JP4361684B2 - Peptide ligands for erythropoietin receptors - Google Patents

Description

【０１４３】
（２）配列番号２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：４１個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２：

【０１４４】
（２）配列番号３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３７個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：cDNA
（xi）配列：配列番号３：

【０１４５】
（２）配列番号４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：６３３８個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４：
【化１】

【化２】

【化３】

【０１４６】
（２）配列番号５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号５：

【０１４７】
（２）配列番号６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：４０個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号６：

【０１４８】
（２）配列番号７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：７５０個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号７：
【化４】

【０１４９】
（２）配列番号８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号８：
【化５】

【０１５０】
（２）配列番号９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号９：
【化６】

【０１５１】
（２）配列番号１０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：６３個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号１０：
【化７】

【０１５２】
（２）配列番号１１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：６０個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号１１：
【化８】

【０１５３】
（２）配列番号１２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１２個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号１２：

【０１５４】
（２）配列番号１３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１３：
【化９】

【０１５５】
（２）配列番号１４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１４：
【化１０】

【０１５６】
（２）配列番号１５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１５：
【化１１】

【０１５７】
（２）配列番号１６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１６：
【化１２】

【０１５８】
（２）配列番号１７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１７：
【化１３】

【０１５９】
（２）配列番号１８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１８：
【化１４】

【０１６０】
（２）配列番号１９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号１９：
【化１５】

【０１６１】
（２）配列番号２０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２０：
【化１６】

【０１６２】
（２）配列番号２１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２１：
【化１７】

【０１６３】
（２）配列番号２２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２２：
【化１８】

【０１６４】
（２）配列番号２３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２３：
【化１９】

【０１６５】
（２）配列番号２４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２４：
【化２０】

【０１６６】
（２）配列番号２５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２５：
【化２１】

【０１６７】
（２）配列番号２６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２６：
【化２２】

【０１６８】
（２）配列番号２７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２７：
【化２３】

【０１６９】
（２）配列番号２８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２８：
【化２４】

【０１７０】
（２）配列番号２９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号２９：
【化２５】

【０１７１】
（２）配列番号３０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３０：
【化２６】

【０１７２】
（２）配列番号３１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３１：
【化２７】

【０１７３】
（２）配列番号３２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３２：
【化２８】

【０１７４】
（２）配列番号３３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３３：
【化２９】

【０１７５】
（２）配列番号３４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３４：
【化３０】

【０１７６】
（２）配列番号３５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３５：
【化３１】

【０１７７】
（２）配列番号３６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３６：
【化３２】

【０１７８】
（２）配列番号３７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３７：
【化３３】

【０１７９】
（２）配列番号３８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３８：
【化３４】

【０１８０】
（２）配列番号３９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号３９：
【化３５】

【０１８１】
（２）配列番号４０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４０：
【化３６】

【０１８２】
（２）配列番号４１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４１：
【化３７】

【０１８３】
（２）配列番号４２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４２：
【化３８】

【０１８４】
（２）配列番号４３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４３：
【化３９】

【０１８５】
（２）配列番号４４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４４：
【化４０】

【０１８６】
（２）配列番号４５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４５：
【化４１】

【０１８７】
（２）配列番号４６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１１４個の塩基対
（Ｂ）型：核酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（xi）配列：配列番号４６：
【化４２】

【０１８８】
（２）配列番号４７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号４７：
【化４３】

【０１８９】
（２）配列番号４８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号４８：
【化４４】

【０１９０】
（２）配列番号４９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号４９：
【化４５】

【０１９１】
（２）配列番号５０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５０：
【化４６】

【０１９２】
（２）配列番号５１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５１：
【化４７】

【０１９３】
（２）配列番号５２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５２：
【化４８】

【０１９４】
（２）配列番号５３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５３：
【化４９】

【０１９５】
（２）配列番号５４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５４：
【化５０】

【０１９６】
（２）配列番号５５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５５：
【化５１】

【０１９７】
（２）配列番号５６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５６：
【化５２】

【０１９８】
（２）配列番号５７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５７：
【化５３】

【０１９９】
（２）配列番号５８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５８：
【化５４】

【０２００】
（２）配列番号５９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号５９：
【化５５】

【０２０１】
（２）配列番号６０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６０：
【化５６】

【０２０２】
（２）配列番号６１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６１：
【化５７】

【０２０３】
（２）配列番号６２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６２：
【化５８】

【０２０４】
（２）配列番号６３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６３：
【化５９】

【０２０５】
（２）配列番号６４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６４：
【化６０】

【０２０６】
（２）配列番号６５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６５：
【化６１】

【０２０７】
（２）配列番号６６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６６：
【化６２】

【０２０８】
（２）配列番号６７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６７：
【化６３】

【０２０９】
（２）配列番号６８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６８：
【化６４】

【０２１０】
（２）配列番号６９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号６９：
【化６５】

【０２１１】
（２）配列番号７０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７０：
【化６６】

【０２１２】
（２）配列番号７１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７１：
【化６７】

【０２１３】
（２）配列番号７２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７２：
【化６８】

【０２１４】
（２）配列番号７３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７３：
【化６９】

【０２１５】
（２）配列番号７４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７４：
【化７０】

【０２１６】
（２）配列番号７５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７５：
【化７１】

【０２１７】
（２）配列番号７６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７６：
【化７２】

【０２１８】
（２）配列番号７７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３７個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７７：
【化７３】

【０２１９】
（２）配列番号７８についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７８：
【化７４】

【０２２０】
（２）配列番号７９についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号７９：
【化７５】

【０２２１】
（２）配列番号８０についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：３８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８０：
【化７６】

【０２２２】
（２）配列番号８１についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８１：

【０２２３】
（２）配列番号８２についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１８個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８２：

【０２２４】
（２）配列番号８３についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１７個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８３：

【０２２５】
（２）配列番号８４についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１６個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８４：

【０２２６】
（２）配列番号８５についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１５個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８５：

【０２２７】
（２）配列番号８６についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１４個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８６：

【０２２８】
（２）配列番号８７についての情報：
（ｉ）配列の特徴：
（Ａ）長さ：１９個のアミノ酸
（Ｂ）型：アミノ酸
（Ｃ）鎖の数：一本鎖
（Ｄ）トポロジー：直鎖状
（ii）配列の種類：ナシ
（xi）配列：配列番号８７：

