WO2014034127A1 - Binding/neutralizing human monoclonal antibodies against botulinum neurotoxin type b - Google Patents

Binding/neutralizing human monoclonal antibodies against botulinum neurotoxin type b Download PDF

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WO2014034127A1
WO2014034127A1 PCT/JP2013/005123 JP2013005123W WO2014034127A1 WO 2014034127 A1 WO2014034127 A1 WO 2014034127A1 JP 2013005123 W JP2013005123 W JP 2013005123W WO 2014034127 A1 WO2014034127 A1 WO 2014034127A1
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Prior art keywords
monoclonal antibody
seq
botulinum neurotoxin
antigen
amino acid
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PCT/JP2013/005123
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French (fr)
Inventor
Yukako Fujinaga
Takuhiro MATSUMURA
Kazuyoshi Ikuta
Ryo MISAKI
Kazuhito Fujiyama
Tomoko Kohda
Shunji Kozaki
Ken-Ichiro Ono
Piyada WANGROONGSARB
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Osaka University
Medical And Biological Laboratories Co., Ltd
Department Of Medical Sciences
Osaka Prefecture University Public Corporation
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Application filed by Osaka University, Medical And Biological Laboratories Co., Ltd, Department Of Medical Sciences, Osaka Prefecture University Public Corporation filed Critical Osaka University
Priority to CN201380044898.6A priority Critical patent/CN104837866B/en
Priority to JP2015510556A priority patent/JP6286420B2/en
Publication of WO2014034127A1 publication Critical patent/WO2014034127A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • 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
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • 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
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to anti-botulinum neurotoxin (botulin) antibodies and methods of using and manufacturing the same.
  • Botulinum neurotoxins are among the most toxic substance known. These toxins can cause botulism and are produced by various bacteria including Clostridium botulinum . Botulinum toxin-producing clostridia are anaerobic gram-positive bacteria. These bacteria form spores and are commonly distributed in the environment. On a molecular level, botulism is caused by the inhibition of acetylcholine release at neuromuscular junctions by the toxin. Seven toxin serotypes (A, B, C, D, E, F, and G) have been identified. They have similar structures corresponding to an approximately 150kD zinc-endopeptidase protein including a 100-kDa heavy chain and a 50-kDa light chain. Toxin serotypes A, B, E, and F are generally associated with botulism in humans.
  • Botulism can be grouped into various categories-foodborne, wound infection, infant intestinal toxemia, adult intestinal toxemia, toxin injection (iatrogenic botulism), and weaponized toxin.
  • the foodborne illness occurs due to ingestion of toxin in food contaminated with toxin-producing bacteria.
  • wound infection a wound is colonized by toxigenic clostridia that then grow and produce toxins in the wound.
  • infant botulism the infant intestine is colonized by toxigenic clostridia, which then produce toxins.
  • Adult intestinal toxemia is similar to infant botulism.
  • Cosmetic or therapeutic injection of botulinum toxin can sometimes lead to botulism.
  • Botulism can also result from injury by weaponized toxin, for example, by inhalation of aerosolized botulinum toxin.
  • Botulism is characterized by symmetric cranial nerve palsies that are followed by descending symmetric flaccid paralysis of voluntary muscles. This paralysis can result in loss of respiration and death. Toxin binding is irreversible. Recovery involves the growth of new nerve terminals. Recovery can be lengthy and involve extended outpatient rehabilitation therapy. The onset of the disease can be swift following contact with the toxin. Treatment usually involves administration of antitoxin, which can help limit the extent of paralysis. Most antitoxin available is horse-derived and is not necessarily specific to a particular type of Botulinum neurotoxin. There exists a need for more targeted forms of antitoxin. There is also a need for effective toxin-neutralizing antibodies without side effects such as serum sickness. There further exists a need for better diagnostic tests given the rapid course of botulism.
  • Another feature of the present invention is to provide methods of inhibiting and treating botulism.
  • a further feature of the present invention is to provide therapeutics without side effects such as serum sickness.
  • the present invention relates to a an isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof having a neutralization activity against a botulinum neurotoxin type B.
  • the monoclonal antibody can include a human monoclonal antibody, a humanized monoclonal antibody, or both.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a progenitor toxin of botulinum neurotoxin type B.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B) or have a specific binding activity to both a light chain and a heavy chain of a botulinum neurotoxin type B (BoNT/B ).
  • Hybridomas that produce the antibodies or fragments thereof of the present invention are also provided by the present invention, as well as methods of producing such hybridomas and method of producing antibodies or fragments thereof from such hybridomas.
  • the present invention further relates to pharmaceutical compositions.
  • the pharmaceutical composition can contain one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of and a pharmacologically acceptable carrier.
  • a kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject is provided by the present invention.
  • the kit can include an isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of the present invention.
  • Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to manufacture a medicament for inhibiting or treating botulism in a human subject is also provided by the present invention.
  • the present invention also relates to a method of inhibiting or treating botulism in a human subject.
  • the method can include administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to the human subject.
  • the method can further include diagnosing the patient with botulinum neurotoxin poisoning.
  • the method can include monitoring for a decrease in at least one symptom of botulism.
  • the method of inhibiting or treating botulism in a human subject can include administering the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both in combination with one or more additional therapies directed to botulism.
  • the combination can act synergistically to inhibit or treat botulism.
  • the present invention still further relates to a method of detecting botulinum neurotoxin type B in a human subject.
  • the method can include contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
  • the method can also include detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.
  • FIG. 1 is a schematic diagram showing how volunteers were immunized and how antibody producing cells were isolated in accordance with the present invention.
  • FIG. 2 is a flow diagram depicting a cell fusion and cloning schedule in accordance with the present invention.
  • FIG. 3 shows nucleotide and amino acid sequences for the M-2 antibody heavy chain in accordance with the present invention.
  • FIG. 4 shows nucleotide and amino acid sequences for the M-2 antibody light chain in accordance with the present invention.
  • FIG. 5 shows nucleotide and amino acid sequences for the M-4 antibody heavy chain in accordance with the present invention.
  • FIG. 6 shows nucleotide and amino acid sequences for light chain of the RM-4LC-16 antibody in accordance with the present invention.
  • FIG. 7 shows nucleotide and amino acid sequences for light chain of the RM-4LC-18 antibody in accordance with the present invention.
  • FIG. 8 shows nucleotide and amino acid sequences for light chain of the RM-4LC-19 antibody in accordance with the present invention.
  • FIG. 9 includes line graphs showing binding of human monoclonal antibodies to BoNT/A or BoNT/B in accordance with the present invention. Binding of human monoclonal antibody (HuMAb) to BoNT was analyzed by ELISA. HuMAbs (bout 0.05-1.0 micrograms/ml) were added to the plate coated with BoNT/A or BoNT/B. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • FIG. 10 includes Western blots for epitope mapping of human monoclonal antibodies in accordance with the present invention.
  • BoNT was separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane.
  • HuMAbs 1.0 micrograms/ml
  • M-2 specifically bound to light chain of BoNT/B.
  • M-4 bound to both of light chain and heavy chain.
  • FIG. 11 is a bar graph showing binding of human monoclonal antibodies to BoNT/B, progenitor toxin, and non-toxic component in accordance with the present invention.
  • Binding of HuMAb to BoNT, progenitor toxin (12S toxin and 16S toxin) or non-toxic component (NAP-16) was analyzed by ELISA.
  • HuMAbs were added to the plate coated with BoNT, progenitor toxin or NAP-16. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • M-2 and M-4 bound to BoNT and progenitor toxins. In contrast, M-2 and M-4 showed faint binding to NAP-16.
  • FIG. 12 is a line graph showing neutralization activity of a combination of human monoclonal antibodies against BoNT/B in accordance with the present invention. 10 LD 50 of BoNT/B was incubated with a mixture of M-2 and M-4 (0.05, 0.1 or 0.5 micrograms each) and injected into mice. All of the mice that received the mixture of M-2 (0.5 micrograms) and M-4 (0.5 micrograms) survived and had no symptoms.
  • FIG. 12 is a line graph showing neutralization activity of a combination of human monoclonal antibodies against BoNT/B in accordance with the present invention. 10 LD 50 of BoNT/B was incubated with a mixture of M-2 and M-4 (0.05, 0.1 or 0.5 micrograms each) and injected into mice. All of the mice that received the mixture of M-2 (0.5 micrograms) and M-4 (0.5 micrograms) survived and had no symptoms.
  • FIG. 12 is a line graph showing neutralization activity of a combination of human monoclonal antibodies against BoNT/B in accord
  • FIG. 13 is a line graph showing neutralization activity of post-administration of human monoclonal antibodies against progenitor toxin (16S toxin) in accordance with the present invention.
  • Mice were orally administrated with progenitor toxin (16S toxin, 10 ng), and subsequently administrated with HuMAbs (M-2 0.5 micrograms + M-4 0.5 micrograms) by i.p. injection at 12, 24, or 36 hours after the oral administration of 16S toxin. Control mice that were not treated with HuMAbs died within 72 hours.
  • post-exposure treatment of HuMAbs provided complete survival at 12 hours after oral administration of 16S toxin, and partial survival at 24 and 36 hours after administration.
  • FIG. 14 includes a bar graph and line graphs showing binding and neutralization of human monoclonal antibodies against BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) in accordance with the present invention.
  • Binding of HuMAbs (M-2 and M-4) to BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) were analyzed by ELISA.
  • HuMAbs (0.5 micrograms/ml) were added to the plate coated with BoNT. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • M-2 and M-4 showed strong binding to BoNT/B2 and BoNT/B6 in addition to BoNT/B1.
  • FIG. 15 includes Western blots and line graphs showing binding of recombinant human monoclonal antibodies to BoNT/B in accordance with the present invention.
  • HEK293 cells were transiently transfected with pQCXIP-hCH and pQCXIH-hCl expression vectors using LIPOFECTAMINE 2000 transfection reagent.
  • Recombinant HuMAbs in the culture supernatant were determined with the anti-human IgG conjugated with HRP.
  • RM-2 and RM-4 Binding of recombinant M-2 (RM-2) and recombinant M-4 (RM-4) to BoNT/B were analyzed by ELISA.
  • Recombinant HuMAb (about 0.01-0.5 micrograms/ml) were added to the BoNT/B coated plate. After washing, bound recombinant HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • RM-2 and RM-4 bound to BoNT/B in the same manner as the M-2 and M-4 derived from hybridoma.
  • BoNT/B1, BoNT/B2 or BoNT/B6 were separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane.
  • HuMAbs 1.0 micrograms/ml was added to the membrane and incubated for 1 h at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 h at room temperature, and then specific bands were visualized with ECL.
  • Recombinant HuMAbs showed the same binding profile as HuMAbs derived from hybidomas.
  • M-2 and RM-2 specifically bound to light chain of BoNT/B.
  • M-4 and RM-4 (RM-4 LC16, RM-4 LC18, RM-4 LC19, have same heavy chain of M-4) bound to both of light chain and heavy chain.
  • M-4 and RM-4 antibodies heavy chain of B2 subtype was detected more clearly than that of B1.Further, heavy chain of B6 subtype was detected more clearly than that of B2.
  • An isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof having a neutralization activity against a botulinum neurotoxin type B is provided in accordance with the present invention.
  • the monoclonal antibody can include a human monoclonal antibody, a humanized monoclonal antibody, or both.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a progenitor toxin of botulinum neurotoxin type B.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a botulinum neurotoxin type B produced by BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof accordingly can have a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B).
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody can be produced by a hybridoma made by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum neurotoxin type B with a fusion partner cell capable of efficient cell fusion.
  • the botulinum neurotoxin type B can be a product of BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  • Any suitable fusion partner cell can be used, for example, a SPYMEG cell.
  • Hybridomas that produce the antibodies or fragments thereof of the present invention are also provided by the present invention.
  • the hybridoma can have the Deposit number of NITE BP-01639 or NITE BP-01640.
  • An isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639 or NITE BP-01640 is also provided.
  • monoclonal antibodies obtained as described above include a monoclonal antibody produced by the hybridoma named "Hybridoma M-2 (M1E9)" (hereinafter, referred to as M-2 antibody) and "Hybridoma M-4 (M4C9)” (hereinafter, referred to as M-4 antibody).
  • the hybridoma was deposited at the NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) under Deposition No. NITE BP-01639 and No. NITE BP-01640 on Jun. 26, 2013. Then, they were transferred to the international deposit under Budapest Treaty under the same No.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can be IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgD, IgE, any fragment thereof, or any combination thereof.
  • the antibody fragment can include, for example, a Fab, a Fab', a F(ab')2, a scFv, a dsFv, or a combination thereof.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can include a heavy chain and/or light chain variable region.
  • the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or 20, a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or 21.
  • CDR1 complementarity determining region
  • CDR2 having a second amino acid sequence comprising SEQ ID NO: 6 or 20
  • CDR3 having a third amino acid sequence comprising SEQ ID NO: 7 or 21.
  • the light chain variable region can include, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
  • CDR1 having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26
  • CDR2 having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27
  • CDR3 having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
  • the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7.
