WO2022186164A1 - Vecteur pour la thérapie génique de la polyarthrite rhumatoïde - Google Patents

Vecteur pour la thérapie génique de la polyarthrite rhumatoïde Download PDF

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WO2022186164A1
WO2022186164A1 PCT/JP2022/008422 JP2022008422W WO2022186164A1 WO 2022186164 A1 WO2022186164 A1 WO 2022186164A1 JP 2022008422 W JP2022008422 W JP 2022008422W WO 2022186164 A1 WO2022186164 A1 WO 2022186164A1
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vector
antibody
gene
virus
rheumatoid arthritis
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Japanese (ja)
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美幸 田仲
祐志 新井
真人 中西
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京都府公立大学法人
リジェネフォーティー株式会社
株式会社NEXT Stage
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to gene therapy virus vectors for diseases and pharmaceutical compositions for disease therapy containing the same.
  • the vector is a viral vector for gene therapy of disease comprising an anti-CD81 antibody gene. More particularly, the vector is for gene therapy of rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • Medication, surgery, and rehabilitation are mainly used for the treatment of RA.
  • Steroids, non-steroidal anti-inflammatory drugs, anti-rheumatic drugs, and biologics have been used in the pharmacotherapy of RA.
  • Many biological agents target inflammatory cytokines and inflammatory cells.
  • gene therapy for inhibiting the pathological process of RA involving inflammatory cytokines and inflammatory cells has been devised and research continues.
  • Synoviolin is one of the causative substances of RA.
  • CD81 one of the tetraspanins, is involved in the regulation of synoviolin expression. Therefore, RA therapy using CD81 siRNA or anti-CD81 monoclonal antibody targeting CD81 has been proposed (Non-Patent Documents 1 to 3). However, gene therapy targeting CD81 has not been performed.
  • gene therapy has problems such as safety issues including side effects, low persistence of expression, and low gene delivery efficiency.
  • the present inventors have conducted intensive studies to solve the above problems, and have found that a virus vector based on Sendai virus in which a gene encoding an anti-CD81 antibody is incorporated is used for gene therapy of RA in animals.
  • the present inventors have completed the present invention based on the fact that a therapeutic effect can be obtained and the safety to animals has also been confirmed.
  • the present invention provides: (1) A rheumatoid arthritis therapeutic vector based on a minus-strand RNA virus, containing a gene encoding an anti-CD81 antibody. (2) The vector according to (1), which is based on a virus belonging to the Paramyxoviridae family. (3) The vector according to (2), wherein the virus belonging to Paramyxoviridae is Sendai virus. (4) The vector according to any one of (1) to (3), which is a stealth type. (5) The vector according to any one of (1) to (4), wherein the anti-CD81 antibody binds to the LEL (large extracellular loop) of CD81.
  • LEL large extracellular loop
  • a pharmaceutical composition for treating RA comprising:
  • an effective and highly safe RA therapeutic vector is provided.
  • the RA therapeutic vector of the present invention remains in the cytoplasm and does not affect host chromosomes, and is highly safe.
  • the RA therapeutic vector of the present invention suppresses the host's immune reaction to the vector and stably maintains its therapeutic effect over a long period of time.
  • the RA therapeutic vector of the present invention also has a high expression efficiency of the anti-CD81 antibody, which is an active ingredient.
  • FIG. 1 is a scheme showing the structure of the viral vector used in Examples.
  • the upper part is a scheme showing the structure of a control vector that does not contain the anti-CD81 antibody gene.
  • the lower part is a scheme showing the structure of the vector containing the anti-CD81 antibody gene.
  • the anti-CD81 antibody H chain and L chain genes are arranged in tandem in the lower CD81 box.
  • FIG. 2 is a graph showing the results of ELISA examination of the expression of anti-CD81 antibody in articular synovial tissue 28 days after the vector of the present invention containing the anti-CD81 antibody gene was injected into the joint cavity of rheumatoid arthritis model rats. be.
  • the vertical axis is absorbance at 450 nm. Each bar represents the mean of absorbance ⁇ standard deviation.
  • Chimera anti-CD81 antibody indicates a system in which a purified chimeric anti-CD81 antibody was added to CD81 LEL-coated wells.
