WO2019128121A1 - 抗破伤风毒素中和抗体及其制备与应用 - Google Patents

抗破伤风毒素中和抗体及其制备与应用 Download PDF

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WO2019128121A1
WO2019128121A1 PCT/CN2018/090491 CN2018090491W WO2019128121A1 WO 2019128121 A1 WO2019128121 A1 WO 2019128121A1 CN 2018090491 W CN2018090491 W CN 2018090491W WO 2019128121 A1 WO2019128121 A1 WO 2019128121A1
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antibody
tetanus toxin
antigen
amino acid
infection
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PCT/CN2018/090491
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English (en)
French (fr)
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廖化新
王月明
袁晓辉
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珠海泰诺麦博生物技术有限公司
广州泰诺迪生物科技有限公司
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Publication of WO2019128121A1 publication Critical patent/WO2019128121A1/zh

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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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the invention belongs to the field of cellular immunology and genetic engineering, and relates to an anti-tetanus toxin neutralizing antibody, and preparation and application thereof.
  • Tetanus disease is an acute disease characterized by invasive body wounds by Clostridium tetani, which breed under anaerobic conditions and produce neurotropic exotoxin, which causes systemic muscle tonic spasm. Severe patients may die from a sore throat or secondary severe lung infection; neonatal and severe patients, the mortality rate can be as high as 20-50%; about 1 million people worldwide die from tetanus each year, most of them occur in underdeveloped countries Or area. The clinical features of tetanus can be divided into four categories: local tetanus, head tetanus, systemic tetanus, and neonatal tetanus.
  • Clostridium tetani is a strict anaerobic bacterium, its spores can resist high temperature and anti-drying, and are resistant to most preservatives, but sensitive to iodine aqueous solution and neutral glutaraldehyde solution, can be killed by these reagents in a short time.
  • Tetanus can produce two exotoxins: one is tetanus hemolytic toxin with hemolysis; the other is tetanus toxin, commonly known as tetanus toxin.
  • Tetanus toxin is a protein produced by Clostridium tetanus and secreted outside the bacterium. It consists of 1315 amino acids with a relative molecular mass of 150,700 Da. It is estimated that the lethal dose to human is 0.25 ng/kg. Tetanus toxin is expressed in the bacterium as a single protein chain that is cleaved by proteases into light and heavy chains linked by disulfide bonds during secretion. According to the role of toxins in the body, tetanus toxin molecules are divided into three parts: A, B, and C. The light chain fragment of the toxin is A fragment, half of the heavy chain N-terminus is B fragment, and the other half is C fragment.
  • the process of tetanus toxin action is generally three steps of binding, introduction and action.
  • the results show that the C fragment of the toxin can bind to the receptor of the toxin.
  • the receptor of the toxin is generally considered to be a ganglioside.
  • the C fragment of the toxin has the function of retrograde axonal transport into the central nervous system and has been used to study subunits.
  • Vaccine can form an ion channel on the artificial phospholipid membrane, and introduce the active fragment of the toxin into the cell;
  • the A fragment molecule of the toxin is a Zn protease, which has protease activity and can cleave the neurotransmitter-transmitting protein on the nerve cell membrane- The vesicle-associated membrane protein, thereby inhibiting the release of neurotransmitters, allows the impulse of excitement to be transmitted continuously, leading to the clinical symptoms of tonic spasm in patients.
  • tetanus toxin is highly toxic, easy to form spores for propagation, complex in culture and separation, low recovery, and dangerous.
  • Humanized/human-derived antibodies prepared by genetic engineering can reduce or eliminate allergic reactions caused by heterologous serum proteins, and overcome the problems of insufficient blood source and potential virus contamination of human immunoglobulin production. The main direction.
  • isolated antibodies or antigen-binding fragments thereof for use in the prevention and treatment of tetanus toxin infections and conditions or disease conditions mediated by Clostridium tetani. Also provided herein are isolated antibodies or antigen-binding fragments thereof for use in diagnosing and/or monitoring tetanus toxin infection and Clostridium tetani infection. Provided herein are isolated antibodies or antigen-binding fragments thereof that immunospecifically bind to and neutralize tetanus toxin.
  • the antibodies and antigen-binding fragments provided herein can specifically bind to a tetanus toxin A fragment and/or a C fragment protein and neutralize tetanus toxin.
  • Provided herein are fully human-derived isolated antibodies or antigen-binding fragments thereof that immunospecifically bind to tetanus toxin, which are free of exogenous virus contamination and are widely applicable to a wide variety of populations.
  • a first aspect of the invention provides an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:
  • variable heavy domain comprising: a VH CDR1 consisting of the amino acid residue sequence set forth in SEQ ID NO. 1; a VH CDR2 comprising the amino acid residue set forth in SEQ ID NO. a sequence consisting of a VH CDR3 consisting of the amino acid residue sequence set forth in SEQ ID NO. 3;
  • variable light chain domain comprising: VLCDR1 consisting of the amino acid sequence set forth in SEQ ID NO. 5; VLCDR2, said VLCDR2 consisting of the amino acid sequence set forth in SEQ ID NO. 6; and VLCDR3, said The VL CDR3 consists of the amino acid sequence set forth in SEQ ID NO.
  • variable heavy chain domain has the amino acid sequence set forth in SEQ ID NO. 9
  • variable light chain domain has the amino acid sequence set forth in SEQ ID NO.
  • anti-tetanus toxin antibody or antigen-binding fragment comprises:
  • amino acid sequence is set forth in SEQ ID NO. 4.
  • the light chain variable region has the amino acid sequence as shown in SEQ ID NO.
  • the antibody or antigen-binding fragment thereof immunospecifically binds to a tetanus toxin A fragment and/or a C fragment and neutralizes tetanus toxin, the antibody or antigen-binding fragment having at least 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 M affinity for tetanus toxin A fragment and / or C fragment or for tetanus toxin.
  • the invention also encompasses sequences having a certain degree of sequence identity or sequence homology to the amino acid sequence of the antibody or antigen-binding fragment or any nucleotide sequence encoding the antibody, for example having at least SEQ ID NO. VH CDR1 of an amino acid sequence of at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity; having SEQ ID NO.2 VH of an amino acid sequence having at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity CDR2; having at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity of SEQ ID NO.
  • VH CDR3 of the amino acid sequence; having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of SEQ ID NO. VL CDR1 of a high sequence identity amino acid sequence; having at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 9 of SEQ ID NO. VL CDR2 of an amino acid sequence of 6%, 97%, 98%, 99% or higher sequence identity; and at least about 80%, 90%, 91%, 92%, 93% of SEQ ID NO. VL CDR3 of the amino acid sequence of 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the anti-tetanus toxin antibody or antigen-binding fragment of the invention is preferably a fully humanized antibody, including but not limited to, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv, dsFv, double A diabody, Fd or Fd' fragment; said antibody or antigen-binding fragment further comprising a peptide linker, preferably said peptide linker comprises between about 1 and 50 amino acids.
  • An isolated antibody or antigen-binding fragment thereof of the invention comprises a conjugate, such as conjugated to polyethylene glycol (PEG); the antibody or antigen-binding fragment provided herein comprises a therapeutic or diagnostic agent, and exemplary diagnostic agents include Not limited to enzymes, fluorescent compounds, electron transfer agents, and radioactive labels.
  • PEG polyethylene glycol
  • a second aspect of the invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the first aspect of the invention.
  • a third aspect of the invention provides a diagnostic composition for tetanus toxin infection and Clostridium tetani infection, comprising:
  • An antibody or antigen-binding fragment of the first aspect of the invention and/or a container.
  • the diagnostic composition comprises a conjugate comprising a tag conjugated to the antibody or antigen-binding fragment of the first aspect of the invention.
