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WO2014070109A1 - Anticorps spécifique de l'entérovirus 71 - Google Patents

Anticorps spécifique de l'entérovirus 71 Download PDF

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WO2014070109A1
WO2014070109A1 PCT/SG2013/000462 SG2013000462W WO2014070109A1 WO 2014070109 A1 WO2014070109 A1 WO 2014070109A1 SG 2013000462 W SG2013000462 W SG 2013000462W WO 2014070109 A1 WO2014070109 A1 WO 2014070109A1
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Prior art keywords
antibody
disease
binding
linked
exemplified
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Hwei-Sing Jimmy Kwang
Qiang JIA
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Temasek Life Sciences Laboratory Ltd
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Temasek Life Sciences Laboratory Ltd
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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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • 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
    • C07K2317/30—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
    • C07K2317/70—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to at least one Enterovirus 71 (EV71 ) specific monoclonal antibody.
  • EV71 Enterovirus 71
  • the antibody may be capable of being used in prophylaxis and therapeutics of EV71 infection.
  • Enteroviruses are a heterogeneous group of pathogens responsible for a broad spectrum of human and nonhuman diseases. Enteroviruses belong to a large genus within the family Picornaviridae; other genera within this family include rhinoviruses, hepatoviruses, cardiovi ruses, and aphthoviruses.
  • the enterovirus genus encompasses polio viruses, coxsackie A viruses (CAV), coxsackie B viruses (CBV), echoviruses, and enteroviruses 68- 71 , as well as a number of uncharacterized enteroviruses isolated from humans and other primates.
  • enteroviral virions comprise an icosahedral capsid with no envelope enclosing a core comprising infectious, single- stranded genomic sense RNA (ssRNA), about 7-8.5 kb in size.
  • Enteroviruses are distinguished from other members of the picornaviridae by their stability in acid and their fecal-oral route of passage and transmission. Virus entry into cells is believed to involve specific cellular receptors.
  • Virion proteins include multiple copies of four capsid proteins (VP1 , VP2, VP3 and VP4).
  • a small protein, VPg (Mr about 24 x 10 3 ), is linked covalently to the 5' terminus of the genomic RNA.
  • Other proteins of the enteroviruses include the predominantly non-structural proteins, P2 and P3.
  • Enterovirus 71 belongs to the human Enterovirus A species of the Enterovirus genus. EV71 is not a zoonotic agent and is the causative agent of a number of neurological diseases including aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, and poliomyelitis-like syndrome.
  • EV71 is also associated with large outbreaks of hand foot and mouth disease (HFMD) in human beings including other enteroviruses such as CA16, CA5, CA9 and Echo 7.
  • HFMD hand foot and mouth disease
  • EV71 has raised the most public concern due to its large scale outbreak in Asia- Pacific over the last decade and its ability in causing severe neurological complications and sometimes fatality.
  • Effective in vivo protection against EV71 infection accomplished by passive transfer of mouse anti-sera (polyclonal antibodies) which is raised against whole virus and synthetic peptides (Foo, Alonso et al. 2007) has been demonstrated.
  • Application of immune sera has also been used for therapy and prophylactic against neonatal enterovirus infection and in immunocompromised individuals (Abzug, Keyserling et al. 1995). However, these methods are not 100% effective in eradicating the virus.
  • IVIG Intravenous immunoglobulin
  • Mab neutralizing monoclonal antibody
  • the epitope may be at least one conformational epitope of EV71.
  • the antibody may be a neutralising antibody. More in particular, the antibody has substantially the immunological binding characteristics of monoclonal antibody Mab10D3. Even more in particular, the antibody may be of subclass immunoglobulin M.
  • the antibody may be at least one human, humanized or chimeric antibody.
  • EV71 may be of genotype selected from the group consisting of A, B1 , B2, B3, B4, B5, C1 , C2, C3, C4 and C5.
  • the antibody may be labelled with at least one radionuclide and/or fluorochrome.
  • the antibody may be linked with at least one drug, anti-viral drug, and/or toxin.
  • a pharmaceutical composition comprising the antibody according to any aspect as exemplified and a pharmaceutically acceptable carrier.
  • an isolated antibody according to any aspect as exemplified for use in medicine there is provided use of at least one antibody according to any aspect as exemplified for the preparation of a medicament for treatment and/or prevention of EV71 and/or at least one EV71 -linked disease.
