Horse antiserum against betacoronavirus

Abstract

To provide horse antiserum for betacoronavirus, polyclonal antibodies obtained from the serum, monoclonal antibodies originating from horses, human-chimeric antibodies, humanized antibodies, and mixtures comprising a plurality of monoclonal antibodies.SOLUTION: The present invention provides horse antiserum against an immunogenic composition comprising an RBD region of a spike protein (S protein) of betacoronavirus, the antiserum being capable of neutralizing binding between the S protein and angiotensin-converting enzyme 2 (ACE2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to equine antisera against betacoronaviruses, and more specifically to equine antisera against betacoronaviruses, polyclonal antibodies obtained therefrom, equine-derived monoclonal antibodies, human chimeric antibodies, and humanized antibodies, as well as mixtures of monoclonal antibodies. [Background technology]

[0002] Coronaviruses, particularly SARS-CoV-2, have caused pandemics, and efforts are underway to develop methods to prevent or treat infection. Antibodies are believed to be a potential treatment for infection, and monoclonal and polyclonal antibodies are being developed. Antibodies have been obtained from a variety of animal species, including cattle, chickens, pigs, and horses. Clinical trials of equine polyclonal antibodies are being conducted to develop 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 the SARS-CoV-2 spike protein, N protein, and spike EM mosaic protein was used as an immunogen to immunize horses. 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. In 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. Summary of the Invention

[0003] The present disclosure provides equine antisera against betacoronaviruses, and more specifically, the present disclosure provides 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.

[0004] According to the present disclosure, for example, the following inventions can be provided. (1) An equine antiserum against an immunogenic composition comprising the 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 contains 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 humanized virtual polyclonal antibody, comprising 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), wherein the portion of the antibody structure comprises a heavy chain variable region and a light chain variable region, or comprises 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 the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2). (8) The composition described in (7) above, wherein the horse is a horse immunized with an immunogenic composition containing the RBD region of the S protein. (9) The composition according to (8), wherein the immunogenic composition comprises a dimerized RBD region. (10) The composition according to (9), wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions. (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, Immunizing a horse with an immunogenic composition containing the RBD region of the spike protein (S protein) of a beta coronavirus to produce antibodies against the RBD region in the horse's body; determining the sequences of the heavy chain variable region and the light chain variable region of each of multiple mRNAs or cDNAs encoding antibodies produced by multiple 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 the obtained clusters; obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region contained 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 sequences, 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, producing monoclonal antibodies from a plurality of antibody-producing cells that each produce a different monoclonal antibody, and obtaining a composition containing the produced 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 that have antibodies against the RBD region in their bodies, and the portion of the antibody structure comprises a heavy chain variable region and a light chain variable region, or comprises 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 the 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 monomeric RBD regions. (18) An immunogenic composition, comprising: It contains the RBD region of the spike protein (S protein) of betacoronavirus, An immunogenic composition, wherein the RBD region forms a dimer and the amount of the dimer is greater than the amount of the RBD region monomer. (19) The immunogenic composition according to (18) above, for use in immunizing a horse. (20) A method for immunizing a horse, comprising: A method comprising administering the immunogenic composition described in (18) above to a horse, thereby causing the horse to produce antibodies against the RBD region.

[0005] (101) The antiserum according to (1) above, wherein the horse is a thoroughbred. (102) The antiserum according to (1) above, wherein the immunogenic composition comprises a dimerized RBD region and the horse is a thoroughbred. (103) The antiserum according to (1), wherein the immunogenic composition contains a dimerized RBD region, the dimerized RBD region is more abundant than the monomeric RBD region, and the horse is a Thoroughbred. (104) The antiserum according to (1) above, wherein the horse has a body weight of 400 kg to 800 kg. (105) The antiserum according to (101) above, wherein the horse has a body weight of 400 kg to 800 kg. (106) The antiserum according to (102) above, wherein the horse has a body weight of 400 kg to 800 kg. (107) The antiserum according to (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 according to (101) above, wherein the horse has a body weight of 500 kg to 700 kg. (110) The antiserum according to (102) above, wherein the horse has a body weight of 500 kg to 700 kg. (111) The antiserum according to (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.

[0006] (201) A composition or pharmaceutical composition comprising a humanized virtual polyclonal antibody. (202) A composition or pharmaceutical composition comprising a humanized virtual polyclonal antibody, 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), wherein the portion of the antibody structure comprises or consists of a heavy chain variable region and a light chain variable region, or comprises or consists of three complementarity-determining regions (CDRs) of the heavy chain variable region and three CDRs of the light chain variable region; A 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 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). (205) The composition or pharmaceutical composition described in (203) 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). (206) The composition or pharmaceutical composition described in (202) above, wherein each 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). (207) The composition or pharmaceutical composition described in (203) above, wherein each 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). (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.

[0007] (301) A method for preparing the composition according to any one of (201) to (210) above, producing monoclonal antibodies from a plurality of antibody-producing cells that each produce a different monoclonal antibody, and obtaining a composition containing the produced 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 that have antibodies against the RBD region in their bodies, and the portion of the antibody structure comprises or consists of a heavy chain variable region and a light chain variable region, or comprises or consists 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 of the S protein to 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 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). (306) The composition or pharmaceutical composition described in (302) above, wherein each 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). (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.

[0008] (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 beta 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 the δ strain of SARS-CoV-2. (408) Any of the above inventions, wherein the beta coronavirus from which the RBD region of the immunogenic composition is derived is the O strain of SARS-CoV-2. (409) Any of the above-described 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).

[0009] (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 betacoronavirus from which the S protein to be bound is derived is SARS-CoV-2. (503) Any of the above-mentioned inventions, wherein the betacoronavirus from which the S protein to be bound is derived is the wild type of 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 the beta 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).