【図面の簡単な説明】
【図１】 図１は、ERB １ファージクローンがキメラIg−EPORプローブと特異的に結合することを確かめるEPOR−特異的ファージELISA からの結果を示す線グラフである。グラフは、Ig−EPORプローブを用いて単離されたファージクローン（ERB1、黒三角形）；抗−IL−８抗体に対して特異的に結合するファージクローン（HPKF、◆）；及びヒトIL−６受容体に対して特異的に結合するファージクローン（IL−６−B26 、黒四角形）について、異なったファージ希釈度でのELISA （O. D. ）により測定されるような、Ig−EPORに対する結合を示す。
【図２】 図２は、異なった濃度（単位/ml ）での組換えEPO がIg−EPORに結合するためにERB1ファージ（◆）と競争するが、しかしIL−６受容体に結合したファージ（●）に対して効果を有さなかったELISA （O. D. ）の結果を示す線グラフである。
【図３Ａ】 図３A は、ERB1発生されたライブラリーを創造するために使用されたアプローチを示す。図３A は、第１の線上のERB1クローンにおけるpIIIタンパク質に融合されたペプチドのアミノ酸配列、及び第２及び第３の線上のERB1クローンの発生されたライブラリーを創造するために使用された冗長性オリゴヌクレオチドの収集物の配列を示す。オリゴヌクレオチドは、ERB1ペプチドにおけるアミノ酸に対応するコドンを示すために、及び配列の3'末端で９個の相補的塩基を用いてのオリゴヌクレオチドのアニーリングを示すために整列された。
【図３Ｂ】 図３B は、ERB1発生されたライブラリーを創造するために使用されたアプローチを示す。図３B は、冗長性オリゴヌクレオチドにおいて“g ”、“a ”、“ｔ”、“ｃ”及び“ｓ”として表される個々の塩基のために冗長性オリゴヌクレオチドを合成するために使用されたヌクレオチド組成物を示す。ERB1クローンにおけるpIIIタンパク質に融合されたペプチドのアミノ酸配列、及びERB1ペプチド配列の変異体をコードしたオリゴヌクレオチドの収集物を創造するために使用されたスキームが示される。
【図４】 図４は、ERB1発生されたライブラリーに由来するファージクローンのアミノ酸配列、及びIg−EPORのためのクローンの相対的親和性を示す。クローンB1〜B21 は、Ig−EPORに結合することによって単離され、そしてファージクローンN1〜N13 は発生されたライブラリーに由来するが、しかし選択工程の間、Ig−EPORに結合しなかった。相対的親和性は、図５A 〜５D に示されるデータの数的な要約である。
【図５Ａ】 図５A は、元のERB1単離物に関して、Ig−EROPのためのいくつかの独立したファージクローンの親和性を示す一連の線グラフである。図５A はクローンB1〜B5及びERB1についての結果を示す。
【図５Ｂ】 図５B は、元のERB1単離物に関して、Ig−EROPのためのいくつかの独立したファージクローンの親和性を示す一連の線グラフである。図５B はクローンB6〜B10 及びERB1についての結果を示す。
【図５Ｃ】 図５c は、元のERB1単離物に関して、Ig−EROPのためのいくつかの独立したファージクローンの親和性を示す一連の線グラフである。図５C はクローンB11 〜B15 及びERB1についての結果を示す。
【図５Ｄ】 図５D は、元のERB1単離物に関して、Ig−EROPのためのいくつかの独立したファージクローンの親和性を示す一連の線グラフである。図５D はB16 〜B21 及びERB1についての結果を示す。
【図６】 図６は、固定されたEPO へのキメラIg−EPORプローブの結合が、添加されるペプチドの不在（◆）及びIL−６受容体に結合する不適切なペプチドの添加（×）に比較して、競争体ペプチド（ペプチドERB1−７、黒四角形；ペプチドERB1−８、●）により阻害される、ペプチド阻害ELISA からの結果を示す線グラフである。
【図７】 図７は、N −末端切断されたERB1−７ペプチド（17−マー、黒四角形；16−マー、黒三角形；15−マー×；14−マー、★）のIg−EPORへの結合に比較して、及びIL−６受容体に結合する不適切なペプチドの添加又は添加されるインヒビターの不在（＋）に比較して、Ig−EPORへのERB １−7 18−マーペプチド（◆）の結合を示す線グラフである。
【図８Ａ】 図８A は、TF−１細胞増幅アッセイからの結果を示す線グラフである。図８A は、組換えEPO の異なった濃度（単位）でのTF−１細胞（450nm でのO. D. ）の増殖についての用量応答曲線を示す。
【図８Ｂ】 図８B は、TF−１細胞増幅アッセイからの結果を示す線グラフである。図８B は、IL−６受容体に結合する不適切なペプチドの添加（×）に比較して、合成ペプチドERB1−７（黒四角形）及びERB1−８（黒三角形）の異なった濃度（μM ）でのTF−１細胞（450nm でのO. D. ）の増殖についての用量応答曲線を示します。[0001]
Field of Invention
The present invention relates to the fields of pharmacology and drug discovery. More specifically, the present invention relates to novel peptide compositions that can bind to and activate human erythropoietin receptors, and erythropoietin receptors using the peptide compositions described above as design templates. Relates to a method for producing a small molecule agonist of
[0002]
Background of the Invention
Drug discovery has traditionally relied on high-throughput screening of large numbers of compounds to identify new drug precursors. More recently, combinatorial libraries constructed by chemical or biological means have expanded the number of compounds available for screening. Randomly directed semi-random sequence biological libraries such as phage-displayed peptide libraries represent a particularly rich source of molecular diversity, and advantageously have the ability to self-replicate.
[0003]
Due to the self-replicating system, research on high affinity preconductors is not limited to members that happen to be in the initial library. As discussed more fully below, the desired characteristics of the initial sequence can be greatly improved by using sequential stages of mutagenesis, affinity selection and amplification. These approaches have recently been used to discover small peptides that can bind several cytokine receptors.
[0004]
Erythropoietin (EPO) is a cytokine that stimulates the formation of red blood cells by inducing the proliferation and differentiation of progenitor cells. Recombinant versions of human EPO are valuable therapeutic agents useful for treating several pathological conditions such as chronic renal failure, the grade or effect of chemotherapy, anemia associated with HIV and rheumatoid arthritis. is there. When used therapeutically, EPO should be administered by intravenous or subcutaneous injection. The fact that EPO is a relatively large glycoprotein adversely affects the cost of manufacture, the pharmacological properties of the molecule and the diversity of this therapeutic agent.
[0005]
The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily members that share the usual structural features such as the extracellular ligand binding domain, a single transmembrane-extension region, and the intracellular cytoplasmic domain. The extracellular domain (ECD) is sufficient to mediate receptor-ligand binding. Thus, synthesizing DNA encoding ECD as a fusion with a secreted protein, for example, to generate a reagent useful for identifying receptor binding molecules by screening a phage display library, This is possible through recombinant DNA techniques.
[0006]
Phage display libraries expressing fusions of random or semi-random peptides and bacteriophage coat proteins represent a conventional version of a combinatorial library that can be screened to identify receptor ligands. Based on phage particle infection and assembly, the random polypeptide is placed superficially for interaction with an antibody or other receptor probe. Since the phage particle contains a nucleic acid encoding the fusion protein, the genetic information identifying the sequence of the fusion protein is physically linked to the fusion protein.
[0007]
The generation and screening of such phage expression libraries is well known and is described, for example, by Sawyer et al., Protein Engineering 4: 947-953 (1991); Akamatsu et al., J. Immunol. 151: 4651-59. (1993); Smith et al., Methods in Enzymol. 217: 228-257 (1993); Clackson et al., Trends Biotechnol. 12: 173-184 (1994); and US Pat. No. 5,427,908 (Dower et al.) Are listed.
[0008]
In one example of a screening method, a soluble form of EPOR was used to probe a phage display library to identify candidate properties that have EPO-like properties. Wrighton et al. Science 273: 458 (1996) describes the use of a fusion protein comprising an EPOR extracellular domain and human placental alkaline phosphatase in a library screening protocol. The library used for this purpose showed cyclic 8-residue peptides as fusions with filamentous phage pVIII coat protein.
[0009]
Subsequently, a high affinity property for EPOR was isolated from a mutagenesis library that showed pIII protein fusion. This approach leads to the identification of several properties that stimulate erythropoietin synthesis in mice. These agonists were disulfide-linked cyclic peptides with minimal consensus sequence YXCXXGPXTWXCXP (SEQ ID NO: 1), where X is a position that can be governed by any of several amino acids.
Despite this progress in the identification of EPOR ligands, there remains a need to identify ligands that display superior receptor-binding properties and ligands that bind to receptors using previously unknown contact sites. ing.
[0010]
Summary of invention
According to one aspect of the present invention, the following formula:
X_n -C-X₁-X₂-G-W-V-G-X_Three-C-X_Four-X_Five-W-X_c
[Where X_n Is the N-terminal peptide of natural α-amino acids 2-4 in length;
X_c Is a C-terminal dipeptide, and
X₁, X₂, X_Three, X_Four, And X_FiveAre independently selected from the group consisting of natural α-amino acids]
An isolated polypeptide capable of binding a human erythropoietin receptor is disclosed.
[0011]
In a preferred embodiment of the isolated polypeptide, the amino terminal X_n X_n1-X_n2-X_n3-X_n4And where X_n1Is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural α-amino acids, or optionally X_n1Is absent; X_n2Is selected from the group consisting of neutral and polar, neutral and hydrophobic, and basic natural α-amino acids, or optionally X_n2X_n1Is absent if is absent; X_n3Is selected from the group consisting of natural α-amino acids; and X_n4Is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural α-amino acids.
[0012]
More preferably, X_n1Is E, G, N, S, D, Q, L, Y or A; X_n2Is F, V, A, K, R, G, S, I or L; X_n3Is H, Q, E, G, D, A or V; and X_n4Is E, G, V, A, P, D, T or M. In another preferred embodiment, X_n1Is absent; X_n2Is F, V, A, K, R, G, S, I or L; X_n3Is H, Q, E, G, D, A or V; and X_n4Is E, G, V, A, P, D, T or M. In one embodiment, X_n1Is an acidic amino acid. In another embodiment, X_n2Is a branched chain amino acid or K 2. In yet another embodiment, X n3 is an acidic amino acid or V 3. In one embodiment, X_n4Is V or G.
[0013]
In a preferred embodiment, X₁Is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably X₁Is R, I, G, Q, L, T or S; and most preferably R or L. In a preferred embodiment, X₂Are neutral and hydrophobic, neutral and polar or basic amino acids; more preferably X₂Is R, P, W, G, L or N; and most preferably R. In a preferred embodiment, X_ThreeAre neutral and polar, or basic amino acids; and are preferably H, Q or N 2.
[0014]
In a preferred embodiment, X_FourIs a neutral and polar, basic or acidic amino acid; more preferably Q, N, S, K or E; and most preferably K 2 or N 2. In a preferred embodiment, X_FiveIs a neutral and polar, neutral and hydrophobic, or acidic amino acid; more preferably V, A, Y, D or E; and most preferably D or E.
[0015]
In a preferred embodiment, the carboxy-terminal X_c X_c1-X_c2And X_c1Are neutral and polar, or neutral and hydrophobic amino acids, and X_c2Are neutral and polar, neutral and hydrophobic, or basic amino acids. In a preferred embodiment, X_c1Is L, I, P, F, M, Q or G; and more preferably L or Q. In a preferred embodiment, X_c2Is M, W, T, S, G, N or R; and more preferably G or R.
[0016]
Another aspect of the invention is an isolated polypeptide having the amino acid sequence of SEQ ID NO: 82.
According to another aspect of the present invention, a cell having an erythropoietin receptor on its surface is contacted with a peptidomimetic of erythropoietin, wherein the peptidomimetic has the formula:
X_n -C-X₁-X₂-G-W-V-G-X_Three-C-X_Four-X_Five-W-X_c
[Where X_n Is the N-terminal peptide of natural alpha-amino acids of 2-4 lengths; X_c Is a C-terminal dipeptide and X₁, X₂, X_Three, X_Four, And X_FiveIs independently selected from the group consisting of natural α-amino acids], and a compound having the general formula
Allows binding of the peptidomimetic and the erythropoietin receptor, thereby initiating activation of the erythropoietin receptor;
Disclosed is a method for activating a human erythropoietin receptor comprising the steps.
[0017]
According to another aspect of the invention, there is provided a method for inhibiting binding of erythropoietin to an erythropoietin receptor, wherein said method comprises:
Following formula:
X_n -C-X₁-X₂-G-W-V-G-X_Three-C-X_Four-X_Five-W-X_c
[Where X_n Is the N-terminal peptide of natural alpha-amino acids of 2-4 lengths; X_c Is a C-terminal dipeptide and X₁, X₂, X_Three, X_Four, And X_FiveIs independently selected from the group consisting of natural α-amino acids] and provides a sufficient amount of a peptidomimetic having the general formula of competing with erythropoietin for binding to the erythropoietin receptor; And
Allowing interaction between the erythropoietin receptor and the peptidomimetic, thereby inhibiting the binding of erythropoietin to the erythropoietin receptor. In a preferred embodiment, the erythropoietin receptor is derived from a human.
[0018]
Another aspect of the present invention is a method for discovering drugs that mimic erythropoietin, wherein said method comprises a phage display library comprising a peptide wherein the fusion protein comprises a random sequence of amino acids and a phage protein. Screening the phage display library for at least one clone that binds to a human erythropoietin receptor probe;
Isolating said clone that binds to a human erythropoietin receptor probe;
Determining the nucleic acid sequence from the clone encoding the peptide contained within the fusion protein;
[0019]
Constructing a phage display library generated by in vitro mutagenesis of the nucleic acid sequence encoding the peptide contained within the fusion protein;
Screening the generated phage display library for clones that bind to a human erythropoietin receptor probe;
Isolating the clone from the generated phage display library;
Determining a nucleic acid sequence from the clone, wherein each individual nucleic acid encodes a peptide contained within the fusion protein of the clone;
Comparing the nucleic acid sequences to identify a consensus amino acid sequence; and
Synthesizing compounds that mimic the consensus amino acid sequence;
Comprising steps.
[0020]
In a preferred embodiment, the synthetic step comprises synthesizing a compound that is an organic compound; preferably a peptide. More preferably, the synthesized peptide is a cyclic peptide.
[0021]
Detailed description of preferred embodiments:
Herein, the inventors disclose a novel peptide capable of binding and activating human EPOR, and a method for producing small molecule analogs of EPO using peptide ligands as design templates. Surprisingly, the primary amino acid sequence of these peptide ligands is not related to the primary amino acid sequence of the true EPO protein.
[0022]
In addition, the peptides disclosed herein are also unrelated to previously isolated EPO binding peptides from phage display libraries. The novel peptides identified herein serve not only as candidate therapeutics, but also as models for designing small molecules with EPO agonist or antagonist activity. Specifically, peptides or other small molecules that mimic the biological properties of EPO provide valuable additions to drug species.
[0023]
A cyclic peptide conjugation that binds to EPOR and mimics the pharmacological activity of erythropoietin has been identified. One EPOR-binding clone was first isolated from an M13 phage library displaying 38 random amino acids fused to the N-terminus of the pIII capsid protein. When using the sequence encoding the EPOR-binding peptide of this initial isolate, a generated library containing various amino acid substitutions over the full length of the peptide can be used to isolate additional EPOR-binding clones. Manufactured and screened using bioseparation methods. The sequence encoding the binding peptide and the non-binding control were determined.
[0024]
Neither the initial isolate nor the EPOR-binding clone isolated from the generated library shared significant sequence similarity with the primary amino acid sequence of EPO. The sequence of EPO-binding clones contained a consensus sequence that was absent from non-binding clones. This consensus sequence (SEQ ID NO: 12) encodes a 12-mer peptide, but additional amino acid residues also contribute to the efficiency of binding to EPOR.
[0025]
Based on the consensus sequence, a truncated peptide of 18 amino acids in length that retains the activity of the longest 38-mer binding peptide was synthesized. Two typical synthetic peptides with amino acid sequences based on the consensus 12-mer sequence inhibited the binding of true EPO to chimeric Ig-EPOR in an in vitro assay. In addition, these derivative peptides also stimulated proliferation of EPO-responsive cells in culture.
[0026]
Cleavage of one of the 18-mer synthetic peptides by one or more N-termini reduced peptide activity in receptor-binding and biological assays. Thus, a long peptide containing at least an 18-mer amino acid sequence shows high affinity for binding to EPOR, but a shorter peptide (eg, having 3 amino acids located on the N-terminal consensus sequence side) Retained the ability to activate EPOR.
[0027]
Briefly, the EPOR ligand disclosed herein (1) creates a chimeric receptor probe that binds to EPO; (2) creates a phage display library that expresses a number of target random peptides; 3) screening the library with a chimeric receptor probe to identify one or more lead peptides having receptor-binding activity; and (4) variants that bind and / or activate the EPO receptor. They have been identified by a multi-step method involving the creation and testing of derivatives of the lead peptide to identify structural features shared by the molecule. The general characteristics of the method used to obtain new EPOR agonists are described below.
[0028]
EPO Chimeric receptor probe with binding activity
Methods for identifying receptor ligands have relied on the binding of sub-fragments of the native receptor ligand that may essentially alter the results of anti-ligand antibodies or screening assays. For example, identifying peptides that represent tertiary structures that are critical structural features of receptor-binding ligands, as linear peptides of only 6-10 amino acids in length can be fitted into antibody combining sites It is difficult.
[0029]
Similarly, sub-fragments of known ligands cannot represent structures in a critical high order that have or represent homologous ligand structural features. For example, the receptor-binding ligand tertiary structure may not be completely contained in the contiguous amino acids of an antibody or other ligand.
[0030]
In order to circumvent unnecessary restrictions on the structure of combinatorial molecules that can be identified in binding assays, the inventors have created probes that contain the extracellular domain of the native receptor. For convenience in screening assays, we have also used probes that not only bind ligands but can also be identified by secondary probes. The inventor has created a chimeric protein incorporating the EPO receptor ("EPOR") and the ligand binding domain of the hinge region, ie, the CH2 and CH3 domains of the murine IgG1 antibody.
[0031]
As shown below, this chimeric receptor ("Ig-EPOR") bound the native ligand and was also recognized by anti-murine IgG. This sequence was required for receptor interaction and also allowed the binding of a novel ligand to the probe that displayed the critical minimal structural features of the native ligand that could be detected using a second antibody. .
Conveniently, the use of a probe comprising the ligand binding domain of the native receptor is as small as the native ligand or as small as the smallest critical structure required for receptor binding. Made it possible to combine structures.
[0032]
Preparation and screening of phage display library
The inventor has used high complexity phage display libraries as a source of molecular diversity for drug discovery. Traditional libraries of organic compounds screen one compound at a time to identify candidate drug molecules, whereas phage display libraries are abundant that can be rapidly screened using high-throughput screening assays. A variety of different sources.
[0033]
