JP2005052021A - Interleukin-18 mutant protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To analyze the three-dimensional structure of interleukin-18 to enable the utilization of the interleukin-18 for screening new medicine candidate substances or the like, and to provide a mutant in which the biological activity of the interleukin-18 is changed. <P>SOLUTION: The following interleukin-18 mutant proteins or their salts. The interleukin-18 mutant protein comprises an amino acid sequence in which at least one amino acid selected from three amino acid residue groups selected from specific amino acid residues is replaced by at least one of other amino acids. The other interleukin-18 mutant protein comprises an amino acid sequence in which one or several amino acids except the amino acids contained in the amino acid residue groups are deleted, replaced or added, and has the lower or higher biological activity of the interleukin-18 protein than that of mild type interleukin-18 protein. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００９２】
方法
サンプル調製。^１５Ｎ標識化および^１５Ｎ／^１３Ｃ標識化野生型ＩＬ−１８タンパク質は、ＧＳＴ融合タンパク質として発現させた。アフィニティークロマトグラフィーによる精製後、因子Ｘａを用いた消化によりＧＳＴタグを除去した。ＮＭＲ測定用のサンプルは、典型的には、５０ ｍＭ Ｔｒｉｓ−ＨＣｌバッファー（ｐＨ ６．０）中の１−２ ｍＭタンパク質、１５０ ｍＭ ＫＣｌおよびＨ_２Ｏ／^２Ｈ_２Ｏ （９０％／１０％）または^２Ｈ_２Ｏ中の１０ ｍＭ ＤＴＴからなった。
【００９３】
各アミノ酸がアラニンに置換された５０種類の突然変異体ＩＬ−１８タンパク質を、野生型タンパク質と同じ方法で調製した。突然変異させるべき残基は、ＩＬ−１／ＩＬ−１８ファミリーメンバーとの配列アライメント、ＩＬ−１８、ＩＬ−１β（ＰＤＢコード： ２Ｉ１Ｂ）およびＩＬ−１β：ＩＬ−１ＲＩ複合体（ＰＤＢコード： １ＩＴＢ）の間の構造比較、および以前に報告されたＩＬ−１に関する突然変異誘発データに基づいて選択された。
ＮＭＲ分光測定。全てのＮＭＲスペクトルは、Ｂｒｕｋｅｒ ＤＭＸ５００、ＤＲＸ５００またはＤＲＸ８００ ＮＭＲ分光計を用いて３０３Ｋで得た。主鎖および側鎖の^１Ｈ、^１３Ｃおよび^１５Ｎ共鳴の帰属を決めるため、一連の三次元実験を実施した^１２。ＶａｌおよびＬｅｕ残基のメチル基の立体特異的帰属の決定は、以前に記述されたように実施した^１３。距離制限は、混合時間を１００ ｍｓとした^１５Ｎ、^１５Ｎ−、^１５Ｎ、^１３Ｃ−または^１３Ｃ ^１３Ｃ−分解４Ｄ ＮＯＥＳＹ実験から得た^１２。
構造計算。得られた主鎖共鳴の帰属は、鎖Ｓ３とＳ４の間のセグメント周辺にマイナーなコンホメーションの存在を示した。これは恐らくＡｌａ４２−Ｐｒｏ４３ペプチド結合のシス／トランス異性化のためであろう。主要な及びマイナーなコンホメーションの間の１Ｈおよび１５Ｎ化学シフトの差は、比較的小さい。本発明者らは主要なコンホメーションについてのみ構造制限を得た。最初に、プログラムＤＹＡＮＡ（バージョン１．５）を用いて、構造計算およびＮＯＥピークの帰属決定を反復的でかつ手作業による方法で実施した^１４。主鎖ねじり角度制限は、ＨＮＨＡ^１２およびＴＡＬＯＳプログラムの^３Ｊ_ＨＮＨαから得た。Ｈｉｓ、Ｐｈｅ、ＴｙｒおよびＴｒｐのねじり角度χ１は、^３Ｊ_Ｃ’Ｃおよび^３Ｊ_ＮＣの結合定数から推定した^１６。手作業によって全体的な折り畳みを決定した後、ＣＡＮＤＩＤアルゴリズムを用いて残りのＮＯＥピークの帰属を決定し^１７、２２８９個の意味のあるＮＯＥ上部距離制限を得た。これらの制限を用いて、ＣＮＳのプログラム（バージョン１．１）によって最終構造を計算した^１８。この段階で、ゆっくり交換しつつある（ｅｘｃｈａｎｇｉｎｇ）主鎖アミドに由来する水素結合制限を、Ｎ−Ｏ原子対には２．８−３．４Åの距離制限として、またＨＮ−Ｏ原子対には１．８−２．４Åの距離制限としてそれぞれ加えた。非立体特異的に割当てられたプロトンは、流動性（ｆｌｏａｔｉｎｇ）キラリティーおよびΣ（ｒ^−６）^−１／６総計として処理された。合計１００個の構造を精巧化し、そして２０個の最もエネルギーの低い構造をＭＯＬＭＯＬ^１９、ＡＱＵＡおよびＰＲＯＣＨＥＣＫ−ＮＭＲソフトウエア２０を用いて分析した^２０（表２）。
【００９４】
【表２】

ＩＦＮ−γ誘導および受容体結合アッセイ。野生型および突然変異体ＩＬ−１８タンパク質を、ＩＦＮ−γ産生を誘導する能力について以前に記述されたように^２１調べた。野生型および突然変異体ＩＬ−１８のＩＬ−１８受容体に対するｉｎ ｖｉｔｒｏアフィニティーを、ＢＩＡＣＯＲＥ３０００ （Ｐｈａｒａｍａｃｉａ Ｂｉｏｓｅｎｓｏｒ ＡＢ）を用いた表面プラスモン共鳴実験によって２５℃で測定した。すなわち、アミン結合法により抗ヒトＩｇＧ Ｆｃ抗体 （Ｒｏｃｋｌａｎｄ）をＣＭ５センサーチップに結合させることによって特異的結合表面を調製した。次に、組換えＩＬ−１８α／ＦｃまたはＩＬ−１８β／Ｆｃキメラタンパク質（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）をチップ上に固定した。結合密度は２００共鳴単位（ＲＵ）までに制限した。ＩＬ−１８サンプルをＨＢＳ−ＥＰ （１０ ｍＭ ＨＥＰＥＳ， ｐＨ ７．４， ＮａＣｌ １５０ ｍＭ， ３．０ ｍＭ ＥＤＴＡ， ０．００５％ （ｖ／ｖ）界面活性剤Ｐ−２０）で希釈した。野生型および突然変異体ＩＬ−１８タンパク質のＩＬ−１８Ｒαに対する解離定数分析のためには、異なる量のサンプルを平衡相が得られるまで２ μｌ／分の速度でセンサーチップ上に注いだ。０．２ Ｍ グリシン−ＨＣｌ （ｐＨ １．５）を６０秒ずつ２回パルスすることによってセンサー表面を再生した。スキャッチャード（Ｓｃａｔｃｈａｒｄ）プロットによって解離定数を測定した。三量体性複合体形成能を評価するため、最初にＩＬ−１８タンパク質をＩＬ−１８Ｒα結合部位が飽和するまでセンサーチップに注いだ。次に、同一量のＩＬ−１８タンパク質をＩＬ−１８Ｒβタンパク質（３．１２−１００ ｎＭ）と混合して注いだ。
モデル化。ＩＬ−１８：ＩＬ−１８Ｒα複合体のモデル化は、ＩＬ−１８の構造およびＩＬ−１β：ＩＬ−１ＲＩ複合体（ＰＤＢコード： １ＩＴＢ）の結晶構造に基づいて行なわれた。モデルの構築および構造検証のためにＭＯＥ^２２を用いた。ＩＬ−１８のＮＭＲ構造およびＩＬ−１８Ｒαのモデル化された構造の座標をＩＬ−１β：ＩＬ−１ＲＩ複合体の上に重ね合わせた。ＩＬ−１８：ＩＬ−１８Ｒα複合体モデルは、Ｃｏｒｎｅｌｌら^２４によって記述された、全原子フォースフィールドを用いるＡＭＢＥＲ ５．０パッケージ^２３を用いた３００ Ｋにおける７０ ｐｓの分子力学計算によって精巧化された。最終構造をさらにエネルギー最小化した。
座標。座標をＰｒｏｔｅｉｎ Ｄａｔａ Ｂａｎｋに寄託した（登録コード：１ＪＯＳ）。その座標を表Ａ及びＢに示す。表Ａには、２０回の構造計算によって出てきた構造の座標を示す。ＮＭＲによる構造計算では異なる計算によって発生した構造を複数提示し、その構造間の収束を精度と見なす。表Ｂには、表Ａの３次元構造座標の平均値に少し化学エネルギーを使っての補正をしたものを示す。
【００９５】
表Ａ
野生型ＩＬ−１８の３次元構造座標（２０回の構造計算によって出てきた構造）

【００９６】
表Ｂ
野生型ＩＬ−１８の３次元構造座標（表Ａの３次元構造座標の平均値に少し化学エネルギーを使っての補正をしたもの）

【００９７】
表Ａは、最初の行（すなわち、ＭＯＤＥＬ Ｘ（Ｘは１〜２０のいずれかの整数）の表示）ならびに最後から１行及び２行（すなわち、ＴＥＲ及びＥＮＤＭＤＬの表示）を除いて、各原子の３次元座標を記述している。また、表Ｂは、最終行（すなわち、ＥＮＤの表示）を除いて、各原子の３次元座標を記述している。１列目のＡＴＯＭはこの行が原子座標の行であることを示し、２列目は、その原子の順番を、３列目はアミノ酸残基等における原子の区別を、４列目はアミノ酸残基等を、５列目はその原子を含むアミノ酸残基の番号を、６、７、８列目はその原子の座標（Å単位）を示している。最終行は、この表の終わりの行であることを示している。なお、本表は当業者にとって一般的に用いられている表記法であるプロテイン・データ・バンクの形式に従って記述している。（９、１０列目はデータベースの形式に合わせるための数字であり、本発明においては特に意味のある数字ではなく、９列目はすべて１．００を、１０列目はすべて０．００を使用した。）
