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.
Highlights
In recent years, antibody engineering aimed at generating next-generation antibody formats such as bispecific antibodies gained massive interest since they can be programmed to possess multiple novel functionalities that cannot be mediated by conventional monoclonal antibodies
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
To obtain anti-EGFR common light chain antibodies, a chicken was immunized with EGFR-ECD as described [37], and the VH encoding genes were amplified and transferred into the yeast expression vector pYD1 [40]
Summary
Antibody engineering aimed at generating next-generation antibody formats such as bispecific antibodies (bsAbs) gained massive interest since they can be programmed to possess multiple novel functionalities that cannot be mediated by conventional monoclonal antibodies (mAbs). The Wang group published an anti-CD3 fragment antigen-binding (Fab) that was C-terminally fused to two camelid single-domain antibodies (VHHs) targeting non-overlapping epitopes on the cancer target HER2. This resulted in a trivalent, biparatopic construct showing potent T cell-mediated cytotoxicity via simultaneous CD3 and HER2 binding, even in low HER2expressing cells [9]. Even though such antibody fragments can mediate impressive cytotoxic effects, their usage in vivo is limited by their short half-life
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