Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus

Fully­human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full­size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully­human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO­S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR­CH­CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT­DHFR­CH­CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT­DHFRCH­CL cassette plasmid. Then, CHO­S/FRT cells, which contain the FRT­site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT­site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti­vaccinia virus activity in the plaque reduction neutralization test in vitro. 

1. Baykov I.K., Khlusevich Ya.A., Matveev A.L., Tikunova N.V. Construction of cassette vector plasmids for production of full-size recombinant antibodies. Vestnik NGU: Seriya biologicheskaya, klinicheskaya meditsina = Herald of the Novosibirsk State University. Series: Biology and clinical medicine. 2013;11(3):56-64. (in Russian)

2. Baykov I.K., Matveev A.L., Stronin O.V., Ryzhikov A.B., Matveyev L.E., Kasakin M.F., Richter V.A., Tikunova N.V. A protective chimeric antibody to tick-borne encephalitis virus. Vaccine. 2014; 32(29):3589-3594.

3. Coroadinha A.S., Schucht R., Gama-Norton L., Wirth D., Hauser H., Carrondo M.J. The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange. J. Biotechnol. 2006;124(2):457-468.

4. Crickard L., Babas T., Seth S., Silvera P., Koriazova L., Crotty S. Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies. PLoS ONE. 2012; 7(11):e48706.

5. Fernandez J., Yaman I., Merrick W.C., Koromilas A., Wek R.C., Sood R., Hensold J., Hatzoglou M. Regulation of internal ribosome entry sitemediated translation by eukaryotic initiation factor-2α phosphorylation and translation of a small upstream open reading frame. J. Biol. Chem. 2002;277(3):2050-2058.

6. Hirata R., Chamberlain J., Dong R., Russell D.W. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat. Biotechnol. 2002;20:735-738.

7. Hopkins R.J., Lane J.M. Clinical efficacy of intramuscular vaccinia immune globulin: a literature review. Clin. Infect. Dis. 2004;39(6): 819-826.

8. Huang Y., Li Y., Wang Y.G., Gu X., Wang Y., Shen B.F. An efficient and targeted gene integration system for high-level antibody expression. J. Immunol. Methods. 2007;322(1-2):28-39.

9. Kameyama Y., Kawabe Y., Ito A., Kamihira M. An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange. Biotechnol. Bioeng. 2010;105(6):1106-1114.

10. Khlusevich Y.A., Morozova V., Pyshnyi D.V., Tikunova N.V. Antibodies against ectromelia virus capable of neutralizing variola virus: generation and application for epitope mapping. FEBS J. 2013; 280(S1):371-372.

11. Khlusevich Y.A., Tikunova N., Morozova V., Bulychev L., Bormotov N., Vlasov V., Sergeev A. Sredstvo dlya neytralizatsii virusa natural’noy ospy [The agent for neutralizing variola virus]. Patent RF No. 2515905, 2014б. (in Russian)

12. Khlusevich Ya., Tikunova N., Morozova V., Grigor’eva A., Baykov I., P’yankov O. Rekombinantnaya plazmidnaya DNK pQE-p35d, obespechivayushchaya sintez rekombinantnogo belka p35d virusa ospy korov, shtamm bakteriy Escherichia coli – produtsent rekombinantnogo belka r35d virusa ospy korov i rekombinantnyy belok r35d virusa ospy korov, ispol’zuemyy dlya sozdaniya test-sistem i konstruirovaniya sub’edinichnykh vaktsin protiv ortopoksvirusnykh infektsiy [Recombinant plasmid DNA pQE-p35d providing synthesis of p35d recombinant protein of cowpox virus, Escherichia coli bacterial strain that is producer of p35d recombinant protein of cowpox virus and p35d recombinant protein of cowpox virus used to engineer test systems and to design orthopoxvirus split vaccines]. Patent RF No. 2511037, 2014а. (in Russian)

13. Kito M., Itami S., Fukano Y., Yamana K., Shibui T. Construction of engineered CHO strains for high-level production of recombinant proteins. Appl. Microbiol. Biotechnol. 2002;60(4):442-448.

14. Kwaks T.H., Barnett P., Hemrika W., Siersma T., Sewalt R.G., Sa tijn D.P., Brons J.F., van Blokland R., Kwakman P., Kruckeberg A.L., Kelder A., Otte A.P. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nat. Biotechnol. 2003;21(5):553-558.

15. Little M., Breitling F., Dübel S., Fuchs P., Braunagel M., Seehaus T., Klewinghaus I. Universal antibody libraries on phage and bacteria. Year Immunol. 1993;7:50-55.

16. Lucas B.K., Giere L.M., DeMarco R.A., Shen A., Chisholm V., Crowley C.W. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucleic Acids Res. 1996;24(9):1774-1779.

17. Matho M.H., Schlossman A., Meng X., Benhnia M.R., Kaever T., Buller M., Doronin K., Parker S., Peters B., Crotty S., Xiang Y., Zajonc D.M. Structural and functional characterization of anti-A33 antibodies reveal a potent cross-species orthopoxviruses neutralizer. PLoS Pathog. 2015;11.

18. McCausland M.M., Benhnia M.R., Crickard L., Laudenslager J., Granger S.W., Tahara T., Kubo R., Koriazova L., Kato S., Crotty S. Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model. Antivir. Ther. 2010;15(4):661-675.

19. Mutskov V., Felsenfeld G. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J. 2004;23(1):138-149.

20. Petrov I.S., Goncharova E.P., Kolosova I.V., Pozdnyakov S.G., Shchelkunov S.N., Zenkova M.A., Vlasov V.V. Antitumor effect of the LIVPGFP recombinant vaccinia virus. Doklady RAN = Proceedings of the Russian Academy of Sciences. 2013;451(5):592-597. DOI 10.7868/ S0869565213240274. (in Russian)

21. Reichert J.M. Antibodies to watch in 2016. MAbs. 2016;8(2):197-204.

22. Reichert J.M. Antibodies to watch in 2017. MAbs. 2017;9(2):167-181.

23. Richards E.J. Chromatin methylation: who’s on first? Curr. Biol. 2002; 12(20):694-695.

24. Tasic B., Miyamichi K., Hippenmeyer S., Dani V.S., Zeng H., Joo W., Zong H., Chen-Tsai Y., Luo L. Extensions of MADM (Mosaic Ana lysis with Double Markers) in mice. PLoS ONE. 2012;7(3):e33332. DOI 10.1371/journal.pone.0033332.

25. Tikunova N., Dubrovskaya V., Morozova V., Yun T., Khlusevich Y., Bormotov N., Laman A., Brovko F., Shvalov A., Belanov E. The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library. Virus Res. 2012;163(1):141-150.

26. Wilson C.J., Guglielmo C., Moua N.D., Tudor M., Grosveld G., Young R.A., Murray P.J. Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse. Anal. Biochem. 2001;296(2):270-278.

27. Wirth D., Gama-Norton L., Riemer P., Sandhu U., Schucht R., Hauser H. Road to precision: recombinase-based targeting technologies for genome engineering. Curr. Opin. Biotechnol. 2007;18(5): 411-409.

28. Zahn-Zabal M., Kobr M., Girod P.A., Imhof M., Chatellard P., de Jesus M., Wurm F., Mermod N. Development of stable cell lines for production or regulated expression using matrix attachment regions. J. Biotechnol. 2001;87(1):29-42.