JP4234992B2 - Human monoclonal antibody against costimulatory molecule AILIM and pharmaceutical use thereof - Google Patents

Description

【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。
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【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]
BACKGROUND 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)); human monoclonal antibody or part thereof, and human monoclonal antibody or part thereof DNA or a part thereof; a cell (including a recombinant cell) that produces the human monoclonal antibody or a part thereof; a human monoclonal antibody or a part thereof produced by the recombinant cell; the human monoclonal antibody or a part thereof A pharmaceutical composition for treating a delayed allergy-related disease comprising an antibody against AILIM; a method for identifying, quantifying or assaying a substance that binds to AILIM or an AILIM ligand;
And a kit used in the method.
[0002]
[Prior art]
A mammal's living body has an immune response system that attempts to eliminate pathogenic microorganisms (viruses, bacteria, parasites, etc.) and 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 is a mechanism of exclusion 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 an elimination mechanism 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 antigen present outside the cell 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), and the former regulates B cell differentiation and antibody production. Together with phagocytic cells, it destroys the antigen. The latter attacks virus-infected cells themselves (Experimental Medicine (separate volume), “Bio Science Term Library [Immunity]”, Yodosha, p.14-17, 1995).
[0004]
This acquisition (activation) of antigen specificity by T cells is due to the recognition of antigens presented by antigen-presenting cells (APC) such as macrophages, B cells or dendritic cells. Be started. The antigen-presenting cell processes (processes) the incorporated antigen, and binds the processed antigen to a major histocompatibility complex (MHC) to present the antigen. The T cell recognizes the processed antigen presented by the antigen-presenting cell through a complex of T cell receptor (TcR) and CD3 antigen (TcR / CD3 complex) on its cell membrane surface, thereby activating the cell activity. Receives a first signal for activation (acquisition of specificity).
[0005]
However, the first signal through the TcR / CD3 complex alone does not cause sufficient activation of the T cell, but also unresponsiveness or inability to respond to any subsequent stimulation. It falls into a condition called clonal anergy. Interleukin-2 (IL-2) autocrine is required to activate and differentiate and proliferate into antigen-specific T cell clones. Since IL-2 and the like are not produced and cell division does not occur, T cells are inactivated. That is, activation of T cells accompanied by 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 a costimulatory signal (costimulatory signal).
[0006]
T cells interact with a molecule other than the MHC on the antigen-presenting cell via a molecule different from the TcR / CD3 complex on the surface of the T cell (cell-cell adhesion). Receives and transmits into cells. This second signal avoids cell anergy (clonal paralysis) and activates the cells.
[0007]
Although there is a part that has not yet been elucidated in detail about the mechanism of the transmission of the second signal between antigen-presenting cells and lymphocytes such as T cells, from the studies so far, CD28 (also known as Tp44, T44, or 9.3 antigen), a cell surface molecule that is expressed mainly on T cells and thymocytes, and cell surface molecules that are expressed on antigen-presenting cells (macrophages, monocytes, dendritic cells, etc.) Interactions 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 cellular cell surface molecule (ie, their It has been shown that cell-cell adhesion via intermolecular bonds is extremely important.
[0008]
Furthermore, the control of T cell activation by this second signal includes CTLA-4 (Cytolytic T lymphocyte associated antigen 4), whose expression is thought to be enhanced depending on the second signal, and Experimentally clarified that the interaction between CD80 (B7-1) and CD86 (B7-2) also plays an important role (ie, cell-cell adhesion through binding between these molecules). Has been. That is, for the control of T cell activation by this second signal transmission, at least the interaction between CD28 and CD80 / CD86, the enhancement of the expression of CTLA-4 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) necessary for T cell activation and avoidance of anergy. This molecule binds to the costimulator molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (in other words, cell-cell adhesion via the bond between these molecules). The second signal transmitted by Stabilizes the mRNA of Th1-type cytokines, and as a result, promotes the production of large quantities 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 the first signal that enters through TcR / CD3, and the expression is also enhanced by the second signal that is entered by the binding of CD28 and CD80. . In response to these signals, CTLA-4 has been shown to act in a suppressive manner on T cell function as opposed to T cell activation by a second signal entering from CD28.
[0010]
Human CD28 and CTLA-4 are type I glycoproteins having molecular weights of 44 kD and 41 to 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 signal transduction molecule into cells.
[0011]
Human CD28 has been shown to form a homodimer by dizulfide binding, while CTLA-4 exists as a monomer. The positions of the CD28 and CTLA-4 genes on the chromosome are “2q33” in humans and “1C” in mice, and each consists of four exons. Human CD28 and CTLA-4 are each composed of 220 and 223 amino acids including the leader sequence, and the amino acid homology between them 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 higher affinity than CD28 for any ligand, the difference being about 20 times. For binding of CD28 and CTLA-4 to CD80 (B7-1), “MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)”, an amino acid sequence structure conserved across animal species, is important It has been revealed that. When CD28 is stimulated, PI3 kinase (phosphoinositide 3 kinase, PI3K) associates with phosphorylated tyrosine residues in the subsequence “YMNM (Tyr-Met-Asn-Met)” in the cell. CD28 has been shown to play an important role in intracellular signaling through this “YxxM” structure. The CTLA4 intracellular domain also has a sequence represented by “YxxM”, that is, “YVKM (Tyr-Val-Lys-Met)”, and SYPs should associate with this sequence after stimulation. It is shown.
[0013]
CD28 is expressed exclusively in thymocytes and peripheral blood T cells, while CTLA-4 has been shown to be expressed specifically on activated T cells (Cell Engineering / Separate Volume "Adhesion Molecular Handbook", Hide Published by Junsha, pp. 93-102, 1994; Id., Pp. 120-136; Experimental medicine, separate volume “BIO SCIENCE Term Library / Immunity”, published by Yochisha, pp. 94-98, 1995; Experimental medicine, separate volume “BIO SCIENCE terminology library / intracellular signal transduction”, published by Yochisha, pp. 58-59, 1997; Japan Clinical, Vol. 55, No. 6, pp. 215-220, 1997 ).
[0014]
In this way, CTLA-4, etc., which are linked together with costimulator molecules (CD28, CD80 (B7-1), CD86 (B7-2), etc.) in the control of T cell function (T cell activation and function suppression) The importance of interactions between multiple molecules has been advocated, and attention has been focused on elucidating the relationship between these molecules and diseases, and trying to treat diseases by controlling the functions of these molecules. It is becoming.
[0015]
As described above, the living body activates the acquired immune response system against antigens that are foreign to the living body (self), but exhibits immune tolerance that does not show an immune response against its own biological components (self antigen). Have. However, if immune tolerance breaks down for some reason, an immune response to the self-antigen occurs, and autoantigen-reactive T cells are induced by the same mechanism as described above, resulting in an immune abnormal state and various autoimmune diseases are caused. .
[0016]
That is, when the immune system of the living body is normal, unstimulated antigen presenting cells (APCs) in normal tissues do not express costimulatory molecules, so that they react with self antigens. Even if T cells are present, self-tolerance is maintained because T cells are in an unresponsive state, but in an immune abnormal state, self-tolerance is caused by excessive or continuous expression of costimulatory molecules. The possibility has been presented that antigen-reactive T cells are activated and autoimmune diseases are induced.
From this point of view, in recent years, many attempts have been made to treat various autoimmune diseases by regulating the transmission of costimulatory signals, for example, the signal transmission between the aforementioned CD28 / CTLA-4-CD80 / CD86. Has been made.
However, while such therapeutic attempts have been made, a detailed elucidation of the mechanism of T cell activation by interaction between costimulator molecules and related molecules has not yet been made. The mechanism could also involve other molecules not yet identified.
[0017]
Recently, as in the case of “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 transmission molecule) that is thought to control the function of activated lymphocytes such as activated T cells has been identified. This molecule is called activation inducible lymphocyte immunomodulatory molecule (AILIM) (human, mouse and rat; Int. Immunol., Vol.12, No.1, p.51-55, 2000), otherwise called ICOS (Inducible co -stimulator) (human; Nature, Vol.397, No.6716, p.263-266, 1999).
[0018]
On the other hand, recently, a novel molecule called B7h, B7RP-1, GL50, or LICOS, which is a ligand (AILIM ligand) that interacts with this costimulatory molecule AILIM (ICOS), has also 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 novel molecules, namely AILIM (ICOS) and B7RP-1 (B7h / GL50 / LICOS), essential for the activation of lymphocytes such as T cells and the functional control of activated T cells as described above The costimulatory signal transmission pathways of CD28 and CD80 (B7-1) / CD86 (B7-2), CTLA4 and CD80 (B7- 1) In addition to the first and second signaling pathways between CD86 (B7-2), there is a new third pathway through the interaction between AILIM (ICOS) and B7RP-1 (B7h). It became clear that there was.
Regarding the biological function of each of the two new molecules, the functional control of lymphocytes such as T cells by the third costimulatory signaling by the molecule, and the relationship between the novel signaling and disease Is currently under active research (J. Immunol., 166 (1), p.1, 2001; J. Immunol., 165 (9), p.5035, 2000; Biochem. Biophys. Res. 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, like “CD28” and “CTLA-4”, transmits a second signal (costimulatory signal) essential for activation of lymphocytes such as T cells, and the signal. In addition to clarifying the biological function of the novel molecule AILIM, which is thought to be a molecule that regulates the function of activated lymphocytes such as activated T cells, and the relationship between the expression of AILIM and diseases, the AILIM By controlling the biological function of the drug by medical and pharmaceutical methods (for example, drugs such as monoclonal antibodies and low molecular weight compounds), the onset of various diseases depending on the expression state of AILIM is suppressed, or The object is to provide methods and medicaments for treatment.
[0021]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the present inventors have conducted extensive research on human antibodies (especially human monoclonal antibodies) against mammalian AILIM (especially human AILIM). A variety of human monoclonal antibodies that bind to human AILIM, in particular human AILIM, by immunizing transgenic mice prepared to produce the antibodies of human AILIM (specifically, the cell membrane fraction of human AILIM-expressing cells) For the first time in the world, the inventors succeeded in producing various monoclonal antibodies that bind to and regulate signal transduction through human AILIM.
[0022]
Since the antibody (especially monoclonal antibody) of the present invention is an antibody derived from a human, a major problem (side effect) in treating an antibody drug comprising an antibody derived from a non-human mammal such as a mouse-derived antibody. Since it does not cause any serious immune rejection of the host due to antigenicity against humans, that is, HAMA (Human anti-mouse antigenicity), it dramatically increases the value of the antibody as a pharmaceutical product. .
[0023]
Therefore, the human antibody (particularly human monoclonal antibody) that binds to the mammalian AILIM (particularly human AILIM) of the present invention and the 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 that express AILIM without causing immune rejection of the host Of cell proliferation, cytokine production by cells expressing AILIM, immune cytolysis of cells expressing AILIM or apoptosis, and activity to induce antibody-dependent cytotoxicity against cells expressing AILIM Etc.) and / or suppress the onset and / or progression of various diseases involving signaling through the AILIM. , Blocking, and is useful as a medicament for the treatment or prevention of the disease.
[0024]
Specifically, the pharmaceutical composition of the present invention controls (suppresses or promotes) the growth (suppression or promotion) of AILIM-expressing cells or the production of cytokines (for example, interferon γ or interleukin 4) by AILIM-expressing cells. And the suppression of various pathological conditions caused by various physiological phenomena involving signaling through 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 (particularly, autoimmune diseases and delayed allergies caused by cellular immunity) (for example, chronic diseases) Rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, chronic inflammatory skin diseases such as lichen planus, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis); arthropathy (eg, Rheumatoid arthritis (RA), osteoarthritis (OA)), inflammation (eg, hepatitis); graft versus host reaction (GVH reaction); graft versus host disease (graft versus Host disease (GVHD); Accompanied by transplantation of tissue (skin, cornea, bone, etc.) or organ (liver, heart, lung, kidney, pancreas, etc.) (allogeneic or xenotransplantation) Immune rejection; immune reaction elicited by a foreign antigen or self-antigen (for example, production of antibodies to the antigen, cell proliferation, cytokine production, etc.); and diseases that may be caused by abnormalities in intestinal immunity (specifically Specifically, it is possible to suppress, prevent and / or treat inflammatory bowel disease (particularly Crohn's disease and ulcerative colitis) and dietary allergy.
Furthermore, in the field of suppression / treatment of immune rejection associated with transplantation of the tissues and organs described above, the pharmaceutical composition of the present invention is applied for suppression of immune rejection in such transplantation therapy. By using in combination with an existing immunosuppressive agent, it is possible to increase the effect of suppressing graft rejection with the existing agent alone.
[0025]
Moreover, the pharmaceutical composition of the present invention can be applied to the treatment or prevention of any inflammation to which various steroidal agents as anti-inflammatory agents are applied.
Examples of inflammatory diseases to which the pharmaceutical composition of the present invention can be applied include inflammation associated with various arthritis (rheumatoid arthritis, osteoarthritis, etc.), pneumonia, hepatitis (including viral hepatitis), and infectious diseases. Inflammation associated with inflammatory bowel disease, enteritis, nephritis (glomerulonephritis, inflammation associated with renal fibrosis), gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis, post-ischemic reperfusion injury (myocardial) Inflammation in ischemia-reperfusion injury, inflammation caused by immune rejection after transplantation of tissues and organs, burns, various skin inflammations (psoriasis, allergic contact dermatitis, lichen planus, a chronic inflammatory skin disease) Etc.), inflammation in multiple organ disorders, post-operative inflammation of PTCA and PTCR, inflammation associated with arteriosclerosis, autoimmune thyroiditis and the like.
In addition, if the method for identifying a substance that binds to AILIM or AILIM ligand, which is one of the present invention, is used, it binds to AILIM or AILIM ligand and regulates signal transduction due to the interaction between the two, as described above. It becomes possible to screen and select a drug (chemically synthesized compound or antibody) having a potential activity capable of treating various diseases.
[0026]
That is, the present invention is as described in the following (1) to (108).
(1) A human antibody that binds to AILIM.
(2) The human antibody according to (1) above, wherein the AILIM is derived from a human.
(3) A human monoclonal antibody or a part thereof that binds to AILIM.
(4) The human monoclonal antibody or a part thereof according to (3), wherein the AILIM is derived from human.
(5) The human monoclonal antibody according to the above (3) or (4), wherein the human monoclonal antibody has an activity of inhibiting signal transduction into cells via AILIM. Department.
(6) The signal transduction inhibitory activity is the following (a) or (b):
(A) the activity of inhibiting the growth of cells expressing AILIM; or
(B) an activity of suppressing cytokine production by cells expressing AILIM,
The human monoclonal antibody or a part thereof according to (5) above, wherein
(7) The human monoclonal antibody or a part thereof according to (6), wherein the cytokine is a cytokine produced by a Th1-type T cell or a Th2-type T cell.
(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 signal transduction into cells via AILIM. Department.
(11) The signal transduction inducing activity is the following (a) or (b):
(A) an activity that induces proliferation of cells expressing AILIM; or
(B) an activity that induces cytokine production by cells expressing AILIM,
The human monoclonal antibody or a part thereof according to (10) above, wherein
(12) The human monoclonal antibody or a part thereof according to (11), wherein the cytokine is a cytokine produced by a Th1-type T cell or a Th2-type T cell.
(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 cytotoxicity to cells expressing AILIM and / or an activity of inducing immune cell lysis or cell death of cells expressing AILIM. The human monoclonal antibody or a part thereof according to (3) or (4) above, wherein
(15) The binding rate constant (ka) between the human monoclonal antibody and AILIM is 1.0 × 10^ThreeThe human monoclonal antibody or a part thereof according to (3) or (4) above, wherein the value is (1 / M.Sec) or more.
(16) The binding rate constant (ka) is 1.0 × 10^FourThe human monoclonal antibody or a part thereof according to (15), which has a numerical value of (1 / M.Sec) or more.
(17) The binding rate constant (ka) is 1.0 × 10^FiveThe human monoclonal antibody or a part thereof according to (16) above, which has a numerical value of (1 / M.Sec) or more.
(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 (3) or (4), which has a numerical value of the following.
(19) The dissociation rate constant (kd) is 1.0 × 10^-Four(1 / Sec) The human monoclonal antibody or a part thereof according to (18), which has the following numerical value.
(20) The dissociation rate constant (kd) is 1.0 × 10^-Five(1 / Sec) The human monoclonal antibody or a part thereof according to (19), which has a numerical value of the following.
(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 (3) or (4) above, which has the following numerical value.
(22) The dissociation constant (Kd) is 1.0 × 10^-7(M) The human monoclonal antibody or a part thereof according to (21), which has the following numerical value.
(23) The dissociation constant (Kd) is 1.0 × 10^-8(M) The human monoclonal antibody or a part thereof according to (22), which has the following numerical value.
(24) The dissociation constant (Kd) is 1.0 × 10^-9(M) The human monoclonal antibody or a part thereof according to (23), which has the following numerical value.
(25) The V region DNA encoding the heavy chain variable region of the human monoclonal antibody is derived from a human immunoglobulin heavy chain V gene segment of either 1-02 or 3-13, The human monoclonal antibody or a part thereof according to 4).
(26) The V region DNA encoding the light chain variable region of the human monoclonal antibody is derived from the human immunoglobulin light chain V gene segment of either L5 or A27, Human monoclonal antibodies or parts thereof.
(27) The V region DNA 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 antibody The human monoclonal antibody according to (25) or (26) above, wherein the V region DNA encoding the light chain variable region is derived from a human immunoglobulin light chain V gene segment of either L5 or A27. An antibody or part thereof.
(28) The V region DNA encoding the heavy chain variable region of the human monoclonal antibody is derived from the human immunoglobulin heavy chain V gene segment 1-22, 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) above, wherein the region DNA is derived from human immunoglobulin light chain V gene segment L5.
(29) The V region DNA 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 encoding the light chain variable region of the human monoclonal antibody The human monoclonal antibody or a part thereof according to (27) above, wherein the region DNA is derived from human immunoglobulin light chain V gene segment A27.
(30) The human monoclonal antibody according to (4) above, wherein the heavy chain variable region of the human monoclonal has an amino acid sequence comprising any one of the following amino acid sequences (a) to (f): part:
(A) the amino acid sequence of amino acid numbers 20 to 117 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 acid numbers 20 to 117 described in SEQ ID NO: 28;
(C) the amino acid sequence of amino acid numbers 20 to 116 described in SEQ ID NO: 32;
(D) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 20 to 116 described in SEQ ID NO: 32;
(E) the amino acid sequence of amino acid numbers 20 to 116 described in SEQ ID NO: 36; or
(F) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 20 to 116 described in SEQ ID NO: 36;
(31) The human monoclonal antibody or a part thereof according to (4) above, 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 acid numbers 20 to 470 described in SEQ ID NO: 28;
(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 acid numbers 20 to 470 described in SEQ ID NO: 28;
(C) the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32;
(D) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32;
(E) the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36; or
(F) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36;
(32) The human monoclonal antibody or the human monoclonal antibody according to (4), wherein the human monoclonal light chain variable region has an amino acid sequence comprising any one of the following amino acid sequences (a) to (f): part:
(A) the amino acid sequence of amino acid numbers 23 to 116 described 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 acid numbers 23 to 116 described in SEQ ID NO: 30;
(C) the amino acid sequence of amino acid numbers 21 to 116 described in SEQ ID NO: 34;
(D) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 21 to 116 described in SEQ ID NO: 34;
(E) the amino acid sequence of amino acid numbers 21 to 116 described in SEQ ID NO: 38; or
(F) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 21 to 116 described 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 an amino acid sequence of any one of (a) to (f) below:
(A) the amino acid sequence of amino acid numbers 23 to 236 described 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 acid numbers 23 to 236 described in SEQ ID NO: 30;
(C) the amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 34;
(D) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 34;
(E) the amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 38; or
(F) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 38;
(34) The human monoclonal antibody or a part thereof according to (4) above, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence comprising the amino acid sequence of amino acid numbers 20 to 117 set forth in SEQ ID NO: 28; and
(B) The light chain variable region has an amino acid sequence including the amino acid sequence of amino acid numbers 23 to 116 described in SEQ ID NO: 30.
(35) The human monoclonal antibody or a part thereof according to (4) above, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has an amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 28; and
(B) The light chain polypeptide has an amino acid sequence of amino acid numbers 23 to 236 described in SEQ ID NO: 30.
(36) The human monoclonal antibody or a part thereof according to (4) above, wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 32; and
(B) The light chain variable region has an amino acid sequence including the amino acid sequence of amino acid numbers 21 to 116 described in SEQ ID NO: 34.
(37) The human monoclonal antibody or a part thereof according to (4), wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has an amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32; and
(B) The light chain polypeptide has the amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 34.
(38) The human monoclonal antibody or a part thereof according to (4), wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain variable region has an amino acid sequence comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 36; and
(B) The light chain variable region has an amino acid sequence comprising the amino acid sequence of amino acid numbers 21 to 116 set forth in SEQ ID NO: 38.
(39) The human monoclonal antibody or a part thereof according to (4), wherein the human monoclonal antibody has the following characteristics (a) and (b):
(A) the heavy chain polypeptide has an amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36; and
(B) The light chain polypeptide has an amino acid sequence of amino acid numbers 21 to 236 described in SEQ ID NO: 38.
(40) The human monoclonal antibody is a monoclonal antibody derived from a transgenic non-human mammal having the ability to produce a human antibody, as described in any one of (3) to (29) above Human monoclonal antibody or part thereof.
(41) The human monoclonal antibody comprises a cell expressing AILIM, a membrane fraction derived from the cell, all or part of a molecule constituting AILIM, or a gene encoding AILIM or a part thereof, The human monoclonal antibody or a part thereof according to (40) above, which is a monoclonal antibody obtained by immunizing a transgenic non-human mammal having the ability to produce.