【００９８】
引用文献
１． Ｏｋａｍｕｒａ， Ｈ． ｅｔ ａｌ． Ｎａｔｕｒｅ． ３７８， ８８−９１ （１９９５）
２．Ｄｉｎａｒｅｌｌｏ， ＣＡ． Ｅｕｒ． Ｃｙｔｏｋｉｎｅ Ｎｅｔｗ． １１， ４８３−６ （２０００）
３． Ｎａｋａｎｉｓｈｉ， Ｋ．， Ｙｏｓｈｉｍｏｔｏ， Ｔ．， Ｔｓｕｔｓｕｉ Ｈ． ＆ Ｏｋａｍｕｒａ， Ｈ． Ｃｙｔｏｋｉｎｅ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ Ｒｅｖ． １２， ５３−７２． （２００１）
４． Ｋｏｎｉｓｈｉ， Ｈ． ｅｔ ａｌ． Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ． ９９：１１３４０−５． （２００２）
５． Ｗｉｇｇｉｎｔｏｎ ＪＭ， ｅｔ ａｌ． Ｊ． Ｉｍｍｕｎｏｌ． １６９：４４６７−４４７４． （２００２）
６． Ｖｉｇｅｒｓ， Ｇ．Ｐ．， Ａｎｄｅｒｓｏｎ， Ｌ．Ｊ．， Ｃａｆｆｅｓ， Ｐ． ＆ Ｂｒａｎｄｈｕｂｅｒ， Ｂ．Ｊ． Ｎａｔｕｒｅ． ３８６， １９０−１９４ （１９９７）
７． Ｓｃｈｒｅｕｄｅｒ， Ｈ． ｅｔ ａｌ． Ｎａｔｕｒｅ． ３８６， １９４−２００ （１９９７）
８． Ｂａｚａｎ， Ｊ．Ｆ．， Ｔｉｍａｎｓ， Ｊ．Ｃ． ＆ Ｋａｓｔｅｌｅｉｎ， Ｒ．Ａ． Ｎａｔｕｒｅ． ３７９， ５９１ （１９９６）
９． Ｋｉｍ， ＳＨ． ｅｔ ａｌ．Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７７， １０９９８−１００３ （２００２）
１０． Ｂｏｒｎ， ＴＬ．， Ｔｈｏｍａｓｓｅｎ， Ｅ．， Ｂｉｒｄ， ＴＡ． ＆ Ｓｉｍｓ， ＪＥ． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７３， ２９４４５−２９４５０ （１９９８）
１１． Ｅｖａｎｓ， Ｒ．Ｊ． ｅｔ ａｌ． Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７０， １１４７７−１１４８３ （１９９５）
１２． Ｃａｖａｎａｇｈ， Ｊ．， Ｆａｉｒｂｒｏｔｈｅｒ， Ｗ．Ｊ．， Ｐａｌｍｅｒ， Ａ．Ｇ．ＩＩＩ． ＆ Ｓｋｅｌｔｏｎ， Ｎ．Ｊ． Ｐｒｏｔｅｉｎ ＮＭＲ
Ｓｐｅｃｔｒｏｓｃｏｐｙ． （Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ， Ｓａｎ Ｄｉｅｇｏ；１９９６）
１３． Ｈｕ， Ｗ． ＆ Ｚｕｉｄｅｒｗｅｇ， Ｅ．Ｒ．Ｐ． Ｊ． Ｍａｇｎ． Ｒｅｓｏｎ． Ｂ， １１３， ７０−７５ （１９９６）
１４． Ｇｕｎｔｅｒｔ， Ｐ．， Ｍｕｍｅｎｔｈａｌｅｒ， Ｃ． ＆ Ｗｕｔｈｒｉｃｈ， Ｋ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． ２７３， ２８３−２９８ （１９９７）
１５． Ｃｏｒｎｉｌｅｓｃｕ， Ｇ．， Ｄｅｌａｇｌｉｏ， Ｆ． ＆ Ｂａｘ， Ａ． Ｊ． Ｂｉｏｍｏｌ． ＮＭＲ． １３， ２８９−３０２． （１９９９）
１６． Ｈｕ， Ｊ．−Ｓ．， Ｇｒｚｅｓｉｅｋ， Ｓ．， ａｎｄ Ｂａｘ， Ａ． （１９９７） Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ． １１９， １８０３−１８０４
１７． Ｈｅｒｒｍａｎｎ， Ｔ．， Ｇｕｎｔｅｒｔ， Ｐ． ＆ Ｗｕｔｈｒｉｃｈ， Ｋ． Ｊ． Ｍｏｌ． Ｂｉｏｌ． ３１９， ２０９−２２７．（２００２）．
１８． Ｂｒｕｎｇｅｒ， Ａ．Ｔ． ｅｔ ａｌ． Ａｃｔａ Ｃｒｙｓｔａｌｌｏｇ． Ｄ５４， ９０５−９２１．（１９９８）
１９． Ｋｏｒａｄｉ， Ｒ．， Ｂｉｌｌｅｔｅｒ， Ｍ． ＆ Ｗｕｅｔｈｒｉｃｈ， Ｋ． Ｊ． Ｍｏｌ． Ｇｒａｐｈ． １４， ５１−５５ （１９９６）
２０． Ｌａｓｋｏｗｓｋｉ， Ｒ．Ａ．， Ｒｕｌｌｍａｎ， Ｊ．Ａ．Ｃ．， ＭａｃＡｒｔｈｕｒ， Ｍ．Ｗ．， Ｋａｐｔｅｉｎ， Ｒ． ＆ Ｔｈｏｒｎｔｏｎ， Ｊ．Ｍ． Ｊ．
Ｂｉｏｍｏｌ． ＮＭＲ ８， ４７７−４８６ （１９９６）
２１． Ｓｈｉｋａｎｏ， Ｈ． ｅｔ ａｌ．Ｃｌｉｎ． Ｅｘｐ． Ａｌｌｅｒｇｙ． ３１， １２６３−１２７０ （２００１）
２２． ＭＯＥ （Ｔｈｅ Ｍｏｌｅｃｕｌａｒ Ｏｐｅｒａｔｉｎｇ Ｅｎｖｉｒｏｎｍｅｎｔ． Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．， Ｍｏｎｔｒｅａｌ； １９９９）
２３． Ｃａｓｅ， Ｄ． Ａ． ｅｔ ａｌ． ＡＭＢＥＲ ５ （Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ｃａｌｉｆｏｒｎｉａ， Ｓａｎ Ｆｒａｎｃｉｓｃｏ； １９９７）
２４． Ｃｏｒｎｅｌｌ， Ｗ． ｅｔ ａｌ．Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ． １１７， ５１７９−５１９７ （１９９５）
【００９９】
【発明の効果】
本発明により、インターロイキン−１８タンパク質の新規な変異体が提供された。この変異体は、インターロイキン−１８タンパク質のアンタゴニストとして利用することができる。
【０１００】
また、本発明により、野生型インターロイキン−１８タンパク質の３次元構造座標が提供された。この座標を利用することにより、新規な薬剤のスクリーニングができるようになった。
【０１０１】
【配列表】

【０１０２】
【配列表フリーテキスト】
配列番号１は、野生型ＩＬ−１８のアミノ酸配列を示す。
【０１０３】
配列番号２は、野生型ＩＬ−１８のＤＮＡ配列を示す。
【０１０４】
配列番号３〜１７は、ＩＬ−１８の変異体のＤＮＡ配列を示す。
【０１０５】
配列番号１８は、ＩＬ−１８の最適化ｃＤＮＡのＤＮＡ配列を示す。
【図面の簡単な説明】
【図１】図１は、ＧＳＴ−ｈＩＬ−１８融合タンパク質の発現ベクター（ｐＧＥＸ−４Ｔ−Ｘａ−ｈＩＬ−１８）を図式的に表わす。
【図２】図２は、ＧＳＴ−ｈＩＬ−１８の発現および精製を示す。ａ） ＩＰＴＧによる誘導の５時間後のＧＳＴ−ｈＩＬ−１８融合タンパク質の発現のＳＤＳ−ＰＡＧＥ。ｂ） 異なる発現温度における封入体の形成。ｃ） ＧＳＴアフィニティーカラムを用いたＧＳＴ−ｈＩＬ−１８の精製。溶出したＧＳＴ−ｈＩＬ−１８ タンパク質は９０％以上の純度を有する。ｓｕｐ：細胞溶解物の上清； ｐｅｌ： 該溶解物のペレット。矢印はＧＳＴ−ｈＩＬ−１８タンパク質を示す。
【図３】図３は、ＧＳＴ−ｈＩＬ−１８融合タンパク質の開裂を示す。種々の濃度のＧＳＨ、ＤＴＴまたは ２−ＭＥを用いた開裂試験のＳＤＳ−ＰＡＧＥ。（−）および（＋）は、それぞれ因子Ｘａの不存在および存在を示す。Ａ： ＧＳＴ−ｈＩＬ−１８ タンパク質；Ｂ： ＧＳＴ タンパク質；Ｃ： ｈＩＬ−１８ タンパク質。
【図４】図４は、ゲル濾過カラム （Ｓｅｐｈａｃｒｙｌ Ｓ−１００， ２６／６０）による精製を示す。ａ）ゲル濾過カラムに流速２ ｍｌ／分で通液し、４ ｍｌごとにフラクションを回収したクロマトグラム。ｂ） Ｐ１およびＰ２に由来する精製サンプルのＳＤＳ−ＰＡＧＥ分析。Ａ： ＧＳＴ タンパク質；Ｂ： ｈＩＬ−１８タンパク質。
【図５】ヒトＩＬ−１８の溶液構造。ａ． ２０個の立体の最終構造の最も適した主鎖重ね合わせ。残基１−１５７の主鎖原子を重ね合わせている。ｂ． ＭＯＬＭＯＬを用いて描いた、ＩＬ−１８の立体のＮＭＲ構造の模式的リボン図。
【図６】ＩＬ−１８の突然変異分析。ａ． ヒトＩＬ−１８およびＩＬ−１βの配列アライメント。その突然変異が生物学的活性の重大な低下を引き起こしたヒトＩＬ−１８およびＩＬ−１βの残基を星印で示す。ヒトＩＬ−１８およびＩＬ−１β（ＰＤＢコード：２Ｉ１Ｂ）の二次構造エレメントをそれぞれ最上部および最下部に示す。保存された残基を四角で囲んで黄色で示す。ｂ． その突然変異が活性の重大な低下を引き起こしたＩＬ−１８残基の表面表示。部位Ｉ、ＩＩおよびＩＩＩの残基をそれぞれ赤色、橙色および青色で示す。ｃ． 溶媒が近づきうる表面上の静電ポテンシャルの分布。青色は陽性ポテンシャルに対応し、赤色は陰性ポテンシャルに対応する。両パネルとも、分子は図５の配向と同一の配向で（左）、および縦軸を中心として１８０度回転させて（右）示してある。ｄ． ＩＬ−１βの構造。その突然変異が活性の重大な低下を引き起こしたＩＬ−１β残基の表面表示。部位ＡおよびＢの残基をそれぞれ赤色および橙色で示す。ｅ． 溶媒が近づきうる表面上の静電ポテンシャルの分布。
【図７】ＩＬ−１８：ＩＬ−１８Ｒα複合体のモデル、ならびにＩＬ−１８、ＩＬ−１８ＲαおよびＩＬ−１８Ｒβの相互作用。ａ． ＩＬ−１β：ＩＬ−１ＲＩ複合体（ＰＤＢコード： １ＩＴＢ）の結晶構造。部位ＡおよびＢのＩＬ−１β残基をそれぞれ赤色および橙色で示す。ｂ． ＩＬ−１８：ＩＬ−１８Ｒα複合体のモデル化された構造。部位Ｉ、ＩＩおよびＩＩＩの残基をそれぞれ赤色、橙色および青色で示す。ｃ． 野生型ＩＬ−１８の固定化ＩＬ−１８ＲαまたはＩＬ−１８Ｒβに対する表面プラスモン共鳴の容量応答曲線。野生型ＩＬ−１８の濃度も示す。ｄ． ＩＬ−Ｒβの、固定化ＩＬ−１８Ｒαと野生型ＩＬ−１８の複合体に対する、または部位ＩＩＩ突然変異体タンパク質に対する、表面プラスモン共鳴の応答曲線。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to interleukin-18 mutant proteins.
