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WO2015088256A1 - Molécules de liaison pouvant neutraliser des virus de la rage - Google Patents

Molécules de liaison pouvant neutraliser des virus de la rage Download PDF

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Publication number
WO2015088256A1
WO2015088256A1 PCT/KR2014/012171 KR2014012171W WO2015088256A1 WO 2015088256 A1 WO2015088256 A1 WO 2015088256A1 KR 2014012171 W KR2014012171 W KR 2014012171W WO 2015088256 A1 WO2015088256 A1 WO 2015088256A1
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Prior art keywords
seq
binding molecule
region
rabies
variable region
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Ceased
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PCT/KR2014/012171
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English (en)
Korean (ko)
Inventor
홍승서
장신재
김판겸
김철민
서지민
안정선
신지영
맹기은
김동조
류수희
박인창
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Celltrion Inc
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Celltrion Inc
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Priority to CN201480067877.0A priority Critical patent/CN105814077B/zh
Publication of WO2015088256A1 publication Critical patent/WO2015088256A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • 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
    • 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
    • C07K2317/70—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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to binding molecules capable of neutralizing rabies virus.
  • Rabies is a viral common infectious disease that primarily affects wildlife and pets, as well as mammals, including humans, causing acute brain disease. It is a fatal disease that occurs almost once in death, and is known to have the highest mortality rate with AIDS. The rabies is spread worldwide, with more than 10 million people receiving treatment after infection each year, with 40,000 to 70,000 deaths each year.
  • Rabies is transmitted from saliva and blood, usually from bites of dogs or cats infected with rabies. It can also be infected by most mammals, including skunks and bats.
  • Rabies virus shows actual onset symptoms after reaching the nerve tissue through the terminal nerve tissue of the body.
  • the human brain has a blood brain barrier that blocks foreign substances, so viruses cannot penetrate, but the rabies virus passes through the blood barrier through the RVG (rabies virus glycoprotein) protein to the central nervous system. (central nervous system) Infects the brain.
  • RVG rabies virus glycoprotein
  • Anti-rabies antibody an anti-rabies immunoglobulin
  • ERIG equine-derived rabies immunoglobulin
  • the problem to be solved by the present invention is to provide a binding molecule having a neutralizing ability by binding to rabies virus.
  • Another object of the present invention is to provide an immunoconjugate in which one or more tags are bonded to the binding molecule.
  • Another object of the present invention is to provide a polynucleotide encoding the binding molecule.
  • Another object of the present invention is to provide an expression vector inserted with a polynucleotide encoding the binding molecule.
  • Another object of the present invention is to provide a cell line transformed with the expression vector.
  • Another object of the present invention is to provide a composition comprising the binding molecule.
  • Another object of the present invention is to provide a kit comprising the binding molecule.
  • Another object of the present invention is to provide a method for diagnosing rabies using the binding molecule.
  • Another object of the present invention is to provide a method for treating and preventing rabies using the binding molecule.
  • Another object of the present invention is to provide a method of producing the binding molecule of the present invention by culturing the cell line.
  • Another object of the present invention is to provide a method for detecting rabies virus using the binding molecule.
  • one embodiment of the present invention has a neutralizing activity when the rabies virus contains a wild type G protein, but the rabies virus is glycine (Gly) valine (Gal), glutamine (Glu) or 34
  • Gly valine
  • Glu glutamine
  • Arg arginine
  • the amino acid position is a numbered position except for a signal peptide of the G protein.
  • the numbering of amino acid positions of G protein is the same.
  • the binding molecule neutralizes the rabies escape virus in which glycine (Gly) at position 34 of the G protein is mutated to valine, glutamine (Glu), or arginine (Arg). It was confirmed that the epitope is at the antigenic site II of the G protein.
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3;
  • variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3;
  • variable region comprising a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 region of SEQ ID NO: 6
  • the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 25 and a variable region of SEQ ID NO: 26.
  • the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 33 and a light chain of SEQ ID NO: 34.
  • another embodiment of the present invention has a neutralizing activity when the rabies virus includes a wild type G protein, but when the rabies virus includes a G protein in which serine (Ser) at position 331 is mutated to proline (Pro) Provided is a binding molecule having no neutralizing activity.