(42) The human monoclonal antibody or a part thereof according to (40) or (41) above, 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 acid numbers 20 to 117 set forth in SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acid numbers 23 to 116 set forth in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 set forth in SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 36; and
(F) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 set forth 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 acid numbers 20 to 470 described in SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acid numbers 23 to 236 set forth in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36; and
(F) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 38;
(45) DNA selected from the group consisting of the following (a) to (f) or a part thereof:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 105 to 386 set forth in SEQ ID NO: 29;
(C) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31;
(D) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33;
(E) a DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35; and
(F) DNA comprising the nucleotide sequence of nucleotide 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) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27;
(B) DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(C) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 described in SEQ ID NO: 31;
(D) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33;
(E) a DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35; and
(F) a DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37;
(47) A vector comprising the DNA of any one of (43) to (46). (48) The vector according to (47), which comprises the DNA of any one of (a) to (c) below:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31; or
(C) DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35.
(49) The vector according to (47), which comprises the DNA of any one of (a) to (c) below:
(A) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 described in SEQ ID NO: 31; or
(C) DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35.
(50) The vector according to (47), which comprises the DNA of any one of (a) to (c) below:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 105 to 386 set forth in SEQ ID NO: 29;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33; or
(C) DNA comprising the nucleotide sequence of nucleotide numbers 93 to 380 described in SEQ ID NO: 37.
(51) The vector according to (47), which comprises the DNA of any one of (a) to (c) below:
(A) DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(B) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33; or
(C) DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37.
(52) The vector according to (47), which comprises the following DNAs (a) and (b):
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 126 to 419 described in SEQ ID NO: 27; and
(B) DNA comprising the nucleotide sequence of nucleotide numbers 105 to 386 described in SEQ ID NO: 29.
(53) The vector according to (47), which comprises the following DNAs (a) and (b):
(A) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(54) The vector according to the above (47), which comprises the following DNAs (a) and (b):
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31; and
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33;
(55) The vector according to (47), which comprises the following DNAs (a) and (b):
(A) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 described in SEQ ID NO: 31; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33;
(56) The vector according to the above (47), which comprises the following DNAs (a) and (b):
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35; and
(B) DNA comprising the nucleotide sequence of nucleotide numbers 93 to 380 described in SEQ ID NO: 37.
(57) The vector according to (47), which comprises the following DNAs (a) and (b):
(A) a DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37;
(58) A cell which produces the human monoclonal antibody according to any one of (3) to (42).
(59) The above (58), wherein the cell is a fusion cell obtained by fusing a mammal-derived B cell and a mammal-derived myeloma cell with the ability to produce the human monoclonal antibody. The cell described.
(60) DNA of the following (a) or a vector containing the DNA, DNA of the following (b) or a vector containing the DNA, or both of the DNAs of the following (a) and (b) or a vector containing the both Transgenic host transformed by introducing into the host:
(A) a DNA encoding a heavy chain polypeptide of a monoclonal antibody that binds to human AILIM or a portion thereof; and
(B) DNA encoding a light chain polypeptide of a monoclonal antibody that binds to human AILIM or a part thereof.
(61) The genetic recombinant host according to (60), wherein the monoclonal antibody is a human monoclonal antibody.
(62) The genetic recombinant host according to (60) or (61) above, wherein the host is a mammalian cell.
(63) The genetic recombinant host according to (60) or (61) above, wherein the host is a fertilized egg of a mammal.
(64) The heavy chain polypeptide according to any one of (60) to (63) above, 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 acid numbers 20 to 117 set forth in SEQ ID NO: 28;
(B) a heavy chain polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 32; and
(C) A heavy chain polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 36.
(65) The heavy chain polypeptide according to any one of (60) to (63) above, 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 acid numbers 20 to 470 described in SEQ ID NO: 28;
(B) a heavy chain polypeptide having an amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32; and
(C) A heavy chain polypeptide having the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36.
(66) The light chain polypeptide according to any one of (60) to (63) above, 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 acid numbers 23 to 116 set forth in SEQ ID NO: 30;
(B) a light chain polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 set forth in SEQ ID NO: 34; and
(C) a light chain polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 set forth in SEQ ID NO: 38.
(67) The light chain polypeptide according to any one of (60) to (63) above, 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 acid numbers 23 to 236 set forth in SEQ ID NO: 30;
(B) a light chain polypeptide having the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 34; and
(C) A light chain polypeptide having the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 38.
(68) The genetic recombinant host according to any one 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; and
(B) a light chain polypeptide comprising amino acids 23 to 116 of SEQ ID NO: 30.
(69) The genetic recombinant host according to any one 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: 28; and
(B) a light chain polypeptide having amino acids 23 to 236 shown in SEQ ID NO: 30;
(70) The genetic recombinant host according to any one 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; and
(B) a light chain polypeptide comprising amino acids 21 to 116 of SEQ ID NO: 34.
(71) The genetic recombinant host according to any one 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; and
(B) a light chain polypeptide having amino acids 21 to 236 shown in SEQ ID NO: 34;
(72) The genetic recombinant host according to any one 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; and
(B) a light chain polypeptide comprising amino acids 21 to 116 of SEQ ID NO: 38.
(73) The recombinant host according to any one 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: 36; and
(B) a light chain polypeptide having amino acids 21 to 236 shown in SEQ ID NO: 38;
(74) The gene according to any one of (60) to (63) above, 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 nucleotide numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31; or
(C) DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35.
(75) The gene according to any one of (60) to (63) above, wherein the DNA encoding the heavy chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27;
(B) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 described in SEQ ID NO: 31; or
(C) DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35.
(76) The gene according to any one of (60) to (63) above, 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 nucleotide numbers 105 to 386 set forth in SEQ ID NO: 29;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33; or
(C) DNA comprising the nucleotide sequence of nucleotide numbers 93 to 380 described in SEQ ID NO: 37.
(77) The gene according to any one of (60) to (63) above, wherein the DNA encoding the light chain polypeptide is any one of the following DNAs (a) to (c): Recombinant host:
(A) DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(B) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33; or
(C) DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37.
(78) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b): Or the genetic recombinant host according to any one of (63) above:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 126 to 419 described in SEQ ID NO: 27; and
(B) DNA comprising the nucleotide sequence of nucleotide numbers 105 to 386 described in SEQ ID NO: 29.
(79) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b): Or the genetic recombinant host according to any one of (63) above:
(A) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(80) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b): Or the genetic recombinant host according to any one of (63) above:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31; and
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33;
(81) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b): Or the genetic recombinant host according to any one of (63) above:
(A) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 set forth in SEQ ID NO: 31; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33;
(82) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b) Or the genetic recombinant host according to any one of (63) above:
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35; and
(B) DNA comprising the nucleotide sequence of nucleotide numbers 93 to 380 described in SEQ ID NO: 37.
(83) The DNA encoding the heavy chain polypeptide is the DNA of the following (a), and the DNA encoding the light chain polypeptide is the DNA of the following (b): Or the genetic recombinant host according to any one of (63) above:
(A) a DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35; and
(B) a DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37;
(84) The genetic recombinant host according to any one of (60) to (62) or any of (64) to (83) (except when the host is a fertilized egg) Produced human monoclonal antibody or part thereof.
(85) A pharmaceutical composition comprising the human antibody according to (1) or (2) above and a pharmaceutically acceptable carrier.
(86) A pharmaceutical composition comprising the human monoclonal antibody or a part thereof according to any one of (3) to (42), and a pharmaceutically acceptable carrier.
(87) A pharmaceutical composition comprising the human monoclonal antibody or a part thereof according to (84), and a pharmaceutically acceptable carrier.
(88) The pharmaceutical composition according to any one of (85) to (87), wherein the pharmaceutical composition is used for inhibiting signal transduction into cells via AILIM.
(89) The pharmaceutical composition according to any one of (85) to (87), wherein the pharmaceutical composition is used for suppressing proliferation of cells expressing AILIM.
(90) The pharmaceutical composition according to any one of (85) to (87), wherein the pharmaceutical composition is used for suppressing the production of cytokines by cells expressing AILIM.
(91) The pharmaceutical composition according to any one 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 cells expressing AILIM.
(93) The pharmaceutical composition according to any one of (85) to (87), wherein the pharmaceutical composition is used for inducing production of cytokines by cells expressing AILIM.
(94) The pharmaceutical composition is used for inducing antibody-dependent cytotoxicity to cells expressing AILIM and / or for inducing immune cell lysis or cell death of cells expressing AILIM. The pharmaceutical composition according to any one of (85) to (87).
(95) A pharmaceutical composition for suppressing, treating or preventing delayed-type allergy, comprising a substance having an activity of controlling signal transduction via AILIM and a pharmaceutically acceptable carrier.
(96) The pharmaceutical composition as described in (95) above, wherein the substance is a protein substance.
(97) The pharmaceutical composition according to (96), wherein the protein substance is any one selected from the following group:
a) an antibody or part thereof that binds to AILIM;
b) a polypeptide comprising all or part of the extracellular region of AILIM;
c) a fusion polypeptide comprising all or part of the extracellular region of AILIM and all or part of the constant region of an immunoglobulin heavy chain;
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 one 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 as described in (95) above, wherein the substance is a non-protein substance.
(102) The pharmaceutical composition according to the above (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 an 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 labeling substance capable of providing a detectable signal;
(C) reacting the insoluble carrier of step (a) and 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 emitted from the labeling substance contained in the complex produced by the reaction of step (c) and a signal emitted from the labeling substance contained in the complex produced by the reaction of step (d) Detecting; and
(F) A step of comparing each of the magnitudes of the signals detected in step (e).
(104) A method for identifying a substance that binds to AILIM or an AILIM ligand, comprising the following steps:
(A) a step of preparing an insoluble carrier on which a polypeptide containing all or part of the extracellular region of the 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) and 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 emitted from the labeling substance contained in the complex produced by the reaction of step (c) and a signal emitted from the labeling substance contained in the complex produced by the reaction of step (d) Detecting; and
(F) A step of comparing each of the magnitudes of the signals detected in step (e).
(105) A polypeptide comprising all or part of the extracellular region of AILIM is composed of a polypeptide comprising all or part of the extracellular region of AILIM and all or part of the constant region of an immunoglobulin heavy chain. The method according to (103) or (104) above, wherein the fusion polypeptide is
(106) The polypeptide containing all or part of the extracellular region of the AILIM ligand is a polypeptide containing all or part of the extracellular region of the AILIM ligand and all or part of the constant region of an immunoglobulin heavy chain The method according to (103) or (104) above, which is a fusion polypeptide comprising:
(107) The (103) to the above, wherein the AILIM is human AILIM
(106) The method according to any one of (106).
(108) The method according to any one of (103) to (107), wherein the AILIM ligand is a human AILIM ligand.
[0027]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail by clarifying the meaning of the words 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, particularly preferably Human, rat, hamster or mouse.
The terms “mammal other than human” and “non-human mammal” used in the present invention are synonymous with each other and mean any mammal other than a human in the mammals defined above.
[0028]
The term “amino acid” used in the present invention means any amino acid that exists in nature, and is preferably represented as follows according to the three-letter code or the one-letter code of the alphabet used to represent the amino acid. Means an amino acid.
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]
As used herein, “AILIM” is an abbreviation for Activation inducible lymphocyte immunomodulatory molecule and means a mammalian cell surface molecule having the structure and function reported in the previous report as described above, and particularly preferably derived from human. (Eg, International Immunology, Vol. 12, No. 1, p. 51-55; GenBank Accession Number: BAA82129 (human); BAA82128 (rat); BAA82127 (rat variant); BAA82126 (mouse) ).
In addition, this AILIM is ICOS (Nature, Vol.397, No.6716, p.263-266, 1999) or JTT-1 antigen / JTT-2 antigen (Japanese Patent Application Publication No. 11-29599); Patent application publication WO98 / 38216)), but these molecules mean the same molecule.
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 above-mentioned costimulatory transmission 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 addition, “AILIM” referred to in the present invention includes a polypeptide having substantially the same amino acid sequence as the amino acid sequence of each mammalian AILIM, particularly preferably the human AILIM described in the aforementioned literature. Peptides are also included. Furthermore, a human AILIM variant similar to the already identified rat-derived AILIM variant (GenBank Accession Number: BAA82127) is also included in “AILIM” in the present invention.
The “AILIM ligand” in the present invention is also defined with the same meaning as described above.
[0031]
Here, the “polypeptide having substantially the same amino acid sequence” means the following mutant polypeptide.
That is, as long as it has substantially the same biological properties as natural AILIM (particularly preferably AILIM derived from human), a plurality of amino acids in the amino acid sequence, preferably 1 to 10 amino acids, particularly preferably Is a mutant polypeptide having an amino acid sequence in which 1 to 5 amino acids are substituted, deleted, and / or modified, and a plurality of amino acids in the naturally occurring AILIM (particularly preferably AILIM derived from human). A mutant polypeptide having an amino acid sequence to which are added, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids.
Further, it may be a mutant polypeptide having a plurality of such substitutions, deletions, modifications, and additions.
The “AILIM ligand” in the present invention is also defined with the same meaning as described above.
AILIM (especially human AILIM) and AILIM ligand (especially human AILIM ligand) in the present invention are known in the technical field such as chemical synthesis method, cell culture method, etc. in addition to gene recombination technology. It can manufacture by using suitably the well-known method or its modification method.
Such amino acid substitutions, deletions, or insertions can be carried out according to conventional methods (Experimental Medicine separate volume, “Gene Engineering Handbook” (1992), etc.).
[0032]
Examples of the method for producing the mutant polypeptide as described above include a synthetic oligonucleotide-directed mutagenesis method (gapped duplex) method, a method of randomly introducing point mutations by nitrite or sulfite treatment, Ba131 enzyme, etc. Examples of methods for preparing deletion mutants, cassette mutation methods, linker scanning methods, misincorporation methods, mismatch primer methods, and DNA segment synthesis methods.
[0033]
The synthetic oligonucleotide-directed mutagenesis (gapped duplex) method can be performed, for example, as follows. A region desired 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 restriction enzyme treatment, mixed with the above single-stranded phage DNA, denatured and annealed to form “gapped duplex DNA”. This is hybridized with a synthetic oligonucleotide into which a mutation is introduced, and a closed circular double-stranded DNA is obtained by the reaction of DNA polymerase and DNA ligase. This DNA is transfected into an Escherichia coli mutS strain that lacks the ability to modify mismatches, and the grown phages are infected with E. coli without a suppressor function, and only phages that do not have an amber mutation are selected.
[0034]
Moreover, the method of introducing the point mutation by nitrous acid utilizes the following principle, for example. Treatment of DNA with nitrous acid deaminates the base, converting adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine. When deaminated DNA is introduced into cells, hypoxanthine forms base pairs with cytosine, uracil forms a base pair with thymine, and `` A: T '' changes to `` G: C ''. “G: C” replaces “A: T”. In practice, nitrous acid-treated single-stranded DNA fragments are hybridized to “gapped duplex DNA”, and then the mutants can be isolated by the same procedure as in the synthetic oligonucleotide-directed mutagenesis (gapped duplex) method. .
[0035]
Further, “AILIM” in the present invention includes “a part” of the AILIM. Here, “part” means a polypeptide containing an arbitrary partial sequence in the amino acid sequence of AILIM defined above.
Preferably, it means the extracellular region of AILIM defined above (particularly preferably human AILIM) or any part thereof.
The “AILIM ligand” in the present invention is also defined with the same meaning as described above.
The “part” of the AILIM (preferably the extracellular region of AILIM or any part thereof) is a gene recombination technique or chemical synthesis according to a known method or its modification method known in the technical field described later. The AILIM (particularly preferably human AILIM) isolated by a cell culture method can be produced by appropriately cleaving with a proteolytic enzyme or the like.
In addition, “a part of the 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 the above-defined AILIM or a part thereof (particularly preferably AILIM derived from a human 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 the above-defined AILIM or a part thereof (particularly preferably, a human-derived AILIM or a part thereof).
More specifically, the variable region of the heavy chain (H chain) and the constant region of the H chain and the constant region of the light chain (L chain) and the constant region of the L chain constituting the immunoglobulin. All regions including human immunoglobulin are derived from genes encoding human immunoglobulins. Examples of the L chain include human κ chain and human λ chain.
[0037]
A human monoclonal antibody that binds to AILIM of the present invention (particularly preferably AILIM derived from human) 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 with characteristics.
More specifically, it is various human monoclonal antibodies having various characteristics 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 or a part thereof according to any of (5) to (42) or (84) of the present invention.
A particularly preferred embodiment is a human monoclonal antibody that binds to human AILIM according to (30) or (39) of the present invention.
[0038]
The “human monoclonal antibody” of the present invention can be prepared by immunizing a human antibody-producing transgenic non-human mammal described below with any of the following immunogens (antigens).
(A) a natural cell or an artificially established cell line that expresses the above-defined AILIM (particularly preferably AILIM derived from human) on the cell surface;
(B) a genetically modified cell produced using a gene recombination technique so that the above-defined AILIM (particularly preferably human-derived AILIM) is expressed on the cell surface;
(C) A cell lysate obtained by solubilizing the cells of (a) or (b) above, or a polypeptide fragment of AILIM (particularly preferably human-derived AILIM) purified from the cell lysate ;
(D) Using genetic recombination technology so that a part of AILIM defined above (particularly preferably AILIM derived from human) (particularly preferably, its extracellular region or any peptide thereof) is expressed as a soluble polypeptide. Genetically modified cells produced by
(E) A culture supernatant obtained by culturing the genetically modified cells of (d) above or an extracellular region polypeptide (soluble AILIM) of AILIM (particularly preferably human-derived AILIM) purified from the culture supernatant Or
(F) A part of chemically synthesized AILIM (particularly preferably human-derived AILIM) (particularly preferably, its extracellular region or any peptide thereof).
[0039]
Furthermore, the human monoclonal antibody of the present invention can be obtained by cDNA encoding each of the heavy chain and the light chain of the human monoclonal antibody of the present invention (preferably a vector containing the cDNA) using gene recombination technology. A “recombinant host” of the present invention, which is a recombinant host obtained by transforming a host and produces a recombinant human monoclonal antibody (wherein the host is a eukaryotic cell other than a fertilized egg ( Mammalian cells such as CHO, lymphocytes and myeloma) are preferred, and can also be obtained from the culture supernatant.
Specifically, the genetic recombinant host according to any one of (60) to (62) or (64) to (80) of the present invention described above (wherein the host is a eukaryotic cell other than a fertilized egg) (Preferably mammalian cells such as CHO, lymphocytes and myeloma).) Can be obtained by culturing.
[0040]
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. IgG (IgG1, IgG2, IgG3, IgG4) is preferable, and IgG1, IgG2, or IgG4 is preferable.
[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 later 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. A monoclonal antibody is prepared by preparing a fused 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, This is produced by selecting a clone that produces a monoclonal antibody having a specific affinity for the antigen used for immunization.
[0042]
More specifically, it can be produced as follows. That is, the immunogen of any one of the above (a) to (c) is used together with Freund's Adjuvant as necessary, and the human antibody-producing transgenic non-human mammal (particularly preferably “human antibody described later”). Production transgenic mice ") are immunized by subcutaneous injection, intramuscular, intravenous, Hoodpad or intraperitoneal injection or transplantation once to several times. Usually, immunization is carried out 1 to 4 times about every 1 to 14 days from 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 times of immunization and the time interval can be appropriately changed depending on the nature of the immunogen used.
[0043]
Fusion cells (hybridomas) that secrete human monoclonal antibodies can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and modification methods according thereto. That is, antibody-producing cells contained in the spleen, lymph nodes, bone marrow, tonsils, etc., preferably spleen obtained from a human antibody-producing transgenic non-human mammal immunized as described above, preferably mice, rats, It is prepared by cell fusion with a non-autoantibody-producing myeloma cell derived from a mammal such as guinea pig, hamster, rabbit or human, more preferably mouse, rat or human.
[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), P3 / X63-Ag8 .U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), NS0, PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1- 2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used.
Screening of a cell producing a monoclonal antibody (for example, a hybridoma) is performed by culturing the cell, for example, in a microtiter plate, and against the immunizing antigen used in the above-described immunization of the culture supernatant of a well in which proliferation has been observed. The reactivity can be measured, for example, by measuring by an enzyme immunoassay such as RIA (radio immunoassay) or ELISA (enzyme-linked immuno-solvent assay).
Production of monoclonal antibodies from hybridomas is carried out in vitro or in vivo in mice, rats, guinea pigs, hamsters or rabbits, preferably mice or rats, more preferably mice ascites, and the resulting culture supernatant. Or by isolation from ascites of mammals.
[0045]
In addition, the monoclonal antibody of the present invention can be produced in large quantities by the following method.
(1) From the hybridoma, a gene (such as cDNA) encoding each of the heavy and light chains of the monoclonal antibody is cloned.
(2) An expression vector is prepared by inserting genes encoding each of the cloned heavy chain and light chain into separate or single vectors.
(3) The vector is introduced into a fertilized egg of a desired non-human mammal (such as a goat). (4) The transgenic fertilized egg is transplanted into the uterus of a foster mother to obtain a chimeric non-human animal.
(5) A transgenic non-human mammal (bovine, goat, wherein a gene encoding each of the heavy chain and light chain is incorporated into an endogenous gene by further mating the chimeric goat with another non-human mammal. Sheep or pigs).
(6) A large amount of 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 encompassed by the present invention. When culturing the monoclonal antibody-producing cells 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 the culture method, and the like. It can be carried out using any nutrient medium derived from a known nutrient medium or a known basal medium such as those used to produce monoclonal antibodies.
[0046]
As the basic medium, for example, low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, and high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium, etc. The basic medium can contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances depending on the purpose.
Isolation and purification of the monoclonal antibody can be carried out by using the above-mentioned culture supernatant or ascites, 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 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 are deleted, substituted or added in the amino acid sequences of the heavy chain and / or light chain constituting the antibody. Also included are human monoclonal antibodies consisting of / or light chains.
[0048]
Here, “several amino acids” mean a plurality of amino acids, specifically 1 to 10 amino acids, preferably 1 to 5 amino acids.
In the amino acid sequence of the human monoclonal antibody of the present invention, partial modification (deletion, substitution, insertion, addition) of the amino acid as described above is introduced by partially modifying the base sequence encoding the amino acid sequence. can do. This partial modification of the base sequence can be introduced by a conventional method using a known site-specific mutagenesis method (Proc. Natl. Acsd. Sci. USA, Vol. 81, p. 5662-5666, 1984).
