biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2025-728biopreparat-728Research ArticleТема номера: ИННОВАЦИОННЫЕ БИОЛОГИЧЕСКИЕ ЛЕКАРСТВЕННЫЕ ПРЕПАРАТЫ: ОТ ФУНДАМЕНТАЛЬНЫХ ИССЛЕДОВАНИЙ К РЕАЛЬНОЙ КЛИНИЧЕСКОЙ ПРАКТИКЕIssue topic INNOVATIVE BIOLOGICAL PRODUCTS: TRANSLATING FUNDAMENTAL RESEARCH INTO REAL CLINICAL PRACTICEscFv-фрагменты рекомбинантных антител к вирусу гриппа: получение и характеристика функциональной активностиProduction and functional activity of scFv fragments obtained from recombinant antibodies against influenza virushttps://orcid.org/0000-0001-8196-3156ПлотниковаМ. А.PlotnikovaM. A.

Плотникова Марина Александровна, канд. биол. наук

ул. Профессора Попова, д. 15/17, Санкт-Петербург, 197022

Marina A. Plotnikova, Cand. Sci. (Biol.)

15/17 Prof. Popov St., St. Petersburg 197022

https://orcid.org/0000-0002-7450-7096ТопороваВ. А.ToporovaV. A.

Топорова Виктория Александровна

ул. Профессора Попова, д. 15/17, Санкт-Петербург, 197022; ул. Миклухо-Маклая, д. 16/10, Москва, 117997

Viktoriya A. Toporova

15/17 Prof. Popov St., St. Petersburg 197022; GSP-7, 16/10 Miklukho-Maklay St., Moscow 117997

https://orcid.org/0000-0001-7560-398XРомановская-РоманькоЕ. А.Romanovskaya-RomankoE. A.

Романовская-Романько Екатерина Андреевна, канд. биол. наук

ул. Профессора Попова, д. 15/17, Санкт-Петербург, 197022

Ekaterina A. Romanovskaya-Romanko, Cand. Sci. (Biol.)

15/17 Prof. Popov St., St. Petersburg 197022

https://orcid.org/0000-0002-8646-6252ШалджянА. А.ShaldzhyanA. A.

Шалджян Арам Арутюнович

ул. Профессора Попова, д. 15/17, Санкт-Петербург, 197022

Aram A. Shaldzhyan

15/17 Prof. Popov St., St. Petersburg 197022

https://orcid.org/0000-0003-0289-6560КлотченкоС. А.KlotchenkoS. A.

Клотченко Сергей Анатольевич, канд. биол. наук

ул. Профессора Попова, д. 15/17, Санкт-Петербург, 197022

Sergey A. Klotchenko, Cand. Sci. (Biol.)

15/17 Prof. Popov St., St. Petersburg 197022

fosfatik@mail.ruФедеральное государственное бюджетное учреждение «Научно-исследовательский институт гриппа имени А.А. Смородинцева» Министерства здравоохранения Российской ФедерацииРоссияSmorodintsev Research Institute of InfluenzaRussian FederationФедеральное государственное бюджетное учреждение «Научно-исследовательский институт гриппа имени А.А. Смородинцева» Министерства здравоохранения Российской Федерации; Федеральное государственное бюджетное учреждение науки Государственный научный центр Российской Федерации Институт биоорганической химии им. Академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наукРоссияSmorodintsev Research Institute of Influenza; Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Federation2026010420262615466Copyright © Плотникова М.А., Топорова В.А., Романовская-Романько Е.А., Шалджян А.А., Клотченко С.А., 20262026Плотникова М.А., Топорова В.А., Романовская-Романько Е.А., Шалджян А.А., Клотченко С.А.Plotnikova M.A., Toporova V.A., Romanovskaya-Romanko E.A., Shaldzhyan A.A., Klotchenko S.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/728ВВЕДЕНИЕ

ВВЕДЕНИЕ. Пассивная иммунотерапия с использованием антител широкого спектра действия является перспективным направлением разработки новых лекарственных средств для борьбы с гриппом. Однако технологии получения, очистки и хранения рекомбинантных антител, пригодных для клинического применения, по-прежнему сопряжены со значительными трудностями. scFv-фрагменты антител (одноцепочечные вариабельные фрагменты) являются более простой, надежной и гибкой альтернативой полноразмерным аналогам антител.