  • the light chain variable region can include a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
  • the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21.
  • CDR1 complementarity determining region
  • CDR2 complementarity determining region having a second amino acid sequence comprising SEQ ID NO: 20
  • CDR3 having a third amino acid sequence comprising SEQ ID NO: 21.
  • the light chain variable region can include, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28.
  • CDR1 complementarity determining region
  • CDR2 second complementarity determining region
  • CDR3 having a sixth amino acid sequence comprising SEQ ID NO: 28.
  • the heavy chain variable region can include SEQ ID NO: 3 or 17
  • the light chain variable region can include SEQ ID NO: 10 or 24.
  • compositions are provided by the present invention.
  • the pharmaceutical composition can contain one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of and a pharmacologically acceptable carrier.
  • the pharmaceutical composition can contain the anti-botulinum neurotoxin type B monoclonal antibody and a pharmacologically acceptable carrier.
  • the pharmaceutical composition can contain two isolated anti-botulinum neurotoxin type B monoclonal antibodies, antigen-binding fragment thereof, or both.
  • the pharmaceutical composition can contain first and second antibodies.
  • the first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (1) an isolated human monoclonal antibody or antigen-binding fragment including a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
  • CDR1 complementarity determining region
  • CDR2 second complementarity determining region
  • CDR3
  • the first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (2) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10.
  • the first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (3) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit No. NITE BP-01639.
  • the first isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above.
  • the second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (4) an isolated human monoclonal antibody or antigen-binding fragment including a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21, and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28.
  • CDR1 complementarity determining region
  • CDR2 second complementarity determining region
  • the second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (5) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 24.
  • the second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (6) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit No. NITE BP-01640.
  • the second isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above.
  • a method for producing an isolated anti-botulinum neurotoxin type B monoclonal antibody is provided by the present invention.
  • the method can include producing a hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum neurotoxin type B with a fusion partner cell capable of efficient cell fusion.
  • the method can further include obtaining a botulinum neurotoxin type B monoclonal antibody from the hybridoma.
  • PBMC peripheral blood mononuclear cell
  • the botulinum neurotoxin type B is produced, for example, by BONT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  • the fusion partner cell can be, for example, a SPYMEG cell.
  • kits for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject can include an isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of the present invention.
  • the kit can also include one or more additional agents for treating and/or detecting botulinum neurotoxin type B poisoning.
  • the kit can also include one or more agents for treating and/or detecting one or more additional types of botulinum neurotoxins, for example, type A.
  • Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to manufacture a medicament for inhibiting or treating botulism in a human subject is also provided by the present invention.
  • a method of inhibiting or treating botulism in a human subject is provided by the present invention.
  • the method can include administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to the human subject.
  • the method can further include diagnosing the patient with botulinum neurotoxin poisoning.
  • the method can include monitoring for a decrease in at least one symptom of botulism.
  • the at least one symptom can include, for example, paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, inability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, general muscle reflex loss, any other symptom of botulism, or any combination thereof.
  • botulism can be inhibited or treated in a human subject, for example, foodborne botulism, wound infection botulism, infant intestinal toxemia botulism, adult intestinal toxemia botulism, iatrogenic botulism, airborne botulism, or any combination thereof.
  • the method of inhibiting or treating botulism in a human subject can include administering the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both in combination with one or more additional therapies directed to botulism.
  • the combination can act synergistically to inhibit or treat botulism.
  • the one or more additional therapies can include, for example, administering a second isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
  • the one or more additional therapies can include, for example, administering an isolated anti-botulinum neurotoxin type A monoclonal antibody, an antigen-binding fragment thereof, or both.
  • the one or more additional therapies can include, for example, one or more antibiotics, for example, a penicillin, or any combination thereof.
  • a method of detecting botulinum neurotoxin type B in a human subject is provided by the present invention.
  • the method can include contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
  • the method can also include detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.
  • the method can further include detecting one or more additional types of botulinum neurotoxins, for example, type A.
  • Anti-botulinum neurotoxin antibodies and polypeptides containing antigen binding fragments thereof are provided as well as methods, uses, compositions, and kits employing the same.
  • the isolated monoclonal antibodies and antigen-binding fragments thereof described and claimed in the present application are not products of nature. These monoclonal antibodies and antigen-binding fragments thereof are the product of a hybridoma or equivalent artificial cell system generated using various laboratory procedures. Methods of using antibodies or antigen-binding fragments thereof for therapeutic, diagnostic, or other purposes, compositions including the same, kits including same, and/or non-naturally occurring antibodies or antibody fragments are not so limited unless explicitly specified.
  • a method of forming an antibody specific to a botulinum neurotoxin or a polypeptide or a fragment thereof is provided.
  • Such a method can contain providing a nucleic acid encoding a botulinum neurotoxin antigen polypeptide or a polypeptide containing an immunologically specific epitope thereof; expressing the polypeptide containing the antigen amino acid sequence or a polypeptide containing an immunologically specific epitope thereof from the isolated nucleic acid; and generating an antibody specific to the polypeptide obtained or a polypeptide containing an antigen binding fragment thereof.
  • An antibody or polypeptide containing an antigen binding fragment thereof produced by the aforementioned method is provided.
  • an isolated antibody or isolated polypeptide containing an antigen binding fragment thereof that specifically binds a botulinum neurotoxin antigen is provided.
  • Such an antibody can be generated using any acceptable method(s) known in the art.
  • the antibodies as well as kits, methods, and/or other aspects of the present invention employing antibodies can include one or more of the following: a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, a monovalent antibody, a diabody, and/or a humanized antibody.
  • Naturally occurring antibody structural units typically contain a tetramer.
  • Each such tetramer can be composed of two identical pairs of polypeptide chains, each pair having one full-length light" (for example, about 1 kDa to 25 kDa) and one full- length "heavy" chain (for example, about 50-70 kDa).
  • the amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function.
  • Human light chains are typically classified as kappa and lambda light chains.
  • Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4.
  • IgM has subclasses including, but not limited to, IgMl and IgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2.
  • variable and constant regions can be joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 or more amino acids. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)).
  • the variable regions of each light/heavy chain pair typically form the antigen binding site.
  • variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope.
  • both light and heavy chain variable regions typically contain the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
  • Antibody fragments include a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab1, F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Fv is an antibody fragment which contains a complete antigen-recognition and -binding site. This region includes a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. A single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can recognize and bind an antigen.
  • Single-chain Fv or “sFv” antibody fragments include the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide can further contain a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • Antibodies can be used as probes, therapeutic treatments and other uses. Antibodies can be made by injecting mice, rabbits, goats, or other animals with the translated product or synthetic peptide fragments thereof. These antibodies are useful in diagnostic assays or as an active ingredient in a pharmaceutical composition.
  • the antibody or polypeptide administered can be conjugated to a functional agent to form an immunoconguate.
  • the functional agent can be a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate), an antibiotic, a nucleolytic enzyme, or any combination thereof.
  • Chemotherapeutic agents can be used in the generation of immunoconjugates, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes, and/or fragments thereof, such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below.
  • immunoconjugates e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or
  • Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricotheeenes. Any appropriate radionucleotide or radioactive agent known in the art or are otherwise available can be used to produce radioconjugated antibodies.
  • Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2- pyridyldithiol)propionate (SPDP); iminothiolane (IT); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL); active esters (such as disuccinimidyl suberate); aldehydes (such as glutareldehyde); bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine); bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine); diisocyanates (such as tolyene 2,6-diisocyanate); bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene); maleimidocaproyl (MC); valine-
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacctic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody, see WO 94/11026.
  • the antibody can be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody- receptor conjugate is administered to the subject, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a "receptor” such as streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • a detectable marker is an agent detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • detectable markers include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, colored particles, biotin, or dioxigenin.
  • a detectable marker often generates a measurable signal, such as radioactivity, fluorescent light, color, or enzyme activity.
  • Antibodies conjugated to detectable agents can be used for diagnostic or therapeutic purposes.
  • detectable agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
  • the detectable substance can be coupled or conjugated either directly to the antibody or indirectly, through an intermediate such as, for example, a linker known in the art, using techniques known in the art. See, e.g., U.S. Patent No. 4,741,900, describing the conjugation of metal ions to antibodies for diagnostic use.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferin, and aequorin.
  • Antibodies useful in practicing the present invention can be prepared in laboratory animals or by recombinant DNA techniques using the following methods.
  • Polyclonal antibodies can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the gene product molecule or fragment thereof in combination with an adjuvant such as Freund's adjuvant (complete or incomplete).
  • an adjuvant such as Freund's adjuvant (complete or incomplete).
  • immunogenic conjugates can be produced recombinantly as fusion proteins.
  • Animals can be immunized against the immunogenic conjugates or derivatives (such as a fragment containing the target amino acid sequence) by combining about 1 mg or about 1 microgram of conjugate (for rabbits or mice, respectively) with about 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. Approximately 7 to 14 days later, animals are bled and the serum is assayed for antibody titer. Animals are boosted with antigen repeatedly until the titer plateaus. The animal can be boosted with the same molecule or fragment thereof as was used for the initial immunization, but conjugated to a different protein and/or through a different cross-linking agent. In addition, aggregating agents such as alum can be used in the injections to enhance the immune response.
  • immunogenic conjugates or derivatives such as a fragment containing the target amino acid sequence
  • the antibody administered can include a chimeric antibody.
  • the antibody administered can include a humanized antibody.
  • the antibody administered can include a completely humanized antibody.
  • the antibodies can be humanized or partially humanized.
  • Non-human antibodies can be humanized using any applicable method known in the art.
  • a humanized antibody can be produced using a transgenic animal whose immune system has been partly or fully humanized. Any antibody or fragment thereof of the present invention can be partially or fully humanized.
  • Chimeric antibodies can be produced using any known technique in the art. See, e.g., U.S. Patent Nos. 5,169,939; 5,750,078; 6,020,153; 6,420,113; 6,423,511; 6,632,927; and 6,800,738.
  • the antibody administered can include a monoclonal antibody, that is, the anti-botulinum neurotoxin antibodies of the present invention that can be monoclonal antibodies.
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • Monoclonal antibodies can be screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal Antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies can be tested for specific immunoreactivity with a translated product and lack of immunoreactivity to the corresponding prototypical gene product.
  • Monoclonal antibodies can be prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g., by fusion with myeloma cells.
  • preperipheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid are fused with myeloma cells.
  • PBMC preperipheral blood mononuclear cell
  • the clones are then screened for those expressing the desired antibody.
  • the monoclonal antibody preferably does not cross-react with other gene products.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods.
  • Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
  • the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the present invention can serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the present invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • Preparation of antibodies using recombinant DNA methods such as the phagemid display method, can be accomplished using commercially available kits, as for example, the Recombinant Phagemid Antibody System available from Pharmacia (Uppsala, Sweden), or the SurfZAP TM phage display system (Stratagene Inc., La Jolla, Califorinia).
  • the present invention provides human monoclonal antibodies (HuMAbs) generated against type A BoNT (BoNT/A) and type B BoNT (BoNT/B) using a murine-human chimera fusion partner cell line, named SPYMEG.
  • hybridoma cell lines, transformed B cell lines, and host cells that produce the monoclonal antibodies of the present invention; the progeny or derivatives of these hybridomas, transformed B cell lines, and host cells; and equivalent or similar hybridomas, transformed B cell lines, and host cells.
  • the antibodies can be diabodies.
  • the term "diabodies' refers to small antibody fragments with two antigen-binding sites, which fragments include a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • VH-VL light-chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains can be forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • the antibody administered can include a single-chain antibody.
  • the antibodies can be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain can be truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • the antibodies can be bispecific. Bispecific antibodies that specifically bind to one protein and that specifically bind to other antigens relevant to pathology and/or treatment are produced, isolated, and tested using standard procedures that have been described in the literature. [See, e.g., Pluckthun & Pack, Immunotechnology, 3:83-105 (1997); Carter, et al., J. Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No. 145, pp. 179-209; Pfreundschuh U.S. Patent No. 5,643,759; Segal, et al., J.
  • the antibodies disclosed herein can be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art. such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See Gabizon et al, J. National Cancer Inst., 81(19): 1484 (1989).
  • the present invention provides a method of inhibiting or treating botulinum poising in a human subject including administering a therapeutically effective amount of the anti-botulinum neurotoxin monoclonal antibody, antigen-binding fragment thereof of the invention to the human subject, or both.
  • the method can further include diagnosing the patient with botulinum neurotoxin poisoning.
  • Anti-botulinum neurotoxin antibodies or antigen-binding fragment thereof of the present invention can be administered to a subject before, during, and/or after diagnosing the patient as having botulinum neurotoxin poisioning.
  • the method can further include monitoring for a decrease in at least one symptom of botulinum neurotoxin poisioning.
  • two or more botulinum neurotoxin antagonists can be administered. At least one of the botulinum neurotoxin antagonists can include a botulinum neurotoxin antagonist. The at least one botulinum neurotoxin antagonist can be combined with one or more additional botulinum neurotoxin antagonists. At least one botulinum neurotoxin antagonist can be administered in combination with one or more additional therapies directed against botulinum neurotoxin poisoning and/or botulinum infection.