[0049]
The “transgenic human antibody-producing non-human mammal” in the present invention, a human antibody-producing transgenic mouse that is a particularly preferred embodiment, can be prepared according to a report (Nature Genetics, Vol. 7, p. 13-21, 1994). Nature Genetics, Vol.15, p.146-156, 1997; Japanese translations of Japanese translation of PCT publication No. Hei 4-504365; Japanese translations of Japanese translation of Heisei 7-509137; Nikkei Science, June, 40-50, 1995 International application publication WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese translations of PCT publication No. 6-500363).
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 the same manner.
[0050]
(1) By replacing at least part of the mouse endogenous immunoglobulin heavy chain gene locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination, the mouse endogenous immunoglobulin heavy chain gene is functionally disabled. 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 (especially the kappa chain gene) ) Is a step of producing a functionally inactivated knockout mouse.
(3) A desired region of a human immunoglobulin heavy chain locus is incorporated into a 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 (especially κ chain) locus is integrated into a mouse chromosome using a vector capable of carrying a large gene such as YAC. Manufacturing step.
(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 is 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 can be used to prevent rearrangement of the locus by replacing an appropriate region of the mouse endogenous immunoglobulin locus with a foreign marker gene (such as a neomycin resistance gene) by homologous recombination. It can be produced by activation. For inactivation using the homologous recombination, for example, a method called positive negative selection (PNS) can be used (Nikkei Science, May, p.52-62). , 1994).
Functional inactivation of an immunoglobulin heavy chain locus can be achieved, for example, by introducing a disorder into part of the J region or C region (eg, Cμ region). In addition, functional inactivation of immunoglobulin light chain (for example, κ chain) can be achieved by introducing a disorder into a region including, for example, a part of J region or C region, or a region spanning J region and C region. It is.
[0052]
Transgenic mice can be obtained by conventional methods commonly used in the production of transgenic animals (see, for example, the latest animal cell experiment manual, published by L.I.C., Chapters 361 to 408, 1990). ). Specifically, for example, an HPRT-negative (hypoxanthating aniline phosphoribosyltransferase gene-deficient) ES cell (embryonic stem cell) derived from a normal mouse blastcyst is used as the human immunoglobulin heavy chain. It fuses by the spheroplast fusion method with the yeast containing the YAC vector in which the gene coding for the locus or the light chain locus or a part thereof and the HPRT gene is inserted. ES cells in which the exogenous gene is integrated on the mouse endogenous gene are selected by the HAT selection method. Subsequently, 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 transplanted into the uterus of another normal mouse as a foster parent. Thus, a chimeric transgenic mouse is born from the temporary parent mouse. A heterotransgenic mouse is obtained by crossing the chimeric transgenic mouse with a normal mouse. By crossing the heterogeneic transgenic mice, homogeneic 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 aforementioned human monoclonal antibody of the present invention, specifically, F (ab ′)₂, Fab ', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody), etc. (Expert Opinion on Therapeutic Patents (Exp. Opin. Ther. Patents), Vol. 6, No. 5, pp. 441-456, 1996).
[0054]
Where `` F (ab ')₂"And" Fab '"are produced by treating immunoglobulin (monoclonal antibody) with proteolytic enzymes such as pepsin or papain, etc., and the disulfide bond existing between the two H chains in the hinge region. It means an antibody fragment 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_L(L chain variable region) and C_LL chain consisting of (L chain constant region), and V_H(H chain variable region) and C_HTwo homologous antibody fragments in which an H chain fragment consisting of γ1 (γ1 region in the H chain constant region) is bound by a disulfide bond at the C-terminal region can be produced. Each of these two homologous antibody fragments is called Fab ′. In addition, when IgG is treated with pepsin, an antibody fragment that is cleaved downstream of the disulfide bond existing between the two heavy chains in the hinge region to produce a slightly larger antibody fragment than the two Fab's connected in the hinge region is produced. Can do. F (ab ') this antibody fragment₂That's it.
[0055]
The “binding rate constant (ka)” in the present invention means a value indicating the strength (degree) of binding of the monoclonal antibody to the target antigen calculated based on the antigen-antibody reaction kinetics. 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 “binding 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, but using a commercially available measurement kit BiacoreX (manufactured by Amersham Pharmacia) or a similar kit, it is easy to follow according to the instruction manual attached to the kit and the experimental operation method. Can be analyzed. The ka value, kd value, and Kd value obtained using the kit are represented by units of 1 / M.Sec, 1 / Sec, and M (mole), respectively. The tested monoclonal antibody shows that it has strong antigen binding activity, so that ka value is large, and has strong neutralizing activity, so that Kd value is small.
[0057]
The human monoclonal antibodies of the present invention include human monoclonal antibodies having ka values, kd values, or Kd values as shown in the following (1) to (3).
(1) Binding rate constant (ka) with human AILIM is 1.0 × 10^Four(1 / M.Sec) or more, preferably 1.0 × 10^Five(1 / M.Sec) A human monoclonal antibody that binds to human AILIM or a part of the above value.
(2) Dissociation rate constant (kd) with human AILIM is 1.0 × 10^-Four(1 / Sec) or less, preferably 1.0 × 10^-Five(1 / Sec) A human monoclonal antibody that binds to human AILIM or a part thereof having the following numerical value.
(3) 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 having reactivity with 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 somewhat as an error range depending on various conditions at the time of measurement, but generally the index hardly fluctuates. It is.
[0058]
The “cell producing the monoclonal antibody” of the present invention or the “genetically recombinant host” of the present invention producing the recombinant human monoclonal antibody (wherein the host is a cell other than a fertilized egg). It means any cell that produces the human monoclonal antibody of the present invention described above.
Specifically, for example, the cells described in any of (1) to (3) below can be mentioned, but this is not restrictive.
(1) A human monoclonal antibody-producing B cell obtained by immunizing the above-described immunogen (antigen) with the above-described human antibody-producing transgenic non-human mammal and collected from the immunized animal.
(2) The aforementioned fused cell (hybridoma) obtained by cell fusion of the human monoclonal antibody-producing B cell thus obtained with a myeloma cell derived from a mammal.
(3) a gene encoding the human monoclonal antibody isolated from the human monoclonal antibody-producing B cell or a human monoclonal antibody-producing fused cell (hybridoma) (either a gene encoding a heavy chain or a gene encoding a light chain) Or a gene obtained by transforming cells other than the B cells and hybridomas (for example, CHO (Chinese hamster ovarian) cells, BHK (Baby hamster kydney) cells, lymphocytes such as myeloma, etc.)). Genetically modified cells that produce recombinant human monoclonal antibodies.
[0059]
Here, the recombinant cell producing the recombinant human monoclonal antibody described in (3) above, that is, the recombinant gene of the human monoclonal antibody produced by the B cell of (1) or the hybridoma of (2) It means a genetically modified cell that produces the body.
The “host” in the “genetical recombination 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. A gene encoding any monoclonal antibody against human AILIM of the present invention (preferably a human monoclonal antibody) (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, the recombinant fertilized egg of the present invention can be obtained. This transgenic fertilized egg is used for the production of transgenic animals in order to prepare a large amount of the above-mentioned desired protein from milk (Nikkei Science, April 1997, pages 78 to 84).
[0060]
“Substance” constituting the present invention, specifically “substance having an activity to control signal transduction via AILIM”, more specifically “inhibition of proliferation of AILIM-expressing cell or cytokine by AILIM-expressing cell” The “substance having the activity to suppress the production of” means a natural substance existing in nature or any substance prepared artificially.
In addition, the “substance” according to the “substance binding to AILIM” and “substance binding to the AILIM ligand” in the present invention means a natural substance existing in nature or any substance artificially prepared.
As used herein, “signal transduction via AILIM” refers to any phenotypic change in cells expressing AILIM (cell proliferation, cell activation, cell inactivation, cell death, and / or AILIM-expressing cells). Signal transduction through AILIM, which leads to changes in the ability to produce any cytokine.
[0061]
The “substance” can be roughly classified into “protein substance” and “non-protein substance”. Examples of the “proteinaceous substance” include polypeptides and antibodies described later (polyclonal antibodies, monoclonal antibodies, or a part of the monoclonal antibodies, particularly preferably the above-mentioned human antibodies).
When the substance is an antibody, it is preferably a monoclonal antibody. When the substance is a monoclonal antibody, 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-described human monoclonal antibody are included.
[0062]
Here, the “recombinant chimeric monoclonal antibody” is a monoclonal antibody produced by genetic engineering, and specifically, the variable region thereof is an immunoglobulin of a non-human mammal (mouse, rat, hamster, etc.). It means a chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody which is a variable region derived from human and whose constant region is a constant region derived from human immunoglobulin.
[0063]
A “human 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 thereof. A complementarity determining region of a hypervariable region derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework region of the variable region being a framework region of a variable region derived from human immunoglobulin, and It means a human monoclonal antibody characterized in that the constant region is a constant region derived from human immunoglobulin.
[0064]
Here, the complementarity-determining regions of the hypervariable regions are the three regions (Complementarity-determining residues; CDR1, CDR2) that are present in the hypervariable region of the variable region of the antibody and that directly bind complementarily to the antigen. , CDR3) and the framework region of the variable region refers to four relatively conserved 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 regions other than part or all of the complementarity determining region of the hypervariable region of a monoclonal antibody derived from a non-human mammal are replaced with the corresponding region of human immunoglobulin.
[0065]
The constant region derived from human immunoglobulin has a unique amino acid sequence depending on isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. In the present invention, the human monoclonal antibody The constant region may be a constant region of human immunoglobulin belonging to any isotype. Preferably, it is a constant region of human IgG. 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, the following polypeptide, a fragment of the polypeptide (oligopeptide), a fusion polypeptide, and any chemical modification thereof are included. Examples of 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 to or absorption in the digestive tract upon 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 comprising all or part of the extracellular region of AILIM and all or part of the constant region of an immunoglobulin heavy chain; or
(3) A polypeptide that binds to AILIM.
[0067]
Examples of the “non-proteinaceous substance” include DNA, RNA, and chemically synthesized compounds.
Here, “DNA” means “antigenic DNA drug useful as an antisense DNA pharmaceutical designed based on the base sequence of DNA (including cDNA and genomic DNA) encoding the above-mentioned AILIM (preferably human AILIM). It means “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 composed 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, more preferably a continuous partial base sequence of 5 to 50 bases, and more. A continuous partial base sequence of 5 to 30 bases is preferable.
[0069]
In addition, when the DNA is used as an antisense drug, it increases blood half-life (stability) when the DNA is administered into a patient's body, increases the permeability of an intracellular membrane, or is administered orally. For the purpose of increasing degradation resistance in the digestive organs or increasing absorption in some cases, it is possible to chemically modify a part of the base sequence of the DNA. Examples of the chemical modification include chemical modifications such as phosphate bond, ribose, nucleobase, sugar moiety, 3 'and / or 5' end in the structure of the oligonucleotide.
Phosphate bond modifications include one or more of these bonds, phosphodiester bonds (D-oligos), phosphorothioate bonds, phosphorodithioate bonds (S-oligos), methylphosphonate bonds (MP-oligos), phosphoramidos. Mention may be made of any of the date bonds, non-phosphate bonds and methylphosphonothioate bonds or combinations thereof. Examples of the modification of ribose include a change to 2′-fluororibose or 2′-O-methylribose. Nucleobase modifications include changes to 5-propynyluracil or 2-aminoadenine.
[0070]
Here, “RNA” refers to “RNA containing a partial base sequence of the RNA or those useful as an antisense RNA drug designed based on the base sequence of RNA encoding the aforementioned AILIM (preferably human AILIM)” Means a chemically modified RNA ". The antisense RNA is hybridized to DNA encoding AILIM, and is then hybridized to DNA or RNA encoding AILIM to thereby transcribe the DNA encoding AILIM into mRNA or to the protein of mRNA. Can be inhibited.
[0071]
Here, the “partial base sequence” means a partial base sequence composed 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, more preferably a continuous partial base sequence of 5 to 50 bases, and more. A continuous partial base sequence of 5 to 30 bases is preferable.
The antisense RNA increases blood half-life when the RNA is administered to a patient's body, increases permeability of the intracellular membrane, or increases or absorbs degradation resistance in the digestive tract when administered orally. For the purpose of increasing the number of RNAs, it is possible to chemically modify a part of the base sequence of the RNA. Examples of the chemical modification include chemical modification applied to the above-described antisense DNA.
[0072]
“Chemically synthesized compound” means any compound excluding the above-mentioned DNA, RNA and protein substances, 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 More preferred are compounds 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), preferably of the polypeptide constituting AILIM. It means the whole or part of the extracellular region (this region may optionally have 1 to 5 amino acids added to its N-terminus and / or C-terminus).
AILIM according to the present invention is a transmembrane molecule that penetrates a cell membrane composed of one or two polypeptide chains.
[0074]
As used herein, the term “transmembrane protein” refers to a membrane that is linked to the membrane by a hydrophobic peptide region that penetrates the lipid bilayer of the membrane 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 extracellular region, a transmembrane region, and a cytoplasmic region. Furthermore, such transmembrane proteins may be homodimers, heterodimers, as monomers, or together with another chain having the same amino acid sequence or a chain having a different amino acid sequence, respectively. Or each receptor and cell surface molecule | numerator are comprised by forming and forming an oligomer (origomer).
[0075]
Here, the “extracellular region” means all or part of a partial structure (partial region) existing on the outer side of the membrane to which the membrane protein is linked, among the whole structure of the transmembrane protein as described above. In other words, all or part of the region other than the region taken into the membrane (transmembrane region) and the region in the cytoplasm following the region in the membrane (intracellular region) means.
[0076]
The “fusion polypeptide” included in the above-mentioned “proteinaceous substance” refers to all or part of the extracellular region of a polypeptide constituting AILIM (preferably human AILIM) and “immunoglobulin (Ig, preferably It is a fusion polypeptide consisting of “all or part of the constant region of the heavy chain of human Ig)”. Preferably, it is 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 extracellular region of AILIM and the hinge region, CH2 domain and CH3 domain of the heavy chain of human IgG It is a fusion polypeptide with the region (Fc). As IgG, IgG1 is preferable. As AILIM, human, mouse or rat (preferably human) AILIM is preferable.
[0077]
Here, “all or part of the constant region of the heavy chain of immunoglobulin (Ig)” is preferably a constant region of a heavy chain (H chain) of a human-derived immunoglobulin, Fc region Or some of them. The immunoglobulin may be an immunoglobulin belonging to any class and subclass, specifically, 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]
Immunoglobulin is a Y-shape in which four chains of two homologous light chains (Light Chain, L chain) and two homologous heavy chains (Heavy chain) are linked by disulfide bonds (SS bonds). Having a structural unit of The light chain is a light chain variable region (V_L) And light chain constant region (C_L). The heavy chain is a heavy chain variable region (V_H) And heavy chain constant region (C_H).
[0079]
The heavy chain constant region is composed of several domains, each with 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 in order from the N-terminus to V_H, CH1 domain, hinge region, CH2 domain and CH3 domain.
[0081]
Similarly, the heavy chain of IgG1 is V_H, Cγ₁1 domain, hinge region, Cγ₁2 domains and Cγ₁It consists of 3 domains. The heavy chain of IgG2 is in order from the N-terminus to V_H, Cγ₂1 domain, hinge region, Cγ₂2 domains and Cγ₂It consists of 3 domains. The heavy chain of IgG3 is V-sequenced from the N-terminus._H, Cγ₃1 domain, hinge region, Cγ₃2 domains and Cγ₃It consists of 3 domains. The heavy chain of IgG4 is V_H, Cγ₄1 domain, hinge region, Cγ₄2 domains and Cγ₄It consists of 3 domains.
[0082]
The heavy chain of IgA consists of V_H, Cα1 domain, hinge region, Cα2 domain and Cα3 domain.
[0083]
Similarly, the heavy chain of IgA1 is V V in order from the N-terminus._H, Cα11 domain, hinge region, Cα₁2 domains and Cα₁It consists of 3 domains. The heavy chain of IgA2 is V-sequenced from the N-terminus._H, Cα₂1 domain, hinge region, Cα₂2 domains and Cα₂It consists of 3 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]
Furthermore, if IgG is taken as an example, when IgG is treated with papain, the disulfide bond existing in the hinge region connecting two heavy chains is cleaved slightly at the N-terminal side, and V_HAnd C_HTwo homologous Fabs in which a heavy chain fragment consisting of 1 and one light chain are linked by a disulfide bond, and a hinge region, C_H2 domains and C_HTwo homologous heavy chain fragments consisting of three domains result in one Fc linked by disulfide bonds (above, “Immunology Illustrated”, 2nd Edition, 65-75, 1992, published by Nanedo, And “The focus of the latest medical science“ recognition mechanism of the immune system ”, pages 4-7, 1991, published by Nankodo etc.).
[0088]
That is, the above-mentioned “part of the constant region of an immunoglobulin heavy chain” means a part of the constant region of an immunoglobulin heavy chain having the above structural characteristics, and preferably lacks the C1 domain. Constant region or Fc region. Specifically, in the case of IgG, IgA or IgD, each region includes a hinge region, C2 domain and C3 domain. In the case of IgM or IgE, each C2 domain, C3 domain and C4 domain The area | region which consists of is mentioned. Particularly preferred examples include human-derived IgG1 Fc regions.
[0089]
Since the above-mentioned fusion polypeptide has a part of the constant region of immunoglolin such as IgG as described above (for example, Fc) as a fusion partner, the property of protein A that specifically binds to the immunoglobulin fragment There is an advantage in that the fusion polypeptide can be purified very easily by using affinity column chromatography. Further, since various antibodies against Fc of various immunoglobulins are provided, immunoassay of the fusion polypeptide can be easily performed using the antibody against Fc.
[0090]
The “polypeptide” included in the definition of “substance” also includes “polypeptide that binds to AILIM”.
The “polypeptide that binds to AILIM” is a molecule called B7h, B7RP-1, GL50 or LICOS (Nature, Vol.402, No.6763, p.827-) which is the “AILIM ligand” defined above. 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), which includes all or part of the polypeptide.
Preferably, a polypeptide comprising all or part of the extracellular region of the ligand (B7h, B7RP-1, GL50, LICOS), or the constant region of the heavy chain of the polypeptide and an immunoglobulin (preferably human immunoglobulin) A fusion polypeptide consisting of all or part of Here, the terms “extracellular region” and “constant region of immunoglobulin heavy chain” 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, a cell culture method, etc. in addition to a gene recombination technique as described later. It can manufacture by using a well-known method or its modification method suitably.
The “antibody” in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody against mammalian AILIM (particularly preferably human AILIM) as defined above, preferably a monoclonal antibody.
Specifically, it is an antibody that binds to AILIM and suppresses the growth of AILIM-expressing cells, or binds to AILIM and has an activity of suppressing the production of interferon γ or interleukin 4 by AILIM-expressing cells.
[0092]
“Delayed type allergy” in the present invention is mediated by cellular immunity (particularly mediated by Th1-type T cells), that is, T cells sensitized by an antigen (memory T cells storing an antigen). A biological body that is allergic and sensitized with an antigen means any allergy that requires approximately 24 to 48 hours to develop an allergic reaction accompanied by inflammation caused by the memory T cells that occurs upon re-contact with the antigen.
This delayed type allergy includes allergy to infectious pathogen antigens such as tuberculin allergy derived from Mycobacterium tuberculosis, Jones-Mote type delayed allergy that appears transiently to trace amounts of proteins, drugs such as picryl chloride, lacquer, etc. Contact allergies to phytotoxins, or allergies related to transplant rejection seen in transplantation of allografts.
[0093]
The “pharmaceutical composition” in the present invention refers to an antibody (preferably human antibody) or a monoclonal antibody (preferably 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”.
As used herein, “pharmaceutically acceptable carrier” refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners. , Flavoring agents, solubilizers or other additives.
By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, solutions, capsules, trowels, elixirs, suspensions, emulsions or 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 prescribed by conventional methods, suppositories for enteric administration, pessaries, and the like.
[0094]
The dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient (such as the above-mentioned protein or antibody) contained in the pharmaceutical composition, etc. The dose can be administered in a range of 10 μg to 1000 mg (or 10 μg to 500 mg) 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 injections, 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. It can be produced by dissolving or suspending in the solution.
[0095]
The thus-prepared injection is administered to a human patient in need of treatment at a rate of 1 μg to 100 mg per kg body weight in a single administration, preferably at a rate of 50 μg to 50 mg per day. Can be administered several times to several times. Examples of administration forms include medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection. Intravenous injection is preferred.
In addition, injections can be prepared as non-aqueous diluents (for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions or emulsions. .
Such sterilization of the injection can be performed by filtration sterilization through a bacteria-retaining filter, blending of a bactericide, or irradiation. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in sterile water for injection or other solvents before use.
[0096]
The pharmaceutical composition comprising the human antibody of the present invention is costimulated via AILIM to cells expressing AILIM without inducing host immune rejection caused by HAMA (human anti-mouse antibody). Various biological reactions involving the transmission of Lee signals (eg, costimulatory signals) (eg, cell growth of cells expressing AILIM, production of cytokines by cells expressing AILIM, and immune cytolysis of cells expressing AILIM) as a pharmaceutical for controlling (eg, cytolysis) or cell death) and / or inhibiting and preventing the onset and / or progression of various diseases involving signaling through the AILIM, and treating or It is useful as a medicine for prevention.
[0097]
In the present invention, “immune cytolysis” means the following biological phenomenon.
Cell lysis occurs not only by binding to killer cells but also via antibodies (particularly cytolytic antibodies). This cytolytic antibody is an antibody that acts on the lysis reaction of cells such as immune cells, tissue cells, or sperm, among other cytotoxic antibodies, and a complement component when the antibody binds to a cell surface antigen. An antibody that damages the cell or induces 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 antibodies to cell surface antigens. Complement first component (C1) is activated by the antibody bound to the surface antigen, and as a result of a series of complement reactions from complement second to ninth components (C2 to C9), a cytotoxic site is formed. The cell contents are released and cell lysis is reached.
[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. In the cytotoxic action of a target cell to be caused, the cytotoxic action requires an antibody in order to induce the cytotoxicity in addition to the effective cell and the target cell.
[0099]
The “mixed lymphocyte reaction” in the present invention is a biological phenomenon abbreviated as MLR. It is also called mixed leukocyte reaction.
