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RU2010116208A - Способ модификации изоэлектрической точки антитела с помощью аминокислотных замен в cdr - Google Patents

Способ модификации изоэлектрической точки антитела с помощью аминокислотных замен в cdr Download PDF

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RU2010116208A
RU2010116208A RU2010116208/10A RU2010116208A RU2010116208A RU 2010116208 A RU2010116208 A RU 2010116208A RU 2010116208/10 A RU2010116208/10 A RU 2010116208/10A RU 2010116208 A RU2010116208 A RU 2010116208A RU 2010116208 A RU2010116208 A RU 2010116208A
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Томоюки ИГАВА (JP)
Томоюки ИГАВА
Хироюки ЦУНОДА (JP)
Хироюки ЦУНОДА
Тацухико ТАТИБАНА (JP)
Тацухико ТАТИБАНА
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Abstract

1. Способ модификации изоэлектрической точки полипептида, содержащего вариабельную область антитела, сохраняя антиген-связывающую активность вариабельной области, который содержит модификацию заряда по меньшей мере одного аминокислотного остатка, расположенного на поверхности гипервариабельного участка (CDR) полипептида. ! 2. Способ по п.1, где полипептид, содержащий вариабельную область антитела, дополнительно содержит FcRn-связывающий домен. ! 3. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой антитело IgG. ! 4. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой химерное антитело, гуманизированное антитело или антитело человека. ! 5. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой полиспецифический полипептид, который связывается по меньшей мере с двумя типами антигенов. ! 6. Способ по п.1, где заряд аминокислотного остатка модифицирован с помощью замены аминокислоты. ! 7. Способ по п.1, где модификация заряда аминокислотного остатка приводит к изменению теоретической изоэлектрической точки на 1,0 или более. ! 8. Способ по п.1, где аминокислотный остаток, расположенный на поверхности области CDR, представляет собой по меньшей мере один аминокислотный остаток, выбранный из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату. ! 9. Полипептид, содержащий вариабельную область антитела с модифицированной изоэлектрической точкой, который получен способом по любому из пп.

Claims (44)

1. Способ модификации изоэлектрической точки полипептида, содержащего вариабельную область антитела, сохраняя антиген-связывающую активность вариабельной области, который содержит модификацию заряда по меньшей мере одного аминокислотного остатка, расположенного на поверхности гипервариабельного участка (CDR) полипептида.
2. Способ по п.1, где полипептид, содержащий вариабельную область антитела, дополнительно содержит FcRn-связывающий домен.
3. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой антитело IgG.
4. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой химерное антитело, гуманизированное антитело или антитело человека.
5. Способ по п.1, где полипептид, содержащий вариабельную область антитела, представляет собой полиспецифический полипептид, который связывается по меньшей мере с двумя типами антигенов.
6. Способ по п.1, где заряд аминокислотного остатка модифицирован с помощью замены аминокислоты.
7. Способ по п.1, где модификация заряда аминокислотного остатка приводит к изменению теоретической изоэлектрической точки на 1,0 или более.
8. Способ по п.1, где аминокислотный остаток, расположенный на поверхности области CDR, представляет собой по меньшей мере один аминокислотный остаток, выбранный из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату.
9. Полипептид, содержащий вариабельную область антитела с модифицированной изоэлектрической точкой, который получен способом по любому из пп.1-8.
10. Способ контролирования фармакокинетических параметров полипептида в плазме, содержащего вариабельную область антитела, который содержит модификацию изоэлектрической точки полипептида способом по любому из пп.1-8.
11. Способ по п.10, где контроль фармакокинетических параметров относится к увеличению или уменьшению любого параметра из клиренса (CL) плазмы, площади под кривой концентрации (AUC), среднего времени удерживания в плазме и времени полужизни в плазме (t1/2).
12. Полипептид, содержащий вариабельную область антитела с контролируемыми фармакокинетическими параметрами в плазме, что достигается способом по п.10.
13. Способ получения полипептида, содержащего вариабельную область антитела с модифицированной изоэлектрической точкой, который содержит:
(a) модификацию нуклеиновой кислоты, кодирующей полипептид, для того, чтобы модифицировать заряд по меньшей мере одного аминокислотного остатка, расположенного на поверхности области CDR полипептида;
(b) культивирование клетки-хозяина для того, чтобы экспрессировать нуклеиновую кислоту; и
(c) сбор полипептида, содержащего вариабельную область антитела, из культуры клетки-хозяина.
14. Способ по п.13, где полипептид, содержащий вариабельную область антитела, дополнительно содержит FcRn-связывающий домен.
15. Способ по п.13, где полипептид, содержащий вариабельную область антитела, представляет собой антитело IgG.
16. Способ по п.13, где полипептид, содержащий вариабельную область антитела, представляет собой химерное антитело, гуманизированное антитело или антитело человека.
17. Способ по п.13, где полипептид, содержащий вариабельную область антитела, представляет собой полиспецифический полипептид, который связывается по меньшей мере с двумя типами антигенов.
18. Способ по п.13, где заряд аминокислотного остатка модифицирован с помощью аминокислотной замены.
19. Способ по п.13, где модификация заряда аминокислотного остатка приводит к изменению теоретической изоэлектрической точки на 1,0 или более.
20. Способ по п.13, где аминокислотный остаток, расположенный на поверхности области CDR, представляет собой по меньшей мере один аминокислотный остаток, выбранный из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату.
21. Полипептид, содержащий вариабельную область антитела с модифицированной изоэлектрической точкой, что достигается способом по любому из пп.13-20.
22. Способ получения полипептида, содержащего вариабельную область антитела с контролируемыми фармакокинетическими параметрами в плазме, который содержит модификацию изоэлектрической точки полипептида, содержащего вариабельную область антитела способом по любому из пп.13-20.
23. Способ по п.22, где контроль фармакокинетических параметров относится к увеличению или уменьшению любого параметра из клиренса (CL) плазмы, площади под кривой концентрации (AUC), среднего времени удерживания в плазме и времени полужизни в плазме (t1/2).
24. Полипептид, содержащий вариабельную область антитела с контролируемыми фармакокинетическими свойствами в плазме, что достигается способом по п.22.
25. Способ получения полиспецифического полипептида, содержащего первый полипептид и второй полипептид, каждый из которых содержит вариабельную область антитела, который содержит:
(a) модификацию нуклеиновой кислоты, кодирующей полипептид, чтобы модифицировать заряд по меньшей мере одного аминокислотного остатка, расположенного на поверхности области CDR первого полипептида и второго полипептида, в частности модификацию обеих или одной нуклеиновой кислоты, кодирующей аминокислотные остатки первого полипептида, или другой нуклеиновой кислоты, кодирующей аминокислотные остатки второго полипептида, чтобы увеличить разность изоэлектрических точек первого полипептида и второго полипептида по сравнению с состоянием до модификации;
(b) культивирование клетки-хозяина для того, чтобы экспрессировать нуклеиновые кислоты; и
(c) сбор полиспецифического антитела из культуры клетки-хозяина.
26. Способ по п.25, где стадию сбора полиспецифического полипептида, содержащего первый полипептид и второй полипептид, из культуры клетки-хозяина осуществляют стандартной хроматографией.
27. Способ по п.25, где нуклеиновая кислота модифицирована так, чтобы пики гомомультимера первого полипептида, гомомультимера второго полипептида и гетеромультимера первого полипептида и второго полипептида более четко разделялись в стандартном хроматографическом анализе по сравнению с состоянием до модификации.
28. Способ по п.25, где полиспецифический полипептид представляет собой полиспецифическое антитело.
29. Полиспецифическое антитело, которое получено способом по п.27.
30. Полиспецифическое антитело по п.29, которое представляет собой биспецифическое антитело.
31. Антитело, изоэлектрическая точка которого модифицирована по сравнению с антителом до модификации, сохраняя антиген-связывающую активность, которое содержит каркасную область человека (FR), константную область человека и CDR, который выбран из группы, состоящей из полученных от человека CDR, CDR, полученных из животных, не относящихся к человеку, и синтетических CDR, где по меньшей мере один аминокислотный остаток, расположенный на поверхности области CDR, отличается зарядом от аминокислотного остатка в соответствующем положении в CDR дикого типа.
32. Антитело по п.31, где константная область человека содержит Fc-домен человека.
33. Антитело по п.31, у которого фармакокинетические параметры в плазме контролируют с помощью модификации изоэлектрической точки.
34. Антитело IgG, изоэлектрическая точка которого модифицирована по сравнению с антителом до модификации аминокислот, где заряд меньшей мере одного аминокислотного остатка выбран из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату.
35. Антитело по п.34, где модифицированная аминокислота выбрана из аминокислотных остатков из одной из нижеследующих групп (a) и (b):
(a) глутаминовая кислота (E) и аспарагиновая кислота (D); и
(b) лизин (K), аргинин (R) и гистидин (H).
36. Полиспецифическое антитело, содержащее первый полипептид и второй полипептид, изоэлектрические точки которых отличаются друг от друга, и по меньшей мере один аминокислотный остаток первого полипептида, выбранный из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату, обладает зарядом.
37. Антитело по п.36, где по меньшей мере один аминокислотный остаток второго полипептида, выбранный из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату, не имеет заряда или обладает противоположным зарядом по отношению к заряду выбранного аминокислотного остатка первого полипептида.
38. Антитело по п.36, в котором аминокислотный остаток с зарядом, и аминокислотный остаток с противоположным зарядом, выбраны в виде комбинации из разных групп:
(a) глутаминовая кислота (E) и аспарагиновая кислота (D); и
(b) лизин (K), аргинин (R) и гистидин (H).
39. Полиспецифическое антитело, содержащее первый полипептид и второй полипептид по п.36, которое дает раздельные пики для гомомультимера первого полипептида и гомомультимера второго полипептида в стандартном хроматографическом анализе.
40. Композиция, содержащая фармацевтически приемлемый носитель и антитело по любому из пп.31-39.
41. Нуклеиновая кислота, кодирующая полипептид, который образует антитело по любому из пп.31-39.
42. Клетка-хозяин, содержащая нуклеиновую кислоту по п.41.
43. Способ получения антитела по любому из пп.31-39, который содержит стадии культивирования клетки-хозяина по п.42 и сбора полипептида из культуры клеток.
44. Способ замены аминокислотного остатка, расположенного на поверхности гипервариабельного участка (CDR) полипептида, содержащего вариабельную область антитела, сохраняя антиген-связывающую активность полипептида, который включает замену по меньшей мере одного аминокислотного остатка, выбранного из аминокислотных остатков в положениях 31, 61, 62, 64 и 65 вариабельной области тяжелой цепи и в положениях 24, 27, 53, 54 и 55 вариабельной области легкой цепи согласно системе нумерации по Кабату.
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