WO2016131128A1 - Anticorps humanisés contre ebola et utilisations de ces derniers - Google Patents

Anticorps humanisés contre ebola et utilisations de ces derniers Download PDF

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WO2016131128A1
WO2016131128A1 PCT/CA2016/000061 CA2016000061W WO2016131128A1 WO 2016131128 A1 WO2016131128 A1 WO 2016131128A1 CA 2016000061 W CA2016000061 W CA 2016000061W WO 2016131128 A1 WO2016131128 A1 WO 2016131128A1
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amino acid
acid sequence
seq
sequence
nos
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Darrell JOHNSTONE
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Cangene Corp
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Cangene Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to Ebolavirus (EBOV), compositions comprising the antibody or antigen-binding portion thereof, methods of producing the antibody or antigen-binding portion thereof and methods of using the antibody or antigen-binding portion thereof.
  • EBOV Ebolavirus
  • the Ebolavirus is a pleomorphic filamentous virus in the Filoviridae family. Infection with EBOV usually causes a severe hemorrhagic fever, with 50-90% lethality. The outbreak frequency of EBOV has also increased recently.
  • RNA genome of EBOV encodes seven genes.
  • the fourth gene, GP actually encodes two unique proteins: a non-structural, dimeric and secreted glycoprotein (called sGP), and a trimeric, virion-attached, membrane embedded envelope glycoprotein, termed GP.
  • sGP non-structural, dimeric and secreted glycoprotein
  • GP trimeric, virion-attached, membrane embedded envelope glycoprotein
  • These glycoproteins share the first 295 amino acids, but have unique C termini as a result of transcriptional editing. The unique C termini result in different patterns of disulfide bonding and different structures as well as different roles in pathogenesis.
  • sGP non-structural, dimeric and secreted glycoprotein
  • GP trimeric, virion-attached, membrane embedded envelope glycoprotein
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds to Ebolavirus (EBOV).
  • the antibody or antigen-binding portion thereof that binds to Ebolavirus is an isolated antibody or antigen-binding portion thereof comprising: (a) a light chain CDR1 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ID NO: 6, 30, 54, 78, 102, 126, 150, 174 or 198; (b) a light chain CDR2 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ID NO: 7, 31 , 55, 79, 103, 127, 151, 175, or 199; (c) a light chain CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ID NO: 8, 32, 56, 80, 104, 128, 152, 176, or 200; (d) a heavy chain
  • the isolated antibody or antigen-binding portion thereof comprises: (a) a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ID NOs: 6, 7 and 8, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ED NOs: 18, 19 and 20, respectively; a light chain FR1, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ID NOs: 268, 269, 270, and 271 , respectively; and a heavy chain FR1 , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence comprising SEQ ED NOs: 272, 273, 274 and 275, respectively; (b) a light chain CDR1, C
  • the antibody or antigen-binding portion thereof that binds to Ebolavirus is an isolated antibody or antigen-binding portion thereof comprising: (a) a light chain CDR1 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 3, 27, 51, 75, 99, 123, 147, 171 or 195; (b) a light chain CDR2 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 4, 28, 52, 76, 100, 124, 148, 172, or 196; (c) a light chain CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 5, 29, 53, 77, 101 , 125, 149, 173, or 197; (d)
  • the isolated antibody or antigen-binding portion thereof comprises: (a) a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NOs: 3, 4, and 5, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NOs: 15, 16, and 17, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NOs: 9, 10, 11, and 12, respectively; and a heavy chain FRl , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising S
  • the antibody or antigen-binding portion thereof comprises: (a) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181, 205, 340, 352, or 364; and (b) a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 1 , 25, 49, 73, 97, 121 , 145, 169, or 193.
  • the antibody or antigen-binding portion thereof comprises: (a) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 13 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 1 ; (b) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 37 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 25; (c) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to an amino acid sequence encoded by a nucleotide sequence comprising SEQ ID NO: 61 and a
  • the isolated antibody or antigen-binding portion thereof that binds to Ebolavirus comprises: (a) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 14, 38, 62, 86, 1 10, 134, 158, 182, 206, 341, 353, or 365; and (b) a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 2, 26, 50, 74, 98, 122, 146, 170 or 194.
  • the isolated antibody or antigen-binding portion thereof comprises: (a) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 14 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 2; (b) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 38 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 26; (c) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 62 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 50; (d) a variable heavy chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO: 86 and a variable light chain comprising an amino acid sequence that has at least about 98% sequence identity to SEQ ID NO:
  • the isolated antibody or antigen-binding portion thereof comprises: (a) a variable heavy chain comprising an amino acid sequence comprising SEQ ID NO: 14 and a variable light chain comprising an amino acid sequence comprising SEQ ID NO: 2; (b) a variable heavy chain comprising an amino acid sequence comprising SEQ ID NO: 38 and a variable light chain comprising an amino acid sequence comprising SEQ ID NO: 26; (c) a variable heavy chain comprising an amino acid sequence comprising SEQ ID NO: 62 and a variable light chain comprising an amino acid sequence comprising SEQ ID NO: 50; (d) a variable heavy chain comprising an amino acid sequence comprising SEQ ID NO: 86 and a variable light chain comprising an amino acid sequence comprising SEQ ID NO: 74; (e) a variable heavy chain comprising an amino acid sequence comprising SEQ ID NO: 110 and a variable light chain comprising an amino acid sequence comprising SEQ ID NO: 98; (f) a variable heavy chain comprising an amino acid
  • the isolated antibody or antigen-binding portion thereof disclosed herein can be a whole immunoglobulin, an scFv, a Fab fragment, an F(ab')2, or a disulfide linked Fv.
  • the isolated antibody of antigen-binding portion thereof binds to the GP subunit of the Ebolavirus. In some embodiments, the antibody or antigen- binding portion thereof binds to the mucin domain of the GP subunit of the Ebolavirus.
  • the Ebolavirus can be Zaire ebolavirus, Sudan ebolavirus, Reston ebolavirus, Tai Forest ebolavirus, or Bundibug o ebolavirus. In some embodiments, the Ebolavirus is Cote d'llude ebolavirus.
  • nucleic acid sequence encoding an antibody or antigen- binding portion thereof disclosed herein.
  • present disclosure also provides an expression vector comprising a promoter operably linked to a nucleotide sequence disclosed herein, such as a nucleic acid sequence encoding an antibody or antigen-binding portion thereof disclosed herein.
  • the expression vector comprises a nucleotide sequence with the following sequences: (a) SEQ ID NOs: 3, 4, 5, 9, 10, 11 , 12, 15, 16, 17, 21 , 22, 23, and 24; (b) SEQ ID NOs: 27, 28, 29, 33, 34, 35, 36, 39, 40, 41 , 45, 46, 47, and 48; (c) SEQ ID NOs: 51, 52, 53, 57, 58, 59, 60, 63, 64, ,65, 69, 70, 71 , and 72; (d) SEQ ID NOs: 75, 76, 77, 81 , 82, 83, 84, 87, 88, 89, 93, 94, 95, and 96; (e) SEQ ID NOs: 99, 100, 101 , 105, 106, 107, 108, 111, 112, 113, 117, 118, 119, and 120, (f) SEQ ID NOs: 123, 124, 125, 129,
  • the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs. 1 , 13, 25, 37, 49, 61 , 73, 85, 97, 109, 121, 133, 145, 157, 169, 181 , 193, 205, 340, 352, and 364.
