This application claims benefit of priority to U.S. Provisional Application Serial No. 62/379,474, filed August 25, 2016, and to U.S. Provisional Application Serial No.62/381,840, filed August 31, 2016, the entire contents of which are hereby incorporated by reference. FEDERAL FUNDING STATEMENT

This invention was made with government support under grant number 1U19AI109711 awarded by the National Institutes of Health, and under grant number HDTRA1-13-1-0034 awarded by the U.S. Defense Threat Reduction Agency (Department of Defense). The government has certain rights in the invention. BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine, infectious disease, and immunology. More particular, the disclosure relates to bispecific antibodies and antibody fragments that neutralize ebolavirus. 2. Background

The development of therapeutics targeting Ebola virus (EBOV) and other filoviruses is a global health priority. The recent success of ZMapp ^TM – a cocktail of three monoclonal antibodies (mAbs) targeting the EBOV surface glycoprotein GP– in reversing advanced Ebola virus disease in non-human primates has underscored the promise of antiviral immunotherapy and spurred efforts to discover and develop additional protective mAbs and cocktails against filoviruses and other viral pathogens (Qui et al., 2014). However, a limitation of ZMapp and most other available candidate mAbs is their narrow antiviral spectrum, which arises from their recognition of variable surface-exposed GP epitopes on extracellular viral particles (virions) and infected cells (Murin et al., 2014). Thus, ZMapp ^TM protects against EBOV, but lacks activity against other filoviruses with known epidemic potential, including the ebolaviruses Bundibugyo virus (BDBV) and Sudan virus (SUDV), and the more divergent marburgviruses. Given the scientific and logistical challenges   inherent in developing a separate mAb cocktail for each filovirus, the generation of broadly- protective anti-filovirus immunotherapies is a highly desirable goal. A number of cross- reactive but weakly neutralizing antibodies targeting filovirus GP surface epitopes have been identified, but are anticipated to have little therapeutic efficacy (Fusco et al., 2015; Wang et al., 2015; Hernandez et al., 2015; Keck et al., 2016). A few antibodies have shown cross- neutralization and protection in rodent models, indicating that cross-species protection by a single molecule is possible; however, such antibodies are exceedingly rare (Keck et al., 2016; Howell et al., 2016; Flyak et al., 2016; Frei et al., 2016; Furuyama et al., 2016; Holtsberg et al., 2016).

An unusual, and underexploited, feature of the cell entry mechanism of filoviruses is the programmed disassembly of GP in endosomes and lysosomes, to reveal highly conserved ‘cryptic’ epitopes (Moller-Tank et al., 2015; Miller et al., 2012; White and Whittaker, 2016). Cleavage and removal of a‘glycan shield,’ comprising the glycan cap and mucin domains of GP, by host endo/lysosomal cysteine proteases unmasks a recessed binding site for its critical intracellular receptor (Chandran et al., 2005; Bornholdt et al., 2015; Wang et al., 2016; Schornberg et al., 2006), the ubiquitous cholesterol transport protein Niemann-Pick C1 (NPC1) (FIGS. 5A-E) (Miller et al., 2012; Carette et al., 2011; Cote et al., 2011; Herbert et al., 2015). Engagement of NPC1’s second luminal domain, domain C, by cleaved GP (GPCL) in late endosomes is required for viral membrane fusion and cytoplasmic escape (Miller et al., 2012; Spence et al., 2016; Aman, 2016). In contrast to the surface GP epitopes bound by most protective mAbs, the cryptic GP _CL receptor-binding site (RBS) is highly conserved among filoviruses (Bornholdt et al., 2015). Indeed, an RBS-specific mAb isolated from a Marburg virus disease survivor, potently blocked GP _CL -NPC1 interaction in vitro, and broadly neutralized viruses bearing in vitro-cleaved GPCL (Bornnoldt et al., 2015; Hashiguchi et al., 2015; Flyak et al., 2015). However, MR72 failed to neutralize infection by uncleaved ebolaviruses, likely because it could not gain access to late endosomes, where the GPCL RBS becomes unmasked (Bornholdt et al., 2015). Therefore, the development of broadly protective immunotherapies targeting the GPCL RBS, or its universal binding partner NPC1, is challenged by an evolved immune evasion mechanism of filoviruses - their sequestration of virus-receptor complexes in late endocytic compartments.   SUMMARY

Thus, in accordance with the present disclosure, there is provided a method of treating a subject infected with a filovirus, or reducing the likelihood of infection of a subject at risk of contracting a filovirus, comprising delivering to said subject an bispecific antibody or bispecific antibody fragment having binding affinity for (a) a first filovirus glycoprotein domain; and (b) either (i) a Neimann-Pick C1 (NPC1) receptor or other surface feature that is targeted to the endosome; or (ii) a second filovirus glycoprotein receptor binding domain that interacts with the NPC1 domain.

The bispecific antibody fragment may be a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab’)2 fragment, or Fv fragment, or incorporated as scFv or Fab in a diabody. Element (b) may be a conserved linear epitope in a glycan cap structure of said glycoprotein. The bispecific antibody or bispecific antibody fragment may be an IgG. The bispecific antibody or bispecific antibody fragment may be a chimeric antibody or a humanized.

The bispecific antibody or bispecific antibody fragment may be administered prior to infection. The bispecific antibody or bispecific antibody fragment may be administered after infection. Delivering may comprise antibody or bispecific antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment. The bispecific antibody or bispecific antibody fragment may cross-react with epitopes on Marburg virus and Ebolavirus.

Also provided is a bispecific antibody or bispecific antibody fragment having binding affinity for (a) a first filovirus glycoprotein domain; and (b) either (i) a Neimann-Pick C1 (NPC1) receptor or other surface feature that is targeted to the endosome; or (ii) a second filovirus glycoprotein receptor binding domain that interacts with the NPC1 domain.

The bispecific antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab’) ₂ fragment, or Fv fragment, or incorporated as scFv or Fab in a diabody. Element (b) may be a conserved linear epitope in a glycan cap structure of said glycoprotein. The bispecific antibody or bispecific antibody fragment may be an IgG. The bispecific antibody or bispecific antibody fragment may be a chimeric antibody or a humanized.

The bispecific antibody or bispecific antibody fragment may comprise a variable heavy chain comprising two distinct variable regions, one having binding affinity for element (a) and one having binding affinity for element (b), and a light chain comprising two distinct   variable regions, one having binding affinity for element (a) and one having binding affinity for element (b). The light and heavy chain variable regions having affinity for element (a) may be located N-terminal to the light and heavy chain variables region having binding affinity for element (b), respectively. The bispecific antibody may be a bivalent antibody comprising a first heavy-light chain binding arm with affinity for element (a), and a second heavy-light chain binding arm with affinity for element (b). The bispecific antibody or bispecific antibody fragment may cross-react with epitopes on Marburg virus and Ebolavirus.

Also provided is a nucleic acid encoding a bispecific antibody or bispecific antibody fragment as described above.

Also provided is a recombinant cell expressing a bispecific antibody or bispecific antibody fragment as described above.

Also provided is a recombinant cell comprising an expression construct as described above.

Also provided is a vaccine formulation comprising a bispecific antibody or bispecific antibody fragment according as described above.

The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The word“about” means plus or minus 5% of the stated number.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

  BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-D. Dual-variable domain Ig (DVD-Ig) molecules combining extracellular ‘delivery’ and endosomal receptor/RBS-binding specificities can recognize both of their respective antigens. (FIG. 1A) Schematic of mAb-548 and MR72 endosomal receptor/RBS-specific mAbs and FVM09 GP-specific delivery mAb (top row), and DVD-Igs engineered to combine them (bottom row). (FIG.1B) A hypothetical mechanism for delivery of DVD-Igs (bottom), but not parent IgGs (top), to the endosomal sites of GP _CL -NPC1 interaction. (FIG. 1C) Kinetic binding curves for DVD-Ig:antigen interactions were determined by BLI. FVM09~548 (left panel) and FVM09~MR72 (right panel) 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. (FIG. 1D) Two-phase binding experiments for the DVD-Igs by BLI. Each DVD-Ig was loaded onto a probe, which was then sequentially dipped in analyte solutions containing EBOV GP and then NPC1–C (FVM09~548) or EBOV GP and then GPCL (FVM09~MR72).

FIGS. 2A-G. DVD-Igs, but not parent IgGs or their mixtures, possess broad neutralizing activity against ebolaviruses. (FIGS. 2A-D) Neutralization of rVSVs encoding eGFP and bearing ebolavirus GP proteins in human U2OS osteosarcoma cells. Virions were pre-incubated with increasing concentrations of each parent IgG, DVD-Ig, or equimolar mixtures of parent IgGs (e.g., FVM09~mAb-548), and then exposed to cells for 12–14 hr 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. (FIGS. 2E-F) Neutralization of authentic filoviruses in human U2OS osteosarcoma cells, measured in microneutralization assays. Infected cells were immunostained for viral antigen at 48 hr post-infection, and enumerated by automated fluorescence microscopy. Averages ± SD (n=3) from 2–3 experiments are shown. (FIG. 2G) Ab concentrations at half-maximal neutralization (IC ₅₀ ± 95% confidence intervals for nonlinear curve fit) are shown.   FIGS. 3A-E. Roles of delivery and endosomal receptor/RBS-targeting specificities in ebolavirus neutralization by DVD-Igs. (FIG. 3A) Neutralizing activity of DVD-Igs against rVSVs bearing WT GP or a GP(E288D/W292R) mutant that abolishes GP-FVM09 binding (see FIG.12). (FIG.3B) Neutralizing activity of DVD- Igs against rVSV-EBOV GP in U2OS cells bearing endogenous levels of NPC1 (NPC1 ^WT ) or ectopically over-expressing NPC1 (NPC1 ^High ). (FIG. 3C) Neutralizing activity of a mutant DVD-Ig protein containing mutated GPCL combining sites (FVM09~MR72 ^Mut ). (FIG.3D) 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 were covalently labeled with the acid-dependent fluorophore pHrodo Red ^TM . Labeled Abs were incubated with rVSV-EBOV GP particles containing an internal fluorescent protein label (mNeongreen), and then exposed to U2OS cells for 30 min at 37 ˚C. Cells were subjected to two-color flow cytometry (see FIG.14), and the virus ^– Ab ⁺ and virus ⁺ Ab ⁺ populations were measured. Averages ± SD (n=4) from 2 independent experiments are shown. (FIG. 3E) FVM09~548 was incubated with mNeongreen-labeled rVSV-EBOV GP particles and then exposed to U2OS cells expressing an NPC1-enhanced blue fluorescent protein-2 fusion protein for 45 min at 37 ˚C. Cells were fixed, permeabilized, and immunostained, and viral particles, Ab, and NPC1 were visualized by fluorescence microscopy (also see FIG.14).

