WO2024085207A1 - Equine antiserum against betacoronavirus - Google Patents

Equine antiserum against betacoronavirus Download PDF

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WO2024085207A1
WO2024085207A1 PCT/JP2023/037814 JP2023037814W WO2024085207A1 WO 2024085207 A1 WO2024085207 A1 WO 2024085207A1 JP 2023037814 W JP2023037814 W JP 2023037814W WO 2024085207 A1 WO2024085207 A1 WO 2024085207A1
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antibody
protein
rbd
horse
antibodies
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PCT/JP2023/037814
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French (fr)
Japanese (ja)
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卓也 角田
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ＦＰＨＳ Ｍｅｄｉｃａｌ 株式会社
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    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present disclosure relates to equine antisera against betacoronaviruses. More specifically, the present disclosure relates to equine antisera against betacoronaviruses, polyclonal antibodies obtained from the sera, equine-derived monoclonal antibodies, human chimeric antibodies, and humanized antibodies, as well as mixtures of multiple monoclonal antibodies.
  • Coronaviruses especially SARS-CoV-2 have caused pandemics, and efforts are underway to develop methods to prevent or treat infection. Prevention or treatment of infection may be possible with antibodies, and monoclonal and polyclonal antibodies are being developed. Antibodies have been obtained from a variety of animal species, including cows, chickens, pigs, and horses. Clinical trials of equine polyclonal antibodies are being conducted to establish treatments for moderately or severely ill patients (NCT04573855, NCT04610502, NCT04494984, NCT04514302, NCT04838821, and NCT04913779).
  • horse antisera against betacoronavirus are provided. More specifically, in accordance with the present disclosure, horse antisera against betacoronavirus, polyclonal antibodies obtained from the serum, equine-derived monoclonal antibodies, human chimeric antibodies, and humanized antibodies, as well as mixtures of multiple monoclonal antibodies, are provided.
  • an equine antiserum against an immunogenic composition comprising the receptor binding domain (RBD region) of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), preferably capable of neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
  • a betacoronavirus preferably a SARS-associated coronavirus, e.g., SARS-CoV-2
  • ACE2 angiotensin-converting enzyme 2
  • a composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies, Contains a plurality of different monoclonal antibodies, Each monoclonal antibody comprises a portion of the structure of a horse antibody directed against the RBD region of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), said portion of the structure of said antibody comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region; The remainder of each monoclonal antibody is derived from a human antibody, A composition, wherein each monoclonal antibody is capable of binding to said S protein and neutralizing the binding of said S protein to angiotensin-converting enzyme 2 (ACE2).
  • S protein spike protein
  • betacoronavirus preferably a SARS-associated coronavirus, e.
  • composition according to (7) above, wherein the horse is a horse that has been immunized with an immunogenic composition comprising the RBD region of the S protein.
  • the composition according to (8) above, wherein the immunogenic composition comprises a dimerized RBD region.
  • the composition according to (9) above, wherein the immunogenic composition contains more dimerized RBD regions than monomers.
  • (11) The composition according to any one of (7) to (10) above, wherein the horse is a thoroughbred.
  • a composition or pharmaceutical composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies.
  • a composition or pharmaceutical composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies, comprising a plurality of different antibodies (preferably a plurality of different monoclonal antibodies);
  • Each monoclonal antibody comprises a portion of the structure of a horse antibody directed against the RBD region of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), said portion of the structure of said antibody comprising or consisting of a heavy chain variable region and a light chain variable region, or comprising or consisting of three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
  • S protein spike protein
  • betacoronavirus preferably a SARS-associated coronavirus, e.g.,
  • composition or pharmaceutical composition according to (202) above which is capable of neutralizing the binding of the S protein to angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • composition or pharmaceutical composition described in (202) above wherein at least one of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • ACE2 angiotensin-converting enzyme 2
  • the method includes producing monoclonal antibodies from a plurality of antibody-producing cells each producing a different monoclonal antibody, and obtaining a composition containing the produced plurality of monoclonal antibodies;
  • Each monoclonal antibody contained in the composition comprises a portion of the structure of an antibody selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, said portion of the structure of the antibody comprising or consisting of a heavy chain variable region and a light chain variable region, or comprising or consisting of three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
  • the remainder of each monoclonal antibody is derived from a human antibody.
  • compositions capable of neutralizing the binding between the S protein and angiotensin converting enzyme 2 (ACE2).
  • ACE2 angiotensin converting enzyme 2
  • ACE2 angiotensin-converting enzyme 2
  • any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is a SARS-associated coronavirus.
  • the betacoronavirus from which the RBD region of the immunogenic composition is derived is SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is wild-type SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is an alpha strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is a ⁇ strain of SARS-CoV-2.
  • the beta coronavirus from which the RBD region of the immunogenic composition is derived is the gamma strain of SARS-CoV-2.
  • the beta coronavirus from which the RBD region of the immunogenic composition is derived is a ⁇ strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is the O strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is a variant of SARS-CoV-2 (e.g., a naturally occurring variant).
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is a SARS-associated coronavirus.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is wild-type SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is an alpha strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is a ⁇ strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the gamma strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the ⁇ strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the O strain of SARS-CoV-2.
  • any of the above-mentioned inventions, wherein the betacoronavirus from which the S protein to be bound is derived is a variant of SARS-CoV-2 (e.g., a naturally occurring variant).
  • any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include four or more types of monoclonal antibodies.
  • Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include five or more types of monoclonal antibodies.
  • Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include six or more types of monoclonal antibodies.
  • Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include seven or more types of monoclonal antibodies.
  • any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include eight or more types of monoclonal antibodies.
  • any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include nine or more types of monoclonal antibodies.
  • FIG. 1 shows the changes in viable cell density (VCD) and cell viability after gene transfer of immunogen-producing cells.
  • Figure 2 shows the results of Western blot of the obtained immunogenic composition. The contents of each lane are as shown in Table 2.
  • FIG. 3 shows neutralizing antibody titers (50% IC) in the serum of horses immunized with an immunogenic composition (wild-type (WT) or a mixture of WT and mutant ⁇ strains (WT&Delta)) containing the immunogen recovered 40 hours after gene transfer.
  • 4 shows the amount of antigen-specific antibodies in the serum of horses immunized with immunogenic compositions (WT or WT&Delta) containing the immunogens collected 40 hours after gene transfer.
  • FIG. 4 shows the time course of the amount of antibodies specific to the immunogen (WT) in the antisera of horses immunized with the immunogen (WT)
  • panel B shows the time course of the amount of antibodies specific to the immunogen (WT) in the antisera of horses immunized with the immunogen (WT&Delta)
  • panel C shows the time course of the amount of antibodies specific to the immunogen (Delta) in the antisera of horses immunized with the immunogen (WT)
  • panel D shows the time course of the amount of antibodies specific to the immunogen (Delta) in the antisera of horses immunized with the immunogen (WT&Delta).
  • FIG. 5 shows the neutralizing antibody titers (50% IC) of horse antisera against various SARS-CoV-2 wild-type (WT) and mutants ( ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ strains).
  • FIG. 6 shows the CBB staining and Western blot (WB) results of the immunogens.
  • FIG. 7 shows a method for setting each region (monomer: A and B, dimer: C, trimer: D, and multimer: E) in densitometry analysis based on the results of Western blot of immunogens.
  • a "subject” refers to a vertebrate, e.g., a mammal, including a human, e.g., a mammal (cat, ferret, bat, and pangolin) that is infected with a coronavirus (e.g., SARS-CoV-2).
  • a coronavirus e.g., SARS-CoV-2
  • the subject may be a subject infected with a coronavirus, an asymptomatic carrier of a coronavirus, or a subject infected with SARS-CoV-2 and developing COVID-19.
  • the subject may be a subject at risk of being infected with a coronavirus, or a subject at risk of being infected with a coronavirus.
  • the subject may be a child (e.g., infant (1-6 years), school-age child (6-12 years), adolescent (12 years or older), or adult (20 years or older).
  • An adult may be an adult aged 30 years or older, 40 years or older, 50 years or older, 60 years or older, or 70 years or older.
  • the coronavirus is preferably a betacoronavirus, and more preferably SARS-CoV-2.
  • coronavirus refers to a virus of the Orthocoronavirus subfamily of the Coronaviridae family of the Nidovirales order, and is a single-stranded positive-strand RNA virus. Coronaviruses are named coronaviruses because they have spike protein protrusions (S proteins) on the surface of the virus and have an appearance similar to the corona of the sun. In humans, they cause respiratory infections, including the common cold. Coronaviruses of the Orthocoronavirus subfamily are broadly classified into alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. SARS-associated coronaviruses are classified as betacoronaviruses.
  • SARS-associated coronaviruses include SARS-CoV and SARS-CoV-2.
  • SARS-CoV-2 has been causing the COVID-19 pandemic since the end of 2019.
  • SARS-associated coronaviruses infect host cells by binding to the ACE2 receptor of the host cell via the S protein.
  • SARS-associated coronaviruses share a common infection mechanism in that they use the ACE2 receptor to infect cells.
  • the S protein of SARS-CoV-2 contains a furin cleavage site inside that increases infectivity and pathogenicity (Andersen et al., Nature Medicine, 26, 450-452, 2020).
  • SARS-CoV-2 refers to the coronavirus that caused the 2020 pandemic.
  • WHO World Health Organization
  • 2019-nCoV the World Health Organization
  • ICTV International Committee on Taxonomy of Viruses
  • Coronaviruses can cause a range of respiratory illnesses from the common cold to severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • the WHO has named the disease caused by this new coronavirus COVID-19.
  • the International Committee on Taxonomy of Viruses considers SARS-CoV-2 to belong to the Betacoronavirus genus and to be the same species (or a sister lineage) as SARS-CoV.
  • the complete genome sequence of the wild strain (or Wuhan strain) of SARS-CoV-2 has been registered with the National Center for Biotechnology Information (NCBI) under Gene Bank registration number: MN908947.3.
  • the virus particle (virion) has a particle size of about 50 to 200 nm, and contains spike protein, nucleocapsid protein, membrane protein, envelope protein, and viral genome RNA, just like common coronaviruses.
  • the nucleocapsid protein forms a complex with RNA, and the lipid-bound spike protein, membrane protein, and envelope protein surround it to form the virion envelope.
  • the spike protein located on the outermost surface of the envelope is thought to bind to the ACE2 receptor on the cell surface and promote infection of the cell.
  • SARS-CoV-2 There are people who do not show symptoms of the disease even if they are infected with SARS-CoV-2, and these are called asymptomatic pathogen carriers. It has been pointed out that asymptomatic pathogen carriers may infect others with the virus they carry. It has been noted that infection with SARS-CoV-2 can reduce or eliminate the sense of smell and/or taste. SARS-CoV-2 can cause severe acute respiratory syndrome.
  • SARS-CoV-2 infection is mainly determined by PCR testing. This PCR test evaluates the presence or absence of SARS-CoV-2 genes in the body by whether or not a band specific to SARS-CoV-2 is amplified.
  • Treatments for SARS-CoV-2 include antiviral drugs against SARS-CoV-2 (e.g., remdesivir), steroid anti-inflammatory drugs (e.g., dexamethasone), and inhibitors of inflammatory cytokines (e.g., IL-6 inhibitors, e.g., anti-IL-6 antibodies, TNF- ⁇ inhibitors, e.g., etanercept).
  • antiviral drugs against SARS-CoV-2 e.g., remdesivir
  • steroid anti-inflammatory drugs e.g., dexamethasone
  • inhibitors of inflammatory cytokines e.g., IL-6 inhibitors, e.g., anti-IL-6 antibodies, TNF- ⁇ inhibitors, e.g., etanercept.
  • the "spike protein” may be a protein encoded by positions 21563 to 25384 of the SARS-CoV-2 genome registered with the National Center for Biotechnology Information (NCBI) under GenBank Accession No. MN908947.3, and the spike protein of SARS-CoV-2 has an amino acid sequence registered with the NCBI under GenBank Accession No. QHD43416.1.
  • the spike protein includes S1 and S2, where S1 is present at positions 13 to 541 of the amino acid sequence, and S2 is present at positions 543 to 1208 of the amino acid sequence.
  • S1 further has an N-terminal domain (NTD) and a receptor binding domain (RBD), where NTD is present at positions 13 to 304 of the amino acid sequence, and RBD is present at positions 319 to 541.
  • the RBD region is known to bind to human angiotensin converting enzyme 2 (ACE2).
  • ACE2 human angiotensin converting enzyme 2
  • SEQ ID NO: 1 an example of the amino acid sequence of the wild-type RBD region is shown in SEQ ID NO: 1
  • SEQ ID NO: 2 an example of the amino acid sequence of the RBD region of the ⁇ strain is shown in SEQ ID NO: 2.
  • S1 and S2 are cleaved in the cell, produced as separate peptides, and form a complex during viral particle formation.
  • the spike protein is also called the S protein.
  • the spike protein forms a trimer, binds to angiotensin converting enzyme 2 (ACE2) expressed in the host cell, and can infect the cell.
  • ACE2 angiotensin converting enzyme 2
  • any spike protein (having an amino acid sequence corresponding to the amino acid sequence registered with NCBI as GenBank registration number: QHD43416.1) possessed by a natural virus (including a mutant virus) can be used.
  • An example of the amino acid sequence of the spike protein of a mutant virus is as follows.
  • ⁇ strain has the following mutations compared to the wild-type strain sequence: HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.
  • ⁇ strain has the following mutations compared to the wild-type strain sequence: L18F, D80A, D215G, LAL242-244del, R246I, K417N, E484K, N501Y, D614G, and A701V.
  • ⁇ strain has the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F.
  • Strain ⁇ This strain has the following mutations compared to the sequence of the wild-type strain: T19R, G142D, EF156-157del, R158G, L452R, T478K, D614G, P681R, and D950N.
  • peptide refers to a polymer of amino acids. A polymer usually has no branching.
  • Partial peptide refers to a portion of a particular peptide. Peptides and partial peptides can be produced from nucleic acids encoding the peptide. Peptides and partial peptides can also be chemically synthesized. Peptides and partial peptides can also be isolated, enriched, or purified. Isolated means that the peptides and partial peptides are at least separated from other components, and purified means that the peptides and partial peptides are at least selectively separated. Enriched means that the peptides and partial peptides are in an increased concentration.
  • composition is a mixture of one or more components.
  • the composition may include, for example, a partial peptide and an aqueous solvent (e.g., water).
  • the composition may further include a pharma- ceutically acceptable excipient.
  • an immunogenic composition is a composition that can induce an immune response in a subject by administering it to the subject.
  • the immunogenic composition may be used to induce an immune response in a subject. Since the immunogenic composition can induce an immune response in a subject, it may be used as a vaccine.
  • the immunogenic composition of the present invention may be used to induce an immune response against a SARS-associated coronavirus (e.g., SARS-CoV-2), or may be used as a vaccine or a therapeutic agent against a SARS-associated coronavirus (e.g., SARS-CoV-2).
  • SARS-associated coronavirus e.g., SARS-CoV-2
  • SARS-CoV-2 a SARS-associated coronavirus
  • treatment includes prophylactic and therapeutic treatment.
  • Therapeutic treatment may be given to an infected virus, and prophylactic treatment may be given to prevent future infection or to delay future infection from causing a coronavirus infection (e.g., COVID-19) or to reduce symptoms of an established coronavirus infection (e.g., COVID-19).
  • Therapeutic treatment may be given to symptomatic patients or asymptomatic pathogen carriers.
  • Prophylactic treatment may be given to uninfected individuals.
  • exogenous or “exogenous” are used interchangeably to refer to the artificial introduction of a gene or nucleic acid into a target cell by genetic engineering or gene transfer or other manipulation, and to the gene or nucleic acid artificially introduced into a target cell, as well as the expressed protein thereof.
  • An exogenous gene may be operably linked to a promoter sequence that drives expression of the gene.
  • endogenous or “endogenous” means that it is inherently present in the cell.
  • a human-derived cell means that the cell is a cell obtained from a human, or that the cell is a cell line obtained by subculturing the cell, e.g., a human cell.
  • An equine-derived antibody means that the antibody is an antibody obtained from a horse, or that the antibody is produced from an antibody-producing cell having a nucleotide sequence encoding an antibody obtained from a horse or a variant thereof, e.g., an equine antibody, a human chimeric antibody thereof, or a humanized antibody thereof.
  • identity refers to the Identity value obtained using EMBOSS Needle (Nucleic Acids Res.; 2015; 43: W580-W584) with the default parameters.
  • an antibody refers to a protein in which a pair of heterodimers formed by a heavy chain (H chain) and a light chain (L chain) stabilized by a disulfide bond are further associated by a disulfide bond to form a heterotetramer structure.
  • An antibody may have specificity for an antigen. Binding with specificity means binding that is not nonspecific adsorption. Specificity can be ensured by immunization of an animal with the antigen. Having specificity may mean having a stronger affinity for an antigen than for at least one or several other proteins.
  • an antibody that has a strong binding affinity for a specific antigen is an antibody that can specifically bind to the antigen.
  • the heavy chain is composed of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain is composed of a light chain variable region VL and a light chain constant region CL.
  • the variable region fragment (Fv) composed of VH and VL is a region that is directly involved in antigen binding and provides diversity to the antibody.
  • the antigen-binding region composed of VL, CL, VH, and CH1 is called the Fab region, and the region composed of the hinge region, CH2, and CH3 is called the Fc region.
  • the region that directly contacts the antigen has a rich diversity of amino acid sequences among antibodies and is called the complementarity-determining region (CDR).
  • the region other than the CDR, which has a nearly constant amino acid sequence among antibodies, is called the framework region (FR).
  • the light chain and heavy chain variable regions each have three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus.
  • the heavy chain variable region usually contains heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, in this order.
  • a light chain variable region typically comprises, in that order, light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody of the present invention may be of any isotype, IgG, IgM, IgA, IgD, or IgE.
  • the antibody may be produced by immunizing a non-human animal such as a mouse, rat, hamster, guinea pig, rabbit, or chicken, or may be a recombinant antibody, a chimeric antibody, a humanized antibody, or a fully humanized antibody.
  • a chimeric antibody refers to an antibody in which fragments of antibodies derived from different species are linked.
  • IgG subclasses include IgG1, IgG2, IgG3, and IgG4.
  • the antibody of the present invention may be of any subclass, but may be, for example, one or more selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, for example, IgG2, IgG3, or IgG4.
  • Humanized antibody refers to an antibody in which the corresponding positions in a human antibody have been replaced with amino acid sequences characteristic of antibodies of non-human origin (i.e., a CDR-grafted antibody), and examples thereof include antibodies having heavy chain CDR1-3 (HCDR1-3, respectively) and light chain CDR1-3 (LCDR1-3, respectively) of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FR) of the heavy and light chains, being derived from a human antibody. Such antibodies are sometimes called CDR-grafted antibodies.
  • the term “humanized antibody” can include human chimeric antibodies and CDR-grafted humanized antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that binds to an antigen.
  • fragments include, but are not limited to, Fab consisting of VL, VH, CL, and CH1 regions, F(ab')2 in which two Fabs are linked by a disulfide bond at the hinge region, Fv consisting of VL and VH, scFv which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker, and bispecific antibodies such as diabody type, scDb type, tandem scFv type, and leucine zipper type.
  • the antibody may be an isolated monoclonal antibody (e.g., an isolated humanized antibody).
  • the monoclonal antibody contained in the pharmaceutical product is usually isolated and formulated as a pharmaceutical product having a composition suitable for administration.
  • isolated means at least separated from other components. “Isolated” is used to include separation from contaminants to an extent that is compatible with a pharmaceutical product.
  • compositions obtained by mixing multiple isolated monoclonal antibodies (and additives, if necessary) is a composition containing isolated monoclonal antibodies.
  • the composition is prepared so as not to cause unacceptable adverse effects (e.g., toxicity and injury) to cells under in vitro conditions (particularly in a system consisting of cells and physiological saline).
  • pharmaceutical compositions are prepared so as not to cause unacceptable side effects (e.g., toxicity) in a subject (particularly a human) when administered to the subject.
  • the betacoronavirus is preferably a SARS-associated coronavirus, more preferably SARS-CoV-2.
  • SARS-CoV-2 is classified into wild type (the original strain isolated in Wuhan, China), alpha strains (B.1.1.7 and Q lineage strains), beta strains (B.1.351 and its descendants), gamma strains (P.1 and its descendants), delta strains (B.1.617.2 and AY lineage strains), epsilon strains (B.1.427 and B.1.429), eta strains (E.1.525), iota strains (B.1.526), mu strains (B.1.621 and B.1.621.1), zeta strains (P.2 and omi).
  • the SARS-CoV-2 may be any one or more of the SARS-CoV-2 strains selected from the group consisting of clonal strains (B.1.1.529 strain, and BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineage strains), and variants thereof (e.g., but not limited to, Omicron strains selected from the group consisting of XBB.1.5, XBB.1.16, EG.5, BA.2.75, CH.1.1, XBB, XBB.1.9.1, XBB.1.9.2, XBB.2.3, and BA.2.86) and other variants.
  • the betacoronavirus may be a wild-type strain, a delta strain, or an Omicron strain of SARS-CoV-2.
  • a horse antiserum is provided.
  • the horse antiserum of the present disclosure can neutralize the binding of the spike protein (S protein) of a beta coronavirus to angiotensin converting enzyme 2 (ACE2).
  • the horse antiserum of the present disclosure can also suppress the infection of a beta coronavirus to ACE2-expressing human cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells). It is possible to obtain an antibody that inhibits the binding of S1 protein to ACE2, to obtain an antibody that inhibits the infection of a beta coronavirus to human cells, and/or to obtain an antibody that inhibits the infection of a beta coronavirus to humans.
  • the above immunogenic composition can also obtain an antibody that can inhibit the infection of a beta coronavirus to human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells).
  • the infection suppression ability can be expressed by a neutralizing antibody titer (IC 50 ).
  • the neutralizing antibody titer can be expressed by the dilution ratio of the obtained antibody solution or serum.
  • the dilution ratio at which the infection suppression ability is 50% can be taken as IC 50 .
  • the IC 50 can be, for example, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, or 50,000 or more.
  • the IC 50 can be, for example, 5,000 to 100,000, 10,000 to 90,000, 20,000 to 80,000, 30,000 to 70,000, or 40,000 to 60,000.
  • the IC 50 can be calculated by a standard method based on the infection-inhibiting ability using a dilution series of the obtained antibody solution or serum.
  • the horse antisera of the present disclosure are induced by an immunogenic composition
  • an immunogenic composition comprising an RBD region of a spike protein (S protein) of a betacoronavirus.
  • the RBD region is an RBD region of an S protein of a SARS-associated coronavirus.
  • the immunogenic composition comprises two different RBD regions. The two different RBD regions can be from different betacoronaviruses, different SARS-associated coronaviruses, or different SARS-CoV-2 strains.
  • the RBD region is an RBD region of an S protein of SARS-CoV-2.
  • the RBD region is selected from the group consisting of wild type (the original strain first isolated in Wuhan, China), alpha (B.1.1.7 and Q lineage strains), beta (B.1.351 and its progeny), gamma (P.1 and its progeny), delta (B.1.617.2 and AY lineage strains), epsilon (B.1.427 and B.1.429), eta (E.1.525), iota (B.1.526), mu (B.1.621 and B.1.621.1), zeta (P.2), and omicron.
  • the RBD region of the S protein of SARS-CoV-2 is any one or more of the following strains selected from the group consisting of strains (B.1.1.529, and strains of the BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 series), and variants thereof (e.g., Omicron strains selected from the group consisting of, but not limited to, XBB.1.5, XBB.1.16, EG.5, BA.2.75, CH.1.1, XBB, XBB.1.9.1, XBB.1.9.2, XBB.2.3, and BA.2.86) and other variants.
  • strains B.1.1.529, and strains of the BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 series
  • variants thereof e.g., Omicron strains selected from the group consisting of, but not limited to, XBB.1.5, XBB.1.16, EG.5, BA.2.75, CH.1.1, XBB, XBB.1.9.1,
  • the RBD region of the spike protein (S protein) of the beta coronavirus forms a dimer.
  • the immunogenic composition comprises a monomer of the RBD region of the spike protein (S protein) of the beta coronavirus.
  • the immunogenic composition comprises a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus.
  • the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus.
  • the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus, and the content of the dimer is greater than the content of the monomer.
  • the presence of a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus and the content of the dimer is greater than the content of the monomer can be determined by Western blotting after electrophoresis in a non-reducing environment (e.g., native SDS-PAGE).
  • a polyclonal antibody against the horse RBD region can be used as the primary antibody.
  • the immunogenic composition can be used to immunize a horse.
  • the present disclosure provides the immunogenic composition for use in immunizing a horse.
  • the present disclosure also provides a method of immunizing a horse, comprising administering the immunogenic composition to the horse.
  • an effective amount of the immunogenic composition is administered to the horse.
  • it is possible to produce antibodies against RBD in the horse obtain antibodies that inhibit the binding of S1 protein to ACE2, obtain antibodies that inhibit the infection of beta coronaviruses in human cells, and/or obtain antibodies that inhibit the infection of beta coronaviruses in humans.
  • the immunogenic composition can also obtain antibodies that can inhibit the infection of beta coronaviruses in human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells).
  • the infection-inhibiting ability can be expressed by neutralizing antibody titer ( IC50 ).
  • the IC 50 can be, for example, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, or 50,000 or more.
  • the IC 50 can be, for example, 5,000 to 100,000, 10,000 to 90,000, 20,000 to 80,000, 30,000 to 70,000, or 40,000 to 60,000.
  • the IC 50 can be calculated by conventional methods.
  • the RBD region of the spike protein (S protein) of betacoronavirus can be expressed by mammalian cells.
  • the cells are suitable for protein expression, such as CHO cells and 293 cells, and variants thereof (e.g., Expi293F cells and ExpiCHO-S cells).
  • the cells are cultured under conditions suitable for protein expression.
  • the RBD region is recovered from the cell culture, for example, within 60 hours, particularly within 2 days after gene introduction (preferably 24 hours or thereafter, more preferably 30 hours or thereafter, even more preferably 36 hours or thereafter, and even more preferably 42 hours or thereafter). In this way, an immunogenic composition containing a large amount of dimers of the RBD region can be obtained.
  • An immunogenic composition containing a monomer and a dimer of the RBD region of the spike protein (S protein) of a beta coronavirus, in which the content of the dimer is greater than the content of the monomer, may have high immunogenicity.
  • SARS-CoV-2 Spike Protein S1 RBD ELISA Kit (Elabscience, E-EL-E605)
  • it may be evaluated as having a protein concentration that is 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, or 9-fold or more of the estimated protein amount measured by the BCA method.
  • the RBD region has the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • the RBD region has a signal peptide at the N-terminus.
  • the signal peptide may be a secretory signal peptide, and those skilled in the art can select and use it as appropriate.
  • a secretory signal peptide for example, a signal peptide present at the N-terminus of an antibody heavy or light chain may be used, for example, a signal peptide of an IgG heavy or light chain may be used, and for example, a signal peptide having the amino acid sequence set forth in SEQ ID NO: 3 may be used.
  • the RBD region may further have a tag peptide for protein purification. The tag for protein purification may be linked, for example, to the C-terminus of the RBD region.
  • the RBD region may have the form of a fusion peptide that includes a signal peptide, an RBD region, and a tag peptide in this order from the N-terminus to the C-terminus.
  • tag peptides include, but are not limited to, FLAG tag, myc tag, V5 tag, S tag, E tag, T7 tag, VSV-G tag, Glu-Glu tag, Strep-tag II, HSV tag, and other tags.
  • the tag peptide can be, for example, 15 amino acids long, 14 amino acids long, 13 amino acids long, 12 amino acids long, 11 amino acids long, 10 amino acids long, 9 amino acids long, 8 amino acids long, 7 amino acids long, or 6 amino acids long.
  • the tag peptide can be, for example, a 6xHis tag as set forth in SEQ ID NO:4.
  • the horse is a Thoroughbred.
  • the direct paternal ancestry of the Thoroughbred is said to be any of the Darley Arabian, the Byerley Turk, and the Godolphin Arabian.
  • the horse is a Thoroughbred with less than 25% Arabian blood.
  • the horse is male or female, for example, 10 to 20 years of age.
  • the horse has a body weight of 400 kg to 800 kg, preferably 500 kg to 700 kg.
  • the horse is a thoroughbred and the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus.
  • the horse is a thoroughbred and the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus, and the dimer content is greater than the monomer content.
  • the equine antiserum of the present disclosure can be obtained by administering the immunogenic composition to a horse (preferably a thoroughbred).
  • the equine antiserum obtained can exhibit high neutralizing antibody titers and can also have high infection-inhibiting ability.
  • a polyclonal antibody contained in horse antiserum is provided.
  • the polyclonal antibody contained in horse antiserum can be obtained by purifying the antibody from the horse antiserum.
  • the purification can be performed, for example, by plasma fractionation, ammonium sulfate fractionation, or caprylic acid precipitation.
  • the purification can be performed, for example, by a column equipped with protein G or protein A that selectively adsorbs the antibody. Purification of the antibody by a column equipped with protein G or protein A can be appropriately performed by a person skilled in the art according to a conventional method.
  • the purified polyclonal antibody is also an isolated polyclonal antibody.
  • the present disclosure also provides a monoclonal antibody related to any one of the antibodies contained in the horse antiserum.
  • the monoclonal antibody can be obtained by various methods. For example, any cell selected from spleen cells, B cells, or plasma cells obtained from a horse administered with an immunogenic composition is collected. The sequence of the mRNA or cDNA encoding the antibody in the collected cells is decoded to determine the amino acid sequence of the antibody, and a gene encoding the antibody having the determined amino acid sequence is introduced into a protein-expressing cell (e.g., Chinese hamster ovary (CHO) cell) to obtain an antibody-producing cell, and a monoclonal antibody can be obtained from the obtained antibody-producing cell.
  • a protein-expressing cell e.g., Chinese hamster ovary (CHO) cell
  • any cell selected from spleen cells, B cells, or plasma cells obtained from a horse may be fused with a myeloma cell to obtain a hybridoma.
  • the sequence of the mRNA or cDNA encoding the antibody in the obtained hybridoma cell is decoded, and the gene encoding the decoded antibody is introduced into a protein-expressing cell (e.g., Chinese hamster ovary (CHO) cell) to obtain an antibody-producing cell, and a monoclonal antibody can be obtained from the obtained antibody-producing cell.
  • a purified monoclonal antibody is also an isolated monoclonal antibody.
  • Monoclonal antibodies can be human chimerized or humanized.
  • human chimerized monoclonal antibodies and humanized monoclonal antibodies can be provided.
  • the present disclosure provides a composition comprising a polyclonal antibody of the present disclosure, a composition comprising a monoclonal antibody of the present disclosure, a composition comprising a human chimerized monoclonal antibody of the present disclosure, and a composition comprising a humanized monoclonal antibody of the present disclosure.
  • the present disclosure also provides a composition comprising multiple monoclonal antibodies. Such a composition may be advantageous, for example, in increasing the types of betacoronaviruses that can be neutralized.
  • compositions are provided that include a plurality of human chimerized monoclonal antibodies. In some embodiments, compositions are provided that include a plurality of humanized monoclonal antibodies. In these embodiments, the plurality of monoclonal antibodies (i.e., the plurality of human chimerized monoclonal antibodies and the plurality of humanized monoclonal antibodies) differ from each other as substances. In some embodiments, the plurality of monoclonal antibodies differ from each other in amino acid sequence. In some embodiments, the plurality of monoclonal antibodies differ from each other in the amino acid sequence of one or more CDRs selected from the group consisting of heavy chain CDR1-3 and light chain CDR1-3.
  • the difference in amino acid sequence can be from 1 amino acid to 10 amino acids.
  • any at least two of the plurality of antibodies can each bind to a different epitope, e.g., do not compete with each other for binding to the RBD region.
  • the antibodies of the present disclosure may bind to the RBD region (preferably a dimer thereof) with a binding dissociation constant (KD value) of, for example, 10 ⁇ 6 or less, 10 ⁇ 7 or less, 10 ⁇ 8 or less, 10 ⁇ 9 or less, 10 ⁇ 10 or less, or 10 ⁇ 11 or less, where a smaller KD value indicates a stronger binding of the antibody to the RBD region (preferably a dimer thereof).
  • KD value binding dissociation constant
  • compositions comprising a virtual polyclonal antibody
  • a virtual polyclonal antibody is an antibody mixture (or a composition comprising the mixture) comprising a plurality of different or diverse monoclonal antibodies (each of which is an isolated monoclonal).
  • a virtual polyclonal antibody comprises a plurality of monoclonal antibodies, but the content of each monoclonal antibody reaches an effective amount.
  • a virtual polyclonal antibody is an antibody mixture that can robustly express a desired antibody function (e.g., can suppress a decrease in binding affinity to an antigen mutant) compared to a single monoclonal antibody, like a polyclonal antibody.
  • at least one monoclonal antibody included in the virtual polyclonal antibody can bind to S protein simultaneously with at least one other monoclonal antibody.
  • a plurality of different humanized antibodies, for example, virtual polyclonal antibodies may each contain an effective amount of two or more monoclonal antibodies.
  • a plurality of different humanized antibodies, for example, virtual polyclonal antibodies may each contain an effective amount of three or more monoclonal antibodies.
  • a plurality of different humanized antibodies, for example, virtual polyclonal antibodies may each contain an effective amount of n or more monoclonal antibodies ⁇ where n is not particularly limited as long as it is a natural number of 2 or more, but may be, for example, a natural number from 2 to 50, 2 to 40, 2 to 30, 2 to 20, or 2 to 10 ⁇ .
  • a plurality of different humanized antibodies may each contain an effective amount of 4 to 10 monoclonal antibodies.
  • Each of the monoclonal antibodies contained in a plurality of different humanized antibodies, for example, virtual polyclonal antibodies, is human chimerized or humanized.
  • a plurality of different humanized antibodies e.g., virtual polyclonal antibodies, Immunizing a horse with any one of the above immunogenic compositions containing the RBD region of a spike protein (S protein) of a beta coronavirus, thereby producing antibodies against the RBD region in the horse's body; determining sequences (e.g., nucleotide sequences or amino acid sequences) of heavy and light chain variable regions of each of a plurality of mRNAs or cDNAs encoding each of the antibodies produced by a plurality of cells obtained from the horse, wherein the cells are selected from the group consisting of spleen cells, B cells, and plasma cells; Clustering the determined sequences (e.g., nucleotide sequences or amino acid sequences) into a plurality of clusters based on their sequence identity; selecting at least q clusters from the top p clusters containing the largest number of nucleotide sequences from among the obtained clusters; obtaining a nucleotide sequence en
  • any two of the multiple antibodies contained in the antibody can be ones that do not compete with each other for binding to the RBD region.
  • a composition comprising a plurality of different humanized antibodies, e.g., a virtual polyclonal antibody
  • Each monoclonal antibody contained in the composition comprises a portion of an antibody structure selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, the portion of the antibody structure comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
  • S protein spike protein
  • CDRs complementarity determining regions
  • the immunogenic composition may be any of the immunogenic compositions described above.
  • a composition comprising a plurality of different humanized antibodies of the present disclosure can be obtained by a method including obtaining an antibody that inhibits binding of S1 protein to ACE2, obtaining an antibody that inhibits infection of human cells by betacoronavirus, and/or obtaining an antibody that inhibits infection of humans by betacoronavirus.
  • a method of treating a betacoronavirus infection in a subject comprising administering to the subject an effective amount of an antiserum, monoclonal antibody, polyclonal antibody, or virtual polyclonal antibody of the present disclosure.
  • An embodiment may be a healthy individual or a subject infected with a betacoronavirus.
  • a subject may be a carrier (subject asymptomatic) of a betacoronavirus infection.
  • a subject may be a subject suffering from an infection with a betacoronavirus.
  • a subject may be a subject suffering from a mild betacoronavirus infection.
  • a subject may be a subject suffering from a moderate betacoronavirus infection.
  • a subject may be a subject suffering from a severe betacoronavirus infection.
  • a subject does not have a coronavirus infection (e.g., a betacoronavirus infection) but has a history of close contact with a betacoronavirus infection.
  • a subject may be a subject infected with a betacoronavirus but does not have a coronavirus infection (e.g., a betacoronavirus infection).
  • composition comprising an antiserum, a monoclonal antibody, a polyclonal antibody, or a plurality of different humanized antibodies of the present disclosure, e.g., a virtual polyclonal antibody, for use in the methods of the present disclosure.
  • composition or pharmaceutical composition comprising an antiserum, a monoclonal antibody, a polyclonal antibody, or a plurality of different humanized antibodies, e.g., virtual polyclonal antibodies, of the present disclosure for use in the methods of the present disclosure.
  • the present disclosure provides for the use of an antiserum, monoclonal antibody, polyclonal antibody, or composition comprising a plurality of different humanized antibodies, e.g., a virtual polyclonal antibody, of the present disclosure in the manufacture of a medicament or composition for use in the methods of the present invention.
  • Example 1 Preparation of antigen
  • a partial peptide of S1 protein was prepared as an antigen.
  • ExpiCHO-S frozen cells were thawed, subcultured repeatedly until cell growth stabilized, and then frozen to obtain frozen cells.
  • ExpiCHO Expression Medium was introduced into an empty, clean Erlenmeyer flask and incubated in a CO2 incubator at 37°C.
  • the cells were seeded at a concentration of 0.2x106 cells/mL to 0.3x106 cells/mL and cultured at 37°C in the presence of 8% CO2 .
  • the viable cell density reached a concentration of 0.4x106 cells/mL to 0.6x106 cells/mL.
  • the RBD region (amino acids 319 to 541) of the S1 protein of SARS-CoV-2 (Wuhan strain) was used as the antigen.
  • the Wuhan strain may be referred to as wild type or WT.
  • a His tag was added to the antigen.
  • the resulting sequence is referred to as S1-RBD-His tag (WT).
  • the S1-RBD-His tag had an IgG ⁇ leader sequence (SEQ ID NO: 3) having the amino acid sequence of SEQ ID NO: 3 added to the N-terminus of the RBD region having the amino acid sequence of SEQ ID NO: 1, and had 6 ⁇ His (His tag; SEQ ID NO: 4) at the C-terminus.
  • an IgG ⁇ leader sequence (SEQ ID NO: 3) was added to the N-terminus of the RBD region (SEQ ID NO: 2) of the S1 protein of SARS-CoV-2 ( ⁇ strain), and a His tag (SEQ ID NO: 4) was added to the C-terminus to obtain S1-RBD-His tag ( ⁇ strain).
  • Each antigen was cloned into the pcDNA3.4-TOPO vector.
  • Each antigen was designed to be driven by the CMV promoter carried in the pcDNA3.4-TOPO vector.
  • the supernatant was collected.
  • the collected supernatant was used as a recombinant protein extract.
  • the protein was subjected to SDS-PAGE, CBB staining or Western blotting (detection was performed with alkaline phosphatase-labeled Anti-6 ⁇ His tag antibody (Abcam, ab49746)). Protein concentrations were quantified by UV absorption or the bicinchoninic acid (BCA) method.
  • Protein expression was as shown in Table 1. As shown in Table 1, the amount of recovered protein was approximately 40-50% in the sample taken 5 days after gene introduction. The recombinant protein in the supernatant was quantified 60 hours after gene introduction. Due to concerns that depth filter filtration would reduce the protein recovery rate, depth filter filtration was not performed in the recovery process, and only 0.22 ⁇ m filter filtration was carried out. The amount of recovered recombinant protein was improved. The recombinant protein in the supernatant was quantified 40 hours after gene introduction. Depth filter filtration was not performed in the recovery process, and only 0.22 ⁇ m filter filtration was carried out.
  • a band of the recombinant protein was confirmed at the desired size. Looking at lanes 10 to 17 in Figure 2, a band was also detected above the desired band (between 50 kDa and 75 kDa). Considering that this band was not observed under reducing conditions and that the size of the band was about twice that of the recombinant protein, it was suggested that this band corresponds to a dimer of the recombinant protein. In lanes 11 and 15 in Figure 2, the band of the dimer was stained darker than the monomer of the recombinant protein, suggesting that the dimer was predominant in terms of abundance compared to the monomer.
  • the recombinant protein recovered 40 hours after gene transfer was quantified by the BCA method and also by ELISA. The results are shown in Table 3.
  • the protein concentration was adjusted by the BCA method as shown in Table 3.
  • the obtained samples with adjusted protein concentrations were subjected to ELISA measurement, and the concentrations estimated by ELISA far exceeded the actual protein concentrations in samples 1 to 4. This suggests that the obtained proteins react extremely strongly with antibodies compared to the protein amount, i.e., their immunogenic activity is remarkably high.
  • Example 2 Induction of antibody production in animals
  • purified S1-RBD-His tag (WT) derived from cells collected 40 hours after gene transfer in Example 1, and a mixture of the purified S1-RBD-His tag (WT) and purified S1-RBD-His tag ( ⁇ strain) were used as immunogens.
  • the immunogen was mixed with Freund's complete adjuvant (DIFCO 263810) or Freund's incomplete adjuvant (DIFCO 263910) to obtain an emulsion.
  • the immunogen was administered at a dose of 5 mg/day in terms of protein amount.
  • the animals used were horses. Thoroughbreds (12-18 years old) of different pedigrees were used as horses. The horses were immunized as follows: On day 0, recombinant protein (5 mg total) mixed with Freund's complete adjuvant was injected into the neck and rump. On day 14, recombinant protein (5 mg total) mixed with Freund's incomplete adjuvant was administered, and on day 28, recombinant protein (5 mg total) diluted with saline was further administered. Small amounts of blood were collected for testing at various time points. In addition, large amounts of blood (5 L) were collected in bags containing 250 mL of 4% cytramine solution on days 56, 63, and 70.
  • the collected blood was left to stand at room temperature for 1 hour to precipitate blood cells, and the serum from which blood cell components had been removed was refrigerated until use.
  • the obtained sera are called anti-WT serum and anti-WT&Delta serum, respectively.
  • the polyclonal antibody obtained from the anti-WT&Delta serum obtained n days after immunization is called LADn
  • the polyclonal antibody obtained from the anti-Delta serum obtained n days after immunization is called LBDn. That is, LBD56 means the polyclonal antibody obtained from the anti-Delta serum obtained 56 days after immunization.
  • Example 3 Testing of obtained serum Serum was separated from the blood (for testing) collected in Example 2 and tested.
  • cPass test The titer of neutralizing antibodies was measured for the obtained serum.
  • the antibody titer was measured using the cPASS SARS-CoV-2 Neutralizing Antibody Detection Kit (GenScript). This kit is designed to mimic virus-host interactions by direct protein-protein interactions in a test tube or in the wells of an ELISA plate using purified receptor binding domain (RBD), a protein derived from the viral spike (S) protein, and the host cell receptor ACE2.
  • RBD receptor binding domain
  • S viral spike
  • S host cell receptor ACE2
  • the neutralizing antibody titer 50% IC (IC 50 ) was calculated by a conventional method. Specifically, the inhibition rate (%) was probit converted, plotted on a graph with the dilution factor and the probit-converted inhibition rate as axes, and fitted with an approximate straight line to determine the dilution rate equivalent to an inhibition rate of 50%.
  • the neutralizing antibody titer of this example is an order of magnitude higher than the neutralizing antibody titers of previous vaccine products.
  • the amount of specific antibodies in the antiserum was quantified by antigen ELISA. The results are shown in Figure 4. The amount of specific antibodies peaked at about 40 to 60 days, and high levels of antibodies continued for a long period thereafter.
  • PVNT Pseudovirus Neutralization Test
  • a pseudovirus in which the envelope glycoprotein in the lentivirus vector was replaced with the S protein was used to simulate infection with SARS-CoV-2.
  • the pseudovirus contained a nucleic acid encoding luciferase, and expressed luciferase in cells when infected with the cells. Therefore, the amount of virus infected into the cells could be estimated from the luminescence intensity by luciferase.
  • the ability of the antibody to suppress infection with the virus was estimated by observing the effect of suppressing luciferase expression.
  • PVNT was performed using SARS-CoV-2 Pseudovirus Neutralization Assay Kit Luc Reporter (SC2087A, SC2087-027, and SC2087-W).
  • the antibody used was a horse polyclonal antibody purified from horse serum by the caprylic acid fractionation method (using 5 v/v % caprylic acid).
  • the neutralizing antibody titer was compared with that of a commercially available polyclonal antibody.
  • the horse serum used was serum obtained by immunizing S1-RBD-His tag (WT).
  • the mouse polyclonal antibody was 40592-MP01 (Sino Biological), a mouse polyclonal antibody against the RBD of S1, the rabbit polyclonal antibody was GTX135356 (GeneTex), a rabbit polyclonal antibody against the spike protein S1, and the human polyclonal antibody was Cov-Neut-S-500 (Ray Biotech), a serum obtained from a coronavirus-positive donor. The results were as shown in Table 5.
  • the recombinant protein used as the immunogen contained many multimers (especially dimers), and the polyclonal antibody obtained from the horse antiserum in this example recognized the multimers well. Furthermore, as shown in Table 8, the mouse polyclonal antibody had weak reactivity to the immunogen, but did not match the horse polyclonal antibody in terms of binding specificity to multimers.
  • the antibodies produced from horses immunized with multimeric RBD showed surprisingly excellent binding properties and neutralizing activity. It is also suggested that immunization of horses is more suitable for producing a diverse group of antibodies, including antibodies with higher affinity to antigens, than immunization of mice. Therefore, it is expected that excellent monoclonal antibodies can be obtained from antibodies produced from horses immunized with multimeric RBD (particularly RBD dimers).
  • the excellent properties of the above polyclonal antibodies are thought to be the effect of multiple antibodies, and therefore it is expected that multiple excellent monoclonal antibodies can be reproducibly obtained from antibodies produced from horses immunized with multimeric RBD (particularly RBD dimers).
  • thoroughbreds are known to be highly genetically homogeneous, and immunization of thoroughbreds is thought to be suitable for maintaining a constant diversity and quality of antibodies produced.

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Abstract

Provided according to the present disclosure is an equine antiserum against a betacoronavirus. More specifically, provided according to the present disclosure are an equine antiserum against a betacoronavirus, a polyclonal antibody obtained from said serum, a monoclonal antibody derived from a horse, a human chimerized antibody and a humanized antibody, and a mixture of multiple monoclonal antibodies.

Description

ベータコロナウイルスに対するウマ抗血清Equine antisera to betacoronavirus
　本開示は、ベータコロナウイルスに対するウマ抗血清に関する。本開示は、より具体的には、ベータコロナウイルスに対するウマ抗血清、当該血清から得られるポリクローナル抗体、ウマに由来するモノクローナル抗体、ヒトキメラ化抗体、およびヒト化抗体、並びに複数のモノクローナル抗体の混合物に関する。 The present disclosure relates to equine antisera against betacoronaviruses. More specifically, the present disclosure relates to equine antisera against betacoronaviruses, polyclonal antibodies obtained from the sera, equine-derived monoclonal antibodies, human chimeric antibodies, and humanized antibodies, as well as mixtures of multiple monoclonal antibodies.
　コロナウイウルス（特にＳＡＲＳ－ＣｏＶ－２）がパンデミックを引き起こし、感染を予防または治療する方法の確立が進められている。感染の予防または治療は、抗体により可能であると考えられ、モノクローナル抗体およびポリクローナル抗体の開発が行われている。抗体は、ウシ、ニワトリ、ブタ、およびウマを含む様々な動物種から取得されている。中等度または重篤な患者に対する治療法確立のために、ウマのポリクローナル抗体に関する臨床試験が実施されている（ＮＣＴ０４５７３８５５、ＮＣＴ０４６１０５０２、ＮＣＴ０４４９４９８４、ＮＣＴ０４５１４３０２、ＮＣＴ０４８３８８２１、およびＮＣＴ０４９１３７７９）。Ｓｃｉｅｎｔｉｆｉｃ　Ｒｅｐｏｒｔｓ　ｖｏｌｕｍｅ　１１，　Ａｒｔｉｃｌｅｎｕｍｂｅｒ：９８２５（２０２１）では、ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質、Ｎタンパク質、およびスパイクＥ－Ｍモザイクタンパク質の混合物が免疫原として用いられ、ウマを当該免疫原で免疫した。Ｓｃｉｅｎｔｉｆｉｃ　Ｒｅｐｏｒｔｓ　ｖｏｌｕｍｅ　１２，　Ａｒｔｉｃｌｅ　ｎｕｍｂｅｒ：　３８９０　（２０２２）では、不活性化ＳＡＲＳ－ＣｏＶ－２ウイルス懸濁液が免疫原として用いられ、ウマを当該免疫原で免疫した。ｉＳｃｉｅｎｃｅ　２４，　１０３３１５，　Ｎｏｖｅｍｂｅｒ　１９，　２０２１では、Ｓ１ドメインとＳ２ドメインの両方を含むスパイクタンパク質エクトドメインの三量体が免疫原として用いられ、ウマを当該免疫原で免疫した。 Coronaviruses (especially SARS-CoV-2) have caused pandemics, and efforts are underway to develop methods to prevent or treat infection. Prevention or treatment of infection may be possible with antibodies, and monoclonal and polyclonal antibodies are being developed. Antibodies have been obtained from a variety of animal species, including cows, chickens, pigs, and horses. Clinical trials of equine polyclonal antibodies are being conducted to establish treatments for moderately or severely ill patients (NCT04573855, NCT04610502, NCT04494984, NCT04514302, NCT04838821, and NCT04913779). In Scientific Reports volume 11, Article number: 9825 (2021), a mixture of spike protein, N protein, and spike E-M mosaic protein of SARS-CoV-2 was used as an immunogen, and horses were immunized with the immunogen. In Scientific Reports volume 12, Article number: 3890 (2022), an inactivated SARS-CoV-2 virus suspension was used as an immunogen, and horses were immunized with the immunogen. iScience 24, 103315, November 19, 2021, a trimer of the spike protein ectodomain containing both the S1 and S2 domains was used as an immunogen, and horses were immunized with the immunogen.
