WO2021000886A1 - 百日咳毒素结合蛋白 - Google Patents

百日咳毒素结合蛋白 Download PDF

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WO2021000886A1
WO2021000886A1 PCT/CN2020/099691 CN2020099691W WO2021000886A1 WO 2021000886 A1 WO2021000886 A1 WO 2021000886A1 CN 2020099691 W CN2020099691 W CN 2020099691W WO 2021000886 A1 WO2021000886 A1 WO 2021000886A1
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seq
single variable
variable domain
immunoglobulin single
immunoglobulin
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PCT/CN2020/099691
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English (en)
French (fr)
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徐霆
汪皛皛
王玲
朱丹明
高丽
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苏州康宁杰瑞生物科技有限公司
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Priority to US17/624,062 priority Critical patent/US20230061378A1/en
Priority to CN202080049059.3A priority patent/CN114466863A/zh
Priority to EP20834925.8A priority patent/EP3995510A4/en
Priority to JP2021577588A priority patent/JP7315259B2/ja
Publication of WO2021000886A1 publication Critical patent/WO2021000886A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1225—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Bordetella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/235—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bordetella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of medical biology, and discloses a single domain antibody against pertussis toxin (PT) and its derivative protein. Specifically, the present invention discloses a pertussis toxin binding protein and its use.
  • PT pertussis toxin
  • Bordetella pertussis (B. pertussis) is a gram-negative bacterium that infects the upper respiratory tract and causes uncontrollable severe cough. According to the World Health Organization, Bordetella pertussis infection causes an estimated 300,000 deaths worldwide each year, mainly in young unvaccinated infants. Babies with whooping cough often need to be hospitalized in a pediatric intensive care unit, and their treatment usually involves mechanical ventilation. Pertussis in adults usually causes a chronic cough. The incidence of whooping cough increases due to exposure of unvaccinated and undervaccinated individuals (including infants who have not been fully vaccinated), individuals whose immunity has weakened over time, and asymptomatic carriers.
  • the present invention provides a pertussis toxin binding protein capable of specifically binding to pertussis toxin and comprising at least one immunoglobulin single variable domain, the at least one immunoglobulin single variable domain comprising selected from CDR1, CDR2 and CDR3:
  • the present invention provides a nucleic acid molecule that encodes the pertussis toxin binding protein of the present invention.
  • the invention provides an expression vector comprising the nucleic acid molecule of the invention operably linked to an expression control element.
  • the present invention provides a recombinant cell which contains the nucleic acid molecule of the present invention or is transformed with the expression vector of the present invention, and is capable of expressing the pertussis toxin binding protein.
  • the present invention provides a method for producing the pertussis toxin binding protein of the present invention, comprising:
  • step b) optionally further purifying and/or modifying the pertussis toxin binding protein obtained from step b).
  • the present invention provides a pharmaceutical composition comprising the pertussis toxin binding protein of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating Bordetella pertussis infection in a subject, the method comprising administering to the subject an effective amount of the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention .
  • the present invention provides a method for preventing Bordetella pertussis infection in a subject, the method comprising to the subject an effective amount of the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention.
  • the present invention provides a method for detecting the presence and/or amount of pertussis toxin in a biological sample, including:
  • the difference in complex formation between the biological sample and the control sample indicates the presence and/or amount of pertussis toxin in the sample.
  • the present invention provides a kit comprising the pertussis toxin binding protein of the present invention.
  • Figure 1 Survival curve of protection against PT toxin at a single dose of 20 ⁇ g/dose.
  • Figure 5 Survival curve of protection against Bacillus pertussis with multiple doses of 20 ⁇ g/dose or 40 ⁇ g/dose.
  • Figure 7 Amino acid sequence alignment of iPT15 humanized variants.
  • Figure 8 Amino acid sequence alignment of iPT42 humanized variants.
  • antibody or “immunoglobulin” used interchangeably herein, whether referring to heavy chain antibodies or conventional 4-chain antibodies, are used as general terms to include full-length antibodies, individual The chain and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domain or VH/VL domain, respectively).
  • sequence as used herein (for example, in terms such as “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence) should generally be understood To include both the related amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.
  • domain refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally speaking, a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without losing the rest of the protein and/or the function of the domain.
  • immunoglobulin domain refers to a spherical region of an antibody chain (for example, a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a spherical region.
  • the immunoglobulin domain is characterized in that it maintains the immunoglobulin folding characteristics of antibody molecules.
  • immunoglobulin variable domain refers to the term "framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3 "Or “FR3” and “framework region 4" or “FR4" four “framework regions” composed of immunoglobulin domains, wherein the framework regions are referred to as “complementarity determining region 1" in the art and hereinafter, respectively Or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are separated by three “complementarity determining regions” or "CDRs".
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the immunoglobulin variable domains have antigen-binding sites to give antibodies specificity for antigens.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding to an epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain in the meaning of the present invention is a "domain antibody”, such as immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is the "VHH domain” of Camelidae (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments and VHH antibodies, are antigen-binding immunity called “heavy chain antibodies” (ie “antibodies lacking light chains")
  • the variable domain of globulin Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature (363,446-448 1993)).
  • the term “VHH domain” is used to compare the variable domain with the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain”) as well as those present in conventional 4-chain antibodies.
  • the light chain variable domain (which is referred to herein as the "VL domain") distinguishes it.
  • the VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain In the context of the present invention, the terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “VHH antibody fragment”, and “VHH antibody” are used interchangeably.
  • the amino acid residues used in the VHH domain of Camelidae can be based on the VH domain given by Kabat et al. According to the general numbering method (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM CDR represents a compromise between Kabat hypervariable region and Chothia structural loop, and is used in Oxford Molecular's AbM antibody modeling software.
  • the "Contact" CDR is based on the analysis of the available complex crystal structure. The residues of the CDRs from each method are described below:
  • the total number of amino acid residues in each CDR may be different, and may not correspond to the total number of amino acid residues indicated by Kabat numbering (ie according to One or more positions of Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering).
  • Kabat numbering ie according to One or more positions of Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering.
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • CDR may include "extended CDR", such as 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 ( LCDR3); 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • extended CDR such as 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 ( LCDR3)
  • HCDR1 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • the total number of amino acid residues in the VHH domain will usually be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domain The other structural and functional properties of the VHH domain and the polypeptides containing it can be summarized as follows:
  • the VHH domain (which has been naturally "designed” to functionally bind to the antigen without the light chain variable domain and interacting with the light chain variable domain) can be used as a single and relatively small function Sexual antigen binding structural unit, domain or polypeptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies. These VH and VL domains themselves are usually not suitable for practical applications as single antigen binding proteins or immunoglobulin single variable domains.
  • the form or another form is combined to provide a functional antigen binding unit (e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFv composed of VH domains covalently linked to VL domains).
  • VHH domains are used as part of a larger polypeptide—provides many advantages over the use of conventional VH and VL domains, scFv, or conventional antibody fragments (eg Fab- or F(ab')2-fragments)
  • Significant advantages Only a single domain is required to bind antigen with high affinity and high selectivity, so that neither two separate domains are required, nor is it necessary to ensure that the two domains exist in an appropriate spatial conformation and configuration (such as scFv generally requires the use of specially designed linkers);
  • VHH domains can be expressed from a single gene without post-translational folding or modification; VHH domains can be easily transformed into a multivalent and multispecific format (formatted); VHH structure The domain is highly soluble and has no tendency to aggregate; the VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without using freezing equipment, thereby saving cost, time and environment ; VHH domain is easy to
  • the VHH domain derived from Camelidae can be processed by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding position in the VH domain of a conventional human 4-chain antibody.
  • Humanization also referred to herein as “sequence optimization", in addition to humanization, “sequence optimization” can also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removing Potential post-translational modification sites).
  • the humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using a method of resurfacing of protein surface amino acids and/or a humanized universal framework CDR grafting (CDR grafting to a universal framework), for example, as exemplified in the embodiments.
  • epitope or the term “antigenic determinant” used interchangeably refers to any epitope on the antigen to which the paratope of an antibody binds.
  • Antigenic determinants usually contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear "Epitope” or "Conformational” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined by, for example, the following method: synthesize a large number of peptides on a solid support at the same time, wherein these peptides correspond to parts of the protein molecule, and make these peptides interact with the support while still connected to the support. Antibody response.
  • conformational epitopes can be identified by, for example, determining the spatial configuration of amino acids by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).
  • Antibodies can be screened competitively for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete with each other or cross-compete with antigen binding. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in the international patent application WO03/48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecule of the present invention for binding to the same epitope on the pertussis toxin.
  • the term “specificity” refers to the number of different types of antigens or epitopes that a specific antigen binding molecule or antigen binding protein (such as the immunoglobulin single variable domain of the present invention) can bind.
  • the specificity of the antigen binding protein can be determined based on its affinity and/or affinity.
  • the affinity expressed by the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the difference between the epitope and the antigen-binding protein The stronger the binding strength between (or, the affinity can also be expressed as the association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner.
  • Avidity is a measure of the binding strength between an antigen binding protein (for example, an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide containing the same) and a related antigen.
  • Avidity is related to both: the affinity between the antigen-binding sites on its antigen-binding protein, and the number of related binding sites present on the antigen-binding protein.
  • pertussis toxin binding protein means any protein capable of specifically binding to pertussis toxin.
  • the pertussis toxin binding protein may include an antibody as defined herein against pertussis toxin.
  • Pertussis toxin binding protein also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • IgSF immunoglobulin superfamily antibodies
  • Pertussis toxin is a multi-subunit protein toxin derived from Bordetella pertussis (B. pertussis), which can specifically act on G protein and inhibit G protein from exerting its signal pathway function.
  • Pertussis toxin contains subunit S1, subunit S2, subunit S3, subunit S4, and subunit S5. Its amino acid sequence is entered in the UniProtKB database, and the corresponding accession numbers are: P04977, P04978, P04979, P0A3R5, and P04981.
  • the "pertussis toxin binding protein" of the present invention may comprise at least one immunoglobulin single variable domain such as VHH that binds to pertussis toxin.
  • the "pertussis toxin binding molecule" of the present invention may comprise 2, 3, 4 or more immunoglobulin single variable domains such as VHH that bind pertussis toxin.
  • the pertussis toxin binding protein of the present invention may include a linker and/or a part with effector functions in addition to the immunoglobulin single variable domain that binds to pertussis toxin, such as a half-life extension part (such as a single immunoglobulin that binds to serum albumin).
  • variable domain and/or fusion partner (such as serum albumin) and/or conjugated polymer (such as PEG) and/or Fc region.
  • fusion partner such as serum albumin
  • conjugated polymer such as PEG
  • the pertussis toxin binding protein of the present invention will be preferably 10 -7 to 10 -10 mol/liter (M), more preferably 10 -8 to 10 -10 mol/liter, as measured in the Biacore or KinExA or Fortibio assay. Even more preferably a dissociation constant (KD) of 10 -9 to 10 -10 or lower, and/or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , more Preferably, an association constant (KA) of at least 10 10 M -1 binds to the antigen to be bound (ie, pertussis toxin). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, the surface plasmon resonance (SPR) assay, Scatchard assay, and/or competitive binding assay described herein (e.g., Radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay.
  • SPR surface plasmon resonance
  • RIA Radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competitive assay sandwich competitive assay.
  • amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as well known and agreed in the art.
  • amino acid difference refers to the insertion, deletion or substitution of a specified number of amino acid residues at a certain position in the reference sequence compared to another sequence.
  • substitution will preferably be a conservative amino acid substitution, which refers to the substitution of an amino acid residue by another amino acid residue with a similar chemical structure, and its impact on the function, activity or other biological properties of the polypeptide Little or almost no effect.
  • the conservative amino acid substitutions are well known in the art.
  • a conservative amino acid substitution is preferably one amino acid in the following groups (i)-(v) is replaced by another amino acid residue in the same group: (i) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) Polar negatively charged residues and (uncharged) amides: Asp, Asn, Glu and Gln; (iii) Polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala Or Pro replacement; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Replace; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is usually measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For the determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Information and Genome Projects, Smith, DW, ed., Academic Press, New York ,1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • the polypeptide or nucleic acid molecule is considered “isolated”.
  • a polypeptide or nucleic acid molecule is considered “isolated” when it has been purified at least 2 times, particularly at least 10 times, more particularly at least 100 times and up to 1000 times or more.
  • suitable techniques e.g. suitable chromatographic techniques, such as polyacrylamide gel electrophoresis
  • the "separated" polypeptides or nucleic acid molecules are preferably substantially homogeneous.
  • Effective amount means that the amount of the pertussis toxin binding protein or pharmaceutical composition of the present invention can reduce the severity of disease symptoms, increase the frequency and duration of the asymptomatic period of the disease, or prevent damage or loss caused by disease pain. can.
  • subject means mammals, especially primates, especially humans.
  • the present invention provides a pertussis toxin binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding pertussis toxin.
  • the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NOs: 40-52.
  • the CDR may be Kabat CDR, AbM CDR, Chothia CDR or Contact CDR. In some embodiments, the CDR is a Kabat CDR.
  • the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 selected from:
  • CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3 (corresponding to the CDR of iPT 3);
  • At least one immunoglobulin single variable domain in the pertussis toxin binding protein of the present invention is VHH.
  • the VHH comprises any amino acid sequence in SEQ ID NO: 40-52.
  • At least one immunoglobulin single variable domain in the pertussis toxin binding protein of the present invention is a humanized VHH.
  • At least one immunoglobulin single variable domain in the pertussis toxin binding protein of the present invention is a humanized VHH, and the humanized VHH includes any of SEQ ID NO: 40-52 A sequence has an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity.
  • the amino acid sequence of the humanized VHH includes one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 40-52. For example, it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.
  • At least one immunoglobulin single variable domain in the pertussis toxin binding protein of the present invention is a humanized VHH, wherein the humanized VHH comprises any of SEQ ID NO: 53-85 An amino acid sequence.
  • the pertussis toxin binding protein comprises an immunoglobulin single variable domain that specifically binds to pertussis toxin.
  • the pertussis toxin binding protein comprises at least two, for example, 2, 3, 4 or more immunoglobulin single variable domains that specifically bind pertussis toxin.
  • the at least two immunoglobulin single variable domains bind to the same epitope or compete for binding or partially compete for binding to the same epitope, for example, the at least two immunoglobulin single variable domains are identical.
  • the at least two immunoglobulin single variable domains bind to different epitopes or do not compete for binding to the same epitope.
  • epitope binning which is the biomembrane interference technology BLI, as illustrated in the examples of this application.
  • the at least two immunoglobulin single variable domains that specifically bind pertussis toxin are directly connected to each other.
  • the at least two immunoglobulin single variable domains that specifically bind pertussis toxin are connected to each other by a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the joint is a flexible joint, such as GGGGS, GS, GAP, (GGGGS)x 3, etc.
  • the pertussis toxin binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein the first immunoglobulin single variable domain is located at all The N-terminus of the second immunoglobulin single variable domain, and
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, CDR3 shown in SEQ ID NO: 24, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39
  • second immunoglobulin The single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5, and CDR3 shown in SEQ ID NO: 6; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 33; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, CDR3 shown in SEQ ID NO: 33, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, and CDR3 shown in SEQ ID NO: 33; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32, CDR3 shown in SEQ ID NO: 33, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15, and the second immunoglobulin
  • the globulin single variable domain comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9; or
  • first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9, and the second immunoglobulin
  • the single variable domain of a globulin comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39; or
  • the first immunoglobulin single variable domain comprises CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 38, and CDR3 shown in SEQ ID NO: 39
  • the second immunoglobulin The globulin single variable domain includes CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8, and CDR3 shown in SEQ ID NO: 9.