FIGS. 4A-B. FVM09~MR72 affords broad post-exposure protection from lethal ebolavirus challenge. (FIG. 4A) BALB/c mice were challenged with mouse-adapted EBOV (EBOV-MA), and then treated with single doses of parent IgG mixtures (300 µg), DVD-Igs (400 µg; adjusted for molecular weight), or vehicle control at 2 days post-challenge. (FIG.4B) Type 1 IFNα /β R ^−/− mice were challenged with WT SUDV, and then treated with two doses of parent IgG mixtures (300 µg per dose), DVD-Igs (400 µg per dose), or vehicle control at 1 and 4 days post-challenge. Statistical values are for comparison of each Ab group to the vehicle group in an unpaired t-test. Only comparisons that reached statistical significance (P<0.05) are shown.

FIGS. 5A-E. mAb-548 targets the GPCL-binding site in NPC1 domain C (NPC1– C), and blocks GPCL:NPC1–C binding in vitro. (FIG. 5A) Surface-shaded representation of the X-ray crystal structure of an EBOV GP _CL :NPC1–C complex (PDB ID: 5F1B) (17). Green, NPC1–C; blue, GP1; orange, GP2; red, GPCL receptor- binding site (RBS) residues; yellow, GP _CL contact residues in NPC1–C. (FIGS.5B-C)   Structure of NPC1–C. GPCL contact residues (yellow) and residues critical for mAb- 548 binding (pink) are highlighted. (FIG. 5D) Kinetic binding curves for mAb- 548:human NPC1–C interaction were determined by biolayer interferometry (BLI). mAb-548 was loaded onto the probe, which was then dipped in solutions containing increasing concentrations of NPC1–C (nM; indicated for each curve). (FIG. 5E) Dose-dependent inhibition of rVSV-EBOV GP _CL :NPC1–C binding by mAb-548 in a competition ELISA.

FIGS. 6A-D. Mapping the mAb-548 binding site in human NPC1–C. (FIG. 6A) Cartoon line diagram of human NPC1 with the second luminal domain, domain C, highlighted. (FIGS. 6B-C) Using a species-specific difference in mAb-548 recognition to map its binding site in human NPC1–C:mAb-548 recognizes human NPC1–C, but not NPC1–C derived from the Russell’s viper (Daboia russellii) in an ELISA (FIG. 6C). A panel of chimeras between human and Russell’s viper NPC1–C, in which human sequences were introduced into the Russell’s viper background (44) (FIG. 6B) were tested for mAb-548 binding by ELISA (FIG. C). This gain-of- function genetic analysis demonstrated that NPC1 residues 502–511 (human numbering) contain residues critical for mAb-548 recognition (FIG.6D). In FIG.6D, amino acid sequence differences are shown in yellow. The inverted orange triangle indicates an N–linked glycosylation site present in Russell’s viper NPC1–C, but not in human NPC1–C. Russell’s viper = SEQ ID NO: 11; Human = SEQ ID NO: 12.

FIGS. 7A-C. Biochemical characterization of DVD-Igs. (FIG. 7A) FVM09~548 and FVM09~MR72 were resolved on SDS-polyacrylamide gels under reducing (R) and nonreducing (NR) conditions, and proteins were visualized by staining with Coomassie Brilliant Blue. A control IgG is shown for comparison. (FIGS.7B-C) Size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis of FVM09~548 (FIG. 7B) and FVM09~MR72 (FIG. 7C). Absorbance (arbitrary units; au) was monitored at 280 nm. m, monomer peak; a, aggregate peak. Calculated (MWcalc) and observed (MWobs) molecular weight estimates from MALS are shown in inset.

FIGS. 8A-B. DVD-Igs broadly recognize ebolavirus rVSV-GP particles in an FVM09-dependent manner. (FIGS. 8A-B) Viral particles were normalized for GP content by ELISA with an ebolavirus GP peptide-specific polyclonal antiserum (45), captured onto plates, and probed with FVM09~548 (FIG. 8A) and FVM09~MR72   (FIG. 8B) in an ELISA. DVD-Ig binding was FVM09-dependent, since only non- specific binding to viral particles was observed with parent IgG MR72 (FIG.8B).

FIGS. 9A-D. DVD-Igs, but not parent IgGs, possess broad neutralizing activity against authentic ebolaviruses. (FIGS.9A-C) Neutralization of authentic filoviruses in human U2OS osteosarcoma cells, measured in microneutralization assays. Infected cells were immunostained for viral antigen at 48 hr post-infection, and enumerated by automated fluorescence microscopy. Averages ± SD (n=3) from 1–2 representative experiments are shown. (FIG. 9D) Ab concentrations at half-maximal neutralization (IC50 ± 95% confidence intervals for nonlinear curve fit) are shown.

FIGS. 10A-D. Duobody molecules combining extracellular ‘delivery’ and endosomal RBS-targeting specificities. (FIG. 10A) Schematic of GPCL-specific and FVM09 (GP-specific) mAbs (top row), and a duobody engineered to combine them (bottom row). (FIG. 10B) Biochemical characterization of IgGs and duobody. FVM09MR72 was resolved on SDS-polyacrylamide gels under reducing (R) and nonreducing (NR) conditions, and proteins were visualized by staining with Coomassie Brilliant Blue. (FIG. 10C) Parent IgGs and the post-exchange product (putative duobody) were analyzed by mass spectrometry (matrix-assisted laser desorption ionization-time of flight; MALDI-TOF). Calculated (MWcalc) and observed (MW _obs ) molecular masses are indicated. nd, not determined. (FIG.10D) Parent IgGs and post-exchange product (putative duobody) were analyzed by ion-exchange chromatography to determine the efficiency of Fab-arm exchange. The duobody eluted at a volume intermediate to those of the parent IgGs, and area-under-the-curve measurements indicated that the efficiency of exchange was≈ 97%.

FIGS. 11A-C. Duobody combining FVM09 and MR72 possesses broad neutralizing activity. (FIGS.11A-C) Neutralizing activity of duobodies against rVSVs encoding eGFP and bearing ebolavirus GP proteins in human U2OS cells. See FIGS. 2A-G for details. Averages ± SD (n=3) from a representative experiment are shown. CtrlMR72 is a control Duobody combining MR72 with an irrelevant IgG (HIV-1 specific IgG, b12).

FIG. 12. Two mutations in glycan cap subdomain of EBOV GP abolish FVM09 IgG binding. rVSV particles bearing EBOV GP(WT) or GP(E288D/W292R) were captured onto plates and probed with FVM09 IgG in an ELISA.

FIGS. 13A-H. Workflow for flow cytometric measurement of Ab internalization into cells in the absence or presence of viral particles. Parent IgGs and DVD-Igs were   covalently labeled with the acid-dependent fluorophore pHrodo Red ^TM . Labeled Abs were incubated with rVSV-EBOV GP particles containing an internal fluorescent protein label (mNeongreen), and then exposed to U2OS cells for 30 min at 37 ˚C. Cells were chilled and then analyzed on a BD LSRII. Cells were first gated on side (FIG. 13A) and forward (FIG. 13B) scatter to gate out debris. Cells were then gated using pulse width/height (FIG. 13C) and nuclear DNA content (DAPI) (FIG. 13D) to identify a singlet population for further analysis. Virus ⁺ (FIG. 13E) and virus ^– (FIG. 13G) subpopulations were identified on the basis of mNeongreen fluorescence, and each was separately analyzed for pHrodo Red fluorescence (FIG.13F–H).

FIG. 14. DVD-Igs, but not parent IgGs, undergo virion- and FVM09-dependent delivery to NPC1 ⁺ cellular compartments. Parent IgGs and DVD-Igs were incubated with mNeongreen-labeled rVSV-EBOV GP particles and then exposed to U2OS cells expressing an NPC1-enhanced blue fluorescent protein-2 (eBFP2) fusion protein for 60 min at 37 ˚C. Cells were fixed, permeabilized, and immunostained, and viral particles, Ab, and NPC1 were visualized by fluorescence microscopy.

FIGS. 15A-C. FVM09 bind to uncleaved EBOV GP with similar affinity at neutral pH and the presumptive acidic pH of early and late endosomes. Kinetic binding curves for FVM09~MR72 DVD-Ig:EBOV GP binding were determined by BLI at pH 7.5 (FIG.15A), pH 6.5 (FIG.15B), and pH 5.5 (FIG.15C). FVM09~MR72 was loaded onto probes at pH 7.5, which were then equilibrated in buffer adjusted to the test pH, and dipped in analyte solutions. Gray lines show curve fits to a 1:1 binding model. See Table 1 for kinetic binding constants.

