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WO2013043125A1 - Anticorps spécifiques d'entérovirus 71 et leurs utilisations - Google Patents

Anticorps spécifiques d'entérovirus 71 et leurs utilisations Download PDF

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Publication number
WO2013043125A1
WO2013043125A1 PCT/SG2012/000344 SG2012000344W WO2013043125A1 WO 2013043125 A1 WO2013043125 A1 WO 2013043125A1 SG 2012000344 W SG2012000344 W SG 2012000344W WO 2013043125 A1 WO2013043125 A1 WO 2013043125A1
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Prior art keywords
antibody
binding
cell line
epitope
hybridoma cell
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English (en)
Inventor
Qiang JIA
Xiao Fang LIM
Jimmy Kwang
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Temasek Life Sciences Laboratory Ltd
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Temasek Life Sciences Laboratory Ltd
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Priority to CN201280043224.XA priority Critical patent/CN104220090A/zh
Publication of WO2013043125A1 publication Critical patent/WO2013043125A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates generally to Enterovirus 71 (EV71 ) specific monoclonal antibodies.
  • the antibodies may be neutralising monoclonal antibodies.
  • 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.
  • EV71 exhibits a wide variation in clinical presentation.
  • Enzyme- Linked Immunosorbent Assay is also commonly used. Recombinant protein capture ELISA (Shin, Li et al. 2000) and IgM capture ELISA (Wang, Lin et al. 2004) which are both available as commercial diagnostic kits are limited by the lack of specificity for EV71 (Hovi and Roivainen 1993), often resulting in false positive results.
  • IVIG Intravenous immunoglobulin
  • the present invention addresses the problems above, and in particular provides at least one novel monoclonal antibody that is specific to at least one EV71 and/or EV71 related disease.
  • the monoclonal antibody may be neutralising.
  • the present invention provides at least one isolated neutralising monoclonal antibody or a fragment thereof of subclass immunoglobulin M that is capable of specifically binding to at least one epitope of EV71.
  • the antibody may be selected from the group consisting of:
  • the epitope according to any aspect of the present invention may comprise, consists of or consists essentially of the amino acid sequence KQEKD (SEQ ID NO:1 ) and/or may be a conformational epitope.
  • the present invention provides at least one isolated monoclonal antibody or a fragment thereof that is capable of specifically binding to at least one epitope of E V71.
  • the antibody may be selected from the group consisting of:
  • the epitope according to any aspect of the present invention may comprise, consists of or consists essentially of the amino acid sequence KQEKD (SEQ ID NO:1 ) or an epitope to which Mab4 specifically binds.
  • the present invention provides at least one isolated hybridoma cell line deposited with CellBank Australia (214 Hawkesbury Road, Westmead NSW 2145) on 27 July 2011 with accession number CBA20110004, CBA20110005, CBA20110006, or CBA20110007.
  • the present invention provides a method of detecting and/or quantifying the presence of EV71 , a method of treating and/or preventing EV71 and/or at least one EV71 -linked disease, the antibody or fragment thereof of the present invention for use as medicine, use of the antibody or fragment thereof of the present invention for the preparation of a medicament, kits, nucleic acids and uses thereof.
  • preferred embodiments of the present invention allow an optimal use of the isolated antibodies to take advantage of their accuracy and specificity to at least one epitope of EV71 .
  • Figure 1 is a photograph of the results obtained from western blot hybridization of Mab51 and Mab53 during characterization.
  • Lane 1 Rhabdomyosarcoma RD cells total cell lysate.
  • Lane 2 Concentrated C4 (Yamagata) virus.
  • Lane 3 GST-tagged VP1 recombinant protein ( ⁇ 61 kDa).
  • Lane 4 GST-tagged VP2 recombinant protein ( ⁇ 56kDa).
  • Lane 5 GST-tagged VP3 recombinant protein ( ⁇ 50kDa).
  • Figure 2 is a photograph of the results obtained from western blot hybridization of Mab51 and Mab53 during epitope mapping.
  • Lane 1 GST-KQEK fusion protein ( ⁇ 25kDa)
  • Lane 2 GST-HKQEKD fusion protein
  • Lane 3 GST-HKQEK fusion protein
  • Lane 4 GST-KQEKD fusion protein
  • Lane 5 GST fusion protein.
  • Figures 3A, 3B and 3C are photographs of the results obtained from western blot hybridization of Mab4 epitope mapping.
  • Figure 3A Lane 1 : VP1 (1-66) GST recombinant protein protein, Lane 2: VP1 (1 -132), Lane 3: VP1 (1 -163), Lane 4: VP1 (1 -177), Lane 5: VP1 (1 -208), Lane 6:(1 -222), Lane 7: VP1 (1 -240), Lane 8: VP1 (1 -260);
  • Figure 3B Lane 1 : GST protein (negative control), Lane 2: A48 (48-297), Lane 3: A32 (32-297), Lane 4: A24 (24-297), Lane 5: A10 (10-297).
  • Figure 3C Lane 1 : GST protein (negative control), Lane 2: Ai2 (12-297), Lane 3: A14 (14-297), Lane 4: A16 (16-297), Lane 5: A18 (18- 297), Lane 6: A18 (20-297), Lane 7: A22 (22-297) and lane 8: A10 (10-297, positive control).
  • Figure 4 is a photograph of the results obtained from western blot hybridization showing cross reactivity of Mab51 and Mab53 with EV71 subgenotypes.
  • Lane 1 A (BrCr), Lane 2: B2 (7423/MS/87), Lane 3: B4 (HFM41 ), Lane 4: B5 (NUH0083), Lane 5: C1 (Y90-3761 ), Lane 6: C4 (75-Yamagata), Lane 7: C5 (3437/SIN/06), Lane 8: RG-C2 (AF286504).
  • Figure 5 is a photograph of the results obtained from immunofluorescence assay (IFA) of CA16 (isolated in Finland, 1994, U05876) infected vero cells with Mab against 3D, Mab51 , Mab53, Mab57 and Mab4.
  • IFA immunofluorescence assay
  • Figure 6 is a photograph of the results obtained from IFA of Mab51 to show cross reactivity with EV71 subgenotypes. Vero cells were infected with EV71 virus strains as depicted at the top-left corner of each image.
  • Figure 7 is a photograph of the results obtained from IFA of Mab53 to show cross reactivity with EV71 subgenotypes. Vero cells were infected with EV71 virus strains as depicted at the top-left corner of each image.
  • Figure 8 is a photograph of the results obtained from IFA of Mab4 to show cross reactivity with EV71 subgenotypes. Vero cells were infected with EV71 virus strains as depicted at the top-left corner of each image.
  • Figure 9 is a photograph of the results obtained from IFA of Mab57 to show cross reactivity with EV71 subgenotypes. Vero cells were infected with EV71 virus strains as depicted at the top-left comer of each image.
  • Figure 10A is a picture of a cross-section of control mice (AG129; challenged with EV71 infection) spinal cord with arrow indicating neuropil vacuolation and neuronal loss without inflammation in the anterior horn (original magnification 100x).
  • Figure 10B is a high-power view of Figure 10A showing neuropil vacuolation and loss of neuronal cells without inflammation (original magnification 400x).
  • Figure 11A is a picture of a cross-section of protected mice (AG129; challenged with EV71 infection and prophylactic protection with Mab 51 ) spinal cord showing no significant pathology (original magnification 40x).
  • Figure 11 B is a picture of a cross-section of spinal cord showing no significant pathology (original magnification 100x).
  • Figure 11 C is a high-power view of Figure 1 1A showing no significant pathology (original magnification 400x).
  • 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 51 " may be used interchangeably with "Mab51 ", and which may be capable of specifically binding to EV71 including but not limited to an epitope of SEQ ID NO:1.
