FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein

Abstract

A phage library displaying 10 10 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD and the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

Establishment of the first Chinese national standard for protein subunit SARS-CoV-2 vaccine

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Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application

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An adaptable, monobody-based biosensor scaffold with FRET output

“…Here, we create an adaptable and genetically encoded switch by applying the AFF mechanism to the 10 th domain of FN3, termed monobody, a small protein that has been evolved by in vitro selection methods to specifically bind ∼30 protein targets 14,15 including epidermal growth factor receptor 16 , maltose-binding protein (MBP) 17 , mixed lineage kinase domain-like protein 18,19 , and severe acute respiratory syndrome coronavirus 2 spike protein 20,21 . Like the antibody light chains they resemble, monobodies achieve molecular recognition by presenting different residues at surface positions (1-3 CDR-like loops as well as several βstrands) while maintaining a constant amino acid sequence at other positions 14 .…”