JP6286420B2 - Binding / neutralizing human monoclonal antibody against botulinum neurotoxin type B - Google Patents

Description

  The present invention relates to anti-botulinum neurotoxin (botulin) antibodies and methods of using and manufacturing the same.

This application claims the benefit of US Provisional Patent Application No. 61 / 695,318, filed August 31, 2012.

Botulinum neurotoxin is one of the most toxic substances known. These toxins can cause botulism and are produced by a variety of bacteria including Clostridium botulinum ( Clostridium botulinum ). Clostridia, which produces botulinum toxin, is an anaerobic gram-positive bacterium. These bacteria form spores and are widely distributed in the environment. At the molecular level, botulism is caused by inhibition of the release of acetylcholine at the neuromuscular junction by the toxin. Seven toxin serotypes (A, B, C, D, E, F and G) have been identified. They have a similar structure corresponding to an approximately 150 kD zinc-endopeptidase protein comprising 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 divided into various categories: food-borne, wound infection, childhood enterotoxemia, adult enterotoxemia, toxin injection (iatrogenic botulism), and weaponized toxins. Foodborne illness is caused by ingestion of toxins in food contaminated with toxin-producing bacteria. In wound infection, the toxin-producing clostridial colonizes the wound and then grows in the wound to produce toxins. In pediatric botulism, toxins are produced after the toxin-producing clostridial colonizes the child's intestines. Adult enterotoxemia is similar to childhood botulism. Cosmetic or therapeutic botulinum toxin injections can cause botulism. Botulism can also be caused by inhalation of weaponized toxins, such as aerosolized botulinum toxin.

  Botulism is characterized by symmetric cranial nerve palsy followed by symmetric descending relaxation paralysis of voluntary muscles. This paralysis can lead to hypopnea and death. Toxin binding is irreversible. Recovery requires new nerve terminal growth. Recovery is prolonged and may involve long outpatient rehabilitation therapy. The onset of the disease can occur immediately after contact with the toxin. Treatment usually involves the administration of antitoxins that can help reduce the degree of paralysis. Most of the available antitoxins are derived from horses and are not necessarily specific for a particular type of botulinum neurotoxin. There is a need for a more targeted form of antitoxin. There is also a need for effective toxin neutralizing antibodies that do not have side effects such as serum sickness. There is a further need for better diagnostic tests when the course of botulism is rapid.

Arimitsu et al., Infect. Immun. 71: 1599-1603, 2003

  Therefore, a feature of the present invention is to provide therapeutic antibodies against botulinum neurotoxins with better efficacy and safety.

  Another feature of the present invention is to provide a method for inhibiting and treating botulism.

  A further feature of the present invention is to provide a treatment without side effects such as serum sickness.

  Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the specification and the appended claims.

  In accordance with the embodiments and broad description provided herein, in order to achieve the objects and other advantages of the present invention, the present invention provides an isolated botulinum neurotoxin monoclonal antibody or botulinum neurotoxin B It relates to an antigen-binding fragment having neutralizing activity against. Monoclonal antibodies can include human monoclonal antibodies, humanized monoclonal antibodies, or both. The isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof may have a neutralizing activity against a type B toxin genus toxin (progenitor toxin). The isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof may have specific binding activity for the light chain of type B botulinum neurotoxin (BoNT / B), or It may have specific binding activity for both the light chain and heavy chain of botulinum neurotoxin type B (BoNT / B). In addition to the hybridoma producing the antibody or fragment thereof of the present invention, a method for producing such a hybridoma and a method for producing an antibody or fragment thereof from such a hybridoma are also provided by the present invention.

  The invention further relates to a pharmaceutical composition. The pharmaceutical composition may contain one or more isolated anti-type B botulinum neurotoxin monoclonal antibodies and antigen-binding fragments thereof and a pharmaceutically acceptable carrier. A kit for at least one of the prevention, treatment and detection of botulinum neurotoxin poisoning type B in a human subject is provided by the present invention. The kit may comprise an isolated anti-type B botulinum neurotoxin monoclonal antibody of the invention, an antigen-binding fragment thereof, or both. Also provided by the present invention is the use of an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both for the manufacture of a medicament for inhibiting or treating botulism in a human subject.

  The invention also relates to a method for inhibiting or treating botulism in a human subject. The method can include administering to the human subject a therapeutically effective amount of an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment, or both. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. The method can include monitoring the alleviation of at least one symptom of botulism. A method of inhibiting or treating botulism in a human subject comprises the isolation of an anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both, and one or more additional treatments for botulism. And administering in combination with. Combination therapy can act synergistically to inhibit or treat botulism.

  The invention further relates to a method for detecting a botulinum neurotoxin type B in a human subject. The method can include contacting a specimen from a human subject with an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both. The method can also include detecting the presence or absence of a botulinum neurotoxin type B in a human subject based on whether the antibody, fragment thereof, or both binds to a 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 further explanation of the invention as claimed. Should be understood.

  The accompanying drawings, which are hereby incorporated by reference, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention.

In the present invention, it is a schematic diagram showing how to immunize a volunteer (volunteer) and how to isolate antibody-producing cells. FIG. 3 is a flow diagram showing a cell fusion and cloning schedule according to the present invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the heavy chain of M-2 antibody by this invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the light chain of M-2 antibody by this invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the heavy chain of M-4 antibody by this invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the light chain of RM-4 LC-16 antibody by this invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the light chain of RM-4 LC-18 antibody by this invention. It is a figure which shows the nucleotide sequence and amino acid sequence about the light chain of RM-4 LC-19 antibody by this invention. FIG. 3 includes a line graph showing the binding of a human monoclonal antibody according to the present invention to BoNT / A or BoNT / B. Binding of human monoclonal antibody (HuMAb) to BoNT was analyzed by ELISA. HuMAb (about 0.05-1.0 microgram / mL) was added to the plates coated with BoNT / A or BoNT / B. After washing, bound HuMAb was detected by anti-human IgG antibody-HRP complex. M-2 and M-4 showed specific binding to BoNT / B. FIG. 4 includes a Western blot for epitope mapping of a human monoclonal antibody according to the present invention. BoNT was separated into Hc and Lc by SDS-PAGE and transferred to a nitrocellulose membrane. HuMAb (1.0 microgram / 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-HRP complex for 1 hour at room temperature, after which specific bands were visualized by ECL. M-2 specifically bound to the BoNT / B light chain. In contrast, M-4 bound to both light and heavy chains. 2 is a bar graph showing the binding of human monoclonal antibodies according to the present invention to BoNT / B, progenitor toxin and non-toxic components. Binding of HuMAb to BoNT, progenitor toxin (12S toxin and 16S toxin) or non-toxic components (NAP-16) was analyzed by ELISA. HuMAb was added to plates coated with BoNT, progenitor toxin or NAP-16. After washing, bound HuMAb was detected by anti-human IgG antibody-HRP complex. M-2 and M-4 bound to BoNT and progenitor toxin. In contrast, M-2 and M-4 showed only slight binding to NAP-16. These results indicate that M-2 and M-4 bind to BoNT in the progenitor toxin as well as BoNT alone, and that M-2 and M-4 specifically bind to BoNT. It is a line graph which shows the neutralization activity with respect to BoNT / B when the human monoclonal antibody by this invention is combined. 10 LD ₅₀ BoNT / B was incubated with a mixture of M-2 and M-4 (0.05 microgram, 0.1 microgram, or 0.5 microgram, respectively) and injected into mice. All mice that received a mixture of M-2 (0.5 microgram) and M-4 (0.5 microgram) survived and presented no symptoms. It is a line graph which shows the neutralization activity after administration of the human monoclonal antibody with respect to the progenitor toxin (16S toxin) by this invention. Progenitor toxin (16S toxin, 10 ng) was orally administered to mice, followed by HuMAb (M-2 0.5 microgram + M-4 0.5 microgram) 12 hours, 24 hours or 36 hours after oral administration of 16S toxin. Administered by intraperitoneal injection. Control mice that were not treated with HuMAb died within 72 hours. In contrast, post-exposure treatment with HuMAb resulted in complete survival at 12 hours after oral administration of 16S toxin and partial survival at 24 and 36 hours after administration. FIG. 2 includes a bar graph and a line graph showing the binding and neutralization of human monoclonal antibodies to BoNT / B2 (111 strain) and BoNT / B6 (Osaka05 strain) according to the present invention. The binding of HuMAb (M-2 and M-4) to BoNT / B2 (111 strain) and BoNT / B6 (Osaka05 strain) was analyzed by ELISA. HuMAb (0.5 microgram / mL) was added to the BoNT coated plate. After washing, bound HuMAb was detected by anti-human IgG antibody-HRP complex. 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 microgram + 0.5 microgram) completely neutralized BoNT / B2 (10 ng) and BoNT / B6 (2.5 ng). FIG. 2 includes a Western blot and line graph showing binding of recombinant human monoclonal antibodies according to the present invention to BoNT / B. HEK293 cells were transiently transfected with pQCXIP-hCH expression vector and pQCXIH-hCl expression vector using LIPOFECTAMINE 2000 transfection reagent. Recombinant HuMAb in the culture supernatant was determined by anti-human IgG-HRP complex. The binding of recombinant M-2 (RM-2) and recombinant M-4 (RM-4) to BoNT / B was analyzed by ELISA. Recombinant HuMAb (approximately 0.01-0.5 microgram / mL) was added to the BoNT / B coated plate. After washing, bound recombinant HuMAb was detected by anti-human IgG antibody-HRP complex. RM-2 and RM-4 bound to BoNT / B in the same way as M-2 and M-4 derived from hybridomas. FIG. 2 includes a Western blot of binding of HuMAb and recombinant HuMAbs (RM-2 and RM-4) derived from hybridomas to BoNT / B (BoNT / B1, BoNT / B2, or BoNT / B6). BoNT / B1, BoNT / B2 or BoNT / B6 was separated into Hc and Lc by SDS-PAGE and transferred to a nitrocellulose membrane. HuMAb (1.0 microgram / 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-HRP complex for 1 hour at room temperature, after which specific bands were visualized by ECL. Recombinant HuMAb showed the same binding profile as HuMAb derived from hybridoma. M-2 and RM-2 specifically bound to the light chain of BoNT / B. In contrast, M-4 and RM-4 (RM-4 LC16, RM-4 LC18, RM-4 LC19 have the same M-4 heavy chain) bound to both the light and heavy chains. The M2 and RM-4 antibodies detected the B2 subtype heavy chain more clearly than the B1 heavy chain. Furthermore, the heavy chain of the B6 subtype was detected more clearly than the heavy chain of B2.

