Humanization of Chicken-derived scFv Using Yeast Surface Display and NGS Data Mining.

Generation of high-affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. We established a generic procedure for the humanization of chicken-derived antibodies. To this end, high-affinity binders of the epidermal growth factor receptor extracellular domain were isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high-affinity binders were grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement were partially randomized. A yeast surface display library comprising approximately 300,000 variants was screened for high-affinity binders in the scFv and Fab format. Next-generation sequencing disclosed humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, our sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high-throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance. This article is protected by copyright. All rights reserved.