ЦЕЛЬ

ЦЕЛЬ. Разработка экспрессионных конструкций для синтеза scFv-фрагментов рекомбинантных антител к вирусу гриппа А и В, получение белковых препаратов scFv и оценка их функциональной активности in vitro.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. Экспрессионные конструкции, кодирующие scFv-фрагменты антител, получали методом ПЦР с использованием перекрывающихся праймеров и методами генной инженерии. Построение 3D-моделей разработанных scFv-фрагментов проводили по первичной аминокислотной последовательности на сервере AlfaFold. Наработку антител проводили в клеточной линии HEK293 в ходе транзиентной экспрессии. Препараты антител очищали из культуральной жидкости методом металл-аффинной хроматографии. Иммуноферментный анализ (ИФА) использовали для изучения вирус-специфической активности антител. Вируснейтрализующую активность антител изучали на монослойной культуре клеток MDCK по цитопатическому действию и регистрировали в реакции гемагглютинации.

РЕЗУЛЬТАТЫ

РЕЗУЛЬТАТЫ. На основе рекомбинантных антител, специфичных к вирусу гриппа А и В, был осуществлен дизайн и предсказана пространственная структура scFv-фрагментов, получены три генетические конструкции для экспрессии белков scFv в культуре эукариотических клеток. scFv-фрагменты были наработаны и очищены методом аффинной хроматографии с никелевым сорбентом в количестве не менее 0,5 мг и концентрации около 1 мг/мл каждого. Методом электрофореза белков в полиакриламидном геле было подтверждено соответствие выделенных scFv-фрагментов ожидаемому значению молекулярной массы — около 28 кДа. Методом ИФА было показано специфическое связывание scFv-фрагментов с различными штаммами вируса гриппа А и B. Установлено, что 50% вируснейтрализующая доза scFv-фрагмента антитела к поверхностному гемагглютинину вируса гриппа (170 нг/мл) сопоставима с нейтрализующей дозой для исходного полноразмерного антитела (179 нг/мл).

ВЫВОДЫ

ВЫВОДЫ. Разработан дизайн и получены scFv-фрагменты двух антител, одного — обладающего широкой нейтрализующей активностью против вируса гриппа A, другого — специфичностью в отношении вируса гриппа В. Благодаря малым размерам scFv-фрагменты могут эффективно проникать через слизистые оболочки при интраназальном введении. Указанное свойство определяет потенциал использования scFv-фрагментов в экстренной профилактике и ранней терапии ОРВИ. Перспективным направлением для усиления нейтрализующей активности является создание биспецифических scFv-фрагментов, способных одновременно нацеливаться на два вирусных эпитопа.

INTRODUCTION

INTRODUCTION. Passive immunotherapy using broad-spectrum antibodies is a promising development vector of new drugs against influenza. However, production process, purification, and storage of clinically suitable recombinant antibodies still face significant challenges. Antibody scFv (single-chain variable fragments) represent a more reliable, flexible, and simpler alternative to full-length antibody analogues.

AIM

AIM. This study aimed to develop expression constructs used to synthesize scFv fragments of recombinant antibodies against influenza A and B viruses, produce scFv protein preparations, and evaluate their in vitro functional activity.

MATERIALS AND METHODS

MATERIALS AND METHODS. Expression constructs encoding antibody scFv fragments were obtained by PCR using overlapping primers and genetic engineering methods. 3D models of the developed scFv fragments were constructed from the primary amino acid sequence using AlfaFold Server. Antibodies were produced in a HEK293 cell line via transient expression. Antibody preparations were purified from the culture fluid by metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) was used to study the virus-specific activity of the antibodies. Virus neutralising activity was studied in Madine-Darbi canine kidney (MDCK) cell monolayer culture based on cytopathic effect and recorded in a haemagglutination assay.

RESULTS

RESULTS. Based on recombinant antibodies specific to influenza A and B viruses, configuration of scFv fragments was designed and predicted, and three genetic constructs were obtained for expression of scFv proteins in a eukaryotic cell culture. The scFv fragments were produced and purified by affinity chromatography using Ni sorbent (no less than 0.5 mg) at a concentration of about 1 mg/mL of each fragment. Protein polyacrylamide gel electrophoresis confirmed that the isolated scFv fragments matched the expected size of approximately 28 kDa. ELISA demonstrated specific binding of scFv fragments to various influenza A and B strains. It was established that a 50% virus neutralising dose of the scFv antibody fragment against the influenza virus surface haemagglutinin (170 ng/mL) is comparable to that of the original full-length antibody (179 ng/mL).

CONCLUSIONS

CONCLUSIONS. The scFv fragments have been designed and obtained for two antibodies; the one has broad neutralising activity against influenza A virus, the other is specific for influenza B virus. Due to their small size, scFv fragments can effectively penetrate mucous membranes upon intranasal administration. This makes scFv fragments potentially useful in the emergency prophylaxis and early therapy of acute respiratory viral infections. A promising decision for enhanced neutralising activity is to create bispecific scFv fragments capable of simultaneously targeting two viral epitopes.

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The authors declare that there are no conflicts of interest present.