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CN108623681B - A kind of neutralizing antibody of anti-tetanus toxin and application - Google Patents

A kind of neutralizing antibody of anti-tetanus toxin and application Download PDF

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Publication number
CN108623681B
CN108623681B CN201810420730.5A CN201810420730A CN108623681B CN 108623681 B CN108623681 B CN 108623681B CN 201810420730 A CN201810420730 A CN 201810420730A CN 108623681 B CN108623681 B CN 108623681B
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tetanus toxin
antibody
seq
infection
neutralizing antibody
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CN108623681A (en
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廖化新
王月明
袁晓辉
郑伟宏
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Changchun BCHT Biotechnology Co.
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GUANGZHOU TAINUODI BIOTECHNOLOGY CO Ltd
Zhuhai Microlab Biotechnology Co Ltd
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Priority to CN201810420730.5A priority Critical patent/CN108623681B/en
Priority to PCT/CN2018/090488 priority patent/WO2019210552A1/en
Publication of CN108623681A publication Critical patent/CN108623681A/en
Priority to US16/958,213 priority patent/US11725046B2/en
Priority to EP18896977.8A priority patent/EP3733699A4/en
Priority to AU2018395100A priority patent/AU2018395100B2/en
Priority to JP2020555284A priority patent/JP7368670B2/en
Priority to BR112020013094-0A priority patent/BR112020013094A2/en
Priority to PCT/CN2018/124958 priority patent/WO2019129214A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

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Abstract

The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin.The neutralizing antibody of anti-tetanus toxin provided by the invention can specifically bind tetanus toxin, thus have extremely strong neutralization activity and very high affine activity, and dosage is low.The neutralizing antibody of anti-tetanus toxin provided by the invention can be used in preventing and treating the disease of tetanus toxin infection and clostridium tetani mediation or the isolated antibody of disease condition, while can also be used to diagnose and/or monitor the isolated antibody of tetanus toxin infection and infection due to Clostridium tetani.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention is polluted without exogenous virus, it is widely portable to various crowds.

Description

A kind of neutralizing antibody of anti-tetanus toxin and application
Technical field
The present invention relates to the neutralizing antibodies and application of gene engineering technology field more particularly to a kind of anti-tetanus toxin.
Background technique
Tetanus toxin is a kind of inhibition that clostridium tetani (Clostridium tetani) is generated under anaerobic The secretory protein of neurotransmitter regulator.Tetanus toxin can cause super reflex response and band muscle spasmus, receive in muscle tonue It contracts on the basis of (myotonia hardens), the strong spasm of paroxysmal, patient with severe symptoms can be lethal, and case fatality rate may be up to 50%.
Prevention and treatment for tetanus toxin generally use horse serum TAT (Tatanus Antitoxin, tetanus Antitoxin) it is treated.But due to the difference of human body constitution and to the acceptance level difference of horse serum TAT, many people can go out The adverse reactions such as existing allergic reaction, anaphylactic shock or serum sickness.Therefore, horse serum TAT is not particularly suited for owner.With biology The progress of medical treatment, HTIG (Human Tetanus Immunoglobulin, Homo-Tet) are progressed into Market.HTIG can overcome adverse reaction when horse serum TAT clinical use, and it is horizontal to greatly improve tetanic prevention and treatment.But Since people's blood source source is because difficult, expensive, there are the danger of exogenous virus pollution, therefore, the industrialized production of HTIG and face Bed application is greatly limited.
In recent years, due to the development of technique for gene engineering, so that preparing humanization/humanized by the method for genetic engineering Antibody is possibly realized.It can reduce or eliminate foreign sera albumen using humanization/human antibody prepared by genetic engineering to cause Allergic reaction, and can overcome source of people immunoglobulin produce inadequate blood source and potential virus pollution possibility the problems such as, at For the Main way studied at present.
Summary of the invention
The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin, and to solve, existing HTIG is expensive, deposits The problem of exogenous virus pollutes.
In a first aspect, the present invention provides a kind of neutralizing antibody of anti-tetanus toxin, comprising:
Variable heavy chain domain comprising VH CDR1, VH CDR2 and VH CDR3 and comprising VLCDR1, VLCDR2 and The variable light chain domain of VLCDR3, wherein
The VH CDR1 includes amino acid sequence shown in SEQ ID NO.1；
The VH CDR2 includes amino acid sequence shown in SEQ ID NO.2；
The VH CDR3 includes amino acid sequence shown in SEQ ID NO.3；
The VL CDR1 includes amino acid sequence shown in SEQ ID NO.4；
The VL CDR2 includes amino acid sequence shown in SEQ ID NO.5；
The VL CDR3 includes amino acid sequence shown in SEQ ID NO.6
It preferably, further include light chain variable shown in heavy chain variable region shown in SEQ ID NO.7 and SEQ ID NO.8 Area.