By mixing leukocytes or lymphocytes from each of two different individuals from allogenic animals and culturing them for several days, these cells blast and DNA synthesis within the cells (ie cell proliferation) Is promoted. This reaction is referred to as MLR (allogenic MLR).
The degree of DNA synthesis (cell proliferation) can be analyzed by stopping one lymphocyte by irradiation or treatment with mitomycin and measuring the degree of DNA synthesis in the other lymphocyte.
The degree of DNA synthesis 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 is a method of cloning cDNA from mRNA encoding the AILIM according to a conventional method, a method of isolating genomic DNA and splicing, and the cDNA. It can be obtained by a method of preparing by PCR using a sequence or mRNA sequence as a template, a method of chemical synthesis, or the like.
Further, DNA encoding the AILIM ligand in 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 cleaving (digesting) the DNA containing each of the DNAs encoding the AILIM thus prepared with an appropriate restriction enzyme. Fragments can be prepared by ligating together with 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 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 and produces the target protein. For the preparation of mRNA, total RNA prepared using a known method such as the guanidine thiocyanate method (Biochemistry, Vol. 18, p. 5294, 1979), the hot phenol method or the AGPC method is used for oligo (dT) cellulose or polyU. -It can be carried out by affinity chromatography using Sepharose or the like.
[0102]
Then, using the obtained mRNA as a template, a known method such as using reverse transcriptase, for example, the method of Okayama et al. (Mol. Cell. Biol., Vol. 2, p. 161, 1982 and the same magazine Vol. 3, p. .280, 1983) and the method of Hoffman et al. (Gene, Vol. 25, p.263, 1983) and the like, and a cDNA strand is synthesized and converted into a double-stranded cDNA. This cDNA is incorporated into a plasmid vector, phage vector or cosmid vector, and transformed into E. coli, or after in vitro packaging, a cDNA library is prepared by transfecting E. coli.
[0103]
The plasmid vector used here is not particularly limited as long as it can be replicated and retained in the host, and any phage vector that can be propagated in the host may be used. Examples of commonly used cloning vectors include pUC19, λgt10, λgt11, and the like. However, when subjected to immunological screening described later, a vector having a promoter capable of expressing a gene encoding the target protein in the host is preferable.
[0104]
Examples of a method for incorporating cDNA into a plasmid include the method described in the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p. 1.53, 1989). Moreover, as a method for incorporating cDNA into a phage vector, the method of Hyunh et al. (DNA Cloning, a practical approach, Vol. 1, p. 49, 1985) and the like can be mentioned. For convenience, a commercially available cloning kit (for example, Takara Shuzo Co., Ltd.) can also be used. The recombinant plasmid and phage vector thus obtained are introduced into a suitable host such as a prokaryotic cell (for example, E. coli: HB101, DH5α, Y1090, DH10B, MC1061 / P3, etc.).
[0105]
As a method for introducing a plasmid into a host, a calcium chloride method, a calcium chloride / rubidium chloride method, or an electroporation method described in (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.1.74, 1989). Etc. Examples of the method of introducing the phage vector into the host include a method of introducing the phage DNA into the grown host after in vitro packaging. In vitro packaging can be performed conveniently by using a commercially available in vitro packaging kit (for example, manufactured by Stratagene, Amersham, etc.).
A method of isolating a cDNA encoding a target protein from a cDNA library prepared by the above method can be performed by combining general cDNA screening methods.
[0106]
For example, after chemically synthesizing oligonucleotides that are thought to correspond to the amino acid sequence of the target protein separately,³²Labeled with P as a probe, known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA, Vol. 72, p.3961, 1975) or plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.2.108, 1989), a method for screening clones containing the target cDNA, PCR primers are prepared, and a specific region of the target protein is amplified by the PCR method. And a method of selecting a clone having a DNA fragment encoding the region.
[0107]
In addition, when a cDNA library prepared 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 to the target protein is used. Clones can be selected. When a large amount of clones are processed, it is preferable to use a screening method using the PCR method.
The base sequence of the DNA thus obtained is a dideoxynucleotide using the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA., Vol74, p.560, 1977) or phage M13. It can be determined by the method of 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 with a restriction enzyme or the like.
[0108]
Examples of the preparation method by isolating DNA encoding the target protein from genomic DNA derived from cells expressing the target protein as described above include the following methods.
The cells are preferably lysed using SDS or proteinase K or the like, and extraction with phenol is repeated to deproteinize DNA. The RNA is preferably digested with ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the resulting 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 all or a part of the gene encoding the target protein is cut out from the clone with a restriction enzyme or the like.
[0109]
Preparation of DNA encoding the target protein by PCR can be performed by a conventional method 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.) Issue, 1992, etc.).
In addition, production of DNA encoding the target protein by chemical synthesis can be performed according to a conventional method based on the base sequence of the target protein.
[0110]
AILIM of the present invention (particularly preferably human AILIM) or a part thereof (preferably extracellular region) is a DNA (genomic DNA containing cDNA or intron) encoding AILIM prepared using the method exemplified above. Each AILIM-encoding DNA fragment is obtained by cleaving with an appropriate restriction enzyme, and these fragments are obtained by linking them with a linker DNA or a tag (Tag), if necessary, using an appropriate DNA polymerase or the like. Using the obtained DNA, a recombinant protein can be prepared by a conventional method using a commonly used gene recombination technique.
The same applies to an AILIM ligand (particularly preferably a human AILIM ligand) or a part thereof (preferably an extracellular region).
Specifically, it is as illustrated 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 a host cell as described later is transformed with the expression vector to obtain a transformant. . The target protein is produced in the culture supernatant by culturing the transformant. The target protein in the culture supernatant can be easily purified using column chromatography or the like.
[0111]
An expression vector used for producing recombinant AILIM (or its extracellular region), a plasmid that is not particularly limited as long as it can be replicated or self-replicated in various prokaryotic and / or eukaryotic hosts, plasmid Vectors and phage vectors are included (Cloning Vectors: A Laboratory Manual, Elsby, New York, 1985).
The expression vector is conveniently prepared by ligating DNA encoding AILIM (or its extracellular region) to a recombination vector (plasmid DNA and bacterial phage DNA) available in the industry by a conventional method. Can do. Specific examples of the recombination vector used include plasmids derived from E. coli such as pBR322, pBR325, pUC12, pUC13 and pUC19, yeast derived plasmids such as pSH19 and pSH15, and Bacillus subtilis derived plasmids such as pUB110, pTP5 and pC194. Etc. are exemplified. Examples of the phage include bacteriophages such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
[0112]
A DNA encoding the AILIM of the present invention (particularly preferably human AILIM) or its soluble extracellular region is expressed and AILIM is expressed on the surface of the host cell, or a soluble extracellular region of AILIM (particularly preferably human AILIM) is produced. 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 Medicine separate volume "Gene Engineering Handbook", 1992, etc.) be able to.
[0113]
When bacteria, particularly Escherichia coli, is used as a host cell, an expression vector generally comprises at least a promoter-operator region, a start codon, DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit.
When yeast, animal cells or insect cells are used as the host, the expression vector should contain at least a promoter, an initiation codon, AILIM of the present invention (particularly preferably human AILIM) or DNA encoding its extracellular region, and a stop codon. preferable. In addition, DNA encoding signal peptide, enhancer sequence, 5 ′ and 3 ′ untranslated regions of gene encoding AILIM of the present invention, splicing junction, polyadenylation site, selectable marker region or replicable unit, etc. May be included. Moreover, the gene amplification gene (marker) normally used according to the objective may be included.
[0114]
The promoter-operator-region for expressing the AILIM of the present invention (particularly preferably human AILIM) or its extracellular region in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). It is a waste. For example, when the host is Escherichia, preferred examples include those containing Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like.
Examples of promoters for expressing the AILIM of the present invention (particularly preferably human AILIM) or the extracellular region thereof in yeast include PH05 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is Bacillus Include SL01 promoter, SP02 promoter, penP promoter and the like. In addition, when the host is a eukaryotic cell such as a mammalian cell, for example, an SV40-derived promoter, a retrovirus promoter, a heat shock promoter and the like can be mentioned. However, it is not particularly limited to these. In addition, the use of an enhancer is an effective method for expression.
[0115]
A suitable initiation codon is exemplified by methionine codon (ATG).
Examples of stop codons include commonly used stop codons (eg, TAG, TAA, TGA).
As the terminator region, a commonly used natural or synthetic terminator can be used.
A replicable unit refers to DNA that has the ability to replicate its entire DNA sequence in a host cell, and includes a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from a natural plasmid) and Synthetic plasmids and the like are included. Suitable plasmids include plasmid pBR322 or an artificial modification thereof (DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) in E. coli, yeast 2μ plasmid, or yeast chromosomal DNA in yeast, Examples of mammalian cells include plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV-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 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.
The gene amplification gene includes dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, aspartate transcarbamylase gene Etc. can be illustrated.
[0117]
The expression vector of the present invention can be prepared by linking at least the above-mentioned promoter, start codon, DNA encoding the protein of the present invention, stop codon and terminator region continuously and circularly to suitable replicable units. it can. At this time, an appropriate DNA fragment (for example, a linker, other restriction enzyme sites, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase, if desired.
The transformed cell of the present invention can be prepared by introducing the above 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-described expression vector and can be transformed, and is a natural cell or artificially established usually used in the technical field of the present invention. Examples include various cells such as recombinant cells (for example, bacteria (Escherichia, Bacillus), yeasts (Saccharomyces, Pichia, etc.), animal cells or insect cells).
Preferred are E. coli or animal cells, specifically E. coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.), mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1 or NIH3T3, etc.) , Rat-derived cells, hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.) and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma cells And Namalwa et al.).
Introduction (transformation (transfection)) of an expression vector into a host cell can be carried out 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) and competent methods (J. Mol. Biol., Vol.56, p.209, 1971), for example, in the case of Saccharomyces cerevisiae, Hinnen et al. (Proc. Natl. Acad. Sci. USA., Vol.75, p.1927, 1978) or lithium method (J. Bacteriol., Vol.153, p.163, 1983) For example, Graham's method (Virology, Vol. 52, p. 456, 1973). In the case of insect cells, for example, Summers et al.'S method (Mol. Cell. Biol., Vol. 3, p. 2156-2165, 1983), respectively.
[0120]
The extracellular region (soluble AILIM) of AILIM of the present invention (particularly preferably human AILIM) is a transformed cell containing the expression vector prepared as described above (hereinafter used to include a transfectant). It can be produced by culturing in a nutrient medium. The same applies to the 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. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean cake, potato extract and the like. In addition, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be included as desired. .
Culturing is performed by methods known in the art. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.
[0121]
In addition, although the specific culture medium and culture | cultivation conditions used according to a host cell below are illustrated, it is not limited to these at all.
When the host is a bacterium, actinomycetes, yeast or filamentous fungus, for example, a liquid medium containing the above nutrient source is suitable. Preferably, the medium has a pH of 5-8.
When the host is E. coli, preferred media 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 culture can be performed usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary.
[0122]
When the host is a Bacillus genus, the treatment can be performed 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's minimum medium (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980), and the pH is 5-8. It is desirable to be. The culture is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration or agitation can be performed.
When the host is an animal cell, for example, a MEM medium (Science, Vol. 122, p. 501, 1952) containing about 5 to 20% fetal bovine serum, a DMEM medium (Virology, Vol. 8, p. 396, 1959), RPMI1640 medium (J. Am. Med. Assoc., Vol.199, p.519, 1967), 199 medium (Proc. Soc. Exp. Biol. Med., Vol.73, p.1, 1950) HamF12 medium or the like can be used. The pH of the medium is preferably about 6-8, and the culture is usually carried out at about 30-40 ° C. for about 15-72 hours, and if necessary, aeration or agitation 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) can be mentioned, and its pH is about 5-8. Is preferred. The culture is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and aeration and agitation can be performed as necessary.
The cells (soluble AILIM) of the AILIM of the present invention (particularly preferably human AILIM) are secreted into the culture supernatant by culturing the above-described transformed cells (particularly animal cells or E. coli). Can be manufactured. That is, a culture filtrate (supernatant) is obtained from the obtained culture by a method such as filtration or centrifugation, and the target protein is obtained according to a conventional method generally used for purifying and isolating natural or synthetic proteins from the culture filtrate. 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 and solvent precipitation, dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate. -Methods using molecular weight differences such as polyacrylamide gel electrophoresis, methods using charges such as ion exchange chromatography and hydroxylapatite chromatography, methods using hydrophobic differences such as reverse phase high performance liquid chromatography, etc. Examples thereof include a method using the difference in isoelectric point such as electric point electrophoresis.
[0125]
On the other hand, if the target protein is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to conventional methods such as filtration or centrifugation to collect the cells or cells and suspend them in an appropriate buffer. After turbidity, for example, cell walls and / or cell membranes such as cells are destroyed by a method such as ultrasonic wave, lysozyme, and freeze-thaw, and then a membrane fraction containing the target protein is obtained by a method such as centrifugation or filtration. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. 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, etc., or plates, test tubes or tubes made of water-insoluble substances such as glass , Beads, balls, filters, membranes, etc., 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 materials An insoluble carrier used for affinity chromatography such as a carrier can be exemplified.
[0127]
Examples of the “labeling substance capable of providing a detectable signal” in the present invention include, for example, enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, radioisotopes, and the like. More specifically, peroxidases (for example, horseradish) peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, etc. Fluorescent substances such as enzymes, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate,^ThreeH,¹⁴C,¹²⁵I or¹³¹A radioisotope such as I, biotin, avidin, or a chemiluminescent substance.
[0128]
Here, the radioisotope and the fluorescent substance can provide a detectable signal alone. On the other hand, enzymes, chemiluminescent substances, biotin, and avidin cannot provide a detectable signal by themselves, and therefore, can 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 enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence 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. Various color-developing substances depending on the substrate are used as necessary.
In the present invention, any of the above-mentioned 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 for identifying a substance that binds to AILIM or an AILIM ligand” of the present invention is based on an immunoassay.
Specifically, the principles of various methods as described in enzyme immunoassay (3rd edition, edited by Yuji Ishikawa et al., Published by Medical School, 1987) can be applied.
Suitable principles include, for example, the one-antibody solid phase method, the two-antibody liquid phase method, the two-antibody solid phase method, the sandwich method, and the one-pot method as described in JP-B-2-39747. Can be mentioned. In addition, as an assay using antigen-antibody reaction, EMIT method (Enzyme multiplied immunoassay technique), enzyme channeling assay (Enzyme channeling immunoassay), enzyme activity modifying substance labeled immunoassay (Enzyme modulator mediated enzyme immunoassay, EMMIA), enzyme inhibitor labeling Also known are an immunoassay, an immunoenzymometric assay, an enzyme enhanced immunoassay, a proximal linkage immunoassay, and the like.
[0130]
In the present invention, any principle of such an immunoassay can be appropriately selected and used according to the purpose, but it is convenient for operation and / or economical convenience, particularly for clinical use. In view of the characteristics, it is preferable to use the principle of the sandwich method, the one-pot method, or the one-antibody solid-phase method, more preferably the principle of the sandwich method or the one-pot method. Particularly preferably, a sandwich method using a multi-well microtiter plate having a large number of wells as typified by a 96-well microplate or a bead having a polypeptide immobilized on its surface and an enzyme such as peroxidase or biotin. This is a one-pot method using a labeled counter part.
[0131]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited only to the embodiments described in the examples.
[0132]
Example 1 Preparation of immunogen
<1-1> Preparation of recombinant cells expressing human AILIM
Other earlier applications (Japanese Patent Application Publication No. 11-29599 and International Patent Application Publication No. WO98 / 38216) and one already published (Int. Immunology, Vol. 12, No. 1), one of the present inventors , p.51-55, 2000), two types of recombinant cells (CHO cells and HPB-ALL cells) that highly express human AILIM were prepared. Specifically, it is as follows.
A cDNA containing the full-length ORF of human AILIM (GenBank Accession Number: AB023135 (cDNA); BAA82129 (amino acid)) was inserted into the vector pEF-neo, and then the recombinant expression vector was converted using Gene Pulser (BioRad). It was introduced into each of 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; manufactured by 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
A mouse anti-human AILIM monoclonal antibody (mouse) named “SA12” previously prepared and reported by the present inventors on the precipitate collected by centrifugation of 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 No. WO 98/38216 (Example 12)) (concentration: diluted with EDTA-BSA / PBS) 10 μg / ml to 100 μl / 10^Fivecells) and reacted at 4 ° C. for 30 minutes. The cells were washed twice with the above EDTA-BSA / PBS (200 μl), then picoerythrin-labeled streptavidin (SA-PE; 100 μl diluted 500-fold) was added and reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed 3 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-Dichinson) to select recombinant HPB-ALL cells that highly express human AILIM did. Sorted cells were cultured until confluent in RPMI1640 medium containing 10% FCS and G418 (1 mg / ml).
[0134]
<1-3> Selection of recombinant CHO cells that highly express human AILIM
The mouse anti-human AILIM monoclonal antibody SA12 (100 μg / ml diluted with EDTA-BSA / PBS) labeled with FITC is added to the precipitate collected by centrifugation of the drug-resistant CHO cells selected in <1-1>. In addition, the mixture was reacted at 4 ° C. for 30 minutes. The cells were washed with the EDTA-BSA / PBS, and 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-Dichinson) to select recombinant CHO cells that highly express human AILIM. Sorted cells were cultured until confluent in RPMI1640 medium containing 10% FCS and G418 (1 mg / ml).
[0135]
<1-4> Preparation of immunogen from HPB-ALL cells expressing human AILIM
The human AILIM highly expressing HPB-ALL cells obtained in <1-2> were centrifuged. The collected precipitate was washed four times with a phosphate buffer (PBS; manufactured by Nikken Biological Laboratories), and then a protease inhibitor-containing buffer (25 mM HEPES (pH 7.4), 10 mM MgCl).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.). The supernatant is then collected, ultracentrifuged (100,000 g, 1 hour, 4 ° C.), the precipitated membrane fraction is collected, suspended in phosphate buffer (1 × 10 × 1 ml PBS).⁷Adjusted to the concentration of the cell-derived membrane fraction) and stored at minus 80 ° C. This suspension of cell membrane fraction was used as an antigen (immunogen) in the production of 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 highly expressing CHO cells obtained in the above <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 Nikken Biological Laboratories), and then a protease inhibitor-containing buffer (25 mM HEPES (pH 7.4), 10 mM MgCl).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.). The supernatant is then collected, ultracentrifuged (100,000 g, 1 hour, 4 ° C.), the precipitated membrane fraction is collected, suspended in phosphate buffer (1 × 10 × 1 ml PBS).⁷Adjusted to the concentration of the cell-derived membrane fraction) and stored at minus 80 ° C. This suspension of cell membrane fraction was used as an antigen (immunogen) in the production of the human antibody of the present invention described later.
[0137]
Example 2 Preparation of anti-human AILIM human monoclonal antibody-producing hybridoma
Production of monoclonal antibodies in this example was carried out using experimental medicine (separate volume), cell engineering handbook (edited by Toshio Kuroki et al., Published by Yodosha, pages 66-74, 1992), and an introduction to monoclonal antibody experimentation (Teihei Ando) Et al., Published by Kodansha, 1991) and the like.
[0138]
As a human AILIM as an immunogen, a cell membrane fraction prepared from a recombinant cell highly expressing human AILIM prepared in Example 1 was used.
As an immunized animal, a human antibody-producing transgenic mouse produced by the above-described method was used (Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146- 156, 1997; JP-T 4-504365 Publication; JP-T 7-509137 Publication; Nikkei Science, June, pages 40-50, 1995, etc.).
The cell culture operation was performed using a multiwell microplate.
[0139]
<2-1> Immunization and hybridoma preparation
Any of the immunogens (100 μl / mouse / single administration) prepared in <1-4> (derived from HPB-ALL) and <1-5> (derived from CHO) is added to the human antibody-producing transgenic mouse. First immunization was performed by injecting into the Hoodpad together with Freund's Complete Adjuvant (ICN / CAPPEL).
One week after the first immunization, booster is given 2 or 3 times by intra-hood pad injection every week, and the immunogens are introduced the day before the lymphocyte acquisition described below. The final immunization was conducted in the same manner.
[0140]
Two days after the final immunization, lymphocytes were obtained from lymph nodes (inguinal and below knee) and spleen removed 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 (Boehringer Mannheim) was added as a fusion agent, and then 10 times An amount of serum-free basic medium EX-CELL301 (manufactured by JRH Bioscience) was added for dilution. Next, the mixed cells are washed with the basic medium, suspended in HAT medium (containing 13.61 mg of hypoxanthine, 176 μg of aminopterin, and 3.88 mg of thymidine per liter of the basic medium), and seeded in a 96-well microplate. The cells were cultured for 10 to 14 days for cell fusion. A number of hybridomas were obtained by this cell fusion.
[0141]
<2-2> Screening of human monoclonal antibody-producing hybridomas
A number of hybridomas prepared in the above <2-1> were screened by the following cell ELISA to select hybridomas producing human monoclonal antibodies against human AILIM.
Recombinant HPB-ALL cells and recombinant CHO cells that highly express human AILIM prepared above (1 × 10 5 each)^FiveCells / well) were seeded in each well of a 96-well microplate for ELISA and cultured at 37 ° C. for 2 days.
Next, the supernatant was discarded, and each hybridoma culture supernatant sample (50 μl / well) was added to each well and allowed to react 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).
Subsequently, for detection of 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 was 50 μl / well in each well). Manufactured by Ameircan Corex; 1% BSA / PBS) and incubated at room temperature for 1 hour.
On the other hand, for detection of the light chain of human immunoglobulin (human monoclonal antibody) contained in the hybridoma supernatant, a peroxidase-labeled goat anti-human immunoglobulin κ chain antibody (1/2000 dilution of 50 μl) 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 3 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 H₂SO_Four(50 μl / well) was added to each well to stop the reaction. Absorbance at a wavelength of 450 nm was measured with a Bio-Rad, Model 3550 Microplate Reader (Model 3550 Microplate Reader, manufactured by BIO-RAD).
As a control test, ELISA was performed in the same manner as 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 No. WO98 / 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> above. .
As a result, a large number of hybridomas producing human monoclonal antibodies having binding ability to human AILIM were selected.