  • the pertussis toxin binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein the first immunoglobulin single variable domain is located at all The N-terminus of the second immunoglobulin single variable domain, and
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 41, 53-63 and the second immunoglobulin single variable domain comprises SEQ ID NO: 44, 64- The amino acid sequence shown in one of 74; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 44, 64-74 and the second immunoglobulin single variable domain comprises SEQ ID NO: 41, 53- The amino acid sequence shown in one of 63; or
  • the first immunoglobulin single variable domain includes the amino acid sequence shown in one of SEQ ID NO: 41, 53-63, and the second immunoglobulin single variable domain includes the amino acid sequence shown in SEQ ID NO: 47 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 47 and the second immunoglobulin single variable domain comprises the amino acid shown in one of SEQ ID NO: 41, 53-63 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 41, 53-63 and the second immunoglobulin single variable domain comprises SEQ ID NO: 52, 75- The amino acid sequence shown in one of 85; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 52, 75-85 and the second immunoglobulin single variable domain comprises SEQ ID NO: 41, 53- The amino acid sequence shown in one of 63; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 42 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 50; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 50 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 42; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 44, 64-74, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 50 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 44, 64-74 and the second immunoglobulin single variable domain comprises SEQ ID NO: 52, 75- The amino acid sequence shown in one of 85; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 50 and the second immunoglobulin single variable domain comprises the amino acid shown in one of SEQ ID NO: 52, 75-85 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 42 and the second immunoglobulin single variable domain comprises the amino acid shown in one of SEQ ID NO: 44, 64-74 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 44, 64-74, and the second immunoglobulin single variable domain comprises the amino acid shown in SEQ ID NO: 42 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 42 and the second immunoglobulin single variable domain comprises the amino acid shown in one of SEQ ID NO: 52, 75-85 Sequence; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO: 52, 75-85, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 42 sequence.
  • the pertussis toxin binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein the first immunoglobulin single variable domain is located at all The N-terminus of the second immunoglobulin single variable domain, and
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 63 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 70; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 63 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 74; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 80 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 74; or
  • the first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 80 and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO: 63.
  • the pertussis toxin binding protein of the present invention comprises an amino acid sequence selected from SEQ ID NO: 87-105.
  • the pertussis toxin binding protein of the present invention in addition to at least one immunoglobulin single variable domain capable of specifically binding pertussis toxin, further comprises an immunoglobulin Fc region.
  • Including the immunoglobulin Fc region in the pertussis toxin binding protein of the present invention can make the binding protein form a dimer molecule and at the same time extend the half-life of the binding protein in vivo.
  • the Fc region useful in the present invention may be derived from immunoglobulins of different subtypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.
  • the immunoglobulin Fc region generally includes the hinge region or part of the hinge region, the CH2 region and the CH3 region of the immunoglobulin constant region.
  • mutations can be introduced on the wild-type Fc sequence to alter the related Fc-mediated activity.
  • the mutations include, but are not limited to: a). mutations that change Fc-mediated CDC activity; b). mutations that change Fc-mediated ADCC activity; or c). mutations that change FcRn-mediated half-life in vivo.
  • Such mutations are described in the following documents: Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al.
  • one, two, three, four, five, six, seven, eight, nine, or ten amino acids in the CH2 region can be mutated to increase or remove Fc-mediated ADCC or CDC activity or to increase or decrease the activity of FcRn. Affinity.
  • the stability of the protein can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the hinge region.
  • mutations can be introduced into the Fc sequence, so that the mutated Fc is more likely to form homodimers or heterodimers.
  • the knob-hole model that uses the steric effect of amino acid side chain groups on the Fc contact interface makes it easier to form heterodimers between different Fc mutations; another example is CN In 102558355A or CN 103388013A, by changing the charge of amino acids of the Fc contact interface, the ionic interaction force between the Fc contact interface is changed, so that different Fc mutation pairs are more likely to form heterodimers (CN 102558355A). Or, it is easier to form homodimers between Fc with the same mutation (CN 103388013A).
  • the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably a human IgG1 Fc region.
  • the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:86.
  • the at least one immunoglobulin single variable domain capable of specifically binding pertussis toxin is directly connected to the immunoglobulin Fc region or is connected through a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the linker is a flexible linker, such as GGGGS, GS, GAP, etc.
  • the immunoglobulin Fc region is located at the C-terminus of the at least one immunoglobulin single variable domain that specifically binds to pertussis toxin.
  • the pertussis toxin binding protein of the present invention comprises a first amino acid sequence selected from one of SEQ ID NOs: 40-85 and 87-105, and a SEQ ID directly connected to the first amino acid sequence or connected through a linker The second amino acid sequence shown in NO: 86, wherein the second amino acid sequence is located at the C-terminus of the first amino acid sequence.
  • the pertussis toxin binding protein of the present invention comprises an amino acid sequence selected from SEQ ID NO: 106-109.
  • the pertussis toxin binding protein of the present invention comprises an immunoglobulin single variable domain that specifically binds to pertussis toxin, which is directly or via a linker connected to the immunoglobulin Fc region, the immunoglobulin Fc region
  • the pertussis toxin binding protein is allowed to form a dimer molecule comprising two pertussis toxin binding domains.
  • Such pertussis toxin binding protein is also called bivalent pertussis toxin binding protein.
  • the dimer is a homodimer.
  • the pertussis toxin binding protein of the present invention comprises two immunoglobulin single variable domains that specifically bind pertussis toxin and an immunoglobulin Fc region which are connected directly or through a linker.
  • the Fc region allows the pertussis toxin binding protein to form a dimer molecule comprising four pertussis toxin binding domains.
  • Such pertussis toxin binding protein is also called tetravalent pertussis toxin binding protein.
  • the dimer is a homodimer.
  • the invention in another aspect, relates to a nucleic acid molecule encoding the pertussis toxin binding protein of the invention.
  • the nucleic acid of the present invention can be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acid of the present invention may also be in the form of a vector, may be present in a vector and/or may be a part of a vector, such as a plasmid, cosmid or YAC.
  • the vector may especially be an expression vector, that is, a vector that provides the pertussis toxin binding protein in vitro and/or in vivo (ie, in a suitable host cell, host organism, and/or expression system).
  • the expression vector usually contains at least one nucleic acid of the present invention, which is operably linked to one or more suitable expression control elements (e.g., promoter, enhancer, terminator, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Specific examples of regulatory elements and other elements useful or necessary for the expression of the pertussis toxin binding protein of the present invention, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.
  • the nucleic acid of the present invention can be prepared or obtained in a known manner (for example, by automated DNA synthesis and/or recombinant DNA technology) based on the information about the amino acid sequence of the polypeptide of the present invention given herein, and/or can be obtained from suitable natural sources. The source is separated.
  • the present invention relates to a recombinant host cell expressing or capable of expressing one or more pertussis toxin binding proteins of the present invention and/or containing the nucleic acid or vector of the present invention.
  • the preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include gram-negative bacterial strains (e.g. Escherichia coli, Proteus and Pseudomonas strains) and gram-positive bacterial strains (e.g. Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).
  • gram-negative bacterial strains e.g. Escherichia coli, Proteus and Pseudomonas strains
  • gram-positive bacterial strains e.g. Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.
  • Suitable fungal cells include cells of Trichoderma, Neurospora and Aspergillus species; or include Saccharomyces (such as Saccharomyces cerevisiae), fission Saccharomyces (Schizosaccharomyces) (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Chinese Cells of species of the genus Hansenula.
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission Saccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Chinese Cells of species of the genus Hansenula Chinese Cells of species of the genus Hansenula.
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells and the like.
  • the present invention can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins.
  • the present invention also provides a method for producing the pertussis toxin binding protein of the present invention.
  • the method generally comprises the following steps:
  • the pertussis toxin binding protein of the present invention can be produced in a cell as described above in an intracellular manner (for example, in the cytoplasm, in the periplasm, or in inclusion bodies), and then isolated from the host cell and optionally further purified; or it can be extracellular (E.g., in the culture medium in which the host cells are grown), then separated from the culture medium and optionally further purified.
  • the pertussis toxin binding protein of the present invention can also be obtained by other protein production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.
  • the present invention provides a composition, such as a pharmaceutical composition, which contains one or a combination of the pertussis toxin binding protein of the present invention formulated with a pharmaceutically acceptable carrier.
  • a composition may comprise one or a combination (e.g., two or more different) pertussis toxin binding proteins of the invention.
  • the pharmaceutical composition of the present invention may contain a combination of antibody molecules that bind to different epitopes on the target antigen (pertussis toxin).
  • the "pharmaceutically acceptable carrier” used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).
  • the active compound ie, antibody molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
  • the pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that maintains the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1 -19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from aliphatic monocarboxylic and dicarboxylic acids, phenyl Salts derived from non-toxic organic acids such as substituted alkanoic acid, hydroxyalkanoic acid, aromatic acid, aliphatic and aromatic sulfonic acid.
  • non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid
  • phenyl Salts derived from non-toxic organic acids such as substituted alkanoic acid, hydroxyalkanoic acid, aromatic acid, aliphatic and aromatic sulfonic acid.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and those derived from alkaline earth metals such as N,N'-dibenzylethylenediamine, N-methylglucamine, and chloroprocaine. , Choline, diethanolamine, ethylenediamine, procaine and other non-toxic organic amine-derived salts.
  • the pharmaceutical composition of the present invention may also contain pharmaceutically acceptable antioxidants.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitate ascorbic acid Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA) ), sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as palmitate ascorbic acid Ester, butylated hydroxy
  • compositions may also contain agents such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the prevention of the presence of microorganisms can be ensured by sterilization procedures or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, etc.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide.
  • Prolonged absorption of injectable drugs can be achieved by adding absorption delaying agents, such as monostearate and gelatin, to the composition.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injections or dispersions.
  • sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injections or dispersions.
  • Conventional media or agents, except for any range incompatible with the active compound, may be in the pharmaceutical composition of the present invention. It is also possible to incorporate supplementary active compounds into the composition.
  • the therapeutic composition must generally be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.
  • the carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • a coating such as lecithin
  • a surfactant proper fluidity can be maintained.
  • Sterile injection can be prepared by mixing the active compound in a suitable solvent in the required amount, and adding one or a combination of the above-listed ingredients as needed, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other required ingredients listed above.
  • the preferred preparation methods are vacuum drying and freeze drying (lyophilization), and the powder of the active ingredient plus any additional required ingredients is obtained from the pre-sterile filtered solution.
  • the amount of active ingredient that can be combined with a carrier material to prepare a single dosage form varies according to the subject to be treated and the specific mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to prepare a single dosage form is generally that amount of the composition that produces a therapeutic effect. Generally, on a 100% basis, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, most preferably about 1% to about 30% of the active ingredient, and pharmaceutically acceptable The carrier is combined.
  • the dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be reduced or increased proportionally as needed by the emergency of the treatment condition. It is particularly advantageous to formulate parenteral compositions in dosage unit form for easy administration and uniform dosage.
  • the dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount of active compound, and the predetermined amount of active compound is calculated in combination with the required pharmaceutical carrier to produce The desired therapeutic effect.
  • the specific description of the dosage unit form of the present invention is limited and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent in the art for formulating such sensitivity to treat individuals Limits of active compounds.
  • the dosage range is about 0.0001 to 100 mg/kg, more usually 0.01 to 30 mg/kg of recipient body weight.
  • the dosage can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight or 30 mg/kg body weight, or within the range of 1-30 mg/kg .
  • Exemplary treatment regimens require dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or starting dosing intervals Slightly shorter (such as once a week to once every three weeks) later dosing interval lengthened (such as once a month to once every 3-6 months).
  • the antibody molecule can also be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dosage and frequency vary according to the half-life of the antibody molecule in the patient.
  • human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies.
  • the dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are given at less frequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete improvement in disease symptoms. Afterwards, it can be administered to the patient in a preventive regimen.
  • the actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be changed to obtain an amount of the active ingredient that can effectively achieve the desired therapeutic response to a specific patient, composition and mode of administration without being toxic to the patient.
  • the dosage level selected depends on a variety of pharmacokinetic factors, including the application of the specific composition of the invention or the activity of its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the specific compound used, and the therapeutic effect. Duration, other drugs, compounds and/or materials used in combination with the specific composition applied, age, gender, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field
  • composition of the present invention can be administered through one or more administration routes using one or more methods known in the art. Those skilled in the art should understand that the route and/or manner of administration will vary according to the desired result.
  • the preferred route of administration of the pertussis toxin binding protein of the present invention includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as injection or infusion.
  • parenteral administration refers to a mode of administration other than enteral and local administration, usually injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions.
  • the pertussis toxin binding protein of the present invention can also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual or topical.
  • non-parenteral routes such as topical, epidermal or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual or topical.
  • the present invention relates to a method of treating a Bordetella pertussis infection in a subject, the method comprising administering (e.g., in an effective amount) the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention to the subject.
  • the method of the present invention relates to a method of preventing Bordetella pertussis infection in a subject, the method comprising administering (for example, in an effective amount) the pertussis toxin binding protein of the present invention or the drug of the present invention combination.
  • the subject is at risk of Bordetella pertussis infection (eg, the patient is a pre-vaccination infant and/or the patient has been exposed to pertussis toxin).
  • the method of the present invention relates to a method of neutralizing pertussis toxin in a subject, the method comprising administering the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention to the subject (for example, in an effective amount).
  • the subject is infected with Bordetella pertussis.
  • the method of the present invention relates to a method for a disease or disorder caused by Bordetella pertussis infection in a subject, the method comprising administering (for example, in an effective amount) the pertussis toxin binding protein of the present invention or The pharmaceutical composition of the present invention.
  • Leukocytosis or increased white blood cell counts are characteristic of Bordetella pertussis infection.
  • the method of the invention includes reducing the white blood cell count in the patient.
  • the method of the invention produces an accelerated regression of leukocytosis.
  • the method of the invention produces a reduction in the maximum white blood cell count during the course of infection.
  • the method of the invention produces neutralization (inhibition or antagonism) of pertussis toxin protein.
  • the pertussis toxin binding protein of the present invention can bind to the pertussis toxin protein so as to partially or completely inhibit one or more biological activities of the pertussis toxin protein.
  • One of the biological activities of pertussis toxin protein that can be inhibited or blocked by neutralizing antibodies is the ability of pertussis toxin protein to bind to cell receptors.
  • the receptor binding region of pertussis toxin protein consists of four polypeptide subunits called subunit S2, subunit S3, subunit S4, and subunit S5, respectively.
  • Examples of cellular receptors bound by the subunits S2, S3, S4 and S5 of the pertussis toxin protein are members of the N-linked sialoglycoprotein family such as fetuin, haptoblobin and transferrin.
  • the pertussis toxin binding protein of the present invention prevents the pertussis toxin protein from binding to its cellular receptor.
  • the pertussis toxin binding protein of the present invention changes the intracellular transport step of pertussis toxin so that the toxin does not reach the cell cytoplasm.
  • Another important activity of the pertussis toxin protein that can be inhibited by the pertussis toxin binding protein of the present invention is the enzymatic activity of the pertussis toxin protein as an ADP ribosylase against the G protein.
  • the subunit S1 of the pertussis toxin protein conferring enzyme activity as an ADP ribosylase is subunit S1.
  • the pertussis toxin protein is pertussis sea cucumber toxin.
  • the pertussis sea cucumber toxin, referred to herein as the pertussis toxin protein contains all five pertussis toxin protein subunits.
  • the pertussis toxin protein is a truncated pertussis toxin protein.
  • the truncated pertussis protein as mentioned herein comprises at least one of pertussis toxin protein subunits (ie, S1, S2, S3, S4, and S5).
  • pertussis toxin protein subunits ie, S1, S2, S3, S4, and S5.
  • S1, S2, S3, S4, and S5 pertussis toxin protein subunits
  • the compositions and methods of the invention are suitable for treating or preventing pertussis infection at any stage.
  • the incubation period of whooping cough is usually 7-10 days, the range is 4-21 days, and rarely can be as long as 42 days.
  • the compositions and methods of the invention increase the length of the incubation period by making infections more difficult to occur.
  • the clinical course of the disease is divided into three stages. The first stage, the catarrhal stage, is characterized by the onset of insidious rhinitis, sneezing, low fever and mild, and occasional cough, similar to a cold. The cough gradually becomes more severe, and after 1-2 weeks, the second or paroxysmal phase begins.
  • the compositions and methods of the invention reduce the length of the catarrhal phase and optionally prevent it from progressing to the paroxysmal phase.
  • the compositions and methods of the present invention treat one or more of rhinitis, sneezing, low fever, and cough. It is during the paroxysmal phase when pertussis is usually suspected. Typically, patients have a large number of bursts or bursts of rapid coughing, apparently due to the difficulty in expelling thick mucus from the tracheobronchial tree. At the end of the paroxysm, a long inhalation effort is usually accompanied by a characteristic loud gasp. During this attack, the patient may become cyanotic.
  • the compositions and methods of the present invention reduce the amount and/or frequency of flare-ups.
  • the compositions and methods of the invention prevent patients from becoming cyanotic. Paroxysmal attacks occur more frequently at night, with an average of 15 attacks every 24 hours. During the first 1 or 2 weeks of this phase, the frequency of attacks increases, remains at the same level for 2 to 3 weeks, and then gradually decreases. The paroxysmal phase usually lasts 1 to 6 weeks, but can last as long as 10 weeks. In various embodiments, the compositions and methods of the invention reduce the length of this stage. During the recovery period, recovery is gradual.
