Producer cell line of an antibody for therapeutic use influences its affinity and antigen-neutralizing activity

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

Copenhague

Congreso; 26 th ESACT Meeting; 2019

Background and noveltyMonoclonal antibodies (mAb) constitute a large and growing subset of the marketed-biotherapeutics glycoproteins. Although mAbs are considered bifunctional molecules since variable (V) and constant (C) regions are independents domains, and glycosylation have no effect on antigen binding, there are several studies suggesting the influence of C regions of different mAbs with identical V regions on the antigen binding activity.We developed a novel humanized single-chain variable fragment (scFv) against human interferon-α2b (hIFN-α2b), which was fused to the human Fcγ1 region (hz.scFv Fc) as a therapeutic candidate for autoimmune diseases. This technology is subject of a UNL-CONICET patent application. In this work, we study the impact of the producer cell line in the affinity constant and antigen-neutralizing ability of this potential biotherapeutic.Experimental approachThe hz.scFv-Fc antibody was produced by CHO-K1, HEK293 and NS0 cells. Affinity constant was measured by competitive ELISA and antigen-neutralizing ability was evaluated by three independent bioassays. N-glycosylation structures were analyzed by hydrophilic chromatography.Results and discussionResults showed significant differences both in affinity and antigen-neutralization ability between these molecules. Surprisingly, hz.scFv-Fc produced by HEK293 cells presented the lowest affinity constant and antigen-neutralizing ability. After analyzing the N-glycosylation pattern, we found that both percentage of occupied N-glycan sites and main oligosaccharide structure differ between them. Although it is necessary to continue with the comparative characterization, the producer cell line influences the antigen binding activity and it should be taken into account to develop a new therapeutic antibody.