[0002]
[Prior art]
Interleukin-18 (IL-18), a cytokine called interferon-gamma inducing factor, enhances IFN-gamma production, Fas-mediated cytotoxicity against natural killer cells, and developmental regulation of helper T lymphocyte type 1 It has pleiotropic immunoregulatory functions (Non-Patent Documents 1 to 3).
[0003]
IL-18, which is functionally similar to IL-12 in terms of IFN-γ production, is associated with severe inflammatory diseases. It has been inferred that abnormal expression of IL-18 is related to serious inflammatory diseases such as autoimmune diseases, allergies or neuropathy (Non-Patent Documents 2 to 4). Successful therapeutic approaches such as tumor suppression in mice using IL-18 have been reported (Non-Patent Document 5). The function of IL-18 in vivo is closely related to the function of IL-12. Thus, IL-18 deficient mice show a phenotype similar to that of IL-12 deficient mice. However, IL-18 and its receptor do not show any structural similarity to IL-12 and its receptor, respectively (Non-patent Documents 2 and 3). In contrast, IL-1β and IL-18 show moderate sequence similarity (17% identity), despite lacking significant functional similarity in terms of IFN-γ production. Furthermore, the polypeptide processing mechanisms of IL-18 and IL-1 share a common feature, namely that both precursor caspase-1 cleavage is essential for maturation (Non-Patent Documents 2 and 3). ). The receptors for both IL-1β and IL-18 belong to the IL-1 receptor family. Thus, intracellular signaling pathways for responding to these cytokines share the same downstream mediators such as TRAF6 and NF-κB2,3. Activation of these receptors requires heterodimerization of two structurally related but distinct immunoglobulin-like chains; the initiation of the IL-18 pathway requires IL- 18 receptor α (IL-18Rα; formerly called IL-1Rrp1) and receptor β (IL-18Rβ; formerly called IL-1AcPL) are required (non-patent literature). 2 and 3), IL-1 receptor type I (IL-1RI) and IL-1 accessory protein (IL-1AcP) are required for initiation of the IL-1 pathway. The interaction between IL-1 and IL-1RI was elucidated by the crystal structure of these complexes (Non-patent Documents 6 and 7). However, the recognition of IL-1AcP by IL-1 and, therefore, the activation mechanism of the IL-1 pathway is still unclear (Non-Patent Documents 2 and 3).
[0004]
Moreover, there is no report regarding the three-dimensional structure of interleukin 18.
[0005]
[Non-Patent Document 1]
Okamura, H .; et al. Nature. 378, 88-91 (1995)
[0006]
[Non-Patent Document 2]
Dinarello, CA. Eur. Cytokine Netw. 11, 483-6 (2000)
[0007]
[Non-Patent Document 3]
Nakanishi, K .; Yoshimoto, T .; Tsutsui H., et al. & Okamura, H .; Cytokine Growth Factor Rev. 12, 53-72. (2001)
[0008]
[Non-Patent Document 4]
Konishi, H .; et al. Proc. Natl. Acad. Sci. USA. 99: 11340-5. (2002)
[0009]
[Non-Patent Document 5]
Wiggington JM, et al. J. et al. Immunol. 169: 4467-4474. (2002)
[0010]
[Non-Patent Document 6]
Vigers, G.G. P. , Anderson, L .; J. et al. , Caffes, P.M. & Brandhuber, B.B. J. et al. Nature. 386, 190-194 (1997)
[0011]
[Non-Patent Document 7]
Schreuder, H.C. et al. Nature. 386, 194-200 (1997)
[0012]
[Problems to be solved by the invention]
The present invention improves the understanding of the biological activity of interleukin-18 (ie, receptor activation of IL-18) by analyzing the three-dimensional structure of interleukin-18, and is used for screening new drug candidates. The purpose is to make it available.
[0013]
Another object of the present invention is to obtain a mutant having altered biological activity of interleukin-18.
[0014]
[Means for Solving the Problems]
The present inventors determined the solution structure of IL-18 as a first step to understand the receptor activation mechanism of IL-18. IL-18 is folded into a structure similar to the β-1 trefoil (trefoil) structure of IL-1. Extensive mutagenesis has revealed that there are three sites important for receptor activation. Two of them function as a binding site for IL-18Rα and are located at a location similar to the IL-1RI binding site of IL-1. On the other hand, the third site is involved in IL-18β binding. These structures and mutagenesis data provide the basis for understanding the heterodimerization of receptor subunits required for receptor activation induced by IL-18. The present invention has been completed based on these findings.
[0015]
The gist of the present invention is as follows.
[1] The interleukin-18 mutant protein or salt thereof described in (a) or (b) below.
(A) In the amino acid sequence of SEQ ID NO: 1, the first amino acid residue group consisting of the 13th arginine, the 17th aspartic acid, the 33rd methionine, the 35th aspartic acid and the 132nd aspartic acid Phenylalanine, 4th lysine, 5th leucine, 8th lysine, 58th arginine, 60th methionine and 104th arginine second amino acid residue group, and 79th lysine, 84th Interleukin-18 comprising an amino acid sequence in which at least one amino acid selected from three amino acid residue groups consisting of a third amino acid residue group consisting of lysine and 98th aspartic acid is substituted with another amino acid Mutant protein
(B) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids other than the amino acids contained in the first to third amino acid residues are deleted, substituted or added; Interleukin-18 mutant protein having a biological activity of leukin-18 protein lower or higher than wild type interleukin-18 protein consisting of the amino acid sequence of SEQ ID NO: 1
[2] DNA encoding the protein according to [1].
[3] A recombinant vector containing the DNA according to [2].
[4] A transformant comprising the recombinant vector according to [3].
[5] A method for producing an interleukin-18 mutant protein comprising culturing a host transformed with the DNA according to [2] and collecting the interleukin-18 mutant protein from the culture.
[6] An antagonist of interleukin-18 protein comprising the interleukin-18 mutant protein or a salt thereof according to [1].
[7] A pharmaceutical composition comprising the interleukin-18 mutant protein or a salt thereof according to [1] as an active ingredient.
[8] An antibody against the interleukin-18 mutant protein or a salt thereof according to [1].
[9] A method for screening a substance that binds to the interleukin-18 protein using all or part of the three-dimensional structure coordinates shown in Table A and / or Table B.
[10] In the amino acid sequence of the wild-type interleukin-18 protein consisting of the amino acid sequence of SEQ ID NO: 1 or the interleukin-18 mutant protein according to claim 1, the 13th amino acid, the 17th amino acid, the 33rd amino acid A first amino acid residue group consisting of the 35th amino acid and the 132nd amino acid, the 2nd amino acid, the 4th amino acid, the 5th amino acid, the 8th amino acid, the 58th amino acid, the 60th amino acid, and Selected from the second amino acid residue group consisting of the 104th amino acid and the three amino acid residue groups consisting of the 79th amino acid, the 84th amino acid and the third amino acid residue group consisting of the 98th amino acid Examining the interaction of at least one amino acid residue with a test substance; Method for searching a substance which binds to interleukin -18 protein.
[11] A DNA represented by the nucleotide sequence of SEQ ID NO: 18.
[0016]
In the present specification, the “mutant protein” means a protein that is different from the reference protein but retains the essential properties of the reference protein. A typical mutant protein differs in amino acid sequence from the reference protein.
