JP6954696B1 - Variants of SARS-CoV-2 Spike protein and their uses - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a variant of a Peplomer protein capable of reducing the inducing ability of an antibody which enhances the binding ability of the Spike protein of SARS-CoV-2 to angiotensin converting enzyme 2. A variant of the SARS-CoV-2 Spike protein, which may be a protein consisting of the following (Sm1) amino acid sequence: (Sm1) Amino acid of the wild SARS-CoV-2 Spike protein (Sw protein). In the sequence, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213rd, and 214th positions in the Spike protein (Ss protein, specific sequence) of the reference SARS-CoV-2. An amino acid sequence having a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 216 and 217. [Selection diagram] Fig. 6

Description

The present invention relates to variants of the SARS-CoV-2 Spike protein and uses thereof.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has been prevalent worldwide since its outbreak was confirmed near Wuhan, China, in November 2019. In addition, the number of people suffering from the new coronavirus infection (COVID-19) caused by SARS-CoV-2 infection and the number of deaths due to COVID-19 is increasing rapidly, and the development of therapeutic agents and vaccines is underway.

In human infection with SARS-CoV-2, binding of the Spike protein to angiotensin converting enzyme 2 (ACE2) is considered to be important in establishing intracellular infection (Non-Patent Document). 1).

Markus Hoffmann et.al., “SARS-CoV-2 Cell Entry Depends on ACE2and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor”, Cell, 2020, vol. 181, pages 271-280

In viruses other than SARS-CoV-2 (hereinafter, also referred to as "SARS2") of the Coronavirus family, an antibody against the virus may cause Fc receptor-dependent enhancement of infection (antibody-dependent enhancement (ADE)). Although known, the mechanism of antibody-dependent enhancement of infection has not been fully elucidated. In addition, Fc receptor-dependent ADE has been confirmed in SARS2. Therefore, when an antibody having an ability to induce infection enhancement is induced in the SARS2 vaccine, there is a concern that administration of the vaccine may conversely lead to aggravation of COVID-19.

Therefore, an object of the present invention is to provide a variant of a Peplomer protein capable of reducing the inducing ability of an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein.

In order to achieve the above object, the variant of the Spike protein of SARS-CoV-2 of the present invention (hereinafter, also referred to as “mutant protein”) is a protein consisting of the following (Sm) amino acid sequence:
(Sm) The amino acid sequence of the following (Sm1), (Sm2), or (Sm3):
(Sm1) In the amino acid sequence of the Spike protein (Sw protein) of wild SARS-CoV-2, the 30th, 32nd, and 64th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, It has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 65, 66, 186, 187, 211, 213, 214, 216, and 217. Amino acid sequence;
In the amino acid sequence of (Sm2) (Sm1), an amino acid sequence having 1 to 127 insertions, additions, substitutions and / or deletions and in which the mutated amino acid of (Sm1) is maintained;
(Sm3) An amino acid sequence having 90% or more identity with respect to the amino acid sequence of (Sm1) and maintaining the mutated amino acid of (Sm1).

The mutant polypeptide of the present invention (hereinafter, also referred to as “muted peptide”) includes a partial polypeptide having a partial sequence of a variant of the Spike protein of the present invention.
The partial polypeptide comprises at least one mutant amino acid in the variant.

The SARS-CoV-2 mutant virus of the present invention (hereinafter, also referred to as “mutant virus”) includes a variant of the Spike protein of the present invention.

The virus-like particles of the present invention (hereinafter, also referred to as “VLP”) include the variants of the Spike protein of the present invention and / or the mutant polypeptides of the present invention.

The nucleic acid of the present invention includes a nucleic acid molecule encoding a variant of the Spike protein of the present invention and / or a nucleic acid molecule encoding the mutant polypeptide of the present invention.

The expression vector of the present invention contains the nucleic acid of the present invention.

The pharmaceutical composition of the present invention is a variant of the Spike protein of the present invention, the mutant polypeptide of the present invention, the mutant virus of the present invention, the virus-like particles of the present invention, the nucleic acid of the present invention, and / or The expression vector of the present invention and a pharmaceutically acceptable carrier are included.

The present invention is a method for producing a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (St protein) of the target SARS-CoV-2, the 30th, 32nd, 64th, 65th, and 66th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A mutation step of introducing a mutation into an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. include.

The present invention is a method for screening a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (Sc protein) of the candidate SARS-CoV-2, the 30th, 32nd, 64th, 65th, and 66th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A Sc protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. It comprises a selection step of selecting as a variant of the Spike protein.

The SARS-CoV-2 infection enhancing marker (hereinafter, also referred to as “first marker”) of the present invention is the Spike protein (Sw protein) of wild SARS-CoV-2, which is the Spike of the reference SARS-CoV-2. From the amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in the protein (Ss protein, SEQ ID NO: 1) An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group.

The SARS-CoV-2 infection-enhancing marker (hereinafter, also referred to as “second marker”) of the present invention competes with the reference antibody for binding of the wild-type SARS-CoV-2 to the Spike protein (Sw protein). It is a competing antibody and
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, 216, and 217.

The method for detecting an infection-enhancing antibody for SARS-CoV-2 of the present invention (hereinafter, also referred to as "first detection method") is a Spike protein (Sw) of wild-type SARS-CoV-2 for a biological sample of a subject. Protein), 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213rd, in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A detection step of detecting an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 214, 216, and 217 is included.

The method for detecting the SARS-CoV-2 infection-enhancing antibody of the present invention (hereinafter, also referred to as “second detection method”) is a wild-type SARS-CoV-2 spike protein (Sw) for a biological sample of a subject. Including a detection step of detecting a competing antibody that competes with a reference antibody for binding to a protein).
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, 216, and 217.

According to the present invention, it is possible to reduce the inducing ability of an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Spike protein.

FIG. 1 is a graph showing the binding of full-length S protein-expressing cells to ACE2 in the presence of each antibody recognition site and each antibody sample in S protein in Example 1. FIG. 2 is a graph showing the binding of full-length S protein-expressing cells to ACE2 in the presence of each antibody sample in Example 1. FIG. 3 is a graph showing the binding of full-length S protein-expressing cells and ACE2 in the presence of each antibody sample in Example 1. FIG. 4 is a graph showing the infection result of the pseudotyped virus expressing the Peplomer protein in Example 2. FIG. 5 is a graph showing the infection result of SARS-CoV-2 in Example 2. FIG. 6 is a graph showing relative values of fluorescence intensity in Example 3. FIG. 7 is a graph showing changes in the fluorescence intensity of the infection-enhancing antibody (clone 8D2, 2210, 2490) in Example 4. FIG. 8 shows the positions of the NTD amino acids commonly recognized by the infection-enhancing antibody in Example 4. FIG. 9 is a histogram showing the amount of antibody bound in Example 5. FIG. 10 is a histogram showing the amount of antibody bound in Example 6. FIG. 11 is a graph showing the relationship between the anti-NTD antibody titer, the anti-RBD antibody titer, the ACE2 binding property, and the infection-enhancing antibody titer in the serum of a SARS-CoV-2 infected person in Example 7. FIG. 12 is a graph showing changes in the infection-enhancing antibody titer, anti-RBD antibody titer, or ACE2-binding property in the serum of a SARS-CoV-2 infected person in Example 8. FIG. 13 is a histogram showing the amount of anti-NTD antibody bound to serum in Example 9. FIG. 14 is a diagram showing the structure of the Spike protein in Example 10. FIG. 15 is a graph showing the antibody titer in serum in Example 11. FIG. 16 is a graph showing the binding property to ACE2 and the infection rate of pseudotyped virus in Example 12. FIG. 17 is a graph showing the binding property to ACE2 in Example 13.

Hereinafter, the present invention will be described with reference to an example. In the following description, the description of each invention can be incorporated into each other unless otherwise specified.

<Definition>
In the present invention, "SARS-CoV-2" means severe acute respiratory syndrome coronavirus 2. The SARS is a virus belonging to the SARS-related coronavirus species (Severe acute respiratory syndrome-related coronavirus) of the betacoronavirus genus ( Betacoronavirus) of the Coronaviridae family. In addition, SARS2 is a virus having a single-stranded positive-strand RNA as a genome.

In the present invention, "spiked protein (S protein)" means a protein or polypeptide having all or part of the amino acid sequence of the spiked protein of SARS-CoV-2. The S protein is a protein that forms virus particles together with nucleocapsid (N) protein, membrane (M) protein, and envelope (E) protein in SARS2, and is arranged outward from the envelope of the virus particles. There is. As mentioned above, the S protein is presumed to be important for establishing intracellular infection via ACE2. Examples of the S protein include a protein consisting of an amino acid sequence registered in Genbank with Accession No .: YP_009724390.1 (reference S protein (Ss protein), SEQ ID NO: 1). In addition, examples of the S protein include proteins encoded by the base sequences 21563 to 25384 of the base sequence (including SEQ ID NO: 2 and stop codon) registered in Genbank with Accession No .: NC_045512.2. .. In the present invention, the S protein may be a protein lacking the amino acid sequence of the intracellular region indicated by the underlined in parentheses in the amino acid sequence of SEQ ID NO: 1. Further, the gene encoding the S protein may be a gene encoding a protein lacking the amino acid sequence of the intracellular region indicated by the underlined in parentheses in the nucleotide sequence of SEQ ID NO: 2. By deleting the intracellular region, a soluble S protein can be produced as the S protein.

Reference S protein (SEQ ID NO: 1)
[ KFDEDDSEPVLKGVKLHYT ]

Nucleotide sequence encoding reference S protein (SEQ ID NO: 2)
5'-[ AAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTACACATAA] -3'

The S protein contains an S1 region containing an N-terminal domain (NTD or NTD region) and a receptor binding domain (RBD or RBD region) and an S2 region from the N-terminal to the C-terminal. Examples of the amino acid sequences of the NTD, RBD and S2 regions in the reference S protein include the amino acid sequences of SEQ ID NOs: 3 to 5.

Reference S protein NTD (SEQ ID NO: 3)
QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRF

RBD of reference S protein (SEQ ID NO: 4)
NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVVG

S2 region of reference S protein (SEQ ID NO: 5)
ILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWY

The amino acid sequences of S proteins other than the reference S protein are, for example, NCBI SARS-CoV-2 Resources (https://www.ncbi.nlm.nih.gov/sars-cov-2/), CoV-GLUE (http). (: //cov-glue.cvr.gla.ac.uk/#/replacement) can be referred to.

