Human antibodies against Ebola virus glycoproteins

Abstract

To provide human antibodies to Ebola virus glycoproteins.SOLUTION: The present invention provides monoclonal antibodies, or antigen-binding fragments thereof, that bind to Ebola virus glycoproteins, pharmaceutical compositions comprising the antibodies, and methods of use. The antibodies of the invention are useful for inhibiting or neutralizing Ebola virus activity, thus providing means of treating or preventing Ebola virus infection in humans. In some embodiments, the invention provides for use of one or more antibodies that bind to the Ebola virus for preventing viral attachment and / or entry into host cells. The antibodies of the invention may be used prophylactically or therapeutically and may be used alone or in combination with one or more other anti-viral agents or vaccines.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antibodies that bind to Ebola virus glycoproteins, pharmaceutical compositions containing these antibodies, and methods of use thereof. [Background technology]

[0002] background Ebola virus (EBOV) and related filoviruses cause severe viral hemorrhagic fever in humans and non-human primates, with a case fatality rate of approximately 90% in human outbreaks (Murin, CD et al., (2014), Proc Natl Acad Sci USA, 111(48):17182-17187). The immune mechanisms mediating protection are under investigation, but to date, no treatments are approved for use in humans.

[0003] The Ebola virus glycoprotein (GP) is the only protein present on the surface of the virus and on infected cells. GP is presumed to be involved in virus-host cell binding and fusion. GP exists in several forms, encoded within two open reading frames. Unedited GP mRNA produces a 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, S164)). sGP forms dimers (Volchkova et al., (1995), Virology, 214:421-430; Falzarano, D. et al., Chembiochem (2006), 7:1605-1611) and is detected in high amounts 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).

[0004] Late in infection, an edited mRNA is produced that has acquired coding capability from a second open reading frame. This edited mRNA encodes a form of GP that contains a transmembrane (TM) domain, allowing this form of GP to be anchored to the cell's plasma membrane and incorporated into virions, where it can function as a functional host cell receptor-binding protein / fusion protein. During the biosynthesis of this form of GP, the protein is proteolytically processed into two products linked together by disulfide bonds. The amino-terminal product is designated GP1 (140 kDa), and the carboxy-terminal truncated product is designated GP2 (26 kDa) (Sanchez et al., (1998), J. Virol., 72:6442-6447).

[0005] Ebola virus GP (EBOV GP) can be a target of protective antibodies, but the role of antibodies in disease resistance is controversial. Negligible serum titers of neutralizing antibodies from convalescent patients, along with conflicting results in achieving protection using experimental transfer of immune serum to animals, have led to speculation regarding the role of neutralizing antibodies in recovery from infection (Peters, CJ and LeDuc, JW, (1999), J. Infect. Dis., 179:1; Mikhailov, VV, (1994), Vopr. Virusol., 39:82; Xu, L. et al., (1998), Nature Med., 4:37). However, in a recent Ebola virus outbreak, a small number of patients who contracted the disease and were treated with a cocktail of monoclonal antibodies (ZMapp) specific for the viral GP recovered from the disease. Furthermore, other patients treated with sera from these patients and from other patients who survived after developing infection also had positive outcomes.

[0006] Several antibodies that bind to Ebola virus GP have been reported (see, e.g., U.S. Patent Nos. 6,630,144, 6,875,433, 7,335,356, and 8,513,391; see also EP 1,539,238, EP 2,350,270, and EP 8,513,391).

[0007] Although technological advances have improved the ability to generate improved Ebola virus antigen(s) vaccine compositions, there remains a need to provide additional sources of protection to combat emerging strains of Ebola virus. 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):190ra179; 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, Several candidate therapeutic agents against Ebola virus are currently under evaluation, including monoclonal antibodies (T.W. et al., (2010), Lancet, 375(9729):1896-1905) and monoclonal antibodies (Saphire, E.O., (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 proven effective (Dye, JM. et al., (2012), Proc Natl Acad Sci USA, 109(13):5034-5039). More recently, a cocktail of three antibodies (ZMapp) is now being produced in tobacco plants and is being developed for human use (Qiu, X. et al., (2014), Nature, 514(7520):47-53). While the concept of a vaccine composition containing an antigen of interest (e.g., GP) to generate neutralizing antibodies in patients is generally considered a good approach, it may not be advantageous for use in patients who have already been exposed to the virus, as it may take several weeks for the body to respond to the vaccine composition. By that time, depending on the level of medical and palliative care available, the patient may already have succumbed to the viral infection. In these patients, or any patient who is unable to mount an effective antibody response, it may be more beneficial to provide a composition that already contains protective antibodies that may target a specific strain of EBOV, or epitopes common to many different strains. Thus, there remains a need in the art to identify new antibodies that can be used to prevent or treat Ebola virus infection. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 6,630,144 [Patent Document 2] U.S. Patent No. 6,875,433 [Patent Document 3] U.S. Patent No. 7,335,356 [Patent Document 4] U.S. Patent No. 8,513,391 [Patent Document 5] European Patent Application Publication No. 1539238 [Patent Document 6] European Patent Application Publication No. 2350270 [Patent Document 7] European Patent Application Publication No. 8513391 [Non-patent literature]

[0009] [Non-Patent Document 1] Murin, CDら, (2014), Proc Natl Acad Sci USA, Volume 111 (No. 48): Pages 17182~17187 [Non-licensed Document 2] Volchkova, (1995), Virology, Volume 214: Pages 421~430 [Non-licensed Document 3] Volchkova, VA, (1998), Virology, Volume 250: Pages 408~414 [Non-licensed Document 4] Sanchezら, (1996), Proc Natl Acad Sci USA, Volume 93: Pages 3602~3607 [Non-licensed Document 5] Sanchez, (1999) J. Infect. Dis. Volume 179 (Supplement 1, S164) [Non-licensed Document 6] Falzarano, D.ら, Chembiochem (2006), Volume 7: Pages 1605~1611 [Non-licensed Document 7] Dolnik, O. (2004), EMBO J, Volume 23: 2175-2184 [Non-licensed Document 8] Sanchez, (1998), J. Virol., Volume 72: 6442-6447 [Non-licensed Document 9] Peters, CJ LeDuc, JW, (1999), J. Infect. Dis., Volume 179, Supplement 1 [Non-licensed Document 10] Mikhailov, VV (1994), Vol. Virusol., 39 volumes, 82 pages [Non-licensed Document 11] Xu, L. (1998), Nature Med., Volume 4: 37 pages; Feldman, H. (2007), PLoS Pathog, Volume 3 (No. 1): e2 pages. [Non-licensed Document 12] Cote, M. (2011), Nature, Volume 477 (No. 7364): Pages 344~348 [Non-licensed Document 13] Johansen, LM et al. (2013), Sci Transl Med, 5(190):190ra179 [Non-Patent Document 14] Warren, TK et al. (2014), Nature, Vol. 508 (No. 7496): pp. 402-405 [Non-Patent Document 15] Geisbert, TW et al. (2006), J. Infect Dis., Vol. 193 (Issue 12): pp. 1650-1657. [Non-Patent Document 16] Geisbert, TW et al. (2010), Lancet, Vol. 375 (No. 9729): pp. 1896-1905. [Non-Patent Document 17] Saphire, EO, (2013), Immunotherapy, Volume 5 (Issue 11): pp. 1221-1233. [Non-Patent Document 18] Wong, G. et al. (2014), Trends Microbiol., 22(8):456-463 [Non-Patent Document 19] Qiu, X et al. (2014), Hum. Vaccin. Immunother., 10(4):964-967 [Non-Patent Document 20] Dye, JM. et al. (2012), Proc Natl Acad Sci USA, 109(13):5034-5039 [Non-Patent Document 21] Qiu, X. et al. (2014), Nature, Vol. 514 (No. 7520): pp. 47-53 Summary of the Invention [Means for solving the problem]

[0010] SUMMARY OF THE INVENTION The present invention provides antibodies and antigen-binding fragments thereof that bind to Ebola virus (EBOV) glycoprotein (GP). The antibodies of the present invention are useful for inhibiting or neutralizing the activity of Ebola virus. In some embodiments, the antibodies are useful for blocking Ebola virus attachment to host cells and / or preventing Ebola virus entry into host cells. In some embodiments, the antibodies function by inhibiting viral cell-to-cell transmission or by killing Ebola virus-infected cells, reducing the production of pathogenic virus. In certain embodiments, the antibodies are useful in preventing, treating, or ameliorating at least one symptom of Ebola virus infection in a subject. In certain embodiments, the antibodies can be administered prophylactically or therapeutically to subjects with or at risk of developing Ebola virus infection. In certain embodiments, compositions containing at least one antibody of the invention can be administered to patients for whom a vaccine is contraindicated or to subjects for whom a vaccine is less effective, such as elderly patients, young patients, patients who may be allergic to any one or more components of the vaccine, or immunocompromised patients who may not respond to the immunogens in the vaccine. In certain embodiments, compositions containing at least one antibody of the invention can be administered to medical staff, hospitalized patients or nursing home residents, or other high-risk patients during an Ebola virus outbreak. In certain embodiments, compositions containing at least one antibody of the invention can be administered as a first-line treatment to patients already exposed to Ebola virus.

[0011] Antibodies of the invention may be full length (e.g., IgG1 or IgG4 antibodies), may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect functionality, for example, to increase persistence in the host or to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, antibodies may be bispecific.

[0012] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to EBOV GP.

[0013] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to Ebola virus (EBOV) and / or Ebola virus glycoprotein (EBOV-GP), and has the following characteristics: (a) comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in 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 in 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) It is a fully human monoclonal antibody; (c) EBOV or virus-like particles (VLPs) expressing EBOV-GP were found to be 10 -7 A dissociation constant (K D ) to join; (d) exhibiting a dissociation half-life (t1 / 2) at pH 5 or pH 6 that is at least three times longer than that at pH 7.4; (e) about 10 -11 Approximately 10 minutes from M -9 Demonstrating neutralization of Zaire Ebola virus with IC50s ranging up to M; (f) demonstrating that binding to cells expressing said EBOV-GP induces antibody-dependent cellular cytotoxicity; (g) cross-reacts with one or more strains of EBOV selected from the group consisting of Zaire 2014, Zaire 1995, Sudan, Bundibugyo, and Côte d'Ivoire; (h) binding to soluble GP (sGP); (i) cross-compete with a reference antibody comprising a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the heavy chain variable region (HCVR) amino acid sequences and light chain variable region (LCVR) amino acid sequences in Table 1. The present invention provides an isolated recombinant antibody or antigen-binding fragment thereof, having one or more of:

[0014] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to EBOV and / or Ebola virus glycoprotein (EBOV-GP), having the following characteristics: (a) It is a fully human monoclonal antibody; (b) EBOV or virus-like particles (VLPs) expressing EBOV-GP were found to be 10 -7 A dissociation constant (K D ) to join; (c) exhibiting a dissociation half-life (t1 / 2) at pH 5 or pH 6 that is at least three times longer than that at pH 7.4; (d) About 10 -11 Approximately 10 minutes from M -9 IC ranging from M 50 Demonstrating neutralization of Zaire Ebola virus in (e) demonstrating that binding to cells expressing the EBOV GP induces antibody-dependent cellular cytotoxicity; (f) cross-reacts with one or more strains of EBOV selected from the group consisting of Zaire 2014, Zaire 1995, Sudan, Bundibugyo, and Côte d'Ivoire; (g) binding to soluble GP (sGP); (h) cross-competing with a reference antibody comprising a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR amino acid sequences and LCVR amino acid sequences in Table 1. The present invention provides an isolated recombinant antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has two or more of:

[0015] Exemplary anti-Ebola virus GP antibodies of the invention are listed in Tables 1 and 2 herein. Table 1 shows the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (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 shows the nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-Ebola virus GP antibodies.

[0016] The present invention provides 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0017] The present invention also provides 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0018] The present invention also provides antibodies or antigen-binding fragments thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising a pair of any of the HCVR amino acid sequences listed in Table 1 and any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-Ebola virus GP antibodies listed in Table 1.

[0019] In 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.

[0020] In one embodiment, the isolated antibody or antigen-binding fragment comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 76, 156, 12, 44, 60, 92, 108, 124, 140, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 78, 158, 14, 46, 62, 94, 110, 126, 142, 174, 190, 206, 222, 238, 254, 270, 286, and 302; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 80, 160, 16, 48, 64, 96, 112, 128, 144, 176, 192, 208, 224, 240, 256, 272, 288, and 304. Includes.

[0021] In 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).

[0022] The present invention also provides an antibody or antigen-binding fragment 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] The present invention also provides 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0024] The present invention also provides 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0025] The present invention also provides an antibody or antigen-binding fragment 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0026] The present invention also provides an antibody or antigen-binding fragment 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0027] The present invention also provides an antibody or antigen-binding fragment 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0028] The present invention also provides 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 and any pair of LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-Ebola virus GP antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 24 / 32 (e.g., H1H17139P), 72 / 80 (e.g., H1H17161P), and 152 / 160 (e.g., H1H17203P).

[0029] The present invention also provides 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 anti-Ebola virus GP antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set 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).

[0030] In related embodiments, the present invention provides antibodies or antigen-binding fragments thereof that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-Ebola virus GP antibodies listed in Table 1. For example, the present invention includes antibodies or antigen-binding fragments thereof that comprise a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18 / 26 (e.g., H1H17139P), 66 / 74 (e.g., H1H17161P); and 146 / 154 (e.g., H1H17203P). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise 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, 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0031] The present invention includes anti-Ebola virus antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al., (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0032] The present invention also provides antibodies and antigen-binding fragments thereof that compete for specific binding to Ebola virus with an antibody or antigen-binding fragment thereof that comprises the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0033] The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to Ebola virus with a reference antibody or antigen-binding fragment thereof, comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0034] The present invention also provides isolated antibodies and antigen-binding fragments thereof that block Ebola virus attachment to and / or entry into host cells.

[0035] In certain embodiments, an antibody or antigen-binding fragment of the invention is bispecific, comprising a first binding specificity for a first epitope of Ebola virus and a second binding specificity for a second epitope of Ebola virus, wherein the first epitope and the second epitope are distinct and non-overlapping. In certain embodiments, the bispecific may comprise a first arm that binds to an epitope of a viral glycoprotein and a second arm that binds to an epitope of a different viral antigen.

[0036] In a second aspect, the present invention provides nucleic acid molecules encoding anti-Ebola virus antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0037] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0038] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0039] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0041] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0042] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0043] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0044] The present invention also provides nucleic acid molecules encoding an HCVR comprising a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-Ebola virus GP antibodies listed in Table 1.

[0045] The present invention also provides nucleic acid molecules encoding an LCVR comprising a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-Ebola virus GP antibodies listed in Table 1.

[0046] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, both of which are derived from the same anti-Ebola virus GP antibody, as listed in Table 1.

[0047] The present invention provides nucleic acid molecules that encode any of the heavy chain amino acid sequences listed in Table 1. The present invention also provides nucleic acid molecules that encode any of the light chain amino acid sequences listed in Table 1.

[0048] In a related aspect, the invention provides recombinant expression vectors capable of expressing polypeptides comprising the heavy or light chain variable region of an anti-Ebola virus GP antibody. For example, the invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., any of the HCVR, LCVR, and / or CDR sequences listed in Table 1. Also included within the scope of the invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing host cells under conditions that permit the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments thus produced.

[0049] In a third aspect, the present invention provides pharmaceutical compositions comprising one or more isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to 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. In one embodiment, the HCVR / LCVR amino acid sequence pair is 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. In one embodiment, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18 / 26, 66 / 74, and 146 / 154.

[0050] In a related aspect, the invention features a composition that is a combination of at least two antibodies of the invention and a pharmaceutically acceptable carrier or diluent.

[0051] In a related aspect, the invention features a composition that is a combination / cocktail of at least three antibodies of the invention and a pharmaceutically acceptable carrier or diluent.

[0052] In one embodiment, the pharmaceutical composition comprises: (a) a first anti-Ebola virus antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1, or an antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1, or an antigen-binding fragment thereof; and (c) a third anti-Ebola virus antibody comprising an HCVR / LCVR amino acid sequence pair listed in Table 1, or an antigen-binding fragment thereof, wherein the first antibody binds to or interacts with a first epitope on the Ebola virus GP, and the second antibody and / or the third antibody bind to or interact with a different epitope on the Ebola virus GP, and (d) a pharmaceutically acceptable carrier or diluent.

[0053] In another related aspect, the invention features a composition that is a combination of an anti-Ebola virus GP antibody and a second therapeutic agent.

[0054] In one embodiment, the second therapeutic agent is any agent that can be advantageously combined with an anti-Ebola virus GP antibody. Exemplary agents that can be advantageously combined with an anti-Ebola virus antibody include, without limitation, other agents that bind to 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 to the Ebola virus but nevertheless inhibit the activity of the virus, including its ability to infect host cells.

[0055] In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first anti-Ebola virus antibody comprising an HCVR / LCVR amino acid sequence pair set forth in Table 1, or an antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody comprising an HCVR / LCVR amino acid sequence pair set forth in Table 1, or an antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on the Ebola virus GP and the second antibody binds to a second epitope on the Ebola virus GP, and the first epitope and the second epitope are distinct and non-overlapping; and (c) a pharmaceutically acceptable carrier or diluent.

[0056] In certain embodiments, the present invention provides pharmaceutical compositions comprising: (a) a first anti-Ebola virus antibody or antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody or antigen-binding fragment thereof, wherein the first and second antibodies do not cross-compete for binding to Ebola virus; and (c) a pharmaceutically acceptable carrier or diluent.

[0057] In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first anti-Ebola virus antibody or antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody or antigen-binding fragment thereof that interacts with a different Ebola virus antigen, wherein the first antibody binds to an epitope on an Ebola virus GP and the second antibody binds to an epitope on a different Ebola virus antigen; and (c) a pharmaceutically acceptable carrier or diluent.

[0058] In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first anti-Ebola virus antibody or antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody or antigen-binding fragment thereof; (c) a third anti-Ebola virus antibody or antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on the Ebola virus GP, and the second antibody and / or the third antibody bind to a different epitope on the Ebola virus GP, and the first epitope, the second epitope, and the third epitope are distinct and non-overlapping; and (d) a pharmaceutically acceptable carrier or diluent.

[0059] In certain embodiments, the present invention provides pharmaceutical compositions comprising: (a) a first anti-Ebola virus antibody or antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody or antigen-binding fragment thereof; (c) a third anti-Ebola virus antibody or 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.

[0060] In certain embodiments, the present invention provides pharmaceutical compositions comprising: (a) a first anti-Ebola virus antibody or antigen-binding fragment thereof; (b) a second anti-Ebola virus antibody or antigen-binding fragment thereof and / or a third anti-Ebola virus antibody or antigen-binding fragment thereof that interact with a different Ebola virus antigen, wherein the first antibody binds to an epitope on an Ebola virus and the second antibody and / or the third antibody bind to an epitope on a different Ebola virus antigen; and (c) a pharmaceutically acceptable carrier or diluent.

[0061] In one embodiment, the pharmaceutical composition comprises a first anti-Ebola virus antibody or antigen-binding fragment thereof that binds to or interacts with one epitope on one strain of Ebola virus, and a second anti-Ebola virus antibody or antigen-binding fragment thereof and / or a third anti-Ebola virus antibody or antigen-binding fragment thereof that binds to or interacts with a second and / or third epitope on the same or a different strain of Ebola virus. The Ebola virus strains that interact with the antibodies of the invention can be selected from the group consisting of the Zaire 2014 (Zaire.2014) strain, the Zaire 1995 (Zaire.1995) strain, the Sudan strain, the Bundibugyo strain, and the Cote d'Ivoire strain, or variants thereof.

[0062] In a related aspect, the invention provides a pharmaceutical composition comprising a first isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Ebola virus GP, the first isolated monoclonal antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO:20, the HCDR2 amino acid sequence of SEQ ID NO:22, the HCDR3 amino acid sequence of SEQ ID NO:24, the LCDR1 amino acid sequence of SEQ ID NO:28, the LCDR2 amino acid sequence of SEQ ID NO:30, and the LCDR3 amino acid sequence of SEQ ID NO:32, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may further comprise a second isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Ebola virus GP, the second isolated monoclonal antibody or antigen-binding fragment thereof comprising the HCDR1 amino acid sequence of SEQ ID NO:68, the HCDR2 amino acid sequence of SEQ ID NO:70, the HCDR3 amino acid sequence of SEQ ID NO:72, the LCDR1 amino acid sequence of 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. The pharmaceutical composition may further comprise a third isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Ebola virus GP, wherein the third isolated monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 148; an HCDR2 amino acid sequence of SEQ ID NO: 150; an HCDR3 amino acid sequence of SEQ ID NO: 152; an LCDR1 amino acid sequence of SEQ ID NO: 156; an LCDR2 amino acid sequence of SEQ ID NO: 158; and an LCDR3 amino acid sequence of SEQ ID NO: 160.

[0063] In certain embodiments, each antibody can be formulated as a separate formulation, and if it is determined that more than one antibody is required to achieve maximum therapeutic efficacy, each of the antibody formulations can be co-administered (concurrently or sequentially) as needed. Alternatively, the antibody cocktail can be co-formulated.

[0064] In certain embodiments, when two or more antibodies are combined together in a single pharmaceutical composition, they may or may not bind to the same epitope or overlapping epitopes on an Ebola virus protein. Additional combination therapies and co-formulations involving the anti-Ebola virus antibodies of the invention are disclosed elsewhere herein.

[0065] In a fourth aspect, the present invention provides a therapeutic method for treating a disease or disorder associated with Ebola virus (e.g., a viral infection in a subject), or the frequency or severity of at least one symptom associated with a viral infection, or at least one symptom associated with EBOV infection, using an anti-Ebola virus GP antibody or antigen-binding portion thereof of the present invention, or a cocktail of at least two or more antibodies of the present invention, comprising administering to a subject in need thereof a therapeutically effective amount of at least two or more antibodies or antigen-binding fragments of the present invention. In one embodiment, the method comprises administering a combination (cocktail) of at least three antibodies of the present invention. In 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. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by inhibition of Ebola virus activity. In certain embodiments, the present invention provides methods for preventing, treating, or ameliorating at least one symptom of Ebola virus infection, comprising administering to a subject in need thereof a therapeutically effective amount of at least one or more anti-Ebola virus GP antibodies or antigen-binding fragments thereof of the present invention.

[0066] In a related aspect, the present invention provides a method for 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 the exposing step results in enhanced protection of the cell from viral infection or cell death. In certain embodiments, the exposing step can be in vitro or in vivo. In one embodiment, the method comprises administering one or more antibodies of the present invention. In one embodiment, the method comprises administering a combination (cocktail) of at least three antibodies of the present invention. In 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.

[0067] In some embodiments, the invention provides methods of ameliorating or reducing the severity, duration, or frequency of occurrence of at least one symptom of Ebola virus infection in a subject by administering one or more anti-Ebola virus GP antibodies of the invention, wherein the at least one symptom is selected from the group consisting of fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

[0068] In certain embodiments, the present invention provides a method of reducing viral load in a subject, comprising administering to the subject an effective amount of one or more antibodies of the present invention or fragments thereof that bind to Ebola virus GP and block Ebola virus binding to and / or entry into host cells.

[0069] In a related aspect, the present invention provides a method for increasing the survival, or likelihood of survival, of a subject infected with EBOV, or a subject exposed to EBOV, or at risk of exposure to or infection with EBOV, comprising administering to a subject in need thereof at least one antibody or antigen-binding fragment of the invention, or a pharmaceutical composition comprising at least one antibody of the invention.

[0070] In one embodiment, the invention provides a method for increasing the survival, or likelihood of survival, of a subject infected with EBOV, or a subject exposed to EBOV, or at risk of exposure to or infection with EBOV, comprising the step of administering an antibody cocktail comprising a mixture of at least two anti-EBOV antibodies of the invention. In one embodiment, the method comprises the step of administering an antibody cocktail comprising a mixture of at least three anti-EBOV antibodies of the invention. In one embodiment, the antibody cocktail administered comprises a mixture of at least three anti-EBOV antibodies of the 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.

[0071] In one embodiment, the subject in need thereof is a subject at risk of being exposed to or acquiring the Ebola virus, wherein the subject is selected from the group consisting of immunocompromised individuals, healthcare workers, people suspected of having been exposed to someone with the Ebola virus, people in physical contact or close physical proximity to infected individuals, hospital personnel, pharmaceutical researchers, maintenance personnel responsible for cleaning hospital equipment or facilities where Ebola patients are being treated, individuals who have visited or are planning to visit an area or country known to have or suspected of having an outbreak of the Ebola virus, and frequent travelers.

[0072] In one embodiment, a subject in need thereof can be administered a pharmaceutical composition comprising at least one anti-EBOV antibody or antigen-binding fragment thereof of the present invention, or at least one antibody or antigen-binding fragment thereof in combination with a second therapeutic agent, which can be selected from the group consisting of an antiviral drug, an anti-inflammatory drug (such as a corticosteroid or a nonsteroidal anti-inflammatory drug), a different antibody against EBOV, a vaccine against EBOV, TKM Ebola (a small interfering RNA targeting the viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), and an interferon.

[0073] In one embodiment, the pharmaceutical composition can be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

[0074] In a related embodiment, enhanced protection may be observed in a mammal exposed to or infected with EBOV when the mammal is treated with a pharmaceutical composition comprising an antibody cocktail comprising at least three antibodies of the invention.

[0075] In one embodiment, the enhanced protection observed can be measured by a decrease in the severity or frequency of at least one symptom associated with EBOV infection, by a decrease in viral load, or by an increase in survival of a mammal infected with EBOV, wherein the at least one symptom can be selected from the group consisting of fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

[0076] Enhanced protection may be observed when the antibody is used alone or when the antibody is used in combination with one or more additional therapeutic agents or anti-EBOV treatment modalities.

[0077] The one or more additional therapeutic agents can be selected from the group consisting of antiviral agents, anti-inflammatory agents (such as corticosteroids and nonsteroidal anti-inflammatory agents), different antibodies against Ebola virus, vaccines against Ebola virus, TKM Ebola (a small interfering RNA targeting viral RNA polymerase) brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), and interferon.

[0078] In one embodiment, the one or more additional therapeutic agents comprise one or more anti-EBOV antibodies.

[0079] In one embodiment, the one or more anti-EBOV antibodies comprise a heavy chain variable region (HCVR) amino acid sequence 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 in Table 1.

[0080] In related embodiments, 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.

[0081] In another related embodiment, the one or more anti-EBOV antibodies comprise a heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18 / 26, 66 / 74 and 146 / 154.

[0082] In some embodiments, one or more antibodies or antigen-binding fragments thereof can be administered prophylactically or therapeutically to subjects who have, are at risk of, or are predisposed to developing Ebola virus infection. At-risk subjects include, but are not limited to, immunocompromised individuals, such as those immunocompromised due to autoimmune disease, or those undergoing immunosuppressive therapy (e.g., after organ transplantation), or those suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), certain forms of anemia in which white blood cells are depleted or destroyed, those undergoing radiation therapy or chemotherapy, or those suffering from inflammatory disorders. Other subjects at risk of developing Ebola virus infection include healthcare workers, or anyone who has physical contact or close proximity with an infected individual, or is exposed to bodily fluids or tissues from an infected individual, and who is at increased risk of developing Ebola virus infection. Additionally, subjects who live, for example, in densely populated cities or in close proximity to subjects with confirmed or suspected Ebola virus infection are at risk of contracting Ebola virus infection due to their proximity to the disease outbreak, or due to their occupational choices, for example, maintenance personnel responsible for cleaning hospital equipment or facilities where Ebola patients are being treated, hospital personnel, pharmaceutical researchers, individuals who have visited or plan to visit areas or countries known to have or suspected of having Ebola virus outbreaks, or frequent travelers.

[0083] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent can be selected from the group consisting of anti-inflammatory drugs (such as corticosteroids and nonsteroidal anti-inflammatory drugs), anti-infective drugs, antiviral drugs, different antibodies against Ebola virus, vaccines against Ebola virus, TKM Ebola (small interfering RNA targeting viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (antisense phosphorodiamidate morpholino oligomer targeting Ebola virus VP24 gene), interferon, antioxidants, and any other drug or other dietary supplements known in the art to be useful for improving at least one symptom of Ebola virus infection or reducing the viral load in patients. In certain embodiments, the second therapeutic agent may be an agent that counteracts or helps reduce any potential side effect(s) associated with the antibody or antigen-binding fragment thereof of the present invention, if such side effect(s) occur. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. In one embodiment, the antibody may be administered as a single intravenous infusion to maximize the concentration of the antibody in the subject's serum. The antibody or fragment thereof may be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight. In certain embodiments, the antibody of the present invention may be administered in one or more doses comprised between 50 mg and 600 mg.

[0084] The invention also encompasses anti-Ebola virus antibodies or antigen-binding fragments thereof of the invention for use in treating a subject having, suspected of having, or who has been exposed to EBOV, or for use in the manufacture of a medicament for treating a disease or disorder in which blocking Ebola virus binding and / or activity would be beneficial.

