EA048493B1 - APPLICATION OF PHARMACEUTICAL COMPOSITIONS OF HUMAN ANTIBODIES TO EBOLA VIRUS GLYCOPROTEIN - Google Patents

Abstract

The present invention relates to the use of a pharmaceutical composition (variants) comprising one or more isolated antibodies for inhibiting the activity of the Ebola virus (EBOV); preventing, treating, or alleviating at least one symptom of an infection caused by EBOV, or reducing the frequency or severity of at least one symptom of an infection caused by EBOV; treating a subject who has EBOV, or increasing the survival or likelihood of survival of a subject suffering from an infection caused by EBOV, or a subject exposed to EBOV or at risk of interacting with or acquiring EBOV; prophylactically treating a subject exposed to EBOV or at risk of interacting with EBOV or at risk of acquiring an infection caused by EBOV, or minimizing the risk of acquiring an infection caused by EBOV in a subject exposed to EBOV or at risk of interacting with EBOV. The pharmaceutical antibody compositions of the present invention can be used prophylactically or therapeutically and can be used alone or in combination with one or more other antiviral agents or vaccines.

Description

Field of technology to which the present invention relates

The present invention relates to antibodies that bind to a glycoprotein of a virus

Ebola, pharmaceutical compositions containing such antibodies, and methods of using them.

Prior art

Ebola virus (EBOV) and related filoviruses cause severe viral hemorrhagic fever in humans and non-human primates, with mortality rates in human outbreak sites of up to approximately 90%. (Murin, C.D. et al., (2014), Proc Natl Acad Sci USA, 111(48): 17182–17187). Research continues into the immune mechanisms that mediate protection, but to date no treatments have been approved for use in humans.

The Ebola virus glycoprotein (GP) is the only protein present on the surface of the virus and on infected cells. It is thought to be responsible for binding and fusion of the virus with host cells. GP exists in several forms. These GPs are encoded in two open reading frames. Unedited GP mRNA yields nonstructural secreted, soluble GP (sGP), which is synthesized early during infection (Volchkova, et al. (1995) Virology 214: 421-430; Volchkova, VA et al., (1998) Virology 250: 408-414; Sanchez, et al (1996) Proc Natl Acad Sci USA, 93: 3602-3607; Sanchez, et al (1999) J. Infect. Dis. 179 (suppl. 1, SI64)). The mentioned sGP forms dimers (Volchkova, et al (1995), Virology 214: 421-430; Falzarano, D. et al., Chembiochem (2006), 7: 1605-1611), and their large amounts are detected in the blood of patients and experimentally infected animals (Sanchez, et al. (1996), Proc Natl Acad Sci USA, 93:3602-3607; Dolnik, O. et al., (2004), EMBO J 23: 2175-2184).

At a later stage of infection, an edited mRNA is produced that acquires coding capacity from the second open reading frame. This edited mRNA encodes a form of GP that contains a transmembrane (TM) domain that allows this form of GP to bind to the cell plasma membrane and be incorporated into virions, where it functions as a functional host cell receptor binding/fusion protein.

During the biosynthesis of this form of GP, the protein is proteolytically processed into two products that are held together by disulfide bonds. The amino-terminal product is called GP1 (140 kDa) and the carboxy-terminal cleavage product is called GP2 (26 kDa) (Sanchez, et al. (1998) J. Virol. 72:64426447).

Ebola virus GP (EBOV GP) may be a target for protective antibodies, but the role of antibodies in disease resistance is controversial. Low serum neutralizing antibody titers in convalescent patients, coupled with inconsistent results in achieving protection with experimental transfer of immune sera into animals, have led to speculation about the role of neutralizing antibodies in recovery from infection (Peters, CJ and LeDuc, JW, (1999), J. Infect. Dis. 179 Suppl 1; Mikhailov, VV, (1994), Vopr. Virusol. 39: 82; Xu, L. et al., (1998), Nature Med. 4: 37). However, in one of the more recent Ebola outbreaks, several patients who had contracted the disease and were treated with a cocktail of monoclonal antibodies (ZMapp) specific to the viral GP recovered from the disease. Moreover, other patients who were treated with serum from such patients and from other patients who had survived the infection also had positive results.

Several antibodies that bind Ebola virus GP have been described (see, e.g., U.S. Patents 6,630,144, 6,875,433, 7,335,356, and 8,513,391. See also EP1539238, EP2350270, and EP8513391).

Although technological advances have increased the potential to produce improved vaccine formulations based on Ebola virus antigen(s), there remains a need to provide additional sources of protection to address emerging Ebola virus strains. Several potential therapeutic agents against Ebola virus are currently being evaluated, including post-exposure vaccines (Feldman, H, et al. (2007), PLos Pathog 3(1): e2), small molecule inhibitors (Cote, M. et al. (2011), Nature, 477 (7364): 344–348; Johansen, LM, et al. (2013), Sci Transl Med 5 (190): 190 ra179; Warren, TK, et al., (2014), Nature, 508(7496): 402–405), siRNA-based therapeutics (Geisbert, TW, et al., (2006), J. Infect Dis. 193(12): 1650–1657; Geisbert, TW et al., (2010), Lancet 375(9729): 1896–1905) and monoclonal antibodies (Saphire, EO, (2013), Immunotherapy 5(11): 1221–1233; Wong, G. et al. (2014), Trends Microbiol. 22(8): 456–463; Qiu, X et al., (2014), Hum. Vaccin. Immunother. 10(4): 964–967). Passive administration of antibodies to non-human primates has been shown to be effective (Dye, JM., et al., (2012), Proc Natl Acad Sci USA 109(13): 5034–5039). Recently, a cocktail of three antibodies (ZMapp) has been produced in tobacco plants and is being developed for use in humans (Qiu, X. et al., (2014), Nature 514(7520): 47-53).

Although the idea of a vaccine composition containing an antigen of interest (e.g. GP) to generate neutralizing antibodies in a patient is generally considered a good approach, it may not be practical for use in patients who have already been exposed to the virus, as it may take several weeks for the body to respond to such a vaccine composition. By that time, the patient may already have died due to the viral infection, depending

- 1 048493 on the level of care and available palliative care. For such patients, or for any patient who is unable to mount an effective antibody response, it may be advantageous to create a composition already containing protective antibodies that can specifically target epitopes common to a particular EBOV strain or to a number of strains.

Accordingly, there is still a need in the field to identify new antibodies that can be used to prevent or treat Ebola virus infection.

Brief summary of the present invention

The present invention relates to antibodies, and antigen-binding fragments thereof, that bind the glycoprotein (GP) of the Ebola virus (EBOV). The antibodies of the present invention are useful for inhibiting or neutralizing the activity of the Ebola virus. According to some embodiments, the antibodies are useful for blocking the attachment of the Ebola virus to host cells and/or preventing its entry into them. According to some embodiments, the function of the antibodies is to inhibit intercellular transmission of the virus or to kill Ebola virus-infected cells by reducing the production of pathogenic virus. According to certain embodiments, the antibodies are useful for preventing, treating, or ameliorating at least one symptom of an Ebola virus infection in a subject. According to certain embodiments, the antibodies can be administered prophylactically or therapeutically to a subject having or at risk of acquiring an Ebola virus infection. According to certain embodiments, compositions comprising at least one antibody of the present invention can be administered to a subject for whom a vaccine is contraindicated or for whom the vaccine is less effective, such as an elderly patient, a very young patient, a patient who may be allergic to any one or more components of the vaccine, or an immunocompromised patient who may not respond to the immunogens in the vaccine. According to certain embodiments, compositions comprising at least one antibody of the present invention can be administered to healthcare personnel, hospitalized patients or nursing home residents, or other high-risk patients during an Ebola virus outbreak. According to certain embodiments, compositions comprising at least one antibody of the present invention can be administered as a first-line therapy to patients who have already been exposed to the Ebola virus.

The antibodies of the present invention may be full-length (e.g., an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment) and may be modified so that functionality is affected, such as to improve persistence in an infected organism or to eliminate residual effector functions (Reddy et al., (2000), J. Immunol. 164: 1925-1933). According to certain embodiments, the antibodies may be bispecific.

According to a first aspect, the present invention relates to isolated recombinant monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to EBOV GP.

According to one embodiment, the present invention relates to an isolated recombinant antibody, or antigen-binding fragment thereof, that specifically binds to Ebola virus (EBOV) and/or Ebola virus glycoprotein (EBOV GP), wherein the antibody has one or more of the following characteristics:

(a) comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 18, 66, 146, 2, 34, 50, 82, 98, 114, 130, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 306, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 26, 74, 154, 10, 42, 58, 90, 106, 122, 138, 170, 186, 202, 218, 234, 250, 266, 282 and 298;

(b) is a fully human monoclonal antibody;

(c) binds to EBOV or a virus-like particle (VLP) expressing EBOV GP with a dissociation constant (KD) of less than 10 ^-7 M, as measured by a surface plasmon resonance assay;

(d) is characterized by at least a 3-fold increase in the half-dissociation period (t1/2) at pH 5 or pH 6 relative to pH 7.4;

(e) is characterized by neutralization of Zaire ebolavirus with an IC ₅₀ ranging from approximately 10 ^-11 M to approximately 10 ^-9 M;

(f) is characterized by binding to cells expressing EBOV GP, triggering antibody-dependent cellular cytotoxicity;

(g) cross-reacts with one or more EBOV strains selected from the group consisting of Zaire.2014, Zaire.1995, Sudan, Bundibugyo and Cote d'Ivoire;

(h) binds to soluble GP (sGP);

- 2 048493 (i) cross-competes with a reference antibody, wherein the reference antibody comprises an amino acid sequence of a heavy chain variable region (HCVR) and a light chain variable region (LCVR) selected from the group consisting of any amino acid sequences

HCVR and LCVR from Table 1.

According to one embodiment, the present invention relates to an isolated recombinant antibody, or antigen-binding fragment thereof, that specifically binds to EBOV and/or Ebola virus glycoprotein (EBOV GP), wherein the antibody has two or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to EBOV or a virus-like particle (VLP) expressing EBOV GP with a dissociation constant (K _D ) less than 10 ^-7 M, as measured by a surface plasmon resonance assay;

(c) is characterized by at least a 3-fold increase in the half-dissociation period (t1/2) at pH 5 or pH 6 relative to pH 7.4;

(d) is characterized by neutralization of Zaire ebolavirus with an IC ₅₀ ranging from approximately 10 ^-11 M to approximately 10 ^-9 M;

(e) is characterized by binding to cells expressing EBOV GP, triggering antibody-dependent cellular cytotoxicity;

(f) cross-reacts with one or more EBOV strains selected from the group consisting of Zaire.2014, Zaire.1995, Sudan, Bundibugyo and Cote d'Ivoire;

(g) binds to soluble GP (sGP);

(h) cross-competes with a reference antibody, wherein the reference antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR and LCVR amino acid sequences from Table 1.

Exemplary anti-Ebola virus GP antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 provides amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-Ebola virus GP antibodies. Table 2 provides nucleic acid sequence identifiers of HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-Ebola virus GP antibodies.

The present invention relates to antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising an HCVR and LCVR amino acid sequence pair (HCVR/LCVR), comprising any of the HCVR amino acid sequences listed in Table 1, paired with any of the LCVR amino acid sequences listed in Table 1. According to some embodiments, the present invention relates to antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained in any of the exemplary Ebola virus GP antibodies listed in Table 1.

According to one embodiment, the isolated antibody or antigen-binding fragment comprises an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26, 66/74, 146/154, 2/10, 34/42, 50/58, 82/90, 98/106, 114/122, 130/138, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, and 306/282.

According to one embodiment, the isolated antibody, or antigen-binding fragment, comprises:

(a) an HCDR1 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 68, 148, 4, 36, 52, 84, 100, 116, 132, 164, 180, 196, 212, 228, 244, 260, 276, 292 and 308;

(b) an HCDR2 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 70, 150, 6, 38, 54, 86, 102, 118, 134, 166, 182, 198, 214, 230, 246, 262, 278, 294 and 310;

(c) an HCDR3 domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 72, 152, 8, 40, 56, 88, 104, 120, 136, 168, 184, 200, 216, 232, 248, 264, 280, 296 and 312;

(d) an LCDR1 domain with an amino acid sequence selected from the group consisting of

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SEQ ID NO: 28, 76, 156, 12, 44, 60, 92, 108, 124, 140, 172, 188, 204, 220, 236, 252, 268, 284 and 300;

(e) an LCDR2 domain with an amino acid sequence selected from the group consisting of

SEQ ID NO: 30, 78, 158, 14, 46, 62, 94, 11, 126, 142, 174, 190, 206, 222, 238, 254, 270, 286 and 302;

(f) an LCDR3 domain with an amino acid sequence selected from the group consisting of SEQ

ID NO: 32, 80, 160, 16, 48, 64, 96, 112, 128, 144, 176, 192, 208, 224, 240, 256, 272, 288 and 304.

According to certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18/26 (H1H17139P), 66/74 (H1H17161P), and 146/154 (H1H17203P).

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3/LCDR3), comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention relates to antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary Ebola virus GP antibodies listed in Table 1. According to certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 24/32 (e.g., H1H17139P), 72/80 (e.g., H1H17161P), and 152/160 (e.g., H1H17203P).

The present invention also relates to antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary Ebola virus GP antibodies listed in Table 1. According to certain embodiments, the set of amino acid sequences HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NOs: 20-22-24-28-30-32 (e.g., H1H17139P), 68-70-72-76-78-80 (e.g., H1H17161P), and 148-150-152-156-158-160 (e.g., H1H17203P).

According to a related embodiment, the present invention relates to antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair that is defined by any of the exemplary Ebola virus GP antibodies listed in Table 1. For example, the present invention relates to antibodies, or antigen-binding fragments thereof, comprising a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3 contained within an HCVR/LCVR amino acid sequence pair selected

- 4 048 493 from the group consisting of SEQ ID NO: 18/26 (e.g. H1H17139P), 66/74 (e.g. H1H17161P) and 146/154 (e.g. H1H17203P). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary notations that can be used to identify CDR boundaries include, for example, the Kabat notation, the Chothia notation and the AbM notation. In general, the Kabat notation is based on sequence variability, the Chothia notation is based on the arrangement of structural loop regions, and the AbM notation is a cross between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol Biol. 273: 927–948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 5(5: 9268–9272 (1989). Publicly available databases are also available for identifying CDR sequences in an antibody.

The present invention relates to antibodies to Ebola virus with a modified glycosylation pattern. According to some embodiments, a modification that removes undesirable glycosylation sites or removes a fucose component present on an oligosaccharide chain may be useful, for example, to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosylation modification may be performed to modify complement-dependent cytotoxicity (CDC).

The present invention also relates to antibodies, and antigen-binding fragments thereof, that compete for specific binding to Ebola virus with an antibody, or antigen-binding fragment thereof, comprising a CDR of HCVR and a CDR of LCVR, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

The present invention also relates to antibodies, and antigen-binding fragments thereof, that cross-compete for binding to Ebola virus with a reference antibody, or antigen-binding fragment thereof, comprising a CDR of HCVR and a CDR of LCVR, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

The present invention also relates to isolated antibodies, and antigen-binding fragments thereof, that block the attachment of Ebola virus to and/or entry into a host cell.

According to certain embodiments, the antibodies, or antigen-binding fragments, of the present invention are bispecific, having a first binding specificity for a first epitope in Ebola virus and a second binding specificity for a second epitope in Ebola virus, wherein the first and second epitopes are distinct and non-overlapping. According to certain embodiments, the bispecific antibody may comprise a first arm that binds to an epitope in a viral glycoprotein and a second arm that binds to an epitope in a different viral antigen.

According to a second aspect, the present invention relates to nucleic acid molecules encoding antibodies to the Ebola virus or portions thereof. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; according to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The present invention also relates to nucleic acid molecules encoding HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as defined by any of the exemplary antibodies to Ebola virus GP listed in Table 1.

The present invention also relates to nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences of LCDR1-LCDR2-LCDR3 is as defined by any of the exemplary antibodies to Ebola virus GP listed in Table 1.

The present invention also relates to nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence with any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. According to certain embodiments according to this aspect of the present invention, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same antibody to the Ebola virus GP listed in Table 1.

The present invention relates to nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in Table 1. The present invention also relates to nucleic acid molecules encoding any of the light chain amino acid sequences listed in Table 1.

According to a related aspect, the present invention relates to recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an antibody to the Ebola virus GP. For example, the present invention relates to recombinant expression vectors comprising any of the above-mentioned nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences listed in Table 1. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of antibodies or antibody fragments, and

- 6 048493 isolation of antibodies and antibody fragments obtained in this manner.

According to a third aspect, the present invention relates to a pharmaceutical composition comprising one or more isolated monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to the Ebola virus GP, and a pharmaceutically acceptable carrier or diluent. The one or more isolated antibodies comprise an HCVR/LCVR amino acid sequence pair selected from the group consisting of the HCVR and LCVR sequences listed in Table 1. According to one embodiment, the amino acid sequence pair HCVR/LCVR is selected from the group consisting of SEQ ID NO: 18/26, 66/74, 146/154, 2/10, 34/42, 50/58, 82/90, 98/106, 114/122, 130/138, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298 and 306/282. According to one embodiment, the amino acid sequence pair HCVR/LCVR is selected from the group consisting of SEQ ID NOs: 18/26, 66/74 and 146/154.

According to a related aspect, the present invention relates to a composition which is a combination of at least two antibodies of the present invention and a pharmaceutically acceptable carrier or diluent.

According to a related aspect, the present invention relates to a composition which is a combination/cocktail of at least three antibodies of the present invention and a pharmaceutically acceptable carrier or diluent.

According to one embodiment, the pharmaceutical composition comprises (a) a first antibody to Ebola virus comprising an HCVR/LCVR amino acid sequence pair as described in Table 1, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus comprising an HCVR/LCVR amino acid sequence pair as described in Table 1, or an antigen-binding fragment thereof; and (c) a third antibody to Ebola virus comprising an HCVR/LCVR amino acid sequence pair as described in Table 1, or an antigen-binding fragment thereof, wherein the first antibody binds or interacts with a first epitope on Ebola virus GP and the second and/or third antibody binds to or interacts with a different epitope on Ebola virus GP, and (d) a pharmaceutically acceptable carrier or diluent.

According to another related aspect, the present invention relates to a composition that is a combination of an antibody to Ebola virus GP and a second therapeutic agent.

According to one embodiment, the second therapeutic agent is any agent that is advantageously combined with an antibody to the Ebola virus GP. Exemplary agents that can be advantageously combined with an antibody to the Ebola virus include, but are not limited to, other agents that bind and/or inhibit the activity of the Ebola virus (including other antibodies or antigen-binding fragments thereof, etc.), and/or agents that do not directly bind the Ebola virus but nevertheless inhibit viral activity, including infectivity of host cells.

According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus comprising an HCVR/LCVR amino acid sequence pair as described in Table 1, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus comprising an HCVR/LCVR amino acid sequence pair as described in Table 1, or an antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on Ebola virus GP and the second antibody binds to a second epitope on Ebola virus GP, wherein the first and second epitopes are distinct and non-overlapping; and (c) a pharmaceutically acceptable carrier or diluent.

According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus, or an antigen-binding fragment thereof, wherein the first antibody does not cross-compete with the second antibody for binding to Ebola virus; and (c) a pharmaceutically acceptable carrier or diluent.

According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus, or an antigen-binding fragment thereof, that interacts with a different antigen of Ebola virus, wherein the first antibody binds to an epitope on GP of Ebola virus, and the second antibody binds to an epitope on a different antigen of Ebola virus; and (c) a pharmaceutically acceptable carrier or diluent.

According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus, or an antigen-binding fragment thereof; (c) a third antibody to Ebola virus, or an antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on Ebola virus GP and the second and/or third antibody binds to a different epitope on Ebola virus GP, wherein the first, second and third epitopes are distinct and non-overlapping; and (d) a pharmaceutically acceptable carrier or diluent.

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According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus, or an antigen-binding fragment thereof; (b) a second antibody to Ebola virus, or an antigen-binding fragment thereof; (c) a third antibody to Ebola virus, or an antigen-binding fragment thereof, wherein the first antibody may or may not cross-compete with the second and/or third antibody for binding to Ebola virus; and (d) a pharmaceutically acceptable carrier or diluent.

According to certain embodiments, the present invention relates to a pharmaceutical composition comprising: (a) a first antibody to Ebola virus, or an antigen-binding fragment thereof; (b) a second and/or third antibody to Ebola virus, or an antigen-binding fragment thereof, that interacts with a different antigen of Ebola virus, wherein the first antibody binds to an epitope on Ebola virus, and the second and/or third antibody binds to an epitope on a different antigen of Ebola virus; and (c) a pharmaceutically acceptable carrier or diluent.

According to one embodiment, the pharmaceutical composition comprises a first antibody to Ebola virus, or an antigen binding fragment thereof, that binds or interacts with one epitope on one strain of Ebola virus, and a second and/or third antibody to Ebola virus, or an antigen binding fragment thereof, that binds or interacts with a second and/or third epitope on the same strain or on a different strain of Ebola virus. The Ebola virus strains that interact with the antibody of the present invention can be selected from the group consisting of the Zaire.2014, Zaire. 1995, Sudan, Bundibugyo and Cote d'Ivoire strains or variants thereof.

According to a related aspect, the present invention relates to a pharmaceutical composition comprising a first isolated monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to Ebola virus GP, wherein the first isolated monoclonal antibody, or an antigen-binding fragment thereof, comprises the amino acid sequence of HCDR1 of SEQ ID NO: 20; the amino acid sequence of HCDR2 of SEQ ID NO: 22; the amino acid sequence of HCDR3 of SEQ ID NO: 24; the amino acid sequence of LCDR1 of SEQ ID NO: 28; the amino acid sequence of LCDR2 of SEQ ID NO: 30 and the amino acid sequence of LCDR3 of SEQ ID NO: 32, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may further comprise a second isolated monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to the Ebola virus GP, wherein the second isolated monoclonal antibody, or an antigen-binding fragment thereof, comprises the amino acid sequence of HCDR1 of SEQ ID NO: 68; the amino acid sequence of HCDR2 of SEQ ID NO: 70; the amino acid sequence of HCDR3 of SEQ ID NO: 72; the amino acid sequence of LCDR1 of SEQ ID NO: 76; the amino acid sequence of LCDR2 of SEQ ID NO: 78 and the amino acid sequence of LCDR3 of SEQ ID NO: 80. The pharmaceutical composition may further comprise a third isolated monoclonal antibody, or an antigen-binding fragment thereof, that specifically binds to the Ebola virus GP, wherein the third isolated monoclonal antibody, or an antigen-binding fragment thereof, comprises the amino acid sequence of HCDR1 of SEQ ID NO: 148; the amino acid sequence of HCDR2 SEQ ID NO: 150; the amino acid sequence of HCDR3 SEQ ID NO: 152; the amino acid sequence of LCDR1 SEQ ID NO: 156; the amino acid sequence of LCDR2 SEQ ID NO: 158 and the amino acid sequence of LCDR3 SEQ ID NO: 160.

According to certain embodiments, each antibody may be formulated as a separate formulation, and if it is determined that more than one antibody is needed to achieve maximum therapeutic efficacy, each of the antibody formulations may be co-administered (simultaneously or sequentially) as needed. Alternatively, a cocktail of antibodies may be co-formulated.

According to certain embodiments, when two or more antibodies are combined in a single pharmaceutical composition, they may or may not bind the same or overlapping epitopes on the Ebola virus protein. Additional combination therapies and co-formulations comprising the Ebola virus antibodies of the present invention are disclosed elsewhere herein.

According to a fourth aspect, the present invention relates to therapeutic methods for treating a disease or disorder associated with Ebola virus (such as a viral infection in a subject), or at least one symptom associated with a viral infection, or the frequency or severity of at least one symptom associated with an infection caused by EBOV, using an anti-Ebola virus GP antibody or antigen-binding portion of an antibody of the present invention, or a cocktail of at least two or more antibodies of the present invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of at least two or more antibodies, or antigen-binding fragments, of the present invention to a subject in need thereof. According to one embodiment, the methods comprise administering a combination (cocktail) of at least three antibodies of the present invention. According to one embodiment, the antibody cocktail comprises

- 8 048 493 three antibodies to EBOV having the amino acid sequence pairs set forth under SEQ ID NOs: 18/26, 66/74 and 146/154. The disorder being treated is any disease or condition that is reduced, ameliorated, inhibited or prevented by inhibiting Ebola virus activity. According to certain embodiments, the present invention relates to methods for preventing, treating or reducing at least one symptom of an Ebola virus infection, the method comprising administering a therapeutically effective amount of at least one or more antibodies to Ebola virus GP, or antigen-binding fragments thereof, of the present invention to a subject in need thereof.

According to a related aspect, the present invention relates to a method of neutralizing infectious EBOV, comprising exposing a cell infected with EBOV to a composition comprising one or more anti-EBOV antibodies, or antigen-binding fragments thereof, wherein such exposing results in enhanced protection of the cell from viral infection or from cell death. According to certain embodiments, the exposing may occur in vitro or in vivo. According to one embodiment, the methods comprise administering one or more antibodies of the present invention. According to one embodiment, the methods comprise administering a combination (cocktail) of at least three antibodies of the present invention. According to one embodiment, the antibody cocktail comprises three anti-EBOV antibodies having the amino acid sequence pairs set forth in SEQ ID NOs: 18/26, 66/74, and 146/154.

According to some embodiments, the present invention relates to methods for alleviating or reducing the severity, duration, or frequency of at least one symptom of an Ebola virus infection in a subject by administering one or more antibodies to Ebola virus GP of the present invention, wherein at least one symptom is selected from the group consisting of fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

According to certain embodiments, the present invention relates to methods for reducing viral load in a subject, the methods comprising administering to the subject an effective amount of one or more antibodies, or fragments thereof, of the present invention that bind Ebola virus GP and block Ebola virus binding to and/or entry into a host cell.

According to a related aspect, the present invention provides a method for increasing the survival or likelihood of survival of a subject suffering from an EBOV infection or a subject exposed to EBOV or at risk of exposure to or acquisition of EBOV, the method comprising administering at least one antibody, or antigen-binding fragment, of the present invention or a pharmaceutical composition comprising at least one antibody of the present invention to a subject in need thereof.

According to one embodiment, the present invention provides a method for increasing the survival or likelihood of survival of a subject suffering from an EBOV infection or a subject exposed to EBOV or at risk of exposure to or acquiring EBOV, the method comprising administering an antibody cocktail comprising a mixture of at least two anti-EBOV antibodies of the present invention. According to one embodiment, the method comprises administering an antibody cocktail comprising a mixture of at least three anti-EBOV antibodies of the present invention. According to one embodiment, the antibody cocktail to be administered comprises a mixture of at least three anti-EBOV antibodies of the present invention, wherein the at least three antibodies comprise the HCVR/LCVR amino acid sequence pairs set forth in SEQ ID NOs: 18/26, 66/74, and 146/154.

According to one embodiment, the subject in need thereof is a subject at risk of exposure to Ebola virus or acquiring an infection caused by Ebola virus, wherein the subject is selected from the group consisting of an immunocompromised individual, a healthcare worker, a person suspected of having been exposed to a person carrying Ebola virus, a person who comes into physical contact with or is in very close proximity to an infected individual, a hospital worker, a pharmaceutical researcher, maintenance personnel responsible for cleaning hospital equipment or a facility in which a patient with an infection caused by Ebola virus has been treated, individuals who have visited or plan to visit an area or country known to have an outbreak of Ebola virus or suspected of having one, and a person who frequently travels.

According to one embodiment, at least one anti-EBOV antibody of the present invention, or antigen-binding fragment thereof, or a pharmaceutical composition comprising at least one antibody of the present invention, or antigen-binding fragment thereof, in combination with a second therapeutic agent can be administered to a subject in need thereof.

- 9 048493 agent. The second therapeutic agent can be selected from the group consisting of an antiviral drug, an anti-inflammatory drug (such as corticosteroids and non-steroidal anti-inflammatory drugs), an anti-EBOV antibody, an EBOV vaccine, Ebola TKM (small interfering RNA that specifically targets the viral RNA polymerase), brincidofovir (SMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomers that specifically targets the VP24 gene of the Ebola virus) and interferons.

According to one embodiment, the pharmaceutical composition can be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally.

According to a related embodiment, enhanced protection can be observed in a mammal exposed to or infected with EBOV when the mammal is treated with a pharmaceutical composition comprising an antibody cocktail that comprises at least three antibodies of the present invention.

According to one embodiment, increased protection can be measured by a decrease in the severity or frequency of at least one symptom associated with an EBOV infection, a decrease in the viral load, or an increase in the survival of an EBOV-infected mammal. The at least one symptom can be selected from the group consisting of fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

Enhanced protection can be observed when the antibody is administered alone or when it is administered in combination with one or more additional EBOV therapeutic agents or treatments.

One or more therapeutic agents may be selected from the group consisting of an antiviral drug, an anti-inflammatory drug (such as corticosteroids and non-steroidal anti-inflammatory drugs), a distinct antibody to Ebola virus, an Ebola virus vaccine, Ebola TKM (small interfering RNA that specifically targets viral RNA polymerase), brincidofovir (SMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomers that specifically targets the VP24 gene of Ebola virus), and interferons.

According to one embodiment, the one or more additional therapeutic agents comprise one or more antibodies to EBOV.

According to one embodiment, one or more antibodies to EBOV comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR and LCVR amino acid sequences from Table 1.

According to a related embodiment, the one or more anti-EBOV antibodies comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298, and 306/282.

According to another embodiment, one or more antibodies to EBOV comprise a pair of heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 18/26, 66/74, and 146/154.

According to certain embodiments, one or more antibodies, or antigen-binding fragments thereof, can be administered prophylactically or therapeutically to a subject who has or is at risk of having or is predisposed to developing an Ebola virus infection. Subjects at risk include, but are not limited to, a person with an immune deficiency, such as a person who has an immune deficiency due to an autoimmune disease, or persons undergoing immunosuppressive therapy (e.g., after an organ transplant), or persons suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), certain forms of anemia that reduce or destroy white blood cells, persons undergoing radiation or chemotherapy, or persons suffering from an inflammatory disorder. Other individuals at risk of acquiring Ebola virus infection include health care workers or any individual who has physical contact with or is in very close proximity to an infected individual or is exposed to bodily fluids or tissues from infected individuals, who also have an increased risk of developing Ebola virus infection. Additionally, an individual is at risk of acquiring Ebola virus infection because of proximity to an outbreak, such as being in a densely populated city or in close proximity to individuals with confirmed or suspected Ebola virus infections, or because of their choice of work, such as cleaning staff

- 10 048493 hospital equipment or facility that treated a patient with Ebola virus infection, a hospital worker, a pharmaceutical researcher, an individual who has visited or plans to visit an area or country known to have an outbreak of Ebola virus disease or suspected of having an outbreak, or a frequent traveler.

According to certain embodiments, an antibody, or antigen-binding fragment thereof, of the present invention is administered in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent may be selected from the group consisting of an anti-inflammatory drug (such as corticosteroids and non-steroidal anti-inflammatory drugs), an anti-infective drug, an antiviral drug, a distinct Ebola virus antibody, an Ebola virus vaccine, ZMapp therapy, Ebola TKM (small interfering RNA that specifically targets viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomers that specifically targets the EBOV VP24 gene), interferons, a dietary supplement such as antioxidants, and any other drug or therapy known in the art to be useful for alleviating at least one symptom of Ebola virus infection or for reducing the viral load in a patient. According to certain embodiments, the second therapeutic agent can be an agent that helps neutralize or reduce any possible side effect(s) associated with the antibody, or antigen-binding fragment thereof, of the present invention, when such side effect(s) occur. The antibody, or fragment thereof, can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. According to one embodiment, the antibody can be administered as a single intravenous infusion to maximize the concentration of the antibody in the serum of the subject. The antibody, or fragment thereof, can be administered at a dose of from about 0.1 mg/kg body weight to about 100 mg/kg body weight of the subject. According to certain embodiments, the antibody of the present invention can be administered in one or more doses containing from 50 mg to 600 mg.

The present invention also relates to an antibody to Ebola virus, or antigen-binding fragment thereof, of the present invention for use in the treatment of a subject who has, or is suspected of having, or who has been exposed to EBOV, or for use in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking the binding and/or activity of Ebola virus.

Other embodiments will become apparent from consideration of the following detailed disclosure.

Brief description of the drawings

Fig. 1: H1H17161P effectively neutralizes live EBOV.

Fig. 2: Shows the interaction of three anti-EBOV antibodies with Ebola GP or soluble Ebola GP (sGP).

Detailed disclosure of the present invention

Before the present methods are disclosed, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used in this description is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Definitions.

Ebola virus or EBOV is a genus of the Filoviridae family known to cause severe and rapidly progressive hemorrhagic fever. There are many different species and strains of Ebola virus based on the nucleotide sequence and location of the outbreak, such as Zaire, Tai Forest (formerly known as Cote d'Ivoire or Ivory Coast), Sudan, Reston, and Bundibugyo. The most deadly forms of the virus are the Zaire and Sudan strains. The Reston strain is the only strain known to infect only non-human primates. The term Ebola virus also includes Ebola virus variants isolated from different Ebola virus isolates.

The amino acid sequence of the full-length Ebola virus glycoprotein, designated

- 11 048493 in this document EBOV GP or Ebola virus GP, is illustrated by the amino acid sequences that can be found under GenBank accession numbers AHX24649.1 (see also SEQ ID NO: 314) and AHX24649.2 (see also SEQ ID NO: 315). The term also encompasses Ebola virus GP or a fragment thereof linked to, for example, a histidine tag (e.g., see accession number AHX24649.1 with a decastigidine tag (SEQ ID NO: 318)), a mouse or human Fc or a signal sequence. The amino acid sequence of soluble GP or sGP is shown under accession number ANH24650 and under SEQ ID NO: 316 (with signal sequence) and under SEQ ID NO: 317 (without signal sequence, but with myc-myc-hexahistidine tag). The amino acid sequence of GP1 starts from the amino terminus of full-length GP at residue 1 and ends at residue 501 of SEQ ID NO: 315. The amino acid sequence of GP2 spans residues 502-676 of full-length GP shown under SEQ ID NO: 315.

As used herein, the term Ebola virus infection or EBOV infection refers to a severe hemorrhagic fever caused by exposure to the virus or an infected animal or infected human patient, or by contact with body fluids or tissues from an animal patient or human patient who has Ebola virus infection. Symptoms associated with Ebola virus infection include fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

As used herein, the term "antibody" is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, linked by disulfide bonds (i.e., whole antibody molecules), as well as multimers thereof (e.g., IgM), or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (comprising the CH1, CH2, and CH3 domains). Each light chain consists of a light chain variable region (LCVR or V _L ) and a light chain constant region ( _CL ). The VH and V _L regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), alternating with regions that are more conserved, called framework regions (FRs). Each VH and V _L comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to certain embodiments, according to the present invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be modified naturally or artificially. An amino acid consensus sequence may be determined based on an analysis of the parallel sequences of two or more CDRs.

It is also possible to replace one or more CDR residues or to delete one or more CDRs. The literature describes antibodies in which one or two CDRs are not essential for binding function. Padlan et al. (1995 FASEB J. 9: 133–139) analyzed the contact sites between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with antigen (see also Vajdos et al. 2002 J Mol Biol 320: 415–428).

CDR residues that do not contact antigen can be identified based on previous studies (e.g., residues H60-H65 in CDRH2 are often not essential), based on Kabat CDR regions outside the Chothia CDR, by molecular modeling, and/or by experiment. If a CDR or its residue(s) is excluded, it is typically replaced by an amino acid occupying the corresponding position in another human antibody sequence or a consensus sequence of such sequences. Substitution positions within the CDRs and amino acids to be replaced can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

The fully human monoclonal antibodies to Ebola virus disclosed herein may contain one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the variable domains of the heavy and light chains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from publicly available antibody sequence databases. The present invention provides antibodies, and antigen-binding fragments thereof, that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and/or CDR regions are mutated at the corresponding residue(s) of the germline sequence from which the antibody was derived, or at the corresponding residue(s) of another human germline sequence, or at a conservative amino acid substitution of the corresponding residue(s) of the germline (such sequence changes being collected

- 12 048493 but referred to herein as germline mutations). One of ordinary skill in the art, given the heavy and light chain variable region sequences disclosed herein, can readily prepare numerous antibodies and antigen-binding fragments that comprise one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only mutated residues occurring in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues occurring in only CDR1, CDR2, or CDR3. According to other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). However, the antibodies of the present invention can comprise any combination of two or more germline mutations in the framework and/or CDR regions, in which, for example, some individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues that are different from the original germline sequence are retained or are mutated to the corresponding residue of the different germline sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or increased antagonist or agonist biological properties (as appropriate), reduced immunogenicity, etc. The present invention relates to antibodies and antigen-binding fragments that are generally produced by this method.

The present invention also relates to fully human monoclonal antibodies to Ebola virus comprising variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention relates to antibodies to Ebola virus having HCVR, LCVR and/or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo), such as in the CDRs and in particular in CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been fused to human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal. This term is not intended to include antibodies isolated from or produced in a human subject.

As used herein, the term "recombinant" refers to antibodies, or antigen-binding fragments thereof, of the present invention that have been created, expressed, isolated, or produced using techniques or methods known in the art, such as recombinant DNA technology, which includes, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in non-human mammals (including transgenic non-human mammals, such as transgenic mice) or in a cellular (e.g., CHO cells) expression system, or isolated from a combinatorial library of recombinant human antibodies.

The term “specifically binds to, or specifically binds to, or the like” means that the antibody, or antigen-binding fragment thereof, forms a complex with the antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 ^-7 M or less (e.g., a lower KD indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. The antibodies described herein that specifically bind to the Ebola virus were detected using a surface plasmon resonance-based method, such as the

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BIACORE™ Moreover, in the context of the present disclosure, multispecific antibodies that bind to one domain of the Ebola virus and one or more additional antigens, or a bispecific antibody that binds to two different regions of the Ebola virus, are still considered antibodies that bind specifically.

The term "high affinity antibody" refers to a mAb having a binding affinity to Ebola virus, expressed as Kd, of at least ^10-7 M, preferably ^10-8 M, more preferably ^10-9 M, even more preferably ^10-10 M, even more preferably ^10-11 M, even more preferably ^10-12 M, as measured by surface plasmon resonance, such as on BIACORE™, or by affinity ELISA in solution.

By the term slow off-rate, Kofi' or kd is meant an antibody that dissociates from the Ebola virus or a virus-like particle expressing the Ebola virus GP with a reaction rate constant of 1x 10 ^-3 s ^-1 or less, preferably 1x 10 ^-4 s ^-1 or less, as determined by surface plasmon resonance, such as on BIACORE™.

As used herein, the terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms antigen-binding fragment of an antibody or antibody fragment refer to one or more fragments of an antibody that retain the ability to bind to the Ebola virus.

According to certain embodiments, the antibody or antibody fragments of the present invention may be conjugated to a component such as a ligand or a therapeutic component (immunoconjugate), such as an antiviral drug, a second antibody to Ebola virus, or any other therapeutic component useful for treating Ebola virus infection.

As used herein, the term "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds Ebola virus, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than Ebola virus).

As used herein, the term blocking antibody or neutralizing antibody (or an antibody that neutralizes the activity of Ebola virus or an antibody antagonist) is intended to refer to an antibody whose binding to Ebola virus results in the inhibition of at least one biological activity of Ebola virus. For example, an antibody of the present invention may prevent or block the attachment of Ebola virus to or entry into a host cell. Additionally, a neutralizing antibody is an antibody that can neutralize, i.e., prevent, inhibit, reduce, interfere with, or interfere with the ability of a pathogen to initiate and/or establish infection in a host. The terms neutralizing antibody and antibody that neutralizes or antibodies that neutralize are used interchangeably herein. Such antibodies can be used alone or in combination, as prophylactic or therapeutic agents with other antiviral agents when appropriately formulated, or in combination with active vaccination, or as a diagnostic tool.

Antibody-dependent cell-mediated cytotoxicity or ADCC is a mechanism of cell-mediated immune defense by which an effector cell of the immune system actively lyses a target cell whose membrane-surface antigens have been bound by specific antibodies, such as those described herein. Thus, it is one mechanism by which, for example, a virus-specific antibody can act to limit the spread of infection. Classical ADCC is mediated by natural killer cells (NK cells), macrophages, neutrophils, and, in some cases, eosinophils.

As used herein, the term surface plasmon resonance refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentrations in a biosensor matrix, such as those used by the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

The term KD as used herein is intended to refer to the equilibrium dissociation constant of a particular interaction between an antibody and an antigen.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. The term epitope also refers to the site on an antigen to which B and/or T cells respond. It also refers to the portion of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes and have residues that contribute directly to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. According to certain embodiments, epitopes may include determinants that are chemically active surface moieties of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups and, according to certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics.

As used herein, the term "cross-competes" means that an antibody, or an antigen-binding fragment thereof, binds to an antigen and inhibits or blocks the binding of another antibody, or an antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, i.e., a first antibody that binds and blocks the binding of a second antibody, and vice versa. According to certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes, such that binding of one inhibits or blocks the binding of the second antibody, such as due to steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, such as by label-free real-time interferometric analysis. In order to determine whether a test antibody cross-competes with the reference antibody to Ebola virus GP of the present invention, the reference antibody is allowed to bind to the GP or peptide of Ebola virus under saturating conditions. Then, the ability of the test antibody to bind to the GP of Ebola virus is evaluated. If the test antibody is able to bind to the GP of Ebola virus after saturating binding with the reference antibody to Ebola virus GP, it can be concluded that the test antibody binds to an epitope different from the reference antibody to Ebola virus. On the other hand, if the test antibody is not able to bind to the GP molecule of Ebola virus after saturating binding with the reference antibody to Ebola virus GP, the test antibody may bind to the same epitope as the epitope bound by the reference antibody to Ebola virus GP of the present invention.

The term "substantial identity" or "substantially identical" with respect to a nucleic acid or fragment thereof indicates that, when optimally aligned by appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity of at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term substantial similarity or substantially similar means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs, with default gap opening penalties applied, are at least 90% identical, even more preferably at least 95%, 98% or 99% identical. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such an adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307–331). Examples of groups of amino acids that have side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix,

- 15 048493 disclosed in Gonnet et al. (1992) Science 256: 1443 45. A moderately conservative substitution is any change that has a non-negative value in the log-likelihood matrix PAM250.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software compares similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) outputs alignment results and percent sequence identity for the regions with the best match between the query and search sequences (Pearson (2000) earlier). Another preferred algorithm for comparing a sequence of the present invention with a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-3402.

By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect in the subject to whom it is administered. The precise amount will depend on the purpose of treatment and can be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention and/or treatment of a disease or disorder, such as a viral infection. The subject may have an Ebola virus infection or be predisposed to developing an Ebola virus infection. Individuals predisposed to developing Ebola virus infection or who may be at increased risk of acquiring Ebola virus infection include individuals with a compromised immune system due to an autoimmune disease, individuals undergoing immunosuppressive therapy (e.g., following an organ transplant), individuals with human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), certain forms of anemia that reduce or destroy white blood cells, individuals undergoing radiation or chemotherapy, or individuals with an inflammatory disorder. In addition, individuals who are young or elderly may be at increased risk. Any person who has physical contact with, or is in very close proximity to, an infected animal or human patient, or is exposed to bodily fluids or tissues from an infected animal or human patient, has an increased risk of developing Ebola virus disease. In addition, a person is at risk of acquiring Ebola virus disease because of proximity to an outbreak, such as being in a densely populated area or in close proximity to individuals with confirmed or suspected Ebola virus infections, or because of their choice of work, such as being a hospital worker, a pharmaceutical researcher, an individual who has visited or plans to visit an area or country known to have an Ebola virus outbreak or a suspected Ebola virus outbreak, or a person who travels frequently.

As used herein, the terms treat, treat or cure refer to reducing or alleviating the severity of at least one symptom or sign of an Ebola virus infection in connection with the administration of a therapeutic agent, such as an antibody of the present invention, to a subject in need thereof. The terms include inhibiting the progression of the disease or the worsening of the infection. The terms also include a positive prognosis for the disease, i.e., the subject may be cured of the infection or may have reduced viral titers or viral titers may be absent when administered a therapeutic agent, such as an antibody of the present invention. The therapeutic agent may be administered to the subject at a therapeutic dose.

The terms prevent, prevent or prevent refer to inhibition of the occurrence of an Ebola virus infection or any symptoms or signs of an Ebola virus infection when an antibody of the present invention is administered. The term includes preventing the spread of infection in a subject exposed to the virus or the risk of acquiring an Ebola virus infection.

As used herein, the term antiviral drug refers to any anti-infective agent or therapeutic agent, whether a chemical component or a biological therapeutic agent, used to treat, prevent, or alleviate a viral infection in a subject. For example, according to the present invention, the antiviral drug may include, but is not limited to, an antibody to Ebola virus (in one embodiment, the antibody to Ebola virus may be different from those described herein), an Ebola virus vaccine, ZMapp therapy, Ebola TKM (small interfering RNA that specifically targets viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomers that specifically targets the VP24 gene of Ebola virus), interferons. According to the present invention, the infection to be treated is caused by Ebola virus.

General disclosure of the present invention

Ebola virus disease is a severe, often fatal disease caused by thread-shaped viral particles that are members of the Filoviridae family. There are several known species of Ebola viruses that can cause disease in humans. These include Zaire, Sudan, Tai Forest (formerly Ivory Coast), and Bundibugyo. The natural reservoir for this virus is unknown, and there are currently no approved therapies or vaccines.

The viral genome consists of a single strand of negative-sense RNA approximately 19 kb in length. Ebola virions contain seven proteins: surface glycoprotein (GP), nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and RNA-dependent RNA polymerase (L). (Feldman, et al. (1992) Virus Res. 24, 1-19).

The only protein present on the surface of the virus is the glycoprotein. Through RNA editing, transcription of the GP gene results in the synthesis of several GP gene-specific mRNAs encoding viral GPs, including nonstructural soluble GP (sGP) and virion surface GP (Volchkova, VA et al., (1998), Virology 250: 408-414). Both GPs are synthesized as a precursor molecule that is proteolytically cleaved by the cellular protease furin during intracellular processing (Volchkov, VE, et al., ((1998), Proc Natl Acad Sci USA 95: 5762-5767). sGP forms dimers, whereas the cleaved carboxy-terminal fragment of the end is a monomer. The protrusions on the viral surface are formed as a trimer of GP1,2. consisting of two subunits GP1 and GP2 linked by a disulfide bond (Volchkova, VA et al., (1998), Virology 250: 408-414; Falzarano, D. et al., (2006), Chembiochem 7: 1605-1611). GP1 is known to mediate viral attachment to host cell, and GP2 is involved in membrane fusion (Sanchez, A. et al., (1996), Proc Natl Acad Sci USA 93: 3602-3607; Alazard-Dany, N. et al. (2006), J. Gen. Virol. 87: 1247-1257).

During EBOV infection, significant amounts of soluble glycoprotein (sGP) are released from virus-infected cells. This form of GP has been shown to bind and block virus-neutralizing antibodies directed against surface or virion GP (Dolnik, O. et al., (2004), EMBO J 23: 2175-2184). Apart from this antibody blockade, the role of soluble GP in viral replication and/or pathogenicity has not been well studied. Recent studies by Escudero-Perez et al. have shown that sGP can bind to and activate uninfected dendritic cells and macrophages and induce the secretion of pro- and anti-inflammatory cytokines. In addition, their results indicate that sGP influences endothelial cell function and may affect vascular permeability. (Escudero-Perez, et al., (2014), PLOS Pathogens, Vol. 10, Issue 11: 1-17) This may explain the impaired inflammatory response in the host following infection and may contribute to the pathogenicity of the virus.

Passive immunotherapy for the prevention or treatment of infectious diseases has been used for over a century, usually in the form of sera from recovered individuals that contain high titers of neutralizing antibodies (Good et al., (1991); Cancer 68: 1415-1421). Today, many purified monoclonal antibodies are currently in preclinical and clinical development for use as antimicrobials (Marasco et al. 2007; Nature Biotechnology 25: 1421-1434). Certain antibodies have been described that bind to the Ebola virus glycoprotein. (See, for example, Audet et. al. (2014), Scientific Reports 4: 6881; Chen, et. al. (2014), ACS Chem Biol. Oct. 17; 9(10): 2263-73; Koellhoffer JF, et. al., (2012), Chembiochem Nov. 26; 13(17): 2549-57; Qiu, X., et. al, Nature (2014) Oct 2;514(7520): 47-53).

The inventors herein have described fully human antibodies and antigen-binding fragments thereof that specifically bind to Ebola virus GP and modulate the interaction of Ebola virus with such cells. Antibodies to Ebola virus GP can bind to Ebola virus with high affinity. According to certain embodiments, the antibodies of the present invention are blocking antibodies, wherein the antibodies can bind to Ebola virus GP and block the attachment of the virus to and/or its entry into host cells. According to certain embodiments, the antibodies of the present invention can block the binding of Ebola virus to cells and thus can inhibit or neutralize viral infection in host cells. According to certain embodiments, the antibodies of the present invention can block the binding of Ebola virus to cells and thus can inhibit or neutralize viral infection in host cells. According to certain embodiments, the antibodies of the present invention can block the binding of Ebola virus to cells and can inhibit or neutralize viral infection in host cells.

- 17 048 493 embodiments, the antibodies of the present invention can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and thus can promote the destruction of cells that carry said virus. According to certain embodiments, the antibodies can exert their effects in two ways, for example, they can neutralize viral infectivity and can mediate ADCC. According to some embodiments, the antibodies can be useful for treating a subject suffering from an infection caused by the Ebola virus. The antibodies, when administered to a subject in need thereof, can reduce an infection caused by a virus, such as the Ebola virus, in the subject. They can be used to reduce viral loads in a subject. They can be used alone or as an adjunct therapy with other therapeutic components or agents known in the art for the treatment of a viral infection. According to certain embodiments, such antibodies can bind to an epitope at the amino terminus of the Ebola virus GP. According to certain embodiments, such antibodies can bind to an epitope at the carboxy terminus of the Ebola virus GP. In addition, the detected antibodies can be used prophylactically (prior to infection) to protect mammals from infection or can be used therapeutically (after infection has been established) to treat a previously established infection or to alleviate at least one symptom associated with said infection.

The full-length amino acid sequence of an illustrative Ebola virus GP is shown under GenBank accession numbers AHX24649.1 and AHX24649.2, and under SEQ ID NOs: 314 and 315, respectively. GP1 occupies amino acid residues 1 to 501 of the full-length GP, and GP2 occupies amino acid residues 502 to 676 of the full-length GP, shown under SEQ ID NO: 314 or SEQ ID NO: 315). The full-length EBOV GP, also shown under accession number AHX24649.1, can be linked to a decahistidine tag, such as shown under SEQ ID NO: 318. Soluble GP (sGP) is shown under GenBank accession number AHX24650.1, as well as under SEQ ID NO: 316 (with attached signal sequence), as well as under SEQ ID NO: 317 (without signal sequence, but containing a myc-myc-his tag).

According to certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as full-length Ebola virus GP, or a recombinant form of Ebola virus GP or fragments thereof, followed by immunization with a secondary immunogen or an immunogenically active fragment of Ebola virus GP. According to certain embodiments, said antibodies are obtained from mice immunized with DNA encoding full-length Ebola virus GP (Zaire.2014, see GenBank KJ660346.2; also SEQ ID NO: 313).

The immunogen may be a biologically active and/or immunogenic fragment of the Ebola virus GP or DNA encoding an active fragment thereof. The fragment may be derived from any portion of the viral GP, including the amino-terminal fragment (e.g., GP1) or the carboxy-terminal fragment (e.g., GP2). Peptides may be modified to include the addition or substitution of certain residues for labeling or for conjugation to carrier molecules such as KLH. For example, a cysteine may be added to either the N- or C-terminus of the peptide, or a linker sequence may be added to produce a peptide for conjugation, for example, to KLH for immunization.

Certain Ebola virus antibodies of the present invention are capable of binding to and neutralizing the activity of Ebola virus, as determined in in vitro or in vivo assays. The ability of the antibodies of the present invention to bind and neutralize the activity of Ebola virus and thus the attachment of the virus to and/or its entry into a host cell and the resulting viral infection can be measured by any standard method known to those skilled in the art, including binding assays or activity assays as described herein.

