Antibody-mediated cell-cell fusion is ACE2-independent.

(A) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, hIgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of HEK293T cells stimulated with 0.1, 0.5, 2.5, 12.5 nM REGN10933 or Isotype Control for 16 hours. Data and blots were representative of four individual repeats; (B) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, hIgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions collected of CB6-stimulated HEK293T cells, treated without or with 2.5 μM EK1C4 for 16 hours. Data and blots are representative of five individual repeats; (C) Fluorescent images of mCherry reporter cell-cell fusion assay, captured at 594 nM from HEK293T cells co-expressing Beta N417 and K417 (reversion) with Cre, mixed with stop-mCherry HEK293T cells and stimulated without or with 12.5 nM CB6 for 16 hours, scale bars are representative of 50 μm; (D) Infectivity in RLU obtained from HEK293T-ACE2 cell lysates infected with SARS-CoV-2 MLV-S-WT pseudotyped particles in the presence of 0.01, 0.1, 1 and 10 μg/mL mouse anti-ACE2 blocking antibody. Data are representative of four repeats; (E) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, anti-ACE2 mIgG, hIgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of HEK293T cells stimulated with 12.5 nM CB6 antibody, without or with 1 μg/mL anti-ACE2 blocking antibody; (F) Luciferase activity (RLU) measured from cell-cell fusion assay and immunoblots showing shedded S1 subunits, hIgG Hc, full-length spike, S1, S2 and cleaved S2’ collected from supernatant and cell lysate fractions of sgControl or sgACE2 HEK293T cells stimulated with 12.5 nM CB6 antibody for 16 hours. Data are representative of three individual repeats; (G) Immunoblots showing shedded S1 subunits, anti-ACE2 mIgG, full-length spike and S1 collected from supernatant and cell lysate fractions of lentivirus transduced A549 cells expressing WT spike, stimulated with 12.5 nM CB6 antibody without or with anti-ACE2 blocking antibody for 16 hours. Blots are representative of two individual repeats; (H) Immunoblots showing shedded S1 subunits, hIgG Hc, full-length spike and S1 collected from supernatant and cell lysate fractions of lentivirus transduced sgControl and sgACE2 A549 cells expressing WT spike, stimulated with 12.5 nM CB6 antibody. Blots are representative of two individual repeats. (TIF)