WO2020029860A1 - 针对狂犬病病毒的双特异性抗体及其用途 - Google Patents

针对狂犬病病毒的双特异性抗体及其用途 Download PDF

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WO2020029860A1
WO2020029860A1 PCT/CN2019/098836 CN2019098836W WO2020029860A1 WO 2020029860 A1 WO2020029860 A1 WO 2020029860A1 CN 2019098836 W CN2019098836 W CN 2019098836W WO 2020029860 A1 WO2020029860 A1 WO 2020029860A1
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amino acid
acid sequence
rabies virus
seq
protein
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PCT/CN2019/098836
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French (fr)
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刘志刚
郝小勃
刘玉兰
郭晶晶
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北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Priority to RU2020130632A priority Critical patent/RU2764740C1/ru
Priority to US17/057,100 priority patent/US11773155B2/en
Publication of WO2020029860A1 publication Critical patent/WO2020029860A1/zh
Priority to ZA2020/07259A priority patent/ZA202007259B/en

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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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This application relates generally to the fields of genetic engineering and antibody drugs; specifically, this application relates to bispecific antibodies against rabies virus and uses thereof.
  • Rabies is an acute infectious disease caused by rabies virus. It is a zoonotic disease and is mainly transmitted in dogs, wolves, cats and other animals, but other mammals such as raccoons, skunks, bats and foxes are also common hosts. Animals spread the virus by biting each other, and most people are infected by being bitten by sick animals. At present, there is no effective treatment for rabies. The mortality rate of people suffering from rabies is almost 100%, and patients usually die from respiratory or circulatory failure within 3-6 days. It is estimated that more than 70,000 people worldwide die each year from the disease, and millions of people require post-exposure treatment.
  • Rabies virus is a bullet-shaped, enveloped, single-stranded RNA virus that belongs to the Rhabdoviridae family and belongs to the genus Rabies virus.
  • the rabies virus genome encodes five viral proteins: RNA-dependent RNA polymerase (L), nucleoprotein (N), phosphorylated protein (P), matrix protein (M) located on the inner side of the viral protein envelope, and outer surface glycoproteins (G).
  • the glycoprotein (G protein) of rabies virus can bind to acetylcholine, which determines the neurophagicity of rabies virus.
  • G protein (62-67kDa) is a type I glycoprotein consisting of 505 amino acids.
  • G protein forms a protrusion covering the outer surface of the envelope of the virus particle.
  • G protein can induce virus neutralizing antibodies.
  • G protein has at least 5 neutralizing epitopes, of which epitope II is a discontinuous spatial epitope, including amino acid residues 34-42 and amino acid residues 198-200, and epitope III is 330-338, which is linear Epitope, about 97% of the reported antibodies recognize epitope II and epitope III, and rabies virus neutralizing antibody CR4098 binds to epitope III. Few antibodies recognize epitope I and epitope IV.
  • the rabies virus neutralizing antibody CR57 recognizes linear epitope I, namely 218-240, and the core binding domain is KLCGVL at positions 226-231.
  • Epitope IV contains residues 251 and 264.
  • Another epitope is a microepitope a that is 3 amino acid residues apart from epitope III and does not overlap epitope III, with only two amino acid residues 342-343.
  • rabies virus vaccines for active treatment are mainly human rabies immunoglobulin (HRIG) and equine rabies immunoglobulin (ERIG). Because ERIG is a heterologous protein to the human body, sometimes the side effects are serious, while HRIG is expensive, the supply is limited, and there is a risk of potential blood-borne pathogen contamination. Therefore, clinical development of new passive treatment drugs for rabies virus is needed.
  • HRIG human rabies immunoglobulin
  • ERIG equine rabies immunoglobulin
  • CL184 a drug based on two monoclonal antibodies
  • CL184 is based on a mixed preparation of two monoclonal antibodies, and the preparation cost is relatively high.
  • the present application provides a bispecific antibody comprising two antigen-binding fragments that bind to different epitopes of the G protein of rabies virus, and the bispecific antibody has activity to neutralize rabies virus.
  • one antigen-binding fragment of the bispecific antibody binds epitope I of the rabies virus G protein and the other antigen-binding fragment binds epitope III of the rabies virus G protein.
  • the antigen-binding fragment that binds epitope I of the rabies virus G protein comprises:
  • the amino acid sequence is HCDR1 of RYTIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDIGAYDYVS, the amino acid sequence is LCDR2 of DATKRPS, and the amino acid sequence is LCDR3 of CSYAGDYTPGVV; or
  • the amino acid sequence is HCDR1 of RYSIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDIDGYDFVS, the amino acid sequence is LCDR2 of DATKRPS, the amino acid sequence is LCDR3 of CSYAGDYTPGVV;
  • the amino acid sequence is HCDR1 of GYTIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDLGGYDFVS, the amino acid sequence is LCDR2 of DATKRPS, and the amino acid sequence is LCDR3 of CSYAGDYTPGVV;
  • amino acid sequences of HCDR and LCDR are defined according to Kabat.
  • the antigen-binding fragment that binds to epitope III of the rabies virus G protein comprises:
  • the amino acid sequence is HCDR1 of SYGMH, the amino acid sequence is HCDR2 of TISYDGSIKDYADSVKG, the amino acid sequence is HCDR3 of GDRTGNLDY, the amino acid sequence is LCDR1 of RASQNIRNALN, the amino acid sequence is LCDR2 of DASTRQS, and the amino acid sequence is LCDR3 of QQNSEFPPT;
  • amino acid sequences of HCDR and LCDR are defined according to Kabat.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope I of the rabies virus G protein is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.
  • amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the rabies virus G protein is shown in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope I of the rabies virus G protein is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID As shown in NO: 25, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the rabies virus G protein is shown in SEQ ID: NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope III of the G protein of rabies virus is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 3; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the G protein of rabies virus is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.
  • the form of the two antigen-binding fragments is independently selected from a single chain antibody (scFv) or a Fab fragment.
  • the antigen-binding fragment that binds epitope I of the rabies virus G protein is a single-chain antibody (scFv), and the antigen-binding fragment that binds epitope III of the rabies virus G protein is a Fab fragment.
  • the bispecific antibody comprises the amino acid sequence shown in one of SEQ ID NO: 32, 33, 34. In some embodiments, the bispecific antibody comprises the amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody according to the first aspect and a pharmaceutically acceptable excipient, diluent, or carrier.
  • the pharmaceutical composition is used to prevent or treat rabies.
  • the application provides the use of the bispecific antibody according to the first aspect in the manufacture of a medicament for preventing or treating rabies.
  • the present application provides a method for preventing or treating rabies, comprising administering to a subject in need thereof the bispecific antibody according to the first aspect or the pharmaceutical composition according to the second aspect.
  • Figure 1 shows the binding capacity of fully human monoclonal antibodies C34m and C11m to inactivated rabies virus by ELISA analysis.
  • Figure 2 shows the inhibition of phage-C11m binding to inactivated rabies virus by ELISA analysis of fully human monoclonal antibodies C34m and C11m.
  • Figure 3 shows the inhibition of phage-C34m binding to inactivated rabies virus by ELISA analysis of fully human monoclonal antibodies C34m and C11m.
  • Figure 4 shows the inhibition of the binding of chimeric antibody CR57-mIgG2a to inactivated rabies virus by ELISA analysis of fully human monoclonal antibodies C11m and C34m.
  • Fig. 5 shows the inhibition of the binding of chimeric antibody CR4098-mIgG2a to inactivated rabies virus by ELISA analysis of fully human monoclonal antibodies C11m and C34m.
  • FIG. 6 shows the binding ability of C11m-scFv-Fc single-chain antibody Fc fusion protein, single-chain antibody Fc fusion protein of three C11m-scFv mutants and fully human monoclonal antibody C34m to inactivated rabies virus.
  • Figure 7 shows the inhibitory effect of the C11m-scFv-Fc single-chain antibody Fc fusion protein and three C11m-scFv mutant single-chain antibody Fc fusion proteins on the binding of the chimeric antibody C34m-mIgG2a to inactivated rabies virus by ELISA analysis.
  • Figure 8 shows the inhibitory effect of the C11m-scFv-Fc single chain antibody Fc fusion protein and the three C11m-scFv mutant single chain antibody Fc fusion proteins on the binding of the chimeric antibody C11m-mIgG2a to inactivated rabies virus by ELISA analysis.
  • Figure 9 shows the ELISA analysis of the binding capacity of various anti-rabies virus G protein antibodies to inactivated rabies virus.
  • Figure 10 shows ELISA analysis of bispecific antibody S2E3-scFv-FcH + C34m-IgG1K, single chain antibody Fc fusion protein S2E3-scFv-Fc and fully human monoclonal antibody C34m against chimeric antibody C34m-mIgG2a and inactivated rabies virus Inhibition of binding.
  • Figure 11 shows ELISA analysis of bispecific antibody S2E3-scFv-FcH + C34m-IgG1K, single-chain antibody Fc fusion protein S2E3-scFv-Fc, and fully human monoclonal antibody C34m against chimeric protein S2E3-scFv-mFc and inactivation. Inhibitory effect of rabies virus binding.
  • Figure 12 shows the binding capacity of anti-rabies virus G protein antibodies C34m and S2E3-scFv-Fc to the G protein mutant of rabies virus strain CVS-11 by ELISA analysis:
  • Figure 12A is an ELISA analysis of C34m and S2E3-scFv-Fc and fusion protein GCVS11-CCD-His binding capacity;
  • Figure 12B is an ELISA analysis of the binding capacity of C34m and S2E3-scFv-Fc and the fusion protein GCVS11-G229E-CCD-His;
  • Figure 12C is an ELISA analysis of C34m and S2E3-scFv-Fc and the fusion protein GCVS11-I338T-CCD-His binding capacity.
  • SEQ ID NO: 1 shows the amino acid sequence of the heavy chain variable region C34mVH of the antibody C34m.
  • SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region C11mVH of the antibody C11m.
  • SEQ ID NO: 3 shows the amino acid sequence of the light chain variable region C34mVK of the antibody C34m.
  • SEQ ID NO: 4 shows the amino acid sequence of the light chain variable region C11mVL of the antibody C11m.
  • SEQ ID NO: 5 shows the amino acid sequence of the human heavy chain constant region CH-IgG1.
  • SEQ ID NO: 6 shows the amino acid sequence of the human light chain constant region CK.
  • SEQ ID NO: 7 shows the amino acid sequence of the homosapiens light chain constant region CL.
  • SEQ ID NO: 8 shows the amino acid sequence of the single chain antibody C34m-scFv.
  • SEQ ID NO: 9 shows the amino acid sequence of the single chain antibody C11m-scFv.
  • SEQ ID NO: 10 shows the amino acid sequence of the Fc segment of human sapiens IgG1.
  • SEQ ID NO: 11 shows the amino acid sequence of the Fc segment of muskmus IgG2a.
  • SEQ ID NO: 12 and SEQ ID NO: 15 show the amino acid sequences of the heavy chain variable region CR57VH and the light chain variable region CR57VL of the monoclonal antibody CR57 against the rabies virus G protein epitope I, respectively.
  • SEQ ID NO: 13 and SEQ ID NO: 16 show the amino acid sequences of the heavy chain variable regions CR4098VH and CR4098VK of the monoclonal antibody CR4098 against the rabies virus G protein epitope III, respectively.
  • SEQ ID NO: 14 shows the amino acid sequence of the constant region of the musculus IgG2a heavy chain.
  • SEQ ID NO: 17 shows the amino acid sequence of the constant region mCL of the musculus light chain.
  • SEQ ID NO: 18 shows the amino acid sequence of the constant region mCK of the musculus light chain.
  • SEQ ID NO: 19 shows the amino acid sequence of an Fc segment (FcK) containing a Knob mutation.
  • SEQ ID NO: 20 shows the amino acid sequence of an Fc segment (FcH) containing a Hole mutation.
  • SEQ ID NO: 21 shows the amino acid sequence of the mutant S1C10-scFv of the single-chain antibody C11m-scFv.
  • SEQ ID NO: 22 shows the amino acid sequence of the mutant S2A1-scFv of the single-chain antibody C11m-scFv.
  • SEQ ID NO: 23 Amino acid sequence of mutant S2E3-scFv of single-chain antibody C11m-scFv.
  • SEQ ID Nos: 24 and 25 show the amino acid sequence of the heavy chain variable region and the light chain variable region of the single chain antibody C11m-scFv mutant S1C10-scFv, respectively.
  • SEQ ID Nos: 26 and 27 show the amino acid sequence of the heavy chain variable region and the light chain variable region of the single-chain antibody C11m-scFv mutant S2A1-scFv, respectively.
  • SEQ ID Nos: 28 and 29 show the amino acid sequence of the heavy chain variable region and the light chain variable region of the mutant S2E3-scFv of the single chain antibody C11m-scFv, respectively.
  • SEQ ID NO: 30 shows the amino acid sequence of C34mVK-CK.
  • SEQ ID NO: 31 shows the amino acid sequence of C34mVH-IgG1K.
  • SEQ ID NO: 32 shows the amino acid sequence of the single-chain antibody Fc fusion protein S2E3-scFv-FcH.
  • SEQ ID NO: 33 shows the amino acid sequence of the single-chain antibody Fc fusion protein S1C10-scFv-FcH.
  • SEQ ID NO: 34 shows the amino acid sequence of the single-chain antibody Fc fusion protein S2A1-scFv-FcH.
  • SEQ ID NO: 35 shows the amino acid sequence of the G protein GCVS11 of the wild-type Rabies virus strain CVS-11.
  • SEQ ID NO: 36 shows the amino acid sequence of the epitope I mutant GCVS11-G229E of the G protein GCVS11 of the rabies virus strain CVS-11.
  • SEQ ID NO: 37 shows the amino acid sequence of the epitope III mutant GCVS11-I338T of the G protein GCVS11 of the rabies virus strain CVS-11.
  • SEQ ID NO: 38 shows the amino acid sequence of the trimeric domain CCD of human coronary protein 1A.
  • SEQ ID NO: 39 shows the amino acid sequence of the fusion protein GCVS11-CCD-His.
  • SEQ ID NO: 40 shows the amino acid sequence of the fusion protein GCVS11-G229E-CCD-His.
  • SEQ ID NO: 41 shows the amino acid sequence of the fusion protein GCVS11-I338T-CCD-His.
  • a new bispecific antibody against rabies virus is provided, a polynucleotide encoding the bispecific antibody, a vector comprising the polynucleotide, comprising the polynucleotide or a vector.
  • Host cell, method for preparing and purifying the bispecific antibody, and medical and biological applications of the bispecific antibody According to the sequence of the variable region of the bispecific antibody provided in the present application, a full-length bispecific antibody molecule can be constructed as a drug for clinical use to prevent or treat rabies.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in a variable region of an immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only whole (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab ', F (ab') 2 , Fv), variants thereof, including antibody portions Fusion proteins, humanized antibodies, chimeric antibodies, diabody, linear antibodies, single chain antibodies, multispecific antibodies (e.g. bispecific antibodies), and any other immunoglobulin containing an antigen recognition site of the desired specificity Modified configuration of protein molecules, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • an intact or full-length antibody contains two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variation region (VH) and first, second, and third constant regions (CH1, CH2, and CH3).
  • Each light chain contains a light chain variation region (VL) and a constant region (CL).
  • a full-length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), but the antibody need not belong to any particular class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • bispecific antibody as used herein is an antibody that has the ability to bind two different antigens.
  • a bispecific antibody may consist of two Fc fragments and two antigen-binding portions, respectively, fused to it.
  • antigen-binding portion or “antigen-binding fragment” are used interchangeably and refer to a portion or region of an entire antibody molecule responsible for binding an antigen.
  • the antigen-binding domain may comprise a heavy chain variation region (VH), a light chain variation region (VL), or both.
  • VH and VL typically contains three complementarity determining regions CDR1, CDR2, and CDR3.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • CDR sequences of VH or VL namely Kabat definition and Chothia definition.
  • Kabat "Sequences of Proteins of Immunological Interface", National Institutes of Health, Bethesda, Md. (1991); A1-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86: 9268-9272 (1989)
  • the sequence of the CDR regions in the VH and VL sequences can be determined according to the Kabat definition or Chothia definition.
  • Kabat is used to define CDR sequences.
  • variable region sequence of a given antibody the CDR region sequence in the variable region sequence can be analyzed in various ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F (ab ') 2 fragment, It can be a bivalent fragment with two Fab 'fragments, which are connected by a disulfide bridge (ie, a dimer of Fab') of the hinge region; (3) VL and VH with one arm of the antibody Fv fragment of the domain; (4) single-chain Fv (scFv), which may be a single peptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may include Two VH domains connected by a peptide linker and two VL domains, which are combined with the two VH domains via a disulfide bridge.
  • a Fab fragment which may be a monovalent fragment having a VL-CL chain and
  • the "antigen-binding portion” includes, but is not limited to, the form of a Fab fragment or the form of a single chain antibody (scFv).
  • single chain antibody scFv, single chain fragment variable
  • VH heavy chain variable region
  • VL light chain variable region
  • a flexible linker is usually designed between the heavy and light chain variable regions so that the heavy and light chain variable regions can be folded into the correct conformation capable of binding the antigen.
  • Fab fragment antigen binding fragment
  • Fab portion refers to an antibody fragment capable of binding to an antigen produced by treating a whole antibody with papain, including a complete light chain (VL- CL), heavy chain variable region, and CH1 fragment (VH-CH1).
  • Monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that make up the population are the same except that naturally occurring mutations may be present in a small number of individuals.
  • Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is the same or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the heavy The remainder of the chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they exhibit the desired Biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
  • epitope is also called an antigenic determinant or an antigenic determinant (AD), and refers to a special chemical group in an antigen molecule that determines the specificity of an antigen.
  • Antigens bind to corresponding antigen receptors on the surface of lymphocytes through antigen epitopes, thereby activating lymphocytes and causing immune responses. Antigens also exert immune effects by specifically binding epitopes to corresponding antibodies or sensitized lymphocytes. The nature, number and spatial configuration of the epitope determine the specificity of the antigen.
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • R is A or G
  • Y is C or T
  • M is A or C
  • K is G or T
  • S is C or G
  • W is A or T
  • H is A or C or T
  • B is C or G or T
  • V for A or C or G
  • D for A or G or T
  • N for A or C or G or T.
  • the present application provides a bispecific antibody comprising two antigen-binding fragments that bind different epitopes of the G protein of rabies virus, and the bispecific antibody has activity to neutralize rabies virus.
  • one antigen-binding fragment of the bispecific antibody binds epitope I of the rabies virus G protein and the other antigen-binding fragment binds epitope III of the rabies virus G protein.
  • the antigen-binding fragment that binds epitope I of the rabies virus G protein comprises:
  • the amino acid sequence is HCDR1 of RYTIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDIGAYDYVS, the amino acid sequence is LCDR2 of DATKRPS, and the amino acid sequence is LCDR3 of CSYAGDYTPGVV; or
  • the amino acid sequence is HCDR1 of RYSIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDIDGYDFVS, the amino acid sequence is LCDR2 of DATKRPS, and the amino acid sequence is LCDR3 of CSYAGDYTPGVV; or
  • the amino acid sequence is HCDR1 of GYTIN, the amino acid sequence is HCDR2 of GIIPIFGTANYAQRFQG, the amino acid sequence is HCDR3 of ENLDNSGTYYYFSGWFDP, the amino acid sequence is LCDR1 of TGTSSDLGGYDFVS, the amino acid sequence is LCDR2 of DATKRPS, and the amino acid sequence is LCDR3 of CSYAGDYTPGVV;
  • amino acid sequences of HCDR and LCDR are defined according to Kabat.
  • the antigen-binding fragment that binds to epitope III of the rabies virus G protein comprises:
  • the amino acid sequence is HCDR1 of SYGMH, the amino acid sequence is HCDR2 of TISYDGSIKDYADSVKG, the amino acid sequence is HCDR3 of GDRTGNLDY, the amino acid sequence is LCDR1 of RASQNIRNALN, the amino acid sequence is LCDR2 of DASTRQS, and the amino acid sequence is LCDR3 of QQNSEFPPT;
  • amino acid sequences of HCDR and LCDR are defined according to Kabat.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope I of the rabies virus G protein is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.
  • amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the rabies virus G protein is shown in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope I of the rabies virus G protein is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID As shown in NO: 25, the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the rabies virus G protein is shown in SEQ ID: NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.
  • the amino acid sequence of the variable region of the heavy chain of the antigen-binding fragment that binds to the epitope III of the G protein of rabies virus is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 3; or
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope I of the G protein of rabies virus is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29.
  • the amino acid sequence of the heavy chain variable region of the antigen-binding fragment that binds to the epitope III of the G protein of rabies virus is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3.
  • the form of the two antigen-binding fragments is independently selected from a single chain antibody (scFv) or a Fab fragment.
