Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail

Abstract

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

Fig. 1. Paired antibody repertoire for human- and mouse-derived SARS-CoV-2 neutralizing antibodies.

(A and B) Variable (V) gene frequencies for paired heavy (x axes) and light (y axes) chains of isolated neutralizing antibodies to SARS-CoV-2 for VI mice (A) (N = 185) and convalescent human donors (B) (N = 68). The color and size of the circles correspond to the number of heavy and light chain pairs present in the repertoires of isolated neutralizing antibodies. Neutralization is defined as >70% with 1:4 dilution of antibody (~2 μg/ml) in VSV-based pseudoparticle neutralization assay.

Fig. 2. Neutralization potency of anti–SARS-CoV-2 spike mAbs.

(A) Serial dilutions of anti-spike mAbs, IgG1 isotype control, and recombinant dimeric ACE2 (hACE2.hFc) were added with pVSV-SARS-CoV-2-S(mNeon) to Vero cells, and mNeon expression was measured 24 hours after infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only infection control. (B) Neutralization potency of anti-spike mAbs, recombinant dimeric ACE2, and IgG1 isotype control against nonreplicating pVSV-SARS-CoV-2-S(mNeon) in Calu-3 cells. (C) Neutralization potency of individual anti-spike mAbs and combinations of mAbs against replicating VSV-SARS-CoV-2-S virus in Vero cells. Cells were infected with a multiplicity of infection (MOI) 1 of the virus and stained for viral protein 24 hours after infection to measure infectivity. (D) Neutralization potency of individual anti-spike mAbs and combinations of mAbs against SARS-CoV-2-S virus in VeroE6 cells.

Fig. 3. HDX-MS determines mAb interaction on spike protein RBD.

3D surface models for the structure of the spike protein RBD domain showing the ACE2 interface and HDX-MS epitope mapping results. RBD residues that make contacts with ACE2 (21, 22) are indicated in yellow (top). RBD residues protected by anti–SARS-CoV2 spike antibodies are indicated with colors that represent the extent of protection, as determined by HDX-MS experiments. RBD residues in purple and blue indicate sites of lesser solvent exchange upon antibody binding that have greater likelihood to be antibody-binding residues. The RBD structure is reproduced from PDB 6M17 (21).

Fig. 4. Complex of REGN10933 and REGN10987 with the SARS-CoV-2 RBD.

(A) 3.9-Å cryo-EM map of the REGN10933-RBD-REGN10987 complex, colored according to the chains in the refined model (B). RBD is colored dark blue; REGN10933 heavy and light chains are green and cyan, respectively; and REGN10987 heavy and light chains are yellow and red, respectively.