Preferably, the neutralizing antibody includes:
With the VH CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.1, And/or with the VL CDR1 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.4；With/ Or,
With the VH CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.2, And/or with the VL CDR2 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.5；With/ Or,
With the VH CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.3, And/or with the VL CDR3 with the sequence at least amino acid sequence of 80% sequence identity shown in the SEQ ID NO.6.
Preferably, the neutralizing antibody immunospecifically combines tetanus toxin, and the neutralizing antibody has at least 10- 6, the affinity to the tetanus toxin and/or the clostridium tetani of 10-7,10-8,10-9 or 10-10M.
Second aspect, the present invention provide a kind of composition, the neutralizing antibody of the anti-tetanus toxin including first aspect.
The third aspect, the present invention provide a kind of nucleic acid molecules, the anti-tetanus poison of the nucleic acid molecule encoding first aspect The neutralizing antibody of element.
Fourth aspect, the present invention provide a kind of expression vector, the nucleic acid molecules including the third aspect.
5th aspect, the present invention provide a kind of recombinant cell, the table of nucleic acid molecules or fourth aspect including the third aspect Up to carrier.
6th aspect, the present invention provide a kind of kit, the neutralizing antibody of the anti-tetanus toxin including first aspect, or The recombination of the expression vector or the 5th aspect of the composition of second aspect or the nucleic acid molecules of the third aspect or fourth aspect is thin Born of the same parents.
7th aspect, the present invention provide a kind of application method, comprising:
The neutralizing antibody of the anti-tetanus toxin of first aspect is in preparation by tetanus toxin infection and clostridium tetani Application in the drug of infection；And/or
The neutralizing antibody of the anti-tetanus toxin of first aspect is in preparation detection tetanus toxin infection and tetanus shuttle Application in the kit of bacterium infection；And/or
The composition of second aspect is in preparation treatment by the drug of tetanus toxin infection and infection due to Clostridium tetani Application；And/or
The composition of second aspect is in the kit of preparation diagnosis tetanus toxin infection and infection due to Clostridium tetani Application；
The disease that the nucleic acid molecules of the third aspect are mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application；
The disease that the expression vector of fourth aspect is mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application；
The disease that the recombinant cell of 5th aspect is mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application.
The technical solution that the embodiment of the present invention provides can include the following benefits:
The present invention provides neutralizing antibody and the application of a kind of anti-tetanus toxin.Anti-tetanus toxin provided by the invention Neutralizing antibody can specifically bind tetanus toxin, thus have extremely strong neutralization activity and very high affine activity, and use It measures low.The neutralizing antibody of anti-tetanus toxin provided by the invention can be used in preventing and treating tetanus toxin infection and break The disease of clostridium mediation of catching cold or the isolated antibody of disease condition, while can also be used to diagnose and/or monitor broken wind toxin The isolated antibody of element infection and infection due to Clostridium tetani.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention No exogenous virus pollution, is widely portable to various crowds.
It should be understood that above general description and following detailed description be only it is exemplary and explanatory, not It can the limitation present invention.
Detailed description of the invention
In order to illustrate more clearly of the technical solution of the application, letter will be made to attached drawing needed in the embodiment below Singly introduce, it should be apparent that, for those of ordinary skills, without any creative labor, It is also possible to obtain other drawings based on these drawings.
Fig. 1 passes through SDS-PAGE (English name: Sodium dodecyl sulfate- to be provided in an embodiment of the present invention Polyacrylamide gelelectrophoresis, Chinese: sodium dodecyl sulfate-polypropylene acyl ammonia gel electrophoresis) The detection of expression result figure for the neutralizing antibody that method detects；
Fig. 2 is provided in an embodiment of the present invention by the detection of Western Blot (Chinese: immunoblotting) method The detection of expression result figure of obtained neutralizing antibody；
Fig. 3 is that the antigen-binding activity of neutralizing antibody provided in an embodiment of the present invention detects figure；
Fig. 4 is the affine Activity determination figure of neutralizing antibody provided in an embodiment of the present invention and tetanus toxin；
Fig. 5 is protective effect of the neutralizing antibody provided in an embodiment of the present invention to the tetanus toxin LD50 of 20 multiple doses Figure；
Fig. 6 is protective effect of the neutralizing antibody provided in an embodiment of the present invention to the tetanus toxin LD50 of 60 multiple doses Figure.
Specific embodiment
In term, antibody refers to the protein molecular for playing the receptor acting of specific recognition antigen, including with specific antigen The immunoglobulin molecules of immune response, including entire antibody, dimerization, trimerization and multimeric antibody；Bispecific antibody；Inosculating antibody Body；Recombination and engineered antibody and its segment, as long as the antibody includes antigen binding domain.Immunoglobulin molecules have weight Chain and light chain, each heavy chain and light chain include constant region and variable region, wherein the variable region of each heavy chain and light chain is referred to as variable Heavy chain (variable heavy chain, VH) and variable light (variable light chain, VL).Heavy chain and light chain Variable region includes four framework regions, is interrupted by three hypervariable regions, also referred to as complementary determining region (complementarity Determining region, CDR), wherein the main function of CDR is the epitope for being integrated to antigen.
It will also be appreciated by those of skill in the art that the amino acid sequence that the present invention covers the tetanus toxin antibody is repaired Decorations.For example, it may be desired to improve the binding affinity and/or other biological characteristics of antibody.Anti-tetanus toxin antibody it Amino acid sequence variation is from introducing appropriate nucleotide variation into anti-tetanus toxin antibody nucleic acid or being prepared by peptide synthesis.It should Equal modifications include the residue deletions and/or insertion and/or substitution that (for example) anti-tetanus toxin antibody amino acid sequence is interior.It carries out For any combination of missing, insertion and substitution to reach final construct, restrictive condition has by the final construct is wanted special Sign.Amino acid variation also can be changed anti-tetanus toxin antibody translation after process, such as change glycosylation site number or Position.
The embodiment of the present invention provides a kind of neutralizing antibody of anti-tetanus toxin, and in embodiments of the present invention, the neutralization is anti- Body is known as antibody TRN1015.Antibody TRN1015 provided in an embodiment of the present invention includes: comprising VH CDR1, VH CDR2 and VH The variable heavy chain domain of CDR3 and variable light chain domain comprising VLCDR1, VLCDR2 and VLCDR3, wherein
VH CDR1 includes amino acid residue sequence shown in SEQ ID NO.1；
VH CDR2 includes amino acid residue sequence shown in SEQ ID NO.2；
VH CDR3 includes amino acid residue sequence shown in SEQ ID NO.3；
VL CDR1 includes amino acid sequence shown in SEQ ID NO.4；
VL CDR2 includes amino acid sequence shown in SEQ ID NO.5；
VL CDR3 includes amino acid sequence shown in SEQ ID NO.6.
In antibody TRN1015 provided in an embodiment of the present invention, the sequence of SEQ ID NO.1-SEQ ID NO.6 is distinguished Are as follows:
SEQ ID NO.1:Gly Phe Gly Ile Gly Ile Tyr Thr；
SEQ ID NO.2:Ile Ser Gly Thr Ser Gln Tyr Ile；
SEQ ID NO.3:Val Ser Gly Ser Ser Leu Asp Tyr；
SEQ ID NO.4:Gln Asn Ile Gly Thr Asn；
SEQ ID NO.5:Gly Ala Ser；
SEQ ID NO.6:Tyr Gln Asn Tyr Asn Ser Pro Lys Thr.
Further, antibody TRN1015 provided in an embodiment of the present invention further includes heavy chain variable region shown in SEQ ID NO.7 With light chain variable region shown in SEQ ID NO.8, wherein the sequence of SEQ ID NO.7 are as follows: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Arg Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ser Val Ser Gly Phe Gly Ile Gly Ile Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Thr Ser Gln Tyr Ile Tyr Tyr Ala Asn Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Ser Leu Tyr Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Val Ser Gly Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Ile Ser Ser；The sequence of SEQ ID NO.8 Are as follows: Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Gly Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Gly Thr Asn Leu Asn Trp Phe Gln Gln Lys Pro Gly Thr Ala Pro Asn Leu Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Thr Phe Thr Leu Thr Ile Asp Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Tyr Gln Asn Tyr Asn Ser Pro Lys Thr Phe Gly Gln Gly Thr Lys Val Asp Leu Lys。
In antibody TRN1015 provided in an embodiment of the present invention, antibody TRN1015 can immunospecifically combine broken Wind toxin element, antibody have at least 10-6、10-7、10-8、10-9Or 10-10M to tetanus toxin.