[0017]
The “interleukin-18 protein” generally means an interleukin-18 protein (wild type) consisting of the amino acid sequence of SEQ ID NO: 1 and mutant proteins thereof.
[0018]
By “biological activity of interleukin-18 protein” is meant the metabolic or physiological function of interleukin-18 protein, including enhanced activity of IFN-γ production, Fas-mediated cells against natural killer cells. In addition to the pleiotropic immunoregulatory function including the cytotoxicity and developmental regulation of helper T lymphocyte type 1, the activity as an antigen and the activity as an immunogen are also included.
[0019]
“Antagonist” refers to a substance that reduces or eliminates the activity of a substance having biological activity.
[0020]
“Antibody” means a protein that is induced in an organism by an immune reaction as a result of antigen stimulation and has an activity of specifically binding to an immunogen (antigen). Examples include antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and Fab or Fab fragments.
[0021]
“Screening” means selecting a target substance, which includes identification, search, evaluation, design, etc. of the target substance.
[0022]
“Interaction” means that a force works between two or more objects (for example, atoms and molecules). Examples of the interaction include hydrophilic interaction (for example, hydrogen bond, salt bridge), hydrophobic interaction (for example, hydrophobic bond), electrostatic interaction, van der Waals interaction, and the like.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
1. Interleukin-18 mutant protein
The present invention provides the following (a) or (b) interleukin-18 mutant protein or a salt thereof.
(A) In the amino acid sequence of SEQ ID NO: 1, the first amino acid residue group consisting of the 13th arginine, the 17th aspartic acid, the 33rd methionine, the 35th aspartic acid and the 132nd aspartic acid Phenylalanine, 4th lysine, 5th leucine, 8th lysine, 58th arginine, 60th methionine and 104th arginine second amino acid residue group, and 79th lysine, 84th Interleukin-18 comprising an amino acid sequence in which at least one amino acid selected from three amino acid residue groups consisting of a third amino acid residue group consisting of lysine and 98th aspartic acid is substituted with another amino acid Mutant protein
(B) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids other than the amino acids contained in the first to third amino acid residues are deleted, substituted or added; Interleukin-18 mutant protein having a biological activity of leukin-18 protein lower or higher than wild type interleukin-18 protein consisting of the amino acid sequence of SEQ ID NO: 1
As the interleukin-18 mutant protein of (a), in the amino acid sequence of SEQ ID NO: 1, the 13th arginine, the 17th aspartic acid, the 33rd methionine, the 35th aspartic acid, the 132nd aspartic acid, 2nd phenylalanine, 4th lysine, 5th leucine, 8th lysine, 58th arginine, 60th methionine, 104th arginine, 79th lysine, 84th lysine and 98th aspartic acid Examples include those in which at least one amino acid is substituted with any one of alanine, valine, isoleucine, proline, tryptophan, glycine, serine, threonine, cysteine, glutamine, asparagine, tyrosine, histidine, or glutamic acid.
The interleukin-18 mutant protein in which the biological activity of the interleukin-18 protein in (b) is lower than that of the wild type interleukin-18 protein can be used as an antagonist of the interleukin-18 protein. The antagonist of interleukin-18 protein can be used for the prevention and / or treatment of diseases caused by interleukin-18 protein (for example, autoimmune diseases, allergies, neuropathies, etc.).
The interleukin-18 mutant protein in which the biological activity of the interleukin-18 protein in (b) is higher than that of the wild-type interleukin-18 protein can be used as an agonist of the interleukin-18 protein. An “agonist” refers to a substance that has the same affinity and / or biological activity of a substance for its receptor. The agonist of interleukin-18 protein can be used for the prevention and / or treatment of diseases (for example, hepatitis C, tumor, etc.) whose symptoms are improved by administration of interleukin-18 protein.
[0024]
The salt of the interleukin-18 mutant protein of the present invention is particularly preferably a pharmacologically acceptable acid addition salt. Pharmacologically acceptable acid addition salts include salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid). Examples thereof include salts with acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid.
[0025]
The interleukin-18 mutant protein and its salt of the present invention can be produced by a known method. For example, as described in 2 below, DNA encoding interleukin-18 mutant protein is obtained, and the obtained DNA is incorporated into an appropriate expression vector and then introduced into an appropriate host to obtain a recombinant protein. By producing it, an interleukin-18 mutant protein can be produced (for example, Satoshi Saigo, Yoko Sano, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experiment Protocol, I, II, III, Maruzen Co., Ltd .: (See Original, Ausubel, FM et al., Short Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., New York).
[0026]
Alternatively, the interleukin-18 mutant protein and salts thereof of the present invention can also be produced according to known peptide synthesis methods.
[0027]
2. DNA encoding interleukin-18 mutant protein
The DNA encoding the interleukin-18 mutant protein of the present invention may be any DNA as long as it contains a base sequence encoding the interleukin-18 mutant protein of the present invention. Examples of the DNA encoding the interleukin-18 mutant protein of the present invention include DNA represented by any one of the nucleotide sequences of SEQ ID NOs: 3 to 17.
[0028]
The DNA encoding the interleukin-18 mutant protein of the present invention can be produced, for example, as follows.
[0029]
MRNA is extracted from healthy human blood and cDNA is synthesized using reverse transcriptase and oligo dT primers. The coding region of mature hIL-18 (residue 157) is amplified by PCR. The obtained PCR product is DNA encoding wild-type hIL-18 protein. One example of the amino acid sequence and base sequence of DNA encoding wild-type hIL-18 protein is shown in SEQ ID NOs: 1 and 2, respectively.
[0030]
DNA encoding the hIL-18 mutant protein can be prepared by mutating the coding region (157 residues) of mature hIL-18 by point mutagenesis. The mutated mature hIL-18 coding region (residue 157) is amplified by PCR. The obtained PCR product is DNA encoding the hIL-18 mutant protein. Examples of the base sequence of DNA encoding hIL-18 mutant protein are shown in SEQ ID NOs: 3-17. DNAs having the nucleotide sequences of SEQ ID NOs: 3 to 17, respectively, in the amino acid sequence of SEQ ID NO: 1, the second phenylalanine, the fourth lysine, the fifth leucine, the eighth lysine, the thirteenth arginine, the seventeenth Aspartic acid, 33rd methionine, 35th aspartic acid, 58th arginine, 60th methionine, 79th lysine, 84th lysine, 98th aspartic acid, 104th arginine and 132th asparagine It encodes an interleukin-18 mutant protein consisting of an amino acid sequence in which the acid is substituted with alanine. The DNA encoding the above hIL-18 mutant protein was prepared based on the wild type hIL-18 gene, but instead of the wild type hIL-18 gene, an optimized cDNA was designed and synthesized, Based on this, DNA encoding the interleukin-18 mutant protein can also be prepared. An example of the optimized cDNA sequence is shown in SEQ ID NO: 18. It has been confirmed that the expression / production of interleukin-18 protein is greatly improved (5 times or more) by using this optimized cDNA.
[0031]
3. Recombinant vector
A recombinant vector containing a DNA encoding the interleukin-18 mutant protein of the present invention can be obtained by a known method (for example, described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Method), by inserting the DNA encoding the interleukin-18 mutant protein of the present invention into an appropriate expression vector.
[0032]
As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, etc. Bacteriophages, animal viruses such as retroviruses and vaccinia viruses, insect pathogenic viruses such as baculoviruses, and the like can be used.
[0033]
A promoter, enhancer, splicing signal, poly A addition signal, selection marker, SV40 replication origin, etc. may be added to the expression vector.
[0034]
The expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercially available, such as pGEX series (Amersham Pharmacia Biotech), pET CBD Fusion System 34b-38b (Novagen), pET Dsb Fusion Systems 39b and 40b (Novagen 41st), FET GST. and 42 (Novagen).
[0035]
4). Transformant
A transformant can be obtained by introducing a recombinant vector containing DNA encoding the interleukin-18 mutant protein of the present invention into a host.
[0036]
Hosts include bacterial cells (eg, Escherichia, Bacillus, Bacillus, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells (eg, CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells, and the like.
[0037]
To introduce a recombinant vector into a host, Molecular Cloning 2nd Edition, J. MoI. Sambrook et al. Cold Spring Harbor Lab. Press, 1989 (for example, calcium phosphate method, DEAE-dextran method, transfection method, microinjection method, lipofection method, electroporation method, transduction method, scrape loading method, shotgun method, etc.) or infection Can be performed.
[0038]
The transformant can be cultured in a medium, and the interleukin-18 mutant protein can be collected from the culture. When the interleukin-18 mutant protein is secreted into the medium, the medium may be recovered, and the interleukin-18 mutant protein may be separated from the medium and purified. When the interleukin-18 mutant protein is produced in the transformed cell, the cell may be lysed, and the interleukin-18 mutant protein may be separated from the lysate and purified.
[0039]
When the interleukin-18 mutant protein is expressed in the form of a fusion protein with another protein (functioning as a tag), the fusion protein is separated and purified, and then treated with factor Xa or an enzyme (enterokinase). By doing so, another protein can be cut | disconnected and the target interleukin-18 mutant protein can be obtained.
[0040]
Separation and purification of the interleukin-18 mutant protein can be performed by known methods. Known separation and purification methods include differences in molecular weight such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that use, methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, methods that use hydrophobic differences such as reversed-phase high-performance liquid chromatography, etc. A method using the difference in isoelectric point, such as electric point electrophoresis, is used.
[0041]
5. Use of interleukin-18 protein as an antagonist
Among the interleukin-18 mutant proteins of the present invention, the interferon γ-inducing activity is low as compared to the wild-type interleukin-18 protein, and the interleukin-18 receptor α subunit (IL-18Rα) Those having low binding activity can be used as antagonists of interleukin-18 protein. The antagonist of interleukin-18 protein can be used for the prevention and / or treatment of diseases caused by interleukin-18 protein (for example, autoimmune diseases, allergies, neuropathies, etc.). To date, since no substance that can be used as an antagonist of interleukin-18 protein has been found, the industrial utility value of the interleukin-18 mutant protein of the present invention is high.
