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WO2013048130A2 - Molécule de liaison pour neutraliser un virus de la rage - Google Patents

Molécule de liaison pour neutraliser un virus de la rage Download PDF

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WO2013048130A2
WO2013048130A2 PCT/KR2012/007795 KR2012007795W WO2013048130A2 WO 2013048130 A2 WO2013048130 A2 WO 2013048130A2 KR 2012007795 W KR2012007795 W KR 2012007795W WO 2013048130 A2 WO2013048130 A2 WO 2013048130A2
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binding molecule
seq
region
polypeptide
rabies
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PCT/KR2012/007795
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English (en)
Korean (ko)
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WO2013048130A3 (fr
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장신재
김판겸
김만수
박현진
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(주)셀트리온
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Priority to CN201280047216.2A priority Critical patent/CN103998059B/zh
Publication of WO2013048130A2 publication Critical patent/WO2013048130A2/fr
Publication of WO2013048130A3 publication Critical patent/WO2013048130A3/fr

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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C07K2317/70—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
    • G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
    • 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

  • Rabies is a viral common infection that primarily affects wild and pet animals, 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 reaches the brain's nerve tissue through the body's nerve tissue and shows the actual onset symptoms.
  • 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 nervous system. central nervous system) infects the brain.
  • RVG rabies virus glycoprotein
  • Rabies can be treated and prevented by post-exposure prevention through immediate topical wound protection and passive (anti-rabies immuno noglobulin: hereinafter referred to as "anti-rabies antibody”) and active (vaccine) immunization.
  • anti-rabies antibodies include human derived rabies immunoglobulin (hereinafter referred to as "HRIG”) and equine derived rabies immunoglobulin (hereinafter referred to as "ERIG").
  • HRIG's supply is difficult and prices are high.
  • HRIG is derived from human blood and has a high risk of infection, such as HIV, and is not high in efficacy because it is a polyclonal antibody.
  • ERIG In the case of ERIG, it is derived from horses and has a lower therapeutic efficiency than HRIG, and therefore is administered to the patient at a higher dose than HRIG. Although it is cheaper than HRIG, it is also not supplied smoothly, and anaphyaxis may occur because the antibody is derived from an individual other than human.
  • HRIG HRIG
  • monoclonal antibodies that can neutralize the rabies virus in post-exposure prophylaxis has been proposed. Rabies-virus neutralizing murine monoclonal antibodies have been developed (Schumacher CL et al., J. Clin. Invest. Vol. 84, p.
  • Another object of the present invention is to provide an immunoconjugate in which one or more tags are attached 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 into which a polynucleotide encoding the binding molecule is inserted.
  • Another object of the present invention to provide a cell line transformed with the expression vector.
  • 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 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 for detecting rabies virus using the binding molecule.
  • the present invention provides a binding molecule having a neutralizing ability to bind to rabies virus.
  • the present invention also provides an immunoconjugate wherein at least one tag is attached to the binding molecule.
  • the present invention also provides an expression vector inserted with a polynucleotide encoding the binding molecule.
  • the present invention also provides a cell line wherein the expression vector is transformed into a host cell to produce the binding molecule of the present invention.
  • the present invention also provides a method of producing the binding molecule of the present invention by culturing the cell line.
  • the present invention also provides a pharmaceutical composition further comprising the binding molecule and a pharmaceutically acceptable excipient.
  • the present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.
  • the present invention also provides a kit for diagnosing rabies comprising the binding molecule.
  • the present invention also provides a kit for the treatment and prevention of rabies comprising the binding molecule.
  • the present invention also provides a method for diagnosing rabies using the binding molecule.
  • the present invention also provides a method for treating and preventing rabies comprising administering to said subject an effective amount of said binding molecule.