The library used in the screening method disclosed below showed fusion of phage M13 with the pIII minor coat protein. The N-terminal domain of the pIII molecule binds to the bacterial F cilia and is required for infection, whereas the C-terminal domain anchors the protein in the phage capsid. Conveniently, it can be tolerated in constructs that include a large size range of fusion pIII proteins, thereby enhancing the molecular diversity of the library. A relatively long random amino acid sequence (about 30 to about 50 residues) can be fused to the pIII protein to display a relatively long peptide comprising the random sequence for screening.
[0034]
A library of long random peptides provides a “sliding window” of adjacent amino acid residues, and also has the potential to provide tertiary structure representing intermittent epitopes in the complex protein. This added level of complexity can be important when screening for molecules that can interact with complex targets, such as cell surface receptors. In addition, a library of long random peptides allows for the formation of multiple contact sites that can bind complex targets.
[0035]
Generation and testing of derivatives of leading peptides
The inventor has identified and isolated an initial isolate from one of the phage display libraries as a leading peptide, and then constituted a molecular variant of that leading peptide and is similar or enhanced. Screened for clones showing high biological activity. That is, the inventor has shown that a phage display library in which nearly all of the codon positions in the 38-mer leader peptide are randomized with the expected 50% frequency to generate a library of mutant molecules. Created. By screening a library of variants, the inventors identified a number of additional peptides that also bound the EPO receptor probe.
[0036]
The alignment of peptide sequences deduced from the mutant DNA sequence allowed the identification of amino acid consensus sequences for molecules with the desired functional characteristics. In this regard, this method of creating a library of variant peptides based on a nucleic acid sequence encoding a leading peptide is referred to as “molecular evolution”. Using this method, the inventor has a mutant clone with an increased ability to interact with the receptor of interest (ie, has an increased binding affinity for EPOR relative to the binding affinity of the lead clone) Clone).
[0037]
Molecular evolution methods have allowed a screen of over 100,000,000 protein variants to identify additional functional peptides. The alignment of the amino acid sequences of these additional peptides and the identification of the consensus sequence served as the basic principle for identifying optimized receptor binding properties. Preferably, these optimized peptides are smaller than the peptides present in the lead isolate, and more preferably, the optimized peptides are smaller than the variants identified by using molecular evolution.
[0038]
Definition
“Chimeric protein” means a non-naturally occurring protein or polypeptide comprising some or all of the amino acid sequence from at least two different proteins or polypeptides, or from one protein or polypeptide, And a non-naturally occurring polypeptide chain. As used herein, a chimeric protein is designed, constructed by genetic engineering, synthesized, or deliberately selected, and at least two domains (ie, at least a first domain and a first domain). Two domains, each domain has some structural and / or functional characteristics not present in other domains).
[0039]
“Directly or indirectly labeled” can include a label component (eg, a radioisotope, dye, or fluorescent or chemiluminescent component) that emits a signal that allows the molecule to be directly detected. , Or through some additional reaction (ie indirectly), which can include a detectable moiety (eg, bound enzyme, ligand, eg, biotin, enzyme substrate, epitope or nucleotide sequence).
By “second molecule” is meant a molecule that can bind to at least a portion of the second domain of a chimeric protein, thereby allowing detection or purification of the chimeric protein.
[0040]
“Hinge region” or “immunoglobulin heavy chain hinge region” refers to the C of many mammalian immunoglobulin heavy chains._H2And C_H1Means one of a family of proline-containing and cysteine-containing amino acid regions present between the regions, or analogues of their amino acid sequences based thereon, wherein the amino- and carboxyl-terminal regions of the hinge are To facilitate separate and simultaneous specific binding to other molecules, they are spatially separated by rotation or kinking in the polypeptide chain.
[0041]
“Ligand” means a molecule or multimeric molecular complex capable of specifically binding to another predetermined molecule or molecular complex (ie, its target). Often, but not necessarily, the ligand is soluble and at the same time its target is identified, for example, by an anchor domain immobilized in the cell membrane.
[0042]
A “receptor” is an at least one molecule or multimeric molecular complex that has an anchor domain identified in the cell membrane and is capable of binding a given molecule or molecular complex in its native environment. Part. Often the receptors are capable of transdncing intracellular signals in response to ligand binding. Many receptors have a particularly high affinity for the ligand when either the receptor or the ligand or both are present in a homomultimeric or heteromultimeric form (eg, dimer).
[0043]
“Solid support” means a biologically insoluble matrix, such as but not limited to cells or bacteriophage particles, or a synthetic insoluble matrix such as acrylamide derivatives, cellulose, nylon, silica. And magnetic particles (but not limited to) (insoluble molecules can be linked or bound).
“Modified” means not naturally occurring or altered in a way that deviates from a naturally occurring compound.
[0044]
“Molecular evolution” refers to a method of producing a library of variant peptides by controlled rate randomization of nucleic acid sequences encoding a leading peptide having the desired functional characteristics.
“Molecule” means a molecule-sized inorganic or organic compound, such as a peptide, protein, store, fat or fatty acid, which may be naturally occurring or synthetically produced.
“Multimeric molecular complex” means a complex comprising at least two molecular components, wherein the individual components are peptides, proteins, nucleic acids, fats or fatty acids, the complex being a covalent bond Held together by non-covalent bonds or other known chemical interactions.
[0045]
Amino acids described by three letter (or one letter) abbreviations are separated according to the nature of their side groups (as described in Genes VB Lews, Oxford University Press, Inc., New York, NY. 1994). ) These groups are as follows: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Trp (W), Phe (F) and Met (M). Including "neutral and hydrophobic" groups; including "Gly (G), Ser (S), Thr (T), Tyr (Y), Cys (C), Glu (Q) and Asn (N)" "Basic and polar" groups; "basic" groups including Lys (K), Arg (R) and His (H); and "acidic" groups including Asp (D) and Glu (E). “Branched chain amino acids” refers to I, L and V.
[0046]
Unless otherwise stated, all chemical and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The general definitions of many terms used herein are Dictionary of Microbiology and Molecular Biology, and ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), and The Harper Collins Dictionary of Provided in Biology (Hale & Marham, 1991, Harper Perennial, New York, NY), and the protocol manual listed below.
[0047]
EPOR Identification of binding peptides
The methods described herein are EPOR binding peptides that can be used as receptor agonists for treating humans and animals and as structural templates for the design of small molecule EPO analogs with similar applicability. Was used to identify. A short peptide or small molecule EPO mimetic that stimulates erythropoiesis will have distinct advantages over recombinant EPO in terms of delivery convenience, pharmacokinetics and production costs.
[0048]
The inventor has found a high complexity (about 10 to identify EPOR binding peptides).⁸ EPO peptide mimetics were identified by screening a phage display library. The library displayed random peptide sequences as pIII fusion proteins expressed on the surface of M13 phage particles. Phages with the desired binding activity are affinity purified from a large excess of unbound phage, and the purified phage DNA is isolated and amplified to determine the amino acid sequence of the displayed peptide. And sequenced.
[0049]
In a typical phage display library, random peptides are displayed on the surface of bacteriophage M13 as N-terminal fusions that can be expressed on major (eg, pVIII) or minor (eg, pIII) coat proteins. . Individual bacteriophage particle display sequences having the desired binding characteristics can be affinity purified and cloned using standard laboratory techniques. Most phage libraries are constructed to display random sequences of only 6-8 amino acids in length as pIII or pVIII fusion proteins.
[0050]
However, longer random peptides displayed on the phage library have a “sliding window” effect (eg, a 38mer peptide contains 32 overlapping hexamers) and the native phage protein to which the peptide is fused. Has the potential to include increased complexity due to the ability to assume unrelated tertiary structures.
[0051]
As disclosed herein, the inventors have used IgG-EPO receptor fusion proteins to identify and isolate early and generated EPO peptide analogs, A phage display library displaying long peptides was screened. Although the methods used below are generally applicable to the discovery of peptides that bind virtually any receptor, in particular any type I cytokine or growth factor receptor, the peptides discovered by this pathway This structure cannot be predicted prior to substantial isolation and sequencing of the clone encoding the peptide.
[0052]
12-mer consensus sequence of amino acids by determining the DN and predicted amino acid sequence in clones isolated from the generated library that are bound to the EPOR probe, and sequences from clones that are not bound to this probe Was identified as present in all of the clones bound to the EPOR probe. The consensus sequence is CXXGWVGXCXXW (where X represents various amino acids).
[0053]
In addition to the 12-mer consensus sequence, the amino acid test of the 38-mer peptide of the binding clone (see Figure 4) shows that the first residue is acidic, or neutral and polar, or neutral. And a hydrophobic amino acid, preferably D, E, G, N, S, Q, L, Y or A and more preferably D.
The second residue is a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably L, V, I, K, A, F, R, G or S, more preferably branched. The amino acid of the chain, most preferably L.
[0054]
The third residue is any of the four general types of amino acids characterized by their side chain properties and is preferably E, D, Q, G, H, V or A, more preferably E or Q.
The fourth residue is an acidic, or neutral and polar, or neutral and hydrophobic amino acid, preferably G, T, V, A, P, M, D or E, and more preferably V or G. It was.
The fifth residue was C in the consensus sequence.
The sixth residue was a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably R, L, I, G, Q, T or S, and more preferably R .
[0055]
The seventh residue was a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably R, G, N, P, W, or L, and more preferably R.
Residues 8-11 are the conserved 12-mer GWVG sequence.
Residue 12 is H, Q or N, preferably H 2 and
The 13th residue was a 12-mer conserved C 2.
The 14th residue was acidic, or basic or neutral and polar, preferably E, K, N, Q or S, more preferably N or K.
[0056]
The fifteenth residue was an acidic, neutral and polar, or neutral and hydrophobic amino acid, preferably D, E, Y, V or Y, and more preferably D 1 or E 2, most preferably D 2.
Residue 16 is the consensus 12-mer W and
The 17th residue was a neutral and polar, or neutral and hydrophobic amino acid, preferably Q, G, L, I, P, F or M, and more preferably L.
[0057]
The eighteenth residue was a basic, or neutral polar, or neutral and hydrophobic amino acid, preferably R, G, N, T, S, M or W, and more preferably R 1 or G 2 .
Residues 19-21 were invariant DEY conserved in all of the peptides due to the mutagenesis scheme used (see Example 7 below).
[0058]
Residues 22-38 of the peptide shown in FIG. 4 also included a subset of the 20 naturally occurring α-amino acids.
In particular residue 22 is A, T, N, S or E, preferably A;
Residue 23 is K, R, S, N or I, preferably K or R; residue 24 is P, I, N, T, Q, K, H, R or E, preferably N Yes;
Residue 25 is P, T, R or H, preferably P;
Residue 26 is I, P, S, G, T, N, R or K, preferably R or G;
Residue 27 is L, S, N, T, Y, H or K, preferably Y;
Residue 28 is P, A, Q, G or S, preferably P;
[0059]
Residue 29 is a branched chain amino acid, such as A, M, F, N, T, E or D, preferably V;
Residue 30 is P, A, V, T, Q, R or E, preferably P;
Residue 31 is P, N, Q, H, K, R or D, preferably P;
Residue 32 is I, G, S, N, T, Q or R, preferably G or S;
Residue 33 is L, V, f, T, Y, N, S, Q, K, H, E or D, and N, Y or K appears most frequently.
[0060]
Residue 34 is L, V, P, A, Y, S, K, R, H, D or E, preferably S;
Residue 35 was L, V, P, M, Y, N, S, R or H, preferably one branched chain amino acid, more preferably L.
Residue 36 was I, N, G, Q, S, T or K, preferably neutral and polar amino acids, and more preferably N 2.
Residue 37 is P, L, T, G, S or R; and
Residue 38 was P, L, A, T, S, H or R. Both residues 37 and 38 were preferably P 2.
[0061]
Those amino acid residues represent the sequence of a population of 22 EPOR binding clones and their amino acid sequences representing binding peptides are shown in FIG. Thus, although certain trends in EPOR binding peptides can be discerned by testing their sequences, particularly for the preferred amino acids for some residues, they are not limiting and for binding clones in FIG. EPOR binding peptides that conserve a conserved 12-mer sequence having a flanking sequence that is not represented by any of the sequences shown are within the scope of the invention.
[0062]
Many of the amino acids that preferably appear at specific residue positions in their generated peptides are nucleic acid having the mutagenesis scheme described in Example 7 (73% of the parent nucleotide and 9% of the other three nucleotides). Was the same as the amino acid present at that position in the starting peptide that could affect the redundancy of the genetic code (eg, R at residue 5 and at residue 25) P).
[0063]
However, for some residues, it selectively changes to the amino acid that appeared at that position (eg, residue 33) and one or more amino acids that were not generally found at that position in non-binding clones There appears to be considerable denaturation of some residues in the binding clones that appear to be. For example, residue 23 in the binding clone is preferably the basic amino acid K 1 or R 2, representing a change from the neutral parent S residue, and K and R were not provided high at residue 23 in the unbound . Similarly, non-binding clones retained a higher percentage of parental clones at residue 31 than binding clones. Thus, in addition to the conserved 12-mer sequence, additional amino acids that are present in the generated binding clone, or preferably not present compared to the non-binding clone, are probably due to the unique EPOR binding of the clone. Contribute.
[0064]
Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described in the following examples. Standard methods that can be used by those skilled in the art to perform the genetic engineering and other methods described herein include the Molecular Cloning: A Laboratory Manual (Sambrook et al. Eds. Cold Spring Harbor Lab Publ. 1989) and Current Protocols in Molecular Biology (Ausubel et al. Eds., Greene Publishing Associates and wiley-Interscience 1987), as well as other well-known literature.
[0065]
Well-known immunoassay methods, such as solid phase immunoassays suitable for rapidly screening large numbers of peptides and other compounds are also known to those skilled in the art (Harlow & Lane, Antibodies, A Laboratory Manual, 1988, Cold Sprinng Harbor Laboratory, Cold Spring Harbor, NY, especially Chapt. 14, pp. 553-612).
The present invention may be better understood by the following examples, which are representative of preferred embodiments, but are not intended to limit the scope of the invention.
[0066]
Example 1 describes the construction of a plasmid cloning vector, pcDNA-IgG, encoding a portion of a chimeric IgG-EPOR protein used to probe a phage display library. This plasmid vector was designed to receive a polynucleotide cassette encoding a portion of the murine IgG1 heavy chain and encoding the ligand-binding domain of EPOR.
[0067]
Example
Example 1.mouse or rat IgG 1 H Chain C _H2 , C _H3 And an expression vector encoding a hinge domain
The plasmid vector, pcDNA 3, comprises neomycin and ampicillin drug resistance (selection marker) genes, multiple CalE1, f1 and SV40 origins, and multiple for HindIII, KpnI, BstXI, EcoRI, EcoRV, NotI, XhoI, XbaI and ApaI. A DNA fragment that contains an endonuclease recognition sequence in a cloning site-containing restriction where it is cloned into multiple cloning site regions can be expressed by using the CMV promoter contained in the vector sequence (Invitrogen Corp., San Diego CA) ).
[0068]
Plasmid pcDNA 3 was digested with NotI and XhoI restriction endonucleases and the digested product was used with TBE buffer (89 mM Tris, pH 8.0, 89 mM boric acid, 2 mM EDTA (ethylenediaminetetraacetic acid)). And separated by electrophoresis on a 1% agarose gel. The largest DNA fragment of the digest was gel purified, ethanol precipitated, pelleted and immediately dried. The dried pellets of purified DNA fragments were resuspended in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) and stored at −20 ° C. This linearized plasmid was used to receive a polynucleotide encoding a portion of a murine immunoglobulin (Ig) heavy chain.
[0069]
Polynucleotides encoding the murine IgG 1 heavy chain constant regions CH2, CH3 and the hinge domain were amplified from genomic DNA using a PCR protocol with primers having the following sequences:
The first strand primers were as follows (SEQ ID NO: 2):
5'-AGCTTCGAGCGGCCGCCGTGCCCAGGGATTGTGGTTGTAAG −3 '
The reverse primer was as follows (SEQ ID NO: 3):
5'-GATCCTCGAGTCATTTACCAGGAGAGTGGGAGAGGCT −3 '