[0010] (601) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes four or more types of monoclonal antibodies. (602) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes five or more types of monoclonal antibodies. (603) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes six or more types of monoclonal antibodies. (604) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes seven or more types of monoclonal antibodies. (605) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes eight or more types of monoclonal antibodies. (606) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes nine or more types of monoclonal antibodies. (607) Any of the above-described inventions, wherein the plurality of monoclonal antibodies includes 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 exhibit significant competition for binding to the RBD region. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 shows changes in viable cell density (VCD) and cell viability after gene transfection of immunogen-producing cells. [Figure 2] Figure 2 shows the results of Western blot of the obtained immunogenic composition. The contents of each lane are as shown in Table 2. [Figure 3] Figure 3 shows the neutralizing antibody titers (50% IC) of serum from horses immunized with an immunogenic composition containing the immunogen (wild-type (WT) or a mixture of WT and mutant δ strains (WT&Delta)) collected 40 hours after gene transfer. [Figure 4] Figure 4 shows the amount of antigen-specific antibody in the serum of horses immunized with immunogen-containing immunogenic compositions (WT or WT&Delta) collected 40 hours after gene transfer. Panel A of Figure 4 shows the time course of the amount of antibody 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 antibody specific to the immunogen (WT) in the antisera of horses immunized with the immunogens (WT&Delta), panel C shows the time course of the amount of antibody 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 antibody specific to the immunogen (Delta) in the antisera of horses immunized with the immunogens (WT&Delta). [Figure 5] Figure 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). [Figure 6] FIG. 6 shows the CBB staining and Western blot (WB) results of the immunogens. [Figure 7] FIG. 7 shows how each region (monomer: A and B, dimer: C, trimer: D, and multimer: E) was set in densitometry analysis based on the Western blot results of the immunogen. Modes for carrying out the invention

[0012] As used herein, a "subject" refers to a vertebrate, e.g., a mammal, including a human, such as a mammal (e.g., cat, ferret, bat, or pangolin) infected by a coronavirus (e.g., SARS-CoV-2). The subject may be a subject infected with a coronavirus, an asymptomatic carrier infected with a coronavirus, or a subject infected with SARS-CoV-2 and developing COVID-19. The subject may be a subject at risk for or at risk of being infected with a coronavirus. The subject may be a child (e.g., infant (1-6 years of age)), a school-age child (6-12 years of age), an adolescent (12 years or older), or an adult (20 years or older). An adult may be 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, more preferably SARS-CoV-2.

[0013] As used herein, "coronavirus" refers to a single-stranded, positive-strand RNA virus in the Orthocoronavirus subfamily of the Coronaviridae family of the Nidovirales order. Coronaviruses are named for their spike protein protrusions (S proteins) on their surface, which resemble the solar corona. In humans, they cause respiratory infections, including the common cold. Orthocoronaviruses are broadly classified into alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. SARS-related coronaviruses are classified as betacoronaviruses. SARS-related coronaviruses include SARS coronavirus (SARS-CoV) and SARS coronavirus 2 (SARS-CoV-2). SARS-CoV-2 has been causing the novel coronavirus disease (COVID-19) pandemic since the end of 2019. SARS-related coronaviruses infect host cells by binding to the ACE2 receptor on the host cell via the S protein. SARS-related 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 within it that enhances infectivity and pathogenicity (Andersen et al., Nature Medicine, 26, 450-452, 2020).

[0014] 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 this virus 2019-nCoV. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) officially named this 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). WHO has named this new coronavirus COVID-19. The International Committee on Taxonomy of Viruses (ICTV) has designated SARS-CoV-2 as belonging to the Betacoronavirus genus and the same species (or sister lineage) as SARS-CoV. The complete genome sequence of the wild-type (or Wuhan) strain of SARS-CoV-2 has been deposited with the National Center for Biotechnology Information (NCBI) under Gene Bank accession number MN908947.3. Virus particles (virions) are approximately 50–200 nm in diameter and, like common coronaviruses, contain spike proteins, nucleocapsid proteins, membrane proteins, and envelope proteins, as well as viral genomic RNA. The nucleocapsid protein forms a complex with RNA, which is then surrounded by lipid-bound spike proteins, membrane proteins, and envelope proteins to form the virion envelope. The spike proteins, located on the outermost surface of the envelope, are thought to bind to ACE2 receptors on the cell surface and promote cell infection. Some SARS-CoV-2 infections do not show symptoms; these are called asymptomatic carriers. It has been noted that asymptomatic carriers may transmit the virus to others. SARS-CoV-2 infection has been reported to cause a decrease or loss of smell and / or taste. SARS-CoV-2 can cause severe acute respiratory syndrome (SAS). Symptoms of SAS include a fever of approximately 40°C, cough, and shortness of breath. The most notable complication is pneumonia. SARS-CoV-2 infection is primarily determined by PCR testing.This PCR test evaluates whether SARS-CoV-2 genes are present in the body by amplifying a SARS-CoV-2-specific band. Treatments for SARS-CoV-2 include antiviral drugs (e.g., remdesivir), steroidal 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).