  • CIA(+)+SeVdp-CD81 PBS was prepared by coating wells of the CIA(+)+SeVdp-antiCD81 group (the group injected with the vector containing the anti-CD81 antibody gene) joint washing (washed with PBS) with CD81 LEL. shows the system added to CIA(+)+SeVdp-control PBS is a system in which the joint washing solution (washed with PBS) of the CIA(+)+SeVdp-control group (group injected with control vector) was added to wells coated with CD81 LEL. show.
  • CIA(+)+SeVdp-CD81 acid elute For CIA(+)+SeVdp-CD81 acid elute, CIA(+)+SeVdp-antiCD81 group (group injected with vector containing anti-CD81 antibody gene) joint lavage fluid (washed with PBS) was added to pH 3.0 gly-HCL. and neutralized to pH 7.0, added to CD81 LEL-coated wells.
  • CIA(+)+SeVdp-control acid elute was obtained by eluting the joint lavage fluid (washed with PBS) of the CIA(+)+SeVdp-control group (group injected with control vector) with pH 3.0 gly-HCL to pH 7.
  • FIG. 3 is a graph showing changes in body weight of rheumatoid arthritis model rats injected into the joint cavity with the vector of the present invention containing the anti-CD81 antibody gene.
  • the vertical axis represents body weight (g).
  • the horizontal axis indicates the number of days after collagen administration. Vector injection was performed immediately after collagen administration.
  • X indicates the body weight of the control vector-injected group.
  • ⁇ indicates the body weight of the group injected with the vector containing the anti-CD81 antibody gene.
  • ⁇ indicates the weight of the normal group.
  • FIG. 4 is a graph showing changes in paw volumes of rheumatoid arthritis model rats injected into the joint cavity with the vector of the present invention containing the anti-CD81 antibody gene.
  • the vertical axis indicates the foot volume (cm3).
  • the horizontal axis indicates the number of days after collagen administration. Vector injection was performed immediately after collagen administration.
  • X indicates the paw volume of the control vector-injected group.
  • ⁇ indicates the paw volume of the group injected with the vector containing the anti-CD81 antibody gene.
  • FIG. 5 is a graph showing changes in clinical scores of rheumatoid arthritis model rats into which the vector of the present invention containing the anti-CD81 antibody gene was introduced into the joint cavity.
  • the horizontal axis indicates the number of days after collagen administration. Vector introduction was performed immediately after collagen administration.
  • X indicates the score of the control vector-injected group.
  • FIG. 6 is a photograph showing swollen paws of rheumatoid arthritis model rats injected into the joint cavity with the vector of the present invention containing the anti-CD81 antibody gene.
  • the upper row shows the paws of rats in the normal group.
  • the middle row shows the paws of rats in the control vector-injected group.
  • the bottom row shows the paws of rats in the group injected with the vector containing the anti-CD81 antibody gene.
  • day15 shows the state of the foot on the 15th day after collagen administration.
  • Day 28 shows the state of the leg on the 15th day after administration of collagen.
  • macroscopic view indicates a magnified image.
  • a radiographic image indicates an X-ray image.
  • FIG. 7 is a microscopic image showing the results of hematoxylin-eosin staining and safranin O staining of a leg joint section of a rheumatoid arthritis model rat injected into the joint cavity with the vector of the present invention containing an anti-CD81 antibody gene.
  • Tissues were harvested and stained 28 days after vector injection.
  • the upper row shows images of the normal group.
  • the middle row shows images of the group injected with the control vector.
  • the bottom row shows images of a group injected with a vector containing the anti-CD81 antibody gene.
  • FIG. 8 shows histological scores of leg joint tissues of rheumatoid arthritis model rats injected into the joint cavity with the vector of the present invention containing the anti-CD81 antibody gene.
  • CIA(+)+SeVdp-control indicates the results of the control vector-injected group.
  • CIA(+)+SeVdp-CD81 shows the results of the group injected with the vector containing the anti-CD81 antibody gene.
  • CIA(-) indicates the results of the normal group. Each bar represents the mean of absorbance ⁇ standard deviation. * indicates p ⁇ 0.05.