FIGS. 16A-F. FVM09~548 lacks neutralizing activity in murine cells, because it binds weakly to the murine (M. musculus) NPC1 ortholog and only poorly blocks GP _CL -NPC1 interaction. (FIGS. 16A-B) Neutralization of rVSV-EBOV GP infection in murine NIH/3T3 cells by the FVM09~548 and FVM09~MR72 DVD-Igs. (FIGS. 16C-D) Kinetic binding curves for FVM09~548:NPC1–C interaction were determined by BLI. FVM09~548 was loaded onto probes, which were then dipped in (FIG. 16C) murine NPC1–C or (FIG. 16D) mantled guereza (C. guereza) non-human primate NPC1–C analyte solutions. (FIGS. 16E-F) Dose-dependent inhibition of rVSV-EBOV GPCL:NPC1–C binding by mAb-548 in a competition ELISA. (FIG. 16C) Murine NPC1–C. (FIG.16D) Mantled guereza NPC1–C. Gray lines show curve fits to a 1:1 binding model. See Table 1 for kinetic binding constants.   FIGS. 17A-B. Amino acid sequence divergence of murine and primate NPC1 orthologs at the GP _CL binding interface. (FIG. 17A) Left, cartoon representation of human NPC1–C (PDB ID: 5F1B) (17). Boxed region is magnified at right, to highlight GP _CL -contacting loops 1 and 2 (balls and sticks). (FIG. 17B) Amino acid sequence alignments of NPC1–C sequences corresponding to loops 1 and 2, and the mAb-548-binding region. Sequence differences are highlighted. Homo sapiens = SEQ ID NO: 13; Macaca mulatta = SEQ ID NO: 14; Colobus guereza = SEQ ID NO: 15; Macaca fascicularis = SEQ ID NO: 16; Mus musculus = SEQ ID NO: 17.

FIGS. 18A-D. Statistical analysis of Ab internalization into cells (FIG.3D and FIGS. 13A-H). Results from the flow cytometric measurement of Ab internalization into cells in FIG. 3D and FIGS. 13A-H were subjected to a two-way analysis of variance (ANOVA). (FIG. 18A) The influence of two independent variables (presence or absence of mNeongreen-labeled rVSV-EBOV GP particles, identity of pHrodo Red-labeled Ab) on the dependent variable (%Ab+ cells) was determined. Ab internalization was significantly higher in virus+ than in virus– cells, and was significantly dependent on both Ab identity, and on the interaction between virus and Ab. df, degrees of freedom. (FIG. 18B) Šídák’s post hoc test was used to compare %Ab+ cells in virus– vs. virus+ cells, for each test Ab. (FIG.18C–D) Dunnett’s post hoc test was used to compare %Ab+ cells obtained for each test Ab vs. the‘no Ab’ control in virus– (FIG. 18C) and virus+ (FIG. 18D) cell populations. FIGS. 19A-B. DVD-Igs specifically neutralize filovirus entry and infection. (FIGS. 19A–B) Neutralization of rVSVs bearing EBOV GP or non-filovirus glycoproteins derived from a rhabdovirus (VSV G) and a hantavirus (Andes virus; ANDV Gn/Gc) by FVM09~548 (FIG. 19A) and FVM09~MR72 (FIG. 19B). Averages ± SD for 3 technical replicates from a representative experiment are shown. Two independent experiments were performed. FIG. 20. DVD-Igs, but not parent IgGs, undergo EBOV VLP- and FVM09-dependent delivery to NPC1+ cellular compartments. Parent IgGs and DVD-Igs were incubated with EBOV GP/VP40 VLPs, and then exposed to U2OS cells expressing an NPC1-enhanced blue fluorescent protein-2 fusion protein for 60 min at 37°C. Cells were fixed, permeabilized, and immunostained, and VLPs, Ab, and NPC1 were visualized by fluorescence microscopy. Images from a representative experiment are shown. Two independent experiments were performed. Scale bar, 20 μm.   DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As discussed above, there is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other epidemic-causing filoviruses. The conserved, essential interaction between the filovirus spike glycoprotein, GP, and its cell entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs, but is ‘stealthed’ by multiple mechanisms, including physical sequestration in late endosomes. Here, the inventors describe a bispecific antibody strategy to target this shielded interaction, in which mAbs specific for NPC1 or the NPC1-binding site in GP are coupled to a mAb directed against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parentAL mAbs, potently neutralized all known ebolaviruses by coöpting viral particles themselves for endosomal delivery, and conferred post-exposure protection against multiple ebolaviruses in vivo. Such‘Trojan horse’ bispecific antibodies have strong potential as broad anti-filovirus immunotherapeutics. These and other aspects of the disclosure are described in detail below. I. Filoviruses

A. Ebolavirus

The genus Ebolavirus is a virological taxon included in the family Filoviridae, order Mononegavirales. The members of this genus are called ebolaviruses. The five known virus species are named for the region where each was originally identified: Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus (originally Côte d'Ivoire ebolavirus), and Zaire ebolavirus.

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424–457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear   import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.

Ebolaviruses were first described after outbreaks of EVD in southern Sudan in June 1976 and in Zaire in August 1976. The name Ebolavirus is derived from the Ebola River in Zaire (now the Democratic Republic of the Congo), the location of the 1976 outbreak, and the taxonomic suffix -virus (denoting a viral genus). This genus was introduced in 1998 as the "Ebola-like viruses.” In 2002 the name was changed to Ebolavirus and in 2010, the genus was emended. Ebolaviruses are closely related to Marburg viruses.

Researchers have now found evidence of Ebola infection in three species of fruit bats. The bats show no symptoms of the disease, indicating that they might be spreading it. Researchers found that bats of three species– Hypsignathus monstrosus, Epomops franqueti, and Myonycteris torquata– had either genetic material from the Ebola virus, known as RNA sequences, or evidence of an immune response to the disease. The bats showed no symptoms themselves. Other hosts are possible as well. 1. Taxonomy

A virus of the family Filoviridae is a member of the genus Ebolavirus if its genome has several gene overlaps, its fourth gene (GP) encodes four proteins (sGP, ssGP, Δ-peptide, and GP1,2) using co-transcriptional editing to express ssGP and GP1,2 and proteolytic cleavage to express sGP and Δ-peptide, peak infectivity of its virions is associated with particles≈805 nm in length, its genome differs from that of Marburg virus by≥50% and from that of Ebola virus by <50% at the nucleotide level, its virions show almost no antigenic cross reactivity with Marburg virions.

The genera Ebolavirus and Marburgvirus were originally classified as the species of the now-obsolete Filovirus genus. In March 1998, the Vertebrate Virus Subcommittee proposed in the International Committee on Taxonomy of Viruses (ICTV) to change the Filovirus genus to the Filoviridae family with two specific genera: Ebola-like viruses and Marburg-like viruses. This proposal was implemented in Washington, D.C., as of April 2001 and in Paris as of July 2002. In 2000, another proposal was made in Washington, D.C., to change the "-like viruses" to "-virus" resulting in today's Ebolavirus and Marburgvirus.

Each species of the genus Ebolavirus has one member virus, and four of these cause Ebola virus disease (EVD) in humans, a type of hemorrhagic fever having a very high case fatality rate; the fifth, Reston virus, has caused EVD in other primates. Zaire ebolavirus is the type species (reference or example species) for Ebolavirus, and has the highest mortality rate   of the ebolaviruses, and is also responsible for the largest number of outbreaks of the five known members of the genus, including the 1976 Zaire outbreak and the outbreak with the most deaths (2014). The five characterized species of the Ebolavirus genus are:

Zaire ebolavirus (ZEBOV). Also known simply as the Zaire virus, ZEBOV has the highest case-fatality rate, up to 90% in some epidemics, with an average case fatality rate of approximately 83% over 27 years. There have been more outbreaks of Zaire ebolavirus than of any other species. The first outbreak took place on 26 August 1976 in Yambuku. Mabalo Lokela, a 44-year-old schoolteacher, became the first recorded case. The symptoms resembled malaria, and subsequent patients received quinine. Transmission has been attributed to reuse of unsterilized needles and close personal contact. The virus is responsible for the 2014 West Africa Ebola virus outbreak, with the largest number of deaths to date.

Sudan ebolavirus (SUDV). Like ZEBOV, SUDV emerged in 1976; it was at first assumed to be identical with ZEBOV. SUDV is believed to have broken out first amongst cotton factory workers in Nzara, Sudan (now in South Sudan), in June 1976, with the first case reported as a worker exposed to a potential natural reservoir. Scientists tested local animals and insects in response to this; however, none tested positive for the virus. The carrier is still unknown. The lack of barrier nursing (or "bedside isolation") facilitated the spread of the disease. The average fatality rates for SUDV were 54% in 1976, 68% in 1979, and 53% in 2000 and 2001.

Reston ebolavirus (RESTV). This virus was discovered during an outbreak of simian hemorrhagic fever virus (SHFV) in crab-eating macaques from Hazleton Laboratories (now Covance) in 1989. Since the initial outbreak in Reston, Virginia, it has since been found in nonhuman primates in Pennsylvania, Texas, and Siena, Italy. In each case, the affected animals had been imported from a facility in the Philippines, where the virus has also infected pigs. Despite its status as a Level-4 organism and its apparent pathogenicity in monkeys, RESTV did not cause disease in exposed human laboratory workers.

Taï Forest ebolavirus (TAFV). Formerly known as "Côte d'Ivoire ebolavirus,” it was first discovered among chimpanzees from the Tai Forest in Côte d'Ivoire, Africa, in 1994. Necropsies showed blood within the heart to be brown; no obvious marks were seen on the organs; and one necropsy displayed lungs filled with blood. Studies of tissues taken from the chimpanzees showed results similar to human cases during the 1976 Ebola outbreaks in Zaire and Sudan. As more dead chimpanzees were discovered, many tested positive for Ebola using molecular techniques. The source of the virus was believed to be the meat of infected western red colobus monkeys (Procolubus badius) upon which the chimpanzees preyed. One of the   scientists performing the necropsies on the infected chimpanzees contracted Ebola. She developed symptoms similar to those of dengue fever approximately a week after the necropsy, and was transported to Switzerland for treatment. She was discharged from hospital after two weeks and had fully recovered six weeks after the infection.

