Citation

  • Authors: Tanaka S. et al.
  • Year: 2021
  • Journal: Sci Rep 11(1)
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: CHO-S
    Description: Chinese hamster ovary cells

Method

CHO-S cells in suspension were cultured in CD-CHO media with 8 mM L-glutamine in shaker flasks at 37 ℃ with 125 rpm rotation and 8% CO2. One day before transfection, CHO-S cells were seeded at a density of 1 × 10^6 cells/mL in 45 mL culture flask. On the day of transfection, 75 µL of FectoPRO® was mixed with 5 mL of 15 µg/mL pcDNA3 plasmid DNA in CD-CHO media and incubated for 10 min at room temperature. The DNA/transfection reagent mixture was added to 45 mL of CHO-S culture and incubated at 37 ℃ with 5% CO2 and 125 rpm rotation. On Day 3, 50 mL of the CD-CHO media with 8 mM L-glutamine was added and the culture incubated for an additional 4 days. ACE2 Decoy IgGs were then purified using HiTrap™ MabSelect SuRe™ column

Abstract

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.

Pubmed