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. 2016 Oct 21;354(6310):350-354.
doi: 10.1126/science.aag3267. Epub 2016 Sep 8.

A "Trojan horse" bispecific-antibody strategy for broad protection against ebolaviruses

Anna Z Wec  1 , Elisabeth K Nyakatura  2 , Andrew S Herbert  3 , Katie A Howell  4 , Frederick W Holtsberg  4 , Russell R Bakken  3 , Eva Mittler  1 , John R Christin  5 , Sergey Shulenin  4 , Rohit K Jangra  1 , Sushma Bharrhan  1 , Ana I Kuehne  3 , Zachary A Bornholdt  6 , Andrew I Flyak  7 , Erica Ollmann Saphire  6   8 , James E Crowe Jr  9   10   11 , M Javad Aman  12 , John M Dye  13 , Jonathan R Lai  14 , Kartik Chandran  15
Affiliations

A "Trojan horse" bispecific-antibody strategy for broad protection against ebolaviruses

Anna Z Wec et al. Science. .

Abstract

There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor-binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such "Trojan horse" bispecific antibodies have potential as broad antifilovirus immunotherapeutics.

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Figures

Fig. 1
Fig. 1. Dual-variable domain Ig (DVD-Ig) molecules combining extracellular delivery and endosomal receptor–RBS–binding specificities can recognize both of their respective antigens
(A) Schematic of mAb-548 and MR72 endosomal receptor or RBS–specific mAbs and FVM09 GP–specific delivery mAb (top row), as well as DVD-Igs engineered to combine them (bottom row). (B) A hypothetical mechanism for delivery of DVD-Igs (bottom), but not parent IgGs (top), to the endosomal sites of GPCL-NPC1 interaction. LE, late endosomes. (C) Kinetic binding curves for DVD-Ig–antigen interactions were determined by BLI. FVM09∼548 (left) and FVM09∼MR72 (right) were loaded onto probes, which were then dipped in analyte solutions (FVM09∼548: EBOV GP and human NPC1-C; FVM09∼MR72: EBOV GP and GPCL). Gray lines show curve fits to a 1:1 binding model. See table S1 for kinetic binding constants. (D) Two-phase binding experiments for the DVD-Igs by BLI. Each DVD-Ig–bearing probe was sequentially dipped in analyte solutions containing EBOV GP and then NPC1-C (FVM09∼548), or EBOV GP and then GPCL (FVM09∼MR72). (C) and (D) Representative curves from two independent experiments are shown.
Fig. 2
Fig. 2. DVD-Igs, but not parent IgGs or their mixtures, have broad neutralizing activity against ebolaviruses
(A to D) Neutralization of rVSVs encoding enhanced green fluorescent protein (eGFP) and bearing filovirus GP proteins in human U2OS osteosarcoma cells. Virions were preincubated with increasing concentrations of each parent IgG, DVD-Ig, or equimolar mixtures of parent IgGs (e.g., FVM09 + mAb-548) (1:1 mixture) and then exposed to cells for 12 to 14 hours at 37°C. Infection was measured by automated counting of eGFP+ cells and normalized to infection obtained in the absence of Ab. TAFV, Taï forest virus; RESTV, Reston virus; MARV, Marburg virus. (E to F) Neutralization of authentic filoviruses in human U2OS osteosarcoma cells, measured in microneutralization assays. Infected cells were immunostained for viral antigen at 48 hours postinfection and enumerated by automated fluorescence microscopy. (A) to (F) Averages ± SD for four to six technical replicates pooled from two or three independent experiments. (G) Data in (A) to (F) were subjected to nonlinear regression analysis to derive Ab concentrations at half-maximal neutralization (IC50 ± 95% confidence intervals for nonlinear curve fit). *IC50 values derived from curves that did not reach 90% neutralization at the highest concentration tested in the experiments are shown.
Fig. 3
Fig. 3. Roles of delivery and endosomal receptor–RBS– targeting specificities in ebolavirus neutralization by DVD-Igs
(A) Neutralizing activity of DVD-Igs against rVSVs bearing WT GP or a GP(E288D/W292R) mutant, in which Asp replaces Glu288 and Arg replaces Trp 292 (see fig. S9). (B) Neutralizing activity of mutant DVD-Igs bearing mAb-548 or MR72 combining sites with mutations in the third VH complementarity determining region (FVM09∼548Mut and FVM09∼MR72Mut, respectively). (C) Neutralizing activity of DVD-Igs against rVSV-EBOV GP in U2OS cells bearing endogenous levels of NPC1 (NPC1WT) or ectopically overexpressing NPC1 (NPC1High). (A) to (C) Averages ± SD for six technical replicates pooled from two independent experiments. (D) Internalization of labeled Abs into cells in the absence or presence of viral particles. A schematic of the experiment is shown at the left. Parent IgGs and DVD-Igs covalently labeled with the acid-dependent fluorophore pHrodo Red were incubated with rVSV-EBOV GP particles and exposed to cells. Virus− Ab+ and virus+ Ab+ populations were measured by flow cytometry. Averages ± SD for four technical replicates pooled from two independent experiments are shown. Group means for the percentage of Ab+ cells were compared by two-factor analysis of variance (ANOVA) (see fig. S11). Šídák's post hoc test was used to compare the capacity of each Ab to internalize into virus− versus virus+ cell populations (****P < 0.0001; ns, not significant). Dunnett's post hoc test was used to compare the internalization of each Ab to that of the “no Ab” control in virus+ cell populations (****P < 0.0001; all other Ab versus no Ab comparisons were not significant). (E) Delivery of Abs to NPC1+ endosomes. FVM09∼548 was incubated with rVSV–EBOV GP particles and exposed to cells expressing an NPC1–enhanced blue fluorescent protein–2 fusion protein. Viral particles, Ab, and NPC1 were visualized by fluorescence microscopy (also see figs. S12 and S13). Representative images from two independent experiments are shown. Scale bar, 20 μm.
Fig. 4
Fig. 4. FVM09∼MR72 affords broad postexposure protection from lethal ebolavirus challenge
(A) BALB/c mice were challenged with mouse-adapted EBOV (EBOV-MA), and then treated with single doses of parent IgG mixtures [300 mg, ∼15 mg per kilogram body weight (mg/kg)] or DVD-Igs (400 μg, ∼20 mg/kg, adjusted for molecular weight), or vehicle (phosphate-buffered saline, PBS) at 2 days postchallenge. (B) Type 1 IFNα/β R−/− mice were challenged with WT SUDV and then treated with two doses of parent IgG mixtures (300 μg total per dose, ∼15 mg/kg); DVD-Igs (400 μg per dose, ∼20 mg/kg); or vehicle (PBS) 1 and 3 days post-challenge. “n” indicates the number of animals per group. Data from single cohorts are shown. Survival in each group was compared with that in the PBS group by log-rank (Mantel-Cox) test (*P < 0.05; ***P < 0.001); all other comparisons to the untreated group were not significant.
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Comment in

  • Hitting Ebola, to the power of two.
    Labrijn AF, Parren PW. Labrijn AF, et al. Science. 2016 Oct 21;354(6310):284-285. doi: 10.1126/science.aaj2036. Science. 2016. PMID: 27846516 No abstract available.

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