Further, antibody TRN1015 provided in an embodiment of the present invention simultaneously further include with the amino acid sequence of antibody or Any nucleotide sequence that person encodes the antibody has the sequence of a degree of sequence identity or sequence homology.Specifically Ground, antibody TRN1015 provided in an embodiment of the present invention further include having and at least 80% sequence of sequence shown in SEQ ID NO.1 simultaneously The VH CDR1 of the amino acid sequence of the column phase same sex, and/or have identical as at least 80% sequence of sequence shown in SEQ ID NO.4 The VL CDR1 of the amino acid sequence of property；And/or with the ammonia at least 80% sequence identity of sequence shown in SEQ ID NO.2 The VH CDR2 of base acid sequence, and/or with the amino acid sequence at least 80% sequence identity of sequence shown in SEQ ID NO.5 The VL CDR2 of column；And/or with the VH at least amino acid sequence of 80% sequence identity of sequence shown in SEQ ID NO.3 CDR3, and/or with the VL CDR3 at least amino acid sequence of 80% sequence identity of sequence shown in SEQ ID NO.6.
Such as: have SEQ ID NO.1 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VH CDR1；
With SEQ ID NO.2 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VH CDR2；
With SEQ IDNO.3 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VH CDR3；
With SEQ ID NO.5 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VL CDR1；
With SEQ ID NO.6 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VL CDR2；
And have SEQ ID NO.7 at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the higher order column phase same sex amino acid sequence VL CDR3.
Antibody TRN1015 provided in an embodiment of the present invention is preferably full humanized antibody, including scFv (single Chain antibody fragment, scFv), Fab, Fab ', F (ab ') 2, Fv, dsFv, double antibody (diabody), Fd or Fd ' segment.But antibody TRN1015 provided in an embodiment of the present invention is not limited to above-mentioned full humanized antibody.Further, originally The antibody TRN1015 that inventive embodiments provide further includes containing peptide linker.More preferably, peptide linker includes about 1-50 amino Acid.
Antibody TRN1015 provided in an embodiment of the present invention further includes conjugate, which includes to be conjugated to antibody The label of TRN1015 can such as be conjugated to polyethylene glycol (PEG).Further, antibody TRN1015 provided in an embodiment of the present invention is also Including therapeutic agent or diagnosticum, e.g., diagnosticum includes but is not limited to enzyme, fluorescent chemicals, electron transfer agent and radioactive label.
The embodiment of the present invention also provides a kind of composition, and the composition includes antibody TRN1015.The embodiment of the present invention provides Composition can be diagnosis composition, or pharmaceutical composition.However, being diagnosis composition or pharmaceutical composition It include antibody TRN1015.
When the composition of the embodiment of the present invention is diagnosis composition, which further includes accommodating antibody The container of TRN1015.Further, which further includes conjugate, wherein conjugate includes to be conjugated to antibody The label of TRN1015, such as detectable part/reagent.Label is directly conjugated to antibody by covalent bond or non-covalent bond On TRN1015.In addition, label can be conjugated to antibody TRN1015 using one or more connection compounds, wherein for that will mark It is well known to those skilled in the art that label, which are conjugated to the technology of antibody TRN1015,.Detectable part/examination as label Agent is preferably selected from by enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emission material One of the group that the paramagnetic metal ion of material and on-radiation forms.
When the composition of the embodiment of the present invention is pharmaceutical composition, which further includes pharmaceutically acceptable Carrier, wherein pharmaceutically acceptable carrier, which refers to, will not cause organism significantly to stimulate and will not eliminate applied chemical combination The bioactivity of object and the carrier of property or diluent, e.g., physiological saline, sterile water, Ringer's solution, buffer salt solution, Portugal The mixture of grape sugar juice, maltodextrin solution, glycerol, ethyl alcohol and two of them or more.If desired, the present invention is real Apply example offer pharmaceutical composition can also include other conventional additives, as antioxidant, buffer, bacteriostatic agent, dispersing agent, Surfactant, adhesive and lubricant, and be configured to injectable formulation, such as aqueous solution, suspension and emulsion, pill, glue Wafer, granule and tablet.
The embodiment of the present invention also provides a kind of nucleic acid molecules, which can encode provided in an embodiment of the present invention anti- Body TRN1015.
The embodiment of the present invention also provides a kind of expression vector, which includes above-mentioned nucleic acid molecules.The present invention is implemented The expression vector that example provides further includes the expression regulation sequence being operatively connected with sequence of nucleic acid molecules.Wherein, the expression carries Body include in prokaryotic cell carry out gene expression carrier, in eukaryocyte carry out gene expression carrier and other The carrier that gene expression is carried out in cell such as mammalian cell, plant cell, yeast cells, insect cell, imports phagocytosis Body, plasmid, clay, minichromosome, virus or retrovirus vector polynucleotides.