Non-limiting illustrative assays for measuring in vitro binding activity are illustrated herein in Example 3. In Example 3, dissociation constants and binding affinities of antibodies to Ebola virus GP for Ebola virus were determined using the Biacore system. In Examples 4 and 7, neutralization assays were used to determine the infectivity of various Ebola virus strains.

The antibodies specific to Ebola GP may not contain additional labels or components, or they may contain an N-terminal or C-terminal label or component. According to one embodiment, the label or component is biotin. In a binding assay, the orientation of the peptide relative to the surface to which the peptide is bound can be determined from the location of the label (if present). For example, if the surface is coated with avidin, the N-terminal biotin-containing peptide will be oriented such that the C-terminal portion is remote with respect to the surface. According to one embodiment, the label can be a radionuclide, a fluorescent dye, or a label detectable in MRI. According to certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays.

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Antigen-binding fragments of antibodies.

Unless otherwise specified, the term "antibody" as used herein is to be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., entire antibody molecules), as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion of an antibody," "antigen-binding fragment of an antibody," and the like include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen-binding fragment of an antibody" or "antibody fragment" refer to one or more fragments of an antibody that retain the ability to specifically bind to the Ebola virus. An antibody fragment may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. According to certain embodiments, the term antigen-binding fragment refers to a polypeptide fragment of a multispecific antigen-binding molecule.

Antigen-binding fragments of an antibody can be obtained, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic cleavage or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and/or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or it can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv molecules (scFv); (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Also encompassed by the term antigen-binding fragment as used herein are other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-fused antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small molecule modular protein (SMIP) immunopreparations, and shark IgNAR variable domains.

Typically, an antigen-binding fragment of an antibody will comprise at least one variable domain. The variable domain may be of any size or amino acid composition, and will typically comprise at least one CDR that is adjacent to or within one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be arranged relative to one another in any suitable order. For example, the variable region may be dimeric and comprise _VH - _VH , _VH - _VL, or _VL - _VL dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric VH or VL domain.

According to certain embodiments, the antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable or constant domains that may be found in an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Cl3; (xi) Vl-Ch1-Ch2; (xii) VL-CH1-CH2-CH3; (xiii) _Vl - _Ch2 - _Ch3 ; and (xiv) _Vl - _Cl . In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to each other or can be linked by a full or partial hinge or linker region. The hinge region can be at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody of the present invention can comprise a homodimer or heterodimer (or other multimer) in any of the above configurations of variable and constant domains in non-covalent association with each other and/or with one or more monomeric VH or VD domains (e.g., via disulfide bond(s)).

As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific

- 19 048493 The antigen-binding fragment of an antibody will typically comprise at least two different variable domains, each variable domain being capable of specifically binding to a separate antigen or to a different epitope on a single antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragment of an antibody of the present invention using standard techniques available in the art.

Obtaining human antibodies.

Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods may be used in the context of the present invention to generate human antibodies that specifically bind to Ebola virus GP. An immunogen may be used to generate antibodies to the Ebola virus, including any of the following. According to certain embodiments, the antibodies of the present invention are obtained from mice immunized with full-length native Ebola virus GP (see, e.g., GenBank accession numbers AHX24649.1 (SEQ ID NO: 314) and AHX24649.2 (SEQ ID NO: 315) or DNA encoding the glycoprotein or a fragment thereof. Alternatively, Ebola virus GP or a fragment thereof can be obtained using standard biochemical techniques and modified and used as an immunogen. According to one embodiment, the immunogen is a recombinantly produced Ebola virus GP or a fragment thereof. According to certain embodiments of the present invention, the immunogen can be a commercially available Ebola virus GP. According to certain embodiments, one or more booster injections can be administered. According to certain embodiments, the booster injections can comprise one or more commercially available Ebola virus GP. According to certain embodiments, the immunogen may be a recombinant Ebola virus GP expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

Using VELOCIMMUNE™ technology (see, for example, patent document US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for creating monoclonal antibodies, high-affinity chimeric antibodies to Ebola virus GP with a human variable region and a murine constant region are initially isolated. VELOCIMMUNE® technology involves creating a transgenic mouse having a genome containing loci for human variable regions of the heavy and light chains operably linked to endogenous loci for the murine constant regions such that the mouse, in response to stimulation with an antigen, produces an antibody containing a human variable region and a murine constant region. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human constant regions of the heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, VELOCIMMUNE® mice are challenged with an antigen of interest and lymphocytes (such as B cells) from the mice that express antibodies are isolated. The lymphocytes can be hybridized with a myeloma cell line to produce immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain can be isolated and fused to the desired isotypic constant regions of the heavy chain and light chain. Such an antibody protein can be produced in a cell such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies with a human variable region and a murine constant region are isolated. As in the experimental section below, the antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to create a fully human antibody of the present invention, such as a wild-type or modified IgG1 or IgG4. Although the constant region selected may vary according to the specific purpose, the variable region should have high-affinity antigen binding and target specificity characteristics.

Bioequivalents.

The Ebola virus GP antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that differ from those of the disclosed antibodies, but which retain the ability to bind Ebola virus. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions or substitutions compared to the original sequence, but are characterized by biological activity that is actually

- 20 048493 is equivalent to the activity of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention encompass sequences that contain one or more additions, deletions or substitutions of nucleotides compared to the disclosed sequence, but which encode an antibody or antibody fragment that is substantially bioequivalent to the antibody or antibody fragment of the present invention.

Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives for which no significant difference is observed in the rate or extent of absorption when administered at the same molar dose under similar experimental conditions, either in a single dose or in multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in their extent of absorption but not in their rate of absorption, and they can still be considered bioequivalent because such differences in absorption rate are intentional and accounted for by labeling, are not significant in achieving effective concentrations of the drug in the body, for example, upon chronic administration, and are considered medically insignificant for the particular finished dosage form being studied.

According to one embodiment, two antigen-binding proteins are bioequivalent if they do not have clinically significant differences in their safety, purity, and activity.

According to one embodiment, two antigen-binding proteins are bioequivalent if a patient can substitute one or more times the reference product for the biological product and vice versa without a predicted increased risk of adverse effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to continued therapy without such substitution.

According to one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action with respect to a condition or conditions of use, provided that such mechanisms are known.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo testing in humans or other mammals in which the concentration of the antibody or its metabolites in blood, blood plasma, blood serum, or other biological fluid is measured as a function of time; (b) in vitro testing that is correlated with, and reasonably predictable from, in vivo bioavailability data in humans; (c) in vivo testing in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a rigorously controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the antibodies of the present invention can be constructed, for example, by making various substitutions of residues or sequences or by removing terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unwanted or abnormal intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies can include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation sites.

Antibodies to Ebola virus containing Fc variants.

According to certain embodiments of the present invention, there are provided antibodies to Ebola virus comprising an Fc domain comprising one or more mutations that enhance or decrease binding of the antibody to the FcRn receptor, such as at acidic pH compared to neutral pH. For example, the present invention provides antibodies to Ebola virus GP comprising a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the half-life of the antibody in serum when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T) and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F) and 434. According to one embodiment, said modification comprises a modification of 428L (e.g., M428L) and 434S (e.g., N434S); modification 428L, 259I (e.g. V259I) and 308F (e.g. V308F); modification 433K (e.g. H433K) and 434 (e.g. 434Y); modification 252, 254 and 256 (e.g.

- 21 048493

252Y, 254T and 256E); modification 250Q and 428L (e.g. T250Q and M428L); and modification 307 and/or

308 (e.g. 308F or 308P). According to another embodiment, said modification includes modification 265A (e.g. D265A) and/or 297A (e.g. N297A).

For example, the present invention relates to antibodies to Ebola virus comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the above mutations in the Fc domains and other mutations within the variable domains of the antibodies disclosed herein are considered to be within the scope of the present invention.

The present invention also relates to antibodies to the Ebola virus comprising a chimeric heavy chain constant region (CH), wherein the chimeric CH region comprises segments derived from CH regions of multiple immunoglobulin isotypes. For example, antibodies of the present invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule, in combination with part or all of a CH3 domain derived from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, antibodies of the present invention comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a hinge region of human IgG1, human IgG2, or human IgG4, combined with a lower hinge sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a hinge region of human IgG1, human IgG2, or human IgG4. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from an upper hinge of human IgG1 or human IgG4 and amino acid residues derived from a lower hinge of human IgG2. An antibody comprising a chimeric CH region as described herein, according to certain embodiments, may have modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Provisional Patent Application No. 61/759,578, filed February 1, 2013.)

Biological characteristics of antibodies.

In general, the function of the antibodies of the present invention is to bind to Ebola virus GP. For example, the present invention provides antibodies and antigen-binding fragments of antibodies that bind Ebola virus GP (e.g., at 25°C or at 37°C) with a KD of less than 10 ^-7 M, as measured by surface plasmon resonance, for example, using the assay format described herein. According to certain embodiments, the antibodies or antigen-binding fragments thereof bind Ebola virus GP with a KD of less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, for example, using the assay format described herein.

The present invention also relates to antibodies and antigen-binding fragments thereof that bind Ebola virus with a half-life (t1/2) of greater than about 3 minutes, as measured by surface plasmon resonance at 25°C, or greater than about 1 minute, as measured by surface plasmon resonance at 37°C, for example, and with at least a 3-fold increase in half-life (t1/2) at pH 5 or pH 6; using the assay format described herein or a substantially similar assay. According to certain embodiments, the antibodies or antigen-binding fragments of the present invention bind Ebola virus with a t1/2 of greater than about 10 minutes, greater than about 30 minutes, greater than about 60 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, or greater than about 1000 minutes as measured by surface plasmon resonance at 25°C or at 37°C, such as using an assay format as described herein (e.g., a mAb capture or antigen capture format), or a substantially similar assay.

The present invention also relates to antibodies, or antigen-binding fragments thereof, that neutralize the infectivity of Ebola virus to its host cells. According to some embodiments, said antibodies are characterized by neutralizing activity against Zaire.2014 VLPs with an IC ₅₀ ranging from about 10 ^-11 M to about 10 ^-9 M. The antibodies of the present invention also cross-react with Ebola virus VLPs comprising GP from different strains of EBOV, including Zaire. 1995, Zaire.2014, Ebola Sudan, Bundibugyo, and Cote d'Ivoire (Ivory Coast). The antibodies of the present invention also mediate ADCC, as shown in Example 5. Moreover, the antibodies of the present invention cross-compete with other antibodies that bind EBOV GP, as shown in Example 6.

According to one embodiment, the present invention relates to an isolated recombinant antibody, or antigen-binding fragment thereof, that specifically binds to Ebola virus GP, wherein said antibody, or fragment thereof, exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to EBOV or a virus-like particle (VLP) expressing Ebola virus glycoprotein with a dissociation constant ( _KD ) of less than ^10-7 M, as measured in a surface plasmon resonance assay; (c) is characterized by at least a 3-fold increase in half-dissociation time (t1/2) at pH 5 or pH 6 relative to pH 7.4; (d) is characterized by neutralization of Zaire ebolavirus with an _IC50 ranging from about ^10-11 M to about ^10-9 M; (e) exhibits antibody-dependent cellular cytotoxicity against Ebola virus-infected cells; (f) cross-reacts with one or more Ebola virus VLP strains selected from the group consisting of Zaire.2014, Zaire. 1995, Sudan, Bundibugyo and Cote d'Ivoire; (g) cross-competes with a reference antibody, wherein the reference antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR and LCVR amino acid sequences from Table 1.

The antibodies of the present invention may have one or more of the above-mentioned biological characteristics, or any combination thereof. Some of the properties of the antibodies of the present invention are summarized below. Other biological characteristics of the antibodies of the present invention will be apparent to those of ordinary skill in the art upon consideration of the present disclosure, including the working examples provided herein.

inAb Properties IC50 Neutralization ADC Connections inAb neutralization I'm alive WITH esGP And virus pseudovirus (M)

N1N17161 R Neutralizer, ADCC-, sGP- 8,ZE-11 Yes No No N1N17139 R Not a neutralizer, ADCC+, sGP+ No No Yes Yes N1N17203 R Neutralizer, ADCC+, sGP- 2E-10 No Yes No

Epitope mapping and related techniques.

The present invention relates to antibodies to Ebola virus that interact with one or more amino acids occurring in the GP of Ebola virus. The epitope to which the antibodies bind may consist of a single continuous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in the GP molecule of Ebola virus (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located in the GP of Ebola virus (e.g., a conformational epitope).

Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody interacts with one or more amino acids in a polypeptide or protein. Exemplary techniques include, for example, standard epitope cross-competition assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis assays, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage assays, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer

- 23 048493 (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen/deuterium exchange, determined by mass spectrometry. In general, the hydrogen/deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to the input, and the exchangeable protons in the amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen exchange back at a slower rate than the exchangeable protons in the amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface can retain deuterium and thus have a relatively higher mass than amino acids not included in the interface. Following dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252–259; Engen and Smith (2001) Anal. Chem. 73: 256A–265A.

The term epitope refers to a site on an antigen to which B and/or T cells respond. Intracellular epitopes may be formed from either nonessential or essential amino acids that come into contact through tertiary structure of the protein. Epitopes formed from nonessential amino acids are usually retained by denaturing solvents, while epitopes formed through tertiary structure are usually lost upon treatment with denaturing solvents. An epitope typically includes at least 3 or, more typically, at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-based antibody assay (MAP), also known as antigen structure-based antibody assay (ASAP), is a method by which large numbers of monoclonal antibodies (mAbs) to a single antigen are classified according to the similarity of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920). Each category may reflect a unique epitope, either clearly distinct from or substantially identical to an epitope represented in another category. This technique allows genetically identical antibodies to be rapidly filtered out so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to separate the antibodies of the present invention into groups of antibodies that bind different epitopes.

According to certain embodiments, the Ebola virus antibodies, or antigen-binding fragments thereof, bind an epitope in any one or more illustrated regions of the Ebola virus GP, either in natural form or recombinantly produced, or a fragment thereof.

The present invention relates to antibodies to Ebola virus that bind to the same epitope or portion of an epitope. Similarly, the present invention also relates to antibodies to Ebola virus GP that compete for binding to Ebola virus GP or a fragment thereof with any of the specific illustrative antibodies described herein. For example, the present invention relates to antibodies to Ebola virus GPP that cross-compete for binding to Ebola virus with one or more antibodies derived from such antibodies as described in Tables 1 and 2.

It can be easily determined by standard methods known in the art whether an antibody binds to the same epitope as a reference antibody to Ebola virus GP or competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as the reference antibody to Ebola virus GP of the present invention, the reference antibody is allowed to bind to GP or a peptide of Ebola virus under saturating conditions. Then, the ability of the test antibody to bind to GP of Ebola virus is evaluated. If the test antibody is able to bind to GP of Ebola virus after saturating binding with the reference antibody to GP of Ebola virus, it can be concluded that the test antibody binds to an epitope different from the reference antibody to Ebola virus. On the other hand, if the test antibody is unable to bind to the Ebola virus GP molecule after saturating binding with the Ebola virus GP reference antibody, the test antibody may bind to the same epitope as the epitope bound by the Ebola virus GP reference antibody of the present invention.

To determine whether an antibody competes for binding with the reference antibody to Ebola virus GP, the binding methodology described above is performed in two adaptations. In the first adaptation, the reference antibody is allowed to bind to Ebola virus GP under saturating conditions, followed by assessment of binding of the test antibody to Ebola virus GP. In the second adaptation, the test antibody is allowed to bind to Ebola virus GP under saturating conditions, followed by assessment of binding of the reference antibody to Ebola virus GP. If in both adaptations only the first (saturating) antibody is bound to Ebola virus GP, then the binding of the reference antibody to Ebola virus GP is assessed.

- 24 048493 (a) a test antibody is capable of binding to the Ebola virus GP, then it is concluded that the test antibody and the reference antibody compete for binding to the Ebola virus GP. It will be understood by one of ordinary skill in the art that an antibody that competes for binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to a matching or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, two antibodies have the same epitope if virtually all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if several amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Further standard experiments (e.g., mutation and peptide binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this kind can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

Immunoconjugates.

The present invention relates to a human monoclonal antibody to Ebola virus GP conjugated to a therapeutic moiety (immunoconjugate), such as an antiviral drug for the treatment of Ebola virus infection. As used herein, the term immunoconjugate refers to an antibody that is chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location on the molecule, so long as it can bind to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. According to one embodiment, said agent may be a second different antibody to the Ebola virus or to the Ebola virus GP. According to certain embodiments, the antibody may be conjugated to an agent that is specific for a cell infected with the virus. The type of therapeutic component that may be conjugated to the antibody to the Ebola virus will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO 05/103081.

Multispecific antibodies.

The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may comprise antigen-binding domains specific for multiple target polypeptides. See, e.g., Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244.

As will be known to one of skill in the art, any of the multispecific antigen-binding molecules of the present invention, or variants thereof, can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression technology).

According to some embodiments, Ebola virus-specific antibodies are designed in a bispecific format (bispecific molecule) in which variable regions that bind to different domains of Ebola virus are linked together to confer specificity for two domains in a single binding molecule. Appropriately designed bispecific molecules can enhance the overall inhibitory potency against an Ebola virus protein by increasing both the specificity and the avidity of binding. Variable regions with specificity for separate domains (e.g., segments of the N-terminal domain), or that can bind to different regions within a single domain, are paired on a scaffold, allowing each region to simultaneously bind to separate epitopes or to different regions within a single domain. In one example of a bispecific molecule, the heavy chain variable regions (VH) from a binding molecule with specificity for a single domain are recombined with the variable

- 25 048 493 light chain (VL) regions from a series of binding molecules with specificity for the second domain to identify unrelated VL partners that can pair with the parent VH without compromising the parent specificity of that VH. Thus, a single VL segment (e.g., VL1) can be combined with two different VH domains (e.g., VH1 and VH2) to create a bispecific molecule consisting of two binding arms (VH1-VL1 and VH2-VL1). The use of a single VL segment reduces the complexity of the system and thus simplifies and increases the efficiency of the cloning, expression, and purification methods used to create the bispecific molecule (see, e.g., USSN13/022759 and US2010/0331527).