  • the administration of two or more therapies, including one or more botulinum neurotoxin antagonists can be simultaneous, sequential, or in combination. Accordingly, when two or more therapies are administered, they need not be administered simultaneously or in the same way or in the same dose.
  • the two or more therapies can be administered in the same composition or in different compositions.
  • the two or more therapies can be administered using the same route of administration or different routes of administration.
  • the therapies can be administered before or after each other.
  • Administration order of the two or more therapies can be alternated.
  • the respective doses of the one or more therapies can be varied over time.
  • the type of one or more therapy can be varied over time.
  • the separation of the two or more administrations can be any time period. If administered multiple times, the length of the time period can vary.
  • the separation between administration of the two or more therapies can be 0 second, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30, minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, three months, six months, 1 year or longer.
  • Two or more botulinum neurotoxin antagonists can act synergistically to treat or reduce botulinum neurotoxin poisoning or a symptom of the same.
  • Symptoms of botulism include, for example, paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, in ability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, and general muscle reflex loss.
  • Botulism can be confirmed in the laboratory by demonstration of toxin in clinical specimens or in samples of ingested foods. Wound cultures yielding the organism are highly suggestive in symptomatic cases. Confirmation of botulism can be performed by mouse bioassay. Neutralization of the paralysis in mice by a particular antitoxin indicates that toxin serotype in clinical sample.
  • a botulinum neurotoxin antagonist can be one or more anti-botulinum neurotoxin antibodies alone or in combination with one or more other botulinum neurotoxin antagonist, for example, a small drug pharmaceutical, or other anti-botulinum neurotoxin therapy.
  • small drug pharmaceuticals include antibiotics such as penicillin.
  • Other anti-botulinum therapies include heptavalent botulinum antitoxin (HBAT).
  • HBAT can contain horse-serum derived antibody fragments to neurotoxins A-G, and can contain, for example, less than about 2.0% intact immunoglobulin, and greater than or equal to about 90% Fab and F(ab')2 antibody fragments.
  • Two or more anti-botulinum neurotoxin antibodies, or at least one anti- botulinum neurotoxin antibody and one or more additional therapies can act synergistically to treat or reduce a botulinum neurotoxin poisoning.
  • Two or more therapies, including one or more anti-botulinum neurotoxin antibody can be administered in synergistic amounts. Accordingly, the administration of two or more therapies can have a synergistic effect on the decrease in one or more symptoms of botulinum neurotoxin poisoning, whether administered simultaneously, sequentially, or in any combination.
  • a first therapy can increase the efficacy of a second therapy greater than if second therapy was employed alone, or a second therapy increases the efficacy of a first therapy, or both.
  • the effect of administering two or more therapies can be such that the effect on decreasing one or more symptoms of botulinum neurotoxin poisoning is greater than the additive effect of each being administered alone.
  • one therapy can enhance the efficacy of one or more other therapy on the decrease in one or more symptoms of a botulinum neurotoxin poisoning, even if the amount of one or more therapy alone would have no substantial effect on one or more symptom of botulinum neurotoxin poisioning.
  • Measurements and calculations of synergism can be performed as described in Teicher, "Assays for In Vitro and In Vivo Synergy," in Methods in Molecular Medicine, vol. 85: Novel Anticancer Drug Protocols, pp. 297-321 (2003) and/or by calculating the combination index (CI) using CalcuSyn software.
  • Exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See, e.g., Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. I.]
  • the attending physician can determine when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician can also adjust treatment to higher levels if the clinical response were not adequate, precluding toxicity.
  • the magnitude of an administrated dose in the management of disorder of interest will vary with the severity of the disorder to be treated and the route of administration. The severity of the disorder can, for example, be evaluated, in part, by standard prognostic evaluation methods.
  • the dose and dose frequency can vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above can be used in veterinary medicine.
  • compositions relevant to the present invention can be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds relevant to the present invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, tablets, dragees, solutions, suspensions and the like, for oral ingestion by a patient to be treated.
  • the therapeutic agent can be prepared in a depot form to allow for release into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150).
  • Depot forms of therapeutic agents can be, for example, an implantable composition containing the therapeutic agent and a porous or non-porous material, such as a polymer, wherein the therapeutic agent is encapsulated by or diffused throughout the material and/or degradation of the non-porous material.
  • the depot is then implanted into the desired location within the body and the therapeutic agent is released from the implant at a predetermined rate.
  • the therapeutic agent that is used in the present invention can be formed as a composition, such as a pharmaceutical composition containing a carrier and a therapeutic compound.
  • Pharmaceutical compositions containing the therapeutic agent can include more than one therapeutic agent.
  • the pharmaceutical composition can alternatively contain a therapeutic agent in combination with other pharmaceutically active agents or drugs.
  • the carrier can be any suitable carrier.
  • the carrier can be a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used with consideration of chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the therapeutic compounds of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • the pharmaceutically acceptable carriers described herein for example, vehicles, adjuvants, excipients, and diluents; are well-known to those skilled in the art and are readily available to the public.
  • the pharmaceutically acceptable carrier can be chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier can be determined in part by the particular therapeutic agent, as well as by the particular method used to administer the therapeutic compound.
  • suitable formulations of the pharmaceutical composition of the present invention There are a variety of suitable formulations of the pharmaceutical composition of the present invention.
  • formulations for oral, aerosol, parenteral, subcutaneous, transdermal, transmucosal, intestinal, intramedullary injections, direct intraventricular, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intraperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. More than one route can be used to administer the therapeutic agent, and in some instances, a particular route can provide a more immediate and more effective response than another route. Depending on the specific disorder being treated, such agents can be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990).
  • Formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations can include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant.
  • Capsule forms can be of the ordinary hard or soft shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients.
  • Lozenge forms can contain the inhibitor in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles containing the inhibitor in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.
  • an inert base such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers can be added.
  • the therapeutic agent can be made into aerosol formulations to be administered via inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressurized preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.
  • Topical formulations are well known to those of skill in the art. Such formulations are particularly suitable in the context of the invention for application to the skin.
  • Injectable formulations are in accordance with the present invention.
  • the parameters for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art [see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622 630 (1986)].
  • the agents of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • Formulations suitable for parenteral administration can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the therapeutic agent can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, poly(ethyleneglycol) 400, glycerol, dimethylsulfoxide, ketals such as 2,2-dimethyl-l,3- dioxolane-4-methanol, ethers, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
  • Oils which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations can contain from about 0.5% to about 25% by weight of the drug in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophilic-lipophilic balance
  • parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition involving the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the therapeutic agent can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art.
  • such agents can be encapsulated into liposomes.
  • Liposomes are spherical lipid bilayers with aqueous interiors. Molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior.
  • the liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intra-cellularly.
  • Materials and methods described for one aspect of the present invention can also be employed in other aspects of the present invention. For example, a material such a nucleic acid or antibody described for use in screening assays can also be employed as therapeutic agents and vice versa.
  • the present invention includes the following aspects/embodiments/features in any order and/or in any combination: 1.An isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof comprising a neutralization activity against a botulinum neurotoxin type B, wherein the monoclonal antibody comprises a human monoclonal antibody, a humanized monoclonal antibody, or both.
  • BONT/B1 strain Okra
  • BoNT/B2 strain 111
  • BoNT/B6 strain Osaka05
  • PBMC peripheral blood mononuclear cell
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising an IgG, a Fab, a Fab', a F(ab')2, a scFv, a dsFv, or a combination thereof.
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising: a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or 21; and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
  • CDR1 complementarity determining region
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising: a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7; and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
  • CDR1 complementarity determining region
  • CDR2 second complementarity determining region
  • the isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising: a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21; and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28.
  • CDR1 complementarity determining region
  • CDR2 second complementarity determining region
  • the botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising: a heavy chain variable region comprising SEQ ID NO: 3 or 17, and a light chain variable region comprising SEQ ID NO: 10 or 24.
  • 14.A hybridoma which has the Deposit number of NITE BP-01639 or NITE BP-01640.
  • a pharmaceutical composition comprising one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, and a pharmacologically acceptable carrier.
  • the pharmaceutical composition comprising the anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect and a pharmacologically acceptable carrier.
  • the pharmaceutical composition comprising two isolated anti-botulinum neurotoxin type B monoclonal antibodies, antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect, comprising: a first isolated human monoclonal antibody or antigen-binding fragment thereof comprising (1) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity
  • a method for producing an isolated anti-botulinum neurotoxin type B monoclonal antibody comprising: producing a hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid with a fusion partner cell capable of efficient cell fusion; and obtaining a botulinum neurotoxin type B monoclonal antibody from the hybridoma.
  • PBMC peripheral blood mononuclear cell
  • the method for producing the isolated anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect wherein the botulinum neurotoxin type B is produced by BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  • the fusion partner cell is a SPYMEG cell.
  • 22.A kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject comprising the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect.
  • 23.A method of inhibiting or treating botulism in a human subject comprising administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect to the human subject.
  • the at least one symptom comprises paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, inability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, general muscle reflex loss, or any combination thereof.
  • 29. The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the combination acts synergistically to inhibit or treat botulism.
  • 30. The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the one or more additional therapies comprise administering a second isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
  • a method of detecting botulinum neurotoxin type B in a human subject comprising; contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect; and detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.
  • the present invention will be further clarified by the following examples, which are intended to be exemplary of, but not limiting, the present invention. These examples demonstrate the surprising and unexpected properties of the claimed antibodies including, for example, their ability to neutralize botulinum poisoning and treat botulism infections. The examples demonstrate development of human monoclonal antibodies against botulinum neurotoxins in accordance with the present invention. The data demonstrates the therapeutic and diagnostic uses of the present invention.
  • HuMAbs (M-2 + M-4) neutralized other subtypes, BoNT/B2 and BoNT/B6, in addition to BoNT/B1.
  • the data obtained from experiments with the M-2 and M-4 human monoclonal antibodies are representative generally of the antibodies and methods of producing and using the same in accordance with present invention.
  • Clostridium botulinum type B strain okra was cultured using a cellophane-tube procedure, and the culture supernatant was obtained.
  • Progenitor toxins (16S toxin and 12S toxin) were purified from culture supernatant using (Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003).
  • BoNT/B1 and NAP-16 were prepared from 16S toxin as described (Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003).
  • BoNT/A1 strain 62A
  • BoNT/B2 strain 111
  • BoNT/B6 strain Osaka05
  • a tetravalent botulinum toxoid vaccine was provided by Dr. M. Takahashi. 12S toxins were purified from each culture supernatants of C. botulinum types A, B, E, F and detoxification with formalin. Detoxification of toxins was tested with mice intra-peritoneally (i.p.).
  • the toxoid preparations of the four types were mixed together and then mixed with aluminum (adjuvant) and thimerosal (preserving agent).
  • the toxoid preparation contained 0.1 mg each of the four types, 0.2 mg of aluminum, 0.001% of thimerosal, and 0.0006% of formalin per ml.
  • a tetravalent botulinum toxoid (types A, B, E, and F) was inoculated to two healthy adult volunteers (4 or 5 times), who gave written informed consents and received physical examination. The toxoid was injected intramuscularly in 0.5 ml doses to healthy individuals only. After 9 and 18 days, peripheral blood samples (10 ml) were collected from each volunteer. Blood samples were collected from each volunteer and the antibody titers of plasma samples against BoNT/A or BoNT/B were tested using ELISA. This protocol was approved by the Institutional Review Board of Osaka University.
  • ELISA enzyme-linked immunosorbent assay
  • HRP horseradish peroxidase
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs were fused with SPYMEG cells at a ratio of 10:1 with polyethylene glycol (Roche).
  • Fused cells were cultured in Dulbecco's modified Eagle medium (DMEM, GIBCO) supplemented with 15% FBS in 96-well plates for about 10-14 days in the presence hypoxanthine-aminopterin-thymidine (HAT, GIBCO).
  • DMEM Dulbecco's modified Eagle medium
  • HAT hypoxanthine-aminopterin-thymidine
  • the first screening of the culture medium for antibodies specific to BoNT/A or BoNT/B was performed by ELISA. As a result, 27 positive wells were obtained from the blood samples collected 9 days after the vaccination, and 8 positive wells from the blood samples collected 18 days after the vaccination. Specific antibody positive wells were next subjected to cell cloning by limiting dilution, and clonal cells were obtained. The second screening was also performed by ELISA. Eight stable hybridoma clones were obtained (Table 2).
  • M-1, M-2, M-4 and S-1 are IgG
  • M-3, M-5 and M-6 are IgM
  • M-7 is IgA (Table 3).
  • IgG antibodies were purified from the supernatant by Protein G column (GE Healthcare). The subclass of each IgG antibodies was determined using IgG subclass human ELISA kit (Invitrogen).
  • the coding region of the H- and L-chains of M-2 and M-4 antibodies was amplified by PCR with KOD-Pus-Neo (Toyobo) and the following primers: 5'-ATGGACTGGACCTGGAGGATCCTC-3' (M-2-H-chain sense primer) (SEQ ID NO: 35), 5'-ATGAAACACCTGTGGTTCTTCCTCCT-3' (M-4-H-chain sense primer) (SEQ ID NO: 36), and 5'-CTCCCGCGGCTTTGTCTTGGCATTA-3' (H-chain antisense primer) (SEQ ID NO: 38); and 5'-ATGGCCTGGWYYCCTCTCYTYCTS-3' (M-4-16-L-chain sense primer) (SEQ ID NO: 38), 5'-ATGSCCTGGGCTCYKCTSCTCCTS-3' (M-2- and M-4-18-L-chain sense primer) (SEQ ID NO: 39), 5'-ATGGCCTGGRYCYCMYTCYWCCTM-3' (
  • PCR products were ligated into pGEM-T Easy Vector (Promega) and their sequences were analyzed using a BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
  • IgG subclass assay revealed that M-2 and M-4 are comprised of IgG1 heavy chain and light (lambda) chain.
  • BoNT/A or BoNT/B 600 ng was separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane (BIO-RAD).
  • the membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween.
  • HuMAbs 1.0 micrograms/ml was added to the membrane and incubated for 1 hour at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 hour at room temperature, and then specific bands were visualized with ECL (PIERCE) (FIGS. 9A and 9B).
  • HuMAb binding of HuMAb to BoNT, progenitor toxin, or non-toxic component (NAP-16) of type B was analyzed by ELISA.
  • HuMAbs were added to the plate coated with BoNT, progenitor toxin, or NAP-16.
  • HuMAbs (0.01 - 0.5 micrograms/ml) were added to the plate coated with BoNT/A or BoNT/B. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • M-2 and M-4 bound to BoNT and progenitor toxins. In contrast, M-2 and M-4 showed faint binding to NAP-16.
  • HuMAbs were incubated with BoNT/B (10 LD 50 ) at room temperature for 1 hour prior to intraperitoneal injection (in a total volume of 500 microliter) into mice (ddY, female, 4 weeks, SLC). 10 LD 50 of BoNT/B was incubated with 100 micrograms of HuMAb or PBS (Cnt) for 1 hour, and injected intraperitoneally into mice. Mice were observed for morbidity and mortality for 3 weeks. Mice injected with BoNT/B + PBS (Cnt) died within 12 hours.
  • BoNT/B 10 LD 50
  • mice injected with BoNT/B + M-4 were partially protected, as evidenced by an increased time-to-death compared to those of control mice.
  • complete protection was observed in mice injected with BoNT/B + M-2 (Table 4).
  • mice were orally administrated with the type B progenitor toxin (16S toxin, 10 ng in a volume of 300 microliters), and subsequently administrated with HuMAbs (M-2 + M-4) by i.p. injection at 12, 24, or 36 hours after the oral administration of 16S toxin. Mice were observed for morbidity and mortality for 3 weeks.
  • HuMAbs M-2 + M-4
  • mice developed symptoms of botulism at 12 hours after oral administration of 16S toxin. Control mice that were not treated with HuAbs died within 72 hours.
  • post-exposure treatment of HuMAbs provided complete survival at 12 hours after oral administration of 16S toxin, and partial survival at 24 and 36 hours after administration (FIG. 13).
  • HuMAbs (M-2 and M-4) to BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) were analyzed by ELISA.
  • HuMAbs (0.5 micrograms/ml) were added to the plate coated with BoNT. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • M-2 and M-4 showed strong binding to BoNT/B2 and BoNT/B6 in addition to BoNT/B1.
  • M-2 + M-4 (0.5 micrograms + 0.5 micrograms) completely neutralized BoNT/B2 (10 ng) and BoNT/B6 (2.5 ng) (FIG. 14).
  • IgG-expression vectors were constructed as follows.
  • pGEM-T Easy Vectors with the variable region gene of H- and L-chains were subjected to PCR to add restriction enzyme sites and a Kozak sequence with the following primer sets (restriction enzyme sites are underlined): 5'-ATTT GCGGCCGC CATGGACTGGACCTGGAGG-3' (M2-H-chain sense primer) (SEQ ID NO: 42), 5'-ATTT GCGGCCGC CATGAAACACCTGTGGTTCTTC-3' (M-4-H-chain sense primer) (SEQ ID NO: 43) and 5'-ATA CTCGAG GGTGCCAGGGGGAAGACCGATG-3' (H-chain antisense primer) (SEQ ID NO:44); and 5'-ATTT GCGGCCGC CATGGCCTGGTTTCCTCTCTTC-3' (M-4-16-L-chain sense primer
  • PCR products of the H- and L-chain coding regions were digested by restriction enzymes, Not I and Xho I, and then ligated to expression vectors, pQCXIP-hCH and pQCXIH-hC, which have a human immunoglobulin-constant region of gamma and lambda chains (MBL), respectively.
  • Not I and Xho I restriction enzymes
  • pQCXIP-hCH and pQCXIH-hC which have a human immunoglobulin-constant region of gamma and lambda chains (MBL), respectively.
  • HEK293 cells were cultured in 5% CO 2 at 37 deg C in MEM containing 10% FBS. Cells grown on 100 mm culture dish were used for transient transfection with pQCXIP-hCH and pQCXIH-hC expression vectors using LIPOFECTAMINE2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After transfection, the medium was removed and cells were washed and incubated in Opti-Pro Serum Free Medium (Gibco) in 5% CO 2 at 37 deg C for 10 days. Recombinant HuMAbs in culture supernatant were determined with the anti-human IgG conjugated with HRP (BIO-RAD). Recombinant HuMAbs were purified from the supernatant by Protein G column (GE Healthcare).
  • RM-2 and RM-4 Binding of recombinant HuMAbs (RM-2 and RM-4; depicted as RM-4 LC16, RM-4 LC18, RM-4 LC19, which have the same heavy chain and light chain of M-4, but have different signal sequences of light chain shown in FIG. 6, 7, 8 respectively) to BoNT/B were analyzed by ELISA.
  • RM-2 and RM-4 (about 0.01-0.5 micrograms/ml) were added to the plate. After washing, bound recombinant HuMAbs were detected by anti-human IgG antibody conjugated with HRP.
  • HRP anti-human IgG antibody conjugated with HRP.
  • RM-2 and RM-4 bound to BoNT/B in the same manner as the M-2 and M-4 derived from hybridoma (FIG. 15).
  • RM-2 + RM-4 0.5 micrograms + 0.5 micrograms neutralized BoNT/B1 (10 LD 50 ) neutralized BoNT/B1 (10 LD 50
  • HuMAbs derived from hybridomas and recombinant HuMAbs (RM-2 and RM-4) to BoNT/B were analyzed by SDS-PAGE.
  • BoNT/B1, BoNT/B2 or BoNT/B6 were separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane.
  • HuMAbs (1.0 micrograms/ml) was added to the membrane and incubated for 1 h at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 h at room temperature, and then specific bands were visualized with ECL.
  • Recombinant HuMAbs showed the same binding profile as HuMAbs derived from hybridomas.
  • M-2 and RM-2 specifically bound to light chain of BoNT/B.
  • M-4 and RM-4 bound to both of light chain and heavy chain.
  • heavy chain of B2 subtype was detected more clearly than that of B1.
  • heavy chain of B6 subtype was detected more clearly than that of B2 (FIG. 16).

Abstract

Isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragments thereof having a neutralization activity against a botulinum neurotoxin type B are provided, including a human monoclonal antibody. Hybridomas that produce such antibodies or fragments thereof are also provided by the present invention, as well as methods of producing such hybridomas and method of producing antibodies or fragments thereof from such hybridomas. Pharmaceutical compositions and kits including antibodies or fragments thereof for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning are further provided. Methods of inhibiting or treating botulism in a human subject are provided, as are methods of detecting botulinum neurotoxin type B.

Description

BINDING/NEUTRALIZING HUMAN MONOCLONAL ANTIBODIES AGAINST BOTULINUM NEUROTOXIN TYPE B
The present invention relates to anti-botulinum neurotoxin (botulin) antibodies and methods of using and manufacturing the same.
RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application No. 61/695,318, filed on August 31, 2012, which is incorporated herein by reference in its entirety.
Botulinum neurotoxins are among the most toxic substance known. These toxins can cause botulism and are produced by various bacteria including Clostridium botulinum. Botulinum toxin-producing clostridia are anaerobic gram-positive bacteria. These bacteria form spores and are commonly distributed in the environment. On a molecular level, botulism is caused by the inhibition of acetylcholine release at neuromuscular junctions by the toxin. Seven toxin serotypes (A, B, C, D, E, F, and G) have been identified. They have similar structures corresponding to an approximately 150kD zinc-endopeptidase protein including a 100-kDa heavy chain and a 50-kDa light chain. Toxin serotypes A, B, E, and F are generally associated with botulism in humans.
Botulism can be grouped into various categories-foodborne, wound infection, infant intestinal toxemia, adult intestinal toxemia, toxin injection (iatrogenic botulism), and weaponized toxin. The foodborne illness occurs due to ingestion of toxin in food contaminated with toxin-producing bacteria. In wound infection, a wound is colonized by toxigenic clostridia that then grow and produce toxins in the wound. In infant botulism, the infant intestine is colonized by toxigenic clostridia, which then produce toxins. Adult intestinal toxemia is similar to infant botulism. Cosmetic or therapeutic injection of botulinum toxin can sometimes lead to botulism. Botulism can also result from injury by weaponized toxin, for example, by inhalation of aerosolized botulinum toxin.
Botulism is characterized by symmetric cranial nerve palsies that are followed by descending symmetric flaccid paralysis of voluntary muscles. This paralysis can result in loss of respiration and death. Toxin binding is irreversible. Recovery involves the growth of new nerve terminals. Recovery can be lengthy and involve extended outpatient rehabilitation therapy. The onset of the disease can be swift following contact with the toxin. Treatment usually involves administration of antitoxin, which can help limit the extent of paralysis. Most antitoxin available is horse-derived and is not necessarily specific to a particular type of Botulinum neurotoxin. There exists a need for more targeted forms of antitoxin. There is also a need for effective toxin-neutralizing antibodies without side effects such as serum sickness. There further exists a need for better diagnostic tests given the rapid course of botulism.
[NPL 1] Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003
It is therefore a feature of the present invention to provide therapeutic antibodies against botulinum neurotoxins having superior efficacy and safety.
Another feature of the present invention is to provide methods of inhibiting and treating botulism.
A further feature of the present invention is to provide therapeutics without side effects such as serum sickness.
Additional features and advantages of the present invention will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the present invention. The objectives and other advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.
To achieve these and other advantages, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention relates to a an isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof having a neutralization activity against a botulinum neurotoxin type B. The monoclonal antibody can include a human monoclonal antibody, a humanized monoclonal antibody, or both. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a progenitor toxin of botulinum neurotoxin type B. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B) or have a specific binding activity to both a light chain and a heavy chain of a botulinum neurotoxin type B (BoNT/B ). Hybridomas that produce the antibodies or fragments thereof of the present invention are also provided by the present invention, as well as methods of producing such hybridomas and method of producing antibodies or fragments thereof from such hybridomas.
The present invention further relates to pharmaceutical compositions. The pharmaceutical composition can contain one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of and a pharmacologically acceptable carrier. A kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject is provided by the present invention. The kit can include an isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of the present invention. Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to manufacture a medicament for inhibiting or treating botulism in a human subject is also provided by the present invention.
The present invention also relates to a method of inhibiting or treating botulism in a human subject. The method can include administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to the human subject. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. The method can include monitoring for a decrease in at least one symptom of botulism. The method of inhibiting or treating botulism in a human subject can include administering the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both in combination with one or more additional therapies directed to botulism. The combination can act synergistically to inhibit or treat botulism.
The present invention still further relates to a method of detecting botulinum neurotoxin type B in a human subject. The method can include contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both. The method can also include detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide a further explanation of the present invention, as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate some of the embodiments of the present invention and together with the description, serve to explain the principles of the present invention.
