WO2019128120A1 - Anticorps neutralisant entièrement humain pour la lutte contre la toxine tétanique - Google Patents

Anticorps neutralisant entièrement humain pour la lutte contre la toxine tétanique Download PDF

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WO2019128120A1
WO2019128120A1 PCT/CN2018/090490 CN2018090490W WO2019128120A1 WO 2019128120 A1 WO2019128120 A1 WO 2019128120A1 CN 2018090490 W CN2018090490 W CN 2018090490W WO 2019128120 A1 WO2019128120 A1 WO 2019128120A1
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antibody
tetanus toxin
tetanus
toxin
infection
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PCT/CN2018/090490
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English (en)
Chinese (zh)
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廖化新
袁晓辉
王月明
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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 WO2019128120A1 publication Critical patent/WO2019128120A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C07K2317/20—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

Definitions

  • the invention belongs to the field of cellular immunology and genetic engineering, and relates to a whole human antibody against tetanus toxin and application thereof.
  • Tetanus is an acute disease caused by Clostridium tetani infection.
  • the main clinical symptoms are closed jaws, and local or systemic muscles are paroxysmal or strong.
  • Clostridium tetanus infects the wound and reproduces under hypoxic conditions and produces the exotoxin, tetanus neurotoxin (total tetanus toxin), which is mostly carried by the bloodstream into the blood system and spreads to the body.
  • the entire neural pathway affects the central nervous system.
  • Clostridium tetani has no invasiveness and only grows in contaminated local tissues.
  • the whole body striated tendon is caused.
  • the toxin is absorbed through the motor endplate, along the nerve fiber space to the anterior horn of the spinal cord, to the brain stem, and can also be absorbed by the lymph, and reaches the central nervous system through the blood vessel.
  • the toxin binds to the ganglionin in the nervous tissue, blocks the inhibitory synaptic terminals of the spinal cord, prevents the release of the impulse-inducing mediators glycine and gamma-aminobutyric acid, thereby disrupting the normal inhibitory impulse between the upper and lower neuron. Delivery, leading to super-reflective reactions (excessive excitability) and striated tendon.
  • tetanus vaccine can completely prevent the clinical symptoms of tetanus, it is still very common to have a tetanus infection due to lack of vaccination or an overdue of immune effects.
  • Tetanus toxin is highly toxic and rapid. When the patient is diagnosed with infection with tetanus, a large number of bacteria and toxins are already present in the body, and the use of antibiotics is too late to save the lives of patients. Therefore, the only effective method for prevention and treatment of tetanus is to timely inject anti-tetanus toxin antibodies, neutralize the toxins to inactivate the toxins, and sterilize the toxins from the body by mediated by antibody receptors. Therefore, the preparation of tetanus toxins with high immunogenicity, high affinity, high specificity and high titer is very important in the art.
  • the main object of the present invention is to provide a fully human monoclonal antibody and antigen-binding fragment thereof against tetanus toxin without high immunogenicity, high affinity, high specificity and high titer. And methods of encoding the sequences thereof, producing their cell lines, and applying them for diagnosis, prevention or treatment.
  • tetanus toxin refers to the secreted protein tetanus toxin produced by Clostridium tetani under anaerobic conditions, which is well known in the art (see the published sequence of the NCBI GENEBANK database, etc.).
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the term “antibody” is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibodies e.g, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD IgE or IgM antibodies.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the term “antibody” is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibodies antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antibody it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • mAb and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, in addition to a natural mutation that may occur spontaneously, A group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • neutralizing antibody refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence of a target virus (eg, the ability to infect a cell).
  • the term "immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the mode of immunization.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (KD) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction that is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • an antibody eg, the monoclonal antibody TRN0011 of the invention
  • an antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or
  • a smaller dissociation equilibrium constant (KD) binds to an antigen (eg, tetanus toxin), for example, as measured using a surface plasmon resonance (SPR) in a BIACORE instrument.
  • SPR surface plasmon resonance
  • the terms “isolated” or “isolated” refer to artificially obtained from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • separation the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease such as a tetanus infection or a disease associated with tetanus infection
  • an effective amount to prevent a disease is an amount sufficient to prevent, arrest, or delay the onset of a disease, such as a tetanus infection or a disease associated with tetanus infection
  • treating an effective amount of a disease is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • a first aspect of the invention provides a monoclonal antibody or antigen-binding fragment thereof which is capable of specifically tetanus toxin, wherein the monoclonal antibody comprises a heavy chain variable region (VH) selected from the group consisting of:
  • the monoclonal antibody comprises a light chain variable region (VL) selected from the group consisting of:
  • VL CDR1 as shown in SEQ ID NO. 5;
  • VLCDR2 as shown in SEQ ID NO. 6;
  • the monoclonal antibody comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO. 4; and/or
  • the amino acid sequence is the light chain variable region (VL) set forth in SEQ ID NO.
  • the antibody comprises all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.
  • the antibody is a fully humanized antibody.
  • the monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or Smaller KD combined with tetanus toxin.
