WO2021105669A1 - Anticorps - Google Patents

Anticorps Download PDF

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Publication number
WO2021105669A1
WO2021105669A1 PCT/GB2020/052998 GB2020052998W WO2021105669A1 WO 2021105669 A1 WO2021105669 A1 WO 2021105669A1 GB 2020052998 W GB2020052998 W GB 2020052998W WO 2021105669 A1 WO2021105669 A1 WO 2021105669A1
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seq
antibody
sequence
antibodies
shows
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PCT/GB2020/052998
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Daniel John Lightwood
Victoria Louise REDGRAVE-O'DOWD
Kerry Louise Tyson
Simon John DRAPER
Francesca Rose DONNELLAN
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Oxford University Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA 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
    • A61P31/14—Antivirals for 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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 invention relates to antibodies binding to the Ebola virus glycoprotein.
  • the present invention also relates to pharmaceutical compositions comprising the antibodies and methods of preventing, ameliorating or treating an Ebola virus infection using such antibodies.
  • VIC Viral Haemorrhagic Fever Immunotherapeutic Consortium
  • case fatality rate in the ZMapp arm was 49%, whereas for patients receiving REGN-3470-3471-3479 and 114 the case fatality rates were 29% and 34% respectively.
  • case fatality rates were 29% and 34% respectively.
  • those patients who were treated soon after infection had a survival rate of 90% (Maxmen, 2019, Nature News doi: 10.1038/d41586-019-02442-6).
  • ZMapp is a cocktail of murine chimeric antibodies (cl3C6, c2G4 and c4G7), one targeting the glycan cap and two to the base of the GP (Qiu et al. 2014, Nature, 514, 47- 53).
  • An antibody of human origin, 114 (to the receptor binding region) was assessed as a monotherapy (Corti et al. 2016, Science, 351: 1339-42).
  • REGN-3470-3471-3479 is an antibody cocktail developed by Regeneron, derived from humanised mice, containing one antibody to the fusion loop, one to the head, and one glycan cap (Pascal et al. 2018, J Infect Dis, 1-15).
  • This cocktail was intentionally chosen to combine antibodies to independent epitopes with neutralisation and immune effector functions, thought to be complementary.
  • Ebola virus that cause fatal haemorrhagic ebola virus disease (EVD) in humans: alongside EBOV, Sudan Ebola virus (SUDV) and Bundibugyo Ebola virus (BDBV) have caused several outbreaks.
  • EBOV haemorrhagic ebola virus disease
  • SUDV Sudan Ebola virus
  • BDBV Bundibugyo Ebola virus
  • Tai Forest Ebola virus (TAFV) caused a single case of non-lethal EVD (Burk et al, FEMS, 2019), and
  • ADI-15946 and its matured variant ADI-23774 bind the base of the GP chalice (Wee et al, Cell Host & Microbe 2019; West et al, Nature Structural & Molecular Biology, 2019), and FVM04 which binds the crest of the receptor binding region (Howell et al, Cell Rep, 2016).
  • Cocktails of antibodies can show greater therapeutic efficacy than monotherapies by both improving overall protection and reducing severity of symptoms (Qiu et al, Nature, 2014; Howell et al, Cell Rep, 2016; Keck et al, J Virol, 2016; Wee et al, Cell Host & Microbe, 2019; Brannan et al, Nature Comm, 2019; Bornholdt et al, Cell Host & Microbe, 2019); in some cases with synergy between non-neutralising and neutralising antibodies (Howell et al, Cell Rep, 2017).
  • cocktail of antibodies can reduce transient viremia in comparison to a monotherapy even if both are protective (Corti et al, Science, 2016) and hence reduce opportunities for the virus to evolve to avoid therapeutic antibodies (Saphire and Aman, Trends in Microbiol., 2016).
  • conserveed epitopes such as those targeted by pan-Ebola virus antibodies may also incur a higher fitness cost to viral escape mutations, decreasing the chances of viral escape from antibodies targeting these regions.
  • single point mutations have been identified that abolish binding by broadly-protective antibodies without affecting viral entry into cells (Wee et al, Cell, 2017), highlighting the need for antibodies to be used in combination therapies.
  • the present invention provides an anti-Ebola virus antibody that recognises an epitope on the Ebola virus glycoprotein comprising one or more residues selected from the group consisting of F132, P133, R134, C135, R136, Y137, V138, H139, K140, V141, S142 and G143 of SEQ ID NO: 22.
  • the invention provides an antibody that binds to the same epitope as, or competes for binding with, an antibody of the invention.
  • the present invention also provides a nucleic acid or a pair of nucleic acids, optionally mRNA, encoding the heavy and light chains of an antibody of the invention, an expression vector comprising the nucleic acid(s) or a pair of expression vectors comprising the pair of nucleic acids, a host cell comprising the expression vector(s) and a method of producing an antibody of the invention, said method comprising culturing the host cell under conditions permitting production of the antibody and recovering the antibody so produced.
  • the invention provides a pharmaceutical composition comprising an antibody of the invention, or a nucleic acid/pair of nucleic acids of the invention.
  • the invention also provides a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering a composition of the invention to a subject in need thereof.
  • the invention provides a composition of the invention for use in a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the composition to a subject in need thereof and use of a composition of the invention in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
  • Figure 1 shows binding of broadly reactive monoclonal antibodies to HEK293 cells transiently transfected with GP from different Ebola virus species or irrelevant antigen (mock). Mean and range of duplicates shown. Binding confirmed in two other experiments (TAFV GP binding for mabs 11886, 11881, 11889, 11892 confirmed in i other experiment only).
  • Figure 2 shows a table of inhibitory concentration at 50% (IC50) and 90% (IC90) values for in vitro neutralisation of S-FLU viruses pseudotyped with GP from EBOV, SUDV and BDBV. Strong: neutralises >90% of virus at tested concentrations; neut: neutralises virus to 90% at tested concentrations; partial: cannot neutralise 90% of virus at tested concentrations; nn: non-neutralising. *reached 90% at highest concentration tested.
  • CA45 is a published broadly neutralising monolconal antibody (Zhao et al, cell, 2017).
  • Figure 3 shows in vitro neutralisation of Ebola virus GP pseudotyped S-FLU viruses by broadly-reactive monoclonal antibodies. Mean and range of duplicates shown for each assay. Results confirmed in independent repeat of experiment.
  • Figure 4 shows that broadly-reactive rabbit antibodies 11897, 11883 and 11889 compete for binding to EBOV GP with monoclonal antibodies with known epitopes in the glycan cap region in a competitive immunofluorescence assay.
  • 21-D8-5A influenza neuraminidase-specific antibody acting as a negative control
  • 6541, 66-4-cl2, c2G4 and c4G7 overlapping base epitopes
  • 6660 and 6662 receptor binding region epitopes
  • 66-3- 9C, 66-3-2C, 040 and cl3C6 different glycan cap epitopes
  • CA45, 6D6, FVM02, and ADI-15878 bind to the conserved fusion loop.
  • Self indicates signal when competed with unbiotinylated version of the same monoclonal antibody. Error bars show mean and 95% confidence intervals for six replicates within assay.
  • Figure 5 shows that broadly-reactive rabbit antibodies 11886 and 11892 compete for binding to EBOV GP with monoclonal antibodies with known epitopes in the base region in a competitive immunofluorescence assay.
  • 21-D8-5A influenza neuraminidase-specific antibody acting as a negative control
  • 6541, 66-4-cl2, c2G4 and c4G7 overlapping base epitopes
  • 6660 and 6662 receptor binding region epitopes
  • 66-3-9C, 66-3-2C, 040 and cl3C6 different glycan cap epitopes
  • CA45, 6D6, FVM02, and ADI-15878 bind to the conserved fusion loop.
  • Self indicates signal when competed with unbiotinylated version of the same monoclonal antibody. Error bars show mean and 95% confidence intervals for six replicates within assay.
  • Figure 6 shows binding of broadly reactive rabbit antibodies to EBOV GP and SUDV GP digested with thermolysin. Closed points: thermolysin treated GP; open points: untreated GP. Error bars show mean and range of 6 replicates per thermolysin treated sample and 3 replicates per undigested sample. 040: glycan cap antibody; 6541: base binding antibody; MR78: receptor binding region antibody that can only bind GP in the absence of the glycan cap.
  • Figure 7 shows logo plots generated by MEME tool from top 100 enriched sequences from panning 9mer and 13mer peptide phage display libraries with antibody 11886.
  • Figure 8 shows alignment of GP sequences with meme-derived motifs from peptide sequences enriched across three experiments and two peptide phage libraries by antibody 11886.
  • Figure 9 shows arginine residues in 11886 binding peptide motifs highlighted on existing electron microscopy structure of EBOV GP. Key arginine residues highlighted in stick representation. Cartoon representation of PDB structure 5KEL (Pallesen et al, Nature Microbiology, 2016).
  • Figure 10 shows 11886 binding peptide motifs highlighted on existing electron microscopy structures of EBOV GP in complex with published fabs.
  • the 11886 binding motif derived from peptide phage display is shown in rectangles.
  • A Cartoon representation of PDB structure 5KEL (Pallesen et al, Nature Microbiology, 2016).
  • B Surface rendering of 5KEL generated in Pymol with cl3C6 fab and c2G4 fab.
  • C Cartoon representation of PDB structure 6MAM (West et al, Nature Structural and Molecular Methods, 2019).
  • D Surface rendering of 6MAM generated in Pymol with ADI-15946 fab: teal.
  • E Surface rendering of 5KEL generated in Pymol with fabs hidden. FVM04 and m2 ID 10 contacts are shown.
  • SEQ ID NO: 1 shows the 11886 CDRL1
  • SEQ ID NO: 2 shows the 11886 CDRL2
  • SEQ ID NO: 3 shows the 11886 CDRL3
  • SEQ ID NO: 4 shows a 11886 CDRL3 variant
  • SEQ ID NO: 5 shows a 11886 CDRL3 variant
  • SEQ ID NO: 6 shows the 11886 CDRH1
  • SEQ ID NO: 7 shows the 11886 CDRH2
  • SEQ ID NO: 8 shows the 11886 CDRH3
  • SEQ ID NO: 9 shows a 11886 CDRH3 variant
  • SEQ ID NO: 10 shows a 11886 CDRH3 variant
  • SEQ ID NO: 11 shows the rabbit 11886 VL (amino acid)
  • SEQ ID NO: 12 shows the rabbit 11886 VL (nucleic acid)
  • SEQ ID NO: 13 shows the rabbit 11886 VH (amino acid)
  • SEQ ID NO: 14 shows the rabbit 11886 VH (nucleic acid)
  • SEQ ID NO: 15 shows a humanised 11886 VL
  • SEQ ID NO: 16 shows a humanised 11886 VL (with CDRL3 variant)
  • SEQ ID NO: 17 shows a humanised 11886 VL (with CDRL3 variant)
  • SEQ ID NO: 18 shows a humanised 11886 VH
  • SEQ ID NO: 19 shows a humanised 11886 VH (with CDRH3 variant)
  • SEQ ID NO: 20 shows a humanised 11886 VH (with CDRH3 variant)
  • SEQ ID NO: 21 shows the Bundibugyo Ebola virus glycoprotein sequence (NBCI reference YP_003815435.1).
