WO2020036135A1 - Particule de type norovirus humain et utilisation associée - Google Patents

Particule de type norovirus humain et utilisation associée Download PDF

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WO2020036135A1
WO2020036135A1 PCT/JP2019/031571 JP2019031571W WO2020036135A1 WO 2020036135 A1 WO2020036135 A1 WO 2020036135A1 JP 2019031571 W JP2019031571 W JP 2019031571W WO 2020036135 A1 WO2020036135 A1 WO 2020036135A1
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gii
antibody
hunov
protein
antigen
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PCT/JP2019/031571
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Japanese (ja)
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慎太郎 佐藤
義和 幸
宏 清野
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国立大学法人東京大学
一般財団法人阪大微生物病研究会
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Publication of WO2020036135A1 publication Critical patent/WO2020036135A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to human norovirus-like particles and uses thereof.
  • Human norovirus is a pathogenic virus that invades cells of the upper small intestine from the duodenum and causes nonbacterial acute gastroenteritis, and belongs to the non-enveloped Calicivirus family.
  • the HuNoV genome is a single-stranded plus-sense RNA of about 7.7 kb.
  • a viral protein called VPg is covalently linked to the 5 'end of the genome and the 3' end is polyadenylated.
  • ORF1, VP1 and VP2 which code for non-structural protein, structural protein 1 and structural protein 2, respectively.
  • HuNoV constructs a truncated icosahedral structure with 90 (by 180 molecules) VP1 dimers.
  • Each VP1 monomer is divided into two domains, a shell domain (S domain) and a protruding domain (P domain), of which the P domain is further divided into P1 and P2 subdomains.
  • P2 recognizes blood group antigens that function as receptors and host infectious agents during infection. Mutations in the P2 subdomain cause changes in binding to blood group antigens (Non-Patent Document 1).
  • HuNoV is classified into three gene groups (GI, GII and GIV) based on the genome sequence, and it is said that there are at least 25 genotypes. Although it is a norovirus that shows high diversity, in recent years, only a small number of strains such as GII, particularly genotype 4 (GII.4) have been responsible for the spread of infection (Non-Patent Document 1). About 20 million HuNoV infections are transmitted annually in the U.S., and about 70,000 are hospitalized. In Japan, there is no clear data, but about 1 to 20,000 inpatients and more than 7 million infections Are estimated to occur every year.
  • VLPs virus like particle
  • GII.4 is the main genotype in the HuNoV outbreak, but recently other genotypes, especially GII.2, GII.3, GII.6 and GII.17, have become more influential (non-patented).
  • References 3-6 Therefore, there is a need for the development of viral therapeutics such as cross-reactive antibodies that inhibit viral infection of several genotypes. However, at present, there is no antibody that inhibits viral infection of multiple genotypes or a single vaccine antigen that can induce it.
  • an object of the present invention is to provide an antigen for producing an antibody that suppresses multiple types of HuNoV infection, use thereof, and an antibody that suppresses multiple types of HuNoV infection.
  • the present inventors have generated antibodies against GII.4 VLP, GII.3 VLP, and GII.17 VLP, and used a line of intestinal epithelial cells derived from iPS cells (iPSCs) to generate several genes for HuNoV. The type was tested to determine whether it inhibited its infection (HuNoV invasion into and / or replication within intestinal epithelial cells). As a result, an antibody prepared using VLPs formed by self-aggregation of the VP1 protein of GII.4 as an antigen was able to suppress the replication of GII.4, but suppressed the replication of HuNoV of other genotypes. Did not.
  • Antibodies prepared using VLPs formed by self-aggregation of the VP1 protein of GII.3 as antigens were able to suppress the replication of GII.3, but inhibited the replication of HuNoV of other genotypes. Did not.
  • an antibody prepared using VLPs formed by self-aggregation of the VP1 protein of GII.17 as antigens not only suppresses GII.17 replication but also effectively suppresses GII.4 replication.
  • the present invention provides an antigen suitable for the purpose of producing an antibody that inhibits HuNoV infection of a plurality of different genotypes (particularly, GII.4 HuNoV and GII.17 HuNoV), and an antigen produced using the antibody. And the like.
  • the present invention includes the following (1) to (10).
  • a method for producing an antibody comprising: (2) The method for producing an antibody according to (1) above, wherein the full length of the VP1 protein of GII.17 HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
  • the antibody or the functional fragment thereof according to the above (4) or (5), wherein the plurality of different genotypes are GII.4 and GII.17.
  • a pharmaceutical composition comprising the antibody or the functional fragment thereof according to any of (4) to (6).
  • a vaccine for suppressing infection of HuNoV of a plurality of different genotypes which contains the full length or part of GII.17 HuNoV VP1 protein as an antigen.
  • the vaccine according to the above (8), wherein the full length of the VP1 protein of GII.17 HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
  • the vaccine according to (8) or (9), wherein the plurality of different genotypes are GII.4 and GII.17.
  • the present invention provides an antigen for producing an antibody that suppresses the replication of HuNoV of a plurality of different genotypes (for example, GII.4 and GII.17).
  • the present invention also provides an antibody against the above antigen.
  • the antibody can suppress HuNoV infection of a plurality of different genotypes (eg, GII.4 and GII.17), and can provide an effective treatment for HuNoV infection.
  • A is the genomic copy number of GII.4 HuNoV (strain 17-231) cultured after pretreatment with anti-GII.4 VLP polyclonal antibody
  • B is GII. 3 shows the results of measuring the genome copy number of GII.17 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection.
  • As a control IgG an unimmunized rabbit-derived IgG was used. Each value is a representative value of three independent experiments and is shown as a mean value ⁇ standard deviation (SD). Effect of anti-GII.3 VLP polyclonal antibody on HuNoV replication.
  • A is the genomic copy number of GII.3 HuNoV cultured after pretreatment with an anti-GII.3 VLP polyclonal antibody
  • B is GII.4 (17B93 strain), which was pretreated and cultured with an anti-GII.3 VLP polyclonal antibody
  • It is the result of measuring the genome copy number of GII.17 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection.
  • As a control IgG an unimmunized rabbit-derived IgG was used. Each value is a representative value of three independent experiments and is shown as a mean value ⁇ standard deviation (SD). Effect of anti-GII.17 VLP polyclonal antibody on HuNoV replication.
  • A shows the genomic copy number of GII.17 HuNoV cultured after pretreatment with anti-GII.17 VLP polyclonal antibody
  • B shows GII.4 (strain 17-231) cultured after pretreatment with anti-GII.17 VLP polyclonal antibody. And 17B93 strain), and the results of measuring the genome copy number of GII.3 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection.
  • As a control IgG an unimmunized rabbit-derived IgG was used. Each value is representative of three independent experiments and is shown as the mean ⁇ SD.
  • a first embodiment of the present invention relates to a method for producing an antibody that inhibits infection of a plurality of different genotypes of HuNoV into intestinal epithelial cells, the method comprising: This is a method for producing an antibody, including immunization as an antigen.
  • the antibody production method according to the first embodiment shows cross-reactivity with HuNoV of different genotypes and inhibits their infection (or A method for producing an antibody that suppresses the VP1 protein, wherein the full length or a part of the VP1 protein of GII.17 HuNoV is used as an antigen.
  • the genotype of HuNoV that the antibody produced by the antibody production method of the present invention inhibits infection is preferably GII.4 or GII.17 belonging to the GII genogroup.
  • the VP1 protein of the antigen GII.17 HuNoV may be its full length, or may be a partial region having antigenicity. It is a protein represented by SEQ ID NO: 3.
  • the antigenic region can be easily selected by preparing a part of the VP1 protein of GII.17 HuNoV and using it as an immunogen to confirm the neutralizing ability of the antibody (the ability to suppress the replication of HuNoV) can do.
  • “GII.17 HuNoV VP1 protein” refers to a protein including the amino acid sequence represented by SEQ ID NO: 3 as well as the amino acid sequence represented by SEQ ID NO: 3 And a protein comprising an amino acid sequence substantially identical to
  • the term "protein containing substantially the same amino acid sequence” refers to the amino acid sequence represented by SEQ ID NO: 3 by about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, 82% or more.
  • the antibody against the protein comprises an amino acid sequence having about 99% amino acid identity and is capable of infecting intestinal epithelial cells with HuNoVs (eg, GII.4 and GII.17) of multiple different genotypes.
  • HuNoVs eg, GII.4 and GII.17
  • a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 refers to one or several (preferably about 1 to 30, more preferably Is composed of an amino acid sequence in which about 1 to 10, more preferably 1 to 5) amino acids have been deleted, substituted, inserted or added, and an antibody against the protein is intestinal tract of HuNoV of a plurality of different genotypes.
  • the VP1 protein of GII.17 HuNoV is obtained by obtaining a nucleic acid (for example, SEQ ID NO: 4 or the like) encoding this protein from a cDNA library or the like, incorporating the nucleic acid into an appropriate expression vector, and transforming an appropriate host cell with the expression vector. It can be prepared by transforming or transfecting, culturing this in an appropriate medium, expressing and purifying the VP1 protein.
