WO2022082918A1 - 针对破伤风毒素的抗体及其用途 - Google Patents

针对破伤风毒素的抗体及其用途 Download PDF

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WO2022082918A1
WO2022082918A1 PCT/CN2020/130111 CN2020130111W WO2022082918A1 WO 2022082918 A1 WO2022082918 A1 WO 2022082918A1 CN 2020130111 W CN2020130111 W CN 2020130111W WO 2022082918 A1 WO2022082918 A1 WO 2022082918A1
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seq
amino acid
acid sequence
variable region
antibody
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French (fr)
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刘志刚
周晓巍
刘玉兰
郝小勃
胡俊杰
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北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Publication of WO2022082918A1 publication Critical patent/WO2022082918A1/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)
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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

Definitions

  • the present invention generally relates to the fields of genetic engineering and antibody drugs; in particular, the present application relates to antibodies against tetanus toxin and uses thereof.
  • Tetanus is an acute and fatal disease caused by the neurotoxin secreted by Clostridium tetani. Humans are also susceptible to neurotoxins secreted by Clostridium tetanus and can develop disease at any age. In economically underdeveloped areas, neonatal tetanus is one of the leading causes of neonatal death. The incubation period for tetanus is usually 3-21 days, with most being around 10 days, but it can range from 1 day to several months depending on wound characteristics, extent and location. The typical clinical manifestations of tetanus are trismus, increased motor nerve center irritability, and partial or general muscle spastic paralysis. Most patients die of asphyxia and systemic organ failure.
  • tetanus Even with modern intensive care, the case fatality rate is very high, especially after severe natural disasters, the mortality rate of tetanus can reach 19% to 31%.
  • the clinical types of tetanus are divided into 3 types: generalized tetanus, localized tetanus and cephalic tetanus, of which generalized tetanus accounts for about 88%, localized tetanus accounts for about 12%, and cephalic tetanus about 1%.
  • Clostridium tetanus is a strictly anaerobic Gram-positive bacterium with flagella but no capsule. Its spores are widely present in fertilized soil, street dust, putrid sludge, animal intestines and the surface of pollutants. Clostridium tetanus mainly enters the human body through wounds. If the wound is narrow and deep, or the wound is infected with aerobic pyogenic bacteria at the same time, under anaerobic conditions, Clostridium tetanus will multiply and produce three kinds of exotoxins. Among them, tetanus toxin causes the characteristic symptoms of tetanus and stimulates the production of protective antibodies 1 .
  • Clostridium tetanus can also invade the body through small abrasions. Clostridium tetanus is divided into 10 serotypes according to the flagellar antigen agglutination reaction, but all serotypes produce the same neurotoxin, and the toxins produced by each type of strain can be neutralized by any type of antitoxin. Tetanus toxin is very toxic, second only to botulinum toxin, and the lethal dose to mice is only 2-6ng/kg.
  • Tetanus toxin also known as Tetanus neurotoxin (Tetanus neurotoxin)
  • Tetanus neurotoxin is a single-chain protein with a relative molecular mass of about 150kDa, consisting of 1315 amino acid residues, which is cleaved into disulfide bonds.
  • light chain fragment A, 50 kDa
  • heavy chain HC, 100 kDa
  • Fragment A is a zinc metalloproteinase that digests vesicle-associated membrane protein-2 (VAMP-2) after entering the cytoplasm, blocking the release of inhibitory neurotransmitters such as glycine and gamma-aminobutyric acid (GABA), causing muscle spastic paralysis 3 .
  • VAMP-2 vesicle-associated membrane protein-2
  • GABA gamma-aminobutyric acid
  • the heavy chain consists of two functional domains, the C-terminal domain (fragment C) binds to the surface of nerve cells, allowing the toxin molecules to be endocytosed into vesicles; the N-terminal domain (fragment B) passes through the vesicle membrane and integrates fragment A. Transported to neuronal cytoplasm 4 .
  • Fragment C is further divided into a C-terminal subdomain (HCC) and an N-terminal subdomain (HCN) 5 .
  • HCC C-terminal subdomain
  • HN N-terminal subdomain
  • the process of the entry of tetanus toxin into nerve cells is not yet fully understood, and the commonly accepted mechanism is dual receptors, one is ganglioside receptors, especially GT1b and GD1b, and the other is protein receptor 6 .
  • Tetanus is a preventable disease, and active and passive immunization can be carried out after trauma.
  • Humoral immunity is protective against tetanus.
  • Neutralizing antibodies bind to the toxin, interfering with the toxin's interaction with receptors on target cells and subsequent internalization into the cell.
  • Passive immunization is clinically recommended for trauma patients with unclear or unimmunized immunization history, incomplete initial immunization, or who have completed initial immunization but whose last boost was more than 5 years old.
  • tetanus antitoxin TAT
  • TAT tetanus antitoxin
  • a skin test is required, and it is easy to cause allergic reactions. The incidence of allergies is 5%-30%, and anaphylactic shock is occasionally seen.
  • the other is human tetanus immune globulin (HIGT), which is collected from plasma or serum with high tetanus antibody titers from blood donors immunized with hepatitis B vaccine and then immunized with tetanus toxoid, and extracted by low temperature ethanol method.
  • HIGT does not require a skin test and can be injected directly. However, because it is a blood product, there is a potential risk of infection with infectious diseases such as hepatitis C and AIDS, and due to source restrictions, the yield is low, the cost is high, and the quality is not uniform between batches 7 .
  • Antibody drugs can overcome some of the above shortcomings, and also have certain advantages, such as stable expression through mammalian cell expression systems, and clear components.
  • the application provides a bispecific antibody comprising a first antigen-binding fragment and a second antigen-binding fragment that bind to different epitopes of tetanus toxin, and the bispecific antibody has activity to neutralize tetanus toxin .
  • the first antigen-binding fragment comprises: HCDR1 with an amino acid sequence of SYWIY (SEQ ID NO: 1) and an amino acid sequence of EINPTNGFANYNEKFKT (SEQ ID NO: 2) or EINPTAGFANYNEKFKT (SEQ ID NO: 2) : 3) or the HCDR2 of EINPTNAFANYNEKFKT (SEQ ID NO: 4), the amino acid sequence is the HCDR3 of HFRFPY (SEQ ID NO: 5), the amino acid sequence is the LCDR1 of RASQDIGSSLT (SEQ ID NO: 6), the amino acid sequence is ATSSLDS (SEQ ID NO: 6) NO:7) LCDR2 and LCDR3 whose amino acid sequence is LQYASSPYT (SEQ ID NO:8); wherein, the HCDR and LCDR amino acid sequences are defined according to Kabat.
  • the second antigen-binding fragment comprises: HCDR1 with the amino acid sequence of DYGVN (SEQ ID NO: 9), HCDR2 with the amino acid sequence of MIWSDGTTDYSSALKS (SEQ ID NO: 10), and the amino acid sequence of HCDR3 of VDGYSHYYAMDY (SEQ ID NO: 11), LCDR1 of amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 of amino acid sequence NAKTLAE (SEQ ID NO: 13) and QHHYGLPFT (SEQ ID NO: 14) LCDR3; wherein the HCDR and LCDR amino acid sequences are as defined by Kabat.
  • the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18 or the amino acid sequence of the variable region of the heavy chain of the first antigen-binding fragment is shown in SEQ ID NO: 16, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 18; or the first The amino acid sequence of the heavy chain variable region of the antigen-binding fragment is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.
  • amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20 Show.
  • the forms of the first and second antigen-binding fragments are independently selected from a single chain antibody (scFv) or a Fab fragment.
  • the application provides a monoclonal antibody that binds to tetanus toxin, comprising:
  • the amino acid sequence is HCDR1 of SYWIY (SEQ ID NO: 1), and the amino acid sequence is HCDR2 of EINPTNGFANYNEKFKT (SEQ ID NO: 2) or EINPTAGFANYNEKFKT (SEQ ID NO: 3) or EINPTNAFANYNEKFKT (SEQ ID NO: 4), and the amino acid sequence is HFRFPY (SEQ ID NO: 5) HCDR3, amino acid sequence LCDR1 of RASQDIGSSLT (SEQ ID NO: 6), amino acid sequence LCDR2 of ATSSLDS (SEQ ID NO: 7) and amino acid sequence LQYASSPYT (SEQ ID NO: 8) LCDR3; wherein the HCDR and LCDR amino acid sequences are as defined by Kabat.
  • the application provides a monoclonal antibody that binds to tetanus toxin, comprising:
  • Amino acid sequence is HCDR1 of DYGVN (SEQ ID NO: 9), amino acid sequence is HCDR2 of MIWSDGTTDYSSALKS (SEQ ID NO: 10), amino acid sequence is HCDR3 of VDGYSHYYAMDY (SEQ ID NO: 11), amino acid sequence is RASENIYSYLA (SEQ ID NO: 11) : the LCDR1 of 12), the LCDR2 of the amino acid sequence NAKTLAE (SEQ ID NO: 13) and the LCDR3 of the amino acid sequence of QHHYGLPFT (SEQ ID NO: 14); wherein, the HCDR and LCDR amino acid sequences are defined according to Kabat.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody described in the first aspect, or the monoclonal antibody described in the second aspect or the third aspect, and a pharmaceutically acceptable excipient or diluent. or carrier.
  • the present application provides the bispecific antibody described in the first aspect, the monoclonal antibody described in the second aspect or the third aspect, or the pharmaceutical composition described in the fourth aspect in preparation for prevention or treatment Use in medicine for tetanus.
  • Figure 1 shows ELISA analysis of the inhibitory effect of anti-TT-Hc monoclonal antibodies S22E2-mlgG2a and S24B8-mlgg2a on the binding of S22E2 purified phage to TT-Hc.
  • Figure 2 shows ELISA analysis of the inhibitory effect of anti-TT-Hc monoclonal antibodies S22E2-mIgG2a and S24B8-mIgG2a on the binding of S24B8 purified phage to TT-Hc.
  • Figure 3 shows ELISA analysis of the inhibitory effect of anti-TT-Hc monoclonal antibodies S22E2-mlgg2a and S24B8-mlgg2a on the binding of TT-Hc to ganglioside GT1b.
  • Figure 4 shows the neutralization of tetanus toxin by anti-tetanus toxin monoclonal antibodies.
  • Figure 5 shows the protection of Balb/c mice from lethal tetanus toxin challenge by the combination of anti-tetanus toxin monoclonal antibodies.
  • Figure 6 shows the inhibitory effect of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K on the binding of TT-Hc to the ganglioside GM1.
  • Figure 7 shows the inhibitory effect of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K on the binding of TT-Hc to ganglioside GD3.
  • Figure 8 shows the inhibitory effect of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K on the binding of TT-Hc to the ganglioside GT1b.
  • Figure 9 shows the results of neutralizing activity of the anti-tetanus toxin bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K.
  • SEQ ID NO: 1 shows the amino acid sequence of HCDR1 of humanized heavy chain variable region mutants S24B8VH-h1, S24B8VH-h2 and S24B8VH-h3.