  • the present invention also provides a method for detecting rabies virus using the binding molecule.
  • binding molecule refers to an intact immunoglobulin comprising a monoclonal antibody, such as a chimeric, humanized or human monoclonal antibody, or an immunoglobulin that binds to an antigen, e.g. 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 may comprise at least two 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, 40 of the amino acid sequence of the binding molecule.
  • At least 50 contiguous amino acid residues at least 50 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, 125 Peptides or polypeptides comprising an amino acid sequence of at least contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
  • binding molecule includes all immunoglobulin classes and subclasses known in the art. Binding molecules can be divided into five major classes IgA, IgD, IgE, IgG, and IgM of intact antibodies, depending on the amino acid sequence of the constant domain of the heavy chain, some of which are for example IgA1, IgA2, IgG1, It can be further divided into subclasses (isotypes) such as IgG2, IgG3 and IgG4.
  • 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 binding molecule may be a naked or unconjugated binding molecule but may be part of an immunoconjugate.
  • 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, agents 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.
  • CDC Center for Disease Control
  • the inventors of the Center for Disease Control received a hybridoma cell that has been demonstrated to be capable of neutralizing a wide range of rabies viruses.
  • the variable region sequence of was obtained.
  • the heavy and light chain variable regions were linked to an IgG1 backbone to prepare a chimeric antibody.
  • the chimeric antibodies prepared as described above were subjected to in vivo and in vitro experiments to test the neutralization ability of various rabies viruses, whereby the monoclonal antibodies of the present invention were infected with rabies viruses derived from a wide range of individuals. It has been found that it can be usefully used to treat
  • the present invention provides a binding molecule that binds to rabies virus and has a neutralizing ability.
  • the binding molecule comprises a CDR (complementarity determining regions) 1 region comprising a polypeptide set forth in SEQ ID NO: 23, a CDR2 region comprising a polypeptide set forth in SEQ ID NO: 24 and a polypeptide described in SEQ ID NO: 25 It characterized in that it comprises a variable region having a CDR3 region.
  • CDR complementarity determining regions
  • the binding molecule has a CDR1 region comprising a polypeptide as set out in SEQ ID NO: 26, a CDR2 region comprising a polypeptide as set out in SEQ ID NO: 27 and a CDR3 region comprising a polypeptide as set out in SEQ ID NO: 28 And a variable region.
  • the binding molecule is a CDR1 region comprising a polypeptide set forth in SEQ ID NO: 23, a CDR2 region comprising a polypentide set forth in SEQ ID NO: 24 and a CDR3 region comprising a polypeptide described in SEQ ID NO: 25
  • the binding molecule may be a Fab fragment, Fv fragment, diabody, triabody, tetrabody, chimeric antibody, humanized antibody or human antibody.
  • the binding molecule is characterized in that it comprises a variable region comprising the polypeptide sequence set forth in SEQ ID NO: 29.
  • the binding molecule is characterized in that it comprises a variable region comprising the polypeptide sequence set forth in SEQ ID NO: 30.
  • the binding molecule is characterized in that it comprises a heavy chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 29 and a light chain variable region comprising a polypeptide sequence as set out in SEQ ID NO: 30.
  • the binding molecule may be a Fab fragment, Fv fragment, diabody, triabody, tetrabody, chimeric antibody, humanized antibody or human antibody.
  • the binding molecule is characterized in that it comprises a heavy region comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 29 and a constant disease comprising a polypeptide sequence as set out in SEQ ID NO: 31.
  • the binding molecule is characterized in that it comprises a light chain comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 30 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO.
  • the binding molecule is a heavy chain comprising a variable region comprising a polypeptide sequence as set out in SEQ ID NO: 29 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 31 and a polypeptide sequence as set out in SEQ ID NO: 30 And a light chain comprising a constant region comprising a variable region comprising a and a polypeptide sequence as set forth in SEQ ID NO: 32.
  • 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 of the present invention may be an antibody.
