• Publisher-DOI

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. A generic procedure is established for the humanization of chicken‐derived antibodies. To this end, high‐affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high‐affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high‐affinity binders in the scFv and Fab formats. Next‐generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the 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.