  • the antigen-binding fragment that binds epitope I of the rabies virus G protein is a single chain antibody (scFv), and the antigen-binding fragment that binds epitope III of the rabies virus G protein is a Fab fragment.
  • the bispecific antibody comprises the amino acid sequence shown in one of SEQ ID NO: 32, 33, 34. In some embodiments, the bispecific antibody comprises the amino acid sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody according to the first aspect and a pharmaceutically acceptable excipient, diluent, or carrier.
  • the pharmaceutical composition is used to prevent or treat rabies.
  • the pharmaceutical composition may further include one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; Preservatives, such as benzoic acid, sorbic acid, and calcium propionate; sweeteners and / or flavoring agents.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifiers such as talc, magnesium stearate, and mineral oil
  • suspending agents such as benzoic acid, sorbic acid, and calcium propionate
  • Preservatives such as benzoic acid, sorbic acid, and calcium propionate
  • sweeteners and / or flavoring agents such as benzoic acid, sorbic acid, and calcium propionate
  • the pharmaceutical composition in the present application can be formulated in the form of tablets, pills, powders, dragees, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.
  • the pharmaceutical composition of the present application can be delivered using any physiologically acceptable mode of administration including, but not limited to: oral administration, parenteral administration, nasal administration, rectal administration Drugs, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use can be formulated in the form of a lyophilized preparation or an aqueous solution by mixing an agent having a desired purity with an optionally pharmaceutically acceptable carrier, excipient, etc. storage.
  • the application provides the use of the bispecific antibody according to the first aspect in the manufacture of a medicament for preventing or treating rabies.
  • the present application provides a method for preventing or treating rabies, comprising administering to a subject in need thereof the bispecific antibody according to the first aspect or the pharmaceutical composition according to the second aspect.
  • the application provides a nucleic acid molecule encoding a bispecific antibody according to the first aspect.
  • the nucleic acid molecule is operably linked to a regulatory sequence, which can be recognized by a host cell transformed with the vector.
  • the application also provides a vector comprising an isolated nucleic acid molecule encoding a bispecific antibody of the present application and a host cell comprising the nucleic acid molecule or vector.
  • the application also provides methods for producing the bispecific antibodies of the application.
  • a method of producing a bispecific antibody includes culturing a host cell to facilitate expression of a nucleic acid.
  • the method of producing a bispecific antibody further comprises recovering the bispecific antibody from the host cell culture medium.
  • Example 1 Preparation and confirmation of monoclonal antibodies against rabies virus G protein
  • the inventors screened and identified two humanized monoclonal antibodies with neutralizing activity using inactivated rabies virus as antigens, named C11m and C34m, respectively, and completed the heavy and light chains of two monoclonal antibodies C11m and C34m Sequence analysis of variable regions. First, based on the variable region sequences of C11m and C34m, fully human monoclonal antibodies C34m and C11m of rabies virus G protein were prepared, respectively.
  • the genes encoding the antibody heavy chain variable region C34mVH (SEQ ID NO: 1) and the antibody light chain variable region C34mVK (SEQ ID NO: 3) were cloned into a fusion encoding a human heavy chain constant region CH- Eukaryotic expression vectors of genes of IgG1 (SEQ ID NO: 5) and light chain constant region CK (SEQ ID NO: 6) (such as pcDNA3.1 of invitrogen company, etc.) to obtain C34m recombinant antibody expression vectors.
  • the genes encoding the antibody heavy chain variable region C11mVH (SEQ ID NO: 2) and the antibody light chain variable region C11mVL (SEQ ID NO: 4) were cloned into a fusion encoding the human heavy chain constant region CH-IgG1 ( SEQ ID NO: 5) and light chain constant region CL (SEQ ID No: 7) gene eukaryotic expression vector (such as pcDNA3.1 of invitrogen company, etc.) to obtain a C11m recombinant antibody expression vector.
  • liposomes such as invitrogen's 293fectin
  • other cationic transfection reagents such as PEI, etc.
  • a single-chain antibody Fc fusion protein (scFv-Fc) against rabies virus G protein was also prepared.
  • a flexible linker peptide GGGGSGGGGSGGGGS was added to the heavy chain variable region and light chain variable region of the monoclonal antibodies C34m and C11m, respectively, to construct a single-chain antibody C34m-scFv (SEQ ID ID NO based on VH-connected peptide-VK format).
  • the mFc recombinant antibody expression vector is transfected into HEK293 cells (such as HEK293F from invitrogen), cultured under serum-free suspension culture conditions for 3-5 days, and then the culture supernatant is harvested by centrifugation and other methods.
  • the culture supernatant of the human IgG1 fully human monoclonal antibody or single-chain antibody Fc fusion protein expressing anti-rabies virus G protein is purified in one step with a ProteinA / G affinity chromatography column (such as GE's MabselectSURE, etc.) . Then use a desalting column (such as Hitrap desaulting from GE) to replace the recombinant antibody storage buffer with PBS buffer (pH 7.0) or other suitable buffer. If necessary, the antibody sample can be sterilized by filtration, and then stored at -20 ° C.
  • a ProteinA / G affinity chromatography column such as GE's MabselectSURE, etc.
  • PBS buffer pH 7.0
  • Example 2 Verification of monoclonal antibodies binding to non-competitive epitopes of rabies virus G protein
  • a 96-well ELISA plate (1 IU / mL, 100 ⁇ L / well) was coated with the prepared inactivated rabies virus (prepared using MRC-5 cells) and coated at 4 ° C. overnight. After blocking with a blocking solution (2% milk-PBST buffer) at 37 ° C for 1 hour, add the equimolar concentration (200 nM) of a 3-fold gradient dilution (8 concentration points in total) prepared in Example 1 The source monoclonal antibodies C34m and C11m were bound for 1 hour at 37 ° C. The ELISA plate was washed with PBST buffer, HRP anti-human IgG (secondary antibody) was added, and bound for 1 hour at 37 ° C.
  • the genes encoding the heavy chain variable region and light chain variable region of the fully human monoclonal antibodies C34m and C11m were cloned into the two-vector presentation system pADK-S and pAG-S, respectively (for the experimental technical process, please refer to Chinese Patent Application No. 201510097117.0 Example 4.1) of No., phage-C34m and phage-C11m exhibiting a single kind of Fab were prepared and purified, and after titer measurement, they were frozen and stored at -20 ° C until use.
  • a 96-well ELISA plate (1 IU / mL, 100 ⁇ L / well) was coated with the prepared inactivated rabies virus (prepared using MRC-5 cells) and coated at 4 ° C. overnight.
  • HRP anti-M13-IgG (secondary antibody) was used to detect the inhibitory effects of fully human monoclonal antibodies C34m and C11m on the ability of phage-C34m and phage-C11m to bind to inactivated rabies virus, respectively.
  • the ELISA analysis results show that the fully human monoclonal antibody C11m can completely block the binding of phage-C11m and inactivated rabies virus, and the fully human monoclonal antibody C34m cannot completely block the phage-C11m and destroy Binding of live rabies virus; fully human monoclonal antibody C34m can completely block the binding of phage-C34m to inactivated rabies virus, and fully human monoclonal antibody C11m cannot block the binding of phage-C34m to inactivated rabies virus, indicating that The fully human monoclonal antibodies C34m and C11m possess non-competitive binding epitopes to the rabies virus venom G protein.
  • the heavy chain variable region CR57VH (SEQ ID NO: 12) encoding the monoclonal antibody CR57 against the rabies virus G protein epitope I and the heavy chain variable region of the monoclonal antibody CR4098 against the rabies virus G protein epitope III were cloned, respectively.
  • CR4098VH (SEQ ID ID NO: 13) gene (Bakker, ABetal. Novel, human, monoclonal, antibody combination, effective, neutralizing, natural, rabies, virus, variants, and individual diversity in vitro, introspective, vitro, escape, mutants. J. Virol.
  • a eukaryotic expression vector such as pcDNA3.1 of invitrogen company fused to a gene encoding a murine IgG2a heavy chain constant region (CH-mIgG2a, SEQ ID NO: 14).
  • the gene encoding the light chain variable region CR57VL (SEQ ID NO: 15) of CR57 was cloned into a eukaryotic expression vector (e.g., pcDNA3 of invitrogen company) fused with the gene encoding the murine light chain constant region mCL (SEQ ID NO: 17).
  • a 96-well ELISA plate (1 IU / mL, 100 ⁇ L / well) was coated with the prepared inactivated rabies virus (prepared using MRC-5 cells) and coated at 4 ° C. overnight.
  • the chimeric antibodies CR57-mIgG2a and CR4098-mIgG2a at a fixed concentration (2.5 ⁇ g / mL) were used to perform a three-fold gradient (equal to 12 concentration points) on all human monoclonal antibodies C11m and C34m at an equimolar concentration (200 nM).
  • Dilute add 100 ⁇ L / well to a 96-well plate, and incubate at 37 ° C for 1 hour.
  • HRP anti-mouse IgG (secondary antibody) was used to detect the inhibitory effects of the fully human monoclonal antibodies C11m and C34m on the ability of the chimeric antibodies CR57-mIgG2a and CR4098-mIgG2a to bind to inactivated rabies virus, respectively.
  • the ELISA analysis results ( Figure 4, Figure 5) show that the fully human monoclonal antibody C11m competes with the chimeric antibody CR57-mIgG2a to bind the rabies virus G protein, but does not compete with the chimeric antibody CR4098-mIgG2a to bind the rabies virus G protein.
  • IEC Ion exchange chromatography
  • Example 1 The gene encoding the single-chain antibody C11m-scFv prepared in Example 1 was cloned into the vector pADscFv-s (for the experimental technical procedure, please refer to Example 1.3 in Chinese Patent Application No. 201510097117.0) to obtain a recombinant expression vector pADscFv-C11m- scFv.
  • pADscFv-C11m-scFv Using overlap extension PCR (overlap PCR) technology, mutations were introduced into the CDR region of the recombinant expression vector pADscFv-C11m-scFv, and a C11m-scFv mutant library with a library capacity exceeding 4.6 ⁇ 10E6 was constructed, in which the key primers needed for amplification were amplified As shown in Table 1.
  • the single-chain antibody Fc fusion protein (scFv-Fc) of the three mutants fused to the Fc segment of human IgG1 was prepared: S1C10-scFv-Fc, S2A1-scFv-Fc, and S2E3-scFv-Fc .
  • a C34m chimeric antibody C34m-mIgG2a fused with murine IgG2a heavy chain constant region (CH-mIgG2a) and light chain constant region (mCK) was prepared, and a murine IgG2a heavy chain constant region (CH-mIgG2a) and light chain constant were fused Region (mCL) C11m chimeric antibody C11m-mIgG2a for functional analysis.
