RU2321630C2 - Гликозилированные антитела (варианты), обладающие повышенной антителозависимой клеточной цитотоксичностью - Google Patents

Гликозилированные антитела (варианты), обладающие повышенной антителозависимой клеточной цитотоксичностью Download PDF

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RU2321630C2
RU2321630C2 RU2004106559/13A RU2004106559A RU2321630C2 RU 2321630 C2 RU2321630 C2 RU 2321630C2 RU 2004106559/13 A RU2004106559/13 A RU 2004106559/13A RU 2004106559 A RU2004106559 A RU 2004106559A RU 2321630 C2 RU2321630 C2 RU 2321630C2
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Abstract

Настоящее изобретение относится к модификации гликозилирования белков для получения полипептидов с улучшенными терапевтическими характеристиками, включая антитела с повышенной антителозависимой клеточной цитотоксичностью. Для получения указанных полипептидов используют клетку-хозяина, модифицированную нуклеиновой кислотой, кодирующей β(1,4)-N-ацетилглюкозаминилтрансферазу III (GnTIII). Полученный полипептид представляет собой, в частности, IgG или его фрагмент. Изобретение раскрывает способ получения полипептида, а также антитела или его фрагмента и белка слияния, полученных указанным способом. Описан фармацевтический состав для увеличения Fc-опосредованной клеточной цитотоксичности, содержащий антитело и носитель, и его использование для лечения рака, а также способ лечения заболевания, связанного с увеличением количества или производства В-клеток с использованием указанного антитела, в частности, против CD20, и в предпочтительном варианте представляющее собой антитело IDEC-C2B8. Изобретение позволяет получать полипептид или антитело, обладающие увеличенной Fc-опосредованной клеточной цитотоксичностью, при использовании которого уменьшается содержание В-клеток в организме пациента. 10 н. и 28 з.п. ф-лы, 21 ил.

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Claims (38)

