WO2022188565A1 - 抗水痘-带状疱疹病毒的抗体及其用途 - Google Patents

抗水痘-带状疱疹病毒的抗体及其用途 Download PDF

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WO2022188565A1
WO2022188565A1 PCT/CN2022/073199 CN2022073199W WO2022188565A1 WO 2022188565 A1 WO2022188565 A1 WO 2022188565A1 CN 2022073199 W CN2022073199 W CN 2022073199W WO 2022188565 A1 WO2022188565 A1 WO 2022188565A1
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amino acid
antibody
acid sequence
seq
variable region
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PCT/CN2022/073199
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French (fr)
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张雪萍
刘志刚
周晓巍
郝小勃
胡俊杰
刘玉兰
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北京智仁美博生物科技有限公司
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Priority to EP22766092.5A priority Critical patent/EP4306539A1/en
Priority to US18/281,207 priority patent/US20240166725A1/en
Priority to JP2023555313A priority patent/JP2024509933A/ja
Publication of WO2022188565A1 publication Critical patent/WO2022188565A1/zh

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    • 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
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/088—Varicella-zoster virus
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application generally relates to the fields of genetic engineering and antibody drugs; in particular, to the field of anti-varicella-zoster virus antibodies and uses thereof.
  • the present application develops a novel anti-varicella-zoster virus antibody and provides the use of the antibody in preventing or treating chickenpox and shingles.
  • VZV Varicella zoster virus
  • VZV belongs to the alpha subfamily of the herpesvirus genus and is an enveloped DNA virus.
  • VZV is the smallest human herpesvirus, with a genome of approximately 125,000 bp in length, including more than 70 open reading frames, encoding a variety of proteins (Davison, A.J., and J.E.Scott. 1986).
  • VZV has at least 6 surface glycoproteins, such as gB (gp II), gC (gp IV), gE (gp I), gH (gp III), gI and gL, and on infected cell membranes, the glycoproteins gE, gB and gH are extremely abundant, and the antibodies they elicit are capable of neutralizing the virus (Davison, A.J., C.M. Edson. 1986). The glycoprotein gE is most abundantly expressed in VZV-infected cells, non-covalently linked to the gI protein, and binds to the Fc segment of immunoglobulin IgG.
  • glycoproteins such as gB (gp II), gC (gp IV), gE (gp I), gH (gp III), gI and gL, and on infected cell membranes, the glycoproteins gE, gB and gH are extremely abundant, and the antibodies they elicit are capable of neutralizing
  • the gB protein is the target of neutralizing antibodies and plays a role in viral entry into host cells, and the amino acid sequence of gB is highly conserved in VZV and herpes simplex virus-1 (HSV-1) (Kapsenberg, J.G. 1964).
  • the gH protein mediates the fusion between the viral envelope and the cell membrane and between the cell membranes, which is conducive to the spread of the virus between cells.
  • the co-expression of gH and gL can be glycosylated and transported to the cell surface (Forghani, B., L. Ni, and C. Grose. 1994).
  • VZV infects human diploid cells and melanoma cells and can also replicate in VERO cells and primary African green monkey kidney cells.
  • VZV adsorbs heparan sulfate proteoglycan on the cell surface, and then binds to the low-affinity second receptor into cells, expresses viral proteins in infected cells, and forms multinucleated giant cells (Zhu, Z., M.D.Gershon.1995). Most viral particles are retained in cytoplasmic vacuoles, and free virus is rarely released extracellularly.
  • VZV has only one serotype with limited genetic diversity, which does not affect its infectivity and virulence.
  • VZV infection is highly species-specific and has no animal reservoir. Although it can infect some non-human primates and small animals such as guinea pigs (Chen, J. 2003) and rats (Sadzot-Delvaux. 1990), it does not disease in these animals. Humans are the only known natural host, and the skin is the main target organ of the virus.
  • VZV can be transmitted by droplets and/or contact, and the primary infection in children mainly causes chickenpox. Chickenpox is prevalent in temperate regions and usually occurs in winter and spring.
  • VZV VZV Specific cellular immunity declines, and the latent virus is activated and replicated in large numbers, transferring to the skin through sensory nerve axons, causing shingles.
  • Shingles arises from reactivation of a latent virus, so only patients who have had a primary infection develop shingles, and the onset is not seasonal.
  • Herpes zoster mainly occurs in people over 45 years old, and the incidence rate increases with age.
  • the incidence rate of herpes zoster in the general population is (3-5) people/1000 people/year, and it increases year by year by 2.5%-5.0 %, the population over the age of 75 can reach 10/1000/year, and the incidence of herpes zoster in children is extremely low (Guess, H.A. 1985).
  • Herpes zoster is more common in patients receiving immunosuppressive therapy and in patients with HIV infection.
  • herpes zoster is accompanied by severe pain, and a common complication is Post Herpetic Neuralgia (PHN), which can last from several months to several years.
  • PPN Post Herpetic Neuralgia
  • VZV infection produces IgG, IgM and IgA antibodies that bind viral proteins including glycoproteins, regulatory proteins, structural proteins and enzymes (Bogger-Goren. 1984), and neutralizing antibodies mediate lysis of infected cells.
  • Neutralizing antibodies that bind to gE and gI proteins require complement for neutralization, while antibodies that bind to gB and gH/gL proteins do not.
  • IgG antibodies persist in the body for a long time, protecting the body from secondary infections, especially glycoprotein antibodies.
  • VZV immunoglobulin injection within 72 hours of VZV exposure in neonates unimmunized pregnant or immunocompromised individuals can suppress infection and viral replication in vivo (Zaia, J.A., 1983), reduce the severity of varicella, and reduce the risk of varicella pneumonia , but the injection of immune globulin in children with acute chickenpox does not clinically improve the course of the disease.
  • Antiviral drugs are effective in reducing the severity of chickenpox and shingles, shortening the duration of the disease, and reducing the incidence of postherpetic neuralgia.
  • the treatment of herpes zoster is currently mainly non-specific antiviral therapy, no specific antiviral drugs.
  • the application provides an antibody against varicella-zoster virus comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, in
  • the HCDR1 amino acid sequence is SGNYWN
  • the HCDR2 amino acid sequence is YISYDGSTYYNPSLKN
  • the HCDR3 amino acid sequence is GYYGYWFAY
  • the LCDR1 amino acid sequence is RASSSVSYMH
  • the LCDR2 amino acid sequence is ATSNLAS
  • the LCDR3 amino acid sequence is QQWSSNPFT; or
  • the HCDR1 amino acid sequence is SGYYWN
  • the HCDR2 amino acid sequence is YISYDGSNNYNTSLKN
  • the HCDR3 amino acid sequence is EDVNYPPYALDY
  • the LCDR1 amino acid sequence is RSSQSLVHSNGNTYLH
  • the LCDR2 amino acid sequence is KVSNRFS
  • the LCDR3 amino acid sequence is SQSTHVPWT; or
  • the HCDR1 amino acid sequence is SYTMS
  • the HCDR2 amino acid sequence is FISNGGDNNYYADTVKG
  • the HCDR3 amino acid sequence is HNGNWGFAY
  • the LCDR1 amino acid sequence is SASSSISSNYLH
  • the LCDR2 amino acid sequence is RTSNLAS
  • the LCDR3 amino acid sequence is QQGSSIPLT; or
  • the HCDR1 amino acid sequence is TYAMH
  • the HCDR2 amino acid sequence is VISYGGGNRYYAASVKG
  • the HCDR3 amino acid sequence is ARDNHYFFGMDV
  • the LCDR1 amino acid sequence is RASQGISSWLA
  • the LCDR2 amino acid sequence is AASSLQS
  • the LCDR3 amino acid sequence is QQANSFPLT, QEGFYFPIN, QQATYFPIN or QQSSYFPIA;
  • HCDR and LCDR amino acid sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, 3, 5, or 7.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 2, 4, 6, 8, 9, 10 or 28.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:2 display
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 4; Or
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO:6;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 8;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 9;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 10;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO:28.
  • the application provides an antibody against varicella-zoster virus, wherein the amino acid sequence of the variable region of the heavy chain of the antibody has at least 90 % identity, and the amino acid sequence of the light chain variable region of the antibody is at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10, and 28.
  • the antibody is a whole antibody, a Fab fragment, an F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a monoclonal antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype or IgG4 subtype.
  • the antibody further comprises a light chain constant region selected from a kappa subtype or a lambda subtype.
  • the antibody binds to the varicella-zoster virus gH/gL protein; and/or the antibody mediates antibody-dependent cell-mediated cytotoxicity (ADCC) ).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the application provides a nucleic acid molecule encoding the antibody of the first aspect or the second aspect.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present application provides the antibody of the first aspect or the second aspect, the nucleic acid molecule of the third aspect, or the pharmaceutical composition of the fourth aspect prepared for the prevention or treatment of chickenpox and shingles. Use in medicine for herpes.
  • the present application provides a method for preventing or treating chickenpox and herpes zoster, comprising administering the antibody of the first aspect or the second aspect, or the pharmaceutical composition of the fourth aspect to an individual in need thereof.
  • Figure 1 shows ELISA analysis of the ability of anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody to block the binding of anti-recombinant protein gH/gL purified phage to recombinant protein gH/gL.