  • SEQ ID NO: 2 shows the amino acid sequence of HCDR2 of the humanized heavy chain variable region mutant S24B8VH-h1.
  • SEQ ID NO: 3 shows the amino acid sequence of HCDR2 of the humanized heavy chain variable region mutant S24B8VH-h2.
  • SEQ ID NO: 4 shows the amino acid sequence of HCDR2 of the humanized heavy chain variable region mutant S24B8VH-h3.
  • SEQ ID NO: 5 shows the amino acid sequence of HCDR3 of humanized heavy chain variable region mutants S24B8VH-h1, S24B8VH-h2 and S24B8VH-h3.
  • SEQ ID Nos: 6-8 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the humanized light chain variable region mutant S24B8VK-h1.
  • SEQ ID NOs: 9-11 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, of the humanized heavy chain variable region mutant S22E2VH-h1.
  • SEQ ID Nos: 12-14 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the humanized light chain variable region mutant S22E2VK-h1.
  • SEQ ID NO: 15 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h1.
  • SEQ ID NO: 16 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h2.
  • SEQ ID NO: 17 shows the amino acid sequence of the humanized heavy chain variable region mutant S24B8VH-h3.
  • SEQ ID NO: 18 shows the amino acid sequence of the humanized light chain variable region mutant S24B8VK-h1.
  • SEQ ID NO: 19 shows the amino acid sequence of the humanized heavy chain variable region mutant S22E2VH-h1.
  • SEQ ID NO: 20 shows the amino acid sequence of the humanized light chain variable region mutant S22E2VK-h1.
  • SEQ ID NO: 21 is shown as the amino acid sequence of S24B8VH-h1+CH-IgG1K.
  • SEQ ID NO: 22 shows the amino acid sequence of S24B8VH-h2+CH-IgG1K.
  • SEQ ID NO: 23 shows the amino acid sequence of S24B8VH-h3+CH-IgG1K.
  • SEQ ID NO: 24 shows the amino acid sequence of S24B8VK-h1+CK.
  • SEQ ID NO: 25 shows the amino acid sequence of S22E2-h1-scFv-FcH1.
  • SEQ ID NO: 26 shows the amino acid sequence of the C-terminal domain recombinant protein (TT-Hc) of the tetanus toxin heavy chain.
  • SEQ ID NO: 27 shows the amino acid sequence of the His-tag.
  • SEQ ID NO: 28 shows the amino acid sequence of the human (homo sapiens) heavy chain constant region IgG1 subtype.
  • SEQ ID NO: 29 shows the amino acid sequence of the murine (mus musculus) heavy chain constant region IgG2a subtype.
  • SEQ ID NO: 30 shows the amino acid sequence of human IgGl subtype mutant IgGlH.
  • SEQ ID NO: 31 shows the amino acid sequence of human IgG1 subtype mutant IgG1K.
  • SEQ ID NO: 32 shows the amino acid sequence of the murine IgG2a subtype mutant mIgG2a-H.
  • SEQ ID NO: 33 shows the amino acid sequence of the murine IgG2a subtype mutant mIgG2aK.
  • SEQ ID NO: 34 shows the amino acid sequence of the human (homo sapiens) light chain constant region kappa isoform.
  • SEQ ID NO: 35 shows the amino acid sequence of the lambda isoform of the human (homo sapiens) light chain constant region.
  • SEQ ID NO: 36 shows the amino acid sequence of the murine (mus musculus) light chain constant region kappa isoform.
  • SEQ ID NO: 37 shows the amino acid sequence of the murine (mus musculus) light chain constant region lambda isoform.
  • SEQ ID NO: 38 shows the nucleotide sequence of primer PmCGR.
  • SEQ ID NO: 39 shows the nucleotide sequence of primer PmCKR.
  • SEQ ID NO: 40 shows the amino acid sequence of the Fc segment (FcK) containing the Knob mutation.
  • SEQ ID NO: 41 shows the amino acid sequence of the Fc segment (FcH1) containing the Hole mutation.
  • Tetanus toxin is a macromolecular protein composed of several domains. In theory, it has multiple epitopes that stimulate humoral immunity. Antibodies that bind to different epitopes can be obtained from immunized individuals. Tetanus toxin fragment C is a domain that binds to the receptor, and antibodies against fragment C have a high probability of having neutralizing activity, and antibodies that block the binding of the toxin to gangliosides GT1b, GD1b and GM1 have a neutralizing effect on the toxin.
  • novel bispecific antibodies and monoclonal antibodies against tetanus toxin polynucleotides encoding the bispecific antibodies or monoclonal antibodies, and vectors comprising the polynucleotides , a host cell comprising said polynucleotide or vector, a method of preparing and purifying said bispecific antibody or monoclonal antibody, and medical and biological applications of said bispecific antibody or monoclonal antibody.
  • a full-length bispecific antibody molecule or monoclonal antibody molecule can be constructed as a drug for clinical prevention or treatment of tetanus.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, including antibody portions fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other immunoglobulins that contain an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • antigen-binding fragments thereof eg, Fab, Fab', F(ab') 2 , Fv
  • variants thereof including antibody portions fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other immunoglobulins that contain an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosy
  • full or full-length antibodies contain two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variant region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variable region (VL) and a constant region (CL).
  • a full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or a subclass of the above), but the antibody need not belong to any particular class.
  • Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domains of the heavy chains.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • bispecific antibody as used herein is an antibody that has the ability to bind two different antigens.
  • Bispecific antibodies can have a variety of structural configurations.
  • a bispecific antibody can consist of two Fc fragments and two antigen-binding moieties fused to each (similar to a natural antibody, except that the two arms bind different antigenic targets or epitopes), and the antigen-binding moiety can be a single Chain antibody (scfv) or Fab fragment in two forms.
  • the two different antigen binding parts of the bispecific antibody each bind to the N-terminus of one Fc fragment, and the antigen binding part configuration of the two arms can have four combinations: scfv + Fab Fragments, Fab fragments + scfv, scfv + scfv and Fab fragments + Fab fragments.
  • the Fc fragment can contain mutations that can ensure the heteromerization of the heavy chain, and KIH technology (knob-in-hole, KIH) is a strategy to solve the heteromerization of the heavy chain.
  • KIH technology refers to the modification of the amino acid sequence of the CH3 region to form a structure that is conducive to the pairing of heterologous half-antibodies, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible.
  • Guidance on KIH techniques can be found, for example, in "An efficient route to human bispecific IgG", A. Margaret Merchant et al., Nature Biotechnology, Volume 16, 1998, which is hereby incorporated by reference in its entirety.
  • the bispecific antibody may be structured such that an antibody that binds a first antigen (eg, in the form of a native antibody) extends from the C-terminus of the CH3 region (possibly via a flexible linker) an antigen-binding portion capable of binding a second antigen.
  • a first antigen eg, in the form of a native antibody
  • antigen binding portion or “antigen-binding fragment” are used interchangeably and refer to a portion or region of an intact antibody molecule responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variant region (VH), a light chain variant region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2, and CDR3.
  • CDRs complementarity determining regions
  • VH or VL the complementarity determining regions
  • Chothia definition See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)).
  • the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In embodiments of the present application, the CDR sequences are defined using Kabat.
  • variable region sequence of a given antibody the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab') 2 fragments, It can be a bivalent fragment with two Fab' fragments connected by a disulfide bridge in the hinge region (ie, a dimer of Fab'); (3) VL and VH with one arm of the antibody Fv fragments of domains; (4) single-chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which can comprise Two VH domains and two VL domains linked by a peptide linker, the two VL domains are combined with the two VH domains via a disulfide bridge.
  • Fab fragments which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain
  • an "antigen-binding portion” includes, but is not limited to, the form of a Fab fragment or the form of a single chain antibody (scFv).
  • single chain antibody single chain fragment variable
  • VH heavy chain variable region
  • VL light chain variable region
  • a flexible linker is usually designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region can fold into the correct conformation capable of binding the antigen.
  • Fab fragment antigen binding fragment
  • Fab portion or similar terms as used herein refers to an antibody fragment capable of binding to an antigen produced by papain treatment of an intact antibody, including intact light chains (VL- CL), heavy chain variable region and CH1 fragment (VH-CH1).
  • Monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals.
  • Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they exhibit the desired (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • binding refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope.
  • the application provides a bispecific antibody comprising a first antigen-binding fragment and a second antigen-binding fragment that bind to different epitopes of tetanus toxin, and the bispecific antibody has activity to neutralize tetanus toxin .
  • the first antigen-binding fragment comprises: HCDR1 with an amino acid sequence of SYWIY (SEQ ID NO: 1) and an amino acid sequence of EINPTNGFANYNEKFKT (SEQ ID NO: 2) or EINPTAGFANYNEKFKT (SEQ ID NO: 2) : 3) or the HCDR2 of EINPTNAFANYNEKFKT (SEQ ID NO: 4), the amino acid sequence is the HCDR3 of HFRFPY (SEQ ID NO: 5), the amino acid sequence is the LCDR1 of RASQDIGSSLT (SEQ ID NO: 6), the amino acid sequence is ATSSLDS (SEQ ID NO: 6) NO:7) LCDR2 and LCDR3 whose amino acid sequence is LQYASSPYT (SEQ ID NO:8); wherein, the HCDR and LCDR amino acid sequences are defined according to Kabat.
  • the second antigen-binding fragment comprises: HCDR1 with the amino acid sequence of DYGVN (SEQ ID NO: 9), HCDR2 with the amino acid sequence of MIWSDGTTDYSSALKS (SEQ ID NO: 10), and the amino acid sequence of HCDR3 of VDGYSHYYAMDY (SEQ ID NO: 11), LCDR1 of amino acid sequence RASENIYSYLA (SEQ ID NO: 12), LCDR2 of amino acid sequence NAKTLAE (SEQ ID NO: 13) and QHHYGLPFT (SEQ ID NO: 14) LCDR3; wherein the HCDR and LCDR amino acid sequences are as defined by Kabat.
  • the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18 or the amino acid sequence of the variable region of the heavy chain of the first antigen-binding fragment is shown in SEQ ID NO: 16, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 18; or the first The amino acid sequence of the heavy chain variable region of the antigen-binding fragment is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.
  • amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20 Show.
  • the forms of the first and second antigen-binding fragments are independently selected from a single chain antibody (scFv) or a Fab fragment.
  • the first antigen-binding fragment is a Fab fragment and the second antigen-binding fragment is a single chain antibody (scFv).
  • the bispecific antibody comprises the amino acid sequence set forth in one of SEQ ID NOs: 21, 22 and 23 and the amino acid sequence set forth in SEQ ID NO:24.