Alternatively, antibodies that bind to multiple domains and a second target, such as, but not limited to, a second distinct antibody to Ebola virus, can be prepared in a bispecific molecule format using the techniques described herein or other techniques known to those skilled in the art. Variable regions of antibodies that bind to different regions can be linked to variable regions that bind to corresponding sites, such as on Ebola virus, to impart specificity to two antigens in a single binding molecule. Accordingly, bispecific molecules of this nature are designed to serve dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for the outer portion of the extracellular domain and paired on a scaffold, allowing each variable region to bind to separate antigens.

An exemplary bispecific antibody format that can be used in the context of the present invention comprises the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other in at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference. According to one embodiment, the first Ig CH3 domain binds to protein A and the second Ig _CH3 domain comprises a mutation that reduces or eliminates binding to protein A, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). Additional modifications that can be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU) for IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU) for IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU) for IgG4 antibodies. The above described variants of the bispecific antibody format are provided within the scope of the present invention.

Other exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv or diabody-based bispecific formats, IgG-scFv hybrids, dual variable domain (DVD) Ig, quadruple hybridoma, knob-in-hole, common light chain (e.g., common light chain with knobs-in-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, IgG with dual acting Fab (DAF), and Mab ² bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references therein, for a review of the aforementioned formats). Bispecific antibodies can also be constructed using, for example, peptide/nucleic acid conjugation, whereby unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valence, and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic administration and dosage forms.

The present invention relates to therapeutic compositions comprising the antibodies to Ebola virus GP, or antigen-binding fragments thereof, of the present invention. The therapeutic compositions of the present invention will be administered with suitable carriers, excipients, and other agents that are included in the formulations to provide improved transport, delivery, stability, etc. A large number of suitable formulations can be found in a pharmacologically familiar to all: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such dosage forms include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-liquid gels, and semi-liquid mixtures containing carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) JPharm Sci Technol 52: 238-311.

The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, conditions, route of administration, etc. When using the antibody of the present invention to treat a disease or disorder in an adult patient or to prevent

- 26 048493 such a disease, it is preferable to administer the antibody of the present invention normally in a single dose of from about 0.1 to about 60 mg/kg body weight, more preferably from about 5 to about 60, from about 10 to about 50, or from about 20 to about 50 mg/kg body weight. Depending on the severity of the condition, the frequency of administration and the duration of treatment can be adjusted. According to certain embodiments, the antibody, or antigen-binding fragment thereof, of the present invention can be administered in an initial dose of at least from about 0.1 mg to about 800 mg, from about 1 to about 500 mg, from about 5 to about 300 mg, or from about 10 to about 200 mg, to about 100 mg, or to about 50 mg. According to certain embodiments, the initial dose may be followed by administration of a second or more subsequent doses of the antibody, or antigen-binding fragment thereof, in an amount that may be about the same as or less than the initial dose, wherein the subsequent doses are separated by at least 1-3 days, at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition can be administered by any suitable route, such as by infusion or injection of a bolus dose of the substance, by absorption through epithelial or mucous membranes (e.g., the oral mucosa, the rectal or small intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration may be systemic or local. The pharmaceutical composition may also be delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249: 1527-1533).

The present document also provides the use of nanoparticles for delivering the antibodies of the present invention. The antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic purposes. Antibody-conjugated nanoparticles and methods for making and using them are described in detail in Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389). The nanoparticles can be designed and conjugated to antibodies contained in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US 8,257,740 or US 8,246,995.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. According to one embodiment, a pump can be used. According to another embodiment, polymeric materials can be used. According to another embodiment, the controlled release system can be placed near the target of the composition, thus making only a portion of the systemic dose necessary.

Injectable forms may include dosage forms for intravenous, subcutaneous, intracutaneous, intradermal, intracranial and intramuscular injections, intravenous drip infusions, etc. Such injectable forms can be prepared by generally known methods. For example, injectable forms can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or salt thereof in a sterile aqueous medium or oil medium commonly used for injections. As an aqueous injection medium, there are, for example, a physiological solution, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g. ethanol), a polyhydric alcohol (e.g. propylene glycol, polyethylene glycol), a nonionic surfactant [e.g. polysorbate 80, HCO-50 (an adduct of polyoxyethylene (50 mol) and hydrogenated castor oil)], etc. As an oil medium, there are, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection preparation thus obtained is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen-type delivery device can be easily used to deliver the pharmaceutical composition of the present invention. Such a pen-type delivery device can be reusable or disposable. In a reusable syringe-type delivery device

- 27 048493 The pen typically uses a replaceable cartridge that contains the pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen-type delivery device can then be reused. A disposable pen-type delivery device does not have a replaceable cartridge. Instead, the disposable pen-type delivery device is supplied filled with the pharmaceutical composition contained in the reservoir of the device. Once the pharmaceutical composition has been removed from the reservoir, the entire device is discarded.

For subcutaneous delivery of the pharmaceutical composition of the present invention, a variety of reusable syringe pens and autoinjector delivery devices are used. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen-type delivery devices useful for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA™ pen (Abbott Labs, Abbott Park, Ill.), to name a few.

Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared in unit dose dosage forms selected in accordance with the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injection preparations (ampoules), suppositories, etc. The amount of antibody contained is usually from about 5 to about 500 mg per unit dose dosage form; especially in the form of an injection preparation, it is preferable that the antibody is contained in an amount of from about 5 to about 100 mg and from about 10 to about 250 mg per dosage form.

Therapeutic applications of antibodies.

The antibodies of the present invention are useful for treating and/or preventing a disease, disorder, or condition associated with Ebola virus infection and/or for alleviating at least one symptom associated with such a disease, disorder, or condition.

According to certain embodiments, the antibodies of the present invention are useful for treating subjects suffering from severe and acute respiratory infection caused by the Ebola virus. According to some embodiments, the antibodies of the present invention are useful for reducing viral titers or reducing viral load in a host. According to one embodiment, an antibody, or antigen-binding fragment thereof, of the present invention can be administered at a therapeutic dose to a patient with an infection caused by the Ebola virus.

One or more antibodies of the present invention can be administered to alleviate or prevent, or reduce the severity of one or more symptoms or conditions of a disease or disorder. The antibodies can be used to alleviate or reduce the severity of at least one symptom of an infection caused by the Ebola virus, including but not limited to fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

Also provided herein is the prophylactic use of one or more antibodies of the invention for subjects at risk of developing an Ebola virus infection, such as an immunocompromised individual, a healthcare worker, a person suspected of having been exposed to an individual carrying the Ebola virus, a person who has physical contact with or is in very close proximity to an infected individual, a hospital worker, a pharmaceutical researcher, a caregiver responsible for cleaning hospital equipment or a facility in which a patient with an Ebola virus infection has been treated, individuals who have visited or plan to visit an area or country known to have an Ebola virus outbreak or is suspected of having one, and a frequent traveler.

According to a further embodiment of the present invention, the present antibodies are used to prepare a pharmaceutical composition for the treatment of patients suffering from an infection caused by the Ebola virus. According to another embodiment of the present

- 28 048493 of the invention, the present antibodies are used as an adjunct therapy to any other agent or any other therapy known to those skilled in the art suitable for the treatment or alleviation of infection caused by the Ebola virus.

Combination therapy agents.

Combination therapy agents may include an antibody to Ebola virus GP of the present invention and any additional therapeutic agent that can be advantageously combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention. The antibodies of the present invention may be synergistically combined with one or more drugs or agents used to treat an infection caused by the Ebola virus.

For example, exemplary agents for treating a viral infection may include, for example, an antiviral drug, an anti-inflammatory drug (e.g., corticosteroids and non-steroidal anti-inflammatory drugs), another EBOV antibody, an Ebola virus vaccine, ZMapp therapy, Ebola TKM (small interfering RNA that specifically targets viral RNA polymerase), brincidofovir (CMX001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomers that specifically target the VP24 gene of the Ebola virus), interferons, or any other palliative therapy for the treatment of Ebola virus infection.

According to some embodiments, the antibodies of the present invention can be combined with a second therapeutic agent to reduce the viral load in a patient with an Ebola virus infection or to alleviate one or more symptoms of said infection.

According to certain embodiments, the second therapeutic agent is another different antibody or antibody cocktail specific for Ebola virus GP, wherein the different antibody or antibodies in the cocktail may or may not bind to the same epitope or an overlapping epitope as the antibody of the present invention. According to certain embodiments, the second therapeutic agent is an antibody to a different protein of Ebola virus. The second antibody may be specific for one or more different proteins of Ebola virus from different strains of the virus. Provided herein is the use of a combination (cocktail) of antibodies of the present invention with neutralizing or inhibitory activity against Ebola virus. According to some embodiments, non-competing antibodies can be combined in a combination and administered to a subject in need thereof to reduce the ability of the Ebola virus to evade mutation. According to some embodiments, antibodies comprising such a combination bind to separate non-overlapping epitopes on GP. Antibodies comprising such a combination can block the attachment of the virus to, and/or its entry into, and/or fusion with host cells. The antibodies can interact with GP from a strain of EBOV selected from Zaire, Sudan, Bundibugyo or Cote d'Ivoire, and when used alone or in combination with any one or more of the above agents, can neutralize any one or more of the above strains of Ebola virus.

The present document also provides the use of a combination of antibodies to Ebola virus GP of the present invention, wherein the combination comprises one or more antibodies that do not cross-compete. According to certain embodiments, the combination comprises a cocktail comprising a mixture of at least three antibodies of the present invention. The antibodies in the cocktail may differ in their ability to neutralize the virus or virus-infected cells, or in their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), or in their ability to bind EBOV soluble glycoprotein (sGP).

The term in combination with as used herein means that the additional therapeutically active component(s) can be administered before, simultaneously with or after the administration of at least one anti-Ebola virus GP antibody of the present invention or a cocktail comprising one or more antibodies of the present invention. The term in combination with also includes sequential or parallel administration of an anti-Ebola virus GP antibody and a second therapeutic agent.

The additional therapeutically active component(s) may be administered to the subject prior to administration of the anti-Ebola virus GP antibody of the present invention. For example, the first component may be considered to be administered prior to the second component if the first component is administered 1 week, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute prior to administration of the second component. According to other embodiments, the additional therapeutically active component(s) may be administered to the subject after administration of the anti-Ebola virus GP antibody of the present invention. For example, the first component may be considered to be administered after the second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes,

- 29,048,493 minutes, after 1 hour, after 2 hours, after 3 hours, after 4 hours, after 5 hours, after 6 hours, after 12 hours, after 24 hours, after 36 hours, after 48 hours, after 60 hours, after 72 hours after the administration of the second component. According to still other embodiments, the additional therapeutically active component(s) can be administered to the subject simultaneously with the administration of the anti-Ebola virus GP antibody of the present invention. Simultaneous administration, in the context of the present invention, includes, for example, administering the anti-Ebola virus GP antibody and the additional therapeutically active component to the subject in the same dosage form or in separate dosage forms administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form can be administered by the same route (e.g., both the anti-Ebola virus GP antibody and the additional therapeutically active component can be administered intravenously, etc.); Alternatively, each dosage form may be administered by a different route (e.g., the anti-Ebola virus GP antibody may be administered intravenously and the additional therapeutically active component may be administered orally). In any case, administration of the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes is considered to be simultaneous administration in the context of the present disclosure. In the context of the present disclosure, administration of the anti-Ebola virus GP antibody before, simultaneously with, or after (as these terms are defined herein above) administration of the additional therapeutically active component is considered to be administration of the anti-Ebola virus GP antibody in combination with the additional therapeutically active component.

The present invention relates to pharmaceutical compositions in which the anti-Ebola virus GP antibody of the present invention is co-formulated with one or more additional therapeutically active components as described elsewhere herein.

Administration schemes.

According to some embodiments, a single dose of the anti-Ebola virus GP antibody of the present invention (or a pharmaceutical composition comprising a combination of the anti-Ebola virus GP antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject in need thereof. According to some embodiments of the present invention, multiple doses of the anti-Ebola virus GP antibody of the present invention (or a pharmaceutical composition comprising a combination of the anti-Ebola virus GP antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject in need thereof over a period of time. The methods according to this aspect of the present invention comprise sequentially administering multiple doses of the anti-Ebola virus GP antibody of the present invention to a subject. As used herein, sequential administration means that each dose of the anti-Ebola virus GP antibody is administered to the subject at different times, such as on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention relates to methods that comprise sequentially administering to a patient a single initial dose of an antibody to Ebola virus GP, followed by one or more secondary doses of an antibody to Ebola virus GP, and optionally followed by one or more tertiary doses of an antibody to Ebola virus GP.

The terms "primary dose," "secondary doses," and "tertiary doses" refer to the temporal sequence of administration of the anti-Ebola virus GP antibody of the present invention. Thus, the primary dose is the dose administered at the beginning of the treatment regimen (also referred to as the initial dose), the secondary doses are the doses administered after the primary dose, and the tertiary doses are the doses administered after the secondary doses. Each of the primary and secondary and tertiary doses may contain the same amount of anti-Ebola virus GP antibody, but may typically differ from each other with respect to the frequency of administration. However, according to certain embodiments, the amount of anti-Ebola virus GP antibody contained in the primary, secondary, and/or tertiary doses varies among them (e.g., is adjusted upward or downward as needed) during the course of treatment. According to certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered less frequently (e.g., maintenance doses).

According to certain exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered 1-48 hours apart (e.g., 1, ¹ _1/2 , 2, 2 ^1/2 , 3, ³ 1/2, 4, 4 1/2, 5, ⁵ 1/2 ^, 6, 6 1/2, 7, 7 1/2, 8, 8 1/2, 9, ⁹ 1/2, 10, 10 1/2, 11, 11%, ¹² , 12%, 13, 13%, 14, 14 1/2, 15, 15 1/2, ¹⁶ , 16 1/2, 17, 17 ^1/2 , 18, 18 ^1/2 , ¹⁹ , 19 ^1/2 , 20, 20%, 21, 21^, 22, 22^, 23, 23%, 24, 24 ¹ ^ 25, 25^, 26, 26% or more) after the immediately preceding dose. As used herein, the phrase immediately preceding dose means, in a sequence of multiple administrations, the dose of anti-Ebola virus GP antibody that is administered to a patient before the administration of the immediately following dose in the sequence, with no intermediate doses.

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Methods according to this aspect of the present invention may comprise administering to the patient any number of secondary or tertiary doses of the antibody to the Ebola virus GP. For example, according to certain embodiments, the patient is administered only one secondary dose. According to other embodiments, the patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Similarly, according to certain embodiments, the patient is administered only one tertiary dose. According to other embodiments, the patient is administered two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses.

According to certain embodiments of the present invention, the frequency with which secondary and/or tertiary doses are administered to the patient may vary throughout the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by the attending physician, depending on the individual needs of the patient, after clinical examination.

Diagnostic applications of antibodies.

The anti-Ebola virus GP antibodies of the present invention can be used to detect and/or measure Ebola virus in a sample, such as for diagnostic purposes. Some embodiments include the use of one or more antibodies of the present invention in assays for detecting a disease or disorder, such as a viral infection. Exemplary diagnostic assays for Ebola virus can comprise, for example, contacting a sample obtained from a patient with an anti-Ebola virus GP antibody of the present invention, wherein the anti-Ebola virus GP antibody is labeled with a detectable label or reporter group, or is used as a capture ligand to selectively isolate Ebola virus from patient samples. Alternatively, an unlabeled anti-Ebola virus GP antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter group may be a radioisotope such as ^3H , ^14C , ^32P , ^35S , or ^125I ; a fluorescent or chemiluminescent component such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, βgalactosidase, horseradish peroxidase, or luciferase. Specific illustrative assays that can be used to detect or measure Ebola virus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in diagnostic assays for Ebola virus in accordance with the present invention include any sample of tissue or fluid obtained from a patient that contains detectable amounts of either Ebola virus or fragments thereof under normal or pathological conditions. Typically, levels of Ebola virus in a particular sample obtained from a healthy patient (e.g., a patient not suffering from an Ebola virus-associated disease) will be measured, initially establishing a baseline or standard level of Ebola virus. Such a baseline level of Ebola virus can then be compared with levels of Ebola virus measured in samples obtained from individuals suspected of having an Ebola virus-associated condition or symptoms associated with such a condition.

The Ebola virus-specific antibodies may not contain additional tags or components, or they may contain an N-terminal or C-terminal tag or component. According to one embodiment, the tag or component is biotin. In a binding assay, the orientation of the peptide relative to the surface to which the peptide is bound can be determined from the location of the tag (if present). For example, if the surface is coated with avidin, the N-terminal biotin-containing peptide will be oriented such that the C-terminal portion is remote relative to the surface.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention and are not intended to limit the scope of the present invention as contemplated by the inventors. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and variations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperatures are in degrees Celsius, room temperature is approximately 25 C, and pressure is atmospheric or near atmospheric.

Example 1.

Obtaining human antibodies to the Ebola virus.

Human antibodies to Ebola virus were produced in a mouse containing DNA encoding the variable regions of the human kappa light and heavy chain immunoglobulin. According to one embodiment, human antibodies to Ebola virus were produced in a VELOCIMMUNE® mouse. According to one embodiment, Veloclmmune® (VI) mice were immunized with DNA encoding full-length Ebola virus GP [Zaire ebolavirus 2014 (GenBank: KJ660346.2)].