  • the binding molecule is confirmed that the serine (Ser) at position 331 of the G protein does not neutralize the rabies escape virus (Escape virus) mutated to proline (Pro) epitope of the G protein It was confirmed that it is at antigenic site III.
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9;
  • variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12
  • the binding molecule As one example, the binding molecule
  • a variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9;
  • variable region comprising a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 region of SEQ ID NO: 12
  • the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 27 and a variable region of SEQ ID NO: 28.
  • the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 35 and a light chain of SEQ ID NO: 36.
  • another embodiment of the present invention has a neutralizing activity when the rabies virus includes a wild type G protein, the valine (Val) at position 210 is glutamic acid (Glu) or glutamic acid (Glu) at position 413 ) Contains a G protein mutated to aspartic acid (Asp) to provide a binding molecule having no neutralizing activity.
  • the binding molecule is a rabies escape virus in which valine (Val) at position 210 of G protein is mutated to glutamic acid (Glu) or glutamic acid at position 413 to aspartic acid (Asp).
  • Glu glutamic acid
  • Asp aspartic acid
  • the G protein may be the G protein of CVS-11, but is not limited to this strain. More specifically, the wild type G protein may comprise the amino acid sequence of SEQ ID NO: 81.
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15;
  • variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18;
  • variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21;
  • variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24
  • It may be a binding molecule, including any one variable region selected from the group consisting of.
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15;
  • variable region comprising a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 region of SEQ ID NO: 18
  • the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 29 and a variable region of SEQ ID NO: 30.
  • the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 37 and a light chain of SEQ ID NO: 38.
  • the binding molecule As one example, the binding molecule
  • variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21;
  • variable region comprising a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 region of SEQ ID NO: 24
  • the binding molecule may be a binding molecule including a variable region of SEQ ID NO: 31 and a variable region of SEQ ID NO: 32.
  • the binding molecule may be a binding molecule comprising a heavy chain of SEQ ID NO: 39 and a light chain of SEQ ID NO: 40.
  • the antigenic site, amino acid position, and epitope form of rabies virus G protein generally known are shown in Table 1.
  • the CDRs of the variable regions were determined by conventional methods according to a system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5 th ), National Institutes of Health, Bethesda) , MD. (1991)]. Although the CDRs used in the present invention were determined using the Kabat method, binding molecules including CDRs determined according to other methods such as the IMGT method, the Chothia method, and the AbM method are also included in the present invention.
  • the binding molecule may be an antibody.
  • the binding molecule may be, but is not limited to, Fab fragments, Fv fragments, diabodies, chimeric antibodies, humanized antibodies, or human antibodies.
  • One embodiment of the present invention provides a fully human antibody that binds to rabies virus.
  • an antibody is used in its broadest sense and is specifically an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from two or more intact antibodies, and a target.
  • Antibodies are proteins produced by the immune system that can recognize and bind specific antigens.
  • the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains).
  • Each antibody mainly has two regions, a variable region and a constant region.
  • the variable region located at the distal portion of the arm of Y binds to and interacts with the target antigen.
  • the variable region comprises a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen.
  • CDR complementarity determining region
  • the constant region located in the tail of Y is recognized and interacted with by the immune system.
  • Target antigens generally have multiple binding sites called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen may have one or more corresponding antibodies.
  • the present invention includes functional variants of the antibody.
  • Antibodies are considered functional variants of the antibodies of the invention if the variants can compete with the antibodies of the invention to specifically bind rabies virus or its G protein.
  • Functional variants include, but are not limited to, derivatives having substantially similar primary structural sequences, including, for example, in vitro or in vivo modifications, chemicals and / or biochemicals. However, they are not found in the parental monoclonal antibodies of the invention.
  • Such modifications include, for example, acetylation, acylation, covalent bonds of nucleotides or nucleotide derivatives, covalent bonds of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxide, methylation, oxidation, PEGylation, proteolysis And phosphorylation and the like.
  • Functional variants may optionally be antibodies comprising an amino acid sequence containing substitutions, insertions, deletions or combinations of one or more amino acids in comparison to the amino acid sequence of the parent antibody.