The embodiment of the present invention also provides a kind of recombinant cell, which includes above-mentioned nucleic acid molecules or expression vector. Recombinant cell can be prokaryotic cell, eukaryocyte, and such as the bacterium including Escherichia coli, streptomycete and salmonella typhimurium is thin Those skilled in the art can be used in the art in born of the same parents, yeast cells, fungal cell, zooblast and plant cell etc. The known any cell that can be used as mammalian host cell.
The embodiment of the present invention also provides a kind of kit, which includes above-mentioned antibody TRN1015 or composition, or Nucleic acid molecules or expression vector or recombinant cell.Kit provided in an embodiment of the present invention can be used in detecting tetanus toxin Infection and infection due to Clostridium tetani.Specifically, kit provided in an embodiment of the present invention can be measured by antibody TRN1015 Tetanus toxin in fluid, cell or tissue sample is horizontal.Meanwhile kit provided in an embodiment of the present invention can also be by institute Tetanus toxin level is measured compared with control level, wherein what is measured is broken compared with the control level of tetanus toxin The raising of wind endotoxin level represents tetanus toxin infection.Preferably, the cell or tissue sample is obtained from human subjects, institute Stating cell or tissue sample includes for blood, urine, saliva, lung phlegm, lavation or lymph sample.
The embodiment of the present invention also provides a kind of application method, which includes:
Antibody TRN1015 is in preparation by the application in the drug of tetanus toxin infection and infection due to Clostridium tetani；With/ Or,
Antibody TRN1015 answering in the kit of preparation detection tetanus toxin infection and infection due to Clostridium tetani With；And/or
Composition is in preparation treatment by the application in the drug of tetanus toxin infection and infection due to Clostridium tetani；With/ Or,
Application of the composition in the kit of preparation diagnosis tetanus toxin infection and infection due to Clostridium tetani；With/ Or,
Nucleic acid molecules are in the drug for the disease that preparation treatment is mediated by tetanus toxin infection and clostridium tetani Using；And/or
Expression vector is in the drug for the disease that preparation treatment is mediated by tetanus toxin infection and clostridium tetani Using；And/or
Recombinant cell is in the drug for the disease that preparation treatment is mediated by tetanus toxin infection and clostridium tetani Using.
The neutralizing antibody (antibody TRN1015) of anti-tetanus toxin provided by the invention can specifically bind broken wind toxin Element, thus there is extremely strong neutralization activity and very high affine activity, and dosage is low.Anti-tetanus toxin provided by the invention The disease or disease condition that neutralizing antibody can be used in preventing and treating tetanus toxin infection and clostridium tetani mediates Isolated antibody, while can also be used to diagnose and/or monitor tetanus toxin infection and the separation of infection due to Clostridium tetani Antibody.In addition, the neutralizing antibody of anti-tetanus toxin provided by the invention is polluted without exogenous virus, it is widely portable to various Crowd.
With reference to the accompanying drawings and examples to the extraction of antibody TRN1015 provided in an embodiment of the present invention, purifying and in It is described in further detail with performance, affine dynamic performance and protection in vivo.The reality of actual conditions is not specified in embodiment Proved recipe method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The screening and purifying of 1 selected by flow cytometry apoptosis B cell of embodiment and anti-tetanus toxin antibody
1, PBMC separation and thick liquid cell sorting
15ml venous blood is acquired with the volunteer of protection antibody from having injected tetanus vaccine and having produced, and is placed in In anticoagulant tube containing heparin.15ml blood sample is isolated into PBMC (peripheral blood with Ficoll (ficoll) Mononuclear cell, peripheral blood mononuclear cells).PBMC count after using BD FACSria flow cytometer from PBMC into The single B cell sorting of row, is placed in 96 hole PCR (Polymerase Chain Reaction, polymerase for the intact B cell of form Chain reaction) in plate, each hole is made to contain a B cell.It is -80 DEG C of refrigerators that the 96 hole PCR plates for being equipped with B cell, which are placed in temperature, In, it saves backup.
2, separation antibody variable region gene
By containing single B cell 96 hole PCR plates in be added 0.5 μM each subtype heavy chain and light chain constant region primers with And Superscript III reverse transcriptase, 1h is incubated under the conditions of temperature is 37 DEG C.After incubation, according to 95 DEG C of 15min； 95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30cycles；72℃10min；4 DEG C of 5min conditions carry out PCR amplification, are expanded Product cDNA.Amplified production cDNA is placed under conditions of temperature is -20 DEG C and saves.
