Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Abstract

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

Fig. 1. S2E12 and S2M11 neutralize SARS-CoV-2 ultrapotently by targeting the RBD.

(A and B) Neutralization of authentic SARS-CoV-2 (SARS-CoV-2-Nluc) by S2E12 (A) and S2M11 (B) IgG or Fab. Symbols are means ± SD of triplicates. Dashed lines indicate IC₅₀ and IC₉₀ values. Average IC₅₀ values are indicated in parentheses below the graphs (determined from two independent experiments). (C to F) ELISA binding of S2M11 (red), S2E12 (blue), or S309 (yellow) mAbs to immobilized SARS-CoV-2 RBD (C), SARS-CoV-2 S (D), SARS-CoV RBD (E), or SARS-CoV S (F). Symbols show means of duplicates. (G) SPR analysis of S2E12 and S2M11 Fab binding to the SARS-CoV-2 RBD or S ectodomain trimer. Experiments were carried out at pH 7.4 (orange) and pH 5.4 (green) and were repeated twice with similar results (one experiment is shown). The apparent equilibrium dissociation constants (K_{D, app}) at pH 7.4 are indicated. White and gray stripes indicate association and dissociation phases, respectively. S2M11 binding to S was fit to two parallel kinetic phases and the resulting K_{D, app #1} and K_{D, app #2} were interpreted as apparent affinities for open RBDs (tertiary epitope) and closed RBDs (quaternary epitope), respectively. This is supported by the similar binding kinetics and affinity of the faster off-rate phase (K_{D, app #1}) with that observed for S2M11 binding to the isolated RBD (compare with table S1 for full fit results). Ab conc, mAb concentration.

Fig. 2. The S2E12 neutralizing mAb recognizes the SARS-CoV-2 RBM.

(A and B) Cryo-EM structure of the prefusion SARS-CoV-2 S ectodomain trimer with three S2E12 Fab fragments bound to three open RBDs viewed along two orthogonal orientations. (C) The S2E12 concave paratope recognizes the convex RBM tip. (D) Close-up view showing selected interactions formed between S2E12 and the SARS-CoV-2 RBD. In (A) to (D), each SARS-CoV-2 S protomer is colored distinctly (cyan, pink, and gold), whereas the S2E12 light- and heavy-chain variable domains are colored magenta and purple, respectively. N-linked glycans are rendered as blue spheres in (A) to (C). Abbreviations for the amino acid residues are as follows: E, Glu; F, Phe; I, Ile; L, Leu; N, Asn; Q, Gln; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3. The S2M11 neutralizing mAb recognizes a quaternary epitope spanning two RBDs and stabilizes S in the closed state.

(A and B) Cryo-EM structure of the prefusion SARS-CoV-2 S ectodomain trimer bound to three S2M11 Fab fragments viewed along two orthogonal orientations. (C and D) The S2M11 binding pose, which involves a quaternary epitope spanning two neighboring RBDs. (E and F) Close-up views showing selected interactions formed between S2M11 and the SARS-CoV-2 RBDs. In (A) to (F), each SARS-CoV-2 S protomer is colored distinctly (cyan, pink, and gold), whereas the S2M11 light- and heavy-chain variable domains are colored magenta and purple, respectively. N-linked glycans are rendered as blue spheres in (A) to (D) and as sticks in (E) and (F). FR, framework.

Fig. 4. S2E12 and S2M11 prevent SARS-CoV-2 S attachment to ACE2 and inhibit membrane fusion, and S2M11 triggers effector functions.

(A) S2E12 (magenta/purple) and ACE2 (dark green) bind overlapping binding sites on the SARS-CoV-2 RBD (blue). (B) S2M11 (magenta/purple) and ACE2 (dark green) bind overlapping binding sites on the SARS-CoV-2 RBD (blue). The red stars indicate steric clashes. (C and D) Binding of the SARS-CoV-2 RBD (C) or S ectodomain trimer (D) alone (gray) or precomplexed with the S2M11 (red), S2E12 (blue), or S309* (yellow) mAbs to the ACE2 ectodomain immobilized at the surface of biosensors analyzed by biolayer interferometry. S309* is an optimized version of the parent S309 mAb (21). KB, kinetic buffer (negative control). (E) Binding of varying concentrations of S2E12 (blue), S2M11 (red), or S309 (yellow) mAbs to full-length S expressed at the surface of CHO cells in the presence of the ACE2 ectodomain (20 μg/ml) analyzed by flow cytometry (one measurement per condition). (F) Cell-cell fusion inhibition assay with Vero E6 cells transfected with SARS-CoV-2 S and incubated with varying concentrations of S2E12 (blue), S2M11 (red), S309 (yellow), or a control mAb. The values are normalized to the percentage of fusion without mAb and to the percentage of fusion of nontransfected cells. (G) FcγRIIIa (high-affinity variant V158) signaling induced by individual mAbs or mAb cocktails. For mAb cocktails, the concentration of the constant mAb was 5 μg/ml. The concentration of the diluted mAb is indicated on the x axis. (H) ADCC using primary NK cells as effectors and SARS-CoV-2 S-expressing CHO cells as targets. The magnitude of NK cell–mediated killing is expressed as the area under the curve (AUC) for each mAb used at concentrations ranging between 0.1 ng/ml and 20 μg/ml. For mAb cocktails, the mAb listed first was kept constant at 5 μg/ml. Each symbol represents one donor; data are combined from two individual experiments. See fig. S6E for curves from a representative donor. (I) ADCP using peripheral blood mononuclear cells (PBMCs) as a source of phagocytic cells (monocytes) and PKH67–fluorescently labeled S-expressing CHO cells as target cells. The y axis indicates percentage of monocytes double-positive for anti-CD14 (monocyte) marker and PKH67. The dashed line indicates the signal detected in the presence of target and effector cells but without mAb (baseline). Each line indicates the data for one PBMC donor. Symbols are means of duplicates. Data are from one experiment. Ab conc, mAb concentration.

Fig. 5. S2E12, S2M11, or cocktails of the two mAbs provide robust in vivo protection against SARS-CoV-2 challenge.

Syrian hamsters were injected with the indicated amount of mAbs 48 hours before intranasal challenge with SARS-CoV-2. (A) Quantification of viral RNA in the lungs 4 days after infection. (B) The concentration of mAbs measured in the serum before infection (day 0) inversely correlates with the viral RNA load in the lung 4 days after infection. (C) Quantification of replicating virus in lung homogenates harvested 4 days after infection using a TCID₅₀ assay. For mAb cocktails, the total dose of an equimolar mixture of both mAbs is indicated.