[0070]
The underlined portion of SEQ ID NO: 2 corresponds to the NotI restriction endonuclease degradation site, and the bold underlined portion of SEQ ID NO: 3 corresponds to the XhoI restriction endonuclease degradation site.
Mouse genomic DNA was prepared from lysates of frozen NIH3T3 cells using standard experimental methods. In short, cells (5 × 10^Five ) Is pelleted by centrifugation, washed with phosphate buffer, hypotonic buffer containing 0.5% (v / v) nonionic detergent (Tween ™ -20) and 10 μg proteinase K (50 mM KCl, 10 mM Tris HCl (pH 8.4), 1.5 mM MgCl₂ ) Resuspended in 100 μl. The mixture was incubated at 56 ° C. for 45 minutes, heated to 95 ° C. for 10 minutes and then stored at 4 ° C.
[0071]
A PCR reaction to amplify the polynucleotide region encoding the murine IgG 1 heavy chain CH2, CH3 and hinge domains was prepared by combining the following reagents in 0.6 ml sterile microtubules in the following order: 10 μl 10 × PCR buffer II (100 mM Tris HCl (pH 8.3), 500 mM KCl), 6 μl 25 mM MgCl₂ 2 μl of 10 mM solution of individual dNTPs, 2.5 μl of 10 nM murine IgG 1 first strand primer (SEQ ID NO: 2), 2.5 μl of 10 nM murine IgG 1 reverse strand primer (SEQ ID NO: 3), 0.5 μl (2.5 units) Thermostable DNA polymerase (AMPLITAQ ™, Perkin Elmer Corp., Foster City CA), 66.5 μl ultrapure water, and one wax bead.
[0072]
After incubating the reaction mixture at 70 ° C. to melt the Max beads, 10 μl of lysate containing genomic DN template was added to the tube. The reaction mixture was incubated for 30 cycles as follows: 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1.5 minutes (Perkin Elmer 4BO Thermal Cycler). The completed reaction mixture was stored at 4 ° C. until use.
[0073]
Amplified DNA from the PCR reaction was gel purified by electrophoresis through a 1% agarose gel in TBE. The band corresponding to the amplified DNA was excised from the gel and eluted in 40 μl of water. The approximately 1 kb amplified IgG 1 gene fragment was then digested with NotI and XhoI restriction endonucleases and the digested product was electrophoresed on a 1% agarose / TBE gel. The approximately 1 kb DNA fragment was again purified from the gel and eluted in 40 μl of water. The yield of the purified fragment was determined by measuring the optical density of the solution at 260 nm (Beckman DU600 spectrometer).
[0074]
XhoI and NotI digested IgG 1 PCR products were incubated overnight at room temperature, 50 mM Tris-HCl (pH 7.8), 10 mM MgCl.₂ Ligation reaction containing about 100 ng of each pcDNA3 vector and IgG 1 amplified DNA fragment in 10 mM dithiothreitol (DTT), 1 mM ATP, 25 μg / ml bovine serum albumin (BSA) and 1 unit of DNA ligase Ligated in XhoI and NotI digested pcDNA3 vector in 20 μl.
[0075]
A 1 μl aliquot of the ligation mixture was used to transform competent E. coli cells (SURE ™ EPICUREAN COLI ™, Stratagene, La Jolla, CA) according to standard experimental methods. Transformants were selected by growth on ampicillin-containing LB plates. Plasmid DNA isolated from several independent clones was digested with NotI and XhoI and lysed on a 1% agarose / TBE analysis gel, the presence of a polynucleotide segment encoding a murine IgG1 invariant and hinge region Investigated about. Plasmid DNA from a clone containing NotI / XhoI was prepared for nucleic acid sequencing.
[0076]
Nucleic acid sequencing of NotI / XhoI inserts was performed using a dideoxy sequencing protocol. The sequencing reaction mixture was developed on a 4% acrylamide denaturing gel containing urea for 10 hours and the complete sequence of the fragments was determined. An insert in which one of the clones named pcDNA3-IgG1 (SEQ ID NO: 4) has the correct sequence (ie, murine IgG 1 C_H2, C_H3Large-scale plasmid preparation was performed.
[0077]
The pcDNA3-IgG1 expression vector was used to create a novel expression plasmid encoding a chimeric EPUR protein useful for probing phage display or other combinatorial libraries for ligands as described in Example 2. pcDNA3-IgG1 vector can be used as receptor DNA for sequences encoding other peptides, thereby allowing easy creation of other chimeric proteins useful in similar types of probing assays Will be understood by those skilled in the art. That is, the pcDNA3-IgG1 vector is a general purpose for producing a chimeric protein containing a portion of a mouse.
[0078]
The following example shows murine IgG 1 C_H2, C_H3And a method used to construct a plasmid expression vector encoding a chimeric protein having a hinge region and a ligand-binding domain of human EPOR.
[0079]
Example 2.EPOR Expression vector encoding a chimeric receptor incorporating a ligand binding domain
A DNA fragment encoding the extracellular portion of human RPOR was obtained by PCR amplification of cDNA synthesized from human leukocyte total RNA. RNA was reverse transcribed in a reaction mixture containing: 1 μg RNA, 12.5 mM individual dNTPs, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 5 mM DTT (dithiothreitol), 20 pmoles of random deoxyribonucleotide hexamer and 100 units of reverse transcriptase (SUPERSCRIPT ™, Life Technologies Gibco / BRL, Grand Island, NY). The mixture was incubated at 42 ° C. for 1 hour, heat treated at 95 ° C. for 5 minutes, and then stored at 4 ° C. until use. PCR reaction was performed using the following primers.
[0080]
First strand primers were as follows:
5'-GATCGGATCCATGGACCACCTCGGGGCGTCCCTC-3 ′ (SEQ ID NO: 5).
The reverse primer was as follows:
5′-AGCTTCGAGCGGCCGCGGGGTCCAGGTCGCTAGGCGTCAG-3 ′ (SEQ ID NO: 6).
The first strand primer (SEQ ID NO: 5) contains, in the amplification product, an ATG translation initiation codon (shown underlined above) and a BamHI recognition sequence (shown in bold above) located immediately upstream of the start codon. Incorporated. The reverse primer (SEQ ID NO: 6) introduced a NotI restriction endonuclease degradation site (shown in bold above) at the downstream end of the amplified fragment.
[0081]
An amplified EPOR DNA fragment that is run under conditions substantially as described in Example 1, but with primers of the sequences of SEQ ID NOs: 5 and 6, and having the sequence of SEQ ID NO: 7 Was generated. The amplified EPOR DNA fragment (ie, the PCR product) and pcDNA3-IgG1 plasmid are digested with BamHI and NotI, respectively, and the large DNA fragment of each reaction is gelled using the method as described in Example 1. Purified. The purified EPOR DNA fragment and plasmid vector were then ligated using ligation conditions substantially as described in Example 1 to produce a chimeric expression vector designated pcDNA3-IgG1-EPOR.
[0082]
This chimeric expression vector was transfected into competent E. coli cells using standard methods substantially as described in Example 1. RNA transcribed from the vector-loaded CMV promoter was translated in the transfected cells to produce a fusion protein having a domain corresponding to murine IgG1 and a cellular domain of human EPOR (ECD). Vector construction was verified by diagnostic restriction digestion and nucleic acid sequencing using standard methods. Large scale plasmid preparation was performed from transformed E. coli clones with pcDNA3-IgG1-EPOR plasmid.
[0083]
The following example describes the method used to produce the chimeric Ig-EPOR protein encoded by the pcDNA3-IgG1-EPOR plasmid in eukaryotic cells. Although COS-7 host cells have been used in these methods, one skilled in the art will appreciate that a variety of other cultured cells can be readily substituted.
[0084]
Example 3.pcDNA3 − IgG1 − EPOR Structure transfection and chimera Ig − EP OR Protein expression
COP-7 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4500 mg / l D-glucose, 584 mg / ml L-glutamine and 10% fetal bovine serum (FBS). In the transfection preparation, the cells are about 2-4 × 10 5 per T −150 in a 20 ml volume.^Five Inoculate with 5 cells and 5% CO to about 50% -70% confluency₂ Grow in an incubator humidified at 37 ° C under atmosphere.
[0085]
Plates are washed with serum free medium and 10 μg pcDNA3-IgG1-EPOR plasmid DNA comprising positively charged and neutral lipids (LIPOFECTAMINE ™, GibcoBRL, Grand Island, NY) in DMEM and 31 μl of standard transfection reagent mixture was added to the cells and then returned to the incubator for 5 hours. Subsequently, the transfection medium was replaced with DMEM and 10% FBS. Stable transfections were drug selected in the presence of G418 using standard experimental methods.
[0086]
After 48-72 hours, the cell-free medium was assayed for the presence of Ig-EPOR protein using the ELISA protocol described in the following example. The results of those assays indicated that the protein in the cell free supernatant contained one component that could bind to pure EPO and another component that could bind to the anti-IgG antibody. That is, the transfectant containing the pcDNA3-IgG1-EPOR plasmid expressed a chimeric protein containing functional IgG1 and EPOR regions.
[0087]
Example 4 describes the ELISA method used to confirm that the chimeric Ig-EPOR protein was secreted from cells transfected with the pcDNA3-IgG1-EPOR plasmid, and the chimeric protein was pure EPO and label The method used to show that the anti-IgG antibodies made can be bound simultaneously is described.
[0088]
Example 4.Chimera Ig − EPOR -To verify protein production and incorporationELISAProtocol
(1)Relative amount of secreted protein
The wells of a standard 96-well plate (IMMULON ™ 2, Dynatech) were coated with goat anti-murine IgG with hAd diluted 1 / 1,000 in PBS. After blocking the wells for non-specific binding with a solution of 1% BSA and 0.05% Tween ™ -20 in PBS, serially diluted samples of cell-free supernatant from the transfection were added to the wells. And allowed contact of the immobilized antibody.
[0089]
After removal of unbound chimeric protein by washing, anti-murine IgG1 detection antibody labeled with horseradish peroxidase (HRP) was added and allowed to bind the IgG portion of the chimeric protein. HRP-labeled antibody bound to the chimeric protein was detected using TMB peroxidase substrate (Kirkegard & Perry, Gaithersburg MD) by standard methods. The activity was calculated by spectrophotometry at 450 nm to 650 nm. That is, the concentration of the chimeric protein was calculated against a standard curve of known antibodies. In particular, measure the absorbance of each diluted solution at 450 nm and subtract the absorbance at 650 nm. Absorbance from non-specific sources (ie cell debris and dust) was excluded.
[0090]
The results of those methods indicated that the protein concentration ranged from 125 to 4,000 ng / ml, depending on the culture confluency and the frequency of supernatant harvest.
When confirming that the transformed cells secreted a protein with the same domain as the anti-murine IgG antibody, the inventors began to confirm that the chimeric protein could also bind the EPO ligand. It was.
[0091]
(2)Of the chimeric receptor EPO -Binding activity
The wells of a 96-well microtiter plate were coated with a 3.1 unit / ml solution of recombinant human EPO (R & D Systems, minneapolis MN) diluted in PBS. The wells were washed 3 times with PBS containing 0.05% (v / v) Tween ™ -20 nonionic surfactant and then 1% (w / v) in PBS (blocking buffer). ) Blocked with BSA and 0.05% Tween ™ -20 to prevent non-specific binding. Excess blocking buffer was removed by washing once.
[0092]
The culture medium was serially diluted with blocking buffer and 50 μl of each diluted solution was added to the coated and blocked wells. Wells receiving undiluted medium were used as controls. A set of uncoated wells also received diluted cell-free medium. The plates were then incubated for 2 hours at room temperature and washed three times as described above.
[0093]
After removal of the unbound chimeric receptor by washing, the bound chimeric protein was assayed essentially as described above with 100 μl of HRP labeled anti-murine IgG antibody. Color progression was initiated by the addition of 100 μl of TMB peroxidase substrate and the extent of peroxidase reaction was measured spectrophotometrically at 450 nm and 650 nm as described above.
The results of those methods indicated that the chimeric receptor protein bound to EPO and could be detected by using a labeled anti-murine IgG antibody. Control wells showed no color progression, whereas wells formed with the EPO / Ig-EPOR complex were clearly blue to purple.
[0094]
The results indicated that the chimeric IgG-EPOR protein was suitable as a probe for identifying EPOR ligands. That is, chimeric Ig-EPOR showed specific binding to EPO and also bound anti-murine IgG antibody.
The following example describes the method used to purify the chimeric Ig-EPOR protein used to identify EPOR ligands.
[0095]
Example 5.Chimera Ig − EPOR Protein purification