  • compositions and methods of the invention accelerate the onset of this stage and/or reduce its duration. Furthermore, in various embodiments, the compositions and methods of the present invention prevent or reduce paroxysmal recurrence, which may occur with subsequent respiratory infections. In various embodiments, the compositions and methods of the present invention prevent or reduce one or more of the following seizures: secondary bacterial pneumonia, neurological complications such as epilepsy and encephalopathy, hypoxia, otitis media, dehydration, pneumothorax, Nasal bleeding, subdural hematoma, hernia, rectal prolapse, difficulty sleeping, urinary incontinence, pneumonia, and rib fractures. Furthermore, in some embodiments, the compositions and methods of the invention reduce or prevent necrotizing bronchiolitis, pneumonia (e.g., from Bordetella pertussis), pulmonary edema, pulmonary hypertension, and death.
  • necrotizing bronchiolitis pneumonia (e.g., from Bordetella pertussis), pulmonary edema, pulmonary
  • the method comprises administering to a subject the pertussis toxin binding protein of the invention or a combination of the pharmaceutical composition of the invention and an antimicrobial agent. It is expected that co-administration of the pertussis toxin binding protein of the present invention or the combination of the pharmaceutical composition of the present invention and an antimicrobial agent produces a synergistic effect.
  • Illustrative antimicrobial agents that can be used in the present invention include, but are not limited to, azithromycin, clarithromycin, erythromycin, trimethoprim-sulfamethoxazole, roxithromycin, ketolide (e.g., telithromycin ), ampicillin, amoxicillin, tetracycline, chloramphenicol, fluoroquinolones (for example, ciprofloxacin, levofloxacin, ofloxacin, moxifloxacin), and cephalosporins.
  • the subject treated by the methods of the invention is a human.
  • the subject is an infant.
  • the subject is a newborn.
  • the subject is a newborn child less than four weeks, less than three weeks, less than two weeks, less than one week, less than six days, less than five days, less than four days, less than three days, less than two days, or less than one day.
  • the person is one month old, two months old, three months old, four months old, five months old, or six months old.
  • the human has a period of about 6 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years old, about 20 to about 25 years old, about 25 to about 30 years old, about 30 to about 35 years old, about 35 to about 40 years old, about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old Years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old , About 90 to about 95 years old or about 95 to about 100 years of age in the range.
  • the method of the present invention prevents Bordetella pertussis infection in a subject previously exposed to Bordetella pertussis bacteria, the method comprising administering the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention to the subject .
  • the method provides an effective preventive treatment in preventing Bordetella pertussis infection in subjects exposed to Bordetella pertussis bacteria.
  • the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention is used for prophylactic applications in subjects who have not been previously vaccinated against the bacteria.
  • the pertussis toxin binding protein of the present invention or the pharmaceutical composition of the present invention can also serve as an adjuvant for vaccination such as DtaP or Tdap.
  • the methods of the present invention treat or prevent Bordetella pertussis infection in subjects who have not been previously vaccinated against the Bordetella pertussis bacteria.
  • the present invention also provides a method for detecting the presence and/or amount of pertussis toxin in a biological sample, which comprises allowing the organism to form a complex between the pertussis toxin binding protein of the present invention and the pertussis toxin.
  • the scientific sample and the control sample contact the pertussis toxin binding protein of the present invention.
  • the formation of the complex is then detected, wherein the difference in complex formation between the biological sample and the control sample is indicative of the presence and/or amount of pertussis toxin in the sample.
  • the pertussis toxin binding protein of the present invention is also conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, tags, etc. that can be used for detection or can be detected by other reagents.
  • the biological sample is a vaccine for preventing Bordetella pertussis infection, for example, the vaccine contains pertussis toxin.
  • the scope of the present invention also includes a kit used in the method of the present invention, which includes the pertussis toxin binding protein of the present invention and instructions for use.
  • the kit may further include at least one reagent for detection.
  • the kit generally includes a label indicating the intended use of the contents of the kit.
  • the term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • Lymphocytes were separated by density gradient centrifugation, total RNA was extracted using the RNA extraction kit provided by QIAGEN, and the SuperScript III FIRST STRANDSUPERMIX kit was used to reverse transcribed all the extracted RNA into cDNA according to the instructions, and the coding was amplified by nested PCR Nucleic acid fragments of the variable region of heavy chain antibodies.
  • the target heavy chain single domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pMECS using restriction endonucleases (purchased from NEB) PstI and NotI.
  • the product was subsequently electrotransformed into E. coli electrocompetent cells TG1, and a phage display library of immune single domain antibodies against PT toxin was constructed and the library was verified. Pave the plate through gradient dilution, and calculate the size of the storage volume to be 1.2 ⁇ 10 8 .
  • 50 clones were randomly selected for sequencing. There were 50 clones with the correct foreign fragment insertion, and the accuracy rate was 100%. By analyzing and comparing the DNA and amino acid sequences of the sequencing clones, it is confirmed that all the sequences are the expected camel VHH sequences, and the diversity can be estimated to be more than 95%.
  • the bacterium liquid collected by centrifugation was spread on a 2TYAG plate and incubated overnight at 37°C. The next day, the E. coli on the plate was eluted and collected, and a total of about 50 mL of bacterial solution was obtained. According to the OD value calculation, about 10 11 Escherichia coli are obtained.
  • Example 1.3 The 32 candidate sequences obtained in Example 1.3 were entrusted to Suzhou Hongxun Biotechnology Co., Ltd. for gene synthesis, and restriction sites were added at both ends.
  • VHH fragment of the PT single domain antibody is double-enzyme cut, fused with the DNA fragment encoding human IgG1FC, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the PT single domain antibody Fc fusion protein in mammals.
  • the vector constructed in 2.1 was transfected into HEK293 cells for transient expression of antibodies.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and added to the PEI (Polyethylenimine) solution required for transformation, each group of plasmid/PEI mixture was added to the HEK293 cell suspension, and placed at 37° C., 5% CO 2 , and cultured at 130 rpm. Four hours later, supplement with EXCELL293 medium, 2mM glutamine, and culture at 130rpm. After 24 hours, add 3.8mM VPA, and after 72 hours add 4g/L glucose.
  • PEI Polyethylenimine
  • PT single domain antibody Fc fusion protein is above 200mg/L, and after one-step purification by Protein A affinity chromatography column, its concentration is mostly stable above 1.0mg/mL, and the purity is also high.
  • the selected PT single domain antibody variable region sequence information is as follows (the boxes from left to right of each sequence show CDR1, CDR2 and CDR3 respectively):
  • the binding kinetics of the PT single-domain antibody Fc fusion protein obtained in the above examples to the recombinant human PT protein is measured by the biofilm interference (Biolayer interferometry, BLI) technology using a molecular interaction instrument.
  • the PT single domain antibody Fc fusion protein obtained in Example 2.2 was diluted to a final concentration of 2.5 ⁇ g/mL and directly solidified on AHC biosensor.
  • PT was diluted to 100nM, 50nM, 25nM, 12.5nM with 0.02% PBST20, respectively , 5 concentrations of 6.25nM, 120s injection, 600s dissociation time, 10mM glycineHCl (pH1.7) regeneration for 5s.
  • the concentration of PT toxin is 434.3 ⁇ g/mL.
  • 10% FBS+F12K was diluted 2-fold from the original concentration, 10 concentrations, 50 ⁇ L per well, placed at 37°C for 1 hour, CHOK1 cells were collected and counted, and the number of cells was adjusted to 3 ⁇ 10 5 cells/mL, each in a 96-well plate Add 50uL to the well, fix with 100% alcohol after 24 hours, stain with 0.1% crystal violet, wash with PBS, observe and take pictures under a microscope.
  • PT-biotin was diluted with 0.02% PBST20 to 10ug/mL and cured on SAbiosensor, and the curing time was 120s.
  • the PT single domain antibody Fc fusion protein was diluted to 100nM with 0.02% PBST20 and divided into two groups.
  • the antibody binding time was 300s, and the regeneration solution was 10mM glycineHCl (pH1.7); the first antibody (saturated antibody) bound to the sensor Up to saturation, then the second antibody (competing antibody) competes with the first antibody at the same concentration, and the percentage is calculated.
  • the percentage calculation formula is Ab2with Ab1/Ab2without Ab1.
  • the measurement results are shown in Table 4 and Table 5.
  • the results show that there may be competition between iPT 13, iPT 20 and iPT 36, iPT7 and iPT12, iPT26 and iPT35, iPT22 and iPT15 may have competition; the other antibodies have no obvious competition relationship and have different antigen binding epitopes.
  • Select 7 antibodies iPT 7, iPT 12, iPT 15, iPT22, iPT26, iPT35, iPT42 According to the overlap of antibody recognition epitope (epitope binning result), they can be divided into 4 groups. After the small overlapping sequences are combined in tandem, they are fused with DNA fragments encoding human IgG1FC and cloned into conventional mammalian expression vectors to obtain recombinant plasmids for the expression of PT tetravalent antibody Fc fusion proteins in mammals.
  • iPT12diFc and iPT15diFc are two identical iPT12 or iPT15 sequences in tandem combination and fused with a human IgG1Fc fragment as a control.
  • the vector constructed in 4.1 was transfected into HEK293 cells for transient expression of antibodies.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and added to the PEI (Polyethylenimine) solution required for transformation, each group of plasmid/PEI mixture was added to the HEK293 cell suspension, and placed at 37° C., 5% CO2, and cultured at 130 rpm. Four hours later, supplement with EXCELL293 medium, 2mM glutamine, and culture at 130rpm. After 24 hours, add 3.8mM VPA, and after 72 hours add 4g/L glucose.
  • PEI Polyethylenimine
  • the transient expression culture supernatant was collected, and purified by Protein A affinity chromatography to obtain the Fc fusion protein of the target PT tetravalent antibody.
  • the purity of the protein was investigated by SDSPAGE and SECHPLC. The expression and purity analysis of each protein are shown in Table 8 below. It can be seen that the expression level of Fc fusion protein of PT tetravalent antibody is above 200mg/L, and after one-step purification by Protein A affinity chromatography column, its concentration is mostly 1.0mg It is relatively stable above /mL, and the SEC results show that the purity is also high.
  • Antibody Expression amount (mg/L) SDSPAGE purity% Proportion of main peak% iPT7n15Fc 281 >95% 98.9% iPT15n7Fc 271 >95% 97.9% iPT7n22Fc 374 >95% 98.0% iPT22n7Fc 320 >95% 97.1% iPT7n42Fc 308 >95% 97.6% iPT42n7Fc 310 >95% 98.7% iPT12n35Fc 316 >95% 98.0% iPT35n12Fc 306 >95% 99.0% iPT15n35Fc 328 >95% 99.3% iPT15n42Fc 320 >95% 93.0% iPT35n42Fc 290 >95% 98.4% iPT12n15Fc 367 >95% 97.9% iPT15n12Fc 359 >95% 98.8% iPT12n42Fc 399 >95% 95
  • Example 5 Identification of the function of the Fc fusion protein of PT tetravalent antibody
  • the Fc fusion protein of the PT tetravalent antibody prepared in Example 4.2 was diluted to 2.5 ⁇ g/mL and cured on the biosensor, cured for 60 seconds, and the cured height was about 0.8 nm.
  • PTpuri was diluted to 5 gradients of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, baseline 60s, binding 120s, dissociation 600s.
  • the dilution solution is 0.02PBST20%
  • the regeneration solution is glycineHCl (pH 1.7)
  • the neutralization solution is the dilution solution
  • the biosensor is AHC. From Table 9, it can be seen that the antibody affinity is very good, especially the antibody iPT15n7Fc has the highest affinity.
  • the concentration of PT toxin is 434.3 ⁇ g/mL.
  • Use 10%FBS+F12K+10%FBS to prepare 12ng/mL, add 50 ⁇ L per well, and use 10% FBS for the FC fusion protein of the PT tetravalent antibody prepared in Example 4.2 +F12K was diluted 2 times from the original concentration, 10 concentrations, 50 ⁇ L per well, placed at 37°C for 1 hour, CHOK1 cells were collected and counted, the number of cells was adjusted to 3 ⁇ 10 5 cells/mL, and 50 ⁇ L was added to each well of a 96-well plate After 24 hours, it was fixed with 100% alcohol, stained with 0.1% crystal violet, washed with PBS, observed and photographed under a microscope. The results are shown in Table 10.
  • the neutralizing activities of the tetravalent antibodies iPT7n15Fc, iPT42n7Fc, iPT12n15Fc, iPT12n42Fc, iPT12diFc, iPT15n12Fc, iPT42n12Fc are better, and iPT7n15Fc, iPT42n7Fc, iPT12n15Fc, iPT12n42Fc, iPT15n12Fc, iPT15n12Fc, iPT15n12Fc and iPT15n12Fc have higher neutralizing activities. It is also higher than the bivalent antibody iPT12Fc. It can be seen that the neutralizing activity of single-domain antibodies that recognize different epitopes is improved.
  • Antibody Completely neutralize the concentration of PTToxin ( ⁇ g/mL) iPT15n7FC 5 ⁇ g/mL partially neutralized iPT7n22FC 5 iPT22n7FC 100 iPT7n42FC 2.5 ⁇ g/mL partially neutralized iPT12n35Fc 100 iPT35n12Fc 100 iPT15n35Fc 20 ⁇ g/mL partially neutralized iPT15n42Fc 100 iPT35n42Fc 100 ⁇ g/mL partially neutralized iPT12Fc 0.42 iPT7n15Fc 0.1 iPT42n7Fc 0.05 iPT12n15Fc 0.111 iPT12n42Fc 0.333 iPT12diFc 0.333 ⁇ g/mL partially neutralized iPT15n12Fc 0.111 iPT15diFc 1 ⁇ g/mL partially neutralized iPT42n12Fc 0.333
  • CHOK1 empty cells and 293F empty cells were resuspended in 3% BSAPBS, the number of cells was adjusted to 6 ⁇ 10 6 cells/mL, and the final concentration of the Fc fusion protein of the PT tetravalent antibody obtained in Example 4.2 was added to 100 ⁇ g/mL at the same time.
  • Negative control and blank control ice bath for 30min. After washing, add Boster's secondary antibody FITC rabbit anti-human IgG antibody, and ice bath for 30 minutes. After washing, the cells were resuspended in 300 ⁇ L of 1% PBSBSA Buffer and tested by flow cytometry. The results are shown in Table 11, showing no non-specific binding.
  • iPT12n42Fc 100 3.46 2.39 iPT12diFc 100 3.57 2.40 iPT15n12Fc 100 3.57 2.33 iPT15diFc 100 3.50 2.40 iPT42n12Fc 100 3.45 2.51 Blank —— 4.64 4.29 Negative —— 6.04 5.43 iPT7n15Fc 100 7.27 5.46 iPT42n7Fc 100 6.44 5.35
  • NIH mice used were divided into groups of 10 each, and PTtoxin was intraperitoneally injected into NIH mice at a dose of 5 ⁇ g/mouse, and a challenge mouse model for evaluating PT toxin antibodies was established by intraperitoneal injection of PTtoxin.
  • the PT tetravalent antibody FC fusion proteins iPT7n15Fc, iPT42n7Fc, iPT15n12Fc, iPT42n12Fc, iPT12diFc were injected intraperitoneally into the challenged mouse model, and the death of the mice was recorded every day.
  • the single-dose injections were 20 ⁇ g/dose and 40 ⁇ g/dose, respectively; the multiple-dose injections were divided into 5 times, the doses were 20 ⁇ g/dose and 40 ⁇ g/dose, grouped
  • the day of the disease was recorded as day 0, and a negative control was made at the same time.
  • the mice were weighed at D0, D5, D10, and D15, the number of white blood cells and lymphocytes were measured, and the survival rate of the mice was recorded every day.
  • mice Compared with the negative control, all antibodies have a protective effect on challenged mice; single-dose and multi-dose administration, antibodies iPT15n12FC, iPT12diFC at a dose of 40 ⁇ g/dose compared to 20 ⁇ g/dose The survival rate of mice increased significantly, and there was no significant difference in other antibodies.
  • the survival curve of single-dose administration of 20 ⁇ g/dose and 40 ⁇ g/dose is shown in Figure 1 and Figure 2, respectively, and multi-dose administration of 20 ⁇ g/dose and 40 ⁇ g/dose are shown in Figure 3 and Figure 4, respectively.