  • SEQ ID NO: 22 shows the Zaire Ebola virus glycoprotein sequence (ATY51135.1)
  • SEQ ID NO: 23 shows the Sudan Ebola virus glycoprotein sequence (NCBI Reference Sequence: YP 138523.1)
  • SEQ ID NO: 24 shows the Tai Forest Ebola virus glycoprotein sequence (NCBI Reference Sequence: YP 003815426.1)
  • SEQ ID NO: 25 shows the Ebola virus glycoprotein consensus motif
  • SEQ ID NOs: 26-33 show 9 and 13 mer peptide motifs which mapped onto Ebola glycoprotein sequences
  • SEQ ID NO: 34 shows the 11897 CDRLl SEQ ID NO: 35 shows the 11897 CDRL2 SEQ ID NO: 36 shows the 11897 CDRL3 SEQ ID NO: 37 shows the 11897 CDRHl SEQ ID NO: 38 shows the 11897 CDRH2 SEQ ID NO: 39 shows a 11897 CDRH2 variant SEQ ID NO: 40 shows a 11897 CDRH2 variant SEQ ID NO: 41 shows a 11897 CDRH2 variant SEQ ID NO: 42 shows the 11897 CDRH3 SEQ ID NO: 43 shows the 11897 rabbit VL (amino acid)
  • SEQ ID NO: 44 shows the 11897 rabbit VL (nucleic acid)
  • SEQ ID NO: 45 shows the 11897 rabbit VH (amino acid)
  • SEQ ID NO: 46 shows the 11897 rabbit VH (nucleic acid)
  • SEQ ID NO: 47 shows a humanised 11897 VL
  • SEQ ID NO: 48 shows a humanised 11897 VH
  • SEQ ID NO: 49 shows a humanised 11897 VH (CDRH2 variant)
  • SEQ ID NO: 50 shows a humanised 11897 VH (CDRH2 variant)
  • SEQ ID NO: 51 shows a humanised 11897 VH (CDRH2 variant)
  • SEQ ID NO: 52 shows the 11878 CDRL1
  • SEQ ID NO: 53 shows the 11878 CDRL2
  • SEQ ID NO: 54 shows the 11878 CDRL3
  • SEQ ID NO: 55 shows the 11878 CDRH1
  • SEQ ID NO: 56 shows the 11878 CDRH2
  • SEQ ID NO: 57 shows the 11878 CDRH3
  • SEQ ID NO: 58 shows the 11878 rabbit VL (amino acid)
  • SEQ ID NO: 59 shows the 11878 rabbit VL (nucleic acid)
  • SEQ ID NO: 60 shows the 11878 rabbit VH (amino acid)
  • SEQ ID NO: 61 shows the 11878 rabbit VH (nucleic acid)
  • SEQ ID NO: 62 shows a humanised 11878 VL
  • SEQ ID NO: 63 shows a humanised 11878 VH
  • SEQ ID NO: 64 shows the 11883 CDRL1
  • SEQ ID NO: 65 shows the 11883 CDRL2
  • SEQ ID NO: 66 shows the 11883 CDRL3
  • 67 shows the 11883 CDRH1
  • SEQ ID NO: 68 shows a 11883 CDRH1 variant
  • SEQ ID NO: 69 shows a 11883 CDRH1 variant
  • SEQ ID NO: 70 shows the 11883 CDRH2 SEQ ID NO: 71 shows a 11883 CDRH2 variant SEQ ID NO: 72 shows a 11883 CDRH2 variant SEQ ID NO: 73 shows the 11883 CDRH3 SEQ ID NO: 74 shows the 11883 rabbit VL (amino acid)
  • SEQ ID NO: 75 shows the 11883 rabbit VL (nucleic acid)
  • SEQ ID NO: 76 shows the 11883 rabbit VH (amino acid)
  • SEQ ID NO: 77 shows the 11883 rabbit VH (nucleic acid)
  • SEQ ID NO: 78 shows a humanised 11883 VL
  • SEQ ID NO: 79 shows a humanised 11883 VH
  • SEQ ID NO: 80 shows a humanised 11883 VH (CDRH1 and 2 variant)
  • SEQ ID NO: 81 shows a humanised 11883 VH (CDRH1 and 2 variant)
  • SEQ ID NO: 82 shows a humanised 11883 VH (CDRH1 and 2 variant)
  • SEQ ID NO: 83 shows the 11889 CDRL1
  • SEQ ID NO: 84 shows the 11889 CDRL2
  • SEQ ID NO: 85 shows the 11889 CDRL3
  • SEQ ID NO: 86 shows the 11889 CDRH1
  • SEQ ID NO: 87 shows a 11889 CDRH1 variant
  • SEQ ID NO: 88 shows the 11889 CDRH2
  • SEQ ID NO: 89 shows a 11889 CDRH2 variant
  • SEQ ID NO: 90 shows the 11889 CDRH3
  • SEQ ID NO: 91 shows the 11889 rabbit VL (amino acid)
  • SEQ ID NO: 92 shows the 11889 rabbit VL (nucleic acid)
  • SEQ ID NO: 93 shows the 11889 rabbit VH (amino acid)
  • SEQ ID NO: 94 shows the 11889 rabbit VH (nucleic acid)
  • SEQ ID NO: 95 shows a humanised 11889 VL
  • SEQ ID NO: 96 shows a humanised 11889 VH
  • SEQ ID NO: 97 shows a humanised 11889 VH (CDRH1 and 2 variant)
  • SEQ ID NO: 98 shows the 11892 CDRL1
  • SEQ ID NO: 99 shows the 11892 CDRL2
  • SEQ ID NO: 100 shows the 11892 CDRL3
  • SEQ ID NO: 101 shows a 11892 CDRL3 variant
  • SEQ ID NO: 102 shows a 11892 CDRL3 variant
  • SEQ ID NO: 103 shows the 11892 CDRH1
  • SEQ ID NO: 104 shows the 11892 CDRH2
  • SEQ ID NO: 105 shows the 11892 CDRH3
  • SEQ ID NO: 106 shows a 11892 CDRH3 variant
  • SEQ ID NO: 107 shows a 11892 CDRH3 variant
  • SEQ ID NO: 108 shows the 11892 rabbit VL (amino acid)
  • SEQ ID NO: 109 shows the 11892 rabbit VL (nucleic acid)
  • SEQ ID NO: 110 shows the 11892 rabbit VH (amino acid)
  • SEQ ID NO: 111 shows the 11892 rabbit VH (nucleic acid)
  • SEQ ID NO: 112 shows a humanised 11892 VL
  • SEQ ID NO: 113 shows a humanised 11892 VL (CDRL3 variant)
  • SEQ ID NO: 172 shows a 6662 variant CDRH2 SEQ ID NO: 173 shows a 6662 variant CDRH2 SEQ ID NO: 174 shows a 6662 variant CDRH3 SEQ ID NO: 175 shows a 6662 variant CDRH3 SEQ ID NO: 176 shows a 6662 variant CDRL3
  • SEQ ID NO: 177 shows a 6662 variant CDRL3
  • SEQ ID NO: 178 shows a 6662 variant CDRL3 Detailed Description of the Invention
  • the present invention relates to antibodies that bind to (recognise) the Ebola virus glycoprotein (GP) and to pharmaceutical compositions comprising such antibodies.
  • Ebola virus species There are currently six Ebola virus species (Zaire, Sudan, Bundibugyo, Reston, Tai Forest and Bombali) and many different strains. Zaire, Sudan, Bundibugyo and Tai Forest cause disease in humans, with Zaire being the most deadly.
  • the Ebola vims glycoprotein is the only virally expressed protein on the vims surface and is critical for attachment to host cells and catalysis of membrane fusion.
  • the glycoprotein is cleaved by furin to form a disulphide-linked GP1-GP2 heterodimer, which assembles as trimers on the vims surface.
  • GP1 contains the receptor-binding site responsible for host cell attachment, the glycan cap and the mucin-like domain, whereas GP2 contains heptad repeats and a transmembrane domain.
  • Ebola vims glycoprotein sequences vary between species. The nucleotide and amino acid sequences of the glycoprotein from various Ebola vims species/strains have been determined, with examples including GenBank: AF086833.2 providing the complete genome of Zaire Mayinga, and GenBank: AAN37507.1, GenBank: AAG40168.1 and GenBank: ATY51135.1 providing examples of Zaire glycoprotein sequences. Accession numbers NC_014373.1 and NC_006432.1 provide the complete genome sequences of Budibugyo and Sudan Gulu.
  • GenBank: AGL73446.1, GenBank: AGL73439.1 and NCBI Reference Sequence: YP 138523.1 provide examples of a Sudan glycoprotein sequence and accession numbers GenBank: AGL73474.1, GenBank: AGL73467.1 and NCBI Reference Sequence: YP 003815435.1 provide examples of Bundibugyo glycoprotein sequences. Accession number NCBI Reference Sequence:
  • YP 003815426.1 provides a Tai Forest glycoprotein sequence.
  • Other sequences are readily available from sequence databases, such as GenBank and UniProt.
  • Antibodies of the invention may be “isolated” antibodies.
  • An isolated antibody is an antibody which is substantially free of other antibodies having different antigenic specificities.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof.
  • An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • various cells of the immune system e.g., effector cells
  • the first component (Clq) of the classical complement system e.g., Clq
  • Antibodies of the invention are typically monoclonal antibodies.
  • An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody or an antigen-binding portion of any thereof.
  • the antibody is a humanised antibody.
  • Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.
  • the antibody molecules of the present invention may comprise a complete antibody molecule having full length heavy and light chains or a fragment or antigen-binding portion thereof.
