WO2010082134A1 - Anticorps combinés destinés au traitement et à la prévention d'une maladie provoquée par bacillus anthracis, ainsi que par les bactéries connexes et par leurs toxines - Google Patents

Anticorps combinés destinés au traitement et à la prévention d'une maladie provoquée par bacillus anthracis, ainsi que par les bactéries connexes et par leurs toxines Download PDF

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Publication number
WO2010082134A1
WO2010082134A1 PCT/IB2010/000146 IB2010000146W WO2010082134A1 WO 2010082134 A1 WO2010082134 A1 WO 2010082134A1 IB 2010000146 W IB2010000146 W IB 2010000146W WO 2010082134 A1 WO2010082134 A1 WO 2010082134A1
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antibody
seq
antibodies
anthracis
iqnlf
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PCT/IB2010/000146
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English (en)
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Herman Groen
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Iq Therapeutics Bv
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Priority to EP10706048A priority Critical patent/EP2387584A1/fr
Priority to CA2749572A priority patent/CA2749572A1/fr
Priority to US12/840,779 priority patent/US20110129460A1/en
Publication of WO2010082134A1 publication Critical patent/WO2010082134A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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 compositions and methods for the treatment and prevention of disease caused by Bacillus anthracis (anthrax) or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves, using a combination of at least two neutralizing monoclonal antibodies.
  • Bacillus anthracis the etiologic agent of anthrax
  • Bacillus anthracis is a gram-positive, rod shaped, aerobic and/or facultative anaerobic, spore-forming bacterium that can cause human disease via the gastrointestinal, cutaneous, or inhalation routes.
  • the incubation period usually varies from 12 hours to 5 days depending upon the dose received.
  • the onset can be longer following inhalation exposure and some reports suggest a delayed onset of several weeks in low dose exposure or following removal of therapeutic intervention.
  • the initial clinical signs and symptoms are nonspecific and may include malaise, headache, fever, nausea, and vomiting. These are followed by a sudden onset of respiratory distress with dyspnea, stridor, cyanosis and chest pain.
  • the onset of respiratory distress is followed by shock and death with high mortality.
  • Anthrax is considered a serious biological terrorist and military threat due to the highly lethal effects when exposure is by inhalation (approaching 100 percent lethality) and the stability of the B. anthracis spore.
  • the virulence of B. anthracis is based on two virulence factors: encapsulation (prevention of phagocytosis) and the production of two interlinked toxins, lethal toxin and edema toxin. Three exoprotein components, protective antigen (PA), lethal factor (LF), and edema factor (EF), interact to form the two toxins.
  • PA combines with lethal factor to produce lethal toxin and with edema factor to produce edema toxin.
  • PA binds to host cells and is cleaved, exposing binding sites for which lethal factor and edema factor compete.
  • the current consensus is that the cleaved PA forms a channel into the cell, allowing lethal toxin (PA-LF) or edema toxin (PA-EF) to enter.
  • PA-LF lethal toxin
  • PA-EF edema toxin
  • the PA monomer consists of four functional domains: domain 1 (residues 1-
  • Domain 1 the amino terminal domain, contains a furin protease cleavage site. Cleavage of Domain 1 releases a 20 kilodalton fragment (PA20) which triggers heptamerization of the remainder of the protein at the cell surface. Domain 2 assists in heptamerization and, along with domain III, forms a heptameric pore on the cell surface that allows binding of LF or EF, enabling endocytosis of the toxin complex into the cell. Domain 4 contains the host cell receptor binding site.
  • PA20 20 kilodalton fragment
  • lethal toxin is responsible for the majority of the tissue damage and systemic shock that occurs as the infection progresses, but the mechanism is not clearly understood. Internalization and translocation of the lethal factor into the cytosol occurs when the PA protein binds to it cell surface receptor.
  • the highly specific LF enzyme has four domains (1-4). Domain III has a hydrophobic core (282-382) and contains a five- tandem repeat 101 amino acid sequence. Assembly and cellular internalization of lethal toxin results in increased permeability to sodium and potassium ions followed by ATP hydrolysis which inhibits macromolecular synthesis and leads to cell death.
  • Bacteria other than B. anthracis may contain B. anthracis virulence genes.
  • other bacteria may contain genes that produce proteins homologous to those of B. anthracis for encapsulation and the production of toxins, such as PA, LF, and EF.
  • An example is the PA protein of Bacillus cereus G9241 and the homologous proteins of B. thuringiensis and C. perfringens (see Hoffmaster et al, Proc. Natl. Acad. Sci. U. S. A. (2004) 101 :8449-8454; Hoffmaster et al, J. Clin. Microbiol. (2006) 44:3352-3360; and Petosa et al., Nature (1997) 385:833-838).
  • AIGIV human polyclonal anthrax immunoglobulin derived from immunized donors.
  • AIGIV has a number of advantages. It provides instant protection, is likely to be effective during mid- to advanced- stage disease, is equally effective against antibiotic-resistant strains, results in minimal adverse reactions, has a prolonged serum half-life, and targets multiple epitopes, making it difficult to subvert its efficacy.
  • AIGIV suffers from several serious drawbacks that prevent its usefulness on a large scale.
  • AIGIV therapy requires the maintenance of stocks of antibodies having high toxin neutralization activity. These stocks must be obtained from an immunologically diverse population of donors, and must be constantly renewed.
  • the present invention provides an alternative approach which utilizes a combination of antibodies with neutralizing activity against both protective antigen and lethal factor for the prevention and treatment of disease caused by Bacillus anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves.
  • the present invention provides methods and compositions for the treatment and prevention of disease caused by bacterial infection, particularly infection by B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves.
  • the methods and compositions of the invention comprise a combination of at least two neutralizing antibodies, preferably monoclonal antibodies, most preferably human monoclonal antibodies, each of which binds to a different bacterial antigen.
  • the antigens are selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homo log of any of the foregoing.
  • the methods and compositions of the invention offer enhanced protection against infection when administered prophylactically and provide an increased probability of survival when administered therapeutically.
  • the approach of combining at least two antibodies having different antigen specificities provides broader protection than a single antibody or single antigen approach.
  • the methods and compositions of the invention are more likely than single antibody approaches to be effective against B. anthracis, including variations in bacterial strains and escape mutants, as well as against other bacteria which produce toxins or toxin components homologous to those produced by B. anthracis.
  • the methods and compositions of the invention advantageously extend the treatment window for subjects exposed to B. anthracis, or to bacteria which produce toxins or toxin components homologous to those produced by B.
  • compositions and methods of the invention also provide significant cost reductions and reduced health risks compared to mass vaccination strategies because the present invention targets treatment to those who have been exposed or are likely to be exposed to B. anthracis toxins, toxin components, or homologs thereof.
  • the invention provides a method for the treatment of disease caused by B.
  • anthracis toxins, toxin components, or homologs thereof, in a subject in need of such treatment comprising administering to the subject at least two neutralizing monoclonal antibodies, or antigen binding fragments thereof, wherein each of the antibodies has affinity for a different bacterial antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homolog of any of the foregoing.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • one of the at least two antibodies has affinity for an epitope of PA within domain 4 of PA, most preferably within amino acid residues 679-693 of domain 4.
  • one of the at least two antibodies has affinity for an epitope of LF within domain 1 of LF.
  • the disease is caused by a bacteria.
  • the disease is caused by a bacteria selected from the group consisting of B. anthracis, B. cereus, B. thuringiensis, and C. perfringens.
  • the disease is caused by a bacterium, or a combination of different bacteria which produce one or more proteins homologous to one or more of the PA, LF, and EF proteins of B. anthracis.
  • the disease results from toxemia caused by one or more bacterial toxins comprising one or more of PA, LF and EF, or a homolog of any of the foregoing.
  • toxemia may occur in the presence or absence of bacteria.
  • the antibodies are human monoclonal antibodies. In another embodiment, the antibodies are humanized monoclonal antibodies.
  • the affinity (K a ) of each antibody for its antigen is from
  • each antibody for its antigen is from 10 9
  • one of the at least two antibodies is an anti-PA antibody which neutralizes the protective antigen protein of B. anthracis, or a homo log thereof.
  • the anti-PA antibody competitively inhibits the binding of the protective antigen protein of B. anthracis, or a homo log thereof, to the monoclonal antibody IQNPA.
  • the anti-PA antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 17 or 18 to the monoclonal antibody IQNPA.
  • the anti-PA antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : KKPGA (SEQ ID NO:5); VH
  • CDR2 SNAIQWVRQAPGQRLEW (SEQ ID NO:6); and VH CDR3: YMELSSLR (SEQ ID NO:6);
  • the anti-PA antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence:
  • VL CDRl LTQSPGTLSLS (SEQ ID NO:8); VL CDR2: SYSSLAW (SEQ ID NO:9); and
  • VL CDR3 GPDFTLTIS (SEQ ID NO:10).
  • the anti-PA antibody comprises six CDRs, each comprising the following amino acid sequence: VH
  • VH CDR2 SEQ ID NO:6
  • VH CDR3 SEQ ID NO:7
  • VL CDRl SEQ ID NO:8
  • VL CDR2 SEQ ID NO:9
  • VL CDR3 SEQ ID NO:10.
  • the anti-PA antibody is the human monoclonal antibody IQNPA.
  • one of the at least two antibodies is an anti-LF antibody which neutralizes the lethal factor protein of B. anthracis, or a homo log thereof.
