してもよい。
【０３３４】
さらに、抗体配列作成装置１００等の分散・統合の具体的形態は明細書および図面に示すものに限られず、その全部または一部を、各種の負荷等に応じた任意の単位で、機能的または物理的に分散・統合して構成することができる（例えば、グリッド・コンピューティングなど）。例えば、各データベースを独立したデータベース装置として独立に構成してもよく、また、処理の一部をＣＧＩ（Ｃｏｍｍｏｎ Ｇａｔｅｗａｙ Ｉｎｔｅｒｆａｃｅ）を用いて実現してもよい。
【０３３５】
【発明の効果】
以上詳細に説明したように、本発明によれば、相補性決定領域情報および／またはフレームワーク領域情報を含む抗体配列情報を取得し、取得された抗体配列情報を予め定められた長さの抗体断片配列情報に分割し、分割された抗体断片配列情報ごとに、一致する他の生物種に関するアミノ酸断片配列情報を検索し、一致するアミノ酸断片配列情報が検索されなかった抗体断片配列情報ごとに、類似するアミノ酸断片配列情報を検索し、検索された各類似するアミノ酸断片配列情報と検索元の抗体断片配列情報とを入れ替えて抗体配列情報を変異し、変異された抗体配列情報について、変異前の抗体配列情報の活性を維持しているかを示す抗体活性維持率を決定し、決定された抗体活性維持率に基づいて、抗体配列情報の変異を決定するので、他の生物種に関するアミノ酸断片配列情報を多く含む、つまり他の生物種において抗原性が低い抗体配列情報を効率よく作成することができ、かつ、本発明によれば、抗体活性維持率に基づいて、抗体配列情報の変異を決定するので、抗体活性に影響を与える部分が変異していない、つまり抗体活性の高い抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３３６】
また、本発明によれば、抗原性はタンパク質の断片配列を単位にＴＣＲにて認識されているという免疫反応のしくみに基づいて、抗体断片配列情報を他の生物種に関するアミノ酸断片配列情報に変異させているので、生物学的事実を反映した抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３３７】
また、本発明によれば、抗体配列情報を、相補性決定領域情報とフレームワーク領域情報とに分割するので、例えば、相補性決定領域情報とフレームワーク領域情報にまたがる抗体断片配列情報を変異の対象に考慮せずに抗体配列情報を作成すことができ、高精度で抗体配列情報を作成することができ、かつ、計算量を効果的に削減することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３３８】
また、本発明によれば、例えば、相補性決定領域情報の関与する抗体断片配列情報が抗原性を持つ可能性が低い場合には、変異の対象をフレームワーク領域情報のみに限定して取り扱うことができるため、相補性決定領域情報は元の配列のままにフレームワーク領域情報のみを他の生物種に関するアミノ酸断片配列情報に変異させた、抗原性が低く、かつ、抗体活性の高い抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。逆に、相補性決定領域情報の関与する抗体断片配列情報が抗原性を持つ可能性がある場合には、変異の対象としてフレームワーク領域情報と相補性決定領域情報の両方を考慮することができる。この際、相補性決定領域情報に関するデータベースをフレームワーク領域情報に関するものとは別に用意することもできる。
【０３３９】
また、本発明によれば、抗体配列情報の先頭から１アミノ酸残基ずつスライドしながら予め定められた長さの抗体断片配列情報に分割するので、異物としてマクロファージに貪食されたタンパク質は任意の位置で分解され断片化された後、ＴＣＲに提示されるという免疫反応のしくみに基づいて、分割される可能性のある抗体断片配列情報を漏れなく作成するので、生物学的事実を反映した抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４０】
また、抗体断片配列情報の予め定められた長さは、９〜２０であるので、異物としてマクロファージに貪食されたタンパク質は任意の位置で９〜２０残基程度の断片配列に分解され断片化された後、ＴＣＲに提示されるという免疫反応のしくみに基づいて、抗体断片配列情報を作成するので、生物学的事実を反映した抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４１】
また、本発明によれば、入れ替える類似するアミノ酸断片配列情報と検索元の抗体断片配列情報との変化量を算出し、算出された変化量に基づいて、抗体活性維持率を決定するので、アミノ酸残基の変化による抗体活性の低下を考慮して、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４２】
すなわち、本発明によれば、アミノ酸残基の変化量が多くなれば抗体活性が失われ、また、アミノ酸残基の変化量が少なくなれば抗体活性が維持される点を考慮し、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４３】
また、本発明によれば、入れ替える類似するアミノ酸断片配列情報および検索元の抗体断片配列情報について、それぞれの構造を比較し、配列の変化が構造の変化にどれだけ寄与したかを示す構造変化寄与率を算出し、算出された構造変化寄与率に基づいて、抗体活性維持率を決定するので、抗体の構造の変化による抗体の抗体活性の低下を考慮して、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４４】
すなわち、本発明によれば、抗体の構造変化が大きくなれば抗体活性が失われ、また、抗体の構造変化が小さくなれば抗体活性が維持される点を考慮し、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４５】
また、本発明によれば、入れ替える類似するアミノ酸断片配列情報および検索元の抗体断片配列情報について、それぞれの配列上で変化したアミノ酸の性質に関する指標情報を算出し、算出された指標情報に基づいて、抗体活性維持率を決定するので、類似するアミノ酸断片配列情報および検索元の抗体断片配列情報について、それぞれの配列上における、例えば、電荷の差やエネルギーの差やアフィニティの差などの指標情報の変化による抗体活性の低下を考慮して、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４６】
すなわち、本発明によれば、類似するアミノ酸断片配列情報および検索元の抗体断片配列情報について、それぞれの配列上における、例えば、電荷の差やエネルギーの差やアフィニティの差などが大きくなれば抗体活性が失われ、一方、それぞれの配列上における、例えば、電荷の差やエネルギーの差やアフィニティの差などが小さくなれば抗体活性が維持される点を考慮し、元の抗体配列の一部を類似する他の生物種に関するアミノ酸断片配列情報に変異させることができ、そのため、他の生物種における抗原性を低下させ、かつ、抗体活性を高く維持させた抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４７】
また、本発明によれば、変異前に対して変異後の抗体配列情報の抗体活性維持率が減少した場合、変異を終了するので、変異前の抗体配列情報の抗体活性が下がるような変異をなくすことができ、これにより、抗原性を維持し、かつ、抗体活性の高い抗体配列情報を効果的に作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４８】
また、本発明によれば、抗体配列情報は、非ヒトに関する抗体配列情報であり、かつ、他の生物種に関するアミノ酸断片配列情報は、ヒトに関するアミノ酸断片配列情報であるので、相補性決定領域情報および／またはフレームワーク領域情報を含む非ヒト抗体配列情報を取得し、取得された非ヒト抗体配列情報を予め定められた長さの非ヒト抗体断片配列情報に分割し、分割された非ヒト抗体断片配列情報ごとに、一致するヒトアミノ酸断片配列情報を検索し、一致するヒトアミノ酸断片配列情報が検索されなかった非ヒト抗体断片配列情報ごとに、類似するヒトアミノ酸断片配列情報を検索し、検索された各類似するヒトアミノ酸断片配列情報と検索元の非ヒト抗体断片配列情報とを入れ替えて非ヒト抗体配列情報を変異し、変異された非ヒト抗体配列情報について、変異前の非ヒト抗体配列情報の活性を維持しているかを示す抗体活性維持率を決定し、決定された抗体活性維持率に基づいて、非ヒト抗体配列情報をヒト化したヒト化抗体配列情報の変異を決定するので、マウスなどに由来する非ヒト抗体に対してヒト由来のアミノ酸断片配列を多く含ませることによりヒト化を行って、ヒトに対する抗原性を低くし、副作用が少ないヒト化抗体配列情報を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３４９】
また、本発明によれば、非ヒト抗体の活性に影響を与える部分を変異させない、つまり非ヒト抗体の抗体活性を高く維持させるようにヒト化抗体配列情報を効率よく作成することができ、これにより、効果的なヒト化抗体を効率よく作成することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３５０】
さらに、本発明によれば、作成されたヒト化抗体配列情報を産業上多くの分野、特に医薬品、医療等の分野で広く利用することができる抗体配列作成装置、抗体配列作成方法、プログラム、および、記録媒体を提供することができる。
【０３５１】
【配列表】

【図面の簡単な説明】
【図１】本発明の基本原理を示す原理構成図である。
【図２】本発明が適用される本システムの構成の一例を示すブロック図である。
【図３】本発明が適用される本システムの取得部１０２ａの構成の一例を示すブロック図である。
【図４】本発明が適用される本システムの分割部１０２ｂの構成の一例を示すブロック図である。
【図５】本発明が適用される本システムの抗体活性維持率決定部１０２ｆの構成の一例を示すブロック図である。
【図６】本実施形態における本システムのメイン処理の一例を示すフローチャートである。
【図７】本実施形態における本システムの領域分割処理の一例を示すフローチャートである。
【図８】本実施形態における本システムのスライド分割処理の一例を示すフローチャートである。
【図９】本実施形態における本システムの変化量基準抗体活性維持率決定処理の一例を示すフローチャートである。
【図１０】本実施形態における本システムの構造変化寄与率基準抗体活性維持率決定処理の一例を示すフローチャートである。
【図１１】本実施形態における本システムの指標情報基準抗体活性維持率決定処理の一例を示すフローチャートである。
【図１２】本実施例における従来技術で用いられた「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。
【図１３】本実施例における従来技術で用いられた「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。
【図１４】本実施例における従来技術でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。
【図１５】本実施例における従来技術でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。
【図１６】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の一例を示す図である。
【図１７】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の一例を示す図である。
【図１８】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のアミノ酸配列の一例を示す図である。
【図１９】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＬ鎖のヒト化状態の別の一例を示す図である。
【図２０】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のＨ鎖のヒト化状態の別の一例を示す図である。
【図２１】本実施例における本発明の方法でヒト化された「ＣＲＡ４」のアミノ酸配列の別の一例を示す図である。
【図２２】本実施例において用いられたマウス抗体「ＴＦ（ＰＤＢコード１ａｈｗ）」のＬ鎖のヒト化状態の一例を示す図である。
【図２３】本実施例において用いられたマウス抗体「ＴＦ（ＰＤＢコード１ａｈｗ）」のＨ鎖のヒト化状態の一例を示す図である。
【図２４】本実施例において用いられたヒト化済みマウス抗体「ＴＦ（ＰＤＢコード１ｊｐｓ）」のＬ鎖のヒト化状態の一例を示す図である。
【図２５】図４１は、本実施例において用いられたヒト化済みマウス抗体「ＴＦ（ＰＤＢコード１ｊｐｓ）」のＨ鎖のヒト化状態の一例を示す図である。
【図２６】本実施例において本発明の方法でヒト化されたマウス抗体「ＴＦ（ＰＤＢコード１ａｈｗ）」のＬ鎖のヒト化状態の一例を示す図である。
【図２７】本実施例において本発明の方法でヒト化されたマウス抗体「ＴＦ（ＰＤＢコード１ａｈｗ）」のＨ鎖のヒト化状態の一例を示す図である。
【図２８】本実施例において本発明の方法でヒト化されたマウス抗体「ＴＦ（ＰＤＢコード１ａｈｗ）」のアミノ酸配列の一例を示す図である。
【図２９】スライド分割の概念の一例を示す図である。
【符号の説明】
１００ 抗体配列作成装置
１０２ 制御部
１０２ａ 取得部
１０２ｂ 分割部
１０２ｃ 一致検索部
１０２ｄ 類似検索部
１０２ｅ 変異部
１０２ｆ 抗体活性維持率決定部
１０２ｇ 変異決定部
１０２ｈ 領域分割部
１０２ｉ スライド分割部
１０２ｊ 変化量算出部
１０２ｋ 変化量基準抗体活性維持率決定部
１０２ｍ 構造変化寄与率算出部
１０２ｎ 構造変化寄与率基準抗体活性維持率決定部
１０２ｐ 指標情報算出部
１０２ｒ 指標情報基準抗体活性維持率決定部
１０４ 通信制御インターフェース部
１０６ 記憶部
１０６ａ 抗体配列情報ファイル
１０６ｂ 抗体断片配列情報ファイル
１０６ｃ 類似アミノ酸断片配列情報ファイル
１０６ｄ 変異抗体断片配列情報ファイル
１０６ｅ 抗体活性維持率ファイル
１０６ｆ 変異決定抗体配列情報ファイル
１０６ｇ アミノ酸断片配列情報データベース
１０８ 入出力制御インターフェース部
１１２ 入力装置
１１４ 出力装置
２００ 外部システム
３００ ネットワーク[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium, and in particular, significantly reduces antigenicity in humans while maintaining the activity of non-human-derived monoclonal antibodies against any antigen. The present invention relates to an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can create a specific antibody.
[0002]
[Prior art]
Non-human-derived monoclonal antibodies can be expected to be effective therapeutic agents because they bind specifically to the antigen causing the disease.
[0003]
However, since non-human-derived monoclonal antibodies have high antigenicity in humans, non-human-derived monoclonal antibodies cannot be frequently administered to humans.
[0004]
Specifically, for example, human anti-mouse antibodies generated against non-human antibodies such as mouse antibodies administered to humans cause dangerous immune responses, so non-human-derived monoclonal antibodies are frequently administered to humans. I can't do it.
[0005]
Further, for example, since a non-human antibody administered to a human can be metabolized as a foreign substance, the half-life of the non-human antibody in human is short, and the expected effect cannot be exhibited.
[0006]
Such dangerous immune responses and foreign metabolism are thought to be mainly due to the antigenicity of non-human antibodies in humans. In order to solve these problems, methods for reducing the antigenicity of non-human-derived antibodies have been developed.
[0007]
One of them is the production of a chimeric antibody (see, for example, Non-Patent Document 1). In this method, the variable region (V region) is derived from the original non-human monoclonal antibody, and the constant region (C region) is a chimeric antibody derived from an appropriate human antibody. .
[0008]
Since the chimeric antibody obtained by this method contains the complete V region of the original non-human antibody, it can be expected to bind to the antigen with the same specificity as the original non-human antibody.
[0009]
Furthermore, the chimeric antibody has a substantially reduced amino acid sequence derived from non-human and is therefore expected to be less antigenic than the original non-human antibody.
[0010]
However, since the V region of the chimeric antibody is still a non-human sequence, dangerous immune responses and foreign metabolism due to the non-human sequence can occur (eg, when the chimeric antibody is administered to a human).
[0011]
Therefore, as a second method for reducing the antigenicity of a non-human antibody, a technique generally called a humanized antibody production method has been developed (see, for example, Non-Patent Document 2, Non-Patent Document 3, etc.) .) This humanized antibody production method is to create a new human antibody V region by transplanting only the complementarity determining region (CDR) of the V region of the non-human antibody into the V region of the human antibody. (A method called “CDR-grafging” by PDL (Protein Design Labs, Inc.) (company name), etc. (see, for example, Patent Document 2)).
[0012]
However, if necessary, in order to bring the CDR structure of the V region of a human antibody closer to the structure of the original non-human antibody, some amino acids of the framework region (FR) supporting the CDR structure The sequence may be grafted from the V region of a non-human antibody to the V region of a human antibody.
[0013]
In the humanized antibody produced by this humanized antibody production method, the only part derived from the non-human amino acid sequence is CDR (and some FRs).
[0014]
In general, a CDR is composed of a hypervariable amino acid sequence, and is originally a site where mutation is severely occurring in humans.
[0015]
Therefore, it is said that this region is unlikely to cause antigenicity even if left as non-human, and humanized antibodies produced by this humanized antibody production method are potential nonhuman antibodies. It is expected that the antigenicity will be further greatly reduced.
[0016]
However, since the humanized antibody prepared by this humanized antibody preparation method contains an amino acid sequence grafted from the V region of the non-human antibody to the V region of the human antibody as part of the FR, the prepared humanized antibody is When administered to humans, there is a risk of creating antibodies against regions having amino acid sequences that do not exist in humans.
[0017]
Therefore, a method for avoiding this has been proposed (see, for example, Patent Document 1).
[0018]
In this method, for the four FRs (FR1 to FR4) necessary for retaining the three-dimensional structure of three CDRs, the FR of a non-human antibody exists in a database with one FR as a unit. Substitutes FR of high human antibody. At this time, several types of FRs are selected from the human antibodies existing in the database and replaced to create highly active humanized antibodies (see, for example, Patent Document 1).
[0019]
[Non-Patent Document 1]
Lobuglio A. et. al, "Mouse / humanchimeric monoantibody in man: kinetics and immune response.", Proc. Natl. Acad. Sci. USA, 86, p. 4220-4224, 1989
[Non-Patent Document 2]
Riechmann L. et. al, "Reshaping human antigens for therapy.", Nature, 322, p. 323-327, 1988
[Non-Patent Document 3]
Sato K. et. al, “Reshaping a human antigen to inhibit the interleukin 6-dependent tumor cell group.”, Cancer Res. 53, p. 851-856, 1993
[Patent Document 1]
International Publication No. 99/51743 Pamphlet
[Patent Document 2]
Japanese Patent No. 28828340
[0020]
[Problems to be solved by the invention]
However, when the content of non-human-derived amino acid sequences is high like a chimeric antibody, the antigenicity is high, and when the content of non-human-derived amino acid sequences is low like a humanized antibody, the activity tends to be low. In other words, there is a problem that “high antibody activity” and “low antigenicity” conflict with each other in the production of antibodies from non-human antibodies.
[0021]
That is, considering the application of antibodies as therapeutic agents, antibodies have two conditions: 1. 1. high activity against an antigen; It is necessary to satisfy that antigenicity against humans is low.
[0022]
In order to satisfy the above two requirements at the same time, a completely new approach is necessary for eliminating antigenicity in humans, but no effective means has been established yet.
[0023]
In addition, for example, according to Jener's network hypothesis, it is said that another antibody against CDR exists and forms a network of antibodies (for example, “Jerne N. K.,“ The immunosystem. ”, Sci. Amer., No. 229, p.52-60, 1973 "," Jerne NK, "Towards a network the of the immunosystem.", Ann. Immunol. (Inst. Pasteur), No. 125. C, p. 373-389, 1974 ”,“ Tadao Tada, “Immunity Semantics”, Seidosha, 1993 ”, etc.), and conventional chimeric antibody / humanized antibody production methods allow humanization of CDRs Antibody is generated so that it does not react with CDR that is a non-human sequence. There is still a problem that there is still a risk that an antibody can be produced.
[0024]
As described above, the conventional system or the like has a number of problems, and as a result, it is inconvenient and inefficient for both the system user and the administrator.
[0025]
The present invention has been made in view of the above problems, and is capable of producing an antibody sequence capable of producing an antibody having a significantly reduced antigenicity in humans while retaining the activity of a non-human-derived monoclonal antibody against any antigen. An object is to provide an apparatus, a method for producing an antibody sequence, a program, and a recording medium.
[0026]
[Means for Solving the Problems]
In order to solve the above-described problems, the present inventor has focused on the mechanism from when a foreign substance enters the body until the immune reaction starts.
[0027]
The mechanism will be briefly described. First, foreign substances are phagocytosed by macrophages and the like. Subsequently, the protein belonging to the foreign substance is decomposed, and the fragment is presented as an antigen peptide to a receptor (TCR) in the helper T cell.
[0028]
A TCR that can initiate an immune response is a protein that is not strongly bound to self-fragments, that is, a protein that has been differentiated by thymic stromal cells that make up the organ of the thymus as to whether "foreign" and "self" can be distinguished properly .
[0029]
TCR distinguishes whether the presented antigenic peptide is a fragment derived from a protein that does not exist. If it is determined that it does not exist here, an immune response to the foreign substance starts by a command from the helper T cell.
[0030]
The present inventor has the following characteristics that are considered important in the mechanism: 1. antigenicity is recognized by protein fragments; I thought that TCR controls the immune response by identifying whether it is a fragment that I do not have.
[0031]
Furthermore, if the property is really important, the present inventor has considered that non-human antibody sequence fragments (fragments of about 9 to 11 residues) in order to eliminate antigenicity from non-humanized antibodies and to humanize them. ) To the fragment sequence of the human fragment.
[0032]
Based on the above circumstances, the present inventor has developed a novel computational antibody design method based on the immune reaction by the antigen presentation mechanism for TCR and the conventional concept of self-assembly (humanization) of sequence fragments. We propose a solution to the above-mentioned problems.
[0033]
In order to achieve such an object, the antibody sequence creation device according to claim 1 acquires an antibody sequence information including complementarity determining region information and / or framework region information, and acquired by the acquiring unit. Dividing means for dividing the antibody sequence information obtained into antibody fragment sequence information of a predetermined length, and amino acid fragment sequences relating to other species that match each of the antibody fragment sequence information divided by the dividing means Match search means for searching for information; similarity search means for searching for similar amino acid fragment sequence information for each antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search means; Replacing each of the similar amino acid fragment sequence information searched by the similarity search means with the antibody fragment sequence information of the search source Maintaining antibody activity for determining the antibody activity maintenance ratio indicating whether the activity of the antibody sequence information before the mutation is maintained for the antibody sequence information mutated by the mutation means for mutating the antibody sequence information and the antibody sequence information mutated by the mutation means It comprises a rate determining means and a mutation determining means for determining a mutation in the antibody sequence information based on the antibody activity maintaining ratio determined by the antibody activity maintaining ratio determining means.
[0034]
According to this apparatus, antibody sequence information including complementarity determining region information and / or framework region information is obtained, and the obtained antibody sequence information is divided into antibody fragment sequence information of a predetermined length, and divided. Search for amino acid fragment sequence information for other matching species for each antibody fragment sequence information, and search for similar amino acid fragment sequence information for each antibody fragment sequence information for which no matching amino acid fragment sequence information was found The antibody sequence information is mutated by replacing each searched similar amino acid fragment sequence information with the antibody fragment sequence information of the search source, and the activity of the antibody sequence information before the mutation is maintained for the mutated antibody sequence information. Antibody activity maintenance rate indicating whether or not the antibody activity is maintained, and the variation in the antibody sequence information is determined based on the determined antibody activity maintenance rate. It is possible to efficiently create antibody sequence information that contains a lot of acid fragment sequence information, that is, low antigenicity in other species, and according to this apparatus, based on the antibody activity maintenance rate, Since the mutation is determined, it is possible to efficiently create antibody sequence information in which the portion that affects the antibody activity is not mutated, that is, the antibody activity is high.
[0035]
Also, according to this device, antibody fragment sequence information is changed to amino acid fragment sequence information on other species based on the mechanism of immune reaction that antigenicity is recognized by TCR in units of protein fragment sequences. Therefore, antibody sequence information reflecting biological facts can be efficiently created.
[0036]
Further, the antibody sequence creation device according to claim 2 is the antibody sequence creation device according to claim 1, wherein the acquisition means includes the antibody sequence information, the complementarity determining region information, the framework region information, and The image forming apparatus further includes area dividing means for dividing the image into two.
[0037]
This more specifically shows an example of the acquisition means. According to this apparatus, antibody sequence information is divided into complementarity determining region information and framework region information. For example, antibody fragment sequence information spanning complementarity determining region information and framework region information is targeted for mutation. Antibody sequence information can be created without consideration, antibody sequence information can be created with high accuracy, and the amount of calculation can be effectively reduced.
[0038]
In addition, according to this apparatus, for example, when the antibody fragment sequence information related to complementarity determining region information is unlikely to have antigenicity, the target of mutation is limited to framework region information only. Therefore, the complementarity-determining region information is the same as the original sequence, but only the framework region information is mutated to the amino acid fragment sequence information on other species, and the antibody sequence information has low antigenicity and high antibody activity. Can be created efficiently. On the other hand, if there is a possibility that antibody fragment sequence information related to complementarity determining region information has antigenicity, both framework region information and complementarity determining region information can be considered as the target of mutation. . At this time, a database relating to complementarity determining region information can be prepared separately from that relating to framework region information.
[0039]
The antibody sequence creation device according to claim 3 is the antibody sequence creation device according to claim 1 or 2, wherein the dividing means slides the amino acid residue from the head of the antibody sequence information by one amino acid residue in advance. It further comprises slide dividing means for dividing the antibody fragment sequence information of a predetermined length.
[0040]
This shows one example of the dividing means more specifically. According to this device, the antibody fragment information is divided into antibody fragment sequence information of a predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information, so that the protein phagocytosed by the macrophage as a foreign substance is decomposed at an arbitrary position. Antibody fragment sequence information that may be split based on the mechanism of the immune reaction that is presented to the TCR after being fragmented. Can be created efficiently.
[0041]
The antibody sequence creation device according to claim 4 is the antibody sequence creation device according to any one of claims 1 to 3, wherein the predetermined length of the antibody fragment sequence information is 9 to It is 20, It is characterized by the above-mentioned.
[0042]
This more specifically shows one example of the predetermined length of the antibody fragment sequence information. According to this apparatus, since the predetermined length of the antibody fragment sequence information is 9 to 20, the protein phagocytosed by the macrophage as a foreign substance is decomposed into a fragment sequence of about 9 to 20 residues at an arbitrary position. Since the antibody fragment sequence information is created based on the mechanism of the immune reaction that is presented to the TCR after being fragmented, the antibody sequence information reflecting biological facts can be efficiently created.
[0043]
The antibody sequence creation device according to claim 5 is the antibody sequence creation device according to any one of claims 1 to 4, wherein the antibody activity maintenance rate determining means is replaced by the mutation means. A change amount calculating means for calculating a change amount between the amino acid fragment sequence information and the antibody fragment sequence information of the search source, and the antibody activity maintenance rate based on the change amount calculated by the change amount calculating means. And a change amount reference antibody activity maintenance rate determination means for determining the change amount.
[0044]
This shows one example of the antibody activity maintenance rate determining means more specifically. According to this apparatus, the amount of change between similar amino acid fragment sequence information to be replaced and the antibody fragment sequence information of the search source is calculated, and based on the calculated amount of change, the antibody activity maintenance rate is determined. In view of the decrease in antibody activity due to changes in amino acids, it is possible to mutate part of the original antibody sequence to amino acid fragment sequence information on other similar species, thus reducing antigenicity in other species. In addition, it is possible to efficiently create antibody sequence information that maintains high antibody activity.
[0045]
That is, according to this apparatus, considering that the antibody activity is lost if the amount of amino acid residue change is large, and that the antibody activity is maintained if the amount of amino acid residue change is small, the original antibody A part of the sequence can be mutated to amino acid fragment sequence information on other species of similar species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently used. Can be well created.
[0046]
The antibody sequence creation device according to claim 6 is the antibody sequence creation device according to any one of claims 1 to 5, wherein the antibody activity maintenance rate determining means is replaced by the mutation means. Compare the structures of the amino acid fragment sequence information and the antibody fragment sequence information of the search source, and calculate the structural change contribution ratio indicating how much the sequence change contributed to the structural change. And a structure change contribution rate reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate based on the structure change contribution rate calculated by the structure change contribution rate calculation means. It is characterized by.
[0047]
This shows one example of the antibody activity maintenance rate determining means more specifically. According to this apparatus, the structures of the similar amino acid fragment sequence information to be replaced and the antibody fragment sequence information of the search source are compared with each other, and the structural change contribution ratio indicating how much the change in the sequence contributed to the change in the structure Since the antibody activity maintenance rate is determined based on the calculated structural change contribution rate, a part of the original antibody sequence is similar in consideration of the decrease in the antibody activity of the antibody due to the change in the antibody structure. It can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created.
[0048]
That is, according to this device, considering that the antibody activity is lost when the antibody structural change is large and the antibody activity is maintained when the antibody structural change is small, a part of the original antibody sequence is considered. Can be mutated into amino acid fragment sequence information on other similar species, so that antibody sequence information with reduced antigenicity and high antibody activity in other species can be efficiently created. Can do.
[0049]
The antibody sequence creation device according to claim 7 is the antibody sequence creation device according to any one of claims 1 to 6, wherein the antibody activity maintenance rate determining means is replaced by the mutation means. For the amino acid fragment sequence information and the antibody fragment sequence information of the search source, index information calculation means for calculating index information regarding the nature of the amino acid changed on each sequence, and the index information calculation means calculated by the index information calculation means It further comprises index information reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate based on the index information.
[0050]
This shows one example of the antibody activity maintenance rate determining means more specifically. According to this apparatus, for similar amino acid fragment sequence information to be replaced and antibody fragment sequence information of the search source, index information relating to the properties of amino acids changed on the respective sequences is calculated, and based on the calculated index information, antibody information is calculated. Since the activity maintenance rate is determined, the similar amino acid fragment sequence information and the search source antibody fragment sequence information, for example, due to changes in index information such as charge difference, energy difference and affinity difference on each sequence In view of the reduction in antibody activity, a portion of the original antibody sequence can be mutated to amino acid fragment sequence information relating to similar other species, thus reducing antigenicity in other species, and Antibody sequence information that maintains high antibody activity can be created efficiently.
[0051]
That is, according to this apparatus, when the amino acid fragment sequence information and the search source antibody fragment sequence information are similar to each other, for example, if the difference in charge, energy or affinity is increased, the antibody activity is increased. On the other hand, considering that the antibody activity is maintained if, for example, the difference in charge, energy or affinity is reduced on each sequence, a part of the original antibody sequence is similar. Can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created. .
[0052]
Further, the antibody sequence creation device according to claim 8 is the antibody sequence creation device according to any one of claims 1 to 7, wherein the mutation determining means includes the antibody sequence after the mutation before the mutation. When the antibody activity maintenance rate of information decreases, the mutation is terminated.
[0053]
This more specifically shows one example of the mutation determining means. According to this apparatus, when the antibody activity maintenance rate of the antibody sequence information after mutation decreases compared to that before mutation, the mutation is terminated, so that the mutation that decreases the antibody activity of the antibody sequence information before mutation is eliminated. Thus, antibody sequence information that maintains antigenicity and has high antibody activity can be effectively created.
[0054]
The antibody sequence creation device according to claim 9 is the antibody sequence creation device according to any one of claims 1 to 8, wherein the antibody sequence information is the antibody sequence information related to a non-human, and The amino acid fragment sequence information relating to the other biological species is the amino acid fragment sequence information relating to humans.
[0055]
This more specifically shows one example of antibody sequence information and amino acid fragment sequence information concerning other species. According to this apparatus, the antibody sequence information is antibody sequence information related to non-humans, and the amino acid fragment sequence information related to other biological species is amino acid fragment sequence information related to humans. Or, obtain non-human antibody sequence information including framework region information, divide the obtained non-human antibody sequence information into non-human antibody fragment sequence information of a predetermined length, and divide the non-human antibody fragment sequence For each piece of information, search for matching human amino acid fragment sequence information, and search for similar human amino acid fragment sequence information for each non-human antibody fragment sequence information for which no matching human amino acid fragment sequence information was searched. Each similar human amino acid fragment sequence information is replaced with the non-human antibody fragment sequence information of the search source to mutate the non-human antibody sequence information. Regarding the antibody sequence information, the antibody activity maintenance rate indicating whether the activity of the non-human antibody sequence information before the mutation is maintained was determined, and the non-human antibody sequence information was humanized based on the determined antibody activity maintenance rate Since the mutation of humanized antibody sequence information is determined, humanization is performed by including many human-derived amino acid fragment sequences in non-human antibodies derived from mice, etc., thereby reducing antigenicity to humans and causing side effects. Humanized antibody sequence information can be efficiently generated.
[0056]
In addition, according to this apparatus, humanized antibody sequence information can be efficiently created so as not to mutate a portion that affects the activity of a non-human antibody, that is, to maintain a high antibody activity of the non-human antibody. Thus, an effective humanized antibody can be efficiently produced.
[0057]
In addition, according to this apparatus, the prepared humanized antibody sequence information can be widely used in many industrial fields, particularly in the fields of pharmaceuticals, medicine, and the like.
[0058]
The present invention also relates to an antibody sequence creation method, wherein the antibody sequence creation method according to claim 10 obtains antibody sequence information including complementarity determining region information and / or framework region information; A dividing step of dividing the antibody sequence information acquired by the acquiring step into antibody fragment sequence information of a predetermined length, and another organism that matches each of the antibody fragment sequence information divided by the dividing step Searching for similar amino acid fragment sequence information for each of the antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search step and searching for the amino acid fragment sequence information related to the species Similarity search step and each of the similar amino acids searched by the similarity search step The mutation step of mutating the antibody sequence information by exchanging the single sequence information and the antibody fragment sequence information of the search source, and the activity of the antibody sequence information before mutation for the antibody sequence information mutated by the mutation step An antibody activity maintenance rate determining step for determining whether the antibody activity is maintained or not, and a variation in the antibody sequence information is determined based on the antibody activity maintenance rate determined by the antibody activity maintenance rate determining step. A mutation determining step.
