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.

Keywords: antibody discovery; biparatopic antibody; chicken-derived; common light chain; yeast display.

Figure 1

Common light chain biparatopic antibodies and schematic representation of the screening procedure. (A) The heterodimerization of the heavy chains is achieved by the Knob-into-Hole technology. Due to the need for correct pairing of heavy and light chains, only 25% of produced antibodies are correctly assembled. (B) The utilization of a common light chain pairing with both heavy chains enabled the circumvention of the light chain pairing problem. (C) Monoclonal antibodies recognize a single epitope of the target antigen and can therefore bind to two individual receptors. (D) Biparatopic antibodies bind to two distinct epitopes of the same antigen. By binding to two receptor molecules, additional epitopes on the target remain exposed allowing for the binding of an additional antibody, leading to crosslinking of the target receptor and receptor clustering. (E) Epitope binning-based FACS screening resulted in two VH domains, addressing orthogonal epitopes while comprising one common light chain. Subsequent reformatting into the Knob-into-Hole format enabled the production of a biparatopic antibody. Created with BioRender.com.

Figure 2

Epitope binning of FEB4 and SEB7. (A) Schematic representation of epitope binning in-tandem setup. His-tagged antigen is loaded onto Ni-NTA biosensors (1), followed by binding of the first antibody until saturation is achieved (2). Subsequently, either the saturation antibody or the completion antibody is applied (3), resulting only in an increment of layer thickness if orthogonal epitopes are addressed. (B) BLI-based binning in-tandem setup investigating epitope recognition of FEB4 and SEB7. Ni-NTA tips were loaded with EGFR-ECD, followed by binding to the first antibody of interest until saturation was achieved. Subsequently, either the second antibody was applied, or a new incubation of the first antibody was performed, which served as control. Created with BioRender.com.

Figure 3

Epitope analysis of FEB4 and SEB7. (A) Schematic representation of EGFR domains and the fragments investigated in this study. (B) YSD-based epitope mapping. Binding of FEB4 and SEB7 to yeast cells expressing different EGFR-derived domains were detected utilizing the goat anti-human Fc PE antibody. (C) Conformational epitope recognition on various truncated versions of EGFR-ECD. Yeast cells expressing the targeted EGFR-derived fragments were either incubated at 4°C or 80°C, respectively, for 30 min. Surface presentation was verified by the anti-c-myc biotin antibody and Streptavidin APC, binding of FEB4 and SEB7 was measured utilizing the anti-human Fc PE antibody.

Figure 4

EGF competition assays. (A) BLI-based EGF competition. Using AHC biosensors, the depicted antibodies were loaded followed by association to EGFR preincubated with varying EGF concentrations. (B) EGF-dependent binding to EGFR⁺⁺⁺ A431 cells. 1×10⁶ A431 cells were stained utilizing antibodies preincubated with or without EGF. Binding to A431 cells was measured by FACS using the goat anti-human Fc PE antibody. Created with BioRender.com.

Figure 5

Clustering assay of the biparatopic antibody. (A) Schematic representation of BLI-based two-site binding assay that can either result in a clean two-site binding or a mixed site binding. (B) BLI-based two-site binding assay. Biotinylated BpAb was loaded onto SAX tips followed by incubation with EGFR-ECD or kinetics buffer, respectively. Subsequently, binding of unmodified BpAb to the complex was analyzed. Each step was aligned separately (C) Schematic representation of ELISA-based two-site binding assay. (D) ELISA of BpAb binned against itself. 50 µl of a 25-nM BpAb solution was coated on a 96-well MaxiSorp MTP (ThermoFisher) overnight, followed by incubation with 200 nM EGFR-ECD and varying concentrations of biotinylated BpAb (1–128 nM). Binding was detected utilizing Streptavidin-HRP and TMB-One solution (Promega). Each concentration was measured in triplicates and analyzed using GraphPad Prism 8. A two-site specific binding fit was applied. Created with BioRender.com.

Figure 6

ADCC Assay of parental and biparatopic antibodies on A431 cells. The Promega ADCC assay was performed utilizing the parental and the biparatopic variants. 12,500 A431 cells were cultured overnight before incubation with a fourfold serial dilution of antibodies (5 µg/ml to 305 pg/ml) and ADCC effector cells for 6 h. Luciferase signal is plotted against the logarithmic antibody concentration. EC₅₀ values: FEB4, 45.8 pM; SEB7, 191.5 pM; FEB4/SEB7, 98.9 pM.