Систему противовирусной защиты можно применить для эффективной иммунотерапии рака

Рис. 1. Механизм действия РНК-липоплексной противораковой вакцины. а — РНК в составе липоплекса адресно проникают в предшественники дендритных клеток, индуцируют их созревание и миграцию к Т-клеткам. Проникновение наночастиц в другие дендритные клетки индуцирует синтез интерферона, который способствует активации Т-клеток. b — трансляция РНК в созревающих дендритных клетках дает антиген, который презентируется Т-клеткам. Захват наночастиц макрофагами дает вторую волну интерферона, в результате чего Т-клетки полностью мобилизуются против специфического антигена. с — мобилизованные Т-клетки атакуют клетки опухоли. Рисунок из синопсиса к обсуждаемой статье в Nature

Немецким ученым удалось создать принципиально новую противораковую вакцину. Они запаковали матричную РНК генов, специфичных для раковых клеток, в особые наночастицы и заставили их адресно проникать в дендритные клетки лимфоидных тканей. В результате дендритные клетки «инструктировали» Т-лимфоциты и направляли их в атаку на раковый антиген, то есть на раковые клетки. Испытания на мышах и даже на пациентах, больных меланомой, показали высокую терапевтическую и профилактическую эффективность новой вакцины.

Методы неоперативного лечения рака постоянно совершенствуются. Но даже такие массовые методы, как радиотерапия и химиотерапия, не всегда достаточно эффективны и избирательны, обладают побочными эффектами и могут вызывать осложнения. Например, химиотерапия цитостатиками очень тяжело переносится пациентами.

В последнее время всё более активно разрабатываются методы иммунотерапии рака и других трудноизлечимых заболеваний. Так, недавно мы обсуждали работу по созданию бивалентных антител применительно к вирусу иммунодефицита человека (Биспецифические антитела могут уничтожать скрытые резервуары ВИЧ-инфекции, «Элементы», 16.02.2016). Этот метод можно приспособить и к адресной доставке Т-киллеров к раковым клеткам. Но на чужеродные антитела, применяемые в таких системах, может развиться нежелательный иммунный ответ. Поэтому активно проводятся исследования, имеющие целью мобилизовать и усилить собственный иммунный ответ организма на раковую опухоль, связанный с Т-лимфоцитами.

Система клеточного иммунитета замечательна своей гибкостью и эффективностью. Одна из важных составляющих этой системы — так называемые антигенпрезентирующие дендритные клетки. Они локализованы в лимфоидных органах (селезенка, лимфоузлы, костный мозг) и способны поглощать чужеродные для организма антигены, расщеплять их на отдельные пептиды и выставлять эти пептиды на своей поверхности для «обозрения» другими клетками, прежде всего Т-клетками. Таким образом дендритные клетки как бы «обучают» Т-клетки и направляют их в атаку на нужный антиген.

Систему противовирусной защиты можно применить для эффективной иммунотерапии рака

Рис. 2. Схематическое изображение липоплекса. Красным цветом обозначены заряженные гидрофильные головки липидов, серым — липидные хвосты, синим — молекулы ДНК или РНК. Липидный компонент в кровотоке защищает нуклеиновую кислоту от разложения нуклеазами. Рисунок с сайта ru.wikipedia.org

Дендритные клетки представляются идеальным инструментом для эффективной инициации и усиления Т-клеточного иммунного ответа на чужеродный антиген. Обычно в организме этот механизм направлен на нейтрализацию вирусных инфекций. Но большому коллективу ученых из нескольких научных учреждений в Германии удалось приспособить его для иммунотерапии раковых опухолей. Важно подчеркнуть, что это весьма нетривиальный результат, потому что иммунная система обычно не воспринимает раковые клетки как врагов (плохо срабатывает распознавание «свой — чужой», потому что раковые клетки, в общем-то, «свои»), а также из-за того, что образование опухоли часто не сопровождается выделением каких-либо специфических антигенов. Более того, раковые клетки довольно долго эволюционируют внутри организма в постоянном контакте с иммунной системой, так что на них действует своего рода отбор на способность противостоять иммунному ответу.

Ученые решили не нагружать дендритные клетки уже готовым антигеном, специфичным для раковых клеток и отсутствующим в зрелых нормальных клетках, а предоставить им возможность самим синтезировать его на основе соответствующих мРНК. Трудность, однако, в том, что в кровотоке свободная РНК быстро разлагается активно работающими нуклеазами. Поэтому для доставки «антигенных» РНК в предшественники дендритных клеток использовались специальные наночастицы — липоплексы (разновидность метода ДНК-вакцинации). Это слоистые структуры, в которых липидные мембраны защищают мРНК (рис. 2).

Первой проблемой, которую требовалось решить, была адресная доставка этих липоплексов в лимфоидные ткани, где локализованы дендритные клетки и активируются Т-клетки. Оказалось, что для этого не нужно добавлять ничего лишнего: ученым удалось подобрать соотношения мРНК и липидов, при которых после внутривенного введения липоплексов экспрессия мРНК наблюдалась практически исключительно в лимфоидных тканях мышей — селезенке, костном мозге и лимфоузлах (рис. 3).