[0143]
<2-3> Primary cloning of hybridoma
According to the following test procedure, a large number of single hybridomas were cloned from a large number of hybridomas (parent strains) that were selected in <2-2> and produced human monoclonal antibodies against human AILIM.
Each of the hybridomas selected in <2-2> was spread on a 24-well (well) microplate, and the number of hybridoma cells in each well was counted by pipetting. Next, 10% fetal bovine serum (FCS; manufactured by Trace Bioscience PTY), 1% penicillin / streptomycin (manufactured by Sigma), 1% HT in EX-CELL301 medium (manufactured by JRH Bioscience) containing 4.0 mM L-Glutamine and lipid. 1 × 10 6 hybridomas in each well with a modified medium prepared by adding Supplement (Gibco BRL) and 2.5% T-STIM Culture Supplement (Collaborative Biomedical Products)^FourThe cells were suspended by diluting to 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, add the cell suspension to each well of multiple 96-well microplates at a concentration of 200 μl / well. In addition, 4 cells / well were set. The modified medium (50 ml or 100 ml) was newly added to the remaining cell suspension and mixed, and the resulting cell suspension was added to each well of another 96-well microplate to give 2 cells / well. .
After culturing for 1 to 2 weeks, formation of single hybridoma colonies was confirmed in many wells.
It was confirmed by the cell ELISA described in <2-2> above that human monoclonal antibodies against human AILIM were produced in the culture supernatant of each well in which the formation of the colonies was confirmed.
[0144]
<2-4> Secondary cloning of hybridoma
According to the same method as in the above <2-3>, each of the multiple hybridoma clones cloned in the above <2-3> was subcloned (secondary cloning).
In this test, the number of cells in each well of the 96-well microplate was adjusted to 1 cell / well.
As a result, a large 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),
In addition, in the following examples including this example, and in the drawings or tables shown as test results in the examples, the above names are used.
[0145]
Example 3 Characterization of monoclonal antibody
<3-1> Analysis of heavy chain and light chain
It was confirmed by the following ELISA and flow cytometry 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 highly expressing human AILIM prepared in Example 1 (3 × 10^FourCells / well) were seeded into each well of a V-bottom microplate and cultured in RPMI 1640 medium containing 10% FCS at 37 ° C.
After incubation, the plate was centrifuged (1,800 rpm, 2 minutes) to precipitate the cells. Then, the supernatant was discarded, and each well was cloned into each of the hybridoma culture supernatant samples (50 μl / well) cloned in <2-4> above or a mouse anti-human AILIM monoclonal antibody SA12 (2 μg / 50 μl) as a control or Anti-KLH human monoclonal antibody (50 μl / well) was added and reacted 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 diluted 1000-fold with the above-mentioned phosphate buffer) was added to each well, the cells were dispersed, and allowed to react in the 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 (from EY Laboratories); or
Biotin-labeled anti-human Igκ antibody (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 diluted 500-fold with the phosphate buffer) was added to each well and allowed to react in the refrigerator for 30 minutes. After the reaction, each well was washed with the phosphate buffer. Subsequently, the phosphate buffer solution (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 to the human AILIM highly expressing HPB-ALL cells in each well was analyzed.
[0147]
In addition, as a control test, tests and analyzes 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> above. .
As a result, it was confirmed that all of the hybridomas cloned in <2-4> are human monoclonal antibodies composed of human-derived heavy chains and human-derived κ light chains.
As an example of the results, the test results for each of the hybridoma clones AIH5D3 (JMab-136), AII289 (JMab-138), and AII394 (JMab-139) are shown in FIG.
[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 <2-4> and analyzed in <3-1> above was determined as 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 anti-human AILIM human monoclonal antibodies were confirmed to be IgG2 / κ.
[0149]
Example 4 Large-scale preparation and purification of 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 is added to Tissue Culture Flask (50 ml, manufactured by FALCON), and FBS (Ultra Low Bovine IgG FBS containing ultra-low bovine immunoglobulin). 5% CO in ASF104 medium (Ajinomoto Co.) containing 10% GIBCO-BRL₂The cells were cultured at 37 ° C. until confluent.
Next, the entire amount of the culture solution was transferred to Tissue Culture Flask (750 ml, manufactured by FALCON), and ASF104 medium (Ajinomoto Co., Inc.) containing 10% of ultra-low concentration bovine immunoglobulin-containing FBS (Ultra Low Bovine IgG FBS, manufactured by GIBCO-BRL). Made), 5% CO₂The cells were cultured at 37 ° C. until 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 centrifugal supernatant was filtered through a Sterilization Filter Unit (manufactured by NALGEN), and the filtrate was recovered.
[0150]
The filtrate was added to a high trap protein G column (HiTrap affinity column Protein G, manufactured by Amersham Pharmacia) equilibrated with a phosphate buffer (30 ml) at 3 ml / min.
Next, after washing the column with phosphate buffer (20 ml), 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute the antibody. Next, the solution (pH 9.0) consisting of 750 mM Tris-HCl was neutralized by adding to the eluate, followed by filtration with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was dialyzed with a phosphate buffer (overnight) and then filtered through a filter (Millipore) to obtain a purified anti-AILIM human monoclonal antibody from each hybridoma.
In addition, the value in A280 was calculated | required using the spectrophotometer, and protein concentration was computed (1A280 = 1.41mg / ml).
[0151]
<4-2> Method 2
Each hybridoma clone prepared in the above <2-4> conditioned to ASF104 medium (manufactured by Ajinomoto Co.) containing 10% Ultra Low Bovine IgG FBS (manufactured by GIBCO-BRL) (1-2 x 10 each)⁶Cells / ml) were seeded on an 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 × 10⁸When the number of cells / ml was reached, the culture supernatant of each hybridoma was collected.
Each culture supernatant 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 phosphate buffer (20 ml), 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute the antibody. Next, the solution (pH 9.0) consisting of 750 mM Tris-HCl was neutralized by adding to the eluate, followed by filtration with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was dialyzed with a phosphate buffer (overnight) and then filtered through a filter (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 purified anti-human AILIM human monoclonal antibodies to human AILIM and the cross-reactivity to mouse AILIM and rat AILIM were analyzed by cell ELISA.
[0153]
<5-1> Probability of ELISA system and calibration curve for IgG antibody concentration measurement
Since any anti-human AILIM human monoclonal antibody purified as described above is an IgG (IgG2) antibody, an ELISA for measuring the concentration of IgG antibody was established.
A goat anti-human IgG (Fc) antibody (1.2 μg / ml in PBS; 100 μl / well; manufactured by Organon Teknika) was added to each well of a 96-well microplate for ELISA (manufactured by Nunc) and incubated at room temperature for 2 hours. Anti-IgG (Fc) antibody was adsorbed on the microplate. Next, the supernatant was discarded, and after washing three times with 0.05% Tween20-containing phosphate buffer (PBS), each well contained PBS containing a blocking reagent (200 μl / well, 0.5% bovine serum albumin (BSA) and 0.1% Tween20). ) And incubated at room temperature for 2 hours to block sites where the anti-IgG (Fc) antibody was not bound. The blocking reagent was then discarded and each well was washed twice with PBS.
[0154]
Various concentrations (0 to 100 ng / ml) of human-derived IgG2 antibody (50 μl / well; manufactured by The Binding Site) as a standard antibody was added to each well of the plate and reacted at room temperature for 2 hours. Excess of the standard antibody was removed and each well was washed 3 times with phosphate buffer containing 0.05% Tween20.
Next, goat anti-human IgG / κ antibody (4,000-fold diluted, 100 μl / well, manufactured by Protos) labeled with peroxidase was added to each well and incubated at room temperature for 1 hour.
The supernatant was discarded, and the microplate was washed three times with a phosphate buffer containing 0.05% Tween 20, and then the substrate buffer (100 μl / well. <Composition>: Ortho-phenylenediamine (O-Phenylenediamine, OPD; 20 mg) / Citrate / phosphate buffer solution (pH 5.0, 50 ml) / 30% hydrogen peroxide solution (15 μl)) was added to each well and incubated at room temperature for about 7 minutes.
Subsequently, 2M sulfuric acid (50 μl / well) was added to each well to stop the reaction. A calibration curve was prepared based on the value obtained by measuring the absorbance at a wavelength of 490 nm with a microplate reader (FIG. 2).
As a control, the assay was performed in the same manner as described above using only the culture solution or only the BSA solution as the 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> Reagent preparation
The reagent used for this cell ELISA was prepared as follows.
<5-2-1-1> Production of recombinant CHO cells highly expressing mouse AILIM
Recombinant CHO cells that highly express mouse AILIM were prepared and obtained in the same manner as in <1-1> and <1-3> above.
After inserting cDNA (GenBank Accession Number: AB023132 (cDNA); BAA82126 (amino acid)) containing the full-length ORF of mouse AILIM into the vector pEF-neo, the recombinant expression vector was obtained using Gene Pulser (BioRad). It was introduced into each of Chinese hamster ovary cells (CHO cells) by electroporation (960 μF, 320 volts) according to a conventional method. Each cell was cultured in RPMI1640 medium containing Geneticin (0.8 mg / ml; manufactured by Gibco BRL) and 10% FCS to select drug-resistant transformed cells, and mouse AILIM high-expressing recombinant CHO cells were obtained.
[0156]
<5-2-1-2> Production of recombinant CHO cells that highly express rat AILIM
Recombinant CHO cells that highly express rat AILIM were prepared and obtained in the same manner as in <1-1> and <1-3> above.
After inserting cDNA (GenBank Accession Number: AB023134 (cDNA); BAA82128 (amino acid)) containing the full-length ORF of rat AILIM into the vector pEF-neo, the recombinant expression vector was converted using Gene Pulser (BioRad). It was introduced into each of Chinese hamster ovary cells (CHO cells) by electroporation (960 μF, 320 volts) according to a conventional method. Each cell was cultured in RPMI1640 medium containing Geneticin (0.8 mg / ml; manufactured by Gibco BRL) and 10% FCS to select drug-resistant transformed cells, and recombinant ACHO cells highly expressing rat AILIM were obtained.
[0157]
<5-2-1-3> Preparation of monoclonal antibody against mouse AILIM
The mouse AILIM-expressing recombinant CHO cells prepared in <5-2-1-1> above are homogenized, ultracentrifuged (100,000 × g), and the centrifugal residue containing the cell membrane fraction is collected and suspended in PBS. Made cloudy. The obtained cell membrane fraction was first immunized (day 0) by injection into a Wistar rat hood pad together with complete Freund's adjuvant. Furthermore, the cell membrane fraction antigen was administered into the footpad at intervals of 7, 7, and 28 days. Lymph node cells were collected 2 days after the last immunization.
The lymph node cells and mouse myeloma cell 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 fusing agent. Monoclonal antibody-producing hybridomas were prepared by cell fusion using these. Hybridomas were selected by culturing in a HAT-containing ASF104 medium (Ajinomoto Co.) containing 10% fetal bovine serum and aminopterin.
[0158]
The reactivity of rat monoclonal antibody produced in the culture supernatant of each hybridoma to mouse AILIM was reacted with each mouse supernatant in the mouse AILIM-expressing CHO cells, and then FITC-labeled anti-rat IgG (manufactured by Cappel). ) And the fluorescence intensity of the cells stained by the reaction was confirmed by measuring with an EPICS-ELITE flow cytometer. As a result, a plurality of hybridomas producing monoclonal antibodies reactive to mouse AILIM were obtained.
One of those hybridomas was named “B10.5”. This hybridoma (10⁶10⁷/0.5 ml / mouse) were injected intraperitoneally into ICR nu / nu mice (female, 7-8 weeks old). Ten to 20 days later, the mouse was opened 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 to AILIM
The concentrations of the anti-human AILIM human monoclonal antibody and control antibody used in the following ELISA were determined based on the ELISA and calibration curve described in <5-1> above.
Recombinant CHO cells highly expressing human AILIM prepared in Example 1, mouse AILIM highly expressing recombinant CHO cells prepared in <5-2-1-1>, and <5-2-1-2> Each of the rat AILIM-expressing recombinant CHO cells prepared in (7 × 10^ThreeCells / well) were seeded in each well of an ELISA 96-well microplate and cultured at 37 ° C. until confluenet.
Next, the supernatant was discarded, and each well was prepared with the various purified anti-human AILIM human monoclonal antibodies or control antibodies prepared above (concentration: 200 μg / ml of the antibody in PBS containing 1% BSA 3 times, 3 times each).²Double, 3^ThreeDouble, 3^FourDouble, 3^FiveDouble, 3⁶Double, 3⁷Double, 3⁸Double, 3⁹Double, 3^TenDouble, 3¹¹Double, and 3¹²Diluted twice. ) Was added at 50 μl / well and reacted at room temperature for 2 hours. Excess monoclonal antibody was discarded and each well was washed 3 times with phosphate buffer containing 1% BSA (Sigma).
Next, an anti-human IgG (Fc) antibody (1,000-fold diluted, 50 μl / well, manufactured by American Quarex) labeled with horseradish peroxidase (Peroxidase) was added to each well and incubated at room temperature for 1 hour.
Excess of the labeled antibody was discarded, and the microplate was washed 3 times with a phosphate buffer containing 1% BSA, and then the substrate buffer (50 μl / well. <Composition>: Ortho-Phenylenediamine, OPD) 20 mg) / citrate / phosphate buffer (pH 5.0, 50 ml) / 30% aqueous hydrogen peroxide (15 μl)) was added to each well and incubated at room temperature for about 7 minutes.
Subsequently, 2M sulfuric acid (50 μl / well) was added to each well to stop the reaction. Absorbance at a wavelength of 490 nm was measured with a microplate reader (Bio-Rad).
[0160]
In addition, ELISA was performed in the same manner as described above using each of the following as the 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 No. WO98 / 38216 (Example 12)).
(2) Rat monoclonal antibody B10.5 against mouse AILIM (described above <5-2-1-3>).
(3) The mouse monoclonal antibody JTT2 against rat AILIM (Biotechnology, Japan Institute of Technology, Ministry of International Trade and Industry, Japan) which is an international depositary organization certified under the Budapest Treaty on October 11, 1996 with the international deposit number FERM BP-5708 Monoclonal antibody produced by a hybridoma deposited internationally at the National Institute of Industrial Science and Technology, Japanese Patent Application Publication No. 11-29599 (Examples 1 and 2) and International Patent Application Publication No. WO98 / 38216 (Examples 1 and 2) ).
(4) A human monoclonal antibody against KLH (keyhole limpet hemocyanin, manufactured by Pierce) prepared above instead of the hybridoma supernatant. As a control test, ELISA was performed in the same manner as described above using each of the following wild-type CHO cells instead of AILIM-expressing recombinant CHO cells.
The results are shown in FIGS.
[0161]
Based on the obtained data, human AILIM (human AILIM high expression recombinant CHO cells), mouse AILIM (mouse AILIM high expression recombinant CHO cells), and rat AILIM (rat AILIM high) of each anti-human AILIM human monoclonal antibody. 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 for human AILIM high expression CHO
AIF 34 (JMab-124): 5.3ng / ml
AIF182 (JMab-126): 3.6ng / ml
AIF348 (JMab-127): 9.1ng / ml
AIF620 (JMab-128): 10.1ng / ml
AIF1052 (JMab-135): 2.0ng / ml
AIH5D3 (JMab-136): 7.5ng / ml
AIH386 (JMab-137): 9.6ng / ml
AII289 (JMab-138): 10.5ng / ml
AII394 (JMab-139): 10.6ng / ml
AII488 (JMab-140): 11.0ng / ml
AIJ 40 (JMab-141): 3.7ng / ml
SA 12: 1.8ng / ml
SG430: 1.2ng / ml
(B) ED50 for mouse AILIM overexpressing CHO
AIF 34 (JMab-124): 42ng / ml
AIF348 (JMab-127): 81ng / ml
AIF620 (JMab-128): 100ng / ml
AII289 (JMab-138): 53ng / ml
AII394 (JMab-139): 60ng / ml
AII488 (JMab-140): 70ng / ml
(C) ED50 for rat AILIM overexpressing CHO
AIF 34 (JMab-124): 45ng / ml
AIF348 (JMab-127): 62ng / ml
AIF620 (JMab-128): 97ng / ml
AII289 (JMab-138): 57ng / ml
AII394 (JMab-139): 90ng / ml
AII488 (JMab-140): 90ng / 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 (above (B) and (C)) have 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)
Binding rate constants (ka), dissociation rate constants (kd) and dissociation constants (Kd) of various purified anti-human AILIM human monoclonal antibodies prepared above with human AILIM were measured using commercially available measurement kit Biacore X (manufactured by Amersham-Pharmacia). ).
[0164]
<6-1> Preparation of antigen for sensor chip fixation
A recombinant chimeric antigen (hereinafter referred to as “human AILIM-IgFc”) comprising an extracellular region of human AILIM and a constant region (Fc) of human IgG1 was prepared as an antigen to be immobilized on the sensor chip of the kit.
The human AILIM-IgFc is another one of the inventors of Tezuka, another earlier application (Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and International Patent Application Publication WO98 / 38216. 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 is applied to a HiTrap affinity column Protein G (Amersham Pharmacia) equilibrated with a phosphate buffer (30 ml) at 3 ml / min. Then, human AILIM-IgFc contained in the culture supernatant was adsorbed onto the column.
[0165]
Next, the column was washed with a phosphate buffer (20 ml), and then 100 mM citrate buffer (pH 2.0) was added to the column at about 1 ml / min to elute human AILIM-IgFc. Subsequently, the eluate was neutralized by adding a solution (pH 9.0) of 750 mM Tris-HCl, and then dialyzed against a phosphate buffer (overnight). Next, the dialyzed solution was filtered through a filter (Millipore) to obtain purified anti-human AILIM-IgFc.
The value at A280 was obtained 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 comprising rat extracellular region of AILIM and human IgG1 constant region (Fc) (rat AILIM-IgFc; Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and international patent application A purified product of published WO 98/38216 (Example 16 (2)) was also prepared in the same manner as above, and as a result, 0.45 mg / ml 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 to the sensor chip were performed according to the instruction manual and the experimental operation method attached to the commercially available measurement kit Biacore X (Amersham-Pharmacia).
Flow Cell-1 attached to the kit contains HBS buffer (containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20, pH 7.0, 5 μl / min). Next, a solution (15 μl) of 0.005M NHS (N-Hydroxysuccinimide) and 0.2M EDC (N-Ethyl-N '-(dimethylaminopropyl) carbodiimide) is added, and CM coated on the sensor chip surface The carboxyl group of was activated.
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 1M Ethanol amine hydrochloride. In the two immobilization operations, the amounts of immobilized human AILIM-IgFc were 2,444 RU (resonance unit) and 2,213 RU, respectively. RU indicates mass per area, 1RU = 1pg / mm²It is.
[0167]
Flow cell 2 (Flow Cell-2) as a reference was capped by processing in the same manner as described above without adding human AILIM-IgFc.
To the flow cell (sensor chip), a phosphate buffer was flowed at a flow rate of 20 μl / min, and each purified anti-human AILIM human monoclonal antibody (10-50 μg / ml, 60 μl) prepared in the above Example was added.
The measurement was performed using a binding phase of 3 minutes and a dissociation phase of 10 minutes as standard conditions. The amount of antibody bound to the antigen and the amount of antibody dissociated from the antigen were measured over time to obtain a sensorgram. The antibody bound to the antigen was dissociated by flowing PBS through the sensor chip at a flow rate of 20 μl / min.
Based on the obtained sensorgram data, using the analysis software (BIAevaluation 3.0) attached to the kit, the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (Kd; Kd = kd / ka) Was calculated.
The antigen affinity and neutralizing activity of mouse monoclonal antibodies SA12 and SG430 against human AILIM prepared in the above example were also analyzed in the same manner as described above.
Each value is shown below.
[0168]
<Clone name> <ka (1 / M.Sec)> <kd [1 / Sec]> <Kd (M)>
AIF 34 (JMab-124) 1.6 × 10^Four            1.0 × 10^-Four        6.3 × 10^-9
AIF182 (JMab-126) 3.2 × 10^Four            2.8 × 10^-Five        8.8 × 10^-Ten
AIF348 (JMab-127) 1.9 × 10^Four            6.4 × 10^-Five        3.4 × 10^-9
AIF620 (JMab-128) 1.1 × 10^Four            1.1 × 10^-Four        1.0 × 10^-8
AIF1052 (JMab-135) 1.6 × 10^Four            6.3 × 10^-Five        3.9 × 10^-9
AIH5D3 (JMab-136) 2.8 × 10^Four            4.9 × 10^-6        1.8 × 10^-Ten
AIH386 (JMab-137) 1.2 × 10^Five            3.1 × 10^-Four        2.6 × 10^-9
AII289 (JMab-138) 3.7 × 10^Four            4.2 × 10^-Five        1.1 × 10^-9
AII394 (JMab-139) 3.1 × 10^Four            2.4 × 10^-Five        7.7 × 10^-Ten
AII488 (JMab-140) 2.3 × 10^Four            3.5 × 10^-Five        1.5 × 10^-9
AIJ 40 (JMab-141) 1.9 × 10^Four            1.9 × 10^-Five        1.0 × 10^-9
SA 12 7.8 × 10^Three            7.9 × 10^-Five        1.0 × 10^-8
SG430 2.2 × 10^Four            1.5 × 10^-Four        6.8 × 10^-9
From these results, it was revealed that any anti-human AILIM human monoclonal antibody and anti-human AILIM mouse monoclonal antibody have extremely high binding affinity and neutralizing activity for human AILIM.
[0169]
Example 7 Costimulatory Signaling Activity of Anti-human AILIM Human Monoclonal Antibody against Human T Cells
Whether or not 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). That is, whether or not it has the ability to control the transmission of a co-stimulatory signal via AILIM into the cell, the cytokines (IFN-γ and IL-4) from human T cells The amount of production and the degree of proliferation of the human T cells were analyzed as indicators.
[0170]
<7-1> Antibody dilution
Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted with a 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 antibodies
In each well of a 96-well microplate (1), either (1) anti-human CD3 monoclonal antibody OKT3 (25 μl / well of 8 μg / ml) or any of various anti-human AILIM human monoclonal antibodies (40 μg / ml to 0.0049 μg) (2) anti-human CD3 monoclonal antibody OKT3 (8 μg / ml was added at 25 μl / well) and incubated at 37 ° C. for 2 hours. The antibody was then discarded and each well washed 3 times with PBS. After washing, 10% FCS-containing RPMI1640 medium (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 antibody of (1) or (2) above.
