WO2020148554A1 - Antibodies to ebola virus glycoprotein - Google Patents

Antibodies to ebola virus glycoprotein Download PDF

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Publication number
WO2020148554A1
WO2020148554A1 PCT/GB2020/050103 GB2020050103W WO2020148554A1 WO 2020148554 A1 WO2020148554 A1 WO 2020148554A1 GB 2020050103 W GB2020050103 W GB 2020050103W WO 2020148554 A1 WO2020148554 A1 WO 2020148554A1
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WIPO (PCT)
Prior art keywords
seq
antibody
antibodies
nos
binding
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PCT/GB2020/050103
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French (fr)
Inventor
Daniel John Lightwood
Victoria Louise REDGRAVE-O'DOWD
Alain TOWNSEND
Pramila RIJAL
Lisa Marie Smith
Simon John DRAPER
Sean Christopher ELIAS
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UCB Biopharma SRL
Oxford University Innovation Limited
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Priority to EP20701856.5A priority Critical patent/EP3911414A1/en
Publication of WO2020148554A1 publication Critical patent/WO2020148554A1/en

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    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies binding to the Ebola virus glycoprotein.
  • the present invention relates to cocktails comprising a mixture of antibodies binding to different epitopes on Ebola virus glycoprotein.
  • the invention also relates to methods of preventing, ameliorating or treating an Ebola virus infection using such antibodies and cocktails.
  • the ZMapp cocktail of murine chimeric antibodies (cl3C6, c2G4 and c4G7), one targeting the glycan cap and two to the base of the glycoprotein, was successful in protecting 100% of non-human primates as late as 5-days post infection (Qiu et al. 2014, Nature, 514, 47-53).
  • the antibodies of human origin, 114 (to the receptor binding region) and 100 (to the base) showed a similarly profound therapeutic effect (Corti et al. 2016, Science, 351 : 1339-42).
  • the ZMapp cocktail was not proven statistically to be protective in human trials during the outbreak in West Africa because of the small number of participants, there was a trend in the direction of improved survival (Group et al.
  • VIC Viral Haemorrhagic Fever Immunotherapeutic Consortium
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies that bind to Ebola virus glycoprotein and a pharmaceutically acceptable carrier or diluent, wherein at least one of the antibodies comprises a set of six CDRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, selected from the group consisting of: SEQ ID NOs: 9-14, SEQ ID NOs: 15-20, SEQ ID NOs: 21-26 and SEQ ID NOs: 27-32.
  • the present invention also provides a nucleic acid or a pair of nucleic acids encoding the heavy and light chains of an antibody of the invention. Furthermore, the invention provides an expression vector comprising the nucleic acid(s) or pair of nucleic acids, a host cell comprising the expression vector and a method of producing an antibody of the invention, comprising culturing the host cell under conditions permitting production of the antibody and recovering the antibody so produced.
  • the invention provides a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering a pharmaceutical composition an antibody of the invention to a subject in need thereof.
  • the invention provides a pharmaceutical composition of the invention for use in a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the
  • the invention provides use of a pharmaceutical composition of the invention in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
  • the invention also provides an antibody cocktail comprising three or more antibodies binding to the Ebola virus glycoprotein, wherein one antibody binds to the glycan cap, one antibody binds to the receptor binding region and one antibody binds to the base.
  • the invention provides a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the antibody cocktail of the invention to a patient in need thereof.
  • the invention provides the antibody cocktail of the invention for use in a method of treating, preventing or ameliorating Ebola virus infection, said method comprising administering the antibody cocktail to a patient in need thereof.
  • the invention provides use of the antibody cocktail of the invention in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
  • Figure 1 List of the monoclonal antibodies with their V H and V l/K genes.
  • Figure 2 Human monoclonal antibodies isolated from vaccinated individuals.
  • A A total of 82 antibodies were isolated from 11 vaccinated volunteers. 38 out of 82 antibodies blocked infection of MDCK-SIAT cells by E-S-FLU.
  • B Example of in vitro microneutralisation of Ebola pseudotyped influenza virus (named as E-S-FLU virus) infecting MDCK-SIAT cells.
  • C Microscopic images showing virus neutralisation by different antibodies at 5 mg/ml concentration (40x Magnification). Cell monolayer shows a confluent monolayer of MDCK-SIAT 1 cells. In the absence of antibody (virus only control), the majority of the cells are infected, which is indicated by eGFP expression.
  • Partial neutralising antibody 66-4-C12 suppressed the virus infection partially, whereas the strong neutraliser 66-6-3 completely suppressed infection by the virus.
  • Figure 3 Identification of Epitopes.
  • Figure 4 Binding of mAb P6 to peptide fragments of large yeast display library.
  • FIG. 5 Effects of thermolysin on glycan cap mAbs.
  • Binding unit mAb binding/ WGA fluorescence.
  • THL Thermolysin
  • RBR Receptor binding region
  • MLD Mucin-like domain
  • SA-AF647 Streptavidin labelled with Alexa fluor 647.
  • FIG. 6 Identification antibodies recognising mucin-like domain (MLD) of the glycoprotein.
  • MLD mucin-like domain
  • Figure 7 Effect of GP specific antibodies on cleavage by thermolysin.
  • GC Glycan cap
  • RBR Receptor binding region
  • THL Thermolysin
  • E- SIAT MDCK-SIAT1 cells expressing Ebola virus glycoprotein.
  • Figure 8 Sequence homology of the glycoproteins used.
  • Figure 9 Twenty out of 82 mAbs are cross-reactive to both Sudan and Bundibugyo GPs.
  • FIG. 10 A) Phylogeny of the antibodies based on VDJ amino acid sequences. More than 23 VH genes have been used altogether. VH3-15 is the most used gene and all nine antibodies that recognize 114-like epitope are encoded by this gene. There is diversity in terms of VH gene use within individuals and within the antibodies to epitopes in glycan cap and base. Tree was drawn using MEGA v7 software and alignment was done using Neighour-joining tree settings. B) CDR3 length and frequency of somatic mutations in the largest set of VH genes.
  • Figure 11 Range of affinity constants for antibodies isolated from vaccinated donors. Calculated affinity constants for a selection of neutralising antibodies compared to established therapeutic antibodies 114 to RBR and 100 to the Base, 6D6 to fusion peptide, cl3C6 to RBR.
  • Figure 12 Selection of antibody cocktails.
  • F Characteristics of antibodies selected for inclusion in cocktail for guinea pig trial.
  • Figure 13 Protection of guinea pigs by antibody mixtures against Ebola virus infection.
  • Figure 14 Viral PCR of the samples from guinea pigs that survived via
  • Figure 16 Liver of animal 19063, 924/17 (group 1). Patchy staining of tissue denoting presence of viral antigen, and often corresponding to focal areas of necrosis.
  • Figure 17 Spleen of animal 000176,930/17. Strong, diffuse, positive staining for viral antigen primarily in the red pulp.
  • FIG. 18 Liver from animal 19666,940/17. Patchy staining of viral antigen.
  • Figure 19 (A) Liver immunohisto chemistry for group 5. (B) Spleen
  • Figure 20 Liver of animal 00436,957/17. Strong, multifocal staining of viral antigen.
  • Figure 21 Spleen of animal 9186,931/17 (group 2) showing congestion of the sinusoids.
  • Figure 22 Spleen of animal 02176,946/17 (group 5) showing scattered, degenerating cells with fragmented nuclei.
  • Figure 23 Spleen of animal 19186,931/17 (group 2) showing infiltration of neutrophils in the red pulp.
  • Figure 24 Spleen of animal 00252,927/17 (group 1) showing scattered lymphocyte loss, likely apoptosis.
  • Figure 25 Spleen of animal 19058,945/17 (group 4) showing reduction of cells in the white pulp.
  • Figure 26 Liver of animal 00252,927/17 (group 1) showing scattered necrotic foci comprising degenerating cells and nuclear debris with scattered mineralised areas.
  • Figure 27 Animal 00252,927/17 (groupl ) showing foci of mineralisation within the necrotic areas.
  • Figure 28 Liver of animal 00252,927/17 (group 1) showing diffusely scattered, macrovesicular vacuo lation in hepatocytes (lipid).
  • Figure 29 HCVR and LCVR sequences of antibodies of the invention.
  • SEQ ID NO: 1 provides the sequence of the 66-3-9C heavy chain variable region.
  • SEQ ID NO: 2 provides the sequence of the 66-3-9C light chain variable region.
  • SEQ ID NO: 3 provides the sequence of the 040 heavy chain variable region.
  • SEQ ID NO: 4 provides the sequence of the 040 light chain variable region.
  • SEQ ID NO: 5 provides the sequence of the 6662 heavy chain variable region.
  • SEQ ID NO: 6 provides the sequence of the 6662 light chain variable region.
  • SEQ ID NO: 7 provides the sequence of the 6541 heavy chain variable region.
  • SEQ ID NO: 8 provides the sequence of the 6541 light chain variable region.
  • SEQ ID NO: 9 provides the sequence of the 66-3-9C HCDR1.
  • SEQ ID NO: 10 provides the sequence of the 66-3-9C HCDR2.
  • SEQ ID NO: 11 provides the sequence of the 66-3-9C HCDR3.
  • SEQ ID NO: 12 provides the sequence of the 66-3-9C LCDR1.
  • SEQ ID NO: 13 provides the sequence of the 66-3-9C LCDR2.
  • SEQ ID NO: 14 provides the sequence of the 66-3-9C LCDR3.
  • SEQ ID NO: 15 provides the sequence of the 040 HCDR1.
  • SEQ ID NO: 16 provides the sequence of the 040 HCDR2.
  • SEQ ID NO: 17 provides the sequence of the 040 HCDR3.
  • SEQ ID NO: 18 provides the sequence of the 040 LCDR1.
  • SEQ ID NO: 19 provides the sequence of the 040 LCDR2.
  • SEQ ID NO: 20 provides the sequence of the 040 LCDR3.
  • SEQ ID NO: 21 provides the sequence of the 6662 HCDR1.
  • SEQ ID NO: 22 provides the sequence of the 6662 HCDR2.
  • SEQ ID NO: 23 provides the sequence of the 6662 HCDR3.
  • SEQ ID NO: 24 provides the sequence of the 6662 LCDR1.
  • SEQ ID NO: 25 provides the sequence of the 6662 LCDR2.
  • SEQ ID NO: 26 provides the sequence of the 6662 LCDR3.
  • SEQ ID NO: 27 provides the sequence of the 6541 HCDR1.
  • SEQ ID NO: 28 provides the sequence of the 6541 HCDR2.
  • SEQ ID NO: 29 provides the sequence of the 6541 HCDR3.
  • SEQ ID NO: 30 provides the sequence of the 6541 LCDR1.
  • SEQ ID NO: 31 provides the sequence of the 6541 LCDR2.
  • SEQ ID NO: 32 provides the sequence of the 6541 LCDR3.
  • SEQ ID NO: 33 provides the sequence of the 6660 HCDR3.
  • SEQ ID NO: 34 provides the sequence of the 6669 HCDR3.
  • SEQ ID NO: 35 provides the sequence of the 6670 HCDR3.
  • SEQ ID NO: 36 provides the sequence of the 6666 HCDR3.
  • SEQ ID NO: 37 provides the sequence of the 6667 HCDR3.
  • SEQ ID NO: 38 provides the sequence of the P7 HCDR3.
  • SEQ ID NO: 39 provides the sequence of the 105 HCDR3.
  • SEQ ID NO: 40 provides the sequence of the 66-6-14 HCDR3.
  • SEQ ID NO: 41 provides the sequence of the 56-4-D4 HCDR3.
  • SEQ ID NOIs: 42-205 provide the heavy and light junction sequences as shown in Figure 1
  • SEQ ID NO: 206 provides the sequence of the 66-3-9C heavy chain.
  • SEQ ID NO: 207 provides the sequence of the 66-3-9C light chain.
  • SEQ ID NO: 208 provides the sequence of the 040 heavy chain.
  • SEQ ID NO: 209 provides the sequence of the 040 light chain.
  • SEQ ID NO: 210 provides the sequence of the 6662 heavy chain.
  • SEQ ID NO: 211 provides the sequence of the 6662 light chain.
  • SEQ ID NO: 212 provides the sequence of the 6541 heavy chain.
  • SEQ ID NO: 213 provides the sequence of the 6541 light chain.
  • SEQ ID NO: 214 provides the sequence of the 6660 heavy chain.
  • SEQ ID NO: 215 provides the sequence of the 6660 light chain.
  • SEQ ID NO: 216 provides the sequence of the 66-3-9C heavy chain.
  • SEQ ID NO: 217 provides the sequence of the 66-3-9C light chain.
  • SEQ ID NO: 218 provides the sequence of the 66-6-3 heavy chain.
  • SEQ ID NO: 219 provides the sequence of the 66-6-3 light chain.
  • SEQ ID Nos: 220-337 provide the heavy and light chain variable regions of the antibodies in Figure 29 (Table 10).
  • SEQ ID NOs: 378-380 provide variant sequences of the 66-3-9C LCDR1
  • SEQ ID NOs: 381-383 provide variant sequences of the 6662 HCDR2
  • SEQ ID NOs: 384 and 385 provide variant sequences of the 6662 HCDR3
  • SEQ ID NOs: 386-388 provide variant sequences of the 6662 LCDR3
  • SEQ ID NOs: 389-392 provide variant sequences of the 6541 HCDR2
  • SEQ ID NO: 393 provides the sequence of the 66-4C-12 HCDR1
  • SEQ ID NO: 394 provides the sequence of the 66-4C-12 HCDR2
  • SEQ ID NO: 395 provides the sequence of the 66-4C-12 HCDR3
  • SEQ ID NO: 396 provides a variant sequence of the 66-4C-12 HCDR3
  • SEQ ID NO: 397 provides the sequence of the 66-4C-12 LCDR1
  • SEQ ID NO: 398 provides the sequence of the 66-4C-12 LCDR2
  • SEQ ID NO: 399 provides the sequence of the 66-4C-12 LCDR3
  • SEQ ID NO: 400 provides the sequence of the 6651 HCDR1
  • SEQ ID NO: 401 provides the sequence of the 6651 HCDR2
  • SEQ ID NO: 402 provides the sequence of the 6651 HCDR3
  • SEQ ID NO: 403 provides the sequence of the 6651 LCDR1
  • SEQ ID NO: 404 provides the sequence of the 6651 LCDR2
  • SEQ ID NO: 405 provides the sequence of the 6651 LCDR3
  • 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, Ta ' i Forest and Bombali) and many different strains. Zaire, Sudan, Bundibugyo and Ta ' i Forest cause disease in humans, with Zaire being the most deadly.
  • the Ebola virus glycoprotein is the only virally expressed protein on the virus 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 virus surface.
  • GP1 contains the receptor-binding site responsible for host cell attachment, the glycan cap and the mucin like domain
  • GP2 contains heptad repeats and a transmembrane domain.
  • Ebola virus glycoprotein sequences vary between species.
  • GenBank: AF086833.2 providing the complete genome of Zaire Mayinga
  • GenBank: AAG40168.1 providing examples of Zaire glycoprotein sequences.
  • Accession numbers NC_014373.1 and NC_006432.1 provide the complete genome sequences ofBudibugyo and Sudan Gulu.
  • GenBank: AGL73446.1 and GenBank: AGL73439.1 provide examples of a Sudan glycoprotein sequence and accession numbers GenBank: AGL73474.1 and GenBank: AGL73467.1 provide examples of Bundibugyo glycoprotein sequences. 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
  • CDR complementarity determining regions
  • FR framework regions
  • the constant regions of the antibodies may mediate the binding of the antibodies
  • 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.
  • 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 human 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 al., 1998, Journal of Immunological Methods, 216, 165-181).
  • Other antibody fragments for use in the present invention include the Fab and Fab’ fragments described in International patent applications WO 2005/003169, WO
  • 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 human IgA, IgD, IgE, IgG or IgM domains.
  • the constant regions are human.
  • human IgG i.e. IgG1, IgG2, IgG3 or IgG4 constant region domains may be used.
  • a human IgGl constant region i.e. IgG1, IgG2, IgG3 or IgG4
  • the light chain constant region may be either lambda or kappa.
  • kappa light chain constant regions may be used with the 040, 6541 and 66-3-9C antibodies and a lambda light chain constant region may be used with the 6662 antibody.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • 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 al, 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al, 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 187 9-18), 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
  • Human antibodies can also be generated using the method described in the Examples below.
  • 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, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework 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 or rat 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 or rat 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 above 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 above are transferred to the human antibody framework.
  • only the specificity determining residues from each of the CDRs described herein above 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 neutralising 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.
  • 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.
  • CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Rabat definition, the Chothia definition, and the AbM definition.
  • the Rabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Rabat and Chothia approaches. See, e.g., Rabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md.
  • An antibody of the invention may contain one, two, three, four, five or six CDR sequences from an antibody heavy and light chain variable region sequence pair of the invention (including those identified in Table 10).
  • An antibody of the invention typically comprises all six (i.e. three heavy and three light chain) CDR sequences from a heavy/light chain variable region sequence pair of the invention.
  • an antibody of the invention may comprise six CDRs contained within a heavy and light chain variable region sequence pair of SEQ ID NOs: 1/2, 3/4, 5/6, or 7/8. These are the heavy and light chain variable region sequence pairs of the 66-3-9C, 040, 6662 and 6541 antibodies of the invention.
  • An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 9 to 11 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 66-3-9C antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 12 to 14
  • LCDDR1/LCDR2/LCDR3 are the LCDR1/LCDR2/LCDR3 sequences of the 66-3-9C antibody of the Examples (as per the Rabat definitions).
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 11.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 9 to 11 and at least one light chain CDR sequence selected from SEQ ID NOS 12 to 14.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 9 to 11 and at least two light chain CDR sequences selected from SEQ ID NOS: 12 to 14.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 9 to 11 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 12 to 14 (LCDR1/LCDR2/LCDR3 respectively).
  • an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 15 to 17
  • HCDR1/HCDR2/HCDR3 respectively. These are the HCDR1/HCDR2/HCDR3 sequences of the 040 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 18 to 20
  • LCDDR1/LCDR2/LCDR3 are the LCDR1/LCDR2/LCDR3 sequences of the 040 antibody of the Examples (as per the Rabat definition).
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 17.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 15 to 17 and at least one light chain CDR sequence selected from SEQ ID NOS 18 to 20.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 15 to 17 and at least two light chain CDR sequences selected from SEQ ID NOS: 18 to 20.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 15 to 17 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 18 to 20 (LCDR1/LCDR2/LCDR3 respectively).
  • the antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 21 to 23 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 6662 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 24 to 26
  • LCDR1/LCDR2/LCDR3 respectively. These are the LCDR1/LCDR2/LCDR3 sequences of the 6662 antibody of the Examples (as per the Rabat definition).
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 23.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 21 to 23 and at least one light chain CDR sequence selected from SEQ ID NOS 24 to 26.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 21 to 23 and at least two light chain CDR sequences selected from SEQ ID NOS: 24 to 26.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 21 to 23 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 24 to 26 (LCDR1/LCDR2/LCDR3 respectively).
  • the antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 27 to 29
  • HCDR1/HCDR2/HCDR3 respectively. These are the HCDR1/HCDR2/HCDR3 sequences of the 6541 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 30 to 32
  • LCDDR1/LCDR2/LCDR3 are the LCDR1/LCDR2/LCDR3 sequences of the 6541 antibody of the Examples (as per the Rabat definition).
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 29.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 27 to 29 and at least one light chain CDR sequence selected from SEQ ID NOS 30 to 32.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 27 to 29 and at least two light chain CDR sequences selected from SEQ ID NOS: 30 to 32.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 27 to 29 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 30 to 32 (LCDR 1 /LCDR2/LCDR3 respectively).
  • An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 393 to 395 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 66-4C-12 antibody of the Examples.
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 397 to 399
  • LCDDR1/LCDR2/LCDR3 are the LCDR1/LCDR2/LCDR3 sequences of the 66-4C-12 antibody of the Examples.
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 395.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 393 to 395 and at least one light chain CDR sequence selected from SEQ ID NOS 397 to 399.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 393 to 395 and at least two light chain CDR sequences selected from SEQ ID NOS: 397 to 399.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 393 to 395 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 397 to 399 (LCDR1/LCDR2/LCDR3 respectively).
  • An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 400 to 402 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 6651 antibody of the Examples.
  • the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 403 to 405
  • LCDDR1/LCDR2/LCDR3 respectively. These are the LCDR1/LCDR2/LCDR3 sequences of the 6651 antibody of the Examples.
  • the antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 402.
  • the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 400 to 402 and at least one light chain CDR sequence selected from SEQ ID NOS 403 to 405.
  • the antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 400 to 402 and at least two light chain CDR sequences selected from SEQ ID NOS: 403 to 405.
  • the antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 400 to 402 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 403 to 405 (LCDR1/LCDR2/LCDR3 respectively).
  • an antibody of the invention may comprise an HCDR3 sequence of one of the antibodies of the Examples (SEQ ID NOs: 33-41).
  • An antibody of the invention may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 1 (the HCVR of 66-3-9C).
  • An antibody of the invention may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 2 (the LCVR of 66-3-9C).
  • An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 2.
  • An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 3 (the HCVR of 040).
  • An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 4 (the LCVR of 040).
  • An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4.
  • An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 5 (the HCVR of 6662).
  • An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 6 (the LCVR of 6662).
  • An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 5 and the light chain variable region sequence of SEQ ID NO: 6.
  • An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 7 (the HCVR of 6541).
  • An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 8 (the LCVR of 6541).
  • An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.
  • SEQ ID NO: 206 presents the complete heavy chain sequence of 66-3-9C and SEQ ID NO: 207 presents the complete light chain sequence of 66-3-9C (kappa light).
  • An antibody of the invention may comprise the 66-3-9C variable regions, the heavy chain constant region from SEQ ID NO: 206 and the light chain constant region from SEQ ID NO: 207.
  • An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 206 and a light chain of SEQ ID NO: 207.
  • SEQ ID NO: 208 presents the complete heavy chain sequence of 040 and SEQ ID NO: 209 presents the complete light chain sequence of 040 (kappa light).
  • An antibody of the invention may comprise the 040 variable regions, the heavy chain constant region from SEQ ID NO: 208 and the light chain constant region from SEQ ID NO: 209.
  • An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 208 and a light chain of SEQ ID NO: 209.
  • SEQ ID NO: 210 presents the complete heavy chain sequence of 6662 and SEQ ID NO: 211 presents the complete light chain sequence of 6662 (lambda light).
  • An antibody of the invention may comprise the 6662 variable regions, the heavy chain constant region from SEQ ID NO: 210 and the light chain constant region from SEQ ID NO: 211.
  • An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 210 and a light chain of SEQ ID NO: 211.
  • SEQ ID NO: 212 presents the complete heavy chain sequence of 6541 and SEQ ID NO: 213 presents the complete light chain sequence of 6541 (kappa light).
  • An antibody of the invention may comprise the 6541 variable regions, the heavy chain constant region from SEQ ID NO: 212 and the light chain constant region from SEQ ID NO: 213.
  • An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 212 and a light chain of SEQ ID NO: 213.
  • An antibody of the invention may also comprise six CDR sequences of a
  • HCVR/LVCR pair as identified in Figure 29 (Table 10). Such CDRs may be identified using the methods described above.
  • an antibody of the invention may comprise a HCVR or LCVR (or a HCVR/LVCR pair) as identified in Table 10.
  • antibodies with the above sequences but engineered for example to (i) remove deamidation and glycosylation sites and/or (ii) iso- asp removal and/or (iii) C-terminal lysine removal and/or N-terminal Q to E exchange.
  • 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 or NT.
  • 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.
  • variant LCDR1 sequences for the 66-3-9C antibody are presented in SEQ ID NOs: 378-380.
  • An antibody of the invention may comprise one of these variant sequences.