  • the single-chain antibody Fc fusion proteins of the three C11m-scFv mutants were respectively tested for their ability to bind the rabies virus G protein and non-competitive epitopes.
  • G protein binding capacity is equivalent; C11m-scFv-Fc single chain antibody Fc fusion protein and three C11m-scFv mutant single chain antibody Fc fusion protein and C34m-mIgG2a non-competitive binding to rabies virus G protein epitope; C11m- The scFv-Fc single-chain antibody Fc fusion protein and three C11m-scFv mutant single-chain antibody Fc fusion proteins and C11m-mIgG2a competitively bind to the rabies virus venom G protein epitope.
  • Antibody name pI value C11m-scFv 7.38 S1C10-scFv 7.13 S2A1-scFv 6.90 S2E3-scFv 6.90
  • the antigen-binding fragments of epitope I and epitope III of the rabies virus G protein were designed into scFv format and Fab format, respectively.
  • a human IgG1 heterodimer based on KIH (Knob-Into-Hole) technology was constructed.
  • the C34m antigen-binding fragment was fused to the N-terminus of the Knob mutation-containing Fc segment (FcK, SEQ ID NO: 19), and the scFv-form C11m mutant antigen-binding fragment (scFv) was fused to the Hole mutation-containing Fc segment (FcH, SEQ ID NO: 20) N-terminus, to construct a bispecific antibody based on the G protein of rabies virus.
  • Three eukaryotic expression vectors expressing S1C10-scFv-FcH, C34mVH-IgG1K, and C34mVK-CK were co-transfected into HEK293F cells with liposomes and cultured in serum-free suspension culture for 3-5 days. , And then harvest the culture supernatant by centrifugation and other methods.
  • the bispecific antibody in the culture supernatant is purified with a ProteinA / G affinity chromatography column (such as GE's Mabselect SURE, etc.), and then the desalting column (such as GE's Hitrap, desaulting, etc.) is used to replace the recombinant antibody storage buffer.
  • the desalted protein solution was purified by size exclusion chromatography (SEC) using Superdex200 (GE) to obtain the bispecific antibody S1C10-scFv-FcH + C34m-IgG1K. If necessary, the antibody sample can be filtered and sterilized, and then stored at -20 ° C.
  • the prepared inactivated rabies virus (prepared using MRC-5 cells) was coated on a 96-well ELISA plate (1 IU / mL, 100 ⁇ L / well) and coated overnight at 4 ° C. After blocking with blocking solution (2% milk-PBST buffer) for 1 hour at 37 ° C, anti-rabies virus G-protein double specificity was added with a 3-fold gradient dilution (12 concentration points) of equal molar concentration (200 nM).
  • Antibodies (S1C10-scFv-FcH + C34m-IgG1K, S2A1-scFv-FcH + C34m-IgG1K, S2E3-scFv-FcH + C34m-IgG1K, S1C10-scFv-Fc, S2A1-scFv-Fc, S2E3-scFv-Fc , C11m-scFv-Fc and C34m), binding for 1 hour at 37 ° C.
  • the ELISA plate was washed with PBST buffer, HRP anti-human IgG (secondary antibody) was added, and bound for 1 hour at 37 ° C.
  • the single-chain antibody S2E3-scFv with a lower pI value was selected, and the chimeric protein S2E3-scFv-mFc was constructed according to the method in Example 1.
  • C34m-mIgG2a and S2E3-scFv- were used at a fixed concentration (2.5 ⁇ g / mL), respectively.
  • mFc was subjected to 3-fold gradient dilution of bispecific antibody S2E3-scFv-FcH + C34m-IgG1K, single chain antibody Fc fusion protein S2E3-scFv-Fc, and fully human monoclonal antibody C34m at an equimolar concentration (200 nM).
  • Example 7 Determination of antibody titer based on anti-rabies virus neutralizing antibody rapid fluorescent focus inhibition test (RFFIT)
  • RFFIT Rapid Fluorescent Focus Inhibition Test
  • wells without standard serum or test antibody were set as virus dilution control wells, and wells without standard serum or test antibody and rabies virus were set as cell control wells.
  • Incubate at 37 ° C for 1 hour add 50 ⁇ L BSR cells (1 ⁇ 10E6 cells / ml) to each well, and culture in 37 ° C, 5% CO 2 incubator. After 24 hours, 50 ⁇ L of cold acetone was added for 30 minutes, and then washed with PBST buffer. Add 50 ⁇ L rabies virus fluorescent antibody working solution to each well and incubate at 37 ° C for 30 min. Wash with PBS buffer, add 80% glycerol to each well, perform fluorescence staining detection under an inverted fluorescence microscope, and calculate the titer of the sample to be tested according to the Reed-Muench method.
  • the rabies virus standard attack strain CVS-11 was used to detect the three antibodies, and the experimental results are shown in Table 3. The results indicate that the bispecific antibody S2E3-scFv-FcH + C34m-IgG1K, the single-chain antibody Fc fusion protein S2E3-scFv-Fc, and the fully human monoclonal antibody C34m all have good neutralizing activity.
  • Example 8 Anti-rabies virus antibody protects golden hamsters from lethal rabies virus attack
  • the protective effect of each group of antibodies was determined by immunoprotection of golden hamsters injected with a lethal dose of rabies virus after immunoglobulin exposure and comparison with commercial products.
  • the golden hind hamster was challenged on the right hind leg (0.2 ml / head) with a rabies virus standard challenge strain CVS-11 of 6.67 lg LD50 / mL.
  • Anti-rabies virus antibodies were given at the same site 24 hours later.
  • the bispecific antibodies S2E3-scFv-FcH + C34m-IgG1K are divided into three groups according to the injection doses 5IU / kg, 20IU / kg, and 30IU / kg; the single-chain antibody Fc is fused separately.
  • Each group was divided into two groups; golden hamsters without injection of anti-rabies virus antibody were used as a control group, a total of 10 groups, 9 per group. Dilute each sample with PBS buffer according to the golden hamster weighing results.
  • the final injection volume of each animal on the ipsilateral side is 100 ⁇ L of sample.
  • the present application selects a single point mutation to perform amino acid mutations on the neutralizing epitopes I and epitopes III of the wild-type G protein of the rabies virus strain CVS-11 to obtain the amino acid sequence of the corresponding escape strain (Novelabies virus-neutralizing epitope recognition) human monoclonal antibody: fine mapping and mutant analysis; Rabies virus: effect on pathogenicity and sequence characterization of rabies virus mutations affectionation antigenic effect site III of the glycoprotein).
  • the obtained fusion protein genes encoding the rabies virus G protein and the two mutants (G229E and I338T) extracellular region and CCD were cloned into eukaryotic expression vectors (e.g., invitrogen company pcDNA3.1, etc.) fused with a His tag at the C-terminus. ), And then use liposomes (such as 293fectin from invitrogen) to transfect the prepared recombinant antigen expression vectors into HEK293 cells, culture them under serum-free suspension culture conditions for 3-5 days, and centrifuge to harvest the culture supernatant. The obtained culture supernatant was concentrated about 10 times with an ultrafiltration centrifuge tube, and then left at -80 ° C until use.
  • eukaryotic expression vectors e.g., invitrogen company pcDNA3.1, etc.
  • liposomes such as 293fectin from invitrogen
  • a 96-well ELISA plate (5 ⁇ g / mL, 100 ⁇ L / well) was coated with the prepared fully human monoclonal antibody C34m and the single-chain antibody Fc fusion protein S2E3-scFv-Fc, respectively, and coated overnight at 4 ° C.
  • the obtained fusion proteins GCVS11-CCD-His (SEQ ID NO: 39), GCVS11-G229E-CCD-His (SEQ ID NO: 40), GCVS11-I338T-CCD-His (SEQ ID NO: 41) concentrated solution was diluted 3 times (a total of 11 concentration gradients), and 100 ⁇ L / well was added to the coated with C34m and S2E3-scFv-Fc In a 96-well plate, incubate at 37 ° C for 1 hour. The binding signal was detected using an HRP-labeled anti-His-tagged antibody (secondary antibody).