  • the expression vector comprises a nucleotide sequence according to SEQ ID NO: 1, 25, 49, 73, 97, 121 , 145, 169, or 193.
  • the expression vector comprises a nucleotide sequence according to SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181 , 205, 340, 352, or 364.
  • the expression vector comprises nucleotide sequences with the following sequences: (a) SEQ ID NOs: 1 and 13; (b) SEQ ID NOs: 25 and 37; (c) SEQ ID NOs: 49 and 61 ; (d) SEQ ID NOs: 73 and 85; (e) SEQ ID NOs: 97 and 109; (f) SEQ ID NOs: 121 and 133; (g) SEQ ID NOs: 145 and 157; (h) SEQ ID NOs: 169 and 181; (i) SEQ ID NOs: 193 and 205; (j) SEQ ID NOs: 73 and 340; (k) SEQ ID NOs: 73 and 352; or (1) SEQ ID NOs: 73 and 364.
  • a host cell comprising an expression vector disclosed herein.
  • the cell is a bacterial, eukaryotic or mammalian cell.
  • the cell can be a COS-1 , COS-7, HEK293, BHK21, CHO, BSC-1 , HepG2, SP2/0, HeLa, myeloma or lymphoma cell.
  • the present disclosure also provides a method of producing an antibody or antigen-binding portion thereof that binds to Ebolavirus disclosed herein.
  • the method comprises culturing a host cell, such as one disclosed herein, and recovering the antibody or antigen-binding portion thereof.
  • compositions comprising an antibody or antigen-binding portion thereof disclosed herein.
  • the composition is a pharmaceutical composition comprising an antibody or antigen-binding portion thereof.
  • the pharmaceutical composition can further comprise a pharmaceutically acceptable carrier.
  • the composition comprises an antibody or antigen-binding portion thereof disclosed herein and one or more other antibodies or antigen-binding portions thereof.
  • the one or more other antibodies or antigen-binding portions thereof can bind a protein produced by a virus in the Filoviridae family.
  • the protein is a glycoprotein.
  • the virus can be Ebolavirus or Marburgvirus .
  • the virus is Zaire ebolavirus, Sudan ebolavirus, Reston ebolavirus, Tai Forest ebolavirus, Cote d 'Iretebolavirus, Bundibugyo ebolavirus, Marburg virus or Ravn virus.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • a method for reducing, treating or preventing an Ebolavirus infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody or antigen- binding portion thereof disclosed herein is provided.
  • a method for reducing, treating or preventing an Ebolavirus infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a composition disclosed herein.
  • the subject is a human.
  • Methods for detecting Ebolavirus in a sample comprises contacting the sample with an antibody or antigen- binding portion thereof disclosed herein.
  • the sample is a cell, tissue, or biological fluid from a subject suspected of having or at risk of a filovirus infection.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to Ebolavirus (EBOV), compositions comprising the antibody or antigen-binding portion thereof, methods of producing the antibody or antigen-binding portion thereof and methods of using the antibody or antigen-binding portion thereof, which are described in further detail below.
  • EBOV Ebolavirus
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated.
  • the use of the alternative should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the terms “include” and “comprise” are used synonymously.
  • an antibody that binds to Ebolavirus (EBOV) is used interchangeably with “an anti-EBOV antibody.”
  • An anti-EBOV antibody refers to a monoclonal or recombinant antibody or antibody fragment that binds to EBOV with specificity.
  • the antibody can be human, humanized or chimeric.
  • An anti-EBOV antibody or antigen-binding portion thereof includes any antibody or substance having a binding domain with the required specificity.
  • an anti-EBOV antibody or antigen-binding portion thereof includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic.
  • a humanized antibody may be a modified antibody having the variable regions of a non-human, e.g., murine, antibody and the constant region of a human antibody.
  • a humanized antibody may also be a modified antibody having the variable regions of a non- human, e.g., murine, antibody and the framework region(s) of a human antibody.
  • the term "humanized” refers to a process of making an antibody or immunoglobulin binding proteins and polypeptides derived from a non-human species ⁇ e.g., mouse or rat) less immunogenic to humans, while still retaining antigen -binding properties of the original antibody, using genetic engineering techniques.
  • the binding domain(s) of an antibody or immunoglobulin binding proteins and polypeptides e.g., light and heavy chain variable regions, Fab, scFv
  • Fab, scFv are humanized.
  • other regions of the antibody or immunoglobulin binding proteins and polypeptides, such as the hinge region and constant region domains can also be humanized.
  • variable region also referred to as “light chain variable domain” or “VL” or VL
  • heavy chain variable region also referred to as “heavy chain variable domain” or “VH” or VH
  • the variable binding regions are made up of discrete, well- defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). In one embodiment, the FRs are humanized.
  • CDRs complementarity determining regions
  • FRs framework regions
  • the FRs are humanized.
  • CL refers to an "immunoglobulin light chain constant region” or a "light chain constant region,” i.e., a constant region from an antibody light chain.
  • CH refers to an "immunoglobulin heavy chain constant region" or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CHI , CH2, and CH3 (IgA, IgD, IgG), or CHI , CH2, CH3, and CH4 domains (IgE, IgM).
  • a "Fab” fragment antigen binding is the part of an antibody that binds to antigens and includes the variable region and CHI domain of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • the six “complementarity determining regions" or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the binding domain referred to as “framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).
  • an antibody, or antigen-binding fragment thereof contains at least one heavy chain variable region and/or at least one light chain variable region.
  • the heavy chain variable region typically contains three CDRs and four framework regions (FRs), arranged from amino- terminus to carboxyl-terminus in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • the Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Nevertheless, application of either definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated.
  • the appropriate amino acids which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody.
  • CDRs can also be determined using IMGT® (the international ImMunoGeneTics information system®) numbering.
  • H heavy chain
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat numbering refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure.
  • antigen-binding portion refers to the domain, region, portion, or site of a protein, polypeptide, oligopeptide, or peptide or antibody or binding domain derived from an antibody that possesses the ability to specifically recognize and bind to an antigen.
  • exemplary binding antigen-binding portions include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab).
  • the binding domain comprises or consists of an antigen binding site (e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
  • an antigen binding site e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
  • FRs alternative framework regions
  • An antibody or antigen-binding portion "specifically binds" an antigen if it binds the antigen with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M "1 , while not significantly binding other components present in a test sample.
  • Ka i.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • the antibody or antigen-binding portion can be classified as "high affinity” or “low affinity.”
  • “High affinity” refer to those antibodies or antigen-binding portions with a Ka of at least about 10 7 M “1 , at least about 10 8 M '1 , at least about 10 9 M '1 , at least about 10 10 M “1 , at least about 10" M '1 , at least about lO 12 M “1 , or at least about 10 13 M “! .
  • “Low affinity” r refer to those antibodies or antigen-binding portions with a Ka of up to 10 7 M “1 , up to 10 6 M “1 , up to 10 5 M "1 .
  • affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 "5 M to 10 '13 M).
  • Kd equilibrium dissociation constant
  • Affinities of refer to those antibodies or antigen-binding portions according to the present disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
  • reference antibody refers to an antibody that is known in the art and which serves the basis for a humanized, chimeric or recombinant antibody.