In the present invention, the "wild-type spike protein" (Sw protein) means a spike protein in which the amino acid residue at a predetermined position is the same as that of the Ss protein based on the amino acid sequence of the Ss protein. do. The predetermined positions are the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th positions in the Ss protein. In the Ss protein, the amino acid residues at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 are N30, respectively. , F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217.

In the present invention, "angiotensin converting enzyme 2" (ACE2) is a zinc metalloprotease belonging to the ACE family, and is considered to function as a regulator of the renin-angiotensin system. In addition, ACE2 is considered to be important for establishing intracellular infection of SARS2 as described above. Examples of the human ACE2 protein include a protein (SEQ ID NO: 6) consisting of an amino acid sequence registered in Genbank with Accession No .: NP_001358344.1. The base sequence encoding the human ACE2 protein includes, for example, a protein encoded by the base sequence (including SEQ ID NO: 7, stop codon) registered in Genbank with Accession No .: NM_001371415.1.

Human ACE2 protein (SEQ ID NO: 6)
MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDDVQTSF

Nucleotide sequence encoding human ACE2 protein (SEQ ID NO: 7)
5'--3'

In the present invention, the "Fc receptor" is generally known as a receptor protein capable of binding to the Fc region of an antibody molecule. In addition, the Fc receptor is considered to play an important role in Fc receptor-dependent ADE. Examples of the Fc receptor include Fc receptors for IgA, IgG, IgE, and IgM, and specific examples thereof include FcγR, FcαR, FcεR, FcμR, and FcRn (fetal Fc receptors). Examples of the FcγR include FcγR1 (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In addition, in the Fc receptor, FcγRII (CD32) is generally considered to be involved in ADE in coronavirus.

In the present invention, a "virus-like particle (VLP)" is, for example, a structure in which at least one attribute is similar to a virus but is not contagious or is shown to be non-contagious. Means the body. In the present invention, the VLP does not have, for example, the genetic information or nucleic acid encoding the protein or polypeptide that the VLP has. The VLP is generally non-communicable because it lacks the genome of the virus from which the VLP is derived.

In the present invention, an "expression vector" (vector) means a recombinant plasmid or virus containing a nucleic acid delivered to a host cell in vitro or in vivo.

In the present invention, the "vaccine" is, for example, an attenuated or inactivated (killed) pathogen, or a preparation of an antigenic determinant (epitope) derived from the pathogen, and antibody immunity or cell-mediated immunity against the pathogen. Means the preparation used to induce. In the present invention, said vaccine may mean, for example, a preparation administered to provide immunity to SARS2 or protection against COVID-19 caused by SARS2. The "vaccine" also means, for example, a suspension or solution of an immunogen that, when administered to a subject, provides an immune response, i.e., immunity that prevents or reduces the severity of the disease caused by the infection. You may. The vaccine can also be referred to as, for example, a "vaccine composition" or a "vaccine formulation".

In the present invention, the "immune response" is an antibody or cell-mediated reaction to an infectious pathogen or disease exhibited by a vertebrate such as a human being, which prevents or ameliorate the infection, or causes a disease symptom caused by the infection. Means a reaction that alleviates at least one. In the case of a reaction with the antibody, the immune response is, for example, neutralization of the infectious agent (eg, immunogen, hereafter, the same) with the antibody; blocking, inhibiting, preventing, or suppressing the invasion of the infectious agent into cells. It means at least one of; inhibition of replication of an infectious pathogen; and / or stimulation of the production of an antibody that protects against infection of the host cell and destruction of the host cell. Also, in the case of a response by the cells, the immune response means, for example, an immune response mediated by T cells and / or other leukocyte cells against an infectious agent or disease. Specifically, said immune response means, for example, prevention or amelioration of an infection or disease by cells, or alleviation of at least one symptom of a disease symptom resulting from the infection. The immune response can also be referred to as, for example, a "defensive response" or a "defensive immune response."

In the present invention, the subject, or the subject to be treated, administered, or treated (hereinafter collectively referred to as "administration subject" or "subject") is not particularly limited. Examples of the subject include humans and non-human animals other than humans. Examples of the non-human animals include mammals such as mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins and sea lions; birds such as chickens, citrus butterflies, ducks and geese; fish; etc. Can be given. The age of the subject is not particularly limited, and examples thereof include newborn babies, infants, toddlers, children, and adults.

In the present invention, "immune activator" means a compound that enhances, modifies, or modifies the resulting immune response when used in combination with a particular immunogen in a formulation or composition. The enhancement, modification, or modification of the immune response means, for example, enhancement, increase, or enhancement of the specificity of at least one of an antibody response and a cell-mediated immune response. The enhancement, modification, or modification of the immune response may mean a reduction, reduction, or suppression of a particular antigen-specific immune response. Examples of the immunogen include the mutant protein, the mutant peptide, SARS-CoV-2 containing the mutant protein, the mutant protein or VLP containing the mutant peptide, the nucleic acid molecule, the expression vector and the like. The immunostimulatory agent includes, for example, an adjuvant.

The antibody response is measured, for example, by measuring the amount of neutralizing antibody and / or serum antibody by the ELISA method, a binding inhibition assay with recombinant ACE2, an infection inhibition assay of SARS2, an infection inhibition assay of SARS2 pseudovirus, and the like. Can be measured. The cell-mediated immune response can be measured, for example, by measuring the response by cytotoxic T cells (CD8 + T cells), antigen presenting cells, helper T cells (CD4 + T cells), dendritic cells and / or other cells. .. The response by T cells is described, for example, by using a T cell assay indexed by a specific marker by, for example, flow cytometry; T cell proliferation assay, T cell damage assay, tetramer assay, and / or ELISPOT assay. It can be measured by measuring the abundance of CD4 + and / or CD8 + cells.

In the present invention, "positive (+)" means a negative control cell that does not express the antigen or a negative control that uses an antibody that does not react with the antigen by an analysis method such as flow cytometry detected by using an antigen-antibody reaction. This means that a higher signal is detected compared to the reaction. Further, in the present invention, "negative (-)" means that a signal equivalent to or less than that of a negative control cell that does not express the antigen or a negative control reaction that uses an antibody that does not react with the antigen is detected. Means.

In the present invention, "effective dose" means an amount of immunogen sufficient to prevent and / or ameliorate infection, for example by inducing an immune response. The "effective dose" may also mean an amount of immunogen sufficient to alleviate the infection or at least one symptom of a disease caused by the infection. The "effective dose" may mean an amount of immunogen sufficient to delay or minimize, reduce, or suppress the onset of an infection or disease. The "effective dose" may mean an amount of immunogen sufficient to provide a therapeutic benefit in the treatment or management of an infection or disease. The "effective dose" may mean an amount of immunogen sufficient to provide therapeutic benefit in the treatment or management of an infection or disease, alone or in combination with other therapies. The "effective dose" may be an amount of immunogen sufficient to enhance the autoimmune response of the subject (eg, human) to subsequent exposure to SARS2 or a disease caused by SARS2. The "effective dose" may be a dose that prevents SARS2 and / or reduces the severity of symptoms caused by SARS2. The "effective dose" can also be referred to as, for example, an "effective amount".

In the present invention, "antibody" means a protein comprising one or more polypeptides, which is substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Immunoglobulin genes include 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 V region. , D region, J region and other genes that can encode innumerable immunoglobulin variable regions. The antibodies include, for example, heavy and light chains. The light chain contains κ and λ and constitutes the κ and λ chains, respectively. The heavy chain comprises γ, μ, α, δ, or ε and constitutes the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. The antibody may be a structural unit of a typical immunoglobulin (antibody) composed of tetramers. In this case, the antibody is composed of two pairs of identical polypeptide chains, each pair consisting of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). In addition, the N-terminus of each chain defines a variable region composed of about 100 to 110 or more amino acids mainly involved in antigen recognition. The antibody may be a full-length immunoglobulin or an antigen-binding fragment thereof.

In the present invention, 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 digesting the full-length immunoglobulin with various peptidases. Examples of the antigen-binding fragment include F (ab') 2, Fab', Fab, Fv (variable fragment of antibody), disulfide-bonded Fv, single-chain antibody (scFv), and polymers thereof.

In the present invention, "complementarity determining regions" (CDRs) mean regions that form antigen-binding sites in the variable regions of immunoglobulins (antibodies). The CDR can also be referred to as, for example, a hypervariable region. The CDR is generally a region in which the variable region of the antibody is particularly highly mutagenic in the primary structure, and is usually separated into three positions on the primary structure. In the antibody, each of the heavy chain variable region and the light chain variable region contains three CDRs (from the N-terminal side, HCDR1, HCDR2, and HCDR3, and from the N-terminal side, LCDR1, LCDR2, and LCDR3). These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to which they bind. In the present invention, the CDR of an antibody can be determined according to Kabat's numbering system (Reference 1 below).
Reference 1: Kabat et.al., “Sequences of Proteins of Immunological Interest”, 1987, US Department of Health and Human Services, NIH, USA

In the present invention, the "variable region" may have, for example, a framework region (FR) in addition to the above-mentioned CDR. The FR is usually separated into four points in the variable region of the antibody. In the antibody, each of the heavy chain variable region and the light chain variable region contains four FRs (from the N-terminal side, HFR1, HFR2, HFR3, and HFR4, and from the N-terminal side, NFR1, NFR2, NFR3, and NFR4). ..

In the present invention, the "antibody" may have, for example, a constant region in addition to the heavy chain variable region and the light chain variable region described above. The constant region is, for example, a human constant region or a mouse constant region. In the case of an antibody (immunoglobulin), the constant region of the heavy chain comprises, for example, the regions CH1, CH2, and CH3, and the constant region of the light chain includes, for example, the region CL. When the antibody of the present invention has the constant region, for example, the heavy chain variable region is bound to at least one of CH1, CH2, and CH3, and the light chain variable region is bound to the CL. The heavy chain variable region is, for example, directly linked to CH1.

In the present invention, "human antibody" means an antibody whose heavy chain and light chain are derived from human immunoglobulin.

In the present invention, the "humanized antibody" means an antibody composed of a variable region composed of a CDR of an antibody derived from a non-human animal and an FR derived from a human antibody, and a constant region derived from a human antibody.

In the present invention, the "chimeric antibody" is an antibody in which at least one of a heavy chain and a light chain is composed of a variable region derived from non-human animal immunoglobulin and a constant region derived from human immunoglobulin, or a variable derived from human immunoglobulin. It means an antibody composed of a region and a constant region derived from non-human animal immunoglobulin.

In the present invention, "treatment" refers to suppressing, preventing, suppressing, preventing, or delaying the onset of a target disease, stopping, suppressing, suppressing, or delaying the progression of the developed target disease or its symptoms, and improving or delaying the onset of the target disease. It may be used in any sense of remission.

In the present invention, "isolated" means the identified and isolated state and / or the state recovered from the component in its natural state. The "isolation" can be carried out, for example, by obtaining at least one purification step.

<Mutant protein>
The present invention provides a mutant protein capable of reducing the inducing ability of an antibody that enhances the ability of SARS2 to bind S protein to ACE2. The variant of the Spike protein of SARS-CoV-2 of the present invention is a protein consisting of the following (Sm) amino acid sequence. The mutant protein of the present invention is derived from the amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. It is characterized by having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group, and other configurations and conditions are not particularly limited.

(Sm) The amino acid sequence of the following (Sm1), (Sm2), or (Sm3):
(Sm1) In the amino acid sequence of the Spike protein (Sw protein) of wild SARS-CoV-2, the 30th, 32nd, and 64th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, It has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 65, 66, 186, 187, 211, 213, 214, 216, and 217. Amino acid sequence;
(Sm2) In the amino acid sequence of (Sm1), an amino acid sequence having one or several insertions, additions, substitutions and / or deletions and maintaining the mutated amino acid of (Sm1);
(Sm3) An amino acid sequence having 70% or more identity with respect to the amino acid sequence of (Sm1) and maintaining the mutated amino acid of (Sm1).

As a result of diligent research, the present inventors have found that there is an antibody that enhances the binding of SARS2 to human cells in an Fc receptor-independent manner in persons infected with SARS2. In addition, as a result of further research, the present inventors have found that specific sites of the S protein of SARS2 (30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th in the Ss protein). Amino acid residues at positions 214, 216, and / or 217, in particular amino acid residues at positions 64, 66, 187, 213, and / or 214, or at positions 64, 66, and 213. The presence of an antibody that recognizes the amino acid residue at the position and / or the 214th position, or an antibody that competes with the antibody, enhances the ability of the S protein to bind to the ACE2 protein expressed on human cells. I found it. It was also confirmed that the enhancement of the binding ability to ACE2 was induced in an Fc receptor-independent manner. Specifically, when an antibody that binds to the specific site binds to the S protein, a change in the structure (conformation) of the S protein occurs, whereby the receptor binding domain of the S protein tends to bind to ACE2. Change to structure. As a result, it was presumed that the Fc receptor-independent binding ability of the S protein to ACE2 was enhanced. In ADE, which is generally known, a virus and an antibody form a complex, and after binding to a cell through the binding between the antibody and the Fc receptor, the virus invades and infects the cell. Caused by. Therefore, the Fc receptor-independent ADE is a novel mechanism ADE discovered for the first time by the present inventors. Then, the present inventors use an antibody in which a mutation is introduced into an amino acid at a specific site in the S protein to enhance the binding ability of the S protein to the ACE2 protein when administered to a subject. We have found that the induction of protein can be suppressed, and have established the present invention. Therefore, according to the mutant protein of the present invention, for example, an antibody that enhances the ability of SARS2 to bind to the ACE2 protein in an Fc receptor-independent manner when used as an active ingredient of a vaccine (hereinafter referred to as an antibody). The inducing ability of (also referred to as "ACE2 binding enhancing antibody") can be reduced (also referred to as suppression or reduction). Therefore, according to the mutant protein of the present invention, for example, the development of Fc receptor-independent ADE can be suppressed, so that the risk of ADE in the vaccinated person can be reduced.

The mutant protein of the present invention may be, for example, an isolated mutant protein or a mutant protein mixed with other substances.

The number of mutant amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of (Sm1) is one. It may be a plurality (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12), and for example, the latter can further suppress the inducing ability of the ACE2 binding enhancing antibody. Is. In the mutant protein, the mutant amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the above (Sm1) It is presumed that the ability to induce the ACE2-binding enhancing antibody can be suppressed more efficiently by increasing the number. When the amino acid sequence of the (Sm1) and the amino acid sequence of the Ss protein are aligned, the mutant amino acid contains an amino acid different from the amino acid at the corresponding position of the Ss protein in the amino acid sequence of (Sm1). means. The alignment can be performed with default parameters using, for example, FASTA, BLAST and the like.

When there is one mutant amino acid in (Sm1), the positions of the mutant amino acids are, for example, positions 30, 32, 64, 65, 66, 186, 187, 211, etc. in the Ss protein. Amino acids corresponding to amino acid residues at positions 213, 214, 216, or 217, or 30th, 32nd, 64th, 66th, 186th, 187th, 211th, 213th, 214th, 216th. It is an amino acid corresponding to the amino acid residue at the position or 217, and can further reduce the inducing ability of more ACE2-binding enhancing antibodies. The amino acid corresponding to the amino acid residue at position or 214, the amino acid corresponding to the amino acid residue at position 64, 66, 187, 213, or 214, or the amino acid corresponding to the amino acid residue at position 64, 66, 213, or 214. The amino acid corresponding to the amino acid residue at the position.

When there are a plurality of mutant amino acids in (Sm1), the positions of the mutant amino acids are, for example, positions 30, 32, 64, 65, 66, 186, 187, 211, etc. in the Ss protein. Amino acids corresponding to any two or more amino acid residues in the amino acids at positions 213, 214, 216, and 217, or at positions 30, 32, 64, 66, 186, 187, and 211. Amino acids corresponding to any two or more amino acid residues in the amino acids at positions 2, 213, 214, 216, and 217, preferably at positions 64, 65, 66, 187, and 213. And the amino acid corresponding to any two or more amino acid residues at position 214, the amino acid corresponding to any two or more amino acid residues at positions 64, 66, 187, 213, and 214, or 64 Amino acids corresponding to any two or more amino acid residues at positions 6, 213, and 214, more preferably (a) positions 64, 65, and / or 66, (b). Amino acids corresponding to the amino acid residues at positions 187, 213, and / or 214, (c) 64, 65, 66, 187, 213, and / or 214, (d) 64, Amino acids corresponding to the amino acid residues at positions 66, 187, 213, and / or 214, or (e) amino acids corresponding to the amino acid residues at positions 64, 66, 213, and / or 214. Yes, more preferably, (1) amino acids corresponding to the amino acid residues at positions 64, 65, 66, 187, 213, and 214, (2) amino acids at positions 64, 66, 187, and 213. , And the amino acid corresponding to the amino acid residue at position 214, or (3) the amino acid corresponding to the amino acid residue at positions 64, 66, 213, and 214.

In the above (Sm1), in the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and / or 217th positions in the Ss protein. Alternatively or additionally, the amino acids corresponding to the amino acid residues at positions 68, 94, 97, 129, 185, and / or 215 may be the positions of the mutant amino acids.

The position of the mutant amino acid in (Sm1) is preferably N30, F32, W64, F65, H66 in the Sw protein and in the Ss protein because, for example, the inducing ability of the ACE2 binding enhancing antibody can be further reduced. , F186, K187, N211, V213, R214, L216, and P217, the amino acids corresponding to one or more amino acid residues, or N30, F32, W64, H66, F186, K187, N211, V213, R214, Amino acids corresponding to one or more amino acid residues in the amino acids L216, and P217, more preferably W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, Alternatively, it is an amino acid corresponding to one or more amino acid residues in the amino acids W64, H66, V213, and R214. The position of the mutant amino acid in the (Sm1) can further reduce, for example, the ability to induce an antibody that enhances the binding ability of the S protein of SARS2 to the ACE2 protein. Therefore, the Ss in the Sw protein is more preferable. Amino acids corresponding to the amino acid residues of W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, or W64, H66, V213, and R214 in proteins.

In (Sm1), in place of or in addition to N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and / or P217 in the Ss protein, I68, S94, K97. , K129, N185, and / or the amino acid corresponding to the amino acid residue of D215 may be the position of the mutant amino acid.

In the present invention, the (Sm1) may be, for example, the same as the amino acid sequence of the Ss protein, that is, may consist of the amino acid sequence of the Ss protein.

In the present invention, the "inducible ability of an ACE2-binding enhancing antibody" is defined by, for example, immunizing a non-human animal using an object for measuring the inducible ability (measurement object), and ACE2 in the blood of the non-human animal. It can be evaluated by examining whether or not the binding-enhancing antibody is induced, and as a specific example, it can be carried out in the same manner as in Example 11 described later. Specifically, first, the measurement target and an optionally immunostimulatory agent are mixed and administered to a non-human animal. Blood is collected from the non-human animal and serum is isolated 2 to 4 weeks after the administration. Then, the ACE2 binding enhancing antibody in the serum is measured. The ACE2 binding enhancing antibody may be measured by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the serum, or in the serum. It may be indirectly measured by measuring whether or not an antibody that recognizes a predetermined position in the Ss protein is present in the antibody of. In the former case, the measurement can be carried out in the same manner as in Example 1 described later, for example. In the latter case, the measurement can be performed, for example, in the same manner as in Example 4 described later.

In the above (Sm2), "1 or several" may be a range in which the mutated amino acid of (Sm1) is maintained, and preferably a range in which an antibody against the S protein or the Sw protein can be induced, that is, , S protein or Sw protein, as long as it is an immunogen of an antibody (for example, a neutralizing antibody) that can bind to the protein. The "1 or several" includes, for example, 1 to 254, 1 to 250, 1 to 240, 1 to 230, 1 to 220, 1 to 210, 1 to 200, and 1 to 190. , 1-180, 1-170, 1-160, 1-150, 1-140, 1-130, 1-127, 1-120, 1-110, 1-100 , 1 to 90, 1 to 80, 1 to 76, 1 to 70, 1 to 63, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 25 , 1 to 20, 1 to 10, 1 to 5, 1, 3, 1, 2, or 1.

In the above (Sm2), the amino acid substitution or the like may be, for example, a conservative substitution (hereinafter, the same applies). The conservative substitution means substituting one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. It is preferable that the "amino acid to be substituted" and the "amino acid to be substituted" are, for example, similar in properties and / or functions. Specifically, for example, it is preferable that the indicators of hydrophobicity and hydrophilicity (hydropathy), the chemical properties such as polarity and electric charge, or the physical properties such as secondary structure are similar. Amino acids or amino acid derivatives having similar properties and / or functions are known in the art, for example. Specific examples include non-polar amino acids (hydrophobic amino acids) such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine and methionine, and polar amino acids (neutral amino acids) include glycine, serine and threonine. , Tyrosine, glutamine, asparagine, cysteine, etc., positively charged amino acids (basic amino) acids include arginine, histidine, lysine, etc., and negatively charged amino acids (acidic amino acids) are aspartic acid, Glutamic acid and the like can be mentioned.

In the above (Sm3), "identity" may be a range in which the mutated amino acid of (Sm1) is maintained, and preferably a range in which an antibody against the S protein or the Sw protein can be induced, that is, S. Any range may be used as an immunogen for an antibody capable of binding to a protein or Sw protein (for example, a neutralizing antibody). The "identity" is, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the occurrence rate (%) of an accurate match of amino acids between the sequences. The "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99. % Or more. The identity can be calculated with default parameters using, for example, analysis software such as BLAST or FASTA (the same applies hereinafter).

In the mutant protein, the mutant amino acid may be an artificially introduced mutation or a mutation isolated from a virus strain of SARS2 or the like. In the former case, the "having a mutation in an amino acid" can be said to be, for example, "a mutation has been introduced into an amino acid", and the "mutated amino acid" can be said to be, for example, a "mutated amino acid". ..

In the case of a mutation in which the mutant amino acid is artificially introduced, the mutant protein can be introduced into an S protein such as a Sw protein or a mutant protein by using a method for introducing an amino acid mutation into a protein. As a specific example, the mutation may be introduced at random (random mutation introduction method) or site-specific (site-specific mutation introduction method). When introducing the random mutation, the mutation can be randomly introduced, for example, by carrying out a PCR reaction on the polynucleotide encoding the S protein or an expression vector containing the same in the presence of manganese. .. When introducing the site-specific mutation, the mutation is, for example, in the polynucleotide encoding the S protein, a polynucleotide (eg, a primer) that allows all types of base pair changes at any particular site. ) Can be introduced by a specific mutagenesis method. As a specific example, the specific mutagenesis method has one or more mismatches between the nucleotide sequence of the polynucleotide encoding the S protein or the expression vector containing the same and the codon encoding the amino acid into which the mutation is to be introduced. It can be introduced by carrying out a PCR reaction using a partially complementary polynucleotide containing a base and a polynucleotide encoding the S protein or an expression vector containing the same. The mutation uses, for example, homologous recombination (DNA shuffling), mutagenesis using a template nucleic acid molecule containing uracil, oligonucleotide-specific mutagenesis, phosphorothioate-modified DNA mutagenesis, and gapped duplex DNA. Mutation induction, point mismatch repair, mutation induction using repair-deficient host strain, deletion mutation induction, mutation induction by whole gene synthesis, mutation induction using double-strand break repair, α-ray, β-ray, γ-ray, and Mutation induction by irradiation treatment such as X-ray, mutagenesis by chemical substance treatment with mutagenesis agents such as ethyl methanesulfonate (EMS) and ethynylnitrosolea (ENU), mutagenesis by heavy ion beam treatment, genome editing technology Examples include the mutagenesis or introduction used.

When the mutant amino acid is a mutation isolated from a virus strain or the like, the mutant amino acid can be isolated from the SARS2 virus strain by analyzing the amino acid sequence of the S protein and identifying the amino acid mutation. ..

In the mutant amino acid, the type of mutation may be any range as long as the function in the Sw protein is modified by the amino acid mutation, and the antibody that enhances the binding ability of the mutated S protein to the ACE2 protein by the amino acid mutation. It is preferable that the range is such that the induction of the protein can be suppressed. Therefore, the "mutation" can be, for example, a modification. Types of said mutations include, for example, amino acid deletions, substitutions, insertions and / or additions. The type of mutation introduced into the mutant protein may be one type or a plurality of types.

It is assumed that the type of mutation is, for example, easy to maintain the immunogenicity of a mutant protein having a mutant amino acid, and suppresses the influence on the induction of production of a neutralizing antibody that intersects with the mutant protein and the Sw protein. Therefore, replacement is preferable. When the mutation is a substitution, the substitution is preferably not the above-mentioned conservative substitution, and in particular, a substitution in which the charge of the side chain of the amino acid is changed is preferable. By introducing such a substitution as a mutation, the ability to induce an antibody that enhances the ability of SARS2 to bind to the ACE2 protein can be further reduced. Specific examples of the substitution include substitution with alanine (alanine substitution) and substitution with a sugar chain modification motif (NX [S / T]).

When the mutation is an alanine substitution, the amino acid sequence of (Sm1) is preferably in the Sw protein, in the Ss protein, N30, F32, W64, F65, H66, F186, K187, N211, V213, R214. , L216, and the amino acid corresponding to one or more amino acid residues in the amino acid of P217, or one or more of the amino acids of N30, F32, W64, H66, F186, K187, N211, V213, R214, L216, and P217. An amino acid sequence having an alanine substitution in the amino acid corresponding to the plurality of amino acid residues, more preferably W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, or W64, An amino acid sequence having an alanine substitution in the amino acid corresponding to one or more amino acid residues in the amino acids of H66, V213, and R214, more preferably W64, F65, H66, K187, V213, and R214, W64, An amino acid sequence having an alanine substitution in the amino acid corresponding to the amino acid residue of H66, K187, V213, and R214, or W64, H66, V213, and R214.

When the mutation is a substitution with a sugar chain modification motif, the mutation replaces the amino acid at position 64 in the Ss protein with N (asparagine) and the amino acid at position 66 with S (serine) or T (threonine). It can be introduced by. As a specific example, the amino acid sequence of (Sm1) is, for example, an amino acid sequence having a substitution of W64N and H66S or W64N and H66T in the Ss protein in the Sw protein.

The substitution may be a combination of alanine substitution and substitution with a sugar chain modification motif. In this case, the amino acid sequence of (Sm1) can be the same amino acid sequence except that W64 and H66 are changed to W64N and H66S, or W64N and H66T in the above-mentioned example of alanine substitution.

The mutations in the mutant amino acids are the amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in the Ss protein. Amino acids corresponding to at least one amino acid residue selected from the group consisting of, or positions 30, 32, 64, 66, 186, 187, 211, 213, 214, 216, and. It is preferable to reduce the binding to an antibody (hereinafter, also referred to as “reference antibody”) that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acid at position 217. Thereby, for example, when the mutant protein of the present invention is used as an active ingredient of a vaccine, the induction of an antibody that recognizes the mutant amino acid can be suppressed, so that the S protein can be more effectively transferred to the ACE2 protein. It is possible to suppress the induction of an antibody that enhances the binding ability.

The mutant protein of the present invention may have mutations in other amino acids in addition to the above-mentioned mutant amino acids. Specifically, the mutant protein is located at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, and 216 in the amino acid sequence of the Ss protein. , And mutations may be introduced into amino acids corresponding to amino acid residues other than position 217. As a specific example, the mutant protein may have a mutation in the amino acid corresponding to the amino acid residue at positions 986 and / or 987 in the Ss protein. In this case, in the mutant protein, the amino acids at positions 986 (K) and 987 (V) in the Ss protein are preferably replaced with proline (P). Thereby, the mutant protein can stabilize the structure, for example.

The mutant protein of the present invention may have a mutation at the furin cleavage site as another example of the mutation of the other amino acids. Specifically, in the mutant protein, for example, a mutation may be introduced into the amino acid corresponding to the amino acid residue at positions 682 to 685 in the Ss protein. In this case, the amino acid sequence after the introduction of the mutation is preferably an amino acid sequence that is not decomposed into furin. As a specific example, the mutant protein may have a mutation in the amino acid corresponding to the amino acid residue at positions 682 and / or 685 in the Ss protein. In this case, in the mutant protein, the amino acid at position 682 (R) and / or position 685 (R) in the Ss protein is preferably replaced with alanine (A), serine (S) or the like. Thereby, for example, the mutant protein can suppress the decomposition into the S1 region and the S2 region, so that the structure can be stabilized.

When the mutant protein of the present invention is a soluble protein, the mutant protein can be prepared by deleting the intracellular domain. The intracellular domain of the mutant protein is, for example, an amino acid corresponding to the amino acid residue at position 1255 to 1273 in the Ss protein.

Examples of the method for confirming the reduction in binding to the reference antibody include a method for detecting the binding between the S protein into which the amino acid mutation has been introduced and the reference antibody in vitro. Examples of the detection method include ELISA, flow cytometry, pull-down assay and the like, and specific examples thereof can be carried out in the same manner as in Example 5 described later. In the confirmation method, for example, a significant difference (eg, 10%, 20%, 30%,) showing binding to the reference antibody as compared with, for example, a test system not containing the reference antibody or a test system containing the control antibody. When a signal of 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more) is detected, it can be evaluated that the binding to the reference antibody is reduced. As the mutant protein having the mutant amino acid, a crudely purified or purified mutant protein may be used, or a host cell such as a cell expressing the mutant protein may be used.

The reference antibody is preferably an antibody that enhances the ability of the Ss protein to bind to the ACE2 protein. Examples of the method for measuring the enhancement of the binding ability include a method of detecting the binding between the Ss protein and the ACE protein in vitro in the presence of the reference antibody. Examples of the detection method include ELISA, flow cytometry, pull-down assay and the like, and specific examples thereof can be carried out in the same manner as in Example 1 described later. In the measurement method, for example, a significant difference (for example, 10%, 20%) showing the binding between the Ss protein and the ACE2 protein is compared with the test system containing the reference antibody or the test system containing the control antibody. , 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more difference), enhances the binding ability of the Ss protein to the ACE2 protein. It can be evaluated that it is. As the Ss protein and / or the ACE2 protein, a crudely purified or purified protein may be used, or a host cell such as a cell expressing the Ss protein or the ACE2 protein may be used.

The reference antibody comprises amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in the S protein. Amino acids corresponding to at least one amino acid residue selected from the group, or positions 30, 32, 64, 66, 186, 187, 211, 213, 214, 216, and 217. Recognizes amino acids corresponding to at least one amino acid residue selected from the group consisting of amino acids of. As the description of the amino acid recognized by the reference antibody, the description of the amino acid to be mutated in (Sm1) of the mutant protein and the description of the antibody in the first marker described later can be incorporated.

Examples of the reference antibody include antibodies containing the following heavy chain variable region (H) and light chain variable region (L). The reference antibody is preferably an antibody capable of enhancing the binding ability of the Ss protein to the ACE2 protein.

(H) Heavy chain variable region:
Amino acid sequence of heavy chain complementarity determining regions (HCDR) 1 containing or consisting of the amino acid sequence of SEQ ID NO: 8 (X ₁ X ₂ YX ₃ X ₄ X ₅ ), amino acid sequence of SEQ ID NO: 9 (X ₆ IX ₇ X ₈ X) _{HCDR2 containing or consisting of 9} X ₁₀ GX ₁₁ X ₁₂ X ₁₃ X ₁₄ YX ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ ), and the amino acid sequence of SEQ ID NO: 10 (X ₂₁ X ₂₂ X ₂₃ X ₂₄ X) ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ X ₃₅ X ₃₆ X ₃₇ X ₃₈ X ₃₉ DX ₄₀ ) is a heavy chain variable region containing or consisting of HCDR3s.
X ₁ is S or T;
X ₂ does not exist or is N;
X ₃ is A, D, or W;
X ₄ is M or W;
X ₅ is G, H, N, or S;
X ₆ is A, D, N, T, V, or W;
X ₇ is G, H, N, or S;
X ₈ is Q, T, or Y;
X ₉ is A, D, S, or N;
X ₁₀ does not exist or is T;
X ₁₁ is D, G, I, N, or S;
X ₁₂ is E, N, P, S or T;
X ₁₃ does not exist or is K;
X ₁₄ is T or Y;
X ₁₅ is A, N, P, or V;
X ₁₆ is D, G, P, or Q;
X ₁₇ is G or S;
X ₁₈ is F, L, or V;
X ₁₉ is K, R, or T;
X ₂₀ is G or S;
X ₂₁ does not exist or is A;
X ₂₂ is D or R;
X ₂₃ is F, P, Q, T, or W;
X ₂₄ does not exist or is D, E, G, or Y;
X ₂₅ is a G, S, Q, W, or Y,;
X ₂₆ is D, F, G, or L;
X ₂₇ is I, L, N, R, or Y;
X ₂₈ is absent or G, L, P, T, W, or Y;
X ₂₉ does not exist or is A, G, Q, S, or T;
X ₃₀ is absent or A, D, G, H, or M;
X ₃₁ does not exist or is S, V, or Y;
X ₃₂ does not exist or is E or S;
X ₃₃ does not exist or is L, S, W, or Y;
X ₃₄ is absent or F, G, T, or Y;
X ₃₅ is absent or A, G, L, or Y;
X ₃₆ does not exist or is A, T, or Y;
X ₃₇ is F or Y;
X- ₃₈ is absent or C or G;
X ₃₉ does not exist or is M;
X ₄₀ is F, I, or V.

The (H) heavy chain variable region is, for example, a heavy chain variable region including HCDR1 containing the amino acid sequence of SEQ ID NO: 8, HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and HCDR3 containing the amino acid sequence of SEQ ID NO: 10. be. The (H) heavy chain variable region is, for example, a heavy chain variable region including HCDR1 consisting of the amino acid sequence of SEQ ID NO: 8, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 10. be.

(L) Light chain variable region:
Light chain complementarity determining regions (LCDRs) 1, comprising or consisting of the amino acid sequence of SEQ ID NO: 11 (X ₄₁ X ₄₂ SX ₄₃ X ₄₄ X ₄₅ X ₄₆ X ₄₇ X ₄₈ X ₄₉ X ₅₀ X _{51), SEQ ID NO:} LCDR2s containing or consisting of 12 amino acid sequences (X ₅₂ X ₅₃ SX ₅₄ X ₅₅ X ₅₆ X ₅₇ ), and the amino acid sequence of SEQ ID NO: 13 (X ₅₈ QX ₅₉ X ₆₀ X ₆₁ X ₆₂ X ₆₃ X ₆₄ X). _A light chain variable region comprising an LCDR3 containing or consisting of 65 X _{66 T):}
X ₄₁ is K, Q, or R;
X ₄₂ does not exist or is A or S;
X ₄₃ does not exist or is Q or V;
X ₄₄ is G or S;
X ₄₅ is absent or I, L, or V;
X ₄₆ does not exist or is L, R, or S;
X ₄₇ does not exist or is H;
X ₄₈ does not exist or is N or S;
X ₄₉ does not exist or is D, N, Y, or W;
X ₅₀ is absent or G or L;
X ₅₁ does not exist or is A, G, or K;
X ₅₂ is A, D, E, G, or K;
X ₅₃ is A or V;
X ₅₄ is N, S, or T;
X ₅₅ is L or R;
X ₅₆ is A, E, F, or Q;
X ₅₇ is S or T;
X ₅₈ is L, M, or Q;
X ₅₉ is A, H, L, S, or Y;
X ₆₀ is G, I, or N;
X ₆₁ is N, S, or Q;
X- ₆₂ does not exist or is F, W, or Y;
X ₆₃ does not exist or is P, S, or W;
X ₆₄ is P or R;
X ₆₅ does not exist or is L;
X ₆₆ does not exist or is I.

The (L) light chain variable region is, for example, a light chain variable region including LCDR1 containing the amino acid sequence of SEQ ID NO: 11, LCDR2 containing the amino acid sequence of SEQ ID NO: 12, and LCDR3 containing the amino acid sequence of SEQ ID NO: 13. be. The (L) light chain variable region includes, for example, LCDR1 consisting of the amino acid sequence of SEQ ID NO: 11, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 13. be.

Examples of the antibody containing the heavy chain variable region (H) and the light chain variable region (L) include the following antibodies (1) to (6). The antibodies (1) to (6) below are antibodies that enhance the binding ability of the Ss protein to the ACE2 protein, that is, ACE2 binding enhancing antibodies, as shown in Examples described later. The following antibodies (1) to (6) are human-derived antibodies.

(1) An antibody containing the following (HA) heavy chain variable region and the following (LA) light chain variable region:
(H1) Includes HCDR1, HCDR2, and HCDR3
HCDR1 is a polypeptide comprising or consisting of the following (H1-A) amino acid sequences.
HCDR2 is a polypeptide comprising or consisting of the following (H2-A) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-A) amino acid sequence:
(H1-A) Amino acid sequence of the following (H1-A1), (H1-A2) or (H1-A3) (H1-A1) Amino acid sequence of SEQ ID NO: 14 (SYAMH) (H1-A2) Amino acid of SEQ ID NO: 14 Amino acid sequence having 70% or more identity with respect to the sequence (H1-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 14. (H2-A) Amino acid sequence of the following (H2-A1), (H2-A2) or (H2-A3) (H2-A1) Amino acid sequence of SEQ ID NO: 15 (VISYDGSNKYYADSVKG) (H2-A2) Amino acid of SEQ ID NO: 15. Amino acid sequence having 70% or more identity with respect to the sequence (H2-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 15. (H3-A) Amino acid sequence of the following (H3-A1), (H3-A2) or (H3-A3) (H3-A1) Amino acid sequence of SEQ ID NO: 16 (DQEWFRELFLFDY) (H3-A2) Amino acid of SEQ ID NO: 16. Amino acid sequence having 70% or more identity with respect to the sequence (H3-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 16. ;
(L1) Includes LCDR1, LCDR2, and LCDR3
LCDR1 is a polypeptide containing or consisting of the following (L1-A) amino acid sequences.
LCDR2 is a polypeptide comprising or consisting of the following (L2-A) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-A) amino acid sequence:
(L1-A) Amino acid sequence of the following (L1-A1), (L1-A2) or (L1-A3) (L1-A1) Amino acid sequence of SEQ ID NO: 17 (RASQGISSWLA) (L1-A2) Amino acid of SEQ ID NO: 17 Amino acid sequence having 70% or more identity with respect to the sequence (L1-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 17 (L2-A) Amino acid of the following (L2-A1), (L2-A2) or (L2-A3) amino acid sequence (L2-A1) Amino acid sequence of SEQ ID NO: 18 (DASSLQS) (L2-A2) Amino acid of SEQ ID NO: 18 Amino acid sequence having 70% or more identity with respect to the sequence (L2-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 18. (L3-A) Amino acid sequence of the following (L3-A1), (L3-A2) or (L3-A3) (L3-A1) Amino acid sequence of SEQ ID NO: 19 (QQANSFPPT) (L3-A2) Amino acid of SEQ ID NO: 19 Amino acid sequence having 70% or more identity with respect to the sequence (L3-A3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 19. ..

The antibody (1) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66, 213, and 214, more preferably in the amino acid sequence of the Ss protein, 64, 66, 186, 187, 211, 213, 214. It is an antibody that recognizes amino acids at positions 216 and 217.

(2) An antibody containing the following (HB) heavy chain variable region and the following (LB) light chain variable region:
(HB) Includes HCDR1, HCDR2, and HCDR3
HCDR1 is a polypeptide comprising or consisting of the following (H1-B) amino acid sequences.
HCDR2 is a polypeptide comprising or consisting of the following (H2-B) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-B) amino acid sequence:
(H1-B) Amino acid of the following (H1-B1), (H1-B2) or (H1-B3) amino acid sequence (H1-B1) Amino acid sequence of SEQ ID NO: 20 (SYWMN) (H1-B2) Amino acid of SEQ ID NO: 20 Amino acid sequence having 70% or more identity with respect to the sequence (H1-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 20. (H2-B) Amino acid sequence of the following (H2-B1), (H2-B2) or (H2-B3) (H2-B1) Amino acid sequence of SEQ ID NO: 21 (NINQDGGEKYYVDSVRG) (H2-B2) Amino acid of SEQ ID NO: 21 Amino acid sequence having 70% or more identity with respect to the sequence (H2-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 21. (H3-B) Amino acid of the following (H3-B1), (H3-B2) or (H3-B3) amino acid sequence (H3-B1) Amino acid sequence of SEQ ID NO: 22 (DPYDLYGDYGGTFDY) (H3-B2) Amino acid of SEQ ID NO: 22 Amino acid sequence having 70% or more identity with respect to the sequence (H3-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 22. ;
(LB) Including LCDR1, LCDR2, and LCDR3,
LCDR1 is a polypeptide containing or consisting of the following (L1-B) amino acid sequences.
LCDR2 is a polypeptide containing or consisting of the following (L2-B) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-B) amino acid sequence:
(L1-B) Amino acid sequence of the following (L1-B1), (L1-B2) or (L1-B3) (L1-B1) Amino acid sequence of SEQ ID NO: 23 (RASQSVSSNLA) (L1-B2) Amino acid of SEQ ID NO: 23 Amino acid sequence having 70% or more identity with respect to the sequence (L1-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 23. (L2-B) Amino acid sequence of the following (L2-B1), (L2-B2) or (L2-B3) (L2-B1) Amino acid sequence of SEQ ID NO: 24 (GASTRAT) (L2-B2) Amino acid of SEQ ID NO: 24 Amino acid sequence having 70% or more identity with respect to the sequence (L2-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 24. (L3-B) Amino acid sequence of the following (L3-B1), (L3-B2) or (L3-B3) (L3-B1) Amino acid sequence of SEQ ID NO: 25 (QQYNNWWRT) (L3-B2) Amino acid of SEQ ID NO: 25 Amino acid sequence having 70% or more identity with respect to the sequence (L3-B3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 25. ..

The antibody (2) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66, 213, and 214, more preferably in the amino acid sequence of the Ss protein, at positions 30, 64, 66, 213, 214, and 216. It is an antibody that recognizes amino acids.

(3) An antibody containing the following (HC) heavy chain variable region and the following (LC) light chain variable region:
(HC) includes HCDR1, HCDR2, and HCDR3,
HCDR1 is a polypeptide comprising or consisting of the following (H1-C) amino acid sequence.
HCDR2 is a polypeptide comprising or consisting of the following (H2-C) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-C) amino acid sequence:
(H1-C) Amino acid sequence of the following (H1-C1), (H1-C2) or (H1-C3) (H1-C1) Amino acid sequence of SEQ ID NO: 26 (SYWMS) (H1-C2) Amino acid of SEQ ID NO: 26 Amino acid sequence having 70% or more identity with respect to the sequence (H1-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 26. (H2-C) Amino acid sequence of the following (H2-C1), (H2-C2) or (H2-C3) (H2-C1) Amino acid sequence of SEQ ID NO: 27 (NINQDGSEKYYVDSVKG) (H2-C2) Amino acid of SEQ ID NO: 27 Amino acid sequence having 70% or more identity with respect to the sequence (H2-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 27. (H3-C) Amino acid sequence of the following (H3-C1), (H3-C2) or (H3-C3) (H3-C1) Amino acid sequence of SEQ ID NO: 28 (DWDYDILTGSWFGAFDI) (H3-C2) Amino acid of SEQ ID NO: 28 Amino acid sequence having 70% or more identity with respect to the sequence (H3-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 28. ;
(LC) Includes LCDR1, LCDR2, and LCDR3
LCDR1 is a polypeptide containing or consisting of the following (L1-C) amino acid sequences.
LCDR2 is a polypeptide containing or consisting of the following (L2-C) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-C) amino acid sequence:
(L1-C) Amino acid sequence of the following (L1-C1), (L1-C2) or (L1-C3) (L1-C1) Amino acid sequence of SEQ ID NO: 29 (RASQGIRNDLG) (L1-C2) Amino acid of SEQ ID NO: 29 Amino acid sequence having 70% or more identity with respect to the sequence (L1-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 29. (L2-C) Amino acid of the following (L2-C1), (L2-C2) or (L2-C3) amino acid sequence (L2-C1) Amino acid sequence of SEQ ID NO: 30 (AASSLQS) (L2-C2) Amino acid of SEQ ID NO: 30 Amino acid sequence having 70% or more identity with respect to the sequence (L2-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 30. (L3-C) Amino acid sequence of the following (L3-C1), (L3-C2) or (L3-C3) (L3-C1) Amino acid sequence of SEQ ID NO: 31 (LQHNSYPLT) (L3-C2) Amino acid of SEQ ID NO: 31 Amino acid sequence having 70% or more identity with respect to the sequence (L3-C3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 31. ..

The antibody of (3) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66-position, 213-position, and 214-position amino acids, and more preferably, in the amino acid sequence of the Ss protein, the 30-position, 32-position, 64-position, 66-position, 68-position, 94-position, and 97 positions. It is an antibody that recognizes amino acids at positions 129, 185, 187, 213, 214, 215, 216, 217, and 258.

(4) An antibody containing the following (HD) heavy chain variable region and the following (LD) light chain variable region:
(HD) Including HCDR1, HCDR2, and HCDR3,
HCDR1 is a polypeptide comprising or consisting of the following (H1-D) amino acid sequence.
HCDR2 is a polypeptide comprising or consisting of the following (H2-D) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-D) amino acid sequence:
(H1-D) Amino acid sequence of the following (H1-D1), (H1-D2) or (H1-D3) (H1-D1) Amino acid sequence of SEQ ID NO: 32 (TYAMN) (H1-D2) Amino acid of SEQ ID NO: 32 Amino acid sequence having 70% or more identity with respect to the sequence (H1-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 32. (H2-D) Amino acid of the following (H2-D1), (H2-D2) or (H2-D3) amino acid sequence (H2-D1) Amino acid sequence of SEQ ID NO: 33 (WINTNTGNPTYAQGFTG) (H2-D2) Amino acid of SEQ ID NO: 33 Amino acid sequence having 70% or more identity with respect to the sequence (H2-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 33. (H3-D) Amino acid sequence of the following (H3-D1), (H3-D2) or (H3-D3) amino acid sequence (H3-D1) Amino acid sequence of SEQ ID NO: 34 (DQDSGYPTYYYYYMDV) (H3-D2) Amino acid of SEQ ID NO: 34 Amino acid sequence having 70% or more identity with respect to the sequence (H3-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 34. ;
(LD) includes LCDR1, LCDR2, and LCDR3.
LCDR1 is a polypeptide containing or consisting of the following (L1-D) amino acid sequence.
LCDR2 is a polypeptide containing or consisting of the following (L2-D) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-D) amino acid sequence:
(L1-D) Amino acid sequence of the following (L1-D1), (L1-D2) or (L1-D3) (L1-D1) Amino acid sequence of SEQ ID NO: 35 (KSSQSLLHSDGK) (L1-D2) Amino acid of SEQ ID NO: 35 Amino acid sequence having 70% or more identity with respect to the sequence (L1-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 35. (L2-D) Amino acid sequence of the following (L2-D1), (L2-D2) or (L2-D3) (L2-D1) Amino acid sequence of SEQ ID NO: 36 (EVSNRFS) (L2-D2) Amino acid of SEQ ID NO: 36 Amino acid sequence having 70% or more identity with respect to the sequence (L2-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 36. (L3-D) Amino acid sequence of the following (L3-D1), (L3-D2) or (L3-D3) (L3-D1) Amino acid sequence of SEQ ID NO: 37 (MQSIQPPLT) (L3-D2) Amino acid of SEQ ID NO: 37 Amino acid sequence having 70% or more identity with respect to the sequence (L3-D3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 37. ..

The antibody (4) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably in the amino acid sequence of the Ss protein, 30th, 32nd, 64th, 66th, 68th, 94th, and 129th. It is an antibody that recognizes amino acids at positions 185, 213, 214, and 258.

(5) An antibody containing the following (HE) heavy chain variable region and the following (LE) light chain variable region:
(HE) Includes HCDR1, HCDR2, and HCDR3
HCDR1 is a polypeptide comprising or consisting of the following (H1-E) amino acid sequences.
HCDR2 is a polypeptide comprising or consisting of the following (H2-E) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-E) amino acid sequence:
(H1-E) Amino acid sequence of the following (H1-E1), (H1-E2) or (H1-E3) (H1-E1) Amino acid sequence of SEQ ID NO: 38 (SYAMH) (H1-E2) Amino acid of SEQ ID NO: 38 Amino acid sequence having 70% or more identity with respect to the sequence (H1-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 38. (H2-E) Amino acid of the following (H2-E1), (H2-E2) or (H2-E3) amino acid sequence (H2-E1) Amino acid sequence of SEQ ID NO: 39 (DISYDGSEKYYADSVKG) (H2-E2) Amino acid of SEQ ID NO: 39 Amino acid sequence having 70% or more identity with respect to the sequence (H2-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 39. (H3-E) Amino acid sequence of the following (H3-E1), (H3-E2) or (H3-E3) (H3-E1) Amino acid sequence of SEQ ID NO: 40 (DFGGDNTAMVEYFFDF) (H3-E2) Amino acid of SEQ ID NO: 40 Amino acid sequence having 70% or more identity with respect to the sequence (H3-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 40. ;
(LE) Including LCDR1, LCDR2, and LCDR3
LCDR1 is a polypeptide containing or consisting of the following (L1-E) amino acid sequences.
LCDR2 is a polypeptide containing or consisting of the following (L2-E) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-E) amino acid sequence:
(L1-E) Amino acid sequence of the following (L1-E1), (L1-E2) or (L1-E3) (L1-E1) Amino acid sequence of SEQ ID NO: 41 (RASQSISSWLA) (L1-E2) Amino acid of SEQ ID NO: 41 Amino acid sequence having 70% or more identity with respect to the sequence (L1-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 41. (L2-E) Amino acid sequence of the following (L2-E1), (L2-E2) or (L2-E3) (L2-E1) Amino acid sequence of SEQ ID NO: 42 (KASSLES) (L2-E2) Amino acid of SEQ ID NO: 42 Amino acid sequence having 70% or more identity with respect to the sequence (L2-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 42. (L3-E) Amino acid sequence of the following (L3-E1), (L3-E2) or (L3-E3) (L3-E1) Amino acid sequence of SEQ ID NO: 43 (QQYNSYSPT) (L3-E2) Amino acid of SEQ ID NO: 43 Amino acid sequence having 70% or more identity with respect to the sequence (L3-E3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 43. ..

The antibody (5) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably, 30th, 32nd, 64th, 66th, 187th, 213th, and 214th in the amino acid sequence of the Ss protein. It is an antibody that recognizes amino acids at positions 216 and 217.

(6) An antibody containing the following (HF) heavy chain variable region and the following (LF) light chain variable region:
(HF) Includes HCDR1, HCDR2, and HCDR3
HCDR1 is a polypeptide comprising or consisting of the following (H1-F) amino acid sequence.
HCDR2 is a polypeptide comprising or consisting of the following (H2-F) amino acid sequence.
Heavy chain variable region in which HCDR3 is a polypeptide comprising or consisting of the following (H3-F) amino acid sequence:
(H1-F) Amino acid of the following (H1-F1), (H1-F2) or (H1-F3) amino acid sequence (H1-F1) Amino acid sequence of SEQ ID NO: 44 (SYDMH) (H1-F2) Amino acid of SEQ ID NO: 44 Amino acid sequence having 70% or more identity with respect to the sequence (H1-F3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 44. (H2-F) Amino acid sequence of the following (H2-F1), (H2-F2) or (H2-F3) (H2-F1) Amino acid sequence of SEQ ID NO: 45 (AIGTAGDTYYPGSVKG) (H2-F2) Amino acid of SEQ ID NO: 45 Amino acid sequence having 70% or more identity with respect to the sequence (H2-F3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 45. (H3-G) Amino acid sequence of the following (H3-G1), (H3-G2) or (H3-G3) (H3-G1) Amino acid sequence of SEQ ID NO: 46 (ADPYQLLGQHYYYGMDV) (H3-G2) Amino acid of SEQ ID NO: 46 Amino acid sequence having 70% or more identity with respect to the sequence (H3-G3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 46. ;
(LF) Including LCDR1, LCDR2, and LCDR3,
LCDR1 is a polypeptide containing or consisting of the following (L1-F) amino acid sequences.
LCDR2 is a polypeptide containing or consisting of the following (L2-F) amino acid sequence.
A light chain variable region in which LCDR3 is a polypeptide comprising or consisting of the following (L3-F) amino acid sequence:
(L1-F) Amino acid sequence of the following (L1-F1), (L1-F2) or (L1-F3) (L1-F1) Amino acid sequence of SEQ ID NO: 47 (QSVSSSYLA) (L1-F2) Amino acid of SEQ ID NO: 47 Amino acid sequence having 70% or more identity with respect to the sequence (L1-F3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 47. (L2-F) Amino acid sequence of the following (L2-F1), (L2-F2) or (L2-F3) (L2-F1) Amino acid sequence of SEQ ID NO: 48 (GASSRAT) (L2-F2) Amino acid of SEQ ID NO: 48 Amino acid sequence having 70% or more identity with respect to the sequence (L2-F3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 48. (L3-F) Amino acid sequence of the following (L3-F1), (L3-F2) or (L3-F3) (L3-F1) Amino acid sequence of SEQ ID NO: 49 (QQYGSSPLIT) (L3-F2) Amino acid of SEQ ID NO: 49 Amino acid sequence having 70% or more identity with respect to the sequence (L3-F3) Amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 49. ..