  • a nucleic acid for example, SEQ ID NO: 4 or the like
  • Examples of host cells for expressing the VP1 protein include bacterial cells (eg, Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans , Pseudomonas putida, etc.); molds (eg, Penicillium ) camembertii , Acremonium chrysogenum, etc.), animal cells, plant cells, baculovirus / insect cells or yeast cells (eg, Saccharomyces cerevisiae and Pichia pastoris ) can be used and expressed in these cells.
  • bacterial cells eg, Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans , Pse
  • an expression vector for expressing the VP1 protein a vector suitable for various host cells can be used.
  • expression vectors include, for example, pBR322, pBR325, pUC118, pET etc. (Escherichia coli host), pEGF-C, pEGF-N etc. (animal cell host), pVL1392, pVL1393 etc. (insect cell host, baculovirus vector), pG -1, Yep13 or pPICZ (yeast cell host) can be used.
  • These expression vectors have a replication origin, a selection marker, and a promoter suitable for each vector, and may include an enhancer, a transcription assembly sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like, if necessary. You may have. Furthermore, in order to facilitate purification of the expressed polypeptide, a base sequence for expressing by fusion of a FLAG tag, a His tag, an HA tag, a GST tag and the like may be inserted into the expression vector. Preparation of the expression vector can be carried out by a method known to those skilled in the art, and can also be carried out using a commercially available kit or the like as appropriate.
  • the cells or cultured cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and / or lysozyme.
  • a soluble extract is obtained by centrifugation or filtration.
  • it is desirable to obtain the VP1 protein expressed in the culture supernatant by collecting the supernatant. From the obtained extract or culture supernatant, a desired protein can be obtained by appropriately combining known separation and purification methods.
  • Known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, methods using mainly differences in molecular weight such as SDS-PAGE, and ionization.
  • a method using a charge difference such as exchange chromatography, a method using a specific affinity such as affinity chromatography (for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • affinity chromatography for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • affinity chromatography for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • affinity chromatography for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used.
  • the antibody produced in the first embodiment is not particularly limited, and may be, for example, a monoclonal antibody, a polyclonal antibody, a nanoantibody, or the like.
  • an antigen can be used for an immunized animal (for example, but not limited to, rabbit, goat, sheep, chicken, guinea pig, mouse, rat or pig).
  • an adjuvant mixture can be prepared.
  • the antigen and / or adjuvant is injected multiple times into the immunized animal subcutaneously or intraperitoneally.
  • Adjuvants include, but are not limited to, for example, complete Freund and monophosphoryl lipid A synthesis-trehalose dicorynomycolate (MPL-TMD).
  • MPL-TMD monophosphoryl lipid A synthesis-trehalose dicorynomycolate
  • serum containing an antibody against VP1 is prepared from the immunized animal, and the desired antibody can be purified by a conventional method (for example, a method using rProtein A-Sepharose).
  • the term “monoclonal” refers to a property of an antibody obtained from a substantially homogeneous population of antibodies, and the antibody is produced by a specific method (eg, a hybridoma method). It does not mean to be done.
  • a method for producing a monoclonal antibody include a hybridoma method (Kohler and Milstein, Nature 256 495 1975) or a recombinant method (US Pat. No. 4,816,567).
  • the monoclonal antibody according to the present invention may be isolated from a phage antibody library (Clackson et al., Nature 352 624-628 1991; Marks et al., J. Mol. Biol. 222 581-597 1991).
  • the preparation method includes, for example, the following four steps: (i) immunizing the VP1 protein of GII.17 HuNoV or a part thereof; Immunize animals, (ii) collect lymphocytes secreting (or potentially secreting) monoclonal antibodies, (iii) fuse lymphocytes with immortalized cells, (iv) secrete desired monoclonal antibodies Select cells.
  • the immunized animal for example, mouse, rat, guinea pig, hamster and the like can be selected.
  • lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hybridoma cells.
  • a fusion agent such as polyethylene glycol
  • the fused cells for example, rat or mouse myeloma cell lines are used.
  • the cells are grown in a suitable medium containing unfused lymphocytes and one or more substrates that inhibit the growth or survival of the immortalized cell line.
  • the usual technique uses parental cells that lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT).
  • hypoxanthine, aminopterin and thymidine inhibit the growth of HGPRT-deficient cells and are added to a medium that allows hybridoma growth (HAT medium).