PCR separation antibody gene: the RT (reverse transcription, reverse transcription) containing 5 μ l in 50uL system is anti- Answer product, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), each subtype heavy chain of dNTPs and 0.5uM with The specific primer of light chain antibody, reaction condition: then 94 DEG C of 5min of initial denaturation carry out 35 PCR cycles, each circulation are as follows: 94 DEG C × 30s, 55 DEG C × 30s, 72 DEG C × 50s, finally with 72 DEG C of extension 7min, obtain pcr amplification product.
3, the expression vector of recombinant antibodies is constructed
2 μ l pcr amplification products are taken to detect through 1% agarose gel electrophoresis.Gel electrophoresis is accredited as the positive, and heavy chain Pairs of antibody variable gene PCR product can be matched with light chain to be connected on pcDNA3.3 carrier using the method that TA is cloned, Connection product is converted in DH5 α competent bacteria, 37 DEG C of overnight incubations on the plate containing ampicillin, immediately picking 10 single colonies carry out PCR with specific primer, reaction condition: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C are annealed 30s, 72 DEG C of extension 1min40s, 28 circulations, last 72 DEG C re-extend 5min；Take 5 μ l PCR products in 1% Ago-Gel Upper carry out electrophoresis detection, identifies the transformant containing heavy chain of antibody or light chain gene in positive transformant.
4, antibody expression
The plasmid of the positive antibody heavy chain and light chain gene massive amplification in bacillus coli DH 5 alpha will be expressed, recombination matter is carried out Grain rapidly extracting.With 293 cell of transfection reagent PolyFect cotransfection, concrete operations are referring to specification.6-8h renews after transfection Fresh culture medium, and in 37 DEG C of 8%CO296h is cultivated in incubator, is collected cell conditioned medium and is detected.
5, the selective mechanisms of antibody are expressed
Using tetanus vaccine as antigen, and 96 hole elisa plates, every hole will be coated with after 0 times of antigen 1 dilution with coating buffer 4 DEG C of 100uL is coated with overnight, closes 2 h with confining liquid room temperature.It is incubated for the transient transfection supernatant of 100uL as 37 DEG C of primary antibody 2h uses HRP/anti-His-tag (1:2000 dilution) as 37 DEG C of incubation 1h of secondary antibody, 100 hole μ l/ of substrate developing solution is added, often After warm avoid light place 5min, with 2M sulfuric acid stopped reaction, tested and analyzed with 450nm wavelength.
6, antibody great expression and purifying
293 cell of expression vector cotransfection of the heavy chain of antibody for having neutralization activity and light chain of experimental identification will be neutralized, is turned 6-8h changes fresh culture after dye, and in 37 DEG C of 8%CO296h is cultivated in incubator.Collect transfection supernatant, 4000rpm centrifugation 1h is purified using albumen (Protein) A affinity chromatography.Antibody is examined using SDS-PAGE and Western Blot Expression and purifying situation.
7, result
As a result as shown in Figure 1, 2, confirm that obtaining purer purpose resists by SDS-PAGE method and Western Blot method Body, antibody light chain and heavy chain after unwinding can be obviously observed.
The neutralization activity of embodiment 2ELISA detection antibody TRN1015
1, experimentation
Using tetanus normaltoxin as antigen, and 96 hole elisa plates, every hole will be coated with after 0 times of antigen 1 dilution with coating buffer In containing the overnight coating that volume is 100 μ, temperature is 4 DEG C, close 2h with confining liquid room temperature.By the antibody of expression and purification TRN1015 is used as 37 DEG C of incubation 2h of primary antibody after being serially diluted, and uses HRP/anti-His-tag (1:2000 dilution) as two Resist 37 DEG C of incubations 1h, room temperature avoid light place 5min after addition substrate developing solution, with 2M sulfuric acid stopped reaction, with 450nm wavelength into Row tests and analyzes.
2, result
As a result as shown in figure 3, the antibody of expression and purification carries out after 10000 dilutions (antibody concentration about are as follows: 0.0002 μ g/ Ml), antibody TRN1015 still is able to neutralize tetanus normaltoxin, therefore, antibody TRN1015 provided in an embodiment of the present invention With extremely strong neutralization activity.
The affine activity dynamics of 3 antibody TRN1015 of embodiment are analyzed
1, prize law is tested
Affinity of antibody test uses prize law, and buffer HBS-EP, CM5 chip is first coupled with proteinA. Then dilution capture antibody makes its concentration 1ug/ml, binding time 50s.By analyte tetanus element element to gradually increase Concentration flows successively through chip, respectively obtains signal curve.Each concentration is recycled as 1, uses 3M MgCl2 after completing 1 circulation Regeneration chip is to be returned to the original state for not capturing antibody, reproduction time 30s.With BiaCore X-100System software Obtained signal curve is analyzed, the affine activity of neutralizing antibody and tetanus toxin provided in an embodiment of the present invention is obtained Detection figure.