[0085] Other embodiments will become apparent upon review of the detailed description of the invention that follows. In certain embodiments, for example, the following are provided: (Item 1) 1. An isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to Ebola virus (EBOV) and / or Ebola virus glycoprotein (EBOV-GP), having the following characteristics: (a) comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in 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 in 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) It is a fully human monoclonal antibody; (c) EBOV or virus-like particles (VLPs) expressing EBOV-GP were found to be 10 -7 A dissociation constant (K D ) to join; (d) exhibiting a dissociation half-life (t1 / 2) at pH 5 or pH 6 that is at least three times longer than that at pH 7.4; (e) about 10 -11 Approximately 10 minutes from M -9 Demonstrating neutralization of Zaire Ebola virus with IC50s ranging up to M; (f) demonstrating that binding to cells expressing said EBOV-GP induces antibody-dependent cellular cytotoxicity; (g) cross-reacts with one or more strains of EBOV selected from the group consisting of Zaire 2014, Zaire 1995, Sudan, Bundibugyo, and Côte d'Ivoire; (h) binding to soluble GP (sGP); (i) cross-compete with a reference antibody comprising a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the heavy chain variable region (HCVR) amino acid sequences and light chain variable region (LCVR) amino acid sequences in Table 1. An isolated recombinant antibody or antigen-binding fragment thereof, having one or more of: (Item 2) 1. An isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to Ebola virus (EBOV) and / or Ebola virus glycoprotein (EBOV-GP), having the following characteristics: (a) It is a fully human monoclonal antibody; (b) EBOV or virus-like particles (VLPs) expressing EBOV-GP were found to be 10 -7 A dissociation constant (K D ) to join; (c) exhibiting a dissociation half-life (t1 / 2) at pH 5 or pH 6 that is at least three times longer than that at pH 7.4; (d) About 10 -11 Approximately 10 minutes from M -9 Demonstrating neutralization of Zaire Ebola virus with IC50s ranging up to M; (e) demonstrating that binding to cells expressing said EBOV-GP induces antibody-dependent cellular cytotoxicity; (f) cross-reacts with one or more strains of EBOV selected from the group consisting of Zaire 2014, Zaire 1995, Sudan, Bundibugyo, and Côte d'Ivoire; (g) binding to soluble GP (sGP); (h) cross-competing with a reference antibody comprising a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR amino acid sequences and LCVR amino acid sequences in Table 1. An isolated recombinant antibody or antigen-binding fragment thereof, having two or more of: (Item 3) 3. The isolated antibody or antigen-binding fragment thereof of either item 1 or 2, comprising an HCVR having an amino acid sequence 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. (Item 4) 4. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 3, comprising an LCVR having an amino acid sequence 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. (Item 5) (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 76, 156, 12, 44, 60, 92, 108, 124, 140, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 78, 158, 14, 46, 62, 94, 110, 126, 142, 174, 190, 206, 222, 238, 254, 270, 286 and 302; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 80, 160, 16, 48, 64, 96, 112, 128, 144, 176, 192, 208, 224, 240, 256, 272, 288, and 304. Item 6. The isolated antibody or antigen-binding fragment thereof according to any one of Items 1 to 4, comprising: 6. The isolated antibody or antigen-binding fragment of any one of items 1 to 5, comprising 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. (Item 7) 7. An isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 6, comprising an HCDR1 amino acid sequence of SEQ ID NO: 20; an HCDR2 amino acid sequence of SEQ ID NO: 22; an HCDR3 amino acid sequence of SEQ ID NO: 24; an LCDR1 amino acid sequence of SEQ ID NO: 28; an LCDR2 amino acid sequence of SEQ ID NO: 30; and an LCDR3 amino acid sequence of SEQ ID NO: 32. (Item 8) 7. An isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 6, comprising an HCDR1 amino acid sequence of SEQ ID NO: 68; an HCDR2 amino acid sequence of SEQ ID NO: 70; an HCDR3 amino acid sequence of SEQ ID NO: 72; an LCDR1 amino acid sequence of SEQ ID NO: 76; an LCDR2 amino acid sequence of SEQ ID NO: 78; and an LCDR3 amino acid sequence of SEQ ID NO: 80. (Item 9) 7. An isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 6, comprising an HCDR1 amino acid sequence of SEQ ID NO: 148; an HCDR2 amino acid sequence of SEQ ID NO: 150; an HCDR3 amino acid sequence of SEQ ID NO: 152; an LCDR1 amino acid sequence of SEQ ID NO: 156; an LCDR2 amino acid sequence of SEQ ID NO: 158; and an LCDR3 amino acid sequence of SEQ ID NO: 160. (Item 10) An isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to EBOV and / or EBOV GP with a reference antibody or antigen-binding fragment comprising: a CDR of an HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and a CDR of an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 11) An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the same epitope on EBOV and / or EBOV GP as a reference antibody or antigen-binding fragment comprising: a CDR of an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1; and a CDR of an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1. (Item 12) 12. The isolated antibody of any one of paragraphs 1 to 11, which prevents Ebola virus from attaching to and / or entering a host cell. (Item 13) 13. A method for 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 according to any one of items 1 to 12, wherein the exposing results in enhanced protection of the cell from viral infection or cell death. (Item 14) 14. The method of claim 13, wherein the infectious EBOV is neutralized in vitro or in vivo. (Item 15) 14. The method of claim 13, wherein the enhanced protection is observed when the antibody is used alone or in combination with one or more additional therapeutic agents or anti-EBOV treatment modalities. (Item 16) 16. The method of claim 15, wherein the one or more additional therapeutic agents are selected from the group consisting of antiviral agents, anti-inflammatory agents (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), different antibodies against EBOV, vaccines against EBOV, TKM Ebola (a small interfering RNA targeting viral RNA polymerase) brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the EBOV VP24 gene), and interferon. (Item 17) 16. The method of claim 15, wherein the one or more additional therapeutic agents comprise one or more anti-EBOV antibodies. (Item 18) 18. The method of item 17, wherein the one or more anti-EBOV antibodies comprise a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the HCVR amino acid sequences and LCVR amino acid sequences in Table 1. (Item 19) 19. The method of claim 18, wherein 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. (Item 20) 20. The method of claim 19, wherein 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: 18 / 26, 66 / 74, and 146 / 154. (Item 21) 13. A pharmaceutical composition comprising one or more isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to EBOV according to any one of items 1 to 12, and a pharmaceutically acceptable carrier or diluent. (Item 22) 22. The pharmaceutical composition of item 21, wherein 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. (Item 23) 23. The pharmaceutical composition of item 22, wherein the HCVR / LCVR amino acid sequence pair is 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. (Item 24) 24. The pharmaceutical composition of item 23, wherein the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18 / 26, 66 / 74 and 146 / 154. (Item 25) 13. A pharmaceutical composition comprising: (a) a first anti-EBOV antibody or antigen-binding fragment thereof; (b) a second anti-EBOV antibody or antigen-binding fragment thereof; and (c) a third anti-EBOV antibody or antigen-binding fragment thereof according to any one of items 1 to 12, wherein the first antibody binds to or interacts with a first epitope on EBOV and the second antibody and / or the third antibody bind to or interact with a different epitope on EBOV; and (d) a pharmaceutically acceptable carrier or diluent. (Item 26) 26. The pharmaceutical composition of item 25, wherein the first anti-EBOV antibody, the second anti-EBOV antibody, and the third anti-EBOV antibody comprise an HCVR / LCVR amino acid sequence pair selected from the group consisting of the HCVR sequences and LCVR sequences listed in Table 1. (Item 27) 26. The pharmaceutical composition of item 25, wherein the epitopes bound to or interacted with the first anti-EBOV antibody or antigen-binding fragment thereof, the second anti-EBOV antibody or antigen-binding fragment thereof, and / or the third anti-EBOV antibody or antigen-binding fragment thereof are distinct and non-overlapping. (Item 28) 26. The pharmaceutical composition of item 25, wherein the first anti-EBOV antibody or antigen-binding fragment thereof binds to or interacts with one epitope on one strain of EBOV, and the second anti-EBOV antibody or antigen-binding fragment thereof and / or third anti-EBOV antibody or antigen-binding fragment thereof bind to or interact with a second and / or third epitope on the same or a different strain of EBOV. (Item 29) 29. The pharmaceutical composition of item 28, wherein the EBOV strain is selected from the group consisting of Zaire 2014 strain, Zaire 1995 strain, Sudan strain, Bundibugyo strain, and Côte d'Ivoire strain. (Item 30) A pharmaceutical composition comprising a first isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EBOV, the first isolated monoclonal antibody or antigen-binding fragment thereof comprising an HCDR1 amino acid sequence of SEQ ID NO: 20; an HCDR2 amino acid sequence of SEQ ID NO: 22; an HCDR3 amino acid sequence of SEQ ID NO: 24; an LCDR1 amino acid sequence of SEQ ID NO: 28; an LCDR2 amino acid sequence of SEQ ID NO: 30; and an LCDR3 amino acid sequence of SEQ ID NO: 32, and a pharmaceutically acceptable carrier or diluent. (Item 31) 31. The pharmaceutical composition of Item 30, further comprising a second isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EBOV, wherein the second isolated monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 68; an HCDR2 amino acid sequence of SEQ ID NO: 70; an HCDR3 amino acid sequence of SEQ ID NO: 72; an LCDR1 amino acid sequence of SEQ ID NO: 76; an LCDR2 amino acid sequence of SEQ ID NO: 78; and an LCDR3 amino acid sequence of SEQ ID NO: 80. (Item 32) 32. The pharmaceutical composition of Item 31, further comprising a third isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EBOV, wherein the third isolated monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 amino acid sequence of SEQ ID NO: 148; an HCDR2 amino acid sequence of SEQ ID NO: 150; an HCDR3 amino acid sequence of SEQ ID NO: 152; an LCDR1 amino acid sequence of SEQ ID NO: 156; an LCDR2 amino acid sequence of SEQ ID NO: 158; and an LCDR3 amino acid sequence of SEQ ID NO: 160. (Item 33) 13. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR and / or LCVR of the antibody of any one of items 1 to 12. (Item 34) A vector comprising the polynucleotide sequence of item 33. (Item 35) A cell expressing the vector described in item 34. (Item 36) 32. A method for preventing, treating, or ameliorating at least one symptom of EBOV infection, or reducing the frequency or severity of at least one symptom of EBOV infection, comprising administering to a subject in need thereof the antibody or antigen-binding fragment of any one of items 1 to 12 or the pharmaceutical composition of items 21 to 31. (Item 37) 37. The method of claim 36, comprising administering an antibody cocktail comprising a mixture of at least two anti-EBOV antibodies. (Item 38) 38. The method of any of items 36 or 37, comprising administering an antibody cocktail comprising a mixture of three anti-EBOV antibodies comprising the HCVR / LCVR amino acid sequence pairs set forth in SEQ ID NOs: 18 / 26, 66 / 74 and 146 / 154. (Item 39) 37. The method of item 36, wherein the at least one symptom is selected from the group consisting of fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding. (Item 40) 37. The method of item 36, wherein the pharmaceutical composition is administered prophylactically or therapeutically to the subject in need thereof. (Item 41) 41. The method of item 40, wherein the subject in need thereof is infected with EBOV or has been exposed to or is at risk of being exposed to or infected with EBOV, and the subject is selected from the group consisting of immunocompromised individuals, healthcare workers, people suspected of having been exposed to someone with the Ebola virus, people in physical contact or close proximity to infected individuals, hospital personnel, pharmaceutical researchers, maintenance personnel responsible for cleaning hospital equipment or facilities where Ebola patients are being treated, individuals who have visited or are planning to visit an area or country known or suspected of having an Ebola virus outbreak, and frequent travelers. (Item 42) 42. The method of any one of items 36 to 41, wherein the antibody or antigen-binding fragment thereof, or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is administered in combination with a second therapeutic agent. (Item 43) 43. The method of claim 42, wherein the second therapeutic agent is selected from the group consisting of antiviral agents, anti-inflammatory agents (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), different antibodies against EBOV, vaccines against EBOV, TKM Ebola (a small interfering RNA targeting viral RNA polymerase) brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), and interferon. (Item 44) 44. The method according to any one of items 36 to 43, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally. (Item 45) 32. A method for increasing the survival or likelihood of survival of a subject infected with EBOV, or a subject exposed to EBOV, or at risk of exposure to or infection with EBOV, comprising administering to a subject in need thereof at least one antibody or antigen-binding fragment of any one of items 1 to 12, or the pharmaceutical composition of any one of items 21 to 31. (Item 46) 46. ​​The method of claim 45, comprising administering an antibody cocktail comprising a mixture of at least two anti-EBOV antibodies. (Item 47) 47. The method of any of items 45 or 46, comprising administering an antibody cocktail comprising a mixture of three anti-EBOV antibodies comprising the HCVR / LCVR amino acid sequence pairs set forth in SEQ ID NOs: 18 / 26, 66 / 74 and 146 / 154. (Item 48) 48. The method of any one of items 45 to 47, wherein the antibody or antigen-binding fragment thereof, or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the antibody cocktail is administered prophylactically or therapeutically to the subject in need thereof. (Item 49) 49. The method of any one of items 45 to 48, wherein said subject in need thereof at risk of exposure to or infection with EBOV is selected from the group consisting of immunocompromised individuals, healthcare workers, people suspected of having been exposed to someone with the Ebola virus, people in physical contact or close physical proximity to infected individuals, hospital personnel, pharmaceutical researchers, maintenance personnel responsible for cleaning hospital equipment or facilities where Ebola patients are being treated, individuals who have visited or plan to visit an area or country known or suspected of having an Ebola virus outbreak, and frequent travelers. (Item 50) 50. The method of any one of items 45 to 49, wherein the antibody or antigen-binding fragment thereof, or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or the antibody cocktail is administered in combination with a second therapeutic agent. (Item 51) 51. The method of claim 50, wherein the second therapeutic agent is selected from the group consisting of an antiviral agent, an anti-inflammatory agent (e.g., a corticosteroid or a nonsteroidal anti-inflammatory agent), a different antibody against EBOV, a vaccine against EBOV, TKM Ebola (a small interfering RNA targeting viral RNA polymerase) brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), and an interferon. (Item 52) 52. The method according to any one of items 45 to 51, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally. [Brief explanation of the drawings]

[0086] [Figure 1] H1H17161P potently neutralizes live EBOV.

[0087] [Figure 2] Interactions of three anti-EBOV antibodies with Ebola GP or Ebola soluble GP (sGP) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0088] Detailed Description Before describing the present methods, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also understood that the terminology used herein 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.

[0089] 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 described herein.

[0090] definition "Ebola virus" or "EBOV" is a genus in the Filoviridae family and is known to cause severe, rapidly progressive hemorrhagic fever. Based on the nucleotide sequence and location of the outbreak, there are many different Ebola virus species and strains, such as Zaire, Tai Forest (formerly known as Côte d'Ivoire or Ivory Coast), Sudan, Reston, and Bundibugyo. The most lethal 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 encompasses variants of Ebola virus isolated from different Ebola virus isolates.

[0091] The amino acid sequence of the full-length Ebola virus glycoprotein, designated herein as "EBOV GP" or "Ebola virus GP," is exemplified by the amino acid sequence found in GenBank under 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 fragments thereof coupled, for example, to a histidine tag (see, e.g., accession number AHX24649.1 (SEQ ID NO:318) with a decahistidine tag), a mouse Fc or a human Fc, or a signal sequence. The amino acid sequence of "soluble GP" or "sGP" is set forth in accession number AHX24650 and as SEQ ID NO:316 (with a signal sequence) and also in SEQ ID NO:317 (without a signal sequence but with a myc-myc-hexahistidine tag). The amino acid sequence of "GP1" begins at residue 1 of the amino terminus of full-length GP and ends at residue 501 of SEQ ID NO: 315. The amino acid sequence of "GP2" extends from residue 502 to residue 676 of full-length GP, shown as SEQ ID NO: 315.

[0092] The term "Ebola virus infection" or "EBOV infection," as used herein, refers to a severe hemorrhagic fever resulting from exposure to the virus, or an infected animal, or an infected human patient, or contact with bodily fluids or tissues from an animal or human patient with an Ebola virus infection. "Symptoms associated with Ebola virus infection" include fever, headache, fatigue, loss of appetite, muscle aches, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

[0093] The term "antibody," as used herein, refers to an immunoglobulin molecule (i.e., a "whole antibody molecule") composed of four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and the heavy chain constant region (domain C H 1. C H2 and C H Each light chain is composed of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) and V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) and interspersed regions that are more conserved called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0094] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies in which one or two CDRs can be allocated for binding are described in the scientific literature. Padlan et al. (1995, FASEB J. 9:133-139) report on published Based on the crystal structures obtained, Padlan analyzed the contact regions between antibodies and their antigens and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have any amino acids that contact the antigen (see also Vajdos et al., 2002, J Mol Biol 320:415-428).

[0095] CDR residues that do not contact the antigen can be identified from regions of the Kabat CDRs outside the Chothia CDRs based on previous testing, molecular modeling, and / or empirically (e.g., residues H60-H65 of CDRH2 are often unnecessary). The CDR or its residue(s) are omitted and typically substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. The substitution positions within the CDRs and the substituting amino acids can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0096] The fully human anti-Ebola virus monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains 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 public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll of the framework and / or CDR residues within a domain are backmutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., the mutated residues are found only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention can contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, and certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as, for example, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are within the scope of the invention.

[0097] The present invention also encompasses fully human anti-Ebola virus monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes anti-Ebola virus antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0098] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the invention may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutated by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. This term encompasses antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. This term is not intended to include antibodies isolated from or generated in human subjects.

[0099] The term "recombinant," as used herein, refers to an antibody or antigen-binding fragment thereof of the present invention that is created, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term also refers to antibodies expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse) or cell (e.g., a CHO cell) expression system, or isolated from a recombinant combinatorial human antibody library.

[0100] Terms such as "specifically binds" or "binds specifically to" mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is defined as binding with an equilibrium dissociation constant of at least about 1 x 10 -7 M or less (e.g., K D(A smaller r indicates tighter 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. As described herein, antibodies that specifically bind to Ebola virus have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, multispecific antibodies that bind to one domain of Ebola virus and one or more additional antigens, or bispecific antibodies that bind to two different regions of Ebola virus, are still considered to be "specifically binding" antibodies as used herein.

[0101] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 to Ebola virus as measured by surface plasmon resonance, e.g., BIACORE™, or by solution affinity ELISA. -7 M; preferably 10 -8 M; more preferably 10 -9 M, and even more preferably 10 -10 M, and even more preferably 10 -11 M, and even more preferably 10 -12 K of M D This refers to a mAb having a binding affinity represented by the formula:

[0102] The term "slow off rate," "Koff," or "kd" refers to the rate at which an antibody binds to an Ebola virus or Ebola virus GP-expressing virus-like particle within 1 x 10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 or less, preferably 1 x 10 -4 s -1 This means that the molecule dissociates with a rate constant equal to or less than that.

[0103] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to Ebola virus.

[0104] In certain embodiments, the antibodies or antibody fragments of the invention can be conjugated to a moiety such as a ligand or therapeutic moiety, such as an antiviral drug, a second anti-Ebola virus antibody, or any other therapeutic moiety useful for treating infection caused by Ebola virus (an "immunoconjugate").

[0105] The term "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody, or fragment thereof, that specifically binds to Ebola virus is substantially free of Abs that specifically bind to antigens other than Ebola virus).

[0106] A "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes Ebola virus activity" or "antagonist antibody"), as used herein, is intended to refer to an antibody that binds to an Ebola virus, thereby resulting in the inhibition of at least one biological activity of the Ebola virus. For example, the antibody of the present invention prevents or blocks the attachment or entry of the Ebola virus into a host cell. Furthermore, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with, the ability of a pathogen to initiate and / or perpetuate infection in a host. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone or in combination with other antiviral agents, in an appropriate formulation, or in connection with active vaccination, as prophylactic or therapeutic agents, or as diagnostic tools.

[0107] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by specific antibodies, such as those described herein. As such, this is one mechanism by which, for example, virus-specific antibodies can act to limit the spread of infection. Classical ADCC is mediated by natural killer cells (NK cells), macrophages, neutrophils, and, in certain instances, eosinophils.

[0108] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0109] "KD The term " ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0110] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. An epitope also refers to the region of an antigen to which an antibody binds. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, consisting of amino acids that are not linear. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural and / or charge characteristics.

[0111] As used herein, the term "cross-compete" refers to an antibody or antigen-binding fragment thereof that binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. This term encompasses bidirectional competition between two antibodies, i.e., a first antibody binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, a first antibody and a second antibody may bind to the same epitope. Alternatively, a first antibody and a second antibody may bind to different but overlapping epitopes, such that the binding of one antibody inhibits or blocks the binding of the second antibody, for example, due to steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, such as a real-time label-free biolayer interference assay. To determine whether a test antibody cross-competes with a reference anti-Ebola virus GP antibody of the present invention, the reference antibody is bound to Ebola virus GP or a peptide under saturating conditions. The ability of the test antibody to bind to Ebola virus GP is then assessed. If the test antibody is able to bind to Ebola virus GP after saturation binding with the reference anti-Ebola virus GP antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Ebola virus GP antibody. On the other hand, if the test antibody is unable to bind to Ebola virus GP after saturation binding with the reference anti-Ebola virus GP antibody, the test antibody may bind to the same epitope as the reference anti-Ebola virus GP antibody of the present invention.

[0112] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, the nucleotide sequence identity is 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. In certain examples, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0113] As applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using default gap weights, e.g., with the programs GAP or BESTFIT, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Non-identical residue positions preferably differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331). (ii) Examples of groups of amino acids with similar side chain chemistries 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: aspartic acid and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions can be made using the PAM250 log-likelihood algorithm disclosed in Gonnet et al. (1992) Science 256:1443-45. A "moderately conservative" replacement is any change that has a positive value in the PAM250 log-likelihood matrix. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0114] The sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity criteria assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) supra). The sequences of the present invention can be compared with a large number of sequences from different organisms. Another preferred algorithm for comparing sequences to a database containing the sequence is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403- 410, and (1997) Nucleic Acids Res. 25:3389-3402.

[0115] The phrase "therapeutically effective amount" means an amount that produces a desired effect upon administration. The precise amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0116] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of reversal, prevention, and / or treatment of a disease or disorder, such as a viral infection. The subject may have an Ebola virus infection or may be predisposed to developing an Ebola virus infection. A subject "predisposed to developing an Ebola virus infection" or "may be at increased risk of contracting an Ebola virus infection" is a subject whose immune system is impaired due to an autoimmune disease, a person undergoing immunosuppressive therapy (e.g., after an organ transplant), a person suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), a person suffering from certain forms of anemia in which white blood cells are depleted or destroyed, a person undergoing radiation therapy or chemotherapy, or a person suffering from an inflammatory disorder. Additionally, very young or elderly subjects are at increased risk. Any person who comes into physical contact or close proximity with an infected animal or human patient, or who is exposed to bodily fluids or tissues from an infected animal or human patient, is at increased risk of contracting an Ebola virus infection. Additionally, subjects living, for example, in densely populated cities or in close proximity to subjects with confirmed or suspected Ebola virus infection are at risk of contracting the Ebola virus due to their proximity to disease outbreaks, or due to their occupational choices, for example, hospital personnel, pharmaceutical researchers, individuals who have visited or plan to visit areas or countries known to have or suspected of having Ebola virus outbreaks, or frequent travelers.

[0117] As used herein, the terms "treat," "treating," or "treatment" refer to reducing or ameliorating the severity of at least one symptom or sign of Ebola virus infection by administering a therapeutic agent, such as an antibody of the invention, to a subject in need thereof. The term encompasses inhibition of disease progression or worsening of infection. The term also encompasses a positive prognosis of the disease, i.e., that administration of a therapeutic agent, such as an antibody of the invention, may result in a subject being spared infection or having a reduced or eliminated viral titer. The therapeutic agent may be administered to the subject in a therapeutic dose.

[0118] The terms "prevent," "preventing," or "prevention" refer to inhibiting the manifestation of Ebola virus infection or any symptom or sign of Ebola virus infection with the administration of an antibody of the invention. This term encompasses preventing the spread of infection in a subject who has been exposed to the virus or who is at risk of having an Ebola virus infection.

[0119] As used herein, the term "antiviral agent" refers to any anti-infective agent or therapy, whether a chemical moiety or a biological therapy, used to treat, prevent, or reverse a viral infection in a subject. For example, in the present invention, antiviral agents include, but are not limited to, antibodies against Ebola virus (in one embodiment, the antibodies against Ebola virus may be different from those described herein), vaccines against Ebola virus, TKM Ebola (a small interfering RNA targeting the viral RNA polymerase), brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), and interferon. In the present invention, the infection being treated is caused by the Ebola virus.

[0120] General Description Ebola virus disease is a severe, often fatal illness caused by filamentous virus particles that are members of the Filoviridae family. Several species of the Ebola virus genus are known that can cause disease in humans. These include Zaire, Sudan, Tai Forest (formerly Ivory Coast), and Bundibugyo. The natural reservoir of the virus is unknown, and to date, there are no approved therapies or vaccines.

[0121] The genome of this virus consists of a single strand of negative-sense RNA approximately 19 kb in length. The Ebola virion contains seven proteins: a surface glycoprotein (GP), a nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and an RNA-dependent RNA polymerase (L) (Feldman et al., 2019). 1992)Virus Res., Vol. 24, pp. 1-19).

[0122] The only proteins present on the surface of the virus are glycoproteins. Due to RNA editing, transcription of the GP gene leads to the synthesis of several GP gene-specific mRNAs that encode viral GPs, including the nonstructural soluble GP (sGP) and the surface virion GP (Volchkova, VA et al., (1998), Virology, 250:408-414). Both GPs are synthesized as precursor molecules that are proteolytically cleaved by the cellular protease furin during intracellular processing (Volchkov, VE et al., (1998), Virology, 250:408-414). (998), Proc Natl Acad Sci USA, 95:5762-5767). sGP forms a dimer, while the cleaved carboxy-terminal fragment is a monomer. The viral surface spikes are composed of two subunits, GP1 and GP2, linked by disulfide bonds. 1,2GP1 is formed as a trimer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 122, 123, 124, 125, 130, 131, 132, 133, 134, 135, 136, 140 GP2 is known to be 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., vol. 87: 1247-1257).

[0123] During EBOV infection, significant amounts of soluble glycoprotein (sGP) are released from virus-infected cells. This form of GP has been shown to bind to and sequester virus-neutralizing antibodies directed against surface or virion GP (Dolnik, O. et al., (2004), EMBO J, 23:2175-2184). Beyond this antibody blockade, the role of soluble GP in viral replication and / or pathogenesis has not been clearly defined. A more recent study by Escudero-Perez et al. showed that sGP can bind to and activate uninfected dendritic cells and macrophages, inducing the secretion of pro- and anti-inflammatory cytokines. Furthermore, Escudero-Perez et al. demonstrated that sGP can affect endothelial cell function and vascular permeability (Escudero-Perez et al., (2014), PLOS Pathogens, 10, 11:1-17). ), which may explain the dysregulated inflammatory host response following infection and may also contribute to viral pathogenesis.

[0124] Passive immunotherapy to prevent or treat infectious diseases, usually in the form of convalescent human serum containing high titers of neutralizing antibodies, has been used for over a century (Good et al. (1991) Cancer 68:1415-1421). Currently, a number of purified monoclonal antibodies are in preclinical and clinical development for use as antimicrobial agents (Marasco et al. , 2007; Nature Biotechnology, 25:1421-1434). Antibodies have been described that bind to Ebola virus glycoproteins (see, e.g., Audet et al. (2014) Scientific Reports 4:6881; Chen et al. (2014) ACS Chem Biol. October 17; 9(10):2263-73; Koellhoffer JF et al. (2012) Chembiochem. November 26; 13(17):2549-57; Qiu, X. et al., Nature (2014) October 2; 514(7520):47-53).

[0125] The present inventors describe fully human antibodies and antigen-binding fragments thereof that specifically bind to Ebola virus GP and modulate the interaction of Ebola virus with these cells. Anti-Ebola virus GP antibodies are capable of binding to Ebola virus with high affinity. In certain embodiments, the antibodies of the present invention are blocking antibodies capable of binding to Ebola virus GP and blocking viral attachment and / or entry into host cells. In certain embodiments, the antibodies of the present invention are capable of blocking Ebola virus binding to cells and thus inhibiting or neutralizing viral infection of host cells. In certain embodiments, the antibodies of the present invention are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) and thus aiding in the destruction of virus-harboring cells. In certain embodiments, the antibodies are capable of acting in both ways, e.g., neutralizing viral infectivity and mediating ADCC. In some embodiments, the antibodies may be useful for treating subjects suffering from Ebola virus infection. When administered to a subject in need thereof, the antibodies can reduce infection by a virus, such as Ebola virus, in the subject. The antibodies can be used to reduce viral load in a subject. The antibodies can be used alone or as adjunctive therapy with other therapeutic moieties or modalities known in the art for treating viral infections. In certain embodiments, these antibodies are capable of binding to an epitope within the amino terminus of Ebola virus GP. In certain embodiments, these antibodies are capable of binding to an epitope within the carboxy terminus of Ebola virus GP. Furthermore, the identified antibodies can be used prophylactically (before infection) to protect a mammal from infection, or therapeutically (after infection has been established) to reverse a previously established infection or to reverse at least one symptom associated with the infection.

[0126] The full-length amino acid sequences of exemplary Ebola virus GPs are also set forth in GenBank under accession numbers AHX24649.1 and AHX24649.2, and as SEQ ID NOs: 314 and 315, respectively. GP1 spans amino acid residues 1-501 of the full-length GP set forth in SEQ ID NO: 314 or 315, and GP2 spans amino acid residues 502 to 676 of the full-length GP. The full-length EBOV GP, also set forth under accession number AHX24649.1, can be coupled to a decahistidine tag, such as that set forth in SEQ ID NO: 318. Soluble GP (sGP), set forth under GenBank accession number AHX24650.1, is also set forth under SEQ ID NO: 316 (with a signal sequence attached) and SEQ ID NO: 317 (without the signal sequence but containing a myc-myc-his tag).

[0127] In certain embodiments, antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as full-length Ebola virus GP, or with a recombinant Ebola virus GP or a fragment thereof, followed by a secondary immunogen or an immunogenically active fragment of Ebola virus GP. In certain embodiments, antibodies are obtained from mice immunized with DNA encoding full-length Ebola virus GP (Zaire 2014, see GenBank KJ660346.2; see also SEQ ID NO: 313). The immunogen can be a biologically active and / or immunogenic fragment of Ebola virus GP, or a DNA encoding an active fragment of Ebola virus GP. Fragments can be derived from any region of the viral GP, including the amino-terminal fragment (e.g., GP1) or the carboxy-terminal fragment (e.g., GP2). Peptides can be modified to include the addition or substitution of certain residues for tagging or conjugation to a carrier molecule such as KLH. For example, cysteines can be added to either the N- or C-terminus of the peptide, and linker sequences can be added to prepare the peptide for conjugation with, for example, KLH, for immunization.

[0128] Certain anti-Ebola virus antibodies of the invention are capable of binding to and neutralizing the activity of Ebola virus as determined by in vitro or in vivo assays. The ability of antibodies of the invention to bind to Ebola virus and neutralize its activity, and thus viral attachment and / or entry into host cells and subsequent viral infection, can be measured using any standard method known to those of skill in the art, including the binding assays described herein or activity assays.

[0129] A non-limiting, exemplary in vitro assay for measuring binding activity is illustrated herein in Example 3. In Example 3, the binding affinity and dissociation constant of anti-Ebola virus GP antibodies to Ebola virus were determined by Biacore. In Examples 4 and 7, neutralization assays were used to determine the infectivity of various strains of Ebola virus.

[0130] Antibodies specific for Ebola virus GP may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if any) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or a label detectable by MRI. In certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays.

[0131] Antigen-binding fragment of an antibody Unless otherwise specified, the term "antibody," as used herein, is understood to encompass an antibody molecule composed of two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "whole antibody molecule"), as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to Ebola virus. An antibody fragment may include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing CDRs, or an isolated CDR. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived, for example, from whole antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, by using chemical or molecular biological techniques to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0132] 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 (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) of antibodies or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0133] Antigen-binding fragments of antibodies generally contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR that is contiguous or in-frame with one or more framework sequences. H Domains and V L In the antigen-binding fragments of the associated domains, V H Domains and V L The domains can be positioned in any suitable arrangement relative to each other. For example, the variable region can be a dimer, with the V H -V H Dimer, V H -V L Dimer or V L -V L Alternatively, the antigen-binding fragment of the antibody contains a monomeric V dimer. H Domain or V L It may contain domains.

[0134] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3;, and (xiv) V L -C LIn any of the variable and constant domain configurations, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above, with the domains non-covalently associated (e.g., by disulfide bond(s)).

[0135] Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Antigen-binding fragments of multispecific antibodies generally comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any of the multispecific antibody formats, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use with antigen-binding fragments of antibodies of the invention using routine techniques available in the art.

[0136] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in connection with the present invention to generate human antibodies that specifically bind to Ebola virus GP. Antibodies against Ebola virus can be generated using immunogens comprising any one of the following: In certain embodiments, antibodies of the invention are obtained from mice immunized with full-length native Ebola virus GP (e.g., see GenBank Accession Nos. AHX24649.1 (SEQ ID NO: 314) and AHX24649.2 (SEQ ID NO: 315) or with DNA encoding the glycoprotein or a fragment thereof. Alternatively, Ebola virus GP or a fragment thereof can be made, modified, and used as an immunogen using standard biochemical techniques. In one embodiment, the immunogen is a recombinantly produced Ebola virus GP or a fragment thereof. In certain embodiments of the invention, the immunogen may be a commercially available Ebola virus GP. In certain embodiments, one or more booster shots can be administered. In certain embodiments, the booster shot may comprise one or more commercially available Ebola virus GP. In certain embodiments, the immunogen may be recombinant Ebola virus GP expressed in E. coli or in any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0137] Using VELOCIMMUNE® technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies against Ebola virus GP with human variable regions and mouse constant regions are first isolated. VELOCIMMUNE® technology involves the generation of transgenic mice with genomes containing human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0138] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (e.g., B cells) expressing antibodies are collected from the mice. Lymphocytes can be fused with myeloma cell lines to prepare immortal hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines producing antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to heavy and light chain constant regions of the desired isotype. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0139] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antibody is characterized and selected for desirable properties, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody of the invention, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the particular use, the variable region is endowed with high-affinity antigen binding and target specificity characteristics.

[0140] bioequivalence The anti-Ebola virus GP antibodies and antibody fragments of the present invention include proteins with amino acid sequences that differ from the amino acid sequences of the described antibodies but retain the ability to bind to Ebola virus. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, DNA sequences encoding the antibodies of the present invention include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention.

[0141] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they show no significant differences in the rate and extent of absorption when administered at the same molar dose, under similar experimental conditions, either in single or multiple doses. Some antibodies may be considered equivalent or pharmaceutical substitutes if their extent of absorption is equivalent but their absorption rate is not, and they can still be considered bioequivalent because such differences in absorption rate are intentional, reflected in the label, not necessary to achieve effective body drug levels, for example, for long-term use, and not medically significant with respect to the particular drug being tested.

[0142] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0143] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biologic without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, compared to continuing treatment without such a switch.

[0144] In one embodiment, two antigen binding proteins are bioequivalent if they both act through a common mechanism(s) of action for the condition(s) in which they are used, to the extent such mechanism(s) are known.

[0145] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Criteria for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effect of the antibody (or its target) as a function of time; and (d) well-conducted clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0146] Bioequivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0147] Anti-Ebola virus antibodies containing Fc variants According to certain embodiments of the present invention, there are provided anti-Ebola virus antibodies comprising an Fc domain containing one or more mutations that enhance or attenuate antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the present invention provides an anti-Ebola virus antibody comprising an Fc domain containing one or more mutations that enhance or attenuate antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. H 2nd area or C HThe present invention encompasses anti-Ebola virus GP antibodies that contain mutations in three regions, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, which have a pH range of about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody 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); a modification at positions 250 and 428 (e.g., L or F); a modification at position 252 (e.g., L / Y / F / W or T), a modification at position 254 (e.g., S or T), and a modification at position 256 (e.g., S / R / Q / E / D or T); or a modification at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or a modification at position 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]); or a modification at positions 250 and / or 428; or a modification at positions 307 or 308 (e.g., 308F, V308F), and a modification at position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0148] For example, the present invention encompasses anti-Ebola virus antibodies comprising an Fc domain containing 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). It is contemplated that all possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are within the scope of the present invention.

[0149] The present invention relates to chimeric heavy chain constant (C H ) region, and chimeric C H The region is C of more than one immunoglobulin isotype H Also encompassed are anti-Ebola virus antibodies that contain segments derived from regions of the C region derived from human IgG1, human IgG2, or human IgG4 molecules. H Chimeric C containing part or all of the 2 domains H region and C derived from a human IgG1, human IgG2, or human IgG4 molecule. H In certain embodiments, the antibodies of the present invention may comprise a chimeric C domain having a chimeric hinge region. HFor example, a chimeric hinge may comprise a combination of an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region and a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge region and amino acid residues derived from a human IgG2 lower hinge region. The chimeric C described herein H Antibodies comprising the region, in certain embodiments, exhibit altered Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Application No. 61 / 759,578, filed February 1, 2013).

[0150] Biological properties of antibodies Generally, the antibodies of the invention function by binding to Ebola virus GP. For example, the invention provides antibodies that bind to Ebola virus GP (e.g., at 25°C or 37°C) in a range of 10 to 120% of the antibody activity, as measured by surface plasmon resonance, e.g., using the assay formats described herein. -7 K less than M D In certain embodiments, the antibody or antigen-binding fragment thereof binds to Ebola virus GP with a K 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, e.g., using an assay format described herein or a substantially similar assay. D Combine with.

[0151] The invention also encompasses antibodies and antigen-binding fragments thereof that bind to Ebola virus using the assay formats defined herein, or substantially similar assays, with a dissociation half-life (t1 / 2) of greater than about 3 minutes at 25°C as measured by surface plasmon resonance, or greater than about 1 minute at 37°C as measured by surface plasmon resonance, and a dissociation half-life (t1 / 2) of at least 3-fold greater at pH 5 or pH 6. In certain embodiments, the antibodies or antigen-binding fragments of the invention bind to Ebola virus with a t 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 37°C, e.g., using an assay format defined herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.

[0152] The present invention also encompasses antibodies or antigen-binding fragments thereof that neutralize the infectivity of Ebola virus to its host cells. In some embodiments, the antibody has a specific binding affinity to Zaire 2014 VLP of about 10 -11 Approximately 10 minutes from M -9 IC ranging from M 50 The antibodies of the invention also cross-react with Ebola virus VLPs containing GPs from various strains of EBOV, including Zaire 1995, Zaire 2014, Ebola Soudan, Bundibugyo, and Côte d'Ivoire (Ivory Coast). The antibodies of the invention also mediate ADCC, as shown in Example 5. Furthermore, the antibodies of the invention cross-compete with other antibodies that bind to EBOV GP, as shown in Example 6.

[0153] In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to Ebola virus GP, having the following characteristics: (a) being a fully human monoclonal antibody; (b) binding to EBOV or virus-like particles (VLPs) expressing Ebola virus glycoproteins at least 10 times as measured by a surface plasmon resonance assay. -7 A dissociation constant (K D (c) exhibiting a dissociation half-life (t) at pH 5 or pH 6 that is at least three times longer than that at pH 7.4; (d) exhibiting a dissociation half-life (t) of about 10 -11 Approximately 10 minutes from M -9 IC ranging from M 50 (e) exhibiting neutralization of Zaire Ebola virus in vitro; (f) exhibiting antibody-dependent cellular cytotoxicity of Ebola virus-infected cells; (g) cross-reacting with one or more strains of Ebola virus VLPs selected from the group consisting of Zaire 2014, Zaire 1995, Sudan, Bundibugyo, and Côte d'Ivoire; and (g) cross-competing with a reference antibody comprising a heavy chain variable region (HCVR) amino acid sequence and a light chain variable region (LCVR) amino acid sequence selected from the group consisting of any of the heavy chain variable region (HCVR) amino acid sequences and light chain variable region (LCVR) amino acid sequences in Table 1.

[0154] The antibodies of the invention may have one or more of the above biological properties, or any combination thereof. Certain properties of the antibodies of the invention are summarized below. Other biological properties of the antibodies of the invention will be apparent to those skilled in the art from a review of this disclosure, including the Examples herein. [Table 10]

[0155] Epitope mapping and related techniques The present invention encompasses anti-Ebola virus antibodies that interact with one or more amino acids found within the Ebola virus GP. The epitope to which the antibody binds can consist of a single, contiguous sequence of three 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 within the Ebola virus GP molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of multiple, non-contiguous amino acids (or amino acid sequences) located within the Ebola virus GP molecule (e.g., a conformational epitope).

[0156] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, 1999). Other methods include conventional cross-blocking assays, such as those described in (Reineke, 2004) Methods Mol. Biol., 248:443-63), alanine scanning mutagenesis analysis, peptide blot analysis (Reineke, 2004) Methods Mol. Biol., 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR. Further methods include epitope excision, epitope extraction, and chemical modification of antigens (Tomer (2000) Prot. Sci., 9:487-496). Another method that can be used to identify the amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves labeling a protein of interest with deuterium and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface are able to retain deuterium and therefore exhibit a higher mass than amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues corresponding 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.

[0157] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from either contiguous amino acids or non-contiguous amino acids arranged by tertiary folding of a protein. Epitopes formed from contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally contains at least three, usually at least five or eight to ten, amino acids in a unique spatial conformation.

[0158] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for categorizing multiple monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920). Each category may reflect a unique epitope that is distinctly different from or partially overlaps with the epitopes represented by other categories. This technique allows for rapid filtering of genetically identical antibodies, thus focusing characterization on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP can be used to separate the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0159] In certain embodiments, the Ebola virus antibody or antigen-binding fragment thereof binds to an epitope within any one or more of the exemplified regions of Ebola virus GP, either in its native form or recombinantly produced, or a fragment thereof.

[0160] The present invention encompasses anti-Ebola virus GP antibodies that bind to the same epitope, or a portion of that epitope. Similarly, the present invention encompasses anti-Ebola virus GP antibodies that compete for binding to Ebola virus GP or a fragment thereof with any of the specific exemplary antibodies described herein. For example, the present invention encompasses anti-Ebola virus GPP antibodies that cross-compete for binding to Ebola virus with one or more antibodies derived from the antibodies described in Tables 1 and 2.

[0161] Whether an antibody binds to the same epitope as a reference anti-Ebola virus GP antibody or competes for binding can be easily determined by using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Ebola virus GP antibody of the present invention, the reference antibody is bound to an Ebola virus GP or peptide under saturating conditions. The ability of the test antibody to bind to an Ebola virus GP molecule is then evaluated. If the test antibody can bind to an Ebola virus GP molecule after saturation binding with the reference anti-Ebola virus GP antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-Ebola virus antibody. On the other hand, if the test antibody cannot bind to an Ebola virus GP molecule after saturation binding with the reference anti-Ebola virus GP antibody, the test antibody may bind to the same epitope as the reference anti-Ebola virus GP antibody of the present invention.

[0162] To determine whether an antibody competes for binding with a reference anti-Ebola virus GP antibody, the above binding method is performed in two directions: in the first direction, the reference antibody is allowed to bind to Ebola virus GP under saturating conditions, and then the binding of the test antibody to Ebola virus GP is assessed. In the second direction, the test antibody is allowed to bind to Ebola virus GP under saturating conditions, and then the binding of the reference antibody to Ebola virus GP is assessed. If only the first (saturating) antibody is able to bind to Ebola virus GP in both directions, it is concluded that the test antibody and the reference antibody compete for binding to Ebola virus GP. As will be understood by those skilled in the art, an antibody that competes for binding with a 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 an overlapping or adjacent epitope.

[0163] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, 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 essentially all of the amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0164] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether the observed lack of binding is due to steric blocking (or another phenomenon). These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0165] Immunoconjugates The present invention encompasses human anti-Ebola virus GP monoclonal antibodies conjugated to a therapeutic moiety, such as an antiviral drug, for treating Ebola virus infection ("immunoconjugates"). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody can be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule as long as it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second, different antibody against Ebola virus or Ebola virus GP. In certain embodiments, the antibody can be conjugated to an agent specific for virus-infected cells. The type of therapeutic moiety that can be conjugated to an anti-Ebola virus GP antibody takes into consideration the condition being treated and the desired therapeutic effect to be achieved. Forming an immunoconjugate Examples of suitable agents for this purpose are known in the art; see, for example, WO05 / 103081.

[0166] 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 contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol., 147:60-69; Kufer et al., 2002, J. Immunol., 147:60-69; See Trends Biotechnol. 22:238-244, 2004.

[0167] 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 techniques) that will be known to those skilled in the art.

[0168] In some embodiments, Ebola virus-specific antibodies are produced in a bispecific format ("bispecific") in which variable regions that bind to distinct domains of Ebola virus are linked to confer dual domain specificity within a single binding molecule. Properly designed bispecifics can enhance overall Ebola virus-protein inhibitory efficacy by increasing both specificity and avidity. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain), or variable regions capable of binding to different regions within a domain, are paired on a structural scaffold that allows each region to simultaneously bind to a separate epitope or different regions within a domain. In one example, for a bispecific, a heavy chain variable region (V) from a binder with specificity for one domain is combined with a heavy chain variable region (V) from a binder with specificity for one domain. H ) with a light chain variable region (V) from a series of binders with specificity for the second domain. L ) and rearrange it to the original V H And that V H Non-cognate V that can be paired without disrupting the original specificity for L Identify the partner. In this way, a single V L Segments (e.g., V L 1) Two different V H Domain (e.g., V H 1 and V H 2) to form two binding "arms" (V H 1-V L 1 and V H 2-V L 1) can be used to generate bispecifics consisting of a single V L The use of segments reduces the complexity of the systems in the cloning, expression, and purification processes used to generate bispecifics, thereby simplifying and increasing efficiency (see, e.g., USSN 13 / 022759 and US2010 / 0331527).

[0169] Alternatively, antibodies that bind to more than one domain and a second target, such as, but not limited to, a second, different anti-Ebola virus antibody, can be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to separate regions can be linked to variable regions that bind to related sites on the Ebola virus, for example, to confer dual antigen specificity within a single binding molecule. A properly designed bispecific of this nature performs dual functions. A variable region with specificity for the extracellular domain is paired with a variable region with specificity for the outside of the extracellular domain, paired on a structural scaffold that allows each variable region to bind to a different antigen.

[0170] An exemplary bispecific antibody format that can be used in connection with the present invention is a first immunoglobulin (Ig) C H 3 domain and second Ig C H 3 domains, where the first Ig C H 3 domain and second Ig C H The three domains differ from each other by at least one amino acid, and 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. H The 3 domain binds to protein A and the second Ig C H The 3 domain contains mutations that reduce or abolish binding to Protein A, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise the modification Y96F (according to IMGT; Y436F according to EU). HAdditional modifications that may be found in 3 include, in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I according to EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Variations on the bispecific antibody format described above are contemplated as falling within the scope of the present invention.

[0171] Other exemplary bispecific formats that can be used in connection with the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 and bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al., 2012, mAbs, Vol. 4:6, pp. 1-11, and the references therein). (See cited references.) Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, when unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that self-assemble into multimeric complexes with defined composition, valency, and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]). (I want to be).

[0172] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-Ebola virus GP antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions according to the present invention are administered with suitable carriers, excipients, and other agents incorporated into the formulation to improve transfer, delivery, tolerability, etc. Many suitable formulations can be found in Remington's Pharmaceuticals, a formulary known to all pharmacologists. Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0173] The antibody dosage may vary depending on the age and size of the subject receiving the antibody, the target disease, condition, route of administration, etc. When the antibody of the present invention is used to treat or prevent a disease or disorder in an adult patient, it is generally advantageous to administer the antibody of the present invention at a single dose of about 0.1 mg to about 60 mg per kg of body weight, more preferably about 5 mg to about 60 mg, about 10 mg to about 50 mg, or about 20 mg to about 50 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention can be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg. In certain embodiments, after the initial dose, a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof can be administered in an amount that can be about the same as or less than the initial dose, where the subsequent doses are separated by at least 1 to 3 days; at least 1 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.

[0174] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. For example, liposomes, microparticles, microcapsules, recombinant cells capable of expressing viral mutants, and receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem., 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0175] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for their preparation and use are described in detail in Arruebo, M. et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications", J. Nanomat. 2009, Vol. 439389, p. 24, doi: 10.1155 / 2009 / 439389). Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995.

[0176] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0177] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like. These can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) addition product of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which can be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The thus-prepared injectable preparations are preferably filled into appropriate ampoules.

[0178] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not use a replaceable cartridge. Instead, the disposable pen delivery device is filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

[0179] A number of reusable pen-type and auto-injector-type delivery devices are adapted for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are certainly not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, 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). Examples of disposable pen delivery devices applicable to subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0180] The above-mentioned pharmaceutical compositions for oral or parenteral use are advantageously prepared in a unit dosage form suitable for adjusting the dose of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories. The amount of antibody contained in a unit dosage form is generally about 5 to about 500 mg. In particular, in the form of injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0181] Therapeutic Uses of Antibodies The antibodies of the invention are useful for treating and / or preventing a disease or disorder or condition associated with Ebola virus infection and / or ameliorating at least one symptom associated with such a disease, disorder or condition.

[0182] In certain embodiments, the antibodies of the present invention are useful for treating subjects suffering from severe and acute respiratory infection caused by Ebola virus. In some embodiments, the antibodies of the present invention are useful in reducing viral titers or viral load in a host. In one embodiment, the antibodies of the present invention or antigen-binding fragments thereof can be administered in therapeutic doses to patients infected with Ebola virus.

[0183] One or more antibodies of the invention can be administered to alleviate or prevent, or to reduce the severity of, one or more symptoms or conditions of a disease or disorder. The antibodies can be used to ameliorate or reduce the severity of at least one symptom of Ebola virus infection, including, but not limited to, fever, headache, fatigue, loss of appetite, muscle pain, diarrhea, vomiting, abdominal pain, dehydration, and unexplained bleeding.

