Anti-ebola binding proteins and methods of use thereof

Abstract

Certain embodiments of the invention provide isolated anti-Ebola single domain antibodies (sdAbs), as well as polypeptides and protein molecules comprising such sdAbs, including Fc fusion thereof, and bispecific anti-Ebola binder protein. Certain embodiments of the invention also provide methods of using these sdAbs, polypeptides and protein molecules for treating an Ebola virus infection.

Description

[0001] ANTI-EBOLA BINDING PROTEINS AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 733,072 that was filed on December 12, 2024 and United States Provisional Application Number 63 / 849,532 that was filed on July 23, 2025. The entire content of the applications referenced above is hereby incorporated by reference herein.

[0004] GOVERNMENT FUNDING

[0005] This invention was made with government support under All 71954 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] The Ebola virus (EBOV), a member of the filovirus family, poses a serious threat to global health due to its high fatality rates and reservoirs in both animals and humans. During the unprecedented outbreak in West Africa from 2014 to 2016, there were 28,652 infections with a case fatality rate of ~ 40%. Smaller outbreaks of EBOV and related filoviruses have occurred periodically. Bats and nonhuman primates are believed to be natural reservoirs for EBOV.

[0008] Additionally, EBOV can remain dormant in the human body for years before reemerging to cause new infections. Currently, there are two FDA-approved human antibody drugs targeting EBOV, but they only modestly reduced the fatality rate to about 35%. Given the long-term coexistence of EBOV with humans and limitations of current therapy, development of effective and accessible treatment strategies is needed.

[0009] SUMMARY OF THE INVENTION

[0010] Certain embodiments of the invention provide an isolated anti -Ebola binder protein (e.g., a single domain antibody (sdAb) such as Nanosota-EB2 and Nanosota-EBl and Fc fusion thereof, or a monospecific or bispecific binder protein comprising one or both sdAb domains) that comprises:

[0011] (1) one or more complementarity determining regions (CDRs) selected from the group consisting of

[0012] (a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GRTFSNDA (SEQ ID NO:9);

[0013] (b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of IDYNGGRT (SEQ ID NO: 10); and

[0014] (c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO: 11); and / or

[0015] (2) one or more CDRs selected from the group consisting of:

[0016] (a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GSTSVIYA (SEQ ID NO:2);

[0017] (b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of ITRGVGST (SEQ ID NO:3); and

[0018] (c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

[0019] Certain embodiments of the invention provide a sdAb-Fc fusion protein comprising an isolated anti-Ebola sdAb as described herein operably linked to an Fc domain amino acid sequence.

[0020] Certain embodiments of the invention provide an isolated anti-Ebola binder protein (e.g., monospecific or bispecific binder) comprising two independently selected sdAb-Fc fusion proteins as described herein, wherein the two Fc polypeptides are linked to form a dimer e.g., homodimer or heterodimer).

[0021] Certain embodiments of the invention provide a composition comprising isolated anti-Ebola binder protein (e.g., a bispecific anti-Ebola binder) as described herein, and a carrier.

[0022] Certain embodiments of the invention provide an isolated polynucleotide(s) comprising a nucleotide sequence encoding an isolated anti-Ebola binder protein as described herein.

[0023] Certain embodiments of the invention provide a vector or a pair of vectors comprising the polynucleotide(s) as described herein.

[0024] Certain embodiments of the invention provide a cell comprising the polynucleotide(s) as described herein or the vector(s) as described herein.

[0025] Certain embodiments of the invention provide anti-Ebola binder protein(s), or mixture thereof.

[0026] Certain embodiments of the invention provide a method of inhibiting the activity of Ebola virus, comprising contacting Ebola virus with an isolated anti-Ebola binder protein as described herein.

[0027] Certain embodiments of the invention provide a method for treating or preventing an Ebola virus infection in a mammal, comprising administering an effective amount of an isolated anti-Ebola binder protein as described herein, to the mammal.

[0028] Certain embodiments of the invention provide an isolated anti-Ebola binder protein as described herein, for the prophylactic or therapeutic treatment of an Ebola virus infection.

[0029] Certain embodiments of the invention provide an isolated anti-Ebola binder protein as described herein, for use in medical therapy or for use in the treatment of an Ebola virus infection in a mammal.

[0030] Certain embodiments of the invention an isolated anti-Ebola binder protein or a composition described herein for the manufacture of a medicament for treating an Ebola virus infection in a mammal.

[0031] Certain embodiments of the invention provide a kit comprising:

[0032] 1) an isolated anti-Ebola binder protein as described herein;

[0033] 2) packaging material; and

[0034] 3) instructions for administering the binder protein to a mammal to treat or prevent an Ebola infection.

[0035] Certain embodiments of the invention provide a method for producing an anti-Ebola binder protein, comprising culturing a host cell as described herein under conditions in which the protein is expressed.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figures 1A-1D. In Vitro Characterization of Two Novel Anti-EBOV Nanobodies, Nanosota-EBl and -EB2. (Fig.lA) Binding affinities between His-tagged nanobodies and two versions of EBOV GP proteins (GP-AM and GPcl; see Fig.7B for their definitions) were measured by surface plasmon resonance (SPR). N. D., binding not detected. (Fig.lB) Binding interactions between His-tagged nanobodies and three versions of the EBOV GP proteins (GP-AM, GPcl and sGP; see Fig.7B for their definitions) were evaluated by ELISA. A450: absorbances at 450 nm. Data are presented as mean ± SEM (n = 3). An unpaired two-tailed Student’s / -test was used to analyze the statistical differences between the indicated groups, with results indicated above each bar. ****p < 0.0001. (Fig.lC) Efficacy of Fc-tagged nanobodies in neutralizing EBOV pseudoviruses. Retroviruses pseudotyped with full-length EBOV GP were used to infect Huh7 cells in the presence of Fc-tagged Nansota-EBl or -EB2 at different concentrations. The efficacy of each nanobody against EBOV pseudoviruses was expressed as the concentration required to neutralize pseudovirus entry by 50% (IC50). Error bars represent SEM (n=3). (Fig.lD) Efficacy of Fc-tagged nanobodies in neutralizing authentic EBOV infection. Authentic EBOV was used to infect Huh7 cells in the presence of Fc-tagged Nanosota-EBl or -EB2 at different concentrations. The efficacy of each nanobody against authentic EBOV infection was expressed as the concentration required to neutralize EBOV infection by 50% (ICso). Error bars represent SEM (n=3). Since Nanosota-EBl-Fc could not fully block viral entry at any of the tested concentrations, the ICso values for Nanosota-EBl-Fc are estimations.

[0038] Figures 2A-2C. Structural Basis for the Anti-EBOV Functions of Nanosota-EBl. (Fig.2A) Cryo-EM structure of EBOV GP-AM complexed with Nanosota-EBl (top view; surface presentation). The three subunits of EBOV GP-AM are colored orange, gray, and green, respectively. Nanosota-EBl is shown in blue. The trimeric GP-AM is bound by two Nanosota-EBl molecules. (Fig.2B) Cryo-EM structure of EBOV GP-AM complexed with Nanosota-EBl (side view). The overall structure is shown in surface presentation, with one GP subunit and one Nanosota-EBl molecule shown in cartoon presentation. Nanosota-EBl binds to the glycan cap of EBOV GP. The glycan cap is colored cyan. The cathepsin cleavage site near the glycan cap is marked by a red circle. (Fig.2C) The binding interface between Nanosota-EBl and the glycan cap. Nanosota-EBl binds to the P 17 strand of the glycan cap, displacing the pi8 strand and pushing it aside to form a loop.

[0039] Figures 3A-3H. Structural Basis for the Anti-EBOV Functions of Nanosota-EB2. (Fig.3A) Cryo-EM structure of EBOV GP-AM complexed with Nanosota-EB2 (top view; surface presentation). The three subunits of EBOV GP-AM are colored orange, gray, and green, respectively. Nanosota-EB2 is shown in blue. The N563 glycan involved in binding Nanosota-EB2 is shown in red. The trimeric GP-AM is bound by three Nanosota-EB2 molecules. (Fig.3B) Cryo-EM structure of EBOV GP-AM complexed with Nanosota-EB2 (side view). The overall structure is shown in surface presentation, with one Nanosota-EB2 molecule and several membrane-fusion elements in GP shown in cartoon presentation, and the N563 glycan shown in sticks. (Fig.3C) The binding interface between Nanosota-EB2 and GP. Nanosota-EB2 recognizes quaternary epitopes, including HR1, fusion loop, N-terminus of GP2, and pi / p2 strands of GP1. (Figs.3D-3H) Detailed interactions between Nanosota-EB2 and N563 glycan, HR1, fusion loop, N-terminus of GP2, and pi / p2 strands of GP1, respectively. Dotted lines indicate hydrogen bonds. Double arrows indicate hydrophobic interactions.

[0040] Figures 4A-4D. Biochemical Mechanisms for the Anti-EBOV Functions of Nanosota-EBl and -EB2. (Fig.4A) Differential scanning fluorimetry (DSF) assay for assessing the impact of nanobodies on the thermal stability of EBOV GP-AM. His-tagged Nanosota-EBl and -EB2 slightly and significantly increased the thermostability of EBOV GP-AM, respectively. Comparisons of the Tm values for GP-AM in the absence or presence of the nanobodies were performed using an unpaired two-tailed Student’s / -test. Error bars represent SEM (n=6). ** / ?<0.01, **** / ?<0.0001. (Fig.4B) DSF assay for assessing the impact of His- tagged Nanosota-EB2 on the thermal stability of EBOV GPcl at lower pHs. Nanosota-EB2 significantly increased the thermostability of EBOV GPcl at low pH. Comparisons of the Tm values for GPcl in the absence or presence of Nanosota-EB2 were performed using an unpaired two-tailed Student’s / -test. Error bars represent SEM (n=6). **** / ?<0.0001. n.s.: not significant. Note that this experiment could not be conducted for Nanosota-EBl because Nanosota-EBl does not bind to EBOV GPcl. (Fig.4C) Glycan cap cleavage assay to evaluate the effect of Nanosota-EBl on the protease sensitivity of the glycan cap, using SDS-PAGE under reducing conditions and Coomassie blue staining. Nanosota-EBl presence slowed the thermolysin L cleavage of GP-AM. (Fig.4D) Glycan cap cleavage assay to evaluate the effect of Nanosota-EBl on the protease sensitivity of the glycan cap, using Western blot to detect the His tag on GP-AM under non-reducing conditions. Nanosota-EB 1 presence again slowed thermolysin L cleavage of GP-AM. Each of the above experiments was performed three times, yielding consistent results.

[0041] Figures 5A-5F. Footprints of Nanosota-EBl and Nanosota-EBl on EBOV GP and Their Binding to the GP of EBOV-Related Ebolaviruses. (Fig.5A) Footprint of Nanosota-EBl on the glycan cap of EBOV GP (surface representation). (Fig.5B) Footprint of Nanosota-EB2 on the quaternary epitopes of EBOV GP (surface representation). Nanobody-contacting residues on EBOV GP are labeled, with residues conserved between EBOV and BDBV shown in orange, and residues differing between the two viruses shown in pink. (Fig.5C) ELISA results showing the binding interactions between Nanosota-EB 1-Fc and GP-AM from EBOV, BDBV, and SUDV. (Fig.5D) ELISA results showing the binding interactions between Nanosota-EB2-Fc and GP-AM from EBOV, BDBV, and SUDV. (Fig.5E) Neutralization efficacy of Fc-tagged nanobodies against BDBV pseudoviruses. (Fig.5F) Neutralization efficacy of Fc-tagged nanobodies against SUDV pseudoviruses.

[0042] Figures 6A-6D. In Vivo Efficacy of Nanosota-EBl-Fc, -EB2-Fc, and Their Cocktail.

[0043] Groups of 10 mice were challenged with EBOV and then treated with the indicated nanobody, nanobody cocktail, or vehicle alone (control). Each nanobody was administered at a total dose of 50 mg / kg via intraperitoneal (IP) injection. For the cocktail, 25 mg / kg of each nanobody was given. Four animals were used for serum collection and subsequently removed from the study. The remaining 6 animals were monitored for 18 days. (Fig.6A) Survival curves were generated using Kaplan-Meier analysis, and comparisons to the vehicle control were made using the Mantel-Cox test. *P<0.05. (Fig.6B) Weight change for each animal was calculated from its starting weight. Statistical analysis was performed for the data collected on Day 6 using an Ordinary One-Way ANOVA. The average weight change of each experimental group was compared to that of the vehicle control. Error bars on Day 6 represent SEM (n=3 for control; n=6 for treatment groups). **P<0.01; ***P<0.001. (Fig.6C) The highest clinical score for each group on each day is displayed. (Fig.6D) Viral loads were measured in mice. Genome copy numbers (GN) were back-calculated from Cq values using a standard curve of synthetic RNA corresponding to the amplicon. Statistical analysis was performed using Ordinary One-Way ANOVA with multiple comparisons, with each experimental group compared to the vehicle control. Error bars represent SEM (n=4). ****P<0.0001.

[0044] Figures 7A-7B. Structural overview of EBOV GP. (Fig.7A) The overall structure of the EBOV GP ectodomain (PDB: 5JQ3). (Fig.7B) Schematic representations of four EBOV GP variants: GP ectodomain, GP-AM, GPcl, and sGP. GP1 is the receptor-binding subunit, and GP2 is the membrane-fusion subunit. RBS: receptor-binding site. MLD: mucin-like domain. HR1: heptad repeat 1. HR2: heptad repeat 2. SP: signal peptide.

[0045] Figures 8A-8D. Surface Plasmon Resonance (SPR) Sensorgrams. (Fig.8A) SPR sensorgrams of binding kinetics between Nanosota-EBl-His and EBOV GP-AM. (Fig.8B) No binding was detected between Nanosota-EBl-His and EBOV GPcl. (Fig.8C) SPR sensorgrams of binding kinetics between Nanosota-EB2-His and EBOV GP-AM. (Fig.8D) SPR sensorgrams of binding kinetics between Nanosota-EB2-His and EBOV GPcl. Ka, kon, and

[0046]

[0047] values are labeled.

[0048] Figure 9. Efficacy of His-tagged nanobodies in neutralizing EBOV pseudoviruses. The assay was conducted as described in Fig. 1C.

[0049] Figure 10. Flow Chart of Cryo-EM Image Processing and Map Reconstruction for the Structures of EBOV GP-AM Complexed with Nanosota-EBl. Representative raw cryo-EM images and 2D classes of the complex are presented. 3D refinement using all the particles in good 3D classes generated a 3.07 A map. The final maps, half-map FSC curves, angular distribution plot, and accompanying local resolution illustrations are enclosed in the dashed black box.

[0050] Figure 11. Flow Chart of Cryo-EM Image Processing and Map Reconstruction for the Structures of EBOV GP-AM Complexed with Nanosota-EBl. Representative raw cryo-EM images and 2D classes of the complex are presented. 3D refinement using all the particles in good 3D classes generated a 3.36 A map. The final maps, half-map FSC curves, angular distribution plot, and accompanying local resolution illustrations are enclosed in the dashed black box.

[0051] Figures 12A-12H. Cryo-EM Densities of Selected Regions in the Structures of EBOV GP-AM Complexed with Nanosota-EBl or Nanosota-EBl. (Figs.l2A-12B) Cryo-EM densities of the interface between Nanosota-EBl and EBOV GP. (Figs.l2C-12H) Cryo-EM densities of the interface between Nanosota-EB2 and EBOV GP. Nanobodies are colored in blue. Different parts of EBOV GP are colored differently. Contact residues are shown as sticks.

[0052] Figures 13A-13D. Nanosota-EBl Binds to a Cryptic Epitope on the EBOV GP Glycan Cap. (Fig.l3A) Left panel: Structure of the EBOV GP glycan cap without a bound ligand (PDB: 9BSV). The glycan cap appears disordered due to its flexibility. Right panel: Structure of the EBOV GP glycan cap bound to Nanosota-EBl. Binding by Nanosota-EBl stabilizes the glycan cap, making it visible. (Fig.l3B) Structure of the EBOV GP glycan cap bound to the human antibody REGN-3470 (PDB: 7TN9). Structural motifs of the glycan cap are labeled. The bound REGN-3470 is depicted as a gray oval. (Fig.l3C) Structure of the EBOV GP glycan cap bound to Nanosota-EBl. Nanosota-EBl disrupts the pi8 strand and interacts with the inner P 17 strand, revealing part of the P 17 / 18 loop. The bound Nanosota-EBl is illustrated as a blue oval. (Fig.l3D) Comparison of the structures of the EBOV GP glycan cap bound to the human antibody EBOV-548 (PDB: 6UYE) and the glycan cap bound to Nanosota-EBl. Both the human antibody and Nanosota-EBl target the P 17 strand but approach it from different orientations.

[0053] Figure 14. Superimposition of N563 Glycan from Different Structures. Three structures of EBOV GP (PDBs: 9BSU, 9BSV, and 7TN9) are superimposed by their HR1 region. The N563 glycan from each of the three structures is colored green, orange, and magenta, respectively.

[0054] Figures 15A-15F. Impacts of Nanosota-EBl and Nanosota-EBl on Thermostability of EBOV GP-AM and GPcl at Different pHs. The positive first derivatives are plotted against temperature. The melting temperatures (Tm) at the peak point in the first derivative curve are shown at the top of each valley. pH values are labeled for each panel. (Fig.l5A) pH7 GP-AM, (Fig.l5B) pH7 GPcl, (Fig.l5C) pH6, (Fig.l5D) pH5.5, (Fig.l5E) pH5, and (Fig.l5F) pH4.5.

[0055] Figures 16A-16B. Glycan Cap Cleavage Assay to Evaluate the Effect of Nanosota-EBl on the Protease Sensitivity of the Glycan Cap. (Fig.l6A) The assay was conducted using SDS-PAGE under reducing conditions and Coomassie blue staining. Nanosota-EB2 presence had no obvious effect on the thermolysin L cleavage of GP-AM. (Fig.l6B) The assay was performed using Western blot to detect the His tag on GP-AM under non-reducing conditions. Nanosota-EB2 presence again had no obvious effect on the thermolysin L cleavage of GP-AM. Each of the above experiments was performed three times, yielding consistent results.

[0056] Figures 17A-17B. Quantification of Western blot results for the glycan cap proteolysis assay. ImageJ (version 1.53a) was used to quantify the GP-AM monomer bands from the Western blot data in Fig.4D and its two replicates for EBl-Fc (Fig.l7A) and in Fig.16B and its two replicates for EB2-Fc (Fig.l7B). Unpaired two-tailed Student’s / -tests were conducted to compare the treatment condition and the control condition at each time point (n=3). ** / ?<0.01. n.s.: not significant.

[0057] Figures 18A-18B. In Vitro Stability of Nanosota-EBl-Fc and Nanosota-EB2-Fc. ELISA ((Fig.l8A) EBl-Fc, and (Fig.l8B) EB2-Fc) was performed to assess the effect of storage conditions on the binding affinity of the Fc-tagged nanobodies to recombinant EBOV GP-AM.

[0058] Figures 19A-19B. Comparison of the Epitopes on EBOV GP Recognized by Nanobodies (Nanosota-EBl and Nanosota-EB2) and Human Antibodies. (Fig.l9A) Epitopes on the glycan cap of GPL PDB IDs of human antibodies are indicated. Epitope residues were analyzed using LigPlot+ v.2.2 and are displayed on the GP monomer in surface mode, with different colors representing different antibodies: blue for Nanosota-EBl, cyan for REGN-3470, green for EBOV-293, and magenta for EBOV-296. (Fig.l9B) Epitopes on GP2. PDB IDs of human antibodies are indicated. Epitope residues are shown in surface mode on the two monomers of the GP trimer, with colors distinguishing antibodies: blue for Nanosota-EB2, cyan for REGN-3479, green for KZ52, and magenta for ADI-15878. An overlay of KZ52 and Nanosota-EB2 on the same GP2 structure reveals a clash, suggesting overlapping binding epitopes.

[0059] Figures 20A-20D. In Vivo Characterization of the Anti-EBOV Functions of Nanosota-EBl, Nanosota-EB2, Their Combination Cocktail, and Their Bispecific Combination in Early-Stage Infection. Interferon-a / p-receptor knockout mice were divided into 5 groups, with 10 mice in each group. All the mice were challenged with 100 PFU live EBOV. Four hours post-infection, mice in different groups received different nanobodies at a dosage of 50 mg / kg through intraperitoneal (IP) injection. These nanobodies included EBl-Fc alone, EB2-Fc alone, a cocktail of EBl-Fc and EB2-Fc (1:1 ratio), and a bispecific nanobody containing one copy of EB1, one copy of EB2, and a human Fc tag. PBS was used as a negative control. For each group of mice, survival rates (Fig. 20A), clinical scores (recorded only for surviving mice) (Fig.20B), body weight changes (recorded only for surviving mice) (Fig.20C), and virus loads (for 4 mice in each group) (Fig.20D) were recorded. The survival rates, clinical scores, and body weight changes were monitored for 18 days, while the virus loads were measured on day 4 post-infection. Comparisons of the viral loads between the control group and each of the treatment groups were performed using an unpaired two-tailed Student’s / -test. Error bars represent SEM (n=4). **** <0.0001. In (Fig.20C), data are presented as mean ± SEM (n values vary depending on the number of surviving mice on each day). P-values for weight change were calculated between the indicated experimental groups using a mixed-effects model with Geisser-Greenhouse correction, based on data collected throughout the entire experiment. ****p < 0.0001; **P < 0.01; *P < 0.05. Fig.20C showed significant difference between bispecific nanobody vs EB2-Fc group (*P); significant difference between bispecific nanobody vs EBl-Fc group (**P); and significant difference between bispecific nanobody vs control group (****p), etc.

[0060] Figures 21A-21C. Biochemical Characterization of Nanosota-EB1 / EB2-Fc Bispecific Nanobody shows synergistic activity of the bispecific nanobody in overcoming EBOV sGP decoy interference. (Fig.21A) Capability of the bispecific nanobody to simultaneously bind to GPcl (glycan cap-cleaved form of GP) and sGP as determined using surface plasmon resonance (SPR). For step 1, the SPR sensor chip was coated with sGP (which contains the EBl-binding site but not the EB2-binding site) (blue curve). For step 2, the bispecific nanobody (red curve) was injected / flowed through until the SPR signal reached a plateau. For step 3, GPcl (which contains the EB2 epitope but not the EB1 epitope) (cyan curve) was flowed through the chip. The data confirmed that the bispecific nanobody can bind sGP and GPcl simultaneously. (Fig.21B) The bispecific nanobody resists sGP diversion when binding to GP, as determined by ELISA. Each of the three nanobodies (EBl-Fc, EB2-Fc, and the bispecific nanobody) binds to GP-AM in the presence of sGP at one of the indicated concentrations. Each of the binding reactions was characterized using the half-maximal effective concentration (ECso), which is defined as the concentration of a nanobody that produces 50% of the maximum possible response signal in the assay. The EC50 values for each binding experiment were calculated, along with the fold changes in the ECso in the presence of sGP relative to the absence of sGP. The data showed that sGP has no impact on the binding affinities of EB2-Fc and the bispecific nanobody to GP but has a significant impact on the binding affinity of EBl-Fc to GP.

[0061] (Fig.21C) The bispecific nanobody resists sGP diversion when neutralizing EBOV pseudovirus entry. Each of the three nanobodies (EBl-Fc, EB2-Fc, and the bispecific nanobody) was used to neutralize EBOV pseudovirus entry in the presence of sGP at one of the indicated concentrations. The ICso values for each pseudovirus entry experiment were calculated, as well as the fold changes in the IC50 in the presence of sGP relative to the absence of sGP.

[0062] Neutralizing potency was expressed as ICso, defined as the concentration required to inhibit pseudovirus entry by 50%. The data showed that sGP has no impact on the neutralizing potencies of EB2-Fc and the bispecific nanobody but has a significant impact on the neutralizing potency of EB 1 -Fc.

[0063] Figure 22. A proposed “anchor, recycle, and block” mechanism for the superior anti-EBOV potency of the bispecific nanobody. EB1 and EB2 are colored blue and red, respectively. The bispecific nanobody molecules can bind to either the EB1 epitope or the EB2 epitope on GP. When bound to the EB2 epitope, the bispecific nanobody molecules are anchored on EBOV particles, resisting diversion by sGP. When bound to the EB1 epitope, the bispecific nanobody molecules are recycled inside endosomes and become available to bind to the EB2 epitope. Both strategies increase the local concentration of EB1 and EB2 in the endosomes, blocking the fusion of viral and endosomal membranes. See Example 2 for more details on this mechanism.

[0064] Figure 23A-23D. Synergistic binding of the bispecific nanobody to EBOV GP and stabilization of the glycan cap. (Fig.23A) Construction of the bispecific nanobody, Nanosota-EB1ZEB2-Fc, by fusing two individual nanobodies, Nanosota-EBl and -EB2, to a human Fc domain. The bispecific nanobody exhibited potent neutralization of authentic EBOV infection in vitro. Huh7 cells were infected with authentic EBOV in the presence of EB1 / EB2-Fc at varying concentrations. Nanobody efficacy was expressed as the concentration required to achieve 50% inhibition of EBOV infection (ICso). Data are presented as mean ± SEM (n = 3).

[0065] (Fig.23B) Cryo-EM structure of the ternary complex consisting of EBOV GP ectodomain, EB1, and EB2. The trimeric GP ectodomain is shown in gray; the three EB1 molecules are shown in blue; and the three EB2 molecules are shown in green. (Fig.23C) Close-up view of the synergistic binding of EB1 and EB2 to EBOV GP. EB2 binding indirectly stabilizes the glycan cap via a hinge region. The proteolysis loop (residues 198-212), which is disordered in the structure, is indicated by a red oval. (Fig.23D) Glycan cap cleavage assay assessing the effect of EB1 / EB2-Fc on glycan cap proteolysis, using Western blot detection of the His tag on GP ectodomain under non-reducing conditions. EB1 / EB2-Fc inhibited GP ectodomain proteolysis after 60 minutes. The experiment was repeated three times with consistent results.

[0066] Figures 24A-24D. In vivo evaluation of the anti-EBOV efficacy of the bispecific nanobody in late-stage infection. (Fig.24A) Interferon-a / p receptor knockout mice were divided into six groups (n=5 per group on Day 0). All mice were challenged with 100 PFU of authentic EBOV. Mice in different groups received a single 50 mg / kg dose of EB1 / EB2-Fc via I. P. injection, administered at the indicated time points post-infection. PBS was used as a negative control. Favipiravir, administered daily at a dose of 100 mg / kg, was included for comparison. For each group, survival rates (Fig.24B), body weight changes (recorded only for surviving mice) (Fig.24C), and clinical scores (recorded only for surviving mice) (Fig.24D) were monitored over 11 days. In (Fig.24C), data are presented as mean ± SEM (n values vary depending on the number of surviving mice on each day). P-values for weight change were calculated between the indicated experimental groups using a mixed-effects model with Geisser-Greenhouse correction, based on data from the first eight days (except for groups 4 vs. 5, where only data from the first six days were used). ****p < 0.0001; *P < 0.05; n.s.: not statistically significant.

[0067] Figures 25A-25D. In vivo evaluation of the anti-EBOV efficacy of the bispecific nanobody administered via the intranasal route. (Fig.25A) Interferon-a / p receptor knockout mice were divided into two groups (n = 5 per group on Day 0). All mice were challenged with 100 PFU of authentic EBOV. Mice in the treatment group received a single 50 mg / kg dose of EB1 / EB2-Fc administered intranasally (I. N.) four hours post-infection. PBS was used as a negative control. For each group, survival rates (Fig.25B), body weight changes (recorded only for surviving mice) (Fig.25C), and clinical scores (recorded only for surviving mice) (Fig.25D) were monitored for 11 days. In (Fig.25C), data are presented as mean ± SEM (n values vary depending on the number of surviving mice on each day). P-values for weight change were calculated between the indicated experimental groups using a mixed-effects model with Geisser-Greenhouse correction, based on data collected throughout the entire experiment. **P < 0.01.