  • the histological scores are as follows: synovial mononuclear cells-0: none, 1: low, 2: moderate, 3: large intrasynovial macrophages-0: none, 1: low, 2: moderate, 3: massive cartilage damage-0: no damage, 1: surface irregularity, 2: partial damage, 3: full-thickness damage Bone erosion-0: none, 1: partial cortical irregularity, 2: mild cortical irregularity 3: moderate cortical irregularity, 4: marked cortical bone damage, 5: cortical fragmentation, 6: full-thickness cortical bone damage
  • the present invention relates to gene therapy virus vectors for diseases and pharmaceutical compositions for disease therapy containing the same.
  • the vector is a viral vector for gene therapy of disease comprising an anti-CD81 antibody gene. More particularly, the vector is for gene therapy of rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • the vector of the present invention may be based on any virus.
  • the vectors of the invention are based on RNA viruses, more preferably negative-strand RNA viruses.
  • the present invention provides a negative-strand RNA virus-based RA therapeutic vector comprising a gene encoding an anti-CD81 antibody.
  • a vector based on a negative-strand RNA virus is a cytoplasmic RNA virus vector.
  • Cytoplasmic RNA viral vectors do not affect host chromosomes because the expression of the gene incorporated therein takes place in the cytoplasm. Therefore, the viral vector of the present invention, which is based on a minus-strand RNA virus, is extremely safe.
  • preferred viral vectors of the present invention are characterized by high efficiency of gene transfer and gene expression, and long-term expression.
  • the viral vector of the present invention based on Paramyxovirus, particularly Sendai virus, has the above characteristics.
  • the virus on which the viral vector of the present invention is based is a minus-strand RNA virus.
  • Minus-strand RNA viruses include Paramyxoviridae, Rhabdoviridae, Filoviridae, and the like.
  • the Paramyxoviridae family includes the subfamilies Paramyxovirinae and Pneumovirinae.
  • the Paramyxovirinae include the genus Paramyxoviridae (Sendai virus, human parainfluenza virus type 1, human parainfluenza virus type 3, etc.), the genus Morbillivirus (measles virus, canine distemper virus, rinderpest virus, etc.), and Rubulaviruses (mumps virus, human parainfluenza virus type 2, Newcastle disease virus, simian parainfluenza virus, etc.) are included.
  • the subfamily Pneumovirinae includes the genus Pneumoviridae (respiratory syncytial virus, murine pneumonia virus, bovine respiratory syncytial virus, etc.).
  • minus-strand virus-based vector may be used in the present invention.
  • viral vectors based on viruses of the family Paramyxoviridae are preferred.
  • Sendai virus-based viral vectors are capable of persistent infection, which is convenient because it results in persistent expression of anti-CD81 antibody in the host.
  • a virus-based vector refers to a vector containing all or part of the genome of the virus, and the genome may be modified.
  • the viral genome may be modified according to purposes such as improvement of safety, improvement of expression efficiency, expansion of gene size that can be introduced, suppression of host immunity induction, and the like. Modification may be artificial or may be due to natural mutation. Modification of the viral genome can be performed using known means and methods. For example, in the case of Sendai virus, the F, HN, and M genes may all be deleted to minimize extracellular release of viral antigens and ensure safety. Codons of the viral genome may be optimized according to the purpose.
  • the viral vector may appropriately contain a marker gene (eg drug resistance gene, GFP gene, etc.) for checking the expression of the transgene.
  • a marker gene eg drug resistance gene, GFP gene, etc.
  • a stealth virus vector may be used.
  • a stealth viral vector is a vector that is difficult or not recognized by the host's innate immune system, and is a vector that can suppress or eliminate the induction of host immunity.
  • Methods for stealthing viral vectors are known, and include, for example, modifying or removing the PAMP structure in the viral genome, adding a factor that suppresses the innate immune system to the viral genome, and the like.
  • Examples of Sendai virus-based stealth vectors include, but are not limited to, the SRVTM vector (manufactured by Tokiwa-Bio Co., Ltd.).
  • CD81 is involved in regulating the expression of synoviolin, the causative agent of RA. Therefore, RA therapy targeting CD81 has been proposed. However, gene therapy for RA targeting CD81 has not been performed. Under such circumstances, the present invention provides a negative-strand RNA virus-based therapeutic vector for RA containing a gene encoding an anti-CD81 antibody, which enables effective and highly safe gene therapy for RA. .