  • functional variants may include truncated forms of amino acid sequences at one or both of the amino terminus or carboxy terminus.
  • the functional variant of the invention may have the same or different, higher or lower binding affinity compared to the parent antibody of the invention, but can still bind to rabies virus or its G protein.
  • the amino acid sequence of the variable region including but not limited to, a framework, Hypervariable region, particularly the CDR 3 region, may be modified.
  • the light or heavy chain region comprises three hypervariable regions, including three CDR regions, and a more conserved region, the framework region (FR).
  • Hypervariable regions include amino acid residues from CDRs and amino acid residues from hypervariable loops.
  • Functional variants within the scope of the present invention are about 50% -99%, about 60% -99%, about 80% -99%, about 90% -99%, about 95% -99%, Or about 97% -99% amino acid sequence identity. Gap or Bestfit known to those skilled in the art can be used in computer algorithms to optimally arrange the amino acid sequences to be compared and define similar or identical amino acid residues.
  • the functional variant can be changed by or obtained by known general molecular biology methods including but not limited to PCR methods, mutagenesis using oligomeric nucleotides, and partial mutagenesis.
  • the rabies virus may be derived from any one selected from the group consisting of dogs, cattle, mongoose, bats, skunks, raccoons, coyotes, foxes and wolves, but is not limited thereto.
  • another embodiment of the present invention provides an immunoconjugate in which at least one tag is additionally bound to the binding molecule.
  • a drug may be further attached to the antibody according to the invention. That is, the antibody according to the present invention can be used in the form of an antibody-drug conjugate to which a drug is bound.
  • ADCs antibody-drug conjugates
  • immunoconjugates for topical delivery of drugs enables targeted delivery of the drug moiety to infected cells. Unacceptable levels of toxicity can result.
  • mAb polyclonal and monoclonal antibodies
  • mAb monoclonal antibodies
  • drug-linking and drug-releasing properties can improve the maximal efficacy and minimal toxicity of ADC.
  • another embodiment of the present invention provides a nucleic acid molecule encoding the binding molecule.
  • Nucleic acid molecules of the invention include all nucleic acid molecules in which the amino acid sequence of an antibody provided herein is translated into a polynucleotide sequence, as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), all of which are also included in the nucleic acid molecules of the present invention.
  • ORF open reading frame
  • another embodiment of the present invention provides an expression vector into which the nucleic acid molecule is inserted.
  • Celltrion's unique expression vector MarEx vector see Korean Patent Registration No. 10-1076602
  • commercially widely used pCDNA vectors F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, T7
  • the introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection or electroporation, but is not limited thereto.
  • An introduction method suitable for the expression vector and the host cell can be selected and used.
  • the vector contains one or more selection markers, but is not limited thereto, and may be selected depending on whether the product is produced using a vector that does not include the selection marker.
  • the selection of the selection marker is chosen by the host cell of interest, which uses methods already known to those skilled in the art and the present invention is not so limited.
  • a tag sequence may be inserted and fused to an expression vector.
  • the tag may include, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag that facilitates purification known to those skilled in the art may be used in the present invention.
  • another embodiment of the present invention provides a cell line wherein the expression vector is transformed into a host cell, thereby binding to rabies virus to produce a binding molecule having a neutralizing ability.
  • the cell line may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin.
  • the mammalian cells include any one selected from the group consisting of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. It is preferable to use one as a host cell, but is not limited thereto, and all cells usable as mammalian host cells known to those skilled in the art are available.
  • another embodiment of the present invention provides a pharmaceutical composition for treating and preventing rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.
  • another embodiment of the present invention provides a rabies diagnostic composition further comprising the binding molecule and a pharmaceutically acceptable excipient.
  • compositions of the present invention may include pharmaceutically acceptable excipients in addition to binding molecules having the ability to neutralize rabies virus.
  • pharmaceutically acceptable excipients are well known to those skilled in the art.
  • the prophylactic and therapeutic compositions of the present invention may include at least one other rabies therapeutic agent, and may also include several types of monoclonal antibodies, thereby exhibiting a synergistic effect on neutralizing activity.