An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof having neutralizing activity against a type B botulinum neurotoxin is provided according to the present invention. Monoclonal antibodies can include human monoclonal antibodies, humanized monoclonal antibodies, or both. The isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof can have a neutralizing activity of a type B botulinum neurotoxin against a progenitor toxin. The isolated anti-type B botulinum neurotoxin monoclonal antibodies or antigen-binding fragments thereof are Clostridium botulinum type B BoNT / B1 (Okra strain), Clostridium botulinum type B BoNT / B2 (111 strain), Clostridium botulinum type B It may have neutralizing activity against botulinum neurotoxin type B produced by BoNT / B6 (Osaka05 strain), or any combination thereof. Thus, an isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof can have specific binding activity for the light chain of type B botulinum neurotoxin (BoNT / B).

Antibodies or fragments thereof can be generated using any suitable technique. For example, the isolated anti-type B botulinum neurotoxin monoclonal antibody is a fusion partner that enables efficient cell fusion of human-derived peripheral blood mononuclear cells (PBMC) immunized with type B botulinum neurotoxin Hybridomas produced by fusing with cells can be produced. Type B botulinum neurotoxins are Clostridium botulinum type B BoNT / B1 (Okra strain), Clostridium botulinum type B BoNT / B2 (111 strain), Clostridium botulinum type B BoNT / B6 (Osaka05 strain), or any of them Can be the product of a combination of Any suitable fusion partner cell, such as SPYMEG cells can be used.

  Hybridomas that produce the antibodies or fragments thereof of the invention are also provided by the invention. For example, the deposit number of the hybridoma can be NITE BP-01639 or NITE BP-01640. Also provided is an isolated monoclonal antibody produced by a hybridoma with a deposit number of NITE BP-01639 or NITE BP-01640. Examples of the monoclonal antibody obtained as described above include a monoclonal antibody produced by a hybridoma called “Hybridoma M-2 (M1E9)” (hereinafter referred to as M-2 antibody) and “Hybridoma M-4 (M4C9)”. And a monoclonal antibody produced by a hybridoma (hereinafter referred to as M-4 antibody). These hybridomas are deposited with the NITE BP-01639 deposit number at the National Institute of Technology and Evaluation (NITE), the Patent Microorganism Deposit Center (Japan, Kazusa-Kamashita 2-5-8 122, Kisarazu City, Chiba Prefecture 292-0818, Japan) Deposited on June 26, 2013 under the number NITE BP-01640. Later, these hybridomas moved to the international deposit under the Budapest Treaty with the same number.

  The isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof is IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgD, IgE, any fragment thereof, or their Any combination can be used. An antibody fragment can comprise, for example, Fab, Fab ′, F (ab ′) 2, scFv, dsFv, or combinations thereof. The isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof can comprise a heavy chain variable region and / or a light chain variable region. For example, the heavy chain variable region comprises a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or SEQ ID NO: 19, a second amino acid sequence comprising SEQ ID NO: 6 or SEQ ID NO: 20. And a second complementarity determining region (CDR2) having a third amino acid sequence comprising SEQ ID NO: 7 or SEQ ID NO: 21. The light chain variable region has, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or SEQ ID NO: 26, a fifth amino acid sequence comprising SEQ ID NO: 13 or SEQ ID NO: 27 A second complementarity determining region (CDR2) and a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or SEQ ID NO: 28 may be included.

  In another example, the heavy chain variable region is a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5, a second complement having a second amino acid sequence comprising SEQ ID NO: 6. A sex determining region (CDR2) and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 may be included. The light chain variable region includes a first complementarity determining region (CDR1) having a fourth amino acid sequence including SEQ ID NO: 12, and a second complementarity determining region (CDR2) having a fifth amino acid sequence including 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 has a first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 19, a second amino acid sequence comprising a second amino acid sequence comprising SEQ ID NO: 20. A complementarity determining region (CDR2) and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21 may be included. The light chain variable region includes, for example, a first complementarity determining region (CDR1) having a fourth amino acid sequence including SEQ ID NO: 26, and a second complementarity determining region having a fifth amino acid sequence including SEQ ID NO: 27 ( CDR2) 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 comprise SEQ ID NO: 3 or SEQ ID NO: 17, and the light chain variable region can comprise SEQ ID NO: 10 or SEQ ID NO: 24.

  A pharmaceutical composition is provided by the present invention. The pharmaceutical composition may contain one or more isolated anti-type B botulinum neurotoxin monoclonal antibodies and antigen-binding fragments thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain an anti-type B botulinum neurotoxin monoclonal antibody and a pharmacologically acceptable carrier. The pharmaceutical composition may contain two isolated anti-type B botulinum neurotoxin monoclonal antibodies, antigen-binding fragments thereof, or both. The pharmaceutical composition may contain a first antibody and a second antibody. Examples of the first isolated human monoclonal antibody or antigen-binding fragment include (1) a first complementarity determining region (CDR1) having a first amino acid sequence containing SEQ ID NO: 5 and SEQ ID NO: 6 A heavy chain variable region comprising a second complementarity determining region (CDR2) having an amino acid sequence of 2 and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7, and SEQ ID NO: A first complementarity determining region (CDR1) having a fourth amino acid sequence comprising 12; a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13; and SEQ ID NO: 14 There may be mentioned an isolated human monoclonal antibody or antigen-binding fragment comprising a light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence. As the first isolated human monoclonal antibody or antigen-binding fragment, for example, (2) an isolated human monoclonal comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10 There may be mentioned antibodies or antigen-binding fragments. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, (3) an isolated monoclonal antibody produced by a hybridoma with deposit number NITE BP-01639. The first isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above. Examples of the second isolated human monoclonal antibody or antigen-binding fragment include (4) a first complementarity determining region (CDR1) having a first amino acid sequence containing SEQ ID NO: 19, and SEQ ID NO: 20 A heavy chain variable region comprising a second complementarity determining region (CDR2) having two amino acid sequences and a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21; and SEQ ID NO: A first complementarity determining region (CDR1) having a fourth amino acid sequence comprising 26, a second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 27, and SEQ ID NO: 28 There may be mentioned an isolated human monoclonal antibody or antigen-binding fragment comprising a light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence. As the second isolated human monoclonal antibody or antigen-binding fragment, for example, (5) an isolated human monoclonal comprising a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 24 There may be mentioned antibodies or antigen-binding fragments. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, (6) an isolated monoclonal antibody produced by a hybridoma with deposit number NITE BP-01640. The second isolated human monoclonal antibody or antigen-binding fragment can include, for example, any combination of the above.

A method of making an isolated anti-type B botulinum neurotoxin monoclonal antibody is provided by the present invention. This method involves creating a hybridoma by fusing human-derived peripheral blood mononuclear cells (PBMC) immunized with a botulinum neurotoxin B with fusion partner cells that enable efficient cell fusion. May be included. The method can further comprise obtaining an anti-type B botulinum neurotoxin monoclonal antibody from the hybridoma. Type B botulinum neurotoxins include, for example, Clostridium botulinum type B BoNT / B1 (Okra strain), Clostridium botulinum type B BoNT / B2 (111 strain), Clostridium botulinum type B BoNT / B6 (Osaka05 strain), or Produced by any combination. The fusion partner cell can be, for example, a SPYMEG cell.