Bundibugyo ebolavirus (BDBV). On November 24, 2007, the Uganda Ministry of Health confirmed an outbreak of Ebola in the Bundibugyo District. After confirmation of samples tested by the United States National Reference Laboratories and the CDC, the World Health Organization confirmed the presence of the new species. On 20 February 2008, the Uganda Ministry officially announced the end of the epidemic in Bundibugyo, with the last infected person discharged on 8 January 2008. An epidemiological study conducted by WHO and Uganda Ministry of Health scientists determined there were 116 confirmed and probable cases the new Ebola species, and that the outbreak had a mortality rate of 34% (39 deaths). 2. Ebola Virus Disease

Symptoms of Ebola virus disease. The incubation period from infection with the virus to onset of symptoms is 2 to 21 days. Humans are not infectious until they develop symptoms. First symptoms are the sudden onset of fever fatigue, muscle pain, headache and sore throat. This is followed by vomiting, diarrhea, rash, symptoms of impaired kidney and liver function, and in some cases, both internal and external bleeding (e.g., oozing from the gums, blood in the stools). Laboratory findings include low white blood cell and platelet counts and elevated liver enzymes.

Diagnosis. It can be difficult to distinguish ebolavirus from other infectious diseases such as malaria, typhoid fever and meningitis. Confirmation that symptoms are caused by ebolavirus infection are made using antibody-capture ELISA, antigen-capture detection tests, serum neutralization test, RT-PCR assay, electron microscopy, and virus isolation by cell culture. Samples from patients are an extreme biohazard risk; laboratory testing on non- inactivated samples should be conducted under maximum biological containment conditions.

Treatment and vaccines. Supportive care-rehydration with oral or intravenous fluids- and treatment of specific symptoms, improves survival. There is as yet no proven treatment available for ebolavrus. However, a range of potential treatments including blood products, immune therapies and drug therapies are currently being evaluated. No licensed vaccines are available yet, but 2 potential vaccines are undergoing human safety testing.

Prevention and control. Good outbreak control relies on applying a package of interventions, namely case management, surveillance and contact tracing, a good laboratory   service, safe burials and social mobilization. Community engagement is key to successfully controlling outbreaks. Raising awareness of risk factors for Ebola infection and protective measures that individuals can take is an effective way to reduce human transmission. Risk reduction messaging should focus on several factors:

reducing the risk of wildlife-to-human transmission from contact with infected fruit bats or monkeys/apes and the consumption of their raw meat;

reducing the risk of human-to-human transmission from direct or close contact with people with Ebola symptoms, particularly with their bodily fluids;

outbreak containment measures including prompt and safe burial of the dead;

identifying people who may have been in contact with someone infected with Ebola and monitoring the health of contacts for 21 days;

the importance of separating the healthy from the sick to prevent further spread; and the importance of good hygiene and maintaining a clean environment

In terms of controlling infection in health-care settings, health-care workers should always take standard precautions when caring for patients, regardless of their presumed diagnosis. These include basic hand hygiene, respiratory hygiene, use of personal protective equipment (to block splashes or other contact with infected materials), safe injection practices and safe burial practices. Health-care workers caring for patients with suspected or confirmed Ebola virus should apply extra infection control measures to prevent contact with the patient’s blood and body fluids and contaminated surfaces or materials such as clothing and bedding. When in close contact (within 1 meter) of patients with EBV, health-care workers should wear face protection (a face shield or a medical mask and goggles), a clean, non-sterile long- sleeved gown, and gloves (sterile gloves for some procedures). Laboratory workers are also at risk. Samples taken from humans and animals for investigation of Ebola infection should be handled by trained staff and processed in suitably equipped laboratories. B. Marburg Virus

Marburg virus is a hemorrhagic fever virus of the Filoviridae family of viruses and a member of the species Marburg marburgvirus, genus Marburgvirus. Marburg virus (MARV) causes Marburg virus disease in humans and nonhuman primates, a form of viral hemorrhagic fever. The virus is considered to be extremely dangerous. The WHO rates it as a Risk Group 4 Pathogen (requiring biosafety level 4-equivalent containment). In the United States, the NIH/National Institute of Allergy and Infectious Diseases ranks it as a Category A Priority Pathogen and the Centers for Disease Control and Prevention lists it as a Category A   Bioterrorism Agent. It is also listed as a biological agent for export control by the Australia Group. In 2009, expanded clinical trials of an Ebola and Marburg vaccine began in Kampala, Uganda.

Marburg virus was first described in 1967. It was noticed during small outbreaks in the German cities Marburg and Frankfurt and the Yugoslav capital Belgrade in the 1960s. German workers were exposed to tissues of infected grivet monkeys (Chlorocebus aethiops) at the city's former main industrial plant, the Behringwerke, then part of Hoechst, and today of CSL Behring. During these outbreaks, 31 people became infected and seven of them died. MARV is a Select Agent.

The virus is one of two members of the species Marburg marburgvirus, which is included in the genus Marburgvirus, family Filoviridae, order Mononegavirales. The name Marburg virus is derived from Marburg (the city in Hesse, Germany, where the virus was first discovered) and the taxonomic suffix virus.

According to the rules for taxon naming established by the International Committee on Taxonomy of Viruses (ICTV), the name Marburg virus is always to be capitalized, but is never italicized, and may be abbreviated (with MARV being the official abbreviation).

Marburg virus was first introduced under this name in 1967. In 2005, the virus name was changed to Lake Victoria marburgvirus, which unfortunately was the same spelling as its species Lake Victoria marburgvirus. However, most scientific articles continued to refer to Marburg virus. Consequently, in 2010, the name Marburg virus was reinstated and the species name changed. A previous abbreviation for the virus was MBGV.

MARV is one of two Marburg viruses that causes Marburg virus disease (MVD) in humans (in the literature also often referred to as Marburg hemorrhagic fever, MHF). The other one is Ravn virus (RAVV). Both viruses fulfill the criteria for being a member of the species Marburg marburgvirus because their genomes diverge from the prototype Marburg marburgvirus or the Marburg virus variant Musoke (MARV/Mus) by <10% at the nucleotide level.

Like all mononegaviruses, marburgvirions contain non-infectious, linear nonsegmented, single-stranded RNA genomes of negative polarity that possess inverse- complementary 3' and 5' termini, do not possess a 5' cap, are not polyadenylated, and are not covalently linked to a protein. Marburgvirus genomes are approximately 19 kb long and contain seven genes in the order 3'-UTR-NP-VP35-VP40-GP-VP30-VP24-L-5'-UTR. The genomes of the two different marburgviruses (MARV and RAVV) differ in sequence.   Like all filoviruses, marburgvirions are filamentous particles that may appear in the shape of a shepherd's crook or in the shape of a "U" or a "6", and they may be coiled, toroid, or branched. Marburgvirions are generally 80 nm in width, but vary somewhat in length. In general, the median particle length of marburgviruses ranges from 795 to 828 nm (in contrast to ebolavirions, whose median particle length was measured to be 974–1,086 nm ), but particles as long as 14,000 nm have been detected in tissue culture. Marburgvirions consist of seven structural proteins. At the center is the helical ribonucleocapsid, which consists of the genomic RNA wrapped around a polymer of nucleoproteins (NP). Associated with the ribonucleoprotein is the RNA-dependent RNA polymerase (L) with the polymerase cofactor (VP35) and a transcription activator (VP30). The ribonucleoprotein is embedded in a matrix, formed by the major (VP40) and minor (VP24) matrix proteins. These particles are surrounded by a lipid membrane derived from the host cell membrane. The membrane anchors a glycoprotein (GP1,2) that projects 7 to 10 nm spikes away from its surface. While nearly identical to ebolavirus virions in structure, marburgvirsus virions are antigenically distinct.

Niemann–Pick C1 (NPC1) cholesterol transporter protein appears to be essential for infection with both Ebola and Marburg virus. Two independent studies reported in the same issue of Nature showed that Ebola virus cell entry and replication requires NPC1. When cells from patients lacking NPC1 were exposed to Ebola virus in the laboratory, the cells survived and appeared immune to the virus, further indicating that Ebola relies on NPC1 to enter cells. This might imply that genetic mutations in the NPC1 gene in humans could make some people resistant to one of the deadliest known viruses affecting humans. The same studies described similar results with Marburg virus, showing that it also needs NPC1 to enter cells. Furthermore, NPC1 was shown to be critical to filovirus entry because it mediates infection by binding directly to the viral envelope glycoprotein and that the second lysosomal domain of NPC1 mediates this binding.

In one of the original studies, a small molecule was shown to inhibit Ebola virus infection by preventing the virus glycoprotein from binding to NPC1. In the other study, mice that were heterozygous for NPC1 were shown to be protected from lethal challenge with mouse-adapted Ebola virus. Together, these studies suggest NPC1 may be potential therapeutic target for an Ebola antiviral drug.