[0184] Also contemplated herein is the prophylactic use of one or more antibodies of the invention in subjects at risk of developing an Ebola virus infection, such as immunocompromised individuals, healthcare workers, individuals suspected of having been exposed to an individual with the Ebola virus, individuals in physical contact with or close physical proximity to an infected individual, hospital personnel, pharmaceutical researchers, maintenance personnel responsible for cleaning hospital equipment or facilities where Ebola patients are being treated, individuals who have visited or plan to visit areas or countries known or suspected of having an Ebola virus outbreak, or frequent travelers.

[0185] In a further embodiment of the invention, the antibodies are used to prepare a pharmaceutical composition for treating a patient suffering from an Ebola virus infection. In another embodiment of the invention, the antibodies are used as an adjunct therapy with any other agent or therapy known to those skilled in the art to be useful for treating or ameliorating an Ebola virus infection.

[0186] Combination therapy Combination therapies include the anti-Ebola virus GP antibodies of the invention and any additional therapeutic agent that is advantageously combined with the antibodies of the invention or biologically active fragments of the antibodies of the invention. The antibodies of the invention can be synergistically combined with one or more drugs or agents used to treat Ebola virus infection.

[0187] For example, exemplary agents for treating viral infections can include, for example, antivirals, anti-inflammatory drugs (such as corticosteroids and nonsteroidal anti-inflammatory drugs), different antibodies against the Ebola virus, vaccines against the Ebola virus, TKM Ebola (a small interfering RNA targeting the viral RNA polymerase) brincidofovir (CMX-001), favipiravir (T-705), BCX-4430, AVI-7537 (an antisense phosphorodiamidate morpholino oligomer targeting the Ebola virus VP24 gene), interferon, or any other palliative therapy for treating an Ebola virus infection.

[0188] In some embodiments, the antibodies of the invention can be combined with a second therapeutic agent to reduce viral load in a patient infected with Ebola virus or to ameliorate one or more symptoms of the infection.

[0189] In certain embodiments, the second therapeutic agent is another, different antibody or antibody cocktail specific to the Ebola virus GP, and the different antibody or antibodies within the cocktail may or may not bind to the same epitope or overlapping epitopes as the antibody of the present invention. In certain embodiments, the second therapeutic agent is an antibody against a different Ebola virus protein. The second antibody may be specific to one or more different Ebola virus proteins derived from different strains of the virus. The use of a combination ("cocktail") of antibodies of the present invention that have neutralizing or inhibitory activity against Ebola virus is contemplated herein. In some embodiments, non-competing antibodies can be administered in combination to a subject in need thereof to reduce the ability of the Ebola virus to escape due to mutations. In some embodiments, the antibodies in the combination bind to distinct, non-overlapping epitopes on the GP. The antibodies in the combination may block viral attachment to and / or entry into and / or fusion with host cells. The antibodies may interact with GP derived from a strain of EBOV selected from Zaire, Sudan, Bundibugyo, or Côte d'Ivoire, and may neutralize any one or more of the described Ebola virus strains when used alone or in combination with any one or more of the above agents.

[0190] Also contemplated herein is the use of combinations of anti-Ebola virus GP antibodies of the invention, wherein the combination comprises one or more antibodies that do not cross-compete. In certain embodiments, the combination comprises a cocktail comprised of a mixture of at least three antibodies of the invention. The antibodies within the cocktail may differ in their ability to neutralize the virus or virus-infected cells, or to mediate antibody-dependent cellular cytotoxicity (ADCC), or to bind to EBOV soluble glycoprotein (sGP).

[0191] As used herein, the term "in combination" means that an additional therapeutically active component(s) can be administered before, simultaneously with, or after administration of at least one anti-Ebola virus GP antibody of the invention, or a cocktail comprised of one or more of the antibodies of the invention. The term "in combination" also encompasses sequential or simultaneous administration of an anti-Ebola virus GP antibody and a second therapeutic agent.

[0192] The additional therapeutically active component(s) can be administered to a subject prior to administration of an anti-Ebola virus GP antibody of the invention. For example, a first component can be considered to be administered "before" a 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 before the second component. In other embodiments, the additional therapeutically active component(s) can be administered to a subject after administration of an anti-Ebola virus GP antibody of the invention. For example, if a first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after the administration of the second component, the first component is considered to be administered "after" the second component. In 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 invention. Administering "concurrently," for purposes of the present invention, includes, for example, administering the anti-Ebola virus GP antibody and the additional therapeutically active component to the subject as a single dosage form or as separate dosage forms administered to the subject within about 30 minutes or less of each other. When administered as 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). Alternatively, each dosage form can be administered by a different route (e.g., the anti-Ebola virus GP antibody can be administered intravenously and the additional therapeutically active component can be administered orally). In any event, administration of the components by the same route as a single dosage form, by the same route as separate dosage forms, or by different routes as separate dosage forms are all considered "administered simultaneously" for purposes of this disclosure.For purposes of this disclosure, administration of an anti-Ebola virus GP antibody "before," "concurrently with," or "after" the administration of an additional therapeutically active component (as these terms are defined herein above) is considered administration of the anti-Ebola virus GP antibody "in combination with" the additional therapeutically active component.

[0193] The present invention encompasses pharmaceutical compositions formulated with the anti-Ebola virus GP antibodies of the invention together with one or more of the additional therapeutically active component(s) described elsewhere herein.

[0194] Dosing regimen According to certain embodiments, a single dose of an anti-Ebola virus GP antibody of the invention (or a pharmaceutical composition comprising a combination of an 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 certain embodiments of the invention, multiple doses of an anti-Ebola virus GP antibody (or a pharmaceutical composition comprising a combination of an anti-Ebola virus GP antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a predetermined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of an anti-Ebola virus GP antibody of the invention to a subject. As used herein, "sequentially administering" means administering each dose of an anti-Ebola virus GP antibody to a subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention encompasses methods comprising sequentially administering to a patient a single initial dose of anti-Ebola virus GP antibody, followed by one or more secondary doses of anti-Ebola virus GP antibody, and optionally, followed by one or more tertiary doses of anti-Ebola virus GP antibody.

[0195] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of the anti-Ebola virus GP antibodies of the present invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-Ebola virus GP antibody but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of anti-Ebola virus GP antibody contained in the initial, secondary, and / or tertiary doses varies from one another during the course of treatment (e.g., adjusted upward or downward as needed). In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered at the beginning of a treatment regimen as "loading doses," followed by subsequent doses administered less frequently (e.g., "maintenance doses").

[0196] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is administered 1 to 48 hours (e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours) after the immediately preceding dose. hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, 22.5 hours, 23 hours, 23.5 hours, 24 hours, 24.5 hours, 25 hours, 25.5 hours, 26 hours, 26.5 hours, or more). The phrase "immediately preceding dose" as used herein refers to the dose of anti-Ebola virus GP antibody administered to a patient in a multiple administration series immediately prior to administration of the next dose in the series, with no intervening doses in between.

[0197] Methods according to this aspect of the invention can include administering to a patient any number of secondary and / or tertiary doses of anti-Ebola virus GP antibodies. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0198] In certain embodiments of the present invention, the frequency with which the secondary and / or tertiary doses are administered to a patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by a physician during the course of treatment according to the needs of an individual patient after clinical testing.

[0199] Diagnostic Uses of Antibodies The anti-Ebola virus GP antibodies of the present invention can be used, for example, for diagnostic purposes, to detect and / or measure Ebola virus in a sample. In some embodiments, the use of one or more antibodies of the present invention in assays for detecting diseases or disorders, such as viral infections, is contemplated. An exemplary diagnostic assay for Ebola virus, for example, involves contacting a sample obtained from a patient with an anti-Ebola virus GP antibody of the present invention, where the anti-Ebola virus GP antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate Ebola virus from the patient sample. Alternatively, an unlabeled anti-Ebola virus GP antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125The antibody may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Certain exemplary 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).

[0200] Samples that can be used in the Ebola virus diagnostic assays of the present invention include any tissue or fluid sample obtained from a patient under normal or pathological conditions that contains a detectable amount of Ebola virus or a fragment thereof. Generally, to first establish a baseline or standard Ebola virus level, the level of Ebola virus in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease associated with Ebola virus) is measured. This baseline Ebola virus level can then be compared with the level of Ebola virus measured in a sample obtained from an individual suspected of having an Ebola virus-associated condition or symptoms associated with such a condition.

[0201] The antibody specific for Ebola virus may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if any) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented so that the C-terminal portion of the peptide is distal from the surface. [Example]

[0202] The following examples are presented 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 what the inventors regard as their invention. While attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

[0203] Example 1 Generation of human antibodies against Ebola virus Human antibodies against Ebola virus were generated in mice containing DNA encoding human immunoglobulin heavy chain variable regions and kappa light chain variable regions. In one embodiment, human antibodies against Ebola virus were generated in VELOCIMMUNE® mice. In one embodiment, VelocImmune® (VI) mice were immunized with DNA encoding full-length Ebola virus GP [Zaire Ebola virus 2014 (GenBank: KJ660346.2)]. Antibodies were generated following an accelerated regimen consisting of two immunizations separated by two weeks. Antibody immune responses were monitored by Ebola virus GP-specific immunoassays. For example, sera were assayed for specific antibody titers against purified full-length EBOV GP, subunit GP proteins (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 methods. For example, when the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to preserve their viability, forming hybridoma cell lines.The hybridoma cell lines were screened and selected to identify cell lines that produce Ebola virus GP-specific antibodies.Using this technique and the various immunogens described above, several chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained.The exemplary antibodies thus generated were named H1M17354N, H2aM17356N, H1M17357N, H2aM17358N, H2aM17359N, and H2aM17360N.

[0204] Anti-Ebola virus antibodies were also isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298. Using this method, several fully human anti-Ebola virus GP antibodies (i.e., antibodies with human variable and constant domains) were obtained. Exemplary antibodies thus generated were designated H1H17134P, H1H17139P, H1H17142P, H1H17151P, H1H17161P, H1H17162P, H1H17193P, H1H17196P, H1H17199P, H1H17203P, H1H17214P, H1H17219P, H1H17223P, and H1H17228P.

[0205] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples below.

[0206] Example 2 Amino acid and nucleotide sequences of the heavy and light chain variable regions Table 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-Ebola virus antibodies of the invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. [Table 1] [Table 2]

[0207] As used herein, antibodies are generally referred to according to the following nomenclature: an Fc prefix (e.g., "H1H," "H2M," etc.), followed by a numerical identifier (e.g., "17139," "17161," etc., as shown in Tables 1 or 2), followed by a "P," "P2," "N," "N2," or "B" suffix. The H1H and H2M prefixes in antibody names used herein indicate the particular Fc region isotype of the antibody. Thus, in accordance with this nomenclature, antibodies may be referred to herein as, for example, "H1H17359N," "H2aM17359N," etc. For example, an "H1M" antibody has a murine IgG1 Fc, and an "H2M" antibody has a murine IgG2 Fc (a or b isotype) (as indicated by the initial "H" in the antibody name, all of the variable regions are fully human). As will be understood by those skilled in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc, etc.), but in either case, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 or 2 will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0208] Example 3 Antibody binding to Ebola virus GP as determined by surface plasmon resonance A. pH-dependent dissociation rate constant at 37°C The binding dissociation rate constant (k) for Ebola virus GP binding to purified anti-Ebola virus GP monoclonal antibody at 37°C. d The dissociation half-life (t) and dissociation half-life (t) were determined using a real-time surface plasmon resonance biosensor assay on a Biacore T200 instrument. The CM4 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) or a monoclonal goat anti-mouse Fc antibody (GE, #BR-1008-38) to capture purified anti-Ebola virus GP mAb. All Biacore binding studies in Example 3A were performed in buffers consisting of 0.01 M NaHPO / NaHPO, 0.15 M NaCl, 0.05% v / v surfactant P20 (PBS-P running buffer), pH 7.4, pH 6.0, and pH 5.0. Low pH chases (low pH A chase was performed to assess whether antibody binding was maintained at low pH. This mimics the conditions encountered by the virus during membrane fusion due to endosomal acidification. Various concentrations of Ebola virus GP with a C-terminal polyhistidine tag (EbolaGP.his; Sino Biologicals, catalog no. 40442-V08B1) prepared in PBS-P running buffer (3-fold serial dilutions ranging from 90 nM to 11.1 nM) were injected over the surface on which anti-Ebola virus GP mAb was captured at a flow rate of 25 μL per minute. The association of Ebola virus GP with the captured monoclonal antibody 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 kinetic dissociation (k d ) Rate constants were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. The dissociation half-life (t) was calculated from the reaction rate constants as follows:

number

[0209] The dissociation rate parameters for Ebola virus GP binding to purified anti-Ebola virus GP mAbs at 37°C are shown in Table 3. [Table 3]

[0210] B. Binding affinity and kinetics at 25°C and 37°C The equilibrium dissociation constant (K) for Ebola virus GP binding to purified anti-Ebola virus GP mAb DThe binding affinity (RI) values ​​were determined using a real-time surface plasmon resonance biosensor on a Biacore 4000 instrument. The CM4 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) or a monoclonal goat anti-mouse Fc antibody (GE, #BR-1008-38) to capture purified anti-Ebola virus GP mAb. 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, and 0.05% v / v surfactant P20 (HBS-ET running buffer). Various concentrations of Ebola virus GP (Sino Biologicals, Cat. No. 40442-V08B1) with a C-terminal polyhistidine tag (ranging from 90 nM to 3.3 nM, 3-fold serial dilutions) prepared in HBS-ET running buffer were injected over the surface with captured anti-Ebola virus GP mAb at a flow rate of 30 μL per minute. The association of Ebola virus GP with the captured monoclonal antibody was monitored for 5 minutes, and the dissociation of Ebola virus GP in HBS-ET running buffer was monitored for 10 minutes. All binding kinetic experiments were performed at 25°C and 37°C. The kinetic association (k a ) rate constant and dissociation (k d The binding-dissociation equilibrium constant (K) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. D ) and dissociation half-life (t1 / 2) were calculated from the reaction rate constants as follows:

number

[0211] The binding kinetic parameters for Ebola virus GP binding to purified anti-Ebola virus GP mAbs at 25°C and 37°C are shown in Tables 4A and 4B. [Table 4A] [Table 4B]

[0212] result As shown in Tables 4A and 4B above, the antibodies bind to Ebola virus GP with K values ​​ranging from 934 pM to 1890 nM at 25°C. D values, K values ​​ranging from 227 pM to 2310 nM at 37 °C D At pH 7.4, antibody dissociation half-life (t) values ​​ranged from 3.0 minutes to over 1155 minutes at 25°C and from 2.0 minutes to over 1155 minutes at 37°C. No decrease in binding was observed at low pH. Some antibodies had increased dissociation half-life (t) values ​​at low pH compared to pH 7.4. Antibodies that have a three-fold or greater increase in dissociation half-life (t½) values ​​at pH 5 and / or pH 6 include H1H17162P, H1H17177P, H1H17150P, H1H17151P, H1H17160P, H1H17161P, H1H17141P, H1H17139P, H1H17210P, H1H17203P, H1H17199P, H1M17348N, H1M17350N, H2aM17360N and H2aM17361N.

[0213] Example 4 Ebola virus pseudoparticle generation and neutralization assays Ebola virus pseudoparticles (also called virus-like particles, or VLPs) were generated by cotransfecting 293T cells with a mixture of Ebola virus GP, a plasmid construct expressing HIV gag-pol, and an HIV proviral vector encoding firefly luciferase. Supernatants containing Ebola virus pseudoparticles were collected 48 hours posttransfection, clarified using centrifugation, aliquoted, and frozen at -80°C. Control pseudoparticles were generated by replacing the Ebola virus GP-expressing plasmid with a plasmid encoding vesicular stomatitis virus glycoprotein (VSVg).