4, result
As a result as shown in Figure 4 and Table 1, neutralizing antibody TRN1015 is to the affinity of tetanus toxin up to 10-9The quantity of mol Grade shows that antibody TRN1015 provided in an embodiment of the present invention has very high affine activity；Moreover, the antibody and between In conjunction with rate (ka) be 3.88E+04Ms-1, dissociation rate (kd) reach 9.40E-05s-1, show the antibody combination/compound Object is with good stability；Also, the antibody concentration has carried out repetitive cycling twice when being 4ug/mL, analysis result is very Close, repetition shows the stability of experiment condition and instrument state, it was demonstrated that the reliability of testing result.
Table 1: the affine Activity determination result of antibody TRN1015 and tetanus toxin
The protection test in vivo of 4 antibody TRN1015 of embodiment
1, the measurement (LD of median lethal dose50)
Prepared tetanus toxin is successively diluted 10 with dilution2, 103, 104, 105, 106, 107, each dilution At least dilute 2ml, take 0.2ml inject mouse, every group 4.Observation 5 days.LD is calculated according to experimental result50, experimental group difference Use 20 times and 60 times of LD50Dosage.
2, TRN1015 detects the protection of small white mouse
Experiment is divided into 3 groups, every group of 4 mouse, every mouse injects 0.4ml, wherein negative control group includes 0.2ml Toxin+0.2ml borate buffered saline；Positive controls include 0.2ml toxin+0.2ml antitoxin；Experimental group includes 0.2ml Toxin+0.2ml monoclonal antibody.
The normal antitoxin diluted is quantitatively drawn in mixing and the monoclonal antibody to be checked of different dilutions is respectively charged into small test tube, The dilution test toxin of equivalent is added in every pipe, is uniformly mixed, jumps a queue, and 37 DEG C of incubation 1h carry out subcutaneous abdomen to small white mouse immediately Injection.When mixing, the suction pipe for drawing normal antitoxin, monoclonal antibody to be checked and toxin must not be used with.
3, result
As a result as shown in Figure 5,6, when the attack of the tetanus toxin of the LD50 agent at 20 times, negative control group mouse for 24 hours- 48h is all dead, positive controls mouse and each experimental group (0.62ug/ml, 1.85ug/ml, 5.56ug/ml, 16.67ug/ml With 50ug/ml dosage) mouse all survives in 7 days；Under the tetanus toxin attack of the LD50 agent at 60 times, negative control group is small For mouse in all death interior for 24 hours, dosage is that the experimental mice of 0.62ug/ml observes whole death, dosage in 120h The experimental mice of 1.85ug/ml is then all dead in 144h, experimental group (5.56ug/ml, 16.67ug/ of middle and high dosage Ml and 50ug/ml dosage) mouse all survives in 7 days.Show that antibody TRN1015 provided in an embodiment of the present invention and standard are anti- The potency (10IU/ml) of toxin quite, can be effectively protected the attack of animal defense lethal dose tetanus toxin, anti-with standard The protectiveness of toxin is almost the same.Meanwhile the actual amount of antibody TRN1015 provided in an embodiment of the present invention is anti-far below standard Toxin, this shows its effect more preferably than normal antitoxin.Thereby, it is possible to illustrate antibody TRN1015 provided in an embodiment of the present invention The vivotoxin of mouse can be effectively neutralized, mouse is protected.
Those skilled in the art will readily occur to of the invention its after considering specification and the disclosure invented here of practice Its embodiment.This application is intended to cover any variations, uses, or adaptations of the invention, these modifications, purposes or Person's adaptive change follows general principle of the invention and including the undocumented common knowledge in the art of the present invention Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are by following Claim is pointed out.
It should be understood that the relational terms of such as " first " and " second " or the like be used merely to an entity or Operation is distinguished with another entity or operation, and without necessarily requiring or implying between these entities or operation, there are any This actual relationship or sequence.The present invention is not limited to the precise structure already described above and shown in the accompanying drawings, And various modifications and changes may be made without departing from the scope thereof.The scope of the present invention is only limited by the attached claims System.