  • mice used were female, 1622g/mouse, 10 rats in each group. The day of grouping was recorded as day 0. The mice were weighed, the number of white blood cells and lymphocytes were measured, and B. pertussis was injected into the NIH mice intraperitoneally. A challenge mouse model for evaluating PT toxin antibodies was established by intraperitoneal injection of B. pertussis.
  • the PT tetravalent antibody FC fusion proteins iPT7n15Fc, iPT42n7Fc, iPT15n12Fc, iPT42n12Fc, iPT12diFc were injected intraperitoneally into the challenged mouse model, and the death of the mice was recorded every day.
  • Two hours after the challenge the injection of PT tetravalent antibody FC fusion protein was started.
  • the multi-dose injection was divided into 5 doses, the doses were 20 ⁇ g/dose and 40 ⁇ g/dose, and a negative control was used at the same time, and then respectively in D0, D5, D10 And D15 weighed the mice, measured the number of white blood cells and lymphocytes, and recorded the survival rate of the mice every day.
  • the humanization method adopts the resurfacing method of protein surface amino acids and the VHH humanization universal framework transplantation method (CDR grafting to a universal framework).
  • the steps of humanization are as follows: homologous modeling of antibody strains iPT7, iPT15 and iPT42, the modeling software is Modeller9.
  • the reference homologous sequence is the NbBcII10 antibody (PDB code: 3DWT), and the relative solvent accessibility of amino acids is calculated based on the three-dimensional structure of the protein. If a certain amino acid of the antibody strains iPT7, iPT15 and iPT42 is exposed in the solvent, it will be replaced with the amino acid at the same position of the reference human antibody 10HQ sequence, and finally all the replacements will be completed.
  • the specific steps of the VHH humanized universal framework transplantation method are as follows: First obtain the universal humanized VHH framework hNbBcII10FGLA (PDB number: 3EAK) designed by Cécile Vincke et al. based on sequence homology.
  • the framework design is based on the nanobody NbBcII10 antibody (PDB number: 3DWT), refer to human antibody 10HQ to humanize protein surface amino acids, and modify the VHH sequence framework 1 (framework1) partial amino acid VLP, VHH sequence framework 2 (framework2) partial amino acid GL, VHH sequence framework 3 (framework3) partial amino acid RSKRAAV and VHH sequence frame 4 (framework4) amino acid L completed.
  • hNbBcII10FGLA directly as the framework, the CDRs were replaced with the CDR regions of antibody strains iPT7, iPT15 and iPT42 to complete the humanization of antibodies.
  • the antibody strains iPT7, iPT15 and iPT42 were humanized, and 11 humanized variants of the antibody strain huPT were obtained.
  • the sequence numbering of these humanized variants and the numbering of amino acid residues therein are compared with Kabat numbering.
  • Figures 6-8 show the results of the alignment of the humanized sequences.
  • Example 8 Preparation of FC fusion protein of huPT single domain antibody using mammalian cells
  • the huPT single-domain antibody coding sequence in Example 7 was genetically synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and restriction sites were added at both ends.
  • VHH fragment of the huPT single domain antibody is double-enzyme digested, fused with the DNA fragment encoding human IgG1FC, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the huPT single domain antibody Fc fusion protein in mammals.
  • the vector constructed in 8.1 was transfected into HEK293 cells for transient expression of antibodies.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and added to the PEI (Polyethylenimine) solution required for transformation, each group of plasmid/PEI mixture was added to the HEK293 cell suspension, and placed at 37° C., 5% CO 2 , and cultured at 130 rpm. Four hours later, supplement with EXCELL293 medium, 2mM glutamine, and culture at 130rpm. After 24 hours, add 3.8mM VPA, and after 72 hours add 4g/L glucose.
  • PEI Polyethylenimine
  • the transient expression culture supernatant is collected, and purified by Protein A affinity chromatography to obtain the target huPT single domain antibody Fc fusion protein.
  • the purity of the protein was investigated by SDSPAGE and SECHPLC. The expression and purity analysis of each protein are shown in Table 14 below. The results show that the all humanized variants of iPT7 and iPT15 are not very different in expression level and purity, which is acceptable.
  • the humanized variant of iPT42 shows a certain stability difference. Among them, the humanized variants v1, v2, v7, v8, v10, v11 are not ideal in terms of expression level and product purity.
  • Example 9 Identification of the function of the humanized huPT single domain antibody Fc fusion protein
  • the plate was coated with recombinant human PT protein at 0.3 ⁇ g/well at 4° C. overnight.
  • the huPT single domain antibody Fc fusion protein obtained in Example 8.2 was a serial dilution series and reacted at room temperature for 2 hours.
  • the secondary antibody goat anti-human IgGFC horseradish peroxidase labeled antibody Sigma was added and reacted at room temperature for 2 hours.
  • add the color developing solution read the 450nm and 650nm wavelength absorbance values, the absorbance value of 450nm wavelength minus the absorbance value of 650nm wavelength is the final absorbance value.
  • the software SotfMax Pro v5.4 was used for data processing and mapping analysis.
  • the concentration of PT toxin is 434.3 ⁇ g/mL.
  • 10 concentrations add 50 ⁇ L to each well, place at 37°C for 1 hour, collect and count CHOK1 cells, adjust the number of cells to 3 ⁇ 10 5 cells/mL, add 50uL to each well of a 96-well plate, and fix with 100% alcohol after 24 hours.
  • Stain with 0.1% crystal violet wash with PBS, observe and take pictures under a microscope.
  • 3 humanized mutations were randomly selected from each of iPT7, 15 and 42 to investigate their neutralizing activity.
  • v1, v2, v7, v8, v10, and v11 with poor stability are removed.
  • CHOK1 empty cells and 293F empty cells were resuspended in 3% BSAPBS, the number of cells was adjusted to 6 ⁇ 10 6 cells/mL, and the final concentration of the Fc fusion protein of the huPT single domain antibody obtained in Example 8.2 was 100 ⁇ g/mL and 10 ⁇ g/mL. mL, set negative control and blank control at the same time, ice bath for 30 minutes. After washing, add Boster's secondary antibody FITC rabbit anti-human IgG antibody, and ice bath for 30 minutes. After washing, the cells were resuspended in 300 ⁇ L of 1% PBSBSA Buffer and tested by flow cytometry. The results are shown in Table 17, which shows that there is no non-specific binding.
  • Example 4 select the humanized antibody sequences obtained and investigated in Examples 7-9, huPT7v11, huPT15v7c, and huPT42v6. According to the overlap of the epitope recognized by the antibody (epitope binning result), the two-by-two series combination , Fused with a DNA fragment encoding human IgG1FC to obtain quadrivalent bispecific antibodies huPT7v11n15v7c-Fc, huPT7v11n15v11c-Fc, huPT42v6n15v11c-Fc and huPT42v6n7v11-Fc.
  • HEK293 cells transiently expressed and purified the Fc fusion protein of the target PT tetravalent antibody.
  • the Fc fusion protein of the PT tetravalent antibody prepared in Example 10 was diluted to 2.5 ⁇ g/mL and solidified on the biosensor, solidified for 60 seconds, and the solidified height was about 0.8 nm.
  • PTpuri was diluted to 5 gradients of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, baseline 60s, binding 120s, dissociation 600s.
  • the dilution solution is 0.02PBST20%
  • the regeneration solution is glycineHCl (pH 1.7)
  • the neutralization solution is the dilution solution
  • the biosensor is AHC.
  • Table 19 The results are shown in Table 19 below.
  • 1B7 and 11E6 are control antibodies that bind to PT toxin and have a neutralizing effect. Refer to the sequence in the patent US10035846 for autonomous cloning and preparation.
  • the concentration of PT toxin is 434.3 ⁇ g/mL, it is prepared with 10%FBS+F-12K+10%FBS to 12ng/mL, 50 ⁇ L is added to each well, and the FC fusion protein of PT tetravalent antibody prepared in Example 4.2 is used for 10 %FBS+F-12K is diluted 2 times from the original concentration, 10 concentrations, 50 ⁇ L per well, placed at 37°C for 1 hour, collect and count CHO-K1 cells, adjust the number of cells to 3 ⁇ 10 5 cells/mL, 96 Add 50 ⁇ L to each well of the well plate, fix with 100% alcohol after 24 hours, stain with 0.1% crystal violet, wash with PBS, observe and take pictures under a microscope.
  • 1B7 and 11E6 are control antibodies that bind to PT toxin and have a neutralizing effect. Refer to the sequence in the patent US10035846 for autonomous cloning and preparation.
  • the results are shown in Table 20.
  • the tetravalent antibodies huPT7n15-Fc, huPT42n7-Fc, huPT42n15-Fc, iPT12n42-Fc, iPT12di-Fc, iPT15n12-Fc, iPT42n12-Fc have good neutralizing activities, and iPT7n15-Fc, iPT42n7-Fc,
  • the neutralizing activities of iPT12n15-Fc, iPT12n42-Fc, iPT15n12-Fc, and iPT42n12-Fc are all higher than iPT15di-Fc and iPT12di-Fc, and also higher than the bivalent antibody iPT12-Fc, showing single domain antibodies that recognize different epitopes After the combination, the neutralizing activity is improved.