  • the bispecific antibody comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the application provides a monoclonal antibody that binds to tetanus toxin, comprising:
  • the amino acid sequence is HCDR1 of SYWIY (SEQ ID NO: 1), and the amino acid sequence is HCDR2 of EINPTNGFANYNEKFKT (SEQ ID NO: 2) or EINPTAGFANYNEKFKT (SEQ ID NO: 3) or EINPTNAFANYNEKFKT (SEQ ID NO: 4), and the amino acid sequence is HFRFPY (SEQ ID NO: 5) HCDR3, amino acid sequence LCDR1 of RASQDIGSSLT (SEQ ID NO: 6), amino acid sequence LCDR2 of ATSSLDS (SEQ ID NO: 7) and amino acid sequence LQYASSPYT (SEQ ID NO: 8) LCDR3; wherein the HCDR and LCDR amino acid sequences are as defined by Kabat.
  • the monoclonal antibody comprises: the heavy chain variable region amino acid sequence set forth in SEQ ID NO:15, and the light chain variable region amino acid sequence set forth in SEQ ID NO:18 or the heavy chain variable region amino acid sequence shown in SEQ ID NO: 16, the light chain variable region amino acid sequence shown in SEQ ID NO: 18; or the heavy chain variable region shown in SEQ ID NO: 17 Region amino acid sequence, such as the light chain variable region amino acid sequence shown in SEQ ID NO: 18.
  • the application provides a monoclonal antibody that binds to tetanus toxin, comprising:
  • Amino acid sequence is HCDR1 of DYGVN (SEQ ID NO: 9), amino acid sequence is HCDR2 of MIWSDGTTDYSSALKS (SEQ ID NO: 10), amino acid sequence is HCDR3 of VDGYSHYYAMDY (SEQ ID NO: 11), amino acid sequence is RASENIYSYLA (SEQ ID NO: 11) : the LCDR1 of 12), the LCDR2 of the amino acid sequence NAKTLAE (SEQ ID NO: 13) and the LCDR3 of the amino acid sequence of QHHYGLPFT (SEQ ID NO: 14); wherein, the HCDR and LCDR amino acid sequences are defined according to Kabat.
  • the monoclonal antibody comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO:19 and the light chain variable region amino acid sequence set forth in SEQ ID NO:20.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody described in the first aspect, or the monoclonal antibody described in the second aspect or the third aspect, and a pharmaceutically acceptable excipient or diluent. or carrier.
  • the pharmaceutical composition is for preventing or treating tetanus.
  • the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers ; suspending agents; preservatives such as benzoic acid, sorbic acid and calcium propionate; sweetening and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifiers such as talc, magnesium stearate, and mineral oil
  • suspending agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives such as benzoic acid, sorbic acid and calcium propionate
  • sweetening and/or flavoring agents etc.
  • compositions of the present application may be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application may be delivered by any physiologically acceptable mode of administration, including but not limited to: oral administration, parenteral administration, nasal administration drug, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • the medicament for therapeutic use may be formulated in the form of a lyophilized formulation or an aqueous solution by admixing the agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, etc. Composition for storage.
  • the present application provides the bispecific antibody described in the first aspect, the monoclonal antibody described in the second aspect or the third aspect, or the pharmaceutical composition described in the fourth aspect in preparation for prevention or treatment Use in medicine for tetanus.
  • the present application provides a method for preventing or treating tetanus, comprising administering to an individual in need the bispecific antibody of the first aspect, the monoclonal antibody of the second aspect or the third aspect, or the The pharmaceutical composition described in the fourth aspect.
  • the application provides nucleic acid molecules encoding the bispecific antibodies of the first aspect, the monoclonal antibodies of the second aspect or the third aspect.
  • the nucleic acid molecule is operably linked to regulatory sequences that can be recognized by host cells transformed with the vector.
  • the application also provides vectors comprising an isolated nucleic acid molecule encoding the bispecific antibody of the first aspect, the monoclonal antibody of the second aspect or the third aspect, and host cells comprising the nucleic acid molecule or vector.
  • the application also provides methods of producing the bispecific antibody of the first aspect, the monoclonal antibody of the second aspect or the third aspect.
  • the method of producing the bispecific antibody of the first aspect, the monoclonal antibody of the second aspect or the third aspect comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing the bispecific antibody of the first aspect, the monoclonal antibody of the second aspect or the third aspect further comprises recovering the bispecific antibody or monoclonal antibody from the host cell culture medium .
  • Example 1 Preparation of recombinant tetanus toxin antigen and recombinant antibody
  • the C-terminal domain recombinant protein (TT-Hc, SEQ ID NO: 26) of the tetanus toxin heavy chain needs to be used in the process of preparing the anti-tetanus toxin monoclonal antibody.
  • This protein is derived from Clostridium tetani and has no post-translational modifications, so it can be expressed using the E. coli expression system.
  • a His tag His, SEQ ID NO: 27 was added to the C-terminus of the recombinant protein, which would be more conducive to the purification of the recombinant protein and the identification of the function of the monoclonal antibody.
  • TT-Hc TT-Hc recombinant protein
  • a gene encoding TT-Hc was designed and synthesized.
  • a suitable eukaryotic expression vector such as pET-22b from Invitrogen, etc.
  • Escherichia coli expressing TT-Hc-His recombinant protein is induced to express by IPTG (such as I8070-1g from Soleibo Company).
  • the cells are collected by centrifugation, etc., and the cells are ultrasonically disrupted with an ultrasonic cell disruptor (eg, VCX130 from Sonics, etc.), and the supernatant is collected by centrifugation and the like.
  • an ultrasonic cell disruptor eg, VCX130 from Sonics, etc.
  • One-step purification of the recombinant protein in the supernatant is performed using a metal chelate affinity chromatography column (such as HisTrap FF from GE).
  • a desalting column such as GE's Hitrap desaulting, etc.
  • samples can be filter sterilized and stored in aliquots at -20°C.
  • nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody were cloned into eukaryotic expression fusing the nucleotide sequences encoding the heavy chain constant region and the light chain constant region, respectively.
  • a vector such as pcDNA3.1 from Invitrogen, etc.
  • the whole antibody is expressed in combination.
  • the heavy chain constant region of the antibody can be of human IgG1 subtype (SEQ ID NO:28), murine IgG2a subtype (SEQ ID NO:29), human IgG1 subtype mutant IgG1H based on KIH (Knob-Into-Hole) technology (SEQ ID NO:30) and IgG1K (SEQ ID NO:31), or murine IgG2a subtype mutants mIgG2a-H (SEQ ID NO:32) and mIgG2aK (SEQ ID NO:33), the light chain constant region may be Human kappa subtype (SEQ ID NO:34), human lambda subtype (SEQ ID NO:35), murine kappa subtype (SEQ ID NO:36) or murine lambda subtype (SEQ ID NO:37).
  • liposomes such as 293fectin from Invitrogen, etc.
  • other transfection reagents such as PEI, etc.
  • transfect the prepared recombinant antibody expression plasmids into HEK293 cells such as HEK293F from Invitrogen
  • culture under serum-free suspension culture conditions 3-5 days.
  • the culture supernatant is harvested by centrifugation, etc., and one-step purification is performed using a ProteinA/G affinity chromatography column (such as Mabselect SURE from GE, etc.).
  • a desalting column such as GE's Hitrap desaulting, etc.
  • PBS pH 7.0
  • antibody samples can be filter sterilized and stored in aliquots at -20°C.
  • mice Using the recombinant protein TT-Hc-His prepared in Example 1 as the antigen, BALB/c mice aged 6-8 weeks were immunized at a dose of 50 ⁇ g/mouse, boosted once every 14 days. Mice were sacrificed at 8 weeks and splenocytes were collected. Mouse spleen lymphocytes were isolated using mouse lymphocyte separation medium (Daktronics Biotechnology Co., Ltd., CAT#DKW33-R0100), and total cell RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430), the isolated lymphocytes were subjected to total RNA extraction.
  • mouse lymphocyte separation medium Daktronics Biotechnology Co., Ltd., CAT#DKW33-R0100
  • total cell RNA extraction kit Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430
  • the first-strand cDNA synthesis kit (Thermo scientific, CAT#K1621) was used to synthesize the variable region of the heavy chain and the variable region of the light chain, respectively.
  • the reverse transcription primers were gene-specific primers.
  • the paired regions are respectively located in the antibody heavy chain constant region and the antibody light chain constant region, and the specific sequences are respectively PmCGR:TGCATTTGAACTCCTTGCC (SEQ ID NO:38) and PmCKR:CCATCAATCTTCCACTTGAC (SEQ ID NO:39).
  • the synthesized cDNA was immediately stored at -70°C for future use.
  • the prepared mouse single-chain antibody nucleotide sequence was cloned into the vector pADSCFV-S (for the experimental technical process, please refer to Example 1 of Chinese Patent Application No. 201510097117.0, the entire content of the above patent application is incorporated herein by reference ) to build the scFv library.
  • the capacity of the antibody library reaches 5 ⁇ 10E8, and the correct rate is 50%.
  • Screening example 2 constructed The phage library displaying mouse single-chain antibodies was screened for three rounds by binding, elution, neutralization, infection, and amplification, and finally two single-chain antibodies S22E2 and S24B8 that specifically bind to human TT-Hc-His were obtained. .
  • nucleotide sequences encoding the heavy chain and light chain of S22E2 and S24B8 were cloned into a real nucleotide sequence fused with the nucleotide sequences encoding the mouse mIgG2a heavy chain constant region and mouse ⁇ light chain constant region, respectively. nuclear expression vector to prepare recombinant murine antibody.
  • the affinity of anti-TT-Hc antibodies was determined by surface plasmon resonance using Biacore X100.
  • Amino Coupling Kit (BR-1000-50), Mouse Antibody Capture Kit (BR-1008-38), CM5 Chip (BR100012) and 10 ⁇ HBS-EP (BR100669) with pH 7.4 and other related reagents and consumables are all available. Purchased from GE healthcare.
  • TT-Hc-His was set up with a series of concentration gradients (eg 6.17nM, 18.5nM, 55.6nM, 167nM and 500nM) and injected from low concentration to high concentration at 30 ⁇ L/min at 25°C with a binding time of 120s , the dissociation time was 3600 s, and the chip surface was regenerated by injecting 10 mM pH2.0 glycine-HCl solution for 30 s at 10 ⁇ L/min.
  • Association rates (K a ) and dissociation rates (K d ) were calculated by fitting association and dissociation sensorgrams by a 1:1 binding model using Biacore X100 evaluation software version 2.0.1.
  • the dissociation equilibrium constant (KD ) is calculated as the ratio Kd /Ka.
  • the fitting results are shown in Table 1.
  • the recombinant protein TT-Hc-His was used to coat a 96-well ELISA plate (3 ⁇ g/mL, 100 ⁇ L/well), and it was coated overnight in a refrigerator at 4°C. Blocked with blocking solution PBS-0.1% Tween 20-3% milk for 1 hour at 37°C.
  • S22E2-mIgG2a and S24B8-mIgG2a murine antibodies were serially diluted with a fixed concentration (1*10 11 cfu/mL) of S22E2 purified phage (S22E2 phage), starting at 50 ⁇ g/mL, 3-fold serial dilution, a total of 11 Concentration titer, 100 ⁇ L/well was added to the blocked 96-well ELISA plate, and incubated at 37°C for 1 hour.