  • Figures A-C are the blocking results of anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibodies S8E1, S8B8 and S7A10, respectively.
  • Figure 2 shows ELISA analysis of the ability of anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody H4H8-L1B7 to block the binding of anti-recombinant protein gH/gL purified phage to recombinant protein gH/gL.
  • Figure 3 shows assessment of ADCC activity of anti-varicella-zoster virus monoclonal antibodies based on Jurkat-Dual-CD16a reporter cells.
  • SEQ ID NO: 1 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody S7A10.
  • SEQ ID NO: 2 shows the amino acid sequence of the light chain variable region of monoclonal antibody S7A10.
  • SEQ ID NO: 3 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody S8E1.
  • SEQ ID NO: 4 shows the amino acid sequence of the light chain variable region of monoclonal antibody S8E1.
  • SEQ ID NO: 5 shows the amino acid sequence of the heavy chain variable region of monoclonal antibody S8B8.
  • SEQ ID NO: 6 shows the amino acid sequence of the light chain variable region of monoclonal antibody S8B8.
  • SEQ ID NO: 7 shows the amino acid sequences of the heavy chain variable regions of monoclonal antibodies H4H8-L1B7, H4H8-L14H8, H4H8-L13C1 and H4H8-L13C7.
  • SEQ ID NO: 8 shows the amino acid sequence of the light chain variable region of monoclonal antibody H4H8-L13C1.
  • SEQ ID NO: 9 shows the amino acid sequence of the light chain variable region of monoclonal antibody H4H8-L13C7.
  • SEQ ID NO: 10 shows the amino acid sequence of the light chain variable region of monoclonal antibodies H4H8-L14H8.
  • SEQ ID NO: 11 shows the amino acid sequence of the gH glycoprotein of Varicella zoster virus.
  • SEQ ID NO: 12 shows the amino acid sequence of the gL glycoprotein of Varicella zoster virus.
  • SEQ ID NO: 13 shows the amino acid sequence of the His-tag.
  • SEQ ID NO: 14 shows the amino acid sequence of the human (homo sapiens) IgGl subtype heavy chain constant region.
  • SEQ ID NO: 15 shows the amino acid sequence of the heavy chain constant region of human (homo sapiens) IgG2 subtype.
  • SEQ ID NO: 16 shows the amino acid sequence of the human (homo sapiens) IgG4 subtype heavy chain constant region.
  • SEQ ID NO: 17 shows the amino acid sequence of the mouse (mus musculus) IgG1 subtype heavy chain constant region.
  • SEQ ID NO: 18 shows the amino acid sequence of the mouse (mus musculus) IgG2a subtype heavy chain constant region.
  • SEQ ID NO: 19 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region.
  • SEQ ID NO: 20 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.
  • SEQ ID NO: 21 shows the amino acid sequence of the mouse (mus musculus) kappa subtype light chain constant region.
  • SEQ ID NO: 22 shows the amino acid sequence of the mouse (mus musculus) lambda subtype light chain constant region.
  • SEQ ID NO: 23 shows the nucleotide sequence of primer PmCGR.
  • SEQ ID NO: 24 shows the nucleotide sequence of primer PmCKR.
  • SEQ ID NO: 25 shows the amino acid sequence of single chain antibody S7A10.
  • SEQ ID NO: 26 shows the amino acid sequence of single chain antibody S8E1.
  • SEQ ID NO: 27 shows the amino acid sequence of single chain antibody S8B8.
  • SEQ ID NO: 28 shows the amino acid sequence of the light chain variable region of monoclonal antibody H4H8-L1B7.
  • novel antibodies against varicella-zoster virus obtained new antibodies against varicella-zoster virus through antibody engineering technology.
  • novel antibodies against varicella-zoster virus nucleic acid molecules encoding said antibodies or antigen-binding fragments thereof, vectors comprising said nucleic acid molecules, and vectors comprising said nucleic acid molecules or vectors Host cells, methods of making and purifying the antibodies, and medical and biological applications of the antibodies.
  • a full-length antibody molecule can be constructed as a medicine for preventing or treating chickenpox and herpes zoster.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, including antibody portions fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other immunoglobulins that contain an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • antigen-binding fragments thereof eg, Fab, Fab', F(ab') 2 , Fv
  • variants thereof including antibody portions fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other immunoglobulins that contain an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosy
  • full or full-length antibodies contain two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variable region (VL) and a constant region (CL).
  • a full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or a subclass of the above), but the antibody need not belong to any particular class.
  • Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant domains of the heavy chains.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen binding domain refers to a portion or region of an intact antibody molecule responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2, and CDR3.
  • CDRs complementarity determining regions
  • CDR1 and CDR3 complementarity determining regions
  • CDR2 and CDR3 are the regions in the variable region that have the greatest impact on the affinity and specificity of antibodies.
  • CDR amino acid sequences of VH or VL There are two common ways of defining the CDR amino acid sequences of VH or VL, the Chothia definition and the kabat definition.
  • Kabat "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al, Proc. Natl. Acad. Sci.
  • the CDR amino acid sequences in the VH and VL amino acid sequences can be determined according to the Chothia definition or the Kabat definition. In embodiments of the present application, CDR amino acid sequences are defined using Kabat.
  • variable region amino acid sequence of a given antibody the middle CDR amino acid sequence of the variable region amino acid sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab') 2 fragments, which may be two A bivalent fragment of a Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (ie, a dimer of the Fab'); (3) an Fv fragment with the VL and VH domains of the one-armed antibody; ( 4) Single-chain Fv (scFv), which can be a single polypeptide chain consisting of VH and VL domains via a peptide linker; and (5) (scFv) 2 , which can comprise two peptides linked by a peptide A symbol-linked VH domain and two VL domains that are combined with the two VH domains via a disulfide bridge.
  • Fab fragments which may be monovalent fragments having a VL-CL chain and
  • binding refers to a non-random binding reaction between two molecules, eg, binding of an antibody to an epitope.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals.
  • the monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding amino acid sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, whereas The remainder of the heavy and/or light chain is identical or homologous to the corresponding amino acid sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided they express desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • the application provides an antibody against varicella-zoster virus comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, in
  • the HCDR1 amino acid sequence is SGNYWN
  • the HCDR2 amino acid sequence is YISYDGSTYYNPSLKN
  • the HCDR3 amino acid sequence is GYYGYWFAY
  • the LCDR1 amino acid sequence is RASSSVSYMH
  • the LCDR2 amino acid sequence is ATSNLAS
  • the LCDR3 amino acid sequence is QQWSSNPFT; or
  • the HCDR1 amino acid sequence is SGYYWN
  • the HCDR2 amino acid sequence is YISYDGSNNYNTSLKN
  • the HCDR3 amino acid sequence is EDVNYPPYALDY
  • the LCDR1 amino acid sequence is RSSQSLVHSNGNTYLH
  • the LCDR2 amino acid sequence is KVSNRFS
  • the LCDR3 amino acid sequence is SQSTHVPWT; or
  • the HCDR1 amino acid sequence is SYTMS
  • the HCDR2 amino acid sequence is FISNGGDNNYYADTVKG
  • the HCDR3 amino acid sequence is HNGNWGFAY
  • the LCDR1 amino acid sequence is SASSSISSNYLH
  • the LCDR2 amino acid sequence is RTSNLAS
  • the LCDR3 amino acid sequence is QQGSSIPLT; or
  • the HCDR1 amino acid sequence is TYAMH
  • the HCDR2 amino acid sequence is VISYGGGNRYYAASVKG
  • the HCDR3 amino acid sequence is ARDNHYFFGMDV
  • the LCDR1 amino acid sequence is RASQGISSWLA
  • the LCDR2 amino acid sequence is AASSLQS
  • the LCDR3 amino acid sequence is QQANSFPLT, QEGFYFPIN, QQATYFPIN or QQSSYFPIA;
  • HCDR and LCDR amino acid sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, 3, 5, or 7.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 2, 4, 6, 8, 9, 10 or 28.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:2 display
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 4; Or
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO:6;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 8;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 9;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 7
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 10;
  • amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO:28.
  • the application provides an antibody against varicella-zoster virus, wherein the amino acid sequence of the variable region of the heavy chain of the antibody has at least 90 % identity, and the amino acid sequence of the light chain variable region of the antibody is at least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10, and 28.
  • the amino acid sequence of the heavy chain variable region of the antibody is at least 90%, 91%, 92%, 93%, or 93% relative to any one of SEQ ID NOs: 1, 3, 5, and 7 %, 94%, 95%, 96%, 97%, 98%, 99% or higher homology.
  • the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology.
  • the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5 and 7 by about 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions.
  • the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10 and 28 Substitutions, deletions and/or additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5 and 7 may also be truncated by about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar function of the heavy chain variable region of the antibody.
  • 1, 2, 3, 4, 5 may also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5 and 7 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, the resulting amino acid sequence still retains similar function of the heavy chain variable region of the antibody.
  • 1, 2, 3 may also be added or deleted to regions other than the C- or N-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 5, and 7 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the altered amino acid sequence maintains substantially similar weight to the antibody The function of the chain variable region.
  • the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10 and 28 may also be truncated by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still remaining similar to the light chain of the antibody can be Variable area function.