  • HAT medium a medium that allows hybridoma growth
  • a hybridoma producing a desired antibody is selected, and the desired monoclonal antibody can be obtained from a medium in which the selected hybridoma grows according to a conventional method.
  • the hybridoma thus prepared can be cultured in vitro or in vivo in ascites of mice, rats, guinea pigs, hamsters, etc., and the target antibody can be prepared from the culture supernatant or ascites.
  • a nanoantibody is a polypeptide comprising a variable domain of the heavy chain of an antibody (VHH).
  • VHH antibody
  • antibodies such as humans are composed of heavy chains and light chains.
  • Camelid animals such as llamas, alpacas and camels produce single-chain antibodies consisting only of heavy chains (heavy chain antibodies).
  • a heavy chain antibody can recognize a target antigen and bind to the antigen, similarly to a normal heavy and light chain antibody.
  • the variable region of a heavy chain antibody is the smallest unit that has binding affinity for an antigen, and this variable region fragment is called a "nanoantibody.”
  • Nanoantibodies have high heat resistance, digestion resistance, and room temperature stability, and can be easily prepared in large quantities by genetic engineering techniques. Nanoantibodies can be prepared, for example, as follows.
  • a camelid animal is immunized with an antigen, the presence or absence of the target antibody is detected from the collected serum, and cDNA is prepared from RNA derived from peripheral blood lymphocytes of the immunized animal in which the desired antibody titer has been detected.
  • a VHH-encoding DNA fragment is amplified from the obtained cDNA and inserted into a phagemid to prepare a VHH phagemid library.
  • a desired nanoantibody can be prepared from the prepared VHH phagemid library through several screenings.
  • a second embodiment of the present invention relates to an antibody that binds to the VP1 protein of GII.17 HuNoV, wherein the antibody inhibits infection of multiple different genotypes of HuNoV into intestinal epithelial cells (hereinafter referred to as “the antibody of the present invention Or a functional fragment thereof.
  • the antibody of the present invention is not particularly limited, and may be, for example, a monoclonal antibody, a polyclonal antibody, or a nanoantibody.
  • the antibody of the present invention may be a recombinant antibody.
  • the recombinant antibody is not particularly limited, and includes, for example, chimeric antibodies (for example, humanized antibodies and human antibodies).
  • a chimeric antibody is an antibody in which a variable region and a constant region derived from different animal species are linked, for example, an antibody in which the variable region of a mouse-derived antibody is linked to a human-derived constant region.
  • Such linked antibodies can easily be constructed by genetic recombination techniques well known to those skilled in the art.
  • the genotype of HuNoV that the antibody of the present invention inhibits infection is preferably GII.4 and GII.17.
  • a functional fragment of the antibody of the present invention is a partial region of the antibody of the present invention, which means an antibody fragment that binds to HuNoV VPL and inhibits its infection or proliferation in intestinal epithelial cells, for example, Fab, Fab ', F (ab') 2, Fv (variable fragment of antibody), single chain antibody (heavy chain, light chain, heavy chain variable region, light chain variable region, nano antibody, etc.), scFv (single chain) Fv), diabody (scFv dimer), dsFv (disulfide-stabilized ⁇ Fv), and peptides containing at least a part of the CDR of the antibody of the present invention.
  • Fab is an antibody fragment having antigen-binding activity in which about half of the N-terminal side of the heavy chain and the entire light chain are bound by disulfide bonds, among fragments obtained by treating an antibody molecule with the protease, papain.
  • Fab is prepared by treating the antibody molecule with papain to obtain a fragment, for example, constructing an appropriate expression vector into which DNA encoding Fab is inserted, and inserting it into an appropriate host cell (for example, CHO cell or the like). After introduction into mammalian cells, yeast cells, insect cells, and the like), and then expressing Fab in the cells.
  • F (ab ') 2 is an antibody fragment having an antigen-binding activity, which is a little larger than a fragment obtained by treating an antibody molecule with the protease pepsin, wherein the Fab is bound via a disulfide bond in the hinge region. It is.
  • F (ab ') 2 can be prepared by treating the antibody molecule with pepsin to obtain a fragment, or by linking the Fab to a thioether bond or a disulfide bond. can do.
  • Fab' is an antibody fragment having antigen-binding activity, which is obtained by cleaving a disulfide bond in the hinge region of F (ab ') 2.
  • scFv is a VH-linker-VL or VL-linker-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker. And an antibody fragment having an antigen-binding activity.
  • the scFv can be prepared by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of the antibody, and using a genetic engineering technique.