Sterile culture supernatant containing the chimeric receptor protein was loaded onto an anti-murine IgG1 affinity column (SEPHAROSE ™ affinity column, Zymed Laboratories, San Francisco CA) equilibrated with PBS. After loading, the column was washed with phosphate buffer solution (PBS) to remove non-specifically bound proteins. The protein bound to anti-IgG was removed from the column by eluting with a buffer containing 0.15 M NaCl and 0.1 M glycine (pH 2.4).
[0096]
Protein containing fractions were pooled, concentrated, and the buffer was exchanged into PBS using standard methods (CENTRICON ™ 50 cartridge, Amicon, Beverly, Mass.). The relative protein concentration of the Ig-EPOR chimera was determined using an IgG sandwich ELISA and known concentrations of antibody, all following standard experimental methods substantially as described in Example 4. Chimeric protein incorporation was determined by PAGE and silver staining (Bio-Red, Hercules CA) on 4% Tris-Glycine precast acrylamide gels and Ig-EPOR protein detected according to standard experimental methods To do this, immunoblotting was performed using HRP-labeled goat anti-murine IgG1.
[0097]
The results of those methods indicated that the major protein species had a molecular weight of about 59 KDa. Contaminated bands and degradation products on the gel were minimized. The binding kinematics investigated by Biacore analysis is based on known affinity values (0.1-1.1 x 10^-9k) in good agreement (Karlsson et al., 1991, J. Immunol. Methods 145 (1-2): 229-240). Ig-EPOR protein could be recovered in μg quantities using the method described above.
[0098]
Using the purified chimeric Ig-EPOR protein as a probe, the present inventors isolated a phage clone bound to EPOR by screening a phage display library. The protein probe is immobilized on magnetic beads, although other solid supports can be used. The magnetic beads were used because the high surface area of the matrix facilitates effective contact between the immobilized probe and the phage, and magnetic separation allows for effective washing between processing steps. It is.
Example 6 describes the method used to isolate a recombinant phage expressing a fusion protein capable of binding a chimeric Ig-EPOR probe.
[0099]
Example 6.Biopanning method and magnetic beads ELISA
Four M13 phage display libraries expressing random peptides as pIII fusion proteins were each screened using biopanning with Ig-EPOR probes. Those libraries designated as XC8, XC13, X38, XC43 were constructed using the method described above (McConnell et al., Molec. Diversity 1: 165-176, 1996). The XC8 and XC13 libraries showed random amino acid sequences of 8 and 13 residues, respectively, with cysteine residues at the ends.
[0100]
The XC38 library expressed a fusion protein showing 37 random amino acids incorporating an invariant alanine at position 22 in the peptide sequence. The XC43 library shows a 43-mer peptide comprising 40 random amino acids surrounding the invariant Gly-Gys-Gly sequence at residues 21, 22 and 23, which is an intramolecular disulfide loop of the indicated peptide Made possible the formation of.
[0101]
The phage library was screened for EPOR binding clones by standard biopanning methods using the chimeric Ig-EPOR protein as a probe. Biopanning was performed using chimeric receptor proteins immobilized on magnetic beads (DYNABEADS ™ M-45 rat anti-murine IgG 1 beads, Dynal, Inc.) by standard methods. Chimeric Ig-EPOR protein (500 ng / ml) contained in 10 ml of clear supernatant from cells transfected with pcDNA3-IgG 1-EPOR plasmid was mixed with 400 μl of magnetic beads and the mixture was shaken gently Incubate overnight at 4 ° C.
[0102]
Subsequently, the beads were blocked for 1 hour in a greenhouse in standard Tris-buffer solution blocking medium (TBS SUPERBLOCK ™, Pierce Chemical, Chicago IL). The beads were further treated for 1 hour at room temperature by the addition of an Fc fragment of murine IgG (10 μg / ml) to block high affinity sites that non-specifically bind the IgG domain of the chimeric probe. The beads were washed three times with TBST (Tris buffer solution containing 0.1% (v / v) Tween ™ -20) and then suspended in 1 ml blocking medium (TBS SUPERBLOCk ™). About 1 × 10^TenPhages in 100 μl aliquots containing plaque forming units (pfu) were added to the beads and the mixture was lightly spun for 2 hours at room temperature.
[0103]
After washing the beads five times with TBST, the phages were eluted with 30 μl of 500 mM glycine buffer (pH 2.2) for 10 minutes at room temperature. The eluted phage was immediately neutralized with 8 μl of 2M Tris base without pH adjustment, mixed and stored at 4 ° C. Phage isolated by biopanning was mixed with an equal volume of eluted phage and an overnight culture of E. coli strain JS5, and the mixture was mixed with 2 × YT (12.0 μg / ml) containing tetracycline (12.0 μg / ml). 20 ml) and amplified by incubating overnight at 37 ° C. with shaking.
[0104]
The resulting phage lysate was freed of cell debris by centrifugation for 5 minutes in microtubules, and 100 μl of the resulting supernatant lysate was used for subsequent biopanning. All phage titrations were performed using 2xYT upper agar and JS5 bacteria plated on agar plates.
[0105]
The results of the library screening method showed that a phage clone isolated from one of the four libraries bound the chimeric Ig-EPOR probe. This clone, designated ERB 1 (for “EPO receptor binding agent 1”), had DNA encoding the pIII fusion protein bound to the probe. The ERB 1 clone contained a polynucleotide having the sequence of SEQ ID NO: 8 fused to the sequence encoding the pIII coat protein. The amino acid sequence of the EPOR-binding peptide contained within the ERB 1 clone was as follows:
[0106]
DFDVCRRGWVGHCKDWLSDEYASNPSYPVPHSYYLNPP (SEQ ID NO: 9). Although the amino acid sequence of the ERB1-encoded peptide is unrelated to the amino acid sequence of EPO, the ERB 1 phage clone specifically bound to the IgEPOR protein.
A magnetic bead ELISA protocol was used to confirm that the ERB 1 phage clone bound to the chimeric Ig-EPOR probe by a specific interaction. The chimeric Ig-EPOR probe was prepared as described above in a sample (100 and 100%) of a 1: 2 dilution of phage lysate (PBS and 1% BSA) bound to magnetic beads representing rat anti-murine IgG1. μl) were combined with the beads and the phage allowed the binding of the immobilized probe for 1 hour at room temperature.
[0107]
The phage used in this method were ERB 1 and two negative control phages: HPKF, a clone that specifically binds to the commercially available anti-IL-8 mAb, and IL6-B26. That is, a clone that specifically binds to the human IL-6 receptor. Unbound phage was removed by washing 4 times with 200 μl aliquots of TBST (25 nM Tris HCl, pH 7.2, 0.15 M NaCl, 0.1% TweenR-20), then 1: 1 in PBS. HRP-conjugated anti-M13 detection antibody (Pharmacia) diluted to 5,000 was added.
[0108]
After incubation (1 hour at room temperature), excess detection antibody is removed by washing, and 10 μl of each washed bead complex is added to a microtiter well (FALCON ™ 3912, MICROTEST III, Beckton Dicknson, Oxnard CA). Individual wells were developed with TMB peroxidase substrate and the extent of the peroxidase reaction was measured spectrophotometrically as described in Example 4.
[0109]
The results shown in FIG. 1 showed that ERB1 phage clones and Ig-EPOR probes were particularly elevated. The inverse relationship between the optical density (OD) read in the ELISA assay and the extent of phage dilution indicates that progressively more diluted samples of ERB1 phage resulted in correspondingly lower binding of anti-M13 detection antibodies showed that. Control methods showed that the ERB 1 clone did not bind to the magnetic beads alone or to the extracellular domain of the receptor for tumor necrosis factor (INFR) or interleukin-6 (IL-6R) ( Results are not shown). Furthermore, control phage with specificity for either anti-IL-8 mAb or IL-6 receptor did not show binding to the Ig-EPOR probe. Therefore, the ERB 1 clone specifically bound to the Ig-EPOR probe.
[0110]
The inventors also confirmed that the interaction between the Ig-EPOR probe and the ERB 1 phage clone included the probe's ligand binding domain. To make this determination, magnetic beads were first coated with a negative control that was a chimeric Ig-EPOR protein, or a chimeric Ig-IL-6 receptor protein, substantially using the method described above (Brown et al.,. 1997, “A versitile expression system for the extracellular somain of type I cytokine receptors” (submitted for publication)).
[0111]
About 1 × 10⁸pfu ERB1 and IL6-B26 phage clones were each mixed with increasing amounts of EPO and the mixture was contacted with the coated beads. After washing to remove unbound phage and EPO, HRP-conjugated anti-M13 detection antibody is added to all samples and incubated (1 hour at room temperature) and then excess The detection antibody was removed by washing. Washed bead complexes (10 μl) were transferred to 96-well microtiter wells, developed with TMB peroxidase, and the reaction was measured spectrophotometrically, substantially as described above.
[0112]
The results shown in FIG. 2 confirmed that both the ERB 1 phage clone and EPO bound to the ligand binding domain of the Ig-EPOR probe. The results indicated that increasing concentrations of EPO competed with ERB 1 for binding to the probe. Increasing the concentration of EPO reduced the number of ERB 1 phage clones bound to the probe. Increasing concentrations of EPO did not inhibit unrelated phage / probe combination binding. Thus, the interaction between the ERB1 phage clone and Ig-EPOR was specific and could be competed with pure EPO ligand. In addition, the chimeric Ig-EPOR probe described herein displayed the binding properties of EPOR, and molecules that bound the chimeric probe were expected to bind to EPOR.
[0113]
Generated to obtain phage clones that are similar to or have a higher affinity for EPOR than the ERB1 clone and to identify subsequences within the ERB1 fusion protein that contribute to EPOR binding A library was created using saturated oligo or nucleotide doping mutagenesis of the original ERB1 sequence, and the generated library was screened using a chimeric Ig-EPOR probe. The following example describes the method used to create and screen a phage display library of 38-mer peptides related to fusion proteins expressed by ERB1 phage clones.
Example 7.ERB1 Evolution library construction and screening
The evolved library was constructed using methods substantially as described above (McCOnnell et al., 1996, Molecular Diversity 1: 165). Briefly, the inventors have found that codons corresponding to individual amino acid positions of the 38-mer ERB1 EPOR-binding peptide are each replaced with random amino acids at a predetermined frequency (provided that the DEY internals near the center of the ERB1 sequence). A collection of oligonucleotides was synthesized so that the amino acid sequence was removed). Using these methods, codons that represent random amino acid substitutions at any single position in the 38-mer peptide, except for the amino acid encoding the DEY tripeptide, occur at a frequency of about 50%. It was predicted.
[0114]
Polynucleotides were synthesized according to the doping scheme shown in FIG. Briefly, two collections of redundant oligonucleotides represented by the oligonucleotides of FIG. 3A (SEQ ID NOs: 10 and 11) are represented by short complements where the two groups of oligonucleotides are present at their 3 ′ ends. The target sequences were synthesized so that they could be annealed to each other. Their complementary sequences encode the internal D-E-Y amino acids of the ERB 1 peptide and thus retain this tripeptide in individual clones of the resulting development library.
[0115]
The complementary strands of the annealed oligonucleotide population were then synthesized by standard in vitro DNA extension methods to create a population of double stranded DNA fragments. Those DNA fragments are cloned into an appropriate M13 vector (eg, McConell et al., MsM1 above) to generate a phage display library that expresses the pIII fusion protein, where each fusion protein is an ERB1 peptide sequence N-terminal 38-mer peptide derived from
[0116]
Due to the first two positions of the individual codons of the ERB1 oligonucleotide, the redundant oligonucleotide is 73% of the same nucleotide relative to the nucleotide used in the position of the ERB1 oligonucleotide sequence and the other three nucleotides. Of 9% of the product. The nucleotide composition corresponding to the individual postponement shown at the bottom in the redundant oligonucleotide sequence is shown in FIG. 3B. The third position of each codon (indicated by “S” in FIGS. 3A and 3B) was synthesized using an equimolar mixture of dGTP and dCTP.
[0117]