FIG. 1 is a schematic diagram showing how volunteers were immunized and how antibody producing cells were isolated in accordance with the present invention. FIG. 2 is a flow diagram depicting a cell fusion and cloning schedule in accordance with the present invention. FIG. 3 shows nucleotide and amino acid sequences for the M-2 antibody heavy chain in accordance with the present invention. FIG. 4 shows nucleotide and amino acid sequences for the M-2 antibody light chain in accordance with the present invention. FIG. 5 shows nucleotide and amino acid sequences for the M-4 antibody heavy chain in accordance with the present invention. FIG. 6 shows nucleotide and amino acid sequences for light chain of the RM-4LC-16 antibody in accordance with the present invention. FIG. 7 shows nucleotide and amino acid sequences for light chain of the RM-4LC-18 antibody in accordance with the present invention. FIG. 8 shows nucleotide and amino acid sequences for light chain of the RM-4LC-19 antibody in accordance with the present invention. FIG. 9 includes line graphs showing binding of human monoclonal antibodies to BoNT/A or BoNT/B in accordance with the present invention. Binding of human monoclonal antibody (HuMAb) to BoNT was analyzed by ELISA. HuMAbs (bout 0.05-1.0 micrograms/ml) were added to the plate coated with BoNT/A or BoNT/B. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP. M-2 and M-4 showed specific binding to BoNT/B. FIG. 10 includes Western blots for epitope mapping of human monoclonal antibodies in accordance with the present invention. BoNT was separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane. HuMAbs (1.0 micrograms/ml) was added to the membrane and incubated for 1 hour at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 hour at room temperature, and then specific bands were visualized with ECL. M-2 specifically bound to light chain of BoNT/B. In contrast, M-4 bound to both of light chain and heavy chain. FIG. 11 is a bar graph showing binding of human monoclonal antibodies to BoNT/B, progenitor toxin, and non-toxic component in accordance with the present invention. Binding of HuMAb to BoNT, progenitor toxin (12S toxin and 16S toxin) or non-toxic component (NAP-16) was analyzed by ELISA. HuMAbs were added to the plate coated with BoNT, progenitor toxin or NAP-16. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP. M-2 and M-4 bound to BoNT and progenitor toxins. In contrast, M-2 and M-4 showed faint binding to NAP-16. These results show that M-2 and M-4 bind to BoNT in the progenitor toxins, as well as BoNT alone, and that M-2 and M-4 specifically bind to BoNT. FIG. 12 is a line graph showing neutralization activity of a combination of human monoclonal antibodies against BoNT/B in accordance with the present invention. 10 LD50 of BoNT/B was incubated with a mixture of M-2 and M-4 (0.05, 0.1 or 0.5 micrograms each) and injected into mice. All of the mice that received the mixture of M-2 (0.5 micrograms) and M-4 (0.5 micrograms) survived and had no symptoms. FIG. 13 is a line graph showing neutralization activity of post-administration of human monoclonal antibodies against progenitor toxin (16S toxin) in accordance with the present invention. Mice were orally administrated with progenitor toxin (16S toxin, 10 ng), and subsequently administrated with HuMAbs (M-2 0.5 micrograms + M-4 0.5 micrograms) by i.p. injection at 12, 24, or 36 hours after the oral administration of 16S toxin. Control mice that were not treated with HuMAbs died within 72 hours. In contrast, post-exposure treatment of HuMAbs provided complete survival at 12 hours after oral administration of 16S toxin, and partial survival at 24 and 36 hours after administration. FIG. 14 includes a bar graph and line graphs showing binding and neutralization of human monoclonal antibodies against BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) in accordance with the present invention. Binding of HuMAbs (M-2 and M-4) to BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) were analyzed by ELISA. HuMAbs (0.5 micrograms/ml) were added to the plate coated with BoNT. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP. M-2 and M-4 showed strong binding to BoNT/B2 and BoNT/B6 in addition to BoNT/B1. In the neutralization test, M-2 + M-4 (0.5 micrograms + 0.5 micrograms) completely neutralized BoNT/B2 (10 ng) and BoNT/B6 (2.5 ng). FIG. 15 includes Western blots and line graphs showing binding of recombinant human monoclonal antibodies to BoNT/B in accordance with the present invention. HEK293 cells were transiently transfected with pQCXIP-hCH and pQCXIH-hCl expression vectors using LIPOFECTAMINE 2000 transfection reagent. Recombinant HuMAbs in the culture supernatant were determined with the anti-human IgG conjugated with HRP. Binding of recombinant M-2 (RM-2) and recombinant M-4 (RM-4) to BoNT/B were analyzed by ELISA. Recombinant HuMAb (about 0.01-0.5 micrograms/ml) were added to the BoNT/B coated plate. After washing, bound recombinant HuMAbs were detected by anti-human IgG antibody conjugated with HRP. RM-2 and RM-4 bound to BoNT/B in the same manner as the M-2 and M-4 derived from hybridoma. FIG. 16 includes Western blots Binding of HuMAbs derived from hybridomas and recombinant HuMAbs (RM-2 and RM-4) to BoNT/B (BoNT/B1, BoNT/B2 or BoNT/B6). BoNT/B1, BoNT/B2 or BoNT/B6 were separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane. HuMAbs (1.0 micrograms/ml) was added to the membrane and incubated for 1 h at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 h at room temperature, and then specific bands were visualized with ECL. Recombinant HuMAbs showed the same binding profile as HuMAbs derived from hybidomas. M-2 and RM-2 specifically bound to light chain of BoNT/B. In contrast, M-4 and RM-4 (RM-4 LC16, RM-4 LC18, RM-4 LC19, have same heavy chain of M-4) bound to both of light chain and heavy chain. By M-4 and RM-4 antibodies, heavy chain of B2 subtype was detected more clearly than that of B1.Further, heavy chain of B6 subtype was detected more clearly than that of B2.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
An isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof having a neutralization activity against a botulinum neurotoxin type B is provided in accordance with the present invention. The monoclonal antibody can include a human monoclonal antibody, a humanized monoclonal antibody, or both. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a progenitor toxin of botulinum neurotoxin type B. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can have a neutralization activity against a botulinum neurotoxin type B produced by BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof accordingly can have a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B).
The antibody or fragment thereof can be produced using any suitable technique. For example, the isolated anti-botulinum neurotoxin type B monoclonal antibody can be produced by a hybridoma made by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum neurotoxin type B with a fusion partner cell capable of efficient cell fusion. The botulinum neurotoxin type B can be a product of BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof. Any suitable fusion partner cell can be used, for example, a SPYMEG cell.
Hybridomas that produce the antibodies or fragments thereof of the present invention are also provided by the present invention. For example, the hybridoma can have the Deposit number of NITE BP-01639 or NITE BP-01640. An isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639 or NITE BP-01640 is also provided. Examples of monoclonal antibodies obtained as described above include a monoclonal antibody produced by the hybridoma named "Hybridoma M-2 (M1E9)" (hereinafter, referred to as M-2 antibody) and "Hybridoma M-4 (M4C9)" (hereinafter, referred to as M-4 antibody). The hybridoma was deposited at the NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (#122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) under Deposition No. NITE BP-01639 and No. NITE BP-01640 on Jun. 26, 2013. Then, they were transferred to the international deposit under Budapest Treaty under the same No.
The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can be IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgD, IgE, any fragment thereof, or any combination thereof. The antibody fragment can include, for example, a Fab, a Fab', a F(ab')2, a scFv, a dsFv, or a combination thereof. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof can include a heavy chain and/or light chain variable region. For example, the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or 20, a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or 21. The light chain variable region can include, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
In another example, the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7. The light chain variable region can include a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
In yet another example, the heavy chain variable region can include a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21. The light chain variable region can include, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28. In a further example, the heavy chain variable region can include SEQ ID NO: 3 or 17, and the light chain variable region can include SEQ ID NO: 10 or 24.
Pharmaceutical compositions are provided by the present invention. The pharmaceutical composition can contain one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of and a pharmacologically acceptable carrier. The pharmaceutical composition can contain the anti-botulinum neurotoxin type B monoclonal antibody and a pharmacologically acceptable carrier. The pharmaceutical composition can contain two isolated anti-botulinum neurotoxin type B monoclonal antibodies, antigen-binding fragment thereof, or both. The pharmaceutical composition can contain first and second antibodies. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (1) an isolated human monoclonal antibody or antigen-binding fragment including a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (2) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (3) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit No. NITE BP-01639. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (4) an isolated human monoclonal antibody or antigen-binding fragment including a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21, and a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (5) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 24. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (6) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit No. NITE BP-01640. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above.
A method for producing an isolated anti-botulinum neurotoxin type B monoclonal antibody is provided by the present invention. The method can include producing a hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum neurotoxin type B with a fusion partner cell capable of efficient cell fusion. The method can further include obtaining a botulinum neurotoxin type B monoclonal antibody from the hybridoma. The botulinum neurotoxin type B is produced, for example, by BONT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof. The fusion partner cell can be, for example, a SPYMEG cell.
A kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject is provided by the present invention. The kit can include an isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of the present invention. The kit can also include one or more additional agents for treating and/or detecting botulinum neurotoxin type B poisoning. The kit can also include one or more agents for treating and/or detecting one or more additional types of botulinum neurotoxins, for example, type A. Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to manufacture a medicament for inhibiting or treating botulism in a human subject is also provided by the present invention.
A method of inhibiting or treating botulism in a human subject is provided by the present invention. The method can include administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both to the human subject. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. The method can include monitoring for a decrease in at least one symptom of botulism. The at least one symptom can include, for example, paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, inability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, general muscle reflex loss, any other symptom of botulism, or any combination thereof. Any type of botulism can be inhibited or treated in a human subject, for example, foodborne botulism, wound infection botulism, infant intestinal toxemia botulism, adult intestinal toxemia botulism, iatrogenic botulism, airborne botulism, or any combination thereof.
The method of inhibiting or treating botulism in a human subject can include administering the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both in combination with one or more additional therapies directed to botulism. The combination can act synergistically to inhibit or treat botulism. The one or more additional therapies can include, for example, administering a second isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both. The one or more additional therapies can include, for example, administering an isolated anti-botulinum neurotoxin type A monoclonal antibody, an antigen-binding fragment thereof, or both. The one or more additional therapies can include, for example, one or more antibiotics, for example, a penicillin, or any combination thereof.
A method of detecting botulinum neurotoxin type B in a human subject is provided by the present invention. The method can include contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both. The method can also include detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B. The method can further include detecting one or more additional types of botulinum neurotoxins, for example, type A.
Anti-botulinum neurotoxin antibodies and polypeptides containing antigen binding fragments thereof are provided as well as methods, uses, compositions, and kits employing the same. Unless otherwise stated, the isolated monoclonal antibodies and antigen-binding fragments thereof described and claimed in the present application are not products of nature. These monoclonal antibodies and antigen-binding fragments thereof are the product of a hybridoma or equivalent artificial cell system generated using various laboratory procedures. Methods of using antibodies or antigen-binding fragments thereof for therapeutic, diagnostic, or other purposes, compositions including the same, kits including same, and/or non-naturally occurring antibodies or antibody fragments are not so limited unless explicitly specified.
A method of forming an antibody specific to a botulinum neurotoxin or a polypeptide or a fragment thereof is provided. Such a method can contain providing a nucleic acid encoding a botulinum neurotoxin antigen polypeptide or a polypeptide containing an immunologically specific epitope thereof; expressing the polypeptide containing the antigen amino acid sequence or a polypeptide containing an immunologically specific epitope thereof from the isolated nucleic acid; and generating an antibody specific to the polypeptide obtained or a polypeptide containing an antigen binding fragment thereof. An antibody or polypeptide containing an antigen binding fragment thereof produced by the aforementioned method is provided. An isolated antibody or isolated polypeptide containing an antigen binding fragment thereof that specifically binds a botulinum neurotoxin antigen is provided. Such an antibody can be generated using any acceptable method(s) known in the art. The antibodies as well as kits, methods, and/or other aspects of the present invention employing antibodies can include one or more of the following: a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, a monovalent antibody, a diabody, and/or a humanized antibody.
Naturally occurring antibody structural units typically contain a tetramer. Each such tetramer can be composed of two identical pairs of polypeptide chains, each pair having one full-length light" (for example, about 1 kDa to 25 kDa) and one full- length "heavy" chain (for example, about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. In light and heavy chains, the variable and constant regions can be joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). The variable regions of each light/heavy chain pair typically form the antigen binding site.
The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically contain the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. MoI. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).
"Antibody fragments" include a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab1, F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Fv" is an antibody fragment which contains a complete antigen-recognition and -binding site. This region includes a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. A single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can recognize and bind an antigen. "Single-chain Fv" or "sFv" antibody fragments include the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further contain a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).
Antibodies can be used as probes, therapeutic treatments and other uses. Antibodies can be made by injecting mice, rabbits, goats, or other animals with the translated product or synthetic peptide fragments thereof. These antibodies are useful in diagnostic assays or as an active ingredient in a pharmaceutical composition.
The antibody or polypeptide administered can be conjugated to a functional agent to form an immunoconguate. The functional agent can be a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate), an antibiotic, a nucleolytic enzyme, or any combination thereof. Chemotherapeutic agents can be used in the generation of immunoconjugates, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes, and/or fragments thereof, such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricotheeenes. Any appropriate radionucleotide or radioactive agent known in the art or are otherwise available can be used to produce radioconjugated antibodies.
Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2- pyridyldithiol)propionate (SPDP); iminothiolane (IT); bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL); active esters (such as disuccinimidyl suberate); aldehydes (such as glutareldehyde); bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine); bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine); diisocyanates (such as tolyene 2,6-diisocyanate); bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene); maleimidocaproyl (MC); valine-citrulline, dipeptide site in protease cleavable linker (VC); 2-amino-5-ureido pentanoic acid PAB=p-aminobenzylcarbamoyl ("self immolative" portion of linker) (Citrulene); N-methyl-valine citrulline where the linker peptide bond has been modified to prevent its cleavage by cathepsin B (Me); maleimidocaproyl-polyethylene glycol, attached to antibody cysteines; N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP); and N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (SMCC). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacctic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody, see WO 94/11026. The antibody can be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody- receptor conjugate is administered to the subject, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionucleotide).
