Клеточная модель для тестирования препаратов, аффинных к PD-L1 человека

Цель исследования — создание и испытание клеточной модели, предназначенной для in vitro и in vivo тестирования специфичности препаратов, аффинных к PD-L1 человека.

Материалы и методы. Генно-модифицированные клетки, экспрессирующие PD-L1 человека (штамм CT26-PD-L1), получали методом ретровирусной трансдукции клеток мышиной карциномы CT26. Активность гена PD-L1 оценивали методом полимеразной цепной реакции в режиме реального времени; экспрессию PD-L1 на клетках выявляли с помощью проточной цитофлуориметрии. Испытания клеток проводили с использованием рекомбинантных однодоменных антител (наноантител) против PD-L1 человека, конъюгированных с радиоизотопами ⁶⁸Ga или ¹⁷⁷Lu. In vitro оценивали содержание иммунореактивной фракции и интернализацию клетками радиоконъюгатов. In vivo клетки CT26-PD-L1 трансплантировали мышам; на 9–14-е сутки вводили радиоиммуноконъюгаты; через 1–48 ч извлекали опухоли и проводили их прямую радиометрию. Контролем в экспериментах служили интактные клетки CT26, не экспрессирующие антиген.

Результаты. Создан штамм CT26-PD-L1 опухолевых клеток мышей, экспрессирующих на мембране PD-L1 человека. При трансплантации интактным мышам BALB/c или сублетально облученным мышам F1(DBA×BALB/c) клетки штамма формировали опухоли. Таким образом, существенным достоинством модели стала возможность проведения in vivo тестирования аффинных к PD-L1 человека препаратов на животных в условиях конвенционального вивария. При введении радиоиммуноконъюгатов мышам радионуклиды накапливались в опухолях из трансплантированных клеток CT26-PD-L1, но не из клеток CT26. Клетки CT26-PD-L1 in vitro интернализировали аффинные к PD-L1 наноантитела. Благодаря высокой плотности молекул-мишеней клетки CT26-PD-L1 позволили в рамках одного тестирования подтверждать специфичность препарата и количественно оценивать мишень-связывающую фракцию конъюгатов.

Заключение. Созданные клетки являются первыми отечественными генно-инженерными клетками, предназначенными для оценки препаратов, аффинных к PD-L1 человека. Результаты испытаний подтвердили пригодность модели для in vitro и in vivo тестирования специфичности нацеленных на PD-L1 человека препаратов.

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