CHO-K1, HEK293 AND NS0 CELL LINES: GLYCOSYLATION IMPACT ON AFFINITY, STABILITY AND NEUTRALIZING ACTIVITY OF A CHIMERIC anti-rhIFN-α2b ANTIBODY

ATTALLAH C; AGUILAR, MF; ETCHEVERRIGARAY, M; OGGERO, M

Río de Janeiro

Simposio; 8° Simposio Latinoamericano de Tecnología de Cultivo de Células Animales (SLATCC); 2018

Federal University of Rio de Janeiro (UFRJ)

The monoclonal antibodies constitute a large subset of the marketed biotherapeutics, most of which are glycosylated, and thus produced in mammalian cells. Complete antibodies are bifunctional molecules, since the variable (V) regions are responsible of antigen binding and the constant (C) regions confer effector properties. However, this immunological dogma is in revision because several studies suggest that C regions of different class or subclasses of antibodies with identical V regions, influence the antigen binding activity (Casadevall, A. and Janda, A. 2012, Janda, A., Bowen, A., et al. 2016) Also, despite the glycosylation pattern strongly influences the antibody effector functions, this feature always was considered not to be important for binding antigen ability (Torres, M., Fernandez-Fuentes, N., et al. 2007). In this work, the impact of the different cell lines on the antigen-antibody affinity constant, the thermal and pH-dependent stability and antigen neutralizing ability of a chimeric anti-rhIFN α2b murine single chain Fv fused to Fcγ1 (scFv-Fc) was studied. The proteins were produced by CHO-K1, HEK293 and NS0 cells. They showed the same stability degree both in association as no-dissociation condition, but differed when they were submitted to thermal analysis where CHO-K1 showed the lowest resistance to temperature. The also showed no significant differences in the affinity constant measured by competitive ELISA. The scFv-Fc neutralizing ability of IFN-a2b activity was measured by inhibition of GFP expression from Mx-GFP HeLa reporter. The inhibition capacity was higher using the molecule produced by CHO cells despite the three antibodies showed no difference in the antigen binding assay. In fact, the neutralizing activity of the same deglycosylated protein was considerably reduced. The present study invites us to critically discuss the choice of the cell line to produce biotherapeutic antibodies.