  • 1, 2 may also be added to the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10 and 28 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, the resulting amino acid sequence still remains similar to the light chain of the antibody function of the variable region.
  • amino acid sequence shown in any one of SEQ ID NOs: 2, 4, 6, 8, 9, 10 and 28 may also be added to a region other than the C-terminus or the N-terminus Deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the altered amino acid sequence remains substantially similar function of the light chain variable region of the antibody.
  • the antibody is a whole antibody, a Fab fragment, an F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody is a monoclonal antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype or IgG4 subtype.
  • the heavy chain constant region is of the IgGl subtype.
  • the antibody further comprises a light chain constant region selected from a kappa subtype or a lambda subtype.
  • the light chain constant region is of the kappa subtype.
  • the antibody binds to the varicella-zoster virus gH/gL protein; and/or the antibody mediates antibody-dependent cell-mediated cytotoxicity (ADCC) ).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the application provides a nucleic acid molecule encoding the antibody of the first aspect or the second aspect.
  • the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat chickenpox and shingles.
  • the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives agents, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as emulsifying agents
  • suspending agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives agents such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents etc.
  • the pharmaceutical compositions herein can be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules, and the like.
  • the pharmaceutical compositions of the present application can be delivered by any physiologically acceptable mode of administration, including, but not limited to: oral, parenteral, nasal, rectal drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • compositions for therapeutic use can be formulated in the form of lyophilized formulations or aqueous solutions by admixing an agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, etc. storage.
  • the present application provides the antibody of the first aspect or the second aspect, the nucleic acid molecule of the third aspect, or the pharmaceutical composition of the fourth aspect prepared for the prevention or treatment of chickenpox and shingles. Use in medicine for herpes.
  • the present application provides a method for preventing or treating chickenpox and herpes zoster, comprising administering the antibody of the first aspect or the second aspect, or the pharmaceutical composition of the fourth aspect to an individual in need thereof.
  • the application also provides isolated nucleic acid molecules encoding the antibodies of the invention, or light or heavy chains thereof, as well as vectors comprising the nucleic acid molecules, host cells comprising the vectors, and methods of producing the antibodies.
  • the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.
  • the method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
  • antibodies described herein specific for varicella-zoster virus can also be used to detect the presence of varicella-zoster virus in biological samples.
  • Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
  • the gH glycoprotein (SEQ ID NO: 11) and gL glycoprotein (SEQ ID NO: 12) are required for the preparation and identification of varicella-zoster virus-specific antibodies.
  • gH and gL can only be effectively secreted and expressed after they are synthesized separately in cells and folded together to form gH/gL dimers, and gH/gL dimers have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc. ), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of recombinant proteins.
  • the synthesized genes encoding gH-His and gL recombinant proteins were cloned into appropriate eukaryotic expression vectors (such as pcDNA3.1 from Invitrogen) using conventional molecular biology techniques, and then liposomes (such as 293fectin from Invitrogen) were used. etc.) or other transfection reagents (such as PEI, etc.) to transfect the prepared recombinant protein expression plasmids into HEK293 cells (such as HEK293F from Invitrogen), and culture them under serum-free suspension culture conditions for 3-5 days. The culture supernatant is then harvested by centrifugation or the like.
  • appropriate eukaryotic expression vectors such as pcDNA3.1 from Invitrogen
  • liposomes such as 293fectin from Invitrogen
  • PEI transfection reagents
  • One-step purification of the recombinant protein in the supernatant is performed using a metal chelate affinity chromatography column (such as HisTrap FF from GE). Then use a desalting column (such as GE's Hitrap desaulting, etc.) to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. If necessary, samples can be filter sterilized and stored in aliquots at -20°C.
  • a metal chelate affinity chromatography column such as HisTrap FF from GE.
  • PBS pH 7.0
  • the antibody heavy chain constant region can be of human IgG1 subtype (SEQ ID NO: 14), human IgG2 subtype (SEQ ID NO: 15), human IgG4 subtype (SEQ ID NO: 16), or The murine IgG1 subtype (SEQ ID NO: 17), the murine IgG2a (SEQ ID NO: 18) subtype, the light chain constant region may be human kappa subtype (SEQ ID NO: 19), human lambda subtype (SEQ ID NO: 19) : 20), murine kappa subtype (SEQ ID NO:21), murine lambda subtype (SEQ ID NO:22).
  • nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody were cloned into eukaryotic expression fusing the nucleotide sequences encoding the heavy chain constant region and the light chain constant region, respectively.
  • a vector (such as pcDNA3.1 from Invitrogen, etc.) is used to express the whole antibody in combination.
  • Use liposomes (such as 293fectin from Invitrogen, etc.) or other transfection reagents (such as PEI, etc.) to transfect the prepared recombinant antibody expression plasmids into HEK293 cells (such as HEK293F from Invitrogen), and culture under serum-free suspension culture conditions.
  • the culture supernatant is harvested by centrifugation, etc., and one-step purification is performed using a ProteinA/G affinity chromatography column (such as Mabselect SURE from GE, etc.). Then use a desalting column (such as GE's Hitrap desaulting, etc.) to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. If necessary, antibody samples can be filter sterilized and stored in aliquots at -20°C.
  • Example 2 Screening of mouse anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • mice Using the recombinant protein gH-His/gL prepared in Example 1 as the antigen, BALB/c mice aged 6-8 weeks were immunized, and the immunization dose was 50 ⁇ g/mouse. The immunization was boosted once every 14 days. Mice were sacrificed at 8 weeks and splenocytes were collected. Mouse spleen lymphocytes were isolated using Mouse Lymphocyte Separation Solution (Daktronics Biotechnology Co., Ltd., CAT#DKW33-R0100). Total RNA was extracted from the isolated lymphocytes using a total cell RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., CAT#DP430).
  • the first-strand cDNA synthesis kit (Thermo scientific, CAT#K1621) was used to synthesize the cDNAs of the variable region of the heavy chain and the variable region of the light chain, respectively.
  • the reverse transcription primers were gene-specific primers.
  • the primer paired regions are respectively located in the antibody heavy chain constant region and the antibody light chain constant region, and the specific sequences are respectively PmCGR:TGCATTTGAACTCCTTGCC (SEQ ID NO:23) and PmCKR:CCATCAATCTTCCACTTGAC (SEQ ID NO:24).
  • the synthesized cDNA was immediately stored at -70°C for future use.
  • the prepared mouse single-chain antibody nucleotide sequence was cloned into the vector pADSCFV-S (for the experimental technical process, please refer to Example 1 of Chinese Patent Application No. 201510097117.0, the entire content of the above patent application is incorporated herein by reference ) to build the scFv library.
  • the capacity of the antibody library reaches 2.13E+08, and the correct rate is 50%.
  • Example 3 Affinity analysis of mouse anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • nucleotide sequences encoding the light and heavy chains of single-chain antibodies that specifically bind to gH/gL were cloned into eukaryotic expression vectors to prepare mouse-human chimeric antibodies in the form of recombinant human IgG1- ⁇ .
  • the affinity of anti-gH/gL antibodies was determined by surface plasmon resonance using a Biacore X100.
  • Amino Coupling Kit (BR-1000-50), Human Antibody Capture Kit (BR-1008-39), CM5 Chip (BR100012) and 10 ⁇ HBS-EP (BR100669) with pH 7.4 and other related reagents and consumables are all available. Purchased from GE healthcare.
  • the recombinant protein gH/gL was set up with a series of concentration gradients (for example, 1.23nM, 3.7nM, 11.1nM, 33.3nM and 100nM), and 30 ⁇ L/min at 25°C was injected from low concentration to high concentration, and the binding time was 120s , the dissociation time was 3600 s, and the chip surface was regenerated by injecting 3M MgCl 2 at 10 ⁇ L/min for 30 s.
  • Association rates (K a ) and dissociation rates (K d ) were calculated by fitting association and dissociation sensorgrams by a 1:1 binding model using Biacore X100 evaluation software version 2.0.1.
  • the dissociation equilibrium constant (K D ) is calculated as the ratio K d /K a .
  • the fitting results are shown in Table 1.
  • Example 4 Epitope analysis of mouse anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • a 96-well ELISA plate (3 ⁇ g/mL, 100 ⁇ L/well) was coated with the recombinant varicella-zoster virus protein gH/gL, and it was coated in a refrigerator at 4°C overnight. Block with blocking solution PBS-0.1% Tween 20-3% milk for 1 hour at 37°C.
  • Antibodies against recombinant protein gH/gL (S7A10, S8B8 and S8E1) were purified with fixed concentrations (5 x 10 10 -1 x 10 11 cfu/mL) of each anti-recombinant protein gH/gL phage (S7A10, S8B8 and S8E1).
  • the initial concentration is 10 ⁇ g/mL, 3-fold gradient dilution, 8-10 concentration gradients, 100 ⁇ L/well is added to the sealed 96-well ELISA plate, and incubated at 37°C for 1 hour.
  • the ELISA plate was washed with PBS-0.1% Tween 20, then HRP anti-M13 secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05T-H) was added, and incubated at 37°C for 1 hour.