The marburgvirus life cycle begins with virion attachment to specific cell-surface receptors, followed by fusion of the virion envelope with cellular membranes and the concomitant release of the virus nucleocapsid into the cytosol. The virus RdRp partially   uncoats the nucleocapsid and transcribes the genes into positive-stranded mRNAs, which are then translated into structural and nonstructural proteins. Marburgvirus L binds to a single promoter located at the 3' end of the genome. Transcription either terminates after a gene or continues to the next gene downstream. This means that genes close to the 3' end of the genome are transcribed in the greatest abundance, whereas those toward the 5' end are least likely to be transcribed. The gene order is therefore a simple but effective form of transcriptional regulation. The most abundant protein produced is the nucleoprotein, whose concentration in the cell determines when L switches from gene transcription to genome replication. Replication results in full-length, positive-stranded antigenomes that are in turn transcribed into negative-stranded virus progeny genome copies. Newly synthesized structural proteins and genomes self-assemble and accumulate near the inside of the cell membrane. Virions bud off from the cell, gaining their envelopes from the cellular membrane they bud from. The mature progeny particles then infect other cells to repeat the cycle. II. Monoclonal Antibodies and Production Thereof

A. General Methods

It will be understood that monoclonal antibodies binding to ebolavirus or the NPC1 receptor will have several applications. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).

The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host, or as described below, the identification of subjects who are immune due to prior natural infection. Antibody-producing cells may be induced to expand by priming with immunogens. A variety of routes can be used to administer such immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.   Somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp.65-66, 1986; Campbell, pp.75-83, 1984).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 ^-6 to 1 x 10- ⁸ . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.

The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl   transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain is also used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single- clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.

mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.   Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. For this, RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 10 ⁴ times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.

Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Patent 4,867,973 which describes antibody-therapeutic agent conjugates. B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity, which in this case is for ebolavirus glycoprotein (GP) and the NPC1 receptor. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. Such antibodies may be produced by the clones discussed below in the Examples section using methods described herein.

In a second aspect, the antibodies may be defined by their variable sequence, which include additional“framework” regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature   conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (f) the amino acids may vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing apply to the nucleic acid sequences set forth as Table 3 and the amino acid sequences of Table 4. C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. The following is a general discussion of relevant techniques for antibody engineering.

Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.

Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected and purified from the 293 or CHO cell supernatant.

The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.   Antibody molecules will comprise fragments (such as F(ab’), F(ab’)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form“chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.

In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ± 1), glutamate (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.   As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. D. Single Chain Antibodies

  A Single Chain Variable Fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv   repertoire (approx. 5 × 10 ⁶ different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the V _H C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.

The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.

In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C- terminus of the light chain via an appropriate chemical bridge).

Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero- bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).

It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo,   preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.

The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-dithiopropionate. The N-hydroxy- succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

U.S. Patent 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.

U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separation techniques.   E. Intrabodies

In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell– such antibodies are known as“intrabodies.” These antibodies may interfere with target function by a variety of mechanism, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript/single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required.

The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell/tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability/membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage diplay and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.

An additional feature that intrabodies may require is a signal for intracellular targeting. Vectors that can target intrabodies (or other proteins) to subcellular regions such as the cytoplasm, nucleus, mitochondria and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997).

By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies may not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell may interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC1 dimer formation. F. Dual Variable Domain Igs

The present disclosure incorporates a technology known as Dual Variable Domain antibodies, or DVD-Ig®. These are antibody-like binding proteins capable of binding multiple targets and methods of making the same. In a particular embodiment, the binding   protein comprises a polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2 is a second variable domain, C is a constant domain, X1 represents an amino acid or polypeptide, X2 represents an Fc region and n is 0 or 1. Further details regarding this technology can be found in U.S. Patent 7,612,181. G. Duobodies

DuoBody technology produces stable bispecific human IgG1 antibodies in a post- production exchange reaction. In a first step, two IgG1s, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgG1 antibodies are purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield (typically >95%) (see Labrijn et al., 2013). H. Purification

In certain embodiments, the antibodies of the present disclosure may be purified. The term“purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term“substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel   filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies is bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art, in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

  It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. III. Active/Passive Immunization and Treatment/Prevention of Ebola Virus Infection The present disclosure provides pharmaceutical compositions comprising anti- ebolavirus/NPC1 bispecific antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In   a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term“carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in“Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

Active vaccines of the present disclosure, as described herein, can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer. Pharmaceutically acceptable salts, include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium,   potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non- human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.

Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. IV. Antibody Conjugates

Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or   polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.

Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging." Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine ²¹¹ , ¹⁴ carbon, ⁵¹ chromium, ³⁶ chlorine, ⁵⁷ cobalt, ⁵⁸ cobalt, copper ⁶⁷ , ¹⁵² Eu, gallium ⁶⁷ , ³ hydrogen, iodine ¹²³ , iodine ¹²⁵ , iodine ¹³¹ , indium ¹¹¹ , ⁵⁹ iron, ³² phosphorus, rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , ⁷⁵ selenium, ³⁵ sulphur, technicium ^99m and/or yttrium ⁹⁰ . ¹²⁵ I is often being preferred for use in certain embodiments, and technicium ^99m and/or indium ¹¹¹ are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well- known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the disclosure may be labeled with technetium ^99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as   metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

Another type of antibody conjugates contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.

Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten- based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.

Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents.

Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3 ^-6 ^-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and   4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Patent 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O’Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation. V. Immunodetection Methods

In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting Ebolavirus and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.

Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of Ebolavirus antibodies directed to specific parasite epitopes in samples also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing Ebolavirus, and contacting the sample with a first antibody in   accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying Ebolavirus or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the Ebolavirus or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the Ebolavirus antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.

The immunobinding methods also include methods for detecting and quantifying the amount of Ebolavirus or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing Ebolavirus or its antigens, and contact the sample with an antibody that binds Ebolavirus or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing Ebolavirus or Ebolavirus antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.

Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to Ebolavirus or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages through the use of a secondary binding ligand   such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a“secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of   the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the Ebolavirus or Ebolavirus antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti- Ebolavirus antibody that is linked to a detectable label. This type of ELISA is a simple“sandwich ELISA.” Detection may also be achieved by the addition of a second anti-Ebolavirus antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the Ebolavirus or Ebolavirus antigen are immobilized onto the well surface and then contacted with the anti- Ebolavirus antibodies of the disclosure. After binding and washing to remove non- specifically bound immune complexes, the bound anti-Ebolavirus antibodies are detected. Where the initial anti-Ebolavirus antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a   second antibody that has binding affinity for the first anti-Ebolavirus antibody, with the second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.

In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

The“suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune   complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of Ebolavirus antibodies in sample. In competition based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample.

Here, the inventors propose the use of labeled Ebolavirus monoclonal antibodies to determine the amount of Ebolavirus antibodies in a sample. The basic format would include contacting a known amount of Ebolavirus monoclonal antibody (linked to a detectable label) with Ebolavirus antigen or particle. The Ebolavirus antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample.   B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins   equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probing. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies. C. Immunohistochemistry

The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.

Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted. D. Immunodetection Kits

In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect Ebolavirus or Ebolavirus antigens, the antibodies may be included in the kit. The   immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to Ebolavirus or Ebolavirus antigen, and optionally an immunodetection reagent.

In certain embodiments, the Ebolavirus antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.

The kits may further comprise a suitably aliquoted composition of the Ebolavirus or Ebolavirus antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. VI. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that   many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1 - Materials and Methods

Cell lines. Human U2OS osteosarcoma cells, 293T human embryonic kidney fibroblast cells, and Freestyle ^TM -293F suspension-adapted HEK-293 cells were obtained from ATCC. U2OS cells were maintained in modified McCoy’s 5A media (Thermo Fisher, Waltham, MA) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 1% GlutaMAX (Thermo Fisher), and 1% penicillin-streptomycin (Thermo Fisher). Vero and 293T cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher) supplemented with the above reagents. All adherent cell lines were maintained in a humidified 37 ˚C, 5% CO2 incubator.293-F cells were maintained in GIBCO FreeStyle™ 293 expression medium (Thermo Fisher) at 37 ˚C and 8% CO ₂ .