  • the antibody includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, and fragments thereof which retain the antigen binding function of the parent antibody.
  • the antibody “Mab57” is capable of specifically binding to EV71 , including but not limited to a conformational epitope comprising at least one capsid protein of EV71 and includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, and fragments thereof which retain the antigen binding function of the parent antibody.
  • the antibody “Mab53” is capable of specifically binding to EV71 including but not limited to at least one epitope of SEQ ID NO:1 and includes monoclonal antibodies, polyclonal antibodies, single-chain antibodies, and fragments thereof which retain the antigen binding function of the parent antibody.
  • the antibody "Mab4" is capable of specifically binding to EV71 including but not limited to at least one epitope and 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 Mab51 , Mab57, Mab53 and Mab4 are encompassed by the invention 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 Mab51 comprises the antigen binding function that enables it to bind to an epitope of SEQ ID NO:1 of a capsid protein of EV71
  • the fragment of a Mab57 comprises the antigen binding function that enables it to bind to a conformational epitope of a capsid protein of EV71
  • the fragment of Mab53 comprises the antigen binding function that enables it to bind to an epitope of SEQ ID NO:1 of a capsid protein of EV71
  • the fragment of Mab4 comprises the antigen binding function that enables it to bind to a specific epitope in EV71.
  • antigen refers to a substance that prompts the generation of antibodies and can cause an immune response. It may be used interchangeably in the present invention 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.
  • 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.
  • 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 wit T cell receptors through amino acids that are continuous in a line. These are known as linear epitopes.
  • a non-limiting example of a linear eptiope is SEQ ID NO:1 and/or the epitope to which Mab4 binds.
  • the term “comprising” is herein defined to be that where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term “comprising” encompasses the more restrictive terms “consisting essentially of” and “consisting of.”
  • the term “derivative,” as used herein, 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.
  • hybridomas 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. Examples of hybridomas are the cell lines with accession number CBA201 10004, CBA201 10005, CBA201 10006, or CBA201 10007.
  • “Immortalised cells” as used herein are also known as transformed cells - i.e. cells whose growth properties have been altered.
  • Immortalised cell lines include but are not limited to NS1 , Jurkat, HeLa, HepG2, SP2/0, Hep-3b and the like.
  • 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.
  • the invention provides a neutralising human monoclonal antibody, wherein the antibody recognises an antigen from EV71.
  • sample as used herein, 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.
  • Mab51 and Mab53 may specifically bind a linear epitope of SEQ ID NO:1
  • Mab57 may specifically bind a conformational epitope on the capsid
  • Mab4 may specifically bind a linear epitope.
  • the term "subject” is herein defined as vertebrate, particularly mammal, more particularly human.
  • the subject may particularly be at least one animal model, e.g., a mouse, rat and the like.
  • animal model e.g., a mouse, rat and the like.
  • the present invention may be practised without undue experimentation according to the method given herein.
  • the methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.
  • the present invention provides isolated 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.
  • 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 according to any aspect of the present application provide at least one anti- EV71 antibody which is capable of neutralising EV71 infection and inhibiting cell-to-cell spread. These antibodies according to any aspect of the present application may be used as prophylactic and/or therapeutic agent(s) for the treatment of E V71 and EV71 associated diseases.
  • 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 linear one of SEQ ID NO:1 or may be a conformational epitope.
  • the conformational epitope may be an intact virus capsid. More in particular, the capsid protein may be VP1 , VP2 VP3, VP4 and/or VP0 precursor.
  • the antibody may have substantially the immunological binding characteristics of monoclonal antibody Mab51.
  • the antibody may be selected from the group consisting of:
  • the antibody may have substantially the immunological binding characteristics of monoclonal antibody Mab57.
  • the antibody may be selected from the group consisting of:
  • These antibodies 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 antibodies specifically bind to a highly conserved epitopes of EV71 for example SEQ ID NO:1 of VP1 and conformational epitope of an intact virus capsid, which are essential for the virus survival, development of drug resistance is most unlikely.