  • the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibodies and fragments and antigen binding portions thereof may be, but are not limited to Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, single domain antibodies (e.g.
  • VH or VL or VHH VH or VL or VHH
  • scFv bi, tri or tetra-valent antibodies
  • Bis-scFv diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above
  • the methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et ak, 1998, Journal of Immunological Methods, 216, 165-181).
  • antibody fragments for use in the present invention include the Fab and Fab’ fragments described in International patent applications WO 2005/003169, WO 2005/003170 and WO 2005/003171 and Fab-dAb fragments described in International patent application W02009/040562.
  • Multi-valent antibodies may comprise multiple specificities or may be monospecific (see for example WO 92/22853 and WO 05/113605 and the DVD-Ig as disclosed in WO 2007/024715, or the so-called (FabFv)2Fc described in WO 2011/030107).
  • An alternative multi-specific antigen-binding fragment comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding the same or a different target (e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for instance, albumin).
  • target e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for instance, albumin.
  • antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.
  • the constant region domains of the antibody molecule of the present invention may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required.
  • the constant region domains may be IgA, IgD, IgE, IgG or IgM domains, typically IgG (i.e. IgGl,
  • IgG2, IgG3 or IgG4 are human.
  • the constant regions are human.
  • human IgG i.e. IgGl, IgG2, IgG3 or IgG4 constant region domains may be used.
  • a human IgGl constant region may be used.
  • the light chain constant region may be either lambda or kappa.
  • Antibodies of the invention may be mono-specific or multi-specific (e.g. bi-specific).
  • a multi-specific antibody comprises at least two different variable domains, wherein each variable domain is capable of binding to a separate antigen or to a different epitope on the same antigen.
  • An antibody of the invention may be a human antibody.
  • the term "human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human antibody derivatives refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. It will also be understood by one skilled in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).
  • Biological molecules such as antibodies or fragments, contain acidic and/or basic functional groups, thereby giving the molecule a net positive or negative charge.
  • the amount of overall “observed” charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure and the environmental conditions of the molecule.
  • the isoelectric point (pi) is the pH at which a particular molecule or surface carries no net electrical charge.
  • the antibody or fragment according to the present disclosure has an isoelectric point (pi) of at least 7.
  • the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9.
  • the pi of the antibody is 8.
  • Antibodies may be obtained by administering polypeptides to an animal, e.g. a non human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.
  • Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et ah, 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et ah, Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).
  • Antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by for example the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843-78481; WO92/02551; W02004/051268 and W02004/106377.
  • the antibodies can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al.
  • Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody.
  • Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts e.g. as described in general terms in EP
  • humanized antibody is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rabbit, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences and also within the CDR sequences.
  • CDR-grafted antibody molecule refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine, rat or rabbit monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody).
  • a donor antibody e.g. a murine, rat or rabbit monoclonal antibody
  • acceptor antibody e.g. a human antibody
  • only one or more of the specificity determining residues from any one of the CDRs described herein are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34).
  • only the specificity determining residues from one or more of the CDRs described herein are transferred to the human antibody framework.
  • only the specificity determining residues from each of the CDRs described herein are transferred to the human antibody framework.
  • any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
  • the CDR-grafted antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity determining residues described above.
  • a CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.
  • human frameworks which can be used are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Rabat et al., supra).
  • KOL and NEWM can be used for the heavy chain
  • REI can be used for the light chain and EU
  • LAY and POM can be used for both the heavy chain and the light chain.
  • human germline sequences may be used; these are available for example at: http://www.vbase2.org/ (see Retter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).
  • the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
  • the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody.
  • a protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91/09967.
  • the invention provides an anti-Ebola virus antibody that recognises (binds to) an epitope on the Ebola glycoprotein comprising one or more residues selected from the group consisting of F132, P133, R134, C135, R136, Y137, V138, H139, K140, V141, S142 and G143 of SEQ ID NO: 22.
  • SEQ ID NO: 22 is the Zaire Ebola virus glycoprotein sequence with accession number ATY51135.1.
  • residue 141 is A and residue 142 is Q (see Figure 8).
  • Antibodies of the invention also therefore typically recognise an epitope comprising
  • antibodies of the invention recognise an epitope comprising one or more residues selected from the group consisting of F132, P133, R134, C135, R136, Y137, V138, H139, K140, A141, Q142 and G143 of SEQ ID NO: 23.
  • antibodies of the invention recognise an epitope comprising one or more residues selected from the group consisting of F132, P133, R134, C135, R136, Y137, V138, H139 and K140 of any one of SEQ ID NOs: 21-24, preferably in all of SEQ ID NOs: 21-24.
  • antibodies of the invention recognise an epitope comprising one or more residues selected from the group consisting of R134, C135 and R136 (of any one of, preferably all of, SEQ ID NOs: 21-24. In some instances antibodies of the invention recognise an epitope comprising R134 and R136, or all three of R134, C135 and R136.
  • the epitope may further comprise one of more residues selected from group consisting of G102, E103, W104, A105, E106, N107 and C108 and/or (b) one of more residues selected from group consisting of R605, W606, G607, G608, T609, C610 and H611 .
  • the residue numbering may be according to SEQ ID NO: 22. However, these residues are conserved in Zaire, Bundibugyo, Sudan and Tai Forest. Therefore the residue numbering may also be according to SEQ ID NO: 21, 23 or 24.
  • the antibodies recognise epitopes which are conserved across species, the antibodies are cross-reactive for all of Zaire, Bundibugyo, Sudan and Tai Forest
  • a routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed.
  • Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis.
  • methods such as hydrogen deuterium exchange, epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Such methods are well known in the art.
  • the antibody epitope may also be determined by electron microscopy.
  • antibody epitopes be determined by x-ray crystallography analysis. Antibodies of the present invention may therefore be assessed through x-ray crystallogray analysis of the antibody bound to the Ebola virus glycorptein. Epitopes may, in particular, be identified in this way by determining residues within 4 ⁇ (possibly 5 A) of an antibody paratope residue.
  • the epitope residues of the 11886 antibody were determined by screening peptide libraries and mapping the peptides bound by the antibody onto the Ebola virus sequences. Such methods (e.g. peptide scanning) may be used in identifying antibodies binding to the regions specified in the claims.
  • an antibody of the invention may bind to peptides comprising the motifs as shown in SEQ ID NOs: 26-33.
  • Peptides may also comprise residue numbers 102-108, 132-143 or 605-611 of any one of SEQ ID NOs: 21-24.
  • An antibody of the invention may in particular comprise one or more CDR sequences selected from the following:
  • SEQ ID Nos: 1, 2, 3, 6, 7 and 8 are the parental rabbit antibody CDR sequences of 11886.
  • SEQ ID Nos: 4, 5, 9 and 10 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOs: 6, 7 and 8/9/10.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOs: 1, 2 and 3/4/5.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 6-10 and at least one light chain CDR sequence selected from SEQ ID NOSs: 1-5.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 6-10 and at least two light chain CDR sequences selected from SEQ ID NOs: 1-5.
  • an antibody of the invention may comprise HCDR3 of SEQ ID NO: 8, 9 or 10.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 13 (the VH of 11886).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 11 (the VL of 11886).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 13 and 11.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 18, 19 or 20. These are humanised sequences of 11886.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 15, 16 or 17. These are also humanised sequences of 11886.
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs: 18, 19 and 20 and a VL selected from the group consisting of SEQ ID NOs: 15, 16 and 17.
  • An antibody of the invention may also comprise one or more CDR sequences selected from the following:
  • SEQ ID Nos: 34, 35, 36, 37, 38 and 42 are the parental rabbit antibody CDR sequences of 11897.
  • SEQ ID Nos: 39, 40 and 31 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 37, 38/39/40/41 and 42.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 34, 35 and 36.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 37-42 and at least one light chain CDR sequence selected from SEQ ID NOS 34-36.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 37-42 and at least two light chain CDR sequences selected from SEQ ID NOS: 34-36.
  • an antibody of the invention may comprise HCDR3 of SEQ ID NO: 42.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 45 (the VH of 11897).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 43 (the VL of 11897).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 45 and 43.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 48, 49, 50 or 51. These are humanised sequences of 11897.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 47. This is also a humanised sequence of 11897.
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs: 48, 49, 50 and 51 and a VL of SEQ ID NO: 47.
  • An antibody of the invention may also comprise one or more CDR sequences selected from the following:
  • SEQ ID NOs: 52-57 are the rabbit antibody CDR sequences of 11878.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 55, 56 and 57.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 52, 53 and 54.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 55-57 and at least one light chain CDR sequence selected from SEQ ID NOS 52-54.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 55-57 and at least two light chain CDR sequences selected from SEQ ID NOS: 52-54.
  • an antibody of the invention may comprise a HCDR3 of SEQ ID NO: 57.
  • An antibody of the invention may in particular comprise the following CDRs: (a) a CDRL1 sequence of SEQ ID NO: 52;
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 60 (the VH of 11878).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 58 (the VL of 11878).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 60 and 58.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 63. This is a humanised sequence of 11878.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 62. This is also a humanised sequence of 11878.
  • an antibody of the invention may comprise a VH of SEQ ID NO: 63 and a VL of SEQ ID NO: 62.
  • an antibody of the invention may comprise one or more CDR sequences selected from the following:
  • SEQ ID Nos: 64, 65, 66, 67, 70 and 73 are the parental rabbit antibody CDR sequences of 11883.
  • SEQ ID Nos: 68, 69, 71 and 72 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOs: 67/68/69, 70/71/72 or 73.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 64, 65 or 66.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 67-73 and at least one light chain CDR sequence selected from SEQ ID NOS 64-66.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 67-73 and at least two light chain CDR sequences selected from SEQ ID NOS: 64-66.
  • an antibody of the invention may comprise a HCDR3 of SEQ ID NO: 73.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 76 (the VH of 11883).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 74 (the VL of 11883).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 76 and 74.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 79, 80, 81 or 82. These are humanised sequences of 11883.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 78. This is a humanised sequence of 11883.
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs: 79, 80, 81 and 82 and a VL of SEQ ID NO: 78.
  • an antibody of the invention may comprise one or more CDR sequences selected from the following:
  • SEQ ID NOs: 83, 84, 85, 86, 88 and 90 are the parental rabbit antibody CDR sequences of 11889.
  • SEQ ID NOs: 87 and 89 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOs:86/87, 88/89 or 90.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOs: 83, 84 or 85.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 86-90 and at least one light chain CDR sequence selected from SEQ ID NOs 83-85.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 86-90 and at least two light chain CDR sequences selected from SEQ ID NOS: 83-85.
  • an antibody of the invention may comprise a HCDR3 of SEQ ID NO: 90.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 93 (the VH of 11889).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 91 (the VL of 11889).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 93 and 91.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 96 or 97.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 95. This is a humanised sequence of 11889.
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs:
  • An antibody of the invention may comprise one or more CDR sequences selected from the following:
  • SEQ ID NOs: 98, 99, 100, 103, 104 and 105 are the parental rabbit antibody CDR sequences of 11892.
  • SEQ ID NOs: 101, 102, 106 and 107 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOs: 103, 104 and 105/106/107.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOs: 98, 99 and 100/101/102.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 103-107 and at least one light chain CDR sequence selected from SEQ ID NOs 98-102.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 103-107 and at least two light chain CDR sequences selected from SEQ ID NOs: 98-102.
  • an antibody of the invention may comprise a HCDR3 of SEQ ID NO: 105, 106 or 107.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 110 (the VH of 11892).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 108 (the VL of 11892).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 110 and 108.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 115, 116 or 117. These are humanised sequences of 11892.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 112, 113 or 114. These are humanised sequences of 11892.
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs: 115-117 and a VL selected from the group consisting of SEQ ID NO: 112-114.
  • An antibody of the invention may comprise one or more CDR sequences selected from the following:
  • SEQ ID NOs: 118, 119, 120, 123, 124 and 125 are the parental rabbit antibody CDR sequences of 11881.
  • SEQ ID NOs: 121, 122, 126 and 127 are humanized variants of the rabbit CDRs.
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOs: 123, 124 and 125/126/127.
  • An antibody of the invention may also comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOs: 118, 119 and 120/121/122.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 123-127 and at least one light chain CDR sequence selected from SEQ ID NOs 118-122.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 123-127 and at least two light chain CDR sequences selected from SEQ ID NOs: 118-122.
  • an antibody of the invention may comprise a HCDR3 of SEQ ID NO: 125, 126 or 127.
  • An antibody of the invention may in particular comprise the following CDRs:
  • an antibody of the invention may comprise the following:
  • An antibody of the invention may comprise a heavy chain variable region sequence of SEQ ID NO: 130 (the VH of 11881).
  • An antibody of the invention may comprise a light chain variable region sequence of SEQ ID NO: 128 (the VL of 11881).
  • An antibody of the invention may comprise a VH and VL sequence pair of SEQ ID NOs: 130 and 128.
  • An antibody of the invention may also comprise a VH of SEQ ID NO: 135, 136 or 137. These are humanised sequences of 11881.
  • An antibody of the invention may comprise a VL of SEQ ID NO: 132, 133 or 134. These are humanised sequences of 1188 T
  • an antibody of the invention may comprise a VH selected from the group consisting of SEQ ID NOs: 135, 136 and 137 and a VL selected from the group consisting of SEQ ID NOs: 132, 133 and 134.
  • one or more sequences may be modified to remove undesirable residues or sites, such as cysteine residues or aspartic acid (D) isomerisation sites or asparagine (N) deamidation sites.
  • undesirable residues or sites such as cysteine residues or aspartic acid (D) isomerisation sites or asparagine (N) deamidation sites.
  • cysteine residues in any one of the sequences may be substituted with another amino acid, such as serine.
  • an asparagine deamidation site may be removed from one or more of the sequences (for example, one or more of the CDRs) by mutating the asparagine residue (N) and/or a neighbouring residue to any other suitable amino acid.
  • an asparagine deamidation site such as NG or NS may be mutated, for example to NA orNT.
  • an aspartic acid isomerisation site may be removed from one or more of the sequences (for example, one or more of the CDRs) by mutating the aspartic acid residue (D) and/or a neighbouring residue to any other suitable amino acid.
  • an aspartic acid isomerisation site such as DG or DS may be mutated, for example to EG, DA or DT.
  • an N-glycosylation site such as NLS may be removed by mutating the asparagine residue (N) to any other suitable amino acid, for example to SLS or QLS.
  • an N-glycosylation site such as NLS may be removed by mutating the serine residue (S) to any other residue with the exception of threonine (T).
  • Antibodies of the invention may include a plurality of the above modifications.
  • the antibody may be or may comprise a variant of one of the specific sequences recited above.
  • a variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.
  • a variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) amino acid substitutions and/or deletions from the specific sequences discussed above.
  • “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features.
  • “Substitution” variants typically involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions.
  • an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid.
  • an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid.
  • Derivatives or variants generally include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected. Derivatives and variants as described above may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
  • Variant antibodies may have an amino acid sequence which has more than about 60%, or more than about 70%, e.g. 75 or 80%, preferably more than about 85%, e.g. more than about 90 or 95% amino acid identity to the amino acid sequences disclosed herein
  • the antibody may be a variant which has more than about 60%, or more than about 70%, e.g. about 75 or 80%, typically more than about 85%, e.g. more than about 90 or 95% amino acid identity to the VH/VL sequences disclosed herein, whilst retaining the exact CDRs disclosed for these sequences.
  • Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the VH/VL sequences disclosed herein (in some circumstances whilst retaining the exact CDRs).
  • This level of amino acid identity is typically seen across the full length of the relevant SEQ ID NO sequence but may be over a part of the sequence, such as across about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.
  • sequence identity refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:
  • Pairwise alignment parameters -Method accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;
  • the present invention thus provides antibodies having specific sequences and variants which maintain the function or activity of the antibody.
  • antibodies of the invention are able to neutralise at least one biological activity of Ebola virus (a neutralising antibody), particularly to neutralise virus infectivity.
  • a neutralising antibody particularly to neutralise virus infectivity.
  • the ability of an antibody to neutralise virus infectivity may be measured using an appropriate assay, particularly using a cell-based neutralisation assay.
  • neutralisation may be determined using an assay for measuring infection of cells using a surrogate virus coated with the Ebola virus glycoprotein. For these surrogate viruses infection of cells is dependent on the Ebola virus glycoprotein.
  • E-S-FLU Ebola virus surrogate as described in the Examples below.
  • This assay utilises a disabled influenza virus core coated with Ebola virus GP.
  • the E-S-FLU encodes eGFP that replaces the hemagglutinin coding sequence so that infected cells fluoresce green.
  • the loss of fluorescent signal e.g. after overnight infection provides a measure of the inhibition of infection by an antibody.
  • antibodies of the invention may be “partial” neutralising antibodies, where inhibition of infection plateaus at 50-90% inhibition or “strong” neutralising antibodies, which achieve > 90% inhibition.
  • Antibody concentrations may be as tested in the Examples/ Figures, for example with an concentration of 5 pg/ml used to determine if any antibody is a “strong” or “partial” neutralising antibody.
  • Neutralisation may also be determined up to a maximum concentration of 50 pg/ml.
  • the 11886 antibody is a strong neutralising antibody for all of Zaire, Sudan and Bundibugyo Ebola virus.
  • Antibodies of the invention may have sequences as described above and be either a strong or partial neutralising antibody.
  • an antibody may have six CDR sequences of 11886 and be a strong neutralising antibody (preferably for all of Zaire,
  • Neutralisation may also be determined using IC50 or IC90 values.
  • IC50 and IC90 values can be determined from the results of a neutralisation assay (as discussed above) using standard methods.
  • An antibody of the invention may for example have an IC50 value of less than (i.e. better than) 10 pg/ml, less than 5 pg/ml, less than 2 pg/ml or less than 1 pg/ml (typically down to 0.1 pg/ml).
  • an antibody of the invention may have an IC50 value of between 0.1 pg/ml and 10 pg/ml, sometimes between 0.1 pg/ml and 5 pg/ml, between 0.1 pg/ml and 2 pg/ml or even between 0.1 pg/ml and 1 pg/ml. In some instances, an antibody of the invention may have an IC50 value of between 0.5 pg/ml and 10 pg/ml, sometimes between 0.5 pg/ml and 5 pg/ml or between 0.5 pg/ml and 2 pg/ml.
  • An antibody of the invention may have an IC90 value of less than 10 pg/ml, optionally less than 5 pg/ml (typically down to 1 pg/ml).
  • an antibody of the invention may have an IC90 value of between 1 pg/ml and 10 pg/ml, for example between 1 pg/ml and 5 pg/ml.
  • Neutralisation ability may be determined for any species of Ebola virus, such as Zaire, as shown in the Examples.
  • an antibody of the invention will have the above IC50/90 values for all of Zaire, Sudan and Budibugyo.
  • An antibody of the invention may have an IC50 value of less than 2 pg/ml for Zaire, less than 2 pg/ml (or preferably less than 1 pg/ml) for Sudan and/or less than 1 pg/ml for Budibugyo. In some instances, an antibody of the invention may therefore have an IC50 value of less than 2 pg/ml for all three species. Typical lower limits are as described above.
  • An antibody of the invention may also have an IC90 of less than 10 pg/ml for Zaire, less than 5 pg/ml for Sudan and less than 5 (or 3 or 2) pg/ml for Budibugyo.
  • An antibody of the invention may therefpre have an IC90 value of less than 10 pg/ml for all three species. Once again, typical lower limits are as described above.
  • IC 50 / IC 90 values may be applied to the sequences described above.
  • an antibody of the invention may have six CDR sequences as described above (particularly the CDRs of 11886) and an IC 50 / IC 90 value as presented above.
  • Antibodies of the invention are also preferably able to provide in vivo protection in Ebola virus infected animals.
  • administration of an antibody of the invention to Ebola virus infected animals may result in a survival rate of greater than 30% or greater than 50%.
  • antibodies of the invention achieve a survival rate of 100%. Survival rates may be determined using routine methods. For example, in in vivo protection may be determined in mice (such as after a dose of 100 pg antibody at day two of infection). In vivo protection may also be determined in guinea pigs (for example, at a dose of 10 mg/kg of each antibody at day three of infection).
  • antibodies of the invention are typically cross-reactive for one or more Ebola virus species, such as Zaire (e.g. Zaire Mayinga and/or Makona), Bundibugyo and Sudan (e.g. Sudan Gulu).
  • Ebola virus species such as Zaire (e.g. Zaire Mayinga and/or Makona), Bundibugyo and Sudan (e.g. Sudan Gulu).
  • Antibodies of the invention are also typically cross-reactive for Tai Forest.
  • antibodies are cross-reactive for all of the above.