  • the prophylactic and therapeutic composition of the present invention may further include at least one other therapeutic or diagnostic agent.
  • therapeutic agents include, but are not limited to, anti-viral agents.
  • agents can be antibodies, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, anti-viral peptides, and the like.
  • compositions of the present invention are sterile and stable under the conditions of manufacture and storage, and may be in powder form for reconstitution in a suitable pharmaceutically acceptable excipient upon or prior to delivery.
  • suitable methods of preparation are vacuum drying and lyophilization, which produce further desired components from the powder of the active ingredient and its presterilized-filtered solution.
  • the compositions of the present invention may be in solution and may be added and / or mixed before or at the time of delivery of the appropriate pharmaceutically acceptable excipient to provide a unit dosage injectable form.
  • the pharmaceutically acceptable excipients used in the present invention are suitable for drug concentration, can maintain a suitable flowability, and can be delayed if necessary.
  • monoclonal antibodies of the invention can be prepared with carriers that prevent their rapid release, such as controlled release formulations, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used in the present invention.
  • Monoclonal antibodies can also be coated with or administered with a substance or compound that prevented the inactivation of the antibody.
  • monoclonal antibodies can be administered with a suitable carrier—liposomes or diluents.
  • Methods of administering the prophylactic and therapeutic compositions of the invention can be divided orally and parenterally, and the preferred route of administration is intravenous but not limited thereto.
  • the oral forms include tablets, troches, medicinal drops, aqueous or oily suspensions, powders or dispersible granules, emulsions, rigid capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, solutions, gels or It may be formulated as a slurry.
  • These formulations include, but are not limited to, pharmaceutical excipients containing inert diluents, granulating or disintegrating agents, binders, brightening agents, preservatives, colorants, flavoring or sweetening agents, vegetable or mineral oils, wetting agents and thickeners.
  • the parenteral form may be in the form of an aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solution or suspension.
  • the solution or suspension may be a drug such as 1,3-butanediol, Ringus's solution, Hanks' solution, isotonic sodium chloride solution, oils, fatty acids, local anesthetics, preservatives, buffers, viscosity or solubility that are nontoxic to the receptor at the dosage and concentration applied.
  • Agents, water soluble antioxidants, oil soluble antioxidants and metal chelating agents may be used to dissolve.
  • the binding molecule of the present invention used in the diagnostic composition is detectably labeled.
  • Various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention.
  • Examples of marker types that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.
  • markers include phosphors (eg, fluresin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, ⁇ -galactosidase, alkaline phosphatase), radioisotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg dioxetane, luminol or acridinium). Labeling methods such as covalent binding of enzymes or biotinyl groups, iodide methods, phosphorylation methods, biotinylation methods and the like are well known in the art.
  • Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions, and the like. Commonly used detection assays include radioisotopes or non-radioisotope methods. These include Western blotting, overlay-assay, Radioimmuno Assay (RIA) and Immunity Radioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (CIA) and Chemiluminoluminescent Immune Assay).
  • radioisotopes or non-radioisotope methods include Western blotting, overlay-assay, Radioimmuno Assay (RIA) and Immunity Radioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (CIA) and Chemiluminoluminescent Immun
  • It provides a rabies diagnostic kit comprising a.
  • It provides a kit for the treatment and prevention of rabies comprising.
  • step b) determining whether rabies infection is analyzed by analyzing the result of step a)
  • It provides a rabies diagnostic method comprising a.
  • It provides a method for providing information for the diagnosis of rabies comprising.
  • another embodiment of the present invention provides a method for treating and preventing rabies, comprising administering to a subject a therapeutically effective amount of said binding molecule.
  • immunity to rabies virus can be conferred within 1, 2, 3 or several days by administering the human monoclonal antibody of the present invention when traveling to a rabies risk zone.
  • It provides a method for detecting a rabies virus comprising a.
  • the subject sample includes, but is not limited to, blood, serum, saliva, sputum, saliva, sweat, tissue or other biological material from a (potentially) infected subject.
  • Sample preparation is possible by the following method.
  • the (potential) subject of infection may be a human subject, but may also be animals suspected of being a carrier of rabies virus.