  • the rabies virus may be derived from an individual such as a bat, a dog, a cow, a mongoose, a skunk, a wolf, and the like, but is not limited thereto.
  • the present invention also provides an immunoconjugate in which at least one tag is attached to the binding molecule.
  • the present invention also provides an expression vector inserted with a nucleic acid molecule encoding the binding molecule.
  • the expression vector Celltrion's unique expression vector, MarEx vector (see patent application 10-2006-0020723), and pCDNA vectors commercially widely used, F, R1, RP1, Col, pBR322, ToL, Ti vectors; Cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ T-even, T2, T3, T7; It is preferable to use an expression vector selected from any one selected from the group consisting of plant viruses, but not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and when selecting an expression vector, a target host may be selected. It depends on the nature of the cell.
  • 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 selected 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.
  • tag sequences can 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.
  • the present invention also provides a cell line wherein the expression vector is transformed into a host cell to produce the binding molecule of the present invention.
  • 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.
  • the present invention also provides a method of producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising the steps of: i) culturing the cell line; And ii) recovering the expressed binding molecule.
  • 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 present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.
  • composition of the present invention may include a pharmaceutically acceptable excipient in addition to the binding molecule having the rabies virus neutralizing ability of the present invention.
  • Pharmaceutically acceptable excipients are well known to those skilled in the art.
  • the prophylactic and therapeutic composition of the present invention may include at least five different rabies therapeutics, and may also include several kinds of monoclonal antibodies, thereby exhibiting a synergistic effect on neutralizing activity.
  • the preventive and therapeutic compositions 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 pharmaceutically acceptable excipient upon or prior to delivery.
  • preferred 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 high drug concentrations, can maintain adequate flowability and delay absorption as 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 granules, emulsions, hard 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 present invention also provides a kit for diagnosing rabies comprising the binding molecule comprising the following steps: i) a binding molecule having the rabies virus neutralizing ability of the present invention; And ii) a container.
  • the present invention also provides a kit for treating and preventing rabies comprising the binding molecule comprising the following steps: i) a binding molecule having the rabies virus neutralizing ability of the present invention; And ii) a container.
  • the present invention also provides a method for diagnosing rabies using the binding molecule, comprising the steps of: i) contacting a sample of a subject with a binding molecule having the rabies virus neutralizing ability of the present invention; And ii) analyzing the results of step i) to determine whether rabies is infected.
  • the present invention also provides a method of treating and preventing rabies, comprising administering to a subject an effective amount of the binding molecule comprising the following steps: A binding molecule having the ability to neutralize rabies virus to a subject identified as rabies infected Administering a therapeutically effective amount.
  • the present invention also provides a method for detecting rabies virus, comprising the steps of: i) contacting a sample of a subject with a binding molecule having the rabies virus neutralizing ability of the present invention; And ii) determining whether the binding molecule specifically binds to the sample of interest.
  • the subject's sample may be a biological sample, including but not limited to blood, serum, tissue or other biological material from a (potentially) infected subject.
  • the (potential) infectious subject 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. Formation of immunological complexes indicative of the presence of rabies virus in the subject sample is detected and measured by appropriate means.
  • Such methods include, but are not limited to, immunoassays such as radioimmunoassay (RIA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE, Western blot analysis.
  • the binding molecule capable of neutralizing the rabies virus of the present invention has a neutralizing ability against various rabies viruses, it is useful for treating and preventing rabies in a patient or animal infected with rabies virus.