[0015] As used herein, the term "spike protein" refers to a protein encoded by positions 21,563 to 25,384 of the SARS-CoV-2 genome, registered with the National Center for Biotechnology Information (NCBI) under GenBank accession number MN908947.3. The SARS-CoV-2 spike protein has an amino acid sequence registered with NCBI under GenBank accession number QHD43416.1. The spike protein includes S1 and S2, with S1 located at positions 13 to 541 of the amino acid sequence, and S2 located at positions 543 to 1,208 of the amino acid sequence. S1 further includes an N-terminal domain (NTD) and a receptor binding domain (RBD), with the NTD located at positions 13 to 304 of the amino acid sequence, and the RBD located at positions 319 to 541. 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 intracellularly and produced as separate peptides, which 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 host cells, and can infect cells. Any spike protein (having an amino acid sequence corresponding to the amino acid sequence registered with NCBI under GenBank accession number QHD43416.1) found in natural viruses (including mutant viruses) can be used as the spike protein. Examples of amino acid sequences of spike proteins of mutant viruses are shown below. α strain: Compared to the wild-type strain sequence, it has the following mutations: HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. β strain: Compared to the wild-type strain sequence, it has the following mutations: L18F, D80A, D215G, LAL242-244del, R246I, K417N, E484K, N501Y, D614G, and A701V. Gamma strains: have the following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F. Strain δ: Compared to the wild-type strain sequence, it has the following mutations: T19R, G142D, EF156-157del, R158G, L452R, T478K, D614G, P681R, and D950N. Strain ο: Compared to the sequence of the wild-type strain, it has the following mutations: 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, and L981F.

[0016] As used herein, "peptide" refers to a polymer of amino acids. A polymer is usually unbranched. "Partial peptide" refers to a portion of a specific 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. Isolation means that peptides and partial peptides are at least separated from other components, and purification means that peptides and partial peptides are at least selectively separated. Enrichment means that the concentration of peptides and partial peptides is increased.

[0017] As used herein, a "composition" refers to a mixture of one or more components. The composition may comprise, for example, a partial peptide and an aqueous solvent (e.g., water). The composition may further comprise a pharmaceutically acceptable excipient. As used herein, an immunogenic composition is a composition that can elicit an immune response in a subject when administered to the subject. The immunogenic composition can be used to induce an immune response in a subject. Because the immunogenic composition can elicit an immune response in a subject, it can be used as a vaccine. For example, the immunogenic composition of the present invention can be used to induce an immune response against a SARS-associated coronavirus (e.g., SARS-CoV-2), or can be used as a vaccine or therapeutic agent against a SARS-associated coronavirus (e.g., SARS-CoV-2).

[0018] As used herein, "treatment" includes preventive and therapeutic treatment. Therapeutic treatment can be performed against an infected virus, and preventive treatment can be performed to prevent future infection, or to delay the onset of a coronavirus infection (e.g., COVID-19) due to future infection, or to reduce the symptoms of an existing coronavirus infection (e.g., COVID-19). Therapeutic treatment can be performed on symptomatic patients or asymptomatic pathogen carriers. Prophylactic treatment can be performed on uninfected individuals.

[0019] As used herein, the terms "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, gene transfer, or other manipulation, as well as to the gene or nucleic acid artificially introduced into a target cell and its expressed protein. An exogenous gene may be operably linked to a promoter sequence that drives expression of the gene. As used herein, "endogenous" or "endogenously" means something that is inherent in the cell.

[0020] As used herein, the term "derived from" refers to the animal species from which a cell or antibody is obtained. For example, a human-derived cell means that the cell is 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 obtained from a horse, or that the antibody is produced from an antibody-producing cell having a nucleotide sequence encoding an equine antibody or a variant thereof, e.g., an equine antibody, a human chimeric antibody thereof, or a humanized antibody thereof.

[0021] As used herein, "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

[0022] As used herein, "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 immunizing 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. Also, for example, having 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 An antibody (having a molecular weight of M or less) is an antibody that can specifically bind to a given antigen. The heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, while the light chain consists of a light chain variable region VL and a light chain constant region CL. Of these, the variable region fragment (Fv) consisting of VH and VL is directly involved in antigen binding and is the region that confers diversity to the antibody. The antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region. Within the variable region, the region that directly contacts the antigen has a high degree of diversity in amino acid sequence among antibodies and is called the complementarity-determining region (CDR). Regions other than the CDRs that have a nearly constant amino acid sequence among antibodies are called the framework region (FR). The light chain and heavy chain variable regions each contain three CDRs, which are called heavy chain CDR1-3 and light chain CDR1-3, respectively, from the N-terminus. The heavy chain variable region typically contains heavy chain FR1, heavy chain CDR1, heavy chain FR2, heavy chain CDR2, heavy chain FR3, heavy chain CDR3, and heavy chain FR4, in that order. The light chain variable region typically contains light chain FR1, light chain CDR1, light chain FR2, light chain CDR2, light chain FR3, light chain CDR3, and light chain FR4, in that order. The antibody may be a monoclonal or polyclonal antibody. Furthermore, the antibody of the present invention may be of any isotype, including IgG, IgM, IgA, IgD, and IgE. It may be produced by immunizing a non-human animal, such as a mouse, rat, hamster, guinea pig, rabbit, or chicken, or it may be a recombinant antibody, or it may be a chimeric antibody, humanized antibody, or fully humanized antibody. A chimeric antibody is an antibody in which antibody fragments derived from different species are linked. IgG subclasses in humans include IgG1, IgG2, IgG3, and IgG4. The antibody of the present invention may be of any subclass, but may also be one or more selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, such as IgG2, IgG3, or IgG4. The term "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). Examples of such antibodies include those that have heavy chain CDRs 1 to 3 (HCDRs 1 to 3, respectively) and light chain CDRs 1 to 3 (LCDRs 1 to 3, respectively) of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FRs) of the heavy and light chains, 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 include, but are not limited to, Fab consisting of VL, VH, CL, and CH1 domains; 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 can be an isolated monoclonal antibody (eg, an isolated humanized antibody).

[0023] As used herein, "isolated" means at least separated from other components. "Isolated" is used in a sense that includes separation from contaminants to an extent that is compatible with pharmaceutical products.

[0024] As used herein, the term "isolated" does not exclude mixing with other substances after isolation. Thus, as used herein, a composition obtained by mixing multiple isolated monoclonal antibodies (and optional additives) is a composition containing an isolated monoclonal antibody. 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). Furthermore, 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.

