EP3583122A1 - Anticorps monoclonaux et cocktails pour le traitement d'infections par le virus ebola - Google Patents

Anticorps monoclonaux et cocktails pour le traitement d'infections par le virus ebola

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Publication number
EP3583122A1
EP3583122A1 EP18754083.6A EP18754083A EP3583122A1 EP 3583122 A1 EP3583122 A1 EP 3583122A1 EP 18754083 A EP18754083 A EP 18754083A EP 3583122 A1 EP3583122 A1 EP 3583122A1
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EP
European Patent Office
Prior art keywords
seq
amino acid
acid sequence
chain variable
variable region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18754083.6A
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German (de)
English (en)
Other versions
EP3583122A4 (fr
Inventor
Zachary A. BORNHOLDT
Larry Zeitlin
Kartik Chandran
Anna WEC
Laura Walker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Albert Einstein College of Medicine
Adimab LLC
Mapp Biopharmaceutical Inc
Original Assignee
Albert Einstein College of Medicine
Adimab LLC
Mapp Biopharmaceutical Inc
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Application filed by Albert Einstein College of Medicine, Adimab LLC, Mapp Biopharmaceutical Inc filed Critical Albert Einstein College of Medicine
Priority claimed from PCT/US2018/018559 external-priority patent/WO2018152452A1/fr
Publication of EP3583122A1 publication Critical patent/EP3583122A1/fr
Publication of EP3583122A4 publication Critical patent/EP3583122A4/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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

