WO2013048130A2 - Binding molecule for neutralizing rabies virus - Google Patents

Binding molecule for neutralizing rabies virus 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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French (fr)
Korean (ko)
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WO2013048130A3 (en
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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/en
Publication of WO2013048130A2 publication Critical patent/WO2013048130A2/en
Publication of WO2013048130A3 publication Critical patent/WO2013048130A3/en

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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
    • 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

The present invention relates to a binding molecule for neutralizing a rabies virus. More specifically, the binding molecule according to the present invention, can neutralize the rabies virus that is derived from species such as bats, dogs, cows, mongooses, skunks, and wolves, and thus can be useful in treating a patient that has contracted the rabies virus derived from a wide variety of species.

Description

광견병 바이러스를 중화시킬 수 있는 결합 분자Binding molecules that can neutralize rabies virus
본 발명은 광견병 바이러스를 중화시킬 수 있는 결합 분자에 관한 것이다. The present invention relates to binding molecules capable of neutralizing rabies virus.
광견병(rabies)은 바이러스성 인수공통 감염병이며, 주로 야생 및 애완 동물 에 영향을 미칠 뿐 아니라 인간을 포함한 포유류에게 영향을 미쳐 급성 뇌 질환의 원인이 된다. 한 번 발생하면 거의 사망에 이르는 치명적인 질병으로, 에이즈와 더불어 치사율이 가장 높은 질병으로 알려져 있다. 이러한 광견병은 전 세계적으로 퍼져 있으며, 매년 천만 명 이상이 감염 후 치료를 받으며 매년 40,000명에서 70,000명이 사망한다.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.
광견병은 타액과 피로 전염되는데, 주로 광견병에 감염된 개 또는 고양이 등에 물리게 되면 발병한다. 또한 스컹크, 박쥐 등 대부분의 포유류에 의해 감염될 수 있다.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.
광견병 바이러스(rabies virus)는 신체의 신경 조직을 통해 뇌신경 조직으로 도달한 뒤 실제 발병 증상을 나타낸다. 원래 인간의 뇌에는 혈액 내 장벽(blood brain barrier)이 존재하여 외부 물질을 차단하기 때문에 바이러스 등이 침투할 수 없으나, 광견병 바이러스는 RVG(rabies virus glycoprotein) 단백질을 통해 혈액 내 장벽을 통과하여 신경계(central nervous system) 뇌를 감염시킨다.Rabies virus reaches the brain's nerve tissue through the body's nerve tissue and shows the actual onset symptoms. Originally, 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.
초기에는 감기와 비슷한 증상 이외에, 물린 부위에 가려움증이나 열을 느낀다. 광견병이 진행되면서 불안감, 공수증(물 등의 액체를 삼키게 되면 근육이 경련을 일으키고 심한 통증을 느껴 물을 두려워하는 증상), 바람에 대한 두려움(바람이 감각 기관을 과민하게 함), 흥분, 마비, 정신 이상 등의 신경 이상 증상이 나타난다. 또한 햇빛에 과민 반응을 일으키기도 한다. 이러한 증상이 관찰된 후 2-7일 뒤에 전신의 신경이나 근육이 마비를 일으켜 혼수 상태에 빠지고, 호흡 장애로 사망하게 된다.In the early stages, in addition to cold-like symptoms, you feel itching or fever at the bite. As rabies progresses, anxiety, rabies (swallowing liquids such as water causes muscle cramps and severe pain, fearing water), fear of wind (wind makes the sense organs sensitive), excitement, paralysis , Neurological symptoms such as mental disorders. It can also cause hypersensitivity to sunlight. Two to seven days after these symptoms are observed, the nerves and muscles of the whole body become paralyzed, leading to coma and dying from breathing problems.
광견병은 노출 후 예방은 즉각적인 국소 상처 보호 및 수동(항-광견병 이뮤 노글로블린: 이하 "anti-rabies antibody"라 칭함) 및 능동(백신) 면역화의 투여를 통해 치료 및 예방될 수 있다. 현재 개발된 anti-rabies antibody는 인간 유래 광견병 항체(human derived rabies immunoglobulin: 이하 "HRIG"라 칭함) 및 말 유래 광견병 항체(equine derived rabies immunoglobulin: 이하 "ERIG"라 칭함)가 있다. HRIG의 경우 원활한 공급이 어려우며, 가격이 고가이다. 또한 인간의 혈액에서 유래한 것으로 HIV 등의 감염 위험성이 높고, 다클론 항체(polyclonal antibody)여서 효능이 높지 않다. ERIG의 경우 말에서 유래하여 HRIG 보다 치료 효율이 낮으며, 이로 인하여 HRIG보다 높은 용량으로 환자에게 투여된다. HRIG 보다 저가이기는 하나 이 역시 원활한 공급이 되지 않고 있는 실정이며, 인간과는 다른 개체에서 유래한 항체임으로 과민증(anaphyaxis)이 올 수 있다. 이러한 단점을 극복하기 위해 노출 후 예방에서 광견병 바이러스를 중화시킬 수 있는 단일클론 항체(monoclonal antibody)의 사용이 제안되었다. 광견병-바이러스 중화 뮤린 단일클론 항체가 개발되었으나(Schumacher CL et al., J. Clin. Invest. Vol. 84, p. 971-975, 1989), 짧은 혈청 반감기, 인간 이펙터 기능을 유도하는 능력 부재 및 인체에서 뮤린 항체에 대한 원치 않는 HAMA(human anti-mouse antibody) 반응이 유도되어 광견병에 감염된 인간 환자에 대한 직접적인 투여에는 제한되어 있다.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. Currently developed 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. In addition, it 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. 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. To overcome this drawback, the use of 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. 971-975, 1989), but short serum half-life, lack of ability to induce human effector function, and Induction of unwanted human anti-mouse antibody (HAMA) responses to murine antibodies in the human body is limited to direct administration to rabies-infected human patients.
이에 광견병 치료를 위하여 혈액에서 유래하지 않아 안정성이 높으며, 배양을 통한 합성으로 대규모 생산 공급이 가능하고, 균일한 품질 확보가 가능한 단일클론 항체의 개발이 시급한 실정이다.Therefore, it is urgent to develop monoclonal antibodies capable of securing large-scale production and uniform quality by synthesizing through culturing because they are not derived from blood for the treatment of rabies.
본 발명의 목적은 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 제공하는 것이다.It is an object of the present invention to provide a binding molecule that binds to rabies virus and has a neutralizing ability.
또한, 본 발명의 다른 목적은 상기 결합 분자에 하나 이상의 태그가 결합된 이뮤노컨쥬게이트를 제공하는 것이다.Another object of the present invention is to provide an immunoconjugate in which one or more tags are attached to the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 암호화하는 폴리뉴클레오티드를 제공하는 것이다.Another object of the present invention is to provide a polynucleotide encoding the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 암호화하는 폴리뉴클레오티드가 삽입된 발현 벡터를 제공하는 것이다.Another object of the present invention is to provide an expression vector into which a polynucleotide encoding the binding molecule is inserted.
또한, 본 발명의 다른 목적은 상기 발현 벡터가 형질전환된 세포주를 제공하는 것이다.In addition, another object of the present invention to provide a cell line transformed with the expression vector.
또한, 본 발명의 다른 목적은 상기 세포주를 배양하여 본 발명의 결합 분자를 생산하는 방법을 제공하는 것이다.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.
또한, 본 발명의 다른 목적은 상기 결합 분자를 포함하는 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition comprising the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 포함하는 키트를 제공하는 것이다.Another object of the present invention is to provide a kit comprising the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 이용한 광견병 진단 방법을 제공하는 것이다.Another object of the present invention is to provide a method for diagnosing rabies using the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 이용한 광견병 치료 및 예방 방법을 제공하는 것이다.In addition, another object of the present invention is to provide a method for treating and preventing rabies using the binding molecule.
또한, 본 발명의 다른 목적은 상기 결합 분자를 이용한 광견병 바이러스 검출 방법을 제공한다. Another object of the present invention is to provide a method for detecting rabies virus using the binding molecule.
상기 목적을 달성하기 위하여, 본 발명은 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 제공한다.In order to achieve the above object, the present invention provides a binding molecule having a neutralizing ability to bind to rabies virus.
또한, 본 발명은 상기 결합 분자에 하나 이상의 태그가 결합된 이뮤노컨쥬게 이트를 제공한다.The present invention also provides an immunoconjugate wherein at least one tag is attached to the binding molecule.
또한, 본 발명은 상기 결합 분자를 암호화하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding the binding molecule.
또한, 본 발명은 상기 결합 분자를 암호화하는 폴리뉴클레오티드가 삽입된 발현 벡터를 제공한다.The present invention also provides an expression vector inserted with a polynucleotide encoding the binding molecule.
또한, 본 발명은 상기 발현 벡터가 숙주 세포에 형질전환되어, 본 발명의 결합 분자를 생산하는 세포주를 제공한다.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.
또한, 본 발명은 상기 세포주를 배양하여 본 발명의 결합 분자를 생산하는 방법을 제공한다.The present invention also provides a method of producing the binding molecule of the present invention by culturing the cell line.
또한, 본 발명은 상기 결합 분자 및 약제학적으로 허용가능한 부형제가 추가적으로 포함된 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition further comprising the binding molecule and a pharmaceutically acceptable excipient.
또한, 본 발명은 상기 결합 분자 및 약제학적으로 허용가능한 부형제가 추가 적으로 포함된 광견병 치료 및 예방용 조성물을 제공한다.The present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.
또한, 본 발명은 상기 결합 분자를 포함하는 광견병 진단용 키트를 제공한다.The present invention also provides a kit for diagnosing rabies comprising the binding molecule.
또한, 본 발명은 상기 결합 분자를 포함하는 광견병 치료 및 예방용 키트를 제공한다.The present invention also provides a kit for the treatment and prevention of rabies comprising the binding molecule.
또한, 본 발명은 상기 결합 분자를 이용한 광견병 진단 방법을 제공한다. The present invention also provides a method for diagnosing rabies using the binding molecule.
또한, 본 발명은 상기 결합 분자를 대상에게 유효량으로 투여하는 단계를 포함하는 광견병 치료 및 예방 방법을 제공한다.The present invention also provides a method for treating and preventing rabies comprising administering to said subject an effective amount of said binding molecule.
또한, 본 발명은 상기 결합 분자를 이용한 광견병 바이러스 검출 방법을 제공한다. The present invention also provides a method for detecting rabies virus using the binding molecule.
