JP3543966B2 - Human monoclonal antibody against costimulatory transfer molecule AILIM and its pharmaceutical use - Google Patents

Description

この結果からいずれの抗ヒトＡＩＬＩＭヒトモノクローナル抗体及び抗ヒトＡＩＬＩＭマウスモノクローナル抗体も、ヒトＡＩＬＩＭに対して極めて高い結合親和性及び中和活性を有していることが明らかとなった。
【０１６９】
実施例７ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体のヒトＴ細胞に対するコスティミュレイトリーシグナル伝達活性
本発明の抗ヒトＡＩＬＩＭヒトモノクローナル抗体が、ヒトＴ細胞反応（ＩＦＮ−γやＩＬ−４などのサイトカインの産生、及び細胞増殖など）を制御（促進及び／または抑制）する能力を有するか否か、即ちＡＩＬＩＭを介したコスティミュレイトリーシグナル（ｃｏ−ｓｔｉｍｕｌａｔｏｒｙ ｓｉｇｎａｌ）の細胞内への伝達の制御能を有するか否かを、ヒトＴ細胞からのサイトカイン（ＩＦＮ−γ及びＩＬ−４）の産生量、並びに該ヒトＴ細胞の増殖の程度を指標に解析した。
【０１７０】
＜７−１＞ 抗体の希釈
抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（ＡＴＣＣ ＣＲＬ−８００１）を、リン酸緩衝液（ＰＢＳ）で最終濃度８μｇ／ｍｌとなるように希釈した。
前記で調製した各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体の各々を、ＰＢＳで最終濃度が４０μｇ／ｍｌとなるように希釈した後、ＰＢＳでさらに各種濃度に希釈した（４０μｇ／ｍｌ乃至０．００４９μｇ／ｍｌ）。
【０１７１】
＜７−２＞ 抗体によりマイクロプレートのコーティング
９６ウェルマイクロプレート（複数枚）の各ウェルに、（１）抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（８μｇ／ｍｌを２５μｌ／ｗｅｌｌ）及び各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のいずれか（４０μｇ／ｍｌ乃至０．００４９μｇ／ｍｌを２５μｌ）、または（２）抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（８μｇ／ｍｌを２５μｌ／ｗｅｌｌ）のみを加え、３７℃で２時間インキュベーションした。次いで、抗体を捨て、各ウェルをＰＢＳで３回洗浄した。洗浄後、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（１００μｌ／ｗｅｌｌ）を加え、３７℃で１時間インキュベーションして、上記（１）または（２）の抗体でプレートの各ウェルをコーティングした。
なお、対照抗体として、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに、下記の各々のモノクローナル抗体を用いて、上記と同様にしてプレートのコーティングを行った。
（１）ヒトＡＩＬＩＭに対するマウスモノクローナル抗体ＳＡ１２または同ＳＧ４３０（日本国特許出願公開１１−２９５９９号公報（実施例１２）及び国際特許出願公開ＷＯ９８／３８２１６号（実施例１２））。
（２）マウス抗ヒトＣＥＴＰモノクローナル抗体ＪＨＣ１（ＪＭａｂ１０９とも別称する。日本特許出願公開第９−２０８００号公報）。
（３）抗ＫＬＨヒトモノクローナル抗体（ＪＭａｂ２３とも別称する。前記実施例）。
これらの抗体コーティングマイクロプレートを以下の試験で用いた。
【０１７２】
＜７−３＞ ヒトＴ細胞懸濁液の調製
健常人（５人；ドナーＡ， Ｂ， Ｃ， Ｄ及びＥ）の各々の末梢血を採取し、ＬｙｍｐｈｏＰｒｅｐ（Ｎｙｃｏｍｅｄ製）を用いた密度勾配遠心により単核球を分画した。実験操作マニュアルに従い、Ｐａｎ−Ｔ ｃｅｌｌ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｍｉｌｔｅｎｙｉ製）及びマグネティックソーターを用いて、該ヒト単核球からヒトＴ細胞を分離取得した。該Ｔ細胞の細胞数を、血球計算盤により計数した。該ヒトＴ細胞を、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中に懸濁しヒトＴ細胞懸濁液（１×１０^６細胞／ｍｌ）を調製した。
【０１７３】
＜７−４＞ 細胞培養
（１）抗ヒトＣＤ３抗体と抗ヒトＡＩＬＩＭ抗体をコートしたマイクロプレートでの培養前記の抗体コーティングマイクロプレートの各ウェルに、ヒトＴ細胞懸濁液（ドナーＡ， Ｂ， Ｃ， Ｄ及びＥ；１００μｌ／ｗｅｌｌ；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ）を加え、ＣＯ_２インキュベーター内で３７℃で３日間培養した。
培養後、後述の試験（ＩＦＮγの定量）での使用の目的で、培養上清（５０μｌ）を分取しマイナス２０℃で保存した。該培養上清のサンプリング後の各々のマイクロプレートを以下の試験に用いた。
（２）抗ヒトＣＤ３抗体のみをコートしたマイクロプレートでの培養
前記の抗体コーティングマイクロプレートの各ウェルに、ヒトＴ細胞懸濁液（ドナーＤ；１００μｌ／ｗｅｌｌ；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ）を加えた後、各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のいずれか（４０μｇ／ｍｌ乃至０．００４９μｇ／ｍｌを２５μｌ）を加え、ＣＯ_２インキュベーター内で３７℃で３日間培養した。
【０１７４】
＜７−５＞ Ｔ細胞増殖活性の測定
前記の培養後の各プレートの各ウェルに、ｍｅｔｈｙｌ［^３Ｈ］ｔｈｙｍｉｄｉｎｅ（０．５μＣｉ／ｗｅｌｌ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を加え、ＣＯ_２インキュベーター内で３７℃で６時間インキュベーションした。培養後、セルハーベスターを用いて、細胞をＧＦ／Ｃフィルター（Ｐａｃｋａｒｄ製）上にトラップした。次いで、フィルターを、４０℃で３時間以上乾燥させた後、Ｍｉｃｒｏｓｃｉｎｔｉ ０（２０μｌ／ｗｅｌｌ；Ｐａｃｋａｒｄ製）を加えた。βカウンター（ＴＯＰ ＣＯＵＮＴ）を用いて、フィルター上にトラップされた細胞に取込まれている^３Ｈの放射活性を測定し、培養後のＴ細胞の増殖の程度を解析した。
結果を図１５乃至図３９に示す。
本試験の結果、抗ヒトＡＩＬＩＭモノクローナル抗体（ヒトモノクローナル抗体またはマウスモノクローナル抗体）を抗ヒトＣＤ３抗体とともにコートしたマイクロプレートを用いた場合には、ヒトＴ細胞の濃度依存的な有意な増殖が認められた。また、該細胞増殖の程度は、ドナー間でいくらかの差が見られた。
一方、抗ヒトＣＤ３抗体のみをコートしたプレートを用いて、抗ヒトＡＩＬＩＭモノクローナル抗体（ヒトモノクローナル抗体またはマウスモノクローナル抗体）を溶液の状態（液相）で細胞培養系に加えて培養した場合には、ヒトＴ細胞の有意な増殖は認められなかった。
【０１７５】
＜７−６＞ Ｔ細胞の培養上清中のＩＦＮγの量の測定
前記＜７−４＞の（１）の各々のＴ細胞の培養系（ドナーＢ及びにＣ）ついて、培養上清に含まれるＩＦＮγの量を、市販のｈｕｍａｎ ＩＦＮγ ＥＬＩＳＡ ＫＩＴ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製；Ｅｎｄｏｇｅｎ製）を用いて測定した。
結果を図４０乃至図４７に示す。
本試験の結果、抗ヒトＡＩＬＩＭモノクローナル抗体（ヒトモノクローナル抗体またはマウスモノクローナル抗体）の濃度に依存した有意なＩＦＮγの産生増加が認められた。
【０１７６】
実施例８ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体の混合リンパ球反応（ＭＬＲ）の制御活性
本発明の抗ヒトＡＩＬＩＭヒトモノクローナル抗体が、Ｔ細胞反応（ＩＦＮ−γやＩＬ−４などのサイトカインの産生、及び細胞増殖など）を制御（促進及び／または抑制）する能力を有するか否か、即ちＡＩＬＩＭを介したコスティミュレイトリーシグナル（ｃｏ−ｓｔｉｍｕｌａｔｏｒｙ ｓｉｇｎａｌ）の細胞内への伝達の制御能を有するか否かを、アロジェニック混合リンパ球反応（ａｌｌｏｇｅｎｉｃ ｍｉｘｅｄ ｌｙｍｐｈｏｃｙｔｅ ｒｅａｃｔｉｏｎ； ａｌｌｏｇｅｎｉｃ ＭＬＲ）におけるＴ細胞の増殖（即ち、細胞内でのＤＮＡ合成）を制御する活性の有無を指標に解析した。
【０１７７】
＜８−１＞ ヒトＰＢＭＣ及びＴ細胞の調製
健常人（７人；ドナーＡ， Ｂ， Ｃ， Ｄ， Ｅ， Ｆ及びＧ）の各々から採取した各々の末梢血（２００ｍｌ）を、マイクロチューブ（５０ｍｌ；Ｆａｌｃｏｎ製）に分注したＬｙｍｐｈｏｐｒｅｐ（１５ｍｌ；Ｎｙｃｏｍｅｄ製）に重層した。次いで、遠心分離（１６００回転、１０分）の後、中間層を回収した。回収した細胞を、リン酸緩衝液で２倍以上に希釈した後、遠心分離（１，８００回転、１０分）して、ＰＢＭＣ（末梢血単核球細胞；２×１０^８〜５×１０^８細胞）を調製した。血球計数板を用いて細胞数を計数し、ＭＬＲ試験に必要な細胞数（１．０８×１０^８細胞／９マイクロプレート）を分取し、氷上に保存した。残りの細胞は、以下のＴ細胞の分離に用いた。
ＰＢＭＣからのＴ細胞の分離には、ＰａｎＴ Ｉｓｏｌａｔｉｏｎキット（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃｈ製）を用いた。該キットの添付の実験操作マニュアルに従って、該残りのＰＢＭＣを、該キットに付属の溶液に添加し、反応させた。次いで、細胞を５ｍＭのＥＤＴＡ及び０．５％ＢＳＡ含有ＰＢＳで洗浄した後、該ＰＢＳ中に再懸濁した。次いで、該細胞懸濁液を、該ＰＢＳで膨潤させたＰｏｓｉｔｉｖｅ Ｓｅｌｅｃｔｉｏｎ Ｃｏｌｕｍｎ ＶＳ＋（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃｈ製）に添加し、非吸着画分を回収した。また、該カラムに該ＰＢＳを添加して、洗浄液を回収した。同様の操作を再度行った。回収液を併せて、Ｔ細胞画分とした。該Ｔ細胞画分を、遠心した後、該ＰＢＳ中に再懸濁した。得られたＴ細胞の細胞数を、血球計数板を用いて計数し、以下の試験に用いた。
【０１７８】
＜８−２＞ 混合リンパ球反応（ＭＬＲ）
先に述べたとおり、Ｔ細胞等のリンパ球の活性化に必要なコスティミュレイトリーシグナルの伝達経路には、既に比較的十分な解析がなされているＣＤ２８とＣＤ８０（Ｂ７−１）／ＣＤ８６（Ｂ７−２）との間のシグナル伝達経路、及びＣＴＬＡ４とＣＤ８０（Ｂ７−１）／ＣＤ８６（Ｂ７−２）との間のシグナル伝達経路の２つの経路が知られている。
即ち、混合リンパ球反応（ＭＬＲ）におけるＴ細胞の増殖は、該既知の２つの経路を介するシグナル伝達によっても誘導される。
従って、本試験では、下記の被験物質を用いて、（１）ＣＴＬＡ４を介するシグナル伝達経路の遮断によるＭＬＲの抑制、（２）ＣＤ８０（Ｂ７−１）／ＣＤ８６（Ｂ７−２）を介するシグナル伝達経路の遮断によるＭＬＲの抑制、（３）ＣＴＬＡ４を介する経路及びＣＤ８０（Ｂ７−１）／ＣＤ８６（Ｂ７−２）を介するシグナル伝達経路の両方の遮断によるＭＬＲの抑制、（４）ＡＩＬＩＭが担う第３のシグナル伝達経路の遮断によるＭＬＲの抑制、及び（５）ＣＴＬＡ４を介する経路及びＡＩＬＩＭを介する経路の両方の遮断によるＭＬＲの抑制の各々について解析した。
【０１７９】
下記を、被験物質として用いた。
（１）抗ヒトＡＩＬＩＭヒトモノクローナル抗体（前記実施例で調製）。
（２）抗ヒトＡＩＬＩＭマウスモノクローナル抗体ＳＡ１２（前記実施例に同じ）。
（３）抗ＫＬＨヒトモノクローナル抗体（陰性対照；前記実施例に同じ）。
（４）マウスＩｇＧ抗体（抗ヒトＣＤ３４；陰性対照；Ｉｍｍｕｎｏｔｅｃｈ製）。
（５）抗ヒトＣＤ８０モノクローナル抗体（Ｐｈａｒｍｉｎｇｅｎ製）と抗ヒトＣＤ８６モノクローナル抗体（Ｐｈａｒｍｉｎｇｅｎ製）との混合物。
（６）ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子（Ａｎｃｅｌｌ製）。
【０１８０】
前記＜８−１＞で得た各ドナーから調製したＰＢＭＣ及びＴ細胞を用いて、下記の６通りの組み合わせによる混合リンパ球反応（ＭＬＲ）を行った。
（ｉ） Ｔ細胞（ドナーＡ）／ＰＢＭＣ（ドナーＤ）
（ｉｉ） Ｔ細胞（ドナーＤ）／ＰＢＭＣ（ドナーＢ）
（ｉｉｉ）Ｔ細胞（ドナーＣ）／ＰＢＭＣ（ドナーＡ）
（ｉｖ） Ｔ細胞（ドナーＥ）／ＰＢＭＣ（ドナーＧ）
（ｖ） Ｔ細胞（ドナーＦ）／ＰＢＭＣ（ドナーＥ）
（ｖｉ） Ｔ細胞（ドナーＧ）／ＰＢＭＣ（ドナーＦ）
試験に用いるＰＢＭＣ及びＴ細胞は、下記の濃度に調整した。
ＰＢＭＣをＰＢＳ中に分散した後、培養皿（６０ｍｍ）移し、放射線照射装置（日立メディコ製）でＸ線照射（５０Ｇｙ）した。細胞を回収して遠心した後、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地に加え、細胞数を、２×１０^５細胞／５０μｌに調整した。
また、上記で得た各々のドナーからのＴ細胞を、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地に加え、細胞数を、１×１０^５細胞／５０μｌに調整した。
【０１８１】
＜８−２−１＞ ＭＬＲに対する抗ヒトＡＩＬＩＭヒトモノクローナル抗体によるＭＬＲの抑制
９６穴Ｕ底マイクロプレートのの各ウェルに１０％ＦＣＳ含有ＰＲＭＩ１６４０培地を加えた後、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地で各種濃度に希釈した抗ヒトＡＩＬＩＭヒトモノクローナル抗体または抗ヒトＡＩＬＩＭマウスモノクローナル抗体ＳＡ１２の溶液を加えた（最終濃度：０、０．３１、１．２５、５及び２０μｇ／ｍｌ）。次いで、Ｔ細胞（５０μｌ）を加え、ＣＯ_２インキュベーター（ＮＡＰＣＯ製）内で、３７℃で１時間培養した。反応終了後、別のドナー由来のＰＢＭＣ（５０μｌ）を加え、ＭＬＲを開始させた。
なお、被験物質として抗ヒトＡＩＬＩＭヒトモノクローナル抗体以外（上記（３）乃至（６））を用いた場合のＭＬＲは、ＰＢＭＣと該被験物質との培養の後に、別のドナー由来のＴ細胞を加えて反応を行った。
培養の５日目に、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地で希釈したトリチウム標識チミジン（^３Ｈ−Ｔｈｙｍｉｄｉｎｅ；２０μｌ；１μＣｉ／ｗｅｌｌ）を添加した後さらに１日培養して。培養後、Ｃｅｌｌ Ｈａｒｖｅｓｔｅｒ（Ｐａｃｋａｒｄ製）を用いて細胞を回収し、βカウンター（ＴＯＰ ＣＯＵＮＴ；Ｐａｃｋａｒｄ製）を用いて、細胞に取込まれている^３Ｈの放射活性を測定し、培養後のＴ細胞の増殖の程度を解析した。
結果を図４８乃至図５９に示した。
【０１８２】
＜８−２−２＞ 予めＣＴＬＡ４を介するシグナル伝達を遮断した系でのＭＬＲに対する抗ヒトＡＩＬＩＭヒトモノクローナル抗体によるＭＬＲの抑制
９６穴Ｕ底マイクロプレートの各ウェルに１０％ＦＣＳ含有ＰＲＭＩ１６４０培地を加えた後、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地で各種濃度に希釈した抗ヒトＡＩＬＩＭヒトモノクローナル抗体または抗ヒトＡＩＬＩＭマウスモノクローナル抗体ＳＡ１２の溶液を加えた（最終濃度：０、０．３１、１．２５、５及び２０μｇ／ｍｌ）。次いで、Ｔ細胞（５０μｌ）を加え、ＣＯ_２インキュベーター（ＮＡＰＣＯ製）内で、３７℃で１時間培養した。
該Ｔ細胞の培養とは別に、別のドナー由来のＰＢＭＣ（１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中）にヒトＣＴＬＡ４−ＩｇＦｃを加え、ＣＯ_２インキュベーター（ＮＡＰＣＯ製）内で、３７℃で１時間培養した。なお、該ＣＴＬＡ４−ＩｇＦｃの濃度は、ＭＬＲの開始の時点で２０μｇ／ｍｌとなるように調整した。
次いで、上記Ｔ細胞の培養系に、該ＰＢＭＣ（５０μｌ）を加え、ＭＬＲを開始させた。なお、被験物質として抗ヒトＡＩＬＩＭヒトモノクローナル抗体以外（上記（３）乃至（５））を用いた場合のＭＬＲは、上記と同様にしてＣＴＬＡ４−ＩｇＦｃとともに培養して得たＰＢＭＣと該被験物質との培養の後に、別のドナー由来のＴ細胞を加えて反応を行った。
培養の５日目に、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地で希釈したトリチウム標識チミジン（^３Ｈ−Ｔｈｙｍｉｄｉｎｅ；２０μｌ；１μＣｉ／ｗｅｌｌ）を添加した後さらに１日培養して。培養後、Ｃｅｌｌ Ｈａｒｖｅｓｔｅｒ（Ｐａｃｋａｒｄ製）を用いて細胞を回収し、βカウンター（ＴＯＰ ＣＯＵＮＴ；Ｐａｃｋａｒｄ製）を用いて、細胞に取込まれている^３Ｈの放射活性を測定し、培養後のＴ細胞の増殖の程度を解析した。
結果を図６０乃至図６９に示した。
【０１８３】
上記２つの試験の結果、下記が明らかとなった。
（１）ＣＴＬＡ４−ＩｇＦｃは、ＣＴＬＡ−４を介するシグナル伝達を遮断することによりアロジェニックＭＬＲによるＴ細胞の増殖が抑制する。
（２）抗ＣＤ８０抗体及び抗ＣＤ８６抗体は、ＣＴＬＡ４及びＣＤ２８のリガンドであるＣＤ８０／ＣＤ８６を介するシグナル伝達を阻害することによりアロジェニックＭＬＲによるＴ細胞の増殖を抑制される。
（３）ＣＴＬＡ４−ＩｇＦｃ、抗ＣＤ８０抗体及び抗ＣＤ８６抗体と同様に、ヒトＡＩＬＩＭに対するモノクローナル抗体が抗体濃度依存的に、ＡＩＬＩＭを介するシグナル伝達によるアロジェニックＭＬＲでのＴ細胞の増殖を有意に抑制する。
この結果は、即ち、Ｔ細胞の活性化に必要なコスティミュレイトリーシグナルの伝達経路には、既知のＣＴＬＡ４／ＣＤ８０／ＣＤ８６を介する経路及びＣＤ２８／ＣＤ８０／ＣＤ８６を介する経路の他に、ＡＩＬＩＭとそのリガンドを介する第３の経路が存在すること、並びに該ＡＩＬＩＭを介するシグナル伝達経路が、ＡＩＬＩＭに対する抗体により阻害されることを示すものである。
さらに、該シグナル伝達におけるＡＩＬＩＭを介する経路の貢献度は、ＣＴＬＡ４／ＣＤ８０／ＣＤ８６を介する経路及びＣＤ２８／ＣＤ８０／ＣＤ８６を介する経路のそれと同程度である可能性が示された。
【０１８４】
実施例９ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体の抗体依存性細胞性障害（ＡＤＣＣ）の誘導活性
抗体が引き起こす生物活性の一つに、抗体依存性細胞性障害（ａｎｔｉｂｏｄｙ−ｄｅｐｅｎｄｅｎｔ ｃｅｌｌｕｌａｒ ｃｙｔｏｔｏｘｉｃｉｔｙ；ＡＤＣＣ）の誘導がある。ＡＤＣＣとは、リンパ球、マクロファージあるいは多形核白血球のような効果細胞（ｅｆｆｅｃｔｏｒ ｃｅｌｌｓ）により引き起こされる標的細胞（ｔａｒｇｅｔ ｃｅｌｌ）の細胞障害作用において、該効果細胞と該標的細胞に加えて、その細胞障害が誘導されるために抗体を必要とする細胞障害作用である。
本発明の抗ヒトＡＩＬＩＭモノクローナル抗体のＡＤＣＣ誘導活性の有無を下記のようにして解析した。
【０１８５】
前記実施例で調製したヒトＡＩＬＩＭ高発現組換えＨＰＢ−ＡＬＬ細胞の培養系に、^５１Ｃｒ（０．１ｍＣｉ／１０^６細胞；Ａｍｅｒｓｈａｍ−Ｐｈａｒｍａｃｉａ製）を添加し、３７℃で２時間インキュベートした後、ＲＰＭＩ１６４０培地で８回洗浄して得た放射性標識細胞を、標的細胞（Ｔａｒｇｅｔ Ｃｅｌｌ）として用いた。
なお、対照試験での使用のために、野生型ヒトＨＰＢ−ＡＬＬ細胞を上記と同様にして放射性標識した。
健常人から採取した末梢血から、Ｌｙｍｐｈｏｓｅｐａｒ Ｉ（ＩＢＬ製）を用いて、ＰＢＭＣ画分を分離した。得られたヒトＰＢＭＣを効果細胞（Ｅｆｆｅｃｔｏｒ ｃｅｌｌ）として用いた。
９６穴Ｕ底マイクロプレート（Ｎｕｎｃ製）に、該標的細胞（１×１０^４ｃｅｌｌｓ／ｗｅｌｌ；２５μｌ／ｗｅｌｌ）を蒔いた。次いで、各ウェルに、５％ＦＢＳ含有ＲＰＭＩ１６４０培地で希釈した各種濃度の抗ヒトＡＩＬＩＭヒトモノクローナル抗体（０．０００１〜１．０μｇ／ｍｌ；２５μｌ／ｗｅｌｌ）、該培地のみ（２５μｌ／ｗｅｌｌ）または１％Ｎｏｎｉｄｅｔ Ｐ−４０（２５μｌ／ｗｅｌｌ；細胞膜溶解活性を有する界面活性剤）を加え、室温で２０分間培養した。
なお、陽性対照として、抗ヒトＡＩＬＩＭ抗体の代わりに抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（ＡＴＣＣ ＣＲＬ−８００１）を用いて上記と同様にして培養した。
【０１８６】
次いで、各ウェルに効果細胞（Ｅ／Ｔ ｒａｔｉｏ＝５０；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ；５０μｌ／ｗｅｌｌ）を加え、５％ＣＯ_２インキュベーター内で３７℃で１６時間培養した。
培養の後、遠心分離（１，５００ｒｐｍ；４℃；１０分）して、遠心上澄を回収した。該遠心上澄中の放射活性をガンマカウンターを用いて測定した。該放射活性は、ＡＤＣＣによる細胞膜の傷害により培養上清中に放出された^５１Ｃｒの量を示す。
培地のみでのアッセイでの放射活性を細胞膜障害率ゼロ（０）とし、またＮｏｎｉｄｅｔ添加でのアッセイでの方社活性を細胞膜障害率１００％とし、抗ＡＩＬＩＭ抗体または抗ＣＤ３抗体により誘導されるＡＤＣＣによる細胞膜障害率（細胞溶解率）（％）を算出した。
結果を図７０及び図７１に示す。
本試験の結果、本発明の抗ヒトＡＩＬＩＭヒトモノクローナル抗体は、濃度依存的なＡＤＣＣ誘導活性を有していた。
【０１８７】
実施例１０ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体の遺伝子配列及びアミノ酸配列の決定及び解析
前記実施例で作製された種々のヒトＡＩＬＩＭに対するヒトモノクローナル抗体を構成する重鎖（Ｈｅａｖｙ Ｃｈａｉｎ）コードするｃＤＮＡ配列、並びに軽鎖（Ｌｉｇｈｔ Ｃｈａｉｎ）をコードするｃＤＮＡ配列を下記のようにして決定するとともに、該遺伝子の構造的特徴を解析した。
前記実施例で作製したヒトＡＩＬＩＭに対するヒトモノクローナル抗体を産生するハイブリドーマ（クローン：ＡＩＨ５Ｄ３（ＪＭａｂ−１３６）， ＡＩＩ２８９（ＪＭａｂ−１３８）及びＡＩＩ３９４（ＪＭａｂ−１３９））の各々から、Ｑｕｉｃｋ Ｐｒｅｐ ｍＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を用いてＰｏｌｙＡ^＋ＲＮＡの抽出、精製した。
該ハイブリドーマを、細胞溶解緩衝液（Ｌｙｓｉｓ Ｂｕｆｆｅｒ）に溶解し、シリンジにより細胞を破壊し、可溶化させた。該可溶化物にＯｌｉｇｏ（ｄＴ） ｒｅｓｉｎを加え緩やかに振盪した。次いで、Ｏｌｉｇｏ（ｄＴ） ｒｅｓｉｎを洗浄後、ＰｏｌｙＡ^＋ＲＮＡをＥｌｌｕｔｉｏｎ Ｂｕｆｆｅｒで溶出させた。溶出したＰｏｌｙＡ^＋ＲＮＡをエタノール沈殿させ、Ｔｒｉｓ−ＥＤＴＡ緩衝液に溶解した。得られたＰｏｌｙＡ^＋ＲＮＡの濃度を、２６０ｎｍの波長での吸光度を測定することにより決定した。
【０１８８】
得られたＰｏｌｙＡ^＋ＲＮＡを鋳型とし、市販のｃＤＮＡ ｓｙｎｔｈｅｓｉｓ キット（ＧＩＢＣＯＢＲＬ製）および合成オリゴＤＮＡ ＮｏｔＩ−Ｔ（配列番号１）をプライマーとして用いたＭ−ＭＬＶ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ法により二本鎖ｃＤＮＡを合成した。
即ち、ハイブリドーマから精製したＰｏｌｙＡ^＋ＲＮＡ（約５μｇ）を鋳型として、該プライマー（約４００ｐｍｏｌｅ）及びＭ−ＭＬＶ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅを加えた溶液中（約５０μｌ）で３７℃で１時間反応させ一本鎖ｃＤＮＡを合成した。次いで、反応溶液（４μｌ）に、ｄＮＴＰ、ＤＮＡ ｐｏｌｙｍｅｒａｓｅ Ｉ、ＲＮａｓｅＨ、ＤＮＡ ｌｉｇａｓｅ、緩衝液及び蒸留水を加えて１６℃で２時間反応させ二本鎖ｃＤＮＡを合成した。得られた二本鎖ｃＤＮＡを、フェノール／クロロホルムで抽出した後、エタノール沈殿させた。
次いで、ＴＥ緩衝液に溶解した該二本鎖ｃＤＮＡ（約５０μｌ）に、ＥｃｏＲＩリンカーＤＮＡ（約３００ｐｍｏｌｅ）及びＤＮＡリガーゼ（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ；３３μｌ；ＴＯＹＯＢＯ製）を加え、１６℃で約８０分反応させて、該ｃＤＮＡにリンカーＤＮＡを連結した。なお、該リンカーＤＮＡは、５’末端をリン酸化したオリゴＤＮＡ（２０ａｄｐ；配列番号２）とオリゴＤＮＡ（２４ａｄｐ；配列番号３）を常法に従ってハイブリダイズさせて調製した二本鎖ＤＮＡを用いた。
【０１８９】
得られた反応物をフェノール／クロロホルムを用いて抽出した後、エタノール沈殿させた。次いで、該ＤＮＡ反応物を、市販の制限酵素ＮｏｔＩ（ＴＯＹＯＢＯ製）を用いて消化した後、市販のＡＴＰ溶液（ＧＩＢＣＯ ＢＲＬ製）とＴ４キナーゼ（ＴＯＹＯＢＯ製）と反応させ（３７℃で３０分）その５’末端をリン酸化した。
得られたＤＮＡ反応物エタノール沈殿した後、ポリアクリルアミドゲル電気泳動で分画し約５００ｂｐから２０００ｂｐのＤＮＡを含むゲルを切り出した。該ゲルの切り出しは、エチジウムブロマイド染色及び紫外線照射下での写真撮影により行った。
切り出したゲルを、粉砕した後ＴＥ緩衝液に懸濁させ、遠心分離して遠心上澄を回収した。
回収した該ＤＮＡと市販のラムダファージベクターλＥＸｃｅｌｌ（０．２５μｇ；ＡｍｅｒｓｈａｍＰｈａｒｍａｃｉａ製）を、市販のＤＮＡ Ｌｉｇａｓｅ（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ；ＴＯＹＯＢＯ製）の存在下で反応させ（１６℃、３０分）連結させた。次に該ＤＮＡ反応物を市販のラムダファージパッケージングキットＧｉｇａｐａｃｋ ＩＩＩ Ｇｏｌｄ（ＳＴＲＡＴＡＧＥＮＥ製）を用いてラムダファージとし、大腸菌ＮＭ５２２を宿主としてｃＤＮＡライブラリーを作製した。なお、全ての操作は、該キットに添付の実験操作説明書に従って行った。
【０１９０】
次いで、プラークハイブリダイゼーション法（マニアティス（Ｍａｎｉａｔｉｓ）ら、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｌａｔｏｒｙ Ｍａｎｕａｌ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ， Ｎｅｗ Ｙｏｒｋ）に従い、該ｃＤＮＡライブラリーのスクリーニングを下記のようにして行った。
該ｃＤＮＡライブラリー（１×１０^４個プラーク）を寒天プレートに蒔き、ハイボンド−Ｎ ナイロンメンブラン（Ｈｙｂｏｎｄ−Ｎ ｎｙｌｏｎ ｍｅｎｂｒａｎｅ、Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を用いてレプリカを作製した。このレプリカを、γ^３２Ｐ−ＡＴＰで標識したプローブを用いて、ハイブリダイゼーション緩衝液中でのプラークハイブリダイゼーションによりスクリーニングした。抗体軽鎖については、プローブＨＩＧＬＣ（配列番号４）を、また抗体重鎖については、プローブＮＨＣｃ２（配列番号５）を用いた。１次スクリーニング及び２次スクリーニングにより同定した陽性クローンの各々をシングルプラークで単離した。
【０１９１】
各々の陽性クローンのファージ溶液を鋳型ＤＮＡとし、一つのＰＣＲ用プライマー及びＴａｑ ＰＣＲキット（ＴＡＫＡＲＡ製）を用いたＰＣＲにより抗体重鎖及び抗体軽鎖の各々を増幅した。抗体軽鎖のプライマーには、ＥｘｃｅｌｌＥ（配列番号６）及びｃｋ１１７（配列番号７）を、抗体重鎖のプライマーにはＥｘｃｅｌｌＥ（配列番号６）及びＮＨＣｃ２（配列番号５）を用いた。ＰＣＲ産物は常法によりアガロースゲル電気泳動にて分画し重鎖及び軽鎖ともに約６００ｂｐの大きさのＤＮＡを含むゲルを切り出した。該ゲルから精製したＤＮＡの塩基配列を、ＤＮＡ Ｓｅｑｕｅｎｃｅｒ（３７３Ａ；ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）、ＡＢＩ ＰＲＩＳＭ Ｓｅｑｕｅｎｃｉｎｇ Ｓｏｆｔｗａｒｅ（ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）及びＡＢＩ ＰＲＩＳＭ Ａｕｔｏ Ａｓｓｅｍｂｌｅｒ（ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）を用いて解析し、各々の陽性クローンが十分な塩基長のＤＮＡを有することを確認した。
【０１９２】
各々の陽性クローンのプラークのλファージを、大腸菌ＮＰ６６に感染させ、プラスミドＤＮＡを放出させた後、アンピシリンを含有するプレート上に蒔きコロニーを形成させた。次いで、該コロニーから常法に従って回収した精製プラスミドＤＮＡで大腸菌ＪＭ１０９を形質転換した。次いで、形質転換細胞をアンピシリンを含む栄養培地に蒔きコロニーを形成させた。
次いで、アンピシリンを含むＬＢ培地中に懸濁させた各々のコロニーの懸濁液を、液体栄養培地中で３７℃で２４時間培養した。培養液から菌体を集め、プラスミド精製キット（Ｑｕｉａｇｅｎ製）を用いてプラスミドＤＮＡを精製した。各々の精製プラスミドＤＮＡを制限酵素ＥｃｏＲＩ／ＮｏｔＩで消化し、ベクターＤＮＡと挿入ＤＮＡ（重鎖ｃＤＮＡまたは軽鎖ｃＤＮＡ）の存在を確認した。
各々の精製プラスミドに挿入されている抗体重鎖または抗体軽鎖の各々をコードするｃＤＮＡの塩基配列を、常法に従って、ＤＮＡ Ｓｅｑｕｅｎｃｅｒ（３７７Ａ；ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）、ＡＢＩ ＰＲＩＳＭ Ｓｅｑｕｅｎｃｉｎｇ Ｓｏｆｔｗａｒｅ（ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）及びＡＢＩ ＰＲＩＳＭ Ａｕｔｏ Ａｓｓｅｍｂｌｅｒ（ＰＥ−Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）を用いて決定した。
なお、本配列決定においては、下記のプライマーを使用した。
【０１９３】
＜重鎖ｃＤＮＡの配列解析に用いたプライマー＞
Ｍ１３Ｒプライマー（配列番号８；ＳＴＲＡＴＡＧＥＮＥ製）、ＥｘｃｅｌｌＥ（配列番号６）、１３６Ｈ（配列番号９）、１３８／９Ｈ（配列番号１０）、ＡＩＬＩＭＨＣ１（配列番号１１）、ＨＣｃ１（配列番号１２）、ＮＨＣｃ２（配列番号５）、ＨＣｃ７（配列番号１３）、ＨＣｃ８（配列番号１４）、ＨＣｃ３（配列番号１５）、ＨＣｃ４（配列番号１６）、ＨＣｃ６（配列番号１７）、ＨＩＧＨＣ（配列番号１８）、ＨＣｃ９（配列番号１９）、ＨＣｃ５（配列番号２０）及びｐｏｌｙＡ（配列番号２１）。
＜軽鎖ｃＤＮＡの配列解析に用いたプライマー＞
Ｍ１３Ｒプライマー（配列番号８；ＳＴＲＡＴＡＧＥＮＥ製）、ＥｘｃｅｌｌＥ（配列番号６）、ＡＩＬＩＭＬＣ１（配列番号２２）、ＡＩＬＩＭＬＣ２（配列番号２３）、ＬＣｃ１（配列番号２４）、ｃｋ１１７（配列番号７）、ＨＩＧＬＣ（配列番号４）、ＬＣｃ２（配列番号２５）、ＨＩＫ（配列番号２６）、及びｐｏｌｙＡ（配列番号２１）。
前記の各々のハイブリドーマが産生するヒトＡＩＬＩＭに対するヒトモノクローナル抗体の重鎖をコードするｃＤＮＡ配列、軽鎖（Ｌｉｇｈｔ Ｃｈａｉｎ）をコードするｃＤＮＡ配列、並びに該各々のｃＤＮＡ配列から演繹されるアミノ酸配列を下記のとおり配列表に示した。
【０１９４】
クローンＡＩＨ５Ｄ３（ＪＭａｂ−１３６）
＜重鎖＞
ＤＮＡ配列 ：配列番号２７（シグナル配列：塩基番号６９乃至１２５、Ｖ領域：塩基番号１２６乃至４１９）
アミノ酸配列：配列番号２８（シグナル配列：アミノ酸番号１乃至１９、可変領域：アミノ酸番号２０乃至１１８を含む。）
＜軽鎖＞
ＤＮＡ配列 ：配列番号２９（シグナル配列：塩基番号３９乃至１０４、Ｖ領域：塩基番号１０５乃至３８６）
アミノ酸配列：配列番号３０（シグナル配列：アミノ酸番号１乃至２２、可変領域：アミノ酸番号２３乃至１１６を含む）
【０１９５】
クローンＡＩＩ２８９（ＪＭａｂ−１３８）
＜重鎖＞
ＤＮＡ配列 ：配列番号３１（シグナル配列：塩基番号９４乃至１５０、Ｖ領域：塩基番号１５１乃至４４１を含む。）
アミノ酸配列：配列番号３２（シグナル配列：アミノ酸番号１乃至１９、可変領域：アミノ酸番号２０乃至１１６を含む。）
＜軽鎖＞
ＤＮＡ配列 ：配列番号３３（シグナル配列：塩基番号２８乃至８７、Ｖ領域：塩基番号８８乃至３７５を含む。）
アミノ酸配列：配列番号３４（シグナル配列：アミノ酸番号１乃至２０、可変領域：アミノ酸番号２１乃至１１６を含む）
【０１９６】
クローンＡＩＩ３９４（ＪＭａｂ−１３９）
＜重鎖＞
ＤＮＡ配列 ：配列番号３５（シグナル配列：塩基番号９６乃至１５２、Ｖ領域：塩基番号１５３乃至４４３）
アミノ酸配列：配列番号３６（シグナル配列：アミノ酸番号１乃至１９、可変領域：アミノ酸番号２０乃至１１６を含む。）
＜軽鎖＞
ＤＮＡ配列 ：配列番号３７（シグナル配列：塩基番号３３乃至９２、Ｖ領域：塩基番号９３乃至３８０）
アミノ酸配列：配列番号３８（シグナル配列：アミノ酸番号１乃至２０、可変領域：アミノ酸番号２１乃至１１６を含む。）
【０１９７】
決定された各々のＤＮＡ配列を基に、遺伝子配列解析ソフトウェアーを用いて、Ｔｏｍｌｉｎｓｏｎらにより作成されたヒトイムノグロブリンの可変領域遺伝子のライブラリーＶ ＢＡＳＥ Ｓｅｑｕｅｎｃｅ（Ｉｍｍｕｎｏｌ． Ｔｏｄａｙ， Ｖｏｌ．１６， Ｎｏ．５， ｐ．２３７−２４２， １９９５）を検索した。
その結果、上記ヒトモノクローナル抗体の重鎖及び軽鎖の各々のＶ領域遺伝子は、各々下記のセグメントから構成されていた。
【０１９８】
＜重鎖Ｖ領域遺伝子＞
クローンＡＩＨ５Ｄ３（ＪＭａｂ−１３６）： １−０２
クローンＡＩＩ２８９（ＪＭａｂ−１３８）： ３−１３
クローンＡＩＩ３９４（ＪＭａｂ−１３９）： ３−１３
＜軽鎖Ｖ領域遺伝子＞
クローンＡＩＨ５Ｄ３（ＪＭａｂ−１３６）： Ｌ５
クローンＡＩＩ２８９（ＪＭａｂ−１３８）： Ａ２７
クローンＡＩＩ３９４（ＪＭａｂ−１３９）： Ａ２７
【０１９９】
実施例１１ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体の遅延型過敏症（ｄｅｌａｙｅｄ−ｔｙｐｅ ｈｙｐｅｒｓｅｎｓｉｔｉｖｉｔｙ；ＤＴＨ）の抑制効果
生体にとって有害な抗原（ウイルス、細菌及び寄生虫などの病原微生物や外来異物など）を排除しようとする生体の免疫応答システムは、自然免疫応答と獲得免疫応答（後天性免疫）に大別される。
前者は、食細胞（多形核白血球、単球、マクロファージなど）による貪食、ナチュラルキラー（ＮＫ）細胞による攻撃、及び補体による抗原のオプソニン化などのような非特異的な認識による排除機構である。
後者の獲得免疫応答は、該抗原に対する特異性を獲得（活性化）したリンパ球（主にＴ細胞、Ｂ細胞）による排除機構である。
獲得免疫応答は、細胞性免疫と体液性免疫にさらに大別される。
細胞性免疫は、抗体依存性の体液性免疫とは異なり、主としてＴ細胞が直接標的である抗原に作用して発現される免疫反応である。細胞性免疫は、ウイルスや腫瘍に対する免疫反応、組織や臓器の移植後に惹起される免疫反応、ある種の薬物アレルギー、及び一部の自己免疫疾患などに関与していることが分かっている。
【０２００】
この細胞性免疫の最も代表的な現象として、ツベルクリンアレルギー（ツベルクリン過敏症とほぼ同義）がよく知られている。ツベルクリンアレルギーは、結核菌の感染にともなって成立する遅延型アレルギーであり、結核菌の培養液中に生成されるツベルクリン蛋白質を生体の皮内に注射して免疫反応を惹起することにより誘導される。
遅延型アレルギーは、抗原により感作されたＴ細胞（抗原を記憶したメモリーＴ細胞）により仲介されるアレルギーであり、抗原に感作された生体は同抗原と再接触した際に起こる当該メモリーＴ細胞による炎症を伴うアレルギー反応に発現に２４乃至４８時間を要することから遅延型と呼ばれている。
遅延型アレルギーの代表例であるツベルクリンアレルギーの現象は、動物が結核菌による感作が成立しているか否かの有無の診断のための「ツベルクリンテスト」に普遍的に応用されている。即ち、結核菌の培養液から精製したツベルクリン蛋白質である精製ツベルクリン（ｐｕｒｉｆｉｅｄ ｐｒｏｔｅｉｎ ｄｅｒｉｖａｔｉｖｅ； ＰＰＤ；一般診断においては、０．０５μｇ／０．１ｍｌ（２．５ＴＵ）を０．１ｍｌ））を動物の皮内に投与し、投与後４８時間に投与部位の皮膚の発赤の長径を測定してその長径により結核菌の感染の有無を診断する方法である。発赤の長径が４ｍｍまでを陰性、４〜９ｍｍを疑陽性、及び１０ｍｍ以上を陽性としている。
【０２０１】
細胞性免疫により惹起される遅延型アレルギーとしては、上記の結核菌由来ツベルクリンアレルギーのような感染病原体抗原に対するアレルギーの他に、微量の蛋白質などに対して一過性に出現するジョーンズ・モート型遅延型アレルギー、塩化ピクリルなどの薬物や漆などの植物毒に対する接触性アレルギー、あるいは同種移植片の移植で見られる移植拒絶に関与するアレルギーなどが挙げられる。
【０２０２】
本試験においては、上述のツベルクリンテストを応用して、抗ＡＩＬＩＭ抗体の遅延型過敏症（遅延型アレルギー）の抑制効果を評価した。試験は下記のようにして行った。
ウシ型結核菌の弱毒生菌であるＢＣＧ（Ｂａｃｉｌｌｅ ｄｅ Ｃａｌｍｅｔｔｅ ｅｔ Ｇｕｅｒｉｎ）の免疫感作が成立しているのカニクイザル（雄、体重：６．０乃至８．５ｋｇ、環境バイリス研究所製）の各々（各群３匹）を塩酸ケタミン（１０ｍｇ／ｋｇ、筋肉内投与）で麻酔した後、下記のいずれかの試験試料を、０．１ｍｌ／１投与部位（６投与部位／匹）の濃度で胸部表皮内に投与した。なお、試料を投与する各部位の間隔を３ｃｍ以上離した。
【０２０３】
（１）抗ヒトＡＩＬＩＭヒトモノクローナル抗体（ＪＭａｂ−１３６；０．２ｍｇ；１０μｇ／１投与部位）とツベルクリン（４μｇ／生理食塩水１ｍｌ）の１：１の混合溶液。
（２）抗ヒトＡＩＬＩＭヒトモノクローナル抗体（ＪＭａｂ−１３６；２ｍｇ；１００μｇ／１投与部位）とツベルクリン（４μｇ／生理食塩水１ｍｌ）の１：１の混合溶液。
（３）対照としてのリン酸緩衝液（ＰＢＳ（−））。
（４）陽性対照としての市販のステロイド抗炎症剤プレドニゾロン（Ｐｒｅｄｎｉｓｏｌｏｎｅ；０．２ｍｇ；１０μｇ／１投与部位）とツベルクリン（４μｇ／生理食塩水１ｍｌ）の１：１の混合溶液。
（５）陰性対照としての抗ＫＬＨヒトモノクローナル抗体（実施例＜２−２＞；０．２ｍｇ；１０μｇ／１投与部位）とツベルクリン（４μｇ／生理食塩水１ｍｌ）の１：１の混合溶液。。
各試料の投与から２４時間後に各投与部位の発赤の長径及び短径を測定し発赤の面積を算出した。
結果を図７２に示した。
この結果、抗ＡＩＬＩＭ抗体を投与したいずれの群においても、対照及び陰性対照に比べ、遅延型アレルギーによる発赤が有意に抑制された。また、その抑制効果は、陽性対照であるステロイド性抗炎症剤と同程度であった。
【０２０４】
実施例１２ 移植片対宿主病（ＧＶＨＤ）患者の各組織でのＡＩＬＩＭの発現の解析
ドナーから同種移植を受けた後に臨床的に急性または慢性の移植片対宿主病（ｇｒａｆｔ ｖｅｒｓｕｓ ｈｏｓｔ ｄｉｓｅａｓｅ；ＧＶＨＤ）と診断されたレシピエントである患者から採取した種々の生検組織でのＡＩＬＩＭの発現の有無を、常法に従って前記実施例で作製した抗ヒトＡＩＬＩＭヒトモノクローナル抗体（ＪＭａｂ−３６）を用いたＨＥ染色法により組織染色することにより分析した。
急性ＧＶＨＤ患者（２８例）の種々組織から採取した３３検体並びに慢性ＧＶＨＤ患者（５例）より５検体について解析した。
その結果、急性ＧＶＨＤ患者については、皮膚組織では２９検体中の１５検体がＡＩＬＩＭ陽性、胃組織では３検体中１検体がＡＩＬＩＭ陽性、また結腸組織では１検体中１検体がＡＩＬＩＭ陽性であった。慢性ＧＶＨＤ患者においては、皮膚組織では３検体中１検体がＡＩＬＩＭ陽性であり、また結腸組織では２検体中２検体がＡＩＬＩＭ陽性であった。さらに、リンパ球細胞の浸潤が著明な検体では、１３検体中１０検体がＡＩＬＩＭ陽性であった。
【０２０５】
実施例１３ 抗ヒトＡＩＬＩＭヒトモノクローナル抗体のサルＴ細胞に対するコスティミュレイトリーシグナル伝達活性
実施例７では、本発明の抗ヒトＡＩＬＩＭヒトモノクローナル抗体が、ヒトＴ細胞反応の制御能、具体的にはＡＩＬＩＭを介したコスティミュレイトリーシグナルの細胞内への伝達を制御することによるヒトＴ細胞を増殖促進させることを実証した。そこで、、本試験においては該ヒトモノクローナル抗体のサルのＴ細胞に対する細胞増殖促進能の有無を実施例７と同様にして解析した。
【０２０６】
＜１３−１＞ 抗体の希釈
抗ヒトＣＤ３モノクローナル抗体ＳＰ３４（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）を、リン酸緩衝液（ＰＢＳ）で最終濃度１μｇ／ｍｌとなるように希釈した。
前記で調製した抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭＡｂ１３６を、ＰＢＳで最終濃度が４０μｇ／ｍｌとなるように希釈した後、ＰＢＳでさらに各種濃度に希釈した（４０μｇ／ｍｌ乃至０．０６４μｇ／ｍｌ）。
【０２０７】
＜１３−２＞ 抗体によりマイクロプレートのコーティング
９６ウェルマイクロプレート（複数枚）の各ウェルに、抗ヒトＣＤ３モノクローナル抗体ＳＰ３４（１μｇ／ｍｌを２５μｌ／ｗｅｌｌ）及び抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭＡｂ１３６（４０μｇ／ｍｌ乃至０．０６４μｇ／ｍｌを２５μｌ）、３７℃で２時間インキュベーションした。次いで、抗体を捨て、各ウェルをＰＢＳで３回洗浄した。洗浄後、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（１００μｌ／ｗｅｌｌ）を加え、３７℃で１時間インキュベーションして、上記抗体でプレートの各ウェルをコーティングした。
なお、対照抗体として、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに、抗ＫＬＨヒトモノクローナル抗体（ＪＭａｂ２３とも別称する。前記実施例）を用いて、上記と同様にしてプレートのコーティングを行った。
これらの抗体コーティングマイクロプレートを以下の試験で用いた。
【０２０８】
＜１３−３＞ サルＴ細胞懸濁液の調製
カニクイザルの末梢血を採取し、ＮｙｃｏＰｒｅｐ１．０７７Ａ（Ｎｙｃｏｍｅｄ製）を用いた密度勾配遠心により単核球を分画した。実験操作マニュアルに従い、抗ヒトＣＤ４抗体Ｍ−Ｔ４７７（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）、抗ヒトＣＤ８抗体ＲＰＡ−Ｔ８（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）、抗マウスＩｇＧマイクロビーズ（Ｍｉｌｔｅｎｙｉ製）及びマグネティックソーターを用いて、該カニクイサル単核球からサルＴ細胞を分離取得した。該Ｔ細胞の細胞数を、血球計算盤により計数した。該サルＴ細胞を、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中に懸濁しサルＴ細胞懸濁液（１×１０^６細胞／ｍｌ）を調製した。
【０２０９】
＜１３−４＞ 細胞培養
（１）抗ヒトＣＤ３抗体と抗ヒトＡＩＬＩＭ抗体をコートしたマイクロプレートでの培養
前記の抗体コーティングマイクロプレートの各ウェルに、サルＴ細胞懸濁液（１００μｌ／ｗｅｌｌ；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ）を加え、ＣＯ_２インキュベーター内で３７℃で２日間培養した。
培養後、各々のマイクロプレートを以下の試験に用いた。
【０２１０】
＜１３−５＞ Ｔ細胞増殖活性の測定
前記の培養後の各プレートの各ウェルに、ｍｅｔｈｙｌ［^３Ｈ］ｔｈｙｍｉｄｉｎｅ（０．５μＣｉ／ｗｅｌｌ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を加え、ＣＯ_２インキュベーター内で３７℃で６時間インキュベーションした。培養後、セルハーベスターを用いて、細胞をＧＦ／Ｃフィルター（Ｐａｃｋａｒｄ製）上にトラップした。次いで、フィルターを、４０℃で３時間以上乾燥させた後、Ｍｉｃｒｏｓｃｉｎｔｉ ０（２０μｌ／ｗｅｌｌ；Ｐａｃｋａｒｄ製）を加えた。βカウンター（ＴＯＰ ＣＯＵＮＴ）を用いて、フィルター上にトラップされた細胞に取込まれている^３Ｈの放射活性を測定し、培養後のＴ細胞の増殖の程度を解析した。
結果を図７３に示す。
本試験の結果、抗ヒトＡＩＬＩＭモノクローナル抗体（ヒトモノクローナル抗体またはマウスモノクローナル抗体）を抗ヒトＣＤ３抗体とともにコートしたマイクロプレートを用いた場合には、サルＴ細胞の濃度依存的な有意な増殖が認められた。
この結果は、また本発明の抗ヒトＡＩＬＩＭヒトモノクローナル抗体が、サルのＡＩＬＩＭにも結合しサルのＡＩＬＩＭの機能を制御する活性を有することを示すものである。
【０２１１】
実施例１４ ＡＩＬＩＭまたはＡＩＬＩＭリガンドに結合する物質を同定、定量する方法の構築
ＡＩＬＩＭ（ＩＣＯＳ）に結合する物質あるいはＡＩＬＩＭリガンド（Ｂ７ｈ／Ｂ７ＲＰ１／ＧＬ５０／ＬＩＣＯＳ）を同定または定量可能なＥＬＩＳＡ（Ｅｎｚｙｍｅ−ｌｉｎｋｅｄ Ｉｍｍｕｎｏ ｓｏｌｖｅｎｔ ａｓｓａｙ）を構築した。
下記に一例として詳述する方法は、当該物質が可溶性ヒトＡＩＬＩＭ（ｈＡＩＬＩＭ−ＩｇＦｃ）と可溶性ヒトＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）の結合を阻害する程度をＥＬＩＳＡにより評価することを原理とする。
【０２１２】
＜１４−１＞ 試料
以下の試料を用いた。
（１）Ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ（Ｓｏｕｔｈｅｒｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ａｓｓｏｃｉａｔｅｓ，Ｉｎｃ．製）。
（２）可溶性ヒトＡＩＬＩＭリガンド（ヒトＢ７ｈの細胞外領域とヒトＩｇＧ１の定常領域とからなる融合タンパク）
次項＜１４−２＞のようにして作製した。
（３）ビオチン標識可溶性ＡＩＬＩＭ−ＩｇＦｃ
本発明者の一人である手塚の他の先の出願（日本国特許出願公開１１−２９５９９号公報（実施例１６（２））及び国際特許出願公開ＷＯ９８／３８２１６号公報（実施例１６（２））の方法により調製したものをさらに精製することにより調製した。
（４）抗ヒトＡＩＬＩＭヒトモノクローナル抗体（ＪＭａｂ−１３６；上記）。
（５）抗ＫＬＨヒトモノクローナル抗体（陰性対照抗体；ＪＭａｂ−２３；上記）。
（６）リン酸緩衝液（ＰＢＳ（−）；日研生物製）。
（７）ＰＲＭＩ１６４０培地（日研生物製）。
（８）牛胎児血清（ＦＣＳ；ＪＲＨ−Ｂｉｏｓｃｉｅｎｃｅ製）。
（９）３０％牛血清アルブミン（ＢＳＡ；Ｓｉｇｍａ製）。
（１０）Ｔｗｅｅｎ２０（ＢｉｏＲａｄ製）。
（１１）ＴＭＢ^＋ ｓｕｂｓｔｒａｔｅ ｃｈｒｏｍｏｇｅｎ （Ｄａｋｏ製）。
【０２１３】
＜１４−２＞ 可溶性ヒトＡＩＬＩＭリガンド（ヒトＢ７ｈの細胞外領域とヒトＩｇＧ１の定常領域との融合蛋白（ｈＢ７ｈ−ＩｇＦｃ））の調製
前述の実施例と同様にしてヒト末梢血由来の単核球からｔｏｔａｌ ＲＮＡを調製した。
調製したｔｏｔａｌ ＲＮＡ（５μｇ）を鋳型とし、Ｓｕｐｅｒｓｃｒｉｐｔ Ｐｒｅａｍｐｌｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ ｆｏｒ Ｆｉｒｓｔ Ｓｔｒａｎｄ ｃＤＮＡ Ｓｙｔｈｅｓｉｓ （ＧＩＢＣＯ−ＢＲＬ製）を用いてｃＤＮＡを合成した。次いで、ヒトＡＩＬＩＭリガンド（ｈＢ７ｈ）の細胞外領域をコードするｃＤＮＡをＰＣＲで増幅するために、末端にＸｈｏＩ切断部位を有する５’プライマー（５’−ＧＡＧＧＴＣＴＣＣＧＣＣＣＴＣＧＡＧＡＴＧＣＧＧＣＴＧＧＧＣＡＧＴＣＣ−３’、配列番号３９）及びＢａｍＨＩ切断部位を有する３’プライマー（５’−ＣＡＣＡＧＧＡＣＡＧＣＣＡＧＧＧＧＡＴＣＣＣＡＣＧＴＧＧＣＣＧＣＧ−３’、配列番号４０）を設計、合成した。得られたｃＤＮＡを鋳型として該プライマーを用いてＰＣＲを行い、両端にＸｈｏＩ及びＢａｍＨＩ切断部位を各々有するヒトＢ７ｈの細胞外領域をコードするｃＤＮＡを含むｃＤＮＡを調製した。得られたＰＣＲ産物をＸｈｏＩ及びＢａｍＨＩで消化し、アガロースゲル電気泳動し、目的の細胞外領域をコードするｃＤＮＡ断片と予測される約７２０ｂｐのサイズのバンドを単離した。単離されたｃＤＮＡ断片を、ＸｈｏＩ及びＢａｍＨＩで切断したプラスミドｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）（Ｓｔｒａｔａｇｅｎｅ製）中にサブクローニングした。自動蛍光ＤＮＡシークエンサー（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製）による配列解析により、該ｃＤＮＡ断片がヒトＢ７ｈの細胞外領域をコードする領域を含むことを確認した。
【０２１４】
一方、融合パートナーとしてのヒトＩｇＧ１のＦｃをコードするＤＮＡは、プラスミ ド（Ｃｅｌｌ， Ｖｏｌ．６１， ｐ．１３０３−１３１３， １９９０参照。マサチューセッツ・ゼネラル・ホ スピタルのシード博士（Ｂ． Ｓｅｅｄ）らにより作製）を、ＢａｍＨＩ及びＸｂａＩで消化することによりＢａｍＨＩ−ＸｂａＩＤＮＡ断片（約１．３ｋｂ）として切り出した。この断片には、ヒトＩｇＧ１のヒンジ領域、Ｃγ１２、及びＣγ１３の各々コードするエクソンが含まれる。
前記のようにして作製したヒトＢ７ｈ（ｈＢ７ｈ）の細胞外領域をコードするＸｈｏＩ−ＢａｍＨＩ断片、及びヒトＩｇＧ１のＦｃ（「ＩｇＦｃ」と略記する）をコードするエクソンを含むＢａｍＨＩ−ＸｂａＩ断片を、ＸｈｏＩ及びＸｂａＩで切断したプラスミドｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）（Ｓｔｒａｔａｇｅｎｅ製）中にサブクローニングした。
次いで、該プラスミドをＸｈｏＩ及びＸｂａＩで消化し、ヒトＢ７ｈの細胞外領域とヒトＩｇＦｃとからなる融合ＤＮＡを含む約１．８ｋｂのＤＮＡ断片を切りだした。この融合ＤＮＡ断片を、Ｔ４ ＤＮＡリガーゼを用いて、発現ベクターｐＭＥ１８Ｓ（Ｍｅｄｉｃａｌ Ｉｍｍｕｎｏｌｏｇｙ， Ｖｏｌ．２０， Ｎｏ．１， ｐ．２７−３２， １９９０、及び実験医学（別冊）：「遺伝子工学ハンドブック」、羊土社、第１０１−１０７頁、１９９２年）のＸｈｏＩ及びＸｂａＩ部位に挿入し、プラスミドｐｈＢ７ｈ−ＩｇＦｃを構築した。
【０２１５】
エレクトロポレーション法により、１０％ウシ胎児血清及びアンピシリンを含むＤＭＥＭ培地中でサブコンフルエントに単層培養したＣＯＳ７細胞（ＡＴＣＣ ＣＲＬ−１６５１）を、プラスミドｐｈＢ７ｈ−ＩｇＦｃで形質転換し、形質転換細胞を取得した。
形質転換細胞を、無血清ＡＳＦ１０４培地中で７２時間培養することによりｈＢ７ｈ−ＩｇＦｃを発現させた。
ＨＢ７ｈ−ＩｇＦｃは、Ｐｒｏｔｅｉｎ Ｇ Ｓｅｐｈａｒｏｓｅアフィニティ−カラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製） を用いて次のように精製した。
前記培養上清を遠心分離して得た遠心上清を、予め結合緩衝液（ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ）で平衡化したＰｒｏｔｅｉｎ Ｇ Ｓｅｐｈａｒｏｓｅアフィニティーカラムに加えた。次いで、カラムを結合緩衝液で洗浄した後、溶出緩衝液（ｅｌｕｔｉｏｎ ｂｕｆｆｅｒ）で溶出させた。溶出液を回収し、リン酸緩衝液で２回以上外液交換することにより透析し、精製ｈＢ７ｈ−ＩｇＦｃを得た。
【０２１６】
＜１４−３＞ 抗体及び可溶性ヒトＡＩＬＩＭ（ｈＡＩＬＩＭ−ＩｇＦｃ）の希釈及び、それらの反応抗ヒトＡＩＬＩＭモノクローナル抗体（ＪＭａｂ−１３６）及び陰性対照抗体である抗ＫＬＨヒトモノクローナル抗体（ＪＭａｂ−２３）は各々、２０μｇ／ｍｌの溶液を１１段階に希釈した後、各サンプル２００μｌに１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（２００μｌ）を加えて混和し、種々濃度の溶液を調製した。調製した種々濃度の溶液の各々に、ビオチン標識ｈＡＩＬＩＭ−ＩｇＦＣ（２μｌ／ｔｕｂｅ；終濃度１μｇ／ｍｌ）を添加、混和した後、室温で３０分間インキュベートした。
【０２１７】
＜１４−４＞ 抗ＡＩＬＩＭモノクローナル抗体によるｈＡＩＬＩＭ−ＩｇＦｃとｈＢ７ｈ−ＩｇＦｃの結合の阻害活性の測定
９６ウェルマイクロプレートにｈＢ７ｈ−ＩｇＦｃ（５０μｌ／ｗｅｌｌ（８００ｎｇ／ｗｅｌｌ））を加え、プレートをシールした後、３７℃で１時間インキュベートした。各ウェルから溶液を除去し、ＰＢＳ（１２０μｌ）で３回洗浄した。次いで、各ウェルに０．５％ＢＳＡ含有ＰＢＳ（１００μｌ／ｗｅｌｌ）を加え未反応部分をブロックし、プレートをシールした後、３７℃で１時間インキュベートした。反応後、溶液を除去し、ＰＢＳ（１２０μｌ）で３回洗浄した。次いで、＜１４−３＞で調製した各試料（５０μｌ／ｗｅｌｌ）を加え、プレートをシールした後３７℃で１時間インキュベートした。各ウェルから溶液を除去し、３７℃に温めた１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（１２０μｌ）で３回洗浄した。次いで、各ウェルに３．７％ホルマリン含有ＰＢＳ（１００μｌ／ｗｅｌｌ）を加え、氷上で５分間インキュベートした。各ウェルから溶液を除去した後、０．１％Ｔｗｅｅｎ２０（１２０μｌ）で３回洗浄した。次いで、Ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ（５０μｌ／ｗｅｌｌ）を加え、プレートをシール後室温で３０分間インキュベートした。各ウェルから溶液を除去し、０．１％Ｔｗｅｅｎ２０含有ＰＢＳ（１２０μｌ）で３回洗浄した。次いで、ＴＭＢ^＋ｓｕｂｓｔｒａｔｅ ｃｈｒｏｍｏｇｅｎ（５０μｌ／ｗｅｌｌ）を加え、室温で２０分間インキュベートした。次いで、２Ｎ硫酸（５０μｌ／ｗｅｌｌ）を加え、反応を停止させ、ＴＨＥＲＭＯ ｍａｘ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ製）を用いて、４５０ｎｍでの吸光度を測定した。
結果を図７４乃至図７６に示す。
この結果、ｈＡＩＬＩＭ−ＩｇＦｃとｈＢ７ｈ−ＩｇＦｃの結合の抗ＡＩＬＩＭ抗体による用量依存的な阻害活性が測定された。
従って、本方法に例示されるようなアッセイ系を用いれば、ＡＩＬＩＭまたはＡＩＬＩＭリガンドに結合する物質（例えば、抗体または合成低分子化合物）をスクリーニングし、同定することができることが示された。
【０２１８】
実施例１５ ＡＩＬＩＭ−ＡＩＬＩＭリガンドを介するコスティミュレイトリーシグナル伝達によるヒトＴ細胞の増殖に対する抗ヒトＡＩＬＩＭヒトモノクローナル抗体の抑制活性
本発明の抗ヒトＡＩＬＩＭモノクローナル抗体が、ヒトＴ細胞上のＡＩＬＩＭを介するシグナル伝達を制御する能力を有するか否かを、ヒトＴ細胞をＡＩＬＩＭリガンド（Ｂ７ｈ／Ｂ７ＲＰ１／ＧＬ５０／ＬＩＣＯＳ）と接触させて誘導される細胞増殖の該抗ヒトＡＩＬＩＭヒトモノクローナル抗体による阻害効果を測定することにより解析した。
【０２１９】
＜１５−１＞ 抗体等の希釈
抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（ＡＴＣＣ ＣＲＬ−８００１）を、リン酸緩衝液（ＰＢＳ）で最終濃度８μｇ／ｍｌとなるように希釈した。
前記で調製した可溶性ヒトＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）を、ＰＢＳで最終濃度が４０μｇ／ｍｌとなるように希釈した後、ＰＢＳでさらに各種濃度に希釈した（４０μｇ／ｍｌ乃至０．００６４μｇ／ｍｌ）。
【０２２０】
＜１５−２＞ 抗体によるマイクロプレートのコーティング
９６ウェルマイクロプレート（複数枚）の各ウェルに、（１）抗ヒトＣＤ３モノクローナル抗体ＯＫＴ３（８μｇ／ｍｌを２５μｌ／ｗｅｌｌ）及びｈＢ７ｈ−ＩｇＦｃ（４０μｇ／ｍｌ乃至０．００６４μｇ／ｍｌを２５μｌ）を加え、３７℃で２時間インキュベーションした。次いで、抗体を捨て、各ウェルをＰＢＳで３回洗浄した。洗浄後、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（１００μｌ／ｗｅｌｌ）を加え、３７℃で１時間インキュベーションして、プレートの各ウェルをコーティングした。
【０２２１】
＜１５−３＞ ヒトＴ細胞懸濁液の調製
健常人の末梢血を採取し、ＬｙｍｐｈｏＰｒｅｐ（Ｎｙｃｏｍｅｄ製）を用いた密度勾配遠心により単核球を分画した。実験操作マニュアルに従い、Ｐａｎ−Ｔ ｃｅｌｌ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ｍｉｌｔｅｎｙｉ製）及びマグネティックソーターを用いて、該ヒト単核球からヒトＴ細胞を分離取得した。該Ｔ細胞の細胞数を、血球計算盤により計数した。該ヒトＴ細胞を、抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６（２０μｇ／ｍｌ）を加えた１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中に懸濁しヒトＴ細胞懸濁液（１×１０^６細胞／ｍｌ）を調製し、室温で３０分間インキュベートした。
なお、陰性対照抗体には、抗ＫＬＨヒトモノクローナル抗体ＪＭＡｂ２３（２０μｇ／ｍｌ）を用いた。
【０２２２】
＜１５−４＞ 細胞培養
前記のとおりにして抗ヒトＣＤ３抗体とｈＢ７ｈ−ＩｇＦｃでコートしたマイクロプレート各ウェルに、ヒトＴ細胞懸濁液（１００μｌ／ｗｅｌｌ；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ）を加え、ＣＯ_２インキュベーター内で３７℃で３日間培養した。
【０２２３】
＜１５−５＞ Ｔ細胞増殖活性の測定
前記の培養後の各プレートの各ウェルに、ｍｅｔｈｙｌ［^３Ｈ］ｔｈｙｍｉｄｉｎｅ（０．５μＣｉ／ｗｅｌｌ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を加え、ＣＯ_２インキュベーター内で３７℃で６時間インキュベーションした。培養後、セルハーベスターを用いて、細胞をＧＦ／Ｃフィルター（Ｐａｃｋａｒｄ製）上にトラップした。次いで、フィルターを、４０℃で３時間以上乾燥させた後、Ｍｉｃｒｏｓｃｉｎｔｉ ０（２０μｌ／ｗｅｌｌ；Ｐａｃｋａｒｄ製）を加えた。βカウンター（ＴＯＰ ＣＯＵＮＴ）を用いて、フィルター上にトラップされた細胞に取込まれている^３Ｈの放射活性を測定し、培養後のヒトＴ細胞の増殖の程度を解析した。
結果を図７７に示す。
本試験の結果、ヒトＢ７ｈ−ＩｇＦｃ濃度依存的なヒトＴ細胞の有意な増殖が観察された（陰性対照抗体を用いた試験）。さらに、当該ヒトＴ細胞の増殖が、抗ヒトＡＩＬＩＭモノクローナル抗体により有意に抑制された。
【０２２４】
実施例１６ ＡＩＬＩＭ−ＡＩＬＩＭリガンドを介するコスティミュレイトリーシグナル伝達によるサルＴ細胞の増殖に対する抗ヒトＡＩＬＩＭヒトモノクローナル抗体の抑制活性
前記実施例１５の試験をヒトＴ細胞の代わりにサルのＴ細胞を用いて同様にして行った。
【０２２５】
＜１６−１＞ 抗体等の希釈
抗ヒトＣＤ３モノクローナル抗体ＳＰ３４（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）を、リン酸緩衝液（ＰＢＳ）で最終濃度１μｇ／ｍｌとなるように希釈した。
前記で調製したヒトＢ７ｈ−ＩｇＦｃを、ＰＢＳで最終濃度が４０μｇ／ｍｌとなるように希釈した後、ＰＢＳでさらに各種濃度に希釈した（４０μｇ／ｍｌ乃至０．００６４μｇ／ｍｌ）。
【０２２６】
＜１６−２＞ 抗体によりマイクロプレートのコーティング
９６ウェルマイクロプレート（複数枚）の各ウェルに、（１）抗ヒトＣＤ３モノクローナル抗体ＳＰ３４（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）（１μｇ／ｍｌを２５μｌ／ｗｅｌｌ）及びヒトＢ７ｈ−ＩｇＦｃ（４０μｇ／ｍｌ乃至０．００６４μｇ／ｍｌを２５μｌ）を加え、３７℃で２時間インキュベーションした。次いで、抗体を捨て、各ウェルをＰＢＳで３回洗浄した。洗浄後、各ウェルに、１０％ＦＣＳ含有ＲＰＭＩ１６４０培地（１００μｌ／ｗｅｌｌ）を加え、３７℃で１時間インキュベーションして、プレートの各ウェルをコーティングした。
【０２２７】
＜１６−３＞ サルＴ細胞懸濁液の調製
カニクイザルの末梢血を採取し、ＮｙｃｏＰｒｅｐ１．０７７Ａ（Ｎｙｃｏｍｅｄ製）を用いた密度勾配遠心により単核球を分画した。実験操作マニュアルに従い、抗ヒトＣＤ４抗体Ｍ−Ｔ４７７（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）、抗ヒトＣＤ８抗体ＲＰＡ−Ｔ８（ＢＤ−Ｐｈａｒｍｉｎｇｅｎ製）、抗マウスＩｇＧマイクロビーズ（Ｍｉｌｔｅｎｙｉ製）及びマグネティックソーターを用いて、該カニクイサル単核球からサルＴ細胞を分離取得した。該Ｔ細胞の細胞数を、血球計算盤により計数した。該サルＴ細胞を、抗ヒトＡＩｌＩＭヒトモノクローナル抗体ＪＭａｂ−１３６（２０μｇ／ｍｌ）を加えた１０％ＦＣＳ含有ＲＰＭＩ１６４０培地中に懸濁しサルＴ細胞懸濁液（１×１０^６細胞／ｍｌ）を調製し、室温で３０分間インキュベートした。
なお、陰性対照抗体として抗ＫＬＨヒトモノクローナル抗体ＪＭａｂ−２３（２０μｇ／ｍｌ）を用いた。
【０２２８】
＜１６−４＞ 細胞培養
前記のとおりにして抗ヒトＣＤ３抗体とｈＢ７ｈ−ＩｇＦｃでコートしたマイクロプレート各ウェルに、サルＴ細胞懸濁液（１００μｌ／ｗｅｌｌ；１×１０^５ｃｅｌｌｓ／ｗｅｌｌ）を加え、ＣＯ_２インキュベーター内で３７℃で３日間培養した。
【０２２９】
＜１６−５＞ Ｔ細胞増殖活性の測定
前記の培養後の各プレートの各ウェルに、ｍｅｔｈｙｌ［^３Ｈ］ｔｈｙｍｉｄｉｎｅ（０．５μＣｉ／ｗｅｌｌ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ製）を加え、ＣＯ_２インキュベーター内で３７℃で６時間インキュベーションした。培養後、セルハーベスターを用いて、細胞をＧＦ／Ｃフィルター（Ｐａｃｋａｒｄ製）上にトラップした。次いで、フィルターを、４０℃で３時間以上乾燥させた後、Ｍｉｃｒｏｓｃｉｎｔｉ ０（２０μｌ／ｗｅｌｌ；Ｐａｃｋａｒｄ製）を加えた。βカウンター（ＴＯＰ ＣＯＵＮＴ）を用いて、フィルター上にトラップされた細胞に取込まれている^３Ｈの放射活性を測定し、培養後のＴ細胞の増殖の程度を解析した。
結果を図７８に示す。
本試験の結果、ヒトＢ７ｈ−ＩｇＦｃ濃度依存的なサルＴ細胞の有意な増殖が観察された（陰性対照抗体を用いた試験）。さらに、当該サルＴ細胞の増殖が、抗ヒトＡＩＬＩＭモノクローナル抗体により有意に抑制された。
【０２３０】
【発明の効果】
本発明のヒトＡＩＬＩＭに結合するヒトモノクローナル抗体は、ヒトに由来する抗体であることから、マウス由来の抗体等の非ヒト哺乳動物由来の抗体からなる抗体医薬品の治療上の大きな問題点（副作用）であったヒトに対する抗原性、即ちＨＡＭＡ（Ｈｕｍａｎ ａｎｔｉ−ｍｏｕｓｅ ａｎｔｉｇｅｎｉｃｉｔｙ）に起因する宿主の重篤な免疫拒絶反応を全く惹起しないことから、抗体の医薬品としての価値を劇的に増大させるものである。
【０２３１】
従って、本発明のＡＩＬＩＭ（特に、ヒトＡＩＬＩＭ）に対するヒトモノクローナル抗体及び該ヒトモノクローナル抗体からなる医薬組成物は、ＨＡＭＡに起因する宿主の免疫拒絶反応を惹起することなく、ＡＩＬＩＭを発現する細胞へのＡＩＬＩＭを介するコスティミュレイトリーシグナル（副刺激シグナル）の伝達が関与する種々の生体反応（例えば、ＡＩＬＩＭを発現する細胞の細胞増殖、ＡＩＬＩＭを発現する細胞によるサイトカインの産生、ＡＩＬＩＭを発現する細胞の免疫細胞溶解（ｉｍｍｕｎｅ ｃｙｔｏｌｙｓｉｓ）若しくは細胞死（ａｐｏｐｔｏｓｉｓ）、及びＡＩＬＩＭを発現する細胞に対する抗体依存性細胞障害を誘導する活性など）を制御するための医薬品として、及び／または該ＡＩＬＩＭを介するシグナル伝達が関与する種々の疾患の発症及び／または進行を抑制、阻止し、該疾患を治療または予防することが可能である。
【０２３２】
具体的には、本発明の抗ＡＩＬＩＭヒトモノクローナル抗体若しくはその一部を活性生物とする医薬品を提供することにより、例えば、自己免疫疾患またはアレルギー性疾患（特には、細胞性免疫により惹起される自己免疫疾患や遅延型アレルギー）に分類される種々の疾患（例えば、慢性関節リウマチ、多発性硬化症、自己免疫性甲状腺炎、アレルギー性接触性皮膚炎、慢性炎症性皮膚疾患である扁平苔癬、全身性エリテマトーデス、インスリン依存性糖尿病及び乾癬など）； 関節症（例えば、関節リウマチ（ｒｈｅｕｍａｔｏｉｄ ａｒｔｈｒｉｔｉｓ； ＲＡ）、変形性関節症（ｏｓｔｅｏａｒｔｈｒｉｔｉｓ； ＯＡ））、炎症（例えば、肝炎）； 移植片対宿主反応（ｇｒａｆｔ ｖｅｒｓｕｓ ｈｏｓｔ ｒｅａｃｔｉｏｎ； ＧＶＨ ｒｅａｃｔｉｏｎ）； 移植片対宿主病（ｇｒａｆｔ ｖｅｒｓｕｓ ｈｏｓｔ ｄｉｓｅａｓｅ； ＧＶＨＤ）； 組織（皮膚、角膜、骨などの組織）若しくは臓器（肝臓、心臓、肺、腎臓、膵臓など）の移植（同種移植または異種移植）に伴う免疫拒絶反応； 外来抗原若しくは自己抗原により惹起される免疫反応（例えば、該抗原に対する抗体の産生、細胞増殖、サイトカインの産生など）； 及び腸管免疫の異常に起因する可能性を有する疾患（具体的には、炎症性腸疾患（特には、クローン病及び潰瘍性大腸炎）や食餌性アレルギー）などの抑制、または治療及び予防が可能となる。
【０２３３】
本発明の医薬組成物はまた、抗炎症薬としての種々のステロイド剤が適用されているような任意の炎症、例えば、種々の関節炎（関節リウマチ、変形性関節症など）に伴う炎症、肺炎、肝炎（ウイルス性の肝炎を含む）、感染症に伴う炎症、炎症性腸疾患、腸炎、腎炎（糸球体腎炎、腎線維症に伴う炎症）、胃炎、血管炎、膵炎、腹膜炎、気管支炎、心筋炎、脳炎、虚血後再潅流障害（心筋虚血再潅流障害など）における炎症、組織や臓器の移植後免疫拒絶に起因する炎症、火傷、種々の皮膚の炎症（乾癬、アレルギー性接触性皮膚炎、慢性炎症性皮膚疾患である扁平苔癬など）、多発性臓器障害における炎症、ＰＴＣＡやＰＴＣＲの術後における炎症、及び動脈硬化症に伴う炎症、自己免疫性甲状腺炎などの治療または予防を可能とする。
さらには、上記の組織や臓器の移植に伴う免疫拒絶反応の抑制／治療分野においては、本発明の医薬組成物を、そのような移植治療での免疫拒絶反応の抑制のために施されている既存の免疫抑制剤と併用することにより当該既存薬単独での移植片拒絶反応の抑制の効果を増大させることが可能である。
また、本発明の１つであるＡＩＬＩＭまたはＡＩＬＩＭリガンドに結合する物質を同定する方法を用いれば、ＡＩＬＩＭまたはＡＩＬＩＭリガンドに結合して、両者の相互作用によるシグナル伝達を調節することにより上述のような種々の疾患を治療し得る潜在的な活性を有する医薬（化学合成化合物や抗体）をスクリーニングして選択することが可能となる。
【０２３４】
【配列表】

【０２３５】
「配列表フリーテキスト」
配列番号：１
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＮｏｔＩ−Ｔ。
配列番号：２
他の情報：人工配列についての記載：人工的に合成したリンカー配列２０ａｄｐ。
配列番号：３
他の情報：人工配列についての記載：人工的に合成したリンカー配列２４ａｄｐ。
配列番号：４
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＩＧＬＣ。
配列番号：５
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＮＨＣｃ２。
配列番号：６
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＥＸｃｅｌｌＥ。
配列番号：７
他の情報：人工配列についての記載：人工的に合成したプライマー配列ｃｋ１１７。
配列番号：８
他の情報：人工配列についての記載：人工的に合成したプライマー配列Ｍ１３Ｒ。
配列番号：９
他の情報：人工配列についての記載：人工的に合成したプライマー配列１３６Ｈ。
配列番号：１０
他の情報：人工配列についての記載：人工的に合成したプライマー配列１３８／９Ｈ。
配列番号：１１
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＡＩＬＩＭＨＣ１。
配列番号：１２
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ１。
配列番号：１３
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ７。
配列番号：１４
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ８。
配列番号：１５
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ３。
配列番号：１６
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ４。
配列番号：１７
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ６。
配列番号：１８
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＩＧＨＣ。
配列番号：１９
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ９。
配列番号：２０
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＣｃ５。
配列番号：２１
他の情報：人工配列についての記載：人工的に合成したプライマー配列ｐｏｌｙＡ。
配列番号：２２
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＡＩＬＩＭＬＣ１。
配列番号：２３
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＡＩＬＩＭＬＣ２。
配列番号：２４
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＬＣｃ１。
配列番号：２５
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＬＣｃ２。
配列番号：２６
他の情報：人工配列についての記載：人工的に合成したプライマー配列ＨＩＫ。
配列番号：３９
他の情報：人工配列についての記載：人工的に合成したプライマー配列。
配列番号：４０
他の情報：人工配列についての記載：人工的に合成したプライマー配列。
【０２３６】
【図面の簡単な説明】
【図１】細胞ＥＬＩＳＡの結果をフローサイトメーターを用いて解析した抗ヒトＡＩＬＩＭヒトモノクローナル抗体に対する抗ヒトＩｇＧ抗体、抗ヒトＩｇκ抗体及び抗ヒトＩｇＦｃ抗体の各々の反応性を示す図。
分図（ａ）乃至（ｌ）は各々下記の試験結果を示す。
分図（ａ）：野生型ＨＰＢ−ＡＬＬ細胞を蒔いたマイクロプレートに一次抗体を未添加で二次抗体としてのビオチン標識抗ヒトＩｇＧ抗体を加えた場合の試験結果。
分図（ｂ）：野生型ＨＰＢ−ＡＬＬ細胞を蒔いたマイクロプレートに一次抗体を未添加で二次抗体としてのビオチン標識抗ヒトＩｇκ抗体を加えた場合の試験結果。
分図（ｃ）： 野生型ＨＰＢ−ＡＬＬ細胞を蒔いたマイクロプレートに一次抗体を未添加で二次抗体としてのビオチン標識抗ヒトＩｇＦｃ抗体を加えた場合の試験結果。
分図（ｄ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６を用い、二次抗体としてビオチン標識抗ヒトＩｇＧ抗体を用いた場合の試験結果。
分図（ｅ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６を用い、二次抗体としてビオチン標識抗ヒトＩｇκ抗体を用いた場合の試験結果。
分図（ｆ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６を用い、二次抗体としてビオチン標識抗ヒトＩｇＦｃ抗体を用いた場合の試験結果。
分図（ｇ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８を用い、二次抗体としてビオチン標識抗ヒトＩｇＧ抗体を用いた場合の試験結果。
分図（ｈ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８を用い、二次抗体としてビオチン標識抗ヒトＩｇκ抗体を用いた場合の試験結果。
分図（ｉ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８を用い、二次抗体としてビオチン標識抗ヒトＩｇＦｃ抗体を用いた場合の試験結果。
分図（ｊ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９を用い、二次抗体としてビオチン標識抗ヒトＩｇＧ抗体を用いた場合の試験結果。
分図（ｋ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９を用い、二次抗体としてビオチン標識抗ヒトＩｇκ抗体を用いた場合の試験結果。
分図（ｌ）：一次抗体として抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９を用い、二次抗体としてビオチン標識抗ヒトＩｇＦｃ抗体を用いた場合の試験結果。
なお、各々の分図中の白抜きカーブは対照抗体である抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図２】抗ヒトＩｇＧ抗体を用いたサンドイッチＥＬＩＳＡにより定量したヒトＩｇＧモノクローナル抗体（標準物質）の検量線を示す図。縦軸は蛍光強度を示し、横軸は該標準物質の濃度を示す。
【図３】各種の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｈｕｍａｎ」なる用語は、該ヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図４】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体または陰性対照としての抗ＫＬＨヒトモノクローナル抗体のヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｈｕｍａｎ」なる用語は、該ヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図５】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｈｕｍａｎ」なる用語は、該ヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図６】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｈｕｍａｎ」なる用語は、該ヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図７】抗マウスＡＩＬＩＭラットモノクローナル抗体のマウスＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｍｏｕｓｅ」なる用語は、該マウスＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図８】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体または陰性対照としての抗ＫＬＨヒトモノクローナル抗体のマウスＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｍｏｕｓｅ」なる用語は、該マウスＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図９】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のマウスＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｍｏｕｓｅ」なる用語は、該マウスＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１０】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のマウスＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｍｏｕｓｅ」なる用語は、該マウスＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１１】抗ラットＡＩＬＩＭマウスモノクローナル抗体のラットＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｒａｔ」なる用語は、該ラットＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１２】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体または陰性対照としての抗ＫＬＨヒトモノクローナル抗体のラットＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｒａｔ」なる用語は、該ラットＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１３】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のラットＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｒａｔ」なる用語は、該ラットＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１４】各種の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のラットＡＩＬＩＭ高発現組換えＣＨＯ細胞または野生型ＣＨＯ細胞への結合活性を示す図。縦軸は組換え該結合活性の指標となる蛍光強度を示し、横軸は加えた抗体の濃度を示す。なお、図中の「ＣＨＯ」なる用語は、該野生型ＣＨＯ細胞への結合試験であることを示し、また「ｒａｔ」なる用語は、該ラットＡＩＬＩＭ高発現組換えＣＨＯ細胞への結合試験であることを示す。
【図１５】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＡ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
【図１６】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＡ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
【図１７】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＢ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図１８】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＢ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図１９】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＢ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図２０】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＣ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図２１】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＣ」由来のＴ細胞の増殖活性を示す図。縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。また、各種表記は下記を意味する。「１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２７。
【図２２】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＣ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６。
【図２３】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＣ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３９。
【図２４】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＣ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４１。
【図２５】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図２６】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２７。
【図２７】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
【図２８】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３９。
【図２９】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４１。
【図３０】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＥ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図３１】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＥ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２７。
【図３２】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＥ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６。
【図３３】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＥ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３９。
【図３４】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＥ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４１。
【図３５】抗ヒトＣＤ３モノクローナル抗体のみをコーティングしたマイクロプレートを用い、且つ抗ヒトＡＩＬＩＭマウスモノクローナル抗体を溶液の状態（液相）で加えた場合の種々の抗ヒトＡＩＬＩＭマウスモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図３６】抗ヒトＣＤ３モノクローナル抗体のみをコーティングしたマイクロプレートを用い、且つ抗ヒトＡＩＬＩＭヒトモノクローナル抗体を溶液の状態（液相）で加えた場合の種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２７。
【図３７】抗ヒトＣＤ３モノクローナル抗体のみをコーティングしたマイクロプレートを用い、且つ抗ヒトＡＩＬＩＭヒトモノクローナル抗体を溶液の状態（液相）で加えた場合の種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６。
【図３８】抗ヒトＣＤ３モノクローナル抗体のみをコーティングしたマイクロプレートを用い、且つ抗ヒトＡＩＬＩＭヒトモノクローナル抗体を溶液の状態（液相）で加えた場合の種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３９。
【図３９】抗ヒトＣＤ３モノクローナル抗体のみをコーティングしたマイクロプレートを用い、且つ抗ヒトＡＩＬＩＭヒトモノクローナル抗体を溶液の状態（液相）で加えた場合の種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、健常人「ドナーＤ」由来のＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４１。
【図４０】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＢ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図４１】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＢ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図４２】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＢ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図４３】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭマウスモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＣ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ＪＨＣ１」は、該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の代わりに陰性対照としての抗ヒトＣＥＴＰモノクローナル抗体を用いた場合の試験結果を示す。
【図４４】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＣ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２７。
【図４５】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＣ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３６。
【図４６】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＣ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３７。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１３９。
【図４７】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレート中で培養した健常人「ドナーＣ」由来のＴ細胞の培養上清中に産生されたＩＦＮ−γの量を示す図。
縦軸はＩＦＮ−γの濃度を示し、横軸は該抗ヒトＡＩＬＩＭマウスモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
また、各種表記は下記を意味する。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ１４１。
【図４８】健常人「ドナーＡ」のＴ細胞を健常人「ドナーＤ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図４９】健常人「ドナーＡ」のＴ細胞を健常人「ドナーＤ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図５０】健常人「ドナーＤ」のＴ細胞を健常人「ドナーＢ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図５１】健常人「ドナーＤ」のＴ細胞を健常人「ドナーＢ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図５２】健常人「ドナーＣ」のＴ細胞を健常人「ドナーＡ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図５３】健常人「ドナーＣ」のＴ細胞を健常人「ドナーＡ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図５４】健常人「ドナーＥ」のＴ細胞を健常人「ドナーＧ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ｃｏｎｔｒｏｌ ｍＩｇＧ」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＤ８０＋８６ Ａｂ」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
【図５５】健常人「ドナーＥ」のＴ細胞を健常人「ドナーＧ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４１。
【図５６】健常人「ドナーＦ」のＴ細胞を健常人「ドナーＥ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ｃｏｎｔｒｏｌ ｍＩｇＧ」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＤ８０＋８６ Ａｂ」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
【図５７】健常人「ドナーＦ」のＴ細胞を健常人「ドナーＥ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４１。
【図５８】健常人「ドナーＧ」のＴ細胞を健常人「ドナーＦ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ｃｏｎｔｒｏｌ ｍＩｇＧ」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＤ８０＋８６ Ａｂ」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
「ＣＴＬＡ４−Ｉｇ」：ヒトＣＴＬＡ４−ＩｇＦｃキメラ分子。
【図５９】健常人「ドナーＧ」のＴ細胞を健常人「ドナーＦ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４１。
【図６０】健常人「ドナーＡ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＤ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図６１】健常人「ドナーＡ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＤ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図６２】健常人「ドナーＤ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＢ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図６３】健常人「ドナーＤ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＢ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図６４】健常人「ドナーＣ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＡ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ＣＤ８０＋８６」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ｍＩｇＧ１」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図６５】健常人「ドナーＣ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＡ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１２４」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２４。
「１２６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２６。
「１２７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２７。
「１２８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１２８。
「１３５」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３５。
「１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３７」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３７。
【図６６】健常人「ドナーＥ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＧ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ｃｏｎｔｒｏｌ ｍＩｇＧ」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＤ８０＋８６ Ａｂ」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図６７】健常人「ドナーＥ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＧ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４１。
【図６８】健常人「ドナーＧ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＦ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の対照被験物質による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ｃｏｎｔｒｏｌ ｍＩｇＧ」：抗ヒトＣＤ３４／ＩｇＧ１マウスモノクローナル抗体。
「ＣＤ８０＋８６ Ａｂ」：抗ＣＤ８０抗体と抗ＣＤ８６抗体との混合物。
「ＳＡ１２」：抗ヒトＡＩＬＩＭマウスモノクローナル抗体。
【図６９】健常人「ドナーＧ」のＴ細胞を、予めヒトＣＴＬＡ４−Ｉｇキメラ分子の存在下で前培養した健常人「ドナーＦ」のＰＢＭＣと培養した場合の混合リンパ球反応（ＭＬＲ）での該Ｔ細胞の増殖試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体による該Ｔ細胞の増殖の抑制効果を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該被験物質の濃度を示す。
なお、図中の各種表記は下記を意味する。
「ａｎｔｉ−ＫＬＨ」：陰性対照としての抗ＫＬＨヒトモノクローナル抗体。
「ＪＭａｂ−１３６」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３６。
「１３８」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３８。
「１３９」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１３９。
「１４０」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４０。
「１４１」：抗ヒトＡＩＬＩＭヒトモノクローナル抗体ＪＭａｂ−１４１。
【図７０】野生型ＣＨＯ細胞を標的細胞として用いた場合の、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体及び対照抗体の各々のＡＤＣＣ誘導活性を示す図。
縦軸は、該抗体によるＡＤＣＣ誘導活性により起こる細胞障害率を示し、横軸は該抗体の濃度を示す。
【図７１】ヒトＡＩＬＩＭ高発現組換えＣＨＯ細胞を標的細胞として用いた場合の、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体及び対照抗体の各々のＡＤＣＣ誘導活性を示す図。
縦軸は、該抗体によるＡＤＣＣ誘導活性により起こる細胞障害率を示し、横軸は該抗体の濃度を示す。
【図７２】抗ＡＩＬＩＭ抗体による遅延型アレルギーの抑制効果を示す図。
縦軸は、遅延型アレルギー発症の指標である発赤の面積を示し、横軸は被験動物に投与した試料の種類を示す。
【図７３】抗ヒトＣＤ３モノクローナル抗体とともに抗ヒトＡＩＬＩＭヒトモノクローナル抗体をコーティングしたマイクロプレートを用いた種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体のコスティミュレイトリーシグナル伝達活性測定試験における、サルＴ細胞の増殖活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の濃度を示す。
なお、図中の「ａｎｔｉ−ＫＬＨ」は、該抗ヒトＡＩＬＩＭヒトモノクローナル抗体の代わりに陰性対照としての抗ＫＬＨヒトモノクローナル抗体を用いた場合の試験結果を示す。
【図７４】種々濃度の可溶性ＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）と可溶性ＡＩＬＩＭ（ＡＩＬＩＭ−ＩｇＦｃ）との結合の陰性対照抗体による阻害活性を示す図。
縦軸は阻害活性の指標である吸光度を示し、横軸は可溶性ＡＩＬＩＭの濃度を示す。
【図７５】種々濃度の可溶性ＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）と可溶性ＡＩＬＩＭ（ＡＩＬＩＭ−ＩｇＦｃ）との結合の抗ＡＩＬＩＭ抗体による阻害活性を示す図。
縦軸は阻害活性の指標である吸光度を示し、横軸は可溶性ＡＩＬＩＭの濃度を示す。
【図７６】可溶性ＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）と可溶性ＡＩＬＩＭ（ＡＩＬＩＭ−ＩｇＦｃ）との結合の種々濃度の抗ＡＩＬＩＭ抗体による阻害活性を示す図。
縦軸は阻害活性の指標である吸光度を示し、横軸は抗ＡＩＬＩＭ抗体の濃度を示す。
【図７７】抗ヒトＣＤ３モノクローナル抗体とともに可溶性ヒトＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）をコーティングしたマイクロプレートを用いたコスティミュレイトリーシグナル伝達活性測定試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体によるヒトＴ細胞の増殖阻害活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は抗体濃度を示す。
【図７８】抗ヒトＣＤ３モノクローナル抗体とともに可溶性ヒトＡＩＬＩＭリガンド（ｈＢ７ｈ−ＩｇＦｃ）をコーティングしたマイクロプレートを用いたコスティミュレイトリーシグナル伝達活性測定試験における、種々の抗ヒトＡＩＬＩＭヒトモノクローナル抗体によるサルＴ細胞の増殖阻害活性を示す図。
縦軸は細胞増殖の程度の指標としての［^３Ｈ］チミジンの細胞内への取込み量を示し、横軸は抗体濃度を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a human antibody that binds to AILIM (activation inducible lymphocyte immunomodulatory molecule; also referred to as ICOS (inducible co-stimulator)); a human monoclonal antibody or a part thereof, and the human monoclonal antibody or a part thereof. DNA or a part thereof; cells producing the human monoclonal antibody or a part thereof (including transgenic cells); human monoclonal antibodies produced by the transgenic cell or a part thereof; the human monoclonal antibody or a part thereof A pharmaceutical composition for treating a delayed allergy-related disease comprising an antibody against AILIM; AILIM or AI Identifying substances that bind to IM ligand, a method for quantifying or assay;
And a kit used in the method.
[0002]
[Prior art]
BACKGROUND ART Living organisms of mammals have an immune response system for eliminating pathogenic microorganisms (viruses, bacteria, parasites, etc.) and foreign foreign substances (hereinafter collectively referred to as “antigens”) that have entered the body. One is called the innate immune response system and the other is called the acquired immune response system. The former are elimination mechanisms by nonspecific recognition, such as phagocytosis by phagocytes (polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack by natural killer (NK) cells, and opsonization of antigen by complement. is there. The latter acquired immune response system is a mechanism of elimination by lymphocytes (mainly T cells and B cells) that have acquired (activated) specificity for the antigen.
[0003]
B cells that have acquired antigen specificity attack the extracellular antigen by producing antibodies specific to the antigen. T cells that have acquired (activated) antigen specificity are classified into helper T cells and cytotoxic T cells (cytotoxic lymphocytes; CTLs), the former regulating B cell differentiation and antibody production. Together with the phagocytes to destroy the antigen. The latter themselves attack virus-infected cells and the like (Experimental Medicine (separate volume), “BioScience Term Library [Immunity]”, Yodosha, pp. 14-17, 1995).
[0004]
The acquisition (activation) of antigen specificity by T cells is achieved by T cells recognizing antigens presented by antigen-presenting cells (APC) such as macrophages, B cells or dendritic cells. Be started. The antigen presenting cell processes (processes) the taken-in antigen, binds the processed antigen to the major histocompatibility complex (MHC), and presents the antigen. T cells recognize the processed antigen presented by the antigen-presenting cell through a complex of a T cell receptor (TcR) and a CD3 antigen (TcR / CD3 complex) on the surface of the cell membrane to activate the cell. Receive the first signal for activation (gain of specificity).
[0005]
However, the first signal alone through the TcR / CD3 complex not only does not result in sufficient activation of T cells, but also becomes unresponsive or unresponsive to any subsequent stimuli. It falls into a condition called clonal paralysis. Autoleukin (autocrine) of interleukin-2 (IL-2) is required to activate T cells to differentiate and proliferate into antigen-specific T cell clones. Since cell division does not occur without production of IL-2 or the like, T cells are in an inactivated state. That is, activation of T cells with production of cytokines such as IL-2 requires a second signal following the first signal via the TcR / CD3 complex. This second signal is called the costimulatory signal (costimulatory signal).
[0006]
T cells interact with another molecule, distinct from the MHC on antigen presenting cells, via a molecule separate from the TcR / CD3 complex on the T cell surface (cell-cell adhesion) to generate this second signal. Receiving and transmitting into cells. The second signal avoids cell anergy (clonal paralysis) and activates the cell.
[0007]
Although the mechanism of transmission of the second signal between antigen presenting cells and lymphocytes such as T cells has not yet been elucidated in detail, according to previous studies, this second signal transmission involves: CD28 (also known as Tp44, T44 or 9.3 antigen), which is a cell surface molecule mainly expressed on T cells and thymocytes, and cell surface molecule expressed on antigen presenting cells (macrophages, monocytes, dendritic cells, etc.) Between CD80 (also known as B7-1, B7, BB1, or B7 / BB1) and CD86 (also known as B7-2 or B70), which is also an antigen-presenting cell-like cell surface molecule (i.e., Cell-cell adhesion through bonding between these molecules) has been shown to be extremely important.
[0008]
Further, in controlling the activation of T cells by the second signal, CTLA-4 (Cytolytic T lymphocyte associated antigen 4), which is considered to be enhanced in expression depending on the second signal, and the TLA It is experimentally evident that the interaction between CD80 (B7-1) and CD86 (B7-2) (ie, cell-cell adhesion through binding between their molecules) also plays an important role. Have been. That is, the regulation of T cell activation by the transmission of the second signal includes at least the interaction between CD28 and CD80 / CD86, the enhancement of CTLA-4 expression which is considered to depend on the interaction, and It has been shown that the interaction between CTLA-4 and CD80 / CD86 is involved.
[0009]