A clonal U2OS cell line stably expressing human NPC1 C–terminally tagged with enhanced blue fluorescent protein-2 (eBFP2) (Ai et al., 2007) was generated as follows. Cells were transfected with a plasmid encoding this construct, and transfected cells were selected with geneticin (Thermo Fisher; 400 µg/mL). Single eBFP2-positive cells were isolated by FACS sorting. For further experimentation, the inventors chose a cell clone expressing NPC1-EBFP2 at a moderate level, and displaying a representative NPC1 distribution in late endosomal/lysosomal vesicles. Generation of mAb-548. To generate hybridomas secreting human NPC1–C-specific mAbs, 5–6-week old female BALB/c mice were immunized with purified human NPC1–C protein (125 µg; intraperitoneal [i.p.] route) in complete Freund’s adjuvant. After 5 weeks and 10 weeks post-immunization, mice were boosted with the same antigen in incomplete Freund’s adjuvant. Nine weeks after the second boost, and on the 4 ^th and 3 ^rd days prior to fusion, mice were boosted with NPC1–C in PBS (100 µg). Mice were sacrificed, and spleen cells were isolated and fused to Ag8.653 or NSO-bcl2 myeloma cells as described previously (Sroubek et al., 2012). Supernatants from growing wells were assayed by ELISA against NPC1–C for IgG binding. Positive colonies were cloned in soft agar, isolated and expanded, and tested as above. One hybridoma, 548, was identified as secreting an IgG2b that strongly recognized human NPC1 in cells by immunofluorescence microscopy, and potently blocked EBOV GPCL:NPC1–C binding at pH 5.5.   5–10 million cells of hybridoma 548 were harvested, and total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. cDNA was prepared using SuperScript® III First-Strand Synthesis System with Mu IgG VH 3ʹ-2 and Mu Igκ VL 3ʹ-1 reverse primers from the Mouse Ig-Primer Set (EMD Millipore, Billerica, MA). Following reverse transcription, the same primer sets were then used in PCR to specifically amplify the variable regions of light- and heavy-chain cDNAs. Reactions yielding the correct band size (~500 bp) were gel-purified and sequenced. Decoded light and heavy chain sequences were submitted for analysis to IgBLAST (Ye et al., 2013). Functional chains were subcloned into pMAZ-IgL and pMAZ-IgH vectors, and the chimeric IgGs were expressed in 293-F cells, as described below. Cloning of IgGs and DVD-Igs. Synthetic genes encoding IgG variable domains were subcloned into linearized pMAZ-IgH and pMAZ-IgL vectors encoding human IgG1 heavy and light chain constant domains using BssHII/BsiWI and BssHII/NheI restriction sites, respectively, as described (Mazor et al., 2007). To generate DVD-Igs, the outer variable domains of the heavy and light chains were appended N-terminally, using short linkers “ASTKGP” and“TVAAP”, respectively, as described (Wu et al., 2007). All insert and junction sequences were confirmed by Sanger sequencing. Antibody expression and purification. pMAZ-IgH and pMAZ-IgL vectors encoding each antibody were co-transfected into 293-F cells using linear polyethylenimine (Polysciences, Warrington, PA). Cell cultures were incubated at 37 °C and 8% CO ₂ for 6 days post-transfection. Cleared cell supernatants were then applied to a Protein A affinity column (~1 mL packed beads per 600 mL culture) (Thermo Scientific). Antibodies were purified using the Gentle Antibody Elution System (Thermo Scientific), according to the manufacturer’s instructions, and subsequently exchanged into 150 mM HEPES (pH 7.4), 200 mM NaCl. For some large-scale preparations, cell supernatants were concentrated by tangential flow filtration prior to Ab purification. Endotoxin levels in the purified antibodies were determined by the Limulus amebocyte lysate test according to the manufacturer’s instructions (Kinetic-QCL kinetic chromogenic LAL assay; Lonza, Walkersville, MD). Generation of Duobodies by controlled Fab-arm exchange (cFAE). Parental IgG1 antibody molecules containing matching point mutations, K409R and F405L (EU numbering convention), in their CH3 domains were separately expressed from respective pMAZ-IgH and pMAZ-IgL vectors in 293-F cells (see above). cFAE was performed as described (43).   Briefly, in a total reaction volume of 1 mL, 0.5 mg of each parental IgG1 was mixed with the reducing agent 2-mercaptoetylamine HCL (2-MEA; Sigma Aldrich; 75 mM) in PBS pH 7.4. Samples were mixed on a rotating laboratory mixer for 5 min at ambient temperature and subsequently incubated at 31 °C for 5 h. To remove 2-MEA and allow for reformation of disulfide bonds, the samples were transferred to Amicon Ultra-15 centrifugal units (30 KDa MWCO; EMD Millipore) and washed 5 times with phosphate-buffered saline (PBS). The samples were then stored at 4 °C, to allow sufficient time for complete re-oxidation to occur. Fab-arm exchange was detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and analysis of exchange efficiency was carried out by cation exchange chromatography on a MonoS 5/50 GL column (GE Healthcare). Samples were injected in 10 µL into 10 mM sodium phosphate (pH 7.0), and eluted in a linear NaCl gradient (0–100 mM; 20 mL). Elution curves for each antibody species were integrated, and the percentage of the FVM09 ^MR72 Duobody that is distinct from either of the parent IgGs, was calculated as 97%. Size Exclusion Chromatography/Multi Angled Light Scattering (SEC/MALS). Static light scattering data were collected on a mini Dawn Treos light scattering instrument (Wyatt Technology, Santa Barbara, CA) coupled with analytical gel filtration (column: Sepax SRT SEC-300; particle size 5 µm, pore size 500 Å, 4.6 x 300 mm) and UV detection at 280 nm. All measurements were performed in PBS at room temperature with a flow rate of 0.5 mL/min. Data were analyzed using the ASTRA software package (Wyatt Technology). Biolayer interferometry (BLI). The Octet Red ^TM system (ForteBio Pall LLC) was used to determine the binding properties of IgGs and DVD-Igs. Anti-human Fc capture sensors were used for initial Ab loading. For single-phase binding experiments, global data fitting to a 1:1 binding model was used to estimate values for the kon (association rate constant), k _off (dissociation rate constant), and K _D (equilibrium dissociation constant). For double-phase binding experiment, Ab was first immobilized first onto an Fc sensor, and then allowed to equilibrate in a solution containing the first antigen. The sensor was then transferred to a second solution containing the second antigen. ELISAs. Cell supernatants containing flag-tagged NPC1–C, or purified flag-tagged NPC1–C proteins (Miller et al., 2012; Ndungo et al., 2016), were normalized for ELISA as described previously, by immunoblotting of SDS-polyacrylamide gels with anti-flag primary antibody (Sigma Aldrich) and anti-mouse Alexa-680 secondary antibody (Thermo Fisher),   and quantification on the LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE) (Ng et al., 2015). The GP content of different rVSV-GP particle preparations was normalized by ELISA with a GP-specific polyclonal antiserum (Misasi et al., 2012), as described previously (Bornholdt et al., 2015).

NPC1–C:Ab and GP:Ab-binding ELISAs were performed as follows. Pre-titrated amounts of purified NPC1–C or rVSV-GP particles were coated onto high-binding 96-well ELISA plates (Corning, Corning NY). Plates were then blocked with PBS containing 3% bovine serum albumin (PBSA), and probed with increasing concentrations of test Ab. Bound Abs were detected with species-specific anti-IgG Abs conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX) and Ultra-TMB colorimetric substrate (Thermo Fisher).

EBOV GP:NPC1–C capture ELISAs were performed as described previously (Bornholdt et al., 2015; Ng et al., 2015; Ndungo et al., 2016). Briefly, ELISA plates were coated with EBOV GP-specific mAb KZ52 (2 µg/mL in PBS), and then blocked with PBSA. Viral particles were cleaved with thermolysin and captured on the plate. Plates were then incubated with increasing concentrations of NPC1–C, and bound NPC1–C was detected with an anti-flag antibody conjugated to horseradish peroxidase and Ultra-TMB substrate (Thermo Fisher). For Ab inhibition studies, NPC1–C (concentration≈ binding IC ₅₀ ) was preincubated with increasing concentrations of test Ab at 37 ˚C for 1 hr, and then added to blocked plates coated with rVSV-EBOV GP _CL . EC ₅₀ values were calculated from binding curves generated by non-linear regression analysis using Prism (GraphPad Software, La Jolla CA). All incubation steps were done at 37 ˚C for 1 hr, or at 4 ˚C, overnight. rVSVs and infections. Recombinant vesicular stomatitis Indiana viruses (rVSVs) expressing eGFP or an mNeongreen-phosphoprotein P (mNG-P) fusion, and EBOV GP in place of VSV G, have been described previously (Kleinfelter et al., 2015; Wong et al., 2010). Similar rVSVs expressing eGFP and representative GP proteins from BDBV, TAFV, SUDV, and RESTV were generated as above. rVSV particles containing GPCL were generated by incubating rVSV-GP particles with thermolysin (200 μg/mL) for 1 hr at 37 ˚C, as described. The protease was inactivated by addition of phosphoramidon (Sigma Aldrich; 1 mM), and reaction mixtures were used immediately. Infectivities of rVSVs were measured by automated enumeration of eGFP ⁺ cells (infectious units; IU) using a CellInsight CX5 imager (Thermo Fisher) at 12–14 hr post-infection.   For Ab neutralization experiments, pre-titrated amounts of rVSV-GP particles (MOI≈ 1 IU per cell) were incubated with increasing concentrations of test Ab at room temp for 1 h, prior to addition to cell monolayers in 96-well plates. Viral infectivities were measured as above. Viral neutralization data were subjected to nonlinear regression analysis to extract EC50 values (4-parameter, variable slope sigmoidal dose-response equation; GraphPad Prism). Authentic filoviruses and infections. The authentic filoviruses Ebola virus/H.sapiens-tc/COD/1995/Kikwit-9510621 (EBOV/Kik-9510621;‘EBOV-Zaire 1995’) (Jahrling et al., 1999), mouse-adapted EBOV (EBOV-MA; derived from Mayinga variant) (35), Sudan virus/H. sapiens-gp-tc/SDN/1976/Boniface-USAMRIID111808 (SUDV/Bon- USAMRIID111808;‘SUDV-Boniface 1976’) (WHO/International Study Team Report, 1978), and Bundibugyo virus/H. sapiens-tc/UGA/2007/Bundibugyo-200706291 (Towner et al., 2008) were used in this study. Antibodies were diluted to indicated concentrations in culture media and incubated with EBOV, SUDV, or BDBV for 1 hr. U2OS cells were exposed to antibody/virus inoculum at an MOI of 0.2 (EBOV, BDBV) or 0.5 (SUDV) plaque-forming unit (PFU)/cell for 1 hr. Antibody/virus inoculum was then removed and fresh culture media was added. At 48 hr post-infection, cells were fixed with formalin, and blocked with 1% bovine serum albumin. EBOV-, SUDV-, or BDBV-infected cells and uninfected controls were incubated with EBOV GP-specific mAb KZ52 (Maruymama et al.,1999), SUDV GP-specific mAb 3C10 (USAMRIID), or ebolavirus GP-specific mAb ADI-15742 (Bornholdt et al., 2016). Cells were washed with PBS prior to incubation with either goat anti-mouse IgG or goat anti-human IgG conjugated to Alexa 488. Cells were counterstained with Hoechst stain (Invitrogen), washed with PBS and stored at 4 °C. Infected cells were quantitated by fluorescence microscopy and automated image analysis. Images were acquired at 20 fields/well with a 20× objective lens on an Operetta high content device (Perkin Elmer, Waltham, MA). Operetta images were analyzed with a customized scheme built from image analysis functions available in Harmony software. Phrodo Red ^TM -labeling and flow cytometry. To analyze Ab internalization into cells during infection, DVD-Igs and parental IgGs were first labeled with the acid-sensitive fluorophore, Phrodo Red. Abs were exchanged into PBS, and covalently conjugated to pHrodo Red, succinimidyl ester (Thermo Fisher), following the manufacturer’s instructions. Labeling reactions contained a 5-fold molar excess of dye over Ab. Following conjugation,   excess unconjugated dye was removed using PD-10 desalting columns (GE healthcare, Wauwatosa, WI), and Ab-pHrodo Red conjugates were exchanged into PBS and concentrated with Amicon Ultra filters (EMD Millipore; membrane NMWL 50 kDa). Protein concentration and degree of labeling were determined as described in the pHrodo Red succinimidyl ester conjugation protocol.