[0214] Ebola pseudoparticle-based neutralization assay The pseudoparticles produced as described above were tested in a neutralization assay. Specifically, antibody dilutions were incubated with Ebola virus pseudoparticles at room temperature for 1 hour. Huh7 cells were detached using 0.02M EDTA, washed, and incubated with the antibody / pseudoparticle mixture for 72 hours. Infection efficiency was quantified by luciferase detection using the BrightGlo® Luciferase Assay (Promega, San Luis Obispo, CA, USA) and light production readings on a Victor® X3 plate reader (Perkin Elmer, Waltham, MA, USA). [Table 5]

[0215] From the data presented in Table 5 above, it can be seen that, using the experimental design described herein, 14 of the 20 anti-Ebola virus antibodies of the present invention exhibited a cytotoxicity of approximately 10 -11 Approximately 10 minutes from M -9 ICs ranging up to M 50 It has been shown to strongly neutralize infectivity.

[0216] Example 5 Antibody-dependent cell-mediated cytotoxicity (ADCC) by anti-Ebola virus antibodies Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured using an antibody-based CD16-based reporter system (Promega ADCC Reporter Bioassay Core Kit, San Luis The ability of the Ebola virus GP-expressing 293 cells to transmit signals via the GFP-binding protein (Ebola virus GP) was tested. After 1 day, diluted GFP-binding protein was added to the GFP-binding protein. -Antibodies produced in cell lines (see U.S. Pat. No. 8,409,838) and effector cells (effector-to-target ratio 1.5:1) were added and incubated overnight. Reporter activity was measured using the BioGlo® Luciferase Assay (Promega, San Luis Obispo, CA, USA), and light production was read using a Victor® X3 plate reader (Perkin Elmer, Waltham, MA, USA). [Table 6]

[0217] The ability of antibodies to mediate ADCC was calculated based on activity compared to the isotype (negative) control. All values ​​greater than 5-fold the negative control were considered positive. The data in Table 6 above show that 17 of the 20 anti-Ebola virus antibodies mediated ADCC.

[0218] Example 6 Octet crossing conflict Binding competition between anti-Ebola virus GP monoclonal antibodies previously determined to bind Ebola virus GP was determined using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor (ForteBio Corp., A Division of Pall Life Sciences). Binding of relevant controls for soluble GP (sGP), GP1, or GP2 was measured in the same assay format, and the response was subtracted from 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% v / v surfactant P20, and 1.0 mg / mL BSA (Octet HBS-ET buffer), with the 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 (Ebola virus GP.h, Sino Biologicals Inc., GenBank AHX24649.1 and SEQ ID NO: 314), approximately 1.0 nm of Ebola virus GP was first captured onto an Octet biosensor (Fortebio Inc., #18-5079) coated with an anti-penta-His antibody by immersing the biosensor in a well containing a 20 μg / mL solution of Ebola virus GP for 3 minutes. The biosensor with the captured antigen was then saturated with a first anti-Ebola virus GP monoclonal antibody (hereafter referred to as mAb-1) by immersing the biosensor in a well containing a 50 μg / mL solution of mAb-1 for 5 minutes. The biosensors were then immersed in wells containing a 50 μg / mL solution of a second anti-Ebola virus GP monoclonal antibody (hereafter referred to as mAb-2) for 3 minutes. All biosensors were washed with Octet HBS-ET buffer between each step of the experiment. Real-time binding responses were monitored throughout the course of the experiment and recorded at the end of every step.The binding response of mAb-2 to Ebola virus GP precomplexed with mAb-1 was compared, and the competitive / non-competitive behavior of different anti-Ebola virus GP monoclonal antibodies was determined using a 50% inhibition threshold. Table 7 clearly defines the relationship between antibodies that compete in both directions, regardless of the order of binding.

[0219] As shown in Table 7, the left column shows the mAb1 antibody captured using the AHC Octet biosensor, and the right column shows the antibody (mAb2) that cross-competes with the mAb1 antibody. [Table 7-1] [Table 7-2] [Table 7-3]

[0220] Example 7 Sequential binding of H1H17203P, H1H17139P, and H1H17161P to Ebola virus glycoproteins Using the information obtained from the cross-competition experiments, sequential binding studies were performed to determine whether the three individual candidate antibodies could simultaneously bind to soluble Ebola virus glycoprotein (GP), thereby confirming that the binding sites on Ebola virus GP were independent for each monoclonal antibody. If so, this information would support the use of these antibodies in a therapeutic cocktail.

[0221] Therefore, in sequential binding experiments, three anti-Ebola virus GP monoclonal antibodies, H1H17203P, H1H17139P, and H1H17161P, were tested for independent, noncompetitive binding to Ebola virus GP. The experiments were performed using a real-time, label-free biolayer interferometry (BLI) assay on an 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% v / v surfactant P20, and 1.0 mg / mL BSA (Octet HBS-ET buffer), with plate shaking at 1000 rpm. To assess whether the three antibodies could simultaneously bind to the captured Ebola virus GP antigen (Ebola virus GP.his, Sino Biologicals) expressed with a C-terminal polyhistidine tag, approximately 0.6 nm of Ebola virus GP.h was first captured on an Octet biosensor (Fortebio Inc., #18-5079) coated with an anti-penta-His antibody by immersing the biosensor in a well containing a 20 μg / mL solution of Ebola virus GP.h for 3 minutes. The biosensor was then saturated with the first anti-Ebola virus GP monoclonal antibody (hereafter referred to as H1H17161P) by immersing it in a well containing a 50 μg / mL solution of REGN H1H17161P for 5 minutes. The biosensor was then immersed in a well containing a 50 μg / mL solution of a second anti-Ebola virus GP monoclonal antibody (hereafter referred to as H1H17139P) for 5 minutes. Finally, 50 μg / mL of a third antibody (hereafter referred to as H1H17161P) was injected for 5 minutes until saturation was reached. Real-time binding responses were monitored throughout the course of the experiment and recorded at the end of every step.

[0222] result The three candidate monoclonal antibodies tested were able to simultaneously bind to Ebola virus GP, indicating that each antibody does not interfere with the Ebola virus GP binding site of the other tested antibodies, suggesting that each antibody binds to or interacts with a different epitope, supporting a role for the use of these three antibodies in therapeutic antibody cocktails.

[0223] Example 8 Binding of anti-Ebola antibodies to different Ebola virus-like particle (VLP) strains We conducted a study to determine whether anti-Ebola virus GP antibodies reacted with virus-like particles (VLPs) containing GPs from other Ebola virus strains. This study included VLPs containing GPs from Bundibugyo NC_014373, Côte d'Ivoire FJ217162, Sudan NC_006432, Zaire 1995, and Zaire 2014 AY354458, as well as a negative control, VSV glycoprotein (VSVg). The study was performed using "MesoScale Discovery" (MSD), a technology that allows for the binding / immobilization of Ebola strain VLPs (expressing Ebola glycoprotein / viral surface protein) to a carbon surface, followed by an ELISA-type binding assay. The goal was to identify the binding profiles of the mAbs with respect to various Ebola strains.

[0224] The assay was performed in a 96-well polypropylene microwell plate by first preparing 1:10 dilutions of supernatants from the various VLPs per well (as described in the table below), adding the dilutions to PBS (50 μl / well), and incubating overnight at 4°C.

[0225] The liquid in the wells was discarded, followed by blocking with 150 μl / well of PBS + 2% BSA and incubation at room temperature for 1 hour. The contents of each well were then discarded, and the wells were washed with PBS using an AquaMax 2000 plate washer specified for MSD. Fifty microliters of primary antibody was diluted in PBS + 1% BSA and incubated at room temperature with shaking at medium speed (5). The contents of the wells were then discarded, and the plate was washed with PBS. Fifty microliters of sulfo-TAG detection reagent (human or mouse Fc) was added to each well at a concentration of 1 μg / ml in PBS + 0.5% BSA and incubated at room temperature for 1 hour with shaking at medium speed (5). The contents of the wells were discarded, and the plate was washed with PBS + 0.5% BSA. 150 μl of 1x Read Buffer without detergent was added to each well, and the plate was read via barcode on a SECTORImager 6000.

[0226] The results, shown in Table 8 below, demonstrate that all of the anti-Ebola virus GP antibodies tested bind to VLPs containing Zaire 2014 GP and VLPs containing Zaire 1995 GP. Certain of the antibodies tested, in addition to binding to the two Zaire strains shown in Table 8, also bind to VLPs containing GPs from other Ebola virus strains. Specifically, in addition to binding to VLPs containing GPs from Zaire 2014 and Zaire 1995, the anti-Ebola virus antibodies designated H1H17161P and H1H17162P bind to VLPs containing GPs from the Sudan strain and VLPs containing GPs from the Bundibugyo strain, while the anti-Ebola virus antibodies designated H2aM17356N and H1H17142P bind to the Bundibugyo and Ivory Coast strains. [Table 8]

[0227] Example 9 In vitro neutralization of live / infectious Ebola virus (EBOV) Antibodies designated H1H17203P, H1H17139P, and H1H17161P were analyzed for their ability to neutralize infectious EBOV in Vero cells. Vero cells were plated in 384-well plates in DMEM-10% FBS and grown at 37°C to approximately 75% confluence. H1H17203P, H1H17139P, and H1H17161P were diluted as indicated. EBOV strains (Mayinga, Kikwit, Makona, and guinea pig-adapted Mayinga) were thawed and appropriately diluted to an MOI of 0.01 to 0.1. A commercially available anti-EBOV antibody designated KZ52 was used as a positive control (Maruyama, T. et al., J Virol 73:6024-6030 (1999)). See the literature (1999). The antibody was incubated with the virus for 1 hour at 37°C. The antibody / virus mixture was then added to the pre-plated cells, and the plates were incubated for 24 hours at 37°C. After the incubation period, the plates were removed from the incubator, inactivated by immersion in 10% neutral-buffered formalin, placed in heat-sealed bags, and stored overnight at 4°C in a BSL-4 container. The plates were washed three times with 1x PBS, and the cells were permeabilized with 25 μl of 0.1% Triton X-100 in 1x PBS at room temperature (RT) for 15–20 minutes. The 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 anti-EBOV GP primary antibody 4F3 (see IBT BIOSERVICES for mouse anti-EBOV GP monoclonal antibody 4F3, catalog number 0201-020) diluted 1:1500 in 1x PBS. Plates were washed twice with 1x PBS for 10-15 minutes. Cells were incubated with anti-mouse secondary antibody conjugated with Alexa-fluor-488 for 1 hour. The secondary antibody was discarded, and plates were washed twice with 1x PBS for 10-15 minutes. Plates were incubated with 25 μl / well of Hoechst (1:50,000 in 1x PBS) for 30 minutes at RT. Plates were imaged by fluorescence microscopy using blue and green fluorescence channels.

[0228] result The results shown in Figure 1 demonstrated that the H1H17161P antibody neutralized live virus and was more potent than the positive control antibody KZ52, whereas the antibodies designated H1H17203P and H1H17139P did not act as neutralizers.

[0229] Example 10 Binding of anti-Ebola antibodies to soluble GP (sGP) The fourth gene in the EBOV genome encodes two unique proteins: a nonstructural, dimeric secreted glycoprotein designated sGP, and a trimeric virion-attached envelope glycoprotein (GP). These two GPs share the first 295 amino acids but have unique C-termini. To determine whether Regeneron's lead mAb binds to sGP, a recombinant sGP.mmh protein was generated in-house (SEQ ID NO: 317). The interferometry-based biosensor Octet HTX was used to determine whether the H1H17203P, H1H17139P, and H1H17161P monoclonal antibodies could bind to the Ebola sGP.mmh protein. The assay format involved capturing H1H17203P, H1H17139P, and H1H17161P on an anti-hFc sensor tip, followed by immersion in a 300 nM solution of Ebola GP.10xhis (SEQ ID NO: 318), sGP.mmh (SEQ ID NO: 317), or hCNTFR (ciliary neurotrophic factor receptor.mmh, a negative control protein). Each mAb was captured at levels between 0.94 and 1.36 nM.

[0230] As shown in Figure 2, all mAbs showed specific binding to Ebola GP.10xhis and no binding to the negative control protein; whereas, only H1H17139 demonstrated specific binding to Ebola sGP.mmh. This finding suggests that the binding epitope of H1H17139 is likely located in a common region within the first 295 amino acids of both sGP and GP; whereas, the other mAbs likely recognize only the C-terminus of Ebola GP.

[0231] Example 11 Binding of additional anti-EBOV GP antibodies to EbolaGP.h, EbolaGP soluble.mmh, and hCNTFR.mmh Further studies were conducted to determine the binding characteristics of additional anti-EBOV GP antibodies of the present invention. Specifically, studies were conducted to determine the ability of these additional antibodies to bind to soluble GP and GP. The studies were performed using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX 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% v / v surfactant P20, and 1.0 mg / mL BSA (Octet HBS-ET buffer), with the plate shaking at 1000 rpm. To assess whether the antibody could bind to Ebola sGP or other Ebola GP reagents, approximately 1.0 nm of anti-Ebola GP mAb was captured onto an Octet biosensor coated with anti-human Fc (Fortebio Inc, #18-5064) antibody by immersing the biosensor in a well containing a 20 μg / mL solution of the mAb for 3 minutes. The biosensor with the captured mAb was tested for binding to the selected protein reagent by immersing it in a well containing a 300 nM solution of Ebola GP protein or an unrelated control for 5 minutes. All biosensors were washed with Octet HBS-ET buffer between each step of the experiment. Real-time binding responses were monitored throughout the course of the experiment and recorded at the end of every step.

[0232] result All values ​​below 0.10 nm were determined to be non-binding antibodies, as shown in Table 9. Based on results to date, all but one of the antibodies tested (H1H17360N) demonstrated binding to EBOV full-length GP, and 13 of the 20 antibodies tested demonstrated binding to soluble GP (sGP). [Table 9]