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Abstract

本申请提供了针对狂犬病病毒的双特异性抗体及其用途。所述双特异性抗体包含结合狂犬病病毒G蛋白不同表位的两个抗原结合片段，并且具有中和狂犬病病毒的活性。

Description

针对狂犬病病毒的双特异性抗体及其用途
相关申请的交叉引用
本申请要求于2018年8月9日递交的中国专利申请第201810901518.0号的优先权，其全部内容通过引用整体并入本文。
技术领域
本申请大体涉及基因工程和抗体药物领域；具体而言，本申请涉及针对狂犬病病毒的双特异性抗体及其用途。
背景技术
狂犬病是狂犬病病毒所致的急性传染病，人兽共患，主要在犬、狼、猫等动物中传播，但是其他哺乳动物如浣熊、臭鼬、蝙蝠和狐狸也是常见的宿主。动物通过互相间的撕咬而传播病毒，人多因被病兽咬伤而感染。目前对于狂犬病尚缺乏有效的治疗手段，人患狂犬病后的病死率几近100％，患者一般在3-6日内因呼吸或循环衰竭而死亡。估计全球每年有超过70000人因该疾病死亡，数百万人需要暴露后治疗。
狂犬病病毒是一种子弹形状的、有包膜的、单链RNA病毒，属于弹状病毒科，狂犬病病毒属。狂犬病病毒的基因组编码5种病毒蛋白质：RNA依赖性RNA聚合酶(L)、核蛋白(N)、磷酸化蛋白质(P)、位于病毒蛋白包膜内侧的基质蛋白(M)及外表面糖蛋白(G)。狂犬病病毒的糖蛋白(G蛋白)能与乙酰胆碱结合，决定了狂犬病病毒的噬神经性。G蛋白(62-67kDa)是由505个氨基酸组成的一种I型糖蛋白，G蛋白形成覆盖病毒粒包膜外表面的突起，研究证实G蛋白能够诱导病毒中和抗体。G蛋白至少有5个中和表位，其中表位II是不连续空间表位，包括34-42位氨基酸残基和198-200位氨基酸残基，表位III位于330-338位，是线性表位，报道的抗体中约97％识别的都是表位II和表位III，狂犬病病毒中和抗体CR4098结合表位III。识别表位I和表位IV的抗体很少，狂犬病病毒中和抗体CR57识别线性表位I，即218-240位，核心结合域是226-231位的KLCGVL。表位IV含251和264位残基。还有一个表位是与表位 III间隔3个氨基酸残基与表位III不重叠的微表位a，只有两个氨基酸残基342-343。
针对狂犬病病毒的预防和治疗，WHO建议：III类暴露以及野生动物咬伤的II类以上的暴露，应同时进行主动和被动免疫治疗，以获得快速的保护作用。当前用于主动治疗的狂犬病病毒疫苗的研发比较成熟，且在国内外都有多种狂犬病病毒疫苗上市，用于狂犬病病毒的主动预防。而用于狂犬病病毒暴露后的被动治疗的药物主要是人狂犬病免疫球蛋白(human rabies immune globulin，HRIG)和马狂犬病免疫球蛋白(Equine rabies immune globulin，ERIG)。ERIG由于对人体是异源蛋白，有时副反应比较严重，而HRIG价格昂贵，供应量有限并且有潜在的血源性病原体污染的风险，因此临床上需要开发新型的狂犬病病毒被动治疗药物。
1989年，Schumacherl等人制备了多株针对狂犬病病毒糖蛋白、核蛋白的鼠源单抗，这些用称之为鸡尾酒单克隆抗体疗法，对小鼠及地鼠的保护性实验表明：该方法不仅具有被动免疫后完全抵抗致死剂量狂犬病病毒的攻击能力，还具有暴露后保护作用。1990年Bernhard Dietzschold等人利用细胞融合技术制备了多株人源抗狂犬病病毒单克隆抗体，其中Mab57表现出对狂犬病病毒糖蛋白较高的亲和力，可广泛中和狂犬病病毒并对狂犬病病毒攻击的小鼠起到保护作用。在获得抗狂犬病病毒抗体基因后，使得利用生物反应器来制备人源抗狂犬病病毒单克隆抗体成为可能。在2005年，Goudsmit等人和Bakker研究小组报道了两株人源性抗狂犬病病毒G蛋白的单抗CR57和CR4098。直接混合使用这两株单抗，并与HRIG进行比较，结果表明，混合单抗与HRIG在暴露后预防能力相当，且与多株狂犬病病毒有良好的交叉反应性，证明了重组人源抗狂犬病病毒单抗的实际可用性，甚至可以替代RIG。目前基于两种单抗的药物CL184已经在开展临床研究，并且在临床试验时未发现任何副作用，安全有效，临床上可能代替RIG用于暴露后预防。但正在临床研究的CL184是基于两种单抗的混合制剂，制备成本较高。
新的针对狂犬病病毒G蛋白的双特异抗体的开发和应用是本领域所需要的。
发明概述
第一方面，本申请提供了双特异性抗体，其包含结合狂犬病病毒G蛋白不同表位的两个抗原结合片段，并且所述双特异性抗体具有中和狂犬病病毒的活性。
在一些实施方案中，双特异性抗体中的一个抗原结合片段结合狂犬病病毒G蛋白的表位I，另一个抗原结合片段结合狂犬病病毒G蛋白的表位III。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段包含：
氨基酸序列为RYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIGAYDYVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
氨基酸序列为RYSIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIDGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
氨基酸序列为GYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDLGGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；
其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段包含：
氨基酸序列为SYGMH的HCDR1，氨基酸序列为TISYDGSIKDYADSVKG的HCDR2，氨基酸序列为GDRTGNLDY的HCDR3，氨基酸序列为RASQNIRNALN的LCDR1，氨基酸序列为 DASTRQS的LCDR2，氨基酸序列为QQNSEFPPT的LCDR3；
其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：24所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：26所示，轻链可变区的氨基酸序列如SEQ ID NO：27所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：28所示，轻链可变区的氨基酸序列如SEQ ID NO：29所示。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：24所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：26所示，轻链可变区的氨基酸序列如SEQ ID NO：27所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：28所示，轻链可变区的氨基酸序列如SEQ ID NO：29所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示。
在一些实施方案中，两个抗原结合片段的形式独立地选自单链抗体 (scFv)或Fab片段。
在一些实施方案中，结合狂犬病病毒G蛋白的表位I的抗原结合片段为单链抗体(scFv)，结合狂犬病病毒G蛋白的表位III的抗原结合片段为Fab片段。在一些实施方案中，所述双特异性抗体包含SEQ ID NO:32、33、34之一所示的氨基酸序列。在一些实施方案中，所述双特异性抗体包含SEQ ID NO:30和SEQ ID NO:31所示的氨基酸序列。
第二方面，本申请提供了药物组合物，其包含第一方面所述的双特异性抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于预防或治疗狂犬病。
第三方面，本申请提供了第一方面所述的双特异性抗体在制备用于预防或治疗狂犬病的药物中的用途。
第四方面，本申请提供了预防或治疗狂犬病的方法，包括向有需要的个体给予第一方面所述的双特异性抗体或第二方面所述的药物组合物。
附图说明
图1显示ELISA分析全人源单克隆抗体C34m和C11m与灭活狂犬病病毒的结合能力。
图2显示ELISA分析全人源单克隆抗体C34m和C11m对噬菌体-C11m与灭活狂犬病病毒结合的抑制作用。
图3显示ELISA分析全人源单克隆抗体C34m和C11m对噬菌体-C34m与灭活狂犬病病毒结合的抑制作用。
图4显示ELISA分析全人源单克隆抗体C11m和C34m对嵌合抗体CR57-mIgG2a与灭活狂犬病病毒结合的抑制作用。
图5显示ELISA分析全人源单克隆抗体C11m和C34m对嵌合抗体CR4098-mIgG2a与灭活狂犬病病毒结合的抑制作用。
图6显示ELISA分析C11m-scFv-Fc单链抗体Fc融合蛋白、3个C11m-scFv突变体的单链抗体Fc融合蛋白以及全人源单克隆抗体C34m与灭活狂犬病病毒的结合能力。
图7显示ELISA分析C11m-scFv-Fc单链抗体Fc融合蛋白和3个 C11m-scFv突变体的单链抗体Fc融合蛋白对嵌合抗体C34m-mIgG2a与灭活狂犬病病毒结合的抑制作用。
图8显示ELISA分析C11m-scFv-Fc单链抗体Fc融合蛋白和3个C11m-scFv突变体的单链抗体Fc融合蛋白对嵌合抗体C11m-mIgG2a与灭活狂犬病病毒结合的抑制作用。
图9显示ELISA分析多种抗狂犬病病毒G蛋白抗体与灭活狂犬病病毒的结合能力。
图10显示ELISA分析双特异性抗体S2E3-scFv-FcH+C34m-IgG1K、单链抗体Fc融合蛋白S2E3-scFv-Fc和全人源单克隆抗体C34m对嵌合抗体C34m-mIgG2a与灭活狂犬病病毒结合的抑制作用。
图11显示ELISA分析双特异性抗体S2E3-scFv-FcH+C34m-IgG1K、单链抗体Fc融合蛋白S2E3-scFv-Fc和全人源单克隆抗体C34m对嵌合蛋白S2E3-scFv-mFc与灭活狂犬病病毒结合的抑制作用。
图12显示ELISA分析抗狂犬病病毒G蛋白抗体C34m和S2E3-scFv-Fc与狂犬病病毒毒株CVS-11的G蛋白突变体的结合能力：图12A为ELISA分析C34m和S2E3-scFv-Fc与融合蛋白GCVS11-CCD-His的结合能力；图12B为ELISA分析C34m和S2E3-scFv-Fc与融合蛋白GCVS11-G229E-CCD-His的结合能力；图12C为ELISA分析C34m和S2E3-scFv-Fc与融合蛋白GCVS11-I338T-CCD-His的结合能力。
序列说明
SEQ ID NO：1显示抗体C34m的重链可变区C34mVH的氨基酸序列。
SEQ ID NO：2显示抗体C11m的重链可变区C11mVH的氨基酸序列。
SEQ ID NO：3显示抗体C34m的轻链可变区C34mVK的氨基酸序列。
SEQ ID NO：4显示抗体C11m的轻链可变区C11mVL的氨基酸序列。
SEQ ID NO：5显示人(homo sapiens)重链恒定区CH-IgG1的氨基酸序列。
SEQ ID NO：6显示人(homo sapiens)轻链恒定区CK的氨基酸序列。
SEQ ID NO：7显示人(homo sapiens)轻链恒定区CL的氨基酸序列。
SEQ ID NO：8显示单链抗体C34m-scFv的氨基酸序列。
SEQ ID NO：9显示单链抗体C11m-scFv的氨基酸序列。
SEQ ID NO：10显示人(homo sapiens)IgG1的Fc段的氨基酸序列。
SEQ ID NO：11显示鼠(mus musculus)IgG2a的Fc段的氨基酸序列。
SEQ ID NO：12和SEQ ID NO：15分别显示针对狂犬病病毒G蛋白表位I的单克隆抗体CR57的重链可变区CR57VH和轻链可变区CR57VL氨基酸序列。
SEQ ID NO：13和SEQ ID NO：16分别显示针对狂犬病病毒G蛋白表位III的单克隆抗体CR4098的重链可变区CR4098VH和CR4098VK的氨基酸序列。
SEQ ID NO：14显示鼠(mus musculus)IgG2a重链恒定区的氨基酸序列。