[0059]
According to this method, antibody sequence information including complementarity determining region information and / or framework region information is obtained, and the obtained antibody sequence information is divided into antibody fragment sequence information of a predetermined length, and divided. Search for amino acid fragment sequence information for other matching species for each antibody fragment sequence information, and search for similar amino acid fragment sequence information for each antibody fragment sequence information for which no matching amino acid fragment sequence information was found The antibody sequence information is mutated by replacing each searched similar amino acid fragment sequence information with the antibody fragment sequence information of the search source, and the activity of the antibody sequence information before the mutation is maintained for the mutated antibody sequence information. Antibody activity maintenance rate indicating whether or not the antibody activity is maintained, and the variation in the antibody sequence information is determined based on the determined antibody activity maintenance rate. It is possible to efficiently create antibody sequence information that contains a lot of acid fragment sequence information, that is, low antigenicity in other species, and according to this method, based on the antibody activity maintenance rate, Since the mutation is determined, it is possible to efficiently create antibody sequence information in which the portion that affects the antibody activity is not mutated, that is, the antibody activity is high.
[0060]
Further, according to this method, antibody fragment sequence information is changed to amino acid fragment sequence information on other species based on the mechanism of immune reaction that antigenicity is recognized by TCR in units of protein fragment sequences. Therefore, antibody sequence information reflecting biological facts can be efficiently created.
[0061]
The antibody sequence creation method according to claim 11 is the antibody sequence creation method according to claim 10, wherein the obtaining step includes the antibody sequence information, the complementarity determining region information, and the framework region information. The method further includes an area dividing step for dividing the image into areas.
[0062]
This more specifically shows an example of the acquisition step. According to this method, antibody sequence information is divided into complementarity determining region information and framework region information. For example, antibody fragment sequence information spanning complementarity determining region information and framework region information is targeted for mutation. Antibody sequence information can be created without consideration, antibody sequence information can be created with high accuracy, and the amount of calculation can be effectively reduced.
[0063]
Further, according to this method, for example, when the antibody fragment sequence information related to complementarity determining region information is unlikely to have antigenicity, the mutation target is limited to the framework region information only. Therefore, the complementarity-determining region information is the same as the original sequence, but only the framework region information is mutated to the amino acid fragment sequence information on other species, and the antibody sequence information has low antigenicity and high antibody activity. Can be created efficiently. On the other hand, if there is a possibility that antibody fragment sequence information related to complementarity determining region information has antigenicity, both framework region information and complementarity determining region information can be considered as the target of mutation. . At this time, a database relating to complementarity determining region information can be prepared separately from that relating to framework region information.
[0064]
The antibody sequence creation method according to claim 12 is the antibody sequence creation method according to claim 10 or 11, wherein the dividing step is performed by sliding the amino acid residues from the beginning of the antibody sequence information by sliding the amino acid residues in advance. The method further includes a slide dividing step of dividing the antibody fragment sequence information of a predetermined length.
[0065]
This more specifically shows an example of the division step. According to this method, since the antibody sequence information is divided into antibody fragment sequence information of a predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information, the protein phagocytosed by the macrophage as a foreign substance is decomposed at an arbitrary position. Antibody fragment sequence information that may be split based on the mechanism of the immune reaction that is presented to the TCR after being fragmented. Can be created efficiently.
[0066]
The antibody sequence creation method according to claim 13 is the antibody sequence creation method according to any one of claims 10 to 12, wherein the predetermined length of the antibody fragment sequence information is 9 to It is 20, It is characterized by the above-mentioned.
[0067]
This more specifically shows one example of the predetermined length of the antibody fragment sequence information. According to this method, since the predetermined length of the antibody fragment sequence information is 9 to 20, the protein phagocytosed by the macrophage as a foreign substance is decomposed into a fragment sequence of about 9 to 20 residues at an arbitrary position. Since antibody fragment sequence information is created based on the mechanism of the immune reaction that is presented to the TCR after being fragmented, antibody sequence information that reflects biological facts can be created efficiently.
[0068]
The antibody sequence creation method according to claim 14 is the antibody sequence creation method according to any one of claims 10 to 13, wherein the antibody activity maintenance rate determination step is replaced by the mutation step. A change amount calculating step for calculating a change amount between the amino acid fragment sequence information and the antibody fragment sequence information of the search source, and the antibody activity maintenance rate based on the change amount calculated by the change amount calculating step. And a change amount reference antibody activity maintenance rate determination step of determining the change amount.
[0069]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this method, the amount of change between similar amino acid fragment sequence information to be replaced and the antibody fragment sequence information of the search source is calculated, and the antibody activity maintenance rate is determined based on the calculated amount of change. In view of the decrease in antibody activity due to changes in amino acids, it is possible to mutate part of the original antibody sequence to amino acid fragment sequence information on other similar species, thus reducing antigenicity in other species. In addition, it is possible to efficiently create antibody sequence information that maintains high antibody activity.
[0070]
That is, according to this method, considering that the antibody activity is lost when the amount of amino acid residue change increases, and the antibody activity is maintained when the amount of amino acid residue change decreases, the original antibody A part of the sequence can be mutated to amino acid fragment sequence information on other species of similar species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently used. Can be well created.
[0071]
The antibody sequence creation method according to claim 15 is the antibody sequence creation method according to any one of claims 10 to 14, wherein the antibody activity maintenance rate determination step is replaced by the mutation step. Compare the structures of the amino acid fragment sequence information and the antibody fragment sequence information of the search source, and calculate the structural change contribution ratio indicating how much the sequence change contributed to the structural change. And a structure change contribution rate reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate based on the structure change contribution rate calculated by the structure change contribution rate calculation step. Features.
[0072]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this method, for similar amino acid fragment sequence information to be replaced and antibody fragment sequence information of the search source, respective structures are compared, and a structural change contribution ratio indicating how much the change in the sequence contributed to the change in the structure is obtained. Since the antibody activity maintenance rate is determined based on the calculated structural change contribution rate, a part of the original antibody sequence is similar in consideration of the decrease in the antibody activity of the antibody due to the change in the antibody structure. It can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created.
[0073]
That is, according to this method, the antibody activity is lost when the structural change of the antibody is increased, and the antibody activity is maintained when the structural change of the antibody is reduced. Can be mutated into amino acid fragment sequence information on other similar species, so that antibody sequence information with reduced antigenicity and high antibody activity in other species can be efficiently created. Can do.
[0074]
The antibody sequence creation method according to claim 16 is the antibody sequence creation method according to any one of claims 10 to 15, wherein the antibody activity maintenance rate determination step is replaced by the mutation step. For the amino acid fragment sequence information and the antibody fragment sequence information of the search source, an index information calculation step for calculating index information regarding the nature of the amino acid changed on each sequence, and the index information calculation step calculated above An index information reference antibody activity maintenance rate determination step of determining the antibody activity maintenance rate based on the index information.
[0075]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this method, for similar amino acid fragment sequence information to be replaced and antibody fragment sequence information of the search source, index information relating to the properties of amino acids changed on the respective sequences is calculated, and based on the calculated index information, antibody information is calculated. Since the activity maintenance rate is determined, the similar amino acid fragment sequence information and the search source antibody fragment sequence information, for example, due to changes in index information such as charge difference, energy difference and affinity difference on each sequence In view of the reduction in antibody activity, a portion of the original antibody sequence can be mutated to amino acid fragment sequence information relating to similar other species, thus reducing antigenicity in other species, and Antibody sequence information that maintains high antibody activity can be created efficiently.
[0076]
That is, according to this method, when the amino acid fragment sequence information and the search source antibody fragment sequence information are similar to each other, for example, if the difference in charge, energy, affinity, etc. increases, the antibody activity On the other hand, considering that the antibody activity is maintained if, for example, the difference in charge, energy or affinity is reduced on each sequence, a part of the original antibody sequence is similar. Can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created. .
[0077]
The antibody sequence creation method according to claim 17 is the antibody sequence creation method according to any one of claims 10 to 16, wherein the mutation determination step includes the antibody sequence after the mutation before the mutation. When the antibody activity maintenance rate of information decreases, the mutation is terminated.
[0078]
This more specifically shows an example of the mutation determination step. According to this method, when the antibody activity maintenance rate of the antibody sequence information after mutation decreases compared to that before the mutation, the mutation is terminated, so that the mutation that decreases the antibody activity of the antibody sequence information before mutation is eliminated. Thus, antibody sequence information that maintains antigenicity and has high antibody activity can be effectively created.
[0079]
The antibody sequence creation method according to claim 18 is the antibody sequence creation method according to any one of claims 10 to 17, wherein the antibody sequence information is the antibody sequence information related to a non-human, and The amino acid fragment sequence information relating to the other biological species is the amino acid fragment sequence information relating to humans.
[0080]
This more specifically shows one example of antibody sequence information and amino acid fragment sequence information concerning other species. According to this method, the antibody sequence information is antibody sequence information related to non-humans, and the amino acid fragment sequence information related to other biological species is amino acid fragment sequence information related to humans. Or, obtain non-human antibody sequence information including framework region information, divide the obtained non-human antibody sequence information into non-human antibody fragment sequence information of a predetermined length, and divide the non-human antibody fragment sequence For each information, search for matching human amino acid fragment sequence information, search for similar human amino acid fragment sequence information for each non-human antibody fragment sequence information for which no matching human amino acid fragment sequence information was searched Each similar human amino acid fragment sequence information is replaced with the non-human antibody fragment sequence information of the search source to mutate the non-human antibody sequence information. Regarding the antibody sequence information, the antibody activity maintenance rate indicating whether the activity of the non-human antibody sequence information before the mutation is maintained was determined, and the non-human antibody sequence information was humanized based on the determined antibody activity maintenance rate Since the mutation of humanized antibody sequence information is determined, humanization is performed by including many human-derived amino acid fragment sequences in non-human antibodies derived from mice, etc., thereby reducing antigenicity to humans and causing side effects. Humanized antibody sequence information can be efficiently generated.
[0081]
In addition, according to this method, humanized antibody sequence information can be efficiently generated so as not to mutate the portion that affects the activity of the non-human antibody, that is, to maintain the antibody activity of the non-human antibody at a high level. Thus, an effective humanized antibody can be efficiently produced.
[0082]
In addition, according to this method, the prepared humanized antibody sequence information can be widely used in many industrial fields, particularly in the fields of pharmaceuticals, medicine, and the like.
[0083]
In addition, the present invention relates to a program, and causes a computer to execute the antibody sequence creation method according to claim 19. The program includes an antibody sequence including complementarity determining region information and / or framework region information. An acquisition step for acquiring information, a dividing step for dividing the antibody sequence information acquired by the acquiring step into antibody fragment sequence information of a predetermined length, and the antibody fragment sequence information divided by the dividing step A matching search step for searching for amino acid fragment sequence information relating to other matching organism species for each match, and the antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search step are similar. A similarity search step for searching the amino acid fragment sequence information, and the similarity search step. A mutation step for mutating the antibody sequence information by replacing each of the similar amino acid fragment sequence information searched by the search and the antibody fragment sequence information of the search source, and the antibody sequence information mutated by the mutation step Based on the antibody activity maintenance rate determined by the antibody activity maintenance rate determination step for determining an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before mutation is maintained, and the antibody activity maintenance rate determination step And a mutation determining step for determining a mutation in the antibody sequence information.
[0084]
According to this program, antibody sequence information including complementarity determining region information and / or framework region information is obtained, and the obtained antibody sequence information is divided into antibody fragment sequence information of a predetermined length, and divided. Search for amino acid fragment sequence information for other matching species for each antibody fragment sequence information, and search for similar amino acid fragment sequence information for each antibody fragment sequence information for which no matching amino acid fragment sequence information was found The antibody sequence information is mutated by replacing each searched similar amino acid fragment sequence information with the antibody fragment sequence information of the search source, and the activity of the antibody sequence information before the mutation is maintained for the mutated antibody sequence information. Antibody activity maintenance rate is determined, and the variation in antibody sequence information is determined based on the determined antibody activity maintenance rate. It is possible to efficiently create antibody sequence information that contains a lot of amino acid fragment sequence information, that is, has low antigenicity in other species, and according to this program, based on the antibody activity maintenance rate, Since the mutation is determined, it is possible to efficiently create antibody sequence information in which the portion that affects the antibody activity is not mutated, that is, the antibody activity is high.
[0085]
In addition, according to this program, antibody fragment sequence information is changed to amino acid fragment sequence information on other species based on the mechanism of immune reaction that antigenicity is recognized by TCR in units of protein fragment sequences. Therefore, antibody sequence information reflecting biological facts can be efficiently created.
[0086]
The program according to claim 20 is the program according to claim 19, wherein the obtaining step divides the antibody sequence information into the complementarity determining region information and the framework region information. Is further included.
[0087]
This more specifically shows an example of the acquisition step. According to this program, antibody sequence information is divided into complementarity determining region information and framework region information. For example, antibody fragment sequence information spanning complementarity determining region information and framework region information is targeted for mutation. Antibody sequence information can be created without consideration, antibody sequence information can be created with high accuracy, and the amount of calculation can be effectively reduced.
[0088]
In addition, according to this program, for example, when antibody fragment sequence information related to complementarity determining region information is unlikely to have antigenicity, the target of mutation is limited to framework region information only. Therefore, the complementarity-determining region information is the same as the original sequence, but only the framework region information is mutated to the amino acid fragment sequence information on other species, and the antibody sequence information has low antigenicity and high antibody activity. Can be created efficiently. On the other hand, if there is a possibility that antibody fragment sequence information related to complementarity determining region information has antigenicity, both framework region information and complementarity determining region information can be considered as the target of mutation. . At this time, a database relating to complementarity determining region information can be prepared separately from that relating to framework region information.
[0089]
The program according to claim 21 is the program according to claim 19 or 20, wherein the dividing step has the predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information. The method further includes a slide dividing step of dividing the antibody fragment sequence information.
[0090]
This more specifically shows an example of the division step. According to this program, since the antibody sequence information is divided into antibody fragment sequence information of a predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information, the protein phagocytosed by the macrophage as a foreign substance is decomposed at an arbitrary position. Antibody fragment sequence information that may be split based on the mechanism of the immune reaction that is presented to the TCR after being fragmented. Can be created efficiently.
[0091]
The program according to claim 22 is the program according to any one of claims 19 to 21, wherein the predetermined length of the antibody fragment sequence information is 9 to 20. And
[0092]
This more specifically shows one example of the predetermined length of the antibody fragment sequence information. According to this program, since the predetermined length of the antibody fragment sequence information is 9 to 20, the protein phagocytosed by the macrophage as a foreign substance is decomposed into a fragment sequence of about 9 to 20 residues at an arbitrary position. Since antibody fragment sequence information is created based on the mechanism of the immune reaction that is presented to the TCR after being fragmented, antibody sequence information that reflects biological facts can be created efficiently.
[0093]
Further, the program according to claim 23 is the program according to any one of claims 19 to 22, wherein the antibody activity maintenance rate determination step replaces the similar amino acid fragment sequence information replaced in the mutation step. A change amount calculation step for calculating a change amount between the antibody fragment sequence information of the search source and the search source, and a change amount criterion for determining the antibody activity maintenance rate based on the change amount calculated by the change amount calculation step And an antibody activity maintenance rate determination step.
[0094]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this program, the amount of change between similar amino acid fragment sequence information to be replaced and the antibody fragment sequence information of the search source is calculated, and the antibody activity maintenance rate is determined based on the calculated amount of change. In view of the decrease in antibody activity due to changes in amino acids, it is possible to mutate part of the original antibody sequence to amino acid fragment sequence information on other similar species, thus reducing antigenicity in other species. In addition, it is possible to efficiently create antibody sequence information that maintains high antibody activity.
[0095]
That is, according to this program, the antibody activity is lost when the amount of amino acid residue change increases, and the antibody activity is maintained when the amount of amino acid residue change decreases. A part of the sequence can be mutated to amino acid fragment sequence information on other species of similar species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently used. Can be well created.
[0096]
Further, the program according to claim 24 is the program according to any one of claims 19 to 23, wherein the antibody activity maintenance rate determination step replaces the similar amino acid fragment sequence information replaced in the mutation step. And for the antibody fragment sequence information of the search source, a structure change contribution rate calculating step for comparing each structure and calculating a structure change contribution rate indicating how much the sequence change contributed to the structure change, It further includes a structure change contribution rate reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate based on the structure change contribution rate calculated by the structure change contribution rate calculation step.
[0097]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this program, the structures of similar amino acid fragment sequences to be replaced and the antibody fragment sequence information of the search source are compared, and the structural change contribution ratio indicating how much the change in the sequence contributed to the change in the structure is obtained. Since the antibody activity maintenance rate is determined based on the calculated structural change contribution rate, a part of the original antibody sequence is similar in consideration of the decrease in the antibody activity of the antibody due to the change in the antibody structure. It can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created.
