Humanization of Chicken‐Derived scFv Using Yeast Surface Display and NGS Data Mining

Abstract

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.

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

“…For therapeutic usage, avian-derived antibodies are unsuitable due to their high immunogenicity, likely resulting in anti-drug-antibodies (ADA), which diminish the effectiveness of therapeutic mAbs ( 62 ). To circumvent this problem, our group previously established a YSD-based humanization strategy ( 55 ). In brief, the CDRs of the chicken-derived antibody are grafted onto a human acceptor framework with simultaneous randomization of Vernier residues, critical for CDR orientation.…”

“…Our group previously showed that chicken-derived immune libraries, exhibiting common light chains can successfully be screened to isolate high affine binders targeting a large epitope space (53) and that these cLC mAbs can be utilized to construct biparatopic/bispecific antibodies exhibiting improved properties compared to their parental, monospecific counterparts (54). As those molecules were still of avian germline origin, we established an expeditious humanization technique, based on CDR-grafting and randomization of Vernier residues, to generate humanized antibodies with parental-like properties, high germline identity and low number of T cell epitopes (55). This strategy was used here for the first time to generate humanized bi-and trispecific molecules starting from avian antibodies.…”

“…All plasmids, as well as their utilization for library generation by homologous recombination, Golden Gate cloning, or for expression, were previously discussed in detail (53)(54)(55). Yeast strains and their handling are described elsewhere (53,102).…”

“…We recently showed that immunization of chickens enables the isolation of common light chain antibodies targeting a broad epitope space (53), and that these antibodies can be assembled in a heterodimerized manner resulting in biparatopic antibodies comprising cLCs (54). Furthermore, we demonstrated a straightforward method for humanization of chicken-derived molecules (55). Here, we describe the affinity maturation of an anti-EGFR cLC-based antibody by light chain shuffling and utilization of the resulting light chain as a new common light chain to generate anti-CD16a and anti-PD-L1 antibodies.…”

Expeditious Generation of Biparatopic Common Light Chain Antibodies via Chicken Immunization and Yeast Display Screening

“…44% homology to the avian H2 VL domain (data not shown), a pairing with a chicken-derived VH would most probably not result in a functional antibody. Our group recently demonstrated a fast and straightforward solution to humanize avian-derived antibodies via CDR loop transplantation onto human scaffolds, randomization of key residues that dictate loop orientation followed by functional screening using yeast surface display (55). Experiments leading to humanized bispecific and biparatopic chicken-derived antibodies are currently in progress.…”