[0068] Figures 26A-26D. Pharmacokinetics of the bispecific nanobody in mice. Human neonatal Fc receptor (hFcRn) transgenic mice were used in this study. (Fig.26A) A log-linear relationship between EB1 / EB2-Fc concentrations and chemiluminescence intensities was established by ELISA and used in calculating the plasma concentrations of EB1 / EB2-Fc.

[0069] (Fig.26B) EB1 / EB2-Fc was administered through the I. P. route to mice (n=3), its plasma concentrations at different time points were recorded, and its plasma half-life was calculated. (Fig.26C) EB1 / EB2-Fc was administered through the I. N. route to mice (n=3) and its plasma concentrations at different time points were recorded. (Fig.26D) The relative bioavailability of I. N. delivery compared to I. P. injection was determined using the ratio of the total exposure to EB1 / EB2-Fc, represented by the area under the curve (AUC) values. Data are presented as mean ± SEM (n=3).

[0070] Figures 27A-27B. In Vitro Stability of the bispecific nanobody. ELISA was performed to evaluate the effect of storage conditions ((Fig.27A) one week incubation, (Fig.27B) two-month incubation) on the binding affinity of EB1 / EB2-Fc to recombinant EBOV GP ectodomain. Data are presented as mean ± SEM (n=3).

[0071] Figure 28. Cryo-EM image processing and map reconstruction workflow for the ternary complex of EBOV GP ectodomain, Nanosota-EBl, and Nanosota-EB2.

[0072] Representative raw cryo-EM images and 2D class averages of the complex are shown. 3D refinement using particles from high-quality 3D classes yielded a 2.92 A map. The final maps, half-map FSC curves, angular distribution plot, and corresponding local resolution maps are shown within the dashed black box.

[0073] Figures 29A-29C. Impacts of sGP on the target-binding affinities of Nanosota-EBl-Fc, Nanosota-EB2-Fc, and Nanosota-EB1 / EB2-Fc. Fig.29A shows EBl-Fc data. Fig.29B shows EB2-Fc data. Fig.29C shows EB1 / EB2-Fc data. See the legend for Fig. 21B for details. Data are presented as mean ± SEM (n=3).

[0074] Figures 30A-30C. Impacts of sGP on the neutralizing potencies of Nanosota-EBl-Fc, Nanosota-EB2-Fc, and Nanosota-EB1 / EB2-Fc. Fig.30A shows EBl-Fc data. Fig.30B shows EB2-Fc data. Fig.30C shows EB1 / EB2-Fc data. See the legend for Fig. 21C for details. Data are presented as mean ± SEM (n=3).

[0075] Figures 31A-31F. Construction of the bispecific nanobody and characterization of its in vitro anti-EBOV potency. (Fig.31A) Construction of the bispecific nanobody Nanosota-EB1 / EB2-Fc by fusing two individual nanobodies, Nanosota-EBl and Nanosota-EB2, to a human Fc domain. (Fig.31B) Efficacy of Nanosota-EB1 / EB2-Fc in neutralizing live EBOV infection. Authentic EBOV (Mayinga strain) was used to infect Huh7 cells in the presence of Nanosota-EB1 / EB2-Fc at different concentrations. Neutralization efficacy was expressed as the concentration required to reduce EBOV infection by 50% (IC50). Error bars represent SEM (n = 3). (Fig.31C)-(Fig.31F) Efficacy of Nanosota-EB1 / EB2-Fc in neutralizing EBOV pseudoviruses. Retroviruses pseudotyped with full-length EBOV GP from four different EBOV strains (Mayinga in Fig.31C, Makona in Fig.31D, Lturi in Fig.31E, Mbandaka in Fig.31F) were used to infect Huh7 cells in the presence of Nanosota-EB1 / EB2-Fc at different concentrations. Neutralization efficacy was expressed as the concentration required to reduce pseudovirus entry by 50% (IC50). Error bars represent SEM (n = 3).

[0076] Figures 32A-32D. Synergistic binding of the bispecific nanobody to EBOV GP and stabilization of the glycan cap. (Fig.32A) Cryo-EM structure of the ternary complex consisting of EBOV GP ectodomain, EB1, and EB2. The trimeric GP ectodomain is shown in gray; the three EB1 molecules are shown in blue; and the three EB2 molecules are shown in green.

[0077] (Fig.32B) Close-up view of the synergistic binding of EB1 and EB2 to EBOV GP. EB2 binding indirectly stabilizes the glycan cap via a hinge region. The proteolysis loop (residues 198-212), which is disordered in the structure, is indicated by a red oval. (Fig.32C) Glycan cap cleavage assay assessing the effect of EB1 / EB2-Fc on glycan cap proteolysis, using Western blot detection of the His tag on GP ectodomain under non-reducing conditions. EB1 / EB2-Fc inhibited GP ectodomain proteolysis after 60 minutes ((boxed). The experiment was repeated three times with consistent results. (Fig.32D) Glycan cap cleavage assay assessing the effect of EB2-Fc on glycan cap proteolysis, using the same assay conditions as in panel (Fig.32C). EB2-Fc inhibited GP ectodomain proteolysis for up to 30 minutes.

[0078] DETAILED DESCRIPTION

[0079] The deadly Ebola virus (EBOV) poses a serious threat to global health and national security. Nanobodies, also referred to as single-domain antibodies (sdAbs) herein, have shown promising therapeutic potential. Described herein include anti-EBOV nanobodies, named Nanosota-EBl and Nanosota-EB2, which target the EBOV glycoprotein (GP protein). EB1 targets the glycan cap of GP1 subunit, while EB2 stabilizes GP2 subunit in its pre-fusion state. Nanosota-EBl and Nanosota-EB2 are the first nanobodies shown to inhibit authentic EBOV. In addition, exemplary novel bispecific binder protein that combines EB1 and EB2 with human Fc tag is also described herein. Unexpectedly, this bispecific nanobody-Fc binder protein provided complete protection to mice challenged with EBOV, outperforming the individual nanobodies and their combination cocktail. By recognizing two epitopes, it effectively countered the virus's secreted GP protein based-decoy strategy. Without wanting to be bound by theory, this bispecific nanobody-Fc protein may operate through an "anchor, recycle, and block" mechanism, increasing the presence of EB1 and EB2 in endosomes through anchoring and recycling, thereby blocking viral entry into cytoplasm. These anti-Ebola binder proteins, including the sdAbs and Fc fusion proteins thereof, such as a bispecific inhibitor described herein, could be powerful tools in combating future EBOV outbreaks.

[0080] The terms “nanobody” and “single-domain antibody” are used interchangeably herein. As used herein, the term “nanobody” or “single-domain antibody” refers to a single monomeric variable antibody domain comprising three complementarity-determining regions (CDRs including CDR1, CDR2, CDR3) and four framework regions (FRs including FR1, FR2, FR3, FR4), such as a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression), which is capable of binding to a specific antigen. The terms “nanobody” and “single-domain antibody” are used herein in its broadest form to include variants that may have various amino acid substitutions (e.g., conservative substitutions) and also functional “fragment” or “antigen binding fragment” of the sdAb as long as the fragment retains binding to the specific antigen.

[0081] As used herein, the term “anti-Ebola binder protein” refers to a protein having binding affinity for an Ebola viral antigen (e.g., Ebola glycoprotein). Such a protein may comprise or consist of a sdAb as described herein, or an antigen binding fragment thereof. In certain embodiments, an anti-Ebola binder protein comprises a sdAb as described herein. In certain embodiments, an anti-Ebola binder protein consists of a sdAb as described herein. In certain embodiments, an anti-Ebola binder protein comprises an antigen binding fragment of a sdAb as described herein. In certain embodiments, an anti-Ebola binder protein consists of an antigen binding fragment of a sdAb as described herein. In certain embodiments, an anti-Ebola binder protein comprises two sdAbs (same or different sdAbs) as described herein.

[0082] Accordingly, certain embodiments of the invention provide an anti-Ebola binder protein as described herein. In certain embodiments, the anti-Ebola binder protein comprises a sdAb, and optionally, one or more polypeptide tags, wherein the sdAb is operably linked to the one or more polypeptide tags (e.g., a monomeric sdAb with an optional His tag and / or HA tag). In certain embodiments, the anti-Ebola binder protein comprises two sdAb-Fc fusion polypeptides that are dimerized via the Fc domain. In certain embodiments, an anti-Ebola binder protein comprises or consists of one or more polypeptide as described in Example 1 or in Table 1 (e.g., Nanosota-EBl, Nanosota-EBl-Fc, Nanosota-EB2, and Nanosota-EB2-Fc).

[0083] In some embodiments, the binder protein (e.g., anti-Ebola sdAb, or sdAb-Fc fusion protein(s)) comprises: (1) one or more complementarity determining region (CDR) sequences as described herein; and / or (2) one or more heavy chain variable region (VHH) sequence as described herein. In some embodiments, the binder protein (e.g., sdAb-Fc fusion) is a monospecific binder protein. In some embodiments, the binder protein is a bispecific binder protein.

[0084] In some embodiments, the binder protein comprises one, two, three or more CDRs as described herein (e.g., Example 1, Table 1). In some embodiments, the binder protein comprises two, or three CDRs as described herein. In some embodiments, the binder protein comprises three CDRs (CDR1, CDR2, and CDR3) as described herein. In some embodiments, the binder protein is a bispecific binder protein that comprises six CDRs as described herein. Accordingly, certain embodiments provide a binder protein comprising:

[0085] (1) one or more complementarity determining regions (CDRs) selected from the group consisting of:

[0086] (a) a CDR1 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of GRTFSNDA (SEQ ID NO: 9);

[0087] (b) a CDR2 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of IDYNGGRT (SEQ ID NO: 10); and

[0088] (c) a CDR3 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO: 11); and / or

[0089] (2) one or more CDRs selected from the group consisting of:

[0090] (a) a CDR1 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of GSTSVIYA (SEQ ID NO:2);

[0091] (b) a CDR2 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of ITRGVGST (SEQ ID NO:3); and

[0092] (c) a CDR3 comprising an amino acid sequence having at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

[0093] In some embodiments, the binder protein comprises one, two, or three CDRs of a clone (e.g., EB2 or EB1) as described herein. In some embodiments, the binder protein comprises two, or three CDRs of a clone described herein. In some embodiments, the binder protein comprises three CDRs (CDR1, CDR2, and CDR3) of a clone described herein. In certain embodiments, the clone is Nanosota-EB2. In certain embodiments, the clone is Nanosota-EBl. In some embodiments, the binder protein comprises three CDRs of a first clone and three CDRs of a second clone.

[0094] In certain embodiments, an anti-Ebola binder protein (e.g., Nanosota-EB2 or Fc fusion) comprises one or more CDRs selected from the group consisting of:

[0095] (a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GRTFSNDA (SEQ ID NO:9);

[0096] (b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of IDYNGGRT (SEQ ID NO: 10); and

[0097] (c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO:11). In certain embodiments, the anti-Ebola binder protein comprises one or more CDRs selected from the group consisting of:

[0098] (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:9;

[0099] (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and

[0100] (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

[0101] In certain embodiments, an anti-Ebola binder protein (e.g., Nanosota-EBl or Fc fusion) comprises one or more CDRs selected from the group consisting of:

[0102] (a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GSTSVIYA (SEQ ID NO:2);

[0103] (b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of ITRGVGST (SEQ ID NO:3); and

[0104] (c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

[0105] In certain embodiments, the anti-Ebola binder protein comprises one or more CDRs selected from the group consisting of:

[0106] (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:2;

[0107] (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:3; and

[0108] (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:4.

[0109] In some embodiments, the binder protein comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of VHH sequences as described herein (e.g., as described in Table 1 below). In some embodiments, an anti-Ebola binder protein comprises a sequence (e.g., a variable domain of a heavy-chain only antibody (VHH)) derived from any of the following sdAbs described herein: Nanosota-EB2, and Nanosota-EBl.

[0110] In certain embodiments, an anti-Ebola binder protein described herein comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of:

[0111] QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQAPGKDREFAAGIDY NGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAARPWSIANLAYTYD SWGQGTQVTVSS (SEQ ID NO:8); and QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVAAITR GVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNARLLVAPPPYEYD YWGQGTQVTVSS (SEQ ID NO:1).

[0112] In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence of any one of SEQ ID NOs: 1 and 8. In some embodiments, an anti-Ebola binder protein consists of the amino acid sequence of any one of SEQ ID NOs: 1 and 8.

[0113] Clone EB2

[0114] In some embodiments, an anti-Ebola binder protein comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, an anti-Ebola binder protein comprises CDRs 1-3 consisting of the amino acid sequences of SEQ ID NOs:9, 10, and 11, respectively.

[0115] In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:8. In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence that 1) has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:8; and 2) comprises SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, an anti-Ebola binder protein consists of the amino acid sequence of SEQ ID NO:8.

[0116] In certain embodiments, an anti-Ebola binder protein (e.g., EB2) binds both GP2 and GP1 (e.g., at HR1, fusion loop, and N-terminus of GP2, and Q 1 / |32 strands of GP1). In certain embodiments, an anti-Ebola binder protein (e.g., EB2) does not bind sGP.

[0117] In certain embodiments, an anti-Ebola binder protein binds GP epitope(s) (e.g., at HR1, fusion loop, and N-terminus of GP2, and Q 1 / |32 strands of GP1) comprising eight or more (e.g., 9, 10, 11, or 12 residues) residues selected from the group consisting of the GP protein residues 34, 45, 505, 506, 507, 508, 528, 529, 563, 564, 567 and 568, wherein residue 563 isN-glycan linked Asn (N563 glycan). In certain embodiments, the GP epitope comprises or consists of GP protein residues 34, 45, 505, 506, 507, 508, 528, 529, 563, 564, 567 and 568, wherein residue 563 is N-glycan linked Asn (N563 glycan).

[0118] In certain embodiments, an anti-Ebola binder protein binds a GP1 epitope (e.g., Q 1 / Q2 strands of GP1) at the GP protein residues 34 and 45.

[0119] In certain embodiments, an anti-Ebola binder protein binds a GP2 epitope (e.g., at HR1, fusion loop, and N-terminus of GP2). In certain embodiments, an anti-Ebola binder protein binds a GP2 epitope comprising six or more (e.g., 7, 8, 9, or 10 residues) residues selected from the group consisting of the GP protein residues 505, 506, 507, 508, 528, 529, 563, 564, 567 and 568, wherein residue 563 is N-glycan linked Asn (N563 glycan). In certain embodiments, an anti-Ebola binder protein stabilizes the N563 glycan.

[0120] In certain embodiments, an anti-Ebola binder protein binds the GP2 HR1 at the GP protein residues 563, 564, 567, and 568, wherein residue 563 is N-glycan linked Asn (N563 glycan).

[0121] In certain embodiments, an anti-Ebola binder protein binds the GP2 N-terminus at the GP protein residues 505, 506, 507, and 508.

[0122] In certain embodiments, an anti-Ebola binder protein binds the GP2 fusion loop at the GP protein residues 528 and 529.

[0123] Clone EB1

[0124] In some embodiments, an anti-Ebola binder protein comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR2 comprising the amino acid sequence of SEQ ID NO:3, and a CDR3 comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, an anti-Ebola binder protein comprises CDRs 1-3 consisting of the amino acid sequences of SEQ ID NOs:2, 3, and 4, respectively.

[0125] In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence that has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1. In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence that 1) has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1; and 2) comprises SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, an anti-Ebola binder protein consists of the amino acid sequence of SEQ ID NO:1.

[0126] In certain embodiments, an anti-Ebola binder protein binds a GP1 epitope. In certain embodiments, an anti-Ebola binder protein binds the GP1 glycan cap.

[0127] In certain embodiments, an anti-Ebola binder protein binds a GP1 epitope comprising six or more (e.g., 7, 8, 9, 10, or 11 residues) residues selected from the group consisting of the GP protein residues 256, 259, 262, 263, 273, 274, 275, 276, 277, 278, and 281. In certain embodiments, the GP1 epitope comprises or consists of 11 residues that are the GP protein residues 256, 259, 262, 263, 273, 274, 275, 276, 277, 278, and 281.

[0128] In certain embodiments, an anti-Ebola binder protein described herein binds, and stabilizes the glycan cap of GP protein, and / or prevents glycan cap cleavage (e.g., by cathepsins). In certain embodiments, an anti-Ebola binder protein described herein binds P-17 strand. In certain embodiments, an anti-Ebola binder protein binds a GP1 P-17 strand epitope comprising four or more (e.g., 5, 6 or 7 residues) residues selected from the group consisting of the GP protein residues 273, 274, 275, 276, 277, 278, and 281. In certain embodiments, the P-17 strand epitope comprises five or more (e.g., 6 or 7 residues) residues selected from the group consisting of the GP protein residues 273, 274, 275, 276, 277, 278, and 281. In certain embodiments, the P-17 strand epitope comprises or consists of 7 residues that are the GP protein residues 273, 274, 275, 276, 277, 278, and 281.

[0129] In certain embodiments, an anti-Ebola binder protein (e.g., EB1 or EB2) binds epitope(s) or residues as described herein (e.g., in Example 1, see Table S2, Table S3, or in the Figures, such as Figure 3, Figure 5).

[0130] In certain embodiments, an isolated anti-Ebola binder protein described herein is an inhibitor of Ebola virus.

[0131] In certain embodiments, the Ebola virus (EBOV) is Zaire ebolavirus (see NCBI reference sequence accession number NC 002549.1), or related strain or variant. In certain embodiments, the Ebola virus is Zaire ebolavirus. In certain embodiments, the Ebola virus is Bundibugyo ebolavirus. In certain embodiments, the Ebola virus is Sudan ebolavirus.

[0132] In certain embodiments, the Ebola virus is Mayinga strain, Makona strain, Lturi strain, or Mbandaka strain (see Makona, NCBI accession number: AIE11800.1; lturi, NCBI accession number: AYN74184.1; Mbandaka, NCBI accession number: WWV92555.1).

[0133] The term “Ebola virus glycoprotein” refers to Ebola virus envelop glycoprotein, which is responsible for target binding and subsequent membrane fusion. Exemplary Ebola virus glycoprotein (GP) is described herein (e.g., in Examples 1 and 2), and known in the art, for example, NCBI accession number Q05320.1.

[0134] The term “secreted glycoprotein” or “sGP” as used herein refers to secreted form of Ebola virus glycoprotein, which includes most regions of GP1 but none of GP2. In certain embodiments, the secreted glycoprotein comprises or consists of residues 1-305 of Ebola virus glycoprotein.

[0135] The term “inhibitor of Ebola” as used herein refers to a binder protein that is capable of inhibiting the function of Ebola virus (e.g., inhibits the fusion of viral and endosomal membranes, and / or the entry or release of viral genomic materials into cells). For example, in certain embodiments, a binder protein as described herein detectably inhibits the biological activity of Ebola virus as measured, e.g., using an assay described herein. In certain embodiments, the binder protein inhibits the biological activity of Ebola virus by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0136] In certain embodiments, an isolated anti-Ebola binder protein described herein (e.g., EB1, EB2, or a bispecific binder described herein) inhibits protease cleavage (e.g., by cathepsins or thermolysin L) of Ebola GP protein glycan cap (e.g., by at least about 20%, 30%, 40%, 50%, 60% or more). In certain embodiments, a binder protein described herein (e.g., Nanosota-EBl, EB2, or a bispecific binder) stabilizes Ebola GP protein glycan cap. In certain embodiments, a binder protein described herein (e.g., a bispecific binder) inhibits protease cleavage of Ebola GP protein glycan cap for at least 15 minutes, 30 minutes, 60 minutes, or longer.

[0137] In certain embodiments, an isolated anti-Ebola binder protein described herein (e.g., Nanosota-EB2, or a bispecific binder described herein) stabilizes or locks GP protein or glycan cap cleaved GP (GPcl) in prefusion configuration, thus inhibiting fusion of viral / endosome membranes or the entry of viral genomic materials into cytoplasm. In certain embodiments, an isolated anti-Ebola binder protein (e.g., Nanosota-EB2, or a bispecific binder) increases the thermostability of GP protein, and / or glycan cap cleaved GP protein by at least 5°C, or more. In certain embodiments, the binder protein increases glycan cap cleaved GP protein melting temperature Tmby at least 5°C, 6°C, 7°C, 8°C, or more (e.g., in both neutral and acidic pH). In certain embodiments, the binder protein increases glycan cap cleaved GP protein Tmby at least 6°C, 7°C, 8°C, 9°C, 10°C, 11°C or more (e.g., in acidic conditions, such as pH4.5-pH6).

[0138] In certain embodiments, an isolated anti-Ebola binder protein described herein binds Ebola virus glycoprotein (GP), GP-AM, and / or GPcl with a dissociation constant Kd value (e.g., as determined by SPR) of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900nM. In certain embodiments, an isolated anti-Ebola binder protein described herein binds Ebola virus glycoprotein (GP), GP-AM, and / or GPcl with a dissociation constant Kd value of about 0.1 to 900nM, 0.3 to 800nM, 0.5 to 600nM, 1 to 500nM, 1 to 400nM, 1 to 300nM, 1 to lOOnM, 1 to 80nM, 2 to 60nM, 2 to 30nM, 1 to 20nM, 1 to lOnM, 1 to 6nM, or 1 to 3nM.

[0139] In certain embodiments, an isolated anti-Ebola binder protein described herein (e.g., Nanosota-EB2, or a bispecific binder described herein) is resistant to the sGP decoy diversion. For example, in certain embodiments, the binding affinity of a binder protein (e.g., EB2-Fc or bispecific binder) for GP protein is not reduced in the presence of sGP, and / or the neutralizing potency of a binder protein against Ebola virus is not reduced in the presence of sGP.

[0140] In certain embodiments, a binder protein described herein specifically binds to Ebola virus or its variant. In certain embodiments, the binder protein is capable of binding to a region that is conserved across two or more strains or variants.

[0141] In certain embodiments, a binder protein has a binding footprint on Ebola GP protein as described herein (e.g., binds one or more residues as described herein, also see Example 1, Table S2 and Table S3).

[0142] In certain embodiments, a binder protein described herein is capable of neutralizing (e.g., neutralize viral entry of genomic materials into cytoplasm) one or more Ebola virus strains or variants.

[0143] Tags

[0144] In certain embodiments, a binder protein described herein is operably linked to at least one detectable agent (e.g., a polypeptide tag such as His6 tag). In certain embodiments, an isolated anti-Ebola binder protein as described herein is operably linked to at least one detectable agent. The location of the detectable agent is not critical, provided that it does not interfere with the function of the binder protein. In certain embodiments, the detectable agent is operably linked to the N-terminus of the binder protein. In certain embodiments, the detectable agent is operably linked to the C-terminus of the binder protein.

[0145] In certain embodiments, a binder protein (e.g., Nanosota-EB2, or EB1) as described in Example 1 is operably linked to a detectable agent described herein.

[0146] In certain embodiments, the at least one detectable agent is a tag, such as an affinity tag or an epitope tag. For example, such a tag may be useful for detecting, isolating and / or purifying the binder protein. In certain embodiments, the tag is a polypeptide tag. Polypeptide tags are known in the art and include, but are not limited to, e.g., a His tag, Myc tag, HA tag or an Fc tag. In certain embodiments, the at least one detectable agent is an Fc tag (e.g., an IgGl, IgG2, IgG3, or IgG4 Fc). For example, as described below, a sdAb may be operably linked to an Fc domain amino acid sequence, to produce a sdAb-Fc fusion protein. In certain embodiments, the at least one detectable agent is a His tag (e.g., His6 tag). In certain embodiments, the at least one detectable agent is a HA tag. In certain embodiments, multiple tags may be operably linked in tandem either directly or via a linker group. In certain embodiments, the detectable agent(s) comprise a HA tag and / or a His tag (e.g., His6 tag). In certain embodiments, the binder protein is directly linked to the detectable agent, such as a polypeptide tag (e.g., through a peptide bond).

[0147] In certain other embodiments, the binder protein is linked to the detectable agent, such as a polypeptide tag, via one or more optional linker group(s). The nature of the linker group is not critical, provided that the linker group does not interfere with the function of the binder protein or the detectable agent. In certain embodiments, the linker group is an amino acid sequence (e.g., a sequence described herein). In certain embodiments, the linker group is an amino acid sequence that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length. In certain embodiments, the linker group is an amino acid sequence about 1 to about 25 amino acids in length, or about 1 to about 20 amino acids in length, or about 1 to about 15 amino acids in length, or about 1 to about 10 amino acids in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length). In certain embodiments, the linker group is glycine rich linker (e.g., having about more than 60% of the amino acid residues in the linker group is glycine). In certain embodiments, the linker group is glycine-serine linker (e.g., GS, GGS, or GGSGGS (SEQ ID NO:20)). In certain embodiments, the linker group is GGQ.

[0148] Accordingly, in certain embodiments, an anti-Ebola binder protein described herein operably linked to a polypeptide tag, comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of:

[0149] QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQAPGKDREFAAGIDYNGGR TAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAARPWSIANLAYTYDSWG QGTQVTVSSGGQHHHHHHGAYPYDVPDYAS (SEQ ID NO: 12); and QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVAAITRGVGS TNYADSVI< GRFTISRDNAI< NTMYLQMNSLI< PEDTAVYYCNARLLVAPPPYEYDYWGQ GTQVTVSSGGQHHHHHHGAYPYDVPDYAS (SEQ ID NO:5).

[0150] In some embodiments, an anti-Ebola binder protein comprises an amino acid sequence of any one of SEQ ID NOs:5, and 12. In some embodiments, an anti-Ebola binder protein consists of the amino acid sequence of any one of SEQ ID NOs:5, and 12.

[0151] In some embodiments, the binder protein is encoded by a polynucleotide comprising a nucleic acid sequence that has at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs:18 and 19.

[0152] In certain embodiments, a sdAb as described herein is a recombinant sdAb. In certain embodiments, a sdAb as described herein is a chimeric sdAb. In certain embodiments, a sdAb as described herein is humanized. In certain embodiments, a sdAb of the invention is a monoclonal sdAb.

[0153] Certain Monospecific or Bispecific Proteins of the Invention

[0154] As described herein, a binder protein may comprise a sdAb of the invention and may optionally be linked to one or more additional polypeptides. For example, in certain embodiments, an isolated binder protein described herein is further linked to one or more antibody domain sequences (e.g., heavy or light chain domain sequences, such as variable or constant domain sequences). Accordingly, certain embodiments provide a protein comprising a sdAb of the invention operably linked to one or more antibody domain sequences. Such protein molecules comprising a sdAb of the invention and one or more additional antibody domains may be referenced herein as an antibody or antibody fragment. Additionally, such molecules may be further modified as described herein (e.g., humanized or to alter its affinity, etc.). In certain embodiments, the one or more antibody domain sequences are derived from an antibody class or isotype as defined herein (e.g., IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl and IgA2), IgD, and IgE).

[0155] In certain embodiments, the sdAb is not linked to a light chain domain. In certain embodiments, the sdAb is not linked to a constant domain region. In certain embodiments, the sdAb is not linked to a CHI region.

[0156] In certain embodiments, an isolated anti-Ebola sdAb described herein is linked (e.g., through a linker or a direct bond, such as a peptide bond) to at least one heavy chain constant region (e.g., 1, 2, or 3). In certain embodiments, the sdAb is linked to two heavy chain constant regions (e.g., a CH2 and CH3 region). In certain embodiments, the sdAb is operably linked to an Fc domain amino acid sequence (e.g., an IgG Fc domain such as IgGl, IgG2, IgG3, or IgG4 Fc domain), to produce a sdAb-Fc fusion protein.