The antibodies of the present invention can be coupled directly or indirectly to a detectable marker by techniques well known in the art. A detectable marker is an agent detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, colored particles, biotin, or dioxigenin. A detectable marker often generates a measurable signal, such as radioactivity, fluorescent light, color, or enzyme activity. Antibodies conjugated to detectable agents can be used for diagnostic or therapeutic purposes. Examples of detectable agents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the antibody or indirectly, through an intermediate such as, for example, a linker known in the art, using techniques known in the art. See, e.g., U.S. Patent No. 4,741,900, describing the conjugation of metal ions to antibodies for diagnostic use. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferin, and aequorin.
Antibodies useful in practicing the present invention can be prepared in laboratory animals or by recombinant DNA techniques using the following methods. Polyclonal antibodies can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the gene product molecule or fragment thereof in combination with an adjuvant such as Freund's adjuvant (complete or incomplete). To enhance immunogenicity, it can be useful to first conjugate the gene product molecule or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, etc. Alternatively, immunogenic conjugates can be produced recombinantly as fusion proteins.
Animals can be immunized against the immunogenic conjugates or derivatives (such as a fragment containing the target amino acid sequence) by combining about 1 mg or about 1 microgram of conjugate (for rabbits or mice, respectively) with about 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. Approximately 7 to 14 days later, animals are bled and the serum is assayed for antibody titer. Animals are boosted with antigen repeatedly until the titer plateaus. The animal can be boosted with the same molecule or fragment thereof as was used for the initial immunization, but conjugated to a different protein and/or through a different cross-linking agent. In addition, aggregating agents such as alum can be used in the injections to enhance the immune response.
The antibody administered can include a chimeric antibody. The antibody administered can include a humanized antibody. The antibody administered can include a completely humanized antibody. The antibodies can be humanized or partially humanized. Non-human antibodies can be humanized using any applicable method known in the art. A humanized antibody can be produced using a transgenic animal whose immune system has been partly or fully humanized. Any antibody or fragment thereof of the present invention can be partially or fully humanized. Chimeric antibodies can be produced using any known technique in the art. See, e.g., U.S. Patent Nos. 5,169,939; 5,750,078; 6,020,153; 6,420,113; 6,423,511; 6,632,927; and 6,800,738.
The antibody administered can include a monoclonal antibody, that is, the anti-botulinum neurotoxin antibodies of the present invention that can be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. Monoclonal antibodies can be screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal Antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies can be tested for specific immunoreactivity with a translated product and lack of immunoreactivity to the corresponding prototypical gene product.
Monoclonal antibodies can be prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g., by fusion with myeloma cells. Preferably, preperipheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid are fused with myeloma cells. The clones are then screened for those expressing the desired antibody. The monoclonal antibody preferably does not cross-react with other gene products. After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the present invention can serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the present invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. Preparation of antibodies using recombinant DNA methods such as the phagemid display method, can be accomplished using commercially available kits, as for example, the Recombinant Phagemid Antibody System available from Pharmacia (Uppsala, Sweden), or the SurfZAPTM phage display system (Stratagene Inc., La Jolla, Califorinia). The present invention provides human monoclonal antibodies (HuMAbs) generated against type A BoNT (BoNT/A) and type B BoNT (BoNT/B) using a murine-human chimera fusion partner cell line, named SPYMEG.
Also included in the present invention are hybridoma cell lines, transformed B cell lines, and host cells that produce the monoclonal antibodies of the present invention; the progeny or derivatives of these hybridomas, transformed B cell lines, and host cells; and equivalent or similar hybridomas, transformed B cell lines, and host cells.
The antibodies can be diabodies. The term "diabodies' refers to small antibody fragments with two antigen-binding sites, which fragments include a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
The antibody administered can include a single-chain antibody. The antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain can be truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
The antibodies can be bispecific. Bispecific antibodies that specifically bind to one protein and that specifically bind to other antigens relevant to pathology and/or treatment are produced, isolated, and tested using standard procedures that have been described in the literature. [See, e.g., Pluckthun & Pack, Immunotechnology, 3:83-105 (1997); Carter, et al., J. Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No. 145, pp. 179-209; Pfreundschuh U.S. Patent No. 5,643,759; Segal, et al., J. Hematotherapy, 4:377-382 (1995); Segal, et al., Immunobiology, 185:390-402 (1992); and Bolhuis, et al., Cancer Immunol. Immunother., 34:1-8 (1991)].
The antibodies disclosed herein can be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art. such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al, J. National Cancer Inst., 81(19): 1484 (1989).
The present invention provides a method of inhibiting or treating botulinum poising in a human subject including administering a therapeutically effective amount of the anti-botulinum neurotoxin monoclonal antibody, antigen-binding fragment thereof of the invention to the human subject, or both. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. Anti-botulinum neurotoxin antibodies or antigen-binding fragment thereof of the present invention can be administered to a subject before, during, and/or after diagnosing the patient as having botulinum neurotoxin poisioning. The method can further include monitoring for a decrease in at least one symptom of botulinum neurotoxin poisioning.
In accordance with the present invention, two or more botulinum neurotoxin antagonists can be administered. At least one of the botulinum neurotoxin antagonists can include a botulinum neurotoxin antagonist. The at least one botulinum neurotoxin antagonist can be combined with one or more additional botulinum neurotoxin antagonists. At least one botulinum neurotoxin antagonist can be administered in combination with one or more additional therapies directed against botulinum neurotoxin poisoning and/or botulinum infection. The administration of two or more therapies, including one or more botulinum neurotoxin antagonists, can be simultaneous, sequential, or in combination. Accordingly, when two or more therapies are administered, they need not be administered simultaneously or in the same way or in the same dose. When administered simultaneously, the two or more therapies can be administered in the same composition or in different compositions. The two or more therapies can be administered using the same route of administration or different routes of administration. When administered at different times, the therapies can be administered before or after each other. Administration order of the two or more therapies can be alternated. The respective doses of the one or more therapies can be varied over time. The type of one or more therapy can be varied over time. When administered at separate times, the separation of the two or more administrations can be any time period. If administered multiple times, the length of the time period can vary. The separation between administration of the two or more therapies can be 0 second, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30, minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 7.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, three months, six months, 1 year or longer.
Two or more botulinum neurotoxin antagonists can act synergistically to treat or reduce botulinum neurotoxin poisoning or a symptom of the same. Symptoms of botulism include, for example, paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, in ability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, and general muscle reflex loss. Botulism can be confirmed in the laboratory by demonstration of toxin in clinical specimens or in samples of ingested foods. Wound cultures yielding the organism are highly suggestive in symptomatic cases. Confirmation of botulism can be performed by mouse bioassay. Neutralization of the paralysis in mice by a particular antitoxin indicates that toxin serotype in clinical sample.
A botulinum neurotoxin antagonist can be one or more anti-botulinum neurotoxin antibodies alone or in combination with one or more other botulinum neurotoxin antagonist, for example, a small drug pharmaceutical, or other anti-botulinum neurotoxin therapy. Examples of small drug pharmaceuticals include antibiotics such as penicillin. Other anti-botulinum therapies include heptavalent botulinum antitoxin (HBAT). HBAT can contain horse-serum derived antibody fragments to neurotoxins A-G, and can contain, for example, less than about 2.0% intact immunoglobulin, and greater than or equal to about 90% Fab and F(ab')2 antibody fragments. Two or more anti-botulinum neurotoxin antibodies, or at least one anti- botulinum neurotoxin antibody and one or more additional therapies can act synergistically to treat or reduce a botulinum neurotoxin poisoning. Two or more therapies, including one or more anti-botulinum neurotoxin antibody, can be administered in synergistic amounts. Accordingly, the administration of two or more therapies can have a synergistic effect on the decrease in one or more symptoms of botulinum neurotoxin poisoning, whether administered simultaneously, sequentially, or in any combination. A first therapy can increase the efficacy of a second therapy greater than if second therapy was employed alone, or a second therapy increases the efficacy of a first therapy, or both. The effect of administering two or more therapies can be such that the effect on decreasing one or more symptoms of botulinum neurotoxin poisoning is greater than the additive effect of each being administered alone. When given in synergistic amounts, one therapy can enhance the efficacy of one or more other therapy on the decrease in one or more symptoms of a botulinum neurotoxin poisoning, even if the amount of one or more therapy alone would have no substantial effect on one or more symptom of botulinum neurotoxin poisioning. Measurements and calculations of synergism can be performed as described in Teicher, "Assays for In Vitro and In Vivo Synergy," in Methods in Molecular Medicine, vol. 85: Novel Anticancer Drug Protocols, pp. 297-321 (2003) and/or by calculating the combination index (CI) using CalcuSyn software.
Exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See, e.g., Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. I.] The attending physician can determine when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician can also adjust treatment to higher levels if the clinical response were not adequate, precluding toxicity. The magnitude of an administrated dose in the management of disorder of interest will vary with the severity of the disorder to be treated and the route of administration. The severity of the disorder can, for example, be evaluated, in part, by standard prognostic evaluation methods. The dose and dose frequency, can vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above can be used in veterinary medicine.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the present invention. With proper choice of carrier and suitable manufacturing practice, the compositions relevant to the present invention, in particular, those formulated as solutions, can be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds relevant to the present invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, tablets, dragees, solutions, suspensions and the like, for oral ingestion by a patient to be treated.
The therapeutic agent can be prepared in a depot form to allow for release into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of therapeutic agents can be, for example, an implantable composition containing the therapeutic agent and a porous or non-porous material, such as a polymer, wherein the therapeutic agent is encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the therapeutic agent is released from the implant at a predetermined rate.
The therapeutic agent that is used in the present invention can be formed as a composition, such as a pharmaceutical composition containing a carrier and a therapeutic compound. Pharmaceutical compositions containing the therapeutic agent can include more than one therapeutic agent. The pharmaceutical composition can alternatively contain a therapeutic agent in combination with other pharmaceutically active agents or drugs.
The carrier can be any suitable carrier. For example, the carrier can be a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used with consideration of chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. In addition to, or in the alternative to, the following described pharmaceutical compositions, the therapeutic compounds of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents; are well-known to those skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier can be chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier can be determined in part by the particular therapeutic agent, as well as by the particular method used to administer the therapeutic compound. There are a variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, transdermal, transmucosal, intestinal, intramedullary injections, direct intraventricular, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intraperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. More than one route can be used to administer the therapeutic agent, and in some instances, a particular route can provide a more immediate and more effective response than another route. Depending on the specific disorder being treated, such agents can be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990).
Formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations can include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard or soft shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can contain the inhibitor in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles containing the inhibitor in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art.
Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added.
The therapeutic agent, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressurized preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. Topical formulations are well known to those of skill in the art. Such formulations are particularly suitable in the context of the invention for application to the skin.
Injectable formulations are in accordance with the present invention. The parameters for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art [see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622 630 (1986)]. For injection, the agents of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Formulations suitable for parenteral administration can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The therapeutic agent can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, poly(ethyleneglycol) 400, glycerol, dimethylsulfoxide, ketals such as 2,2-dimethyl-l,3- dioxolane-4-methanol, ethers, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
The parenteral formulations can contain from about 0.5% to about 25% by weight of the drug in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition involving the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
The therapeutic agent can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents can be encapsulated into liposomes. Liposomes are spherical lipid bilayers with aqueous interiors. Molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intra-cellularly. Materials and methods described for one aspect of the present invention can also be employed in other aspects of the present invention. For example, a material such a nucleic acid or antibody described for use in screening assays can also be employed as therapeutic agents and vice versa.
The present invention includes the following aspects/embodiments/features in any order and/or in any combination:
1.An isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof comprising a neutralization activity against a botulinum neurotoxin type B, wherein the monoclonal antibody comprises a human monoclonal antibody, a humanized monoclonal antibody, or both.
2.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, wherein the monoclonal antibody or antigen-binding fragment thereof further comprises a neutralization activity against a progenitor toxin of botulinum neurotoxin type B.
3.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a neutralization activity against a botulinum neurotoxin type B produced by BONT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
4.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof according to any preceding or following embodiment/feature/aspect, wherein the monoclonal antibody or antigen-binding fragment thereof has a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B).
5.The isolated anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the human monoclonal antibody is produced by a hybridoma made by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid with a fusion partner cell capable of efficient cell fusion.
6.The isolated anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the botulinum neurotoxin type B is a product of BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
7.The isolated anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the fusion partner cell is a SPYMEG cell.
8.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect comprising an IgG, a Fab, a Fab', a F(ab')2, a scFv, a dsFv, or a combination thereof.
