Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody

Abstract

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

Fig. 1. Engineering SARS-CoV-2 antibodies for enhanced neutralization breadth and potency.

(A) Flow cytometry plots from the terminal round of selection, showing binding of parental antibodies (light blue) and affinity maturation library antibodies (dark blue) to the SARS-CoV-2 S1 protein at 1 nM. Gates indicate the yeast populations sorted for antibody sequencing and characterization. (B) Dot plots of Fab binding affinities (left) and MLV–SARS-CoV-2 pseudovirus neutralization IC₅₀s (right) of parental antibodies and affinity-matured progeny. Clinical-stage SARS-CoV-2 antibodies are shown for comparison. (C) Heatmap showing the neutralization IC₅₀s of the indicated antibodies against authentic SARS-CoV, WIV-1-nLuc, SHC014-nLuc, SARS-CoV-2-nLuc, and SARS-CoV-2 on either HeLa-hACE2 or Vero target cells. SARS-CoV, WIV-1-nLuc, SHC014-nLuc, and SARS-CoV-2 nLuc assays were performed on Vero target cells. N.D., not determined; N.A., not applicable due to maximal neutralization plateau at <50% neutralization. (D) Authentic SARS-CoV-2 neutralization titrations performed using either HeLa-hACE2 (left) or Vero (right) target cells. The curves were fit by nonlinear regression. Error bars represent SD. All data are representative of at least two independent experiments.

Fig. 2. Breadth of antibody binding to diverse sarbecoviruses and circulating SARS-CoV-2 variants.

(A) Phylogenetic tree of 57 sarbecoviruses constructed via MAFFT (Multiple Alignment using Fast Fourier Transform) and maximum likelihood analysis of RBD subdomain 1 amino acid sequences extracted from the European Nucleotide Archive and GISAID database. Representative sarbecovirus RBDs selected for further study are denoted in bold and colored according to their canonical phylogenetic lineages. (B) Heatmap of antibody and recombinant hACE2 binding to yeast-displayed RBDs from 17 representative sarbecoviruses, grouped by phylogenetic lineages. K_D^App values were calculated by normalized nonlinear regression fitting. N.B., nonbinder under the conditions of this assay. (C) Antibody binding to naturally occurring SARS-CoV-2 RBD variants displayed on the surface of yeast. SARS-CoV-2 sequences were retrieved from the GISAID database on 14 July 2020 (n = 63,551 sequences). Antibody binding signal was normalized to RBD expression and calculated as percent binding of the variant relative to the WT SARS-CoV-2 RBD, assessed at their respective K_D^App concentrations for the WT construct. The prevalence of each variant, calculated from deposited GSAID sequences on 9 December 2020 (n = 211,539 sequences), is shown as a percentage of the total number of sequences analyzed. (D) Correlation between the number of resistant SARS-CoV-2 variants and percentage of clade 1 sarbecovirus RBDs recognized. All data are representative of two independent experiments.

Fig. 3. ADG-2 binds to a conserved RBD epitope overlapping the hACE2 binding site.

(A) Schematic showing the generation and selection of a mutagenized, yeast surface–displayed SARS-CoV-2 RBD library to identify mutations that knock down ADG-2 binding. (B) Heatmap showing mutations that abrogate binding of ADG-2 to the SARS-CoV-2 RBD. S309 and CR3022, which bind nonoverlapping epitopes distinct from the ADG-2 binding site, are included to control for mutations that globally disrupt the conformation of the RBD. Values indicate percent antibody or recombinant hACE2-Fc binding to the mutant SARS-CoV-2 RBD relative to the WT SARS-CoV-2 RBD, assessed at their respective EC₈₀ concentrations (80% effective concentrations) for the WT RBD construct. (C) Protein sequence alignment of representative sarbecovirus RBDs, with sequences colored by percentage sequence identity and conservation shown as a bar plot. Positions delineating the receptor binding motif are based on the SARS-CoV-2 RBD. Residues determined to be important for ADG-2 binding, on the basis of the data shown in (B), are denoted in red. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (D) Cryo-EM reconstruction of the SARS-CoV-2 RBD bound by ADG-2, with ADG-2 knockdown mutations (blue) and the hACE2 binding site (black outline) highlighted. (E) Structures of previously reported antibodies (bold), representing frequently observed SARS-CoV-2 nAb classes 1 to 4, are overlaid on the ADG-2 structure (D), with additional representative SARS-CoV-2 nAbs listed.

Fig. 4. ADG-2 triggers Fc-mediated effector functions.

The indicated antibodies were assessed for the ability to induce Fc-mediated effector functions against RBD-coated targets at varying concentrations. (A) Primary human NK cells were analyzed for surface expression of CD107a, indicating degranulation (left), and the production of interferon-γ (IFNγ) (middle) or tumor necrosis factor–α (TNFα) (right) after incubation with antibody-RBD immune complexes for 5 hours. IgG, immunoglobulin G. (B) Antibody-mediated phagocytosis of RBD-coated fluorescent beads by differentiated THP-1 monocytes (left) or HL-60 neutrophils (right) was measured after incubation with immune complexes for 18 hours. Hu., human. (C) Antibody-mediated complement deposition was measured by detection of complement component C3 onto RBD-coated fluorescent beads after incubation of guinea pig complement with immune complexes for 20 min. MFI, mean fluorescence intensity. Error bars represent SD. All data are representative of two independent experiments.

Fig. 5. Prophylactic and therapeutic administration of ADG-2 protects mice from SARS-CoV– and SARS-CoV-2–associated disease.

Efficacy of prophylactic treatment with ADG-2 in (A) SARS-CoV–MA15 and (B) SARS-CoV-2–MA10 challenge models. A single dose of ADG-2 or sham treatment was delivered intraperitoneally 12 hours before infection. Mouse body weight and respiratory function were monitored for 4 days. Gross lung hemorrhage scores were determined on day 2 (MA15) or day 4 (MA10) after infection, and lung viral titers were measured on days 2 and 4 after infection. (C) Therapeutic treatment with ADG-2 or sham treatment 12 hours after SARS-CoV-2–MA10 infection. Mouse body weight, respiratory function, gross hemorrhage scores (day 2), and lung viral titers (days 2 and 4) were assessed as described above. Statistical comparisons were made using Mann-Whitney U tests or two-sided t tests with Holm-Sidak corrections for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). Dotted lines indicate the limit of detection. Horizontal bars indicate mean or geometric mean.