[0042]
6). Pharmaceutical composition
The interleukin-18 mutant protein and the salt thereof of the present invention can be improved in symptoms caused by interleukin-18 protein (for example, autoimmune disease, allergy, neuropathy, etc.) or administration of interleukin-18 protein. It can be used for the prevention and / or treatment of diseases found (eg, hepatitis C, tumors, etc.).
[0043]
The interleukin-18 mutant protein of the present invention or a salt thereof alone or in combination with a pharmacologically acceptable carrier, diluent or excipient, can be used as a pharmaceutical composition of an appropriate dosage form for mammals (for example, , Human, rabbit, dog, cat, rat, mouse) orally or parenterally. The dose varies depending on the administration subject, target disease, symptom, administration route, and the like. For example, when used for the prevention / treatment of an excessive immune response (eg, hay fever) in adults, interleukin-18 is used. The mutant protein or a salt thereof as a single dose is usually about 0.1 to 10.0 μg / kg body weight, preferably about 1.0 μg / kg body weight at a frequency of about 3 times a week, preferably 2 It may be administered by intravenous injection (preferably, continuously or every other day) about once a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
[0044]
Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition can be produced by a conventional method, and may contain a carrier, diluent or excipient usually used in the pharmaceutical field. For example, examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.
[0045]
Examples of the composition for parenteral administration include injections and suppositories, and injections include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and intravenous injections. It is good to be. Such an injection is prepared by a conventional method, that is, by dissolving, suspending or emulsifying interleukin-18 mutant protein or a salt thereof in a sterile aqueous or oily liquid usually used for injection. Aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other adjuvants, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, Polyethylene glycol), nonionic surfactants (for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) added of hydrogenated castor oil)) and the like. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration can be prepared by mixing interleukin-18 mutant protein or a salt thereof with an ordinary suppository base.
[0046]
The above oral or parenteral pharmaceutical composition may be prepared in dosage unit form so as to be compatible with the dose of the active ingredient. Such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 0.125 to 1.000 mg, especially injections, for each dosage unit form. Is preferably 0.125 to 1.000 mg, and other dosage forms are preferably 0.125 to 1.000 mg of interleukin-18 mutant protein or a salt thereof.
[0047]
Moreover, said pharmaceutical composition may contain another active ingredient, unless an unfavorable interaction is produced by blending with interleukin-18 mutant protein or a salt thereof.
[0048]
7). antibody
The antibody against the interleukin-18 mutant protein or salt thereof of the present invention can be used for detection and / or quantification of the interleukin-18 mutant protein or salt thereof of the present invention.
[0049]
The antibody against the interleukin-18 mutant protein of the present invention or a salt thereof can be obtained by administering a fragment containing the interleukin-18 mutant protein of the present invention or a salt thereof or an epitope thereof to an animal using a conventional protocol. can get.
[0050]
The antibody of the present invention may be any of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, and a humanized antibody.
[0051]
In order to produce a polyclonal antibody, it can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (protein antigen) and a carrier protein is prepared, administered (immunized) to an animal, and an antibody-containing substance against the protein of the present invention is collected from the immunized animal, and the antibody is separated and purified. Can be manufactured. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from blood, ascites, etc. of an immunized animal, preferably blood. Polyclonal antibodies can be separated and purified by immunoglobulin separation and purification methods (for example, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger, ultracentrifugation method, gel filtration method, A specific purification method in which only an antibody is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the antibody is obtained by dissociating the binding.
[0052]
Monoclonal antibodies are described in G. Nature (1975) 256: 495, Science (1980) 208: 692-. Koehler and C.H. It can be produced by the Milstein hybridoma method. That is, after immunizing an animal, antibody-producing cells are isolated from the spleen of the immunized animal and fused with myeloma cells to prepare monoclonal antibody-producing cells. In addition, cell lines that produce antibodies that react specifically with the interleukin-18 mutant protein or salt thereof but do not substantially cross-react with other antigenic proteins may be isolated. This cell line can be cultured, and the desired monoclonal antibody can be obtained from the culture. Purification of the monoclonal antibody can be performed according to the above-described method for separating and purifying immunoglobulin.
[0053]
Techniques for making single chain antibodies are described in US Pat. No. 4,946,778.
[0054]
Techniques for making humanized antibodies are described in Biotechnology 10, 1121-, 1992; Biotechnology 10, 169-, 1992.
[0055]
8). Screening method
The present invention provides the three-dimensional structural coordinates of wild type interleukin 18. The three-dimensional structural coordinates of wild-type interleukin 18 are shown in Table A and Table B. All or part of the three-dimensional structural coordinates shown in Table A and / or Table B can be used to screen for substances that bind to interleukin-18 protein.
[0056]
The substance that binds to interleukin-18 protein may be either a natural product or a synthetic product, and may be either a high molecular compound or a low molecular compound.
[0057]
A substance that binds to an interleukin-18 protein can modulate the biological activity of the interleukin-18 protein. Substances that reduce or eliminate the biological activity of interleukin-18 protein by binding to interleukin-18 protein are diseases caused by interleukin-18 protein (for example, autoimmune diseases, allergies, neuropathies, etc.) ) Can be used for prevention and / or treatment. Substances that maintain or improve the biological activity of interleukin-18 protein by binding to interleukin-18 protein are diseases whose symptoms are improved by administration of interleukin-18 protein (eg, hepatitis C). , Tumor etc.) and / or treatment.
[0058]
For example, using all or part of the three-dimensional structural coordinates shown in Table A and / or Table B, a substance that binds to interleukin-18 protein can be identified, searched, evaluated, or designed by a computer.
[0059]
The computer used for screening is not particularly limited as long as the screening program operates.
[0060]
As a screening program, DOCK (Science, 1992, 257, 1078), Gold4, Glide, FlexX (J. Mol. Biol., 1996, 261, 470), AutoDock (J. Comput. Chem., 1998, 19, 1639), ICM (J. Comput. Chem., 1994, 15, 488), Ludi, and the like.
[0061]
A substance that binds to the interleukin-18 protein selected by the screening may be produced by a known technique according to the type of the substance.
[0062]
Next, biological activity (for example, interferon γ-inducing activity), interaction with interleukin-18 protein receptor (for example, dissociation constant for IL-18Rα) may be examined. Methods for examining interferon γ-inducing activity and dissociation constants for IL-18Rα are described in the examples below.
[0063]
9. Search method for substances that bind to interleukin-18 protein
In the amino acid sequence of the wild type interleukin-18 protein consisting of the amino acid sequence of SEQ ID NO: 1 or the interleukin-18 mutant protein of the present invention, the 13th amino acid, the 17th amino acid, the 33rd amino acid, the 35th amino acid And the first amino acid residue group consisting of the 132nd amino acid, the second amino acid, the fourth amino acid, the fifth amino acid, the eighth amino acid, the 58th amino acid, the 60th amino acid and the 104th amino acid. A second amino acid residue group, and at least one amino acid residue selected from the three amino acid residue groups consisting of the third amino acid residue group consisting of the 79th amino acid, the 84th amino acid and the 98th amino acid By examining the interaction between the group and the test substance, interleukin- Substance binding to the 8 protein can be searched.
[0064]
The test substance may be either a natural product or a synthetic product, and may be either a high molecular compound or a low molecular compound.
[0065]
A substance that binds to an interleukin-18 protein can modulate the biological activity of the interleukin-18 protein. Substances that reduce or eliminate the biological activity of interleukin-18 protein by binding to interleukin-18 protein are diseases caused by interleukin-18 protein (for example, autoimmune diseases, allergies, neuropathies, etc.) ) Can be used for prevention and / or treatment. Substances that maintain or improve the biological activity of interleukin-18 protein by binding to interleukin-18 protein are diseases whose symptoms are improved by administration of interleukin-18 protein (eg, hepatitis C). , Tumor etc.) and / or treatment.
[0066]
The interaction between the amino acid residue at the above site in the amino acid sequence of the wild-type interleukin-18 protein or the interleukin-18 mutant protein of the present invention and the test substance is determined by X-ray crystal structure analysis, nuclear magnetic resonance (NMR), Three-dimensional structure analysis such as neutron diffraction, a method of observing the shape of a complex such as an electron microscope or an atomic force microscope, three-dimensional structures of interleukin-18 proteins shown in Table A and / or Table B (or known three-dimensional structures) It is possible to use a technique such as a docking study in which a stable binding mode with a test substance having an arbitrary structure is simulated by a computer.
[0067]
For example, using the NMR signal as an index, a substance that binds to the interleukin-18 protein can be searched by the following procedures (1) to (4).
(1) 13th amino acid, 17th amino acid, 33 in the amino acid sequence of the wild type interleukin-18 protein or fragment thereof comprising the amino acid of SEQ ID NO: 1 or the interleukin-18 mutant protein or fragment thereof of the present invention; 1st amino acid residue group consisting of amino acid 35, amino acid 35 and amino acid 132, 2nd amino acid, 4th amino acid, 5th amino acid, 8th amino acid, 58th amino acid, 60th amino acid A second amino acid residue group consisting of the amino acid of 104 and the 104th amino acid, and three amino acid residue groups consisting of the third amino acid residue group consisting of the 79th amino acid, 84th amino acid and 98th amino acid An isotope capable of NMR observation of at least one selected amino acid residue (for example,¹³C,¹⁵Prepare a label labeled with N).
(2) Obtain an NMR signal of the labeled protein or fragment thereof prepared in (1). (3) An NMR signal is obtained after contacting the test substance with the labeled protein prepared in (1) or a fragment thereof.
(4) Based on the difference between the NMR signal obtained in (2) and the NMR signal obtained in (3), the interaction between the test substance and the interleukin-18 protein is evaluated.