[0157] Thus, certain embodiments of the invention provide a sdAb-Fc-fusion protein comprising a sdAb of the invention operably linked to a Fc domain amino acid sequence. In certain embodiments, the sdAb and Fc domain amino acid sequence are directly linked, e.g., through a peptide bond. In certain embodiments, the sdAb and Fc domain amino acid sequence are linked through an amino acid linking group. In certain embodiments, the Fc domain amino acid sequence is a human IgGl Fc domain amino acid sequence. In certain embodiments, the Fc domain amino acid sequence has at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 15. In certain embodiments, the Fc domain amino acid sequence comprises SEQ ID NO: 15. In certain embodiments, the Fc domain amino acid sequence consists of SEQ ID NO: 15.

[0158] In certain embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of:

[0159] (a) Q VQLQESGGGLVQ AGGSLRLSC VHSGRTF SNDAMAWFRQAPGKDREF AA GIDYNGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAR PWSIANLAYTYDSWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 13);

[0160] (b) Q VQLQESGGGLVQ AGGSLRLSC VHSGRTF SNDAMAWFRQ APGKDREF AA GIDYNGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAR PWSIANLAYTYDSWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 14);

[0161] (c) QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVA AITRGVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNAR LLVAPPPYEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 6); and

[0162] (d) QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVA AITRGVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNAR LLVAPPPYEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 7).

[0163] In some embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence of any one of SEQ ID NOs:6, 7, 13, and 14. In some embodiments, the sdAb-Fc fusion protein consists of an amino acid sequence of any one of SEQ ID NOs:6, 7, 13, and 14.

[0164] Certain embodiments of the invention also provide multivalent sdAbs (e.g., bivalent, trivalent, tetravalent, pentavalent or higher valence multivalent sdAbs). Thus, certain embodiments of the invention provide a binder protein comprising two or more independently selected sdAbs as described herein, wherein the sdAbs are operably linked to each other (e.g., to form a dimer, trimer, tetramer, pentamer or higher valence multimer sdAb). In certain embodiments, a multivalent sdAb or binding protein as described herein is a homo-multimer (e.g., dimer, trimer, tetramer or pentamer). In certain embodiments, a multivalent sdAb or binder protein as described herein is a hetero-multimer (e.g., dimer, trimer, tetramer or pentamer).

[0165] In certain embodiments, the two or more sdAbs are operably linked via a linker group (e.g., a peptide linker group), disulfide bond(s) and / or by non-covalent interactions. In certain embodiments, the two or more sdAbs are operably linked via oligomerization of tag polypeptides (e.g., multimerization tags, such as a dimerization tags, trimerization tags, tetramerization tags, etc.).

[0166] In certain embodiments, the two or more sdAbs are operably linked via a linker group. The nature of the linker group is not critical, provided that the linker group does not interfere with the function of the sdAbs. In certain embodiments, the linker group is a peptide linker group. In certain embodiments, the peptide linker is a glycine-serine rich linker.

[0167] In certain embodiments, two independently selected sdAbs are linked via a linker group (e.g., a peptide linker group) to form dimeric sdAb. In certain embodiments, three independently selected sdAbs are linked via two linker groups (e.g., two peptide linker groups) to form a trimeric sdAb. In certain embodiments, four independently selected sdAbs are linked via three linker groups (e.g., three peptide linker groups) to form a tetrameric sdAb. In certain embodiments, five independently selected sdAbs are linked via four linker groups (e.g., four peptide linker groups) to form a pentameric sdAb.

[0168] In certain embodiments, the two or more sdAbs are operably linked via oligomerization of tag polypeptides. For example, a sdAb as described herein may be operably linked to a tag polypeptide to form a sdAb-tag fusion protein, wherein the tag polypeptide is capable of oligomerizing. Accordingly, two or more sdAb-tag fusion proteins may be operably linked to form a dimer, trimer, tetramer, pentamer or a higher valence multimer via polypeptide tag-mediated oligomerization.

[0169] In certain embodiments, the sdAb and tag polypeptide are linked through a peptide linker to form the sdAb-tag fusion protein. In certain embodiments, a sdAb and tag polypeptide are directly linked without an intervening peptide linker to form the sdAb-tag fusion protein.

[0170] In certain embodiments, the tag polypeptide is a human Fc sequence, a human collagen XVIII trimerization domain or a coiled-coil peptide derived from human cartilage oligomeric matrix protein COMP48, which is capable of forming a multimer, such as a pentamer.

[0171] In certain embodiments, the tag polypeptide is a human Fc sequence. Accordingly, certain embodiments of the invention provide a binding protein comprising: two independently selected sdAb-Fc fusion proteins as described herein, wherein the two Fc polypeptides are linked to form a dimer (e.g., homodimer or heterodimer linked by a covalent bond, such as a disulfide bond, or by non-covalent interactions such as electrostatic interactions, hydrogen bonding, etc.).

[0172] In certain embodiments, the binding protein is a monospecific binder.

[0173] In certain embodiments, the two sdAb-Fc fusion proteins are the same. In certain embodiments, sdAb-Fc fusion proteins as described herein can form homo-dimers. In certain embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 13. In certain embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence of SEQ ID NO: 13. In certain embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:6. In certain embodiments, the sdAb-Fc fusion protein comprises an amino acid sequence of SEQ ID NO:6.

[0174] In certain embodiments, the binding protein is a bispecific binder.

[0175] In certain embodiments, the two sdAb-Fc fusion proteins are different. In certain embodiments, sdAb-Fc fusion proteins as described herein can form hetero-dimers. In certain embodiments, sdAb-Fc fusion proteins as described herein can form bispecific hetero-dimers having binding affinities for different Ebola virus epitopes.

[0176] In certain embodiments, a bispecific binder protein comprises a first sdAb domain (e.g., EB2 VHH sequence) and a second sdAb domain (e.g., EB1 VHH sequence). In certain embodiments, a bispecific binder comprises a first sdAb domain that binds cleaved GP protein (GPcl) and a second sdAb domain that binds sGP. In certain embodiments,

[0177] the first sdAb domain comprises

[0178] (a) a CDR1 comprising an amino acid sequence of SEQ ID NO:9;

[0179] (b) a CDR2 comprising an amino acid sequence of SEQ ID NO: 10; and (c) a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; and the second sdAb domain comprises

[0180] (a) a CDR1 comprising an amino acid sequence of SEQ ID NO:2;

[0181] (b) a CDR2 comprising an amino acid sequence of SEQ ID NO:3; and (c) a CDR3 comprising an amino acid sequence of SEQ ID NO:4.

[0182] In certain embodiments, a bispecific binder protein comprises: a first sdAb domain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to:

[0183] QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQAPGKDREFAAGIDYNGGR TAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAARPWSIANLAYTYDSWG QGTQVTVSS (SEQ ID NO: 8); and

[0184] a second sdAb domain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to:

[0185] QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVAAITRGVGS TNYADSVI< GRFTISRDNAI< NTMYLQMNSLI< PEDTAVYYCNARLLVAPPPYEYDYWGQ GTQVTVSS (SEQ ID NO:1).

[0186] In certain embodiments, a bispecific binder comprises Fc domain amino acid sequences comprising mutation(s) that promote heterodimerization. Technologies, such as “Knobs into Holes”, for making bispecific binder proteins are known in the art and described herein. For example, a bispecific binder protein described herein may comprise a first sdAb-Fc fusion protein comprising a mutation (e.g., Y407T) in the Fc tag, and a second sdAb-Fc fusion protein comprising another mutation (e.g., T366Y) in the Fc tag.

[0187] In certain embodiments, a bispecific binder protein comprises:

[0188] a first sdAb-Fc fusion protein comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to:

[0189] QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQAPGKDREFAAGIDYNGGR TAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAARPWSIANLAYTYDSWG QGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 14); and

[0190] a second sdAb-Fc fusion protein comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to:

[0191] QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVAAITRGVGS TNYADSVI< GRFTISRDNAI< NTMYLQMNSLI< PEDTAVYYCNARLLVAPPPYEYDYWGQ GTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO:7).

[0192] In certain other embodiments, a single sdAb of the invention is operably linked to an Fc dimer.

[0193] In certain embodiments, a protein molecule as described herein is further operably linked to a detectable agent (e.g., a detectable agent described herein).

[0194] In certain embodiments, a binder protein (e.g., sdAb, or sdAb-Fc) has good thermostability at room temperature or above. For example, in certain embodiments, the target antigen binding ability (e.g., as determined by SPR or ELISA) of the binder protein is maintained at about at least 75%, 80%, 85%, 90%, 95%, or 100% binding ability after 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days (e.g., 7 days), 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks storage at about 20-37°C (e.g., 20°C, 25°C, 30°C, 35°C, or 37 °C), as compared to that of a control binder protein (e.g., same binder protein after storage at -80°C or -20°C).

[0195] In certain embodiments, a binder protein described herein administered to a subject reduces viral load (e.g., viral genome copies number) in the subject by at least 103to 104-fold (e.g., 3 x 103to 104-fold, or by 103-fold, by 104-fold), as compared to that of a control (e.g., vehicle control, or no treatment).

[0196] In certain embodiments, a binder protein described herein administered to a subject could delay the onset of post-infection weight loss by at least 1, 2, 3 days, or more. In certain embodiments, a binder protein described herein administered to a subject could maintain the body weight of the subject, wherein weight loss (e.g., at day 6, day 7, day 8 or day 9 postinfection) is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (e.g., in certain embodiments, the subject experiences no post-infection weight loss).

[0197] Certain embodiments of the invention also provide a protein molecule comprising a sdAb as described herein. The terms “protein”, “protein molecule”, and "polypeptide" are used interchangeably herein. In certain embodiments, the term “protein” or “protein molecule” may refer to a single polypeptide or may refer to two or more polypeptides, wherein the two or more polypeptides may be linked by a covalent (e.g., disulfide bridge) or non-covalent interactions.

[0198] As used herein, the term “antibody” includes a single-chain variable fragment (scFv), a dimer of a sdAb, a sdAb-Fc fusion protein or dimer thereof, humanized, fully human or chimeric antibodies, single-chain antibodies, diabodies, and antigen-binding fragments of antibodies that do not contain the Fc region (e.g., Fab fragments). In certain embodiments, the antibody is a camelid antibody, human antibody or a humanized antibody. A “humanized” antibody contains only the three CDRs (complementarity determining regions) and sometimes a few carefully selected “framework” residues (the non-CDR portions of the variable regions) from each donor antibody variable region recombinantly linked onto the corresponding frameworks and constant regions of a human antibody sequence. A “fully humanized antibody” is created in a hybridoma from mice genetically engineered to have only human-derived antibody genes or by selection from a phage-display library of human-derived antibody genes.

[0199] As used herein, the term "monoclonal sdAb" or “monoclonal antibody” refers to a sdAb / antibody obtained from a group of substantially homogeneous sdAbs / antibodies, that is, a sdAb / antibody group wherein the sdAbs / antibodies constituting the group are homogeneous except for naturally occurring mutants that exist in a small amount. Monoclonal sdAbs / antibodies are highly specific and interact with a single antigenic site. Furthermore, each monoclonal sdAb / antibody targets a single antigenic determinant (epitope) on an antigen, as compared to common polyclonal sdAb / antibody preparations that typically contain various sdAbs / antibodies against diverse antigenic determinants. In addition to their specificity, monoclonal sdAbs / antibodies are advantageous in that they are typically produced from hybridoma cultures not contaminated with other immunoglobulins.

[0200] The adjective "monoclonal" indicates a characteristic of antibodies and sdAbs obtained from a substantially homogeneous group of antibodies / sdAbs, and does not specify antibodies / sdAbs produced by a particular method. For example, a monoclonal sdAb to be used in the present invention can be produced by, for example, hybridoma methods (Kohler and Milstein, Nature 256:495, 1975) or recombination methods (U. S. Pat. No. 4,816,567). The monoclonal sdAbs used in the present invention can be also isolated from a phage sdAb library (Clackson et al., Nature 352:624-628, 1991; Marks et al., J. Mol. Biol. 222:581-597, 1991). The monoclonal sdAbs of the present invention may be linked to other antibody domain sequences. Therefore, the resulting polypeptides / protein molecules may be "chimeric" immunoglobulins, wherein a part of the polypeptide is derived from a specific species or a specific antibody class or subclass, and the remaining portion is derived from another species, or another antibody class or subclass. Furthermore, mutant sdAbs, as well as mutant polypeptides / protein molecules comprising a sdAb of the invention, are also comprised in the present invention (U. S. Pat. No.

[0201] 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984).

[0202] As used herein, the term “mutant sdAb” or "mutant antibody" refers to a sdAb / antibody comprising a variant amino acid sequence in which one or more amino acid residues have been altered. For example, the variable region of a sdAb / antibody can be modified to improve its biological properties, such as antigen binding. Such modifications can be achieved by site-directed mutagenesis (see Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)), PCR-based mutagenesis, cassette mutagenesis, and the like. Such mutants comprise an amino acid sequence which is at least 70% identical to the amino acid sequence of the heavy chain variable region of the sdAb, more specifically at least 75%, even more specifically at least 80%, still more specifically at least 85%, yet more specifically at least 90%, and most specifically at least 95% identical. Such mutants also comprise an amino acid sequence which is at least 70% identical to the amino acid sequence of a heavy or light chain variable region of the antibody, more specifically at least 75%, even more specifically at least 80%, still more specifically at least 85%, yet more specifically at least 90%, and most specifically at least 95% identical. As used herein, the term “sequence identity” is defined as the percentage of residues identical to those in the sdAb’s / antibody's original amino acid sequence, determined after the sequences are aligned and gaps are appropriately introduced to maximize the sequence identity as necessary.

[0203] Specifically, the identity of one nucleotide sequence or amino acid sequence to another can be determined using the algorithm BLAST, by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Programs such as BLASTN and BLASTX were developed based on this algorithm (Altschul et al., J. Mol. Biol. 215: 403-410, 1990). To analyze nucleotide sequences according to BLASTN based on BLAST, the parameters are set, for example, as score=100 and wordlength=12. On the other hand, parameters used for the analysis of amino acid sequences by BLASTX based on BLAST include, for example, score=50 and wordlength=3. Default parameters for each program are used when using the BLAST and Gapped BLAST programs. Specific techniques for such analyses are known in the art (see the website of the National Center for Biotechnology Information (NCBI), Basic Local Alignment Search Tool (BLAST); www.ncbi.nlm.nih.gov).

[0204] Polyclonal and monoclonal sdAbs / antibodies can be prepared by methods known to those skilled in the art.

[0205] In another embodiment, antibodies or antibody fragments (e.g., sdAbs) can be isolated from an antibody / sdAb phage library, produced by using the technique reported by McCafferty et al. (Nature 348:552-554 (1990)). Clackson et al. (Nature 352:624-628 (1991)), Marks et al. (J. Mol. Biol. 222:581-597 (1991)) and Muyldermans et al. (Annual Review of Biochemistry Volume 82, pp 775-797(2013)) reported on the respective isolation of mouse, camelid and human antibodies from phage libraries. There are also reports that describe the production of high affinity (nM range) human antibodies based on chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), and combinatorial infection and in vivo recombination, which are methods for constructing large-scale phage libraries (Waterhouse et al., Nucleic Acids Res. 21:2265-2266 (1993)). These technologies can also be used to isolate monoclonal sdAbs / antibodies, instead of using conventional hybridoma technology for monoclonal sdAb / antibody production.

[0206] SdAbs / antibodies to be used in the present invention can be purified by a method appropriately selected from known methods, such as the protein A-Sepharose method, hydroxyapatite chromatography, salting-out method with sulfate, ion exchange chromatography, and affinity chromatography, or by the combined use of the same.

[0207] The present invention may use recombinant sdAbs / antibodies, produced by gene engineering. The genes encoding the sdAbs / antibodies obtained by a method described above are isolated from B cells or hybridomas. The genes are inserted into an appropriate vector, and then introduced into a host (see, e.g., Carl, A. K. Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies, Published in the United Kingdom by Macmillan Publishers Ltd, 1990). The present invention provides the nucleic acids encoding the sdAbs / antibodies of the present invention, and vectors comprising these nucleic acids. Specifically, using a reverse transcriptase, cDNAs encoding the variable region(s) (V region) of the sdAbs / antibodies are synthesized from the mRNAs of B cells or hybridomas. After obtaining the DNAs encoding the variable region(s) of interest, they are optionally ligated with DNAs encoding desired constant regions (C regions), and the resulting DNA constructs are inserted into expression vectors. Alternatively, the DNAs encoding the variable region(s) may be inserted into expression vectors comprising the DNAs of the C regions. These are inserted into expression vectors so that the genes are expressed under the regulation of an expression regulatory region, for example, an enhancer and promoter. Then, host cells are transformed with the expression vectors to express the sdAbs / antibodies. The present invention provides cells expressing sdAbs / antibodies of the present invention. The cells expressing sdAbs / antibodies of the present invention include cells and hybridomas transformed with a gene of such a sdAb / antibody.

[0208] The sdAbs / antibodies of the present invention also include sdAbs / antibodies which comprise complementarity-determining regions (CDRs), or regions functionally equivalent to CDRs. The term "functionally equivalent" refers to comprising amino acid sequences similar to the amino acid sequences of CDRs of any of the monoclonal sdAbs isolated in the Examples. The term " CDR" refers to a region in a sdAb / antibody variable region (also called " V region"), and determines the specificity of antigen binding. The H chain and L chain (if present) each have three CDRs, designated from the N terminus as CDR1, CDR2, and CDR3. There are four regions flanking these CDRs: these regions are referred to as "framework," and their amino acid sequences are highly conserved. The CDRs can be transplanted into other sdAbs / antibodies, and thus a recombinant antibody can be prepared by combining CDRs with the framework of a desired sdAb / antibody. One or more amino acids of a CDR can be modified without losing the ability to bind to its antigen. For example, one or more amino acids in a CDR can be substituted, deleted, and / or added.

[0209] In certain embodiments, an amino acid residue is mutated into one that allows the properties of the amino acid side-chain to be conserved. Examples of the properties of amino acid side chains comprise: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W). The letters within parenthesis indicate the one-letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues is deleted, added, and / or substituted can retain the original biological activity (Mark D. F. et al., Proc. Natl. Acad. Sci. U. S. A. 81:5662-5666 (1984); Zoller M. J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. U. S. A. 79: 6409-6413 (1982)). The number of mutated amino acids is not limited, but in general, the number falls within 40% of amino acids of each CDR, and specifically within 35%, and still more specifically within 30% (e.g., within 25%). The identity of amino acid sequences can be determined as described herein.

[0210] In the present invention, recombinant sdAbs / antibodies artificially modified to reduce heterologous antigenicity against humans can be used. Examples include chimeric sdAbs / antibodies and humanized sdAbs / antibodies. These modified sdAbs / antibodies can be produced using known methods. A chimeric antibody includes an antibody comprising variable and constant regions of species that are different to each other, for example, an antibody comprising the antibody heavy chain and light chain variable regions of a nonhuman mammal such as a mouse, and the antibody heavy chain and light chain constant regions of a human. Additionally, a chimeric antibody or polypeptide may be produced by combining a sdAb of the invention with constant regions that are of different species to each other. Such an antibody (e.g., a cam elid-human chimeric antibody) can be obtained by (1) ligating a DNA from the different regions; (2) incorporating this into an expression vector; and (3) introducing the vector into a host for production of the antibody.

[0211] A humanized sdAb / antibody, which is also called a reshaped human sdAb / antibody, may be obtained by replacing a CDR of a human antibody with an H or L chain CDR of a sdAb / antibody of a nonhuman mammal such as a mouse or camelid. Conventional genetic recombination techniques for the preparation of such antibodies are known (see, for example, Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta Curr. Op. Struct. Biol. 2: 593-596 (1992)). Specifically, a DNA sequence designed to ligate a CDR of a mouse / camelid antibody with the framework regions (FRs) of a human antibody is synthesized by PCR, using several oligonucleotides constructed to comprise overlapping portions at their ends. A humanized antibody can be obtained by (1) ligating the resulting DNA to a DNA that encodes a human antibody constant region; (2) incorporating this into an expression vector; and (3) transfecting the vector into a host to produce the antibody (see, European Patent Application No. EP 239,400, and International Patent Application No. WO 96 / 02576). Human antibody FRs that are ligated via the CDR are selected where the CDR forms a favorable antigen-binding site. The humanized antibody may comprise additional amino acid residue(s) that are not included in the CDRs introduced into the recipient antibody, nor in the framework sequences. Such amino acid residues are usually introduced to more accurately optimize the antibody's ability to recognize and bind to an antigen. For example, as necessary, amino acids in the framework region of a variable region may be substituted such that the CDR of a reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

[0212] Isotypes of sdAb-fusion proteins or antibodies comprising a sdAb of the present invention, or antibody fragments thereof, are not limited. The isotypes include, for example, IgG (IgGl, IgG2, IgG3, and IgG4), IgM, IgA (IgAl and IgA2), IgD, and IgE.

[0213] As described herein, a sdAb of the present invention may be operably linked to one or more antibody domain sequences (e.g., a sdAb-Fc fusion protein). Therefore, such polypeptides / protein molecules comprising a sdAb of the invention and one or more additional antibody domains may be referenced herein as an antibody or antibody fragment. The term "antibody fragment" refers to a portion of a full-length antibody, and generally to a fragment comprising an antigen-binding domain or a variable region. Such antibody fragments include, for example, single domain antibody (sdAb), Fab, F(ab')2, Fv, single-chain Fv (scFv) which comprises a heavy chain Fv and a light chain Fv coupled together with an appropriate linker, diabody (diabodies), linear antibodies, and multispecific antibodies prepared from antibody fragments. Previously, antibody fragments were produced by digesting natural antibodies with a protease; currently, methods for expressing them as recombinant antibodies using genetic engineering techniques are also known (see Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); Brennan et al., Science 229:81 (1985); Co, M. S. et al., J. Immunol., 1994, 152, 2968-2976; Better, M. & Horwitz, A. H., Methods in Enzymology, 1989, 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A., Methods in Enzymology, 1989, 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology, 1989, 121, 663-669; Bird, R. E. et al., TIBTECH, 1991, 9, 132-137).

[0214] The sdAbs / antibodies can be purified to homogeneity. The sdAbs / antibodies can be isolated and purified by a method routinely used to isolate and purify proteins. The sdAbs / antibodies can be isolated and purified by the combined use of one or more methods appropriately selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectro-focusing, for example (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography. These chromatographic methods can be practiced using liquid phase chromatography, such as HPLC and FPLC. Columns to be used in affinity chromatography include protein A columns and protein G columns. For example, protein A columns include Hyper D, POROS, and Sepharose F. F. (Pharmacia).

[0215] SdAbs / antibodies can also be purified by utilizing antigen binding, using carriers on which antigens have been immobilized.

[0216] The sdAbs / antibodies of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U. S. A), and may comprise pharmaceutically acceptable carriers and / or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the sdAbs / antibodies of the invention, and pharmaceutically acceptable carriers and / or additives. Exemplary carriers include surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, and corrigents. However, the carriers that may be employed in the present invention are not limited to this list. In fact, other commonly used carriers can be appropriately employed: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, and so on. The composition may also comprise other low-molecular-weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it can comprise an isotonic solution comprising, for example, physiological saline, dextrose, and other adjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which can also contain an appropriate solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol and PEG), and non-ionic detergent (polysorbate 80 and HCO-50).

[0217] If necessary, sdAbs / antibodies of the present invention may be encapsulated in microcapsules (microcapsules made of hydroxy cellulose, gelatin, polymethylmethacrylate, and the like), and made into components (encapsulated or as a surface functionalization moiety) of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nano-capsules) (for example, see " Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Moreover, methods for making sustained-release drugs are known, and these can be applied for the sdAbs / antibodies of the present invention (Langer et al., J.

[0218] Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech. 12: 98-105 (1982); U. S. Pat. No.

[0219] 3,773,919; EP Patent Application No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP: 133,988).

[0220] Nucleic Acids, Expression Cassettes, Vectors and Cells

[0221] Certain embodiments of the invention provide an isolated nucleic acid encoding a binder protein as described herein (e.g., a sdAb, or sdAb-Fc), or a pair of isolated polynucleotides that comprise:

[0222] a first isolated polynucleotide comprising a nucleotide sequence encoding the first sdAb domain as described herein, or the first sdAb-Fc fusion protein as described herein; and

[0223] a second isolated polynucleotide comprising a nucleotide sequence encoding the second sdAb domain as described herein, or the second sdAb-Fc fusion protein as described herein.

[0224] Certain embodiments of the invention provide an isolated nucleic acid comprising a sequence that has at least about 80% e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 18 or 19. In certain embodiments, the isolated nucleic acid comprises or consists of SEQ ID NO: 18 or 19. In certain embodiments, the isolated nucleic acid comprises or consists of SEQ ID NO: 18 or 19. In certain embodiments, the isolated nucleic acid comprises of consists of SEQ ID NO: 18 or 19.

[0225] In certain embodiments, the nucleic acid further comprises a promoter.

[0226] Certain embodiments of the invention provide an expression cassette comprising a nucleic acid as described herein and a promoter.

[0227] Certain embodiments of the invention provide a vector (e.g., a phagemid, Adeno-associated viruses (AAV)) comprising a nucleic acid or an expression cassette as described herein. Certain embodiments of the invention provide a pair of vectors as described herein, that comprise a first vector comprising the first isolated polynucleotide as described herein and a second vector comprising the second isolated polynucleotide as described herein.

[0228] Certain embodiments of the invention provide a cell comprising a nucleic acid, expression cassette or vector as described herein. In certain embodiments, the cell is a bacterial cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human mammalian cell. In certain embodiments, the cell is a human embryonic kidney (HEK) 293 cell. In certain embodiments, the cell is a 293F cell. In certain embodiments, the cell is a 293T cell. In certain embodiments, the cell is a human embryonic retinal (PER. C6) cell. In certain embodiments, the cell is a HT-1080 cell. In certain embodiments, the cell is a Huh-7 cell. In certain embodiments, the cell is a non-human mammalian cell. In certain embodiments, the cell is a Monkey kidney epithelial (Vero) cell. In certain embodiments, the cell is a Chinese Hamster Ovary (CHO) cell. In certain embodiments, the cell is a baby hamster kidney (BHK) cell. In certain embodiments, the cell is a non-mammalian cell. In certain embodiments, the cell is an insect cell. In certain embodiments, the cell is a yeast cell.

[0229] Certain embodiments of the invention provide a phage particle comprising a vector as described herein.

[0230] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991); Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell. Probes, 8:91 (1994). A "nucleic acid fragment" is a fraction of a given nucleic acid molecule.

[0231] Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0232] By “portion” or “fragment,” as it relates to a nucleic acid molecule, sequence or segment of the invention, when it is linked to other sequences for expression, is meant a sequence having at least 80 nucleotides, more specifically at least 150 nucleotides, and still more specifically at least 400 nucleotides. If not employed for expressing, a “portion” or “fragment” means at least 9, specifically 12, more specifically 15, even more specifically at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention.

[0233] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid ( / .<., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, culture medium may represent less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By "fragment" or "portion" is meant a full length or less than full length of the nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein.

[0234] " Naturally occurring" is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.

[0235] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g, 81%-84%, at least 85%, e.g, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0236] “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0237] “Recombinant DNA molecule” is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (3rdedition, 2001).

[0238] The terms "heterologous DNA sequence," "exogenous DNA segment" or "heterologous nucleic acid," each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.

[0239] A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0240] " Wild-type" refers to the normal gene, or organism found in nature without any known mutation.

[0241] “Genome” refers to the complete genetic material of an organism.

[0242] A “vector" is defined to include, inter alia, any plasmid, cosmid, viral vector, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0243] " Cloning vectors" typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.

[0244] " Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0245] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0246] The term " RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. " Messenger RNA" (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.

[0247] " Regulatory sequences" and "suitable regulatory sequences" each refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. As is noted above, the term "suitable regulatory sequences" is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive promoters, tissue-specific promoters, development-specific promoters, inducible promoters and viral promoters.

