Chicken-derived antibodies emerged as a promising tool for diagnostic and therapeutic usage. Due to the phylogenetic distance between birds and mammals, chicken immunization campaigns with human antigens result in a chicken antibody (IgY) repertoire targeting epitopes not addressed by rodent-derived antibodies. However, this phylogenetic distance accounts for a low homology of IgY molecules to human antibodies, resulting in potential immunogenicity and thus excluding IgYs from therapeutic applications. Herein, we describe a straightforward method to efficiently humanize chicken-derived antibodies by a CDR-grafting-based approach, including a simultaneous randomization of key residues (Vernier residues). Utilizing yeast surface display (YSD) and fluorescence-activated cell sorting (FACS), yeast cells displaying functional humanized scFvs and Fab variants are isolated, and subsequent next-generation sequencing (NGS) enables the identification of humanized antibody variants with restored affinity and beneficial protein characteristics.
Keywords: CDR grafting; Chicken antibody; Fluorescence-activated cell sorting; Humanization; Next-generation sequencing; Vernier residues; Yeast surface display.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.