  • the anti-LF antibody competitively inhibits the binding of the lethal factor protein of B. anthracis, or a homo log thereof, to the monoclonal antibody IQNLF.
  • the anti-LF antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 19 to the monoclonal antibody IQNLF.
  • the anti-LF antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : VQPGG (SEQ ID NO: 11), VH
  • the anti-LF antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : TQSPDFQSVSP (SEQ ID NO:14), VL CDR2: SSLHWYQ (SEQ ID NO: 15), and VL CDR3: DFTLTINSL (SEQ ID NO: 16).
  • VL variable light chain domain
  • the antibody comprises six CDRs, each comprising the following amino acid sequence: VH CDRl : SEQ ID NO:11, VH CDR2: SEQ ID NO:12, VH CDR3: SEQ ID NO: 13, VL CDRl : SEQ ID NO:14, VL CDR2: SEQ ID NO:15, and VL CDR3: SEQ ID NO:16.
  • the anti-LF antibody is the human monoclonal antibody IQNLF.
  • one of the at least two neutralizing monoclonal antibodies is an anti-PA antibody which neutralizes the protective antigen protein of B. anthracis, or a homolog thereof, and the other antibody is an anti-LF antibody which neutralizes the lethal factor protein of B. anthracis, or a homolog thereof.
  • the antibodies are the human monoclonal antibodies, IQNPA and IQNLF.
  • each antibody is administered at a dose of from 1 to 20 mg/kg body weight of the subject. In another embodiment, one antibody is administered at a dose of from 1 to 10 mg/kg body weight of the subject. In another embodiment, one antibody is administered at a dose of from 2.5 to 15 mg/kg body weight of the subject. In one embodiment, the doses of the at least two antibodies are administered separately. In another embodiment, the doses of the at least two antibodies are administered at substantially the same time. In certain embodiments, each dose is in a separate composition. In other embodiments, the doses are contained in the same composition.
  • the antibodies are administered to the subject after the subject's exposure to B. anthracis toxins, toxin components, or homologs thereof. In one embodiment, the antibodies are administered to the subject between 0 and 48 hours after the subject's exposure to B. anthracis toxins, toxin components, or homologs thereof. In another embodiment, the antibodies are administered to the subject 48 hours after the subject's exposure to B. anthracis toxins, toxin components, or homologs thereof. Such exposure may be in the form of exposure to B. anthracis or to a bacterium that produces toxins or toxin components homologous to those produced by B. anthracis. In an alternative embodiment, such exposure is in the form of exposure to the B.
  • the method further comprises administering to the subject an antibacterial agent.
  • the antibacterial agent is levofloxacin, ciprofloxacin, or doxycycline.
  • the invention also provides a method for the prevention of disease caused by
  • B. anthracis toxins, toxin components, or homo logs thereof, in a subject in need of such prevention comprising administering to the subject at least two neutralizing monoclonal antibodies, or antigen binding fragments thereof, wherein each of the antibodies has affinity for a different bacterial antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homo log of any of the foregoing, and wherein the antibodies are administered prior to the subject's exposure to the B. anthracis toxins, toxin components, or homologs thereof.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • the disease caused by a bacteria In one embodiment, the disease is caused by a bacteria selected from the group consisting of B. anthracis, B. cereus, B. thuringiensis, and C. perfringens. In other embodiments, the disease is caused by a bacterium, or a combination of different bacteria, which produce factors homologous to one or more of the PA, LF, and EF proteins of B. anthracis. In another embodiment, the disease results from toxemia caused by one or more bacterial toxins comprising one or more of PA, LF and EF, or a homo log of any of the foregoing. In accordance with this embodiment, toxemia may occur in the presence or absence of bacteria. [29] In one embodiment, the antibodies are human monoclonal antibodies. In another embodiment, the antibodies are humanized monoclonal antibodies. [30] In one embodiment, the affinity (K a ) of each antibody for its antigen is from
  • the affinity (Ka) of each antibody for its antigen is from 10 9 M ⁇ tO lO 10 M "1 .
  • one of the at least two antibodies is an anti-PA antibody which neutralizes the protective antigen protein of B. anthracis, or a homo log thereof.
  • the anti-PA antibody competitively inhibits the binding of the protective antigen protein of B. anthracis, or a homolog thereof, to the monoclonal antibody IQNPA.
  • the anti-PA antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 17 or 18 to the monoclonal antibody IQNPA.
  • the anti-PA antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : KKPGA (SEQ ID NO:5); VH CDR2: SNAIQWVRQAPGQRLEW (SEQ ID NO:6); and VH CDR3: YMELSSLR (SEQ ID NO: 7).
  • VH CDRl variable heavy chain domain
  • KKPGA SEQ ID NO:5
  • VH CDR2 SNAIQWVRQAPGQRLEW
  • VH CDR3 YMELSSLR
  • the anti-PA antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : LTQSPGTLSLS (SEQ ID NO:8); VL CDR2: SYSSLAW (SEQ ID NO:9); and VL CDR3: GPDFTLTIS (SEQ ID NO:10).
  • VL CDRl LTQSPGTLSLS
  • VL CDR2 SYSSLAW
  • VL CDR3 GPDFTLTIS (SEQ ID NO:10).
  • the anti-PA antibody comprises six CDRs, each comprising the following amino acid sequence: VH CDRl : SEQ ID NO:5, VH CDR2: SEQ ID NO:6, VH CDR3: SEQ ID NO:7, VL CDRl : SEQ ID NO:8, VL CDR2: SEQ ID NO:9, and VL CDR3: SEQ ID NO:10.
  • the anti-PA antibody is the human monoclonal antibody IQNPA.
  • one of the at least two antibodies is an anti-LF antibody which neutralizes the lethal factor protein of B. anthracis, or a homo log thereof.
  • the anti-LF antibody competitively inhibits the binding of the lethal factor protein of B. anthracis, or a homo log thereof, to the monoclonal antibody IQNLF.
  • the anti-LF antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 19 to the monoclonal antibody IQNLF.
  • the anti-LF antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : VQPGG (SEQ ID NO: 11), VH CDR2: SYAMSWVRQAPGKGLEW (SEQ ID NO: 12), and VH CDR3: YMQMNSL (SEQ ID NO: 13).
  • VH variable heavy chain domain
  • CDR VQPGG
  • VH CDR2 SYAMSWVRQAPGKGLEW
  • VH CDR3 YMQMNSL
  • the anti-LF antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : TQSPDFQSVSP (SEQ ID NO:14), VL CDR2: SSLHWYQ (SEQ ID NO: 15), and VL CDR3: DFTLTINSL (SEQ ID NO: 16).
  • VL CDRl TQSPDFQSVSP
  • VL CDR2 SSLHWYQ
  • VL CDR3 DFTLTINSL
  • the antibody comprises six CDRs, each comprising the following amino acid sequence: VH CDRl : SEQ ID NO:11, VH CDR2: SEQ ID N0:12, VH CDR3: SEQ ID NO: 13, VL CDRl : SEQ ID NO:14, VL CDR2: SEQ ID NO:15, and VL CDR3: SEQ ID NO:16.
  • the anti-LF antibody is the human monoclonal antibody IQNLF.
  • one of the at least two monoclonal antibodies is an anti-
  • each antibody is administered at a dose of from 1 to 20 mg/kg body weight of the subject. In another embodiment, one antibody is administered at a dose of from 1 to 10 mg/kg body weight of the subject. In another embodiment, one antibody is administered at a dose of from 2.5 to 15 mg/kg body weight of the subject. In one embodiment, the doses of the at least two antibodies are administered separately. In another embodiment, the doses of the at least two antibodies are administered at substantially the same time. In certain embodiments, each dose is in a separate composition. In other embodiments, the doses are contained in the same composition.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least two neutralizing monoclonal antibodies, or antigen binding fragments thereof, wherein each of the antibodies has affinity for a different bacterial antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homo log of any of the foregoing, and a pharmaceutically acceptable excipient or carrier.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • the composition comprises an anti-PA antibody which neutralizes the protective antigen protein of B. anthracis, or a homo log thereof.
  • the anti-PA antibody competitively inhibits the binding of the protective antigen protein of B. anthracis, or a homo log thereof, to the monoclonal antibody IQNPA.
  • the anti-PA antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 17 or 18 to the monoclonal antibody IQNPA.
  • the anti-PA antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : KKPGA (SEQ ID NO:5); VH CDR2:
  • the anti-PA antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : LTQSPGTLSLS (SEQ ID NO:8); VL CDR2: SYSSLAW (SEQ ID NO:9); and VL CDR3: GPDFTLTIS (SEQ ID NO: 10).
  • VL variable light chain domain
  • the anti-PA antibody comprises six CDRs, each comprising the following amino acid sequence: VH CDRl : SEQ ID NO:5, VH CDR2: SEQ ID NO:6, VH CDR3: SEQ ID NO:7, VL CDRl : SEQ ID NO:8, VL CDR2: SEQ ID NO:9, and VL CDR3: SEQ ID NO:10.
  • the anti-PA antibody is the human monoclonal antibody IQNPA.
  • the composition comprises an anti-LF antibody which neutralizes the lethal factor protein of B. anthracis, or a homolog thereof.
  • the anti-LF antibody competitively inhibits the binding of the lethal factor protein of B. anthracis, or a homo log thereof, to the monoclonal antibody IQNLF.
  • the anti-LF antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 19 to the monoclonal antibody IQNLF.
  • the anti-LF antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : VQPGG (SEQ ID NO: 11), VH CDR2: SYAMSWVRQAPGKGLEW (SEQ ID NO: 12), and VH CDR3: YMQMNSL (SEQ ID NO: 13).
  • VH variable heavy chain domain
  • CDR VQPGG
  • VH CDR2 SYAMSWVRQAPGKGLEW
  • VH CDR3 YMQMNSL
  • the anti-LF antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : TQSPDFQSVSP (SEQ ID NO:14), VL CDR2: SSLHWYQ (SEQ ID NO: 15), and VL CDR3: DFTLTINSL (SEQ ID NO: 16).
  • VL CDRl TQSPDFQSVSP
  • VL CDR2 SSLHWYQ
  • VL CDR3 DFTLTINSL
  • the antibody comprises six CDRs, each comprising the following amino acid sequence: VH CDRl : SEQ ID NO:11, VH CDR2: SEQ ID NO:12, VH CDR3: SEQ ID NO: 13, VL CDRl : SEQ ID NO:14, VL CDR2: SEQ ID NO:15, and VL CDR3: SEQ ID NO:16.
  • the composition comprises the monoclonal IQNPA antibody and the monoclonal IQNLF antibody.
  • one of the at least two neutralizing monoclonal antibodies in the composition is an anti-PA antibody which neutralizes the protective antigen protein of B. anthracis, or a homolog thereof, and the other antibody is an anti-LF antibody which neutralizes the lethal factor protein of B. anthracis, or a homolog thereof.
  • the antibodies are the human monoclonal antibodies, IQNPA and IQNLF.
  • the composition further comprises at least one antibacterial agent.
  • the at least one antibacterial agent is selected from ciprofloxacin, doxycycline, or levofloxacin.
  • Figure 1 Kaplan-Meier curves representing time-to-death and survival data for each group of animals in Example 1.4 (Pre-Exposure Efficacy).
  • Figure 2 Pharmacokinetic profiles of IQNPA and IQNLF antibodies as determined in diluted rabbit serum. Concentrations were back-calculated from the ELISA values using a 4 parameter fit method and then expressed as ng/mL in 100% rabbit serum.
  • Figure 3 Kaplan-Meier curves representing time-to-death and survival data for each group of animals in Example 1.5 (Post-Exposure Efficacy - Experiment 1).
  • IQNPA red, Groups 1-3; IQNLF: blue, Groups 4-6; IQNPA+IQNLF: green, Groups 8-12; control: gray, Group 7.
  • Figure 4 Kaplan-Meier curves representing time-to-death and survival data for each group of animals in Example 1.6 (Post-Exposure Efficacy - Experiment 2).
  • Figure 6 Estimated logistic regression curves for the IQNLF and combined treatments in Example 1.6. Points show the proportion of animals that survived for each group.
  • Figure 8 Kaplan-Meier curves representing time-to-death and survival data for each group in Example 1.7.
  • the methods and compositions of the invention offer enhanced protection against bacterial infection or toxemia (which may occur in the presence or absence of a bacterial infection) caused by B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves, when administered before exposure and provide an increased probability of survival when administered following exposure to the bacteria, bacterial toxins, or their component proteins.
  • the invention combines at least two neutralizing monoclonal antibodies, each having a different antigen specificity.
  • each of the at least two antibodies has affinity for a different bacterial antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • At least one of the antibodies binds to an epitope of the PA protein of B. anthracis, or a homo log thereof, that includes one or more amino acids within one of the following groups of amino acids (with reference to Genebank Accession No. P13423): Group 1 (amino acids 121-150); Group 2 (amino acids 143-158); Group 3 (amino acids 421-440); Group 4 (amino acids 339-359) and Group 5 (amino acids 678-697).
  • At least one of the antibodies binds to an epitope of PA that includes one or more amino acids within at least one of the following groups of amino acids (with reference to Genebank Accession No. P13423): Group 6 (Phe-342, Phe-343, Asp-344); Group 7 (Trp-375, Met-379, and Leu-381); Group 8 (Phe-581, Phe-583, Ile-591, Leu-595, and Ile-603); Group 9 (Pro-213, Leu-216, Phe-231, Leu-232, Pro-234, Ile-236, Ile-239, Trp- 255, and Phe-265) and Group 10 (Asn-686 and any residue from Lys-708 to Asn-722).
  • At least one of the antibodies binds to an epitope of LF that includes one or more amino acids within any of domains 1 to 4 of the LF protein of B. anthracis, or a homo log thereof.
  • Epitopes may comprise or consist of one or more linear polypeptide fragments of a protein.
  • neutralizes or “neutralizing” in the context of antibodies against a bacterium, or against a bacterial toxin or its component, means that the antibody inhibits the ability of the bacterium or the toxin to cause disease.
  • the neutralizing activity of an antibody derives from its ability to bind to a bacterial antigen, particularly a bacterial protein necessary for virulence.
  • the antibodies may neutralize, for example, by preventing or reversing the assembly of toxin components to form a functional toxin, or by disabling the toxin or toxin component from exerting its biological activity.
  • an antibody may inhibit cleavage of the PA monomer, or it may inhibit the formation of the PA heptamer, or the antibody may block the binding of LF or EF to the PA heptamer.
  • the neutralizing activity of an antibody can be measured, for example, as the ability of the antibody to block entry of the bacteria into cells, to block replication of the bacteria within cells, to enhance the uptake and/or intracellular killing of the bacteria by cells of the immune system, such as macrophages, as well as the ability of the antibody to prevent or ameliorate the clinical symptoms of disease caused by bacterial infection and/or toxemia in a mammal.
  • the neutralizing activity of an antibody against a bacterial toxin can also be measured more directly, for example, using a toxin neutralization assay.
  • a toxin neutralization assay Such assays are known in the art and are described, for example in Albrecht et al, Infect. Immunity, (2007) 75:5425-5433 and Li et al, J. Immunol. Methods, (2008) 333:89-106.
  • the term "homolog” refers to a protein having an amino acid sequence which differs from the sequence of the corresponding B. anthracis protein, PA, LF, or EF, but in which the differences are such that the protein retains the function and/or antigenic character of the corresponding B. anthracis protein.
  • a homolog of PA, LF, or EF may be produced by a bacteria other than B. anthracis. Homology is typically determined on the basis of sequence similarity or sequence identity.
  • a homologous protein is one which shares at least 70%, at least 80%, at least 90%, or at least 95% sequence identity over its entire length to a B. anthracis protein selected from PA, LF, and EF. Most preferably, the homolog is at least 98% identical over its entire length to the corresponding B. anthracis protein. In other embodiments, the homologous protein shares high sequence identity to a B. anthracis protein selected from PA, LF, and EF, over one or more regions smaller than its entire length.
  • a PA homolog shares at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the PA protein of B. anthracis in one or more functional domains selected from the group consisting of domain 1 (residues 1-258), domain 2 (residues 259-487), domain 3 (residues 488-595), and domain 4 (residues 596-735), with reference to the amino acid sequence of the PA protein of B. anthracis given in GENBANK ACCESSION NO: P 13423.
  • an LF homolog shares at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the LF protein of B. anthracis in one or more functional domains selected from the group consisting of domain 1, 2, 3, and 4, with reference to the amino acid sequence of the LF protein of B. anthracis given in GENBANK ACCESSION NO: YP O 16503.
  • derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified amino acid.
  • Derivatives or analogs include, e.g. , molecules including regions that are substantially homologous to the PA, LF, or EF proteins, in various embodiments, by at least about 70%, 80%, or 95%, 98%, or even 99% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done using sequence analysis software, such as, for example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, with the default parameters therein.
  • non-identical positions are preferably, but not necessarily, conservative substitutions of the corresponding residue(s) in the reference sequence.
  • Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Conservative amino acid changes also refer to changes between amino acids of broadly similar molecular properties, e.g, substitutions within the aliphatic group alanine, valine, leucine and isoleucine.
  • a substitution of glycine for an aliphatic amino acid is also a conservative substitution.
  • Other conservative substitutions include those within the sulfur- containing group methionine and cysteine.
  • Preferred conservative substitution groups are aspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine; and lysine-arginine.
  • a substitution other than a conservative amino acid substitution is made outside of a functional domain of the reference protein, e.g., outside of domains 1-4 of either PA or LF.
  • a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide.
  • a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide, which is 50% identical to the reference polypeptide over its entire length.
  • other polypeptides will meet the same criteria.
  • bacterial strains other than B. anthracis may contain B. anthracis virulence genes.
  • other bacterial strains may contain genes that produce virulence proteins which are the same or homologous to those proteins of B. anthracis which are responsible for virulence.
  • other bacterial strains may produce proteins identical or homologous to the PA, LF, or EF proteins produced by B. anthracis.
  • the antibodies for use in the methods and compositions of the invention include antibodies that neutralize bacteria other than B. anthracis.
  • the dual antibody approach of the present invention can thus be used in the prophylaxis and treatment of disease caused by such other bacteria, including, but not limited to, B.
  • the antibodies for use in the methods and compositions of the invention bind to at least one, and most preferably two, of the B. anthracis toxin components, PA, LF, and EF, or a homo log of any of the foregoing.
  • the methods and compositions of the invention provide a combination of at least two antibodies, each antibody having affinity for a different antigen selected from the B.
  • anthracis toxin components PA, LF, and EF, or a homo log of any of the foregoing.
  • at least one antibody has affinity for PA, or a homolog thereof, and another antibody has affinity for LF, or a homolog thereof.
  • the antibodies for use in the methods and compositions of the invention are monoclonal antibodies.
  • the terms "antibody” and “antibodies” refer to fully human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, CDR-grafted antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, and antigen-binding fragments of any of the foregoing.
  • the antibodies include immunoglobulin molecules and antigen-binding active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Such fragments may or may not be fused to another immunoglobulin domain including, but not limited to, an Fc region or fragment thereof.
  • fusion products may be generated, including but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.
  • Immunoglobulin molecules can be of any type, including, IgG, IgE, IgM, IgD, IgA and IgY, and of any class, including IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ), or of any subclass.
  • the monoclonal antibodies for use in the methods and compositions of the invention are IgG antibodies.
  • the antibodies for use in the methods and compositions of the invention bind to an antigen selected from PA, LF, or EF, or homologs thereof.
  • an antibody for use in the methods and compositions of the invention binds with high affinity to the protective antigen (PA) or the lethal factor protein (LF) of B. anthracis, B. cereus, B. thuringiensis, C. perfringens, or a homolog of any of the foregoing.
  • PA protective antigen
  • LF lethal factor protein
  • Affinity is a measure of the strength of binding between an antibody and an antigen. Affinity can be expressed in several ways. One way is in terms of the dissociation constant (IQ) of the interaction. IQ can be measured by routine methods, include equilibrium dialysis or by directly measuring the rates of antigen-antibody dissociation and association, the koff and k on rates, respectively (see e.g., Nature, 1993 361 :186-87). The ratio of k o ff/k on cancels all parameters not related to affinity, and is equal to the dissociation constant IQ (see, generally, Davies et ah, Annual Rev Biochem, 1990 59:439-473). Thus, a smaller Kd means a higher affinity.
  • a high affinity antibody for use in the compositions and methods of the invention is an antibody that binds to an antigen of B. anthracis with a K d in the picomolar (pM, 10 ⁇ 12 M) or nanomolar (nM, 10 ⁇ 9 M) range, or with a K a of at least 10 7 M “1 or, preferably, from 10 9 M "1 to 10 10 M "1 .
  • the antibody binds with a IQ of from 1 to 100 pM, from
  • the antibody binds with a IQ from 1 to 100 nM, from 100 to 250 nM, from 250 to 500 nM, or from 500 to 1000 nM.
  • the antibody binds with a IQ from 1 to 200 pM or from 1 to 200 nM.
  • the antibody binds to the antigen with an affinity constant (IQ) of at least 10 7 M “1 , preferably with a IQ of from 10 7 M “1 to 10 8 M “1 , from 10 8 M “1 to 10 9 M “1 , from 10 9 M “1 to 10 10 M “1 , or from 10 10 M “1 to 10 11 M “1 .
  • IQ affinity constant
  • at least one antibody of the combination binds to its antigen with an affinity of from 10 9 M "1 to 10 10 M "1 .
  • the monoclonal antibodies useful in the methods and compositions of the invention include chimeric, human, and humanized antibodies, and antigen-binding fragments thereof, which exhibit low toxicity when administered to a subject, preferably a human subject.
  • Toxicity in the context of antibody therapy in a human subject includes, for example, a human anti-murine antibody response (where the antibody is murine) and a human anti-chimeric antibody response (where the antibody is chimeric).
  • the antibodies are monoclonal human or humanized antibodies, or antigen-binding fragements thereof.
  • Antigen-binding fragments of the antibodies include, for example, Fab, Fab',
  • F(ab')2 and Fv fragments are produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • an antigen-binding fragment is a dimer of heavy chains (a camelised antibody), a single-chain Fvs (scFv), a disulfide-linked Fvs (sdFv), a Fab fragment, or a F(ab') fragment.
  • the antibodies for use in the methods and compositions of the invention are monoclonal antibodies.
  • a monoclonal antibody is derived from a substantially homogeneous population of antibodies specific to a particular antigen, which population contains substantially similar epitope binding sites.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof.
  • Methods for monoclonal antibody production are well known in the art.
  • a monoclonal antibody for use in the methods and compositions of the invention is produced using hybridoma technology.
  • a human antibody is one in which all of the sequences arise from human genes.
  • Human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring, which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Human antibodies can also be derived from phage display of human antibody fragments.
  • phage display methods functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding variable heavy and variable light domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues).
  • the DNA encoding the variable heavy and variable light domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector.
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • the phage used in these methods are typically filamentous phage including fd and Ml 3.
  • Phage expressing an antigen binding domain that binds to the antigen epitope of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Examples of phage display methods include those disclosed in Brinkman et al, 1995, J. Immunol. Methods 182:41-50; Ames et al, 1995, J. Immunol. Methods 184:177; Kettleborough et al, 1994, Eur. J. Immunol. 24:952-958; Persic et al, 1997, Gene 187:9; Burton et al, 1994, Adv.
  • a humanized antibody is an antibody which comprises a framework region having substantially the same amino acid sequence as human receptor immunoglobulin and a complementarity determing region ("CDR") having substantially the same amino acid sequence as a non-human donor immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fv) in which all or substantially all of the CDR regions correspond to those of the non-human donor immunoglobulin ⁇ i.e., the donor antibody) and all or substantially all of the framework regions of the human acceptor immunoglobulin.
  • the acceptor may comprise or consist of a consensus sequence of human immunoglobulins.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibody will contain a light chain and at least the variable domain of a heavy chain.
  • the antibody also may include the CHl, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the donor and acceptor sequences, e.g., the donor CDR or the acceptor framework may be mutagenized by substitution, insertion or deletion of at least one residue. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the acceptor framework and donor CDR sequences, more often 90%, and most preferably greater than 95%.
  • a humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (see e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Patent Nos.
  • a chimeric antibody comprises non-human variable region sequences and human constant region sequences.
  • a chimeric antibody may be monovalent, divalent or polyvalent.
  • a monovalent chimeric antibody is a dimer formed by a chimeric heavy chain associated through disulfide bridges with a chimeric light chain.
  • a divalent chimeric antibody is a tetramer formed by two heavy-light chain dimers associated through at least one disulfide bridge.
  • a polyvalent chimeric antibody can also be produced, for example, by employing a heavy chain constant region that aggregates (e.g., from an IgM heavy chain).
  • a "camelised” antibody is one having a functional antigen binding site comprising only the heavy chain variable domains (VH), rather than the conventional antigen binding site which comprises both the heavy and the light chain variable domains (VL).
  • a camelised antibody comprises one or two VH domains and no VL domains.
  • a camelised antibody comprises two VH domains.
  • Methods for making camelised antibodies are known in the art. See, for example, Riechmann et al., J. Immunol. Methods, 1999 231 :25-38, and U.S. Patent Application Publication Nos. US 2004137570 and US 2004142432.
  • the antibodies for use in the methods and compositions of the invention may be produced by recombinant expression using techniques known in the art.
  • the nucleic acid sequences used for recombinant expression are those described in U.S. Patent Application Publication No. 20060258842, published November 16, 2006, and in Albrecht et al., Infection and Immunity 2007 75:5425-5433.
  • a combination of at least two antibodies is administered to a subject in need of treatment or prevention of disease caused by B. anthracis, or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves.
  • the antibodies of the combination may bind to the same or a different bacterial antigen, however at least two antibodies of the combination bind to a different bacterial antigen.
  • each of the at least two antibodies binds to a different antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homo log of any of the foregoing.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • the antibodies suitable for use in the methods and compositions of the invention are preferably human monoclonal antibodies.
  • Human monoclonal antibodies suitable for use in the claimed methods include the anti-PA and anti-LF antiobides described, for example, in U.S. Patent Application Publication No. 20060258842, published November 16, 2006, and in Albrecht et al, Infection and Immunity 2007 75:5425-5433.
  • at least one antibody is an anti-PA antibody which binds to the protective antigen (PA) of B.
  • the antibody binds to PA with a Ka of from 10 9 M ⁇ tO 10 10 M “1 , or from 10 10 M ⁇ tO 10 11 M “1 .
  • at least one antibody is an anti-EF antibody which binds to the edema factor protein (EF) of B.
  • anthracis or a homo log thereof, with an affinity (K a ) of at least 10 7 M “1 , preferably with a K 3 of from 10 7 M ⁇ tO 10 8 M “1 , from 10 8 M “ ⁇ 10 9 M “1 , from 10 9 M “1 to 10 10 M “1 , or from 10 10 M “1 to 10 11 M “1 .
  • the antibody binds to EF with a K a of from 10 9 M “1 to 10 10 M “1 , or from 10 10 M ⁇ tO 10 11 M “1 .
  • At least one antibody is an anti-LF antibody which binds to the lethal factor protein (LF) of B. anthracis, or a homolog thereof, with an affinity (K 3 ) of at least 10 7 M “1 , preferably with a K a of from 10 7 M “1 to 10 8 M “1 , from 10 8 M “1 to 10 9 M “1 , from 10 9 M “1 to 10 10 M “1 , or from 10 10 M “1 to 10 11 M “1 .
  • the antibody binds to LF with a Ka of from 10 9 M ⁇ tO 10 10 M "1 , or from 10 10 M ⁇ tO 10 11 M “1 .
  • the antibodies of the combination are the antibodies IQNPA and IQNLF described in U.S. Patent Application Publication No. 20060258842, published November 16, 2006, and in Albrecht et ah, Infection and Immunity 2007 75:5425-5433.
  • the IQNPA antibody binds to the B. anthracis protective antigen (PA), specifically to domain IV of the PA protein.
  • the IQNLF antibody binds to B. anthracis lethal factor (LF), specifically to domain I of the LF protein.
  • PA B. anthracis protective antigen
  • LF B. anthracis lethal factor
  • Hybridoma fusions were screened for expression of anti-PA- and anti-LF-specific antibodies by enzyme-linked immunosorbent assays (ELISAs).
  • ELISAs enzyme-linked immunosorbent assays
  • Candidate anti-PA and anti-LF antibodies were isotyped using a human Ig subclass ELISA kit (Invitrogen, Carlsbad, CA).
  • the IQNPA and IQNLF antibodies are produced by stable hybridoma cell lines designated and , respectively. The hybridomas and , were deposited on , pursuant to the requirements of the Budapest
  • IQNLF antibodies are provided below. [86] >IQNPA H.gamma. amino acid sequence: (SEQ ID NO: 1)
  • At least one antibody of the combination is an anti-PA antibody which neutralizes the protective antigen (PA) and comprises a heavy chain amino acid sequences comprising SEQ ID NO: 1.
  • the anti-PA antibody comprises a light chain amino acid sequence comprising SEQ ID NO: 2.
  • the anti-PA antibody comprises a heavy chain amino acid sequence comprising SEQ ID NO: 1 and a light chain amino acid sequence comprising SEQ ID NO: 2.
  • at least one antibody of the combination is an anti-LF antibody which neutralizes the lethal factor protein (LF) and comprises a heavy chain amino acid sequence comprising SEQ ID NO: 3.
  • the anti-LF antibody comprises a light chain amino acid sequence comprising SEQ ID NO: 4.
  • the anti-LF antibody comprises a heavy chain amino acid sequence comprising SEQ ID NO: 3 and a light chain amino acid sequence comprising SEQ ID NO: 4.
  • the anti-PA antibody comprises a variable heavy chain domain (VH) having three complementarity determining regions (CDR), each CDR comprising the following amino acid sequence: VH CDRl : KKPGA (SEQ ID NO:5); VH CDR2: SNAIQWVRQAPGQRLEW (SEQ ID NO:6); and VH CDR3: YMELSSLR (SEQ ID NO: 7).
  • VH CDRl variable heavy chain domain
  • KKPGA SEQ ID NO:5
  • VH CDR2 SNAIQWVRQAPGQRLEW
  • VH CDR3 YMELSSLR
  • the anti-PA antibody comprises a variable light chain domain (VL) having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : LTQSPGTLSLS (SEQ ID NO:8); VL CDR2: SYSSLAW (SEQ ID NO:9); and VL CDR3: GPDFTLTIS (SEQ ID NO:10).
  • VL variable light chain domain
  • the anti-PA antibody comprises all six of the preceding CDRs.
  • the anti-LF antibody comprises a VH domain having three CDRs, each CDR comprising the following amino acid sequence: VH CDRl : VQPGG (SEQ ID NO:11); VH CDR2: SYAMSWVRQ APGKGLEW (SEQ ID NO: 12); and VH CDR3: YMQMNSL (SEQ ID NO:13).
  • the anti-LF antibody comprises a VL domain having three CDRs, each CDR comprising the following amino acid sequence: VL CDRl : TQSPDFQSVSP (SEQ ID NO: 14); VL CDR2: SSLHWYQ (SEQ ID NO: 15); and VL CDR3: DFTLTINSL (SEQ ID NO: 16).
  • the anti-LF antibody comprises all six of the preceding CDRs.
  • the anti-PA antibody binds to a protective antigen (PA) polypeptide comprising or consisting of the following amino acid sequence: NNIAVGADES VVKEAHREVI NSSTEGLLLN IDKDIRKILS GYIVEIEDTE GLKEVINDRYDMLNISSLRQ DGKTFIDFKK YNDKLPLYIS NPNYKVNVYA VTKENTIINP SENGDTSTNG IKKILIFSKK GYEIG (SEQ ID NO: 17).
  • PA protective antigen
  • the anti-PA antibody binds to a protective antigen (PA) polypeptide comprising or consisting of the following amino acid sequence: TNIYT VLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN SSTEGLLLNI DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY NDKLPLYISN PNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG (SEQ ID NO: 18).
  • PA protective antigen
  • the anti-PA antibody competitively inhibits the binding of the monoclonal antibody IQNPA to the protective antigen protein of B. anthracis, or a homolog thereof.
  • the anti-PA antibody competitively inhibits the binding of a polypeptide comprising SEQ ID NO: 17 or 18 to the monoclonal antibody IQNPA.
  • the anti-LF antibody binds to a lethal factor (LF) polypeptide comprising or consisting of the following amino acid sequence: ERNKTQEEHLK EIMKHIVKIE VKGEEAVKKE AAEKLLEKVP SDVLEMYKAI GGKIYIVDGD ITKHISLEAL SEDKKKIKDI YGKDALLHEH YVYAKEGYEP VLVIQSSEDY VENTEKALNV YYEIGKILSR DILSKINQPY QKFLDVLNTI KNASDSDGQD LLFTNQLKEH PTDFSVEFLE QNSNEVQEVF AKAFAYYIEP QHRDVLQLYA PEAFNYMDKF NEQEINLSLE ELKDQ (SEQ ID NO: 19).
  • LF lethal factor
  • the anti-LF antibody competitively inhibits the binding of the monoclonal antibody IQNLF to the lethal factor protein of B. anthracis, or a homolog thereof. In another embodiment, the anti-LF antibody competitively inhibits binding of the monoclonal antibody IQNLF to a polypeptide comprising SEQ ID NO: 19.
  • Methods for determining antibody specificity and affinity by competitive inhibition are known in the art, for example, such methods can be found in Harlow, et al. , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N. Y., (1992, 1993); and Muller, Meth. Enzymol. 92:589 601 (1983).
  • the antibodies for use in the methods and compositions of the invention are isolated or purified.
  • An “isolated” or “purified” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • antibody that is substantially free of cellular material includes preparations having less than about 30%, or about 20%, or about 10%, or about 5%, or about 1% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • the antibody When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, e.g., culture medium represents less than about 20%, or about 10%, or about 5%, or about 1% of the volume of the protein preparation.
  • culture medium represents less than about 20%, or about 10%, or about 5%, or about 1% of the volume of the protein preparation.
  • the antibody When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, e.g., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. Accordingly such preparations of antibody have less than about 30%, or about 20%, or about 10%, or about 5%, or about 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest.
  • compositions comprising a combination of at least two of the antibodies described above in Section 1.1.
  • a composition comprising the antibodies is suitable for administration to a human subject.
  • the composition is a pharmaceutical composition comprising at least two antibodies, an anti-PA antibody and an anti-LF antibody, and one or more pharmaceutically acceptable carriers or excipients.
  • the composition is formulated as a liquid. In another embodiment, the composition is lyophilized.
  • excipient broadly refers to a biologically inactive substance used in combination with the active agents, i.e., the antibodies, of the composition.
  • An excipient can be used, for example, as a solubilizing agent, a stabilizing agent, a surfactant, a demulcent, a viscosity agent, a diluent, an inert carrier, a preservative, a binder, a disintegrant, a coating agent, a flavoring agent, or a coloring agent.
  • at least one excipient is chosen to provide one or more beneficial physical properties to the composition, such as increased stability and/or solubility of the active agent(s).
  • a "pharmaceutically acceptable" excipient is one that has been approved by a state or federal regulatory agency for use in animals, and preferably for use in humans, or is listed in the U.S. Pharmacopia, the European Pharmacopia or another generally recognized pharmacopia for use in animals, and preferably for use in humans.
  • Examples of carriers that may be used in the compositions of the present invention include water, mixtures of water and water-miscible solvents, such as Cl- to Cl- alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water-soluble polymers, natural products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia, starch derivatives, such as starch acetate and hydroxypropyl starch, and also other synthetic products, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, such as neutral Carbopol, or mixtures of those polymers.
  • water-miscible solvents such as Cl- to Cl- alkanols
  • vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water-soluble polymers
  • natural products such as gelatin
  • excipients include certain inert proteins such as albumins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as aspartic acid (which may alternatively be referred to as aspartate), glutamic acid (which may alternatively be referred to as glutamate), lysine, arginine, glycine, and histidine; fatty acids and phospholipids such as alkyl sulfonates and caprylate; surfactants such as sodium dodecyl sulphate and polysorbate; nonionic surfactants such as such as TWEEN®, PLURONICS®, or a polyethylene glycol (PEG) designatied 200, 300, 400, or 600; a Carbowax designated 1000, 1500, 4000, 6000, and 10000; carbohydrates such as glucose, sucrose, mannose, maltose, trehalose, and dextrione, amino acids and amino acids such as aspartic acid (which may alternatively be referred to as aspartate),
  • the present invention provides methods for the prevention and treatment of disease caused by B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves, in a subject in need thereof by administering at least two neutralizing monoclonal antibodies to the subject, each having a different antigen specificity.
  • Each of the at least two antibodies has affinity for a different bacterial antigen selected from the protective antigen (PA), lethal factor (LF), and edema factor (EF) of B. anthracis, or a homo log of any of the foregoing.
  • at least one antibody is an anti-protective antigen (PA) antibody.
  • prophylactic treatment is administered to a subject either prophylactically or therapeutically.
  • a subject in need of prophylactic treatment is one who has been exposed to B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or to the toxins or toxin components themselves, but has not developed any clinical signs of infection (post-exposure prophylaxis), or one who is likely to be exposed in the near future (pre-exposure prophylaxis).
  • prophylactic treatment is administered to a subject who is asymptomatic following exposure.
  • prophylactic treatment is administered to a subject prior to exposure.
  • prophylactic treatment results in an inhibition or delay in the onset or progression of at least one clinical symptom associated with the bacterial infection.
  • a subject in need of therapeutic treatment is one who already presents with one or more clinical symptoms of bacterial infection.
  • therapeutic treatment is administered to a subject following exposure who presents with one or more clinical signs or symptoms of the bacterial infection or toxemia, which may occur in the absence of bacteria.
  • the invention also provides methods for increasing the survival odds for a subject who has been exposed to B. anthracis or a bacterium which produces toxins or toxin components homologous to the virulence factors produced by B. anthracis or to the toxins or toxin components themselves, in the absence of bacteria, by administering a combination of at least two antibodies to the subject, preferably an anti-PA antibody and an anti-LF antibody.
  • the antibodies are administered as part of a therapeutic regimen that includes antibiotics, preferably ciprofloxacin and/or doxycycline. Combination therapy with antibiotics is discussed in more detail below in Section 1.2.2.
  • the antibodies of the present invention can be administered to a subject either separately or together.
  • the antibodies are administered at the same time or at substantially the same time.
  • the subject may be any mammal, including, for example, a mouse, a rat, a rabbit, a dog, a pig, a non-human primate, or a human.
  • the subject is human.
  • one or more of the antibodies is administered in combination with one or more additional therapeutic agents, preferably one or more antibiotics, as described below in Section 1.2.2.
  • the dosage administered will vary depending upon known factors such as the pharmacodynamic characteristics of the particular antibodies, the mode and route of administration, and the age, health, and weight of the subject.
  • Dosage preferably reflects the total amount of antibody administered to the subject.
  • Exemplary doses include 1 to 20 mg of antibody per kg (mg/kg) of body weight or about 1 to 10 mg/kg of body weight.
  • the total amount of antibody administered is about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, about 12 mg/kg, about 14 mg/kg, about 16 mg/kg, about 18 mg/kg, or about 20 mg/kg of the subject's body weight.
  • the amount of each antibody administered will usually be different, and depends on the efficacy of the particular combination of antibodies. The effective amount of each antibody is determined using routine methods for determining optimal dose and efficacy.
  • the amount of each antibody administered will be in the range of 1 to 20 mg/kg, preferably 1 to 10 mg/kg, and most preferably 1 to 5 mg/kg body weight.
  • the antibody combination comprises the IQNPA and IQNLF antibodies, wherein the IQNPA antibody is administered at a dosage of from 1 to 5 mg/kg body weight and the IQNLF antibody is administered at a dosage of from 1 to 10 mg/kg.
  • the IQNPA antibody is administered at a dosage of 2.5 mg/kg.
  • the total amount of antibody administered is from 1 to 10 mg/kg.
  • the antibodies for use in the methods of the invention are preferably formulated for intravenous, intramuscular, or subcutaneous administration.
  • the antibodies are formulated for administration by injection through another route, such as intradermal or transdermal.
  • the antibodies are formulated for intravenous administration.
  • the antibodies may be formulated for any suitable route of administration.
  • An effective amount of the antibodies is the amount sufficient to reduce the severity of the disease caused by B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis, or disease caused by the toxins or toxin components themselves, the amount sufficient to prevent the incidence or advancement of the disease, or the amount sufficient to enhance or improve the therapeutic effect(s) of another therapy or therapeutic agent.
  • the effective amount is the amount sufficient to prevent mortality or to expand the treatment window for a subject who has been exposed to B. anthracis or a bacterium which produces toxins or toxin components homologous to those produced by B. anthracis or to the toxins or toxin components themselves, in the absence of bacteria.
  • the antibodies of the present invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents.
  • the dosage administered will vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • dosing regimens that can be used in the methods of the invention include, but are not limited to, once daily, three times weekly (intermittent), weekly, or every 14 days.
  • dosing regimens include, but are not limited to, monthly dosing or dosing every 6-8 weeks.
  • pre-exposure treatment the regimen includes dosing once every 2 to 4 weeks.
  • postexposure treatment a single dose is administered as soon as possible following exposure.
  • the regimen further includes another dose about 2 weeks following exposure.
  • the regimen includes dosing once a week for 4 to 8 weeks following exposure.
  • the antibodies are administered as part of a therapeutic regimen which includes antibacterial agents.
  • Antibacterial agents including antibiotics, that can be used in combination with the antibodies of the invention include, without limitation, aminoglycoside antibiotics, glycopeptides, amphenicol antibiotics, ansamycin antibiotics, cephalosporins, cephamycins oxazolidinones, penicillins, quinolones, streptogamins, tetracyclins, and analogs thereof.
  • the antibody combinations of the invention are administered as part of a therapeutic regimen that includes ciprofloxacin and doxycycline.
  • the regimen includes administration of an antibacterial agent at a dosage of from 10-50 mg/kg/day, preferably 20, 25, 30, or 35 mg/kg/day, for 30-90 days, preferably for 60-90 days.
  • the antibacterial agent is selected from the group consisting of ampicillin, amoxicillin, ciprofloxacin, gentamycin, kanamycin, neomycin, penicillin G, streptomycin, sulfanilamide, and vancomycin.
  • the antibacterial agent is selected from the group consisting of azithromycin, cefonicid, cefotetan, cephalothin, cephamycin, chlortetracycline, clarithromycin, clindamycin, cycloserine, dalfopristin, doxycycline, erythromycin, linezolid, mupirocin, oxytetracycline, quinupristin, rifampin, spectinomycin, and trimethoprim
  • Additional, non-limiting examples of antibacterial agents for use in combination with the antibodies of the invention include the following: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol
  • Additional examples include cycloserine, mupirocin, tuberin amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, and 2,4 diaminopyrimidines (e.g., brodimoprim).
  • the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with an antibody composition of the invention.
  • the composition is an aqueous formulation.
  • the composition is lyophilized.
  • the liquid or lyophilized composition is sterile.
  • the kit comprises a liquid or lyophilized composition of the invention, in one or more containers, and one or more other prophylactic or therapeutic agents useful for the treatment of a bacterial infection or toxemia.
  • the one or more other prophylactic or therapeutic agents may be in the same container as the antibody composition, or in one or more other containers.
  • the one or more other prophylactic or therapeutic agents comprises an antibiotic, preferably ciprofloxacin and/or doxycycline.
  • the kit further comprises instructions for use in the treatment of anthrax (e.g., using the antibody compositions of the invention alone or in combination with another prophylactic or therapeutic agent), as well as side effects and dosage information for one or more routes of administration.
  • anthrax e.g., using the antibody compositions of the invention alone or in combination with another prophylactic or therapeutic agent
  • side effects and dosage information for one or more routes of administration.
  • a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g. CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • this invention provides kits for the packaging and/or storage and/or use of the antibody composition described herein, as well as kits for the practice of the methods described herein.
  • the kits can be designed to facilitate one or more aspects of shipping, use, and storage.
  • IQNPA and IQNLF when administered prior to exposure, to protect against death due to inhalational anthrax in New Zealand White rabbits.
  • the average aerosol challenge dose for this study was 132 ⁇ 21 LD50s with a range of 99 - 199 LD50s.
  • Body weight, body temperature, clinical observations, and bacteremia were all examined during the course of this study. The data, discussed in more detail below, demonstrated that both antibodies were able to prolong survival following infection with B. anthracis. Treatment did not affect the weight gain or loss seen in animals following infection, nor was there any significant change in body temperature during the course of infection (data not shown).
  • Pre-treatment with either IQNLF or IQNPA prolonged survival to 5.8 and 8.12 days, respectively, on average.
  • the average time to death was 4.41 days.
  • both antibodies increased the survival rate following exposure. While no animals survived in the untreated control group, 100% of animals survived for the 14 day study period in the groups receiving either 5 mg/kg or 2.5 mg/kg IQNPA. 75% survival was seen in the groups receiving either 10 mg/kg or 1.25 mg/kg IQNPA as well as in the group receiving 10 mg/ml IQNLF.
  • Figure 1 shows a Kaplan-Meier curve representing time-to-death and survival data for each group.
  • the survival data are tabulated below in Table 1.
  • Bacteremia sample could not be processed for one animal because sample was lost due to broken tube.
  • Bacteremia sample could not be drawn for animal K94338. Therefore, there were only three animals in this group where presence of bacteremia could be determined at all time points.
  • IQNPA 10.0, 5.0, 2.5, 1.25, or 0.625 mg/kg IQNPA or 10.0, 5.0, 2.5, 1.25, or 0.625 IQNLF (see Table 1, supra).
  • Groups 1-5 received the IQNPA antibody at doses ranging from 10.0 mg/kg to 0.625 mg/kg.
  • Animals in group 6 received buffer only as a control.
  • Groups 7-11 received the IQNLF antibody at doses ranging from 10.0 mg/kg to 0.625 mg/kg. All four rabbits in each group received the indicated doses.
  • Aerosol Challenge This study required two aerosol challenge days with 22 rabbits challenged per day. The overall average dose for the two study days was 132 LD50s with an average challenge dose of 133 ⁇ 23 LD50s for the first day and 131 ⁇ 19 LD50s for the second day. The mass-median aerodynamic diameter for challenge material aerosols on day one was 1.17 ⁇ m and the mass-median aerodynamic diameter for challenge material aerosols on day two was 1.18 ⁇ m. Rabbits were transported into the BL-3 facility 6 days prior to challenge to allow time for acclimation.
  • Blood Collection Blood was drawn from the medial auricular artery or the marginal ear vein. Oil of wintergreen (topical) or acepromazine (1-5 mg/kg) subcutaneous Iy) was used to facilitate blood sampling via the ear. Amounts of blood collected were within the guidelines established by the Battelle IACUC, derived in part from the Canadian Guide to the Care and Use of Experimental Animals.
  • Bacteremia Blood collected in EDTA tubes on study days 0, 2, 14 and/or time of death were cultured, by streaking ⁇ 40 ⁇ l of whole blood over blood agar plates, to determine the presence or absence of B. anthracis.
  • IQNPA and IQNLF when administered after exposure to B. anthracis, either alone or in combination, to protect against death due to inhalational anthrax in rabbits.
  • a body temperature increase of about 2 degrees Farenheit is observed at the start of the symptomatic period, which occurs about 25-29 hours post-exposure.
  • the average aerosol challenge dose for this study was 132 ⁇ 30 LD50s, with an average challenge dose of 144 ⁇ 28 LD50s for the first day of challenges and an average dose of 119 ⁇ 31 LD50s for the second day of challenges.
  • Body weight, body temperature, clinical observations and bacteremia were all examined during the course of this study.
  • the data demonstrate that both the IQNPA and IQNLF antibodies increase survival when administered 24 hours following exposure to inhalational anthrax.
  • the data further demonstrate that the combination of both antibodies resulted in an increased probability of survival compared to either antibody administered alone.
  • IQNPA doses 2.5 mg/kg and 1.25 mg/kg, resulted in 50% and 33% survival, respectively.
  • the two highest doses of IQNLF (15 mg/kg and 7.5 mg/kg) resulted in 66% survival and the lowest dose (3.75 mg/kg) resulted in 33% survival. None of the animals in the control group survived.
  • Table 3 shows the survival data and results of the Fisher's Exact Test for each treatment group. [143] The data further show that when administered in combination, the two antibodies are capable of working in a coordinated manner to eradicate infection. Importantly, 50% survival was observed in the Group 11 even at doses as low as 0.625 mg/kg IQNPA and 1.88 mg/kg IQNLF. The data also show that survival is dose-dependent.
  • Figure 3 shows the Kaplan-Meier curves for each group.
  • Table 5 shows the proportion of animals that were bacteremic at any time point during the 14 day study period with a 95 percent binomial confidence interval. About half (51% or 37/72) of the challenged animals were bacteremic on day 1. All but three of the animals that died or were euthanized prior to study day 14 were bacteremic. The majority of the surviving animals were not bacteremic on day 14 as determined by blood culture. Of these surviving animals, 33% (12/36) were bacteremic at some time point. [146] For Group 1, which received 5.0 mg/kg IQNPA, 3/6 animals were bacteremic on study days 1 and 2, but negative at the end of study (day 14). The other three animals in this group were not bacteremic at any time point.
  • Test System Seventy-two (36 male and 36 female) specific pathogen free
  • New Zealand white rabbits (purchased from Covance Laboratories) weighing between 2.0 to 4.0 kg at the time of randomization that were in good health were placed on study. Six additional rabbits were housed as extras until the completion of the study. Prior to challenge, rabbits were assigned to one of twelve groups (six rabbits per group) based on animal weights, one of three aerosol challenge days (two rabbits per group per day) and a challenge order per day. Randomization was based on animal weights. The study was continued for 14 days.
  • Aerosol Challenge This study required two aerosol challenge days with 36 rabbits challenged per day. Rabbits were transported into the BL-3 facility 6 days prior to challenge to allow time for acclimation. On Study Day 0, rabbits were placed into a plethysmography chamber and passed into a Class III cabinet system, and aerosol challenged with a targeted dose of 100 LD50s B. anthracis (Ames strain) spores aerosolized by a Collision nebulizer. Aerosol concentrations of B. anthracis were quantified by determination of cfu. effluent streams collected directly from an animal exposure port by an in-line impinger (Model 7541, Ace Glass Incorporated).
  • B. anthracis the rabbits were administered one of two antibodies at varying doses, or in combination (see Table 3, supra).
  • Groups 1-3 received IQNPA at doses ranging from 5 mg/kg to 1.25 mg/kg.
  • Groups 4-6 received IQNLF at doses ranging from 15.0 mg/kg to 3.75 mg/kg.
  • Group 7 received buffer only as a control.
  • Groups 8-12 received decreasing doses of the combination treatment (IQNPA + IQNLF). All six rabbits in each group received the indicated doses as indicated by signed paperwork filled out during the dosing process. All treatments were via a single bolus dose.
  • Blood samples were collected on days -1, 1 (prior to treatment), 2 and 14, or at time of death. Blood was drawn from the marginal ear vein according. Oil of wintergreen (topical) or acepromazine (1-5 mg/kg subcutaneously) was utilized to facilitate blood sampling via the ear. Amounts of blood collected fell within the guidelines established by the Battelle IACUC, derived in part from the Canadian Guide to the Care and Use of Experimental Animals.
  • Bacteremia Blood collected in EDTA tubes on study days -1, 1, 2,
  • the main objective of this study was to further examine the efficacy of the combined treatment of IQNLF and IQNPA against inhalational anthrax infection.
  • the target infectious dose for this study was 100 LD50s; the average aerosol challenge dose for the study was 91 ⁇ 27 with a range of 47 - 149 LD50s.
  • the log-rank test applied to the time-to- death data showed that the pooled control group was significantly less protected than groups treated with combined antibodies when time to death was considered in addition to the overall survival rates.
  • IQNLF alone did not provide as high a level of protection as the combined treatment.
  • Figure 4 is a Kaplan-Meier curve representing time-to-death and survival data for each group.
  • Table 6 summarizes the survival data for each group. Confidence intervals for the survival rates and the results of Fisher's exact test comparisons of survival rates to the control group are also provided. According to the unadjusted p values from Fisher's exact test, treatment group 4 (IQNLF, 2.5 mg/kg) as well as groups 8 through 11 (all groups with combined antibody doses of IQNPA and IQNLF) had significantly higher survival rates than the pooled control group (group 6). When a Bonferroni Holm adjustment was used to control the overall level of significance at 0.05, the same groups (4 and 8 through 11) had a significantly higher survival rate than the control group.
  • Table 8 shows the estimates and p values for the effects included in the final logistic regression model that models survival with effects for the base 10 log transformed combined antibody dose and treatment (IQNLF or IQNPA + IQNLF).
  • the overall effect for treatment was statistically significant at the 0.05 level (p value ⁇ 0.0001) indicating that survival rates differed among the two treatments (IQNLF and combined IQNPA+IQNLF).
  • Table 9 shows the odds ratios for the model.
  • Table 10 shows the estimates and p values for the effects included in the logistic regression model fitted to the base 10 log transformed IQNLF dose and an indicator for treatment (IQNLF or IQNPA+IQNLF).
  • Table 11 presents the odds ratios from the model.
  • IQNLF as stand alone treatment is not very protective.
  • the IQNPA+IQNLF combination is very successful in protecting from death due to anthrax challenge 24 hrs before treatment.
  • the combination treatment provided a 15 times higher chance of survival in this post-exposure aerosol challenge model.
  • the increased protection by the IQNPA+IQNLF combination was significant, there was no dose dependency (all but one combination group demonstrated 87.5% survival).
  • the results of this study suggest that the IQNPA+IQNLF combination represents an improvement over the IQNLF treatment alone.
  • Table 12 illustrates the proportion of animals that were bacteremic at any time point post-challenge along with the 95 percent confidence interval. All animals surviving to study day 14 were negative for bacteria on study day 14. However, only 32% (11/34) of these surviving animals were positive at any time point. All except two animals that died or were euthanized prior to study day 14, were positive. While only 2.7% (1/36) of the animals were bacteremia positive on study day 7, 32.8% (25/76) of the animals were bacteremia positive on study day 2 and fifty-three percent (42/80) of the animals were bacteremia positive just prior to treatment.
  • Table 13 Frequency Table of Animals Bacteremic at Any Time Point Versus Survival Status (Alive or Dead)
  • Test System Eighty (40 male and 40 female) specific pathogen free New
  • Aerosol Challenge This study required two aerosol challenge days with 40 rabbits challenged per day. The first 40 rabbits were challenged and treated and followed for 14 days and then the second 40 rabbits arrived, were challenged, treated, and followed for 14 days. Thus, there were two separate randomizations performed. Prior to challenge day A, rabbits were assigned to one of six groups based on animal study day -8 weights, and a challenge order per day. The day of aerosol challenge was considered Day 0. The first group of 40 rabbits was randomized according to Table 14A and the second group of 40 rabbits was randomized according to Table 14B. All rabbits to be challenged on the second of the two challenge days were randomized by Study Day -7 weights. Rabbits were transported into the BL3 facility immediately upon arrival for quarantine.
  • rabbits were placed into a plethysmography chamber and passed into a Class III cabinet system, and challenged with a targeted aerosol dose of 100 LD50s B. anthracis (Ames strain) spores.