  • the subject sample may first be manipulated to make it more suitable for the detection method.
  • the binding molecule or immunoconjugate of the invention is contacted with the subject sample under conditions that allow the formation of an immunological complex between the binding molecule and the rabies virus or its antigenic component present in the subject sample.
  • immunological complexes indicative of the presence of rabies virus in the subject sample is detected and measured by appropriate means.
  • immunoassays such as radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE, Western blot analysis.
  • variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3, and a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6
  • Binding molecules comprising a variable region comprising a region;
  • variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12
  • Binding molecules comprising a variable region comprising a region;
  • variable region comprising a CDR1 region of SEQ ID NO: 13, a CDR2 region of SEQ ID NO: 14, and a CDR3 region of SEQ ID NO: 15, and a CDR1 region of SEQ ID NO: 16, a CDR2 region of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 18 Binding molecules comprising a variable region comprising a region;
  • variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 of SEQ ID NO: 24
  • Binding molecules comprising variable regions comprising regions
  • compositions comprising two or more binding molecules selected from the group consisting of:
  • the cocktail composition is
  • variable region comprising a CDR1 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR3 region of SEQ ID NO: 3, and a CDR1 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6
  • Binding molecules comprising a variable region comprising a region;
  • variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12
  • Binding molecules comprising a variable region comprising a region.
  • the cocktail composition is
  • variable region comprising a CDR1 region of SEQ ID NO: 7, a CDR2 region of SEQ ID NO: 8, and a CDR3 region of SEQ ID NO: 9, and a CDR1 region of SEQ ID NO: 10, a CDR2 region of SEQ ID NO: 11, and a CDR3 of SEQ ID NO: 12 Binding molecules comprising a variable region comprising a region;
  • variable region comprising a CDR1 region of SEQ ID NO: 19, a CDR2 region of SEQ ID NO: 20, and a CDR3 region of SEQ ID NO: 21, and a CDR1 region of SEQ ID NO: 22, a CDR2 region of SEQ ID NO: 23, and a CDR3 of SEQ ID NO: 24
  • Binding molecules comprising a variable region comprising a region.
  • binding molecule refers to an intact immunoglobulin, or an immunoglobulin that binds to an antigen, including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, eg For example, it refers to a variable domain comprising an immunoglobulin fragment that competes with an intact immunoglobulin for binding to a rabies virus or a G protein (Glycoprotein) or fragment thereof outside the virus. Regardless of the structure, the antigen-binding fragment binds to the same antigen recognized by intact immunoglobulins.
  • An antigen-binding fragment comprises two or more contiguous amino acid residues of the binding molecule, five or more contiguous amino acid residues, ten or more contiguous amino acid residues, and 15 or more contiguous amino acid residues. At least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, 70 or more contiguous amino acid residues, 80 or more contiguous amino acid residues, 90 or more contiguous amino acid residues, 100 or more contiguous amino acid residues, 125 or more contiguous amino acid residues, 150 or more contiguous amino acid residues, 175 or more contiguous amino acid residues, 200 At least 2 consecutive amino acid residues, or at least 250 consecutive amino acid residues It may comprise a peptide or polypeptide comprising the amino acid sequence.
  • Antigen-binding fragments are especially Fab, F (ab '), F (ab') 2 , Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single- Chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of immunoglobulin sufficient to bind a particular antigen to the polypeptide, and the like It includes.
  • the fragments may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins or may be produced genetically by recombinant DNA techniques. Production methods are well known in the art.
  • the term "pharmaceutically acceptable excipient” refers to an inert material that is combined into an active molecule, such as a drug, agent, or binding molecule, to produce an acceptable or convenient dosage form.
  • Pharmaceutically acceptable excipients are nontoxic or are excipients that are acceptable to the recipient for their intended use, at least in the doses and concentrations in which the toxicity is used, and with other components of the formulation including drugs, preparations or binding powders. It is compatible.
  • the term "therapeutically useful amount” refers to the amount of the binding molecule of the invention effective for the prophylaxis or treatment of or before or after exposure to the rabies virus.