  • a fourth aspect of the invention provides a DNA molecule comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention.
  • a fifth aspect of the invention provides an expression vector, which comprises the DNA molecule sequence of the fourth aspect of the invention, and an expression control sequence operably linked to the sequence.
  • the expression vector includes, but is not limited to, a vector for gene expression in prokaryotic cells, and a vector for gene expression in eukaryotic cells.
  • a sixth aspect of the invention provides a recombinant cell comprising the DNA molecule of the fourth aspect of the invention, or the expression vector of the fifth aspect of the invention.
  • the recombinant cells may be prokaryotic cells or eukaryotic cells.
  • a seventh aspect of the invention provides the application of any of the following, comprising:
  • the kit for detecting tetanus toxin infection and Clostridium tetani infection comprising (a) determining a tetanus toxin in a fluid, cell or tissue sample using the antibody or antigen-binding fragment thereof provided herein Level; (b) Comparison of the measured levels of tetanus toxin with the control level, wherein an increase in the level of tetanus toxin determined represents a tetanus toxin infection compared to the control level of tetanus toxin.
  • the cell or tissue sample is obtained from a human subject, including but not limited to blood, urine, saliva, pulmonary sputum, lavage or lymphoid samples.
  • TRN0012 anti-tetanus toxin neutralizing antibodies or antigen-binding fragments thereof
  • Figure 1 is a graph showing the results of SDS-PAGE and Western Blot detection of expressed antibodies; wherein, Figure A is an SDS-PAGE diagram; and Figure B is a Western Blot diagram;
  • Figure 2 is a diagram showing the detection of antigen binding activity of the antibody of the present invention.
  • Figure 3 is a graph showing the affinity activity of the antibody of the present invention and tetanus toxin;
  • Figure 4 is a graph showing the protective effect of the antibody of the present invention on mice in vivo.
  • antibody refers to a protein molecule that functions as a receptor that specifically recognizes an antigen, including immunoglobulin molecules that immunoreact with a particular antigen, including whole antibodies, dimeric, trimeric, and multimeric antibodies; Bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof, as long as the antibody comprises an antigen binding region.
  • IgA, IgD, IgE, IgM, and IgG have subtypes including IgG1, IgG2, IgG3, and IgG4.
  • An antibody fragment refers to a fragment that has an antigen binding effect, including Fab, Fab', F(ab')2, and Fv.
  • the Fab has a light chain and heavy chain variable region, a light chain constant region, and a first heavy chain constant region (CH1 domain), and includes an antigen binding site.
  • Fab' differs from Fab in that it has a hinge region comprising at least a cysteine residue at the C-terminal region of the heavy chain CH1 domain.
  • Fv variable fragment
  • dsFv Double-stranded Fv
  • scFv single-chain Fv
  • antibody fragments can be obtained using proteases (for example, Fab fragments can be obtained by cleaving whole antibodies with papain, and F(ab')2 fragments can be obtained by pepsin cleavage of whole antibodies).
  • proteases for example, Fab fragments can be obtained by cleaving whole antibodies with papain, and F(ab')2 fragments can be obtained by pepsin cleavage of whole antibodies.
  • antibody fragments can be constructed by genetic recombination techniques.
  • the term "monoclonal antibody” refers to an antibody molecule having a single molecular composition obtained from a population of substantially identical antibodies. This monoclonal antibody shows a single binding specificity and affinity for a particular epitope.
  • immunoglobulins have heavy and light chains. Each heavy and light chain comprises a constant region and a variable region (the region is also referred to as a "domain").
  • the light and heavy chain variable regions comprise four framework regions that are interrupted by three hypervariable regions, also referred to as “complementarity determining regions” (hereinafter referred to as "CDRs").
  • the CDR is primarily responsible for binding to the epitope of the antigen.
  • the CDRs of each chain are typically CDR1, CDR2 and CDR3, numbered consecutively from the N-terminus, and are typically also identified by the strand in which the particular CDR is located.
  • the monoclonal antibodies of the invention also include functional variants of the antibodies that bind to tetanus toxin or a fragment thereof and have neutralizing activity against the subtype or fragment.
  • a functional variant includes, but is not limited to, a chemically and/or biochemical modification, such as in vitro or in vivo, that is substantially similar in the primary structural sequence but does not comprise a parental monoclonal antibody of the invention.
  • Sex derivatives include, for example, acetylation, acylation, covalent attachment of nucleotide or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, Hydroxylation, methylation, oxidation, PEGylation, proteolytic treatment, phosphorylation, and the like.
  • the functional variant may be a monoclonal antibody comprising an amino acid sequence comprising a substitution, insertion, deletion or combination of one or more amino acids as compared to the amino acid sequence of the parent monoclonal antibody.
  • the functional variant may comprise a truncation of the amino acid sequence at one or both of the amino terminus or the carboxy terminus.
  • the functional variants according to the invention may have the same or different, higher or lower binding affinities than the parent monoclonal antibodies, but are still capable of binding to tetanus toxin or a fragment thereof.
  • a functional variant according to the invention may have an increased or decreased binding affinity for tetanus toxin or a fragment thereof as compared to a parent monoclonal antibody.
  • the amino acid sequence of the variable region including, but not limited to, the framework region, the hypervariable region, and in particular the CDR3 region is modified.
  • the light or heavy chain region comprises three hypervariable regions (including three CDRs) and a more conserved region (so called framework regions (FR)).
  • Highly variable regions include amino acid residues from the CDRs and amino acid residues from the hypervariable loop.
  • Computer algorithms known to those skilled in the art such as Gap or Bestfit, can be used to optimally align the amino acid sequences to be compared and define similar or identical amino acid residues.
  • the parental monoclonal antibody or portion thereof can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis, and site-directed mutagenesis. Or obtain functional variants by organic synthesis.
  • amino acid sequence modifications of the tetanus toxin antibodies may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an anti-tetanus toxin antibody are prepared by introducing appropriate nucleotide changes into the anti-tetanus toxin antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of an anti-tetanus toxin antibody. Any combination of deletions, insertions, and substitutions is made to arrive at the final construct, with the proviso that the final construct has the desired characteristics. Amino acid changes can also alter the post-translational process of anti-tetanus toxin antibodies, such as changing the number or location of glycosylation sites.
  • the expression vector of the present invention is not particularly limited, but may be capable of including mammalian cells (for example, human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (
  • it can be a vector comprising at least one selectable marker operably linked to a suitable promoter such that the polynucleotide can be expressed in the host cell.
  • the vector may comprise a polynucleotide introduced into a phage, plasmid, cosmid, minichromosome, viral or retroviral vector.
  • the recombinant cells for introduction into an expression vector in the present invention include prokaryotic cells and eukaryotic cells, and the above cells include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium; yeast cells; Red yeast; insect cells such as fly or Noctuida Sf9 cells; animal cells such as Chinese hamster ovary cells, SP2/0, human lymphoid mother cells, COS, NSO, 293T, Bowes melanoma cells, HT-1080, BHK ( Baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc.; and plant cells. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.
  • introducing refers to the delivery of a vector comprising a polynucleotide encoding a monoclonal antibody into a host cell.
  • This introduction can be by various methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polyamine transduce transfection, electroporation, microinjection, lipid Plastid-mediated transfection, liposome fusion, lipofection and protoplast fusion.
  • transfection refers to the delivery of a desired material into a cell by infection with viral particles.
  • the vector can be introduced into a host cell by gene bombardment. In the present invention, introduction and transfection can be used interchangeably.
  • the diagnostic composition of the invention comprises at least one detectable label, such as a detectable moiety/reagent.
  • the tag can be non-covalently conjugated to a monoclonal antibody of the invention.
