Cell line impact on antigen binding activity of a therapeutic humanized scFv-Fc antibody

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

Newport, Rhode Island

Congreso; 14th Protein Expression in Animal Cells (PEACe) Conference; 2019

Protein Expression in Animal Cells (PEACe) Conference

Introduction:Monoclonal antibodies (mAbs) constitute a large and growing subset of the marketed-biotherapeutics glycoproteins. It is widely assumed that antibodies are bifunctional molecules, where the variable (V) regions are responsible for antigen binding and the constant (C) regions confer effector properties, being both domains functionally independent. However, there are several studies suggesting that the C regions of different class or subclasses of antibodies with identical V regions influence the antigen binding activity. Nevertheless, although the glycosylation pattern strongly influences the effector functions, it seems not having effect in the binding antigen ability. We developed a novel humanized single-chain variable fragment (scFv) against human interferon-α2b (hIFN-α2b) fused to the human Fcγ1 region (hz.scFv Fc) as a therapeutic candidate for autoimmune diseases. This engineered antibody was obtained from a chimeric scFv-Fc (mouse scFv). In this work, we evaluated the impact of the producer cell line on affinity constant and antigen-neutralizing ability of this novel molecule in order to choose the best cell system depending on therapeutic use.Methods: The hz.scFv-Fc antibody was produced in CHO-K1, HEK293 and NS0 cells and a partially de-glycosylated scFv-Fc was generated from HEK293 produced variant.Affinity constants were measured by competitive ELISA and the neutralization of IFN biological activity was evaluated by three independent bioassays (antiviral, antiproliferative and using a reporter gene cell line). N-glycosylation structures were analyzed by hydrophilic chromatography. By way of comparison, all assays were performed including the chimeric scFv-Fc (qm.scFv-Fc) produced in CHO-K1 cells.Results and conclusions: Results showed significant differences in both affinity and antigen neutralization ability between these molecules. Although hz.scFv-Fc produced in HEK293 cells presented the lowest affinity constant between different cell produced antibodies, the partially deglycosylated molecule presented the worst properties. After analyzing the N-glycosylation pattern, we found that both, the percentage of occupied N-glycan sites and the structure of the main oligosaccharides differ depending on the host cells. These results partially agree with preliminary studies of anti-hIFN-α2b chimeric scFv Fc molecules produced in the different cell systems that suggest the influence of some post translational modification on the antibodies that affect the neutralization ability performance. Additionally, the evaluation of a partially deglycosylated hz.scFv-Fc showed the importance of glycans on antigen-binding activity. These results evidence that the producer cell line influence the mAb affinity and neutralizing action and not only its immunogenicity, leading to the conclusion that the host cells should be carefully taken into account in order to develop a new therapeutic antibody.