[0248] "5' non-coding sequence" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., Mol. Biotech., 3:225 (1995).

[0249] "3' non-coding sequence" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0250] The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.

[0251] The term "mature" protein refers to a post-translationally processed polypeptide without its signal peptide. " Precursor" protein refers to the primary product of translation of an mRNA. " Signal peptide" refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide.

[0252] " Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. " Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. " Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0253] The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e. further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0254] Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and / or an initiator.

[0255] " Constitutive expression" refers to expression using a constitutive or regulated promoter. " Conditional" and "regulated expression" refer to expression controlled by a regulated promoter.

[0256] As used herein, the term "operably linked" refers to a linkage of two elements in a functional relationship. For example, “operably linked” may refer to a linkage of polynucleotide elements or polypeptide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence ( / .<., that the coding sequence or functional RNA is under the transcriptional control of the promoter).

[0257] Coding sequences can be operably -linked to regulatory sequences in sense or antisense orientation. “Operably-linked” also refers to the association two chemical moieties so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function.

[0258] " Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0259] " Transcription stop fragment" refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of terminating transcription. Examples of transcription stop fragments are known to the art. " Translation stop fragment" refers to nucleotide sequences that contain one or more regulatory signals, such as one or more termination codons in all three frames, capable of terminating translation. Insertion of a translation stop fragment adjacent to or near the initiation codon at the 5' end of the coding sequence will result in no translation or improper translation. Excision of the translation stop fragment by site-specific recombination will leave a site-specific sequence in the coding sequence that does not interfere with proper translation using the initiation codon.

[0260] The terms "c / .s-acting sequence" and "c / .s-acting element" refer to DNA or RNA sequences whose functions require them to be on the same molecule.

[0261] The terms " / ra / r.s-acting sequence" and " / ra / r.s-acting element" refer to DNA or RNA sequences whose function does not require them to be on the same molecule.

[0262] The following terms are used to describe the sequence relationships between two or more sequences (e.g., nucleic acids, polynucleotides or polypeptides): (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity," and (e) "substantial identity."

[0263] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA, gene sequence or peptide sequence, or the complete cDNA, gene sequence or peptide sequence.

[0264] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a sequence, wherein the sequence in the comparison window may comprise additions or deletions ( / .<., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0265] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, CAB IOS, 4:11 (1988); the local homology algorithm of Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, JMB, 48:443 (1970); the search-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988); the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).

[0266] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al., Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151 (1989); Corpet et al., Nucl. Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155 (1992); and Pearson et al., Meth. Mol. Biol., 24:307 (1994). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., JMB, 215:403 (1990); Nucl. Acids Res., 25:3389 (1990), are based on the algorithm of Karlin and Altschul supra.

[0267] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (available on the world wide web at ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

[0268] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more specifically less than about 0.01, and most specifically less than about 0.001.

[0269] To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389 (1997).

[0270] Alternatively, PSLBLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSLBLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See the world wide web at ncbi.nlm.nih.gov. Alignment may also be performed manually by visual inspection.

[0271] For purposes of the present invention, comparison of sequences for determination of percent sequence identity to another sequence may be made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.

[0272] (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

[0273] Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art.

[0274] Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0275] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions ( / .<., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0276] (e)(i) The term "substantial identity" of sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, and at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, at least 95%.

[0277] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1°C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. (e)(ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

[0278] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0279] As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0280] " Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of

[0281] post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.

[0282] By "variant" polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C -terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0283] Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Kunkel et al., Meth. Enzymol., 154:367 (1987); U. S. Patent No. 4,873,192; Walker and Gaastra, Techniques in Mol. Biol. (MacMillan Publishing Co. (1983), and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found. 1978). Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.

[0284] Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the polypeptides of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired activity. The deletions, insertions, and substitutions of the polypeptide sequence encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.

[0285] Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic:

[0286] Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations."

[0287] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0288] " Transformed," "transgenic," and "recombinant" refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook and Russell, supra. See also Innis et al., PCR Protocols, Academic Press (1995); and Gelfand, PCR Strategies, Academic Press (1995); and Innis and Gelfand, PCR Methods Manual, Academic Press (1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. For example, "transformed," "transformant," and "transgenic" cells have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0289] Compositions and Kits

[0290] Certain embodiments provide a composition comprising an anti-Ebola binder protein as described herein, or a vector, or a cocktail mixture thereof, as described herein and a carrier. In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In certain embodiments, the composition comprises a bispecific anti-Ebola binder protein.

[0291] In certain embodiments, the composition is a cocktail mixture comprising two anti-Ebola binder proteins that each bind to non-overlapping regions (e.g., different epitopes) on the Ebola glycoprotein. In certain embodiments, the composition is a cocktail mixture comprising two anti-Ebola binder proteins (e.g., sdAbs or sdAb-Fc) as described herein.

[0292] In certain embodiments, the composition comprises two Ebola binder protein selected from the group consisting of

[0293] (a) an isolated anti-Ebola binder protein comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1, 5 or 6; and (b) an isolated anti-Ebola binder protein comprising an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:8, 12 or 13. In certain embodiments, the composition comprises two Ebola binder protein selected from the group consisting of:

[0294] (a) an isolated anti-Ebola binder protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13; and

[0295] (b) an isolated anti-Ebola binder protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:6.

[0296] In certain embodiments, the composition comprises two or more binder proteins that are: (a) Nanosota-EB2, or Nanosota-EB2-Fc as described herein; and

[0297] (b) Nanosota-EBl, or Nanosota-EBl-Fc as described herein.

[0298] In certain embodiments, the composition is a liquid composition. In certain embodiments, the composition is a solid composition (e.g., powder or lyophilized formulation). In certain embodiments, the composition is a lyophilized composition that further comprises one or more excipients selected from the group consisting of a cryo-lyoprotectant (e.g., trehalose, sucrose) and a bulking agent (e.g., mannitol, glycine). In certain embodiments, the solid composition may be reconstituted (e.g., with water, saline or Dextrose solution) prior to use.

[0299] Certain embodiments also provide a kit comprising an isolated anti-Ebola binder protein as described herein, or a vector, or a cocktail mixture thereof, as described herein, packaging material, and instructions for administering the binder protein / vector, to a mammal to treat an Ebola infection. In certain embodiments, the kit further comprises at least one additional therapeutic agent. In certain embodiments, the at least one additional therapeutic agent is useful for preventing or treating a viral infection or inflammation. In certain embodiments, the at least one additional therapeutic agent is an antibody or a sdAb.

[0300] In certain embodiments, the kit further comprises a syringe (e.g., a pre-filled syringe) or a vial comprising the composition as described herein. In certain embodiments, the kit further comprises an atomizer nozzle for nasal or pulmonary delivery, wherein the atomizer nozzle is or could be fitted with the syringe or vial to produce a spray or mist. In certain embodiments, the kit further comprises an inhaler device. In certain embodiments, the kit further comprises a nasal spray device. In certain embodiments, the kit further comprises a needle that is or could be fitted with the syringe (e.g., to deliver subcutaneous, intradermal, or intramuscular injection).

[0301] Methods of Use

[0302] Certain embodiments provide a method of inhibiting the activity of Ebola virus, comprising contacting Ebola virus with an isolated anti-Ebola binder protein as described herein. In certain embodiments, the fusion of viral and endosome membranes, viral entry or release of viral genomic materials into cytoplasm is inhibited. Thus, certain embodiments also provide a method for inhibiting Ebola virus entry, comprising contacting Ebola virus with an isolated binder protein as described herein.

[0303] In certain embodiments, a binder protein descirbed herein (e.g., Nanosota-EBl) inhibits the cleavage of the virus glycoprotein glycan cap by host protease(s), thereby preventing the exposure of the receptor-binding site (RBS) necessary for binding to the Niemann-Pick Cl (NPC1) receptor.

[0304] In certain embodiments, a binder protein descirbed herein (e.g., Nanosota-EB2) blocks the virus’s membrane fusion machinery from penetrating host cells, thereby preventing viral spread.

[0305] In certain embodiments, the Ebola virus is contacted in vitro. In certain embodiments, the Ebola virus is contacted in vivo. In certain embodiments, the Ebola virus is contacted extracellularly. In certain embodiments, the Ebola virus is contacted intracellularly (e.g., intracellularly delivered or expressed sdAb binds virus within an infected cell). Methods for measuring the activity of Ebola virus are known in the art and described herein. For example, in certain embodiments, an assay described herein may be used. In certain embodiments, a binder protein of the invention inhibits the activity of Ebola virus by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% as compared to a control.

[0306] In certain embodiments, a binder protein of the invention inhibits the activity of an ebolavirus (live virus or pseudovirus of an Ebola strain or variant) with a ICso potency of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 ng / mL. In certain embodiments, a binder protein of the invention inhibits the activity of an Ebola (live virus or pseudovirus) with ICso potency of about 1 to 200 ng / mL, 1 to 6 ng / mL, 3 to 10 ng / mL, 2 to 120 ng / mL, 2 to 60 ng / mL, 3 to 110 ng / mL, 30 to 60 ng / mL, or 40 to 50 ng / mL. In certain embodiments, a binder protein of the invention inhibits the activity of an Ebola (live virus or pseudovirus) with ICso potency of about 0.5 to 50 ng / mL, 0.8 to 50 ng / mL, 0.8 to 30 ng / mL, 0.8 to 20 ng / mL, 0.8 to 17 ng / mL, 1 to 50 ng / mL, 1 to 47 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, 1 to 17 ng / mL, 1 to 12 ng / mL, or 1 to 10 ng / mL.

[0307] In certain embodiments, a binder protein of the invention (e.g., a bispecific binder described herein) inhibits the activity of an ebolavirus with a ICso potency of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, or 60 ng / mL as determined in an authentic EBOV infection in vitro assay as described herein. In certain embodiments, a binder protein of the invention (e.g., a bispecific binder described herein) has an ICso of about 20 to 60 ng / mL, 25 to 50 ng / mL, 26 to 45 ng / mL, 25 to 35 ng / mL, or 26 to 33 ng / mL (e.g., 29 ng / mL).

[0308] In certain embodiments, a binder protein described herein has a blood circulating halflife of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In certain embodiments, a binder protein described herein has a blood circulating half-life of about 2-10, 3-9, or 4-7 days.

[0309] Certain embodiments also provide a method for treating or preventing Ebola virus infection in a mammal, comprising administering an effective amount of an isolated anti-Ebola virus binder protein, or a vector as described herein to the mammal.

[0310] In certain embodiments, the method further comprises administering at least one additional therapeutic agent to the mammal. In certain embodiments, the at least one additional therapeutic agent is useful for treating a viral infection or inflammation. In certain embodiments, the at least one additional therapeutic agent is an antibody or a sdAb.

[0311] Certain embodiments provide an isolated anti-Ebola binder protein, or vector as described herein for the prophylactic or therapeutic treatment of Ebola virus infection.

[0312] Certain embodiments provide the use of an isolated anti-Ebola binder protein or vector as described herein to prepare a medicament for the treatment of Ebola virus infection in a mammal.

[0313] Certain embodiments provide an isolated anti-Ebola virus binder protein or vector as described herein for use in medical therapy.

[0314] Certain embodiments of the invention provide a method for producing an anti-Ebola binder protein, comprising culturing a host cell as described herein under conditions in which the protein is expressed.

[0315] Administration

[0316] For in vivo use, a protein molecule as described herein (e.g., a sdAb of the invention, or a polypeptide or protein molecule comprising such a sdAb), or a vector, or a cocktail mixture thereof, as described herein is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, one or more protein molecules or vectors of the invention may be present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease, as measured using a representative assay). A pharmaceutical composition comprises one or more such protein molecules or vectors in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition. In certain embodiments, an Ebola virus binder protein or composition described herein could be administered to an animal (e.g., mammal such as human) in need of before or after an Ebola virus infection, for example, for prevention or treatment of Ebola virus infection. In certain embodiments, an Ebola virus binder protein or composition described herein could be administered to an animal about 3, 2, 1 week(s) or 6, 5, 4, 3, 2, 1 day(s) or 20, 15, 10, 5, 1 hour(s) before an Ebola virus infection or before potential exposure to Ebola virus. In certain embodiments, an Ebola virus binder protein or composition described herein could be administered to an animal about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hour(s), 1, 2, 3, 4, 5, 6 day(s), or 1, 2, 3 week(s) after suspected or confirmed Ebola virus infection. In certain embodiments, an Ebola virus binder protein or composition described herein could be administered to an animal about 1, 2, 3, or 4 day(s) after suspected or confirmed Ebola virus infection.

[0317] In certain embodiments, an Ebola virus binder protein (e.g., a sdAb, a sdAb-Fc, or a protein comprising sdAb domain(s)) is administered to an animal (e.g., mammal such as human) in need of at a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 mg / kg. In certain embodiments, an Ebola virus binder protein (e.g., a sdAb, a sdAb-Fc, or a protein comprising sdAb domain(s)) is administered to an animal (e.g., mammal such as human) in need of at a dosage range of about 0.1 to 50, 0.5 to 40, 1 to 30, 2 to 25, 3 to 20, 4 to 18, 5 to 16, 10 to 50, or 25 to 50 mg / kg.

[0318] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of a protein molecule or vector either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0319] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder, such as an Ebola virus infection. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized ( / .<., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. " Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. In certain embodiments, the present protein molecules / vectors may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the protein molecule / vector may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of a protein molecule / vector of the present invention. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of protein molecule / vector in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0320] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the protein molecule / vector, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the protein molecule / vector may be incorporated into sustained-release preparations and devices.

[0321] The protein molecule or a vector as described herein may also be administered subcutaneously, intradermally, intranasally, intramuscularly, intravenously or intraperitoneally by infusion or injection. The protein molecule or a vector as described herein may also be administered via intranasal and / or pulmonary delivery (e.g., delivered as a spray or mist).

[0322] Additionally, the protein molecule or vector may be administered by local injection, such as by intrathecal injection, epidural injection or peri-neural injection using a scope. Solutions of the protein molecule or vector may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0323] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the protein molecule or vector that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0324] Sterile injectable solutions are prepared by incorporating the protein molecule or vector in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the protein molecule or vector plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0325] For topical administration, the present protein molecules / vectors may be applied in pure form, / .<., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0326] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present protein molecules / vectors can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0327] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0328] Examples of useful dermatological compositions that can be used to deliver the protein molecules / vectors of the present invention to the skin are known to the art; for example, see Jacquet et al. (U. S. Pat. No. 4,608,392), Geria (U. S. Pat. No. 4,992,478), Smith et al. (U. S. Pat. No. 4,559,157) and Wortzman (U. S. Pat. No. 4,820,508).

[0329] Useful dosages of the protein molecules or vectors of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Pat. No. 4,938,949.

[0330] The amount of a protein molecule or vector of the present invention required for use in treatment will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0331] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0332] Protein molecules or vectors of the invention can also be administered in combination with other therapeutic agents and / or treatments, such as other agents or treatments that are useful for the treatment of an Ebola virus infection. In certain embodiments such an agent is an antibody or a sdAb. Additionally, one or more protein molecules or vectors of the invention, may be administered (e.g., a combination of sdAbs, polypeptides, protein molecules and / or vectors may be administered). Accordingly, one embodiment the invention also provides a composition comprising a protein molecule or vector of the invention, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising a protein molecule or vector of the invention, at least one other therapeutic agent, packaging material, and instructions for administering a protein molecule or vector of the invention, and the other therapeutic agent or agents to an animal to treat an Ebola virus infection. As used herein, the term “therapeutic agent” refers to any agent or material that has a beneficial effect on the mammalian recipient.

[0333] The invention will now be illustrated by the following non-limiting Examples.

[0334] EXAMPLE 1. Discovery of Nanosota-EB 1 and -EB2 as Novel Nanobody Inhibitors Against Ebola Virus Infection

[0335] The Ebola filovirus (EBOV) poses a serious threat to global health and national security. Nanobodies, a type of single-domain antibody, have demonstrated promising therapeutic potential. We identified two anti-EBOV nanobodies, Nanosota-EB 1 and Nanosota-EB2, which specifically target the EBOV glycoprotein (GP). Cryo-EM and biochemical data revealed that Nanosota-EB 1 binds to the glycan cap of GP1, preventing its protease cleavage, while Nanosota-EB2 binds to important membrane-fusion elements in GP2, stabilizing it in the prefusion state. Nanosota-EB2 is a potent neutralizer of EBOV infection in vitro and offers excellent protection in a mouse model of EBOV challenge, while Nanosota-EB 1 provides moderate neutralization and protection. Nanosota-EB 1 and Nanosota-EB2 are the first nanobodies shown to inhibit authentic EBOV. Combined with our newly developed structure-guided in vitro evolution approach, they lay the foundation for nanobody -based therapies against EBOV and other viruses within the ebolavirus genus.

[0336] Introduction

[0337] The Ebola virus (EBOV), a member of the ebolavirus genus in the filovirus family, poses a serious threat to global health and national security due to its high fatality rate. During the unprecedented outbreak in West Africa from 2014 to 2016, there were 28,652 infections with a case fatality rate of ~ 40% [1, 2], Smaller outbreaks of EBOV and related filoviruses have occurred periodically [3], Although bats have been suggested as a potential reservoir, intact EBOV has yet to be isolated from bats [4], Additionally, EBOV can remain dormant in the human body for years before reemerging to cause new infections [5], Given the long-term coexistence of EBOV with humans, developing effective and accessible treatment strategies is a significant priority.

[0338] The GP protein of EBOV guides viral entry into human cells and is a primary target for neutralizing antibodies [6], GP is presented on the virus surface as a homotrimer, having three copies of the receptor-binding subunit GP1 and a trimeric stalk formed by the membrane-fusion subunit GP2 (Fig.7A), which also anchors the trimer in the viral membrane via a transmembrane region [7], GP1 contains a receptor-binding site (RBS) that is concealed by a glycan cap and a mucin-like domain (MLD) [6, 8], GP2 features a fusion peptide and two heptad repeat regions (HR1 and HR2) [9], During molecular maturation, GP is cleaved by the human protease furin at the GP1 / GP2 boundary, but GP1 and GP2 remain associated in a metastable "pre-fusion" state. During cell entry, GP1 interacts with cell-surface factors to facilitate viral attachment to host cells, after which the virus is taken up by endocytosis

[0010] , Inside the endosomes, cathepsins remove the glycan cap and MLD, exposing the RBS

[0011] , The RBS then binds to its host receptor Niemann-Pick Cl (NPC1) on the endosomal membranes [12, 13], Subsequently, GP2 transitions to its "post-fusion" state, the lowest-energy conformation, where HR1 and HR2 form a six-helix bundle, allowing the fusion peptide to merge the viral and host membranes [14, 15], An effective anti-EBOV antibody therapy may inhibit protease cleavage, block receptor binding, or prevent the structural transition of EBOV GP. A challenge in anti-EBOV antibody therapy is that EBOV secretes large amounts of sGP, a secreted form of GP that includes most regions of GP1 but none of GP2, as a decoy to divert GP1 -targeting antibodies

[0016] , The primary transcript of the GP gene is sGP mRNA (-75%). Occasionally, the viral polymerase pauses near the sGP stop codon, causing a frameshift that leads to the production of full-length GP mRNA (-25%)

[0017] , Effective anti-EBOV antibody therapy may block GP-guided viral entry and resist sGP diversion simultaneously.

[0339] Currently, two FDA-approved human antibody drugs target EBOV GP [18, 19], One of these, Ebanga, is a single antibody that targets the RBS and blocks receptor binding, though it may be susceptible to viral escape due to epitope mutations. Additionally, there has been a case of acute Ebola virus disease (EVD) relapse in a survivor from an earlier EBOV outbreak who had previously been treated with Ebanga

[0020] , In contrast, Inmazeb, a cocktail of three antibodies that control infection by targeting three distinct epitopes, makes viral escape more difficult

[0021] , One of the three antibodies targets the RBS to block receptor binding, another targets the glycan cap and inhibits protease cleavage of the glycan cap, and the third targets GP2, although its mechanism of action has not been fully characterized. Both of these approved antibody drugs have reduced the fatality rate of EBOV infections to around 35%, including in some late-stage cases [18, 19], Furthermore, some EVD survivors show evidence of viral persistence, where the large size of conventional antibodies may hinder access to immune sanctuary sites [21-23], Moreover, the high costs of production, transport, and storage for human antibodies, coupled with their injection-only administration, present significant barriers.

[0340] Currently, no FDA-approved drugs or vaccines exist for other members of the ebolavirus genus, such as Bundibugyo ebolavirus (BDBV) and Sudan ebolavirus (SUDV), which continue to pose serious threats to global health and national security [24, 25], Adapting anti-EBOV human antibodies to combat related filoviruses can be challenging. Thus, there is an urgent need to develop potent, small, cost-effective, and broadly accessible therapies for EBOV infections, with potential applicability to other filoviruses.

[0341] Nanobodies are single-domain antibodies derived from the heavy-chain-only antibodies produced by camelid animals and sharks [26, 27], Their single-domain structure offers numerous advantages over conventional antibodies in antiviral applications. For instance, nanobodies exhibit excellent epitope accessibility and tissue permeability [28, 29], which enhance their antiviral efficacy and may benefit EVD survivors with viral persistence.

[0342] Moreover, nanobodies can be produced in high yields, demonstrate good in vitro thermostability, and are cost-effective to manufacture, transport, and store

[0030] , They can also be administered intranasally [31, 32], making them a promising option for needle-free therapies. Additionally, due to their high homology to human germline antibodies, nanobodies show minimal toxicity and immunogenicity in humans [28, 29], In 2019, the first nanobody -based therapeutic was FDA-approved to treat a blood clotting disorder

[0033] , During the COVID-19 pandemic, we developed a series of nanobody inhibitors, known as the Nanosota series, which demonstrated exceptional potency and broad-spectrum activity against SARS-CoV-2 variants [30, 32, 34, 35], Importantly, we recently developed a novel structure-guided in vitro evolution approach for nanobodies

[0036] , allowing rapid adaptation of nanobodies to target different viral variants. Previously, only one study reported a nanobody targeting pseudoviruses packaged with the EBOV GP protein

[0037] , To date, no nanobody inhibitors have been developed or evaluated against authentic EBOV either in vitro or in vivo.

[0343] In this study, we identified two nanobodies, Nanosota-EBl and Nanosota-EB2, which bind to distinct epitopes on the EBOV GP protein. Using cryo-EM and biochemical assays, we investigated their mechanisms of action and assessed their anti-EBOV efficacy both in vitro and in a mouse model. These nanobodies represent a promising, cost-effective, and accessible therapeutic option for EBOV infections. Combined with our recently developed nanobody evolution approach, they provide a foundation for nanobody-based therapies targeting EBOV and related filoviruses.

[0344] Results

[0345] Discovery and in vitro characterization of Nanosota-EBl and -EB2

[0346] To identify nanobodies targeting EBOV, we produced a recombinant version of the EBOV GP ectodomain lacking the mucin-like domain (MLD). The MLD, located at the top of each GP monomer, shields the receptor-binding site (RBS) from immune recognition. Its removal does not affect the overall GP structure but enhances GP ectodomain expression

[0038] , This modified GP was named GP-AM (Fig.7B). An alpaca was immunized with this protein, and peripheral blood mononuclear cells were collected to construct a phage display library containing the alpaca’s nanobodies. Using GP-AM as bait, we screened the library and identified six nanobodies that bound to GP-AM. Among these, two nanobodies, named Nanosota-EBl and Nanosota-EB2 (abbreviated as EB 1 and EB2, respectively), exhibited the highest affinity for GP-AM based on initial ELISA results. We expressed and purified the His-tagged nanobodies (EBl-His and EB2-His) from bacteria, achieving expression yields of over 20 mg / L of medium for each. Moreover, we expressed and purified the Fc-tagged versions (EBl-Fc and EB2-Fc) from mammalian cells, with expression yields exceeding 50 mg / L of medium for each. These purified nanobodies were subsequently used for functional and structural characterization.

[0347] We characterized the binding of EB1 and EB2 to the EBOV GP protein. First, we used surface plasmon resonance (SPR) to evaluate the binding affinity of the His-tagged nanobodies to GP-AM. Both EBl-His and EB2-His demonstrated high-affinity binding to GP-AM, with dissociation constants (Kd) in the nanomolar range (Fig. 1A, Fig.8). Second, SPR was performed to measure the binding affinity of the nanobodies to a further cleaved GP ectodomain (GPcl), which lacks both the MLD and the glycan cap (Fig.7B). The results showed that EBl-His does not bind to GPcl, whereas EB2-His retains high-affinity binding with a Kd value in the nanomolar range (Fig. 1 A, Fig.8), suggesting that EB1 interacts with the glycan cap, unlike EB2. Additionally, we used ELISA to assess the binding of the His-tagged nanobodies to sGP, the secreted form of GP that includes most of GP1 but none of GP2 (Fig.7B), comparing it to GP-AM and GPcl. The results revealed that EBl-His binds to sGP, while EB2-His does not (Fig. IB). Taken together, these findings indicate that EB1 targets the glycan cap of GP1 and sGP, whereas EB2 does not, despite both nanobodies exhibiting strong binding to GP-AM.

[0348] Next, we evaluated the efficacy of EB1 and EB2 in neutralizing EBOV entry in vitro. Lentiviruses pseudotyped with the full-length EBOV GP (including the glycan cap and MLD), referred to as EBOV pseudoviruses, were used to measure their entry into human cells in the presence of the nanobodies. Both His-tagged and Fc-tagged nanobodies were tested. The results showed that EBl-Fc and EB2-Fc neutralized EBOV pseudovirus entry with moderate and high potency, achieving ICso values of 110 ng / ml and 3.1 ng / ml, respectively (Fig. 1C). Furthermore, the Fc-tagged nanobodies exhibited stronger neutralization than their His-tagged counterparts (Fig.9), likely due to increased valency provided by the Fc tag. We also tested the Fc-tagged nanobodies against authentic EBOV infection in human cells. EBl-Fc and EB2-Fc neutralized authentic EBOV infection with low and high potency, achieving ICso values of 47 pg / ml and 43 ng / ml, respectively (Fig. ID). The -1000-fold reduction in EBl-Fc’s neutralizing efficacy against authentic EBOV infection assay compared to pseudovirus entry assay is likely due to the large amounts of sGP secreted by EBOV-infected cells. In contrast, the neutralizing efficacy of EB2-Fc decreased by only ~ 10-fold in the same comparison, reflecting a calibration difference between the pseudovirus and authentic EBOV neutralization assays, as EB2 does not interact with sGP. Overall, these findings confirm that EB1 is a moderate inhibitor of EBOV entry, with its activity further diminished against authentic EBOV due to the effects of sGP. Conversely, EB2 is a highly effective inhibitor of both EBOV pseudovirus entry and authentic EBOV infection, with its efficacy unaffected by sGP.

[0349] Structural basis for anti-EBOV functions of Nanosota-EBl and -EB2

[0350] To investigate the structural basis of the nanobodies' inhibition of EBOV entry, we determined the cryo-EM structures of EBOV GP-AM in complex with EBl-His and EB2-His, respectively (Fig.10, Fig.l 1, Fig.12, Table SI). The structure of GP-AM in complex with EB1 revealed that the trimeric GP-AM is engaged by two EB1 molecules (Fig. 2A, Fig. 2B). EB1 binds to the top of the glycan cap, burying 662 A2of surface area at the interface (Fig. 2C). In the absence of EB1, the glycan cap density is not visible, indicating that it is disordered (Fig.13 A). However, upon EB1 binding, the glycan cap becomes ordered (Fig.13 A). The human antibody REGN-3470 targets the glycan cap by binding to the outermost P-18 strand (Fig.13B). Unlike REGN-3470, EB1 displaces the P-18 strand, pushing it aside and exclusively interacting with the inner P-17 strand (Fig.l3C). Another human antibody, EBOV-548, also targets the glycan cap by displacing the P-18 strand and binding to the inner P-17 strand. However, EB1 and EBOV-548 bind to the glycan cap from different orientations (Fig.l3D). These structural findings suggest that, similar to glycan cap-targeting human antibodies

[0039] , EB1 may inhibit EBOV entry by stabilizing the glycan cap, potentially preventing its cleavage by cathepsins.