  • Antibody Minimum neutralization concentration huPT7n15-Fc 0.025 ⁇ g/mL huPT42n7-Fc 0.05 ⁇ g/mL huPT42n15-Fc 0.1 ⁇ g/mL 1B7 1.6 ⁇ g/mL 11E6 0.8 ⁇ g/mL 1B7+11E6 (0.4+0.4) ⁇ g/mL huPT7n15-Fc+huPT42n7-Fc (0.00625+0.00625) ⁇ g/mL huPT7n15-Fc+huPT42n15-Fc (0.0125+0.0125) ⁇ g/mL
  • the biological membrane interference technology BLI: epitope binning is used to compare the binding epitopes of the PT single domain antibody Fc fusion protein of the present invention and the control antibody.
  • PT-biotin was diluted with 0.02% PBST20 to 10ug/mL and cured on the SAbiosensor, and the curing time was 120s.
  • the PT single domain antibody Fc fusion protein was diluted to 100nM with 0.02% PBST20 and divided into two groups.
  • the antibody binding time was 300s, and the regeneration solution was 10mM glycineHCl (pH1.7); the first antibody (saturated antibody) bound to the sensor Up to saturation, then the second antibody (competing antibody) competes with the first antibody at the same concentration, and the percentage is calculated.
  • the percentage calculation formula is Ab2with Ab1/Ab2without Ab1.
  • the control antibody (11E6, 1B7, sequence source patent US10035846) is Ab1, and the subsequent antibody is Ab2.
  • a control antibody was added to the Ab2 group as a reference for the complete overlap of the epitope.

Abstract

提供了针对百日咳毒素（PT）的单域抗体、人源化单域抗体、其衍生蛋白及其用途。

Description

百日咳毒素结合蛋白 技术领域
本发明涉及医药生物领域，公开了针对百日咳毒素(PT)的单域抗体及其衍生蛋白。具体而言，本发明公开了一种百日咳毒素结合蛋白及其用途。
发明背景
百日咳博德特氏杆菌(Bordetella pertussis，B.pertussis)是感染上呼吸道、引起不可控制的剧烈咳嗽的革兰氏阴性细菌。根据世界卫生组织，百日咳博德特氏杆菌感染每年造成全世界估计300,000例死亡，主要在年幼的未接种的婴儿中。患有百日咳的婴儿往往需要在儿科重症监护病房住院，并且他们的治疗通常涉及机械通气。成人中的百日咳通常导致慢性咳嗽。百日咳的发病率由于未接种疫苗和疫苗接种不足的个体(包括尚未完全接种的婴儿)、免疫力已随时间推移减弱的个体以及无症状携带者的暴露而增加。
据报道，之前的百日咳疫苗不提供长期保护。不存在用于百日咳的批准的治疗疫苗。抗生素治疗对百日咳的病程不具有主要作用，因为虽然治疗可从呼吸道消除百日咳博德特氏杆菌细菌，但它不中和百日咳毒素蛋白。此外，在发展中世界，获得现有百日咳疫苗(然而有缺陷的)是不一致的和非常困难的。
因此，仍然需要针对百日咳的更有效的疗法。
发明简述
在一方面，本发明提供一种百日咳毒素结合蛋白，其能够特异性结合百日咳毒素且包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，SEQ ID NO:3所示的CDR3；
(2)SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:5所示的CDR3；
(3)SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；
(4)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所示的CDR3；
(5)SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；
(6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2，SEQ ID NO:18所示 的CDR3；
(7)SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2，SEQ ID NO:21所示的CDR3；
(8)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；
(9)SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2，SEQ ID NO:27所示的CDR3；
(10)SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2，SEQ ID NO:30所示的CDR3；
(11)SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；
(12)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:36所示的CDR3；
(13)SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3。
在一方面，本发明提供一种核酸分子，其编码本发明的百日咳毒素结合蛋白。
在一方面，本发明提供一种表达载体，其包含与表达调控元件可操作地连接的本发明的核酸分子。
在一方面，本发明提供一种重组细胞，其包含本发明的核酸分子或以本发明的表达载体转化，并能够表达所述百日咳毒素结合蛋白。
在一方面，本发明提供一种产生本发明的百日咳毒素结合蛋白的方法，包括：
a)在允许所述百日咳毒素结合蛋白表达的条件下培养本发明的重组细胞；
b)从得自步骤a)的培养物回收由所述重组细胞表达的百日咳毒素结合蛋白；及
c)任选进一步纯化和/或修饰得自步骤b)的百日咳毒素结合蛋白。
在一方面，本发明提供一种药物组合物，其包含本发明的百日咳毒素结合蛋白以及药学上可接受的载体。
在一方面，本发明提供一种一种治疗对象中百日咳博德特氏杆菌感染的方法，所述方法包括向所述对象施用有效量的本发明的百日咳毒素结合蛋白或本发明的药物组合物。
在一方面，本发明提供一种一种在对象中预防百日咳博德特氏杆菌感染的方法，所述方法包括向对象有效量的本发明的百日咳毒素结合蛋白或本发明的药物组合物。
在一方面，本发明提供一种检测生物学样品中百日咳毒素的存在和/或量的方法，包括：
(a)在百日咳毒素结合蛋白与百日咳毒素之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的百日咳毒素结合蛋白；
(b)检测复合物的形成，
其中所述生物学样品与对照样品之间复合物形成的差异指示样品中百日咳毒素的存在和/或量。
在一方面，本发明提供一种试剂盒，其包含本发明的百日咳毒素结合蛋白。
附图简述
图1.单剂量给药20μg/dose时针对PT毒素保护的生存曲线。
图2.单剂量给药40μg/dose时针对PT毒素保护的生存曲线。
图3.多剂量给药20μg/dose时针对PT毒素保护的生存曲线。
图4.多剂量给药40μg/dose时针对PT毒素保护的生存曲线。
图5.多剂量给药20μg/dose或40μg/dose时针对百日咳杆菌保护的生存曲线。
图6.iPT7人源化变体氨基酸序列比对。
图7.iPT15人源化变体氨基酸序列比对。
图8.iPT42人源化变体氨基酸序列比对。
发明详述
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人,“Molecular Cloning:A Laboratory Manual”(第2版),第1-3卷,Cold Spring Harbor Laboratory Press(1989)；Lewin,“Genes IV”，Oxford University Press,New York,(1990)；及Roitt等人,“Immunology”(第2版),Gower Medical Publishing,London,New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature 363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、以及“VHH抗体”可互换使用。
例如Riechmann及Muyldermans,J.Immunol.Methods 231,25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，可以根据Kabat等人给出的VH结构域的一般编号法来编号(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。例如，Chothia CDR指的是结构环的位置(Chothia and Lesk,J.Mol.Biol.196:901-917(1987))。AbM CDR代表的是Kabat超变区和Chothia结构环的折中，并且在Oxford Molecular's AbM抗体建模软件中使用。“接触(Contact)”CDR则基于对可获得的复合物晶体结构的分析。来自每种方法的CDR的残基描述如下：
环 Kabat AbM Chothia Contact
LCDR1 L24-L34 L24-L34 L26-L32 L30-L36
LCDR2 L50-L56 L50-L56 L50-L52 L46-L55
LCDR3 L89-L97 L89-L97 L91-L96 L89-L96
HCDR1(Kabat编号) H31-H35B H26-H35B H26-H32 H30-H35B
HCDR1(Chothia编号) H31-H35 H26-H35 H26-H32 H30-H35
HCDR2 H50-H65 H50-H58 H53-H55 H47-H58
HCDR3 H95-H102 H95-H102 H96-H101 H93-H101
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
例如，CDR可以包括“扩展的(extended)CDR”，例如：在VL中的24-36或24-34(LCDR1)，46-56或50-56(LCDR2)以及89-97或89-96(LCDR3)；在VH中的26-35(HCDR1)，50-65或49-65(HCDR2)以及93-102、94-102或95-102(HCDR3)。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分VHH结构域与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域—单独或作为较大多肽的一部分—提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；VHH结构域可容易地改造成多价及多特异性格式(格式化)；VHH结构域高度可溶且无聚集趋势；VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从 而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.,Journal of Immunological Methods,240(2000)185–195；Li et al.,J Biol Chem.,287(2012)13713–13721；Deffar et al.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”(本文中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。人源化可以使用蛋白表面氨基酸人源化(resurfacing)的方法和/或人源化通用框架CDR移植法(CDR grafting to a universal framework)完成，例如，如实施例中所示例。
如本文所用，术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA 81:3998-4002；Geysen等人(1986)Molec.Immunol.23:709-715中。类似地，构象表位可通过诸如通过例如x射线结晶学及2维核磁共振确定氨基酸的空间构形加以鉴别。参见例如Epitope Mapping Protocols(同上)。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的抗体分子竞争结合百日咳毒素上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋 白的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合蛋白上的抗原结合位点之间的亲和力，以及存在于抗原结合蛋白上的相关结合位点的数目。
如本文所用，术语“百日咳毒素结合蛋白”意指任何能够特异性结合百日咳毒素的蛋白。百日咳毒素结合蛋白可以包括针对百日咳毒素的如本文定义的抗体。百日咳毒素结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
“百日咳毒素”来源于百日咳博德特氏杆菌(Bordetella pertussis，B.pertussis)的一种多亚基蛋白毒素，其能特异性作用于G蛋白，抑制G蛋白发挥信号通路的功能。百日咳毒素包含亚基S1、亚基S2、亚基S3、亚基S4和亚基S5，其氨基酸序列被录入在UniProtKB数据库中，对应的登录号分别为：P04977、P04978、P04979、P0A3R5和P04981。
本发明的“百日咳毒素结合蛋白”可以包含至少一个结合百日咳毒素的免疫球蛋白单一可变结构域如VHH。在一些实施方案中，本发明的“百日咳毒素结合分子”可以包含2、3、4或更多个结合百日咳毒素的免疫球蛋白单一可变结构域如VHH。本发明的百日咳毒素结合蛋白除结合百日咳毒素的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本发明的“百日咳毒素结合蛋白”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本发明的百日咳毒素结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(KD)，和/或以至少10 7M -1、优选至少10 8M -1、更优选至少10 9M -1，更优选至少10 10M -1的缔合常数(KA)结合所要结合的抗原(即百日咳毒素)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨 基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“分离的”多肽或核酸分子优选基本上为均质的。
“有效量”意指本发明的百日咳毒素结合蛋白或药物组合物的量能导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本发明的百日咳毒素结合蛋白
本发明提供了一种百日咳毒素结合蛋白，其包含至少一个能够特异性结合百日咳毒素的免疫球蛋白单一可变结构域。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:40-52中任一所示的VHH中的CDR1、CDR2和CDR3。所述CDR可以是Kabat CDR、AbM CDR、Chothia CDR或Contact CDR。在一些实施方案中，所述CDR是Kabat CDR。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，SEQ ID NO:3所示的CDR3(对应于iPT 3的CDR)；
(2)SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:5所示的CDR3(对应于iPT 7的CDR)；
(3)SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3(对应于iPT 12的CDR)；
(4)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所示的CDR3(对应于iPT 13的CDR)；
(5)SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3(对应于iPT 15的CDR)；
(6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2，SEQ ID NO:18所示的CDR3(对应于iPT 20的CDR)；
(7)SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2，SEQ ID NO:21所示的CDR3(对应于iPT 21的CDR)；
(8)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3(对应于iPT 22的CDR)；
(9)SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2，SEQ ID NO:27所示的CDR3(对应于iPT 26的CDR)；
(10)SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2，SEQ ID NO:30所示的CDR3(对应于iPT 28的CDR)；
(11)SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3(对应于iPT 35的CDR)；
(12)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:36所示的CDR3(对应于iPT 36的CDR)；
(13)SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3(对应于iPT 42的CDR)。
在一些实施方案中，本发明的百日咳毒素结合蛋白中的至少一个免疫球蛋白单一可变结构域是VHH。在一些实施方案中，所述VHH包含SEQ ID NO:40-52中任一氨基酸序列。
在一些实施方案中，本发明的百日咳毒素结合蛋白中的至少一个免疫球蛋白单一可变结构域是人源化的VHH。
在一些实施方案中，本发明的百日咳毒素结合蛋白中的至少一个免疫球蛋白单一可变结构域是人源化的VHH，所述人源化的VHH包含与SEQ ID NO:40-52中任一序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。在一些实施方案中，所述人源化的VHH的氨基酸序列与SEQ ID NO:40-52中任一相比包含一或多个氨基酸取代，优选保守氨基酸取代。例如，包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代。
在一些实施方案中，本发明的百日咳毒素结合蛋白中的至少一个免疫球蛋白单一可变结构域是人源化的VHH，其中所述人源化的VHH包含SEQ ID NO:53-85中任一氨基酸序列。
在一些实施方案中，所述百日咳毒素结合蛋白包含一个特异性结合百日咳毒素的免疫球蛋白单一可变结构域。
在一些实施方案中，所述百日咳毒素结合蛋白包含至少两个，例如2、3、4或更多个特异性结合百日咳毒素的免疫球蛋白单一可变结构域。
在一些实施方案中，所述至少两个免疫球蛋白单一可变结构域结合相同表位或竞争结合或部分竞争结合相同表位，例如，所述至少两个免疫球蛋白单一可变结构域是相同的。
在一些实施方案中，所述至少两个免疫球蛋白单一可变结构域结合不同表位或不竞争结合相同表位。
两个抗体或免疫球蛋白单一可变结构域是否结合或竞争结合相同表位可以通过生物膜干涉技术BLI进行epitope binning来确定，如本申请实施例所示例。
在一些实施方案中，所述至少两个特异性结合百日咳毒素的免疫球蛋白单一可变结构域直接相互连接。
在一些实施方案中，所述至少两个特异性结合百日咳毒素的免疫球蛋白单一可变结构域通过接头相互连接。所述接头可以是长1-20或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如GGGGS、GS、GAP、(GGGGS)x 3等。
在一些实施方案中，所述百日咳毒素结合蛋白包含第一免疫球蛋白单一可变结构域和第二免疫球蛋白单一可变结构域，其中所述第一免疫球蛋白单一可变结构域位于所述第二免疫球蛋白单一可变结构域的N端，且
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示 的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所 示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3。