  • the reference antibody may be a non-human, (e.g. , murine), human, humanized, chimeric and / or recombinant antibody or antibody-like polypeptide.
  • Treatment refers to either a therapeutic treatment or prophylactic/preventative treatment.
  • a therapeutic treatment may improve at least one symptom of disease in an individual receiving treatment or may delay worsening of a progressive disease in an individual, or prevent onset of additional associated diseases.
  • Ameliorating or reducing or reduction of infection can include but is not limited to delaying the onset of the infection, attenuating the symptoms of the infection, shortening the duration of the infection, reducing the viral titer in a patient (eg. in the blood), or slowing the progression of the infection.
  • Filovirus infections encompassed by the present application include, but are not limited to, Marburgvirus and Ebolavirus.
  • a “therapeutically effective amount,” “therapeutically effective dose” or “effective dose” refers to that amount of the antibody or compound sufficient to result in amelioration of one or more symptoms of the disease being treated.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered serially or simultaneously.
  • One or more specific therapeutic molecules may be administered according to methods of the invention, each in an effective dose. The effective dose can be determined empirically through dose studies.
  • terapéuticaally effective amount refers to an amount that prevents infection with EBOV, prevents disease associated with EBOV infection, reduces the number and/or severity of symptoms of an EBOV infection, stops or limits the spread of EBOV, and/or shortens the duration of an EBOV infection.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.”
  • the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form.
  • nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleic acid As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., mRNA
  • analogs of the DNA or RNA generated using nucleotide analogs e.g., mRNA
  • expression vector refers to a nucleic acid molecule, linear or circular, comprising one or more expression units.
  • an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers.
  • Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.
  • sequence identity refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of "identical” positions.
  • the number of "identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity.” Percentage of "sequence identity” is determined by comparing two optimally aligned sequences over a comparison window.
  • the comparison window for nucleic acid sequences can be, for instance, at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length.
  • the comparison window for polypeptide sequences can be, for instance, at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length.
  • the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant.
  • An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" positions between the reference and comparator sequences.
  • Sequence identity between two sequences can be determined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of September 1 , 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873- 5877, 1993).
  • BLASTN for nucleotide sequence comparison
  • BLASTP for polypeptide sequence comparison
  • Two nucleotide or amino acid sequences are considered to have "substantially similar sequence identity” or “substantial sequence identity” if the two sequences have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity relative to each other.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to Ebolavirus (EBOV), compositions comprising the antibody or antigen-binding portion thereof, methods of producing the antibody or antigen-binding portion thereof and methods of using the antibody or antigen-binding portion thereof.
  • EBOV Ebolavirus
  • an anti-EBOV antibody or antigen- binding portion thereof include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH domains; (ii) the Fd fragment consisting of the VH and CH domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody (e.g., linked by a disulfide bond); (iv) the dAb fragment (Ward, E. S.
  • an "anti-EBOV antibody” is a recombinant anti-EBOV antibody or polypeptide.
  • Recombinant anti-EBOV antibodies and polypeptides include, for instance, Fc fusions, toxin fusions, fusions to enzymatic activities, minibodies, diabodies, linear antibodies, single chain antibodies, bispecific antibody fragments, scFv and Fab fragments.
  • a recombinant anti-EBOV antibody includes a molecule or polypeptide that incorporates an amino acid sequence derived from an anti-EBOV antibody and which is capable of binding EBOV with specificity.
  • Recombinant anti-EBOV antibodies include molecules that are optimized, for instance, for stability, solubility, in vitro and in vivo binding.
  • an anti-EBOV antibody is a diabody.
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associated with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer. See WO94/13804 which is incorporated by reference in its entirety.
  • an anti-EBOV antibody is a scFv.
  • a scFv is constructed by joining a variable heavy chain and a variable light chain with a linker using recombinant methods. The linker that enables the VH and VL regions to be made as a single chain protein. See, for instance, Bird et al, 1988, Science 242:423-426 and Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883.
  • the scFv comprises VH and VL regions that are identical or derived from a reference anti-EBOV antibody.
  • an anti-EBOV antibody or antigen-binding portion thereof is an Fv.
  • An Fv is an antibody fragment which contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • the six CDRs confer antigen-binding specificity to the antibody, However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fv comprises VH and VL regions that are identical or derived from a reference anti-EBOV antibody.
  • an anti-EBOV antibody is a single chain polypeptide comprising, from amino to carboxyl terminus, a binding domain ⁇ e.g. , scFv), an immunoglobulin hinge region and an immunoglobulin constant region.
  • a binding domain ⁇ e.g. , scFv
  • an immunoglobulin hinge region ⁇ e.g. , an immunoglobulin constant region.
  • an immunoglobulin constant region also known as a small modular immunopharmaceutical (SMIP)
  • SMIP small modular immunopharmaceutical
  • the anti-EBOV antibody or antigen-binding portion thereof can be a recombinant polypeptide, fusion protein or immunoconjugate that binds EBOV and comprise an antibody fragment or are derived in part from a monoclonal or polyclonal anti-EBOV antibody.
  • an anti-EBOV antibody or antigen-binding portion thereof is a molecule or polypeptide that is derived from a reference anti-EBOV antibody and is capable of binding with specificity to the same epitope as the reference anti-EBOV antibody.
  • the epitope is on the GP subunit of EBOV.
  • the anti- EBOV antibody or antigen-binding portion thereof binds to the GP subunit of the Ebolavirus.
  • the GP subunit can be of the Zaire ebolavirus, Sudan ebolavirus, Reston ebolavirus, Tai Forest ebolavirus, or Bundibugyo ebolavirus.
  • An anti-EBOV derived from a reference anti-EBOV antibody can include a molecule or polypeptide comprising at least about 10 contiguous amino acids, at least about 20 contiguous amino acids or at least about 50 or more contiguous amino acids as the reference anti-EBOV antibody.
  • an anti-EBOV antibody or antigen-binding portion thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1 , light chain CDR2, and/or light chain CDR3 with the same amino acid sequence as a reference anti-EBOV antibody.
  • an anti-EBOV antibody or antigen-binding portion thereof comprises a heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and/or light chain CDR3 that has an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of the respective heavy chain CDRl, heavy chain CDR2, heavy chain CDR3, light chain CDRl, light chain CDR2, and/or light chain CDR3 of a reference anti-EBOV antibody.
  • an anti-EBOV antibody or antigen-binding portion thereof comprises a VH and/or VL with the same amino acid sequence as a reference anti- EBOV molecule.
  • an anti-EBOV antibody or antigen-binding portion thereof comprises a VH and/or VL that has an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence of the respective VH and/or VL of a reference anti-EBOV molecule.
  • the reference anti-EBOV molecule is a murine anti-EBOV antibody.
  • the reference anti-EBOV molecule is the murine anti-EBOV antibody CAN7G1.
  • the reference anti-EBOV molecule is the murine anti-EBOV antibody CAN8G1.
  • the reference anti-EBOV molecule is the murine anti-EBOV antibody CAN9G1.
  • the CDRs, VHs and VLs of these murine antibodies are provided in Table 2.