  • the tag can also be directly conjugated to a monoclonal antibody by a covalent bond.
  • the tag can be conjugated to the monoclonal antibody described above using one or more ligation compounds. Techniques for conjugating a tag to a monoclonal antibody are well known to those skilled in the art.
  • the detectable moiety/agent as a label is preferably selected from the group consisting of, but not limited to, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting material, and a non-radioactive paramagnetic metal ion.
  • an enzyme e.g., a prosthetic group
  • a fluorescent material e.g., a fluorescent material
  • luminescent material e.g., a luminescent material
  • bioluminescent material e.g., a radioactive material
  • a positron emitting material e.g., positron emitting material
  • the pharmaceutical composition of the present invention includes the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound.
  • the pharmaceutically acceptable carrier which can be used to formulate the composition of the present invention in the form of a liquid solution includes physiological saline, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerin, ethanol, and a mixture of two or more. If desired, the compositions of the present invention may also contain other conventional additives such as antioxidants, buffers and bacteriostats.
  • composition of the present invention may further comprise a diluent, a dispersing agent, a surfactant, a binder and a lubricant to configure it into an injectable preparation such as an aqueous solution, a suspension and an emulsion, a pill, a capsule, a granule. And tablets.
  • the pharmaceutical compositions may be in the form of various oral or parenteral formulations.
  • the pharmaceutical compositions are formulated using conventional diluents or excipients including fillers, fillers, binders, wetting agents, disintegrating agents, and surfactants.
  • Solid oral preparations include tablets, pills, powders, granules, capsules and the like. These solid preparations can be prepared by mixing at least one compound with one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin or the like.
  • lubricants such as magnesium stearate or talc may also be employed.
  • liquid oral preparations include suspensions, solutions, emulsions and syrups and the like.
  • Formulations for parenteral administration include sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, lyophilizates, suppositories, and the like.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used as the nonaqueous solvent and suspending agent.
  • the main components of the suppository may include witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric fat, glycerin gelatin and the like.
  • the pharmaceutical composition may have any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, nonaqueous solutions, suspensions, emulsions, lyophilized preparations And suppositories.
  • compositions of the invention may be administered in a pharmaceutically effective amount.
  • “Pharmaceutically effective amount” means a reasonable benefit/risk ratio in an amount sufficient to treat the disease for any medical treatment.
  • the effective dosage level of the composition may be based on the type of the subject, the severity of the disease, the age and sex of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the time of treatment, It is determined by the drugs used in combination with the composition and other known factors in the medical field.
  • the pharmaceutical compositions of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • the composition can be administered in one or more dosage forms. Given all of the above factors, it is critical to apply the composition at a minimum amount that is capable of exhibiting maximum effect without causing side effects, which can be readily determined by one skilled in the art.
  • PBMC mononuclear cells
  • 96-well plates containing single B cells were added to 0.5 ⁇ M constant region primers of each subtype heavy chain and light chain and Superscript III reverse transcriptase, and incubated at 37 ° C for 1 h; PCR amplification was carried out under the following conditions: 95 ° C for 15 min; 95 ° C 1 min, 55 ° C 1 min, 72 ° C 1 min, 30 cycles; 72 ° C 10 min; 4 ° C 5 min.
  • the product cDNA was stored at -20 °C.
  • PCR isolation of antibody gene 50 ⁇ L system containing 5 ⁇ l of RT reaction product, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), dNTPs, and 0.5 uM specific primers for each subtype heavy chain and light chain antibody, reaction conditions: pre- Denaturation at 94 ° C for 5 min, then 35 PCR cycles, each cycle: 94 ° C ⁇ 30 s, 55 ° C ⁇ 30 s, 72 ° C ⁇ 50 s, and finally extended at 72 ° C for 7 min.
  • the PCR product was identified by 1% agarose gel electrophoresis.
  • the reaction conditions were: pre-denaturation at 94 ° C for 3 min, denaturation at 94 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min 40 s, 28 One cycle was repeated for a further 5 min at 72 ° C; 5 ⁇ l of the PCR product was electrophoresed on a 1% agarose gel, and transformants containing the antibody heavy or light chain genes were identified in the positive transformants.
  • a plasmid expressing a positive antibody heavy chain and a light chain gene was amplified in a large amount in Escherichia coli DH5 ⁇ , and a recombinant plasmid was rapidly extracted. 293 cells were co-transfected with the transfection reagent PolyFect. For details, see the instructions. Fresh medium was changed 6-8 h after transfection, and cultured in a 37 ° C 8% CO 2 incubator for 96 h, and the cell supernatant was collected for detection.
  • the tetanus vaccine was used as an antigen, and the antigen was diluted 10-fold with a coating solution, and then coated with a 96-well ELISA plate, and coated at 100 ⁇ l per well at 4 ° C overnight, and blocked with a blocking solution at room temperature for 2 h.
  • 100 ⁇ l of the transient transfection supernatant was incubated as a primary antibody at 37 ° C for 2 h, and incubated with HRP/anti-His-tag (1:2000 dilution) as a secondary antibody at 37 ° C for 1 h, and the substrate coloring solution was added at 100 ⁇ l/well. After standing for 5 min, the reaction was quenched with 2 M sulfuric acid and analyzed by a wavelength of 450 nm.
  • the neutralizing activity of the antibody heavy chain and light chain expression vector identified by the neutralization experiment were co-transfected into 293 cells, and the fresh medium was changed 6-8 hours after transfection, and cultured in a 37 ° C 8% CO 2 incubator for 96 hours. .
  • the transfected supernatant was collected, centrifuged at 4000 rpm for 1 h, and purified by Protein A affinity chromatography.
  • the expression and purification of the antibody were examined by SDS-PAGE and Western Blot.
  • the tetanus standard toxin was used as an antigen, and the antigen was diluted 10-fold with a coating solution, and then coated with a 96-well ELISA plate, and coated with 100 ⁇ l of each well at 4 ° C overnight, and blocked with a blocking solution at room temperature for 2 hours.
  • the purified antibody TRN0012 was serially diluted and incubated as a primary antibody at 37 ° C for 2 h, and incubated with HRP/anti-His-tag (1:2000 dilution) as a secondary antibody at 37 ° C for 1 h, and the substrate was added to the color development solution and protected from light at room temperature. After standing for 5 min, the reaction was quenched with 2 M sulfuric acid and analyzed by a wavelength of 450 nm.
  • the TRN0012 antibody was still able to neutralize the tetanus standard toxin and had extremely strong neutralizing activity.
  • the CM5 chip is coupled to the capture molecule to activate the surface of the dextran of the chip, and the amount of coupling is determined by the injection time.
  • the prepared human-derived anti-tetanus neutralizing antibody was used as a ligand, and the calculated signal value was used to determine the injection concentration and contact time of the monoclonal antibody.
  • the tetanus standard toxin was diluted with HBS-EP buffer as an analyte, and the analyte was sequentially flowed through the chip at a gradually increasing concentration to obtain a signal curve. Each concentration was used as one cycle, and after completion of one cycle, the chip was regenerated with 10 mmol/L of glycine-hydrochloric acid to return to the state of the original unbound antigen. Analysis was performed using BiaCore X-100 System software.
  • the neutralizing antibody TRN0012 has an affinity for tetanus toxin of the order of 10 -8 mol, indicating that the antibody has a high affinity activity.
  • the experiment was divided into 3 groups of 4 mice each, and 0.4 ml was injected into each mouse.
  • the negative control group included 0.2 ml of toxin + 0.2 ml of borate buffered saline; the positive control group included 0.2 ml of toxin + 0.2 ml.
  • mice The results are shown in Fig. 4.