  A kit for at least one of the prevention, treatment and detection of botulinum neurotoxin poisoning type B in a human subject is provided by the present invention. The kit may comprise an isolated anti-type B botulinum neurotoxin monoclonal antibody of the invention, an antigen-binding fragment thereof, or both. The kit may also include one or more additional agents for treating and / or detecting botulinum neurotoxin poisoning type B. The kit may also include one or more agents for treating and / or detecting one or more additional types, such as type A botulinum neurotoxin. Also provided by the present invention is the use of an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both for the manufacture of a medicament for inhibiting or treating botulism in a human subject.

  Methods of inhibiting or treating botulism in human subjects are provided by the present invention. The method can include administering to the human subject a therapeutically effective amount of an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. The method can include monitoring the alleviation of at least one symptom of botulism. At least one symptom includes, for example, paralysis, symmetric cranial nerve palsy, symmetric descending relaxation paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, respiratory arrest, insufficiency, swallowing, weak voice, drooping eyelids, facial paralysis, pupil Fixation, dilated pupil, foggy vision, floppy neck, generalized relaxation, generalized weakness, double vision, dysarthria, vocalization disorder, dysphagia, no sweat, mucosal erythema, postural hypotension, nausea, constipation Urinary retention, dizziness, dry mouth, sore throat, suppressed, depression, generalized muscle reflex loss, any other symptom of botulism, or any combination thereof . For the subject, any type of botulism, such as food-borne botulism, wound infection botulism, childhood enterotoxemia botulism, adult enterotoxemia botulism, iatrogenic botulism, Air mediated botulism, or any combination thereof can be inhibited or treated.

  A method of inhibiting or treating botulism in a human subject comprises isolating an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both, and one or more additional therapies for botulism. Administration in combination may be included. Combination therapy can act synergistically to inhibit or treat botulism. The one or more additional therapies can include, for example, administering a second isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both. One or more additional therapies can include, for example, administering an isolated anti-type Botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both. The one or more additional therapies may include, for example, one or more antibiotics, such as penicillin, or any combination thereof.

  A method of detecting botulinum neurotoxin B in a human subject is provided by the present invention. The method can include contacting a specimen from a human subject with an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both. The method can also include detecting the presence or absence of a botulinum neurotoxin type B in a human subject based on whether the antibody, fragment thereof, or both binds to a botulinum neurotoxin type B. The method may further comprise detecting one or more additional types, such as type A botulinum neurotoxin.

  Provided are polypeptides containing anti-botulinum neurotoxin antibodies and antigen-binding fragments thereof, and methods, uses, compositions and kits using them. Unless otherwise specified, the isolated monoclonal antibodies and antigen-binding fragments thereof described and claimed in this application are not natural products. These monoclonal antibodies and antigen-binding fragments thereof are the products of hybridomas or equivalent artificial cell lines produced using various experimental methods. Antibodies or antigen-binding fragments thereof for therapeutic, diagnostic or other purposes, compositions containing them, kits containing them, and / or methods of using non-naturally occurring antibodies or antibody fragments are explicitly specified. It is not limited to such a thing unless it is.

  Methods are provided for forming antibodies specific for botulinum neurotoxins or polypeptides or fragments thereof. Such a method comprises preparing a nucleic acid encoding a botulinum neurotoxin antigen polypeptide or a polypeptide containing an immunologically specific epitope thereof, and a polypeptide containing the antigen amino acid sequence from the isolated nucleic acid or the immunity thereof. Expressing a polypeptide containing a scientifically specific epitope and generating a polypeptide containing an antibody or antigen-binding fragment thereof specific for the resulting polypeptide. A polypeptide containing an antibody or antigen-binding fragment thereof produced by the above-described method is provided. An isolated polypeptide containing an isolated antibody or antigen-binding fragment thereof that specifically binds to a botulinum neurotoxin antigen is provided. Such antibodies can be generated using any acceptable method (s) known in the art. Antibodies and kits, methods and / or other embodiments of the invention using antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, monovalent antibodies, diabodies, and / or humanized antibodies. One or more may be included.

  Naturally occurring antibody structural units usually contain a tetramer. Each such tetramer has one full-length “light” chain (eg, about 1 kDa to 25 kDa) and one full-length “heavy” chain (eg, about 50-70 kDa) for each pair. It can be composed of two identical polypeptide chain pairs. The amino terminal portion of each chain usually contains a variable region of about 100 amino acids to 110 amino acids or more that are usually involved in antigen recognition. The carboxy terminus of each chain usually defines a constant region that can participate in effector functions. Human light chains are usually classified as kappa and lambda light chains. Heavy chains are usually classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses including but not limited to IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. In the light and heavy chains, the variable and constant regions are connected by a “J” region of about 12 amino acids or more, and the heavy chain further comprises a “D” region of about 10 amino acids or more. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)). The variable regions of each light / heavy chain pair usually form the antigen binding site.

  A variable region usually exhibits the same general structure of relatively conserved framework regions (FRs) joined by three complementarity determining regions or hypervariable regions, also called CDRs. The CDRs of the two strands of each pair are usually aligned by the framework region, which allows binding to specific epitopes. Both the light chain and heavy chain variable regions usually contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains from the N-terminus to the C-terminus. The assignment of amino acids to each domain is usually the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987 ), Chothia et al., Nature 342: 878-883 (1989).

  “Antibody fragments” comprise a portion of an intact antibody, eg, 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]); Chain antibody molecules; as well as multispecific antibodies formed from antibody fragments. By papain digestion of the antibody, two identical antigen-binding fragments, called “Fab” fragments, each having a single antigen-binding site, and a remaining “Fc” fragment, the designation reflecting its ability to crystallize easily Occurs. Pepsin treatment yields an F (ab ′) 2 fragment that has two antigen binding sites and is still capable of cross-linking with antigen. “Fv” is an antibody fragment that contains a complete antigen-recognition and -binding site. This region includes a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. A single variable domain (or half of the Fv containing only three CDRs specific for the antigen) can recognize and bind the antigen. “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further contain a polypeptide linker between the VH domain and the VL domain, allowing the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

  The antibodies can be used for probes, therapeutic treatments and other applications. Antibodies can be generated by injecting translation products or synthetic peptide fragments thereof into mice, rabbits, goats or other animals. These antibodies are useful in diagnostic assays or as active ingredients in pharmaceutical compositions.

  The antibody or polypeptide to be administered can be complexed with a functional agent to form an immunoconjugate. Functional agents are cytotoxic such as chemotherapeutic agents, toxins (eg, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin), or radioisotopes (ie, radioconjugates). It can be an agent, antibiotic, nucleolytic enzyme, or any combination thereof. Chemotherapeutic agents such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents and / or fragments thereof, antibiotics And small molecule toxins, including toxins such as fragments and / or variants thereof, or enzymatically active toxins of bacterial, fungal, plant or animal origin, and various antitumor agents or anticancers disclosed below Agents can be used to generate immunoconjugates. Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria toxin A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A Chain, abrin A chain, modesin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S) (Momordica charantia) inhibitors, Kursin, Crotin, saponaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. Any suitable radionucleotide or radioactive agent known or otherwise available in the art can be used to produce antibodies that are radioconjugated.

  Conjugates of antibodies and cytotoxic agents are available for various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP); iminothiolane (IT); Derivatives (dimethyl adipimidate HcL, etc.); active esters (disuccinimidyl suberate, etc.); aldehydes (glutaraldehyde, etc.); bis-azide compounds (bis (p-azidobenzoyl) hexanediamine, etc.); bis-diazonium derivatives ( Bis- (p-diazonium benzoyl) -ethylenediamine etc.); Diisocyanate (triene 2,6-diisocyanate etc.); Bis-active fluorine compound (1,5-difluoro-2,4-dinitrobenzene etc.); Maleimide Caproyl (MC); valine-citrulline, a dipeptide in the protease cleavage linker Site (VC); 2-amino-5-ureidopentanoic acid PAB (= p-aminobenzylcarbamoyl) ("self immolative" part of the linker) (Citrulene); N-methyl-valine citrulline (here In which the linker peptide bond is modified to prevent cleavage by cathepsin B) (Me); maleimidocaproyl-polyethylene glycol that binds to the cysteine of the antibody; N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP) And N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). For example, the immunotoxin ricin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). 14C (Carbon-14) labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the complex formation of radionucleotide and antibody. See WO94 / 11026. The antibody can form a complex with a “receptor” (such as streptavidin) that is used for tumor pre-targeting, and after the antibody-receptor complex is administered to the subject, a clarifier is used to circulate blood. An unbound complex is removed from and then a “ligand” (eg, avidin) is complexed with a cytotoxic agent (eg, a radionucleotide).