  • the EV71 -linked disease may at least be one disease selected from the group consisting of aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, poliomyelitis-like syndrome, and Hand, Foot and Mouth disease.
  • an isolated antibody according to any aspect as exemplified for use in treatment of and/or prevention of EV71 and/or at least one EV71 -linked disease.
  • the EV71 -linked disease may at least be one disease selected from the group consisting of aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, poliomyelitis-like syndrome, and Hand, Foot and Mouth disease.
  • a method of treatment and/or prevention of EV71 and/or at least one EV71 -linked disease comprising administering to a subject in need thereof at least one antibody or a fragment thereof according to any aspect as exemplified.
  • the EV71 -linked disease may at least be one disease selected from the group consisting of aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, poliomyelitis-like syndrome, and Hand, Foot and Mouth disease.
  • a method of detecting and/or quantifying the presence and distribution of at least one EV71 infected cell in a subject comprising:
  • the step of detecting may comprise contacting the sample with a binding protein that contains or is conjugated to a detectable element.
  • the antibody may be immobilized onto a solid surface.
  • the binding protein may contain a radioactive atom, is conjugated to a fluorescent molecule, or is conjugated to an enzyme.
  • a kit for diagnosing EV71 and/or at least one EV71 -linked disease comprising at least one antibody or a fragment thereof according to any aspect as exemplified.
  • the EV71 -linked disease may at least be one disease selected from the group consisting of aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, poliomyelitis-like syndrome, and Hand, Foot and Mouth disease.
  • Figure 1 are results obtained from an immunofluorescence assay of CA16 (isolated in Finland, 1994, U05876) infected Vero cells with Mab against 3D and Mab10D3.
  • A-B Fluorescence microscopy
  • C-D merged image of bright field and fluorescence microscopy.
  • Figure 2 are results of immunofluorescence assays showing cross reactivity of Mab10D3 to EV71 subgenotypes. Vero cells were infected with EV71 virus strains as depicted at the top-left corner of each image.
  • Figure 3 are results of immunofluorescence assays showing that escape mutants of different EV71 genogroups are no longer recognized by Mab10D3. Escape mutants of Mab10D3 were created by incubating EV71 virus strains A, B4, C2 and C4 with an excess of Mab, followed by sequential incubation of RD cells for 4 days until cytopathic effects (CPE) was observed. Escaped viruses were then collected and added to fresh RD cells.
  • CPE cytopathic effects
  • CPE was visible after 2 dpi (brightfield images in third row) when cells were fixed and labeled with Mab10D3 followed by FITC coupled secondary antibody. Cells infected with wild-type viruses served as positive controls (first row). Mab10D3 staining was abolished in all four escape mutants carrying either the mutation P59L or E67D (second row).
  • Figure 4 are results of immunofluorescence assays showing that P59 and E67 of EV71 VP3 protein are essential for Mab10D3 binding.
  • RG viruses were engineered using the wild-type B4 backbone. The escape mutations P59L and E67D were introduced individually (RGV1 , RGV2) and together (RGV3).
  • RD cells were infected with wt B4, RGV B4 and mutated RG viruses for 2 days.
  • CPE was visible for all viruses 2 dpi when cells were fixed and labeled with Mab10D3 followed by FITC coupled secondary antibody. While B4 wt and B4 RGV gave a clear IFA signal, the signal was abolished in all three mutated RGVs, showing that Mab10D3 binding relies on both P59 and E67.
  • Figure 5 are results of immunofluorescence assays showing that P59 and E67 of EV71 VP3 protein are essential for Mab10D3 mediated virus neutralization.
  • the loss of neutralization of mutated RG viruses was established in a microneutralization assay.
  • 100 TCID 5 0 of wt B4 RGV and mutated RGVs were mixed with serial dilutions of Mab10D3 or positive control Mab51. While 10D3 neutralized the wt RGV efficiently, no neutralization was observed for the mutated RGVs, indicating that P59 and E67 of VP3 are essential for Mab binding and neutralization.
  • the positive control Mab51 which recognizes a conserved linear epitope on VP1 , efficiently neutralized all RGVs.
  • administering means delivering using any of the various methods and delivery systems known to those skilled in the art.