[0025] <SARS-associated coronavirus as a preferred embodiment> In all aspects and embodiments of the present disclosure, the betacoronavirus is preferably a SARS-associated coronavirus, more preferably SARS-CoV-2. The SARS-CoV-2 may be any one or more of the following SARS-CoV-2 strains: wild-type strain (the original strain isolated in Wuhan, China), alpha strains (B.1.1.7 and Q-series strains), beta strains (B.1.351 and its descendants), gamma strains (P.1 and its descendants), delta strains (B.1.617.2 and AY-series 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 omicron strains (B.1.1.529 and BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5-series strains), and variants thereof 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.

[0026] Antisera of the Present Disclosure According to the present disclosure, an equine antiserum is provided. The equine antiserum of the present disclosure can neutralize the binding of the spike protein (S protein) of betacoronavirus to angiotensin-converting enzyme 2 (ACE2). The equine antiserum of the present disclosure can also inhibit the infection of ACE2-expressing human cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells) by betacoronavirus. It is possible to obtain antibodies that inhibit the binding of S1 protein to ACE2, to obtain antibodies that inhibit the infection of human cells by betacoronavirus, and / or to obtain antibodies that inhibit the infection of humans by betacoronavirus. The above immunogenic composition can also obtain antibodies that can inhibit the infection of human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells) by betacoronavirus. The infection-inhibitory ability can be measured by the neutralizing antibody titer (IC 50 The neutralizing antibody titer can be expressed by the dilution factor of the antibody solution or serum obtained. The dilution factor at which the infection suppression ability is 50% is called the IC 50 It can be said that 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. 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. 50 can be calculated by a standard method based on the infection-inhibitory activity using a dilution series of the obtained antibody solution or serum.

[0027] In one embodiment, the equine antisera of the present disclosure are induced by an immunogenic composition comprising the RBD region of a spike protein (S protein) of a betacoronavirus. In one embodiment, the RBD region is the RBD region of the S protein of a SARS-associated coronavirus. In one embodiment, the immunogenic composition comprises two different RBD regions. The two different RBD regions can be derived from different betacoronaviruses, different SARS-associated coronaviruses, or different SARS-CoV-2 strains. In one embodiment, the RBD region is the RBD region of the S protein of SARS-CoV-2. In one embodiment, the RBD region is selected from wild-type strains (the original strain first isolated in Wuhan, China), alpha strains (strain B.1.1.7 and Q-series strains), beta strains (strain B.1.351 and its descendants), gamma strains (strain P.1 and its descendants), delta strains (strain B.1.617.2 and AY-series strains), epsilon strains (strain B.1.427 and B.1.429), eta strains (strain E.1.525), iota strains ( The RBD region of the S protein of any one or more SARS-CoV-2 strains selected from the group consisting of: B.1.1.526 strain), Mu strain (B.1.621 strain and B.1.621.1 strain), Zeta strain (P.2 strain) and Omicron strain (B.1.1.529 strain and BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 series strains), and variants thereof and other variants.

[0028] In one embodiment, the RBD region of the spike protein (S protein) of a betacoronavirus forms a dimer. In one embodiment, the immunogenic composition comprises a monomer of the RBD region of the spike protein (S protein) of a betacoronavirus. In one embodiment, the immunogenic composition comprises a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus. In a preferred embodiment, 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 immunogenic composition comprises a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus, and the content of the dimer is higher 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 a betacoronavirus, and the content of the dimer is higher than the content of the monomer, can be determined by Western blotting after electrophoresis under a non-reducing environment (e.g., native SDS-PAGE). A polyclonal antibody against the horse RBD region can be used as the primary antibody.

[0029] The immunogenic composition can be used to immunize a horse. Accordingly, the present disclosure provides the immunogenic composition for use in immunizing a horse. The present disclosure also provides a method for immunizing a horse, the method comprising administering the immunogenic composition to the horse. In these embodiments, an effective amount of the immunogenic composition is administered to the horse. In this manner, 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 betacoronaviruses in human cells, and / or obtain antibodies that inhibit the infection of betacoronaviruses in humans. The immunogenic composition can also obtain antibodies that can inhibit the infection of betacoronaviruses in human ACE2-positive cells (e.g., alveolar epithelial cells, particularly type II alveolar epithelial cells). The infection-inhibitory ability can be measured by the neutralizing antibody titer (IC 50 ) can be expressed as IC 50can 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. 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. 50 can be calculated by a conventional method.

[0030] The RBD region of the spike protein (S protein) of betacoronavirus can be expressed in mammalian cells. The cells are suitable for protein expression, such as CHO cells, 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 transfer (preferably 24 hours or later, more preferably 30 hours or later, even more preferably 36 hours or later, and even more preferably 42 hours or later). This preferably results in an immunogenic composition rich in RBD region dimers.

[0031] An immunogenic composition containing a monomer and a dimer of the RBD region of the spike protein (S protein) of a betacoronavirus, in which the dimer content is higher than the monomer content, may have high immunogenicity. For example, when quantified by ELISA (SARS-CoV-2 Spike Protein S1 RBD ELISA Kit (Elabscience, E-EL-E605)) according to the manufacturer's instructions, the composition may be evaluated as having a protein concentration that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold higher than the estimated protein amount measured by the BCA method.