  • These antibodies of the present invention provide several advantages including being capable of use as drugs or vaccines for HFMD.
  • the antibodies according to any aspect of the present invention may be available in large quantities, prepared either in hybridoma supernatant or ascites fluid. There may also be a constant and renewable source of monoclonal antibodies available, with any one of the hybridoma cell lines according to any aspect of the present invention.
  • the defined epitope of the antibodies according to any aspect of the present invention 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.
  • the antibodies according to any aspect of the present invention may be purified using the protocol disclosed in Li, Mao et al., 2009.
  • the neutralizing antibodies according to any aspect of the present invention may result in a complete protection from cytopathic effects (CPE) of EV71 .
  • CPE cytopathic effects
  • 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 according to this invention binds an epitope of VP1 of EV71 of SEQ ID NO:1. This region of the epitope may be highly conserved and thus mutations seldom occur.
  • the neutralizing antibody according to this invention binds 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 according to any aspect of the present invention may comprise the immunological binding characteristics of monoclonal antibody Mab51 or Mab57.
  • These immunological binding characteristics of Mab51 are produced by hybridoma Mab51 , deposited with the CellBank Australia, 214 Hawkesbury Road, Westmead NSW 2145, Australia on 27 July 201 1 , in accordance with the provisions of the Budapest Treaty, and assigned Accession Number CBA201 10005.
  • These immunological binding characteristics of Mab57 are produced by hybridoma Mab57, deposited with the CellBank Australia, 214 Hawkesbury Road, Westmead NSW 2145, Australia on 27 July 201 1 , in accordance with the provisions of the Budapest Treaty, and assigned Accession Number CBA20110007.
  • the hybridoma provides a continuous source of the mAbs and binding proteins of the invention.
  • the present invention provides an isolated monoclonal antibody or a fragment thereof that may be capable of specifically binding to at least one linear epitope.
  • the epitope may be of SEQ ID NO:1 or the epitope to which Mab4 specifically binds or fragment thereof.
  • the antibodies according to any aspect of the present invention may be capable of recognising a whole spectrum of EV71 viruses and at the same time and/or may be capable of differentiating EV71 from CA16 infection. These antibodies may be of high specificity and sensitivity.
  • the antibody according to an aspect of the present invention may comprise the immunological binding characteristics of monoclonal antibody Mab53 or Mab4.
  • These immunological binding characteristics of Mab53 are produced by hybridoma Mab53, deposited with the CellBank Australia, 214 Hawkesbury Road, Westmead NSW 2145, Australia on 27 July 2011 , in accordance with the provisions of the Budapest Treaty, and assigned Accession Number CBA20110006.
  • the hybridoma provides a continuous source of Mab53 and binding proteins of the invention.
  • Mab4 immunological binding characteristics of Mab4 are produced by hybridoma Mab4, deposited with the CellBank Australia, 214 Hawkesbury Road, Westmead NSW 2145, Australia on 27 July 2011 , in accordance with the provisions of the Budapest Treaty, and assigned Accession Number CBA20110004.
  • the hybridoma provides a continuous source of Mab4 and binding proteins of the invention.
  • the antibody according to one aspect of the present invention may be selected from the group consisting of: (a) antibody produced by hybridoma cell line CBA20110006 or CBA20110004;
  • the antibody according to any aspect of the present invention 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 of the present invention 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 Mab51 , Mab57, Mab53 or Mab4 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. Techniques have been developed for the production of 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 of the invention.
  • 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 of the present invention 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.
  • Mab5 , Mab57, Mab53 or Mab4 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, doxycycline, halofanthne, mefloquine, primaquine, pyrimethamine, quinine, quinidine, and sulfadoxine; anti-biotic drugs such as ampicillin, azithromycin
  • the antibodies according to any aspect of the present invention 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 according to any aspect of the present invention may be a noninvasive method and may also be useful for studying several immunological aspects of EV71.
