Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

Abstract

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

Keywords: Ebolavirus; antibody synergy; antibody therapeutics; cooperative neutralization; ebolavirus infection; epitope mapping; glycoprotein; molecular mimicry; neutralizing antibodies; viral antibodies.

Graphical abstract

Figure 1

Candidate Cocktail Human mAbs rEBOV-520 and EBOV-548 Exhibit Differential Fc-Mediated Activities, Broadly Neutralize ebolaviruses, and Differentially Recognize the GP (A) In vitro killing capacity curves for IgG1-engineered variants of mAbs determined using SNAP-tagged EBOV GP-expressing 293F cell line as a target and human PBMCs as source of effector cells. The dotted line indicates assay background. (B) EBOV, BDBV, or SUDV neutralization. Viruses encoding enhanced green fluorescent protein (eGFP) were incubated with increasing concentrations of purified mAbs and infection was determined at 3 days after inoculation by measuring eGFP fluorescence in cells. (C) Binding of candidate mAbs to intact cell-surface-displayed EBOV GP (solid shapes) or cleaved EBOV GP_CL (open shapes). Fluorescently labeled mAbs were incubated with a suspension of cells from a Jurkat cell line that was stably transduced with EBOV GP (Jurkat-EBOV GP), or the same cells treated with thermolysin to cleave GP (Jurkat-EBOV GP_CL); binding was assessed by flow cytometry. Dotted lines (black) indicate a dynamic range of mAb binding to the GP. Dashed lines show estimated curve slopes based on a constraint for saturating binding values. Mean ± SD (n = 3) from at least two independent experiments are shown in (A) to (C). See also Figure S1 and Table S1.

Figure 2

MAbs in the Cocktail Cooperate, Enabling Enhanced GP Binding, Virus Neutralization, and In Vivo Protection (A) 2D class averages of Fab-EBOV GP complexes by negative stain EM demonstrate simultaneous binding of rEBOV-520 (orange) and rEBOV-548 (blue) to the GP. (B) Binding to the Jurkat-EBOV GP was assessed by flow cytometry using Alexa Fluor 647 (AF647)-labeled mAb rEBOV-520 or rEBOV-548 alone (solid shape), or AF647-labeled mAb titrated into a fixed concentration (20 μg/mL) of unlabeled partner mAb (open shape) as indicated. Saturated binding was estimated as in Figure 1C. Effect from mAb composition was assessed by two-way ANOVA. Arrows show comparisons to estimate fold-increase in binding. (C) Concentration-dependent potentiation of GP binding by partner mAb was assessed as in (B) using a single concentration of labeled mAb alone or the same labeled mAb in the mixture with increasing concentrations of unlabeled partner mAb as indicated. Fold increase in binding to the GP is shown with numbers in orange. (D) rEBOV-548 potentiated neutralization of SUDV by rEBOV-520. Virus was incubated with increasing concentrations of rEBOV-520 alone (gray), or rEBOV-520 titrated into a sub-neutralizing concentration (20 μg/mL) of rEBOV-548 (green). Percent SUDV neutralization by rEBOV-548 alone is shown with dotted line. p value was estimated from a comparison of IC₅₀ values (Student’s t test). (E) Cooperative enhancement of protection against SUDV infection in mice by rEBOV-548. Stat1^−/− mice were inoculated with WT SUDV and treated at 1 dpi with 10 mg/kg rEBOV-520 alone, 10 mg/kg of rEBOV-548 alone, 20 mg/kg rEBOV-520/rEBOV-548 cocktail (1:1 mixture of each mAb), or mAb DENV 2D22 (control). Survival (top), combined clinical scores (middle), and combined body weights (bottom) of surviving mice are shown. Indicated groups were compared using Mantel-Cox test. A “+” indicates an animal found dead prior to reaching the pre-determined clinical score. Data are pooled from two to three independent experiments. Mean ± SD (n = 3) from at least two independent experiments are shown in (B)–(D). See also Figure S2 and Table S2.

Figure 3

The Cocktail mAbs Recognize Relatively Conserved Epitopes on the Glycan Cap and Base Regions of the GP (A) Negative stain EM reconstruction of rEBOV-548 Fab was overlaid onto a reconstruction of rEBOV-520 (EMDB-7955). A single protomer of EBOV GP is shown fit into GP density (Protein Database [PDB]: 5JQ3). (B) Mutations to alanine in indicated residues that reduced EBOV-548 binding (< 25% of binding to WT EBOV GP, magenta bars) but did not affect binding of control mAbs BDBV425 or rEBOV-520 (gray bars) were identified (top). The exception is the W275A mutation that reduced binding of BDBV425, because W275 is part of the BDBV425 epitope on the GP. Error bars represent the mean and range (half of the maximum minus minimum values) of at least two replicates. Identified epitope residues are shown on EBOV GP trimer (PDB: 5JQ3) in magenta (bottom). (C) Crystal structure of EBOV GP_CL in complex with rEBOV-520 Fab. EBOV GP_CL is shown in surface representation. GP1 colored in cyan and GP2 colored in yellow. rEBOV-520 Fab is shown in cartoon representation. The heavy chain (HC) colored dark orange and the light chain (LC) colored light orange. The approach angle of ADI-15946 is indicated with a violet dashed arrow. (D) The footprints of rEBOV-520 or ADI-15946 Fab on EBOV GP_CL (represented as in C) are shown in red or violet, respectively. Their shared footprint is shown in blue. The location of the non-conserved EBOV GP residue N506 is indicated within a dashed white circle. See also Table S3.