[0351] The structure of GP-AM in complex with EB2-His revealed that each trimeric GP-AM molecule is bound by three EB2 molecules (Fig. 3 A). Each EB2 binds to relatively conserved quaternary epitopes on GP, including HR1, the fusion loop, the N-terminus of GP2, and the P1 / P2 strands of GP1, forming a large, buried interface of 966 A2(Figs. 3B-3C). All three complementarity-determining regions (CDRs) of EB2 strongly interact with an N-linked glycan on Asn563 (N563 glycan) of HR1, which is important for GP2's stability and membrane fusion function, burying 316 A2at the interface (Fig. 3D). In the absence of EB2, the N563 glycan is less ordered, with fewer sugar rings visible (Fig.14). Similarly, while the human antibody REGN-3479 also binds to the N563 glycan, its stabilization of the glycan is less pronounced, as evidenced by fewer visible sugar rings compared to EB2 (Fig.14)

[0039] , EB2 stabilizes the glycan through strong hydrophobic interactions and hydrogen bonding (Fig. 3D), pulling the glycan closer to itself (Fig.14). This results in a significant positional shift of the N563 glycan, a phenomenon observed for the first time. Additionally, the CDR3 of EB2 engages with the N-terminus of GP2 (Fig. 3E). In the absence of EB2, the residues at GP2's N-terminus are flexible, with only a few residues visible. EB2 secures the N-terminus through strong hydrogen bonds, enhancing its visibility and locking it in place. The CDR3 also interacts with HR1 via hydrophobic interactions and hydrogen bonds (Fig. 3F) and binds to the P 1 / |32 strands of GP1 through hydrophobic interactions (Fig. 3G). Notably, EB2 forms interactions not only with a single GP subunit but also bridges two distinct GP subunits. For example, CDR3 of EB2 interacts with the fusion loop from a different GP subunit through hydrophobic interactions and hydrogen bonds (Fig. 3H). These unique and robust interactions between EB2 and multiple GP epitopes suggest two mechanisms by which EB2 inhibits EBOV entry. First, EB2 stabilizes the N563 glycan and the N-terminus of GP2 while bridging two GP2 subunits and connecting GP1 and GP2, thereby enhancing the stability of the pre-fusion GP. Second, EB2 engages key membrane-fusion elements, including HR1 and the fusion peptide, locking them in their prefusion conformations.

[0352] To validate the structural data, we conducted two biochemical assays. First, we examined how each nanobody affects the thermostability of GP-AM and GPcl by measuring the thermal shifts induced by EBl-His and EB2-His (Figs. 4A-4B; Fig.15). At neutral pH, EB1 and EB2, both of which bind to GP-AM, increased its thermostability slightly (by 1°C) and significantly (by 6°C), respectively (Fig. 4A). At the same pH, EB2, which binds to GPcl, significantly enhanced GPcl's thermostability (Fig. 4B), whereas EB1, which does not bind to GPcl, had no significant effect. At acidic pH levels (pH 4.5-6), which are physiologically relevant to endosomes, EB2 significantly increased GPcl's thermostability by 11°C (Fig. 4B). This suggests that EB2 strongly binds to and locks the prefusion GPcl even after cathepsin-mediated glycan cap cleavage in the endosomes. As a result, EB2 binding increases the energy barrier for GPcl to transition from its prefusion to post-fusion state, thereby blocking EBOV cell entry. This thermostability assay, the first conducted for GP -targeting antibodies / nanobodies, indicates that REGN-3479, which shares an overlapping epitope with EB2, may utilize a similar neutralization mechanism. Second, we investigated the effect of each nanobody on the protease sensitivity of GP-AM (Fig. 4; Fig.16; Fig.17). Previous studies have demonstrated that the bacterial protease thermolysin mimics endosomal proteases by cleaving the glycan cap and mucin domain from GP1 [40, 41], GP-AM was treated with thermolysin L in the presence or absence of EB1 and EB2, and the glycan cap cleavage rate was monitored. Reducing SDS-PAGE analysis showed that the GP1 band disappeared completely after thermolysin L treatment in the absence of EB1 but remained nearly intact in its presence (Fig. 4C). Similarly, non-reducing Western blot analysis revealed that the GP-AM band faded after thermolysin L treatment without EB1 but persisted longer when EB1 was present (Fig. 4D; Fig.l7A). These findings indicate that EB1 delays protease-mediated glycan cap cleavage, whereas this effect is less pronounced with EB2 (Fig.16; Fig.l7B). This observation aligns with previous research on glycan cap-binding human antibodies

[0039] , In summary, EB1 reduces GP’s protease sensitivity, while EB2 stabilizes GP, supporting the findings from our structural analysis.

[0353] To evaluate the anti-ebolavirus spectrum of EB1 and EB2, we analyzed their specific interactions with EBOV GP. Among the EBOV residues directly interacting with EB1, two differ from those in BDBV, and five differ from those in SUDV (Fig. 5A, Table S2). Similarly, among the EBOV residues directly interacting with EB2, three differ from those in BDBV, and four differ from those in SUDV (Fig. 5B, Table S3). These differences suggest that neither EB1 nor EB2 serves as a strong cross-inhibitor of BDBV or SUDV. To validate the structural data, we conducted two biochemical assays. ELISA results demonstrated that EB1 binds less strongly to BDBV GP-AM compared to EBOV GP-AM and does not significantly bind to SUDV GP-AM (Fig. 5C), whereas EB2 fails to significantly bind GP-AM from either BDBV or SUDV (Fig. 5D). Pseudovirus entry assays further revealed that EB1 weakly neutralizes BDBV pseudoviruses but does not neutralize SUDV pseudoviruses (Fig. 5E), while EB2 does not neutralize pseudoviruses from either BDBV or SUDV (Fig. 5F). Thus, neither EB1 nor EB2 acts as a potent inhibitor of BDBV or SUDV. However, using our newly developed structure-guided in vitro evolution approach

[0036] , EB1 and EB2 can potentially be engineered into pan-ebolavirus nanobody therapeutics.

[0354] In vivo efficacy of Nanosota-EBl, -EB2, and their cocktail for treatment of disease We assessed the therapeutic efficacy of Fc-tagged constructs - EBl-Fc, EB2-Fc, and their combination - in treating Ebola virus disease using a mouse model. Previous studies have shown that Fc-tagged nanobodies offer several advantages over His-tagged nanobodies as antiviral therapeutic candidates. These advantages include increased valency and enhanced neutralization potency, as well as a significantly longer in vivo half-life due to their size exceeding the kidney clearance threshold

[0030] , Despite this, Fc-tagged nanobodies are still only half the size of human antibodies. Importantly, they maintain a single-domain structure for target binding, exhibit high in vitro stability

[0030] , and have the potential for intranasal administration

[0032] , The interferon-a / p receptor knockout (IFNAR) mouse model has been widely validated as a robust system for evaluating GP -targeting antibodies, as it reliably mimics the disease progression observed in humans infected with wild-type EBOV [42, 43], In this study, we investigated the anti -EBOV efficacy of EBl-Fc and EB2-Fc in the IFNAR mouse model.

[0355] For in vivo testing, groups of 10 mice were treated 4 hours post- virus challenge with either individual nanobodies or a cocktail of EBl-Fc and EB2-Fc at a dosage of 50 mg / kg via intraperitoneal (IP) injection. Four days post-challenge, 4 mice were euthanized for blood sample collection, while the remaining 6 were monitored for disease progression (Fig. 6). In the vehicle-treated group, 5 of 6 mice died by day 6. Similarly, in the EBl-Fc group, 5 of 6 mice also died, but survival was extended by 3 days, with deaths occurring by day 9. In contrast, only 1 of 6 mice died in the EB2-Fc and cocktail groups, with deaths delayed until day 8 and day 9, respectively, indicating significantly improved survival compared to the vehicle control (P=0.011, Mantel-Cox test) (Fig. 6A). In addition to survival, weight loss, a key disease indicator, was also improved. Weight loss onset was delayed by 1, 2, and 3 days in the EBl-Fc, EB2-Fc, and cocktail groups, respectively, compared to the vehicle control, with significant differences across treatments on day 6 (Fig. 6B). By day 9, weight loss in the vehicle, EBl-Fc, and cocktail-treated groups converged at 16-19%, whereas the EB2-Fc group showed less weight loss at 10% (Fig. 6B). These delays in weight loss were correlated with improved clinical scores based on behavioral and appearance indicators (Fig. 6C). Viral loads in serum were measured on day 4, just before the peak serum viral load in this model

[0043] , Viral genome numbers were quantified by qPCR of RNA extracted from serum. All treatment groups showed significant but similar reductions in viral genome numbers, ranging from 3 x 103to 104fold compared to the vehicle group (Fig. 6D). Overall, the results demonstrated that EB2-Fc and the cocktail were highly effective in controlling the disease, while EBl-Fc provided modest but significant benefits.

[0356] In vitro stability of Nanosota-EBl and -EB2

[0357] We assessed the in vitro stability of EBl-Fc and EB2-Fc by incubating them at four temperatures (-80°C, 4°C, 25°C, and 37°C) for one week, followed by measuring their remaining GP-AM-binding capacity using ELISA (Fig.18). Using -80°C as the reference, both EBl-Fc and EB2-Fc preserved nearly all of their GP-AM-binding capacity at the other three temperatures, including 37°C. As previous EBOV outbreaks occurred in warm regions of Africa with limited access to cold storage, this high in vitro stability offers these two anti-EBOV nanobodies significant advantages, potentially lowering storage and transportation costs.

[0358] Discussion

[0359] There remains a clear need for new treatments for filoviruses, despite of the availability of two FDA-approved human antibody drugs (Ebanga and Inmazeb) and several non-FDA-approved human antibodies (ZMapp, FVM04 / CA45, MBP134AF, rEBOV-520 / 548, rEBOV- 442 / 515, 1C3 / 1C11) for EBOV infections [44-51], Current antibody-based therapies face significant challenges, including further reducing fatality rates, addressing viral persistence in some survivors, lowering production, transportation, and storage costs, and exploring more accessible administration routes beyond injection. Furthermore, human antibody drugs are not easily adaptable for targeting other EBOV-related filoviruses, such as Bundibugyo ebolavirus (BDBV) and Sudan ebolavirus (SUDV). Nanobodies, with their modular single-domain structure, offer advantages such as easier production, transportation, and storage, the potential for intranasal administration, and the ability to address viral persistence. Notably, nanobodies can be rapidly adapted to target other viral variants or related viruses using a structure-based in vitro evolution strategy that we recently developed

[0036] , In this study, we developed two nanobodies, Nanosota-EBl and -EB2, that target EBOV and have potential applications for other viruses within the ebolavirus genus. As the first nanobodies identified to combat authentic filoviruses, Nanosota-EBl and -EB2 establish a foundation for nanobody -based therapies against filoviruses.

[0360] Both Nanosota-EBl and -EB2 target the EBOV glycoprotein (GP). EB1 binds to and stabilizes the glycan cap in GP1, slowing its protease-mediated cleavage and acting as a moderate inhibitor of EBOV cell entry, a property also observed in glycan cap-binding human antibodies

[0039] , Furthermore, EBl-Fc has the potential to act as an effector for ADCC and other antiviral immune responses, such as neutrophil activation, phagocytosis, and complement activation, against EBOV infection in vivo [52-54], However, EBl-Fc exhibited moderate efficacy in the mouse model, only delaying disease onset. This outcome is likely attributed to EBl’s moderate neutralization ability. In contrast, EB2 binds to GP2, targeting the membrane fusion loop, HR1, and the N563 glycan, locking them in their prefusion conformation. Our findings also reveal that EB2 significantly enhances the stability of GP, a feature not previously explored for human antibody drugs. By stabilizing GP2 in its prefusion state, EB2 prevents the transition to the post-fusion conformation, introducing a mechanism previously not described for human antibodies. As a result, EB2-Fc demonstrated strong anti-EBOV activity both in vitro and in vivo.

[0361] Several human antibodies have been identified that bind to the glycan cap or GP2 of EBOV GP (Fig.19). Among the three antibodies in the FDA-approved cocktail Inmazeb, REGN-3471 and REGN-3479 target regions similar to those bound by EB1 and EB2, respectively. However, EB1 binds to a unique loop in the glycan cap that differs from the binding site of REGN-3471, though both reduce protease cleavage of GPl’s glycan cap. Additionally, EB2's interaction with key membrane fusion elements in GP2 provides significant stabilization of GP2, a mechanism not previously reported for human antibodies. Among non-FDA-approved antibodies, EBOV-293 and EBOV-296 also bind to the glycan cap, while KZ52 and ADI-15878 target GP2 [6, 55, 56], Compared to REGN-3471 and REGN-3479, the epitopes recognized by these non-FDA-approved antibodies differ more but still overlap with those targeted by EB1 and EB2. This overlap is highlighted by the structural clash observed between KZ52 and EB2 when their binding sites are overlaid on GP2 (Fig.l9B). Overall, EB1 and EB2 share some overlapping epitopes with several GP -targeting human antibodies, yet our structural and biochemical analyses reveal novel insights into their mechanisms of action.

[0362] Although extensive research on anti-EBOV human antibodies has been conducted, this study is among the most comprehensive, encompassing nanobody discovery, structural determination, molecular mechanism elucidation, in vitro antiviral assays, animal challenge studies, and comparisons with human antibodies. Notably, it identifies the first two nanobody inhibitors effective against authentic EBOV. Both nanobodies demonstrate significant promise as anti-EBOV inhibitors, particularly Nanosota-EB2, which exhibits high potency in neutralizing EBOV both in vitro and in mouse models. In addition to their antiviral efficacy, these nanobodies are expected to possess excellent therapeutic qualities, consistent with those previously reported for other nanobodies. While a full evaluation of the therapeutic properties of EB1 and EB2 lies beyond the scope of this Example, it highlights that both Fc-tagged nanobodies can be efficiently produced in mammalian cells and exhibit excellent in vitro stability. These properties could help reduce production, storage, and transportation costs.

[0363] Although neither nanobody is highly effective against two related ebolaviruses, BDBV and SUDV, the small number of residue differences between these viruses suggests that our recently developed structure-guided in vitro evolution approach can adapt these nanobodies to neutralize BDBV and SUDV

[0036] , This strategy enables the engineering and optimization of existing nanobody inhibitors with known binding epitopes and mechanisms for related viruses, eliminating the need to re-immunize animals to generate new nanobodies from scratch. In summary, Nanosota-EBl and Nanosota-EB2 establish a strong foundation for the development of nanobody-based therapies against EBOV and related filoviruses.

[0364] Materials and methods

[0365] Cell lines, plasmids and virus

[0366] HEK293T and Huh7 cells (American Type Culture Collection (ATCC)) were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units / mL penicillin, and 100 pg / mL streptomycin. Expi293F cells (ThermoFisher) for protein expression were grown in Expi293 Expression Medium (ThermoFisher). TGI E. coli and SS320 E. coli (Lucigen) for phage display were grown in 2YT medium. No commonly misidentified cell lines were used.

[0367] EBOV GP gene (strain Zaire; NCBI Reference Sequence NC 002549.1), BDBV GP gene (GenBank: FJ217161.1), and SUDV GP gene (NCBI Reference Sequence NC 006432.1) were synthesized (GenScript). For pseudovirus packaging, the gene encoding one of the full-length GP proteins was cloned into the pcDNA3.1(+) vector with a C-terminal C9 tag sequence. For protein expressions, the gene encoding each of the GP ectodomains without MLD, named GP-AM (residues 1-632, excluding residues 312-463), was cloned into the Lenti-CMV vector (Vigene Biosciences) with a C-terminal foldon trimerization motif sequence followed by a His tag sequence. The gene encoding EBOV sGP (residues 1-305) was cloned into the Lenti-CMV vector with a C-terminal His tag sequence. Plasmids encoding Fc-tagged nanobodies were cloned into the Lenti-CMV vector with an N-termina tPA signal peptide sequence and a C-terminal human IgGl Fc tag sequence (GenBank: AEV43323.1).

[0368] Authentic, replication-competent EBOV (strain H.sapiens-tc / COD / 1976 / Yambuku-Mayinga) was used to infect Huh7 cells for in vitro assays and interferon-a / p-receptor knockout mice for in vivo assays. Experiments involving infectious EBOV were conducted in approved Biosafety Level 4 laboratories at the National Institute of Allergy and Infectious Diseases (for in vitro assays) and National Emerging Infectious Diseases Laboratories (NEIDL) at Boston University (for in vivo testing).

[0369] Alpaca immunization

[0370] Induced nanobody phage display libraries were constructed as previously described

[0057] , Briefly, an alpaca was immunized subcutaneously in the scapular region with 300 ug EBOV GP-AM followed by 3 additional immunizations (2 week intervals) of 150 ug EBOV GP-AM.

[0371] Construction of induced nanobody phage display library

[0372] Following immunization of the alpaca, blood was drawn, peripheral blood mononuclear cells (PBMCs) were isolated from 35 mL blood and a cDNA library was constructed from the PBMC RNA by reverse transcription using oligo dT primers and Superscript IV reverse transcriptase (ThermoFisher). A nested PCR strategy was used to amplify coding regions of nanobody fragments. The resulting PCR fragments were cloned into a modified pADL22 vector (Antibody Design Labs). The nanobody phage display library with a size of 3 x 108was constructed following the manufacturer’s protocols (Antibody Design Labs).

[0373] Screening of nanobody phage display library

[0374] Screening of nanobody phage display library was conducted as previously described

[0032] , Briefly, three rounds of bio-panning were performed to identify strong nanobody binders to GP-AM. For this purpose, 20 pg of purified GP-AM was coated on an immune tube overnight. The coated tube was then blocked with 5% milk and incubated with 500 pl of phages for 1 hour. After washing, the retained phages were eluted and used to infect TGI E. coli. The infected TGI E. coli were used to amplify phages, which were then employed for the next round of biopanning. After the third round, the eluted phages were used to infect ss320 E. coli and then spread onto 2YT agar plates. Single colonies were picked, and nanobody expression was induced using 1 mM IPTG. The supernatants were then subjected to ELISA to identify nanobodies that bound to GP-AM.

[0375] Protein expression and purification

[0376] His-tagged nanobodies were expressed and purified from the periplasm of ss320 E. coli as previously described

[0032] , Briefly, expression of the proteins was induced using 1 mM IPTG. Cell pellets were collected and re-suspended in 15 ml TES buffer (0.2 M Tris pH 8, 0.5 mM EDTA, 0.5 M sucrose), shaken on ice for 1 hour, diluted with 40 ml % TES buffer (TES buffer at % concentration for each component), and then shaken on ice for another hour. The proteins in the supernatant were sequentially purified using a Ni-NTA column and a Superdex200 gel filtration column (Cytiva).

[0377] EBOV GP-AM, EBOV sGP, and Fc-tagged nanobodies were expressed and purified from mammalian cells as previously described [32, 58], Briefly, the plasmids encoding each of the above proteins were transiently transfected into the Expi293F cells using polyethylenimine (PEI, Polysciences). 3 days post transfection, the proteins were harvested from the supernatants of cell culture medium. Subsequently, His-tagged EBOV GP-AM and EBOV sGP were purified on a Ni-NTA column and purified further on a Superose200 gel filtration column (Cytiva); Fc-tagged nanobodies were purified on a Protein A column and then purified further on a Supedex200 gel filtration column (Cytiva). To produce EBOV GPcl (GP ectodomain with both MLD and the glycan cap removed), 3 mg EBOV GP-AM was cleaved with 15 pg thermolysin L (Sigma- Aldrich) at room temperature overnight and then purified on a Superose200 gel filtration column (Cytiva). BDBV GP-AM and SUDV GP-AM were expressed and purified using the same procedure as EBOV GP-AM.

[0378] To prepare the complexes of EBOV GP-AM and individual nanobodies, EBOV GP-AM and each of the His-tagged nanobodies (with the nanobodies in excess) were incubated at room temperature for 1 hour and then were purified on a gel filtration Superose 6 increase 10 / 300 GL column (Cytiva).

[0379] ELISA ELISA was conducted to detect the binding between recombinant GP-AM and each of the nanobodies from the supernatant of ss320 E. coli as previously described

[0032] , Briefly, ELISA plates were coated with one of the recombinant EBOV GP proteins and were then incubated sequentially with the supernatant of ss320 E. coli (containing HA-tagged nanobodies) and HRP-conjugated anti-HA antibody (1:1,000) (Sigma-Aldrich). ELISA substrate (Invitrogen) was added and then the reactions were stopped using IN H2SO4. Absorbances at 450 nm (A450) were measured using a Synergy LX Multi-Mode Reader (BioTek).

[0380] ELISA was performed to detect the binding between each of the recombinant nanobodies (Nanosota-EBl and Nanosota-EB2) and each of the recombinant GP proteins (GP-AM, GPcl, and sGP from EBOV and GP-AM from BDBV and SUDV). The procedure was the same as described above, except that recombinant HA-tagged nanobodies (also His-tagged) replaced the supernatant of ss320 E. coli.

[0381] ELISA was also performed to evaluate the effect of storage conditions on the binding affinity of the Fc-tagged nanobodies to recombinant EBOV GP-AM. Briefly, ELISA plates were coated with recombinant EBOV GP-AM, followed by the addition of serially diluted nanobody samples. An HRP-conjugated anti-Fc antibody (1:3,000) (Sigma-Aldrich) was then applied. The remaining steps followed the procedure described above.

[0382] Surface plasmon resonance

[0383] Surface plasmon resonance (SPR) was carried out to measure the binding affinity between each of the recombinant nanobodies (Nanosota-EBl and -EB2) and each of the recombinant EBOV GP proteins (GP-AM and GPcl) using Biacore S200 system (Cytiva) as previously described

[0032] , Briefly, one of the recombinant EBOV GP proteins was immobilized on a CM5 sensor chip (Cytiva) through chemical crosslinking. Serial dilutions of one of the His-tagged nanobodies were injected at different concentrations. The resulting data were analyzed using Biacore Evaluation Software (Cytiva).

[0384] Glycan cap cleavage

[0385] The cleavage of the GP glycan cap was performed as previously described, with modifications [13, 39], Briefly, 60 pg of EBOV GP-AM complexed with either Nanosota-EBl -His or Nanosota-EB2-His (100 pg) was treated with 0.25 pg of thermolysin L (Sigma-Aldrich) at 37°C for varying durations (5, 15, or 30 minutes). A control sample of 60 pg of EBOV GP-AM alone was also prepared. At each time point, aliquots were immediately mixed with SDS-PAGE loading buffer (under reducing conditions) and boiled for 10 minutes to halt the reactions. All samples were then analyzed by SDS-PAGE and Coomassie blue staining. To further confirm the cleavage of the GP glycan cap, Western blot analysis was performed following an extended incubation period. In this procedure, 60 pg of EBOV GP-AM complexed with either Nanosota- EBl-Fc or Nanosota-EB2-Fc (100 pg) was treated with 0.25 pg of thermolysin L (Sigma-Aldrich) at 37°C for different durations (15, 30, or 60 minutes). At each time point, aliquots were immediately mixed with SDS-PAGE loading buffer (under non-reducing conditions) and boiled for 10 minutes to stop the reactions. GP-AM was detected using an anti -His tag antibody (Sigma-Aldrich, 1:1,000).

[0386] Pseudovirus cell entry assay

[0387] The EBOV pseudovirus entry assay was conducted to measure the neutralizing potencies of nanobodies against EBOV pseudoviruses, as previously described

[0030] , Briefly, EBOV pseudoviruses were produced by co-transfecting HEK293T cells with a pcDNA3.1(+) plasmid encoding the full-length EBOV GP, a lentiviral packaging plasmid psPAX2, and a reporter plasmid plenti-CMV-luc. After 72 hours, the pseudoviruses were collected, incubated with each of the nanobodies at different concentrations at 37 °C for 1 hour, and then used to infect Huh7 cells. After an additional 48 hours, the cells were lysed. Portions of the cell lysates were transferred to new plates, a luciferase substrate was added, and Relative Light Units (RLUs) were measured using an EnSpire plate reader (PerkinElmer). The efficacy of each nanobody was determined and expressed as the concentration required to inhibit pseudovirus entry by 50% (ICso).

[0388] BDB V and SUDV pseudovirus entry assays were performed following the same procedure as the EBOV pseudovirus entry assay, except that BDBV GP and SUDV GP were used in place of EBOV GP, respectively.

[0389] Authentic EBOV neutralization assay

[0390] The neutralizing potency of the nanobodies was evaluated using authentic EBOV.

[0391] Nanobodies were diluted to specified concentrations in cell culture medium and incubated with the virus for 60 minutes. Infection levels were assessed using an immunofluorescence assay, where Hoechst staining (ThermoFisher) was used to visualize cell nuclei, with nuclei counts serving as a proxy for cell counts. The efficacy of each nanobody was determined by calculating the concentration required to reduce infected cell counts by 50% (ICso) compared to the virus-exposed control group without nanobody treatment.

[0392] Mouse efficacy study

[0393] The efficacy of the nanobody treatments was evaluated in an interferon-a / p-receptor knockout (IFNAR-KO) mouse model (strain B6(Cg)-Ifnarltml.2Ees / J; Jackson Laboratories) as previously described

[0059] , All animal procedures were conducted by the Animal Studies Core at the National Emerging Infectious Disease Laboratory at Boston University under biosafety level 4 (ABSL-4) conditions. Briefly, 8- to 12-week-old mice were randomly assigned to groups of 10, with equal numbers of males and females in each group. All mice were challenged with 100 PFU of EBOV in a 0.1 mL volume of PBS buffer via intraperitoneal (IP) injection on day 0. Treatments, dissolved in Dulbecco’s phosphate-buffered saline (PBS, Gibco), were administered once via IP injection 4 hours post-infection. Group 1 received Nanosota-EBl-Fc at 50 mg / kg, group 2 received Nanosota-EB2-Fc at 50 mg / kg, group 3 received a combination of Nanosota-EBl-Fc and EB2-Fc at a total dosage of 50 mg / kg (25 mg / kg of each nanobody), and group 4 received PBS alone. All animals were monitored daily for survival, weight, and clinical signs, with observations conducted twice daily during the critical phase (days 3-10). Scoring criteria for humane endpoints were previously defined based on correlative analysis of factors associated with disease outcomes, including eye appearance, general appearance, responsiveness, and body weight loss (>11%). Animals meeting three of the four criteria during two consecutive observation periods were humanely euthanized following American Veterinary Medical Association (AVMA) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. Animals experiencing 20% body weight loss or agonal states were also euthanized.

[0394] On day 4, four animals (two males and two females) per group were randomly selected, euthanized, and serum samples collected. These animals were excluded from survival analyses. The remaining animals continued in the study under the same conditions until day 18, at which point the study concluded.

[0395] Day 4 was selected for viral load measurement based on a previous study indicating that viral titers had not yet reached the plateau phase on this day

[0043] , making it an ideal time point for comparisons. Furthermore, untreated mice began to succumb by day 5, making later time points unsuitable for measuring viral load. Serum samples collected on day 4 were inactivated using Trizol LS (Invitrogen) and analyzed for viral genome copy numbers (GN). More specifically, total RNA was extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) following the manufacturer’s instructions. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies). EBOV-specific primers (IDT, 5’-CATGCGTACCAGGGAGATTAC-3’ and 5’-ACTCCATCACGCTTCTTGAC-3’) and a probe (IDT, 5’- / 56 FAM / TCAAGTATT / ZEN / TGGAAGGGCACGGGT / 3IABkFQ / -3’) were used in a reverse transcriptase quantitative PCR (RT-qPCR) assay. The assay was performed using the Luna One-Step Universal Probe RT-qPCR Kit (New England Biolabs) on a BioRad instrument, with analysis conducted using BioRad Maestro software. A standard curve was generated using an EBOV RNA standard (transcribed from a PCR-derived EBOV NP fragment template) through 10-fold serial dilutions in water. Each sample, including standards, was run in duplicate along with a non-targeting control. The average values from duplicates were used to calculate genome copy numbers in the serum samples using the standard curve.