  • Administering can be performed, for example, intraperitoneally, intracerebrally, intravenously, orally, transmucosally, subcutaneously, transdermal ⁇ , intradermally, intramuscularly, topically, parenterally, via implant, intrathecally, intralymphatically, intralesionally, pericardially, or epidurally.
  • An agent or composition may also be administered in an aerosol, such as for pulmonary and/or intranasal delivery.
  • Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the term “antibody” refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific epitope. Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody.
  • the term “monoclonal antibody” may be referred to as "Mab”.
  • the antibody “monoclonal antibody 10D3” may be used interchangeably with "Mab10D3".
  • the antibody includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, and fragments thereof which retain the antigen binding function of the parent antibody.
  • antibody fragment refers to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Fragments of the Mab10D3 are encompassed by that which is exemplified so long as they retain the desired affinity of the full-length antibody. In particular, it may be shorter by at least one amino acid.
  • the fragment of Mab10D3 comprises the antigen binding function that enables it to bind to a specific epitope of EV71.
  • antigen refers to a substance that prompts the generation of antibodies and can cause an immune response. It may be used interchangeably in that which is exemplified with the term "immunogen".
  • immunogens are those substances that elicit a response from the immune system, whereas antigens are defined as substances that bind to specific antibodies.
  • An antigen or fragment thereof may be a molecule (i.e. an epitope) that makes contact with a particular antibody.
  • numerous regions of the protein may induce the production of antibodies (i.e. elicit the immune response), which bind specifically to the antigen (given regions or three-dimensional structures on the protein).
  • the antigen may include but is not limited to a capsid protein and/or non-structural proteins of EV71.
  • epitope refers to a consecutive sequence of from about 5 to about 13 amino acids which form an antibody binding site.
  • the epitope in the form that binds to the Mab or binding protein may be in a denatured protein that is substantially devoid of tertiary structure.
  • the epitope may be a conformational epitope.
  • a "conformational epitope” is herein defined as a sequence of subunits (usually, amino acids) composing an antigen that comes in direct contact with a receptor of the immune system.
  • a receptor Whenever a receptor interacts with an undigested antigen, the surface amino acids that come in contact may not be continuous with each other if the protein is unwound. Such discontinuous amino acids that come together in three dimensional conformation and interact with the receptor's paratope are called conformational epitopes. In contrast, if the antigen is digested, small segments called peptides are formed, which bind with major histocompatibility complex molecules, and then later with T cell receptors through amino acids that are continuous in a line. These are known as linear epitopes.
  • derivative refers to the chemical modification of at least one EV71 epitope.
  • Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • humanized antibody refers to at least one antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • hybridoma refers to cells that have been engineered to produce a desired antibody in large amounts.
  • B cells are removed from the spleen of an animal that has been challenged with the relevant antigen and fused with at least one immortalized cell. This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), will multiply rapidly and indefinitely and will produce at least one antibody.
  • Immortalised cells as used herein are also known as transformed cells - i.e. cells whose growth properties have been altered. This does not necessarily mean that these are “cancer” or “tumour” cells, i.e. able to form a tumour if introduced into an experimental animal, although in some cases they may do. Immortalised cell lines include but are not limited to NS1 , Jurkat, HeLa, HepG2, SP2/0, Hep-3b and the like.
  • the term "immunological binding characteristics" of a Mab or related binding protein in all of its grammatical forms, refers to the specificity, affinity and cross-reactivity of the Mab or binding protein for its antigen.
  • isolated is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • neutralising antibody is herein defined as an antibody that can neutralise the ability of that pathogen to initiate and/or perpetuate an infection in a host. That which is exemplified provides a neutralising human monoclonal antibody, wherein the antibody recognises an antigen from EV71.
  • “Pharmaceutically acceptable carriers” can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients.
  • auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients.
  • the instant compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.
  • sample is used in its broadest sense.
  • a biological sample suspected of containing nucleic acids encoding at least one EV71 derived peptide, or fragments thereof, or EV71 itself may comprise a bodily fluid, an extract from a cell, chromosome, organelle, or membrane isolated from a cell, a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solid support), a tissue, a tissue print and the like.
  • the terms “specific binding” or “specifically binding” refer to the interaction between a protein or peptide and an agonist, an antibody, or an antagonist. In particular, the binding is between an antigen and an antibody. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigen or epitope). For example, if an antibody is specific for epitope "A", the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • subject is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a mouse, rat and the like.
