STUDIES OF THE INFLUENCE OF DIFFERENT EUKARYOTIC VECTORS’ DESIGN ON THE EXPRESSION OF RECOMBINANT ANTIBODY IgA1 ISOTYPE

Production and application of therapeutic monoclonal antibodies take second place in the global pharmaceutical market after vaccines. Nowadays the IgG1 is one of the prevailing monoclonal antibodies type for the therapeutic use that are produced in CHO cells. Recently the possibility of developing therapeutic drugs based on antibodies of IgA-isotype has demonstrated a broad range of effector functions on human mucous membranes, which has caused a significant interest in this research field. In our research about level of IgA1 antibody expression in mammal cells, we designed a set of bipromoter (CMV, EF1α) vectors containing variable regions of heavy chain (VH) and light chain (VL) genes from human monoclonal antibody FI6v3 against influenza A virus, constant regions IgA1 of heavy and light chains of human antibody (with or without introns). The vectors differed in mutual orientation of promoters and presence or lack of introns. Two versions of the full-length light and heavy chains were cloned in eukaryotic expressing vector in “head-head”/ “head-tail” orientations and then transfected into HEK-293T and CHO/dhfr-cells. The level of antibody production was determined using ELISA test for transfection of CHO DG44 and HEK-293T cell culture. The results of the experiments showed a significant increase in the production of FI6v3-IgA1 antibodies when eukaryotic cells were transfected with a plasmid pBiPr-ABIgA1FI6-Iht containing introns in constant regions of IgA1 and with orientation of transcription in the “head-tail” direction.

1. Beyer T., Lohse S., Berger S., Peipp M., Valerius T., Dechant M. Serum-free production and purification of chimeric IgA antibodies // J. Immunol. Methods. 2009. Vol. 346. N 1–2. P. 26–37.

2. Woof J.M., Kerr M.A. The function of immunoglobulin A in immunity // J. Pathol. 2006. Vol. 208. N 2. P. 270–282.

3. Woof J.M., Russell M.W. Structure and function relationships in IgA // Mucosal Immunol. 2011. Vol. 4. N 6. P. 590–597.

4. Lohse S., Derer S., Beyer T., Klauz K., Peipp M., Leusen J.H.W., Van de Winkel J.G.J, Dechant M., Valerius T. Recombinant dimeric IgA epidermal growth factor receptor mediate effective tumor cell killing // J. Immunol. 2011. Vol. 18. N 6. P.3770–3778.

5. Muramatsu M., Yoshida R., Miamoto H., Tomabechi D., Masahiro K. Heterosubtypic antiviral activity of hemagglutinin- specific antibodies induced by intranasal immunization with inactivated influenza viruses in mice // PLoS One. 2013. Vol. 8. N 8. e71534.

6. Van Riet E., Ainai A, Suzuki T., Hasegawa H. Mucosal IgA responses in influenza virus infections; thoughts for vaccine design // Vaccine. 2012. Vol. 30. N 40. P. 5893–5959.

7. Blutt S.E., Miller A.D., Salmon L., Metzger D.W., Conner M.E. IgA is important for clearance and critical for protection from rotavirus infection // Mucosal Immunol. 2012. Vol. 5. N 6. P. 712–719.

8. Corti D., Voss J., Gamblin S. J., et al. A neutralizing antibody selected from plasma cells that binding to group1 and group2 influenza A hemagglutinin // Science. 2011. Vol. 33. N 6044. P. 850–856.

9. Wurm F.M. Production of recombinant protein therapeutics in cultivated mammalian cells // Nat. Biotechnol. 2004. Vol. 22. N.11. P. 1393–1398.

10. Schlatter S., Stansfield S.H., Dinnis D.M., Racher A.J., Birch J.R., James D.C. On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells // Biotechnol. Prog. 2005. Vol. 21. N.1. P. 122–133.

11. Jostock T., Vanhove M., Brepoels E., van Gool R., Daukandt M., Wehnert A., van Hegelsom R., Dransfield D., Sexton D., Devlin M., Ley A., Hoogenboom H.R., Müllberg J. Rapid generation of functional human IgG antibodies derived from Fab on-phage display libraries // J. Immunol. Methods. 2004. Vol. 289. N. 1–2. P. 65–80.

12. Li J., Menzel C., Meier D.,Zhang C., Dübel S., Jostock T. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies // J. Immunol. Methods. 2007. Vol. 318. N 1–2. P. 113–124.

13. Chusainow J., Yang Y.S., Yeo J. H.M., Toh P.C., Asvadi P., Wong N.S., Yap M.G. A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer? // Biotechnol. Bioeng. 2009. Vol. 102. N 4. P. 1182–1194.

14. Van Craenenbroeck K., Vanhoenacker P., Haegeman G. Episomal vectors for gene expression in mammalian cells // Eur. J. Biochem. 2000. Vol. 267. N 18. P. 5665–5678.

15. Bout A., Halford D., Jones A. Efficient production of IgA in recombinant mammalian cells. Patent EP1508576A1, 2005.

16. Corti D., Voss J., Gamblin S.J., et al. A neutralizing antibody selected from plasma cells that binding to group1 and group2 influenza A hemagglutinin // Science.2011. Vol. 333. N 6044. P. 850–856.

17. Radko B.V., Boitchenko V.E., Nedospasov S.A., Korobko V.G. Characterization of the genes encoding variable light and heavy chains of the high-affinity monoclonal antibody against human tumor necrosis factor // Russ. J. Immunol. 2002. Vol. 7. N 4. P. 371–374.

18. Алиев Т.К., Балабашин Д.С., Долгих Д.А., Кирпичников М.П., Панина А.А., Топорова В.А. Рекомбинантная плазмидная ДНК, кодирующая химерное антитело против фактора некроза опухоли-альфа человека, линия эукариотических клеток — продуцент химерного антитела и способ получения химерного антитела. Патент № 2555533, Россия, 23.05.2013.

19. Balabashin D., Kovalenko E., Toporova V., Aliev T., Panina A., Svirshchevskaya E., Dolgikh D., Kirpichnikov M. Production of anti tnf-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains // Cytotechnology. 2015. Vol. 67. N 5. P. 761–772.

20. Aliev T.K., Dement’yeva I.G., Bokov M.N., Pozdnyakova L.P., Sveshnikov P.G., Toporova V.A., Rybchenko V.S., Dolgikh D.A., Kirpichnikov M.P. Development and properties of recombinant proteins based on the broadly neutralizing antibody to influenza A virus // Moscow Univ. Biol. Sci. Bull. 2016. Vol.71. N 2. P. 87–92.