  • variable sequence WFRQPPG GLEWLAHIWWTDD YYNPALKSRLTI region S DTSNNQVVLTMTNMDPVDTATYYCARIGYDGP
  • variable sequence QQQPE GPRYLM L KDGSHSKGDGIPDRFSGSSS region GAERYLTISSLQSEDEADYYCGVGDTIKEQFVYVF
  • variable sequence QQQPLKAP YVMELK DGSHSTGDGIPDRFSGSSS region GADRYLWISNIQPEDEAMYICGVGDTI EQFVYVF
  • Variable 341 sequence SKDTSNNQVVLTMTNMDPVDTATYYCARIGYDGP region
  • an anti-EBOV antibody or antigen-binding portion thereof is a humanized antibody of a murine anti-EBOV antibody.
  • the humanized anti-EBOV antibody or antigen-binding fragment thereof can comprise one or more CDRs of a murine anti-EBOV antibody and one or more human FRs.
  • the humanized anti- EBOV antibody or antigen-binding fragment thereof comprises one or more CDRs of the murine anti-EBOV antibody CAN7G1 , CAN8G1, or CAN9G1.
  • the humanized anti-EBOV antibody or antigen-binding fragment thereof comprises one or more human FRs from Table 2.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a light chain CDR1 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 6, 30, 54, 78, 102, 126, 150, 174 or 198; a light chain CDR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 7, 31, 55, 79, 103, 127, 151 , 175, or 199; a light chain CDR3 comprising an amino acid sequence comprising
  • a humanized anti-EBOV antibody or antigen-binding portion thereof further comprises a light chain FRl comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 268, 276, 284, 292, 300, 308, 316, 324, or 332; a light chain FR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • a light chain FR3 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 270, 278, 286, 294, 302, 310, 318, 326, or 334; a light chain FR4 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • a heavy chain FR1 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NOs: 272, 280, 288, 296, 304, 312, 320, 328, 336, 376, 380, or 384; a heavy chain FR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 273, 281, 289, 2
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises: a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 6, 7 and 8, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 18, 19 and 20, respectively; a light chain FR1, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%
  • a humanized anti-EBOV antibody comprises a light chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 30, 31 , and 32, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 42, 43, and 44, respectively; a light chain FR1 , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%, 8
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 54, 55, and 56, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 66, 67, and 68, respectively; a light chain FR1 , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%,
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 78, 79 and 80, respectively; a heavy chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 90, 91 and 92, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%, 87%
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 102, 103, and 104, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 114, 1 15, and 116, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%
  • a humanized anti-EBOV antibody comprises a light chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 126, 127, and 128, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 138, 139, and 140, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%
  • the humanized anti-EBOV antibody comprises a light chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 150, 151, and 152, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 162, 163, and 164, respectively; a light chain FRl , FR2, FR3, and FR4 comprismg an amino acid sequence that has at least about 85%, 86%
  • a humanized anti-EBOV antibody comprises a light chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 174, 175, and 176, respectively; a heavy chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 186, 187, and 188, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%,
  • a humanized anti-EBOV antibody comprises a light chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 198, 199 and 200, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 210, 21 1 , and 212, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about
  • a humanized anti-EBOV antibody comprises a light chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ E) NOs: 78, 79, and 80, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 345, 346, and 347, respectively; a light chain FRl , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 78, 79, and 80, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 357, 358, and 359, respectively; a light chain FRl , FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 8
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 78, 79, and 80, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence comprising SEQ ID NOs: 369, 370, and 371, respectively; a light chain FRl, FR2, FR3, and FR4 comprising an amino acid sequence that has at least about 85%, 86%,
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ED NO: 14, 38, 62, 86, 1 10, 134, 158, 182, 206, 341 , 353, or 365; and/or a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 26, 50, 74, 98, 122, 146, 170 or 194.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof can comprise a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 62 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 50.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 86 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 10 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 98.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 134 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 158 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 146.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 182 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 170.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 206 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 194.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 341 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 353 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%o, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 365 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 74.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a light chain CDR1 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 3, 27, 51 , 75, 99, 123, 147, 171 or 195; a light chain CDR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 4, 28, 52, 76, 100, 124, 148,
  • a humanized anti-EBOV antibody or antigen-binding portion thereof further comprises a light chain FR1 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ED NO: 9, 33, 57, 81 , 105, 129, 153, 177, or 201 ; a light chain FR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 10, 34, 58, 82
  • a heavy chain FR2 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 22, 46, 70, 94, 118, 142, 166, 190, 214,
  • a heavy chain FR3 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 23, 47, 71, 95, 119, 143, 167, 191 , 215,
  • a heavy chain FR4 comprising an amino acid sequence comprising a sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 24, 48, 72, 96, 120, 144, 168, 192, 216, 351, 363, or 375.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises: a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 3, 4, and 5, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 15, 16, and 17, respectively; a light chain FRl , FR2, FR3,
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 27, 28, and 29, respectively; a heavy chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 39, 40, and 41 , respectively; a light chain FRl , FR2, FR3, and FR4 comprising an amino acid sequence encoded by a
  • a humanized anti-EBOV antibody comprises a light chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 51, 52, and 53, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 63, 64, and 65, respectively; a light chain FR1, FR2, FR3 comprising an amino acid sequence
  • a humanized anti-EBOV antibody comprises a light chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 75, 76, and 77, respectively; a heavy chain CDRl , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 87, 88, and 89, respectively; a light chain FR1, FR2,
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 99, 100, and 101 , respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 111 , 1 12, and 113, respectively; a light chain FRl, FR2, FR3, and
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 123, 124 and 125,, respectively; a heavy chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 135, 136, and 137, respectively; a light chain FRl , FR2, FR3, and
  • the humanized anti-EBOV antibody comprises a light chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 147, 148 and 149, respectively; a heavy chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 159, 160, and 161 , respectively; a light chain FR1 , FR2,
  • a humanized anti-EBOV antibody comprises a light chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 171, 172, and 173, respectively; a heavy chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 183, 184, and 185, respectively; a light chain FR1 , FR2, FR3,
  • a humanized anti-EBOV antibody comprises a light chain CDRl, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 195, 196 and 197, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 207, 208, and 209, respectively; a light chain FRl , FR2,
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 75, 76, and 77, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 342, 343, and 344, respectively; a light chain FRl, FR2, FR3,
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 75, 76, and 77, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 354, 355, and 356, respectively; a light chain FRl, FR2, FR3, and
  • a humanized anti-EBOV antibody comprises a light chain CDR1, CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 75, 76, and 77, respectively; a heavy chain CDR1 , CDR2, and CDR3 comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NOs: 366, 367, and 368, respectively; a light chain FRl , FR2, FR
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181 , 205, 340, 352, or 364; and/or a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1, 25, 49, 73, 97, 121 , 145,
  • a humanized anti-EBOV antibody or antigen-binding portion thereof can comprise a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 13 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 25.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 61 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 49.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 85 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 73.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 109 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 97.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 133 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 121.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 157 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 145.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 181 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 169.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 205 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 193.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 340 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 73.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 352 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 73.
  • a humanized anti-EBOV antibody or antigen-binding portion thereof comprises a VH comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 364 and a VL comprising an amino acid sequence that has at least about 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 73
  • a multispecific molecule comprising a humanized anti-EBOV antibody or antigen-binding portion thereof disclosed herein.