이하, 본 발명에서 사용되는 용어를 아래와 같이 정의한다. Hereinafter, terms used in the present invention are defined as follows.
본 발명에서 사용되는 "결합 분자"라는 용어는 키메라, 인간화 또는 인간 단일클론 항체와 같은 단일클론 항체를 포함하는 온전한(intact) 이뮤노글로블린(immunoglobulin), 또는 항원에 결합하는 이뮤노글로블린, 예를 들면 광견병 바이러스 또는 바이러스 외부의 G 단백질(Glycoprotein) 또는 그의 단편과의 결합을 위해 온전한(intact) 이뮤노글로블린과 경쟁하는 이뮤노글로블린 단편을 포함하는 가변성 도메인을 뜻한다. 구조와는 상관없이 항원-결합 단편은 온전한(intact) 이뮤노글로블린에 의해 인식된 동일한 항원과 결합된다. 항원-결합 단편은 결합 분자의 아미노산 서열의 2개 이상의 연속(contiguous) 아미노산 잔기, 20개 이상의 연속 아미노산 잔기, 25개 이상의 연속 아미노산 잔기, 30개 이상의 연속 아미노산 잔기, 35개 이상의 연속 아미노산 잔기, 40개 이상의 연속 아미노산 잔기, 50개 이상의 연속 아미노산 잔기, 60개 이상의 연속 아미노산 잔기, 70개 이상의 연속 아미노산 잔기, 80개 이상의 연속 아미노산 잔기, 90개 이상의 연속 아미노산 잔기, 100개 이상의 연속 아미노산 잔기, 125개 이상의 연속 아미노산 잔기, 150개 이상의 연속 아미노산 잔기, 175개 이상 연속 아미노산 잔기, 200개 이상의 연속 아미노산 잔기, 또는 250개 이상의 연속 아미노산 잔기의 아미노산 서열을 포함하는 펩티드 또는 폴리펩티드를 포함할 수 있다.As used herein, the term "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 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.
본 발명에서 사용되는 "결합 분자"라는 용어는 당업계에 알려진 모든 이뮤노글로블린 종류(class)와 아강(subclass)을 포함한다. 결합 분자는 중쇄의 불변 도메인의 아미노산 서열에 따라서 온전한(intact) 항체의 다섯 개의 주요 종류 IgA, IgD, IgE, IgG, 및 IgM으로 나뉠 수 있으며, 이들 중 일부는 예를 들면 IgA1, IgA2, IgG1, IgG2, IgG3 및 IgG4와 같은 아강(이소타입)으로 추가로 나뉠 수 있다.As used herein, the term "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.
항원-결합 단편은 특히 Fab, F(ab'), F(ab')2, Fv, dAb, Fd, 상보성 결정 영역(CDR) 단편, 단일-쇄 항체(scFv), 2가(bivalent) 단일-쇄 항체, 단일-쇄 파지 항체, 디아바디(diabody), 트리아바디(triabody), 테트라바디(tetrabody), 폴리펩티드로의 특정 항원에 결합하기에 충분한 이뮤노글로브린의 하나 이상의 단편을 함유하는 폴리펩티드 등을 포함한다. 상기 단편은 합성으로 또는 완전한 이뮤노글로블린의 효소적 또는 화학적 분해에 의해 생성되거나, 재조합 DNA 기술에 의해 유전공학적으로 생성될 수 있다. 생성 방법은 당업계에 잘 알려져 있다. 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.
결합 분자는 있는 그대로의(naked) 또는 컨쥬게이트되지 않은 결합 분자일 수 있지만 이뮤노컨쥬게이트의 일부일 수 있다. The binding molecule may be a naked or unconjugated binding molecule but may be part of an immunoconjugate.
본 발명에서 사용되는 "약제학적으로 허용가능한 부형제"라는 용어는 용인가능한 또는 편리한 투약 형태를 제조하기 위한 약물, 제제 또는 결합 분자와 같은 활성 분자로 조합되는 불활성 물질을 의미한다. 약제학적으로 허용가능한 부형제는 비독성이거나, 적어도 독성이 사용된 용량 및 농도에서 수용자에게 이의 의도된 용도를 위해 허용될 수 있는 부형제이고, 약물, 제제 또는 결합 분제를 포함하는 제형화의 다른 성분과 양립할 수 있다.As used herein, 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.
본 발명에서 사용되는 "치료학적으로 유용한 양"이라는 용어는 광견병 바이러스의 노출 전 또는 노출 후에 예방 또는 치료에 유효한 본 발명의 결합 분자의 양을 나타낸다.As used herein, 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.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 미국질병관리본부(Center for Disease Control: 이하 "CDC"라 칭함)에서 광범위한 광견병 바이러스에 중화 능력을 보인다고 검증된 하이브리도마 세포를 전달받아 이를 대상으로 마우스 타입 단일클론 항체의 중쇄 및 경쇄의 가변 영역 서열을 확보하였다. 이후 IgG1 백본(backbone)에 상기 중쇄 및 경쇄 가변 영역을 연결하여 키메릭 항체를 제조하였다. 이후 위와 같이 제조된 키메릭 항체를 in vivo 및 in vitro 실험을 수행하여 다양한 광견병 바이러스를 대상으로 중화 능력 시험을 수행하였으며, 이를 통하여 본 발명의 단일클론 항체가 광범위한 개체에서 유래된 광견병 바이러스에 감염된 환자를 치료하는데 유용하게 이용될 수 있음이 확인되었다.The inventors of the Center for Disease Control (hereinafter referred to as "CDC") 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. Thereafter, the heavy and light chain variable regions were linked to an IgG1 backbone to prepare a chimeric antibody. Subsequently, 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
이에, 본 발명은 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 제공한다.Accordingly, the present invention provides a binding molecule that binds to rabies virus and has a neutralizing ability.
본 발명에 있어서, 상기 결합 분자는 서열번호 23으로 기재되는 폴리펩티드를 포함하는 CDR(complementarity determining regions) 1 영역, 서열번호 24로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 25로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 가변 영역을 포함하는 것을 특징으로 한다. In the present invention, 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.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 26으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 27로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 28로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 가변 영역을 포함하는 것을 특징으로 한다. Also in the present invention, 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.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 23으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 24로 기재되는 폴리펜티드를 포함하는 CDR2 영역 및 서열번호 25로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 중쇄 가변 영역 및 서열번호 26으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 27로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 28로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 경쇄 가변 영역을 포함하는 것을 특징으로 한다. 상기 결합 분자는 Fab 절편, Fv 절편, 디아바디(diabody), 트리아바디(triabody), 테트라바디(tetrabody), 키메라 항체, 인간화 항체 또는 인간 항체일 수 있다. Also in the present invention, 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 A light chain variable region having a heavy chain variable region having a CDR1 region comprising a polypeptide represented by SEQ ID NO: 26, a CDR2 region comprising a polypeptide represented by SEQ ID NO: 27, and a CDR3 region comprising a polypeptide represented by SEQ ID NO: 28 It is characterized by including. The binding molecule may be a Fab fragment, Fv fragment, diabody, triabody, tetrabody, chimeric antibody, humanized antibody or human antibody.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역을 포함하는 것을 특징으로 한다.In addition, in the present invention, the binding molecule is characterized in that it comprises a variable region comprising the polypeptide sequence set forth in SEQ ID NO: 29.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역을 포함하는 것을 특징으로 한다.In the present invention, the binding molecule is characterized in that it comprises a variable region comprising the polypeptide sequence set forth in SEQ ID NO: 30.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 중쇄 가변 영역 및 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 경쇄 가변 영역을 포함하는 것을 특징으로 한다. 상기 결합 분자는 Fab 절편, Fv 절편, 디아바디(diabody), 트리아바디(triabody), 테트라바디(tetrabody), 키메라 항체, 인간화 항체 또는 인간 항체일 수 있다. Also in the present invention, 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.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 31로 기재되는 폴리펩티드 서열을 포함하는 불변 병역을 포함하는 중쇄를 포함하는 것을 특징으로 한다. In addition, in the present invention, 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.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 32로 기재되는 폴리펩티드 서열을 포함하는 불변 영역을 포함하는 경쇄를 포함하는 것을 특징으로 한다.Also in the present invention, 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.
또한 본 발명에 있어서, 상기 결합 분자는 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 31로 기재되는 폴리펩티드 서열을 포함하는 불변 병역을 포함하는 중쇄 및 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 32로 기재되는 폴리펩오티드 서열을 포함하는 불변 영역을 포함하는 경쇄를 포함하는 것을 특징으로 한다. Also in the present invention, 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.
본 발명에 있어서, 가변영역의 CDR은 Kabat 등에 의해 고안된 시스템에 따라 통상적인 방법으로 결정되었다(문헌[Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD. (1991)] 참조). 본 발명에 사용된 CDR 결정은 Kabat 방법을 사용했지만, 이외에 IMGT 방법, Chothia 방법, AbM 방법 등 다른 방법에 따라 결정된 CDR을 포함하는 결합분자도 본 발명에 포함된다.In the present invention, 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.
본 발명의 상기 결합 분자는 항체일 수 있다. The binding molecule of the present invention may be an antibody.
또한 상기 광견병 바이러스는 박쥐, 개, 소, 몽구스, 스컹크, 늑대 등의 개체에서 유래될 수 있지만, 이것에 한정되는 것은 아니다. In addition, 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.
또한, 본 발명은 상기 결합 분자에 하나 이상의 태그가 결합된 이뮤노컨쥬게이트를 제공한다.The present invention also provides an immunoconjugate in which at least one tag is attached to the binding molecule.
또한, 본 발명은 상기 결합 분자를 암호화하는 핵산 분자를 제공한다.The present invention also provides a nucleic acid molecule encoding the binding molecule.
본 발명의 핵산 분자는 본 발명에서 제공하는 항체의 아미노산 서열을 당업자에게 알려진 바와 같이 폴리뉴클레오티드 서열로 번역된 핵산 분자 모두를 포함한다. 그러므로 ORF(open reading frame)에 의한 다양한 폴리뉴클레오티드 서열이 제조될 수 있으며 이 또한 모두 본 발명의 핵산 분자에 포함된다.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.
또한, 본 발명은 상기 결합 분자를 암호화하는 핵산 분자가 삽입된 발현 벡터를 제공한다.The present invention also provides an expression vector inserted with a nucleic acid molecule encoding the binding molecule.