  • the negative control mice all died within 24 to 48 hours, except for the experimental group with an antibody concentration of 1.85 ⁇ g/mL, and other experimental groups (0.62 ⁇ g/mL, 5.56 ⁇ g/mL, 16.67).
  • Mice in the ⁇ g/mL, 50 ⁇ g/mL and positive control groups survived, comparable to the standard antitoxin titer (10 IU/ml), and effectively protected the animal against the lethal dose of tetanus toxin attack, with standard antitoxin
  • the protection is basically the same.
  • the actual amount of the monoclonal antibody of the present invention is much lower than that of the standard antitoxin, which indicates that the effect is better than the standard antitoxin. This indicates that the antibody TRN0012 can effectively neutralize the toxins in mice and protect mice.
  • the invention provides an anti-tetanus toxin neutralizing antibody, and the preparation and application thereof, the antibody and the antigen-binding fragment provided by the invention can specifically bind the tetanus toxin A fragment and/or the C fragment protein and neutralize the tetanus toxin .
  • the present invention provides a fully human-derived isolated antibody or antigen-binding fragment thereof which immunospecifically binds to tetanus toxin, has no foreign virus contamination, can be widely applied to various populations, and has strong industrial applicability.

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Abstract

抗破伤风毒素中和体及其制备与应用，所述抗体可以用于预防、治疗和诊断破伤风感染和/或治疗破伤风梭菌感染介导的一种或多症状。还提供了产生免疫特异性地结合破伤风毒素的抗体的方法。

Description

抗破伤风毒素中和抗体及其制备与应用
本申请要求于2017年12月29日提交中国专利局、申请号为201711486693X、发明名称为“抗破伤风毒素中和抗体及其制备与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明本发明属于细胞免疫学、基因工程领域，涉及一种抗破伤风毒素中和抗体及其制备与应用。
背景技术
破伤风(Tetanus)疾病是由破伤风梭菌(Clostridium tetani)侵入机体伤口后，在厌氧环境下繁殖，产生嗜神经外毒素而引起全身肌肉强直性痉挛为特点的急性病。重型患者可因喉痉挛或继发严重肺部感染而死亡；新生儿及重型患者，病死率可高达20-50％；全球每年约有100万人死于破伤风，大部分发生在不发达国家或地区。破伤风的临床特征具体可以分为四类：局部破伤风、头部破伤风、全身性破伤风、以及新生儿破伤风。
破伤风梭菌为严格厌氧菌，其芽胞可抗高温抗干燥，对大多数防腐剂由抗性，但对碘的水溶液及中性戊二醛溶液敏感，可在短时间内被这些试剂杀灭。破伤风杆菌可以产生两种外毒素：一种是具有溶血作用的破伤风溶血毒素；另一种是破伤风痉挛毒素，即常说的破伤风毒素。
破伤风毒素是由破伤风梭状杆菌产生并分泌至菌体外的一种蛋白质，由1315个氨基酸组成，相对分子质量为150700Da，推测对人的致死剂量为0.25ng/kg。破伤风毒素在菌体内表达后是单一的一条蛋白质链，在分泌过程中被蛋白酶裂解成由二硫键链接的轻链和重链。根据毒素在体内的作用，破伤风毒素分子分为A、B、C三个部分，毒素的轻链片段为A片段，重链N末端的一半为B片段，另一半为C片段。
破伤风毒素作用过程一般为结合、导入和作用三步。研究结果表明毒素的C片段可以和毒素的受体结合，毒素的受体一般认为是神经节苷脂，毒素的C片段具有逆行轴突运输进入中枢神经系统的功能，已被用于研究亚单位疫苗；B片段可以在人工磷脂膜上形成离子通道，将毒素的活性片段导入到细胞内；毒素的 A片段分子是Zn蛋白酶，具有蛋白酶活性，可以裂解神经细胞膜上的传送神经递质的蛋白质-囊泡相关膜蛋白，从而抑制神经递质的释放，使兴奋的冲动不停传递，导致患者产生强直性痉挛的临床症状。然而，从破伤风毒素中直接提取和纯化C片段亦有许多弊端：破伤风毒素毒性高，易形成芽胞进行传播，培养、分离过程复杂、回收率不高，且具有一定危险性。
最早破伤风类类毒素作为免疫制剂用于破伤风的预防和治疗，但是有相当量的不良反应发生(如致敏性强等)。20世纪60年代，欧美发达国家相继研制出人破伤风免疫球蛋白(HTIG)。我国自80年代初期开始了HTIG的生产，目前HTIG尚不能满足市场需求，主要还是由马血清TAT占领着市场，人源化的HTIG克服了马血清TAT临床使用的过敏反应等不良反应，大大提升了破伤风的防治水平。但由于人血源来源因难、价格昂贵，存在外源病毒污染的危险，使其工业化生产和临床应用受到了很大限制。
目前，国外已有对鼠源性单克隆抗体进行改造以制备人源化基因工程破伤风抗毒素等的研究报道，但仍处于实验室研究阶段。近年来，由于基因工程技术的发展，使利用基因工程的方法制备人源化/人源性抗体成为可能。利用基因工程制备的人源化/人源性抗体可降低或消除异种血清蛋白引起的过敏反应，又可克服人源免疫球蛋白生产的血源不足及潜在病毒污染的可能等问题，成为目前研究的主要方向。
发明内容
为了弥补现有技术的不足，本文提供用于预防和治疗破伤风毒素感染以及破伤风梭菌介导的疾病或疾病状况的分离的抗体或其抗原结合片段。本文还提供用于诊断和/或监测破伤风毒素感染以及破伤风梭菌感染的分离的抗体或其抗原结合片段。本文提供免疫特异性地结合并中和破伤风毒素的分离的抗体或其抗原结合片段。本文所提供的抗体和抗原结合片段可以特异性结合破伤风毒素A片段和/或C片段蛋白以及中和破伤风毒素。本文提供免疫特异性地结合破伤风毒素的全人源的分离的抗体或其抗原结合片段，无外源病毒污染，可广泛适用于各种人群。
本发明的第一方面提供了抗体或其抗原结合片段，所述抗体或其抗原结合片段包括：
可变重链结构域，包含：VH CDR1，所述VH CDR1由SEQ ID NO.1所示的氨基酸残基序列组成；VH CDR2，所述VH CDR2由SEQ ID NO.2所示的氨基酸残基序列组成；VH CDR3，所述VH CDR3由SEQ ID NO.3所示的氨基酸残基序列组成；以及