As a control antibody, instead of the anti-human AILIM human monoclonal antibody, each of the following monoclonal antibodies was used to coat the plate 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 No. WO98 / 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-mentioned example).
These antibody-coated microplates were used in the following tests.
[0172]
<7-3> Preparation of human T cell suspension
Peripheral blood of each healthy person (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). Human T cells were separated and obtained from the human mononuclear cells using a Pan-T cell Isolation Kit (Miltenyi) and a magnetic sorter according to the experimental operation manual. The number of T cells was counted with a hemocytometer. The human T cells are suspended in RPMI1640 medium containing 10% FCS, and the human T cell suspension (1 × 10 6 is added).⁶Cells / ml) was prepared.
[0173]
<7-4> Cell culture
(1) Culture in microplate coated with anti-human CD3 antibody and anti-human AILIM antibody In each well of the antibody-coated microplate, human T cell suspension (donor A, B, C, D and E; 100 μl) / well; 1 × 10^Fivecells / well) and CO₂The cells were cultured at 37 ° C. for 3 days in an incubator.
After the culture, the culture supernatant (50 μl) was collected and stored at −20 ° C. for the purpose of use in the test described later (quantification of IFNγ). Each microplate after sampling of the culture supernatant was used for the following tests.
(2) Culture on a microplate coated only with anti-human CD3 antibody
In each well of the antibody-coated microplate, a human T cell suspension (donor D; 100 μl / well; 1 × 10 6^Fivecells / well), and then add one of various anti-human AILIM human monoclonal antibodies (40 μg / ml to 0.0049 μg / ml in 25 μl), and add CO₂The 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 [^ThreeH] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia)₂Incubated for 6 hours at 37 ° C. in an incubator. After the culture, the cells were trapped on a GF / C filter (manufactured by Packard) using a cell harvester. Next, after the filter was dried at 40 ° C. for 3 hours or more, Microscinti 0 (20 μl / well; manufactured by Packard) was added. It is taken up by the cells trapped on the filter using the β counter (TOP COUNT)^ThreeThe 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 anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) and anti-human CD3 antibody was used, significant proliferation-dependent growth of human T cells was observed. It was. In addition, the degree of cell proliferation was somewhat different between donors.
On the other hand, when using a plate coated only with anti-human CD3 antibody and culturing anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) in the state of solution (liquid phase) in the cell culture system, 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 of the T cell culture systems (donors B and C) of <7-4>, the amount of IFNγ contained in the culture supernatant was determined using a commercially available human IFNγ ELISA KIT (manufactured by Amersham Pharmacia; Endogen ).
The results are shown in FIGS.
As a result of this test, a significant increase in IFNγ production depending on the concentration of anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) was observed.
[0176]
Example 8 Control activity of mixed lymphocyte reaction (MLR) of anti-human AILIM human monoclonal antibody
Whether or not the anti-human AILIM human monoclonal antibody of the present invention has the ability to control (promote and / or suppress) T cell responses (production of cytokines such as IFN-γ and IL-4, and cell proliferation); In other words, whether or not it has the ability to control the transmission of co-stimulatory signals via AILIM into cells is determined by T in allogenic mixed lymphocyte reaction (allogenic MLR). The presence or absence of an activity that controls cell proliferation (ie, intracellular DNA synthesis) was analyzed as an indicator.
[0177]
<8-1> Preparation of human PBMC and T cells
Lymphoprep (15 ml) dispensed each peripheral blood (200 ml) collected from each of healthy individuals (7 people; donors A, B, C, D, E, F and G) into microtubes (50 ml; manufactured by Falcon) Layered on Nycomed). Subsequently, after centrifugation (1600 rpm, 10 minutes), the intermediate layer was recovered. The collected cells are diluted 2 times or more with a phosphate buffer, and then centrifuged (1,800 rpm, 10 minutes) to obtain PBMC (peripheral blood mononuclear cells; 2 × 10⁸~ 5 × 10⁸Cells). Count the number of cells using a hemocytometer and count the number of cells required for the MLR test (1.08 x 10⁸Cells / 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, the 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 allowed to react. The cells were then washed with PBS containing 5 mM EDTA and 0.5% BSA and then resuspended in the PBS. Next, the cell suspension was added to the Positive Selection Column VS + (Miltenyi Biotech) swollen with the PBS, and the non-adsorbed fraction was collected. In addition, the PBS was added to the column to recover the washing solution. The same operation was performed again. The collected solution was combined to obtain a T cell fraction. The T cell fraction was centrifuged and then resuspended in the PBS. The number of obtained T cells was counted using a hemocytometer and used for the following tests.
[0178]
<8-2> Mixed lymphocyte reaction (MLR)
As mentioned earlier, CD28 and CD80 (B7-1) / CD86, which have already been comparatively well analyzed for costimulatory signal transmission pathways necessary for the activation of lymphocytes such as T cells. 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 study, using the following test substances, (1) suppression of MLR by blocking the signal transduction pathway via CTLA4, (2) signal transduction via CD80 (B7-1) / CD86 (B7-2) Suppression of MLR by blocking the pathway, (3) Suppression of MLR by blocking both the pathway through CTLA4 and the signaling pathway through CD80 (B7-1) / CD86 (B7-2), (4) AILIM MLR suppression by blocking 3 signaling pathways and (5) MLR suppression by blocking both CTLA4-mediated pathway and AILIM-mediated pathway were analyzed.
[0179]
The following were used as test substances.
(1) Anti-human AILIM human monoclonal antibody (prepared in the above examples).
(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 previous 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 the PBMC and T cells prepared from each donor obtained in <8-1>, a mixed lymphocyte reaction (MLR) was performed by the following 6 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 for the test were adjusted to the following concentrations.
After PBMC was dispersed in PBS, the culture dish (60 mm) was transferred, and X-ray irradiation (50 Gy) was performed with a radiation irradiation apparatus (manufactured by Hitachi Medical Corporation). After collecting and centrifuging the cells, the cells were added to RPMI1640 medium containing 10% FCS, and the number of cells was adjusted to 2 × 10.^FiveCells / 50 μl were adjusted.
In addition, T cells from each donor obtained above were added to RPMI1640 medium containing 10% FCS, and the number of cells was changed to 1 × 10 6.^FiveCells / 50 μl were adjusted.
[0181]
<8-2-1> Suppression of MLR by anti-human AILIM human monoclonal antibody against MLR
A solution of anti-human AILIM human monoclonal antibody or anti-human AILIM mouse monoclonal antibody SA12 diluted in various concentrations with RPMI1640 medium containing 10% FCS after adding PRMI1640 medium containing 10% FCS to each well of a 96-well U-bottom microplate (Final concentrations: 0, 0.31, 1.25, 5 and 20 μg / ml). T cells (50 μl) are then added and CO₂The cells were cultured for 1 hour at 37 ° C. in an incubator (manufactured by NAPCO). After the reaction was completed, PLR (50 μl) from another donor was added to start MLR.
In addition, MLR when using other than anti-human AILIM human monoclonal antibody as the test substance (above (3) to (6)), add T cells from another donor after culturing PBMC and the test substance. The reaction was performed.
On the fifth day of the culture, each well was diluted with tritiated thymidine (RPMI1640 medium containing 10% FCS).^Three(H-Thymidine; 20 μl; 1 μCi / well), and then further cultured for 1 day. After culturing, the cells are collected using Cell Harvester (manufactured by Packard) and taken up into the cells using a β counter (TOP COUNT; manufactured by Packard).^ThreeThe radioactivity of H was measured, and the degree of proliferation of T cells after culture was analyzed.
The results are shown in FIGS. 48 to 59.
[0182]
<8-2-2> Inhibition of MLR by anti-human AILIM human monoclonal antibody against MLR in a system that previously blocked CTLA4-mediated signal transduction
Add PRMI1640 medium containing 10% FCS to each well of a 96-well U-bottom microplate, and then add anti-human AILIM human monoclonal antibody or anti-human AILIM mouse monoclonal antibody SA12 diluted to various concentrations in RPMI1640 medium containing 10% FCS. (Final concentrations: 0, 0.31, 1.25, 5 and 20 μg / ml). T cells (50 μl) are then added and CO₂The cells were cultured for 1 hour at 37 ° C. in an incubator (manufactured by NAPCO).
Separately from the T cell culture, human CTLA4-IgFc was added to PBMC (in RPMI1640 medium containing 10% FCS) derived from another donor, and CO 2₂The cells were cultured for 1 hour at 37 ° C. in an incubator (manufactured by NAPCO). The CTLA4-IgFc concentration 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 initiate MLR. The MLR when a test substance other than an anti-human AILIM human monoclonal antibody (above (3) to (5)) is used is the same as described above. PLR obtained by culturing with CTLA4-IgFc 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 was diluted with tritiated thymidine (RPMI1640 medium containing 10% FCS).^Three(H-Thymidine; 20 μl; 1 μCi / well), and then further cultured for 1 day. After culturing, the cells are collected using Cell Harvester (manufactured by Packard) and taken up into the cells using a β counter (TOP COUNT; manufactured by Packard).^ThreeThe 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 T cell proliferation by allogenic MLR by blocking signal transduction via CTLA-4.
(2) The anti-CD80 antibody and the anti-CD86 antibody suppress the proliferation of T cells by allogenic MLR by inhibiting the signal transduction through CD80 / CD86, which is a ligand of CTLA4 and CD28.
(3) Similar to CTLA4-IgFc, anti-CD80 antibody and anti-CD86 antibody, monoclonal antibody against human AILIM significantly suppresses T cell proliferation in allogenic MLR due to signal transduction via AILIM in an antibody concentration-dependent manner .
This result indicates that, in addition to the known CTLA4 / CD80 / CD86 and CD28 / CD80 / CD86 pathways, AILIM signaling pathways for costimulatory signals required for T cell activation And the presence of a third pathway through its ligand, and the signal transduction pathway through AILIM is inhibited by an antibody against AILIM.
Furthermore, the contribution of the pathway through AILIM in the signal transduction could be comparable to that of the pathway through CTLA4 / CD80 / CD86 and the pathway through CD28 / CD80 / CD86.
[0184]
Example 9 Induction activity of antibody-dependent cellular cytotoxicity (ADCC) of anti-human AILIM human monoclonal antibody
One of the biological activities caused by antibodies is induction of antibody-dependent cellular cytotoxicity (ADCC). ADCC is a cytotoxic effect of a target cell 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 in order for the damage to be induced.
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 human AILIM high-expression recombinant HPB-ALL cell culture system prepared in the above Example,⁵¹Cr (0.1mCi / 10⁶Cells; Amersham-Pharmacia) were added, incubated at 37 ° C. for 2 hours, and then washed with RPMI1640 medium 8 times, and radiolabeled cells obtained as target cells were used as target cells.
For use in the control test, wild-type human HPB-ALL cells were radiolabeled in the same manner as described above.
The PBMC fraction was separated from peripheral blood collected from healthy individuals using Lymphosepar I (IBL). The obtained human PBMC was used as an effector cell.
To a 96-well U-bottom microplate (Nunc), the target cells (1 × 10 6^Fourcells / well; 25 μl / well). Then, each well was mixed with various concentrations of anti-human AILIM human monoclonal antibody (0.0001-1.0 μg / ml; 25 μl / well) diluted with RPMI1640 medium containing 5% FBS, the medium alone (25 μl / well) or 1% Nonidet P -40 (25 μl / well; surfactant having cell membrane lytic activity) was added and incubated at room temperature for 20 minutes.
As a positive control, the anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was used instead of the anti-human AILIM antibody and cultured in the same manner as described above.
[0186]
Then, in each well, effect cells (E / T ratio = 50; 1 × 10 6^Fivecells / well; 50 μl / well), 5% CO₂The cells were cultured at 37 ° C. for 16 hours in an incubator.
After culturing, the supernatant was collected by centrifugation (1,500 rpm; 4 ° C .; 10 minutes). Radioactivity in the supernatant was measured using a gamma counter. The radioactivity was released into the culture supernatant by ADCC cell membrane damage.⁵¹Indicates the amount of Cr.
ADCC induced by anti-AILIM antibody or anti-CD3 antibody, with radioactivity in the medium-only assay set to zero (0) cell membrane damage rate, and with nonidet addition, the company activity in the assay with 100% cell membrane damage rate 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 Gene sequence and amino acid sequence of anti-human AILIM human monoclonal antibody
Column determination and analysis
The heavy chain (Heavy Chain) -encoding cDNA sequence and the light chain (Light Chain) -encoding cDNA sequence constituting the human monoclonal antibodies against various human AILIMs prepared in the above examples are determined as follows. The structural features of the gene were analyzed.
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 example, Quick Prep mRNA Purification Kit kit PolyA using (Amersham Pharmacia)⁺RNA was extracted and purified.
The hybridoma was dissolved in a cell lysis buffer (Lysis Buffer), and the cells were disrupted by a syringe and solubilized. Oligo (dT) resin was added to the solubilized product and gently shaken. Next, after washing Oligo (dT) resin, PolyA⁺RNA was eluted with Ellution Buffer. Eluted PolyA⁺RNA was ethanol precipitated and dissolved in Tris-EDTA buffer. Obtained PolyA⁺The concentration of RNA was determined by measuring the absorbance at a wavelength of 260 nm.
[0188]
Obtained PolyA⁺Double-stranded cDNA was synthesized by M-MLV Reverse Transcriptase method using RNA as a template and commercially available cDNA synthesis kit (GIBCOBRL) and synthetic oligo DNA NotI-T (SEQ ID NO: 1) as primers.
That is, PolyA purified from hybridoma⁺Using RNA (about 5 μg) as a template, single-stranded cDNA was synthesized by reacting 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 solution and distilled water were added to the reaction solution (4 μl) and reacted at 16 ° C. for 2 hours to synthesize double-stranded cDNA. The obtained double-stranded cDNA was extracted with phenol / chloroform and then ethanol precipitated.
Next, EcoRI linker DNA (about 300 pmole) and DNA ligase (Ligation High; 33 μl; manufactured by TOYOBO) are added to the double-stranded cDNA (about 50 μl) dissolved in TE buffer, and reacted at 16 ° C. for about 80 minutes. The linker DNA was ligated to the cDNA. As the linker DNA, double-stranded DNA prepared by hybridizing oligo DNA (20adp; SEQ ID NO: 2) phosphorylated at the 5 ′ end and oligo DNA (24adp; SEQ ID NO: 3) according to a conventional method was used. .
[0189]
The resulting reaction product was extracted with phenol / chloroform and ethanol precipitated. 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 (GIBCO BRL) and T4 kinase (manufactured by TOYOBO) (at 37 ° C. for 30 minutes). Its 5 ′ end was phosphorylated.
The obtained DNA reaction product was precipitated with ethanol and fractionated by polyacrylamide gel electrophoresis to cut out a gel containing about 500 bp to 2000 bp DNA. The gel was cut out by ethidium bromide staining and photography under ultraviolet irradiation.
The excised gel was pulverized, suspended in TE buffer, and centrifuged to collect the centrifugal supernatant.
The recovered DNA and a commercially available lambda phage vector λEXcell (0.25 μg; manufactured by Amersham Pharmacia) were reacted (16 ° C., 30 minutes) in the presence of a commercially available DNA Ligase (Ligation High; manufactured by TOYOBO). Next, the DNA reaction product was converted into lambda phage using a commercially available lambda phage packaging kit Gigapack III Gold (manufactured by STRATAGENE), and a cDNA library was prepared using Escherichia coli NM522 as a host. All operations were performed according to the experimental operation manual attached to the kit.
[0190]
Subsequently, the cDNA library was screened as follows according to the plaque hybridization method (Maniatis et al., Molecular Cloning: A Labolatory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
The cDNA library (1 × 10^FourEach plaque was spread on an agar plate, and a replica was prepared using Hybond-N nylon membrane (manufactured by Amersham Pharmacia). This replica³²Screening was performed by plaque hybridization in hybridization buffer using a probe labeled with P-ATP. The probe HIGLC (SEQ ID NO: 4) was used for the antibody light chain, and the probe NHCc2 (SEQ ID NO: 5) was used for the antibody heavy chain. Each positive clone identified by primary and secondary screening was isolated in a single plaque.
[0191]
Each antibody heavy chain and antibody light chain were amplified by PCR using a phage solution of each positive clone as template DNA and one PCR primer and Taq PCR kit (manufactured by TAKARA). ExcellE (SEQ ID NO: 6) and ck117 (SEQ ID NO: 7) were used as the antibody light chain primers, and ExcellE (SEQ ID NO: 6) and NHCc2 (SEQ ID NO: 5) were used as the antibody heavy chain primers. The PCR product was fractionated by agarose gel electrophoresis by a conventional method, and a gel containing about 600 bp in size of DNA was cut out for both heavy and light chains. Analysis of DNA sequence purified from the gel using DNA Sequencer (373A; manufactured by PE-Applied Biosystems), ABI PRISM Sequencing Software (manufactured by PE-Applied Biosystems) and ABI PRISM Auto Assembler (manufactured by PE-Applied Biosystems) Then, it was confirmed that each positive clone had DNA having a sufficient base length.
[0192]
Plaque λ phage of each positive clone was infected with E. coli NP66 to release plasmid DNA, and then seeded on a plate containing ampicillin to form colonies. Subsequently, E. coli JM109 was transformed with the purified plasmid DNA collected from the colony according to a conventional method. Subsequently, the transformed cells were spread on a nutrient medium containing ampicillin to form colonies.
Then, the suspension of each colony suspended in LB medium containing ampicillin was cultured in liquid nutrient medium at 37 ° C. for 24 hours. Bacteria were collected from the culture solution and plasmid DNA was purified using a plasmid purification kit (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.
According to a conventional method, the nucleotide sequence of cDNA encoding each of the antibody heavy chain or antibody light chain inserted in each purified plasmid is converted into DNA Sequencer (377A; manufactured by PE-Applied Biosystems), ABI PRISM Sequencing Software (PE- Applied Biosystems) and ABI PRISM Auto Assembler (PE-Applied Biosystems).
In this sequencing, the following primers were used.
[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 ( Sequence number 5), HCc7 (sequence number 13), HCc8 (sequence number 14), HCc3 (sequence number 15), HCc4 (sequence number 16), HCc6 (sequence number 17), HIGHC (sequence number 18), HCc9 (sequence) 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 hybridoma, the cDNA sequence encoding the light chain, and the amino acid sequence deduced from each cDNA sequence are as follows: As 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 (including signal sequence: amino acid numbers 1 to 19, variable region: amino acid numbers 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 (including signal sequence: amino acid numbers 1 to 22, variable region: amino acid numbers 23 to 116)
[0195]
Clone AII289 (JMab-138)
<Heavy chain>
DNA sequence: SEQ ID NO: 31 (including signal sequence: base numbers 94 to 150, V region: base numbers 151 to 441)
Amino acid sequence: SEQ ID NO: 32 (including signal sequence: amino acid numbers 1 to 19, variable region: amino acid numbers 20 to 116)
<Light chain>
DNA sequence: SEQ ID NO: 33 (including signal sequence: base numbers 28 to 87, V region: base numbers 88 to 375)
Amino acid sequence: SEQ ID NO: 34 (signal sequence: including amino acid numbers 1 to 20, variable region: including amino acid numbers 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 (including signal sequence: amino acid numbers 1 to 19, variable region: amino acid numbers 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 (including signal sequence: amino acid numbers 1 to 20, variable region: amino acid numbers 21 to 116)
[0197]
Based on each determined DNA sequence, a library of human immunoglobulin variable region genes V BASE Sequence (Immunol. Today, Vol. 16, No. 16) prepared by Tomlinson et al. Using gene sequence analysis software. 5, p.237-242, 1995).
As a result, the V region genes of the heavy chain and light chain of the human monoclonal antibody were each 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 Delayed-type hypersensitivity of anti-human AILIM human monoclonal antibody
hypersensitivity (DTH)
Living body immune response systems that try to eliminate antigens harmful to the body (such as pathogenic microorganisms such as viruses, bacteria, and parasites and foreign substances) are roughly divided into innate immune responses and acquired immune responses (acquired immunity). .
The former is a mechanism of exclusion 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 an elimination mechanism by lymphocytes (mainly T cells and B cells) that have acquired (activated) specificity for the antigen.
Acquired immune responses are further divided into cellular immunity and humoral immunity.
Unlike antibody-dependent humoral immunity, cellular immunity is an immune reaction that is expressed mainly by acting on antigens to which T cells are directly targeted. 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]
Tuberculin allergy (substantially synonymous with tuberculin hypersensitivity) is well known as the most typical phenomenon of this cellular immunity. Tuberculin allergy is a delayed-type allergy that is caused by infection with M. tuberculosis, and is induced by injecting a tuberculin protein produced in the culture of M. tuberculosis into the skin of the body to induce an immune response. .
Delayed type allergy is an allergy mediated by T cells sensitized by an antigen (memory T cells storing the antigen), and the memory T that occurs when an organism sensitized to the antigen re-contacts the antigen. Since it takes 24 to 48 hours to develop an allergic reaction accompanied by inflammation by cells, it is called a delayed type.
The phenomenon of tuberculin allergy, which is a representative example of delayed type allergy, is 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) 0.1 ml)) is administered intradermally to the 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 M. tuberculosis infection is diagnosed based on the major axis. Redness of major axis up to 4mm is negative, 4-9mm is false positive, and 10mm or more is positive.
[0201]
Delayed type allergy caused by cellular immunity includes allergy to infectious agent antigens such as tuberculosis-derived tuberculin allergy as described above, as well as Jones-Mote type delay that appears transiently for trace amounts of proteins. Type allergies, contact allergies to drugs such as picryl chloride and phytotoxins such as lacquer, or allergies related to transplant rejection seen in transplantation of allografts.
[0202]
In this test, the above-described tuberculin test was applied to evaluate the effect of anti-AILIM antibody on delayed type hypersensitivity (delayed type allergy). The test was conducted as follows.