  • an anti-CD81 antibody is an antibody that specifically recognizes CD81, binds to it, and suppresses or eliminates the activity of CD81.
  • Anti-CD81 antibodies recognize all or part of CD81.
  • CD81 has extracellular loops, one of which is called LEL (large extracellular loop).
  • LEL large extracellular loop
  • the anti-CD81 antibody may recognize and bind the LEL of CD81.
  • the anti-CD81 antibody may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
  • anti-CD81 antibody refers to an anti-CD81 monoclonal antibody.
  • the anti-CD81 antibody used in the present invention can be obtained using a peptide having the amino acid sequence of all or part of CD81 as an immunogen. Since the amino acid sequence of CD81 is known, those skilled in the art can prepare partial peptides of CD81 using known genetic engineering techniques and/or synthetic chemical techniques.
  • CD81 or a partial peptide thereof is administered to an animal for immunization.
  • Methods and means for immunizing animals are known to those skilled in the art. Animals are immunized by administering CD81 or partial peptides thereof by known routes such as subcutaneous injection, intradermal injection, intravenous injection, and direct injection into the spleen. Examples of animals include, but are not limited to, rats, mice, guinea pigs, rabbits, monkeys, and the like.
  • an adjuvant such as Freund's complete or incomplete adjuvant. The types and usage of adjuvants are also known.
  • hybridomas are known to those skilled in the art.
  • Cell fusion methods are also known, and include, but are not limited to, methods using polyethylene glycol, methods using Sendai virus, and electrofusion methods.
  • Hybridomas producing the antibody of interest are screened and cloned.
  • Methods for selecting hybridomas and screening methods for antibody-producing hybridomas are known to those skilled in the art.
  • a method for measuring antibody activity can also be appropriately selected by those skilled in the art according to the properties of the antibody of interest.
  • Methods for cloning hybridomas are also known. In addition to the methods described above, methods for preparing desired monoclonal antibodies are known, and for example, phage display methods may be used.
  • anti-CD81 antibodies include a monoclonal antibody produced by a hybridoma deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center and given accession number NITE P-02092, and an independent administrative agency, National Institute of Technology for Product Evaluation. Examples include, but are not limited to, monoclonal antibodies produced by hybridomas deposited at the Organization Patent Microorganisms Depositary Center and assigned accession number NITE P-02093.
  • references to anti-CD81 antibodies include variants, variants, chimeric antibodies, humanized antibodies, fully human antibodies, and fragments thereof (e.g., Fv, scFv, dsFv, Fab, Fab', F(ab')2, single chain antibodies, etc.) and the like.
  • Mutants, modifications, chimeric antibodies, humanized antibodies, fully human antibodies, and fragments of antibodies are known, and can be obtained using known methods.
  • Mutants, modifications, chimeric antibodies, humanized antibodies, fully human antibodies, and fragments of anti-CD81 antibodies maintain the characteristics of the original anti-CD81 antibody. These are preferably, for example, those capable of treating RA as or more potently as the original anti-CD81 antibody. These may recognize the same epitope as the original anti-CD81 antibody, or may recognize a different epitope.
  • the amino acid sequences of the three complementarity determining regions of the heavy chain (heavy chain CDR1, CDR2, CDR3) and the three complementarity determining regions of the light chain (light chain CDR1, CDR2, CDR3) of the mutant or variant are
  • the amino acid sequences of the heavy chain CDR1, CDR2, CDR3 and the light chain CDR1, CDR2, CDR3 of the heavy chain of the original anti-CD81 antibody may be the same or different.
  • the difference in the amino acid sequences of the complementarity determining regions is preferably 1, 2 or 3 residues per CDR. Such differences may also be substitutions between cognate amino acids. Cognate amino acids are known to those of skill in the art.
  • amino acid designations are in three letter system: “Gly, Ala”, “Lys, Arg, His”, “Asp, Glu”, “Asn, Gln”, “Ser, Thr”, “Cys, Met”, “Phe, Trp, Tyr”, “Val, Leu, Ile”.