CD28 has been shown to be a costimulator molecule that transmits a second signal (costimulatory signal) required for T cell activation and avoidance of anergy. The binding of this molecule to the costimulator molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (in other words, cell-cell adhesion via binding between those molecules) Signal stabilizes mRNA for Th1-type cytokines, and thus promotes the production of large amounts of Th1-type cytokines such as IL-2, IFNγ and TNFα from T cells. On the other hand, it is known that the expression of CTLA-4 is induced by a first signal entering through TcR / CD3, and the expression is also enhanced by the second signal entering by binding of CD28 and CD80. . CTLA-4 has been shown to receive these signals and act suppressively on T cell function as opposed to T cell activation by a second signal coming from CD28.
[0010]
Human CD28 and CTLA-4 are type I glycoproteins with molecular weights of 44 kD and 41-43 kD, respectively. Both have one immunoglobulin-like domain, belong to the immunoglobulin superfamily, and have both functions as an intercellular adhesion molecule and a function as a signal transduction molecule into cells.
[0011]
It has been shown that human CD28 forms a homodimer via disulfide bonds, while CTLA-4 exists as a monomer. The positions on the chromosome of the genes for CD28 and CTLA-4 are both "2q33" in humans and "1C" in mice, and each consists of four exons. Human CD28 and CTLA-4 are composed of 220 and 223 amino acids, respectively, including the leader sequence, and their amino acid homology is about 20 to 30%.
[0012]
The ligands for CD28 and CTLA-4 have been elucidated to be CD80 (B7-1) and CD86 (B7-2) in humans and mice. CTLA-4 has a higher affinity for both ligands than CD28, with a difference of about 20-fold. For the binding of CD28 and CTLA-4 to CD80 (B7-1), "MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)", which is an amino acid sequence structure conserved across animal species, is important. It has been revealed that Further, when CD28 is stimulated, PI3 kinase (phosphoinositide 3 kinase, PI3K) associates with a phosphorylated tyrosine residue in the intracellular partial sequence “YMNM (Tyr-Met-Asn-Met)”. It has been shown that CD28 plays an important role in intracellular signal transduction via this “YxxM” structure. In addition, the intracellular region of CTLA4 also has a sequence represented by “YxxM”, that is, “YVKM (Tyr-Val-Lys-Met)”, and SYP associates with this sequence after stimulation. It is shown.
[0013]
It has been shown that CD28 is expressed exclusively on thymocytes and peripheral blood T cells, while CTLA-4 is specifically expressed on activated T cells. Jun-sha, pp. 93-102, 1994; Journal, pp. 120-136; Experimental Medicine, separate volume, "BIO SCIENCE Terminology Library / Immunity", published by Yodosha, pp. 94-98, 1995; Experimental Medicine, separate volume, "BIO SCIENCE Terminology Library, Intracellular Signaling", published by Yodosha, pp. 58-59, 1997; Japan Clinical Laboratory, Vol. 55, No. 6, pp. 215-220, 1997. ).
[0014]
Thus, costimulator molecules (such as CD28, CD80 (B7-1) and CD86 (B7-2)) and associated CTLA-4, etc., in controlling T cell functions (T cell activation and function suppression) The importance of the interaction between multiple molecules has been proposed. Attempts to elucidate the relationship between these molecules and diseases and to treat diseases by controlling the functions of those molecules have been attracting attention. It is becoming.
[0015]
As described above, a living body activates an adaptive immune response system against an antigen that is foreign to the living body (self), but has an immune tolerance that does not show an immune response against its own biological component (self antigen). Have. However, when immune tolerance is disrupted for any reason, an immune response to self-antigen occurs, and self-antigen-reactive T cells are induced by the same mechanism as described above, resulting in an immune abnormality and various autoimmune diseases. .
[0016]
That is, when the immune system of the living body is in a normal state, unstimulated antigen presenting cells (APCs) of normal tissues do not express costimulatory molecules, and thus, for example, self-antigen reactivity which reacts with self antigens Even if T cells are present, self tolerance is maintained because the T cells are in an unresponsive state, but in an immune abnormal state, self-tolerance is caused by excess or continuous expression of costimulatory molecules. It has been suggested that antigen-reactive T cells can be activated to cause autoimmune diseases.
From such a viewpoint, in recent years, many attempts have been made to treat various autoimmune diseases by controlling the transmission of costimulatory signals, for example, the signal transmission between CD28 / CTLA-4-CD80 / CD86. It has been done.
However, while such therapeutic attempts have been made, the mechanism of activation of T cells by the interaction between costimulator molecules and related molecules has not been elucidated yet, and The mechanism could also involve other unidentified molecules.
[0017]
Recently, similarly to the above-mentioned "CD28" and "CTLA-4", transmission of a second signal (costimulatory signal) essential for activation of lymphocytes such as T cells, and in conjunction with the signal, A new costimulatory molecule (costimulatory molecule) that is thought to control the function of activated lymphocytes such as activated T cells has been identified. This molecule is called AILIM (activation inducible lymphocyte immunomodulatory molecule) (human, mouse and rat; Int. Immunol., Vol. 12, No. 1, p. 51-55, 2000), and is also referred to as ICOSd (coId). -Human stimulator) (human; Nature, Vol. 397, No. 6716, p. 263-266, 1999).
[0018]
On the other hand, very recently, a novel molecule called B7h, B7RP-1, GL50 or LICOS which is a ligand (AILIM ligand) interacting with the costimulatory molecule AILIM (ICOS) has been identified (Nature). , Vol.402, No.6763, p.827-832, 1999; Nature Medicine, Vol.5, No.12, p.1365-1369, 1999; J. Immunology, Vol.164, p.1653-1657, 2000; Curr. Biol., Vol. 10, No. 6, p. 333-336, 2000).
[0019]
Identification of these two new molecules, AILIM (ICOS) and B7RP-1 (B7h / GL50 / LICOS), is essential for the activation of lymphocytes such as T cells and the control of the functions of activated T cells as described above. Are signaling pathways between CD28 and CD80 (B7-1) / CD86 (B7-2), and CTLA4 and CD80 (B7-). 1) In addition to the first and second pathways that are signaling pathways between / CD86 (B7-2), a new third pathway through the interaction between AILIM (ICOS) and B7RP-1 (B7h) It turned out to be.
Biological function of each of these two new molecules, regulation of lymphocyte functions such as T cells by third costimulatory signal transduction by the molecules, and relevance of the novel signal transduction to disease Are currently being vigorously studied (J. Immunol., 166 (1), p. 1, 2001; J. Immunol., 165 (9), p. 5035, 2000; Biochem. Commun., 276 (1), p.335, 2000; Immunity, 13 (1), p.95, 2000; J. Exp. Med., 192 (1), p.53, 2000; Eur. J. Immunol., 30 (4), p.1040, 2000, International Patent Application Publication WO 01/15732. .
[0020]
[Problems to be solved by the invention]
That is, the present invention provides the transmission of a second signal (costimulatory signal) essential for activation of lymphocytes such as T cells, as well as the aforementioned “CD28” and “CTLA-4”, and the signal. Clarifies the biological function of AILIM, a novel molecule considered to be a molecule that controls the function of activated lymphocytes such as activated T cells in conjunction with AILIM, and the relationship between AILIM expression and disease. By controlling the biological function of AILIM by medical and pharmacological techniques (for example, drugs such as monoclonal antibodies and low molecular weight compounds) to suppress the onset of various diseases depending on the state of AILIM expression, or It is intended to provide methods and medicaments for treatment.
[0021]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on human antibodies (especially human monoclonal antibodies) against mammalian AILIM (especially human AILIM) in order to achieve the above-mentioned object. Various human monoclonal antibodies, particularly human AILIM, that bind to human AILIM, are immunized with human AILIM (specifically, a cell membrane fraction of human AILIM-expressing cells) with transgenic mice prepared to produce For the first time in the world to produce various monoclonal antibodies that bind to AILIM and regulate signaling through human AILIM.
[0022]
Since the antibody of the present invention (particularly, monoclonal antibody) is an antibody derived from a human, a major problem (side effect) in the treatment of an antibody drug comprising an antibody derived from a non-human mammal such as an antibody derived from a mouse is caused. Does not cause severe immunorejection of the host due to antigenicity against humans, that is, HAMA (Human anti-mouse antigenicity), thus dramatically increasing the value of antibodies as pharmaceuticals. .
[0023]
Therefore, the human antibody (particularly human monoclonal antibody) that binds to mammalian AILIM (particularly human AILIM) of the present invention and a pharmaceutical composition comprising the human antibody (particularly human monoclonal antibody) are derived from HAMA. Various biological reactions involving the transmission of costimulatory signals (costimulatory signals) via AILIM to cells expressing AILIM without causing immune rejection of the host Cell proliferation, production of cytokines by AILIM-expressing cells, immune lysis or apoptosis of AILIM-expressing cells, and activity of inducing antibody-dependent cellular damage to AILIM-expressing cells And other drugs to control In addition, the present invention is useful as a medicament for suppressing or preventing the onset and / or progression of various diseases in which signal transduction via AILIM is involved, and for treating or preventing the diseases.
[0024]
Specifically, the pharmaceutical composition of the present invention controls (suppresses or promotes) the growth (suppression or promotion) of AILIM-expressing cells or controls (suppresses or promotes) the production of cytokines (eg, interferon γ or interleukin 4) by AILIM-expressing cells. ) To allow the suppression of various pathological conditions caused by various physiological phenomena involving signal transduction via AILIM, and the treatment or prevention of various diseases. By using the pharmaceutical composition of the present invention, for example, various diseases classified into autoimmune diseases or allergic diseases (in particular, autoimmune diseases caused by cell-mediated immunity and delayed allergy) (for example, chronic diseases) Rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, lichen planus, a chronic inflammatory skin disease, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis); arthrosis (eg, Rheumatoid arthritis (RA), osteoarthritis (OA), inflammation (eg, hepatitis); graft-versus-host reaction; GVH reaction vs. graft disease; host disease e; GVHD); immunorejection reaction associated with transplantation (allograft or xenograft) of tissue (tissue such as skin, cornea, bone, etc.) or organ (liver, heart, lung, kidney, pancreas, etc.); foreign antigen or autoantigen (E.g., production of antibodies to the antigen, cell proliferation, production of cytokines, etc.); and diseases that may be caused by abnormal intestinal immunity (specifically, inflammatory bowel diseases (especially Can inhibit, prevent and / or treat Crohn's disease and ulcerative colitis) and dietary allergies.
Furthermore, in the field of suppressing / treating immune rejection associated with the transplantation of tissues and organs, the pharmaceutical composition of the present invention is used for suppressing immune rejection in such transplantation treatment. When used in combination with an existing immunosuppressant, it is possible to increase the effect of suppressing transplant rejection using the existing drug alone.
[0025]
Further, the pharmaceutical composition of the present invention can be applied to treatment or prevention of any inflammation to which various steroids as anti-inflammatory drugs are applied.
The inflammatory diseases to which the pharmaceutical composition of the present invention can be applied include, for example, inflammation associated with various arthritis (rheumatoid arthritis, osteoarthritis, etc.), pneumonia, hepatitis (including viral hepatitis), infectious disease Associated inflammation, inflammatory bowel disease, enteritis, nephritis (inflammation associated with glomerulonephritis, renal fibrosis), gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis, reperfusion injury after ischemia (myocardium Inflammation due to ischemia-reperfusion injury), inflammation due to immune rejection after transplantation of tissues or organs, burns, various skin inflammations (psoriasis, allergic contact dermatitis, lichen planus, a chronic inflammatory skin disease) ), Inflammation in multiple organ disorders, inflammation after PTCA or PTCR surgery, inflammation associated with arteriosclerosis, autoimmune thyroiditis, and the like.
In addition, by using the method for identifying a substance that binds to AILIM or AILIM ligand, which is one of the present invention, it binds to AILIM or AILIM ligand and regulates signal transduction by the interaction between the two, as described above. It becomes possible to screen and select a drug (a chemically synthesized compound or an antibody) having a potential activity capable of treating various diseases.
[0026]
That is, the invention is as described in the following (1) to (108) of the invention.
(1) A human antibody that binds to AILIM.
(2) The human antibody according to (1), wherein the AILIM is derived from a human.
(3) A human monoclonal antibody that binds to AILIM or a part thereof.
(4) The human monoclonal antibody or a part thereof according to (3), wherein the AILIM is derived from a human.
(5) The human monoclonal antibody according to the above (3) or (4), wherein the human monoclonal antibody has an activity of inhibiting AILIM-mediated signal transduction into cells. Department.
(6) The inhibitory activity of the signal transduction is the following (a) or (b):
(A) an activity of suppressing the growth of AILIM-expressing cells; or
(B) an activity of suppressing cytokine production by cells expressing AILIM;
The human monoclonal antibody or a part thereof according to the above (5), wherein
(7) The human monoclonal antibody or a part thereof according to (6), wherein the cytokine is a cytokine produced by Th1 type T cells or Th2 type T cells.
(8) The human monoclonal antibody or a part thereof according to (7), wherein the cytokine is interferon γ or interleukin 4.
(9) The human monoclonal antibody or a part thereof according to (5), wherein the human monoclonal antibody has an activity of suppressing a mixed lymphocyte reaction.
(10) The human monoclonal antibody according to the above (3) or (4), wherein the human monoclonal antibody has an activity of inducing AILIM-mediated signal transduction into cells. Department.
(11) The activity of inducing the signal transduction is the following (a) or (b):
(A) an activity of inducing the proliferation of cells expressing AILIM;
(B) an activity of inducing the production of cytokines by cells expressing AILIM;
The human monoclonal antibody or a part thereof according to the above (10), wherein
(12) The human monoclonal antibody or a part thereof according to (11), wherein the cytokine is a cytokine produced by Th1 type T cells or Th2 type T cells.
(13) The human monoclonal antibody or a part thereof according to (12), wherein the cytokine is interferon γ or interleukin 4.
(14) The human monoclonal antibody has an activity of inducing antibody-dependent cellular cytotoxicity against AILIM-expressing cells and / or an activity of inducing immune cell lysis or cell death of AILIM-expressing cells. The human monoclonal antibody according to the above (3) or (4) or a part thereof.
(15) The binding rate constant (ka) between the human monoclonal antibody and AILIM is 1.0 × 10 ³ The human monoclonal antibody or a part thereof according to the above (3) or (4), which has a numerical value of (1 / M.Sec) or more.
(16) The binding rate constant (ka) is 1.0 × 10 ⁴ The human monoclonal antibody or a part thereof according to the above (15), wherein the numerical value is not less than (1 / M.Sec).
(17) The binding rate constant (ka) is 1.0 × 10 ⁵ The human monoclonal antibody or a part thereof according to the above (16), wherein the numerical value is not less than (1 / M.Sec).
(18) The dissociation rate constant (kd) between the human monoclonal antibody and AILIM is 1.0 × 10 ^-3 (1 / Sec) The human monoclonal antibody or a part thereof according to the above (3) or (4), which has the following numerical value.
(19) The dissociation rate constant (kd) is 1.0 × 10 ^-4 (1 / Sec) The human monoclonal antibody or a part thereof according to the above (18), which has the following numerical value.
(20) The dissociation rate constant (kd) is 1.0 × 10 ^-5 (1 / Sec) The human monoclonal antibody or a part thereof according to the above (19), which has the following numerical value.
(21) The dissociation constant (Kd) between the human monoclonal antibody and AILIM is 1.0 × 10 ^-6 (M) The human monoclonal antibody or a part thereof according to the above (3) or (4), which has the following numerical values.
(22) The dissociation constant (Kd) is 1.0 × 10 ^-7 (M) The human monoclonal antibody or a part thereof according to the above (21), which has the following numerical values.
(23) The dissociation constant (Kd) is 1.0 × 10 ^-8 (M) The human monoclonal antibody or a part thereof according to the above (22), which has the following numerical values.
(24) The dissociation constant (Kd) is 1.0 × 10 ^-9 (M) The human monoclonal antibody or a part thereof according to the above (23), which has the following numerical values.
(25) The DNA of the human monoclonal antibody, wherein the DNA of the V region encoding the variable region of the heavy chain is derived from any of human immunoglobulin heavy chain V gene segments of 1-02 or 3-13. The human monoclonal antibody or a part thereof according to 4).
(26) The DNA according to (4), wherein the DNA of the V region encoding the light chain variable region of the human monoclonal antibody is derived from either the L5 or A27 human immunoglobulin light chain V gene segment. Or a human monoclonal antibody thereof.
(27) the DNA of the V region encoding the heavy chain variable region of the human monoclonal antibody is derived from the human immunoglobulin heavy chain V gene segment of either 1-02 or 3-13, and The human monoclonal according to (25) or (26), wherein the DNA of the V region encoding the light chain variable region is derived from a human immunoglobulin light chain V gene segment of either L5 or A27. Antibody or part thereof.
(28) The DNA of the V region encoding the heavy chain variable region of the human monoclonal antibody is derived from the human immunoglobulin heavy chain V gene segment 1-02, and the V region encoding the light chain variable region of the human monoclonal antibody. The human monoclonal antibody or a part thereof according to (27), wherein the DNA of the region is derived from a human immunoglobulin light chain V gene segment L5.
(29) The DNA of the V region encoding the heavy chain variable region of the human monoclonal antibody is derived from the human immunoglobulin heavy chain V gene segment 3-13, and the V region encodes the light chain variable region of the human monoclonal antibody. The human monoclonal antibody or a part thereof according to (27), wherein the DNA of the region is derived from human immunoglobulin light chain V gene segment A27.
(30) The human monoclonal antibody or the human monoclonal antibody thereof according to (4), wherein the heavy chain variable region of the human monoclonal has an amino acid sequence containing any one of the following amino acids (a) to (f): part:
(A) the amino acid sequence of amino acids 20 to 117 shown in SEQ ID NO: 28;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 117 shown in SEQ ID NO: 28;
(C) the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 32;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 116 described in SEQ ID NO: 32;
(E) the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 36; or
(F) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 36.
(31) The human monoclonal antibody or a part thereof according to (4), wherein the human monoclonal heavy chain polypeptide has any one of the following amino acid sequences (a) to (f):
(A) the amino acid sequence of amino acids 20 to 470 described in SEQ ID NO: 28;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 470 shown in SEQ ID NO: 28;
(C) the amino acid sequence of amino acids 20 to 470 described in SEQ ID NO: 32;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 470 of SEQ ID NO: 32;
(E) the amino acid sequence of amino acids 20 to 470 of SEQ ID NO: 36; or
(F) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 20 to 470 described in SEQ ID NO: 36.
(32) The human monoclonal antibody or the human monoclonal antibody thereof according to (4), wherein the light chain variable region of the human monoclonal has an amino acid sequence containing any one of the following amino acids (a) to (f): part:
(A) the amino acid sequence of amino acids 23 to 116 shown in SEQ ID NO: 30;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added in the amino acid sequence of amino acids 23 to 116 shown in SEQ ID NO: 30;
(C) the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 34;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 34;
(E) the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 38;
(F) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 38;
(33) The human monoclonal antibody or a part thereof according to (4), wherein the human monoclonal light chain polypeptide has any one of the following amino acid sequences (a) to (f):
(A) the amino acid sequence of amino acids 23 to 236 described in SEQ ID NO: 30;
(B) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 23 to 236 shown in SEQ ID NO: 30;
(C) the amino acid sequence of amino acids 21 to 236 described in SEQ ID NO: 34;
(D) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 34;
(E) the amino acid sequence of amino acids 21 to 236 of SEQ ID NO: 38; or
(F) an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 38.
(34) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence containing the amino acid sequence of amino acids 20 to 117 shown in SEQ ID NO: 28;
(B) the light chain variable region has an amino acid sequence containing the amino acid sequence of amino acids 23 to 116 shown in SEQ ID NO: 30;
(35) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 28;
(B) the light chain polypeptide has the amino acid sequence of amino acids 23 to 236 shown in SEQ ID NO: 30;
(36) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence containing the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 32;
(B) the light chain variable region has an amino acid sequence containing the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 34;
(37) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 32;
(B) the light chain polypeptide has the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 34;
(38) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence including the amino acid sequence of amino acids 20 to 116 as set forth in SEQ ID NO: 36;
(B) the light chain variable region has an amino acid sequence containing the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 38;
(39) The human monoclonal antibody according to (4) or a part thereof, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 36;
(B) the light chain polypeptide has the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 38;
(40) The method according to any one of (3) to (29), wherein the human monoclonal antibody is a monoclonal antibody derived from a transgenic non-human mammal capable of producing a human antibody. Human monoclonal antibodies or parts thereof.
(41) The human monoclonal antibody is used to convert a cell expressing AILIM, a membrane fraction derived from the cell, all or a part of a molecule constituting AILIM, or a gene encoding AILIM or a part thereof to a human antibody. The human monoclonal antibody or a part thereof according to the above (40), which is a monoclonal antibody obtained by immunizing a transgenic non-human mammal capable of producing.
(42) The human monoclonal antibody or a part thereof according to the above (40) or (41), wherein the transgenic non-human mammal is a transgenic mouse.