Систему противовирусной защиты можно применить для эффективной иммунотерапии рака

Рис. 3. b — зависимость места экспрессии мРНК от соотношения «липид/РНК» (числа над изображениями мышей). Три правых группы изображений и круговые диаграммы показывают, что при определенном соотношении РНК и липидов мРНК экспрессируется практически исключительно в лимфоидных тканях. Spleen — селезенка, lungs — легкие, liver — печень. g — экспрессия мРНК люциферазы, введенной в кровь в составе липоплексов (нижний ряд), происходит изолированно в лимфоузлах (LN) и костном мозге (Bones). Свободная РНК (верхний ряд) разрушается в кровотоке, не достигая лимфоидных органов. Рисунок из обсуждаемой статьи в Nature

Для исследования биологических эффектов — ответа организма на экспрессию мРНК в дендритных клетках — сначала провели модельные эксперименты. Липоплексы нагрузили РНК, кодирующей гемагглютинин вируса гриппа. В результате внутривенного введения таких частиц в селезенке наблюдалось созревание дендритных клеток и активация различных типов Т-клеток. Это сопровождалось характерной для противовирусного иммунного ответа активацией экспрессии интерферона альфа. Если же липоплексы были нагружены мРНК, кодирующими фрагмент овальбумина, которым были мечены клетки меланомы, или кодирующими гликопротеин gp70 вируса лейкемии мышей, то наблюдалась продукция Т-клеток против этих антигенов и клеток. В результате трехкратного введения таких липоплексов иммунный ответ «запоминался» и обеспечивал защиту после трансплантации соответствующих опухолевых клеток. В то же время неиммунизированные мыши погибали в течение 30 дней после трансплантации.

Для определения эффективности такого лечения на мышах был испытан ряд раковых моделей. Так, трехкратное введение липоплексов приводило к полному излечению меланомных метастаз в легких в течение 20 дней после последней иммунизации (рис. 4). Сходные результаты были получены и на других моделях, а также на обезьянах. Ни у мышей, ни у обезьян при введении липоплексов не наблюдалось никаких неприемлемых побочных эффектов.

Систему противовирусной защиты можно применить для эффективной иммунотерапии рака

Рис. 4. Подавление роста агрессивных опухолей у мышей в результате введения вакцины мРНК-липоплекс. Мышам вводили культивируемые клетки колоректального рака мышей СТ26, в которые был введен ген люциферазы. Эти клетки экспрессируют антиген gp70, которого нет в нормальных клетках. Верхний ряд в левой группе изображений — контроль, нижний ряд — мыши после регулярной иммунизации соответствующей вакциной. Вверху в правой группе изображений — легкие мышей контрольной и иммунизированной, внизу — биолюминесцентное изображение этих легких (см. Биолюминецентный имиджинг раковых клеток in vivo). Рисунок из обсуждаемой статьи в Nature

Результаты детальных экспериментов, проведенных на модельных животных, позволили авторам получить разрешение и провести испытания нового метода на пациентах, больных далеко продвинутой меланомой. Несмотря на то что выборка мала (испытания были проведены только на трех пациентах), полученные результаты впечатляют и выглядят многообещающими. Липоплексы были нагружены мРНК четырех различных антигенов, свойственных клеткам меланомы, и введены пациентам: сначала малая доза, а затем четыре увеличенных дозы еженедельно. Инъекции переносились пациентами достаточно хорошо, вызывая лишь симптомы простуды. У всех пациентов наблюдалось усиление синтеза интерферона альфа и резкое увеличение продукции Т-клеток против введенных антигенов. У одного пациента наблюдалсь регрессия метастазов в лимфатических узлах. У второго пациента, у которого до иммунизации были удалены метастазы, в течение семи месяцев (вплоть до момента публикации статьи) новых метастазов не наблюдалось. У третьего пациента, у которого до иммунизации было найдено восемь метастазов в легких, их дальнейшего роста не происходило.

Таким образом, авторам удалось создать принципиально новую вакцину на основе мРНК, которая обладает высоким терапевтическим потенциалом. Впервые в истории создания подобных вакцин были проведены испытания не только на модельных животных, но и на больных, показавшие высокую противораковую эффективность (хотя, подчеркнем еще раз, данные пока есть всего по трем пациентам). Такую вакцину можно приготовить быстро, она сравнительно недорога, а мРНК может кодировать практически любой опухолевый антиген. В общем же, описанный подход к иммунотерапии с помощью мРНК-наночастиц открывает новые перспективы в лечении рака.

Разумеется, предложенную систему иммунотерапии нужно еще продолжать исследовать. Надо выяснить ее применимость к другим типам рака, а также испытать целый ряд мРНК антигенов, которые экспрессируются в опухолях, но не в нормальных зрелых клетках. Следует также исследовать способность других клеток иммунной системы (нейтрофилов, моноцитов) поглощать наночастицы и активироваться под их влиянием. Работа предстоит еще очень большая, но хочется верить, что она даст хорошие результаты.

Источник: elementy.ru

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