The antibody (6) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably in the amino acid sequence of the Ss protein, 30th, 32nd, 64th, 66th, 129th, 212th, 213. It is an antibody that recognizes amino acids at positions 214, 215, 216, 217, and 237.

In each CDR of the antibodies (1) to (6), "identity" is, for example, the degree of identity when the sequences to be compared are properly aligned, and the exact match of amino acids between the sequences. Means the appearance rate (%) of. The "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, respectively. Or 99% or more.

In each of the CDRs of the antibodies (1) to (6), "one or several" relating to deletion, substitution, insertion and / or addition (hereinafter referred to as "substitution, etc.") is, for example, 1 to 1, respectively. 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, 1 piece.

The substitution may be, for example, the above-mentioned conservative substitution.

In each of the antibodies, the region other than the CDR is not particularly limited as long as it is a sequence capable of maintaining the structure as an antibody and exerting a function. For example, a mouse-derived sequence, a human-derived sequence, or another animal-derived sequence. , Their chimeric sequences, and / or artificial sequences can be used. Examples of the region other than the CDR include a framework region (FR), a stationary region, and the like. When each of the antibodies contains a constant region, the amino acid sequences of the constant regions of the heavy chain and the light chain can be, for example, those described in References 2 to 4 below.
Reference 2: S. Huck et.al., “Sequence of a human immunoglobulin gamma 3 heavy chain constant region gene: comparison with the other human C gamma genes.”, 1986, Nucleic Acids Res, vol. 14, No. 4 , pages 1779-1789
Reference 3: PA Hieter et.al., “Evolution of human immunoglobulin kappa J region genes.”, 1982, J. Biol. Chem., Vol. 257, pages 1516-1522
Reference 4: Philip A. Hieter et.al., “Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments.”, 1980, Cell, vol. 22, pages 197-207

In each of the antibodies, the following amino acid sequence can be exemplified as the amino acid sequence of the FR region.

FR region (HFR1-4) in heavy chain variable region of (HA)
HFR1: Amino acid sequence of SEQ ID NO: 50
QVQLVESGGGVVQPGRSLRLSCAASGFTFS (SEQ ID NO: 50)
HFR2: Amino acid sequence of SEQ ID NO: 51
WVRQAPGKGLEWVA (SEQ ID NO: 51)
HFR3: Amino acid sequence of SEQ ID NO: 52
RFTISRDNSKNTLYLQMNSLRVEDTAVYYCAR (SEQ ID NO: 52)
HFR4: Amino acid sequence of SEQ ID NO: 53
WGQGTLVTVSS (SEQ ID NO: 53)

FR region (LFR1-4) in the light chain variable region of (LA)
LFR1: Amino acid sequence of SEQ ID NO: 54
DIQMTQSPSSVSASVGDRVTITC (SEQ ID NO: 54)
LFR2: Amino acid sequence of SEQ ID NO: 55
WYQQKPGKAPKLLIY (SEQ ID NO: 55)
LFR3: Amino acid sequence of SEQ ID NO: 56
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 56)
LFR4: Amino acid sequence of SEQ ID NO: 57
FGPGTKVDIK (SEQ ID NO: 57)

FR region (HFR1-4) in heavy chain variable region of (HB)
HFR1: Amino acid sequence of SEQ ID NO: 58
EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 58)
HFR2: Amino acid sequence of SEQ ID NO: 59
WVRQAPGKGLEWVA (SEQ ID NO: 59)
HFR3: Amino acid sequence of SEQ ID NO: 60
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 60)
HFR4: Amino acid sequence of SEQ ID NO: 61
WGQGTLVTVSS (SEQ ID NO: 61)

FR region (LFR1-4) in the light chain variable region of (LB)
LFR1: Amino acid sequence of SEQ ID NO: 62
EIVLTQSPATLSVSPGERATLSC (SEQ ID NO: 62)
LFR2: Amino acid sequence of SEQ ID NO: 63
WYQQKPGQAPRLLIY (SEQ ID NO: 63)
LFR3: Amino acid sequence of SEQ ID NO: 64
GIPARFSGSGSGTDFTLTISSLQSEDFALYYC (SEQ ID NO: 64)
LFR4: Amino acid sequence of SEQ ID NO: 65
FGQGTKVEIN (SEQ ID NO: 65)

FR region (HFR1-4) in heavy chain variable region of (HC)
HFR1: Amino acid sequence of SEQ ID NO: 66
EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 66)
HFR2: Amino acid sequence of SEQ ID NO: 67
WVRQAPGKGLEWVA (SEQ ID NO: 67)
HFR3: Amino acid sequence of SEQ ID NO: 68
RFTISRDNAKNSLYLQVNSLRAEDTAVYYCAR (SEQ ID NO: 68)
HFR4: Amino acid sequence of SEQ ID NO: 69
WGQGTTVTVSS (SEQ ID NO: 69)

FR region (LFR1-4) in the light chain variable region of (LC)
LFR1: Amino acid sequence of SEQ ID NO: 70
DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 70)
LFR2: Amino acid sequence of SEQ ID NO: 71
WYQQKPGKAPKRLIY (SEQ ID NO: 71)
LFR3: Amino acid sequence of SEQ ID NO: 72
GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 72)
LFR4: Amino acid sequence of SEQ ID NO: 73
FGGGTKVEIK (SEQ ID NO: 73)

FR region (HFR1-4) in heavy chain variable region of (HD)
HFR1: Amino acid sequence of SEQ ID NO: 74
QVQLVQSGSELKKPGASVKVSCKASGYTFT (SEQ ID NO: 74)
HFR2: Amino acid sequence of SEQ ID NO: 75
WVRQAPGQGLEWMG (SEQ ID NO: 75)
HFR3: Amino acid sequence of SEQ ID NO: 76
RFVFSLDTSVNTAFLHIGSLKAEDTAVYYCAR (SEQ ID NO: 76)
HFR4: Amino acid sequence of SEQ ID NO: 77
WGKGTTVTVSS (SEQ ID NO: 77)

FR region (LFR1-4) in the light chain variable region of (LD)
LFR1: Amino acid sequence of SEQ ID NO: 78
DIVMTQTPLSLSVTPGQPASISC (SEQ ID NO: 78)
LFR2: Amino acid sequence of SEQ ID NO: 79
TYLYWYLQKPGQSPQLLIY (SEQ ID NO: 79)
LFR3: Amino acid sequence of SEQ ID NO: 80
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 80)
LFR4: Amino acid sequence of SEQ ID NO: 81
FGGGTKVEIK (SEQ ID NO: 81)

FR region (HFR1-4) in heavy chain variable region of (HE)
HFR1: Amino acid sequence of SEQ ID NO: 82
QVQLVESGGGVVQPGRSLRLSCAASGFTFS (SEQ ID NO: 82)
HFR2: Amino acid sequence of SEQ ID NO: 83
WVRQAPGKGLEWVA (SEQ ID NO: 83)
HFR3: Amino acid sequence of SEQ ID NO: 84
RFTIYRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 84)
HFR4: Amino acid sequence of SEQ ID NO: 85
WGQGTLVTVSS (SEQ ID NO: 85)

FR region (LFR1-4) in the light chain variable region of (LE)
LFR1: Amino acid sequence of SEQ ID NO: 86
DIQMTQSPSTLSASVGDRVTITC (SEQ ID NO: 86)
LFR2: Amino acid sequence of SEQ ID NO: 87
WYQQKPGKAPKLLIY (SEQ ID NO: 87)
LFR3: Amino acid sequence of SEQ ID NO: 88
GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 88)
LFR4: Amino acid sequence of SEQ ID NO: 89
FGQGTKVEIK (SEQ ID NO: 89)

FR region (HFR1-4) in heavy chain variable region of (HF)
HFR1: Amino acid sequence of SEQ ID NO: 90
EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 90)
HFR2: Amino acid sequence of SEQ ID NO: 91
WVRQATGKGLEWVS (SEQ ID NO: 91)
HFR3: Amino acid sequence of SEQ ID NO: 92
RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR (SEQ ID NO: 92)
HFR4: Amino acid sequence of SEQ ID NO: 93
WGQGTTVTVSS (SEQ ID NO: 93)

FR region (LFR1-4) in the light chain variable region of (LF)
LFR1: Amino acid sequence of SEQ ID NO: 94
EIVLTQSPGTLSLSPGERATLSCRAS (SEQ ID NO: 94)
LFR2: Amino acid sequence of SEQ ID NO: 95
WYQQKPGQAPRLLIY (SEQ ID NO: 95)
LFR3: Amino acid sequence of SEQ ID NO: 96
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 96)
LFR4: Amino acid sequence of SEQ ID NO: 97
FGQGTRLEIK (SEQ ID NO: 97)

Each FR may have, for example, an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added. The "1 or several" can be referred to the description of "1 or several" in the CDR. Further, each FR may have an amino acid sequence having 70% or more identity with respect to the amino acid sequence of each FR. The description of "identity" in the CDR can be incorporated as the "identity".

The antibody containing the heavy chain variable region of (H) and the light chain variable region of (L) is, for example, the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D). Examples thereof include 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The following antibodies (A) to (F) are antibodies belonging to the antibodies (1) to (6) above, respectively.

The antibody (A) contains, as a heavy chain variable region, a polypeptide containing or consisting of the following amino acid sequence (H-A). In addition, the antibody (A) contains a polypeptide containing or consisting of the following amino acid sequence (LA) as a light chain variable region.