  • the concentrations of B. anthracis inhaled by the rabbits was determined from the number of B. anthracis spores collected directly from an animal exposure port by an in-line impinger (Model 7541, Ace Glass Incorporated). Serial dilutions of impinger samples were plated and enumerated. The inhaled dose was calculated using the number of CFU/liter of air multiplied by the respiratory volume of the rabbits.
  • the overall average dose for the two aerosol challenge days was 91 ⁇ 27 LD50s with an average challenge dose of 115 ⁇ 34 LD50s for the first day of challenges and an average dose of 66 ⁇ 19 LD50s for the second day of challenges.
  • the mass-median aerodynamic diameter for challenge material aerosols on day one was 1.18 ⁇ m and the mass-median aerodynamic diameter for challenge material aerosols on day two was 1.15 ⁇ m (as determined with an Aerodynamic Particle Sizer (APS model 3321, TSI Inc, St. Paul, MN)).
  • Table 14A Study Design Part I
  • IQNPA dose concentration remained the same at 2.5 mg/kg while the IQNLF dose varied accordingly
  • Bacteremia Blood collected in EDTA tubes on days -7, just prior to treatment, 2, 14 and/or at time of death was cultured by streaking ⁇ 40 ⁇ l of whole blood over blood ager plates, to determine the presence or absence of B. anthracis bacteremia.
  • Sera Collection and Shipment Approximately 2.0 ml of whole blood was collected into SST tubes on study days -7, 14 or at time of death. This blood was processed and the serum collected. Serum was then filtered and checked for sterility for shipment to IQ Corporation for serological analysis. When possible, a terminal sample was taken from any animal found dead or found to be moribund prior to euthanasia.
  • IQNPA and IQNLF antibodies could extend the window for treatment following inhalational infection with B. anthracis.
  • the IQNPA and IQNLF antibodies were given alone or in combination at 24, 32, 40, and 48 hours post-challenge.
  • the target infectious dose for this study was 100 LD50s; the overall average dose for the three study days was 100 ⁇ 25 LD50s with an average challenge dose of 91 ⁇ 28 LD50s for the first day of challenges and an average dose of 102 ⁇ 19 LD50s for the second day of challenges and an average dose of 106 ⁇ 29 LD50s for the third day of challenge.
  • While survival was the key objective of this study, several other parameters including temperature, body weight, clinical observations, and bacteremia were also examined.
  • Test System Seventy-two (36 male and 36 female) New Zealand white rabbits (purchased from Covance Laboratories), specific pathogen free (SPF), that weighed between 2.0 to 4.0 kg at the time of randomization and were in good health were placed on study. Seventy-eight rabbits were ordered. As all animals were free of malformations and illness, the replacement animals were not required, and were transferred to a training protocol and used for training purposes.
  • Aerosol Challenge This study required three aerosol challenge days with 22 rabbits challenged per day. Rabbits were transported into the BL-3 facility 3 days prior to challenge to allow time for acclimation. On Study Day 0, rabbits were placed into a plethysmography chamber and passed into a Class III cabinet system, and challenged with a targeted aerosol dose of 100 LD50s B. anthracis (Ames strain) spores. The concentrations of B. anthracis inhaled by the rabbits is determine from the number of B. anthracis collected directly from an animal exposure port by an in-line impinger (Model 7541, Ace Glass Incorporated).
  • the inhaled dose was calculated using the number of CFU/liter of air multiplied by the respiratory volume of the rabbits.
  • the overall average dose for the three study days was 100 ⁇ 25 LD50s with an average challenge dose of 91 ⁇ 28 LD50s for the first day of challenges and an average dose of 102 ⁇ 19 LD50s for the second day of challenges and an average dose of 106 ⁇ 29 for the third challenge day.
  • the mass-median aerodynamic diameter for challenge material aerosols on day one was 1.16 ⁇ m and the mass- median aerodynamic diameter for challenge material aerosols on day two was 1.15 ⁇ m and the mass-median aerodynamic diameter for challenge material aerosols on day three was 1.12 ⁇ m (as determined with an Aerodynamic Particle Sizer (APS model 3321, TSI Inc, St. Paul, MN)).
  • IQNPA 2.5 mg/kg
  • IQNLF 7.5 mg/kg
  • a combination of the two see Table 15, supra.
  • Doses were given via a single bolus intravenous injection. Briefly, groups one through three received 2.5 mg/kg of IQNPA at the indicated times post-challenge, group six received buffer only as a control, and groups eight through eleven received 7.5 mg/kg IQNLF at 24, 32, 40, and 48 hours post-challenge. All rabbits were treated according to Table 15. The study director visually verified doses prior to being administered to ensure the required dose levels were given.
  • Rabbits were administered 2.5 mg/kg IQNPA at 24, 32, and 40 hours post-challenge; 7.5 mg/kg IQNLF was administered at 24, 32, and 40 post-challenge; 5.0 + 7.5 mg/kg IQNPA + IQNLF was administered 24, 32, 40, and 48 hours after being challenged with B. anthracis. All treatments were via a single bolus dose.
  • Post-challenge body temperatures were monitored from a single transponder chip (rump). All temperature readings were taken prior to treatment with acepromazine.
  • Animal Weights Animals were weighed once daily for the duration of the study beginning 10 days prior to the first challenge day. Weights were used to determine the amount of acepromazine to be administered prior to temperature transponder implantation. Study day 0 weights were used to determine the required amount of test/control article to be administered.
  • Blood was drawn from the marginal ear vein. Oil of wintergreen (topical) or acepromazine (1-5 mg/kg subcutaneously) was utilized to facilitate blood sampling via the ear. Amounts of blood collected fell within the guidelines established by the Battelle IACUC, derived in part from the Canadian Guide to the Care and Use of Experimental Animals.
  • Bacteremia Blood collected in EDTA tubes on days -1, just prior to treatment, 2, 14 and/or time of death were cultured, by streaking ⁇ 40 ⁇ l of while blood over blood ager plates, to determine the presence or absence of B. anthracis bacteremia.
  • Sera Collection and Shipment Approximately 2.0 ml of whole blood was collected into SST tubes (see Blood collection). This blood was processed and the serum collected. Serum was then filtered, and checked for sterility for shipment to IQ Corporation for serological analysis. When possible, a terminal sample was taken from any animal found dead or found to be moribund prior to euthanasia.
  • IQNPA+IQNLF antibody treatment represents a statistically significant improvement over both treatment with either antibody alone.
  • the rabbits will be challenged with 100 times the LD50 equivalent of Bacillus anthracis Ames spores (100xLD50) by inhalation or intranasal instillation.
  • Antibodies will be administered via a single injection of either (i) IQNPA alone, (ii) IQNLF alone, or (iii) either a single injection containing both IQNP A+I QNLF or two separate injections of IQNPA and IQNLF alone given at about the same time. Injections will be either subcutaneous, intramuscular, or intravenous.
  • Levofloxacin (7 mg/kg/d for 6 consecutive days) will be administered at the same time as exposure to anthrax to demonstrate that the model can be modulated with agents other than monoclonal antibodies.
  • Time of antibody treatment is the moment where the rabbits start to become symptomatic.
  • the symptomatic phase (as measure by the presence of PA in blood and the presence of bacteremia) generally starts between 25 and 29 hrs after exposure.
  • the results of these studies should demonstrate the added benefit of IQNLF with respect to survival.
  • the start of the Levofloxacin treatment is at the time the rabbits become symptomatic.
  • Time of antibody treatment is the moment where the rabbits start to become symptomatic.
  • the symptomatic phase (as measure by the presence of PA in blood and the presence of bacteremia) generally starts between 25 and 29 hrs after exposure. The results should indicate that no significant negative impact of the antibiotic on the effect to the antibodies.
  • Time of antibody treatment is the moment 48 hrs after exposure.
  • the symptomatic phase (as measure by the presence of PA in blood and the presence of bacteremia) generally starts between 25 and 29 hrs after exposure, and 48 hrs is well into this symptomatic phase.

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Abstract

La présente invention concerne des procédés et compositions destinés à la prévention et au traitement d'une maladie provoquée par B. anthracis ou par une bactérie qui produit des toxines ou des composants de toxines homologues aux facteurs de virulence produits par B. anthracis, ou en l'absence des bactéries, par les toxines ou par les composants des toxines eux-mêmes. Les procédés et compositions de l'invention comprennent une combinaison d'au moins deux anticorps, de préférence des anticorps monoclonaux, et plus préférablement des anticorps monoclonaux humains, dont chacun se lie avec une haute affinité à un antigène bactérien différent.
PCT/IB2010/000146 2009-01-14 2010-01-14 Anticorps combinés destinés au traitement et à la prévention d'une maladie provoquée par bacillus anthracis, ainsi que par les bactéries connexes et par leurs toxines WO2010082134A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP10706048A EP2387584A1 (fr) 2009-01-14 2010-01-14 Anticorps combinés destinés au traitement et à la prévention d'une maladie provoquée par bacillus anthracis, ainsi que par les bactéries connexes et par leurs toxines
CA2749572A CA2749572A1 (fr) 2009-01-14 2010-01-14 Anticorps combines destines au traitement et a la prevention d'une maladie provoquee par bacillus anthracis, ainsi que par les bacteries connexes et par leurs toxines
US12/840,779 US20110129460A1 (en) 2009-01-14 2010-07-21 Combination Antibodies For The Treatment And Prevention Of Disease Caused By Bacillus Anthracis And Related Bacteria And Their Toxins

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US14450709P 2009-01-14 2009-01-14
US61/144,507 2009-01-14

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