  • Example 5 Neutralizing activity of mouse anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • the neutralizing activity of the antibodies was determined by the reduction of plaque formation in MRC5 cells infected with the antibody mixed with VZV virus.
  • MRC-5 cells were seeded at 1.5 ⁇ 10 5 cells/well in a 6-well cell culture plate, and cultured for 72 hours to form a monolayer of cells. When 90% confluent, the culture medium supernatant was removed, and virus maintenance solution (MEM containing 2% serum) was added. medium), 400 ⁇ L per well.
  • VZV Dilute VZV (Oka) to 2000pfu/mL with virus maintenance solution, that is, after mixing with the antibody in equal volume, each 100 ⁇ L of VZV contains 100pfu; use virus diluent to dilute the antibody 2-fold gradient, the initial concentration is 20 ⁇ g/mL, 8 A concentration gradient, that is, the antibody concentration after mixing with VZV in equal volume is 10 ⁇ g/mL, 5 ⁇ g/mL, 2.5 ⁇ g/mL, 1.25 ⁇ g/mL, 0.62 ⁇ g/mL, 0.31 ⁇ g/mL, 0.16 ⁇ g/mL and 0.08 ⁇ g/mL mL.
  • VZV was mixed with an equal volume of antibody and placed at 25°C for 1 hour to infect MRC5 cells in a 6-well plate, 100 ⁇ L per well. Adsorb at 37°C for 1 hour, aspirate and discard the supernatant, wash twice with PBS, add 3 mL of maintenance solution to each well, maintain the culture at 37°C for 8 days, remove the supernatant, wash once with PBS, and add 1 mL of Coomassie brilliant blue staining solution to each well. , after 10 minutes of action, washed once with PBS, placed in a bright place to count plaques, and calculated the antibody concentration (PRNT50) that reduced plaques by 50% by Reed and Muench method. The experimental results are shown in Table 2.
  • Example 6 Anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody inhibits the activity of virus transmission between cells
  • Antibodies (S7A10, S8B8, and S8E1) were added 24 hours after VZV virus infection of MRC5 cells, and the activity of the antibodies to inhibit virus-to-cell transmission was determined by the reduction of plaque formation.
  • MRC-5 cells were seeded at 1.5 ⁇ 10 5 cells/well in a 6-well cell culture plate, cultured for 72 hours to form a monolayer of cells, 90% confluent, removed the culture supernatant, and added virus maintenance solution (MEM medium containing 2% serum) ), 400 ⁇ L per well. Dilute VZV to 1000 pfu/mL with virus maintenance solution, and infect MRC5 cells in a 6-well plate with 100 ⁇ L per well, that is, infect 100 pfu of VZV per well.
  • the cells were adsorbed at 37°C for 1 hour, the supernatant was discarded, washed twice with PBS, 3 mL of maintenance solution was added to each well, and the culture was maintained at 37°C for 24 hours.
  • the antibody was diluted 2-fold with virus diluent, the initial concentration was 400 ⁇ g/mL, 8 concentration gradients were added, and MRC5 cells infected with VZV were added to the 6-well plate, 30 ⁇ L per well, that is, the antibody concentration in the culture medium was 4 ⁇ g/mL. , 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL and 0.125 ⁇ g/mL.
  • the culture was maintained at 37°C for 7 days, the supernatant was removed, washed once with PBS, and 1 mL of Coomassie brilliant blue staining solution was added to each well. After 10 minutes, the cells were washed once with PBS, placed in a bright place to count plaques, and the plaque reduction was calculated by 50. % antibody concentration.
  • the experimental results are shown in Table 3.
  • Example 7 Screening of fully human anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • the recombinant protein gH-His/gL prepared in Example 1 was used as the antigen, and the solid-phase screening strategy was used (for the experimental protocol, please refer to Phage Display: General Experimental Guide/(US) Clarkson, T.), (US) Lohman ( Edited by Lowman, H.B.); translated by Ma Lan et al. Chemical Industry Press, 2008.5, the entire contents of the above documents are incorporated herein by reference) Screening of natural human phage antibody library (for experimental technical procedures, please refer to Chinese Patent Application No.
  • Example 1 the entire content of the above patent application is incorporated herein by reference), and finally a fully human single-chain antibody H4H8-L1B7 that specifically binds to the recombinant protein gH-His/gL was obtained.
  • Example 8 Epitope analysis of fully human anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • the light and heavy chains encoding H4H8-L1B7 were cloned into a eukaryotic expression vector to prepare a fully human monoclonal antibody in the form of recombinant human IgG1- ⁇ .
  • the phages (S7A10, S8E1, S8B8 and H4H8-L1B7) were purified against recombinant protein gH/gL with a fixed concentration (5 ⁇ 10 10 -1 ⁇ 10 11 cfu/mL) of each anti-recombinant protein gH/gL, respectively.
  • the antibody (H4H8-L1B7-IgG1) was serially diluted, and the blocking of human anti-recombinant protein gH/gL monoclonal antibody H4H8-L1B7-IgG1 was determined.
  • the results of ELISA analysis are shown in Figure 2.
  • the H4H8-L1B7-IgG1 monoclonal antibody can completely block the binding signal of H4H8-L1B7 phage and S8E1 phage to the recombinant protein gH/gL.
  • the binding signal of /gL did not have any effect.
  • Example 9 Construction and screening of light chain mutation library of fully human anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody H4H8-L1B7
  • the LCDR3 mutant library of L1B7 (SEQ ID NO: 28) was screened for 2 rounds by a solid-phase screening strategy, and finally 3 light chains were obtained. Mutants L13C1 (SEQ ID NO:8), L13C7 (SEQ ID NO:9) and L14H8 (SEQ ID NO:10).
  • H4H8-L14H8, H4H8-L13C1 and H4H8-L13C7 were cloned into eukaryotic expression vectors to prepare fully human monoclonal antibodies in the form of recombinant human IgG1- ⁇ .
  • Example 10 Neutralizing activity of anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody
  • Example 11 Activity of anti-varicella-zoster virus recombinant protein gH/gL monoclonal antibody to inhibit virus transmission between cells
  • Example 6 the activity of monoclonal antibodies H4H8-L14H8, H4H8-L13C1 and H4H8-L13C7 for inhibiting the spread of viruses between cells was determined. The experimental results are shown in Table 7.
  • Example 12 Anti-Varicella-Zoster Virus Monoclonal Antibody Mediates ADCC Effects on Infected Cells
  • the inventors of the present application constructed the Jurkat-Dual-CD16a reporter gene cell line by transferring the CD16a plasmid into the jurkat-dual cells of Invivogen Company.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the activation signal is transferred to the downstream NF- ⁇ B pathway through the CD16a molecule, and finally ADCC activity is detected by luciferase .
  • Jurkat-Dual-CD16a cells and VZV-infected target cells were mixed at an effector-to-target ratio of 15:1.
  • the molecules to be tested H4H8-L14H8-IgG1, H4H8-L13C1-IgG1, H4H8-L1B7-IgG1 and isotype control IgG1 antibodies were started at a final concentration of 60 ⁇ g/mL, and were diluted 3-fold, with a total of 10 concentration points, mixed with the above cells. co-cultivate.