[0032] In one embodiment, the immunogenic composition comprising the RBD region of the spike protein (S protein) of a betacoronavirus comprises an RBD region expressed in ExpiCHO-S cells and recovered within 48 hours of gene transfer. In a preferred embodiment, the RBD region has the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0033] In a preferred embodiment, the RBD region has a signal peptide at its N-terminus. The signal peptide may be a secretory signal peptide, and those skilled in the art can select and use it as appropriate. Examples of secretory signal peptides include signal peptides present at the N-terminus of an antibody heavy or light chain, such as the signal peptide of an IgG heavy or light chain, and a signal peptide having the amino acid sequence set forth in SEQ ID NO: 3. 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 comprising, from the N-terminus to the C-terminus, a signal peptide, an RBD region, and a tag peptide, in this order. 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, and HSV tag. The tag peptide can be, for example, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids in length. The tag peptide can be, for example, a 6xHis tag set forth in SEQ ID NO:4.

[0034] In some embodiments, the horse is a Thoroughbred. The direct paternal ancestry of a Thoroughbred is said to be any of the Darley Arabian, Byerley Turk, and Godolphin Arabian. In some embodiments, the horse is a Thoroughbred with less than 25% Arabian blood. In some embodiments, the horse is, for example, 10 to 20 years old, male or female. In some embodiments, the horse weighs between 400 kg and 800 kg, preferably between 500 kg and 700 kg.

[0035] 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 content of the dimer is higher than the content of the monomer.

[0036] The equine antiserum of the present disclosure can be obtained by administering the immunogenic composition to a horse (preferably a thoroughbred). The obtained equine antiserum can exhibit a high neutralizing antibody titer and can also have a high infection-inhibiting ability.

[0037] <Monoclonal and polyclonal antibodies of the present disclosure> According to the present disclosure, polyclonal antibodies contained in horse antisera are provided. Polyclonal antibodies contained in horse antisera can be obtained by purifying antibodies from horse antisera. Purification can be performed, for example, by plasma fractionation, ammonium sulfate fractionation, or caprylic acid precipitation. Alternatively, purification can be performed, for example, using a column equipped with Protein G or Protein A, which selectively adsorbs antibodies. Purification of antibodies using a column equipped with Protein G or Protein A can be performed appropriately by those skilled in the art using conventional methods. A purified polyclonal antibody is also an isolated polyclonal antibody.

[0038] The present disclosure also provides a monoclonal antibody related to any one of the antibodies contained in the horse antiserum. Monoclonal antibodies can be obtained by various methods. For example, any cell selected from spleen cells, B cells, or plasma cells obtained from a horse administered 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. A gene encoding the antibody having the determined amino acid sequence is then introduced into protein-expressing cells (e.g., Chinese hamster ovary (CHO) cells) to obtain antibody-producing cells, and a monoclonal antibody can be obtained from the obtained antibody-producing cells. In one embodiment, any cell selected from spleen cells, B cells, or plasma cells obtained from the horse can be fused with myeloma cells to obtain a hybridoma. The sequence of the mRNA or cDNA encoding the antibody in the obtained hybridoma cells is decoded, and the decoded gene encoding the antibody is then introduced into protein-expressing cells (e.g., Chinese hamster ovary (CHO) cells) to obtain antibody-producing cells, and a monoclonal antibody can be obtained from the obtained antibody-producing cells. A purified monoclonal antibody is also an isolated monoclonal antibody.

[0039] 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.

[0040] Thus, the present disclosure provides compositions comprising polyclonal antibodies of the present disclosure, monoclonal antibodies of the present disclosure, human chimerized monoclonal antibodies of the present disclosure, and humanized monoclonal antibodies of the present disclosure. The present disclosure also provides compositions comprising multiple monoclonal antibodies. Such compositions may be advantageous, for example, in increasing the number of betacoronaviruses that can be neutralized.

[0041] In certain embodiments, a composition comprising a plurality of human chimerized monoclonal antibodies is provided. In certain embodiments, a composition comprising a plurality of humanized monoclonal antibodies is also provided. 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 certain embodiments, the plurality of monoclonal antibodies differ from each other in amino acid sequence. In certain 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 CDRs 1-3 and light chain CDRs 1-3. In certain embodiments, the difference in amino acid sequence can be from 1 to 10 amino acids. In certain preferred embodiments, at least two of the plurality of antibodies can bind to different epitopes, e.g., do not compete with each other for binding to the RBD region.

[0042] The antibody of the present disclosure can bind to the RBD region (preferably a dimer thereof), for example, 10 -6 Below, 10 -7 Below, 10 -8 Below, 10 -9 Below, 10 -10 Less than or equal to 10 -11 They can bind with the following binding dissociation constants (KD values): Here, a smaller KD value means that the antibody binds more strongly to the RBD region (preferably a dimer thereof).

[0043] <Compositions containing the 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 composition) comprising multiple or diverse monoclonal antibodies. A virtual polyclonal antibody contains multiple monoclonal antibodies, but the content of each monoclonal antibody reaches an effective amount. As a result, like a polyclonal antibody, a virtual polyclonal antibody is an antibody mixture that can robustly express desired antibody functions (e.g., can suppress a decrease in binding affinity for antigen mutants) compared to a single monoclonal antibody.

[0044] A virtual polyclonal antibody may, for example, comprise two or more monoclonal antibodies, each in an effective amount. A virtual polyclonal antibody may, for example, comprise three or more monoclonal antibodies, each in an effective amount. A virtual polyclonal antibody may, for example, comprise n or more monoclonal antibodies, each in an effective amount {where n is a natural number of 2 or greater, and 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 virtual polyclonal antibody may, for example, comprise 4 to 10 monoclonal antibodies, each in an effective amount. Each of the monoclonal antibodies contained in the virtual polyclonal antibody is human chimeric or humanized.

[0045] Virtual polyclonal antibodies are Immunizing a horse with any of the 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 the sequences (e.g., nucleotide sequences or amino acid sequences) of the heavy chain variable regions and light chain variable regions of each of multiple mRNAs or cDNAs encoding antibodies produced by multiple 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 the plurality of clusters obtained; obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region contained 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 sequences, 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.