SEQ ID NO：17显示鼠(mus musculus)轻链恒定区mCL的氨基酸序列。
SEQ ID NO：18显示鼠(mus musculus)轻链恒定区mCK的氨基酸序列。
SEQ ID NO：19显示含有Knob突变的Fc段(FcK)的氨基酸序列。
SEQ ID NO：20显示含有Hole突变的Fc段(FcH)的氨基酸序列。
SEQ ID NO：21显示单链抗体C11m-scFv的突变体S1C10-scFv的氨基酸序列。
SEQ ID NO：22显示单链抗体C11m-scFv的突变体S2A1-scFv的氨基酸序列。
SEQ ID NO：23单链抗体C11m-scFv的突变体S2E3-scFv的氨基酸序列。
SEQ ID NO：24和25分别显示单链抗体C11m-scFv的突变体S1C10-scFv的重链可变区的氨基酸序列和轻链可变区的氨基酸序列。
SEQ ID NO：26和27分别显示单链抗体C11m-scFv的突变体S2A1-scFv的重链可变区的氨基酸序列和轻链可变区的氨基酸序列。
SEQ ID NO：28和29分别显示单链抗体C11m-scFv的突变体S2E3-scFv的重链可变区的氨基酸序列和轻链可变区的氨基酸序列。
SEQ ID NO：30显示C34mVK-CK的氨基酸序列。
SEQ ID NO：31显示C34mVH-IgG1K的氨基酸序列。
SEQ ID NO：32显示单链抗体Fc融合蛋白S2E3-scFv-FcH的氨基酸序列。
SEQ ID NO：33显示单链抗体Fc融合蛋白S1C10-scFv-FcH的氨基酸序列。
SEQ ID NO：34显示单链抗体Fc融合蛋白S2A1-scFv-FcH的氨基酸序列。
SEQ ID NO：35显示野生型狂犬病病毒(Rabies virus)毒株CVS-11的G蛋白GCVS11的氨基酸序列。
SEQ ID NO：36显示狂犬病病毒毒株CVS-11的G蛋白GCVS11的表位I突变体GCVS11-G229E的氨基酸序列。
SEQ ID NO：37显示狂犬病病毒毒株CVS-11的G蛋白GCVS11的表位III突变体GCVS11-I338T的氨基酸序列。
SEQ ID NO：38显示人(homo sapiens)冠状蛋白1A的三聚体结构域CCD的氨基酸序列。
SEQ ID NO：39显示融合蛋白GCVS11-CCD-His的氨基酸序列。
SEQ ID NO：40显示融合蛋白GCVS11-G229E-CCD-His的氨基酸序列。
SEQ ID NO：41显示融合蛋白GCVS11-I338T-CCD-His的氨基酸序列。
发明详细描述
本申请的发明人通过抗体工程技术得到了新的针对狂犬病病毒的双特异性抗体。在本申请的多个方面，提供了新的针对狂犬病病毒的双特异性抗体，编码所述双特异性抗体的多核苷酸、包含所述多核苷酸的载 体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该双特异性抗体的方法及所述双特异性抗体的医学和生物学应用。根据本申请提供的双特异性抗体的可变区的序列，可构建全长的双特异性抗体分子作为药物在临床上用于预防或治疗狂犬病。
除非另外指明，本申请的实施采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab') 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“双特异性抗体”是具有结合两种不同抗原能力的抗 体。例如，双特异性抗体可以由两个Fc片段以及分别与其融合的两个抗原结合部分组成。
如本文所用术语“抗原结合部分”或“抗原结合片段”可互换使用，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
对于一般抗体而言，抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv) 2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
在双特异性抗体构建中，“抗原结合部分”包括但不限于Fab片段的形式或单链抗体(scFv)的形式。
如本文所用术语“单链抗体(scFv,single chain fragment variable)”是指一般利用基因工程技术构建的单链结构的抗体，包含重链可变区 (VH)和轻链可变区(VL)的一条多肽链。在重链可变区和轻链可变区之间通常会设计一段柔性的连接肽(linker)以便重链可变区和轻链可变区可以折叠成为能够结合抗原的正确构象。
如本文所用术语“Fab(fragment antigen binding)片段”、“Fab部分”或类似的术语是指完整的抗体用木瓜蛋白酶处理后产生的能够与抗原结合的抗体片段，包括完整的轻链(VL-CL)、重链可变区和CH1片段(VH-CH1)。
如本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(美国专利号4,816,567；和Morrison等人，Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
如本文所用术语“表位”又称抗原决定簇或抗原决定基(antigenic determinant，AD)，是指抗原分子中决定抗原特异性的特殊化学基团。抗原通过抗原表位与相应的淋巴细胞表面的抗原受体结合，从而激活淋巴细胞，引起免疫应答；抗原也借表位与相应抗体或致敏淋巴细胞发生特异性结合而发挥免疫效应。抗原表位的性质、数目和空间构型决定抗原的特异性。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
在本文给出的核酸序列中涉及兼并碱基(除了A、T、C、G常规碱基之外)的使用，其含义与本领域技术人员通常理解的相同。例如，R代表A或G；Y代表C或T，M代表A或C；K代表G或T；S代表C或G；W代表A或T；H代表A或C或T；B代表C或G或T；V代表A或C或G；D代表A或G或T；N代表A或C或G或T。
第一方面，本申请提供了双特异性抗体，其包含结合狂犬病病毒G 蛋白不同表位的两个抗原结合片段，并且所述双特异性抗体具有中和狂犬病病毒的活性。
在一些实施方案中，双特异性抗体中的一个抗原结合片段结合狂犬病病毒G蛋白的表位I，另一个抗原结合片段结合狂犬病病毒G蛋白的表位III。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段包含：
氨基酸序列为RYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIGAYDYVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
氨基酸序列为RYSIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIDGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
氨基酸序列为GYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDLGGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；
其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段包含：
氨基酸序列为SYGMH的HCDR1，氨基酸序列为TISYDGSIKDYADSVKG的HCDR2，氨基酸序列为GDRTGNLDY的HCDR3，氨基酸序列为RASQNIRNALN的LCDR1，氨基酸序列为DASTRQS的LCDR2，氨基酸序列为QQNSEFPPT的LCDR3；
其中，HCDR和LCDR氨基酸序列根据Kabat定义。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：24所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：26所示，轻链可变区的氨基酸序列如SEQ ID NO：27所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：28所示，轻链可变区的氨基酸序列如SEQ ID NO：29所示。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示。
在一些实施方案中，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：24所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：26所示，轻链可变区的氨基酸序列如SEQ ID NO：27所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示；或者
所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：28所示，轻链可变区的氨基酸序列如SEQ ID NO：29所示，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示。
在一些实施方案中，两个抗原结合片段的形式独立地选自单链抗体(scFv)或Fab片段。
在一些实施方案中，结合狂犬病病毒G蛋白的表位I的抗原结合片段 为单链抗体(scFv)，结合狂犬病病毒G蛋白的表位III的抗原结合片段为Fab片段。在一些实施方案中，所述双特异性抗体包含SEQ ID NO:32、33、34之一所示的氨基酸序列。在一些实施方案中，所述双特异性抗体包含SEQ ID NO:30和SEQ ID NO:31所示的氨基酸序列。
第二方面，本申请提供了药物组合物，其包含第一方面所述的双特异性抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于预防或治疗狂犬病。
在一些实施方案中，所述药物组合物还可包含下述物质中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第三方面，本申请提供了第一方面所述的双特异性抗体在制备用于预防或治疗狂犬病的药物中的用途。
第四方面，本申请提供了预防或治疗狂犬病的方法，包括向有需要的个体给予第一方面所述的双特异性抗体或第二方面所述的药物组合物。
在其他方面，本申请提供了核酸分子，其编码第一方面所述的双特异性抗体。在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
本申请还提供包含编码本申请双特异性抗体的分离的核酸分子的载体以及包含所述核酸分子或载体的宿主细胞。
在其他方面，本申请还提供产生本申请双特异性抗体的方法。在一些实施方案中，产生双特异性抗体的方法包括培养宿主细胞以便于表达核酸。在一些实施方案中，产生双特异性抗体的方法还包括从宿主细胞培养基中回收双特异性抗体。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1：抗狂犬病病毒G蛋白单克隆抗体的制备和确认
发明人以灭活的狂犬病病毒为抗原筛选并鉴定出两个具有中和活性的人源性单克隆抗体，分别命名为C11m和C34m，并完成两个单抗C11m和C34m的重链和轻链可变区的序列分析。首先根据C11m和C34m的可变区序列，分别制备抗狂犬病病毒G蛋白全人源单克隆抗体C34m和C11m。具体而言，分别将编码抗体重链可变区C34mVH(SEQ ID NO：1)和抗体轻链可变区C34mVK(SEQ ID NO：3)的基因克隆至融合有编码人重链恒定区CH-IgG1(SEQ ID NO：5)和轻链恒定区CK(SEQ ID NO：6)的基因的真核表达载体(如invitrogen公司的pcDNA3.1等)，得到C34m重组抗体表达载体。另外，分别将编码抗体重链可变区C11mVH(SEQ ID NO：2)和抗体轻链可变区C11mVL(SEQ ID NO：4)的基因克隆至融合有编码人重链恒定区CH-IgG1(SEQ ID NO：5)和轻链恒定区CL(SEQ ID NO：7)的基因真核表达载体(如invitrogen公司的pcDNA3.1等)，得到C11m重组抗体表达载体。然后利用脂质体(如invitrogen公司的293fectin)或其他阳离子转染试剂(如PEI等)分别将制备的C34m重组抗体表达载体和C11m重组抗体表达载体转染入HEK293细胞(如invitrogen公司的HEK293F细胞)，在无血清悬浮培养条件下培养3-5天，然后通过离心等方式收获培养上清。
