Lois Lequesne, PhD
Albert Einstein College of Medicine
Leveraging the Natural Diversity of the Bovine Antibody Repertoire for the Discovery of Cysteine-Rich Peptide via Yeast Display
Cattle diversify their antibody repertoire through a unique mechanism involving a cysteine-rich ultralong complementarity-determining region 3 of the heavy chain (UL-CDRH3). At 40–70 amino acids, versus 8–16 in humans, UL-CDRH3s comprise at least 10% of the bovine repertoire. They form a distinctive “stalk-and-knob” structure with a ~5-kDa, cysteine-rich knob containing 3–5 disulfide bonds. The knob can be expressed independently and alone mediates antigen recognition. In size, length, and structure, these knobs resemble cysteine-rich peptides (CRPs) from plants and animal venoms, which combine structural stability with specific binding to ion channels, or microbial antigens.Unlike CRPs, UL-CDRH3 knobs undergo somatic hypermutation and can be readily isolated and characterized.
To harness this sub-repertoire, we built a semi-synthetic yeast-display library of UL-CDRH3s grafted onto an scFv scaffold, containing ~10⁸ variants. The library aims to identify binders to cryptic or recessed epitopes and non-immunogenic targets, ultimately enabling the production of stable, antigen-specific peptides independent of the scFv scaffold. As a proof of concept, we panned the library against SARS-CoV-2 spike and human transferrin receptor 1 proteins, isolating hundreds of binders to each target with diverse knob sequences and binding phenotypes. These findings support the discovery of CRP-like binders to challenging targets for therapeutic, diagnostic, and research applications.
Cattle diversify their antibody repertoire through a unique mechanism involving a cysteine-rich ultralong complementarity-determining region 3 of the heavy chain (UL-CDRH3). At 40–70 amino acids, versus 8–16 in humans, UL-CDRH3s comprise at least 10% of the bovine repertoire. They form a distinctive “stalk-and-knob” structure with a ~5-kDa, cysteine-rich knob containing 3–5 disulfide bonds. The knob can be expressed independently and alone mediates antigen recognition. In size, length, and structure, these knobs resemble cysteine-rich peptides (CRPs) from plants and animal venoms, which combine structural stability with specific binding to ion channels, or microbial antigens.Unlike CRPs, UL-CDRH3 knobs undergo somatic hypermutation and can be readily isolated and characterized.
To harness this sub-repertoire, we built a semi-synthetic yeast-display library of UL-CDRH3s grafted onto an scFv scaffold, containing ~10⁸ variants. The library aims to identify binders to cryptic or recessed epitopes and non-immunogenic targets, ultimately enabling the production of stable, antigen-specific peptides independent of the scFv scaffold. As a proof of concept, we panned the library against SARS-CoV-2 spike and human transferrin receptor 1 proteins, isolating hundreds of binders to each target with diverse knob sequences and binding phenotypes. These findings support the discovery of CRP-like binders to challenging targets for therapeutic, diagnostic, and research applications.