  • the ELISA plate was washed with PBS-0.1% Tween20, and then HRP anti-M13 secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05T-H) was added, and incubated at 37°C for 1 hour. Wash the ELISA plate with PBS-0.1% Tween20, add OPD substrate chromogenic solution, stop the color development with 1M H 2 SO 4 after 5-10 minutes, and use a microplate reader to measure the 492nm/630nm dual-wavelength optical density value. The results of ELISA analysis (Fig.
  • S22E2-mIgG2a and S24B8-mIgG2a were serially diluted with a fixed concentration of S24B8 purified phage (S24B8 phage), and the binding signals of S24B8 phage and TT-Hc were detected with HRP anti-M13 secondary antibody.
  • the ELISA results ( Figure 2) showed that S24B8-mlgG2a could completely inhibit the binding signal of S24B8 phage and TT-Hc recombinant protein, but S22E2-mlgG2a had no effect on the binding of S24B8 phage and TT-Hc.
  • the anti-TT-Hc monoclonal antibodies S22E2-mIgG2a and S24B8-mIgG2a bind to different epitopes of TT-Hc.
  • Example 5 Tetanus toxin monoclonal antibody inhibits the binding of TT-Hc to gangliosides
  • Tetanus toxin can bind to specific receptors on the surface of nerve cells through the Hc domain.
  • a dual receptor mechanism is generally accepted, one is a ganglioside receptor and the other is a protein receptor.
  • the ganglioside GT1b was diluted with methanol to 10 ⁇ g/mL, 100 ⁇ L/well was coated on a 96-well ELISA plate, and left at room temperature overnight to evaporate the methanol. Block with blocking solution PBS-0.1% Tween20-1% BSA for 1 hour at 37°C.
  • TT-Hc was serially diluted with PBS, the initial concentration was 100 ⁇ g/mL, 3-fold serial dilution, a total of 7 concentration gradients; the anti-TT-Hc mouse monoclonal antibody was diluted with PBS to 40 ⁇ g/mL, with TT-Hc After mixing in equal volume, 100 ⁇ L/well was added to the blocked 96-well ELISA plate, and a TT-Hc control group without antibody was set at the same time, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween20, HRP anti-TT polyclonal antibody (prepared in the laboratory) was added, 100 ⁇ L/well, and incubated at 37° C.
  • Antibody neutralizing activity was determined by injecting Balb/c mice after mixing the antibody with a lethal dose of tetanus toxin in vitro. Tetanus toxin was mixed with anti-tetanus toxin monoclonal antibody or antitoxin, placed at 37°C for 1 hour, and injected into the right hind leg muscle of Balb/c mice.
  • the injection dose of tetanus toxin is 20 ⁇ LD 50 / animal; the injection dose of commercial equine tetanus immunoglobulin (F(ab') 2 ) (marked as TAT, Shanghai Sailun Biotechnology Co., Ltd.) is 1IU/
  • the injection dose of monoclonal antibodies S22E2 and S24B8 was 10 ⁇ g/mouse, and the dose of S24B8 and S22E2 in combination was 1.25 ⁇ g/mouse of each antibody; Balb/c mice injected with 20 ⁇ LD 50 of tetanus toxin were used as control group, a total of 5 groups, 8 animals/group.
  • Dilute tetanus toxin to 800 ⁇ LD 50 /mL with boric acid buffer that is, after mixing with antibody in equal amount, each 50 microliter injection volume contains 20 ⁇ LD 50
  • dilute equine tetanus immunoglobulin with boric acid buffer to 40IU/mL (i.e., 1IU per 50 microliter injection volume after mixing with tetanus toxin in equal amounts)
  • dilute antibodies S22E2 and S24B8 to 0.4mg/mL (or 0.1mg/mL) with borate buffer i.e.
  • S22E2 Or S24B8 is used alone, mixed with tetanus toxin in equal amounts and contains 10 ⁇ g per 50 ⁇ l injection; S24B8 and S22E2 are used in combination, and the two antibodies mixed in equal amounts are mixed with tetanus toxin in equal amounts and injected in every 50 ⁇ l The amount each contains 1.25 ⁇ g). Each animal was injected with 50 ⁇ L of tetanus toxin and antibody mixture. The experimental results are shown in Figure 4: all Balb/c mice in the control group died of tetanus within 3 days of challenge.
  • Example 7 Anti-tetanus toxin monoclonal antibody combination protects Balb/c mice from lethal tetanus toxin challenge
  • Balb/c mice were immunized before immunoglobulin exposure, and then the antibody-injected mice were challenged with a lethal dose of tetanus toxin to determine the protective effect of the antibody.
  • the injection dose of monoclonal antibodies S22E2 and S24B8 was 10 ⁇ g/mouse, and the injection dose of S24B8 and S22E2 combination group was 1.25 ⁇ g/mouse of each antibody; Balb/c mice injected with 20 ⁇ LD 50 of tetanus toxin were used as control group, a total of 4 groups, 8 animals/group.
  • Tetanus toxin was diluted to 400 x LD 50 /mL with borate buffer (ie, 20 x LD 50 per 50 microliter injection volume); antibodies S22E2 and S24B8 were diluted to 0.2 mg/mL (or 0.05 in borate buffer) mg/mL) (i.e., S22E2 or S24B8 alone, containing 10 ⁇ g per 50 ⁇ l injection; S24B8 and S22E2 in combination, the two antibodies were mixed in equal amounts and each containing 1.25 ⁇ g per 50 ⁇ l injection).
  • mice were given 50 ⁇ L of anti-tetanus toxin antibody, and Balb/c mice were challenged with tetanus toxin in the right hind leg muscle 24 hours later, and each animal was injected with 50 ⁇ L of tetanus toxin.
  • the experimental results are shown in Figure 5: all Balb/c mice in the control group died of tetanus within 4 days of the challenge. Under the experimental conditions, the combination of S24B8 and S22E2 could completely protect the Balb/c mice, and all the mice were completely protected during the experimental period. survive.
  • the appropriate J region gene sequence was selected to provide framework region 4 (FR4).
  • This template can be selected based on a variety of factors, such as: relative overall length of the antibody, size of the CDRs, amino acid residues located at the junction between the framework regions (FR) and hypervariable regions (CDR) of the antibody, homology to the overall sequence sex, etc.
  • the template chosen may be a mixture of multiple sequences or may be a consensus template in order to maintain the proper conformation of the parental complementarity determining regions (CDRs) as much as possible.
  • the humanization of the S22E2 murine monoclonal antibody employed a classical framework grafting strategy.
  • the light and heavy chain variable regions of S22E2 were subjected to CDR transplantation to obtain a humanized heavy chain variable region mutant S22E2VH-h1 (SEQ ID NO: 19) and a humanized light chain variable region mutant S22E2VK -h1 (SEQ ID NO: 20).
  • variable region genes of the antibodies were designed and synthesized, cloned into a suitable eukaryotic gene expression vector, and the whole antibody was prepared with reference to Example 1.
  • Three humanized versions of S24B8 were designated S24B8-h1, S24B8-h2 and S24B8-h3.
  • Humanized S22E2 was named S22E2-h1.
  • Biacore X100 was used to detect the binding affinity of humanized anti-TT-Hc antibody to TT-Hc.
  • the fitting results are shown in Table 2.
  • the heavy chain constant regions of each humanized antibody were of the human IgG1 subtype.
  • the antigen-binding fragments S24B8-h3 and S22E2-h1 which bind to different epitopes of the tetanus toxin Hc protein, were designed into Fab form and scFv, respectively. Format, construction of human IgG1 heterodimer based on KIH (Knob-Into-Hole) technology.
  • the S24B8-h3 antigen-binding fragment in Fab form is fused to the N-terminus of the Fc segment containing Knob mutation (FcK, SEQ ID NO: 40), and the antigen-binding fragment of S22E2-h1 in scFv format is fused to the Fc segment containing Hole mutation.
  • FcH1, SEQ ID NO: 41 N-terminal to construct a bispecific antibody against tetanus toxin protein (Hc).
  • the constructed eukaryotic expression vectors expressing S22E2-h1-scFv-FcH1, expressing S24B8VH-h3+CH-IgG1K and expressing S24B8VK-h1+CK were co-transfected into HEK293F cells by liposome, and suspended in serum-free cells. After culturing under the culture conditions for 3-5 days, the culture supernatant is harvested by centrifugation or the like.
  • the bispecific antibody in the culture supernatant is purified with a ProteinA/G affinity chromatography column (such as Mabselect SURE of GE, etc.), and then the recombinant antibody storage buffer is replaced by a desalting column (such as Hitrap desaulting of GE, etc.). PBS buffer (pH 7.0) or other suitable buffer.
  • the desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K. If necessary, antibody samples can be filter sterilized and stored in aliquots at -20°C.
  • Example 5 the ability of anti-TT-Hc antibody to inhibit the binding of TT-Hc to three gangliosides GD3, GT1b and GM1a was examined.
  • the ELISA results show that the monoclonal antibody S22E2-h1-scFv-FcH1 and the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K inhibit the interaction of TT-Hc with the three gangliosides
  • the binding has obvious inhibitory effect, and the monoclonal antibody S24B8-h3-IgG1 does not affect the binding of TT-Hc to the three gangliosides.
  • the injection dose of tetanus toxin is 20 ⁇ LD 50 per animal
  • the injection dose of monoclonal antibodies S24B8-h3 and S22E2-h1 alone is 10 ⁇ g per animal
  • the combined use of monoclonal antibodies S24B8-h3 and S22E2-h1 The injection dose of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was 2.4 ⁇ g/mouse; Balb/c mice injected with 20 ⁇ LD 50 of tetanus toxin were used as control group, a total of 5 groups, 8 animals/group.
  • +S24B8h3-IgG1K was diluted to 0.096 mg/mL (ie, 2.4 ⁇ g per 50 ⁇ l injection volume after mixing with tetanus toxin in equal amounts). Each animal was injected with 50 ⁇ L of tetanus toxin and antibody mixture.
  • the titer of the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was determined based on the method for determining the neutralizing activity of the antibody in Example 6, and the test product was compared with the standard product.
  • Dilution of tetanus toxin dilute tetanus toxin with boric acid buffer to contain 40 test volumes per milliliter (L+1/10) (that is, after mixing with antibodies in equal amounts, each 50 microliter injection volume contains 1 test volume (L+1/10)).
  • Dilution of human tetanus immunoglobulin standard dilute human tetanus immunoglobulin standard to 4 IU/mL with boric acid buffer (ie, 1/10 IU per 50 microliter injection volume after mixing with toxin in equal amount).
  • the bispecific antibody S22E2h1-scFv-FcH1+S24B8h3-IgG1K was diluted in boronic acid buffer into 5 dilutions, 14.40 ⁇ g/mL, 16.80 ⁇ g/mL, 20 ⁇ g/mL, 23.6 ⁇ g/mL, and 27.6 ⁇ g/mL (that is, after mixing with toxin in equal amount, each 50 microliter injection volume contains 0.36 ⁇ g, 0.42 ⁇ g, 0.50 ⁇ g, 0.59 ⁇ g and 0.69 ug of double antibody), and the dilution interval is 15%.