此外，还制备了抗狂犬病病毒G蛋白的单链抗体Fc融合蛋白 (scFv-Fc)。具体而言，分别在单克隆抗体C34m和C11m的重链可变区和轻链可变区间添加柔性连接肽GGGGSGGGGSGGGGS，构建基于VH-连接肽-VK形式的单链抗体C34m-scFv(SEQ ID NO：8)和C11m-scFv(SEQ ID NO：9)，然后分别将编码单链抗体C34m-scFv和C11m-scFv的基因克隆至融合有编码人IgG1的Fc段(Fc，SEQ ID NO：10)或鼠IgG2a的Fc段(mFc，SEQ ID NO：11)的基因的真核表达载体(如invitrogen公司的pcDNA3.1等)，得到单链抗体C34m-scFv-Fc/mFc重组抗体表达载体和单链抗体C11m-scFv-Fc/mFc重组抗体表达载体。利用脂质体(如invitrogen公司的293fectin)或其他阳离子转染试剂(如PEI等)分别将制备的单链抗体C34m-scFv-Fc/mFc重组抗体表达载体和单链抗体C11m-scFv-Fc/mFc重组抗体表达载体转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天，然后通过离心等方式收获培养上清。
将收获的表达抗狂犬病病毒G蛋白的人IgG1全人源单克隆抗体或单链抗体Fc融合蛋白的培养上清用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组抗体保存缓冲液置换为PBS缓冲液(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2：单克隆抗体结合狂犬病病毒G蛋白非竞争性表位的验证
使用制备的灭活狂犬病病毒(采用MRC-5细胞制备)包被96孔ELISA板(1IU/mL，100μL/孔)，4℃包被过夜。利用封闭液(2％牛奶-PBST缓冲液)在37℃封闭1小时后，分别加入等摩尔浓度起始(200nM)3倍梯度稀释(共8个浓度点)的实施例1中制备的全人源单克隆抗体C34m和C11m，37℃结合1小时。用PBST缓冲液洗涤ELISA板，加入HRP抗人IgG(二抗)，37℃结合1小时。然后用PBST缓冲液洗涤ELISA板，并加入OPD底物显色液，5-10分钟后用1M的H 2SO 4溶液终止显色，使用酶标仪在490nm/630nm双波长测定光密度值。 ELISA分析结果(图1)显示，全人源单克隆抗体C34m和C11m均能与包被的灭活狂犬病病毒结合，且结合能力相当。
分别将编码全人源单克隆抗体C34m和C11m的重链可变区和轻链可变区的基因克隆至双载体呈现系统pADK-S和pAG-S(实验技术流程可参见中国专利申请第201510097117.0号中的实施例4.1)，制备并纯化呈现单一种类Fab的噬菌体-C34m和噬菌体-C11m，进行滴度测定后冻存于-20℃待用。使用制备的灭活狂犬病病毒(采用MRC-5细胞制备)包被96孔ELISA板(1IU/mL，100μL/孔)，4℃包被过夜。分别用固定浓度(1×10E12cfu/mL)的噬菌体-C34m和噬菌体-C11m对等摩尔浓度起始(200nM)的实施例1中制备的全人源单克隆抗体C34m和C11m进行3倍梯度稀释(共8个浓度点)，以100μL/孔加入96孔板中，37℃孵育1小时。使用HRP抗M13-IgG(二抗)检测全人源单克隆抗体C34m和C11m分别对噬菌体-C34m和噬菌体-C11m与灭活狂犬病病毒结合能力的抑制作用。ELISA分析结果(图2、图3)显示，全人源单克隆抗体C11m能完全阻断噬菌体-C11m与灭活狂犬病病毒的结合，全人源单克隆抗体C34m不能完全阻断噬菌体-C11m与灭活狂犬病病毒的结合；全人源单克隆抗体C34m能完全阻断噬菌体-C34m与灭活狂犬病病毒的结合，全人源单克隆抗体C11m不能阻断噬菌体-C34m与灭活狂犬病病毒的结合，表明全人源单克隆抗体C34m和C11m与狂犬病病毒毒G蛋白拥有非竞争性结合表位。
实施例3：抗狂犬病病毒G蛋白单克隆抗体表位的确认
分别克隆编码针对狂犬病病毒G蛋白表位I的单克隆抗体CR57的重链可变区CR57VH(SEQ ID NO：12)和针对狂犬病病毒G蛋白表位III的单克隆抗体CR4098的重链可变区CR4098VH(SEQ ID NO：13)的基因(Bakker,A.B.et al.Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants.J.Virol.79,9062–9068；美国专利申请US 9005624 B2)至融合有编码鼠IgG2a重链恒定区(CH-mIgG2a，SEQ ID NO：14)的基因的真核表达载体(如invitrogen公司的 pcDNA3.1等)。克隆编码CR57的轻链可变区CR57VL(SEQ ID NO：15)的基因至融合有编码鼠轻链恒定区mCL(SEQ ID NO：17)的基因的真核表达载体(如invitrogen公司的pcDNA3.1等)，以及克隆编码CR4098的轻链可变区CR4098VK(SEQ ID NO：16)的基因至融合有编码鼠轻链恒定区mCK(SEQ ID NO：18)的真核表达载体(如invitrogen公司的pcDNA3.1等)，参照实施例1中的方法分别构建CR57的嵌合抗体CR57-mIgG2a和CR4098的嵌合抗体CR4098-mIgG2a。
使用制备的灭活狂犬病病毒(采用MRC-5细胞制备)包被96孔ELISA板(1IU/mL，100μL/孔)，4℃包被过夜。分别用固定浓度(2.5μg/mL)的嵌合抗体CR57-mIgG2a和CR4098-mIgG2a对等摩尔浓度起始(200nM)的全人源单克隆抗体C11m和C34m进行3倍梯度(共12个浓度点)稀释，以100μL/孔加入96孔板中，37℃孵育1小时。使用HRP抗鼠IgG(二抗)检测全人源单克隆抗体C11m和C34m分别对嵌合抗体CR57-mIgG2a和CR4098-mIgG2a与灭活狂犬病病毒结合能力的抑制作用。ELISA分析结果(图4、图5)显示：全人源单克隆抗体C11m与嵌合抗体CR57-mIgG2a竞争性结合狂犬病病毒G蛋白，但是不与嵌合抗体CR4098-mIgG2a竞争性结合狂犬病病毒G蛋白，表明全人源单克隆抗体C11m特异性结合狂犬病病毒G蛋白表位I。全人源单克隆抗体C34m与嵌合抗体CR4098-mIgG2a竞争性结合狂犬病病毒G蛋白，但是不与嵌合抗体CR57-mIgG2a竞争性结合狂犬病病毒G蛋白，表明全人源单克隆抗体C34m特异性结合狂犬病病毒G蛋白表位III。
实施例4：筛选pI值更合适的C11m-scFv突变体
基于KIH策略的双特异抗体更容易形成异源二聚体，但是在大规模的制备过程中仍然很难完全阻止同源二聚体的形成。离子交换色谱(IEC)是双特异性抗体纯化过程中利用Protein A一步纯化后常用的去除杂质的方法，同源二聚体在离子交换树脂中保留的时间取决于抗体分子的等电点(pI)，因此可以通过改变等电点的方式提高抗体在下游制备过程中的效率。
将实施例1中制备的编码单链抗体C11m-scFv的基因克隆至载体 pADscFv-s中(实验技术流程可参见中国专利申请第201510097117.0号中的实施例1.3)，得到重组表达载体pADscFv-C11m-scFv。利用重叠延伸PCR(overlap PCR)技术，在重组表达载体pADscFv-C11m-scFv的CDR区中引入突变，构建了库容量超过4.6×10E6的C11m-scFv突变体库，其中扩增所需的关键引物如表1所示。将灭活狂犬病病毒(采用MRC-5细胞制备)作为抗原，对C11m-scFv突变体库进行三轮筛选。最后鉴定出3个结合特性提高且预测pI值(http://www.bioinformatics.org/sms2/protein_iep.html)降低的突变体S1C10-scFv(SEQ ID NO：21)、S2A1-scFv(SEQ ID NO：22)、S2E3-scFv(SEQ ID NO：23)，各突变体的pI值如表2所示。
参照实施例1中的方法制备上述3个突变体与人IgG1的Fc段融合的单链抗体Fc融合蛋白(scFv-Fc)：S1C10-scFv-Fc、S2A1-scFv-Fc和S2E3-scFv-Fc。同时制备融合有鼠IgG2a重链恒定区(CH-mIgG2a)和轻链恒定区(mCK)的C34m嵌合抗体C34m-mIgG2a，以及融合有鼠IgG2a重链恒定区(CH-mIgG2a)和轻链恒定区(mCL)的C11m的嵌合抗体C11m-mIgG2a，用于功能分析。参照实施例2和实施例3中的方法，分别对3个C11m-scFv突变体的单链抗体Fc融合蛋白进行结合狂犬病病毒G蛋白能力和非竞争性表位验证。ELISA分析结果如图6、图7和图8所示，表明3个突变体的单链抗体Fc融合蛋白与狂犬病病毒G蛋白的结合能力明显提高，并且和全人源单克隆抗体C34m与狂犬病病毒G蛋白的结合能力相当；C11m-scFv-Fc单链抗体Fc融合蛋白及3个C11m-scFv突变体的单链抗体Fc融合蛋白和C34m-mIgG2a非竞争性结合狂犬病病毒G蛋白表位；C11m-scFv-Fc单链抗体Fc融合蛋白及3个C11m-scFv突变体的单链抗体Fc融合蛋白和C11m-mIgG2a竞争性结合狂犬病病毒毒G蛋白表位。
表1.构建C11m-scFv突变库所需的关键引物
Figure PCTCN2019098836-appb-000001
Figure PCTCN2019098836-appb-000002
表2.C11m-scFv及其突变体的pI值
抗体名称 pI值
C11m-scFv 7.38
S1C10-scFv 7.13
S2A1-scFv 6.90
S2E3-scFv 6.90
实施例5：双特异性抗体的制备
将针对狂犬病病毒G蛋白的表位I和表位III的抗原结合片段分别设计成scFv形式和Fab形式，构建基于KIH(Knob-Into-Hole)技术的人IgG1异二聚体，即将Fab形式的C34m抗原结合片段融合在含有Knob突变的Fc段(FcK，SEQ ID NO：19)的N端，将scFv形式的C11m突变体的抗原结合片段(scFv)融合在含有Hole突变的Fc段(FcH，SEQ ID NO：20)的N端，构建基于狂犬病病毒G蛋白的双特异性抗体。
分别将构建的表达S1C10-scFv-FcH、表达C34mVH-IgG1K和表达C34mVK-CK的3个真核表达载体利用脂质体共转染入HEK293F细胞，在无血清悬浮培养条件下培养3-5天，然后通过离心等方式收获培养上清。培养上清中的双特异性抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行纯化，然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组抗体保存缓冲液置换为PBS缓冲液(pH7.0)或者其它合适的缓冲液。将脱盐后蛋白溶液通过尺寸排阻层析(SEC)使用Superdex200(GE)纯化得到双特异性抗体S1C10-scFv-FcH+C34m-IgG1K。必要时，可以对抗体样品进行过滤除 菌，然后分装保存于-20℃。
类似地，制备S2A1-scFv-FcH+C34m-IgG1K和S2E3-scFv-FcH+C34m-IgG1K两种双特异性抗体。
实施例6：双特异性抗体功能验证