Labeled IgGs and DVD-Igs (final concentration, 29 nM) were incubated for 1 hr at room temp, in the absence or presence of rVSV(mNG-P)-EBOV GP particles (3.4×10 ⁴ infectious units per well). Virus, Ab, and virus-Ab mixtures were then added to wells of a pre-chilled 6-well plate containing confluent monolayers of U2OS cells. Viral particles were ‘spinoculated’ onto cells by centrifugation of the plates at 1,500 ×g in a swing-out rotor. Unbound virus was removed by washing with cold PBS, fresh culture media was added, and cells were allowed to internalize viral particles and/or Abs at 37 ˚C. After 30 min, cells were returned to ice, washed with cold PBS, and then harvested using cold trypsin-EDTA (Thermo Fisher). Harvested cells were kept on ice, filtered through mesh caps at 4 ˚C, and analyzed for virus (mNG-P) and Ab (pHrodo Red) fluorescence on a BD LSRII flow cytometer, using the gating scheme described in FIGS.13A-H. Immunofluorescence microscopy and co-immunostaining. To investigate transport of parent IgGs and DVD-Igs into target cells, rVSV(mNG-P)-EBOV GP particles (Kleinfelter et al., 2015) were preincubated with Abs (100 nM) for 1 hr at room temp. VSV-antibody complexes were diluted into imaging buffer (20 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM glucose, 2% FBS), and spinoculated onto pre-chilled U2OS cells on coverslips stably expressing NPC1-EBFP2 as described above. Unbound virus was removed by washing with cold PBS. Cells were then placed in warm imaging buffer, and allowed to internalize viral particles and/or Abs for 1 hr at 37 °C. Cells were fixed with 3.7% paraformaldehyde and permeabilized with PBS/0.1% Triton X-100. NPC1 was detected by a primary anti-GFP Ab and a secondary anti-mouse Alexa 405 antibody-fluorophore conjugate (Thermo Scientific). Internalized Abs were visualized by staining with a secondary anti-human Alexa 555 antibody-fluorophore conjugate (Thermo Scientific). Imaging was performed on an Axio Observer Z1 widefield fluorescence microscope (Zeiss Inc., Thornwood, NY) equipped with a 63×/1.4 numerical aperture oil immersion objective, and images were captured with a ORCA-Flash4.0 LT digital CMOS camera (Hamamatsu Photonics, Bridgewater, NJ).   Animal challenge studies. Female BALB/c mice (Jackson Labs, Bar Harbor, ME) were challenged via the intraperitoneal (i.p.) route with EBOV-MA (100 PFU; ~3,000 LD ₅₀ ). Mice were treated i.p.2 days post-challenge with PBS vehicle, 300 µg of parent IgG mixtures (150 µg each), or 400 µg DVD-Ig (dose adjusted to account for the higher molecular weight of DVD-Igs). Mice were observed daily for clinical signs of disease and lethality. Daily observations were increased to a minimum of twice daily while mice were exhibiting signs of disease. Moribund mice were humanely euthanized on the basis of IACUC- approved criteria.

6–8 week old male and female Type 1 IFN α/β receptor knockout mice (Type 1 IFNα /β R−/−) (Jackson Labs) were challenged with WT SUDV (1000 PFU i.p.). Mice were treated i.p. 1 and 4 days post-challenge with PBS vehicle, 300 µg of parent IgG mixtures (150 µg each) or 400 µg DVD-Ig, and monitored as above. In both studies, an unpaired t-test was used to compare the survival of each treatment group to the vehicle control group. Animal welfare statement. Generation of murine hybridomas and murine challenge studies were conducted under IACUC-approved protocols in compliance with the Animal Welfare Act, PHS Policy, and other applicable federal statutes and regulations relating to animals and experiments involving animals. The facilities where this research was conducted (Albert Einstein College of Medicine and USAMRIID) are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and adhere to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011.

Statistical analysis. Dose-response neutralization curves were fit to a logistic equation by nonlinear regression analysis. 95% confidence intervals (95% CI) for the extracted IC ₅₀ parameter were estimated under the assumption of normality. Statistical comparison of means among multiple independent groups in FIG.3D was carried out by two- factor analysis of variance (ANOVA) with Šídák’s and Dunnett’s post hoc tests to correct for multiple comparisons (see FIGS. 18A-D). Analysis of survival curves in FIGS. 4A-B was performed with the Mantel-Cox (log-rank) test. Testing level (alpha) was 0.05 for all statistical tests. All analyses were carried out in GraphPad Prism. Example 2 - Results

Here, the inventors envisioned a strategy for bispecific antibody engineering in which the shielded filovirus-receptor interaction could be targeted by a‘Trojan horse’ mechanism. The inventors reasoned that, by coupling receptor/RBS-targeting mAbs to a delivery mAb   directed against a broadly-conserved epitope in uncleaved GP, viral particles themselves could be coöpted to transport these bispecific antibodies (bsAbs) to endosomes, where interactions between GPCL and NPC1 should be vulnerable (FIGS.1A-B).

To block the filovirus-receptor interaction, the inventors chose mAbs targeting both its viral and host facets: MR72, a human mAb that broadly recognizes the GP _CL RBS, as discussed above; and mAb-548, a novel murine mAb that engages human NPC1 domain C (NPC1–C). mAb-548 bound, with picomolar affinity, to an NPC1–C epitope that overlaps its binding interface with GPCL, and blocked GPCL-NPC1–C association in vitro at pH 5.5, the presumptive pH of virus-receptor engagement in late endosomes (FIGS. 5A-D and 6A-D). However, like MR72, mAb-548 possessed little or no neutralizing activity against infection by uncleaved viruses (FIG. 2A & FIGS. 9A-D), consistent with the lack of NPC1 at the cell surface (19). To deliver mAb-548 and MR72 to endosomes, the inventors chose the broadly reactive macaque mAb FVM09 as an initial candidate (FIG. 1A). FVM09 recognizes a conserved linear epitope in the GP glycan cap of all five known ebolaviruses with high affinity, but it lacks neutralizing activity, and confers limited protection in a murine post- exposure model of EBOV challenge (6).

The heavy (VH) and light chain (VL) variable domains of FVM09 were fused to the N–termini of the heavy (HC) and light chains (LC) of mAb-548 or MR72 using the dual variable domain design strategy (DVD-Ig ^TM ) (Wu et al., 2007). Although many bsAb engineering modalities exist (Spiess et al., 2015), the DVD-Ig format was chosen as an initial test case because it allows bivalent binding of both combining sites, but does not employ long, flexible polypeptide linker domains that may be susceptible to proteolysis or immunogenic presentation. The FVM09~548 and FVM09~MR72 DVD-Igs expressed readily in HEK293 suspension cells and migrated as a disulfide-bonded complex (apparent molecular weight, M _r >200K) in SDS-polyacrylamide gels (FIG. 7A). Size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS) analysis of the DVD-Igs indicated a monodisperse population of monomers, with some higher aggregate present (FIG.7B).

The inventors first measured the binding affinities of the DVD-Igs by biolayer interferometry (BLI) (FIG. 1C and Table 1). Each DVD-Ig could bind to EBOV GP, via the FVM09‘outer’ variable domains, with no appreciable loss of affinity relative to the parent FVM09 IgG. FVM09~548 could also recognize human NPC1–C, via its‘inner’ variable domains, with a K _D of <1 pM. Presentation of the MR72 variable domains in the DVD-Ig format also resulted in retention of subnanomolar affinity toward GPCL. Finally, the inventors   conducted two-phase binding studies in which each DVD-Ig, immobilized on the biosensor probe, was first exposed to EBOV GP, and then to NPC1-C or GP _CL (FIG.1D). The inventors found that each DVD-Ig could simultaneously bind both of its targets, indicating that there were no steric restrictions to engagement of both combining sites.

The inventors next tested the DVD-Igs for their capacity to neutralize infection in human cells by recombinant vesicular stomatitis viruses bearing EBOV GP (rVSV-EBOV GP) (FIG.2A-B). rVSVs provide a highly validated surrogate system to study filovirus entry and Ab-mediated neutralization under biosafety level 2 conditions (Takada et al., 1997; Bale et al., 2012). Both FVM09~548 and FVM09~MR72 potently neutralized rVSV-EBOV GP in a dose-dependent manner, with low-nanomolar IC50 values. By contrast, the parental mAbs FVM09, mAb-548, and MR72 had little or no neutralizing activity, as expected. Additionally, equimolar mixtures of the‘delivery’ IgG FVM09 with each receptor/RBS-targeting IgG (mAb-548 or MR72) also did not neutralize infection (FIGS. 2A-B). Therefore, viral neutralization by the DVD-Igs requires the physical linkage of delivery and receptor/RBS- binding specificities.