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Antibodies were generated using an accelerated schedule of 2 immunizations separated by 2 weeks. The antibody response was monitored using an immunoassay specific for Ebola virus GP. For example, sera were assayed for specific antibody titers to purified full-length EBOV GP, GP protein subunits (GP1 and GP2), and virus-like particles (VLPs) expressing EBOV GP. Antibody-producing clones were isolated using both B-cell sorting technology (BST) and hybridoma techniques. For example, when the desired immune response was achieved, splenocytes were harvested and hybridized with murine myeloma cells to maintain their viability and generate hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produced antibodies specific for Ebola virus GP. Using this technique and the various immunogens described above, several chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were prepared; the illustrative antibodies thus obtained were designated H1M17354N, H2aM17356N, H1M17357N, H2aM17358N, H2aM17359N, and H2aM17360N.

Antibodies to Ebola virus have also been directly isolated from antigen-positive mouse B cells without hybridization to myeloma cells, as described in U.S. Patent No. 7,582,298. Several fully human antibodies to Ebola virus GP (i.e., antibodies possessing human variable domains and human constant domains) have been produced using this method; The illustrative antibodies obtained in this manner were designated H1H17134P, H1H17139P, H1H17142P, H1H17151P, H1H17161P, H1H17162P, H1H17193P, H1H17196P, H1H17199P, H1H17203P, H1H17214P, H1H17219P, H1H17223P, and H1H17228P.

The biological properties of illustrative antibodies produced according to the methods of the present example are described in detail in the following examples.

Example 2.

Amino acid and nucleotide sequences of the variable regions of the heavy and light chains.

Table 1 shows the identifiers of the amino acid sequences of the variable regions of the heavy and light chains and the CDRs of selected antibodies to the Ebola virus according to the present invention. The identifiers of the corresponding nucleic acid sequences are shown in Table 2.

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Table 1

Amino acid sequence identifiers______________

SEQ ID NO: Shtitel designation HCVR UCDR1 UCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 N1N17134P 1 1 5 5 10 12 14 16 N1N17139P 18 >0 >2 >4 >6 >8 10 12 N1N17142P 14 16 18 10 12 14 16 18 N1N17151P 50 12 54 56 58 50 52 54 N1N17161P 56 58 70 72 74 76 78 50 N1N17162P 52 14 56 58 )0 )2 )4 )6 N1N17193R )8 100 102 104 106 108 110 112 N1N17196P 114 116 118 120 122 124 126 128 N1N17199R 130 132 134 136 138 140 142 144 N1N17203P 146 148 150 152 154 156 158 160 N1N17214P 162 164 166 168 170 172 174 176 N1N17219R 178 180 182 184 186 188 190 192 N1N17223P 194 196 198 >00 >02 >04 206 >08 N1N17228P BY >12 >14 >16 >18 >20 >22 >24 H1H17354N >26 >28 >30 >32 >34 >36 >38 >40 H1H17356N >42 >44 >46 >48 >50 >52 >54 >56 H1H17357N >58 >60 >62 >64 >66 >68 >70 >72 H1H17358N2 >74 >76 >78 >80 >82 >84 >86 >88 H1H17359N >90 >92 >94 >96 298 300 302 304 H1H17360N 106 108 110 112 282 284 286 288 H1M17354N >26 >28 >30 >32 >34 >36 >38 >40 H2aM17356N >42 >44 >46 >48 >50 >52 >54 >56 H1M17357N >58 >60 >62 >64 >66 >68 >70 >72 H2aM17358N >74 >76 >78 >80 >82 >84 >86 >88 H2aM17359N >90 >92 >94 >96 298 300 302 304 H2aM17360N 106 108 110 112 282 284 286 288

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Table 2

Nucleic acid sequence identifiers

3EQ ID NO: Shtitel designation acvR SCDRl EICDR2 aCDR3 LCVR LCDR1 LCDR2 LCDR3 N1N17134P I 1 5 7 ) 11 13 15 N1N17139P 17 19 ’1 23 25 27 29 51 N1N17142P 13 15 17 19 11 13 15 17 N1N17151P 19 11 53 55 57 59 51 53 N1N17161P 55 57 59 71 73 75 77 79 N1N17162P AND 53 55 57 59 P )3 )5 N1N17193R J7 )9 101 103 105 107 109 111 N1N17196P 113 115 117 119 121 123 125 127 N1N17199R 129 131 133 135 137 139 141 143 N1N17203P 145 147 149 151 153 155 157 159 N1N17214P 161 163 165 167 169 171 173 175 N1N17219R 177 179 181 183 185 187 189 191 N1N17223P 193 195 197 199 201 203 205 207 N1N17228P 209 ’ll 213 215 217 219 221 223 H1H17354N 225 ’27 ’29 231 233 235 237 239 H1H17356N 241 243 245 247 249 251 253 255 H1H17357N 257 259 261 263 265 267 269 271 H1H17358N2 273 275 277 279 281 283 285 287 H1H17359N 289 291 293 295 297 299 301 303 H1H17360N 105 107 109 111 281 283 285 287 H1M17354N 225 227 229 231 233 235 237 239 H2aM17356N 241 243 245 247 249 251 253 255 H1M17357N 257 259 261 263 265 267 269 271 H2aM17358N 273 275 277 279 281 283 285 287 H2aM17359N 289 291 293 295 297 299 301 303 H2aM17360N 105 107 109 111 281 283 285 287

As used herein, antibodies are generally designated according to the following nomenclature: an Fc prefix (e.g., H1H, H2M, etc.), followed by a numeric identifier (e.g., 17139, 17161, etc., as shown in Table 1 or 2), followed by a suffix of P, P2, N, N2, or B. The prefixes H1H and H2M in the antibody designations used herein indicate a particular isotype of the Fc region of the antibody. Thus, according to this nomenclature, an antibody may be referred to herein as, for example, H1H17359N, H2aM17359N, etc. For example, antibody H1M has a murine IgG1 Fc and antibody H2M has a murine IgG2 Fc (isotype a or b) (all variable regions are fully human, as indicated by the first H in the antibody designation). As will be appreciated by those skilled in the art, an antibody having a particular Fc isotype may be converted into an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc may be converted into an antibody with a human IgG4, etc.), but in either case, the variable domains (including the CDRs), which are designated by the numerical identifiers shown in Table 1 or 2, will remain unchanged and are expected to have identical or substantially similar antigen binding properties, regardless of the nature of the Fc domain.

Example 3.

Antibody binding to Ebola virus GP as determined by surface plasmon resonance.

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A. pH-dependent rate constant of dissociation reaction at 37°C.

Binding dissociation reaction rate constants (kd) and half-dissociation times (t1/2) for binding of Ebola virus GP to purified anti-Ebola virus GP mAbs at 37°C were determined using real-time surface plasmon resonance biosensor analysis on a Biacore T200. The Biacore CM4 sensor surface was derivatized by amine coupling with mouse anti-human Fc mAb (GE, #BR-1008-39) or goat anti-mouse Fc mAb (GE, #BR-1008-38) to capture purified anti-Ebola virus GP mAbs. All Biacore binding studies in Example 3A were performed in a buffer consisting of 0.01 M _Na2HPO4 / _NaH2PO4 , _0.15 M NaCl, 0.05% ( _vol /vol) surfactant P20 (PBS-P running buffer) at pH 7.4, 6.0, and 5.0. A low pH run was performed to assess whether the antibodies retained binding at low pH. This was intended to mimic the conditions the virus would encounter during membrane fusion when the endosome was acidified. Ebola virus GP with a C-terminal polyhistidine tag (EbolaGP.his; Sino Biologicals, Cat# 40442-V08B1) prepared in PBS-P running buffer (ranging from 90 nM to 11.1 nM, 3-fold dilutions) at different concentrations was injected onto the surface with captured Ebola virus GP mAb at a flow rate of 25 μl/min. The association of Ebola virus GP with the captured mAb was monitored for 5 min, and the dissociation of Ebola virus GP in PBS-P running buffer was monitored for 6 min. All dissociation rate constant experiments were performed at 37°C. The reaction dissociation rate constants (k _d ) were determined by fitting the real-time sensorgrams to a 1:1 binding model using the Scrubber 2.0 s function fit software. The half-dissociation times (t1/2) were calculated from the reaction rate constants as follows:

1п(2) .½ (4,,,,.)= ⁶⁰ *kd

The dissociation rate parameters for binding of Ebola virus GP to purified anti-Ebola virus GP mAbs at 37°C are shown in Table 3.

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Table 3

pH dependence of half-dissociation periods at 37°C

t7 ₂ Ratio pH7,4 / pH7,4 / Captured shAЬ pH6,0 pH5,0 H1H1238N0 control NB NB N1N17162P 0,3 0,3 N1N17177P 0,2 0,2 N1N17193R 1,1 0.8 N1N17196P 1,0 1,0 N1N17150R 0,2 0,2 N1N17151P 0,03 0,01 N1N17160P 0,2 0,4 N1N17161P 0,2 0,2 N1N17214P 1,0 1,0 N1N17219R 1,0 1,0 N1N17223P 0,4 0,4 N1N17228P 0,6 0,5 N1N17142P 0,5 0,5 N1N17141P 0,3 0,3 N1N17139P 0,2 0,2 N1N17134P 0,6 0,6 N1N17211P 6,7 3.0 N1N17210P 0,2 0,2 N1N17203P 0,2 0,1 N1N17199R 0,4 0,1 H1M17348N 0,3 0,4 H1M17349N 2.7 6.9 H1M17350N 0,1 1,1 H1M17351N NB NB H1M17352N 1,1 1,1 H1M17353N 1,6 0,3 H1M17354N 1,3 1,3 H1M17357N 0.8 0,5 H2aM17355N 0,5 0,6 H2aM17356N 0.9 0.9 H2aM17358N 0,7 0,4 H2aM17359N 0.8 0,7 H2aM17360N 0,2 0,5 H2aM17361N 0,2 0,5

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NB - under the assay conditions tested, there was no detectable binding.

B. Affinity and kinetics of binding at 25°C and 37°C.

Equilibrium dissociation constants (KD values) for binding of Ebola virus GP to purified anti-Ebola virus GP mAbs were determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. The Biacore CM4 sensor surface was derivatized by coupling at the amino group with a mouse monoclonal anti-human Fc antibody (GE, #BR-1008-39) or a goat monoclonal anti-mouse Fc antibody (GE, #BR-1008-38) to capture purified anti-Ebola virus GP mAbs. All Biacore binding studies in Example 3B were performed in a buffer consisting of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol % P20 surfactant (HBS-ET running buffer). Ebola virus GP with a C-terminal polyhistidine tag (Sino Biologicals, Cat# 40442-V08B1) prepared in HBS-ET running buffer (ranging from 90 nM to 3.3 nM, 3-fold dilutions) at various concentrations were injected onto the anti-Ebola virus GP mAb-captured surface at a flow rate of 30 μl/min. The association of Ebola virus GP with the captured monoclonal antibody was monitored for 5 min and the dissociation of Ebola virus GP in HBS-ET running buffer was monitored for 10 min. All binding kinetics experiments were performed at 25°C and 37°C. The association ( _ka ) and dissociation (kd) reaction rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c function fit software. Equilibrium binding dissociation constants (KD) and half-dissociation times (t1/2) were calculated from the reaction rate constants as follows:

kd 1p(2)

Ko (M) = and 1½ (min) = 60 * kd

The binding kinetic parameters for the binding of Ebola virus GP to purified anti-Ebola virus GP mAbs at 25°C and 37°C are shown in Tables 4A and 4B.

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Table 4A

Kinetics of binding at 25°C

mAb ka (1/Mc) kd (1/c) KD(M) tl/2 (min.) N1N17162P 2D8E+04 > IE-5 4,60E-10 < 1155 N1N17177P ZD8E+OZ 1D2E-05 3,53E-09 1030.3 N1N17193R 4.58E+03 l,08E-04 2,36E-08 106.6 N1N17196P 2.56E+04 > IE-5 3.91E-10 < 1155 N1N17150R 2D6E+04 5,42E-05 2,51E-09 213.1 N1N17151P l,26E+04 9,44E-05 7,49E-09 122.3 N1N17160P 6.85E+04 3,76E-03 5,48E-08 3.1 N1N17161P 5.29E+04 > IE-5 l,89E-10 < 1155 N1N17214P 3.76E+04 > IE-5 2,66E-10 < 1155 N1N17219R ZD1E+04 2,90E-05 9,34E-10 398.3 N1N17223P 3.00E+04 6,08E-05 2,03E-09 190,0 N1N17228P 4.49E+04 l,69E-03 3,76E-08 6.9 N1N17142P 2.00E+04 2,81E-05 l,41E-09 410.7 N1N17141P l,98E+04 9,69E-05 4,90E-09 119.2 N1N17139P 2.29E+04 l,63E-04 7,13E-09 70.8 N1N17134P 7.65E+04 9,41E-04 l,23E-08 12.3 N1N17211P 3.33E+04 2D4E-04 6,43E-09 54.0 N1N17210P l,09E+02 2,06E-04 l,89E-06 56,0 N1N17203P 2.78E+04 l,68E-04 6.04E-09 68.7 N1N17199R l,25E+04 2,36E-04 l,89E-08 49,0 H1M17348N IC IC IC IC H1M17349N 7.03E+04 8,69E-04 l,24E-08 13.3 H1M17350N IC IC IC IC H1M17351N NB NB NB NB H1M17352N IC IC IC IC H1M17353N IC IC IC IC H1M17354N 4.94E+04 ZD6E-OZ 6,39E-08 3.7 H1M17357N IC IC IC IC H2aM17355N l,44E+04 > IE-5 6,96E-10 < 1155 H2aM17356N 2D8E+04 9,57E-05 4,40E-09 120.7 H2aM17358N 3.22E+02 2,01E-04 6,23E-07 57.5 H2aM17359N 3.82E+03 l,95E-04 5,09E-08 59.4 H2aM17360N 2.30E+04 l,06E-05 4,63E-10 1086,5 H2aM17361N l,22E+02 l,25E-04 l,02E-06 92.5

NB - under the assay conditions tested, there was no detectable binding.

IC - binding sensorgram, insufficient for approximation.

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Table 4B

Binding kinetics at 37°C

inAb ka (1/Mc) kd (1/c) KD(M) tl/2 (min.) N1N17162P 3.47E+04 > IE-5 2,88E-10 < 1155 N1N17177P l,68E+04 l,62E-04 9,62E-09 71.3 N1N17193R l,58E+04 5,03E-04 3,18E-08 22.9 N1N17196P 3,16E+04 > IE-5 3,17E-10 < 1155 N1N17150R 3,18E+04 3,94E-05 l,24E-09 292.8 N1N17151P 2.26E+04 3,83E-04 l,70E-08 30.2 N1N17160P 5.72E+04 5,63E-03 9,85E-08 2,1 N1N17161P 4.39E+04 > IE-5 2,28E-10 < 1155 N1N17214P 3.67E+04 l,54E-04 4,20E-09 74.9 N1N17219R 4.41E+04 > IE-5 2,27E-10 < 1155 N1N17223P 3.51E+04 2,42E-04 6,89E-09 47.7 N1N17228P 7.32E+04 3,83E-03 5,23E-08 3 N1N17142P 2,60E+04 l,74E-04 6,68E-09 66.6 N1N17141P 2.65E+04 2,92E-04 l,10E-08 39.6 N1N17139P 2.48E+04 5,12E-04 2,06E-08 22.5 N1N17134P 6.99E+04 4,69E-04 6,70E-09 24.6 N1N17211P l,90E+04 7,31E-04 3,84E-08 15.8 N1N17210P 6,19E+02 6,12E-04 9,89E-07 18.9 N1N17203P 3.85E+04 l,19E-03 3,09E-08 9.7 N1N17199R 3.04E+04 l,28E-03 4,22E-08 9 H1M17348N IC IC IC IC H1M17349N l,77E+04 l,93E-03 l,09E-07 6 H1M17350N 4.84E+02 1,00E-03 2,07E-06 H,5 H1M17351N NB NB NB NB H1M17352N 4.09E+04 l,55E-03 3,80E-08 7.4 H1M17353N 2,33E+02 5,38E-04 2,31E-06 21,5 H1M17354N 5.08E+04 5,73E-03 l,13E-07 2 H1M17357N 2.35E+04 l,84E-03 7,81E-08 6.3 H2aM17355N l,99E+04 2,06E-04 l,03E-08 56.2 H2aM17356N 7.26E+03 2,50E-04 3,44E-08 46.2 H2aM17358N l,07E+04 5,67E-04 5,28E-08 20.4 H2aM17359N l,54E+04 3,52E-04 2,29E-08 32.8 H2aM17360N 2.43E+04 3,37E-04 l,39E-08 34.3 H2aM17361N l,83E+04 4,15E-04 2,27E-08 27.8

NB - under the assay conditions tested, there was no detectable binding.

IC - binding sensorgram, insufficient for approximation.

Results.

As can be seen from Tables 4A and 4B above, the antibodies bound to Ebola virus GP with a

- 39 048493 our KD ranged from 934 pM to 1890 nM at 25°C and from 227 pM to 2310 nM at 37°C. At pH 7.4, the antibodies exhibited t1/2 values ranging from 3.0 to greater than 1155 minutes at 25°C and from 2.0 minutes to greater than 1155 minutes at 37°C. No loss of binding was observed at low pH. Some antibodies exhibited elevated t1/2 values at low pH relative to pH 7.4. The antibodies with a 3-fold or more increase in half-dissociation period (t1/2) at pH 5 and/or pH 6 included H1H17162P, H1H17177P, H1H17150P, H1H17151P, H1H17160P, H1H17161P, H1H17141P, H1H17139P, H1H17210P, H1H17203P, H1H17199P, H1M17348N, H1M17350N, H2aM17360N, and H2aM17361N.