[0098]
That is, according to this program, considering that the antibody activity is lost when the antibody structural change is large and the antibody activity is maintained when the antibody structural change is small, a part of the original antibody sequence is considered. Can be mutated into amino acid fragment sequence information on other similar species, so that antibody sequence information with reduced antigenicity and high antibody activity in other species can be efficiently created. Can do.
[0099]
Further, the program according to claim 25 is the program according to any one of claims 19 to 24, wherein the antibody activity maintenance rate determining step is replaced with the similar amino acid fragment sequence information replaced in the mutation step. And for the antibody fragment sequence information of the search source, based on the index information calculation step for calculating index information on the properties of amino acids changed on each sequence, and the index information calculated by the index information calculation step, An index information reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate.
[0100]
This more specifically shows one example of the antibody activity maintenance rate determination step. According to this program, for similar amino acid fragment sequence information to be replaced and antibody fragment sequence information of the search source, index information regarding the property of the amino acid changed on each sequence is calculated, and based on the calculated index information, antibody information is calculated. Since the activity maintenance rate is determined, the similar amino acid fragment sequence information and the search source antibody fragment sequence information, for example, due to changes in index information such as charge difference, energy difference and affinity difference on each sequence In view of the reduction in antibody activity, a portion of the original antibody sequence can be mutated to amino acid fragment sequence information relating to similar other species, thus reducing antigenicity in other species, and Antibody sequence information that maintains high antibody activity can be created efficiently.
[0101]
That is, according to this program, for similar amino acid fragment sequence information and search source antibody fragment sequence information, if the difference in charge, energy, affinity, etc. on each sequence increases, antibody activity On the other hand, considering that the antibody activity is maintained if, for example, the difference in charge, energy or affinity is reduced on each sequence, a part of the original antibody sequence is similar. Can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created. .
[0102]
The program according to claim 26 is the program according to any one of claims 19 to 25, wherein the mutation determination step maintains the antibody activity of the antibody sequence information after mutation relative to that before mutation. When the rate decreases, the mutation is terminated.
[0103]
This more specifically shows an example of the mutation determination step. According to this program, if the antibody activity maintenance rate of the antibody sequence information after mutation decreases compared to that before mutation, the mutation is terminated, so that there is no mutation that reduces the antibody activity of the antibody sequence information before mutation. Thus, antibody sequence information that maintains antigenicity and has high antibody activity can be effectively created.
[0104]
The program according to claim 27 is the program according to any one of claims 19 to 26, wherein the antibody sequence information is the antibody sequence information related to non-human and the other biological species. The amino acid fragment sequence information on is the amino acid fragment sequence information on human.
[0105]
This more specifically shows one example of antibody sequence information and amino acid fragment sequence information concerning other species. According to this program, the antibody sequence information is antibody sequence information related to non-humans, and the amino acid fragment sequence information related to other biological species is amino acid fragment sequence information related to humans. Or, obtain non-human antibody sequence information including framework region information, divide the obtained non-human antibody sequence information into non-human antibody fragment sequence information of a predetermined length, and divide the non-human antibody fragment sequence For each information, search for matching human amino acid fragment sequence information, search for similar human amino acid fragment sequence information for each non-human antibody fragment sequence information for which no matching human amino acid fragment sequence information was searched Non-human antibody sequence information is mutated by replacing each similar human amino acid fragment sequence information with the search source non-human antibody fragment sequence information. For non-human antibody sequence information, determine the antibody activity maintenance rate indicating whether the activity of the non-human antibody sequence information before mutation is maintained, and based on the determined antibody activity maintenance rate, determine the non-human antibody sequence information as human Since the humanized antibody sequence information mutation is determined, humanization is performed by including many human-derived amino acid fragment sequences in non-human antibodies derived from mice, etc., to reduce antigenicity to humans. Thus, humanized antibody sequence information with few side effects can be created efficiently.
[0106]
In addition, according to this program, humanized antibody sequence information can be efficiently created so as not to mutate the portion that affects the activity of the non-human antibody, that is, to maintain the antibody activity of the non-human antibody at a high level. Thus, an effective humanized antibody can be efficiently produced.
[0107]
In addition, according to this program, the created humanized antibody sequence information can be widely used in many industrial fields, particularly in fields such as pharmaceuticals and medicine.
[0108]
Further, the present invention relates to a recording medium, and the recording medium according to claim 28 records the program according to any one of claims 19 to 27.
[0109]
According to this recording medium, the program according to any one of claims 19 to 27 can be realized using a computer, and the same effects as those of these methods can be obtained.
[0110]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of an antigenic exclusion antibody production apparatus, antigenic exclusion antibody production method, program, and recording medium according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments. In this embodiment, a case where a mouse antibody sequence is humanized will be described as an example. However, the present invention is not limited to this, and an antibody sequence suitable for another species is created from the antibody sequence of a certain species. May be.
[0111]
[Outline of the present invention]
Hereinafter, the outline of the present invention will be described, and then the configuration and processing of the present invention will be described in detail. FIG. 1 is a principle configuration diagram showing the basic principle of the present invention.
[0112]
The present invention generally has the following basic features.
[0113]
First, antibody sequence information including complementarity determining region information and / or framework region information is acquired (step S-1).
[0114]
Here, in step S-1, the acquired antibody sequence information may be divided into complementarity determining region information and framework region information. That is, it may be divided into complementarity determining region information and framework region information, and the following processing may be performed separately.
[0115]
Next, the acquired antibody sequence information is divided into antibody fragment sequence information having a predetermined length (step S-2).
[0116]
Here, in step S-2, the predetermined length of the antibody fragment sequence information is, for example, about 9 to 20 sequences.
[0117]
In step S-2, antibody fragment sequence information having a predetermined length may be divided (slide division) while sliding one amino acid residue at a time from the beginning of the antibody sequence information.
[0118]
Here, the concept of slide division will be described with reference to FIG. FIG. 29 is a diagram illustrating an example of the concept of slide division.
[0119]
First, the start position of the slide division is set to the head position of the antibody sequence information, and it is divided from the set position into antibody fragment sequence information having a predetermined length (“9” in FIG. 29). Next, the start position of the next slide division is set to a position slid by one amino acid residue from the head position, and the antibody fragment sequence information having a predetermined length is divided from the set position.
[0120]
In this way, the process of dividing into antibody fragment sequence information of a predetermined length is repeated while sliding the slide splitting start position by one amino acid residue. When the slide split start position is set to the last position of the antibody sequence information, the slide split is ended. This completes the explanation of the concept of slide division.
[0121]
Next, for each divided antibody fragment sequence information, amino acid fragment sequence information relating to other matching biological species is searched (step S-3).
[0122]
Here, the “other biological species” may be, for example, a biological species derived from information different from the antibody sequence information, and is a target for antibody medical treatment using the antibody designed according to the present invention, such as human Or any other species.
[0123]
Next, for each antibody fragment sequence information for which the amino acid fragment sequence information relating to other matching species is not searched, the amino acid fragment sequence information relating to other similar species is searched (step S-4).
[0124]
Here, in step S-3 and step S-4, each divided antibody fragment sequence information is used as a query sequence, for example, “nr (non-redundant) -aa (GenPept, GenPept-upd, SwissProt , SwissProt-upd, PIR, PRF, databases from which duplicates of the same sequence are removed), “Human Ig, TcR”, “GenPept”, “SwissProt”, “PIR”, “PRF”, etc. Thus, the amino acid fragment sequence information regarding other matching species and the amino acid fragment sequence information regarding other similar species may be searched using an existing similar sequence search method such as BLAST or FASTA.
[0125]
Subsequently, the antibody sequence information is mutated by exchanging the searched amino acid fragment sequence information about each similar other species and the search source antibody fragment sequence information (step S-5).
[0126]
Next, an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before the mutation is maintained is determined for the mutated antibody sequence information (step S-6).
[0127]
Here, in step S-6, the determination of the antibody activity maintenance rate is carried out with respect to the amino acid fragment sequence information regarding other mutated similar organism species and the antibody fragment sequence information affected by the mutation with step S-3 and Similarly, a search for amino acid fragment sequence information relating to other matching species may be performed, and the antibody activity maintenance rate may be determined based on the search result.
[0128]
Further, in step S-6, the amount of change between the amino acid fragment sequence information related to other similar species to be replaced in step S-5 and the antibody fragment sequence information of the search source is calculated, and based on the calculated amount of change. The antibody activity maintenance rate may be determined.
[0129]
The amount of change may be, for example, the number of amino acid residue differences between the amino acid fragment sequence information regarding other similar species to be replaced and the antibody fragment sequence information of the search source, the degree of similarity, and the like.
[0130]
Further, in step S-6, the structures of the amino acid fragment sequence information and other antibody fragment sequence information on other similar species to be replaced in step S-5 are compared, and the change in the sequence is the change in the structure. It is also possible to calculate a structure change contribution ratio that indicates how much has been contributed to and to determine the antibody activity maintenance ratio based on the calculated structure change contribution ratio.
[0131]
The structure may be a secondary structure or a tertiary structure.
[0132]
For prediction of the secondary structure, for example, 1. "Chou-Fasman method", 2. 2. “GOR (Garnier-Osguthorpe-Robson) method”; 3. “Nearest Neighbor Method” “Secondary structure prediction method using neural network model (for example,“ Qian N. and Seijnowski T. J., “Predicting the secondary structure of global proteins using neural network. 202. J. M. -884, 1988 ", and websites that perform neural network analysis such as" PHD: http://www.embl-heidelberg.de/predictprotein/predictprotein.html "). “Secondary structure prediction methods based on hidden Markov models (for example,“ Stultz CM, and White JV, and Smith TF, “Structural analysis based on state-space modeling.”, Protein Sc. 2, p. 305-314, 1993 ", etc.)", etc., may be used.
[0133]
For prediction of the tertiary structure, for example, 1. "Homology modeling method", 2. “Threading method”; “3D-1D method”; 4. “Ab initio method”; Existing methods for tertiary structure prediction such as “fragment assembly method (see, for example,“ Proteins: Structure, Function, and Genetics, Supplement, 3, 1999 ”) may be used.
[0134]
In step S-6, index information on the nature of the amino acid changed on each sequence is obtained for amino acid fragment sequence information on other similar species to be replaced in step S-5 and antibody fragment sequence information on the search source. The antibody activity maintenance rate may be determined based on the calculated index information.
[0135]
The index information may be, for example, information on the charge of the amino acid sequence, information on energy (for example, electrostatic energy), information on affinity, and the like.
[0136]
For calculating the index information, for example, the charge of a positively charged charged amino acid (lysine, arginine) is 1, and the charge of a negatively charged charged amino acid (glutamic acid, aspartic acid) is − A method may be used in which the charge of amino acids other than 1 is 0.
[0137]
In addition, for the calculation of the index information, for example, each amino acid residue is calculated by the existing quantum chemical calculation method based on the three-dimensional structure information of the protein registered in the protein structure database or the three-dimensional structure information obtained by the simulation method. A method for determining the electric charge of each of them may be used.
[0138]
The index information is calculated by, for example, an energy calculation method such as molecular mechanics, molecular dynamics, molecular orbital method, density functional method, or fragment MO method (Chemical Physics Letters, Volume 336, Issues 1-2, 9 March). , P.163-170, 2001), a method of calculating electrostatic energy, or a method of calculating affinity.
[0139]
Moreover, you may use combining these each method suitably for calculation of the said parameter | index information.
[0140]
In addition, the antibody activity maintenance rate may be determined using the presence rate of amino acid fragment sequence information regarding other species included in the antibody sequence information after mutation relative to that before mutation.
[0141]
Subsequently, the variation | mutation of antibody sequence information is determined based on the antibody activity maintenance rate about the determined antibody sequence information after each variation | mutation (step S-7).
[0142]
Here, in step S-7, based on the determined antibody activity maintenance rate for the antibody sequence information after each mutation, for example, the variation in the antibody sequence information with the highest antibody activity maintenance rate may be determined.
[0143]
In step S-7, for example, antibody sequence information mutation may be optimized using optimization techniques such as Monte Carlo method, simulated annealing, genetic algorithm, etc. to determine antibody sequence information mutation. .
[0144]
Moreover, in step S-7, when the antibody activity maintenance rate of the antibody sequence information after mutation decreases compared to before mutation, the mutation may be terminated.
[0145]
The antibody sequence information may be antibody sequence information related to a non-human such as a mouse, for example, and the amino acid fragment sequence information related to the other species may be amino acid fragment sequence information related to a human.
[0146]
[System configuration]
Next, the configuration of this system will be described. FIG. 2 is a block diagram showing an example of the configuration of the system to which the present invention is applied, and conceptually shows only the portion related to the present invention in the configuration. This system generally connects the antibody sequence creation device 100 and an external system 200 that provides an external database such as sequence information and structure information, an external program such as a homology search, and the like through a network 300 so that they can communicate with each other. Configured.
[0147]
In FIG. 2, a network 300 has a function of mutually connecting the antibody sequence creation device 100 and the external system 200, and is, for example, the Internet.
[0148]
In FIG. 2, an external system 200 is mutually connected to the antibody sequence creation apparatus 100 via a network 300, and external programs such as homology search and motif search for sequence information and structure information are provided to the user. It has a function of providing a website to be executed.
[0149]
Here, the external system 200 may be configured as a WEB server, an ASP server, or the like, and the hardware configuration may be configured by an information processing apparatus such as a commercially available workstation or a personal computer and an accessory device thereof. Good. Each function of the external system 200 is realized by a CPU, a disk device, a memory device, an input device, an output device, a communication control device, and the like in the hardware configuration of the external system 200 and a program for controlling them.
[0150]
In FIG. 2, the antibody sequence creation device 100 schematically includes a control unit 102 such as a CPU that comprehensively controls the entire antibody sequence creation device 100, a communication device such as a router connected to a communication line (not shown). ) Connected to the communication control interface unit 104, the input / output control interface unit 108 connected to the input device 112 and the output device 114, and the storage unit 106 for storing various databases and tables. These units are communicably connected via an arbitrary communication path. Further, the antibody sequence creation device 100 is communicably connected to the network 300 via a communication device such as a router and a wired or wireless communication line such as a dedicated line.
[0151]
Various databases and tables (antibody sequence information file 106a to amino acid fragment sequence information database 106g) stored in the storage unit 106 are storage means such as a fixed disk device, and various programs, tables and files used for various processes, Stores database and web page files.
[0152]
Among these components of the storage unit 106, the antibody sequence information file 106a is an antibody sequence information storage unit that stores antibody sequence information acquired by the acquisition unit 102a described later.
[0153]
The information stored in the antibody sequence information file 106a is composed of the acquired antibody sequence information.
[0154]
The antibody fragment sequence information file 106b is antibody fragment sequence information storage means for storing antibody fragment sequence information divided by the dividing unit 102b described later.
[0155]
The information stored in the antibody fragment sequence information file 106b is configured by associating antibody fragment identification information for uniquely identifying antibody fragment sequence information with antibody fragment sequence information.
[0156]
The similar amino acid fragment sequence information file 106c is similar amino acid fragment sequence information storage means for storing similar amino acid fragment sequence information related to other species searched by the similarity search unit 102d described later.
[0157]
The information stored in the similar amino acid fragment sequence information file 106c corresponds to the above antibody fragment identification information, similar amino acid fragment identification information for uniquely identifying similar amino acid fragment sequence information regarding other species, and other information It is constructed by associating similar amino acid fragment sequence information on biological species with each other.
[0158]
The mutant antibody fragment sequence information file 106d stores similar amino acid fragment sequence information related to other biological species mutated by the mutating unit 102e described later and antibody fragment sequence information affected by the mutation. Information storage means.
[0159]
The information stored in the mutant antibody fragment sequence information file 106d includes the antibody fragment identification information and / or the similar amino acid fragment identification information, the similar amino acid fragment sequence information regarding other mutated species and / or the mutation. It is configured to correlate with the affected antibody fragment sequence information.
[0160]
The antibody activity maintenance rate file 106e is antibody activity maintenance rate storage means for storing the antibody activity maintenance rate determined by the antibody activity maintenance rate determination unit 102f described later.
[0161]
The information stored in the antibody activity maintenance rate file 106e is configured by associating the similar amino acid fragment identification information with the antibody activity maintenance rate.
[0162]
The mutation-determined antibody sequence information file 106f is a mutation-determined antibody sequence information storage unit that stores antibody sequence information whose mutation has been determined by a mutation determination unit 102g described later.
[0163]
The information stored in the mutation-determining antibody sequence information file 106f is composed of mutated antibody sequence information.