  • At least one neutralizing monoclonal antibody that may be capable of neutralising EV71 infection.
  • monoclonal antibodies and related binding proteins that bind specifically to EV71.
  • Monoclonal antibodies also known as "Mabs” may be a substantially homogeneous population of antibodies derivable from a single antibody-producing cell. Thus, all antibodies in the population may be identical and may have the same specificity for a given epitope. The specificity of the Mab responses provides a basis for an effective diagnostic reagent.
  • Monoclonal antibodies and binding proteins derived therefrom also have utility as therapeutic agents.
  • the antibodies as exemplified may provide at least one anti-EV71 antibody which is capable of neutralising EV71 infection and inhibiting cell-to-cell spread.
  • the isolated monoclonal antibody or a fragment thereof of subclass immunoglobulin M may be a neutralising monoclonal antibody or a fragment thereof that may be capable of specifically binding to at least one epitope of EV71.
  • the epitope may be a conformational epitope.
  • (c) antibody that binds to an antigen capable of binding the antibody produced by the hybridoma cell line CBA20120021 The antibody as exemplified may be capable of blocking viral mechanisms to spread within a host. They effectively neutralize cell-free virus particles and inhibit the direct cell-to-cell spread of the virus. Since the antibody specifically binds to a highly conserved epitope of EV71 , which is essential for the virus survival, development of drug resistance against the antibody may be most unlikely. This region of the epitope may be highly conserved and thus mutations seldom occur.
  • the major advantage of using Mabs over immune sera is its potential availability in large quantities, prepared either in hybridoma supernatant or ascites fluid.
  • the defined epitope of the antibodies as exemplified also allows for mechanistic study of its virus neutralization ability to be easily performed.
  • These antibodies may also be easily purified by affinity chromatography, using any method known in the art. For example, but not limiting, the antibodies as exemplified may be purified using the protocol disclosed in Li, Mao et al., 2009.
  • the neutralizing antibodies as exemplified may result in a complete protection from cytopathic effects (CPE) of EV71. These antibodies may be capable of effective in vivo protection against EV71 infection. The efficacy and specificity of these antibodies are shown in the Examples.
  • the neutralizing antibody as exemplified may bind to at least one conformational epitope of EV71. This is advantageous as epitopes usually exist in nature in a three dimensional conformation and the antibodies may thus be more efficient and effective in detecting the presence of EV71 and/or subsequently neutralizing the effect of EV71. These antibodies may thus be able to bind and to recognize viral antigens without prior treatments of a tissue section.
  • the conformational epitope may comprise at least one capsid protein and/or at least one non-structural protein.
  • the capsid protein may be an intact virus capsid protein.
  • the capsid protein may comprise one or more proteins selected from the group consisting of VP1 , VP2 VP3, VP4 and VPO precursor.
  • the antibody as exemplified may comprise the immunological binding characteristics of monoclonal antibody Mab10D3.
  • These immunological binding characteristics of Mab10D3 are produced by hybridoma Mab10D3, deposited with deposited with the CellBank Australia, 214 Hawkesbury Road, Westmead NSW 2145, Australia on 1 September 2011 , in accordance with the provisions of the Budapest Treaty, and assigned Accession Number CBA20120021.
  • the hybridoma provides a continuous source of the Mabs and binding proteins as exemplified.
  • the antibody may be of subclass immunoglobulin M.
  • the antibody as exemplified may be capable of recognizing EV71 of any genotype.
  • the antibody may be capable of recognizing EV71 of a genotype selected from the group consisting of A, B1 , B2, B3, B4, B5, C1 , C2, C3, C4 and C5.
  • the Mabs as exemplified may be produced by any technique that provides for the production of antibody molecules by continuous cell lines in culture. Such methods include, but are not limited to, the hybridoma technique originally developed in 1975 by Kohler and Milstein, as well as the trioma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985). Human antibodies can be used and can be obtained by using human hybridomas (Cote et al., 1983).
  • chimeric antibodies By splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • the genes from a mouse antibody molecule such as Mab10D3 can be spliced together with genes from a human antibody molecule of appropriate biological activity.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine Mab and a human immunoglobulin constant region. Chimeric antibodies are also those that contain a human Fc portion and a murine (or other non-human) Fv portion.
  • humanized antibodies e.g., US 5,585,089 and/or US 5,225,539, which are incorporated herein by reference in their entirety.