Example 4.

Creation of Ebola virus pseudoparticles and neutralization research.

Ebola virus pseudoparticles (also called virus-like particles or VLPs) were generated by co-transfecting 293T cells with a mixture of plasmid constructs expressing Ebola virus GP, HIV gag-pol, and an HIV viral vector encoding firefly luciferase. Supernatants containing Ebola virus pseudoparticles were collected 48 h post-transfection, clarified by centrifugation, aliquoted, and frozen at -80°C. Control pseudoparticles were generated by replacing the plasmid expressing Ebola virus GP with a plasmid encoding vesicular stomatitis virus glycoprotein (VSVg).

Ebola virus pseudoparticle-based neutralization assay

The pseudoparticles generated as described above were tested in neutralization assays. Specifically, antibodies at various dilutions were incubated with Ebola virus pseudoparticles for 1 h at room temperature. Huh7 cells were detached with 0.02 M EDTA, washed, and incubated with antibody/pseudoparticle mixtures for 72 h. Infection efficiency was quantified by detecting luciferase activity using the BrightGlo® Luciferase Assay (Promega, San Luis Obispo, CA, USA) and read in a Victor® X3 Plate Reader (Perkin Elmer, Waltham, MA, USA) by light production.

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Table 5

Neutralization of VLP Zaire 2014

GO AV The corresponding hybridoma is GO AЬ Neutralizer VLP Zaire 2014 TC50 (Μ) N1N17134P - - N1N17139P - - N1N17142P + 1,59Ε-09 N1N17151P + 1,51Ε-09 N1N17161P + 2,55E-10 N1N17162P + 2,86E-10 N1N17193P - - N1N17196P + l,68E-09 N1N17199R - - N1N17203P + 8,68E-10 N1N17214P + 8.99E-10 N1N17219R + 6,95E-10 N1N17223P + l,58E-09 N1N17228P + 3,26E-09 H1M17354N H1M17354N - - H2aM17356N H2aM17356N + 4,77E-09 H1M17357N H1M17357N - - H2aM17358N H2aM17358N + 4,68E-09 H2aM17359N H2aM17359N + 3,36E-09 H2aM17360N H2aM17360N + 3.75E-09

The data shown in Table 5 above indicate that 14 of the 20 Ebola virus antibodies of the present invention, when used in the experimental design described herein, effectively neutralized infectivity with an IC ₅₀ ranging from about ^10-11 M to about ^10-9 M.

Example 5.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by antibodies to Ebola virus.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was tested by the ability of antibodies to signal through a CD16-based reporter system (ADCC Reporter Bioassay Core Kit, Promega, San Luis Obispo, CA, USA). 293 cells expressing Ebola virus GP were plated. One day later, diluted antibodies raised in fuc cell lines (see US Patent No. 8,409,838) and effector cells (1.5:1 effector to target ratio) were added and incubated overnight. Reporter activity was measured by the BioGlo® luciferase assay (Promega, San Luis Obispo, CA, USA) and read in a Victor® X3 plate reader (Perkin Elmer, Waltham, MA, USA) by light production.

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Table 6

ADCC Results

ADCC reporter bioassay SH AЬ Clone ID ADCC- activity N1N17134P + N1N17139P + N1N17142P + N1N17151P + N1N17161P - N1N17162P - N1N17193R + N1N17196P + N1N17199R + N1N17203P + N1N17214P + N1N17219R - N1N17223P + N1N17228P + H1M17354N HCAF05C08- 22 + H2aM17356N HCAF08C07- 09 + H1M17357N HCAF09D11- 13 + H2aM17358N HCAF12C05- 14 + H2aM17359N HCAF12C06- 26 + H2aM17360N HCAF12G09- 07 +

The ability of antibodies to mediate ADCC was calculated by activity relative to an isotype (negative) control. Any value greater than 5 times the negative control was considered positive. The data in Table 6 above indicate that 17 of the 20 Ebola antibodies mediated ADCC.

Example 6.

Cross-competition using Octet.

Competition for binding between anti-Ebola virus GP mAbs previously shown to bind to Ebola virus GP was determined by a label-free, real-time biolayer interferometric assay (BLI) using the Octet HTX biosensor (ForteBio Corp., a division of Pall Life Sciences). Binding of appropriate controls to soluble GP (sGP), GP1, or GP2 was measured in the same assay format and subtracted from the result obtained for the Ebola virus GP reagent of interest for each mAb tested. The entire experiment was performed at 25°C in a buffer consisting of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol% P20 surfactant, 1.0 mg/ml BSA (HBS-ET buffer for Octet), with plate shaking at 1000 rpm. To assess whether the two antibodies could compete with each other for binding to their respective epitopes on Ebola virus GP, the expression

- 42 048493 with a C-terminal polyhistidine tag (Ebola virus GP.h, Sino Biologicals Inc., also see GenBank AHX24649.1 and SEQ ID NO: 314), approximately ~1.0 μg of Ebola virus GP was first captured onto anti-penta-His antibody-coated Octet biosensors (Fortebio Inc, #18-5079) by immersing the biosensors in wells containing 20 μg/mL Ebola virus GP solution for 3 min. The antigen-captured biosensors were then saturated with a primary anti-Ebola virus GP monoclonal antibody (hereafter referred to as mAb-1) by immersing in wells containing 50 μg/mL mAb-1 solution for 5 min. The biosensors were then immersed in wells containing 50 μg/mL of a second monoclonal antibody to Ebola virus GP (hereafter referred to as mAb-2) for 3 min. Between each step of the experiment, all biosensors were washed in HBS-ET buffer for Octet. The binding response was monitored in real time during the experiment and the binding response was recorded at the end of each step. The binding response of mAb-2 to Ebola virus GP pre-complexed with mAb-1 was compared and the competitive/non-competitive nature of the different monoclonal antibodies to Ebola virus GP was determined using the 50% inhibition threshold. Table 7 details the ratios of antibodies competing in both directions, regardless of the binding order.

As shown in Table 7, the left column shows the mAb1 antibodies that are captured by the ANS Octet biosensors, and the right column shows the antibodies (mAb2) that cross-compete with the mAbl antibody.

Table 7

Cross-competition of antibodies to Ebola virus GP for binding to Ebola virus GP First mAb (mAb-1) captured by ANS Octet biosensors Н1Н17160Р Antibodies mAb-2, for which competition with mAb-Ι was observed H1H17160P, H1M17354N, H1M17357N, H1H17228P, H1H17203P H1M17354N Н1Н17160Р, H1M17354N, H1M17357N, Н1Н17228Р, Н1Н17203Р H1M17357N Н1Н17228Р Н1Н17160Р, H1M17354N, H1M17357N, Н1Н17228Р, Н1Н17203Р N1N17160R, H1M17354N, H1M17357N, N1N17228R, N1N17203R, Н1Н17151Р, Н1Н17142Р, Н1Н17177Р, H2aM17359N N1N17160R, H1M17354N, H1M17357N, N1N17228R, N1N17203R, N1N17203P N1N17151P Н1Н17151Р, Н1Н17142Р, Н1Н17177Р, H2aM17359N, Н1Н17214Р, Н1Н17199Р, H2aM17358N, H2aM17360N, H1M17352N H1H17228P, H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N,

- 43 048493

H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17353N, H1H17223P, H1H17196P, H1H17193P H1H17142P H1H17228P, H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1M17353N, H1H17223P, H1H17196P, H1H17193P H1H17177P H1H17228P, H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N, H1H17141P, H1H17223P, H1H17196P, H1H17139P, H1H17193P, H1M17350N H2aM17359N H1H17228P, H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N H2aM17356N, H2aM17361N, H2aM17355N, 1H17211P, H1M17348N, H1M17353N, H1H17141P, H1H17223P H1H17139P, H1H17193P, H1M17350N H1H17214P H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N H1H17141P, H1H17223P, H1H17139P H1H17193P, H1M17350N H1H17199P H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N, H1H17141P, H1H17139P, H1H17193P, H1M17350N H2aM17358N H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N

- 44 048493

H1H17139P, H1H17193P, H1M17350N H2aM17360N H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N H1M17353N, H1H17139P, H1H17193P, H1M17350N H1M17352N H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N, H1H17139P, H1H17193P, H1M17350N H2aM17356N H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N H2aM17361N, H2aM17355N, H1H17211P, H1M17348N H1M17353N, H1H17139P, H1H17193P, H1M17350N H2aM17361N H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N, H1H17139P, H1H17193P, H1M17350N H2aM17355N H1H17203P, H1H17151P, H1H17142P, H1H17177P, H2aM17359N H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N H1M17353N, H1H17193P, H1M17350N H1H17211P H1H17151P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N, H1M17353N H1H17193P, H1M17350N H1M17348N H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N H2aM17355N, H1H1721 IP, H1M17348N, H1M17353N, H1H17193P H1M17350N H1M17353N H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P H1H17199P, H2aM17358N, H2aM17360N, H1M17352N H2aM17356N, H2aM17361N, H2aM17355N, H1H17211P, H1M17348N,

- 45 048493

H1M17353N, Н1Н17141Р, Н1Н17223Р, Н1Н17196Р, Н1Н17139Р, Н1Н17193Р N1N17141P Н1Н17177Р, H2aM17359N, Н1Н17214Р, Н1Н17199Р, H1M17353N N1N17141R, N1N17223R, N1N17196R, N1N17139R N1N17223P Н1Н17151Р, Н1Н17142Р, Н1Н17177Р, H2aM17359N, Н1Н17214Р, H1M17353N N1N17141R, N1N17223R, N1N17196R, N1N17139R N1N17196P Н1Н17151Р, Н1Н17142Р, Н1Н17177Р, H1M17353N, Н1Н17141Р, N1N17223R, N1N17196R, N1N17139R N1N17139P Н1Н17177Р, H2aM17359N, Н1Н17214Р, Н1Н17199Р, H2aM17358N H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H1M17353N H1H17141P, H1H17223P, H1H17196P, H1H17139P N1N17193R H1H17151P, H1H17142P, H1H17177P, H2aM17359N, H1H17214P, H1H17199P H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N H2aM17355N, H1H17211P, H1M17348N, H1M17353N, H1H17193P, H1M17350N H1M17350N H1H17177P, H2aM17359N, H1H17214P, H1H17199P, H2aM17358N, H2aM17360N, H1M17352N, H2aM17356N, H2aM17361N, H2aM17355N H1H17211P, H1M17348N, H1H17193P, H1M17350N N1N17219R H1H17219P, H1H17150P, H1H17161P N1N17150R H1H17219P, H1H17150P, H1H17161P N1N17161P H1H17219P, H1H17150P, H1H17161P H1M17349N H1M17349N N1N17134P H1H17134P N1N17162P H1H17162P N1N17210P H1H17210P

Example 7.

Sequential binding of H1H17203P, H1H17139P and H1H17161P to the Ebola virus glycoprotein.

Using the information obtained from the cross-competition experiments, a sequential binding study was performed to determine whether the three individual candidate antibodies were able to simultaneously bind to the soluble glycoprotein (GP) of Ebola virus, thereby confirming that the binding sites on Ebola virus GP are independent for each monoclonal antibody. If so, this information would support the use of these antibodies in a therapeutic cocktail.

In this regard, sequential binding experiments for three anti-Ebola virus GP mAbs to Ebola virus GP, H1H17203P, H1H17139P, and H1H17161P, were performed for independent and non-competitive binding to Ebola virus GP. This experiment was performed by label-free real-time biolayer interferometric assay (BLI) using the Octet RED biosensor (ForteBio Corp., a division of Pall Life Sciences). The entire experiment was performed at 25°C in a buffer consisting of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol % P20 surfactant, 1.0 mg/mL BSA (HBS-ET buffer for Octet), with plate shaking at 1000 rpm. To assess whether three antibodies could simultaneously bind to captured Ebola virus GP antigen expressed with a C-terminal polyhistidine tag (Ebola virus GP.his, Sino Biologicals), approximately ~0.6 nM of Ebola virus GP.h was first captured on a biosensor

- 46,048,493 anti-penta-His coated biosensors (Fortebio Inc, #18-5079) were coated by immersing the biosensors in wells containing 20 μg/mL Ebola virus GP.h solution for 3 minutes. The antigen-captured biosensors were then saturated with the primary anti-Ebola virus GP monoclonal antibody (later referred to as H1H17161P) by immersing them in wells containing 50 μg/mL REGN H1H17161P solution for 5 minutes. The biosensors were then immersed in wells containing 50 μg/mL Ebola virus GP monoclonal antibody (later referred to as H1H17139P) solution for 5 minutes. Finally, 50 μg/ml of the third antibody (later called H1H17161P) was injected for 5 min until saturation was reached. The binding response was monitored in real time during the experiment and the binding response was recorded at the end of each step.

Results.

The three candidate monoclonal antibodies tested were able to bind simultaneously to ebolavirus GP, indicating that each antibody did not interfere with the binding site on ebolavirus GP of the other antibodies tested, indicating that they bound or interacted with different epitopes. This substantiates the relevance for the use of these three antibodies in a therapeutic antibody cocktail.

Example 8.

Binding of Ebola virus antibodies to different strains of Ebola virus-like particles (VLPs).

A study was conducted to determine whether antibodies to Ebola virus GP would react with virus-like particles (VLPs) containing GP from other Ebola virus strains. Included in this study were VLPs containing GP from Bundibugyo NC_014373, Cote d'Ivoire FJ217162, Sudan NC_006432, Zaire. 1995, Zaire.2014 AY354458, and a negative control, VSV glycoprotein (VSVg). The study was performed using MesoScale Discovery (MSD) technology, which allows binding/immobilization of Ebola virus strain VLPs (which express Ebola virus glycoproteins/viral surface proteins) to a carbon surface, followed by ELISA-type binding assay. The objective was to identify binding profiles of mAbs against different Ebola virus strains.

The assay was performed in 96-well polypropylene microwell plates by first preparing 1:10 dilutions of supernatants from different VLPs/well (as indicated in the table below) and adding the dilutions to PBS (50 µl/well) and incubating at 4°C overnight.

The liquid in the wells was discarded followed by blocking with 150 μl/well in PBS + 2% BSA and incubation for one hour at room temperature. The contents of each well were then discarded and the wells were washed with PBS using an AquaMax2000 immunoassay plate washer for MSD. Fifty microliters of primary antibody was diluted in PBS + 1% BSA and incubated at room temperature with shaking at intermediate speed (5). The contents of the wells were then discarded and the plates were washed with PBS. Fifty microliters of sulfamethodoxylate detection reagent (human or mouse Fc) at a concentration of 1 μg/ml in PBS + 0.5% BSA was added to each well and incubated at room temperature for one hour with shaking at intermediate speed (5). The well contents were discarded and the plates were washed with PBS + 0.5% BSA. 150 µl of 1X surfactant-free reading buffer was added to each well and the plates were read on a SECTORImager6000 from the barcode.

The results, which are shown in Table 8 below, indicate that all of the anti-Ebola virus GP antibodies tested bound to VLPs containing GP from Zaire 2014 and Zaire 1995. Some of the antibodies tested, in addition to binding to the two Zaire strains, bound to VLPs containing GP from other Ebola virus strains, as shown in Table 8. Specifically, in addition to binding to VLPs containing GP from Zaire 2014 and Zaire 1995, the anti-Ebola virus antibodies designated H1H17161P and H1H17162P bound to VLPs containing GP from the Sudan and Bundibugyo strains, while the anti-Ebola virus antibodies designated H2aM17356N and H1H17142P bound to the Bundibugyo and Cote d'Ivoire strains.

- 47 048493

Table 8

Cross-reactivity of Ebola virus antibodies with GP ________from different Ebola virus strains____________

Binding of Ebola virus antibody to VLPs containing GP from different Ebola virus strains AbPID Sudan Bundibugyo Ivory Coast Zaire 2014 Zaire 1995 N1N17161P + + — + + N1N17139P — — — + + N1N17203P — — — + + N1N17219R — — — + + N1N17162P + + — + + N1N17199R — — — + + N1N17193R — — — + + H1M17354N — — — + + H1M17357N — — — + + N1N17134P — — — + + H1H17360N — — — + + H1H17358N2 - - - + + H2aM17356N — + + + + N1N17223P — — — + + N1N17196P — — — + + N1N17151P — — — + + N1N17142P — + + + + N1N17214P — — — + + N1N17228P — — — + + H2aM17359N — — — + +

Example 9.

In vitro neutralization of live/infectious Ebola virus (EBOV).

Antibodies designated H1H17203P, H1H17139P, and H1H17161P were assayed for their ability to neutralize infectious EBOV in Vero cells. Vero cells were seeded in 384-well plates in DMEM-10% FBS and allowed to grow to approximately 75% confluence at 37°C. H1H17203P, H1H17139P, and H1H17161P were diluted as previously described. EBOV strains (Mayinga, Kikwit, Makona, and guinea pig-adapted Mayinga) were thawed and appropriately diluted to MOIs of 0.01 to 0.1. A commercially available anti-EBOV antibody designated KZ52 was used as a positive control. (See Maruyama, T. et al., J Virol 73, 6024–6030 (1999). Antibodies were incubated with virus for 1 hour at 37°C. The antibody/virus mixture was then added to the pre-seeded cells and the plates were incubated at 37°C for 24 hours. After the incubation period, the plates were removed from the incubator and inactivated by immersion in 10% neutral buffered formalin, placed in a sealed bag and stored at 4°C overnight in BSL-4. The plates were washed 3 times in 1X-PBS and the cells were permeabilized at room temperature (RT) with 25 μl 0.1% Triton X-100 in 1X-PBS for 15–20 minutes. Triton X was discarded and the plates were blocked with 3.5% BSA in 1X-PBS for 1 hour at RT. Plates were treated overnight at 4°C with EBOV GP 4F3 primary antibody (see IBT BIOSERVICES for EBOV GP mouse monoclonal antibody 4F3, catalog #0201-020) diluted 1:1500 in 1X-PBS. Plates were washed in 1X-PBS for 10-15 minutes and the procedure was repeated two more times. Cells were incubated for 1 hour with Alexa-fluor-488-conjugated anti-mouse secondary antibody.

