Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates

Abstract

Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases.

Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses.

Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent.

Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

Figure 1.

REGN3470, REGN3471, and REGN3479 bind to separate epitopes on the Ebola virus (EBOV) glycoprotein (GP). (A) Sensorgram demonstrating simultaneous binding of all 3 monoclonal antibodies (mAbs) on EBOV GP.10xhis captured on a CM5 sensor chip. Real-time increases in resonance are shown after the sequential addition of EBOV GP.10xhis, followed by REGN3479, REGN3470, and REGN3471 (graph foreshortened at indicated vertical lines to facilitate presentation). (B) Graph showing the maximal Response units (RU) values obtained from the experimental format shown in (A), for all possible orders of addition of the 3 mAbs. (C) Negative-stain electron microscopy of REGN3470, REGN3471, and REGN3479 bound to EBOV GPΔTM. Fragment antigen-binding agents (Fabs) bound to EBOV GPΔTM were examined by single-particle, negative-stain electron microscopy (EM). Far left panels show representative 2-dimensional reference-free class averages of Fab:GP complexes. Central panels show side views (parallel to the viral surface) and top views (perpendicular to the viral surface, down the 3-fold axis of symmetry) of reconstructions of Fabs bound to EBOV GPΔTM (in white), with Fabs segmented and colored purple (top, REGN3471), green (middle, REGN3470), and orange (bottom, REGN3479). Right panels show combined reconstructions of REGN3471, REGN3470, and REGN3479 on a single EBOV GPΔTM, demonstrating the relative locations of the epitopes on GP from the 3 different competition groups. Maps were aligned onto EBOV GPΔTM from the c13C6:c4G7 reconstruction (EMDB 6152). Bottom right, model of the targeted epitopes showing additional detail of the predicted membrane-bound GP.

Figure 2.

REGN3470, REGN3471, and REGN3479 monoclonal antibodies (mAbs) display different functional properties and protect guinea pigs from lethal Ebola virus (EBOV) disease. (A) Neutralization of virus infection of Huh7 cells challenged with EBOV Zaire 2014 glycoprotein (GP) or vesicular stomatitis virus (VSV) GP-pseudotyped lentiviruses by REGN3470, REGN3471, and REGN3479, alone or in a 1:1:1 cocktail. (B) Activation of FcγRIIIa signaling in engineered Jurkat cells mixed with HEK293 target cells expressing EBOV Zaire 2014 GP in the presence of REGN3470, REGN3471, and REGN3479, alone or in a 1:1:1 cocktail. (C) Survival and clinical scores of guinea pigs infected with 1000 plaque-forming units of guinea pig-adapted EBOV given a single dose of REGN3470, REGN3471, REGN3479, or a control mAb (6 animals per group) at day 1 postinfection.

Figure 3.

Three doses of REGN3470-3471-3479 cocktail protect rhesus macaques from disease after infection with the Kikwit strain of Ebola virus. (A) Survival plots, (B) signs of clinical disease, and (C) viral load data for animals treated with placebo (6 animals) or with 3 doses of 50 mg/kg (1:1:1) of REGN3470-3471-3479 cocktail on days 5, 8, and 11 postinfection (9 animals).

Figure 4.

REGN3470-3471-3479 cocktail protects rhesus macaques from disease after infection with the Kikwit strain of Ebola virus using various doses/dosing regimens. (A) Survival, (B) signs of clinical disease, and viral load by (C) plaque assay or (D) genome copies for animals in rhesus macaques treated with placebo (4 animals), 3 doses of 50 mg/kg (1:1:1) of cocktail on days 5, 8, and 11 postinfection (4 animals), 2 doses of 50 mg/kg (1:1:1) of REGN3470-3471-3479 cocktail on days 5 and 8 (5 animals), or 1 dose of 150 mg/kg (1:1:1) of cocktail on day 5 (5 animals).

Figure 5.

A single dose of REGN3470-3471-3479 cocktail protects rhesus macaques from a lethal infection with the Kikwit strain of Ebola virus. (A) Survival and (B and C) animal temperature data for rhesus macaques treated with placebo (4 animals) or a single dose of 10 mg/kg (9 animals), 50 mg/kg (9 animals), 100 mg/kg (9 animals), or 150 mg/kg (9 animals) (1:1:1) of REGN3470-3471-3479 cocktail on day 5 postinfection.