RU2018136607A - Способы и составы для производства лимфоцитов и их регулируемого увеличения - Google Patents

Способы и составы для производства лимфоцитов и их регулируемого увеличения Download PDF

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RU2018136607A
RU2018136607A RU2018136607A RU2018136607A RU2018136607A RU 2018136607 A RU2018136607 A RU 2018136607A RU 2018136607 A RU2018136607 A RU 2018136607A RU 2018136607 A RU2018136607 A RU 2018136607A RU 2018136607 A RU2018136607 A RU 2018136607A
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incompetent
recombinant retrovirus
polypeptide
receptor
domain
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RU2018136607A
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RU2018136607A3 (ru
RU2755059C2 (ru
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Грегори Ян Фрост
Джеймс Джозеф ОНУФФЕР
Гьябе Х. ГУЙБИНГА
Анирбан Кунду
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Ф1 Онколоджи, Инк.
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Claims (36)

1. Способ генной модификации Т-клетки, включающий:
взаимодействие Т-клетки пациента ex vivo с некомпетентным репликативным рекомбинантным ретровирусом для образования реакционной смеси для трансдукции,
причем некомпетентный репликативный рекомбинантный ретровирус содержит:
a) псевдотипирующий элемент на его поверхности, который способен связываться с Т-клеткой и облегчает мембранное слияние с ним некомпетентного репликативного рекомбинантного ретровируса; и
b) полинуклеотид, содержащий одну или несколько единиц транскрипции, функционально связанных с промотором, активным в Т-клетках, причем одна или несколько единиц транскрипции кодируют первый сконструированный сигнальный полипептид, содержащий первый рецептор химерного антигена, обладающего антигенспецифической областью для нацеливания; трансмембранный домен; и внутриклеточный активирующий домен,
причем Т-клетка взаимодействует ex vivo с некомпетентным репликативным рекомбинантным ретровирусом в интервале от 15 минут до 14 часов до того, как Т-клетка вымывается из реакционной смеси для трансдукции, и
причем указанное взаимодействие облегчает мембранное слияние Т-клетки с некомпетентным репликативным рекомбинантным ретровирусом для получения генетически модифицированной Т-клетки.
2. Способ по п. 1, отличающийся тем, что некомпетентный репликативный рекомбинантный ретровирус является лентивирусом, который дополнительно содержит мембранносвязанный полипептид, способный связываться с CD3.
3. Способ по п. 2, отличающийся тем, что мембранносвязанный полипептид, способный связываться с CD3, является антителом к CD3, и при этом взаимодействие Т-клеток с некомпетентным репликативным рекомбинантным ретровирусом включает взаимодействие мононуклеарных клеток периферической крови (РВМС), содержащих Т-клетки с некомпетентным репликативным рекомбинантным ретровирусом.
4. Способ по п. 3, отличающийся тем, что антитело к CD3 является ГИФ-якорным антителом к CD3.
5. Способ по п. 3, отличающийся тем, что псевдотипирующий элемент содержит оболоченный белок вируса везикулярного стоматита, полипептид вируса кори F, или полипептид вируса кори Н и/или его функциональный фрагмент, и некомпетентный репликативный рекомбинантный ретровирус дополнительно содержит мембранносвязанный полипептид, способный связываться с CD28.
6. Способ по п. 1, отличающийся тем, что одна или несколько единиц транскрипции кодируют второй сконструированный сигнальный полипептид, включающий лимфопролиферативный элемент, который стимулирует пролиферацию и/или выживание Т-клеток, и при этом указанная генно-модифицированная клетка способна выживать в культуре в течение по меньшей мере 7 дней в отсутствие IL-2.
7. Способ по п. 6, отличающийся тем, что лимфопролиферативный элемент содержит конститутивно активный полипептид рецептора цитокина или его фрагмент, и при этом экспрессия конститутивно активного полипептида рецептора цитокина или его фрагмента регулируется.
8. Способ по п. 7, отличающийся тем, что лимфопролиферативный элемент содержит внутриклеточный сигнальный домен рецептора IL-7, внутриклеточный сигнальный домен рецептора IL-12, внутриклеточный сигнальный домен рецептора IL-15, внутриклеточный сигнальный домен рецептора IL-21, или внутриклеточный сигнальный домен рецептора рецептора-ловушки трансформирующего фактора роста β.
9. Способ по п. 6, отличающийся тем, что первый сконструированный сигнальный полипептид и второй сконструированный сигнальный полипептид экспрессируются из одного транскрипта на обратной стороне вирусного генома, экспрессированного из вирусного LTR или его функционального фрагмента.