The secondary antibody was discarded and the plates were washed in 1X-PBS for 10-15 min and repeated two more times. The plates were incubated with 25 µl/well Hoechst (1:50,000 in 1X-PBS) for 30 min at RT. The plates were visualized by fluorescence microscopy using the blue and green fluorescence channels.

Results.

From the results shown in Fig. 1, it is evident that the antibody to H1H17161P neutralized the live virus and was more active than the positive control antibody KZ52, but the antibodies designated as H1H17203P and H1H17139P did not act as neutralizers.

Example 10.

Binding of Ebola virus antibodies to soluble GP (sGP).

The fourth gene in the EBOV genome encodes two unique proteins, a nonstructural, dimeric secreted glycoprotein called sGP and a trimeric, virion-attached envelope glycoprotein (GP). The two GPs share the first 295 amino acids but have unique C-termini. To determine whether Regeneron's mAb candidates bind to sGP, recombinant sGP.mmh protein (SEQ ID NO: 317) was produced in-house. The Octet HTX interferometric biosensor was used to determine whether mAbs H1H17203P, H1H17139P, and H1H17161P could bind to the Ebola virus sGP.mmh protein. The assay format involved capturing H1H17203P, H1H17139P, and H1H17161P onto hFc sensor tips followed by immersion in 300 nM solutions of Ebola virus GP.10xhis (SEQ ID NO: 318), sGP.mmh (SEQ ID NO: 317), or hCNTFR (ciliary neurotrophic factor receptor.mmh, which served as a negative control protein). Each mAb was captured at levels ranging from 0.94 to 1.36 nM.

As shown in Fig. 2, all mAbs showed specific binding to Ebola virus GP.10xhis and no binding to the negative control protein; while only H1H17139 showed specific binding to Ebola virus sGP.mmh. These results suggest that the binding epitope of H1H17139 is likely located in a common region within the first 295 amino acids of both sGP and GP; while the other mAbs probably recognized only the C-terminus of Ebola virus GP.

Example 11.

Binding of additional antibodies to EBOV GP to Ebola virus GP.h, soluble Ebola virus GP.mmh, and hCNTFR.mmh.

An additional study was conducted to determine the binding characteristics of additional EBOV GP antibodies of the present invention; in particular, a study was conducted to determine the ability of these additional antibodies to bind to soluble GP and GP. This study was performed using a label-free real-time biolayer interferometric assay (BLI) using an Octet HTX biosensor (ForteBio Corp., a division of Pall Life Sciences). The entire experiment was carried out at 25°C in a buffer consisting of 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05 vol % P20 surfactant, 1.0 mg/mL BSA (HBS-ET buffer for Octet), with plate shaking at 1000 rpm. To assess whether the antibodies could bind to Ebola virus sGP or other Ebola virus GP reagents, approximately ~1.0 nM of anti-Ebola virus GP antibodies were captured onto anti-human Fc coated Octet biosensors (Fortebio Inc, #18-5064) by immersing the biosensors in wells containing 20 μg/mL mAb solutions for 3 min. The mAb-captured biosensors were tested for binding to the selected protein reagents by immersing in wells containing 300 nM Ebola virus GP proteins or irrelevant controls for 5 min. Between each step of the experiment, all biosensors were washed in Octet HBSET buffer. The binding response was monitored in real time throughout the experiment and the binding response was recorded at the end of each step.

Results.

As shown in Table 9, any value below 0.10 nM was defined as non-binding antibody. Based on the results available to date, all but one of the antibodies tested (H1H17360N) were observed to bind to full-length EBOV GP, and thirteen of the twenty antibodies tested were observed to bind to soluble GP (sGP).

-

Claims (39)

Table 9 Binding of anti-Ebola virus GP antibodies to Ebola virus GP.h, soluble Ebola virus GP.mmh, and hCNTFR.mmh Antibody number csGP binding 300 nM sGP Ebola virus (F2) bind (nM) 300 nM GP.h Ebola virus bind (nM) 300 nM hCNTFR.mmh (negative control) bind (nM) H1H17161P No 0.00 0.55 -0.01 Н1Н17139Р Yes 0.19 0.55 0.01 H1H17203P No 0.02 0.61 0.01 H1H17219P No 0.01 0.68 0.01 H1H17162P No 0.03 0.49 0.02 Н1Н17199Р Yes 0.33 0.38 0.00 Н1Н17193Р Yes 0.26 0.33 0.02 H1M17354N Yes 0.18 0.71 0.02 H1M17357N No 0.10 0.56 0.01 H1H17134P No 0.01 0.70 -0.01 H1H17360N No 0.09 0.09 0.03 H1H17358N2 Yes 0.30 0.35 0.01 H1H17356N Yes 0.22 0.23 0.02 Н1Н17223Р Yes 0.31 0.61 0.02 N1N17196R Yes 0.25 0.62 0.01 Н1Н17151Р Yes 0.33 0.43 -0.02 Н1Н17142Р Yes 0.28 0.34 0.01 Н1Н17214Р Yes 0.38 0.52 0.01 Н1Н17228Р Yes 0.35 0.58 0.00 H1H17359N Yes 0.39 0.51 0.00 CLAUSE OF THE INVENTION 1. The use of a pharmaceutical composition in a method for inhibiting the activity of the Ebola virus (EBOV), wherein the pharmaceutical composition comprises one or more isolated antibodies or antigen-binding fragments thereof that specifically bind to EBOV and/or Ebola virus glycoprotein (EBOV-GP), selected from the group consisting of: (i) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 20; the amino acid sequence of HCDR2 of SEQ ID NO: 22; the amino acid sequence of HCDR3 of SEQ ID NO: 24; the amino acid sequence of LCDR1 of SEQ ID NO: 28; the amino acid sequence of LCDR2 of SEQ ID NO: 30 and the amino acid sequence of LCDR3 of SEQ ID NO: 32; (ii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 68; the amino acid sequence of HCDR2 of SEQ ID NO: 70; the amino acid sequence of HCDR3 of SEQ ID NO: 72; the amino acid sequence of LCDR1 of SEQ ID NO: 76; the amino acid sequence of LCDR2 of SEQ ID NO: 78; and the amino acid sequence of LCDR3 of SEQ ID NO: 80; and (iii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 148; the amino acid sequence of HCDR2 of SEQ ID NO: 150; the amino acid sequence of HCDR3 of SEQ ID NO: 152; the amino acid sequence of LCDR1 of SEQ ID NO: 156; the amino acid sequence of LCDR2 of SEQ ID NO: 158; and the amino acid sequence of LCDR3 of SEQ ID NO: 160. - 50 048493 2. The use according to claim 1, wherein the pharmaceutical composition is used in a method for inhibiting intercellular transmission of EBOV. 3. The use according to claim 1, wherein the pharmaceutical composition is used in a method for destroying EBOV-infected cells. 4. The use according to claim 1, wherein the pharmaceutical composition is used in a method for reducing the production of pathogenic EBOV. 5. The use according to claim 1, wherein the pharmaceutical composition is used in a method for inhibiting the penetration of EBOV into a cell. 6. The use according to claim 1, wherein the infectious EBOV is neutralized in vitro or in vivo. 7. The use according to claim 1, wherein the composition is used in combination with one or more additional therapeutic agents or treatments against EBOV. 8. The use according to claim 7, wherein one or more additional therapeutic agents are selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody to EBOV, an EBOV vaccine, Ebola TCM, brincidofovir, favipiravir, BCX-4430, AVI-7537 and interferons. 9. The use according to claim 1, wherein the pharmaceutical composition comprises a combination of two or more antibodies to EBOV selected from the group consisting of: (a) (i) and (ii); (b) (i) and (iii); (c) (ii) and (iii); and (d) (i), (ii) and (iii). 10. Use according to claim 1, wherein the virus is neutralized in vivo, and the composition is administered to a subject in need thereof subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally. 11. Use of a pharmaceutical composition in a method for preventing, treating or alleviating at least one symptom of an infection caused by EBOV, or reducing the frequency or severity of at least one symptom of an infection caused by EBOV, wherein the pharmaceutical composition comprises one or more isolated antibodies or antigen-binding fragments thereof that specifically bind to the Ebola virus (EBOV) and/or the Ebola virus glycoprotein (EBOVGP), selected from the group consisting of: (i) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 20; the amino acid sequence of HCDR2 of SEQ ID NO: 22; the amino acid sequence of HCDR3 of SEQ ID NO: 24; the amino acid sequence of LCDR1 of SEQ ID NO: 28; the amino acid sequence of LCDR2 of SEQ ID NO: 30 and the amino acid sequence of LCDR3 of SEQ ID NO: 32; (ii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 68; the amino acid sequence of HCDR2 of SEQ ID NO: 70; the amino acid sequence of HCDR3 of SEQ ID NO: 72; the amino acid sequence of LCDR1 of SEQ ID NO: 76; the amino acid sequence of LCDR2 of SEQ ID NO: 78; and the amino acid sequence of LCDR3 of SEQ ID NO: 80; and (iii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 148; the amino acid sequence of HCDR2 of SEQ ID NO: 150; the amino acid sequence of HCDR3 of SEQ ID NO: 152; the amino acid sequence of LCDR1 of SEQ ID NO: 156; the amino acid sequence of LCDR2 of SEQ ID NO: 158; and the amino acid sequence of LCDR3 of SEQ ID NO: 160. 12. The use according to claim 11, wherein at least one symptom is selected from the group consisting of fever, headache, fatigue, loss of appetite, myalgia, diarrhea, vomiting, abdominal pain, dehydration and unexplained bleeding. 13. Use according to claim 11, wherein the pharmaceutical composition is formulated for prophylactic or therapeutic administration. 14. Use according to claim 11, wherein the pharmaceutical composition is used for therapeutic purposes in combination with a second therapeutic agent. 15. The use according to claim 14, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody to EBOV, a vaccine against EBOV, small interfering RNAs that specifically target viral RNA polymerase, brincidofovir, favipiravir, BSH-4430, antisense phosphorodiamidate morpholino oligomers that specifically target the VP24 gene of the Ebola virus, and interferons. 16. The use according to claim 14, wherein the second therapeutic agent is an anti-inflammatory agent selected from corticosteroids and non-steroidal anti-inflammatory drugs. 17. Use according to claim 11, wherein the pharmaceutical composition is formulated for subcutaneous, intravenous, intradermal, intramuscular, intranasal or oral administration. - 51 048493 18. The use according to claim 11, wherein the pharmaceutical composition comprises a combination of two or more antibodies to EBOV selected from the group consisting of: (a) (i) and (ii); (b) (i) and (iii); (c) (ii) and (iii); and (d) (i), (ii) and (iii). 19. Use of a pharmaceutical composition in a method of treating a subject that has EBOV, or increasing the survival or likelihood of survival of a subject suffering from an infection caused by EBOV, or a subject exposed to EBOV or at risk of exposure to or acquiring EBOV, wherein the pharmaceutical composition comprises one or more isolated antibodies or antigen-binding fragments thereof that specifically bind to Ebola virus (EBOV) and/or Ebola virus glycoprotein (EBOV-GP), selected from the group consisting of: (i) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 20; the amino acid sequence of HCDR2 of SEQ ID NO: 22; the amino acid sequence of HCDR3 of SEQ ID NO: 24; the amino acid sequence of LCDR1 of SEQ ID NO: 28; the amino acid sequence of LCDR2 of SEQ ID NO: 30 and the amino acid sequence of LCDR3 of SEQ ID NO: 32; (ii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 68; the amino acid sequence of HCDR2 of SEQ ID NO: 70; the amino acid sequence of HCDR3 of SEQ ID NO: 72; the amino acid sequence of LCDR1 of SEQ ID NO: 76; the amino acid sequence of LCDR2 of SEQ ID NO: 78; and the amino acid sequence of LCDR3 of SEQ ID NO: 80; and (iii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 148; the amino acid sequence of HCDR2 of SEQ ID NO: 150; the amino acid sequence of HCDR3 of SEQ ID NO: 152; the amino acid sequence of LCDR1 of SEQ ID NO: 156; the amino acid sequence of LCDR2 of SEQ ID NO: 158; and the amino acid sequence of LCDR3 of SEQ ID NO: 160. 20. Use according to claim 19, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof. 21. The use according to claim 19, wherein the composition is used in combination with one or more additional therapeutic agents or treatments against EBOV. 22. The use according to claim 21, wherein one or more additional therapeutic agents are selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody to EBOV, an EBOV vaccine, Ebola TCM, brincidofovir, favipiravir, BCX-4430, AVI-7537 and interferons. 23. The use according to claim 22, wherein the second therapeutic agent is an anti-inflammatory agent selected from corticosteroids and non-steroidal anti-inflammatory drugs. 24. Use according to claim 19, wherein the pharmaceutical composition is formulated for subcutaneous, intravenous, intradermal, intramuscular, intranasal or oral administration. 25. The use according to claim 19, wherein the pharmaceutical composition comprises a combination of two or more antibodies to EBOV selected from the group consisting of: (a) (i) and (ii); (b) (i) and (iii); (c) (ii) and (iii); and (d) (i), (ii) and (iii). 26. Use of a pharmaceutical composition in a method for prophylactically treating a subject exposed to EBOV or at risk of exposure to EBOV or at risk of acquiring an infection caused by EBOV, or minimizing the risk of acquiring an infection caused by EBOV in a subject exposed to EBOV or at risk of exposure to EBOV, wherein the pharmaceutical composition comprises one or more isolated antibodies or antigen-binding fragments thereof that specifically bind to Ebola virus (EBOV) and/or Ebola virus glycoprotein (EBOV-GP), selected from the group consisting of: (i) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 20; the amino acid sequence of HCDR2 of SEQ ID NO: 22; the amino acid sequence of HCDR3 of SEQ ID NO: 24; the amino acid sequence of LCDR1 of SEQ ID NO: 28; the amino acid sequence of LCDR2 of SEQ ID NO: 30 and the amino acid sequence of LCDR3 of SEQ ID NO: 32; (ii) an antibody or antigen-binding fragment thereof comprising the amino acid sequence of HCDR1 of SEQ ID NO: 68; the amino acid sequence of HCDR2 of SEQ ID NO: 70; the amino acid sequence of HCDR3 of SEQ ID NO: 72; the amino acid sequence of LCDR1 - 52 048493 SEQ ID NO: 76; the LCDR2 amino acid sequence of SEQ ID NO: 78 and the LCDR3 amino acid sequence of SEQ ID NO: 80; and (iii) an antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO: 148; the HCDR2 amino acid sequence of SEQ ID NO: 150; the HCDR3 amino acid sequence of SEQ ID NO: 152; the LCDR1 amino acid sequence of SEQ ID NO: 156; the LCDR2 amino acid sequence of SEQ ID NO: 158 and the LCDR3 amino acid sequence of SEQ ID NO: 160. 27. The use according to claim 26, wherein the subject is selected from the group consisting of an immunocompromised individual, a healthcare worker, a person suspected of having been exposed to an individual carrying the Ebola virus, a person who comes into physical contact with or is in very close proximity to an infected individual, a hospital worker, a pharmaceutical researcher, maintenance personnel responsible for cleaning hospital equipment or a facility in which a patient with an Ebola virus infection has been treated, individuals who have visited or are planning to visit an area or country known to have an outbreak of the Ebola virus or suspected of having one, and a frequent traveler. 28. Use according to claim 26, wherein the subject is a subject for whom the vaccine is contraindicated or for whom the vaccine is less effective. 29. Use according to paragraph 26, wherein the subject is elderly or very young. 30. Use according to paragraph 26, wherein at the time of administration of the antibody or its antigen-binding fragment the subject does not have an infection or has reduced viral titers or viral titers are absent. 31. The use according to claim 26, wherein the subject is exposed to EBOV as a result of physical contact with an infected individual or being in very close proximity to such an infected individual. 32. Use according to paragraph 26, wherein the subject is a worker in a hospital in a territorial area or country known to have an outbreak of EBOV or suspected of having one. 33. The use according to claim 26, wherein EBOV is the Zaire strain. 34. The use according to claim 26, wherein the composition is used in combination with one or more additional therapeutic agents or treatments against EBOV. 35. The use according to claim 34, wherein one or more additional therapeutic agents are selected from the group consisting of an antiviral drug, an anti-inflammatory drug, a different antibody to EBOV, an EBOV vaccine, brincidofovir, favipiravir and interferons. 36. The use according to claim 34, wherein the second therapeutic agent is selected from the group consisting of TKM Ebola, CMX-001, T-705, BCX-4430 and AVI-7537. 37. The use according to claim 34, wherein the second therapeutic agent is an anti-inflammatory agent selected from corticosteroids and non-steroidal anti-inflammatory drugs. 38. Use according to paragraph 34, wherein the pharmaceutical composition is formulated for subcutaneous, intravenous, intradermal, intramuscular, intranasal or oral administration. 39. The use according to claim 26, wherein the pharmaceutical composition comprises a combination of two or more antibodies to EBOV selected from the group consisting of: (a) (i) and (ii); (b) (i) and (iii); (c) (ii) and (iii); and (d) (i), (ii) and (iii). -