  The antibodies of the invention can be conjugated directly or indirectly to a marker detectable by techniques known in the art. A detectable marker is an agent detectable by, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, high electron density reagents, enzymes, colored particles, biotin or dioxygenin. Detectable markers often generate measurable signals such as radioactivity, fluorescence, color or enzyme activity. Antibodies that complex with a detectable agent 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 used in various positron emission tomography, and non-radiating paramagnetic metal ions Is mentioned. The detectable substance may be conjugated or complexed directly with the antibody or indirectly using techniques known in the art, eg, via an intermediate such as a linker known in the art. can do. See, for example, US Pat. No. 4,741,900, which describes the formation of a complex between a metal ion and an antibody for diagnostic use. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-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, examples of luminescent materials include luminol, Examples include luciferin and aequorin.

  Antibodies useful for practicing the present invention can be prepared in laboratory animals or by DNA recombination methods using the methods described below. Polyclonal antibodies can be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the gene product molecule or fragment thereof with an adjuvant, such as Freund's adjuvant (complete or incomplete). In order to increase immunogenicity, first a bifunctional substance or derivatizing agent such as maleimidobenzoylsulfosuccinimide ester (complex formation via cysteine residues), N-hydroxysuccinimide (via lysine residues) , Glutaraldehyde, succinic anhydride, SOCl, etc., and a gene product molecule or fragment containing the target amino acid sequence and a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, It may be useful to complex with bovine thyroglobulin, or soybean trypsin inhibitor. Alternatively, the immunogenic complex can be made recombinantly as a fusion protein.

  An animal is injected with about 1 mg or about 1 microgram of the conjugate (for rabbits or mice, respectively) in combination with about 3 volumes of complete Freund's adjuvant and the solution is injected intradermally at multiple sites. Immunity against sex complexes or derivatives (such as fragments containing the target amino acid sequence) can be conferred. Approximately 7-14 days later, animals are bled and the serum antibody titer is chemically analyzed. The animals are boosted with the antigen repeatedly until the titer reaches a plateau. The animal can be boosted with the same molecule or fragment thereof used for the initial immunization, but may be complexed with different proteins and / or via different cross-linking agents. In addition, aggregating agents such as alum can be used in the injection solution to enhance the immune response.

  The antibody to be administered can comprise a chimeric antibody. The antibody to be administered can comprise a humanized antibody. The antibody to be administered can comprise a fully humanized antibody. The antibody may be humanized or partially humanized. Non-human antibodies can be humanized using any applicable method known in the art. Humanized antibodies can be produced using transgenic animals whose immune system is partially or fully humanized. Any antibody of the invention or fragment thereof may be partially or fully humanized. Chimeric antibodies can be produced using any technique known in the art. For example, see U.S. Pat. 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 to be administered can include a monoclonal antibody, ie, an anti-botulinum neurotoxin antibody of the present invention, which can be a monoclonal antibody. Monoclonal antibodies can be prepared using the hybridoma method as described in Kohler and Milstein, Nature, 256: 495 (1975). In hybridoma methods, mice, hamsters or other suitable host animals are usually immunized with an immunizing agent to produce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. Monoclonal antibodies 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). Can be screened as follows. Monoclonal antibodies can be tested for specific immunoreactivity with the translation product and loss of immunoreactivity with the corresponding prototype gene product.

  Monoclonal antibodies can be prepared by recovering spleen cells from the immunized animal and immortalizing the cells in a conventional manner, for example, by fusion with myeloma cells. Preferably, human-derived preperipheral blood mononuclear cells (PBMC) immunized with botulinum toxoids are fused with myeloma cells. Clones are then screened for those expressing the desired antibody. Monoclonal antibodies preferably do not cross-react with other gene products. After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution 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, hybridoma cells can be grown in vivo in mammalian ascites. Monoclonal antibodies secreted by subclones should be isolated or purified from culture media or ascites by conventional immunoglobulin purification methods such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Can do.

  Monoclonal antibodies can also be produced by DNA recombination methods as described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily obtained using conventional techniques (for example, by using an oligonucleotide probe capable of specifically binding to the gene encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the present invention can be a preferred source of such DNA. After isolation, the DNA can be incorporated into an expression vector, which is then a host such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would otherwise not produce immunoglobulin proteins Transfecting cells results in the synthesis of monoclonal antibodies in recombinant host cells. DNA is covalently linked, for example, by replacing the coding sequences of the human heavy and light chain constant domains with homologous mouse sequences or to immunoglobulin coding sequences in whole or in part of the coding sequences of non-immunoglobulin polypeptides. You may modify by doing. By replacing the constant domain of the antibody of the present invention with such a non-immunoglobulin polypeptide, or replacing the variable domain of one antigen-binding site of the antibody of the present invention, a chimeric bivalent antibody can be produced. . Preparation of an antibody using a DNA recombination method such as a phagemid display method is performed by, for example, Recombinant Phagemid Antibody System available from Pharmacia (Uppsala, Sweden), or SurfZAP ™ phage display system (Stratagene Inc., La Jolla, CA). Can be achieved using commercially available kits such as The present invention provides a human monoclonal antibody (HuMAb) produced against type A BoNT (BoNT / A) and type B BoNT (BoNT / B) using a mouse-human chimeric fusion partner cell line called SPYMEG.

  Furthermore, hybridoma cell lines, transformed B cell lines, and host cells that produce the monoclonal antibodies of the present invention, and their hybridomas, transformed B cell lines, and progeny or derivatives of host cells, and equivalent or similar hybridomas, traits Converted B cell lines and host cells are included in the present invention.

  The antibody can be a diabody. The term “diabody” refers to a small antibody fragment comprising two antigen binding sites. This fragment contains a heavy chain variable domain (VH) linked 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 two domains on the same chain, the domain can be paired with the complementary domain of another chain to generate two antigen-binding sites. . Diabodies are described in more detail in, for example, EP 404,097, WO 93/11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). Yes.

  The antibody to be administered can comprise a single chain antibody. The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. Heavy chains can generally be cleaved at any point in the Fc region 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 the antibody producing that fragment, particularly the Fab fragment, can be accomplished using conventional methods known in the art.

  The antibody can be bispecific. Producing bispecific antibodies that specifically bind to one protein and specifically bind to other antigens associated with disease states and / or treatments using standard techniques described in the literature; Isolate and test (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. Pat.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 antibodies were prepared using Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980), And by methods known in the art as described in US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in US Pat. 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 into filters of a predetermined pore size to obtain liposomes of the desired diameter. The Fab ′ fragment of the antibody of the present invention can be complexed with liposomes by a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982). 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 relates to a method of inhibiting or treating botulinum poisoning in a human subject, wherein the anti-botulinum neurotoxin monoclonal antibody of the present invention, an antigen-binding fragment thereof, A method is provided comprising administering both. The method can further include diagnosing the patient with botulinum neurotoxin poisoning. The anti-botulinum neurotoxin antibodies or antigen-binding fragments thereof of the present invention can be administered to a subject before, during and / or after diagnosis of a patient with botulinum neurotoxin poisoning. The method can further include monitoring the alleviation of at least one symptom of botulinum neurotoxin poisoning.

  According to the present invention, two or more botulinum neurotoxin antagonists can be administered. At least one of the botulinum neurotoxin antagonists may comprise a botulinum neurotoxin antagonist. 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 therapeutic agents for botulinum neurotoxin poisoning and / or botulinum infection. Administration of two or more therapeutic agents including one or more botulinum neurotoxin antagonists may be simultaneous administration, sequential administration, or combined administration. Thus, when two or more therapeutic agents are administered, it need not be administered simultaneously or in the same manner or at the same dose. When administered simultaneously, two or more therapeutic agents may be administered in the same composition or in different compositions. Two or more therapeutic agents may be administered using the same route of administration or different routes of administration. When administered separately, the therapeutic agents may be administered before or after each other. The order of administration of the two or more therapeutic agents can vary. Each dose of one or more therapeutic agents can vary over time. The type of one or more therapeutic agents can be changed over time. In the case of administration at multiple times (at separate times), the interval between two or more administrations may be any period. When administered multiple times, the length of the period can be varied. The interval between administration of two or more therapeutic agents is 0 seconds, 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, 1 month, 6 weeks, 8 weeks, 3 months, 6 months, 1 year or longer.

  Two or more botulinum neurotoxin antagonists can act synergistically to treat or reduce botulinum neurotoxin poisoning or symptoms thereof. Symptoms of botulism include, for example, paralysis, symmetric cranial nerve palsy, symmetric paralytic relaxation paralysis, symmetric paralysis of voluntary muscles, pharyngeal collapse, breathlessness, insufficiency, swallowing, low voice, drooping eyelids, facial paralysis, Pupil fixation, dilated pupil, foggy, hanging neck, generalized relaxation, generalized weakness, double vision, dysarthria, dysphonia, dysphagia, no sweat, mucosal erythema, postural hypotension, nausea, constipation, urinary retention , Dizziness, dry mouth, sore throat, stunted growth, strangulation reflex, and loss of systemic muscle reflexes. Botulism can be confirmed in the laboratory by demonstration of toxins in clinical laboratory materials or ingested food samples. Biological wound cultures are suggestive in symptomatic cases. Confirmation of botulism can be performed by a mouse bioassay. Neutralization of mouse paralysis by certain antitoxins suggests a serotype of the toxin in clinical samples.