(43) DNA encoding a polypeptide selected from the group consisting of the following (a) to (f) or a part thereof:
(A) a polypeptide comprising the amino acid sequence of amino acids 20 to 117 of SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acids 23 to 116 shown in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 36;
(F) a polypeptide comprising the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 38;
(44) DNA encoding a polypeptide selected from the group consisting of the following (a) to (f) or a part thereof:
(A) a polypeptide comprising the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acids 23 to 236 shown in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acids 21 to 236 of SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acids 20 to 470 of SEQ ID NO: 36;
(F) a polypeptide comprising the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 38;
(45) DNA selected from the group consisting of the following (a) to (f) or a part thereof: (a) DNA containing the nucleotide sequence of base numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of base numbers 105 to 386 described in SEQ ID NO: 29;
(C) a DNA comprising the nucleotide sequence of base numbers 151 to 441 described in SEQ ID NO: 31;
(D) a DNA comprising the base sequence of base numbers 88 to 375 described in SEQ ID NO: 33;
(E) a DNA comprising the nucleotide sequence of base numbers 153 to 443 described in SEQ ID NO: 35;
(F) a DNA comprising the nucleotide sequence of base numbers 93 to 380 described in SEQ ID NO: 37;
(46) DNA selected from the group consisting of the following (a) to (f) or a part thereof: (a) DNA having a base sequence of base numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of base numbers 39 to 749 shown in SEQ ID NO: 29;
(C) a DNA having the nucleotide sequence of base numbers 94 to 1506 described in SEQ ID NO: 31;
(D) a DNA having the nucleotide sequence of base numbers 28 to 738 shown in SEQ ID NO: 33;
(E) a DNA having the nucleotide sequence of base numbers 96 to 1508 described in SEQ ID NO: 35;
(F) a DNA having the nucleotide sequence of base numbers 33 to 743 described in SEQ ID NO: 37;
(47) A vector comprising the DNA according to any one of (43) to (46).
(48) The vector according to (47), comprising the DNA of any of the following (a) to (c):
(A) a DNA comprising the nucleotide sequence of base numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of base numbers 151 to 441 described in SEQ ID NO: 31;
(C) a DNA comprising the nucleotide sequence of base numbers 153 to 443 described in SEQ ID NO: 35;
(49) The vector according to (47), comprising the DNA of any of the following (a) to (c):
(A) DNA having a base sequence of base numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of base numbers 94 to 1506 described in SEQ ID NO: 31;
(C) a DNA having the nucleotide sequence of base numbers 96 to 1508 described in SEQ ID NO: 35;
(50) The vector according to (47), comprising the DNA of any one of the following (a) to (c):
(A) a DNA comprising the nucleotide sequence of nucleotides 105 to 386 described in SEQ ID NO: 29;
(B) a DNA comprising the nucleotide sequence of base numbers 88 to 375 described in SEQ ID NO: 33; or
(C) a DNA comprising the nucleotide sequence of base numbers 93 to 380 shown in SEQ ID NO: 37;
(51) The vector according to the above (47), which comprises any one of the following DNAs (a) to (c):
(A) a DNA having the nucleotide sequence of base numbers 39 to 749 shown in SEQ ID NO: 29;
(B) DNA having a nucleotide sequence of base numbers 28 to 738 shown in SEQ ID NO: 33; or
(C) a DNA having the nucleotide sequence of base numbers 33 to 743 described in SEQ ID NO: 37;
(52) The vector according to the above (47), comprising the DNA of the following (a) and (b):
(A) a DNA comprising the nucleotide sequence of base numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of base numbers 105 to 386 described in SEQ ID NO: 29;
(53) The vector according to the above (47), comprising the DNA of the following (a) and (b):
(A) a DNA having the nucleotide sequence of base numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of base numbers 39 to 749 shown in SEQ ID NO: 29;
(54) The vector according to (47), comprising the DNA of the following (a) and (b):
(A) a DNA comprising the nucleotide sequence of base numbers 151 to 441 described in SEQ ID NO: 31;
(B) a DNA comprising the base sequence of base numbers 88 to 375 described in SEQ ID NO: 33;
(55) The vector according to the above (47), comprising the DNA of the following (a) and (b):
(A) a DNA having the nucleotide sequence of base numbers 94 to 1506 described in SEQ ID NO: 31;
(B) a DNA having the nucleotide sequence of base numbers 28 to 738 shown in SEQ ID NO: 33;
(56) The vector according to the above (47), comprising the DNA of the following (a) and (b):
(A) a DNA comprising the nucleotide sequence of base numbers 153 to 443 described in SEQ ID NO: 35;
(B) a DNA comprising the nucleotide sequence of base numbers 93 to 380 described in SEQ ID NO: 37;
(57) The vector according to the above (47), comprising the DNA of the following (a) and (b):
(A) DNA having the nucleotide sequence of base numbers 96 to 1508 described in SEQ ID NO: 35;
(B) a DNA having the nucleotide sequence of base numbers 33 to 743 described in SEQ ID NO: 37;
(58) A cell that produces the human monoclonal antibody according to any one of (3) to (42).
(59) The cell according to (58), wherein the cell is a fused cell obtained by fusing mammalian-derived B cells with mammalian-derived myeloma cells for the ability to produce the human monoclonal antibody. The cells as described.
(60) The DNA of the following (a) or a vector containing the DNA, the DNA of the following (b) or a vector containing the DNA, or the DNA of both the following (a) and (b) or a vector containing both DNAs Genetically modified host transformed by introducing into a host:
(A) DNA encoding a heavy chain polypeptide of a monoclonal antibody that binds to human AILIM or a part thereof;
(B) DNA encoding a light chain polypeptide of a monoclonal antibody that binds to human AILIM or a part thereof.
(61) The recombinant host according to (60), wherein the monoclonal antibody is a human monoclonal antibody.
(62) The genetically modified host according to (60) or (61), wherein the host is a mammalian cell.
(63) The genetically modified host according to (60) or (61), wherein the host is a fertilized egg of a mammal.
(64) The heavy chain polypeptide according to any one of (60) to (63), wherein the heavy chain polypeptide is a heavy chain polypeptide selected from the group consisting of the following (a) to (c). Genetically modified host:
(A) a heavy chain polypeptide comprising the amino acid sequence of amino acids 20 to 117 of SEQ ID NO: 28;
(B) a heavy chain polypeptide comprising the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 32;
(C) a heavy chain polypeptide comprising the amino acid sequence of amino acids 20 to 116 shown in SEQ ID NO: 36;
(65) The heavy chain polypeptide according to any one of (60) to (63), wherein the heavy chain polypeptide is a heavy chain polypeptide selected from the group consisting of the following (a) to (c). Genetically modified host:
(A) a heavy chain polypeptide having the amino acid sequence of amino acids 20 to 470 as set forth in SEQ ID NO: 28;
(B) a heavy-chain polypeptide having the amino acid sequence of amino acids 20-470 as set forth in SEQ ID NO: 32;
(C) a heavy-chain polypeptide having the amino acid sequence of amino acids 20-470 as set forth in SEQ ID NO: 36.
(66) The light chain polypeptide according to any one of (60) to (63), wherein the light chain polypeptide is a light chain polypeptide selected from the group consisting of the following (a) to (c): Genetically modified host:
(A) a light chain polypeptide comprising the amino acid sequence of amino acids 23 to 116 shown in SEQ ID NO: 30;
(B) a light chain polypeptide comprising the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 34;
(C) a light chain polypeptide comprising the amino acid sequence of amino acids 21 to 116 shown in SEQ ID NO: 38;
(67) The light chain polypeptide according to any one of (60) to (63), wherein the light chain polypeptide is a light chain polypeptide selected from the group consisting of the following (a) to (c). Genetically modified host:
(A) a light chain polypeptide having the amino acid sequence of amino acids 23 to 236 shown in SEQ ID NO: 30;
(B) a light chain polypeptide having the amino acid sequence of amino acids 21 to 236 of SEQ ID NO: 34;
(C) a light chain polypeptide having the amino acid sequence of amino acids 21 to 236 shown in SEQ ID NO: 38;
(68) The genetically modified host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are the following (a) and (b), respectively. :
(A) a heavy chain polypeptide comprising amino acids 20 to 117 of SEQ ID NO: 28;
(B) a light chain polypeptide comprising amino acids 23 to 116 shown in SEQ ID NO: 30;
(69) The recombinant host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are (a) and (b), respectively. :
(A) a heavy chain polypeptide having amino acids 20 to 470 of SEQ ID NO: 28;
(B) a light chain polypeptide having the amino acids 23 to 236 of SEQ ID NO: 30;
(70) The genetically modified host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are the following (a) and (b), respectively. :
(A) a heavy chain polypeptide comprising amino acids 20 to 116 of SEQ ID NO: 32;
(B) a light chain polypeptide comprising the amino acids 21 to 116 of SEQ ID NO: 34;
(71) The genetically modified host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are the following (a) and (b), respectively. :
(A) a heavy chain polypeptide having amino acids 20 to 470 of SEQ ID NO: 32;
(B) a light chain polypeptide having the amino acids 21 to 236 of SEQ ID NO: 34;
(72) The genetically modified host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are the following (a) and (b), respectively. :
(A) a heavy chain polypeptide comprising amino acids 20 to 116 of SEQ ID NO: 36;
(B) a light chain polypeptide comprising the amino acids 21 to 116 of SEQ ID NO: 38;
(73) The genetically modified host according to any of (60) to (63), wherein the heavy chain polypeptide and the light chain polypeptide are the following (a) and (b), respectively. :
(A) a heavy-chain polypeptide having the amino acids 20-470 of SEQ ID NO: 36;
(B) a light chain polypeptide having the amino acids 21 to 236 of SEQ ID NO: 38;
(74) The gene according to any one of (60) to (63), wherein the DNA encoding the heavy chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) a DNA comprising the nucleotide sequence of base numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of base numbers 151 to 441 described in SEQ ID NO: 31;
(C) a DNA comprising the nucleotide sequence of base numbers 153 to 443 described in SEQ ID NO: 35;
(75) The gene according to any one of (60) to (63), wherein the DNA encoding the heavy chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) DNA having a base sequence of base numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of base numbers 94 to 1506 described in SEQ ID NO: 31;
(C) a DNA having the nucleotide sequence of base numbers 96 to 1508 described in SEQ ID NO: 35;
(76) The gene according to any one of (60) to (63), wherein the DNA encoding the light chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) a DNA comprising the nucleotide sequence of nucleotides 105 to 386 described in SEQ ID NO: 29;
(B) a DNA comprising the nucleotide sequence of base numbers 88 to 375 described in SEQ ID NO: 33; or
(C) a DNA comprising the nucleotide sequence of base numbers 93 to 380 shown in SEQ ID NO: 37;
(77) The gene according to any one of (60) to (63), wherein the DNA encoding the light chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) a DNA having the nucleotide sequence of base numbers 39 to 749 shown in SEQ ID NO: 29;
(B) DNA having a nucleotide sequence of base numbers 28 to 738 shown in SEQ ID NO: 33; or
(C) a DNA having the nucleotide sequence of base numbers 33 to 743 described in SEQ ID NO: 37;
(78) The DNA according to (60), wherein the DNA encoding the heavy chain polypeptide is a DNA of the following (a), and the DNA encoding the light chain polypeptide is a DNA of the following (b). Or the genetically modified host according to any of (63):
(A) a DNA comprising the nucleotide sequence of base numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of base numbers 105 to 386 described in SEQ ID NO: 29;
(79) The DNA according to (60), wherein the DNA encoding the heavy chain polypeptide is the following DNA (a), and the DNA encoding the light chain polypeptide is the following DNA (b). Or the genetically modified host according to any of (63):
(A) a DNA having the nucleotide sequence of base numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of base numbers 39 to 749 shown in SEQ ID NO: 29;
(80) The DNA according to (60), wherein the DNA encoding the heavy chain polypeptide is a DNA of the following (a), and the DNA encoding the light chain polypeptide is a DNA of the following (b): Or the genetically modified host according to any of (63):
(A) a DNA comprising the nucleotide sequence of base numbers 151 to 441 described in SEQ ID NO: 31;
(B) a DNA comprising the base sequence of base numbers 88 to 375 described in SEQ ID NO: 33;
(81) The DNA according to (60), wherein the DNA encoding the heavy chain polypeptide is a DNA of the following (a), and the DNA encoding the light chain polypeptide is a DNA of the following (b). Or the genetically modified host according to any of (63):
(A) DNA having a nucleotide sequence of base numbers 94 to 1506 described in SEQ ID NO: 31;
(B) a DNA having the nucleotide sequence of base numbers 28 to 738 shown in SEQ ID NO: 33;
(82) The DNA according to the above (60), wherein the DNA encoding the heavy chain polypeptide is the following DNA (a), and the DNA encoding the light chain polypeptide is the following DNA (b). Or the genetically modified host according to any of (63):
(A) a DNA comprising the nucleotide sequence of base numbers 153 to 443 described in SEQ ID NO: 35;
(B) a DNA comprising the nucleotide sequence of base numbers 93 to 380 described in SEQ ID NO: 37;
(83) The DNA according to (60), wherein the DNA encoding the heavy chain polypeptide is a DNA of the following (a), and the DNA encoding the light chain polypeptide is a DNA of the following (b): Or the genetically modified host according to any of (63):
(A) a DNA having the nucleotide sequence of base numbers 96 to 1508 described in SEQ ID NO: 35;
(B) a DNA having the nucleotide sequence of base numbers 33 to 743 described in SEQ ID NO: 37;
(84) The genetically modified host according to any one of (60) to (62) or (64) to (83) (except when the host is a fertilized egg) A human monoclonal antibody or a part thereof to be produced.
(85) A pharmaceutical composition comprising the human antibody according to (1) or (2) and a pharmaceutically acceptable carrier.
(86) A pharmaceutical composition comprising the human monoclonal antibody or a part thereof according to any of (3) to (42), and a pharmaceutically acceptable carrier.
(87) A pharmaceutical composition comprising the human monoclonal antibody according to (84) or a part thereof, and a pharmaceutically acceptable carrier.
(88) The pharmaceutical composition according to any of (85) to (87), wherein the pharmaceutical composition is used for inhibiting AILIM-mediated signal transduction into cells.
(89) The pharmaceutical composition according to any of (85) to (87), wherein the pharmaceutical composition is used for suppressing the growth of AILIM-expressing cells.
(90) The pharmaceutical composition according to any of (85) to (87), wherein the pharmaceutical composition is used for suppressing cytokine production by cells expressing AILIM.
(91) The pharmaceutical composition according to any of (85) to (87), wherein the pharmaceutical composition is used for inducing signal transduction into cells via AILIM.
(92) The pharmaceutical composition according to any one of (85) to (87), wherein the pharmaceutical composition is used for inducing proliferation of AILIM-expressing cells.
(93) The pharmaceutical composition according to any of (85) to (87), wherein the pharmaceutical composition is used for inducing cytokine production by cells expressing AILIM.
(94) The pharmaceutical composition is used for inducing antibody-dependent cytotoxicity against AILIM-expressing cells and / or for inducing immune cell lysis or cell death of AILIM-expressing cells. The pharmaceutical composition according to any one of the above (85) to (87).
(95) A pharmaceutical composition comprising a substance having an activity of controlling AILIM-mediated signal transduction and a pharmaceutically acceptable carrier, for suppressing, treating or preventing delayed-type allergy.
(96) The pharmaceutical composition according to (95), wherein the substance is a proteinaceous substance.
(97) The pharmaceutical composition according to (96), wherein the proteinaceous substance is any one selected from the following group:
a) an antibody or a portion thereof that binds to AILIM;
b) a polypeptide comprising all or part of the extracellular region of AILIM;
c) a fusion polypeptide consisting of all or part of the extracellular region of AILIM and all or part of the constant region of the immunoglobulin heavy chain; and
d) A polypeptide that binds to AILIM.
(98) The pharmaceutical composition according to (97), wherein the antibody that binds to AILIM is the human antibody according to (1) or (2).
(99) The pharmaceutical composition according to (97), wherein the antibody that binds to AILIM is the human monoclonal antibody according to any of (3) to (42).
(100) The pharmaceutical composition according to (97), wherein the antibody against AILIM is the human monoclonal antibody according to (84).
(101) The pharmaceutical composition according to the above (95), wherein the substance is a non-protein substance.
(102) The pharmaceutical composition according to (101), wherein the non-proteinaceous substance is DNA, RNA or a chemically synthesized compound.
(103) A method for identifying a substance that binds to AILIM or AILIM ligand, comprising the following steps:
(A) preparing an insoluble carrier on which a polypeptide containing all or part of the extracellular region of AILIM is immobilized;
(B) preparing a polypeptide comprising all or part of the extracellular region of the AILIM ligand labeled with a label capable of producing a detectable signal;
(C) reacting the insoluble carrier of step (a) with the polypeptide of step (b);
(D) reacting the insoluble carrier of step (a), the polypeptide of step (b) and the substance in any order;
(E) the signal generated by the labeling substance contained in the complex produced by the reaction of step (c) and the signal produced by the labeling substance contained in the complex produced by the reaction of step (d) are respectively Detecting; and
(F) comparing each of the signal magnitudes detected in step (e).
(104) A method for identifying a substance that binds to AILIM or AILIM ligand, comprising the following steps:
(A) preparing an insoluble carrier on which a polypeptide containing all or a part of the extracellular region of AILIM ligand is immobilized;
(B) preparing a polypeptide comprising all or part of the extracellular region of AILIM labeled with a labeling substance capable of producing a detectable signal;
(C) reacting the insoluble carrier of step (a) with the polypeptide of step (b);
(D) reacting the insoluble carrier of step (a), the polypeptide of step (b) and the substance in any order;
(E) the signal generated by the labeling substance contained in the complex produced by the reaction of step (c) and the signal produced by the labeling substance contained in the complex produced by the reaction of step (d) are respectively Detecting; and
(F) comparing each of the signal magnitudes detected in step (e).
(105) A polypeptide comprising all or a part of the extracellular region of AILIM is obtained by combining a polypeptide comprising all or a part of the extracellular region of AILIM with all or a part of a constant region of a heavy chain of immunoglobulin. The method according to (103) or (104), wherein the method is a fusion polypeptide.
(106) a polypeptide containing all or a part of the extracellular region of the AILIM ligand, a polypeptide containing all or a part of the extracellular region of the AILIM ligand, and all or a part of a constant region of a heavy chain of an immunoglobulin; The method according to (103) or (104), which is a fusion polypeptide consisting of:
(107) The above (103) to the above (103), wherein the AILIM is human AILIM.
The method according to any one of (106).
(108) The method according to any of (103) to (107), wherein the AILIM ligand is a human AILIM ligand.
[0027]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail by clarifying the meanings of phrases used in the present invention.
The `` mammal '' in the present invention means human, cow, goat, rabbit, mouse, rat, hamster, guinea pig, and the like, preferably human, rabbit, rat, hamster or mouse, and particularly preferably, A human, rat, hamster or mouse.
The terms “non-human mammal” and “non-human mammal” used in the present invention are synonymous with each other, and refer to any mammal other than a human in the above-defined mammals.
[0028]
The term `` amino acid '' used in the present invention means any naturally occurring amino acid, and is preferably represented as follows according to the three-letter or single-letter notation of the alphabet used to represent amino acids. Amino acids.
Glycine (Gly / G), alanine (Ala / A), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), serine (Ser / S), threonine (Thr / T), asparagine Acid (Asp / D), glutamic acid (Glu / E), asparagine (Asn / N), glutamine (Glu / Q), lysine (Lys / K), arginine (Arg / R), cysteine (Cys / C), methionine (Met / M), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), histidine (His / H), proline (Pro / P).
[0029]
The term “AILIM” as used in the present invention is an abbreviation of Activation inducible lymphocyte immunomodulatory molecule, and refers to a mammalian cell surface molecule having the structure and function reported as described above, and particularly preferably a human-derived molecule. (E.g., International Immunology, Vol. 12, No. 1, p. 51-55; GenBank Accession Number: BAA82129 (human); BAA82128 (rat); BAA82127 (rat mutant); BAA82126 (mouse) ).
This AILIM can be obtained from ICOS (Nature, Vol. 397, No. 6716, p. 263-266, 1999) or JTT-1 antigen / JTT-2 antigen (Japanese Patent Application Publication No. H11-29599; International). Although they are also referred to as Patent Application Publication No. WO98 / 38216, those molecules mean the same molecule as each other.
The “AILIM ligand” in the present invention means a molecule called B7h, B7RP-1, GL50 or LICOS, which is a cell surface molecule that interacts with the costimulatory molecule AILIM (ICOS) (Nature, Vol. 402, No. 6763, p.827-832, 1999; Nature Medicine, Vol.5, No. 12, p.1365-1369, 1999; J. Immunology, Vol.164, p.1653-1657, 2000; Curr Biol., Vol.10, No.6, p.333-336, 2000).
[0030]
In the present invention, "AILIM" includes a polyamino acid sequence having substantially the same amino acid sequence as the amino acid sequence of each mammalian AILIM, particularly preferably human AILIM, described in the above-mentioned publication. Peptides are also included. Furthermore, a human AILIM mutant similar to the already identified rat-derived AILIM mutant (GenBank Accession Number: BAA82127) is also included in the “AILIM” of the present invention.
Further, “AILIM ligand” in the present invention is defined with the same meaning as described above.
[0031]
Here, “a polypeptide having substantially the same amino acid sequence” means a mutant polypeptide as described below.
That is, a plurality of amino acids in the amino acid sequence, preferably 1 to 10 amino acids, particularly preferably, as long as they have substantially the same biological properties as natural AILIM (particularly preferably human-derived AILIM). Is a mutant polypeptide having an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted and / or modified, and a plurality of amino acids in the amino acid sequence of the natural type AILIM (particularly preferably human-derived AILIM). A mutant polypeptide having an amino acid sequence to which an amino acid is added, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids.
Furthermore, a mutant polypeptide having a plurality of such substitutions, deletions, modifications and additions may be used.
Further, “AILIM ligand” in the present invention is defined with the same meaning as described above.
The AILIM (particularly human AILIM) and AILIM ligand (particularly human AILIM ligand) in the present invention are known in the technical field such as a chemical synthesis method, a cell culture method and the like, in addition to a gene recombination technique. It can be produced by appropriately using a known method or its modification method.
Such amino acid substitution, deletion, or insertion can be performed according to a conventional method (Experimental Medicine Supplement, “Genetic Engineering Handbook” (1992), etc.).
[0032]
Examples of the method for producing the mutant polypeptide as described above include, for example, a synthetic oligonucleotide-directed mutagenesis method (gapped duplex), a method of randomly introducing a point mutation by nitrous acid or sulfurous acid treatment, and a Ba131 enzyme. , A cassette mutation method, a linker scanning method, a misincorporation method, a mismatch primer method, a DNA segment synthesis method, and the like.
[0033]
The synthetic oligonucleotide-directed mutagenesis (gapped duplex) method can be performed, for example, as follows. A region to be mutagenized is cloned into an M13 phage vector having an amber mutation to prepare a single-stranded phage DNA. The RFI DNA of the M13 vector having no amber mutation is linearized by treatment with a restriction enzyme, mixed with the single-stranded phage DNA described above, denatured, and annealed to form “gapped duplex DNA”. A synthetic oligonucleotide having a mutation introduced therein is hybridized, and a closed circular double-stranded DNA is obtained by a reaction between DNA polymerase and DNA ligase. This DNA is transfected into an Escherichia coli mutS strain deficient in mismatch modification ability, the grown phage is infected into Escherichia coli having no suppressor function, and only phages having no amber mutation are selected.
[0034]
In addition, a method for introducing a point mutation with nitrite utilizes, for example, the following principle. When DNA is treated with nitrous acid, the base is deaminated, adenine becomes hypoxanthin, cytosine becomes uracil, and guanine becomes xanthine. When the deaminated DNA is introduced into cells, hypoxanthine and cytosine form hypoxanthine and adenine and xanthine form thymine at the time of DNA replication, so "A: T" becomes "G: C" and "A: T" becomes "G: C". “G: C” replaces “A: T”. Actually, a single-stranded DNA fragment treated with nitrite is hybridized to “gapped duplex DNA”, and the mutant strain may be isolated by the same procedure as in the synthetic oligonucleotide-directed mutagenesis (gapped duplex) method. .
[0035]
Further, “AILIM” in the present invention includes “part” of the AILIM. Here, “part” means a polypeptide containing any partial sequence in the amino acid sequence of AILIM defined above.
Preferably, it refers to the extracellular region of AILIM as defined above (particularly preferably human AILIM) or any part thereof.
Further, “AILIM ligand” in the present invention is also defined with the same meaning as described above.
The “part” of AILIM (preferably, the extracellular region of AILIM or any part thereof) may be obtained by a genetic recombination technique or a chemical synthesis method according to a known method known in the art described below or a modification method thereof. It can be produced by a suitable method, or can be produced by appropriately cleaving AILIM (particularly preferably human AILIM) isolated by a cell culture method using a proteolytic enzyme or the like.
The “part of AILIM ligand” in the present invention can also be produced by the same method as described above.
[0036]
The “human antibody” of the present invention is a human antibody that binds to AILIM as defined above or a part thereof (particularly preferably, human-derived AILIM or a part thereof). Specifically, it means a human-derived polyclonal antibody (human polyclonal antibody, human antiserum) or a human-derived monoclonal antibody (human monoclonal antibody). The "human monoclonal antibody" of the present invention is a human monoclonal antibody that binds to AILIM or a part thereof (particularly preferably, AILIM or a part thereof derived from human) as defined above.
More specifically, the variable region (Variable region) of the heavy chain (H chain) and the constant region of the H chain (Constant Region) constituting the immunoglobulin and the variable region of the light chain (L chain) and the constant region of the L chain are defined. The entire region including the human immunoglobulin is derived from the gene encoding human immunoglobulin. Examples of the L chain include a human κ chain and a human λ chain.
[0037]
The human monoclonal antibody that binds to the AILIM of the present invention (particularly preferably human-derived AILIM) or a part thereof is described in any of (5) to (42) or (84) of the present invention as described above. It is a human monoclonal antibody having characteristics.
More specifically, there are various human monoclonal antibodies having various properties and industrial utility as described in the examples and drawings below.
A preferred embodiment of the human monoclonal antibody of the present invention is a human monoclonal antibody that binds to AILIM described in any of (5) to (42) or (84) of the present invention or a part thereof.
A particularly preferred embodiment is a human monoclonal antibody that binds to human AILIM described in (30) or (39) of the present invention.
[0038]
The "human monoclonal antibody" of the present invention can be prepared by immunizing any of the following immunogens (antigens) to a human antibody-producing transgenic non-human mammal described below.
(B) a natural cell or an artificially established cell line that expresses AILIM as defined above (particularly preferably human-derived AILIM) on the cell surface;
(B) a transgenic cell produced using a transgenic technique so as to express AILIM as defined above (particularly preferably human-derived AILIM) on the cell surface;
(C) a cell lysate obtained by solubilizing the cells of (a) or (b), or a polypeptide fragment of AILIM (particularly preferably human-derived AILIM) purified from the cell lysate;
(D) Using a gene recombination technique to express a part (particularly preferably, the extracellular region or any peptide thereof) of AILIM (particularly preferably human-derived AILIM) as defined above as a soluble polypeptide. Genetically modified cells produced by;
(E) A culture supernatant obtained by culturing the genetically modified cells of (d) or an extracellular domain polypeptide of AILIM (particularly preferably human-derived AILIM) purified from the culture supernatant (soluble AILIM) Or
(F) Part of AILIM (particularly preferably human-derived AILIM) that is chemically synthesized (particularly preferably, its extracellular region or any peptide thereof).
[0039]
Further, the human monoclonal antibody of the present invention can be obtained by using a cDNA (preferably a vector containing the cDNA) encoding each of the heavy chain and light chain of such a human monoclonal antibody of the present invention using a gene recombination technique. A genetically modified host obtained by transforming a host, wherein the “genetically modified host” of the present invention producing a recombinant human monoclonal antibody (where the host is a eukaryotic cell other than a fertilized egg) CHO, mammalian cells such as lymphocytes and myeloma, etc.) can also be obtained from the culture supernatant by culturing.
Specifically, the genetically modified host according to any one of (60) to (62) or (64) to (80) of the present invention (where the host is a eukaryotic cell other than a fertilized egg) (Preferably mammalian cells such as CHO, lymphocytes and myeloma).).
[0040]
Further, the human monoclonal antibody of the present invention may be a human monoclonal antibody having any isotype of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE. Preferably, it is IgG (IgG1, IgG2, IgG3, IgG4), preferably IgG1, IgG2 or IgG4.
[0041]
The human monoclonal antibody of the present invention immunizes a human antibody-producing transgenic non-human mammal, such as a human antibody-producing transgenic mouse described below, with any of the immunogens (antigens) described in (a) to (f) above. The monoclonal antibody can be produced according to an existing general production method.
That is, for example, the human antibody-producing transgenic non-human mammal is immunized with the antigen together with Freund's Adjuvant as necessary. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. In addition, a monoclonal antibody is prepared by preparing a fusion cell (hybridoma) from the antibody-producing cell obtained from the immunized animal and a myeloma cell (myeloma cell) having no autoantibody-producing ability, cloning the hybridoma, It is produced by selecting a clone that produces a monoclonal antibody showing a specific affinity for the antigen used for immunization.
[0042]
More specifically, it can be produced as follows. That is, the human antibody-producing transgenic non-human mammal (particularly preferably described below) is combined with the immunogen of any one of the above (a) to (c) together with Freund's Adjuvant, if necessary, together with Freund's Adjuvant. Immunization is carried out by subcutaneous, intramuscular, intravenous, intravenous, intraperitoneal or intraperitoneal injection or transplantation of human antibody-producing transgenic mice "). Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of immunizations and the time interval can be appropriately changed depending on the nature of the immunogen used.
[0043]
The preparation of a fusion cell (hybridoma) secreting a human monoclonal antibody can be performed according to the method of Koehler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and a modification method analogous thereto. That is, spleen, lymph node, bone marrow or tonsil, etc., obtained from a human antibody-producing transgenic non-human mammal immunized as described above, and antibody-producing cells preferably contained in the spleen, preferably mice, rats, It is prepared by cell fusion with mammals such as guinea pig, hamster, rabbit or human, more preferably mouse, rat or human derived myeloma cells having no autoantibody-producing ability.
[0044]
Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (ATCC No. CRL-1580), P3 / NSI / 1-Ag4-1 (NS-1), and P3 / X63-Ag8. . U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), NS0, PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag. 2.3. And human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15.
Screening for cells producing monoclonal antibodies (eg, hybridomas) involves culturing the cells, eg, in a microtiter plate, and using the culture supernatant of the wells in which growth has been observed against the immunizing antigen used in the immunization described above. The reactivity can be measured by, for example, measuring an enzyme immunoassay such as RIA (radio immunoassay) or ELISA (enzyme-linked immuno-solvent assay).
Production of the monoclonal antibody from the hybridoma is carried out in vitro on the hybridoma or in vivo in a mouse, rat, guinea pig, hamster or rabbit or the like, preferably in a mouse or rat, more preferably in ascites of a mouse, and the obtained culture supernatant. Alternatively, it can be performed by isolating from ascites of a mammal.
[0045]
Further, the monoclonal antibody of the present invention can be produced in large quantities by the following method.
(1) Cloning a gene (such as cDNA) encoding each of the heavy and light chains of the monoclonal antibody from the hybridoma.
(2) An expression vector is prepared by inserting the genes encoding each of the cloned heavy and light chains separately or into a single vector.
(3) introducing the vector into a fertilized egg of a desired non-human mammal (such as a goat);
(4) The fertilized egg into which the gene has been introduced is transplanted into the uterus of a foster mother to obtain a chimeric non-human animal.
(5) The transgenic non-human mammal (cow, goat, or chick) in which the gene encoding each of the heavy and light chains has been incorporated into an endogenous gene by further mating the chimeric goat with another non-human mammal. Make sheep or pigs).
(6) A large amount of a monoclonal antibody derived from the human monoclonal antibody gene is obtained from the milk of the transgenic non-human mammal (Nikkei Science, April 1997, pages 78 to 84).
Human monoclonal antibodies produced by this method are also included in the present invention. When the monoclonal antibody-producing cells are cultured in vitro, the hybridoma is grown, maintained, and stored in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culture method. It can be carried out using a known nutrient medium as used for producing monoclonal antibodies or any nutrient medium derived and prepared from a known basal medium.
[0046]
As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium, etc. The basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.
For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites is obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.
[0047]
The human monoclonal antibody of the present invention includes a heavy chain having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of each of the heavy chain and / or light chain constituting the antibody. Also included are human monoclonal antibodies consisting of a light chain.
[0048]
Here, "several amino acids" means a plurality of amino acids, specifically 1 to 10 amino acids, and preferably 1 to 5 amino acids.
The above-mentioned partial modification (deletion, substitution, insertion, addition) of an amino acid into the amino acid sequence of the human monoclonal antibody of the present invention is introduced by partially modifying the base sequence encoding the amino acid sequence. can do. This partial modification of the nucleotide sequence can be carried out by a conventional method using a known site-specific mutagenesis method (Site specific mutagenesis) (Proc. Natl. Acsd. Sci. USA, Vol. 81, p. 5662-5666, 1984).
[0049]
The “transgenic human antibody-producing non-human mammal” of the present invention, a particularly preferred embodiment of a human antibody-producing transgenic mouse can be produced according to a previously reported report (Nature Genetics, Vol. 7, p. 13-21, 1994). Nature Genetics, Vol. 15, p. 146-156, 1997; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; Nikkei Science, June, pp. 40-50, 1995. International Application Publication No. WO 94/25585; Nature, Vol. 368, p. 856-859, 1994;
Specifically, the human antibody-producing transgenic mouse can be produced, for example, by using a technique comprising the following steps. Other human antibody-producing transgenic non-human mammals can be produced in a similar manner.
[0050]
(1) The mouse endogenous immunoglobulin heavy chain gene is functionally disabled by substituting at least a part of the mouse endogenous immunoglobulin heavy chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination. The step of producing an activated knockout mouse.
(2) By replacing at least a part of the mouse endogenous immunoglobulin light chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination, the mouse endogenous immunoglobulin light chain gene (particularly the κ chain gene) B) producing knockout mice that have been functionally inactivated.
(3) The desired region of the human immunoglobulin heavy chain locus is integrated into the mouse chromosome using a vector capable of carrying a large gene, such as a yeast artificial chromosome (YAC) vector. Producing a transgenic mouse.
(4) A transgenic mouse in which a desired region of a human immunoglobulin light chain (particularly κ chain) locus is integrated into a mouse chromosome using a vector capable of carrying a large gene represented by YAC or the like. The process of making.
(5) By crossing the knockout mouse and the transgenic mouse of (1) to (4) in any order, both the mouse endogenous immunoglobulin heavy chain locus and the mouse endogenous immunoglobulin light chain locus function. Producing a transgenic mouse that has been specifically inactivated and has both the desired region of the human immunoglobulin heavy chain locus and the desired region of the human immunoglobulin light chain locus integrated on the mouse chromosome.
[0051]
The knockout mouse is constructed so that the appropriate region of the mouse endogenous immunoglobulin locus is replaced by a foreign marker gene (such as a neomycin resistance gene) by homologous recombination so that the locus cannot be rearranged (rearranged). It can be produced by activating. For the inactivation using the homologous recombination, for example, a method called positive negative selection (Positive Negative Selection; PNS) can be used (Nikkei Science, May, p. 52-62). , 1994).
Functional inactivation of the immunoglobulin heavy chain locus can be achieved, for example, by introducing a disorder into a portion of the J or C region (eg, the Cμ region). Functional inactivation of an immunoglobulin light chain (for example, a κ chain) can be achieved by, for example, introducing a disorder into a part of the J region or the C region, or a region including a region spanning the J region and the C region. It is.
[0052]
The transgenic mouse can be prepared by a conventional method commonly used in the production of transgenic animals (for example, see the latest manual for animal cell experiments, published by LCI, Chapter 7, pp. 361 to 408, 1990). ). Specifically, for example, an HPRT-negative (lacking the hypoxanthine guanine phosphoribosyltransferase gene) ES cell (embryonic stem cell) derived from a normal mouse blastcyst is blasted with the human immunoglobulin heavy chain. The gene encoding the locus or the light chain locus or a part thereof and the yeast containing the YAC vector into which the HPRT gene has been inserted are fused by spheroplast fusion. ES cells in which the exogenous gene has been integrated on the mouse endogenous gene are selected by the HAT selection method. Next, the selected ES cells are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384). , 1980; U.S. Pat. No. 4,873,191). The blastocyst is implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse is obtained. By breeding the heterogeneic transgenic mice, homogenic transgenic mice can be obtained according to Mendel's law.
[0053]
The "part of the monoclonal antibody" in the present invention means a partial region of the above-described human monoclonal antibody of the present invention, and specifically, F (ab ') ₂ , Fab ′, Fab, variable fragment of antibody (Fv), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain opinion opponent eponymology). Ther. Patents), Vol. 6, No. 5, pp. 441-456, 1996).
[0054]
Here, "F (ab ') ₂ "And" Fab '"are produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain to form a disulfide bond existing between two H chains in the hinge region. Antibody fragments produced by digestion before and after. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond existing between the two heavy chains in the hinge region, resulting in V.V. _L (L chain variable region) and C _L (L chain constant region) and V chain _H (H chain variable region) and C _H Two homologous antibody fragments in which an H chain fragment composed of γ1 (the γ1 region in the H chain constant region) is linked by a disulfide bond at the C-terminal region can be produced. Each of these two homologous antibody fragments is called Fab '. When IgG is treated with pepsin, it is cleaved downstream of a disulfide bond existing between two H chains in the hinge region to produce an antibody fragment slightly larger than the two Fab's connected in the hinge region. Can be. This antibody fragment is expressed as F (ab ') ₂ That.
[0055]
The “binding rate constant (ka)” in the present invention means a value indicating the strength (degree) of the binding of the monoclonal antibody to the target antigen, calculated based on the kinetics of the antigen-antibody reaction. The “dissociation rate constant (kd)” means a value indicating the strength (degree) of dissociation of the monoclonal antibody from the target antigen, calculated based on the antigen-antibody reaction kinetics. The “dissociation constant (Kd)” is a value obtained by dividing the “dissociation rate constant (kd)” value by the “dissociation rate constant (ka)” value. These constants are used as indices indicating the affinity of the monoclonal antibody for the antigen and the neutralizing activity of the antigen.
[0056]
The constants can be analyzed according to various methods. However, a commercially available measurement kit, BiacoreX (manufactured by Amersham Pharmacia) or a similar kit, can be easily used according to the instruction manual and experimental operation method attached to the kit. Can be analyzed. The ka value, kd value and Kd value determined using the kit are respectively 1 / M. It is expressed by units of Sec, 1 / Sec and M (mole). The higher the ka value of the tested monoclonal antibody, the stronger the antigen binding activity, and the lower the Kd value, the stronger the neutralizing activity.
[0057]
The human monoclonal antibody of the present invention includes a human monoclonal antibody having a ka value, a kd value or a Kd value as shown in the following (1) to (3).
(1) The binding rate constant (ka) with human AILIM is 1.0 × 10 ⁴ (1 / M.Sec) or more, preferably 1.0 × 10 ⁵ A human monoclonal antibody that binds to human AILIM or a part thereof having a value of (1 / M.Sec) or more.
(2) The dissociation rate constant (kd) with human AILIM is 1.0 × 10 ^-4 (1 / Sec) or less, preferably 1.0 × 10 ^-5 (1 / Sec) A human monoclonal antibody that binds to human AILIM or a part thereof having the following value.
(3) The dissociation constant (Kd) with human AILIM is 1.0 × 10 ^-7 (1 / Sec) or less, preferably 1.0 × 10 ^-8 (M) or less, more preferably 1.0 × 10 ^-9 (M) A human monoclonal antibody reactive to human AILIM or a part thereof having the following numerical values.
It should be noted that the values of ka, kd, and Kd described above are expected to vary slightly depending on various conditions at the time of measurement, but may vary as an error range. It is.
[0058]
The "cell producing a monoclonal antibody" of the present invention or the "genetically modified host" of the present invention producing a recombinant human monoclonal antibody (here, the host is a cell other than a fertilized egg) It means any cell that produces the above-described human monoclonal antibody of the present invention.
Specifically, for example, cells described in any of the following (1) to (3) can be mentioned, but are not limited thereto.