  • the antibodies are capable of binding to the glycoprotein from these species/strains. Binding can be measured, for example, using Surface Plasmon Resonance.
  • An antibody may be cross-reactive if it retains 100% of its binding capability.
  • An antibody may also be cross-reactive with lower retention of binding, such as retaining at least 50% or at least 30% binding capability across one or all species.
  • a measure of binding would be the K D value.
  • antibodies of the invention may be cross-reactive if they have a K D value of less than 1 pM for more than one species (antibodies may have a K D value of less than 1 mM for more than one species, such as Zaire, Bundibugyo, Tai Forest and Sudan).
  • Antibodies of the invention may have any combination of one or more of the above properties.
  • Antibodies of the invention may bind to the same epitope, or compete for binding to Ebola virus glycoprotein, with any one of the reference antibodies described above (i.e. in particular with antibodies with the heavy and light chain variable regions described above). Methods for identifying antibodies binding to the same epitope, or cross-competing with one another, are discussed below.
  • the present invention also provides an isolated nucleic sequence encoding the heavy and/or light chain variable regions(s) of an antibody molecule of the present invention, or the full heavy and/or light chain (in some instances a pair of nucleic acids encoding the heavy and light chain variable regions or full heavy/light chains).
  • Nucleic acid sequences which encode an antibody molecule of the present invention may be DNA or RNA (for example mRNA).
  • Nucleic acid sequences which encode an antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. For example, nucleic acids sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.
  • the invention also provides expression vectors comprising the nucleic acid sequences(s).
  • a host cell comprising one or more cloning or expression vectors comprising one or more nucleic acid sequences encoding an antibody of the present invention.
  • Any suitable host cell/vector system may be used for expression of the nucleic acid sequences encoding the antibody molecule of the present invention.
  • Bacterial, for example E. coli, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used.
  • Suitable mammalian host cells include CHO, or myeloma.
  • antibodies may be produced in CHO cells, modified CHO cells (to produce afucosylated antibodies) or HEK-293 cells.
  • the present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell containing a vector of the present invention under conditions suitable for leading to expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.
  • the invention also provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition of the invention may comprise one or more nucleic acids (as described above) encoding an antibody of the invention.
  • the nucleic acids may for example be mRNA.
  • the pharmaceutical composition may comprises one or more additional anti-Ebola virus antibodies, or sequences encoding one or more additional anti-Ebola virus antibodies. It is preferable that the antibodies do not cross-compete with one another, particularly that the antibodies bind to non-overlapping epitopes on the Ebola virus glycoprotein.
  • a pharmaceutical composition of the invention may comprise any of the antibodies described above, alone or in any combination (or sequences encoding any of the antibodies described above).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions of the invention may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.
  • Pharmaceutically acceptable carriers comprise aqueous carriers or diluents.
  • suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline.
  • suitable aqueous carriers include water, buffered water and saline.
  • other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • compositions of the invention may comprise additional therapeutic ingredients, for example additional anti-viral agents.
  • Anti-viral agents may bind to Ebola virus and inhibit viral activity. Alternatively, anti-viral agents may not bind directly to Ebola virus but still affect viral activity/infectivity.
  • An anti-viral agent could be a further anti-Ebola antibody, which binds somewhere other than the glycoprotein.
  • the additional therapeutic ingredient could also be an anti-inflammatory agent, such as a corticosteroid or a non-steroidal anti-inflammatory drug.
  • the additional therapeutic agent could also be an anti-Ebola vaccine.
  • the pharmaceutical composition may be administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally or orally.
  • the invention also provides anti-Ebola virus antibody cocktails, particularly a cocktail comprising two or more, typically three or more antibodies to the Ebola virus glycoprotein.
  • an “antibody cocktail” generally refers to a combination/mixture of antibodies within the same composition, i.e. a single pharmaceutical composition comprising the antibodies.
  • the invention also includes the combined use of different anti-Ebola virus antibodies in separate pharmaceutical compositions.
  • the pharmaceutical compositions may comprise the antibodies per se, or nucleic acid sequences (e.g. mRNA) encoding the antibodies.
  • the antibodies may bind to the Ebola virus glycoprotein from any of the species/strains discussed above.
  • a cocktail of the invention may comprise two or more antibodies (or sequences encoding the antibodies).
  • a cocktail of the invention comprises two or more antibodies binding to different regions of the Ebola virus glycoprotein.
  • a cocktail may comprise two or more antibodies binding to at least two of the following regions of the glycoprotein: glycan cap, b17-18 loop, receptor binding region and base.
  • a cocktail of the invention may comprise three or four antibodies binding to the Ebola virus glycoprotein.
  • the skilled person would also readily be able to determine the binding site (epitope) of an antibody using standard techniques, such as those described above. The skilled person could also readily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art.
  • test antibody binds to the same epitope as a reference antibody of the invention
  • the reference antibody is allowed to bind to a protein or peptide under saturating conditions.
  • the ability of a test antibody to bind to the protein or peptide is assessed. If the test antibody is able to bind to the protein or peptide following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody.
  • the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention.
  • an antibody competes for binding with a reference antibody the above-described binding methodology is performed in two orientations.
  • a first orientation the reference antibody is allowed to bind to a protein/peptide under saturating conditions followed by assessment of binding of the test antibody to the protein/peptide molecule.
  • the test antibody is allowed to bind to the protein/peptide under saturating conditions followed by assessment of binding of the reference antibody to the protein/peptide. If, in both orientations, only the first (saturating) antibody is capable of binding to the protein/peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein/peptide.
  • an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.
  • Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et ak, Cancer Res,
  • two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Additional routine experimentation e.g., peptide mutation and binding analyses
  • peptide mutation and binding analyses can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding.
  • steric blocking or another phenomenon
  • this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
  • antibody epitopes include hydrogen/deuterium exchange, X-ray crystallography and peptide display libraries (as described in the Examples). A combination of these techniques may be used to determine the epitope of the test antibody.
  • the antibodies bind non-overlapping epitopes, or do not cross-compete with one another. This can be determined using the methods described above.
  • One or more of the antibodies included in the cocktails of the invention may be neutralising antibodies (in other words, one or more of the antibodies may be individually neutralising). In some instances, all of the antibodies in the cocktail may be neutralising antibodies. Such neutralising antibodies are described above.
  • one or more of the antibodies in the cocktail individually enhance survival of animals infected with Ebola virus. In some instances, all of the antibodies in the cocktail enhance survival of Ebola virus infected animals.
  • administering results in a survival rate of at least 50%.
  • administration of an antibody cocktail of the invention to Ebola virus infected animals results in a 100% survival rate.
  • survival rates may be determined in mice (e.g. with a single dose of 100 pg of antibody at day 2 of infection). Survival rates may also be determined in infected guinea pigs.
  • Antibodies may be administered at day three following infection. Such experiments may be conducted at a dose of 10 mg/kg of each antibody, or in some instances at a total dose (for all antibodies) of 5 mg/kg.
  • a cocktail may therefore result in a survival rate of at least 50% at a dose of 10 mg/kg of each antibody or at a total dose of 5 mg/kg.
  • a cocktail may result in a 100% survival rate at a dose of 10 mg/kg of each antibody or at a total dose of 5 mg/kg.
  • one or more (for example, one, two, three or four) antibodies in the cocktail cross-react with different Ebola virus species, for example Zaire and/or Sudan and/or Bundibugyo. It is most preferred that all of the antibodies in the cocktail cross-react with all of these species. Cross-reactivity is discussed above.
  • the cocktail may include any of the antibodies of the invention as defined above.
  • the cocktail of the invention may include an antibody based on 11886 (i.e. with the sequences described above).
  • additional antibodies included in the cocktail could be one or more antibodies selected from 66-3-9C, 040 and 6662.
  • an antibody in the cocktail may comprise one or more of the following CDR sequences:
  • CDR sequences of the 66-3-9C antibody are CDR sequences of the 66-3-9C antibody.
  • the antibody comprises six CDRs as set out above.
  • An antibody in the cocktail may also comprise a VH of SEQ ID NO: 144 and/or a VL of SEQ ID NO: 145. These are the VH and VL sequences of the 66-3 -9C antibody. Furthermore, an antibody may comprise a heavy chain of SEQ ID NO: 162 and a light chain of SEQ ID NO: 163. These are heavy and light chain sequences of 66-9-3 C.
  • An antibody in the cocktail may also comprise one or more of the following CDR sequences:
  • CDR sequences of the 040 antibody are CDR sequences of the 040 antibody.
  • the antibody comprises six CDRs as set out above.
  • An antibody in the cocktail may also comprise a VH of SEQ ID NO: 152 and/or a VL of SEQ ID NO: 153. These are the VH and VL sequences of the 040 antibody. Furthermore, an antibody may comprise a heavy chain of SEQ ID NO: 164 and a light chain of SEQ ID NO: 165. These are heavy and light chain sequences of 040.
  • An antibody in the cocktail may also comprise one or more of the following CDR sequences:
  • CDR sequences of the 6662 antibody typically comprises six CDRs as set out above.
  • An antibody in the cocktail may also comprise a VH of SEQ ID NO: 160 and/or a VL of SEQ ID NO: 161. These are the VH and VL sequences of the 6662 antibody.
  • an antibody may comprise a heavy chain of SEQ ID NO: 166 and a light chain of SEQ ID NO: 167. These are heavy and light chain sequences of 6662.
  • an antibody of the invention may be used in a cocktail in combination with an antibody based on 66-3-9C, 040 and 6662 (i.e. with the above sequences).
  • the antibodies may be formulated using a pharmaceutically acceptable carrier or diluent, as discussed above.
  • the antibodies, nucleic acids, pharmaceutical composition and cocktails of the invention may be used for the treatment, prevention or amelioration of Ebola virus infection.
  • the antibodies may be used for the treatment of disease associated with Ebola virus and/or to decrease the viral load.
  • Ebola virus disease develops after infection with Ebola virus and the subsequent incubation period.
  • Early symptoms of Ebola virus infection are fatigue fever, myalgia, headache, sore throat, which are followed by vomiting, diarrhoea, exanthema, renal and hepatic dysfunction, external haemorrhage and other symptoms.
  • Antibodies, pharmaceutical composition and cocktails of the invention may be used to ameliorate or reduce the severity, duration or frequency of one or more symptoms associated with Ebola virus infection.
  • the symptom may be fever, headache, fatigue, loss of appetite, myalgia, diarrhoea, vomiting, abdominal pain, dehydration and/or bleeding.