The interaction between GP _CL and NPC1 is critical and conserved across all filoviruses (Bornholdt et al., 2015; Miller et al., 2012; Ng et al., 2014; 2015), and FVM09 (and the DVD-Igs) recognize GP proteins derived from all five known ebolaviruses (FIGS. 8A-B) (Keck et al., 2016). Accordingly, the inventors postulated that the DVD-Igs would exhibit broad neutralizing activity. The inventors found that rVSVs bearing representative GP proteins derived from the four other ebolaviruses were all sensitive to neutralization by both DVD-Igs (FIGS. 2C–D), with IC50 values in the low-nanomolar range (FIG. 2G). rVSVs bearing GP proteins from the divergent marburgvirus clade were not neutralized by either DVD-Ig (data not shown), consistent with the known specificity of FVM09 towards ebolavirus but not marburgvirus epitopes (Keck et al., 2016). To determine whether these findings also extended to authentic filoviruses, the inventors tested the DVD-Igs against infectious EBOV, BDBV, and SUDV in microneutralization assays under BSL4 containment (FIGS. 2E–F). Each of the filoviruses was neutralized by both DVD-Igs with single-digit nanomolar potency, whereas the individual parent IgGs demonstrated little or no antiviral effect even at >300 nM (FIG. 2G and FIGS. 9A-D). Thus, both receptor- and RBS-targeting DVD-Igs possess in vitro activity against all five ebolaviruses, including the three associated with human disease outbreaks (EBOV, BDBV, SUDV).   The success of antibody therapeutics has fueled the development of a wide array of optimized bsAb architectures, several of which (including the DVD-Ig) are in clinical trials (Spiess et al., 2015). To explore the generality of the inventors’ strategy toward other bispecific modalities, the inventors used‘controlled Fab-arm exchange’ between half- molecules of FVM09 and MR72 IgGs to engineer an FVM09 ^ MR72 bispecific Duobody ^TM (Labrijn et al., 2013) (FIGS. 10A-D and FIG. 11A-C). FVM09 ^MR72 broadly neutralized rVSVs bearing ebolavirus GPs, albeit with reduced potency against SUDV GP, possibly due to its loss of bivalent recognition of GP and/or GPCL. Nonetheless, these results illustrate that endosomal targeting of the ebolavirus host-receptor interaction is amenable to other bispecific antibody engineering formats.

The observation that bsAbs combining two non-neutralizing antibodies could confer potent antiviral neutralization strongly suggested critical roles for both delivery and receptor/RBS-binding specificities. To explicitly test this hypothesis, the inventors first examined the activity of the DVD-Igs against rVSV-EBOV GP particles containing two point mutations in the FVM09 epitope that together abolish GP-FVM09 association (FIG. 3A & FIG.12). The neutralization potencies of both DVD-Igs were reduced by≈99% against this mutant virus. Second, the inventors evaluated the roles of the NPC1- and GPCL-binding functions by engineering DVD-Igs bearing 548 and MR72 variable domains with mutations in the third VH complementarity determining region (CDR-H3) that abrogate binding. The resulting FVM09~548 ^Mut and FVM09~MR72 ^Mut were completely bereft of neutralizing activity (FIG. 3B). Third, the inventors reasoned that if the NPC1-binding function of FVM09~548 were required, then high levels of NPC1 in cells should reduce its potency by saturating available mAb-548 combining sites. Accordingly, the inventors compared the capacities of the DVD-Igs to neutralize rVSV-EBOV GP in isogenic cell lines bearing normal or supraphysiological levels of NPC1 (FIG. 3C). NPC1 over-expression completely abrogated viral neutralization by FVM09~548, whereas it had no discernible effect on neutralization by FVM09~MR72. The latter result is consistent with the higher affinity of the GPCL/ ^GPCL complex (55 pM; Table 1, experiment 6) relative to the GPCL/NPC1-C complex (150 µM; from ref. (17)). Therefore, viral neutralization requires that (i) both DVD-Igs bind to the GP glycan cap in extracellular viral particles via their FVM09 variable domains; and that (ii) each DVD-Ig also bind to its respective cryptic endosomal epitope in cells (NPC1 or the GPCL RBS).   The hypothesis that the bsAbs could harness extracellular virions for their delivery to endosomal sites of filovirus-receptor interaction in the context of natural infection formed the basis of our experimental approach. Accordingly, the inventors directly evaluated the internalization of DVD-Igs and their parent IgGs into cellular endosomes (FIG. 3D & FIGS. 13A-H). Each Ab was covalently labeled with the acid-dependent fluorescent probe pHrodo Red ^TM , and exposed to cells, either alone, or following preincubation with mNeongreen- labeled fluorescent rVSV-EBOV GP particles (23, 34). After 30 min at 37 ˚C, cells were measured for both virus- and Ab-associated fluorescence by flow cytometry. Virus-negative cells displayed little or no Ab signal, indicating that neither the DVD-Igs nor their parent IgGs could internalize into cells in the absence of viral particles. By contrast, virus-positive cells were strongly positive for the DVD-Igs, but not for the parent mAb-548 and MR72 IgGs. Concordantly, examination of cells by fluorescence microscopy revealed that only FVM09 and the DVD-Igs could efficiently colocalize with viral particles in NPC1 ⁺ late endosomes (FIG.3E & FIG.14). These results, together with the capacity of FVM09 to bind EBOV GP with high affinity between pH 5.5–7.5 (FIGS. 15A-C & Table 1, experiments 9– 11), suggest that virion-bsAb complexes remain associated in early endosomes and traffic together to late endosomes, where proteolytic removal of the GP glycan cap would be expected to dislodge FVM09, and where mAb-548 and MR72 could engage their respective cellular and viral targets. Collectively, therefore, these findings strongly support a two-step ‘deliver-and-block’ mechanism for ebolavirus neutralization by FVM09~548 and FVM09~MR72.

Finally, the inventor evaluated the protective efficacy of the DVD-Igs in two murine models of lethal ebolavirus challenge. Because all of the inventors’ prior experiments were conducted in human cells, they first tested in vitro neutralization activity in murine NIH/3T3 cells (FIGS. 16A-C). Whereas FVM09~MR72 retained full activity, FVM09~548 exhibited poor neutralization in murine cells (FIGS. 16A–B), which could be readily explained by its dramatically reduced binding affinity for the house mouse NPC1 ortholog (FIG. 16C), and consequent reduced capacity to block GP _CL -NPC1 interaction (FIG. 16E). The discrepancy between binding of mAb- ^ ^ ^ to mouse and human NPC1 likely arises from species- dependent amino acid sequence differences in the mAb-548-binding region of NPC1–C (FIGS. 17A-B). This loss in NPC1 recognition and blocking potency did not extend to an NPC1 ortholog derived from a nonhuman primate (NHP), the mantled guereza (FIGS. 16D- F), whose NPC1–C sequence in the mAb-548-binding region is identical to those of humans,   rhesus macaques, and crab-eating (cynomolgus) macaques (FIGS.17A-B). The latter provide the two NHP models of filovirus challenge currently in use. Therefore, while host species- specific differences in NPC1 binding may affect FVM09~548’s efficacy in mice, they are unlikely to do so in NHPs and humans.

Both DVD-Igs were tested for their capacity to protect WT BALB/c mice, following a lethal challenge with a mouse-adapted EBOV variant (EBOV-MA) (FIG. 4A) (Bray et al., 1998). Mice were dosed at 2 days post-challenge with DVD-Igs, mixtures of parent IgGs, or vehicle, and monitored for 28 days. FVM09~MR72 afforded a high level of survival (70%) relative to the vehicle control group, whereas no statistically significant survival over the vehicle group was recorded for FVM09~548 and the parent IgG mixtures. The inventors also evaluated the DVD-Igs for post-exposure protection against a lethal human SUDV isolate in the immunocompromised Type 1 IFNα/β R ^−/− mouse model (FIG. 4B) (Holtsberg et al., 2016; Labrijn et al., 2013). Mice were dosed with antibody at 1 and 4 days post-challenge, and monitored as above. FVM09~MR72 was fully protective, and FVM09~548 yielded a statistically significant level of partial protection, relative to the vehicle control group. By contrast, no significant protection was observed with the parent IgG mixtures. Together, these findings provide evidence that a bsAb targeting the critical intracellular virus-receptor interaction can confer broad protection against lethal ebolavirus challenge, even under stringent conditions of post-exposure treatment. The poor in vivo efficacy of the receptor- targeting bsAb, FVM09~548, was consistent with its reduced capacity to inhibit viral interaction with murine NPC1 (FIGS.16A-F), lending support to the proposed two-step mode of action. Despite this considerable handicap, FVM09~548 provided partial post-exposure protection in the SUDV challenge model, suggesting that it could be more effective in NHP and human hosts bearing compatible NPC1 proteins.

Recent antibody discovery efforts have demonstrated the existence of conserved GP surface epitopes that can elicit broadly reactive mAbs with cross-protective potential (Howell et al., 2016; Flyak et al., 2016; Frei et al., 2016; Furuyama et al., 2016; Holtsberg et al., 2016; Bornholdt et al., 2016). Herein, the inventors describe a complementary strategy to generate broadly-protective Abs that target highly conserved epitopes at the intracellular filovirus- receptor interface, which are normally shielded from GP-specific mAbs. Because the‘cryptic epitope’-targeting components of the bsAbs engineered in this study block endosomal receptor binding by all known filoviruses (Bornholdt et al., 2015) (this study), next- generation molecules combining them with appropriate delivery mAbs of viral or cellular   origin may afford true pan-filovirus coverage. This‘Trojan horse’ bispecific antibody approach may also find utility against other viral pathogens known to use intracellular receptors (e.g., Lassa virus (Jae et al., 2016)), or more generally, to target entry-related virus structural rearrangements that occur only in the endo/lysosomal pathway.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.   VII. REFERENCES

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