Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization

Abstract

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1–4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

“…We characterized Fab-S cryo-EM structures (overall resolutions from 3.4-3.8Å) of potent hNAbs (C002, C104, C119, and C121) predicted to compete with ACE2 binding 5 , which varied in their V gene segment usage and CDRH3 lengths ( Table 1). Fab-S cryo-EM structures of these class 2 hNAbs showed bound RBDs in both "up" or "down" conformations, consistent with observations of similar hNAbs from nsEM 5,12 and single-particle cryo-EM studies 10,34,41 . By contrast, the C144-S structure showed Fabs bound only to "down" RBDs ( Fig.…”

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies

“…Our biochemical and cell-based experimental results showed that the full-length IgG nAbs have greater binding ability, neutralization ability, and induce more S1 shedding than Fab. Similar results were also obtained in other study (27) reported, most of which used nAb in Fab-form, especially those solved with X-ray crystallography. Our results suggest that the full-length IgG-form, which is more biologically relevant than Fab, should be used for the structural determination as much as possible to understand the structural and functional features of nAbs.…”

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

“…To obtain pre-fusion spike ectodomain, methods similar to the previous reports were used. 33 , 49 The expression plasmid, provided by the McLellan lab, was used in a transient transfection with 100 mL, high-density Chinese Hamster Ovary (ExpiCHO, Thermo Fisher) culture following the “High Titer” protocol provided by Thermo Fisher. Six to nine days post-transfection, the supernatant was collected with centrifugation at 4,000xg at room temperature.…”

“…To obtain pre-fusion spike ectodomain, methods similar to the previous reports were used. 33,49 The processing package. 51 Micrographs were manually curated, CTF was estimated utilizing patches and particles were picked with a Gaussian blob.…”