  • a multispecific molecule has specificity for at least two different antigens or epitopes. While such a molecule generally binds to two antigens (i.e., bispecific molecule or antibody), the term "multispecific molecule" in the present invention encompasses an anti-EBOV antibody having specificity for two or more (such as three) antigens.
  • a multispecific molecule comprising an anti-EBOV antibody or antigen-binding portion thereof binds at least one epitope of EBOV.
  • a multispecific molecule may comprise a full length antibody or a fragment of such an antibody.
  • the anti-EBOV antibody is a scFv dimer or diabody rather than whole antibody.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites.
  • the multivalent or multispecific anti-EBOV antibody comprises two dimerized single chain polypeptides.
  • each single chain polypeptide comprises, from amino to carboxyl terminus, a first binding domain (e.g., scFv), an immunoglobulin hinge region, an immunoglobulin constant region (with or without a CHI region), a c-terminus linker, and a second binding domain.
  • the c-terminus linker may comprise, for instance, an amino acid linker derived from an amino acid sequence of an immunoglobulin hinge region (e.g., an immunoglobulin "core" hinge region) or an amino acid sequence derived from a stalk region of a type II C lectin (e.g., NKG2A, NKG2D).
  • the c-terminus linker comprises an amino acid sequence such as (A 4 S) 3 or (G4S) 3 .
  • the single chain polypeptide may also comprise a heterodimerization domain so that each single chain polypeptide dimerizes with a different single chain polypeptide such that a heterodimer is formed with up to four different binding domains.
  • the disclosure also includes nucleic acids (e.g., DNA or RNA) encoding an anti- EBOV antibody or antigen-binding portion thereof described herein.
  • Nucleic acids of the disclosure include nucleic acids having a region that is substantially identical to a polynucleotide as listed in Table 2.
  • Nucleic acids of the disclosure also include complementary nucleic acids.
  • the nucleic acid sequences provided herein can be exploited using codon optimization, degenerate sequence, silent mutations, and other DNA techniques to optimize expression in a particular host, and the present disclosure encompasses such sequence modifications.
  • Nucleic acids encoding an anti-EBOV antibody or antigen-binding portion thereof described herein can be propagated by placing the nucleic acids in a vector.
  • the choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.
  • a nucleic acid encoding an antibody or antigen-binding portion thereof disclosed herein a nucleic acid molecule encoding the polypeptide, operably linked to regulatory sequences that control transcriptional expression in an expression vector, is introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
  • the gene product encoded by a polynucleotide of the disclosure is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems.
  • the disclosure includes an expression vector comprising a nucleic acid segment, wherein the nucleic acid segment may comprise a nucleotide sequence with the following sequences: (a) SEQ ID NOs: 3, 4, 5, 9, 10, 1 1, 12, 15, 16, 17, 21 , 22, 23, and 24; (b) SEQ ID NOs: 27, 28, 29, 33, 34, 35, 36, 39, 40, 41 , 45, 46, 47, and 48; (c) SEQ ID NOs: 51 , 52, 53, 57, 58, 59, 60, 63, 64, 65, 69, 70, 71 , and 72; (d) SEQ ID NOs: 75, 76, 77, 81 , 82, 83, 84, 87, 88, 89, 93, 94, 95, and 96; (e) SEQ ID NOs: 99, 100, 101 , 105, 106, 107, 108, 111 , 112, 113, 117, 118, 119, and 120; (e) S
  • an expression vector comprises a nucleotide sequence with the following sequences: (a) SEQ ID NOs: 1 and 13; (b) SEQ ID NOs: 25 and 37; (c) SEQ ID NOs: 49 and 61 ; (d) SEQ ID NOs: 73 and 85; (e) SEQ ID NOs: 97 and 109; (f) SEQ ID NOs: 121 and 133; (g) SEQ ID NOs: 145 and 157; (h) SEQ ID NOs: 169 and 181 ; (i) SEQ ID NOs: 193 and 205; (j) SEQ ID NOs: 73 and 340; (k) SEQ ID NOs: 73 and 352; (1) SEQ ID NOs: 73 and 364; a nucleotide sequence that has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9
  • a host cell comprising an expression vector disclosed herein. Accordingly, antibodies and antigen-binding portions thereof disclosed herein can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacterial, eukaryotic or mammalian cells, such as cultured higher eukaryotic cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells. Cultured mammalian cells are suitable hosts for production of recombinant proteins for use within the present disclosure.
  • suitable mammalian host cells include COS-1 , COS-7, HEK293, BHK21 , CHO, BSC-1 , HepG2, SP2/0, HeLa, myeloma or lymphoma cell.
  • Other mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21 , BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO- Kl ; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); see also, e.g., Chasin et al, Som.
  • rat pituitary cells GH1 ; ATCC CCL82
  • HeLa S3 cells ATCC CCL2.2
  • rat hepatoma cells H-4-II-E
  • SV40- transformed monkey kidney cells COS-1 ; ATCC CRL 1650
  • murine embryonic cells NIH-3T3; ATCC CRL 1658. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, Virginia.
  • the present disclosure also provides a composition comprising an anti-EBOV antibody or antigen-binding portion thereof and one or more other antibodies or antigen- binding portions thereof, wherein the one or more other antibodies or antigen-binding portions thereof binds a protein produced by a virus in the Filoviridae family.
  • the one or more other antibodies or antigen-binding portions thereof can bind a glycoprotein, such as GP.
  • the one or more other antibodies or antigen-binding portions thereof binds Ebolavirus or Marburgvirus, such as Zaire ebolavirus, Sudan ebolavirus, Reston ebolavirus, Tai Forest ebolavirus, Bundibugyo ebolavirus, Cote d 'Irete ebolavirus, Marburg virus or Ravn virus.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • a composition comprising an anti-EBOV antibody or antigen-binding portion thereof disclosed herein with another antibody or antigen-binding portion thereof act synergistically when administered to a subject in need.
  • “synergy” or a “synergistic” response refers to an activity or improvement (e.g., prevents infection with EBOV, prevents disease associated with EBOV infection, reduces the number and/or severity of symptoms of an EBOV infection, stops or limits the spread of EBOV, and/or shortens the duration of an EBOV infection at a rate) that is greater than the sum of the effect of each therapy as a monotherapy.
  • synergy can be shown in vitro, ex vivo and in vivo.
  • the antibody or antigen-binding portion thereof may be used in the preparation of a medicament or pharmaceutical composition for administration (either therapeutic or prophylactic) to a subject in need of such treatment.
  • the medicament or pharmaceutical composition is prepared by mixing the antibody or antigen-binding portion thereof with a pharmaceutically acceptable carrier.
  • the resulting composition is pharmacologically acceptable if its administration can be tolerated by a recipient patient.
  • another aspect of the present disclosure is a pharmaceutical composition comprising an anti-EBOV antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise a pharmaceutically acceptable carrier, diluent, excipient, and/or other additives, such as water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like.
  • Additives used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present invention.
  • compositions will vary according to the route of administration selected ⁇ e.g. , solution, emulsion).
  • An appropriate composition comprising the active agent to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers [1122]
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous, oleaginous suspension, dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, vegetable oils, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the form should be sterile and must be fluid to the extent that easy syringability exists.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • compositions useful for administration may be formulated with uptake or absorption enhancers to increase their efficacy.