  • the diluted human tetanus immune globulin standard solution and the bispecific antibody solutions of different dilutions were mixed with the same amount of diluted tetanus toxin, and injected immediately after combining at 37°C for 1 hour.
  • the highest dose of bispecific antibody that died at the same time as the control group was 0.42 ⁇ g, and the calculated titer of the bispecific antibody was 238IU. /mg.

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Abstract

提供针对破伤风毒素的双特异性抗体、单克隆抗体及其用途，所述双特异性抗体包含结合破伤风毒素不同表位的第一抗原结合片段和第二抗原结合片段，并且具有中和破伤风毒素的活性。所述单克隆抗体结合破伤风毒素，并且具有中和破伤风毒素的活性。

Description

针对破伤风毒素的抗体及其用途
相关申请的交叉引用
本申请要求于2020年10月21日递交的中国专利申请第202011128926.0号的优先权，其全部内容通过引用整体并入本文。
发明领域
本发明通常涉及基因工程和抗体药物领域；具体而言，本申请涉及针对破伤风毒素的抗体及其用途。
发明背景
破伤风是由破伤风梭状芽孢杆菌分泌的神经毒素引起的急性、致死性疾病，人兽共患，最易感动物为奇蹄目的马、骡、驴等。人类对由破伤风梭状芽孢杆菌分泌的神经毒素也易感，可以在任何年龄发病。在经济不发达地区，新生儿破伤风是新生儿死亡的主要因素之一。破伤风的潜伏期通常为3-21天，大多数在10天左右，但根据伤口特征、范围和部位，也可能为1天至数月。典型的破伤风临床表现为牙关紧闭，运动神经中枢应激性增高，局部或全身肌肉痉挛性麻痹。患者多死于窒息及全身器官衰竭，即便有了现代加强监护的条件，病例死亡率也非常高，特别是在严重自然灾害后，破伤风死亡率可达19％至31％。破伤风的临床类型分为3种：全身型破伤风、局部型破伤风和头部型破伤风，其中全身型破伤风约占88％，局部型破伤风约占12％，头部型破伤风约占1％。
破伤风梭状芽孢杆菌是严格厌氧的革兰氏阳性菌，有鞭毛无荚膜，其芽孢广泛存在于施肥土壤、街道尘土、腐臭污泥、动物肠道和污染物体表面。破伤风梭状芽孢杆菌主要通过伤口进入人体，若伤口狭且深，或者伤口同时感染了嗜氧的化脓菌，在厌氧条件下破伤风梭状芽孢杆菌会大量繁殖并产生三种外毒素，其中破伤风毒素引起破伤风的 特征性症状，刺激保护性抗体产生 1。但也有约20％的患者未发现明显的侵入伤口，推测破伤风梭状芽孢杆菌也能通过小的擦伤面侵入机体。破伤风梭状芽孢杆菌根据鞭毛抗原凝集反应分为10个血清型，但所有血清型都产生同一种神经毒素，各型菌株所产生的毒素可以被任何一型抗毒素中和。破伤风毒素的毒性非常强，仅次于肉毒毒素，对小鼠的致死剂量仅为2-6ng/kg。
破伤风毒素(Tetanus toxin)，又称为破伤风神经毒素(Tetanus neurotoxin)，其是单链蛋白，相对分子质量为约150kDa，由1315个氨基酸残基组成，被切割成由二硫键连接的轻链(片段A，50kDa)和重链(HC，100kDa)，从而变成活性形式 2。片段A是锌金属蛋白酶，进入胞质后消化囊泡相关膜蛋白-2(VAMP-2)，阻断甘氨酸和γ-氨基丁酸(GABA)等抑制性神经递质释放，引起肌肉痉挛性麻痹 3。重链包括两个功能性结构域，C末端结构域(片段C)与神经细胞表面结合，使得毒素分子内吞入囊泡；N末端结构域(片段B)则穿过囊泡膜将片段A转运至神经元胞浆中 4。片段C又分为C末端亚结构域(HCC)和N末端亚结构域(HCN) 5。破伤风毒素进入神经细胞的过程尚未完全清楚，目前普遍接受的是双受体机制，一种是神经节苷脂受体，尤其是GT1b和GD1b，另一种是蛋白受体 6。
破伤风属于可预防性疾病，创伤后可以进行主动免疫和被动免疫。体液免疫对破伤风具有保护作用。中和抗体与毒素结合，干扰毒素与靶细胞上的受体作用以及随后内化进入细胞的过程。临床上对于免疫史不清或未免疫、未完成初始免疫、完成初始免疫但最近一次加强超过5年的外伤患者都建议进行被动免疫。目前临床上用于预防和治疗破伤风的被动免疫制剂有两种，一种是破伤风抗毒素(TAT)，其为用破伤风类毒素免疫的马血浆经酶消化、盐析制成，使用前需皮试，容易引起过敏反应，过敏发生率为5％-30％，偶见过敏性休克。另一种是人破伤风免疫球蛋白(HIGT)，由乙型肝炎疫苗免疫后再经破伤风类毒素免疫的献血员中采集破伤风抗体效价高的血浆或血清，经低温乙醇法提取。HIGT使用时无需皮试，可直接注射，但由于其属于血液制品，存在感染丙肝、艾滋病等传染病的潜在风险，而且受来源限制， 产量较低，成本较高，且批次间质量不均一 7。
抗体药物能够克服以上部分缺点，并且还具有一定优势，例如可以通过哺乳动物细胞表达系统稳定表达，成分明确等。
基于临床需求，探索和研发可以中和破伤风毒素，预防和治疗破伤风的抗体具有重要的医学意义。
发明概述
第一方面，本申请提供了双特异性抗体，其包含结合破伤风毒素不同表位的第一抗原结合片段和第二抗原结合片段，并且所述双特异性抗体具有中和破伤风毒素的活性。
在第一方面的一些实施方案中，所述第一抗原结合片段包含：氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述第二抗原结合片段包含：氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列 如SEQ ID NO：18所示；或者所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：16所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示；或者所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：17所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示。
在第一方面的一些实施方案中，所述第二抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：19所示，轻链可变区的氨基酸序列如SEQ ID NO：20所示。
在第一方面的一些实施方案中，所述第一抗原结合片段和第二抗原结合片段的形式独立地选自单链抗体(scFv)或Fab片段。
在第二方面，本申请提供了结合破伤风毒素的单克隆抗体，其包含：
氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第三方面，本申请提供了结合破伤风毒素的单克隆抗体，其包含：
氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
第四方面，本申请提供了药物组合物，其包含第一方面所述的双特异性抗体、或者第二方面或第三方面所述的单克隆抗体以及药学可接受的赋形剂、稀释剂或载体。
第五方面，本申请提供了第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体、或者第四方面所述的药物组合物在制备用于预防或治疗破伤风的药物中的用途。
附图简述
图1显示ELISA分析抗TT-Hc单克隆抗体S22E2-mIgG2a和S24B8-mIgG2a对S22E2纯化噬菌体与TT-Hc结合的抑制作用。
图2显示ELISA分析抗TT-Hc单克隆抗体S22E2-mIgG2a和S24B8-mIgG2a对S24B8纯化噬菌体与TT-Hc结合的抑制作用。
图3显示ELISA分析抗TT-Hc单克隆抗体S22E2-mIgG2a和S24B8-mIgG2a对TT-Hc与神经节苷脂GT1b结合的抑制作用。
图4显示了抗破伤风毒素单克隆抗体对破伤风毒素的中和作用。
图5显示了抗破伤风毒素单克隆抗体联用对Balb/c小鼠避免致死性破伤风毒素攻击的保护作用。
图6显示了双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K对TT-Hc与神经节苷脂GM1结合的抑制作用。
图7显示了双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K对TT-Hc与神经节苷脂GD3结合的抑制作用。
图8显示了双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K对TT-Hc与神经节苷脂GT1b结合的抑制作用。
图9显示了抗破伤风毒素双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K的中和活性结果。
序列说明
SEQ ID NO：1显示人源化的重链可变区突变体S24B8VH-h1、S24B8VH-h2和S24B8VH-h3的HCDR1的氨基酸序列。
SEQ ID NO：2显示人源化的重链可变区突变体S24B8VH-h1的 HCDR2的氨基酸序列。
SEQ ID NO：3显示人源化的重链可变区突变体S24B8VH-h2的HCDR2的氨基酸序列。
SEQ ID NO：4显示人源化的重链可变区突变体S24B8VH-h3的HCDR2的氨基酸序列。
SEQ ID NO：5显示人源化的重链可变区突变体S24B8VH-h1、S24B8VH-h2和S24B8VH-h3的HCDR3的氨基酸序列。
SEQ ID NO：6-8分别显示了人源化的轻链可变区突变体S24B8VK-h1的LCDR1、LCDR2和LCDR3的氨基酸序列。
SEQ ID NO：9-11分别显示人源化的重链可变区突变体S22E2VH-h1的HCDR1、HCDR2和HCDR3的氨基酸序列。
SEQ ID NO：12-14分别显示人源化轻链可变区突变体S22E2VK-h1的LCDR1、LCDR2和LCDR3的氨基酸序列。
SEQ ID NO：15显示人源化的重链可变区突变体S24B8VH-h1的氨基酸序列。
SEQ ID NO：16显示人源化的重链可变区突变体S24B8VH-h2的氨基酸序列。
SEQ ID NO：17显示人源化的重链可变区突变体S24B8VH-h3的氨基酸序列。
SEQ ID NO：18显示人源化轻链可变区突变体S24B8VK-h1的氨基酸序列。
SEQ ID NO：19显示人源化的重链可变区突变体S22E2VH-h1的氨基酸序列。
SEQ ID NO：20显示人源化轻链可变区突变体S22E2VK-h1的氨基酸序列。
SEQ ID NO：21显示为S24B8VH-h1+CH-IgG1K的氨基酸序列。
SEQ ID NO：22显示S24B8VH-h2+CH-IgG1K的氨基酸序列。
SEQ ID NO：23显示S24B8VH-h3+CH-IgG1K的氨基酸序列。
SEQ ID NO：24显示S24B8VK-h1+CK的氨基酸序列。
SEQ ID NO：25显示S22E2-h1-scFv-FcH1的氨基酸序列。
SEQ ID NO：26显示破伤风毒素重链的C末端结构域重组蛋白(TT-Hc)的氨基酸序列。
SEQ ID NO：27显示His标签的氨基酸序列。
SEQ ID NO：28显示人(homo sapiens)重链恒定区IgG1亚型的氨基酸序列。
SEQ ID NO：29显示鼠(mus musculus)重链恒定区IgG2a亚型的氨基酸序列。
SEQ ID NO：30显示人IgG1亚型突变体IgG1H的氨基酸序列。
SEQ ID NO：31显示人IgG1亚型突变体IgG1K的氨基酸序列。
SEQ ID NO：32显示鼠IgG2a亚型突变体mIgG2a-H的氨基酸序列。
SEQ ID NO：33显示鼠IgG2a亚型突变体mIgG2aK的氨基酸序列。
SEQ ID NO：34显示人(homo sapiens)轻链恒定区κ亚型的氨基酸序列。
SEQ ID NO：35显示人(homo sapiens)轻链恒定区λ亚型的氨基酸序列。
SEQ ID NO：36显示鼠(mus musculus)轻链恒定区κ亚型的氨基酸序列。
SEQ ID NO：37显示鼠(mus musculus)轻链恒定区λ亚型的氨基酸序列。
SEQ ID NO：38显示引物PmCGR的核苷酸序列。
SEQ ID NO：39显示引物PmCKR的核苷酸序列。
SEQ ID NO：40显示含有Knob突变的Fc段(FcK)的氨基酸序列。
SEQ ID NO：41显示含有Hole突变的Fc段(FcH1)的氨基酸序列。
发明详述