在一些实施方案中，所述百日咳毒素结合蛋白包含第一免疫球蛋白单一可变结构域和第二免疫球蛋白单一可变结构域，其中所述第一免疫球蛋白单一可变结构域位于所述第二免疫球蛋白单一可变结构域的N端，且
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:47所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:47所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列。
在一些实施方案中，所述百日咳毒素结合蛋白包含第一免疫球蛋白单一可变结构域和第二免疫球蛋白单一可变结构域，其中所述第一免疫球蛋白单一可变结构域位于所述第二免疫球蛋白单一可变结构域的N端，且
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:63所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:70所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:63所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:74所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:80所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:74所示氨基酸序列；或
所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:80所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:63所示氨基酸序列。
在一些实施方案中，本发明的百日咳毒素结合蛋白包含选自SEQ ID NO:87-105的氨基酸序列。
在一些实施方案中，本发明的百日咳毒素结合蛋白，除了至少一个能够特异性结合百日咳毒素的免疫球蛋白单一可变结构域外，还包含免疫球蛋白Fc区。在本发明的百日咳毒素结合蛋白中包含免疫球蛋白Fc区可以使所述结合蛋白形成二聚体分子，同时延长所述结合蛋白的体内半衰期。可用于本发明的Fc区可以来自不同亚型的免疫球蛋 白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。免疫球蛋白Fc区一般而言包括免疫球蛋白恒定区的铰链区或部分铰链区、CH2区和CH3区。
在一些实施方案中，可以在野生型的Fc序列上引入突变用于改变Fc介导的相关活性。所述突变包括但不限于：a).改变Fc介导的CDC活性的突变；b).改变Fc介导的ADCC活性的突变；或c).改变FcRn介导的体内半衰期的突变。此类突变描述于下列文献中：Leonard G Presta,Current Opinion in Immunology 2008,20:460–470；Esohe E.Idusogie et al.,J Immunol 2000,164:4178-4184；RAPHAEL A.CLYNES et al.,Nature Medicine,2000,Volume 6,Number 4:443-446；Paul R.Hinton et al.,J Immunol,2006,176:346-356。例如，可以通过突变CH2区上的1、2、3、4、5、6、7、8、9或10个氨基酸用于增加或去除Fc介导的ADCC或CDC活性或是增强或减弱FcRn的亲和力。此外，可以通过突变铰链区的1、2、3、4、5、6、7、8、9或10个氨基酸增加蛋白的稳定性。
在一些实施方案中，Fc序列上可以引入突变，从而使得突变的Fc更容易形成同二聚体或者异二聚体。如Ridgway,Presta et al.1996以及Carter 2001中提到的利用Fc接触界面氨基酸侧链基团空间作用的knob-hole模型，使得不同Fc突变之间更容易形成异二聚体；再比如如CN 102558355A或者CN 103388013A中，通过改变Fc接触界面氨基酸所带的电荷，进而改变Fc接触界面之间的离子相互作用力，使得不同的Fc突变对之间更容易形成异二聚体(CN 102558355A)，亦或是具有相同突变的Fc之间更容易形成同二聚体(CN 103388013A)。
所述免疫球蛋白Fc区优选是人免疫球蛋白Fc区，更优选是人IgG1的Fc区。在一些具体实施方案中，所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO:86。
在一些实施方案中，本发明的百日咳毒素结合蛋白中，所述至少一个能够特异性结合百日咳毒素的免疫球蛋白单一可变结构域与免疫球蛋白Fc区直接连接或通过接头连接。所述接头可以是长1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如GGGGS、GS、GAP等。在一些实施方案中，所述免疫球蛋白Fc区位于所述至少一个特异性结合百日咳毒素的免疫球蛋白单一可变结构域的C端。
在一些实施方案中，本发明的百日咳毒素结合蛋白包含选自SEQ ID NO:40-85和87-105之一的第一氨基酸序列，以及与第一氨基酸序列直接连接或通过接头连接的SEQ ID NO:86所示的第二氨基酸序列，其中第二氨基酸序列位于第一氨基酸序列的C端。
在一些实施方案中，本发明的百日咳毒素结合蛋白包含选自SEQ ID NO:106-109的氨基酸序列。
在一些实施方案中，本发明的百日咳毒素结合蛋白包含一个特异性结合百日咳毒素的免疫球蛋白单一可变结构域，其直接或通过接头与免疫球蛋白Fc区连接，所述免疫球蛋白Fc区允许所述百日咳毒素结合蛋白形成包含两个百日咳毒素结合结构域的二聚 体分子。这样的百日咳毒素结合蛋白也称为二价百日咳毒素结合蛋白。在一些实施方案中，所述二聚体是同二聚体。
在一些实施方案中，本发明的百日咳毒素结合蛋白包含直接或通过接头相互连接的两个特异性结合百日咳毒素的免疫球蛋白单一可变结构域和一个免疫球蛋白Fc区，所述免疫球蛋白Fc区允许所述百日咳毒素结合蛋白形成包含四个百日咳毒素结合结构域的二聚体分子。这样的百日咳毒素结合蛋白也称为四价百日咳毒素结合蛋白。在一些实施方案中，所述二聚体是同二聚体。
核酸、载体、宿主细胞
在另一方面中，本发明涉及编码本发明的百日咳毒素结合蛋白的核酸分子。本发明的核酸可为RNA、DNA或cDNA。根据本发明的一个实施方案，本发明的核酸是基本上分离的核酸。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供百日咳毒素结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本发明的百日咳毒素结合蛋白的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本发明的核酸可基于关于本文给出的本发明的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本发明涉及表达或能够表达一种或多种本发明的百日咳毒素结合蛋白和/或含有本发明的核酸或载体的重组宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、 COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本发明还提供产生本发明的百日咳毒素结合蛋白的方法，所述方法通常包含以下步骤：
-在允许表达本发明的百日咳毒素结合蛋白的条件下培养本发明的宿主细胞；及
-从培养物回收由所述宿主细胞表达的百日咳毒素结合蛋白；及
-任选进一步纯化和/或修饰本发明的百日咳毒素结合蛋白。
本发明的百日咳毒素结合蛋白可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本发明的百日咳毒素结合蛋白的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
然而，本发明的百日咳毒素结合蛋白也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
药物组合物
另一方面，本发明提供一种组合物，例如药物组合物，其含有与药学上可接受的载体配制在一起的一种或组合的本发明的百日咳毒素结合蛋白。这样的组合物可以包含一种或组合的(例如两种或多种不同的)本发明的百日咳毒素结合蛋白。例如，本发明的药物组合物可以含有结合靶抗原(百日咳毒素)上的不同表位的抗体分子组合。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用。
本发明的药物组合物可包含一种或多种药学上可接受的盐。“药学上可接受的盐”是指保持了亲代化合物的所需生物活性而不引起任何不期望的毒理学作用的盐(参见如Berge，S.M.等(1977)J.Pharm.Sci.66：1-19)。这样的盐的例子包括酸加成盐和碱加成盐。酸加成盐包括那些由诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等无毒性无机酸衍生的盐，以及由诸如脂族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳族酸、脂族和芳族磺酸等无毒性有机酸衍生的盐。碱加成盐包括那些由诸如钠、钾、镁、钙等碱土金属衍生的盐，以及由诸如N，N’-二苄基乙二胺、N-甲基葡糖胺、氯普 鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等无毒性有机胺衍生的盐。
本发明的药物组合物也可含有药学上可接受的抗氧化剂。药学上可接受的抗氧化剂的例子包括：(1)水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠，亚硫酸钠等；(2)油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚等；和(3)金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等。
这些组合物还可含有如防腐剂、润湿剂、乳化剂和分散剂。
可以通过灭菌程序或通过包含诸如对羟基苯甲酸酯、氯代丁醇、苯酚山梨酸等各种抗细菌剂和抗真菌剂确保防止存在微生物。在很多情况下，组合物中优选包含等渗剂，例如，糖、多元醇例如甘露糖醇、山梨糖醇或氧化钠。通过在组合物中加入延迟吸收剂，例如单硬脂酸盐和明胶，可实现注射型药物延长的吸收。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些用于药学活性物质的介质和试剂的使用是本领域公知的。常规介质或试剂，除了任何与活性化合物不相容的范围外，都可能在本发明的药物组合物中。还可以向组合物中掺入补充的活性化合物。
治疗性组合物一般必须是无菌的并且在制备和贮存条件下稳定的。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液态聚乙二醇等)及其合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散液的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分中的一种或其组合，接着无菌微过滤，可制备无菌注射液。通常，通过将活性化合物掺入到含有基本分散介质和上面所列其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其预先无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合制备单一剂量形式的活性成分的量根据所治疗的对象和特定给药方式而不同。可以与载体材料组合制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
可以调节剂量方案以提供最佳的期望的反应(例如，治疗反应)。例如，可以施用单一推注，可以随时间施用几次分开的剂量，或者根据治疗状况的紧急情况所需，可以按比例减小或增加剂量。特别有利的是将肠胃外组合物配制成容易给药并且剂量均匀的剂量单位形式。此处使用的剂量单位形式是指适合作为单位剂量用于所治疗的对象的物理不连续单位；每个单位含有预定量的活性化合物，经计算该预定量的活性化合物与需要的药物载体组合产生所需的治疗效果。对本发明剂量单位形式的具体说明限定于且直接 依赖于(a)活性化合物的独特特性和要达到的特定治疗效果，和(b)本领域中固有的对于配制这种用于治疗个体敏感性的活性化合物的限制。
对于抗体分子的给药而言，剂量范围为约0.0001至100mg/kg，更通常为0.01至30mg/kg受者体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重、10mg/kg体重、20mg/kg体重或30mg/kg体重，或在1-30mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短(如每周一次至每三周一次)后期给药间隔加长(如每月一次至每3-6个月一次)。
或者，抗体分子也可以作为持续释放制剂来给药，在此情况中需要频率较低的给药。剂量和频率根据抗体分子在患者中的半衰期而不同。通常，人抗体表现出最长的半衰期，之后是人源化抗体、嵌合抗体和非人类抗体。给药剂量和频率根据处理是预防性的还是治疗性的而不同。在预防性应用中，在长时间内以较不频繁的间隔给予相对较低的剂量。有些患者在余生中持续接受处理。在治疗性应用中，有时需要以较短的间隔给予较高的剂量，直到疾病的进展减轻或停止，优选直到患者表现为疾病症状部分或完全改善。之后，可以以预防性方案给患者给药。
本发明药物组合物中活性成分的实际剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物或其酯、盐或酰胺的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素
本发明的组合物可以利用本领域公知的一种或多种方法通过一种或多种给药途径给药。本领域技术人员应当理解，给药途径和/或方式根据期望的结果而不同。本发明百日咳毒素结合蛋白的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外给药途径，例如注射或输注。本文使用的短语“肠胃外给药”是指除肠和局部给药以外的给药模式，通常是注射，包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
或者，本发明的百日咳毒素结合蛋白也可以通过非肠胃外途径给药，如局部、表皮或粘膜途径给药，例如，鼻内、经口、阴道、直肠、舌下或局部。
疾病预防和治疗
本发明涉及治疗对象中百日咳博德特氏杆菌感染的方法，所述方法包括向所述对象(例如以有效量)施用本发明的百日咳毒素结合蛋白或本发明的药物组合物。
另一方面，本发明的方法涉及一种在对象中预防百日咳博德特氏杆菌感染的方法，所述方法包括向对象(例如以有效量)施用本发明的百日咳毒素结合蛋白或本发明的药物 组合物。在一些实施方案中，所述对象处于百日咳博德特氏杆菌感染的风险(例如，所述患者是疫苗接种前婴儿和/或所述患者已暴露于百日咳毒素)。
另一方面，本发明的方法涉及一种在对象中中和百日咳毒素的方法，所述方法包括向对象(例如以有效量)施用本发明的百日咳毒素结合蛋白或本发明的药物组合物。在一些实施方案中，所述对象被百日咳博德特氏杆菌感染。
另一方面，本发明的方法涉及一种在对象中百日咳博德特氏杆菌感染引起的疾病或病症的方法，所述方法包括向对象(例如以有效量)施用本发明的百日咳毒素结合蛋白或本发明的药物组合物。
白细胞增多或白血细胞计数升高是百日咳博德特氏杆菌感染的特征。在一个实施方案中，本发明的方法包括降低患者中的白血细胞计数。在一个实施方案中，本发明的方法产生白细胞增多的消退加速。在另一个实施方案中，本发明的方法产生在感染过程期间最大白血细胞计数的降低。
在一个实施方案中，本发明的方法产生百日咳毒素蛋白的中和(抑制或拮抗)。例如，本发明的百日咳毒素结合蛋白可结合百日咳毒素蛋白以便部分或完全抑制百日咳毒素蛋白的一种或多种生物活性。中和抗体可抑制或阻断的百日咳毒素蛋白的生物活性之一是百日咳毒素蛋白结合细胞受体的能力。百日咳毒素蛋白的受体结合区由分别被称为亚基S2、亚基S3、亚基S4和亚基S5的四个多肽亚基组成。由百日咳毒素蛋白的亚基S2、S3、S4和S5结合的细胞受体的实例是N连接的唾液酸糖蛋白家族的成员如胎球蛋白、结合珠蛋白(haptoblobin)和转铁蛋白。在一个示例性实施方案中，本发明的百日咳毒素结合蛋白防止百日咳毒素蛋白结合其细胞受体。在另一个实施方案，本发明的百日咳毒素结合蛋白改变百日咳毒素的细胞内运输步骤，以使得所述毒素不会到达细胞胞质。百日咳毒素蛋白的可通过本发明的百日咳毒素结合蛋白抑制的另一种重要活性是百日咳毒素蛋白作为针对G蛋白的ADP核糖基酶的酶活性。百日咳毒素蛋白中赋予作为ADP核糖基酶的酶活性的亚基是亚基S1。在一些实施方案中，百日咳毒素蛋白是百日咳海参毒素。在本文被称为百日咳毒素蛋白的百日咳海参毒素包含所有五个百日咳毒素蛋白亚基。在其他实施方案中，百日咳毒素蛋白是截短的百日咳毒素蛋白。如在本文提及的截短的百日咳蛋白包含百日咳毒素蛋白亚基(即，S1、S2、S3、S4和S5)中的至少一个。各种形式的百日咳毒素蛋白在现有技术中已有描述。
在各种实施方案中，本发明的组合物和方法适用于治疗或预防百日咳感染的任何阶段。例如，百日咳的潜伏期通常是7–10天，范围是4–21天，并且很少可以是长达42天。在各种实施方案中，本发明的组合物和方法通过使感染更难以发生来增加潜伏期的长度。该疾病的临床病程被分成三个阶段。第一阶段，卡他性阶段，特征在于鼻炎的发病隐袭、打喷嚏、低热和温和、偶尔咳嗽，类似于感冒。咳嗽逐渐变得更严重，并且在1-2周之后，第二或阵发性阶段开始。在各种实施方案中，本发明的组合物和方法减少卡他性阶段的长度并且任选地防止它进展至阵发性阶段。在各种实施方案中，本发明的组合物和方法治疗鼻炎、打喷嚏、低热和咳嗽中的一种或多种。其正是在通常疑似诊断 百日咳的阵发性阶段期间。典型地，患者具有大量、快速咳嗽的突发或阵发，显然是由于将稠厚粘液从气管支气管树驱逐出的困难所致。在阵发结束时，长吸气努力通常伴随特征性高声喘息。在这种发作中，患者可能变得发绀。儿童和婴幼儿尤其出现非常不适和痛苦。在发作之后通常是呕吐和疲惫。在各种实施方案中，本发明的组合物和方法减少阵发的量和/或频率。在各种实施方案中，本发明的组合物和方法防止患者变得发绀。阵发性发作更频繁地在夜间发生，平均每24小时15次发作。在此阶段的最初1或2周，发作频率增加，对于2至3周保持在相同水平，且然后逐渐降低。阵发性阶段通常持续1至6周，但可能持续长达10周。在各种实施方案中，本发明的组合物和方法减少此阶段的长度。在恢复期，恢复是逐渐的。咳嗽变得阵发性降低且在2至3周消失。在各种实施方案中，本发明的组合物和方法加速此阶段的发作和/或减少其持续时间。此外，在各种实施方案中，本发明的组合物和方法预防或减少阵发的复发，所述复发可能与随后呼吸道感染一起发生。在各种实施方案中，本发明的组合物和方法预防或减少一种或多种以下发作：继发性细菌性肺炎、神经系统并发症如癫痫和脑病、缺氧、中耳炎、脱水、气胸、鼻出血、硬脑膜下血肿、疝、直肠脱垂、睡眠困难、尿失禁、肺炎以及肋骨骨折。此外，在一些实施方案中，本发明的组合物和方法减少或预防坏死性细支气管炎、肺炎(例如，来自百日咳博德特氏杆菌)、肺水肿、肺高血压症和死亡。
在另一个实施方案中，所述方法包括向对象施用本发明的百日咳毒素结合蛋白或本发明的药物组合物与抗微生物剂的组合。预期共同施用本发明的百日咳毒素结合蛋白或本发明的药物组合物与抗微生物剂的组合产生协同作用。可用于本发明的说明性抗微生物剂包括但不限于阿奇霉素、克拉霉素、红霉素、三甲氧苄二氨嘧啶-磺胺甲噁唑、罗红霉素、酮内脂(例如，泰利霉素)、氨苄西林、阿莫西林、四环素、氯霉素、氟喹诺酮(例如，环丙沙星、左氧氟沙星、氧氟沙星、莫西沙星)以及头孢菌素。
在各种实施方案中，本发明的方法治疗的对象是人。在一个实施方案中，对象是婴儿。在一个实施方案中，对象是新生儿。在另一个实施方案中，对象是小于四周、小于三周、小于两周、小于一周、小于六天、小于五天、小于四天、小于三天、小于两天或小于一天的新生儿。在一些实施方案中，所述人是一个月大、两个月大、三个月大、四个月大、五个月大或六个月大。在一些实施方案中，人具有在约6至约18个月、约18至约36个月、约1至约5岁、约5至约10岁、约10至约15岁、约15至约20岁、约20至约25岁、约25至约30岁、约30至约35岁、约35至约40岁、约40至约45岁、约45至约50岁、约50至约55岁、约55至约60岁、约60至约65岁、约65至约70岁、约70至约75岁、约75至约80岁、约80至约85岁、约85至约90岁、约90至约95岁或约95至约100岁范围内的年龄。
另一方面，本发明的方法预防先前暴露于百日咳博德特氏杆菌细菌的对象的百日咳博德特氏杆菌感染，该方法包括向对象施用本发明的百日咳毒素结合蛋白或本发明的药物组合物。在各种实施方案中，该方法提供在预防暴露于百日咳博德特氏杆菌细菌的对象的百日咳博德特氏杆菌感染方面的有效预防性治疗。
在一些实施方案中，本发明的百日咳毒素结合蛋白或本发明的药物组合物用于先前尚未针对所述细菌疫苗接种的对象中的预防性应用。
预期本发明的百日咳毒素结合蛋白或本发明的药物组合物还可充当用于疫苗接种如DtaP或Tdap的佐剂。此外，在各种实施方案中，本发明的方法治疗或预防先前尚未针对百日咳博德特氏杆菌细菌疫苗接种的对象的百日咳博德特氏杆菌感染。
检测
