S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice

J Virol. 2024 Jun 13;98(6):e0003824. doi: 10.1128/jvi.00038-24. Epub 2024 May 20.


Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics.

Importance: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Keywords: CAR-NK; COVID; SARS-CoV-2 virus; chimeric antigen receptor (CAR); natural killer (NK) cell.

MeSH terms

  • Angiotensin-Converting Enzyme 2* / genetics
  • Angiotensin-Converting Enzyme 2* / immunology
  • Angiotensin-Converting Enzyme 2* / metabolism
  • Animals
  • COVID-19* / immunology
  • COVID-19* / therapy
  • COVID-19* / virology
  • Humans
  • Killer Cells, Natural* / immunology
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / immunology
  • Receptors, Chimeric Antigen* / metabolism
  • SARS-CoV-2* / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • Spike Glycoprotein, Coronavirus* / genetics
  • Spike Glycoprotein, Coronavirus* / immunology
  • Spike Glycoprotein, Coronavirus* / metabolism
  • Viral Load*


  • Angiotensin-Converting Enzyme 2
  • Receptors, Chimeric Antigen
  • Spike Glycoprotein, Coronavirus
  • ACE2 protein, human
  • Single-Chain Antibodies
  • spike protein, SARS-CoV-2

Supplementary concepts

  • SARS-CoV-2 variants