Citation
- Authors: Zhao F. et al.
- Year: 2022
- Journal: iScience 25 104914
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Biotinylated proteins were made by co-transfecting Avitagged RBD plasmids with a BirA expression plasmid and into Expi293 cells using FectoPRO. After incubation for 10 min at RT, transfection mixture was added to Expi293 cells at a density of ~ 3 million cells/mL. After 24h of transfection, cells were fed with D-(+)- glucose solution and 300 mM of sterile sodium valproic acid solution. After 5 days of transfection, supernatants were harvested and filtered with a 0.22 mm membrane.
Abstract
The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we used a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduces the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity-matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for antiviral indications would benefit from affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.