[0164]
In addition, the amino acid fragment sequence information database 106g includes amino acid fragment sequence information relating to a biological species different from the biological species in the antibody sequence information acquired by the acquisition unit 102a described later, or an antibody using an antibody designed according to the present invention. It is the database which stored the amino acid fragment sequence information regarding the biological species used as a medicine target beforehand.
[0165]
The amino acid fragment sequence information database 106g may be an external amino acid fragment sequence information database that is accessed via the Internet, and may be copied, stored with original sequence information, It may be an in-house database created by adding unique annotation information or the like.
[0166]
In addition, as other information, the storage unit 106 of the antibody sequence creation device 100 stores various Web data for providing a website to the external system 200, a CGI program, a match search unit 102c and a similarity search unit described later. Prediction of secondary structure and tertiary structure of the above-described protein, for example, used in a program for executing a similar sequence search method such as BLAST or FASTA used in 102d, or a structural change contribution ratio calculation unit 102m described later For example, a program for executing a method for determining the charge of each amino acid residue, an energy calculation method, an affinity calculation method, and the like used in the index information calculation unit 102p described later. ing.
[0167]
The Web data includes data for displaying various Web pages to be described later, and the data is formed as a text file described in HTML or XML, for example. In addition, a part file, a work file, and other temporary files for creating the Web data are also stored in the storage unit 106.
[0168]
In addition, if necessary, audio to be transmitted to the external system 200 is stored as an audio file such as WAVE format or AIFF format, and still images or moving images are stored as image files such as JPEG format or MPEG2 format. You can do it.
[0169]
In FIG. 2, the communication control interface unit 104 performs communication control between the antibody sequence creation device 100 and the network 300 (or a communication device such as a router). That is, the communication control interface unit 104 has a function of communicating data with other terminals via a communication line.
[0170]
In FIG. 2, the input / output control interface unit 108 controls the input device 112 and the output device 114. Here, as the output device 114, in addition to a monitor (including a home TV), a speaker can be used (hereinafter, the output device 114 may be described as a monitor). As the input device 112, a keyboard, a mouse, a microphone, or the like can be used. The monitor also realizes a pointing device function in cooperation with the mouse.
[0171]
In FIG. 2, the control unit 102 has a control program such as an OS (Operating System), a program defining various processing procedures, and an internal memory for storing necessary data. Information processing for executing various processes is performed. The control unit 102 includes an acquisition unit 102a, a division unit 102b, a match search unit 102c, a similarity search unit 102d, a mutation unit 102e, an antibody activity maintenance rate determination unit 102f, and a mutation determination unit 102g in terms of functional concept. Has been.
[0172]
Among these, the acquisition unit 102a is an acquisition unit that acquires antibody sequence information including complementarity determining region information and / or framework region information. Here, as illustrated in FIG. 3, the acquisition unit 102a further includes an area dividing unit 102h.
[0173]
FIG. 3 is a block diagram showing an example of the configuration of the acquisition unit 102a of the system to which the present invention is applied, and conceptually shows only the portion related to the present invention in the configuration.
[0174]
In FIG. 3, an area dividing unit 102h is an area dividing unit that divides antibody sequence information into complementarity determining area information and framework area information.
[0175]
Returning again to FIG. 2, the dividing unit 102b is a dividing unit that divides the antibody sequence information into antibody fragment sequence information having a predetermined length. Here, as shown in FIG. 4, the dividing unit 102b further includes a slide dividing unit 102i.
[0176]
FIG. 4 is a block diagram showing an example of the configuration of the dividing unit 102b of the system to which the present invention is applied, and conceptually shows only the portion related to the present invention in the configuration.
[0177]
In FIG. 4, a slide dividing unit 102i is a slide dividing unit that divides antibody fragment sequence information having a predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information.
[0178]
Returning to FIG. 2 again, the match search unit 102c is a match search means for searching for amino acid fragment sequence information regarding other matching species for each antibody fragment sequence information.
[0179]
The similarity search unit 102d is a similarity search unit that searches for amino acid fragment sequence information related to other similar species for each antibody fragment sequence information.
[0180]
The mutation unit 102e is a mutation unit that mutates the antibody sequence information by exchanging the amino acid fragment sequence information on each similar other species and the search source antibody fragment sequence information.
[0181]
The antibody activity maintenance rate determination unit 102f is an antibody activity maintenance rate determination unit that determines an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before the mutation is maintained for the mutated antibody sequence information. Here, as shown in FIG. 5, the antibody activity maintenance rate determination unit 102f includes a change amount calculation unit 102j, a change amount reference antibody activity maintenance rate determination unit 102k, a structure change contribution rate calculation unit 102m, and a structure change contribution rate reference antibody. It further includes an activity maintenance rate determination unit 102n, an index information calculation unit 102p, and an index information reference antibody activity maintenance rate determination unit 102r.
[0182]
FIG. 5 is a block diagram showing an example of the configuration of the antibody activity maintenance rate determination unit 102f of the present system to which the present invention is applied, and conceptually shows only the portion related to the present invention in the configuration.
[0183]
In FIG. 5, a change amount calculation unit 102j is a change amount calculation unit that calculates a change amount between amino acid fragment sequence information relating to another similar species to be replaced and antibody fragment sequence information of a search source.
[0184]
The change amount reference antibody activity maintenance rate determination unit 102k is change amount reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate based on the calculated change amount.
[0185]
Further, the structure change contribution rate calculation unit 102m compares the structures of amino acid fragment sequence information on other similar species to be replaced and the antibody fragment sequence information of the search source, and how much the change in the sequence corresponds to the change in the structure. It is a structure change contribution rate calculation means for calculating a structure change contribution rate indicating whether or not a contribution has been made.
[0186]
The structure change contribution rate reference antibody activity maintenance rate determination unit 102n is a structure change contribution rate reference antibody activity maintenance rate determination unit that determines the antibody activity maintenance rate based on the calculated structure change contribution rate.
[0187]
In addition, the index information calculation unit 102p calculates index information regarding the amino acid fragment sequence information regarding other similar species to be replaced and the antibody fragment sequence information of the search source to calculate index information regarding the property of the amino acid changed on each sequence. Means.
[0188]
The index information reference antibody activity maintenance rate determination unit 102r is an index information reference antibody activity maintenance rate determination unit that determines the antibody activity maintenance rate based on the calculated index information.
[0189]
Returning to FIG. 2 again, the mutation determining unit 102g is a mutation determining unit that determines a variation in antibody sequence information based on the antibody activity maintenance rate.
[0190]
Details of processing performed by each of these units will be described later.
[0191]
[System processing]
Next, an example of the processing of the system according to the present embodiment configured as described above will be described in detail with reference to FIGS.
[0192]
[Main processing]
First, details of the main process will be described with reference to FIG. FIG. 6 is a flowchart showing an example of main processing of the system according to the present embodiment.
[0193]
First, the antibody sequence creation device 100 acquires the antibody sequence information including the complementarity determining region information and / or the framework region information by the processing of the acquiring unit 102a, and stores it in a predetermined region of the antibody sequence information file 106a ( Step SA-1).
[0194]
Here, the acquisition unit 102a may divide the antibody sequence information acquired in step SA-1 into complementarity determination region information and framework region information by the processing of the region division unit 102h (region division processing). . That is, it may be divided into complementarity determining region information and framework region information, and the following processing may be performed separately.
[0195]
Here, the details of the region dividing process performed in the region dividing unit 102h will be described with reference to FIG.
[0196]
FIG. 7 is a flowchart showing an example of area division processing of the system according to the present embodiment.
[0197]
First, the acquiring unit 102a divides the acquired antibody sequence information into complementarity determining region information and framework region information by the processing of the region dividing unit 102h, and stores it in a predetermined region of the antibody sequence information file 106a. (Step SB-1).
[0198]
This completes the region division process.
[0199]
Returning to FIG. 6 again, the antibody sequence creation device 100 divides the antibody sequence information acquired in step SA-1 into antibody fragment sequence information of a predetermined length by the processing of the dividing unit 102b (step SA). -2).
[0200]
Here, in Step SA-2, the predetermined length of the antibody fragment sequence information is, for example, about 9 to 20 sequences.
[0201]
Here, the dividing unit 102b performs a process of the slide dividing unit 102i, and slides the antibody sequence information acquired in step SA-1 by one amino acid residue from the beginning, and antibody fragment sequence information having a predetermined length. (Slide division processing).
[0202]
Here, details of the slide division processing performed in the slide division unit 102i will be described with reference to FIG.
[0203]
FIG. 8 is a flowchart illustrating an example of the slide division processing of the system according to the present embodiment.
[0204]
First, the dividing unit 102b sets 1 to information “i” indicating the position from the head of the antibody sequence information by the processing of the slide dividing unit 102i (step SC-1).
[0205]
Next, the dividing unit 102b obtains antibody fragment sequence information having a predetermined length from the i-th amino acid residue of the antibody sequence information obtained in step SA-1 by the processing of the slide dividing unit 102i. It stores in a predetermined area of the antibody fragment sequence information file 106b (step SC-2).
[0206]
Next, the dividing unit 102b increments by adding 1 to i by the processing of the slide dividing unit 102i (step SC-3).
[0207]
Next, the dividing unit 102b determines whether or not it is the last of the antibody sequence information based on i incremented in step SC-3 by the processing of the slide dividing unit 102i (step SC-4).
[0208]
If it is determined as “not last” in step SC-4, the process returns to step SC-2. If it is determined as “last”, the process ends.
[0209]
This completes the slide division process.
[0210]
Referring back to FIG. 6 again, the antibody sequence creation device 100 obtains amino acid fragment sequence information on other matching species for each antibody fragment sequence information divided in step SA-2 by the process of the match search unit 102c. A search is made from the fragment sequence information database 106g (step SA-3).
[0211]
Here, the “other biological species” may be, for example, a biological species derived from information different from the antibody sequence information, and is a target for antibody medical treatment using the antibody designed according to the present invention, such as human Or any other species.
[0212]
Next, the antibody sequence creation device 100 performs similar processing for each antibody fragment sequence information for which amino acid fragment sequence information relating to another species that matches in step SA-3 was not searched by the processing of the similarity search unit 102d. The amino acid fragment sequence information on the biological species is searched from the amino acid fragment sequence information database 106g and stored in a predetermined area of the similar amino acid fragment sequence information file 106c (step SA-4).
[0213]
Here, in step SA-3 and step SA-4, each divided antibody fragment sequence information is used as a query sequence, for example, “nr-aa”, “Human Ig, TcR”, “GenPept”, From existing amino acid fragment sequence databases such as “SwissProt”, “PIR”, “PRF” and the like, amino acid fragment sequence information regarding other matching species and amino acid fragment sequence information regarding other similar species, for example, BLAST, You may search using the existing similar sequence search methods, such as FASTA.
[0214]
Subsequently, the antibody sequence creation device 100 replaces the amino acid fragment sequence information on each similar other species searched in step SA-4 with the search source antibody fragment sequence information by the processing of the mutation unit 102e. The sequence information is mutated, and the replaced amino acid fragment sequence information regarding other similar species and the antibody fragment sequence information affected by the mutation are stored in a predetermined region of the mutant antibody fragment sequence information file 106d (step SA- 5).
[0215]
Next, the antibody sequence creation device 100 indicates whether the activity of the antibody sequence information before mutation is maintained for each antibody sequence information mutated in step SA-5 by the processing of the antibody activity maintenance rate determination unit 102f. The antibody activity maintenance rate is determined and stored in a predetermined area of the antibody activity maintenance rate file 106e (step SA-6).
[0216]
Here, in step SA-6, the determination of the maintenance ratio of the antibody activity is carried out with respect to the amino acid fragment sequence information regarding other mutated similar species and the antibody fragment sequence information affected by the mutation. As in the case of No. 3, amino acid fragment sequence information relating to other biological species that match may be searched, and the antibody activity maintenance rate may be calculated based on the search result.
[0217]
In addition, the antibody activity maintenance rate determination unit 102f includes the amino acid fragment sequence information related to other similar species to be replaced in step SA-5 by the processing of the change amount calculation unit 102j and the change amount reference antibody activity maintenance rate determination unit 102k. The amount of change with the antibody fragment sequence information of the search source may be calculated, and the antibody activity maintenance rate may be determined based on the calculated amount of change (change amount reference antibody activity maintenance rate determination process).
[0218]
Details of the change amount reference antibody activity maintenance rate determination process performed in the change amount calculation unit 102j and the change amount reference antibody activity maintenance rate determination unit 102k will be described with reference to FIG.
[0219]
FIG. 9 is a flowchart showing an example of the change amount reference antibody activity maintenance rate determination process of the system according to the present embodiment.
[0220]
First, the antibody activity maintenance rate determination unit 102f performs a change in the amount of change between the amino acid fragment sequence information related to another similar species to be replaced in step SA-5 and the search source antibody fragment sequence information by the processing of the change amount calculation unit 102j. Is calculated (step SD-1).
[0221]
Here, the amount of change may be, for example, the number of amino acid residue differences between the amino acid fragment sequence information related to other similar species to be replaced and the antibody fragment sequence information of the search source, the degree of similarity, and the like.
[0222]
Next, the antibody activity maintenance rate determination unit 102f determines the antibody activity maintenance rate based on the amount of change calculated in step SD-1 by the processing of the change amount reference antibody activity maintenance rate determination unit 102k, and the antibody activity The data is stored in a predetermined area of the maintenance rate file 106e (step SD-2).
[0223]
Thus, the variation reference antibody activity maintenance rate determination process is completed.
[0224]
In step SA-6 of FIG. 6, the antibody activity maintenance rate determination unit 102f performs processing in step SA-5 by the processing of the structure change contribution rate calculation unit 102m and the structure change contribution rate reference antibody activity maintenance rate determination unit 102n. Compare the structures of amino acid fragment sequences for other similar species to be replaced and the antibody fragment sequence information of the search source, and calculate how much the change in the sequence contributed to the change in the structure. Then, the antibody activity maintenance rate may be determined based on the calculated structure change contribution rate (structural change contribution rate reference antibody activity maintenance rate determination process).
[0225]
Here, the structure change contribution rate reference antibody activity maintenance rate determination process performed in the structure change contribution rate calculation unit 102m and the structure change contribution rate reference antibody activity maintenance rate determination unit 102n will be described with reference to FIG.
[0226]
FIG. 10 is a flowchart showing an example of the structure change contribution rate reference antibody activity maintenance rate determination process of the system according to the present embodiment.
[0227]
First, the antibody activity maintenance rate determination unit 102f performs the process of the structural change contribution rate calculation unit 102m, with respect to the amino acid fragment sequence information regarding other similar species to be replaced in step SA-5 and the antibody fragment sequence information of the search source. Each structure is compared, and a structure change contribution ratio indicating how much the change in the sequence contributes to the change in the structure is calculated (step SE-1).
[0228]
Here, the structure may be either a secondary structure or a tertiary structure.
[0229]
For prediction of the secondary structure, for example, 1. "Chou-Fasman method", 2. 2. “GOR (Garnier-Osguthorpe-Robson) method”; 3. “Nearest Neighbor Method” “Secondary structure prediction method using neural network model (for example,“ Qian N. and Seijnowski T. J., “Predicting the secondary structure of global proteins using neural network. 202. J. M. -884, 1988 ", and websites that perform neural network analysis such as" PHD: http://www.embl-heidelberg.de/predictprotein/predictprotein.html "). “Secondary structure prediction methods based on hidden Markov models (for example,“ Stultz CM, and White JV, and Smith TF, “Structural analysis based on state-space modeling.”, Protein Sc. 2, p. 305-314, 1993 ", etc.)", etc., may be used.
[0230]
For prediction of the tertiary structure, for example, 1. "Homology modeling method", 2. “Threading method”; “3D-1D method”; 4. “Ab initio method”; An existing method for tertiary structure prediction such as “fragment assembly method (see, for example,“ Proteins: StructureFunction, and Genetics, Supplement, 3, 1999 ”) may be used.
[0231]
Next, the antibody activity maintenance rate determination unit 102f determines the antibody activity maintenance rate based on the structure change contribution rate calculated in step SE-1 by the processing of the structure change contribution rate reference antibody activity maintenance rate determination unit 102n. And stored in a predetermined area of the antibody activity maintenance ratio file 106e (step SE-2).
[0232]
This completes the structure change contribution rate reference antibody activity maintenance rate determination process.
[0233]
Further, in step SA-6 of FIG. 6, the antibody activity maintenance rate determination unit 102f is replaced by the similar processing in step SA-5 by the processing of the index information calculation unit 102p and the index information reference antibody activity maintenance rate determination unit 102r. For the amino acid fragment sequence information on the species and the antibody fragment sequence information of the search source, index information on the nature of the amino acid changed on each sequence is calculated, and the antibody activity maintenance rate is determined based on the calculated index information (Index information reference antibody activity maintenance rate determination process).
