CN104837866B - Human monoclonal antibodies combination/neutralization Type B botulic neurotoxin - Google Patents

Human monoclonal antibodies combination/neutralization Type B botulic neurotoxin Download PDF

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CN104837866B
CN104837866B CN201380044898.6A CN201380044898A CN104837866B CN 104837866 B CN104837866 B CN 104837866B CN 201380044898 A CN201380044898 A CN 201380044898A CN 104837866 B CN104837866 B CN 104837866B
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determining region
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amino acid
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CN104837866A (en
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藤永由佳子
松村拓大
生田和良
三崎亮
藤山和仁
幸田知子
小崎俊司
小野健郎
小野健一郎
王荣萨·毗亚达
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Medical and Biological Laboratories Co Ltd
Osaka University NUC
Osaka Prefecture University
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Osaka University NUC
Osaka Prefecture University
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Abstract

Provide the anti-Type B botulic neurotoxin monoclonal antibody and its antigen-binding fragment of separation, the neutralization activity with anti-Type B botulic neurotoxin, including human monoclonal antibodies；The present invention also provides antibody as generation or the hybridomas of its segment, and generate the method for these hybridomas and generate antibody or the method for its segment from these hybridomas；Pharmaceutical composition and kit including antibody or its segment are additionally provided for preventing, treating and detecting at least one of Type B botulic neurotoxin poisoning in human experimenter；The method for providing inhibition or treating botulismus in human experimenter, similarly, the method for providing Type B botulic neurotoxin in detection human experimenter.

Description

Human monoclonal antibodies combination/neutralization Type B botulic neurotoxin
Technical field
The present invention relates to anti-botulic neurotoxin (botulinum neurotoxin) (botulinum toxin (botulin)) Antibody and the method that uses and manufacture them.
[related application]
This application claims the equity for the U.S. Provisional Patent Application No.61/695318 that August in 2012 is submitted on the 31st, the Shens Please it is incorporated by herein by quoting.
Background technology
Botulic neurotoxin is known most toxic substance.These toxin can lead to botulismus (botulism), include by various bacteriumsClostridium botulinum (Clostridium botulinum)It generates.Generate meat poisoning The clostridium of bacillus toxin is anaerobic gram positive bacteria.These bacteriums form spore, and are typically distributed on ring In border.On a molecular scale, botulismus is the inhibition by being discharged by toxin acetylcholine caused by myoneural junction It is caused.Have determined 7 kinds of toxin serotypes (A, B, C, D, E, F and G).They have correspond to include 100-kDa heavy chains with The similar structure of the zinc endopeptidase albumen of the about 150kD of 50-kDa light chains.Toxin serotypes A, B, E and F are usually and the mankind Middle botulismus is related.
Botulismus is segmented into different classes of-food-borne, wound infection, baby's enterotoxemia, adult's enterotoxemia, poison Element injection (iatrogenic botulismus) and weaponization toxin.Food origin disease is since intake is generated the germ contamination of toxin Toxin in food and occur.In wound infection, the malicious clostruidium of wound aggregation production, then its grow in wound with Generate toxin.In baby's botulismus, malicious clostruidium is produced in baby intestinal aggregation, and then it generates toxin.Adult's intestines Toxaemia is similar to baby's botulismus.Beauty or treatment injection botulin toxin there may come a time when to lead to botulismus.Meat poisoning Poisoning injures caused by can also resulting from weaponization toxin, for example, the botulin toxin by sucking aerosolization.
The characteristics of botulismus is symmetrical parmlysis of cranial nerve, and then voluntary muscle successively decreases symmetrical flaccid paralysis.This paralysis Cause to lose breathing and death.It is irreversible that toxin, which combines,.Restore the growth for being related to new nerve endings.Recovery may be very long , and it is related to extended outpatient rehabilitation treatment.After toxin exposure, the breaking-out of the disease can be rapid.Treatment usually relates to And antitoxin is applied, it can help to limit the degree of paralysis.Available most of antitoxins are horse source property, and not necessarily It is exclusively used in certain types of botulic neurotoxin.In the presence of antitoxic needs to more targeted form.Also Have to being free from side effects such as the needs of the effective toxin neutralizing antibody of serum sickness.In view of botulismus development is so fast, Further there are the needs to better diagnostic test.
Reference listing
Non-patent literature
[non-patent literature 1] Arimitsu etc., Infect.Immun.71：1599-1603,2003
Invention content
(technical problem)
Therefore, feature of this invention is that providing the anti-botulic neurotoxin with superior efficacy and safety Therapeutic antibodies.
The method that another feature of the invention is to provide inhibition and treats botulismus.
The further feature of the present invention is to provide the therapeutic agent being free from side effects such as serum sickness.
The other feature and advantage of the present invention will be illustrated partly in the following description, and part is according to specification Will be apparent, or can be through the invention implementation and learn.Objectives and other advantages of the present invention will be by illustrating The element that is particularly pointed out in book and the attached claims and combination are realized and are reached.
(technical solution for solving technical problem)
It is being such as embodied here and extensive retouch in order to realize these and other advantage, and purpose according to the present invention It states, the present invention relates to the anti-Type B botulic neurotoxin monoclonal antibody of separation or its antigen-binding fragments, have anti-B The neutralization activity of type botulic neurotoxin.Monoclonal antibody may include human monoclonal antibodies, Humanized monoclonal antibodies, Or both.The anti-Type B botulic neurotoxin monoclonal antibody or its antigen-binding fragment of separation can have anti-Type B meat poisoning The neutralization activity of the precursor toxin of bacillus neurotoxin.The anti-Type B botulic neurotoxin monoclonal antibody of separation or it is anti- Former binding fragment can have to Type B botulic neurotoxin (botulinum neurotoxin type B, BoNT/B) The specific binding activity of light chain or with to the special of the light chain of Type B botulic neurotoxin (BoNT/B) and both heavy chains Property combine activity.The present invention also provides the hybridomas (Hybridomas) of the antibody or its segment that generate the present invention, and are formed The method of such hybridoma, and generate antibody or the method for its segment from such hybridoma.
The invention further relates to pharmaceutical compositions.Pharmaceutical composition can include the anti-Type B meat poisoning bar of one or more separation Bacterium neurotoxin monoclonal antibody and its antigen-binding fragment and pharmaceutically acceptable carrier.The present invention is provided to pre- Anti-, treatment and the kit for detecting at least one of Type B botulic neurotoxin poisoning in human experimenter.Kit can With including the present invention the anti-Type B botulic neurotoxin monoclonal antibody of separation, its antigen-binding fragment, or both.This Invention also provide the anti-Type B botulic neurotoxin monoclonal antibody of separation, its antigen-binding fragment, or both be used to prepare Purposes for inhibiting or treating the drug of botulismus in human experimenter.
The invention further relates to the methods for inhibiting or treating botulismus in human experimenter.Method may include to the mankind by Examination person applies the anti-Type B botulic neurotoxin monoclonal antibody of the separation of therapeutically effective amount, its antigen-binding fragment or two Person.Method can also include diagnosis patient's botulic neurotoxin poisoning.Method may include monitoring botulismus at least A kind of reduction of symptom.The method for inhibiting or treating botulismus in human experimenter may include using the anti-Type B meat detached Bacillus venenosus neurotoxin monoclonal antibody, its antigen-binding fragment, or both and combine one or more for botulismus In addition treatment.Joint can act synergistically to inhibit or treat botulismus.
The invention further relates to the methods of Type B botulic neurotoxin in detection human experimenter.Method may include by Sample from human experimenter with detach anti-Type B botulic neurotoxin monoclonal antibody, its antigen-binding fragment or The two contacts.Method can also include based on antibody, its segment, or both whether examine in conjunction with Type B botulic neurotoxin Survey the existence or non-existence of Type B botulic neurotoxin in human experimenter.
It should be understood that foregoing general description and it is subsequent detailed description be all only exemplary with it is illustrative, it is intended to carry For claimed invention is explained further.
Attached drawing includes in this application and to form the part of the application, illustrates some specific embodiments of the present invention, and And it is used to explain the principle of the present invention together with specification.
Brief Description Of Drawings
Fig. 1 is to show how volunteer according to the present invention is immunized and generates the cell of antibody and how to detach Schematic diagram.
Fig. 2 is the flow chart for describing cell fusion and clone-time table according to the present invention.
Fig. 3 shows the nucleotide sequence and amino acid sequence of the heavy chain of M-2 antibody according to the present invention.
Fig. 4 shows the nucleotide sequence and amino acid sequence of the light chain of M-2 antibody according to the present invention.
Fig. 5 shows the nucleotide sequence and amino acid sequence of the heavy chain of M-4 antibody according to the present invention.
Fig. 6 shows the nucleotide sequence and amino acid sequence of the light chain of RM-4LC-16 antibody according to the present invention.
Fig. 7 shows the nucleotide sequence and amino acid sequence of the light chain of RM-4LC-18 antibody according to the present invention.
Fig. 8 shows the nucleotide sequence and amino acid sequence of the light chain of RM-4LC-19 antibody according to the present invention.
Fig. 9 includes the line chart for showing human monoclonal antibodies combination BoNT/A or BoNT/B according to the present invention.People Dan Ke Grand antibody (HuMAb) is analyzed in conjunction with BoNT by ELISA.HuMAbs (about 0.05-1.0 mcg/mls), which is added to, to be coated with The plate of BoNT/A or BoNT/B.After washing, in conjunction with HuMAbs by be coupled HRP anti-human IgG antibodies be detected.M-2 and M-4 shows the specific binding to BoNT/B.
Figure 10 includes that the west of the epitope mapping according to the present invention for human monoclonal antibodies turns stain (Western Blot it) analyzes.BoNT is separated into Hc and Lc by SDS-PAGE, and is transferred on nitrocellulose filter.HuMAbs (1.0 micrograms/ Milliliter) film is added to, and incubate 1 hour at room temperature.It is after washing, the anti-human IgG antibodies of film and coupling HRP are warm at room temperature It educates 1 hour, then specific band is visualized by ECL.M-2 specifically binds the light chain of BoNT/B.In contrast, M-4 is tied Close both light chain and heavy chain.
Figure 11 is the column for showing human monoclonal antibodies combination BoNT/B according to the present invention, precursor toxin and non-toxic components Shape figure.HuMAb combinations BoNT, precursor toxin (12S toxin and 16S toxin) or non-toxic components (NAP-16) are carried out by ELISA Analysis.HuMAbs adds to the plate for being coated with BoNT, precursor toxin or NAP-16.After washing, in conjunction with HuMAbs pass through be coupled HRP Anti-human IgG antibodies be detected.M-2 and M-4 combinations BoNT and precursor toxin.In contrast, M-2 and M-4 are shown to NAP- 16 faint combination.These show M- the result shows that the BoNT in M-2 and M-4 combination precursor toxin and individual BoNT 2 and M-4 specifically binds BoNT.
Figure 12 is the line chart combined to the neutralization activity of BoNT/B for showing human monoclonal antibodies according to the present invention. 10LD50The mixture of BoNT/B and M-2 and M-4 (each 0.05,0.1 or 0.5 microgram) incubate and be injected into mouse.It is all The mouse for receiving the mixture of M-2 (0.5 microgram) and M-4 (0.5 microgram) survives and does not have symptom.
Figure 13 is to show that the neutralization to precursor toxin (16S toxin) after human monoclonal antibodies application according to the present invention is lived The line chart of property.To Mouse oral apply precursor toxin (16S toxin, 10 nanograms), then 12 after 16S toxin is administered orally, HuMAbs (0.5 micrograms of M-20.5 micrograms+M-4) was applied by intraperitoneal injection (i.p.injection) in 24 or 36 hours.That The control mice not handled a bit with HuMAbs is dead in 72 hours.In contrast, treatment is taking orally after the exposure of HuMAbs Complete survival was provided using 12 hours after 16S toxin, provides within 24 hours and 36 hours part survival after application.
Figure 14 includes showing that human monoclonal antibodies according to the present invention combine and neutralize BoNT/B2 (bacterial strain 111) and BoNT/ The column diagram and line chart of B6 (bacterial strain Osaka05).HuMAbs (M-2 and M-4) combines BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05) is analyzed by ELISA.HuMAbs (0.5 mcg/ml) adds to the plate for being coated with BoNT.Washing Afterwards, in conjunction with HuMAbs by be coupled HRP anti-human IgG antibodies be detected.M-2 and M-4 is shown other than to BoNT/B1 also BoNT/B2 and BoNT/B6 strengths are combined.In neutralization test, M-2+M-4 (+0.5 microgram of 0.5 microgram) complete neutralization BoNT/ B2 (10 nanogram) and BoNT/B6 (2.5 nanogram).
Figure 15 includes that the west of display recombination human monoclonal antibodies combination BoNT/B according to the present invention turns stain analysis and folding Line chart.PQCXIP-hCH and pQCXIH-hCl expression vectors are transiently transfected with 2000 transfection reagents of LIPOFECTAMINE HEK293 cells.Recombination HuMAbs in culture supernatant is measured with the anti-human igg of coupling HRP.Recombinate M-2 (RM-2) and Recombinating M-4 (RM-4) and combining BoNT/B is analyzed by ELISA.Recombination HuMAb (about 0.01-0.5 mcg/mls) is added to It is coated with the plate of BoNT/B.After washing, in conjunction with recombination HuMAbs by be coupled HRP anti-human IgG antibodies be detected.RM-2 With RM-4 by with from the M-2 and M-4 of hybridoma it is identical in a manner of combine BoNT/B.
Figure 16 includes to the HuMAbs and recombination HuMAbs (RM-2 and RM-4) and BoNT/B (BoNT/ from hybridoma B1, BoNT/B2 or BoNT/B6) combination carry out west turn stain analysis.BoNT/B1, BoNT/B2 or BoNT/B6 pass through SDS- PAGE is separated into Hc and Lc, and is transferred on nitrocellulose filter.HuMAbs (1.0 mcg/ml) adds to the film, and in room temperature It is lower to incubate 1 hour.After washing, the anti-human IgG antibodies of film and coupling HRP are incubated 1 hour at room temperature, then specific band It is visualized by ECL.Recombination HuMAbs shows combination profile identical with the HuMAbs of hybridoma is derived from.M-2 and RM-2 is special The opposite sex combines the light chain of BoNT/B.In contrast, (RM-4LC16, RM-4LC18, RM-4LC19 are having the same by M-4 and RM-4 The heavy chain of M-4) it is attached to both light chain and heavy chain.By M-4 and RM-4 antibody, the heavy chain of the heavy chain ratio B1 of B2 hypotypes detects It is clearer.In addition, the heavy chain of the heavy chain ratio B2 of B6 hypotypes detects clearer.
Specific implementation mode
(detailed description of the invention)
According to the present invention, anti-Type B botulic neurotoxin monoclonal antibody or its antigen binding of a kind of separation are provided Segment, the neutralization activity with anti-Type B botulic neurotoxin.Monoclonal antibody may include human monoclonal antibodies, Ren Yuan Change monoclonal antibody or the rwo.The anti-Type B botulic neurotoxin monoclonal antibody or its antigen-binding fragment of separation can With the neutralization activity of the precursor toxin with anti-Type B botulic neurotoxin.The anti-Type B botulic neurotoxin list of separation Clonal antibody or its antigen-binding fragment can have anti-by Type BClostridium botulinumBoNT/B1 (bacterial strain Okra), B TypeClostridium botulinumBoNT/B2 (bacterial strain 111), Type BClostridium botulinumBoNT/B6 (bacterial strains Osaka05) or it arbitrarily combines neutralization activity of the Type B botulic neurotoxin generated.Therefore the anti-Type B meat poisoning of separation Bacillus neurotoxin monoclonal antibody or its antigen-binding fragment can have to Type B botulic neurotoxin (BoNT/B) The specific binding activity of light chain.
Antibody or its segment can be generated using any suitable technology.For example, the anti-Type B clostridium botulinum Nervous toxicity of separation Plain monoclonal antibody can be generated by hybridoma, and the hybridoma is immune come Type B botulic neurotoxin of using by oneself by merging People peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) and can effective cell melt It is prepared by the fusion partner cells of conjunction.Type B botulic neurotoxin can be Type BClostridium botulinumBoNT/ B1 (bacterial strain Okra), Type BClostridium botulinumBoNT/B2 (bacterial strain 111), Type BClostridium botulinumBoNT/ B6 (bacterial strain Osaka05) or its product arbitrarily combined.Any suitable fusion partner cells, such as SPYMEG can be used Cell.
The present invention also provides the hybridomas of the antibody or its segment that generate the present invention.For example, hybridoma can have NITE The preserving number of BP-01639 or NITE BP-01640.The monoclonal antibody that separation is also provided, by with NITE BP-01639 or The hybridoma of NITE BP-01640 preserving numbers generates.The example of the monoclonal antibody obtained as described above includes by entitled " hybridization (hreinafter referred to as M-2 is anti-for the monoclonal antibody that the hybridoma of tumor M-2 (M1E9) " (" Hybridoma M-2 (M1E9) ") generates Body) and by entitled " hybridoma M-4 (M4C9) " (" Hybridoma M-4 (M4C9) ") hybridoma generate monoclonal antibody (hreinafter referred to as M-4 antibody).These hybridomas are on June 26th, 2013 with preserving number NITE BP-01639 and NITE BP- 01640 is deposited in independent administrative legal person's products assessment technique basal disc organization's patent Organism Depositary (the NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation) (day Total honest and clean foot 2-5-8, No. 122 room, postcode 292-0818 in Jinshi City Mu Geng of this Chiba county).Then, according to budapest treaty, it With identical preserving number shift for international accession.
Separation anti-Type B botulic neurotoxin monoclonal antibody or its antigen-binding fragment can be IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgD, IgE, its any segment or its arbitrary combination.Antibody fragment can To include such as Fab, Fab ', F (ab ') 2, scFv, dsFv or its arbitrary combination.The anti-Type B botulic neurotoxin of separation Monoclonal antibody or its antigen-binding fragment may include heavy chain and/or light chain variable region.For example, heavy chain variable region can wrap It includes：With including SEQ ID NO：First complementary determining region (complementarity of 5 or 19 the first amino acid sequence Determining region) (CDR1), have include SEQ ID NO：Second complementation of 6 or 20 the second amino acid sequence is determined Determine area (CDR2), have to include SEQ ID NO：The third complementary determining region (CDR3) of 7 or 21 third amino acid sequence.Light chain Variable region may include：For example, with including SEQ ID NO：First complementary determining region of 12 or 26 tetramino acid sequence (CDR1), it includes SEQ ID NO to have：The second complementary determining region (CDR2) of 13 or 27 pentaamino acid sequence and have Including SEQ ID NO：The third complementary determining region (CDR3) of 14 or 28 the 6th amino acid sequence.
In another example, heavy chain variable region may include：With including SEQ ID NO：5 the first amino acid sequence First complementary determining region (CDR1), have include SEQ ID NO：Second complementary determining region of 6 the second amino acid sequence (CDR2), it includes SEQ ID NO to have：The third complementary determining region (CDR3) of 7 third amino acid sequence.Light chain variable region can To include：With including SEQ ID NO：The first complementary determining region (CDR1) of 12 tetramino acid sequence, have include SEQ ID NO：The second complementary determining region (CDR2) of 13 pentaamino acid sequence and with including SEQ ID NO：14 the 6th ammonia The third complementary determining region (CDR3) of base acid sequence.
In another example, heavy chain variable region may include：With including SEQ ID NO：19 the first amino acid sequence The first complementary determining region (CDR1), have include SEQ ID NO：Second complementary determining region of 20 the second amino acid sequence (CDR2) and with including SEQ ID NO：The third complementary determining region (CDR3) of 21 third amino acid sequence.Light chain variable Area may include：For example, with including SEQ ID NO：The first complementary determining region (CDR1), the tool of 26 tetramino acid sequence Have including SEQ ID NO：The second complementary determining region (CDR2) of 27 pentaamino acid sequence and with including SEQ ID NO： The third complementary determining region (CDR3) of 28 the 6th amino acid sequence.In further example, heavy chain variable region may include SEQ ID NO：3 or 17, light chain variable region may include SEQ ID NO：10 or 24.
The present invention provides pharmaceutical composition.Pharmaceutical composition can include the anti-Type B clostridium botulinum of one or more separation Neurotoxin monoclonal antibody and its antigen-binding fragment and pharmaceutically acceptable carrier.Pharmaceutical composition can include anti- Type B botulic neurotoxin monoclonal antibody and pharmaceutically acceptable carrier.Pharmaceutical composition can include two kinds of separation Anti- Type B botulic neurotoxin monoclonal antibody, its antigen-binding fragment, or both.Pharmaceutical composition can include the One and secondary antibody.First human monoclonal antibodies detached or antigen-binding fragment may include：For example, (1) include heavy chain can Become the human monoclonal antibodies or antigen-binding fragment of the separation of area and light chain variable region, heavy chain variable region includes：With including SEQ ID NO：The first complementary determining region (CDR1) of 5 the first amino acid sequence, have include SEQ ID NO：6 the second amino acid The second complementary determining region (CDR2) of sequence and with including SEQ ID NO：The third complementation of 7 third amino acid sequence is determined Determine area (CDR3), light chain variable region includes：With including SEQ ID NO：The first of 12 tetramino acid sequence is complementary to be determined Area (CDR1), have include SEQ ID NO：The second complementary determining region (CDR2) of 13 pentaamino acid sequence and with packet Include SEQ ID NO：The third complementary determining region (CDR3) of 14 the 6th amino acid sequence.First separation human monoclonal antibodies or Antigen-binding fragment may include：For example, (2) include the separation of heavy chain variable region and light chain variable region human monoclonal antibodies or Antigen-binding fragment, heavy chain variable region include SEQ ID NO：3, light chain variable region includes SEQ ID NO：10.First separation Human monoclonal antibodies or antigen-binding fragment may include：For example, the monoclonal antibody of (3) separation, by with preserving number The hybridoma of NITE BP-01639 generates.First separation human monoclonal antibodies or antigen-binding fragment may include for example on The arbitrary combination stated.Second human monoclonal antibodies detached or antigen-binding fragment may include：For example, (4) include heavy chain can Become the human monoclonal antibodies or antigen-binding fragment of the separation of area and light chain variable region, heavy chain variable region includes：With including SEQ ID NO：The first complementary determining region (CDR1) of 19 the first amino acid sequence, have include SEQ ID NO：20 the second amino The second complementary determining region (CDR2) of acid sequence and with including SEQ ID NO：The third of 21 third amino acid sequence is complementary It determines area (CDR3), light chain variable region includes：With including SEQ ID NO：First complementation of 26 tetramino acid sequence is determined Determine area (CDR1), have to include SEQ ID NO：The second complementary determining region (CDR2) of 27 pentaamino acid sequence and have Including SEQ ID NO：The third complementary determining region (CDR3) of 28 the 6th amino acid sequence.The human monoclonal antibodies of second separation Or antigen-binding fragment may include：For example, (5) include the human monoclonal antibodies of the separation of heavy chain variable region and light chain variable region Or antigen-binding fragment, heavy chain variable region include SEQ ID NO：17, light chain variable region includes SEQ ID NO：24.Second separation Human monoclonal antibodies or antigen-binding fragment may include：For example, the monoclonal antibody of (6) separation, by with preserving number The hybridoma of NITE BP-01640 generates.Second separation human monoclonal antibodies or antigen-binding fragment may include for example on The arbitrary combination stated.
The present invention is provided to generate the method for the anti-Type B botulic neurotoxin monoclonal antibody of separation.This method May include by merging come the peripheral blood mononuclear cells (PBMC) of the immune people of Type B botulic neurotoxin of using by oneself and can The fusion partner cells of effective cell fusion generate hybridoma.This method may further include from hybridoma and obtain Type B meat Bacillus venenosus neurotoxin monoclonal antibody.Type B botulic neurotoxin, such as pass through Type BClostridium botulinum's BoNT/B1 (bacterial strain Okra), Type BClostridium botulinumBoNT/B2 (bacterial strain 111), Type BClostridium botulinum's BoNT/B6 (bacterial strain Osaka05) or its arbitrary combination generate.Fusion partner cells can be such as SPYMEG cells.
The present invention is provided to prevent, treat and detect in human experimenter in the poisoning of Type B botulic neurotoxin At least one kit.Kit may include the present invention separation anti-Type B botulic neurotoxin monoclonal antibody, Its antigen-binding fragment, or both.Kit can also include for treating and/or detecting in Type B botulic neurotoxin One or more other reagents of poison.Kit can also include for treating and/or detecting one or more other types Such as one or more reagents of the botulic neurotoxin of A types.The present invention also provides the anti-Type B clostridium botulinum of separation nerves Toxin monoclone antibody, its antigen-binding fragment, or both be used to prepare inhibition or treatment human experimenter in botulismus use Drug purposes.