可变轻链结构域，包含：VLCDR1，所述VLCDR1由SEQ ID NO.5所示的氨基酸序列组成；VLCDR2，所述VLCDR2由SEQ ID NO.6所示的氨基酸序列组成；以及VLCDR3，所述VL CDR3由SEQ ID NO.7所示的氨基酸序列组成。
进一步，可变重链结构域具有如SEQ ID NO.9所示的氨基酸序列，可变轻链结构域具有如SEQ ID NO.10所示的氨基酸序列。
进一步，所述抗破伤风毒素抗体或抗原结合片段包含：
重链可变区，氨基酸序列如SEQ ID NO.4所示；以及
轻链可变区，氨基酸序列如SEQ ID NO.8的所示。
进一步，所述抗体或其抗原结合片段免疫特异性地结合破伤风毒素A片段和/或C片段并中和破伤风毒素，所述抗体或抗原结合片段具有至少10 -6、10 -7、10 -8、10 -9或10 -10M的对破伤风毒素A片段和/或C片段或对破伤风毒素的亲和力。
本发明同时涵盖与所述的抗体或抗原结合片段的氨基酸序列或者编码该抗体的任何核苷酸序列具有一定程度的序列一致性或序列同源性的序列，例如具有SEQ ID NO.1至少或至少约80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VH CDR1；具有SEQ ID NO.2至少或至少约80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VH CDR2；具有SEQ IDNO.3至少或至少约80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VH CDR3；具有SEQ ID NO.5至少或至少约80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VL CDR1；具有SEQ ID NO.6至少或至少约80％、90％、91％、 92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VL CDR2；以及具有SEQ ID NO.7至少或至少约80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高序列相同性的氨基酸序列的VL CDR3。
本发明的抗破伤风毒素抗体或抗原结合片段优选为全人源化抗体，包括(但不限于)单链Fv(scFv)、Fab、Fab′、F(ab’)2、Fv、dsFv、双抗体(diabody)、Fd或Fd’片段；所述抗体或抗原结合片段还包含肽接头，优选的，所述肽接头包含约1-50个氨基酸。
本发明分离的抗体或其抗原结合片段包括缀合物，如可缀合至聚乙二醇(PEG)；本发明提供的抗体或抗原结合片段包含治疗剂或诊断剂，示例性诊断剂包括但不限于酶、荧光化合物、电子转移剂和放射性标记。
本发明的第二方面提供了一种药物组合物，所述药物组合物包括本发明第一方面所述的抗体或抗原结合片段。
本发明的第三方面提供了一种破伤风毒素感染以及破伤风梭菌感染的诊断组合物，包括：
本发明第一方面所述的抗体或抗原结合片段；和/或容器。
进一步，所述诊断组合物包括缀合物，所述缀合物包含缀合至本发明第一方面所述的抗体或抗原结合片段的标签。
本发明的第四方面提供了一种DNA分子，所述DNA包含编码本发明个第一方面所述的抗体或其抗原结合片段。
本发明的第五方面提供了一种表达载体，所述表达载体本发明第四方面所述的DNA分子序列以及与所述序列操作性相连的表达调控序列。其中，所述表达载体包括(但不限于)在原核细胞中进行基因表达的载体、在真核细胞中进行基因表达的载体。
本发明的第六方面提供了一种重组细胞，所述重组细胞包含本发明第四方面所述的DNA分子，或本发明第五方面所述的表达载体。其中，重组细胞可以是原核细胞、真核细胞。
本发明的第七方面提供了如下任一项所述的应用，包括：
1)本发明的第一方面所述的抗体抗原结合片段在制备治疗由破伤风毒素感染以及破伤风梭菌感染的药物中的应用；
2)本发明的第一方面所述的抗体或抗原结合片段在制备检测破伤风毒素感染以及破伤风梭菌感染的试剂盒中的应用；
3)本发明的第二方面所述的权利要求4所述的药物组合物在制备治疗由破伤风毒素感染以及破伤风梭菌感染的药物中的应用；
4)本发明的第三方面所述的诊断组合物在制备诊断破伤风毒素感染以及破伤风梭菌感染的试剂盒中的应用；
5)本发明的第四方面所述的DNA分子在制备治疗由破伤风毒素感染以及破伤风梭菌介导的疾病的药物中的应用；
6)本发明的第五方面所述的表达载体在制备治疗由破伤风毒素感染以及破伤风梭菌介导的疾病的药物中的应用；
7)本发明的第六方面所述的宿主细胞在制备治疗由破伤风毒素感染以及破伤风梭菌介导的疾病的药物中的应用。
进一步，2)所述的试剂盒检测破伤风毒素感染以及破伤风梭菌感染的方法，包括(a)利用本文所提供的抗体或其抗原结合片段测定流体、细胞或组织样品中的破伤风毒素水平；(b)将所测定破伤风毒素水平与对照水平比较，其中与破伤风毒素的对照水平相比，所测定的破伤风毒素水平的提高代表破伤风毒素感染。优选的，所述细胞或组织样品获得自人类对象，所述细胞或组织样品包括(但不限于)为血液、尿、唾液、肺痰、灌洗或淋巴样品。
本文提供抗破伤风毒素中和抗体或其抗原结合片段，所述抗破伤风毒素抗体或其抗原结合片段称为TRN0012。
附图说明
图1是SDS-PAGE和Western Blot检测表达的抗体的结果图；其中，图A是SDS-PAGE图；图B是Western Blot图；
图2是本发明的抗体的抗原结合活性检测图；
图3是本发明的抗体与破伤风毒素的亲和活性检测图；
图4是体内检测本发明的抗体对小鼠的保护作用图。
具体的实施方式
除非另有定义，本文使用的所有技术术语和科学术语具有与本发明所属领域技术人员通常理解的相同的意义。除非另有说明，整个公开中提到的所有专利、专利申请、公开的申请和出版物、Genebank序列、数据库、网站和其他公开的材料整体援引加入本文。如果本文的术语有多种定义，则以本节中的定义为准。当提及URL或者其他这样的标识符或地址时，应当理解这类标识符可以改变，并且在网络上特定的信息可以交流，但是通过搜索网络可以找到等同的信息。对其引用证明这类信息的可得性和公开传播。
抗体
如本文所用，术语“抗体”是指起到特异性识别抗原的受体作用的蛋白分子，包括与特定抗原免疫反应的免疫球蛋白分子，包括整个抗体、二聚、三聚和多聚抗体；双特异性抗体；嵌合抗体；重组和工程抗体，和其片段，只要该抗体包括抗原结合区即可。
全抗体包括IgA、IgD、IgE、IgM和IgG，IgG具有包括IgG1、IgG2、IgG3和IgG4在内的亚型。抗体片段是指具有结合抗原作用的片段，包括Fab、Fab'、F(ab')2和Fv。Fab具有轻链和重链可变区、轻链恒定区和第一重链恒定区(CH1域)，并包括一抗原结合位点。Fab'与Fab的不同在于它具有铰链区，在重链CH1域的C末端区至少包括半胱氨酸残基。通过Fab'铰链区的半胱氨酸残基间的二硫键制备F(ab')2抗体。Fv(可变片段)是指仅具有重链可变区和轻链可变区的最小抗体片段。双链Fv(dsFv)具有通过二硫键连接到轻链可变区的重链可变区，单链Fv(scFv)通常具有通过肽接头连接到轻链可变区的重链可变区。可以使用蛋白酶获得此类抗体片段(例如，可以通过用木瓜蛋白酶裂解全抗体获得Fab片段，可以通过胃蛋白酶裂解全抗体获得F(ab')2片段)。优选地，可以通过基因重组技术构建抗体片段。
术语“单克隆抗体”是指具有单一分子组成的抗体分子，获自一群基本相同的抗体。该单克隆抗体显示出对特定表位的单一结合特异性和亲和性。典型地，免疫球蛋白具有重链和轻链。各重链和轻链包含恒定区和可变区(区域也称为"域")。轻链和重链可变区包含四个框架区，被三个超变区打断，也称为"互补决定区"(下文称为“CDR”)。CDR主要负责结合到抗原的表位。各链的CDR通常为CDR1、CDR2和CDR3，从N末端开始连续编号，通常也用特定CDR所处的链标识。
本发明的单克隆抗体还包括该抗体的功能性变异体，所述功能性变异体可结合至破伤风毒素或其片段，且具有抗所述亚型或片段的中和活性。
