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

Abstract

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings.

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

“…Biolayer interferometric measurements revealed no epitope drift, as dFEB4-1 targeted the same epitope as the parental FEB4 mAb, exhibiting an overlapping epitope with matuzumab, but not with cetuximab (Supplementary Figure 4). These results are in accordance with previous binning experiments performed with the parental antibody variant (54). Additionally, BLI measurements showed that EGF interferes with the ability of dFEB4-1 to bind EGFR (Figure 3A), comparable to the parental molecule or cetuximab (54).…”

“…Recently, we isolated a chicken-derived EGFR-specific antibody termed FEB4 from an immune library comprising a restricted light chain diversity. The respective antibody showed affinity in the lower-double-digit nanomolar range, targeted a conformational epitope on EGFR domain III, exhibited an overlapping binding site with matuzumab, and inhibited EGF binding to its receptor (54). Due to its ability to block EGF binding to its receptor, as well as targeting a proximal binding site to the intensively investigated matuzumab epitope, we chose FEB4 as a starting molecule.…”

“…In order to generate a light chain library, the VL genes were amplified from cDNA derived from the EGFR-immunized chicken from which the FEB4 VH gene originated (54,56). The genes for VL domains were inserted into a pYD1-derived vector encoding a human lambda CL by homologous recombination in BJ5464 yeast.…”

“…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).…”

Recombinant Antibody Production Using a Dual-Promoter Single Plasmid System

“…Assembly of the MD expression constructs was conducted with 75 ng destination vector and equimolar amounts of the respective fragments, 20 U BbsI-HF, 10 U Esp3I, and 200 U T4-DNA ligase (NEB, Frankfurt, Germany) for 30 cycles (1 min; 16 • C; 37 • C). For the reference constructs, VH and VL genes were amplified incorporating SapI restriction sites and then inserted into a pTT5-derived vector utilizing CH1-CH2-CH3 or κ/λ entry vectors using GGC as described before [24,25]. PCR reactions were performed utilizing Q5 polymerase (NEB) according to the manufacturer's protocol and purified using the Wizard SV Gel and PCR Clean-up System (Promega, Walldorf, Germany).…”