　本開示によれば、ベータコロナウイルスに対するウマ抗血清が提供される。本開示によれば、より具体的には、ベータコロナウイルスに対するウマ抗血清、当該血清から得られるポリクローナル抗体、ウマに由来するモノクローナル抗体、ヒトキメラ化抗体、およびヒト化抗体、並びに複数のモノクローナル抗体の混合物が提供される。 In accordance with the present disclosure, horse antisera against betacoronavirus are provided. More specifically, in accordance with the present disclosure, horse antisera against betacoronavirus, polyclonal antibodies obtained from the serum, equine-derived monoclonal antibodies, human chimeric antibodies, and humanized antibodies, as well as mixtures of multiple monoclonal antibodies, are provided.
　本開示によれば、例えば以下の発明が提供され得る。
（１）ベータコロナウイルス（好ましくは、ＳＡＲＳ関連コロナウイルス、例えば、ＳＡＲＳ－ＣｏＶ－２）のスパイクタンパク質（Ｓタンパク質）の受容体結合ドメイン（ＲＢＤ領域）を含む免疫原性組成物に対するウマの抗血清であって、好ましくは、Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、抗血清。
（２）免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、上記（１）に記載の抗血清。
（３）免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、上記（２）に記載の抗血清。
（４）ウマが、サラブレッド個体である、上記（１）～（３）のいずれかに記載の抗血清。
（５）ウマが、サラブレッド個体である、上記（３）に記載の抗血清。
（６）上記（１）～（５）のいずれかに記載の抗血清から精製されたポリクローナル抗体を含む組成物。
（７）複数の異なるヒト化抗体、例えば、ヒト化バーチャルポリクローナル抗体を含む組成物であって、
　複数の異なるモノクローナル抗体を含み、
　各モノクローナル抗体は、ベータコロナウイルス（好ましくは、ＳＡＲＳ関連コロナウイルス、例えば、ＳＡＲＳ－ＣｏＶ－２）のスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域に対するウマ抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含むか、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含み、
　各モノクローナル抗体の残りの部分は、ヒト抗体に由来し、
　各モノクローナル抗体は、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、組成物。
（８）前記ウマが、Ｓタンパク質のＲＢＤ領域を含む免疫原性組成物により免疫されたウマである、上記（７）に記載の組成物。
（９）前記免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、上記（８）に記載の組成物。
（１０）免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、上記（９）に記載の組成物。
（１１）ウマが、サラブレッド個体である、上記（７）～（１０）のいずれかに記載の組成物。
（１２）ウマが、サラブレッド個体である、上記（１０）に記載の組成物。
（１３）上記（７）～（１２）のいずれかに記載の組成物の調製方法であって、
　ウマをベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫し、ウマの体内に前記ＲＢＤ領域に対する抗体を産生させることと、
　前記ウマから取得された複数の細胞が産生する抗体それぞれをコードする複数ｍＲＮＡまたはｃＤＮＡそれぞれの重鎖可変領域および軽鎖可変領域の配列を決定することと、ここで、前記細胞は、脾臓細胞、Ｂ細胞および形質細胞からなる群から選択される細胞であり、
　決定されたヌクレオチド配列をその配列同一性に基づいて複数のクラスタにクラスタリングすることと、
　取得された複数のクラスタのうち、含まれるヌクレオチド配列数が多いクラスタの上位１０位から少なくとも２つのクラスタを選択することと、
　選択されたクラスタそれぞれに含まれる重鎖可変領域および軽鎖可変領域を含むヒトキメラ抗体をコードするヌクレオチド配列か、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含むヒト化抗体をコードするヌクレオチド配列を得ることと、
　得られたヌクレオチド配列それぞれを有する複数の抗体産生細胞から、前記ヒトキメラ抗体またはヒト化抗体を産生させ、前記ヒトキメラ抗体またはヒト化抗体を含む複数の抗体の混合物を得ることと、
を含む、方法。
（１４）上記（７）～（１２）のいずれかに記載の組成物の調製方法であって、
　異なるモノクローナル抗体をそれぞれ産生する複数の抗体産生細胞からモノクローナル抗体を産生させ、産生された複数のモノクローナル抗体を含む組成物を得ることを含み、
　組成物に含まれる各モノクローナル抗体は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫され、体内にＲＢＤ領域に対する抗体群を有するウマにおいて検出される抗体群から選択される抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含むか、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含み、
　各モノクローナル抗体の残りの部分は、ヒト抗体に由来し、
　各モノクローナル抗体は、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、方法。
（１５）ウマが、サラブレッド個体である、上記（１３）または（１４）に記載の方法。
（１６）免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、上記（１３）～（１５）のいずれかに記載の方法。
（１７）免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、上記（１６）に記載の方法。
（１８）免疫原性組成物であって、
　ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含み、
　前記ＲＢＤ領域は、二量体を形成しており、かつ、二量体の量が、ＲＢＤ領域の単量体の量よりも多い、免疫原性組成物。
（１９）ウマを免疫することに用いるための、上記（１８）に記載の免疫原性組成物。
（２０）ウマを免疫する方法であって、
　上記（１８）に記載の免疫原性組成物をウマに投与することを含み、これにより、ウマにＲＢＤ領域に対する抗体を産生させる、方法。 According to the present disclosure, for example, the following inventions can be provided.
(1) An equine antiserum against an immunogenic composition comprising the receptor binding domain (RBD region) of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), preferably capable of neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(2) The antiserum described in (1) above, wherein the immunogenic composition comprises a dimerized RBD region.
(3) The antiserum according to (2) above, wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions.
(4) The antiserum according to any one of (1) to (3) above, wherein the horse is a thoroughbred.
(5) The antiserum according to (3) above, wherein the horse is a thoroughbred.
(6) A composition comprising a polyclonal antibody purified from the antiserum according to any one of (1) to (5) above.
(7) A composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies,
Contains a plurality of different monoclonal antibodies,
Each monoclonal antibody comprises a portion of the structure of a horse antibody directed against the RBD region of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), said portion of the structure of said antibody comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
The remainder of each monoclonal antibody is derived from a human antibody,
A composition, wherein each monoclonal antibody is capable of binding to said S protein and neutralizing the binding of said S protein to angiotensin-converting enzyme 2 (ACE2).
(8) The composition according to (7) above, wherein the horse is a horse that has been immunized with an immunogenic composition comprising the RBD region of the S protein.
(9) The composition according to (8) above, wherein the immunogenic composition comprises a dimerized RBD region.
(10) The composition according to (9) above, wherein the immunogenic composition contains more dimerized RBD regions than monomers.
(11) The composition according to any one of (7) to (10) above, wherein the horse is a thoroughbred.
(12) The composition according to (10) above, wherein the horse is a thoroughbred.
(13) A method for preparing the composition according to any one of (7) to (12) above, comprising the steps of:
Immunizing a horse with an immunogenic composition containing the RBD region of a spike protein (S protein) of a beta coronavirus to produce antibodies against the RBD region in the horse's body;
determining sequences of heavy and light chain variable regions of each of a plurality of mRNAs or cDNAs encoding antibodies produced by a plurality of cells obtained from the horse, wherein the cells are selected from the group consisting of spleen cells, B cells, and plasma cells;
clustering the determined nucleotide sequences into a plurality of clusters based on their sequence identity;
selecting at least two clusters from the top 10 clusters containing the largest number of nucleotide sequences from among the obtained clusters;
obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region comprised in each of the selected clusters, or a nucleotide sequence encoding a humanized antibody comprising three complementarity determining regions (CDRs) of a heavy chain variable region and three CDRs of a light chain variable region;
producing the human chimeric or humanized antibodies from a plurality of antibody-producing cells each having the obtained nucleotide sequence, thereby obtaining a mixture of a plurality of antibodies containing the human chimeric or humanized antibodies;
A method comprising:
(14) A method for preparing the composition according to any one of (7) to (12) above, comprising the steps of:
The method includes producing monoclonal antibodies from a plurality of antibody-producing cells each producing a different monoclonal antibody, and obtaining a composition containing the produced plurality of monoclonal antibodies;
Each monoclonal antibody contained in the composition comprises a portion of an antibody structure selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, the portion of the antibody structure comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
The remainder of each monoclonal antibody is derived from a human antibody,
A method, wherein each monoclonal antibody is capable of neutralizing the binding of said S protein to angiotensin-converting enzyme 2 (ACE2).
(15) The method according to (13) or (14) above, wherein the horse is a thoroughbred.
(16) The method according to any one of (13) to (15) above, wherein the immunogenic composition comprises a dimerized RBD region.
(17) The method according to (16) above, wherein the immunogenic composition contains more dimerized RBD regions than monomers.
(18) An immunogenic composition comprising:
It contains the RBD region of the spike protein (S protein) of a betacoronavirus,
An immunogenic composition, wherein the RBD region forms a dimer and the amount of the dimer is greater than the amount of the monomer of the RBD region.
(19) The immunogenic composition according to (18) above, for use in immunizing a horse.
(20) A method for immunizing a horse, comprising the steps of:
A method comprising administering to a horse the immunogenic composition described in (18) above, thereby causing the horse to produce antibodies against the RBD region.
（１０１）ウマがサラブレッド個体である、上記（１）に記載の抗血清。
（１０２）免疫原性組成物が、二量体を形成したＲＢＤ領域を含み、かつ、ウマがサラブレッド個体である、上記（１）に記載の抗血清。
（１０３）免疫原性組成物が、二量体を形成したＲＢＤ領域を含み、単量体に対して、二量体を形成したＲＢＤ領域をより多く含み、かつ、ウマがサラブレッド個体である、上記（１）に記載の抗血清。
（１０４）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（１）に記載の抗血清。
（１０５）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（１０１）に記載の抗血清。
（１０６）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（１０２）に記載の抗血清。
（１０７）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（１０３）に記載の抗血清。
（１０８）ウマが、５００ｋｇから７００ｋｇの体重を有する、上記（１）に記載の抗血清。
（１０９）ウマが、５００ｋｇから７００ｋｇの体重を有する、上記（１０１）に記載の抗血清。
（１１０）ウマが、５００ｋｇから７００ｋｇの体重を有する、上記（１０２）に記載の抗血清。
（１１１）ウマが、５００ｋｇから７００ｋｇの体重を有する、上記（１０３）に記載の抗血清。
（１１２）上記いずれかの抗血清から精製されたポリクローナル抗体を含む組成物。
（１１３）上記いずれかの抗血清から精製されたポリクローナル抗体を含む医薬組成物。 (101) The antiserum described in (1) above, wherein the horse is a thoroughbred.
(102) The antiserum described in (1) above, wherein the immunogenic composition comprises a dimerized RBD region and the horse is a thoroughbred.
(103) The antiserum described in (1) above, wherein the immunogenic composition contains a dimerized RBD region and contains more dimerized RBD region than monomeric RBD region, and the horse is a thoroughbred.
(104) The antiserum described in (1) above, wherein the horse has a body weight of 400 kg to 800 kg.
(105) The antiserum described in (101) above, wherein the horse has a body weight of 400 kg to 800 kg.
(106) The antiserum described in (102) above, wherein the horse has a body weight of 400 kg to 800 kg.
(107) The antiserum described in (103) above, wherein the horse has a body weight of 400 kg to 800 kg.
(108) The antiserum described in (1) above, wherein the horse has a body weight of 500 kg to 700 kg.
(109) The antiserum described in (101) above, wherein the horse has a body weight of 500 kg to 700 kg.
(110) The antiserum described in (102) above, wherein the horse has a body weight of 500 kg to 700 kg.
(111) The antiserum described in (103) above, wherein the horse has a body weight of 500 kg to 700 kg.
(112) A composition comprising a polyclonal antibody purified from any of the above antisera.
(113) A pharmaceutical composition comprising a polyclonal antibody purified from any of the above antisera.
（２０１）複数の異なるヒト化抗体、例えば、ヒト化バーチャルポリクローナル抗体を含む組成物または医薬組成物。
（２０２）複数の異なるヒト化抗体、例えば、ヒト化バーチャルポリクローナル抗体を含む組成物または医薬組成物であって、
　複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）を含み、
　各モノクローナル抗体は、ベータコロナウイルス（好ましくは、ＳＡＲＳ関連コロナウイルス、例えば、ＳＡＲＳ－ＣｏＶ－２）のスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域に対するウマ抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含む（ｃｏｍｐｒｉｓｅ）か、もしくは、からなり（ｃｏｎｓｉｓｔ　ｏｆ）、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含むか、もしくは、からなり、
　各モノクローナル抗体の残りの部分は、ヒト抗体に由来する、組成物または医薬組成物。
（２０３）前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（２０２）に記載の組成物または医薬組成物。
（２０４）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）の少なくとも１つが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（２０２）に記載の組成物または医薬組成物。
（２０５）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）の少なくとも１つが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（２０３）に記載の組成物または医薬組成物。
（２０６）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）のそれぞれが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（２０２）に記載の組成物または医薬組成物。
（２０７）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）のそれぞれが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（２０３）に記載の組成物または医薬組成物。
（２０８）ウマが、サラブレッド個体である、上記（２０１）～（２０７）のいずれかに記載の組成物または医薬組成物。
（２０９）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（２０１）～（２０７）のいずれかに記載の組成物または医薬組成物。
（２１０）ウマが、４００ｋｇから８００ｋｇの体重を有するサラブレッド個体である、上記（２０１）～（２０７）のいずれかに記載の組成物または医薬組成物。 (201) A composition or pharmaceutical composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies.
(202) A composition or pharmaceutical composition comprising a plurality of different humanized antibodies, e.g., humanized virtual polyclonal antibodies,
comprising a plurality of different antibodies (preferably a plurality of different monoclonal antibodies);
Each monoclonal antibody comprises a portion of the structure of a horse antibody directed against the RBD region of the spike protein (S protein) of a betacoronavirus (preferably a SARS-associated coronavirus, e.g., SARS-CoV-2), said portion of the structure of said antibody comprising or consisting of a heavy chain variable region and a light chain variable region, or comprising or consisting of three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
The composition or pharmaceutical composition, wherein the remainder of each monoclonal antibody is derived from a human antibody.
(203) The composition or pharmaceutical composition according to (202) above, which is capable of neutralizing the binding of the S protein to angiotensin converting enzyme 2 (ACE2).
(204) The composition or pharmaceutical composition described in (202) above, wherein at least one of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(205) The composition or pharmaceutical composition described in (203) above, wherein at least one of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(206) The composition or pharmaceutical composition described in (202) above, wherein each of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(207) The composition or pharmaceutical composition described in (203) above, wherein each of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding between the S protein and angiotensin-converting enzyme 2 (ACE2).
(208) The composition or pharmaceutical composition according to any one of (201) to (207) above, wherein the horse is a thoroughbred.
(209) The composition or pharmaceutical composition according to any one of (201) to (207) above, wherein the horse has a body weight of 400 kg to 800 kg.
(210) The composition or pharmaceutical composition according to any one of (201) to (207) above, wherein the horse is a thoroughbred individual having a body weight of 400 kg to 800 kg.
（３０１）上記（２０１）～（２１０）のいずれかに記載の組成物の調製方法であって、
　異なるモノクローナル抗体をそれぞれ産生する複数の抗体産生細胞からモノクローナル抗体を産生させ、産生された複数のモノクローナル抗体を含む組成物を得ることを含み、
　組成物に含まれる各モノクローナル抗体は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫され、体内にＲＢＤ領域に対する抗体群を有するウマにおいて検出される抗体群から選択される抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含む（ｃｏｍｐｒｉｓｅ）か、もしくは、からなり（ｃｏｎｓｉｓｔ　ｏｆ）、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含むか、もしくは、からなり、
　各モノクローナル抗体の残りの部分は、ヒト抗体に由来する、方法。
（３０２）前記組成物が、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（３０１）に記載の方法。
（３０３）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）の少なくとも１つが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（３０１）に記載の組成物または医薬組成物。
（３０４）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）の少なくとも１つが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（３０２）に記載の組成物または医薬組成物。
（３０５）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）のそれぞれが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（３０１）に記載の組成物または医薬組成物。
（３０６）前記複数の異なる抗体（好ましくは複数の異なるモノクローナル抗体）のそれぞれが、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、上記（３０２）に記載の組成物または医薬組成物。
（３０７）ウマが、サラブレッド個体である、上記（３０１）～（３０６）のいずれかに記載の組成物または医薬組成物。
（３０８）ウマが、４００ｋｇから８００ｋｇの体重を有する、上記（３０１）～（３０６）のいずれかに記載の組成物または医薬組成物。
（３０９）ウマが、４００ｋｇから８００ｋｇの体重を有するサラブレッド個体である、上記（３０１）～（３０６）のいずれかに記載の組成物または医薬組成物。 (301) A method for preparing the composition according to any one of (201) to (210) above, comprising the steps of:
The method includes producing monoclonal antibodies from a plurality of antibody-producing cells each producing a different monoclonal antibody, and obtaining a composition containing the produced plurality of monoclonal antibodies;
Each monoclonal antibody contained in the composition comprises a portion of the structure of an antibody selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, said portion of the structure of the antibody comprising or consisting of a heavy chain variable region and a light chain variable region, or comprising or consisting of three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
The remainder of each monoclonal antibody is derived from a human antibody.
(302) The method according to (301) above, wherein the composition is capable of neutralizing the binding between the S protein and angiotensin converting enzyme 2 (ACE2).
(303) The composition or pharmaceutical composition described in (301) above, wherein at least one of the plurality of different antibodies (preferably a plurality of different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(304) The composition or pharmaceutical composition described in (302) above, wherein at least one of the plurality of different antibodies (preferably a plurality of different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(305) The composition or pharmaceutical composition described in (301) above, wherein each of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding between the S protein and angiotensin-converting enzyme 2 (ACE2).
(306) The composition or pharmaceutical composition described in (302) above, wherein each of the multiple different antibodies (preferably multiple different monoclonal antibodies) is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
(307) The composition or pharmaceutical composition according to any one of (301) to (306) above, wherein the horse is a thoroughbred.
(308) The composition or pharmaceutical composition according to any one of (301) to (306) above, wherein the horse has a body weight of 400 kg to 800 kg.
(309) The composition or pharmaceutical composition according to any one of (301) to (306) above, wherein the horse is a thoroughbred individual having a body weight of 400 kg to 800 kg.
（４０１）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ関連コロナウイルスである、上記いずれかに記載の発明。
（４０２）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２である、上記いずれかに記載の発明。
（４０３）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２の野生型である、上記いずれかに記載の発明。
（４０４）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のα株である、上記いずれかに記載の発明。
（４０５）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のβ株である、上記いずれかに記載の発明。
（４０６）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のγ株である、上記いずれかに記載の発明。
（４０７）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のδ株である、上記いずれかに記載の発明。
（４０８）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のο株である、上記いずれかに記載の発明。
（４０９）免疫原性組成物のＲＢＤ領域が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２の変種（例えば、天然に生じた変種）である、上記いずれかに記載の発明。 (401) Any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is a SARS-associated coronavirus.
(402) Any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is SARS-CoV-2.
(403) Any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is wild-type SARS-CoV-2.
(404) Any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is an alpha strain of SARS-CoV-2.
(405) Any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is a β strain of SARS-CoV-2.
(406) Any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is the gamma strain of SARS-CoV-2.
(407) Any of the above-mentioned inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is a δ strain of SARS-CoV-2.
(408) Any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is the O strain of SARS-CoV-2.
(409) Any of the above-mentioned inventions, wherein the betacoronavirus from which the RBD region of the immunogenic composition is derived is a variant of SARS-CoV-2 (e.g., a naturally occurring variant).
（５０１）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ関連コロナウイルスである、上記いずれかに記載の発明。
（５０２）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２である、上記いずれかに記載の発明。
（５０３）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２の野生型である、上記いずれかに記載の発明。
（５０４）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のα株である、上記いずれかに記載の発明。
（５０５）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のβ株である、上記いずれかに記載の発明。
（５０６）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のγ株である、上記いずれかに記載の発明。
（５０７）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のδ株である、上記いずれかに記載の発明。
（５０８）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２のο株である、上記いずれかに記載の発明。
（５０９）結合対象であるＳタンパク質が由来するベータコロナウイルスが、ＳＡＲＳ－ＣｏＶ－２の変種（例えば、天然に生じた変種）である、上記いずれかに記載の発明。 (501) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is a SARS-associated coronavirus.
(502) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is SARS-CoV-2.
(503) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is wild-type SARS-CoV-2.
(504) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is an alpha strain of SARS-CoV-2.
(505) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is a β strain of SARS-CoV-2.
(506) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the gamma strain of SARS-CoV-2.
(507) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the δ strain of SARS-CoV-2.
(508) Any of the above-mentioned inventions, wherein the beta coronavirus from which the S protein to be bound is derived is the O strain of SARS-CoV-2.
(509) Any of the above-mentioned inventions, wherein the betacoronavirus from which the S protein to be bound is derived is a variant of SARS-CoV-2 (e.g., a naturally occurring variant).
（６０１）複数のモノクローナル抗体が、４種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０２）複数のモノクローナル抗体が、５種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０３）複数のモノクローナル抗体が、６種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０４）複数のモノクローナル抗体が、７種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０５）複数のモノクローナル抗体が、８種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０６）複数のモノクローナル抗体が、９種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０７）複数のモノクローナル抗体が、１０種類以上のモノクローナル抗体を含む、上記いずれかに記載の発明。
（６０８）複数のモノクローナル抗体の少なくとも２つが、ＲＢＤ領域への結合に関して有意な競合を示さない、上記（６０１）～（６０７）のいずれかに記載の発明。 (601) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include four or more types of monoclonal antibodies.
(602) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include five or more types of monoclonal antibodies.
(603) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include six or more types of monoclonal antibodies.
(604) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include seven or more types of monoclonal antibodies.
(605) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include eight or more types of monoclonal antibodies.
(606) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include nine or more types of monoclonal antibodies.
(607) Any of the above-mentioned inventions, wherein the multiple monoclonal antibodies include 10 or more types of monoclonal antibodies.
(608) The invention described in any one of (601) to (607) above, wherein at least two of the monoclonal antibodies do not show significant competition for binding to the RBD region.
