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Generation and Characterization of Human Interferon-beta Neutralizing Humanized Antibody


Humanization of antibodies for the development of novel therapeutic agents with low immunogenicity remains a topical problem in modern science. In the present work we describe the humanization of murine antibody B16 which binds and neutralizes human interferon-beta using the CDR-grafting method. Based on amino acid sequences of humanized and murine antibodies we constructed models of variable domains, analyzed, and compared them. The genes of humanized antibody hB16 and chimeric antibody chB16 were expressed in transient CHO cells. Antibodies were recovered from conditioned media, purified using affinity chromatography, and their properties were studied by biochemical and immunochemical methods. It was proven that humanized antibody hB16 possesses the same properties as murine mAb B16. This humanized antibody hB16 will be used in further work in order to obtain therapeutic immune complex composed of human interferon-beta and bispecific antibody which binds interferon-beta and the ErbB2 receptor.

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  1. Hwang, W.Y. and Foote, J., Methods, 2005, vol. 36, pp. 3–10.

    CAS  Article  PubMed  Google Scholar 

  2. Scott, A.M., Lee, F., Hopkins, W., Cebon, J.S., Wheatley, J.M., and Liu, Z., J. Clin. Oncol., 2001, vol. 19, pp. 3976–3987.

    CAS  Article  PubMed  Google Scholar 

  3. Buist, M.R., Kenemans, P., van Kamp, G.J., and Haisma, H.J., Cancer Immunol. Immunother., 1995, vol. 40, pp. 24–30.

    CAS  Article  PubMed  Google Scholar 

  4. Roque-Navarro, L., Mateo, C., Lombardero, J., Mustelier, G., Fernández, A., Sosa, S., Morrison, S.L., and Pérez, R., Hybridoma Hybridomics, 2004, vol. 22, pp. 245–257.

    CAS  Article  Google Scholar 

  5. Richards, J., Auger, J., Peace, D., Gale, D., Michel, J., Koons, A., Haverty, T., Zivin, R., Jolliffe, L., and Bluestone, J.A., Cancer Res., 1999, vol. 59, pp. 2096–2101.

    CAS  PubMed  Google Scholar 

  6. Almagro, J. and Franson, J., BioScience, 2008, vol. 13, pp. 1619–1633.

    CAS  Google Scholar 

  7. Hale, G. and Phillips, M., Biochem. Soc. Transact., 1995, vol. 23, pp. 1057–1063.

    CAS  Article  Google Scholar 

  8. Safdari, Y., Farajniaa, S., Asgharzadehb, M., and Khalili, M., Biotech. Genet. Eng. Rev., 2013, vol. 29, pp. 175–186.

    CAS  Article  Google Scholar 

  9. Makabe, K., Nakanishi, N., Tsumoto, K., Tanaka, Y., Kondo, H., Umetsu, M., Sone, Y., Asano, R., and Kumagai, I., J. Biol. Chem., 2008, vol. 283, pp. 1156–1166.

    CAS  Article  PubMed  Google Scholar 

  10. Wedemayer, G.J., Patten, PA., Wang, L.H., Schultz, P.G., and Stevens, R.C., Science, 1997, vol. 276, pp. 1665–1669.

    CAS  Article  Google Scholar 

  11. Zimmermann, J., Oakman, E.L., Thorpe, I.F., Shi, X., Abbyad, P., Brooks, C.L., Boxer, S.G., and Romesberg, F.E., Proc. Natl. Acad. Sci. U. S. A., 2006, vol. 103, pp. 13 722–13 727.

    Article  Google Scholar 

  12. Kabat, E.A., Sequences of Immunological Interest, 5 ed., Bethesda, Md, USA: Public Health Service, NIH, 1991.

    Google Scholar 

  13. Ilina, E.N., Solopova, O.N., Balabashin, D.S., Larina, M.V., Aliev, T.K., Grebennikova, T.V., Losich, M.A., Zaykova, O.N., Sveshnikov, P.G., Dolgikh, D.A., and Kirpichnikov, M.P., Russ. J. Bioorg. Chem., 2019, vol. 45, pp. 59–68.

    Article  Google Scholar 

  14. Rizner, T.L., Biochem. Mol. Biol. Educ., 2014, vol. 42, pp. 152–159.

    CAS  Article  PubMed  Google Scholar 

  15. Friguet, B., Chaffotte, A.F., Djavadi-Ohaniance, L., and Goldberg, M.E., J. Immunol. Methods, 1985, vol. 77, pp. 305–319.

    CAS  Article  Google Scholar 

  16. aspx?mediaformatid=10061&destinationid=10016& assetid=14782

  17. Supino, R., In vitro Toxicity Testing Protocols, Humana Press, 1995, pp. 137–149.

    Google Scholar 

  18. Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.

    CAS  Article  PubMed  Google Scholar 

  19. 15596026.

  20. Weitzner, B.D., Jeliazkov, J.R., Lyskov, S., Marze, N., Kuroda, D., Frick, R., Bryfogle, A.A., Biswas, N., and Gray, J.J., Nat. Protoc., 2017, vol. 12, pp. 401–416.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Abraham, M.J., Murtola, T., Schulz, R., Pall, S., Smith, J.C., Hess, B., and Lindahl, E., SoftwareX, 2015, vols. 1–2, pp. 19–25.

    Article  Google Scholar 

  22. Humphrey, W., Dalke, A., and Schulten, K., J. Mol. Graph., 1996, vol. 14, p. 3338.

    Article  Google Scholar 

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The work was performed using the equipment of the Center for the collective use of ultrahigh-performance computing resources of Moscow State University.


The work was supported by the Ministry of Healthcare of the Russian Federation (agreement no. 075-15-2019-1385 of 19.06.2019, unique project identifier RFMEFI60417X0189).

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Correspondence to V. S. Rybchenko.

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Mononuclear peripheral blood cells were obtained from healthy donors’ blood. All donors provided voluntary informed consent.

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Authors declare they have no conflicts of interest.

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Translated by N. Onishchenko

Abbreviations: CDR, complementarity determining region; SDR, specificity determining region; FR, framework regions; VH and VL, variable domains of heavy and light chains of immunoglobulins; CH, constant domains of heavy chain of immunoglobulins; Cκ and Сλ, constant domains of light chains of immunoglobulins; mAbs, monoclonal antibodies; PBMC, peripheral blood mononuclear cells; IFN-β, interferon-beta; PBS(T), phosphate-buffered saline supplemented with Tween-20; MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide; V, volume.

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Rybchenko, V.S., Panina, A.A., Novoseletsky, N.V. et al. Generation and Characterization of Human Interferon-beta Neutralizing Humanized Antibody. Russ J Bioorg Chem 46, 778–786 (2020).

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  • humanized antibodies
  • chimeric antibodies
  • antibody structure modeling
  • СНО
  • interferon-beta