9.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, comprising:
a heavy chain variable region comprising
a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or 19,
a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or 20, and
a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or 21; and
a light chain variable region comprising
a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26,
a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27, and
a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
10.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, comprising:
a heavy chain variable region comprising
a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5,
a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and
a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7; and
a light chain variable region comprising
a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12,
a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and
a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
11.The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, comprising:
a heavy chain variable region comprising
a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19,
a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and
a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21; and
a light chain variable region comprising
a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26,
a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and
a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28.
12.The botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, comprising:
a heavy chain variable region comprising SEQ ID NO: 3 or 17, and
a light chain variable region comprising SEQ ID NO: 10 or 24.
13.The botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the antibody is IgG1.
14.A hybridoma which has the Deposit number of NITE BP-01639 or NITE BP-01640.
15.An isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639 or NITE BP-01640..
16.A pharmaceutical composition comprising one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of any preceding or following embodiment/feature/aspect, and a pharmacologically acceptable carrier.
17.The pharmaceutical composition comprising the anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect and a pharmacologically acceptable carrier.
18.The pharmaceutical composition comprising two isolated anti-botulinum neurotoxin type B monoclonal antibodies, antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect, comprising:
a first isolated human monoclonal antibody or antigen-binding fragment thereof comprising
(1) an isolated human monoclonal antibody or antigen-binding fragment comprising
a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and
a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14,
(2) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10,
(3) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639,
or any combination thereof; and
a second isolated human monoclonal antibody or antigen-binding fragment thereof comprising
(4) an isolated human monoclonal antibody or antigen-binding fragment thereof comprising
a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21, and
a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28,
(5) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 24,
(6) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01640,
or any combination thereof.
19.A method for producing an isolated anti-botulinum neurotoxin type B monoclonal antibody comprising:
producing a hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid with a fusion partner cell capable of efficient cell fusion; and
obtaining a botulinum neurotoxin type B monoclonal antibody from the hybridoma.
20.The method for producing the isolated anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the botulinum neurotoxin type B is produced by BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
21.The method for producing the anti-botulinum neurotoxin type B monoclonal antibody of any preceding or following embodiment/feature/aspect, wherein the fusion partner cell is a SPYMEG cell.
22.A kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject comprising the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect.
23.A method of inhibiting or treating botulism in a human subject comprising administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect to the human subject.
24.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, further comprising diagnosing the patient with botulinum neurotoxin poisoning.
25.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, further comprising monitoring for a decrease in at least one symptom of botulism.
26.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the at least one symptom comprises paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, inability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, general muscle reflex loss, or any combination thereof.
27.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the botulism comprises foodborne botulism, wound infection botulism, infant intestinal toxemia botulism, adult intestinal toxemia botulism, iatrogenic botulism, airborne botulism, or any combination thereof.
28.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect is administered in combination with one or more additional therapies directed to botulism.
29.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the combination acts synergistically to inhibit or treat botulism.
30.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the one or more additional therapies comprise administering a second isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
31.The method of inhibiting or treating botulism in a human subject of any preceding or following embodiment/feature/aspect, wherein the one or more additional therapies comprise administering an isolated anti-botulinum neurotoxin type A monoclonal antibody, an antigen-binding fragment thereof, or both.
32.Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect to manufacture a medicament for inhibiting or treating botulism in a human subject.
33.A method of detecting botulinum neurotoxin type B in a human subject comprising;
contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of any preceding or following embodiment/feature/aspect; and
detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.
The present invention will be further clarified by the following examples, which are intended to be exemplary of, but not limiting, the present invention. These examples demonstrate the surprising and unexpected properties of the claimed antibodies including, for example, their ability to neutralize botulinum poisoning and treat botulism infections. The examples demonstrate development of human monoclonal antibodies against botulinum neurotoxins in accordance with the present invention. The data demonstrates the therapeutic and diagnostic uses of the present invention.
These examples demonstrate that the M-2 antibody specifically bound to the light chain of BoNT/B and showed a potent neutralization activity in the mouse bioassay (approximately >= 100 LD50/mg). Furthermore, the combination of M-2 and M-4 was able to completely neutralize BoNT/B with a potency of greater than 10,000 LD50/mg. This potency is most effective ever and over 18 times greater than the BoNT/B neutralization standard devised for the commercially available BABYBIG human anti-BoNT antiserum (540 LD50/mg). In the post-exposure treatment model, HuMAbs (M-2 + M-4) provided complete protection against lethal dose of BoNT/B when treated within 12 hours of oral administration of toxins. HuMAbs (M-2 + M-4) neutralized other subtypes, BoNT/B2 and BoNT/B6, in addition to BoNT/B1. The data obtained from experiments with the M-2 and M-4 human monoclonal antibodies are representative generally of the antibodies and methods of producing and using the same in accordance with present invention.
Clostridium botulinum type B strain okra was cultured using a cellophane-tube procedure, and the culture supernatant was obtained. Progenitor toxins (16S toxin and 12S toxin) were purified from culture supernatant using (Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003). BoNT/B1 and NAP-16 were prepared from 16S toxin as described (Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003). BoNT/A1 (strain 62A), BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) were provided by Dr. S. Kozaki. A tetravalent botulinum toxoid vaccine was provided by Dr. M. Takahashi. 12S toxins were purified from each culture supernatants of C. botulinum types A, B, E, F and detoxification with formalin. Detoxification of toxins was tested with mice intra-peritoneally (i.p.). The toxoid preparations of the four types were mixed together and then mixed with aluminum (adjuvant) and thimerosal (preserving agent). The toxoid preparation contained 0.1 mg each of the four types, 0.2 mg of aluminum, 0.001% of thimerosal, and 0.0006% of formalin per ml.
An immunization schedule was followed as depicted in FIG. 1. A tetravalent botulinum toxoid (types A, B, E, and F) was inoculated to two healthy adult volunteers (4 or 5 times), who gave written informed consents and received physical examination. The toxoid was injected intramuscularly in 0.5 ml doses to healthy individuals only. After 9 and 18 days, peripheral blood samples (10 ml) were collected from each volunteer. Blood samples were collected from each volunteer and the antibody titers of plasma samples against BoNT/A or BoNT/B were tested using ELISA. This protocol was approved by the Institutional Review Board of Osaka University.
An enzyme-linked immunosorbent assay (ELISA) was performed as follows. The 96-well plates (Falcon) were coated with 100 microliters of BoNT/A or (and) BoNT/B at final concentration of 3 micrograms/ml in PBS for 2 hours at 37 deg C. The wells were washed with PBS containing 0.05% Tween (PBS-T) and blocked with 0.2% BSA (Sigma)/PBS-T for overnight at 4 deg C. Human serum samples were diluted twofold serially and 50 microliters of each dilution was added to a well and incubated for 2 hours at 37 deg C. After washing, 50 microliters of anti-human IgG conjugated with horseradish peroxidase (HRP) (BIO-RAD) was added and incubated for 2 hours at 37 deg C. After washing, plates were incubated with substrate solution (o-phenylendiamine, nacalai tesque) for 30 min at 37 deg C and absorbance values at OD492 were measured. The ELISA titer was expressed in the highest dilution factor showing an absorbance higher than double of negative control serum. Antibody titers were elevated in two volunteers 9 or 18 days after the vaccination (Table 1).
 Table1
Figure JPOXMLDOC01-appb-I000001
A cell fusion, screening, and cloning procedure was carried out as depicted in Fig. 2. Peripheral blood samples were collected from volunteers 9 or 18 days after the last immunization, and the peripheral blood mononuclear cells (PBMCs) were purified by Ficoll (GE Healthcare) gradient centrifugation. The PBMCs were fused with SPYMEG cells at a ratio of 10:1 with polyethylene glycol (Roche). Fused cells were cultured in Dulbecco's modified Eagle medium (DMEM, GIBCO) supplemented with 15% FBS in 96-well plates for about 10-14 days in the presence hypoxanthine-aminopterin-thymidine (HAT, GIBCO). After HAT selection, the first screening of the culture medium for antibodies specific to BoNT/A or BoNT/B was performed by ELISA. As a result, 27 positive wells were obtained from the blood samples collected 9 days after the vaccination, and 8 positive wells from the blood samples collected 18 days after the vaccination. Specific antibody positive wells were next subjected to cell cloning by limiting dilution, and clonal cells were obtained. The second screening was also performed by ELISA. Eight stable hybridoma clones were obtained (Table 2).
Table2
Figure JPOXMLDOC01-appb-I000002
Isotype analysis showed that M-1, M-2, M-4 and S-1 are IgG, M-3, M-5 and M-6 are IgM, and M-7 is IgA (Table 3).
Table3
Figure JPOXMLDOC01-appb-I000003


Each stable hybridoma was cultured in serum free medium (GIBCO), and then the supernatant was collected. Isotypes of HuMAbs in culture supernatant were determined with the anti-human IgG conjugated with HRP (BIO-RAD), anti-human IgM conjugated with HRP (BIOSOURCE INTERNATIONAL) or anti-human IgA conjugated with HRP (Invitrogen) in Western blot. IgG antibodies were purified from the supernatant by Protein G column (GE Healthcare). The subclass of each IgG antibodies was determined using IgG subclass human ELISA kit (Invitrogen).
Cloning and sequencing of variable region gene of monoclonal antibodies was performed. The sequences of heavy chain and light chain variable regions determined are shown in Figures 3-8. Total RNA was extracted from the hybridoma using an RNeasy Mini Kit (Qiagen) and produced by RT-PCR using a SUPERSCRIPT(R) VILOTM cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. The coding region of the H- and L-chains of M-2 and M-4 antibodies was amplified by PCR with KOD-Pus-Neo (Toyobo) and the following primers: 5'-ATGGACTGGACCTGGAGGATCCTC-3' (M-2-H-chain sense primer) (SEQ ID NO: 35), 5'-ATGAAACACCTGTGGTTCTTCCTCCT-3' (M-4-H-chain sense primer) (SEQ ID NO: 36), and 5'-CTCCCGCGGCTTTGTCTTGGCATTA-3' (H-chain antisense primer) (SEQ ID NO: 38); and 5'-ATGGCCTGGWYYCCTCTCYTYCTS-3' (M-4-16-L-chain sense primer) (SEQ ID NO: 38), 5'-ATGSCCTGGGCTCYKCTSCTCCTS-3' (M-2- and M-4-18-L-chain sense primer) (SEQ ID NO: 39), 5'-ATGGCCTGGRYCYCMYTCYWCCTM-3' (M-4-19-L-chain sense primer) (SEQ ID NO: 40), and 5'-TGGCAGCTGTAGCTTCTGTGGGACT-3' (L-chain antisense primer) (SEQ ID NO: 41). After incubation with TaKaRa Ex Taq(R) (Takara), PCR products were ligated into pGEM-T Easy Vector (Promega) and their sequences were analyzed using a BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). IgG subclass assay revealed that M-2 and M-4 are comprised of IgG1 heavy chain and light (lambda) chain.
BoNT/A or BoNT/B (600 ng) was separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane (BIO-RAD). The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween. HuMAbs (1.0 micrograms/ml) was added to the membrane and incubated for 1 hour at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 hour at room temperature, and then specific bands were visualized with ECL (PIERCE) (FIGS. 9A and 9B).
To determine epitopes for the HuMAbs, Western blot analysis was performed. The results show that M-2 specifically bound to light chain of BoNT/B (FIG. 10). In contrast, M-4 bound to both of the light chain and heavy chain (FIG. 10).
Binding of HuMAb to BoNT, progenitor toxin, or non-toxic component (NAP-16) of type B was analyzed by ELISA. HuMAbs were added to the plate coated with BoNT, progenitor toxin, or NAP-16. HuMAbs (0.01 - 0.5 micrograms/ml) were added to the plate coated with BoNT/A or BoNT/B. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP. M-2 and M-4 bound to BoNT and progenitor toxins. In contrast, M-2 and M-4 showed faint binding to NAP-16. These results show that M-2 and M-4 bind to BoNT in the progenitor toxins, as well as BoNT alone, and that M-2 and M-4 specifically bind to BoNT. M-2 and M-4 showed specific binding to BoNT/B (FIG. 11).
The neutralization activity of HuMAbs was tested by mouse bioassay. HuMAbs were incubated with BoNT/B (10 LD50) at room temperature for 1 hour prior to intraperitoneal injection (in a total volume of 500 microliter) into mice (ddY, female, 4 weeks, SLC). 10 LD50 of BoNT/B was incubated with 100 micrograms of HuMAb or PBS (Cnt) for 1 hour, and injected intraperitoneally into mice. Mice were observed for morbidity and mortality for 3 weeks. Mice injected with BoNT/B + PBS (Cnt) died within 12 hours. In contrast, mice injected with BoNT/B + M-4 were partially protected, as evidenced by an increased time-to-death compared to those of control mice. On the other hand, complete protection was observed in mice injected with BoNT/B + M-2 (Table 4).
 Table4
Figure JPOXMLDOC01-appb-I000004

The combination of HuMAbs was tested for synergy. 10 LD50 of BoNT/B was incubated with a mixture of M-2 and M-4 (0.5 micrograms each, in a volume of 500 microliters) and injected into mice. All of the mice that received the HuMAbs survived and had no symptoms (FIG. 12).