1. Клетка-хозяин, модифицированная для получения полипептида, имеющего увеличенную Fc-опосредованную цитотоксичность, путем экспрессии по меньшей мере одной нуклеиновой кислоты, кодирующей β(1,4)-N-ацетилглюкозаминилтрансферазу III (GnT III), при этом указанный полипептид представляет собой целую молекулу антитела, фрагмент антитела и белок слияния, который содержит область, эквивалентную Fc-области иммуноглобулина, и при этом указанная GnT III экспрессируется в количестве, достаточном для увеличения доли указанных полипептидов, содержащих раздвоенные гибридные олигосахариды или галактозилированные комплексные олигосахариды, или их смеси в Fc-области.
2. Клетка-хозяин по п.1, отличающаяся тем, что указанный полипептид представляет собой IgG или его фрагмент.
3. Клетка-хозяин по п.1, отличающаяся тем, что указанный полипептид представляет собой IgGI или его фрагмент.
4. Клетка-хозяин по п.1, отличающаяся тем, что указанный полипептид представляет собой белок слияния, который содержит область, эквивалентную Fc-области IgG человека.
5. Клетка-хозяин по п.1, отличающаяся тем, что молекулу нуклеиновой кислоты, содержащую по меньшей мере один ген, кодирующий GnT III, вводят в указанную клетку-хозяина.
6. Клетка-хозяин по п.1, отличающаяся тем, что указанная клетка-хозяин модифицирована таким образом, что активируется ген эндогенной GnT III.
7. Клетка-хозяин по п.6, отличающаяся тем, что указанная эндогенная GnT III активируется вставкой элемента ДНК, который увеличивает генную экспрессию в хромосоме хозяина.
8. Клетка-хозяин по п.6, отличающаяся тем, что указанную клетку-хозяина отбирают таким образом, чтобы она содержала мутацию, стимулирующую экспрессию эндогенной GnT III.
9. Клетка-хозяин по п.8, отличающаяся тем, что указанная клетка-хозяин представляет собой клетку-мутант lec 10 СНО.
10. Клетка-хозяин по п.1, отличающаяся тем, что указанная клетка-хозяин представляет собой клетку СНО, клетку ВНК, клетку NSO, клетку SP2/0, клетку YO миеломы, клетку РЗХ63 миеломы мыши, клетку PER или клетку PER.C6 или клетку гибридомы.
11. Клетка-хозяин по п.10, отличающаяся тем, что указанный полипептид представляет собой антитело против CD20.
12. Клетка-хозяин по п.10, отличающаяся тем, что указанное антитело против CD20 представляет собой IDEC-C2B8.
13. Клетка-хозяин по п.10, отличающаяся тем, что указанная клетка-хозяин представляет собой клетку SP2/0.
14. Клетка-хозяин по п.13, отличающаяся тем, что указанное антитело представляет собой химерное моноклональное антитело chG250 против гипернефромы человека.
15. Клетка-хозяин по п.5, отличающаяся тем, что указанный по меньшей мере один ген, кодирующий GnT III, вводят в хромосому указанной клетки-хозяина.
16. Клетка-хозяин по п.6, отличающаяся тем, что указанная эндогенная GnT III активируется вставкой промоторного элемента, транспозона или ретровирусного элемента в хромосому клетки-хозяина.
17. Клетка-хозяин по п.1, отличающаяся тем, что содержит также по меньшей мере одну трансфецированную нуклеиновую кислоту, кодирующую молекулу антитела, фрагмент антитела или белок слияния, который включает область, эквивалентную Fc-области иммуноглобулина.
18. Клетка-хозяин по п.1, отличающаяся тем, что указанная по меньшей мере одна нуклеиновая кислота, кодирующая GnT III, функционально связана с конститутивным промоторным элементом.
19. Клетка-хозяин по п.17, отличающаяся тем, что указанная клетка-хозяин содержит по меньшей мере одну трансфецированную нуклеиновую кислоту, кодирующую антитело против CD-20, химерное моноклональное антитело chCE7 против нейробластомы человека, химерное моноклональное антитело chG250 против гипернефромы человека, химерное моноклональное антитело ING-1 против карциномы ободочной кишки, легких и молочной железы человека, гуманизированное моноклональное антитело 3622W94 против антигена 17-1А человека, гуманизированное антитело А33 против опухоли ободочной и прямой кишки, антитело R24, направленное против ганглиозида GD3, против меланомы человека, или химерное моноклональное антитело SF-25 против плоскоклеточной карциономы человека, антитело против EGFR человека, антитело против EGFRvIII человека, антитело против PSMA человека, антитело против PSCA человека, антитело против CD22 человека, антитело против CD30 человека. антитело против CD33 человека, антитело против CD38 человека, антитело против CD40 человека, антитело против CD45 человека, антитело против CD52 человека, антитело против CD138 человека, вариант антитела против HLA-DR человека, антитело против ЕрСАМ человека, антитело против СЕА человека, антитело против MUC1 человека, антитело против капсидного белка MUC1 человека, антитело против арберрантно гликозилированного MUC1 человека, антитело против вариантов фибронектина человека, содержащих домен ED-B, и антитело против HER2/neu человека.
20. Способ получения полипептида, содержащего раздвоенные гибридные олигосахариды или галактозилированные комплексные или их смесь в Fc-области, включающий культивирование клетки-хозяина по пп.1-19 при условиях, которые позволяют получать указанный полипептид, имеющий увеличенную Fc-опосредованную клеточную цитотоксичность.
21. Способ по п.20, отличающийся тем, что включает выделение указанного полипептида, имеющего увеличенную, Fc-опосредованную клеточную цитотоксичность.
22. Способ по п.20, отличающийся тем, что указанная клетка-хозяин содержит по меньшей мере одну нуклеиновую кислоту, кодирующую белок слияния, содержащий область, эквивалентную Fc-области иммуноглобулина.
23. Способ по п.20, отличающийся тем, что более 50% олигосахаридов в Fc-области указанного полипептида являются раздвоенными.
24. Способ по п.20, отличающийся тем, что более 70% олигосахаридов в Fc-области указанного полипептида являются раздвоенными.
25. Способ по п.20, отличающийся тем, что содержание раздвоенных гибридных олигосахаридов или галактозилированных комплексных олигосахаридов или их смесей в Fc-области превышает содержание раздвоенных комплексных олигосахаридов в Fc-области указанных полипептидов.
26. Способ по п.20, отличающийся тем, что указанный полипептид является антителом IDEC-C2B8 против CD20, и антитела IDEC-C2B8, образованные указанной клеткой-хозяином, согласно анализу MALDI/TOF-MS имеют спектр гликозилирования, который практически эквивалентен показанному на фиг.2Е.
27. Способ по п.20, отличающийся тем, что указанный полипептид является моноклональным антителом chG250, и антитела chG250, образованные указанной клеткой-хозяином, согласно анализу MALDI/TOF-MS имеют спектр гликозилирования, который практически эквивалентен показанному на фиг.7D.
28. Антитело, имеющее повышенную антителозависимую клеточную цитотоксичность (ADCC), полученное способом по п.21.
29. Антитело по п.28, отличающееся тем, что указанное антитело выбирают из группы, включающей IDEC-C2B8, chCE7, ch-G250, гуманизированное моноклональное антитело против HER2, ING-1, 3622W94, SF-25, А33 и R24.
30. Фрагмент антитела, содержащий область, эквивалентную Fc-области иммуноглобулина, имеющий увеличенную Fc-опосредованную клеточную цитотоксичность, полученный способом по п.21.
31. Белок слияния, содержащий область, эквивалентную Fc-области иммуноглобулина, имеющий увеличенную Fc-опосредованную клеточную цитотоксичность и полученный способом по п.21.
32. Фармацевтический состав для увеличения Fc-опосредованной клеточной цитотоксичности, содержащий антитело по п.28, и фармацевтически допустимый носитель.
33. Фармацевтический состав для увеличения Fc-опосредованной клеточной цитотоксичности, содержащий фрагмент антитела по п.30, и фармацевтически допустимый носитель.
34. Фармацевтический состав для увеличения Fc-опосредованной клеточной цитотоксичности, содержащий слитый белок по п.31, и фармацевтически допустимый носитель.
35. Способ лечения рака, включающий введение терапевтически эффективной дозы фармацевтического состава по пп.32-34 пациенту, который нуждается в таком лечении.
36. Способ лечения заболевания, связанного с увеличением количества или производства В-клеток, основанный на уменьшении содержания В-клеток и включающий введение терапевтически эффективной дозы антитела пациенту, который нуждается в таком лечении, при этом усовершенствование заключается в введении терапевтически эффективной для уменьшения числа В-клеток дозы антитела, полученной способом по п.28.
37. Способ по п.36, отличающийся тем, что указанное антитело представляет собой моноклональное антитело против CD20.
38. Способ по п.37, отличающийся тем, что указанное антитело против CD20 представляет собой антитело IDEC-C2B8.
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