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Abstract

提供了针对水痘-带状疱疹病毒的抗体、编码所述抗体的核酸分子、包含所述核酸分子的表达载体、包含所述核酸分子或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体的应用。

Description

抗水痘-带状疱疹病毒的抗体及其用途
相关申请的交叉引用
本申请要求于2021年3月10日递交的中国专利申请第202110259834.4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，涉及抗水痘-带状疱疹病毒抗体领域及其用途。本申请开发了新的抗水痘-带状疱疹病毒抗体，并提供了该抗体在预防或治疗水痘和带状疱疹中的用途。
背景技术
水痘-带状疱疹病毒(Varicella zoster virus，VZV)属疱疹病毒属α亚科，是有被膜的DNA病毒。VZV是最小的人疱疹病毒，基因组全长约125,000bp，包括70多种开放读码框，编码多种蛋白质(Davison,A.J.,and J.E.Scott.1986)。VZV至少有6种表面糖蛋白，如gB(gp II)、gC(gp IV)、gE(gp I)、gH(gp III)、gI和gL，在受感染的细胞膜上，糖蛋白gE、gB和gH极为丰富，它们诱生的抗体均能中和病毒(Davison,A.J.,C.M.Edson.1986)。糖蛋白gE在VZV感染细胞中表达丰度最高，与gI蛋白非共价连接，结合免疫球蛋白IgG的Fc段。gB蛋白是中和抗体的靶点，在病毒侵入宿主细胞的过程中发挥作用，gB的氨基酸序列在VZV和单纯疱疹病毒-1(HSV-1)中高度保守(Kapsenberk,J.G.1964)。gH蛋白介导病毒被膜与细胞膜以及细胞膜之间的融合，利于病毒在细胞间的扩散，gH与gL共表达才能糖基化并转运到细胞表面(Forghani,B.,L.Ni,and C.Grose.1994)。
VZV感染人二倍体细胞和黑色素瘤细胞，也能在VERO细胞和原代非洲绿猴肾细胞中复制。VZV通过吸附细胞表面的硫酸肝素蛋 白聚糖，进而与低亲和力的第二受体结合进入细胞，在感染细胞内表达病毒蛋白，形成多核巨细胞(Zhu,Z.,M.D.Gershon.1995)。多数病毒颗粒保留在细胞质液泡中，很少向胞外释放游离的病毒。
VZV只有一个血清型，遗传多样性有限，且这种遗传多样性不影响其感染性和毒力。VZV感染具有高度的种属特异性，没有动物储存宿主，虽然能感染一些非人灵长类动物以及豚鼠(Chen,J.2003)和大鼠(Sadzot-Delvaux.1990)等小动物，但不会引起这些动物发病。人是唯一已知自然宿主，皮肤是病毒的主要靶器官。VZV可经飞沫和(或)接触传播，儿童初次感染主要引起水痘。水痘在温带地区流行，一般在冬春季发病。感染水痘后产生终身免疫，二次感染的病例很罕见(Gershon,A.A.1984)。感染者体内残余的VZV可沿感觉神经轴突逆行，或经感染的T细胞与神经元细胞的融合，转移到脊髓后根神经节或颅神经节内并潜伏，当机体免疫力降低时，VZV特异性细胞免疫下降，潜伏的病毒被激活并大量复制，通过感觉神经轴突转移到皮肤，引起带状疱疹。带状疱疹源于潜伏病毒的重新激活，因此只有发生过原发感染的患者才会患带状疱疹，而且发病没有季节性。带状疱疹主要在45岁以上人群中发病，发病率随着年龄的增高而增加，普通人群带状疱疹的发病率为(3-5)人/1000人/年，并逐年递增2.5％-5.0％，75岁以上人群可以达到10人/1000人/年，儿童带状疱疹发病率极低(Guess,H.A.1985)。带状疱疹在接受免疫抑制剂治疗的患者以及感染HIV的患者中较常见。带状疱疹除皮损外伴随剧烈疼痛，常见并发症为带状疱疹后神经痛(Post Herpetic Neuralgia，PHN)，可持续数月至数年。
VZV感染产生IgG、IgM和IgA抗体，这些抗体结合包括糖蛋白、调节蛋白、结构蛋白和酶在内的病毒蛋白(Bogger-Goren.1984)，中和抗体介导感染细胞的裂解。结合gE和gI蛋白的中和抗体发挥中和作用需要补体，而结合gB、gH/gL蛋白的抗体则不需要补体。初次感染水痘后体内IgG抗体长期存在，保护机体免受二次感染，尤其是糖蛋白抗体。新生儿、未免疫妊娠者或者免疫功能低下者暴露VZV后72小时内注射VZV免疫球蛋白可以抑制感染和体内病毒复制(Zaia, J.A.,1983)，减轻水痘的严重程度，降低水痘性肺炎的风险，但是急性水痘患儿注射免疫球蛋白在临床上不能改善疾病病程。抗病毒药物能有效降低水痘和带状疱疹的严重程度，缩短病程，降低带状疱疹后神经痛的发病率。带状疱疹的治疗目前主要是非特异性抗病毒治疗，无特异性抗病毒药物。
因此，基于临床需求，探索和研发抗水痘-带状疱疹的抗体具有重要的生物学和医学意义。
发明概述
第一方面，本申请提供了针对水痘-带状疱疹病毒的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区以及含LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
所述HCDR1氨基酸序列为SGNYWN，所述HCDR2氨基酸序列为YISYDGSTYYNPSLKN，所述HCDR3氨基酸序列为GYYGYWFAY，所述LCDR1氨基酸序列为RASSSVSYMH，所述LCDR2氨基酸序列为ATSNLAS，所述LCDR3氨基酸序列为QQWSSNPFT；或者
所述HCDR1氨基酸序列为SGYYWN，所述HCDR2氨基酸序列为YISYDGSNNYNTSLKN，所述HCDR3氨基酸序列为EDVNYPPYALDY，所述LCDR1氨基酸序列为RSSQSLVHSNGNTYLH，所述LCDR2氨基酸序列为KVSNRFS，所述LCDR3氨基酸序列为SQSTHVPWT；或者
所述HCDR1氨基酸序列为SYTMS，所述HCDR2氨基酸序列为FISNGGDNNYYADTVKG，所述HCDR3氨基酸序列为HNGNWGFAY，所述LCDR1氨基酸序列为SASSSISSNYLH，所述LCDR2氨基酸序列为RTSNLAS，所述LCDR3氨基酸序列为QQGSSIPLT；或者
所述HCDR1氨基酸序列为TYAMH，所述HCDR2氨基酸序列为VISYGGGNRYYAASVKG，所述HCDR3氨基酸序列为ARDNHYFFGMDV，所述LCDR1氨基酸序列为RASQGISSWLA， 所述LCDR2氨基酸序列为AASSLQS，所述LCDR3氨基酸序列为QQANSFPLT、QEGFYFPIN、QQATYFPIN或QQSSYFPIA；
其中HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、5或者7所示。
在第一方面的一些实施方案中，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、4、6、8、9、10或者28所示。
在第一方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:10所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示。
第二方面，本申请提供了针对水痘-带状疱疹病毒的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:1、3、5和7中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:2、4、6、8、9、10和28中任何一项具有至少90％的一致性。
在第一方面和第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面和第二方面的一些实施方案中，所述抗体为单克隆抗 体。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体结合水痘-带状疱疹病毒gH/gL蛋白；和/或所述抗体介导抗体依赖性的细胞介导的细胞毒作用(ADCC)。
第三方面，本申请提供了核酸分子，其编码第一方面或第二方面所述的抗体。
第四方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的抗体以及药学上可接受的赋形剂、稀释剂或载体。
第五方面，本申请提供了第一方面或第二方面所述的抗体、第三方面所述的核酸分子、或第四方面所述的药物组合物在制备用于预防或治疗水痘和带状疱疹的药物中的用途。
第六方面，本申请提供了预防或治疗水痘和带状疱疹的方法，包括向有需要的个体给予第一方面或第二方面所述的抗体、或者第四方面所述的药物组合物。
附图说明
图1显示ELISA分析抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体阻断抗重组蛋白gH/gL纯化噬菌体与重组蛋白gH/gL结合的能力。其中，图A-图C分别为抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体S8E1、S8B8和S7A10的阻断结果。
图2显示ELISA分析抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体H4H8-L1B7阻断抗重组蛋白gH/gL纯化噬菌体与重组蛋白gH/gL结合的能力。
图3显示基于Jurkat-Dual-CD16a报告基因细胞评估抗水痘-带状疱疹病毒单克隆抗体的ADCC活性。
序列说明
SEQ ID NO:1显示单克隆抗体S7A10的重链可变区的氨基酸序列。
SEQ ID NO:2显示单克隆抗体S7A10的轻链可变区的氨基酸序列。
SEQ ID NO:3显示单克隆抗体S8E1的重链可变区的氨基酸序列。
SEQ ID NO:4显示单克隆抗体S8E1的轻链可变区的氨基酸序列。
SEQ ID NO:5显示单克隆抗体S8B8的重链可变区的氨基酸序列。
SEQ ID NO:6显示单克隆抗体S8B8的轻链可变区的氨基酸序列。
SEQ ID NO:7显示单克隆抗体H4H8-L1B7、H4H8-L14H8、H4H8-L13C1和H4H8-L13C7的重链可变区的氨基酸序列。
SEQ ID NO:8显示单克隆抗体H4H8-L13C1的轻链可变区的氨基酸序列。
SEQ ID NO:9显示单克隆抗体H4H8-L13C7的轻链可变区的氨基酸序列。
SEQ ID NO:10显示单克隆抗体H4H8-L14H8的轻链可变区的氨基酸序列。
SEQ ID NO:11显示水痘-带状疱疹病毒(Varicella zoster virus)的gH糖蛋白的氨基酸序列。
SEQ ID NO:12显示水痘-带状疱疹病毒(Varicella zoster virus)的gL糖蛋白的氨基酸序列。
SEQ ID NO:13显示His标签的氨基酸序列。
SEQ ID NO:14显示人(homo sapiens)IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:15显示人(homo sapiens)IgG2亚型重链恒定区的氨基酸序列。
SEQ ID NO:16显示人(homo sapiens)IgG4亚型重链恒定区的氨基酸序列。
SEQ ID NO:17显示小鼠(mus musculus)IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:18显示小鼠(mus musculus)IgG2a亚型重链恒定区的氨基酸序列。
SEQ ID NO:19显示人(homo sapiens)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:20显示人(homo sapiens)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:21显示小鼠(mus musculus)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:22显示小鼠(mus musculus)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:23显示引物PmCGR的核苷酸序列。