  • enhancers include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85: 1282-1285, 1996) and Oliyai and Stella ⁇ Ann. Rev. Pharmacol. Toxicol, 32:521-544, 1993).
  • the present disclosure provides methods for reducing, preventing, or treating an EBOV infection in a subject in need thereof.
  • a subject in need thereof includes a subject that has been infected with EBOV, is showing symptoms consistent with an EBOV infection, is exhibiting an EBOV infection, is suspected of having an EBOV infection, or is at risk of developing an EBOV infection.
  • a method of treating an EBOV infection or outbreak comprising administering a therapeutically or prophylactically effective amount of an anti- EBOV antibody or antigen-binding portion thereof to an individual in need of such treatment.
  • the antibodies or antigen-binding portion thereof may be formulated into a pharmaceutical product for providing treatment for individuals for EBOV infection, comprising a therapeutically effective amount of said antibody or antigen-binding portion.
  • an effective amount of the antibody or antigen-binding portion thereof may be formulated into a pharmaceutical product for treating an individual who has been exposed to or infected with EBOV, who is at risk of EBOV infection, or who is displaying symptoms of an EBOV infection.
  • a therapeutically effective amount can be determined by the skilled person.
  • the therapeutically effective dosage of the pharmaceutical composition can be determined readily by the skilled artisan, for example, from animal studies. In addition, human clinical studies can be performed to determine the preferred effective dose for humans by a skilled artisan. The precise dose to be employed will also depend on the route of administration.
  • the antibodies and antigen-binding portions provided herein may be administered via enteral (including without limitation oral administration and rectal administration) or parenteral (including without limitation intravenous administration, intramuscular administration, and aerosol delivery) administration.
  • enteral including without limitation oral administration and rectal administration
  • parenteral including without limitation intravenous administration, intramuscular administration, and aerosol delivery
  • Additional exemplary appropriate methods for administration of the antibodies and antigen-binding fragments provided herein include nasal, buccal, vaginal, ophthalmic, subcutaneous, intraperitoneal, intraarterial, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, sublingual, oral mucosal, bronchial, lymphatic, intra-uterine, integrated on an implantable device such as a suture or in an implantable device such as an implantable polymer, intradural, intracortical, or dermal.
  • compositions would normally be administered as pharmaceutically acceptable compositions as described herein.
  • the antibodies or antigen-binding portions thereof may be administered to the subject once per day, or in multiple doses per day. In one embodiment, the antibodies or antigen-binding portions thereof are administered to the subject until symptoms improve or resolve and/or until the subject is no longer at risk of EBOV infection.
  • the term "subject” or “patient” refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non- human primates such as chimpanzees and other apes and monkey species. Farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats (including cotton rats) and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples.
  • the terms "mammals” and “animals” are included in this definition. Both adult and newborn individuals are intended to be covered.
  • the methods and compositions provided herein are methods and compositions for treating EBOV infections in human subjects.
  • a dosage of antibody which is in the range of from about 1 ⁇ g kg body weight of individual to 1 g/kg body weight. It is of note that many factors are involved in determining what is a therapeutically effective dose or effective amount such as, for example but by no means limited to, the patient's age, weight, sex and general condition. Effective amounts may also vary according to the quality of the preparation and the severity of the infection or outbreak. Accordingly, it is noted that one of skill in the art will be able to determine what constitutes an "effective amount" based on a particular set of circumstances without undue experimentation.
  • the antibody or antigen-binding portion thereof is "protective” or “neutralizing” and accordingly on administration will hinder the spread of the virus.
  • the antibodies and antigen-binding portions thereof provided herein interfere either with viral attachment, entry and/or unpackaging once inside the cell. Accordingly, in some embodiments, administering an effective amount to an individual in need of such treatment will result in at least one of the following: reduced viral load, reduction in severity of symptoms associated with the EBOV infection, and reduced or slowed viral reproduction.
  • the antibody or antigen-binding portion thereof described herein may be used in a method for detecting EBOV in a sample suspected of containing EBOV.
  • the antibody or antigen-binding portion thereof described herein may be used in a method for diagnosing a filovirus infection.
  • Such methods are well known in the art and a wide variety of suitable methods will be readily apparent to one of skill in the art. Such methods may involve contacting the sample to be investigated with the antibody or antigen-binding fragment thereof under conditions suitable for binding, and then detecting the bound antibody or fragment.
  • the sample may be, for example, a biological sample, such as cells, tissue, biological fluid or the like or may be an environmental sample such as a soil or water sample or a food sample such as canned goods, meats and the like.
  • a biological sample such as cells, tissue, biological fluid or the like
  • an environmental sample such as a soil or water sample or a food sample such as canned goods, meats and the like.
  • Other suitable samples will be readily apparent to one of skill in the art.
  • detection antibodies must show high specificity and avidity for their antigenic target. As such, showing that a monoclonal antibody or antigen-binding fragment thereof reacts with the antigenic target derived from a highly purified or in vitro prepared sample does not guarantee that the antibody has sufficient specificity for use with biological sample. That is, the antibody must have sufficient specificity that it will not produce false positives or react with antigens from related, viruses.
  • suitable tests for determining utility as a diagnostic or as a neutralizing mAb include but are by no means limited to negative neutralization and or negative detection of a non-EBOV, or C-ELISA data showing competition of binding with the mouse mAbs that is being detected thereby showing that the mAbs can be used to show that an immune response to EBOV has occurred in patient/animal sera, meaning that they were exposed/infected (abrogation of binding by human antibodies).
  • biological material such as blood, mucus or stool with could be spiked or enriched with the virus and the monoclonal antibodies used to detect added virus in the sample, which would in turn determine limits of detection as well as other parameters of the monoclonal antibodies.
  • Biological samples from experimentally infected animals could also be used to determine the utility of the mAbs at different stages of the infection cycle.
  • At least one of the detection antibodies is mixed with a biological sample under suitable conditions to promote binding of the at least one detection antibody with the antigenic target if the antigenic target is present in the biological sample. Binding of the detection antibody to an antigenic target within the sample is then detected using means known in the art, for example, by use of a labelled secondary antibody or other means discussed herein and/or known in the art.
  • the detection antibody e.g., an anti-EBOV antibody disclosed herein
  • the detection antibody is labelled.
  • V gene sequencing for CAN9G1 was performed by first isolating RNA from the CAN9G1 parental hybridoma clonal cell line using the RNAeasy Mini Kit. A panel of specific primers for each variable gene group was used to amplify the cDNA in a single step RT-PCR reaction to generate cDNA encoding the heavy and light chain variable domains (VH and VL) of CAN9G1. The cDNAs were cloned and sequenced using standard techniques. After sequencing and identification of the variable gene, subgroup specific leader primers were designed to remove potential mutations from degenerate primers in the original primer panel to ensure sequence identity of the full variable gene sequence.
  • the cDNA sequences encoding the VL and VH of CAN9G1 are presented as SEQ ID NO: 249 and SEQ ID NO: 257, respectively, and the amino acid sequences are shown as SEQ ID NO: 250 and SEQ ID NO: 258, respectively.