 Example9
In a post-exposure treatment model, mice were orally administrated with the type B progenitor toxin (16S toxin, 10 ng in a volume of 300 microliters), and subsequently administrated with HuMAbs (M-2 + M-4) by i.p. injection at 12, 24, or 36 hours after the oral administration of 16S toxin. Mice were observed for morbidity and mortality for 3 weeks. In the post-exposure treatment model, mice developed symptoms of botulism at 12 hours after oral administration of 16S toxin. Control mice that were not treated with HuAbs died within 72 hours. In contrast, post-exposure treatment of HuMAbs provided complete survival at 12 hours after oral administration of 16S toxin, and partial survival at 24 and 36 hours after administration (FIG. 13).
Binding of HuMAbs (M-2 and M-4) to BoNT/B2 (strain 111) and BoNT/B6 (strain Osaka05) were analyzed by ELISA. HuMAbs (0.5 micrograms/ml) were added to the plate coated with BoNT. After washing, bound HuMAbs were detected by anti-human IgG antibody conjugated with HRP. M-2 and M-4 showed strong binding to BoNT/B2 and BoNT/B6 in addition to BoNT/B1. In the neutralization test, M-2 + M-4 (0.5 micrograms + 0.5 micrograms) completely neutralized BoNT/B2 (10 ng) and BoNT/B6 (2.5 ng) (FIG. 14).
For expression of recombinant HuMAbs in mammalian cells (HEK293 cells) and testing of the same, IgG-expression vectors were constructed as follows. pGEM-T Easy Vectors with the variable region gene of H- and L-chains were subjected to PCR to add restriction enzyme sites and a Kozak sequence with the following primer sets (restriction enzyme sites are underlined): 5'-ATTTGCGGCCGCCATGGACTGGACCTGGAGG-3' (M2-H-chain sense primer) (SEQ ID NO: 42), 5'-ATTTGCGGCCGCCATGAAACACCTGTGGTTCTTC-3' (M-4-H-chain sense primer) (SEQ ID NO: 43) and 5'-ATACTCGAGGGTGCCAGGGGGAAGACCGATG-3' (H-chain antisense primer) (SEQ ID NO:44); and 5'-ATTTGCGGCCGCCATGGCCTGGTTTCCTCTCTTC-3' (M-4-16-L-chain sense primer) (SEQ ID NO:45), 5'-ATTTGCGGCCGCCATGGCCTGGGCTCTGCT-3' (M-2- and M-4-18-L-chain sense primer) (SEQ ID NO: 46), 5'-ATTTGCGGCCGCCATGGCCTGGGTCTCATT-3' (M-4-19-L-chain sense primer) (SEQ ID NO: 47), and 5'-ATACTCGAGGGCGGGAACAGAGTGACCGTGG-3' (L-chain antisense primer) (SEQ ID NO: 48). PCR products of the H- and L-chain coding regions were digested by restriction enzymes, Not I and Xho I, and then ligated to expression vectors, pQCXIP-hCH and pQCXIH-hC, which have a human immunoglobulin-constant region of gamma and lambda chains (MBL), respectively.
HEK293 cells were cultured in 5% CO2 at 37 deg C in MEM containing 10% FBS. Cells grown on 100 mm culture dish were used for transient transfection with pQCXIP-hCH and pQCXIH-hC expression vectors using LIPOFECTAMINE2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. After transfection, the medium was removed and cells were washed and incubated in Opti-Pro Serum Free Medium (Gibco) in 5% CO2 at 37 deg C for 10 days. Recombinant HuMAbs in culture supernatant were determined with the anti-human IgG conjugated with HRP (BIO-RAD). Recombinant HuMAbs were purified from the supernatant by Protein G column (GE Healthcare).
Binding of recombinant HuMAbs (RM-2 and RM-4; depicted as RM-4 LC16, RM-4 LC18, RM-4 LC19, which have the same heavy chain and light chain of M-4, but have different signal sequences of light chain shown in FIG. 6, 7, 8 respectively) to BoNT/B were analyzed by ELISA. RM-2 and RM-4 (about 0.01-0.5 micrograms/ml) were added to the plate. After washing, bound recombinant HuMAbs were detected by anti-human IgG antibody conjugated with HRP. As the results, RM-2 and RM-4 bound to BoNT/B in the same manner as the M-2 and M-4 derived from hybridoma (FIG. 15). In the neutralization test, RM-2 + RM-4 (0.5 micrograms + 0.5 micrograms) neutralized BoNT/B1 (10 LD50) (Tables 5A and 5B).
Table5A
Figure JPOXMLDOC01-appb-I000005

Table5B
Figure JPOXMLDOC01-appb-I000006

Binding of HuMAbs derived from hybridomas and recombinant HuMAbs (RM-2 and RM-4) to BoNT/B (BoNT/B1, BoNT/B2 or BoNT/B6) were analyzed by SDS-PAGE. BoNT/B1, BoNT/B2 or BoNT/B6 were separated into Hc and Lc by SDS-PAGE, and transferred to the nitrocellulose membrane. HuMAbs (1.0 micrograms/ml) was added to the membrane and incubated for 1 h at room temperature. After washing, the membrane was incubated with anti-human IgG antibody conjugated with HRP for 1 h at room temperature, and then specific bands were visualized with ECL. Recombinant HuMAbs showed the same binding profile as HuMAbs derived from hybridomas. M-2 and RM-2 specifically bound to light chain of BoNT/B. In contrast, M-4 and RM-4 bound to both of light chain and heavy chain. By M-4 and RM-4 antibodies, heavy chain of B2 subtype was detected more clearly than that of B1. Further, heavy chain of B6 subtype was detected more clearly than that of B2 (FIG. 16).
Applicants specifically incorporate the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the present specification and practice of the present invention disclosed herein. It is intended that the present specification and examples be considered as exemplary only with a true scope and spirit of the invention being indicated by the following claims and equivalents thereof.

Claims (33)

  1. An isolated anti-botulinum neurotoxin type B monoclonal antibody or an antigen-binding fragment thereof comprising a neutralization activity against a botulinum neurotoxin type B, wherein the monoclonal antibody comprises a human monoclonal antibody, a humanized monoclonal antibody, or both.
  2. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof further comprises a neutralization activity against a progenitor toxin of botulinum neurotoxin type B.
  3. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a neutralization activity against a botulinum neurotoxin type B produced by BONT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  4. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof has a specific binding activity to a light chain of a botulinum neurotoxin type B (BoNT/B).
  5. The isolated anti-botulinum neurotoxin type B monoclonal antibody of claim 1, wherein the human monoclonal antibody is produced by a hybridoma made by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid with a fusion partner cell capable of efficient cell fusion.
  6. The isolated anti-botulinum neurotoxin type B monoclonal antibody of claim 5, wherein the botulinum neurotoxin type B is a product of BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  7. The isolated anti-botulinum neurotoxin type B monoclonal antibody of claim 5, wherein the fusion partner cell is a SPYMEG cell.
  8. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising an IgG, a Fab, a Fab', a F(ab')2, a scFv, a dsFv, or a combination thereof.
  9. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising:
    a heavy chain variable region comprising
    a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or 19,
    a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or 20, and
    a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or 21; and
    a light chain variable region comprising
    a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or 26,
    a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or 27, and
    a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or 28.
  10. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising:
    a heavy chain variable region comprising
    a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5,
    a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and
    a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7; and
    a light chain variable region comprising
    a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12,
    a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and
    a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14.
  11. The isolated anti-botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising:
    a heavy chain variable region comprising
    a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19,
    a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and
    a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21; and
    a light chain variable region comprising
    a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26,
    a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and
    a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28.
  12. The botulinum neurotoxin type B monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising:
    a heavy chain variable region comprising SEQ ID NO: 3 or 17, and
    a light chain variable region comprising SEQ ID NO: 10 or 24.
  13. The botulinum neurotoxin type B monoclonal antibody of claim 1, wherein the antibody is IgG1.
  14. A hybridoma which has the Deposit number of NITE BP-01639 or NITE BP-01640.
  15. An isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639 or NITE BP-01640.
  16. A pharmaceutical composition comprising one or more of the isolated anti-botulinum neurotoxin type B monoclonal antibody and antigen-binding fragment thereof of claim 1, and a pharmacologically acceptable carrier.
  17. The pharmaceutical composition comprising the anti-botulinum neurotoxin type B monoclonal antibody of claim 16 and a pharmacologically acceptable carrier.
  18. The pharmaceutical composition comprising two isolated anti-botulinum neurotoxin type B monoclonal antibodies, antigen-binding fragment thereof, or both of claim 16, comprising:
    a first isolated human monoclonal antibody or antigen-binding fragment thereof comprising
    (1) an isolated human monoclonal antibody or antigen-binding fragment thereof comprising
    a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and
    a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14,
    (2) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10,
    (3) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01639,
    or any combination thereof; and
    a second isolated human monoclonal antibody or antigen-binding fragment thereof comprising
    (4) an isolated human monoclonal antibody or antigen-binding fragment thereof comprising
    a heavy chain variable region comprising a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 20, and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21, and
    a light chain variable region comprising a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28,
    (5) an isolated human monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 17, and a light chain variable region comprising SEQ ID NO: 24,
    (6) an isolated monoclonal antibody, which is produced by the hybridoma having Deposit number of NITE BP-01640,
    or any combination thereof.
  19. A method for producing an isolated anti-botulinum neurotoxin type B monoclonal antibody comprising:
    producing a hybridoma by fusing a peripheral blood mononuclear cell (PBMC) from a human being immunized by a botulinum toxoid with a fusion partner cell capable of efficient cell fusion; and
    obtaining a botulinum neurotoxin type B monoclonal antibody from the hybridoma.
  20. The method for producing the isolated anti-botulinum neurotoxin type B monoclonal antibody of claim 19, wherein the botulinum neurotoxin type B is produced by BoNT/B1 (strain Okra) of Clostridium botulinum type B, BoNT/B2 (strain 111) of Clostridium botulinum type B, BoNT/B6 (strain Osaka05) of Clostridium botulinum type B, or any combination thereof.
  21. The method for producing the anti-botulinum neurotoxin type B monoclonal antibody of claim 19, wherein the fusion partner cell is a SPYMEG cell.
  22. A kit for at least one of the prevention, the treatment, and the detection of botulinum neurotoxin type B poisoning in a human subject comprising the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of claim 1.
  23. A method of inhibiting or treating botulism in a human subject comprising administering a therapeutically effective amount of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of claim 1 to the human subject.
  24. The method of inhibiting or treating botulism in a human subject of claim 23, further comprising diagnosing the patient with botulinum neurotoxin poisoning.
  25. The method of inhibiting or treating botulism in a human subject of claim 23, further comprising monitoring for a decrease in at least one symptom of botulism.
  26. The method of inhibiting or treating botulism in a human subject of claim 25, wherein the at least one symptom comprises paralysis, symmetric cranial nerve palsies, symmetric descending flaccid paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, inability to suck, inability to swallow, weakened voice, ptosis, facial paralysis, fixed pupils, dilated pupils, blurred vision, floppy neck, generalized flaccidity, generalized weakness, diplopia, dysarthria, dysphonia, dysphagia, anhidrosis, mucosal erythema, postural hypotension, nausea, constipation, urinary retention, dizziness, dry mouth, sore throat, suppressed, gag reflex, general muscle reflex loss, or any combination thereof.
  27. The method of inhibiting or treating botulism in a human subject of claim 23, wherein the botulism comprises foodborne botulism, wound infection botulism, infant intestinal toxemia botulism, adult intestinal toxemia botulism, iatrogenic botulism, airborne botulism, or any combination thereof.
  28. The method of inhibiting or treating botulism in a human subject of claim 23, wherein the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of claim 1 is administered in combination with one or more additional therapies directed to botulism.
  29. The method of inhibiting or treating botulism in a human subject of claim 28, wherein the combination acts synergistically to inhibit or treat botulism.
  30. The method of inhibiting or treating botulism in a human subject of claim 28, wherein the one or more additional therapies comprise administering a second isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both.
  31. The method of inhibiting or treating botulism in a human subject of claim 28, wherein the one or more additional therapies comprise administering an isolated anti-botulinum neurotoxin type A monoclonal antibody, an antigen-binding fragment thereof, or both.
  32. Use of the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of claim 1 to manufacture a medicament for inhibiting or treating botulism in a human subject.
  33. A method of detecting botulinum neurotoxin type B in a human subject comprising;
    contacting a sample from the human subject with the isolated anti-botulinum neurotoxin type B monoclonal antibody, an antigen-binding fragment thereof, or both of claim 1; and
    detecting the presence or absence of botulinum neurotoxin type B in the human subject based on whether the antibody, the fragment thereof, or both binds botulinum neurotoxin type B.

PCT/JP2013/005123 2012-08-31 2013-08-29 Binding/neutralizing human monoclonal antibodies against botulinum neurotoxin type b WO2014034127A1 (en)

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