[0234]
Here, the index information reference antibody activity maintenance rate determination process performed in the index information calculation unit 102p and the index information reference antibody activity maintenance rate determination unit 102r will be described with reference to FIG.
[0235]
FIG. 11 is a flowchart showing an example of the index information reference antibody activity maintenance rate determination process of the system according to the present embodiment.
[0236]
First, the antibody activity maintenance rate determination unit 102f performs the processing of the index information calculation unit 102p, with respect to the amino acid fragment sequence information and other search source antibody fragment sequence information regarding other similar species to be replaced in step SA-5. Index information relating to the properties of amino acids changed on the sequence is calculated (step SF-1).
[0237]
Here, the index information may be, for example, information about the charge of the amino acid sequence, information about energy (for example, electrostatic energy), information about affinity, and the like.
[0238]
For calculating the index information, for example, the charge of a positively charged charged amino acid (lysine, arginine) is 1, and the charge of a negatively charged charged amino acid (glutamic acid, aspartic acid) is − A method may be used in which the charge of amino acids other than 1 is 0.
[0239]
In addition, for the calculation of the index information, for example, each amino acid residue is calculated by the existing quantum chemical calculation method based on the three-dimensional structure information of the protein registered in the protein structure database or the three-dimensional structure information obtained by the simulation method. A method for determining the electric charge of each of them may be used.
[0240]
The index information is calculated by, for example, an energy calculation method such as molecular mechanics, molecular dynamics, molecular orbital method, density functional method, or fragment MO method (Chemical Physics Letters, Volume 336, Issues 1-2, 9 March). , P.163-170, 2001), a method of calculating electrostatic energy, or a method of calculating affinity.
[0241]
Moreover, you may use combining these each method suitably for calculation of the said parameter | index information.
[0242]
Next, the antibody activity maintenance rate determination unit 102f determines the antibody activity maintenance rate based on the index information calculated in step SF-1 by the processing of the index information reference antibody activity maintenance rate determination unit 102r, and the antibody activity The data is stored in a predetermined area of the maintenance rate file 106e (step SF-2).
[0243]
This completes the index information reference antibody activity maintenance rate determination process.
[0244]
Returning to FIG. 6 again, the antibody sequence creation device 100 performs the processing of the mutation determination unit 102g, based on the antibody activity maintenance rate for the antibody sequence information after each mutation determined in step SA-6. Are determined and stored in a predetermined area of the mutation-determined antibody sequence information file 106f (step SA-7).
[0245]
Here, in Step SA-7, based on the determined antibody activity maintenance rate for the antibody sequence information after each mutation, for example, a variation in antibody sequence information having the highest antibody activity maintenance rate may be determined.
[0246]
Further, in step SA-7, for example, antibody sequence information mutation may be optimized using optimization techniques such as Monte Carlo method, simulated annealing, genetic algorithm, etc. to determine the antibody sequence information mutation. .
[0247]
Moreover, in step SA-7, when the antibody activity maintenance rate of the antibody sequence information after the mutation is decreased with respect to that before the mutation, the mutation may be terminated.
[0248]
Here, the antibody sequence information described above is antibody sequence information related to a non-human such as a mouse, for example, and the amino acid fragment sequence information related to the other biological species may be amino acid fragment sequence information related to a human.
[0249]
This completes the main process.
[0250]
[Calculation example]
Next, an example of a calculation method for determining antibody mutation according to the present invention will be described.
Here, in the calculation method for determining the mutation of the antibody according to the present invention, the antibody activity maintenance rate is obtained using the abundance of amino acid fragment sequence information on other species included in the antibody sequence information after the mutation with respect to the mutation. Will be described as an example.
[0251]
Here, the abundance ratio is defined as shown in Equation 1 below, for example.
[0252]
[Expression 1]

Here, ΔE is the abundance rate, i is the number of the antibody fragment sequence information (i is an integer in the range of 1 to n−f + 1 (n is the length of the antibody sequence information, f is the length of the antibody fragment sequence information) And δexist (j) is the abundance of amino acid fragment sequence information on other species that matches the j-th antibody fragment sequence information after mutation relative to the pre-mutation, and sim (s ′ , S) is an antibody activity maintaining function (in this embodiment, “1” is set) of the antibody fragment sequence information “s” before mutation and the amino acid fragment sequence information “s ′” after mutation, and exist (s ', J) and exist (s, j) match the abundance of amino acid fragment sequence information on other species that matches the j-th antibody fragment sequence information after mutation and the j-th antibody fragment sequence information before mutation Amino acid fragment sequence information on other living species Is the existence rate.
[0253]
Exist (s ′, j) and exist (s, j) are, for example, when amino acid fragment sequence information relating to other species that matches the antibody fragment sequence information “s ′” (“s”) exists. “1” may be given, and “0” may be given when it does not exist.
[0254]
That is, ΔE is obtained by mutating the antibody fragment sequence information “s” before mutation to amino acid fragment sequence information related to another species, and the amino acid fragment sequence information related to the mutated other species and the variation. This means a change in the abundance of amino acid fragment sequence information relating to other biological species that matches the antibody fragment sequence information “s ′” affected by the above.
[0255]
Specifically, when ΔE is negative, the amino acid fragment sequence information related to other species contained in the antibody sequence information after mutation has decreased compared to before mutation, that is, after mutation compared to before mutation. Antibody sequence information means that antigenicity against other species has increased.
[0256]
On the other hand, when ΔE is positive, the amino acid fragment sequence information on other species included in the antibody sequence information after mutation has increased compared to before mutation, that is, the antibody sequence after mutation compared to before mutation. Information means that antigenicity against other species is reduced.
[0257]
The above-described antibody activity maintenance function sim (s ′, s) is set to “1” in the present embodiment, for example. However, other constant values may be used, and the following antibody activity maintenance function is determined. You may define based on the method.
[0258]
Here, three examples of the method of determining the antibody activity maintenance function sim (s ′, s) will be described.
[0259]
(1) First, the antibody activity maintenance function may be determined based on the number of amino acid residue differences between s ′ and s.
[0260]
Specifically, for example, s ′ (with amino acid fragment sequence information regarding other matching species) is “ACDEFGHHI”, and s (without amino acid fragment sequence information regarding other matching species) is “YCDEFGHHIK”. In some cases, “S” (s ′, s) = 1 ”because“ A ”and“ Y ”differ by one in the two fragment sequences.
[0261]
(2) In addition, if the mutation is predicted to be different in the amino acid difference between s ′ and s (previously obtained as information using a fragment structure prediction method or the like), sim (s ′, The value of s) may be small, otherwise it may be large.
[0262]
Specifically, for example, s ′ (with amino acid fragment sequence information regarding other matching species) is “ACDEFGHIK”, and s (without amino acid fragment sequence information regarding other matching species) is “GCDEFGHHIK”. In some cases, one of “A” and “G” is different, but the structural change contribution rate from “A” to “G”, which is information acquired in advance, is “diff (A, G ) = 0.5 ”, sim (s ′, s) is, for example, the inverse of diff“ 1 / 0.5 ”, that is,“ sim (s ′, s) = 1 / 0.5 ”.
[0263]
Specifically, for example, s ′ (with amino acid fragment sequence information regarding other matching species) is “ACDEFGHIK”, and s (without amino acid fragment sequence information regarding other matching species) is “ACDEFWHIK”. ”, One of“ G ”and“ W ”is different, but the structural change contribution rate from“ G ”to“ W ”, which is information acquired in advance, is“ diff (G , W) = 3.0 ”,“ sim (s ′, s) = 1 / 3.0 ”.
[0264]
(3) Furthermore, if the amino acid difference between s ′ and s is a mutation that is predicted to contribute greatly to affinity (previously obtained as information), the value of sim (s ′, s) is set to a large value. Otherwise, it can be small.
[0265]
This completes the description of the calculation method for determining antibody mutations.
[0266]
[Example]
Next, the method of the present invention is applied to humanization of mouse antibody sequences, and it is verified by using the following examples whether the method of the present invention may lead to a correct result.
[0267]
First, in this example, the calculation / display of the humanized state was performed according to the following procedure (hereinafter referred to as the humanized state calculating / displaying procedure).
[0268]
1. The antibody sequence information creation device 100 acquires mouse antibody sequence information by processing of the acquisition unit 102a, and stores it in a predetermined area of the antibody sequence information file 106a.
[0269]
2. The antibody sequence information creating apparatus 100 is configured to slide the acquired mouse antibody sequence information by one amino acid residue from the beginning by the processing of the slide dividing unit 102i, and the mouse antibody fragment sequence information having a predetermined length of “9”. And is stored in a predetermined area of the antibody fragment sequence information file 106b.
[0270]
3. The antibody sequence information creation device 100 converts each divided mouse antibody fragment sequence information into a query sequence by the process of the match search unit 102c, and uses an external database (hereinafter referred to as “human” in non-reduntant sequence data). nr.Human ”)) or“ Human, Ig TcR ”database and search for matching human fragment sequence information using BLAST.
[0271]
4). As a result of the process of the matching search unit 102c, the antibody sequence information creation apparatus 100 sets “1” when the search is performed and “0” when the search is not performed. Append.
[0272]
5. The antibody sequence information creation device 100 calculates the presence rate of human fragment sequence information by the following formula 2 for each amino acid residue of the mouse antibody sequence information by the processing of the control unit 102.
[Expression 2]

Here, E (i) is a human fragment at an amino acid residue related to residue number i (i is an integer taking a value of 1 to n (n is the length of mouse antibody sequence information)). This is the abundance of sequence information, f is the length of the predetermined mouse antibody fragment sequence information (in this example, “9”), and exist (s, j) is the jth amino acid residue. This is the presence rate of human fragment sequence information that matches the mouse antibody fragment sequence information that has a group at the head (in this example, “1” is given if it exists, and “0” is given if it does not exist).
[0273]
That is, E (i) means the abundance of human fragment sequence information that matches each mouse antibody fragment sequence information (the number matches f) containing the i-th amino acid residue. The possible values are 0 to f (in this embodiment, “9”).
[0274]
6). The antibody sequence information creation device 100 displays the calculated human fragment existence rate for each residue on the output device 114 in a graph by the processing of the control unit 102.
[0275]
This concludes the description of the humanized state calculation / display procedure. The graph described below is displayed according to the humanized state calculation / display procedure.
[0276]
[Example 1: Humanized state of mouse monoclonal antibody (hereinafter referred to as “CRA4”) derived from mouse hybridoma cell “CRA4” used in the prior art and humanized]
Next, the humanized state of the humanized FcR mouse antibody “CRA4” used in JP-A-7-165799 and JP-A-9-191886 (hereinafter referred to as conventional technology) and humanized, This will be described with reference to FIGS. Note that the database in the match search is “nr.Human”.
[0277]
FIG. 12 is a diagram showing an example of the humanized state of the L chain of “CRA4” used in the prior art in this example. Moreover, FIG. 13 is a figure which shows an example of the humanized state of the heavy chain of "CRA4" used by the prior art in a present Example. Moreover, FIG. 14 is a figure which shows an example of the humanization state of the L chain of "CRA4" humanized by the prior art in a present Example. Furthermore, FIG. 15 is a figure which shows an example of the humanization state of the heavy chain of "CRA4" humanized by the prior art in a present Example.
[0278]
Here, the items displayed in the graph will be described by taking FIG. 12 as an example. The same applies to the graphs shown below.
[0279]
First, in FIG. 12, the horizontal axis represents the residue number, and the vertical axis represents the presence rate of the human fragment for each amino acid residue. Moreover, the part shown in black is a part showing that the matching human fragment sequence information exists, and the part shown in gray is a part showing that there was not. In addition, the horizontal bar shown at the top of the graph indicates the CDR range.
[0280]
As shown in FIGS. 12 and 13, CRA4 before humanization has a small area indicated by black, that is, a state in which the presence rate of human fragments is small.
[0281]
Also, as shown in FIGS. 12 to 15, when looking at the shape of the graph, there is a clear difference in characteristics between the mouse antibody and the humanized antibody. For example, in a well-humanized antibody, except for the CDR portion, the abundance of human fragments for each amino acid residue is kept at the maximum value (= fragment sequence length “9”). The humanized antibody is characterized in that the portion related to the CDR draws a “V-shape” from the center of the CDR.
[0282]
This is the end of the description of the first embodiment.
[0283]
[Example 2: Humanized state of antibody humanized in the present invention (part 1)]
Next, the humanized state of the antibody humanized in the present invention will be described with reference to FIGS.
[0284]
(2-1. Humanized state of CRA4 (part 1))
First, the humanized state of CRA4 used in the prior art will be described with reference to FIGS. In the humanization of CRA4 by the method of the present invention, the match search unit 102c and the similarity search unit 102d performed a search for “nr.Human”. The antibody activity maintenance rate determination unit 102f mutated all the similar human acid fragment sequence information. Furthermore, the mutation determining unit 102g terminated the mutation when the antibody sequence maintenance rate after the mutation decreased with respect to that before the mutation.
[0285]
FIG. 16 is a diagram showing an example of the humanized state of the L chain of “CRA4” humanized by the method of the present invention in this example. Moreover, FIG. 17 is a figure which shows an example of the humanization state of the heavy chain of "CRA4" humanized by the method of this invention in a present Example. Furthermore, FIG. 18 is a figure which shows an example of the amino acid sequence of "CRA4" humanized by the method of this invention in a present Example.
[0286]
Here, the amino acid sequences shown in the upper part of FIG. 18 are CRA4 ("mouse" shown in FIG. 18) used in the prior art in the L chain and the H chain and CRA4 humanized by the method of the present invention (in FIG. 18). Amino acid sequence of “humanized (new)”).
[0287]
In addition, the amino acid sequences shown in the lower part of FIG. 18 are CRA4 humanized by the prior art in the L chain and the H chain (“humanized (con)” shown in FIG. 18) and humanized CRA4 by the method of the present invention. Amino acid sequence ("humanized (new)" shown in FIG. 18).
[0288]
In FIG. 18, “*” attached to the “dif” line is a symbol indicating that the amino acid is a different amino acid residue, and “L” attached to the “cdr” line. "To" J "are symbols indicating amino acid residues contained in the CDR site.
[0289]
As shown in FIGS. 16 to 18, it is understood that the L chain is almost humanized except for the CDR site, but in the H chain, one CDR site is further humanized. This result is the same as the result of “CRA4” humanized by the prior art shown in FIGS. 14 and 15.
[0290]
In the abundance ratios shown in FIGS. 16 and 17, and in FIG. 14 and FIG. 15, the overall abundance ratio values are not so different, but the differences are large when the respective antibody sequences are compared (see FIG. 18). Recognize.
[0291]
In the H chain of CRA4 humanized by the prior art, the difference in amino acid residues was large, and there were as many as 18 mutation differences. There were also differences in the absolute number of mutations, and the number of mutations in CRA4 humanized by the prior art was 19 for the L chain and 22 for the H chain (see FIG. 18).
[0292]
On the other hand, the number of mutations of CRA4 humanized by the method of the present invention was 13 for the L chain and 13 for the H chain, almost halved. In view of the fact that the possibility of a decrease in antibody activity generally increases when a large number of mutations are introduced, the method of the present invention is likely to avoid a decrease in antibody activity.
[0293]
(2-2. Humanized state of CRA4 (part 2))
Next, the humanized state of CRA4 used in the prior art will be described with reference to FIGS. In the humanization of CRA4 by the method of the present invention, the match search unit 102c and the similarity search unit 102d searched the external database “nr.Human”. The antibody activity maintenance rate determination unit 102f mutated the human acid fragment sequence information having the highest similarity. Furthermore, the mutation determination unit 102g did not terminate the mutation even when the antibody activity maintenance rate after the mutation decreased with respect to that before the mutation.
[0294]
FIG. 19 is a view showing another example of the humanized state of the L chain of “CRA4” humanized by the method of the present invention in this example. Moreover, FIG. 20 is a figure which shows another example of the humanization state of the heavy chain of "CRA4" humanized by the method of this invention in a present Example. Furthermore, FIG. 21 is a figure which shows another example of the amino acid sequence of "CRA4" humanized by the method of this invention in a present Example.
[0295]
As shown in FIGS. 19 to 21, as in the results of FIGS. 16 to 18, it is understood that the L chain is almost humanized except for the CDR site, and that one CDR site is humanized in the H chain. it can.
[0296]
In addition, in the abundance ratios shown in FIGS. 19 and 20, FIG. 14 and FIG. 15, the value of the overall abundance ratio does not change so much, but the difference is large when the respective sequences are compared (see FIG. 21). Recognize.