(1) Human monoclonal antibody-producing B cells which are obtained by immunizing the above-described human antibody-producing transgenic non-human mammal with the immunogen (antigen) as defined above, and which can be collected from the immunized animal.
(2) The aforementioned fused cells (hybridomas) obtained by cell fusion of the human monoclonal antibody-producing B cells thus obtained with mammalian myeloma cells.
(3) a gene encoding the human monoclonal antibody (either a gene encoding a heavy chain or a gene encoding a light chain) isolated from the human monoclonal antibody-producing B cells or human monoclonal antibody-producing fusion cells (hybridoma) Or both genes) and a gene obtained by transforming a cell other than the B cell and the hybridoma (eg, a CHO (Chinese hamster ovarian) cell, a BHK (Baby hamster childy) cell, a lymphocyte such as myeloma, etc.). Genetically modified cells that produce recombinant human monoclonal antibodies.
[0059]
Here, the transgenic cells producing the transgenic human monoclonal antibody according to the above (3), that is, the transgenic human monoclonal antibody produced by the B cell of the above (1) or the hybridoma of the above (2) Genetically modified cells that produce the body.
The “host” in the “genetically modified host” of the present invention includes not only cells derived from various mammals as described above, but also any non-human mammal (goat, pig, sheep, cow, etc.). Fertilized eggs are also included. This fertilized egg contains a gene encoding any monoclonal antibody (preferably, a human monoclonal antibody) against human AILIM of the present invention (either a gene encoding a heavy chain or a gene encoding a light chain, or both genes). ) Is introduced using an expression vector or the like to obtain the transgenic fertilized egg of the present invention. This transgenic fertilized egg is used for producing a transgenic animal in order to prepare the above-mentioned desired protein from milk in large quantities (Nikkei Science, April 1997, pages 78 to 84).
[0060]
The "substance" constituting the present invention, specifically, "a substance having an activity of controlling AILIM-mediated signal transduction", more specifically, "a cytokine that suppresses the growth of AILIM-expressing cells or is produced by AILIM-expressing cells" The term "substance having an activity of inhibiting the production of" refers to a natural substance existing in nature or any substance prepared artificially.
Further, the “substance” relating to the “substance that binds to AILIM” and the “substance that binds to AILIM ligand” in the present invention also means a natural substance existing in the natural world or an artificially prepared substance.
Here, “signal transduction via AILIM” refers to any change in phenotype (cell proliferation, cell activation, cell inactivation, cell death, and / or AILIM-expressing cells) in cells expressing AILIM. (A change in the ability to produce any of the cytokines).
[0061]
The “substance” can be broadly classified into “proteinaceous substance” and “non-proteinaceous substance”. Examples of the "proteinaceous substance" include a polypeptide and an antibody (polyclonal antibody, monoclonal antibody or a part of the monoclonal antibody, particularly preferably the above-described human antibody) described below.
When the substance is an antibody, it is preferably a monoclonal antibody. When the substance is a monoclonal antibody, it includes not only a monoclonal antibody derived from a non-human mammal but also a recombinant chimeric monoclonal antibody, a recombinant human monoclonal antibody, and the above-mentioned human monoclonal antibodies.
[0062]
Here, the “recombinant chimeric monoclonal antibody” is a monoclonal antibody produced by genetic engineering, and specifically, the variable region thereof is a non-human mammal (mouse, rat, hamster, etc.) immunoglobulin. A chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody, wherein the variable region is derived from a human and the constant region is a human immunoglobulin-derived constant region.
[0063]
The “human-type monoclonal antibody (CDR-grafted antibody)” is a monoclonal antibody produced by genetic engineering, and specifically, a part or all of the complementarity determining region of the hypervariable region. A non-human mammal (mouse, rat, hamster, etc.) is a complementarity determining region of a hypervariable region derived from a monoclonal antibody, the framework region of the variable region is a human immunoglobulin-derived variable region framework region, and A humanized monoclonal antibody, wherein the constant region is a human immunoglobulin-derived constant region.
[0064]
Here, the complementarity-determining regions of the hypervariable region are three regions (Complementarity-determining resin; CDR1 and CDR2) which are present in the hypervariable region in the variable region of the antibody and are directly and complementary to the antigen. , CDR3), and the framework region of the variable region refers to four relatively conserved regions (Framework regions; FR1, FR2, FR3, FR4) intervening before and after the three complementarity determining regions.
In other words, it means a monoclonal antibody in which all but a part or all of the complementarity determining region of the hypervariable region of a non-human mammal-derived monoclonal antibody has replaced the corresponding region of human immunoglobulin.
[0065]
The human immunoglobulin-derived constant region has an amino acid sequence unique to each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. In the present invention, the humanized monoclonal antibody May be a human immunoglobulin constant region belonging to any isotype. Preferably, it is a human IgG constant region. Further, the framework region of the variable region derived from human immunoglobulin is not limited.
[0066]
When the “substance” of the present invention is a polypeptide, it includes a polypeptide described below, a fragment (oligopeptide) of the polypeptide, a fusion polypeptide, and a chemically modified product thereof. Oligopeptides include peptides consisting of 5 to 30 amino acids, preferably 5 to 20 amino acids. The chemical modification can be designed according to various purposes such as an increase in blood half-life when administered to a living body or an increase in resistance or absorption to degradation in the digestive tract during oral administration. .
Specific examples of the polypeptide include those described below.
(1) a polypeptide comprising all or part of the extracellular region of AILIM;
(2) a fusion polypeptide consisting of all or part of the extracellular region of AILIM and all or part of the constant region of the heavy chain of immunoglobulin; or
(3) A polypeptide that binds to AILIM.
[0067]
Such "non-proteinaceous substances" include DNA, RNA and chemically synthesized compounds.
Here, “DNA” refers to “a DNA of the aforementioned AILIM (preferably human AILIM)” which is useful as an antisense DNA drug designed based on the base sequence of DNA (including cDNA and genomic DNA). DNA containing a partial base sequence or chemically modified DNA obtained by chemically modifying them. " That is, the antisense DNA can inhibit transcription of the DNA encoding AILIM into mRNA or translation of the mRNA into protein by hybridizing to DNA or RNA encoding AILIM.
[0068]
Here, the “partial base sequence” means a partial base sequence consisting of an arbitrary number of bases at an arbitrary site. Examples of the partial base sequence include a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, and more preferably a continuous partial base sequence of 5 to 50 bases. Preferably, a partial base sequence of 5 to 30 bases is used.
[0069]
When the DNA is used as an antisense drug, the DNA has an increased half-life in blood (stability) when administered to the body of a patient, an increased permeability of the intracellular membrane, or an increased oral administration. A part of the base sequence of the DNA can be chemically modified for the purpose of increasing the resistance to degradation in the digestive tract or increasing the absorption in such cases. The chemical modification includes, for example, chemical modification of a phosphate bond, ribose, nucleobase, sugar moiety, 3 ′ and / or 5 ′ end in the structure of the oligonucleotide.
As the modification of the phosphate bond, one or more of the bonds may be a phosphodiester bond (D-oligo), a phosphorothioate bond, a phosphorodithioate bond (S-oligo), a methylphosphonate bond (MP-oligo), a phosphoramido bond. Changes to any or a combination of a date bond, a non-phosphate bond and a methylphosphonothioate bond can be included. Examples of the modification of ribose include a change to 2′-fluororibose or 2′-O-methylribose. Modifications of nucleobases include changes to 5-propynyluracil or 2-aminoadenine.
[0070]
As used herein, “RNA” refers to “RNA containing a partial nucleotide sequence of the RNA or a RNA thereof containing a partial nucleotide sequence of the RNA useful as an antisense RNA drug designed based on the nucleotide sequence of the RNA encoding AILIM (preferably human AILIM)”. Chemically modified RNA ”. The antisense RNA hybridizes to DNA encoding AILIM, and thereby hybridizes to DNA or RNA encoding AILIM to transcribe the DNA encoding AILIM to mRNA or to a protein of the mRNA. Translation can be inhibited.
[0071]
Here, the “partial base sequence” means a partial base sequence consisting of an arbitrary number of bases at an arbitrary site. Examples of the partial base sequence include a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, and more preferably a continuous partial base sequence of 5 to 50 bases. Preferably, a partial base sequence of 5 to 30 bases is used.
The antisense RNA increases the blood half-life when the RNA is administered to a patient, increases the permeability of the inner cell membrane, or increases or absorbs degradation resistance in the digestive tract when administered orally. A part of the base sequence of the RNA can be chemically modified for the purpose of increasing the number of RNAs. Examples of the chemical modification include a chemical modification as applied to the above-described antisense DNA.
[0072]
"Chemically synthesized compound" means any compound other than the above-mentioned DNA, RNA and proteinaceous substances, and is a compound having a molecular weight of about 100 to about 1000 or less, preferably a compound having a molecular weight of about 100 to about 800. And more preferably a compound having a molecular weight of about 100 to about 600.
[0073]
The “polypeptide” included in the definition of the “substance” means a part (fragment) of a polypeptide chain constituting AILIM (preferably human AILIM), and preferably a part of a polypeptide constituting AILIM. It means the whole or a part of the extracellular region (the region may optionally have 1 to 5 amino acids added to its N-terminal and / or C-terminal).
AILIM according to the present invention is a transmembrane molecule that penetrates a cell membrane constituted by one or two polypeptide chains.
[0074]
As used herein, the term “transmembrane protein” refers to a cell that is linked to a membrane by a hydrophobic peptide region that penetrates the lipid bilayer once or several times, as seen in many receptors or cell membrane surface molecules. It refers to a protein having a structure composed of three main regions: an outer region (extracellular region), a transmembrane region (transmembrane region), and a cytoplasmic region. Further, such transmembrane proteins can be homodimers, heterodimers or heterodimers, as monomers or with another chain having the same amino acid sequence or a chain having a different amino acid sequence, respectively. Alternatively, each receptor or cell surface molecule is constituted by the presence of an oligomer.
[0075]
Here, the “extracellular region” refers to all or a part of the partial structure (partial region) existing on the outer side of the membrane to which the membrane protein is connected, of the entire structure of the cell membrane transmembrane protein as described above. In other words, all or part of the region other than the region taken up in the membrane (transmembrane region) and the region present in the cytoplasm following the region in the membrane (intracellular region) means.
[0076]
The “fusion polypeptide” included in the aforementioned “proteinaceous substance” refers to all or a part of the extracellular region of the polypeptide constituting AILIM (preferably human AILIM) and “immunoglobulin (Ig, preferably, All or part of the constant region of the heavy chain of human Ig). " It is preferably a fusion polypeptide of the extracellular region of AILIM and a part of the constant region of the heavy chain of human IgG, and particularly preferably the hinge region, CH2 domain and CH3 domain of the extracellular region of AILIM and the heavy chain of human IgG. And a fusion polypeptide with a region (Fc) consisting of In addition, IgG1 is preferable as IgG. As AILIM, human, mouse or rat (preferably human) AILIM is preferable.
[0077]
Here, “all or a part of the constant region of the heavy chain of immunoglobulin (Ig)” is preferably a constant region (Constant region) of a heavy chain (Heavy Chain, H chain) of an immunoglobulin derived from human, and an Fc region. Or mean some of them. The immunoglobulin may be of any class and subclass, and specifically includes IgG (IgG1, IgG2, IgG3 and IgG4), IgM, IgA (IgA1 and IgA2), IgD and IgE. Can be mentioned. Preferably, it is IgG (IgG1, IgG2, IgG3 or IgG4) or IgM. Particularly preferred examples in the present invention are immunoglobulins belonging to human-derived IgG (IgG1, IgG2, IgG3 or IgG4).
[0078]
The immunoglobulin is a Y-shape in which four homologous light chains (Light Chain, L chain) and two homologous heavy chains (Heavy Chain, H chain) are linked by a disulfide bond (SS bond). Having the following structural unit. The light chain comprises a light chain variable region (V _L ) And the light chain constant region (C _L ). The heavy chain has a heavy chain variable region (V _H ) And the heavy chain constant region (C _H ).
[0079]
The heavy chain constant region is comprised of several domains each having a unique amino acid sequence for each class (IgG, IgM, IgA, IgD and IgE) and subclass (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). .
[0080]
The heavy chains of IgG (IgG1, IgG2, IgG3 and IgG4) are V _H , CH1 domain, hinge region, CH2 domain and CH3 domain.
[0081]
Similarly, the heavy chain of IgG1 has V _H , Cγ ₁ 1 domain, hinge region, Cγ ₁ 2 domains and Cγ ₁ It consists of three domains. The heavy chain of IgG2 is composed of V _H , Cγ ₂ 1 domain, hinge region, Cγ ₂ 2 domains and Cγ ₂ It consists of three domains. The heavy chain of IgG3 is V _H , Cγ ₃ 1 domain, hinge region, Cγ ₃ 2 domains and Cγ ₃ It consists of three domains. The heavy chain of IgG4 has V _H , Cγ ₄ 1 domain, hinge region, Cγ ₄ 2 domains and Cγ ₄ It consists of three domains.
[0082]
The heavy chain of IgA is V _H , Cα1 domain, hinge region, Cα2 domain and Cα3 domain.
[0083]
Similarly, the heavy chain of IgA1 _H , Cα11 domain, hinge region, Cα ₁ 2 domains and Cα ₁ It consists of three domains. The heavy chain of IgA2 is composed of V _H , Cα ₂ 1 domain, hinge region, Cα ₂ 2 domains and Cα ₂ It consists of three domains.
[0084]
The heavy chain of IgD consists of V _H , Cδ1 domain, hinge region, Cδ2 domain and Cδ3 domain.
[0085]
The heavy chain of IgM consists of V _H , Cμ1 domain, Cμ2 domain, Cμ3 domain and Cμ4 domain, and has no hinge region as found in IgG, IgA and IgD.
[0086]
The heavy chain of IgE is V _H , Cε1 domain, Cε2 domain, Cε3 domain and Cε4 domain, and has no hinge region as found in IgG, IgA and IgD.
[0087]
Further, taking IgG as an example, when IgG is treated with papain, it is cleaved slightly at the N-terminal side of the disulfide bond present in the hinge region connecting the two heavy chains, and _H And C _H 1 and two homologous Fabs in which one light chain and one light chain are linked by a disulfide bond, and a hinge region, C _H 2 domains and C _H Two homologous heavy chain fragments consisting of three domains generate one Fc linked by a disulfide bond (hereinafter, "Immunological Illustrated", 2nd edition, original, 65-75, 1992, published by Nankodo, And "Focus on the latest medical science:" Recognition mechanism of the immune system "", pages 4 to 7, 1991, published by Nankodo)
[0088]
That is, the “part of the constant region of the immunoglobulin heavy chain” described above means a part of the constant region of the immunoglobulin heavy chain having the above-described structural characteristics, and preferably lacks the C1 domain. It is a constant region or Fc region. Specifically, in the case of IgG, IgA or IgD, a region consisting of each hinge region, C2 domain and C3 domain is mentioned, and in the case of IgM or IgE, respective C2 domain, C3 domain and C4 domain Region. Particularly preferred examples include a human-derived IgG1 Fc region.
[0089]
Since the above fusion polypeptide has a part of the constant region (for example, Fc) of immunoglobulin such as IgG as described above as a fusion partner, the property of protein A that specifically binds to the immunoglobulin fragment is used. There is an advantage in that the fusion polypeptide can be extremely easily purified by using affinity column chromatography using. Further, since various antibodies against Fc of various immunoglobulins are provided, immunoassay of the fusion polypeptide can be easily performed using antibodies against the Fc.
[0090]
“Polypeptide” included in the definition of “substance” also includes “polypeptide that binds to AILIM”.
As the “polypeptide that binds to AILIM”, a molecule referred to as “AILIM ligand” defined above, such as B7h, B7RP-1, GL50 or LICOS (Nature, Vol. 402, No. 6763, p. 827-) Nature Medicine, Vol.5, No.12, p.1365-1369, 1999; J. Immunology, Vol.164, p.1653-1657, 2000; Curr.Biol., Vol.10, No.832, 1999; 6, p. 333-336, 2000).
Preferably, a polypeptide containing all or a part of the extracellular region of the ligand (B7h, B7RP-1, GL50, LICOS), or a constant region of the heavy chain of the polypeptide and an immunoglobulin (preferably a human immunoglobulin) Is a fusion polypeptide consisting of all or part of Here, the terms “extracellular region” and “immunoglobulin heavy chain constant region” have the same meaning as described above.
[0091]
The above-mentioned polypeptide, a part (fragment) of the polypeptide and a fusion polypeptide are known in the technical field such as a chemical synthesis method and a cell culture method, in addition to the gene recombination technique described below. It can be produced by appropriately using a known method or a modification method thereof.
The “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody against the mammalian AILIM (particularly preferably human AILIM) as defined above, and is preferably a monoclonal antibody.
Specifically, it is an antibody that binds to AILIM and suppresses the growth of AILIM-expressing cells, or has an activity to bind to AILIM and suppress the production of interferon γ or interleukin 4 by AILIM-expressing cells.
[0092]
The term “delayed allergy” in the present invention is mediated by cellular immunity (particularly mediated by Th1 type T cells), that is, mediated by antigen-sensitized T cells (memory T cells storing antigens). A living being allergic and sensitized to an antigen means any allergy that requires approximately 24-48 hours to develop an allergic reaction accompanied by inflammation by the memory T cells that occurs upon re-contact with the antigen.
Examples of this delayed allergy include allergies to infectious pathogen antigens such as tuberculosis allergy derived from Mycobacterium tuberculosis, Jones-Mote delayed allergy that appears transiently for trace amounts of proteins, drugs such as picryl chloride, and lacquer. Contact allergy to phytotoxin, or allergy related to transplant rejection seen in allograft transplantation.
[0093]
The “pharmaceutical composition” in the present invention is an antibody (preferably a human antibody) or a monoclonal antibody (preferably a human monoclonal antibody) that binds to the AILIM of the present invention (particularly preferably human AILIM) or a part thereof as an active ingredient, or It means a composition useful as a pharmaceutical comprising a part thereof and a "pharmaceutically acceptable carrier".
Here, "pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners, thickeners. , A flavoring agent, a solubilizer or other additives.
By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, liquids, capsules, troches, elixirs, suspensions, emulsions, syrups, etc. A composition can be prepared.
These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions containing one or more active substances and formulated in a conventional manner, suppositories and pessaries for enteral administration.
[0094]
The dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, treatment time, or the type of active ingredient (such as the protein or antibody) contained in the pharmaceutical composition. The dose can be administered in the range of 10 μg to 1000 mg (or 10 μg to 500 mg) per dose per person. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.
In particular, in the case of an injection, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. Can be produced by dissolving or suspending as described above.
[0095]
The injection thus produced is applied to a human patient in need of treatment at a rate of 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg per day, in a single administration. It can be administered once to several times. Examples of the administration form include medically appropriate administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection. Preferably it is an intravenous injection.
Injectables can also be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions and emulsions. .
Such injections can be sterilized by filtration sterilization through a bacteria retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of preparation for use. That is, it can be used as a sterile solid composition by freeze-drying or the like, dissolved in sterile distilled water for injection or another solvent before use.
[0096]
The pharmaceutical composition comprising the human antibody of the present invention can be used to provide AILIM-mediated costimulation to AILIM-expressing cells without inducing host immune rejection caused by HAMA (human anti-mouse antibody). A variety of biological reactions involving the transmission of Lie signals (co-stimulatory signals), such as cell proliferation of AILIM-expressing cells, production of cytokines by AILIM-expressing cells, and immunocytolysis of AILIM-expressing cells (immune (Cytolysis) or cell death (apoptosis) and / or the like, and / or suppresses or prevents the onset and / or progression of various diseases involving signal transduction through the AILIM, thereby treating or preventing the diseases. Yes as a preventive drug It is.
[0097]
“Immune cytolysis” in the present invention means the following biological phenomenon.
Cytolysis of cells occurs not only when it is caused by binding to killer cells but also through antibodies (particularly, cytolytic antibodies). The cytolytic antibody is an antibody that acts on the lytic reaction of cells such as immune cells, tissue cells or sperm, among cytotoxic antibodies, and complements the antibody when the antibody binds to a cell surface antigen. Are antibodies that damage the cell or induce cell lysis in the presence of.
This immune cell lysis is induced by the action of complement components in addition to the specific binding of the antibody to the cell surface antigen. The first component of complement (C1) is activated by the antibody bound to the surface antigen, and a subsequent series of complement reactions from the second to ninth components of complement (C2 to C9) forms a cytotoxic site. The cell contents are released, leading to cell lysis.
[0098]
“Antibody-dependent cellular cytotoxicity” in the present invention is a biological phenomenon also abbreviated as ADCC, and is caused by effector cells such as lymphocytes, macrophages or polymorphonuclear leukocytes. The induced cytotoxicity of a target cell is a cytotoxicity that requires an antibody in order to induce the cytotoxicity in addition to the effected cell and the target cell.
[0099]
“Mixed lymphocyte reaction” in the present invention is a biological phenomenon abbreviated as MLR. It is also called a mixed leukocyte reaction.
Mixing leukocytes or lymphocytes from each of two different individuals from allogeneic animals and culturing them for several days causes their cells to blast and synthesize DNA within the cells (ie, cell growth) Is promoted. This reaction is called MLR (allogenic MLR).
The degree of DNA synthesis (cell proliferation) can be analyzed by stopping one lymphocyte by irradiation or treating with mitomycin and measuring the degree of DNA synthesis in the other lymphocyte.
The degree of synthesis of the DNA can be analyzed by measuring the amount of thymidine labeled with a radioactive substance such as tritium into the cell nucleus.
[0100]
The DNA encoding AILIM (particularly preferably human AILIM) used in the present invention can be obtained by a method of cloning cDNA from mRNA encoding AILIM, a method of isolating genomic DNA and splicing it, or a method of cDNA. It can be obtained by a method of preparing by PCR using a sequence or mRNA sequence as a template, or a method of chemical synthesis.
The DNA encoding the AILIM ligand of the present invention can be obtained in the same manner as described above.
The DNA encoding the AILIM of the present invention (particularly preferably human AILIM) is obtained by digesting (digesting) the DNA containing each DNA encoding the AILIM thus prepared with an appropriate restriction enzyme. The fragment can be prepared by ligating the fragment together with a linker DNA or a tag (Tag), if necessary, using an appropriate DNA polymerase or the like. The same applies to DNA encoding AILIM ligand.
[0101]
Examples of a method for cloning cDNA encoding AILIM (particularly preferably human AILIM; hereinafter referred to as a target protein) from mRNA include the following methods.
The same applies to DNA encoding AILIM ligand.
First, mRNA encoding a target protein is prepared from a tissue or cell that expresses / produces the target protein. For preparation of mRNA, total RNA prepared by a known method such as a guanidine thiocyanate method (Biochemistry, Vol. 18, p. 5294, 1979), a hot phenol method or an AGPC method is used to prepare oligo (dT) cellulose or poly-U. -It can be carried out by subjecting to affinity chromatography using Sepharose or the like.
[0102]
Next, a known method such as, for example, using reverse transcriptase using the obtained mRNA as a template, for example, the method of Okayama et al. (Mol. Cell. Biol., Vol. 2, p. 161, 1982 and the same journal, Vol. 3, p. 280, 1983) or the method of Hoffman et al. (Gene, Vol. 25, p. 263, 1983) and the like, and the cDNA is converted into double-stranded cDNA. This cDNA is incorporated into a plasmid vector, a phage vector or a cosmid vector, and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli to prepare a cDNA library.
[0103]
The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector can be used as long as it can be propagated in the host. Examples of commonly used cloning vectors include pUC19, λgt10, λgt11 and the like. However, when subjected to the immunological screening described below, a vector having a promoter capable of expressing a gene encoding a target protein in a host is preferable.
[0104]
Methods for incorporating cDNA into a plasmid include, for example, the method described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 1.53, 1989). Examples of the method for incorporating cDNA into a phage vector include the method of Hyunh et al. (DNA Cloning, a practical approach, Vol. 1, p. 49, 1985). For convenience, a commercially available cloning kit (for example, a product of Takara Shuzo Co., Ltd.) can also be used. The thus obtained recombinant plasmid or phage vector is introduced into a suitable host such as a prokaryotic cell (for example, E. coli: HB101, DH5α, Y1090, DH10B or MC1061 / P3).
[0105]
Examples of a method for introducing a plasmid into a host include a calcium chloride method or a calcium chloride / rubidium chloride method described in (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 1.74, 1989). Ration method and the like. Examples of a method for introducing a phage vector into a host include a method in which phage DNA is packaged in vitro and then introduced into a grown host. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (eg, manufactured by Stratagene, Amersham, etc.).
The method of isolating the cDNA encoding the target protein from the cDNA library prepared by the above method can be performed by combining general cDNA screening methods.
[0106]
For example, after separately synthesizing an oligonucleotide that is considered to correspond to the amino acid sequence of the target protein, ³² Labeled with P to form a probe, a known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA, Vol. 72, p. 3961, 1975) or a plaque hybridization method (Molecular) Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 2.108, 1989), a method for screening a clone containing a target cDNA, a PCR primer, and a specific region of the target protein by a PCR method. A method of amplifying and selecting a clone having a DNA fragment encoding the region can be used.
[0107]
When a cDNA library prepared by using a vector capable of expressing cDNA (for example, a phage vector such as λgt11) is used, an antigen-antibody reaction using an antibody reactive with the target protein is used. Clones can be selected. When a large number of clones are processed, it is preferable to use a screening method using a PCR method.
The nucleotide sequence of the DNA thus obtained was determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA., Vol. 74, p. 560, 1977) or a dideoxynucleotide using phage M13. Synthetic chain termination (Sanger et al., Proc. Natl. Acad. Sci. USA., Vol. 74, p. 5463-5467, 1977). The gene encoding the target protein can be obtained by cutting out all or part of the gene from the clone obtained as described above using restriction enzymes and the like.
[0108]
In addition, as a preparation method by isolating DNA encoding the target protein from genomic DNA derived from cells expressing the target protein as described above, for example, the following method is exemplified.
The cells are preferably lysed using SDS or proteinase K and the like, and DNA extraction is performed by repeating extraction with phenol. The RNA is digested, preferably by ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the obtained DNA fragment is amplified with an appropriate phage or cosmid to prepare a library. Then, a clone having the target sequence is detected by, for example, a method using a radiolabeled DNA probe, and the whole or a part of the gene encoding the target protein is cut out from the clone with a restriction enzyme or the like and obtained.
[0109]
The DNA encoding the target protein can be prepared by PCR using a known mRNA or cDNA of the target protein as a template ("Gene amplification PCR method, basic and new development", Kyoritsu Shuppan Co., Ltd.) Published, 1992).
The production of DNA encoding the target protein by chemical synthesis can be carried out according to a conventional method based on the base sequence of the target protein.
[0110]
The AILIM of the present invention (particularly preferably human AILIM) or a part thereof (preferably the extracellular region) is obtained by preparing a DNA encoding AILIM (genomic DNA containing cDNA or intron) prepared using the method exemplified above. The DNA fragments encoding the AILIM are obtained by cleaving each with an appropriate restriction enzyme, and these fragments are ligated together with a linker DNA or a tag (Tag), if necessary, using an appropriate DNA polymerase or the like. The recombinant DNA can be prepared as a recombinant protein by a conventional method using a commonly used gene recombination technique.
The same applies to AILIM ligand (particularly preferably human AILIM ligand) or a part thereof (preferably extracellular region).
Specifically, it is as exemplified below. That is, the DNA prepared as described above is inserted into a vector as described in detail below to prepare an expression vector, and the expression vector is used to transform a host cell as described below to obtain a transformant. . By culturing the transformant, the target protein is produced in the culture supernatant. The target protein in the culture supernatant can be easily purified using column chromatography or the like.
[0111]
There are no particular limitations on the expression vector used for producing recombinant AILIM (or its extracellular region), as long as it can replicate and maintain or self-proliferate in various prokaryotic and / or eukaryotic host cells. And phage vectors (Cloning Vectors: A Laboratory Manual, Elsby, New York, 1985).
The expression vector is prepared by simply ligating a DNA encoding AILIM (or an extracellular region thereof) to a vector for recombination (plasmid DNA and bacterial phage DNA) available in the art in a conventional manner. Can be. Specific examples of the recombination vector used include plasmids derived from Escherichia coli such as pBR322, pBR325, pUC12, pUC13 and pUC19, plasmids derived from yeast such as pSH19 and pSH15, and plasmids derived from Bacillus subtilis such as pUB110, pTP5 and pC194. And the like. Examples of the phage include bacteriophage such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nucleopolyhedrovirus.
[0112]
Expression of AILIM of the present invention (particularly preferably human AILIM) or a DNA encoding the soluble extracellular region thereof, and expression of AILIM on the surface of a host cell, or production of soluble extracellular region of AILIM (particularly preferably human AILIM) For purposes, plasmid vectors are useful. As a plasmid vector, a gene encoding the AILIM (particularly preferably human AILIM) or its soluble extracellular region is expressed in various prokaryotic and / or eukaryotic host cells to produce these polypeptides. There is no particular limitation as long as it has a function. For example, pMAL C2, pcDNA3.1 (-), pEF-BOS (Nucleic Acid Research, Vol. 18, p. 5322, 1990, etc.) or pME18S (Experimental Medical Supplement "Genetic Engineering Handbook", 1992, etc.) and the like can be mentioned. be able to.
[0113]
When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally comprises at least a promoter-operator region, an initiation codon, a DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit.
When a yeast, animal cell or insect cell is used as a host, the expression vector may contain at least a promoter, an initiation codon, AILIM of the present invention (particularly preferably human AILIM) or a DNA encoding the extracellular region thereof, and a stop codon. preferable. In addition, DNA encoding a signal peptide, an enhancer sequence, 5 ′ and 3 ′ untranslated regions of the gene encoding AILIM of the present invention, a splicing junction, a polyadenylation site, a selectable marker region or a replicable unit, etc. May be included. Further, it may contain a gene amplification gene (marker) usually used depending on the purpose.
[0114]
The promoter-operator-region for expressing the AILIM of the present invention (particularly preferably human AILIM) or its extracellular region in bacteria includes a promoter, an operator and a Shine-Dalgarno (SD) sequence (eg, AAGG). It is a thing. For example, when the host is a bacterium belonging to the genus Escherichia, those containing a Trp promoter, a lac promoter, a recA promoter, a λPL promoter, an lpp promoter, a tac promoter and the like are preferably exemplified.
Examples of a promoter for expressing the AILIM of the present invention (particularly preferably human AILIM) or its extracellular region in yeast include a PH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter. Examples include SL01 promoter, SP02 promoter, penP promoter and the like. When the host is a eukaryotic cell such as a mammalian cell, examples thereof include an SV40-derived promoter, a retrovirus promoter, and a heat shock promoter. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression.
[0115]
A suitable initiation codon is, for example, a methionine codon (ATG).
Examples of the stop codon include common stop codons (for example, TAG, TAA, and TGA).
As the terminator region, a commonly used natural or synthetic terminator can be used.
A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, including native plasmids, artificially modified plasmids (DNA fragments prepared from native plasmids) and Includes synthetic plasmids and the like. Suitable plasmids include E. coli. In E. coli, plasmid pBR322 or an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) is used. In yeast, yeast 2μ plasmid or yeast chromosomal DNA is used. In mammalian cells, plasmid pRSVneo ATCC 37198 is used. And plasmid pSV2dhfr ATCC 37145, plasmid pdBV-MMTneo ATCC 37224, plasmid pSV2neo ATCC 37149, plasmid pSV2bsr, and the like.
As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, for example, can be used.
[0116]
As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include antibiotic resistance genes such as tetracycline, ampicillin, and kanamycin.
Examples of the gene amplification gene include a dihydrofolate reductase (DHFR) gene, a thymidine kinase gene, a neomycin resistance gene, a glutamate synthase gene, an adenosine deaminase gene, an ornithine decarboxylase gene, a hygromycin-B-phosphotransferase gene, and an aspartate transcarbamylase gene. And the like.
[0117]
The expression vector of the present invention can be prepared by ligating at least the above-mentioned promoter, start codon, DNA encoding the protein of the present invention, stop codon and terminator region to an appropriate replicable unit in a continuous and circular manner. it can. At this time, if necessary, an appropriate DNA fragment (for example, a linker or another restriction enzyme site) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
The transformed cell of the present invention can be prepared by introducing the above-described expression vector into a host cell.
[0118]
The host cell used in the present invention is not particularly limited as long as it is compatible with the above-mentioned expression vector and can be transformed, and natural cells or artificially established cells usually used in the technical field of the present invention are used. Various cells such as recombinant cells (for example, bacteria (genus Escherichia, genus Bacillus), yeast (genus Saccharomyces, genus Pichia), animal cells or insect cells, etc.) are exemplified.
Escherichia coli or animal cells are preferred, and specifically, Escherichia coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.), and mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1, or NIH3T3, etc.) , Rat-derived cells, hamster-derived cells (such as BHK and CHO), monkey-derived cells (such as COS1, COS3, COS7, CV1 and Velo) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells) And Namalwa etc.).
Introduction (transformation (transfection)) of an expression vector into a host cell can be performed using a conventionally known method.
[0119]
For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., Vol. 69, p. 2110, 1972), the protoplast method (Mol. Gen.). Genet., Vol. 168, p. 111, 1979) or the competent method (J. Mol. Biol., Vol. 56, p. 209, 1971), in the case of Saccharomyces cerevisiae, for example, Hinnen et al. Natl. Acad. Sci. USA., Vol. 75, p. 1927, 1978) or the lithium method (J. Bacteriol., Vol. 153, p. 163, 1983). For example, the method of Graham (Virology, Vol. 52, p. 456, 1973), and in the case of insect cells, for example, the method of Summers et al. (Mol. Cell. Biol., Vol. 3, p. 2156-2165, 1983).
[0120]
The extracellular region (soluble AILIM) of AILIM (especially preferably human AILIM) of the present invention is a transformed cell containing the expression vector prepared as described above (hereinafter referred to as including a transfectant). It can be produced by culturing in a nutrient medium. The same applies to AILIM ligand.
The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, and so on. Examples include soybean meal and potato extract. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. .
The culturing is performed by a method known in the art. Culture conditions, such as temperature, pH of the medium, and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.
[0121]
Specific examples of the culture medium and culture conditions used according to the host cell are shown below, but the invention is not limited thereto.
When the host is a bacterium, an actinomycete, a yeast or a filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8.
If the host is E. coli. In the case of E. coli, preferred mediums include LB medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p. 431, 1972), YT medium and the like. In such a case, the cultivation can be carried out usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary.
[0122]
When the host is a bacterium belonging to the genus Bacillus, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary.
When the host is yeast, examples of the medium include Burkholder minimum culture (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980). Desirably. The cultivation is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and stirring can be performed.
When the host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum (Science, Vol. 122, p. 501, 1952) or a DMEM medium (Virology, Vol. 8, p. 396). 1959), RPMI 1640 medium (J. Am. Med. Assoc., Vol. 199, p. 519, 1967), 199 medium (Proc. Soc. Exp. Biol. Med., Vol. 73, p. 1, 1950). , HamF12 medium and the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.
[0123]
When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p. 8404, 1985), etc., and the pH thereof is about 5 to 8 It is preferred that The cultivation is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring may be performed.
The cell (soluble AILIM) of the AILIM (especially preferably human AILIM) of the present invention can be secreted into the culture supernatant by culturing the above-mentioned transformed cells (especially animal cells or Escherichia coli). Can be manufactured. That is, the obtained culture is filtered or centrifuged to obtain a culture filtrate (supernatant), and the target protein is purified from the culture filtrate according to a conventional method generally used for purifying and isolating a natural or synthetic protein. Purify and isolate.
[0124]
Isolation and purification methods include, for example, a method utilizing specific affinity such as affinity column chromatography, a method utilizing solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate -Methods utilizing differences in molecular weight such as polyacrylamide gel electrophoresis, methods utilizing charges such as ion exchange chromatography and hydroxylapatite chromatography, methods utilizing differences in hydrophobicity such as reverse phase high performance liquid chromatography, etc. A method using a difference between isoelectric points such as electric point electrophoresis is exemplified.
[0125]
On the other hand, when the target protein is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect the cells or cells and suspended in an appropriate buffer. After turbidity and disruption of cell walls such as cells and / or cell membrane by methods such as ultrasonic waves, lysozyme, and freeze-thawing, a membrane fraction containing the target protein is obtained by methods such as centrifugation and filtration. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. Then, the crude solution can be isolated and purified by using a conventional method as exemplified above.
[0126]
The “insoluble carrier” in the present invention means a support for supporting a polypeptide by physical adsorption or chemical bonding. For example, (1) plastics made of polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or the like, or plates, test tubes, tubes, or the like made of a substance insoluble in water such as glass, etc. , Beads, balls, filters, or membranes, and (2) cellulose-based carriers, agarose-based carriers, polyacrylamide-based carriers, dextran-based carriers, polystyrene-based carriers, polyvinyl alcohol-based carriers, polyamino acid-based carriers, or porous silica-based carriers Examples include insoluble carriers used for affinity chromatography such as carriers.
[0127]
Examples of the "labeling substance capable of providing a detectable signal" in the present invention include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, radioisotopes, and the like. More specifically, peroxidase (for example, horseradish) peroxydase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, etc. Enzymes, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothio A fluorescent substance such as Aneto, ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ A radioisotope such as I, biotin, avidin, or a chemiluminescent substance.
[0128]
Here, the radioisotope and the fluorescent substance alone can provide a detectable signal. On the other hand, enzymes, chemiluminescent substances, biotin and avidin alone cannot provide a detectable signal and, therefore, react with one or more other substances to provide a detectable signal. For example, in the case of an enzyme, at least a substrate is required, and various substrates are used depending on the method for measuring the enzyme activity (colorimetric method, fluorescent method, bioluminescent method, chemiluminescent method, etc.). For example, in the case of peroxidase, hydrogen peroxide is used as a substrate. In the case of biotin, at least avidin or enzyme-modified avidin is generally reacted, but not limited thereto. If necessary, various coloring substances depending on the substrate may be used.
In the present invention, any of the above labeling substances can be used, but in view of the high detection sensitivity or quantitative sensitivity and the convenience of operation, it is preferable to label with an enzyme such as peroxidase or biotin. .
[0129]
The “method of identifying a substance that binds to AILIM or AILIM ligand” of the present invention is based on an immunoassay.
Specifically, the principles of various methods described in the enzyme immunoassay (3rd edition, edited by Eiji Ishikawa et al., Published by Medical Shoin, 1987) can be applied.
Preferable examples of applicable principles include, for example, a one-antibody solid phase method, a two-antibody liquid phase method, a two-antibody solid phase method, a sandwich method, and a one-pot method as described in JP-B-2-39747. It can be mentioned as. Examples of an assay utilizing an antigen-antibody reaction include an EMIT method (Enzyme multiplied immunoassay technique), an enzyme channeling assay (Enzyme channeling immunoassay), an enzyme activity-modifying substance-labeled immunoassay, an enzyme activity modified enzyme immunoassay, and an enzyme activity-enhanced immunoassay. Immunoassay (Enzyme inhibitor immunoassay), Immunoenzyme metric assay (Immunoenzymometric assay), Enzyme enhanced immunoassay and Proximal linkage immunoassay (Pyximal linkage immunoassay P) oximal linkage immunoassay), etc. are also known.
[0130]
In the present invention, any of the principles of such an immunoassay can be appropriately selected and used depending on the purpose, but it is convenient in operation and / or economical convenience, especially in clinical use. In consideration of the nature, it is preferable to use the principle of the sandwich method, the one-pot method, or the one-antibody solid phase method, and more preferably, the principle of the sandwich method or the one-pot method. Particularly preferably, a sandwich method using a multiwell microtiter plate having a large number of wells represented by a 96-well microplate, or a bead having a polypeptide immobilized on its surface and an enzyme such as peroxidase or biotin is used. This is a one-pot method using a labeled counterpart.
[0131]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but it is needless to say that the present invention is not limited to only the embodiments described in the Examples.