SEQ ID NO:24显示引物PmCKR的核苷酸序列。
SEQ ID NO:25显示单链抗体S7A10的氨基酸序列。
SEQ ID NO:26显示单链抗体S8E1的氨基酸序列。
SEQ ID NO:27显示单链抗体S8B8的氨基酸序列。
SEQ ID NO:28显示单克隆抗体H4H8-L1B7的轻链可变区的氨基酸序列。
发明详述
本申请的发明人通过抗体工程技术得到了新的针对水痘-带状疱疹病毒抗体。在本申请的多个方面，提供了新的针对水痘-带状疱疹病毒抗体，编码所述抗体或其抗原结合片段的核酸分子、包含所述核酸分子的载体、包含所述核酸分子或载体的宿主细胞、制备和纯化所述抗体的方法及所述抗体的医学和生物学应用。根据本申请提供的抗体的可变区的氨基酸序列，可构建全长的抗体分子作为药物用于预防或治疗水痘和带状疱疹。
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靶标的免疫球蛋白分子。靶标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。每个重链含有重链可变区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链可变区(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“抗原结合片段或抗原结合部分”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可变区(VH)、轻链可变区(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氨基酸序列有两种常见的定义方式，即Chothia定义和kabat定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md. (1991)；A1-Lazikani等人，J.Mol.Biol.273:927-948(1997)；以及Martin等人，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区氨基酸序列，可以根据Chothia定义或者Kabat定义来确定VH和VL氨基酸序列中的CDR氨基酸序列。在本申请的实施方案中，利用Kabat定义CDR氨基酸序列。
对于给定抗体的可变区氨基酸序列，可以通过多种方式分析可变区氨基酸序列的中CDR氨基酸序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
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。
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。
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氨基酸序列相同或同源，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氨基酸序列相同或同源，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))。
第一方面，本申请提供了针对水痘-带状疱疹病毒的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区以及含 LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
所述HCDR1氨基酸序列为SGNYWN，所述HCDR2氨基酸序列为YISYDGSTYYNPSLKN，所述HCDR3氨基酸序列为GYYGYWFAY，所述LCDR1氨基酸序列为RASSSVSYMH，所述LCDR2氨基酸序列为ATSNLAS，所述LCDR3氨基酸序列为QQWSSNPFT；或者
所述HCDR1氨基酸序列为SGYYWN，所述HCDR2氨基酸序列为YISYDGSNNYNTSLKN，所述HCDR3氨基酸序列为EDVNYPPYALDY，所述LCDR1氨基酸序列为RSSQSLVHSNGNTYLH，所述LCDR2氨基酸序列为KVSNRFS，所述LCDR3氨基酸序列为SQSTHVPWT；或者
所述HCDR1氨基酸序列为SYTMS，所述HCDR2氨基酸序列为FISNGGDNNYYADTVKG，所述HCDR3氨基酸序列为HNGNWGFAY，所述LCDR1氨基酸序列为SASSSISSNYLH，所述LCDR2氨基酸序列为RTSNLAS，所述LCDR3氨基酸序列为QQGSSIPLT；或者
所述HCDR1氨基酸序列为TYAMH，所述HCDR2氨基酸序列为VISYGGGNRYYAASVKG，所述HCDR3氨基酸序列为ARDNHYFFGMDV，所述LCDR1氨基酸序列为RASQGISSWLA，所述LCDR2氨基酸序列为AASSLQS，所述LCDR3氨基酸序列为QQANSFPLT、QEGFYFPIN、QQATYFPIN或QQSSYFPIA；
其中HCDR和LCDR氨基酸序列根据Kabat定义。
在第一方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、5或者7所示。
在第一方面的一些实施方案中，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、4、6、8、9、10或者28所示。
在第一方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所 述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:10所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示。
第二方面，本申请提供了针对水痘-带状疱疹病毒的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:1、3、5和7中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:2、4、6、8、9、10和28中任何一项具有至少90％的一致性。
在第二方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列与SEQ ID NO:1、3、5和7中任何一项具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。
在第二方面的一些实施方案中，所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:2、4、6、8、9、10和28中任何一项具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。
在第二方面的一些实施方案中，所述抗体的重链可变区的氨基酸序列与SEQ ID NO:1、3、5和7中任何一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、缺失和/或添加。
在第二方面的一些实施方案中，所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:2、4、6、8、9、10和28中任何一项所示的氨基酸序列相差约1、2、3、4、5、6、7、8、9或10个氨基酸的取代、 缺失和/或添加。
在第二方面的一些实施方案中，SEQ ID NO:1、3、5和7中任何一项所示的氨基酸序列的C端或N端区域还可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，而仍然保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中，还可以在SEQ ID NO:1、3、5和7中任何一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，得到的氨基酸序列仍然保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中，还可以在SEQ ID NO:1、3、5和7中任何一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，只要改变后的氨基酸序列基本上保持类似的所述抗体的重链可变区的功能。
在第二方面的一些实施方案中，SEQ ID NO:2、4、6、8、9、10和28中任何一项所示的氨基酸序列的C端或N端区域还可以被截短约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，而仍然保持类似的所述抗体的轻链可变区的功能。
在第二方面的一些实施方案中，还可以在SEQ ID NO:2、4、6、8、9、10和28中任何一项所示的氨基酸序列的C端或N端区域添加1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，得到的氨基酸序列仍然保持类似的所述抗体的轻链可变区的功能。
在第二方面的一些实施方案中，还可以在SEQ ID NO:2、4、6、8、9、10和28中任何一项所示的氨基酸序列的C端或N端以外的区域添加或缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25或更多个氨基酸，只要改变后的氨基酸序列基本上保持类似的所述抗体的轻链可变区的功能。
在第一方面和第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面和第二方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面和第二方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面的一些具体实施方案中，所述重链恒定区为IgG1亚型。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面和第二方面的一些具体实施方案中，所述轻链恒定区为κ亚型。
在第一方面和第二方面的一些实施方案中，所述抗体结合水痘-带状疱疹病毒gH/gL蛋白；和/或所述抗体介导抗体依赖性的细胞介导的细胞毒作用(ADCC)。
第三方面，本申请提供了核酸分子，其编码第一方面或第二方面所述的抗体。
在一些实施方案中，所述核酸分子可操作地连接到调控氨基酸序列，调控氨基酸序列可以被用所述载体转化过的宿主细胞识别。
第四方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的抗体以及药学上可接受的赋形剂、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水痘和带状疱疹。
在一些实施方案中，所述药物组合物还可包含下述中的一种或多种：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。
第五方面，本申请提供了第一方面或第二方面所述的抗体、第三方面所述的核酸分子、或第四方面所述的药物组合物在制备用于预防或治疗水痘和带状疱疹的药物中的用途。
第六方面，本申请提供了预防或治疗水痘和带状疱疹的方法，包括向有需要的个体给予第一方面或第二方面所述的抗体，或者第四方面所述的药物组合物。
在其他方面，本申请还提供编码本发明抗体或其轻链或重链的分离的核酸分子以及包含所述核酸分子的载体、包含所述载体的宿主细胞以及产生所述抗体的方法。在一些实施方案中，所述核酸分子可操作地连接到调控氨基酸序列，调控氨基酸序列可以被用所述载体转化过的宿主细胞识别。在一些实施方案中，产生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，产生抗体的方法还包括从宿主细胞培养基中回收抗体。
此外，本文所述的特异性针对水痘-带状疱疹病毒的抗体也可用于检测生物样品中水痘-带状疱疹病毒的存在。基于抗体的检测方法在本领域是众所周知的，并且包括例如ELISA、免疫印迹、放射免疫试验、免疫荧光、免疫沉淀以及其它相关技术。