  • Diabody is an antibody fragment obtained by dimerizing scFv and has a bivalent antigen-binding activity.
  • the divalent antigen-binding activities may be the same antigen-binding activity or one may be a different antigen-binding activity.
  • the diabody obtains cDNAs encoding the heavy chain variable region and light chain variable region of the antibody, constructs a cDNA expressing the scFv in which the heavy chain variable region and the light chain variable region are linked by a peptide linker, and performs genetic engineering. It can be produced by a technique.
  • DsFv refers to a polypeptide in which one amino acid residue in each of the heavy chain variable region and the light chain variable region has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted for the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody.
  • dsFv can be prepared by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of an antibody, constructing a DNA encoding the dsFv, and performing genetic engineering techniques.
  • the peptide containing the CDR is configured to include at least one region or more of the heavy chain or light chain CDRs (CDR1 to 3). Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • a DNA encoding the heavy chain or light chain CDR of the antibody is constructed and inserted into an expression vector.
  • the type of the vector is not particularly limited, and may be appropriately selected depending on the type of the host cell to be subsequently introduced. These can be produced by introducing them into an appropriate host cell (eg, a mammalian cell such as a CHO cell, a yeast cell, an insect cell, etc.) in order to express them as an antibody.
  • an appropriate host cell eg, a mammalian cell such as a CHO cell, a yeast cell, an insect cell, etc.
  • the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the
  • the third embodiment of the present invention is a medicine containing the antibody of the present invention or a functional fragment thereof (hereinafter, also referred to as “the medicine of the present invention”).
  • the medicament of the present invention has a pharmacological effect as a prophylactic and / or therapeutic agent for infections caused by HuNoV.
  • the medicament of the present invention may be in a form in which the antibody of the present invention as an active ingredient or the functional fragment thereof itself is administered, but generally, in addition to the antibody of the present invention as an active ingredient or the functional fragment thereof, Alternatively, it is desirable to administer in the form of a pharmaceutical composition containing two or more pharmaceutical additives (hereinafter also referred to as “the pharmaceutical composition of the present invention”). Further, the pharmaceutical composition according to the embodiment of the present invention may further contain other known drugs.
  • the medicament or the pharmaceutical composition according to the embodiment of the present invention is not particularly limited, and may be in the form of a tablet, capsule, granule, powder, syrup, suspension, suppository, ointment, cream, gel. Preparations, patches, inhalants or injections. These preparations are prepared according to a conventional method. In the case of liquid preparations, they may be dissolved or suspended in water or another suitable solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the antibody of the present invention or a functional fragment thereof is prepared by dissolving the same in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, An agent may be added.
  • Formulations for oral or parenteral administration are provided in any formulation form.
  • Formulation forms include, for example, oral administration agents in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, intravenous administration, intramuscular It can be prepared in the form of injections, drops, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants or suppositories for administration or subcutaneous administration. Injections, drops, and the like can be prepared as a powdery dosage form such as a lyophilized form, and dissolved in an appropriate aqueous medium such as physiological saline before use.
  • the type of the pharmaceutical additive used in the production of the medicament or the pharmaceutical composition according to the embodiment of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method of producing the medicament or the pharmaceutical composition depends on the form. Thus, a person skilled in the art can select as appropriate. Inorganic or organic substances, or solid or liquid substances can be used as pharmaceutical additives. Generally, for example, 0.1% to 99.9% by weight, 1% to 1% by weight based on the weight of the active ingredient. 95.0% by weight, or between 1% and 90.0% by weight.
  • lactose glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium as examples of pharmaceutical additives , Ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysodium Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants
  • the active ingredient and excipient components for example, lactose, starch, microcrystalline cellulose, calcium lactate or silicic acid anhydride and the like, or a powder, or A binder such as sucrose, hydroxypropylcellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose or calcium carboxymethylcellulose are added, and the mixture is granulated by wet or dry granulation to give granules.
  • these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc.
  • granules or tablets are coated with an enteric base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer to form an enteric coated preparation, or coated with ethyl cellulose, carnauba wax or hardened oil to obtain a sustained release preparation. You can also.
  • an enteric base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer
  • enteric coated preparation or coated with ethyl cellulose, carnauba wax or hardened oil to obtain a sustained release preparation.
  • To produce capsules powders or granules are filled in hard capsules, or the active ingredient is used as it is, or is dissolved in glycerin, polyethylene glycol, sesame oil or olive oil, and then coated with gelatin to form soft capsules. be able to.