This doping scheme produced nucleotide triplets encoding the original amino acid at each position of the ERB1 38 -mer peptide about 50% of that time. The remaining 50% of the time, ie the codons that did not encode the original amino acid, were replaced with a random mixture of the other 19 amino acids.
[0118]
The resulting ERB1 evolution library was screened by biopanning, substantially as described in Example 6, to identify clones that bound the chimeric Ig-EPOR probe. Several random phage clones were also isolated from the evolved library to represent unbound clones. Approximately 88% of clones selected from the panned group are positive for EPOR binding, as determined by the results of the EPOR-specific magnetic bead ELISA test (as described in Example 6). All of the randomly selected clones from the non-group were negative for EPOR binding.
[0119]
This is because randomly selected clones in the generated library are unlikely to bind EPOR, but clones selected by the panning method showed very high frequency of EPOR binding. Accordingly, the screening methods described herein are useful for identifying and isolating phage clones that exhibit ligand functionality; in this case, the clones can bind to EPOR.
[0120]
A total of 48 binding (panned group) and 24 non-binding (non-panned group) clones were isolated from the generated library. The DNA sequence encoding the 38-mer region of the fusion peptide of those clones was determined by dideoxy sequencing using standard methods. Among those 72 isolates, 34 unique sequences were found (21 sequences for the panned group and 13 sequences for the non-panned group).
[0121]
Those 34 unique DNA sequences are disclosed in SEQ ID NOs: 13-46, and the amino acid sequences predicted to be encoded by those sequences are disclosed in SEQ ID NOs: 47-80. Their peptide sequences are shown in FIG. A total of 21 different binding clones, designated B1-B21 (SEQ ID NO: 47-67), were isolated from the first 48 selected binding clones; and as N1-N13 (SEQ ID NO: 68-80) The 13 named different non-binding clones were isolated from the first 24 non-binding clones selected.
[0122]
From the aligned peptide sequences of EPOR-binding and non-binding clones, a consensus sequence that was present in all binding clones and absent in non-binding clones was determined. The consensus sequence is CXXGWVGXCXXW (SEQ ID NO: 12), where X is the various amino acids, if the mutagenesis scheme used (see Figure 3A) eliminates the DEY tripeptide present in all of the clones. Indicates an acceptable position). Invariant residues in the consensus sequence found in binding clones are shown in bold in FIG. In non-binding clones, some invariant residues were also present (underlined in FIG. 4), but there was no complete motif of the consensus sequence.
[0123]
All of the bound and unbound clones contained the same pIII protein sequence, and the binding of the phage clone to the EPOR probe did not depend on the interaction between the probe and the pIII sequence. Instead, the binding of the phage clone to the Ig-EPOR probe should be due to the additional peptide sequence of the fusion protein (eg, those sequences shown for clones B1-B21 in FIG. 4). Thus, a peptide comprising a 12-mer consensus sequence bound to the ligand binding domain of human EPOR, and a peptide comprising the sequence of SEQ ID NO: 12 represented an EPO peptide mimetic.
[0124]
In order to better characterize phage isolates with binding specificity for EPOR, we compared the relative affinities of the binding clones shown in FIG. 4 using a phage ELISA method. The phage stock of the binding clones was dropped in triplicate to accurately determine the pfu / ml concentration for each lysate. Different isolates were then assayed by ELISA using standardized phage concentrations, so that all clones were tested with an equivalent number of phage. The optical density in the ELISA corresponding to the 50% maximum response in the ERB1 test was then determined for each of the different phage isolates.
[0125]
Optical density per phage at 50% ERB 1 maximum response x 1 x 10⁸ Gave a value of 1.0 for ERB1 and represented a standard for measuring the relative binding affinity of B 1 -B21 isolates. The normalized relative affinity ("Rel. Aff.") For each clone is shown to the right of the peptide sequence of the clone in FIG. For each of the non-binding clones, its relative affinity is listed as <0.1 since they are negative for EPOR binding.
[0126]
5A-5D graphically show the ELISA results for clones B1-B2. Several clones, including B7 and B8, bound chimeric Ig-EPOR protein with an affinity about 20 times higher than that of the parent ERB 1 clone. Other clones including B3, B15 and B19 bind EPOR with affinity similar to ERB1, and the two clones (B20 and B21) have somewhat lower binding affinity compared to ERB1. Had.
[0127]
Its different relative affinities are probably due to amino acid differences between different clones. Thus, many binding clones isolated from the generated library bound to the ligand binding domain of the human EPO receptor with an increased affinity compared to the ERB1 parental clone. All of the binding clones had a peptide containing the 12-mer consensus sequence identified by SEQ ID NO: 12.
The following example describes the method used above to show that a synthetic peptide having an amino acid sequence derived from the sequence of the clone competes with pure EPO for binding to Ig-EPOR.
[0128]
Example 8.Peptide inhibition ELISA
This example shows that a synthetic peptide comprising the consensus sequence of SEQ ID NO: 12 competes with EPO for EPOR binding. Since the consensus sequence is located on the amino-terminal side chain of the conserved DEY residue, the peptide is synthesized based on the amino-terminal sequences of two typical clones that exhibit high affinity binding for the chimeric Ig-EPOR probe. It was done. The peptide is synthesized in a circular form and contains a sequence corresponding to the amino terminal 18 amino acids of B7 and B8 clones.
[0129]
A synthetic peptide having the sequences DREGCRRGWVGQCKAWFN (SEQ ID NO: 81) and DVEACGGGWVGHCNYWLR (SEQ ID NO: 82) was synthesized in a cyclic (disulfide linked) form using standard methods (Peninsula Laboratories, Belmont, CA). The 18-mer peptide sequences are derived from B7 and B8 clones, and these peptides are referred to herein as “ERB1-7” (SEQ ID NO: 81) and “ERB1-8” (SEQ ID NO: 82), respectively. The The structures of the two purified peptides, each 95% or more pure as judged by HPLC, were confirmed by mass spectrometry.
[0130]
The peptides were tested separately in the EPO-specific sandwich ELISA protocol for their ability to inhibit Ig-EPOR protein binding on EPO-coated polystyrene plates. The results shown in FIG. 6 indicate that ERB1-7 and ERB1-8 synthetic peptides competed with EPO to bind to the chimeric Ig-EPOR protein. Both peptides have an IC of about 45 nM in the assay.₅₀Had a value.
[0131]
This is where a synthetic 18-mer peptide (see FIG. 4) derived from the sequence of the binding clone, including the consensus 12-mer sequence identified above, is removed from the contents of the M13pIII fusion protein. Even proved to have joined EPOR. The results show that the consensus 12-mer sequence (SEQ ID NO: 12) and an 18-mer peptide comprising 4 amino acids on the amino acid terminal side of the consensus sequence and 2 amino acids on the carboxyl terminus are EPOR binding compositions. Support a conclusion. Such synthetic peptides are EPO mimetics.
[0132]
In order to identify the size limit of functional EPO peptidomimetics, a shorter form of ERB1-7 was synthesized and tested to determine the relationship between peptide size and receptor binding activity. As described in the following examples, a series of N-terminal truncations of ERB1-7 were synthesized and tested in the EPO specific inhibition ELISA described above.
[0133]
Example 9.12 -Residues outside of the consensus are synthetic peptides EPOR Required for high-affinity interaction between
This example shows that amino acid residues located outside the limits of the 12-mer consensus sequence identified above contribute to the ability of the synthetic peptide to bind the Ig-EPOR probe. Synthetic peptides corresponding to the sequence of ERB1-7 but lacking either 1, 2, 3 or 4 N-terminal amino acids were tested in a peptide inhibition ELISA protocol. The following peptides were synthesized and tested substantially as described in Example 8:
[0134]
18-mer peptide ERB1-7 consisting of DREGCRRGWVGQCKAWFN (SEQ ID NO: 81);
17-mer peptide 7N-1 consisting of REGCRRGWVGQCKAWFN (SEQ ID NO: 83);
16-mer peptide 7N-2 consisting of EGCRRGWVGQCKAWFN (SEQ ID NO: 84);
15-mer peptide 7N-3 consisting of GCRRGWVGQCKAWFN (SEQ ID NO: 85);
14-mer peptide 7N-4 consisting of CRRGWVGQCKAWFN (SEQ ID NO: 86).
[0135]
The results shown in FIG. 7 show that other amino acids of the 12-mer consensus contribute to effective competition with EPO to bind to the Ig-EPOR protein. That is, the IC for the 18-mer₅₀The value is about 45 mM, whereas the 17-mer peptide has a reduced IC of 91 nM.₅₀showed that. Furthermore, the cut is about 3700nM IC₅₀A 16-mer with Additional cleaves (ie, 15-mer and 14-mer peptides) completely abolished EPOR binding activity.
[0136]
The cleavage of ERB1-7 peptide not only interferes with the physical interaction between the peptide and Ig-EPOR, but also interferes with the biological activity of the peptide, as described in Example 10. Therefore, the consensus 12-mer peptide given by SEQ ID NO: 12 is required, but not sufficient to create a synthetic peptide with high affinity for EPOR.
The following example describes a method used to illustrate that a synthetic peptide conjugated with EPOR also showed EPO-like pharmacological activity in an in vitro cell culture system.
[0137]
Example Ten.ERB1 And the generated peptide is EPO -Stimulate proliferation in responding cell lines
To assess the biological activity of two representative synthetic peptides containing 12-mer consensus sequences, the peptides ERB1-7 and ERB1-8 were tested for proliferation of the EPO-responsive cell line (TF-1). We tested for their ability to stimulate. One skilled in the art will appreciate that the TF-1 cell line is useful for detecting and quantifying EPO biological activity (although other EPO-responsive cells can also be substituted). The TF-1 cell line was established from a heparinized bone marrow aspirate isolated from a 35 year old male with pancytopenia (Kitamura et al., 1989, J. Physiol. 140: 323).
[0138]
TF-1 cells were grown in RPMI 1640 with 10% BCS and 5 ng / ml CM-CSF. After washing twice with PBS to remove residual GM-CSF, 5,000 cells are placed in individual wells of a 96-well microtiter plate in the presence of 0.5 units of EPO / ml and standard The cells were cultured under conditions for 3 days (Kitamura et al., 1989, Blood 73: 375-380).
[0139]
Growth in response to EPO was determined by standard XXT assay (sodium 3 '-(-1- (phenylaminocarbonyl) -3,4-tetrazolium) -bis (4-methoxy-6-nitro) benzenesulfonic acid hydration. Product-based assay; Promega, Madison WI). According to a similar method, ERB1-7 (SEQ ID NO: 81) or ERB1-8 synthetic peptide (SEQ ID NO: 82) was used instead of EPO. Also, as a negative control, an IL-6 peptide having the following sequence was tested:
GGAFCEAVGCGPDRNFYGG (SEQ ID NO: 87). Peptide concentrations were tested over a 1,000 culture concentration range of 0.02 to 20 μM.
[0140]
The results shown in FIGS. 8A and 8B show that the synthetic peptide conjugated with EPOR also showed EPO-like pharmacological activity in biological assays. A positive control, ie purified EPO, confirmed that TF-1 cells were EPO responsive. TF-1 cells with half maximal response (ED50) in EPO ranging from 0.05 to 0.1 units / ml proliferated (FIG. 8A). Both peptides were EPOR agonists that produced a proliferative response in a dose-dependent manner in TF-1 cells at concentrations of 1-10 μM (FIG. 8B). In contrast, inappropriate IL-6 peptides that did not bind EPOR did not stimulate TF-1 cell proliferation at any tested concentration up to 20 μM.
[0141]
This indicated that non-specific interactions in the assay did not contribute to the growth results found by the synthetic EPO mimetic. Thus, a synthetic peptide containing the consensus 12-mer SEQ ID NO: 12 and flanking amino acids showed biological activity similar to EPO. While particular embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that the embodiments are illustrative and do not limit the scope of the invention.
[0142]
[Sequence Listing]
(1) General information:
(I) Applicant: McCONNELL, Stephen, J. and SPINELLA, Dominic, G.
(Ii) Title of invention: Peptide ligand for ectropoietin receptor
(Iii) Number of sequences: 87
(2) Information about SEQ ID NO: 1
(I) Sequence features:
(A) Length: 14 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 1:

