Identification of Pyroglutamate and O-linked Glycosylation on Anti-IL17A Peptide Anti-IL22 Antibody Bispecific Genetic Fusion

摘要

In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N-termini of an anti-IL22 antibody, through either the light chain, heavy chain, or both chains (Fig 1.). While the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay. In-Vitro serum stability analysis followed by immunoprecipitation and mass spectroscopic identification revealed instability in the N-terminal peptide portion of the molecule (Fig.2). When a glutamine residue was introduced at the N-terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was partially restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide (Fig.3). Subsequent removal of this posttranslational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein (Fig.4). These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptideantibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule.