SARS-CoV-2 VARIANT-SPECIFIC RECOGNITION ANTIBODY

Abstract

To provide a novel antibody capable of binding to SARS-CoV-2.SOLUTION: An antibody or antigen-binding fragment thereof of the present disclosure is an antibody or antigen-binding fragment thereof against spike protein of SARS-CoV-2, wherein the antibody is selected from the group consisting of (Ca) and (Cb) defined below: (Ca) an antibody comprising amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in specific sequences, and amino acid sequences of light chain CDR1, light chain CDR2, and light chain CDR3 represented in other specific sequences different from the foregoing sequences; and (Cb) a variant of the antibody (a) comprising one or more substitutions, insertions, additions, and / or deletions in the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and / or the light chain CDR3.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present disclosure relates to an antibody against the spike protein of SARS-CoV-2 or an antigen-binding fragment thereof and uses thereof.

Background Art

[0002] SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, hereinafter also referred to as "SARS2") has spread worldwide after its occurrence was confirmed near Wuhan City, China in November 2019. In addition, for SARS2, mutant strains such as the Omicron strain have emerged, and the epidemic has repeated. As a method for detecting SARS2 infection, antigen testing of spike proteins using antibodies is generally performed.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, an object of the present disclosure is to provide a new antibody capable of binding to SARS-CoV-2.

Means for Solving the Problems

[0004] To achieve the above object, the antibody or antigen-binding fragment thereof of the present disclosure is an antibody or antigen-binding fragment thereof against the spike protein of SARS-CoV-2, wherein the antibody is selected from the group consisting of the following (Ca) and (Cb): (Ca) an antibody comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 4, 5, and 6, respectively; (Cb) a variant of the (Ca) antibody comprising one substitution, insertion, addition, or deletion in the entirety of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3.

[0005] The detection reagent for the spike protein of SARS-CoV-2 of the present disclosure contains the antibody of the present disclosure or its antigen-binding fragment.

[0006] The reagent for use in testing the likelihood of contracting SARS-CoV-2 of the present disclosure contains the antibody of the present disclosure or its antigen-binding fragment.

[0007] The method for detecting the spike protein of SARS-CoV-2 of the present disclosure includes a contacting step of contacting a sample with the antibody or its antigen-binding fragment according to any one of claims 1 to 5, and a detecting step of detecting a complex of the spike protein of SARS-CoV-2 in the sample and the antibody or its antigen-binding fragment.

[0008] The method for testing the likelihood of contracting SARS-CoV-2 of the present disclosure includes a contacting step of contacting a biological sample of a subject with the antibody or its antigen-binding fragment according to any one of claims 1 to 5, and a measuring step of measuring the expression level of the spike protein of SARS-CoV-2 in the biological sample.

Advantages of the Invention

[0009] According to the present disclosure, a new antibody capable of binding to SARS-CoV-2 can be provided.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0011] <Definition> In this specification, "SARS-CoV-2" (hereinafter also referred to as "SARS2") means severe acute respiratory syndrome coronavirus 2. The said SARS is a virus belonging to the genus Betacoronavirus of the family Coronaviridae ( Coronaviridae ) and belonging to the species Severe acute respiratory syndrome-related coronavirus. Also, SARS2 is a virus having single-stranded positive-strand RNA as its genome. Betacoronavirus )

[0012] In this specification, "spike protein" (hereinafter also referred to as "S protein") means a protein or polypeptide having all or part of the amino acid sequence of the spike protein of SARS-CoV-2. The said S protein is a protein that forms virus particles together with the nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein in SARS2, and is arranged outward from the envelope of the said virus particles. The said S protein is presumed to be important for establishing infection into cells via ACE2. The said S protein includes an S1 region containing an N-terminal domain (NTD or NTD domain) and a receptor-binding domain (RBD or RBD domain), and an S2 region, from the N-terminus towards the C-terminus. The said NTD domain is a region corresponding to amino acids 1 to 318 in the amino acid sequence of the S protein of the origin strain described later. Also, the said RBD domain is a region corresponding to amino acids 319 to 541 in the amino acid sequence of the S protein of the origin strain. The said S2 region is a region corresponding to amino acids 542 to 1273 in the amino acid sequence of the S protein of the origin strain.

[0013] As used herein, the "original strain of SARS-CoV-2" or "original strain" refers to the strain defined as the Wuhan-Hu-1 reference strain sequence with Accession No.: MN908947.3 in GenBank. The amino acid sequence of the S protein of the original strain may be a protein (SEQ ID NO: 9) consisting of the amino acid sequence registered in Genbank with Accession No.: QHD43416.1.

[0014] Amino acid sequence of the S protein of the original strain (SEQ ID NO: 9)

[0015] As used herein, the term "SARS-CoV-2 Omicron strain" or "Omicron strain" refers to a variant strain (B.1.1.529) of SARS-CoV-2. The amino acid sequence of the S protein (partial sequence) of the Omicron strain may be exemplified by the protein (SEQ ID NO: 10) consisting of the amino acid sequence registered in Genbank under Accession No.: UNJ82271.1. The amino acid sequence of the partial sequence is encoded by the genomic sequence registered in Genbank under Accession No.: ON023332.1. The amino acid sequence of the S protein (partial sequence) of the Omicron strain may be exemplified by, for example, Accession No.: WHZ81008.1, WIG86805.1, etc. in Genbank. Also, the amino acid sequence of each partial sequence is encoded by the genomic sequences registered under OR055807.1 and OR086387.1, respectively. The S protein of the Omicron strain contains, for example, amino acid mutations of G339D, S477N, S371L, T478K, S373P, E484A, S375F, Q493R, K417N, G496S, N440K, Q498R, G446S, N501Y, and Y505H with respect to the S protein of the original strain.

[0016] Amino acid sequence of the S protein (partial sequence) of the Omicron strain (SEQ ID NO: 10)

[0017] As used herein, "protein", "polypeptide", "oligopeptide", or "peptide" are used interchangeably and refer to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The length of the protein can be of any length.

[0018] As used herein, "polynucleotide", "oligonucleotide", or "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The polynucleotide may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The polynucleotide may be composed of natural nucleotides, modified or artificial nucleotides, or both.

[0019] As used herein, "gene" refers to a factor that defines a genetic trait, and "gene" may refer to "polynucleotide", "oligonucleotide", and "nucleic acid".

[0020] As used herein, the "corresponding" amino acid or nucleic acid means an amino acid or nucleotide that has, or is predicted to have, a similar function to a given amino acid or nucleotide or portion in a reference polypeptide or polynucleotide in a polypeptide or polynucleotide being compared.

[0021] As used herein, "antibody" means a protein comprising one or more polypeptides substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene, and includes a molecule or molecules capable of binding to a specific epitope on an antigen. The immunoglobulin gene includes, for example, genes encoding constant regions such as κ, λ, α (including α1 and α2), γ (including γ1, γ2, γ3, and γ4), δ, ε, and μ, and genes capable of encoding numerous immunoglobulin variable regions such as V regions, D regions, and J regions. The antibody includes, for example, heavy chains and light chains. The light chains include κ and λ, and constitute the κ chain and the λ chain, respectively. The heavy chains include γ, μ, α, δ, or ε, and constitute IgG (for example, IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, and IgE, which are classes of immunoglobulins, respectively. The antibody may be a structural unit of a typical immunoglobulin (antibody) composed of a tetramer. In this case, the antibody is composed of pairs of two identical polypeptide chains, and each pair is composed of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). Also, the N-terminus of each chain defines a variable region composed of about 100-110 or more amino acids mainly involved in antigen recognition. When the antibody is derived from a chicken, the class of the antibody may be IgY.

[0022] As used herein, the "complementary determining region" (CDR) refers to the region in the variable region of an immunoglobulin (antibody) that forms the antigen-binding site. The CDR can also be referred to as, for example, a hypervariable region. The CDR is generally also a region in the variable region of the antibody, particularly a region with high primary structure variability, and is usually separated into three locations in the primary structure. In the antibody, the heavy chain variable region and the light chain variable region each contain three CDRs (from the N-terminal side, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and from the N-terminal side, light chain CDR1, light chain CDR2, and light chain CDR3). These sites are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind. As used herein, the CDR of an antibody may be determined according to the Kabat numbering system (Kabat et al., "Sequences of Proteins of Immunological Interest", 1987, US Department of Health and Human Services, NIH, USA). Also, as used herein, the CDR of an antibody may be determined by adopting the definition by Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol., 1987; 196: 901-917).

[0023] As used herein, the "antigen-binding fragment" is, for example, a polypeptide containing a part of an antibody, and more specifically, a polypeptide containing the variable region. The antigen-binding fragment can be produced, for example, by digestion of the full-length immunoglobulin with various peptidases.

[0024] As used herein, the form of the "antibody" may be a full-length immunoglobulin (an antibody having an Fc region and a Fab region, a full-length antibody), or F(ab')2, Fab', Fab, Fv antibody (variable fragment of antibody), disulfide-bonded Fv (dsFv), single-chain antibody (scFv), and polymers thereof (e.g., diabody), etc. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be, for example, a chimeric antibody, a humanized antibody, or a fully humanized antibody. The antibody may be, for example, a bispecific or oligo specific antibody.

[0025] As used herein, "F(ab’) 2 antibody" is, for example, an antibody containing two sites corresponding to Fab among the fragments obtained by treating an antibody containing a Fab region and an Fc region with the protease pepsin. The F(ab’) 2 can be obtained, for example, by treating an antibody against the spike protein of SARS-CoV-2 of the present disclosure (hereinafter, also referred to as "S protein antibody") containing a Fab region and an Fc region with the protease pepsin.

[0026] As used herein, "Fab’ antibody" is, for example, an antibody obtained by cleaving the disulfide bond in the hinge region of an F(ab’) 2 antibody. The Fab’ antibody can be obtained, for example, by treating an F(ab’) 2 antibody with the reducing agent dithiothreitol.

[0027] As used herein, "Fab antibody" is, for example, an antibody in which approximately the first half of the N-terminal side of the heavy chain and the entire light chain are bound via some disulfide bonds among the fragments obtained by treating an antibody containing a Fab region and an Fc region with the protease papain. The Fab antibody can be obtained, for example, by treating an S protein antibody of the present disclosure containing a Fab region and an Fc region with the protease papain.

[0028] As used herein, an "Fv antibody" is an antibody that contains an antigen recognition site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain by non-covalent bond. In this configuration, the three CDRs of each variable domain can interact with each other to form an antigen binding site on the surface of the VH-VL dimer.

[0029] As used herein, a "disulfide bond Fv" (dsFv) is an antibody in which polypeptides with cysteine residues introduced into VH and VL are linked via a disulfide bond between the cysteine residues. The positions at which the cysteine residues are introduced can be selected, for example, based on the method shown by Reiter et al. (Reiter et al., “Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv”, Protein Eng. 1994 May;7(5):697-704) and based on the three-dimensional structure prediction of the antibody.

[0030] As used herein, an "scFv antibody" means an antibody in which VH and VL are linked via a peptide linker. The scFv antibody can be produced, for example, by obtaining cDNAs encoding VH and VL of the S protein antibody of the present disclosure, constructing a polynucleotide encoding VH-peptide linker-VL, incorporating the polynucleotide into a vector, and using cells for expression.

[0031] As used herein, a "diabody" is an antibody having bivalent antigen-binding activity. The bivalent antigen-binding activity may be the same antigen activity or one of them may be a different antigen-binding activity. The diabody can be produced, for example, by constructing a polynucleotide encoding scFv such that the length of the amino acid sequence of the peptide linker is 8 residues or less, incorporating the resulting polynucleotide into a vector, and using cells for expression.

[0032] As used herein, a "monoclonal antibody" is an antibody that corresponds to substantially a single epitope or is substantially identical, for example, excluding antibodies in which individual antibodies constituting a population have mutations that can occur naturally in small amounts. The monoclonal antibody may be prepared, for example, by the hybridoma method or a similar method, or by phage display using a phage antibody library.

[0033] As used herein, the “chimeric antibody” means an antibody in which at least one of the heavy chain and the light chain is composed of a variable region derived from a non-human animal immunoglobulin and a constant region derived from a human immunoglobulin, or an antibody composed of a variable region derived from a human immunoglobulin and a constant region derived from a non-human animal immunoglobulin. The chimeric antibody can be prepared, for example, by genetic recombination technology. As a specific example, when preparing a chicken-human chimeric antibody, the method for preparing the chimeric antibody can be, for example, by ligating a chicken leader sequence and a variable region sequence to a sequence encoding a human antibody constant region (for example, Nahoko Nishibori et al., “Expression vectors for chicken-human chimeric antibodies”, Biologicals. 2004 Dec;32(4): p. 213-8). Specifically, the preparation method may be, for example, by ligating the chicken leader sequence and the variable region sequence present in the cloned cDNA to the sequence encoding the human antibody constant region already present in the expression vector of mammalian cells. Further, the preparation method may be to ligate the chicken leader sequence and the variable region sequence present in the cloned cDNA to the sequence encoding the human antibody constant region and then ligate it to the mammalian cell expression vector. The fragment of the human antibody constant region can be that of the heavy chain constant region of any human antibody and the light chain constant region of a human antibody. As a specific example, the fragment of the human antibody constant region includes, for example, Cγ1, Cγ2, Cγ3 or Cγ4 as the human heavy chain. Further, the fragment of the human antibody constant region includes, for example, Cλ or Cκ as the human light chain.

[0034] As used herein, the term "humanized antibody" refers to an antibody comprising a variable region composed of CDRs from an antibody derived from a non-human animal and framework regions (FRs) from a human antibody, and a constant region from a human antibody. The humanized antibody can be prepared, for example, by CDR grafting (Ozaki et al., “Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells”, Blood, 1999 Jun 1;93(11):3922-3930.), re-surfacing (Roguska et al., “Humanization of murine monoclonal antibodies through variable domain resurfacing”, Proc. Natl. Acad. Sci. USA, 1994 Feb 1;91(3):969-973), or FR shuffling (Damschroder et al., “Framework shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical properties”, Mol Immunol., 2007 Apr;44(11):3049-3060, Epub 2007 Jan 22), etc. When using chicken-derived CDRs, the humanized antibody can be prepared, for example, with reference to the method described in JP-A-2006-241026. In the preparation of the humanized antibody, in order to modify or improve the binding to the antigen, the amino acid residues of the human FR may be substituted with the corresponding residues from the CDR donor antibody. Substitution of the amino acid residues of the human FR can be carried out, for example, by methods well known in the art (see, for example, Riechmann et al., “Reshaping human antibodies for therapy”, Nature, 1988 Mar 24; 332 (6162) :323-327).

[0035] As used herein, the "antibody against the spike protein of SARS-CoV-2" means an antibody that binds to the spike protein of SARS-CoV-2. The binding by the S protein antibody is preferably specific binding, and more preferably specific binding to the S protein of the Omicron strain. Further, it is preferable that the binding of the S protein antibody to the S protein of the original strain is suppressed. The form of the S protein antibody can refer to the description of the form of the aforementioned antibody.

[0036] As used herein, the "label" means something for identifying a target molecule or substance from other molecules or substances. Examples of the label include fluorescent labels such as fluorescent dyes or fluorescent substances; chemiluminescent labels; radioisotopes (RI); and the like.

[0037] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means the treatment, cure, prevention, inhibition, remission, improvement of a disease, condition, or disorder, or the arrest, inhibition, reduction, or delay of the progression of a disease, condition, or disorder. As used herein, "prevention" means a decrease in the likelihood of onset of a disease or condition, or a delay in the onset of a disease or condition. The above "treatment" may be, for example, treatment of a patient suffering from a target disease or treatment of a model animal of the target disease.

[0038] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can refer to the description of other disclosures.

[0039] <S protein antibody> In one aspect, the present disclosure discloses an antibody against the spike protein of SARS-CoV-2 or an antigen-binding fragment thereof. The S protein antibody of the present disclosure is an antibody against the spike protein of SARS-CoV-2 or an antigen-binding fragment thereof, and the antibody is selected from the group consisting of the following (Ca) and (Cb).

[0040] (Ca) An antibody ((Ca) antibody) comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 4, 5, and 6, respectively (Cb) A variant of the (Ca) antibody ((Cb) antibody) comprising one or several substitutions, insertions, additions, or deletions in the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and / or the light chain CDR3

[0041] In the (Ca) antibody, the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 are as follows. Also, in the (Ca) antibody, the nucleotide sequences encoding heavy chain CDR1-3 and light chain CDR1-3 are, for example, as follows

[0042] [Table 1]

[0043] In each CDR of the (Cb) antibody, "one or several" may be, for example, within the range where the variant of the (Ca) antibody binds to the S protein, particularly the S protein of the Omicron strain. The "one or several" is, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 1, preferably 1 to 2 or 1. In the present disclosure, the numerical range of the number of amino acids, the number of bases, etc. discloses all positive integers belonging to the range. That is, for example, the description of "1 to 5" means the disclosure of all of "1, 2, 3, 4, 5" (the same applies hereinafter).

[0044] In the (Cb) antibody, "one or several" may be the number of substitutions, insertions, additions or deletions (hereinafter also collectively referred to as "mutations") in each CDR, that is, each of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, or may be the number of mutations in all CDRs, that is, all of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3. Preferably, it is the number of all mutations in all CDRs.

[0045] The (Cb) antibody preferably contains 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 1 substitution, insertion, addition or deletion in total of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.

[0046] In the (Cb) antibody, the "substitution, insertion, addition or deletion" is preferably substitution, insertion or deletion, or addition of at least one of the N-terminus and C-terminus of the CDR. The substitution is preferably a conservative substitution (the same shall apply hereinafter). The "conservative substitution" means substituting one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially modify the function of the protein. The "amino acid to be substituted" and the "amino acid to be substituted with" are preferably similar in properties and / or functions, for example. Specifically, for example, it is preferable that chemical properties such as indices of hydrophobicity and hydrophilicity (hydropathy), polarity, charge, etc., or physical properties such as secondary structure, etc. are similar. Amino acids or amino acid derivatives with similar properties and / or functions are known in the art, for example. As specific examples, non-polar amino acids (hydrophobic amino acids) include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc., polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc., amino acids with a positive charge (basic amino acids) include arginine, histidine, lysine, etc., and amino acids with a negative charge (acidic amino acids) include aspartic acid, glutamic acid, etc.

[0047] In the antibody or antigen-binding fragment thereof of the present disclosure, the antibody may be an antibody or antigen-binding fragment thereof selected from the group consisting of the following (Va), (Vb), and (Vc).

[0048] (Va) An antibody ((Va) antibody) comprising the amino acid sequences of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8 (Vb) A variant ((Vb) antibody) of the (Va) antibody comprising the amino acid sequences of a heavy chain variable region containing one or several substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region containing one or several substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 8 (Vc) A mutant ((Vc) antibody) of an antibody ((Va)) comprising an amino acid sequence of a heavy chain variable region having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 8

[0049] In the above-mentioned ((Va)) antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region are as follows. Also, in the above-mentioned ((Va)) antibody, the nucleotide sequences encoding the amino acid sequences of the heavy chain variable region and the light chain variable region are, for example, as follows. In the heavy chain variable region of the following ((Va)) antibody, the amino acid sequence or nucleotide sequence enclosed in parentheses and underlined corresponds to heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively, from the N-terminus. In the light chain variable region of the following ((Va)) antibody, the amino acid sequence or nucleotide sequence enclosed in parentheses and underlined corresponds to light chain CDR1, light chain CDR2, and light chain CDR3, respectively, from the N-terminus.

[0050]

Table 2

[0051] In the above-mentioned ((Vb)) antibody, "one or several" may be, for example, within the range of binding to the S protein, particularly the S protein of the Omicron strain. In the heavy chain variable region of the above-mentioned ((Vb)) antibody, "one or several" may be, for example, 1 to 22, 1 to 21, 1 to 16, 1 to 15, 1 to 11, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, preferably 1 to 2 or 1. Also, in the light chain variable region of the above-mentioned ((Vb)) antibody, "one or several" may be, for example, 1 to 21, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, preferably 1 to 2 or 1.

[0052] In the (Vb) antibody, the position of the mutation may be, for example, in the CDR or in the FR, but preferably in the FR. When the position of the mutation in the (Vb) antibody is in the FR, the (Vb) antibody contains one or several substitutions, insertions, additions or deletions in the framework region of the antibody.

[0053] In the (Vb) antibody, the "substitution, insertion, addition or deletion" is preferably a substitution, insertion or deletion, or an addition at at least one of the N-terminus and C-terminus of the CDR. The substitution is preferably a conservative substitution.

[0054] In the (Vb) antibody, the amino acid sequences of the CDRs of the heavy chain variable region and the light chain variable region are preferably conserved. In this case, in the antibody of (Vb), the amino acid sequences corresponding to the amino acid sequences of each CDR represented by (Ca) are conserved.

[0055] In the (Vc) antibody, the "identity" is, for example, the degree of identity when the sequences to be compared are properly aligned, and means the occurrence rate (%) of exact matches of amino acids between the sequences. The identity can be calculated with default parameters using analysis software such as BLAST, FASTA, etc. (the same applies hereinafter).

[0056] In the (Vc) antibody, the "identity" only needs to be within the range of binding to the S protein, particularly the S protein of the Omicron strain. In the heavy chain variable region of the (Vc) antibody, the "identity" is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. In the light chain variable region of the (Vc) antibody, the "identity" is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

[0057] In the (Vc) antibody, the amino acids different from the (Va) antibody may be present in the CDR or in the FR, but are preferably present in the FR.

[0058] In the (Vc) antibody, it is preferable that the amino acid sequences of the CDRs of the heavy chain variable region and the light chain variable region are conserved. In this case, in the antibody of (Vc), the amino acid sequences corresponding to the amino acid sequences of the respective CDRs represented by (Ca) are conserved.

[0059] The S protein antibody of the present disclosure preferably specifically binds to, for example, the spike protein of the Omicron strain. The specific binding can be evaluated, for example, by comparing the binding of the S protein antibody to the S protein of the origin strain with the binding of the S protein antibody to the S protein of the Omicron strain. Specifically, first, for the S protein antibody, according to Example 1(5) described later, by the ELISA method using the trimer of the S protein, the binding to the S protein of the origin strain and the S protein of the Omicron strain is evaluated. Then, when the binding of the S protein antibody to the S protein of the Omicron strain is relatively high based on the binding to the S protein of the origin strain, preferably, based on the absorbance indicating the binding to the S protein of the origin strain, when the absorbance indicating the binding to the S protein of the Omicron strain is 2 times or more, 5 times or more, 10 times or more, the S protein antibody can be evaluated as specifically binding to the S protein of the Omicron strain.

[0060] The S protein antibody of the present disclosure preferably has, for example, suppressed binding to the spike protein of the SARS-CoV-2 original strain. The suppression of the binding to the S protein of the original strain can be evaluated, for example, by the binding of the S protein antibody to the S protein of the original strain. Specifically, first, for the S protein antibody, according to Example 1(5) described later, it can be carried out by evaluating the binding to the S protein of the original strain by the ELISA method using the trimer of the S protein.

[0061] The binding to the S protein may be evaluated, for example, by the dissociation constant for the protein to be bound. In this case, the dissociation constant (KD) can be measured, for example, in accordance with Example 1(6) described later. And when the binding ability of the target antibody to the S protein is 1×10 -6 or less, 1×10 -7 or less, 1×10 -8 or less, or 1×10 -9 or less, the target antibody can be evaluated as binding to the S protein. The specific binding to the S protein of the Omicron strain can be evaluated, for example, by the KD (KD 1 ) of the S protein antibody to the S protein of the Omicron strain and the KD (KD 0 ) of the S protein antibody to the S protein of the original strain. Specifically, when the KD 1 of the target antibody is 1×10 -6 or less, 1×10 -7 or less, 1×10 -8 or less, or 1×10 -9 or less, and the KD 0 of the target antibody is 1×10 -3 or more, 1×10 -2 or more, or 1×10 -1 or more (including the case where the dissociation constant cannot be evaluated), the target antibody can be evaluated as specifically binding to the S protein of the Omicron strain.

[0062] The administration target of the antibody or antigen-binding fragment thereof of the present disclosure is, for example, a human or a non-human animal (for example, cow, pig, sheep, mouse, rat, rabbit, horse, monkey, etc.). The dosage of the antibody or antigen-binding fragment thereof of the present disclosure is, for example, a therapeutically effective amount. The administration route of the antibody or antigen-binding fragment thereof of the present disclosure is, for example, oral administration or parenteral administration. Examples of the parenteral administration include intravenous administration and nasal administration.

[0063] The antibody or antigen-binding fragment thereof of the present disclosure can bind to the S protein, particularly the S protein of the Omicron strain. Therefore, the antibody or antigen-binding fragment thereof of the present disclosure can be suitably used, for example, for the detection of SARS2.

[0064] <Nucleic acid> In another aspect, the present disclosure discloses a nucleic acid encoding the S protein antibody of the present disclosure or an antigen-binding fragment thereof. The nucleic acid of the present disclosure encodes the S protein antibody of the present disclosure or an antigen-binding fragment thereof. According to the nucleic acid of the present disclosure, the S protein antibody of the present disclosure or an antigen-binding fragment thereof can be produced by genetic engineering techniques.

[0065] The nucleic acid of the present disclosure can be obtained, for example, by the following method. First, after extracting RNA from a hybridoma producing the S protein antibody of the present disclosure or an antigen-binding fragment thereof, cDNA is synthesized using reverse transcriptase. For the obtained cDNA, the cDNA is amplified using primers for sequences conserved in the variable regions of the heavy chain gene and the light chain gene, and the DNA base sequence information can be determined from the obtained amplified fragment. Further, from the obtained base sequence information, a DNA encoding the S protein antibody can be obtained by chemically synthesizing the sequence of the variable region or a part thereof and binding it to the sequence containing the constant region.

[0066] The nucleic acid of the present disclosure may be functionally linked to a vector such as a plasmid vector.

[0067] <Transformant> In another aspect, the present disclosure discloses a transformant (host cell) containing a nucleic acid encoding the S protein antibody of the present disclosure or an antigen-binding fragment thereof. The transformant of the present disclosure contains the nucleic acid of the present disclosure. According to the transformant of the present disclosure, the S protein antibody of the present disclosure or an antigen-binding fragment thereof can be produced.

[0068] Examples of the transformant include prokaryotic cells such as Escherichia coli and Bacillus subtilis; eukaryotic cells, etc., preferably eukaryotic cells, and more preferably cells derived from mammals. Examples of the cells derived from mammals include Chinese hamster ovary cells (CHO cells), COS, myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, etc.

[0069] The nucleic acid may be a vector containing the nucleic acid. The vector can be appropriately selected according to, for example, the type of transformant. Examples of vectors that can be expressed in mammalian-derived cells include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, pREP4; viral vectors such as pDON-AI DNA (manufactured by Takara Bio Inc.); and the like.

[0070] The S protein antibody or antigen-binding fragment thereof of the present disclosure can be recovered and purified (isolated) from, for example, the culture solution of the transformant. For the separation and purification of the antibody and the like, the separation and purification methods commonly used for ordinary proteins can be used. For the purification, for example, chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, etc. can be appropriately selected and used alone or in combination to separate and purify the antibody.

[0071] <Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition that can be used for diseases associated with SARS-CoV2. The pharmaceutical composition of the present disclosure contains the S protein antibody or antigen-binding fragment thereof of the present disclosure. According to the pharmaceutical composition of the present disclosure, as described below, it may be used for the treatment of SARS-CoV-2. Also,

[0072] Since the pharmaceutical composition of the present disclosure can bind to the RBD domain of the S protein of SARS-CoV-2, for example, it is expected to be suitably used for the preventive treatment of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and / or the treatment of infectious diseases caused by SARS-CoV-2. Therefore, the pharmaceutical composition of the present disclosure is expected to be suitably used, for example, for subjects infected with (patients) SARS-CoV-2 or subjects suspected of being infected.

[0073] The pharmaceutical composition of the present disclosure is expected to be suitably used, for example, as an administration target for a patient with SARS-CoV-2 or a subject suspected of having SARS-CoV-2.

[0074] <Treatment method> In another aspect, the present disclosure provides a treatment method that can be implemented for an infectious disease caused by SARS-CoV-2. The preventive treatment method for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or the treatment method for an infectious disease caused by SARS-CoV-2 of the present disclosure includes an administration step of administering the S protein antibody or an antigen-binding fragment thereof of the present disclosure. According to the treatment method of the present disclosure, it is expected that the treatment for an infectious disease caused by SARS-CoV-2 can be suitably implemented.

[0075] <Detection reagent> In another aspect, the present disclosure discloses a detection reagent that can be used for the detection of the spike protein of SARS-CoV-2. The detection reagent for the spike protein of SARS-CoV-2 of the present disclosure includes the antibody of the present disclosure or an antigen-binding fragment thereof. According to the detection reagent of the present disclosure, the S protein, particularly the S protein of the Omicron strain, can be suitably detected. Therefore, according to the detection reagent of the present disclosure, for example, the Omicron strain can be detected.

[0076] In the detection reagent of the present disclosure, the antibody or an antigen-binding fragment thereof may be labeled.

[0077] The detection reagent of the present disclosure can also be used, for example, for detecting the presence or absence of the S protein or the expression level of the S protein, that is, for qualitative analysis or quantitative analysis. Therefore, the detection reagent of the present disclosure can also be referred to as an analytical reagent, for example.

[0078] <Test reagent> In another aspect, the present disclosure discloses a reagent that can be used for testing the likelihood of contracting SARS-CoV-2. The reagent for testing the likelihood of contracting SARS-CoV-2 of the present disclosure includes the antibody of the present disclosure or an antigen-binding fragment thereof. According to the test reagent of the present disclosure, it is possible to detect a subject who is likely to contract SARS2, particularly the Omicron strain. The test reagent of the present disclosure can also be used, for example, as a companion diagnostic for a pharmaceutical composition used in the treatment of the infectious disease (COVID-19) of SARS2. Therefore, the test reagent of the present disclosure can also be referred to as, for example, a companion diagnostic for COVID-19.

[0079] The detection reagent or test reagent of the present disclosure may constitute a kit. In this case, the detection kit or test kit of the present disclosure includes the antibody of the present disclosure or an antigen-binding fragment thereof and other components. Examples of the other components include other reagents such as buffer solutions and instruction manuals.

[0080] <Detection method> In another aspect, the present disclosure discloses a detection method capable of detecting the S protein, particularly the S protein of the Omicron strain. The detection method of the spike protein of SARS-CoV-2 of the present disclosure includes a contacting step of contacting a sample with the antibody of the present disclosure or an antigen-binding fragment thereof and / or a detection reagent for the S protein of the present disclosure, and a detection step of detecting a complex of the spike protein of SARS-CoV-2 in the sample and the antibody or an antigen-binding fragment thereof. According to the detection method of the present disclosure, the presence or absence of the S protein in the sample and / or the abundance of the S protein in the sample can be detected. Therefore, according to the detection method of the present disclosure, for example, the Omicron strain can be detected.

[0081] The sample may be, for example, a biological sample. The living body is, for example, a living body of a human or a non-human animal (such as a cow, a pig, a sheep, a mouse, a rat, a rabbit, a horse, a monkey, etc.). The biological sample may be, for example, a body fluid, a cell, a tissue, an organ, etc. The sample may be, for example, liquid or solid. When the sample is solid, in the detection method of the present disclosure, it is preferable to mix the solid sample with a liquid to prepare a liquid sample prior to the detection step.

[0082] The contacting step is a step of contacting the antibody or its antigen-binding fragment of the present disclosure, and / or the detection reagent for the S protein of the present disclosure. In the contacting step, the method of contacting the sample with the antibody or its antigen-binding fragment is not particularly limited, and it is preferably carried out, for example, in a liquid. The contacting may be carried out, for example, by mixing the sample with the antibody or its antigen-binding fragment. The liquid may be, for example, water, physiological saline, a buffer solution, etc. The conditions at the time of contact in the contacting step can be set, for example, as general conditions under which binding between an antibody and an antigen occurs.

[0083] The detecting step is a step of detecting a complex of the S protein in the sample and the antibody or its antigen-binding fragment. By detecting the presence or absence of the binding between the two, for example, the presence or absence of the S protein in the sample can be analyzed (qualitatively), and by detecting the degree of binding (binding amount) between the two, for example, the amount of the S protein in the sample can be analyzed (quantitatively).

[0084] In the detection step, if the binding between the S protein and the antibody or its antigen-binding fragment cannot be detected, it can be determined that the S protein does not exist in the sample. If the binding is detected, it can be determined that the S protein exists in the sample. In addition, in the detection step, a correlation between the abundance of the S protein and the binding amount is determined in advance, and based on the correlation, the expression level of the S protein in the sample can also be analyzed from the binding amount. Therefore, the detection method of the present disclosure can also be referred to as an analysis method, for example.

[0085] The method for detecting the binding between the S protein and the antibody or its antigen-binding fragment is not particularly limited. For example, a conventionally known method for detecting the binding between substances can be adopted.

[0086] <Use> The present disclosure is the S protein antibody or its antigen-binding fragment of the present disclosure for use in a prophylactic treatment method for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a treatment method for an infectious disease caused by SARS-CoV-2. In addition, the present disclosure is the use of the S protein antibody or its antigen-binding fragment of the present disclosure in the manufacture of a pharmaceutical composition for use in a prophylactic treatment method for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a treatment method for an infectious disease caused by SARS-CoV-2.

Examples

[0087] Hereinafter, the present disclosure will be described in detail using examples, but the present invention is not limited to the embodiments described. In the following examples, "mol / l" may be denoted as "M". Also, unless otherwise specified, commercially available reagents and kits were used according to the attached protocols.

[0088] [Example 1] Using the spike protein of the SARS-CoV-2 Omicron strain as an antigen, antibodies were produced, and it was confirmed that the binding to the SARS-CoV-2 original strain was suppressed, but antibodies that bind to the SARS-CoV-2 Omicron strain were produced.

[0089] (1) Preparation of antigen DNA encoding the amino acid sequence of a partial sequence (SEQ ID NO: 19) of the S protein RBD domain derived from the SARS-CoV-2 Omicron strain (SEQ ID NO: 20, manufactured by Thermo Fisher Scientific) and DNA encoding a secretion signal sequence derived from the yeast Saccharomyces cerevisiae (SEQ ID NO: 21) were fused, and a plasmid was constructed by placing the fused DNA downstream of the artificial promoter of Pichia pastoris. In the plasmid, the fused DNA was inserted so that a His×6 tag was added to the C-terminus of the RBD domain. After introducing the plasmid into the genome of a Pichia pastoris strain, the obtained colonies were inoculated into a test tube containing DAPG (2,4-Diacetylphloroglucinol, CAS No.: 2161-86-6, manufactured by Santa Cruz, Cat. No.: Sc-206518)-BMDY medium and cultured with shaking for 24 hours (30 °C, 170 rpm). The composition of the BMDY medium was 10 g / l yeast extract, 20 g / l hipolypeptone (manufactured by Fujifilm Wako Pure Chemical Corporation, Cat. No.: 390-02116), 13.4 g / l yeast nitrogen base without amino acids (manufactured by BD Biosciences), 0.4 mg / l biotin (manufactured by Nacalai Tesque), 100 mM potassium phosphate buffer (pH 6.0), and 20 g / l glucose. The DAPG-BMDY medium was prepared by adding DAPG to the BMDY medium at a concentration of 10 μM. After culturing for 24 hours, the culture solution was centrifuged (3500 rpm, 5 minutes), and the supernatant was discarded. The yeast pellet obtained by centrifugation was suspended in BMDY medium (test tube) and then cultured with shaking for 60 hours (18 °C, 170 rpm). After the culturing, the culture solution was centrifuged, and the RBD domain was purified from the supernatant using an affinity column (Capturem (trademark) His-Tagged Purification Maxiprep Columns, manufactured by Takara Bio). After washing the column with a 20 mM imidazole solution, the RBD domain was eluted from the column using a 500 mM imidazole solution.The obtained eluate was desalted using a PD10 column (manufactured by Cytiva), dissolved in PBS (manufactured by Nacalai Tesque, Cat. No.: 27575-31), and an antigen solution was obtained.

[0090] Partial sequence of the RBD domain (SEQ ID NO: 19) ITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGGHHHHHH

[0091] Nucleotide sequence encoding the partial sequence of the RBD domain (SEQ ID NO: 20) 5'-atcaccaacttgtgcccattcgacgaggttttcaacgctactagattcgcttccgtttacgcctggaacagaaagagaatctccaactgcgttgctgactactccgtcttgtataacctggctccattcttcaccttcaagtgctacggtgtttccccaactaagttgaacgacctgtgtttcactaacgtctacgccgactccttcgttattagaggtgacgaggttagacagatcgctccaggtcaaactggtaatatcgctgactacaactacaagctgccagacgacttcaccggttgtgttattgcttggaactccaacaagctggactccaaggtttccggtaactacaattacctgtaccgtctgttcagaaagtccaacctgaagccattcgagagagacatctccaccgaaatctaccaagctggtaacaagccatgtaacggtgttgccggtttcaactgttacttcccattgagatcctacagcttccgtccaacttacggtgttggtcatcagccatacagagtcgtcgttttgtccttcgagttgttgcatgctccagctactgtttgcggtccaaagaagtccactaacctggtcaagaacaagggtggtggtcatcatcatcaccatcactag-3'

[0092] Nucleotide sequence encoding a secretion signal sequence (SEQ ID NO: 21) 5'-atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgctgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaaga-3'

[0093] (2) Immunization The prepared antigen was used to immunize chickens. Three 34-day-old Borris Brown chickens were used. After adding 0.5 ml of D-PBS (manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 045-29795) to 1 ml of the antigen solution (antigen concentration: 1 mg / ml), it was mixed with 1.5 ml of adjuvant to prepare a total of 3 ml of immunogen. At the first immunization, Freund's Complete Adjuvant (manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 014-09541) was used as the adjuvant, and at the second and subsequent immunizations, Freund's Incomplete Adjuvant (manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 011-09551) was used. The obtained immunogen was intraperitoneally administered to each chicken at a dose of 1 ml per time. At the final immunization, 333 μg of the antigen was administered via the wing vein. Three days after the final immunization, after collecting all the blood from each chicken, the spleen was collected. The RNA extracted from the collected spleen was stored at -80°C.

[0094] (3) Confirmation of the antibody titer of the final antiserum After preparing the antiserum from the whole blood collection, the binding ability of the antiserum to the RBD domain of the S protein was confirmed by ELISA. First, a solid-phase solution was prepared to a concentration of 5 μg / ml BSA (manufactured by NEB, Cat No.: B9001S) and 1 μg / ml SARS-CoV2-B.1.1.529 (Omicron strain) Spike RBD Protein (manufactured by Cosmo Bio, Cat No.: SIN-40592-V08H121-100). The obtained solid-phase solution was added to an Immuno Plate MAXISORP NUNC-IMMUNO PLATE (manufactured by Thermo Fisher Scientific, Cat No.: 442404) and left standing at 4°C overnight (about 8 hours; the same applies hereinafter) to immobilize the BSA or RBD domain on the plate. After the immobilization, the solid-phase solution was discarded, and then blocking was performed at 37°C for 1 hour using a 25% blocking solution. The 25% blocking solution was prepared by dissolving Block Ace (manufactured by KAC, Cat No.: UKB80) in PBS to a concentration of 25%. The antiserum was serially diluted 4-fold by 10 times (1-fold, 10-fold, 100-fold, or 1000-fold) using a 10% blocking solution to prepare sample solutions. The 10% blocking solution was prepared by dissolving Block Ace in PBS to a concentration of 10%. After the blocking, each well was washed with PBS-T, and then the sample solution was added to each well and reacted at 37°C for 1 hour. The composition of PBS-T is 137 mM NaCl, 8.9 mM Na 2 HPO 4 ·12H 2 O, 2.7 mM KCl, 1.5 mM KH 2 PO 4、0.05% Tween-20 was used. After the reaction, each well was washed with PBS-T, and a secondary antibody (HRP-anti-chicken-IgG, manufactured by KPL, Cat No.: 5220-0373) diluted 1000-fold was added, and the reaction was carried out at 37°C for 1 hour. Next, after washing each well with the PBS-T, a chromogenic dye (KPL SureBlue™, manufactured by KPL, Cat. No.: 5120-0074) was added and allowed to develop color at room temperature (about 25°C, the same hereinafter) for 30 minutes. After the color development, a reaction stop solution (KPL TMB stop solution, manufactured by KPL, Cat No.: 5150-0019) was added to stop the reaction. For each well of the plate, the absorbance at 450 nm - 650 nm was measured using a multiplate reader Cytation5 (manufactured by BioTek, Cat No.: GEN5). The results of the ELISA using the antiserum are shown in Figure 1.

[0095] Figure 1 is a graph showing the antibody titers of the antiserum against each antigen. In Figure 1, the horizontal axis represents the dilution ratio of the antiserum, and the vertical axis represents the absorbance value representing the antibody titer. As shown in Figure 1, it was confirmed that in the antiserum, the antibody titer against the S protein of the Omicron strain was higher than that against BSA. That is, it was confirmed that an antiserum with low binding affinity to BSA but high binding affinity to the S protein of the Omicron strain was obtained.

[0096] (4) Acquisition of antibodies Using an mRNA extraction kit (manufactured by Promega, Cat No.: Z5310), the mRNA of the collected chicken spleen was extracted. The RNA was used to synthesize cDNA by RT-PCR using the PrimeScript II 1st Strand cDNA Synthesis Kit (manufactured by TAKARA, Cat No.: 6210A). Subsequently, an scFv phage library was prepared from the cDNA with reference to the method described in "Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814" (Reference 1). Panning of the scFv phage library was performed using a plate immobilized with SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein. The panning was carried out with reference to the method described in Reference 1 above. 200 μg of SARS-CoV-2 WT RBD (SEQ ID NO: 22, derived from the secreted expression of Pichia pastoris at Kobe University, hereinafter also referred to as "SARS-CoV-2 WT RBD (original strain)") was prepared to 1 mg / ml, and an absorption operation was performed at each panning step. The SARS-CoV-2 WT RBD (original strain) has a ×6His tag linked to the C-terminus of the RBD domain via a ×3 glycine linker. The binding property of the library was confirmed by ELISA. A solid-phase solution was prepared such that BSA was 5 μg / ml, SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein was 1 μg / ml, and SARS-CoV2 WT RBD (original strain) was 1 μg / ml. After adding the obtained solid-phase solution to the immunoplate, it was left standing at 4°C overnight (about 8 hours) to immobilize the BSA or RBD domain on the plate. After immobilization, the solid-phase solution was discarded, and then blocking was performed at 37°C for 1 hour using the 25% blocking solution. After blocking, each well was washed with PBS-T, and then the re-expressed supernatant of the scFv library at each panning step was added to each well and reacted at 37°C for 1 hour.After the reaction, each well was washed with PBS-T, and then a secondary antibody (HRP-anti-mouse-IgG, manufactured by KPL, Cat. No.: 5220-0341) diluted 1000-fold was added and reacted at 37°C for 1 hour. Next, after washing each well with the PBS-T, the color-developing reagent was added and allowed to develop color at room temperature for 30 minutes. After the color development, the reaction stop solution was added to stop the reaction. For each well of the plate, the absorbance at 450 nm - 650 nm was measured using the multiplate reader. The results of ELISA using the scFv library at each panning stage are shown in Figure 2.

[0097] SARS-CoV-2 WT RBD (SEQ ID NO: 22) ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKGGGHHHHHH

[0098] Figure 2 is a graph showing the binding ability of the library at each panning stage to each antigen. In Figure 2, the horizontal axis indicates the number of panning cycles, and the vertical axis indicates the absorbance value representing the binding ability of the library. As shown in Figure 2, it was confirmed that the binding ability to the S protein (RBD (Omicron)) of the Omicron strain increased with each panning cycle. Also, as shown in Figure 2, the binding ability to the S protein (RBD(WT)) of the original strain also increased, but it was confirmed that the increase in its binding ability was suppressed to a lower level compared to the S protein of the Omicron strain. On the other hand, it was confirmed that the binding ability to BSA was suppressed at a low level even after multiple panning cycles. That is, by repeating the panning cycles, it was found that an scFv library with high binding ability to the S protein of the Omicron strain but suppressed binding ability to the S protein of the original strain and BSA was obtained.

[0099] After screening the 11th round of the phage display library, clones were obtained that had binding affinity for the SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein but not for the SARS-CoV-2 WT RBD (original strain). By performing sequence analysis of the obtained clones, the nucleotide sequences were identified. Also, from the nucleotide sequences of the clones, the amino acid sequences of the heavy chain variable region (SEQ ID NO: 7) and the light chain variable region (SEQ ID NO: 8) of the clones were determined (Table 2). Further, from the amino acid sequences, the amino acid sequences of heavy chain CDR1 to CDR3 and light chain CDR1 to 3 shown in Table 1 were determined based on Kabat numbering. Using the DNA strand encoding the scFv antibody of the clone as a template, the variable regions of the antibody genes of the heavy chain and the light chain derived from chicken were amplified by PCR. Thereafter, the amplified DNA was subcloned into a vector in which the constant regions of the chicken heavy chain (C-terminal His-tag) and the light chain were pre-incorporated. The obtained constructs of the heavy chain and the light chain were transiently expressed in 293 cells (manufactured by Thermo Fisher Scientific, Cat. No.: A14528). From the culture solution containing the obtained antibody, a monoclonal antibody (Omi-7 IgY) was purified using Ni-Sepharose (trademark) excel (manufactured by Thermo Fisher Scientific, Cat. No.: 17-3712-01). Thereafter, the purified antibody solution was buffer-exchanged to PBS using PD-10 (manufactured by Cytiva).

[0100] (5) Confirmation of antibody titer of the purified antibody SARS-CoV-2 Spike trimer (from the original strain) (manufactured by Acro Biosystems, Cat. No.: SPN-C52H9) and SARS-CoV-2 Spike trimer Omicron / B.1.1.529 (from the Omicron strain) (Acro Biosystems, Cat. No.: SPN-C52Hz) were each prepared into immobilization solutions at a concentration of 1 μg / ml. After adding the obtained immobilization solutions to the immunoplates, they were left standing at 4°C overnight to immobilize the trimers on the plates. After the immobilization, the immobilization solutions were discarded, and then blocking was performed at 37°C for 1 hour using 25% blocking solution. Serial dilutions of the primary antibody Omi-7 IgY were performed in six steps by a factor of 10 from 100 ng / ml (100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 ng / ml) using 10% blocking solution to prepare sample solutions. After the blocking, each well was washed with PBS-T, and then the sample solutions were added to each well and reacted at 37°C for 1 hour. After the reaction, each well was washed with PBS-T, and a 1000-fold diluted secondary antibody (HRP-anti-chicken-IgG) was added and reacted at 37°C for 1 hour. Next, after washing each well with PBS-T, the chromogenic dye was added and allowed to develop color at room temperature for 30 minutes. After the color development, the reaction stop solution was added to stop the reaction. For each well of the plate, the absorbance at 450 nm - 650 nm was measured using the microplate reader. These results are shown in Figure 3.

[0101] Figure 3 is a graph showing the antibody titers of the purified antibody against each antigen. In Figure 3, the horizontal axis represents the dilution factor of the purified antibody, and the vertical axis represents the absorbance value representing the antibody titer. As shown in Figure 3, it was confirmed that Omi-7 IgY had its binding to the S protein of the original strain suppressed, while its binding affinity to the S protein of the Omicron strain was extremely high. That is, it was confirmed that Omi-7 IgY is an antibody that does not bind to the S protein of the original strain but has a high binding affinity to the S protein of the Omicron strain.

[0102] (6) Confirmation of the affinity of the purified antibody The affinity of the purified Omi-7 IgY antibody was examined. Specifically, the affinity was measured using the single-cycle kinetics method. For the measurement, a Series S Biotin CAPture Kit (manufactured by Cytiva, 28920234) was used. As the running buffer, HBS-EP+ buffer (manufactured by Cytiva, BR100669) was used. For biotinylated Omi-7 in which the Omi-7 IgY antibody was biotinylated, it was adjusted to 2.5 nM using the running buffer HBS-EP+ buffer (manufactured by Cytiva, BR100669) to obtain a ligand solution. The Biotin CAPture Reagent was diluted 40-fold with the running buffer. SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein (manufactured by Sino Biological, 40592-V08H121) and SARS-CoV2 WT RBD (original strain) (manufactured by Sino Biological, 40592-V08H) were each diluted with the running buffer, and five 3-fold dilution series points starting from 300 nM were prepared to obtain an analyte solution. Also, the running buffer was used as the blank solution. The diluted Biotin CAPture Reagent was added to Flow cell 1 and 2 at a flow rate of 2 μl / min for 300 seconds. Next, the ligand solution was added to Flow cell 2 under the conditions of a flow rate of 10 μl / min for 180 seconds. After the addition, the blank solution or the analyte solution was added to Flow cell 1 and 2 under the conditions of a flow rate of 30 μl / min for 120 seconds. The dissociation time was set to 60 seconds. Thereafter, the Regeneration buffer was added at a flow rate of 10 μl / min for 120 seconds to dissociate the ligand antibody. For the analysis, Biacore (trademark) Insight Evaluation Software (manufactured by Cytiva) was used, and the binding parameters were calculated by a 1:1 binding model. These results are shown in Table 3 below.

[0103] Table 3 below is a table showing the affinity of the Omi-7 IgY antibody for the SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein and the SARS-CoV2 WT RBD (original strain). As shown in Table 3 below, the Omi-7 IgY antibody showed no affinity for the SARS-CoV2 WT RBD (original strain), but was found to show high affinity for the SARS-CoV-2 B.1.1.529 (Omicron strain) Spike RBD Protein.

[0104] [Table 3]

[0105] (6) Confirmation of the binding of the purified antibody by Western blotting After preparing the SARS-CoV-2 Spike trimer and the SARS-CoV-2 Spike trimer Omicon / B.1.1.529 to 10 ng / lane, they were applied to a electrophoresis gel (NuPAGE 4-12% BIS-Tris Gel, manufactured by Thermo Fisher Scientific, NP0329BOX). The buffer for SDS-PAGE used MOPS Buffer (manufactured by Thermo Fisher Scientific, Cat No.: NP0001) and electrophoresis was performed at 125 V for 90 minutes. Using an iBlot2 Gel Transfer Device (manufactured by Thermo Fisher Scientific, Cat. No.: IB21001), the electrophoresed gel was transferred to a membrane (iBlot2 PVDF Mini Stacks, manufactured by Thermo Fisher Scientific, Cat. No.: IB240002). The transferred membrane was blocked with a blocking solution (Blocking One, manufactured by Nacalai Tesque, Cat. No.: 03953-95) for 30 minutes at room temperature. Omi-7 IgY was diluted with the blocking solution to 100 ng / ml and then reacted with the membrane at 4°C overnight. After the reaction, the membrane was washed 3 times with TBS-T (50 mM Tris-HCl (pH 7.6) 0.05% Tween-20) for 5 minutes each. After the washing, a secondary antibody (HRP-anti-chicken-IgG) diluted 10,000-fold was added and reacted at room temperature for 1 hour. The blocking solution was used for diluting the secondary antibody. Next, the membrane was washed 3 times with the TBS-T for 5 minutes each. The membrane was reacted with a luminescence reagent (ECL(™) Prime Western Blotting Detection Regents, manufactured by Cytiva, Cat. No.: RPN2232) and then photographed using LuminoGraph (manufactured by ATTO, WSE-6100) under the condition of an exposure time of 1 second. For the detection of the His tag, Anti 6*His, His-Tag (manufactured by PGI, HRP-66005) was used. The results of Western blotting are shown in Figure 4.

[0106] Figure 4 is a Western blotting photograph showing the binding property of the purified antibody. In Figure 4, each lane shows the results of SARS-CoV-2 Spike trimer (1) and SARS-CoV-2 Spike trimer Omicron / B.1.1.529 (2) from left to right. As shown in Figure 4, a band was confirmed around 250 kD in lane 2, which was not confirmed in lane 1. From these, it was confirmed that Omi-7 IgY is an antibody that does not bind to the S protein of the original strain and shows high binding to the S protein of the Omicron strain.

[0107] As described above, the present disclosure has been described with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0108] <Supplementary Note> Some or all of the above embodiments and examples can be described as follows, but are not limited thereto. (Supplementary Note 1) An antibody or an antigen-binding fragment thereof against the spike protein of SARS-CoV-2, wherein the antibody is an antibody or an antigen-binding fragment thereof selected from the group consisting of the following (Ca) and (Cb): (Ca) An antibody comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 shown in SEQ ID NOs: 4, 5, and 6, respectively; (Cb) A variant of the (Ca) antibody comprising one substitution, insertion, addition, or deletion in the whole of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3. (Supplementary Note 2) The antibody is an antibody or an antigen-binding fragment thereof according to Supplementary Note 1, selected from the group consisting of the following (Va), (Vb), and (Vc): An antibody comprising the amino acid sequences of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8; (Vb) A variant of the (Va) antibody, comprising the amino acid sequences of a heavy chain variable region containing 1 to 11 substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region containing 1 to 10 substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 8; (Vc) A variant of the (Va) antibody, comprising the amino acid sequences of a heavy chain variable region having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 8. (Appendix 3) The antibody is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, bispecific or oligo-specific antibody, single-chain antibody, single-chain antibody (scFv), diabody, sc(Fv) 2 , and an antibody selected from scFv-Fc, the antibody or antigen-binding fragment thereof according to Appendix 1 or 2. (Appendix 4) The antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 3, wherein the binding to the spike protein of the SARS-CoV-2 original strain is suppressed. (Appendix 5) The antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 4, which specifically binds to the spike protein of the SARS-CoV-2 Omicron strain. (Appendix 6) A detection reagent for the spike protein of SARS-CoV-2, comprising the antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 5. (Appendix 7) A reagent for use in testing the likelihood of SARS-CoV-2 infection, comprising the antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 5. (Appendix 8) A contacting step of contacting a sample with the antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 5; A method for detecting the spike protein of SARS-CoV-2, comprising a detection step of detecting a complex of the spike protein of SARS-CoV-2 in the sample and the antibody or its antigen-binding fragment. (Appendix 9) A contacting step of contacting a biological sample of a subject with the antibody or its antigen-binding fragment according to any one of Appendices 1 to 5, A test method for the possibility of SARS-CoV-2 infection, comprising a measuring step of measuring the expression level of the spike protein of SARS-CoV-2 in the biological sample. (Appendix 10) The test method according to Appendix 9, comprising a comparing step of comparing the expression level of the spike protein of SARS-CoV-2 in the biological sample with a reference value. (Appendix 11) The test method according to Appendix 10, wherein the reference value is the expression level of the spike protein of SARS-CoV-2 in a biological sample of a healthy subject or a biological sample of a SARS-CoV-2 patient.

Industrial Applicability

[0109] As described above, according to the present disclosure, a new antibody capable of binding to SARS-CoV-2 can be provided. Therefore, the present disclosure is extremely useful in, for example, the fields of inspection and medicine.

Claims

1. An antibody or antigen-binding fragment thereof against the spike protein of SARS-CoV-2, The antibody is an antibody or an antigen-binding fragment thereof selected from the group consisting of (Ca) and (Cb) below: (Ca) an antibody comprising the amino acid sequences of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain CDR1, light chain CDR2 and light chain CDR3 shown in SEQ ID NOs: 4, 5 and 6, respectively; (Cb) A variant of the antibody (Ca) comprising a single substitution, insertion, addition or deletion throughout the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is selected from the group consisting of (Va), (Vb), and (Vc): (Va) an antibody comprising the amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 8; (Vb) an antibody mutant (Va) comprising an amino acid sequence of a heavy chain variable region having 1 to 11 substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 7 and an amino acid sequence of a heavy chain variable region having 1 to 10 substitutions, insertions, additions or deletions in the amino acid sequence shown in SEQ ID NO: 8; (Vc) An antibody mutant (Va) comprising an amino acid sequence of a heavy chain variable region having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

8.

3. The antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a bispecific or oligospecific antibody, a single chain antibody, a single chain antibody (scFv), a diabody, a sc(Fv), ２ The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is an antibody selected from the group consisting of scFv-Fc and scFv-Fc.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which has suppressed binding to the spike protein of the SARS-CoV-2 origin strain.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which specifically binds to the spike protein of the Omicron strain of SARS-CoV-2.

6. A detection reagent for the spike protein of SARS-CoV-2, comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. A reagent for use in testing for possible infection with SARS-CoV-2, comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. A contacting step of contacting a sample with the antibody or antigen-binding fragment thereof of claim 1 or 2; detecting a complex between the spike protein of SARS-CoV-2 and the antibody or antigen-binding fragment thereof in the sample.

9. A contacting step of contacting a biological sample from a subject with the antibody or antigen-binding fragment thereof according to claim 1 or 2; A method for testing the possibility of SARS-CoV-2 infection, comprising a measuring step of measuring the expression level of spike protein of SARS-CoV-2 in the biological sample.

10. The test method according to claim 9, comprising a comparison step of comparing the expression level of SARS-CoV-2 spike protein in the biological sample with a reference value.

11. The test method according to claim 10, wherein the reference value is the expression level of spike protein of SARS-CoV-2 in a biological sample of a healthy person or a biological sample of a SARS-CoV-2 patient.

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