  • the neutralizing antibodies to the tetanus toxin of the present invention also include functional variants thereof. If a variant of a monoclonal antibody competes with a monoclonal antibody of the invention to specifically bind to a fragment of tetanus toxin, they are considered to be functional variants of the monoclonal antibodies of the invention. Specifically, if a functional variant binds to tetanus toxin or a fragment thereof and has neutralizing activity against the subtype or fragment, the functional variant is considered to be a functional variant of the invention.
  • Functional variants include, but are not limited to, chemically and/or biochemically modified derivatives that are substantially similar in the primary structural sequence but that are not included in the parental monoclonal antibodies of the invention, such as in vitro or in vivo.
  • modifications 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.
  • Functional variants intended to fall within the scope of the invention have at least about 50 to 99%, preferably at least about 60 to 99%, more preferably at least about 80 to 99%, or even more, of the parent monoclonal antibody as defined herein.
  • Amino acid sequence homology of at least about 90 to 99%, especially at least about 95 to 99%, and particularly at least about 97 to 99% is preferred.
  • 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.
  • a second aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the first aspect of the invention.
  • the nucleic acid molecule of the present invention comprises all of the nucleic acid molecules obtained by translating the amino acid sequence of the antibody of the present invention into a polynucleotide sequence by a method known to those skilled in the art.
  • a plurality of polynucleotide sequences having an open reading frame (ORF) can be prepared, and the prepared polynucleotide sequence is included within the scope of the nucleic acid molecule of the present invention.
  • a third aspect of the invention provides an expression vector comprising the nucleic acid molecule of the second aspect of the invention.
  • the expression vector of the present invention includes, but is not limited to, a MarEx expression vector produced by Celltrion Inc. (Korea); a commercially available pCDNA vector; F, R1, RP1, Col, pBR322, ToL, Ti carrier; 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. 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.
  • 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 a selectable 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.
  • 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.
  • a fourth aspect of the invention provides a host cell comprising the nucleic acid molecule of the second aspect of the invention, or the expression vector of the third aspect of the invention.
  • Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin.
  • mammalian cells it is preferably selected from, but not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, and HEK293T.
  • One of the groups consisting of cells serves as a host cell. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.
  • a method of producing a monoclonal antibody or antigen-binding fragment thereof of the present invention which comprises culturing a host cell of the present invention under suitable conditions, and recovering the monoclonal antibody of the present invention from the cell culture or Antigen-binding fragment.
  • a fifth aspect of the invention provides a fifth aspect of the invention provides a pharmaceutical composition comprising the antibody of the first aspect of the invention.
  • composition further comprises a pharmaceutically acceptable carrier.
  • compositions of the invention 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.
  • a sixth aspect of the invention provides the application of any of the following:
  • nucleic acid molecule of the second aspect of the invention for the preparation of a medicament against tetanus toxin or Clostridium tetanium infection;
  • the invention also provides a kit comprising a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the kit further comprises a second antibody that specifically recognizes a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the second antibody further comprises a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials and enzymes (e.g., horseradish peroxidase), and the like.
  • the invention also provides a method of detecting the presence or level of tetanus toxin live Clostridium tetani in a sample comprising the use of a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal antibody or antigen-binding fragment thereof of the 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 invention provides a method of diagnosing whether a subject is infected with a tetanus toxin or a Clostridium tetani infection, comprising: detecting tetanus toxin or tetanus using the monoclonal antibody or antigen-binding fragment thereof of the invention Clostridium infection is present in samples from the subject.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the method further comprises detecting a monoclonal antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label.
  • a monoclonal antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting the presence or level of tetanus toxin or Clostridium tetanium in a sample, Or to diagnose whether the subject is infected with tetanus.
  • the invention provides a method for neutralizing the virulence of tetanus toxin in a sample comprising contacting a sample comprising tetanus toxin with a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • a monoclonal antibody of the invention or an antigen binding fragment thereof 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).
  • an anti-tetanus toxin monoclonal antibody or antigen-binding fragment thereof of the invention for the preparation of a diagnostic agent for Clostridium tetani infection.
  • 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.
  • Amino acid sequence inserts include amino and/or carboxyl terminal fusions of one residue in length to polypeptides containing one hundred or more residues, as well as intrasequence inserts having single or multiple amino acid residues.
  • terminal inserts include anti-tetanus toxin antibodies having an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide.
  • Other insertional variants of the anti-tetanus toxin antibody molecule include fusions of the N-terminal or C-terminal end of the anti-tetanus toxin antibody with an enzyme (eg, for ADEPT) or a polypeptide that increases the serum half-life of the antibody.
  • the present invention encompasses sequences having a certain degree of sequence identity or sequence homology to the amino acid sequence of the antibody or any nucleotide sequence encoding the antibody.
