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RU2011133819A - Композиции модифицированных антител, способы их получения и применения - Google Patents

Композиции модифицированных антител, способы их получения и применения Download PDF

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Publication number
RU2011133819A
RU2011133819A RU2011133819/10A RU2011133819A RU2011133819A RU 2011133819 A RU2011133819 A RU 2011133819A RU 2011133819/10 A RU2011133819/10 A RU 2011133819/10A RU 2011133819 A RU2011133819 A RU 2011133819A RU 2011133819 A RU2011133819 A RU 2011133819A
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Russia
Prior art keywords
activated antibody
target
tissue
binding
antibody according
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RU2011133819/10A
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English (en)
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RU2636046C2 (ru
Inventor
Нэнси Э. СТАГЛИАНО
Джеймс У. УЭСТ
Катрин КАМАТ
Пол Х. БЕССЕТТ
Фред ГЛАК
Джейсон СЭЙДЖЕРТ
Патрик ДОХЕРТИ
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САЙТОМКС ТЕРАПЬЮТИКС, ЭлЭлСи
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Abstract

1. Активируемое антитело, содержащее:антитело или его антигенсвязывающий фрагмент (АВ), связывающиеся с молекулой-мишенью с равновесной константой диссоциации, равной примерно 100 нМ или меньше;маскирующую часть (ММ), способную ингибировать связывание АВ с молекулой-мишенью в нерасщепленном состоянии, где:(А) ММ является полипептидом длиной около 2-40 аминокислот,(В) ММ связывается с АВ с равновесной константой диссоциации, которая больше равновесной константы диссоциации АВ в отношении молекулы-мишени,(С) полипептидная последовательность ММ отличается от молекулы-мишени, при этом полипептидная последовательность ММ не более, чем на 50% идентична природному партнеру связывания АВ, и(D) ММ не препятствует связыванию АВ или не конкурирует с АВ за связывание в расщепленном состоянии;расщепляемую часть (СМ), которая представляет собой полипептид длиной до 15 аминокислот, где СМ действует в качестве субстрата для протеазы, локализованной вместе с молекулой-мишенью в первой ткани, где такая протеаза может расщеплять СМ в активируемом антителе, при этом СМ расположена в активируемом антителе так, что в нерасщепленном состоянии связывание активируемого антитела с мишенью снижено до равновесной константы диссоциации, которая по меньшей мере в 100 раз больше равновесной константы диссоциации немодифицированного АВ, связывающегося с мишенью, тогда как в расщепленном состоянии АВ может связываться с молекулой-мишенью в присутствии мишени; идва сливающих пептида (LP1, LP2), каждый из которых является пептидом длиной около 1-20 амиинокислот, причем LP1 и LP2 необязательно должны быть идентичны друг другу;активируемое антитело, стру�

Claims (18)