[0046] To ensure that the included antibodies each bind to a different epitope, any two of the included antibodies may not compete with each other for binding to the RBD region.

[0047] In one aspect, the virtual polyclonal antibody comprises: A method comprising producing monoclonal antibodies from a plurality of antibody-producing cells that each produce a different monoclonal antibody, and obtaining a composition containing the produced 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 that have antibodies against the RBD region in their bodies, and the portion of the antibody structure comprises a heavy chain variable region and a light chain variable region, or comprises 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 is capable of neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2). It can be produced by the following method.

[0048] In the method of generating antibodies, the immunogenic composition can be any of the immunogenic compositions described above.

[0049] The virtual polyclonal antibodies disclosed herein can be used to obtain antibodies that inhibit the binding of S1 protein to ACE2, to obtain antibodies that inhibit infection of human cells by betacoronavirus, and / or to obtain antibodies that inhibit infection of humans by betacoronavirus.

[0050] The present disclosure provides a method for treating a betacoronavirus infection in a subject, the method 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. The subject may be a carrier (a subject who does not exhibit symptoms) of a betacoronavirus infection. The subject may be a subject suffering from an infection with a betacoronavirus. In some embodiments, the subject may be a subject suffering from a mild betacoronavirus infection. In some embodiments, the subject may be a subject suffering from a moderate betacoronavirus infection. In some embodiments, the subject may be a subject suffering from a severe betacoronavirus infection. In some embodiments, the subject does not have a coronavirus infection (e.g., a betacoronavirus infection) but has a history of close contact with a betacoronavirus infection. In some embodiments, the subject may be a subject infected with a betacoronavirus but does not have a coronavirus infection (e.g., a betacoronavirus infection).

[0051] According to the present disclosure, there is provided an antiserum, monoclonal antibody, polyclonal antibody, or virtual polyclonal antibody of the present disclosure for use in the methods of the present disclosure.

[0052] According to the present disclosure, there is provided a composition or pharmaceutical composition comprising an antiserum, monoclonal antibody, polyclonal antibody, or virtual polyclonal antibody of the present disclosure for use in the methods of the present disclosure.

[0053] The present disclosure provides the use of an antiserum, monoclonal antibody, polyclonal antibody, or 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]

[0054] Example 1: Antigen preparation In this example, a partial peptide of the S1 protein was prepared as an antigen.

[0055] (1) Cell preparation Frozen ExpiCHO-S cells were thawed and subcultured repeatedly until cell growth stabilized. The cells were then frozen again to obtain frozen cells. Specifically, ExpiCHO Expression Medium was added to an empty, clean Erlenmeyer flask and incubated in a CO2 incubator at 37°C. 0.2 x 10 6 cells / mL to 0.3 x 10 6 The cells were seeded at a density of 0.4 × 10 cells / mL and cultured at 37°C in the presence of 8% CO2. 6 cells / mL to 0.6 x 10 6 The concentration reached 100 cells / mL. One vial of the frozen cells was thawed and subcultured three times. It was confirmed 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 of the time before freezing (doubling time before freezing: 17.7 hours, doubling time at the third passage after freezing: 18.9 hours). It was also confirmed that there was no contamination by bacteria, etc. The obtained cells were used in the Examples described below. All cell cultures for antigen preparation were carried out at 37°C and 8% CO2.

[0056] (2) Preparation of antigen expression vector The antigen used was the RBD region (amino acids 319 to 541) of the S1 protein of SARS-CoV-2 (Wuhan strain). Herein, the Wuhan strain is sometimes 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 was constructed by adding an IgGκ leader sequence (SEQ ID NO: 3) having the amino acid sequence of SEQ ID NO: 3 to the N-terminus of the RBD region having the amino acid sequence of SEQ ID NO: 1, and a 6×His tag (His tag; SEQ ID NO: 4) to 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.

[0057] (3) Gene transfer into cells The cells obtained by (1) above (0.4 × 10 6 cells / mL to 0.6 x 10 6 The cell viability of the cells (at a concentration of 1000 cells / mL) was confirmed to be 95% or higher. The cells obtained in (1) above were transfected into an Erlenmeyer flask containing ExpiCHO Expression Medium. The vector obtained in (2) above was transfected into the cells using ExpiFectamine CHO Reagent. The day after gene transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added. The cells were harvested 5 days, 60 hours, and 40 hours after gene transfection, and cell viability and protein expression levels were confirmed.

[0058] (4) Confirmation of recombinant protein expression (4-1) Purification of recombinant proteins A portion of the culture medium containing the cells collected at each time point was washed and sonicated. The supernatant was collected by centrifugation. 100 μL of Ni Sepharose 6 Fast Flow resin was equilibrated with buffer. The supernatant was added to the resin and stirred at 4°C for 1 hour. The resin was then centrifuged. The resin was then suspended in buffer and centrifuged three times. The supernatant was removed, and the resin was suspended in elution buffer. After centrifugation, the supernatant was collected. The collected supernatant was used as the recombinant protein extract. Proteins were subjected to SDS-PAGE followed by CBB staining or Western blotting (detection was performed using alkaline phosphatase-conjugated anti-6xHis tag antibody (Abcam, ab49746)). Protein concentration was quantified by UV absorption or bicinchoninic acid (BCA) assay.

[0059] (4-2) Preparation of cell culture supernatant The culture supernatant was filtered through a depth filter and a 0.22 μm filter. The depth filter was a Millistak+™ HC pod filter, DOHC, 0.027 μm. 2 The supernatant was filtered twice using a Merck filter. A 0.22 μm bottle-top filter (Thermo Fisher, 597-4520) was used as the 0.22 μm filter. Protein concentration was quantified by ELISA (SARS-CoV-2 Spike Protein S1 RBD ELISA Kit (Elabscience, E-EL-E605)) according to the manufacturer's instructions.