  The botulinum neurotoxin antagonist is one or more anti-botulinum neurotoxin antibodies alone, or in combination with one or more other botulinum neurotoxin antagonists, eg, small drugs, or other anti-botulinum neurotoxin therapeutics Can be. Examples of small pharmaceuticals include antibiotics such as penicillin. Other anti-botulinum therapeutic agents include heptavalent botulinum antitoxin (HBAT). HBAT contains horse serum-derived antibody fragments against neurotoxins A to G, for example, containing less than about 2.0% intact immunoglobulins and about 90% or more Fab and F (ab ') 2 antibody fragments. obtain. Two or more anti-botulinum neurotoxin antibodies or at least one anti-botulinum neurotoxin antibody and one or more additional therapeutic agents can act synergistically to treat or reduce botulinum neurotoxin addiction. Two or more therapeutic agents, including one or more anti-botulinum neurotoxin antibodies, can be administered in synergistic amounts. Thus, administration of two or more therapeutic agents has a synergistic effect on alleviating one or more symptoms of botulinum neurotoxin poisoning, whether administered simultaneously, sequentially, or in any combination. obtain. The primary treatment can greatly increase the effectiveness of the secondary treatment than when the secondary treatment is used alone, or the secondary treatment can increase the effectiveness of the primary treatment Or both. The administration effect of two or more therapeutic agents may be such that the effect on alleviating one or more symptoms of botulinum neurotoxin poisoning is greater than the additive effect when each is administered alone. When given in a synergistic amount, the amount of one or more therapeutic agents alone has no substantial effect on one or more symptoms of botulinum neurotoxin poisoning, but one therapeutic agent is a botulinum nerve. The effectiveness of one or more other therapeutic agents for reducing one or more symptoms of toxin addiction can be increased. The measurement and calculation of synergy is 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 a combination index (CI) using CalcuSyn software.

  The exact formulation design, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (eg, Fingl et. Al., In The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. I See.) The attending physician can decide when to stop, stop, or adjust the dose due to toxicity or organ dysfunction. Conversely, the attending physician can adjust the treatment to a higher level if the clinical response is inadequate and non-toxic. The magnitude of an administrated dose in managing a subject's disorder depends on the severity of the disorder being treated and the route of administration. The severity of the disorder can be partially evaluated by, for example, standard prognostic evaluation methods. The dose and frequency of administration can vary according to the age, weight and response of the individual patient. Programs equivalent to those described above can be used in veterinary medicine.

  It is within the scope of the present invention to use a pharmaceutically acceptable carrier to formulate the compounds herein disclosed for practice of the present invention in dosages suitable for systemic administration. . By selecting an appropriate carrier and carrying out a suitable production, the composition related to the present invention, particularly one formulated as a solution, can be administered parenterally by intravenous injection or the like. The compounds can be readily formulated in dosage forms suitable for oral administration using pharmaceutically acceptable carriers known in the art. With such a carrier, the compound related to the present invention can be used as a tablet, pill, capsule, liquid, gel, syrup, slurry, tablet, dragee, solution, suspension for oral intake by the patient to be treated. Etc. can be blended.

  The therapeutic agent can be prepared in a depot form to allow for controlled release into the body as a function of time and location in the body (see, eg, US Pat. No. 4,450,150). The depot form of the therapeutic agent can be, for example, an implantable composition containing the therapeutic agent and a porous or non-porous material such as a polymer, where the therapeutic agent is encapsulated by the material. Or dispersed throughout the material and / or by degradation of the non-porous material. The depot is then implanted in the desired location within the body and the therapeutic agent is released from the implant at a predetermined rate.

  The therapeutic agent used in the present invention can be formed as a composition such as a pharmaceutical composition containing a carrier and a therapeutic compound. A pharmaceutical composition containing a therapeutic agent may include two or more therapeutic agents. Alternatively, the pharmaceutical composition may contain a therapeutic agent together with other pharmaceutically active agents or drugs.

  The carrier can be any suitable carrier. For example, the carrier can be a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier is any of those conventionally used in view of solubility and physicochemical properties such as loss of reactivity to the active compound (s), and the route of administration. There may be. In addition to or instead of the pharmaceutical composition described below, the therapeutic compound of the method of the invention can be formulated as an inclusion complex such as a cyclodextrin inclusion complex or a liposome.

  The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are known to those skilled in the art and are generally readily available. A pharmaceutically acceptable carrier can be one that is chemically inert to the active agent (s) and has no deleterious side effects or toxicity under the conditions of use. The choice of carrier can be made in part by the particular therapeutic agent and the particular method used to administer the therapeutic compound. There are a variety of suitable formulations of the pharmaceutical composition of the present invention. Oral administration, aerosol administration, parenteral administration, subcutaneous administration, transdermal administration, transmucosal administration, enteral administration, intramedullary injection, direct intraventricular administration, intravenous administration, intranasal administration, intraocular administration, intramuscular administration, The following formulations for 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 cases a particular route may result in a faster and more effective response than another route. Depending on the specific disorder being treated, such agents can be formulated and administered systemically or locally. Formulation and administration methods can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990).

  Formulations suitable for oral administration include: (a) an effective amount of an inhibitor dissolved in a liquid solution, for example, a diluent such as water, saline or orange juice; (b) a predetermined amount of each as a solid or granule Capsules, sachets, tablets, lozenges and lozenges, (c) powders, (d) suitable liquid suspensions, and (e) suitable emulsions containing the active ingredients may be included. Liquid formulations may include water and diluents such as alcohols such as ethanol, benzyl alcohol and polyethylene alcohol, with or without the addition of pharmaceutically acceptable surfactants. Capsule forms can be of the usual hard or soft shell gelatin type, containing for example surfactants, lubricants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch. Tablet form is 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, buffers, disintegrants, wetting agents, preservatives, flavoring agents, and other pharmacologically compatible excipients Or a plurality may be included. Lozenge forms include inhibitors in flavoring agents, usually sucrose and acacia or tragacanth, and inert bases such as gelatin and glycerin, or aromatic tablets containing inhibitors in sucrose and acacia, emulsions, gels, etc. In addition, excipients as known in the art can be included.

  Pharmaceutical preparations that can be used in the oral cavity include push-type capsules made of gelatin and sealed soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-in capsules may contain the active ingredient in admixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

  The therapeutic agent can be an aerosol formulation for inhalation administration alone or in combination with other suitable components. These aerosol formulations can be incorporated into acceptable high pressure gases such as dichlorodifluoromethane, propane, nitrogen and the like. These aerosol formulations can also be formulated as non-pressurized pharmaceuticals, such as in a nebulizer or atomizer. Such spray formulations can also be used to spray the mucosa. Topical formulations are known to those skilled in the art. Such formulations are particularly suitable for application to the skin in the present invention.

  Injectable formulations are in accordance with the present invention. Effective pharmaceutical carrier parameters for injectable compositions are known to those skilled in the art (eg, Pharmaeutics and Pharmacy Practice, JB 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 Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to penetrate the barrier are used in the formulation. Such penetrants are generally known in the art.

  Formulations suitable for parenteral administration may contain antioxidants, buffers, bacteriostats and solutes, making the formulations isotonic with the blood of the intended recipient. Isotonic sterile injectable solutions and aqueous and non-aqueous sterile suspensions may be included, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Add therapeutic agents, pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifiers, and other pharmaceutical adjuvants With or without addition, physiologically acceptable diluents in pharmaceutical carriers such as water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol Or polyethylene glycol, poly (ethylene glycol) 400, glycerol, dimethyl sulfoxide, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ether, oil, fatty acid, fatty acid ester or glyceride, or alcohol Including chill fatty acid glycerides, it may be administered in sterile solution or liquid mixture.

  Oils that can be used in parenteral formulations include petroleum, animal oils, vegetable oils or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Fatty acids suitable 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.

  Soaps suitable for use in parenteral formulations include alkali metal, ammonium and triethanolamine salts of fatty acids, and suitable detergents include (a) eg dimethyldialkylammonium halides and alkylpyridinium halides. And (b) anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; and (c) eg fatty Nonionic detergents such as aliphatic amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers, and (d) amphiphilic substances such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts Cleaning agent and (e) it It includes a mixture of.

  Parenteral formulations may contain about 0.5% to about 25% drug by weight in solution. Preservatives and buffers can be used. Such compositions may contain one or more nonionic surfactants having a hydrophilic lipophilic balance (HLB) of about 12 to about 17 to minimize or eliminate inflammation at the injection site. Good. The amount of surfactant in such formulations is usually in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan oleic acid monoester, and high molecular weight adducts of ethylene oxide and hydrophobic bases formed by condensation of propylene oxide and propylene glycol. . Parenteral formulations can be given in single-dose or multiple-dose sealed containers such as ampoules and vials and require only the addition of sterile liquid excipients, such as water, for injection just prior to use. Can be stored under freeze-dry (freeze-dry) conditions. 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. Pessaries, tampons, creams, gels, pastes, foams or spray formulations containing formulations suitable for vaginal administration, as well as active ingredients as known to be suitable in the art Can be given as.