The present invention provides the method for inhibiting or treating botulismus in human experimenter.Method may include to human subjects Person applies the anti-Type B botulic neurotoxin monoclonal antibody of the separation of therapeutically effective amount, its antigen-binding fragment or two Person.Method can also include diagnosis patient's botulic neurotoxin poisoning.Method may include monitoring botulismus at least A kind of reduction of symptom.At least one symptom may include：For example, paralysis, symmetrical parmlysis of cranial nerve, the flaccid paralysis of Rearrangments Paralysis, voluntary muscle symmetrically paralyse, it is pharyngeal collapse, breathe stopping, can not sucking, can not swallow, sound reduction, ptosis, facial paralysis, Pupil fixes, mydriasis, eye-blurred, soft neck, whole-body muscle relaxation, general weakness, diplopia, dysarthrosis, dysphonia, Dysphagia, lossless, erythroplakia, postural hypotension, nausea, constipation, the retention of urine, dizziness, dry, sore-throat, development are not (suppressed), pharyngeal reflex, the muscular atrophy of whole body reflectivity, other symptoms of botulismus or its arbitrary combination entirely.It can be with Inhibit or treat any kind of botulismus in human experimenter, for example, in food-borne botulismus, wound infection meat poisoning Poison, baby's enterotoxemia botulismus, adult's enterotoxemia botulismus, iatrogenic botulismus, air borne botulismus, Or its arbitrary combination.
The method for inhibiting or treating botulismus in human experimenter may include that the anti-Type B meat poisoning bar of separation is administered in combination Bacterium neurotoxin monoclonal antibody, its antigen-binding fragment, or both controlled with for the one or more other of botulismus It treats.Joint can act synergistically to inhibit or treat botulismus.One or more other treatments may include：For example, applying With second separation anti-Type B botulic neurotoxin monoclonal antibody, its antigen-binding fragment, or both.It is one or more In addition treatment may include：For example, the anti-A type botulic neurotoxin monoclonal antibody of application separation, its antigen binding Segment, or both.One or more other treatments may include for example one or more antibiotic, for example, penicillin or its Meaning combination.
The method that the present invention provides Type B botulic neurotoxin in detection human experimenter.Method may include in the future From the sample of human experimenter with detach anti-Type B botulic neurotoxin monoclonal antibody, its antigen-binding fragment or two Person contacts.Method can also include based on antibody, its segment, or both whether detect in conjunction with Type B botulic neurotoxin The existence or non-existence of Type B botulic neurotoxin in human experimenter.Method can also include that detection is one or more another The botulic neurotoxin of outer type, such as A types.
Anti- botulic neurotoxin antibody and the polypeptide containing its antigen-binding fragment are provided, and use their side Method, purposes, composition and kit.Unless otherwise indicated, the monoclonal antibody described in the application with claimed separation And its antigen-binding fragment is not natural products.These monoclonal antibodies and its antigen-binding fragment are hybridomas or use various The product for the equivalent artificial cell system that experimental method generates.Using antibody or its antigen-binding fragment for treating, diagnosing Or the antibody or anti-that the method including their compositions including their kit, and/or non-natural of other purposes generate Body segment is not limited thereto, unless expressly stated.
The method formed to the antibody or its polypeptide or segment of botulic neurotoxin specificity is provided.Such method May include：The nucleic acid of coding botulic neurotoxin antigen polypeptide or the polypeptide containing its immunologic specificity epitope is provided； Contain the polypeptide of antigen amino acid sequence or polypeptide containing its immunologic specificity epitope from the expression of nucleic acid of separation；With generation pair The antibody of the polypeptid specificity of acquisition, or the polypeptide containing its antigen-binding fragment.The antibody generated by preceding method is provided Or the polypeptide containing its antigen-binding fragment.The antibody of the separation of specific binding botulic neurotoxin antigen is provided or is divided From the polypeptide containing its antigen-binding fragment.It can be generated in this way using any acceptable method as known in the art Antibody.Under antibody and kit, the method and/or other aspects using antibody of the present invention may include one or more Row substance：Polyclonal antibody, monoclonal antibody, chimeric antibody, single-chain antibody, univalent antibody, double antibody, and/or humanization are anti- Body.
Naturally-produced antibody structural unit generally comprises the tetramer.Each such tetramer can be identical by two pairs Polypeptide chain forms, each pair of to have overall length " light " chain (for example, about 1kDa to 25kDa) and overall length " weight " chain (for example, about 50kDa- 70kDa).What the amino terminus portion of every chain generally included about 100 to 110 or more amino acid is generally responsible for antigen recognizing Variable region.The carboxy-terminal sections of every chain generally define the constant region that may be responsible for effector function.People's light chain usually divides For κ and lambda light chain.Heavy chain is generally divided into μ, 6, γ, α or ε, and the isotype for limiting antibody be respectively IgM, IgD, IgG, IgA and IgE.IgG has several subclass, including but not limited to IgG1, IgG2, IgG3 and IgG4.IgM have subclass, including but It is not limited to IgM1 and IgM2.Similarly IgA points are subclass, including but not limited to IgA1 and IgA2.In light chain and heavy chain, it can be changed Area can be connected with constant region by area " J " of about 12 or more amino acid, and heavy chain further includes about 10 or more amino Area " D " of acid.See, for example,：The 7th chapter of basic immunology (Fundamental Immunology Ch.7.Paul, W., ed., 2nd ed.Raven Press, N.Y (1989)).The variable region of each light/heavy chain pair is usually formed antigen binding site.
Variable region usually display is by three hypervariable regions, also referred to as complementary determining region (complementarity Determining region) or CDR connections relatively conservative framework region (framework region, FR) it is identical logical Normal structure.The CDR of two chains from every a pair is usually aligned by framework region, can be attached to specificity epitope. From N-terminal to C-terminal, light chain and heavy chain variable region generally comprise structural domain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The distribution of amino acid to each structural domain is typically the Kabat sequences (Kabat according to interested albumen in immunology Sequences of Proteins of Immunological Interest.National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia＆Lesk J.MoI.Biol.196：901-917 (1987)；Chothia et al., Nature342：The restriction of 878-883 (1989).
" antibody fragment " includes a part for complete antibody, the antigen binding such as complete antibody or variable region.Antibody fragment Example include：Fab, Fab1, F (ab ') 2 and Fv segments；Double antibody；Linear antibodies (Zapata et al., Protein Eng.8(10)：1057-1062[1995])；Single-chain antibody molecules；With the multi-specificity antibody formed by antibody fragment.Pawpaw egg White enzymic digestion antibody generates two identical antigen-binding fragments, referred to as " Fab " segment, and respectively there are one individual antigen knots for tool Site and remaining " Fc " segment are closed, name reflects the ability for being easy to crystallization.Pepsin generates a F (ab ') 2 segment, it has there are two antigen binding site and still is able to crosslinking antigen." Fv " is containing there are one complete antigens to know Other and binding site antibody fragment.This region includes that a heavy-chain variable domains and a light variable domains are tight Close, Non-covalent binding dimer.Individual variable domains (or half of the Fv only containing 3 antigentic specificity CDR) can To identify and combine antigen." scFv " or " sFv " antibody fragment includes VH the and VL structural domains of antibody, wherein these structural domains It is present in single polypeptide chain.Fv polypeptides can be further contained between VH and VL structural domains and so that sFv is formed for resisting The peptide linker for the desired structure that original combines.For the summary of sFv, referring to Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).
Antibody may be used as probe, therapeutic treatment and other purposes.Antibody can by by translation product or its synthesis Peptide fragment injection mouse, it is prepared by rabbit, goat or other animals.These antibody are in diagnostic analysis or as medicine group The active constituent closed in object is useful.
The antibody or polypeptide of application can be coupled to functional agent to form immune conjugate.Functional agent can be cytotoxic agent Such as chemotherapeutics, toxin (for example, bacterium, fungi, plant or animal origin enzymatic activity toxin or its segment) or radiation Property isotope (that is, radiation conjugate), antibiotic, nucleolytic enzyme or its arbitrary combine.It can be in the generation of immune conjugate Using chemotherapeutics, for example, amethopterin (methotrexate), adriamycin (adriamycin), vinca alkaloids (vinca Alkaloids) (vincristine (vincristine), vincaleukoblastinum (vinblastine), Etoposide (etoposide)), more It is soft than star (doxorubicin), melphalan (melphalan), mitomycin C (mitomycin C), Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalators, enzyme, and/or its segment, such as nucleolytic enzyme (nucleolytic enzyme), antibiotic and toxin such as small molecule toxins or bacterium, fungi, plant or animal origin Enzymatic activity toxin, including its segment and/or variant, and the various antineoplastics or anticarcinogen that are disclosed below.It can use Enzymatic activity toxin and its segment include：For example, diphtheria A chains (diphtheria A chain), diphtheria toxin it is non-binding Active fragment, exotoxin A chain (exotoxin A chain) (come from pseudomonas aeruginosa (Pseudomonas Aeruginosa)), ricin A chain (ricin A chain), abrin A chains (abrin A chain), capsule lotus root poison Plain A chains (modeccin A chain), α-broom song toxalbumin (alpha-sarcin), Aleurites fordii proteins (Aleurites fordii Protein), China pink fibroin (dianthin protein), dyers' grapes albumen (Phytolacca americana Protein) (PAPI, PAPII and PAP-S), momordica charantia inhibitor (momordica charantia inhibitor), Jatropha curcas Toxalbumin (curcin), crotin (crotin), crystal soda grass inhibitor (saponaria officinalis Inhibitor), gelonin (gelonin), mitogillin (mitogellin), Restrictocin (restrictocin), phenol Mycin (phenomycin), enomycin (enomycin) and Trichothecenes toxin (trichothecenes).This field It is known or in other ways available any suitable radioactive nucleotides or radioreagent can be used for generate radioactivity The antibody of coupling.
The conjugate of antibody and cytotoxic agent can use a variety of bifunctional protein coupling agent such as N- succinimidos -3- (2- pyridines dimercapto) propionic ester (N-succinimidyl-3- (2-pyridyldithiol) propionate, SPDP)；Imido Base sulfane (iminothiolane, IT)；Dual-function derivative (such as the dimethyl imidic acid of imidoate (imidoester) Ester hydrochloride (dimethyl adipimidate HCL))；Active ester (such as disuccinimidyl suberate (disuccinimidyl suberate))；Aldehyde (such as glutaraldehyde (glutaraldehyde))；Double azido compound (such as it is double (to azidobenzoyl) hexamethylene diamine (bis (p-azidobenzoyl) hexanediamine))；Dual azepine derivatives (such as Bis- (to diazo benzoyl)-ethylenediamines (bis- (p-diazoniumbenzoyl)-ethylenediamine))；Two isocyanides Acid esters (such as toluene 2,6- diisocyanate (tolyene 2,6-diisocyanate))；Double activated fluorine compounds (such as 1, Bis- fluoro- 2,4- dinitrobenzenes (1,5-difluoro-2,4-dinitrobenzene) of 5-)；Maleimidocaproyl (maleimidocaproyl, MC)；Valine-citrulline, double peptide site (valine- of protease cracking joint Citrulline, VC)；2- amino -5- ureido pentanoic acids (2-amino-5-ureido pentanoic acid) p- amino of PAB= Benzylamino formyl (p-aminobenzylcarbamoyl) (" falling off certainly " part of connector) (Citrulene)；N- methyl- Valine citrulling (N-methyl-valine citrulline), wherein connector peptide bond have been modified to prevent it from being organized Cathepsin B cracks (Me)；Maleimidocaproyl-polyethylene glycol is connected to antibody cysteine；N- succinimidos 4- (2- pyridines dimercapto) valerate (N-Succinimidyl 4- (2-pyridylthio) pentanoate, SPP)；With N- ambers - 1 carboxylate (N-succinimidyl 4- (N- of imide 4- (N- maleimidomehyls) hexamethylene Maleimidomethyl) cyclohexane-l carboxylate, SMCC) it is prepared.For example, ricin immunotoxin It can be according to Vitetta et al., Science, 238：It is prepared by the method described in 1098 (1987).Carbon-14 marks 1- isothiocyanatobenzyl -3- methyl diethyl pentetic acids (l-isothiocyanatobenzyl-3- Methyldiethylene triaminepentaacctic acid, MX-DTPA) it is a kind of be illustratively used for radioactivity Nucleotide is coupled to the chelating agent of antibody, referring to WO 94/11026.Antibody can be coupled to " receptor " (such as Streptavidin) use It is targeted in advance in tumour, wherein antibody-receptor conjugate is applied to subject, then uses scavenger to be removed from cycle unbonded Conjugate, then application is coupled to " ligand " (such as avidin) of cytotoxic agent (for example, radioactive nucleotides).