図１は、免疫原産生細胞の遺伝子導入後の生細胞密度（ＶＣＤ）および細胞生存率の変化を示す。FIG. 1 shows the changes in viable cell density (VCD) and cell viability after gene transfer of immunogen-producing cells. 図２は、得られた免疫原性組成物のウェスタンブロットの結果を示す。各レーンの内容は、表２に示される通りである。Figure 2 shows the results of Western blot of the obtained immunogenic composition. The contents of each lane are as shown in Table 2. 図３は、遺伝子導入４０時間後に回収された免疫原を含む免疫原性組成物（野生型（ＷＴ）またはＷＴと変異型δ株との混合（ＷＴ＆Ｄｅｌｔａ））により免疫されたウマの血清の中和抗体価（５０％ＩＣ）を示す。FIG. 3 shows neutralizing antibody titers (50% IC) in the serum of horses immunized with an immunogenic composition (wild-type (WT) or a mixture of WT and mutant δ strains (WT&Delta)) containing the immunogen recovered 40 hours after gene transfer. 図４は、遺伝子導入４０時間後に回収された免疫原を含む免疫原性組成物（ＷＴまたはＷＴ＆Ｄｅｌｔａ）により免疫されたウマの血清の抗原特異的抗体量を示す。図４のパネルＡは、免疫原（ＷＴ）で免疫されたウマの抗血清中の免疫原（ＷＴ）に特異的な抗体の量の経時的推移を示し、パネルＢは、免疫原（ＷＴ＆Ｄｅｌｔａ）で免疫されたウマの抗血清中の免疫原（ＷＴ）に特異的な抗体の量の経時的推移を示し、パネルＣは、免疫原（ＷＴ）で免疫されたウマの抗血清中の免疫原（Ｄｅｌｔａ）に特異的な抗体の量の経時的推移を示し、パネルＤは、免疫原（ＷＴ＆Ｄｅｌｔａ）で免疫されたウマの抗血清中の免疫原（Ｄｅｌｔａ）に特異的な抗体の量の経時的推移を示す。4 shows the amount of antigen-specific antibodies in the serum of horses immunized with immunogenic compositions (WT or WT&Delta) containing the immunogens collected 40 hours after gene transfer. Panel A of FIG. 4 shows the time course of the amount of antibodies specific to the immunogen (WT) in the antisera of horses immunized with the immunogen (WT), panel B shows the time course of the amount of antibodies specific to the immunogen (WT) in the antisera of horses immunized with the immunogen (WT&Delta), panel C shows the time course of the amount of antibodies specific to the immunogen (Delta) in the antisera of horses immunized with the immunogen (WT), and panel D shows the time course of the amount of antibodies specific to the immunogen (Delta) in the antisera of horses immunized with the immunogen (WT&Delta). 図５は、各種ＳＡＲＳ－ＣｏＶ－２野生型（ＷＴ）及び変異体（α株、β株、γ株、δ株、およびο株）に対するウマ抗血清の中和抗体価（５０％ＩＣ）を示す。FIG. 5 shows the neutralizing antibody titers (50% IC) of horse antisera against various SARS-CoV-2 wild-type (WT) and mutants (α, β, γ, δ, and ο strains). 図６は、免疫原のＣＢＢ染色およびウェスタンブロット（ＷＢ）結果を示す。FIG. 6 shows the CBB staining and Western blot (WB) results of the immunogens. 図７は、免疫原のウェスタンブロット結果に基づくデンシトメトリ解析における各領域（単量体：ＡおよびＢ、二量体：Ｃ、三量体Ｄ、および多量体：Ｅ）の設定方法を示す。FIG. 7 shows a method for setting each region (monomer: A and B, dimer: C, trimer: D, and multimer: E) in densitometry analysis based on the results of Western blot of immunogens.
発明を実施する形態MODE FOR CARRYING OUT THE DISCLOSURE
　本明細書では、「対象」とは、脊椎動物であり、例えば、ヒトを含む哺乳類、例えば、コロナウイルス（例えば、ＳＡＲＳ－ＣｏＶ－２）が感染する哺乳類（ネコ、フェレット、コウモリ、およびセンザンコウ）であり得る。対象は、コロナウイルスに感染した対象であり得、コロナウイルスに感染した無症状病原体保有者であり得、ＳＡＲＳ－ＣｏＶ－２に感染し、ＣＯＶＩＤ－１９を発症した対象であり得る。対象は、コロナウイルスに感染した可能性（リスク）を有する対象であり得、または、コロナウイルスに感染する可能性（リスク）を有する対象であり得る。対象は、小児（例えば、幼児（生後１～６年）、学童（生後６～１２年）、青年（生後１２年～）、成人（生後２０年～）であり得る。成人は、３０歳以上、４０歳以上、５０歳以上、６０歳以上、または７０歳以上の成人であり得る。本明細書では、コロナウイルスは、好ましくは、ベータコロナウイルスであり、より好ましくは、ＳＡＲＳ－ＣｏＶ－２である。 As used herein, a "subject" refers to a vertebrate, e.g., a mammal, including a human, e.g., a mammal (cat, ferret, bat, and pangolin) that is infected with a coronavirus (e.g., SARS-CoV-2). The subject may be a subject infected with a coronavirus, an asymptomatic carrier of a coronavirus, or a subject infected with SARS-CoV-2 and developing COVID-19. The subject may be a subject at risk of being infected with a coronavirus, or a subject at risk of being infected with a coronavirus. The subject may be a child (e.g., infant (1-6 years), school-age child (6-12 years), adolescent (12 years or older), or adult (20 years or older). An adult may be an adult aged 30 years or older, 40 years or older, 50 years or older, 60 years or older, or 70 years or older. As used herein, the coronavirus is preferably a betacoronavirus, and more preferably SARS-CoV-2.
　本明細書では、「コロナウイルス」は、ニドウイルス目コロナウイルス科オルトコロナウイルス亜科のウイルスであり、一本鎖プラス鎖ＲＮＡウイルスである。コロナウイルスは、ウイルス表面にスパイクタンパク質の突起（Ｓタンパク質）を有し、外観が太陽のコロナに似ていることからコロナウイルスと命名された。ヒトでは、風邪を含む呼吸器感染症を引き起こす。オルトコロナウイルス亜科のコロナウイルスは、アルファコロナウイルス、ベータコロナウイルス、ガンマコロナイウルス、およびデルタコロナウイルスに大別される。ＳＡＲＳ関連コロナウイルスは、ベータコロナウイルスに分類されている。ＳＡＲＳ関連コロナウイルスには、ＳＡＲＳコロナイウルス（ＳＡＲＳ－ＣｏＶ）およびＳＡＲＳコロナイウルス２（ＳＡＲＳ－ＣｏＶ－２）が含まれる。ＳＡＲＳ－ＣｏＶ－２は、２０１９年末から新型コロナウイルス感染症（ＣＯＶＩＤ－１９）のパンデミックを引き起こしている。ＳＡＲＳ関連コロナウイルスは、Ｓタンパク質により宿主細胞のＡＣＥ２受容体に結合して宿主細胞に感染する。ＡＣＥ２受容体を利用して細胞に感染する点で、ＳＡＲＳ関連コロナウイルスは共通した感染機構を有する。ＳＡＲＳ－ＣｏＶ－２のＳタンパク質は、内部に感染力と病原性を高めるフーリン切断部位が存在する（Ａｎｄｅｒｓｅｎ　ｅｔ　ａｌ．，　Ｎａｔｕｒｅ　Ｍｅｄｉｃｉｎｅ，　２６，　４５０－４５２，　２０２０）。 As used herein, "coronavirus" refers to a virus of the Orthocoronavirus subfamily of the Coronaviridae family of the Nidovirales order, and is a single-stranded positive-strand RNA virus. Coronaviruses are named coronaviruses because they have spike protein protrusions (S proteins) on the surface of the virus and have an appearance similar to the corona of the sun. In humans, they cause respiratory infections, including the common cold. Coronaviruses of the Orthocoronavirus subfamily are broadly classified into alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. SARS-associated coronaviruses are classified as betacoronaviruses. SARS-associated coronaviruses include SARS-CoV and SARS-CoV-2. SARS-CoV-2 has been causing the COVID-19 pandemic since the end of 2019. SARS-associated coronaviruses infect host cells by binding to the ACE2 receptor of the host cell via the S protein. SARS-associated coronaviruses share a common infection mechanism in that they use the ACE2 receptor to infect cells. The S protein of SARS-CoV-2 contains a furin cleavage site inside that increases infectivity and pathogenicity (Andersen et al., Nature Medicine, 26, 450-452, 2020).
　本明細書では、「ＳＡＲＳ－ＣｏＶ－２」は、２０２０年に引き起こされたパンデミックの原因となったコロナウイルスである。２０２０年１月７日に世界保健機関（ＷＨＯ）は、このウイルスを２０１９－ｎＣｏＶと暫定的に命名した。また、同年２月１１日に国際ウイルス分類委員会（ＩＣＴＶ）はこのウイルスをＳＡＲＳ－ＣｏＶ－２と正式に命名した。コロナウイルスは、一般的な風邪から重症急性呼吸器症候群（ＳＡＲＳ）や中東呼吸器症候群（ＭＥＲＳ）などの重篤な呼吸器疾患を引き起こし得る。ＷＨＯは、この新型コロナウイルスによる疾患をＣＯＶＩＤ－１９と命名している。国際ウイルス分類委員会は、ＳＡＲＳ－ＣｏＶ－２を、ベータコロナウイルス属に属し、ＳＡＲＳ－ＣｏＶと同じ種（又はその姉妹系統）であるとしている。ＳＡＲＳ－ＣｏＶ－２の野生株（または武漢株）の完全ゲノム配列は、Ｇｅｎｅ　Ｂａｎｋ登録番号：ＭＮ９０８９４７．３として米国国立生物工学情報センター（ＮＣＢＩ）に登録されている。ウイルス粒子（ビリオン）は、５０～２００ｎｍ程度の粒径を有し、一般的なコロナウイルスと同じようにスパイクタンパク質、ヌクレオキャプシドタンパク質、膜タンパク質、およびエンベロープタンパク質とウイルスゲノムＲＮＡを含む。ヌクレオキャプシドタンパク質がＲＮＡと複合体を形成し、その周囲に脂質と結合したスパイクタンパク質、膜タンパク質、およびエンベロープタンパク質が取り囲んで、ビリオンのエンベロープが形成される。エンベロープの最外表面に位置するスパイクタンパク質は、細胞表面のＡＣＥ２受容体に結合して細胞への感染を促進すると考えられている。ＳＡＲＳ－ＣｏＶ－２に感染しても疾患の症状が現れない者が存在し、これを無症状病原体保有者という。無症状病原体保有者は、その保有するウイルスを他者に感染させる可能性があることが指摘されている。ＳＡＲＳ－ＣｏＶ－２による感染によって、嗅覚および／または味覚が低下するまたは喪失することが指摘されている。ＳＡＲＳ－ＣｏＶ－２は、重症急性呼吸症候群を生じさせることがある。重症急性呼吸器症候群では、主症状として、４０℃程度の発熱、咳、息切れが報告されている。顕著な合併症は、肺炎である。ＳＡＲＳ－ＣｏＶ－２の感染の有無は、主にＰＣＲ検査によって判定されている。このＰＣＲ検査は、ＳＡＲＳ－ＣｏＶ－２特異的にバンドを増幅するか否かによって、体内にＳＡＲＳ－ＣｏＶ－２の遺伝子が存在するか否かを評価するものである。ＳＡＲＳ－ＣｏＶ－２に対する治療としては、ＳＡＲＳ－ＣｏＶ－２に対する抗ウイルス薬（例えば、レムデシビル）、ステロイド系抗炎症薬（例えば、デキサメタゾン）、および炎症性サイトカインの阻害剤（例えば、ＩＬ－６阻害剤、例えば、抗ＩＬ－６抗体、ＴＮＦ－α阻害剤、例えば、エタネルセプト）が挙げられる。 As used herein, "SARS-CoV-2" refers to the coronavirus that caused the 2020 pandemic. On January 7, 2020, the World Health Organization (WHO) provisionally named the virus 2019-nCoV. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) formally named the virus SARS-CoV-2. Coronaviruses can cause a range of respiratory illnesses from the common cold to severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The WHO has named the disease caused by this new coronavirus COVID-19. The International Committee on Taxonomy of Viruses considers SARS-CoV-2 to belong to the Betacoronavirus genus and to be the same species (or a sister lineage) as SARS-CoV. The complete genome sequence of the wild strain (or Wuhan strain) of SARS-CoV-2 has been registered with the National Center for Biotechnology Information (NCBI) under Gene Bank registration number: MN908947.3. The virus particle (virion) has a particle size of about 50 to 200 nm, and contains spike protein, nucleocapsid protein, membrane protein, envelope protein, and viral genome RNA, just like common coronaviruses. The nucleocapsid protein forms a complex with RNA, and the lipid-bound spike protein, membrane protein, and envelope protein surround it to form the virion envelope. The spike protein located on the outermost surface of the envelope is thought to bind to the ACE2 receptor on the cell surface and promote infection of the cell. There are people who do not show symptoms of the disease even if they are infected with SARS-CoV-2, and these are called asymptomatic pathogen carriers. It has been pointed out that asymptomatic pathogen carriers may infect others with the virus they carry. It has been noted that infection with SARS-CoV-2 can reduce or eliminate the sense of smell and/or taste. SARS-CoV-2 can cause severe acute respiratory syndrome. In severe acute respiratory syndrome, the main symptoms reported are fever of about 40°C, coughing, and shortness of breath. A notable complication is pneumonia. The presence or absence of SARS-CoV-2 infection is mainly determined by PCR testing. This PCR test evaluates the presence or absence of SARS-CoV-2 genes in the body by whether or not a band specific to SARS-CoV-2 is amplified. Treatments for SARS-CoV-2 include antiviral drugs against SARS-CoV-2 (e.g., remdesivir), steroid anti-inflammatory drugs (e.g., dexamethasone), and inhibitors of inflammatory cytokines (e.g., IL-6 inhibitors, e.g., anti-IL-6 antibodies, TNF-α inhibitors, e.g., etanercept).
　本明細書では、「スパイクタンパク質」は、ＧｅｎＢａｎｋ登録番号：ＭＮ９０８９４７．３として米国国立生物工学情報センター（ＮＣＢＩ）に登録されたＳＡＲＳ－ＣｏＶ－２ゲノムの２１５６３番目～２５３８４番目にコードされるタンパク質であり得、上記ＳＡＲＳ－ＣｏＶ－２のスパイクタンパク質は、ＧｅｎＢａｎｋ登録番号：ＱＨＤ４３４１６．１としてＮＣＢＩに登録されたアミノ酸配列を有する。スパイクタンパク質は、Ｓ１とＳ２を含み、Ｓ１は、上記アミノ酸配列の１３～５４１位に存在し、Ｓ２は、上記アミノ酸配列の５４３～１２０８位に存在する。Ｓ１はさらに、Ｎ末端ドメイン（ＮＴＤ）と受容体結合ドメイン（ＲＢＤ）を有し、ＮＴＤは、上記アミノ酸配列の１３～３０４位に存在し、ＲＢＤは、３１９～５４１位に存在する。ＲＢＤ領域は、ヒトアンジオテンシン転換酵素２（ＡＣＥ２）に結合することが知られる。ここで、野生型ＲＢＤ領域のアミノ酸配列の一例は配列番号１に示され、δ株のＲＢＤ領域のアミノ酸配列の一例は配列番号２に示される。Ｓ１とＳ２は、細胞内で切断され、別々のペプチドとして産生され、ウイルス粒子形成時に複合体を形成する。スパイクタンパク質は、Ｓタンパク質とも呼ばれる。スパイクタンパク質は、三量体を形成し、宿主細胞に発現するアンジオテンシン転換酵素２（ＡＣＥ２）に結合し、細胞に感染し得る。スパイクタンパク質としては、天然のウイルス（例えば、変異ウイルスを含む）が有するスパイクタンパク質（ＧｅｎＢａｎｋ登録番号：ＱＨＤ４３４１６．１としてＮＣＢＩに登録されたアミノ酸配列に対応するアミノ酸配列を有する）は、いずれも用いることができる。変異ウイルスのスパイクタンパク質のアミノ酸配列の例は以下の通りである。
　α株：上記野生株の配列に対してＨＶ６９－７０ｄｅｌ，　Ｙ１４４ｄｅｌ，　Ｎ５０１Ｙ，　Ａ５７０Ｄ，　Ｄ６１４Ｇ，　Ｐ６８１Ｈ，Ｔ７１６Ｉ，　Ｓ９８２Ａ，　およびＤ１１１８Ｈの変異を有する。
　β株：上記野生株の配列に対してＬ１８Ｆ，　Ｄ８０Ａ，　Ｄ２１５Ｇ，　ＬＡＬ２４２－２４４ｄｅｌ，　Ｒ２４６Ｉ，　Ｋ４１７Ｎ，　Ｅ４８４Ｋ，　Ｎ５０１Ｙ，　Ｄ６１４Ｇ，　およびＡ７０１Ｖの変異を有する。
　γ株：Ｌ１８Ｆ，　Ｔ２０Ｎ，　Ｐ２６Ｓ，　Ｄ１３８Ｙ，　Ｒ１９０Ｓ，　Ｋ４１７Ｔ，　Ｅ４８４Ｋ，　Ｎ５０１Ｙ，　Ｄ６１４Ｇ，　Ｈ６５５Ｙ，　Ｔ１０２７Ｉ，　およびＶ１１７６Ｆの変異を有する。
　δ株：上記野生株の配列に対してＴ１９Ｒ，　Ｇ１４２Ｄ，　ＥＦ１５６－１５７ｄｅｌ，　Ｒ１５８Ｇ，　Ｌ４５２Ｒ，　Ｔ４７８Ｋ，　Ｄ６１４Ｇ，　Ｐ６８１Ｒ，　およびＤ９５０Ｎの変異を有する。
　ο株：上記野生株の配列に対してＡ６７Ｖ，　ＨＶ６９－７０ｄｅｌ，　Ｔ９５Ｉ，　Ｇ１４２Ｄ，　ＶＹＹ１４３－１４５ｄｅｌ，　Ｎ２１１ｄｅｌ，　Ｌ２１２Ｉ，　ｉｎｓ２１４ＥＰＥ，　Ｇ３３９Ｄ，　Ｓ３７１Ｌ，　Ｓ３７３Ｐ，　Ｓ３７５Ｆ，　Ｋ４１７Ｎ，　Ｎ４４０Ｋ，　Ｇ４４６Ｓ，　Ｓ４７７Ｎ，　Ｔ４７８Ｋ，　Ｅ４８４Ａ，　Ｑ４９３Ｒ，　Ｇ４９６Ｓ，　Ｑ４９８Ｒ，　Ｎ５０１Ｙ，　Ｙ５０５Ｈ，　Ｔ５４７Ｋ，　Ｄ６１４Ｇ，　Ｈ６５５Ｙ，　Ｎ６７９Ｋ，　Ｐ６８１Ｈ，　Ｎ７６４Ｋ，　Ｄ７９６Ｙ，　Ｎ８５６Ｋ，　Ｑ９５４Ｈ，　Ｎ９６９Ｋ，　およびＬ９８１Ｆの変異を有する。 As used herein, the "spike protein" may be a protein encoded by positions 21563 to 25384 of the SARS-CoV-2 genome registered with the National Center for Biotechnology Information (NCBI) under GenBank Accession No. MN908947.3, and the spike protein of SARS-CoV-2 has an amino acid sequence registered with the NCBI under GenBank Accession No. QHD43416.1. The spike protein includes S1 and S2, where S1 is present at positions 13 to 541 of the amino acid sequence, and S2 is present at positions 543 to 1208 of the amino acid sequence. S1 further has an N-terminal domain (NTD) and a receptor binding domain (RBD), where NTD is present at positions 13 to 304 of the amino acid sequence, and RBD is present at positions 319 to 541. The RBD region is known to bind to human angiotensin converting enzyme 2 (ACE2). Here, an example of the amino acid sequence of the wild-type RBD region is shown in SEQ ID NO: 1, and an example of the amino acid sequence of the RBD region of the δ strain is shown in SEQ ID NO: 2. S1 and S2 are cleaved in the cell, produced as separate peptides, and form a complex during viral particle formation. The spike protein is also called the S protein. The spike protein forms a trimer, binds to angiotensin converting enzyme 2 (ACE2) expressed in the host cell, and can infect the cell. As the spike protein, any spike protein (having an amino acid sequence corresponding to the amino acid sequence registered with NCBI as GenBank registration number: QHD43416.1) possessed by a natural virus (including a mutant virus) can be used. An example of the amino acid sequence of the spike protein of a mutant virus is as follows.
α strain: has the following mutations compared to the wild-type strain sequence: HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.
β strain: has the following mutations compared to the wild-type strain sequence: L18F, D80A, D215G, LAL242-244del, R246I, K417N, E484K, N501Y, D614G, and A701V.
γ strain: has the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F.
Strain δ: This strain has the following mutations compared to the sequence of the wild-type strain: T19R, G142D, EF156-157del, R158G, L452R, T478K, D614G, P681R, and D950N.
Strain O: A67V, HV69-70del, T95I, G142D, VYY143-145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, It has the mutations N969K and L981F.
　本明細書では、「ペプチド」は、アミノ酸のポリマーを意味する。ポリマーは、通常分岐を有しない。「部分ペプチド」とは、特定のペプチドの一部を意味する。ペプチドおよび部分ペプチドは、当該ペプチドをコードする核酸から製造され得る。ペプチドおよび部分ペプチドはまた、化学合成され得る。ペプチドおよび部分ペプチドはまた、単離、濃縮、または精製されている。単離とは、ペプチドおよび部分ペプチドを少なくとも他の成分から分離することを意味し、精製とは、ペプチドおよび部分ペプチドを少なくとも選択的に分離することを意味する。濃縮は、ペプチドおよび部分ペプチドの濃度が高まっていることを意味する。 As used herein, "peptide" refers to a polymer of amino acids. A polymer usually has no branching. "Partial peptide" refers to a portion of a particular peptide. Peptides and partial peptides can be produced from nucleic acids encoding the peptide. Peptides and partial peptides can also be chemically synthesized. Peptides and partial peptides can also be isolated, enriched, or purified. Isolated means that the peptides and partial peptides are at least separated from other components, and purified means that the peptides and partial peptides are at least selectively separated. Enriched means that the peptides and partial peptides are in an increased concentration.
　本明細書では、「組成物」とは、１以上の成分の混合物である。組成物は、例えば、部分ペプチドと水性溶媒（例えば、水）を含み得る。組成物は、薬学的に許容可能な賦形剤をさらに含んでいてもよい。本明細書では、免疫原性組成物とは、対象に投与することによって当該対象の体内で免疫反応を惹起することができる組成物である。免疫原性組成物は、対象において免疫反応を誘発することに用いられ得る。免疫原性組成物は、対象において免疫反応を惹起することができるので、ワクチンとして用いることができる。例えば、本発明の免疫原性組成物は、ＳＡＲＳ関連コロナウイルス（例えば、ＳＡＲＳ－ＣｏＶ－２）に対する免疫反応を誘発することに用いられ得、または、ＳＡＲＳ関連コロナウイルス（例えば、ＳＡＲＳ－ＣｏＶ－２）に対するワクチンとして、もしくは治療薬として用いられ得る。 As used herein, a "composition" is a mixture of one or more components. The composition may include, for example, a partial peptide and an aqueous solvent (e.g., water). The composition may further include a pharma- ceutically acceptable excipient. As used herein, an immunogenic composition is a composition that can induce an immune response in a subject by administering it to the subject. The immunogenic composition may be used to induce an immune response in a subject. Since the immunogenic composition can induce an immune response in a subject, it may be used as a vaccine. For example, the immunogenic composition of the present invention may be used to induce an immune response against a SARS-associated coronavirus (e.g., SARS-CoV-2), or may be used as a vaccine or a therapeutic agent against a SARS-associated coronavirus (e.g., SARS-CoV-2).
　本明細書では、「処置」は、予防的処置および治療的処置を含む。治療的処置は、感染したウイルスに対してなされ得、予防的処置は、将来の感染を予防するため、または将来の感染によりコロナウイルス感染症（例えば、ＣＯＶＩＤ－１９）が発症するのを遅延させ、もしくは発症したコロナウイルス感染症（例えば、ＣＯＶＩＤ－１９）の症状を低減するためになされ得る。治療的処置は、症状を有する患者または無症状病原体保有者に対してなされ得る。予防的処置は、無感染者に対してなされ得る。 As used herein, "treatment" includes prophylactic and therapeutic treatment. Therapeutic treatment may be given to an infected virus, and prophylactic treatment may be given to prevent future infection or to delay future infection from causing a coronavirus infection (e.g., COVID-19) or to reduce symptoms of an established coronavirus infection (e.g., COVID-19). Therapeutic treatment may be given to symptomatic patients or asymptomatic pathogen carriers. Prophylactic treatment may be given to uninfected individuals.
　本明細書中、「外来性」又は「外来的」とは、遺伝子又は核酸を遺伝子操作又は遺伝子導入等の操作により目的の細胞内に人為的に導入すること、及び目的の細胞内に人為的に導入された遺伝子又は核酸、並びにそれら発現タンパク質を指す用語として交換可能に使用される。外来性の遺伝子は、遺伝子の発現を駆動するプロモーター配列に作動可能に連結されうる。本明細書中、「内在性」又は「内在的」とは、細胞に元々備わったものであることを意味する。 As used herein, "exogenous" or "exogenous" are used interchangeably to refer to the artificial introduction of a gene or nucleic acid into a target cell by genetic engineering or gene transfer or other manipulation, and to the gene or nucleic acid artificially introduced into a target cell, as well as the expressed protein thereof. An exogenous gene may be operably linked to a promoter sequence that drives expression of the gene. As used herein, "endogenous" or "endogenous" means that it is inherently present in the cell.
　本明細書中、「由来」とは、細胞または抗体が得られた動物種を示すために用いられる。例えば、ヒト由来細胞は、当該細胞がヒトから得られた細胞であること、又は当該細胞を継代培養して得られた細胞株であることを意味し、例えば、ヒト細胞であることを意味する。また、ウマ由来抗体は、当該抗体がウマから得られた抗体であること、又はウマから得られた抗体もしくはその改変体をコードするヌクレオチド配列を有する抗体産生細胞から産生される抗体を意味し、例えば、ウマ抗体、そのヒトキメラ化抗体、またはそのヒト化抗体であることを意味する。 In this specification, "derived from" is used to indicate the animal species from which the cells or antibodies were obtained. For example, a human-derived cell means that the cell is a cell obtained from a human, or that the cell is a cell line obtained by subculturing the cell, e.g., a human cell. An equine-derived antibody means that the antibody is an antibody obtained from a horse, or that the antibody is produced from an antibody-producing cell having a nucleotide sequence encoding an antibody obtained from a horse or a variant thereof, e.g., an equine antibody, a human chimeric antibody thereof, or a humanized antibody thereof.
　本明細書における「同一性」とは、ＥＭＢＯＳＳ　Ｎｅｅｄｌｅ（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．；２０１５；４３：Ｗ５８０－Ｗ５８４）を用いて、デフォルトで用意されているパラメータによって得られたＩｄｅｎｔｉｔｙの値を意味する。前記のパラメータは以下のとおりである。
　Ｇａｐ　Ｏｐｅｎ　Ｐｅｎａｌｔｙ　＝　１０
　Ｇａｐ　Ｅｘｔｅｎｄ　Ｐｅｎａｌｔｙ　＝　０．５
　Ｍａｔｒｉｘ　＝　ＥＢＬＯＳＵＭ６２
　Ｅｎｄ　Ｇａｐ　Ｐｅｎａｌｔｙ　＝　ｆａｌｓｅ In this specification, the term "identity" refers to the Identity value obtained using EMBOSS Needle (Nucleic Acids Res.; 2015; 43: W580-W584) with the default parameters. The parameters are as follows:
Gap Open Penalty = 10
Gap Extend Penalty = 0.5
Matrix = EBLOSUM62
End Gap Penalty = false
　本明細書では、「抗体」は、ジスルフィド結合で安定化された重鎖（Ｈ鎖）と軽鎖（Ｌ鎖）で形成されたヘテロ二量体の一対が、さらにジスルフィド結合で会合してヘテロ四量体の構造をとるタンパク質をいう。抗体は、抗原に対して特異性を有し得る。特異性を有する結合とは、非特異的吸着ではない結合を意味する。特異性は、動物への抗原の免疫によって担保され得る。特異性を有するとは、他の少なくとも１つまたは幾つかのタンパク質に対するよりも、抗原に対して強い親和性を有することを意味し得る。また、例えば、特定の抗原に強い結合親和性を有する（例えば、ＫＤ値が、１０－７Ｍ以下、１０－８Ｍ以下、１０－９Ｍ以下、１０－１０Ｍ以下、１０－１１Ｍ以下、または１０－１２Ｍ以下である）抗体は、当該抗原に対して特異的に結合し得る抗体である。重鎖は、重鎖可変領域ＶＨ、重鎖定常領域ＣＨ１、ＣＨ２、ＣＨ３、及びＣＨ１とＣＨ２の間に位置するヒンジ領域からなり、軽鎖は、軽鎖可変領域ＶＬと軽鎖定常領域ＣＬとからなる。この中で、ＶＨとＶＬからなる可変領域断片（Ｆｖ）が、抗原結合に直接関与し、抗体に多様性を与える領域である。また、ＶＬ、ＣＬ、ＶＨ、ＣＨ１からなる抗原結合領域をＦａｂ領域と呼び、ヒンジ領域、ＣＨ２、ＣＨ３からなる領域をＦｃ領域と呼ぶ。
　可変領域のうち、直接抗原と接触する領域は抗体間でアミノ酸配列が多様性に富み、相補性決定領域（ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ－ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ：　ＣＤＲ）と呼ばれる。ＣＤＲ以外の抗体間でアミノ酸配列がほぼ一定の領域をフレームワーク（ｆｒａｍｅｗｏｒｋ　ｒｅｇｉｏｎ：　ＦＲ）と呼ぶ。軽鎖と重鎖の可変領域には、それぞれ３つのＣＤＲが存在し、それぞれＮ末端側から順に、重鎖ＣＤＲ１～３及び軽鎖ＣＤＲ１～３と呼ばれる。重鎖可変領域は、通常、重鎖ＦＲ１、重鎖ＣＤＲ１、重鎖ＦＲ２、重鎖ＣＤＲ２、重鎖ＦＲ３、重鎖ＣＤＲ３、および重鎖ＦＲ４をこの順番で含む。軽鎖可変領域は、通常、軽鎖ＦＲ１、軽鎖ＣＤＲ１、軽鎖ＦＲ２、軽鎖ＣＤＲ２、軽鎖ＦＲ３、軽鎖ＣＤＲ３、および軽鎖ＦＲ４をこの順番で含む。
　抗体は、モノクローナル抗体であっても、ポリクローナル抗体であってもよい。また、本発明の抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥのいずれのアイソタイプであってもよい。マウス、ラット、ハムスター、モルモット、ウサギ、ニワトリなどの非ヒト動物を免疫して作製したものであってもよいし、組換え抗体であってもよく、キメラ抗体、ヒト化抗体、完全ヒト化抗体等であってもよい。キメラ型抗体とは、異なる種に由来する抗体の断片が連結された抗体をいう。ＩｇＧのサブクラスとしては、ヒトでは、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、およびＩｇＧ４が挙げられる。本発明の抗体は、いずれのサブクラスでもあり得るが、例えば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３およびＩｇＧ４からなる群から選択される１以上であり得、例えば、ＩｇＧ２であり得、例えば、ＩｇＧ３であり得、例えば、ＩｇＧ４であり得る。
　「ヒト化抗体」とは、非ヒト由来の抗体に特徴的なアミノ酸配列で、ヒト抗体の対応する位置を置換した抗体（すなわち、ＣＤＲグラフト抗体）を意味し、例えば、マウス又はラットを免疫して作製した抗体の重鎖ＣＤＲ１～３（それぞれＨＣＤＲ１～３）及び軽鎖ＣＤＲ１～３（それぞれＬＣＤＲ１～３）を有し、重鎖及び軽鎖のそれぞれ４つのフレームワーク領域（ＦＲ）を含むその他のすべての領域がヒト抗体に由来するものが挙げられる。かかる抗体は、ＣＤＲ移植抗体と呼ばれる場合もある。用語「ヒト化抗体」は、ヒトキメラ抗体およびＣＤＲグラフトヒト化抗体を含み得る。
　本明細書において、抗体の「抗原結合フラグメント」とは、抗体のフラグメントであって、抗原に結合するフラグメントをいう。具体的には、ＶＬ、ＶＨ、ＣＬ及びＣＨ１領域からなるＦａｂ；２つのＦａｂがヒンジ領域でジスルフィド結合によって連結されているＦ（ａｂ’）２；ＶＬ及びＶＨからなるＦｖ；ＶＬ及びＶＨを人工のポリペプチドリンカーで連結した一本鎖抗体であるｓｃＦｖのほか、ｄｉａｂｏｄｙ型、ｓｃＤｂ型、ｔａｎｄｅｍ　ｓｃＦｖ型、ロイシンジッパー型などの二重特異性抗体等が挙げられるが、これらに限定されない。
　本発明において、抗体は、単離されたモノクローナル抗体（例えば、単離されたヒト化抗体）であり得る。医薬品に含まれるモノクローナル抗体は、通常は、単離されており、投与に適する組成を有する医薬品として製剤化されている。 In the present specification, the term "antibody" refers to a protein in which a pair of heterodimers formed by a heavy chain (H chain) and a light chain (L chain) stabilized by a disulfide bond are further associated by a disulfide bond to form a heterotetramer structure. An antibody may have specificity for an antigen. Binding with specificity means binding that is not nonspecific adsorption. Specificity can be ensured by immunization of an animal with the antigen. Having specificity may mean having a stronger affinity for an antigen than for at least one or several other proteins. In addition, for example, an antibody that has a strong binding affinity for a specific antigen (for example, a KD value of 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less) is an antibody that can specifically bind to the antigen. The heavy chain is composed of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and the light chain is composed of a light chain variable region VL and a light chain constant region CL. Among them, the variable region fragment (Fv) composed of VH and VL is a region that is directly involved in antigen binding and provides diversity to the antibody. The antigen-binding region composed of VL, CL, VH, and CH1 is called the Fab region, and the region composed of the hinge region, CH2, and CH3 is called the Fc region.
In the variable region, the region that directly contacts the antigen has a rich diversity of amino acid sequences among antibodies and is called the complementarity-determining region (CDR). The region other than the CDR, which has a nearly constant amino acid sequence among antibodies, is called the framework region (FR). The light chain and heavy chain variable regions each have three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus. The heavy chain variable region usually contains heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, in this order. A light chain variable region typically comprises, in that order, light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4.
The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody of the present invention may be of any isotype, IgG, IgM, IgA, IgD, or IgE. The antibody may be produced by immunizing a non-human animal such as a mouse, rat, hamster, guinea pig, rabbit, or chicken, or may be a recombinant antibody, a chimeric antibody, a humanized antibody, or a fully humanized antibody. A chimeric antibody refers to an antibody in which fragments of antibodies derived from different species are linked. In humans, IgG subclasses include IgG1, IgG2, IgG3, and IgG4. The antibody of the present invention may be of any subclass, but may be, for example, one or more selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, for example, IgG2, IgG3, or IgG4.
"Humanized antibody" refers to an antibody in which the corresponding positions in a human antibody have been replaced with amino acid sequences characteristic of antibodies of non-human origin (i.e., a CDR-grafted antibody), and examples thereof include antibodies having heavy chain CDR1-3 (HCDR1-3, respectively) and light chain CDR1-3 (LCDR1-3, respectively) of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FR) of the heavy and light chains, being derived from a human antibody. Such antibodies are sometimes called CDR-grafted antibodies. The term "humanized antibody" can include human chimeric antibodies and CDR-grafted humanized antibodies.
As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that binds to an antigen. Specific examples of such fragments include, but are not limited to, Fab consisting of VL, VH, CL, and CH1 regions, F(ab')2 in which two Fabs are linked by a disulfide bond at the hinge region, Fv consisting of VL and VH, scFv which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker, and bispecific antibodies such as diabody type, scDb type, tandem scFv type, and leucine zipper type.
In the present invention, the antibody may be an isolated monoclonal antibody (e.g., an isolated humanized antibody). The monoclonal antibody contained in the pharmaceutical product is usually isolated and formulated as a pharmaceutical product having a composition suitable for administration.
　本明細書では、「単離」とは、少なくとも他の成分から分離されていることを意味する。「単離」は、医薬製品に適合する程度の夾雑物からの分離を含む意味で用いられる。 As used herein, "isolated" means at least separated from other components. "Isolated" is used to include separation from contaminants to an extent that is compatible with a pharmaceutical product.
　本明細書では、単離は、単離後に他の物質と混合することを排除する用語ではない。したがって、本明細書では、単離された複数のモノクローナル抗体（及び必要に応じて添加剤）を混合して得られる組成物は、単離されたモノクローナル抗体を含む組成物である。但し、組成物は、インビトロの条件下で（特に細胞と生理食塩水とからなる系において）細胞に許容されない悪影響（例えば、毒性および傷害）を生じさせないように調製されている。また、医薬組成物は、対象（特にヒト）に投与したときに当該対象に許容されない副作用（例えば、毒性）を生じさせないように調製されている。 As used herein, isolation does not exclude mixing with other substances after isolation. Thus, as used herein, a composition obtained by mixing multiple isolated monoclonal antibodies (and additives, if necessary) is a composition containing isolated monoclonal antibodies. However, the composition is prepared so as not to cause unacceptable adverse effects (e.g., toxicity and injury) to cells under in vitro conditions (particularly in a system consisting of cells and physiological saline). In addition, pharmaceutical compositions are prepared so as not to cause unacceptable side effects (e.g., toxicity) in a subject (particularly a human) when administered to the subject.
＜好ましい態様としてのＳＡＲＳ関連コロナウイルス＞
　本開示の全ての側面および態様において、ベータコロナウイルスは、好ましくは、ＳＡＲＳ関連コロナウイルスであり、より好ましくは、ＳＡＲＳ－ＣｏＶ－２である。ＳＡＲＳ－ＣｏＶ－２は、野生株（中国武漢で単離されたオリジナル株）、アルファ株（Ｂ．１．１．７株及びＱ系列株）、ベータ株（Ｂ．１．３５１株およびその子孫株）、ガンマ株（Ｐ．１株およびその子孫株）、デルタ株（Ｂ．１．６１７．２株およびＡＹ系列株）、イプシロン株（Ｂ．１．４２７株およびＢ．１．４２９株）、イータ株（Ｅ．１．５２５株）、イオタ株（Ｂ．１．５２６株）、ミュー株（Ｂ．１．６２１株およびＢ．１．６２１．１株）、ゼータ株（Ｐ．２株）およびオミクロン株（Ｂ．１．１．５２９株、並びにＢＡ．１、ＢＡ．１．１、ＢＡ．２、ＢＡ．３、ＢＡ．４、およびＢＡ．５系列株）、並びにこれらの変種（特に限定されないが例えば、ＸＢＢ．１．５、ＸＢＢ．１．１６、ＥＧ．５、ＢＡ．２．７５、ＣＨ．１．１、ＸＢＢ、ＸＢＢ．１．９．１、ＸＢＢ．１．９．２、ＸＢＢ．２．３、およびＢＡ．２．８６からなる群から選択されるオミクロン株）およびその他の変種からなる群から選択されるいずれか１以上のＳＡＲＳ－ＣｏＶ－２であり得る。本開示の全ての側面および態様において、ベータコロナウイルスは、ＳＡＲＳ－ＣｏＶ－２の野生株であり得、デルタ株であり得、またはオミクロン株であり得る。 Preferred embodiments of SARS-associated coronavirus
In all aspects and embodiments of the present disclosure, the betacoronavirus is preferably a SARS-associated coronavirus, more preferably SARS-CoV-2. SARS-CoV-2 is classified into wild type (the original strain isolated in Wuhan, China), alpha strains (B.1.1.7 and Q lineage strains), beta strains (B.1.351 and its descendants), gamma strains (P.1 and its descendants), delta strains (B.1.617.2 and AY lineage strains), epsilon strains (B.1.427 and B.1.429), eta strains (E.1.525), iota strains (B.1.526), mu strains (B.1.621 and B.1.621.1), zeta strains (P.2 and omi). The SARS-CoV-2 may be any one or more of the SARS-CoV-2 strains selected from the group consisting of clonal strains (B.1.1.529 strain, and BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineage strains), and variants thereof (e.g., but not limited to, Omicron strains selected from the group consisting of XBB.1.5, XBB.1.16, EG.5, BA.2.75, CH.1.1, XBB, XBB.1.9.1, XBB.1.9.2, XBB.2.3, and BA.2.86) and other variants. In all aspects and embodiments of the present disclosure, the betacoronavirus may be a wild-type strain, a delta strain, or an Omicron strain of SARS-CoV-2.
＜本開示の抗血清＞
　本開示によれば、ウマの抗血清が提供される。本開示のウマの抗血清は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる。本開示のウマ抗血清はまた、ベータコロナウイルスのＡＣＥ２発現ヒト細胞（例えば、肺胞上皮細胞、特にＩＩ型肺胞上皮細胞）への感染を抑制することができる。Ｓ１タンパク質のＡＣＥ２への結合を阻害する抗体を得ること、ベータコロナウイルスのヒト細胞への感染を阻害する抗体を得ること、および／またはベータコロナウイルスのヒトへの感染を阻害する抗体を得ることができる。上記免疫原性組成物はまた、ベータコロナウイルスのヒトＡＣＥ２陽性細胞（例えば、肺胞上皮細胞、特にＩＩ型肺胞上皮細胞）への感染を抑制できる抗体を得ることができる。感染抑制力は、中和抗体価（ＩＣ５０）により表され得る。中和抗体価は、得られた抗体溶液または血清の希釈倍率により表すことができる。そして、感染抑制力が５０％となる希釈倍率をＩＣ５０とすることができる。ＩＣ５０は、例えば、５，０００以上、１０，０００以上、１５，０００以上、２０，０００以上、２５，０００以上、３０，０００以上、３５，０００以上、４０，０００以上、４５，０００以上、または５０，０００以上であり得る。ＩＣ５０は、例えば、５，０００～１００，０００、１０，０００～９０，０００、２０，０００～８０，０００、３０，０００～７０，０００、または４０，０００～６０，０００であり得る。ＩＣ５０は、得られた抗体溶液または血清の希釈系列を用いた感染抑制力に基づいて常法により算出することができる。 Antisera of the Present Disclosure
According to the present disclosure, a horse antiserum is provided. The horse antiserum of the present disclosure can neutralize the binding of the spike protein (S protein) of a beta coronavirus to angiotensin converting enzyme 2 (ACE2). The horse antiserum of the present disclosure can also suppress the infection of a beta coronavirus to ACE2-expressing human cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells). It is possible to obtain an antibody that inhibits the binding of S1 protein to ACE2, to obtain an antibody that inhibits the infection of a beta coronavirus to human cells, and/or to obtain an antibody that inhibits the infection of a beta coronavirus to humans. The above immunogenic composition can also obtain an antibody that can inhibit the infection of a beta coronavirus to human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells). The infection suppression ability can be expressed by a neutralizing antibody titer (IC 50 ). The neutralizing antibody titer can be expressed by the dilution ratio of the obtained antibody solution or serum. The dilution ratio at which the infection suppression ability is 50% can be taken as IC 50 . The IC 50 can be, for example, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, or 50,000 or more. The IC 50 can be, for example, 5,000 to 100,000, 10,000 to 90,000, 20,000 to 80,000, 30,000 to 70,000, or 40,000 to 60,000. The IC 50 can be calculated by a standard method based on the infection-inhibiting ability using a dilution series of the obtained antibody solution or serum.
　ある態様では、本開示のウマ抗血清は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物により誘導される。ある態様では、ＲＢＤ領域は、ＳＡＲＳ関連コロナウイルスのＳタンパク質のＲＢＤ領域である。ある態様では、免疫原性組成物は、２つの異なるＲＢＤ領域を含む。２つの異なるＲＢＤ領域は、異なるベータコロナウイルス、異なるＳＡＲＳ関連コロナウイルス、または異なるＳＡＲＳ－ＣｏＶ－２株に由来し得る。ある態様では、ＲＢＤ領域は、ＳＡＲＳ－ＣｏＶ－２のＳタンパク質のＲＢＤ領域である。ある態様では、ＲＢＤ領域は、野生株（中国武漢で最初に単離されたオリジナル株）、アルファ株（Ｂ．１．１．７株及びＱ系列株）、ベータ株（Ｂ．１．３５１株およびその子孫株）、ガンマ株（Ｐ．１株およびその子孫株）、デルタ株（Ｂ．１．６１７．２株およびＡＹ系列株）、イプシロン株（Ｂ．１．４２７株およびＢ．１．４２９株）、イータ株（Ｅ．１．５２５株）、イオタ株（Ｂ．１．５２６株）、ミュー株（Ｂ．１．６２１株およびＢ．１．６２１．１株）、ゼータ株（Ｐ．２株）およびオミクロン株（Ｂ．１．１．５２９株、並びにＢＡ．１、ＢＡ．１．１、ＢＡ．２、ＢＡ．３、ＢＡ．４、およびＢＡ．５系列株）、並びにこれらの変種（特に限定されないが例えば、ＸＢＢ．１．５、ＸＢＢ．１．１６、ＥＧ．５、ＢＡ．２．７５、ＣＨ．１．１、ＸＢＢ、ＸＢＢ．１．９．１、ＸＢＢ．１．９．２、ＸＢＢ．２．３、およびＢＡ．２．８６からなる群から選択されるオミクロン株）およびその他の変種からなる群から選択されるいずれか１以上のＳＡＲＳ－ＣｏＶ－２のＳタンパク質のＲＢＤ領域である。 In one aspect, the horse antisera of the present disclosure are induced by an immunogenic composition comprising an RBD region of a spike protein (S protein) of a betacoronavirus. In one aspect, the RBD region is an RBD region of an S protein of a SARS-associated coronavirus. In one aspect, the immunogenic composition comprises two different RBD regions. The two different RBD regions can be from different betacoronaviruses, different SARS-associated coronaviruses, or different SARS-CoV-2 strains. In one aspect, the RBD region is an RBD region of an S protein of SARS-CoV-2. In one aspect, the RBD region is selected from the group consisting of wild type (the original strain first isolated in Wuhan, China), alpha (B.1.1.7 and Q lineage strains), beta (B.1.351 and its progeny), gamma (P.1 and its progeny), delta (B.1.617.2 and AY lineage strains), epsilon (B.1.427 and B.1.429), eta (E.1.525), iota (B.1.526), mu (B.1.621 and B.1.621.1), zeta (P.2), and omicron. The RBD region of the S protein of SARS-CoV-2 is any one or more of the following strains selected from the group consisting of strains (B.1.1.529, and strains of the BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 series), and variants thereof (e.g., Omicron strains selected from the group consisting of, but not limited to, XBB.1.5, XBB.1.16, EG.5, BA.2.75, CH.1.1, XBB, XBB.1.9.1, XBB.1.9.2, XBB.2.3, and BA.2.86) and other variants.
　ある態様では、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域は、二量体を形成している。ある態様では、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体を含む。ある態様では、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の二量体を含む。ある好ましい態様では、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含む。ある好ましい態様では、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含み、かつ、二量体の含有量が単量体の含有量よりも多い。ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含み、かつ、二量体の含有量が単量体の含有量よりも多いことは、非還元環境下での電気泳動（例えば、ネイティブＳＤＳ－ＰＡＧＥ）後のウェスタンブロッティングにより決定することができる。１次抗体としてはウマのＲＢＤ領域に対するポリクローナル抗体を用いることができる。 In one embodiment, the RBD region of the spike protein (S protein) of the beta coronavirus forms a dimer. In one embodiment, the immunogenic composition comprises a monomer of the RBD region of the spike protein (S protein) of the beta coronavirus. In one embodiment, the immunogenic composition comprises a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus. In one preferred embodiment, the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus. In one preferred embodiment, the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus, and the content of the dimer is greater than the content of the monomer. The presence of a monomer and a dimer of the RBD region of the spike protein (S protein) of the beta coronavirus and the content of the dimer is greater than the content of the monomer can be determined by Western blotting after electrophoresis in a non-reducing environment (e.g., native SDS-PAGE). A polyclonal antibody against the horse RBD region can be used as the primary antibody.
　上記免疫原性組成物は、ウマを免疫することに用いることができる。したがって、本開示では、ウマを免疫することに用いるための、上記免疫原性組成物が提供される。また、本開示では、ウマを免疫する方法であって、上記免疫原性組成物をウマに投与することを含む方法が提供される。これらの態様においては、免疫原性組成物の有効量がウマに投与される。このようにすることで、ウマにおいてＲＢＤに対する抗体を産生させること、Ｓ１タンパク質のＡＣＥ２への結合を阻害する抗体を得ること、ベータコロナウイルスのヒト細胞への感染を阻害する抗体を得ること、および／またはベータコロナウイルスのヒトへの感染を阻害する抗体を得ることができる。上記免疫原性組成物はまた、ベータコロナウイルスのヒトＡＣＥ２陽性細胞（例えば、肺胞上皮細胞、特にＩＩ型肺胞上皮細胞）への感染を抑制できる抗体を得ることができる。感染抑制力は、中和抗体価（ＩＣ５０）により表され得る。ＩＣ５０は、例えば、５，０００以上、１０，０００以上、１５，０００以上、２０，０００以上、２５，０００以上、３０，０００以上、３５，０００以上、４０，０００以上、４５，０００以上、または５０，０００以上であり得る。ＩＣ５０は、例えば、５，０００～１００，０００、１０，０００～９０，０００、２０，０００～８０，０００、３０，０００～７０，０００、または４０，０００～６０，０００であり得る。ＩＣ５０は、常法により算出することができる。 The immunogenic composition can be used to immunize a horse. Thus, the present disclosure provides the immunogenic composition for use in immunizing a horse. The present disclosure also provides a method of immunizing a horse, comprising administering the immunogenic composition to the horse. In these aspects, an effective amount of the immunogenic composition is administered to the horse. In this way, it is possible to produce antibodies against RBD in the horse, obtain antibodies that inhibit the binding of S1 protein to ACE2, obtain antibodies that inhibit the infection of beta coronaviruses in human cells, and/or obtain antibodies that inhibit the infection of beta coronaviruses in humans. The immunogenic composition can also obtain antibodies that can inhibit the infection of beta coronaviruses in human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells). The infection-inhibiting ability can be expressed by neutralizing antibody titer ( IC50 ). The IC 50 can be, for example, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 35,000 or more, 40,000 or more, 45,000 or more, or 50,000 or more. The IC 50 can be, for example, 5,000 to 100,000, 10,000 to 90,000, 20,000 to 80,000, 30,000 to 70,000, or 40,000 to 60,000. The IC 50 can be calculated by conventional methods.
　ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域は、哺乳動物細胞により発現させることができる。細胞は、タンパク質の発現に適した細胞であり、例えば、ＣＨＯ細胞、および２９３細胞、並びにこれらからの改変体（例えば、Ｅｘｐｉ２９３Ｆ細胞およびＥｘｐｉＣＨＯ－Ｓ細胞）が挙げられる。細胞は、タンパク質発現に適した条件下で培養される。ＲＢＤ領域は、遺伝子導入後、例えば、６０時間以内、特に２日以内に（好ましくは２４時間経過時またはその後、より好ましくは３０時間経過時またはその後、さらに好ましくは３６時間経過時またはその後、さらにより好ましくは４２時間経過時またはその後に）細胞培養物中から回収される。このようにすることで、好ましくは、ＲＢＤ領域の二量体を多く含む免疫原性組成物を得ることができる。 The RBD region of the spike protein (S protein) of betacoronavirus can be expressed by mammalian cells. The cells are suitable for protein expression, such as CHO cells and 293 cells, and variants thereof (e.g., Expi293F cells and ExpiCHO-S cells). The cells are cultured under conditions suitable for protein expression. The RBD region is recovered from the cell culture, for example, within 60 hours, particularly within 2 days after gene introduction (preferably 24 hours or thereafter, more preferably 30 hours or thereafter, even more preferably 36 hours or thereafter, and even more preferably 42 hours or thereafter). In this way, an immunogenic composition containing a large amount of dimers of the RBD region can be obtained.
　ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含む免疫原性組成物であって、二量体の含有量が単量体の含有量よりも多い免疫原性組成物は、高い免疫原性を有し得る。例えば、ＥＬＩＳＡ法（ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅ　Ｐｒｏｔｅｉｎ　Ｓ１　ＲＢＤ　ＥＬＩＳＡ　Ｋｉｔ（Ｅｌａｂｓｃｉｅｎｃｅ，　Ｅ－ＥＬ－Ｅ６０５））によって製造者マニュアルにしたがって定量したときに、ＢＣＡ法で測定されるタンパク質量推定値の２倍以上、３倍以上、４倍以上、５倍以上、６倍以上、７倍以上、８倍以上、または９倍以上のタンパク質濃度を有すると評価され得る。 An immunogenic composition containing a monomer and a dimer of the RBD region of the spike protein (S protein) of a beta coronavirus, in which the content of the dimer is greater than the content of the monomer, may have high immunogenicity. For example, when quantified by an ELISA method (SARS-CoV-2 Spike Protein S1 RBD ELISA Kit (Elabscience, E-EL-E605)) according to the manufacturer's manual, it may be evaluated as having a protein concentration that is 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, or 9-fold or more of the estimated protein amount measured by the BCA method.
　ある態様では、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物は、ＥｘｐｉＣＨＯ－Ｓ細胞により発現され、遺伝子導入から４８時間以内に回収されたＲＢＤ領域を含む。ある好ましい態様では、ＲＢＤ領域は、配列番号１または２に記載のアミノ酸配列を有する。 In one embodiment, the immunogenic composition comprising the RBD region of the spike protein (S protein) of a betacoronavirus comprises an RBD region expressed by ExpiCHO-S cells and harvested within 48 hours of gene transfer. In one preferred embodiment, the RBD region has the amino acid sequence set forth in SEQ ID NO: 1 or 2.
　ある好ましい態様では、ＲＢＤ領域は、Ｎ末端にシグナルペプチドを有する。シグナルペプチドは、分泌シグナルペプチドであり得、当業者であれば、適宜選択して用いることができる。分泌シグナルペプチドとしては、例えば、抗体の重鎖または軽鎖のＮ末端に存在するシグナルペプチドを用いることができ、例えば、ＩｇＧの重鎖または軽鎖のシグナルペプチドを用いることができ、例えば、配列番号３に記載のアミノ酸配列を有するシグナルペプチドを用いることができる。ある好ましい態様では、ＲＢＤ領域は、タンパク質精製用のタグペプチドをさらに有していてもよい。タンパク質精製用のタグは、例えば、ＲＢＤ領域のＣ末端に連結され得る。したがって、ある好ましい態様では、ＲＢＤ領域は、Ｎ末端からＣ末端に向けてシグナルペプチド、ＲＢＤ領域、およびタグペプチドをこの順番で含む融合ペプチドの形態を有し得る。タグペプチドとしては、特に限定されないが、ＦＬＡＧタグ、ｍｙｃタグ、Ｖ５タグ、Ｓタグ、Ｅタグ、Ｔ７タグ、ＶＳＶ－Ｇタグ、Ｇｌｕ－Ｇｌｕタグ、Ｓｔｒｅｐ－ｔａｇＩＩ、ＨＳＶタグなどのタグが挙げられる。タグペプチドは、例えば、１５アミノ酸長であり、１４アミノ酸長であり、１３アミノ酸長であり、１２アミノ酸長であり、１１アミノ酸長であり、１０アミノ酸長であり、９アミノ酸長であり、８アミノ酸長であり、７アミノ酸長であり、または６アミノ酸長であり得る。タグペプチドは、例えば、配列番号４に記載の６×Ｈｉｓタグであり得る。 In a preferred embodiment, the RBD region has a signal peptide at the N-terminus. The signal peptide may be a secretory signal peptide, and those skilled in the art can select and use it as appropriate. As the secretory signal peptide, for example, a signal peptide present at the N-terminus of an antibody heavy or light chain may be used, for example, a signal peptide of an IgG heavy or light chain may be used, and for example, a signal peptide having the amino acid sequence set forth in SEQ ID NO: 3 may be used. In a preferred embodiment, the RBD region may further have a tag peptide for protein purification. The tag for protein purification may be linked, for example, to the C-terminus of the RBD region. Thus, in a preferred embodiment, the RBD region may have the form of a fusion peptide that includes a signal peptide, an RBD region, and a tag peptide in this order from the N-terminus to the C-terminus. Examples of tag peptides include, but are not limited to, FLAG tag, myc tag, V5 tag, S tag, E tag, T7 tag, VSV-G tag, Glu-Glu tag, Strep-tag II, HSV tag, and other tags. The tag peptide can be, for example, 15 amino acids long, 14 amino acids long, 13 amino acids long, 12 amino acids long, 11 amino acids long, 10 amino acids long, 9 amino acids long, 8 amino acids long, 7 amino acids long, or 6 amino acids long. The tag peptide can be, for example, a 6xHis tag as set forth in SEQ ID NO:4.
　ある態様では、ウマは、サラブレッドである。サラブレッドの直系父系先祖は、ダーレーアラビアン、バイアリーターク、およびゴドルフィンアラビアンのいずれかであるとされる。ある態様では、ウマは、アラブ血量が２５％未満であるサラブレッドである。ある態様では、ウマは、例えば、１０歳から２０歳のオスまたはメスである。ある態様では、ウマは、４００ｋｇから８００ｋｇの体重、好ましくは５００ｋｇから７００ｋｇの体重を有する。 In some embodiments, the horse is a Thoroughbred. The direct paternal ancestry of the Thoroughbred is said to be any of the Darley Arabian, the Byerley Turk, and the Godolphin Arabian. In some embodiments, the horse is a Thoroughbred with less than 25% Arabian blood. In some embodiments, the horse is male or female, for example, 10 to 20 years of age. In some embodiments, the horse has a body weight of 400 kg to 800 kg, preferably 500 kg to 700 kg.
　ある好ましい態様では、ウマは、サラブレッドであり、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含む。ある好ましい態様では、ウマは、サラブレッドであり、免疫原性組成物は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域の単量体と二量体を含み、かつ、二量体の含有量が単量体の含有量よりも多い。 In a preferred embodiment, the horse is a thoroughbred and the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus. In a preferred embodiment, the horse is a thoroughbred and the immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus, and the dimer content is greater than the monomer content.
　本開示のウマ抗血清は、上記免疫原性組成物をウマ（好ましくはサラブレッド）に投与することにより取得され得る。取得されたウマ抗血清は、高い中和抗体価を示し得、また、高い感染阻害能を有しうる。 The equine antiserum of the present disclosure can be obtained by administering the immunogenic composition to a horse (preferably a thoroughbred). The equine antiserum obtained can exhibit high neutralizing antibody titers and can also have high infection-inhibiting ability.
＜本開示のモノクローナル抗体およびポリクローナル抗体＞
　本開示によれば、ウマ抗血清に含まれるポリクローナル抗体が提供される。ウマ抗血清に含まれるポリクローナル抗体は、ウマ抗血清から抗体を精製することによって取得され得る。精製は、例えば、血漿分画法、硫安画法、またはカプリル酸沈殿法により行うことができる。あるいは、精製は、例えば、抗体を選択的に吸着するプロテインＧまたはプロテインＡを備えたカラムにより行ってもよい。プロテインＧまたはプロテインＡを備えたカラムによる抗体の精製は、常法に従って当業者であれば適宜実施することができる。精製されたポリクローナル抗体は、単離ポリクローナル抗体でもある。 Monoclonal and polyclonal antibodies of the present disclosure
According to the present disclosure, a polyclonal antibody contained in horse antiserum is provided. The polyclonal antibody contained in horse antiserum can be obtained by purifying the antibody from the horse antiserum. The purification can be performed, for example, by plasma fractionation, ammonium sulfate fractionation, or caprylic acid precipitation. Alternatively, the purification can be performed, for example, by a column equipped with protein G or protein A that selectively adsorbs the antibody. Purification of the antibody by a column equipped with protein G or protein A can be appropriately performed by a person skilled in the art according to a conventional method. The purified polyclonal antibody is also an isolated polyclonal antibody.
　本開示によればまた、ウマ抗血清に含まれる抗体のいずれか１つの抗体に関するモノクローナル抗体が提供される。モノクローナル抗体は、様々な方法により取得され得る。一例として例えば、免疫原性組成物を投与されたウマから得られる脾臓細胞、Ｂ細胞または形質細胞から選択されるいずれかの細胞を回収する。回収された細胞中で抗体をコードするｍＲＮＡまたはｃＤＮＡの配列を解読して抗体のアミノ酸配列を決定し、決定されたアミノ酸配列を有する抗体をコードする遺伝子をタンパク質発現細胞（例えば、チャイニーズハムスター卵巣（ＣＨＯ）細胞）に導入することによって抗体産生細胞を得て、得られた抗体産生細胞からモノクローナル抗体が得られ得る。ある態様では、ウマから得られる脾臓細胞、Ｂ細胞または形質細胞から選択されるいずれかの細胞とミエローマ細胞を細胞融合させてハイブリドーマを得てもよい。得られたハイブリドーマ細胞中における抗体をコードするｍＲＮＡまたはｃＤＮＡの配列を解読し、解読された抗体をコードする遺伝子をタンパク質発現細胞（例えば、チャイニーズハムスター卵巣（ＣＨＯ）細胞）に導入することによって抗体産生細胞を得て、得られた抗体産生細胞からモノクローナル抗体が得られ得る。精製されたモノクローナル抗体は、単離モノクローナル抗体でもある。 The present disclosure also provides a monoclonal antibody related to any one of the antibodies contained in the horse antiserum. The monoclonal antibody can be obtained by various methods. For example, any cell selected from spleen cells, B cells, or plasma cells obtained from a horse administered with an immunogenic composition is collected. The sequence of the mRNA or cDNA encoding the antibody in the collected cells is decoded to determine the amino acid sequence of the antibody, and a gene encoding the antibody having the determined amino acid sequence is introduced into a protein-expressing cell (e.g., Chinese hamster ovary (CHO) cell) to obtain an antibody-producing cell, and a monoclonal antibody can be obtained from the obtained antibody-producing cell. In one aspect, any cell selected from spleen cells, B cells, or plasma cells obtained from a horse may be fused with a myeloma cell to obtain a hybridoma. The sequence of the mRNA or cDNA encoding the antibody in the obtained hybridoma cell is decoded, and the gene encoding the decoded antibody is introduced into a protein-expressing cell (e.g., Chinese hamster ovary (CHO) cell) to obtain an antibody-producing cell, and a monoclonal antibody can be obtained from the obtained antibody-producing cell. A purified monoclonal antibody is also an isolated monoclonal antibody.
　モノクローナル抗体は、ヒトキメラ化またはヒト化することができる。したがって、本開示によれば、ヒトキメラ化したモノクローナル抗体およびヒト化されたモノクローナル抗体が提供され得る。 　Monoclonal antibodies can be human chimerized or humanized. Thus, in accordance with the present disclosure, human chimerized monoclonal antibodies and humanized monoclonal antibodies can be provided.
　したがって、本開示によれば、本開示のポリクローナル抗体を含む組成物、本開示のモノクローナル抗体を含む組成物、本開示のヒトキメラ化モノクローナル抗体を含む組成物、および本開示のヒト化モノクローナル抗体を含む組成物が提供される。本開示によればまた、複数のモノクローナル抗体を含む組成物が提供される。このような組成物は、中和できるベータコロナウイルスの種類を増加させることなどにおいて有利であり得る。 Thus, the present disclosure provides a composition comprising a polyclonal antibody of the present disclosure, a composition comprising a monoclonal antibody of the present disclosure, a composition comprising a human chimerized monoclonal antibody of the present disclosure, and a composition comprising a humanized monoclonal antibody of the present disclosure. The present disclosure also provides a composition comprising multiple monoclonal antibodies. Such a composition may be advantageous, for example, in increasing the types of betacoronaviruses that can be neutralized.
　ある態様では、複数のヒトキメラ化モノクローナル抗体を含む組成物が提供される。ある態様ではまた、複数のヒト化モノクローナル抗体を含む組成物が提供される。これらの態様では、複数のモノクローナル抗体（すなわち、複数のヒトキメラ化モノクローナル抗体および複数のヒト化モノクローナル抗体）は、物質として互いに相違する。ある態様では、複数のモノクローナル抗体は、アミノ酸配列において互いに相違する。ある態様では、複数のモノクローナル抗体は、重鎖ＣＤＲ１～３および軽鎖ＣＤＲ１～３からなる群から選択される１以上のＣＤＲのアミノ酸配列において互いに相違する。ある態様では、アミノ酸配列における相違は、１アミノ酸から１０アミノ酸であり得る。ある好ましい態様では、複数の抗体のうちのいずれか少なくとも２つはそれぞれ、異なるエピトープに結合し得る、例えば、ＲＢＤ領域への結合に対して互いに競合することがない。 In some embodiments, compositions are provided that include a plurality of human chimerized monoclonal antibodies. In some embodiments, compositions are provided that include a plurality of humanized monoclonal antibodies. In these embodiments, the plurality of monoclonal antibodies (i.e., the plurality of human chimerized monoclonal antibodies and the plurality of humanized monoclonal antibodies) differ from each other as substances. In some embodiments, the plurality of monoclonal antibodies differ from each other in amino acid sequence. In some embodiments, the plurality of monoclonal antibodies differ from each other in the amino acid sequence of one or more CDRs selected from the group consisting of heavy chain CDR1-3 and light chain CDR1-3. In some embodiments, the difference in amino acid sequence can be from 1 amino acid to 10 amino acids. In some preferred embodiments, any at least two of the plurality of antibodies can each bind to a different epitope, e.g., do not compete with each other for binding to the RBD region.
　本開示の抗体は、上記ＲＢＤ領域（好ましくは、その２量体）に対して、例えば、１０－６以下、１０－７以下、１０－８以下、１０－９以下、１０－１０以下、または１０－１１以下の結合解離定数（ＫＤ値）で結合し得る。ここで、ＫＤ値は小さいほど、抗体がＲＢＤ領域（好ましくは、その２量体）に対して強く結合することを意味する。 The antibodies of the present disclosure may bind to the RBD region (preferably a dimer thereof) with a binding dissociation constant (KD value) of, for example, 10 −6 or less, 10 −7 or less, 10 −8 or less, 10 −9 or less, 10 −10 or less, or 10 −11 or less, where a smaller KD value indicates a stronger binding of the antibody to the RBD region (preferably a dimer thereof).
＜本開示のバーチャルポリクローナル抗体を含む組成物＞
　本開示のある態様では、バーチャルポリクローナル抗体を含む組成物が提供される。バーチャルポリクローナル抗体とは、複数の異なる、または多様なモノクローナル抗体（それぞれ単離されたモノクローナルである）を含む抗体混合物（または当該混合物を含む組成物）である。バーチャルポリクローナル抗体においては、複数のモノクローナル抗体が含まれるが、それぞれのモノクローナル抗体の含有量は、有効量に達する。バーチャルポリクローナル抗体は、これにより、ポリクローナル抗体のように、単一のモノクローナル抗体と比較して、ロバストに所望の抗体機能を発現することができる（例えば、抗原変異体に対する結合親和性の低下を抑制することができる）抗体混合物である。ある好ましい態様では、バーチャルポリクローナル抗体に含まれる少なくとも１つのモノクローナル抗体は、少なくとも１つの他のモノクローナル抗体と、同時にＳタンパク質に結合することができる。 Compositions Comprising Virtual Polyclonal Antibodies of the Present Disclosure
In one aspect of the present disclosure, a composition comprising a virtual polyclonal antibody is provided. A virtual polyclonal antibody is an antibody mixture (or a composition comprising the mixture) comprising a plurality of different or diverse monoclonal antibodies (each of which is an isolated monoclonal). A virtual polyclonal antibody comprises a plurality of monoclonal antibodies, but the content of each monoclonal antibody reaches an effective amount. As a result, a virtual polyclonal antibody is an antibody mixture that can robustly express a desired antibody function (e.g., can suppress a decrease in binding affinity to an antigen mutant) compared to a single monoclonal antibody, like a polyclonal antibody. In one preferred aspect, at least one monoclonal antibody included in the virtual polyclonal antibody can bind to S protein simultaneously with at least one other monoclonal antibody.
　複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体は、例えば、それぞれ有効量の２以上のモノクローナル抗体を含む。複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体は、例えば、それぞれ有効量の３以上のモノクローナル抗体を含む。複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体は、例えば、それぞれ有効量のｎ以上のモノクローナル抗体を含む｛ここで、ｎは、２以上の自然数であれば特に限定されないが例えば、２～５０、２～４０、２～３０、２～２０、または２～１０までの自然数であり得る｝。複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体は、例えば、それぞれ有効量の４～１０のモノクローナル抗体を含み得る。複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体に含まれるモノクローナル抗体はそれぞれ、ヒトキメラ化またはヒト化されている。 　A plurality of different humanized antibodies, for example, virtual polyclonal antibodies, may each contain an effective amount of two or more monoclonal antibodies. A plurality of different humanized antibodies, for example, virtual polyclonal antibodies, may each contain an effective amount of three or more monoclonal antibodies. A plurality of different humanized antibodies, for example, virtual polyclonal antibodies, may each contain an effective amount of n or more monoclonal antibodies {where n is not particularly limited as long as it is a natural number of 2 or more, but may be, for example, a natural number from 2 to 50, 2 to 40, 2 to 30, 2 to 20, or 2 to 10}. A plurality of different humanized antibodies, for example, virtual polyclonal antibodies, may each contain an effective amount of 4 to 10 monoclonal antibodies. Each of the monoclonal antibodies contained in a plurality of different humanized antibodies, for example, virtual polyclonal antibodies, is human chimerized or humanized.
　複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体は、
　ウマをベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む上記いずれかの免疫原性組成物で免疫し、ウマの体内に前記ＲＢＤ領域に対する抗体を産生させることと、
　前記ウマから取得された複数の細胞が産生する抗体それぞれをコードする複数ｍＲＮＡまたはｃＤＮＡそれぞれの重鎖可変領域および軽鎖可変領域の配列（例えば、ヌクレオチド配列またはアミノ酸配列）を決定することと、ここで、前記細胞は、脾臓細胞、Ｂ細胞および形質細胞からなる群から選択される細胞であり、
　決定された配列（例えば、ヌクレオチド配列またはアミノ酸配列）をその配列同一性に基づいて複数のクラスタにクラスタリングすることと、
　取得された複数のクラスタのうち、含まれるヌクレオチド配列数が多いクラスタの上位ｐ個から少なくともｑ個のクラスタを選択することと、
　選択されたクラスタそれぞれに含まれる重鎖可変領域および軽鎖可変領域を含むヒトキメラ抗体をコードするヌクレオチド配列か、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含むヒト化抗体をコードするヌクレオチド配列を得ることと、
　得られたヌクレオチド配列それぞれを有する複数の抗体産生細胞から、前記ヒトキメラ抗体またはヒト化抗体を産生させ、前記ヒトキメラ抗体またはヒト化抗体を含む複数の抗体の混合物を得ることと、
を含む方法により作製することができる。上記において、ｐは、１０～５０までの自然数であり得、ｑは、２～ｐまでの自然数であり得る。ある態様では、ｐは１０であり、ｑは２～１０のいずれかの自然数である。ある態様では、ｐは２０であり、ｑは２～２０のいずれかの自然数である。ある態様では、ｐとｑは等しく、４～１０までの自然数であり得る。 A plurality of different humanized antibodies, e.g., virtual polyclonal antibodies,
Immunizing a horse with any one of the above immunogenic compositions containing the RBD region of a spike protein (S protein) of a beta coronavirus, thereby producing antibodies against the RBD region in the horse's body;
determining sequences (e.g., nucleotide sequences or amino acid sequences) of heavy and light chain variable regions of each of a plurality of mRNAs or cDNAs encoding each of the antibodies produced by a plurality of cells obtained from the horse, wherein the cells are selected from the group consisting of spleen cells, B cells, and plasma cells;
Clustering the determined sequences (e.g., nucleotide sequences or amino acid sequences) into a plurality of clusters based on their sequence identity;
selecting at least q clusters from the top p clusters containing the largest number of nucleotide sequences from among the obtained clusters;
obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region comprised in each of the selected clusters, or a nucleotide sequence encoding a humanized antibody comprising three complementarity determining regions (CDRs) of a heavy chain variable region and three CDRs of a light chain variable region;
producing the human chimeric or humanized antibodies from a plurality of antibody-producing cells each having the obtained nucleotide sequence, thereby obtaining a mixture of a plurality of antibodies containing the human chimeric or humanized antibodies;
In the above, p can be a natural number from 10 to 50, and q can be a natural number from 2 to p. In one embodiment, p is 10, and q is a natural number from 2 to 10. In one embodiment, p is 20, and q is a natural number from 2 to 20. In one embodiment, p and q are equal and can be natural numbers from 4 to 10.
　含まれる複数の抗体が、それぞれ異なるエピトープに結合することを確からしくするために、含まれる複数の抗体のうちのいずれか２つは、ＲＢＤ領域への結合に関して互いに競合しないものとすることができる。 To ensure that the multiple antibodies contained in the antibody bind to different epitopes, any two of the multiple antibodies contained in the antibody can be ones that do not compete with each other for binding to the RBD region.
　ある態様では、複数の異なるヒト化抗体を含む組成物、例えば、バーチャルポリクローナル抗体は、
　異なるモノクローナル抗体をそれぞれ産生する複数の抗体産生細胞からモノクローナル抗体を産生させ、産生された複数のモノクローナル抗体を含む組成物を得ることを含む方法であって、
　組成物に含まれる各モノクローナル抗体は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫され、体内にＲＢＤ領域に対する抗体群を有するウマにおいて検出される抗体群から選択される抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含むか、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含み、
　各モノクローナル抗体の残りの部分は、ヒト抗体に由来し、
　各モノクローナル抗体は、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、方法
により作製することができる。 In one aspect, a composition comprising a plurality of different humanized antibodies, e.g., a virtual polyclonal antibody, is
A method for producing monoclonal antibodies from a plurality of antibody-producing cells each producing a different monoclonal antibody, and obtaining a composition containing the produced monoclonal antibodies, comprising:
Each monoclonal antibody contained in the composition comprises a portion of an antibody structure selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, the portion of the antibody structure comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
The remainder of each monoclonal antibody is derived from a human antibody,
Each monoclonal antibody can be produced by a method capable of neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
　抗体を作製する方法において、免疫原性組成物は、上記のいずれかの免疫原性組成物であり得る。 In the method for producing an antibody, the immunogenic composition may be any of the immunogenic compositions described above.
　本開示の複数の異なるヒト化抗体を含む組成物、例えば、バーチャルポリクローナル抗体は、Ｓ１タンパク質のＡＣＥ２への結合を阻害する抗体を得ること、ベータコロナウイルスのヒト細胞への感染を阻害する抗体を得ること、および／またはベータコロナウイルスのヒトへの感染を阻害する抗体を得ることを含む方法により取得することができる。 A composition comprising a plurality of different humanized antibodies of the present disclosure, e.g., a virtual polyclonal antibody, can be obtained by a method including obtaining an antibody that inhibits binding of S1 protein to ACE2, obtaining an antibody that inhibits infection of human cells by betacoronavirus, and/or obtaining an antibody that inhibits infection of humans by betacoronavirus.
　本開示によれば、対象においてベータコロナウイルスによる感染症を処置する方法であって、当該対象に本開示の抗血清、モノクローナル抗体、ポリクローナル抗体、またはバーチャルポリクローナル抗体の有効量を投与することを含む、方法が提供される。態様は、健常者、またはベータコロナウイルスに感染した対象であり得る。対象は、ベータコロナウイルスに感染したキャリア（症状を呈しない対象）であり得る。対象は、ベータコロナウイウルスによる感染症に罹患した対象であり得る。ある態様では、対象は、軽度のベータコロナウイルス感染症に罹患した対象であり得る。ある態様では、対象は、中等度のベータコロナウイルス感染症に罹患した対象であり得る。ある態様では、対象は、重度のベータコロナウイルス感染症に罹患した対象であり得る。ある態様では、対象は、コロナウイルス感染症（例えば、ベータコロナウイルス感染症）を発症していないが、ベータコロナウイルス感染症との濃厚接触歴を有する。ある態様では、対象は、コロナウイルス感染症（例えば、ベータコロナウイルス感染症）を発症していないが、ベータコロナウイルスに感染した対象であり得る。 According to the present disclosure, a method of treating a betacoronavirus infection in a subject is provided, comprising administering to the subject an effective amount of an antiserum, monoclonal antibody, polyclonal antibody, or virtual polyclonal antibody of the present disclosure. An embodiment may be a healthy individual or a subject infected with a betacoronavirus. A subject may be a carrier (subject asymptomatic) of a betacoronavirus infection. A subject may be a subject suffering from an infection with a betacoronavirus. In an embodiment, a subject may be a subject suffering from a mild betacoronavirus infection. In an embodiment, a subject may be a subject suffering from a moderate betacoronavirus infection. In an embodiment, a subject may be a subject suffering from a severe betacoronavirus infection. In an embodiment, a subject does not have a coronavirus infection (e.g., a betacoronavirus infection) but has a history of close contact with a betacoronavirus infection. In an embodiment, a subject may be a subject infected with a betacoronavirus but does not have a coronavirus infection (e.g., a betacoronavirus infection).
　本開示によれば、本開示の方法において用いるための、本開示の抗血清、モノクローナル抗体、ポリクローナル抗体、または複数の異なるヒト化抗体を含む組成物、例えば、バーチャルポリクローナル抗体が提供される。 In accordance with the present disclosure, there is provided a composition comprising an antiserum, a monoclonal antibody, a polyclonal antibody, or a plurality of different humanized antibodies of the present disclosure, e.g., a virtual polyclonal antibody, for use in the methods of the present disclosure.
　本開示によれば、本開示の方法において用いるための、本開示の抗血清、モノクローナル抗体、ポリクローナル抗体、または複数の異なるヒト化抗体、例えば、バーチャルポリクローナル抗体を含む組成物または医薬組成物が提供される。 In accordance with the present disclosure, there is provided a composition or pharmaceutical composition comprising an antiserum, a monoclonal antibody, a polyclonal antibody, or a plurality of different humanized antibodies, e.g., virtual polyclonal antibodies, of the present disclosure for use in the methods of the present disclosure.
　本開示によれば、本発明の方法において用いるための医薬または組成物の製造における、本開示の抗血清、モノクローナル抗体、ポリクローナル抗体、または複数の異なるヒト化抗体を含む組成物、例えば、バーチャルポリクローナル抗体の使用が提供される。 The present disclosure provides for the use of an antiserum, monoclonal antibody, polyclonal antibody, or composition comprising a plurality of different humanized antibodies, e.g., a virtual polyclonal antibody, of the present disclosure in the manufacture of a medicament or composition for use in the methods of the present invention.
実施例１：抗原の調製
　本実施例では、抗原としてＳ１タンパク質の部分ペプチドを調製した。 Example 1: Preparation of antigen In this example, a partial peptide of S1 protein was prepared as an antigen.
（１）細胞の調製
　ＥｘｐｉＣＨＯ－Ｓ凍結細胞を融解し、細胞増殖が安定するまで継代培養を繰り返し、さらに細胞を凍結して、凍結細胞を得た。具体的には、空の清潔な三角フラスコにＥｘｐｉＣＨＯ　Ｅｘｐｒｅｓｓｉｏｎ　Ｍｅｄｉｕｍを導入し、３７℃のＣＯ２インキュベータ内で保温した。０．２×１０６細胞／ｍＬから０．３×１０６細胞／ｍＬの濃度で播種し、３７℃、８％ＣＯ２存在下で培養した。生細胞密度が０．４×１０６細胞／ｍＬから０．６×１０６細胞／ｍＬの濃度に達した。凍結細胞のうち１つのバイアルを融解し、３回の継代培養を行って、融解直後の細胞生存率が１００％であること、遅滞期（ｌａｇ　ｐｈａｓｅ）が７日以内であること、対数増殖期における倍加時間が凍結前の±２時間以内（凍結前の倍加時間：１７．７時間、凍結後３継代目の倍加時間：１８．９時間）であること確認した。雑菌等による汚染がないことも確認された。得られた細胞を後述する実施例で用いた。全ての抗原調製のための細胞培養は３７℃、８％ＣＯ２条件下で実施した。 (1) Preparation of cells ExpiCHO-S frozen cells were thawed, subcultured repeatedly until cell growth stabilized, and then frozen to obtain frozen cells. Specifically, ExpiCHO Expression Medium was introduced into an empty, clean Erlenmeyer flask and incubated in a CO2 incubator at 37°C. The cells were seeded at a concentration of 0.2x106 cells/mL to 0.3x106 cells/mL and cultured at 37°C in the presence of 8% CO2 . The viable cell density reached a concentration of 0.4x106 cells/mL to 0.6x106 cells/mL. One vial of the frozen cells was thawed and subcultured three times to confirm that the cell viability immediately after thawing was 100%, the lag phase was within 7 days, and the doubling time in the logarithmic growth phase was within ±2 hours before freezing (doubling time before freezing: 17.7 hours, doubling time in the third passage after freezing: 18.9 hours). It was also confirmed that there was no contamination by bacteria or the like. The obtained cells were used in the examples described below. All cell cultures for antigen preparation were carried out under 37°C and 8% CO2 conditions.
（２）抗原発現ベクターの調製
　抗原としては、ＳＡＲＳ－ＣｏＶ－２（武漢株）のＳ１タンパク質のＲＢＤ領域（３１９番目から５４１番目のアミノ酸）を用いた。本明細書では、武漢株を野生型またはＷＴと呼ぶことがある。抗原にＨｉｓタグを付加した。得られた配列をＳ１－ＲＢＤ－Ｈｉｓタグ（ＷＴ）と称する。Ｓ１－ＲＢＤ－Ｈｉｓタグは、配列番号１に記載のアミノ酸配列を有するＲＢＤ領域のＮ末端に配列番号３に記載のアミノ酸配列を有するＩｇＧκリーダー配列（配列番号３）を付加し、Ｃ末端に６×Ｈｉｓ（Ｈｉｓタグ；配列番号４）を有した。同様にしてＳＡＲＳ－ＣｏＶ－２（δ株）のＳ１タンパク質のＲＢＤ領域（配列番号２）のＮ末端にＩｇＧκリーダー配列（配列番号３）を付加し、Ｃ末端にＨｉｓタグ（配列番号４）を付加してＳ１－ＲＢＤ－Ｈｉｓタグ（δ株）を得た。抗原それぞれをｐｃＤＮＡ３．４－ＴＯＰＯベクターにクローニングした。抗原それぞれは、ｐｃＤＮＡ３．４－ＴＯＰＯベクターに搭載されたＣＭＶプロモーターにより駆動されるように設計された。 (2) Preparation of antigen expression vector The RBD region (amino acids 319 to 541) of the S1 protein of SARS-CoV-2 (Wuhan strain) was used as the antigen. In this specification, the Wuhan strain may be referred to as wild type or WT. A His tag was added to the antigen. The resulting sequence is referred to as S1-RBD-His tag (WT). The S1-RBD-His tag had an IgGκ leader sequence (SEQ ID NO: 3) having the amino acid sequence of SEQ ID NO: 3 added to the N-terminus of the RBD region having the amino acid sequence of SEQ ID NO: 1, and had 6×His (His tag; SEQ ID NO: 4) at the C-terminus. Similarly, an IgGκ leader sequence (SEQ ID NO: 3) was added to the N-terminus of the RBD region (SEQ ID NO: 2) of the S1 protein of SARS-CoV-2 (δ strain), and a His tag (SEQ ID NO: 4) was added to the C-terminus to obtain S1-RBD-His tag (δ strain). Each antigen was cloned into the pcDNA3.4-TOPO vector. Each antigen was designed to be driven by the CMV promoter carried in the pcDNA3.4-TOPO vector.
（３）細胞への遺伝子導入
　上記（１）により得られた細胞（０．４×１０６細胞／ｍＬから０．６×１０６細胞／ｍＬの濃度に達した）の細胞生存率が９５％以上であることを確認した。ＥｘｐｉＣＨＯ　Ｅｘｐｒｅｓｓｉｏｎ　Ｍｅｄｉｕｍを含む三角フラスコに、上記（１）により得られた細胞を導入した。ＥｘｐｉＦｅｃｔａｍｉｎｅ　ＣＨＯ　Ｒｅａｇｅｎｔを用いて、上記（２）で得られたベクターを細胞に導入した。遺伝子導入の翌日に、ＥｘｐｉＦｅｃｔａｍｉｎｅ　ＣＨＯ　Ｅｎｈａｎｃｅｒ及びＥｘｐｉＣＨＯ　Ｆｅｅｄを添加した。遺伝子導入操作から５日目、６０時間後、および４０時間後に細胞を回収し、細胞生存率及びタンパク質発現量を確認した。 (3) Gene transfer into cells The cell viability of the cells obtained by (1) above (which reached a concentration of 0.4×10 6 cells/mL to 0.6×10 6 cells/mL) was confirmed to be 95% or more. The cells obtained by (1) above were transferred into an Erlenmeyer flask containing ExpiCHO Expression Medium. The vector obtained by (2) above was transferred into the cells using ExpiFectamine CHO Reagent. The day after gene transfer, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added. The cells were collected 5 days, 60 hours, and 40 hours after the gene transfer operation, and the cell viability and protein expression level were confirmed.
（４）組換えタンパク質発現の確認
（４－１）組換えタンパク質の精製
　上記それぞれの時点で回収した細胞を含む培養液の一部を洗浄し、超音波破砕処理した。遠心分離して上清を回収した。Ｎｉ　Ｓｅｐｈａｒｏｓｅ　６　Ｆａｓｔ　Ｆｌｏｗ樹脂の１００μＬを緩衝液で平衡化した。樹脂に対して上記上清を添加して４℃で１時間攪拌した。樹脂を遠心分離に供した。その後、樹脂をさらに緩衝液に懸濁し、樹脂をさらに遠心分離に供するサイクルを３回実施した。上清を除去し、樹脂に溶出緩衝液を添加して懸濁した。遠心分離後、上清を回収した。回収した上清を組換えタンパク質抽出液とした。タンパク質は、ＳＤＳ－ＰＡＧＥ後、ＣＢＢ染色またはウェスタンブロット（検出は、アルカリフォスファターゼ標識Ａｎｔｉ－６×Ｈｉｓ　ｔａｇ抗体（Ａｂｃａｍ，　ａｂ４９７４６））に供した。タンパク質濃度は、紫外線吸収法またはビシンコニン酸（ＢＣＡ）法によって定量した。 (4) Confirmation of recombinant protein expression (4-1) Purification of recombinant protein A part of the culture containing the cells collected at each of the above time points was washed and subjected to ultrasonic disruption. The supernatant was collected by centrifugation. 100 μL of Ni Sepharose 6 Fast Flow resin was equilibrated with a buffer. The above supernatant was added to the resin and stirred at 4 ° C. for 1 hour. The resin was subjected to centrifugation. Thereafter, the resin was further suspended in a buffer and the resin was further subjected to centrifugation for three cycles. The supernatant was removed, and the resin was suspended in an elution buffer. After centrifugation, the supernatant was collected. The collected supernatant was used as a recombinant protein extract. The protein was subjected to SDS-PAGE, CBB staining or Western blotting (detection was performed with alkaline phosphatase-labeled Anti-6 × His tag antibody (Abcam, ab49746)). Protein concentrations were quantified by UV absorption or the bicinchoninic acid (BCA) method.
（４－２）細胞培養上清の調製
　培養上清をデプスフィルターおよび０．２２μｍフィルターによりろ過した。デプスフィルターとしては、Ｍｉｌｌｉｓｔａｋ＋（商標）ＨＣポッドフィルター，Ｄ０ＨＣ，　０，０２７ｍ２（メルク）を用いて、上清を２回のろ過に供した。０．２２μｍフィルターとしては、０．２２μｍボトルトップフィルター（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ，　５９７－４５２０）を用いた。タンパク質濃度は、ＥＬＩＳＡ法（ＳＡＲＳ－ＣｏＶ－２　Ｓｐｉｋｅ　Ｐｒｏｔｅｉｎ　Ｓ１　ＲＢＤ　ＥＬＩＳＡ　Ｋｉｔ（Ｅｌａｂｓｃｉｅｎｃｅ，　Ｅ－ＥＬ－Ｅ６０５））によって製造者マニュアルにしたがって定量した。 (4-2) Preparation of cell culture supernatant The culture supernatant was filtered through a depth filter and a 0.22 μm filter. As the depth filter, the supernatant was subjected to two filtrations using Millistak+™ HC pod filter, D0HC, 0.027 m 2 (Merck). As the 0.22 μm filter, a 0.22 μm bottle top filter (Thermo Fisher, 597-4520) was used. The protein concentration was quantified by ELISA (SARS-CoV-2 Spike Protein S1 RBD ELISA Kit (Elabscience, E-EL-E605)) according to the manufacturer's manual.
　結果は、以下の通りであった。まず、細胞生存率および細胞密度の経時変化は図１に示される通りであった。図１に示されるように、生細胞密度および細胞生存率は、遺伝子導入６０時間後までは良好であったが、その後、大きく低下した。 The results were as follows. First, the changes in cell viability and cell density over time were as shown in Figure 1. As shown in Figure 1, the viable cell density and cell viability were good up until 60 hours after gene transfer, but then dropped significantly.
　タンパク質発現については、表１に示される通りであった。表１に示されるように、遺伝子導入後５日目の試料では、回収タンパク質量が、約４０～５０％であった。遺伝子導入６０時間後の上清中の組換えタンパク質を定量した。デプスフィルターろ過によるタンパク質回収率の低下を懸念し、回収工程では、デプスフィルターろ過をせず、０．２２μｍフィルターろ過のみを実施した。回収された組換えタンパク質量が改善した。遺伝子導入４０時間後の上清中の組換えタンパク質を定量した。回収工程では、デプスフィルターろ過をせず、０．２２μｍフィルターろ過のみを実施した。 Protein expression was as shown in Table 1. As shown in Table 1, the amount of recovered protein was approximately 40-50% in the sample taken 5 days after gene introduction. The recombinant protein in the supernatant was quantified 60 hours after gene introduction. Due to concerns that depth filter filtration would reduce the protein recovery rate, depth filter filtration was not performed in the recovery process, and only 0.22 μm filter filtration was carried out. The amount of recovered recombinant protein was improved. The recombinant protein in the supernatant was quantified 40 hours after gene introduction. Depth filter filtration was not performed in the recovery process, and only 0.22 μm filter filtration was carried out.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　各サンプルを還元剤で処理し、または還元剤で処理せずにＳＤＳ－ＰＡＧＥに供し、その後、ウェスタンブロッティングを実施した。１次抗体としては、ウマポリクローナル抗体（ＬＢＤ５６）を用い、２次抗体としては、Ａｎｔｉ－Ｈｏｒｓｅ　ＩｇＧ（Ｈ＋Ｌ）（Ｐｒｏｔｅｉｎｔｅｃｈ　Ｃａｔ．　Ｎｏ．　ＳＡ００００１－１３）を用いた。ウェスタンブロットの結果は、図２に示される通りであった。図２中の各レーンは表２に示される通りであった。 Each sample was subjected to SDS-PAGE with or without treatment with a reducing agent, and then Western blotting was performed. Horse polyclonal antibody (LBD56) was used as the primary antibody, and Anti-Horse IgG (H+L) (Proteintech Cat. No. SA00001-13) was used as the secondary antibody. The results of the Western blot are shown in Figure 2. Each lane in Figure 2 is as shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図２に示されるように、目的のサイズに組換えタンパク質のバンドが確認された。図２のレーン１０～１７に着目すると、目的のバンドより上（５０ｋＤａと７５ｋＤａの間の領域）にもバンドが検出された。還元条件下では、このバンドは認められないこと、およびバンドのサイズが組換えタンパク質の約２倍であることを考慮すると、このバンドは、組換えタンパク質の二量体に対応することが示唆された。図２のレーン１１および１５では、組換えタンパク質の単量体よりも二量体のバンドが濃く染色されており、その存在量に関して、単量体に対して二量体が優勢であることが示唆された。このことから、遺伝子導入１日後では発現量が少なすぎ、２日目には発現量が増えると共に二量体が増え、３日目以降は発現量が増えるものの単量体の割合が増加することが明らかになった。この結果から組換えタンパク質の回収は、遺伝子導入４０時間後から６０時間後に行うことが好ましいと理解された。 As shown in Figure 2, a band of the recombinant protein was confirmed at the desired size. Looking at lanes 10 to 17 in Figure 2, a band was also detected above the desired band (between 50 kDa and 75 kDa). Considering that this band was not observed under reducing conditions and that the size of the band was about twice that of the recombinant protein, it was suggested that this band corresponds to a dimer of the recombinant protein. In lanes 11 and 15 in Figure 2, the band of the dimer was stained darker than the monomer of the recombinant protein, suggesting that the dimer was predominant in terms of abundance compared to the monomer. This revealed that the expression level was too low one day after gene introduction, that the expression level increased and the dimer increased on the second day, and that the expression level increased from the third day onwards, but the proportion of the monomer increased. From this result, it was understood that it is preferable to collect the recombinant protein 40 to 60 hours after gene introduction.
　遺伝子導入４０時間後に回収した組換えタンパク質をＢＣＡ法で定量し、ＥＬＩＳＡでも定量した。結果は、表３に示される通りであった。 The recombinant protein recovered 40 hours after gene transfer was quantified by the BCA method and also by ELISA. The results are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　ＢＣＡ法によりタンパク質濃度を表３に記載される通りに調製した。得られたタンパク質濃度調整済みサンプルをＥＬＩＳＡ測定に供したところ、ＥＬＩＳＡで推定された濃度は、サンプル番号１～４において現実のタンパク質濃度をはるかに凌駕した。このことは、得られたタンパク質が、タンパク質量に比して抗体と極めて強く反応すること、すなわち、その免疫原活性が顕著に高いことを示唆する。 The protein concentration was adjusted by the BCA method as shown in Table 3. The obtained samples with adjusted protein concentrations were subjected to ELISA measurement, and the concentrations estimated by ELISA far exceeded the actual protein concentrations in samples 1 to 4. This suggests that the obtained proteins react extremely strongly with antibodies compared to the protein amount, i.e., their immunogenic activity is remarkably high.
実施例２：動物における抗体産生の誘導
　本実施例では、実施例１において遺伝子導入４０時間後に回収された細胞に由来する精製Ｓ１－ＲＢＤ－Ｈｉｓタグ（ＷＴ）、前記精製Ｓ１－ＲＢＤ－Ｈｉｓタグ（ＷＴ）と精製Ｓ１－ＲＢＤ－Ｈｉｓタグ（δ株）の混合物のそれぞれを免疫原として用いた。免疫原は、フロイント完全アジュバント（ＤＩＦＣＯ　２６３８１０）またはフロイント不完全アジュバント（ＤＩＦＣＯ　２６３９１０）と混合し、エマルションを得た。免疫原投与量は、タンパク質量換算で５ｍｇ／日であった。 Example 2: Induction of antibody production in animals In this example, purified S1-RBD-His tag (WT) derived from cells collected 40 hours after gene transfer in Example 1, and a mixture of the purified S1-RBD-His tag (WT) and purified S1-RBD-His tag (δ strain) were used as immunogens. The immunogen was mixed with Freund's complete adjuvant (DIFCO 263810) or Freund's incomplete adjuvant (DIFCO 263910) to obtain an emulsion. The immunogen was administered at a dose of 5 mg/day in terms of protein amount.
　動物は、ウマとした。ウマとしては、それぞれ異なる血統を有するサラブレッド個体（１２～１８歳）を用いた。ウマへの免疫は以下のように行った。０日目にフロイント完全アジュバントと混合した組換えタンパク質（総量５ｍｇ）を頸部および臀部に注射した。１４日目にフロイント不完全アジュバントと混合した組換えタンパク質（総量５ｍｇ）を投与し、２８日目に、生理食塩水で希釈した組換えタンパク質（総量５ｍｇ）をさらに投与した。様々な時点で検査用の少量採血を実施した。加えて、５６日目、６３日目、および７０日目にそれぞれ４％のチトラミン液２５０ｍＬを入れたバックで５Ｌの大量採血を行った。採取された血液は、室温で１時間静置して血球細胞を沈殿させ、血球成分を除いた血清を使用時まで冷蔵保存した。取得された血清を抗ＷＴ血清および抗ＷＴ＆Ｄｅｌｔａ血清とそれぞれ呼ぶ。また、免疫ｎ日後に得られた抗ＷＴ＆Ｄｅｌｔａ血清からポリクローナル抗体をＬＡＤｎとよび、免疫ｎ日後に得られた抗Ｄｅｌｔａ血清から得られたポリクローナル抗体をＬＢＤｎとよぶ。すなわち、ＬＢＤ５６は、免疫５６日後に得られた抗Ｄｅｌｔａ血清から得られたポリクローナル抗体を意味する。 The animals used were horses. Thoroughbreds (12-18 years old) of different pedigrees were used as horses. The horses were immunized as follows: On day 0, recombinant protein (5 mg total) mixed with Freund's complete adjuvant was injected into the neck and rump. On day 14, recombinant protein (5 mg total) mixed with Freund's incomplete adjuvant was administered, and on day 28, recombinant protein (5 mg total) diluted with saline was further administered. Small amounts of blood were collected for testing at various time points. In addition, large amounts of blood (5 L) were collected in bags containing 250 mL of 4% cytramine solution on days 56, 63, and 70. The collected blood was left to stand at room temperature for 1 hour to precipitate blood cells, and the serum from which blood cell components had been removed was refrigerated until use. The obtained sera are called anti-WT serum and anti-WT&Delta serum, respectively. In addition, the polyclonal antibody obtained from the anti-WT&Delta serum obtained n days after immunization is called LADn, and the polyclonal antibody obtained from the anti-Delta serum obtained n days after immunization is called LBDn. That is, LBD56 means the polyclonal antibody obtained from the anti-Delta serum obtained 56 days after immunization.
実施例３：得られた血清の試験
　実施例２で採取した血液（検査用）から血清分離を行い、試験した。 Example 3: Testing of obtained serum Serum was separated from the blood (for testing) collected in Example 2 and tested.
（１）ｃＰａｓｓ試験
　得られた血清について中和抗体の力価を測定した。抗体力価は、ｃＰＡＳＳ　ＳＡＲＳ－ＣｏＶ－２中和抗体検出キット（ＧｅｎＳｃｒｉｐｔ）を用いて測定された。このキットは、精製した受容体結合ドメイン（ＲＢＤ）、ウイルススパイク（Ｓ）タンパク質由来のタンパク質、宿主細胞受容体ＡＣＥ２を用いて、試験管内またはＥＬＩＳＡプレートのウェル内でタンパク質－タンパク質の直接相互作用により、ウイルス－宿主間の相互作用を模倣するよう設計されている。中和抗体価５０％ＩＣ（ＩＣ５０）は、常法により算出した。具体的には、阻害率（％）をプロビット変換し、希釈倍率とプロビット変換された阻害率とを軸とするグラフにプロットし、近似直線でフィッティングして阻害率５０％に相当する希釈率として求めた。 (1) cPass test The titer of neutralizing antibodies was measured for the obtained serum. The antibody titer was measured using the cPASS SARS-CoV-2 Neutralizing Antibody Detection Kit (GenScript). This kit is designed to mimic virus-host interactions by direct protein-protein interactions in a test tube or in the wells of an ELISA plate using purified receptor binding domain (RBD), a protein derived from the viral spike (S) protein, and the host cell receptor ACE2. The neutralizing antibody titer 50% IC (IC 50 ) was calculated by a conventional method. Specifically, the inhibition rate (%) was probit converted, plotted on a graph with the dilution factor and the probit-converted inhibition rate as axes, and fitted with an approximate straight line to determine the dilution rate equivalent to an inhibition rate of 50%.
　試験の結果は、図３に示される通りであった。図３に示される通り、良好な中和抗体の産生が確認され、中和抗体価は４２日目でほぼプラトーに達した。このｃＰａｓｓ試験における推定中和抗体価（５０％ＩＣ；希釈倍率で表される）は、５６日目に５１，６５５であり、６３日目に３９，５８１であり、７０日目に２８，９９８であった。表４にはワクチン接種による獲得中和抗体価を示す。この結果は、血統の異なるウマで共通していたことから、サラブレッドに共通した特性であることが示唆された。 The test results are shown in Figure 3. As shown in Figure 3, good production of neutralizing antibodies was confirmed, with the neutralizing antibody titer reaching a plateau on day 42. The estimated neutralizing antibody titers (50% IC; expressed as dilution factor) in this cPass test were 51,655 on day 56, 39,581 on day 63, and 28,998 on day 70. Table 4 shows the neutralizing antibody titers acquired by vaccination. This result was common to horses of different bloodlines, suggesting that this is a common characteristic among thoroughbreds.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　本実施例の中和抗体力価は、これまでのワクチン製品による中和抗体価と比較して、桁違いの中和抗体価をもたらす。 The neutralizing antibody titer of this example is an order of magnitude higher than the neutralizing antibody titers of previous vaccine products.
　抗原ＥＬＩＳＡにより抗血清中の特異的抗体量を定量した。結果は、図４に示される通りであった。特異的抗体量は、約４０日から６０日にピークを有したが、その後も高い抗体量が長期に持続した。 The amount of specific antibodies in the antiserum was quantified by antigen ELISA. The results are shown in Figure 4. The amount of specific antibodies peaked at about 40 to 60 days, and high levels of antibodies continued for a long period thereafter.
（２）ＳＡＲＳ－ＣｏＶ－２変異株に対する中和抗体価
　ＳＡＲＳ－ＣｏＶ－２のＷＴ株、α株、β株、γ株、δ株、およびο（オミクロン）株のそれぞれに対する中和抗体価を上記（１）と同様に試験した。結果は、図５に示される通りであった。図５に示されるように、いずれの抗血清も様々な変異株に対して中和抗体を誘導した。また、ウマ血清は、臨床的な感染者（ヒト）血清よりも高い中和抗体価を示した。 (2) Neutralizing antibody titers against SARS-CoV-2 mutant strains Neutralizing antibody titers against SARS-CoV-2 WT, α, β, γ, δ, and ο (omicron) strains were tested in the same manner as in (1) above. The results are shown in Figure 5. As shown in Figure 5, all antisera induced neutralizing antibodies against various mutant strains. In addition, horse serum showed a higher neutralizing antibody titer than clinically infected (human) sera.
（３）シュードウイルス中和試験（ＰＶＮＴ）
　ＰＶＮＴでは、ＳＡＲＳ－ＣｏＶ－２の感染を模擬するために、レンチウイルスベクター中のエンベロープ糖タンパク質をＳタンパク質に置き換えたシュードウイルスを用いた。シュードウイルスは、ルシフェラーゼをコードする核酸を含んでおり、細胞に感染するとルシフェラーゼを細胞内で発現した。したがって、ウイルスの細胞への感染量は、ルシフェラーゼによる発光強度により推定できた。ＰＶＮＴでは、抗体がルシフェラーゼ発現を抑制する効果を観察することで、ウイルスに対する感染抑制能を推定した。ＰＶＮＴは、ＳＡＲＳ－ＣｏＶ－２　Ｐｓｅｕｄｏｖｉｒｕｓ　Ｎｅｕｔｒａｌｉｚａｔｉｏｎ　Ａｓｓａｙ　Ｋｉｔ　Ｌｕｃ　Ｒｅｐｏｒｔｅｒ（ＳＣ２０８７Ａ、ＳＣ２０８７－０２７、及びＳＣ２０８７－Ｗ）を用いて実施した。抗体としては、ウマ血清からカプリル酸分画法（５ｖ／ｖ％カプリル酸を用いる）により精製したウマポリクローナル抗体を用いた。 (3) Pseudovirus Neutralization Test (PVNT)
In PVNT, a pseudovirus in which the envelope glycoprotein in the lentivirus vector was replaced with the S protein was used to simulate infection with SARS-CoV-2. The pseudovirus contained a nucleic acid encoding luciferase, and expressed luciferase in cells when infected with the cells. Therefore, the amount of virus infected into the cells could be estimated from the luminescence intensity by luciferase. In PVNT, the ability of the antibody to suppress infection with the virus was estimated by observing the effect of suppressing luciferase expression. PVNT was performed using SARS-CoV-2 Pseudovirus Neutralization Assay Kit Luc Reporter (SC2087A, SC2087-027, and SC2087-W). The antibody used was a horse polyclonal antibody purified from horse serum by the caprylic acid fractionation method (using 5 v/v % caprylic acid).
　結果は図５に示される通りであった。図５に示されるように、上記で得られたウマ血清は、変異株によらず、様々な変異株に対して高い中和抗体価を示した。また、ウマ血清の中和抗体価は、コロナウイルスのＲＮＡワクチン（モデルナ社製またはファイザー社製）を２回または３回投与されたヒトの血清の中和抗体力価よりも高かった。 The results are shown in Figure 5. As shown in Figure 5, the horse serum obtained as described above showed high neutralizing antibody titers against various mutant strains, regardless of the mutant strain. In addition, the neutralizing antibody titers of the horse serum were higher than the neutralizing antibody titers of human serum that had been administered two or three times with a coronavirus RNA vaccine (Moderna or Pfizer).
（４）市販ポリクローナル抗体との比較
　市販ポリクローナル抗体と中和抗体価を比較した。ウマ血清は、Ｓ１－ＲＢＤ－Ｈｉｓタグ（ＷＴ）を免疫して得られた血清を用いた。マウスポリクローナル抗体は、Ｓ１のＲＢＤに対するマウスポリクローナルである４０５９２－ＭＰ０１（Ｓｉｎｏ　Ｂｉｏｌｏｇｉｃａｌ）とし、ウサギポリクローナル抗体は、スパイクタンパク質Ｓ１に対するウサギポリクローナル抗体であるＧＴＸ１３５３５６（ＧｅｎｅＴｅｘ）とし、ヒトポリクローナル抗体は、コロナウイルス陽性ドナーから得られた血清であるＣｏｖ－Ｎｅｕｔ－Ｓ－５００（Ｒａｙ　Ｂｉｏｔｅｃｈ）とした。結果は、表５に示される通りであった。 (4) Comparison with Commercially Available Polyclonal Antibodies The neutralizing antibody titer was compared with that of a commercially available polyclonal antibody. The horse serum used was serum obtained by immunizing S1-RBD-His tag (WT). The mouse polyclonal antibody was 40592-MP01 (Sino Biological), a mouse polyclonal antibody against the RBD of S1, the rabbit polyclonal antibody was GTX135356 (GeneTex), a rabbit polyclonal antibody against the spike protein S1, and the human polyclonal antibody was Cov-Neut-S-500 (Ray Biotech), a serum obtained from a coronavirus-positive donor. The results were as shown in Table 5.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表５に示されるように、上記ウマ血清は高い中和抗体価と比活性を示した。 As shown in Table 5, the above horse sera exhibited high neutralizing antibody titers and specific activity.
（５）抗原の多量体特性と、抗血清の抗原への結合特性
　実施例１で得られた抗原のウェスタンブロット（ＷＢ）結果に基づきバンドの濃さをデンシトメトリにより解析した。ＷＢは、１次抗体としてウマ抗血清から得られたポリクローナル抗体（ＬＢＤ５６、抗体希釈率１：１０００）または市販のマウスポリクローナル抗体（Ｓｉｎｏ　Ｂｉｏｌｏｇｉｃａｌ社製の　ＬｏｔＮｏ．　４０５９２－ＭＰ０１、抗体希釈率１：１００）を用い、２次抗体としてＧｏａｔ　Ｆ（ａｂ’）２　Ａｎｔｉ－Ｍｏｕｓｅ　ＩｇＧ　（Ｆａｂ’）２　（ＨＲＰ）（Ａｂｃａｍ，　ａｂ９８６５９）を用いた。クマシーブリリアントグリーン（ＣＢＢ）染色の結果およびウェスタンブロッティングの結果は図６に示される通りであった。各レーンの詳細は以下表６の通りであった。 (5) Multimeric characteristics of antigens and binding characteristics of antisera to antigens Based on the results of Western blot (WB) of the antigens obtained in Example 1, the band densities were analyzed by densitometry. For WB, a polyclonal antibody obtained from horse antiserum (LBD56, antibody dilution ratio 1:1000) or a commercially available mouse polyclonal antibody (Lot No. 40592-MP01, antibody dilution ratio 1:100, manufactured by Sino Biological Co., Ltd.) was used as the primary antibody, and Goat F(ab')2 Anti-Mouse IgG (Fab')2 (HRP) (Abcam, ab98659) was used as the secondary antibody. The results of Coomassie Brilliant Green (CBB) staining and Western blotting were as shown in FIG. 6. Details of each lane are as shown in Table 6 below.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　次に、得られたウェスタンブロット像に対してデンシトメトリ解析を実施した。具体的には、ウェスタンブロットで得られた像をコンピュータに取り込んで、画像解析によりバンドの濃さを数値化した。測定値について比を算出した。結果は、表７および８に示される通りであった。 Next, densitometry analysis was performed on the obtained Western blot images. Specifically, the images obtained by Western blot were imported into a computer and the band intensities were quantified by image analysis. The ratios of the measured values were calculated. The results are shown in Tables 7 and 8.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　表７に示されるように、免疫原として用いた組換えタンパク質は、多量体（特にダイマー）を多く含み、かつ、本実施例のウマ抗血清から得られたポリクローナル抗体は、多量体を良好に認識した。また、表８に示されるように、マウスポリクローナル抗体は、免疫原に対する反応性が弱かったが、多量体への結合特異性に関して、ウマポリクローナル抗体には及ばなかった。 As shown in Table 7, the recombinant protein used as the immunogen contained many multimers (especially dimers), and the polyclonal antibody obtained from the horse antiserum in this example recognized the multimers well. Furthermore, as shown in Table 8, the mouse polyclonal antibody had weak reactivity to the immunogen, but did not match the horse polyclonal antibody in terms of binding specificity to multimers.
　多量体ＲＢＤ（特にＲＢＤの二量体）で免疫されたウマから産生される抗体は、驚くべき優れた結合特性および中和活性を示した。また、ウマへの免疫は、マウスへの免疫と比較して、より抗原への親和性の高い抗体を含む多様な抗体群の産生に適していることが示唆される。したがって、多量体ＲＢＤ（特にＲＢＤの二量体）で免疫されたウマから産生される抗体からは、優れたモノクローナル抗体を取得することができると期待できる。上記ポリクローナル抗体の優れた特性は、複数の抗体による効果と考えられ、したがって、多量体ＲＢＤ（特にＲＢＤの二量体）で免疫されたウマから産生される抗体からは、優れた複数のモノクローナル抗体を再現性をもって取得できると期待できる。なお、サラブレッドは遺伝学的に均質性が高いことで知られており、サラブレッドへの免疫は、産生される抗体の多様性および品質を一定に保つ上で適していると考えられる。 The antibodies produced from horses immunized with multimeric RBD (particularly RBD dimers) showed surprisingly excellent binding properties and neutralizing activity. It is also suggested that immunization of horses is more suitable for producing a diverse group of antibodies, including antibodies with higher affinity to antigens, than immunization of mice. Therefore, it is expected that excellent monoclonal antibodies can be obtained from antibodies produced from horses immunized with multimeric RBD (particularly RBD dimers). The excellent properties of the above polyclonal antibodies are thought to be the effect of multiple antibodies, and therefore it is expected that multiple excellent monoclonal antibodies can be reproducibly obtained from antibodies produced from horses immunized with multimeric RBD (particularly RBD dimers). Furthermore, thoroughbreds are known to be highly genetically homogeneous, and immunization of thoroughbreds is thought to be suitable for maintaining a constant diversity and quality of antibodies produced.
（６）得られた血清の毒性
　得られた血清をラットに静脈内投与して毒性を検査した。ラット（Ｃｒｌ：ＣＤ（ＳＤ）、オス、７週齢、体重１４０～２２０ｇ）を体重の層別無作為化により３群（各５匹）にわけた。想定される臨床用量の１３倍（９３ｍｇ／ｋｇ）もしくは２６倍（１８６ｍｇ／ｋｇ）の血清または生理食塩水を静脈投与した。投与１３日後、生理食塩水を投与した対照群と比較して、異常の有無を調べた。前例で一般状態に異常は認められず、体重推移にも異常はなかった。ラットの解剖によっても、前例において異常は認められず、器官重量についても血清に起因すると考えられる変化は認められなかった。 (6) Toxicity of the obtained serum The obtained serum was intravenously administered to rats to examine its toxicity. Rats (Crl:CD (SD), male, 7 weeks old, weighing 140-220 g) were randomly divided into three groups (five rats each) by stratification according to weight. Serum or saline was administered intravenously at 13 times (93 mg/kg) or 26 times (186 mg/kg) the expected clinical dose. Thirteen days after administration, the presence or absence of abnormalities was examined in comparison with the control group administered saline. No abnormalities were observed in the general condition of the previous case, and no abnormalities were observed in the weight transition. No abnormalities were observed in the previous case when the rats were dissected, and no changes in organ weights that could be attributed to serum were observed.

Claims (20)

  1. 　ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）の受容体結合ドメイン（ＲＢＤ領域）を含む免疫原性組成物に対するウマの抗血清であって、Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、抗血清。 An equine antiserum against an immunogenic composition containing the receptor binding domain (RBD region) of the spike protein (S protein) of a betacoronavirus, which is capable of neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).
  2. 　免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、請求項１に記載の抗血清。 The antiserum of claim 1, wherein the immunogenic composition comprises a dimerized RBD region.
  3. 　免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、請求項２に記載の抗血清。 The antiserum of claim 2, wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions.
  4. 　ウマが、サラブレッド個体である、請求項１～３のいずれか一項に記載の抗血清。 The antiserum according to any one of claims 1 to 3, wherein the horse is a thoroughbred.
  5. 　ウマが、サラブレッド個体である、請求項３に記載の抗血清。 The antiserum of claim 3, wherein the horse is a thoroughbred.
  6. 　請求項１～５のいずれか一項に記載の抗血清から精製されたポリクローナル抗体を含む組成物。 A composition comprising a polyclonal antibody purified from the antiserum according to any one of claims 1 to 5.
  7. 　ヒト化バーチャルポリクローナル抗体を含む組成物であって、
    　複数の異なるモノクローナル抗体を含み、
    　各モノクローナル抗体は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）の受容体結合ドメイン（ＲＢＤ領域）に対するウマ抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含むか、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含み、
    　各モノクローナル抗体の残りの部分は、ヒト抗体に由来し、
    　各モノクローナル抗体は、前記Ｓタンパク質に結合することができ、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、組成物。     1. A composition comprising a humanized virtual polyclonal antibody,
    Contains a plurality of different monoclonal antibodies,
    Each monoclonal antibody comprises a portion of the structure of an equine antibody directed against the receptor binding domain (RBD region) of the spike protein (S protein) of a betacoronavirus, said portion of the structure of said antibody comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
    The remainder of each monoclonal antibody is derived from a human antibody,
    A composition, wherein each monoclonal antibody is capable of binding to said S protein and neutralizing the binding of said S protein to angiotensin converting enzyme 2 (ACE2).
  8. 　前記ウマが、Ｓタンパク質のＲＢＤ領域を含む免疫原性組成物により免疫されたウマである、請求項７に記載の組成物。 The composition of claim 7, wherein the horse is a horse immunized with an immunogenic composition comprising the RBD region of the S protein.
  9. 　前記免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、請求項８に記載の組成物。 The composition of claim 8, wherein the immunogenic composition comprises a dimerized RBD region.
  10. 　免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、請求項９に記載の組成物。 The composition of claim 9, wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions.
  11. 　ウマが、サラブレッド個体である、請求項７～１０のいずれか一項に記載の組成物。 The composition according to any one of claims 7 to 10, wherein the horse is a thoroughbred.
  12. 　ウマが、サラブレッド個体である、請求項１０に記載の組成物。 The composition of claim 10, wherein the horse is a thoroughbred.
  13. 　請求項７～１２のいずれか一項に記載の組成物の調製方法であって、
    　ウマをベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫し、ウマの体内に前記ＲＢＤ領域に対する抗体を産生させることと、
    　前記ウマから取得された複数の細胞が産生する抗体それぞれをコードする複数ｍＲＮＡそれぞれの重鎖可変領域および軽鎖可変領域の配列を決定することと、ここで、前記細胞は、脾臓細胞、Ｂ細胞および形質細胞からなる群から選択される細胞であり、
    　決定されたヌクレオチド配列をその配列同一性に基づいて複数のクラスタにクラスタリングすることと、
    　取得された複数のクラスタのうち、含まれるヌクレオチド配列数が多いクラスタの上位１０位から少なくとも２つのクラスタを選択することと、
    　選択されたクラスタそれぞれに含まれる重鎖可変領域および軽鎖可変領域を含むヒトキメラ抗体をコードするヌクレオチド配列か、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含むヒト化抗体をコードするヌクレオチド配列を得ることと、
    　得られたヌクレオチド配列それぞれを有する複数の抗体産生細胞から、前記ヒトキメラ抗体またはヒト化抗体を産生させ、前記ヒトキメラ抗体またはヒト化抗体を含む複数の抗体の混合物を得ることと、
    を含む、方法。     A process for preparing a composition according to any one of claims 7 to 12, comprising the steps of:
    Immunizing a horse with an immunogenic composition comprising an RBD region of a spike protein (S protein) of a beta coronavirus, thereby producing antibodies against the RBD region in the horse's body;
    determining sequences of heavy chain variable regions and light chain variable regions of each of a plurality of mRNAs encoding antibodies produced by a plurality of cells obtained from the horse, wherein the cells are selected from the group consisting of spleen cells, B cells, and plasma cells;
    clustering the determined nucleotide sequences into a plurality of clusters based on their sequence identity;
    selecting at least two clusters from the top 10 clusters containing the largest number of nucleotide sequences from among the obtained clusters;
    obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region comprised in each of the selected clusters, or a nucleotide sequence encoding a humanized antibody comprising three complementarity determining regions (CDRs) of a heavy chain variable region and three CDRs of a light chain variable region;
    producing the human chimeric or humanized antibodies from a plurality of antibody-producing cells each having the obtained nucleotide sequence, thereby obtaining a mixture of a plurality of antibodies containing the human chimeric or humanized antibodies;
    A method comprising:
  14. 　請求項７～１２のいずれか一項に記載の組成物の調製方法であって、
    　異なるモノクローナル抗体をそれぞれ産生する複数の抗体産生細胞からモノクローナル抗体を産生させ、産生された複数のモノクローナル抗体を含む組成物を得ることを含み、
    　組成物に含まれる各モノクローナル抗体は、ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）のＲＢＤ領域を含む免疫原性組成物で免疫され、体内にＲＢＤ領域に対する抗体群を有するウマにおいて検出される抗体群から選択される抗体の構造の一部を含み、前記抗体の構造の一部は、重鎖可変領域および軽鎖可変領域を含むか、または、重鎖可変領域の３つの相補性決定領域（ＣＤＲ）および軽鎖可変領域の３つのＣＤＲを含み、
    　各モノクローナル抗体の残りの部分は、ヒト抗体に由来し、
    　各モノクローナル抗体は、前記Ｓタンパク質とアンジオテンシン転換酵素２（ＡＣＥ２）との結合を中和することができる、方法。     A process for preparing a composition according to any one of claims 7 to 12, comprising the steps of:
    The method includes producing monoclonal antibodies from a plurality of antibody-producing cells each producing a different monoclonal antibody, and obtaining a composition containing the produced plurality of monoclonal antibodies;
    Each monoclonal antibody contained in the composition comprises a portion of an antibody structure selected from a group of antibodies detected in horses that have been immunized with an immunogenic composition containing the RBD region of the spike protein (S protein) of a betacoronavirus and have antibodies against the RBD region in their bodies, the portion of the antibody structure comprising a heavy chain variable region and a light chain variable region, or comprising three complementarity determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region;
    The remainder of each monoclonal antibody is derived from a human antibody,
    A method, wherein each monoclonal antibody is capable of neutralizing the binding of said S protein to angiotensin-converting enzyme 2 (ACE2).
  15. 　ウマが、サラブレッド個体である、請求項１３または１４に記載の方法。 The method of claim 13 or 14, wherein the horse is a thoroughbred.
  16. 　免疫原性組成物が、二量体を形成したＲＢＤ領域を含む、請求項１３～１５のいずれか一項に記載の方法。 The method according to any one of claims 13 to 15, wherein the immunogenic composition comprises a dimerized RBD region.
  17. 　免疫原性組成物が、単量体に対して、二量体を形成したＲＢＤ領域をより多く含む、請求項１６に記載の方法。 The method of claim 16, wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions.
  18. 　免疫原性組成物であって、
    　ベータコロナウイルスのスパイクタンパク質（Ｓタンパク質）の受容体結合ドメイン（ＲＢＤ領域）を含み、
    　前記ＲＢＤ領域は、二量体を形成しており、かつ、二量体の量が、ＲＢＤ領域の単量体の量よりも多い、免疫原性組成物。     1. An immunogenic composition comprising:
    It contains the receptor binding domain (RBD region) of the spike protein (S protein) of a betacoronavirus,
    An immunogenic composition, wherein the RBD region forms a dimer and the amount of the dimer is greater than the amount of the monomer of the RBD region.
  19. 　ウマを免疫することに用いるための、請求項１８に記載の免疫原性組成物。 The immunogenic composition of claim 18 for use in immunizing a horse.
  20. 　ウマを免疫する方法であって、
    　請求項１８に記載の免疫原性組成物をウマに投与することを含み、これにより、ウマにＲＢＤ領域に対する抗体を産生させる、方法。

          1. A method of immunizing a horse, comprising:
    20. A method comprising administering to a horse the immunogenic composition of claim 18, thereby causing the horse to produce antibodies against the RBD region.
PCT/JP2023/037814 2022-10-20 2023-10-19 Equine antiserum against betacoronavirus WO2024085207A1 (en)

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