  • the invention comprises not only the administration of antibodies themselves, but also nucleic acid sequences encoding the antibodies (typically mRNA).
  • the invention relates to the administration of the antibodies/compositions to a human subject in need thereof.
  • administration to non-human animals such as rats, rabbits, sheep, pigs, cows, cats, dogs is also contemplated.
  • the subject may be at risk of exposure to Ebola virus infection, such as a healthcare worker or a person who has come into contact with an infected individual.
  • a subject may have visited or be planning to visit a country known or suspected of having an Ebola outbreak.
  • a subject may also be at greater risk, such as an immunocompromised individual (for example an individual receiving immunosuppressive therapy or an individual suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • the antibodies, nucleic acids, compositions and cocktails of the invention may be administered therapeutically or prophylactically.
  • the antibodies, nucleic acids, pharmaceutical compositions and cocktails may be administered subcutatneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially, Typically, the antibodies, nucleic acids, pharmaceutical compositions and cocktails are administered intravenously or subcutaneously.
  • the dose of an antibody may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration.
  • Antibodies may be administered at a dose of about 0.1 mg/kg body weight to a dose of about 100 mg/kg body weight, such as at a dose of about 5 mg/kg to about 10 mg/kg.
  • Antibodies may also be administered at a dose of about 50 mg/kg, 10 mg/kg or about 5 mg/kg body weight.
  • a cocktail of the invention may for example be administered at a dose of about 5 mg/kg to about 10 mg/kg for each antibody, or at a dose of about 10 mg/kg or about 5 mg/kg for each antibody.
  • a cocktail may be administered at a dose of about 5 mg/kg total (e.g. a dose of 1.67 mg/kg of each antibody in a three antibody cocktail).
  • the initial dose may be followed by administration of a second or plurality of subsequent doses.
  • the second and subsequent doses may be separated by an appropriate time.
  • the antibodies of the invention are typically used in a single pharmaceutical composition/cocktail (co-formulated).
  • the invention also generally includes the combined use of antibodies of the invention (in separate preparations/compositions).
  • “In combination with” means that a first antibody may be administered prior to, concurrent with or after a second (or subsequent) antibody.
  • “Concurrent” with includes administration both in single and separate dosage forms, where such separate dosage forms may be administered e.g. within 30 minutes or less of one another.
  • Prior to may include administration e.g. one week before, 48 hours before or 24 hours before.
  • “After” may include e.g. 24 hours after, 48 hours after, or 72 hours after.
  • the dosage forms may be administered by the same route, or by different routes.
  • “In combination with” also includes sequential or concomitant administration.
  • Adherent rabbit fibroblast cell line cultured in 5-stack CellSTACK® Culture Chambers were lifted using StemProTM AccutaseTM Cell Dissociation Reagent (Gibco, A1110501), washed and resuspended in Earle’s Balanced Salts (Sigma, E3024).
  • Cells were transfected using electroporation (160-170V, 20 seconds, 5 amps) with 3x10 L 7 cells in 600 pL per GenePulser electroporation cuvette (BioRad, #1652088).
  • Cells were transfected with EBOV GP (NP 066246.1), SUDV GP (YP 138523.1), BDBV GP (YP_003815435.1) or TAFV GP (YP_003815426.1). Cells were recovered in 50 mL media (RPME 10% FBS /2mM glutamine), transferred to T175 tissue culture flask and incubated at 37°C, 5% CO2 overnight. Cells were lifted the next day, counted and cell viability measured.
  • EBOV GP NP 066246.1
  • SUDV GP SUDV GP
  • BDBV GP BDBV GP
  • TAFV GP YP_003815426.1
  • One female New Zealand White rabbit was immunised four times subcutaneously at two week intervals with rabbit fibroblast cells expressing Ebola virus glycoproteins.
  • Each dose consisted of 2.5xl0 6 cells transfected with EBOV GP, SUDV GP, BDBV GP and TAFV GP (lxlO 7 cells/dose total) in 500 mE PBS.
  • First dose only was adjuvanted with complete Freund’s adjuvant delivered at a separate site. Serum was taken on day of each vaccination and presence of antigen specific IgG confirmed by binding of serum IgG to GPs transiently transfected HEK cells assessed using an iQUE flow cytometer system (Intellicyt).
  • Lymph nodes were harvested 14 days after final vaccination.
  • B cell culture screening was performed using a method similar to that described by Tickle et al. (Tickle et al. 2015, J Biomol Screen, 20: 492-7). Rabbit B cell cultures were prepared using 50 x 96-well plates at a cell density of approximately 5000 cells per well. After 6 days culture, screening was performed.
  • Ebola virus glycoprotein-binding antibodies in B cell culture supernatants was determined using a homogeneous fluorescence-based binding assay performed on a Mirrorball® fluorescence cytometer device using MDCK cells stably transfected to express surface glycoprotein from EBOV or SUDV B cell supernatants were simultaneously counter screened for binding to the parental MDCK SIAT-1 cells.
  • EBOV and SUDV GP expressing cells were stained with DiO and untransfected parental cells were stained with Dil (Vybrant Multicolour cell-labelling kit, Invitrogen, V22889).
  • Binding of B cell culture supernatant was revealed with Alexa Fluor® 647 AffmiPure F(ab') 2 Fragment Rabbit Anti-Human IgG, Fey fragment specific antibody (Jackson, 309- 606-008).
  • a homogeneous fluorescence-based binding assay was performed on an Applied Biosystems 8200 cellular detection system device using parental MDCK SIAT-1 cells, and stably transfected EBOV GP and SUDV GP expressing MDCK SIAT-1 cell lines thawed from aliquots stored in LN2. Binding was revealed with a Alexa Fluor® 647 AffmiPure F(ab') 2 Fragment Goat Anti-Rabbit IgG, Fc fragment specific antibody (Jackson, 111-606-046). Additionally, supernatants were screened in a flow cytometric assay using suspension ExpiHEK293 cells transiently transfected to express EBOV GP or SUDV GP.
  • ExpiHEK293 cells were transfected two days prior to use in the binding assay using ExpiFectamine 293 (Invitrogen) with EBOV GP, SUDV GP or an irrelevant antigen podoplanin as a negative control. Transfection enhancers were added the next day. On the third day, expression of surface Ebola virus glycoproteins was confirmed using known broadly-reactive Ebola virus GP human mAh and Alexa Fluor® 647 AffmiPure F(ab') 2 Fragment Goat Anti-Human IgG, Fey fragment specific antibody (Jackson, 109- 606-170).
  • podoplanin was confirmed with an Anti -Podoplanin mAh PE conjugate (Biolegend, 337004). After confirmation of antigen expression, approximately 10,000 transiently transfected cells/well were incubated with 10 pL B cell culture supematant/well in 384 well plates. After washing, cells were incubated with Alexa Fluor® 647 AffmiPure F(ab') 2 Fragment Goat Anti-Rabbit IgG, Fc fragment specific antibody (Jackson, 111-606-046). After washing, fluorescence was detected using an iQUE flow cytometer system (Intellicyt).
  • Antibody was purified from cell culture supernatant via Protein A affinity chromatography utilising an AKTA Pure system (GE Healthcare) with CETAC autosampler. Antibodies were purified from 30 mL of culture supernatant using 2ml HiTrap MabSelect SuRe Protein A column (GE Healthcare, Life Sciences) and eluted in 0.1M sodium citrate, pH 3.4. Eluted fractions were neutralised with 2 M Tris/HCl pH 8.0. Pooled peak fractions were buffer exchanged into PBS pH 7.4 and concentrated using Amicon Ultra Spin columns with a 30KDa cut off membrane (Millipore, UFC905008) and centrifugation at 4000 g, before sterile filtering.
  • AKTA Pure system GE Healthcare
  • CETAC autosampler CETAC autosampler. Antibodies were purified from 30 mL of culture supernatant using 2ml HiTrap MabSelect SuRe Protein A column (GE Healthcare, Life Sciences) and elute
  • ExpiHEK293 cells were transfected two days prior to use in the binding assay using ExpiFectamine 293 (Invitrogen) with EBOV GP, SUDV GP, BDBV GP or TAFV GP (same strains as used for immunisations for antibody generation) or an irrelevant antigen podoplanin as a negative control. Transfection enhancers were added the next day.
  • Results are presented in Figure 1. All broadly reactive mAbs showed titratable binding to all four species of GP tested and no reactivity to mock cells transfected with an irrelevant antigen.
  • Example 5 In vitro Ebola virus glycoprotein pseudotyped S-FLU virus microneutralisation assay
  • This assay measures the ability of an antibody to prevent the infection of MDCK SIAT-1 cells by an S-FLU pseudotype virus coated with GP from an Ebola virus (Xiao, J., et al, J Virol., 2018 Jan 30;92(4)).
  • the virus is replication incompetent and contains no viral RNA or DNA encoding the GP allowing it to be handled at BSL-2.
  • a GFP reporter gene in the virus replaces the hemagglutinin coding sequence, making infected MDCK SIAT-1 cells detectable by fluorescence. Loss of fluorescence signal indicates neutralisation of the virus and prevention of infection.
  • virus diluted in 50 pL viral growth media (VGM: DMEM, 0.1% BSA, lOmM HEPES, Penicillin/Streptomycin 100U each, 2mM Glutamine) was incubated with antibody diluted 50 pL in phosphate buffered saline (PBS) at titrated concentration for 2 hours, 37°C, 5% C02, before addition of 100 pL MDCK SIAT-1 cells in VGM (3c10 L 5 cells/mL). Virus was used at a dilution previously determined to give maximum infection of cells.
  • VGM viral growth media
  • PBS phosphate buffered saline
  • the panel of novel broadly reactive monoclonal antibodies were used to pan two different random combinatorial linear peptide libraries. Peptide sequences enriched by each mAb were analysed and motifs aligned with GP sequences to identify potential partial epitopes. In each experiment two rounds of panning against each mAb was conducted with decreasing concentrations of antibody. Round 2 panning also included a subtractive step where phage were incubated with non-GP specific rabbit IgG and unbound phage transferred to incubate with the broadly reactive GP mAb. All panning steps occurred with mAb adhered to a plastic surface. After Round 2, phagemid DNA sequences encoding peptide sequences were barcoded via PCR and subjected to Next Generation Sequencing (NGS).