  Agents for intracellular administration can be administered using techniques known to those skilled in the art. For example, such agents can be encapsulated in liposomes. Liposomes are spherical lipid bilayers with an aqueous interior. Molecules present in the aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposome contents are protected from the external microenvironment and are efficiently delivered to the cytoplasm by fusing the liposome with the cell membrane. Furthermore, due to its hydrophobicity, small organic molecules can be administered directly into cells. The materials and methods described in one aspect of the invention can also be used in other aspects of the invention. For example, materials such as nucleic acids or antibodies described for use in screening assays can also be used as therapeutic agents, and vice versa.

The invention encompasses the following aspects / embodiments / features in any order and / or in any combination:
1. An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof having neutralizing activity against type B botulinum neurotoxin, wherein the monoclonal antibody comprises a human monoclonal antibody, a humanized monoclonal antibody, or both An isolated anti-type B botulinum neurotoxin monoclonal antibody or an antigen-binding fragment thereof.
2. An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof of any of the above / below embodiments / features / aspects further comprising neutralizing activity of a type B botulinum neurotoxin against a progenitor toxin.
3. B produced by Clostridium botulinum B-type BONT / B1 (Okra strain), Clostridium botulinum B-type BoNT / B2 (111 strain), Clostridium botulinum B-type BoNT / B6 (Osaka05 strain), or any combination thereof An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof of any of the above / below embodiments / features / aspects, which has neutralizing activity against type botulinum neurotoxin.
4). An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above or below embodiments / features / aspects having specific binding activity to the light chain of a type B botulinum neurotoxin (BoNT / B) or Its antigen-binding fragment.
5). Human monoclonal antibodies are produced by hybridomas made by fusing human-derived peripheral blood mononuclear cells (PBMC) immunized with botulinum toxoids with fusion partner cells that allow efficient cell fusion An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects.
6). Botulinum neurotoxin B is Clostridium botulinum B type BONT / B1 (Okra strain), Clostridium botulinum B type BoNT / B2 (111 strain), Clostridium botulinum B type BoNT / B6 (Osaka05 strain), or any of them An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects, which is the product of a combination of:
7). An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects, wherein the fusion partner cell is a SPYMEG cell.
8). An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above or below embodiments / features / aspects, including IgG, Fab, Fab ′, F (ab ′) 2, scFv, dsFv, or combinations thereof Or an antigen-binding fragment thereof.
9. A first complementarity determining region (CDR1) having a first amino acid sequence comprising SEQ ID NO: 5 or SEQ ID NO: 19;
A second complementarity determining region (CDR2) having a second amino acid sequence comprising SEQ ID NO: 6 or SEQ ID NO: 20;
A third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7 or SEQ ID NO: 21;
A heavy chain variable region comprising:
A first complementarity determining region (CDR1) having a fourth amino acid sequence comprising SEQ ID NO: 12 or SEQ ID NO: 26;
A second complementarity determining region (CDR2) having a fifth amino acid sequence comprising SEQ ID NO: 13 or SEQ ID NO: 27;
A third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14 or SEQ ID NO: 28;
A light chain variable region comprising:
An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof of any of the above / below embodiments / features / aspects.
10. 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,
A heavy 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,
A third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14;
A light chain variable region comprising:
An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof of any of the above / below embodiments / features / aspects.
11. 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;
A third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21;
A heavy 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;
A third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28;
A light chain variable region comprising:
An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof of any of the above / below embodiments / features / aspects.
12 A heavy chain variable region comprising SEQ ID NO: 3 or SEQ ID NO: 17,
A light chain variable region comprising SEQ ID NO: 10 or SEQ ID NO: 24;
A botulinum neurotoxin monoclonal antibody B of any of the above or below embodiments / features / aspects, or an antigen-binding fragment thereof, comprising:
13. A Botulinum neurotoxin monoclonal antibody of any of the above or below embodiments / features / aspects that is IgG1.
14 A hybridoma whose deposit number is NITE BP-01639 or NITE BP-01640.
15. An isolated monoclonal antibody produced by a hybridoma having a deposit number of NITE BP-01639 or NITE BP-01640.
16. Comprising one or more isolated anti-type B botulinum neurotoxin monoclonal antibodies or antigen-binding fragments thereof of any of the above or below embodiments / features / aspects, and a pharmaceutically acceptable carrier. Pharmaceutical composition.
17. A pharmaceutical composition according to any of the above or below embodiments / features / aspects, comprising an anti-type B botulinum neurotoxin monoclonal antibody and a pharmacologically acceptable carrier.
18. A pharmaceutical composition of any of the above / below embodiments / features / aspects comprising two isolated anti-type B botulinum neurotoxin monoclonal antibodies, antigen-binding fragments thereof, or both,
(1) 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 heavy chain variable region comprising a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7;
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 SEQ ID NO: 14 A light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising:
An isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(2) an isolated human monoclonal antibody or antigen-binding fragment 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 produced by a hybridoma whose deposit number is NITE BP-01639, or
Any combination of them,
A first isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(4) 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 heavy chain variable region comprising a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21;
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 SEQ ID NO: 28 A light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising:
An isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(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 produced by a hybridoma whose deposit number is NITE BP-01640, or
Any combination of them,
A second isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
A pharmaceutical composition comprising:
19. A method for producing an isolated anti-type B botulinum neurotoxin monoclonal antibody comprising:
Creating a hybridoma by fusing a human-derived peripheral blood mononuclear cell (PBMC) immunized with a botulinum toxoid with a fusion partner cell that allows efficient cell fusion;
Obtaining a botulinum neurotoxin monoclonal antibody type B from the hybridoma;
Including a method.
20. Botulinum neurotoxin B is Clostridium botulinum B type BONT / B1 (Okra strain), Clostridium botulinum B type BoNT / B2 (111 strain), Clostridium botulinum B type BoNT / B6 (Osaka05 strain), or any of them A method of making an isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects produced by the combination of:
21. A method of producing an anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects, wherein the fusion partner cell is a SPYMEG cell.
22. Prevention, treatment of type B botulinum neurotoxin poisoning in a human subject comprising an isolated anti-type B botulinum neurotoxin monoclonal antibody, antigen-binding fragment thereof, or both of any of the above / below embodiments / features / aspects And / or a kit for detection.
23. A method of inhibiting or treating botulism in a human subject, comprising a therapeutically effective amount of any of the above / below embodiments / features / aspects of an isolated anti-type B botulinum neurotoxin monoclonal antibody, Administering the antigen-binding fragment, or both, to a human subject.
24. A method of inhibiting or treating botulism in a human subject of any of the above / below embodiments / features / aspects, further comprising diagnosing the patient as having botulinum neurotoxin poisoning.
25. A method of inhibiting or treating botulism in a human subject of any of the above / below embodiments / features / aspects, further comprising monitoring the alleviation of at least one symptom of botulism.
26. At least one symptom is paralysis, symmetric cranial nerve palsy, symmetric paralytic relaxation palsy, voluntary symmetric paralysis, pharyngeal collapse, respiratory arrest, insufficiency, swallowing, weak voice, drooping eyelids, facial paralysis, pupil fixation, Pupil dilation, foggy, drooping, generalized relaxation, generalized weakness, double vision, dysarthria, dysphonia, dysphagia, mucus erythema, postural hypotension, nausea, constipation, urinary retention, dizziness Inhibit botulism in a human subject of any of the above / below embodiments / features / aspects, including dry mouth, sore throat, stunted growth, strangulation reflex, loss of systemic muscle reflex, or any combination thereof How to treat.
27. Botulism, food-borne botulism, wound-infected botulism, childhood enterotoxemia botulism, adult enterotoxemia botulism, iatrogenic botulism, air-borne botulism, Or a method of inhibiting or treating botulism in a human subject of any of the above / below embodiments / features / aspects, including any combination thereof.
28. An isolated anti-type B botulinum neurotoxin monoclonal antibody of any of the above / below embodiments / features / aspects, an antigen-binding fragment thereof, or both, and one or more additional therapies for botulism A method of inhibiting or treating botulism in a human subject of any of the above / below embodiments / features / aspects administered in combination.
29. A method of inhibiting or treating botulism in a human subject of any of the above-described embodiments / features / aspects above, wherein the combination therapy acts synergistically to inhibit or treat botulism.
30. Any of the above or below embodiments, wherein the one or more additional therapies comprise administering a second isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both A method of inhibiting or treating botulism in a human subject of / feature / aspect.
31. Any of the above / below embodiments / features / wherein the one or more additional therapies comprise administering an isolated anti-type A botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both A method of inhibiting or treating botulism in a human subject of embodiments.
32. An isolated anti-type B botulinum neurotoxin monoclonal antibody, antigen-binding fragment thereof, or both of any of the embodiments / features / aspects described above for the manufacture of a medicament for inhibiting or treating botulism in a human subject Use of.
33. A method for detecting botulinum neurotoxin type B in a human subject comprising:
Contacting a sample from a human subject with any of the above-described or below-described embodiments / features / aspects of an isolated anti-type B botulinum neurotoxin monoclonal antibody, an antigen-binding fragment thereof, or both;
Detecting the presence or absence of a botulinum neurotoxin type B in a human subject based on whether the antibody, fragment thereof, or both binds to a botulinum neurotoxin type B;
Including a method.