本申请的发明人对针对破伤风抗体药物进行了深入研发，并通过抗体工程技术得到了新的针对破伤风毒素的双特异性抗体和单克隆抗体。破伤风毒素是大分子蛋白，由数个结构域组成，理论上有多个激发体液免疫的表位，从免疫个体中可以获得结合不同表位的抗体。破伤风毒素片段C是结合受体的结构域，针对片段C的抗体大概率具有中和 活性，阻断毒素与神经节苷脂GT1b、GD1b和GM1结合的抗体，对毒素具有中和作用。
在本申请的多个方面，提供了新的针对破伤风毒素的双特异性抗体和单克隆抗体，编码所述双特异性抗体或单克隆抗体的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化所述双特异性抗体或单克隆抗体的方法及所述双特异性抗体或单克隆抗体的医学和生物学应用。根据本申请提供的双特异性抗体或单克隆抗体的可变区的序列，可构建全长的双特异性抗体分子或单克隆抗体分子作为药物在临床上用于预防或治疗破伤风。
除非另外指明，本申请的实施采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab') 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要 的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“双特异性抗体”是具有结合两种不同抗原能力的抗体。双特异性抗体可以具有多种结构构型。例如，双特异性抗体可以由两个Fc片段以及分别与其融合的两个抗原结合部分组成(与天然抗体相似，区别在于两个臂结合不同的抗原靶标或表位)，抗原结合部可以为单链抗体(scfv)或Fab片段两种形式。当针对给定的两种抗原时，双特异性抗体的两个不同的抗原结合部各自与一个Fc片段的N端结合，两个臂的抗原结合部配置可以具有四种组合方式：scfv+Fab片段、Fab片段+scfv、scfv+scfv和Fab片段+Fab片段。Fc片段可以包含能够确保重链异聚化的突变，KIH技术(knob-in-hole，KIH)是解决重链异聚化的一种策略。通常，KIH技术是指通过改造CH3区的氨基酸序列，形成有利于异种半抗体相互配对的结构，可以在构成双特异性抗体的同时又尽可能地保持正常抗体的结构。关于KIH技术的指导，例如可参见“An efficient route to human bispecific IgG”,A.Margaret Merchant et al.,Nature Biotechnology,Volume 16,1998，通过引用的方式将该文献全文并入本文。此外，双特异性抗体的结构可以是结合第一抗原的抗体(例如天然抗体形式)从CH 3区的C端延伸出(可以通过柔性连接子)能结合第二抗原的抗原结合部分。
如本文所用术语“抗原结合部分”或“抗原结合片段”可互换使用，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)； A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
对于一般抗体而言，抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv) 2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
在双特异性抗体构建中，“抗原结合部分”包括但不限于Fab片段的形式或单链抗体(scFv)的形式。
如本文所用术语“单链抗体(scFv,single chain fragment variable)”是指一般利用基因工程技术构建的单链结构的抗体，包含重链可变区(VH)和轻链可变区(VL)的一条多肽链。在重链可变区和轻链可变区之间通常会设计一段柔性的连接肽(linker)以便重链可变区和轻链可变区可以折叠成为能够结合抗原的正确构象。
如本文所用术语“Fab(fragment antigen binding)片段”、“Fab部分”或类似的术语是指完整的抗体用木瓜蛋白酶处理后产生的能够与抗原结合的抗体片段，包括完整的轻链(VL-CL)、重链可变区和CH1片段(VH-CH1)。
如本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚 类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(美国专利号4,816,567；和Morrison等人，Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
第一方面，本申请提供了双特异性抗体，其包含结合破伤风毒素不同表位的第一抗原结合片段和第二抗原结合片段，并且所述双特异性抗体具有中和破伤风毒素的活性。
在第一方面的一些实施方案中，所述第一抗原结合片段包含：氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述第二抗原结合片段包含：氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列 如SEQ ID NO：18所示；或者所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：16所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示；或者所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：17所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示。
在第一方面的一些实施方案中，所述第二抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：19所示，轻链可变区的氨基酸序列如SEQ ID NO：20所示。
在第一方面的一些实施方案中，所述第一抗原结合片段和第二抗原结合片段的形式独立地选自单链抗体(scFv)或Fab片段。
在第一方面的一些实施方案中，所述第一抗原结合片段为Fab片段，所述第二抗原结合片段为单链抗体(scFv)。
在第一方面的一些实施方案中，所述双特异性抗体包含SEQ ID NO:21、22和23之一所示的氨基酸序列以及SEQ ID NO:24所示的氨基酸序列。
在第一方面的一些实施方案中，所述的双特异性抗体包含SEQ ID NO:25所示的氨基酸序列。
在第二方面，本申请提供了结合破伤风毒素的单克隆抗体，其包含：
氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第二方面的一些实施方案中，所述单克隆抗体包含：如SEQ ID NO：15所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻 链可变区氨基酸序列；或者如SEQ ID NO：16所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻链可变区氨基酸序列；或者如SEQ ID NO：17所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻链可变区氨基酸序列。
在第三方面，本申请提供了结合破伤风毒素的单克隆抗体，其包含：
氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在第三方面的一些实施方案中，所述单克隆抗体包含如SEQ ID NO：19所示的重链可变区氨基酸序列，如SEQ ID NO：20所示的轻链可变区氨基酸序列。
第四方面，本申请提供了药物组合物，其包含第一方面所述的双特异性抗体、或者第二方面或第三方面所述的单克隆抗体以及药学可接受的赋形剂、稀释剂或载体。
在第四方面的一些实施方案中，所述药物组合物用于预防或治疗破伤风。
在第四方面的一些实施方案中，所述药物组合物还可包含下述物质中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在第四方面的一些实施方案中，可以将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在第四方面的一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服 给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在第四方面的一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第五方面，本申请提供了第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体、或者第四方面所述的药物组合物在制备用于预防或治疗破伤风的药物中的用途。
第六方面，本申请提供了预防或治疗破伤风的方法，包括向有需要的个体给予第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体、或者第四方面所述的药物组合物。
在其他方面，本申请提供了核酸分子，其编码第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体。在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
本申请还提供包含编码第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体的分离的核酸分子的载体以及包含所述核酸分子或载体的宿主细胞。
在其他方面，本申请还提供产生第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体的方法。在一些实施方案中，产生第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体的方法包括培养宿主细胞以便于表达核酸。在一些实施方案中，产生第一方面所述的双特异性抗体、第二方面或第三方面所述的单克隆抗体的方法还包括从宿主细胞培养基中回收双特异性抗体或单克隆抗体。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1：重组破伤风毒素抗原以及重组抗体的制备
1.1.重组破伤风毒素抗原的制备
制备抗破伤风毒素单克隆抗体的过程中需要用到破伤风毒素重链的C末端结构域重组蛋白(TT-Hc，SEQ ID NO:26)。这个蛋白源于破伤风梭菌，不存在翻译后修饰，因而可以利用大肠杆菌表达系统进行表达。此外，在这个重组蛋白的C端添加了His标签(His，SEQ ID NO:27)，将更有利于重组蛋白的纯化和单克隆抗体功能的鉴定。
根据Uniprot数据库的TT-Hc重组蛋白的氨基酸序列，设计并合成编码TT-Hc的基因(包含His标签)。利用常规的分子生物学技术将合成的基因克隆至合适的真核表达载体(如invitrogen公司的pET-22b等)，表达质粒转化大肠杆菌感受态(如索莱宝公司的BL21(DE3)等)，表达TT-Hc-His重组蛋白的大肠杆菌用IPTG(如索莱宝公司的I8070-1g等)诱导表达。然后通过离心等方式收集菌体，用超声波细胞破碎仪(如Sonics公司的VCX130等)超声破碎菌体，通过离心等方式收集上清。利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对上清中的重组蛋白进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时，可以对样品进行过滤除菌，然后分装保存于-20℃。
1.2.重组抗体制备
利用常规的分子生物学手段，将编码抗体重链可变区和轻链可变区的核苷酸序列分别克隆至融合有编码重链恒定区和轻链恒定区核苷酸序列的真核表达载体(如invitrogen公司的pcDNA3.1等)中，组合表达全抗体。抗体的重链恒定区可以是人IgG1亚型(SEQ ID NO：28)、鼠IgG2a亚型(SEQ ID NO：29)、基于KIH(Knob-Into-Hole)技术的人IgG1亚型突变体IgG1H(SEQ ID NO：30)和IgG1K(SEQ ID NO：31)、或者鼠IgG2a亚型突变体mIgG2a-H(SEQ ID NO：32)和mIgG2aK(SEQ ID NO：33)，轻链恒定区可以是人κ亚型(SEQ ID NO：34)、人λ亚型(SEQ ID NO：35)、鼠κ亚型(SEQ ID NO：36)或者鼠λ亚型(SEQ ID NO：37)。
利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组抗体表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清，利用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2:小鼠免疫以及免疫库的构建