[0143]
(2) Information about SEQ ID NO: 2
(I) Sequence features:
(A) Length: 41 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 2:

[0144]
(2) Information about SEQ ID NO: 3
(I) Sequence features:
(A) Length: 37 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: cDNA
(Xi) Sequence: SEQ ID NO: 3:

[0145]
(2) Information about SEQ ID NO: 4:
(I) Sequence features:
(A) Length: 6338 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 4:
[Chemical 1]

[Chemical formula 2]

[Chemical 3]

[0146]
(2) Information about SEQ ID NO: 5
(I) Sequence features:
(A) Length: 34 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 5

[0147]
(2) Information about SEQ ID NO: 6:
(I) Sequence features:
(A) Length: 40 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 6

[0148]
(2) Information about SEQ ID NO: 7
(I) Sequence features:
(A) Length: 750 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 7
[Formula 4]

[0149]
(2) Information about SEQ ID NO: 8:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 8
[Chemical formula 5]

[0150]
(2) Information about SEQ ID NO: 9:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 9:
[Chemical 6]

[0151]
(2) Information about SEQ ID NO: 10:
(I) Sequence features:
(A) Length: 63 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 10:
[Chemical 7]

[0152]
(2) Information about SEQ ID NO: 11
(I) Sequence features:
(A) Length: 60 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 11:
[Chemical 8]

[0153]
(2) Information about SEQ ID NO: 12:
(I) Sequence features:
(A) Length: 12 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 12:

[0154]
(2) Information about SEQ ID NO: 13:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 13:
[Chemical 9]

[0155]
(2) Information about SEQ ID NO: 14:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 14:
Embedded image

[0156]
(2) Information about SEQ ID NO: 15:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 15
Embedded image

[0157]
(2) Information about SEQ ID NO: 16:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 16
Embedded image

[0158]
(2) Information about SEQ ID NO: 17:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 17:
Embedded image

[0159]
(2) Information about SEQ ID NO: 18:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 18
Embedded image

[0160]
(2) Information about SEQ ID NO: 19:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 19
Embedded image

[0161]
(2) Information about SEQ ID NO: 20:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 20:
Embedded image

[0162]
(2) Information about SEQ ID NO: 21:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 21:
Embedded image

[0163]
(2) Information about SEQ ID NO: 22:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 22:
Embedded image

[0164]
(2) Information about SEQ ID NO: 23:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 23:
Embedded image

[0165]
(2) Information about SEQ ID NO: 24:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 24:
Embedded image

[0166]
(2) Information about SEQ ID NO: 25:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 25:
Embedded image

[0167]
(2) Information about SEQ ID NO: 26:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 26:
Embedded image

[0168]
(2) Information about SEQ ID NO: 27:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 27
Embedded image

[0169]
(2) Information about SEQ ID NO: 28:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 28:
Embedded image

[0170]
(2) Information about SEQ ID NO: 29:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 29:
Embedded image

[0171]
(2) Information about SEQ ID NO: 30:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 30:
Embedded image

[0172]
(2) Information about SEQ ID NO: 31:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 31
Embedded image

[0173]
(2) Information about SEQ ID NO: 32:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 32:
Embedded image

[0174]
(2) Information about SEQ ID NO: 33:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 33:
Embedded image

[0175]
(2) Information about SEQ ID NO: 34:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 34:
Embedded image

[0176]
(2) Information about SEQ ID NO: 35:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 35:
Embedded image

[0177]
(2) Information about SEQ ID NO: 36:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 36:
Embedded image

[0178]
(2) Information about SEQ ID NO: 37:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 37:
Embedded image

[0179]
(2) Information about SEQ ID NO: 38:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 38:
Embedded image

[0180]
(2) Information about SEQ ID NO: 39:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 39:
Embedded image

[0181]
(2) Information about SEQ ID NO: 40:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 40:
Embedded image

[0182]
(2) Information about SEQ ID NO: 41:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 41
Embedded image

[0183]
(2) Information about SEQ ID NO: 42:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 42:
Embedded image

[0184]
(2) Information about SEQ ID NO: 43:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 43:
Embedded image

[0185]
(2) Information about SEQ ID NO: 44:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 44:
Embedded image