  • the inventors of the present invention inoculated rabies vaccine to 15 healthy adult subjects and collected blood to separate PBMCs, and selected candidate antibodies through phage display and B cell sorting using the isolated PBMCs. After measuring the basic titers of the selected candidate antibodies, the antibodies showing a certain level of neutralization ability are representative of each continent and animal that are prevalent in the world at the Center for Disease Control (CDC). In vivo and in vitro experiments were performed on four antibodies that have been shown to neutralize rabies virus, and neutralization ability tests were performed on a variety of rabies viruses. It has been found that it can be usefully used to treat patients infected with rabies virus.
  • Binding molecules capable of neutralizing the rabies virus of the present invention have been found to possess neutralizing ability against various rabies viruses, and thus are useful for the treatment and prevention of rabies.
  • FIG. 1 depicts human antibody expression vectors comprising the heavy and light chain genes of the present invention.
  • Figure 2 shows the ELISA results using the G-protein of rabies virus.
  • Figure 3 is a graph showing the survival rate of the mouse against the SV2 virus in animal experiments.
  • Volunteers in this example consisted of healthy adults vaccinated against the rabies vaccine, which was approved by the Institutional Review Board (IRB). Volunteers confirmed negative responses to other infectious viruses, ie VDRL and HBsAg, and negative responses to anti-HCV and anti-HIV antibodies.
  • a total of the volunteers received rabies vaccination once a year, and those who had not been vaccinated previously received 3 shots.
  • the vaccinated rabies vaccine was Verorab® (Sanofi Pasteur).
  • Two weeks after the last inoculation approximately 50 ml of whole blood was obtained and the peripheral blood mononuclear cells (PBMC) were isolated using the Ficoll-Paque PLUS (GE Healthcare) method.
  • PBMC peripheral blood mononuclear cells
  • variable region of the light and heavy chains of the antibody was amplified by PCR (polymerase chain reaction) using high fidelity Taq polymerase (Roche) and degenerative primer set from the synthesized cDNA. Amplified DNA fragments were isolated by gel extraction kit (Qiagen) after 1% agarose gel electrophoresis. Using the isolated variable region fragment as a template, the variable heavy chain region and the light chain variable region were linked to each other and amplified by scFv-type genes. Separated.
  • the amplified scFv gene was digested with SfiI (Roche) restriction enzyme for 12 hours and then isolated using 1% agarose gel electrophoresis and gel extraction kit (Qiagen). Phagemid vector was also cleaved with SfiI restriction enzyme, isolated, mixed with the scFv gene, T4 DNA ligase (Roche), and reacted at 16 ° C. for 12 hours.
  • the reaction solution was mixed with ER2738 competent cell (Lucigen) and transformed by electroporation.
  • the transformed ER2738 was added to VCSM13 helper phage (Agilent Technologies) for 12 hours after shaking culture to prepare a phage library.
  • rabies virus was incubated in BHK-21 or Vero cells for 3 or 4 days, after which culture medium was obtained, and the virus was concentrated using an ultracentrifuge.
  • the concentrated virus isolated the G protein located on the surface of the virus using octyl beta glucopytanoside chemicals.
  • the isolated protein body was quantitatively and qualitatively analyzed by ELISA.
  • the phage library culture prepared in Example 1-2 was centrifuged to remove host cells, 4% PEG and 0.5 M NaCl were added and centrifuged at 9000 rpm for 15 minutes to settle the phage and remove the supernatant.
  • the precipitated phages were diluted in 1% BSA / TBS and placed in an antigen-binding ELISA plate and reacted at room temperature for 2 hours. After removing the reaction solution, the ELISA plate was washed with PBS containing 0.05% tween20, 60 ⁇ l of 0.1 M glycine-HCl (pH 2.2) was added to remove the antigen-binding phage, and 2 M Tris (pH 9.1) was used. Neutralized. The detached phage was infected with ER2738, incubated with VCSM13 helper phage, and used for the next panning.
  • Candidate antibody segments were selected after two or three panning.
  • Example 1-4 phage enzyme immunoassay
  • a portion of the infected ER2738 was plated onto LB plates before adding helper phage to obtain colonies.