Each of the cynomolgus monkeys (male, body weight: 6.0 to 8.5 kg, manufactured by Environmental Billis Research Institute) in which immunization with BCG (Bacille de Calmette et Guerin), a live attenuated strain of M. bovine, has been established 3) were anesthetized with ketamine hydrochloride (10 mg / kg, intramuscular administration), and then any of the following test samples were administered into the thoracic epidermis at a concentration of 0.1 ml / 1 administration site (6 administration sites / animal) did. In addition, the interval between each site to which the sample was administered 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 / saline 1 ml).
(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 / saline 1 ml).
(3) Phosphate buffer (PBS (−)) as a control.
(4) A 1: 1 mixed solution of commercially available steroidal anti-inflammatory agent prednisolone (Prednisolone; 0.2 mg; 10 μg / 1 administration site) and tuberculin (4 μg / saline 1 ml) as a positive control.
(5) A 1: 1 mixed solution of anti-KLH human monoclonal antibody (Example <2-2>; 0.2 mg; 10 μg / 1 administration site) and tuberculin (4 μg / saline 1 ml) as a negative control. .
24 hours after administration of each sample, the major axis and minor axis of redness at each administration site were measured, and the area of redness was calculated.
The results are shown in FIG.
As a result, in any group administered with the anti-AILIM antibody, redness due to delayed allergy was significantly suppressed as compared with the control and negative control. Moreover, the inhibitory effect was comparable with the steroidal anti-inflammatory agent which is a positive control.
[0204]
Example 12 Analysis of AILIM expression in each tissue of a patient with graft-versus-host disease (GVHD)
Expression of AILIM in various biopsy tissues taken from recipient patients who were clinically diagnosed with acute versus chronic graft versus host disease (GVHD) after receiving an allogeneic transplant from a donor The presence or absence of this was analyzed by tissue staining by the HE staining method using the anti-human AILIM human monoclonal antibody (JMab-36) prepared in the above Example according to a conventional method.
We analyzed 33 samples collected from various tissues of acute GVHD patients (28 cases) and 5 samples from chronic GVHD patients (5 cases).
As a result, for acute GVHD patients, 15 out of 29 samples in skin tissue were AILIM positive, 1 out of 3 samples in stomach tissue were AILIM positive, and 1 out of 1 sample in colon tissue was AILIM positive. In chronic GVHD patients, 1 of 3 specimens were AILIM positive in skin tissue, and 2 of 2 specimens were AILIM positive in colon tissue. Furthermore, 10 specimens out of 13 specimens with marked lymphocyte infiltration were positive for AILIM.
[0205]
Example 13 Costimulatory signaling activity of monkey T cells of anti-human AILIM human monoclonal antibody
In Example 7, the anti-human AILIM human monoclonal antibody of the present invention controls human T cell reaction, specifically, by controlling the transmission of costimulatory signals into cells via AILIM. It was demonstrated to promote proliferation of T cells. Therefore, in this test, the presence or absence of the ability of the human monoclonal antibody to promote cell proliferation 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 (BD-Pharmingen) was diluted with a 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> Microplate coating with antibodies
In each well of a 96-well microplate, anti-human CD3 monoclonal antibody SP34 (1 μg / ml in 25 μl / well) and anti-human AILIM human monoclonal antibody JMAb136 (40 μg / ml to 0.064 μg / ml in 25 μl), 37 Incubated for 2 hours at ° C. The antibody was then discarded and each well washed 3 times with PBS. After washing, 10% FCS-containing RPMI1640 medium (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.
As a control antibody, an anti-KLH human monoclonal antibody (also referred to as JMab23, also referred to as Example) was used instead of the anti-human AILIM human monoclonal antibody, and the plate was coated 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
Peripheral blood of cynomolgus monkey was collected, and mononuclear cells were fractionated by density gradient centrifugation using NycoPrep1.077A (manufactured by Nycomed). According to the experimental operation manual, using the anti-human CD4 antibody M-T477 (BD-Pharmingen), anti-human CD8 antibody RPA-T8 (BD-Pharmingen), anti-mouse IgG microbeads (Miltenyi) and a magnetic sorter, Monkey T cells were isolated from cynomolgus monocytes. The number of T cells was counted with a hemocytometer. The monkey T cells were suspended in RPMI1640 medium containing 10% FCS, and monkey T cell suspension (1 × 10 6⁶Cells / ml) was prepared.
[0209]
<13-4> Cell culture
(1) Culture on microplate coated with anti-human CD3 antibody and anti-human AILIM antibody In each well of the antibody-coated microplate, monkey T cell suspension (100 μl / well; 1 × 10 6^Fivecells / well) and CO₂The cells were cultured for 2 days at 37 ° C. in an incubator.
After incubation, 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 [^ThreeH] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia)₂Incubated for 6 hours at 37 ° C. in an incubator. After the culture, the cells were trapped on a GF / C filter (manufactured by Packard) using a cell harvester. Next, after the filter was dried at 40 ° C. for 3 hours or more, Microscinti 0 (20 μl / well; manufactured by Packard) was added. It is taken up by the cells trapped on the filter using the β counter (TOP COUNT)^ThreeThe 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 study, when a microplate coated with anti-human AILIM monoclonal antibody (human monoclonal antibody or mouse monoclonal antibody) together with anti-human CD3 antibody was used, significant proliferation-dependent monkey T cell proliferation was observed. It 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 substances that bind to AILIM or AILIM ligand
An ELISA (Enzyme-linked Immunosolvent Assay) that can identify or quantify substances that bind to AILIM (ICOS) or AILIM ligands (B7h / B7RP1 / GL50 / LICOS) was constructed.
The method described in detail below as an example is based on the principle of evaluating by ELISA the extent to which the substance inhibits the binding between soluble human AILIM (hAILIM-IgFc) and soluble human AILIM ligand (hB7h-IgFc).
[0212]
<14-1> Sample
The following samples were used.
(1) Streptavidin-HRP (manufactured by Southern Biotechnology Associates, Inc.).
(2) Soluble human AILIM ligand (a fusion protein consisting of the extracellular region of human B7h and the constant region of human IgG1)
It was fabricated as in the next item <14-2>.
(3) Biotin-labeled soluble AILIM-IgFc
Another earlier application of Tezuka, one of the present inventors (Japanese Patent Application Publication No. 11-29599 (Example 16 (2)) and International Patent Application Publication No. WO98 / 38216 (Example 16 (2)) ) Was prepared by further purification.
(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 solution (PBS (-); manufactured by Nikken Bio).
(7) PRMI1640 medium (manufactured by Nikken Bio).
(8) Fetal bovine serum (FCS; manufactured by JRH-Bioscience).
(9) 30% bovine serum albumin (BSA; manufactured by Sigma).
(10) Tween20 (manufactured by BioRad).
(11) TMB⁺ substrate chromogen (Dako).
[0213]
<14-2> Preparation of soluble human AILIM ligand (fusion protein of human B7h extracellular region and human IgG1 constant region (hB7h-IgFc))
Total RNA was prepared from mononuclear cells derived from human peripheral blood in the same manner as in the previous examples.
Using the prepared total RNA (5 μg) as a template, cDNA was synthesized using Superscript Preamplification System for First Strand cDNA Sythesis (GIBCO-BRL). Next, in order to amplify the cDNA encoding the extracellular region of human AILIM ligand (hB7h) by PCR, a 5 ′ primer (5′-GAGGTCTCCGCCCTCGAGATGCGGCTGGGCAGTCC-3 ′, SEQ ID NO: 39) having an XhoI cleavage site at the end and BamHI cleavage A 3 ′ primer having a site (5′-CACAGGACAGCCAGGGGATCCCACGTGGCCGCG-3 ′, SEQ ID NO: 40) was designed and synthesized. PCR was performed using the obtained cDNA as a template and the primers, and cDNA containing cDNA encoding the extracellular region of human B7h having XhoI and BamHI cleavage sites at both ends was prepared. 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 predicted to be a cDNA fragment encoding the target extracellular region was isolated. The isolated cDNA fragment was subcloned into plasmid pBluescript II SK (+) (Stratagene) cut with XhoI and BamHI. By sequence analysis using an automatic fluorescent DNA sequencer (Applied Biosystems), it was confirmed that the cDNA fragment contained a region encoding the extracellular region of human B7h.
[0214]
On the other hand, the DNA encoding the Fc of human IgG1 as a fusion partner is described in Plasmid (see Cell, Vol.61, p.1303-1313, 1990. Dr. B. Seed et al., Massachusetts General Hospital). Was cut out as a BamHI-XbaI DNA fragment (about 1.3 kb) by digesting with BamHI and XbaI. This fragment contains exons encoding each of the hinge region of human IgG1, Cγ12, and Cγ13.
An XhoI-BamHI fragment encoding the extracellular region of human B7h (hB7h) prepared as described above, and a BamHI-XbaI fragment containing an exon encoding Fc of human IgG1 (abbreviated as “IgFc”), And subcloned into the plasmid pBluescript II SK (+) (Stratagene) cut with XbaI.
Subsequently, the plasmid was digested with XhoI and XbaI, and an about 1.8 kb DNA fragment containing a fusion DNA composed of the extracellular region of human B7h and human IgFc was cut out. This fusion DNA fragment was transformed into expression vector pME18S (Medical Immunology, Vol. 20, No. 1, p. 27-32, 1990, and experimental medicine (separate volume): “Gene Engineering Handbook”, sheep using T4 DNA ligase. The plasmid phB7h-IgFc was constructed by inserting it into the XhoI and XbaI sites of Sodosha, pp. 101-107 (1992).
[0215]
By electroporation, COS7 cells (ATCC CRL-1651) monolayer cultured in DMEM medium containing 10% fetal bovine serum and ampicillin were transformed with 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 as follows using a Protein G Sepharose affinity column (manufactured by Amersham Pharmacia).
Centrifugal supernatant obtained by centrifuging the culture supernatant was added to a Protein G Sepharose affinity column previously equilibrated with a binding buffer. The column was then washed with binding buffer and then eluted with an elution buffer. The eluate was collected and dialyzed by exchanging the external solution twice or more with phosphate buffer to obtain purified hB7h-IgFc.
[0216]
<14-3> dilution of antibody and soluble human AILIM (hAILIM-IgFc), and their reactive anti-human AILIM monoclonal antibody (JMab-136) and negative control antibody anti-KLH human monoclonal antibody (JMab-23) After diluting the 20 μg / ml solution into 11 stages, 200 μl of each sample was mixed with 10% FCS-containing RPMI1640 medium (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 of various concentrations, mixed, and incubated at room temperature for 30 minutes.
[0217]
<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 3 times with PBS (120 μl). Subsequently, PBS containing 0.5% BSA (100 μl / well) was added to each well to block the unreacted portion, the plate was sealed, and incubated at 37 ° C. for 1 hour. After the reaction, the solution was removed and washed 3 times with PBS (120 μl). Next, each sample (50 μl / well) prepared in <14-3> was added, the plate was sealed, and incubated at 37 ° C. for 1 hour. The solution was removed from each well and washed 3 times with RPMI 1640 medium (120 μl) containing 10% FCS warmed to 37 ° C. Subsequently, PBS containing 3.7% formalin (100 μl / well) was added to each well and incubated on ice for 5 minutes. After removing the solution from each well, it was washed 3 times with 0.1% Tween20 (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 3 times with PBS containing 0.1% Tween20 (120 μl). Then TMB⁺Substrate chromogen (50 μl / well) was added and incubated at room temperature for 20 minutes. Subsequently, 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. 74 to 76.
As a result, the dose-dependent inhibitory activity of hAILIM-IgFc and hB7h-IgFc binding by anti-AILIM antibody was measured.
Therefore, it was shown that substances (for example, antibodies or synthetic low molecular weight compounds) that bind to AILIM or AILIM ligand can be screened and identified by using an assay system as exemplified in this 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
Whether the anti-human AILIM monoclonal antibody of the present invention has the ability to control signal transduction via AILIM on human T cells is determined by contacting human T cells with AILIM ligand (B7h / B7RP1 / GL50 / LICOS). Analysis was performed by measuring the inhibitory effect of the anti-human AILIM human monoclonal antibody on the induced cell proliferation.
[0219]
<15-1> Antibody dilution
Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) was diluted with a 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> Microplate coating with antibodies
Add (1) anti-human CD3 monoclonal antibody OKT3 (8 μg / ml in 25 μl / well) and hB7h-IgFc (40 μg / ml to 0.0064 μg / ml in 25 μl) to each well of a 96-well microplate (multiple plates), Incubated for 2 hours at 37 ° C. The antibody was then discarded and each well washed 3 times with PBS. After washing, 10% FCS-containing RPMI1640 medium (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 healthy persons was collected, and mononuclear cells were fractionated by density gradient centrifugation using LymphoPrep (Nycomed). Human T cells were separated and obtained from the human mononuclear cells using a Pan-T cell Isolation Kit (Miltenyi) and a magnetic sorter according to the experimental operation manual. The number of T cells was counted with a hemocytometer. The human T cells were suspended in 10% FCS-containing RPMI1640 medium supplemented with anti-human AILIM human monoclonal antibody JMab136 (20 μg / ml) and human T cell suspension (1 × 10 6⁶Cells / ml) was prepared and incubated for 30 minutes at room temperature.
As a negative control antibody, anti-KLH human monoclonal antibody JMAb23 (20 μg / ml) was used.
[0222]
<15-4> Cell culture
In each well of the microplate coated with anti-human CD3 antibody and hB7h-IgFc as described above, human T cell suspension (100 μl / well; 1 × 10 6^Fivecells / well) and CO₂The 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 [^ThreeH] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia)₂Incubated for 6 hours at 37 ° C. in an incubator. After the culture, the cells were trapped on a GF / C filter (manufactured by Packard) using a cell harvester. Next, after the filter was dried at 40 ° C. for 3 hours or more, Microscinti 0 (20 μl / well; manufactured by Packard) was added. It is taken up by the cells trapped on the filter using the β counter (TOP COUNT)^ThreeThe 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 dependent on human B7h-IgFc concentration 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 monkey T cell proliferation by costimulatory signaling through AILIM-AILIM ligand
The test of Example 15 was conducted in the same manner using monkey T cells instead of human T cells.
[0225]
<16-1> Dilution of antibodies, etc.
Anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) was diluted with a 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 antibodies
In each well of a 96-well microplate (multiple plates), (1) anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) (1 μg / ml is 25 μl / well) and human B7h-IgFc (40 μg / ml to 0.0064 μg / well) 25 μl of ml) was added and incubated at 37 ° C. for 2 hours. The antibody was then discarded and each well washed 3 times with PBS. After washing, 10% FCS-containing RPMI1640 medium (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
Peripheral blood of cynomolgus monkey was collected, and mononuclear cells were fractionated by density gradient centrifugation using NycoPrep1.077A (manufactured by Nycomed). According to the experimental operation manual, using the anti-human CD4 antibody M-T477 (BD-Pharmingen), anti-human CD8 antibody RPA-T8 (BD-Pharmingen), anti-mouse IgG microbeads (Miltenyi) and a magnetic sorter, Monkey T cells were isolated from cynomolgus monocytes. The number of T cells was counted with a hemocytometer. The monkey T cells were suspended in 10% FCS-containing RPMI1640 medium supplemented with anti-human AIlIM human monoclonal antibody JMab-136 (20 μg / ml) and monkey T cell suspension (1 × 10 6⁶Cells / ml) was prepared and incubated for 30 minutes at room temperature.
As a negative control antibody, anti-KLH human monoclonal antibody JMab-23 (20 μg / ml) was used.
[0228]
<16-4> Cell culture
In each well of the microplate coated with anti-human CD3 antibody and hB7h-IgFc as described above, monkey T cell suspension (100 μl / well; 1 × 10 6^Fivecells / well) and CO₂The 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 [^ThreeH] thymidine (0.5 μCi / well; manufactured by Amersham Pharmacia)₂Incubated for 6 hours at 37 ° C. in an incubator. After the culture, the cells were trapped on a GF / C filter (manufactured by Packard) using a cell harvester. Next, after the filter was dried at 40 ° C. for 3 hours or more, Microscinti 0 (20 μl / well; manufactured by Packard) was added. It is taken up by the cells trapped on the filter using the β counter (TOP COUNT)^ThreeThe 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 human B7h-IgFc concentration 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】
Since the human monoclonal antibody that binds to human AILIM of the present invention 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 a mouse-derived antibody. Since it does not cause any serious immune rejection of the host due to antigenicity against humans, that is, HAMA (Human anti-mouse antigenicity), it dramatically increases the value of the antibody as a pharmaceutical product. .
[0231]
Therefore, the human monoclonal antibody against AILIM of the present invention (particularly human AILIM) and the pharmaceutical composition comprising the human monoclonal antibody can be applied to cells expressing AILIM without inducing host immune rejection caused by HAMA. Various biological reactions involving the transmission of costimulatory signals (costimulatory signals) via AILIM (eg, cell growth of cells expressing AILIM, production of cytokines by cells expressing AILIM, cells expressing AILIM) Signaling and / or signaling through AILIM, for example, to control immune cytolysis or apoptosis of cells and activity to induce antibody-dependent cytotoxicity against AILIM-expressing cells It is possible to suppress or prevent the onset and / or progression of various diseases involving the disease and to treat or prevent the diseases .
[0232]
Specifically, by providing a pharmaceutical comprising the anti-AILIM human monoclonal antibody of the present invention or a part thereof as an active organism, for example, autoimmune disease or allergic disease (particularly, self-induced by cellular immunity) Various diseases classified as immune diseases and delayed allergies (eg 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 reaction); graft versus host disease (GVHD); tissue (tissue such as skin, cornea, bone) or organ Immune rejection associated with transplantation (allotransplantation or xenotransplantation) of liver, heart, lung, kidney, pancreas, etc .; immune reaction elicited by a foreign antigen or autoantigen (for example, production of antibody against the antigen, cell proliferation, Cytokines, etc.); and suppression of diseases that may result from abnormalities of intestinal immunity (specifically, inflammatory bowel diseases (especially Crohn's disease and ulcerative colitis) and dietary allergies), Or treatment and prevention are possible.
[0233]
The pharmaceutical composition of the present invention can also be used in any inflammation to which various steroids as anti-inflammatory agents are applied, such as inflammation associated with various arthritis (rheumatoid arthritis, osteoarthritis, etc.), pneumonia, Hepatitis (including viral hepatitis), inflammation associated with infection, inflammatory bowel disease, enteritis, nephritis (inflammation associated with glomerulonephritis, renal fibrosis), gastritis, vasculitis, pancreatitis, peritonitis, bronchitis, myocardium Inflammation, encephalitis, inflammation in post-ischemic reperfusion injury (such as myocardial ischemia reperfusion injury), inflammation caused by immune rejection after tissue or organ transplantation, burns, various skin inflammations (psoriasis, allergic contact skin) Treatment of or prevention of inflammation in multiple organ disorders, inflammation after PTCA or PTCR surgery, inflammation associated with arteriosclerosis, autoimmune thyroiditis, etc. Make it possible.
Furthermore, in the field of suppression / treatment of immune rejection associated with transplantation of the tissues and organs described above, the pharmaceutical composition of the present invention is applied for suppression of immune rejection in such transplantation therapy. By using in combination with an existing immunosuppressive agent, it is possible to increase the effect of suppressing graft rejection with the existing agent alone.
In addition, if the method for identifying a substance that binds to AILIM or AILIM ligand, which is one of the present invention, is used, it binds to AILIM or AILIM ligand and regulates signal transduction due to the interaction between the two, as described above. It becomes possible to screen and select a drug (chemically synthesized compound or antibody) having a potential activity capable of treating various diseases.
[0234]
[Sequence Listing]

[0235]
"Sequence Listing Free Text"
SEQ ID NO: 1
Other information: Description of the artificial sequence: Artificially synthesized primer sequence NotI-T.
SEQ ID NO: 2
Other information: Description of the artificial sequence: Artificially synthesized linker sequence 20adp.
SEQ ID NO: 3
Other information: Description of artificial sequences: Artificially synthesized linker sequence 24adp.
SEQ ID NO: 4
Other information: Description of the artificial sequence: Artificially synthesized primer sequence HIGLC.
SEQ ID NO: 5
Other information: Description of the artificial sequence: Artificially synthesized primer sequence NHCc2.
SEQ ID NO: 6
Other information: Description of artificial sequences: 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 sequences: Artificially synthesized primer sequence HCc8.
SEQ ID NO: 15
Other information: Description of artificial sequences: 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 the artificial sequence: Artificially synthesized primer sequence HCc9.
SEQ ID NO: 20
Other information: Description of artificial sequence: Primer sequence HCc5 synthesized artificially.
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 graph showing the reactivity of anti-human IgG antibody, anti-human Igκ antibody and anti-human IgFc antibody with respect to anti-human AILIM human monoclonal antibody, which was obtained by analyzing the results of cell ELISA using a flow cytometer.
Fractions (a) to (l) show the following test results, respectively.
(A): Test results when a primary antibody was not added to a microplate seeded with wild-type HPB-ALL cells, and a biotin-labeled anti-human IgG antibody as a secondary antibody was added.
(B): Test results when a primary antibody is not added to a microplate seeded with wild-type HPB-ALL cells and a biotin-labeled anti-human Igκ antibody is added as a secondary antibody.
(C): Test results when a primary antibody is not added to a microplate seeded with wild-type HPB-ALL cells and a biotin-labeled anti-human IgFc antibody is added as a secondary antibody.
(D): Test results when anti-human AILIM human monoclonal antibody JMab-136 is used as the primary antibody and biotin-labeled anti-human IgG antibody is used as the secondary antibody.
(E): Test results when anti-human AILIM human monoclonal antibody JMab-136 is used as the primary antibody and biotin-labeled anti-human Igκ antibody is used as the secondary antibody.
Fracture (f): Test results when anti-human AILIM human monoclonal antibody JMab-136 is used as the primary antibody and biotin-labeled anti-human IgFc antibody is used as the secondary antibody.
Fracture (g): Test results when anti-human AILIM human monoclonal antibody JMab-138 was used as the primary antibody and biotin-labeled anti-human IgG antibody was used as the secondary antibody.
(H): Test results when anti-human AILIM human monoclonal antibody JMab-138 is used as the primary antibody and biotin-labeled anti-human Igκ antibody is used as the secondary antibody.