  • an antibody may comprise a LCDR1 of one of SEQ ID NOs: 378-380 and then HCDR1, HCDR2, HCDR3, LCDR2 and LCDR3 sequences of SEQ ID NOs: 9, 10, 11, 13 and 14 respectively.
  • Variant HCDR2 sequences for the 6662 antibody are presented in SEQ ID NOs: 381, 382 and 383.
  • Variant HCDR3 sequences for the 6662 antibody are presented in SEQ ID NOs: 384 and 385.
  • Variant LCDR2 sequences for the 6662 antibody are presented in SEQ ID NOs: 386, 387 and 388.
  • An antibody of the invention may comprise one or more of these variant CDR sequences.
  • an antibody may comprise a HCDR1 sequence of SEQ ID NO: 21, a HCDR2 sequence of SEQ ID NO: 22, 381, 382 or 383, a HCDR3 sequence of SEQ ID NO: 23, 284 or 285, a LCDR1 sequence of SEQ ID NO: 24, a LCDR2 sequence of SEQ ID NO: 25 and a LCDR3 sequence of SEQ ID NO: 26, 386, 387 or 388.
  • An a antibody of the invention may comprise any combination of the above CDRs.
  • Variant HCDR2 sequences for the 6541 antibody are presented in SEQ ID NOs: 389- 392.
  • An antibody of the invention may comprise one of these variant sequences.
  • an antibody may comprise a HCDR2 of one of SEQ ID NOs: 389-392 and HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 27, 29, 30, 31 and 32 respectively.
  • a variant HCDR3 sequence for the 66-4-C12 antibody is presented in SEQ ID NO: 396.
  • An antibody of the invention may comprise this variant sequence.
  • an antibody may comprise a HCDR3 of SEQ ID NO: 396 and HCDR1 , HCDR2, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NOs: 393, 394, 397, 398 and 399 respectively.
  • 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 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 (particularly the HCVR/LCVR sequences). Furthermore, 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
  • HCVR/LCVR sequences disclosed herein whilst retaining the exact CDRs disclosed for these sequences. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%,
  • 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 el 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 these chains.
  • 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 virus expressing the Ebola virus glycoprotein.
  • E-S-FLU Ebola virus surrogate as described in the Examples below.
  • This assay utilises a disable 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 a maximum antibody concentration of 5 mg/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 mg/ml.
  • the 6660, 125 and 66-6-3 antibodies are strong neutralising antibodies and the 66-4-C12 was an example of a partial neutralising antibody.
  • Antibodies of the invention may have sequences as described above and be either a strong or partial neutralising antibody.
  • an antibody of the invention may have the six CDR sequences of 6660, 125 or 66-6-3 and be a strong neutralising antibody.
  • Neutralisation may also be determined using IC 50 or IC 90 values.
  • IC 50 and IC 90 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 IC 50 value of less than (i.e. better than) 10 mg/ml, less than 5 mg/ml, less than 1.25 mg/ml, less than 1 mg/ml or less than 0.75 mg/ml (typically down to 0.1 mg/ml).
  • an antibody of the invention may have an IC 50 value of between 0.1 mg/ml and 10 mg/ml, sometimes between 0.1 mg/ml and 5 mg/ml or even between 0.1 mg/ml and 1 mg/ml.
  • an antibody of the invention may have an IC 50 value of between 1 mg/ml and 10 mg/ml, sometimes between 1 mg/ml and 5 mg/ml.
  • An antibody of the invention may have an IC 90 value of less than 10 mg/ml, optionally less than 5 mg/ml (typically down to 0.6 mg/ml).
  • an antibody of the invention may have an IC 90 value of between 0.6 mg/ml and 10 mg/ml, for example between 1 mg/ml and 5 mg/ml.
  • These 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 and an IC 50 / IC 90 value as presented above.
  • Neutralisation ability may be determined for any species of Ebola virus, such as Zaire, as shown in the Examples.
  • an antibody of the invention may have an affinity constant (K D ) value for the glycoprotein monomer of 50 nM or less, 25 nM or less of 10 nM or less.
  • An antibody of the invention may have an affinity constant (K D ) value for the glycoprotein trimer of 50 nM or less, 10 nM or less of 1 nM or less.
  • any antibody may have (a) the six CDR sequences of the 6541 antibody and a K D for the monomeric glycoprotein of 50 nM or less and a K D for the trimeric glycoprotein of 50 nM or less; (b) the six CDR sequences of the 040 antibody and a K D for the monomeric glycoprotein of 25 nM or less and a K D for the trimeric glycoprotein of 10 nM or less or (c) the six CDR sequences of the 66-3-9C antibody and a K D for the monomeric glycoprotein of 25 nM or less and a K D for the trimeric glycoprotein of 1 nM or less.
  • Affinity constants are typically determined using Surface Plasmon Resonance (Biacore) at 25 °C.
  • 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.
  • in vivo protection may be determined in mice administered a single 100 mg dose of antibody at day two of infection. In vivo protection may also be determined in guinea pigs as described in the Examples. In such experiments, antibodies may be administered, for example, at a dose of 10 mg/kg of each antibody at day three of infection.
  • antibodies of the invention may be 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).
  • Zaire e.g. Zaire Mayinga and/or Makona
  • Bundibugyo and Sudan e.g. Sudan Gulu
  • 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 as described in the Examples.
  • 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
  • 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 mM 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 and Sudan). K D values may be determined as described above.
  • the antibodies preferably also retain their neutralisation capabilities and their protection capabilities in the other species.
  • 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 DNA 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.
  • DNA 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, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.
  • a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the present invention.
  • Any suitable host cell/ vector system may be used for expression of the DNA 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
  • a pharmaceutical composition comprising one or more antibodies that bind to the Ebola virus glycoprotein, such as one or more antibodies of the invention as described above, and a pharmaceutically acceptable carrier or diluent. 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.
  • at least one of the antibodies may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences contained within a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs:1/2, 3/4, 5/6 and 7/8.
  • at least one of the antibodies may comprise a set of six CDRs selected from the group consisting of SEQ ID NOs: 9-14, 15-20, 21-26 and 27-32.
  • At least one of the antibodies may comprise a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8. As discussed above, these are the sequences of the 66-3-9C, 040, 6662 and 6541 antibodies of examples.
  • the pharmaceutical composition may comprise at least two or three antibodies as described above (with any of the sequences as described above).
  • the composition comprises four antibodies as described above (with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences contained within the HCVR/LCVR sequence pairs of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8, with six CDRs of SEQ ID NOs: 9-14, 15-20, 21-26 and 27-32 or with HCVR/LCVR sequence pairs of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8).
  • the antibodies may also comprise a constant region as 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.
  • aqueous carriers examples include water, buffered water and saline.
  • suitable aqueous carriers include water, buffered water and saline.
  • suitable aqueous 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 lipid-based 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,
  • an“antibody cocktail” generally refers to a combination/mixture of antibodies within the same composition, i.e. a single
  • composition comprising the antibodies.
  • the invention also includes the combined use of different anti-Ebola virus antibodies in separate pharmaceutical compositions.
  • the antibodies may bind to the Ebola virus glycoprotein from any of the species/strains discussed above.
  • the sequences of such glycoproteins would be well known to the skilled person.
  • a cocktail of the invention may comprise two or more 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, receptor binding region and base.
  • a cocktail of the invention may comprise three antibodies binding to the Ebola virus glycoprotein.
  • the present invention provides an antibody cocktail comprising three or more antibodies binding to the Ebola virus glycoprotein, wherein one antibody binds to the glycan cap, one antibody binds to the receptor binding region and one antibody binds to the base.
  • one antibody recognises an epitope in the glycan cap, one antibody recognises an epitope in the receptor binding region and one antibody recognises an epitope in the base.
  • the cocktail further comprises an antibody binding to (recognising an epitope within) the b17-18 loop.
  • the antibody preferably recognises an epitope where all of the epitope residues are within the specified region. However, in some instances it is sufficient that only some of the epitope residues are within the specified region.
  • 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.
  • the above-described binding methodology is performed in two orientations. In 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. In a second orientation, the test antibody is allowed to bind to the
  • 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 al., Cancer Res, 1990:50:1495-1502).
  • 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.
  • the antibodies described below in the Examples may be used as reference antibodies.
  • antibody epitopes include hydro gen/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 (see above). 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 (for example with a single dose of 100 mg of antibody at day 2 of infection).
  • Survival rates may also be determined in infected guinea pigs as described in the Examples.
  • 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.
  • a cocktail includes the 66-3-9C, 040, 6662 and 6541 antibodies, or antibodies derived from these antibodies (for example comprising the CDR and
  • an antibody in the cocktail may comprise a HCDR3 sequence of any one of SEQ ID NOs: 33-41.
  • the antibodies are monoclonal antibodies.
  • the antibodies may be chimeric antibodies, CDR-grafted antibodies, nanobodies or humanised antibodies.
  • the antibodies are human antibodies.
  • the antibodies may also be antigen-binding fragments (see above). Furthermore, the antibodies may comprise a constant region as described above.
  • 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 al, 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al, 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 187 9-18), 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.
  • 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, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework 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 or rat 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 or rat 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 above 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 above are transferred to the human antibody framework.
  • only the specificity determining residues from each of the CDRs described herein above 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 neutralising 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.
  • 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 antibodies may be formulated using a pharmaceutically acceptable carrier or diluent, as discussed above.
  • the antibodies, 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 ebolavirus 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 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
  • HIV immunodeficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • the antibodies, compositions and cocktails of the invention may be administered therapeutically or prophylactically.
  • the antibodies, pharmaceutical compositions and cocktails may be administered subcutatneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially, Typically, the antibodies, 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.
  • the invention includes use the combined use of the 040, 66-3-9C, 6662 and 6541 antibodies (or antibodies comprising sequences from these antibodies; see above).
  • Example 1 Isolation and expression of monoclonal antibodies (mAbs) from vaccinated donors
  • Example 2 Screening of Monoclonal Antibodies for binding and neutralisation MDCK-SIAT1 cells (Matrosovich et al. 2003; J Virol, 77: 8418-25 ) were transduced to express the glycoprotein from Ebola Zaire wt/GIN/2014/Kissidougou-C15 (KJ660346.1) as described (Xiao et al. 2018, J Virol, 92). MDCK-SIAT1 cells were used in these experiments because, unlike other cell lines, they tolerate high levels of stable expression of EBOV GP, can pseudotype an influenza core, and are readily infected by our EBOV surrogate E-S -FLU (Xiao et al, 2018).
  • E-S-FLU Ebola surrogate E-S-FLU
  • Ebola GP Ebola GP
  • E-S-FLU encodes a fluorescent protein eGFP that replaces the hemagglutinin coding sequence so that infected cells fluoresce green.
  • Figure 2D shows a summary of the 38 neutralising antibodies compared to a set of control antibodies described in the literature including KZ52 (Maruyama et al. 1999, J Virol, 73: 6024-30; Lee et al. 2008, Nature, 454: 177-82); c4G7, c2G4, cl3C6 - the three components of ZMapp (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7), 100 and 114 (Corti et al. 2016, Science, 351: 1339-42 ; Misasi et al. 2016, Science, 351: 1343-6) and 6D6 (Furuyama et al. 2016, Sci Rep, 6: 20514).
  • Example 3 The relationship between in vitro neutralisation and protection in vivo in mice
  • a set of the first 24 antibodies isolated were tested for protection of mice (single dose of 100 mg at day 2 of infection with a mouse-adapted Ebola Mayinga in groups of 10 as part of the work of the Viral Haemorrhagic Fever Immunotherapeutic Consortium (VIC) (Saphire 2018, Cell, 9, 938-952).
  • VIP Viral Haemorrhagic Fever Immunotherapeutic Consortium
  • P6 glycan cap
  • RBR glycan cap/receptor binding region
  • Table 3 Summary of Yeast Display assay with EBOV mAbs
  • Antibodies to Glycan Cap could be divided into three overlapping groups.
  • the antibodies cross- inhibited the binding only of other GC specific antibodies ( Figure 3A).
  • P6 was defined as glycan cap specific by electron microscopy and alanine scanning (Saphire et al. 2018, Cell, 9, 938-952), and both P6 and 040 by sequencing of a protein fragment (amino acids 228- 281 of GP1) expressed in yeast that was bound by these antibodies ( Figure 3B).
  • the epitope bound by these two antibodies was removed by thermo lysin cleavage of GP expressed on MDCK-SIAT1 cells ( Figure 3C).
  • the antibodies inhibited the binding of both GC specific antibodies and RBR specific antibodies exemplified by 66- 3-2C ( Figure 3A), which suggests they bind to an epitope that overlaps these two regions.
  • the third group was defined by antibody 66-3-9C that bound to a small conserved peptide within the b17- 18 disordered loop of the glycan cap (amino acids 286-293 GEWAFWET) expressed in yeast ( Figure 3B iii). 66-3-9C recognises a similar epitope to that bound by the macaque-derived mAb FVM09 (Keck et al. 2016, J Virol, 90: 279-91).
  • the RBR is highly conserved in species of Ebolavirus and Marburgvirus, and therefore offers an attractive target for therapeutic antibodies (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7, Hashiguchi et al., 2015, Cell, 160, 904-912, Flyak et al. 2015, Cell, 160, 893-903,
  • the RBR is partially protected by the glycan cap and mucin-like domain, and binding of EBOV GP to its receptor site on domain C of the NPC1 protein occurs only after the GC and MLD have been removed by cathepsin or thermolysin cleavage
  • mAbs to the RBR were defined by competition for binding to complete GP with human mAb 114. It was showed that 114 bound the GP1 core fragment 102-230 in the yeast expression assay ( Figure 3A and 3B). Mab 114 competed for binding with a subset of neighbouring GC specific antibodies ( Figure 3A). However, in contrast to the GC specific antibodies, 114 and similar antibodies retain binding after release of the GC and MLD following exposure of E-SIAT cells to thermo lysin digestion as expected (Corti et al., 2016, 219, Misasi et al., 2016) ( Figure 3C and 6). Nine mAbs showed this pattern and were placed into the RBR binding group ( Figure 3).
  • Antibodies to the Base/Fusion Loop were defined by competition for binding with the defined antibodies KZ52 (base) (Lee et al. 2008, Nature, 454: 177-82); c2G4 and c4G7 (base) (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7); 100 (Misasi et al. 2016, Science, 351: 1343-6); and 6D6 (fusion loop) (Furuyama et al. 2016, Sci Rep, 6: 20514). Many of these antibodies cross-inhibited each other, but sub-groups were discernible.
  • biotinylated 6541 and 66-4-C12 were both inhibited by the characterised base antibodies KZ52 and 100, but 6541 and 66-4-C12 failed to inhibit each other, suggesting that they bound to non-overlapping sites in the base region (Figure 3A).
  • Antibodies 6541, 66-4-C12 and 66-6-3 competed for binding with the fusion loop specific murine mAb 6D6, which suggested then binding footprints may overlap with the fusion peptide. Binding of base-region specific antibodies to thermo lysin treated cells was typically either unaffected or enhanced (Figure 3C).
  • Mucin Like Domain (MLD) dependent antibodies The binding of 6/82 antibodies to MDCK-SIAT1 cells expressing GP lacking the MLD (amino acids 313-463) was reduced by comparison with cells expressing full-length GP ( Figure 6). Control antibodies to GP1 head (c13C6) and base (KZ52 and c4G7) bound the MLD deleted and full length GP equally. None of the six MLD dependent antibodies were neutralising. Antibodies 66- 3-9C (specific for the b17- 18 loop sequence (GEWAFWET) also lost binding to the MLD deleted GP, and the binding of one base antibody 66-4-C12 was reduced (although was not affected by thermolysin cleavage).
  • GEWAFWET b17- 18 loop sequence
  • Example 5 Antibodies to the glycan cap that block thermolysin cleavage
  • FIG. 7A shows binding of selected GC, RBR and Base specific antibodies after cleavage by thermolysin.
  • the three antibodies to the Glycan cap (P6, 040 and 66-3-9C) lose binding after thermolysin treatment, whereas the epitopes bound by RBR (114) and base (66-4-C12) specific antibodies were not affected ( Figure 7A and 5). The result was confirmed for seven additional GC specific antibodies ( Figure 5).
  • Thermolysin digestion achieved complete removal of these epitopes as shown (i) by reduction of the binding of these antibodies to the level of a negative control specific for influenza ( Figure 5) and (ii) the appearance of the epitope recognised by MR78 that binds to EBOV GP only after removal of the glycan cap (Flyak et al (2015) and Bornholdt et al (2016). The effect of allowing the GC specific antibodies to bind was noted, followed by treatment with thermolysin. The mAbs P6 and 040 remained bound despite the
  • thermolysin effect (Figure 3). This effect was confirmed (Figure 6) for five additional neutralising antibodies to the GC (66-3-7C, 66-3-2C, 141, 66-3-4A and 125). Evidence for cleavage was provided by the loss of the epitope bound by 66-3-9C specific for the b17- 18 loop (GWAFWET) ( Figure 3B) and appearance of the epitope bound by MR78 that binds to EBOV GP after removal of the glycan cap (Flyak et al, 2015) ( Figure 5).
  • pan-Ebola antibodies to the fusion loop can also be isolated (Zhao et al. 2017, Cell, 169: 891-904 el5; Furuyama et al. 2016, Sci Rep, 6: 20514; Wee et al. 2017, Cell, 169: 878-90 e15).
  • Example 8 VII 3-15 and Vl. 1-40 antibodies to the Receptor Binding Region
  • Table 4 Receptor binding region antibodies that are blocked by mAb 114.
  • GC Glycan Cap
  • RBR Receptor binding region
  • IFL Internal fusion loop.
  • Example 10 Selection of antibody cocktails for protection in guinea pigs
  • Figure 12 Seven antibodies (Figure 12) were selected to test for therapeutic protection in guinea pigs against the EBOV Mayinga variant of Ebola virus as described (Dowall et al. 2016, J Infect Dis, 213: 1124-33). Three antibody cocktails were formed from these antibodies - two EBOV-specific cocktails and one containing antibodies that cross-reacted in binding to BDBV and SUDV GPs.
  • the three cocktails were tested at a dose of 10 mg/Kg of each antibody (Table 7, groups 1-3). This dose was selected partly on our experience with protective antibodies in influenza infection, also because the affinities of the antibodies and binding assays in vitro suggested that 10 mg/kg would be saturating.
  • the first cocktail which was expected to be most potent, was also tested at 5 mg/Kg total (equivalent to 1.67 mg/Kg of each antibody) (group 4) for comparison to ZMapp given at the same dose. This dose was not expected to 100% curative and provided an opportunity to test for equivalence to ZMapp.
  • the first and second cocktails differed in the RBR mAh, where 6662 (which was highly protective in the murine challenge) replaced 6660.
  • the third cocktail (group 3) was composed of four mAbs that cross-react in bindingto EBOV, SUDV and BDBV glycoproteins.
  • mAbs specific for epitopes in GC, RBR and Base it included 66-3-9C specific for the b17-18 Loop because of its similarity to FMV09 (Howell et al. 2017, Cell Rep, 19: 413-24) that provided a synergistic therapeutic effect.
  • Controls were ZMapp at a dose of 5 mg/Kg and PBS.
  • Groups 1 and 2 were EBOV specific (cross-reacting between Mayinga and Makona strains), and Group 3 showed additional partial neutralisation of S-FLU coated in BDBV and SUDV GPs ( Figure 12B- E).
  • the control antibody 6D6 strongly neutralised S-FLU coated in GPs from all of the Ebolavirus species.
  • Example 11 Therapeutic protection of guinea pigs by antibody cocktails
  • the group 1 cocktail (125 + 6660 + 66-6-3) resulted in 4/6 guinea pigs surviving ( Figure 13).
  • Survival in group 2 (125 + 6662 + 66-6-3), which differed from group 1 only in replacement of the RBR specific mAb 6660 with 6662 was only 1/6 animals.
  • treatment with the cross -reactive cocktail comprised of four independently binding antibodies (040 + 66-3-9C + 6662 + 6541) resulted in 100% survival without weight loss or clinical signs (Figure 13).
  • Group 1- Antibody cocktail (125 + 6660 + 66-6-3): Microscopic lesions, together with detection of viral antigen, were observed in the liver and spleen of two out of the six animals (19063 and 00252); these two animals were euthanised early for welfare reasons. Furthermore, viral antigen was detected in the liver ( Figure 16) and spleen (graded 2-3 severity). The remaining animals survived to the study end point. In the liver and spleen of animal 99705 (922/17), minimal changes were noted and viral antigen staining was absent. In the liver and spleen of the three remaining animals, there were no microscopic changes or detection of viral antigen.
  • Group 2- Antibody cocktail (125 + 6662 + 66-6-3): Five out of six animals were euthanised between days 6 and 9 for welfare reasons. Microscopic changes referable to viral infection, as well as detection of viral antigen, was noted in both the liver (minimal to moderate) and spleen (minimal to marked) ( Figure 17) of all these animals. Animal 18897 survived to the study end point and the liver and spleen appeared normal with no viral antigen detected.
  • Group 3- Antibody cocktail (040 + 6662 + 6541 + 66-3-9C): All animals survived to the study end point. Neither microscopic changes nor presence of viral antigen were observed in the liver or spleen of any animal.
  • Group 4 Antibody cocktail (Zaire-specific): Three out of six animals survived to the study end point (00484, 19160 and 19335). Only one minimal change was noted in the spleen of animal 00484, and viral antigen was not detected in either the liver or spleen of any of these animals. The remaining three animals were euthanised between days 7 and 8 pc; microscopic changes and viral antigen were noted in the liver (mild) ( Figure 18) and spleen (minimal to marked) of all animals.
  • Group 5 Antibody cocktail (ZMapp): Three animals survived to the study end point (02155, 19088 and 19210); neither microscopic changes nor viral antigen were detected in either the liver or spleen. The remaining animals were euthanised early between 6 and 8 days pc and microscopic changes were noted in the liver (minimal to moderate) and spleen (minimal to marked) in all animals, as well as viral antigen (1-3 severity). In animal 00003, changes observed in the liver were focal ( Figure 19). All animals were euthanised early, between 6 and 8 days pc; microscopic changes, and viral antigen, were detected in both the liver (minimal to moderate) ( Figure 20) and spleen (minimal to marked) of all animals. In animal 00532, changes observed in the liver were focal and comprised macrophage infiltration and necrosis.
  • a novel assay was developed to screen the antibodies for neutralisation. This uses a single cycle influenza core with the hemagglutinin coding sequence replaced with eGFP for detection of infected cells, and coated in the Ebola GP by pseudotyping (Xiao et al. 2018, Cell, 169: 891-904 el5). As this virus can replicate only for a single cycle, and contains no genetic information from Ebola, it can be handled in more convenient containment conditions than Ebola virus. In collaboration with the Viral Haemorrhagic Fever Immuno therapeutic Consortium it was established that the neutralisation assay correlated reasonably with therapeutic activity in mice. The assay was used to narrow the choice of antibodies to combine in therapeutic cocktails.
  • Antibodies to epitopes in the glycan cap, Receptor binding region (RBR) and Base/Fusion loop can all neutralise in vitro and protect in vivo (Saphire 2018, Cell, 9, 938-952). Suggested mechanisms include blockade of NPC1 binding, prevention of cathepsin cleavage, interference with fusion, and Fc dependent interactions with host cells (Saphire 2018, Cell, 9, 938-952, Saphire and Aman 2016, Trends Microbiol, 24: 684-86). Certain Base binding antibodies have been shown to prevent cathepsin cleavage (Shedlock et al.
  • thermolysin as a cathepsin surrogate
  • Antibodies that bind the glycan cap can neutralise in vitro and provide protection in vivo, but it is not clear how this can occur if the epitope is removed by cathepsin cleavage before GPcl binds to NPC1 (Saphire 2018, Cell, 9, 938-952, Saphire and Aman 2016, Trends Microbiol, 24: 684-86). It was found that two neutralising and protective antibodies to the glycan cap, P6 and 040, had the property that once bound to GP expressed on the membrane of transduced cells, the GP became resistant to cleavage by thermolysin.
