CONICET | Buscador de Institutos y Recursos Humanos

"Background and novelty: Monoclonal antibodies are the main subset of marketed-biotherapeutics. Although they are considered bifunctional molecules, it was reported that constant regions of different IgGs with identical variable regions influence both their effector properties and their antigen-binding activity. While the glycosylation pattern strongly influences the effector functions, it was believed to have no effect on the antigen-binding ability. In this work, we evaluated the impact of the N-glycosylation on the antigen-neutralizing ability of a chimeric antibody.Experimental approach: A chimeric anti-human IFN-α2b murine single-chain Fv fused to human Fcγ1 (scFv-Fc) was produced in CHO-K1, HEK293 and NS0 cells (scFv-FcCHO, scFv-FcHEK and scFv-FcNS0, respectively). A partially de-glycosylated scFv-FcCHO (scFv-FcDEG) was generated. The neutralization of the IFN biological activity was evaluated by three independent bioassays. N-glycosylation structures were analyzed by hydrophilic chromatography.Results and discussion: The scFv-Fc(s) showed differences in their IFN-neutralizing ability. The molecule with the best neutralizing activity was scFv-FcCHO, which presented the highest N-glycan site occupation mainly with G1F structures. The scFv-FcNS0 showed the lowest neutralizing activity with 70% of occupied sites (mainly with G2F structures) respect to the scFv-Fc CHO. The scFv-FcHEK showed a medium neutralizing activity with 30% of occupied sites mainly with G0F structures. Apparently, both the quality andthe quantity of N-glycans influence the neutralizing ability of scFv-Fc(s). In accordance with this evidence, scFv-FcDEG showed a considerably reduced neutralizing ability. Thus, glycosylation has to be considered in order to develop therapeutic antibodies, not only for its impact on antibody effector properties but also its influence on functional antigen-binding."

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