  • the active ingredient may be used as necessary, such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, a pH adjuster such as sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, etc. Dissolve in distilled water for injection together with an isotonic agent, and aseptically filter and fill into ampoules, or add mannitol, dextrin, cyclodextrin or gelatin, and freeze-dry in vacuo to obtain a working-soluble injection. Is also good.
  • reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil or the like may be added to the active ingredient and emulsified in water to prepare an injection emulsion.
  • the active ingredient is dissolved by humidification together with a suppository base such as cocoa butter, fatty acid tri-, di- and monoglycerides or polyethylene glycol, and then poured into a mold and cooled, or the active ingredient is cooled. After dissolving in polyethylene glycol or soybean oil or the like, it may be coated with a gelatin film.
  • a suppository base such as cocoa butter, fatty acid tri-, di- and monoglycerides or polyethylene glycol
  • the dose and the number of times of administration of the medicament or the pharmaceutical composition according to the embodiment of the present invention are not particularly limited, and the purpose of preventing and / or treating the deterioration and progression of the target disease, the type of the disease, the weight and age of the patient, and the like Can be appropriately selected by the judgment of the doctor or pharmacist according to the conditions of In general, the daily dose for oral administration in adults is about 0.01 to 1,000 mg (weight of active ingredient), and it can be administered once or several times a day or every few days . When used as an injection, it is desirable to continuously or intermittently administer a daily dose of 0.001 to 100 mg (weight of the active ingredient) to an adult.
  • the medicament or the pharmaceutical composition according to the embodiment of the present invention is prepared as a sustained-release preparation such as an implant and a delivery system encapsulated in microcapsules using a carrier capable of preventing immediate removal from the body.
  • a carrier capable of preventing immediate removal from the body.
  • biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acids can be used. Such materials can be easily prepared by those skilled in the art.
  • a suspension of liposomes can be used as a pharmaceutically acceptable carrier.
  • Liposomes are prepared through a filter of appropriate pore size to a size suitable for use as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE). Can be purified by
  • the medicament or the pharmaceutical composition according to the embodiment of the present invention may be provided in the form of a kit together with instructions such as an administration method.
  • the medicament or the pharmaceutical composition contained in the kit is manufactured from a material that maintains the activity of the active ingredient effectively for a long period of time, does not cause the agent or the like to adsorb to the inside of the container, and does not alter the components. Supplied by container.
  • a sealed glass ampoule may include a buffer or the like encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
  • the kit may be accompanied by instructions for use. Instructions for use of the kit may be printed on paper or the like, or may be stored and supplied on an electromagnetically readable medium such as a CD-ROM or DVD-ROM.
  • a fourth embodiment of the present invention provides a vaccine for suppressing infection of a plurality of different genotypes of HuNoV, which contains the full length or a part of the VP1 protein of GII.17 HuNoV as an antigen (hereinafter referred to as “the vaccine of the present invention”). ").
  • the vaccine of the present invention may comprise one or more adjuvants, such as complete Freund's or incomplete Freund's adjuvant, cholera toxin, heat-labile Escherichia coli toxin, aluminum hydroxide, potassium alum, saponin or a derivative thereof, muramyl dipeptide, mineral oil or Vegetable oils, novasomes or non-ionic block copolymers, DEAE dextran and the like can be included.
  • adjuvants such as complete Freund's or incomplete Freund's adjuvant, cholera toxin, heat-labile Escherichia coli toxin, aluminum hydroxide, potassium alum, saponin or a derivative thereof
  • a pharmaceutically acceptable carrier must be a compound that does not adversely affect the health of the animal being vaccinated.
  • a pharmaceutically acceptable carrier is, for example, sterile water or a buffer.
  • the vaccine of the present invention can be administered by a conventional active immunization method, and may be administered by injection, orally, or by a transmucosal method such as nasal.
  • the vaccine preparation of the present invention can be administered in a single dose by a method suitable for the dosage form in an amount effective for preventing or treating HuNoV infection (an amount sufficient to induce immunity in animals against HuNoV challenge). Or it can be administered multiple times.
  • the vaccine can be administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally, or mucosally, such as intranasally or sublingually.
  • the vaccine preparation of the present invention can be used by mixing with other antigen components.
  • the dose and frequency of administration of the vaccine preparation may vary depending on the administration subject, but protective immunity can be induced by administering the vaccine containing several tens of ⁇ g of the antigen once every several weeks, once every several weeks.
  • a fifth embodiment of the present invention comprises administering to a patient a medicament or pharmaceutical composition of the present invention or a vaccine of the present invention, wherein a plurality of different genotypes of HuNoV (eg, GII.4 and GII.17).