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule. Both chimeric and humanized antibodies may be monoclonal. Such human or humanized chimeric antibodies may be preferred for use in in vivo diagnosis or therapy of human diseases or disorders.
  • Antibody fragments that contain the idiotype of the antibody molecule can be generated by known techniques. For example, such can be produced by pepsin digestion of the antibody molecule; the Fab fragments can be generated by reducing the disulfide bridges of the F(ab)2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Such antibody fragments can be generated from any of the polyclonal or monoclonal antibodies as exemplified.
  • screening for the desired antibody can be accomplished by techniques known in the art.
  • these techniques may include but are not limited to radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme, radioisotope labels or the like), western blots, precipitation reactions, agglutination assays (gel agglutination assays, hemagglutination assays or the like), immunofluorescence assays, Immunoelectrophoresis assays and the like.
  • the antibody binding may be detected by detecting a label on the primary antibody.
  • the primary antibody may be detected by detecting binding of a secondary antibody or other reagent to the primary antibody.
  • the secondary antibody may be labeled.
  • the antibody may be labelled with at least one radionuclide and/or fluorochrome in order to improve targeting of EV71 in vivo in at least a diagnostic and/or therapeutic capacity. For example, detecting EV71 by Positron Emission Tomography (PET) may be used.
  • PET Positron Emission Tomography
  • the antibody labeled with the radionuclide may enable better targeting of EV71 for detection and/or treatment.
  • the antibody may be further labelled with at least one drug, anti-viral drug and/or toxin for treatment of EV71.
  • the antibodies may be used to deliver drugs to the infected cells with a high degree of specificity to suppress viral infections.
  • the drugs may include but are not limited to ribavirin and or other potent inhibitors of EV71 infection such as bovine or human lactoferrins and the like.
  • the antibody as exemplified linked with at least one anti-viral drug may increase the drug availability for EV71 infected cells which may enhance the efficiency of the drug and also reduce side effects usually caused by the anti-viral drugs. This may lead to a new treatment for EV71 possibly reducing other complications associated with EV71 infection.
  • Mab10D3 may be linked with at least one traceable agent and may be potentially used to quantify EV71 infected cells in vitro or in vivo.
  • the traceable agent may be any biological or chemical component which is traceable.
  • the traceable agent may include, but is not limited to environmental agents, blood markers, antigens, pesticides, drugs, chemicals, toxins, PCBS, PBBS, lead, neurotoxins, blood electrolytes, metabolites, analytes, NA+, K+, CA+, urea nitrogen, creatinine, biochemical blood markers and components, ChE, AChE, BuChe, tumour markers, PSA, PAP, CA 125, CEA, AFP, HCG, CA 19-9, CA 15-3, CA 27-29, NSE, hydroxybutyrate, acetoacetate, anti-malarial drugs such as amodiaquine, artemether, artemisinin, artesunate, atovaquone, cinchonine, cinchonidine, chloroquine,
  • the antibodies as exemplified may be used in methods known in the art relating to the detection or localization of EV71.
  • these methods may include but are not limited to Western blotting, ELISA, radioimmunoassay, immunofluorescence assay, immunohistochemical assay, and the like.
  • the method of detecting and/or quantifying as exemplified may be a non-invasive method and may also be useful for studying several immunological aspects of EV71.
  • a method of detecting and/or quantifying the presence and distribution of at least one EV71 infected cell in a subject comprising:
  • the step of detecting may comprise contacting the sample with a binding protein that contains or is conjugated to a detectable element.
  • the antibody as exemplified may be immobilized onto a solid surface.
  • the binding protein may contain a radioactive atom, may be conjugated to a fluorescent molecule, or may be conjugated to an enzyme.
  • kits for the qualitative and/or quantitative determination of EV71.
  • kits may contain at least the Mab or related binding protein as exemplified, means for detecting immunospecific binding of the Mab or related binding protein to EV71 in a biological sample, and instructions for use, depending upon the method selected, e.g., "competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain positive and negative controls. They may be configured to be used with automated analyzers or automated immunohistochemical slide staining instruments.
  • an assay kit as exemplified may further comprise a second antibody or binding protein that may be labelled or may be provided for attachment to a solid support (or attached to a solid support).
  • a second antibody or binding protein may be, for example, one that binds to EV71.
  • Such second antibodies or binding proteins may be polyclonal or monoclonal antibodies.