상기 발현 벡터로는 셀트리온 고유의 발현 벡터인 MarEx 벡터(특허출원 10-2006-0020723 참조) 및 상업적으로 널리 사용되는 pCDNA 벡터, F, R1, RP1, Col, pBR322, ToL, Ti 벡터; 코스미드; 람다, 람도이드(lambdoid), M13, Mu, p1 P22, Qμ T-even, T2, T3, T7 등의 파아지; 식물 바이러스로 이루어진 군으로부터 선택된 어느 하나에서 선택된 발현 벡터를 이용하는 것이 바람직하나 이에 한정되는 것은 아니며, 당업자에게 발현 벡터로 알려진 모든 발현 벡터는 본 발명에 사용 가능하며, 발현 벡터를 선택할 때에는 목적으로 하는 숙주 세포의 성질에 따른다. 숙주세포로의 벡터 도입시 인산칼슘 트랜스펙션, 바이러스 감염, DEAE-덱스트란 조절 트랜스펙션, 리포펙타민 트랜스펙션 또는 전기천공법에 의해 수행될 수 있으나 이에 한정되는 것은 아니며 당업자는 사용하는 발현 벡터 및 숙주 세포에 알맞은 도입 방법을 선택하여 이용할 수 있다. 바람직하게 벡터는 하나 이상의 선별 마커를 함유하나 이에 한정되지 않으며, 선별 마커를 포함하지 않은 벡터를 이용하여 생산물 생산 여부에 따라 선별이 가능하다. 선별 마커의 선택은 목적하는 숙주 세포에 의해 선별되며, 이는 이미 당업자에게 알려진 방법을 이용하므로 본 발명은 이에 제한을 두지 않는다. As 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, Qμ 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. Preferably, 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.
본 발명의 핵산 분자를 정제를 용이하게 하기 위하여 태그 서열을 발현 벡터 상에 삽입하여 융합시킬 수 있다. 상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되는 것은 아니며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다. In order to facilitate purification of the nucleic acid molecules of the present invention, 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.
또한, 본 발명은 상기 발현 벡터가 숙주 세포에 형질전환되어, 본 발명의 결합 분자를 생산하는 세포주를 제공한다.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.
본 발명에 있어서, 상기 세포주는 포유동물, 식물, 곤충, 균류 또는 세포성 기원의 세포를 포함할 수 있지만 이에 한정되지 않는다. 상기 포유동물 세포로는 CHO 세포, F2N 세포, CSO 세포, BHK 세포, 바우스(Bowes) 흑색종 세포, HeLa 세포, 911 세포, AT1080 세포, A549 세포, HEK 293 세포 및 HEK293T 세포로 이루어진 군에서 선택된 어느 하나를 숙주 세포로 사용하는 것이 바람직하나 이에 한정되지 않으며, 당업자에게 알려진 포유동물 숙주세포로 사용 가능한 세포는 모두 이용 가능하다.In 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.
또한, 본 발명은 하기 단계를 포함하는 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 생산하는 방법을 제공한다: i) 상기 세포주를 배양하는 단계; 및 ii) 발현된 결합 분자를 회수하는 단계.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.
또한, 본 발명은 상기 결합 분자 및 약제학적으로 허용가능한 부형제가 추가적으로 포함된 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition further comprising the binding molecule and a pharmaceutically acceptable excipient.
본 발명의 조성물은 광견병 바이러스 중화 능력을 가지는 결합 분자 이외에 약제학적으로 허용 가능한 부형제를 포함할 수 있다. 약제학적으로 허용 가능한 부형제는 당업자에게 이미 잘 알려져 있다. 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.
또한, 본 발명은 상기 결합 분자 및 약제학적으로 허용가능한 부형제가 추가적으로 포함된 광견병 치료 및 예방용 조성물을 제공한다.The present invention also provides a composition for the treatment and prevention of rabies further comprising the binding molecule and a pharmaceutically acceptable excipient.
본 발명의 조성물은 본 발명의 광견병 바이러스 중화 능력 가지는 결합 분자 이외에 약제학적으로 허용 가능한 부형제를 포함할 수 있다. 약제학적으로 허용 가능한 부형제는 당업자에게 이미 잘 알려져 있다. The 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.
또한 본 발명의 예방 및 치료용 조성물은 적어도 5개의 다른 광견병 치료제를 포함할 수 있으며, 또한 여러 종류의 단일클론 항체를 포함할 수 있으며, 이를 통해 중화 활성에 상승 작용을 나타낼 수 있다. In addition, 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.
아울러 본 발명의 예방 및 치료용 조성물을 추가로 적어도 하나의 다른 치료제 또는 진단제를 포함할 수 있다. 상기 치료제로는 항-바이러스 약제를 포함하나 이에 한정되는 것은 아니다. 이러한 약제로는 항체, 소분자, 유기 또는 무기 화합물, 효소, 폴리뉴클레오티드 서열, 항-바이러스성 펩티드 등일 수 있다. In addition, the preventive and therapeutic compositions of the present invention may further include at least one other therapeutic or diagnostic agent. Such therapeutic agents include, but are not limited to, anti-viral agents. Such agents can be antibodies, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, anti-viral peptides, and the like.
본 발명의 예방 및 치료용 조성물은 제조 및 저장 조건하에서 무균하고 안정하며, 전달시 또는 전달 전에 적절한 약제학적으로 허용 가능한 부형제 중에 재구성을 위해 분말 형태로 존재할 수 있다. 살균성 주사용액 제조를 위한 살균 분말의 경우, 바람직한 제조 방법은 활성 구성성분의 분말과 이의 미리 살균-여과된 용액으로부터 추가의 원하는 구성성분을 생산하는 진공 건조 및 동결 건조이다. 본 발명의 조성물은 용액 상태일 수 있고, 적절한 약제학적으로 허용 가능한 부형제가 단위 투여량 주사형을 제공하기 위해 전달되기 전 또는 전달 시에 첨가 및/또는 혼합될 수 있다. 바람직하게 본 발명에 사용되는 약제학적으로 허용 가능한 부형제는 고 약물농도에 알맞고, 알맞은 흐름성을 유지할 수 있고, 필요에 따라 흡수가 지연될 수 있다.The prophylactic and therapeutic 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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. Preferably the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, can maintain adequate flowability and delay absorption as necessary.
본 발명의 예방 및 치료용 조성물 투여의 최적 경로 선택은 조성물 내의 활성 분자들의 물리-화학적 특성, 임상적 상황의 긴급성 및 원하는 치료용 효과에 대한 활성 분자의 플라즈마 농도의 관계를 포함하는 여러 인자들에 의해 영향을 받는다. 예를 들어 본 발명의 단일클론 항체는 임플란트 및 마이크로캡슐화 전달 시스템을 포함하는, 조절된 방출 제형과 같은 그들의 신속한 방출을 막은 담체와 함께 제조될 수 있다. 에틸렌 비닐 아세테이트, 다가무수물, 폴리글리콜산, 콜라겐, 폴리오르토에스테르 및 폴리락트산과 같은 생분해성, 생체적합성 폴리머가 본 발명에 사용될 수 있다. 또한 단일클론 항체는 항체의 불활성화를 막은 물질 또는 화합물로 코팅되거나 함께 투여될 수 있다. 예를 들어, 단일클론 항체는 적합한 담체-리포좀 또는 희석제-와 함께 투여될 수 있다.Selection of the optimal route of administration of the prophylactic and therapeutic compositions of the present invention involves several factors including the relationship of the physico-chemical properties of the active molecules in the composition, the urgency of the clinical situation and the plasma concentration of the active molecule to the desired therapeutic effect Affected by For example, 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. For example, monoclonal antibodies can be administered with a suitable carrier—liposomes or diluents.
본 발명의 예방 및 치료용 조성물의 투여 방법은 경구 및 비경구로 나뉠 수 있으며, 바람직한 투여 경로는 정맥 내이나 이에 한정되는 것은 아니다.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.
상기 경구 형태로는 정제, 트로키제, 약용 드롭스제, 수성 또는 유성 현탁제, 산제 또는 분산과립제, 에멀젼제, 강성 캡슐제, 연성 젤라틴 캡슐제, 시럽제 또는 엘릭서제, 필제, 당의정, 액제, 겔제 또는 슬러리제로서 제제화될 수 있다. 이들 제형은 불활성 희석제, 과립화 또는 붕해제, 결합제, 광택제, 보존제, 착색제, 풍미제 또는 감미제, 식물성유 또는 미네랄유, 습윤제 및 점증제를 함유하는 약제학적 부형제를 포함하나 이에 한정되는 것은 아니다.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.
상기 비경구 형태로는 수성 또는 비수성 등장성 살균 비독성 주사 또는 주입 용액 또는 현탁액의 형태일 수 있다. 용액 또는 현탁액은 적용된 투여량 및 농도에서 수용체에 비독성인 1,3-부탄디올, 링거스 용액, 행크스 용액, 등장성 염화나트륨 용액과 같은 약제, 오일, 지방산, 국소마취제, 보존제, 완충액, 점도 또는 용해도 증가제, 수용성 항산화제, 유용성 항산화제 및 금속 킬레이트화제를 포함할 수 있다.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.
또한, 본 발명은 하기의 단계를 포함하는 상기 결합 분자를 포함하는 광견병 진단용 키트를 제공한다: i) 본 발명의 광견병 바이러스 중화 능력을 가진 결합 분자; 및 ii) 용기. 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.
또한, 본 발명은 하기의 단계를 포함하는 상기 결합 분자를 포함하는 광견병 치료 및 예방용 키트를 제공한다: i) 본 발명의 광견병 바이러스 중화 능력을 가진 결합 분자; 및 ii) 용기. 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.
또한, 본 발명은 하기의 단계를 포함하는 상기 결합 분자를 이용한 광견병 진단 방법을 제공한다: i) 대상의 시료와 본 발명의 광견병 바이러스 중화 능력을 가진 결합 분자를 접촉시키는 단계; 및 ii) 상기 단계 i)의 결과를 분석하여 광견병 감염 여부를 판별하는 단계.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.
또한, 본 발명은 하기의 단계를 포함하는 상기 결합 분자를 대상에게 유효량으로 투여하는 단계를 포함하는 광견병 치료 및 예방 방법을 제공한다: 광견병에 감염되었다고 확인된 대상에게 광견병 바이러스 중화 능력을 가진 결합 분자를 치료학적으로 유효한 양으로 투여하는 단계.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.
또한, 본 발명은 하기의 단계를 포함하는 광견병 바이러스를 검출하는 방법을 제공한다: i) 대상의 시료와 본 발명의 광견병 바이러스 중화 능력을 가진 결합 분자를 접촉시키는 단계; 및 ii) 상기 결합 분자가 대상 시료에 특이적으로 결합하는지 측정하는 단계. 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.