The antibody of the present invention can directly or indirectly be coupled to detectable marker by technology well known in the art.It can examine Survey marker is one kind for example by spectroscopy, photochemistry, biochemistry, immunochemistry or the detectable reagent of chemical means. Useful detectable marker includes but not limited to fluorescent dye, chemiluminescence compound, radioactive isotope, electron dense bodies Reagent, enzyme, coloured particle, biotin or digoxin (digoxigenin).Detectable marker often generates measurable letter Number, such as radioactivity, fluorescence, color or enzymatic activity.The antibody for being coupled to detectable reagent can be used for diagnosing or therapeutic purposes. The example of detectable reagent includes various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, makes With the positron emitting metal and on-radiation paramagnetic metal ion of various positron emission tomography arts.It is detectable Substance can use techniques known in the art directly or by intermediary such as connector for example known in the art indirectly with it is anti- Body is coupled or combines.See, for example, U.S. Patent No. 4741900, describes and metallic ion coupled used to antibody for diagnosing On the way.The example of suitable enzyme includes horseradish peroxidase, alkaline phosphatase, beta galactosidase and acetylcholinesterase；It closes The example of suitable prosthetic group complexes includes Streptavidin/biotin and avidin/biotin；Suitable fluorescent material Example include umbelliferone (umbelliferone), fluorescein (fluorescein), fluorescein isothiocynate (fluorescein isothiocyanate), rhodamine (rhodamine), dichlorotriazine ammonia fluorescein (dichlorotriazinylamine fluorescein), dansyl Cl (dansyl chloride) and phycoerythrin (phycoerythrin)；One example of luminescent material includes luminol (luminol)；The example of bioluminescent material includes Luciferin (luciferin) and aequorin (aequorin).
Useful antibody can use following method in laboratory animal or pass through recombinant DNA skill in the practice of the present invention It is prepared by art.Polyclonal antibody can be by in animal multiple subcutaneous (subcutaneous, sc) or peritonaeum (intraperitoneal, ip) injects gene outcome molecule or its segment parallel connection combination adjuvant such as Freund's adjuvant is (complete or endless It is cultivated entirely).In order to enhance immunogenicity, using difunctional or derivative reagent, for example, maleimidobenzoyl is thio Succinimide ester (maleimidobenzoyl sulfosuccinimide ester) (being coupled by cysteine residues), N-hydroxysuccinimide (passing through lysine residue), glutaraldehyde, succinic anhydride, thionyl chloride (socl) etc., will contain first The gene outcome molecule or segment of target amino acid sequence are coupled in waiting for immune species, such as Keyhole limpet hemocyanin (keyhole limpet hemocyanin), seralbumin (serum albumin), bovine thyroid element Globulin (bovine thyroglobulin) or soybean trypsin inhibitor (soybean trypsin inhibitor), May be useful.Selectively, immunogenic conjugate can be used as fusion protein recombination to generate.
Immunogenic conjugate or derivative (such as segment containing target amino acid sequence) can be used, about 3 times of bodies are passed through Long-pending Freund's complete adjuvant combines about 1 milligram or about 1 microgram conjugate (respectively for rabbit or mouse) and multiple intradermal injections should Solution, animal is immunized.After about 7 to 14 days, acquires the blood of animal and measure the antibody titer of serum.Reinforced repeatedly with antigen Immune animal reaches until titre at titre platform.Can with identical molecule used or its segment is initially immunized to animal Booster immunization is carried out, but it is coupled from different albumen couplings and/or by different crosslinking agents.In addition, agglutinant such as alum can So as to enhance immune response in injection to improve.
The antibody of application may include chimeric antibody.The antibody of application may include humanized antibody.The antibody of application can With the antibody including full-length human.Antibody can be humanization or part-humanised.Non-human antibody can use ability Known any applicable method carries out humanization in domain.Humanized antibody can use immune system partially or completely The transgenic animals of humanization generate.Any antibody or its segment of the present invention can be partially or completely humanization.It is embedding Closing antibody can use any of technology in this field to generate.See, for example, U.S. Patent No. 5169939, No. 5750078, No. 6020153, No. 6420113, No. 6423511, No. 6632927 and No. 6800738.
The antibody of application may include monoclonal antibody, that is to say, that can be the anti-meat poisoning of the present invention of monoclonal antibody Bacillus neurotoxin antibody.Monoclonal antibody can use hybridoma method such as by Kohler and Milstein, Nature, and 256： It is prepared by those of 495 (1975) description method.In hybridoma method, mouse, hamster or other suitable host animals It is usually immunized with immunoreagent, generates or can generate the lymph for specifically binding the antibody of immunoreagent is thin to cause Born of the same parents.Selectively, lymphocyte can be immunized in vitro.Monoclonal antibody can as 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 are screened described in (1986).The specific immunity that monoclonal antibody and translation product can be tested is anti- Ying Xinghe lacks with corresponding prototype gene product immunoreactivity.
Monoclonal antibody can by from immune animal recycle splenocyte and in a usual manner, for example by with myeloma Cell fusion makes cell infinite multiplication be prepared.Preferably, make come the periphery for the immune people of Clostridium botulinum toxoid that uses by oneself Blood monocyte (PBMC) is merged with myeloma cell.Then clone is screened to obtain gram of those expression expectation antibodies It is grand.Preferably with other gene outcomes cross reaction does not occur for monoclonal antibody.After desired hybridoma is determined, clone It can be subcloned by limited dilution method, and be grown by standard method.Suitable culture for this purpose Base includes：For example, Dahl Burke Improved Eagle Medium (Dulbecco ' s Modified Eagle ' s Medium) and RPMI-1640 culture mediums.Selectively, hybridoma can grown as ascites in the mammalian body.By sub- gram The monoclonal antibody of grand secretion can pass through conventional immune globulins purification process such as such as Protein A-agarose, hydroxyapatite layer Analysis, gel electrophoresis, dialysis or affinity chromatography carry out isolated or purified from culture medium or ascites.
Monoclonal antibody can also be by recombinant DNA method, such as the side those of described in U.S. Patent No. 4816567 It is prepared by method.The DNA for encoding the monoclonal antibody of the present invention can use conventional method (for example, by using being capable of specificity In conjunction with the oligonucleotide probe of the gene of the heavy chain and light chain of encoding murine antibody) it is easy to carry out separation and sequencing.The present invention Hybridoma can be as the preferred source of this DNA.Once DNA, can be placed in expression vector, then by it by separation Be transfected into will not otherwise generate immunoglobulin host cell such as monkey COS cells, Chinese hamster ovary (CHO) it is thin In born of the same parents or myeloma cell, to obtain the synthesis of monoclonal antibody in recombinant host cell.DNA can also for example, pass through by The coded sequence of people's heavy chain and light chain constant domain replaces homologous mouse sequence or more by that will be used for non-immunoglobulin The all or part of the coded sequence of peptide is covalently bond to immunoglobulin coding sequence and is modified.This non-immunoglobulin Polypeptide could alternatively be the constant domain of the antibody of the present invention, or could alternatively be an antigen binding of the antibody of the present invention The variable domains in site, to form chimeric bivalent antibody.Preparing antibody using recombinant DNA method such as phage display method can To use commercially available kit, such as from the available recombination phasmid antibody forming systems of Pharmacia (Uppsala, Sweden), or SurfZAPTMPhage display system (Stratagene Inc., La Jolla, Califorinia) is completed.The present invention uses Mouse-people's chimera fusion partner cells system, entitled SPYMEG, to provide anti-A type BoNT (BoNT/A) and Type B BoNT (BoNT/B) Human monoclonal antibodies (HuMAbs).
Further include in the present invention：Generate the hybridoma cell line of monoclonal antibody, the B cell system of conversion and the place of the present invention Chief cell；These hybridomas, the filial generation of the B cell system of conversion and host cell or derivative；And equivalent or similar hybridization Tumor, the B cell system of conversion and host cell.
Antibody can be double antibody.Term " double antibody " refers to that there are two the small antibody fragment of antigen binding site, pieces for tool Section includes the heavy-chain variable domains (VH) for the light variable domains (VL) being connected in identical polypeptide chain (VH-VL). By using the too short connector without allowing to match between two structural domains on same chain, structural domain and another can be forced The complementary domain of chain matches, and forms two antigen binding sites.Double antibody be described in more detail below such as EP404097, W093/11161 and Hollinger et al., Proc.Natl.Acad.Sci.USA, 90：In 6444-6448 (1993).
The antibody of application may include single-chain antibody.Antibody can be univalent antibody.The method for preparing univalent antibody is this Known to field.For example, a kind of method is related to recombinantly expressing light chain immunoglobulin and modifies heavy chain.Heavy chain usually can be in Fc Any point in area is blocked, to prevent heavy chain to be crosslinked.Selectively, relevant cysteine residues are substituted for other amino acid Residue or removal, to prevent being crosslinked.In-vitro method is also suitable for preparing univalent antibody.Antibody digestion is to generate its segment, especially It is Fab segments, can be completed using routine techniques as known in the art.
Antibody can be bispecific.It is generated, detached and measured using the standard method having been described in the literature and is special The opposite sex combines an albumen and specifically binds the bispecific antibody with pathology and/or treatment-related other antigens.(referring to Such as：Pluckthun＆Pack, Immunotechnology, 3：83-105(1997)；Carter, et al., J.Hematotherapy, 4：463-470(1995)；Renner＆Pfreundschuh, Immunological Reviews, 1995, No.145, pp.179-209；Pfreundschuh U.S. Patent No. 5643759；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)).
Immunoliposome can be made in antibody disclosed here.Liposome containing antibody passes through methods known in the art It is prepared, such as in Epstein et al., Proc.Natl.Acad.Sci.USA, 82：3688(1985)；Hwang et Al., Proc.Natl.Acad.Sci.USA77：4030(1980)；And it is retouched in U.S. Patent No. 4485045 and No. 4544545 It states.Circulation time extended liposome is disclosed in U.S. Patent No. 5013556.Particularly useful liposome can pass through Lipid composition of the reverse phase evaporation containing phosphatidyl choline, cholesterol and PEG derivatization phospholipid acyl ethanol amines (PEG-PE) It generates.Liposome can be squeezed out by limiting the filter in aperture, to obtain the liposome with desired diameter.The antibody of the present invention Fab ' segments can be reacted through disulfide exchange and be coupled to liposome, such as description in Martin et al., J.Biol.Chem., 257：In 286-288 (1982).Chemotherapeutics (such as Doxorubicin) is optionally included in liposome.Referring to Gabizon et al, J.National Cancer Inst., 81 (19)：1484(1989).
The present invention provides the method for inhibiting or treating botulismus in human experimenter, including controls human experimenter's application Treat the anti-botulic neurotoxin monoclonal antibody of a effective amount of present invention, its antigen-binding fragment, or both.Method may be used also To include diagnosis patient's botulic neurotoxin poisoning.The anti-botulic neurotoxin antibody of the present invention or its antigen binding Segment can diagnosis patient have botulic neurotoxin poisoning before, be applied to subject in the middle and/or later.Method It can also include the reduction of at least one symptom of monitoring botulic neurotoxin poisoning.
According to the present invention it is possible to using two or more botulic neurotoxin antagonists.At least one meat poisoning bar Bacterium neurotoxin antagonist may include botulic neurotoxin antagonist.At least one botulic neurotoxin antagonist One or more other botulic neurotoxin antagonists can be combined.At least one botulic neurotoxin antagonist It can be poisoned to botulic neurotoxin with combined needle and/or one or more other medicines of clostridium botulinum infection are applied With.Can be simultaneously using the medicine of two or more, including one or more botulic neurotoxin antagonists , sequence or it is united.Therefore, when two or more medicines of application, they need not be simultaneously or with phase Tongfang Formula is applied with same dose.When being administered simultaneously, two or more medicines can be with same composition or different groups Close object application.Two or more medicines can be applied using identical administration route or different administration routes.When When applying in different times, medicine can be applied before or after each other.Two or more medicines are applied It can be replaced with sequence.The respective dosage of one or more medicines can change over time.One or more medicines The type of object can change over time.When being applied in splitting time, when the separation applied two or more times can be any Between the period.If multiple applications, the length of time cycle can change.Point between the application of two or more medicines Every can be 0 second, 1 second, 5 seconds, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, After 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, After 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 Week, 8 weeks, 3 months, 6 months, 1 year or longer.
Two or more botulic neurotoxin antagonists can act synergistically to treat or reduce clostridium botulinum god Through toxin poisoning or its symptom.The symptom of botulismus includes：For example, paralysis, symmetrical parmlysis of cranial nerve, Rearrangments are flaccid Paralysis, voluntary muscle symmetrically paralyse, it is pharyngeal collapse, breathe stopping, can not sucking, can not swallow, sound reduction, ptosis, face Paralysis, pupil are fixed, mydriasis, eye-blurred, soft neck, whole-body muscle relaxation, general weakness, diplopia, dysarthrosis, pronounce barrier Hinder, dysphagia, lossless, erythroplakia, postural hypotension, nausea, constipation, the retention of urine, dizziness, dry, sore-throat, development Incomplete, pharyngeal reflex and whole body reflectivity muscular atrophy.Botulismus can be in the lab by proving clinical sample or intake Toxin in the sample of food confirms.It is that height prompts Symptomatic situation that wound culture, which generates organism,.Botulismus Confirmation can be carried out by Mouse bioassay.Show that toxin serotypes by paralysing in specific antitoxin and in mouse There are in clinical sample.