具体地，如果功能性变异体包括(但并不限于)：在一级结构序列中基本类似、但包含本发明的亲本单克隆抗体中没有的例如体外或体内的化学和/或生物化学的改性的衍生物。这些改性包括例如乙酰化、酰化、核苷酸或核苷酸衍生物的共价连接、脂质或脂质衍生物的共价连接、交联、二硫键的形成、糖基化、羟基化、甲基化、氧化、聚乙二醇化、蛋白水解处理、磷酸化等。
可选择地，功能性变异体可为如下的单克隆抗体：与亲本单克隆抗体的氨基酸序列相比，包括含有一个或多个氨基酸的取代、插入、缺失或其组合的氨基酸序列。进一步地，功能性变异体可在氨基末端或羧基末端的其中一端或两端包括氨基酸序列的截短。与亲本单克隆抗体相比，根据本发明的功能性变异体可能具有相同或不同、较高或较低的结合亲和力，但仍能够键合至破伤风毒素或其片段。例如，与亲本单克隆抗体相比，根据本发明的功能性变异体对破伤风毒素或其片段可具有升高或降低的结合亲和力。
优选地，包括但并不限于构架区域、高可变区域、尤其是CDR3区域的可变区域的氨基酸序列被改性。通常，轻链或重链区域包括三个高可变区域(包括三个CDR)和更保守的区域(所谓的构架区域(FR))。高可变区域包括来自CDR的氨基酸残基和来自高可变环的氨基酸残基。可将本领域技术人员已知的计算机算法诸如Gap或Bestfit用于最优化地比对要对比的氨基酸序列，且定义相似或相同的氨基酸残基。可通过本领域已知的通用的分子生物学方法(包括PCR、寡核苷酸定点诱变(oligonucleotide-directed mutagenesis)和定点诱变(site-directed mutagenesis))改变亲本单克隆抗体或其部分，或通过有机合成方法获 得功能性变异体。
本领域技术人员还将理解的是，本发明涵盖所述破伤风毒素抗体的氨基酸序列修饰。举例而言，可需要改良抗体之结合亲和力及/或其它生物学特性。抗破伤风毒素抗体之氨基酸序列变体是由向抗破伤风毒素抗体核酸中引入适当核苷酸变化或由肽合成制备。该等修饰包括(例如)抗破伤风毒素抗体氨基酸序列内之残基缺失及/或插入及/或取代。进行缺失、插入及取代之任何组合以达成最终构建体，其限制条件为该最终构建体具有所要特征。氨基酸变化亦可改变抗破伤风毒素抗体之转译后过程，诸如改变糖基化位点之数目或位置。
表达载体
本发明对表达载体没有特别的限制，但可以是能在包括哺乳动物细胞(例如，人、猴、兔、大鼠、仓鼠或小鼠细胞)、植物细胞、酵母细胞、昆虫细胞和细菌细胞(如大肠杆菌(E.coli))在内的真核或原核细胞内复制和/或表达多核苷酸的载体。较佳地，它可以是载体，包括至少一选择性标记，可操作地连接到合适的启动子，以致可以在宿主细胞内表达多核苷酸。例如，载体可以包括导入噬菌体、质粒、粘粒、微型染色体、病毒或反转录病毒载体的多核苷酸。
重组细胞
本发明中的用于导入表达载体的重组细胞包括原核细胞和真核细胞，上述细胞包含(但并不限于)细菌细胞，如大肠杆菌，链霉菌和鼠伤寒沙门氏菌；酵母细胞；真菌细胞如毕赤酵母；昆虫细胞如果蝇或夜蛾Sf9细胞；动物细胞，如中国仓鼠卵巢细胞，SP2/0，人淋巴样母细胞，COS，NSO，293T，Bowes黑素瘤细胞，HT-1080，BHK(幼仓鼠肾细胞)，HEK(人胚肾细胞)，PERC.6(人视网膜细胞)等；和植物细胞。在本领域中可使用本领域的技术人员已知的可用作哺乳动物宿主细胞的任何细胞。
术语“导入”是指将包括编码单克隆抗体的多核苷酸的载体递送入宿主细胞。此导入可以通过本领域中已知的各种方法，包括磷酸钙-DNA共沉淀，DEAE-葡聚糖介导的转染，聚凝胺介导的转染，电穿孔，显微注射，脂质体介导的转染，脂质体融合，脂转染和原生质体融合进行。此外，转染是指用病毒颗粒通过感染将期望材料递送入细胞。此外，该载体可以通过基因轰击导入宿主细胞。在本发 明中，导入与转染可以互换使用。
诊断组合物
本发明中的诊断组合物包括至少一种可检测的标签，诸如可检测的部分/试剂。标签可非共价地缀合至本发明的单克隆抗体。标签还可通过共价键直接缀合至单克隆抗体。可选择地，标签可利用一种或多种连接化合物缀合至上述单克隆抗体。用于将标签缀合至单克隆抗体的技术对本领域的技术人员是公知的。作为标签的可检测的部分/试剂优选为选自由(但并不限于)酶、辅基、荧光材料、发光材料、生物发光材料、放射性材料、正电子发射材料和非放射性的顺磁金属离子组成的组中的一种。
药物组合物
本发明中的药物组合物包括本发明的单克隆抗体以及药学上可接受的载体。
药学上可接受的载体是指不会对生物体引起显著刺激且不会消除所施用化合物的生物活性和性质的载体或稀释剂。
可用于将本发明组合物配制为液体溶液形式的药学上可接受的载体包括生理盐水，无菌水，林格氏溶液，缓冲盐溶液，葡萄糖溶液，麦芽糊精溶液，甘油，乙醇，和其中两种或更多种的混合物。如果需要，本发明的组合物也可含有其它常规添加剂，如抗氧化剂，缓冲剂和抑菌剂。此外，本发明组合物还可包含稀释剂，分散剂，表面活性剂，粘合剂和润滑剂，以将它配置成可注射制剂，如水性溶液，悬浮液和乳剂，丸剂，胶囊剂，颗粒剂和片剂。
药物组合物可以是各种口服或胃肠外制剂形式。使用包括填料，填充剂，粘合剂，润湿剂，崩解剂，和表面活性剂在内的常规稀释剂或赋形剂配制药物组合物。固体口服制剂包括片剂，丸剂，粉剂，颗粒剂，胶囊等。这些固体制剂可通过将至少一种化合物与一种或多种赋形剂，例如，淀粉，碳酸钙，蔗糖，乳糖，明胶等混合来制备。除了简单的赋形剂，也可使用润滑剂如硬脂酸镁或滑石。此外，液体口服制剂包括悬浮液，溶液，乳剂和糖浆等。除了通常用作简单稀释剂的水和液体石蜡外，还可包括各种赋形剂，例如，润湿剂，甜味剂，香料，防腐剂等。肠胃外给药的制剂包括灭菌水溶液，非水溶剂，悬浮剂，乳剂，冻干剂，栓剂等。丙二醇，聚乙二醇，植物油如橄榄油，可注射酯如油酸乙酯等可以用作 非水溶剂和悬浮剂。栓剂主要成分可以包括witepsol，聚乙二醇，吐温61，可可脂，月桂脂，甘油明胶等。
药物组合物可具有选自下组的任意一种制剂：片剂，丸剂，粉末，颗粒，胶囊，悬浮液，溶液，乳剂，糖浆，灭菌水溶液，非水溶液，悬浮液，乳液，冻干制剂和栓剂。
本发明的药物组合物可以药学有效量施用。“药学有效量”是指其用量足以治疗疾病，以适用于任何医学治疗的合理的利益/风险比率。组合物的有效剂量水平可以根据受试者的类型、疾病的严重程度、受试者的年龄和性别、药物活性、对药物的敏感性、给药时间，给药途径、排泄率、治疗时间、与组合物联用的药物和医疗领域中其他已知因素来确定。本发明的药物组合物可单独使用或与其它治疗剂组合施用，并且可以与常规的治疗剂依次或同时给药。可采用一种或多种剂型中施用组合物。考虑所有上述因素，在能够表现出最大效果而不引起副作用的最小量下施用组合物至关重要，该最小量可由本领域技术人员容易地确定。
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1 流式细胞仪分选B细胞以及抗破伤风毒素抗体的筛选与纯化
1、PBMC分离和浆细胞分选
从注射了破伤风疫苗并产生了保护性抗体的志愿者身上采集100ml静脉血于含有肝素的抗凝管中。将100ml血样用Ficoll分离单个核细胞(PBMC)；细胞计数后利用BD FACSria流式细胞仪从PBMC进行分选，将形态完好的单个细胞置于96孔PCR板中，使每个孔含有一个记忆性B细胞，-80℃冰箱保存备用。
2、分离抗体可变区基因
将含有单个B细胞的96孔板加入0.5μM的各亚型重链与轻链的恒定区引物与Superscript III反转录酶，37℃孵育1h；按以下条件进行PCR扩增：95℃15min； 95℃1min，55℃1min，72℃1min，30cycles；72℃10min；4℃5min。产物cDNA-20℃保存。
PCR分离抗体基因：50uL体系中含有5μl的RT反应产物，HotStarTaq Plus酶(Invitrogen，Carlsbad，CA)，dNTPs，及0.5uM的各亚型重链与轻链抗体的特异性引物，反应条件：预变性94℃5min，然后进行35个PCR循环，每个循环为：94℃×30s，55℃×30s，72℃×50s，最后用72℃延伸7min。PCR产物用1％的琼脂糖凝胶电泳进行鉴定。