  • the viral vector of the present invention can be obtained by inserting a gene encoding an anti-CD81 antibody into a viral vector.
  • Gene integration into a viral vector can be performed by a known method. Depending on the size and sequence of the gene to be integrated, the genome structure of the viral vector to be used, the type of base virus, etc., the location of integration and the number of genes to be integrated can be determined.
  • a gene encoding the entire anti-CD81 antibody may be incorporated into the viral vector, or a portion thereof may be incorporated into the viral vector, as long as the expressed anti-CD81 antibody has a therapeutic effect on RA.
  • genes encoding the entire H chain and the entire L chain of an anti-CD81 antibody may be tandemly incorporated into a viral vector.
  • the viral vector thus obtained can be used for the treatment of RA.
  • the viral vector of the present invention is administered to humans, it may also be administered to animals other than humans.
  • An anti-CD81 antibody gene suitable for the animal to be administered can be selected.
  • chimeric, humanized, or fully humanized anti-CD81 antibodies are preferably expressed.
  • the present invention provides a pharmaceutical composition for treating RA, comprising the vector of the present invention.
  • the administration of the pharmaceutical composition of the present invention may be local administration (eg, injection into the affected joint, application of patch, gel, ointment, etc.) or systemic administration (eg, infusion, etc.).
  • the pharmaceutical compositions of the invention are administered by injection into the affected joint.
  • the dosage form of the pharmaceutical composition of the present invention can be selected according to the route of administration, and can be, for example, an injection.
  • Methods for manufacturing each dosage form are known.
  • a method for producing an injection may include the following steps: weighing of components, mixing/dissolving with a carrier, filter sterilization, filling into a container, heat-sealing, foreign matter inspection, and packaging/labeling.
  • carriers include, but are not limited to, water for injection, saline, and the like.
  • Injectables may be in the form of vials, ampoules, prefilled syringes.
  • the injection may be in the form of a solution, a suspension, or a solid injection obtained by freeze-drying the ingredients. Injections may contain additives such as buffers, tonicity agents, stabilizers and preservatives.
  • a single dose of the pharmaceutical composition of the present invention is typically about 1 ml to about 5 ml (for a solution containing 7.0 x 108 CIU/mL vector), but the patient's age, weight, It can be determined or changed by a doctor as appropriate, taking into consideration the severity of symptoms, other treatments being received, and the like.
  • the pharmaceutical composition of the present invention may be administered once to several times every few weeks, or once to several times every few months.
  • the administration interval of the pharmaceutical composition of the present invention can also be appropriately determined and changed by a doctor.
  • the present invention in further aspects, provides: A method of treating rheumatoid arthritis in a patient comprising administering to the patient a negative-strand RNA virus-based vector containing a gene encoding an anti-CD81 antibody; Use of a negative-strand RNA virus-based vector containing a gene encoding an anti-CD81 antibody for the manufacture of a therapeutic agent for rheumatoid arthritis and for the treatment of rheumatoid arthritis containing a gene encoding an anti-CD81 antibody A vector based on a negative-strand RNA virus.
  • Example 1 Construction of RA therapeutic vector (a) Control vector (SeVdp-control vector: the vector in the upper part of Fig. 1) In the following experiments, SRVTM control Vector (Tokiwa Bio Co., Ltd.) was used as a control vector.
  • SEQ ID NO: 1 shows the base sequence of the region containing the EGFP gene, chimeric anti-CD81 antibody H chain gene, chimeric anti-CD81 antibody L chain gene, and PuroP gene in the lower part of FIG.
  • the SeVdp-control vector and SeVdp-antiCD81 vector are Sendai virus-based stealth RNA vectors.
  • Example 2 Expression of anti-CD81 antibody in animal joints (1) Rheumatoid arthritis model rat Type II collagen (CII; Collagen Research Center, Tokyo, Japan) and Freund incomplete adjuvant (FIA; Sigma-Aldrich, Saint Louis, MO, USA) A 1:1 mixture was emulsified and 200 ⁇ L was made by intradermal injection at the base of the tail.