[0132]
Example 1 Preparation of immunogen
<1-1> Preparation of recombinant cells expressing human AILIM
One of the present inventors, Tezuka's other earlier applications (Japanese Patent Application Publication No. 11-29599 and International Patent Application Publication No. WO 98/38216) and a previously published report (Int. Immunology, Vol. 12, No. 1). Pp. 51-55, 2000), two types of recombinant cells (CHO cells and HPB-ALL cells) that highly express human AILIM were prepared. The details are as follows.
After inserting a cDNA containing the full-length ORF of human AILIM (GenBank Accession Number: AB02135 (cDNA); BAA82129 (amino acid)) into the vector pEF-neo, the recombinant expression vector is transfected using Gene Pulser (manufactured by BioRad). The cells were introduced into Chinese hamster ovary cells (CHO cells) and human thymoma cell line HPB-ALL by electroporation (960 μF, 320 volts) according to a conventional method. Each cell was cultured in RPMI1640 medium containing Geneticin (0.8 mg / ml; Gibco BRL) and 10% FCS to select drug-resistant transformed cells.
[0133]
<1-2> Selection of recombinant HPB-ALL cells that highly express human AILIM
The mouse anti-human AILIM monoclonal antibody named “SA12” previously created and reported by the present inventors (mouse) was added to the precipitate obtained by centrifuging the drug-resistant HPB-ALL cells selected in <1-1> above. (Anti-human JTT-1 antigen monoclonal antibody) (Japanese Patent Application Publication No. 11-29599 (Example 12) and International Patent Application Publication WO 98/38216 (Example 12)) (concentration: diluted with EDTA-BSA / PBS) 10 μg / ml to 100 μl / 10 ⁵ cells) and reacted at 4 ° C. for 30 minutes. After the cells were washed twice with the above-mentioned EDTA-BSA / PBS (200 μl), picoerythrin-labeled streptavidin (SA-PE; 500-fold diluted 100 μl) was added, and reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed three times with EDTA-BSA / PBS to prepare a cell dispersion.
The expression state of human AILIM in each cell contained in the cell dispersion is analyzed using a flow cytometer FACSort (manufactured by Beckton-Dickinson) to select recombinant HPB-ALL cells that highly express human AILIM. did. The selected cells were cultured in RPMI1640 medium containing 10% FCS and G418 (1 mg / ml) until confluent.
[0134]
<1-3> Selection of recombinant CHO cells overexpressing human AILIM
The mouse anti-human AILIM monoclonal antibody SA12 (100 μg / ml diluted with EDTA-BSA / PBS) labeled with FITC was added to the precipitate collected by centrifugation of the drug-resistant CHO cells selected in <1-1>. The reaction was performed at 4 ° C. for 30 minutes. After washing the cells with the above EDTA-BSA / PBS, EDTA-BSA / PBS (500 μl) was added to prepare a cell dispersion.
The expression state of human AILIM in each cell contained in the cell dispersion was analyzed using a flow cytometer FACSort (manufactured by Beckton-Dickinson), and recombinant CHO cells overexpressing human AILIM were selected. The selected cells were cultured in RPMI1640 medium containing 10% FCS and G418 (1 mg / ml) until confluent.
[0135]
<1-4> Preparation of immunogen from human AILIM high-expressing HPB-ALL cells
The human AILIM high-expressing HPB-ALL cells obtained in <1-2> above were centrifuged. The collected precipitate was washed four times with a phosphate buffer (PBS; manufactured by Niken Institute for Biological Research), and then a buffer containing a protease inhibitor (25 mM HEPES (pH 7.4), 10 mM MgCl 2). ₂ , 0.25 M Sucrose, and protease inhibitors (10 U / ml Aprotinine, 2 μg / ml Pepstatin, 50 μg / ml Leupeptin, and 0.35 mg / ml PMSF). The cell suspension was crushed with a potter-type homogenizer and centrifuged at a low speed (1,500 rpm, 10 minutes, 4 ° C.). Next, the supernatant was collected and ultracentrifuged (100,000 g, 1 hour, 4 ° C.), and the precipitated membrane fraction was collected and suspended by adding a phosphate buffer solution (1 × 10 5 per 1 ml of PBS). ⁷ (Adjusted to the concentration of the cell-derived membrane fraction) Stored at minus 80 ° C. This suspension of the cell membrane fraction was used as an antigen (immunogen) in preparing the human antibody of the present invention described later.
[0136]
<1-5> Preparation of immunogen from human AILIM highly expressing CHO cells
The human AILIM-overexpressing CHO cells obtained in <1-3> were dispersed with a cell scraper, and then centrifuged. The collected precipitate was washed four times with a phosphate buffer (PBS; manufactured by Niken Institute for Biological Research), and then a buffer containing a protease inhibitor (25 mM HEPES (pH 7.4), 10 mM MgCl 2). ₂ , 0.25 M Sucrose, and protease inhibitors (10 U / ml Aprotinine, 2 μg / ml Pepstatin, 50 μg / ml Leupeptin, and 0.35 mg / ml PMSF). The cell suspension was crushed with a potter-type homogenizer and centrifuged at a low speed (1,500 rpm, 10 minutes, 4 ° C.). Next, the supernatant was collected and ultracentrifuged (100,000 g, 1 hour, 4 ° C.), and the precipitated membrane fraction was collected and suspended by adding a phosphate buffer solution (1 × 10 5 per 1 ml of PBS). ⁷ (Adjusted to the concentration of the cell-derived membrane fraction) Stored at minus 80 ° C. This suspension of the cell membrane fraction was used as an antigen (immunogen) in preparing the human antibody of the present invention described later.
[0137]
Example 2 Preparation of hybridoma producing anti-human AILIM human monoclonal antibody
The preparation of the monoclonal antibody in this example is described in Experimental Medicine (separate volume), Cell Engineering Handbook (edited by Toshio Kuroki et al., Published by Yodosha, pp. 66-74, 1992) and an introduction to monoclonal antibody experimental procedures (Tamie Ando) Ed., Published by Kodansha, 1991).
[0138]
As the human AILIM as the immunogen, a cell membrane fraction prepared from the human AILIM high-expressing recombinant cells prepared in Example 1 was used.
As an animal to be immunized, a human antibody-producing transgenic mouse produced by the method described above was used (Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146). 156, 1997; JP-T 4-504365; JP-T 7-509137; Nikkei Science, June, pages 40 to 50, 1995 and the like).
The cell culture operation was performed using a multiwell microplate.
[0139]
<2-1> Immunization and preparation of hybridoma
To the human antibody-producing transgenic mouse, one of the immunogens (100 μl / mouse / single administration) prepared with <1-4> (derived from HPB-ALL) and <1-5> (derived from CHO) was used. Immunization was carried out for the first time (day 0) by injecting in a hood pad together with Freund's Complete Adjuvant (ICN / CAPPEL) with Complete Freund's Adjuvant.
One week after the initial immunization, one of the two types of immunogens is boosted twice or three times by injection into the footpad, and the same immunogen is also used two days before the lymphocyte acquisition described below. The final immunization was performed in the same manner.
[0140]
Two days after the final immunization, lymphocytes were obtained from the lymph nodes (groin and below the knee) and spleen extracted from each immunized transgenic mouse. The lymphocytes and mouse myeloma cells P3 / X63-AG8.653 (ATCC No .: CRL-1580) were mixed at a ratio of 5: 1, and polyethylene glycol 1500 (manufactured by Boehringer Mannheim) was added as a fusion agent, and then 10-fold. A volume of serum-free basal medium EX-CELL301 (manufactured by JRH Bioscience) was added for dilution. Next, after washing the mixed cells with the basal medium, the cells are suspended in a HAT medium (containing 13.61 mg of hypoxanthine, 176 μg of aminopterin, and 3.88 mg of thymidine per 1 L of the basal medium), and the cells are washed in a 96-well microplate. And fused for 10 to 14 days. Many hybridomas were obtained by this cell fusion.
[0141]
<2-2> Screening of hybridoma producing human monoclonal antibody
A large number of hybridomas prepared in the above <2-1> were screened by the following cell ELISA (cell ELISA) to select hybridomas producing a human monoclonal antibody against human AILIM.
Each of the recombinant HPB-ALL cells and recombinant CHO cells (1 × 10 ⁵ Cells / well) were plated in each well of a 96-well ELISA microplate and cultured at 37 ° C. for 2 days.
Next, the supernatant was discarded, and a culture supernatant sample (50 μl / well) of each hybridoma was added to each well, and the mixture was reacted for 1 hour. After the reaction, the sample was discarded, and each well was washed three times with PBS containing 1% BSA (manufactured by Sigma).
Next, in order to detect the heavy chain of human immunoglobulin (human monoclonal antibody) contained in the hybridoma supernatant, a peroxidase-labeled goat anti-human immunoglobulin (Fc) antibody (1/2000 dilution: 50 μl / well) was added to each well. ; Amercan Corex; 1% BSA / PBS) and incubated at room temperature for 1 hour.
On the other hand, in order to detect the light chain of human immunoglobulin (human monoclonal antibody) contained in the hybridoma supernatant, a peroxidase-labeled goat anti-human immunoglobulin κ chain antibody (50 μl diluted 1/2000) was added to each well. In addition, the mixture was incubated at room temperature for 15 minutes.
[0142]
After removing the anti-human IgFc antibody or anti-human Igκ antibody from each well of the microplate, the plate was washed three times with PBS containing 1% BSA, and then tetramethylbenzidine (3, 3 ', 5, 5',- tetramethylbenzidine (TMB), 100 μl / well, manufactured by BIO-RAD) was added to each well and incubated at room temperature for 15 minutes.
Then 1N H ₂ SO ₄ (50 μl / well) was added to each well to stop the reaction. The absorbance at a wavelength of 450 nm was measured using a Bio-Rad, Model 3550 Microplate Reader (Model 3550 Microplate Reader, manufactured by BIO-RAD).
In addition, as a control test, ELISA was performed in the same manner as described above using each of the following.
(1) Wild-type HPB-ALL cells instead of human AILIM-expressing recombinant HPB-ALL cells.
(2) Wild-type CHO cells instead of human AILIM-expressing recombinant CHO cells.
(3) Mouse monoclonal antibody against human AILIM instead of hybridoma supernatant (SA12 or SG430; Japanese Patent Application Publication No. 11-29599 (Example 12) and International Patent Application Publication WO 98/38216 (Example 12)) .
(4) A human monoclonal antibody against KLH (keyhole limpet hemocyanin, manufactured by PIERCE) instead of the hybridoma supernatant.
The anti-KLH human monoclonal antibody was prepared by immunizing the aforementioned human antibody-producing transgenic mouse with KLH (keyhole limpet hemocyanin, manufactured by PIERCE) according to the same method as in <2-1>. .
As a result, a large number of hybridomas producing human monoclonal antibodies having a binding property to human AILIM were selected.
[0143]
<2-3> Primary cloning of hybridoma
By the following test operation, many types of single hybridomas were cloned from a large number of hybridomas (parent strains) selected in the above <2-2> and producing a human monoclonal antibody against human AILIM.
Each of the hybridomas selected in <2-2> was seeded on a 24-well (well) microplate, and the number of hybridoma cells in each well was counted by pipetting. Next, 10% fetal calf serum (FCS; Trace Bioscience PTY), 1% penicillin / streptomycin (Sigma), 1% in EX-CELL301 medium (manufactured by JRH Bioscience) containing 4.0 mM L-Glutamine and lipids Hybridomas in each well were adjusted to 1 × 10 5 with a modified medium prepared by adding HT Supplement (manufactured by Gibco BRL) and 2.5% T-STIM Culture Supplement (manufactured by Collaborative Biomedical Products). ⁴ The cells were diluted and suspended in cells / ml.
After adding the cell suspension (300 μl or 600 μl) of each well to the modified medium (150 ml or 300 ml) and mixing well, the cell suspension was added to each well of a plurality of 96-well microplates at a concentration of 200 μl / well. In addition, it was set to 4 cells / well. After the above-mentioned modified medium (50 ml or 100 ml) was newly added to the remaining cell suspension and mixed, the obtained cell suspension was added to each well of another plurality of 96-well microplates to obtain 2 cells / well. .
After 1-2 weeks of culture, the formation of a single hybridoma colony was confirmed in many wells.
The production of a human monoclonal antibody against human AILIM in the culture supernatant of each well in which the formation of the colonies was confirmed was confirmed by the cell ELISA described in <2-2> above.
[0144]
<2-4> Secondary cloning of hybridoma
According to the same method as in <2-3>, each of the multiple hybridoma clones cloned in <2-3> was subcloned (secondary cloning).
In this test, the number of cells in each well of a 96-well microplate was adjusted to 1 cell / well.
As a result, a number of single hybridoma clones producing human monoclonal antibodies against human AILIM were obtained. Some of the clones are as follows.
(Clone name)
AIF34 (JMab124), AIF182 (JMab-126), AIF348 (JMab-127),
AIF620 (JMab-128), AIF1052 (JMab-135), AIH5D3 (JMab-136),
AIH386 (JMab-137), AII289 (JMab-138), AII394 (JMab-139),
AII488 (JMab-140), AIJ40 (JMab-141),
The above names are used in any of the following examples including the present example, and in the drawings or tables shown as test results in the examples.
[0145]
Example 3 Characteristic analysis of monoclonal antibody
<3-1> Analysis of heavy chain (light chain) and light chain (light chain)
The following ELISA and flow cytometry confirmed that the monoclonal antibody against human AILIM produced by each hybridoma clone cloned in <2-4> was a human monoclonal antibody.
Recombinant HPB-ALL cells that highly express human AILIM prepared in Example 1 (3 × 10 ⁴ Cells / well) were plated in each well of a V-bottom microplate and cultured at 37 ° C. in RPMI 1640 medium containing 10% FCS.
After the culture, the plate was centrifuged (1,800 rotations, 2 minutes) to precipitate the cells. Next, the supernatant was discarded, and in each well, a culture supernatant sample (50 μl / well) of each hybridoma cloned in the above <2-4> or a mouse anti-human AILIM monoclonal antibody SA12 (2 μg / 50 μl) as a control or An anti-KLH human monoclonal antibody (50 μl / well) was added and allowed to react in a refrigerator for 30 minutes. After the reaction, the sample was discarded, and each well was washed with a phosphate buffer (0.5% BSA-PBS containing 5 mM EDTA).
[0146]
Next, one of the following secondary antibodies (50 μl / well of a 1000-fold dilution with the above phosphate buffer) was added to each well, and the cells were dispersed and reacted in a refrigerator for 30 minutes.
(Secondary antibody)
Biotin-labeled anti-human IgG antibody (Zymed);
Biotin-labeled anti-human IgG antibody (Protos);
Biotin-labeled anti-human IgFc antibody (manufactured by EY Laboratories); or
Biotin-labeled anti-human Igκ antibody (manufactured by Vector).
After the reaction, the secondary antibody was discarded, and each well of the plate was washed with the phosphate buffer. Next, picoerythrin-labeled streptavidin (Streptavidin-PE; manufactured by Pharmingen; 50 μl / well of a 500-fold dilution with the phosphate buffer) was added to each well, and the mixture was reacted in a refrigerator for 30 minutes. After the reaction, each well was washed with the phosphate buffer. Next, the phosphate buffer (200 μl / well) was added to each well to disperse the cells.
The binding (reactivity) of the monoclonal antibody to human AILIM contained in the culture supernatant of each hybridoma clone with respect to human AILIM high-expressing HPB-ALL cells in each well was analyzed.
[0147]
In addition, as a control test, the test and analysis were performed in the same manner as described above using each of the following.
(1) Wild-type HPB-ALL cells instead of human AILIM-expressing recombinant HPB-ALL cells.
(2) A human monoclonal antibody against KLH (keyhole limpet hemocyanin, manufactured by PIERCE) instead of the hybridoma supernatant.
The anti-KLH human monoclonal antibody was prepared by immunizing the aforementioned human antibody-producing transgenic mouse with KLH (keyhole limpet hemocyanin, manufactured by PIERCE) according to the same method as in <2-1>. .
As a result, it was confirmed that each of the hybridomas cloned in <2-4> was a human monoclonal antibody composed of a human-derived heavy chain and a human-derived κ light chain.
As an example of the results, FIG. 1 shows the test results for each of the hybridoma clones AIH5D3 (JMab-136), AII289 (JMab-138), and AII394 (JMab-139).
[0148]
<3-2> Determination of human monoclonal antibody isotype
The human monoclonal antibody isotype against human AILIM produced by each of the hybridoma clones cloned in the above <2-4> and analyzed in the above <3-1> was determined using a human monoclonal antibody isotype determination kit (American Corex) And the operation was determined according to the experimental operation protocol attached to the kit.
All the anti-human AILIM human monoclonal antibodies were confirmed to be IgG2 / κ.
[0149]
Example 4 Large-scale preparation and purification of a human monoclonal antibody against human AILIM (anti-human AILIM human monoclonal antibody)
<4-1> Method 1
Each hybridoma clone producing the anti-human AILIM human monoclonal antibody prepared in <2-4> above was added to Tissue Culture Flash (50 ml, manufactured by FALCON), and FBS containing ultra-low concentration bovine immunoglobulin (Ultra Low Bovine IgG FBS) was added. , GIBCO-BRL) in an ASF104 medium (manufactured by Ajinomoto) containing 10% ₂ The culture was continued at 37 ° C. until the mixture became confluent.
Next, the entire amount of the culture solution was transferred to a Tissue Culture Flask (750 ml, manufactured by FALCON), and an ASF104 medium (Ajinomoto Co.) containing 10% of an ultra-low concentration bovine immunoglobulin-containing FBS (Ultra Low Bovine IgG FBS, manufactured by GIBCO-BRL) was used. 5% CO ₂ The culture was continued at 37 ° C. until the mixture became confluent.
After culturing for 10 to 20 days, the culture supernatant of each hybridoma was collected, transferred to a 50 ml Polypropylene Conical Tube (manufactured by FALCON), and centrifuged (500 × g, 5 minutes). Next, the centrifuged supernatant was filtered through a Sterilization Filter Unit (manufactured by NALGEN), and the filtrate was collected.
[0150]
The filtrate was added at 3 ml / min to a HiTrap affinity column Protein G (manufactured by Amersham Pharmacia) equilibrated with a phosphate buffer (30 ml).
Next, after washing the column with a phosphate buffer (20 ml), a 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute the antibody. Next, a solution (pH 9.0) composed of 750 mM Tris-HCl was added to the eluate for neutralization, followed by filtration with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was dialyzed (overnight) with a phosphate buffer, and then filtered through a filter (manufactured by Millipore) to obtain a purified anti-AILIM human monoclonal antibody from each hybridoma.
The value at A280 was determined using a spectrophotometer, and the protein concentration was calculated (1A280 = 1.41 mg / ml).
[0151]
<4-2> Method 2
Each of the hybridoma clones prepared in the above <2-4> adapted to an ASF104 medium (manufactured by Ajinomoto Co.) containing 10% Ultra Low Bovine IgG FBS (manufactured by GIBCO-BRL) (each 1 to 2 × 10 ⁶ Per ml) was inoculated into Integra Cell Line 1000 (INTEGRA CL1000, manufactured by Integra Bioscience) and cultured. After culturing for 7 to 10 days, the number of cultured cells is about 1 × 10 ⁸ At the time of reaching the number of cells / ml, the culture supernatant of each hybridoma was recovered.
Each of the culture supernatants was added to a HiTrap affinity column Protein G (HiTrap affinity column Protein G, manufactured by Amersham Pharmacia) at 3 ml / min, equilibrated with a phosphate buffer (30 ml).
Next, after washing the column with a phosphate buffer (20 ml), a 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute the antibody. Next, a solution (pH 9.0) composed of 750 mM Tris-HCl was added to the eluate for neutralization, followed by filtration with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was dialyzed (overnight) with a phosphate buffer, and then filtered through a filter (manufactured by Millipore) to obtain a purified anti-AILIM human monoclonal antibody from each hybridoma.
[0152]
Example 5 Reactivity of anti-human AILIM human monoclonal antibody to human AILIM and cross-reactivity to mouse AILIM and rat AILIM
The reactivity of the various anti-human AILIM human monoclonal antibodies purified above to human AILIM and cross-reactivity to mouse AILIM and rat AILIM were analyzed by cell ELISA.
[0153]
<5-1> Probability of ELISA System and Measurement of Calibration Curve for IgG Antibody Concentration Measurement
Since any of the purified anti-human AILIM human monoclonal antibodies was an IgG (IgG2) antibody, an ELISA for measuring the concentration of the IgG antibody was established.
A goat anti-human IgG (Fc) antibody (1.2 μg / ml in PBS; 100 μl / well; Organon Teknika) was added to each well of a 96-well ELISA microplate (Nunc) and incubated at room temperature for 2 hours. The anti-IgG (Fc) antibody was adsorbed on a microplate. Next, the supernatant was discarded, and the well was washed three times with a phosphate buffer solution (PBS) containing 0.05% Tween 20, and then the blocking reagent (200 μl / well, 0.5% bovine serum albumin (BSA) and 0.1%) was added to each well. % Tween 20) was added and incubated at room temperature for 2 hours to block the site where the anti-IgG (Fc) antibody was not bound. The blocking reagent was then discarded and each well was washed twice with PBS.
[0154]
To each well of the plate, various concentrations (0 to 100 ng / ml) of human-derived IgG2 antibody (50 μl / well; manufactured by The Binding Site) were added as standard antibodies, and reacted at room temperature for 2 hours. Except for the excess of the standard antibody, each well was washed three times with a phosphate buffer containing 0.05% Tween20.
Then, a goat anti-human IgG / κ antibody (diluted 4,000 times, 100 μl / well, manufactured by Protos) labeled with peroxidase (Peroxidase) was added to each well, and incubated at room temperature for 1 hour.
After discarding the supernatant and washing the microplate three times with a phosphate buffer containing 0.05% Tween 20, the substrate buffer (100 μl / well. <Composition>: orthophenylenediamine (O-Phenylenediamine, OPD; 20 mg) ) / Citrate / phosphate buffer (pH 5.0, 50 ml) / 30% hydrogen peroxide (15 μl)) was added to each well, and the mixture was incubated at room temperature for about 7 minutes.
Then, 2 M sulfuric acid (50 μl / well) was added to each well to stop the reaction. A calibration curve was created based on the value obtained by measuring the absorbance at a wavelength of 490 nm with a microplate reader (FIG. 2).
As a control, an assay was performed in the same manner as described above, using only the culture solution or the BSA solution as a test substance.
[0155]
<5-2> Analysis of reactivity of various purified anti-human AILIM human monoclonal antibodies to human AILIM, and cross-reactivity to mouse AILIM and rat AILIM
<5-2-1> Preparation of reagent
Reagents used for this cell ELISA were prepared as follows.
<5-2-1-1> Preparation of recombinant CHO cells overexpressing mouse AILIM
In the same manner as in <1-1> and <1-3>, recombinant CHO cells overexpressing mouse AILIM were produced and obtained.
After inserting the cDNA containing the full-length ORF of mouse AILIM (GenBank Accession Number: AB021322 (cDNA); BAA82126 (amino acid)) into the vector pEF-neo, the recombinant expression vector is transformed using Gene Pulser (manufactured by BioRad). The cells were introduced into each of Chinese hamster ovary cells (CHO cells) by electroporation (960 μF, 320 volts) according to a conventional method. Each of the cells was cultured in RPMI1640 medium containing Geneticin (0.8 mg / ml; Gibco BRL) and 10% FCS to select drug-resistant transformed cells, and mouse AILIM-highly expressing recombinant CHO cells were obtained. .
[0156]
<5-2-1-2> Preparation of recombinant CHO cells overexpressing rat AILIM
In the same manner as in <1-1> and <1-3>, recombinant CHO cells overexpressing rat AILIM were produced and obtained.
After the cDNA containing the full-length ORF of rat AILIM (GenBank Accession Number: AB0213134 (cDNA); BAA82128 (amino acid)) is inserted into the vector pEF-neo, the recombinant expression vector is inserted into a Gene Pulser (manufactured by BioRad). The cells were introduced into each of Chinese hamster ovary cells (CHO cells) by electroporation (960 μF, 320 volts) according to a conventional method. Each of the cells was cultured in RPMI1640 medium containing Geneticin (0.8 mg / ml; Gibco BRL) and 10% FCS to select drug-resistant transformed cells and to obtain rat AILIM high-expressing recombinant CHO cells. .
[0157]
<5-2-1-3> Preparation of monoclonal antibody against mouse AILIM
The mouse AILIM high-expressing recombinant CHO cells prepared in the above <5-2-1-1> are homogenized, ultracentrifuged (100,000 × g), and the centrifugation residue containing the cell membrane fraction is collected. Was suspended. The obtained cell membrane fraction was firstly immunized (day 0) by injecting into a foot pad of Wistar rat together with complete Freund's adjuvant. Further, the cell membrane fraction antigen was administered to the footpad at intervals of 7, 14, and 28 days. Two days after the last immunization, lymph node cells were collected.
The lymph node cells and mouse myeloma cells PAI (JCR No. B0113; Res. Disclosure, Vol. 217, p. 155, 1982) were mixed at a ratio of 5: 1, and polyethylene glycol 4000 (manufactured by Boehringer Mannheim) was used as a fusion agent. A hybridoma producing a monoclonal antibody was prepared by cell fusion using the same. Hybridomas were selected by culturing in HAT-containing ASF104 medium (manufactured by Ajinomoto) containing 10% fetal bovine serum and aminopterin.
[0158]
The reactivity of the rat monoclonal antibody produced in the culture supernatant of each hybridoma to mouse AILIM was determined by reacting each culture supernatant with the mouse AILIM-expressing CHO cells, followed by FITC-labeled anti-rat IgG (manufactured by Cappel). ) Was confirmed by measuring the fluorescence intensity of cells stained by the reaction with EPICS-ELITE flow-satometer. As a result, a plurality of hybridomas producing a monoclonal antibody reactive to mouse AILIM were obtained.
One of those hybridomas was named "B10.5". This hybridoma (10 ⁶ To 10 ⁷ Per 0.5 ml / mouse) was injected intraperitoneally into ICR nu / nu mice (female, 7-8 weeks old). After 10 to 20 days, the mice were laparotomized under anesthesia, and a large amount of rat anti-mouse AILIM monoclonal antibody B10.5 (IgG1) was prepared from ascites collected according to a conventional method.
[0159]
<5-2-2> Reactivity of human, mouse and rat for AILIM
The concentrations of the anti-human AILIM human monoclonal antibody and the control antibody used in the following ELISA were determined based on the ELISA and the calibration curve of the above <5-1>.
Recombinant CHO cells highly expressing human AILIM prepared in Example 1, mouse AILIM high expressing recombinant CHO cells prepared in <5-2-1-1>, and <5-2-1-2> Of each of the rat AILIM highly expressing recombinant CHO cells (7 × 10 ³ Cells / well) were plated in each well of a 96-well ELISA microplate and cultured at 37 ° C. until confluent.
Next, the supernatant was discarded, and various wells of the purified anti-human AILIM human monoclonal antibody prepared above or a control antibody (concentration: 200 μg / ml of the antibody, 3 times each with PBS containing 1% BSA, were added to each well. ² Times, 3 ³ Times 3 ⁴ Times, 3 ⁵ Times 3 ⁶ Times, 3 ⁷ Times, 3 ⁸ Times, 3 ⁹ Times, 3 ¹⁰ Times 3 ¹¹ Double and 3 ¹² One-fold dilution. ) Was added at 50 μl / well and reacted at room temperature for 2 hours. Excess monoclonal antibody was discarded, and each well was washed three times with a phosphate buffer containing 1% BSA (manufactured by Sigma).
Next, to each well, an anti-human IgG (Fc) antibody (1,000-fold diluted, 50 μl / well, manufactured by American Qualex) labeled with horseradish peroxidase (Peroxidase) was added, and incubated at room temperature for 1 hour.
The excess labeled antibody is discarded, and the microplate is washed three times with a phosphate buffer containing 1% BSA, and then washed with a substrate buffer (50 μl / well. <Composition>: ortho-phenylenediamine (O-Phenylenediamine, OPD). ; 20 mg) / citrate / phosphate buffer (pH 5.0, 50 ml) / 30% hydrogen peroxide (15 μl) was added to each well, and incubated at room temperature for about 7 minutes.
Then, 2 M sulfuric acid (50 μl / well) was added to each well to stop the reaction. The absorbance at a wavelength of 490 nm was measured with a microplate reader (manufactured by Bio-Rad).
[0160]
In addition, ELISA was performed in the same manner as described above using each of the following as the above-mentioned control antibody.
(1) Mouse monoclonal antibody SA12 or SG430 against human AILIM (Japanese Patent Application Publication No. 11-29599 (Example 12) and International Patent Application Publication WO 98/38216 (Example 12)).
(2) Rat monoclonal antibody B10.5 against mouse AILIM (the above <5-2-1-3>).
(3) Mouse monoclonal antibody JTT2 against rat AILIM (International Depository recognized under the Budapest Treaty on October 11, 1996 with International Deposit No. FERMBP-5708) Monoclonal antibodies produced by hybridomas deposited internationally at the Technical Research Institute, Japanese Patent Application Publication No. 11-29599 (Examples 1 and 2) and International Patent Application Publication No. WO 98/38216 (Examples 1 and 2). .
(4) A human monoclonal antibody against KLH (keyhole limpethemocyanin, manufactured by PIERCE) instead of the hybridoma supernatant.
As a control test, ELISA was performed in the same manner as above using wild-type CHO cells in place of AILIM-expressing recombinant CHO cells.
The results are shown in FIGS.
[0161]
Based on the obtained data, each of the anti-human AILIM human monoclonal antibodies human AILIM (human AILIM highly expressing recombinant CHO cells), mouse AILIM (mouse AILIM high expressing recombinant CHO cells), and rat AILIM (rat AILIM high The 50% effective concentration (ED50: ng / ml) as the degree of reactivity to each of the expressed recombinant CHO cells) was calculated as follows.
(A) ED50 against human AILIM highly expressing CHO
AIF 34 (JMab-124): 5.3 ng / ml
AIF182 (JMab-126): 3.6 ng / ml
AIF348 (JMab-127): 9.1 ng / ml
AIF620 (JMab-128): 10.1 ng / ml
AIF1052 (JMab-135): 2.0 ng / ml
AIH5D3 (JMab-136): 7.5 ng / ml
AIH386 (JMab-137): 9.6 ng / ml
AII289 (JMab-138): 10.5 ng / ml
AII394 (JMab-139): 10.6 ng / ml
AII488 (JMab-140): 11.0 ng / ml
AIJ 40 (JMab-141): 3.7 ng / ml
SA 12: 1.8 ng / ml
SG430: 1.2 ng / ml
(B) ED50 against CHO with high expression of mouse AILIM
AIF 34 (JMab-124): 42 ng / ml
AIF348 (JMab-127): 81 ng / ml
AIF620 (JMab-128): 100 ng / ml
AII289 (JMab-138): 53 ng / ml
AII394 (JMab-139): 60 ng / ml
AII488 (JMab-140): 70 ng / ml
(C) ED50 against rat AILIM high expressing CHO
AIF 34 (JMab-124): 45 ng / ml
AIF348 (JMab-127): 62 ng / ml
AIF620 (JMab-128): 97 ng / ml
AII289 (JMab-138): 57 ng / ml
AII394 (JMab-139): 90 ng / ml
AII488 (JMab-140): 90 ng / ml
[0162]
As a result, it was revealed that the anti-human AILIM human monoclonal antibody of the present invention has significant specificity for human AILIM.
Furthermore, it is clear that the six anti-human AILIM human monoclonal antibodies ((B) and (C) above) show reactivity (binding and cross-reactivity) with both mouse AILIM and rat AILIM. became.
[0163]
Example 6 Measurement of affinity and neutralizing activity of anti-human AILIM human monoclonal antibody for antigen (human AILIM)
The binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (Kd) of the various purified anti-human AILIM human monoclonal antibodies prepared above with human AILIM were determined using a commercially available measurement kit Biacore X (manufactured by Amersham-Pharmacia). ).
[0164]
<6-1> Preparation of antigen for fixing sensor chip
As an antigen to be immobilized on the sensor chip of the kit, a recombinant chimeric antigen comprising an extracellular region of human AILIM and a constant region (Fc) of human IgG1 (hereinafter, referred to as “human AILIM-IgFc”) was prepared.
The human AILIM-IgFc is disclosed in Tezuka's other earlier application (Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and International Patent Application Publication No. WO 98/38216, which is one of the present inventors. The product prepared by the method of Example 16 (2) was further purified.
The culture supernatant of the recombinant cell producing the human AILIM-IgFc was applied to a HiTrap protein column G (HiTrap affinity column Protein G, manufactured by Amersham Pharmacia) at 3 ml / min, equilibrated with a phosphate buffer (30 ml). Then, human AILIM-IgFc contained in the culture supernatant was adsorbed to the column.
[0165]
Next, after washing the column with a phosphate buffer (20 ml), 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute human AILIM-IgFc. Next, a solution (pH 9.0) consisting of 750 mM Tris-HCl was added to the eluate for neutralization, followed by dialysis (overnight) with a phosphate buffer. Next, the dialyzed solution was filtered through a filter (manufactured by Millipore) to obtain purified anti-human AILIM-IgFc.
The value at A280 was determined using a spectrophotometer, and the protein concentration was calculated (1A280 = 1 mg / ml). As a result, 0.28 mg / ml human AILIM-IgFc was obtained.
In addition, a chimeric protein consisting of an extracellular region of rat AILIM and a constant region (Fc) of human IgG1 (rat AILIM-IgFc; Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and an international patent application A purified product of WO98 / 38216 (Example 16 (2)) was also prepared in the same manner as described above, and as a result, 0.45 mg / ml of rat AILIM-IgFc was obtained.
[0166]
<6-2> Measurement of affinity and neutralizing activity
Operations other than the immobilization of the antigen (human AILIM-IgFc) described below on the sensor chip were performed according to the instruction manual attached to the commercially available measurement kit Biacore X (manufactured by Amersham-Pharmacia) and the experimental operation method.
HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.0) is contained in Flow Cell 1 (Flow Cell-1) attached to the kit. At a flow rate of 5 μl / min. Then, a solution (15 μl) consisting of 0.005 M NHS (N-hydroxysuccinimide) and 0.2 M EDC (N-Ethyl-N ′-(dimethylaminopropyl) carbodiimide) is added, and the sensor chip surface is added. Activated the carboxyl group of the CM coated on the substrate.
Next, 23 μl of human AILIM-IgFc (10 μg / ml; dissolved in 10 mM sodium acetate buffer (pH 5.0)) was added to immobilize the human AILIM-IgFc on the sensor chip. Unreacted activated carboxyl groups were then blocked by adding 35 μl of 1 M ethanolamine hydrochloride. In the two immobilization operations, the amount of immobilized human AILIM-IgFc was 2,444 RU (resonance unit) and 2,213 RU, respectively. RU indicates the mass per area, and 1 RU = 1 pg / mm ² It is.
[0167]
Flow cell 2 (Flow Cell-2) as a reference was capped by treating in the same manner as above without adding human AILIM-IgFc.
A phosphate buffer was flowed through the flow cell (sensor chip) at a flow rate of 20 μl / min, and each of the purified anti-human AILIM human monoclonal antibodies (10 to 50 μg / ml, 60 μl) prepared in the above Examples was added.
The measurement was carried out with the binding phase being 3 minutes and the dissociation phase being 10 minutes as standard conditions. The amount of the antibody bound to the antigen and the amount of the antibody dissociated from the antigen were measured over time to obtain a sensorgram. The dissociation of the antibody bound to the antigen was performed by flowing PBS through the sensor chip at a flow rate of 20 μl / min.
Based on the obtained sensorgram data, the binding rate constant (ka), dissociation rate constant (kd), and dissociation constant (Kd; Kd; Kd = kd / ka) were determined using analysis software (BIAevaluation 3.0) attached to the kit. Was calculated.
The antigen affinity and neutralizing activity of the mouse monoclonal antibodies SA12 and SG430 against human AILIM prepared in the above Examples were also analyzed in the same manner as described above.
Each value is shown below.
[0168]

These results revealed that each of the anti-human AILIM human monoclonal antibody and the anti-human AILIM mouse monoclonal antibody had extremely high binding affinity and neutralizing activity for human AILIM.
[0169]
Example 7 Costimulatory Signaling Activity of Anti-Human AILIM Human Monoclonal Antibody on Human T Cells
Whether the anti-human AILIM human monoclonal antibody of the present invention has the ability to control (promote and / or suppress) human T cell responses (production of cytokines such as IFN-γ and IL-4 and cell proliferation, etc.) In other words, whether or not it has the ability to control the transmission of a co-stimulatory signal into cells via AILIM is determined by determining the cytokine (IFN-γ and IL-4) from human T cells. Analysis was performed using the amount of production and the degree of proliferation of the human T cells as indices.
[0170]
<7-1> Antibody dilution
Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted with phosphate buffer (PBS) to a final concentration of 8 μg / ml.
Each of the various anti-human AILIM human monoclonal antibodies prepared above was diluted with PBS to a final concentration of 40 μg / ml, and further diluted with PBS to various concentrations (40 μg / ml to 0.0049 μg / ml). ).
[0171]
<7-2> Microplate coating with antibody
In each well of a 96-well microplate (plurality), one of (1) an anti-human CD3 monoclonal antibody OKT3 (25 μl / well of 8 μg / ml) and various anti-human AILIM human monoclonal antibodies (40 μg / ml to 0. 0049 μg / ml at 25 μl) or (2) only the anti-human CD3 monoclonal antibody OKT3 (8 μg / ml at 25 μl / well) and incubated at 37 ° C. for 2 hours. The antibody was then discarded and each well was washed three times with PBS. After washing, RPMI1640 medium containing 10% FCS (100 μl / well) was added to each well, and incubated at 37 ° C. for 1 hour, to coat each well of the plate with the antibody (1) or (2).
In addition, each of the following monoclonal antibodies was used as a control antibody instead of the anti-human AILIM human monoclonal antibody, and the plate was coated in the same manner as described above.
(1) Mouse monoclonal antibody SA12 or SG430 against human AILIM (Japanese Patent Application Publication No. 11-29599 (Example 12) and International Patent Application Publication WO 98/38216 (Example 12)).
(2) Mouse anti-human CETP monoclonal antibody JHC1 (also referred to as JMab109; Japanese Patent Application Publication No. 9-20800).
(3) Anti-KLH human monoclonal antibody (also referred to as JMab23; the above Examples).
These antibody coated microplates were used in the following tests.
[0172]
<7-3> Preparation of human T cell suspension
Peripheral blood of each of healthy persons (5 persons; donors A, B, C, D and E) was collected, and mononuclear cells were fractionated by density gradient centrifugation using LymphoPrep (manufactured by Nycomed). According to the experimental operation manual, human T cells were separated and obtained from the human mononuclear cells using a Pan-T cell Isolation Kit (manufactured by Miltenyi) and a magnetic sorter. The number of the T cells was counted by a hemocytometer. The human T cells were suspended in RPMI1640 medium containing 10% FCS, and a human T cell suspension (1 × 10 4 ⁶ Cells / ml) were prepared.
[0173]
<7-4> Cell culture
(1) Culture on microplate coated with anti-human CD3 antibody and anti-human AILIM antibody Human T cell suspension (donors A, B, C, D and E; 100 μl) was added to each well of the antibody-coated microplate. / Well; 1 × 10 ⁵ cells / well) and add CO ₂ The cells were cultured at 37 ° C. for 3 days in an incubator.
After the culture, the culture supernatant (50 μl) was separated and stored at −20 ° C. for use in the test described below (quantification of IFNγ). Each microplate after the sampling of the culture supernatant was used for the following tests.
(2) Culture on microplate coated only with anti-human CD3 antibody
A human T cell suspension (donor D; 100 μl / well; 1 × 10 5) was added to each well of the antibody-coated microplate. ⁵ cells / well), and any of various anti-human AILIM human monoclonal antibodies (25 μl of 40 μg / ml to 0.0049 μg / ml) was added. ₂ The cells were cultured at 37 ° C. for 3 days in an incubator.