  • the amino acid sequences of the three light chain complementarity determining regions LCDR1 , LCDR2, and LCDR3 are presented as SEQ ID NO: 254, SEQ ID NO: 255, and SEQ ID NO: 256, respectively, and the HCDRl , HCDR2, and HCDR3 regions are presented as SEQ ID NO: 262, SEQ ID NO: 263, and SEQ ID NO: 264, respectively.
  • V gene sequencing for the CAN8G1 parental hybridoma clonal cell line follows the same procedure as described above for CAN9G1.
  • the cDNA sequences encoding the VL and VH of CAN8G1 are presented as SEQ ID NO: 233 and SEQ ID NO: 241 , respectively, and the amino acid sequences are shown as SEQ ID NO: 234 and SEQ ID NO: 242, respectively.
  • the amino acid sequences of the three light chain complementarity determining regions LCDR1 , LCDR2, and LCDR3 are presented as SEQ ID NO: 238, SEQ ID NO: 239, and SEQ ID NO: 240, respectively, and the HCDRl , HCDR2, and HCDR3 regions are presented as SEQ ID NO: 246, SEQ ID NO: 247, and SEQ ID NO: 248, respectively.
  • V gene sequencing for the CAN7G1 parental hybridoma clonal cell line follows the same procedure as described above for CAN9G1.
  • the cDNA sequences encoding the VL and VH of CAN7G1 are presented as SEQ ID NO: 217 and SEQ ID NO: 225, respectively, and the amino acid sequences are shown as SEQ ID NO: 218 and SEQ ID NO: 226, respectively.
  • the amino acid sequences of the three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 are presented as SEQ ID NO: 222, SEQ ID NO: 223, and SEQ ID NO: 224, respectively, and the HCDRl , HCDR2, and HCDR3 regions are presented as SEQ ID NO: 230, SEQ ID NO: 231 , and SEQ ID NO: 232, respectively.
  • Human germline heavy and light chain variable domains with maximum identity alignment with the murine sequences were identified in the NCBI databases for use as identify acceptor frameworks.
  • the human germline alleles selected were IGHV3-30-15/ IGHJ4-01 (VH chain) and IGLV4-69-01/ IGLJl-01 (VK chain). These human germline alleles were identified as best matching and used as an acceptor framework for grafting the CDRs. All 6 CDRs (SEQ ID NOs: 259-261 and 251 -253) corresponding to heavy and light chains were inserted into the human framework regions to encode complementarity determining regions for heavy and light chains (SEQ ID NOs: 254-256 and 262-264).
  • the cdrCAN9Gl VL and VH regions are presented as SEQ ID NOs: 193 and 194 (nucleotide and amino acid sequences of cdrCAN9Gl VL) and SEQ ID NOs: 205 and 206 (nucleotide and amino acid sequences of cdrCAN9Gl VH).
  • Antibodies huCAN9Gl and rehuCAN9Gl "Human engineered" were generated by identifying the closest human germline allele for CAN9G1 mAbs VH and Vk, individually. These were then designed for use as acceptor frameworks, resulting in the VH and VL sequences of huCAN9G l.
  • SEQ ID NOs: 145 and 146 nucleotide and amino acid sequences of huCAN9Gl VL, respectively
  • SEQ ID NOs: 157 and 158 nucleotide and amino acid sequences of huCAN9Gl VH, respectively.
  • the rehuCAN9Gl mAb was further resurfaced by substitution(s) made on surface exposed amino acids to correspond to the adopted human frameworks without disruption of the CDRs.
  • SEQ ED NOs: 169 and 170 nucleotide and amino acid sequences of rehuCAN9Gl VL
  • SEQ ID NOs: 181 and 182 nucleotide and amino acid sequences of rehuCAN9Gl VH.
  • the humanized IgG/k versions of the CAN8G1 murine mAb were created as above for CAN9G1.
  • the human germline alleles selected were hIGHV2-5-08/hIGHJ4- 01 (VH chain) and GKV3-15-01/hIGKJ4-02(VK chain). All 6 CDRs (SEQ ID NOs: 135- 137 and 123-125) corresponding to heavy and light chains were inserted into the human framework regions to encode complementarity determining regions for heavy and light chains (SEQ ID NOs: 138-140 and 126-128).
  • the cdrCAN8Gl VL and VH regions are presented as SEQ ID NOs: 121 and 122 (nucleotide and amino acid sequences of cdrCAN8Gl VL) and SEQ ID NOs: 133 and 134 (nucleotide and amino acid sequences of cdrCAN8Gl VH).
  • the huCAN8Gl sequences are presented as SEQ ID NOs: 73 and 74 (nucleotide and amino acid sequences of huCAN8Gl VL) and SEQ ID NOs: 85 and 86 (nucleotide and amino acid sequences of huCAN8Gl VH).
  • the rehuCAN8Gl sequences are presented as SEQ ID NOs 97 and 98 (nucleotide and amino acid sequences of rehuCAN8Gl VL) and SEQ ID NOs 109 and 110 (nucleotide and amino acid sequences of rehuCAN8Gl VH).
  • the humanized IgG/k versions of the CAN8G1 murine mAb were created as above for CAN9G1.
  • the human germlme alleles selected were IGHV1-3-01/ IGHJ4-01 (VH chain) and IGKV3-11-01/ IGKJ4-01 (VK chain). All 6 CDRs (SEQ ID NOs: 63-65 and 51- 53) corresponding to heavy and light chains were inserted into the human framework regions to encode complementarity determining regions for heavy and light chains (SEQ ID NOs: 66- 68 and 54-56).
  • the cdrCAN7Gl VL and VH regions are presented as SEQ ID NOs: 49 and 50 (nucleotide and amino acid sequences of cdrCAN7Gl VL) and SEQ ID NOs: 61 and 62 (nucleotide and amino acid sequences of cdrCAN7Gl VH).
  • the huCAN7Gl sequences are presented as SEQ ID NOs: 1 and 2 (nucleotide and amino acid sequences of huCAN7Gl VL) and SEQ ID NOs: 13 and 14 (nucleotide and amino acid sequences of huCAN7Gl VH).
  • rehuCAN7Gl sequences are presented as SEQ ID NOs: 25 and 26 (nucleotide and amino acid sequences of rehuCAN7Gl VL) and SEQ ID NOs: 37 and 38 (nucleotide and amino acid sequences of rehuCAN7Gl VH).
  • SEQ ID NOs: 25 and 26 nucleotide and amino acid sequences of rehuCAN7Gl VL
  • SEQ ID NOs: 37 and 38 nucleotide and amino acid sequences of rehuCAN7Gl VH.
  • VH and VL regions for the humanized Ebola mAbs described in Example 2 are cloned into vectors for expression as full- sized humanized antibodies having human IgG constant regions.
  • the VH and VL regions of the parent murine antibody (CAN9G1 , CAN8G1 , and CAN7G1) are also cloned into human constant region vectors for expression as mouse-human chimeric antibodies.
  • Humanized Ebola mAbs were produced by transient transfections in 293F, CHO- S or CHOK1S-V cells.
  • 293F, CHO-S or CHOK1S-V cells were counted using a Haemocytometer in the presence of Trypan Blue, then passaged into transfection medium (293F cells remain in FreeStyle 293 Expression medium; CHO cells are transferred into DMEM/F12 supplemented with 10% FBS and L-Glutamine) at a concentration of 6-8x10 5 cells/ml and incubated 24 hours at 37°C, 8% CO2 and in a shaking incubator at 100 rpm.