3、构建重组抗体的表达载体
取2μl扩增产物经1％琼脂糖凝胶电泳检测。将凝胶电泳鉴定为阳性，且重链与轻链可匹配成对的抗体可变区基因PCR产物利用TA克隆的方法连接到pcDNA3.3载体上，将连接产物转化DH5α感受态细菌中，在含有氨苄青霉素的平板上37℃培养过夜，随即挑取10个单菌落用特异性引物进行PCR，反应条件：94℃预变性3min，94℃变性30s，55℃退火30s，72℃延伸1min40s，28个循环，最后72℃再延伸5min；取5μl PCR产物在1％琼脂糖凝胶上进行电泳检测，在阳性转化子中鉴定出了含有抗体重链或轻链基因的转化子。
4、抗体表达
将表达阳性抗体重链和轻链基因的质粒在大肠杆菌DH5α中大量扩增，进行重组质粒快速提取。用转染试剂PolyFect共转染293细胞，具体操作参见说明书。转染后6-8h换新鲜培养基，并在37℃8％CO 2培养箱中培养96h，收集细胞上清进行检测。
5、表达抗体的筛选检测
以破伤风疫苗为抗原，并用包被液将抗原10倍稀释后包被96孔ELISA板，每孔100μl 4℃过夜包被，用封闭液常温封闭2个h。将100μl的瞬时转染上清作为一抗37℃孵育2h，用HRP/anti-His-tag(1:2000稀释)作为二抗37℃孵育1h，加入底物显色液100μl/孔，常温避光放置5min后，用2M硫酸中止反应，用450nm波长进行检测分析。
6、抗体大量表达与纯化
将中和实验鉴定的有中和活性的抗体重链与轻链的表达载体共转染293细 胞，转染后6-8h换新鲜培养基，并在37℃8％CO 2培养箱中培养96h。收集转染上清，4000rpm离心1h，利用蛋白(Protein)A亲和层析法进行纯化。利用SDS-PAGE和Western Blot检验抗体的表达及纯化情况。
7、结果
结果如图1所示，证实得到较纯的目的抗体，可清晰观察到解链后的抗体轻、重链。
实施例2 ELISA检测TRN0012抗体的中和活性
以破伤风标准毒素为抗原，并用包被液将抗原10倍稀释后包被96孔ELISA板，每孔100μl 4℃过夜包被，用封闭液常温封闭2h。将表达纯化的抗体TRN0012进行系列稀释后作为一抗37℃孵育2h，用HRP/anti-His-tag(1:2000稀释)作为二抗37℃孵育1h，加入底物显色液后常温避光放置5min，用2M硫酸中止反应，用450nm波长进行检测分析。
2、结果
结果如图2所示，表达纯化的抗体进行50，000稀释后(抗体浓度约为：0.0002μg/ml)，TRN0012抗体仍然可以与中和破伤风标准毒素，具有极强的中和活性。
实施例3 TRN0012抗体的亲和活性动力学分析
1、偶联量的确定
CM5芯片偶联捕获分子，活化芯片的葡聚糖表面，以进样时间确定偶联量。
2、配体的捕获
将制备的全人源抗破伤风痉挛中和抗体作为配体，以计算得到的信号值确定单抗的进样浓度及接触时间。
3、TRN0012抗体与抗原的亲和力检测
用HBS-EP缓冲液稀释破伤风标准毒素作为分析物，将分析物以逐渐增高的浓度依次流过芯片，分别得到信号曲线。每个浓度作为1个循环，完成1次循环 后用10mmol/L的甘氨酸－盐酸再生芯片以回复到原始未结合抗原的状态。用BiaCore X-100System软件进行分析。
4、结果
结果如图3及下表所示，中和抗体TRN0012对破伤风毒素的亲和力达10 -8mol的数量级，表明该抗体具有很高的亲和活性。
Ligand Analyte ka(1/Ms) kd(1/s) KD(M)
TRN1012 Tetanus toxin 8.06E+04 3.28E-04 4.07E-09
实施例4 TRN0012抗体的体内保护实验
1、半数致死量的测定(LD 50)
将配制好的毒素用稀释液依次稀释10 2，10 3，10 4，10 5，10 6，10 7，每个稀释度至少稀释2ml，取0.2ml注射小鼠，每组4只。观察5天。根据实验结果计算出LD 50，实验组使用20倍于LD 50量。
2、TRN0012对小白鼠的保护检测
将实验分为3组，每组4只小鼠，每只小鼠注射0.4ml，其中，阴性对照组包括0.2ml毒素+0.2ml硼酸盐缓冲盐水；阳性对照组包括0.2ml毒素+0.2ml抗毒素；实验组包括0.2ml毒素+0.2ml单抗。
混合定量吸取已稀释之标准抗毒素及不同稀释度之待检单抗分别装入小试管中，每管加入等量之稀释试验毒素，混合均匀，加塞，37℃结合1h，立即对小白鼠进行腹部皮下注射。混合时，吸取标准抗毒素、待检单抗及毒素的吸管不得混用。
4、结果
结果如图4所示，阴性对照组小白鼠于24～48h之内全部死亡，而除了抗体浓度为1.85μg/mL的实验组以外，其他实验组(0.62μg/mL、5.56μg/mL、16.67μg/mL、50μg/mL)和阳性对照组的小鼠则全部存活，与标准抗毒素的效价(10IU/ml)相当，能有效地保护动物防御致死剂量破伤风毒素的攻击，与标准抗毒素的保护性基本一致。并且，本发明单抗的实际用量远低于标准抗毒素，这表明其效果比标 准抗毒素更佳。说明抗体TRN0012能够有效的中和小鼠的体内毒素，保护小鼠。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。
工业实用性
本发明提供了一种抗破伤风毒素中和抗体及其制备与应用，本发明所提供的抗体和抗原结合片段可以特异性结合破伤风毒素A片段和/或C片段蛋白以及中和破伤风毒素。本发明提供免疫特异性地结合破伤风毒素的全人源的分离的抗体或其抗原结合片段，无外源病毒污染，可广泛适用于各种人群，具有很强的工业实用性。
Figure PCTCN2018090491-appb-000001
Figure PCTCN2018090491-appb-000002
Figure PCTCN2018090491-appb-000003
Figure PCTCN2018090491-appb-000004
Figure PCTCN2018090491-appb-000005

Claims (10)

  1. 一种抗破伤风毒素抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括：
    可变重链结构域，包含：VH CDR1，所述VH CDR1由SEQ ID NO.1所示的氨基酸残基序列组成；VH CDR2，所述VH CDR2由SEQ ID NO.2所示的氨基酸残基序列组成；VH CDR3，所述VH CDR3由SEQ ID NO.3所示的氨基酸残基序列组成；以及
    可变轻链结构域，包含：VLCDR1，所述VLCDR1由SEQ ID NO.5所示的氨基酸序列组成；VLCDR2，所述VLCDR2由SEQ ID NO.6所示的氨基酸序列组成；以及VLCDR3，所述VLCDR3由SEQ ID NO.7所示的氨基酸序列组成。
  2. 权利要求1的抗破伤风毒素抗体或抗原结合片段，其特征在于，包含：
    重链可变区，氨基酸序列如SEQ ID NO.4所示；以及
    轻链可变区，氨基酸序列如SEQ ID NO.8的所示。
  3. 根据权利要求1或2所述的抗破伤风毒素抗体或抗原结合片段，其特征在于，所述抗体或其抗原结合片段免疫特异性地结合破伤风毒素A片段和/或C片段并中和破伤风毒素，所述抗体或抗原结合片段具有至少10 -6、10 -7、10 -8、10 -9或10 -10M的对破伤风毒素A片段和/或C片段或对破伤风毒素的亲和力。
  4. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-3任一项所述的抗体或抗原结合片段，以及药学可接受的载体或赋形剂。
  5. 一种破伤风毒素感染或破伤风梭菌感染的诊断组合物，其特征在于，包括：
    权利要求1-3任一项所述的单克隆抗体；和/或容器。
  6. 根据权利要求5所述的诊断组合物，其特征在于，所述组合物包括缀合物，所述缀合物包含缀合至权利要求1-3任一项所述的抗体或抗原结合片段的标签。