  • FIG. 1 shows chimeric antibody expression vectors comprising the heavy and light chain genes of the present invention.
  • Figure 2 shows the results of in vivo animal experiments using the Chinese dog rabies virus (Rv342).
  • # 62-71-3 clone was shown to be specifically potent against virus that had low neutralizing capacity in # 2-21-23 disclosed in patent application 10-2011-0024332, and was transferred from US CDC to Celltrion, # 62- Chimeric monoclonal antibodies were prepared using the variable region of 71-3 clone and the constant region of human type antibody.
  • Hybridoma cells # 62-71-3 were received from the US CDC, and the cells were cultured in IMDM medium (Invitrogen 12440-053) to which 5% fetal bovine serum (FBS; Sigma, 12003C) was added. During the culture period, mycoplasma contamination was examined using the Mycoplasma PCR ELISA kit (Roche, 11663925910), and it was confirmed that there was no mycoplasma in the culture.
  • IMDM medium Invitrogen 12440-053
  • FBS fetal bovine serum
  • 5 'RACE CDS primer A 5'-(T) 25 GC-3 ': SEQ ID NO: 1
  • a oligonucleotide (5'- AAG CAG TGG TAT CAA CGC AGA GTA CGT GGG -3 ': SEQ ID NO: 2) and reverse transcriptase were added and mixed, and reverse transcription was performed at 42 ° C. for 90 minutes and 70 ° C. for 10 minutes.
  • hybridoma cell-derived cDNA having a specific sequence at the 5 'end was synthesized.
  • the cDNA fragments including the entire variable regions of the obtained heavy and light chains were cloned into TA vectors in the TOPO TA cloning kit (Invitrogen, K4500), and then sequenced.
  • polymerase chain reaction product of the variable region and the constant region was used as a template, and PCR was performed under the same conditions as described above using HC F1 and HC R2 primers to secure a heavy chain connected to the variable region and the constant region.
  • polymerase chain reaction was carried out in the same manner using LC F1 and LC R2 primers to secure a light chain having a variable region and a constant region.
  • FreeStyle TM Max (Invitrogen, 16447-100), a cationic polymer, was used for transient transfection of cells and transduction was performed according to the manufacturer's instructions.
  • F2N cells KCTC 11309BP
  • EX-CELL 293 Serum free media Sigma, 14571C: hereinafter referred to as "EX-CELL 293 medium”
  • EX-CELL 293 medium EX-CELL 293 Serum free media
  • the medium was replaced with 50 ml (100 ml total) using two 250 ml shaker flasks at a concentration of 0.8 ⁇ 10 6 cells per ml.
  • 125 ⁇ g of pCT234 DNA containing the chimeric antibody gene and 125 ⁇ l of FreeStyle TM Max reagent were diluted in 2 ml volume using OptiPRO SFM II (Invitrogen, 12309) medium, and then mixed lightly. Immediately diluted FreeStyle TM Max reagent solution was mixed with a solution containing DNA, and then reacted at room temperature for 17 minutes. During the 17 min reaction at room temperature, 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.
  • Selected chimeric antibody # 13-6 was diluted to an appropriate concentration (10 times less than the maximum stock solution) and neutralization experiments were performed on representative rabies virus, which was performed via RFFIT. The results are shown in Table 7.
  • the chimeric antibody of the present invention was found to have neutralizing ability against rabies virus derived from bat (AZ Bat, TN 269, CA 3860), dog (Thai Dog, 002 Phil, Dog Phil, China Dog2005, Rv 342). .
  • Example 4-2 In vivo animal experiment
  • the experimental group of animals was divided into five groups, 1. group injected with Rv342 virus only, 2. group injected with Rv342 virus and vaccine (Human diploid Imovax ® Sanofi Pasteur), 3. Rv342 virus and 62-71-3 key Group injected with Merrick Antibody # 17, 4.Rv342 virus, 62-71-3 chimeric antibody # 17 and vaccine (Human diploid Imovax ® Sanofi Pasteur), 5.Rv342 virus and Human Rabies Immune Globulin (HRIG) , Imogam ® Rabies-HT, Sanofi Pasteur).
  • HRIG Human Rabies Immune Globulin
  • Rv342 virus was diluted 1/100 based on MICLD50 / ml to intramuscularly injected 50 ul, and the vaccine was injected with 50 ul of vaccine virus strain of at least about 2.5 IU per ml (0, 3, 7, 14 day), Chimeric antibody # 17 was injected with 50 ul of 0.614 mg / mL, which corresponds to about 20 IU / kg, equivalent to the amount injected with HRIG.
  • Vaccine, chimeric antibody # 17 and HRIG were injected 24 hours after Rv342 virus injection.
  • Virus and 62-71-3 chimeric antibody # 17 survived most of the observations for 45 days (91.7%), but only virus (group 1) or virus and vaccine injection (group 1) Group 2) all died.
  • the virus, 62-71-3 chimeric antibody # 17 and vaccine injection (group 4) also showed 100% survival.
  • HRIG which is currently used as a therapeutic agent, was injected in the same amount as 62-71-3 chimeric antibody # 17, and the survival rate was 33.3%.