  • the present invention provides a method of detecting and/or quantifying the presence and distribution of at least one EV71 infected cell in a subject, the method 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 according to any aspect of the present invention 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 according to any aspect of the present invention, 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 of the invention 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.
  • Mab51 , Mab57, Mab53 or Mab4 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 according to any aspect of the present invention provide convenient, highly specific and sensitive means for detecting EV71 .
  • One such means is the ELISA format.
  • Each of Mab51 , Mab57, Mab53 or Mab4 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
  • Other immunological methods to detect EV71 viruses include, for example, dot-blot and in situ hybridization.
  • the present invention provides at least one method of treatment and/or protection of EV71 and/or at least one EV71 -linked disease, the method comprising administering to a subject in need thereof at least one antibody or a fragment thereof according to any aspect of the present invention.
  • the antibody of the present invention 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.
  • the present invention provides at least one antibody or a fragment thereof according to any aspect of the present invention for use in medicine.
  • the present invention provides at least one use of the antibody or a fragment thereof according to any aspect of the present invention for the preparation of a medicament for treatment of EV71 and/or at least one EV71 -linked disease.
  • 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.
  • the present invention provides at least one pharmaceutical composition comprising an antibody according to any aspect of the present invention and a pharmaceutically acceptable carrier.
  • the EV71 mAbs according to any aspect of the present invention 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 according to any aspect of the present invention 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 according to any aspect of the present invention 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 of according to any aspect of the present invention may be used for direct detection of EV71 viral particles from blister or oral swabs.
  • EXAMPLE 1 Preparation and purification of Mab51, Mab57, Mab53 or Mab4 from whole EV71 virus.
  • Hybridomas secreting specific Mabs were derived from BALB/c mice which had been immunized twice intramuscularly with purified EV71 -B5 strain 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 spienocytes were fused to the SP2/0 myioma 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 PBS 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.
  • 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.
  • Mab51 and Mab53 belong to subclass IgM and IgG respectively as identified with an isotyping Kit (GE). As shown in Figure 1 , Mab51 and Mab53 could detect EV71 viral protein expression in infected vero cells as shown by western blotting. Western blotting of sucrose- gradient purified EV71 virus particles from the C4 strain revealed that the ab51 and Mab53 are specific for VP1 as shown in Figure 1 .
  • the VP1 protein was fragmented into GST-tagged continual overlapping peptides and cloned into pGex-4T-1 vector and expressed in E. coli BL21 cells and tested in Western blots using Mab51 and Mab53 separately. Gradual reduction of the fragment length led to the identification of a linear epitope of Mab51 and Mab53 which mapped to amino acid positions 215-219 of the VP1 protein.
  • the minimal epitope consists of the five amino acids KQEKD (SEQ ID NO:1 ) as shown in Figure 2.
  • the VP1 protein was fragmented into GST- tagged continual overlapping peptides and cloned into pGex-4T-1 vector and expressed in E. coli BL21 cells and tested in Western blots using Mab4. The results are shown in Figure 3.
  • VP1 capsid protein was expressed as eight C-terminal truncated proteins. All the eight fragments carrying the intact N-terminal sequence were recognized by Mab4.
  • the epitope of Mab4 is located at the N-terminal region of VP1 protein.
  • the epitope of Mab4 was found within amino acids 1 -66.
  • fragment A protein as shown in Figure 3A was expressed as four N-terminal truncated proteins denoted as A10, A24, A32 and A48. From the western blot results shown in Figure 3B, Mab4 could only recognize A10 fragment which encompass amino acid residue 10 to 297. This implied that the epitope of Mab4 was within amino acids 10 to 24.