Claims

1. A first nucleic acid molecule comprising a polynucleotide sequence encoding a heavy chain variable region (HCVR) of an antibody that specifically binds to Ebola virus (EBOV) and / or Ebola virus glycoprotein (EBOV-GP); a second nucleic acid molecule comprising a polynucleotide sequence encoding the light chain variable region (LCVR) of said antibody; a set of nucleic acid molecules comprising: (i) a heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SEQ ID NO: 20; a HCDR2 amino acid sequence of SEQ ID NO: 22; a HCDR3 amino acid sequence of SEQ ID NO: 24; a light chain complementarity determining region (LCDR) 1 amino acid sequence of SEQ ID NO: 28; a LCDR2 amino acid sequence of SEQ ID NO: 30; and a LCDR3 amino acid sequence of SEQ ID NO: 32; (ii) an HCDR1 amino acid sequence of SEQ ID NO: 68; an HCDR2 amino acid sequence of SEQ ID NO: 70; an HCDR3 amino acid sequence of SEQ ID NO: 72; an LCDR1 amino acid sequence of SEQ ID NO: 76; an LCDR2 amino acid sequence of SEQ ID NO: 78; and an LCDR3 amino acid sequence of SEQ ID NO: 80; (iii) an HCDR1 amino acid sequence of SEQ ID NO: 148; an HCDR2 amino acid sequence of SEQ ID NO: 150; an HCDR3 amino acid sequence of SEQ ID NO: 152; an LCDR1 amino acid sequence of SEQ ID NO: 156; an LCDR2 amino acid sequence of SEQ ID NO: 158; and an LCDR3 amino acid sequence of SEQ ID NO: 160; (iv) an HCDR1 amino acid sequence of SEQ ID NO: 4; an HCDR2 amino acid sequence of SEQ ID NO: 6; an HCDR3 amino acid sequence of SEQ ID NO: 8; an LCDR1 amino acid sequence of SEQ ID NO: 12; an LCDR2 amino acid sequence of SEQ ID NO: 14; and an LCDR3 amino acid sequence of SEQ ID NO: 16; (v) an HCDR1 amino acid sequence of SEQ ID NO: 36; an HCDR2 amino acid sequence of SEQ ID NO: 38; an HCDR3 amino acid sequence of SEQ ID NO: 40; an LCDR1 amino acid sequence of SEQ ID NO: 44; an LCDR2 amino acid sequence of SEQ ID NO: 46; and an LCDR3 amino acid sequence of SEQ ID NO: 48; (vi) an HCDR1 amino acid sequence of SEQ ID NO: 52; an HCDR2 amino acid sequence of SEQ ID NO: 54; an HCDR3 amino acid sequence of SEQ ID NO: 56; an LCDR1 amino acid sequence of SEQ ID NO: 60; an LCDR2 amino acid sequence of SEQ ID NO: 62; and an LCDR3 amino acid sequence of SEQ ID NO: 64; (vii) an HCDR1 amino acid sequence of SEQ ID NO: 84; an HCDR2 amino acid sequence of SEQ ID NO: 86; an HCDR3 amino acid sequence of SEQ ID NO: 88; an LCDR1 amino acid sequence of SEQ ID NO: 92; an LCDR2 amino acid sequence of SEQ ID NO: 94; and an LCDR3 amino acid sequence of SEQ ID NO: 96; (viii) an HCDR1 amino acid sequence of SEQ ID NO: 100; an HCDR2 amino acid sequence of SEQ ID NO: 102; an HCDR3 amino acid sequence of SEQ ID NO: 104; an LCDR1 amino acid sequence of SEQ ID NO: 108; an LCDR2 amino acid sequence of SEQ ID NO: 110; and an LCDR3 amino acid sequence of SEQ ID NO: 112; (ix) an HCDR1 amino acid sequence of SEQ ID NO: 116; an HCDR2 amino acid sequence of SEQ ID NO: 118; an HCDR3 amino acid sequence of SEQ ID NO: 120; an LCDR1 amino acid sequence of SEQ ID NO: 124; an LCDR2 amino acid sequence of SEQ ID NO: 126; and an LCDR3 amino acid sequence of SEQ ID NO: 128; (x) an HCDR1 amino acid sequence of SEQ ID NO: 132; an HCDR2 amino acid sequence of SEQ ID NO: 134; an HCDR3 amino acid sequence of SEQ ID NO: 136; an LCDR1 amino acid sequence of SEQ ID NO: 140; an LCDR2 amino acid sequence of SEQ ID NO: 142; and an LCDR3 amino acid sequence of SEQ ID NO: 144; (xi) an HCDR1 amino acid sequence of SEQ ID NO: 164; an HCDR2 amino acid sequence of SEQ ID NO: 166; an HCDR3 amino acid sequence of SEQ ID NO: 168; an LCDR1 amino acid sequence of SEQ ID NO: 172; an LCDR2 amino acid sequence of SEQ ID NO: 174; and an LCDR3 amino acid sequence of SEQ ID NO: 176; (xii) an HCDR1 amino acid sequence of SEQ ID NO: 180; an HCDR2 amino acid sequence of SEQ ID NO: 182; an HCDR3 amino acid sequence of SEQ ID NO: 184; an LCDR1 amino acid sequence of SEQ ID NO: 188; an LCDR2 amino acid sequence of SEQ ID NO: 190; and an LCDR3 amino acid sequence of SEQ ID NO: 192; (xiii) an HCDR1 amino acid sequence of SEQ ID NO: 196; an HCDR2 amino acid sequence of SEQ ID NO: 198; an HCDR3 amino acid sequence of SEQ ID NO: 200; an LCDR1 amino acid sequence of SEQ ID NO: 204; an LCDR2 amino acid sequence of SEQ ID NO: 206; and an LCDR3 amino acid sequence of SEQ ID NO: 208; (xiv) an HCDR1 amino acid sequence of SEQ ID NO: 212; an HCDR2 amino acid sequence of SEQ ID NO: 214; an HCDR3 amino acid sequence of SEQ ID NO: 216; an LCDR1 amino acid sequence of SEQ ID NO: 220; an LCDR2 amino acid sequence of SEQ ID NO: 222; and an LCDR3 amino acid sequence of SEQ ID NO: 224; (xv) an HCDR1 amino acid sequence of SEQ ID NO: 228; an HCDR2 amino acid sequence of SEQ ID NO: 230; an HCDR3 amino acid sequence of SEQ ID NO: 232; an LCDR1 amino acid sequence of SEQ ID NO: 236; an LCDR2 amino acid sequence of SEQ ID NO: 238; and an LCDR3 amino acid sequence of SEQ ID NO: 240; (xvi) an HCDR1 amino acid sequence of SEQ ID NO: 244; an HCDR2 amino acid sequence of SEQ ID NO: 246; an HCDR3 amino acid sequence of SEQ ID NO: 248; an LCDR1 amino acid sequence of SEQ ID NO: 252; an LCDR2 amino acid sequence of SEQ ID NO: 254; and an LCDR3 amino acid sequence of SEQ ID NO: 256; (xvii) an HCDR1 amino acid sequence of SEQ ID NO: 260; an HCDR2 amino acid sequence of SEQ ID NO: 262; an HCDR3 amino acid sequence of SEQ ID NO: 264; an LCDR1 amino acid sequence of SEQ ID NO: 268; an LCDR2 amino acid sequence of SEQ ID NO: 270; and an LCDR3 amino acid sequence of SEQ ID NO: 272; (xviii) an HCDR1 amino acid sequence of SEQ ID NO: 276; an HCDR2 amino acid sequence of SEQ ID NO: 278; an HCDR3 amino acid sequence of SEQ ID NO: 280; an LCDR1 amino acid sequence of SEQ ID NO: 284; an LCDR2 amino acid sequence of SEQ ID NO: 286; and an LCDR3 amino acid sequence of SEQ ID NO: 288; (xix) an HCDR1 amino acid sequence of SEQ ID NO: 292; an HCDR2 amino acid sequence of SEQ ID NO: 294; an HCDR3 amino acid sequence of SEQ ID NO: 296; an LCDR1 amino acid sequence of SEQ ID NO: 300; an LCDR2 amino acid sequence of SEQ ID NO: 302; and an LCDR3 amino acid sequence of SEQ ID NO: 304; or (xx) an HCDR1 amino acid sequence of SEQ ID NO: 308; an HCDR2 amino acid sequence of SEQ ID NO: 310; an HCDR3 amino acid sequence of SEQ ID NO: 312; an LCDR1 amino acid sequence of SEQ ID NO: 284; an LCDR2 amino acid sequence of SEQ ID NO: 286; and an LCDR3 amino acid sequence of SEQ ID NO:

288. A set of nucleic acid molecules comprising:

2. The set of nucleic acid molecules described in claim 1, wherein the antibody 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.

3. A set of nucleic acid molecules described in claim 2, wherein the antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 66 / 74, 18 / 26, and 146 / 154.

4. A set of nucleic acid molecules described in claim 3, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 66 / 74.

5. A set of nucleic acid molecules described in claim 3, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 18 / 26.

6. A set of nucleic acid molecules described in claim 3, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 146 / 154.

7. A vector comprising a set of nucleic acid molecules described in claim 1 or claim 2.

8. A composition comprising a first vector containing a first nucleic acid molecule described in claim 1 and a second vector containing a second nucleic acid molecule described in claim 1.

9. A cell comprising the vector described in claim 7 or the composition described in claim 8.

10. A first nucleic acid molecule comprising a polynucleotide sequence encoding a heavy chain variable region (HCVR) of an antibody that specifically binds to Ebola virus (EBOV) and / or Ebola virus glycoprotein (EBOV-GP); a second nucleic acid molecule comprising a polynucleotide sequence encoding the light chain variable region (LCVR) of said antibody; a cell comprising the antibody (i) a heavy chain complementarity determining region (HCDR) 1 amino acid sequence of SEQ ID NO: 20; a HCDR2 amino acid sequence of SEQ ID NO: 22; a HCDR3 amino acid sequence of SEQ ID NO: 24; a light chain complementarity determining region (LCDR) 1 amino acid sequence of SEQ ID NO: 28; a LCDR2 amino acid sequence of SEQ ID NO: 30; and a LCDR3 amino acid sequence of SEQ ID NO: 32; (ii) an HCDR1 amino acid sequence of SEQ ID NO: 68; an HCDR2 amino acid sequence of SEQ ID NO: 70; an HCDR3 amino acid sequence of SEQ ID NO: 72; an LCDR1 amino acid sequence of SEQ ID NO: 76; an LCDR2 amino acid sequence of SEQ ID NO: 78; and an LCDR3 amino acid sequence of SEQ ID NO: 80; (iii) an HCDR1 amino acid sequence of SEQ ID NO: 148; an HCDR2 amino acid sequence of SEQ ID NO: 150; an HCDR3 amino acid sequence of SEQ ID NO: 152; an LCDR1 amino acid sequence of SEQ ID NO: 156; an LCDR2 amino acid sequence of SEQ ID NO: 158; and an LCDR3 amino acid sequence of SEQ ID NO: 160; (iv) an HCDR1 amino acid sequence of SEQ ID NO: 4; an HCDR2 amino acid sequence of SEQ ID NO: 6; an HCDR3 amino acid sequence of SEQ ID NO: 8; an LCDR1 amino acid sequence of SEQ ID NO: 12; an LCDR2 amino acid sequence of SEQ ID NO: 14; and an LCDR3 amino acid sequence of SEQ ID NO: 16; (v) an HCDR1 amino acid sequence of SEQ ID NO: 36; an HCDR2 amino acid sequence of SEQ ID NO: 38; an HCDR3 amino acid sequence of SEQ ID NO: 40; an LCDR1 amino acid sequence of SEQ ID NO: 44; an LCDR2 amino acid sequence of SEQ ID NO: 46; and an LCDR3 amino acid sequence of SEQ ID NO: 48; (vi) an HCDR1 amino acid sequence of SEQ ID NO: 52; an HCDR2 amino acid sequence of SEQ ID NO: 54; an HCDR3 amino acid sequence of SEQ ID NO: 56; an LCDR1 amino acid sequence of SEQ ID NO: 60; an LCDR2 amino acid sequence of SEQ ID NO: 62; and an LCDR3 amino acid sequence of SEQ ID NO: 64; (vii) an HCDR1 amino acid sequence of SEQ ID NO: 84; an HCDR2 amino acid sequence of SEQ ID NO: 86; an HCDR3 amino acid sequence of SEQ ID NO: 88; an LCDR1 amino acid sequence of SEQ ID NO: 92; an LCDR2 amino acid sequence of SEQ ID NO: 94; and an LCDR3 amino acid sequence of SEQ ID NO: 96; (viii) an HCDR1 amino acid sequence of SEQ ID NO: 100; an HCDR2 amino acid sequence of SEQ ID NO: 102; an HCDR3 amino acid sequence of SEQ ID NO: 104; an LCDR1 amino acid sequence of SEQ ID NO: 108; an LCDR2 amino acid sequence of SEQ ID NO: 110; and an LCDR3 amino acid sequence of SEQ ID NO: 112; (ix) an HCDR1 amino acid sequence of SEQ ID NO: 116; an HCDR2 amino acid sequence of SEQ ID NO: 118; an HCDR3 amino acid sequence of SEQ ID NO: 120; an LCDR1 amino acid sequence of SEQ ID NO: 124; an LCDR2 amino acid sequence of SEQ ID NO: 126; and an LCDR3 amino acid sequence of SEQ ID NO: 128; (x) an HCDR1 amino acid sequence of SEQ ID NO: 132; an HCDR2 amino acid sequence of SEQ ID NO: 134; an HCDR3 amino acid sequence of SEQ ID NO: 136; an LCDR1 amino acid sequence of SEQ ID NO: 140; an LCDR2 amino acid sequence of SEQ ID NO: 142; and an LCDR3 amino acid sequence of SEQ ID NO: 144; (xi) an HCDR1 amino acid sequence of SEQ ID NO: 164; an HCDR2 amino acid sequence of SEQ ID NO: 166; an HCDR3 amino acid sequence of SEQ ID NO: 168; an LCDR1 amino acid sequence of SEQ ID NO: 172; an LCDR2 amino acid sequence of SEQ ID NO: 174; and an LCDR3 amino acid sequence of SEQ ID NO: 176; (xii) an HCDR1 amino acid sequence of SEQ ID NO: 180; an HCDR2 amino acid sequence of SEQ ID NO: 182; an HCDR3 amino acid sequence of SEQ ID NO: 184; an LCDR1 amino acid sequence of SEQ ID NO: 188; an LCDR2 amino acid sequence of SEQ ID NO: 190; and an LCDR3 amino acid sequence of SEQ ID NO: 192; (xiii) an HCDR1 amino acid sequence of SEQ ID NO: 196; an HCDR2 amino acid sequence of SEQ ID NO: 198; an HCDR3 amino acid sequence of SEQ ID NO: 200; an LCDR1 amino acid sequence of SEQ ID NO: 204; an LCDR2 amino acid sequence of SEQ ID NO: 206; and an LCDR3 amino acid sequence of SEQ ID NO: 208; (xiv) an HCDR1 amino acid sequence of SEQ ID NO: 212; an HCDR2 amino acid sequence of SEQ ID NO: 214; an HCDR3 amino acid sequence of SEQ ID NO: 216; an LCDR1 amino acid sequence of SEQ ID NO: 220; an LCDR2 amino acid sequence of SEQ ID NO: 222; and an LCDR3 amino acid sequence of SEQ ID NO: 224; (xv) an HCDR1 amino acid sequence of SEQ ID NO: 228; an HCDR2 amino acid sequence of SEQ ID NO: 230; an HCDR3 amino acid sequence of SEQ ID NO: 232; an LCDR1 amino acid sequence of SEQ ID NO: 236; an LCDR2 amino acid sequence of SEQ ID NO: 238; and an LCDR3 amino acid sequence of SEQ ID NO: 240; (xvi) an HCDR1 amino acid sequence of SEQ ID NO: 244; an HCDR2 amino acid sequence of SEQ ID NO: 246; an HCDR3 amino acid sequence of SEQ ID NO: 248; an LCDR1 amino acid sequence of SEQ ID NO: 252; an LCDR2 amino acid sequence of SEQ ID NO: 254; and an LCDR3 amino acid sequence of SEQ ID NO: 256; (xvii) an HCDR1 amino acid sequence of SEQ ID NO: 260; an HCDR2 amino acid sequence of SEQ ID NO: 262; an HCDR3 amino acid sequence of SEQ ID NO: 264; an LCDR1 amino acid sequence of SEQ ID NO: 268; an LCDR2 amino acid sequence of SEQ ID NO: 270; and an LCDR3 amino acid sequence of SEQ ID NO: 272; (xviii) an HCDR1 amino acid sequence of SEQ ID NO: 276; an HCDR2 amino acid sequence of SEQ ID NO: 278; an HCDR3 amino acid sequence of SEQ ID NO: 280; an LCDR1 amino acid sequence of SEQ ID NO: 284; an LCDR2 amino acid sequence of SEQ ID NO: 286; and an LCDR3 amino acid sequence of SEQ ID NO: 288; (xix) an HCDR1 amino acid sequence of SEQ ID NO: 292; an HCDR2 amino acid sequence of SEQ ID NO: 294; an HCDR3 amino acid sequence of SEQ ID NO: 296; an LCDR1 amino acid sequence of SEQ ID NO: 300; an LCDR2 amino acid sequence of SEQ ID NO: 302; and an LCDR3 amino acid sequence of SEQ ID NO: 304; or (xx) an HCDR1 amino acid sequence of SEQ ID NO: 308; an HCDR2 amino acid sequence of SEQ ID NO: 310; an HCDR3 amino acid sequence of SEQ ID NO: 312; an LCDR1 amino acid sequence of SEQ ID NO: 284; an LCDR2 amino acid sequence of SEQ ID NO: 286; and an LCDR3 amino acid sequence of SEQ ID NO:

288. including, cells.

11. The cell described in claim 10, wherein the antibody 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.

12. The cell described in claim 11, wherein the antibody comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 66 / 74, 18 / 26, and 146 / 154.

13. The cell described in claim 12, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 66 / 74.

14. The cell described in claim 12, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 18 / 26.

15. The cell described in claim 12, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of sequence numbers 146 / 154.

16. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to EBOV and / or EBOV-GP, the method comprising culturing a cell described in any one of claims 10 to 15 under conditions that allow the production of the antibody or antigen-binding fragment thereof, and optionally recovering the produced antibody or antigen-binding fragment thereof.

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