Nature | Vol 584 | 20 August 2020 | 445plasma from four previously described individuals with recent
laboratory-confirmed SARS-CoV-2 infection^5 showed that COV2-2196-
and COV2-2130-like antibody responses are subdominant in these indi-
viduals (Extended Data Fig. 4).
As COV2-2196 and COV2-2130 did not compete for binding to the SRBD,
we assessed whether these monoclonal antibodies synergize for virus
neutralization—a phenomenon that has been observed previously for
SARS-CoV monoclonal antibodies^12. We tested combination responses
(Fig. 2c) in an FRNT using SARS-CoV-2, and compared the values
obtained experimentally with the expected responses calculated by
synergy-scoring models^28. The comparison revealed that the combina-
tion of COV2-2196 and COV2-2130 antibodies was synergistic, with an
overall synergy δ-score of 17.4 (where any score greater than 10 indicates
synergy; Fig. 2d). In particular, a combined monoclonal antibody dose
of 79 ng ml−1 (16 ng ml−1 of COV2-2196 and 63 ng ml−1 of COV2-2130) had
the same activity as 250 ng ml−1 of each individual antibody (Fig. 2c).
This finding shows that by using a cocktail of two antibodies, the dose
of each antibody can be reduced by more than threefold to achieve the
same potency of virus neutralization in vitro.
We next defined the epitopes that are recognized by representa-
tive monoclonal antibodies in the two major competition-binding
groups that synergize for neutralization. We used mutagenesis to
determine critical residues in the SRBD for the binding of neutralizing
monoclonal antibodies (Fig. 3a, Extended Data Fig. 5). These studies
showed that F486 or N487 are critical residues for the binding of COV2-
2196, and N487 is a critical residue for COV2-2165—two antibodies that
compete with one another for binding. Likewise, mutagenesis studies
for COV2-2130 using K444A and G447R mutants suggested that these
residues (K444 and G447) are critical for recognition (Fig. 3a). Previous
structural studies have defined the interaction between the SRBD and
human ACE2^29 (Fig. 3b). Most of the interacting residues in the SRBD
are contained within a 60-amino-acid linear peptide that defines the
human ACE2 recognition motif (Fig. 3c). We next tested the binding of
human monoclonal antibodies to this minimal peptide and found that
potent neutralizing members of the largest group of antibodies from
the competition-binding assay—including COV2-2196, COV2-2165 and
COV2-2832—recognized this peptide (Fig. 3c), suggesting that these
monoclonal antibodies make critical contacts within the human ACE2
recognition motif.
We used negative-stain electron microscopy of the S2Pecto trimer
in complex with antigen-binding fragments (Fabs) to determine the
structural epitopes for several monoclonal antibodies (Fig. 3d, e, Sup-
plementary Table 2). The potently neutralizing antibodies COV2-2196
and COV2-2165 bound to the human ACE2 recognition motif of the SRBD
and recognized the ‘open’ conformational state of the S2Pecto trimer,
in which the RBD rotates upward to expose the residues that mediate
ACE2 interaction^30 ,^31 (Fig. 3d). COV2-2130, which represents a different
competition-binding group, bound to the RBD in the S2Pecto trimer in
the ‘closed’ position (Fig. 3d). Because COV2-2196 and COV2-2130 did
not compete for binding, we attempted to make complexes of both
Fabs bound at the same time to the S2Pecto trimer. We found that both
Fabs bound simultaneously when the S2Pecto trimer was in the open
position, indicating that COV2-2130 can recognize the SRBD in both
conformations (Fig. 3e). Overlaying the structure of the two-Fab com-
plex with that of the SRBD–CR3022 complex^27 , we observed that these
antibodies bind to three distinct sites on the SRBD, as predicted by our
competition-binding studies (Fig. 3f).