  • Mab10D3 are highly efficacious in detecting and/or neutralising the effect of EV71. They may be used separately and/or together for efficient and effective early detection of EV71.
  • the antibodies as exemplified provide convenient, highly specific and sensitive means for detecting EV71.
  • One such means is the ELISA format.
  • Mab10D3 can be used as a capture antibody, either alone or in combination. If used alone, the selected antibody can be used as the capture antibody and that same antibody conjugated with horseradish peroxidase (HRP) can be used as detecting antibody.
  • HRP horseradish peroxidase
  • EV71 viruses include, for example, dot-blot and in situ hybridization.
  • At least one method of treatment and/or protection of EV71 and/or at least one EV71-linked disease comprising administering to a subject in need thereof at least one antibody or a fragment thereof as exemplified.
  • the antibody as exemplified may be administered in combination with other similar antibodies targeting different EV71 epitopes.
  • the viruses may thus be given no opportunity to adapt to this form of therapy.
  • EV71 -linked diseases include but are not limited to aseptic meningitis, encephalitis, cranial nerve palsies, Guillan-Barre syndrome, poliomyelitis-like syndrome, Hand, Foot and Mouth disease and the like.
  • At least one pharmaceutical composition comprising an antibody as exemplified and a pharmaceutically acceptable carrier.
  • the EV71 Mabs as exemplified have advantages over other current methodologies as diagnostic tools.
  • the Mabs are highly specific for EV71 which is still not well understood in the field of virology.
  • Such highly specific Mabs represent a breakthrough in the field of EV71 diagnosis.
  • the Mabs as exemplified may recognize all, or essentially all, of the EV71 genotypes.
  • These Mabs also provide a safe and convenient diagnostic approach for the detection of EV71.
  • the antibodies are useful for diagnosis and for the preparation of recombinant antibodies for treatment, and as such will be very useful tools in restraining a potential EV71 outbreak.
  • reagents which can be used in a diagnostic assay to rapidly and accurately distinguish EV71 from CA16, and which can be used to identify the strain of EV71 specifically.
  • the Mabs as exemplified may be used to rapidly and accurately identify the strain of EV71 and/or differentiate between EV71 and CA16.
  • Such reagents and methods will allow the clinician to improve the speed and accuracy of processing large numbers of clinical samples.
  • Such reagents and methods will also aid the clinician in patient management, eliminate unnecessary tests, improve the speed and accuracy of diagnosis and prognosis, help control EV71 infection, and reduce the use of unnecessary antibiotics.
  • the Mabs as exemplified n may be used for direct detection of EV71 viral particles from blister or oral swabs.
  • RNA Interference Technology From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engeike, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.
  • Hybridomas secreting specific Mabs were derived from BALB/c mice which had been immunized twice intramuscularly with purified EV71 , B4 strain (5865/SIN/000009,GenBank Accession number AF316321 ) in 0.1 ml of PBS, emulsified with an equal volume of adjuvant (SEPPIC, France). An intraperitoneal booster of the same dose of virus was given three days before splenocytes were fused to the SP2/0 myeloma cells which were purchased from ATCC. Hybridomas identified to produce specific antibody, were cloned by limiting dilution and expanded in 75cm 2 flasks.
  • hybridoma suspension was harvested and cell debris pelleted by centrifugation at 400 g for 10 min, followed by collection of the supernatant and storage at -20°C.
  • Mab concentrations were determined spectrophotometrically (Nanodrop, DE, USA).
  • 96-well, round-bottom microtiter plates (Nunc, Roskilde, Demark) were coated with 500ng "1 ⁇ g/well of capture antibody in 100 ⁇ of carbonate buffer (73 mM sodium bicarbonate and 30 mM sodium carbonate, pH 9.7) overnight at 4°C or 37°C for 2 h.
  • carbonate buffer 73 mM sodium bicarbonate and 30 mM sodium carbonate, pH 9.7
  • the plates were washed twice with PBST, followed by two washes with PBST after each incubation with antibody or antigen.
  • the antibody-coated plates were blocked with 100 ⁇ of blocking buffer (PBS containing 5% milk) for 1 h at room temperature and then incubated at 37°C for 1 h with 100 ⁇ of virus-containing samples diluted in PBST.