Sequence table
<110>Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd
Guangzhou Tylenol enlightening Biotechnology Co., Ltd
<120>a kind of neutralizing antibody of anti-tetanus toxin and application
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Claims (9)

1. a kind of neutralizing antibody of anti-tetanus toxin characterized by comprising
Variable heavy chain domain comprising VH CDR1, VH CDR2 and VH CDR3 and include VLCDR1, VLCDR2 and VLCDR3 Variable light chain domain, wherein
The amino acid sequence of the VH CDR1 is as shown in SEQ ID NO.1；
The amino acid sequence of the VH CDR2 is as shown in SEQ ID NO.2；
The amino acid sequence of the VH CDR3 is as shown in SEQ ID NO.3；
The amino acid sequence of the VL CDR1 is as shown in SEQ ID NO.4；
The amino acid sequence of the VL CDR2 is as shown in SEQ ID NO.5；
The amino acid sequence of the VL CDR3 is as shown in SEQ ID NO.6.
2. neutralizing antibody according to claim 1, which is characterized in that further include weight chain variable shown in SEQ ID NO.7 Light chain variable region shown in area and SEQ ID NO.8.
3. neutralizing antibody according to claim 1, which is characterized in that the neutralizing antibody immunospecifically combines broken Wind toxin element A segment and/or C segment and/or neutralization tetanus toxin, the neutralizing antibody have at least 10-6M to described broken The affinity of wind toxin element and/or the clostridium tetani.
4. a kind of composition, which is characterized in that the neutralization including anti-tetanus toxin of any of claims 1-3 is anti- Body.
5. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecule encoding is of any of claims 1-3 resist it is broken The neutralizing antibody of wind toxin element.
6. a kind of expression vector, which is characterized in that including the nucleic acid molecules described in claim 5.
7. a kind of recombinant cell, which is characterized in that including the nucleic acid molecules or table as claimed in claim 6 described in claim 5 Up to carrier.
8. a kind of kit, which is characterized in that the neutralization including anti-tetanus toxin of any of claims 1-3 is anti- Nucleic acid molecules described in body or composition as claimed in claim 4 or claim 5 or expression as claimed in claim 6 carry Body or recombinant cell as claimed in claim 7.
9. a kind of application method characterized by comprising
The neutralizing antibody of anti-tetanus toxin of any of claims 1-3 preparation by tetanus toxin infection and Application in the drug of infection due to Clostridium tetani；And/or
The neutralizing antibody of anti-tetanus toxin of any of claims 1-3 preparation detection tetanus toxin infection with And the application in the kit of infection due to Clostridium tetani；And/or
Composition as claimed in claim 4 is in preparation treatment by the drug of tetanus toxin infection and infection due to Clostridium tetani Application；And/or
Composition as claimed in claim 4 is in the kit of preparation diagnosis tetanus toxin infection and infection due to Clostridium tetani Application；And/or
The disease that nucleic acid molecules described in claim 5 are mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application；And/or
The disease that expression vector as claimed in claim 6 is mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application；And/or
The disease that recombinant cell as claimed in claim 7 is mediated in preparation treatment by tetanus toxin infection and clostridium tetani Drug in application.
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CN201810420730.5A CN108623681B (en) 2018-05-04 2018-05-04 A kind of neutralizing antibody of anti-tetanus toxin and application
PCT/CN2018/090488 WO2019210552A1 (en) 2018-05-04 2018-06-08 Neutralizing antibody against tetanus toxin and use thereof
AU2018395100A AU2018395100B2 (en) 2017-12-29 2018-12-28 Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof
EP18896977.8A EP3733699A4 (en) 2017-12-29 2018-12-28 FULLY HUMANIZED MONOCLONAL NEUTRALIZING ANTIBODY FOR TETANUS TOXIN AND ITS USE
US16/958,213 US11725046B2 (en) 2017-12-29 2018-12-28 Human neutralizing anti-tetanus toxin monoclonal antibody and its applications
JP2020555284A JP7368670B2 (en) 2017-12-29 2018-12-28 Completely natural human neutralizing monoclonal antibody against tetanus toxin and its application
BR112020013094-0A BR112020013094A2 (en) 2017-12-29 2018-12-28 TOTALLY HUMAN NATURAL NEUTRALIZING MONOCLONAL ANTIBODY AGAINST TETANIC TOXIN AND ITS APPLICATIONS
PCT/CN2018/124958 WO2019129214A1 (en) 2017-12-29 2018-12-28 Completely humanized monoclonal neutralizing antibody for tetanus toxin and application thereof

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CN112225813B (en) * 2020-10-21 2021-12-21 北京智仁美博生物科技有限公司 Antibodies against tetanus toxin and uses thereof

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CN1634991A (en) * 2003-12-30 2005-07-06 龚小迪 Human anti-tetanotoxin monoclonal antibody, method for preparation and application
CN101220096A (en) * 2003-12-30 2008-07-16 龚小迪 Preparation and application of humanized anti-spasmotoxin monoclone antibody
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