title: Pyroglutamate and O-linked glycan determine functional production of anti-IL17A and anti-IL22 peptide-antibody bispecific genetic fusions.
abstract: Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. 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, the heavy chain, or both chains. Although 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 due to the N-terminal degradation. 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 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. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. 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 peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule.
authors.affiliation: Pfizer BioTherapeutics Research and Development, Cambridge, Massachusetts 02140, USA. Xiaotian.Zhong@pfizer.com
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kieras
authors.forename: Eric
authors.initials: E
authors.lastname: Sousa
authors.forename: Aaron
authors.initials: A
authors.lastname: D'Antona
authors.forename: J Christian
authors.initials: JC
authors.lastname: Baber
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.forename: Joel
authors.initials: J
authors.lastname: Desharnais
authors.forename: Lauren
authors.initials: L
authors.lastname: Wood
authors.forename: Deborah
authors.initials: D
authors.lastname: Luxenberg
authors.forename: Mark
authors.initials: M
authors.lastname: Stahl
authors.forename: Ronald
authors.initials: R
authors.lastname: Kriz
authors.forename: Laura
authors.initials: L
authors.lastname: Lin
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.forename: Lori J
authors.initials: LJ
authors.lastname: Fitz
authors.forename: Jill F
authors.initials: JF
authors.lastname: Wright
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000235:genetics*; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D020381:Interleukin-17 / Q000276:immunology*; D007378:Interleukins / Q000276:immunology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry*; D000097043:Interleukin-22
chemical_list: D018033:Antibodies, Bispecific; D020381:Interleukin-17; D007378:Interleukins; D011134:Polysaccharides; D011761:Pyrrolidonecarboxylic Acid; D000097043:Interleukin-22