Fig 1.
SC31 binds SARS-CoV-2 SP and neutralizes virus through inhibition of binding to ACE2.
(A) Neutralization of 100 TCID50 of infectious virus by SC31 IgG1 compared to control IgG1 or IgG purified from donor serum, represented as a percentage relative to uninfected and “virus only” controls. (B) Binding affinity of SC31 IgG to purified wild type SP or RBD as measured by ELISA. (C) Inhibition of wild-type SP or RBD binding to cells expressing membrane-bound ACE2 by SC31 IgG1 at different concentrations as measured by flow cytometry, expressed as a percentage relative to “no antibody” and “no viral protein” controls. (D) Binding affinity of SC31 as determined by ELISA to purified wild-type SP (blue) and SP mutants that either do not affect SC31 or ACE2 binding (green), affect SC31 but not ACE2 binding (purple) or affect both SC31 and ACE2 binding (red). (E) Binding affinity of purified wild-type and mutant SP to hACE2-expressing CHO cells based on fluorescence intensity measured by flow cytometry. Statistical significance was determined versus wild-type SP for each mutant by one-way ANOVA: ** p<0.01, *** p<0.001, **** p<0.0001. (F). Locations of single amino acid mutations (green/purple/red) on a crystal structure of RBD showing the interaction of the RBM (cyan) with ACE2 N-terminal helix (blue). All results represent the mean of three independent replicates with bars showing standard error.
Fig 2.
(A) Activation of ADCC signaling by SC31 or Fc null-binding LALA variant at various concentrations incubated with FcγRIIIa-expressing reporter cell line and SP- or mock-transfected HEK293, as determined by luciferase expression. Statistical significance in comparison to the highest SC31 concentration of 4 μg/ml was determined by one-way ANOVA. (B) Specific binding of SC31 to SP-transfected HEK293 cells in comparison to mock-transfected cells or fluorophore stain only (Ctrl stain).
Fig 3.
SC31 requires Fc effector functions for optimal therapeutic benefit.
SARS-CoV-2 infected K18-hACE2 mice, 10 mice per group, were dosed with either 20 mg/kg SC31 IgG1 or LALA variant antibodies at 6 hpi. Half of mice (n = 5) were sacrificed at 3 dpi to ascertain lung viral load and cytokine levels in addition to serum antibody titers; the remaining mice were monitored for weight changes and survival. Lung viral load in IgG1-, LALA-treated or untreated mice at 3 dpi as measured by (A) qRT-PCR or (B) cell culture, the limit of detection (LOD) is indicated by the dotted line. Disease progression in infected mice as indicated by (C) weight loss and (D) survival. (E) Lung cytokine mRNA expression determined by qRT-PCR represented as fold-change over uninfected mice. (F) Sera IFNγ protein levels determined by sandwich ELISA. Each point represents one mouse and all error bars show standard error. (G and H) Lack of ADE of SARS-CoV-2 pseudovirus infection based on luciferase reporter gene expression following co-incubation with SC31 or LALA variant in (G) THP-1 and (H) Raji as compared to ACE2-expressing CHO cells. (I) Retention of SC31 binding affinity for wild-type SP between pH 4.5–7.0 as measured by ELISA. Results represent the mean of three or four independent replicates with error bars showing standard error. Statistical significance was determined using two-way ANOVA with Fisher’s LSD test for viral load, D5-7 weight loss or cytokines/chemokines and Chi-Square for D15 survival percentage, ns–p>0.05, * p<0.05, ***<0.001 vs. untreated.
Fig 4.
Dose-and time-dependent therapeutic benefit of SC31.
Groups of 10 SARS-Cov-2 infected K18-hACE2 mice were treated with SC31 IgG1 or isotype control at the indicated doses 6 hpi. Half of the mice (n = 5) were sacrificed at 3 dpi to assess lung viral load; the remaining mice were monitored for weight changes and survival. Lung viral load as measured by (A) qRT-PCR and (B) cell culture, the limit of detection (LOD) is indicated by the dotted line. Disease progression is indicated by (C) weight loss and (D) survival. For the time-dependent study, SC31 IgG1 was administered at 20 mg/kg at the indicated time points. Half of the mice (n = 5) were sacrificed at 3 dpi to assess lung viral load and cytokine levels in addition to serum antibody titers. Remaining mice were monitored for weight changes and survival. Lung viral load as measured by (E) qRT-PCR and (F) cell culture, the limit of detection (LOD) is indicated by the dotted line. Disease progression in infected mice as indicated by (G) weight loss and (H) survival. (I) Lung cytokine mRNA expression determined by qRT-PCR and represented as fold-change over uninfected mice. Each point represents one individual mouse and all error bars show standard error. Statistical significance was determined using two-way ANOVA with Fisher’s LSD test for viral load, D5-6 weight loss or cytokines/chemokines and Chi-Square for D15 survival percentage, ns–p>0.05, * p<0.05, **p<0.01, ***p<0.001 vs. untreated.
Fig 5.
SC31 minimizes disease after SARS-CoV-2 challenge in Golden Syrian hamsters and Indian Rhesus Macaques.
Twelve hamsters (6 male/6 female) were intranasally challenged with 5×105 TCID50 SARS-CoV-2; 6 animals were treated with SC31 antibody or vehicle at 4 hpi. The viral load of nasal swabs (Nasal Sw) collected daily was determined by (A) qRT-PCR and (B) cell culture; dotted line represents the lower limit of detection (LOD) for the assay. (C) Viral load in hamster lungs harvested 7 dpi as determined by qRT-PCR. (D) Disease progression, as indicated by weight loss, was prevalent in vehicle controls. (E) Harvested lungs from vehicle control-treated (left) and SC31-treated (right) hamsters at 7 dpi; images taken following removal of the left lobe for viral load analysis; representative images are shown. (F) Histopathology examination of fixed lung tissue from vehicle control-treated (left) and SC31-treated (right) hamster at 7 dpi; representative images are shown. Indian rhesus macaques (12 male/12 female) were challenged with 1×106 TCID50 SARS-CoV-2 via combined intratracheal/intranasal (mucosal atomization) delivery. Viral loads in Nasal Sw (G-H) and BAL fluid samples (I-J) collected at early, mid, and late stages of disease were determined by qRT-PCR and cell culture. For all panels, error bars show standard error.