Isolation of Common Light Chain Antibodies from Immunized Chickens Using Yeast Biopanning and Fluorescence‐Activated Cell Sorting

Abstract

The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)‐based chicken monoclonal antibodies from an anti‐epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence‐activated cell sorting (FACS). For the selection of high‐affinity antibodies, a yeast cell library presenting cLC‐comprising fragment antigen binding (Fab) fragments is panned against hEGFR‐overexpressing A431 cells. The resulting cell–cell‐complexes are sorted by FACS resulting in gradual enrichment of EGFR‐binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC‐based bispecific antibodies against native mammalian receptors.

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

“…Gene expression was controlled by the galactose-inducible promotor (GAL1). For the soluble expression of full-length chimeric antibodies, pTT5-derived vectors ( 40 ) were utilized, encoding either the heavy or the light chain constant domains. Biparatopic variants were expressed using pTT5-derived vectors encoding the full-length chimeric antibody with either a knob or hole mutation ( 12 ) within the CH3 sequence, and a C-terminal His- or Twin-StrepII-Tag, respectively.…”

“…Plasmids were isolated utilizing the Wizard ® Plus SV Miniprep Kit (Promega) and sequenced at Microsynth Seqlab (Göttingen). Resulting VH genes were amplified using Q5 polymerase (NEB), according to the manufacturer’s protocol, incorporating SapI sites and were subsequently subcloned into pTT5-derived vectors by Golden Gate cloning, as described previously ( 40 ). For soluble expression, Expi293F™ (Thermo Fisher, A14527) cells were transfected, harvested and secreted proteins purified by Protein A as described elsewhere ( 23 ).…”

“…Recently, our group established a fluorescence-activated cell sorting (FACS)-based yeast surface display (YSD) technology for the isolation of high affine chickenderived antibodies (36)(37)(38)(39). Additionally, we demonstrated the potential of incorporating a common light chain (cLC) within the screening procedure while maintaining affinity and a broad epitope coverage (40). In this study, we describe the isolation and characterization of the first biparatopic common light chain antibody targeting the extracellular domain of epidermal growth factor receptor (EGFR-ECD) ( Figures 1C, D) that is derived from immunized chickens by combining a novel epitope binning-based screening strategy with the knob-into-hole technology for the generation of bispecific heavy chain pairs.…”

DataSheet_1.pdf

“…Gene expression was controlled by the galactoseinducible promotor (GAL1). For the soluble expression of fulllength chimeric antibodies, pTT5-derived vectors (40) were utilized, encoding either the heavy or the light chain constant domains. Biparatopic variants were expressed using pTT5-derived vectors encoding the full-length chimeric antibody with either a knob or hole mutation (12) within the CH3 sequence, and a C-terminal His-or Twin-StrepII-Tag, respectively.…”

“…The used yeast strains and their handling, as well as the utilized libraries, were described previously (40). Screening rounds were performed utilizing either a BD Influx FACS Cell sorter or a Sony SH800S.…”