[0060] 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 to 60 hours after gene transfer, but then decreased significantly.

[0061] Protein expression was as shown in Table 1. As shown in Table 1, the amount of recovered protein in the sample taken 5 days after gene transfection was approximately 40-50%. The recombinant protein in the supernatant was quantified 60 hours after gene transfection. Due to concerns that depth filter filtration might reduce protein recovery, filtration through a 0.22 μm filter was used in the recovery step, without depth filter filtration. This improved the amount of recovered recombinant protein. The recombinant protein in the supernatant was quantified 40 hours after gene transfection. The recovery step was performed in 0.22 μm filter filtration, without depth filter filtration.

[0062] [Table 1]

[0063] Each sample was subjected to SDS-PAGE with or without treatment with a reducing agent, followed by Western blotting. A 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. The lanes in Figure 2 are as shown in Table 2.

[0064] [Table 2]

[0065] As shown in Figure 2, a band of the recombinant protein was confirmed at the desired size. Focusing on lanes 10–17 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 its size was approximately twice that of the recombinant protein, this band was suggested to correspond to a dimer of the recombinant protein. In lanes 11 and 15 in Figure 2, the dimer band was stained more intensely than the recombinant protein monomer, suggesting that the dimer was predominant over the monomer in terms of abundance. This revealed that the expression level was too low one day after gene transfection, but on day 2, the expression level increased as the dimer increased, and from day 3 onwards, the expression level increased, but the proportion of the monomer increased. These results suggest that recombinant protein recovery should be performed between 40 and 60 hours after gene transfection.

[0066] The recombinant protein collected 40 hours after transfection was quantified by BCA and ELISA. The results are shown in Table 3.

[0067] [Table 3]

[0068] Protein concentrations were adjusted by the BCA method as shown in Table 3. The resulting samples with adjusted protein concentrations were subjected to ELISA measurement. The concentrations estimated by ELISA far exceeded the actual protein concentrations in samples 1 to 4. This suggests that the resulting proteins reacted extremely strongly with antibodies compared to the protein amount, i.e., their immunogenic activity was significantly high.

[0069] Example 2: Induction of antibody production in animals In this example, purified S1-RBD-His tagged (WT) cells derived from cells recovered 40 hours after gene transfer in Example 1 and a mixture of the purified S1-RBD-His tagged (WT) and purified S1-RBD-His tagged (δ strain) were used as immunogens. The immunogens were mixed with Freund's complete adjuvant (DIFCO 263810) or Freund's incomplete adjuvant (DIFCO 263910) to obtain emulsions. The immunogen dose was 5 mg / day in protein equivalent.

[0070] Horses were used. Thoroughbreds (12–18 years old) of different pedigrees were used. Immunization of the horses was performed as follows: On day 0, 5 mg of recombinant protein mixed with Freund's complete adjuvant was injected into the neck and rump. On day 14, 5 mg of recombinant protein mixed with Freund's incomplete adjuvant was administered, and on day 28, 5 mg of recombinant protein diluted with saline was administered. Small blood samples were collected for testing at various time points. In addition, 5 L of large blood samples were collected on days 56, 63, and 70 using bags containing 250 mL of 4% cytramine solution. The collected blood was left at room temperature for 1 hour to allow blood cells to settle, and the serum, after removal of blood cells, was stored refrigerated until use. The obtained sera were designated anti-WT serum and anti-WT&Delta serum, respectively. Furthermore, polyclonal antibodies obtained from anti-WT&Delta serum obtained n days after immunization are referred to as LADn, and polyclonal antibodies obtained from anti-Delta serum obtained n days after immunization are referred to as LBDn. That is, LBD56 refers to polyclonal antibodies obtained from anti-Delta serum obtained 56 days after immunization.

[0071] Example 3: Testing of the obtained serum Serum was separated from the blood (for testing) collected in Example 2 and tested.

[0072] (1) cPass Exam The neutralizing antibody titers were measured for the obtained sera. Antibody titers were measured using the cPASS SARS-CoV-2 Neutralizing Antibody Detection Kit (GenScript). This kit is designed to mimic virus-host interactions through 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. Neutralizing antibody titers of 50% IC (IC 50 ) was calculated by the usual method. Specifically, the inhibition rate (%) was probit-converted, and the dilution rate was plotted on a graph with the dilution ratio and the probit-converted inhibition rate as the axis. The dilution rate corresponding to an inhibition rate of 50% was calculated by fitting with an approximate straight line.

[0073] The test results are shown in Figure 3. As shown in Figure 3, good neutralizing antibody production 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 obtained by vaccination. These results were consistent across horses of different bloodlines, suggesting that this is a common characteristic among Thoroughbreds.

[0074] [Table 4]

[0075] The neutralizing antibody titer of this example is an order of magnitude higher than the neutralizing antibody titers of previous vaccine products.

[0076] The amount of specific antibody in the antiserum was quantified by antigen ELISA. The results are shown in Figure 4. The amount of specific antibody peaked between about 40 and 60 days after the antigen ELISA, but continued to be high for a long period of time.

[0077] (2) Neutralizing antibody titers against SARS-CoV-2 mutant strains Neutralizing antibody titers against the SARS-CoV-2 wild-type, α, β, γ, δ, and ο (omicron) strains were tested in the same manner as described above (1). The results are shown in Figure 5. As shown in Figure 5, all antisera induced neutralizing antibodies against various mutant strains. Furthermore, horse serum showed higher neutralizing antibody titers than sera from clinically infected individuals (humans).