  • virus particles from the different EV71 subgenogroups were of sucrose purified. ECL reagent was used for developing. RD cells were infected with virus and the supernatant was collected after 48 h when more than 90% of the cells showed cytopathic effect. Cell debris were removed by a clarification spin and microfiltration through a 0.2 ⁇ cut-off filter, followed by concentration by ultraspin at 100'OOOg for 3 h. Virus particles were purified by centrifugation in a 20% to 60% sucrose gradient for 3h at 27'000g. Several bands were detected and the virus band at the 40 to 60% sucrose meniscus was collected. Western blotting using Mab51 and Mab53 separately as primary antibodies was performed and SEQ ID NO:1 protein bands were detected in the viral fractions as shown in Figure 4.
  • Mab51 , Mab53 and Mab4 can thus be used as a universal detection antibody for EV71.
  • the neutralization results for Mab57 and Mab53 against EV71 subgenotypes were confirmed using in vitro microneutralisation methods known in the art. The results are shown in Table 2 below.
  • Mab51 was positive in vitro micro neutralization of all sub genotypes of EV71 .
  • the dilution titer is up to 1 :1024.
  • Mab57 was positive in vitro micro neutralization of some different sub genotypes of EV71 , B4, B5, C1 , C2, C3 and C5.
  • the dilution titer is up to 1 :4096.
  • mice pre-treated (prophylactic studies) with Mab51 did not display any of the disease manifestations and remained healthy throughout the experiment.
  • 2-week-old immunodeficient AG129 mice were susceptible to infection with the non mouse-adapted EV71 strain HFM 41 via the intraperitoneal route of inoculation and anti-EV71 antibody Mab51 was able to confer 100% protection against the lethal EV71 challenge at a dose of 10 ⁇ g/g of body weight. The results are shown in Table 3 and Figures 10 and 1 1 .
  • Figure 10 shows the cross- section of the spinal cord with arrow indicating neuropil vacuolation and neuronal loss without inflammation in the anterior horn in AG129 mice challenged with EV71 infection.
  • Figure 1 1 shows the cross-section of spinal cord showing no significant pathology when AG129 mice challenged with EV71 infection were given prophylactic protection with Mab51 .
  • Mab51 was thus capable of conferring protection against EV71 infection and protecting against all pathologic changes in infected mice. Isotype control Prophylactic studies

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Abstract

L'invention concerne au moins un anticorps isolé ou un fragment de celui-ci, l'anticorps ou le fragment de celui-ci étant apte à se lier spécifiquement à au moins un peptide issu d'EV71.
PCT/SG2012/000344 2011-09-20 2012-09-20 Anticorps spécifiques d'entérovirus 71 et leurs utilisations Ceased WO2013043125A1 (fr)

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WO2014070109A1 (fr) * 2012-10-29 2014-05-08 Temasek Life Sciences Laboratory Limited Anticorps spécifique de l'entérovirus 71
EP3082862A4 (fr) * 2013-12-16 2017-08-02 MAB Explorations Sdn Bhd Anticorps spécifiques des entérovirus qui infectent les humains
US10125187B2 (en) 2013-12-16 2018-11-13 Mab Explorations Sdn Bhd Antibodies specific for enteroviruses that infect humans
TWI700295B (zh) * 2013-12-16 2020-08-01 馬來西亞商Ｍａｂ探索私人有限公司 對會感染人類之腸病毒具有專一性之抗體
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US10822397B2 (en) 2013-12-16 2020-11-03 Mab Explorations Sdn Bhd Antibodies specific for enteroviruses that infect humans
CN104569428A (zh) * 2014-12-30 2015-04-29 浙江普康生物技术股份有限公司 一种肠道病毒71型灭活疫苗抗原酶联免疫检测试剂盒
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
CN110687291A (zh) * 2019-10-30 2020-01-14 中国食品药品检定研究院 一种柯萨奇病毒a16型病毒抗原检测试剂盒
CN111172048A (zh) * 2019-12-27 2020-05-19 深圳康泰生物制品股份有限公司 重组汉逊酵母表达的ca16病毒样颗粒的粗纯工艺、ca16病毒疫苗及其制备方法

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