  • the colonies formed in the culture medium were shaken and cultured.
  • VCSM13 helper phage was added and shaken at 37 ° C for at least 12 hours.
  • the culture medium was centrifuged to remove host cells and a supernatant containing phage was prepared.
  • phage enzyme immunoassay For phage enzyme immunoassay, G-protein was adsorbed on 96-well microtiter plates and 150 ⁇ l of 3% BSA / PBS was added and reacted at 37 ° C. for 1 hour. The prepared phage supernatant was diluted 1: 1 with 6% BSA / PBS and placed in each well and left at 37 ° C. for 2 hours. Each well was washed three times with PBS containing 0.05% Tween 20. Then, anti-M13 antibody labeled with horseradish peroxidase was added and allowed to stand at 37 ° C for 1 hour.
  • PBMC peripheral blood mononuclear cells
  • cytokines IL-4, IL-6, IL-21, CD40L
  • Flow cytometry was performed to isolate B cells expressing antibodies specific to rabies virus in cultured PBMCs.
  • Flow cytometry was performed by labeling PBMCs with fluorescently labeled antibodies or antigens in the presence of an Fc ⁇ R blocker, and then separating only the labeled cells using a flow cytometer (FACS aria II, BD biosciences) and placing them in 96-well PCR plates (applied biosystems).
  • Antibodies or antigens used for labeling are as follows.
  • FITC-labeled rabies virus G protein was constructed to obtain B cells expressing rabies virus specific antibodies.
  • the above-described purified rabies virus G protein was experimented manually using a FITC antibody labeling kit (Pierce) manufactured by Pierce, to obtain a FITC-labeled rabies virus G protein.
  • CDNA was synthesized using a superscript III first strand synthesis system kit manufactured by Invitrogen in single cells separated from each well of a 96 well plate. Synthesis was performed according to the manual provided in the kit. The process of obtaining antibody genes from the synthesized cDNA was performed by modifying the method described in Thomas Tiller et al (Journal of Immunological Methods, 2008). In brief, the amplification of the heavy and light chain genes was carried out by PCR through the first PCR process, and the amplification of the heavy and the light chain genes was performed again using the PCR product obtained through the first PCR through the nested PCR. .
  • the bands of the heavy and light chain genes were identified, incised with a blade, the agarose fragment containing DNA was transferred to a 1.5ml tube, and the DNA was purified from agarose using a PCR purification kit (Qiagen). The bay was dissolved and purified. The purified and heavy and kappa light chains were cloned into a PCDNA3.1 vector containing a portion of the antibody after treatment with Not 1 and Age1 restriction enzymes. Lambda light chains were cloned into a modified PCDNA 3.1 vector containing a portion of the antibody after treatment with Age1, Xho1 restriction enzymes as well.
  • the genes of the candidate antibodies selected in Examples 1-4, 1-5, and 1-6 were expressed in animal cell expression vectors, followed by ELISA using the antibodies secreted into the culture medium. G-protein of rabies virus was attached to the ELISA plate and expressed antibody was added. After washing off the unbound antibody, candidate antibodies bound to the antigen were selected using an anti-human antibody conjugated with horseradish peroxidase.
  • Example 1-8 Selection of Candidate Antibodies Using Virus Neutralization Ability
  • Example 1-7 about 80 candidate antibodies showing some high binding properties were tested for basic viral activity using CVS-11.
  • Conformational epitope means a binding site having a tertiary protein structure.
  • PCR2.1 TA cloning vectors containing the obtained heavy and light chains were treated with the restriction enzymes Nhe I and Pme I to obtain the heavy and light chain genes, and then the obtained heavy and light chain genes were respectively treated with the same restriction enzyme.
  • pCT145 and pCT146 vectors were inserted.
  • the pCT145 and pCT146 vectors are Celltrion-specific vectors designed to clone the heavy and light chains of antibodies, respectively.
  • a restriction enzyme Pac I was added to the pCT145 vector including the heavy chain gene.
  • pCT188 a vector containing both a heavy chain and a light chain transcription unit was selected and named pCT188 (see FIG. 1, Korean Patent Registration No. 10-1076602, Patent Holder: Celltrion Co., Ltd.).