以实施例1中制备的重组蛋白TT-Hc-His为抗原，免疫6-8周龄的BALB/c小鼠，免疫剂量为50μg/只小鼠，每14天加强免疫1次，初免后8周处死小鼠并收集脾细胞。使用小鼠淋巴细胞分离液(达科为生物技术股份有限公司，CAT#DKW33-R0100)对小鼠脾脏淋巴细胞进行分离，利用细胞总RNA提取试剂盒(天根生化科技(北京)有限公司，CAT#DP430)，将分离的淋巴细胞进行总RNA的提取。以提取的总RNA为模板，利用第一链cDNA合成试剂盒(Thermo scientific，CAT#K1621)分别合成抗体重链可变区和轻链可变区，反转录引物采取基因特异性引物，引物配对区分别位于抗体重链恒定区和抗体轻链恒定区，具体序列分别为PmCGR:TGCATTTGAACTCCTTGCC(SEQ ID NO：38)和PmCKR:CCATCAATCTTCCACTTGAC(SEQ ID NO：39)。将合成的cDNA立即存放于-70℃保存备用。然后以反转录得到的cDNA为模板，参考文献(Krebber A,Bornhauser S,Burmester J,et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.J Immunol Methods.1997；201(1):35-55，通过引用方式将上述文献的全部内容并入本文中)合成引物，并利用PCR分别扩增编码鼠抗体VH和VK的核苷酸序列，然后利用重叠延伸PCR技术，构建单链抗体(scFv)基因。最后将制备的小鼠单链抗体核苷酸序列克隆至载体pADSCFV-S(实验技术流程可参见中国专利申请第201510097117.0号的实施例1，通过引用方式将上述专利申请的全部内容并入本文中)，构建scFv库。抗体库的库容达到5×10E8， 正确率为50％。
实施例3：小鼠免疫库的筛选和初步鉴定
3.1.小鼠免疫库的筛选
参照文献(中国专利申请第201510097117.0号，通过引用方式将上述专利申请的全部内容并入本文中)，以实施例1制备的重组人TT-Hc-His为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.)，(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5)筛选实施例2构建的展示小鼠单链抗体的噬菌体库，通过结合、洗脱、中和、感染、扩增的方式共进行三轮筛选，最终获得两株特异结合人TT-Hc-His的单链抗体S22E2和S24B8。
利用常规的分子生物学手段，将编码S22E2和S24B8的重链及轻链核苷酸序列分别克隆至融合有编码小鼠mIgG2a重链恒定区和小鼠κ轻链恒定区核苷酸序列的真核表达载体，制备重组鼠源抗体。
3.2.重组抗TT-Hc单克隆抗体的亲和力分析
利用Biacore X100通过表面等离子共振技术测定抗TT-Hc抗体的亲和力。氨基偶联试剂盒(BR-1000-50)、鼠抗体捕获试剂盒(BR-1008-38)，CM5芯片(BR100012)和pH7.4的10×HBS-EP(BR100669)等相关试剂和耗材均购自GE healthcare。依照试剂盒中的说明书，用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlorid，EDC)和N-羟基琥珀酰亚胺(N-Hydroxysuccinimide，NHS)对羧基化CM5芯片表面进行活化，将抗鼠IgG(Fc)抗体(捕获抗体)用10mM pH5.0乙酸钠稀释至25μg/mL，之后以流速10μL/min注射以实现大约高达10000个响应单位(RU)的偶联量。注射捕获抗体之后，注射1M乙醇胺以封闭未反应的基团。对于动力学测量，稀释抗TT-Hc抗体至0.5-1μg/mL，10μL/min注射，保证50RU左右的抗体被抗鼠Fc的抗体捕获。然后将TT-Hc-His设置一系列的浓度梯度(例如6.17nM、18.5nM、55.6nM、167nM和500nM)，于25℃下以30μL/min从低浓度到高浓度进行注射，结合时间为120s，解离时间为3600s，以 10μL/min注射10mM pH2.0的甘氨酸-HCl溶液30s对芯片表面进行再生。使用Biacore X100评估软件2.0.1版，通过1:1结合模型拟合结合和解离传感图来计算结合速率(K a)和解离速率(K d)。以比率K d/K a计算解离平衡常数(KD)。拟合结果如表1所示。
表1.重组抗TT-Hc单克隆抗体结合TT-Hc-his亲和力常数
  K a K d KD
S22E2-mIgG2a 6.424E+4 1.589E-4 2.473E-9
S24B8-mIgG2a 5.488E+4 1.015E-5 1.849E-10
实施例4：抗破伤风毒素单克隆抗体的表位分析
使用重组蛋白TT-Hc-His包被96孔ELISA板(3μg/mL，100μL/孔)，4℃冰箱包被过夜。利用封闭液PBS-0.1％Tween20-3％牛奶在37℃封闭1小时。用固定浓度(1*10 11cfu/mL)的S22E2纯化噬菌体(S22E2噬菌体)对S22E2-mIgG2a和S24B8-mIgG2a鼠源抗体进行梯度稀释，起始浓度50μg/mL，3倍梯度稀释，共11个浓度滴度，100μL/孔加入封闭好的96孔ELISA板中，37℃孵育1小时。使用PBS-0.1％Tween20洗涤ELISA板，然后加入HRP抗M13二抗(北京义翘神州科技股份有限公司，11973-MM05T-H)，37℃孵育1小时。使用PBS-0.1％Tween20洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，使用酶标仪测定492nm/630nm双波长光密度值。ELISA分析结果(图1)显示，S22E2-mIgG2a可以完全抑制S22E2噬菌体和TT-Hc重组蛋白的结合信号，但S24B8-mIgG2a对S22E2噬菌体和TT-Hc的结合没有影响。
类似地，用固定浓度的S24B8纯化噬菌体(S24B8噬菌体)对S22E2-mIgG2a和S24B8-mIgG2a进行梯度稀释，用HRP抗M13二抗检测S24B8噬菌体和TT-Hc的结合信号。ELISA结果(图2)显示，S24B8-mIgG2a可以完全抑制S24B8噬菌体和TT-Hc重组蛋白的结合信号，但S22E2-mIgG2a对S24B8噬菌体和TT-Hc的结合基本没有影响。
综上所述，抗TT-Hc单克隆抗体S22E2-mIgG2a和S24B8-mIgG2a结合TT-Hc的不同表位。
实施例5：破伤风毒素单克隆抗体抑制TT-Hc与神经节苷脂的结合
破伤风毒素可以通过Hc结构域与神经细胞表面的特异性受体结合，目前普遍接受的是双受体机制，一种是神经节苷脂受体，另一种是蛋白受体。将神经节苷脂GT1b，用甲醇稀释至10μg/mL，100μL/孔包被96孔ELISA板，室温放置过夜使甲醇挥发。使用封闭液PBS-0.1％Tween20-1％BSA在37℃封闭1小时。用PBS对TT-Hc进行梯度稀释，起始浓度100μg/mL，3倍梯度稀释，共7个浓度梯度；将抗TT-Hc鼠源单克隆抗体用PBS稀释至40μg/mL，与TT-Hc等体积混合后100μL/孔加入封闭好的96孔ELISA板中，同时设置不加抗体的TT-Hc对照组，37℃孵育1小时。使用PBS-0.1％Tween20洗涤ELISA板，加入HRP抗TT多克隆抗体(实验室制备)，100μL/孔，37℃孵育1小时。使用PBS-0.1％Tween20洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，使用酶标仪测定492nm/630nm双波长光密度值。ELISA分析结果(图3)显示，以TT-Hc的结合曲线作为基准，加入S22E2-mIgG2a后结合曲线明显右移，说明S22E2-mIgG2a可以有效抑制TT-Hc和神经节苷脂GT1b的结合，而S24B8-mIgG2a不抑制TT-Hc与神经节苷脂的结合，相反具有一定的促进作用。
实施例6：抗破伤风毒素单克隆抗体的中和活性
通过将抗体和致死剂量破伤风毒素体外混合后注射Balb/c小鼠确定抗体中和活性。将破伤风毒素与抗破伤风毒素单克隆抗体或抗毒素混合，37℃放置1小时，注射入Balb/c小鼠右后腿肌肉。破伤风毒素注射剂量为20×LD 50/只；商业化来源的马破伤风免疫球蛋白(F(ab') 2)(标记为TAT，上海赛伦生物技术股份有限公司)注射剂量为1IU/只，单克隆抗体S22E2和S24B8的注射剂量均为10μg/只，S24B8与S22E2联用的剂量为各抗体1.25μg/只；将注射20×LD 50的破伤风毒素的Balb/c小鼠作为对照组，共计5组，8只/组。用硼酸缓冲液将破伤风毒素稀释至800×LD 50/mL(即与抗体等量混合后每50微升注射量中含20×LD 50)；用硼酸缓冲液将马破伤风免疫球蛋白稀释至40IU/mL(即与破伤风毒素等量混合后每50微升注射量中含1IU)；用硼酸缓冲液将抗体S22E2和S24B8稀释至0.4mg/mL(或0.1mg/mL)(即S22E2或S24B8单用，与破伤风毒素等量混 合后每50微升注射量中含10μg；S24B8与S22E2联用，等量混合的两种抗体再与破伤风毒素等量混合后每50微升注射量中各含1.25μg)。每只动物注射量为50μL的破伤风毒素和抗体混合物。实验结果如图4所示：对照组的所有Balb/c小鼠都在攻击3天内死于破伤风，实验条件下，单克隆抗体S24B8或S22E2能部分中和毒素，延长小鼠存活期，而S24B8与S22E2联用可以完全中和毒素，小鼠在实验期内全部存活，与马破伤风免疫球蛋白TAT的中和活性一致。
实施例7：抗破伤风毒素单克隆抗体联用保护Balb/c小鼠避免致死性破伤风毒素攻击
Balb/c小鼠进行免疫球蛋白暴露前免疫，再对已注射抗体的小鼠进行致死剂量破伤风毒素的攻击，确定抗体的保护效果。单克隆抗体S22E2和S24B8的注射剂量均为10μg/只，S24B8与S22E2联用组抗体注射剂量为各抗体1.25μg/只；将注射20×LD 50的破伤风毒素的Balb/c小鼠作为对照组，共计4组，8只/组。用硼酸缓冲液将破伤风毒素稀释至400×LD 50/mL(即每50微升注射量中含20×LD 50)；用硼酸缓冲液将抗体S22E2和S24B8稀释至0.2mg/mL(或0.05mg/mL)(即S22E2或S24B8单用，每50微升注射量中含10μg；S24B8与S22E2联用，两种抗体等量混合后每50微升注射量中各含1.25μg)。给予小鼠50μL抗破伤风毒素抗体，并在24小时后使用破伤风毒素对Balb/c小鼠进行右后腿肌肉攻击，每只动物注射量为50μL破伤风毒素。实验结果如图5所示：对照组的所有Balb/c小鼠都在攻击4天内死于破伤风，实验条件下，S24B8与S22E2联用能完全保护Balb/c小鼠，在实验期内全部存活。
实施例8：破伤风毒素单克隆抗体的人源化及脱氨基位点突变
8.1.S24B8鼠单克隆抗体的人源化
对鼠源单克隆抗体S24B8进行人源化研究以降低其免疫原性。人源化方案采取经典的框架移植策略(参见Tan P,Mitchell DA,Buss TN,Holmes MA,Anasetti C,Foote J."Superhumanized"antibodies:reduction of immunogenic potential by complementarity-determining region grafting with  human germline sequences:application to an anti-CD28.J Immunol.2002；169(2):1119-1125，通过引用方式将上述文献的全部内容并入本文中)。将编码S24B8的重链可变区和轻链可变区的基因分别与IMGT数据库中的人抗体胚系基因序列相比较，选择合适的胚系基因序列以提供抗体的框架区1至3(FR1+FR2+FR3)，选择合适的J区基因序列以提供框架区4(FR4)。这个模板可以根据多种因素选出，如：抗体的相对总长度、CDR的大小、位于抗体框架区(FR)和超变区(CDR)之间连接处的氨基酸残基、序列整体的同源性等。所选的模板可以是多个序列的混合物或者可以是共有模板，目的是尽可能维持亲本互补决定区(CDR)的合适构象。同时，为避免抗体超变区(CDR)中脱氨基位点NG可能带来的蛋白异质性，对人源化后的抗体重链可变区进行了突变设计。最终得到了3个人源化的重链可变区突变体S24B8VH-h1(SEQ ID NO：15)、S24B8VH-h2(SEQ ID NO：16)、S24B8VH-h3(SEQ ID NO：17)和1个人源化的轻链可变区突变体S24B8VK-h1(SEQ ID NO：18)。
8.2.S22E2鼠单克隆抗体的人源化
S22E2鼠单克隆抗体的人源化采用经典的框架移植策略。参照实施例8.1，对S22E2的轻重链可变区进行CDR移植得到人源化的重链可变区突变体S22E2VH-h1(SEQ ID NO：19)和人源化轻链可变区突变体S22E2VK-h1(SEQ ID NO：20)。
根据S24B8和S22E2人源化抗体的氨基酸序列，设计并合成抗体的可变区基因，克隆至合适的真核基因表达载体，参照实施例1制备全抗体。S24B8的三个人源化版本命名为S24B8-h1、S24B8-h2和S24B8-h3。人源化S22E2命名为S22E2-h1。