  • “homology” may be equivalent to “consistency”. ".
  • 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 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 implants, transdermal patches, and microencapsulated 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 antibody may be coated with a material or compound that prevents inactivation of the antibody, or concurrently with such a material or compound.
  • a monoclonal 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.
  • Figure 1 is a diagram showing the results of SDS-PAGE and Western Blot detection of the antibody of the present invention; wherein, Figure A is an SDS-PAGE diagram; and Figure B is a Western Blot diagram;
  • Figure 2 is a graph showing the results of detection of neutralizing activity of the antibody and tetanus standard toxin of the present invention
  • Figure 3 is a graph showing the affinity activity of the antibody of the present invention and the tetanus standard toxin;
  • Fig. 4 is a graph showing the results of the in vivo protection test of the antibody of the present invention.
  • PBMC tetanus toxoid
  • plasma cells were then sorted from PBMC using a BD FACSria flow cytometer, and well-formed individual cells were placed in 96-well PCR plates, each containing a memory B cell. Store at -80 °C for later use.
  • 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 total human antibody gene was PCR amplified using the above cDNA and corresponding primers as templates.
  • PCR parameter setting 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.
  • DH5 ⁇ competent bacteria were transformed with the above ligation product, and cultured on a plate containing ampicillin at 37 ° C overnight.
  • the anti-tetanus toxin full 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. After 96 hours of culture, the expression of HRP-labeled goat anti-human IgG anti-tetanus toxin full human antibody and specific recognition of the antibody to tetanus standard toxin antigen were performed by conventional ELISA.
  • An expression vector for the heavy chain and light chain of the TRN0011 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. 4; the antibody light chain variable region is 293 cells were co-transfected with the amino acid sequence shown in SEQ ID NO. 8 and fresh medium was changed 6-8 hours after transfection and cultured for 96 hours in a 37 ° C 8% CO 2 incubator. The transfection supernatant was collected, centrifuged, and purified by Protein A affinity chromatography.
  • the binding activity of the expressed purified antibody was detected by the same ELISA method as mentioned above: the tetanus standard toxin was used as the antigen, and the antigen was 10-fold diluted with the coating solution, and then coated with a 96-well ELISA plate, 100 ⁇ l per well, 4 ° C. Overnight coating was blocked with blocking solution at room temperature for 2 h.
  • the antibody of the present invention was diluted in a ratio and incubated as a primary antibody at 37 ° C for 2 h, and incubated with HRP/anti-His-tag (diluted 1:2000) as a secondary antibody at 37 ° C for 1 h, and a substrate coloring solution of 100 ⁇ l/well was added. After standing at room temperature for 5 minutes in the dark, the reaction was stopped with 2 M sulfuric acid, and colorimetric with a wavelength of 450 nm, and the results were analyzed.
  • the TNA0011 antibody was able to neutralize the antigen and had extremely strong neutralizing activity.
  • the anti-human FC secondary antibody was first coupled, and the anti-tetanus monoclonal antibody was captured. Finally, different concentrations of tetanus toxin protein were used as the analyte.
  • the tetanus standard toxin 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. Each concentration was used as one cycle, and after completion of one cycle, the chip was regenerated with 3 mol/L of magnesium chloride to return to the state of the original unbound antigen. Affinity and kinetic analysis of mAb binding to tetanus toxoid (antigen) was performed using BiaCore X-100 System software.
  • the monoclonal antibody to be tested was diluted with a diluent to 100 ⁇ g/ml (monotherapy concentration > 1 mg/ml), that is, the concentration of the monoclonal antibody per 50 ml injection amount was 50 ⁇ g/ml after mixing with the toxin.
  • the standard antitoxin is mixed with physiological saline solution and neutral glycerin (autoclaved at 116 ° C for 10 minutes), and then diluted with a diluent to make 0.5 IU per 1 ml (ie 5 IU/10); 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 LD 50 was calculated based on the experimental results, and the experimental group used 20 times the amount of LD 50 .
  • the diluted standard antitoxin and the different dilutions of the monoclonal antibody to be tested were separately mixed into small test tubes, and an equal amount of the diluted test toxin was added to each tube, uniformly mixed, stoppered, and combined at 37 ° C for 1 hour, and immediately injected.
  • mice Twenty-eight healthy mice were randomly divided into 7 groups. 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.
  • Results The results are shown in Fig. 4.
  • the negative control mice all died within 48 hours, and the mice of the experimental group with the concentration of the monoclonal antibody as low as 0.62 ⁇ g/mL survived. It shows that the dose of the monoclonal antibody of the present invention is 0.62 ⁇ g/mL, which is equivalent to the standard anti-toxin titer (10 IU/ml), and can effectively protect the animal against the lethal dose of tetanus toxin attack, which is basically consistent with the protection of the standard antitoxin.
  • the actual amount of the monoclonal antibody of the present invention is much lower than that of the standard antitoxin, which indicates that the anti-tetanus toxin full human monoclonal antibody of the present invention has an extremely potent potency effect against tetanus toxin.
  • the present invention provides an anti-tetanus toxoid monoclonal antibody comprising the heavy chain CDRs 1 to 3 regions represented by SEQ ID NOS. 1 to 3 and the light chain CDRs 1 to 3 regions represented by SEQ ID NOS.
  • the use of antibodies against tetanus toxin in the prevention and treatment of diseases associated with tetanus infection and their use in the detection of tetanus infection are also disclosed.
  • the antibody against tetanus toxin provided by the invention is a whole human monoclonal antibody, has no immunogenicity, has good specificity, high affinity and high titer, and solves the prior art for prevention and treatment of tetanus infection.
  • the short board problem has strong industrial applicability.