1. Активируемое антитело, содержащее:
антитело или его антигенсвязывающий фрагмент (АВ), связывающиеся с молекулой-мишенью с равновесной константой диссоциации, равной примерно 100 нМ или меньше;
маскирующую часть (ММ), способную ингибировать связывание АВ с молекулой-мишенью в нерасщепленном состоянии, где:
(А) ММ является полипептидом длиной около 2-40 аминокислот,
(В) ММ связывается с АВ с равновесной константой диссоциации, которая больше равновесной константы диссоциации АВ в отношении молекулы-мишени,
(С) полипептидная последовательность ММ отличается от молекулы-мишени, при этом полипептидная последовательность ММ не более, чем на 50% идентична природному партнеру связывания АВ, и
(D) ММ не препятствует связыванию АВ или не конкурирует с АВ за связывание в расщепленном состоянии;
расщепляемую часть (СМ), которая представляет собой полипептид длиной до 15 аминокислот, где СМ действует в качестве субстрата для протеазы, локализованной вместе с молекулой-мишенью в первой ткани, где такая протеаза может расщеплять СМ в активируемом антителе, при этом СМ расположена в активируемом антителе так, что в нерасщепленном состоянии связывание активируемого антитела с мишенью снижено до равновесной константы диссоциации, которая по меньшей мере в 100 раз больше равновесной константы диссоциации немодифицированного АВ, связывающегося с мишенью, тогда как в расщепленном состоянии АВ может связываться с молекулой-мишенью в присутствии мишени; и
два сливающих пептида (LP1, LP2), каждый из которых является пептидом длиной около 1-20 амиинокислот, причем LP1 и LP2 необязательно должны быть идентичны друг другу;
активируемое антитело, структура которого организована следующим образом в направлении от N-конца к С-концу в нерасщепленном состоянии: (ММ)-LP1-(CM)-LP2-(AB) или (АВ)-LP2-(CM)-L1-(MM),
где присоединение ММ снижает способность антитела связываться с его мишенью таким образом, что константа диссоциации (Kd) АВ, связанного с ММ, в отношении мишени по меньшей мере в 100 раз больше Kd АВ, не связанного с ММ.
2. Активируемое антитело по п.1, в котором в присутствии молекулы-мишени ММ снижает способность АВ связываться с молекулой-мишенью по меньшей мере на 90% при нерасщепленной СМ по сравнению с расщепленной ММ при определении с помощью анализа замещения мишени in vitro.
3. Активируемое антитело по п.1, где его антигенсвязывающий фрагмент выбран из группы, состоящей из Fab'-фрагмента, F(ab')2-фрагмента, scFv, scab, dAb, антитела с одним доменом тяжелой цепи и антитела с одним доменом легкой цепи.
4. Активируемое антитело по п.1, в котором АВ представляет собой адалимумаб, цетуксимаб, панитумумаб, инфликсимаб, эфализумаб, ипилимумаб, тремелимумаб, адекатумумаб, Hu5c8, алемтузумаб, ранибизумаб, тозитумомаб, ибритумомаб тиуксетан, ритуксимаб, инфликсимаб, бевацизумаб или фигитумумаб или происходит из указанных антител.
5. Активируемое антитело по п.1, в котором мишень представляет собой Notch 1, TNFальфа, EGFR, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, трансферрин, gp130, VCAM-1, CD44, DLL4 или IL4.
6. Активируемое антитело по п.1, в котором ММ является субстратом для фермента, выбираемого из группы, состоящей из uPA, легумаина, плазмина, TMPRSS-3/4, MMP-9, MT1-MMP, катепсина, каспазы, эластазы нейтрофилов человека, бета-секретазы или PSA.
7. Активируемое антитело по п.1, в котором ММ не более, чем на 25% идентична по аминокислотной последовательности или не более, чем на 10% идентична по аминокислотной последовательности в отношении молекулы-мишени АВ, и в котором необязательно ММ содержит консенсусную последовательность, выбираемую из CISPRGC, C(N/P)H(HVF)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG, xCxxYQCLxxxxxx, XXQPxPPRVXX, PxPGFPYCxxxx, xxxxQxxPWPP, GxGxCYTILExxCxxxR, GxxxCYxIxExxCxxxx, GxxxCYxIxExWCxxxx, xxxCCxxYxIxxCCxxx и xxxxxYxILExxxxx.
8. Активируемое антитело по п.1, в котором присоединение ММ снижает способность АВ связываться с его мишенью так, что константа диссоциации (Kd) АВ, связанного с ММ, в отношении мишени по меньшей мере в 1000 раз больше или по меньшей мере в 10000 раз больше Kd АВ, не связанного с ММ, в отношении мишени.
9. Активируемое антитело по п.1, где сродство связывания активируемого антитела с его мишенью является более высоким в первой ткани по сравнению со связыванием активируемого антитела с его мишенью во второй ткани, где первая ткань является больной тканью, и где необязательно больная ткань представляет собой ткань молочной железы, ткань головы, ткань шеи, ткань легкого, ткань поджелудочной железы, ткань нервной системы, ткань печени, ткань предстательной железы, ткань мочеполовой системы или ткань шейки матки.
10. Активируемое антитело по п.1, дополнительно содержащее по меньшей мере одно из следующего:
(i) второе АВ, для которого мишень выбрана из группы, состоящей из Notch 1, EGFR, TNFальфа, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, трансферрина, gp130, VCAM-1, CD44, DLL4 и IL4; или
(ii) по меньшей мере первую СМ и вторую СМ, где первая СМ расщепляется первым расщепляющим средством в ткани-мишени, а вторая СМ расщепляется вторым расщепляющим средством в ткани-мишени.
11. Активируемое антитело по п.10, в котором первая СМ, вторая СМ, первое расщепляющее средство или второе расщепляющее средство обладают по меньшей мере одной из следующих характеристик:
(i) первое расщепляющее средство и второе расщепляющее средство представляют собой один и тот же фермент, при этом первая СМ и вторая СМ являются разными субстратами для фермента;
(ii) первое расщепляющее средство и второе расщепляющее средство являются разными ферментами;
(iii) первое расщепляющее средство и второе расщепляющее средство расположены вместе в первой ткани или
(iv) первая СМ и вторая СМ расщепляются по меньшей мере одним расщепляющим средством в первой ткани.
12. Активируемое антитело по п.1, в котором по меньшей мере один из LP1 и LP2 содержит аминокислотную последовдательность, выбираемую из (GS)n, (GSGGS)n (SEQ ID NQ:12), (GGGS)n (SEQ ID NQ:13), GGSG (SEQ ID NQ:14), GGSGG (SEQ ID NQ:15), GSGSG (SEQ ID NQ:16), GSGGG (SEQ ID NQ:17), GGGSG (SEQ ID NQ:18) и GSSSG (SEQ ID NQ:19), где n является целым числом, равным по меньшей мере единице.
13. Активируемое антитело по п.1, дополнительно содержащее детектируемую часть, необязательно диагностическое средство, или дополнительно содержащее терапевтическое средство, конъюгированное с АВ, необязательно противоопухолевое средство.
14. Применение активируемого антитела по любому из предшествующих пунктов для лечения у субъекта опухоли, аутоиммунного заболевания или воспалительного заболевания или для ингибирования у субъекта ангиогенеза.
15. Применение активируемого антитела по любому из предшествующих пунктов для получения лекарственного средства для лечения у субъекта опухоли, аутоиммунного заболевания или воспалительного заболевания или для получения лекарственного средства для ингибирования у субъекта ангиогенеза.
16. Способ получения активируемого антитела по п.1, включающий стадии:
(а) получения антитела или его антигенсвязывающего фрагмента (АВ), маскирующей части (ММ), расщепляемой части (СМ), первого сшивающего пептида (LP1) и второго сшивающего пептида (LP2);
(b) связывание ММ с СМ при помощи первого пептидного линкера (LP1) и
(с) связывание АВ с СМ при помощи второго пептидного линкера (LP2);
при этом структура активируемого антитела организована следующим образом в направлении от N-конца к С-концу в нерасщепленном состоянии: (ММ)-LP1-(CM)-LP2-(AB) или (АВ)-LP2-(CM)-L1-(MM).
17. Молекула выделенной нуклеиновой кислоты, кодирующая активируемое антитело по п.1.
18. Способ получения активируемого антитела по п.1 посредством культивирования клетки в условиях, вызывающих экспрессию активируемого антитела, где клетка содержит вектор, содержащий молекулу выделенной нуклеиновой кислоты, кодирующую активируемое антитело.
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