10. Способ по п. 6, отличающийся тем, что способ дополнительно содержит:
забор крови, содержащей Т-клетку пациента до взаимодействия Т-клетки ex vivo с некомпетентным репликативным рекомбинантным ретровирусом; и
введение генно-модифицированной Т-клетки пациенту, причем генно-модифицированная Т-клетка не увеличивается ex vivo после указанного взаимодействия и до введения пациенту.
11. Способ по п. 6, отличающийся тем, что взаимодействие производится в интервале от 15 минут до 8 часов.
12. Способ по п. 1, отличающийся тем, что Т-клетка является покоящейся Т-клеткой.
13. Способ по п. 12, отличающийся тем, что псевдотипирующий элемент содержит мембранносвязанный полипептид, способный связываться с CD3.
14. Репликация некомпетентного рекомбинантного ретровируса, содержащего:
а) псевдотипирующий элемент на его поверхности, который способен связываться с Т-клеткой и облегчает мембранное слияние с ней некомпетентного репликативного рекомбинантного ретровируса; и
b) полинуклеотид, содержащий одну или несколько единиц транскрипции, функционально связанных с промотором, активным в Т-клетках, причем одна или несколько единиц транскрипции кодируют первый сконструированный сигнальный полипептид, содержащий первый рецептор химерного антигена, обладающего антигенспецифической областью для нацеливания; трансмембранный домен и внутриклеточный активирующий домен; и второй сконструированный сигнальный полипептид, включающий лимфопролиферативный элемент, который стимулирует пролиферацию и/или выживание Т-клеток, когда клетка выдерживается в культуре в течение по меньшей мере 7 дней в отсутствие IL-2.
15. Некомпетентный репликативный рекомбинантный ретровирус по п. 14, отличающийся тем, что некомпетентный репликативный рекомбинантный ретровирус является лентивирусом, который дополнительно содержит мембранносвязанный полипептид, способный связываться с CD3.
16. Некомпетентный репликативный рекомбинантный ретровирус по п. 15, отличающийся тем, что мембранносвязанный полипептид, который способен связываться с CD3, является антителом к CD3.
17. Некомпетентный репликативный рекомбинантный ретровирус по п. 16, отличающийся тем, что антитело к CD3 является ГИФ-якорным антителом к CD3.
18. Некомпетентный репликативный рекомбинантный ретровирус по п. 14, отличающийся тем, что псевдотипирующий элемент содержит оболоченный белок вируса везикулярного стоматита, полипептид вируса кори F, или полипептид вируса кори Н и/или его функциональный фрагмент, и некомпетентный репликативный рекомбинантный ретровирус дополнительно содержит мембранносвязанный полипептид, способный связываться с CD28.
19. Некомпетентный репликативный рекомбинантный ретровирус по п. 14, отличающийся тем, что лимфопролиферативный элемент содержит конститутивно активный полипептид рецептора цитокина или его фрагмент, и причем экспрессия конститутивно активного полипептида рецептора цитокина или его фрагмента регулируется.
20. Некомпетентный репликативный рекомбинантный ретровирус по п. 14, отличающийся тем, что лимфопролиферативный элемент содержит внутриклеточный сигнальный домен рецептора IL-7, внутриклеточный сигнальный домен рецептора IL-12, внутриклеточный сигнальный домен рецептора IL-15, внутриклеточный сигнальный домен рецептора IL-21, или внутриклеточный сигнальный домен рецептора рецептора-ловушки трансформирующего фактора роста β.
21. Некомпетентный репликативный рекомбинантный ретровирус по п. 14, отличающийся тем, что первый сконструированный сигнальный полипептид и второй сконструированный сигнальный полипептид экспрессируются из одного транскрипта на обратной стороне вирусного генома, экспрессированного из вирусного LTR или его функционального фрагмента.
22. Выделенный полинуклеотид для регулирования экспрессии целевого полинуклеотида, содержащий:
полинуклеотид, кодирующий целевой полинуклеотид, функционально связанный с промотором и рибопереключателем, в котором рибопереключатель содержит:
a) домен аптамера, способный связывать нуклеозидный аналог противовирусного препарата, и имеющей восстановленную связь с гуанином или 2'-дезоксигуанозином относительно нуклеозидного аналога противовирусного препарата; и
b) домен функционального переключателя, способный регулировать экспрессию целевого полинуклеотида, при котором связывание нуклеозидного аналога аптамерным доменом индуцирует или подавляет регуляторную активность экспрессии домена функционального переключателя, тем самым регулируя экспрессию целевого полинуклеотида.
23. Выделенный полинуклеотид по п. 22, отличающийся тем, что нуклеозидным аналогом противовирусного препарата является ацикловир или пенцикловир.
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