[0396] Thermal stability assay

[0397] To evaluate the impact of nanobodies on the thermal stability of EBOV GP, differential scanning fluorimetry (DSF) experiments were conducted as previously described, with modifications

[0060] , Briefly, 20 pg of EBOV GP-AM was incubated with 4 pg of Nanosota-EBl-His or Nanosota-EB2-His separately at room temperature for 30 minutes. Each sample was then mixed with 1.25 pl of Protein Thermal Shift Dye (ThermoFisher) diluted in 10 pl of PBS buffer in a 96-well MicroAmp optical qPCR plate. For the control group, 20 pg of EBOV GP-AM was incubated with Protein Thermal Shift Dye alone. Measurements were performed using a PCR instrument (Applied Biosystems) with a temperature ramp from 25 to 99°C at a rate of 0.05°C / s. Data were collected using QuantStudio real-time PCR software. The negative first derivative of fluorescence was plotted against temperature, and the melting temperature (Tm) was determined as the peak point in the first derivative curve.

[0398] Cryo-EM data collection

[0399] 4 pl of purified complexes of EBOV GP-AM and individual nanobody at -1.25 pM were used for grid preparation. Each of the complexes was applied to freshly glow-discharged Quantifoil Rl.2 / 1.3 300-mesh copper grids (EM Sciences) and blotted for 4 seconds at 22°C under 100% chamber humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM data were collected using Latitude-S (Gatan) equipped with a K3 direct electron detector and with a Biocontinuum energy filter (Gatan). For the GP-AM / Nanosota-EB 1 complex, movies were collected at a nominal magnification of 81,000x (corresponding to 1.1 A per pixel). For the GP-AM / Nanosota-EB2 complex, movies were collected at a nominal magnification of 130,000x (corresponding to 0.664 A per pixel). Statistics of cryo-EM data collection are summarized in SI Table.

[0400] Cryo-EM data processing, model building and refinement

[0401] Cryo-EM data were processed using cryoSPARC v3.3.2

[0061] , and the procedures are outlined in S4 Fig and S5 Fig. Briefly, dose-fractionated movies were subjected to Patch motion correction with MotionCor2

[0062] and Patch CTF estimation with CTFFIND-4.1.13

[0063] , Particles were then picked using the Blob picker in cryoSPARC v3.3.2 and subjected to the Remove Duplicate Particles tool. Junk particles were removed through three rounds of 2D classifications. Particles from the good 2D classes were used for ab-initio reconstruction of four maps. The initial models were set as the starting references for heterogeneous refinement (3D classification). The good 3D classes were then subjected to further homogeneous, non-uniform, and CTF refinements to generate the final maps, applying Cl and C3 symmetry for the GP-AM / Nanosota-EBl complex and GP-AM / Nanosota-EB2 complex, respectively. Map resolutions were determined using gold-standard Fourier shell correlation (FSC) at 0.143 between the two half-maps.

[0402] Initial model building of the EBOV GP / nanobody complex was performed in Coot-0.8.9

[0064] using 5JQ7 as the starting model. The initial model of each nanobody was predicted using SWISS-MODEL (swissmodel.expasy.org / ) and then fitted into the density map. Several rounds of refinement in Phenix-1.16

[0065] and manual building in Coot-0.8.9 were performed until the final reliable models were obtained. Model and map statistics are summarized in SI Table. Figures were generated using UCSF Chimera X v0.93

[0066] , EBOV GP and nanobody contact residues were analyzed using LigPlot, and the EBOV GP and nanobody buried interfaces were analyzed using PDBePISA (ebi.ac.uk / pdbe / pisa / ). Representative structures in density are presented in S6 Fig.

[0403] Statistical analysis

[0404] For the mouse efficacy study, GraphPad Prism (version 10.3.0) was used for data analysis and statistical assessments. Survivorship curves were generated using the Kaplan-Meier method, and comparisons with the vehicle control were performed using the Mantel-Cox test. Percent weight change for each individual animal was calculated relative to its starting weight. P -values for percent weight change were calculated using a One-Way ANOVA on data collected on day 6, comparing each experimental group to the vehicle control. Clinical scores were recorded as the highest score for each group on each day. Viral loads were analyzed using standard methods, with genome numbers (GN) expressed per milliliter. GN / mL values were log-transformed, and P-values were calculated using an Ordinary One-Way ANOVA with multiple comparisons on data collected on day 4, comparing each experimental group to the vehicle control.

[0405] For all other statistical analyses, unpaired two-tailed Student’s / -tests were performed. Please refer to Figure Legends for details.

[0406] Table SI: Cryo-EM Data Collection, Refinement, and Validation Statistics EBOV GP / EBOV GP /

[0407] Nanosoia -EB 1 Nanosota-EB 2 (EMD-44872) (EMD-44873)

[0408]

[0409] (PDB 9BSU) (PDB 9BSVi Data collection and processing

[0410] Magnification 81,009 130,000

[0411] Voltage (kV) 300 300:

[0412] Electron exposure (e—2) 45,00 50.00

[0413] Defocus range (pin) -1,0 -2.0 -1,0 ~ -2.0

[0414] Pixel size (A) 1.1 0,654

[0415] Symmetry imposed Cl C3

[0416] Initial particle images (no.) 1,170,747 585,013

[0417] Final particle images (no.) 482,250 404,158

[0418] Map resolution (A) 3,4 3.1

[0419] FSC threshold ' 0,143 0.143

[0420] Map resolution range (A) 2, 8-4.8 2.6-3.4 Refinement

[0421] Initial model used (PDB code) 5JQ7 5JQ?

[0422] Model resolution (A) 3,5 3,2

[0423] FSC threshold 0.5 0.5

[0424] Model resolution range (A) 32,3-3.3 26,5-3.0

[0425] Map sharpening B factor (A2) -119.2 -144,7

[0426] Model composition

[0427] Non-hydrogen atoms 10227 9882

[0428] Protein residues 1287 1248 Ligands 6 21

[0429] B fectois (A )

[0430] Protein 45,26 1 8,00 Nucleotide

[0431] Ligand 39.39 120.44

[0432] Rm.s. deviations

[0433] Bond lengths (A) 0,004 0.005

[0434] Bond angles (*) 0,777 0,704

[0435] Validation

[0436] MolProbity score 1,89 1,77

[0437] Clashscore 8,78 7.83

[0438] Poor retainers (%) 0.19 0.69 Ramachandran plot

[0439] Favored ( 4) 93.75 95.10

[0440] Allowed (%) 6.02 4.17

[0441]

[0442] Disallowed (%) 0,24 0.74 Table S2: Detailed Interactions Between EBOV GP and Nanosota-EBl. Residues that differ between EBOV and other ebolaviruses are shown in the table.

[0443] Nanosota-EB 1 binding footprint Corresponding residues in

[0444] other ebolaviruses

[0445] Domain Nanobody residues EBOV GP BDBV GP SUDV GP

[0446] interacting rvidt residues residues residues

[0447] EBOV GP GPi -Glycan cap, «2

[0448] LeulOl Leu256 Conserved Conseived

[0449] GPi -Glycan cap, a? Val3G Tln259 Conserved Conserved GPl-Glycan cap, c / 2

[0450] Ser29 1311262 Conserved Leu262

[0451] GPI -Glycan cap, o2

[0452] Ser29, Ser27 Mt 26' Assi263 His263

[0453] GPi-Glycaa cap, 017 Pro i 05 Le«273 Conserved Conseived GPl-Glycan cap. (317

[0454] Tyi-107 Ile274 Conserved Conserved

[0455] GPl-Glycaa cap, pl 7 Pro 104, Vai 102,

[0456] Tip275 Conserved Conseived

[0457] Ala IO- GPi -Glycan cap. (317 TyrI09, Tyrl07,

[0458] Lys276 Conserved Thr276

[0459] ValH>2

[0460] GPl-Glycan cap. [317 Tyrl09, LeulOl Val277 Conseived Lesi27~

[0461] GPl-Glycan cap, 017 Lets 101. TyrHS Asn27S Conserved Asp278

[0462] GPi -Glycan cap, [317

[0463] Tyr32 Ile28I Val281 Conseived

[0464]

[0465] Table S3: Detailed Interactions Between EBOV GP and Nanosota-EBl. Residues that differ between EBOV and other ebolaviruses are shown in the table.

[0466] Naaosota-EB2 binding footprint Corresponding residues in

[0467] other ebolaviruses

[0468] Domain Nanobody residues EBOV GP BDBV GP SUDV GP interacting with EBOV GP residues residues residues

[0469] Monomer A (GP2.

[0470] Asp Hl Gly52S Conseived Conseived

[0471] fusion loop!

[0472] Monomer A (GP2,

[0473] Pro 100 Leu529 Conseived He529

[0474] fits ion loop’)

[0475] Monomer B (GPI.

[0476] Leu 106 Pro34 Conseived Conseived

[0477] hl-p2 strands)

[0478] Monomer B (GP 1,

[0479] Leu 106 VaM5 Conserved Conseived

[0480] pi -p2 strands)

[0481] Monomer B (GP2,

[0482] AIaiO4. AsnlOS, TyrlOS Glu564 Conseived Conserved

[0483] HRiA)

[0484] Monomer B (GP2,

[0485] TyrlOS, Thrl09 G111567 Conseived Conseived

[0486] HRIA)

[0487] Monomer B (GP2.

[0488] TyrlOS A13568 Conseived Conserved

[0489] HRIA)

[0490] Monomer B (GP2, Ser.'O, Asu.31. Asa52. ASN563

[0491] HR1 A, ASN563 Tyr53. Asri54, Arg 109, Glycan Conseived Conserved glycan)

[0492] TrpiOL SerlO2, Ilel03

[0493] Monomer B (GP2. N

[0494] Tyr60, Asa 105 Val505 Lys5SS ThroOS

[0495] terminus)

[0496] Monomer B (GP2. N

[0497] Arg57 Asn506 Argi’06 Lys5Q6

[0498] terminus)

[0499] Monomer B (GP2, N Arg57, Hel03 Ala5O7 Thr5O7 Conseived terminus)

[0500] Monomer B (GP2, N Arg57 G1115O8 Conserved Thr508

[0501]

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[0599] EXAMPLE 2. Discovery of A Novel Bispecific Nanobody Against Ebola Virus Infection As described in Example 1, we have identified the first anti -EBOV nanobodies, named Nanosota-EBl and Nanosota-EB2, which target the EBOV GP protein. EB1 targets the glycan cap of GP1, while EB2 stabilizes GP2 in its pre-fusion state. Notably, we developed a novel bispecific nanobody -Fc fusion protein that combines EB1 and EB2 with human Fc tag (SEQ ID NOs:7 and 14). This bispecific binding protein comprising EB1 and EB2 nanobodies provided complete protection to mice challenged with EBOV, outperforming the individual nanobodies and their combination cocktail. By recognizing two epitopes, it effectively countered the virus's decoy strategy. We further showed that the bispecific nanobody recognized two epitopes on the GP and successfully resisted sGP diversion. To explain the remarkable superior potency of this bispecific nanobody, we propose that this bispecific nanobody may operate through an "anchor, recycle, and block" mechanism, increasing the presence of EB1 and EB2 in endosomes through anchoring and recycling, thereby blocking viral entry. This bispecific nanobody inhibitor could be a powerful tool in combating future EBOV outbreaks and represent a potent and cost-effective treatment option for EBOV infections.

[0600] Results

[0601] In vivo characterization of Nanosota-EBl, -EB2 and their bispecific combination

[0602] We assessed the effectiveness of EB1 and EB2 in neutralizing EBOV using a mouse model. We selected EBl-Fc and EB2-Fc for in vivo testing for several reasons. Fc-tagged nanobodies benefit from an extended in vivo half-life because they exceed the size threshold for renal clearance (G. Ye etal., eLife 10, e64815,2021). The Fc tag also enhances the antiviral activity of nanobodies by increasing their valency. Furthermore, previous research indicates that antibodies binding to certain epitopes on the EBOV GP can induce a strong antibody-dependent cell-mediated cytotoxicity (ADCC) effect in vivo, adding another potential advantage to using an Fc tag (B. M. Gunn et al., Cell host & microbe 24, 221-233. e225, 2018). Please note that Fc-tagged nanobodies retain their single-domain antigen-binding structure, ensuring efficient epitope binding. In addition to testing individual EBl-Fc and EB2-Fc, we also tested a cocktail of EBl-Fc and EB2-Fc to evaluate their combined effectiveness against EBOV in vivo. Our goal was to develop a nanobody drug that can be easily manufactured, target multiple epitopes on GP, and inhibit EBOV using multiple mechanisms. To this end, we also developed a bispecific nanobody that combines the efficacy of both EB1 and EB2 into a single protein molecule. This bispecific nanobody includes one copy each of EB1 and EB2, both linked to a dimeric human Fc tag. We introduced a mutation to the Fc tag to promote the formation of a hetero-Fc dimer containing both EB1 and EB2. Thus, EBl-Fc, EB2-Fc, the EB1-Fc / EB2-Fc cocktail, and the EB1 / EB2-Fc bispecific nanobody were all evaluated head-to-head, along with a negative control, for their anti-EBOV potency in vivo.

[0603] Unexpected results emerged from the mouse challenge experiment. Interferon-a / p-receptor knockout mice were divided into five different groups (10 mice per group) and mice in all five groups were challenged with live EBOV. Four hours post infection, mice in different groups received different nanobodies at a dosage of 50 mg / Kg through intraperitoneal (IP) injection. First, we monitored the mortality rates for each group (Fig. 20A). In the negative control group, 83.3% of the mice succumbed by day 6. In the EBl-Fc group, mortality was also 83.3%, but the deaths occurred on day 9, indicating an improvement of 3 days over the control group. In the EB2-Fc group, only 16.7% of the mice died by day 8, marking a significant enhancement in survival rate compared to the control group. Similarly, in the EB1-Fc / EB2-Fc cocktail group, 16.7% mortality was observed by day 9, showing slight benefits over the EB2-Fc group alone. Remarkably, in the EB1 / EB2-Fc bispecific group, all mice survived. Second, we also tracked the body weights and clinical scores of the mice (Figs. 20B-20C). The control group experienced the most significant weight loss (around 20%) and had the worst clinical scores (clinical score=4) around Day 9. Mice treated with EBl-Fc displayed slight improvements, while those in the EB2-Fc group and the EB1-Fc / EB2-Fc cocktail group showed substantial improvements in both metrics compared to the control group. Strikingly, mice in the EB1ZEB2-Fc bispecific nanobody group maintained their weight and exhibited no clinical symptoms. Lastly, we measured virus titers on day 4 (Fig. 20D). All treatment groups show significant (3.5-4 Logs) reduction of the virus titers compared to the control group. Overall, the results showed that the EB1 / EB2-Fc bispecific nanobody demonstrated the greatest efficacy, surpassing EBl-Fc, EB2-Fc, and the EB1-Fc / EB2-Fc cocktail in inhibiting EBOV in vivo.

[0604] We further characterized the EB1 / EB2-Fc bispecific nanobody using three biochemical assays. First, we investigated whether the bispecific nanobody can bind to both the EB1 and EB2 epitopes. We performed SPR by coating sGP onto the sensor chip and sequentially adding the bispecific nanobody and GPcl (Fig. 21 A). The results showed that the bispecific nanobody binds to sGP (which contains the EB1 epitope but not the EB2 epitope) and GPcl (which contains the EB2 epitope but not the EB1 epitope) simultaneously (Fig. 21 A), confirming that the bispecific nanobody can bind to both the EB1 and EB2 epitopes on the GP. Second, we examined whether sGP impacts the binding of the bispecific nanobody to GP. We performed an ELISA between the bispecific nanobody and GP-AM in the absence or presence of sGP (Fig. 2 IB). The results showed that sGP had no impact on the binding of the bispecific nanobody to GP (Fig. 21B; Fig. 29C). In comparison, sGP significantly impacted the binding of EBl-Fc but not EB2-Fc to GP (Fig. 21B; Figs. 29A-29B). Lastly, we analyzed whether sGP affects the neutralizing potency of the bispecific nanobody. We evaluated the potency of the bispecific nanobody in neutralizing EBOV pseudovirus entry in the absence or presence of sGP (Fig. 21C). The results showed that sGP had no impact on the neutralizing potency of the bispecific nanobody (Fig.21C; Fig. 30C). In contrast, sGP significantly impacted the neutralizing potency of EBl-Fc but not EB2-Fc (Fig. 21C; Figs. 30A-30B). In summary, these data demonstrate that the bispecific nanobody binds to both the EB1 and EB2 epitopes on GP, its binding to GP is not diverted by the sGP decoy, and its potency in neutralizing Ebola entry is not affected by the sGP decoy. Therefore, the bispecific nanobody binds to both the EB1 and EB2 epitopes and effectively resists sGP diversion.

[0605] Discussion

[0606] Many of current human antibodies target GP1 and are easily deceived by the sGP decoy strategy used by EBOV. Human antibodies are generally expensive to produce, transport, and store, and they rely on injections only. Therefore, there is an urgent need for more potent, cost-effective, smaller-sized, and easy-to-administer drugs to combat EBOV infections. In this study, we discovered a bispecific nanobody inhibitor that potentially meets all these criteria.

[0607] As described herein, we discovered two individual nanobodies, Nanosota-EBl and -EB2, that target the Ebola GP. EB1 binds to and stabilizes the glycan cap, slowing down its cleavage by proteases, and is a moderate inhibitor of EBOV entry. Moreover, EBl-Fc is potentially a strong ADCC effector against EBOV infection in vivo (E. O. Saphire et al., Cell 174, 938-952. e913, 2018 and C. D. Murin et al., PNAS 111, 17182-17187, 2014). However, EBl-Fc did not show strong anti-EBOV potency in vivo compared to other experimental groups, as it can be diverted by the large amount of sGP secreted by live EBOV. On the other hand, EB2 binds to the critical membrane-fusion elements in GP2, including HR1, the fusion peptide, and the N563 glycan, locking them in their prefusion conformation. By stabilizing GP in its prefusion conformation, EB2 also slows down the protease cleavage of the glycan cap. Additionally, EB2 does not bind to or get diverted by sGP, making it an ideal inhibitor of EBOV entry. As a result, EB2-Fc demonstrated strong anti -EBOV potency both in vitro and in vivo.

[0608] To overcome the potential limitations of individual nanobodies and their combination cocktail, we aimed to maximize the strong neutralizing function of EB2, retain the strong ADCC effect of EB1, and reduce the decoy effect of sGP. The EB1ZEB2-Fc bispecific nanobody proved to be the most effective, demonstrating significantly better anti-EBOV potency in vivo than EBl-Fc, EB2-Fc, and their combination cocktail. We further showed that the bispecific nanobody binds to both the EB 1 and EB2 epitopes on GP, and its target binding and neutralizing potency are not affected by sGP. Thus, we propose the following mechanism for its superior anti-EBOV potency in vivo (Fig. 22): The bispecific nanobody molecules can bind to either the EB1 epitope or the EB2 epitope on GP, entering endosomes attached to GP along with EBOV particles. During viral entry, the bispecific nanobody molecules bound to the EB2 epitope on GP remain anchored on EBOV particles and are not diverted by sGP (they can even bind both GP and sGP simultaneously), while a significant number of the bispecific nanobody molecules bound to the EB1 epitope are diverted by sGP. Inside the endosomes, cathepsins cleave the glycan cap on GP1, releasing the remaining bispecific nanobody molecules originally attached to the EB1 epitope. These released bispecific nanobody molecules then become available to bind to the EB2 epitope on GP2 that was previously unoccupied. These recycled bispecific nanobody molecules assist the previously attached ones (including those bound to the EB2 epitope on GP and the EB1 epitope on sGP simultaneously) in stabilizing GP2, blocking its membrane fusion function. Therefore, the bispecific nanobody operates via an "anchor, recycle, and block" mechanism: anchoring both EB1 and EB2 (particularly EB2) to prevent sGP diversion, recycling them inside endosomes, and blocking EBOV entry through additional copies of the highly neutralizing EB2 (and to a lesser extent, the moderately neutralizing EB1).

[0609] Our bispecific nanobody offers several advantages over previously discovered anti-EBOV human antibodies. First, the three human antibodies in the FDA-approved cocktail drug Inmazeb (REGN3471, REGN3470, and REGN3479) bind to regions near the RBS in GP1, the glycan cap in GP1, and the membrane-fusion elements in GP2, respectively (V. Rayaprolu et al., Cell host & microbe 31, 260-272. e267, 2023). However, both GP1 -binding REGN3471 and REGN3470 are subjected to sGP diversion, limiting their contributions to EBOV inhibition. In contrast, our GP1 -binding EB1 can mitigate sGP diversion through the bispecific nanobody strategy. Furthermore, our main anti-EBOV neutralizer EB2 alone demonstrated significantly stronger anti-EBOV potency in the EBOV pseudovirus assay than REGN3479 (V. Rayaprolu et al., Cell host & microbe 31, 260-272. e267, 2023), likely due to the unique interaction patterns between EB2 and GP2. (Note that we could not obtain the Inmazeb cocktail for side-by-side experimental comparisons). Importantly, the bispecific nanobody strategy enhanced EB2’s in vivo anti -EBOV potency by resisting sGP diversion and recycling additional copies of EB2 inside endosomes. Second, the bispecific design has been used before for two human antibodies, where an RBS-binding human antibody and a glycan cap-binding human antibody were used to construct bispecific antibodies (A. Z. Wee et al., Science 354, 350-354, 2016). However, both of these human antibodies bind to GP1 and are subjected to sGP diversion. Indeed, the best bispecific human antibody only protected mice from EBOV infection with a fatality rate of 30% (A. Z. Wee etal., Science 354, 350-354, 2016). Our bispecific nanobody differs from the bispecific human antibody by including a super potent inhibitor that binds to the membranefusion elements in GP2, diminishing the decoy effect of sGP while maximizing the neutralizing effect of the GP2 binder. Hence, the anti-EBOV mechanism of our bispecific nanobody differs from the previous bispecific human antibody, and our bispecific nanobody demonstrated superior potency in the mouse model. Additionally, nanobodies, including Fc-tagged nanobodies, have many advantages over human antibodies as antiviral therapeutics due to their cost-effectiveness and potential for intranasal administration (G. Ye etal.. Journal of virology 97, e01448-01423, 023; and G. Ye et al., eLife 10, e64815, 2021). The cost-effectiveness of nanobodies is crucial during a large virus outbreak, while the intranasal administration route is critical for rapid and convenient needle-free therapy outside of clinical settings. Our bispecific nanobody will be further developed to meet the urgent need for new anti-EBOV drugs for the sake of global health and national security.

[0610] Data availability

[0611] The atomic models and corresponding cryo-EM density maps have been deposited into the PDB and the Electron Microscopy Data Bank (EMDB), respectively, with accession numbers 9BSU and EMDB-44872 (EBOV GP complexed with Nanosota-EBl), and 9BSV and EMDB-44873 (EBOV GP complexed with Nanosota-EB2).

[0612] Materials and methods

[0613] Cell lines, plasmids and virus

[0614] Cell lines, plasmid and virus are as described in Example 1. A “Knobs-into-holes” strategy was employed to prepare the Nanosota-EB1 / EB2-Fc bispecific nanobody, with two mutations, T366Y and Y407T, being introduced into the Fc region of the Lenti-CMV-Nanosota-EB1 and Lenti-CMV-Nanosota-EB2 construct, respectively (J. B. Ridgway, et al., Protein Engineering, Design and Selection 9, 617-621, 1996).

[0615] Protein expression and purification Protein expression and purification methods are as described in Example 1. For the expression of Nanosota-EB1 / EB2-Fc bispecific nanobody, equal amounts of the expression plasmids for Nanosota-EBl (containing mutation T366Y in the Fc region of the vector) and Nanosota-EB2 (containing mutation Y407T in the Fc region of the vector) were co-transfected into the Expi293F cells.

[0616] ELISA ELISA protocol is as described in Example 1. ELISA was also carried out to assess the impact of recombinant sGP on the binding interactions between recombinant GP-AM and three different recombinant nanobodies: Nanosota-EBl -Fc, Nanosota-EB2-Fc, and Nanosota-EB1 / EB2-Fc bispecific nanobody. The ELISA plates were coated with GP-AM, and each nanobody, serially diluted (starting at 10 pg / ml and followed by 4-fold dilutions), was added. Afterward, sGP was added at two different concentrations (1 pg / ml and 5 pg / ml). Following incubation, the procedure was carried out as described above. Each of the binding reactions was characterized using the half-maximal effective concentration (ECso), which is defined as the concentration of a nanobody that produces 50% of the maximum possible response in the assay.

[0617] Surface plasmon resonance

[0618] Surface plasmon resonance (SPR) protocol is as described in Example 1. SPR was also conducted to detect the simultaneous binding of the Nanosota-EB1 / EB2-Fc bispecific nanobody to sGP and GPcl. Recombinant sGP was immobilized on a CM5 sensor chip (Cytiva) through chemical crosslinking. Recombinant bispecific nanobody at 40 pg / ml was injected, followed by the injection of GPcl at 40 pg / ml. SPR signals were recorded.

[0619] Pseudovirus entry assay

[0620] The EBOV pseudovirus entry assay was conducted to measure the neutralizing potencies of nanobodies against EBOV pseudoviruses, as previously described (Q. Geng et al., PLoS Pathog. 17, el009897, 2021). Briefly, EBOV pseudoviruses were produced by co-transfecting HEK293T cells with a pcDNA3.1(+) plasmid encoding the full-length EBOV GP, a helper plasmid psPAX2, and a reporter plasmid plenti-CMV-luc. After 72 hours, the pseudoviruses were collected, incubated with each of the nanobodies at different concentrations at 37 °C for 1 hour, and then used to infect Huh7 cells. After an additional 48 hours, the cells were lysed. Portions of the cell lysates were transferred to new plates, a luciferase substrate was added, and Relative Light Units (RLUs) were measured using an EnSpire plate reader (PerkinElmer). The efficacy of each nanobody was determined and expressed as the concentration required to inhibit pseudovirus entry by 50% (ICso). To examine the impact of sGP on the neutralizing potency of the nanobodies, we conducted the EBOV pseudovirus entry assay as described above, with the addition of recombinant sGP at two different concentrations (5 pg / ml and 20 pg / ml).

[0621] Mouse protection study

[0622] The efficacy of the nanobodies was evaluated in an interferon-a / p-receptor knockout (IFNAR-KO) mouse model as previously described (B. A. Rhein et al., PLoS Pathog 11, el005263, 2015). Briefly, 50 mice with an average weight of 21 grams were randomized into 5 groups (n=10). The virus and the nanobodies were all administered through intraperitoneal (IP) injection. All mice received 100 PFU EBOV in a volume of 100 pl on day 0. Four hours postinfection, group 1 mice received Nanosota-EBl -Fc at a dosage of 50 mg / kg, group 2 mice received Nanosota-EB2-Fc at a dosage of 50 mg / kg, group 3 mice received a cocktail of Nanosota-EBl -Fc and EB2-Fc at a total dosage of 50 mg / kg (25 mg / kg of each nanobody), group 4 mice received Nanosota-EB1 / EB2-Fc bispecific nanobody at a dosage of 50 mg / kg, and group 5 received PBS. Survival rates, body weights, and clinical scores of the mice were recorded for 18 days. Body weights and clinical scores were recorded for the surviving mice. On day 4 post-infection, 4 mice from each group were sacrificed and sera samples were collected for viral load determination. For viral load determination, samples collected were inactivated using Trizol, RNA was then extracted, and RT-qPCR was performed to detect the viral load.