  • the selected vector was extracted using Endofree plasmid maxi kit (QIAGEN, Germany, 12362), and finally the nucleotide sequence of the antibody was confirmed by sequencing using some of the extracted DNA.
  • FreeStyle TM Max (Invitrogen, 16447-100), a cationic polymer, was used for transient transduction in cells, and transduction was performed according to the manufacturer's instructions. The day before transduction, centrifugation of F2N cells (refer to Korean Patent No. 10-1005967, patent holder: Celltrion) grown on EX-CELL 293 Serum free media (Sigma, 14571C: hereinafter referred to as "EX-CELL 293 medium").
  • the medium was replaced with FreeStyle293 serum free media (Gibco, 12338), and 50 ml (100 ml total) were inoculated using two 250 ml shaker flasks at a concentration of 0.8 ⁇ 10 6 cells per ml.
  • 125 ⁇ g of the pCT178 DNA containing the antibody gene and 125 ⁇ l of the FreeStyle TM Max reagent were diluted in 2 ml volume using OptiPRO SFM II (Invitrogen, 12309) medium, and the mixture was gently mixed.
  • OptiPRO SFM II Invitrogen, 12309
  • the number of inoculated F2N cells to be used for transduction was measured, and the cell concentration was diluted to 1.0 ⁇ 10 6 cells using the FreeStyle293 medium.
  • transduction was performed by treating the F2N cells with a mixture solution of DNA and FreeStyle TM Max reagent. The day after transduction, the same amount of EX-CELL 293 medium was added to the transduced cells and cultured for 7 days to produce monoclonal antibodies.
  • virus 4-5 clones which were found to be infected or increased incidence, even though the virus obtained from CVS-11 was found to be uninfected or in small amounts was gradually increased.
  • RNA was isolated using a QIAamp Viral RNA Mini kit (QIAGEN, 52904).
  • RNA was used as a template to synthesize cDNA using SuperScript III First-strand Synthesis system for RT-PCR (Invitrogen, 18080-051), followed by amplification with Takara ExTaq (Takara, RR001A), followed by sequencing. Proceeded. As a result of sequencing, the epitope and mutated nucleotide and amino acid information of each antibody are shown in Table 9.
  • CR4098 antibodies were obtained from patents (see US7579446) and the National Center for Biotechnology Information (NCBI) database, and were cloned into pCT146 expression vectors (see Example 2) synthesized in the engineering (oil globules at Daejeon). Delivered to Celltrion. The sequence of the synthesized part was confirmed by restriction digestion analysis and DNA sequencing. CR4098 antibodies were produced in a short run fashion in the F2N78 cell line as in Example 3.
  • RNA was isolated using a QIAamp Viral RNA Mini kit (QIAGEN, 52904).
  • RNA was used as a template to synthesize cDNA using SuperScript III First-strand Synthesis system for RT-PCR (Invitrogen, 18080-051), followed by amplification with Takara ExTaq (Takara, RR001A), followed by sequencing. Proceeded. Sequencing results showed that the altered sequence of escaped mutant virus for the CR4098 antibody was in N336K.
  • Example 6-2 Neutralization test against escape virus produced by CR4098 antibody
  • escape virus (N336K) produced by the CR4098 antibody it was confirmed whether the antibody of the present invention has a neutralizing ability.
  • FIG. 1 is a graph showing the survival rate of the mouse for the SV2 virus (SV1 ⁇ SV6) of the total six viruses in animal experiments.

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Abstract

La présente invention concerne des molécules de liaison pouvant neutraliser des virus de la rage. Plus particulièrement, les molécules de liaison de la présente invention présentent une aptitude à neutraliser des virus de la rage dérivés de sujets tels qu'un chien, une vache, une mangouste, une chauve-souris, une mouffette, un raton laveur, un coyote, un renard et un loup et peuvent donc être utilisées favorablement pour traiter des patients infectés par des virus de la rage dérivés d'un large éventail de sujets.
PCT/KR2014/012171 2013-12-12 2014-12-11 Molécules de liaison pouvant neutraliser des virus de la rage Ceased WO2015088256A1 (fr)

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