在另一方面本发明还提供检测生物学样品中百日咳毒素的存在和/或量的方法，包括在本发明的百日咳毒素结合蛋白与百日咳毒素之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的百日咳毒素结合蛋白。然后检测复合物的形成，其中所述生物学样品与对照样品之间复合物形成的差异指示样品中百日咳毒素的存在和/或量。
在一些实施方案中，本发明的百日咳毒素结合蛋白还缀合有可用于检测或可被其他试剂检测到的荧光染料、化学物质、多肽、酶、同位素、标签等。
在一些实施方案中，所述生物学样品是用于预防百日咳博德特氏杆菌感染的疫苗，例如，所述疫苗包含百日咳毒素。
试剂盒
本发明的范围内还包括用于本发明的方法的试剂盒，该试剂盒包括本发明的百日咳毒素结合蛋白，以及使用说明。该试剂盒可以进一步包括至少一种用于检测的试剂。试剂盒一般包括表明试剂盒内容物的预期用途的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1：针对百日咳毒素(Pertussis Toxin，PT)的重链单域抗体的筛选
1.1文库的构建：
免疫前收集5mL双峰驼动脉血于真空采血管，收集上清作为免疫前血清。初次免疫，选取一只健康的2岁新疆双峰驼，取300μg重组百日咳毒素蛋白作为抗原与完全弗氏佐剂等体积均匀混合，使蛋白完全乳化后，对双峰驼采用颈部肌肉多点注射；后期免疫，每次采用等量的抗原与不完全弗氏佐剂等体积均匀混合；每周免疫一次，后期共对双峰驼免疫六次。最后一次免疫结束时，收集5mL双峰驼动脉血于真空采血管内，收集上清作为免疫后血清，利用ELISA的方法检测免疫前后动物血清中抗体的有变化，免疫后血清中抗体明显增多，由此判断由抗原导致的免疫效果符合实验要求。
利用密度梯度离心法分离出淋巴细胞，使用QIAGEN公司提供的RNA提取试剂盒提取总RNA，使用SuperScript III FIRST STRANDSUPERMIX试剂盒按照说明书将提取的RNA全部反转录为cDNA，用巢式PCR扩增编码重链抗体的可变区的核酸片段。
回收目标重链单域抗体核酸片段，并使用限制性内切酶(购自NEB)PstI及NotI将其克隆进入噬菌体展示用载体pMECS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建抗PT毒素的免疫单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为1.2×10 8。为检测文库的插入率，随机挑选50个克隆测序检测，具有正确的外源片段插入的克隆为50个，正确率为100％。通过对测序克隆的DNA和氨基酸序列分别分析比对，证实所有序列均为预期的骆驼VHH序列，可估测其多样性在95％以上。
1.2针对百日咳毒素(PT)抗体的淘洗
用重组百日咳毒素蛋白PT10μg/孔包被平板，4℃放置过夜。第二天用2％脱脂奶室温封闭2小时后，加入实施例1.1构建的噬菌体文库，100μL噬菌体(约10 10pfu)，在室温下作用2小时。之后用PBST(PBS中含有0.05％吐温20)洗25遍，以洗掉不结合的噬菌体。最后用Glycine(100mM,pH 2.0)将与PT蛋白特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，离心收集菌液涂布于2TYAG平板上，37℃培养过夜。次日，将平板上的大肠杆菌洗脱收集，共得到菌液约50mL。根据OD值计算共得到约10 11大肠杆菌。
1.3针对PT的重链单域抗体的高通量测序筛选序列：
取实施例1.2中获得的大肠杆菌2mL委托苏州泓迅科技有限公司进行高通量测序。
本次高通量测序共得到286521个抗PT单域抗体的序列，挑选其中丰度较高的前50个序列，再根据抗PT单域抗体中CDR1、CDR2、CDR3区各自单独丰度，以及两两组合丰度情况，挑选出32个序列作为候选序列。
实施例2：用哺乳动物细胞制备PT单域抗体的FC融合蛋白
2.1制备PT单域抗体的Fc融合质粒
将实施例1.3中获得的32个候选序列委托苏州泓讯生物科技有限公司进行基因合成，并在两端加上酶切位点。
双酶切PT单域抗体VHH片段，与编码人IgG1FC的DNA片段融合，并克隆至常规哺乳动物表达载体，获得用于在哺乳动物中表达PT单域抗体Fc融合蛋白的重组质粒。
2.2制备PT单域抗体的Fc融合蛋白
将2.1构建获得载体转染至HEK293细胞进行抗体的瞬时表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI 混合物分别加入HEK293细胞悬液中，放置在37℃，5％CO 2，130rpm中培养。四小时后补加EXCELL293培养基，2mM谷氨酰胺，130rpm培养。24小时后加3.8mM VPA，72小时后加入4g/L葡萄糖。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标PT单域抗体Fc融合蛋白。蛋白通过SDSPAGE以及SECHPLC初步考察纯度。部分蛋白表达情况和纯度分析见下表1：
表1
抗体 表达量(mg/L) SDSPAGE纯度％ 主峰比例％
iPT3Fc 380 >95％ 99.3
iPT7Fc 511 >95％ 98.5
iPT12Fc 553 >95％ 98.8
iPT13Fc 568 >95％ 97.7
iPT15Fc 426 >95％ 99.3
iPT20Fc 473 >95％ 98.9
iPT21Fc 445 >95％ 98.3
iPT22Fc 529 >95％ 98.9
iPT26Fc 439 >95％ 98.9
iPT28Fc 226 >95％ 99
iPT35Fc 355 >95％ 99.6
iPT36Fc 417 >95％ 98.4
iPT42Fc 334 >95％ 97.3
可见，PT单域抗体Fc融合蛋白表达量均在200mg/L以上，且经过Protein A亲和层析柱一步纯化后，其浓度大多在1.0mg/mL以上比较稳定，纯度也很高。
所选PT单域抗体可变区序列信息如下(每个序列从左到右的方框分别示出CDR1、CDR2和CDR3)：
Figure PCTCN2020099691-appb-000001
Figure PCTCN2020099691-appb-000002
实施例3：鉴定PT单域抗体Fc融合蛋白的功能
3.1检测PT单域抗体Fc融合蛋白的亲和力(生物膜干涉技术BLI)
上述实施例获得的PT单域抗体Fc融合蛋白针对重组人PT蛋白的结合动力学，通过生物膜干涉(Biolayerinterferometry，BLI)技术，使用分子相互作用仪测量。将实施例2.2所得的PT单域抗体Fc融合蛋白稀释至终浓度2.5μg/mL直接固化到AHC biosensor上，对动力学测量，将PT用0.02％PBST20分别稀释至100nM、50nM、25nM、12.5nM、6.25nM5个浓度，进样120s，解离时间为600s，10mM glycineHCl(pH1.7)再生5s。使用简单一对一Languir结合模型(Octet K2数据分析软件9.0版(Data analysis 9.0))，计算结合速率(kon)和解离速率(kdis)。平衡解离常数(kD)以比率kdis/kon计算。
测量的部分PT单域抗体Fc融合蛋白对PT的亲和力结果见表2。部分候选抗体亲和力较差而被排除(数据未显示)。
表2
Figure PCTCN2020099691-appb-000003
Figure PCTCN2020099691-appb-000004
3.2鉴定PT单域抗体FC融合蛋白对百日咳毒素的中和活性
PT毒素浓度为434.3μg/mL，用10％FBS+F12K+10％FBS将其配制成12ng/mL，每孔加入50μL，根据实施例3.1的结果挑选亲和力好的PT单域抗体FC融合蛋白用10％FBS+F12K自原始浓度开始2倍稀释，10个浓度，每孔加入50μL，37℃放置1小时，收集CHOK1细胞并计数，调整细胞数至3×10 5cells/mL，96孔板每孔加入50uL，24小时后100％酒精固定，0.1％结晶紫染色，PBS洗涤，显微镜下观察并拍照。
结果如下表3显示：在100μg/mL的浓度下，iPT13Fc、iPT21Fc、iPT28Fc和iPT36Fc展示出部分中和活性；iPT3Fc、iPT7Fc、iPT12Fc、iPT15Fc、iPT20Fc、iPT22Fc、iPT26Fc、iPT35Fc、iPT42Fc有比较高的中和活性。部分候选抗体中和活性较差而被排除(数据未显示)。
表3
样品 完全中和浓度(μg/mL)
iPT3Fc 50
iPT7Fc 1.56
iPT12Fc 1.125
iPT13Fc 100μg/mL部分中和活性
iPT15Fc 1.56
iPT20Fc 50
iPT21Fc 100μg/mL部分中和活性
iPT22Fc 3.13
iPT26Fc 1.56
iPT28Fc 100
iPT35Fc 2.25
iPT36Fc 100
iPT42Fc 12.5
3.3检测PT单域抗体Fc融合蛋白与PT结合的不同表位(生物膜干涉技术BLI：epitope binning)
利用intandem方法，将PT-biotin用0.02％PBST20稀释至10ug/mL固化到SAbiosensor上，固化时间为120s。PT单域抗体Fc融合蛋白用0.02％PBST20稀释至100nM，将其分成两组，抗体结合时间均为300s，再生液为10mM glycineHCl(pH1.7)；第一个抗体(饱和抗体)结合到sensor上至饱和状态，之后第二个抗体(竞争抗体)以同样浓度与第一个抗体进行竞争，计算百分比。百分比计算公式为Ab2with Ab1/Ab2without Ab1。
测量结果见表4和表5。结果表明：iPT 13、iPT 20和iPT 36之间可能存在竞争，iPT7和iPT12、iPT26和iPT35、iPT22和iPT15之间可能存在竞争；其余抗体均没有明 显竞争关系，具有不同的抗原结合表位。
表4
Ab1\Ab2 iPT5FC iPT3FC iPT7FC iPT13FC iPT20FC iPT26FC iPT36FC
iPT5FC 8.10％ 85.50％ 66.39％ 48.14％ 105.13％ 19.11％ 104.26％
iPT3FC 104.30％ 29.80％ 42.11％ 43.99％ 96.72％ 18.41％ 93.65％
iPT7FC 71.50％ 63.40％ 11.15％ 55.60％ 60.00％ 70.80％ 58.70％
iPT13FC 79.00％ 79.75％ 74.50％ 22.90％ 0.10％ 76.50％ 0.116
iPT20FC 87.10％ 92.90％ 93.00％ 49.30％ 6.72％ 95.30％ 0.0359
iPT26FC 49.60％ 54.20％ 45.60％ 26.70％ 13.80％ 31.40％ 28.00％
iPT36FC 91.60％ 91.15％ 92.40％ 51.80％ 20.30％ 92.10％ 8.12％
表5
Ab1\Ab2 iPT7 iPT26 iPT12 iPT15 iPT22 iPT35 iPT42
iPT7FC 11.97％ 79.48％ 17.43％ 79.76％ 41.72％ 26.26％ 79.59％
iPT26FC 117.29％ 6.85％ 110.26％ 102.65％ 51.00％ 12.80％ 93.59％
iPT12FC 16.29％ 16.08％ 11.88％ 86.81％ 53.34％ 97.95％ 79.00％
iPT15FC 115.91％ 16.60％ 108.40％ 15.40％ 6.73％ 88.28％ 79.85％
iPT22FC 82.67％ 13.39％ 78.47％ 32.60％ 7.10％ 71.23％ 84.19％
iPT35FC 118.80％ 3.67％ 119.64％ 71.08％ 30.57％ 9.72％ 98.61％
iPT42FC 110.00％ 10.72％ 105.09％ 77.60％ 45.61％ 85.17％ 10.48％
3.4检测PT单域抗体Fc融合蛋白与空细胞的非特异结合
CHOK1空细胞和293F空细胞重悬于3％BSAPBS，调整细胞数至6×10 6cells/mL，分别加入实施例2.2所得的PT单域抗体Fc融合蛋白终浓度为100μg/mL，同时设置阴性对照和空白对照，冰浴30min。洗涤后加入博士德生物二抗FITC兔抗人IgG antibody，冰浴30min。洗涤后将细胞重悬于300μL的1％PBSBSA Buffer中，流式细胞仪进行检测。结果见表6，抗体的MFI值与阴性对照和空白对照相差不大，均没有非特异结合。
表6
  浓度(μg/mL) MFI(CHO) MFI(293)
Blank —— 2.62 2.08
Negative —— 3.09 2.38
二抗 —— 3.01 2.67
iPT3Fc 100ug/mL 2.77 2.8
iPT7Fc 100ug/mL 2.68 2.6
iPT13Fc 100ug/mL 2.81 2.6
iPT21Fc 100ug/mL 2.76 2.64
iPT20Fc 100ug/mL 2.89 2.77
iPT26Fc 100ug/mL 2.89 2.77
iPT36Fc 100ug/mL 2.68 2.67
Figure PCTCN2020099691-appb-000005
Figure PCTCN2020099691-appb-000006
3.6检测PT单域抗体Fc融合蛋白的热稳定性
利用UNCHAINED LABS公司的蛋白热稳定性检测仪，测定微量体积的蛋白样品。升温程序：起始温度15℃，升温速率0.3℃/min，终点温度95℃。记录每个温度、每个波长下样品的荧光吸光度值，软件拟合变性温度Tm值为置信波长BCM下一阶导数的最高点；起始聚合温度Tagg值为静态光散射SLS，473nm下一阶导数的十分之一值。Tm值越高，说明该蛋白越稳定。结果见下表7，可知抗体Tm值基本均高于60℃，稳定性均很好。
表7
Figure PCTCN2020099691-appb-000007
实施例4：用哺乳动物细胞制备PT四价抗体的FC融合蛋白
4.1制备PT四价抗体的Fc融合质粒
挑选7个抗体iPT 7、iPT 12、iPT 15、iPT22、iPT26、iPT35、iPT42，根据抗体识别抗原表位(epitope binning结果)的重叠性，可将其分为4组，将识别抗原表位的重叠性小的序列两两串联组合后，与编码人IgG1FC的DNA片段融合，并克隆至常规哺乳动物表达载体，获得用于在哺乳动物中表达PT四价抗体Fc融合蛋白的重组质粒希望通过这种组合提高其活性；另外，iPT12diFc和iPT15diFc分别为两个相同iPT12或iPT15序列串联组合后，与人IgG1Fc片段融合，作为对照。
4.2制备PT四价抗体的Fc融合蛋白
将4.1构建获得载体转染至HEK293细胞进行抗体的瞬时表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，5％CO2，130rpm中培养。四小时后补加EXCELL293培养基，2mM谷氨酰胺，130rpm培养。24小时后加3.8mM VPA，72小时后加入4g/L葡萄糖。培养5～6天后，收集瞬时表达培养上清液，通过Protein A 亲和层析法，纯化得到目标PT四价抗体的Fc融合蛋白。蛋白通过SDSPAGE以及SECHPLC初步考察纯度。各蛋白表达情况和纯度分析见下表8，可见，PT四价抗体的Fc融合蛋白表达量均在200mg/L以上，且经过Protein A亲和层析柱一步纯化后，其浓度大多在1.0mg/mL以上比较稳定，SEC结果显示纯度也很高。
表8
抗体 表达量(mg/L) SDSPAGE纯度％ 主峰比例％
iPT7n15Fc 281 >95％ 98.9％
iPT15n7Fc 271 >95％ 97.9％
iPT7n22Fc 374 >95％ 98.0％
iPT22n7Fc 320 >95％ 97.1％
iPT7n42Fc 308 >95％ 97.6％
iPT42n7Fc 310 >95％ 98.7％
iPT12n35Fc 316 >95％ 98.0％
iPT35n12Fc 306 >95％ 99.0％
iPT15n35Fc 328 >95％ 99.3％
iPT15n42Fc 320 >95％ 93.0％
iPT35n42Fc 290 >95％ 98.4％
iPT12n15Fc 367 >95％ 97.9％
iPT15n12Fc 359 >95％ 98.8％
iPT12n42Fc 399 >95％ 95.2％
iPT42n12Fc 254 >95％ 97.4％
iPT15diFc 344 >95％ 99.1％
iPT12diFc 257 >95％ 95.8％
实施例5：鉴定PT四价抗体的Fc融合蛋白的功能
5.1检测PT四价抗体的Fc融合蛋白的亲和力(生物膜干涉技术BLI)
将实施例4.2中制备的PT四价抗体的Fc融合蛋白稀释至2.5μg/mL固化到biosensor上，固化60s，固化高度约0.8nm。PTpuri稀释至100nM、50nM、25nM、12.5nM、6.25nM5个梯度，基线60s，结合120s，解离600s。稀释液为0.02PBST20％，再生液为glycineHCl(pH1.7)，中和液为稀释液，biosensor为AHC，由表9可知，抗体亲和力均很好，尤其是抗体iPT15n7Fc亲和力最高。
表9
Figure PCTCN2020099691-appb-000008
Figure PCTCN2020099691-appb-000009
5.2鉴定PT四价抗体的Fc融合蛋白对百日咳毒素的中和活性
PT毒素浓度为434.3μg/mL，用10％FBS+F12K+10％FBS将其配制成12ng/mL，每孔加入50μL，将实施例4.2制备的PT四价抗体的FC融合蛋白用10％FBS+F12K自原始浓度开始2倍稀释，10个浓度，每孔加入50μL，37℃放置1小时，收集CHOK1细胞并计数，调整细胞数至3×10 5cells/mL，96孔板每孔加入50μL，24小时后100％酒精固定，0.1％结晶紫染色，PBS洗涤，显微镜下观察并拍照。结果见表10，四价抗体iPT7n15Fc、iPT42n7Fc、iPT12n15Fc、iPT12n42Fc、iPT12diFc、iPT15n12Fc、iPT42n12Fc中和活性较好，且iPT7n15Fc、iPT42n7Fc、iPT12n15Fc、iPT12n42Fc、iPT15n12Fc、iPT42n12Fc的中和活性均高于iPT15diFc和iPT12diFc，也高于二价抗体iPT12Fc，可见识别不同抗原表位的单域抗体组合之后，其中和活性有所提高。
表10
抗体 完全中和PTToxin的浓度(μg/mL)
iPT15n7FC 5μg/mL部分中和
iPT7n22FC 5
iPT22n7FC 100
iPT7n42FC 2.5μg/mL部分中和
iPT12n35Fc 100
iPT35n12Fc 100
iPT15n35Fc 20μg/mL部分中和
iPT15n42Fc 100
iPT35n42Fc 100μg/mL部分中和
iPT12Fc 0.42
iPT7n15Fc 0.1
iPT42n7Fc 0.05
iPT12n15Fc 0.111
iPT12n42Fc 0.333
iPT12diFc 0.333μg/mL部分中和
iPT15n12Fc 0.111
iPT15diFc 1μg/mL部分中和
iPT42n12Fc 0.333
5.3检测PT四价抗体的Fc融合蛋白与空细胞的非特异结合
CHOK1空细胞和293F空细胞重悬于3％BSAPBS，调整细胞数至6×10 6cells/mL，分别加入实施例4.2获得的PT四价抗体的Fc融合蛋白终浓度为100μg/mL，同时设置阴性对照和空白对照，冰浴30min。洗涤后加入博士德生物二抗FITC兔抗人IgG antibody，冰浴30min。洗涤后将细胞重悬于300μL的1％PBSBSA Buffer中，流式细胞仪进行检测。结果见表11，显示均没有非特异结合。
表11
样品名称 浓度(μg/mL) MFI(293) MFI(CHO)
Blank —— 2.62 2.08
Negative —— 3.09 2.38
iPT12n15Fc 100 3.40 2.40
iPT12n42Fc 100 3.46 2.39
iPT12diFc 100 3.57 2.40
iPT15n12Fc 100 3.57 2.33
iPT15diFc 100 3.50 2.40
iPT42n12Fc 100 3.45 2.51
Blank —— 4.64 4.29
Negative —— 6.04 5.43
iPT7n15Fc 100 7.27 5.46
iPT42n7Fc 100 6.44 5.35
实施例6 PT四价抗体的Fc融合蛋白体内药效研究
6.1 PT毒素保护实验
将所用的NIH小鼠分组，每组10只，将PTtoxin腹腔注射至NIH小鼠，剂量为5μg/只，通过腹腔注射PTtoxin建立用于评价PT毒素抗体的攻毒小鼠模型。
本实验通过将PT四价抗体FC融合蛋白iPT7n15Fc、iPT42n7Fc、iPT15n12Fc、iPT42n12Fc、iPT12diFc，腹腔注射至攻毒小鼠模型，每天记录小鼠死亡情况。攻毒后2小时，开始注射PT四价抗体FC融合蛋白，单剂量注射分别为20μg/dose和40μg/dose；多剂量注射共分5次给药，剂量为20μg/dose和40μg/dose，分组的当天记为第0天，同时做阴性对照，然后分别在D0、D5、D10和D15给小鼠称重、测白细胞和淋巴细胞数，并每天记录小鼠存活率。
结果见下表12，与阴性对照相比，所有抗体对攻毒小鼠均有保护作用；单剂量和多剂量给药，抗体iPT15n12FC、iPT12diFC在给药剂量40μg/dose时比20μg/dose时，小鼠存活率明显上升，其他抗体没有明显差别。生存曲线单剂量给药20μg/dose和40μg/dose分别见图1、图2，多剂量给药20μg/dose和40μg/dose分别见图3、图4。
表12
Figure PCTCN2020099691-appb-000010
6.2细菌保护实验
将所用的NIH小鼠均为雌性，1622g/只，每组10只，分组的当天记为第0天，对小鼠称重、测白细胞和淋巴细胞数，腹腔注射百日咳杆菌至NIH小鼠，通过腹腔注射百日咳杆菌建立用于评价PT毒素抗体的攻毒小鼠模型。