Figure 4

The Binding Site of rEBOV-520 Includes the 3₁₀ Pocket That Is Fully Exposed in GP_CL but Masked by the β17-β18 Loop in Intact GP (A) An enlarged view showing occupation of the 3₁₀ pocket of the GP by rEBOV-520 CDRH3 residues. Ten residues of the CDRH3 tip are shown in orange cartoon. EBOV GP_CL is shown in surface representation with GP1 in cyan and GP2 in yellow. The 3₁₀ pocket residues involved in the interface with rEBOV-520 CDRH3 and that showed a decrease in hydrogen-deuterium binding to the uncleaved GP as determined by HDX-MS, are mapped onto the surface of GP1 (light red) and GP2 (light yellow). (B) Individual alanine mutation of six residues in the β17-β18 loop that increased binding by mAb EBOV-520 (gray bars) but did not affect binding of a recombinant form of the GP base mAb KZ52 (white bars) were identified (top). Mean and range (half of the maximum minus minimum values) of at least two replicate data points are shown. Positions of identified residues in the β17-β18 loop (green) are shown on EBOV GP (PDB: 5JQ3) with gray spheres (bottom). See also Figure S3.

Figure 5

Cooperativity in the Cocktail of rEBOV-520 and rEBOV-548 Is Mediated by Structural Remodeling of the GP Glycan Cap (A) Cryo-EM structure of EBOV GP ΔMucΔTM (GP1 in cyan and GP2 in yellow) bound to rEBOV-548 Fab (HC in dark blue and LC in light blue) and rEBOV-520 Fab (HC in orange and LC in tan). Shown is a side view (left) and top view (right) in relation to the viral membrane. Fab constant domains were excluded by masking. (B) The crystal structure of apo-EBOV GP ΔMucΔTM (PDB: 5JQ3) with the β18-β18′ region (red) and the β17-β18 loop (green). On the right is the EBOV ΔMucΔTM-rEBOV-520-rEBOV-548 cryo-EM structure; the rEBOV-548 CDRH3 loop is shown in blue. (C) Interaction of the rEBOV-548 LC residue Q27_L1 with GP1 residue D117. (D) Enlarged view of the rEBOV-520 epitope (orange, yellow, and cyan) overlaid with the unliganded structure of EBOV GP ΔMucΔTM (gray and green). (E) The β17-β18 loop and the base of the α1 helix in the glycan cap present in the unliganded GP structure (gray and green) overlaid with rEBOV-520 bound GP structure (orange). The regions of interference with rEBOV-520 binding are shown with red crosses. (F) The position of the α1 helix in the rEBOV-520-rEBOV-548 bound GP structure (cyan) overlaid with that of the unliganded GP helix (magenta). (G and H) A cartoon of proposed cooperativity mechanism for the GP binding by the cocktail in which rEBOV-520 alone binds weakly (G), but in the presence of changes caused by rEBOV-548 engagement, rEBOV-520 binds strongly (H). See also Figures S4–S6, Table S4, and Video S1.

Figure 6

MAbs rEBOV-520 and rEBOV-548 Bind to the GP in a Manner That Mimics Interactions in Unliganded GP (A) rEBOV-548 CDRH3 loop residues that make contacts along the β17 sheet in GP1 (from K272–K276), forming several hydrogen bonds within an extended beta sheet in the glycan cap (black dotted lines) and mimic this interaction by the β18-β18′ region in the unliganded structure are shown. Another key contact shown is with W275, which is cradled within a hydrophobic pocket formed by the tip of the CDRH3 at W108 and H112. (B) Interaction of the rEBOV-520 CDRH3 loop residue W100 with the 3₁₀ pocket residue N512 in GP1 that mimics interaction by the β17-β18 loop residue in the unliganded structure, and additional interactions when the glycan cap is intact (Y108 with K510 in GP2, Y106 with T77 and a hydrophobic patch in GP1, and T104 with the base of the α1 helix at P250 in GP1) are shown.

Figure 7

The mAb Cocktail Protects Nonhuman Primates against EVD Animals received a lethal dose of the EBOV Kikwit isolate intramuscularly (i.m.) on day 0 and were treated with total 30 mg/kg of the cocktail (1:1 mixture of each mAb) intravenously on days 3 and 6 after infection (n = 5). The contemporaneous control was an untreated NHP challenged with the virus (n = 1). One experiment was performed. (A) Kaplan-Meier survival plot. (B) Clinical score. (C) Kinetics of blood viral load as determined by qRT-PCR. (D) Selected blood chemistry measurements. Abbreviations are as follows: ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; CRE, creatinine. (E) Plaque assay measurement of infectious virus load in various peripheral tissues of treated NHPs (day 28 after infection). Tissues from succumbed untreated NHP (day 6 after infection) used as a control. The < symbol indicates infections virus was not detected. (F) Concentration of human mAbs that was determined in serum of five treated and one control NHPs at indicated time points after virus challenge. mAb treatment times are indicated with blue dotted lines and orange arrows. Orange curves indicate treated and black curves indicate untreated animals in (A)–(D). Ten historical controls (gray) are shown for comparative purposes in (A). The black dotted line in (B) indicates the clinical score threshold for euthanasia. The black dotted line in (C) indicates the limit of detection (LOD) for genome equivalents (GEQ); each measurement represents the mean of technical duplicates. See also Figure S7 and Tables S5 and S6.