  • Group 3 was selected firstly on the level of cross-reactivity in binding between the GPs from the three Ebola virus species Zaire, Bundibugyo and Sudan, and secondly on their neutralisation and mouse protection. It is notable that cross -reactive antibodies can be found that bind glycan cap, RBR and base/fusion loop. Treatment at day three of infection with the cross-reactive cocktail of four antibodies that included 040 to Glycan Cap, 6662 to the RBR, 6541 to the Base, and 66-3-9C to the b17-18 loop resulted in 100% protection from a Zaire Mayinga Ebola virus without weight loss or clinical signs. Viral RNA was not detected in the tissues of these animals at post mortem on day 21 post infection, which implies that the selection of resistant variants did not occur. .
  • the effectiveness of this combination could not have been predicted from the in vitro neutralisation or murine protection results with individual antibodies.
  • the b17- 18 Loop specific antibody 66-3-9C is closely related to the FVM09 antibody (Keck et al.
  • FVM09 is specific for the conserved exposed loop between beta strands 17 and 18 with the sequence GEWAFWET, and by itself is not neutralising and was only weakly protective in vivo.
  • FVM09 in mixtures enhanced the binding and neutralisation by base antibody 2G4, and the GC specific antibody m8C4 (Holtsberg et al. 2016, J Virol, 90: 266-78; Howell et al. 2017, Cell Rep, 19: 413-24).
  • FVM09 enhanced protection by the GC antibody m8C4 (Howell et al.
  • the guinea pigs were implanted with a temperature and identity chip during a five day acclimatization period.
  • HEK293T human embryonic kidney
  • MDCK-SIAT Mesod Darby Canine kidney - sialltransferase cells
  • Both the cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 I.U./mL penicillin and 100 mg/mL streptomycin. They were incubated at 37 °C with 5% C02.
  • DMEM Modified Eagle Medium
  • MDCK-SIAT cells expressing Ebola GP and MLD-deleted GP MDCK-SIAT 1 cells were purchased from ECACC. The codon optimised sequence was ordered from Geneart. The GP genes were cloned in to a Lenti virus vector pHR-SIN (Demaison et al. 2002, Hum Gene Ther, 13: 803-13). MDCK-SIAT1 cell lines (Matrosovich et al. 2003, J Virol, 77: 8418-25) were transduced with disabled Lenti virus produced in HEK293T cells to express the glycoprotein. Transduced cells were stained with specific monoclonal antibodies and FACS sorted for maximal surface expression. MLD-deleted GP had amino acids 313-463 removed.
  • Ebola pseudotyped influenza viruses were generated as previously described in detail (Xiao et al. 2018). In brief, pseudotyped viruses were propagated and grown in MDCK-SIAT 1 cell lines transduced with disabled lentivirus to express the surface Ebola glycoprotein.
  • Ebola virus strain Yambuku-Ecran, previously known as ME718 (Kuhn et al.
  • Antibodies were isolated by FACS sorting, PCR and antibody variable gene cloning of a single B cell plasmablast of a vaccinated human individual using the protocol described by Tiller et al 2008 and Smith et al 2009 with modifications. Briefly, PBMC were incubated with a cocktail of antibodies to CD3 (PB; UCHT1; BD Pharmingen), CD20 (APC-H7; 2H7; BD Pharmingen), CD19 (FITC; H1B19; BD Pharmingen), CD27 (PE- Cy7, M-T271; BD Pharmingen), CD38 (PE-Cy5, HIT2; BD Pharmingen) and IgG
  • Ebola GP protein (10 mg/mL) and a known biotin- labelled anti-MLD antibody (10 mg/mL) were used to sort antigen specific B cell plasmab lasts.
  • Single cells with the phenotype of CD3- CD20-/low, CD19+, CD27++, CD38++ ,IgG+ were sorted on a FACS Aria III cell sorter (BD Biosciences). Single cells were sorted into 96-well PCR plates containing lysis buffer followed by single cell RT- PCR. Nested PCR was slightly modified to existing methods (Tiller et al, Smith et al). Overlapping bases (approx.
  • PCR products were purified in a Qiagen 96-well system and the inserts were assembled with cut plasmid in the Gibson mix (NEB). Two mL of assembled product was used to transform 10 mL DH5a E. Coli (NEB, C2987) in 96-well plates. Three colonies for each heavy and light chain were grown in a 96-well plate format and purified using Qiagen Turbo 96 miniprep kit. Plasmids were eluted using 100 mL TE buffer. Transfection of 293T cells with heavy and light plasmids ( ⁇ 200 ng of each with 120 mg/mL linear PEI, in 250 mL total volume) and immunofluorescence assays were also performed in a 96-well tissue culture plate.
  • 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). Human B cell cultures were prepared using 132 x 96-well plates at a cell density of approximately 5000 cells per well. After 7-days culture, screening was performed. Briefly, the presence of Ebola glycoprotein-binding antibodies in B cell culture supernatants was determined using a homogeneous fluorescence-based binding assay performed on a Applied Biosystems 8200 cellular detection system device using MDCK cells stably transfected to express surface Ebola glycoprotein.
  • Binding was revealed with a goat anti-human IgG Fey-specific Dylight 649 conjugate (Jackson). Following primary screening, positive supernatants containing reactive antibody were consolidated on 96-well bar-coded master plates and B cells in cell culture plates frozen at -80°C. Master plates were then screened in a further homogeneous fluorescence binding assay to confirm that the antibodies bound the Ebola glycoprotein- expressing MDCK-SIAT1 cells and not the parental MDCK-SIAT1 cells.
  • the Fluorescent Foci method (US Patent 7993864/ Europe EP1570267B1; (Clargo et al. 2014, MAbs, 6: 143-59) utilizing Ebola glycoprotein-expressing MDCK-SIAT1 cells was used to identify and isolate antigen-specific B cells from positive wells, and specific antibody variable region genes were recovered from single cells by reverse transcription (RT)-PCR using heavy and light chain variable region-specific primers.
  • PCR primers contained restriction sites at the 3’ and 5’ ends allowing cloning of the variable region into a human IgGl (VH), human kappa (VK) or human lambda (Vk) mammalian expression vector.
  • Heavy and light chain constructs were co -transfected into Expi293F cells using Expifectamine 293 (Invitrogen) and recombinant antibody expressed. After 6 days expression, supernatants were harvested and antibody rescreened for selectivity using the specificity assays described above. Antibody was purified from conditioned media using affinity chromatography and characterized further.
  • Immunofluo rescene assay was done to screen the binding of antibodies in culture supernatant to Ebola glycoprotein.
  • a 96-well plate was coated overnight with stable transduced MDCK-SIAT1 cells expressing Ebola glycoprotein (E-SIAT cells).
  • Antibody supernatant 50 mL was incubated with a monolayer of E-SIAT cells. After 1 h incubation at RT, plates were washed with PBS.
  • a secondary antibody goat anti-human IgG conjugated with Alexa Fluor647 (A21445; Thermo Fisher; 1:400) or FITC (H10301; Life technologies; 1:160) was added to well and let incubate for 1 h in dark. Plated were then washed and fixed with 1% formalin. Fluorescence was observed under the fluorescence microscope and quantified using the Clariostar platereader (BMG Labtech). GP binding antibodies and influenza antibodies were used as positive and negative controls respectively.
  • Elution pools were neutralised by adding 2 M Tris/HCl pH 8.0 and absorbance read at 280 nm (Cary UV Spectrophotometer). Samples were then buffer exchanged into PBS pH 7.4 using Amicon Ultra Spin columns with a 30K cut off membrane (Millipore, UFC905008) and centrifugation at 4000 g. Absorbance was read at 280 nm and samples supplied at >1.0 mg/mL. Further analysis was done by size exclusion on a UPLC (Acquity) with a BEH200; 1.7 mM, 4.6 mm X 300 mm column (176003905, Waters) and developed with an isocratic gradient of 0.2 M phosphate, pH 7.0 at 0.3 mL/min.
  • UPLC Acquity
  • Monoclonal antibodies used for Guinea Pig protection were expressed in 293T cells and affinity purified by Absolute Antibody Ltd. and provided at 15 mg/mL which were aliquoted and kept at -80 °C until used.
  • Ebola pseudo typed influenza virus E-S-FLU
  • VGM virus growth medium
  • MDCK-SIAT1 cells 3x104
  • Virus only and medium only controls for maximum and minimum signals were included. Percent infection was calculated based on the wells containing virus only and medium only. Inhibitory concentration at 50% and 90% was derived by linear interpolation.
  • Biotin-labeled antibody and competing mAb were mixed and transferred to a monolayer of E-SIAT cells. After 1 h incubation, cells were washed. A second layer of Extravidin-FITC (E2761; Sigma; 1:400) or
  • Extravidin Peroxidase E2886; Sigma; 1:1600
  • Streptavidin-Alexa Fluor 647 S21374; Thermo Fisher; 1:400
  • Extravidin-Peroxidase E2886; Sigma; 1:1600
  • Streptavidin-Alexa Fluor 647 S21374; Thermo Fisher; 1:400
  • OPD substrate P9187; Sigma
  • Streptavidin Alexa-Fluor 647 preferable because it gave a better signal to background ratio in the plate reader. Mean and 90% confidence interval of eight replicate measurements were calculated.
  • Epitope mapping of the mAbs was carried out based on the yeast surface display (YSD) library as previously described (Zuo et al. 2011 , J Biol Chem, 286: 33511-9; Guo et al. 2015, J Acquir Immune Defic Syndr, 68: 502-10). Briefly, the combinatorial fragment library of Zaire Ebola GP was constructed and displayed on the surface of yeast for antibody staining and Fluorescence-activated cell sorting (FACS). Specifically, the full- length GP gene was digested and PCR-reassembled into a range of 100-900 bp fragments, the reassembled fragments were gel purified and cloned into yeast surface display vector.
  • YSD yeast surface display
  • the cloned products were then transformed into competent yeast cell line EBY100 using electroporation.
  • the yeast library was induced and incubated with each of the Ebola mAbs and positive sorted by FACS using Aria III (BD, USA).
  • the sorted positive yeast clones displaying the respective antigenic fragments were harvested and the plasmids encoding the corresponding fragments were extracted and subjected to sequencing and sequence analysis.
  • Binding kinetics of antibodies to both monomeric and trimeric Ebola glycoprotein ectodomain was assessed by SPR using a Biacore 3000 instrument (GE Healthcare), whereby antibody was captured on a CM5 chip (GE Healthcare) via immobilized anti human IgG Fc specific polyclonal antibody, followed by successive titration of
  • Affinity purified polyclonal goat F(ab)2 anti-human IgG Fc (Jackson, 109-006-098) was immobilized following activation of test and reference flow cells by injection of 50 mL of a fresh mixture of 50 mM N-hydroxysuccimide and 200 mM l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide at a flow rate of 10 mL/min.
  • Polyclonal at 50 mg/mL in 10 mM acetate pH 5.0 buffer was injected (50 mL) over the test flow cell and both test and reference flow cell surfaces were then deactivated with a 50 mL pulse of 1 M
  • Binding assays were carried out at 25°C in HBS-EP running buffer (10 mM
  • HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05 % Surfactant P20, GE Healthcare.
  • Antibodies were diluted to 10 nM in HBS-EP and concentrations of monomeric and trimeric GP were prepared in HBS-EP between 5 and 500 nM. The latter were tested separately for each antibody in a series of sensorgram cycles, where 10 mL of antibody was injected at 10 mL/min followed by 90 mL of GP at a flow rate of 30 mL/min to generate an association phase of 180 sec.
  • Sensograms were analysed using the BIAevaluation Software (version 4.1.1, GE Healthcare). After subtraction of respective buffer control and antibody blank cycles, kinetic parameters describing association and dissociation rate constants were determined using the Langmuir binding model. Affinity constants were calculated from the mean log KD values determined over 5 concentrations of glycoprotein.
  • GP proteolysis by cathepsins removes the mucin-like domain and glycan cap, and is essential for binding of GP to NPC1 receptor.
  • Thermolysin mimics cathepsins and the proteolytic activity is active at physiological pH whereas cathepsins require strongly acidic pH, which is toxic to living cells (Dube et al. 2009, J Virol, 83: 2883-91) (Bale et al. 2011, PLoS Negl Trap Dis, 5: el395).
  • Thermolysin (P1512; Sigma) was dissolved in HM buffer (20 mM HEPES, 130 mM NaCl and 20 mM MES and pH adjusted to 7.5).
  • thermolysin treated GP 25 mg/mL antibody was incubated with cells for 1 h. Alexa fluor 647 conjugated anti-human IgG (A21445; Life Technologies; 1:400) was used for binding detection in FACS (Attune; Life Technologies). Antibody binding to untreated GP expressing cells handled in similar manner was done in parallel for binding
  • All guinea pigs were challenged with 103 TCID50 of guinea pig adapted Ebola virus in a volume of 200 mL via the subcutaneous route. During the course of the study, weights and temperatures were collected at least once daily and clinical scores assessed at least twice a day. Animals which met predefined endpoints (20% weight loss; 10% weight and moderate clinical signs; or immobility) were culled by a Schedule 1 approved method. Antibody cocktails were prepared a day before administration. Antibodies were delivered 3 days post-challenge to six animals per group via the intraperitoneal route in a volume of 2 ml. As a positive control, ZMapp was given a dose of 5 mg/Kg per animal. Untreated animals were given 2 mL PBS.
  • PCR and Histology of guinea pig tissue Group identifiers, treatments received, animal identifiers, histology numbers, and duration from challenge to euthanasia are detailed in Table 9 .
  • Each animal was assigned a histology number. Tissue samples were processed to paraffin wax; sections were cut at approximately 3-5 mm thick, stained with haematoxylin and eosin (HE) and examined microscopically. In addition, sections from each animal were stained for Ebola viral antigen using the Leica BondMax (Leica Biosystems) and the Leica Bond Polymer Refine Red Detection Kit (Leica Biosystems). An antigen retrieval step was included for 10 minutes using the Bond Enzyme Pre -treatment Kit, Enzyme 3 (3 drops). A rabbit polyclonal, anti-Ebola viral antibody (IBT Bioservices) (dilution 1:2000) was incubated with the slides for 60 mins. Alkaline phosphatase and haematoxylin
  • Tissue sections were examined by light microscopy and microscopic lesions attributable to Ebola viral infection scored subjectively using a scale comprising minimal, mild, moderate and marked.
  • frequency of staining was scored subjectively using‘1‘(positively stained areas observed occasionally);‘2’
  • IMS Client (vl2H2) was used to capture, store and export digital images.
  • the gene family usage of the variable region of the human IgG heavy- and light- chains was analysed using IMGT/V -Quest. Phylogeny tree was drawn based on MUSCLE alignment and Neighbour joining settings using MEGA version 7.
  • SEQ ID NO: 33 (6660 HCDR3)
  • SEQ ID NO: 180 (66-6-16 heavy junction)
  • SEQ ID NO: 215 (6660 lambda light chain)
  • SEQ ID NO: 382 (6662 variant HCDR2)
  • SEQ ID NO: 383 (6662 variant HCDR2)
  • SEQ ID NO: 384 (6662 variant HCDR3)

Abstract

The invention relates to antibodies that bind to the Ebola virus glycoprotein, particularly to antibody cocktails comprising a number of such antibodies. The antibodies and cocktails may be used to treat, prevent or ameliorate Ebola virus infection.

Description

ANTIBODIES TO EBOLA VIRUS GLYCOPROTEIN
Field of the Invention
The present invention relates to antibodies binding to the Ebola virus glycoprotein. In particular, the present invention relates to cocktails comprising a mixture of antibodies binding to different epitopes on Ebola virus glycoprotein. The invention also relates to methods of preventing, ameliorating or treating an Ebola virus infection using such antibodies and cocktails. Background of the Invention
The Ebola virus (EBOV) outbreak in 2013-2016 in West Africa resulted in 28,616 cases and 11,310 deaths as of June 10, 2016 after which the end of Ebola transmission was declared by the WHO. A new outbreak is in progress in the Democratic Republic of Congo, which had claimed 33 lives to date. Mixtures of monoclonal antibodies to the Ebola virus glycoprotein from convalescent humans (Maruyama et al. 1999, J Virol, 73: 6024-30; Flyak et al. 2016, Cell, 164: 392-405; Corti et al. 2016, Science, 351: 1339-42; Bornholdt et al. 2016, Science, 351: 1078-83; Wee et al. 2017, Cell, 169: 878-90 el5;
Flyak et al. 2018, Nat Microbiol, 3: 670-77; Gilchuk et al. 2018, Immunity, 49, 363-374), humanised mice (Pascal et al. 2018, J Infect Dis, 1-15), hyper- immunised macaques (Keck et al. 2016 J Virol, 90: 279-91 ; Zhao et al. 2017 J Biol Chem, 286: 33511-9) and wild-type mice (Furuyama et al. 2016 Sci Rep, 6: 20514; Marzi et al. 2012 PLoS One, 7: e36192; Wilson et al. 2000 Science, 287: 1664-6 ; Qiu et al. 2012 PLoS Negl Trop Dis, 6: el575 ; Pettitt et al. 2013 Sci Transl Med, 5: 199ral3; Takada et al. 2007 Vaccine, 25: 993-9) can be therapeutic in various animal models.
The ZMapp cocktail of murine chimeric antibodies (cl3C6, c2G4 and c4G7), one targeting the glycan cap and two to the base of the glycoprotein, was successful in protecting 100% of non-human primates as late as 5-days post infection (Qiu et al. 2014, Nature, 514, 47-53). The antibodies of human origin, 114 (to the receptor binding region) and 100 (to the base) showed a similarly profound therapeutic effect (Corti et al. 2016, Science, 351 : 1339-42). Although the ZMapp cocktail was not proven statistically to be protective in human trials during the outbreak in West Africa because of the small number of participants, there was a trend in the direction of improved survival (Group et al. 2016; Prevail II Writing Group et al. 2016, N Engl J Med, 375: 1448-56). Another new cocktail form Regeneron, derived from humanised mice, containing one antibody to the fusion loop, one to the head, and one glycan cap was also protective in primates (Pascal et al. 2018, J Infect Dis, 1-15) and has recently been approved for emergency experimental use during the 2018 outbreak in the Democratic Republic of Congo. This cocktail was intentionally chosen to combine antibodies to independent epitopes with neutralisation and immune effector functions, thought to be complementary. These results encourage the development of optimised cocktails of antibodies for use in human disease caused by the complete range of Ebola species.
A recent comprehensive study of monoclonal antibodies collected from laboratories across the globe by the Viral Haemorrhagic Fever Immunotherapeutic Consortium (VIC) emphasised the variety of independent epitopes on the viral glycoprotein that can be bound by protective antibodies, and the range of antibody dependent mechanisms that can contribute to protection in vivo (Saphire 2018, Cell, 9, 938-952). The VIC study established that neutralisation in vitro was a strong indicator of the protective potential of an antibody, but in addition revealed that multiple Fc recruited functions also contributed to protection.
In these examples the great majority of therapeutic antibodies were isolated from animals or humans after multiple or prolonged exposures to the Ebola glycoprotein. These levels of exposure are thought to select for high affinity antibodies through the acquisition of multiple adaptive somatic mutations after repeated rounds of competitive selection of B cells in the germinal centres of lymph nodes during affinity maturation (reviewed in (Eisen and Chakraborty 2013, Proc Natl Acad Sci U S A, 110: 7-8; Oropallo and Cerutti 2014, Trends Immunol, 35: 287-9)). Structural analysis of human monoclonal antibodies to influenza hemagglutinin, isolated from each stage of this process, suggests that the useful mutations pre-configure the tertiary structure of the binding loops of the antibody to minimize the energy cost of binding. This can increase both the on-rate, and reduce the off- rate of the antibody to achieve affinities in the nano- to pico- molar range associated with virus neutralisation (Schmidt et al. 2013, Proc Natl Acad Sci U S A, 110: 264-9).
This explanatory framework suggests that antibodies with the required specificity and kinetics for neutralisation may be present early in an immune response, but at a lower frequency than after full affinity maturation has taken place. If an efficient screening system is applied to finding them, antibodies with the required properties for therapy should be available from donors responding to an antigen for the first time. Human volunteers in experimental vaccine trials offer a convenient source of such antibodies without the difficulties of sample availability, or concerns about persistent virus or other pathogens in venous samples from convalescent donors.
Summary of the Invention
The present invention provides a pharmaceutical composition comprising one or more antibodies that bind to Ebola virus glycoprotein and a pharmaceutically acceptable carrier or diluent, wherein at least one of the antibodies comprises a set of six CDRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, selected from the group consisting of: SEQ ID NOs: 9-14, SEQ ID NOs: 15-20, SEQ ID NOs: 21-26 and SEQ ID NOs: 27-32.
The present invention also provides a nucleic acid or a pair of nucleic acids encoding the heavy and light chains of an antibody of the invention. Furthermore, the invention provides an expression vector comprising the nucleic acid(s) or pair of nucleic acids, a host cell comprising the expression vector and a method of producing an antibody of the invention, comprising culturing the host cell under conditions permitting production of the antibody and recovering the antibody so produced.
In addition, the invention provides a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering a pharmaceutical composition an antibody of the invention to a subject in need thereof. Similarly, the invention provides a pharmaceutical composition of the invention for use in a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the
pharmaceutical composition to a subject in need thereof. Likewise, the invention provides use of a pharmaceutical composition of the invention in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
The invention also provides an antibody cocktail comprising three or more antibodies binding to the Ebola virus glycoprotein, wherein one antibody binds to the glycan cap, one antibody binds to the receptor binding region and one antibody binds to the base.
Furthermore, the invention provides a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the antibody cocktail of the invention to a patient in need thereof. Similarly, the invention provides the antibody cocktail of the invention for use in a method of treating, preventing or ameliorating Ebola virus infection, said method comprising administering the antibody cocktail to a patient in need thereof. Likewise, the invention provides use of the antibody cocktail of the invention in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection. Brief Description of the Figures
Figure 1: List of the monoclonal antibodies with their VH and Vl/K genes.
Figure 2: Human monoclonal antibodies isolated from vaccinated individuals. (A) A total of 82 antibodies were isolated from 11 vaccinated volunteers. 38 out of 82 antibodies blocked infection of MDCK-SIAT cells by E-S-FLU. (B) Example of in vitro microneutralisation of Ebola pseudotyped influenza virus (named as E-S-FLU virus) infecting MDCK-SIAT cells. (C) Microscopic images showing virus neutralisation by different antibodies at 5 mg/ml concentration (40x Magnification). Cell monolayer shows a confluent monolayer of MDCK-SIAT 1 cells. In the absence of antibody (virus only control), the majority of the cells are infected, which is indicated by eGFP expression. Partial neutralising antibody 66-4-C12 suppressed the virus infection partially, whereas the strong neutraliser 66-6-3 completely suppressed infection by the virus. D) In vitro neutralising concentration of monoclonal antibodies shown as IC50 and IC90. Out of 82 antibodies, 38 are neutralising. Neutralisation IC50 ranges from 100 ng/ml to 10 mg/ml. Partial neutralising antibodies have no IC90 values. Almost half of the antibodies have titres comparable to that of the antibodies published in the literature. Measurements were taken as an average of duplicates and experiments were repeated at least twice, each point represents an independent result. The % value in brackets is the survival of mice treated at day 2 of infection with 100 mg of antibody in the VIC study. E) Association between treatment at day 2 of infection with human mAbs that neutralise ³ 50% E-S-FLU and survival P<0.0001 (2 tailed Fisher’s exact test).