대상의 시료는 (잠재적) 감염 대상으로부터의 혈액, 혈청, 조직 또는 다른 생물학적 물질을 포함하지만, 이것에 한정되지는 않는 생물학적 샘플일 수 있다. (잠재적) 감염 대상은 인간 대상일 수 있지만, 또한 광견병 바이러스의 담체로서 의심되는 동물들일 수 있다. 대상 시료는 먼저 그것을 검출 방법에 더 적절하게 만들기 위해 조작될 수 있다. 바람직하게는, 본 발명의 결합 분자 또는 이뮤노컨쥬게이트는 결합 분자와 광견병 바이러스 또는 대상 시료에 존재하는 그것의 항원성 성분 사이의 면역학적 복합체의 형성을 허용하는 조건 하에서 대상 시료와 접촉된다. 대상 시료 내의 광견병 바이러스의 존재를 나타내는 면역학적 복합체의 형성은 적절한 수단에 의해 검출되고 측정된다. 이러한 방법으로 방사면역측정법(RIA), ELISA, 면역형광법, 면역 조직 화학, FACS, BIACORE, 웨스턴 블롯 분석과 같은 면역분석이 있지만, 이것에 한정되는 것은 아니다.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. Preferably, 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.
본 발명의 광견병 바이러스를 중화시킬 수 있는 결합 분자는, 다양한 광견병 바이러스를 대상으로 중화 능력을 보유하고 있음을 확인하였으므로, 광견병 바이러스에 감염된 환자 또는 동물을 대상으로 광견병 치료 및 예방에 유용하다.Since 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.
도 1은 본 발명의 중쇄 및 경쇄 유전자를 포함하는 키메릭 항체 발현 벡터를 도시한 것이다. 1 shows chimeric antibody expression vectors comprising the heavy and light chain genes of the present invention.
도 2는 중국 개 광견병 바이러스(Rv342)를 이용한 in vivo 동물실험 결과를 도시한 것이다.Figure 2 shows the results of in vivo animal experiments using the Chinese dog rabies virus (Rv342).
이하 본 발명을 실시예에 따라 상세히 설명한다. 하기의 실시예들은 본 발명을 구체적으로 설명하기 위한 것으로 본 발명이 이들에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are intended to illustrate the present invention in detail, but the present invention is not limited thereto.
실시예 1: 하이브리도마(hybridoma) 세포 선별Example 1 Hybridoma Cell Selection
미국질병관리본부(Center for Disease Control: 이하 "CDC"라 칭함)에서 보관중인 하이브리도마 세포에 대한 이전 실험 및 기록에 의거하여 특정 하이브리도마 세포를 선별한 후, 클론의 배양배지에서의 Fab 타이터(titer)를 RFFIT(Rapid Fluorescent Focus Inhibition Test)(Smith, J. S. et al., Geneva: World Health Oragnization, pp.181-191, 1996; 및 centers for Disease Control and Prevention, Morb. Mortal. Weekly Rep. 49(RR-1), 1-21, 1999) 방법으로 측정하였으며, 그 결과는 표 1에 기재하였다. 선별된 하이브리도마 세포의 클론 이름은 #62-71-3이다. 상기 선별된 하이브리도마 세포로부터 정제하여 단일클론 항체를 수득한 후 타이터 관련 실험으로 RFFIT를 수행하였다. Based on previous experiments and records of hybridoma cells stored in the Center for Disease Control (CDC), specific hybridoma cells were selected and then Fabs in the culture medium of the clones. Titers are described in Rapid Fluorescent Focus Inhibition Test (RFFIT) (Smith, JS et al ., Geneva: World Health Oragnization , pp. 181-191, 1996; and centers for Disease Control and Prevention, Morb. Mortal.Weekly Rep 49 (RR-1), 1-21, 1999), and the results are shown in Table 1. The clone name of the selected hybridoma cells is # 62-71-3. Purification from the selected hybridoma cells yielded monoclonal antibodies followed by RFFIT in titer-related experiments.
표 1 정제된 클론을 이용한 RFFIT 결과
항체명 타이터 (IU/mL) IU/mg
62-71-3 480 (5.7 IU/mL) 1.4 IU/mg
Table 1 RFFIT Results Using Purified Clones
Antibody Name Titer (IU / mL) IU / mg
62-71-3 480 (5.7 IU / mL) 1.4 IU / mg
표 1의 #62-71-3 클론을 상대로 미국 CDC에서 보관 중인 전 세계 광견병 바이러스에 대한 RFFIT를 수행하였다. 미국 CDC에서는 전 세계의 약 50 여종에 해당하는 광견병 바이러스를 보유하고 있으며, 바이러스의 리스트는 아래 표 2와 같다. RFFIT for rabies virus stored in the US CDC against the # 62-71-3 clone of Table 1 was performed. The US CDC has about 50 species of rabies viruses worldwide, and the list of viruses is shown in Table 2 below.
표 2 미국 CDC 보유 광견병 바이러스 리스트
No. 광견병 바이러스 종류 SRIG 62-71-3 IU/mg
1 CVS-11 125 170 3.3
2 Mongoose RSA 90 170 4.6
3 CASK 90 <5 0
4 Tunissia dog 125 700 13.7
5 Gabon dog 125 45 0.9
6 TXFX 125 440 8.6
7 THAI DOG 125 625 12.2
8 Sonora dog 125 900 17.6
9 002 Philippines 125 125 2.4
10 DR MX xxx xxx xxx
11 DR Brazil 62.5 <5 0
12 Dog Phi 125 85 1.7
13 WA Bat 125 45 0.9
14 3860 CA Bat 125 250 4.9
15 Dog Arg 125 700 13.7
16 TX SK 90 <5 0
17 RAC 90 <5 0
18 China 2005 62.5 145 5.7
19 Rv342 China 62.5 95 3.7
20 TX Coyote 90 1000 27.1
21 rv61 62.5 <5 0
22 AL Bat 125 440 8.6
23 LC NY xxx xxx xxx
24 Bat Ef 125 125 2.4
25 C1434 125 350 6.8
26 ABV (SM 4476) 125 <5 0
27 Wu ABLV 125 480 9.4
28 AZ Bat 125 540 10.5
29 VA 399 125 540 10.5
30 TN 410 125 125 2.4
31 TN 132 125 ≥1400 ≥27.3
32 SK 4384 90 <5 0
33 AK FX 125 xxx 0
34 857r 62.5 230 9.0
35 I-148 62.5 360 14.0
36 Mong PR 125 250 4.9
37 Gray FX-AZ 125 210 4.1
38 NC SK 90 800 21.7
39 323R 90 230 6.2
40 RVHN xxx xxx xxx
41 MI 1625 125 ≥1400 ≥27.3
42 I-151 62.5 <5 0
43 TN 269 125 625 12.2
44 Sri Lanka 62.5 60 2.3
45 Myotis 125 1100 21.5
TABLE 2 U.S. CDC-owned rabies virus list
No. Rabies virus types SRIG 62-71-3 IU / mg
One CVS-11 125 170 3.3
2 Mongoose rsa 90 170 4.6
3 CASK 90 <5 0
4 Tunissia dog 125 700 13.7
5 Gabon dog 125 45 0.9
6 TXFX 125 440 8.6
7 THAI DOG 125 625 12.2
8 Sonora dog 125 900 17.6
9 002 Philippines 125 125 2.4
10 DR MX xxx xxx xxx
11 DR Brazil 62.5 <5 0
12 Dog phi 125 85 1.7
13 WA Bat 125 45 0.9
14 3860 CA Bat 125 250 4.9
15 Dog arg 125 700 13.7
16 TX SK 90 <5 0
17 RAC 90 <5 0
18 China 2005 62.5 145 5.7
19 Rv342 China 62.5 95 3.7
20 TX Coyote 90 1000 27.1
21 rv61 62.5 <5 0
22 AL Bat 125 440 8.6
23 LC NY xxx xxx xxx
24 Bat ef 125 125 2.4
25 C1434 125 350 6.8
26 ABV (SM 4476) 125 <5 0
27 Wu ABLV 125 480 9.4
28 AZ Bat 125 540 10.5
29 VA 399 125 540 10.5
30 TN 410 125 125 2.4
31 TN 132 125 ≥1400 ≥27.3
32 SK 4384 90 <5 0
33 AK FX 125 xxx 0
34 857r 62.5 230 9.0
35 I-148 62.5 360 14.0
36 Mong PR 125 250 4.9
37 Gray FX-AZ 125 210 4.1
38 NC SK 90 800 21.7
39 323R 90 230 6.2
40 RVHN xxx xxx xxx
41 MI 1625 125 ≥1400 ≥27.3
42 I-151 62.5 <5 0
43 TN 269 125 625 12.2
44 Sri lanka 62.5 60 2.3
45 Myotis 125 1100 21.5
그 결과, 표 2에 기재된 광견병 바이러스에 대하여 #62-71-3 클론의 RFFIT 결과 값을 아래 표 3에 기재하였다. 수치가 높을수록 높은 중화효능을 나타낸다.As a result, RFFIT result values of # 62-71-3 clone for rabies virus described in Table 2 are listed in Table 3 below. The higher the value, the higher the neutralizing effect.