[0623] Table 1. Nanosota-EBl and Nanosota-EB2 Sequences

[0624] SEQ Sequences Comments

[0625] ID NO:

[0626] 1 QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQA Nanosota-EBl VHH PGKQRELVAAI TRGVGS TNYADSVKGRFT I SRDNAKNTMY sequence LQMNSLKPEDTAVYYCNARLLVAPPPYEYDYWGQGTQVTV SS

[0627] 2 GSTSVIYA Nanosota-EBl: CDR-H1 3 I TRGVGS T Nanosota-EBl: CDR-H2 4 NARLLVAPPPYEYDY Nanosota-EBl: CDR-H3 5 QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQA Nanosota-EBl VHH PGKQRELVAAI TRGVGS TNYADSVKGRFT I SRDNAKNTMY sequence with linker LQMNSLKPEDTAVYYCNARLLVAPPPYEYDYWGQGTQVTV (bold) and tag

[0628] S S GGQ HHHHHHGA YP YDVPD YAS including Hise

[0629] ( italics )

[0630] 6 QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQA Nanosota-EBl VHH PGKQRELVAAI TRGVGS TNYADSVKGRFT I SRDNAKNTMY sequence with IgGl LQMNSLKPEDTAVYYCNARLLVAPPPYEYDYWGQGTQVTV Fc domain SSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI sequence (bold)

[0631]

[0632] SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0633] KTI SKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQA Nanosota-EBl VHH PGKQRELVAAI TRGVGS TNYADSVKGRFT I SRDNAKNTMY sequence with IgGl LQMNSLKPEDTAVYYCNARLLVAPPPYEYDYWGQGTQVTV Fc domain SSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI sequence (bold) having T366Y mutation SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0634] (bo Id / under lined) EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTI SKAKGQPRE PQVYTLPPSREEMTKNQVSLYCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQA Nanosota-EB2 VHH PGKDREFAAGIDYNGGRTAYTDSVKGRFTISRDNAKNTVY sequence LQMNSLKPEDTAVYSCAARPWS IANLAYTYDSWGQGTQVT VSS GRTFSNDA Nanosota-EB2: CDR-H1 IDYNGGRT Nanosota-EB2: CDR-H2 AARPWS IANLAYT YDS Nanosota-EB2: CDR-H3 QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQA Nanosota-EB2 VHH PGKDREFAAGIDYNGGRTAYTDSVKGRFTISRDNAKNTVY sequence with linker LQMNSLKPEDTAVYSCAARPWS IANLAYTYDSWGQGTQVT (bold) and tag

[0635] VS SGGQHHHHHHGA YP YDVPD YAS including Hise

[0636] ( italics ) QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQA Nanosota-EB2 VHH PGKDREFAAGIDYNGGRTAYTDSVKGRFTISRDNAKNTVY sequence with IgGl LQMNSLKPEDTAVYSCAARPWS IANLAYTYDSWGQGTQVT Fc domain VSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM sequence (bold) ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQA Nanosota-EB2 VHH PGKDREFAAGIDYNGGRTAYTDSVKGRFTISRDNAKNTVY sequence with IgGl LQMNSLKPEDTAVYSCAARPWS IANLAYTYDSWGQGTQVT Fc domain VSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM sequence (bold) having Y407T mutation ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR

[0637] (bo Id / under lined) EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR Exemplary IgGl Fc TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domain sequence YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT

[0638]

[0639] I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0640] 16 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR IgGl Fc domain TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ sequence having YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT T366Y mutation I SKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPS (underlined) DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0641] 17 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR IgGl Fc domain TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ sequence having YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT Y407T mutation I SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS (underlined) DIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0642] 18 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGGCAGGTGCAGG An exemplary nucleic CTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAG acid encoding CACCAGCGTTATCTATGCCATGGGCTGGTACCGCCAGGCT Nanosota-EBl VHH CCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTACTCGTG sequence GTGTTGGTAGTACAAACTATGCAGACTCCGTGAAGGGCCG ATT GAG C AT C T C C AGAGAC AAT G C C AAGAAC AC GAT G TAT CTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGT ATTACTGTAACGCACGACTATTGGTAGCTCCTCCACCTTA TGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTC TCCTCA

[0643] 19 CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGG An exemplary nucleic CTGGGGGCTCTCTGAGACTCTCCTGTGTACATTCCGGACG acid encoding CACGTTCAGTAACGATGCCATGGCCTGGTTCCGCCAGGCT Nanosota-EB2 VHH CCAGGGAAGGACCGTGAGTTTGCAGCAGGTATTGATTACA sequence ATGGCGGTAGGACAGCCTATACAGACTCCGTGAAGGGCCG ATT GAG C AT T T C C AGAGAC AAC G C C AAGAAC AC G G T G T AT CTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTT ATTCCTGTGCAGCACGCCCCTGGAGTATAGCGAACTTAGC TTATACTTATGACTCCTGGGGCCAGGGGACCCAGGTCACC GTCTCCTCA

[0644]

[0645] Example 3. A Highly Potent and Broadly Accessible Bispecific Nanobody for the Treatment of Ebola Virus Infections

[0646] Ebola virus (EBOV) causes recurring outbreaks, with a case fatality rate of -40%.

[0647] Currently approved vaccine and antibody therapies face major limitations, including only modest reductions in mortality and restricted accessibility due to their reliance on injectionbased delivery and cold-chain transport and storage. To address these challenges, in this Example we developed a bispecific nanobody, Nanosota-EB1 / EB2-Fc (SEQ ID NOs:7 and 14), comprising two camelid-derived single-domain antibodies (Nanosota-EB 1 and Nanosota-EB2) that target distinct epitopes on the EBOV glycoprotein (GP), fused to a human Fc domain. This bispecific nanobody exhibited synergistic inhibition of GP function and effectively overcame the virus’s decoy mechanism. A single dose provided strong protection in EBOV-infected mice, including those in late-stage infection. It was also effective when administered intranasally, offering a needle-free delivery option. Furthermore, its high stability in vitro indicates that it can be deployed without refrigeration. Taken together, this novel bispecific nanobody represents a promising next-generation therapeutic for EBOV, combining high potency with broad accessibility.

[0648] Introduction

[0649] Ebola virus (EBOV) is one of the deadliest known pathogens, with a case fatality rate of -40%. It causes severe hemorrhagic fever, a major factor contributing to its high mortality. To date, EBOV has infected more than 33,000 people, including 28,652 cases during the unprecedented West African outbreak from 2014 to 2016 (7, 2). Smaller outbreaks of EBOV and related filoviruses continue to occur regularly (3). A recent outbreak (2025) in the Democratic Republic of the Congo underscores the persistent risk. It is believed that EBOV has natural reservoirs, although the virus has yet to be definitively identified in the wild (4).

[0650] Moreover, EBOV can persist in the human body for years in a dormant state before reactivating and causing new infections (5). These factors together contribute to the virus’s recurrent emergence. Given its high lethality and long-term persistence in human hosts, EBOV remains a serious threat to global health and national security.

[0651] Current interventions for EBOV infections have major limitations. The only FDA-approved vaccine reduces the fatality rate to approximately 25% - a level that remains unacceptably high (6). Additionally, the two FDA-approved antibody therapies lower mortality to around 35%, including in some late-stage cases - a cause for concern (7, S). Both the vaccine and antibody therapies require injection-based administration and cold-chain transport and storage, severely limiting their accessibility - especially in remote, resource-limited, and warmclimate regions where EBOV outbreaks typically occur (9). As a result of these logistical challenges, only 41% of eligible patients received antibody therapies during recent outbreaks in the Democratic Republic of Congo and Guinea (70). These limitations underscore the urgent need for highly potent and broadly accessible therapies to effectively combat EBOV infections. The EBOV glycoprotein (GP) is the primary target for neutralizing antibodies, as it mediates viral entry into human cells (77). On the viral surface, GP exists in a metastable “pre-fusion” trimeric form, having three copies each of the receptor-binding subunit GP1 and the membranefusion subunit GP2 (72). GP1 contains the receptor-binding site (RBS), which is shielded by a glycan cap and a mucin-like domain (MLD) (77, 73). GP2 contains a fusion peptide and two heptad repeat regions (HR1 and HR2) {14). To initiate infection, GP1 first binds to host cell surface factors, leading to EBOV internalization via endocytosis. Within endosomes, the glycan cap and MLD are removed by proteolytic cleavage, exposing the RBS (75). The exposed RBS then binds to its cellular receptor, Niemann-Pick Cl (NPC1), on the endosomal membrane {16, 17). This interaction triggers a dramatic conformational change in GP2, transitioning it to its most stable “post-fusion” form and enabling fusion of the viral and endosomal membranes {18, 19). Antibodies targeting EBOV GP can potentially interfere with any of these entry steps. In addition, antibodies that recognize exposed GP epitopes, particularly those distal to the viral membrane, can recruit Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) by bridging GP and Fey receptors (FcyRs) on immune cells, thereby activating antiviral responses (PubMed IDs:30096313, 33852832, 22143789, 30092199).

[0652] However, a major challenge for antibody -based therapies is the presence of soluble GP (sGP), a decoy protein encoded by the virus and secreted by infected cells. sGP contains most of GP1 but lacks GP2, and can bind and sequester GP1 -targeting antibodies, diverting them away from the functional GP on the viral surface {20). Therefore, effective anti-EBOV antibodies must not only block GP function but also avoid being diverted by sGP. Antibodies that interfere with GP function while limiting diversion by sGP are therefore likely to be potent neutralizers of EBOV entry. Additionally, antibodies that recognize exposed GP epitopes can contribute to protection by engaging Fc-dependent antiviral effector mechanisms, such as ADCC.

[0653] Nanobodies are single-domain antibodies derived from the heavy-chain-only antibodies of camelid animals (27, 22). Their unique structure offers several therapeutic advantages for antiviral applications. Nanobodies can achieve high antiviral potency, owing to their excellent epitope accessibility and tissue penetration {23, 24). They are also cost-effective to produce, transport, and store, and have the potential for intranasal administration {25, 26). In addition, they exhibit minimal host toxicity due to their high specificity for viral targets, and minimal immunogenicity in humans due to their strong similarity to human germline antibodies (27, 28). In 2019, the FDA approved the first nanobody -based therapeutic for the treatment of a blood clotting disorder {29). Since the onset of COVID- 19, nine nanobody inhibitors have been developed, collectively known as the Nanosota series, that target the SARS-CoV-2 spike protein {30-34). Herein, we developed two nanobodies, Nanosota-EBl and Nanosota-EB2, which are the first nanobodies ever generated against EBOV {35). These two nanobodies bind distinct epitopes on EBOV GP and inhibit its function through different mechanisms: Nanosota-EBl recognizes the glycan cap of GP1, slowing proteolytic cleavage and delaying exposure of the RBS, and its highly exposed epitope suggests substantial ADCC potential; in contrast, Nanosota-EB2 targets key membrane-fusion elements in GP2, blocking the conformational transition required for membrane fusion, but its less exposed epitope suggests limited ADCC potential. These two nanobodies laid a foundation for nanobody -based therapies to treat EBOV infections. Together, these nanobodies establish a foundation for nanobody-based therapies to treat EBOV infection.

[0654] Despite their promise as next-generation anti-EBOV therapeutics, several key questions remain regarding Nanosota-EBl and Nanosota-EB2: regarding whether they can synergistically block GP function, whether their antiviral potency can be maximized through a bispecific design, and whether their unique structural properties can broaden their accessibility. These questions are addressed in this Example 3, and Example 4 below.

[0655] Results

[0656] Design and in vitro characterization of anti-EBOV bispecific nanobody

[0657] Of the two anti-EBOV nanobodies discovered herein, Nanosota-EBl binds to the glycan cap of GP1, slowing proteolytic cleavage and delaying exposure of RBS. In contrast, Nanosota-EB2 targets critical membrane-fusion elements in GP2, blocking its structural transition and preventing membrane fusion (35). Fc-tagged EB2 (EB2-Fc) potently neutralizes both EBOV pseudoviruses (retroviral particles pseudotyped with EBOV GP) and authentic EBOV (ICso = 47 ng / mL), while EBl-Fc shows moderate neutralizing potency against EBOV pseudoviruses but only weak neutralization against authentic virus, likely due to diversion by sGP, which binds EB1 but not EB2. To evaluate whether EB1 and EB2 can synergistically inhibit EBOV, we designed a bispecific nanobody - Nanosota-EB1 / EB2-Fc - combining both nanobodies fused to a human IgG Fc domain (Fig. 23 A). To promote formation of the bispecific nanobody, two Fc-region mutations were introduced: T366Y into the EBl-Fc construct and Y407T into the EB2-Fc construct (36). This "knob-into-hole" strategy facilitates Fc heterodimerization, leading to highly efficient heterodimer formation (>90%). We expressed EB1 / EB2-Fc (SEQ ID NOs:7 and 14) in 293 mammalian cells, achieving a yield exceeding 50 mg / L of culture medium. The purified EB1 / EB2-Fc binds both sGP (via EB1) and GP lacking the glycan cap (via EB2) and also displays remarkable in vitro stability (see below for both properties), confirming successful heterodimer assembly and aligning with previous extensive analyses showing that the knob / hole heterodimer is far more stable than either knob / knob or hole / hole homodimers.

[0658] We then assessed its in vitro potency in neutralizing authentic EBOV. Theoretically, as a hybrid, EB1 / EB2-Fc would be expected to show intermediate potency between EBl-Fc and EB2-Fc. However, EB1 / EB2-Fc outperformed both, demonstrating synergistic inhibition of authentic EBOV with an ICso of 29 ng / mL (Fig. 23 A). To understand the synergistic inhibition of EBOV infection by the bispecific nanobody, we determined the cryo-EM structure of EBOV GP in complex with both EB1 and EB2. Our previous cryo-EM studies of GP bound to individual nanobodies revealed that each trimeric GP was bound by either two EB1 molecules or three EB2 molecules (35). The limited occupancy of EB1 (only two per trimer) was likely due to the flexibility of the EB1 -bound glycan cap, rather than weak binding affinity — since EB1 binds GP with a Kd of 2.77 nM, indicating strong interaction. Surprisingly, in the GP / EB1 / EB2 ternary complex structure, each trimeric GP was bound by three EB1 molecules and three EB2 molecules (Fig. 23B; Fig. 28; Table S4). This finding suggests that the simultaneous binding of EB1 and EB2 stabilizes the glycan cap, allowing full EB1 occupancy and demonstrating synergistic binding of both nanobodies to GP. Further structural analysis indicated that EB2 binding to GP2 indirectly stabilizes the glycan cap via a hinge region (Fig. 23C). Prior studies have shown that glycan cap stabilization interferes with its proteolytic cleavage (35, 37). To evaluate the functional consequence of this synergistic action by EB1 and EB2, we conducted a glycan cap proteolysis assay in the presence of EB1 / EB2-Fc across various incubation times (Fig. 23D). Our previous work showed that EB1-Fc effectively inhibited glycan cap proteolysis for up to 30 minutes (35). In contrast, the current study found that EB1 / EB2-Fc maintained efficient inhibition even after 60 minutes (Fig. 23D). These results demonstrate that the bispecific nanobody is more effective than EBl-Fc at inhibiting glycan cap proteolysis, owing to the synergistic action of EB1 and EB2.

[0659] To explore additional mechanisms underlying the synergistic inhibition of EBOV infection by the bispecific nanobody, we investigated how EB1 / EB2-Fc interacts with GP in the presence of the viral decoy sGP using three complementary assays. First, we assessed whether EB1 / EB2-Fc, when bound to sGP, can still engage GPcl (the glycan cap-cleaved form of GP). Using surface plasmon resonance (SPR), we immobilized sGP on a sensor chip and sequentially introduced EB1 / EB2-Fc followed by GPcl. The resulting SPR signals confirmed that EB1 / EB2-Fc can simultaneously bind sGP (which contains the EB1 epitope but not the EB2 epitope) and GPcl (which contains the EB2 epitope but not the EB1 epitope), indicating that sGP -bound EB1 / EB2-Fc remains capable of engaging the EB2 epitope on GPcl (Fig. 21A). Second, we evaluated whether sGP interferes with EB1 / EB2-Fc binding to GP containing the glycan cap. ELISA was used to measure EB1 / EB2-Fc binding to GP in the presence or absence of sGP, with fold changes in EC so values used to quantify the effect. The results showed that sGP had no significant impact on EB1 / EB2-Fc binding. In contrast, sGP markedly reduced the binding of EBl-Fc, but not EB2-Fc, to GP (Fig. 21B; Fig. 29). Lastly, we examined whether sGP affects the neutralizing potency of EB1 / EB2-Fc. An EBOV pseudovirus entry assay (using retroviruses pseudotyped with full-length EBOV GP) was conducted to assess neutralizing activity in the presence or absence of sGP. Fold changes in pseudovirus entry efficiency, expressed as ICso values, were used to evaluate the effect. The results demonstrated that sGP did not significantly impair the neutralization potency of EB1 / EB2-Fc (Fig. 21C; Fig. 30). By comparison, sGP significantly diminished the neutralizing potency of EBl-Fc, but not EB2-Fc. In summary, these findings demonstrate that the bispecific nanobody binds both the EB1 and EB2 epitopes on GP, resists sGP-mediated diversion, and retains its neutralizing activity in the presence of the sGP decoy. Thus, EB1 / EB2-Fc achieves synergistic inhibition through a second mechanism: overcoming sGP-mediated immune evasion.

[0660] In vivo characterization of anti-EBOV bispecific nanobody

[0661] We evaluated the in vivo efficacy of the bispecific nanobody against authentic EBOV using a stringent mouse model. In this model, wild-type EBOV was used to infect interferon-a / p receptor knockout mice, which are immunocompromised and therefore develop severe symptoms and experience high mortality upon infection. A single dose of EB1 / EB2-Fc was administered intraperitoneally (I P.) at 50 mg / kg, which has been shown to be well tolerated owing to the high safety profile of nanobodies, four hours post-infection. For reference, the FDA-approved antibody cocktail Inmazeb, which consists of three monoclonal antibodies, is administered at a recommended dose of 50 mg / kg for each antibody (38), (150 mg / kg total) to maximize therapeutic benefit in patients, reflecting the high case fatality rate of EBOV infection. In a previous study using the same treatment conditions, the combination of EBl-Fc and EB2-Fc improved survival from 17% in the untreated control group to 83%, while also significantly reducing weight loss and clinical scores (35) (Fig. 20). This performance was slightly better than EB2-Fc alone, which achieved an 83% survival rate but with earlier symptom onset. EBl-Fc alone delayed symptom onset but did not improve survival. In the current study, EB1 / EB2-Fc provided complete protection, achieving 100% survival with no weight loss or clinical symptoms (Fig. 20). These results demonstrate that the bispecific nanobody offers superior in vivo anti-EBOV efficacy compared to the individual nanobodies.

[0662] We also measured serum virus titers on day 4. In our previous study, both EBl-Fc and EB2-Fc produced substantial reductions in viral titers, by approximately 3 to 4 logs relative to the control group. In the current study, EB1 / EB2-Fc similarly reduced virus titers by ~3.5 logs compared with controls (Fig. 20D), but did not distinguish itself from the individual nanobodies by this measure. However, this readout has inherent limitations. Because control mice began to succumb by day 5, day 4 was the latest common sampling time, and this early measurement likely underestimates terminal viral loads, which typically surge immediately before death. Moreover, these nanobodies are viral entry inhibitors that block new infection cycles, each requiring roughly 30 h per cell; thus, day-4 virus titers capture only the early phase of inhibition and are not strictly correlated with the peak viraemia that occurs later. Therefore, although both the bispecific nanobody and the individual nanobodies dramatically reduce serum virus titers, survival and clinical signs are more informative metrics for distinguishing anti-EBOV potency among the nanobodies in this lethal infection mouse model.

[0663] We next assessed the therapeutic potential of the bispecific nanobody in late-stage EBOV infection. A single 50 mg / kg dose of EB1ZEB2-Fc was administered on Day 1, 3, 4, or 5 post-infection. In the untreated group, all mice succumbed before showing high weight loss and clinical scores (Fig. 24). EB1 / EB2-Fc treatment on Day 1 provided near-complete protection, resulting in 100% survival and minimal weight loss and clinical symptoms. Treatment on Day 3 offered strong protection, with 80% survival and mild weight loss and symptoms. A Day 4 dose provided significant protection (40% survival), while treatment on Day 5 offered no benefit. For comparison, favipiravir, a small-molecule EBOV inhibitor (39), was administered daily starting on Day 0. Despite the ongoing dosing, its protective effect matched that of a single EB1 / EB2-Fc dose given on Day 4 in terms of survival, but with greater weight loss and more severe clinical scores (Fig. 24). Overall, the bispecific nanobody showed substantial efficacy even when administered at later stages of infection.

[0664] Finally, we tested the in vivo efficacy of the bispecific nanobody delivered intranasally. A single 50 mg / kg dose of EB1 / EB2-Fc was administered via the intranasal (I. N.) route four hours post-infection. This treatment increased survival from 20% in the untreated group to 80%, while also minimizing weight loss and clinical scores (Fig. 25). These findings confirm that the bispecific nanobody retains strong in vivo anti-EBOV activity even when administered intranasally. They further suggest that, although a I. P. dose of 50 mg / kg of the bispecific nanobody was used in the mouse challenge experiments in accordance with FDA guidance, a substantially lower systemic exposure (corresponding to approximately 5.4% of the 50 mg / kg I. P. dose achieved via the I. N. route; see below for the bioavailability of I. N. compared with I. P. delivery) is sufficient to confer robust protection in EBOV -challenged mice.

[0665] Pharmacokinetics and in vitro stability of anti-EBOV bispecific nanobody

[0666] To evaluate the pharmacokinetics (PK) of the bispecific nanobody, we measured its halflife in mice. First, we established a log-linear relationship between EB1 / EB2-Fc concentrations and chemiluminescence intensities using ELISA (Fig. 26A). A single dose of EB1 / EB2-Fc was then I. P. administered at 20 mg / kg. A key factor influencing the half-life of Fc-tagged proteins is the binding affinity between the Fc region and the neonatal Fc receptor (FcRn) in animal models (40). While human Fc binds well to murine FcRn (41), it is most compatible with its native receptor, human FcRn (hFcRn). Therefore, hFcRn transgenic mice were used in this study. In these mice, plasma EB1 / EB2-Fc levels peaked at 216 pg / mL 24 hours post-injection and gradually declined to 55.3 pg / mL over the following 7 days (Fig. 26B). Based on the PK profile, the estimated plasma half-life of EB1 / EB2-Fc was 7.8 days.

[0667] To evaluate the bioavailability of intranasal (LN.) delivery, a single 50 mg / kg dose of EB1 / EB2-Fc was administered to hFcRn transgenic mice via the I. N. route. Plasma EB1 / EB2-Fc levels peaked at 18.1 pg / mL 24 hours post-injection and gradually declined to 8.9 pg / mL over the following 3 days (Fig. 26C). The total exposure to the bispecific nanobody, represented by the area under the curve (AUC), was calculated for both I. P. and I. N. administrations. Based on these AUC values, the relative bioavailability of I. N. delivery compared to I. P. was determined to be 5.4% (Fig. 26D).

[0668] To assess in vitro stability, EB1 / EB2-Fc was incubated for either one week or two months at -80°C, 4°C, 25°C, or 37°C, and its binding to EBOV GP was evaluated by ELISA. Using -80°C storage as the reference, EB1 / EB2-Fc retained full binding activity after one week and nearly full activity after two months at 37°C (Fig. 27). These findings demonstrate that EB1 / EB2-Fc possesses excellent in vitro stability.

[0669] Discussion

[0670] EBOV remains a major threat to global health and national security due to its high fatality rates and recurring outbreaks. Although two antibody therapies and one vaccine have been approved, these interventions offer only modest reductions in mortality and are hindered by their reliance on injectable administration, high production costs, and cold-chain logistics (6-8). These limitations are particularly problematic in regions where EBOV outbreaks occur, which have warm climates and limited access to healthcare facilities or refrigeration (9). To overcome these challenges, we aimed to develop novel anti-EBOV therapeutics with both high potency and broad accessibility.

[0671] We engineered a bispecific nanobody, Nanosota-EB1 / EB2-Fc, by fusing two individual nanobodies, Nanosota-EBl and -EB2, to a human Fc tag. This design was intended to maximize the antiviral activities of EB1 and EB2, which respectively target glycan cap proteolysis and membrane fusion functions of EBOV GP. EB1 also carries strong ADCC potential because its binding epitope on GP is highly exposed. Structural and biochemical analyses demonstrated that the bispecific nanobody synergistically inhibits GP function by more effectively blocking glycan cap cleavage and successfully evading the sGP decoy mechanism. The synergistic, cooperative binding of EB1 and EB2 stabilizes the glycan cap and prevents its proteolytic cleavage, thereby limiting exposure of the RBS. As a result, although the bispecific nanobody combines a strong neutralizer (EB2) with a weak neutralizer (EB1) and might be expected to exhibit intermediate activity, it instead displays neutralizing potency matching that of EB2-Fc (containing two copies of EB2) alone in vitro. Meanwhile, the nanobody resists the sGP decoy by retaining its ability to bind GP even in the presence of sGP, thus overcoming viral immune evasion. While a cocktail of EB1 and EB2 can also block glycan cap cleavage, it cannot evade the sGP decoy. In contrast, the bispecific nanobody offers advantages over antibody cocktails in terms of manufacturing, stability, delivery, and logistics. The bispecific nanobody preserves the strong neutralizing activity of EB2 and the proteolysis interference activity of EB1, both of which are manifested in vitro and in vivo, and it also retains the strong ADCC potential of EB1, which is manifested only in vivo. The modular, single-domain nature of nanobodies makes them especially well suited for bispecific fusion with Fc tags - unlike conventional antibodies, which often face steric challenges in bispecific formats. Overall, this design leverages nanobody modularity to effectively target critical and unique mechanisms of EBOV entry and immune evasion.

[0672] Due to its novel mechanisms of action, the bispecific nanobody shows strong promise as a highly potent therapeutic candidate against EBOV. In our stringent preclinical model using immunocompromised mice, EBOV infection resulted in 80-100% mortality, with fatalities typically beginning around Day 5 post-infection. A single dose of the bispecific nanobody I. P. administered at early stages (4 hours or Day 1 post-infection) provided complete or nearcomplete protection, achieving 100% survival with minimal weight loss and clinical symptoms. At Day 3, a single dose still offered strong protection with 80% survival and small weight loss and clinical symptoms. Even at Day 4, one day before mice succumb, the nanobody provided significant protection, with 40% survival and reduced weight loss and clinical symptoms.

[0673] Although we were unable to obtain FDA-approved antibody drugs for direct comparison, we tested Favipiravir, a known EBOV polymerase inhibitor (39), as a benchmark. When administered daily, Favipiravir produced the same survival rate as a single Day 4 dose of the bispecific nanobody but resulted in worse weight loss and clinical symptoms, highlighting both the stringency of our preclinical model and the superior potency of the bispecific nanobody. These results demonstrate that the bispecific nanobody offers strong protection against both early and late EBOV infections in a stringent preclinical model, though further validation in other models, particularly non-human primates, is needed.

[0674] Despite having nearly identical neutralizing potency to EB2-Fc in vitro, the bispecific nanobody outperforms EB2-Fc in vivo. Because its in vitro potency already captures the synergy between EB1 and EB2, the superior in vivo activity of the bispecific nanobody over EB2-Fc likely arises from additional antiviral effects, such as antibody-dependent cellular cytotoxicity (ADCC), that are present in the bispecific nanobody but absent in EB2-Fc. By combining an exposed epitope targeting arm (EB1) with a human IgG Fc domain, the bispecific nanobody should be able to engage Fc-mediated antiviral mechanisms, including ADCC, through simultaneous binding of the exposed GP epitope and Fey receptors (FcyRs) on immune cells. However, in the current study this Fc-mediated potential may not have been fully manifested because two factors likely dampened these effects: (i) production in human Expi293F cells, which typically yields Fc glycans associated with reduced ADCC, and (ii) the limited crossspecies affinity of human Fc for mouse FcyRs. To evaluate the full contribution of Fc-driven antiviral mechanisms, we will express the bispecific nanobody in CHO cells to generate an ADCC-enhanced Fc variant and assess its efficacy in human FcyR transgenic mice. These future studies will determine whether Fc-driven activity can further augment the already potent anti-EBOV efficacy of the bispecific nanobody reported here, thereby strengthening its potential as a next-generation anti-EBOV therapeutic.