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。
实施例
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1：重组蛋白的制备
制备和鉴定水痘-带状疱疹病毒特异性抗体的过程中需要用到gH糖蛋白(SEQ ID NO:11)和gL糖蛋白(SEQ ID NO:12)。gH和gL只有在细胞内分别合成，一起发生折叠形成gH/gL二聚体后才能有效的分泌表达，且gH/gL二聚体有大量的翻译后修饰(如糖基化或二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，在gH糖蛋白的C端添加His标签(SEQ ID NO:13)，更有利于重组蛋白的纯化和单克隆抗体功能的鉴定。根据Uniprot数据库中水痘-带状疱疹病毒Oka株的gH糖蛋白和gL糖蛋白的氨基酸序列，设计并合成编码gH(包含His标签)和gL重组蛋白的基因。利用常规的分子生物学技术分别将合成的编码gH-His和gL重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清。利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对上清中的重组蛋白进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时，可以对样品进行过滤除菌，然后分装保存于-20℃。
在制备重组抗体时，抗体重链恒定区可以是人IgG1亚型(SEQ ID NO:14)、人IgG2亚型(SEQ ID NO:15)、人IgG4亚型(SEQ ID NO:16)、或者鼠IgG1亚型(SEQ ID NO:17)、鼠IgG2a(SEQ ID NO:18)亚型、轻链恒定区可以是人κ亚型(SEQ ID NO:19)、人λ亚型(SEQ ID NO:20)、鼠κ亚型(SEQ ID NO:21)、鼠λ亚型(SEQ ID NO：22)。利用常规的分子生物学手段，将编码抗体重链可变区和轻链可变区的核苷酸序列分别克隆至融合有编码重链恒定区和轻链恒定区核苷酸序列的真核表达载体(如invitrogen公司的pcDNA3.1等)， 组合表达全抗体。利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组抗体表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-5天。然后通过离心等方式收获培养上清，利用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2：鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的筛选
2.1小鼠免疫及免疫抗体库的制备
以实施例1中制备的重组蛋白gH-His/gL为抗原，免疫6-8周龄的BALB/c小鼠，免疫剂量为50μg/只小鼠，每14天加强免疫1次，初免后8周处死小鼠并收集脾细胞。使用小鼠淋巴细胞分离液(达科为生物技术股份有限公司，CAT#DKW33-R0100)对小鼠脾脏淋巴细胞进行分离。利用细胞总RNA提取试剂盒(天根生化科技(北京)有限公司，CAT#DP430)，将分离的淋巴细胞进行总RNA的提取。以提取的总RNA为模板，利用第一链cDNA合成试剂盒(Thermo scientific，CAT#K1621)分别合成抗体重链可变区和轻链可变区的cDNA，反转录引物采取基因特异性引物，引物配对区分别位于抗体重链恒定区和抗体轻链恒定区，具体序列分别为PmCGR:TGCATTTGAACTCCTTGCC(SEQ ID NO:23)和PmCKR:CCATCAATCTTCCACTTGAC(SEQ ID NO:24)。将合成的cDNA立即存放于-70℃保存备用。然后以反转录得到的cDNA为模板，参考文献(Krebber A,Bornhauser S,Burmester J,et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.J Immunol Methods.1997；201(1):35-55，通过引用方式将上述文献的全部内容并入本文中)合成引物，并利用PCR分别扩增编码鼠抗体 VH和VK的核苷酸序列，然后利用重叠延伸PCR技术，构建单链抗体(scFv)基因。最后将制备的小鼠单链抗体核苷酸序列克隆至载体pADSCFV-S(实验技术流程可参见中国专利申请第201510097117.0号的实施例1，通过引用方式将上述专利申请的全部内容并入本文中)，构建scFv库。抗体库的库容达到2.13E+08，正确率为50％。
2.2抗水痘-带状疱疹病毒重组蛋白gH/gL小鼠单链抗体的筛选
参照文献(实验技术流程可参见中国专利申请第201510097117.0号，通过引用方式将上述专利申请的全部内容并入本文中)，以实施例1制备的重组蛋白gH-His/gL为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5，通过引用方式将上述文献的全部内容并入本文中)筛选上述构建的展示小鼠单链抗体的噬菌体库，通过结合、洗脱、中和、感染、扩增的方式共进行三轮筛选，最终获得多株序列不同，但均能特异性结合重组蛋白gH-His/gL的单链抗体，包括克隆S7A10(氨基酸序列如SEQ ID NO:25所示)、S8E1(氨基酸序列如SEQ ID NO:26所示)和S8B8(氨基酸序列如SEQ ID NO:27所示)。
实施例3：鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的亲和力分析
利用常规分子生物学方法，将编码特异性结合gH/gL的单链抗体的轻重链的核苷酸序列克隆至真核表达载体，制备重组人IgG1-κ形式鼠-人嵌合抗体。
利用Biacore X100通过表面等离子共振技术测定抗gH/gL抗体的亲和力。氨基偶联试剂盒(BR-1000-50)、人抗体捕获试剂盒(BR-1008-39)、CM5芯片(BR100012)和pH7.4的10×HBS-EP(BR100669)等相关试剂和耗材均购自GE healthcare。依照试剂 盒中的说明书，用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride，EDC)和N-羟基琥珀酰亚胺(N-Hydroxysuccinimide，NHS)对羧基化CM5芯片表面进行活化，将抗人IgG(Fc)抗体(捕获抗体)用10mM pH5.0的乙酸钠稀释至25μg/mL，之后以流速10μL/min注射以实现大约多至10000个响应单位(RU)的偶联量。注射捕获抗体之后，注射1M的乙醇胺以封闭未反应的基团。对于动力学测量，稀释抗gH/gL抗体至0.5-1μg/mL，10μL/min注射，保证50RU左右的抗体被抗人Fc的抗体捕获。然后将重组蛋白gH/gL设置一系列的浓度梯度(例如1.23nM、3.7nM、11.1nM、33.3nM和100nM)，于25℃下30μL/min从低浓度到高浓度进行注射，结合时间为120s，解离时间为3600s，以10μL/min注射3M的MgCl 2 30s对芯片表面进行再生。使用Biacore X100评估软件第2.0.1版，通过1:1结合模型拟合结合和解离传感图来计算结合速率(K a)和解离速率(K d)。以比率K d/K a计算解离平衡常数(K D)。拟合结果如表1所示。
表1.重组抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体结合水痘-带状疱疹病毒重组蛋白gH/gL的亲和力常数
  K a K d K D
S8B8-IgG1 4.488E+5 2.068E-4 4.609E-10
S7A10-IgG1 1.721E+5 9.531E-4 5.539E-9
S8E1-IgG1 6.695E+4 2.995E-4 4.473E-9
实施例4：鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的表位分析
使用水痘-带状疱疹病毒重组蛋白gH/gL包被96孔ELISA板(3μg/mL，100μL/孔)，4℃冰箱包被过夜。利用封闭液PBS-0.1％吐温20-3％牛奶在37℃封闭1小时。用固定浓度(5×10 10-1×10 11cfu/mL)的各个抗重组蛋白gH/gL纯化噬菌体(S7A10、S8B8和S8E1)分别对抗重组蛋白gH/gL的抗体(S7A10、S8B8和S8E1)进行梯度稀释，起始浓度为10μg/mL，3倍梯度稀释，8-10个浓度梯度，100μL/孔加入封闭好的96孔ELISA板中，37℃孵育1小时。使用PBS-0.1％吐温20 洗涤ELISA板，然后加入HRP抗M13二抗(北京义翘神州科技股份有限公司，11973-MM05T-H)，37℃孵育1小时。使用PBS-0.1％吐温20洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，使用酶标仪测定492nm/630nm双波长光密度值。ELISA分析结果如图1所示，S8E1-IgG1单克隆抗体不能阻断S8B8噬菌体、S7A10噬菌体与重组蛋白gH/gL的结合(图1A)；S8B8-IgG1单克隆抗体可以部分阻断S8E1噬菌体与重组蛋白gH/gL的结合信号，但是对S7A10噬菌体与重组蛋白gH/gL的结合没有影响(图1B)；S7A10-IgG1单克隆抗体不能阻断S8B8噬菌体、S7A10噬菌体与重组蛋白gH/gL的结合(图1C)。
实施例5：鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的中和活性
通过抗体与VZV病毒混合后感染MRC5细胞形成空斑减少确定抗体(S7A10、S8B8和S8E1)中和活性。MRC-5细胞以1.5×10 5个/孔接种6孔细胞培养板，培养72小时成单层细胞，90％铺满时，去除培养液上清，加入病毒维持液(含2％血清的MEM培养基)，每孔400μL。用病毒维持液将VZV(Oka)稀释至2000pfu/mL，即与抗体等体积混合后每100μL含100pfu的VZV；用病毒稀释液将抗体进行2倍梯度稀释，起始浓度是20μg/mL，8个浓度梯度，即与VZV等体积混合后抗体浓度为10μg/mL、5μg/mL、2.5μg/mL、1.25μg/mL、0.62μg/mL、0.31μg/mL、0.16μg/mL和0.08μg/mL。VZV与抗体等体积混合，25℃放置1小时，感染6孔板的MRC5细胞，每孔100μL。37℃吸附1小时，吸弃上清，PBS洗2次，每孔加入3mL的维持液，37℃维持培养8天，将上清去掉，PBS清洗一次，每孔加入1mL的考马斯亮蓝染色液，作用10分钟后，PBS清洗1次，置于明亮处计数空斑，用Reed和Muench法计算空斑减少50％的抗体浓度(PRNT50)。实验结果如表2所示。
表2.鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的中和活性
抗体 S7A10 S8B8 S8E1
PRNT50(μg/mL) 0.34 0.38 0.38
实施例6：抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体抑制病毒在细胞间传播的活性
VZV病毒感染MRC5细胞后24小时加入抗体(S7A10、S8B8和S8E1)，通过形成空斑减少确定抗体抑制病毒在细胞间传播的活性。MRC-5细胞以1.5×10 5个/孔接种6孔细胞培养板，培养72小时成单层细胞，90％铺满，去培养上清，加入病毒维持液(含2％血清的MEM培养基)，每孔400μL。用病毒维持液将VZV稀释至1000pfu/mL，感染6孔板的MRC5细胞，每孔100μL，即每孔感染100pfu的VZV。37℃吸附1小时，吸弃上清，PBS洗2次，每孔加入3mL的维持液，37℃维持培养24小时。用病毒稀释液将抗体进行2倍梯度稀释，起始浓度是400μg/mL，8个浓度梯度，加入6孔板感染了VZV的MRC5细胞，每孔30μL，即培养液中抗体浓度为4μg/mL、2μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL和0.125μg/mL。37℃维持培养7天，将上清去掉，PBS清洗一次，每孔加入1mL的考马斯亮蓝染色液，作用10分钟后，PBS清洗1次，置于明亮处计数空斑，计算空斑减少50％的抗体浓度。实验结果如表3所示。