8.3.人源化S24B8和S22E2的亲和力测定
参考实施例3.2，使用人抗体捕获试剂盒(BR-1008-39)，用Biacore X100检测人源化抗TT-Hc抗体结合TT-Hc的亲和力，拟合结果如表2所示。各人源化抗体的重链恒定区均为人IgG1亚型。
表2.重组抗TT-Hc单克隆抗体结合TT-Hc-His亲和力常数
  K a K d KD
S22E2-IgG1 2.042E+5 1.872E-4 9.163E-10
S22E2-h1-IgG1 2.68E+5 1.535E-4 5.728E-10
S24B8-h1-IgG1 7.866E+4 <1E-5 -
S24B8-h2-IgG1 4.447E+4 1.713E-5 3.852E-10
S24B8-h3-IgG1 7.175E+4 1.178E-5 1.642E-10
实施例9：双特异性抗体的制备
基于抗破伤风毒素单克隆抗体S24B8和S22E2联用的中和活性优于单独使用，将结合破伤风毒素Hc蛋白不同表位的抗原结合片段S24B8-h3和S22E2-h1分别设计成Fab形式和scFv形式，构建基于KIH(Knob-Into-Hole)技术的人IgG1异二聚体。即将Fab形式的S24B8-h3抗原结合片段融合在含有Knob突变的Fc段(FcK，SEQ ID NO：40)的N端，将scFv形式的S22E2-h1的抗原结合片段融合在含有Hole突变的Fc段(FcH1，SEQ ID NO：41)的N端，构建抗破伤风毒素蛋白(Hc)的双特异性抗体。
分别将构建的表达S22E2-h1-scFv-FcH1、表达S24B8VH-h3+CH-IgG1K和表达S24B8VK-h1+CK的3个真核表达载体利用脂质体共转染入HEK293F细胞，在无血清悬浮培养条件下培养3-5天，然后通过离心等方式收获培养上清。培养上清中的双特异性抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行纯化，然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组抗体保存缓冲液置换为PBS缓冲液(pH7.0)或者其它合适的缓冲液。将脱盐后蛋白溶液通过尺寸排阻层析(SEC)使用Superdex200(GE)纯化得到双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例10：双特异性抗体的功能验证
10.1.抑制TT-Hc与神经节苷脂的结合
参照实施例5，检测抗TT-Hc抗体对TT-Hc与三种神经节苷脂GD3、GT1b和GM1a结合的抑制能力。ELISA结果(图6、图7和图8)显示： 单克隆抗体S22E2-h1-scFv-FcH1和双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K对TT-Hc与三种神经节苷脂的结合均具有明显的抑制作用，单克隆抗体S24B8-h3-IgG1不影响TT-Hc与三种神经节苷脂的结合。
10.2.中和活性
参考实施例6，破伤风毒素注射剂量为20×LD 50/只,单克隆抗体S24B8-h3和S22E2-h1单独使用的注射剂量为10μg/只，单克隆抗体S24B8-h3和S22E2-h1联合使用的注射剂量为各1.2μg/只，双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K的注射剂量为2.4μg/只；将注射20×LD 50的破伤风毒素的Balb/c小鼠作为对照组，共计5组，8只/组。用硼酸缓冲液将破伤风毒素稀释至800×LD 50/mL(即与抗体等量混合后每50微升注射量中含20×LD 50)；用硼酸缓冲液将抗体S22E2-h1和S24B8-h3稀释至0.4mg/mL(或0.096mg/mL)(即S22E2-h1或S24B8-h3单用，与破伤风毒素等量混合后每50微升注射量中含10μg；S24B8-h3与S22E2-h1联用，等量混合的两种抗体再与破伤风毒素等量混合后每50微升注射量中各含1.2μg)单克隆抗体，用硼酸缓冲液将双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K稀释至0.096mg/mL(即与破伤风毒素等量混合后每50微升注射量中含2.4μg)。每只动物注射量为50μL破伤风毒素和抗体混合物。实验结果如图9所示：对照组的所有Balb/c小鼠都在攻击3天内死于破伤风，S24B8-h3与S22E2-h1联用组的Balb/c小鼠，以及双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K组的Balb/c小鼠在实验期内全部存活，双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K的中和活性优于单克隆抗体。
实施例11：抗体效价测定
基于实施例6中测定抗体中和活性的方法测定双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K的效价，将供试品与标准品进行对比实验，推算出每1mg供试品中所含的抗破伤风毒素抗体国际单位数(IU)。破伤风毒素的稀释：用硼酸缓冲液将破伤风毒素稀释至每毫升含 40个试验量(L+1/10)(即与抗体等量混合后每50微升注射量中含1个试验量(L+1/10))。人破伤风免疫球蛋白标准品的稀释：用硼酸缓冲液将人破伤风免疫球蛋白标准品稀释至4IU/mL(即与毒素等量混合后每50微升注射量中含1/10IU)。用硼酸缓冲液将双特异性抗体S22E2h1-scFv-FcH1+S24B8h3-IgG1K稀释成5个稀释度，依次为14.40μg/mL、16.80μg/mL、20μg/mL、23.6μg/mL和27.6μg/mL(即与毒素等量混合后每50微升注射量中含双抗0.36μg、0.42μg、0.50μg、0.59μg和0.69ug)，稀释度的间隔为15％。稀释后的人破伤风免疫球蛋白标准品溶液及不同稀释度的双特异性抗体溶液，分别与等量的稀释破伤风毒素混合，37℃结合1小时后立即注射，每只小鼠肌肉注射量50μL。每日观察小鼠1次，记录发病及死亡情况。结果显示，对照组的所有Balb/c小鼠都在攻毒72小时死于破伤风，与对照组小鼠同时死亡的双特异性抗体最高剂量组是0.42μg，计算双抗的效价为238IU/mg。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
参考文献
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2.Yousefi M,Tahmasebi F,Younesi V,Razavi A,Khoshnoodi J,Bayat AA,Abbasi E,Rabbani H,Jeddi-tehrani M,Shokri F(2014a)Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C.J Immunotoxicol 11:28–34.
3.Ashton AC,Li Y,Doussau F,Weller U,Dougan G,Poulain B,Dolly JO(1995)Tetanus toxin inhibits neuroexocytosis even when its Zn21-dependent protease activity is removed.J Biol Chem 270:31386–31390.
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Claims (12)

  1. 双特异性抗体，其包含结合破伤风毒素不同表位的第一抗原结合片段和第二抗原结合片段，并且所述双特异性抗体具有中和破伤风毒素的活性。
  2. 如权利要求1所述的双特异性抗体，其中所述第一抗原结合片段包含：氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义。
  3. 如权利要求1所述的双特异性抗体，其中所述第二抗原结合片段包含：氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义。
  4. 如权利要求2所述的双特异性抗体，其中
    所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示；或者
    所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：16所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示；或者
    所述第一抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：17所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示。
  5. 如权利要求3所述的双特异性抗体，其中所述第二抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：19所示，轻链可变区的氨基酸序列如SEQ ID NO：20所示。
  6. 如权利要求2-5中任一项所述的双特异性抗体，其中所述第一抗原结合片段和第二抗原结合片段的形式独立地选自单链抗体(scFv)或Fab片段；
    优选地，所述第一抗原结合片段为Fab片段，所述第二抗原结合片段为单链抗体(scFv)；
    更优选地，所述双特异性抗体包含SEQ ID NO:21、22和23之一所示的氨基酸序列以及SEQ ID NO:24所示的氨基酸序列；和/或
    所述的双特异性抗体包含SEQ ID NO:25所示的氨基酸序列。
  7. 结合破伤风毒素的单克隆抗体，其包含：
    氨基酸序列为SYWIY(SEQ ID NO：1)的HCDR1，氨基酸序列为EINPTNGFANYNEKFKT(SEQ ID NO：2)或EINPTAGFANYNEKFKT(SEQ ID NO：3)或EINPTNAFANYNEKFKT(SEQ ID NO：4)的HCDR2，氨基酸序列为HFRFPY(SEQ ID NO：5)的HCDR3，氨基酸序列为RASQDIGSSLT(SEQ ID NO：6)的LCDR1，氨基酸序列为ATSSLDS(SEQ ID NO：7)的LCDR2和氨基酸序列为LQYASSPYT(SEQ ID NO：8)的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义；
    优选地，所述单克隆抗体包含：
    如SEQ ID NO：15所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻链可变区氨基酸序列；或者
    如SEQ ID NO：16所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻链可变区氨基酸序列；或者
    如SEQ ID NO：17所示的重链可变区氨基酸序列，如SEQ ID NO：18所示的轻链可变区氨基酸序列。
  8. 结合破伤风毒素的单克隆抗体，其包含：
    氨基酸序列为DYGVN(SEQ ID NO：9)的HCDR1，氨基酸序列为MIWSDGTTDYSSALKS(SEQ ID NO：10)的HCDR2，氨基酸序列为VDGYSHYYAMDY(SEQ ID NO：11)的HCDR3，氨基酸序列为RASENIYSYLA(SEQ ID NO：12)的LCDR1，氨基酸序列为NAKTLAE(SEQ ID NO：13)的LCDR2和氨基酸序列为QHHYGLPFT(SEQ ID NO：14)的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义；
    优选地，所述单克隆抗体包含如SEQ ID NO：19所示的重链可变区氨基酸序列，如SEQ ID NO：20所示的轻链可变区氨基酸序列。
  9. 药物组合物，其包含权利要求1-6中任一项所述的双特异性抗体、或者权利要求7或8所述的单克隆抗体以及药学可接受的赋形剂、稀释剂或载体。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗破伤风。
  11. 权利要求1-6中任一项所述的双特异性抗体、权利要求7或8所述的单克隆抗体、或者权利要求9或10所述的药物组合物在制备用于预防或治疗破伤风的药物中的用途。
  12. 预防或治疗破伤风的方法，其包括向有需要的个体给予权利要求1-6中任一项所述的双特异性抗体、权利要求7或8所述的单克隆 抗体、或者权利要求9或10所述的药物组合物。
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