Next, we tested the prophylactic efficacy of COV2-2196 or COV2-2130
monotherapy or a combination of both COV2-2196 and COV2-2130 in a
model of SARS-CoV-2 infection in BALB/c mice. In this model (Fig. 4a),
mice are first treated with an anti-IFNAR1 antibody and then transduced
with an adenovirus that expresses human ACE2 (AdV-hACE2), which
results in susceptibility to infection with SARS-CoV-2, viral replica-
tion and severe bronchopneumonia^32. The mice were treated with a
single dose of COV2-2196 or COV2-2130, a cocktail of COV2-2196 and
COV2-2130, or an isotype control monoclonal antibody one day before
intranasal challenge with a 4 × 10^5 plaque-forming unit (PFU) dose of
SARS-CoV-2. Prophylaxis with COV2-2196, COV2-2130 or their combi-
nation prevented severe SARS-CoV-2-induced weight loss in the mice
during the first week of infection (Fig. 4b). Viral RNA levels were reduced
significantly at 7 days post-infection (dpi) in the lung and in distant sites
including the heart and spleen (Fig. 4c). The expression of cytokine and96
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0 020406080100COV2-2130 (ng ml–1)COV2-2196 (ng ml–1)Neutralization (%)COV2-2196rCR3022
COV2-2196 0 95 99
COV2-2050 –2 89 99
COV2-2068 0 98 97
COV2-2072 –5 89 100
COV2-2098 4 92 97
COV2-2113 –4 96 97
COV2-2082 52 95
COV2-2094 34 89
rCR3022 96 0 26
COV2-2103 97 4 95
COV2-2130 102 90 93
COV2-2096 69 93 100Reference antibodyACE2
blocking (%)Antibody binding in presence of reference antibodyNo competitionCompetitionblockingACE20124816316 3 125 250100
500
–50–100COV2-2196 (ng ml–1)048163163125250COV2-2130 (ng ml–1)G-scoreacdCOV2-2196COV2-2072
COV2-2499COV2-3025
COV2-2381COV2-2479
COV2-2096COV2-2832
COV2-2050COV2-2130
COV2-2819COV2-2955
COV2-2094COV2-2835
COV2-2082COV2-2539
COV2-2165COV2-2562
COV2-2752COV2-2733
COV2-2268COV2-2308
COV2-2780COV2-2068
COV2-2813COV2-2015
COV2-2807COV2-2258
COV2-2812COV2-2098
COV2-2841COV2-211^3
COV2-2919COV2-2354
COV2-2677COV2-2103
COV2-2828COV2-2353
COV2-2676COV2-2489Competing antibodyb
COV2-2130 COV2-2196 rCR3022Full competitionPartial competitionNo competitionBinding of indicated reference antibody (%)Fig. 2 | Epitope mapping of monoclonal antibodies by competition-binding
analysis and synergistic neutralization by a pair of monoclonal antibodies.
a, Left, monoclonal antibody binding to the SRBD in the presence of reference
monoclonal antibodies COV2-2196 or rCR3022. Values in squares are the per
cent binding of the monoclonal antibody in the presence of the competing
monoclonal antibody relative to a mock-competition control. Black squares,
full competition (<33% relative binding); white squares, no competition (>67%
relative binding). Right, biolayer-interferometry-based competition binding
assay measuring the ability of monoclonal antibodies to prevent the binding
of human ACE2. Values are the per cent blocking of human ACE2 by the
monoclonal antibody. Red indicates high blocking activity. b, Competition
of the panel of neutralizing monoclonal antibodies with reference monoclonal
antibodies COV2-2130, COV2-2196 or rCR3022. Binding of reference
monoclonal antibodies to trimeric S2Pecto was measured in the presence of
saturating competitor monoclonal antibody in a competition ELISA and
normalized to binding in the presence of r2D22. Black, full competition (<25%
binding of reference antibody); grey, partial competition (25–60% binding of
reference antibody); white, no competition (>60% binding of reference
antibody). c, Neutralization dose–response matrix of wild-type SARS-CoV-2 by
COV2-2196 and COV2-2130. Axes denote the concentration of each monoclonal
antibody, with the per cent neutralization shown in each square. Data are from
a representative experiment performed in technical triplicate and repeated
twice. The white-to-red heat map denotes 0% neutralization to 100%
neutralization, respectively. d, Synergy map calculated on the basis of the
SARS-CoV-2 neutralization in c. δ-score is a synergy score. Red colour indicates
areas in which synergistic neutralization was observed; black box indicates the
area of maximum synergy between the two monoclonal antibodies.