[0068]
Alternatively, a substance (compound) that interacts with the amino acid residue at the above site in the amino acid sequence of the wild-type interleukin-18 protein or the interleukin-18 mutant protein of the present invention can be obtained from a chemical structure database (for example, “Cambridge”). Crystal Database ”,“ RCSB Protein Database ”, Chemical Abstract, etc.). In this case, the three-dimensional structural coordinates of the wild-type interleukin-18 protein of the present invention or a part thereof is used. In its conformation, the point of being able to bind to interleukin-18 protein is characterized with respect to the advantage of interaction with one or more functional groups. A database of chemical structures is then searched to search for candidate compounds containing one or more functional groups that can interact favorably with the interleukin-18 protein based on this characterization. Compounds with structures that best fit the point of favorable interaction with the interleukin-18 protein conformation are thus identified. Such a search can be performed on a computer using software such as DOCK (J. Med. Chem. (1986) 29, 2149-2153), SYBYL (Tripos).
[0069]
【Example】
Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention, and do not limit the scope of the present invention.
[0070]
[Example 1]
Human interleukin 18 (hIL-18) was expressed using E. coli and purified by affinity chromatography and gel filtration column chromatography. Yields in nutrient medium and minimal medium were 1.1 mg / L and 0.8 mg / L, respectively.
[0071]
Materials and methods
In E. coli hIL-18 Of a plasmid that expresses
MRNA was extracted from blood samples obtained from healthy volunteers and cDNA was synthesized using reverse transcriptase (MMLV) and oligo dT primers for 60 minutes at 72 ° C. The coding region of mature hIL-18 (residue 157) was amplified by PCR and the amplified fragment was cloned into a T vector (Invitrogen). Primers were designed to insert a BamHI restriction site and a factor Xa cleavage site immediately before the mature hIL-18 sequence and an EcoRI restriction site after the stop codon. The resulting amplification product was again cloned into the T vector, cut with BamHI and EcoRI, and then purified by electrophoresis. The purified product was subcloned into the pGEX-4T-1 vector (Pharmacia) and the DNA sequence of this clone was confirmed by bidirectional sequencing. This clone was named pGEX-4T-Xa-hIL-18 (FIG. 1).
[0072]
Expression
BL-21 (DE3) (Novagen) was transformed with pGEX-4T-Xa-hIL-18 according to the manufacturer's instructions. The protein was expressed as described below. That is, each transformed colony was cultured in 5 ml LB medium (10 g tryptone per liter medium, 5 g yeast extract and 10 g NaCl) at 100 nm containing 100 μg / ml ampicillin. Shake at 37 ° C. until the optical density (OD600) reached 0.5. Next, protein production was induced by adding isopropylthio-β-D-galactoside (IPTG) at a final concentration of 1 mM. Prior to the addition of IPTG, 1 ml of each culture was transferred to a separate sterile tube and incubated separately until the next step. After inducing the protein for 3 hours, 20 μl of each culture was used as a sample for expression analysis by SDS-PAGE. Colonies with the highest expression level measured by SDS-PAGE were selected. The 1 ml culture that had been incubated and not treated with IPTG was then used as seed for the next overnight culture. Cultivation was performed overnight (about 12 hours) using 200 ml of LB medium containing 100 μg / ml ampicillin in a 500 ml flask at 37 ° C. with shaking at 100 rpm. The resulting medium was centrifuged at 5,000 g for 10 minutes and the E. coli pellet was transferred into 2 L of LB medium containing 100 μg / ml ampicillin. The culture was incubated at 37 ° C. until OD600 reached 0.45 and cooled to 25 ° C. in an ice water bath. When OD600 reached 0.5, IPTG (final concentration 1 mM) was added to the medium. The culture was incubated at 25 ° C. with shaking at 100 rpm for 5 hours. The resulting culture was centrifuged at 5,000 g for 10 minutes and the E. coli pellet was stored at −80 ° C. until used for purification.
[0073]
Purification
The bacterial cell pellet was added to 10 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 400 mM KCl, 10 mM 2-mercaptoethanol (1 mM) per gram of the pellet containing 1 mM PefaBloc (Roche). 2-ME), 1 mM EDTA). Thawed cells were completely lysed by sonication in an ice-water bath for 10 minutes (with a cycle of 10 seconds and 50 seconds apart), then centrifuged at 300,000 g for 30 minutes at 4 ° C. The clear lysate was filtered through 0.45 μm filter and 0.2 μm filter (Millipore), and the filtrate was passed through a GST affinity column (bed volume: 10 ml, Pharmacia). Next, 200 ml of lysis buffer, 10 ml of wash 1 buffer (50 mM Tris-HCl, pH 8.0, 400 mM KCl, 10 mM 2-ME, 1 mM EDTA, 0.5% Triton-X-100) ), 100 ml lysis buffer, 10 ml wash 2 buffer (50 mM Tris-HCl, pH 8.0, 1 M KCl, 10 mM 2-ME, 1 mM EDTA), and column with 100 ml lysis buffer Was washed. 10 ml of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM glutathione (GSH)) was incubated with the GST column for 10 minutes and then slowly eluted. This procedure was repeated 5 times. The amount of protein in the recovered eluate was measured, and the protein-containing fraction was concentrated by centrifugation (3,000 g, 4 ° C.) using Centriplus-30 (Amicon) until the amount of the solution became less than 1 ml. High salt buffer (50 mM Tris-HCl, pH 8.0, 1.5 M NaCl) and 100 mM EDTA (pH 8.0) at a rate of 10% (v / v) and 1% (v / v), respectively. Added to the concentrated protein solution. Human factor Xa (Funakoshi) was added at a rate of 1% (w / w) based on the final weight of the protein and incubated at 4 ° C. for 12 hours to cleave the GST fusion protein. The amount of this fusion protein was calculated according to the manufacturer's instructions. D. It was 0.5 mg / ml at 280. The cleaved protein was gel filtered using Sephacryl S-100 26/60 (Pharmacia) and gel filtration buffer (50 mM phosphoric acid, pH 6.0, 150 mM KCl, 10 mM 2-ME, 1 mM EDTA). . The desired fractions are collected, mixed with PefaBloc (final concentration 1 mM), concentrated by centrifugation (6,000 g, 4 ° C.) using Centricon-10 (Amicon) to a volume of about 300 μl, and Stored at 4 ° C. until used in further experiments. The concentration of purified hIL-18 protein was estimated using the absorbance constant of hIL-18 (6160).
[0074]
Expression in minimal medium
Expression in minimal medium was performed using the following composition: 0.1 M phosphate buffer (pH 7.0); 9 mM CaC1₂, 100 mM MgSO₄, 5 mM FeCl₃  And a divalent cation consisting of 15 mM sodium citrate; 10 mM CuSO₄, 80 mM manganese chloride and 10 mM zinc sulfate; 1.5 mg / ml thiamine hydrochloride, and 100 mg / ml ampicillin. As the only nitrogen and carbon sources, ammonium chloride (1.9 mM) and 0.25% glucose were added, respectively. Water and phosphate buffer were first treated in an autoclave, then the remaining ingredients were added by filter sterilization. The expression and purification protocol was the same as described in the section on culture in LB medium.
[0075]
result
Expression and purification
Typically, the culture was performed 3 times with 2 L, and a total of 6 L was cultured. The medium showed OD600 = 1.0 after 5 hours of induction. A 15 g E. coli pellet was then obtained from the 6 L culture. Production of the fusion protein in LB medium was clearly observed as a band with the expected molecular weight (43 kDa) as a result of SDS-PAGE (FIG. 2a).
[0076]
The fusion protein was highly expressed in E. coli, but most of the protein precipitated as inclusion bodies when expressed at 37 ° C. (FIG. 2b). Several induction temperatures were tried to obtain maximum yield. At low temperatures such as 15 ° C., no inclusion bodies were formed, but the expression level was very low. Compromising between expression levels and solubility, induction at 25 ° C resulted in the best yield. We used this temperature in subsequent experiments (Figure 2b).
[0077]
In the first step, GST-hIL-18 fusion protein was captured using a GST affinity column. The eluted protein contained GST-hIL-18 with a purity of about 90%. The amount of protein eluted was approximately 20 mg (Figure 2c). The fusion protein was concentrated and cleaved with 200 μg factor Xa. The reducing agent affected the cleavage ability of factor Xa. We have optimized the concentration and type of agent used. GSH and 2ME did not interfere with the cleavage compared to DTT (FIG. 3). Cleavage was complete in about 16 hours at 4 ° C.
[0078]
The cleaved protein was subjected to gel filtration chromatography and showed two major peaks (P1 and P2) (FIG. 4a). P1 corresponded to GST protein and P2 corresponded to IL-18 (FIG. 4b). The yield of IL-18 protein was 6.6 mg (1.1 mg / L) from a 6 L culture. As estimated by SDS-PAGE, the protein had a purity of approximately 98%.
[0079]
When using minimal medium, OD600 = 0.85 was shown 5 hours after induction. The expression level of the fusion protein in the minimal medium was similar to the expression level in the LB medium. Subsequent protein purification indicated that approximately the same amount of protein (0.8 mg / L) was produced as was produced in LB medium.
[0080]
[Example 2]
As a first step in understanding receptor activation by IL-18, we determined the solution structure of human IL-18 using NMR spectroscopy. The structure shows that the IL-18 β-trefoil fold is similar to that of IL-1 family members (IL-1β and IL-1RA, etc.), but the surface residues are quite different. We also constructed point mutants for 50 residues exposed on the surface of IL-18. Receptor binding and cellular response analysis using these mutants revealed the presence of three sites; two of these are important for IL-18Rα binding and the third site is It is involved in cellular responses but not IL-18Rα binding. Comparing the structure of IL-18 and the receptor binding site with those of IL-1 family members, we propose a model for the interaction between IL-18 and IL-18Rα.