  • NGS Next Generation Sequencing
  • Biopanning was carried out with both libraries against each monoclonal antibody with two rounds of selection and additional subtractive panning against species relevant IgG to reduce amplification of phage bound to portions of the antibodies other than the CDRs.
  • a control human antibody with known GP epitope previously identified via yeast display technologies was used to pan phage libraries in parallel to rabbit monoclonal antibodies to validate method.
  • R1 output bacteria were scraped (a small aliquot of each was flash frozen and stored at -80°C), grown at 37°C to OD-0.5 and rescued using M13K07 helper phage (NEB, N0315S). Rescued cultures were resuspended in 2TY- Carbenicillin/Kanamycin without glucose and grown overnight at 30°C to produce phage for R2 panning. Next day phage were precipitated using 20% PEG80002.5 M NaCl, resuspended in l-2ml PBS and blocked with 6% Milk/PBS for a R2 biopanning including a subtraction step.
  • R2 phagemid DNA was extracted and purified from bacterial glycerol stocks; 100 pL of each bacterial library was thawed and phagemid DNA purified using QIAprep Spin Miniprep kit (QIAGEN, 27104). DNA was eluted in 50 pL H2O.
  • PCR amplicon DNA containing peptide sequences of interest (9mer or 13mer) was generated using R2 phagemid DNA as template and barcoded primers were used that added DNA encoded barcodes to distinguish libraries generated by panning against different antibodies and that added PI adaptor sequences designed to enable attachment of DNA to beads for emulsion PCR required for Ion Torrent sequencing platform.
  • barcoded primers were used that added DNA encoded barcodes to distinguish libraries generated by panning against different antibodies and that added PI adaptor sequences designed to enable attachment of DNA to beads for emulsion PCR required for Ion Torrent sequencing platform.
  • 9mer phagemid libraries a single step PCR was utilised.
  • 13mer phagemid libraries a preparatory PCR was required to incorporate an adaptor sequence. Between PCRs samples were column purified using QiaQuick PCR Purification kit (QIAGEN) to remove primers.
  • PCR products were separated by gel electrophoresis using low melting point agarose and bands at ⁇ 350bp were excised and purified using Nucleospin columns (Machery- Nagel, 740609-250). DNA concentration was quantified using A260 (NanoDrop spectrophotometer) and a Bio analysesr 2100 high sensitivity DNA chip. 100 ng of DNA from each individually barcoded library was pooled. Pooled DNA was purified using magnetic AMPure XP beads (Beckman Coulter, A63880) and DNA concentration quantified using A260 (NanoDrop spectrophotometer).
  • NGS data was processed as described in Naqid et al, Scientific Reports, 2016, 6: 24232-24232.
  • a two proportion Z-score analysis (Zhang et al, PNAS, 2011) was conducted to identify peptides specifically enriched by mAh of interest.
  • each set of peptides enriched by a mAh of interest was compared to those enriched by mAh 11886.
  • Peptides enriched by mAh 11886 were compared to those enriched by human GP -binding antibody 66-3-9C.
  • each set of peptides enriched by a target mAh of interest was compared to those enriched by a control non-GP specific rabbit IgG which was panned against the library in parallel to the target GP -binding mAbs.
  • Z-score takes into account the frequency of a peptide sequence in the total number of sequences in the sample, as well as the ratio of the number of copies of a sequence in the sample isolated against the target antibody and number of copies of the same sequence in the sample isolated against the control antibody.
  • nl number of peptide sequences obtained with the target sample
  • n2 number of peptide sequences obtained with the control sample
  • pi the number of sequences obtained for a specific peptide against the target sample/nl
  • p2 the number of sequences obtained for a specific peptide against the control sample/n2
  • Peptides were ranked based on Z-score i.e. for specific enrichment in the total reads from Ion torrent sequencing by target mAh of interest. The top 100 peptides were used in all further analysis.
  • Motifs in the top 100 ranked peptides by z-score were identified using the Multiple EM for Motif Elicitation (MEME) algorithm (Bailey et al, Nucleic Acids Research , 1994). For MEME analysis it was assumed that each motif was expected to occur zero or one time per sequence (zoop). Algorithm was constrained such that motifs occur in a minimum number of ten peptide sequences. Motifs with an E value ⁇ 0.05 were considered significant.
  • Peptide motifs were aligned with GP sequences from EBOV (ATY51135), SUDV (YP_138523.1), BDBV (YP_003815435.1) and TAFV (YP_003815426.1) using Clustal Omega algorithm and MegAlignPro software (Version: 11.2.1).
  • This assay was used to determine if a GP antibody of interest competed for binding with GP antibodies of known epitope.
  • a panel of antibodies including >1 mAh for each epitope bin was used to confirm competition and place new antibodies of interest in one of three epitope bins; base, glycan cap or receptor binding region.
  • Antibodies cl3C6, c4G7, c2G4, CA45, 6D6, FVM02 and ADI15878 have epitopes published in the literature by other groups.
  • Antibodies 6541, 66-4-C12, 6660, 6662, 66-3- 9C, 66-3 -2C and 040 are fully human antibodies with epitopes determined via multiple assays as previously described (Rijal, P., et al, Cell Reports, 2019).
  • Antibody 21-D8-5A is an influenza neuraminidase-specific antibody acting as a negative control.
  • Antibodies 6541, 66-4-C12, c2G4 and c4G7 represent different overlapping base epitopes.
  • Antibodies 6660 and 6662 are designated as RBR binding. 66-3-9C, 66-3-2C, 040 and cl3C6 represent different GC epitopes. CA45, 6D6, FVM02, and ADI15878 bind to the conserved fusion loop. It is noted all novel antibodies tested all competed with cl3C6, irrespective of the other mAbs they competed in the panel, hence any apparent competition with cl3C6 is likely not meaningful.
  • Antibodies were biotinylated using EZ-Link Sulfo-NHS-Biotin kit (Thermo Scientific, 21326). Free biotin was removed using Zeba desalting 7K columns. Biotinylation of mAbs confirmed via dot blot revealed with ExtrAvidin-alkaline phosphatase conjugate (Sigma, E2636) andNBT/BCIP development.
  • MDCK SIAT-1 cells stably transfected to express EBOV GP were seeded at a density of 3xl0 A 5/per well in 100 pL D10 media and incubated for 18 hours, 37°C, 5% CO2.
  • Biotinylated mAbl at 5 pg/mL in PBS and unbiotinylated mAb2 at 50 pg/mL in PBS were mixed in equal volume prior to addition to cells.
  • Degree of competition was determined by: (X-Minimum binding)/(Maximum binding - Minimum binding), where ‘X’ is binding of the biotinylated mAb in presence of competing mAb, ‘minimum binding’ is the signal from the biotinylated mAb in the presence of self (unbiotinylated) and ‘maximum binding’ is the signal from the biotinylated mAb in presence of a non competing non-GP mAb.
  • Ebola virus GP is cleaved by cathepsins in the endosome in a process necessary for binding of GP to the human receptor NPC-C1.
  • Thermolysin mimics cathepsin cleavage of GP by removing the glycan cap and mucin-like domains revealing more of the receptor binding region and leaving base epitopes intact.
  • thermolysin (Sigma, P1512) in HM buffer (20mM MES/1M HEPES/5M NaCl in dH20) or HM buffer alone for lhr,
  • MR78 is a published receptor binding region antibody that cannot bind Ebola virus GP unless the glycan cap domain has been removed (Hashiguchi et al, Cell, 2015); in this assay MR78 directly labelled with Alexa Fluor 647 was used as positive control for digestion of GP by thermolysin.
  • 11886 is a broadly neutralising antibody and can neutralise all three pseudotyped viruses tested (Figure 3). IC50 values are within 2.5 fold of those of CA45 in the same assay against EBOV, SUDV and BDBV GP pseudotypes ( Figure 2). This assay suggests that compared to CA45, 11886 neutralisation of the EBOV S-FLU pseudotype is less potent, but neutralisation of SUDV S-FLU pseudotype is more potent.
  • the BDBV S-FLU pseudotype neutralisation profile for both mAbs is highly similar. Neutralisation of the EBOV S-FLU pseudotype in vitro has shown strong though incomplete correlation with protection against EBOV in a mouse challenge model (Rijal et al, Cell Rep, 2019).
  • binding of 11886 to GP is not completely independent of the presence of the glycan cap ( Figure 6). Binding of 11886 to EBOV GP is reduced to background after digestion of the GP by thermolysin. Binding to SUDV GP is partially reduced after thermolysin digestion. This indicates an intermediate epitope between the glycan cap and chalice base that is not part of the glycan cap, but that is affected by thermolysin digestion. This distinguishes the epitope of this antibody from other base mAbs such as 11892 or 6541 and from true glycan cap antibodies such as 11897 or 040.
  • 11886 was used to pan two different random combinatorial linear peptide libraries across three independent experiments. Z score analysis was applied to compare sequences enriched by 11886 and a control antibody. The top one hundred sequences specifically enriched by 11886 were analysed for motifs using the MEME tool (Bailey et al, Nucleic Acids Research, 1994) with an arginine-cysteine-arginine (RCR) motif highly represented with similar motifs enriched across experiments and in the independent libraries ( Figure 7).
  • Motifs derived from peptides enriched by 11886 predominantly align with a conserved portion of the GP sequences in GP1 ( Figure 8 ) that are not in the glycan cap domain, but the exterior of the receptor binding chalice ( Figure 10A, B) (GP residues 102-108 and 132-143, with motif of most confidence: R134, C135, R136).
  • peptides enriched by 11886 also contain an additional cysteine with spacing consistent with formation of an intra-peptide disulphide bond and a non-linear peptide.
  • This structural feature of the peptides is consistent with a turn structure seen in the GP in the middle of the arginine-cysteine-arginine (RCR) motif ( Figure 9).
  • 11886 does not strongly compete known fusion loop antibodies tested, especially those that are known to bind the fusion loop tip; the assay does indicate some, although not strong, competition with ADI-15878 (Figure 5B).
  • ADI-15878 has a published epitope spanning the paddle of fusion loop and contacts the GP predominantly in GP2, and the residues identified as involved in binding of this mAh to GP do not include those identified for 11886 by peptide phage display (Wee et al, Cell, 2017; West et al, mBio, 2018).
  • the partial competition seen may be due to the angle that these mAbs bind rather than overlapping binding footprint on the GP.
  • epitope of 11886 as defined by the peptide mapping is distinct form epitopes in the literature for other broadly protective mAbs.
