Fundamental Research into Enhancing Fab Functionality by Protein Engineering

Abstract

Antigen-binding fragments (Fab) are a type of antibody fragment that contains an antigen-binding site in a therapeutic antibody format. To further improve their utility as therapeutic antibodies, this study aimed to enhance Fab functionality through protein engineering. A Fab expression system using the yeast Pichia pastoris was constructed, and recombinant Fabs were efficiently prepared. Second, a Fab mutant suitable for conjugation with polyethylene glycol (PEG) was generated to increase the serum half-life of the Fab. The interchain disulfide bond normally formed at the C-terminus (H: Cys224-L: Cys214) was shifted to a novel position (H: Cys177-L: Cys160), allowing a free cysteine residue at the C-terminus to be used for site-directed PEGylation without conformational destabilization of the Fab. The prepared PEGylated Fab displayed an increased serum half-life. Several additional sites for the introduction of interchain disulfide bonds, which contribute to conformational stability, have been identified in the Fab constant region, and a Fab with an N-glycosylation site introduced at position 178 of its heavy chain (H: L178N) was expressed in P. pastoris. The high-mannose type N-glycan attached to Fab showed the inhibited Fab aggregation under pH shift-induced stress, and the immunogenicity of the glycosylated Fab was lower than that of the wild-type Fab. These protein engineering results are expected to contribute to the design of Fab molecules with increased functional value and greater safety.