[0081]
Structure of IL-18
With the exception of the structure of residues 34-43, the structure of IL-18 was fully apparent. IL-18 consists of three sets, one short α-helix (H1) and one 3₁₀It consists of 12 strands (S1-S12) that form a twisted 4-stranded sheet with a helix (H2). These three β sheets are packed together to form a β trefoil fold (FIG. 5). The loop (residues 34-42) extends out of the protein body and is characterized by a small heteronuclear NOE (nuclear overhauser effect) value (0.49 +/− 0.025) and by a lack of long-range NOE Seems to be flexible.
[0082]
Despite moderate sequence identity (17%), the overall organization of IL-18 is such as that of IL-1β (FIG. 6a), as previously predicted based on the conservation of hydrophobic residues. Shows striking similarities with the composition of IL-1 family members⁸. The fact that the root mean square displacement of 70 Cα atoms in the secondary structure element between IL-18 and IL-1β is 1.60Å is related to that IL-18 and IL-1β belong to the same structural class It shows that it is a protein. However, the length and conformation of several segments between chains S3-S4, S4-S5, S7-S8 and S11-S12 are substantially different between IL-18 and IL-1.
[0083]
Contact site of IL-18 receptor α
To identify residues that are important for IL-18 function, we generated a series of mutants and analyzed their ability to induce IFN-γ production and bind IL-18Rα ( Table 1). We selected 50 residues that covered most of the residues exposed on the surface of IL-18. Of the 50 mutant proteins generated, 15 showed the ability to induce significantly reduced IFN-γ production. For each of the mutants that showed a substantial decrease in activity,¹⁵Prepare a protein labeled with N,¹H-¹⁵The N correlation spectrum was compared with that of wild type IL-18. None of these mutants showed any evidence that the natural fold was destroyed by the mutation.
[0084]
Twelve of the 15 mutants that showed a reduction in IFN-γ production inducing ability also showed a significant reduction in receptor binding activity (Table 1). These residues form two separate surface patches, designated site I and site II (FIG. 6b left). Site I, formed by 5 residues (Arg13, Asp17, Met33, Asp35 and Asp132), is located on one side of the core barrel of the β-trefoil fold, while 7 residues (Phe2, Site II formed by Lys4, Leu5, Lys8, Arg58, Met60 and Arg104) is located at the top of the β barrel. Recently, Kim et al. Showed that mutations that reverse the charge of Glu6 or Lys53 alter the biological activity of IL-18.⁹. These two residues are located on the same surface as site II. This suggests that the residues forming site II are important for receptor binding.
[0085]
The structure and mutation data of the IL-1β: IL-1RI complex showed that IL-1β has two interface sites (site A and site B) to IL-1RI (FIG. 6d). Site A touches the two amino-terminal immunoglobulin domains of the extracellular portion of the receptor, and site B touches the third immunoglobulin site (FIG. 7a). Comparison of the tertiary and primary structures of IL-18 and IL-1β indicated that IL-18 site I and site II correspond to IL-1β site A and site B, respectively. This is because these sites are equivalently located in terms of sequence and tertiary structure.
[0086]
These considerations indicate that we use the IL-1β: IL-1RI complex (PDB code: 1ITB) and IL-18 coordinates to model the IL-18: IL-18Rα complex shown in FIG. Made it possible to build. Modeling the tertiary structure of IL-18Rα is fairly simple because of the 22% sequence identity between the IL-1RI and IL-18Rα extracellular domains and the conservation of characteristic amino acids (such as cysteine). It was done. The resulting model of the IL-18: IL-18Rα complex is very similar to the crystal structure of IL-1β: IL-1RI and is consistent with the mutation data presented here for IL-18.
[0087]
Electrostatic complementarity may explain the specificity of IL-18 and IL-1β receptor binding. The molecular surface at site I of IL-18 and its periphery is negatively charged. On the other hand, the molecular surface of IL-18Rα in contact with this site in the model is highly positively charged (FIGS. 6b and 6c). Mutation studies showed that the charge on IL-18 was important for receptor binding: replacement of IL-18 with Asp17, Asp35, or Asp132 with alanine significantly suppressed receptor binding. In contrast, site A of IL-1β is positively charged (FIG. 6e) and the IL-1RI interface is negatively charged. The electrostatic state of the receptor binding site of cytokines may contribute to receptor discrimination.
[0088]
Possible binding sites for IL-18 receptor β
Mutagenesis studies also revealed that residues Lys79, Lys84 and Asp98 are functionally important, but are not closely related to binding to IL-18Rα. Each mutant of these residues was able to bind IL-18Rα but lost IFN-γ-inducing activity (Table 1). These residues are clustered at the bottom of the barrel and are located on the opposite side of site II (referred to as site III) (FIG. 6b). In a model of the IL-18: IL-18Rα complex, site III of IL-18 is exposed to the solvent and points in the opposite direction to IL-18Rα (FIG. 7b). Thus, the notion that alanine substitution of the residue forming site III has little effect on IL-18Rα binding is consistent with this model (Table 1, FIG. 7b).
[0089]
So what is the function of the residues that form site III? We assume that these residues are important for binding to IL-18Rβ. Association of IL-18Rβ and IL-18Rα is essential for initiation of IL-18 receptor signaling^{2, 3}. IL-18 is thought to induce the association of IL-18Rα and IL-18Rβ, and facilitate the homotypic interaction of their cytoplasmic domains to initiate the signaling process.³. Truncated IL-18Rβ lacking the cytoplasmic domain has been shown to suppress IL-18 signaling.¹⁰.
[0090]
In order to determine if site III is involved in the association of IL-18Rα and IL-18Rβ, we performed surface plasmon resonance measurements. IL-18Rβ alone cannot bind to IL-18 or IL-18Rα (FIGS. 7c and d). However, in the presence of IL-18, IL-18Rβ can bind to IL-18Rα immobilized on the sensor, and IL-18Rβ associates with the IL-18: IL-18Rα complex to form a tertiary complex. It shows forming (FIG. 7d). Next, we examined the effect of mutations in the residues forming site III on the formation of the IL-18: IL-18Rα: IL-18Rβ tertiary complex. Mutations of either Lys79, Lys84 or Asp98 cause a significant decrease in the degree of association of IL-18Rβ with the IL-18: IL-18Rα complex. This suggests that the residue at site III is important for tertiary complex formation (FIG. 7d). These residues of IL-18 have charged side chains and form characteristic positive and negatively charged surface patches (FIG. 6b, c). Since the contact site of IL-18 with IL-18Rα and the primary structure of the two receptor subunits of IL-18 are similar to those of IL-1β, by analogy, the IL-1β residue Lys77 , Leu82 and Glu96, which correspond to the residues that make up site III of IL-18, can form an interfacial surface with IL-1AcP. The surface then mediates the association of IL-1AcP with the IL-1RI: IL-1β complex.
[0091]
[Table 1]

[0092]
Method
Sample preparation.¹⁵N labeling and¹⁵N /¹³C-labeled wild type IL-18 protein was expressed as a GST fusion protein. After purification by affinity chromatography, the GST tag was removed by digestion with factor Xa. Samples for NMR measurements are typically 1-2 mM protein, 150 mM KCl and H in 50 mM Tris-HCl buffer (pH 6.0).₂O /²H₂O (90% / 10%) or²H₂It consisted of 10 mM DTT in O.
[0093]
Fifty mutant IL-18 proteins with each amino acid replaced with alanine were prepared in the same manner as the wild type protein. Residues to be mutated are sequence alignment with IL-1 / IL-18 family members, IL-18, IL-1β (PDB code: 2I1B) and IL-1β: IL-1RI complex (PDB code: 1ITB) ), And based on previously reported mutagenesis data for IL-1.
NMR spectroscopy. All NMR spectra were obtained at 303K using a Bruker DMX500, DRX500 or DRX800 NMR spectrometer. Main chain and side chain¹H,¹³C and¹⁵To determine the assignment of N resonances, a series of three-dimensional experiments were performed.¹². Determination of the stereospecific assignment of the methyl group of Val and Leu residues was performed as previously described.¹³. For distance limitation, the mixing time was 100 ms.¹⁵N,¹⁵N-,¹⁵N,¹³C- or¹³C¹³Obtained from C-decomposed 4D NOESY experiment¹².
Structural calculation. The resulting main chain resonance assignments indicated the presence of minor conformations around the segment between chains S3 and S4. This is probably due to cis / trans isomerization of the Ala42-Pro43 peptide bond. The difference in 1H and 15N chemical shifts between the major and minor conformations is relatively small. We obtained structural limitations only for the main conformation. First, using the program DYANA (version 1.5), structural calculations and NOE peak assignment determinations were performed in an iterative and manual manner.¹⁴. Main chain torsion angle limit is HANHA¹²And the TALOS program³J_HNHObtained from α. The twist angle χ1 of His, Phe, Tyr and Trp is³J_C'Cand³J_NCEstimated from the coupling constant of¹⁶. After manually determining the overall fold, the CANDID algorithm is used to determine the attribution of the remaining NOE peaks.¹⁷, 2289 meaningful NOE upper distance limits were obtained. Using these restrictions, the final structure was calculated by the CNS program (version 1.1).¹⁸. At this stage, the hydrogen bond limitation derived from the main chain amide that is slowly exchanging is a distance limit of 2.8-3.4−３ for the N—O atom pair and for the HN—O atom pair. Each was added as a distance limit of 1.8-2.4 km. Protons assigned non-stereospecifically have a floating chirality and Σ (r^-6)^-1/6Processed as a grand total. Refined a total of 100 structures, and the 20 lowest energy structures MOLMOL¹⁹, AQUA and PROCHECK-NMR software 20²⁰(Table 2).