Botulic neurotoxin antagonist can be individual one or more anti-botulic neurotoxin antibody or Combine one or more other botulic neurotoxin antagonists, for example, small pharmaceutical preparation or other anti-clostridium botulinums The medicine of neurotoxin.The example of small pharmaceutical preparation includes antibiotic, such as penicillin.The treatment of other anti-clostridium botulinums Drug includes septivalency clostridium botulinum antitoxin (heptavalent botulinum antitoxin, HBAT).HBAT can contain For the antibody fragment in the horse serum source of neurotoxin A-G, and can include：For example, less than about 2.0% complete is exempted from Epidemic disease globulin, and 2 antibody fragment of Fab and F (ab ') greater than or equal to about 90%.Two or more anti-clostridium botulinum nerves The antibody and one or more other medicines of the antibody of toxin or at least one anti-botulic neurotoxin can be with Synergistic effect, to treat or reduce botulic neurotoxin poisoning.Two or more medicines, including it is one or more Anti- botulic neurotoxin antibody, can be to cooperate with dosage to apply.Therefore, the application of two or more medicines, nothing It, can be in one or more symptoms that botulic neurotoxin is poisoned by being simultaneously, sequence or applied with any combinations There is synergistic effect in reduction.First medicine can increase the effect of the second medicine, if being more than the second medicine The effect of object is used alone or the second medicine increases by the first medicine, or both.Using two or more medicines The effect of object can be such that the effect in the one or more symptoms for reducing botulic neurotoxin poisoning is big In the addition effect that each is administered alone.When to cooperate with dosage to give, a kind of medicine can enhance one or more another The effect of outer medicine in the reduction for one or more symptoms that botulic neurotoxin is poisoned, even if a kind of or more The amount of kind medicine individually will not have remarkable efficacy to one or more symptoms that botulic neurotoxin is poisoned.Collaboration is made Measurement and calculating, can be such as in Teicher, " Assays for In Vitro and In Vivo Synergy, " in Methods in Molecular Medicine, vol.85：Novel Anticancer Drug Protocols, pp.297- It is described in 321 (2003) and/or by using CalcuSyn softwares calculate association index (combination index, CI it) carries out.
Exact preparation, administration route and dosage can be selected by solo practitioner depending on the state of an illness of patient.(referring to example Such as：Fingl et.al., in The Pharmacological Basis ofTherapeutics, 1975, Ch.1 p.I.) it is main Attending doctor can determine due to toxicity or organ dysfunction and when terminate, interrupts or adjust administration.On the contrary, if excluding Toxicity, clinical response are not enough, and attending physician can also adjust treatment and arrive higher level.In the pipe of interested disease The amplitude of dosage will change with the severity of disease to be treated and administration route in reason.The severity of disease can It is evaluated by standard prognostic evaluation method with such as part.Dosage and administration frequency can according to age of individual patient, Weight and reaction and change.It can be used in veterinary science with similar program described above.
Using pharmaceutically acceptable carrier to be configured to be suitable for for putting into practice the compound of the present invention by disclosed here The dosage form of Formulations for systemic administration is within the scope of the present invention.By proper choice of carrier and suitable manufacturing practice, with this hair Bright relevant composition, especially those compositions for being configured to solution, can with parenteral administration, such as by be injected intravenously into Row application.These compounds can be readily formulated into using pharmaceutically acceptable carrier well known in the art to be suitable for taking orally The dosage form of administration.Such carrier makes compound related to the present invention be formulated as tablet, pill, capsule, liquid, gel Agent, syrup, paste, dragee, solution, suspension etc. are used for patient's orally ingestible to be treated.
Therapeutic agent can be prepared in the form of sustained release drug (depot), to allow relative to the position in time and body (see, for example, U.S. Patent No. 4450150) in the body applied to it of control release.The sustained release drug form of therapeutic agent can To be：For example, the implanted composition containing therapeutic agent and porous or pore-free material such as polymer, wherein therapeutic agent pass through material And/or the degradation encapsulation or diffusion of pore-free material.Then sustained release drug is implanted to the desired locations in body, and therapeutic agent is from implantation material It discharges at a predetermined rate.
Therapeutic agent used in this invention is set to be formed as composition, such as drug containing carrier and therapeutic compound Composition.Pharmaceutical composition containing therapeutic agent may include more than one therapeutic agent.Pharmaceutical composition can be selectively Including therapeutic agent and combining other forms of pharmacologically active agents or drug.
Carrier can be any suitable carrier.For example, carrier can be pharmaceutically acceptable carrier.Relative to drug Composition, carrier can consider Chemical Physics Consideration, such as dissolubility and lack reactivity with reactive compound, And pass through those conventional use of carriers of administration route.In addition to pharmaceutical composition beyond the region of objective existence described below, or selectively, this hair The therapeutic compound of bright method can be configured to inclusion compound, such as cyclodextrin inclusion compound or liposome.
Pharmaceutically acceptable carrier described here, such as mediator (vehicle), adjuvant, excipient and diluent, It is well known to those skilled in the art, and the public is readily available.Pharmaceutically acceptable carrier can be to changing Activating agent is inert, carrier with no harmful side-effects or toxicity under conditions of use on.The selection of carrier, can portion Ground is divided to be determined by specific therapeutic agent and by the ad hoc approach for application therapeutic compound.With a variety of present invention's The suitable preparation of pharmaceutical composition.For taking orally, spraying, parenteral, subcutaneous, percutaneous, transmucosal, intestines, intramedullary injection, directly Intra-ventricle, intravenous, intranasal, intraocular, intramuscular, intra-arterial, in intrathecal, peritonaeum, the following preparation of rectum and vagina administration be to show Example property, and be in no way intended to limit.More than one approach can be used to be used to apply therapeutic agent, and in some cases, one Kind specific approach can be provided than another way more rapidly with more effective response.According to the specific disease for the treatment of, this A little medicaments can be prepared and administered either systemically or locally.Technology for preparing and being administered can be in Remington ' s It is found in Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990).
Preparation suitable for oral medication may include：(a) liquid solution, for example, be dissolved in diluent such as water, brine or A effective amount of inhibitor of orange juice；(b) capsule, sachet, tablet, pastille (lozenges) and lozenge (troches), respectively Contain the active constituent of predetermined amount with solid or particle form；(c) powder；(d) suspension in suitable liquid；And (e) suitable lotion.Liquid preparation may include diluent, such as water and alcohol such as ethyl alcohol, benzyl alcohol and polyvinyl alcohol, simultaneously It is added or is added without pharmaceutically acceptable surfactant.Capsule form can be containing such as surfactant, lubricant, And inert filler, such as the common hard or soft-shelled gelatin type of lactose, sucrose, calcium phosphate and cornstarch.Tablet form can be with Including lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, Arabic gum, gelatin, melon That glue, silica colloidal, croscarmellose sodium, talcum powder, magnesium stearate, calcium stearate, zinc stearate, tristearin Acid and other excipient, colorant, diluent, buffer, disintegrant, wetting agent, preservative, flavoring agent and other pharmacy Above compatible excipient is one or more.It is usually sucrose and I that pastille (lozenges) form, which can contain in flavoring agent, Inhibitor in primary glue or tragacanth, and in inert base such as gelatin and glycerine or sucrose and gum arabic, emulsion, solidifying The pastille (pastille) for including inhibitor in glue etc. also includes excipient known in the art other than inhibitor.
The pharmaceutical preparation that can be administered orally includes capsule (push-fit capsule) of slippaging made of gelatin, and The capsule of gelatin and plasticizer sealing soft as made of glycerine or D-sorbite.Capsule of slippaging can include and filler is such as newborn The active constituent of sugar, adhesive such as starch, and/or lubricant such as talcum powder or magnesium stearate and optional stabilizer mixing. In soft capsule, reactive compound can be dissolved or suspended in suitable liquid such as fat oil, atoleine or liquid macrogol In.Furthermore it is possible to which stabilizer is added.
Therapeutic agent individually or the other suitable components of joint, can be made via inhalation aerosol formulation.These During aerosol formulation can be placed in the acceptable propellant of pressurization such as dicholorodifluoromethane, propane, nitrogen.They also may be used To be configured to the drug for non-pressurised preparation such as in sprayer or atomizer.It is viscous that this spray formulation can be also used for spraying Film.Topical formulations are well known for those of ordinary skill in the art.Such preparation is particularly suitable for the present invention's Skin is applied in context.
Injectable formulation meets the present invention.The parameter of active drug carrier for Injectable composition, for this Field those of ordinary skill in the art be it is well known (see, for example,：Pharmaceutics and Pharmacy Practice, J.B.Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622630 (1986)).When for injecting, reagent of the invention can be prepared in aqueous solution, preferably the buffer solution of PHYSIOLOGICALLY COMPATIBLE such as Hunk In family name's solution (Hanks ' s solution), Ringer's solution (Ringer ' s solution) or normal saline buffer solution.For Such mucosal is used in the formulation for the suitable bleeding agent of barrier to be infiltrated.Such bleeding agent usually exists It is known in this field.
The preparation for being suitable for parenteral administration may include aqueous and without aqueous isotonic sterile injection solution, can contain anti- Oxidant, buffer, bacteriostatic agent and the solute for making the blood of preparation and target recipient isotonic, and may include suspending agent, Solubilizer, thickener, stabilizer and preservative aqueous and without aqueous sterile suspensions.Therapeutic agent can in pharmaceutical carrier It is applied in physiologically acceptable diluent, the diluent such as sterile liquid or including water, brine, glucose solution and correlation Sugar juice, alcohol such as ethyl alcohol or hexadecanol, dihydric alcohol such as propylene glycol or polyethylene glycol, poly(ethylene glycol) 400, glycerine, dimethyl Sulfoxide, ketal such as 2,2- dimethyl -1,3-dioxolane -4- methanol, ether, oil, aliphatic acid, aliphatic ester or glyceride or second The mixture of the liquid of acylated fatty glyceride, is added or is added without pharmaceutically acceptable surfactant, such as soaps (soap) or detergent, suspending agent such as pectin, carbomer, methylcellulose, hydroxypropyl methyl cellulose or carboxymethyl cellulose, Or emulsifier and other medicinal adjuvants.
Oil can use in parenteral administration, including oil, animal oil, vegetable oil or synthetic oil.The specific example of oil Including peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, paraffin oil and mineral oil.For parenteral administration Suitable aliphatic acid includes oleic acid, stearic acid and isostearic acid.Ethyl oleate and isopropyl myristate are suitable aliphatic acid The example of ester.
Include fatty alkali metal salt, ammonium salt and triethanolamine salt for using the suitable soaps in parenteral administration, Suitable detergent includes：(a) cationic detergent, such as such as dimethyl dialkyl ammonium halide and alkylpyridinium halides are (b) cloudy Cationic detergent, such as such as alkyl, aryl and alkene sulfonate, alkyl, alkene, ether and monoglyceride sulfates and sulfo group amber Amber hydrochlorate, (c) non-ionic octoxynol detergent, such as such as fatty amine oxide, fatty acid alkanol amides and polyoxyethylene polypropylene are copolymerized Object, (d) amphoteric ion detergent, such as such as alkyl-Beta-alanine ester and 2- alkyl imidazoline quaternary ammonium salts, and (e) it is mixed Close object.
Parenteral administration can contain the drug of about 0.5 weight % to about 25 weight % in the solution.Anti-corrosion can be used Agent and buffer solution.In order to reduce or eliminate the stimulation on injection site to the maximum extent, this kind of composition, which can contain, to be had about One or more nonionic surfactants of the Hydrophilic Lipophilic Balance (HLB) of 12 to about 17.Table in such preparation The amount of face activating agent will be usually in the range of about 5 weight % to about 15 weight %.Suitable surfactant includes poly- second two Dehydration of alcohols sorbitan fatty acid ester such as dehydrated sorbitol mono-fatty acid ester and ethylene oxide and passes through propylene oxide and the third two Alcohol is condensed the high molecular weight adducts for the hydrophobic matrix to be formed.Parenteral administration can reside in unit dose or multi-dose sealing It in container such as ampoule bottle and bottle, and being stored under the conditions of freeze-drying (freeze-drying), when for injecting, being related to using It is preceding that sterile liquid excipient such as water is added immediately.Interim injection solution and suspension can be from the nothings of previously described type It is prepared by bacterium powder end, particle and tablet.
Suppository can be made by being mixed with various matrix, such as emulsified bases or water-soluble base in therapeutic agent.Suitable for the moon The preparation of canal drug administration can be rendered as vaginal suppository, suppository, cream, gelling agent, paste, foaming agent or spray, except activity Outside ingredient, also contain these carriers appropriate as known in the art.
Being intended to the reagent applied into the cell can be administered using technology well known within the skill of those ordinarily skilled.Example Such as, such reagent can be encapsulated in liposome.Liposome is the spherical lipid bilayer for having aqueous interior.It is present in water-soluble Molecule in liquid enters aqueous interior when liposome is formed.Liposomal contents were both protected from external microenvironment, And because of liposome and cell membrane fusion, liposomal contents are efficiently transferred in cytoplasm.Further, since they are dredged Aqueous, organic molecule can be applied directly into the cell.It can also for the material and method of one aspect of the present invention description Other aspects for the present invention.For example, description is also used as treating for screening material such as nucleic acid or antibody in analysis Agent, vice versa.
The present invention includes random order and/or any combination of aspect/specific embodiment/feature below：
1, the anti-Type B botulic neurotoxin monoclonal antibody or its antigen-binding fragment of a kind of separation, including anti-Type B The neutralization activity of botulic neurotoxin, wherein the monoclonal antibody includes that human monoclonal antibodies, Humanized monoclonal are anti- Body, or both.
2, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment further include anti-Type B meat poisoning bar The neutralization activity of the precursor toxin of bacterium neurotoxin.
3, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment include resisting by Type BMeat poisoning shuttle Shape bacillusBoNT/B1 (bacterial strain Okra), Type BClostridium botulinumBoNT/B2 (bacterial strain 111), Type BMeat poisoning shuttle Shape bacillusBoNT/B6 (bacterial strain Osaka05) or neutralization of Type B botulic neurotoxin for generating of its arbitrary combination Activity.
4, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment have to Type B clostridium botulinum The specific binding activity of the light chain of neurotoxin (BoNT/B).
5, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the human monoclonal antibodies by merging come Clostridium botulinum toxoid of using by oneself by being exempted from Hybridoma prepared by the peripheral blood mononuclear cells (PBMC) of the people of epidemic disease and the fusion partner cells for capableing of effective cell fusion It generates.
6, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the Type B botulic neurotoxin is Type BClostridium botulinum's BoNT/B1 (bacterial strain Okra), Type BClostridium botulinumBoNT/B2 (bacterial strain 111), Type BClostridium botulinum's BoNT/B6 (bacterial strain Osaka05) or its product arbitrarily combined.
7, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, wherein the fusion partner cells are SPYMEG cells.
8, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, including IgG, Fab, Fab ', F (ab ') 2, scFv, dsFv or its arbitrary combination.
9, the anti-Type B botulic neurotoxin list of the separation of specific embodiment/feature/aspect of any above or below Clonal antibody or its antigen-binding fragment, including：
Heavy chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 5 or 19 the first amino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 6 or 20 the second amino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 7 or 21 third amino acid sequence；With
Light chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 12 or 26 tetramino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 13 or 27 pentaamino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 14 or 28 the 6th amino acid sequence.
10, the anti-Type B botulic neurotoxin of the separation of specific embodiment/feature/aspect of any above or below Monoclonal antibody or its antigen-binding fragment, including：
Heavy chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 5 the first amino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 6 the second amino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 7 third amino acid sequence；With
Light chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 12 tetramino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 13 pentaamino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 14 the 6th amino acid sequence.
11, the anti-Type B botulic neurotoxin of the separation of specific embodiment/feature/aspect of any above or below Monoclonal antibody or its antigen-binding fragment, including：
Heavy chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 19 the first amino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 20 the second amino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 21 third amino acid sequence；With
Light chain variable region, including：
With including SEQ ID NO：The first complementary determining region (CDR1) of 26 tetramino acid sequence,
With including SEQ ID NO：The second complementary determining region (CDR2) of 27 pentaamino acid sequence, and
With including SEQ ID NO：The third complementary determining region (CDR3) of 28 the 6th amino acid sequence.
12, the Type B botulic neurotoxin monoclonal of specific embodiment/feature/aspect of any above or below is anti- Body or its antigen-binding fragment, including：
Heavy chain variable region, including SEQ ID NO：3 or 17, and
Light chain variable region, including SEQ ID NO：10 or 24.
13, the Type B botulic neurotoxin monoclonal of specific embodiment/feature/aspect of any above or below is anti- Body, wherein the antibody is IgG1.
14, a kind of hybridoma, the preserving number with NITE BP-01639 or NITE BP-01640.
15, a kind of monoclonal antibody of separation, by the preserving number with NITE BP-01639 or NITE BP-01640 Hybridoma generates.
16, a kind of pharmaceutical composition includes specific embodiment/feature/aspect of one or more any above or belows Separation anti-Type B botulic neurotoxin monoclonal antibody or its antigen-binding fragment and pharmaceutically acceptable carrier.
17, the pharmaceutical composition of specific embodiment/feature/aspect of any above or below, including anti-Type B clostridium botulinum Neurotoxin monoclonal antibody and pharmaceutically acceptable carrier.
18, the pharmaceutical composition of specific embodiment/feature/aspect of any above or below includes the anti-B of two kinds of separation Type botulic neurotoxin monoclonal antibody, its antigen-binding fragment, or both, including：
The human monoclonal antibodies or its antigen-binding fragment of first separation, including：
(1) human monoclonal antibodies or antigen-binding fragment detached, including：
Heavy chain variable region, including：With including SEQ ID NO：First complementary determining region of 5 the first amino acid sequence (CDR1), it includes SEQ ID NO to have：The second complementary determining region (CDR2) of 6 the second amino acid sequence and with including SEQ ID NO：The third complementary determining region (CDR3) of 7 third amino acid sequence, and
Light chain variable region, including：With including SEQ ID NO：First complementary determining region of 12 tetramino acid sequence (CDR1), it includes SEQ ID NO to have：The second complementary determining region (CDR2) of 13 pentaamino acid sequence and with including SEQ ID NO：The third complementary determining region (CDR3) of 14 the 6th amino acid sequence,
(2) human monoclonal antibodies or antigen-binding fragment detached, including heavy chain variable region and light chain variable region are described heavy Chain variable region includes SEQ ID NO：3, the light chain variable region includes SEQ ID NO：10,
(3) monoclonal antibody detached is generated by the hybridoma with preserving number NITE BP-01639,
Or its arbitrary combination；With
The human monoclonal antibodies or its antigen-binding fragment of second separation, including：
(4) human monoclonal antibodies or antigen-binding fragment detached, including：
Heavy chain variable region, including：With including SEQ ID NO：First complementary determining region of 19 the first amino acid sequence (CDR1), it includes SEQ ID NO to have：The second complementary determining region (CDR2) of 20 the second amino acid sequence and with including SEQ ID NO：The third complementary determining region (CDR3) of 21 third amino acid sequence, and
Light chain variable region, including：With including SEQ ID NO：First complementary determining region of 26 tetramino acid sequence (CDR1), it includes SEQ ID NO to have：The second complementary determining region (CDR2) of 27 pentaamino acid sequence and with including SEQ ID NO：The third complementary determining region (CDR3) of 28 the 6th amino acid sequence,
(5) human monoclonal antibodies or antigen-binding fragment detached, including heavy chain variable region and light chain variable region are described heavy Chain variable region includes SEQ ID NO：17, the light chain variable region includes SEQ ID NO：24,
(6) monoclonal antibody detached is generated by the hybridoma with preserving number NITE BP-01640,
Or its arbitrary combination.
19, a kind of method for generating the anti-Type B botulic neurotoxin monoclonal antibody of separation, including：
By merging come the peripheral blood mononuclear cells (PBMC) of the immune people of Clostridium botulinum toxoid of using by oneself and can be effective The fusion partner cells of cell fusion generate hybridoma；With
Type B botulic neurotoxin monoclonal antibody is obtained from the hybridoma.
20, the anti-Type B clostridium botulinum for generating separation of specific embodiment/feature/aspect of any above or below The method of neurotoxin monoclonal antibody, wherein the Type B botulic neurotoxin passes through Type BClostridium botulinum's BoNT/B1 (bacterial strain Okra), Type BClostridium botulinumBoNT/B2 (bacterial strain 111), Type BClostridium botulinum's BoNT/B6 (bacterial strain Osaka05) or its arbitrary combination generate.
21, specific embodiment/feature/aspect of any above or below for generating anti-Type B clostridium botulinum Nervous toxicity The method of plain monoclonal antibody, wherein the fusion partner cells are SPYMEG cells.
22, a kind of to be used to prevent, treat and detect in human experimenter in the poisoning of Type B botulic neurotoxin at least A kind of kit, including specific embodiment/feature/aspect of any above or below separation anti-Type B clostridium botulinum god Through toxin monoclone antibody, its antigen-binding fragment, or both.
23, a kind of inhibition or the method for treating botulismus in human experimenter, including human experimenter's application is treated The anti-Type B botulic neurotoxin Dan Ke of the separation of specific embodiment/feature/aspect of a effective amount of any above or below Grand antibody, its antigen-binding fragment, or both.
24, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison further includes diagnosis patient's botulic neurotoxin poisoning.
25, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison further includes the reduction for at least one symptom for monitoring botulismus.
26, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein at least one symptom include paralysis, it is symmetrical parmlysis of cranial nerve, Rearrangments flaccid paralysis, random Flesh symmetrically paralyses, it is pharyngeal collapse, breathe stopping, can not sucking, can not swallow, sound reduction, ptosis, facial paralysis, pupil it is solid Fixed, mydriasis, eye-blurred, soft neck, whole-body muscle relaxation, general weakness, diplopia, dysarthrosis, dysphonia, swallow it is tired Difficult, lossless, erythroplakia, postural hypotension, nausea, constipation, the retention of urine, dizziness, dry, sore-throat, hypoplasia, pharynx are anti- Penetrate, the muscular atrophy of whole body reflectivity or its arbitrary combine.
27, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein the botulismus includes food-borne botulismus, wound infection botulismus, baby's enterotoxemia meat poisoning Poisoning, adult's enterotoxemia botulismus, iatrogenic botulismus, air borne botulismus or its arbitrary combination.
28, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein the anti-Type B clostridium botulinum of the separation of specific embodiment/feature/aspect of any above or below Neurotoxin monoclonal antibody, its antigen-binding fragment, or both, combined needle controls the one or more other of botulismus It treats and applies.
29, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein the joint synergy is to inhibit or treat botulismus.
30, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein one or more other treatments include applying the anti-Type B botulic neurotoxin of the second separation Monoclonal antibody, its antigen-binding fragment, or both.
31, in the inhibition of specific embodiment/feature/aspect of any above or below or treatment human experimenter in meat poisoning The method of poison, wherein one or more other treatments include the anti-A type botulic neurotoxin Dan Ke using separation Grand antibody, its antigen-binding fragment, or both.
32, the anti-Type B botulic neurotoxin of the separation of specific embodiment/feature/aspect of any above or below Monoclonal antibody, its antigen-binding fragment, or both be used to prepare for inhibiting or treating botulismus in human experimenter The purposes of drug.
33, a kind of method detecting Type B botulic neurotoxin in human experimenter, including：
By the separation of specific embodiment/feature/aspect of sample and any above or below from human experimenter Anti- Type B botulic neurotoxin monoclonal antibody, its antigen-binding fragment, or both contact；With
Based on antibody, its segment, or both whether in conjunction with Type B botulic neurotoxin detect B in human experimenter The existence or non-existence of type botulic neurotoxin.
The present invention will be furtherd elucidate by being intended to illustrative rather than the limitation present invention following example.These examples It proves in the surprising and unexpected characteristic of antibody claimed, including such as their neutralization clostridium botulinum The ability of poison and treatment botulismus infection.These examples prove the people Dan Ke of anti-botulic neurotoxin according to the present invention The exploitation of grand antibody.Data prove treatment and the diagnostic uses of the present invention.
These examples prove the light chain of M-2 antibody specificity combinations BoNT/B, and are shown in Mouse bioassay The neutralization activity of strength (is approximately more than equal to 100LD50/ milligram).In addition, the joint of M-2 and M-4 can be more than 10, 000LD50The effect complete neutralization BoNT/B of/milligram.This effect is most effective, is more than to resist for the anti-BoNT of commercially available BABYBIG people Serum (540LD50/ milligram) design BoNT/B in and 18 times of standard.It treats in model, is administered when in toxin oral after exposure When being treated in 12 hours, HuMAbs (M-2+M-4) provides the complete protection to lethal dose BoNT/B.In addition to BoNT/B1, HuMAbs (M-2+M-4) also neutralizes other hypotypes, BoNT/B2 and BoNT/B6.From the reality with M-2 and the human monoclonal antibodies of M-4 The data for testing middle acquisition typically represent antibody according to the present invention and generation and use their method.