  • blocking buffer PBS containing 5% milk
  • Virus binding was detected by incubation with 100 ⁇ of horseradish peroxidase-conjugated detection Mab (in-house labeling; Pierce) for 1 h at 37°C. Chromogen development was mediated by the addition of 100 ⁇ of freshly prepared substrate solution (o-phenylenediamine-dihydrochloride; Sigma). The reaction was stopped by adding 0.1 N sulfuric acid, and the optical density at 490 nm was recorded. The detection limit was determined as the optical density value that gave a signal-to-noise ratio of 3. Characterization of Mab 0D3
  • Mab10D3 belongs to subclass IgM as identified with an isotyping Kit (GE) and possessed conformational epitope, showing positive signal in EV71 infected Vera cells. Mab10D3 reacted with conformational epitope and neutralizes all genotypes of EV71 strains ( Figure 2).
  • GE isotyping Kit
  • Mab10D3 of has demonstrated in vitro neutralization activity against all representative EV71 subgenotypes (A, B1-5, C1-5) up to a dilution of 1 :2048 in vitro. Prophylactic and therapeutic studies of .Mab10D3 in mouse have showed 100% in vivo protection against EV71 infection.
  • Neutralization activity of monoclonal antibodies was determined by in vitro microneutralization assay in RD cells. Two-fold serial monoclonal antibody dilutions (50 ⁇ each) were mixed with equal volume of 200 TCID50 of virus, and incubated at 37°C for 1 h. The antibody-virus mixtures were then added to the wells of the microtiter plates containing RD cells. The highest dilution of monoclonal antibody that inhibited virus growth was considered as the neutralization antibody titer and was determined after incubation at 37 ° C for 96 h. Each assay was performed independently for three times.
  • Mab10D3 which is of isotype IgM gave a complete protection from cytopathic effects (CPE) at a dilution up to 1 :2048 in an in vitro micro-neutralization assay for heterologous EV71 strains and 100% in vivo protection against EV71 infection in Bal b/c mice.
  • CPE cytopathic effects
  • This experiment was carried out by using Bal b/c mice.
  • mice 7days mice were injected with Mab10D3 at 10 ⁇ g/g of body weight one day prior to lethal challenge with 10 7 plaque forming units (PFU) of mouse-adapted EV71 strain HFM41.
  • a control group was injected with an irrelevant mouse IgM antibody (isotype control) before the lethal challenge. While the control animals which received an isotype antibody developed severe limb paralysis as early as day 6 post-infection, mice pre-treated (prophylactic studies) with Mab10D3 did not display any of the disease manifestations and remained healthy throughout the experiment.
  • mice were susceptible to infection with the mouse-adapted EV71 strain HFM41 via the intraperitoneal route of inoculation and anti- EV71 antibody Mab10D3 was able to confer 100% protection against the lethal EV71 challenge at a dose of 10 ⁇ g/g of body weight.
  • the epitope of Mab10D3 was found to be conformational as the antibody did not react with any capsid protein in a Western blot. Hence, the epitope could not be mapped by the conventional fashion of truncated peptides.
  • Epitope mapping of Mab10D3 was instead performed by escape mutant selection. Wild-type EV71 viruses from different subgenogroups (A, B4, C2, C4) were incubated with an excess of Mab10D3 on RD cells. If no cytopathic effects (CPE) was visible after 4 days, supernatants were filtered and added to fresh RD cells. This process was repeated until CPE was evident. 1 to 3 cycles were necessary to isolate escape mutants for all subgenogroups.
  • CPE cytopathic effects
  • the escape mutants were called E1-3/B4 (three individual experiments using EV71-B4 virus), E/A (EV71-A), E/C2 (EV71-C2), and E/C4 (EV71-C4), their Tissue Culture Infective Dose (TCID 50 ) was measured by end-point dilution assay and they were tested for reactivity with Mab10D3 in an I FA.
  • RD cells were infected with an equal amount of either wild-type virus as positive controls or escaped viruses and observed for 2 days until CPE was visible (Figure 3, third row). After incubation with Mab10D3, there was a clear signal for the wt viruses ( Figure 3, first row) but no signal for any of the corresponding escape mutants ( Figure 3, second row).
  • Microneutralization assays were conducted with 100 TCID50 of wild-type viruses, escape mutants and mutated RG viruses against the ab10D3 and VP1 linear neutralizing Mab51 as a positive control..