[0675] In addition to its antiviral potency, the bispecific nanobody shows strong promise as a widely accessible therapeutic. Existing vaccines and antibody treatments are limited by their reliance on injection and cold-chain logistics. In contrast, our nanobody was highly effective when delivered intranasally (I. N.), providing strong protection in EBOV-infected mice and enabling needle-free administration in home or field settings. The nanobody’s human IgG Fc tag increases its molecular size beyond the renal clearance threshold (2S), resulting in a serum halflife of 7.8 days in mice - suitable for single-dose therapeutic use. Despite this increase in size, the Fc-tagged bispecific nanobody remains about half the size of a full human IgG antibody, which substantially enhances its intranasal delivery efficiency. Pharmacokinetic analysis showed that I. N. delivery achieved 5.4% bioavailability relative to I. P. injection, with plasma concentrations reaching 18.1 pg / mL at 24 hours. The combination of an 18.1 pg / mL plasma concentration at 24 hours after I. N. delivery and a 7.8-day half-life ensures that plasma levels remain well above the 29 ng / mL ICso of the bispecific nanobody for an extended period, accounting for its strong in vivo efficacy via the I. N. route. Moreover, the nanobody demonstrated exceptional in vitro stability. After incubation at 37°C for two months, it retained nearly all of its target-binding activity, indicating that transport and storage without cold-chain infrastructure is feasible. This in vitro stability likely stems from its heavy-chain-only structure, which avoids the denaturation issues commonly seen in conventional antibodies with separate heavy and light chains. Taken together, the bispecific nanobody’s needle-free delivery and coldchain independence make it far more accessible than current vaccines and antibody therapeutics - especially in regions affected by EBOV outbreaks, where limited clinical infrastructure, warm weather, and lack of refrigeration present major logistical barriers.

[0676] Although the bispecific nanobody exhibits broad-spectrum anti-EBOV activity by potently neutralizing four major EBOV strains, nanobody resistance mutations may emerge during treatment and compromise its efficacy. A key advantage of bispecific nanobodies is that they engage two distinct epitopes on the same GP, so the virus would need to acquire simultaneous mutations at both sites to escape neutralization, an event that is far less likely than resistance to a monospecific nanobody.

[0677] Like antibody cocktails, nanobody cocktails have several advantages over single nanobodies as potential anti-EBOV therapeutics, including synergistic inhibition of EBOV entry and a reduced risk of viral escape. Our previous study showed that a cocktail of EBl-Fc and EB2-Fc (25 mg / kg each) provided similar protection to EB2-Fc alone (50 mg / kg) in EBOV-challenged mice, indicating that synergy in neutralizing EBOV entry is present but limited and weaker than that achieved by the bispecific nanobody. This synergy likely arises from cooperative inhibition of glycan cap proteolysis by the two nanobodies. However, because of the sGP decoy effect, not all EBl-Fc molecules successfully bind GP on viral particles, which restricts their ability to block glycan cap proteolysis and to mediate ADCC. This explains why the bispecific nanobody provides complete protection in EBOV-challenged mice during early-stage infection, whereas the EB1-Fc / EB2-Fc cocktail, like EB2-Fc alone, confers strong but incomplete protection. Moreover, because the bispecific nanobody is a single protein molecule, manufacturing, storage, transport, and product quality control are substantially easier than for cocktails. Overall, the bispecific nanobody offers several advantages over nanobody cocktails as a potential anti-EBOV therapeutic.

[0678] In summary, a bispecific nanobody was developed that targets two distinct epitopes on EBOV GP, enabling synergistic inhibition of viral entry, immune evasion and Fc-mediated antiviral effector mechanisms. The nanobody demonstrated strong protective efficacy in a stringent EBOV mouse model, remained effective even when administered at late stages of infection or via the intranasal route, and exhibited excellent in vitro stability. With its potent antiviral activity, needle-free delivery capability, and cold-chain independence, this novel bispecific nanobody represents a promising next-generation therapeutic candidate for future EBOV outbreak preparedness and response.

[0679] Data availability: The atomic model and corresponding cryo-EM density map of the ternary complex comprising EBOV GP, Nanosota-EBl, and Nanosota-EB2 have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank, respectively, under accession numbers 9P6X and EMDB-71328.

[0680] Table S4: Cryo-EM data collection, model refinement and validation statistics.

[0681] EBOV GP / EB1 / EB2

[0682] ternary complex

[0683] Data collection and processing

[0684] Magnification 130,000

[0685] Voltage (kV) 300

[0686] Electron exposure (e— / A2) 50.00

[0687] Defocus range (pin) - 1.0 - -2.0

[0688] Pixel size (A) 0.664

[0689] Symmetry imposed C3

[0690] Initial particle images (no.) 3,765,265

[0691] Final particle images (no.) 62,575

[0692] Map resolution (A) 2.92

[0693] FSC threshold 0.143

[0694] Map resolution range (A) 2.6— 7.2

[0695] Model refinement

[0696] Initial model used (PDB code) 9BSU / 9BSV

[0697] Model resolution (A) 3.1

[0698] FSC threshold 0.5

[0699] Model resolution range (A) 43.3-2.8

[0700] Map sharpening B factor (A2) -83.5

[0701] Model composition

[0702] Non-hydrogen atoms 13954

[0703] Protein residues 1770

[0704] Ligands 20

[0705] B factors (A2)

[0706] Protein 59.40

[0707] Nucleotide

[0708] Ligand 69.53

[0709] R.m.s. deviations

[0710] Bond lengths (A) 0.005

[0711] Bond angles (° ) 1.031

[0712] Validation

[0713] MolProbity score 1.58

[0714] Clashscore 4.27

[0715] Poor retainers (%) 0.21

[0716] Ramachandran plot

[0717] Favored (%) 94.59

[0718] Allowed (%) 5.01

[0719] Disallowed (%) 0.40

[0720] Materials and methods

[0721] Cell lines, plasmids, and virus

[0722] Cell lines, plasmid and virus are as described in Example 1 and Example 2.

[0723] Protein expression and purification

[0724] The EBOV GP ectodomain, EBOV sGP, and Fc-tagged nanobodies were expressed and purified from mammalian cells as previously described (26, 42). Briefly, plasmids encoding each of these proteins were transiently transfected into Expi293F cells using polyethylenimine (PEI; Polysciences). For expression of Nanosota-EB1 / EB2-Fc, equal amounts of expression plasmids for EBl-Fc (carrying the T366Y mutation in the Fc region) and EB2-Fc (carrying the Y407T mutation in the Fc region) were co-transfected into Expi293F cells. Three days posttransfection, all proteins were harvested from the culture supernatants. The EBOV GP ectodomain and sGP were purified using aNi-NTA affinity column followed by further purification on a Superose 6 increase 10 / 300 gel filtration column (Cytiva). Fc-tagged nanobodies were purified using a Protein A column followed by further purification on a Superdex 200 increase 10 / 300 gel filtration column (Cytiva). To generate EBOV GPcl (glycan cap-cleaved form of GP ectodomain), 3 mg of EBOV GP ectodomain was digested with 15 pg of thermolysin L (Sigma- Aldrich) overnight at room temperature and then purified on a Superose 6 increase 10 / 300 column (Cytiva).

[0725] To prepare the ternary complex of EBOV GP ectodomain, EB1, and EB2, the GP ectodomain was incubated with an excess of His-tagged EB1 and EB2 at room temperature for 1 hour. The complex was then purified using a Superose 6 increase 10 / 300 column (Cytiva). Cryo-EM data collection, data processing, model building and refinement

[0726] 4 pL of the purified ternary complex of EBOV GP ectodomain, EB1, and EB2 (-1.25 pM) was applied to freshly glow-discharged Quantifoil Rl.2 / 1.3 300-mesh copper grids (EM Sciences). Grids were blotted for 4 seconds at 22°C under 100% chamber humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM data were collected using a Latitude-S system (Gatan) equipped with a K3 direct electron detector and a Biocontinuum energy filter (Gatan). Movies were recorded at a nominal magnification of 130,000 / , corresponding to a pixel size of 0.664 A. Data collection statistics are summarized in Table SI.

[0727] Cryo-EM data were processed using cryoSPARC v3.3.2 (43), with the workflow summarized in Fig. 28. Briefly, dose-fractionated movies were aligned using Patch motion correction with MotionCor2 (44) and CTF parameters were estimated using Patch CTF estimation with CTFFIND-4.1.13 (45). Particles were picked using the Blob picker in cryoSPARC v3.3.2. Junk particles were eliminated through three rounds of 2D classification. Particles from well-defined 2D classes were used for ab initio reconstruction into three maps. These initial models served as starting references for heterogeneous refinement (3D classification). The best 3D classes were then refined using homogeneous, non-uniform, and CTF refinement procedures to generate the final maps, with C3 symmetry applied for the ternary complex. Map resolutions were determined using gold-standard Fourier shell correlation (FSC) at the 0.143 threshold between the two half-maps.

[0728] Initial model building of the ternary complex was performed in Coot vO.8.9 (46), using the GP ectodomain from PDB entry 9BSU as the starting model. The initial nanobody models were taken from PDB entries 9BSU and 9BSV, respectively, and fitted into the density map. Multiple rounds of refinement in Phenix vl.16 (47) and manual adjustments in Coot were carried out to generate the final model. Model and map statistics are summarized in Table SI. All structural figures were prepared using UCSF ChimeraX v0.93 (4S).

[0729] Glycan cap cleavage

[0730] Proteolysis of the GP glycan cap was performed as previously described (35). Briefly, 60 pg of EBOV GP ectodomain complexed with EB1 / EB2-Fc was treated with 0.25 pg of thermolysin L (Sigma- Aldrich) at 37 °C for varying durations (5, 15, 30, or 60 minutes). As a control, 60 pg of EBOV GP ectodomain alone was treated under the same conditions. At each time point, aliquots were immediately mixed with SDS-PAGE loading buffer and boiled for 10 minutes to terminate the reaction. All samples were then analyzed by non-reducing SDS-PAGE.

[0731] Surface plasmon resonance

[0732] Surface plasmon resonance (SPR) was performed to assess the simultaneous binding of EB1 / EB2-Fc to sGP and GPcl. Recombinant sGP was immobilized on a CM5 sensor chip (Cytiva) via chemical crosslinking. EB1 / EB2-Fc (40 pg / mL) was first injected, followed by injection of GPcl at the same concentration. SPR signals were recorded to monitor binding. ELISA to evaluate the effect of sGP on the bispecific nanobody binding to EBOV GP An ELISA was performed to assess the effect of recombinant sGP on the binding of three nanobodies — EBl-Fc, EB2-Fc, and the bispecific EB1 / EB2-Fc — to recombinant EBOV GP ectodomain. ELISA plates were coated with GP ectodomain, and serial dilutions of each nanobody (starting at 10 pg / mL with 4-fold dilution steps) were added. Recombinant sGP was then introduced at two concentrations (1 pg / mL and 5 pg / mL). After incubation, a horseradish peroxidase (HRP)-conjugated anti-Fc antibody (1:3,000; Sigma-Aldrich) was added. The ELISA substrate (Invitrogen) was then applied, and the reactions were stopped using IN EESCk Absorbance at 450 nm (A450) was measured using a Synergy LX Multi-Mode Reader (BioTek). Binding interactions were quantified by calculating the half-maximal effective concentration (EC so), defined as the concentration of nanobody required to elicit 50% of the maximum signal.

[0733] In vitro stability of the bispecific nanobody

[0734] EB1 / EB2-Fc was incubated at various temperatures for either 1 week or 2 months.

[0735] ELISA was then performed to assess its residual target-binding activity, as previously described (35). Briefly, ELISA plates were coated with recombinant EBOV GP ectodomain, followed by the addition of serially diluted EB1 / EB2-Fc. Subsequent steps were carried out as described in the ELISA procedure above.

[0736] Neutralizing potency of the bispecific nanobody against EBOV pseudoviruses

[0737] The EBOV pseudovirus entry assay was performed as described in Example 2.

[0738] Neutralizing potency of the bispecific nanobody against authentic EBOV

[0739] The neutralizing potency of EB1ZEB2-Fc against authentic EBOV was assessed as previously described (35). EB1 / EB2-Fc was diluted to specified concentrations in cell culture medium and incubated with the virus for 60 minutes. Infection levels were evaluated using an immunofluorescence assay. Cell nuclei were stained with Hoechst dye (ThermoFisher), and nuclei counts were used as a proxy for total cell counts. The efficacy of EB1 / EB2-Fc was determined by calculating the concentration required to reduce the number of infected cells by 50% (ICso) relative to the virus-only control group.

[0740] Anti-EBOV efficacy of the bispecific nanobody in mice

[0741] The in vivo efficacy of EB1 / EB2-Fc against EBOV was evaluated in an interferon-a / p receptor knockout (IFNAR-KO) mouse model across three experiments.

[0742] In the first experiment, authentic EBOV and nanobodies were administered via I. P. injection as described in Example 2. All mice were challenged with 100 PFU of EBOV in 100 pL on Day 0. Four hours post-infection, mice received one of the indicated nanobodies at a dose of 50 mg / kg. Survival, body weight, and clinical scores were monitored for 18 days. On day 4, four animals per group (2 males and 2 females) were randomly selected, euthanized, and serum was collected; these animals were excluded from the survival, body weight, and clinical score analyses. The remaining animals continued in the study under the same conditions until day 18, when the experiment was terminated.

[0743] To measure serum viral loads, sera were inactivated using Trizol LS (Invitrogen) and used to determine genome copy number (GN). Total RNA was isolated with the Direct-zol RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies). EBOV-specific primers (IDT, 5'-CATGCGTACCAGGGAGATTAC-3', 5'-ACTCCATCACGCTTCTTGAC-3') and an EBOV-specific probe (IDT, 5'- / 56-FAM / TCAAGTATT / ZEN / TGGAAGGGCACGGGT / 3IABkFQ / -3') were used for reversetranscription quantitative PCR (RT-qPCR) with the Luna One-Step Universal Probe RT-qPCR Kit (New England Biolabs), following the manufacturer’s recommendations, on a Bio-Rad instrument using Bio-Rad Maestro software for analysis. A standard curve was generated from a synthetic EBOV RNA standard using 10-fold serial dilutions in water. Each sample, including standards, was run in duplicate along with a non-targeting control. Duplicate values were averaged, and the standard curve was used to back-calculate GN for the serum samples.

[0744] In the second experiment, 30 mice were randomized into 6 groups (n=5). EBOV, EB1 / EB2-Fc, and favipiravir were administered via I. P. injection. All mice received 100 PFU of EBOV in 100 pL on Day 0. EB1 / EB2-Fc was administered at a dose of 50 mg / kg at the following time points post-infection: 4 hours (group 1), Day 1 (group 2), Day 3 (group 3), or Day 4 (group 4). Group 5 (untreated control) received PBS at 4 hours post-infection, and group 6 received favipiravir daily at a dose of 100 mg / kg. Survival, body weight, and clinical scores were monitored for 11 days.

[0745] In the third experiment, 10 mice were randomized into 2 groups (n=5). EBOV was administered via I. P. injection, while EB1 / EB2-Fc was delivered intranasally (I. N.). All mice were infected with 100 PFU of EBOV in 100 pL on Day 0. Four hours post-infection, mice in the treatment group received 50 mg / kg of EB1 / EB2-Fc via the I. N. route, while control mice received PBS. Survival, body weight, and clinical scores were monitored for 11 days.

[0746] Statistical analysis of mouse study

[0747] GraphPad Prism (version 10.3.0) was used for all data analysis and statistical evaluations. Survival curves were generated using the Kaplan-Meier method. Percent weight change for each animal was calculated relative to its baseline weight. P-values for weight change were calculated between the indicated experimental groups using a mixed-effects model with Geisser-Greenhouse correction. Clinical scores were recorded as the highest score observed for each group on each day. Viral loads were quantified and expressed as genome numbers per milliliter (GN / mL). GN / mL values were log-transformed, and P values were determined by ordinary one-way ANOVA with multiple comparisons on day 4, comparing each experimental group with the vehicle control.

[0748] Pharmacokinetics of the bispecific nanobody in mice

[0749] Human FcRn transgenic mice (Jackson Laboratory) received EB1 / EB2-Fc either via I. P. at a dose of 20 mg / kg or via the intranasal (I. N.) route at 50 mg / kg. At 24-168 hours postadministration, small blood samples were collected from the tail vein and plasma was stored at -80 °C until analysis.

[0750] A calibration curve for quantifying EB1 / EB2-Fc in plasma was established, demonstrating a log-linear relationship between plasma nanobody concentrations and chemiluminescence intensities, which was used for subsequent ELISA measurements. To minimize interference from endogenous plasma proteins, plasma samples were diluted at least 100-fold in PBS prior to ELISA. ELISA was performed to measure EB1 / EB2-Fc concentrations in mouse plasma. Plates were coated with donkey anti -human IgGFc antibody (Jackson ImmunoResearch) at 4°C overnight. After washing with PBS, plates were blocked with 2% BSA at room temperature for 1 hour. Following PBST (PBS + 0.05% Tween) washes, mouse plasma samples were added and incubated at room temperature for 1 hour. Plates were then washed again with PBST and incubated with HRP-conjugated goat anti-human IgG Fc antibody (1:20,000 dilution; Jackson ImmunoResearch) for 1 hour at room temperature. After sequential washes with PBST and PBS, Super Signal Femto substrate (ThermoFisher) was added for 1 minute, and chemiluminescence was measured using a BioTek Synergy Hl plate reader (Agilent).

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[0789] 39...

Claims

CLAIMSWhat is claimed is:

1. An isolated anti-Ebola binder protein comprising:(1) one or more complementarity determining regions (CDRs) selected from the group consisting of:(a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GRTFSNDA (SEQ ID NO: 9);(b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of IDYNGGRT (SEQ ID NO: 10); and(c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO: 11); and / or(2) one or more CDRs selected from the group consisting of:(a) a CDR1 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of GSTSVIYA (SEQ ID NO:2);(b) a CDR2 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of ITRGVGST (SEQ ID NO:3); and(c) a CDR3 comprising an amino acid sequence having at least 75% sequence identity to an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

2. The isolated anti-Ebola binder protein of claim 1, comprising one or more CDRs selected from the group consisting of:(a) a CDR1 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of GRTFSNDA (SEQ ID NO: 9);(b) a CDR2 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of IDYNGGRT (SEQ ID NOTO); and(c) a CDR3 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO: 11).

3. The isolated anti-Ebola binder protein of claim 1, comprising one or more CDRs selected from the group consisting of:(a) a CDR1 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of GSTSVIYA (SEQ ID NO:2);(b) a CDR2 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of ITRGVGST (SEQ ID NO:3); and(c) a CDR3 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

4. The isolated anti-Ebola binder protein of claim 2, comprising:(a) a CDR1 comprising an amino acid sequence of GRTFSNDA (SEQ ID NO:9);(b) a CDR2 comprising an amino acid sequence of IDYNGGRT (SEQ ID NOTO); and(c) a CDR3 comprising an amino acid sequence of AARPWSIANLAYTYDS (SEQ ID NO: 11).

5. The isolated anti-Ebola binder protein of claim 3, comprising:(a) a CDR1 comprising an amino acid sequence of GSTSVIYA (SEQ ID NO:2);(b) a CDR2 comprising an amino acid sequence of ITRGVGST (SEQ ID NO:3); and(c) a CDR3 comprising an amino acid sequence of NARLLVAPPPYEYDY (SEQ ID NO:4).

6. The isolated anti-Ebola binder protein of claim 1, comprising an amino acid sequence that has at least 80% sequence identity to:QVQLQESGGGLVQAGGSLRLSCVHSGRTFSNDAMAWFRQAPGKDREFAAGIDY NGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAARPWSIANLAYTYD SWGQGTQVTVSS (SEQ ID NO:8); or QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVAAITR GVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNARLLVAPPPYEYD YWGQGTQVTVSS (SEQ ID NO:1).

7. The isolated anti-Ebola binder protein of claim 1, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:8, or SEQ ID NO: 1.

8. The isolated anti-Ebola binder protein of claim 2, comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 8.

9. The isolated anti-Ebola binder protein of claim 3, comprising an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 1.

10. The isolated anti-Ebola binder protein according to any one of claims 1-9, wherein thebinder protein comprises an anti-Ebola single-domain antibody (sdAb) that is linked to at least one polypeptide tag through a peptide bond or a polypeptide linker.

11. The isolated anti-Ebola binder protein of claim 10, wherein the at least one polypeptide tag is operably linked to the C-terminus of the sdAb.

12. The isolated anti-Ebola binder protein of any one of claims 10-11, wherein the at least one polypeptide tag comprises a His tag (e.g., a Hise tag), HA tag, Myc tag, or Fc tag.

13. The isolated anti-Ebola binder protein of claim 12, wherein the at least one polypeptide tag comprises a Fc tag.

14. The isolated anti-Ebola binder protein of claim 13, wherein the Fc tag is a human IgGl, IgG2, IgG3, or IgG4 Fc.

15. The isolated anti-Ebola binder protein of claim 14, wherein the Fc tag comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 15.

16. The isolated anti-Ebola binder protein of claim 15, comprising an amino acid sequence that has at least 90% sequence identity to any one of:(a) Q VQLQESGGGLVQ AGGSLRLSC VHSGRTF SNDAMAWFRQAPGKDREF AA GIDYNGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAR PWSIANLAYTYDSWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 13);(b) Q VQLQESGGGLVQ AGGSLRLSC VHSGRTF SNDAMAWFRQ APGKDREF AA GIDYNGGRTAYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAR PWSIANLAYTYDSWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 14);(c) QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVA AITRGVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNAR LLVAPPPYEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 6); and(d) QVQLQESGGGQVQAGGSLRLSCAASGSTSVIYAMGWYRQAPGKQRELVA AITRGVGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNAR LLVAPPPYEYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO:7).

17. The isolated anti -Ebola binder protein of claim 16, comprising an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs:6, 7, 13, and 14.

18. The isolated anti-Ebola binder protein of claim 17, comprising an amino acid sequence of any one of SEQ ID NOs: 6, 7, 13, and 14.

19. The isolated anti -Ebola binder protein of any one of claims 1-18, wherein the binder protein is a bispecific binder protein comprising a first sdAb domain and a second sdAb domain.

20. The isolated anti -Ebola binder protein of claim 19, whereinthe first sdAb domain comprises(a) a CDR1 comprising an amino acid sequence of SEQ ID NO:9;(b) a CDR2 comprising an amino acid sequence of SEQ ID NO: 10; and (c) a CDR3 comprising an amino acid sequence of SEQ ID NO: 11; and the second sdAb domain comprises(a) a CDR1 comprising an amino acid sequence of SEQ ID NO:2;(b) a CDR2 comprising an amino acid sequence of SEQ ID NO:3; and (c) a CDR3 comprising an amino acid sequence of SEQ ID NO:4.

21. The isolated anti -Ebola binder protein of claim 20, whereinthe first sdAb domain comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 8; andthe second sdAb domain comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 1.

22. The isolated anti -Ebola binder protein of claim 21, whereinthe first sdAb domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 8; andthe second sdAb domain comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1.

23. The isolated anti -Ebola binder protein of claim 22, whereinthe first sdAb domain comprises an amino acid sequence of SEQ ID NO:8; and the second sdAb domain comprises an amino acid sequence of SEQ ID NO:1.

24. The isolated anti -Ebola binder protein of any one of claims 1-23 (e.g., any one of claims 1-17), comprising two independently selected sdAb-Fc fusion proteins, wherein the two Fc tag polypeptides are linked to form a dimer (e.g., homodimer or heterodimer).

25. The isolated anti -Ebola binder protein of any one of claims 1-18 and 24, comprising two sdAb-Fc fusion proteins that are the same.

26. The isolated anti-Ebola binder protein of claim 25, comprising a sdAb-Fc fusion protein that comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13.

27. The isolated anti-Ebola binder protein of claim 26, comprising a sdAb-Fc fusion protein that comprises an amino acid sequence of SEQ ID NO: 13.

28. The isolated anti-Ebola binder protein of claim 25, comprising a sdAb-Fc fusion protein that comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:6.

29. The isolated anti-Ebola binder protein of claim 28, comprising a sdAb-Fc fusion protein that comprises an amino acid sequence of SEQ ID NO:6.

30. The isolated anti-Ebola binder protein of claim 24, wherein the two sdAb-Fc fusion proteins are different.

31. The isolated anti-Ebola binder protein of claim 30, comprising a first sdAb-Fc fusion protein that comprises a Fc tag having Y407T and a second sdAb-Fc fusion protein thatcomprises a Fc tag having T366Y.

32. The isolated anti-Ebola binder protein of any one of claims 30-31, wherein the first sdAb-Fc fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 14 and the second sdAb-Fc fusion protein comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:7.

33. The isolated anti-Ebola binder protein of any one of claims 30-32, wherein the first sdAb-Fc fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14 and the second sdAb-Fc fusion protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7.

34. The isolated anti-Ebola binder protein of any one of claims 30-33, wherein the first sdAb-Fc fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14 and the second sdAb-Fc fusion protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7.

35. The isolated anti-Ebola binder protein of any one of claims 30-34, wherein the first sdAb-Fc fusion protein comprises an amino acid sequence of SEQ ID NO: 14 and the second sdAb-Fc fusion protein comprises an amino acid sequence of SEQ ID NO:7.

36. A composition comprising the isolated anti-Ebola binder protein according to any one of claims 1-35, and a carrier.

37. The composition of claim 36, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

38. The composition of claim 36 or 37, which comprises two or more proteins selected from the group consisting of:(a) the isolated anti-Ebola binder protein as described in any one of claims 2, 4, 8, and 26-27; and(b) the isolated anti-Ebola binder protein as described in any one of claims 3, 5, 9, and 28-29.

39. An isolated polynucleotide comprising a nucleotide sequence encoding an isolated antiEbola binder protein of any one of claims 1-35, or a pair of isolated polynucleotides that comprise a first isolated polynucleotide comprising a nucleotide sequence encoding the first sdAb domain as described in any one of claims 20-23 or the first sdAb-Fc fusion protein as described in any one of claims 31-35, and a second isolated polynucleotide comprising a nucleotide sequence encoding the second sdAb domain as described in any one of claims 20-23 or the second sdAb-Fc fusion protein as described in any one of claims 31-35.

40. A vector comprising the polynucleotide(s) of claim 39, or a pair of vectors that comprise a first vector comprising the first isolated polynucleotide as described in claim 39 and a second vector comprising the second isolated polynucleotide as described in claim 39.

41. A cell comprising the polynucleotide(s) of claim 39 or the vector(s) of claim 40.

42. A method of inhibiting the activity of Ebola virus, comprising contacting Ebola virus with an isolated anti -Ebola binder protein of any one of claims 1-35.

43. A method for treating or preventing an Ebola virus infection in a mammal, comprising administering an effective amount of an isolated anti-Ebola binder protein of any one of claims 1-35 to the mammal.

44. The method of claim 43, further comprising administering at least one additional therapeutic agent to the mammal.

45. The use of an isolated anti-Ebola binder protein of any one of claims 1-35, to prepare a medicament for the treatment of an Ebola virus infection in a mammal.

46. An isolated anti-Ebola binder protein of any one of claims 1-35 or a composition of any one of claims 36-38 for use in the treatment of an Ebola virus infection in a mammal.

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