  • CII Collagen Research Center, Tokyo, Japan
  • FIA Freund incomplete adjuvant
  • the SeVdp-control vector was injected into the joint cavity of rheumatoid arthritis model rats, and 48 hours later, it was confirmed that the synovial tissue of the joint was actually infected with the virus and green fluorescence was emitted.
  • a rheumatoid arthritis model rat was created by administering collagen. Immediately after collagen administration, SeVdp-antiCD81 vector (7.0 ⁇ 106 CIU) or SeVdp-control vector (7.0 ⁇ 106 CIU) was injected into the joint cavity of rheumatoid arthritis model rats, and joint synovial tissue was collected 28 days after vector administration. , were examined by ELISA for the expression of anti-CD81 antibodies.
  • Example 3 Therapeutic effect in rheumatoid arthritis model animals
  • SeVdp-antiCD81 vector or SeVdp-control vector was injected into the joint cavity of rheumatoid arthritis model rats, body weight change and degree of joint swelling (leg volume), and clinical Scores were examined over time. The results are shown in Figures 3, 4 and 5, respectively.
  • the paws were observed with a magnifying glass and X-ray.
  • the results are shown in FIG. Twenty-eight days after vector administration, foot joint tissues were collected, and the sections were stained with hematoxylin-eosin and safranin-O.
  • Histological scores were obtained from hematoxylin and eosin staining and Safranin O staining of leg joint tissue taken 28 days after vector administration. The results are shown in FIG.
  • the present invention is useful in the manufacture of pharmaceuticals and research reagents.
  • SEQ ID NO: 1 represents the base sequence of the region containing the EGFP gene, chimeric anti-CD81 antibody H chain gene, chimeric anti-CD81 antibody L chain gene, and PuroP gene inserted into the rheumatoid arthritis therapeutic vector of the present invention. show.
  • bases 1-10 are the transcription initiation signal
  • bases 49-768 are the EGFP gene
  • bases 810-819 are the transcription termination signal
  • bases 823-832 are the transcription initiation signal
  • bases 872-2263 are the chimeric anti-CD81 antibody.
  • bases 2299-2308 are transcription termination signals
  • bases 2312-2321 are transcription initiation signals
  • bases 2360-3079 are chimeric anti-CD81 antibody light chain genes
  • bases 3121-3130 are transcription termination signals
  • bases 3134-3143 is the transcription initiation signal
  • bases 3182-3781 are the PuroR gene
  • bases 3817-3826 are the transcription termination signal.

Abstract

L'invention concerne : un vecteur pour le traitement de la polyarthrite rhumatoïde basé sur un virus à ARN À brin négatif, qui comprend un gène codant pour un anticorps anti-CD81 ; et une composition pharmaceutique pour le traitement de la polyarthrite rhumatoïde qui comprend le vecteur viral.
PCT/JP2022/008422 2021-03-01 2022-02-28 Vecteur pour la thérapie génique de la polyarthrite rhumatoïde WO2022186164A1 (fr)

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JP2017057198A (ja) * 2015-09-15 2017-03-23 中西　徹 Ｃｄ８１ ｌｅｌ特異的モノクローナル抗体

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NAKANISHI TOHRU: "Development of novel antibody therapy targeting tetraspanin CD81", 16 July 2015 (2015-07-16), XP055964292, Retrieved from the Internet <URL:https://kaken.nii.ac.jp/file/KAKENHI-PROJECT-23590102/23590102seika.pdf> *
NAKANISHI TOHRU: "Development of novel gene therapy for rheumatoid arthritis", 19 February 2021 (2021-02-19), XP055964296, Retrieved from the Internet <URL:https://kaken.nii.ac.jp/ja/file/KAKENHI-PROJECT-17K08379/17K08379seika.pdf> [retrieved on 20220923] *
YAMASAKI TSUTOMU, ARAI YUJI, NAKAGAWA SHUJI, KUBO TOSHIKAZU, NAKANISHI TOHRU: "Construction of an Expression Vector Containing Mouse-Rat Chimeric Genes Encoding a Therapeutic Antibody against CD81", JOURNAL OF HARD TISSUE BIOLOGY, vol. 28, no. 3, 1 January 2019 (2019-01-01), pages 239 - 244, XP055964279, ISSN: 1341-7649, DOI: 10.2485/jhtb.28.239 *

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