  The invention will be further clarified by the following examples, which are intended to be illustrative of the invention and not limiting. These examples demonstrate the surprising and unexpected properties of the claimed antibodies, including, for example, the ability to neutralize botulism and treat botulism infections. These examples demonstrate the expression of human monoclonal antibodies against botulinum neurotoxins according to 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 BoNT / B light chain and showed potent neutralizing activity (approximately> = 100 LD ₅₀ / mg) in mouse bioassays. Is done. Furthermore, the combination of M-2 and M-4 was able to completely neutralize BoNT / B with potency exceeding 10,000 LD ₅₀ / mg. This potency is the most effective to date and is more than 18 times greater than the BoNT / B neutralization standard (540 LD ₅₀ / mg) considered for the commercial BABYBIG human anti-BoNT antiserum. In a post-exposure treatment model, HuMAb (M-2 + M-4) provided complete protection against lethal doses of BoNT / B when treated within 12 hours after oral administration of the toxin. HuMAb (M-2 + M-4) neutralized BoNT / B1 and other serotypes BoNT / B2 and BoNT / B6. Data obtained in experiments with M-2 and M-4 human monoclonal antibodies generally represent antibodies according to the present invention, and methods for making and using antibodies.

Example 1

Clostridium botulinum type B Okra strain was cultured using the cellophane tube method to obtain a culture supernatant. Progenitor toxin (16S toxin and 12S toxin) was purified from the culture supernatant using Non-Patent Document 1. BoNT / B1 and NAP-16 were prepared from 16S toxin as described in Non-Patent Document 1. BoNT / A1 (62A strain), BoNT / B2 (111 strain) and BoNT / B6 (Osaka05 strain) were provided by Dr. S. Kozaki. A tetravalent botulinum toxoid vaccine was provided by Dr. M. Takahashi. The 12S toxin was purified from each culture supernatant of A, B, E, and F C. botulinum and detoxified with formalin. Toxin detoxification was tested intraperitoneally (ip) using mice. The four types of toxoid preparations were mixed together and then mixed with aluminum (adjuvant) and thimerosal (preservative). The toxoid preparation contained 0.1 mg, 0.2 mg aluminum, 0.001% thimerosal, and 0.0006% formalin, respectively, per mL.

  The immunization schedule followed that shown in FIG. Tetravalent botulinum toxoid (type A, type B, type E and type F) was given informed consent in writing and inoculated (4 or 5 times) into two healthy adult volunteers who had undergone physical examination . Toxoid was injected intramuscularly only in healthy individuals at a dose of 0.5 mL. After 9 and 18 days, peripheral blood samples (10 mL) were collected from each volunteer. Blood samples were taken from each volunteer and the antibody titer of plasma samples against BoNT / A or BoNT / B was tested using ELISA. This protocol was approved by the Osaka University Clinical Trial Review Board.

  Enzyme-linked immunosorbent assay (ELISA) was performed as follows. A 96-well plate (Falcon) was coated with 100 microliters of BoNT / A and / or BoNT / B at a final concentration of 3 micrograms per mL of PBS at 37 ° C. for 2 hours. Wells were washed with PBS containing 0.05% Tween (PBS-T) and blocked overnight at 4 ° C. with 0.2% BSA (Sigma) / PBS-T. Human serum samples were serially diluted 2-fold and 50 microliters of each dilution was added to the wells and incubated at 37 ° C. for 2 hours. After washing, 50 microliters of anti-human IgG-horseradish peroxidase (HRP) complex (BIO-RAD) was added and incubated at 37 ° C. for 2 hours. After washing, the plate was incubated with a substrate solution (o-phenylenediamine, Nacalai Tesque, Inc.) at 37 ° C. for 30 minutes, and the absorbance value of OD492 was measured. ELISA titers were expressed as the highest dilution factor showing an absorbance higher than twice that of the negative control serum. Antibody titers were higher in 2 volunteers 9 or 18 days after vaccination (Table 1).

Example 2
Cell fusion, screening and cloning methods were performed as shown in FIG. Peripheral blood samples were collected from volunteers 9 or 18 days after the last immunization and peripheral blood mononuclear cells (PBMC) were purified by Ficoll (GE Healthcare) gradient centrifugation. PBMC were fused with SPYMEG cells in a 10: 1 ratio using polyethylene glycol (Roche). The fused cells were cultured in 96-well plates in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 15% FBS in the presence of hypoxanthine-aminopterin-thymidine (HAT, GIBCO) for about 10-14 days. . After selection by HAT, a first screening of the culture medium was performed by ELISA for antibodies specific for BoNT / A or BoNT / B. As a result, 27 positive wells were obtained from blood samples taken 9 days after vaccination and 8 positive wells were obtained from blood samples taken 18 days after vaccination. Next, cell cloning by limiting dilution was performed on positive wells of specific antibodies to obtain clonal cells. The second screening was also performed by ELISA. Eight stable hybridoma clones were obtained (Table 2).

  Isotype analysis shows 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).

  After each stable hybridoma was cultured in serum-free medium (GIBCO), the supernatant was collected. The isotype of HuMAb in the culture supernatant was determined by anti-human IgG-HRP complex (BIO-RAD), anti-human IgM-HRP complex (BIOSOURCE INTERNATIONAL), or anti-human IgA-HRP complex (Invitrogen) in Western blot. Were determined. IgG antibody was purified from the supernatant by Protein G column (GE Healthcare). The subclass of each IgG antibody was determined using an IgG subclass human ELISA kit (Invitrogen).

Example 3
Cloning and sequencing of the variable region gene of the monoclonal antibody was performed. The determined heavy chain variable region and light chain variable region sequences are shown in FIGS. Total RNA was extracted from the hybridoma using the RNeasy Mini Kit (Qiagen) and prepared by RT-PCR using the SUPERSCRIPT ™ VILO ™ cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. The H-2 and L-4 coding regions of the M-2 and M-4 antibodies are labeled with KOD-Pus-Neo (Toyobo) and the following primer: 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) and was amplified by PCR. After incubation with TaKaRa Ex Taq ™ (Takara Bio Inc.), the PCR products were ligated into pGEM-T Easy Vector (Promega), and their sequences were transferred to BigDye Terminator v3.1 Cycle Sequencing Kit and ABI Prism Analysis was performed using 3100 Genetic Analyzer (Applied Biosystems). IgG subclass assays revealed that M-2 and M-4 are composed of IgG1 heavy and light (lambda) chains.

Example 4
BoNT / A or BoNT / B (600 ng) was separated into Hc and Lc by SDS-PAGE and transferred to a nitrocellulose membrane (BIO-RAD). Membranes were blocked with 5% skim milk in Tris buffered saline with 0.05% Tween. HuMAb (1.0 microgram / 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-HRP complex for 1 hour at room temperature, and then specific bands were visualized by ECL (PIERCE) (FIGS. 9A and 9B).

Example 5
Western blot analysis was performed to determine the epitope for HuMAb. These results indicate that M-2 specifically bound to the BoNT / B light chain (FIG. 10). In contrast, M-4 bound to both the light and heavy chains (Figure 10).

Example 6
Binding of HuMAb to type B BoNT, progenitor toxin or non-toxic component (NAP-16) was analyzed by ELISA. HuMAbs were added to plates coated with BoNT, progenitor toxin or NAP-16. HuMAb (0.01-0.5 microgram / mL) was added to the BoNT / A or BoNT / B coated plates. After washing, bound HuMAb was detected by anti-human IgG antibody-HRP complex. M-2 and M-4 bound to BoNT and progenitor toxin. In contrast, M-2 and M-4 showed slight binding to NAP-16. These results indicate that M-2 and M-4 bind not only to BoNT alone but also to BoNT in the progenitor toxin, and that M-2 and M-4 specifically bind to BoNT. M-2 and M-4 showed specific binding to BoNT / B (FIG. 11).

Example 7
HuMAb neutralizing activity was tested by mouse bioassay. HuMAbs were incubated with BoNT / B (10 LD ₅₀ ) for 1 hour at room temperature prior to intraperitoneal injection (total volume 500 microliters) into mice (ddY, female, 4 weeks old, SLC). 10 LD ₅₀ of BoNT / B was incubated for 1 hour with 100 micrograms of HuMAb or PBS (control: Cnt) and injected intraperitoneally into mice. Mice morbidity and mortality were observed over 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 from increased time-to-death over control mice. On the other hand, complete protection was observed in mice injected with BoNT / B + M-2 (Table 4).