  • ⁇ treatment '' refers to preventing or alleviating the progress and worsening of the disease state in a patient already infected with HuNoV, and thereby preventing or alleviating the progress and worsening of HuNoV infection. Is the action to be taken.
  • ⁇ prevention '' means, for a patient who may be infected with HuNoV, preventing the infection in advance, and thereby a treatment aimed at preventing the onset of HuNoV infection in advance. .
  • VLP VLP and polyclonal antibody against VLP
  • the virus was roughly purified from stool containing HuNoV donated by Osaka National Institute of Health and Safety, and a virus genome was prepared therefrom. Primers were set outside the VP1 ORFs of GII.4, GII.3 and GII.17 on the prepared genome, each ORF region was amplified by PCR, and the nucleotide sequence of the amplified product was determined. The ORF of each VP1 was cloned into pFastBac Dual Expression Vector (Invitrogen).
  • the amino acid sequence of GII.4 VP1 and the nucleic acid sequence encoding the same are shown in SEQ ID NOS: 1 and 2, respectively, and the amino acid sequence of GII.17 VP1 and the nucleic acid sequence encoding the same are shown in SEQ ID NOs: 3 and 4, respectively.
  • the amino acid sequence of GII.3 VP1 and the nucleic acid sequence encoding it are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. After confirming that the sequence was correct, each construct was used for producing a recombinant baculovirus of the Bac-to-Bac expression system (Invitrogen).
  • High Five cells (Invitrogen) were infected with each recombinant baculovirus at a MOI (multiplicity of infection) of 7 pfu (plaque-forming units) / cell.
  • MOI multiple of infection
  • the culture supernatant was collected and centrifuged at 20,000 g for 1 hour.
  • the obtained supernatant was ultracentrifuged at 100,000 g for 2 hours, and the precipitated VLP was suspended in PBS.
  • the concentrated VLPs were layered on a 10% -60% sucrose density gradient and purified by ultracentrifugation at 100,000 g for 1 hour.
  • VLPs passed through the sucrose density gradient were dialyzed three times against 2 L of PBS to remove sucrose in the sample.
  • VLP was concentrated with AmiconUltra 30-kDa centrifugal filter (Millipore).
  • VLP GII.4, GII.3 or GII.17
  • complete Freund's adjuvant 100-200 ⁇ g
  • each rabbit received a booster injection with 100-200 ⁇ g of VLP mixed with incomplete Freund's adjuvant.
  • rabbits were bled for serum preparation.
  • the IgG fraction of the serum was purified by rProtein A-Sepharrose Fast flow (GE Healthcare) and then dialyzed 3 times against 2 L of PBS.
  • the obtained cell suspension was added to a 5-fold volume of 10% calf serum / basic medium [Advanced DMEM / F12 (Gibco) in 10 mM HEPES (pH 7.3, Gibco), 2 mM Glutamax (Gibco) and 100 units / mL Penicillin plus 100 ⁇ g / mL streptmycin (Gibco) was added], and the mixture was centrifuged at 440 g for 5 minutes to collect the cells.
  • the collected intestinal epithelial cells were cultured on ice on a 20% organoid culture medium supplemented with 10 ⁇ M Y-27632 [a medium obtained by adding the following to a basic medium.
  • an organoid culture medium (+10 ⁇ M Y-27632) was added to each well.
  • the average passage ratio was 1:16 or 3 ⁇ 10 4 / well.
  • the medium was replaced with a fresh organoid medium every 2-3 days. Passaging was performed every 5-7 days.
  • intestinal epithelial cells that had been removed from the wells and suspended in 100 ⁇ L of an organoid culture medium (+10 ⁇ M Y-27632) in a 2.5% Matrigel-coated 96-well plate or Transwells (Corning3470) were used for 2 ⁇ 10 5 Seeded at a cell density of 4 / well.
  • an organoid culture medium (+10 ⁇ M Y-27632) in a 2.5% Matrigel-coated 96-well plate or Transwells (Corning3470) were used for 2 ⁇ 10 5 Seeded at a cell density of 4 / well.
  • 600 ⁇ L of an organoid culture medium was added to the lower well. After culturing in a 5% CO 2 incubator at 37 ° C. for 2 days, the medium was used as a differentiation medium [a medium obtained by adding the following to a basic medium.
  • RN CM 12.5% RN CM (Takahashi et al., Stem Cell Reports 10: 314-328 2018), 1 ⁇ B-27, 50 ng / mL mouse EGF and 500 nM A83-01].