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Abstract

La présente invention concerne une molécule de liaison conçue pour neutraliser un virus de la rage. De manière plus spécifique, la molécule de liaison selon l'invention permet de neutraliser le virus de la rage qui est dérivé d'espèces telles que les chauves-souris, les chiens, les vaches, les mangoustes, les mouffettes et les loups, et peut ainsi être utilisé pour traiter un patient qui a contracté le virus de la rage dérivé d'une grande variété d'espèces.
PCT/KR2012/007795 2011-09-30 2012-09-27 Molécule de liaison pour neutraliser un virus de la rage WO2013048130A2 (fr)

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CN201280047216.2A CN103998059B (zh) 2011-09-30 2012-09-27 用于中和狂犬病病毒的结合分子

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KR20110099685 2011-09-30
KR10-2011-0099685 2011-09-30

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WO2013048130A2 true WO2013048130A2 (fr) 2013-04-04
WO2013048130A3 WO2013048130A3 (fr) 2013-05-23

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WO2020089742A1 (fr) 2018-11-02 2020-05-07 Cadila Healthcare Limited Anticorps monoclonaux antirabiques et cocktail correspondant

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KR101739313B1 (ko) * 2013-12-12 2017-05-24 (주)셀트리온 광견병 바이러스를 중화시킬 수 있는 결합 분자
US10722571B2 (en) * 2015-06-10 2020-07-28 Celltrion Inc. Rabies virus G protein epitope, and rabies virus neutralising binding molecule that binds specifically thereto

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US5695757A (en) * 1993-05-26 1997-12-09 Thomas Jefferson University Methods for treating post-exposure rabies and anti-rabies compositions
US20060216300A1 (en) * 2003-06-13 2006-09-28 Thomas Jefferson University Recombinant antibodies and compositions and methods for making and using the same
US20070072177A1 (en) * 2004-05-27 2007-03-29 Crucell Holland B.V. Binding molecules capable of neutralizing rabies virus and uses thereof
US20090041777A1 (en) * 2005-02-02 2009-02-12 Thomas Jr William D Human antibodies against rabies and uses thereof

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US8748567B2 (en) * 2006-05-22 2014-06-10 Children's Medical Center Corporation Method for delivery across the blood brain barrier
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CN102643343B (zh) * 2011-02-17 2015-03-25 长春百克生物科技股份公司 一种人源抗狂犬病毒糖蛋白基因工程抗体及其制备与应用

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US5695757A (en) * 1993-05-26 1997-12-09 Thomas Jefferson University Methods for treating post-exposure rabies and anti-rabies compositions
US20060216300A1 (en) * 2003-06-13 2006-09-28 Thomas Jefferson University Recombinant antibodies and compositions and methods for making and using the same
US20070072177A1 (en) * 2004-05-27 2007-03-29 Crucell Holland B.V. Binding molecules capable of neutralizing rabies virus and uses thereof
US20090041777A1 (en) * 2005-02-02 2009-02-12 Thomas Jr William D Human antibodies against rabies and uses thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020089742A1 (fr) 2018-11-02 2020-05-07 Cadila Healthcare Limited Anticorps monoclonaux antirabiques et cocktail correspondant
EP3873526B1 (fr) * 2018-11-02 2024-09-04 Zydus Lifesciences Limited Anticorps monoclonaux anti-rage et cocktail associés

Also Published As

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CN103998059A (zh) 2014-08-20
KR101495019B1 (ko) 2015-02-24
KR20130036150A (ko) 2013-04-11
CN103998059B (zh) 2016-01-20
WO2013048130A3 (fr) 2013-05-23

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