(I): Test results when anti-human AILIM human monoclonal antibody JMab-138 was used as the primary antibody and biotin-labeled anti-human IgFc antibody was used as the secondary antibody.
Fracture (j): Test results when anti-human AILIM human monoclonal antibody JMab-139 is used as the primary antibody and biotin-labeled anti-human IgG antibody is used as the secondary antibody.
(K): Test results when anti-human AILIM human monoclonal antibody JMab-139 is used as the primary antibody and biotin-labeled anti-human Igκ antibody is used as the secondary antibody.
Fracture (l): Test results when anti-human AILIM human monoclonal antibody JMab-139 is used as the primary antibody and biotin-labeled anti-human IgFc antibody is used as the secondary antibody.
In addition, the white curve in each fractional graph shows the test results when using the anti-KLH human monoclonal antibody as a control antibody.
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 is a graph showing the binding activity of various anti-human AILIM mouse monoclonal antibodies to human AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates a binding test to the wild-type CHO cell, and the term “human” is a binding test to the human AILIM high-expression recombinant CHO cell. It shows that.
FIG. 4 is a view showing the binding activity of various anti-human AILIM human monoclonal antibodies or anti-KLH human monoclonal antibodies as a negative control to human AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates a binding test to the wild-type CHO cell, and the term “human” is a binding test to the human AILIM high-expression recombinant CHO cell. It shows that.
FIG. 5 is a view showing the binding activity of various anti-human AILIM human monoclonal antibodies to human AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates a binding test to the wild-type CHO cell, and the term “human” is a binding test to the human AILIM high-expression recombinant CHO cell. It shows that.
FIG. 6 shows the binding activity of various anti-human AILIM human monoclonal antibodies to human AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates a binding test to the wild-type CHO cell, and the term “human” is a binding test to the human AILIM high-expression recombinant CHO cell. It shows that.
FIG. 7 shows the binding activity of an anti-mouse AILIM rat monoclonal antibody to mouse AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “mouse” is the binding test to the mouse AILIM high-expressing recombinant CHO cell. It shows that.
FIG. 8 shows the binding activity of various anti-human AILIM human monoclonal antibodies or anti-KLH human monoclonal antibodies as negative controls to mouse AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “mouse” is the binding test to the mouse AILIM high-expressing recombinant CHO cell. It shows that.
FIG. 9 shows the binding activity of various anti-human AILIM human monoclonal antibodies to mouse AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “mouse” is the binding test to the mouse AILIM high-expressing recombinant CHO cell. It shows that.
FIG. 10 shows the binding activity of various anti-human AILIM human monoclonal antibodies to mouse AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “mouse” is the binding test to the mouse AILIM high-expressing recombinant CHO cell. It shows that.
FIG. 11 is a view showing the binding activity of an anti-rat AILIM mouse monoclonal antibody to rat AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “rat” is the binding test to the rat AILIM high-expression recombinant CHO cell. It shows that.
FIG. 12 shows the binding activity of various anti-human AILIM human monoclonal antibodies or anti-KLH human monoclonal antibodies as negative controls to rat AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “rat” is the binding test to the rat AILIM high-expression recombinant CHO cell. It shows that.
FIG. 13 shows the binding activity of various anti-human AILIM human monoclonal antibodies to rat AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “rat” is the binding test to the rat AILIM high-expression recombinant CHO cell. It shows that.
FIG. 14 is a view showing binding activities of various anti-human AILIM human monoclonal antibodies to rat AILIM high-expressing recombinant CHO cells or wild-type CHO cells.
The vertical axis indicates the fluorescence intensity that is an indicator of the recombinant activity, and the horizontal axis indicates the concentration of the added antibody.
In the figure, the term “CHO” indicates that the test is binding to the wild-type CHO cell, and the term “rat” is the binding test to the rat AILIM high-expression recombinant CHO cell. It shows that.
FIG. 15 shows a healthy subject “donor A” in a costimulatory signaling activity measurement test 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
FIG. 16 shows a healthy subject “donor A” in a costimulatory signaling 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
FIG. 17 shows a healthy person “donor B” in a measurement of costimulatory signaling 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 18 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 CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results 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 costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results 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 costimulatory signaling activity measurement test 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 21 shows a healthy subject “donor C” in a costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows a healthy subject “donor C” in a costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 costimulatory signaling activity measurement test 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 26 shows a healthy person “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 CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 of costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows a healthy person “donor D” in a costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows a healthy person “donor D” in a costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 subject “Donner E” in a costimulatory signaling activity measurement test 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 31 shows a healthy subject “donor E” in a test of costimulatory signaling activity of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows a healthy subject “donor E” in a test of 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 proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows a healthy subject “donor E” in a costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 costimulatory signaling activity measurement test of various anti-human AILIM human monoclonal antibodies using a microplate coated with an anti-human CD3 monoclonal antibody and an anti-human AILIM human monoclonal antibody. The figure which shows the proliferative activity of the T cell derived from.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 35 shows the costimulation of various anti-human AILIM mouse monoclonal antibodies when a microplate coated only with anti-human CD3 monoclonal antibody is used and anti-human AILIM mouse monoclonal antibody is added in solution (liquid phase). The figure which shows the proliferative activity of the T cell derived from a healthy person "donor D" in the Rayleigh signal transduction activity measurement test.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine incorporation into cells is shown, and the horizontal axis indicates the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 36 shows the costimulation of various anti-human AILIM human monoclonal antibodies when a microplate coated only with anti-human CD3 monoclonal antibody is used and anti-human AILIM human monoclonal antibody is added in a solution state (liquid phase). The figure which shows the proliferative activity of the T cell derived from a healthy person "donor D" in the Rayleigh signal transduction activity measurement test.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows the costimulation of various anti-human AILIM human monoclonal antibodies when a microplate coated only with anti-human CD3 monoclonal antibody is used and anti-human AILIM human monoclonal antibody is added in a solution state (liquid phase). The figure which shows the proliferative activity of the T cell derived from a healthy person "donor D" in the Rayleigh signal transduction activity measurement test.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows the costimulation of various anti-human AILIM human monoclonal antibodies when a microplate coated only with anti-human CD3 monoclonal antibody is used and anti-human AILIM human monoclonal antibody is added in a solution state (liquid phase). The figure which shows the proliferative activity of the T cell derived from a healthy person "donor D" in the Rayleigh signal transduction activity measurement test.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 shows the costimulation of various anti-human AILIM human monoclonal antibodies when a microplate coated only with anti-human CD3 monoclonal antibody is used and anti-human AILIM human monoclonal antibody is added in solution (liquid phase). The figure which shows the proliferative activity of the T cell derived from a healthy person "donor D" in the Rayleigh signal transduction activity measurement test.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 healthy human “donor B” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM mouse monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 41 shows the amount of IFN-γ produced in the culture supernatant of healthy human “donor B” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results 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 healthy human “donor B” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results 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 healthy human “donor C” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM mouse monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
In the figure, “JHC1” indicates the test results when an anti-human CETP monoclonal antibody as a negative control was used instead of the anti-human AILIM mouse monoclonal antibody.
FIG. 44 shows the amount of IFN-γ produced in the culture supernatant of healthy human “donor C” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 healthy human “donor C” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, 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 healthy human “donor C” -derived T cells cultured in a microplate coated with anti-human CD3 monoclonal antibody and anti-human AILIM human monoclonal antibody. FIG.
The vertical axis represents the concentration of IFN-γ, and the horizontal axis represents the concentration of the anti-human AILIM mouse monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
Moreover, various notations mean the following.
“140”: anti-human AILIM human monoclonal antibody JMab140.
“141”: anti-human AILIM human monoclonal antibody JMab141.
FIG. 48 shows the results of various control test substances in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor A” were cultured with PBMC of a healthy person “donor D”. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 49 shows various anti-human AILIM humans in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor A” are cultured with PBMC of a healthy person “donor D”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor D” are cultured with PBMC of a healthy person “donor B”, according to various control test substances. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 51 shows various anti-human AILIM humans in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor D” are cultured with PBMC of a healthy person “donor B”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 T cells in a mixed lymphocyte reaction (MLR) in which healthy human “donor C” T cells were cultured with healthy donor “donor A” PBMC according to various control test substances. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 53 shows various anti-human AILIM humans in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor C” are cultured with PBMC of a healthy person “donor A”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 T cells in a mixed lymphocyte reaction (MLR) cultured with healthy donor “donor E” T cells from healthy donor “donor G” according to various control test substances. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 an anti-CD80 antibody and an anti-CD86 antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
FIG. 55 shows various anti-human AILIM humans in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor E” are cultured with PBMC of a healthy person “donor G”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 T cells in a mixed lymphocyte reaction (MLR) when cultured in healthy human “donor F” T cells with PBMC of healthy human “donor E”. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 an anti-CD80 antibody and an anti-CD86 antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
FIG. 57 shows various anti-human AILIM humans in a proliferative test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor F” are cultured with PBMC of a healthy person “donor E”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 T cells in a mixed lymphocyte reaction (MLR) cultured with healthy donor “donor G” T cells from healthy donor “donor F” according to various control test substances. The figure which shows the inhibitory effect of this T cell proliferation.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 an anti-CD80 antibody and an anti-CD86 antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
“CTLA4-Ig”: human CTLA4-IgFc chimeric molecule.
FIG. 59 shows various anti-human AILIM humans in a proliferation test of T cells in a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor G” are cultured with PBMC of a healthy person “donor F”. The figure which shows the inhibitory effect of the proliferation of this T cell by a monoclonal antibody.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor A” were cultured with PBMC of a healthy person “donor D” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various control test substances in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 61 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor A” were cultured with PBMC of a healthy person “donor D” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various anti-human AILIM human monoclonal antibodies in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor D” were cultured with PBMC of a healthy person “donor B” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various control test substances in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 63 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor D” were cultured with PBMC of a healthy person “donor B” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various anti-human AILIM human monoclonal antibodies in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor C” were cultured with PBMC of a healthy person “donor A” previously cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the proliferation of this T cell by various control test substances in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates the concentration of the test substance.
The various notations in the figure mean the following.
“CD80 + 86”: a mixture of an anti-CD80 antibody and an anti-CD86 antibody.
“MIgG1”: anti-human CD34 / IgG1 mouse monoclonal antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 65 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor C” are cultured with PBMC of a healthy person “donor A” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the proliferation of this T cell by various anti-human AILIM human monoclonal antibodies in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor E” were cultured with PBMC of a healthy person “donor G” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various control test substances in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 an anti-CD80 antibody and an anti-CD86 antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 67 shows a mixed lymphocyte reaction (MLR) when healthy human “donor E” T cells were cultured with PBMC of normal human “donor G” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule. The figure which shows the inhibitory effect of the proliferation of this T cell by various anti-human AILIM human monoclonal antibodies in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor G” were cultured with PBMC of a healthy person “donor F” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various control test substances in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 an anti-CD80 antibody and an anti-CD86 antibody.
“SA12”: anti-human AILIM mouse monoclonal antibody.
FIG. 69 shows a mixed lymphocyte reaction (MLR) when T cells of a healthy person “donor G” were cultured with PBMC of a healthy person “donor F” pre-cultured in the presence of a human CTLA4-Ig chimeric molecule in advance. The figure which shows the inhibitory effect of the proliferation of this T cell by various anti-human AILIM human monoclonal antibodies in the proliferation test of this T cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis indicates 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 represents the rate of cytotoxicity caused by ADCC-inducing activity of the antibody, and the horizontal axis represents the concentration of the antibody.
FIG. 71 shows ADCC-inducing activities of various anti-human AILIM human monoclonal antibodies and control antibodies when human AILIM high-expressing recombinant CHO cells are used as target cells.
The vertical axis represents the rate of cytotoxicity caused by ADCC-inducing activity of the antibody, and the horizontal axis represents the concentration of the antibody.
FIG. 72 is a graph showing the effect of suppressing delayed allergy by anti-AILIM antibody.
The vertical axis indicates the area of redness, which is an index of the onset of delayed type allergy, and the horizontal axis indicates the type of sample administered to the test animal.
FIG. 73: Proliferation of monkey T cells in costimulatory signaling 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 activity.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] thymidine uptake into cells is shown, and the horizontal axis represents the concentration of the anti-human AILIM human monoclonal antibody.
Note that “anti-KLH” in the figure indicates the test results when an anti-KLH human monoclonal antibody was used as a negative control instead of the anti-human AILIM human monoclonal antibody.
FIG. 74 shows the inhibitory activity of a negative control antibody on the binding of various concentrations of soluble AILIM ligand (hB7h-IgFc) and soluble AILIM (AILIM-IgFc).
The vertical axis represents absorbance, which is an index of inhibitory activity, and the horizontal axis represents the concentration of soluble AILIM.
FIG. 75 shows the inhibitory activity of anti-AILIM antibodies on the binding of various concentrations of soluble AILIM ligand (hB7h-IgFc) and soluble AILIM (AILIM-IgFc).
The vertical axis represents absorbance, which is an index of inhibitory activity, and the horizontal axis represents the concentration of soluble AILIM.
FIG. 76 shows the inhibitory activity of various concentrations of anti-AILIM antibody on the binding of soluble AILIM ligand (hB7h-IgFc) and soluble AILIM (AILIM-IgFc).
The vertical axis represents absorbance, which is an index of inhibitory activity, and the horizontal axis represents the concentration of anti-AILIM antibody.
FIG. 77. Human T with various anti-human AILIM human monoclonal antibodies in a costimulatory signaling activity measurement test using a microplate coated with soluble human AILIM ligand (hB7h-IgFc) together with anti-human CD3 monoclonal antibody. The figure which shows the proliferation inhibitory activity of a cell.
The vertical axis is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis represents antibody concentration.
FIG. 78: Monkey T with various anti-human AILIM human monoclonal antibodies in a costimulatory signaling 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 is an index of the degree of cell proliferation [^ThreeH] Thymidine is taken up into cells, and the horizontal axis represents antibody concentration.

Claims (6)

DNA encoding a polypeptide selected from the group consisting of the following (a) to (f):
(A) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 117 set forth in SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acid numbers 23 to 116 set forth in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 set forth in SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 116 set forth in SEQ ID NO: 36; and
(F) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 116 described in SEQ ID NO: 38 ;
Or a part thereof, where the part is F (ab ′) 2 , Fab ′ , Fab , Fv , sFv , dsFv or dAb of the human monoclonal antibody described in any of ( i ) to ( iv ) below: Encodes the heavy or light chain of the fragment :
(I) a human monoclonal antibody which binds to human AILIM, dissociation constant between AILIM (Kd) is, 1.0 X 10 ^-9 (M) Human monoclonal antibodies are the following number;
A human monoclonal antibody that binds to (ii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) human monoclonal antibodies that are more numbers;
A human monoclonal antibody that binds to (iii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, a dissociation rate constant of AILIM (kd) is, A human monoclonal antibody having a numerical value of 1.0 × 10 ⁻⁴ ( 1 / Sec ) or less; and
A human monoclonal antibody that binds to (iv) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) or more is a number, the dissociation rate constant of AILIM (kd) is the value of ^{1.0 X 10 -4 (1 / Sec} ) or less, a human monoclonal antibody,
However, the fragments, the dissociation constant of the AILIM (Kd) is 1.0 X 10 ^-9 (M) following numbers, further having any of the features of the following (a ') to (c'):
(A ') having an activity of inhibiting mixed lymphocyte reaction;
( B ′) has an activity of transmitting a costimulatory signal to the T cell and inducing proliferation of the T cell or production of interferon γ from the T cell; or
( C ′) It has an activity of inducing cell damage to cells expressing AILIM . DNA encoding a polypeptide selected from the group consisting of the following (a) to (f):
(A) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 28;
(B) a polypeptide comprising the amino acid sequence of amino acid numbers 23 to 236 set forth in SEQ ID NO: 30;
(C) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 32;
(D) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 34;
(E) a polypeptide comprising the amino acid sequence of amino acid numbers 20 to 470 described in SEQ ID NO: 36; and
(F) a polypeptide comprising the amino acid sequence of amino acid numbers 21 to 236 set forth in SEQ ID NO: 38 ;
Or a part thereof, where the part is F (ab ′) 2 , Fab ′ , Fab , Fv , sFv , dsFv or dAb of the human monoclonal antibody described in any of ( i ) to ( iv ) below: Encodes the heavy or light chain of the fragment :
(I) a human monoclonal antibody which binds to human AILIM, dissociation constant between AILIM (Kd) is, 1.0 X 10 ^-9 (M) Human monoclonal antibodies are the following number;
A human monoclonal antibody that binds to (ii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) human monoclonal antibodies that are more numbers;
A human monoclonal antibody that binds to (iii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, a dissociation rate constant of AILIM (kd) is, A human monoclonal antibody having a numerical value of 1.0 × 10 ⁻⁴ ( 1 / Sec ) or less; and
A human monoclonal antibody that binds to (iv) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) or more is a number, the dissociation rate constant of AILIM (kd) is the value of ^{1.0 X 10 -4 (1 / Sec} ) or less, a human monoclonal antibody,
However, the fragments, the dissociation constant of the AILIM (Kd) is 1.0 X 10 ^-9 (M) following numbers, further having any of the features of the following (a ') to (c'):
(A ') having an activity of inhibiting mixed lymphocyte reaction;
( B ′) having an activity of transmitting a costimulatory signal to the T cell and inducing proliferation of the T cell or production of interferon γ from the T cell; or
( C ′) It has an activity of inducing cell damage to cells expressing AILIM . DNA selected from the group consisting of the following (a) to (f):
(A) a DNA comprising the nucleotide sequence of nucleotide numbers 126 to 419 described in SEQ ID NO: 27;
(B) a DNA comprising the nucleotide sequence of nucleotide numbers 105 to 386 set forth in SEQ ID NO: 29;
(C) a DNA comprising the nucleotide sequence of nucleotide numbers 151 to 441 set forth in SEQ ID NO: 31;
(D) a DNA comprising the nucleotide sequence of nucleotide numbers 88 to 375 described in SEQ ID NO: 33;
(E) a DNA comprising the nucleotide sequence of nucleotide numbers 153 to 443 described in SEQ ID NO: 35; and
(F) a DNA comprising the nucleotide sequence of nucleotide numbers 93 to 380 described in SEQ ID NO: 37 ;
Or a part thereof, where the part is F (ab ′) 2 , Fab ′ , Fab , Fv , sFv , dsFv or dAb of the human monoclonal antibody described in any of ( i ) to ( iv ) below: Encodes the heavy or light chain of the fragment :
(I) a human monoclonal antibody which binds to human AILIM, dissociation constant between AILIM (Kd) is, 1.0 X 10 ^-9 (M) Human monoclonal antibodies are the following number;
A human monoclonal antibody that binds to (ii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) human monoclonal antibodies that are more numbers;
A human monoclonal antibody that binds to (iii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, a dissociation rate constant of AILIM (kd) is, A human monoclonal antibody having a numerical value of 1.0 × 10 ⁻⁴ ( 1 / Sec ) or less; and
A human monoclonal antibody that binds to (iv) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) or more is a number, the dissociation rate constant of AILIM (kd) is the value of ^{1.0 X 10 -4 (1 / Sec} ) or less, a human monoclonal antibody,
However, the fragments, the dissociation constant of the AILIM (Kd) is 1.0 X 10 ^-9 (M) following numbers, further having any of the features of the following (a ') to (c'):
(A ') having an activity of inhibiting mixed lymphocyte reaction;
( B ′) having an activity of transmitting a costimulatory signal to the T cell and inducing proliferation of the T cell or production of interferon γ from the T cell; or
( C ′) It has an activity of inducing cell damage to cells expressing AILIM . DNA selected from the group consisting of the following (a) to (f):
(A) a DNA having the nucleotide sequence of nucleotide numbers 69 to 1481 described in SEQ ID NO: 27;
(B) DNA having the nucleotide sequence of nucleotide numbers 39 to 749 described in SEQ ID NO: 29;
(C) a DNA having the nucleotide sequence of nucleotide numbers 94 to 1506 described in SEQ ID NO: 31;
(D) a DNA having the nucleotide sequence of nucleotide numbers 28 to 738 described in SEQ ID NO: 33;
(E) a DNA having the nucleotide sequence of nucleotide numbers 96 to 1508 described in SEQ ID NO: 35; and
(F) a DNA having the nucleotide sequence of nucleotide numbers 33 to 743 described in SEQ ID NO: 37 ;
Or a part thereof, where the part is F (ab ′) 2 , Fab ′ , Fab , Fv , sFv , dsFv or dAb of the human monoclonal antibody described in any of ( i ) to ( iv ) below: Encodes part heavy or light chain of a fragment :
(I) a human monoclonal antibody which binds to human AILIM, dissociation constant between AILIM (Kd) is, 1.0 X 10 ^-9 (M) Human monoclonal antibodies are the following number;
A human monoclonal antibody that binds to (ii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) human monoclonal antibodies that are more numbers;
A human monoclonal antibody that binds to (iii) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, a dissociation rate constant of AILIM (kd) is, A human monoclonal antibody having a numerical value of 1.0 × 10 ⁻⁴ ( 1 / Sec ) or less; and
A human monoclonal antibody that binds to (iv) human AILIM, dissociation constant between AILIM (Kd) is the value of 1.0 X 10 ^-9 (M) or less, binding rate constant of AILIM (ka) is, ^{1.0 X 10 4 (1 / M.Sec} ) or more is a number, the dissociation rate constant of AILIM (kd) is the value of ^{1.0 X 10 -4 (1 / Sec} ) or less, a human monoclonal antibody,
However, the fragments, the dissociation constant of the AILIM (Kd) is 1.0 X 10 ^-9 (M) following numbers, further having any of the features of the following (a ') to (c'):
(A ') having an activity of inhibiting mixed lymphocyte reaction;
( B ′) has an activity of transmitting a costimulatory signal to the T cell and inducing proliferation of the T cell or production of interferon γ from the T cell; or
( C ′) It has an activity of inducing cell damage to cells expressing AILIM . Vector comprising a DNA or a part thereof described in any one of claims 1 to 4. Transformed recombinant host by DNA or vector of the part or claim 5, wherein as described in any one of claims 1 to 4 is introduced into the host.

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