  • the P1 structural gene region of each escape mutant was sequenced and compared to its parental strain. Viral RNA was extracted and purified with the QIAamp viral RNA isolation kit from filtered RD cell culture supematants containing wt and escape mutant virus. Reverse transcription was then carried out using gene- and strain-specific primers followed by PCR amplification of 2 overlapping portions of the P1 gene using the primer pairs listed in Table 5. After gel purification the cDNAs were sequenced directly and the sequences were analyzed. In the six escape mutants two mutations were identified in the structural gene VP3.
  • mutants E1 -3/B4 derived from the parental strain B4 had a glutamate to aspartate substitution at amino acid position 67 of VP3, while the other three mutants E/A, E/C2, E/C4 derived from A, C2, and C4 subgenogroups had a proline to leucine substitution at amino acid position 59 of VP3 (Table 6).
  • rimer pairs use or t e amp cat on o t e reg on.
  • an EV71-B4 virus consisting of the EV71-B4 (5865/SIN/000009; GenBank Accession number AF316321 ;SEQ ID NO:1 ) sequence by utilizing a human RNA polymerase I driven reverse genetics system was engineered.
  • the two VP3 mutations P59L and E67D were then introduced alone (RG/B4-P59L, RG/B4- E67D) or in tandem (RG/B4-PE59,67LD) into the wild-type RG virus (B4 RGV) by site- directed mutagenesis.
  • the RG viruses were then rescued in RD cells and passage 2 viruses were used in subsequent experiments.
  • RD cells were infected with B4-wt as positive control, RG/B4-wt virus or the mutated RG viruses.
  • the cells were fixed 2 dpi when CPE was clearly observed ( Figure 4, brightfield). While both original viruses (B4-wt and RG/B4-wt) were clearly detected by Mab10D3, no fluorescence was visible for the mutated RGVs carrying either a single or double mutation.
  • Mab10D3 was unable to neutralize the mutated RG viruses in an in vitro microneutralization assay, while the neutralization titer of Mab10D3 against RG/B4-wt reached 2 ⁇ 6 which was the same as for B4-wt.
  • Mab51 against the linear neutralizing epitope KQEKD on VP1 was incubated with the mutated RG viruses.
  • the VP1 epitope was unaffected by our mutagenesis, Mab51 was still able to efficiently neutralize all RG viruses ( Figure 5 and Table 4). Accordingly, it appears that two escape mutations P59L and E67D are solely responsible for the abolishment of Mab10D3 binding to the VP3 protein and neutralization of EV71 virus.
  • RNA Interference The Nuts & Bolts of siRNA Technology, DNA Press, 2003;

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Abstract

Cette invention concerne au moins un anticorps ou fragment d'anticorps isolé, l'anticorps ou fragment d'anticorps étant capable de se lier spécifiquement à au moins un peptide dérivé de l'EV71. En particulier l'anticorps ou fragment d'anticorps est capable de se lier à un épitope conformationnel sur la capside VP3.
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CN107266566A (zh) * 2017-06-06 2017-10-20 中国医学科学院病原生物学研究所 人源抗ev71病毒中和性抗体e1及其应用
CN110551211A (zh) * 2018-05-30 2019-12-10 福又达生物科技股份有限公司 含抗肠病毒71型vp1蛋白单克隆抗体的检测试剂盒
TWI688653B (zh) * 2018-01-30 2020-03-21 長庚醫療財團法人林口長庚紀念醫院 人源抗腸病毒７１型單株抗體的製法、產物及其應用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107266566A (zh) * 2017-06-06 2017-10-20 中国医学科学院病原生物学研究所 人源抗ev71病毒中和性抗体e1及其应用
CN107266566B (zh) * 2017-06-06 2019-07-12 中国医学科学院病原生物学研究所 人源抗ev71病毒中和性抗体e1及其应用
TWI688653B (zh) * 2018-01-30 2020-03-21 長庚醫療財團法人林口長庚紀念醫院 人源抗腸病毒７１型單株抗體的製法、產物及其應用
CN110551211A (zh) * 2018-05-30 2019-12-10 福又达生物科技股份有限公司 含抗肠病毒71型vp1蛋白单克隆抗体的检测试剂盒
CN110551211B (zh) * 2018-05-30 2022-05-24 福又达生物科技股份有限公司 含抗肠病毒71型vp1蛋白单克隆抗体的检测试剂盒

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