표 3 미국 CDC에서 수행된 RFFIT 결과
No. 광견병 바이러스 종류 SRIG 62-71-3 IU/mg
1 CVS-11 125 170 3.3
2 Mongoose RSA 90 170 4.6
3 CASK 90 <5 0
4 Tunissia dog 125 700 13.7
5 Gabon dog 125 45 0.9
6 TXFX 125 440 8.6
7 THAI DOG 125 625 12.2
8 Sonora dog 125 900 17.6
9 002 Philippines 125 125 2.4
10 DR MX xxx xxx xxx
11 DR Brazil 62.5 <5 0
12 Dog Phi 125 85 1.7
13 WA Bat 125 45 0.9
14 3860 CA Bat 125 250 4.9
15 Dog Arg 125 700 13.7
16 TX SK 90 <5 0
17 RAC 90 <5 0
18 China 2005 62.5 145 5.7
19 Rv342 China 62.5 95 3.7
20 TX Coyote 90 1000 27.1
21 rv61 62.5 <5 0
22 AL Bat 125 440 8.6
23 LC NY xxx xxx xxx
24 Bat Ef 125 125 2.4
25 C1434 125 350 6.8
26 ABV (SM 4476) 125 <5 0
27 Wu ABLV 125 480 9.4
28 AZ Bat 125 540 10.5
29 VA 399 125 540 10.5
30 TN 410 125 125 2.4
31 TN 132 125 ≥1400 ≥27.3
32 SK 4384 90 <5 0
33 AK FX 125 xxx 0
34 857r 62.5 230 9.0
35 I-148 62.5 360 14.0
36 Mong PR 125 250 4.9
37 Gray FX-AZ 125 210 4.1
38 NC SK 90 800 21.7
39 323R 90 230 6.2
40 RVHN xxx xxx xxx
41 MI 1625 125 ≥1400 ≥27.3
42 I-151 62.5 <5 0
43 TN 269 125 625 12.2
44 Sri Lanka 62.5 60 2.3
45 Myotis 125 1100 21.5
TABLE 3 RFFIT Results Performed on US CDC
No. Rabies virus types SRIG 62-71-3 IU / mg
One CVS-11 125 170 3.3
2 Mongoose rsa 90 170 4.6
3 CASK 90 <5 0
4 Tunissia dog 125 700 13.7
5 Gabon dog 125 45 0.9
6 TXFX 125 440 8.6
7 THAI DOG 125 625 12.2
8 Sonora dog 125 900 17.6
9 002 Philippines 125 125 2.4
10 DR MX xxx xxx xxx
11 DR Brazil 62.5 <5 0
12 Dog phi 125 85 1.7
13 WA Bat 125 45 0.9
14 3860 CA Bat 125 250 4.9
15 Dog arg 125 700 13.7
16 TX SK 90 <5 0
17 RAC 90 <5 0
18 China 2005 62.5 145 5.7
19 Rv342 China 62.5 95 3.7
20 TX Coyote 90 1000 27.1
21 rv61 62.5 <5 0
22 AL Bat 125 440 8.6
23 LC NY xxx xxx xxx
24 Bat ef 125 125 2.4
25 C1434 125 350 6.8
26 ABV (SM 4476) 125 <5 0
27 Wu ABLV 125 480 9.4
28 AZ Bat 125 540 10.5
29 VA 399 125 540 10.5
30 TN 410 125 125 2.4
31 TN 132 125 ≥1400 ≥27.3
32 SK 4384 90 <5 0
33 AK FX 125 xxx 0
34 857r 62.5 230 9.0
35 I-148 62.5 360 14.0
36 Mong PR 125 250 4.9
37 Gray FX-AZ 125 210 4.1
38 NC SK 90 800 21.7
39 323R 90 230 6.2
40 RVHN xxx xxx xxx
41 MI 1625 125 ≥1400 ≥27.3
42 I-151 62.5 <5 0
43 TN 269 125 625 12.2
44 Sri lanka 62.5 60 2.3
45 Myotis 125 1100 21.5
(SRIG: Standard Rabies Immuno Globulin)(SRIG: Standard Rabies Immuno Globulin)
(xxx: 수행하지 않음)(xxx: do not perform)
#62-71-3 클론은 특허출원 10-2011-0024332에 개시된 #2-21-23에서 중화능력이 작았던 바이러스에 대해서 특이적으로 효능을 보여, 미국 CDC로부터 셀트리온에 전달되었으며, #62-71-3 클론의 가변영역과 인간타입 항체의 불변영역을 이용하여 키메릭 단일클론 항체를 제조하였다. # 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.
실시예 2: 하이브리도마 세포(hybridoma cell)가 분비하는 키메릭 항체(Example 2 Chimeric Antibodies Secreted by Hybridoma Cells chimericchimeric 항체)에 대한  Antibody) cDNAcDNA 확보 secure
2-1. 세포 배양2-1. Cell culture
미국 CDC로부터 하이브리도마 세포 #62-71-3이 입고되었으며, 위의 세포들을 5% 소태아 혈청(FBS; Sigma, 12003C)이 첨가된 IMDM 배지(Invitrogen 12440-053)에서 배양하였다. 배양기간 동안, Mycoplasma PCR ELISA kit(Roche, 11663925910)를 이용하여, mycoplasma 오염 여부를 조사하였으며, 배양액에 mycoplasma가 없음을 확인하였다. 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.
2-2. 하이브리도마 세포가 분비하는 마우스 타입 항체의 중쇄와 경쇄 가변영역 2-2. Heavy and light chain variable regions of mouse-type antibodies secreted by hybridoma cells DNADNA 확보 secure
배양 중인 하이브리도마 #62-71-3에서 RNeasy plus mini kit(Qiagen, 74134)를 사용하여 전체 RNA를 분리하였다. 분리한 1 ㎍의 전체 RNA를 주형 (template)으로 SMARTer RACE cDNA Amplification kit(Clontech, 634923)을 사용하여, cDNA의 5' 말단 고속 증폭 중합효소 연쇄반응(5' end RACE PCR; Rapid Amplification of cDNA Ends Polymerase Chain Reaction)를 진행하여, 5' 말단에 특정 서열을 가지는 cDNA 를 합성하였다. cDNA 합성 반응 조건은 아래와 같다. 먼저, 1 ㎍의 전체 RNA를 5' RACE CDS primer A (5'-(T)25GC-3' : 서열번호 1)와 혼합한 후, 72℃에서 3분 및 42℃에서 2분 반응 후 SMARTer II A Oligonucleotide (5'- AAG CAG TGG TAT CAA CGC AGA GTA CGT GGG -3' : 서열번호 2)와 역전사 효소를 첨가하여 혼합하고, 42℃에서 90 분, 70℃에서 10분 동안 역전사 반응을 수행함으로써, 5' 말단에 특정 서열을 가지는 하이브리도마 세포 유래 cDNA를 합성하였다. 상기 하이브리도마 세포 유래 cDNA를 주형으로, Universal Primer A mix (Long 5'-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3 : 서열번호 3, Short 5'-CTA ATA CGA CTC ACT ATA GGG C-3' : 서열번호 4)와 중쇄 IgG2b의 불변영역에 특이적인 서열을 대상으로 하는 안티센스 프라이머(5'-GCTGGACAGGGATCCAGAGTTCCAAGTCACAGTC-3' : 서열번호 5)와 경쇄의 k 사슬의 불변영역의 특이적인 서열을 대상으로 하는 안티센스 프라이머(5'-cgt ct tgg tca acg tga ggg tgc tgc t-3' : 서열번호 6)를 각각 이용하여 94℃에서 30초 열 변성 후, 72℃ 3분으로 5 회 반복, 94℃에서 30초 열 변성, 70℃ 30초, 72℃ 3분 5 회 반복, 94℃에서 30초 열 변성, 68℃ 30초, 72℃ 3분, 27 회 반복하는 조건으로 중합효소 연쇄반응을 통해, 하이브리도마 세포 #62-71-3 세포에서 발현하는 항체의 가변영역 전체를 포함하는 cDNA를 확보하였다.Total RNA was isolated from hybridoma # 62-71-3 in culture using RNeasy plus mini kit (Qiagen, 74134). 5 'end RACE PCR (Rapid Amplification of cDNA Ends) of cDNA using a SMARTer RACE cDNA Amplification kit (Clontech, 634923) as a template Polymerase Chain Reaction) was performed to synthesize cDNA having a specific sequence at the 5 'end. cDNA synthesis reaction conditions are as follows. First, 1 μg of total RNA was mixed with 5 'RACE CDS primer A (5'-(T) 25 GC-3 ': SEQ ID NO: 1), followed by 3 minutes at 72 ° C and 2 minutes at 42 ° C. II 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. Thus, hybridoma cell-derived cDNA having a specific sequence at the 5 'end was synthesized. As a template of the hybridoma cell-derived cDNA, Universal Primer A mix (Long 5'-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3: SEQ ID NO: 3, Short 5'-CTA ATA CGA CTC) ACT ATA GGG C-3 ': SEQ ID NO: 4) and the antisense primer (5'-GCTGGACAGGGATCCAGAGTTCCAAGTCACAGTC-3': SEQ ID NO: 5) targeting the constant region specific region of the heavy chain IgG2b and the constant region of the k chain of the light chain Heat denatured at 94 ° C for 30 seconds using antisense primers (5'-cgt ct tgg tca acg tga ggg tgc tgc t-3 ': SEQ ID NO: 6) targeting specific sequences, respectively, followed by 5 minutes at 72 ° C for 3 minutes. Repeated times, 30 seconds thermal denaturation at 94 ℃, 70 ℃ 30 seconds, 72 ℃ 3 minutes 5 repetition, 30 seconds thermal denaturation at 94 ℃, 68 ℃ 30 seconds, 72 ℃ 3 minutes, 27 times repeating conditions Through the chain reaction, cDNA including the entire variable region of the antibody expressed in hybridoma cell # 62-71-3 cells was obtained.
확보한 중쇄와 경쇄 각각의 가변영역 전체를 포함하는 cDNA 단편을 TOPO TA cloning kit(Invitrogen, K4500)에 있는 TA 벡터에 클로닝한 후, 염기서열을 분석하였다. 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.
2-3. 키메릭 항체(chimeric antibody)를 암호화 하는 중쇄와 경쇄 cDNA 제작2-3. Construction of heavy and light chain cDNAs encoding chimeric antibodies
중첩 중합효소 연쇄반응(overlapping PCR)을 이용하여, 마우스 타입 항체 사슬의 가변영역 DNA 서열을 인간 타입(human type) 항체의 불변영역에 연결하는 키메릭 항체를 제작하였다. 먼저 중첩 중합효소 연쇄반응을 진행하기 위하여, 표 4에 기재된 프라이머를 이용하여, 95℃에서 5분 열 변성 후, 95℃에서 1분, 57℃에서 1분, 72℃에서 1분의 조건으로 35회 반복하여, 마우스 타입의 경쇄(카파 사슬)와 중쇄(감마 사슬) 가변영역에 대한 중합효소 연쇄반응 산물과 인간 타입의 경쇄(카파 사슬)와 중쇄(감마 사슬) 불변영역에 대한 중합효소 연쇄반응 산물을 확보하였다. 이후 가변영역과 불변영역의 중합효소 연쇄반응 산물을 주형으로 하고, HC F1과 HC R2 프라이머를 이용하여, 앞서 언급한 것과 같은 조건으로 PCR을 진행하여 가변영역과 불변영역이 연결된 중쇄를 확보하였다. 경쇄의 경우도 LC F1과 LC R2 프라이머를 이용하여 같은 방식으로 중합효소 연쇄반응을 진행하여 가변영역과 불변영역이 연결된 경쇄를 확보하였다.Using overlapping PCR, chimeric antibodies were constructed that link the variable region DNA sequence of a mouse type antibody chain to the constant region of a human type antibody. First, in order to proceed with the overlapping polymerase chain reaction, using the primers described in Table 4, after 5 minutes of thermal denaturation at 95 ° C, 1 minute at 95 ° C, 1 minute at 57 ° C, and 1 minute at 72 ° C. Repeated times, polymerase chain reaction products for the mouse type light (kappa chain) and heavy (gamma chain) variable regions and polymerase chain reaction for the human type light (kappa chain) and heavy (gamma chain) constant regions. The product was obtained. Thereafter, the 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. In the case of the light chain, 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.