表3.鼠抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体抑制病毒在细胞间传播的活性
抗体 S7A10 S8B8 S8E1
PRNT50(μg/mL) 0.93 3.21 2.82
实施例7：全人源抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的筛选
以实施例1制备的重组蛋白gH-His/gL为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森 (Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5，通过引用方式将上述文献的全部内容并入本文中)筛选天然人噬菌体抗体库(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，通过引用方式将上述专利申请的全部内容并入本文中)，最终获得1株特异性结合重组蛋白gH-His/gL的全人源单链抗体H4H8-L1B7。
实施例8：全人源抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的表位分析
利用常规分子生物学方法，将编码H4H8-L1B7的轻重链克隆至真核表达载体，制备重组人IgG1-κ形式全人源单克隆抗体。
参照实施例4，用固定浓度(5×10 10-1×10 11cfu/mL)的各个抗重组蛋白gH/gL纯化噬菌体(S7A10、S8E1、S8B8和H4H8-L1B7)分别对抗重组蛋白gH/gL的抗体(H4H8-L1B7-IgG1)进行梯度稀释，测定人抗重组蛋白gH/gL单克隆抗体H4H8-L1B7-IgG1对抗重组蛋白gH/gL噬菌体结合重组蛋白gH/gL的阻断。ELISA分析结果见图2，H4H8-L1B7-IgG1单克隆抗体可以完全阻断H4H8-L1B7噬菌体和S8E1噬菌体与重组蛋白gH/gL的结合信号，对其它2种噬菌体(S7A10和S8B8)与重组蛋白gH/gL的结合信号没有任何影响。
实施例9：全人源抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体H4H8-L1B7轻链突变库的构建和筛选
9.1 H4H8-L1B7轻链突变库的构建及筛选
基于双载体噬菌体展示系统，利用轻链CDR突变的策略(具体操作可参照申请人之前提交的中国专利第201510097117.0号中的实施例5，通过引用方式将上述专利申请的全部内容并入本文中)对H4H8-L1B7单克隆抗体进行体外亲和力成熟。利用经典重叠延伸PCR方法构建了库容量超过5.5E+07的LCDR3突变库，突变库设计方案见表4。
表4.LCDR3突变库设计方案
原始氨基酸 突变设计氨基酸 简并密码子
Q QELVRG SDG
Q QELVRG SDG
A GVADITNS RNT
N FISTYN WHT
N FISTYN WHT
F FISTYN WHT
P FSLPAV BYC
L LSITAV DYC
T STAYND DMT
以重组蛋白gH-His/gL为抗原，以H4H8-L1B7重链为基础，利用固相筛选策略对L1B7(SEQ ID NO:28)的LCDR3突变库进行了2轮筛选，最终获得3个轻链突变体L13C1(SEQ ID NO:8)、L13C7(SEQ ID NO:9)和L14H8(SEQ ID NO:10)。
9.2 H4H8-L1B7轻链突变体亲和力分析
利用常规分子生物学方法，将编码H4H8-L14H8、H4H8-L13C1和H4H8-L13C7的轻重链克隆至真核表达载体，制备重组人IgG1-κ形式全人源单克隆抗体。
参照实施例3，利用Biacore X100评估软件第2.0.1版对H4H8-L1B7轻链突变体亲和力进行了分析，结果见表5。
表5.H4H8-L1B7轻链突变体结合重组蛋白gH/gL的亲和力常数
  K a K d K D
H4H8-L13C1-IgG1 2.601E+5 1.281E-4 4.925E-10
H4H8-L13C7-IgG1 2.494E+5 1.211E-4 4.855E-10
H4H8-L14H8-IgG1 3.666E+5 1.062E-4 2.896E-10
实施例10：抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的中和活性
参照实施例5，测定单克隆抗体H4H8-L14H8、H4H8-L13C1和H4H8-L13C7的中和活性。实验结果如表6所示。
表6.抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体的中和活性
  H4H8-L14H8 H4H8-L13C1 H4H8-L13C7
PRNT50(μg/mL) 0.016 0.016 >0.4
实施例11：抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体抑制病毒在细胞间传播的活性
参照实施例6，测定单克隆抗体H4H8-L14H8、H4H8-L13C1和H4H8-L13C7的抑制病毒在细胞间传播的活性。实验结果如表7所示。
表7.抗水痘-带状疱疹病毒重组蛋白gH/gL单克隆抗体抑制病毒在细胞间传播的活性
  H4H8-L14H8 H4H8-L13C1 H4H8-L13C7
PRNT50(μg/mL) 0.31 0.28 >2
实施例12：抗水痘-带状疱疹病毒单克隆抗体介导对感染细胞的ADCC作用
本申请发明人通过将CD16a质粒转入Invivogen公司的jurkat-dual细胞中,构建了Jurkat-Dual-CD16a报告基因细胞株。当抗体依赖性的细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity，ADCC)效应发生时，活化信号通过CD16a分子转至下游的NF-κB通路，最后通过萤光素酶检测ADCC活性。水痘-带状疱疹病毒(VZV)以MOI＝0.01感染T75细胞培养瓶中90％铺满的MRC5细胞，镜下观察，待瓶中细胞有50％-60％发生病变(感染96小时)，经胰酶消化后，收集细胞。将Jurkat-Dual-CD16a细胞和感染了VZV的靶细胞(MRC-5细胞)以效靶比15:1的比例混合。待测分子H4H8-L14H8-IgG1、H4H8-L13C1-IgG1、H4H8-L1B7-IgG1和同型对照IgG1抗体分别以终浓度60μg/mL起始，3倍梯度稀释，共10个浓度点， 与上述混合细胞共培养。孵育20h后，离心取50μL上清，酶标仪中设置好自动上样程序，在每孔中加入50μL荧光素酶检测液quanti-luc(invivogen,货号rep-qlc2)并进行全波长检测测定萤光强度。基于Jurkat-Dual-CD16a报告基因体系的ADCC活性结果显示(图3)，H4H8-L14H8-IgG1和H4H8-L13C1-IgG1的ADCC活性好于H4H8-L1B7-IgG1。
上文对本申请的各项发明的示例性实施方案进行了描述，但是，在不脱离本申请的实质和范围的情况下，本领域技术人员能够对本申请描述的示例性实施方案进行修改或改进，由此得到的变形方案或等同方案也属于本申请的范围。
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Claims (12)

  1. 针对水痘-带状疱疹病毒的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区以及含LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
    所述HCDR1氨基酸序列为SGNYWN，所述HCDR2氨基酸序列为YISYDGSTYYNPSLKN，所述HCDR3氨基酸序列为GYYGYWFAY，所述LCDR1氨基酸序列为RASSSVSYMH，所述LCDR2氨基酸序列为ATSNLAS，所述LCDR3氨基酸序列为QQWSSNPFT；或者
    所述HCDR1氨基酸序列为SGYYWN，所述HCDR2氨基酸序列为YISYDGSNNYNTSLKN，所述HCDR3氨基酸序列为EDVNYPPYALDY，所述LCDR1氨基酸序列为RSSQSLVHSNGNTYLH，所述LCDR2氨基酸序列为KVSNRFS，所述LCDR3氨基酸序列为SQSTHVPWT；或者
    所述HCDR1氨基酸序列为SYTMS，所述HCDR2氨基酸序列为FISNGGDNNYYADTVKG，所述HCDR3氨基酸序列为HNGNWGFAY，所述LCDR1氨基酸序列为SASSSISSNYLH，所述LCDR2氨基酸序列为RTSNLAS，所述LCDR3氨基酸序列为QQGSSIPLT；或者
    所述HCDR1氨基酸序列为TYAMH，所述HCDR2氨基酸序列为VISYGGGNRYYAASVKG，所述HCDR3氨基酸序列为ARDNHYFFGMDV，所述LCDR1氨基酸序列为RASQGISSWLA，所述LCDR2氨基酸序列为AASSLQS，所述LCDR3氨基酸序列为QQANSFPLT、QEGFYFPIN、QQATYFPIN或QQSSYFPIA；
    其中HCDR和LCDR氨基酸序列根据Kabat定义。
  2. 根据权利要求1所述的抗体，其中所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、5或者7所示。
  3. 根据权利要求1所述的抗体，其中所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、4、6、8、9、10或者28所示。
  4. 根据权利要求1所述的抗体，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:10所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示。
  5. 针对水痘-带状疱疹病毒的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:1、3、5和7中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:2、4、6、8、9、10和28中任何一项具有至少90％的一致性。
  6. 根据权利要求1-5中任一项所述的抗体，其中
    所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)，优选地，所述抗体为全人源抗体；和/或
    所述抗体为单克隆抗体；和/或
    所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区，优选地，所述重链恒定区为IgG1亚型；和/或
    所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区，优选地，所述轻链恒定区为κ亚型。
  7. 如权利要求1-6中任一项所述的抗体，其中
    所述抗体结合水痘-带状疱疹病毒gH/gL蛋白；和/或
    所述抗体介导抗体依赖性的细胞介导的细胞毒作用(ADCC)。
  8. 核酸分子，其编码权利要求1-7中任一项所述的抗体。
  9. 药物组合物，其包含权利要求1-7中任一项所述的抗体以及药学上可接受的赋形剂、稀释剂或载体。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗水痘和带状疱疹。
  11. 权利要求1-7中任一项所述的抗体、权利要求8所述的核酸分子、或权利要求9或10所述的药物组合物在制备用于预防或治疗水痘和带状疱疹的药物中的用途。
  12. 预防或治疗水痘和带状疱疹的方法，其包括向有需要的个体给予权利要求1-7中任一项所述的抗体、或权利要求9或10所述的药物组合物。
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