Figure 3: Identification of Epitopes. A) Antibody epitopes defined by cross- inhibition. Known antibodies 114, ZMapp (cl3C6, c2G4, c4G7), 100 and KZ52 were used as guide mAbs to define groups of antibodies by cross-inhibition of binding. Antibodies 66-4-A8 (MLD-dependent EBOV antibody) and Influenza antibodies were used as non inhibiting controls. Inhibition values were calculated as defined in STAR methods. Results are the average of repeated experiments and 4-8 replicates were measured in each experiment. B) Yeast display library. A selection of mAbs were screened for binding a yeast display library of fragments. Mabs 040 (i), 114 (ii), 66-3-9C (iii). The fragments recognized are represented on the structure of the KZ52 -GP complex PDB 3CSY (iv). C) Binding of mAbs to Ebola GP on transduced MDCK-SIAT 1 cells after thermo lysin digestion. Log of difference of binding of mAbs to thermolysin treated E-SIAT and E- SIAT is shown: Log [(Binding geometric mean fluorescence to thermolysin treated E-
SIAT cells) - (Binding geometric mean fluorescence to untreated E-SIAT cells)]. An arbitrary 0.2 is used as a cut-off line to define mAbs affected by thermo lysin digestion, with reference to mAb 114, which is known to be resistant to thermo lysin digestion (Misasi et al. 2016, Science, 351, 1343-1346). D) Assignment of neutralising or protective antibodies to their epitopes based on cross-inhibition assays i) Mucin-like domain dependent antibodies. Six antibodies lost binding to the MLD deleted GP. 66-3-9C (specific for the b17-18 loop) and the base antibody 66-4-C12 showed reduced binding ii) Glycan Cap and Receptor Binding Region: P6-like (Glycan Cap), 114-like (Receptor Binding Region) and others in-between these epitopes. P6-like and 114-like are independent epitopes and do not block each other. Antibodies on the left (P6-like and neighbours) are sensitive to thermo lysin cleavage. Antibodies on the right (114-like) are resistant to thermolysin cleavage or have enhanced binding after cleavage iii) Antibodies to Base. These antibodies are blocked by defined base antibodies and are resistant to thermolysin digestion.
Figure 4: Binding of mAb P6 to peptide fragments of large yeast display library.
Figure 5: Effects of thermolysin on glycan cap mAbs. A) Binding of mAbs to MDCK-SIAT1 cells expressing Ebola virus GP (E-SIAT). B) Binding of mAbs to thermolysin treated E-SIAT cells. C) Binding of glycan cap mAbs before cleavage by thermolysin. These experiments were done on adherent cells without trypsin to detach cells. The binding fluorescence was read using Clariostar plate reader and Wheat-germ agglutinin (WGA) was used to normalise for the number of cells. Data shown are
Mean±SD of three replicates. Binding unit = mAb binding/ WGA fluorescence. THL: Thermolysin; RBR: Receptor binding region; MLD: Mucin-like domain; SA-AF647: Streptavidin labelled with Alexa fluor 647.
Figure 6: Identification antibodies recognising mucin-like domain (MLD) of the glycoprotein. MLD (amino acids 314-463) was removed from the EBOV GP gene and expressed on the surface of MDCK-SIAT1 cells. Antibody binding to the GP on MDCK- SIAT1 cells (E-SIAT) and E(DMLD)-SIAT cells was compared.
Figure 7: Effect of GP specific antibodies on cleavage by thermolysin. A) Binding by glycan cap antibodies is sensitive to thermolysin cleavage but binding by RBR and base antibodies is not. GC: Glycan cap, RBR: Receptor binding region, THL: Thermolysin, E- SIAT: MDCK-SIAT1 cells expressing Ebola virus glycoprotein. B) Some glycan cap antibodies block cleavage of GP by thermolysin.
Figure 8: Sequence homology of the glycoproteins used. Figure 9: Twenty out of 82 mAbs are cross-reactive to both Sudan and Bundibugyo GPs. A) Monoclonal antibodies were compared by indirect immunofluorescence by FACS for binding to MDCK-SIAT1 cells expressing GPs from different Ebola virus species. B) Venn diagram showing the frequencies of cross-reactive antibodies. Twenty of 84 showed some level of cross-reactivity between the three species of GP. C) A selection of EBOZ reactive antibodies were compared for binding to Bundibugyo and Sudan virus GPs.
Figure 10. A) Phylogeny of the antibodies based on VDJ amino acid sequences. More than 23 VH genes have been used altogether. VH3-15 is the most used gene and all nine antibodies that recognize 114-like epitope are encoded by this gene. There is diversity in terms of VH gene use within individuals and within the antibodies to epitopes in glycan cap and base. Tree was drawn using MEGA v7 software and alignment was done using Neighour-joining tree settings. B) CDR3 length and frequency of somatic mutations in the largest set of VH genes.
Figure 11: Range of affinity constants for antibodies isolated from vaccinated donors. Calculated affinity constants for a selection of neutralising antibodies compared to established therapeutic antibodies 114 to RBR and 100 to the Base, 6D6 to fusion peptide, cl3C6 to RBR.
Figure 12: Selection of antibody cocktails. A) Neutralisation of pseudotyped virus by seven mAbs selected for inclusion in three antibody cocktails. Antibodies were selected on four criteria: 1) Neutralisation, 2) ability to protect mice from Ebola infection, 3) mutually exclusive binding to separate epitopes on GP and 4) Cross -reactivity with Bundibugyo and Sudan GP. B-E) Neutralisation of pseudotyped viruses by the selected antibody cocktails. F) Characteristics of antibodies selected for inclusion in cocktail for guinea pig trial.
Figure 13: Protection of guinea pigs by antibody mixtures against Ebola virus infection. A) Survival curves for the guinea pigs treated with the antibody cocktails on day 3 of infection. B) Body weight (column 1) , Body temperature (column 2) and Clinical scores (column 3).
Figure 14: Viral PCR of the samples from guinea pigs that survived via
immunotherapy or died from infection.
Figure 15: Lesion severity and IHC scores
Figure 16: Liver of animal 19063, 924/17 (group 1). Patchy staining of tissue denoting presence of viral antigen, and often corresponding to focal areas of necrosis. Figure 17: Spleen of animal 000176,930/17. Strong, diffuse, positive staining for viral antigen primarily in the red pulp.
Figure 18: Liver from animal 19666,940/17. Patchy staining of viral antigen.
Figure 19: (A) Liver immunohisto chemistry for group 5. (B) Spleen
immunohisto chemistry for group 5.
Figure 20: Liver of animal 00436,957/17. Strong, multifocal staining of viral antigen.
Figure 21: Spleen of animal 9186,931/17 (group 2) showing congestion of the sinusoids.
Figure 22: Spleen of animal 02176,946/17 (group 5) showing scattered, degenerating cells with fragmented nuclei.
Figure 23: Spleen of animal 19186,931/17 (group 2) showing infiltration of neutrophils in the red pulp.
Figure 24: Spleen of animal 00252,927/17 (group 1) showing scattered lymphocyte loss, likely apoptosis.
Figure 25: Spleen of animal 19058,945/17 (group 4) showing reduction of cells in the white pulp.
Figure 26: Liver of animal 00252,927/17 (group 1) showing scattered necrotic foci comprising degenerating cells and nuclear debris with scattered mineralised areas.
Figure 27: Animal 00252,927/17 (groupl ) showing foci of mineralisation within the necrotic areas.
Figure 28: Liver of animal 00252,927/17 (group 1) showing diffusely scattered, macrovesicular vacuo lation in hepatocytes (lipid).
Figure 29: HCVR and LCVR sequences of antibodies of the invention.
Brief Description of the Sequence Listing
SEQ ID NO: 1 provides the sequence of the 66-3-9C heavy chain variable region.
SEQ ID NO: 2 provides the sequence of the 66-3-9C light chain variable region.
SEQ ID NO: 3 provides the sequence of the 040 heavy chain variable region.
SEQ ID NO: 4 provides the sequence of the 040 light chain variable region.
SEQ ID NO: 5 provides the sequence of the 6662 heavy chain variable region.
SEQ ID NO: 6 provides the sequence of the 6662 light chain variable region.
SEQ ID NO: 7 provides the sequence of the 6541 heavy chain variable region. SEQ ID NO: 8 provides the sequence of the 6541 light chain variable region. SEQ ID NO: 9 provides the sequence of the 66-3-9C HCDR1.
SEQ ID NO: 10 provides the sequence of the 66-3-9C HCDR2.
SEQ ID NO: 11 provides the sequence of the 66-3-9C HCDR3.
SEQ ID NO: 12 provides the sequence of the 66-3-9C LCDR1.
SEQ ID NO: 13 provides the sequence of the 66-3-9C LCDR2.
SEQ ID NO: 14 provides the sequence of the 66-3-9C LCDR3.
SEQ ID NO: 15 provides the sequence of the 040 HCDR1.
SEQ ID NO: 16 provides the sequence of the 040 HCDR2.
SEQ ID NO: 17 provides the sequence of the 040 HCDR3.
SEQ ID NO: 18 provides the sequence of the 040 LCDR1.
SEQ ID NO: 19 provides the sequence of the 040 LCDR2.
SEQ ID NO: 20 provides the sequence of the 040 LCDR3.
SEQ ID NO: 21 provides the sequence of the 6662 HCDR1.
SEQ ID NO: 22 provides the sequence of the 6662 HCDR2.
SEQ ID NO: 23 provides the sequence of the 6662 HCDR3.
SEQ ID NO: 24 provides the sequence of the 6662 LCDR1.
SEQ ID NO: 25 provides the sequence of the 6662 LCDR2.
SEQ ID NO: 26 provides the sequence of the 6662 LCDR3.
SEQ ID NO: 27 provides the sequence of the 6541 HCDR1.
SEQ ID NO: 28 provides the sequence of the 6541 HCDR2.
SEQ ID NO: 29 provides the sequence of the 6541 HCDR3.
SEQ ID NO: 30 provides the sequence of the 6541 LCDR1.
SEQ ID NO: 31 provides the sequence of the 6541 LCDR2.
SEQ ID NO: 32 provides the sequence of the 6541 LCDR3.
SEQ ID NO: 33 provides the sequence of the 6660 HCDR3.
SEQ ID NO: 34 provides the sequence of the 6669 HCDR3.
SEQ ID NO: 35 provides the sequence of the 6670 HCDR3.
SEQ ID NO: 36 provides the sequence of the 6666 HCDR3.
SEQ ID NO: 37 provides the sequence of the 6667 HCDR3.
SEQ ID NO: 38 provides the sequence of the P7 HCDR3.
SEQ ID NO: 39 provides the sequence of the 105 HCDR3.
SEQ ID NO: 40 provides the sequence of the 66-6-14 HCDR3.
SEQ ID NO: 41 provides the sequence of the 56-4-D4 HCDR3. SEQ ID NOIs: 42-205 provide the heavy and light junction sequences as shown in Figure 1
SEQ ID NO: 206 provides the sequence of the 66-3-9C heavy chain.
SEQ ID NO: 207 provides the sequence of the 66-3-9C light chain.
SEQ ID NO: 208 provides the sequence of the 040 heavy chain.
SEQ ID NO: 209 provides the sequence of the 040 light chain.
SEQ ID NO: 210 provides the sequence of the 6662 heavy chain.
SEQ ID NO: 211 provides the sequence of the 6662 light chain.
SEQ ID NO: 212 provides the sequence of the 6541 heavy chain.
SEQ ID NO: 213 provides the sequence of the 6541 light chain.
SEQ ID NO: 214 provides the sequence of the 6660 heavy chain.
SEQ ID NO: 215 provides the sequence of the 6660 light chain.
SEQ ID NO: 216 provides the sequence of the 66-3-9C heavy chain.
SEQ ID NO: 217 provides the sequence of the 66-3-9C light chain.
SEQ ID NO: 218 provides the sequence of the 66-6-3 heavy chain.
SEQ ID NO: 219 provides the sequence of the 66-6-3 light chain.
SEQ ID NOs: 220-337 provide the heavy and light chain variable regions of the antibodies in Figure 29 (Table 10).
SEQ ID NOs: 378-380 provide variant sequences of the 66-3-9C LCDR1 SEQ ID NOs: 381-383 provide variant sequences of the 6662 HCDR2
SEQ ID NOs: 384 and 385 provide variant sequences of the 6662 HCDR3 SEQ ID NOs: 386-388 provide variant sequences of the 6662 LCDR3
SEQ ID NOs: 389-392 provide variant sequences of the 6541 HCDR2
SEQ ID NO: 393 provides the sequence of the 66-4C-12 HCDR1
SEQ ID NO: 394 provides the sequence of the 66-4C-12 HCDR2
SEQ ID NO: 395 provides the sequence of the 66-4C-12 HCDR3
SEQ ID NO: 396 provides a variant sequence of the 66-4C-12 HCDR3
SEQ ID NO: 397 provides the sequence of the 66-4C-12 LCDR1
SEQ ID NO: 398 provides the sequence of the 66-4C-12 LCDR2
SEQ ID NO: 399 provides the sequence of the 66-4C-12 LCDR3
SEQ ID NO: 400 provides the sequence of the 6651 HCDR1
SEQ ID NO: 401 provides the sequence of the 6651 HCDR2
SEQ ID NO: 402 provides the sequence of the 6651 HCDR3
SEQ ID NO: 403 provides the sequence of the 6651 LCDR1 SEQ ID NO: 404 provides the sequence of the 6651 LCDR2
SEQ ID NO: 405 provides the sequence of the 6651 LCDR3
Detailed Description of the Invention
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
In addition as used in this specification and the appended claims, the singular forms “a”,“an”, and“the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to“an amino acid sequence” includes two or more such sequences, and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Anti-Ebola virus antibodies
The present invention relates to antibodies that bind to (recognise) the Ebola virus glycoprotein (GP) and to pharmaceutical compositions comprising such antibodies. There are currently six Ebola virus species (Zaire, Sudan, Bundibugyo, Reston, Ta'i Forest and Bombali) and many different strains. Zaire, Sudan, Bundibugyo and Ta'i Forest cause disease in humans, with Zaire being the most deadly.
The Ebola virus glycoprotein is the only virally expressed protein on the virus 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 virus surface. As discussed further below, 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 virus glycoprotein sequences vary between species. The nucleotide and amino acid sequences of the glycoprotein from various Ebola virus species/strains have been determined, with examples including GenBank: AF086833.2 providing the complete genome of Zaire Mayinga, and GenBank: AAN37507.1 and GenBank: AAG40168.1 providing examples of Zaire glycoprotein sequences. Accession numbers NC_014373.1 and NC_006432.1 provide the complete genome sequences ofBudibugyo and Sudan Gulu.
Accession numbers GenBank: AGL73446.1 and GenBank: AGL73439.1 provide examples of a Sudan glycoprotein sequence and accession numbers GenBank: AGL73474.1 and GenBank: AGL73467.1 provide examples of Bundibugyo glycoprotein sequences. 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.
The term“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).
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.
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. Typically, the antibody is a human 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), scFv, bi, tri or tetra -valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126- 1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other 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). Such antibody fragments are described in International Patent Application Publication No, WO 2015/197772, which is hereby incorporated by reference in its entirety and particularly with respect to the discussion of antibody fragments
These 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, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. Typically, the constant regions are human. In particular, human IgG (i.e. IgG1, IgG2, IgG3 or IgG4) constant region domains may be used. Typically, a human IgGl constant region.
The light chain constant region may be either lambda or kappa. For example, kappa light chain constant regions may be used with the 040, 6541 and 66-3-9C antibodies and a lambda light chain constant region may be used with the 6662 antibody.
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.
The term“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. In one embodiment the antibody or fragment according to the present disclosure has an isoelectric point (pi) of at least 7. In one embodiment the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pi of the antibody is 8. Programs such as ** ExPASY http://www.expasy.ch/tools/pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607), may be used to predict the isoelectric point of the antibody or fragment.
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 al, 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al, 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 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
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
0546073, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474 and EP 0463151. Human antibodies can also be generated using the method described in the Examples below.
The term“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, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
As used herein, the term‘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 or rat monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.
When the CDRs or specificity determining residues are grafted, 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. Suitably, 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. Thus, provided in one embodiment is a neutralising CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.
Examples of human frameworks which can be used are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Rabat et al, supra). For example, 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. Alternatively, 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). In a CDR-grafted antibody, 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.
Also, in a CDR-grafted antibody, 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.
Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Rabat definition, the Chothia definition, and the AbM definition. In general terms, the Rabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Rabat and Chothia approaches. See, e.g., Rabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
An antibody of the invention may contain one, two, three, four, five or six CDR sequences from an antibody heavy and light chain variable region sequence pair of the invention (including those identified in Table 10). An antibody of the invention typically comprises all six (i.e. three heavy and three light chain) CDR sequences from a heavy/light chain variable region sequence pair of the invention. In particular, an antibody of the invention may comprise six CDRs contained within a heavy and light chain variable region sequence pair of SEQ ID NOs: 1/2, 3/4, 5/6, or 7/8. These are the heavy and light chain variable region sequence pairs of the 66-3-9C, 040, 6662 and 6541 antibodies of the invention.
An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 9 to 11 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 66-3-9C antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 12 to 14
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 66-3-9C antibody of the Examples (as per the Rabat definitions).
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 11.
Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 9 to 11 and at least one light chain CDR sequence selected from SEQ ID NOS 12 to 14. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 9 to 11 and at least two light chain CDR sequences selected from SEQ ID NOS: 12 to 14. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 9 to 11 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 12 to 14 (LCDR1/LCDR2/LCDR3 respectively).
In an alternative, an antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 15 to 17
(HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 040 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 18 to 20
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 040 antibody of the Examples (as per the Rabat definition).
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 17.
Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 15 to 17 and at least one light chain CDR sequence selected from SEQ ID NOS 18 to 20. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 15 to 17 and at least two light chain CDR sequences selected from SEQ ID NOS: 18 to 20. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 15 to 17 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 18 to 20 (LCDR1/LCDR2/LCDR3 respectively).
The antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 21 to 23 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 6662 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a
combination of Rabat and Chothia).
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 24 to 26
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 6662 antibody of the Examples (as per the Rabat definition).
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 23.
Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 21 to 23 and at least one light chain CDR sequence selected from SEQ ID NOS 24 to 26. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 21 to 23 and at least two light chain CDR sequences selected from SEQ ID NOS: 24 to 26. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 21 to 23 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 24 to 26 (LCDR1/LCDR2/LCDR3 respectively).
In an alternative, the antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 27 to 29
(HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 6541 antibody of the Examples (these are as per the Rabat definitions, except HCDR1 which is a combination of Rabat and Chothia).
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 30 to 32
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 6541 antibody of the Examples (as per the Rabat definition).
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 29.
Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 27 to 29 and at least one light chain CDR sequence selected from SEQ ID NOS 30 to 32. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 27 to 29 and at least two light chain CDR sequences selected from SEQ ID NOS: 30 to 32. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 27 to 29 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 30 to 32 (LCDR 1 /LCDR2/LCDR3 respectively).
An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 393 to 395 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 66-4C-12 antibody of the Examples.
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 397 to 399
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 66-4C-12 antibody of the Examples.
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 395.
Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 393 to 395 and at least one light chain CDR sequence selected from SEQ ID NOS 397 to 399. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 393 to 395 and at least two light chain CDR sequences selected from SEQ ID NOS: 397 to 399. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 393 to 395 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 397 to 399 (LCDR1/LCDR2/LCDR3 respectively).
An antibody of the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 400 to 402 (HCDR1/HCDR2/HCDR3 respectively). These are the HCDR1/HCDR2/HCDR3 sequences of the 6651 antibody of the Examples.
Furthermore, the antibody of the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 403 to 405
(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3 sequences of the 6651 antibody of the Examples.
The antibody of the invention suitably comprises at least a HCDR3 sequence of SEQ ID NO: 402. Typically, the antibody of the invention comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 400 to 402 and at least one light chain CDR sequence selected from SEQ ID NOS 403 to 405. The antibody of the invention may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 400 to 402 and at least two light chain CDR sequences selected from SEQ ID NOS: 403 to 405. The antibody of the invention typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 400 to 402 (HCDR1/HCDR2/HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 403 to 405 (LCDR1/LCDR2/LCDR3 respectively).
In some instances, an antibody of the invention may comprise an HCDR3 sequence of one of the antibodies of the Examples (SEQ ID NOs: 33-41).
An antibody of the invention may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 1 (the HCVR of 66-3-9C). An antibody of the invention may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 2 (the LCVR of 66-3-9C). An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 2.
An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 3 (the HCVR of 040). An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 4 (the LCVR of 040). An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4.
An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 5 (the HCVR of 6662). An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 6 (the LCVR of 6662). An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 5 and the light chain variable region sequence of SEQ ID NO: 6.
An antibody of the invention may comprise a HCVR sequence of SEQ ID NO: 7 (the HCVR of 6541). An antibody of the invention may comprise a LCVR sequence of SEQ ID NO: 8 (the LCVR of 6541). An antibody of the invention typically comprises the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.
SEQ ID NO: 206 presents the complete heavy chain sequence of 66-3-9C and SEQ ID NO: 207 presents the complete light chain sequence of 66-3-9C (kappa light). An antibody of the invention may comprise the 66-3-9C variable regions, the heavy chain constant region from SEQ ID NO: 206 and the light chain constant region from SEQ ID NO: 207. An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 206 and a light chain of SEQ ID NO: 207.
SEQ ID NO: 208 presents the complete heavy chain sequence of 040 and SEQ ID NO: 209 presents the complete light chain sequence of 040 (kappa light). An antibody of the invention may comprise the 040 variable regions, the heavy chain constant region from SEQ ID NO: 208 and the light chain constant region from SEQ ID NO: 209. An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 208 and a light chain of SEQ ID NO: 209.
SEQ ID NO: 210 presents the complete heavy chain sequence of 6662 and SEQ ID NO: 211 presents the complete light chain sequence of 6662 (lambda light). An antibody of the invention may comprise the 6662 variable regions, the heavy chain constant region from SEQ ID NO: 210 and the light chain constant region from SEQ ID NO: 211. An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 210 and a light chain of SEQ ID NO: 211.
SEQ ID NO: 212 presents the complete heavy chain sequence of 6541 and SEQ ID NO: 213 presents the complete light chain sequence of 6541 (kappa light). An antibody of the invention may comprise the 6541 variable regions, the heavy chain constant region from SEQ ID NO: 212 and the light chain constant region from SEQ ID NO: 213. An antibody of the invention may also comprise a heavy chain of SEQ ID NO: 212 and a light chain of SEQ ID NO: 213.
An antibody of the invention may also comprise six CDR sequences of a
HCVR/LVCR pair as identified in Figure 29 (Table 10). Such CDRs may be identified using the methods described above. Furthermore, an antibody of the invention may comprise a HCVR or LCVR (or a HCVR/LVCR pair) as identified in Table 10.
Also included in the invention are antibodies with the above sequences but engineered for example to (i) remove deamidation and glycosylation sites and/or (ii) iso- asp removal and/or (iii) C-terminal lysine removal and/or N-terminal Q to E exchange.
In one example one or more sequences (for example one or more CDRs) provided herein may be modified to remove undesirable residues or sites, such as cysteine residues or aspartic acid (D) isomerisation sites or asparagine (N) deamidation sites.
For example, one or more cysteine residues in any one of the sequences (for example, in any one of the CDRs) may be substituted with another amino acid, such as serine. In one example, 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. In one example an asparagine deamidation site such as NG or NS may be mutated, for example to NA or NT.
In one example, 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. In one example an aspartic acid isomerisation site such as DG or DS may be mutated, for example to EG, DA or DT.
In one example, 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.
In one example 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.
In particular, variant LCDR1 sequences for the 66-3-9C antibody are presented in SEQ ID NOs: 378-380. An antibody of the invention may comprise one of these variant sequences. For example, an antibody may comprise a LCDR1 of one of SEQ ID NOs: 378-380 and then HCDR1, HCDR2, HCDR3, LCDR2 and LCDR3 sequences of SEQ ID NOs: 9, 10, 11, 13 and 14 respectively.