  • Ebolaviruses are members of the family Filoviridae which infect humans and non- human primates (NHPs) causing hemorrhagic fever with mortality rates up to 90%. Ebolaviruses include Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Tai Forest virus (TAFV), which are causative agents of the hemorrhagic fever [1, 2].
  • EBOV Ebola virus
  • SUDV Sudan virus
  • BDBV Bundibugyo virus
  • RESTV Reston virus
  • TAFV Tai Forest virus
  • a summary of the ebolaviruses can be found in Burk, et. al., Neglected Filoviruses. FEMS Microbiology Reviews, 40, 494-519 (May, 2016), and the differences between the viruses have been well characterized and well known in the art.
  • filovirus glycoprotein (GP)-specific neutralizing antibodies can reduce mortality following experimental inoculation of animals with a lethal dose of EBOV [3-9].
  • the primary target of these neutralizing antibodies, the filovirus surface GP is a trimer composed of three heavily glycosylated GP1-GP2 heterodimers.
  • the GP1 subunit can be divided further into base, head, glycan cap and mucin-like domains [10]. During viral entry, the mucin-like domain and glycan cap mediate binding to multiple host attachment factors present on the cell membrane.
  • the GP After the virus enters the host cell by macropinocytosis [1 1, 12] the GP is cleaved by host proteases that remove approximately 80% of the mass of the GP1 subunit, including the mucin-like domain and glycan cap [13, 14]. After cleavage of GP in the endosome, the receptor binding sites on GP become exposed, and the GP1 head then is able to bind its receptor, the Niemann-Pick CI ( PC1) protein [13, 15, 16]. Subsequent conformational changes in GP facilitate fusion between viral and endosomal membranes.
  • PC1 Niemann-Pick CI
  • KZ52 the first reported human EBOV GP-specific monoclonal antibody (mAb), was obtained from a phage display library that was constructed from bone marrow RNA obtained from a survivor [19]. KZ52 binds a site at the base of the GP and neutralizes EBOV, most likely by blocking cleavage and/or inhibiting the conformational changes required for fusion of viral and endosomal membranes [10]. Some murine Abs also have been reported to bind to the base region of Ebola virus GPs [20, 21].
  • mAbs that are capable of neutralizing Ebola viruses both in vitro and in vivo.
  • the disclosed human antibodies possess pan- ebolavirus cross-reactivity and cross-neutralizing activity, and are thus capable of binding and neutralizing all known species of the Ebola virus.
  • novel monoclonal antibodies capable of binding to and neutralizing an Ebola virus in a patient.
  • said monoclonal antibodies bind to GP proteins from ebolaviruses belonging to at least two different species, thereby neutralizing the infectivity of viral particles or targeting infected cells for destruction.
  • monoclonal antibodies comprising the following heavy and light chain CDR3 amino acid sequences:
  • mAb PE-24-heavy CDR3 SEQ ID No. 3
  • mAb PE-24-light CDR3 SEQ ID No. 4
  • the critical residues in PE-87 and PE-24 heavy chain CDR3 are D95, W99, and Y100C (Kabat numbering).
  • Methods (below) from the peripheral B cells of a survivor of a filovirus infection is modified so that the VH and VL region nucleotide sequences encode modified V region amino acids that confer enhanced binding capabilities to the mAb.
  • a method of preparing a recombinant antibody comprising: providing a nucleotide sequence selected from the group consisting of
  • immunoreactive fragments of any of the herein described monoclonal antibodies are prepared using means known in the art, for example, by preparing nested deletions using enzymatic degradation or convenient restriction enzymes.
  • compositions for the treatment of Ebola comprising: a therapeutically effective combination of a first monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 12, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 14, and affinity matured variants thereof; anda
  • glycoprotein glycoprotein
  • compositions for the treatment of Ebola comprising: a therapeutically effective combination of a first monoclonal antibody or antigen binding fragment selected from a list consisting of:
  • a monoclonal antibody or antigen binding fragment compri sing a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 15, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 18, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment compri sing a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 21 , and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 23, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 29, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 31, and affinity matured variants thereof,
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 33, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 35, and affinity matured variants thereof,
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 1 1 , and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13, and affinity matured variants thereof; and a pharmaceutically acceptable excipient or carrier; wherein said first monoclonal antibody or antigen binding fragment binds at least two species of the Flivovirus glycoprotein.
  • the first monoclonal antibody or antigen binding fragment comprises predominantly a single glycoform.
  • compositions for the treatment of Ebola comprising: a therapeutically effective combination of a first monoclonal antibody or antigen binding fragment is selected from a list consisting of:
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 12, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 14, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 15, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 18, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 21, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 23, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 29, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 31, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment compri sing a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 33, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 35, and affinity matured variants thereof;
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 1 1 , and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 13, and affinity matured variants thereof; a second monoclonal antibody or antigen binding fragment, wherein said second monoclonal antibody or antigen binding fragment binds the Ebola glycoprotein; and a pharmaceutically acceptable excipient or carrier.
  • composition wherein at least one of the first monoclonal antibody or antigen binding fragment and the second antibody or antigen binding fragment comprises predominantly a single glycoform.
  • said therapeutically effective combination further comprises a third monoclonal antibody or antigen binding fragment that binds to the Ebola glycoprotein.
  • said first monoclonal antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 12, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 14, and affinity matured variants thereof: and wherein said second monoclonai antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 15, and affinity matured variants thereof; and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 18, and affinity matured variants thereof.
  • said therapeutically effective combination further comprises a third monoclonal antibody or antigen binding fragment, wherein said third antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ. ID NO: 21 , and affinity matured variants thereof, and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 23, and affinity matured variants thereof.
  • compositions for the treatment of Ebola comprising: a therapeutically effective combination of a first monoclonal antibody or antigen binding fragment is selected from a list consisting of:
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region at least 90% identical to a heavy chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 53, a CDR2 comprising the amino acid sequence as set forth in SEQ. ID NO: 54, and a CDR3 comprising the amino acid sequence as set forth in SEQ. ID NO: 55, and affinity matured variants thereof; and a light chain variable region at least 90% identical to a light chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 56, a CDR2 comprising the amino acid sequence as set forth in SEQ.
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region at least 90% identical to a heavy chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 41, a CDR2 comprising the amino acid sequence as set forth in SEQ. ID NO: 42, and a CDR3 comprising the amino acid sequence as set forth in SEQ, ID NO: 43, and affinity matured variants thereof; and a light chain variable region at least 90% identical to a light chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ.
  • a CDR2 comprising the amino acid sequence as set forth in SEQ. ID NO: 45
  • a CDR3 comprising the amino acid sequence as set forth in SEQ. ID NO: 46, and affinity matured variants thereof; a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region at least 90% identical to a heavy chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ, ID NO: 47, a CDR2 comprising the amino acid sequence as set forth in SEQ. ID NO: 48, and a CDR3 comprising the amino acid sequence as set forth in SEQ.
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region at least 90% identical to a heavy chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 65, a CDR2 comprising the amino acid sequence as set forth in SEQ. ID NO: 66, and a CDR3 comprising the amino acid sequence as set forth in SEQ. ID NO: 67, and affinity matured variants thereof; and a light chain variable region at least 90% identical to a light chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 68, a CDR2 comprising the amino acid sequence as set forth in SEQ.
  • a monoclonal antibody or antigen binding fragment comprising a heavy chain variable region at least 90%> identical to a heavy chain variable region comprising a CDR1 comprising the amino acid sequence as set forth in SEQ. ID NO: 71, a CDR2 comprising the amino acid sequence as set forth in SEQ, ID NO: 72, and a CDR3 comprising the amino acid sequence as set forth in SEQ.
  • compositions further comprising a second monoclonal antibody or antigen binding fragment, wherein said second monoclonal antibody or antigen binding fragment binds the Ebola glycoprotein.
  • Fiivovirus glycoprotein It is still another embodiment of the present invention to provide such a composition, wherein the first monoclonal antibody or antigen binding fragment compiises predominantly a single glycoform.
  • composition wherein the predominantly single glycoform is one of GnGn, G1/G2, and NaNa.
  • Figure 1 shows a neutralization curve for affinity matured variants of one embodiment of the present invention.
  • Figure 2 shows the binding sites on the EBOV-GP of various embodiments of the monoclonal antibodies of the present invention.
  • Figure 3 shows the location of the mutations that result in escape mutant resistance to two monoclonal antibodies of the present invention.
  • Figure 4 shows neutralization assays preformed against the escape mutants.
  • Figure 5 shows survival data for ebolavirus infected guinea pigs treated with certain embodiments of the present invention.
  • Figure 6 shows immune system response data from ebolavirus infected guinea pigs treated with certain embodiments of the present invention.
  • Figure 7 shows survival data for ebolavirus infected guinea pigs treated with certain embodiments of the present invention.
  • Figure 8 shows survival data for ebolavirus infected guinea pigs treated with certain embodiments of the present invention.
  • Figure 13 shows a neutralization curves for certain embodiments of the present invention created using differing production methods.
  • Table 2 shows the efficiency of anti-GP antibody isolation from peripheral B cells.
  • Table 3 shows the cross-reactivity of candidate pan-ebolavirus mAbs against different ebolavirus species. Reactivity was measured by ELISA.
  • Table 4 shows the in vitro neutralization activity and affinities of candidate pan- ebolavirus mAbs.
  • Table 5 shows that mice infected with EBOV and subsequently treated with the monoclonal antibodies described above showed increased survival compared to mice treated with PBS.
  • Table 6 is a summary of rVSV-GP neutralization by cross-neutralizing human mAbs.
  • Table 7 is a summary of authentic ebolavirus neutralization by cross-neutralizing human mAbs.
  • Table 8 shows K D values for recognition of EBOV GP TM by mature PE-87 bearing the indicated mutations in the CDR-H3 loop were determined by BLI. 95% confidence intervals are reported for each binding constant. IC50 values for neutralization of rVSVs bearing ebolavirus GPs by mature PE-87 bearing the indicated mutations in the CDR-H3 loop.
  • Table 9 shows the mAb protection of mice after challenge with EBOV or SUDV.
  • NAb neutralizing antibody
  • diagnosis antibody or “detection antibody” or “detecting antibody” refers to an antibody, for example, a monoclonal antibody, capable of detecting the presence of an antigenic target within a sample.
  • diagnostic antibodies preferably have high specificity for their antigenic target.
  • human antibodies refer to antibodies that were isolated from the B cells of a human or directly from the sequence of serum antibodies.
  • a “therapeutically effective” treatment refers a treatment that is capable of producing a desired effect. Such effects include, but are not limited to, enhanced survival, reduction in presence or severity of symptoms, reduced time to recovery, and prevention of initial infection.
  • “Therapeutically effective” permutations of a mAb may enhance any of the above characteristics in a manner that is detectable by routine analysis of patient data.
  • such therapeutically effective mutations include mutations that improve the stability, solubility, or production of the mAb, including mutations to the framework regions of the mAb sequence.
  • 'immunoreactive fragment refers in this context to an antibody fragment reduced in length compared to the wild-type or parent antibody which retains an acceptable degree or percentage of binding activity to the target antigen. As will be appreciated by one of skill in the art, what is an acceptable degree will depend on the intended use.
  • a mAb has "pan-Ebola” binding characteristics if it is capable of binding to at least 2, but preferable more, ebolavirus species.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as kappa and lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • Within each isotype there may be subtypes, such as IgGi, IgG 2 , IgG 3 , IgG 4 , etc.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. The particular identity of constant region, the isotype, or subtype does not impact the present invention.
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 From N- terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with well known conventions [Kabat "Sequences of Proteins of Immunological Interest" National Institutes of Health, Bethesda, Md. s 1987 and 1991; Chothia, et al., J. Mol. Biol. 196:901-917 (1987); Chothia, et al., Nature 342:878-883 (1989)].
  • glycoengineered variants of the monoclonal antibodies that contain predominantly a single glycoform.
  • a predominantly single glycoform is any glycoform that represents more than half (e.g. greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of all glycoforms present in the antibody solution.
  • the RAMP system has been used for glycoengineering of antibodies, antibody fragments, idiotype vaccines, enzymes, and cytokines. Dozens of antibodies have been produced in the RAMP system by Mapp (5, 6) and others (7, 8). These have predominantly been IgGs but other isotypes, including IgM (9, 10), have been glycoengineered. Glycoengineering has also been extended to human enzymes in the RAMP system (11, 12). Since the RAMP system has a rapid turn-around time from Agrobacterium infection to harvest and purification (13) patient specific idiotype vaccines have been used in clinical trials for non-Hodgkins lymphoma (7).
  • recombinant Agrobacterium containing a mAb cDNA is used for infection of N. benthamiana in combination with the appropriate glycosylation Agrobacteria to produce the desired glycan profile.
  • wild-type glycans i.e. native, plant- produced glycosylation
  • wild-type N. benthamiana is inoculated with only the Agrobacterium containing the anti-M2e cDNA.
  • GnGn glycan the same Agrobacterium is used to inoculate plants that contain little or no fucosyl or xylosyl transfrases ( XF plants).
  • XF plants are inoculated with the Agrobacterium containing the mAb cDNA as well as an Agrobacterium containing the cDNA for -l,4-galactosyltransferase expression contained on a binary Agrobacterium vector to avoid recombination with the TMV and PVX vectors (14).
  • sialylated glycans six additional genes are introduced in binary vectors to reconstitute the mammalian sialic acid biosynthetic pathway.
  • the genes are UDP-N- acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, CMP-N-acetylneuraminic acid synthetase, CMP-NeuAc transporter, ⁇ -1,4- galactosylatransferase, and a2,6-sialyltransferase (14).
  • Glycanalysis of glycoengineered mAbs involved release of N-linked glycans by digestion with N-glycosidase F (PNGase F), and subsequent derivatization of the free glycan is achieved with anthranilic acid (2-AA).
  • PNGase F N-glycosidase F
  • 2-AA anthranilic acid
  • the 2-AA-derivatized oligosaccharide is separated from any excess reagent via normal-phase FIPLC.
  • the column is calibrated with 2-AA-labeled glucose homopolymers and glycan standards.
  • the test samples and 2-AA-labeled glycan standards are detected fluorometrically.
  • Glycoforms are assigned either by comparing their glucose unit (GU) values with those of the 2-AA-labeled glycan standards or by comparing with the theoretical GU values (15). Confirmation of glycan structure was accomplished with LC/MS.
  • GU glucose unit
  • LC/MS LC/MS
  • GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FCyRIII. Biotechnoi Bioeng 74:288-294]), yeast cells (such as Pichia pastor is [Gerngross T. Production of complex human glycoproteins in yeast. Adv Exp Med Biol. 2005; 564]) and bacterial cells (such as E. Coif) have been used produce such mABs.
  • mAbs designated PE-24, PE-87, PE-47, PE-16, PE-64 and PE-05, which have surprisingly exhibited pan-Ebola neutralizing characteristics.
  • the preferred antibodies of the present invention comprise mAbs with amino acid sequences sufficiently identical to referenced amino acid sequencees.
  • amino acid sequences sufficiently identical to referenced amino acid sequencees.
  • “sufficiently identical” is intended an amino acid sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99% or greater sequence identity compared to a reference sequence using one of the alignment programs known in the art.
  • VL amino acids to yield mAb PE-47 (Modifications are shown in Bold, CDR sequences are Underlined).
  • mAb PE-47 VH amino acids SEQ ID No. 12 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGEGLEWVGRIKSKTDG GTIDYAAPVKGRFTISRDDSKNTVYLQMTSLKTEDTAVYYCTTYTEDMQYFDWLLRGG ETFDYWGQGTLVTVS SEQ ID No. 12 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGEGLEWVGRIKSKTDG GTIDYAAPVKGRFTISRDDSKNTVYLQMTSLKTEDTAVYYCTTYTEDMQYFDWLLRGG ETFDYWGQGTLVTVS SEQ ID No. 12 EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGEGLEWVGRIKSKTDG GTIDYAAPVKGRFTISRDDSKNTVYLQMTSLKTEDTAVYYCTTYTEDMQYFDWLLRGG ETFDYWGQGTLVTVS S
  • PE-87 VH amino acids SEQ ID No. 15
  • PE-87 VH nucleotides SEQ ID No. 16
  • PE-87 VH amino acid sequence is: SEQ ID No. 17 (alterations shown in Bold and Underlined)
  • PE-87 VL amino acids SEQ ID No.18
  • PE-87 VL nucleotides SEQ ID No. 19
  • An alternative PE-87 VL amino acid sequence is: SEQ ID No. 20 (alterations shown in Bold and Underlined)
  • PE-24 VH amino acids SEQ ID No. 21
  • PE-24 VH nucleotides SEQ ID No. 22
  • PE-24 VL amino acids SEQ ID No. 23
  • PE-24 VL nucleotides SEQ ID No. 24
  • PE-47 VH amino acids SEQ ID No. 25
  • PE-47 VH nucleotides SEQ ID No. 26
  • PE-47 VL amino acids SEQ ID No.27
  • PE-47 VL nucleotides SEQ ID No. 28
  • PE-16 VH amino acids SEQ ID No.29
  • PE-16 VH nucleotides SEQ ID No.30
  • PE-16 VL amino acids SEQ ID No. 31
  • PE-16 VL nucleotides SEQ ID No. 32
  • PE-05 VH amino acids SEQ ID No. 33
  • PE-05 VH nucleotides SEQ ID No.34
  • PE-05 VL amino acids SEQ ID No. 35
  • PE-05 VL nucleotides SEQ ID No. 36
  • PE-64 VH amino acids SEQ ID No. 37
  • PE-64 VH nucleotides SEQ ID No. 38
  • PE-64 VL amino acids SEQ ID No. 39
  • PE-64 VL nucleotides SEQ ID No. 40
  • the above mAb sequences are affinity matured to enhance binding or otherwise improve the therapeutic efficacy of the antibody.
  • optimization of antibodies was performed via a light chain diversification protocol, and then by introducing diversities into the heavy chain and light chain variable regions as described below:
  • CDRL1 and CDRL2 selection The CDRL3 of a single antibody was recombined into a premade library with CDRL1 and CDRL2 variants of a diversity of 1 x 10 8 and selections were performed with one round of MACS and four rounds of FACS. For each FACS round the libraries were affinity pressured using titrating amounts of an ebolavirus GP (for example, SUDV GP) and sorting was performed in order to obtain a population with the desired characteristics.
  • an ebolavirus GP for example, SUDV GP
  • VH Mut selection The heavy chain variable region (VH) was mutagenized via error prone PCR. The library was then created by transforming this mutagenized VH and the heavy chain expression vector into yeast already containing the light chain plasmid of the parent. Selections were performed similar to previous cycles using FACS sorting for two rounds. For each FACS round the libraries were affinity pressured using titrating amounts of Sudan GP and sorting was performed in order to obtain a population with the desired characteristics.
  • ADI-23774 (PE-47) was generated by combining the most improved HC (from the VH mut selection) with the most improved LC (from the L1/L2 selection).
  • Figure 1 illustrates the enhanced neutralization potential of the parent (PE-64), best VH mutant, best VL mutant, and best VH/VL mutant (PE-47).
  • Endosomally generated GPCL species is the presumptive final target of these mAbs. Strikingly, GP cleavage to GPCL enhanced the antiviral potencies of PE-64, PE-87, and PE-47 by 50-200 fold. Together, these results suggest that the broadly neutralizing mAbs PE- 87 and PE-47 differ from previously described monospecific mAbs (KZ52, c2G4, and 4G7), in their ability to target and neutralize a cleaved GP species that is generated deep in the endocytic pathway. Conversely, the latter mAbs appear to act principally at and/or prior to the GP->GPCL cleavage step.
  • PE-64 displayed a dual behavior, and may act both upstream, to block GP cleavage, and downstream, to target one or more GPcL-like species at or near the membrane fusion step.
  • wild type (WT) BALB/c mice were exposed to mouse- adapted EBOV (EBOV-MA), and then administered a single dose of each mAb at 2 days postinfection (300 ⁇ g/animal).
  • Cross-neutralizing mAbs were highly (>80%) protective against EBOV in this stringent post-exposure setting, with little or no weight loss apparent in mAb-treated animals.
  • Figure 2 illustrates negative stain EM reconstructions of broadly neutralizing ebolavirus mAbs.
  • a structure of ebolavirus GP (based on PDB IDs:5JQ3) displaying the antigenic surfaces and corresponding structural regions of interest.
  • the disordered mucin domain (dashed lines), GP1, GP2, fusion loop, glycan cap in, CHR2 region and the N563-linked glycan.
  • Top and side views are shown for negative stain EM 3D reconstructions of Fab models of PE-87, PE-47, PE-24 and PE-16 (shown in dark gray) in complex with EBOV GP.
  • mice were exposed to WT SUDV, and then dosed with each NAb on days 1 and 3 post-infection (300 ⁇ g/animal/dose).
  • the pan-ebolavirus mAbs PE-87 and PE-47 afforded >95% survival and greatly reduced weight loss, relative to the PBS control group.
  • PE-16 and PE-64 both weak SUDV neutralizers, provided little or no protection against SUDV.
  • the mAbs of the present invention have been shown to provide complete protection to a non-human primate model of Ebola virus challenge.
  • a group of rhesus macaque monkeys were treated with either one dose of an NAb cocktail (comprising 25mg/kg each of PE-87 and PE-47) or two doses of the same NAb cocktail (one at 4 days post infection, comprising 50mg/kg of the NAbs, and another at 7 days post infection, comprising 25mg/kg of the NAbs).
  • an NAb cocktail comprising 25mg/kg each of PE-87 and PE-47
  • two doses of the same NAb cocktail one at 4 days post infection, comprising 50mg/kg of the NAbs, and another at 7 days post infection, comprising 25mg/kg of the NAbs.
  • EBOV infection was uniformly lethal, with the all PBS-treated animals succumbing by the 7 th day post infection.
  • every animal from the NAb treatment groups survived, with no detectable viral
  • the NAb cocktail of PE-87 and PE-47 (also refered to herein as MBP134) was further tested as follows. First, escape mutants that were resistant to the individual components of MBP134 were generated. Escape mutant selections were performed by serial passage of rVSV-GP particles in the presence of test antibody. Briefly, serial 3 -fold dilutions of virus were preincubated for one hour with a concentration of antibody corresponding to the IC90 value derived from neutralization assays, and then added to confluent monolayers of Vero cells in 12-well plates, in duplicate.
  • Figure 3 illustrates the mutations to the rVSV-GP and their relative locations within the three-dimentional structure of the viral glycoprotein for the two escape mutants that were most resistant to PE-47 (MBP047) and PE-87 (MBP087) respectively.
  • the PE-87 escape mutant contained a G528E substitution
  • the PE-47 escape mutant contained a N514D substitution.
  • Figure 4 illustrates the dose response curves of the above-mentioned escape mutants and the wild-type SUDV virus to concentrations PE-47 and PE-87.
  • the escape mutations which provided resistance to one mAb resulted in significantly enhanced neutralization by the other.
  • a combination of multiple antibodies is provided which significantly reduce the risk of viral resistance development.
  • antibodies comprising a substantially single glycan and lacking fucose show enhanced efficacy in patients.
  • fucosylated and afucosylated versions of the cocktail were used to treat guinea pigs challenged with a lethal dose of EBOV. All guinea pigs were healthy and immune competent as per vendor's representation. All guinea pigs were drug and test naive. Animals were monitored daily for food and water consumption and given environmental enrichment according to the guidelines for the species. Cleaning of the animals was completed three times per week which included a complete cage and bedding material change.
  • Figure 5 illustrates the survival curves of the afucosylated vs. fucosylated MBP134 at various doses.
  • the afucosylated cocktail showed dramatically improved survival, even at the lowest dosage tested.
  • blood drawn from the animals showed significantly increased immune reactions in response to treatment with afucosylated PE-47 and PE-87, as compared to their fucosylated counterparts and other anti-EBOV mAbs cl3C6 (also afucosylated) and 2G12, as illustrated in Figure 6.
  • a monoclonal antibody that substantially lacks fucose.
  • the increase dosage of the monoclonal antibodies at later dates post infection allows the host animals to overcome the increased viral load associated with the infection.
  • a patient is treated with an effective dose of a monoclonal antibody or combination of monoclonal antibodies.
  • An effective dose includes, but is not limited to, O.Olmg/kg, 0.05mg/kg, O.
  • Ferrets were anesthetized by intramuscular injection with a ketamine-acepromazine-xylazine cocktail prior to all procedures.
  • transponder chips Bio-Medic Data Systems
  • Subjects were challenged intranasally with a lethal dose of 1000 plaque-forming units (PFU) of ZEBOV strain Kikwit, SEBOV strain Gulu, or BDBV and treated with MBP134-N at the times and dosing shown in Figure 9.
  • PFU plaque-forming units
  • Kikwit ZEBOV strain Kikwit
  • SEBOV strain Gulu SEBOV strain Gulu
  • BDBV BDBV
  • rhesus macaques were infected with a lethal dose of EBOV/Kikwit and treated with the monoclonal antibodies of the present invention.
  • Rhesus macaques at UTMB were challenged by intramuscular injection (EVI) with 1,000 PFU of EBOV/Kikwit.
  • EVI intramuscular injection
  • the macaques were given physical examinations and blood was collected at the time of viral challenge; and on days 4, 7, 10, 14, 21, and 28 after challenge.
  • the macaques were monitored daily and scored for disease progression with an internal filovirus scoring protocol approved by the UTMB Institutional Animal Care and Use Committee (IACUC) in accordance with state and federal statutes and regulations relating to experiments involving animals and by the UTMB Institutional Biosafety Committee.
  • IACUC Animal Care and Use Committee
  • the scoring changes measured from baseline included posture/activity level; attitude/behavior; food and water intake; weight; respiration; and disease manifestations, such as visible rash, hemorrhage, ecchymosis, or flushed skin, with increased scores resulting in euthanasia.
  • the monoclonal antibodies of the present invention provide protection from ebolavirus challenge in different species of primate.
  • Cynomolgus monkeys at UTMB were challenged by intramuscular injection (FM) with 1,000 PFU of BDBV (200706291 Kenya isolate, Vero E6 passage 2).
  • Control animals (n 3) were untreated. All the animals were given physical examinations and blood was collected at the time of viral challenge; and on days 4, 7, 10, 14, 21, and 28 after challenge (or at time of euthanasia).
  • any of the above described antibodies may be formulated into a pharmaceutical treatment for providing passive immunity for individuals suspected of or at risk of developing hemorrhagic fever comprising a therapeutically effective amount of said antibody.
  • the pharmaceutical preparation may include a suitable excipient or carrier. See, for example, Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed. As will be apparent to one knowledgeable in the art, the total dosage will vary according to the weight, health and circumstances of the individual as well as the efficacy of the antibody. While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.
  • HBSS Hank's Balanced Salt Solution
  • Ficoll-Paque Plus GE Healthcare
  • the B cell layer was removed from the density gradient by pipette, washed twice in HBSS by centrifugation at 400xg, frozen at 6.5x106 cells/ml in a 1 : 1 mixture of FBS (Life Technologies) and cryoprotective medium (Lonza) and stored under liquid nitrogen. Plasma was collected from the top layer of the density gradient and stored at -80°C until use.
  • a high-binding ELISA plate was coated with 1 ⁇ g/ml of EBOV rGPATM (IBT Biosciences) diluted in PBS overnight at 4°C. After washing, wells were blocked with 1% BSA in PBS and 0.05% Tween-20 for 2 hours at room temperature. Wells were washed and serial dilutions of human plasma (diluted in blocking buffer) were added and incubated for 1.5 hours at room temperature. As positive and negative controls, serial dilutions of mAb KZ52 (IBT Biosciences) or an irrelevant human mAb, respectively, were added to appropriate wells.
  • HRP-conjugated donkey anti-human IgG Jackson ImmunoResearch
  • HRP- conjugated goat anti-human IgA Southern Biotech
  • Purified B cells were stained using anti-human IgM (BV605), IgD (BV605), IgG (BV421), CD 8 (APC-Cy7), CD14 (AF700), CD19 (PerCP-Cy5.5), CD20 (PerCP-Cy5.5) and biotinylated EBOV GPATM.
  • Biotinylated GPATM was used at a concentration of 50 nM and detected using streptavidin-APC (Life Technologies) at a dilution of 1 :500.
  • Single cells were sorted on a MoFlo cytometer (Beckman-Coulter) into 96-well PCR plates (BioRad) containing 20 ⁇ /well of lysis buffer [5 ⁇ of 5X first strand cDNA buffer (Invitrogen), 0.5 ⁇ RNaseOUT (Invitrogen), 1.25 ⁇ dithiothreitol (Invitrogen), 0.625 ⁇ NP-40 (New England Biolabs), and 12.6 ⁇ dH20]. Plates were immediately frozen on dry ice before storage at 80°C. Amplification and cloning of antibody variable genes
  • Single B cell PCR was performed essentially as previously described [27]. Briefly, IgH, Igk and IgK variable gene transcripts were amplified by RT-PCR and nested PCR reactions using cocktails of primers specific for IgG [27]. The primers used in the second round of PCR contained 40 base pairs of 5' and 3' homology to the cut expression vectors to allow for cloning by homologous recombination into Saccharomyces cerevisiae [28]. PCR products were cloned into S. cerevisiae using the lithium acetate method for chemical transformation [29]. Each transformation reaction contained 20 ⁇ of unpurified heavy chain and light chain PCR product and 200 ng of cut heavy and light chain plasmids. Individual yeast colonies were picked for sequencing and down-stream
  • Antibodies used for binding experiments, competition assays, neutralization assays, and structural studies were expressed in Saccharomyces cerevisiae cultures grown in 24 well plates. After 6 days of growth, the yeast cell culture supernatant was harvested by centrifugation and subject to purification. IgGs used in protection experiments were expressed by transient co- transfection of heavy and light chain plasmids into HEK293 cells. One day prior to transfection, HEK293 cells were passaged at 2.0 - 2.5 X 106 cells/ ml.
  • transfection On the day of transfection, cells were pelleted by centrifuging at 400 g for 5 min, and cell pellets were resuspended in fresh FreeStyle F17 medium at a density of 4 X 106 cells/ ml and returned to the incubator.
  • a transfection mixture was prepared by first diluting the plasmid DNA preparations in FreeStyle F17 medium (1.33 ⁇ g total plasmid DNA per ml of culture).
  • Transfection agent PEIproTM (Polyplus Transfection, Illkirch, France)
  • PEIproTM Polyplus Transfection, Illkirch, France
  • the digestion was terminated by the addition of iodoacetamide, and the Fab and Fc mixtures were passed over Protein A agarose to remove Fc fragments and undigested IgG.
  • the flowthrough of the Protein A resin was then passed over Capture SelectTM IgG-CHl affinity resin (Therm oFischer Scientific), and eluted with 200 mM acetic acid / 50 mM NaCl pH 3.5 into l/8th volume 2M Hepes pH 8.0.
  • Fab fragments then were buffer-exchanged into PBS pH 7.0.
  • EBOV GPATM was biotinylated using EZ-LinkTM Sulfo- HS-LC-Biotin (Life Technologies) followed by a desalting step by a ZebaTM Spin Desalting Column (Life Technologies).
  • IgG binding to the different GP antigens was determined by BLI measurements using a ForteBio Octet HTX instrument (Pall Life Sciences). For high-throughput KD screening, IgGs were immobilized on AHQ sensors (Pall Life Sciences) and exposed to 100 nM antigen in PBS containing 0.1% BSA (PBSF) for an association step, followed by a dissociation step in PBSF buffer. Data was analyzed using the ForteBio Data Analysis Software 7. The data was fit to a 1 : 1 binding model to calculate an association and dissociation rate, and KD was calculated using the ratio kd/ka.
  • ELISA plates were coated with 50 ⁇ PBS containing 4 ⁇ g/mL EBOV GP antigens for 1 h at room temperature. After washing, wells were blocked with 3% BSA for 1 h at room temperature. After removal of the blocking solution, mAbs were applied to the plates at a concentration of 0.2 ⁇ g/ml and incubated at room temperature for 1 h. After washing, binding was detected with an anti-human HRP-conjugated secondary antibody and TMB substrate. Optical density was read at 450 nm.
  • Antibody competition assays were performed essentially as previously described [31]. Antibody competition was measured by the ability of a control anti-EBOV GP Fab to inhibit binding of yeast surface-expressed anti-GP IgGs to GPAmuc. 50 nM biotinylated GPAmuc was pre-incubated with 1 ⁇ competitor Fab for 30 min at RT and then added to a suspension of yeast- expressed anti-GP IgG. Unbound antigen was removed by washing with PBSF. After washing, bound antigen was detected using Streptavidin Alexa Fluor 633 at a 1 :500 dilution (Life Technologies) and analyzed by flow cytometry using a BD FACS Canto II. Results are expressed as the fold reduction in antigen binding in the presence of competitor Fab relative to an antigen- only control.
  • Virus-specific neutralizing antibody responses were titrated essentially as previously described [32]. Briefly, plasma or antibodies were diluted serially in Minimal Essential Medium (Corning Cellgro, Manassas, VA) containing 5% heat-inactivated fetal bovine serum (Gibco-Invitrogen, Gaithersburg, MD), IX Anti-Anti (Gibco-Invitrogen, Gaithersburg, MD) (MEM complete) and incubated 1 hour at 37°C with virus. After incubation, the antibody-virus or plasma-virus mixture was added in duplicate to 6-well plates containing 90-95% confluent monolayers of Vero E6 cells. Plates were incubated for 1 hour at 37°C with gentle rocking every 15 minutes.
  • Minimal Essential Medium (Corning Cellgro, Manassas, VA) containing 5% heat-inactivated fetal bovine serum (Gibco-Invitrogen, Gaithersburg, MD), IX Anti-Anti (Gibco-Invitrog
  • mice The lethal mouse-adapted EBOV mouse model was developed at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) by serial passages of EBOV (Zaire) in progressively older suckling mice [38].
  • Female BALB/c mice Female BALB/c mice, aged 6 to 8 weeks, were purchased from Charles River Laboratory. Upon arrival, mice were housed in microisolator cages in an animal biosafety level 4 containment area and provided chow and water ad libitum. On day 0, mice were infected intraperitoneally (i.p.) with 100 p.f.u. of mouse-adapted EBOV. Two days post-infection, groups of mice (10 mice per group) were treated i.p.
  • mice received PBS. Mice were monitored daily (twice daily if there were clinical signs of disease) for 28 days post-infection. Group weights were taken on days 0-14, and on days 21 and 28 post-infection. Survival was compared using the log-rank test in GraphPad PRISM 5. Differences in survival were considered significant when the P value was less than 0.05. Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals.
  • mice were given 100 ug of the indicated antibody, or PBS, two days post infection. * Average weight change from the pre-injection baseline to the peak of clinical disease. Mice were weighed as groups.
  • mice 4-5 week old, IFNa/bR KO mice will be inoculated I P. with SUDV (1000 pfu). Experimental group will be treated with mAbs (0.3 ml volume) at indicated dose on days 1 and 4 post-infection. Control mice will vehicle control LP. (0.3 ml volume) on the same schedule as experimental mice. Mice will be observed daily for 21 days for moribund condition. Moribund mice will be promptly euthanized (LAW SOP AC- 11-07) when they meet euthanasia criteria (score sheet).
  • Vero African grivet monkey cells and 293T human embryonic kidney fibroblast cells were maintained in high-glucose Dulbecco's modified Eagle medium (DMEM; Thermo Fisher) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 1% GlutaMAX (Thermo Fisher), and 1% penicillin-streptomycin (Thermo Fisher). Cells were maintained in a humidified 37°C, 5% C02 incubator.
  • DMEM Dulbecco's modified Eagle medium
  • VSV Vesicular stomatitis virus
  • BDBV BDBV/H.sap/UGA/07/But-811250
  • SUDV/Boneface SUDV/C.por- lab/SSD/76/Boneface
  • RESTV RESTV/M.fas-tc/USA/89/Phi89-AZ-1435
  • LLOV LLOV/M.sch-wt/ESP/03/Asturias-Bat86
  • VSV pseudotypes bearing eGFP and GP proteins from TAFV TAFV/H.sap- tc/CIV/94/CDC807212
  • MARV MARV/H.sap-tc/KEN/80/Mt. Elgon-Musoke
  • cleaved viral particles bearing GPCL were first generated by incubation with thermolysin (200 ⁇ g/mL, pH 7.5, 37°C for 1 h; Sigma-Aldrich) or recombinant human cathepsin L (CatL, 2 ng/ ⁇ , pH 5.5, 37°C for 1 h; R&D Systems), as described previously [1]. Reactions were stopped by removal onto ice and addition of phosphoramidon (1 mM) or E-64 (10 ⁇ ), respectively, and viral particles were used immediately for infectivity assays. A recombinant, soluble GPATM protein [5] was also essentially as described above.
  • Viral infectivities were measured by automated counting of eGFP+ cells (infectious units; RJ) using a Celllnsight CX5 imager (Thermo Fisher) at 12-14 h post-infection.
  • eGFP+ cells infectious units
  • CX5 imager Celllnsight CX5 imager
  • Viral neutralization data were subjected to nonlinear regression analysis to derive EC50 values (4-parameter, variable slope sigmoidal dose-response equation; GraphPad Prism).
  • IC50 nM
  • mAb concentration that affords half-maximal neutralization of viral infectivity.
  • Recombinant mAbs from the human EBOV disease survivor, as well as germline- reverted (IGL) mAb constructs and WT:IGL chimeras of PE-87 were expressed in Saccharomyces cerevisiae and purified from cell supernatants by protein A affinity chromatography, as described previously [5].
  • Other recombinant mAbs were produced in 293F cells by transient transfection, and purified by protein A affinity chromatography, as described previously [3].
  • the viral lipid envelopes of rVSV-EBOV GP particles were labeled with biotin using a function-spacer-lipid construct (FSL-biotin) (Sigma-Aldrich) for 1 h at pH 7.5 and 37°C, as described [2].
  • FSL-biotin function-spacer-lipid construct
  • Biotinylated viral particles bearing GPCL were generated by incubation with thermolysin, and then captured onto high-binding 96-well ELISA plates precoated with recombinant streptavidin (0.65 ⁇ g/mL; Sigma-Aldrich). Plates were then blocked with PBSA, and incubated with serial dilutions of test mAbs.
  • Washed plates were then incubated with a pre-titrated concentration of soluble, FLAG epitope-tagged, PC1 domain C (NPC1-C) protein [9], and bound PC1-C was detected with an anti-FLAG antibody conjugated to horseradish peroxidase (Sigma-Aldrich). All incubations were performed for 1 h at 37°C. ELISAs and immunoblots to detect mAb inhibition of GP cleavage
  • escape mutant selections were performed by serial passage of rVSV-GP particles in the presence of test mAb. Briefly, serial 3-fold dilutions of virus were preincubated with a concentration of mAb corresponding to the IC90 value derived from neutralization assays, and then added to confluent monolayers of Vero cells in 12-well plates, in duplicate. Infection was allowed to proceed to completion (>90% cell death by eye), and supernatants were harvested from the infected wells that received the highest dilution (i.e., the least amount) of viral inoculum. Following three subsequent passages under mAb selection with virus-containing supernatants as above, supernatants from passage 4 were tested for viral neutralization escape.
  • Antibody Fabs and a EBOV GP TM ectodomain protein were prepared as described previously [5], and incubated at a ratio of 10: 1 (Fab:GP) overnight at 4°C. Complexes were then deposited onto a carbon-coated copper mesh grid, and stained with 1% uranyl formate. Samples were imaged on a Tecnai F 12 microscope using the automated image acquisition software Leginon [10]. Images were collected with a Tietz 4K CMOS detector at 52,000 ⁇ magnification, resulting in a final pixel size of 2.05A at the specimen level. Images were automatically uploaded to and processed within our Appion database [11].
  • the OctetRedTM system (ForteBio, Pall LLC) was used to determine the binding properties of different IgGs to various forms of EBOV GP.
  • Anti-human Fc (AHC) capture sensors (ForteBio) were used for initial mAb loading at 25 mg/mL in l x kinetics buffer (PBS supplemented with 0.002% Tween-20 and 1 mg/mL of BSA). Binding to GP was performed across two-fold serial dilutions of EBOV GP TM or GPCL. The baseline and dissociation steps were carried out in the l x kinetics buffer as per the instrument manufacturer's recommendations.
  • a l x pH 5.5 kinetics buffer 50 mM sodium citrate dihydrate[pH 5.5], 150 mM sodium chloride, 0.002% Tween-20 and 1 mg/mL BSA was used in place of the PBS-based l x kinetic buffer for all steps.
  • a global data fitting to a 1 : 1 binding model was used to estimate values for the k on (association rate constant), k 0 ff (dissociation rate constant), and K D (equilibrium dissociation constant).
  • mice 10-12 week old female BALB/c mice (Jackson Labs) were challenged via the intraperitoneal (i.p.) route with EBOV-MA (100 PFU; -3,000 LD50). Mice were treated i.p. 2 days post-challenge with PBS vehicle or 300 ⁇ g of each mAb (0.3 mL volume, -15 mg mAb/kg). Animals were observed daily for clinical signs of disease and lethality. Daily observations were increased to a minimum of twice daily while mice were exhibiting signs of disease. Moribund mice were humanely euthanized on the basis of IACUC-approved criteria.
  • Type 1 IFN ⁇ / ⁇ receptor knockout mice (Type 1 IFNa/ ⁇ R / ) (Jackson Labs) were challenged with WT SUDV (1000 PFU i.p.). Animals were treated i.p. 1 and 4 days post-challenge with PBS vehicle or 300 ⁇ g (-15 mg mAb/kg) per dose, and monitored and euthanized as above. Table 9: Activity in mouse models
  • Murine challenge studies were conducted under IACUC-approved protocols in compliance with the Animal Welfare Act, PHS Policy, and other applicable federal statutes and regulations relating to animals and experiments involving animals.
  • the dfacility where these studies was conducted (USAMRIID) isaccredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and adhere to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 201 1.
  • Ferret challenge studies were approved by the Animal Care Committee (ACC) of the Canadian Science Centre for Human and Animal Health (CSCHAH) in Winnipeg, Canada, in accordance with guidelines from the Canadian Council on Animal Care (CCAC).
  • Lander, G.C., et al., Appion an integrated, database-driven pipeline to facilitate EM image
  • Nanbo, A., et al. Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner.
  • Murin, CD., et al. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc Natl Acad Sci U S A, 2014. 111(48): p. 17182-7. Volchkov, V.E., et al., Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A, 1998. 95(10): p. 5762-7.

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Abstract

L'invention concerne des compositions et des procédés pour la prévention et le traitement d'infection par le virus Ebola. Selon certains modes de réalisation de la présente invention, des anticorps monoclonaux sensiblement similaires à ceux décrits dans la description, ainsi que des variants mûrs par affinité de ceux-ci, seuls ou en combinaison, fournissent une efficacité thérapeutique chez un patient contre de multiples espèces du virus Ebola.
EP18754083.6A 2017-02-17 2018-02-17 Anticorps monoclonaux et cocktails pour le traitement d'infections par le virus ebola Pending EP3583122A4 (fr)

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Inventor name: BORNHOLDT, ZACHARY, A.

Inventor name: WALKER, LAURA

Inventor name: WEC, ANNA

Inventor name: ZEITLIN, LARRY

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Effective date: 20201014

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Ipc: C07K 16/10 20060101AFI20201008BHEP

Ipc: A61K 39/395 20060101ALI20201008BHEP

Ipc: A61P 31/14 20060101ALI20201008BHEP

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Owner name: MAPP BIOPHARMACEUTICAL, INC.

Owner name: ADIMAB, LLC

Owner name: ALBERT EINSTEIN COLLEGE OF MEDICINE