[0174]
<7-5> Measurement of T cell proliferation activity
In each well of each plate after the culture, methyl [ ³ H] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia) was added, and CO was added. ₂ Incubation was performed at 37 ° C. for 6 hours in an incubator. After the culture, the cells were trapped on a GF / C filter (Packard) using a cell harvester. Next, the filter was dried at 40 ° C. for 3 hours or more, and then Microscinti 0 (20 μl / well; manufactured by Packard) was added. Being taken up by cells trapped on the filter using a β counter (TOP COUNT) ³ The radioactivity of H was measured and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIGS.
As a result of this test, when a microplate coated with an anti-human AILIM monoclonal antibody (a human monoclonal antibody or a mouse monoclonal antibody) together with an anti-human CD3 antibody was used, concentration-dependent significant proliferation of human T cells was observed. Was. Also, the degree of cell proliferation showed some difference between donors.
On the other hand, when a plate coated with only the anti-human CD3 antibody is used and the anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) is added to the cell culture system in a solution state (liquid phase) and cultured, No significant proliferation of human T cells was observed.
[0175]
<7-6> Measurement of the amount of IFNγ in the culture supernatant of T cells
For each T cell culture system (donor B and C) of <7-4> (1), the amount of IFNγ contained in the culture supernatant was measured using a commercially available human IFNγ ELISA KIT (manufactured by Amersham Pharmacia; Endogen). Was used for the measurement.
The results are shown in FIGS.
As a result of this test, a significant increase in the production of IFNγ depending on the concentration of the anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) was observed.
[0176]
Example 8 Control activity of anti-human AILIM human monoclonal antibody on mixed lymphocyte reaction (MLR)
Whether the anti-human AILIM human monoclonal antibody of the present invention has the ability to control (promote and / or suppress) T cell responses (such as production of cytokines such as IFN-γ and IL-4 and cell proliferation); That is, whether or not it has an ability to control the transmission of a co-stimulatory signal into cells via AILIM is determined by T in the allogeneic mixed lymphocyte reaction (allelic MLR). The analysis was performed using the presence or absence of an activity to control cell growth (that is, intracellular DNA synthesis) as an index.
[0177]
<8-1> Preparation of human PBMC and T cells
Peripheral blood (200 ml) collected from each of healthy persons (7; donors A, B, C, D, E, F, and G) was dispensed into Lymphoprep (15 ml) into a microtube (50 ml; Falcon). ; Nycomed). Next, after centrifugation (1600 rpm, 10 minutes), the intermediate layer was recovered. The collected cells were diluted two-fold or more with a phosphate buffer, and then centrifuged (1,800 rpm, 10 minutes) to obtain PBMC (peripheral blood mononuclear cell; 2 × 10 6 ⁸ ~ 5 × 10 ⁸ Cells) were prepared. The number of cells was counted using a hemocytometer, and the number of cells required for the MLR test (1.08 × 10 ⁸ (Cells / 9 microplates) were collected and stored on ice. The remaining cells were used for the following T cell isolation.
For the separation of T cells from PBMC, a PanT Isolation kit (Miltenyi Biotech) was used. According to the experimental operation manual attached to the kit, the remaining PBMC was added to the solution attached to the kit and reacted. The cells were then washed with PBS containing 5 mM EDTA and 0.5% BSA before being resuspended in the PBS. Next, the cell suspension was added to Positive Selection Column VS + (Miltenyi Biotech) swollen with the PBS, and a non-adsorbed fraction was collected. Further, the PBS was added to the column, and the washing solution was collected. The same operation was performed again. The recovered solution was combined to obtain a T cell fraction. The T cell fraction was resuspended in the PBS after centrifugation. The number of the obtained T cells was counted using a hemocytometer and used for the following tests.
[0178]
<8-2> Mixed lymphocyte reaction (MLR)
As described above, the pathways of the costimulatory signals required for the activation of lymphocytes such as T cells are CD28 and CD80 (B7-1) / CD86, which have been relatively sufficiently analyzed. Two pathways are known: a signaling pathway between (B7-2) and a signaling pathway between CTLA4 and CD80 (B7-1) / CD86 (B7-2).
That is, T cell proliferation in the mixed lymphocyte reaction (MLR) is also induced by signal transduction via the two known pathways.
Therefore, in this test, the following test substances were used to (1) suppress the MLR by blocking the CTLA4-mediated signal transduction pathway, and (2) signal transduce via CD80 (B7-1) / CD86 (B7-2). (3) Suppression of MLR by blocking both pathways via CTLA4 and signaling pathway via CD80 (B7-1) / CD86 (B7-2), (4) AILIM 3 were analyzed for MLR suppression by blocking the signaling pathway, and (5) MLR suppression by blocking both the CTLA4-mediated pathway and the AILIM-mediated pathway.
[0179]
The following were used as test substances.
(1) Anti-human AILIM human monoclonal antibody (prepared in the above example).
(2) Anti-human AILIM mouse monoclonal antibody SA12 (same as in the above example).
(3) Anti-KLH human monoclonal antibody (negative control; same as in the above example).
(4) Mouse IgG antibody (anti-human CD34; negative control; manufactured by Immunotech).
(5) A mixture of an anti-human CD80 monoclonal antibody (Pharmingen) and an anti-human CD86 monoclonal antibody (Pharmingen).
(6) Human CTLA4-IgFc chimeric molecule (manufactured by Ancell).
[0180]
Using PBMC and T cells prepared from each donor obtained in <8-1>, a mixed lymphocyte reaction (MLR) was performed in the following six combinations.
(I) T cells (donor A) / PBMC (donor D)
(Ii) T cells (donor D) / PBMC (donor B)
(Iii) T cells (donor C) / PBMC (donor A)
(Iv) T cells (donor E) / PBMC (donor G)
(V) T cells (donor F) / PBMC (donor E)
(Vi) T cells (donor G) / PBMC (donor F)
PBMC and T cells used in the test were adjusted to the following concentrations.
After dispersing PBMC in PBS, it was transferred to a culture dish (60 mm) and irradiated with X-rays (50 Gy) using a radiation irradiation device (manufactured by Hitachi Medical). After the cells were collected and centrifuged, the cells were added to RPMI1640 medium containing 10% FCS, and the number of cells was increased to 2 × 10 ⁵ Adjusted to cells / 50 μl.
Further, the T cells from each donor obtained above were added to RPMI1640 medium containing 10% FCS, and the number of cells was reduced to 1 × 10 ⁵ Adjusted to cells / 50 μl.
[0181]
<8-2-1> Suppression of MLR by anti-human AILIM human monoclonal antibody against MLR
After adding PRMI1640 medium containing 10% FCS to each well of a 96-well U-bottom microplate, a solution of an anti-human AILIM human monoclonal antibody or anti-human AILIM mouse monoclonal antibody SA12 diluted to various concentrations with RPMI1640 medium containing 10% FCS. Was added (final concentrations: 0, 0.31, 1.25, 5, and 20 μg / ml). Then, T cells (50 μl) were added and CO ₂ The cells were cultured at 37 ° C. for 1 hour in an incubator (NAPCO). After completion of the reaction, PBMC from another donor (50 μl) was added to start MLR.
The MLR when a test substance other than an anti-human AILIM human monoclonal antibody ((3) to (6) above) was used was obtained by adding T cells derived from another donor after culturing PBMC and the test substance. The reaction was performed.
On the fifth day of the culture, each well contained a tritium-labeled thymidine diluted with RPMI1640 medium containing 10% FCS ( ³ After adding H-Thymidine (20 μl; 1 μCi / well), the cells were further cultured for one day. After the culture, the cells are collected using a Cell Harvester (manufactured by Packard) and incorporated into the cells using a β counter (TOP COUNT; manufactured by Packard). ³ The radioactivity of H was measured and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIGS.
[0182]
<8-2-2> Inhibition of MLR by anti-human AILIM human monoclonal antibody against MLR in a system in which signal transduction via CTLA4 was previously blocked
After adding PRMI1640 medium containing 10% FCS to each well of a 96-well U-bottom microplate, a solution of an anti-human AILIM human monoclonal antibody or anti-human AILIM mouse monoclonal antibody SA12 diluted to various concentrations with RPMI1640 medium containing 10% FCS was added. (Final concentrations: 0, 0.31, 1.25, 5 and 20 μg / ml). Then, T cells (50 μl) were added and CO ₂ The cells were cultured at 37 ° C. for 1 hour in an incubator (NAPCO).
Separately from the culture of the T cells, human CTLA4-IgFc was added to PBMC (in RPMI1640 medium containing 10% FCS) from another donor, and COB was added. ₂ The cells were cultured at 37 ° C. for 1 hour in an incubator (NAPCO). The concentration of the CTLA4-IgFc was adjusted to 20 μg / ml at the start of MLR.
Next, the PBMC (50 μl) was added to the T cell culture system to start MLR. The MLR in the case where a test substance other than the anti-human AILIM human monoclonal antibody (the above (3) to (5)) was used was PBMC obtained by culturing together with CTLA4-IgFc in the same manner as above, and the test substance. After culturing, the reaction was performed by adding T cells from another donor.
On the fifth day of the culture, each well contained a tritium-labeled thymidine diluted with RPMI1640 medium containing 10% FCS ( ³ After adding H-Thymidine (20 μl; 1 μCi / well), the cells were further cultured for one day. After the culture, the cells are collected using a Cell Harvester (manufactured by Packard) and incorporated into the cells using a β counter (TOP COUNT; manufactured by Packard). ³ The radioactivity of H was measured and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIGS.
[0183]
As a result of the above two tests, the following became clear.
(1) CTLA4-IgFc suppresses TLA-induced proliferation of T cells by blocking signal transduction via CTLA-4.
(2) Anti-CD80 antibody and anti-CD86 antibody suppress the proliferation of T cells by allogeneic MLR by inhibiting signal transduction via CD80 / CD86 which is a ligand of CTLA4 and CD28.
(3) Similar to CTLA4-IgFc, anti-CD80 antibody and anti-CD86 antibody, a monoclonal antibody against human AILIM significantly suppresses T cell proliferation in allogeneic MLR by AILIM-mediated signal transduction in an antibody concentration-dependent manner. .
This result indicates that, in addition to the known CTLA4 / CD80 / CD86-mediated pathway and the CD28 / CD80 / CD86-mediated pathway, AILIM is included in the costimulatory signal transmission pathway required for T cell activation. And that there is a third pathway via the ligand and that the signaling pathway via the AILIM is inhibited by antibodies to AILIM.
Furthermore, the contribution of the AILIM-mediated pathway to the signaling was shown to be likely to be similar to that of the CTLA4 / CD80 / CD86-mediated pathway and CD28 / CD80 / CD86-mediated pathway.
[0184]
Example 9 Activity of Anti-Human AILIM Human Monoclonal Antibody to Induce Antibody-Dependent Cellular Damage (ADCC)
One of the biological activities caused by antibodies is the induction of antibody-dependent cellular cytotoxicity (ADCC). ADCC is a cytotoxic effect of target cells caused by effector cells such as lymphocytes, macrophages or polymorphonuclear leukocytes. In addition to the effector cells and the target cells, ADCC It is a cytotoxic effect that requires antibodies to induce damage.
The presence or absence of ADCC-inducing activity of the anti-human AILIM monoclonal antibody of the present invention was analyzed as follows.
[0185]
In the culture system of human AILIM high-expressing recombinant HPB-ALL cells prepared in the above Examples, ⁵¹ Cr (0.1 mCi / 10 ⁶ After adding cells (Amersham-Pharmacia) and incubating at 37 ° C. for 2 hours, the cells were washed eight times with RPMI1640 medium, and the resulting radiolabeled cells were used as target cells (Target Cell).
In addition, for use in a control test, wild-type human HPB-ALL cells were radioactively labeled in the same manner as described above.
The PBMC fraction was separated from peripheral blood collected from a healthy person using Lymphospar I (manufactured by IBL). The obtained human PBMC was used as an effector cell.
The target cells (1 × 10 6) were placed on a 96-well U-bottom microplate (manufactured by Nunc). ⁴ cells / well; 25 μl / well). Then, in each well, various concentrations of anti-human AILIM human monoclonal antibody (0.0001 to 1.0 μg / ml; 25 μl / well) diluted with 5% FBS-containing RPMI1640 medium, the medium alone (25 μl / well) or 1% % Nonidet P-40 (25 μl / well; a surfactant having cell membrane lytic activity) was added, and the cells were cultured at room temperature for 20 minutes.
As a positive control, anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was used in place of the anti-human AILIM antibody and cultured in the same manner as described above.
[0186]
Then, effector cells (E / T ratio = 50; 1 × 10 ⁵ cells / well; 50 μl / well) and 5% CO ₂ The cells were cultured at 37 ° C. for 16 hours in an incubator.
After the culture, centrifugation (1,500 rpm; 4 ° C .; 10 minutes) was performed to collect the centrifugal supernatant. The radioactivity in the supernatant was measured using a gamma counter. The radioactivity was released into the culture supernatant due to cell membrane damage by ADCC ⁵¹ Indicates the amount of Cr.
ADCC induced by anti-AILIM antibody or anti-CD3 antibody, the radioactivity in the assay using the medium alone was assumed to be zero (0), and the activity in the assay with the addition of Nonidet was assumed to be 100%. The cell membrane damage rate (cell lysis rate) (%) was calculated.
The results are shown in FIGS. 70 and 71.
As a result of this test, the anti-human AILIM human monoclonal antibody of the present invention had a concentration-dependent ADCC-inducing activity.
[0187]
Example 10 Determination and Analysis of Gene Sequence and Amino Acid Sequence of Anti-human AILIM Human Monoclonal Antibody
The cDNA sequence encoding the heavy chain (Heavy Chain) and the cDNA sequence encoding the light chain (Light Chain) constituting the human monoclonal antibodies against various human AILIMs prepared in the above Examples were determined as follows. The structural characteristics of the gene were analyzed.
A Quick Prep mRNA Purification Kit kit from each of the hybridomas (clone: AIH5D3 (JMab-136), AII289 (JMab-138) and AII394 (JMab-139)) that produce human monoclonal antibodies against human AILIM prepared in the above Examples (Amersham Pharmacia) using PolyA ⁺ RNA was extracted and purified.
The hybridoma was lysed in a cell lysis buffer (Lysis Buffer), the cells were disrupted with a syringe, and the cells were solubilized. Oligo (dT) resin was added to the lysate, and the mixture was gently shaken. Then, after washing Oligo (dT) resin, PolyA ⁺ RNA was eluted with Elution Buffer. PolyA eluted ⁺ RNA was ethanol precipitated and dissolved in Tris-EDTA buffer. PolyA obtained ⁺ The concentration of RNA was determined by measuring the absorbance at a wavelength of 260 nm.
[0188]
PolyA obtained ⁺ Using RNA as a template, a double-stranded cDNA was synthesized by the M-MLV Reverse Transcriptase method using a commercially available cDNA synthesis kit (GIBCOBRL) and a synthetic oligo DNA NotI-T (SEQ ID NO: 1) as primers.
That is, PolyA purified from a hybridoma ⁺ Using RNA (about 5 μg) as a template, a single-stranded cDNA was synthesized by reaction at 37 ° C. for 1 hour in a solution (about 50 μl) containing the primer (about 400 pmole) and M-MLV Reverse Transcriptase. Next, dNTP, DNA polymerase I, RNase H, DNA ligase, buffer and distilled water were added to the reaction solution (4 μl), and the mixture was reacted at 16 ° C. for 2 hours to synthesize double-stranded cDNA. The obtained double-stranded cDNA was extracted with phenol / chloroform and then precipitated with ethanol.
Next, EcoRI linker DNA (about 300 pmole) and DNA ligase (Ligation High; 33 μl; manufactured by TOYOBO) were added to the double-stranded cDNA (about 50 μl) dissolved in TE buffer, and reacted at 16 ° C. for about 80 minutes. A linker DNA was ligated to the cDNA. The linker DNA used was a double-stranded DNA prepared by hybridizing an oligo DNA (20 adp; SEQ ID NO: 2) phosphorylated at the 5 ′ end with an oligo DNA (24 adp; SEQ ID NO: 3) according to a conventional method. .
[0189]
The obtained reaction product was extracted with phenol / chloroform and then precipitated with ethanol. Next, the DNA reaction product is digested with a commercially available restriction enzyme NotI (manufactured by TOYOBO), and then reacted with a commercially available ATP solution (manufactured by GIBCO BRL) and T4 kinase (manufactured by TOYOBO) (at 37 ° C. for 30 minutes). The 5 'end was phosphorylated.
After ethanol precipitation of the obtained DNA reaction product, fractionation was performed by polyacrylamide gel electrophoresis, and a gel containing DNA of about 500 bp to 2000 bp was cut out. The gel was cut out by ethidium bromide staining and photographing under ultraviolet irradiation.
The excised gel was crushed, suspended in a TE buffer, centrifuged, and the centrifuged supernatant was collected.
The recovered DNA and a commercially available lambda phage vector λEXcell (0.25 μg; manufactured by Amersham Pharmacia) were reacted (16 ° C., 30 minutes) and ligated in the presence of a commercially available DNA Ligase (Ligation High; manufactured by TOYOBO). Next, the DNA reaction product was used as a lambda phage using a commercially available lambda phage packaging kit Gigapack III Gold (manufactured by STRATAGENE), and a cDNA library was prepared using E. coli NM522 as a host. All operations were performed in accordance with the experimental operation manual attached to the kit.
[0190]
The cDNA library was then screened according to the plaque hybridization method (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) and screening of the cDNA library described below.
The cDNA library (1 × 10 ⁴ Individual plaques were sown on an agar plate, and replicas were prepared using Hybond-N nylon membrane (manufactured by Amersham Pharmacia). This replica is called γ ³² Screening was performed by plaque hybridization in a hybridization buffer using a probe labeled with P-ATP. Probe HIGLC (SEQ ID NO: 4) was used for the antibody light chain, and probe NHCc2 (SEQ ID NO: 5) was used for the antibody heavy chain. Each positive clone identified by the primary and secondary screens was isolated in a single plaque.
[0191]
Using the phage solution of each positive clone as template DNA, each of the antibody heavy chain and the antibody light chain was amplified by PCR using one PCR primer and Taq PCR kit (TAKARA). ExcellE (SEQ ID NO: 6) and ck117 (SEQ ID NO: 7) were used as antibody light chain primers, and ExcellE (SEQ ID NO: 6) and NHCc2 (SEQ ID NO: 5) were used as antibody heavy chain primers. The PCR product was fractionated by agarose gel electrophoresis according to a conventional method, and a gel containing DNA of about 600 bp in both heavy and light chains was cut out. The nucleotide sequence of the DNA purified from the gel was analyzed using DNA Sequencer (373A; manufactured by PE-Applied Biosystems), ABI PRISM Sequencing Software (manufactured by PE-Applied Biosystems) and ABI PRISM Then, it was confirmed that each positive clone had a DNA having a sufficient base length.
[0192]
The λ phage of the plaque of each positive clone was infected into Escherichia coli NP66 to release the plasmid DNA, and then plated on a plate containing ampicillin to form a colony. Next, Escherichia coli JM109 was transformed with the purified plasmid DNA recovered from the colony according to a conventional method. Then, the transformed cells were plated on a nutrient medium containing ampicillin to form colonies.
Next, the suspension of each colony suspended in the LB medium containing ampicillin was cultured in a liquid nutrient medium at 37 ° C. for 24 hours. Cells were collected from the culture solution, and plasmid DNA was purified using a plasmid purification kit (manufactured by Quiagen). Each purified plasmid DNA was digested with restriction enzymes EcoRI / NotI, and the presence of vector DNA and inserted DNA (heavy chain cDNA or light chain cDNA) was confirmed.
The nucleotide sequence of the cDNA encoding each of the antibody heavy chain or antibody light chain inserted into each purified plasmid was determined according to a conventional method using DNA Sequencer (377A; manufactured by PE-Applied Biosystems), ABI PRISM Sequencing Software (PE- Applied Biosystems) and ABI PRISM Auto Assembler (PE-Applied Biosystems).
The following primers were used in this sequencing.
[0193]
<Primers used for sequence analysis of heavy chain cDNA>
M13R primer (SEQ ID NO: 8; manufactured by STRATAGENE), ExcellE (SEQ ID NO: 6), 136H (SEQ ID NO: 9), 138 / 9H (SEQ ID NO: 10), AILIMHC1 (SEQ ID NO: 11), HCc1 (SEQ ID NO: 12), NHCc2 ( SEQ ID NO: 5), HCc7 (SEQ ID NO: 13), HCc8 (SEQ ID NO: 14), HCc3 (SEQ ID NO: 15), HCc4 (SEQ ID NO: 16), HCc6 (SEQ ID NO: 17), HIGHC (SEQ ID NO: 18), HCc9 (SEQ ID NO: 18) No. 19), HCc5 (SEQ ID NO: 20) and polyA (SEQ ID NO: 21).
<Primers used for light chain cDNA sequence analysis>
M13R primer (SEQ ID NO: 8; manufactured by STRATAGENE), ExcellE (SEQ ID NO: 6), AILIMLC1 (SEQ ID NO: 22), AILIMLC2 (SEQ ID NO: 23), LCc1 (SEQ ID NO: 24), ck117 (SEQ ID NO: 7), HIGLC (SEQ ID NO: 7) 4), LCc2 (SEQ ID NO: 25), HIK (SEQ ID NO: 26), and polyA (SEQ ID NO: 21).
The cDNA sequence encoding the heavy chain of the human monoclonal antibody against human AILIM produced by each of the hybridomas, the cDNA sequence encoding the light chain (Light Chain), and the amino acid sequence deduced from each cDNA sequence are shown below. The results are shown in the sequence listing.
[0194]
Clone AIH5D3 (JMab-136)
<Heavy chain>
DNA sequence: SEQ ID NO: 27 (signal sequence: base numbers 69 to 125, V region: base numbers 126 to 419)
Amino acid sequence: SEQ ID NO: 28 (signal sequence: including amino acids 1 to 19, variable region: including amino acids 20 to 118)
<Light chain>
DNA sequence: SEQ ID NO: 29 (signal sequence: base numbers 39 to 104, V region: base numbers 105 to 386)
Amino acid sequence: SEQ ID NO: 30 (signal sequence: including amino acids 1 to 22, variable region: including amino acids 23 to 116)
[0195]
Clone AII289 (JMab-138)
<Heavy chain>
DNA sequence: SEQ ID NO: 31 (signal sequence: base numbers 94 to 150, V region: including base numbers 151 to 441)
Amino acid sequence: SEQ ID NO: 32 (signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 116)
<Light chain>
DNA sequence: SEQ ID NO: 33 (signal sequence: including base numbers 28 to 87, V region: including base numbers 88 to 375)
Amino acid sequence: SEQ ID NO: 34 (signal sequence: including amino acids 1 to 20, variable region: including amino acids 21 to 116)
[0196]
Clone AII394 (JMab-139)
<Heavy chain>
DNA sequence: SEQ ID NO: 35 (signal sequence: base numbers 96 to 152, V region: base numbers 153 to 443)
Amino acid sequence: SEQ ID NO: 36 (signal sequence: including amino acids 1 to 19, variable region: including amino acids 20 to 116)
<Light chain>
DNA sequence: SEQ ID NO: 37 (signal sequence: base numbers 33 to 92, V region: base numbers 93 to 380)
Amino acid sequence: SEQ ID NO: 38 (signal sequence: including amino acids 1 to 20, variable region: including amino acids 21 to 116)
[0197]
Based on the determined DNA sequences, using a gene sequence analysis software, a human immunoglobulin variable region gene library V BASE Sequence (Immunol. Today, Vol. 16, No. 16) prepared by Tomlinson et al. 5, p. 237-242, 1995).
As a result, the V region genes of each of the heavy and light chains of the human monoclonal antibody were composed of the following segments.
[0198]
<Heavy chain V region gene>
Clone AIH5D3 (JMab-136): 1-02
Clone AII289 (JMab-138): 3-13
Clone AII394 (JMab-139): 3-13
<Light chain V region gene>
Clone AIH5D3 (JMab-136): L5
Clone AII289 (JMab-138): A27
Clone AII394 (JMab-139): A27
[0199]
Example 11 Inhibitory effect of anti-human AILIM human monoclonal antibody on delayed-type hypersensitivity (DTH)
The immune response system of a living body that eliminates antigens harmful to the living body (pathogenic microorganisms such as viruses, bacteria and parasites and foreign foreign substances) is roughly classified into an innate immune response and an acquired immune response (acquired immunity). .
The former are elimination mechanisms by nonspecific recognition, such as phagocytosis by phagocytes (polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack by natural killer (NK) cells, and opsonization of antigen by complement. is there.
The latter acquired immune response is a mechanism of elimination by lymphocytes (mainly T cells and B cells) that have acquired (activated) specificity for the antigen.
The adaptive immune response is further classified into cell-mediated immunity and humoral immunity.
Cell-mediated immunity, unlike antibody-dependent humoral immunity, is an immune response that is primarily expressed by T cells acting directly on their target antigen. Cellular immunity has been shown to be involved in immune responses to viruses and tumors, immune responses elicited after transplantation of tissues and organs, certain drug allergies, and some autoimmune diseases.
[0200]
As the most typical phenomenon of this cellular immunity, tuberculin allergy (substantially synonymous with tuberculin hypersensitivity) is well known. Tuberculin allergy is a delayed allergy that is established with the infection of Mycobacterium tuberculosis, and is induced by injecting the tuberculin protein produced in the culture solution of Mycobacterium tuberculosis into the skin of a living body to elicit an immune response. .
Delayed allergy is an allergy mediated by T cells sensitized by an antigen (memory T cells that store the antigen). The allergic reaction accompanied by inflammation by cells requires 24-48 hours to develop, and is therefore called delayed type.
The phenomenon of tuberculin allergy, which is a typical example of delayed allergy, has been universally applied to the “tuberculin test” for diagnosing whether or not an animal has been sensitized by M. tuberculosis. That is, purified tuberculin (purified protein derivative; PPD; in general diagnosis, 0.05 μg / 0.1 ml (2.5 TU) is 0.1 ml, 0.1 ml) is an animal skin. In this method, the major axis of redness of the skin at the administration site is measured 48 hours after administration, and the presence or absence of Mycobacterium tuberculosis is diagnosed based on the major axis. The major axis of redness is negative up to 4 mm, false positive in 4 to 9 mm and positive in 10 mm or more.
[0201]
Delayed allergy induced by cell-mediated immunity includes, in addition to allergy to infectious pathogen antigens such as tuberculosis allergy derived from Mycobacterium tuberculosis, Jones-Mote type delay that appears transiently for trace proteins and the like. Examples include type allergies, contact allergies to drugs such as picryl chloride and phytotoxins such as lacquer, and allergies involved in transplant rejection seen in allograft transplantation.
[0202]
In this test, the above-described tuberculin test was applied to evaluate the inhibitory effect of the anti-AILIM antibody on delayed-type hypersensitivity (delayed-type allergy). The test was performed as follows.
Each of cynomolgus monkeys (male, body weight: 6.0 to 8.5 kg, manufactured by Environmental Baylis Institute) that have been immunized with BCG (Bacille de Calmette et Guerin), which is an attenuated live strain of M. bovis After anesthetizing (3 animals in each group) with ketamine hydrochloride (10 mg / kg, intramuscular administration), any of the following test samples was added to the chest at a concentration of 0.1 ml / 1 administration site (6 administration sites / animal). Administered intraepidermally. In addition, the space | interval of each site | part which administered a sample was separated by 3 cm or more.
[0203]
(1) A 1: 1 mixed solution of anti-human AILIM human monoclonal antibody (JMab-136; 0.2 mg; 10 μg / 1 administration site) and tuberculin (4 μg / 1 ml of physiological saline).
(2) A 1: 1 mixed solution of anti-human AILIM human monoclonal antibody (JMab-136; 2 mg; 100 μg / 1 administration site) and tuberculin (4 μg / 1 ml of physiological saline).
(3) Phosphate buffer (PBS (-)) as a control.
(4) A 1: 1 mixed solution of a commercially available steroid anti-inflammatory drug prednisolone (Prednisolone; 0.2 mg; 10 μg / 1 administration site) and tuberculin (4 μg / 1 ml of physiological saline) as a positive control.
(5) A 1: 1 mixed solution of an anti-KLH human monoclonal antibody (Example <2-2>; 0.2 mg; 10 μg / 1 administration site) and tuberculin (4 μg / 1 ml of physiological saline) as a negative control. .
Twenty-four hours after the administration of each sample, the major axis and minor axis of redness at each administration site were measured to calculate the area of redness.
The results are shown in FIG.
As a result, in any of the groups to which the anti-AILIM antibody was administered, redness due to delayed allergy was significantly suppressed as compared with the control and the negative control. The inhibitory effect was comparable to that of the steroidal anti-inflammatory drug as a positive control.
[0204]
Example 12 Analysis of AILIM expression in each tissue of a graft-versus-host disease (GVHD) patient
Expression of AILIM in various biopsies from recipient patients who have been clinically diagnosed with acute or chronic graft versus host disease (GVHD) after receiving an allogeneic transplant from a donor Was analyzed by tissue staining by HE staining using the anti-human AILIM human monoclonal antibody (JMab-36) prepared in the above example according to a conventional method.
33 samples collected from various tissues of acute GVHD patients (28 cases) and 5 samples from chronic GVHD patients (5 cases) were analyzed.
As a result, in acute GVHD patients, 15 out of 29 samples of skin tissue were AILIM-positive, 1 of 3 samples of stomach tissue were AILIM-positive, and 1 of 1 sample of colon tissue was AILIM-positive. In chronic GVHD patients, one out of three skin tissue samples was AILIM positive, and two out of two colon tissue samples were AILIM positive. Furthermore, among the samples in which lymphocyte cell infiltration was remarkable, 10 out of 13 samples were AILIM-positive.
[0205]
Example 13 Costimulatory Signaling Activity of Anti-human AILIM Human Monoclonal Antibody on Monkey T Cells
Example 7 demonstrates that the anti-human AILIM human monoclonal antibody of the present invention is capable of controlling human T cell responses, specifically, controlling the transmission of costimulatory signals into cells via AILIM. It was demonstrated that T cells promoted proliferation. Thus, in this test, the presence or absence of the ability of the human monoclonal antibody to promote cell growth on monkey T cells was analyzed in the same manner as in Example 7.
[0206]
<13-1> Antibody dilution
Anti-human CD3 monoclonal antibody SP34 (manufactured by BD-Pharmingen) was diluted with phosphate buffer (PBS) to a final concentration of 1 µg / ml.
The anti-human AILIM human monoclonal antibody JMAb136 prepared above was diluted with PBS to a final concentration of 40 μg / ml, and further diluted with PBS to various concentrations (40 μg / ml to 0.064 μg / ml).
[0207]
<13-2> Coating of microplate with antibody
In each well of a 96-well microplate (plural), anti-human CD3 monoclonal antibody SP34 (1 μg / ml at 25 μl / well) and anti-human AILIM human monoclonal antibody JMAb136 (40 μg / ml to 0.064 μg / ml at 25 μl), Incubated at 37 ° C for 2 hours. The antibody was then discarded and each well was washed three times with PBS. After washing, RPMI1640 medium containing 10% FCS (100 μl / well) was added to each well, incubated at 37 ° C. for 1 hour, and each well of the plate was coated with the above antibody.
In addition, instead of the anti-human AILIM human monoclonal antibody, an anti-KLH human monoclonal antibody (also referred to as JMab23; the above-mentioned Example) was used as a control antibody to coat the plate in the same manner as described above.
These antibody coated microplates were used in the following tests.
[0208]
<13-3> Preparation of monkey T cell suspension
Cynomolgus monkey peripheral blood was collected, and mononuclear cells were fractionated by density gradient centrifugation using NycoPrep 1.077A (Nycomed). According to the experimental operation manual, the anti-human CD4 antibody M-T477 (manufactured by BD-Pharmingen), the anti-human CD8 antibody RPA-T8 (manufactured by BD-Pharmingen), anti-mouse IgG microbeads (manufactured by Miltenyi) and a magnetic sorter were used. Monkey T cells were separated and obtained from cynomolgus monocytes. The number of the T cells was counted by a hemocytometer. The monkey T cells were suspended in RPMI1640 medium containing 10% FCS, and a monkey T cell suspension (1 × 10 ⁶ Cells / ml) were prepared.
[0209]
<13-4> Cell culture
(1) Culture on microplate coated with anti-human CD3 antibody and anti-human AILIM antibody
A monkey T cell suspension (100 μl / well; 1 × 10 ⁵ cells / well) and add CO ₂ The cells were cultured at 37 ° C. for 2 days in an incubator.
After the culture, each microplate was used for the following tests.
[0210]
<13-5> Measurement of T cell proliferation activity
In each well of each plate after the culture, methyl [ ³ H] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia) was added, and CO was added. ₂ Incubation was performed at 37 ° C. for 6 hours in an incubator. After the culture, the cells were trapped on a GF / C filter (Packard) using a cell harvester. Next, the filter was dried at 40 ° C. for 3 hours or more, and then Microscinti 0 (20 μl / well; manufactured by Packard) was added. Being taken up by cells trapped on the filter using a β counter (TOP COUNT) ³ The radioactivity of H was measured and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIG.
As a result of this test, when a microplate coated with an anti-human AILIM monoclonal antibody (a human monoclonal antibody or a mouse monoclonal antibody) together with an anti-human CD3 antibody was used, significant proliferation dependent on the concentration of monkey T cells was observed. Was.
This result also shows that the anti-human AILIM human monoclonal antibody of the present invention has an activity of binding to monkey AILIM and controlling the function of monkey AILIM.
[0211]
Example 14 Construction of a method for identifying and quantifying a substance that binds to AILIM or an AILIM ligand
An ELISA (Enzyme-linked Immuno solvent assay) capable of identifying or quantifying a substance that binds to AILIM (ICOS) or an AILIM ligand (B7h / B7RP1 / GL50 / LICOS) was constructed.
The method described in detail below as an example is based on the principle that the extent to which the substance inhibits the binding between soluble human AILIM (hAILIM-IgFc) and soluble human AILIM ligand (hB7h-IgFc) is evaluated by ELISA.
[0212]
<14-1> Sample
The following samples were used.
(1) Streptavidin-HRP (Southern Biotechnology Associates, Inc.).
(2) Soluble human AILIM ligand (fusion protein consisting of extracellular region of human B7h and constant region of human IgG1)
It was produced as in the following section <14-2>.
(3) Biotin-labeled soluble AILIM-IgFc
Tezuka's other earlier application as one of the present inventors (Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and International Patent Application Publication WO98 / 38216 (Example 16 (2)) ) Was prepared by further purifying the product prepared by the method of (1).
(4) Anti-human AILIM human monoclonal antibody (JMab-136; above).
(5) Anti-KLH human monoclonal antibody (negative control antibody; JMab-23; above).
(6) Phosphate buffer (PBS (-); manufactured by Nikken Biological).
(7) PRMI1640 medium (manufactured by Niken Bio Inc.).
(8) Fetal calf serum (FCS; manufactured by JRH-Bioscience).
(9) 30% bovine serum albumin (BSA; manufactured by Sigma).
(10) Tween 20 (manufactured by BioRad).
(11) TMB ⁺ Substrate chromagen (manufactured by Dako).
[0213]
<14-2> Preparation of soluble human AILIM ligand (fusion protein (hB7h-IgFc) of extracellular region of human B7h and constant region of human IgG1)
Total RNA was prepared from human peripheral blood-derived mononuclear cells in the same manner as in the above Examples.
Using the prepared total RNA (5 μg) as a template, cDNA was synthesized using Superscript Preamplification System for First Strand cDNA Synthesis (manufactured by GIBCO-BRL). Next, in order to amplify the cDNA encoding the extracellular region of the human AILIM ligand (hB7h) by PCR, a 5 ′ primer (5′-GAGGTCTCCCCCTCGAGATGCGGCTGGGCAGTCC-3 ′, SEQ ID NO: 39) having a XhoI cleavage site at the end and BamHI cleavage A 3 'primer having a site (5'-CACGAGCAGCCCAGGGGATCCCACGTGGCCGCG-3', SEQ ID NO: 40) was designed and synthesized. PCR was performed using the obtained cDNA as a template and the primers to prepare a cDNA containing a cDNA encoding the extracellular region of human B7h having XhoI and BamHI cleavage sites at both ends. The obtained PCR product was digested with XhoI and BamHI, and subjected to agarose gel electrophoresis, and a band having a size of about 720 bp, which was predicted to be a cDNA fragment encoding the extracellular region of interest, was isolated. The isolated cDNA fragment was subcloned into plasmid pBluescript II SK (+) (Stratagene) digested with XhoI and BamHI. Sequence analysis using an automatic fluorescent DNA sequencer (Applied Biosystems) confirmed that the cDNA fragment contained a region encoding the extracellular region of human B7h.
[0214]
On the other hand, DNA encoding the Fc of human IgG1 as a fusion partner is described in Plasmid (see Cell, Vol. 61, p. 1303-1313, 1990. Seed by Dr. B. Seed of Massachusetts General Hospital). Was digested with BamHI and XbaI to cut out a BamHI-XbaI DNA fragment (about 1.3 kb). This fragment contains exons encoding each of the hinge region, Cγ12, and Cγ13 of human IgG1.
The XhoI-BamHI fragment encoding the extracellular region of human B7h (hB7h) prepared as described above, and the BamHI-XbaI fragment containing the exon encoding Fc of human IgG1 (abbreviated as “IgFc”) were replaced with XhoI. And subcloned into plasmid pBluescript II SK (+) (Stratagene) cut with XbaI.
Next, the plasmid was digested with XhoI and XbaI to cut out a DNA fragment of about 1.8 kb containing a fusion DNA consisting of the extracellular region of human B7h and human IgFc. This fusion DNA fragment was ligated with T4 DNA ligase to express the expression vector pME18S (Medical Immunology, Vol. 20, No. 1, p. 27-32, 1990, and Experimental Medicine (separate volume): "Genetic Handbook" Plasmid phB7h-IgFc was constructed by insertion into the XhoI and XbaI sites of Tsuchiya, pp. 101-107 (1992).
[0215]
By electroporation, COS7 cells (ATCC CRL-1651) cultured in a subconfluent monolayer in a DMEM medium containing 10% fetal bovine serum and ampicillin were transformed with the plasmid phB7h-IgFc to obtain transformed cells. did.
The transformed cells were cultured in serum-free ASF104 medium for 72 hours to express hB7h-IgFc.
HB7h-IgFc was purified using a Protein G Sepharose affinity column (manufactured by Amersham Pharmacia) as follows.
The centrifuged supernatant obtained by centrifuging the culture supernatant was added to a Protein G Sepharose affinity column which had been equilibrated with a binding buffer in advance. The column was then washed with binding buffer and eluted with elution buffer. The eluate was collected and dialyzed by exchanging the external solution with a phosphate buffer at least twice to obtain purified hB7h-IgFc.
[0216]
<14-3> Dilution of antibody and soluble human AILIM (hAILIM-IgFc) and their reaction anti-human AILIM monoclonal antibody (JMab-136) and negative control antibody anti-KLH human monoclonal antibody (JMab-23) , 20 μg / ml solution was diluted in 11 steps, and 200 μl of each sample was added with and mixed with RPMI 1640 medium containing 10% FCS (200 μl) to prepare solutions of various concentrations. Biotin-labeled hAILIM-IgFC (2 μl / tube; final concentration 1 μg / ml) was added to each of the prepared solutions having various concentrations, mixed, and then incubated at room temperature for 30 minutes.
[0219]
<14-4> Measurement of inhibitory activity of hAILIM-IgFc and hB7h-IgFc binding by anti-AILIM monoclonal antibody
HB7h-IgFc (50 μl / well (800 ng / well)) was added to a 96-well microplate, the plate was sealed, and then incubated at 37 ° C. for 1 hour. The solution was removed from each well and washed three times with PBS (120 μl). Then, PBS (100 μl / well) containing 0.5% BSA was added to each well to block unreacted portions, the plate was sealed, and the plate was incubated at 37 ° C. for 1 hour. After the reaction, the solution was removed and washed three times with PBS (120 μl). Next, each sample (50 μl / well) prepared in <14-3> was added, and the plate was sealed and incubated at 37 ° C. for 1 hour. The solution was removed from each well, and washed three times with RPMI1640 medium (120 μl) containing 10% FCS warmed to 37 ° C. Next, 3.7% formalin-containing PBS (100 μl / well) was added to each well and incubated on ice for 5 minutes. After removing the solution from each well, the wells were washed three times with 0.1% Tween 20 (120 μl). Next, Streptavidin-HRP (50 μl / well) was added, and the plate was sealed and incubated at room temperature for 30 minutes. The solution was removed from each well and washed three times with PBS (120 μl) containing 0.1% Tween 20. Then, TMB ⁺ Substrate chromagen (50 μl / well) was added and incubated at room temperature for 20 minutes. Next, 2N sulfuric acid (50 μl / well) was added to stop the reaction, and the absorbance at 450 nm was measured using THERMO max (manufactured by Molecular Devices).