  • the medium was replaced with a differentiation medium with or without 0.03% porcine bile (Sigma). Thereafter, the cells cultured for 2 days were used for HuNoV infection.
  • the cells were washed twice with 150 ⁇ L of the basal medium.
  • a differentiation medium containing or not containing 0.03% bile was added to 100 ⁇ L cells, and pipetted twice, and then collected as a control sample.
  • 100 ⁇ L of differentiation medium with or without 0.03% bile was added to each well and then cultured in 5% CO 2 for 72 hours.
  • GII.4 Antibody prepared using VLP as an antigen Compared with VLP used as an antigen, VP1 of the virus strain (17-231) contained a single amino acid mutation (D235E) in the P1 domain.
  • Incubation of .4 HuNoV with anti-GII.4 VLP antibody completely inhibited its replication in intestinal epithelial cells (FIG. 1A).
  • FIG. 1A Incubation of .4 HuNoV with anti-GII.4 VLP antibody completely inhibited its replication in intestinal epithelial cells (FIG. 1A).
  • FIG. 1A intestinal epithelial cells
  • the anti-GII.4 VLP polyclonal antibody prepared here was unable to inhibit the replication of GII.3, GII.6 and GII.17 HuNoV (FIG. 1B). This result suggests that GII.4 VLP cannot be used as an antigen to induce neutralizing antibodies that inhibit the replication of other genotypes of HuNoV.
  • Antibody prepared using GII.17 VLP as an antigen Pretreatment of GII.17 HuNoV with an anti-GII.17 VLP antibody inhibited the replication of GII.17 HuNoV in intestinal epithelial cells (FIG. 3A). Next, when the effect of the anti-GII.17 VLP antibody on the replication of HuNoV of a genotype other than GII.17 was examined, the anti-GII.17 VLP antibody also inhibited the replication of GII.4 HuNoV (FIG. 3B).
  • GII.17 VLP is highly likely to inhibit the infection of HuNoV of other genotypes classified as GII and has the ability as a multivalent vaccine antigen.
  • the present invention provides an antigen suitable for the purpose of preparing an antibody that inhibits HuNoV infection of a plurality of different genotypes, and the antibody. Therefore, the present invention is expected to be used in the medical field.

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Abstract

La présente invention a pour objet de fournir un antigène permettant de générer un anticorps pour inhiber l'infection par de multiples types de HuNov, une utilisation associée, et un anticorps pour inhiber l'infection par de multiples types de HuNov. Plus particulièrement, la présente invention concerne : un procédé de production d'un anticorps pour inhiber l'infection de cellules épithéliales intestinales par différents génotypes de norovirus humain (HuNoV), comprenant l'immunisation à l'aide d'une protéine VP1 de GII.17 HuNoV pleine longueur ou d'une partie de celle-ci en tant qu'antigène ; et un anticorps pour inhiber l'infection de cellules épithéliales intestinales par une pluralité de génotypes différents de HuNoV.
PCT/JP2019/031571 2018-08-13 2019-08-09 Particule de type norovirus humain et utilisation associée WO2020036135A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022244860A1 (fr) * 2021-05-20 2022-11-24 デンカ株式会社 Anticorps anti-norovirus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110070260A1 (en) * 2009-09-09 2011-03-24 Baric Ralph S Multivalent Immunogenic Compositions Against Noroviruses and Methods of Use
US20160102136A1 (en) * 2013-05-09 2016-04-14 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Single-domain vhh antibodies directed to norovirus gi.1 and gii.4 and their use
WO2018181866A1 (fr) * 2017-03-31 2018-10-04 国立大学法人東京大学 Anticorps de norovirus
JP2019149996A (ja) * 2018-03-06 2019-09-12 株式会社イーダブルニュートリション・ジャパン 貝類のノロウイルス不活化方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110070260A1 (en) * 2009-09-09 2011-03-24 Baric Ralph S Multivalent Immunogenic Compositions Against Noroviruses and Methods of Use
US20160102136A1 (en) * 2013-05-09 2016-04-14 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Single-domain vhh antibodies directed to norovirus gi.1 and gii.4 and their use
WO2018181866A1 (fr) * 2017-03-31 2018-10-04 国立大学法人東京大学 Anticorps de norovirus
JP2019149996A (ja) * 2018-03-06 2019-09-12 株式会社イーダブルニュートリション・ジャパン 貝類のノロウイルス不活化方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022244860A1 (fr) * 2021-05-20 2022-11-24 デンカ株式会社 Anticorps anti-norovirus

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