Embodiment
Embodiment 1
Type BMeat poisoning fusiform brood cell liver bacteriumBacterial strain Okra uses cellophane tube program (cellophane-tube Procedure it) is cultivated, obtains culture supernatant.Precursor toxin (16S toxin and 12S toxin) uses (Arimitsu et Al., Infect.Immun.71：1599-1603,2003) it is purified from culture supernatant.As (Arimitsu et al., Infect.Immun.71：1599-1603,2003) the slave 16S toxin of description prepares BoNT/B1 and NAP-16.BoNT/A1 (bacterium Strain 62A), BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05) provide by doctor S.Kozaki.Tetravalence clostridium botulinum Toxoid vaccine is provided by doctor M.Takahashi.Respective training of the 12S toxin from A, B, E, F type clostridium botulinum Foster supernatant carries out purifying and with formalin detoxification.It is detoxicated to be detected using (i.p.) in mouse peritoneal.Four types Toxoid prepared product mixes, and is then mixed with aluminium (adjuvant) and thimerosal (preservative).Every milliliter of toxoid prepared product Contain each 0.1 milligram of four types, 0.2 milligram of aluminium, 0.001% thimerosal and 0.0006% formalin.
Immunization schedule is followed as described in Fig. 1.Tetravalence Clostridium botulinum toxoid (A, B, E and F type) is inoculated in two Adult human's (4 or 5 times) of a health, has submitted Written informed consent and has received physical examination.Toxoid only with O.5ml dosage intramuscular injection is to healthy individuals.After 9 and 18 days, from each volunteer's peripheral blood sample (10 milliliters).Blood Sample tests the antibody titer of plasma sample anti-BoNT/A or BoNT/B using ELISA method from each volunteer.The reality Proved recipe case is ratified by the human trial committee of Osaka University.
Enzyme-linked immunosorbent assay (ELISA) carries out as follows.With 100 microlitres of BoNT/A or (and) BoNT/B is in PBS In the ultimate density of 3 micrograms/ml 96 hole plates (Falcon) are coated at 37 DEG C 2 hours.By hole with containing 0.05% tween PBS (PBS-T) is washed, and is closed overnight at 4 DEG C with 0.2%BSA (Sigma)/PBS-T.Twice of serial dilution of human serum sample, will 50 microlitres of each dilution is added in hole and is incubated 2 hours at 37 DEG C.After washing, 50 microlitres of coupling horseradish peroxide is added The anti-human igg (BIO-RAD) of compound enzyme (HRP) simultaneously incubates 2 hours at 37 DEG C.After washing, by plate substrate solution (adjacent benzene two Amine, nacalai tesque) it is incubated 30 minutes at 37 DEG C, measure the absorbance value at OD492.ELISA titres are with highest Extension rate indicates that display is more than twice of absorbance of negative control sera.After inoculation 9 or 18 days in two volunteer's moderate resistances Body titre increases (table 1).
Table 1
The ELISA titres of the blood plasma for the people volunteer that table 1. is inoculated with clostridium botulinum tetravalence toxoid
ELISA titres indicate that display is more than twice of absorbance of negative control sera with highest extension rate.After inoculation The antibody titer raising in two volunteers in 9 or 18 days.
Embodiment 2
Cell fusion, screening and Cloning processes are carried out as described in fig. 2.9 or 18 after last time is immune It, peripheral blood sample from volunteer, peripheral blood mononuclear cells (PBMC) by Ficoll (GE Healthcare) gradients from The heart is purified.PBMC and SPYMEG cells are merged with 10 to 1 ratio with polyethylene glycol (Roche).The cell of fusion is 96 In hypoxanthine-amino in supplemented with the Dahl Burke Improved Eagle Medium of 15%FBS (DMEM, GIBCO) in orifice plate It is cultivated in the presence of petrin-thymidine (HAT, GIBCO) about 10-14 days.It is selected by HAT, culture medium pair is carried out with ELISA The first time of BoNT/A or the antibody of BoNT/B specificity screens.As a result, from being obtained in the blood sample of acquisition in 9 days after inoculation 27 positive holes are obtained, 8 positive holes are obtained from the blood sample of acquisition in 18 days after inoculation.Specific antibody positive hole then leads to It crosses limiting dilution and carries out cell clone, the cell cloned.Programmed screening is also carried out by ELISA.Obtain 8 it is stable Hybridoma clone (table 2).
Table 2
The result of table 2. first time and programmed screening
For isotype analysis shows 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).
Table 3
The determination of the isotype of table 3.HuMAbs
8 hybridoma clones are obtained from two volunteers, 4 hybridoma clones generate IgG, and 3 hybridoma clones generate IgM, 1 hybridoma clone generate IgA.
M-1, M-2, M-4 and S-1 are IgG, and M-3, M-5 and M-6 are IgM, and M-7 is IgA.
Each stable hybridoma culture in serum free medium (GIBCO), then collects supernatant.In culture supernatant The anti-human igg (BIO-RAD) of HuMAbs isotypes coupling HRP in liquid, the anti-human IgM (BIOSOURCE for being coupled HRP INTERNATIONAL) or the anti-human IgA (Invitrogen) of coupling HRP turns to be measured in stain analysis in west.IgG antibody It is purified from supernatant by Protein G column (GE Healthcare).The subclass of each IgG antibody uses IgG subclass people ELISA kit (IgG subclass human ELISA kit, Invitrogen) is measured.
Embodiment 3
The clone for having carried out the variable region gene of monoclonal antibody and sequencing.The sequence of determining heavy chain and light chain variable region It is shown in Fig. 3~Fig. 8.Total serum IgE uses RNeasy Mini kits (Qiagen) from hybridoma according to the explanation of manufacturer Extraction, uses SUPERSCRIPT(R)VILOTM cDNA synthetic agent box (Invitrogen) is generated by RT-PCR.M-2 and M- The code area of the H- and L- chains of 4 antibody is expanded by PCR with KOD-Pus-Neo (Toyobo) and following primer：5′- ATGGACTGGACCTGGAGGATCCTC-3 ' (M-2-H- chains sense primer) (SEQ ID NO：35), 5 '- ATGAAACACCTGTGGTTCTTCCTCCT-3 ' (M-4-H- chains sense primer) (SEQ ID NO：And 5 ' -36) CTCCCGCGGCTTTGTCTTGGCATTA-3 ' (H- chains antisense primer) (SEQ ID NO：38)；With 5 '- ATGGCCTGGWYYCCTCTCYTYCTS-3 ' (M-4-16-L- chains sense primer) (SEQ ID NO：38), 5 '- ATGSCCTGGGCTCYKCTSCTCCTS-3 ' (M-2- and M-4-18-L- chains sense primer) (SEQ ID NO：39), 5 '- ATGGCCTGGRYCYCMYTCYWCCTM-3 ' (M-4-19-L- chains sense primer) (SEQ ID NO：And 5 ' -40) TGGCAGCTGTAGCTTCTGTGGGACT-3 ' (L- chains antisense primer) (SEQ ID NO：41).With TaKaRa Ex Taq(R) (Takara) after incubating, PCR product is connected to pGEM-T Easy carriers (Promega), their sequence uses BigDye 3100 genetic analyzer of Terminator v3.1 cycle sequencings kits and ABI Prism (Applied Biosystems) into Analysis is gone.IgG subclass analysis shows that M-2 and M-4 is made of IgG1 heavy chains and light (λ) chain.
Embodiment 4
BoNT/A or BoNT/B (600 nanogram) are separated into Hc and Lc by SDS-PAGE, and are transferred to nitrocellulose filter (BIO-RAD) on.Film is closed with containing 5% skimmed milk power in the Tris buffered salines containing 0.05%Tween.HuMAbs (1.0 mcg/ml) is added to the film, and incubates 1 hour at room temperature.After washing, the anti-human igg of film and coupling HRP is resisted Body incubates 1 hour at room temperature, then specific band ECL (PIERCE) visualizations (Fig. 9 A and 9B).
Embodiment 5
In order to determine the epitope for HuMAbs, carries out west and turn stain analysis.The result shows that M-2 specifically binds BoNT/B Light chain (Figure 10).In contrast, M-4 is attached to both light chain and heavy chain (Figure 10).
Embodiment 6
HuMAb combination Type Bs BoNT, precursor toxin or non-toxic components (NAP-16) are analyzed by ELISA.HuMAbs It is added to the plate of coating BoNT, precursor toxin or NAP-16.HuMAbs (0.01 mcg/ml~0.5 mcg/ml) is added To the plate of coating BoNT/A or BoNT/B.After washing, in conjunction with HuMAbs by be coupled HRP anti-human IgG antibodies be detected.M- 2 and M-4 is attached to BoNT and precursor toxin.In contrast, M-2 and M-4 shows the faint combination to NAP-16.These results Show the BoNT and individual BoNT and M-2 and M-4 specific bindings BoNT that M-2 and M-4 are attached in precursor toxin. M-2 and M-4 display specific binding BoNT/B (Figure 11).
Embodiment 7
The neutralization activity of HuMAbs is tested by Mouse bioassay.It is (total with 500 microlitres in intraperitoneal injection Volume) it arrives before mouse (ddY, female, 4 weeks, SLC), HuMAbs and BoNT/B (10LD50) incubate 1 hour at room temperature. 10LD50BoNT/B and 100 microgram HuMAb or PBS (Cnt) incubate 1 hour, and in intraperitoneal injection to mouse.Observe mouse Morbidity and mortality 3 weeks.The mouse for injecting BoNT/B+PBS (Cnt) is dead in 12 hours.In contrast, BoNT/B is injected The mouse of+M-4 is partly protected, and such as compared with control mice, is proved by the increased death time.On the other hand, completely It protects and is observed (table 4) in the mouse of injection BoNT/B+M-2.
Table 4
The neutralization activity of the anti-BoNT/B of the HuMAb in Mouse bioassay of table 4.
The neutralization activity of the anti-BoNT/B of HuMAbs is determined by Mouse bioassay.10LD50BoNT/B and 100 Microgram HuMAb or PBS (Cnt) are incubated 1 hour, and in intraperitoneal injection to mouse.The mouse of injection BoNT/B+PBS (Cnt) exists It is dead in 12 hours.In contrast, the mouse for injecting BoNT/B+M-4 is partly protected, such as compared with control mice, by increasing Death time proved.On the other hand, complete protection is observed in the mouse of injection BoNT/B+M-2.
Embodiment 8
Combining for HuMAbs has carried out synergistic effect test.10LD50BoNT/B and M-2 and M-4 mixture (each 0.5 microgram, in 500 microlitres of volume) it incubates, and be injected into mouse.All mouse survivals for receiving HuMAbs and do not have Symptom (Figure 12).
Embodiment 9
It treats in model after exposure, Mouse oral applies Type B precursor toxin, and (16S toxin, 10 nanograms are in 300 microlitres of bodies In product), then by after 16S toxin is administered orally intraperitoneal injection in 12,24 or 36 hours apply HuMAbs (M-2+M-4). Observe mouse invasion rate and the death rate 3 weeks.It treats in model after exposure, 12 hours after 16S toxin is administered orally, mouse production The symptom of raw botulismus.Control mice not with HuAbs treatments is dead in 72 hours.In contrast, HuMAbs's is sudden and violent Treatment provides all survivals in 12 hours after 16S toxin is administered orally after dew, and providing part within 24 and 36 hours after application survives (Figure 13).
Embodiment 10
HuMAbs (M-2 and M-4) combine BoNT/B2 (bacterial strain 111) and BoNT/B6 (bacterial strain Osaka05) by ELISA into Row analysis.HuMAbs (0.5 mcg/ml) is added to the plate of coating BoNT.After washing, in conjunction with HuMAbs pass through be coupled HRP Anti-human IgG antibodies be detected.In addition to BoNT/B1, M-2 and M-4 show that also strength combines BoNT/B2 and BoNT/B6.In In experiment, M-2+M-4 (+0.5 microgram of 0.5 microgram) complete neutralization BoNT/B2 (10 nanogram) and BoNT/B6 (2.5 nanogram) (figures 14)。
Embodiment 11
For the expression recombination HuMAbs in mammalian cell (HEK293 cells) and them are tested, IgG expression vectors It is built as follows.Following primer pair (the restriction enzyme of the pGEM-T Easy carriers of variable region gene with H- and L- chains Enzyme site is indicated with underscore) PCR is carried out to add restriction endonuclease sites and Kozak sequences：5′- ATTTGCGGCCGCCATGGACTGGACCTGGAGG-3 ' (M2-H- chains sense primer) (SEQ ID NO：42), 5 '- ATTTGCGGCCGCCATGAAACACCTGTGGTTCTTC-3 ' (M-4-H- chains sense primer) (SEQ ID NO：And 5 ' -43) ATACTCGAGGGTGCCAGGGGGAAGACCGATG-3 ' (H- chains antisense primer) (SEQ ID NO：44)；With 5 '- ATTTGCGGCCGCCATGGCCTGGTTTCCTCTCTTC-3 ' (M-4-16-L- chains sense primer) (SEQ ID NO：45), 5 '- ATTTGCGGCCGCCATGGCCTGGGCTCTGCT-3 ' (M-2- and M-4-18-L- chains sense primer) (SEQ ID NO：46), 5′-ATTTGCGGCCGCCATGGCCTGGGTCTCATT-3 ' (M-4-19-L- chains sense primer) (SEQ ID NO：And 5 ' -47) ATACTCGAGGGCGGGAACAGAGTGACCGTGG-3 ' (L- chains antisense primer) (SEQ ID NO：48).H- and L- chain encodings area PCR product restriction enzyme Not I and Xho I digestions, be then attached to expression vector pQCXIP-hCH and pQCXIH- In hC, it is respectively provided with the human immunoglobulin(HIg) constant region (MBL) of γ and λ chains.