Variant HCDR2 sequences for the 6662 antibody are presented in SEQ ID NOs: 381, 382 and 383. Variant HCDR3 sequences for the 6662 antibody are presented in SEQ ID NOs: 384 and 385. Variant LCDR2 sequences for the 6662 antibody are presented in SEQ ID NOs: 386, 387 and 388. An antibody of the invention may comprise one or more of these variant CDR sequences. For example, an antibody may comprise a HCDR1 sequence of SEQ ID NO: 21, a HCDR2 sequence of SEQ ID NO: 22, 381, 382 or 383, a HCDR3 sequence of SEQ ID NO: 23, 284 or 285, a LCDR1 sequence of SEQ ID NO: 24, a LCDR2 sequence of SEQ ID NO: 25 and a LCDR3 sequence of SEQ ID NO: 26, 386, 387 or 388. An a antibody of the invention may comprise any combination of the above CDRs.
Variant HCDR2 sequences for the 6541 antibody are presented in SEQ ID NOs: 389- 392. An antibody of the invention may comprise one of these variant sequences. For example, an antibody may comprise a HCDR2 of one of SEQ ID NOs: 389-392 and HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 27, 29, 30, 31 and 32 respectively.
A variant HCDR3 sequence for the 66-4-C12 antibody is presented in SEQ ID NO: 396. An antibody of the invention may comprise this variant sequence. For example, an antibody may comprise a HCDR3 of SEQ ID NO: 396 and HCDR1 , HCDR2, LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NOs: 393, 394, 397, 398 and 399 respectively.
The antibody may be or may comprise a variant of one of the specific sequences recited above. For example, 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. For example, 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. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:
Figure imgf000024_0001
'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 (particularly the HCVR/LCVR sequences). Furthermore, 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
HCVR/LCVR 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 HCVR/LCVR 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.
In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson el al., 1994, supra) with the following parameters:
Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;
Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.
The present invention thus provides antibodies having specific sequences and variants which maintain the function or activity of these chains.
With regards to function, in some instances antibodies of the invention are able to neutralise at least one biological activity of Ebola virus (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. In the invention, neutralisation may be determined using an assay for measuring infection of cells using virus expressing the Ebola virus glycoprotein.
An example of such an assay uses the E-S-FLU Ebola virus surrogate as described in the Examples below. This assay utilises a disable 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.
In a neutralisation assay, 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 a maximum antibody concentration of 5 mg/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 mg/ml.
As shown in Figure 2B, the 6660, 125 and 66-6-3 antibodies are strong neutralising antibodies and the 66-4-C12 was an example of a partial neutralising antibody.
Antibodies of the invention may have sequences as described above and be either a strong or partial neutralising antibody. For example, an antibody of the invention may have the six CDR sequences of 6660, 125 or 66-6-3 and be a strong neutralising antibody.
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 mg/ml, less than 5 mg/ml, less than 1.25 mg/ml, less than 1 mg/ml or less than 0.75 mg/ml (typically down to 0.1 mg/ml). For example in some instances an antibody of the invention may have an IC50 value of between 0.1 mg/ml and 10 mg/ml, sometimes between 0.1 mg/ml and 5 mg/ml or even between 0.1 mg/ml and 1 mg/ml. In some instances, an antibody of the invention may have an IC50 value of between 1 mg/ml and 10 mg/ml, sometimes between 1 mg/ml and 5 mg/ml.
An antibody of the invention may have an IC90 value of less than 10 mg/ml, optionally less than 5 mg/ml (typically down to 0.6 mg/ml). For example, an antibody of the invention may have an IC90 value of between 0.6 mg/ml and 10 mg/ml, for example between 1 mg/ml and 5 mg/ml. These IC50/ IC90 values may be applied to the sequences described above. For example, an antibody of the invention may have six CDR sequences as described above and an IC50/ IC90 value as presented above. Neutralisation ability may be determined for any species of Ebola virus, such as Zaire, as shown in the Examples.
In terms of binding kinetics, an antibody of the invention may have an affinity constant (KD) value for the glycoprotein monomer of 50 nM or less, 25 nM or less of 10 nM or less. An antibody of the invention may have an affinity constant (KD) value for the glycoprotein trimer of 50 nM or less, 10 nM or less of 1 nM or less. These values may be applied to the sequences described above. For example, any antibody may have (a) the six CDR sequences of the 6541 antibody and a KD for the monomeric glycoprotein of 50 nM or less and a KD for the trimeric glycoprotein of 50 nM or less; (b) the six CDR sequences of the 040 antibody and a KD for the monomeric glycoprotein of 25 nM or less and a KD for the trimeric glycoprotein of 10 nM or less or (c) the six CDR sequences of the 66-3-9C antibody and a KD for the monomeric glycoprotein of 25 nM or less and a KD for the trimeric glycoprotein of 1 nM or less.
Affinity constants are typically determined using Surface Plasmon Resonance (Biacore) at 25 °C.
Antibodies of the invention are also preferably able to provide in vivo protection in Ebola virus infected animals. For example, 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%. Ideally, antibodies of the invention achieve a survival rate of 100%. Survival rates may be determined using routine methods.
An experiment for assessing in vivo protection in mice is described in the
Examples. In the invention, in vivo protection may be determined in mice administered a single 100 mg dose of antibody at day two of infection. In vivo protection may also be determined in guinea pigs as described in the Examples. In such experiments, antibodies may be administered, for example, at a dose of 10 mg/kg of each antibody at day three of infection.
As discussed further below, antibodies of the invention may be 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). Preferably, antibodies are cross-reactive for all of the above. In other words, the antibodies are capable of binding to the glycoprotein from these species/strains. Binding can be measured, for example, using Surface Plasmon Resonance as described in the Examples. 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 KD value. For example, antibodies of the invention may be cross-reactive if they have a KD value of less than 1 mM for more than one species (antibodies may have a KD value of less than 1 mM for more than one species, such as Zaire, Bundibugyo and Sudan). KD values may be determined as described above.
The antibodies preferably also retain their neutralisation capabilities and their protection capabilities in the other species.
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 DNA 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.
DNA 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, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.
General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to“Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.
Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding an antibody of the present invention. Any suitable host cell/ vector system may be used for expression of the DNA 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. Typically, 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.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising one or more antibodies that bind to the Ebola virus glycoprotein, such as one or more antibodies of the invention as described above, and a pharmaceutically acceptable carrier or diluent. 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.
Numerous methods may be used to determine whether antibodies cross-compete or bind to non-overlapping epitopes. Such methods are utilised in the Examples and are discussed further below.
A pharmaceutical composition of the invention may comprise any of the antibodies described above. For example, in a pharmaceutical composition of the invention at least one of the antibodies may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences contained within a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs:1/2, 3/4, 5/6 and 7/8. Similarly, at least one of the antibodies may comprise a set of six CDRs selected from the group consisting of SEQ ID NOs: 9-14, 15-20, 21-26 and 27-32. At least one of the antibodies may comprise a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8. As discussed above, these are the sequences of the 66-3-9C, 040, 6662 and 6541 antibodies of examples.
The pharmaceutical composition may comprise at least two or three antibodies as described above (with any of the sequences as described above). Preferably, the composition comprises four antibodies as described above (with HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences contained within the HCVR/LCVR sequence pairs of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8, with six CDRs of SEQ ID NOs: 9-14, 15-20, 21-26 and 27-32 or with HCVR/LCVR sequence pairs of SEQ ID NOs: 1/2, 3/4, 5/6 and 7/8). The antibodies may also comprise a constant region as described above. As used herein, "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.
The pharmaceutical 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.
Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of 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. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Therapeutic 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.
Pharmaceutical 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.
Antibody cocktails
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. As used herein, an“antibody cocktail” generally refers to a combination/mixture of antibodies within the same composition, i.e. a single
pharmaceutical composition comprising the antibodies. As described elsewhere herein, the invention also includes the combined use of different anti-Ebola virus antibodies in separate pharmaceutical compositions.
The antibodies may bind to the Ebola virus glycoprotein from any of the species/strains discussed above. The sequences of such glycoproteins would be well known to the skilled person.
A cocktail of the invention may comprise two or more antibodies. Typically, a cocktail of the invention comprises two or more antibodies binding to different regions of the Ebola virus glycoprotein. For example, a cocktail may comprise two or more antibodies binding to at least two of the following regions of the glycoprotein: glycan cap, receptor binding region and base. In some instances, a cocktail of the invention may comprise three antibodies binding to the Ebola virus glycoprotein.
Specifically, the present invention provides an antibody cocktail comprising three or more antibodies binding to the Ebola virus glycoprotein, wherein one antibody binds to the glycan cap, one antibody binds to the receptor binding region and one antibody binds to the base. In other words, one antibody recognises an epitope in the glycan cap, one antibody recognises an epitope in the receptor binding region and one antibody recognises an epitope in the base. Preferably, the cocktail further comprises an antibody binding to (recognising an epitope within) the b17-18 loop. The antibody preferably recognises an epitope where all of the epitope residues are within the specified region. However, in some instances it is sufficient that only some of the epitope residues are within the specified region.
The skilled person would readily be able to determine the amino acid numbering for each of these domains of the glycoprotein based on the published information for the various species/strains.
The skilled person would also readily be able to determine the binding site
(epitope) of an antibody using standard techniques, such as those described in the
Examples of the application. 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.
For example, to determine if a 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. Next, 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 On the other hand, if the test antibody is not able to bind to protein or peptide following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention.
To determine if an antibody competes for binding with a reference antibody, the above-described binding methodology is performed in two orientations. In 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. In a second orientation, 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. As will be appreciated by the skilled person, 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 al., Cancer Res, 1990:50:1495-1502). Alternatively, 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) 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. Experiments of 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. The antibodies described below in the Examples may be used as reference antibodies.
Other techniques that may be used to determine antibody epitopes include hydro gen/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.
In the antibody cocktails of the invention it is preferred that 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.
It is also typical that one or more of the antibodies in the cocktail individually enhance survival of animals infected with Ebola virus (see above). In some instances, all of the antibodies in the cocktail enhance survival of Ebola virus infected animals.
Typically, administration of an antibody cocktail of the invention to Ebola virus infected animals results in a survival rate of at least 50%. Preferably, administration of an antibody cocktail of the invention to Ebola virus infected animals results in a 100% survival rate.
As described in the Examples, survival rates may be determined in mice (for example with a single dose of 100 mg of antibody at day 2 of infection).
Survival rates may also be determined in infected guinea pigs as described in the Examples. 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.
Furthermore, it is advantageous if 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 further above. Antibodies included in the cocktails of the invention may be any of those described above. In some instances, a cocktail includes the 66-3-9C, 040, 6662 and 6541 antibodies, or antibodies derived from these antibodies (for example comprising the CDR and
HCVR/LCVR sequences described above). In other instances, an antibody in the cocktail may comprise a HCDR3 sequence of any one of SEQ ID NOs: 33-41.
As described above, the antibodies are monoclonal antibodies. The antibodies may be chimeric antibodies, CDR-grafted antibodies, nanobodies or humanised antibodies. Typically, the antibodies are human antibodies.
The antibodies may also be antigen-binding fragments (see above). Furthermore, the antibodies may comprise a constant region as described above.
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 al, 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al, 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 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108. 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 0546073, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474 and EP 0463151. Human antibodies can also be generated using the method described in the Examples below.
The term“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, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
As used herein, the term‘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 or rat monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.
When the CDRs or specificity determining residues are grafted, 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. Suitably, 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. Thus, provided in one embodiment is a neutralising CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.
Examples of human frameworks which can be used are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Rabat et al, supra). For example, 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. Alternatively, 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).
In a CDR-grafted antibody, 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.
Also, in a CDR-grafted antibody, 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 antibodies may be formulated using a pharmaceutically acceptable carrier or diluent, as discussed above.
Therapeutic uses
The antibodies, pharmaceutical composition and cocktails of the invention may be used for the treatment, prevention or amelioration of Ebola virus infection. In other words, 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 ebolavirus 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.
Typically, the invention relates to the administration of the antibodies/compositions to a human subject in need thereof. However, 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).
The antibodies, compositions and cocktails of the invention may be administered therapeutically or prophylactically.
As discussed above, the antibodies, pharmaceutical compositions and cocktails may be administered subcutatneously, intravenously, intradermally, orally, intranasally, intramuscularly or intracranially, Typically, the antibodies, 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. Alternatively, 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.
As discussed above, the antibodies of the invention are typically used in a single pharmaceutical composition/cocktail (co-formulated). However, 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. For example, the invention includes use the combined use of the 040, 66-3-9C, 6662 and 6541 antibodies (or antibodies comprising sequences from these antibodies; see above).
The same applies for administration of additional therapeutic agents, which are discussed above. These may be administered in combination with
antibodies/pharmaceutical compositions/cocktails of the invention.
The following examples are presented below so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention. The examples are not intended to limit the scope of what the inventors regard as their invention.
Examples
Example 1: Isolation and expression of monoclonal antibodies (mAbs) from vaccinated donors
Volunteers were vaccinated with chAD3 EBO Z (chimpanzee adenovirus 3 encoding Ebola Mayinga glycoprotein (AF086833) (Stanley et al. 2014, Nat Med, 20:
1126-9) and boosted with MVA-BN Filo Modified Vaccinia Ankara encoding the glycoproteins from Ebola virus Mayinga (ABX75367.1), Sudan Gulu (AAU43887.1) and Marburg virus Musoke (ABA87127.1) viruses, and the nucleoprotein from Tai Forest virus (ACI28629.1), produced by Bavarian Nordic (Ewer et al. 2016, N Engl J Med, 374: 1635- 46). A total of 82 antibodies were isolated from plasmablasts or memory B cells isolated at day 7 and day 28 respectively (Figure 1 (Table 1)), following booster vaccination with the MVA-BN-Filo from 11 vaccinated volunteers (Figure 2A), as described in the Methods section.
Example 2: Screening of Monoclonal Antibodies for binding and neutralisation MDCK-SIAT1 cells (Matrosovich et al. 2003; J Virol, 77: 8418-25 ) were transduced to express the glycoprotein from Ebola Zaire wt/GIN/2014/Kissidougou-C15 (KJ660346.1) as described (Xiao et al. 2018, J Virol, 92). MDCK-SIAT1 cells were used in these experiments because, unlike other cell lines, they tolerate high levels of stable expression of EBOV GP, can pseudotype an influenza core, and are readily infected by our EBOV surrogate E-S -FLU (Xiao et al, 2018).
Initial screening was by detection of binding by indirect immunofluorescence. Unlike other cell lines, MDCK-SIAT1 cells tolerate high levels of stable expression of Ebola GP (Xiao et al. 2018; J Virol, 92).
Antibodies were tested for blockade of infection by our Ebola surrogate E-S-FLU, which contains a disabled influenza core coated with Ebola GP (Xiao et al. 2018, J Virol, 92 ). E-S-FLU encodes a fluorescent protein eGFP that replaces the hemagglutinin coding sequence so that infected cells fluoresce green. Thirty-eight of 82 antibodies inhibited infection by E-S-FLU as defined by loss of eGFP fluorescence after over-night infection by at least 50% (Figure 2A-C). “Partial” neutralising antibodies (inhibition of infection plateaus at 50%-90% inhibition) were distinguished from“strong” neutralising (which achieve >/=90% inhibition of infection) as shown in figure 2B. Figure 2D, shows a summary of the 38 neutralising antibodies compared to a set of control antibodies described in the literature including KZ52 (Maruyama et al. 1999, J Virol, 73: 6024-30; Lee et al. 2008, Nature, 454: 177-82); c4G7, c2G4, cl3C6 - the three components of ZMapp (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7), 100 and 114 (Corti et al. 2016, Science, 351: 1339-42 ; Misasi et al. 2016, Science, 351: 1343-6) and 6D6 (Furuyama et al. 2016, Sci Rep, 6: 20514).
Example 3: The relationship between in vitro neutralisation and protection in vivo in mice
A set of the first 24 antibodies isolated (14 showing >50% neutralisation and 10 non-neutralising) were tested for protection of mice (single dose of 100 mg at day 2 of infection with a mouse-adapted Ebola Mayinga in groups of 10 as part of the work of the Viral Haemorrhagic Fever Immunotherapeutic Consortium (VIC) (Saphire 2018, Cell, 9, 938-952). This revealed that the 14 antibodies showing > 50% neutralisation provided overall 65.7% survival (range 10%- 100%), whereas the 10 non-neutralising antibodies provided 31% overall survival (range 10%-50%); two tailed P <0.0001 by Fisher’s exact test (Table 2). Table 2: Association between treatment at day 2 of infection with human mAbs that neutralise >/= 50% E-S-FLU and survival P <0.0001 (2 tailed Fisher’s exact test).
Figure imgf000040_0001
These results confirmed that neutralisation detected in the assay was associated with a therapeutic effect in mice, but also emphasised that some neutralising antibodies fail to protect, and some non-neutralising antibodies can protect, at least partially.
Example 4: Diversity of epitopes recognised
The epitopes recognised by antibodies were defined with four assays: 1)
competitive inhibition of binding by antibodies with defined specificities including P6 (glycan cap) (defined by alanine scanning and electron microscopy in the VIC study where P6 = VIC 82); cl3C6 (glycan cap/receptor binding region (RBR)) (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7); 114 (RBR) (Misasi et al. 2016, Science, 351: 1343- 6); KZ52 (base) (Lee et al. 2008, Nature, 454: 177-82), c2G4 and c4G7 (base) (Murin et al.
2014, Proc Natl Acad Sci U S A, 111: 17182-7); 100 (base) (Misasi et al. 2016, Science, 351: 1343-6); and 6D6 (fusion loop) (Furuyama et al. 2016, Sci Rep, 6: 20514) as guide antibodies (Figure 3A); 2) a sub-group of antibodies mapped using a yeast surface display antigenic library expressing GP fragments (Figure 3B, Table 3).
Table 3 : Summary of Yeast Display assay with EBOV mAbs
Figure imgf000040_0002
Figure imgf000041_0001
3) Binding to thermo lysin digested GP to mimic cathepsin removal of glycan cap and mucin-like domain (MLD) (Figure 3C, 3D). 4) binding to MDCK-SIAT1 cells transduced to express MLD-deleted GP (amino acids 313-463 deleted) (Figure 6). With this combination of tests seven clusters of antibody binding sites could be distinguished: three clusters in the glycan cap; one in the RBR; two in the base/fusion loop; and one in the MLD (summarised in figure 3D).
Antibodies to Glycan Cap: MAbs to the glycan cap could be divided into three overlapping groups. In the first group (exemplified by P6 and 040) the antibodies cross- inhibited the binding only of other GC specific antibodies (Figure 3A). P6 was defined as glycan cap specific by electron microscopy and alanine scanning (Saphire et al. 2018, Cell, 9, 938-952), and both P6 and 040 by sequencing of a protein fragment (amino acids 228- 281 of GP1) expressed in yeast that was bound by these antibodies (Figure 3B). The epitope bound by these two antibodies was removed by thermo lysin cleavage of GP expressed on MDCK-SIAT1 cells (Figure 3C). In the second group the antibodies inhibited the binding of both GC specific antibodies and RBR specific antibodies exemplified by 66- 3-2C (Figure 3A), which suggests they bind to an epitope that overlaps these two regions. The third group was defined by antibody 66-3-9C that bound to a small conserved peptide within the b17- 18 disordered loop of the glycan cap (amino acids 286-293 GEWAFWET) expressed in yeast (Figure 3B iii). 66-3-9C recognises a similar epitope to that bound by the macaque-derived mAb FVM09 (Keck et al. 2016, J Virol, 90: 279-91). The disordered loop is juxtaposed to the surface footprint bound by the base antibody KZ52 (Keck et al. 2016, J Virol, 90: 279-91), which may explain why KZ52 blocked the binding of biotinylated 66-3-9C (figure 3A). FVM09 was found to synergise with certain other mAbs to the GC and base for in vitro neutralisation and protection in vivo (Howell et al, 2017). Mutually enhanced binding was noted between the base antibodies c2G4 and c4G7 and 66- 3-9C (Figure 3 A) was noted. However, this mutually enhanced binding did not translate to enhanced neutralisation by mixtures of these antibodies in an in vitro assay. The great majority of GC specific antibodies defined by the competition assay lost binding to thermolysin treated GP expressed on MDCK-SIAT1 cells (Figure 3C).
Antibodies to the Receptor Binding Region: The RBR is highly conserved in species of Ebolavirus and Marburgvirus, and therefore offers an attractive target for therapeutic antibodies (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7, Hashiguchi et al., 2015, Cell, 160, 904-912, Flyak et al. 2015, Cell, 160, 893-903,
Bornholdt et al. 2016, Science, 351, 1078-1083, Corti et al. 2016, Science, 351, 1339- 1342). The RBR is partially protected by the glycan cap and mucin-like domain, and binding of EBOV GP to its receptor site on domain C of the NPC1 protein occurs only after the GC and MLD have been removed by cathepsin or thermolysin cleavage
(Chandran et al, 2005, Science, 308, 1643-1645, Schornberg et al., 2006, J. Virol. 80, 4174-4178, Miller et al. 2012, EMBO J, 31, 1947-1960, Cote et al, 2011, Nature, 477, 344- 348). Some antibodies like MR72 and MR191, that block the EBOV receptor binding site, also require removal of the GC to neutralise EBOV efficiently. Others, like 114, which was powerfully therapeutic in vivo (Corti et al., 2016, Misasi et al., 2016, Science, 351, 1343-1346) can bind either to the complete GP or to cleaved GP. mAbs to the RBR were defined by competition for binding to complete GP with human mAb 114. It was showed that 114 bound the GP1 core fragment 102-230 in the yeast expression assay (Figure 3A and 3B). Mab 114 competed for binding with a subset of neighbouring GC specific antibodies (Figure 3A). However, in contrast to the GC specific antibodies, 114 and similar antibodies retain binding after release of the GC and MLD following exposure of E-SIAT cells to thermo lysin digestion as expected (Corti et al., 2016, 219, Misasi et al., 2016) (Figure 3C and 6). Nine mAbs showed this pattern and were placed into the RBR binding group (Figure 3).
Antibodies to the Base/Fusion Loop: These antibodies were defined by competition for binding with the defined antibodies KZ52 (base) (Lee et al. 2008, Nature, 454: 177-82); c2G4 and c4G7 (base) (Murin et al. 2014, Proc Natl Acad Sci U S A, 111: 17182-7); 100 (Misasi et al. 2016, Science, 351: 1343-6); and 6D6 (fusion loop) (Furuyama et al. 2016, Sci Rep, 6: 20514). Many of these antibodies cross-inhibited each other, but sub-groups were discernible. For instance, biotinylated 6541 and 66-4-C12 were both inhibited by the characterised base antibodies KZ52 and 100, but 6541 and 66-4-C12 failed to inhibit each other, suggesting that they bound to non-overlapping sites in the base region (Figure 3A). Antibodies 6541, 66-4-C12 and 66-6-3 competed for binding with the fusion loop specific murine mAb 6D6, which suggested then binding footprints may overlap with the fusion peptide. Binding of base-region specific antibodies to thermo lysin treated cells was typically either unaffected or enhanced (Figure 3C).
Mucin Like Domain (MLD) dependent antibodies: The binding of 6/82 antibodies to MDCK-SIAT1 cells expressing GP lacking the MLD (amino acids 313-463) was reduced by comparison with cells expressing full-length GP (Figure 6). Control antibodies to GP1 head (c13C6) and base (KZ52 and c4G7) bound the MLD deleted and full length GP equally. None of the six MLD dependent antibodies were neutralising. Antibodies 66- 3-9C (specific for the b17- 18 loop sequence (GEWAFWET) also lost binding to the MLD deleted GP, and the binding of one base antibody 66-4-C12 was reduced (although was not affected by thermolysin cleavage).
Example 5: Antibodies to the glycan cap that block thermolysin cleavage
During experiments with thermolysin some antibodies to the glycan cap, once bound, remained bound after exposure of GP-transduced MDCK-SIAT1 (E-SIAT) cells to thermolysin treatment. . Figure 7A shows binding of selected GC, RBR and Base specific antibodies after cleavage by thermolysin. The three antibodies to the Glycan cap (P6, 040 and 66-3-9C) lose binding after thermolysin treatment, whereas the epitopes bound by RBR (114) and base (66-4-C12) specific antibodies were not affected (Figure 7A and 5). The result was confirmed for seven additional GC specific antibodies (Figure 5).
Thermolysin digestion achieved complete removal of these epitopes as shown (i) by reduction of the binding of these antibodies to the level of a negative control specific for influenza (Figure 5) and (ii) the appearance of the epitope recognised by MR78 that binds to EBOV GP only after removal of the glycan cap (Flyak et al (2015) and Bornholdt et al (2016). The effect of allowing the GC specific antibodies to bind was noted, followed by treatment with thermolysin. The mAbs P6 and 040 remained bound despite the
thermolysin effect (Figure 3). This effect was confirmed (Figure 6) for five additional neutralising antibodies to the GC (66-3-7C, 66-3-2C, 141, 66-3-4A and 125). Evidence for cleavage was provided by the loss of the epitope bound by 66-3-9C specific for the b17- 18 loop (GWAFWET) (Figure 3B) and appearance of the epitope bound by MR78 that binds to EBOV GP after removal of the glycan cap (Flyak et al, 2015) (Figure 5).
In these experiments the E-SIAT cells were first trypsinised to detach them from plastic before exposure to thermolysin. The experiment was repeated without thermolysin in plastic plates. Thermolysin treatment resulted in the loss of binding sites for nine GC specific antibodies tested and exposed the binding site for MR78, which binds in the RBR region (Hashiguchi et al (2016), Flyak et al (2015), Bornholdt et al (2016) (Figure 5B). When E-SIAT cells were treated with thermolysin in the presence of GC specific antibodies, these antibodies retained their binding, with the exception of the 66-3-9C to the b17-18 loop (Figure 5C).
These results suggested that the P6 and 040 antibodies prevented cleavage of GP by thermolysin. A similar observation has been made recently with the GC specific monoclonal antibody EBOV-442 (Gilchuk et al. 2018, Immunity).
Example 6: Cross-reactivity of the antibodies
SUDV Gulu and MARV GP proteins were expressed by the booster MVA vaccine, which may have stimulated cross-reactive clones. Cross-reactivity of the collection of antibodies to EBOV Zaire Mayinga 1976 (AF086833) and to other Ebola species - BDBV (NC_014373.1) and Sudan Gulu (NC_006432.1) was investigated. Although there is only 55% and 65% sequence homology to Sudan and Bundibugyo GP protein sequences (Figure 8), it is notable that of 82 antibodies selected for binding to ZEBOV glycoprotein, 20 were cross-reactive in binding to some level on the glycoproteins of Bundibugyo and Sudan species expressed in MDCK-SIAT1 cell (Figure 9). Of the subset of neutralising or protective antibodies examples that were cross-reactive in binding to Bundibugyo and Sudan GP specific for the base (66-4-C12, 6651, 6541), Glycan Cap (040), the b17-18 loop amino acids 286-293 (66-3-9C), and the Receptor Binding region (6662) (Figure 9C) were identified. It is evident that pan-Ebola antibodies to the fusion loop can also be isolated (Zhao et al. 2017, Cell, 169: 891-904 el5; Furuyama et al. 2016, Sci Rep, 6: 20514; Wee et al. 2017, Cell, 169: 878-90 e15).
Example 7: Diversity in gene usage and affinity maturation
Twenty-three VH genes encoded the 82 antibodies from 11 donors, making the VH gene use very diverse within the collection and within the individual donors (Figure 3 A, Figure 10). Almost equal numbers of antibodies have used the Kappa (n=39) or Lambda (n=43) light chain locus. VH 3-15 was the most used germline gene and it encoded 20 antibodies in 10 donors (Figure 1).
Most of the antibody sequences have high identity to their germline genes with an average of five (range of two to eight) somatic mutations giving rise to amino acid changes (Figure 10). This is expected for antibodies that have been recently stimulated with minimal engagement in the germinal centre reaction, as was seen in the response to vaccination with H7 hemagglutinin (Thornburg et al. 2016, J Clin Invest, 126: 1482-94). The number of somatic mutations in these Ebola antibodies is lower than that of influenza antibodies derived from individuals that are likely to have been repeatedly exposed (Huang et al. 2015, J Clin Invest, 125: 2631-45 ; Pappas et al. 2014, Nature, 516: 418-22. ;
Wrammert et al. 2011, J Exp Med, 208: 181-93).
Example 8: VII 3-15 and Vl. 1-40 antibodies to the Receptor Binding Region
Overall there was no particular favoured VH gene identified among neutralising antibodies, which can be seen in the response to some viruses such as pdmHlNl influenza (Jackson et al. 2014, Cell Host Microbe, 16: 105-14) and dengue (Parameswaran et al. 2013, Cell Host Microbe, 13: 691-700 ) for which signature VH genes were identified. Interestingly however, out of a total of 39 mAbs for which the epitopes were identified (6 to MLD, 14 to glycan cap , 10 to RBR and 9 to base), all ten mAbs that competed with the RBR specific antibody 114 (VH 3-13 / VK 1-27) are encoded by VH 3-15 and Vl 1-40 genes and none by other VH genes. These ten VH 3-15 RBR mAbs come from six donors (Table 4, Figure 10). One out of ten, 6662, cross-reacted in binding on EBOV, SUDV and BDBV GPs (Figure 9).
Table 4: Receptor binding region antibodies that are blocked by mAb 114.
Figure imgf000046_0001
One out of ten, 6662, cross-reacted in binding on Zaire, Bundibugyo and Sudan GPs (Figure 9). Example 9: Binding Kinetics compared to established therapeutic antibodies
In view of the relative temporal immaturity of the collection of the binding kinetics were measured (Table 5) and the binding affinity constants calculated (Figure 11) for a selection of neutralising antibodies and compared these to established therapeutic antibodies: 114 to RBR and 100 to Base (Corti et al. 2016, Science, 351: 1339-42; Misasi et al. 2016), 6D6 to fusion peptide (Furuyama et al. 2016), cl3C6 to RBR (Murin et al. 2014, Science, 351: 1343-6).The kinetics of the antibodies were similar in range to the control antibodies (Table 6). The measurements for 114, 100 and cl3C6 gave very similar association rates, but somewhat faster dissociation rates to those of Misasi (Misasi et al. 2016, Science, 351: 1343-6). This may be related to the use of MLD deleted molecules by Misasi, and complete GP in these measurements. Table 5. Binding Kinetics of a set of antibodies compared to established therapeutic antibodies 114 to RBR and 100 to Base, 6D6 to fusion peptide, cl3C6 to RBR.
Figure imgf000047_0001
GC: Glycan Cap; RBR: Receptor binding region; IFL: Internal fusion loop.
Table 6: Comparison of our measurement of kinetics with published values (Misasi et al. 2016).
Figure imgf000047_0002
Figure imgf000048_0001
Example 10: Selection of antibody cocktails for protection in guinea pigs
Seven antibodies (Figure 12) were selected to test for therapeutic protection in guinea pigs against the EBOV Mayinga variant of Ebola virus as described (Dowall et al. 2016, J Infect Dis, 213: 1124-33). Three antibody cocktails were formed from these antibodies - two EBOV-specific cocktails and one containing antibodies that cross-reacted in binding to BDBV and SUDV GPs. The selection of the antibodies was based on four characteristics - 1) neutralisation in our E-S-FLU assay because this correlated with protection in mice in the VIC study, 2) binding independently to non-overlapping epitopes in GC, RBR and base (to reduce the likelihood of selecting antibody resistant variants during treatment) 3) protection in mice, 4) cross-reactivity for Bundibugyo and Sudan GPs (Table 7). Table 7: Antibodies selected for inclusion in therapeutic cocktails
Figure imgf000048_0002
Figure imgf000049_0001
The three cocktails were tested at a dose of 10 mg/Kg of each antibody (Table 7, groups 1-3). This dose was selected partly on our experience with protective antibodies in influenza infection, also because the affinities of the antibodies and binding assays in vitro suggested that 10 mg/kg would be saturating. The first cocktail, which was expected to be most potent, was also tested at 5 mg/Kg total (equivalent to 1.67 mg/Kg of each antibody) (group 4) for comparison to ZMapp given at the same dose. This dose was not expected to 100% curative and provided an opportunity to test for equivalence to ZMapp. The first and second cocktails differed in the RBR mAh, where 6662 (which was highly protective in the murine challenge) replaced 6660. The third cocktail (group 3) was composed of four mAbs that cross-react in bindingto EBOV, SUDV and BDBV glycoproteins. In addition to mAbs specific for epitopes in GC, RBR and Base, it included 66-3-9C specific for the b17-18 Loop because of its similarity to FMV09 (Howell et al. 2017, Cell Rep, 19: 413-24) that provided a synergistic therapeutic effect. Controls were ZMapp at a dose of 5 mg/Kg and PBS.
When tested as cocktails in the neutralisation assay Groups 1 and 2 were EBOV specific (cross-reacting between Mayinga and Makona strains), and Group 3 showed additional partial neutralisation of S-FLU coated in BDBV and SUDV GPs (Figure 12B- E). The control antibody 6D6 strongly neutralised S-FLU coated in GPs from all of the Ebolavirus species.
Example 11: Therapeutic protection of guinea pigs by antibody cocktails
Guinea pigs were challenged with 103 TCID50 of guinea pig adapted Ebola virus in 200 mL volume subcutaneously and treated on day three with the antibody mixtures by intraperitoneal injection in 2 mL volume, at the Porton Down high containment facility. Animals were monitored for temperature and clinical signs and were culled if they reached 10% weight loss and clinical score >/=3, or weight loss of >/= 20% similar to a previously described study (Dowall et al. 2016, Genome Biol, 15: 540).
The group 1 cocktail (125 + 6660 + 66-6-3) resulted in 4/6 guinea pigs surviving (Figure 13). The same cocktail given at lower dose (1.67 mg/Kg each) in group 4 resulted in 3/6 animals surviving, which was the same as ZMapp at the same dose. However this cannot be interpreted as possible equivalence as a higher dose of ZMapp (5 mg/animal) resulted in complete survival in this model when given by the intravenous route (Dowall et al. 2016, Genome Biol, 15: 540). Survival in group 2 (125 + 6662 + 66-6-3), which differed from group 1 only in replacement of the RBR specific mAb 6660 with 6662 was only 1/6 animals. In group 3, treatment with the cross -reactive cocktail comprised of four independently binding antibodies (040 + 66-3-9C + 6662 + 6541) resulted in 100% survival without weight loss or clinical signs (Figure 13).
Animals which met humane endpoints all had high levels of detectable Ebola virus RNA in blood, liver and spleen. Animals which survived until the end of the study (21 days post-challenge) had no detectable viral RNA (Figure 14). These results were confirmed by in situ hybridisation analysis of samples and the absence of Ebola virus- induced lesions and histological changes in surviving animals compared to those in non survivors (see Example 12).
Example 12 - Histology
The types and severity of lesions that were attributable to infection with Ebola virus are detailed in Figure 15. In addition, the results of the immunohistochemical staining are shown in Figure 15. The microscopic changes associated with Ebola virus infection observed were similar to those reported in previous studies. Specific changes that were scored subjectively are described below.
Group 1- Antibody cocktail (125 + 6660 + 66-6-3): Microscopic lesions, together with detection of viral antigen, were observed in the liver and spleen of two out of the six animals (19063 and 00252); these two animals were euthanised early for welfare reasons. Furthermore, viral antigen was detected in the liver (Figure 16) and spleen (graded 2-3 severity). The remaining animals survived to the study end point. In the liver and spleen of animal 99705 (922/17), minimal changes were noted and viral antigen staining was absent. In the liver and spleen of the three remaining animals, there were no microscopic changes or detection of viral antigen.
Group 2- Antibody cocktail (125 + 6662 + 66-6-3): Five out of six animals were euthanised between days 6 and 9 for welfare reasons. Microscopic changes referable to viral infection, as well as detection of viral antigen, was noted in both the liver (minimal to moderate) and spleen (minimal to marked) (Figure 17) of all these animals. Animal 18897 survived to the study end point and the liver and spleen appeared normal with no viral antigen detected. Group 3- Antibody cocktail (040 + 6662 + 6541 + 66-3-9C): All animals survived to the study end point. Neither microscopic changes nor presence of viral antigen were observed in the liver or spleen of any animal.
Group 4 Antibody cocktail (Zaire-specific): Three out of six animals survived to the study end point (00484, 19160 and 19335). Only one minimal change was noted in the spleen of animal 00484, and viral antigen was not detected in either the liver or spleen of any of these animals. The remaining three animals were euthanised between days 7 and 8 pc; microscopic changes and viral antigen were noted in the liver (mild) (Figure 18) and spleen (minimal to marked) of all animals.
Group 5 Antibody cocktail (ZMapp): Three animals survived to the study end point (02155, 19088 and 19210); neither microscopic changes nor viral antigen were detected in either the liver or spleen. The remaining animals were euthanised early between 6 and 8 days pc and microscopic changes were noted in the liver (minimal to moderate) and spleen (minimal to marked) in all animals, as well as viral antigen (1-3 severity). In animal 00003, changes observed in the liver were focal (Figure 19). All animals were euthanised early, between 6 and 8 days pc; microscopic changes, and viral antigen, were detected in both the liver (minimal to moderate) (Figure 20) and spleen (minimal to marked) of all animals. In animal 00532, changes observed in the liver were focal and comprised macrophage infiltration and necrosis.
Acute, histological changes, attributable to infection with Ebola virus, as well as detection of viral antigen, were observed in the spleen and liver of a proportion of animals in Groups 1, 2, 4, 5 and 6. Groups 2 [Antibody cocktail (125 + 6662 + 66-6-3)] and 6 (PBS control) were the most severely affected, with all animals succumbing to infection early in Group 6 and five out of six animals in Group 2.
Protection against disease was afforded to all animals in Group 3 [Antibody cocktail (040 + 6662 + 6541 + 66-3-9C)]. In this group, there was an absence of microscopic changes referable to infection with Ebola virus in the liver and spleen of all animals, as well as the absence of viral antigen. In Group 1 [Antibody cocktail (125+ 6660 + 66-6-3)], two animals were euthanised early, and microscopic lesions and viral antigen were detected in both liver and spleen. The remaining animals in this group survived to the study end point and did not have microscopic lesions; this suggests that partial protection against disease was seen in four out of six animals. Groups 4 [Antibody cocktail (Zaire specific)] and 5 [Antibody cocktail (ZMapp)] were similar in that three out of six animals in each group did not have lesions or viral antigen detected in the liver and spleen, whereas the remaining three animals were euthanised early, with lesions and viral antigen present in both tissues. Therefore, protection against disease was observed in 50% of animals in both groups.
In conclusion, administration of antibody cocktail 040 + 6662 + 6541 + 66-3-9C gave the best protection against Ebola viral infection in guinea pigs when administered three days after challenge.
Discussion of Examples
The antibody response to Ebola GP in vaccinated donors was very diverse both in terms of the range of the >23 VH genes used to generate the 82 antibodies (Figure 1,
Figure 10), and the diversity of epitopes detected (Figure 7). Antibodies to the glycan cap were most abundant (Flyak et al. 2016, Cell, 164: 392-405), but antibodies to the RBR, Base and Mucin Like Domain were also common. The range of specificities, cross- reactivity with other Ebola species, and binding kinetics of the antibodies from vaccinees, despite being relatively immature with few somatic mutations, were comparable to antibodies isolated from convalescent humans, or multiply immunised mice or macaques (Saphire and Aman 2016, Trends Microbiol, 24: 684-86; Zhao et al. 2017, Cell, 169: 891 - 904 el5 ; Pascal et al. 2018, J Infect Dis ; Misasi et al. 2016, Science, 351: 1343-6). The diversity in the epitopes recognised in the response of vaccinees to Ebola GP contrasts with the response to influenza haemagglutinin, where in some individuals after repeated exposures over years the response can be focused onto a localised patch of the HA molecular surface (Huang et al. 2015, J Clin Invest, 125: 2631-45; Linderman et al. 2014, Proc Natl Acad Sci U S A, 111: 15798-803).
A novel assay was developed to screen the antibodies for neutralisation. This uses a single cycle influenza core with the hemagglutinin coding sequence replaced with eGFP for detection of infected cells, and coated in the Ebola GP by pseudotyping (Xiao et al. 2018, Cell, 169: 891-904 el5). As this virus can replicate only for a single cycle, and contains no genetic information from Ebola, it can be handled in more convenient containment conditions than Ebola virus. In collaboration with the Viral Haemorrhagic Fever Immuno therapeutic Consortium it was established that the neutralisation assay correlated reasonably with therapeutic activity in mice. The assay was used to narrow the choice of antibodies to combine in therapeutic cocktails.
The mechanisms by which antibodies protect against Ebola virus in vivo are not fully understood. Antibodies to epitopes in the glycan cap, Receptor binding region (RBR) and Base/Fusion loop can all neutralise in vitro and protect in vivo (Saphire 2018, Cell, 9, 938-952). Suggested mechanisms include blockade of NPC1 binding, prevention of cathepsin cleavage, interference with fusion, and Fc dependent interactions with host cells (Saphire 2018, Cell, 9, 938-952, Saphire and Aman 2016, Trends Microbiol, 24: 684-86). Certain Base binding antibodies have been shown to prevent cathepsin cleavage (Shedlock et al. 2010, Virology, 401: 228-35; Misasi et al. 2016, Science, 351: 1343-6; Wee et al. 2017, Cell, 169: 878-90 el5), and the epitopes bound by base antibodies are usually retained after cleavage by thermolysin (as a cathepsin surrogate) (Figure 3C). Antibodies that bind the glycan cap can neutralise in vitro and provide protection in vivo, but it is not clear how this can occur if the epitope is removed by cathepsin cleavage before GPcl binds to NPC1 (Saphire 2018, Cell, 9, 938-952, Saphire and Aman 2016, Trends Microbiol, 24: 684-86). It was found that two neutralising and protective antibodies to the glycan cap, P6 and 040, had the property that once bound to GP expressed on the membrane of transduced cells, the GP became resistant to cleavage by thermolysin. This effect was specific, because the antibody 66-3-9C bound to the b17-18 loop on the glycan cap did not prevent cleavage by thermolysin (Figure 7). If the GP was treated with thermolysin first, all three antibodies showed greatly reduced binding (Figure 3C). A similar observation was made recently for the GC specific MAb EBOV-442 (Gilchuk et al. 2018, Immunity, 49, 363- 374). Although not definitive, these results suggest that some antibodies bound to the glycan cap may neutralise also through prevention of cathepsin cleavage.
Due to the high mutagenic frequency of Ebola virus (Carroll et al. 2015, Nature, 524: 97-101; Alfson et al. 2015, J Virol, 90: 2345-55), monoclonal antibodies, even when given in combination, can select resistant mutants during treatment of non-human primates (Kugelman et al. 2015, Cell Rep, 12: 2111-20). Therefore the aim was to find sets of neutralising antibodies that bound independently to sites in the glycan cap, receptor binding region and base of GP, to limit the selection of resistant mutants and maximise the likelihood of combining several mechanisms of neutralisation (Saphire 2018, Cell, 9, 938- 952, Saphire and Aman 2016, Trends Microbiol, 24: 684-86). From 82 antibodies isolated from 11 vaccinated donors, three antibody cocktails were formed. Group 1 and 2 were selected for their apparent strength of neutralisation in vitro and protection in the mouse infection assay performed within the VIC study. Neither of these cocktails provided complete protection and therefore it was possible that resistant viruses were selected.
Group 3 was selected firstly on the level of cross-reactivity in binding between the GPs from the three Ebola virus species Zaire, Bundibugyo and Sudan, and secondly on their neutralisation and mouse protection. It is notable that cross -reactive antibodies can be found that bind glycan cap, RBR and base/fusion loop. Treatment at day three of infection with the cross-reactive cocktail of four antibodies that included 040 to Glycan Cap, 6662 to the RBR, 6541 to the Base, and 66-3-9C to the b17-18 loop resulted in 100% protection from a Zaire Mayinga Ebola virus without weight loss or clinical signs. Viral RNA was not detected in the tissues of these animals at post mortem on day 21 post infection, which implies that the selection of resistant variants did not occur. .
The effectiveness of this combination could not have been predicted from the in vitro neutralisation or murine protection results with individual antibodies. The b17- 18 Loop specific antibody 66-3-9C is closely related to the FVM09 antibody (Keck et al.
2016, J Virol, 90: 279-91; Howell et al. 2017, Cell Rep, 19: 413-24) isolated from cynomolgus macaques. FVM09 is specific for the conserved exposed loop between beta strands 17 and 18 with the sequence GEWAFWET, and by itself is not neutralising and was only weakly protective in vivo. However, FVM09 in mixtures enhanced the binding and neutralisation by base antibody 2G4, and the GC specific antibody m8C4 (Holtsberg et al. 2016, J Virol, 90: 266-78; Howell et al. 2017, Cell Rep, 19: 413-24). In vivo FVM09 enhanced protection by the GC antibody m8C4 (Howell et al. 2017, Cell Rep, 19: 413-24) and a fusion loop-binding antibody FVM02p (Keck et al. 2016, J Virol, 90: 279-91). 66-3- 9C is specific for the same sequence (Figure 3B), also does not neutralise in vitro, and provided only modest protection in vivo in the mouse (4/10 survivors). We also saw reciprocal enhanced binding between 66-3-9C and the base antibodies c2G4 and c4G7, but this did not extend to enhanced neutralisation in our assay. However, the profound therapeutic effect of the mixture of antibodies containing 66-3-9C suggests it may have had a synergistic effect in vivo. Antibodies to the conserved loop between the b17- 18 may be a generally useful addition to therapeutic mixtures of antibodies.
Materials
Table 8: Resources table:
Figure imgf000054_0001
Figure imgf000055_0003
Figure imgf000055_0002
Figure imgf000055_0004
Figure imgf000055_0001
Figure imgf000055_0005
Figure imgf000056_0001
Figure imgf000057_0003
Figure imgf000057_0004
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000057_0005
Human PBMC samples used in this study are covered by the REC 14/SC/1256 (Ewer et al. 2016, N Engl J Med, 374: 1635-46).
Animal studies were performed under Containment Level 4 conditions with all procedures being undertaken according the United Kingdom Animals (Scientific
Procedures) Act 1986. Studies were conducted under Establishment License reference PEL PCD 70/1707 with Project License PPL 30/3247. Studies were approved by the Public Health England ethics committee and the Project License approved by a UK Home Office inspector. Guinea Pig Challenge Studies with EBOV : This experiment was performed in the category 4 lab of the Public Health England, Porton Down. Thirty-six female guinea pigs were purchased from a home office approved breeder and supplier (Marshall
Bio Resources). The guinea pigs were implanted with a temperature and identity chip during a five day acclimatization period.
Cell Lines: HEK293T (human embryonic kidney) was obtained from the Sir William Dunn School of Pathology, Oxford, and MDCK-SIAT (Madin Darby Canine kidney - sialltransferase) cells were obtained from ATCC. Both the cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 I.U./mL penicillin and 100 mg/mL streptomycin. They were incubated at 37 °C with 5% C02.
MDCK-SIAT cells expressing Ebola GP and MLD-deleted GP: MDCK-SIAT 1 cells were purchased from ECACC. The codon optimised sequence was ordered from Geneart. The GP genes were cloned in to a Lenti virus vector pHR-SIN (Demaison et al. 2002, Hum Gene Ther, 13: 803-13). MDCK-SIAT1 cell lines (Matrosovich et al. 2003, J Virol, 77: 8418-25) were transduced with disabled Lenti virus produced in HEK293T cells to express the glycoprotein. Transduced cells were stained with specific monoclonal antibodies and FACS sorted for maximal surface expression. MLD-deleted GP had amino acids 313-463 removed.
Viruses: Ebola pseudotyped influenza viruses (S-FLU) were generated as previously described in detail (Xiao et al. 2018). In brief, pseudotyped viruses were propagated and grown in MDCK-SIAT 1 cell lines transduced with disabled lentivirus to express the surface Ebola glycoprotein.
Ebola virus (strain Yambuku-Ecran, previously known as ME718 (Kuhn et al.
2014, Genome Announc, 2) adapted to cause lethal disease in guinea pigs through sequential passage. The virus was passaged five times to achieve lethality in guinea pigs (Dowall et al. 2014, Genome Biol, 15: 540).
Methods
Isolation of mAbs from plasmablasts
Antibodies were isolated by FACS sorting, PCR and antibody variable gene cloning of a single B cell plasmablast of a vaccinated human individual using the protocol described by Tiller et al 2008 and Smith et al 2009 with modifications. Briefly, PBMC were incubated with a cocktail of antibodies to CD3 (PB; UCHT1; BD Pharmingen), CD20 (APC-H7; 2H7; BD Pharmingen), CD19 (FITC; H1B19; BD Pharmingen), CD27 (PE- Cy7, M-T271; BD Pharmingen), CD38 (PE-Cy5, HIT2; BD Pharmingen) and IgG
(BV605, G18-145; BD Pharmingen). For a few sorts, Ebola GP protein (10 mg/mL) and a known biotin- labelled anti-MLD antibody (10 mg/mL) were used to sort antigen specific B cell plasmab lasts. Single cells with the phenotype of CD3- CD20-/low, CD19+, CD27++, CD38++ ,IgG+ were sorted on a FACS Aria III cell sorter (BD Biosciences). Single cells were sorted into 96-well PCR plates containing lysis buffer followed by single cell RT- PCR. Nested PCR was slightly modified to existing methods (Tiller et al, Smith et al). Overlapping bases (approx. 20 nucleotides) were added on to existing 5' and 3' primers without interfering the restriction sites, which could be used as a back-up, to enable digestion free Gibson cloning. PCR products were purified in a Qiagen 96-well system and the inserts were assembled with cut plasmid in the Gibson mix (NEB). Two mL of assembled product was used to transform 10 mL DH5a E. Coli (NEB, C2987) in 96-well plates. Three colonies for each heavy and light chain were grown in a 96-well plate format and purified using Qiagen Turbo 96 miniprep kit. Plasmids were eluted using 100 mL TE buffer. Transfection of 293T cells with heavy and light plasmids (~200 ng of each with 120 mg/mL linear PEI, in 250 mL total volume) and immunofluorescence assays were also performed in a 96-well tissue culture plate.
Isolation of mAbs from memory B cells
PBMCs harvested 28 days after vaccination boost from nine volunteers was collected. 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). Human B cell cultures were prepared using 132 x 96-well plates at a cell density of approximately 5000 cells per well. After 7-days culture, screening was performed. Briefly, the presence of Ebola glycoprotein-binding antibodies in B cell culture supernatants was determined using a homogeneous fluorescence-based binding assay performed on a Applied Biosystems 8200 cellular detection system device using MDCK cells stably transfected to express surface Ebola glycoprotein. Binding was revealed with a goat anti-human IgG Fey-specific Dylight 649 conjugate (Jackson). Following primary screening, positive supernatants containing reactive antibody were consolidated on 96-well bar-coded master plates and B cells in cell culture plates frozen at -80°C. Master plates were then screened in a further homogeneous fluorescence binding assay to confirm that the antibodies bound the Ebola glycoprotein- expressing MDCK-SIAT1 cells and not the parental MDCK-SIAT1 cells.
The Fluorescent Foci method (US Patent 7993864/ Europe EP1570267B1; (Clargo et al. 2014, MAbs, 6: 143-59) utilizing Ebola glycoprotein-expressing MDCK-SIAT1 cells was used to identify and isolate antigen-specific B cells from positive wells, and specific antibody variable region genes were recovered from single cells by reverse transcription (RT)-PCR using heavy and light chain variable region-specific primers. PCR primers contained restriction sites at the 3’ and 5’ ends allowing cloning of the variable region into a human IgGl (VH), human kappa (VK) or human lambda (Vk) mammalian expression vector. Heavy and light chain constructs were co -transfected into Expi293F cells using Expifectamine 293 (Invitrogen) and recombinant antibody expressed. After 6 days expression, supernatants were harvested and antibody rescreened for selectivity using the specificity assays described above. Antibody was purified from conditioned media using affinity chromatography and characterized further.
Immunofluorescence Binding Assays
Immunofluo rescene assay was done to screen the binding of antibodies in culture supernatant to Ebola glycoprotein. A 96-well plate was coated overnight with stable transduced MDCK-SIAT1 cells expressing Ebola glycoprotein (E-SIAT cells). Antibody supernatant (50 mL) was incubated with a monolayer of E-SIAT cells. After 1 h incubation at RT, plates were washed with PBS. A secondary antibody goat anti-human IgG conjugated with Alexa Fluor647 (A21445; Thermo Fisher; 1:400) or FITC (H10301; Life technologies; 1:160) was added to well and let incubate for 1 h in dark. Plated were then washed and fixed with 1% formalin. Fluorescence was observed under the fluorescence microscope and quantified using the Clariostar platereader (BMG Labtech). GP binding antibodies and influenza antibodies were used as positive and negative controls respectively.
Expression and Purification of Antibody
At Weatherall Institute: For the GP binding antibody clones, heavy and light chain plasmids were maxi-prepped. HEK293T cells were co -transfected with heavy and light plasmids. Antibody supernatants were harvested after 4-5 days and purified using Protein A sepharose (P3391; Sigma). The column was washed with Tris buffered saline (TBS) and eluted with citrate buffer pH 3.0. Eluted fractions were neutralised by adding 1 M Tris pH 8 and absorbance read at 280 nm (Nanodrop, Life Technologies).
Batch purifications were performed on small scale (<50ml) antibody supernatants using MabSelect SuRe resin slurry (GE Healthcare, 17-5438-01) and an in-house vacuum manifold system. The resin was washed in phosphate buffered saline (PBS) pH7.4 (Sigma Aldrich Chemicals) and slurry was added to each sample prior to a lh incubation at room temperature on a roller mixer. Supernatants were transferred to the vacuum system and a vacuum applied, the resin was then washed with PBS pH 7.4 and eluted in 0.1 M sodium citrate pH 3.4. Elution pools were neutralised by adding 2 M Tris/HCl pH 8.0 and absorbance read at 280 nm (Cary UV Spectrophotometer). Samples were then buffer exchanged into PBS pH 7.4 using Amicon Ultra Spin columns with a 30K cut off membrane (Millipore, UFC905008) and centrifugation at 4000 g. Absorbance was read at 280 nm and samples supplied at >1.0 mg/mL. Further analysis was done by size exclusion on a UPLC (Acquity) with a BEH200; 1.7 mM, 4.6 mm X 300 mm column (176003905, Waters) and developed with an isocratic gradient of 0.2 M phosphate, pH 7.0 at 0.3 mL/min. For SDS-PAGE analysis, 3-4 mg was loaded onto a Novex Tris glycine 4-20% gel (Thermo fisher) with a Markl2™ unstained standard (LC5677, Thermo fisher) then stained by Coomassie blue.
Monoclonal antibodies used for Guinea Pig protection were expressed in 293T cells and affinity purified by Absolute Antibody Ltd. and provided at 15 mg/mL which were aliquoted and kept at -80 °C until used.
Virus Neutralisation Assays
Neutralisation assays were done as previously described (Xiao et al. 2018). Ebola pseudo typed influenza virus (E-S-FLU) diluted in virus growth medium (VGM) was incubated with mAbs in serial dilution in PBS for 2h at 37 °C. MDCK-SIAT1 cells (3x104) diluted in VGM were then added to each well. Cells were incubated for 20-24 h at 37 °C, 5% C02. Next day, the eGFP fluorescence was read using Clariostar platereader (BMG Labtech). Virus only and medium only controls for maximum and minimum signals were included. Percent infection was calculated based on the wells containing virus only and medium only. Inhibitory concentration at 50% and 90% was derived by linear interpolation. Epitope Mapping using Competitive Binding Assays
Competition binding assay was performed to find out if a known/reference antibody with a defined binding site is blocked by the testing antibody and vice versa. Antibody was biotinylated using EZ-link Sulfo-NHS-LC-biotin (21327; Life
Technologies). Biotin-labeled antibody and competing mAb (in 10-fold excess over biotin- mAb) were mixed and transferred to a monolayer of E-SIAT cells. After 1 h incubation, cells were washed. A second layer of Extravidin-FITC (E2761; Sigma; 1:400) or
Extravidin Peroxidase (E2886; Sigma; 1:1600) or Streptavidin-Alexa Fluor 647 (S21374; Thermo Fisher; 1:400) was then added for 1 hour. Cells were washed three times and then binding was detected. When fluorescent second layer, cells were fixed in 1% formalin and fluorescence was quantified on the Clariostar Plate reader. When Extravidin-Peroxidase was used, signal was developed by adding OPD substrate (P9187; Sigma) and the reaction stopped with 50 mΐ 1 M H2S04. We found Streptavidin Alexa-Fluor 647 preferable because it gave a better signal to background ratio in the plate reader. Mean and 90% confidence interval of eight replicate measurements were calculated. Self-blocking (minimum signal), PBS (background fluorescence) and a non-binding antibody (influenza mAb or a mAb to the Mucin domain; maximum signal) were used as controls. The competition was measured as: (X- Min binding)/(Maximum binding - Minimum binding), where X= Binding of the biotinylated mAb in presence of competing mAb, Minimum binding = Blocking of the biotinylated mAb by self or background binding, Maximum binding = Binding of biotinylated mAb in presence of Flu or MLD mAb.
Epitope Mapping using Yeast Peptide-Display Assays
Epitope mapping of the mAbs was carried out based on the yeast surface display (YSD) library as previously described (Zuo et al. 2011 , J Biol Chem, 286: 33511-9; Guo et al. 2015, J Acquir Immune Defic Syndr, 68: 502-10). Briefly, the combinatorial fragment library of Zaire Ebola GP was constructed and displayed on the surface of yeast for antibody staining and Fluorescence-activated cell sorting (FACS). Specifically, the full- length GP gene was digested and PCR-reassembled into a range of 100-900 bp fragments, the reassembled fragments were gel purified and cloned into yeast surface display vector. The cloned products were then transformed into competent yeast cell line EBY100 using electroporation. The yeast library was induced and incubated with each of the Ebola mAbs and positive sorted by FACS using Aria III (BD, USA). The sorted positive yeast clones displaying the respective antigenic fragments were harvested and the plasmids encoding the corresponding fragments were extracted and subjected to sequencing and sequence analysis.
Binding Kinetics using Surface Plasmon Resonance (SPR)
Binding kinetics of antibodies to both monomeric and trimeric Ebola glycoprotein ectodomain was assessed by SPR using a Biacore 3000 instrument (GE Healthcare), whereby antibody was captured on a CM5 chip (GE Healthcare) via immobilized anti human IgG Fc specific polyclonal antibody, followed by successive titration of
glycoprotein. Resulting sensorgrams were analysed to determine association and dissociation rate constants over a range of GP concentrations.
Affinity purified polyclonal goat F(ab)2 anti-human IgG Fc (Jackson, 109-006-098) was immobilized following activation of test and reference flow cells by injection of 50 mL of a fresh mixture of 50 mM N-hydroxysuccimide and 200 mM l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide at a flow rate of 10 mL/min. Polyclonal at 50 mg/mL in 10 mM acetate pH 5.0 buffer was injected (50 mL) over the test flow cell and both test and reference flow cell surfaces were then deactivated with a 50 mL pulse of 1 M
ethanolamine.HCl pH 8.5.
Binding assays were carried out at 25°C in HBS-EP running buffer (10 mM
HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05 % Surfactant P20, GE Healthcare). Antibodies were diluted to 10 nM in HBS-EP and concentrations of monomeric and trimeric GP were prepared in HBS-EP between 5 and 500 nM. The latter were tested separately for each antibody in a series of sensorgram cycles, where 10 mL of antibody was injected at 10 mL/min followed by 90 mL of GP at a flow rate of 30 mL/min to generate an association phase of 180 sec. After monitoring a dissociation phase of 300 sec the chip was regenerated at the end of each cycle by successive injections of 10 mL 40 mM HC1, 5 mL 5 mM NaOH and 10 mL 40 mM HC1. Sensorgrams provided a record of response unit difference between the test and reference flow cells. For each antibody buffer control cycles were interspersed between GP cycles to allow for drift correction; also, antibody blank cycles were run at each GP concentration to allow correction for any non-specific binding of glycoprotein
Sensograms were analysed using the BIAevaluation Software (version 4.1.1, GE Healthcare). After subtraction of respective buffer control and antibody blank cycles, kinetic parameters describing association and dissociation rate constants were determined using the Langmuir binding model. Affinity constants were calculated from the mean log KD values determined over 5 concentrations of glycoprotein.
Thermolysin Digestion
It is known that GP proteolysis by cathepsins removes the mucin-like domain and glycan cap, and is essential for binding of GP to NPC1 receptor. Thermolysin mimics cathepsins and the proteolytic activity is active at physiological pH whereas cathepsins require strongly acidic pH, which is toxic to living cells (Dube et al. 2009, J Virol, 83: 2883-91) (Bale et al. 2011, PLoS Negl Trap Dis, 5: el395). Thermolysin (P1512; Sigma) was dissolved in HM buffer (20 mM HEPES, 130 mM NaCl and 20 mM MES and pH adjusted to 7.5). Thermolysin faintly precipitates in HM buffer at higher concentration and was clarified in the micro fuge. EBOV glycoprotein expressing cells were trypsinised from the flask and treated with 0.25 mg/mL thermolysin diluted in HM buffer containing 2 mM CaC12 for 1 hour at 37 °C. Then cells were washed with PBS and passed through a cell strainer as cells tend to form clumps after thermolysin treatment. To analyse antibody binding to thermolysin treated GP, 25 mg/mL antibody was incubated with cells for 1 h. Alexa fluor 647 conjugated anti-human IgG (A21445; Life Technologies; 1:400) was used for binding detection in FACS (Attune; Life Technologies). Antibody binding to untreated GP expressing cells handled in similar manner was done in parallel for binding
comparison. Log difference of binding geometric mean fluorescence intensity was calculated.
Treatment of Ebola virus infected guinea-pigs
All guinea pigs were challenged with 103 TCID50 of guinea pig adapted Ebola virus in a volume of 200 mL via the subcutaneous route. During the course of the study, weights and temperatures were collected at least once daily and clinical scores assessed at least twice a day. Animals which met predefined endpoints (20% weight loss; 10% weight and moderate clinical signs; or immobility) were culled by a Schedule 1 approved method. Antibody cocktails were prepared a day before administration. Antibodies were delivered 3 days post-challenge to six animals per group via the intraperitoneal route in a volume of 2 ml. As a positive control, ZMapp was given a dose of 5 mg/Kg per animal. Untreated animals were given 2 mL PBS.
PCR and Histology of guinea pig tissue Group identifiers, treatments received, animal identifiers, histology numbers, and duration from challenge to euthanasia are detailed in Table 9 .
Table 9: Histology group identifiers
Figure imgf000065_0001
Each animal was assigned a histology number. Tissue samples were processed to paraffin wax; sections were cut at approximately 3-5 mm thick, stained with haematoxylin and eosin (HE) and examined microscopically. In addition, sections from each animal were stained for Ebola viral antigen using the Leica BondMax (Leica Biosystems) and the Leica Bond Polymer Refine Red Detection Kit (Leica Biosystems). An antigen retrieval step was included for 10 minutes using the Bond Enzyme Pre -treatment Kit, Enzyme 3 (3 drops). A rabbit polyclonal, anti-Ebola viral antibody (IBT Bioservices) (dilution 1:2000) was incubated with the slides for 60 mins. Alkaline phosphatase and haematoxylin
counterstains were used to visualise the slide. Appropriate positive and negative, tissue and reagent controls were included.
Tissue sections were examined by light microscopy and microscopic lesions attributable to Ebola viral infection scored subjectively using a scale comprising minimal, mild, moderate and marked. For immunohisto chemical staining, frequency of staining was scored subjectively using‘1‘(positively stained areas observed occasionally);‘2’
(positively stained areas observed frequently); and‘3’ (numerous, positively stained areas); ‘4’-predominance of tissue stained positively.
The Pathologist was blinded to the treatments and groups.
Microsoft Word was used to compile the report. IMS Client (vl2H2) was used to capture, store and export digital images.
Gene family usage of IgG genes
The gene family usage of the variable region of the human IgG heavy- and light- chains was analysed using IMGT/V -Quest. Phylogeny tree was drawn based on MUSCLE alignment and Neighbour joining settings using MEGA version 7.
Quantification and Statistical Analysis
For animal studies, Kaplan Meier survival curves were analysed with the log-rank (Mantel-Cox) test using Prism 7 software (GraphPad Software). P values of less than 0.05 were considered significant. For neutralisation, each data point is an average of duplicates from one experiment. Several data points from many experimental repeats are shown in the graph. MN titres were expressed as 50% or 90% maximal effective concentrations derived by linear interpolation from neighbouring points in the titration curve. Geometric Mean Fluorescence Intensity (GMI) was quantified for FACS binding using FlowJo (Treestar). Sequence listing
SEQ ID NO: 1 (66-3-9C VH)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSSITTSSS YIYYAYSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARFLGYSYGTNYYYYG MDVWGQGTTVTVSS
SEQ ID NO: 2 (66-3-9C VL)
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLTFGGGTKVEIK
SEQ ID NO: 3 (040 VH)
QVQLVESGGGFVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISGSGG TTYYAPSVKGRFTISRDNSKNTLYLEMITLRAEDTATYFCANSYYYHSSGLLIRWD DMDVWGQGTTVTVSS
SEQ ID NO: 4 (040 VL)
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHSSYPWTFGQGTKVEIK
SEQ ID NO: 5 (6662 VH)
EVQLVESGGGLVKPGGSLRLSCAASGFGFSSAWMNWVRQAPGKGLEWVGRIKSKTD
GGKTDYAAPVKGRFIMSRDDSKNTLYLQMNSLKTEDAGVYYCTTRIVLNGMDVWGQ
GTLVTVSS
SEQ ID NO: 6 (6662 VL)
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYANNNR PSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSRLSDGVVFGGGTKLTVL
SEQ ID NO: 7 (6541 VH)
EVQLVQWGAGLLKPSETLSLTCAVYGGSFSGNYWTWIRQPPGKGLEWIGEINHSGS
TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATKMGGYGDYWHWYFDL
WGRGTMVTVSS
SEQ ID NO: 8 (6541 VL)
EVVMTQSPATLSVSPGERATLSCRASQSLFTNLAWYQQKPGQAPRLLIYGASTRAT GIPARFSGSGSGTDFSLTISRLQSEDFAFYYCQQYNDWPLTFGGGTKVDIK
SEQ ID NO: 9 (66-3-9C HCDRl )
GFTFSSYNMN
SEQ ID NO: 10 (66-3-9C HCDR2)
SITTSSSYIYYAYSVKG
SEQ ID NO: 11 (66-3-9C HCDR3) FLGYSYGTNYYYYGMDV
SEQ ID NO: 12 (66-3-9C LCDRl)
RSSQSLLHSDGYNYLD
SEQ ID NO: 13 (66-3-9C LCDR2)
LGSNRAS
SEQ ID NO: 14 (66-3-9C LCDR3)
MQALQTLT
SEQ ID NO: 15 (040 HCDRl)
GFTFSSYAMS
SEQ ID NO: 16 (040 HCDR2)
TISGSGGTTYYADSVKG
SEQ ID NO: 17 (040 HCDR3)
SYYYHSSGLLIRWDDMDV
SEQ ID NO: 18 (040 LCDRl)
RASQGIRNDLG
SEQ ID NO: 19 (040 LCDR2)
AASSLQS
SEQ ID NO: 20 (040 LCDR3)
LQHSSYPWT
SEQ ID NO: 21 (6662 HCDRl)
GFGFSSAWMN
SEQ ID NO: 22 (6662 HCDR2)
RIKSKTDGGKTDYAAPVKG
SEQ ID NO: 23 (6662 HCDR3)
RIVLNGMDV
SEQ ID NO: 24 (6662 LCDRl)
TGSSSNIGAGYDVH
SEQ ID NO: 25 (6662 LCDR2)
ANNNRPS
SEQ ID NO: 26 (6662 LCDR3)
QSYDSRLSDGVV
SEQ ID NO: 27 (6541 HCDRl)
GGSFSGNYWT
SEQ ID NO: 28 (6541 HCDR2) EINHSGSTNYNPSLKS
SEQ ID NO: 29 (6541 HCDR3)
KMGGYGDYWHWYFDL
SEQ ID NO: 30 (6541 LCDRl)
RASQSLFTNLA
SEQ ID NO: 31 (6541 LCDR2)
GASTRAT
SEQ ID NO: 32 (6541 LCDR3)
QQYNDWPLT
SEQ ID NO: 33 (6660 HCDR3)
TAGVVCLHGSSCVGV
SEQ ID NO: 34 (6669 HCDR3)
TTEVVGV
SEQ ID NO: 35 (6670 HCDR3)
TTRVSAPFDI
SEQ ID NO: 36 (6666 HCDR3)
TTRWVYNGMDV
SEQ ID NO: 37 (6667 HCDR3)
NTVRSGADY
SEQ ID NO: 38 (P7 HCDR3)
TTGGRDMA
SEQ ID NO: 39 (105 HCDR3)
TTNFWSGRDYMDV
SEQ ID NO: 40 (66-6-14 HCDR3)
IPTAYYDYVWGSYRSDY
SEQ ID NO: 41 (56-4-D4 HCDR3)
TTRLTPNRLRWTQTEYYYYYTMDV
SEQ ID NO: 42 (140 heavy junction)
CARHFHYYDSSGSETDYW
SEQ ID NO: 43 (140 light junction)
CQSADSNRTYSDVVF
SEQ ID NO: 44 (142 heavy junction)
CAGGSSWLYYMDVW
SEQ ID NO: 45 (142 light junction) CQQYGSSKTF
SEQ ID NO: 46 (6672 heavy junction)
CARVNVKFGELLPPYYFDYW
SEQ ID NO: 47 (6672 light junction)
CSSYTSSNSYVF
SEQ ID NO: 48 (141 heavy junction)
CARDSQEVFGVLIMAYYDYYYMDVW
SEQ ID NO: 49 (141 light junction)
CQQYNNRRTF
SEQ ID NO: 50 (139 heavy junction)
CARALLVPAAMPWSVLFYYFGMDVW
SEQ ID NO: 51 (139 light junction)
CCSYAGSSTFNYVF
SEQ ID NO: 52 (143 heavy junction)
CARGGHYCTDGVCYSLPYFMDVW
SEQ ID NO: 53 (143 light junction)
CQQYGSSLLTF
SEQ ID NO: 54 (6659 heavy junction)
CTTVGVHIWNFGYW
SEQ ID NO: 55 (6659 light junction)
CQSYDSRLSDNMIF
SEQ ID NO: 56 (6660 heavy junction)
CTAGVVCLHGSSCVGVW
SEQ ID NO: 57 (6660 light junction)
CQSYDSRLRDFWVF
SEQ ID NO: 58 (6664 heavy junction)
CTTGVVCLDRSSCVGVW
SEQ ID NO: 59 (6664 light junction)
CQSYDSSLSDFWVF
SEQ ID NO: 60 (6669 heavy junction)
CTTEVVGVW
SEQ ID NO: 61 (6669 light junction)
CQSYDSSLRDFWVF
SEQ ID NO: 62 (6655 heavy junction) CAREVTMIVVEWYFDLW
SEQ ID NO: 63 (6655 light junction)
CQQYGSSPPITF
SEQ ID NO: 64 (6661 heavy junction)
CARENLWFGELLSHYDYYYAMDVW
SEQ ID NO: 65 (6661 light junction)
CGTWDSSLSAYVF
SEQ ID NO: 66 (6663 heavy junction)
CARDSLWLGDLLGGDFYYGMDVW
SEQ ID NO: 67 (6663 light junction)
CCSYAGSSPYVF
SEQ ID NO: 68 (6650 heavy junction)
CAKERSRSWYTWASGYFEYW
SEQ ID NO: 69 (6650 light junction)
CQQRSNWPRTF
SEQ ID NO: 70 (6656 heavy junction)
CARRYFDWLLTPIDYYGLGVW
SEQ ID NO: 71 (6656 light junction)
CMQALQTPGFTF
SEQ ID NO: 72 (6654 heavy junction)
CAKDILGGEFWSGSCSGW
SEQ ID NO: 73 (6654 light junction)
CQQSYSIPYTF
SEQ ID NO: 74 (6652 heavy junction)
CARGEETSDIVVLPADAFDIW
SEQ ID NO: 75 (6652 light junction)
CLQHNSYPRTF
SEQ ID NO: 76 (6653 heavy junction)
CASPGAIHSSDWYYYLAMDVW
SEQ ID NO: 77 (6653 light junction)
CQQYNNWPPITF
SEQ ID NO: 78 (6651 heavy junction)
CARVESWQLGAFDIW
SEQ ID NO: 79 (6651 light junction) CQQYNDWPRTF
SEQ ID NO: 80 (6666 heavy junction)
CTTRWVYNGMDVW
SEQ ID NO: 81 (6666 light junction)
CQSYDSSLRDSVVF
SEQ ID NO: 82 (6670 heavy junction)
CTTRVSAPFDIW
SEQ ID NO: 83 (6670 light junction)
CQSYDSRLRDHVVF
SEQ ID NO: 84 (6665 heavy junction)
CTTRWVYNGMDVW
SEQ ID NO: 85 (6665 light junction)
CQSYDSSLRDSVVF
SEQ ID NO: 86 (6667 heavy junction)
CNTVRSGADYW
SEQ ID NO: 87 (6667 light junction)
CQSYDSRLSDNWVF
SEQ ID NO: 88 (6668 heavy junction)
CAKDIGNYEFWSGYFDYW
SEQ ID NO: 89 (6668 light junction)
CQQYNNWPPAF
SEQ ID NO: 90 (176 heavy junction)
CARGTMARGLILHYYGMDVW
SEQ ID NO: 91 (176 light junction)
CSSYAGSNNFYVF
SEQ ID NO: 92 (040 heavy junction)
CANSYYYHSSGLLIRWDDMDVW
SEQ ID NO: 93 (040 light junction)
CLQHSSYPWTF
SEQ ID NO: 94 (18-1-D3 heavy junction)
CTTYREEGLDYW
SEQ ID NO: 95 (18-1-D3 light junction)
CQSPDISGPYVVF
SEQ ID NO: 96 (P7 heavy junction) CTTGGRDMAW
SEQ ID NO: 97 (P7 light junction)
CQSYDARLRDNWVF
SEQ ID NO: 98 (P6 heavy junction)
CARDPLSDGFLSLSNHFYYGMDVW
SEQ ID NO: 99 (P6 light junction)
CQQCGSSPPYTF
SEQ ID NO: 100 (18-1-DlO heavy junction)
CARGASSDPNYYYAMDVW
SEQ ID NO: 101 (18-1-DlO light junction)
CQQANSLPLTF
SEQ ID NO: 102 (18-1-A9 heavy junction)
CARLGGRVVKVVHAIWFDPW
SEQ ID NO: 103 (18-1-A9 light junction)
CGTWDNSPIF
SEQ ID NO: 104 (19-2-12C heavy junction)
CTADCVLLWFGDLLYCGMDVW
SEQ ID NO: 105 (19-2-12C light junction)
CQHYGSSPTF
SEQ ID NO: 106 (45-4-A5 heavy junction)
CIPPPMVRGVTLDYW
SEQ ID NO: 107 (45-4-A5 light junction)
CQSYDSRLSDHWVF
SEQ ID NO: 108 (45-4-B10 heavy junction)
CARDPLSDGFLSLSNHFYYGMDVW
SEQ ID NO: 109 (45-4-B10 light junction)
CQQYNDWPRTF
SEQ ID NO: 110 (116 heavy junction)
CAVGFDSNGWSNW
SEQ ID NO: 111 (116 light junction)
CQSGDSSGTYPGVVI
SEQ ID NO: 112 (094 heavy junction)
CARGRREMQLVRGSYYYYYYMDVW
SEQ ID NO: 113 (094 light junction) CQAWDSSTSLF
SEQ ID NO: 114 (133 heavy junction)
CTRGRREMQLVRGSYYYYYYMDVW
SEQ ID NO: 115 (133 light junction)
CQAWDSSTSLF
SEQ ID NO: 116 (6535 heavy junction)
CARASYCGGDCYFLLSSRFDYW
SEQ ID NO: 117 (6535 light junction)
CQVWHSSSDHVVF
SEQ ID NO: 118 (6673 heavy junction)
CSTRYSSAWFNDYW
SEQ ID NO: 119 (6673 light junction)
CQSYDSSLRDNWVF
SEQ ID NO: 120 (105 heavy junction)
CTTNFWSGRDYMDVW
SEQ ID NO: 121 (105 light junction)
CQSYDSSLRDHVVF
SEQ ID NO: 122 (132 heavy junction)
CTTQRGVTDYW
SEQ ID NO: 123 (132 light junction)
CQSYDSSLRDSCVF
SEQ ID NO: 124 (121 heavy junction)
CAREYCSGGTCYLDYW
SEQ ID NO: 125 (121 light junction)
CQAWDSSTVVF
SEQ ID NO: 126 (125 heavy junction)
CARVRFCSGASCASVYLLDLWYFDLW
SEQ ID NO: 127 (125 light junction)
CQQLDSYPLTF
SEQ ID NO: 128 (56-3-3D heavy junction)
CARSRYGDFWSGYCDYW
SEQ ID NO: 129 (56-3-3D light junction)
CQQYGSSLPYTF
SEQ ID NO: 130 (56-3-7A heavy junction) CARDYVRWMATIGDAFDIW
SEQ ID NO: 131 (56-3-7A light junction)
CSSYAGSNNFDVVF
SEQ ID NO: 132 (56-3-9A heavy junction)
CARPLIATRHGRGTSWYFDLW
SEQ ID NO: 133 (56-3-9A light junction)
CQSYDSSLSYIF
SEQ ID NO: 134 (56-3-lC heavy junction)
CARVRRPIMVRGVDFDYW
SEQ ID NO: 135 (56-3-lC light junction)
CQQGYSILGALTF
SEQ ID NO: 136 (56-3-11D heavy junction)
CAREDTGVAWAPYYYGMDVW
SEQ ID NO: 137 (56-3-llD light junction)
CQQYNNWPARAF
SEQ ID NO: 138 (56-4-D4 heavy junction)
CTTRLTPNRLRWTQTEYYYYYTMDVW
SEQ ID NO: 139 (56-4-D4 light junction)
CQSYDSSRVLF
SEQ ID NO: 140 (56-3-7D heavy junction)
CARSKWELLWVVGTNFYYGMDVW
SEQ ID NO: 141 (56-3-7D light junction)
CQSYDTSLGGSVF
SEQ ID NO: 142 (56-3-9C heavy junction)
CARDGKRITISGVLIPLYWFFDLW
SEQ ID NO: 143 (56-3-9C light junction)
CQQYGSSITF
SEQ ID NO: 144 (6539 heavy junction)
CARDGGRVLLYFGESGFDPW
SEQ ID NO: 145 (6539 light junction)
CQQANSFPLTF
SEQ ID NO: 146 (66-6-10 heavy junction)
CARDDFWSGITW
SEQ ID NO: 147 (66-6-10 light junction) CQQYNNWWTF
SEQ ID NO: 148 (66-3-9A heavy junction)
CARHDSSGYDAFDIW
SEQ ID NO: 149 (66-3-9A light junction)
CQSSDSSATYVVF
SEQ ID NO: 150 (66-3-7B heavy junction)
CARDWLHSRSPRAGNWFDPW
SEQ ID NO: 151 (66-3-7B light junction)
CQQYGGSPTF
SEQ ID NO: 152 (6537 heavy junction)
CARDLLWFGEEPHWYFDLW
SEQ ID NO: 153 (6537 light junction)
CQSYDSSLSGSVF
SEQ ID NO: 154 (6532 heavy junction)
CTTGSYLSYHSGSGYLDIW
SEQ ID NO: 155 (6532 light junction)
CQSYDSRLNDNCVF
SEQ ID NO: 156 (66-3-4A heavy junction)
CTTDQRFEGPVPASNQWLVGYYHYYGMDVW
SEQ ID NO: 157 (66-3-4A light junction)
CQQYNSNPITF
SEQ ID NO: 158 (66-6-14 heavy junction)
CIPTAYYDYVWGSYRSDYW
SEQ ID NO: 159 (66-6-14 light junction)
CQSYDSSLRDAYVF
SEQ ID NO: 160 (66-3-2B heavy junction)
CARALPSMVRGVIYYYYGMDVW
SEQ ID NO: 161 (66-3-2B light junction)
CQQYGSSFTF
SEQ ID NO: 162 (66-3-9C heavy junction)
CARFLGYSYGTNYYYYGMDVW
SEQ ID NO: 163 (66-3-9C light junction)
CMQALQTLTF
SEQ ID NO: 164 (66-6-19 heavy junction) CAREGSSSTILTPVDYYYGMDVW
SEQ ID NO: 165 (66-6-19 light junction)
CMQGLQTPPAL
SEQ ID NO: 166 (6544 heavy junction)
CANISDWLLVWRYFDFW
SEQ ID NO: 167 (6544 light junction)
CQQYNNWPPWTF
SEQ ID NO: 168 (66-4-A8 heavy junction)
CAKDREGRSNVGAICSFW
SEQ ID NO: 169 (66-4-A8 light junction)
CQQYYSTPLTF
SEQ ID NO: 170 (66-6-15 heavy junction)
CVKPQGYYDSSGYSRAFDIW
SEQ ID NO: 171 (66-6-15 light junction)
CQAWDSSTAVF
SEQ ID NO: 172 (66-6-20 heavy junction)
CAKDYDFWSGSRHFDYW
SEQ ID NO: 173 (66-6-20 light junction)
CGTWDSSLSAGVF
SEQ ID NO: 174 (66-3-9B heavy junction)
CAKKGGVWGSYAWYFDLW
SEQ ID NO: 175 (66-3-9B light junction)
CQQYGGSPTF
SEQ ID NO: 176 (66-4-C12 heavy junction)
CAKGPMISYYAWFDPW
SEQ ID NO: 177 (66-4-C12 light junction)
CQQYNFYASWTF
SEQ ID NO: 178 (66-6-12 heavy junction)
CAKDLGSGSYAALFDYW
SEQ ID NO: 179 (66-6-12 light junction)
CQQYYSSPPFF
SEQ ID NO: 180 (66-6-16 heavy junction)
CAKEMSTYFNGVDVW SEQ ID NO: 181 (66-6-16 light junction)
CAAWSYSLNGRVF
SEQ ID NO: 182 (66-6-3 heavy junction)
CARDITVARLYGMDVW
SEQ ID NO: 183 (66-6-3 light junction)
CAAWDDSLTGRVF
SEQ ID NO: 184 (6536 heavy junction)
CAREHLDFRSGYASWLVGDFVYW
SEQ ID NO: 185 (6536 light junction)
CNSRDSSGNHLGVF
SEQ ID NO: 186 (6533 heavy junction)
CARDRAIAALGDLLYYYYGMDVW
SEQ ID NO: 187 (6533 light junction)
CGTWDSSLSSWVF
SEQ ID NO: 188 (6545 heavy junction)
CARDWPLWNDFWSGYLAYW
SEQ ID NO: 189 (6545 light junction)
CQQYNNWPPLTF
SEQ ID NO: 190 (66-3-7C heavy junction)
CARGGDIVVVVTSTDYYGMDVW
SEQ ID NO: 191 (66-3-7C light junction)
CLQHNSYPRTF
SEQ ID NO: 192 (66-3-2C heavy junction)
CARGSETGGRGLEWLLYTSPYFDYW
SEQ ID NO: 193 (66-3-2C light junction)
CQQSYSTPPYTF
SEQ ID NO: 194 (6541 heavy junction)
CATKMGGYGDYWHWYFDLW
SEQ ID NO: 195 (6541 light junction)
CQQYNDWPLTF
SEQ ID NO: 196 (6538 heavy junction)
CASMNTFGGIMDPFDYW
SEQ ID NO: 197 (6538 light junction)
CSSYTSSSTYVF SEQ ID NO: 198 (66-6-13 heavy junction)
CARPYTSGWSGVDYW
SEQ ID NO: 199 (66-6-13 light junction)
CQQRSHWPPELTF
SEQ ID NO: 200 (66-3-8B heavy junction)
CAGSRYYYYGMDVW
SEQ ID NO: 201 (66-3-8B light junction)
CQQYGSSPLTF
SEQ ID NO: 202 (6662 heavy junction)
CTTRIVLNGMDVW
SEQ ID NO: 203 (6662 light junction)
CQSYDSRLSDGVVF
SEQ ID NO: 204 (6658 heavy junction)
CARVVQIDSS11LVPAAINDYYYGMDVW
SEQ ID NO: 205 (6658 light junction)
CCSYAGSSTYVF
SEQ ID NO: 206 (66-3-9C heavy chain)
MGWSCI ILFLVATATGVHSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWV
RQAPGKGLEWVSSITTSSSYIYYAYSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
YYCARFLGYSYGTNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 207 (66-3-9C kappa light chain)
MGWSCI ILFLVATATGVHSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNY LDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
SEQ ID NO: 208 (040 heavy chain) MGWSCI ILFLVATATGVHSQVQLVESGGGFVQPGGSLRLSCAASGFTFSSYAMSWV
RQAPGKGLEWVSTISGSGGTTYYADSVKGRFTISRDNSKNTLYLEMITLRAEDTAT
YFCANSYYYHSSGLLIRWDDMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 209 (040 kappa light chain)
MGWSCI ILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHSS YPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC
SEQ ID NO: 210 (6662 heavy chain)
MGWSCI ILFLVATATGVHSEVQLVESGGGLVKPGGSLRLSCAASGFGFSSAWMNWV RQAPGKGLEWVGRIKSKTDGGKTDYAAPVKGRFIMSRDDSKNTLYLQMNSLKTEDA GVYYCTTRIVLNGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 211 (6662 lambda light chain)
MGWSCI ILFLVATATGVHSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHW YQQLPGTAPKLLIYANNNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY DSRLSDGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG STVEKTVAPTECS
SEQ ID NO: 212 (6541 heavy chain) MGWSCI ILFLVATATGVHSEVQLVQWGAGLLKPSETLSLTCAVYGGSFSGNYWTWI
RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVY
YCATKMGGYGDYWHWYFDLWGRGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 213 (6541 kappa light chain)
MGWSCI ILFLVATATGVHSEVVMTQSPATLSVSPGERATLSCRASQSLFTNLAWYQ QKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFSLTISRLQSEDFAFYYCQQYND WPLTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC
SEQ ID NO: 214 (6660 heavy chain)
MGWSCI ILFLVATATGVHSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWV
RQAPGKGLEWIGRIKSKTDGGPTDYAAPVTGRFTISRDDSKNTLFLQMNSLKTEDT
AVYYCTAGVVCLHGSSCVGVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 215 (6660 lambda light chain)
MGWSCI ILFLVATATGVHSQSVLTQPPSVSGAPGQRVTISCSGSYSNIGAGYDVYW YQQVPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY DSRLRDFWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG STVEKTVAPTECS
SEQ ID NO: 216 (125 heavy chain) MGWSCI ILFLVATATGVHSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWS
WIRQPPGKGLEWIGHIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTA
VYYCARVRFCSGASCASVYLLDLWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 217 (125 kappa light chain)
MGWSCI ILFLVATATGVHSDIQMTQSPSFLSASVGDRVTITCRASQGIRSYLAWYQ QKPGKAPKLLIYAASTLQSGVPSRFSGTGSGTEFTLTISSLQPEDFATYYCQQLDS YPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC
SEQ ID NO: 218 (66-6-3 heavy chain)
MGWSCI ILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMSWV RQAPGKGLEWVSGI IGNGDVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKDITVARLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 219 (66-6-3 lambda light chain)
MGWSCI ILFLVATATGVHSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSYTVNWY QQLPGTAPKLLIYRNDQRPSGVPERFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLTGRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGST VEKTVAPTECS
SEQ ID NOs: 220-377 - see Table 10
SEQ ID NO: 378 (66-3-9C variant LCDRl)
RSSQSLLHSEGYNYLD SEQ ID NO: 379 (66-3-9C variant LCDRl)
RSSQSLLHSDSYNYLD
SEQ ID NO: 380 (66-3-9C variant LCDRl)
RSSQSLLHSDAYNYLD
SEQ ID NO: 381 (6662 variant HCDR2)
RIKSKTEGGKTDYAAPVKG
SEQ ID NO: 382 (6662 variant HCDR2)
RIKSKTDSGKTDYAAPVKG
SEQ ID NO: 383 (6662 variant HCDR2)
RIKSKTDAGKTDYAAPVKG
SEQ ID NO: 384 (6662 variant HCDR3)
RIVLNSMDV
SEQ ID NO: 385 (6662 variant HCDR3)
RIVLNAMDV
SEQ ID NO: 386 (6662 variant LCDR3)
QSYDSRLSEGVV
SEQ ID NO: 387 (6662 variant LCDR3)
QSYDSRLSDSVV
SEQ ID NO: 388 (6662 variant LCDR3)
QSYDSRLSDAVV
SEQ ID NO: 389 (6541 variant HCDR2)
EIYHSGSTNYNPSLKS
SEQ ID NO: 390 (6541 variant HCDR2)
EINHAGSTNYNPSLKS
SEQ ID NO: 391 (6541 variant HCDR2)
EINHGGSTNYNPSLKS
SEQ ID NO: 392 (6541 variant HCDR2)
EITHSGSTNYNPSLKS
SEQ ID NO: 393 (66-4C-12 HCDRl)
GFTFSSYAMS
SEQ ID NO: 394 (66-4C-12 HCDR2)
GISGPGGGTYYADSVKG
SEQ ID NO: 395 (66-4C-12 HCDR3) GPMISYYAWFDP
SEQ ID NO: 396 (66-4C-12 variant HCDR3)
GPMISYYAWFEP
SEQ ID NO: 397 (66-4C-12 LCDRl)
RASQSISSWLA
SEQ ID NO: 398 (66-4C-12 LCDR2)
DASSLES
SEQ ID NO: 399 (66-4C-12 LCDR3)
QQYNFYASWT
SEQ ID NO: 400 (6651 HCDRl)
GGSISTSSYYWG
SEQ ID NO: 401 (6651 HCDR2)
NIFYSGSTYSNPSLKS
SEQ ID NO: 402 (6651 HCDR3)
VESWQLGAFDI
SEQ ID NO: 403 (6651 LCDRl)
RASQSVSSNLA
SEQ ID NO: 404 (6651 LCDR2)
GASTRAT
SEQ ID NO: 405 (6651 LCDR3)
QQYNDWPRT

Claims

1. A pharmaceutical composition comprising one or more antibodies that bind to Ebola virus glycoprotein and a pharmaceutically acceptable carrier or diluent, wherein at least one of the antibodies comprises a set of six CDRs, HCDR1, HCDR2, HCDR3,
LCDR1, LCDR2, LCDR3, selected from the group consisting of: SEQ ID NOs: 9-14, SEQ ID NOs: 15-20, SEQ ID NOs: 21-26 and SEQ ID NOs: 27-32.
2. The pharmaceutical composition of claim 1, wherein the antibodies bind to non- overlapping epitopes.
3. The pharmaceutical composition of claim 1 or 2, wherein at least one of the antibodies comprises a HCVR and LCVR pair selected from the group consisting of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6 and SEQ ID NOs: 7 and 8.
4 The pharmaceutical composition of any one of claims 1-3, which comprises two or three antibodies as defined in claim 1 or 3.
5. The pharmaceutical composition of any one of claims 1-4, which comprises four antibodies as defined in claim 1 or 3.
6. The pharmaceutical composition of claim 6, which comprises antibodies comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 9-14, SEQ ID NOs: 15-20, SEQ ID NOs: 21-26 and SEQ ID NOs: 27-32, optionally antibodies comprising the HCVR and LCVR pairs of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, and 7 and 8.
7. The pharmaceutical composition of any one of the preceding claims, wherein the one or more antibodies comprise an IgGl, IgG2, IgG3 or IgG4 constant region, optionally a human IgGl, IgG2, IgG3 or IgG4 constant region.
8. A nucleic acid or a pair of nucleic acids encoding the heavy and light chains of an antibody as defined in any one of claims 1, 3 or 7.
9. An expression vector comprising the nucleic acid(s) of claim 8 or a pair of expression vectors comprising the pair of nucleic acids of claim 8.
10. A host cell comprising the expression vector(s) of claim 9.
11. A method of producing an antibody of any one of claims 1 , 3 or 7 comprising culturing the host cell of claim 10 under conditions permitting production of the antibody and recovering the antibody so produced.
12. A method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering a pharmaceutical composition of any one of claims 1-7 to a subject in need thereof.
13. A pharmaceutical composition of any one of claims 1 -7 for use in a method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the pharmaceutical composition to a subject in need thereof.
14. Use of a pharmaceutical composition of any one of claims 1-7 in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
15. An antibody cocktail comprising three or more antibodies binding to the Ebola virus glycoprotein, wherein one antibody binds to the glycan cap, one antibody binds to the receptor binding region and one antibody binds to the base.
16. The antibody cocktail of claim 15, which further comprises an antibody binding to the b17-18 loop of the Ebola virus glycoprotein.
17. The antibody cocktail of claim 15 or 16, wherein one of more of the antibodies individually has an IC50 value of less than 1.25 mg/ml, optionally less than 0.75 mg/ml, as determined in an assay for measuring infection of cells using virus expressing the Ebola virus glycoprotein.
18. The antibody cocktail of any one of claims 15-17, wherein one or more of the antibodies individually provides at least 30% protection in Ebola virus infected animals.
19. The antibody cocktail of any one of claims 15-17, wherein one or more of, optionally all of, the antibodies are cross-reactive for Ebola strains Zaire, Bundibugyo and Sudan.
20. The antibody cocktail of any one of claims 15-19, which comprises one or more antibodies as defined in any one of claim 1 or 3.
21. The antibody cocktail of claim 20, wherein the antibodies comprise:
(a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 9-14, 15-20, 21-26 and 27-32; or
(b) HCVR and LCVR sequence pairs of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6 and SEQ ID NOs: 7 and 8.
22. The antibody cocktail of any one of claims 15-19, which comprises an antibody comprising:
(a) an HCDR sequence of one of SEQ ID NOs: 33-41;
(b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 of SEQ ID NOs: 393, 394, 395, 397, 398 and 399 or 400, 401, 402, 403, 404 and 405; or
(d) HCVR and LCVR sequence pairs as identified in Table 10.
23. The antibody cocktail of any one of claims 15-22, wherein administration of the cocktail to Ebola virus infected animals results in at least a 50% survival rate.
24. A method of treating, preventing or ameliorating Ebola virus infection, the method comprising administering the antibody cocktail of any one of claims 15-23 to a patient in need thereof.
25. The antibody cocktail of any one of claims 15-23 for use in a method of treating, preventing or ameliorating Ebola virus infection, said method comprising administering the antibody cocktail to a patient in need thereof.
26. Use of the antibody cocktail of any one of claims 15-23 in the manufacture of a medicament for treating, preventing or ameliorating Ebola virus infection.
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