  • Freestyle Max Transfection Agent was diluted 1/16 in Optimem before being added to 312.5 ⁇ g of the appropriate DNA also diluted in Optimem.
  • DNA/Freestyle Max Transfection Agent mix is incubated at room temperature for 20 minutes and added to 250 x 10 6 cells in FreeStyle 293 Expression Medium (no DMSO) for 293F cells or DMEM/F12 + 10% FBS + 5 mM L-Glutamine that had been treated for 3 hours with 1% DMSO for CHO cells.
  • the culture was harvested after incubation at 37°C/5% C0 2 /125 rpm in a shaking incubator by centrifuging the culture at 2500 rpm for 30 minutes, removing the supernatant and filtering it through a 0.22 ⁇ bottle top filter.
  • the supernatant was concentrated by spin cell concentrator equipped with a 50 kDa membrane to a final volume of -100 mL.
  • the concentrated supernatant was purified by Protein G purification on the FPLC or by using Protein G GraviTrap columns (manual purification system).
  • the purified sample was buffer exchanged by spin-cell concentrator equipped with a 50 kDa membrane into D-PBS and concentrated down to a final volume of 1-2 mL.
  • the final protein concentration was determined by BCA using the Pierce BCA Kit.
  • An ELISA was performed to test the binding of the humanized Ebola mAbs described in Example 3 against multiple strains of Ebola GP or derivatives thereof (GPe, GP ectodomain; GPeAmuc, GPe with mucin domain removed; GPeAmucAtm, GPe with both mucin and transmembrane domains removed) and to determine if they are cross -reactive to various strains of Marburg virus (MARV) GP or derivatives thereof (GPe, GPeAmuc, GPeAmucAtm).
  • the ELISA plate was coated with 200ng/well of antigen.
  • the wells were blocked with 5% skim milk then probed with 60 ⁇ serially diluted humanized mAbs (starting at 17.0ug/mL to 2.1ug/mL). After washing plates, binding was detected with commercial goat anti-human HRP conjugate antibody. Where appropriate, development and detection were carried out with anti-murine HRP-conjugated secondary Ab at the appropriate dilution for positive controls, murine mAbs, and negative (IgG Control mAb) controls were also run. The plate was read at 405nm after minimum 10 minutes incubation with ABTS substrate.
  • Results huCAN9Gl was tested, along with the murine versions (muCAN8Gl and muCAN9Gl) for binding by ELISA to EBOV Zaire GPe and/or EBOV Sudan GPe.
  • Table 3 lists the ELISA results in table form. The results show the binding of muCAN8Gl , muCAN9Gl and huCAN9Gl binding to EBOV Zaire GPe, as well as muCAN8Gl binding to EBOV Sudan GPe.
  • a 4-12% gradient SDS-PAGE gel is run for 1.5 hours at 200 volts with a combination of MARV and EBOV GP proteins. The gel is then transferred to a nitrocellulose membrane for a minimum of 1 hour at 45 volts. The membrane is blocked overnight at 4°C with 5% skim milk in IxTBST. The next day the humanized Ebola mAbs (l°Ab) described in Example 3 are diluted in 2.5% skim milk in IxTBST at concentrations ranging from 2 ⁇ g/mL to 5 depending on the antibody and used to probe the membrane containing the transferred proteins for 2 hours at room temperature (RT).
  • RT room temperature
  • the membranes are then washed with I TBST to remove unbound l °Ab and probed with anti-human IgG-HRP (2°Ab) at a dilution of 1 :4000 to 1 :5000 for 1.5 hours at RT. Where appropriate, development and detection are carried out with anti-murine HRP-conjugated secondary Abs at the appropriate dilution for controls.
  • VSV Vesicular stomatitis virus
  • EBOV GP Vesicular stomatitis virus
  • VSV pseudovirions containing a GFP gene in place of the VSV G gene (VSVAG) and bearing the glycoprotein of EBOV are generated as previously described (Takeda, A. et al. Proc Natl Acad Sci USA, 1997. 94(26): 14764-14769).
  • VSVAG-GP full-length EBOV GP
  • VSVAG-GPAmuc mucin-deleted ⁇ 257-425 GP
  • Pseudovirions are incubated with anti-VSV G mAb for 1 hour at RT, then incubated with 2.5, 10 or 50 ⁇ g/mL of each humanized Ebola mAb in DMEM-10%FBS for an additional hour. Pseudovirion/mAb complexes are added to Vero cells at a multiplicity of infection (MOI) of 0.01. After 48 hours, infection was evaluated by counting GFP- expressing cells.
  • MOI multiplicity of infection
  • mice are treated intraperitoneally (IP) with purified humanized Ebola mAbs described in Example 3, non-relevant IgG, or PBS alone. The mice are then challenged IP with 1000 plaque- forming units (p.f.u.) mouse-adapted EBOV. Mice are monitored for clinical signs of infections for 28 days post-exposure at which point the study ends and the mice are euthanized.
  • IP intraperitoneally
  • p.f.u. plaque- forming units

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Abstract

La présente invention concerne un anticorps, ou la partie se liant à l'antigène de ce dernier, qui se lie à Ebolavirus. L'anticorps ou la partie se liant à l'antigène de ce dernier peut posséder un ou plusieurs CDR murins et une ou plusieurs régions charpente humaines. L'invention concerne également des compositions comprenant l'anticorps ou la partie se liant à l'antigène de ce dernier, des procédés de production de l'anticorps ou de la partie se liant à l'antigène de ce dernier, et des méthodes d'utilisation de l'anticorps ou de la partie se liant à l'antigène de ce dernier.
PCT/CA2016/000061 2015-02-19 2016-02-19 Anticorps humanisés contre ebola et utilisations de ces derniers Ceased WO2016131128A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023147293A3 (fr) * 2022-01-25 2023-09-28 The Trustees Of The University Of Pennsylvania Compositions et méthodes comprenant des récepteurs antigéniques chimériques (car) anti-cd38

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018649A2 (fr) * 2002-08-23 2004-03-04 United States Army Medical Research Institute Of Infectious Diseases Anticorps monoclonaux et regions determinantes de complementarite se liant a des glycoproteine ebola
WO2009094755A1 (fr) * 2008-02-01 2009-08-06 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Anticorps monoclonaux contre les virus d'ebola et de marburg
WO2015127136A2 (fr) * 2014-02-19 2015-08-27 Jody Berry Anticorps monoclonaux anti-ebola

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018649A2 (fr) * 2002-08-23 2004-03-04 United States Army Medical Research Institute Of Infectious Diseases Anticorps monoclonaux et regions determinantes de complementarite se liant a des glycoproteine ebola
WO2009094755A1 (fr) * 2008-02-01 2009-08-06 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Health Anticorps monoclonaux contre les virus d'ebola et de marburg
WO2015127136A2 (fr) * 2014-02-19 2015-08-27 Jody Berry Anticorps monoclonaux anti-ebola

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023147293A3 (fr) * 2022-01-25 2023-09-28 The Trustees Of The University Of Pennsylvania Compositions et méthodes comprenant des récepteurs antigéniques chimériques (car) anti-cd38

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