표 4 프라이머 정보
Primer명칭 설명 염기 서열 서열번호
HC F1 마우스 타입 중쇄의 가변영역에 대한 센스 프라이머 5'-CTG GGC TAG CGC CAC CAT GGC TGT CCT GGC ATT ACT CTT CTG-3' 7
HC R1 마우스 타입 중쇄의 가변영역에 대한 안티센스 프라이머 5'-GGG CCC TTG GTG GAG GCT GAG GAG ACT GTG AGA GTG GTG CC-3' 8
HC F2 인간 타입 중쇄의 불변영역에 대한 센스 프라이머 5'-CTC ACA GTC TCC TCA GCC TCC ACC AAG GGC CC-3' 9
HC R2 인간 타입 중쇄의 불변영역에 대한 안티센스 프라이머 5'-AGCTTTGTTTAAACCACTATCATTTACCCGGAGACAGGGAGA-3' 10
LC F1 마우스 타입 경쇄의 가변영역에 대한 센스 프라이머 5'-GCTGGCTAGCGCCACCATGATGTCCTCTGCTCAGTTCCTTG-3' 11
LC R1 마우스 타입 경쇄의 가변영역에 대한 안티센스 프라이머 5'-GGTGCAGCCACAGTTCGTTTTATTTCCAGCTTGGTCCCCC-3' 12
LC F2 인간 타입 경쇄의 불변영역에 대한 센스 프라이머 5'-GGAAATAAAACGAACTGTGGCTGCACCATC-3' 13
LC R2 인간 타입 경쇄의 불변영역에 대한 안티센스 프라이머 5'-AGCTTTGTTTAAACAGTCTACTAACACTCTCCCCTGTTGAAGCTCT-3' 14
Table 4 Primer Information
Primer Name Explanation Base sequence SEQ ID NO:
HC F1 Sense primers for variable regions of mouse type heavy chains 5'-CTG GGC TAG CGC CAC CAT GGC TGT CCT GGC ATT ACT CTT CTG-3 ' 7
HC R1 Antisense Primer for Variable Regions of Mouse Type Heavy Chains 5'-GGG CCC TTG GTG GAG GCT GAG GAG ACT GTG AGA GTG GTG CC-3 ' 8
HC F2 Sense Primer for the Constant Region of Human Type Heavy Chains 5'-CTC ACA GTC TCC TCA GCC TCC ACC AAG GGC CC-3 ' 9
HC R2 Antisense Primer for the Constant Region of Human Type Heavy Chains 5'-AGCTTTGTTTAAACCACTATCATTTACCCGGAGACAGGGAGA-3 ' 10
LC F1 Sense primers for variable regions of mouse type light chains 5'-GCTGGCTAGCGCCACCATGATGTCCTCTGCTCAGTTCCTTG-3 ' 11
LC R1 Antisense Primers for Variable Regions of Mouse Type Light Chains 5'-GGTGCAGCCACAGTTCGTTTTATTTCCAGCTTGGTCCCCC-3 ' 12
LC F2 Sense Primer for the Constant Region of Human Type Light Chains 5'-GGAAATAAAACGAACTGTGGCTGCACCATC-3 ' 13
LC R2 Antisense Primer for Constant Regions of Human Type Light Chains 5'-AGCTTTGTTTAAACAGTCTACTAACACTCTCCCCTGTTGAAGCTCT-3 ' 14
2-4. 2-4. 키메릭Chimeric 항체 발현 벡터 제작 Antibody Expression Vector Construction
확보된 중쇄와 경쇄 유전자에 제한효소 Nhe I과 Pme I을 처리하여 확보한 후, 확보된 중쇄 유전자와 경쇄 유전자를 각각 동일한 제한효소로 처리된 pCT184 벡터와 pCT146 벡터에 삽입하였다. pCT184 및 pCT146 벡터는 각각 항체의 중쇄와 경쇄를 클로닝하기 위해 제작된 셀트리온 고유의 벡터이다. 이후, 중쇄 전사단위(프로모터-중쇄 유전자-폴리A)와 경쇄 전사단위(프로모터-경쇄 유전자-폴리A)를 같이 포함하는 발현 벡터를 제작하기 위하여, 중쇄 유전자를 포함하는 pCT184 벡터에 제한효소 Pac I과 Asc I을 처리하여 중쇄 전사단위를 확보한 다음, 경쇄 유전자를 포함하는 pCT146 벡터에 동일한 제한효소를 처리하여 중쇄 전사단위를 삽입하였다. 이후 제한효소를 이용하여, 중쇄 전사단위와 경쇄 전사단위를 동시에 포함하는 벡터를 선별하여 pCT234로 명명 하였다(도 1 참조). 선별된 벡터는 Endofree plasmid maxi kit(QIAGEN, Germany, 12362)를 이용하여 추출되었으며, 추출된 DNA 중 일부를 이용하여 염기서열 분석을 통해 최종적으로 항체의 염기서열을 확인하였다. 확인된 서열은 아래 표 5와 같다. 가변영역의 CDR은 Kabat 등에 의해 고안된 시스템에 따라 통상적인 방법으로 결정되었다(문헌[Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD. (1991)] 참조).The obtained heavy and light chain genes were obtained by treating the restriction enzymes Nhe I and Pme I, and then the obtained heavy and light chain genes were inserted into the pCT184 vector and pCT146 vector treated with the same restriction enzyme, respectively. The pCT184 and pCT146 vectors are Celltrion-specific vectors designed to clone the heavy and light chains of antibodies, respectively. Subsequently, in order to construct an expression vector including both a heavy chain transcription unit (promoter-heavy chain gene-polyA) and a light chain transcription unit (promoter-light chain gene-polyA), restriction enzyme Pac I was added to the pCT184 vector containing the heavy chain gene. After treating Asc I and heavy chain transcription units, the same restriction enzyme was inserted into the pCT146 vector including the light chain gene to insert the heavy chain transcription unit. Then, using a restriction enzyme, a vector containing both a heavy chain and a light chain transcription unit was selected and named pCT234 (see FIG. 1). 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. The confirmed sequences are shown in Table 5 below. CDRs of 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)). Reference).
표 5 마우스 타입 단일클론 항체 서열 정보
서열 종류 서열 서열번호
중쇄 가변영역 CDR1폴리뉴클레오티드 GGCCATGGTGTAAAC 15
중쇄 가변영역 CDR2폴리뉴클레오티드 ATAATATGGGCTGATGGAACCACAAACTATAATTCAGCTCTCAAATCC 16
중쇄 가변영역 CDR3폴리뉴클레오티드 GAGGGGGACATCTCGGGCTACTACTTTGACTAC 17
경쇄 가변영역 CDR1 폴리뉴클레오티드 AGGCCAAGTCAAGACATTAACAATTATTTAAGT 18
경쇄 가변영역 CDR2 폴리뉴클레오티드 TACTACACATCAAGATTACACTCA 19
경쇄 가변영역 CDR3 폴리뉴클레오티드 CAGCAGGGTAATACGCTTCCTCCCACG 20
중쇄 가변영역 폴리뉴클레오티드 ATGGCTGTCCTGGCATTACTCTTCTGCCTGGTAACATTCCCAAGCTGTATCCTTTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGTTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGTTTCTCATTAACCGGCCATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATAATATGGGCTGATGGAACCACAAACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGTTACTACTGTGCCAGAGAGGGGGACATCTCGGGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 21
경쇄 가변영역 폴리뉴클레오티드 ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATGTCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCACTTGCAGGCCAAGTCAAGACATTAACAATTATTTAAGTTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAGGAAGATTTTGCCACTTACTTTTGTCAGCAGGGTAATACGCTTCCTCCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 22
중쇄 가변영역 CDR1폴리펩티드 GHGVN 23
중쇄 가변영역 CDR2폴리펩티드 IIWADGTTNYNSALKS 24
중쇄 가변영역 CDR3폴리펩티드 EGDISGYYFDY 25
경쇄 가변영역 CDR1 폴리펩티드 RPSQDINNYLS 26
경쇄 가변영역 CDR2 폴리펩티드 YTSRLHS 27
경쇄 가변영역 CDR3 폴리펩티드 QQGNTLPPT 28
중쇄 가변영역 폴리펩티드 MAVLALLFCLVTFPSCILSQVQLKESGPGLLAPSQSLSITCTVSGFSLTGHGVNWVRQPPGKGLEWLGIIWADGTTNYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTASYYCAREGDISGYYFDYWGQGTTLTVSS 29
경쇄 가변영역 폴리펩티드 MMSSAQFLGLLLLCFQGTRCDVQMTQTTSSLSASLGDRVTITCRPSQDINNYLSWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPPTFGGGTKLEIK 30
중쇄 불변영역폴리펩티드 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31
경쇄 불변영역폴리펩티드 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 32
Table 5 Mouse Type Monoclonal Antibody Sequence Information
Sequence type order SEQ ID NO:
Heavy chain variable region CDR1 polynucleotide GGCCATGGTGTAAAC 15
Heavy chain variable region CDR2 polynucleotide ATAATATGGGCTGATGGAACCACAAACTATAATTCAGCTCTCAAATCC 16
Heavy chain variable region CDR3 polynucleotide GAGGGGGACATCTCGGGCTACTACTTTGACTAC 17
Light Chain Variable Region CDR1 Polynucleotides AGGCCAAGTCAAGACATTAACAATTATTTAAGT 18
Light Chain Variable Region CDR2 Polynucleotides TACTACACATCAAGATTACACTCA 19
Light Chain Variable Region CDR3 Polynucleotides CAGCAGGGTAATACGCTTCCTCCCACG 20
Heavy chain variable region polynucleotide ATGGCTGTCCTGGCATTACTCTTCTGCCTGGTAACATTCCCAAGCTGTATCCTTTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGTTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGTTTCTCATTAACCGGCCATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATAATATGGGCTGATGGAACCACAAACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGTTACTACTGTGCCAGAGAGGGGGACATCTCGGGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 21