[0297]
And in the H chain of CRA4 humanized by the prior art, the difference in amino acid residues was large, and there were as many as 19 mutation differences. In addition, there was a difference in the absolute number of mutations, and the number of mutations of CRA4 humanized by the prior art was 19 for the L chain and 22 for the H chain.
[0298]
On the other hand, the number of mutations of CRA4 humanized by the method of the present invention was 13 for the L chain and 13 for the H chain, almost halved. In view of the fact that the possibility of a decrease in antibody activity generally increases when a large number of mutations are introduced, the method of the present invention is likely to avoid a decrease in antibody activity.
[0299]
This is the end of the description of the second embodiment.
[0300]
[Consideration for Example 1 and Example 2]
In mouse antibodies (see FIGS. 12 and 13), the presence of human fragments is small. If this protein is randomly fragmented in humans, it is very unlikely that the fragment is derived from humans (ie, it can be said to be antigenic).
[0301]
On the other hand, in humanized antibodies (known humanized antibodies (see FIGS. 14 and 15), Example 1 (see FIGS. 16 and 17), and Example 2 (see FIGS. 19 and 20)), the presence of human fragments is generally Except for CDR (and its vicinity), all human fragments involving a certain amino acid residue are in a state where no non-human-derived fragment exists. Under this condition, it can be said that almost all human-derived fragments can be obtained by random fragmentation at sites other than the CDR (and its vicinity) (that is, the antigenicity is low).
[0302]
The problem of achieving both reduction of antigenicity and maintenance of activity due to mutation is a major problem when preparing humanized antibodies. In general, there is a trade-off between antigenicity reduction and activity maintenance. That is, the antigenicity is lowered by the mutation to humanization, but when the mutation increases, the probability of decreasing the activity is expected to increase. Thus, for example, the number of mutations from mouse to humanized antibody can be considered as an example of an indicator of decreased activity. Here, it is considered that the number of mutations increases = the possibility of a decrease in activity increases.
[0303]
Let us compare the humanization method according to the present invention (FIGS. 16, 17, 19, 20) with the existing method (FIGS. 14, 15). Although the humanization graph shows little difference, it can be seen that the humanized sequences are different (see FIGS. 18 and 19).
[0304]
Looking at the difference in sequence variation, it can be seen that the humanization method of the present invention has fewer mutations than the conventional method. The situation of humanization is the same but the number of mutations is small, which means that the antigenicity is low but the activity is likely to be maintained. In other words, as far as this index is concerned, it can be seen that both antigenicity and activity maintenance are improved (particularly in terms of activity maintenance) as compared with the conventional method.
[0305]
In the present invention, in order to further consider the maintenance of antibody activity due to mutation, for example, if there is a human fragment sequence that maintains antibody activity, the human fragment sequence is mutated, or the human fragment sequence that affects antibody activity includes Introduces a mechanism to prevent mutations.
[0306]
Specifically, in the present invention, for example, a sequence distance that correlates with maintenance of antibody activity, that is, an inter-sequence distance (for example, sequence similarity) from the viewpoint of maintaining antibody activity is defined. be able to.
[0307]
Further, in the present invention, the similarity of the antibody activity maintenance function may be defined using a matrix such as PAM30 used in BLAST and the like, and the matrix is further redefined for activity maintenance. Also good.
[0308]
Specifically, in the present invention, the above-described antibody activity maintenance function “sim (s ′, s)” or the like may be determined.
[0309]
In the present invention, for example, by using index information related to amino acid characteristics such as charge information, energy and affinity, correlation information between fragment sequences and structure information, detailed structure information such as structure modeling, and the like, more antibody activity It is also possible to determine an antibody activity maintenance function that can maintain the above.
[0310]
Next, let us consider the antigenicity of CDR. It can be seen that the CDRs of the known humanized antibodies, humanized antibodies in Examples 1 and 2 (and the vicinity thereof) are V-shaped (FIGS. 14, 15, 16, 17, 19, 20). ). This indicates that the fragment data near the CDR does not exist on “nr.Human”.
[0311]
With respect to the absence of CDRs on `` nr.Human '' even in known humanized antibodies, 1. CDRs tend to be exempt from antigenicity, so humanization for this part need not be considered, 2. A CDR is a sequence obtained by a gene whose germline sequence has been rearranged, and therefore may not be present in a normal nr database. Therefore, regarding the antigenicity of CDR, there are two interpretations that “human CDR fragment data” dedicated to CDR may be used instead of “nr.Human”.
[0312]
Although the conventional method is considered to take the position of 1. Basically, the framework of the present invention is different from the conventional method in which CDR is not mutated, and by using “human CDR fragment data”, the CDR site is also human. It is possible to In other words, in our method, it is possible to mutate the CDR.
[0313]
The handling of CDR varies depending on whether you think from the viewpoint of 1 or 2 above, but the direction in which humanization should be promoted depends on how deeply this site is involved in antigenicity. Depends on whether
[0314]
If the CDR site is not very involved in antigenicity, in the present invention, for example, humanization does not require much consideration of CDR (position 1 above). That is, in the present invention, humanized antibodies with fewer mutations may be prepared by dividing antibody sequence information into CDRs and framework regions and subjecting only the framework regions to humanization.
[0315]
On the other hand, if CDR is deeply involved in antigenicity, in the present invention, for example, a CDR fragment database such as the above-mentioned “human CDR fragment data” may be first prepared. By referring to this database, antigenicity can be eliminated more effectively.
[0316]
Regarding the maintenance of antibody activity, CDR is often a site that directly binds to an antigen, and mutations at this site can directly cause inactivation, and thus humanization of the CDR site requires careful handling. Therefore, in the present invention, in addition to the above-described antibody activity maintenance function, an antibody activity maintenance function that is more sensitive to activity may be introduced, and for example, a three-dimensional modeling method or the like may be introduced explicitly.
[0317]
[Example 3: Humanized state of antibody humanized in the present invention (part 2)]
Next, the humanized state of the anti-tissue factor antibody humanized by the method of the present invention will be described with reference to FIGS. The example antibody sequence is the sequence of PDB code “1ahw” for mouse antibodies, and the sequence of “1jps” for known humanized antibodies.
[0318]
FIG. 22 is a diagram showing an example of the humanized state of the L chain of the mouse antibody “1ahw” used in this Example. FIG. 23 is a diagram showing an example of the humanized state of the heavy chain of the mouse antibody “1ahw” used in this example.
[0319]
FIG. 24 is a diagram showing an example of the humanized state of the L chain of the known humanized mouse antibody “1 jps” used in this example. FIG. 25 is a diagram showing an example of the humanized state of the H chain of the known humanized antibody “1 jps” used in this example.
[0320]
FIG. 26 is a diagram showing an example of the humanized state of the L chain of the humanized antibody “humanized (new) 1ahw” prepared by the method of the present invention in this example. FIG. 27 is a diagram showing an example of the humanized state of the H chain of the humanized antibody “humanized (new) 1ahw” prepared by the method of the present invention in this example.
[0321]
Further, FIG. 28 is a diagram showing an example of the amino acid sequence of the humanized antibody “humanized (new) 1ahw” prepared by the method of the present invention in this example.
[0322]
As far as FIG. 22 to FIG. 25 are viewed, it seems that the known humanized antibody “1jps” is also effectively humanized. However, when FIG. 24 to FIG. 27 are viewed, the human prepared by the method of the present invention is used. It can be understood that the humanized antibody “humanized (new) 1ahw” (see FIGS. 26 and 27) is further humanized in the fragment sequence near the CDR. From this, it can be understood that the method of the present invention makes it possible to produce a humanized antibody that maintains high antibody activity and effectively eliminates antigenicity.
[0323]
This is the end of the description of the third embodiment.
[0324]
[Other embodiments]
Although the embodiments of the present invention have been described so far, the present invention can be applied to various different embodiments in addition to the above-described embodiments within the scope of the technical idea described in the claims. May be implemented.
[0325]
For example, the case where the antibody sequence creation device 100 performs processing in a stand-alone form has been described as an example. However, processing is performed in response to a request from a client terminal configured in a separate casing from the antibody sequence creation device 100, You may comprise so that the process result may be returned to the said client terminal.
[0326]
In addition, among the processes described in the embodiment, all or part of the processes described as being automatically performed can be performed manually, or all of the processes described as being performed manually are all performed. Alternatively, a part can be automatically performed by a known method.
[0327]
In addition, the processing procedures, control procedures, specific names, information including parameters such as various registration data and search conditions, screen examples, and database configurations shown in the above documents and drawings, unless otherwise specified. It can be changed arbitrarily.
[0328]
In addition, regarding the antibody sequence creation device 100, each illustrated component is functionally conceptual and does not necessarily need to be physically configured as illustrated.
For example, the processing functions provided in each part or each device of the antibody sequence creation device 100, particularly the processing functions performed by the control unit 102, are all or any part thereof are assigned to a CPU (Central Processing Unit) and the CPU. It can be realized by a program that is interpreted and executed, or can be realized as hardware by wired logic. The program is recorded on a recording medium described later, and is mechanically read by the antibody sequence creation device 100 as necessary.
[0329]
That is, in the storage unit 106 such as a ROM or an HD, a computer program for giving instructions to the CPU and performing various processes in cooperation with an OS (Operating System) is recorded. The computer program is executed by being loaded into a RAM or the like, and constitutes the control unit 102 in cooperation with the CPU. Further, this computer program may be recorded in an application program server connected to the antibody sequence creation device 100 via an arbitrary network 300, and may be downloaded in whole or in part as necessary. It is.
[0330]
The program according to the present invention can also be stored in a computer-readable recording medium. Here, the “recording medium” is an arbitrary “portable physical medium” such as a flexible disk, a magneto-optical disk, a ROM, an EPROM, an EEPROM, a CD-ROM, an MO, and a DVD, and is incorporated in various computer systems. Program in a short time, such as a communication line or carrier wave when transmitting a program via any “fixed physical medium” such as ROM, RAM, HD, or a network such as LAN, WAN, or the Internet The “communication medium” that holds
[0331]
The “program” is a data processing method described in an arbitrary language or description method, and may be in any format such as source code or binary code. The “program” is not necessarily limited to a single configuration, but is distributed in the form of a plurality of modules and libraries, or in cooperation with a separate program represented by an OS (Operating System). Including those that achieve the function. Note that a well-known configuration and procedure can be used for a specific configuration for reading a recording medium, a reading procedure, an installation procedure after reading, and the like in each device described in the embodiment.
[0332]
Various databases and the like (antibody sequence information file 106a to amino acid fragment sequence information database 106g) stored in the storage unit 106 are memory means such as RAM and ROM, fixed disk devices such as hard disks, storage means such as flexible disks and optical disks. It stores various programs, tables, files, databases, web page files, etc. used for various processes and website provision.
[0333]
The antibody sequence creation apparatus 100 connects a peripheral device such as a printer, a monitor or an image scanner to an information processing apparatus such as an information processing terminal such as a known personal computer or workstation, and the method of the present invention is applied to the information processing apparatus. You may implement | achieve by mounting the software (a program, data, etc. are included) which implement | achieve.
[0334]
Furthermore, the specific form of dispersion / integration of the antibody sequence creation device 100 or the like is not limited to that shown in the specification and drawings, and all or a part thereof is functional or arbitrary in any unit corresponding to various loads. It can be physically distributed and integrated (for example, grid computing). For example, each database may be independently configured as an independent database device, and a part of the processing may be realized by using CGI (Common Gateway Interface).
[0335]
【The invention's effect】
As described above in detail, according to the present invention, antibody sequence information including complementarity determining region information and / or framework region information is acquired, and the acquired antibody sequence information is an antibody of a predetermined length. Divided into fragment sequence information, for each divided antibody fragment sequence information, search for amino acid fragment sequence information for other matching species, for each antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched, Search for similar amino acid fragment sequence information, mutate the antibody sequence information by replacing each searched similar amino acid fragment sequence information with the antibody fragment sequence information of the search source, and for the mutated antibody sequence information, Since the antibody activity maintenance rate indicating whether the activity of the antibody sequence information is maintained is determined, and the mutation of the antibody sequence information is determined based on the determined antibody activity maintenance rate. A lot of amino acid fragment sequence information relating to other species, that is, antibody sequence information that is low in antigenicity in other species can be created efficiently, and according to the present invention, the antibody activity maintenance rate is based on Since the mutation of the antibody sequence information is determined, the part that affects the antibody activity is not mutated, that is, the antibody sequence creation device, the antibody sequence creation method, which can efficiently create antibody sequence information with high antibody activity, A program and a recording medium can be provided.
[0336]
Also, according to the present invention, antibody fragment sequence information is mutated to amino acid fragment sequence information on other species based on the mechanism of immune reaction that antigenicity is recognized by TCR in units of protein fragment sequences. Therefore, it is possible to provide an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can efficiently create antibody sequence information reflecting biological facts.
[0337]
In addition, according to the present invention, antibody sequence information is divided into complementarity determining region information and framework region information. For example, antibody fragment sequence information spanning complementarity determining region information and framework region information is mutated. Antibody sequence creation device that can create antibody sequence information without considering the target, can create antibody sequence information with high accuracy, and can effectively reduce the amount of calculation A method, a program, and a recording medium can be provided.
[0338]
Further, according to the present invention, for example, when antibody fragment sequence information related to complementarity determining region information is unlikely to have antigenicity, the target of mutation is limited to framework region information only. Therefore, the complementarity-determining region information is the same as the original sequence, but only the framework region information is mutated to the amino acid fragment sequence information on other species, and the antibody sequence information has low antigenicity and high antibody activity. An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium can be provided. On the other hand, if there is a possibility that antibody fragment sequence information related to complementarity determining region information has antigenicity, both framework region information and complementarity determining region information can be considered as the target of mutation. . At this time, a database relating to complementarity determining region information can be prepared separately from that relating to framework region information.
[0339]
Further, according to the present invention, since the antibody sequence information is divided into antibody fragment sequence information of a predetermined length while sliding one amino acid residue from the beginning of the antibody sequence information, the protein phagocytosed by the macrophage as a foreign substance can be located at any position. Antibody fragment sequence information that may be split based on the mechanism of the immune reaction that is displayed in the TCR after being decomposed and fragmented by An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can efficiently create information can be provided.
[0340]
Further, since the predetermined length of the antibody fragment sequence information is 9 to 20, the protein phagocytosed by the macrophage as a foreign substance is decomposed and fragmented into a fragment sequence of about 9 to 20 residues at an arbitrary position. After that, since the antibody fragment sequence information is created based on the mechanism of the immune reaction that is presented in the TCR, the antibody sequence creation device and the antibody that can efficiently create the antibody sequence information that reflects the biological facts An array creation method, a program, and a recording medium can be provided.
[0341]
Further, according to the present invention, the amount of change between the similar amino acid fragment sequence information to be replaced and the antibody fragment sequence information of the search source is calculated, and the antibody activity maintenance rate is determined based on the calculated amount of change. In view of the decrease in antibody activity due to residue changes, part of the original antibody sequence can be mutated to amino acid fragment sequence information for other similar species, thus reducing antigenicity in other species. It is possible to provide an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can efficiently create antibody sequence information that is reduced and maintains antibody activity high.
[0342]
That is, according to the present invention, the antibody activity is lost when the amount of amino acid residue change is increased, and the antibody activity is maintained when the amount of amino acid residue change is decreased. A part of the sequence can be mutated to amino acid fragment sequence information on other species of similar species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently used. An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can be well created can be provided.
[0343]
Further, according to the present invention, similar amino acid fragment sequence information to be replaced and antibody fragment sequence information to be searched are compared with each other, and the contribution of the structural change indicating how much the change in the sequence contributed to the structural change. Since the antibody activity maintenance rate is determined based on the calculated rate of change in structure and the calculated rate of change in structure, a part of the original antibody sequence is considered in consideration of the decrease in the antibody activity of the antibody due to the change in the structure of the antibody. It is possible to mutate to amino acid fragment sequence information on other similar species, and therefore efficiently create antibody sequence information that reduces antigenicity and maintains high antibody activity in other species. An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium can be provided.
[0344]
That is, according to the present invention, the antibody activity is lost when the structural change of the antibody is increased, and the antibody activity is maintained when the structural change of the antibody is reduced. Can be mutated into amino acid fragment sequence information on other similar species, so that antibody sequence information with reduced antigenicity and high antibody activity in other species can be efficiently created. An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium can be provided.
