Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

Nat Chem Biol. 2021 Jan;17(1):113-121. doi: 10.1038/s41589-020-00679-1. Epub 2020 Oct 20.

Abstract

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2 / antagonists & inhibitors
  • Angiotensin-Converting Enzyme 2 / chemistry*
  • Angiotensin-Converting Enzyme 2 / genetics
  • Angiotensin-Converting Enzyme 2 / immunology
  • Animals
  • Antibodies, Neutralizing / chemistry*
  • Antibodies, Neutralizing / genetics
  • Antibodies, Neutralizing / immunology
  • Antibodies, Viral / chemistry*
  • Antibodies, Viral / genetics
  • Antibodies, Viral / immunology
  • Binding Sites, Antibody / genetics
  • Binding Sites, Antibody / immunology
  • Chlorocebus aethiops
  • Cryoelectron Microscopy
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Peptide Library
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • SARS-CoV-2
  • Single-Chain Antibodies / chemistry*
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • Spike Glycoprotein, Coronavirus / antagonists & inhibitors
  • Spike Glycoprotein, Coronavirus / chemistry*
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology
  • Vero Cells

Substances

  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Peptide Library
  • Single-Chain Antibodies
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2