[0094]
[Table 2]

IFN-γ induction and receptor binding assay. Wild-type and mutant IL-18 proteins were previously described for their ability to induce IFN-γ production.²¹Examined. In vitro affinity of wild type and mutant IL-18 to the IL-18 receptor was measured at 25 ° C. by surface plasmon resonance experiments using a BIACORE 3000 (Pharmacia Biosensor AB). Specifically, a specific binding surface was prepared by binding an anti-human IgG Fc antibody (Rockland) to a CM5 sensor chip by an amine binding method. Next, recombinant IL-18α / Fc or IL-18β / Fc chimeric protein (R & D Systems) was immobilized on the chip. The binding density was limited to 200 resonance units (RU). IL-18 samples were diluted with HBS-EP (10 mM HEPES, pH 7.4, NaCl 150 mM, 3.0 mM EDTA, 0.005% (v / v) surfactant P-20). For dissociation constant analysis of IL-18Rα of wild type and mutant IL-18 protein, different amounts of sample were poured onto the sensor chip at a rate of 2 μl / min until an equilibrium phase was obtained. The sensor surface was regenerated by pulsing 0.2 M glycine-HCl (pH 1.5) twice for 60 seconds. The dissociation constant was measured by a Scatchard plot. In order to evaluate the ability to form a trimeric complex, IL-18 protein was first poured onto the sensor chip until the IL-18Rα binding site was saturated. The same amount of IL-18 protein was then mixed and poured with IL-18Rβ protein (3.12-100 nM).
Modeling. The IL-18: IL-18Rα complex was modeled based on the structure of IL-18 and the crystal structure of the IL-1β: IL-1RI complex (PDB code: 1ITB). MOE for model construction and structural verification²²Was used. The coordinates of the NMR structure of IL-18 and the modeled structure of IL-18Rα were superimposed on the IL-1β: IL-1RI complex. The IL-18: IL-18Rα complex model is described by Cornell et al.²⁴AMBER 5.0 package using all-atom force field described by²³Was refined by molecular mechanics calculation at 70 Kps at 300 K. The final structure was further energy minimized.
Coordinate. Coordinates were deposited with the Protein Data Bank (registration code: 1 JOS). The coordinates are shown in Tables A and B. Table A shows the coordinates of the structure obtained by 20 structural calculations. In the structure calculation by NMR, a plurality of structures generated by different calculations are presented, and convergence between the structures is regarded as accuracy. Table B shows the average value of the three-dimensional structural coordinates in Table A corrected slightly using chemical energy.
[0095]
Table A
Three-dimensional structure coordinates of wild type IL-18 (structures obtained by 20 structural calculations)

[0096]
Table B
Wild-type IL-18 3D structure coordinates (average of 3D structure coordinates in Table A corrected with a little chemical energy)

[0097]
Table A shows each atom except for the first row (ie, MODEL X (where X is an integer from 1 to 20)) and the last row 1 and 2 (ie, TER and ENDMDL). The three-dimensional coordinates are described. Table B describes the three-dimensional coordinates of each atom except for the last line (that is, display of END). ATOM in the first column indicates that this row is a row of atomic coordinates, the second column shows the order of the atoms, the third column shows the distinction of atoms in amino acid residues, etc., and the fourth column shows the amino acid residues. The fifth column shows the number of the amino acid residue containing the atom, and the sixth, seventh and eighth columns show the coordinates of the atom (in units of Å). The last line indicates the last line of this table. This table is described according to the format of the protein data bank, which is a notation commonly used by those skilled in the art. (9th and 10th columns are numbers to match the format of the database. In the present invention, the numbers are not particularly meaningful, and the 9th column uses all 1.00 and the 10th column uses all 0.00. did.)
[0098]
Cited references
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[0099]
【The invention's effect】
According to the present invention, a novel variant of interleukin-18 protein was provided. This mutant can be used as an antagonist of interleukin-18 protein.
[0100]
In addition, according to the present invention, the three-dimensional structural coordinates of the wild type interleukin-18 protein are provided. By using these coordinates, it became possible to screen for new drugs.
[0101]
[Sequence Listing]

[0102]
[Sequence Listing Free Text]
SEQ ID NO: 1 shows the amino acid sequence of wild type IL-18.
[0103]
SEQ ID NO: 2 shows the DNA sequence of wild type IL-18.
[0104]
SEQ ID NOs: 3-17 show the DNA sequences of IL-18 variants.
[0105]
SEQ ID NO: 18 shows the DNA sequence of IL-18 optimized cDNA.
[Brief description of the drawings]
FIG. 1 schematically represents an expression vector for a GST-hIL-18 fusion protein (pGEX-4T-Xa-hIL-18).
FIG. 2 shows GST-hIL-18 expression and purification. a) SDS-PAGE of expression of GST-hIL-18 fusion protein 5 hours after induction with IPTG. b) Formation of inclusion bodies at different expression temperatures. c) Purification of GST-hIL-18 using a GST affinity column. The eluted GST-hIL-18 protein has a purity of 90% or more. sup: cell lysate supernatant; pel: pellet of the lysate. Arrow indicates GST-hIL-18 protein.
FIG. 3 shows cleavage of GST-hIL-18 fusion protein. SDS-PAGE of cleavage tests with various concentrations of GSH, DTT or 2-ME. (-) And (+) indicate the absence and presence of factor Xa, respectively. A: GST-hIL-18 protein; B: GST protein; C: hIL-18 protein.
FIG. 4 shows purification by gel filtration column (Sephaacryl S-100, 26/60). a) A chromatogram in which a fraction is collected every 4 ml after passing through a gel filtration column at a flow rate of 2 ml / min. b) SDS-PAGE analysis of purified samples from P1 and P2. A: GST protein; B: hIL-18 protein.
FIG. 5: Solution structure of human IL-18. a. The most suitable main chain superposition of 20 steric final structures. The main chain atoms of residues 1-157 are superimposed. b. The schematic ribbon figure of the solid NMR structure of IL-18 drawn using MOLMOL.
FIG. 6: Mutation analysis of IL-18. a. Sequence alignment of human IL-18 and IL-1β. Residues of human IL-18 and IL-1β where the mutation caused a significant decrease in biological activity are indicated with an asterisk. The secondary structural elements of human IL-18 and IL-1β (PDB code: 2I1B) are shown at the top and bottom, respectively. Conserved residues are shown in yellow surrounded by a square. b. Surface representation of the IL-18 residue whose mutation caused a significant decrease in activity. Residues at sites I, II and III are shown in red, orange and blue, respectively. c. Distribution of electrostatic potential on the surface where the solvent can approach. Blue corresponds to positive potential and red corresponds to negative potential. In both panels, the molecules are shown in the same orientation as in FIG. 5 (left) and rotated 180 degrees about the vertical axis (right). d. Structure of IL-1β. Surface representation of the IL-1β residue whose mutation caused a significant decrease in activity. Residues at sites A and B are shown in red and orange, respectively. e. Distribution of electrostatic potential on the surface where the solvent can approach.
FIG. 7: Model of IL-18: IL-18Rα complex and interaction of IL-18, IL-18Rα and IL-18Rβ. a. Crystal structure of IL-1β: IL-1RI complex (PDB code: 1ITB). The IL-1β residues at sites A and B are shown in red and orange, respectively. b. Modeled structure of IL-18: IL-18Rα complex. Residues at sites I, II and III are shown in red, orange and blue, respectively. c. Capacitance response curve of surface plasmon resonance for wild-type IL-18 to immobilized IL-18Rα or IL-18Rβ. The concentration of wild type IL-18 is also shown. d. Response curve of surface plasmon resonance for IL-Rβ, for immobilized IL-18Rα and wild-type IL-18 complex, or for site III mutant protein.

Claims (11)

The following (a) or (b) interleukin-18 mutant protein or a salt thereof.
(A) In the amino acid sequence of SEQ ID NO: 1, the first amino acid residue group consisting of the 13th arginine, the 17th aspartic acid, the 33rd methionine, the 35th aspartic acid and the 132nd aspartic acid Phenylalanine, 4th lysine, 5th leucine, 8th lysine, 58th arginine, 60th methionine and 104th arginine second amino acid residue group, and 79th lysine, 84th Interleukin-18 comprising an amino acid sequence in which at least one amino acid selected from three amino acid residue groups consisting of a third amino acid residue group consisting of lysine and 98th aspartic acid is substituted with another amino acid In the amino acid sequence of the protein of mutant protein (b) (a) It consists of an amino acid sequence in which one or several amino acids other than those contained in the first to third amino acid residues are deleted, substituted or added, and the biological activity of the interleukin-18 protein is SEQ ID NO: 1. Interleukin-18 mutant protein having lower or higher wild-type interleukin-18 protein consisting of the amino acid sequence of DNA encoding the protein according to claim 1. A recombinant vector containing the DNA according to claim 2. A transformant comprising the recombinant vector according to claim 3. A method for producing an interleukin-18 mutant protein comprising culturing a host transformed with the DNA according to claim 2 and collecting the interleukin-18 mutant protein from the culture. An interleukin-18 protein antagonist comprising the interleukin-18 mutant protein or a salt thereof according to claim 1. A pharmaceutical composition comprising the interleukin-18 mutant protein according to claim 1 or a salt thereof as an active ingredient. The antibody with respect to the interleukin-18 mutant protein or its salt of Claim 1. A method for screening a substance that binds to interleukin-18 protein using all or part of the three-dimensional structural coordinates shown in Table A and / or Table B. In the amino acid sequence of the wild type interleukin-18 protein consisting of the amino acid sequence of SEQ ID NO: 1 or the interleukin-18 mutant protein according to claim 1, the 13th amino acid, the 17th amino acid, the 33rd amino acid, the 35th amino acid The first amino acid residue group consisting of the amino acid and the 132nd amino acid, the second amino acid, the fourth amino acid, the fifth amino acid, the eighth amino acid, the 58th amino acid, the 60th amino acid and the 104th amino acid A second amino acid residue group consisting of amino acids, and at least one selected from three amino acid residue groups consisting of a third amino acid residue group consisting of the 79th amino acid, the 84th amino acid and the 98th amino acid Interrogation, which involves examining the interaction between amino acid residues and test substances Method for searching a substance binding to the down -18 protein. DNA represented by the nucleotide sequence of SEQ ID NO: 18.

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