Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Fig. 1. PBMC single cell sorting strategy and antibody characterization. a Memory B cells were selected from convalescent patient PBMCs by gating on CD3-CD19+CD20+CD10-IgD-IgG+ cells (Supplementary Fig. 1; upper panel) and single cells sorted by binding to fluorescently labeled SARS-CoV-2 RBD or trimeric spike initially (left panel). Red dots represent the B cells for which monoclonal antibodies were amplified and characterized (Supplementary Fig. 1; Sort 1). In addition, using b mutant RBD protein (mutations colored in orange), single cells were sorted based on FACS epitope bins (a, right panels, Supplementary Fig. 1; Sort 3). c Monoclonal antibodies analyzed, showing affinity for SARS-CoV-2 RBD (ancestral strain), neutralization potential and epitope class (initial FACS epitope bin and validation). nn = non neutralizing. In vitro neutralization of live (d) ancestral and (e) Delta VOC SARS-CoV-2 virus. n = 4 technical replicates, data are presented as mean values. Source data are provided as a Source Data file.

Fig. 2. Structural characterization of the broadly neutralizing class 1 antibody GAR05. a Cryo-EM structure of GAR05 Fab bound to trimeric spike (3.27 Å resolution) showing full antibody Fab occupancy of all the RBDs in the “up” conformation. Two perspectives are shown b Structure of GAR05 bound to SARS-CoV-2 RBD based on the X-ray crystal structure, outlining the antibody bound to the ACE2 “saddle” of the RBD (ACE2 interface shaded black). c Interaction of the CDR regions of the VH and VL domains with the RBD saddle and the Y505 side chain of the RBD. d Comparison of the ACE2 interface on the surface of RBD and the GAR05 epitope, showing high overlap within the ACE2 saddle region. The large, buried surface area of GAR05 (1275 Å2) indeed blankets many key residues identified in VOCs, colored in yellow, yet remarkably still binds all VOCs with high affinity.

Fig. 3. Structural identification of a novel class 6 epitope. a Cryo-EM structure of spike trimer (three shades of grey surface) complexed with GAR03 (blue cartoon and transparent surface) revealing a single GAR03 antibody Fab bound to trimeric spike (resolution 3.39 Å). Binding displaces the S1 domain (N-terminal domain and RBD) of one of the protomers (disordered and not visible in the cryo-EM map), with the GAR03 Fab binding to the RBD of the adjacent protomer. Middle and right panels show two perspectives of the RBD-GAR03 crystal structure. The binding interface is dominated by the GAR03 light chain (light blue cartoon; CDRs L1, L2, and L3) with more minor contribution from the heavy chain (dark blue; CDR H3). The ACE2 interface of the RBD (grey surface and cartoon) is shaded black, whilst Omicron VOC mutation positions are indicated by yellow sticks and surface, and N343 linked carbohydrate is shown as sticks. b Crystal structure of GAR12 (heavy and light chains colored dark and light orange) in complex with RBD (grey cartoon and surface, with ACE2 interface colored black, and Omicron VOCs colored yellow). Two perspectives are shown. c Global juxtaposition of GAR03 (blue surfaces)…

Fig. 4. Protection of K18-hACE2 mice from live viral challenge. Animals were injected interperitoneally with 30 mg/kg of GAR05, GAR12, GAR20 or human IgG1 isotype control 3 days prior to viral challenge (-3 dpi) for prophylactic studies (a–c, g–i) or +1 dpi post challenge for therapeutic studies (d–f). Mice were challenged (d0) with 1 × 103 PFU of intranasal ancestral SARS-CoV-2 or Delta VOC (B.1.167.2) virus and monitored for 6 days, and euthanized (d6) for tissue collection. Measurements of weight (percentage loss from initial weight, a [n = 4 mice per group measured on 6 consecutive days. 2-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05], d [n = 6 mice per group measured on 6 consecutive days. A single isotype control mouse was removed from the study on day 5 as it reached a humane endpoint. Mixed-effects analysis with Šídák’s multiple comparisons test. *p = 0.0149], g [n = 7 istoype control mice and n = 6 GAR05 treated mice measured on 6 consecutive days. 2-way ANOVA with Tukey’s multiple comparisons test. **p = 0.0049]), clinical score (weight loss, eye closure, appearance of fur and posture, and respiration, b/e/h), and viral titers (lung homogenates; plaque assay, c…

Fig. 5. Neutralization of the Omicron VOCs. a SARS-CoV-2 neutralization (live virus), ancestral (A.2.2), Omicron VOCs BA1, BA2, BA5 (n = 4 technical replicates). b IC50 values (ng/ml) reported for S309, GAR05 and GAR12 on ancestral and Omicron VOCs. nn = non-neutralizing. Data are presented as mean values + /- SD for measurements performed over a range of concentrations. Source data are provided as a Source Data file.