GLYCOSYLATION IMPACT ON NEUTRALIZING ACTIVITY OF A CHIMERIC ANTIBODY

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

Copenhague

Congreso; 26 th ESACT Meeting; 2019

Background and novelty: IgGs 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 the antigen-binding activity. While the glycosylation pattern strongly influences the effector functions, it seems to have no effect on the binding antigen ability. In this work, we evaluated the impact of the N glycosylation on 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. Antigen-neutralizing ability was evaluated by three independent bioassays. N glycosylation structures were analyzed by hydrophilic chromatography. Results and discussion: The scFc Fc(s) evaluated showed differences in their antigen neutralizing ability. The molecule with the best neutralizing activity was scFv FcCHO, which presented the highest number of occupied N glycan sites mainly with G1F structures. The scFv FcNS0 showed the lowest neutralizing activity with 70% of occupied sited respect to the scFv FcCHO mainly with G2F structures, and the scFv FcHEK had a medium neutralizing activity with 30% of occupied sites mainly with G0F structures. Apparently, both the quality, as well as the quantity, of N glycans impact on the neutralizing ability of scFv Fc(s). In accordance with this evidence, scFv FcDEG showed a considerably reduced neutralizing ability. Thus, this work has high impact in order to develop therapeutic antibodies since the glycosylation should be taken into account for its influence on functional antigen binding and not only for its impact on the antibody effector properties.