[0186]
(2) Information about SEQ ID NO: 45:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 45:
Embedded image

[0187]
(2) Information about SEQ ID NO: 46:
(I) Sequence features:
(A) Length: 114 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Xi) Sequence: SEQ ID NO: 46:
Embedded image

[0188]
(2) Information about SEQ ID NO: 47:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 47:
Embedded image

[0189]
(2) Information about SEQ ID NO: 48:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 48:
Embedded image

[0190]
(2) Information about SEQ ID NO: 49:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 49:
Embedded image

[0191]
(2) Information about SEQ ID NO: 50:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 50:
Embedded image

[0192]
(2) Information about SEQ ID NO: 51:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 51:
Embedded image

[0193]
(2) Information about SEQ ID NO: 52:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 52:
Embedded image

[0194]
(2) Information about SEQ ID NO: 53:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 53:
Embedded image

[0195]
(2) Information about SEQ ID NO: 54:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 54:
Embedded image

[0196]
(2) Information about SEQ ID NO: 55:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 55:
Embedded image

[0197]
(2) Information about SEQ ID NO: 56:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 56:
Embedded image

[0198]
(2) Information about SEQ ID NO: 57:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 57:
Embedded image

[0199]
(2) Information about SEQ ID NO: 58:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 58
Embedded image

[0200]
(2) Information about SEQ ID NO: 59:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 59:
Embedded image

[0201]
(2) Information about SEQ ID NO: 60:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 60:
Embedded image

[0202]
(2) Information about SEQ ID NO: 61:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 61:
Embedded image

[0203]
(2) Information about SEQ ID NO: 62:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 62:
Embedded image

[0204]
(2) Information about SEQ ID NO: 63:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 63:
Embedded image

[0205]
(2) Information about SEQ ID NO: 64:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 64:
Embedded image

[0206]
(2) Information about SEQ ID NO: 65:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 65:
Embedded image

[0207]
(2) Information about SEQ ID NO: 66:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 66
Embedded image

[0208]
(2) Information about SEQ ID NO: 67:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 67:
Embedded image

[0209]
(2) Information about SEQ ID NO: 68:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 68:
Embedded image

[0210]
(2) Information about SEQ ID NO: 69:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 69:
Embedded image

[0211]
(2) Information about SEQ ID NO: 70:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 70:
Embedded image

[0212]
(2) Information about SEQ ID NO: 71:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 71:
Embedded image

[0213]
(2) Information about SEQ ID NO: 72:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 72:
Embedded image

[0214]
(2) Information about SEQ ID NO: 73:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 73:
Embedded image

[0215]
(2) Information about SEQ ID NO: 74:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 74
Embedded image

[0216]
(2) Information about SEQ ID NO: 75:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 75:
Embedded image

[0217]
(2) Information about SEQ ID NO: 76:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 76:
Embedded image

[0218]
(2) Information about SEQ ID NO: 77:
(I) Sequence features:
(A) Length: 37 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 77:
Embedded image

[0219]
(2) Information about SEQ ID NO: 78:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 78:
Embedded image

[0220]
(2) Information about SEQ ID NO: 79:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 79:
Embedded image

[0221]
(2) Information about SEQ ID NO: 80:
(I) Sequence features:
(A) Length: 38 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 80
Embedded image

[0222]
(2) Information about SEQ ID NO: 81:
(I) Sequence features:
(A) Length: 18 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 81

[0223]
(2) Information about SEQ ID NO: 82
(I) Sequence features:
(A) Length: 18 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 82:

[0224]
(2) Information about SEQ ID NO: 83:
(I) Sequence features:
(A) Length: 17 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 83:

[0225]
(2) Information about SEQ ID NO: 84:
(I) Sequence features:
(A) Length: 16 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 84:

[0226]
(2) Information about SEQ ID NO: 85:
(I) Sequence features:
(A) Length: 15 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 85:

[0227]
(2) Information about SEQ ID NO: 86:
(I) Sequence features:
(A) Length: 14 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 86:

[0228]
(2) Information about SEQ ID NO: 87:
(I) Sequence features:
(A) Length: 19 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: pear
(Xi) Sequence: SEQ ID NO: 87:

[Brief description of the drawings]
FIG. 1 is a line graph showing results from an EPOR-specific phage ELISA that confirms that an ERB 1 phage clone specifically binds to a chimeric Ig-EPOR probe. Graph shows phage clones isolated using Ig-EPOR probe (ERB1, black triangle); phage clones that specifically bind to anti-IL-8 antibody (HPKF, ◆); and human IL-6 Phage clones that specifically bind to the receptor (IL-6-B26, black squares) show binding to Ig-EPOR as measured by ELISA (OD) at different phage dilutions.
FIG. 2 shows that recombinant EPO at different concentrations (units / ml) competes with ERB1 phage (♦) to bind to Ig-EPOR, but binds to IL-6 receptor. It is a line graph which shows the result of ELISA (OD) which had no effect on (●).
FIG. 3A shows the approach used to create an ERB1-generated library. FIG. 3A shows the amino acid sequence of the peptide fused to the pIII protein in the ERB1 clone on the first line, and the redundancy used to create the generated library of ERB1 clones on the second and third lines. The sequence of the oligonucleotide collection is shown. The oligonucleotides were aligned to show codons corresponding to amino acids in the ERB1 peptide and to show annealing of the oligonucleotide with 9 complementary bases at the 3 ′ end of the sequence.
FIG. 3B shows the approach used to create the ERB1-generated library. FIG. 3B was used to synthesize redundant oligonucleotides for the individual bases represented as “g”, “a”, “t”, “c” and “s” in the redundant oligonucleotide. The nucleotide composition is shown. The amino acid sequence of the peptide fused to the pIII protein in the ERB1 clone and the scheme used to create a collection of oligonucleotides encoding variants of the ERB1 peptide sequence are shown.
FIG. 4 shows the amino acid sequence of phage clones derived from the ERB1-generated library and the relative affinity of the clones for Ig-EPOR. Clones B1-B21 were isolated by binding to Ig-EPOR and phage clones N1-N13 were derived from the generated library but did not bind to Ig-EPOR during the selection process. Relative affinity is a numerical summary of the data shown in Figures 5A-5D.
FIG. 5A is a series of line graphs showing the affinity of several independent phage clones for Ig-EROP with respect to the original ERB1 isolate. FIG. 5A shows the results for clones B1-B5 and ERB1.
FIG. 5B is a series of line graphs showing the affinity of several independent phage clones for Ig-EROP with respect to the original ERB1 isolate. FIG. 5B shows the results for clones B6-B10 and ERB1.
FIG. 5c is a series of line graphs showing the affinity of several independent phage clones for Ig-EROP with respect to the original ERB1 isolate. FIG. 5C shows the results for clones B11-B15 and ERB1.
FIG. 5D is a series of line graphs showing the affinity of several independent phage clones for Ig-EROP with respect to the original ERB1 isolate. FIG. 5D shows the results for B16-B21 and ERB1.
FIG. 6 shows the absence of added peptide (♦) and addition of inappropriate peptide binding to IL-6 receptor (×) when chimeric Ig-EPOR probe binding to immobilized EPO. Is a line graph showing the results from a peptide inhibition ELISA that is inhibited by a competitor peptide (peptide ERB1-7, black square; peptide ERB1-8, ●).
FIG. 7 shows the N-terminal truncated ERB1-7 peptide (17-mer, black square; 16-mer, black triangle; 15-mer ×; 14-mer, *) to Ig-EPOR. ERB 1-7 18-mer peptide to Ig-EPOR (compared to binding and compared to the addition of inappropriate peptide binding to IL-6 receptor or absence of added inhibitor (+)) It is a line graph which shows the coupling | bonding of ().
FIG. 8A is a line graph showing results from a TF-1 cell amplification assay. FIG. 8A shows a dose response curve for the growth of TF-1 cells (OD at 450 nm) at different concentrations (units) of recombinant EPO.
FIG. 8B is a line graph showing the results from the TF-1 cell amplification assay. FIG. 8B shows different concentrations (μM) of synthetic peptides ERB1-7 (black squares) and ERB1-8 (black triangles) compared to the addition of inappropriate peptides that bind to the IL-6 receptor (×). Shows the dose response curve for the growth of TF-1 cells (OD at 450 nm) in

Claims (22)

Following formula:
X _n -C ₁ -X ₁ -X ₂ -GWVGX ₃ -C ₂ -X ₄ -X ₅ -WX _c
[Where X _n is X _n1 -X _n2 -X _n3 -X _n4 , where
X _n1 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural α-amino acids, or, optionally, X _n1 is absent;
X _n2 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and basic natural α-amino acids, or optionally, when X _n1 is absent, X _n2 is absent;
X _n3 is selected from the group consisting of natural α-amino acids; and
X _n4 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural α-amino acids;
X ₁ is a neutral and hydrophobic, neutral and polar, or basic amino acid;
X ₂ is a neutral and hydrophobic, neutral and polar or basic amino acid;
X ₃ is a neutral and polar or basic amino acid;
X ₄ is a neutral and polar, basic or acidic amino acid;
X ₅ is a neutral and polar, neutral and hydrophobic, or acidic amino acid; and
X _c is X _c1 -X _c2 and X _c1 is neutral and polar, or neutral and hydrophobic amino acid, and X _c2 is neutral and polar, neutral and hydrophobic, or basic Is an amino acid]
In represented, C ₁ and C ₂ that are cyclized Oyori intramolecular disulfide bond between, human erythropoietin isolated polypeptide capable of binding to Chin receptor. X _n1 is E, G, N, S, D, Q, L, Y or A;
X _n2 is F, V, A, K, R, G, S, I or L;
X _n3 is H, Q, E, G, D, A or V; and
X _n4 is E, G, V, A, P, D, T or M;
2. The isolated polypeptide of claim 1. X _n1 is absent;
X _{n 2 is} F, V, A, K, R, G, S, I or L;
X _n3 is H, Q, E, G, D, A or V; and
X _n4 is E, G, V, A, P, D, T or M;
2. The isolated polypeptide of claim 1. 2. The isolated polypeptide of claim 1, wherein _Xn1 is an acidic amino acid. 2. The isolated polypeptide according to claim 1, wherein _Xn2 is a branched chain amino acid or K2. The isolated polypeptide according to claim 1, wherein X _n3 is an acidic amino acid or V 2. The isolated polypeptide of claim 1, wherein _Xn4 is V or G. The isolated polypeptide according to claim 1, wherein X ₁ is R, I, G, Q, L, T or S. The isolated polypeptide of claim 8, wherein X ₁ is R ₁ or L 2. The isolated polypeptide according to claim 1, wherein X ₂ is R, P, W, G, L or N. The isolated polypeptide of claim 10, wherein X ₂ is R. The isolated polypeptide according to claim 1, wherein X ₃ is H, Q or N 2. The isolated polypeptide according to claim 1, wherein X ₄ is Q, N, S, K or E. The isolated polypeptide of claim 13, wherein X ₄ is K or N. The isolated polypeptide according to claim 1, wherein X ₅ is V, A, Y, D or E. The isolated polypeptide according to claim 15, wherein X ₅ is D 1 or E 2. The isolated polypeptide according to claim 1, wherein _Xc1 is L, I, P, F, M, Q or G. 18. An isolated polypeptide according to claim 17, wherein X _c1 is L 1 or Q 2. 2. The isolated polypeptide according to claim 1, wherein _Xc2 is M, W, T, S, G, N or R. _{20. The} isolated polypeptide of claim 19, wherein _Xc2 is G 1 or R 2.   An isolated polypeptide having the amino acid sequence of SEQ ID NO: 81 and cyclized by intramolecular disulfide bonds.   An isolated polypeptide having the amino acid sequence of SEQ ID NO: 82 and cyclized by intramolecular disulfide bonds.

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