Light chain variable region polynucleotides ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATGTCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCACTTGCAGGCCAAGTCAAGACATTAACAATTATTTAAGTTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAGGAAGATTTTGCCACTTACTTTTGTCAGCAGGGTAATACGCTTCCTCCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 22
Heavy chain variable region CDR1 polypeptide GHGVN 23
Heavy chain variable region CDR2 polypeptide IIWADGTTNYNSALKS 24
Heavy chain variable region CDR3 polypeptide EGDISGYYFDY 25
Light chain variable region CDR1 polypeptide RPSQDINNYLS 26
Light chain variable region CDR2 polypeptide YTSRLHS 27
Light chain variable region CDR3 polypeptides QQGNTLPPT 28
Heavy chain variable region polypeptide MAVLALLFCLVTFPSCILSQVQLKESGPGLLAPSQSLSITCTVSGFSLTGHGVNWVRQPPGKGLEWLGIIWADGTTNYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTASYYCAREGDISGYYFDYWGQGTTLTVSS 29
Light chain variable region polypeptide MMSSAQFLGLLLLCFQGTRCDVQMTQTTSSLSASLGDRVTITCRPSQDINNYLSWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPPTFGGGTKLEIK 30
Heavy chain constant region polypeptide ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31
Light Chain Constant Region Polypeptides RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 32
실시예EXAMPLE 3: 일시적 형질도입 방법에 의한  3: by transient transduction method 키메릭Chimeric 항체의 생산 Production of antibodies
세포 내 일시적 형질도입(transient trasfection)을 위하여 양이온성 폴리머(cationic polymer)인 FreeStyleTM Max(Invitrogen, 16447-100)를 사용하였으며, 제조사의 사용설명서에 따라 형질도입을 수행하였다. 형질도입 전날, EX-CELL 293 Serum free media(Sigma, 14571C: 이하 "EX-CELL 293 배지"라 기재함)에서 자라는 F2N 세포(KCTC 11309BP)를 원심 분리하여, FreeStyle293 serum free media(Gibco, 12338)로 배지를 교체하였으며, ㎖ 당 0.8×106 개의 세포 농도로 250 ㎖ shaker flask 두 개를 이용하여 50 ㎖씩(총 100 ㎖)접종하였다. 형질 도입 당일 날, 키메릭 항체 유전자를 포함하는 pCT234 DNA 125 ㎍과 FreeStyleTM Max 시약 125 ㎕를 각각 OptiPRO SFM II (Invitrogen, 12309) 배지를 이용하여 2 ㎖ volume으로 희석한 후, 가볍게 섞어 주었다. 즉시 희석된 FreeStyleTM Max 시약 용액을 DNA가 희석되어 있는 용액에 섞은 다음, 상온에서 17분 반응하였다. 상온에서 17분 반응하는 동안, 형질 도입에 사용할 접종된 F2N 세포의 수를 측정하고, 그 FreeStyle293 배지를 이용하여, 세포 농도를 1.0×106 개가 되도록 희석하였다. 17분 후, DNA와 FreeStyleTM Max 시약 혼합 용액을 F2N 세포에 처리함으로써 형질도입을 진행하였다. 형질도입 다음 날, 동량의 EX-CELL 293 배지를 형질도입된 세포에 첨가하여 7일 동안 배양함으로써 단일클론 항체를 생산하였다.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. On the day before transduction, F2N cells (KCTC 11309BP) grown in EX-CELL 293 Serum free media (Sigma, 14571C: hereinafter referred to as "EX-CELL 293 medium") were centrifuged and FreeStyle293 serum free media (Gibco, 12338). 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. On the day of transfection, 125 μg of pCT234 DNA containing the chimeric antibody gene and 125 μl of FreeStyle ™ Max reagent were diluted in 2 ml volume using OptiPRO SFM II (Invitrogen, 12309) medium, and then mixed lightly. Immediately diluted FreeStyle ™ 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. After 17 minutes, transduction was performed by treating the F2N cells with a mixture solution of DNA and FreeStyle ™ 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.
실시예 4: 키메릭 단일클론 항체 효능 확인Example 4: Confirmation of Chimeric Monoclonal Antibody Efficacy
실시예 4-1: in vitro 실험Example 4-1: in vitro experiment
실시예 3에서 생산된 단일클론 항체에서 6개의 키메라 형태의 후보를 수득하였으며, 그 중 타이터가 가장 높은 키메릭 항체 #13-6이 선별되었다(표 6 참조). Six chimeric forms of candidates were obtained from the monoclonal antibodies produced in Example 3, of which chimeric antibody # 13-6 with the highest titer was selected (see Table 6).
표 6 키메릭 형태 단일클론 항체의 농도
단일클론 항체 타이터 (IU/mL) IU/mg
키메릭 항체 #1-262-71-3 5 (0.05) 0.05
키메릭 항체 #1-662-71-3 5 (0.05) 0.05
키메릭 항체 #3-262-71-3 5 (0.05) 0.05
키메릭 항체 #3-662-71-3 5 (0.05) 0.05
키메릭 항체 #8-662-71-3 50 (0.48) 4.8
키메릭 항체 #13-662-71-3 ≥1400 (13.3) ≥133
Table 6 Concentration of Chimeric Monoclonal Antibodies
Monoclonal antibody Titer (IU / mL) IU / mg
Chimeric Antibody # 1-262-71-3 5 (0.05) 0.05
Chimeric Antibody # 1-662-71-3 5 (0.05) 0.05
Chimeric Antibody # 3-262-71-3 5 (0.05) 0.05
Chimeric Antibody # 3-662-71-3 5 (0.05) 0.05
Chimeric Antibody # 8-662-71-3 50 (0.48) 4.8
Chimeric Antibody # 13-662-71-3 ≥1400 (13.3) ≥133
선별된 키메릭 항체 #13-6를 적절한 농도로 희석하여(최대 원액의 10배 이하로 희석) 대표적인 광견병 바이러스를 대상으로 중화능력 실험을 수행하였으며, 이는 RFFIT를 통해 수행되었다. 그 결과는 표 7에 기재하였다. 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.
표 7
광견병 바이러스 종류 SRIG 키메릭 항체 #13-662-71-3 IU/mg
AZ Bat 250 ≥1400 ≥11.2
TN 269 250 ≥1400 ≥11.2
CA 3860 250 1100 8.8
Thai Dog 250 1200 9.6
002 Phil 250 540 4.3
Dog Phil 250 1300 10.4
China Dog2005 250 1300 10.4
Rv 342 250 ≥1400 ≥11.2
TABLE 7
Rabies virus types SRIG Chimeric Antibody # 13-662-71-3 IU / mg
AZ Bat 250 ≥1400 ≥11.2
TN 269 250 ≥1400 ≥11.2
CA 3860 250 1100 8.8
Thai dog 250 1200 9.6
002 Phil 250 540 4.3
Dog phil 250 1300 10.4
China Dog2005 250 1300 10.4
Rv 342 250 ≥1400 ≥11.2
그 결과, 본 발명의 키메릭 항체는 박쥐(AZ Bat, TN 269, CA 3860), 개(Thai Dog, 002 Phil, Dog Phil, China Dog2005, Rv 342) 유래 광견병 바이러스에 중화능력이 있음을 확인하였다.As a result, 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). .
실시예 4-2: in vivo 동물실험Example 4-2: In vivo animal experiment
상기 실시예들에서 선별된 62-71-3 키메릭 항체 #17이 in vivo에서 광견병 바이러스에 대한 치료 효과를 가지는지 조사하기 위하여 다음과 같이 시리아 햄스터(Syrian hamster)를 이용하여 동물실험을 진행하였다. 본 동물실험에서는 중국 개 광견병 바이러스 Rv342를 사용하였다. In order to investigate whether the 62-71-3 chimeric antibody # 17 selected in the above examples has a therapeutic effect against rabies virus in vivo, animal experiments were performed using a Syrian hamster as follows. . Chinese animal rabies virus Rv342 was used in this animal experiment.
동물 실험 그룹은 총 5개로 나누어 졌으며, 이는 1. Rv342 바이러스만 주입한 그룹, 2. Rv342 바이러스와 백신(Human diploid Imovax® Sanofi Pasteur)을 주입한 그룹, 3. Rv342 바이러스와 62-71-3 키메릭 항체 #17을 주입한 그룹, 4. Rv342 바이러스와 62-71-3 키메릭 항체 #17 그리고 백신(Human diploid Imovax® Sanofi Pasteur)을 주입한 그룹, 5. Rv342 바이러스와 Human Rabies Immune Globulin (HRIG, Imogam® Rabies-HT, Sanofi Pasteur)을 주입한 그룹으로 이루어 졌다. Rv342 바이러스는 MICLD50/ml에 근거하여 1/100 희석하여 50 ul를 근육주사를 하였고, 백신은 1ml 당 약 2.5 IU 이상의 백신 바이러스 주를 50 ul를 주사하였고 (0, 3, 7, 14 day), 키메릭 항체 #17은 0.614 mg/mL 중 50 ul을 주사하였고 이는 약 20 IU/kg에 해당되며, HRIG 주입한 양과 동일하다. 백신과 키멕릭 항체 #17 그리고 HRIG은 Rv342 바이러스 주사 24시간 후에 각각 주사하였다. 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). 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.
실험 결과는 표 8 및 도 2와 같다. 바이러스와 62-71-3 키메릭 항체 #17를 주입한 경우(그룹 3) 관찰기간인 45일까지 대부분 생존하였으나 (91.7%), 바이러스만 주입하거나(그룹 1) 바이러스와 백신을 주입한 경우(그룹 2)에는 모두 사망하였다. 바이러스와 62-71-3 키메릭 항체 #17 그리고 백신을 주입한 경우(그룹 4)에도 100%의 생존율을 나타냈다. 또한 현재 치료제로 사용되는 HRIG을 62-71-3 키메릭 항체 #17와 동일한 양으로 주입했음에도 33.3%의 생존율을 보였다.The experimental results are shown in Table 8 and FIG. Virus and 62-71-3 chimeric antibody # 17 (group 3) 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. In addition, 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%.
표 8 in vivo 동물실험 결과
동물실험 그룹 처리 총 햄스터 수 생존 햄스터 수 생존율*
1 Rv342 바이러스 6 0 0%
2 Rv342 바이러스 + 백신 6 0 0%
3 Rv342 바이러스 + 62-71-3 키메릭 항체 #17 12 11 91.7%
4 Rv342 바이러스 + 62-71-3 키메릭 항체 #17 + 백신 12 12 100%
5 Rv342 바이러스 + HRIG 9 3 33.3%
Table 8 in vivo animal test results
Animal testing group process Total hamsters Survival Hamster Count Survival rate *
One Rv342 virus 6 0 0%
2 Rv342 Virus + Vaccine 6 0 0%
3 Rv342 Virus + 62-71-3 Chimeric Antibody # 17 12 11 91.7%
4 Rv342 Virus + 62-71-3 Chimeric Antibody # 17 + Vaccine 12 12 100%
5 Rv342 virus + HRIG 9 3 33.3%
* 관찰기간: 바이러스 감염 후 45일* Observation period: 45 days after virus infection
지금까지 예시적인 실시 태양을 참조하여 본 발명을 기술하였지만, 본 발명이 속하는 기술분야의 당업자는 본 발명의 범주를 벗어나지 않고서도 다양한 변화를 실시할 수 있으며, 그의 요소들을 등가물로 대체할 수 있음을 알 수 있을 것이다. 따라서, 본 발명이 본 발명을 실시하는데 계획된 최상의 양식으로서 개시된 특정 실시 태양으로 국한되는 것이 아니며, 본 발명이 첨부된 특허청구범위의 속하는 모든 실시 태양을 포함하는 것으로 해석되어야 한다. While the present invention has been described with reference to exemplary embodiments, it is understood that those skilled in the art can make various changes without departing from the scope of the present invention, and that elements can be replaced by equivalents. You will know. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out the invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims (25)

  1. 카바트(Kabat) 방법에 따라, 서열번호 23으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 24로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 25로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.According to the Kabat method, a variable having a CDR1 region comprising a polypeptide as set out in SEQ ID NO: 23, a CDR2 region comprising a polypeptide as set out in SEQ ID NO: 24 and a CDR3 region comprising a polypeptide as set out in SEQ ID NO: 25 A binding molecule having a neutralizing ability to bind to rabies virus, comprising a region.
  2. 카바트(Kabat) 방법에 따라, 서열번호 26으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 27로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 28로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.According to the Kabat method, a variable having 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 A binding molecule having a neutralizing ability to bind to rabies virus, comprising a region.
  3. 카바트(Kabat) 방법에 따라, 서열번호 23으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 24로 기재되는 폴리펜티드를 포함하는 CDR2 영역 및 서열번호 25로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 중쇄 가변 영역 및 서열번호 26으로 기재되는 폴리펩티드를 포함하는 CDR1 영역, 서열번호 27로 기재되는 폴리펩티드를 포함하는 CDR2 영역 및 서열번호 28로 기재되는 폴리펩티드를 포함하는 CDR3 영역을 가지는 경쇄 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.According to the Kabat method, 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 A light chain variable region having a heavy chain variable region and a CDR1 region comprising a polypeptide set forth in SEQ ID NO: 26, a CDR2 region comprising a polypeptide set forth in SEQ ID NO: 27 and a CDR3 region comprising a polypeptide set forth in SEQ ID NO: 28 A binding molecule that binds to rabies virus and has a neutralizing ability.
  4. 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A binding molecule having a neutralizing ability to bind to rabies virus, comprising a variable region comprising a polypeptide sequence set forth in SEQ ID NO: 29.
  5. 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A binding molecule having a neutralizing ability to bind to rabies virus, comprising a variable region comprising a polypeptide sequence set forth in SEQ ID NO: 30.
  6. 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 중쇄 가변 영역 및 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 경쇄 가변 영역을 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A binding molecule having a neutralizing ability to bind to rabies virus, comprising 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.
  7. 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 31로 기재되는 폴리펩티드 서열을 포함하는 불변 병역을 포함하는 중쇄를 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A binding molecule having a neutralizing ability to bind to rabies virus, comprising a heavy chain 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.
  8. 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 32로 기재되는 폴리펩티드 서열을 포함하는 불변 영역을 포함하는 경쇄를 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A binding molecule having a neutralizing ability to bind to rabies virus, comprising a light chain comprising a variable region comprising a polypeptide sequence as set forth in SEQ ID NO: 30 and a constant region comprising a polypeptide sequence as set out in SEQ ID NO: 32.
  9. 서열번호 29로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 31로 기재되는 폴리펩티드 서열을 포함하는 불변 병역을 포함하는 중쇄 및 서열번호 30으로 기재되는 폴리펩티드 서열을 포함하는 가변 영역 및 서열번호 32로 기재되는 폴리펩오티드 서열을 포함하는 불변 영역을 포함하는 경쇄를 포함하는, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자.A variable region comprising a polypeptide region as set out in SEQ ID NO: 29 and a heavy chain comprising a constant region comprising a polypeptide sequence as set out in SEQ ID NO. 31 and a variable region comprising a polypeptide sequence as set out in SEQ ID NO. A binding molecule having a neutralizing ability to bind to rabies virus, comprising a light chain comprising a constant region comprising a polypeptide sequence as described.
  10. 제 1항 내지 제 9항 중 어느 한 항에 있어서, 상기 결합 분자는 항체인 것을 특징으로 하는 결합 분자.10. The binding molecule of claim 1, wherein the binding molecule is an antibody.
  11. 제 3항 또는 제 6항에 있어서, 상기 결합 분자는 Fab 절편, Fv 절편, 디아바디(diabody), 트리아바디(triabody), 테트라바디(tetrabody), 키메라 항체, 인간화 항체 또는 인간 항체인 것을 특징으로 하는 결합 분자.The method of claim 3 or 6, wherein the binding molecule is a Fab fragment, Fv fragment, diabody (diabody), triabody (triabody), tetrabody (tetrabody), chimeric antibody, humanized antibody or human antibody, characterized in that Binding molecule.
  12. 제 1항 내지 제 9항 중 어느 한 항에 있어서, 상기 광견병 바이러스는 박쥐, 개, 소, 몽구스, 스컹크 및 늑대로 이루어진 군으로부터 선택된 어느 하나의 개체에서 유래된 것을 특징으로 하는 결합 분자. The binding molecule of claim 1, wherein the rabies virus is derived from any one individual selected from the group consisting of bats, dogs, cattle, mongoose, skunks, and wolves.
  13. 제 1항 내지 제 9항 중 어느 한 항의 결합 분자에 추가적으로 하나 이상의 태그가 결합된 이뮤노컨쥬게이트.An immunoconjugate in which at least one tag is additionally bound to the binding molecule of any one of claims 1 to 9.
  14. 제 1항 내지 제 9항 중 어느 한 항의 결합 분자를 암호화하는 핵산 분자.A nucleic acid molecule encoding the binding molecule of any one of claims 1 to 9.
  15. 제 14항의 핵산 분자가 삽입된 발현 벡터. An expression vector in which the nucleic acid molecule of claim 14 is inserted.
  16. 제 15항의 발현 벡터가 숙주 세포에 형질전환되어, 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 생산하는 세포주.A cell line wherein the expression vector of claim 15 is transformed into a host cell to produce a binding molecule that binds to rabies virus and has a neutralizing ability.
  17. 제 16항에 있어서, 상기 숙주 세포는 CHO 세포, F2N 세포, CSO 세포, BHK 세포, 바우스(Bowes) 흑색종 세포, HeLa 세포, 911 세포, AT1080 세포, A549 세포, HEK 293 세포 및 HEK293T 세포로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 세포주.The method of claim 16, wherein the host cell is composed 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 Cell line, characterized in that any one selected from the group.
  18. i) 제 16항의 세포주를 배양하는 단계; 및i) culturing the cell line of claim 16; And
    ii) 발현된 결합 분자를 회수하는 단계ii) recovering the expressed binding molecule
    를 포함하는 광견병 바이러스에 결합하여 중화 능력을 가지는 결합 분자를 생산하는 방법.A method of producing a binding molecule having a neutralizing ability by binding to a rabies virus comprising a.
  19. 제 1항 내지 제 9항 중 어느 한 항의 결합 분자 및 약제학적으로 허용가능한 부형제가 추가적으로 포함된 약학적 조성물.A pharmaceutical composition further comprising the binding molecule of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.
  20. 제 1항 내지 제 9항 중 어느 한 항의 결합 분자 및 약제학적으로 허용가능한 부형제가 추가적으로 포함된 광견병 치료 및 예방용 조성물.A composition for the treatment and prevention of rabies, further comprising the binding molecule of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.
  21. i) 제 1항 내지 제 9항 중 어느 한 항의 결합 분자; 및 i) the binding molecule of any one of claims 1-9; And
    ii) 용기ii) containers
    를 포함하는 광견병 진단용 키트.Rabies diagnostic kit comprising a.
  22. i) 제 1항 내지 제 9항 중 어느 한 항의 결합 분자; 및i) the binding molecule of any one of claims 1-9; And
    ii) 용기ii) containers
    를 포함하는 광견병 치료 및 예방용 키트.Rabies treatment and prevention kit comprising a.
  23. i) 대상의 시료와 제 1항 내지 제 9항 중 어느 한 항의 결합 분자를 접촉시키는 단계; 및i) contacting a sample of interest with a binding molecule of any one of claims 1 to 9; And
    ii) 상기 단계 i)의 결과를 분석하여 광견병 감염 여부를 판별하는 단계ii) determining the result of rabies by analyzing the result of step i)
    를 포함하는 광견병 진단 방법.Rabies diagnostic method comprising a.
  24. 광견병에 감염되었다고 확인된 대상에게 제 1항 내지 제 9항 중 어느 한 항의 결합 분자를 치료학적으로 유효한 양으로 투여하는 단계A therapeutically effective amount of a binding molecule of any one of claims 1 to 9 is administered to a subject identified as infected with rabies.
    를 포함하는 광견병 치료 및 예방 방법.Rabies treatment and prevention method comprising a.
  25. i) 대상의 시료와 제 1항 내지 제 9항 중 어느 한 항의 결합 분자를 접촉시키는 단계; 및i) contacting a sample of interest with a binding molecule of any one of claims 1 to 9; And
    ii) 상기 결합 분자가 대상 시료에 특이적으로 결합하는지 측정하는 단계ii) determining whether the binding molecule specifically binds to the target sample
    를 포함하는 광견병 바이러스를 검출하는 방법.Method for detecting a rabies virus comprising a.
PCT/KR2012/007795 2011-09-30 2012-09-27 Binding molecule for neutralizing rabies virus WO2013048130A2 (en)

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