HEK293 cells are in 5%C02In cultivated in the MEM containing 10%FBS at 37 DEG C.Made according to the explanation of manufacturer With LIPOFECTAMINE2000 transfection reagents (Invitrogen), the cell pQCXIP- grown on 100 millimeters of culture dishes HCH and pQCXIH-hC expression vectors are transiently transfected.After transfection, culture medium is removed, wash cell and Opti-Pro without In 5%C0 in blood serum medium (Gibco)2In incubated 10 days at 37 DEG C.Recombination HuMAbs in culture supernatant is even The anti-human igg (BIO-RAD) of connection HRP is measured.Recombinate HuMAbs by Protein G column (GEHealthcare) from supernatant into Row purifying.
Recombinate HuMAbs (RM-2 and RM-4；It is portrayed as RM-4LC16, RM-4LC18, RM-4LC19, is had identical as M-4 Heavy chain and light chain, but with the unlike signal sequence of light chain being respectively displayed in Fig. 6,7,8) pass through in conjunction with BoNT/B ELISA is analyzed.RM-2 and RM-4 (about 0.01 mcg/ml~0.5 mcg/ml) are added to plate.After washing, in conjunction with Recombination HuMAbs is detected by being coupled the anti-human IgG antibodies of HRP.As a result, RM-2 and RM-4 with from hybridizing The identical mode of M-2 and M-4 of tumor is attached to BoNT/B (Figure 15).In neutralization test, (0.5 microgram+0.5 is micro- by RM-2+RM-4 Gram) neutralize BoNT/B1 (10LD50) (table 5A and 5B).
Table 5A
Recombinate the neutralization activity of the anti-BoNT/B of HuMAb
The neutralization activity of the anti-BoNT/B of recombination HuMAbs is determined by Mouse bioassay.10LD50BoNT/B It incubates, and is injected into mouse with the mixture of M-2 (RM-2) and M-4 (RM-4) (each 0.5 microgram).M-2, RM-2, M-4 and All combinations of RM-4 all neutralize BoNT/B.
Table 5B
Recombinate the neutralization activity of the anti-BoNT/B of HuMAb
The neutralization activity of the anti-BoNT/B of recombination HuMAbs is determined by Mouse bioassay.10LD50BoNT/B It incubates, and is injected into mouse with the mixture of M-2 (RM-2) and M-4 (RM-4) (each 0.5 microgram).M-2, RM-2, M-4 and All combinations of RM-4 all neutralize BoNT/B.
Embodiment 12
HuMAbs and recombination HuMAbs (RM-2 and RM-4) from hybridoma combine BoNT/B (BoNT/B 1, BoNT/ B2 or BoNT/B6) it is analyzed by SDS-PAGE.BoNT/B1, BoNT/B2 or BoNT/B6 are separated into Hc by SDS-PAGE And Lc, and be transferred on nitrocellulose filter.HuMAbs (1.0 mcg/ml) is added to the film, and it is small to incubate 1 at room temperature When.After washing, the anti-human IgG antibodies of film and coupling HRP are incubated 1 hour at room temperature, then specific band can by ECL Depending on change.Recombination HuMAbs shows combination profile identical with the HuMAbs of hybridoma is derived from.M-2 and RM-2 specific bindings The light chain of BoNT/B.In contrast, M-4 and RM-4 are attached to both light chain and heavy chain.Pass through M-4 and RM-4 antibody, B2 hypotypes Heavy chain ratio B1 heavy chain detection it is clearer.In addition, the heavy chain of the heavy chain ratio B2 of B6 hypotypes detects clearer (Figure 16).
The full content of the bibliography of all references is clearly merged into the displosure content by applicant.In addition, working as Quantity, concentration or other values or parameter are given as the list of range, preferred scope or a row preferred upper limit value and preferred lower limit value When going out, it is understood that specifically disclose by any pair of of any range limit or preferred value and any range lower limit or preferred value All ranges formed, no matter whether range is separately disclosed.It, unless otherwise mentioned, should when this text reference numberical range Range is intended to include its endpoint and all integers within the scope of this and score.But it is not meant to that the scope of the present invention is limited to The particular value enumerated when confining spectrum.
The implementation of this specification and the present invention for disclosing from here considers that other embodiments of the present invention is for this field It is obvious for technical staff.Mean that this specification and embodiment are considered merely as illustratively, true model of the invention It encloses and spirit is indicated by following following claims and its equivalent.

Claims (15)

1. a kind of anti-Type B botulic neurotoxin monoclonal antibody of separation or its antigen-binding fragment, including anti-Type B meat poisoning The neutralization activity of bacillus neurotoxin, wherein the monoclonal antibody be selected from human monoclonal antibodies, Humanized monoclonal antibodies or The two,
The anti-Type B botulic neurotoxin monoclonal antibody of the separation or its antigen-binding fragment are characterised by comprising：
Heavy chain variable region comprising：
First complementary determining region (CDR1), the amino acid sequence such as SEQ ID NO of first complementary determining region (CDR1):Shown in 5,
Second complementary determining region (CDR2), the amino acid sequence such as SEQ ID NO of second complementary determining region (CDR2):Shown in 6, With
Third complementary determining region (CDR3), the amino acid sequence such as SEQ ID NO of the third complementary determining region (CDR3):Shown in 7； With
Light chain variable region comprising：
First complementary determining region (CDR1), the amino acid sequence such as SEQ ID NO of first complementary determining region (CDR1):12 institutes Show,
Second complementary determining region (CDR2), the amino acid sequence such as SEQ ID NO of second complementary determining region (CDR2):13 institutes Show, and
Third complementary determining region (CDR3), the amino acid sequence such as SEQ ID NO of the third complementary determining region (CDR3):14 institutes Show.
2. a kind of anti-Type B botulic neurotoxin monoclonal antibody of separation or its antigen-binding fragment, including anti-Type B meat poisoning The neutralization activity of bacillus neurotoxin, wherein the monoclonal antibody be selected from human monoclonal antibodies, Humanized monoclonal antibodies or The two, the anti-Type B botulic neurotoxin monoclonal antibody of the separation or its antigen-binding fragment are characterized in that, are wrapped It includes：
Heavy chain variable region comprising：
First complementary determining region (CDR1), the amino acid sequence such as SEQ ID NO of first complementary determining region (CDR1):19 institutes Show,
Second complementary determining region (CDR2), the amino acid sequence such as SEQ ID NO of second complementary determining region (CDR2):20 institutes Show, and
Third complementary determining region (CDR3), the amino acid sequence such as SEQ ID NO of the third complementary determining region (CDR3):21 institutes Show；With
Light chain variable region comprising：
First complementary determining region (CDR1), the amino acid sequence such as SEQ ID NO of first complementary determining region (CDR1):26 institutes Show,
Second complementary determining region (CDR2), the amino acid sequence such as SEQ ID NO of second complementary determining region (CDR2):27 institutes Show, and
Third complementary determining region (CDR3), the amino acid sequence such as SEQ ID NO of the third complementary determining region (CDR3):28 institutes Show.
3. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or 2 or its antigen binding Segment, including：
Heavy chain variable region, amino acid sequence such as SEQ ID NO：Shown in 3, and
Light chain variable region, amino acid sequence such as SEQ ID NO：Shown in 10；Alternatively,
Heavy chain variable region, amino acid sequence such as SEQ ID NO：Shown in 17, and
Light chain variable region, amino acid sequence such as SEQ ID NO：Shown in 24.
4. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or 2 or its antigen binding Segment is selected from IgG, Fab, Fab', F (ab') 2, scFv, dsFv or its arbitrary combination.
5. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 3 or its antigen binding fragment Section is selected from IgG, Fab, Fab', F (ab') 2, scFv, dsFv or its arbitrary combination.
6. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 1 or 2 or its antigen binding Segment, wherein the antibody is IgG1.
7. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 3 or its antigen binding fragment Section, wherein the antibody is IgG1.
8. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 4 or its antigen binding fragment Section, wherein the antibody is IgG1.
9. the anti-Type B botulic neurotoxin monoclonal antibody of separation according to claim 5 or its antigen binding fragment Section, wherein the antibody is IgG1.
10. a kind of hybridoma, the preserving number with NITE BP-01639 or NITE BP-01640.
11. a kind of monoclonal antibody of separation or its antigen-binding fragment, by with NITE BP-01639 or NITE BP- The hybridoma of 01640 preserving number generates.
12. a kind of pharmaceutical composition, including one or more separation according to claim 1~9, any one of 11 Anti- Type B botulic neurotoxin monoclonal antibody and its antigen-binding fragment and pharmaceutically acceptable carrier.
13. pharmaceutical composition according to claim 12, the anti-Type B botulic neurotoxin Dan Ke selected from two kinds of separation Grand antibody, its antigen-binding fragment, or both, including：
The human monoclonal antibodies or its antigen-binding fragment of first separation, including：
(1) human monoclonal antibodies or its antigen-binding fragment detached, including：
Heavy chain variable region, including：First complementary determining region (CDR1), the amino acid sequence of first complementary determining region (CDR1) is such as SEQ ID NO:Shown in 5；Second complementary determining region (CDR2), the amino acid sequence such as SEQ of second complementary determining region (CDR2) ID NO:Shown in 6；With third complementary determining region (CDR3), the amino acid sequence such as SEQ ID of the third complementary determining region (CDR3) NO:Shown in 7, and
Light chain variable region, including：First complementary determining region (CDR1), the amino acid sequence of first complementary determining region (CDR1) is such as SEQ ID NO:Shown in 12；Second complementary determining region (CDR2), the amino acid sequence such as SEQ of second complementary determining region (CDR2) ID NO:Shown in 13；With third complementary determining region (CDR3), the amino acid sequence such as SEQ of the third complementary determining region (CDR3) ID NO:Shown in 14,
(2) human monoclonal antibodies or antigen-binding fragment detached, including heavy chain variable region and light chain variable region, the heavy chain can Become the amino acid sequence such as SEQ ID NO in area:Shown in 3, the amino acid sequence such as SEQ ID NO of the light chain variable region:10 institutes Show,
(3) monoclonal antibody detached is generated by the hybridoma with preserving number NITE BP-01639,
Or its arbitrary combination；With
The human monoclonal antibodies or its antigen-binding fragment of second separation, including：
(4) human monoclonal antibodies or its antigen-binding fragment detached, including：
Heavy chain variable region, including：First complementary determining region (CDR1), the amino acid sequence of first complementary determining region (CDR1) is such as SEQ ID NO:Shown in 19；Second complementary determining region (CDR2), the amino acid sequence such as SEQ of second complementary determining region (CDR2) ID NO:Shown in 20；With third complementary determining region (CDR3), the amino acid sequence such as SEQ of the third complementary determining region (CDR3) ID NO:Shown in 21, and
Light chain variable region, including：First complementary determining region (CDR1), the amino acid sequence of first complementary determining region (CDR1) is such as SEQ ID NO:Shown in 26；Second complementary determining region (CDR2), the amino acid sequence such as SEQ of second complementary determining region (CDR2) ID NO:Shown in 27；With third complementary determining region (CDR3), the amino acid sequence such as SEQ of the third complementary determining region (CDR3) ID NO:Shown in 28,
(5) human monoclonal antibodies or antigen-binding fragment detached, including heavy chain variable region and light chain variable region, the heavy chain can Become the amino acid sequence such as SEQ ID NO in area:Shown in 17, the amino acid sequence such as SEQ ID NO of the light chain variable region:24 institutes Show,
(6) monoclonal antibody detached is generated by the hybridoma with preserving number NITE BP-01640,
Or its arbitrary combination.
14. one kind is for preventing, treating and detecting at least one of Type B botulic neurotoxin poisoning in human experimenter Kit, include the anti-Type B botulic neurotoxin list of the separation according to claim 1~9, any one of 11 Clonal antibody, its antigen-binding fragment.
15. a kind of anti-Type B botulic neurotoxin Dan Ke of separation according to claim 1~9, any one of 11 Grand antibody, its antigen-binding fragment, or both be used to prepare drug for inhibiting or treating botulismus in human experimenter Purposes.
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