[0078] (3) Pseudovirus Neutralization Test (PVNT) To mimic SARS-CoV-2 infection, a pseudovirus was used in which the envelope glycoprotein of a lentiviral vector was replaced with the S protein. The pseudovirus contained nucleic acid encoding luciferase, which was expressed intracellularly upon infection. Therefore, the amount of virus that infected a cell could be estimated by measuring the intensity of luciferase luminescence. PVNT was performed using the 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).

[0079] The results are shown in Figure 5. As shown in Figure 5, the horse serum obtained above exhibited high neutralizing antibody titers against various mutant strains, regardless of the mutant strain. Furthermore, the neutralizing antibody titers of the horse serum were higher than those of human serum that had been administered two or three times with a coronavirus RNA vaccine (Moderna or Pfizer).

[0080] (4) Comparison with commercially available polyclonal antibodies The neutralizing antibody titers were compared with those of commercially available polyclonal antibodies. Horse serum obtained by immunization with S1-RBD-His tag (WT) was used. 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. The human polyclonal antibody was Cov-Neut-S-500 (Ray Biotech), a serum obtained from a coronavirus-positive donor. The results are shown in Table 5.

[0081] [Table 5]

[0082] As shown in Table 5, the horse sera exhibited high neutralizing antibody titers and specific activities.

[0083] (5) The multimeric nature of the antigen and the binding properties of the antiserum to the antigen Based on the Western blot (WB) results for the antigens obtained in Example 1, band intensities were analyzed by densitometry. For WB, a polyclonal antibody derived from horse antiserum (LBD56, antibody dilution 1:1000) or a commercially available mouse polyclonal antibody (Sino Biological Co., Ltd., Lot No. 40592-MP01, antibody dilution 1:100) 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 are shown in Figure 6. Details of each lane are as shown in Table 6 below.

[0084] [Table 6]

[0085] Next, the Western blot images were analyzed by densitometry. 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.

[0086] [Table 7]

[0087] [Table 8]

[0088] As shown in Table 7, the recombinant protein used as the immunogen contained many multimers (especially dimers), and the polyclonal antibodies obtained from the horse antisera in this example recognized the multimers well. Furthermore, as shown in Table 8, the mouse polyclonal antibodies showed weak reactivity to the immunogen, but their binding specificity for multimers was inferior to that of the horse polyclonal antibodies.

[0089] (6) Toxicity of the obtained serum The obtained serum was administered intravenously to rats for toxicity testing. Rats (Crl:CD(SD), male, 7 weeks old, weighing 140-220g) were randomly divided into three groups (five rats per group) stratified by weight. Serum or saline was administered intravenously at 13 times (93mg / kg) or 26 times (186mg / kg) the expected clinical dose. Thirteen days after administration, the animals were compared with the control group administered saline to check for any abnormalities. No abnormalities were observed in the general condition or weight transitions in any of the rats. Necropsy of the rats also revealed no abnormalities, and no changes in organ weights that could be attributed to the serum were observed.

Claims

1. An equine antiserum against an immunogenic composition comprising the 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. The antiserum of claim 1 , wherein the immunogenic composition comprises a dimerized RBD region.

3. 3. The antiserum of claim 2, wherein the immunogenic composition contains more dimerized RBD regions than monomeric RBD regions.

4. The antiserum according to any one of claims 1 to 3, wherein the horse is a thoroughbred.

5. The antiserum of claim 3, wherein the horse is a thoroughbred.

6. A composition comprising a polyclonal antibody purified from the antiserum of any one of claims 1 to 5.

7. 1. A composition comprising a humanized virtual polyclonal antibody, comprising a plurality of different monoclonal antibodies, Each monoclonal antibody comprises a portion of the structure of an equine antibody directed against the RBD region of the spike protein (S protein) of a betacoronavirus, 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 composition, wherein each monoclonal antibody is capable of binding to the S protein and neutralizing the binding of the S protein to angiotensin-converting enzyme 2 (ACE2).

8. The composition of claim 7, 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 of claim 8 , wherein the immunogenic composition comprises a dimerized RBD region.

10. The composition of claim 9 , wherein the immunogenic composition comprises more dimerized RBD regions than monomeric RBD regions.

11. The composition according to any one of claims 7 to 10, wherein the horse is a thoroughbred.

12. 11. The composition of claim 10, wherein the horse is a thoroughbred.

13. A method for preparing a composition according to any one of claims 7 to 12, 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 the sequences of the heavy chain variable region and the light chain variable region of each of multiple mRNAs encoding antibodies produced by multiple 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 the obtained plurality of clusters; obtaining a nucleotide sequence encoding a human chimeric antibody comprising a heavy chain variable region and a light chain variable region contained 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 sequences, thereby obtaining a mixture of a plurality of antibodies containing the human chimeric or humanized antibodies; A method comprising:

14. A method for preparing a composition according to any one of claims 7 to 12, comprising the steps of: producing monoclonal antibodies from a plurality of antibody-producing cells that each produce a different monoclonal antibody, and obtaining a composition containing the produced 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 that have antibodies against the RBD region in their bodies, and the portion of the antibody structure comprises a heavy chain variable region and a light chain variable region, or comprises 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, The method, wherein each monoclonal antibody is capable of neutralizing the binding of said S protein to angiotensin-converting enzyme 2 (ACE2).

15. 15. The method of claim 13 or 14, wherein the horse is a thoroughbred.

16. The method of any one of claims 13 to 15, wherein the immunogenic composition comprises a dimerized RBD region.

17. 17. The method of claim 16, wherein the immunogenic composition comprises more dimerized RBD regions than monomeric RBD regions.

18. 1. An immunogenic composition comprising: It contains the RBD region of the spike protein (S protein) of a beta coronavirus, An immunogenic composition, wherein the RBD region forms a dimer, and the amount of the dimer is greater than the amount of the RBD region monomer.

19. 20. The immunogenic composition of claim 18 for use in immunizing a horse.

20. 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.

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