The results are shown in FIGS.
As a result, the dose-dependent inhibitory activity of the binding between hAILIM-IgFc and hB7h-IgFc by the anti-AILIM antibody was measured.
Therefore, it was shown that a substance (for example, an antibody or a synthetic small molecule compound) that binds to AILIM or an AILIM ligand can be screened and identified using an assay system as exemplified in the present method.
[0218]
Example 15 Inhibitory activity of anti-human AILIM human monoclonal antibody on proliferation of human T cells by costimulatory signaling through AILIM-AILIM ligand
To determine whether the anti-human AILIM monoclonal antibody of the present invention has the ability to control AILIM-mediated signaling on human T cells, contact human T cells with an AILIM ligand (B7h / B7RP1 / GL50 / LICOS). The analysis was performed by measuring the inhibitory effect of the anti-human AILIM human monoclonal antibody on the induced cell proliferation.
[0219]
<15-1> Dilution of antibody, etc.
Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted with phosphate buffer (PBS) to a final concentration of 8 μg / ml.
The soluble human AILIM ligand (hB7h-IgFc) prepared above was diluted with PBS to a final concentration of 40 μg / ml, and further diluted with PBS to various concentrations (40 μg / ml to 0.0064 μg / ml). .
[0220]
<15-2> Coating of microplate with antibody
(1) Anti-human CD3 monoclonal antibody OKT3 (25 μl / well of 8 μg / ml) and hB7h-IgFc (25 μl of 40 μg / ml to 0.0064 μg / ml) were added to each well of a 96-well microplate (plurality). , 37 ° C for 2 hours. The antibody was then discarded and each well was washed three times with PBS. After washing, RPMI1640 medium containing 10% FCS (100 μl / well) was added to each well, and incubated at 37 ° C. for 1 hour to coat each well of the plate.
[0221]
<15-3> Preparation of human T cell suspension
Peripheral blood of a healthy person was collected, and mononuclear cells were fractionated by density gradient centrifugation using LymphoPrep (manufactured by Nycomed). According to the experimental operation manual, human T cells were separated and obtained from the human mononuclear cells using a Pan-T cell Isolation Kit (manufactured by Miltenyi) and a magnetic sorter. The number of the T cells was counted by a hemocytometer. The human T cells were suspended in RPMI1640 medium containing 10% FCS to which anti-human AILIM human monoclonal antibody JMab136 (20 μg / ml) was added, and a human T cell suspension (1 × 10 4 ⁶ Cells / ml) were prepared and incubated at room temperature for 30 minutes.
The anti-KLH human monoclonal antibody JMAb23 (20 μg / ml) was used as a negative control antibody.
[0222]
<15-4> Cell culture
A human T cell suspension (100 μl / well; 1 × 10 4) was added to each well of the microplate coated with the anti-human CD3 antibody and hB7h-IgFc as described above. ⁵ cells / well) and add CO ₂ The cells were cultured at 37 ° C. for 3 days in an incubator.
[0223]
<15-5> Measurement of T cell proliferation activity
In each well of each plate after the culture, methyl [ ³ H] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia) was added, and CO was added. ₂ Incubation was performed at 37 ° C. for 6 hours in an incubator. After the culture, the cells were trapped on a GF / C filter (Packard) using a cell harvester. Next, the filter was dried at 40 ° C. for 3 hours or more, and then Microscinti 0 (20 μl / well; manufactured by Packard) was added. Being taken up by cells trapped on the filter using a β counter (TOP COUNT) ³ The radioactivity of H was measured and the degree of proliferation of human T cells after culture was analyzed.
The results are shown in FIG.
As a result of this test, significant proliferation of human T cells in a human B7h-IgFc concentration-dependent manner was observed (test using a negative control antibody). Furthermore, the proliferation of the human T cells was significantly suppressed by the anti-human AILIM monoclonal antibody.
[0224]
Example 16 Inhibitory activity of anti-human AILIM human monoclonal antibody on proliferation of monkey T cells by costimulatory signaling through AILIM-AILIM ligand
The test of Example 15 was performed in the same manner using monkey T cells instead of human T cells.
[0225]
<16-1> Dilution of antibody, etc.
Anti-human CD3 monoclonal antibody SP34 (manufactured by BD-Pharmingen) was diluted with phosphate buffer (PBS) to a final concentration of 1 µg / ml.
The human B7h-IgFc prepared above was diluted with PBS to a final concentration of 40 μg / ml, and further diluted with PBS to various concentrations (40 μg / ml to 0.0064 μg / ml).
[0226]
<16-2> Microplate coating with antibody
(1) Anti-human CD3 monoclonal antibody SP34 (manufactured by BD-Pharmingen) (1 μg / ml at 25 μl / well) and human B7h-IgFc (40 μg / ml to 0.0064 μg) / Ml (25 µl) and incubated at 37 ° C for 2 hours. The antibody was then discarded and each well was washed three times with PBS. After washing, RPMI1640 medium containing 10% FCS (100 μl / well) was added to each well, and incubated at 37 ° C. for 1 hour to coat each well of the plate.
[0227]
<16-3> Preparation of monkey T cell suspension
Cynomolgus monkey peripheral blood was collected, and mononuclear cells were fractionated by density gradient centrifugation using NycoPrep 1.077A (Nycomed). According to the experimental operation manual, the anti-human CD4 antibody M-T477 (manufactured by BD-Pharmingen), the anti-human CD8 antibody RPA-T8 (manufactured by BD-Pharmingen), anti-mouse IgG microbeads (manufactured by Miltenyi) and a magnetic sorter were used. Monkey T cells were separated and obtained from cynomolgus monocytes. The number of the T cells was counted by a hemocytometer. The monkey T cells were suspended in RPMI1640 medium containing 10% FCS to which anti-human AIIM human monoclonal antibody JMab-136 (20 μg / ml) was added, and a monkey T cell suspension (1 × 10 6 ⁶ Cells / ml) were prepared and incubated at room temperature for 30 minutes.
In addition, anti-KLH human monoclonal antibody JMab-23 (20 μg / ml) was used as a negative control antibody.
[0228]
<16-4> Cell culture
A monkey T cell suspension (100 μl / well; 1 × 10 5) was added to each well of the microplate coated with the anti-human CD3 antibody and hB7h-IgFc as described above. ⁵ cells / well) and add CO ₂ The cells were cultured at 37 ° C. for 3 days in an incubator.
[0229]
<16-5> Measurement of T cell proliferation activity
In each well of each plate after the culture, methyl [ ³ H] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia) was added, and CO was added. ₂ Incubation was performed at 37 ° C. for 6 hours in an incubator. After the culture, the cells were trapped on a GF / C filter (Packard) using a cell harvester. Next, the filter was dried at 40 ° C. for 3 hours or more, and then Microscinti 0 (20 μl / well; manufactured by Packard) was added. Being taken up by cells trapped on the filter using a β counter (TOP COUNT) ³ The radioactivity of H was measured and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIG.
As a result of this test, significant proliferation of monkey T cells dependent on the concentration of human B7h-IgFc was observed (test using a negative control antibody). Furthermore, the proliferation of the monkey T cells was significantly suppressed by the anti-human AILIM monoclonal antibody.
[0230]
【The invention's effect】
The human monoclonal antibody that binds to human AILIM of the present invention is a human-derived antibody. Therefore, a major problem in the therapeutic treatment of an antibody drug comprising an antibody derived from a non-human mammal such as a mouse-derived antibody (side effect). Does not cause severe immunorejection of the host due to antigenicity against humans, that is, HAMA (Human anti-mouse antigenicity), thus dramatically increasing the value of antibodies as pharmaceuticals. .
[0231]
Therefore, the human monoclonal antibody against AILIM (particularly human AILIM) of the present invention and a pharmaceutical composition comprising the human monoclonal antibody can be used to induce AILIM-expressing cells without inducing host immune rejection caused by HAMA. Various biological reactions involving transmission of costimulatory signals (costimulatory signals) via AILIM (eg, cell proliferation of cells expressing AILIM, production of cytokines by cells expressing AILIM, cells expressing AILIM) As a pharmaceutical agent for controlling immune cytolysis or cell death (apoptosis) and the activity of inducing antibody-dependent cellular cytotoxicity against AILIM-expressing cells, and / or signal transduction via the AILIM But It is possible to suppress or prevent the onset and / or progression of various diseases involved, and treat or prevent the diseases.
[0232]
Specifically, by providing a pharmaceutical product using the anti-AILIM human monoclonal antibody of the present invention or a part thereof as an active organism, for example, an autoimmune disease or an allergic disease (particularly, an autoimmune disease or autoimmune disease caused by cell-mediated immunity) is provided. Various diseases classified as immune diseases and delayed allergies (for example, rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, lichen planus, a chronic inflammatory skin disease, Systemic lupus erythematosus, insulin-dependent diabetes and psoriasis, etc .; arthropathy (eg, rheumatoid arthritis; RA, osteoarthritis; OA), inflammation (eg, hepatitis); graft-versus-host reaction (Graft versus host reaction; GVH re graft versus host disease (GVHD); transplantation of tissue (tissue such as skin, cornea, bone, etc.) or organ (liver, heart, lung, kidney, pancreas, etc.) (allograft or xenograft) ) Associated with the antigen; an immune response elicited by a foreign antigen or a self-antigen (eg, production of antibodies to the antigen, cell proliferation, production of cytokines, etc.); and diseases possibly caused by abnormal intestinal immunity. (Specifically, it becomes possible to suppress, treat, or prevent inflammatory bowel disease (particularly, Crohn's disease and ulcerative colitis) and dietary allergy).
[0233]
The pharmaceutical composition of the present invention may also be used for any inflammation, such as when various steroids are applied as anti-inflammatory drugs, for example, inflammation associated with various arthritis (rheumatoid arthritis, osteoarthritis, etc.), pneumonia, Hepatitis (including viral hepatitis), inflammation associated with infectious diseases, inflammatory bowel disease, enteritis, nephritis (inflammation associated with glomerulonephritis, renal fibrosis), gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocardium Inflammation due to inflammation, encephalitis, post-ischemic reperfusion injury (such as myocardial ischemia-reperfusion injury), inflammation due to immune rejection after transplantation of tissues or organs, burns, various skin inflammations (psoriasis, allergic contact skin Inflammation, lichen planus, which is a chronic inflammatory skin disease), inflammation in multiple organ disorders, inflammation after PTCA or PTCR surgery, inflammation associated with arteriosclerosis, and autoimmune thyroiditis. Make it possible.
Furthermore, in the field of suppressing / treating immune rejection associated with the transplantation of tissues and organs, the pharmaceutical composition of the present invention is used for suppressing immune rejection in such transplantation treatment. When used in combination with an existing immunosuppressant, it is possible to increase the effect of suppressing transplant rejection using the existing drug alone.
In addition, by using the method for identifying a substance that binds to AILIM or AILIM ligand, which is one of the present invention, it binds to AILIM or AILIM ligand and regulates signal transduction by the interaction between the two, as described above. It becomes possible to screen and select a drug (a chemically synthesized compound or an antibody) having a potential activity capable of treating various diseases.
[0234]
[Sequence list]

[0235]
"Sequence List Free Text"
SEQ ID NO: 1
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence NotIT.
SEQ ID NO: 2
Other information: description of artificial sequence: artificially synthesized linker sequence 20adp.
SEQ ID NO: 3
Other information: Description of artificial sequence: Artificially synthesized linker sequence 24adp.
SEQ ID NO: 4
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence HIGLC.
SEQ ID NO: 5
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence NHCc2.
SEQ ID NO: 6
Other information: Description of artificial sequence: Artificially synthesized primer sequence EXcellE.
SEQ ID NO: 7
Other information: description of artificial sequence: artificially synthesized primer sequence ck117.
SEQ ID NO: 8
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence M13R.
SEQ ID NO: 9
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence 136H.
SEQ ID NO: 10
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence 138 / 9H.
SEQ ID NO: 11
Other information: description of artificial sequence: artificially synthesized primer sequence AILIMHC1.
SEQ ID NO: 12
Other information: description of artificial sequence: artificially synthesized primer sequence HCc1.
SEQ ID NO: 13
Other information: description of artificial sequence: artificially synthesized primer sequence HCc7.
SEQ ID NO: 14
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence HCc8.
SEQ ID NO: 15
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence HCc3.
SEQ ID NO: 16
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence HCc4.
SEQ ID NO: 17
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence HCc6.
SEQ ID NO: 18
Other information: Description of artificial sequence: Artificially synthesized primer sequence HIGHC.
SEQ ID NO: 19
Other information: Description of artificial sequence: Artificially synthesized primer sequence HCc9.
SEQ ID NO: 20
Other information: description of artificial sequence: artificially synthesized primer sequence HCc5.
SEQ ID NO: 21
Other information: Description of artificial sequence: artificially synthesized primer sequence polyA.
SEQ ID NO: 22
Other Information: Description of Artificial Sequence: Artificially synthesized primer sequence AILIMLC1.
SEQ ID NO: 23
Other information: Description of artificial sequence: artificially synthesized primer sequence AILIMLC2.
SEQ ID NO: 24
Other information: Description of artificial sequence: artificially synthesized primer sequence LCc1.
SEQ ID NO: 25
Other information: description of artificial sequence: artificially synthesized primer sequence LCc2.
SEQ ID NO: 26
Other information: description of artificial sequence: artificially synthesized primer sequence HIK.
SEQ ID NO: 39
Other information: description of artificial sequence: artificially synthesized primer sequence.
SEQ ID NO: 40
Other information: description of artificial sequence: artificially synthesized primer sequence.
[0236]
[Brief description of the drawings]
FIG. 1 is a diagram showing the reactivity of an anti-human IgG antibody, an anti-human Igκ antibody, and an anti-human IgFc antibody with respect to an anti-human AILIM human monoclonal antibody obtained by analyzing the results of a cell ELISA using a flow cytometer.
(A) to (l) show the following test results, respectively.
Diagram (a): Test results when a biotin-labeled anti-human IgG antibody as a secondary antibody was added to a microplate on which wild-type HPB-ALL cells had been seeded without adding a primary antibody.
Separation diagram (b): Test results when a biotin-labeled anti-human Igκ antibody as a secondary antibody was added to a microplate on which wild-type HPB-ALL cells had been seeded without adding a primary antibody.
Separation diagram (c): Test results when a biotin-labeled anti-human IgFc antibody as a secondary antibody was added to a microplate on which wild-type HPB-ALL cells had been seeded without adding a primary antibody.
Separation diagram (d): Test results using anti-human AILIM human monoclonal antibody JMab-136 as primary antibody and biotin-labeled anti-human IgG antibody as secondary antibody.
Panel (e): Test results using anti-human AILIM human monoclonal antibody JMab-136 as primary antibody and biotin-labeled anti-human Igκ antibody as secondary antibody.
Separation diagram (f): Test results using anti-human AILIM human monoclonal antibody JMab-136 as the primary antibody and biotin-labeled anti-human IgFc antibody as the secondary antibody.
Separation diagram (g): Test results using anti-human AILIM human monoclonal antibody JMab-138 as the primary antibody and biotin-labeled anti-human IgG antibody as the secondary antibody.
Separation diagram (h): Test results using anti-human AILIM human monoclonal antibody JMab-138 as primary antibody and biotin-labeled anti-human Igκ antibody as secondary antibody.
Separation diagram (i): Test results using anti-human AILIM human monoclonal antibody JMab-138 as primary antibody and biotin-labeled anti-human IgFc antibody as secondary antibody.
Separation diagram (j): Test results using anti-human AILIM human monoclonal antibody JMab-139 as the primary antibody and biotin-labeled anti-human IgG antibody as the secondary antibody.
Separation diagram (k): Test results using anti-human AILIM human monoclonal antibody JMab-139 as the primary antibody and biotin-labeled anti-human Igκ antibody as the secondary antibody.
Separation diagram (l): Test results using anti-human AILIM human monoclonal antibody JMab-139 as primary antibody and biotin-labeled anti-human IgFc antibody as secondary antibody.
The open curves in each diagram show the test results when an anti-KLH human monoclonal antibody as a control antibody was used.
FIG. 2 is a diagram showing a calibration curve of a human IgG monoclonal antibody (standard substance) quantified by sandwich ELISA using an anti-human IgG antibody. The vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the concentration of the standard substance.
FIG. 3 shows the binding activities of various anti-human AILIM mouse monoclonal antibodies to human AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates a test for binding to the wild-type CHO cells, and the term "human" indicates a test for binding to the human AILIM-highly expressing recombinant CHO cells. It indicates that.
FIG. 4 shows the binding activity of various anti-human AILIM human monoclonal antibodies or an anti-KLH human monoclonal antibody as a negative control to human AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates a test for binding to the wild-type CHO cells, and the term "human" indicates a test for binding to the human AILIM-highly expressing recombinant CHO cells. It indicates that.
FIG. 5 shows the binding activities of various anti-human AILIM human monoclonal antibodies to human AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates a test for binding to the wild-type CHO cells, and the term "human" indicates a test for binding to the human AILIM-highly expressing recombinant CHO cells. It indicates that.
FIG. 6 shows the binding activities of various anti-human AILIM human monoclonal antibodies to human AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates a test for binding to the wild-type CHO cells, and the term "human" indicates a test for binding to the human AILIM-highly expressing recombinant CHO cells. It indicates that.
FIG. 7 shows the binding activity of anti-mouse AILIM rat monoclonal antibody to mouse AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "mouse" is a binding test to the mouse AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 8 shows the binding activity of various anti-human AILIM human monoclonal antibodies or an anti-KLH human monoclonal antibody as a negative control to mouse AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "mouse" is a binding test to the mouse AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 9 shows the binding activities of various anti-human AILIM human monoclonal antibodies to mouse AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "mouse" is a binding test to the mouse AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 10 shows the binding activities of various anti-human AILIM human monoclonal antibodies to mouse AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "mouse" is a binding test to the mouse AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 11 shows the binding activity of an anti-rat AILIM mouse monoclonal antibody to rat AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "rat" is a binding test to the rat AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 12 is a graph showing the binding activity of various anti-human AILIM human monoclonal antibodies or an anti-KLH human monoclonal antibody as a negative control to rat AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "rat" is a binding test to the rat AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 13 shows the binding activities of various anti-human AILIM human monoclonal antibodies to rat AILIM-highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "rat" is a binding test to the rat AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 14 shows the binding activities of various anti-human AILIM human monoclonal antibodies to rat AILIM highly expressing recombinant CHO cells or wild-type CHO cells. The vertical axis indicates the fluorescence intensity as an indicator of the recombinant binding activity, and the horizontal axis indicates the concentration of the added antibody. The term "CHO" in the figure indicates that the test is a binding test to the wild-type CHO cells, and the term "rat" is a binding test to the rat AILIM high-expressing recombinant CHO cells. It indicates that.
FIG. 15 shows a healthy subject “donor A” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM mouse monoclonal antibodies using a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM mouse monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ [H] Thymidine is shown to be taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody.
FIG. 16 shows a healthy subject “donor A” in a costimulation signal transduction activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
FIG. 17 shows a healthy subject “donor B” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM mouse monoclonal antibodies using a microplate coated with an anti-human AILIM mouse monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ [H] Thymidine is shown to be taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody. "JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
[FIG. 18] A healthy subject “donor B” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody. In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 19 shows a healthy subject “donor B” in a test for measuring the costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody. In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 20 shows a healthy subject “donor C” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM mouse monoclonal antibodies using a microplate coated with an anti-human AILIM mouse monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody. "JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 21 shows a healthy subject “donor C” in a test for measuring the costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "". The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody. In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody. Various notations mean the following. “124”: anti-human AILIM human monoclonal antibody JMab124.
"126": anti-human AILIM human monoclonal antibody JMab126.
"127": anti-human AILIM human monoclonal antibody JMab127.
FIG. 22 shows a healthy subject “donor C” in a costimulation signal transmission activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“128”: anti-human AILIM human monoclonal antibody JMab128.
"135": anti-human AILIM human monoclonal antibody JMab135.
"136": anti-human AILIM human monoclonal antibody JMab136.
FIG. 23. A healthy human “donor C” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“137”: anti-human AILIM human monoclonal antibody JMab137.
“138”: anti-human AILIM human monoclonal antibody JMab138.
“139”: anti-human AILIM human monoclonal antibody JMab139.
FIG. 24 shows a healthy subject “donor C” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 25 shows a healthy subject “donor D” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM mouse monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM mouse monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ [H] Thymidine is shown to be taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody.
"JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 26 shows healthy subjects “donor D” in a test for measuring the costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibodies together with anti-human CD3 monoclonal antibodies. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“124”: anti-human AILIM human monoclonal antibody JMab124.
"126": anti-human AILIM human monoclonal antibody JMab126.
"127": anti-human AILIM human monoclonal antibody JMab127.
FIG. 27 shows a healthy subject “donor D” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“128”: anti-human AILIM human monoclonal antibody JMab128.
"135": anti-human AILIM human monoclonal antibody JMab136.
"136": anti-human AILIM human monoclonal antibody JMab137.
FIG. 28. A healthy subject “donor D” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“137”: anti-human AILIM human monoclonal antibody JMab137.
“138”: anti-human AILIM human monoclonal antibody JMab138.
“139”: anti-human AILIM human monoclonal antibody JMab139.
FIG. 29. A healthy subject “donor D” in a test for measuring the costimulation signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 30 shows a healthy human “donor E” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM mouse monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM mouse monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ [H] Thymidine is shown to be taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody.
"JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 31 shows a healthy subject “donor E” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“124”: anti-human AILIM human monoclonal antibody JMab124.
"126": anti-human AILIM human monoclonal antibody JMab126.
"127": anti-human AILIM human monoclonal antibody JMab127.
FIG. 32. A healthy human “donor E” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“128”: anti-human AILIM human monoclonal antibody JMab128.
"135": anti-human AILIM human monoclonal antibody JMab135.
"136": anti-human AILIM human monoclonal antibody JMab136.
[FIG. 33] A healthy subject “donor E” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] Thymidine is taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“137”: anti-human AILIM human monoclonal antibody JMab137.
"138": anti-human AILIM human monoclonal antibody JMab138.
“139”: anti-human AILIM human monoclonal antibody JMab139.
FIG. 34 shows a healthy subject “donor E” in a test for measuring the costimulatory signal transduction activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. The figure which shows the proliferation activity of the T cell derived from "".
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 35. Costimulation of various anti-human AILIM mouse monoclonal antibodies when using a microplate coated with only anti-human CD3 monoclonal antibody and adding the anti-human AILIM mouse monoclonal antibody in the form of a solution (liquid phase) The figure which shows the proliferative activity of T cell derived from a healthy person "donor D" in a rate signal transmission activity measurement test.
The vertical axis represents [ ³ [H] Thymidine is shown to be taken up into cells, and the horizontal axis shows the concentration of the anti-human AILIM mouse monoclonal antibody.
"JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 36. Costimulation of various anti-human AILIM human monoclonal antibodies when using a microplate coated with only anti-human CD3 monoclonal antibody and adding the anti-human AILIM human monoclonal antibody in a solution state (liquid phase) The figure which shows the proliferative activity of T cell derived from a healthy person "donor D" in a rate signal transmission activity measurement test.
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“124”: anti-human AILIM human monoclonal antibody JMab124.
"126": anti-human AILIM human monoclonal antibody JMab126.
"127": anti-human AILIM human monoclonal antibody JMab127.
FIG. 37. Costimulation of various anti-human AILIM human monoclonal antibodies when using a microplate coated with only anti-human CD3 monoclonal antibody and adding the anti-human AILIM human monoclonal antibody in a solution state (liquid phase) The figure which shows the proliferative activity of T cell derived from a healthy person "donor D" in a rate signal transmission activity measurement test.
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“128”: anti-human AILIM human monoclonal antibody JMab128.
"135": anti-human AILIM human monoclonal antibody JMab135.
"136": anti-human AILIM human monoclonal antibody JMab136.
FIG. 38. Costimulation of various anti-human AILIM human monoclonal antibodies when a microplate coated with only the anti-human CD3 monoclonal antibody is used and the anti-human AILIM human monoclonal antibody is added in a solution state (liquid phase). The figure which shows the proliferative activity of T cell derived from a healthy person "donor D" in a rate signal transmission activity measurement test.
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“137”: anti-human AILIM human monoclonal antibody JMab137.
“138”: anti-human AILIM human monoclonal antibody JMab138.
“139”: anti-human AILIM human monoclonal antibody JMab139.
FIG. 39. Costimulation of various anti-human AILIM human monoclonal antibodies when using a microplate coated with only anti-human CD3 monoclonal antibody and adding the anti-human AILIM human monoclonal antibody in the form of a solution (liquid phase) The figure which shows the proliferative activity of T cell derived from a healthy person "donor D" in a rate signal transmission activity measurement test.
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 40 shows the amount of IFN-γ produced in the culture supernatant of T cells from healthy donor “donor B” cultured in a microplate coated with an anti-human AILIM mouse monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
"JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 41 shows the amount of IFN-γ produced in the culture supernatant of T cells from a healthy “donor B” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 42 shows the amount of IFN-γ produced in the culture supernatant of T cells from healthy donor “donor B” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 43 shows the amount of IFN-γ produced in the culture supernatant of T cells from healthy donor “donor C” cultured in a microplate coated with an anti-human AILIM mouse monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
"JHC1" in the figure shows the test results when an anti-human CETP monoclonal antibody was used as a negative control instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 44 shows the amount of IFN-γ produced in the culture supernatant of T cells from healthy donor “donor C” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“124”: anti-human AILIM human monoclonal antibody JMab124.
"126": anti-human AILIM human monoclonal antibody JMab126.
"127": anti-human AILIM human monoclonal antibody JMab127.
FIG. 45 shows the amount of IFN-γ produced in the culture supernatant of T cells derived from healthy donor “donor C” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“128”: anti-human AILIM human monoclonal antibody JMab128.
"135": anti-human AILIM human monoclonal antibody JMab135.
"136": anti-human AILIM human monoclonal antibody JMab136.
FIG. 46 shows the amount of IFN-γ produced in the culture supernatant of T cells from healthy donor “donor C” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“137”: anti-human AILIM human monoclonal antibody JMab137.
“138”: anti-human AILIM human monoclonal antibody JMab138.
“139”: anti-human AILIM human monoclonal antibody JMab139.
FIG. 47 shows the amount of IFN-γ produced in the culture supernatant of T cells derived from healthy donor “donor C” cultured in a microplate coated with an anti-human AILIM human monoclonal antibody together with an anti-human CD3 monoclonal antibody. FIG.
The vertical axis indicates the concentration of IFN-γ, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 48 shows various control test substances in a T lymphocyte proliferation test (MLR) in a mixed lymphocyte reaction (MLR) when T cells of a healthy donor “donor A” were cultured with PBMC of a healthy donor “donor D”. The figure which shows the suppression effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 49. Various anti-human AILIM humans in a proliferation test of T lymphocytes in a mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor A” were cultured with PBMC of healthy donor “donor D” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 50 shows various control test substances in a T lymphocyte proliferation test in a mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor D” were cultured with PBMC of healthy donor “donor B”. The figure which shows the suppression effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 51. Various anti-human AILIM humans in a T lymphocyte proliferation test in a mixed lymphocyte reaction (MLR) when T cells of a healthy human “donor D” were cultured with PBMC of a healthy human “donor B” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 52 shows various control test substances in a proliferation test of T cells of a healthy donor “donor C” in a mixed lymphocyte reaction (MLR) when cultured with PBMCs of a healthy donor “donor A”. The figure which shows the suppression effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 53. Various anti-human AILIM humans in a T lymphocyte proliferation test in a mixed lymphocyte reaction (MLR) when T cells of a healthy human “donor C” were cultured with PBMC of a healthy human “donor A” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 54 shows various control test substances in a proliferation test of T cells of a healthy subject “donor E” in a mixed lymphocyte reaction (MLR) when cultured with PBMCs of a healthy subject “donor G”. The figure which shows the suppressive effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Control mIgG": anti-human CD34 / IgG1 mouse monoclonal antibody.
"CD80 + 86 Ab": a mixture of anti-CD80 and anti-CD86 antibodies.
"SA12": anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
FIG. 55. Various anti-human AILIM humans in a proliferation test of T cells of a healthy donor “donor E” in a mixed lymphocyte reaction (MLR) when cultured with PBMCs of a healthy donor “donor G” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-136": anti-human AILIM human monoclonal antibody JMab-136.
“138”: anti-human AILIM human monoclonal antibody JMab-138.
"139": anti-human AILIM human monoclonal antibody JMab-139.
“140”: anti-human AILIM human monoclonal antibody JMab-140.
"141": anti-human AILIM human monoclonal antibody JMab-141.
FIG. 56 shows various control test substances in a proliferation test of T cells of a healthy subject “donor F” in a mixed lymphocyte reaction (MLR) when cultured with PBMCs of a healthy subject “donor E”. The figure which shows the suppressive effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Control mIgG": anti-human CD34 / IgG1 mouse monoclonal antibody.
"CD80 + 86 Ab": a mixture of anti-CD80 and anti-CD86 antibodies.
"SA12": anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
FIG. 57. Various anti-human AILIM humans in a proliferation test of T lymphocytes in a mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor F” were cultured with PBMC of healthy donor “donor E” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-136": anti-human AILIM human monoclonal antibody JMab-136.
“138”: anti-human AILIM human monoclonal antibody JMab-138.
"139": anti-human AILIM human monoclonal antibody JMab-139.
“140”: anti-human AILIM human monoclonal antibody JMab-140.
"141": anti-human AILIM human monoclonal antibody JMab-141.
FIG. 58 shows various control test substances in a proliferation test of T cells of a healthy donor “donor G” in a mixed lymphocyte reaction (MLR) when cultured with PBMC of a healthy donor “donor F”. The figure which shows the suppressive effect of the proliferation of the said T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Control mIgG": anti-human CD34 / IgG1 mouse monoclonal antibody.
"CD80 + 86 Ab": a mixture of anti-CD80 and anti-CD86 antibodies.
"SA12": anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: a human CTLA4-IgFc chimeric molecule.
FIG. 59. Various anti-human AILIM humans in a proliferation test of T cells of a healthy human “donor F” in a mixed lymphocyte reaction (MLR) when cultured with PBMCs of a healthy human “donor F” The figure which shows the suppressive effect of the said T cell proliferation by a monoclonal antibody.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-136": anti-human AILIM human monoclonal antibody JMab-136.
“138”: anti-human AILIM human monoclonal antibody JMab-138.
“139”: anti-human AILIM human monoclonal antibody JMab-139.
“140”: anti-human AILIM human monoclonal antibody JMab-140.
"141": anti-human AILIM human monoclonal antibody JMab-141.
FIG. 60: Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor A” were cultured with PBMC of healthy donor “donor D” previously cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the suppression effect of the proliferation of the said T cell by various control test substances in the said proliferation test of this T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 61. Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor A” were cultured with PBMC of healthy donor “donor D” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the said T cell proliferation by various anti-human AILIM human monoclonal antibodies in the said T cell proliferation test.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 62: Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor D” were cultured with PBMC of healthy donor “donor B” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the suppression effect of the proliferation of the said T cell by various control test substances in the said proliferation test of this T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 63: Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor D” were cultured with PBMCs of healthy donor “donor B” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the said T cell proliferation by various anti-human AILIM human monoclonal antibodies in the said T cell proliferation test.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 64. Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor C” were cultured with PBMC of healthy donor “donor A” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the suppression effect of the proliferation of the said T cell by various control test substances in the said proliferation test of this T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"CD80 + 86": mixture of anti-CD80 and anti-CD86 antibodies.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 65. Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor C” were cultured with PBMC of healthy donor “donor A” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the said T cell proliferation by various anti-human AILIM human monoclonal antibodies in the said T cell proliferation test.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-124": anti-human AILIM human monoclonal antibody JMab-124.
“126”: anti-human AILIM human monoclonal antibody JMab-126.
“127”: anti-human AILIM human monoclonal antibody JMab-127.
“128”: anti-human AILIM human monoclonal antibody JMab-128.
“135”: anti-human AILIM human monoclonal antibody JMab-135.
"136": anti-human AILIM human monoclonal antibody JMab-136.
"137": anti-human AILIM human monoclonal antibody JMab-137.
FIG. 66. Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor E” were cultured with PBMC of healthy donor “donor G” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the suppression effect of the proliferation of the said T cell by various control test substances in the said proliferation test of this T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Control mIgG": anti-human CD34 / IgG1 mouse monoclonal antibody.
"CD80 + 86 Ab": a mixture of anti-CD80 and anti-CD86 antibodies.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 67. Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor E” were cultured with PBMC of healthy donor “donor G” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the said T cell proliferation by various anti-human AILIM human monoclonal antibodies in the said T cell proliferation test.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-136": anti-human AILIM human monoclonal antibody JMab-136.
“138”: anti-human AILIM human monoclonal antibody JMab-138.
“139”: anti-human AILIM human monoclonal antibody JMab-139.
“140”: anti-human AILIM human monoclonal antibody JMab-140.
"141": anti-human AILIM human monoclonal antibody JMab-141.
FIG. 68: Mixed lymphocyte reaction (MLR) when T cells of healthy donor “donor G” were cultured with PBMC of healthy donor “donor F” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the suppression effect of the proliferation of the said T cell by various control test substances in the said proliferation test of this T cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Control mIgG": anti-human CD34 / IgG1 mouse monoclonal antibody.
"CD80 + 86 Ab": a mixture of anti-CD80 and anti-CD86 antibodies.
"SA12": anti-human AILIM mouse monoclonal antibody.
FIG. 69 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy human “donor G” were cultured with PBMC of a healthy human “donor F” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the said T cell proliferation by various anti-human AILIM human monoclonal antibodies in the said T cell proliferation test.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the concentration of the test substance.
The various notations in the figure mean the following.
"Anti-KLH": anti-KLH human monoclonal antibody as a negative control.
"JMab-136": anti-human AILIM human monoclonal antibody JMab-136.
“138”: anti-human AILIM human monoclonal antibody JMab-138.
“139”: anti-human AILIM human monoclonal antibody JMab-139.
“140”: anti-human AILIM human monoclonal antibody JMab-140.
"141": anti-human AILIM human monoclonal antibody JMab-141.
FIG. 70 shows ADCC inducing activities of various anti-human AILIM human monoclonal antibodies and control antibodies when wild-type CHO cells are used as target cells.
The vertical axis shows the cytotoxicity caused by ADCC inducing activity by the antibody, and the horizontal axis shows the concentration of the antibody.
FIG. 71 shows ADCC-inducing activities of various anti-human AILIM human monoclonal antibodies and control antibodies when using human AILIM-highly expressing recombinant CHO cells as target cells.
The vertical axis shows the cytotoxicity caused by ADCC inducing activity by the antibody, and the horizontal axis shows the concentration of the antibody.
FIG. 72 is a graph showing the inhibitory effect of a delayed-type allergy by an anti-AILIM antibody.
The vertical axis indicates the area of redness which is an index of the onset of delayed allergy, and the horizontal axis indicates the type of sample administered to the test animal.
FIG. 73. Proliferation of monkey T cells in a test for measuring the costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with anti-human AILIM human monoclonal antibody together with anti-human CD3 monoclonal antibody The figure which shows activity.
The vertical axis represents [ ³ H] The amount of thymidine incorporated into cells is shown, and the horizontal axis shows the concentration of the anti-human AILIM human monoclonal antibody.
In the figure, "anti-KLH" indicates the test result when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 74 is a graph showing the inhibitory activity of a binding of soluble AILIM ligand (hB7h-IgFc) to soluble AILIM (AILIM-IgFc) at various concentrations by a negative control antibody.
The vertical axis indicates the absorbance as an index of the inhibitory activity, and the horizontal axis indicates the concentration of soluble AILIM.
FIG. 75 is a graph showing the inhibitory activity of the binding of soluble AILIM ligand (hB7h-IgFc) to soluble AILIM (AILIM-IgFc) at various concentrations by an anti-AILIM antibody.
The vertical axis indicates the absorbance as an index of the inhibitory activity, and the horizontal axis indicates the concentration of soluble AILIM.
FIG. 76 shows the inhibitory activities of soluble AILIM ligand (hB7h-IgFc) and soluble AILIM (AILIM-IgFc) by various concentrations of anti-AILIM antibody.
The vertical axis indicates the absorbance as an index of the inhibitory activity, and the horizontal axis indicates the concentration of the anti-AILIM antibody.
FIG. 77: Human T by various anti-human AILIM human monoclonal antibodies in a test for measuring the costimulatory signal transduction activity using a microplate coated with a soluble human AILIM ligand (hB7h-IgFc) together with an anti-human CD3 monoclonal antibody The figure which shows the proliferation inhibitory activity of a cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the antibody concentration.
FIG. 78. Monkey T with various anti-human AILIM human monoclonal antibodies in a costimulatory signal transduction activity measurement test using a microplate coated with a soluble human AILIM ligand (hB7h-IgFc) together with an anti-human CD3 monoclonal antibody The figure which shows the proliferation inhibitory activity of a cell.
The vertical axis represents [ ³ H] The amount of thymidine taken up into cells is shown, and the horizontal axis shows the antibody concentration.

Claims (6)

Binds to a ligand (AILIM ligands) interacting with AILIM or said AILIM or the identification of substances which inhibit their binding, or a method of assessing the extent of inhibition of the substance, characterized in that it comprises the following steps And how to:
(A) preparing an insoluble carrier on which a polypeptide containing all or a part of the extracellular region of AILIM is immobilized;
(B) preparing a polypeptide comprising all or part of the extracellular region of the AILIM ligand labeled with a label capable of producing a detectable signal;
(C) reacting the insoluble carrier of step (a) with the polypeptide of step (b);
(D) reacting the insoluble carrier of step (a), the polypeptide of step (b) and the substance in any order;
(E) a signal generated by the labeling substance contained in the complex produced by the reaction of step (c) and a signal produced by the labeling substance contained in the complex produced by the reaction of step (d). Detecting; and (f) comparing each of the signal magnitudes detected in step (e). Binds to a ligand (AILIM ligands) interacting with AILIM or said AILIM or the identification of substances which inhibit their binding, or a method of assessing the extent of inhibition of the substance, characterized in that it comprises the following steps And how to:
(A) preparing an insoluble carrier on which a polypeptide containing all or a part of the extracellular region of AILIM ligand is immobilized;
(B) preparing a polypeptide comprising all or part of the extracellular region of AILIM labeled with a labeling substance capable of producing a detectable signal;
(C) reacting the insoluble carrier of step (a) with the polypeptide of step (b);
(D) reacting the insoluble carrier of step (a), the polypeptide of step (b) and the substance in any order;
(E) a signal generated by the labeling substance contained in the complex produced by the reaction of step (c) and a signal produced by the labeling substance contained in the complex produced by the reaction of step (d). Detecting; and (f) comparing each of the signal magnitudes detected in step (e). A polypeptide comprising all or a part of the extracellular region of AILIM is a fusion polypeptide comprising a polypeptide comprising all or a part of the extracellular region of AILIM and all or a part of a constant region of a heavy chain of an immunoglobulin. The method according to claim 1 or 2, wherein the method is a peptide. The polypeptide containing all or a part of the extracellular region of the AILIM ligand is composed of a polypeptide containing all or a part of the extracellular region of the AILIM ligand and all or a part of the constant region of a heavy chain of an immunoglobulin. The method according to claim 1 or 2, wherein the method is a fusion polypeptide. The method according to any one of claims 1 to 4, wherein the AILIM is human AILIM. The method according to any one of claims 1 to 5, wherein the AILIM ligand is a human AILIM ligand.

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