  7. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1-3中任一项的抗体或抗原结合片段。
  8. 一种表达载体，其特征在于，包含有权利要求7所述的DNA分子序列以及与所述序列操作性相连的表达调控序列。
  9. 一种重组细胞，其特征在于，含有权利要求7所述的DNA分子或权利要求8所述的表达载体。
  10. 如下任一项所述的应用，其特征在于，包括：
    1)权利要求1-3任一项所述的抗体抗原结合片段在制备治疗由破伤风毒素感染或破伤风梭菌感染的药物中的应用；
    2)权利要求1-3任一项所述的抗体或抗原结合片段在制备检测破伤风毒素感染或破伤风梭菌感染的试剂盒中的应用；
    3)权利要求4所述的药物组合物在制备治疗由破伤风毒素感染或破伤风梭菌感染的药物中的应用；
    4)权利要求5或6所述的诊断组合物在制备诊断破伤风毒素感染或破伤风梭菌感染的试剂盒中的应用；
    5)权利要求7所述的DNA分子在制备治疗由破伤风毒素感染或破伤风梭菌感染的药物中的应用；
    6)权利要求8所述的表达载体在制备治疗由破伤风毒素感染或破伤风梭菌感染的药物中的应用；
    7)权利要求9所述的宿主细胞在制备治疗由破伤风毒素感染或破伤风梭菌感染的药物中的应用。
PCT/CN2018/090491 2017-12-29 2018-06-08 抗破伤风毒素中和抗体及其制备与应用 WO2019128121A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2815280C1 (ru) * 2020-10-21 2024-03-13 Бейдзин Уиздомаб Байотекнолоджи Ко., Лтд. Антитело против столбнячного токсина и его применение

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3733699A4 (en) * 2017-12-29 2022-06-08 Zhuhai Trinomab Biotechnology Co., Ltd. FULLY HUMANIZED MONOCLONAL NEUTRALIZING ANTIBODY FOR TETANUS TOXIN AND ITS APPLICATION
CN113416249B (zh) * 2020-10-21 2023-04-07 北京智仁美博生物科技有限公司 针对破伤风毒素的组合物

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0257195A (ja) * 1988-08-19 1990-02-26 Morinaga & Co Ltd 抗破傷風毒素ヒト型モノクローナル抗体、それを利用した破傷風毒素中和剤及びヒト型モノクローナル抗体産生ハイブリドーマ
EP0562132A1 (en) * 1992-03-23 1993-09-29 SCHWEIZERISCHES SERUM- & IMPFINSTITUT BERN Monoclonal anti-tetanus toxin antibodies and pharmaceutical compositions containing them
CN1634991A (zh) * 2003-12-30 2005-07-06 龚小迪 人源抗破伤风毒素单克隆抗体及其制备方法和用途
KR100624011B1 (ko) * 2005-04-20 2006-09-19 주식회사 녹십자 항-파상풍 단일클론항체 및 이를 생산하는 하이브리도마세포주
CN102206275A (zh) * 2011-04-27 2011-10-05 上海生物制品研究所 抗破伤风毒素单克隆中和抗体，其组合物及其用途
CN102875674A (zh) * 2011-10-27 2013-01-16 成都蓉生药业有限责任公司 一种抗破伤风毒素抗体及其制备方法和用途
CN105153305A (zh) * 2015-06-26 2015-12-16 安泰吉（北京）生物技术有限公司 一种全人源抗破伤风毒素单克隆抗体及其衍生物制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003285874A1 (en) * 2002-10-16 2004-05-04 Amgen Inc. HUMAN ANTI-IFN-Gamma NEUTRALIZING ANTIBODIES AS SELECTIVE IFN-Gamma PATHWAY INHIBITORS
CN102584993A (zh) * 2012-02-24 2012-07-18 湖南康润药业有限公司 一种高效价人破伤风免疫血浆及其制备工艺

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0257195A (ja) * 1988-08-19 1990-02-26 Morinaga & Co Ltd 抗破傷風毒素ヒト型モノクローナル抗体、それを利用した破傷風毒素中和剤及びヒト型モノクローナル抗体産生ハイブリドーマ
EP0562132A1 (en) * 1992-03-23 1993-09-29 SCHWEIZERISCHES SERUM- & IMPFINSTITUT BERN Monoclonal anti-tetanus toxin antibodies and pharmaceutical compositions containing them
CN1634991A (zh) * 2003-12-30 2005-07-06 龚小迪 人源抗破伤风毒素单克隆抗体及其制备方法和用途
KR100624011B1 (ko) * 2005-04-20 2006-09-19 주식회사 녹십자 항-파상풍 단일클론항체 및 이를 생산하는 하이브리도마세포주
CN102206275A (zh) * 2011-04-27 2011-10-05 上海生物制品研究所 抗破伤风毒素单克隆中和抗体，其组合物及其用途
CN102875674A (zh) * 2011-10-27 2013-01-16 成都蓉生药业有限责任公司 一种抗破伤风毒素抗体及其制备方法和用途
CN105153305A (zh) * 2015-06-26 2015-12-16 安泰吉（北京）生物技术有限公司 一种全人源抗破伤风毒素单克隆抗体及其衍生物制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MEIJER, P.J., AY866984.1: HOMO SAPIENS CLONE 60A02 ANTI-TETANUS TOXOID IMMUNOGLOBULIN LIGHT CHAIN VARIABLE REGION (IGL@) MRNA, PARTIAL CDS, 26 July 2016 (2016-07-26) *
MEIJER, P.J.: "Isolation of Human Antibody Repertoires with Preservation of the Natural Heavy and Light Chain Pairing", J. MOL. BIOL., 2 March 2006 (2006-03-02), XP024950964 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2815280C1 (ru) * 2020-10-21 2024-03-13 Бейдзин Уиздомаб Байотекнолоджи Ко., Лтд. Антитело против столбнячного токсина и его применение

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