[0345]
Further, according to the present invention, for similar amino acid fragment sequence information to be replaced and antibody fragment sequence information of the search source, index information regarding the property of the amino acid changed on each sequence is calculated, and based on the calculated index information Since the antibody activity maintenance rate is determined, similar amino acid fragment sequence information and search source antibody fragment sequence information, for example, index information such as charge difference, energy difference and affinity difference on each sequence In view of the decrease in antibody activity due to the change, a part of the original antibody sequence can be mutated to amino acid fragment sequence information on other similar species, thus reducing antigenicity in other species, And an antibody sequence creation device, an antibody sequence creation method capable of efficiently creating antibody sequence information maintaining high antibody activity, Programs, and it can provide a recording medium.
[0346]
That is, according to the present invention, for similar amino acid fragment sequence information and search source antibody fragment sequence information, for example, if the difference in charge, energy, affinity, etc. on each sequence increases, antibody activity On the other hand, considering that the antibody activity is maintained if, for example, the difference in charge, energy or affinity is reduced on each sequence, a part of the original antibody sequence is similar. Can be mutated to amino acid fragment sequence information relating to other species, so that antibody sequence information that reduces antigenicity and maintains high antibody activity in other species can be efficiently created. An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium can be provided.
[0347]
In addition, according to the present invention, when the antibody activity maintenance rate of the antibody sequence information after mutation is decreased compared to that before the mutation, the mutation is terminated, so that a mutation that reduces the antibody activity of the antibody sequence information before the mutation is performed. This provides an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can effectively create antibody sequence information that maintains antigenicity and has high antibody activity. can do.
[0348]
Further, according to the present invention, the antibody sequence information is antibody sequence information related to non-humans, and the amino acid fragment sequence information related to other biological species is amino acid fragment sequence information related to humans. And / or obtaining non-human antibody sequence information including framework region information, dividing the obtained non-human antibody sequence information into non-human antibody fragment sequence information of a predetermined length, and dividing the non-human antibody Search for matching human amino acid fragment sequence information for each fragment sequence information, search for similar human amino acid fragment sequence information for each non-human antibody fragment sequence information for which no matching human amino acid fragment sequence information was searched, and search Each similar human amino acid fragment sequence information was replaced with the non-human antibody fragment sequence information of the search source, and the non-human antibody sequence information was mutated. For human antibody sequence information, determine the antibody activity maintenance rate indicating whether the activity of the non-human antibody sequence information before mutation is maintained, and humanize the non-human antibody sequence information based on the determined antibody activity maintenance rate Since the humanized antibody sequence information mutation is determined, humanization is performed by including many human-derived amino acid fragment sequences with respect to non-human antibodies derived from mice and the like, and the antigenicity to humans is lowered, An antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can efficiently create humanized antibody sequence information with few side effects can be provided.
[0349]
In addition, according to the present invention, humanized antibody sequence information can be efficiently generated so as not to mutate a portion that affects the activity of a non-human antibody, that is, to maintain a high antibody activity of the non-human antibody. Thus, an antibody sequence creation device, an antibody sequence creation method, a program, and a recording medium that can efficiently create an effective humanized antibody can be provided.
[0350]
Furthermore, according to the present invention, the prepared humanized antibody sequence information can be widely used in many industrial fields, in particular, in the fields of medicine, medicine, etc., an antibody sequence creation device, an antibody sequence creation method, a program, and A recording medium can be provided.
[0351]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a principle configuration diagram showing a basic principle of the present invention.
FIG. 2 is a block diagram showing an example of the configuration of the system to which the present invention is applied.
FIG. 3 is a block diagram showing an example of the configuration of an acquisition unit 102a of the system to which the present invention is applied.
FIG. 4 is a block diagram showing an example of the configuration of a dividing unit 102b of the present system to which the present invention is applied.
FIG. 5 is a block diagram showing an example of the configuration of an antibody activity maintenance rate determination unit 102f of the present system to which the present invention is applied.
FIG. 6 is a flowchart showing an example of main processing of the system in the present embodiment.
FIG. 7 is a flowchart illustrating an example of area division processing of the system according to the present exemplary embodiment.
FIG. 8 is a flowchart illustrating an example of a slide division process of the system according to the present embodiment.
FIG. 9 is a flowchart showing an example of a change amount reference antibody activity maintenance rate determination process of the system according to the present embodiment.
FIG. 10 is a flowchart showing an example of structure change contribution rate reference antibody activity maintenance rate determination processing of the system in the present embodiment.
FIG. 11 is a flowchart showing an example of index information reference antibody activity maintenance rate determination processing of the system in the present embodiment.
FIG. 12 is a diagram showing an example of the humanized state of the L chain of “CRA4” used in the prior art in this example.
FIG. 13 is a diagram showing an example of a humanized state of the “CRA4” H chain used in the prior art in this example.
FIG. 14 is a diagram showing an example of the humanized state of the L chain of “CRA4” humanized by the prior art in this example.
FIG. 15 is a diagram showing an example of a humanized state of the heavy chain of “CRA4” humanized by the prior art in this example.
FIG. 16 is a diagram showing an example of the humanized state of the L chain of “CRA4” humanized by the method of the present invention in this example.
FIG. 17 is a diagram showing an example of a humanized state of the heavy chain of “CRA4” humanized by the method of the present invention in this example.
FIG. 18 shows an example of the amino acid sequence of “CRA4” humanized by the method of the present invention in this example.
FIG. 19 is a diagram showing another example of the humanized state of the L chain of “CRA4” humanized by the method of the present invention in this example.
FIG. 20 is a view showing another example of the humanized state of the heavy chain of “CRA4” humanized by the method of the present invention in this example.
FIG. 21 is a view showing another example of the amino acid sequence of “CRA4” humanized by the method of the present invention in this example.
FIG. 22 is a diagram showing an example of the humanized state of the L chain of the mouse antibody “TF (PDB code 1ahw)” used in this Example.
FIG. 23 is a diagram showing an example of a humanized state of the H chain of the mouse antibody “TF (PDB code 1ahw)” used in this Example.
FIG. 24 is a diagram showing an example of the humanized state of the L chain of the humanized mouse antibody “TF (PDB code 1 jps)” used in this Example.
FIG. 41 is a diagram showing an example of a humanized state of the H chain of the humanized mouse antibody “TF (PDB code 1 jps)” used in this Example.
FIG. 26 is a diagram showing an example of the humanized state of the L chain of the mouse antibody “TF (PDB code 1ahw)” humanized by the method of the present invention in this Example.
FIG. 27 is a diagram showing an example of the humanized state of the H chain of the mouse antibody “TF (PDB code 1ahw)” humanized by the method of the present invention in this example.
FIG. 28 is a view showing an example of an amino acid sequence of a mouse antibody “TF (PDB code 1ahw)” humanized by the method of the present invention in this example.
FIG. 29 is a diagram illustrating an example of a concept of slide division.
[Explanation of symbols]
100 Antibody sequence generator
102 Control unit
102a acquisition unit
102b Dividing part
102c Match search part
102d Similarity search part
102e mutation
102f Antibody activity maintenance rate determination unit
102g mutation determination part
102h Area division unit
102i Slide division part
102j Change amount calculation unit
102k change amount reference antibody activity maintenance rate determination unit
102m Structural change contribution rate calculator
102n Structural change contribution rate reference antibody activity maintenance rate determination unit
102p index information calculation unit
102r Index information reference antibody activity maintenance rate determination unit
104 Communication control interface unit
106 Storage unit
106a Antibody sequence information file
106b Antibody fragment sequence information file
106c Similar amino acid fragment sequence information file
106d Mutant antibody fragment sequence information file
106e antibody activity maintenance rate file
106f Mutation determination antibody sequence information file
106g Amino acid fragment sequence information database
108 Input / output control interface
112 Input device
114 output device
200 External system
300 network

Claims (28)

Obtaining means for obtaining antibody sequence information including complementarity determining region information and / or framework region information;
Dividing means for dividing the antibody sequence information acquired by the acquisition means into antibody fragment sequence information of a predetermined length;
For each of the antibody fragment sequence information divided by the dividing means, a match search means for searching for amino acid fragment sequence information regarding other matching species,
Similar search means for searching for similar amino acid fragment sequence information for each antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search means;
Mutating means for mutating the antibody sequence information by replacing each similar amino acid fragment sequence information searched by the similarity search means and the antibody fragment sequence information of the search source,
About the antibody sequence information mutated by the mutation means, antibody activity maintenance rate determination means for determining an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before mutation is maintained;
Based on the antibody activity maintenance rate determined by the antibody activity maintenance rate determination means, a mutation determination means for determining a mutation in the antibody sequence information;
An antibody sequence creation device comprising: The acquisition means is
Region dividing means for dividing the antibody sequence information into the complementarity determining region information and the framework region information;
The antibody sequence creation device according to claim 1, further comprising: The dividing means is
A slide splitting means for splitting the antibody fragment sequence information of the predetermined length while sliding by one amino acid residue from the beginning of the antibody sequence information;
The antibody sequence creation device according to claim 1, further comprising: The predetermined length of the antibody fragment sequence information is:
9-20,
The antibody sequence creation device according to any one of claims 1 to 3. The antibody activity maintenance rate determining means is:
Change amount calculating means for calculating a change amount between the similar amino acid fragment sequence information to be replaced by the mutation means and the antibody fragment sequence information of the search source;
Based on the change amount calculated by the change amount calculation means, a change amount reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate;
The antibody sequence creation device according to any one of claims 1 to 4, further comprising: The antibody activity maintenance rate determining means is:
Structural changes indicating how much the change in the sequence contributed to the change in the structure by comparing the structures of the similar amino acid fragment sequence information to be replaced by the mutation means and the antibody fragment sequence information from the search source. A structural change contribution rate calculating means for calculating a contribution rate;
Based on the structure change contribution rate calculated by the structure change contribution rate calculation means, the structure change contribution rate reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate;
The antibody sequence creation device according to claim 1, further comprising: The antibody activity maintenance rate determining means is:
Index information calculating means for calculating index information related to the properties of amino acids changed on each sequence for the similar amino acid fragment sequence information to be replaced by the mutation means and the antibody fragment sequence information of the search source,
Based on the index information calculated by the index information calculation means, index information reference antibody activity maintenance rate determination means for determining the antibody activity maintenance rate,
The antibody sequence production device according to any one of claims 1 to 6, further comprising: The mutation determining means includes
If the antibody activity maintenance rate of the antibody sequence information after mutation has decreased relative to that before mutation, terminating the mutation,
The antibody sequence creation device according to any one of claims 1 to 7, wherein: The antibody sequence information is the antibody sequence information related to a non-human, and the amino acid fragment sequence information related to the other species is the amino acid fragment sequence information related to a human,
The antibody sequence creation device according to any one of claims 1 to 8, wherein: Obtaining antibody sequence information including complementarity determining region information and / or framework region information;
A dividing step of dividing the antibody sequence information acquired by the acquiring step into antibody fragment sequence information of a predetermined length;
For each of the antibody fragment sequence information divided by the division step, a match search step for searching amino acid fragment sequence information regarding other matching species,
For each antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search step, a similar search step for searching for similar amino acid fragment sequence information;
A mutation step of mutating the antibody sequence information by replacing each of the similar amino acid fragment sequence information searched by the similarity search step with the antibody fragment sequence information of the search source,
For the antibody sequence information mutated by the mutation step, an antibody activity maintenance rate determination step for determining an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before the mutation is maintained;
Based on the antibody activity maintenance rate determined by the antibody activity maintenance rate determination step, a mutation determination step for determining a mutation in the antibody sequence information,
A method for producing an antibody sequence comprising the steps of: The acquisition step is
A region dividing step for dividing the antibody sequence information into the complementarity determining region information and the framework region information;
The antibody sequence production method according to claim 10, further comprising: The above dividing step is
A slide splitting step for splitting the antibody fragment sequence information of the predetermined length while sliding by one amino acid residue from the beginning of the antibody sequence information;
The method for producing an antibody sequence according to claim 10 or 11, further comprising: The predetermined length of the antibody fragment sequence information is:
9-20,
The method for producing an antibody sequence according to any one of claims 10 to 12, wherein: The antibody activity maintenance rate determination step includes:
A change amount calculating step for calculating a change amount between the similar amino acid fragment sequence information to be replaced in the mutation step and the antibody fragment sequence information of the search source;
Based on the change amount calculated by the change amount calculation step, a change amount reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate;
The method for producing an antibody sequence according to any one of claims 10 to 13, further comprising: The antibody activity maintenance rate determination step includes:
Structural changes that indicate how much the change in the sequence contributed to the change in the structure by comparing the structures of the similar amino acid fragment sequence information and the antibody fragment sequence information from which the search was performed in the mutation step. A structural change contribution rate calculating step for calculating a contribution rate;
Based on the structure change contribution rate calculated by the structure change contribution rate calculation step, a structure change contribution rate reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate;
The method for producing an antibody sequence according to any one of claims 10 to 14, further comprising: The antibody activity maintenance rate determination step includes:
An index information calculating step for calculating index information relating to the properties of amino acids changed on each sequence for the similar amino acid fragment sequence information to be replaced in the mutation step and the antibody fragment sequence information of the search source;
Based on the index information calculated by the index information calculation step, an index information reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate,
The method for producing an antibody sequence according to any one of claims 10 to 15, further comprising: The mutation determination step includes
If the antibody activity maintenance rate of the antibody sequence information after mutation has decreased relative to that before mutation, terminating the mutation,
The method for producing an antibody sequence according to any one of claims 10 to 16, wherein: The antibody sequence information is the antibody sequence information related to a non-human, and the amino acid fragment sequence information related to the other species is the amino acid fragment sequence information related to a human,
The method for producing an antibody sequence according to any one of claims 10 to 17, wherein: Obtaining antibody sequence information including complementarity determining region information and / or framework region information;
A dividing step of dividing the antibody sequence information acquired by the acquiring step into antibody fragment sequence information of a predetermined length;
For each of the antibody fragment sequence information divided by the division step, a match search step for searching amino acid fragment sequence information regarding other matching species,
For each antibody fragment sequence information for which the matching amino acid fragment sequence information was not searched by the match search step, a similar search step for searching for similar amino acid fragment sequence information;
A mutation step of mutating the antibody sequence information by replacing each of the similar amino acid fragment sequence information searched by the similarity search step with the antibody fragment sequence information of the search source,
For the antibody sequence information mutated by the mutation step, an antibody activity maintenance rate determination step for determining an antibody activity maintenance rate indicating whether the activity of the antibody sequence information before the mutation is maintained;
Based on the antibody activity maintenance rate determined by the antibody activity maintenance rate determination step, a mutation determination step for determining a mutation in the antibody sequence information,
A program for causing a computer to execute a method for producing an antibody sequence comprising The acquisition step is
A region dividing step for dividing the antibody sequence information into the complementarity determining region information and the framework region information;
The program according to claim 19, further comprising: The above dividing step is
A slide splitting step for splitting the antibody fragment sequence information of the predetermined length while sliding by one amino acid residue from the beginning of the antibody sequence information;
The program according to claim 19 or 20, further comprising: The predetermined length of the antibody fragment sequence information is:
9-20,
The program according to any one of claims 19 to 21, characterized in that: The antibody activity maintenance rate determination step includes:
A change amount calculating step for calculating a change amount between the similar amino acid fragment sequence information to be replaced in the mutation step and the antibody fragment sequence information of the search source;
Based on the change amount calculated by the change amount calculation step, a change amount reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate;
The program according to claim 19, further comprising: The antibody activity maintenance rate determination step includes:
Structural changes that indicate how much the change in the sequence contributed to the change in the structure by comparing the structures of the similar amino acid fragment sequence information and the antibody fragment sequence information from which the search was performed in the mutation step. A structural change contribution rate calculating step for calculating a contribution rate;
Based on the structure change contribution rate calculated by the structure change contribution rate calculation step, a structure change contribution rate reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate;
The program according to claim 19, further comprising: The antibody activity maintenance rate determination step includes:
An index information calculating step for calculating index information relating to the properties of amino acids changed on each sequence for the similar amino acid fragment sequence information to be replaced in the mutation step and the antibody fragment sequence information of the search source;
Based on the index information calculated by the index information calculation step, an index information reference antibody activity maintenance rate determination step for determining the antibody activity maintenance rate,
The program according to claim 19, further comprising: The mutation determination step includes
If the antibody activity maintenance rate of the antibody sequence information after mutation has decreased relative to that before mutation, terminating the mutation,
The program according to any one of claims 19 to 25. The antibody sequence information is the antibody sequence information related to a non-human, and the amino acid fragment sequence information related to the other species is the amino acid fragment sequence information related to a human,
The program according to any one of claims 19 to 26, wherein: A computer-readable recording medium, wherein the program according to any one of claims 19 to 27 is recorded.

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