Example 8
The synergistic effect of the combination of HuMAb was tested. 10 LD ₅₀ 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 mice ingested HuMAb survived and showed no symptoms (FIG. 12).

Example 9
In a post-exposure treatment model, mice were orally administered type B progenitor toxin (16S toxin, 10 ng in a volume of 300 microliters) and then intraperitoneally 12 hours, 24 hours or 36 hours after oral administration of 16S toxin HuMAb (M-2 + M-4) was administered by injection. Mice morbidity and mortality were observed over 3 weeks. In the post-exposure treatment model, mice developed botulism 12 hours after oral administration of 16S toxin. Control mice that were not treated with HuAb died within 72 hours. In contrast, post-exposure treatment with HuMAb resulted in complete survival at 12 hours after oral administration of 16S toxin and partial survival at 24 and 36 hours after administration (FIG. 13).

Example 10
The binding of HuMAb (M-2 and M-4) to BoNT / B2 (111 strain) and BoNT / B6 (Osaka05 strain) was analyzed by ELISA. HuMAb (0.5 microgram / mL) was added to the BoNT coated plate. After washing, bound HuMAb was detected by anti-human IgG antibody-HRP complex. 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 microgram + 0.5 microgram) completely neutralized BoNT / B2 (10 ng) and BoNT / B6 (2.5 ng) (Figure 14).

Example 11
For expression of recombinant HuMAb in mammalian cells (HEK293 cells) and testing of recombinant HuMAb, an IgG expression vector was constructed as follows. PGEM-T Easy vector having H chain and L chain variable region genes was subjected to PCR, and restriction enzyme sites and Kozak sequences were added using 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) (SEQ ID NO: 45), 5'- ATTT GCGGCCGC CATGGCCTGGGCTCTGCT-3 ′ (M-2 and M-4-18-L chain sense primer) (SEQ ID NO: 46), 5′-ATTT GCGGCCGC CATGGCCTGGGTCTCATT-3 ′ (M-4-19-L chain sense primer) ( SEQ ID NO: 47) and 5′-ATA CTCGAG GGCGGGAACAGAGTGACCGTGG-3 ′ (light chain antisense primer) ( SEQ ID NO: 48). After digesting the PCR products of the H chain coding region and L chain coding region with restriction enzymes Not I and Xho I, expression vectors pQCXIP-hCH and pQCXIH-hC (MBL) having human immunoglobulin constant regions of gamma chain and lambda chain, respectively. ).

HEK293 cells were cultured in MEM containing 10% FBS at 37 ° C. and 5% CO ₂ . Cells grown in 100 mm culture dishes 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, the cells were washed and cultured in Opti-Pro serum-free medium (Gibco) at 37 ° C. for 10 days in 5% CO ₂ . Recombinant HuMAb in the culture supernatant was determined by anti-human IgG-HRP complex (BIO-RAD). Recombinant HuMAb was purified from the supernatant by Protein G column (GE Healthcare).

Recombinant HuMAbs (RM-2 and RM-4; RMs having the same heavy and light chains as M-4 but with different light chain signal sequences as shown in FIGS. 6, 7 and 8, respectively. -4 LC16, RM-4 LC18, RM-4 LC19) and BoNT / B were analyzed by ELISA. RM-2 and RM-4 (approximately 0.01-0.5 microgram / mL) were added to the plate. After washing, bound recombinant HuMAb was detected by anti-human IgG antibody-HRP complex. As a result, RM-2 and RM-4 bound to BoNT / B in the same way as M-2 and M-4 derived from hybridomas (FIG. 15). In the neutralization test, RM-2 + RM-4 (0.5 microgram + 0.5 microgram) neutralized BoNT / B1 (10 LD ₅₀ ) (Table 5A and Table 5B).

Example 12
Binding between HuMAb and recombinant HuMAb (RM-2 and RM-4) derived from hybridoma and BoNT / B (BoNT / B1, BoNT / B2 or BoNT / B6) was analyzed by SDS-PAGE. BoNT / B1, BoNT / B2 or BoNT / B6 was separated into Hc and Lc by SDS-PAGE and transferred to a nitrocellulose membrane. HuMAb (1.0 microgram / 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-HRP complex for 1 hour at room temperature, after which specific bands were visualized by ECL. Recombinant HuMAb showed the same binding profile as HuMAb derived from hybridoma. M-2 and RM-2 specifically bound to the light chain of BoNT / B. In contrast, M-4 and RM-4 bound to both light and heavy chains. The M2 and RM-4 antibodies detected the B2 subtype heavy chain more clearly than the B1 heavy chain. Furthermore, the heavy chain of the B6 subtype was detected more clearly than the heavy chain of B2 (FIG. 16).

  Applicants specifically incorporate the entire contents of all cited references in this disclosure. Furthermore, if an amount, concentration or other value or parameter is given as either a range, a preferred range, or a list of preferred upper and lower limits, this is true regardless of whether the ranges are disclosed separately. It is to be understood that any range consisting of any combination of any upper range limit or preferred value and any lower range limit or preferred value is specifically disclosed. Where a numerical range is referred to herein, unless otherwise specified, the range is intended to include its endpoints and all integers and decimals 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 invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be illustrative only and that the true scope and spirit of the invention be indicated by the appended claims and their equivalents.

Claims (12)

An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof having neutralizing activity against a type B botulinum neurotoxin, comprising:
The monoclonal antibody,
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,
A heavy 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,
A third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 14;
Including a light chain variable region,
An isolated anti-type B botulinum neurotoxin monoclonal antibody or an antigen-binding fragment thereof. An isolated anti-type B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof having neutralizing activity against a type B botulinum neurotoxin, comprising:
The monoclonal antibody is
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;
A third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21;
A heavy 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;
A third complementarity determining region (CDR3) having a sixth amino acid sequence comprising SEQ ID NO: 28;
Including a light chain variable region,
An isolated anti-type B botulinum neurotoxin monoclonal antibody or an antigen-binding fragment thereof. A heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 10, or
A heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 24;
Including, B botulinum neurotoxin monoclonal antibody or antigen-binding fragment thereof. The isolated anti-type B botulinum neurotoxin monoclonal antibody according to any one of claims 1 to 3, comprising any one selected from IgG, Fab, Fab ', F (ab') 2, scFv, and dsFv Its antigen-binding fragment. The B-type botulinum neurotoxin monoclonal antibody according to any one of claims 1 to 3 , wherein the monoclonal antibody is IgG1.   A hybridoma whose deposit number is NITE BP-01639 or NITE BP-01640.   An isolated monoclonal antibody produced by a hybridoma having a deposit number of NITE BP-01639 or NITE BP-01640. A pharmaceutical composition comprising the anti-type B botulinum neurotoxin monoclonal antibody and / or an antigen-binding fragment thereof according to any one of claims 1 to 3, and a pharmacologically acceptable carrier. 9. A pharmaceutical composition according to claim 8 , comprising the isolated anti-type B botulinum neurotoxin monoclonal antibody according to claim 7 and a pharmacologically acceptable carrier. 10. A pharmaceutical composition according to claim 8 or 9 , comprising first and second two isolated anti-type B botulinum neurotoxin monoclonal antibodies, antigen-binding fragments thereof, or both.
(1) 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 heavy chain variable region comprising a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 7;
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 SEQ ID NO: 14 A light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising:
An isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(2) an isolated human monoclonal antibody or antigen-binding fragment 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 produced by a hybridoma whose deposit number is NITE BP-01639, or
Any combination of them,
A first isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(4) 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 heavy chain variable region comprising a third complementarity determining region (CDR3) having a third amino acid sequence comprising SEQ ID NO: 21;
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 SEQ ID NO: 28 A light chain variable region comprising a third complementarity determining region (CDR3) having a sixth amino acid sequence comprising:
An isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
(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 produced by a hybridoma whose deposit number is NITE BP-01640, or
Any combination of them,
A second isolated human monoclonal antibody or antigen-binding fragment thereof, comprising
A pharmaceutical composition comprising: A botulinum neurotoxin type B in a human subject comprising one or more selected from the isolated anti-type botulinum neurotoxin monoclonal antibody and antigen-binding fragment thereof according to any one of claims 1 to 3 and 7. A kit for preventing, treating and / or detecting addiction. A method for detecting botulinum neurotoxin type B in a blood sample taken from a human comprising the following steps :
Blood samples taken from 1) the person, one or more selected from the isolated anti-B botulinum neurotoxin monoclonal antibodies and antigen-binding fragments thereof of any of claims 1 to 3 and 7 In contact with ;
2) Detecting the presence or absence of botulinum neurotoxin type B in the blood sample collected from the human .