Citation

  • Authors: Weidenbacher PA. et al.
  • Year: 2022
  • Journal: Nat Chem Biol
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi

Method

All proteins were expressed in Expi293F cells. Expi293F cells were cultured in media containing 66% Freestyle/33% Expi media (Thermo Fisher Scientific) and grown in TriForest polycarbonate shaking flasks at 37 °C in 8% CO2. The day before transfection, cells were spun down and resuspended to a density of 3 × 106 cells per ml in fresh media. The next day, cells were diluted and transfected at a density of approximately 3–4 × 106 cells per ml. Transfection mixtures were made by adding the following components: mirA-prepped or maxi-prepped DNA, culture media and FectoPro (Polyplus) would be added to cells to a ratio of 0.5–0.8 µg:100 µl:1.3 µl:900 µl. For example, for a 100-ml transfection, 50–80 µg of DNA would be added to 10 ml of culture media, and then 130 µl of FectoPro would be added to this. After mixing and a 10-minute incubation, the resultant transfection cocktail would be added to 90 ml of cells. The cells were harvested 3–5 days after transfection by spinning the cultures at >7,000g for 15 minutes. Supernatants were filtered using a 0.22-µm filter. To determine hCoV protein expression, spun-down Expi293F supernatant was used without further purification. For proteins containing a biotinylation tag (Avi-Tag), Expi293F cells containing a stable BirA enzyme insertion were used, resulting in spontaneous biotinylation during protein expression.

Abstract

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

Pubmed