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Abstract

L'invention concerne un anticorps neutralisant entièrement humain pour la lutte contre la toxine tétanique. Plus particulièrement, l'invention concerne un anticorps monoclonal pour la lutte contre une anatoxine tétanique, ayant des régions CDR1-3 de chaîne lourde telles que représentées dans SEQ ID NO. 1 à 3 et des régions CDR1-3 de chaîne légère telles que représentées dans les SEQ ID NO. 5 à 7. L'invention concerne également l'utilisation d'un anticorps pour lutter contre la toxine tétanique dans la prévention et le traitement de maladies liées à une infection par le tétanos, et l'utilisation dans la détection d'une infection par le tétanos. L'anticorps contre la toxine tétanique est un anticorps monoclonal entièrement humain, sans immunogénicité et avec une bonne spécificité, une affinité élevée et une valence élevée.
PCT/CN2018/090490 2017-12-29 2018-06-08 Anticorps neutralisant entièrement humain pour la lutte contre la toxine tétanique WO2019128120A1 (fr)

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BR112020013094A2 (pt) * 2017-12-29 2021-03-30 Zhuhai Trinomab Biotechnology Co., Ltd. Anticorpo monoclonal neutralizante totalmente humano nativo contra toxina tetânica e suas aplicações
CN111848791B (zh) * 2020-08-25 2021-12-21 中国人民解放军军事科学院军事医学研究院 一种抗破伤风毒素的全人源中和抗体及应用

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