• Authors: Miersch S. et al.
  • Year: 2021
  • Journal: J Mol Biol 433 167177
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi


Ab production and purification: - IgG and tetravalent Abs were produced in Expi293F cells by transient transfection, by diluting heavy and light chain construct DNA in OptiMem serum-free media (Gibco) before the addition of and incubation with FectoPro (Polyplus Transfection) for 10 min. - For IgG expression, equivalent amounts of plasmids encoding heavy chain or light chain were transfected, whereas for tetravalent formats, a ratio of 2:1 light chain to heavy chain plasmids was used. - Following addition of the DNA complex to Expi293F cells and a 5-day expression period, Abs were purified using rProtein A Sepharose (GE Healthcare), then buffer exchanged and concentrated using Amicon Ultra-15 Centrifugal Filter devices (Millipore). - IgGs were stored in PBS (Gibco), and tetravalent Abs were stored in 10 mM L-Histidine, 0.9% sucrose, 140 mM NaCl, pH 6.0.


Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.