Способ получения рекомбинантных антител, продуцируемых клеточной линией, трансдуцированной рекомбинантными аденовирусами

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.Цели. Разработать технологию получения рекомбинантных антител в суспензионной культуре клеток человека HEK293 с помощью трансдукции рекомбинантными аденовирусами человека пятого серотипа, несущими гены, экспрессирующие тяжелые и легкие цепи антител, на примере двух противогриппозных антител широкого спектра действия 27F3 и CR9114.Методы. Рекомбинантные аденовирусы Ad5-27F3-H, Ad5-CR9114-H и Ad5-27F3-L, несущие ген тяжелой цепи антитела 27F3, ген тяжелой цепи антитела CR9114 и ген легкой цепи 27F3, были получены с помощью набора AdEasy™ Adenoviral vector system. Для накопления препаративных количеств рекомбинантных антител r27F3 и rCR9114 суспензионную клеточную линию HEK293 трансдуцировали рекомбинантными аденовирусами, несущими гены тяжелых и легких цепей антител, и культивировали клетки в биореакторе волнового типа. Рекомбинантные антитела очищали из культуральной жидкости хроматографическим методом. Молекулярную массу полученных антител анализировали с помощью белкового электрофореза, их способность взаимодействовать с вирусами гриппа А и В методом вестерн-блот анализа, а способность нейтрализовать вирусы гриппа А и В с помощью реакции вирус-нейтрализации.Результаты. Отработана методика накопления и очистки рекомбинантных антител r27F3 и CR9114 из культуральной жидкости суспензионной культуры клеток человека после трансдукции ее рекомбинантными аденовирусами, несущими гены тяжелых и легких цепей этих антител. Показана способность антитела r27F3 взаимодействовать с вирусами гриппа А подгруппы 1 (кроме вируса грипп А субтипа H2) и подгруппы 2 и нейтрализовать их. Показана способность антитела rCR9114 взаимодействовать с вирусами гриппа А подгруппы 1 и вирусами гриппа В, а также нейтрализовать вирусы гриппа А подгруппы 1.Выводы. Отработана технология получения рекомбинантных антител в суспензионной культуре клеток HEK293 с помощью трансдукции рекомбинантными аденовирусами, несущими гены, экспрессирующие тяжелые и легкие цепи антител, и показана их специфичность

Stimulation of Dectin-1 and Dectin-2 during Parenteral Immunization, but Not Mincle, Induces Secretory IgA in Intestinal Mucosa

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-κB, NFAT, and AP-1) to various extents in murine RAW 264.7 macrophages, even though similar pathways are activated. The agonists also elicit differential expression of maturation markers in bone marrow-derived dendritic cells, as well as differential cytokine secretion from these cells and from splenic mononuclear cells. In vivo assays also show that agonists of Dectin-1 and Dectin-2, but not Mincle, induce heavy IgA secretion in intestinal mucosa even when delivered parenterally. Strikingly, this effect appears to be formulation-independent. Collectively, the data suggest that adjuvants based on Dectin-1 and Dectin-2 agonists may significantly improve the efficacy of parenteral vaccines by inducing robust local immune reactions in intestinal mucosa

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection