WO2019129214A1 - 针对破伤风毒素的全人源单克隆中和抗体及其应用 - Google Patents

针对破伤风毒素的全人源单克隆中和抗体及其应用 Download PDF

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WO2019129214A1
WO2019129214A1 PCT/CN2018/124958 CN2018124958W WO2019129214A1 WO 2019129214 A1 WO2019129214 A1 WO 2019129214A1 CN 2018124958 W CN2018124958 W CN 2018124958W WO 2019129214 A1 WO2019129214 A1 WO 2019129214A1
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Prior art keywords
seq
neutralizing antibody
variable region
monoclonal neutralizing
chain variable
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PCT/CN2018/124958
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English (en)
French (fr)
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廖化新
王月明
袁晓辉
郑伟宏
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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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Priority claimed from CN201711486693.XA external-priority patent/CN108314730B/zh
Priority claimed from CN201711482969.7A external-priority patent/CN108218984B/zh
Priority claimed from CN201711486732.6A external-priority patent/CN108314731B/zh
Priority claimed from CN201810420730.5A external-priority patent/CN108623681B/zh
Application filed by 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 filed Critical 珠海泰诺麦博生物技术有限公司
Priority to BR112020013094-0A priority Critical patent/BR112020013094A2/pt
Priority to AU2018395100A priority patent/AU2018395100A1/en
Priority to EP18896977.8A priority patent/EP3733699A4/en
Priority to US16/958,213 priority patent/US11725046B2/en
Priority to JP2020555284A priority patent/JP7368670B2/ja
Publication of WO2019129214A1 publication Critical patent/WO2019129214A1/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)
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the invention belongs to the field of cellular immunology and genetic engineering, and particularly relates to a fully human monoclonal neutralizing antibody against tetanus toxin 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 a light and heavy chain 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.
  • the main object of the present invention is to provide a fully human monoclonal monoclonal antibody against tetanus that is non-immunogenic, high affinity, specific, high titer, and free from foreign virus contamination.
  • the present invention provides a fully human monoclonal neutralizing antibody or antigen-binding fragment thereof against tetanus toxin, characterized in that said monoclonal neutralizing antibody comprises at least one CDR comprising three a heavy chain variable region and at least one light chain variable region comprising three CDRs;
  • amino acid sequences of the heavy chain variable regions CDR1, CDR2, CDR3 are set forth in SEQ ID NOs: 1, 2, 3, 9, 10, 11, 19, 20, 21, 27, 28, 29;
  • the amino acid sequences of the variable regions CDR1, CDR2, CDR3 are set forth in SEQ ID NOs: 4, 5, 6, 13, 14, 15, 23, 24, 25, 30, 31, 32; or substituted, deleted or added one or a plurality of amino acids; or an amino acid sequence having at least 80% homology to the amino acid sequence represented by the variable regions CDR1, CDR2, CDR3, and having the same or similar function;
  • the amino acid sequence of the heavy chain variable region CDR1, CDR2, CDR3 of the monoclonal neutralizing antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;
  • the amino acid sequence of CDR2 and CDR3 is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;
  • the amino acid sequence of the heavy chain variable region CDR1, CDR2, CDR3 of the monoclonal neutralizing antibody is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; light chain variable region CDR1 SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; wherein the variable heavy domain of the monoclonal neutralizing antibody has SEQ ID NO.
  • the amino acid sequence shown, the variable light chain domain has the amino acid sequence set forth in SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region CDR1, CDR2, CDR3 of the monoclonal neutralizing antibody is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21;
  • the amino acid sequences of CDR2 and CDR3 are selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25;
  • the amino acid sequence of the heavy chain variable region CDR1, CDR2, CDR3 of the monoclonal neutralizing antibody is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29; light chain variable region CDR1
  • the amino acid sequences of CDR2 and CDR3 are selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.
  • sequence of the heavy chain variable region of the monoclonal neutralizing antibody is as shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 33, or substituted, deleted or added One or more amino acids; or an amino acid sequence having at least 80% homology to the amino acid sequence indicated by the heavy chain variable region, and having the same or similar function;
  • sequence of the light chain variable region is SEQ ID NO: 8, SEQ ID NO. 18, SEQ ID NO: 26, SEQ ID NO: 34, or substituted, deleted or added one or more amino acids; or at least 80% identical to the amino acid sequence shown in the light chain variable region Amino acid sequences that are derived and functionally identical or similar;
  • sequence of the heavy chain variable region of the monoclonal neutralizing antibody is set forth in SEQ ID NO: 7
  • sequence of the variable region of the antibody light chain is set forth in SEQ ID NO: 8.
  • the sequence of the heavy chain variable region of the monoclonal neutralizing antibody is set forth in SEQ ID NO: 17, and the sequence of the light chain variable region is set forth in SEQ ID NO: 18.
  • sequence of the heavy chain variable region of the monoclonal neutralizing antibody is set forth in SEQ ID NO: 22, and the sequence of the light chain variable region is set forth in SEQ ID NO: 26;
  • the sequence of the heavy chain variable region of the monoclonal neutralizing antibody is set forth in SEQ ID NO: 33, and the sequence of the light chain variable region is set forth in SEQ ID NO:34.
  • the monoclonal neutralizing antibody or antigen-binding fragment thereof of the present invention immunospecifically binds to tetanus toxin, and the monoclonal neutralizing antibody or antigen-binding fragment thereof is separated by an equilibrium of not higher than 10 -6 M The constant is dissociated from tetanus toxin and/or Clostridium tetani.
  • the monoclonal neutralizing antibody of the present invention has a heavy chain type of IgG1, IgG2, IgG3 or Ig4. More preferably, it is IgG1.
  • the monoclonal neutralizing antibody of the present invention has a light chain type of kappa or lambda.
  • the monoclonal neutralizing antibodies of the present invention include not only variable regions but also constant regions.
  • the constant region of the monoclonal neutralizing antibody of the present invention is any one of human IgG1, IgG2, IgG3 or IgG4.
  • 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 Antibody (diabody), Fd or Fd' fragment.
  • the monoclonal neutralizing antibody or antigen-binding fragment of the present invention further comprises a peptide linker, preferably, the peptide linker comprises from about 1 to 50 amino acids.
  • the present invention provides a conjugate comprising the monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, further comprising conjugation to the monoclonal neutralizing agent of the present invention
  • a tag of an antibody or antigen-binding fragment thereof such as a detectable moiety/reagent.
  • the tag is directly conjugated to the monoclonal neutralizing antibody or antigen-binding fragment thereof of the present invention by a covalent bond or a non-covalent bond.
  • the tag can be conjugated to a monoclonal neutralizing antibody or antigen-binding fragment thereof of the invention using one or more linking compounds.
  • Detectable moieties/agents as labels include free enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials, non-radioactive paramagnetic metal ions.
  • the present invention provides a conjugate comprising the monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, further comprising covalently linked to the monoclonal neutralizing antibody described above or A chemical label or biomarker on the antigen-binding fragment.
  • the chemical label comprises an isotope label, an immunotoxin label, a chemical drug label
  • the biomarker comprises a biotin label, an avidin label, an enzyme label, a fluorescent compound, an electron transfer agent.
  • the invention provides a conjugate comprising a monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, and/or a conjugate as described above, and/or a front
  • the combination is formed by coupling with a solid medium or a semi-solid medium.
  • solid or semi-solid media such as ELISA plates, magnetic or non-magnetic immunospheres or immunoparticles
  • cell separation media examples of such cell separation media, such as polyvinylpyrrolidone coated colloidal silica , polysaccharides and sodium diatrizoate or derivatives thereof.
  • the present invention provides a composition comprising the monoclonal neutralizing antibody or other antigen-binding fragment described above, and/or the conjugate described above, and/or Or a combination as described above, and/or a conjugate as described above.
  • the composition includes a pharmaceutical composition, a diagnostic composition.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier means a carrier or diluent which does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, such as physiological saline, sterile water, Ringer's solution, A buffered saline solution, a glucose solution, a maltodextrin solution, glycerin, ethanol, and a mixture of two or more thereof.
  • the pharmaceutical composition provided by the embodiments of the present invention may further include other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent, a dispersing agent, a surfactant, a binder, and a lubricant, and is configured as an injectable preparation.
  • aqueous solutions, suspensions and emulsions, pills, capsules, granules and tablets Such as aqueous solutions, suspensions and emulsions, pills, capsules, granules and tablets.
  • compositions of the invention must be sterile and stable under the conditions of manufacture and storage.
  • the preferred methods of preparation are vacuum drying and freeze drying, vacuum drying and freeze drying to produce activity from pre-sterilized solutions of the active ingredient and other desired ingredients.
  • the compositions of the present invention may be in solution, and may be added and/or mixed with a suitable pharmaceutically acceptable excipient prior to delivery or delivery to provide an injectable unit dosage form.
  • the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain proper fluidity, and delay absorption if desired.
  • monoclonal neutralizing antibodies of the invention can be prepared with carriers wherein the carrier will protect them against rapid release (such as a controlled release formulation) comprising an implant, a transdermal patch, and microencapsulation Delivery system.
  • Biodegradable, biocompatible polymers can be used in the present invention, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • the monoclonal neutralizing antibody may be coated with a material or a compound that prevents the antibody from being inactivated, or administered simultaneously with such a material or compound.
  • a monoclonal neutralizing antibody can be administered with a suitable carrier such as a liposome or diluent.
  • the administration route of the pharmaceutical composition of the present invention can be divided into oral administration and parenteral administration.
  • the preferred route of administration is intravenous, but is not limited thereto.
  • Oral dosage forms can be formulated into tablets, troches, lozenges, aqueous or oily suspensions, dispersions of powders or granules, emulsions, hard capsules, soft capsules, syrups or elixirs, pills, dragees, liquids, gels Or ointment.
  • These formulations may contain pharmaceutical excipients including, but not limited to, granulating and disintegrating agents, binding agents, lubricants, preservatives, colorants, flavoring or sweetening agents, vegetable oils or minerals. Oil, wetting agent, and thickener.
  • Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile non-toxic injectable solutions or suspensions.
  • the solution or suspension may include reagents and dosages that are non-toxic to the recipient, such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oil, Fatty acids, topical anesthetics, preservatives, buffers, agents for increased viscosity or solubility, water soluble antioxidants, oil soluble antioxidants, and metal chelators.
  • Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile non-toxic injectable solutions or suspensions.
  • the solution or suspension may include agents that are non-toxic to the dosage and concentration employed, such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonicity.
  • agents that are non-toxic to the dosage and concentration employed such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonicity.
  • the pharmaceutical composition may also include one or more additional therapeutic agents.
  • the therapeutic agent can comprise antibodies, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, and the like.
  • the invention provides a test product comprising a diagnostically effective amount of a monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, and/or a conjugate as described above, and/or The conjugates described above, and/or the conjugates described above, and/or the compositions previously described.
  • the test product provided by the present invention can be used for detecting tetanus toxin infection and Clostridium tetani infection.
  • the detection products provided by the present invention measure the level of tetanus toxin in a fluid, cell or tissue sample by an antibody.
  • the detection product provided by the embodiment of the present invention is also capable of comparing the measured level of tetanus toxin with the control level, wherein the increase in the level of tetanus toxin measured represents a tetanus toxin infection compared to the control level of tetanus toxin.
  • the cell or tissue sample is obtained from a human subject, the blood or urine, saliva, pulmonary sputum, lavage or lymphoid sample.
  • the present invention provides a DNA molecule encoding the monoclonal neutralizing antibody or antigen-binding fragment thereof described above.
  • the DNA molecule sequence can be obtained by any method known in the art. For example, if the nucleotide sequence of the antibody is known, the DNA molecule sequence encoding the antibody can be assembled from the chemically synthesized oligonucleotide.
  • a DNA molecule encoding an antibody can be produced from a nucleic acid from a suitable source. If a clone containing a DNA molecule encoding a specific antibody cannot be obtained, but the sequence of the antibody molecule is known, a DNA molecule encoding the immunoglobulin can be obtained by chemical synthesis, or 3' and 5 can be utilized with the sequence. 'End-hybridizable synthetic primers are obtained by PCR amplification of a DNA molecule encoding the immunoglobulin from a suitable source, or by cloning using an oligonucleotide probe specific for the specific gene sequence.
  • a DNA molecule of a globulin for example, a cDNA clone encoding the antibody is identified from a cDNA library.
  • the amplified DNA molecule produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.
  • the present invention provides a recombinant expression vector comprising the DNA molecule described above.
  • Expression vectors for constructing the recombinant expression vector of the present invention include, but are not limited to, MarEx expression vectors produced by Celltrion Inc. (Korea); commercially available pCDNA vectors; F, R1, RP1, Col, pBR322, ToL , Ti vector; cosmid; phage, such as lambda phage, lambda phage, M13 phage, Mu phage, P1 phage, P22 phage, Q ⁇ phage, T-even phage, T2 phage, T4 phage, T7 phage, etc.; plant virus.
  • MarEx expression vectors produced by Celltrion Inc. (Korea); commercially available pCDNA vectors; F, R1, RP1, Col, pBR322, ToL , Ti vector; cosmid; phage, such as lambda phage, lambda phage, M13 phage, Mu phage, P1 phage, P22 phage
  • any of a variety of expression vectors known to those skilled in the art can be used in the present invention, and the choice of expression vector depends on the nature of the host cell chosen.
  • Introduction of the vector into the host cell can be achieved by, but not limited to, calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection or electroporation, and any of skill in the art Introduction methods suitable for the expression vector and host cell used can be selected and used.
  • the above vector comprises one or more selection markers, but is not limited thereto, and a carrier that does not include a selection marker may also be used.
  • the choice of selection marker can depend on the host cell of choice (as is well known to those skilled in the art), but this is not critical to the invention.
  • a tag sequence can be inserted into an expression vector.
  • tags include, but are not limited to, six histidine tags, hemagglutinin tags, myc tags, or FLAG tags. Any label known to those skilled in the art to facilitate purification can be used in the present invention.
  • the present invention provides a recombinant cell transformed or transfected into a host cell via the expression vector described above.
  • Host cells useful in the present invention include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (e.g., E. coli, Bacillus subtilis (B).
  • microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (e.g., E. coli, Bacillus subtilis (B).
  • a yeast transformed with a recombinant yeast expression vector containing an antibody coding sequence such as Saccharomyces, Pichia
  • a recombinant viral expression vector containing an antibody coding sequence eg, a baculovirus
  • Infected insect cell system a plant transformed with a recombinant viral expression vector (eg, cauliflower mosaic virus (CaMV); tobacco mosaic virus (TMV)-infected or recombinant plasmid expression vector (eg, Ti plasmid) containing an antibody coding sequence) a cellular system
  • a recombinant carrying a promoter derived from the genome of a mammalian cell such as a metallothionein promoter
  • a promoter derived from a mammalian virus for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter
  • a mammalian cell system eg, COS, CHO, BHK,
  • the present invention also provides a method for detecting the presence or level of tetanus toxin and Clostridium tetanium in a sample, which comprises using the monoclonal neutralizing antibody of the present invention or an antigen-binding fragment thereof.
  • the monoclonal neutralizing antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal neutralizing antibody or antigen-binding fragment thereof of the present invention using a second antibody carrying a detectable label.
  • the method can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample or a sample from a patient).
  • the present invention also provides a method of diagnosing whether a subject is infected with tetanus toxin or Clostridium tetani infection, comprising: detecting using the monoclonal neutralizing antibody of the present invention or an antigen-binding fragment thereof The presence of tetanus toxin or Clostridium tetani infection in a sample from the subject.
  • the monoclonal neutralizing antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal neutralizing antibody or antigen-binding fragment thereof of the present invention using a second antibody carrying a detectable label.
  • a method for neutralizing the virulence of tetanus toxin in a sample comprising: a sample comprising tetanus toxin and a monoclonal neutralizing antibody or antigen thereof of the present invention Combine fragment contacts.
  • a sample comprising tetanus toxin and a monoclonal neutralizing antibody or antigen thereof of the present invention Combine fragment contacts.
  • Such methods can be used for therapeutic purposes, or for non-therapeutic purposes (eg, the sample is a cell sample, not a patient or a sample from a patient).
  • the present invention also provides a monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, and/or a conjugate as described above, and/or a conjugate as described above, and / or the use of the previously described conjugates, and/or the compositions described above, in the preparation of a tetanus toxin or Clostridium tetani infection detection product.
  • the detection product is a kit, an ELISA plate, or a chip.
  • the present invention also provides a monoclonal neutralizing antibody or antigen-binding fragment thereof as described above, and/or a conjugate as described above, and/or a conjugate as described above, and / or the use of a conjugate as described above, and/or a composition as described above, in the manufacture of a medicament for the prevention or treatment of a disease caused by tetanus toxin or Clostridium tetani infection.
  • the present invention further provides the DNA molecule described above, the recombinant expression vector described above, and the recombinant cell described above, which are produced by preventing or treating tetanus toxin or Clostridium tetani infection.
  • the application of disease drugs are provided.
  • 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.
  • monoclonal neutralizing antibody refers to a monoclonal antibody having neutralizing activity
  • a 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 strand 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 neutralizing antibodies of the invention also include functional variants of the antibodies which bind to tetanus toxin or a fragment thereof and which have neutralizing activity against the subtype or fragment.
  • a functional variant includes, but is not limited to, chemical and/or biochemical, such as in vitro or in vivo, that is substantially similar in the primary structural sequence but that is not included in the parental monoclonal neutralizing antibody of the invention.
  • Modified derivative 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 neutralizing antibody comprising an amino acid sequence comprising a substitution, insertion, deletion or combination of one or more amino acids compared to the amino acid sequence of the parent monoclonal neutralizing 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 neutralizing 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 compared to a parent monoclonal neutralizing 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.
  • Parental monoclonal neutralizing antibodies or their parental antibodies can be altered by general molecular biology methods known in the art, including PCR, oligonucleotide-directed mutagenesis, and site-directed mutagenesis Functional variants are obtained in part or 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 invention develops a specific whole-human anti-tetanus toxin monoclonal neutralizing antibody by a high-throughput whole human monoclonal antibody research and development comprehensive technology platform.
  • the whole human anti-tetanus toxin monoclonal neutralizing antibody developed by the invention can be used for the prevention and treatment of Clostridium typhimurium infection.
  • the fully human anti-tetanus toxin monoclonal neutralizing antibody developed by the invention can be used for the detection of Clostridium tetani infection.
  • the monoclonal anti-tetanus toxin monoclonal neutralizing antibody developed by the invention has strong affinity with tetanus toxin and has strong neutralizing activity, is safe and efficient, and has no foreign virus pollution, and can be widely applied to each The population has strong industrial applicability.
  • Figure 1 shows an electropherogram for purification of TRN0010 antibody expression by SDS-PAGE and Western blot, wherein: A: SDS-PAGE; B: Western blot;
  • FIG. 2 shows an electropherogram of purification of TRN0012 antibody expression by SDS-PAGE and Western Blot, wherein: A: SDS-PAGE; B: Western Blot;
  • FIG. 3 shows an electropherogram for purification of TRN0011 antibody expression by SDS-PAGE and Western Blot, wherein A: SDS-PAGE map; B: Western Blot map;
  • FIG. 4 shows an electropherogram of purification of TRN1015 antibody expression by SDS-PAGE and Western Blot, wherein: A: SDS-PAGE map; B: Western Blot map;
  • Figure 5 is a graph showing the results of detecting the neutralizing activity of the TRN0010 antibody by ELISA
  • Figure 6 is a graph showing the results of detecting the neutralizing activity of the TRN0012 antibody by ELISA
  • Figure 7 is a graph showing the results of detecting the neutralizing activity of the TRN0011 antibody by ELISA
  • Figure 8 is a graph showing the results of detecting the neutralizing activity of the TRN1015 antibody by ELISA
  • Figure 9 is a graph showing the results of affinity detection of the TRN0010 antibody.
  • Figure 10 is a graph showing the results of affinity detection of the TRN0012 antibody
  • Figure 11 is a graph showing the results of affinity detection of the TRN0011 antibody
  • Figure 12 is a graph showing the results of affinity detection of the TRN1015 antibody
  • Figure 13 is a graph showing the results of protective effects of the TRN0010 antibody on animals
  • Figure 14 is a graph showing the results of protective effects of the TRN0012 antibody on animals.
  • Figure 15 is a graph showing the results of protective effects of the TRN0011 antibody on animals.
  • Figure 16 shows a protective effect of the TRN1015 antibody on a 20-fold dose of tetanus toxin LD50;
  • Figure 17 shows a protective effect of the TRN1015 antibody on a 60-fold dose of tetanus toxin LD50.
  • PBMC tetanus toxoid
  • 96-well plates containing single B cells were added to 0.5 ⁇ M constant region primers of each subtype of heavy and light chain and Superscript III reverse transcriptase, and incubated at 37 ° C for 1 hour; PCR amplification was performed according to the following parameters: 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.
  • the anti-tetanus toxin full human antibody gene was PCR amplified using the above cDNA and corresponding primers as a template.
  • the 50 ⁇ L system contained 5 ⁇ l of RT reaction product, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), dNTPs, and 0.5 ⁇ M specific primers for each of the subtype heavy and light chain antibodies.
  • the reaction conditions were: pre-denaturation at 94 ° C for 5 min. Then 35 PCR cycles were performed, 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.
  • DH5 ⁇ competent bacteria were transformed with the above ligation product, and cultured on a plate containing ampicillin at 37 ° C overnight.
  • 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.
  • the anti-tetanus toxin fully human antibody heavy/light chain expression vector was co-transfected into 293 cells using the DNA transfection kit PolyFect, and the empty cell control of the untransfected plasmid was used. See the instruction manual for specific operations. After 96 hours of culture, the expression of the HRP-labeled goat anti-human IgG anti-tetanus toxin full human antibody and the specific recognition of the antibody against tetanus vaccine (antigen) were performed by conventional ELISA.
  • 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 hours.
  • 100 ⁇ L of 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.
  • An expression vector for the heavy chain and light chain of the TRN0010 antibody having neutralizing activity identified by the neutralization assay (wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 7; the antibody light chain variable region is The amino acid sequence is as shown in SEQ ID NO: 8) co-transfected into 293 cells;
  • the neutralizing activity identified by the neutralization assay is the expression vector of the TRN0012 antibody heavy and light chain (wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 17; the antibody light chain variable region The amino acid sequence is as shown in SEQ ID NO: 18) co-transfected into 293 cells;
  • the neutralizing activity identified by the neutralization assay is the expression vector of the TRN0011 antibody heavy and light chain (wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 22; the antibody light chain variable region is The amino acid sequence is as shown in SEQ ID NO: 26) co-transfected into 293 cells;
  • An expression vector for the heavy chain and light chain of the TRN1015 antibody having neutralization activity identified by the neutralization assay (wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33; the antibody light chain variable region is The amino acid sequence is as shown in SEQ ID NO: 34) co-transfected into 293 cells;
  • the tetanus 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 was diluted in ratio and then 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 in the dark, the reaction was stopped with 2 M sulfuric acid, and colorimetric detection was carried out at a wavelength of 450 nm, and the results were analyzed.
  • the CM5 chip is coupled to the capture molecule, and then the surface of the dextran of the chip is activated, and the amount of coupling is determined by the injection time.
  • the molecular capture ligand is captured by the CM5 chip: the prepared whole human anti-tetanus neutralizing antibody is used as the preparation Ligand.
  • the tetanus toxoid was diluted with HBS-EP buffer as an analyte, and the analyte was sequentially passed through the chip at a gradually increasing concentration to obtain a signal curve.
  • the neutralizing antibody of the present invention has an equilibrium dissociation constant of less than 10 -9 mol for tetanus toxin, indicating that it has a high affinity.
  • the monoclonal antibody to be tested was diluted with a diluent to 100 ⁇ g/ml (monotherapy concentration > 1 mg/ml), and diluted with a diluent at a ratio of 3 times, and used.
  • the standard antitoxin is mixed with physiological lysate and neutral glycerin (autoclaved at 116 ° C for 10 minutes) and then diluted with diluent to make 0.5 IU per ml (ie 5 IU/10); ie with toxin After equal mixing, the injection volume contained IU/10 per 0.4 ml.
  • the standard antitoxin stock solution should not be less than 0.5ml.
  • the lyophilized powdery toxin purchased from the laboratory was mixed with normal glycerol (sterilized by autoclaving at 116 ° C for 10 minutes), and then the toxin was diluted with the diluent to the amount used.
  • the prepared toxin was diluted sequentially with dilutions of 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , diluted at least 2 ml per dilution, and 0.2 ml of injected mice were used, 4 in each group. Observe for 5 days.
  • the LD50 was calculated based on the experimental results, and the experimental group used 20 times the amount of LD50 or even 60 times the amount of LD50.
  • mice Take 140 healthy mice, 4 in each group.
  • the above mixture was subcutaneously injected into the abdomen of 18 to 22 g of mice, and each injection was 0.4 ml (negative control group included 0.2 ml of toxin + 0.2 ml of borate buffered saline; positive control group included 0.2 ml of toxin + 0.2 ml of antitoxin;
  • the experimental group included 0.2 ml of toxin + 0.2 ml of monoclonal antibody). Observe once a day in the morning and afternoon, and record the incidence and death of the mice for one week.
  • the monoclonal antibody of the present invention can effectively neutralize toxins in mice, protect mice, and have in vivo activity.
  • Figure 17 shows that in the challenge of a larger dose of tetanus toxin (60 times LD50), the mice in the negative control group died within 24 hours, and the mice in the low dose group (0.62 ug/ml, 1.85 ug/ml). After 7 days of death, the mice in the middle and high doses of the experimental group (5.56 ug/ml, 16.67 ug/ml and 50 ug/ml dose) all survived within 7 days. It is shown that the monoclonal antibody of the present invention can protect against the attack of high dose of toxin, indicating that the monoclonal antibody of the present invention has an extremely strong neutralizing ability in vivo.

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Abstract

本发明公开了针对破伤风毒素的全人源单克隆中和抗体。本发明通过高通量全人源单克隆抗体研发综合技术平台开发出了全人源抗破伤风毒素单克隆中和抗体。本发明的单克隆中和抗体能用于破伤风梭菌感染的预防和治疗、用于破伤风梭菌感染的检测。本发明研发的全人源抗破伤风毒素单克隆中和抗体与破伤风毒素具有极强的亲和力、且具有极强的中和活性，安全且高效，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。

Description

针对破伤风毒素的全人源单克隆中和抗体及其应用
本申请要求于2017年12月29日提交中国专利局、申请号为2017114867326、发明名称为“一种针对破伤风毒素的全人源单克隆中和抗体及其应用”；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”；申请号为2017114829697、发明名称为“一种抗破伤风毒素的全人源中和抗体”；2018年5月4日提交中国专利局、申请号为201810420730.5、发明名称为“一种抗破伤风毒素的中和抗体及应用”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
本发明属于细胞免疫学、基因工程领域，具体涉及针对破伤风毒素的全人源单克隆中和抗体及其应用。
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，本发明的主要目的在于提供一种无免疫原性、亲和力高、特异性强、效价高、不存在外源病毒污染的针对破伤风毒素的全人源单 克隆中和抗体及其抗原结合片段，以及编码它们的序列，产生它们的细胞株，和应用它们进行诊断、预防或治疗的方法和用途。
根据本发明的一个方面，本发明提供了一种针对破伤风毒素的全人源单克隆中和抗体或其抗原结合片段，其特征在于，所述单克隆中和抗体包含至少一个含有三个CDR的重链可变区和至少一个含有三个CDR的轻链可变区；
其中所述重链可变区CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO:1、2、3、9、10、11、19、20、21、27、28、29所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO:4、5、6、13、14、15、23、24、25、30、31、32所示；或者经取代、缺失或添加一个或多个氨基酸；或与所述可变区CDR1、CDR2、CDR3所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
优选地，
所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示；
所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15所示；其中，所述单克隆中和抗体的可变重链结构域具有如SEQ ID NO.12所示的氨基酸序列，可变轻链结构域具有如SEQ ID NO.16所示的氨基酸序列。
所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25所示；
所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32所示。
进一步，所述单克隆中和抗体重链可变区的序列如SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:22、SEQ ID NO:33所示，或者经取代、缺失或添加一个或 多个氨基酸；或与重链可变区所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；轻链可变区的序列如SEQ ID NO:8、SEQ ID NO.18、SEQ ID NO:26、SEQ ID NO:34所示，或者经取代、缺失或添加一个或多个氨基酸；或与轻链可变区所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
优选地，
所述单克隆中和抗体重链可变区的序列如SEQ ID NO:7所示，抗体轻链可变区的序列如SEQ ID NO:8所示；
所述单克隆中和抗体重链可变区的序列如SEQ ID NO:17所示，轻链可变区的序列如SEQ ID NO:18所示；
所述单克隆中和抗体重链可变区的序列如SEQ ID NO:22所示，轻链可变区的序列如SEQ ID NO:26所示；
所述单克隆中和抗体重链可变区的序列如SEQ ID NO:33所示，轻链可变区的序列如SEQ ID NO:34所示。
进一步，本发明的所述单克隆中和抗体或其抗原结合片段免疫特异性地结合破伤风毒素，所述单克隆中和抗体或其抗原结合片段以不高于10 -6M的平衡解离常数与破伤风毒素和/或破伤风梭菌解离。
优选的，本发明的所述单克隆中和抗体，其重链类型为IgG1、IgG2、IgG3或Ig4。更优选为IgG1。
优选的，本发明所述单克隆中和抗体，其轻链类型为κ或λ。
本发明所述的单克隆中和抗体不仅包括可变区，还包括恒定区。
优选的，本发明所述的单克隆中和抗体的恒定区为人IgG1、IgG2、IgG3或IgG4中的任意一种。
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。
本发明的单克隆中和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。
根据本发明的又一个方面，本发明提供了一种缀合物，缀合物包含前面所述的单克隆中和抗体或其抗原结合片段，还包括缀合至本发明所述单克隆中和抗体或其抗原结合片段的标签，如可检测的部分/试剂。标签通过共价键或非共价键直接缀合至本发明所述单克隆中和抗体或其抗原结合片段。另外，标签可利用一种或多种连接化合物缀合至本发明所述单克隆中和抗体或其抗原结合片段上。其中，用于将标签缀合至本发明所述单克隆中和抗体或其抗原结合片段上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/试剂包括自由酶、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、非放射性的顺磁金属离子。
根据本发明的又一个方面，本发明提供了一种结合物，包含前面所述的单克隆中和抗体或其抗原结合片段，还包含共价连接到前面所述的单克隆中和抗体或其抗原结合片段上的化学标记或生物标记。
优选的，所述化学标记包括同位素标记、免疫毒素标记、化学药物标记；所述生物标记包括生物素标记、亲和素标记、酶标记、荧光化合物、电子转移剂。
根据本发明的又一个方面，本发明提供了一种偶联物，其由前面所述的单克隆中和抗体或其抗原结合片段、和/或前面所述的缀合物、和/或前面所述的结合物与固体介质或半固体介质偶联形成。
固体介质或半固体介质的实例，如，酶标板、磁性或非磁性免疫微球或免疫颗粒，细胞分离介质，所述细胞分离介质的实例，如聚乙烯吡咯烷酮包被的胶质二氧化硅，多聚糖和泛影酸钠或其衍生物。
根据本发明的另一个方面，本发明提供了一种组合物，所述组合物包含前面所述的单克隆中和抗体或其他抗原结合片段、和/或前面所述的缀合物、和/或前面所述的结合物、和/或前面所述的偶联物。
所述组合物包括药物组合物、诊断组合物。
所述药物组合物还包括药学上可接受的载体。其中，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，如，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，本发明实施例提供的药物组合物还可以包括其它常规添加剂，如抗氧化剂、缓冲剂、抑菌剂、分散剂、表面活性剂、粘合剂和润滑剂，并配置成可注射制剂，如水性溶液，悬浮液和乳剂，丸剂，胶囊剂，颗粒剂和片剂。
本发明的药物组合物在制造和储存条件下必须是无菌和稳定的。在用于制备无菌可注射溶液的无菌粉末的情况下，制备的优选方法是真空干燥和冷冻干燥，真空干燥和冷冻干燥从活性成分和其它期望成分的预先无菌过滤过的溶液产生活性成分和其它期望成分的粉末。可选择地，本发明的组合物可在溶液中，且在递送之前或递送时可加入和/或混合适当的药学上可接受的赋形剂以提供可注射的单位剂型。优选地，本发明中使用的药学上可接受的赋形剂适用于高药物浓度，可保持适当的流动性，且如果需要可延迟吸收。
本发明的药物组合物的最佳给药途径的选择会受到几个因素的影响，包含组合物中活性分子的物理化学性质、临床表现的紧迫性和活性分子的血浆浓度与期望的治疗效果之间的关系。例如，可与载剂一起制备本发明的单克隆中和抗体，其中载剂将保护它们以防止快速释放(诸如控释制剂)，该载剂包含植入物、透皮贴剂和微胶囊化的递送系统。可在本发明中使用生物可降解的、生物相容的聚合物，诸如乙烯醋酸乙烯酯、聚酸酐、聚乙醇酸、胶原、聚正酯和聚乳酸。进一步地，单克隆中和抗体可包被有防止抗体失活的材料或化合物、或与这样的材料或化合物同时给药。例如，单克隆中和抗体可与适当的载剂(例如脂质体或稀释剂)一起给药。
本发明的药物组合物的给药途径可分成口服给药和胃肠外给药。优选的给药途径是静脉注射，但并不限于此。
口服剂型可被配制成片剂、锭剂、糖锭、水性或油性悬浮液、分散的粉末或颗粒、乳剂、硬胶囊、软胶胶囊、糖浆或酏剂、丸剂、糖衣丸、液体、凝胶或膏剂。这些制剂可包含药学赋形剂，其中药学赋形剂包括但并不限于：成粒剂和崩解剂，结合剂，润滑剂，防腐剂，着色剂、调味剂或甜味剂，植物油或矿物油，湿润剂，和增稠剂。
由于胃肠外给药的制剂可为水性或非水性的等渗无菌无毒的注射或灌注溶液或悬浮液的形式。所述溶液或悬浮液可包括所采用的剂量和浓度对接受者无毒的试剂，诸如1，3-丁二醇、林格氏溶液、汉克溶液、等渗的氯化钠溶液、油、脂肪酸、局部的麻醉剂、防腐剂、缓冲液、粘度或溶解性增高的试剂、水溶性的抗氧化剂、油溶性的抗氧化剂和金属螯合剂。
由于胃肠外给药的制剂可为水性或非水性的等渗无菌无毒的注射或灌注溶液或悬浮液的形式。所述溶液或悬浮液可包括所采用的剂量和浓度对接受者无毒的试剂，诸如1，3-丁二醇、林格氏溶液(Ringer’s solution)、汉克溶液(Hank’s solution)、等渗的氯化钠溶液、油、脂肪酸、局部的麻醉剂、防腐剂、缓冲液、粘度或溶解性增高的试剂、水溶性的抗氧化剂、油溶性的抗氧化剂和金属螯合剂。
所述药物组合物还可包括一种或多种其它治疗剂。治疗剂可包含抗体、小分子、有机或无机化合物、酶、多核苷酸序列等。
根据本发明的又一个方面，本发明提供了一种检测产品，包括前面所述的诊断有效量的单克隆中和抗体或其抗原结合片段、和/或前面所述的结合物、和/或前面所述的结合物、和/或前面所述的偶联物、和/或前面所述的组合物。
本发明提供的检测产品能够用于检测破伤风毒素感染以及破伤风梭菌感染。具体地，本发明提供的检测产品通过抗体测定流体、细胞或组织样品中的破伤风毒素水平。同时，本发明实施例提供的检测产品还能够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为血液、尿、唾液、肺痰、灌洗或淋巴样品。
根据本发明的又一个方面，本发明提供了一种编码前面所述的单克隆中和抗体或其抗原结合片段的DNA分子。
可以利用本领域任一已知的方法得到DNA分子序列，例如，如果已知抗体的核苷酸序列，就可以从化学合成的寡核苷酸装配出编码所述抗体的DNA分子序列。
或者，可以从来自合适来源的核酸中产生编码抗体的DNA分子。如果无法得到含有编码特定抗体的DNA分子的克隆，但已知所述抗体分子的序列，那么 可以通过化学合成得到编码所述免疫球蛋白的DNA分子，或利用与所述序列的3’和5’末端可杂交的合成引物从合适的来源通过PCR扩增得到编码所述免疫球蛋白的DNA分子，或通过利用特异于所述特定基因序列的寡核苷酸探针通过克隆得到编码所述免疫球蛋白的DNA分子，以例如从cDNA文库中鉴定出编码所述抗体的cDNA克隆。然后可以利用本领域任一熟知的方法将PCR所产生的扩增DNA分子克隆到可复制的克隆载体内。
根据本发明的又一个方面，本发明提供了一种重组表达载体，包括前面所述的DNA分子。
构建本发明的重组表达载体的表达载体包括(但不限于)由Celltrion Inc.(韩国)生产的MarEx表达载体；市场上可广泛买到的pCDNA载体；F、R1、RP1、Col、pBR322、ToL、Ti载体；粘粒；噬菌体，诸如λ噬菌体、λ形噬菌体、M13噬菌体、Mu噬菌体、P1噬菌体、P22噬菌体、Qμ噬菌体、T-偶数噬菌体、T2噬菌体、T4噬菌体、T7噬菌体等；植物病毒。本发明中可使用本领域技术人员已知的各种表达载体的任意一种，且表达载体的选择依赖于所选择的宿主细胞的性质。宿主细胞中载体的导入可通过(但并不限于)磷酸钙转染、病毒感染、DEAE-葡聚糖介导的转染、脂质体转染或电穿孔实现，且本领域的任何技术人员可选择和使用适用于所用的表达载体和宿主细胞的导入方法。优选地，上述载体包含一种或多种选择标记，但并不限于此，且还可使用不包含选择标记的载体。选择标记的选择可依赖于选择的宿主细胞(如本领域的技术人员公知的)，但这对于本发明并不是关键性的。
为了促进本发明的DNA分子的纯化，可将标签(tag)序列插入表达载体中。标签的实例包括(但不限于)六个组氨酸标签、血凝素标签、myc标签或FLAG标签。可在本发明中使用本领域的技术人员已知的促进纯化的任何标签。
根据本发明的又一个方面，本发明提供了一种重组细胞，所述重组细胞经由前面所述的表达载体转化或转染宿主细胞而来。
在本发明中可使用本领域技术人员已知的可用作宿主细胞的任何细胞。可用于本发明的宿主细胞包括但不限于微生物，例如经含有抗体编码序列的重组噬菌体DNA、质粒DNA或粘粒DNA表达载体转化的细菌(例如大肠杆菌(E.coli)、 枯草芽孢杆菌(B.subtilis))；经含有抗体编码序列的重组酵母表达载体转化的酵母菌例如酵母属(Saccharomyces)、毕赤酵母属(Pichia))；经含有抗体编码序列的重组病毒表达载体(例如杆状病毒)感染的昆虫细胞系统；经重组病毒表达载体(例如花椰菜花叶病毒(CaMV)；烟草花叶病毒(TMV)感染的或经含有抗体编码序列的重组质粒表达载体(例如Ti质粒)转化的植物细胞系统；或携带含有来源于哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或来源于哺乳动物病毒的启动子(例如，腺病毒晚期启动子、痘苗病毒7.5K启动子)的重组表达构建体的哺乳动物细胞系统(例如COS、CHO、BHK、293、3T3细胞)。
根据本发明的又一个方面，本发明还提供了检测破伤风毒素和破伤风梭菌在样品中的存在或其水平的方法，其包括使用本发明的单克隆中和抗体或其抗原结合片段。在一个优选的实施方案中，本发明的单克隆中和抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆中和抗体或其抗原结合片段。所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品或者来自患者的样品)。
根据本发明的又一个方面，本发明还提供了诊断受试者是否感染了破伤风毒素或破伤风梭菌感染的方法，其包括：使用本发明的单克隆中和抗体或其抗原结合片段检测破伤风毒素或破伤风梭菌感染在来自所述受试者的样品中的存在。在一个优选的实施方案中，本发明的单克隆中和抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆中和抗体或其抗原结合片段。
根据本发明的又一个方面，本发明还提供了用于中和样品中破伤风毒素的毒力的方法，其包括，将包含破伤风毒素的样品与本发明的单克隆中和抗体或其抗原结合片段接触。此类方法可以用于治疗目的，或非治疗目的(例如所述样品是细胞样品，而不是患者或来自患者的样品)。
根据本发明的又一个方面，本发明还提供了前面所述的单克隆中和抗体或其抗原结合片段、和/或前面所述的缀合物、和/或前面所述的结合物、和/或前面所述的偶联物、和/或前面所述的组合物在制备破伤风毒素或破伤风梭菌感染检测产品中的应用。
优选地，所述检测产品是试剂盒、酶标板、或芯片。
根据本发明的又一个方面，本发明还提供了前面所述的单克隆中和抗体或其抗原结合片段、和/或前面所述的缀合物、和/或前面所述的结合物、和/或前面所述的偶联物、和/或前面所述的组合物在制备预防或治疗破伤风毒素或破伤风梭菌感染引起的疾病药物中的应用。
根据本发明的又一个方面，本发明还提供了前面所述的DNA分子、前面所述的重组表达载体、前面所述的重组细胞在制备预防或治疗破伤风毒素或破伤风梭菌感染引起的疾病药物中的应用。
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“单克隆中和抗体”是指具有中和活性的单克隆抗体，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。
本发明的单克隆中和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中和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中和抗体：与亲本单克隆中和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。与亲本单克隆中和抗体相比，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，与亲本单克隆中和抗体相比，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))改变亲本单克隆中和抗体或其部分，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。
本发明优点和有益效果如下：
本发明通过高通量全人源单克隆抗体研发综合技术平台开发出了特异性的全人源抗破伤风毒素单克隆中和抗体。
本发明研发的全人源抗破伤风毒素单克隆中和抗体能够用于破伤风梭菌感染的预防和治疗。
本发明研发的全人源抗破伤风毒素单克隆中和抗体能够用于破伤风梭菌感染的检测。
本发明研发的全人源抗破伤风毒素单克隆中和抗体与破伤风毒素具有极强的亲和力、且具有极强的中和活性，安全且高效，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。
附图说明
图1显示利用SDS-PAGE和Western blot检测TRN0010抗体表达纯化的电泳图，其中，A：SDS-PAGE图；B：Western blot图；
图2显示利用SDS-PAGE和Western Blot检测TRN0012抗体表达纯化的电泳图，其中，A：SDS-PAGE图；B：Western Blot图；
图3显示利用SDS-PAGE和Western Blot检测TRN0011抗体表达纯化的电泳图，其中，A：SDS-PAGE图；B：Western Blot图；
图4显示利用SDS-PAGE和Western Blot检测TRN1015抗体表达纯化的电泳图，其中，A：SDS-PAGE图；B：Western Blot图；
图5显示利用ELISA检测TRN0010抗体的中和活性的结果图；
图6显示利用ELISA检测TRN0012抗体的中和活性的结果图；
图7显示利用ELISA检测TRN0011抗体的中和活性的结果图；
图8显示利用ELISA检测TRN1015抗体的中和活性的结果图；
图9显示TRN0010抗体的亲和性检测结果图；
图10显示TRN0012抗体的亲和性检测结果图；
图11显示TRN0011抗体的亲和性检测结果图；
图12显示TRN1015抗体的亲和性检测结果图；
图13显示TRN0010抗体对动物保护作用的结果图；
图14显示TRN0012抗体对动物保护作用的结果图；
图15显示TRN0011抗体对动物保护作用的结果图；
图16显示TRN1015抗体对20倍剂量的破伤风毒素LD50的保护作用图；
图17显示TRN1015抗体对60倍剂量的破伤风毒素LD50的保护作用图。
具体实施方式
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 全人源抗破伤风毒素单克隆中和抗体的制备
1、分选细胞
从注射了1500IU破伤风类毒素(疫苗)的健康志愿者身上采集血液样本。然后用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分选出浆细胞，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1小时；按以下参数进行PCR扩增：95℃15min；95℃1min，55℃1min，72℃1min，30cycles；72℃10min；4℃5min。产物cDNA-20℃保存。
以上述cDNA和相应引物为模板，PCR扩增抗破伤风毒素全人源抗体基因。50μL体系中含有5μl的RT反应产物，HotStarTaq Plus酶(Invitrogen，Carlsbad，CA)，dNTPs，及0.5μM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、抗破伤风毒素全人源单克隆中和抗体真核表达载体的构建
(1)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克隆的方法插入pcDNA3.3(+/-)表达载体(购自Invitrogen公司)的单克隆酶切位点，构建抗破伤风毒素全人源单克隆中和抗体的表达载体。
(2)用上述连接产物转化DH5α感受态细菌，在含有氨苄青霉素的平板上37℃培养过夜。
(3)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；
(4)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。
结果显示，本发明构建的抗破伤风毒素单抗重链/轻链的重组表达载体其序列正确。
4、破伤风痉挛毒素全人源单克隆中和抗体的表达和鉴定
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。利用DNA转染试剂盒PolyFect将抗破伤风毒素全人源抗体重链/轻链表达载体共转染293细胞，以未转染质粒的空细胞对照。具体操作参见说明书。培养96h后，采用常规ELISA法，用HRP标记的羊抗人IgG抗破伤风毒素全人源抗体的表达及抗体对破伤风疫苗(抗原)的特异性识别。
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100uL 4℃过夜包被，用封闭液常温封闭2h。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。
结果显示，共转染表达质粒载体的293细胞成功表达全人源抗体，能有效识别破伤风疫苗(抗原)，而未转染表达质粒的293细胞培养上清不能识别破伤风疫苗(抗原)。即瞬时转染293成功表达了能特异性识别破伤风疫苗(抗原)的抗破伤风毒素全人源抗体。
5、破伤风毒素全人源单克隆中和抗体批量生产与纯化
将中和实验鉴定的有中和活性的编号为TRN0010抗体重链与轻链的表达载体(其中，重链可变区的氨基酸序列如SEQ ID NO:7所示；抗体轻链可变区的氨基酸序列如SEQ ID NO:8所示)共转染293细胞；
将中和实验鉴定的有中和活性的编号为TRN0012抗体重链与轻链的表达载体(其中，重链可变区的氨基酸序列如SEQ ID NO:17所示；抗体轻链可变区的氨基酸序列如SEQ ID NO:18所示)共转染293细胞；
将中和实验鉴定的有中和活性的编号为TRN0011抗体重链与轻链的表达载体(其中，重链可变区的氨基酸序列如SEQ ID NO:22所示；抗体轻链可变区的氨基酸序列如SEQ ID NO:26所示)共转染293细胞；
将中和实验鉴定的有中和活性的编号为TRN1015抗体重链与轻链的表达载体(其中，重链可变区的氨基酸序列如SEQ ID NO:33所示；抗体轻链可变区的氨基酸序列如SEQ ID NO:34所示)共转染293细胞；
转染后6-8小时换新鲜培养基，并在37℃8％CO 2培养箱中培养96小时。收集转染上清，4000rpm离心1小时，利用蛋白(Protein)A亲和层析法进行纯化。利用SDS-PAGE和Western Blot实验检验抗体的表达及纯化情况，结果见图1-4，证实得到较纯蛋白，并可清晰观察到解链后的抗体轻、重链，注：图1-4A和图1-4B的1泳道代表的未解链蛋白，图1-4A和图1-4B的2泳道代表的蛋白Marker，图1-4A和图1-4B的3泳道代表的解链蛋白。
实施例2 单克隆中和抗体的中和活性检测
以破伤风毒素为抗原，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。将表达纯化的抗体按比例稀释后作为一抗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100μl/孔，常温避光放置5min后，用2M硫酸中止反应，用450nm波长进行比色检测，分析结果。
结果：
结果如图5-8所示，把表达纯化的抗体进行不低于10,000倍稀释后，抗体浓度约低至0.0002μg/ml，表达纯化的抗体依然可以与抗原结合，显示其具有极强的结 合活性和中和活性。
实施例3 单克隆中和抗体的亲和性检测
先进行CM5芯片偶联捕获分子，再活化芯片的葡聚糖表面，以进样时间确定偶联量，最后利用CM5芯片捕获分子捕获配体：将制备的全人源抗破伤风中和抗体作为配体。破伤风类毒素用HBS-EP缓冲液稀释作为分析物，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软件进行单抗与破伤风类毒素(抗原)结合的亲和力和动力学分析。
结果如图9-12和表1所示，本发明的中和抗体对破伤风毒素的平衡解离常数小于10 -9mol，表明该其具有很高的亲和活力。
表1全人源抗破伤风毒素单克隆中和抗体的亲和力测定结果
抗体名称 ka(1/Ms) kd(1/s) KD(M)
TRN0010 4.15E+04 1.53E-04 3.68E-09
TRN0012 8.06E+04 3.28E-04 4.07E-09
TRN0011 4.27E+04 3.87E-05 9.05E-10
TRN1015 3.88E+04 9.40E-05 2.43E-09
实施例4 抗破伤风毒素单克隆中和抗体对动物的保护作用实验
(1)待检抗破伤风毒素单抗的稀释
用稀释液将待检单抗稀释成100μg/ml(单抗浓度＞1mg/ml)，用稀释液以3倍倍比稀释，备用。
(2)标准抗毒素稀释：
标准抗毒素用生理盐水溶解液与中性甘油(经116℃10分钟高压蒸汽灭菌)等量混合，然后用稀释液稀释，使每ml含0.5IU(即5个IU/10)；即与毒素等量混合后每0.4ml注射量中含IU/10。标准抗毒素原液的一次吸取量不应少于0.5ml。
(3)毒素稀释：
自中检所购买的冻干粉状毒素应用生理盐水溶解液与中性甘油(经116℃10分钟高压蒸汽灭菌)等量混合，然后用稀释液稀释毒素至使用量。
(4)半数致死量的测定(LD50)
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LD50，实验组使用20倍于LD50量甚至60倍于LD50量。
(5)单抗效价测定
混合定量吸取已稀释之标准抗破伤风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(20倍于LD50量或60倍于LD50量)，混合均匀，加塞，37℃结合1小时，立即注射。
取健康实验小白鼠140只，每组4只。将上述中混合物分别皮下注射体重为18～22g小白鼠腹部，每只注射0.4ml(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)。每日上、下午各观察一次，连续一周，记录小白鼠发病与死亡情况。
结果如图13-16所示，在20倍LD50剂的破伤风毒素攻击时，阴性对照组小鼠在48h内全部死亡，而除了TRN0010抗体浓度为1.85μg/mL的实验组小鼠在存活7天后死亡，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均全部存活。说明低剂量单抗低至0.62μg/mL可与10IU/ml的标准抗毒素的效价相当，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。本发明的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。说明本发明的单抗能够有效的中和小鼠的体内毒素，保护小鼠，具有体内活性。
图17结果显示，在更大剂量的破伤风毒素(60倍LD50)攻击下，阴性对照组小鼠在24h内全部死亡，低剂量组(0.62ug/ml、1.85ug/ml)的实验小鼠在7天内死亡，中、高剂量的实验组(5.56ug/ml、16.67ug/ml和50ug/ml剂量)小鼠在7天内全部存活。表明本发明的单抗能够防御高剂量毒素的攻击，说明了本发明的单抗的体内中和能力极强。
虽然以上仅描述了本发明的具体实施方式范例，但是本领域的技术人员应当 理解，这些仅是举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均落入本发明的保护范围。

Claims (12)

  1. 一种针对破伤风毒素的全人源单克隆中和抗体或其抗原结合片段，其特征在于，所述单克隆中和抗体包含至少一个含有三个CDR的重链可变区和至少一个含有三个CDR的轻链可变区；
    其中所述重链可变区CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO:1、2、3、9、10、11、19、20、21、27、28、29所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO:4、5、6、13、14、15、23、24、25、30、31、32所示；或者经取代、缺失或添加一个或多个氨基酸；或与所述可变区CDR1、CDR2、CDR3所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
    优选地，
    所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6所示；
    所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15所示；
    所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25所示；
    所述单克隆中和抗体的重链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29所示；轻链可变区CDR1、CDR2、CDR3的氨基酸序列选自SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32所示。
  2. 根据权利要求1所述的单克隆中和抗体或其抗原结合片段，其特征在于，所述单克隆中和抗体重链可变区的序列如SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:22、SEQ ID NO:33所示，或者经取代、缺失或添加一个或多个氨基酸；或 与重链可变区所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；轻链可变区的序列如SEQ ID NO:8、SEQ ID NO.18、SEQ ID NO:26、SEQ ID NO:34所示，或者经取代、缺失或添加一个或多个氨基酸；或与轻链可变区所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
    优选地，
    所述单克隆中和抗体重链可变区的序列如SEQ ID NO:7所示，抗体轻链可变区的序列如SEQ ID NO:8所示；
    所述单克隆中和抗体重链可变区的序列如SEQ ID NO:17所示，轻链可变区的序列如SEQ ID NO:18所示；
    所述单克隆中和抗体重链可变区的序列如SEQ ID NO:22所示，轻链可变区的序列如SEQ ID NO:26所示；
    所述单克隆中和抗体重链可变区的序列如SEQ ID NO:33所示，轻链可变区的序列如SEQ ID NO:34所示。
  3. 根据权利要求1或2所述的单克隆中和抗体或其抗原结合片段，其特征在于，所述单克隆中和抗体或其抗原结合片段免疫特异性地结合破伤风毒素，所述单克隆中和抗体或其抗原结合片段以不高于10 -6M的平衡解离常数与破伤风毒素和/或破伤风梭菌解离。
  4. 一种编码权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段的DNA分子。
  5. 一种重组表达载体，包括权利要求4所述的DNA分子。
  6. 一种重组细胞，所述重组细胞经由权利要求5所述的重组表达载体转化或转染宿主细胞而来。
  7. 一种结合物，其特征在于，所述结合物包含权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段以及共价连接到所述单克隆中和抗体或其抗原结合片段上的化学标记或生物标记；优选地，所述化学标记包括同位素标记、免疫毒素标记、化学药物标记；所述生物标记包括生物素标记、亲和素标记、酶标记、荧光化合物、电子转移剂。
  8. 一种偶联物，其特征在于，所述偶联物由权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段、和/或权利要求7所述的结合物与固体介质或半固体介质偶联形成。
  9. 一种组合物，其特征在于，所述组合物包括权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段、和/或权利要求7所述的结合物、和/或权利要求8所述的偶联物。
  10. 一种检测产品，其特征在于，所述检测产品包括权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段、和/或权利要求7所述的结合物、和/或权利要求8所述的偶联物、和/或权利要求9所述的组合物；优选地，所述检测产品包括试剂盒、酶标板、或芯片。
  11. 一种方法，所述方法包括使用权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段；所述方法为以下方法中的任一种：
    (1)检测破伤风毒素和破伤风梭菌在样品中存在或其水平的方法；
    (2)诊断受试者是否感染了破伤风毒素或破伤风梭菌感染的方法；
    (3)用于中和样品中破伤风毒素的毒力的方法。
  12. 一种应用，所述应用为以下应用中的任一种：
    (1)权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段、和/或权利要求7所述的结合物、和/或权利要求8所述的偶联物、和/或权利要求9所述的组合物在制备破伤风毒素或破伤风梭菌感染检测产品中的应用；
    (2)权利要求1-3中任一项所述的单克隆中和抗体或其抗原结合片段、和/或权利要求7所述的结合物、和/或权利要求8所述的偶联物、和/或权利要求9所述的组合物在制备预防或治疗破伤风毒素或破伤风梭菌感染引起的疾病药物中的应用；
    (3)权利要求4所述的DNA分子、权利要求5所述的重组表达载体、权利要求6所述的重组细胞在制备预防或治疗破伤风毒素或破伤风梭菌感染引起的疾病药物中的应用。
PCT/CN2018/124958 2017-12-29 2018-12-28 针对破伤风毒素的全人源单克隆中和抗体及其应用 WO2019129214A1 (zh)

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