  • the location of the 11886 binding region is proximal to, but not the same as, the binding region of fab ADI-15946 ( Figure 10D, PDB:6MAM).
  • the contacts made by ADI- 15946 involve the regions 71-77 (GP1), 251-303) (GC) and 508-514 (GP2) with a K510E escape mutant sufficient for complete escape of the virus from this antibody (Wee et al, Cell, 2017; West et al, Nature Structural & Molecular Biology, 2019).
  • a cocktail of afucosylated versions of ADI-23774 (a derivative of ADI-15946) and ADI-15878 named MBP134AF has been shown to be protective against EBOV, SUDV and BDBV challenge in non-human primates (Bomholdt et al, Cell Rep, 2019).
  • Antibody 11886 represents a broadly neutralising antibody that targets an epitope distinct to those in MBP134AF and thus a potential additional component to such cocktails as their design continues to develop.
  • Antibody FVM04 which has shown to be protective in EBOV and SUDV animal models and was able to partially neutralise BDBV in vitro binds to the crest of the chalice at the receptor binding region (Howell et al, Cell Rep, 2016).
  • the key residues for binding of this mAh identified by alanine scanning (K115, D117, and G118) ( Figure 10E) are not those identified for 18866 binding and sit higher up the chalice crest than the RCR motif.
  • Antibody m2 ID 10 is a broadly reactive, though non-neutralising mAb, that binds to GP from EBOV, SUDV (although with lower affinity) BDBV, RESTV as well as a related filovirus, Marburg virus (Holtsberg et al, J Virol, 2016). Like 11886 it also has an epitope on the outside of the receptor binding chalice; however, this antibody shows improved binding after removal of the glycan cap and its epitope constitutes residues 81-90 placing its epitope on the opposite side of the GP1 monomer to the RCR binding motif of 11886 ( Figure 10E).
  • antibody 11886 represents a novel broadly-reactive broadly-neutralising antibody that has an epitope distinct from those already published.
  • the current data suggest the epitope of this antibody sits on the chalice between the crest epitope represented by FVM04, and above the fusion loop antibodies and base epitope of ADI- 15946.
  • Antibodies were humanised in silico by grafting the CDRs from the rabbit antibody V-regions onto human germline antibody V-region frameworks. In order to recover the activity of the antibody, a number of framework residues from the rabbit V-regions were also retained in the humanised sequences. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanised antibodies. WO91/09967).
  • the CDRs grafted from the donor to the acceptor sequence are as defined by Rabat (Rabat et al., 1987), with the exception of CDRH1 where the combined Chothia/Rabat definition is used (see Adair et al., 1991 Humanised antibodies. WO91/09967).
  • VH genes of rabbit antibodies are shorter than the selected human VH acceptor genes.
  • framework 1 of the VH regions of rabbit antibodies When aligned with the human acceptor sequences, framework 1 of the VH regions of rabbit antibodies typically lack the N-terminal residue, which is retained in the humanised antibody.
  • Framework 3 of the rabbit antibody VH regions also typically lack one or two residues (75, or 75 and 76) in the loop between beta sheet strands D and E: in the humanised antibodies the gap is filled with the corresponding residues from the selected human acceptor sequence.
  • Human V-region IGRV1-5 plus JR4 J-region was chosen as the acceptor for antibody 11886 light chain CDRs.
  • one or more of the following framework residues from the 11886 VK gene may be retained at positions 2 and 3 (Rabat numbering): Valine (V2) and Valine (V3), respectively.
  • CDRL3 may be mutated to remove a Cysteine residue (CDRL3 variants; SEQ ID NOs: 4 and 5).
  • CDRH3 Human V-region IGHV3-66 plus JH5 J-region (IMGT, http://www.imgt.org/) was chosen as an acceptor for the heavy chain CDRs of antibody 11886.
  • donor residues one or more of the following framework residues from the 11886 VH gene (donor residues) may be retained at positions 23, 24, 48, 49, 71, 73 and 78 (Rabat numbering): Isoleucine (123), Valine (V24), Isoleucine (148), Glycine (G49), Lysine (R71), Alanine (A73) and Valine (V78), respectively.
  • CDRH3 may be mutated to modify a potential Aspartic Acid-Proline hydrolysis site (CDRH3 variants; SEQ ID NOs: 9 and 10).
  • Human V-region IGRV1D-13 plus JR4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 11897 light chain CDRs.
  • donor residue one or more of the following framework residues from the 11897 VR gene (donor residue) may be retained at positions 2, 3 and 70 (Rabat numbering): Valine (V2), Valine (V3) and Glutamine (Q70), respectively.
  • Human V-region IGHV3-30-3 plus JH4 J-region (IMGT, http://www.imgt.org/) was chosen as an acceptor for the heavy chain CDRs of antibody 11897.
  • donor residues one or more of the following framework residues from the 11897 VH gene (donor residues) may be retained at positions 24, 47, 48, 49, 73 and 78 (Rabat numbering): Valine (V24), Tyrosine (Y47), Isoleucine (148), Glycine (G49), Serine (S73) and Valine (V78), respectively.
  • CDRH2 may be mutated to remove a potential N-linked glycosylation site (CDRH2 variants; SEQ ID NOs: 39-41).
  • Human V-region IGRV1-5 plus JR4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 11878 light chain CDRs.
  • donor residue one or more of the following framework residues from the 11878 VK gene (donor residue) may be retained at positions 2, 3 and 38 (Kabat numbering): Valine (V2), Valine (V3) and Leucine (L38), respectively.
  • Human V-region IGHV3-66 plus JH4 J-region was chosen as an acceptor for the heavy chain CDRs of antibody 11878.
  • one or more of the following framework residues from the 11878 VH gene may be retained at positions 24, 48, 49, 71, 73 and 78 (Kabat numbering): Valine (V24), Isoleucine (148), Glycine (G49), Lysine (K71), Serine (S73) and Valine (V78), respectively.
  • Human V-region IGKV1D-13 plus JK4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 11883 light chain CDRs.
  • donor residue one or more of the following framework residues from the 11883 VK gene (donor residue) may be retained at positions 2, 3 and 71 (Kabat numbering): Valine (V2), Valine (V3) and Tyrosine (Y71), respectively.
  • IMGT Human V-region IGHV3-72 plus JH5 J-region
  • donor residues one or more of the following framework residues from the 11883 VH gene (donor residues) may be retained at positions 48, 49, 71, 73 and 78 (Kabat numbering): Isoleucine (148), Alanine (A49), Lysine (K71), Serine (S73) and Valine (V78), respectively.
  • CDRHl and CDRH2 may be mutated to remove Cysteine residues (CDRHl variants and CDRH2 variants, respectively; SEQ ID NOs: 68, 69, 71 and 72).
  • Human V-region IGKV1D-13 plus JK4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 11889 light chain CDRs.
  • donor residue one or more of the following framework residues from the 11889 VK gene (donor residue) may be retained at positions 2, 3 and 66 (Kabat numbering): Valine (V2), Valine (V3) and Arginine (R66), respectively.
  • CDRH1 and CDRH2 may be mutated to remove Cysteine residues (CDRH1 variants and CDRH2 variants, respectively; SEQ ID NOs: 87 and 89).
  • IGKV1-12 plus JK4 J-region was chosen as the acceptor for antibody 11892 light chain CDRs.
  • donor residue one or more of the following framework residues from the 11892 VK gene (donor residue) may be retained at positions 2 and 3 (Rabat numbering): Valine (V2) and Valine (V3), respectively.
  • CDRL3 may be mutated to remove a Cysteine residue (CDRL3 variants; SEQ ID NOs: 101 and 102).
  • CDRH3 Human V-region IGHV3-66 plus JH5 J-region (IMGT, http://www.imgt.org/) was chosen as an acceptor for the heavy chain CDRs of antibody 11892.
  • donor residues one or more of the following framework residues from the 11892 VH gene (donor residues) may be retained at positions 24, 48, 49, 71, 73, 76 and 78 (Rabat numbering): Valine (V24), Isoleucine (148), Glycine (G49), Lysine (R71), Serine (S73), Threonine (T76) and Valine (V78), respectively.
  • CDRH3 may be mutated to modify a potential Aspartic Acid-Proline hydrolysis site (CDRH3 variants; SEQ ID NOs: 106 and 107).
  • Human V-region IGRV1-12 plus JR4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 11881 light chain CDRs.
  • one or more of the following framework residues from the 11881 VR gene may be retained at positions 2 and 3 (Rabat numbering): Valine (V2) and Valine (V3), respectively.
  • CDRL3 may be mutated to remove a Cysteine residue (CDRL3 variants; SEQ ID NOs: 121 and 122).
  • CDRH3 Human V-region IGHV3-66 plus JH5 J-region (IMGT, http://www.imgt.org/) was chosen as an acceptor for the heavy chain CDRs of antibody 11881.
  • donor residues one or more of the following framework residues from the 11881 VH gene (donor residues) may be retained at positions 24, 48, 49, 71, 73, 76 and 78 (Rabat numbering): Valine (V24), Isoleucine (148), Glycine (G49), Lysine (K71), Serine (S73), Threonine (T76) and Valine (V78), respectively.
  • CDRH3 may be mutated to modify a potential Aspartic Acid-Proline hydrolysis site (CDRH3 variants; SEQ ID NOs: 126 and 127).
  • GKRCRGVDC SEQ ID NO: 31 - 9mer2 motif 2
  • GRCRSSV SEQ ID NO: 32 - 13mer motif 1
  • VAGYAGYGYAFYDAFEP SEQ ID NO: 127 - 11881 CDRH3 variant
  • AASSLQS SEQ ID NO: 151 (040 LCDR3)
  • SEQ ID NO: 163 (66-3-9C kappa light chain) MGWSCIILFLVATATGVHSDIVMTQSPLSLPVTPGEPAS ISCRSSQSLLHSDGYNYLDWYL QKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 164 (040 heavy chain)
  • RSSQSLLHSDSYNYLD SEQ ID NO: 170 (66-3-9C variant LCDR1)

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Abstract

L'invention concerne des anticorps qui se lient à la glycoprotéine du virus Ebola, en particulier des cocktails d'anticorps comprenant de tels anticorps. Les anticorps et les cocktails peuvent être utilisés pour traiter, prévenir ou réduire une infection par le virus Ebola.
PCT/GB2020/052998 2019-11-29 2020-11-25 Anticorps WO2021105669A1 (fr)

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WO2023039612A1 (fr) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Protéines de liaison à l'antigène trem2 et leurs utilisations
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