将制备的灭活狂犬病病毒(采用MRC-5细胞制备)包被96孔ELISA板(1IU/mL，100μL/孔)，4℃包被过夜。利用封闭液(2％的牛奶-PBST缓冲液)在37℃封闭1小时后，分别加入等摩尔浓度起始(200nM)3倍梯度稀释(共12个浓度点)的抗狂犬病病毒G蛋白双特异性抗体(S1C10-scFv-FcH+C34m-IgG1K、S2A1-scFv-FcH+C34m-IgG1K、S2E3-scFv-FcH+C34m-IgG1K、S1C10-scFv-Fc、S2A1-scFv-Fc、S2E3-scFv-Fc、C11m-scFv-Fc和C34m)，37℃结合1小时。用PBST缓冲液洗涤ELISA板，并加入HRP抗人IgG(二抗)，37℃结合1小时。然后用PBST缓冲液洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，使用酶标仪在490nm/630nm双波长测定光密度值。ELISA分析结果(图9)显示，双特异性抗体与灭活狂犬病病毒的结合能力高于单链抗体Fc融合蛋白以及全人源单克隆抗体C34m与灭活狂犬病病毒的结合能力。
挑选具有较低pI值的单链抗体S2E3-scFv，参照实施例1中方法，构建嵌合蛋白S2E3-scFv-mFc，分别用固定浓度(2.5μg/mL)的C34m-mIgG2a和S2E3-scFv-mFc对等摩尔浓度起始(200nM)的双特异性抗体S2E3-scFv-FcH+C34m-IgG1K、单链抗体Fc融合蛋白S2E3-scFv-Fc和全人源单克隆抗体C34m进行3倍梯度稀释(共12个浓度点)，以100μL/孔加入96孔板中，37℃孵育1小时。HRP抗鼠IgG(二抗)检测结合信号。ELISA分析结果(图10和图11)显示，双特异性抗体S2E3-scFv-FcH+C34m-IgG1K能够抑制嵌合抗体S2E3-scFv-mFc和嵌合抗体C34m-mIgG2a与狂犬病病毒疫苗的结合，即针对狂犬病毒G蛋白的双特异抗体S2E3-scFv-FcH+C34m-IgG1K拥有两个非竞争性的结合表位，能够同时结合狂犬病病毒G蛋白表位I和表位III。
实施例7：基于抗狂犬病病毒中和抗体快速荧光灶抑制实验(RFFIT)进行抗体效价测定
根据抗狂犬病病毒中和抗体快速荧光灶抑制实验(Rapid Fluorescent Focus Inhibition Test，RFFIT)，将标准血清(0.5IU/mL)和预稀释的供试抗体(双特异性抗体S2E3-scFv-FcH+C34m-IgG1K、单链抗体Fc融合蛋白S2E3-scFv-Fc和全人源单克隆抗体C34m，10μg/mL)3倍梯度系列稀释，在96孔板中每孔加50μL标准血清或供试抗体，每个样品设置3个重复孔，每孔加入50μL经适当比例稀释的中和用狂犬病病毒。同时设置不加标准血清或供试抗体的孔作为病毒稀释对照孔，设置不加标准血清或供试抗体以及狂犬病毒的孔作为细胞对照孔。37℃孵育1小时，每孔加入50μL BSR细胞(1x10E6个/ml),于37℃，5％CO 2培养箱培养。24小时后加入50μL冷丙酮固定30min，然后使用PBST缓冲液洗涤。每孔加入50μL狂犬病病毒荧光抗体工作液，37℃孵育30min。PBS缓冲液洗涤，每孔加入80％甘油，倒置荧光显微镜下进行荧光染色检测，根据Reed-Muench方法计算待测样品效价。
用狂犬病病毒标准攻击毒株CVS-11分别对3种抗体进行检测，实验结果见表3。结果标明，双特异性抗体S2E3-scFv-FcH+C34m-IgG1K、单链抗体Fc融合蛋白S2E3-scFv-Fc和全人源单克隆抗体C34m均具有较好的中和活性。
表3.RFFIT检测各抗体效价
Figure PCTCN2019098836-appb-000003
实施例8：抗狂犬病病毒抗体保护金黄色地鼠避免致死性狂犬病病毒攻击
通过对已注射致死剂量狂犬病病毒的金黄色地鼠进行免疫球蛋白暴露后免疫保护，与商业化产品进行比较，确定各组抗体的保护效果。
使用6.67lgLD50/mL的狂犬病病毒标准攻击毒株CVS-11对金黄色地鼠进行右后腿攻击(0.2ml/只)。并在24小时后在相同部位给予抗狂犬病病毒抗体。按注射剂量IU/kg进行分组，其中将双特异性抗体S2E3-scFv-FcH+C34m-IgG1K按注射剂量5IU/kg、20IU/kg和30IU/kg分为三组；分别将单链抗体Fc融合蛋白S2E3-scFv-Fc、全人源单克隆抗体C34m和商业化来源(同路生物制药有限公司)的人狂犬病病毒免疫球蛋白(简称同路生物免疫球蛋白)按注射剂量5IU/kg和20IU/kg各分为两组；未注射过抗狂犬病病毒抗体的金黄色地鼠作为对照组，共计10组，9只/组。根据金黄色地鼠称重结果用PBS缓冲液对各样品进行稀释，为方便注射，最终每只动物同侧注射量为100μL样品。
实验结果如表4所示：对照组的所有金黄色地鼠都在攻击2周内死于狂犬病，而在治疗组的金黄色地鼠都具有较高的存活率，其中本申请的抗狂犬病病毒抗体的保护效果均不弱于同路生物免疫球蛋白注射液。
表4.金黄色地鼠暴露后保护实验结果
Figure PCTCN2019098836-appb-000004
实施例9抗狂犬病病毒G蛋白抗体结合表位的确认
为了确定抗狂犬病毒中和性全人源单克隆抗体C34m和C11m-scFv突变体的单链抗体Fc融合蛋白S2E3-scFv-Fc的识别表位，测试狂犬病病毒糖蛋白中和性表位I-III中的氨基酸替换对抗狂犬病病毒G蛋白抗体结合能力的影响。研究证实对狂犬病病毒G蛋白的表位I-III中任一个进行突变，可以使得狂犬病病毒G蛋白的突变表位能够逃逸结合该表位的抗体的识别。本申请选择单点突变对狂犬病病毒毒株CVS-11野生型G蛋白的中和性表位I和表位III进行了氨基酸突变以获得对应逃逸株的氨基酸序列(Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody:fine mapping and escape mutant analysis；Rabies virulence:effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein)。利用常规分子生物学方法，PCR扩增得到编码野生型的狂犬病病毒毒株CVS-11的G蛋白GCVS11(SEQ ID NO：35)及其表位I突变体GCVS11-G229E(SEQ ID NO：36)和表位III突变体GCVS11-I338T(SEQ ID NO：37)的胞外区基因，分别在它们的C端融合编码人冠状蛋白1A的三聚体结构域CCD(SEQ ID NO：38)的基因，以保证分泌的糖蛋白更好地形成三聚体，维持它们的天然蛋白构象和免疫原性。将得到的编码狂犬病病毒G蛋白以及两种突变体(G229E和I338T)胞外区与CCD的融合蛋白基因分别克隆至C端融合有His标签的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin)分别将制备的重组抗原表达载体转染入HEK293细胞，在无血清悬浮培养条件下培养3-5天，离心过滤收获培养上清。将得到的培养上清利用超滤离心管浓缩约10倍后，放置-80℃备用。
分别用制备的全人源单克隆抗体C34m和单链抗体Fc融合蛋白S2E3-scFv-Fc包被96孔ELISA板(5μg/mL，100μL/孔)，4℃包被过夜。利用封闭液(2％的牛奶-PBST缓冲液)在37℃封闭1小时后，分别将得到的融合蛋白GCVS11-CCD-His(SEQ ID NO：39)、GCVS11-G229E-CCD-His(SEQ ID NO：40)、GCVS11-I338T-CCD-His (SEQ ID NO：41)浓缩液进行3倍梯度稀释(共11个浓度梯度)，以100μL/孔加入到包被有C34m和S2E3-scFv-Fc的96孔板中，37℃孵育1小时。使用HRP标记的抗His标签抗体(二抗)检测结合信号。
ELISA分析结果(如图12A、12B、12C)显示，C34m和S2E3-scFv-Fc与融合蛋白GCVS11-CCD-His结合的能力相当(图12A)；融合蛋白GCVS11-G229E-CCD-His与S2E3-scFv-Fc的结合能力明显低于与C34m的结合能力(图12B)，表明S2E3-scFv-Fc的结合表位为狂犬病病毒G蛋白表位I；类似地，融合蛋白GCVS11-I338T-CCD-His与C34m的结合能力明显低于与S2E3-scFv-Fc的结合能力(图12C)，表明C34m的结合表位为狂犬病病毒G蛋白表位III。

Claims (12)

  1. 双特异性抗体，其包含结合狂犬病病毒G蛋白不同表位的两个抗原结合片段，并且所述双特异性抗体具有中和狂犬病病毒的活性。
  2. 如权利要求1所述的双特异性抗体，其中一个抗原结合片段结合狂犬病病毒G蛋白的表位I，另一个抗原结合片段结合狂犬病病毒G蛋白的表位III。
  3. 如权利要求2所述的双特异性抗体，所述结合狂犬病病毒G蛋白的表位I的抗原结合片段包含：
    氨基酸序列为RYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIGAYDYVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
    氨基酸序列为RYSIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDIDGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；或者
    氨基酸序列为GYTIN的HCDR1，氨基酸序列为GIIPIFGTANYAQRFQG的HCDR2，氨基酸序列为ENLDNSGTYYYFSGWFDP的HCDR3，氨基酸序列为TGTSSDLGGYDFVS的LCDR1，氨基酸序列为DATKRPS的LCDR2，氨基酸序列为CSYAGDYTPGVV的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义。
  4. 如权利要求2所述的双特异性抗体，其中所述结合狂犬病病毒G蛋白的表位III的抗原结合片段包含：
    氨基酸序列为SYGMH的HCDR1，氨基酸序列为TISYDGSIKDYADSVKG的HCDR2，氨基酸序列为GDRTGNLDY的HCDR3，氨基酸序列为RASQNIRNALN的LCDR1，氨基酸序列为DASTRQS的LCDR2，氨基酸序列为QQNSEFPPT的LCDR3；
    其中，HCDR和LCDR氨基酸序列根据Kabat定义。
  5. 如权利要求3所述的双特异性抗体，其中
    所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：24所示，轻链可变区的氨基酸序列如SEQ ID NO：25所示；或者
    所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：26所示，轻链可变区的氨基酸序列如SEQ ID NO：27所示；或者
    所述结合狂犬病病毒G蛋白的表位I的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：28所示，轻链可变区的氨基酸序列如SEQ ID NO：29所示。
  6. 如权利要求4所述的双特异性抗体，其中
    所述结合狂犬病病毒G蛋白的表位III的抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：1所示，轻链可变区的氨基酸序列如SEQ ID NO：3所示。
  7. 如权利要求2-6中任一项所述的双特异性抗体，其中所述两个抗原结合片段的形式独立地选自单链抗体(scFv)或Fab片段。
  8. 如权利要求7所述的双特异性抗体，其中所述结合狂犬病病毒G蛋白的表位I的抗原结合片段为单链抗体(scFv)，所述结合狂犬病病毒G蛋白的表位III的抗原结合片段为Fab片段；
    优选地，所述双特异性抗体包含SEQ ID NO:32、33、34之一所示的氨基酸序列，和/或所述双特异性抗体包含SEQ ID NO:30和SEQ ID  NO:31所示的氨基酸序列。
  9. 药物组合物，其包含权利要求1-8中任一项所述的双特异性抗体和药学可接受的赋形剂、稀释剂或载体。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗狂犬病中的用途。
  11. 权利要求1-8中任一项所述的双特异性抗体在制备用于预防或治疗狂犬病的药物中的用途。
  12. 预防或治疗狂犬病的方法，其包括向有需要的个体给予权利要求1-8中任一项所述的双特异性抗体，或权利要求9或10所述的药物组合物。
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