本实验通过将PT四价抗体FC融合蛋白iPT7n15Fc、iPT42n7Fc、iPT15n12Fc、iPT42n12Fc、iPT12diFc，腹腔注射至攻毒小鼠模型，每天记录小鼠死亡情况。攻毒后2小时，开始注射PT四价抗体FC融合蛋白，多剂量注射共分5次给药，剂量分别为 20μg/dose和40μg/dose，同时做阴性对照，然后分别在D0、D5、D10和D15给小鼠称重、测白细胞和淋巴细胞数，并每天记录小鼠存活率。
结果显示见表13，与阴性对照相比，所有抗体对攻毒小鼠均有保护作用，其中抗体iPT7n15FC和iPT42n7Fc均有很好的保护作用，抗体iPT7n15FC和iPT15n12Fc在给药剂量40μg/dose时比20μg/dose，小鼠存活率明显上升。生存曲线见图5。
表13
Figure PCTCN2020099691-appb-000011
实施例7：PT单域抗体的人源化
人源化方法采用蛋白表面氨基酸人源化(resurfacing)的方法及VHH人源化通用框架移植法(CDR grafting to a universal framework)完成。
人源化步骤如下：对抗体株iPT7、iPT15和iPT42进行同源建模，建模软件为Modeller9。参考同源序列为NbBcII10抗体(PDB编号为：3DWT)，并根据蛋白三维结构计算氨基酸的相对溶剂可及性(relative solvent accessibility)。如抗体株iPT7、iPT15和iPT42的某个氨基酸暴露在溶剂内，则替换为参考人抗体10HQ序列相同位置的氨基酸，最终完成全部替换。
VHH人源化通用框架移植法具体步骤如下：首先获取Cécile Vincke等人根据序列同源性设计完成的通用性人源化VHH框架hNbBcII10FGLA(PDB编号为：3EAK)，该框架设计基于纳米抗体NbBcII10抗体(PDB编号为：3DWT)，参考人源抗体10HQ进行蛋白表面氨基酸人源化，并改造VHH序列框架1(framework1)的部分氨基酸VLP、VHH序列框架2(framework2)的部分氨基酸GL、VHH序列框架3(framework3)的部分氨基酸RSKRAAV和VHH序列框架4(framework4)的氨基酸L完成。直接使用hNbBcII10FGLA作为框架，将CDR替换为抗体株iPT7、iPT15和iPT42的CDR区，完成抗体的人源化。
对抗体株iPT7、iPT15和iPT42进行人源化，分别获得11种抗体株huPT的人源化变体。这些人源化变体的序列编号以及其中的氨基酸残基编号比照Kabat编号，图6-8显示人源化序列的比对结果。
实施例8：用哺乳动物细胞制备huPT单域抗体的FC融合蛋白
8.1制备huPT单域抗体的Fc融合质粒
将实施例7中的huPT单域抗体编码序列由苏州泓讯生物科技有限公司进行基因合 成，并在两端加上酶切位点。
双酶切huPT单域抗体VHH片段，与编码人IgG1FC的DNA片段融合，并克隆至常规哺乳动物表达载体，获得用于在哺乳动物中表达huPT单域抗体Fc融合蛋白的重组质粒。
8.2制备huPT单域抗体的Fc融合蛋白
将8.1构建获得载体转染至HEK293细胞进行抗体的瞬时表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，5％CO 2，130rpm中培养。四小时后补加EXCELL293培养基，2mM谷氨酰胺，130rpm培养。24小时后加3.8mM VPA，72小时后加入4g/L葡萄糖。培养5～6天后，收集瞬时表达培养上清液，通过Protein A亲和层析法，纯化得到目标huPT单域抗体Fc融合蛋白。蛋白通过SDSPAGE以及SECHPLC初步考察纯度。各蛋白表达情况和纯度分析见下表14。结果表明，iPT7以及iPT15的所有人源化变体在表达水平和纯度上差异都不是特别大，可以接受。而iPT42的人源化变体表现出一定的稳定性差异。其中人源化变体v1、v2、v7、v8,、v10、v11在表达水平和产物纯度上都不理想。
表14
Figure PCTCN2020099691-appb-000012
Figure PCTCN2020099691-appb-000013
实施例9：鉴定人源化huPT单域抗体Fc融合蛋白的功能
9.1 huPT单域抗体Fc融合蛋白对PT结合能力鉴定(ELISA法)
用重组人PT蛋白包被平板0.3μg/孔4℃过夜，实施例8.2所得的huPT单域抗体Fc融合蛋白的梯度稀释系列，室温下反应2小时。洗涤之后加入二抗山羊抗人IgGFC辣根过氧化物酶标记抗体(Sigma)，室温反应2小时。洗涤之后加入显色液，读取450nm和650nm波长吸收值，450nm波长的吸光度值减去650nm波长的吸光度值为最终的吸光度值。应用软件SotfMax Pro v5.4进行数据处理和作图分析，通过四参数拟合，得到huPT抗体对PT蛋白的结合曲线及EC 50值，以反映抗体对FIX的亲和能力。结果见表15。可以看出各个人源化变体对PT毒素蛋白的结合能力都类似。
表15
Figure PCTCN2020099691-appb-000014
Figure PCTCN2020099691-appb-000015
9.2鉴定人源化PT抗体FC融合蛋白对百日咳毒素的中和活性
PT毒素浓度为434.3μg/mL，用10％FBS+F12K+10％FBS将其配制成12ng/mL，每孔加入50μL，PT抗体FC融合蛋白用10％FBS+F12K自原始浓度开始2倍稀释，10个浓度，每孔加入50μL，37℃放置1小时，收集CHOK1细胞并计数，调整细胞数至3×10 5cells/mL，96孔板每孔加入50uL，24小时后100％酒精固定，0.1％结晶紫染色，PBS洗涤，显微镜下观察并拍照。iPT7、15以及42各选随机取3个人源化突变考察其中和活性。其中iPT42的变体中，去除了稳定性较差的v1、v2、v7、v8、v10、v11。
结果如下表16显示，所选的人源化变体，其细胞毒素中和活性都与母本抗体相当，或者略优于母本。
表16
样品 最低中和浓度(ug/ml)
iPT15-Fc <1
huPT15V3c-ld-Fc <1
huPT15V7c-ld-Fc <1
huPT15V11c-ld-Fc <1
iPT7-Fc <1
huPT7V1-ld-Fc <0.5
huPT7V4-ld-Fc <0.5
huPT7V11-ld-Fc <0.5
iPT42-Fc ～32
huPT42V4-ld-Fc ～32
huPT42V6-ld-Fc ～16
huPT42V9-ld-Fc ～32
9.3检测huPT单域抗体Fc融合蛋白与空细胞的非特异结合
CHOK1空细胞和293F空细胞重悬于3％BSAPBS，调整细胞数至6×10 6cells/mL，分别加入实施例8.2获得的huPT单域抗体的Fc融合蛋白终浓度为100μg/mL和10μg/mL，同时设置阴性对照和空白对照，冰浴30min。洗涤后加入博士德生物二抗FITC兔抗人IgG antibody，冰浴30min。洗涤后将细胞重悬于300μL的1％PBSBSA Buffer中，流式细胞仪进行检测。结果见表17，显示均没有非特异结合。
表17
Figure PCTCN2020099691-appb-000016
Figure PCTCN2020099691-appb-000017
9.4检测huPT单域抗体Fc融合蛋白的热稳定性
利用UNCHAINED LABS公司的蛋白热稳定性检测仪，测定微量体积的蛋白样品。升温程序：起始温度15℃，升温速率0.3℃/min，终点温度95℃。记录每个温度、每个波长下样品的荧光吸光度值，软件拟合变性温度Tm值为置信波长BCM下一阶导数的最高点；起始聚合温度Tagg值为静态光散射SLS，473nm下一阶导数的十分之一值。Tm值越高，说明该蛋白越稳定。结果见下表18，可知抗体Tm值均高于60℃，稳定性均很好。
表18
  Tm1 Tagg
huPT7v4-Fc 60.8 58.5
huPT7v7-Fc 60.4 58.0
huPT7v2-Fc 60.6 58.2
huPT7v1-Fc 60.3 58.1
huPT7v11-Fc 62.1 60.0
huPT42v4-Fc 64.5 62.0
huPT42v6-Fc 67.7 72.8
huPT42v9-Fc 64.2 66.6
huPT15V1c-ld-Fc 66.8 69.6
huPT15V3c-ld-Fc 66.5 69.2
huPT15V7c-ld-Fc 66.8 69.5
huPT15V11c-ld-Fc 67.9 72.0
实施例10：制备四价的人源化PT抗体的Fc融合蛋白
根据实施例4的方法，选取实施例7～9中获得并考察的人源化抗体序列，huPT7v11、huPT15v7c、huPT42v6，根据抗体识别抗原表位(epitope binning结果)的重叠性，两两串联组合后，与编码人IgG1FC的DNA片段融合，获得四价双特异性抗体huPT7v11n15v7c-Fc，huPT7v11n15v11c-Fc，huPT42v6n15v11c-Fc以及huPT42v6n7v11-Fc。
HEK293细胞瞬时表达并纯化得到目标PT四价抗体的Fc融合蛋白。
实施例11进一步考察四价的人源化PT抗体的Fc融合蛋白的活性
11.1考察对PT毒素的亲和力
将实施例10中制备的PT四价抗体的Fc融合蛋白稀释至2.5μg/mL固化到biosensor上，固化60s，固化高度约0.8nm。PTpuri稀释至100nM、50nM、25nM、12.5nM、6.25nM5个梯度，基线60s，结合120s，解离600s。稀释液为0.02PBST20％，再生液为glycineHCl(pH1.7)，中和液为稀释液，biosensor为AHC，结果见下表19。其中1B7与11E6为对照抗体，结合PT毒素并有中和效果。参照专利US10035846中的序列自主克隆并制备。
表19
样品 KD(M) kon(1/Ms) kdis(1/s)
huPT7n15-Fc 8.84E-11 3.75E+05 3.32E-05
huPT42n7-Fc 2.00E-10 4.59E+05 9.18E-05
huPT42n15-Fc <1.0E-12 3.37E+05 <1.0E-07
1B7 <1.0E-12 3.87E+05 <1.0E-07
11E6 <1.0E-12 3.11E+05 <1.0E-07
11.2考察四价人源化PT抗体Fc融合蛋白的中和活性并与对照抗体比较
PT毒素浓度为434.3μg/mL，用10％FBS+F-12K+10％FBS将其配制成12ng/mL，每孔加入50μL，将实施例4.2制备的PT四价抗体的FC融合蛋白用10％FBS+F-12K自原始浓度开始2倍稀释，10个浓度，每孔加入50μL，37℃放置1小时，收集CHO-K1细胞并计数，调整细胞数至3×10 5cells/mL，96孔板每孔加入50μL，24小时后100％酒精固定，0.1％结晶紫染色，PBS洗涤，显微镜下观察并拍照。1B7与11E6为对照抗体，结合PT毒素并有中和效果。参照专利US10035846中的序列自主克隆并制备。
结果见表20，四价抗体huPT7n15-Fc、huPT42n7-Fc、huPT42n15-Fc、iPT12n42-Fc、iPT12di-Fc、iPT15n12-Fc、iPT42n12-Fc中和活性较好，且iPT7n15-Fc、iPT42n7-Fc、iPT12n15-Fc、iPT12n42-Fc、iPT15n12-Fc、iPT42n12-Fc的中和活性均高于iPT15di-Fc 和iPT12di-Fc，也高于二价抗体iPT12-Fc，可见识别不同抗原表位的单域抗体组合之后，其中和活性有所提高。
表20
抗体 最小中和浓度
huPT7n15-Fc 0.025μg/mL
huPT42n7-Fc 0.05μg/mL
huPT42n15-Fc 0.1μg/mL
1B7 1.6μg/mL
11E6 0.8μg/mL
1B7+11E6 (0.4+0.4)μg/mL
huPT7n15-Fc+huPT42n7-Fc (0.00625+0.00625)μg/mL
huPT7n15-Fc+huPT42n15-Fc (0.0125+0.0125)μg/mL
实施例12 PT单域抗体Fc融合蛋白与对照抗体的表位比较
本实施例采用生物膜干涉技术BLI：epitope binning比较本发明的PT单域抗体Fc融合蛋白与对照抗体的结合表位。
具体而言，利用intandem方法，将PT-biotin用0.02％PBST20稀释至10ug/mL固化到SAbiosensor上，固化时间为120s。PT单域抗体Fc融合蛋白用0.02％PBST20稀释至100nM，将其分成两组，抗体结合时间均为300s，再生液为10mM glycineHCl(pH1.7)；第一个抗体(饱和抗体)结合到sensor上至饱和状态，之后第二个抗体(竞争抗体)以同样浓度与第一个抗体进行竞争，计算百分比。百分比计算公式为Ab2with Ab1/Ab2without Ab1。其中对照抗体(11E6，1B7，序列来源专利US10035846)为Ab1，后续抗体为Ab2。同时在Ab2组中也加入对照抗体，作为抗原表位完全重叠的参考。
测量结果见下表21。结果表明：除iPT12与11E6对照抗体抗原表位有重叠，iPT7与11E6对照抗体的抗原表位部分重叠之外，其余抗体均与两个对照抗体没有明显竞争关系，具有完全不重叠的抗原结合表位。
表21
Figure PCTCN2020099691-appb-000018

Claims (24)

  1. 百日咳毒素结合蛋白，其能够特异性结合百日咳毒素且包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (1)SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2，SEQ ID NO:3所示的CDR3；
    (2)SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:5所示的CDR3；
    (3)SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；
    (4)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所示的CDR3；
    (5)SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；
    (6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2，SEQ ID NO:18所示的CDR3；
    (7)SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2，SEQ ID NO:21所示的CDR3；
    (8)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；
    (9)SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2，SEQ ID NO:27所示的CDR3；
    (10)SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2，SEQ ID NO:30所示的CDR3；
    (11)SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；
    (12)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:36所示的CDR3；
    (13)SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3。
  2. 权利要求1的百日咳毒素结合蛋白，其中所述免疫球蛋白单一可变结构域是VHH。
  3. 权利要求2的百日咳毒素结合蛋白，其中所述VHH是人源化的VHH。
  4. 权利要求2的百日咳毒素结合蛋白，其中所述VHH包含SEQ ID NO:40-52中任一氨基酸序列。
  5. 权利要求3的百日咳毒素结合蛋白，其中所述VHH包含与SEQ ID NO:40-52中任一序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
  6. 权利要求3的百日咳毒素结合蛋白，其中所述人源化的VHH包含SEQ ID NO:53-85中任一氨基酸序列。
  7. 权利要求1-6任一项的百日咳毒素结合蛋白，其包含至少两个所述免疫球蛋白单一可变结构域。
  8. 权利要求7的百日咳毒素结合蛋白，其中所述至少两个免疫球蛋白单一可变结构域结合相同表位或竞争结合相同表位，例如，所述至少两个免疫球蛋白单一可变结构域是相同的。
  9. 权利要求7的百日咳毒素结合蛋白，其中所述至少两个免疫球蛋白单一可变结构域结合不同表位或不竞争结合相同表位。
  10. 权利要求9的百日咳毒素结合蛋白，其包含第一免疫球蛋白单一可变结构域和第二免疫球蛋白单一可变结构域，其中所述第一免疫球蛋白单一可变结构域位于所述第二免疫球蛋白单一可变结构域的N端，且
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID  NO:38所示的CDR2，SEQ ID NO:39所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2，SEQ ID NO:6所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2，SEQ ID NO:33所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2，SEQ ID NO:15所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2，SEQ ID NO:39所示的CDR3；或
    其中所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:37所示的CDR1，SEQ ID  NO:38所示的CDR2，SEQ ID NO:39所示的CDR3，且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2，SEQ ID NO:9所示的CDR3。
  11. 权利要求10的百日咳毒素结合蛋白，其中
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:47所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:47所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:41、53-63之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:50所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:44、64-74之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列且所述第 二免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列；或
    所述第一免疫球蛋白单一可变结构域包含SEQ ID NO:52、75-85之一所示氨基酸序列且所述第二免疫球蛋白单一可变结构域包含SEQ ID NO:42所示氨基酸序列。
  12. 权利要求1-11任一项的百日咳毒素结合蛋白，其还包含免疫球蛋白Fc区。
  13. 权利要求12的百日咳毒素结合蛋白，其中所述免疫球蛋白Fc区是人免疫球蛋白Fc区，优选是人IgG1的Fc区。
  14. 权利要求13的百日咳毒素结合蛋白，其中所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO:86。
  15. 权利要求12-14中任一项的百日咳毒素结合蛋白，其包含选自SEQ ID NO:106-109的氨基酸序列。
  16. 核酸分子，其编码权利要求1-15中任一项的百日咳毒素结合蛋白。
  17. 表达载体，其包含与表达调控元件可操作地连接的权利要求16的核酸分子。
  18. 重组细胞，其包含权利要求16的核酸分子或以权利要求17的表达载体转化，并能够表达所述百日咳毒素结合蛋白。
  19. 产生权利要求1-15中任一项的百日咳毒素结合蛋白的方法，包括：
    a)在允许所述百日咳毒素结合蛋白表达的条件下培养权利要求18的重组细胞；
    b)从得自步骤a)的培养物回收由所述重组细胞表达的百日咳毒素结合蛋白；及
    c)任选进一步纯化和/或修饰得自步骤b)的百日咳毒素结合蛋白。
  20. 药物组合物，其包含权利要求1-15任一项的百日咳毒素结合蛋白以及药学上可接受的载体。
  21. 一种治疗对象中百日咳博德特氏杆菌感染的方法，所述方法包括向所述对象施用有效量的权利要求1-15任一项的百日咳毒素结合蛋白或权利要求20的药物组合物。
  22. 一种在对象中预防百日咳博德特氏杆菌感染的方法，所述方法包括向对象有效量的权利要求1-15任一项的百日咳毒素结合蛋白或权利要求20的药物组合物。
  23. 一种检测生物学样品中百日咳毒素的存在和/或量的方法，包括：
    (a)在百日咳毒素结合蛋白与百日咳毒素之间能够形成复合物的条件下，使所述生物学样品和对照样品接触权利要求1-15任一项的百日咳毒素结合蛋白；
    (b)检测复合物的形成，
    其中所述生物学样品与对照样品之间复合物形成的差异指示样品中百日咳毒素的存在和/或量。
  24. 一种试剂盒，其包含权利要求1-15任一项的百日咳毒素结合蛋白。
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