(HA) following (H-A1), (H -A2) or (H-A3) the amino acid sequence (H-A1) the amino acid sequence sequence of SEQ ID NO: 98 Number of 98: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRVEDTAVYYCAR DQEWFRELFLFDY WGQGTLVTVSS
(H-A2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 98 (H-A3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 98. , Inserted and / or added amino acid sequence

(LA) Amino acid sequence of the following (L-A1), (L-A2) or (L-A3) (L-A1) Amino acid sequence of SEQ ID NO: 99 SEQ ID NO: 99: DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKLLIY DASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFP
(L-A2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 99 (L-A3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 99. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-A1) is, for example, a sequence containing the amino acid sequences of (H1-A1) of HCDR1, (H2-A1) of HCDR2, and (H3-A1) of HCDR3. The amino acid sequence of (H-A2) includes, for example, the amino acid sequences of (H1-A1) of HCDR1, (H2-A1) of HCDR2, and HCDR3 (H3-A1), and the amino acid sequence of SEQ ID NO: 98. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-A3) includes, for example, the amino acid sequences of (H1-A1) of HCDR1, (H2-A1) of HCDR2, and HCDR3 (H3-A1), and the amino acid sequence of SEQ ID NO: 98. In an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added.

The amino acid sequence of (L-A1) is, for example, a sequence containing the amino acid sequences of (L1-A1) of LCDR1, (L2-A1) of LCDR2, and (L3-A1) of LCDR3. The amino acid sequence of (L-A2) includes, for example, the amino acid sequences of (L1-A1) of LCDR1, (L2-A1) of LCDR2, and (L3-A1) of LCDR3, and the amino acid of SEQ ID NO: 99. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-A3) includes, for example, the amino acid sequences of (L1-A1) of LCDR1, (L2-A1) of LCDR2, and (L3-A1) of LCDR3, and the amino acid of SEQ ID NO: 99. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (A) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66, 213, and 214, more preferably in the amino acid sequence of the Ss protein, 64, 66, 186, 187, 211, 213, 214. It is an antibody that recognizes amino acids at positions 216 and 217.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-A1), the light chain variable region is the above (L-A1), and the antibody of this combination is hereinafter referred to as "clone". Also called "2210". As will be described later, the clone 2210 recognizes the amino acids at positions 64, 66, 186, 187, 211, 213, 214, 216, and 217 in the amino acid sequence of the Ss protein. It is an antibody.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibody (B) contains a polypeptide containing or consisting of the following (H-B) amino acid sequence as a heavy chain variable region. In addition, the antibody (B) contains a polypeptide containing or consisting of the following (L-B) amino acid sequence as a light chain variable region.

(HB) below (H-B1), (H -B2) or (H-B3) of the amino acid sequence (H-B1) SEQ ID NO: 100 amino acid sequence SEQ ID NO 100: EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMN WVRQAPGKGLEWVA NINQDGGEKYYVDSVRG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DPYDLYGDYGGTFDY WGQGTLVTVSS
(H-B2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 100 (H-B3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 100. , Inserted and / or added amino acid sequence

(LB) Amino acid sequence of the following (L-B1), (L-B2) or (L-B3) (L-B1) Amino acid sequence of SEQ ID NO: 101 SEQ ID NO: 101: EIVLTQSPATLSVSPGERATLSC RASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTDFTLTISSLQSEDFALYYC QQYNNWW
(L-B2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 101 (L-B3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 101. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-B1) is, for example, a sequence containing the amino acid sequences of (H1-B1) of HCDR1, (H2-B1) of HCDR2, and (H3-B1) of HCDR3. The amino acid sequence of (H-B2) includes, for example, the amino acid sequences of (H1-B1) of HCDR1, (H2-B1) of HCDR2, and HCDR3 (H3-B1), and the amino acid sequence of SEQ ID NO: 100. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-B3) includes, for example, the amino acid sequences of (H1-B1) of HCDR1, (H2-B1) of HCDR2, and HCDR3 (H3-B1), and the amino acid sequence of SEQ ID NO: 100. In, one or several amino acids may be deleted, substituted, inserted and / or added amino acid sequences.

The amino acid sequence of (L-B1) is, for example, a sequence containing the amino acid sequences of (L1-B1) of LCDR1, (L2-B1) of LCDR2, and (L3-B1) of LCDR3. The amino acid sequence of (L-B2) includes, for example, the amino acid sequences of (L1-B1) of LCDR1, (L2-B1) of LCDR2, and (L3-B1) of LCDR3, and the amino acid of SEQ ID NO: 101. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-B3) includes, for example, the amino acid sequences of (L1-B1) of LCDR1, (L2-B1) of LCDR2, and (L3-B1) of LCDR3, and the amino acid of SEQ ID NO: 101. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (B) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66, 213, and 214, more preferably in the amino acid sequence of the Ss protein, at positions 30, 64, 66, 213, 214, and 216. It is an antibody that recognizes amino acids.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-B1), the light chain variable region is the above (L-B1), and the antibody of this combination is hereinafter referred to as "clone". Also called "2490". The antibody of the clone 2490 is an antibody that recognizes the amino acids at positions 30, 64, 66, 213, 214, and 216 in the amino acid sequence of the Ss protein, as described later.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibody (C) contains a polypeptide containing or consisting of the following (H-C) amino acid sequence as a heavy chain variable region. In addition, the antibody (C) contains a polypeptide containing or consisting of the following (L-C) amino acid sequence as a light chain variable region.

(HC) following (H-C1), (H -C2) or (H-C3) amino acid sequence (H-C1) SEQ ID NO: 102 amino acid sequence SEQ ID NO 102: EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINQDGSEKYYVDSVKG RFTISRDNAKNSLYLQVNSLRAEDTAVYYCAR DWDYDILTGSWFGAFDI WGQGTTVTVSS
(H-C2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 102 (H-C3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 102. , Inserted and / or added amino acid sequence

(LC) Amino acid sequence of the following (L-C1), (L-C2) or (L-C3) (L-C1) Amino acid sequence of SEQ ID NO: 103 SEQ ID NO: 103: DIQMTQSPSSLSASVGDRVTITC RASQGIRNDLG WYQQKPGKAPKRLIY AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQ
(L-C2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 103 (L-C3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 103. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-C1) is, for example, a sequence containing the amino acid sequences of (H1-C1) of HCDR1, (H2-C1) of HCDR2, and (H3-C1) of HCDR3. The amino acid sequence of (H-C2) includes, for example, the amino acid sequences of (H1-C1) of HCDR1, (H2-C1) of HCDR2, and HCDR3 (H3-C1), and the amino acid sequence of SEQ ID NO: 102. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-C3) includes, for example, the amino acid sequences of (H1-C1) of HCDR1, (H2-C1) of HCDR2, and HCDR3 (H3-C1), and the amino acid sequence of SEQ ID NO: 102. In, one or several amino acids may be deleted, substituted, inserted and / or added amino acid sequences.

The amino acid sequence of (L-C1) is, for example, a sequence containing the amino acid sequences of (L1-C1) of LCDR1, (L2-C1) of LCDR2, and (L3-C1) of LCDR3. The amino acid sequence of (L-C2) includes, for example, the amino acid sequences of (L1-C1) of LCDR1, (L2-C1) of LCDR2, and (L3-C1) of LCDR3, and the amino acid of SEQ ID NO: 107. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-C3) includes, for example, the amino acid sequences of (L1-C1) of LCDR1, (L2-C1) of LCDR2, and (L3-C1) of LCDR3, and the amino acid of SEQ ID NO: 107. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (C) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66-position, 213-position, and 214-position amino acids, and more preferably, in the amino acid sequence of the Ss protein, the 30-position, 32-position, 64-position, 66-position, 68-position, 94-position, and 97 positions. It is an antibody that recognizes amino acids at positions 129, 185, 187, 213, 214, 215, 216, 217, and 258.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-C1), the light chain variable region is the above (L-C1), and the antibody of this combination is hereinafter referred to as "clone". Also called "8D2". As will be described later, the clone 8D2 is located at positions 30, 32, 64, 66, 68, 94, 97, 129, 185, 187, and 213 in the amino acid sequence of the Ss protein. , 214, 215, 216, 217, and 258.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibody (D) contains a polypeptide containing or consisting of the following (HD) amino acid sequence as a heavy chain variable region. In addition, the antibody (D) contains a polypeptide containing or consisting of the following (L-D) amino acid sequence as a light chain variable region.

(HD) below (H-D1), (H -D2) or (H-D3) of the amino acid sequence (H-D1) SEQ ID NO: 104 amino acid sequence SEQ ID NO 104: QVQLVQSGSELKKPGASVKVSCKASGYTFT TYAMN WVRQAPGQGLEWMG WINTNTGNPTYAQGFTG RFVFSLDTSVNTAFLHIGSLKAEDTAVYYCAR DQDSGYPTYYYYYMDV WGKGTTVTVSS
(H-D2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 104 (H-D3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 104. , Inserted and / or added amino acid sequence

(LD) below (L-D1), (L -D2) or (L-D3) of the amino acid sequence (L-D1) of SEQ ID NO: 105 amino acid sequence SEQ ID NO 105: DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSDGK TYLYWYLQKPGQSPQLLIY EVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIQPPLT FGGGTKVEIK
(L-D2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 105 (L-D3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 105. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-D1) is, for example, a sequence containing the amino acid sequences of (H1-D1) of HCDR1, (H2-D1) of HCDR2, and (H3-D1) of HCDR3. The amino acid sequence of (H-D2) includes, for example, the amino acid sequences of (H1-D1) of HCDR1, (H2-D1) of HCDR2, and HCDR3 (H3-D1), and the amino acid sequence of SEQ ID NO: 104. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-D3) includes, for example, the amino acid sequences of (H1-D1) of HCDR1, (H2-D1) of HCDR2, and HCDR3 (H3-D1), and the amino acid sequence of SEQ ID NO: 104. In an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added.

The amino acid sequence of (L-D1) is, for example, a sequence containing the amino acid sequences of (L1-D1) of LCDR1, (L2-D1) of LCDR2, and (L3-D1) of LCDR3. The amino acid sequence of (L-D2) includes, for example, the amino acid sequences of (L1-D1) of LCDR1, (L2-D1) of LCDR2, and (L3-D1) of LCDR3, and the amino acid of SEQ ID NO: 105. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-D3) includes, for example, the amino acid sequences of (L1-D1) of LCDR1, (L2-D1) of LCDR2, and (L3-D1) of LCDR3, and the amino acid of SEQ ID NO: 105. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (D) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably in the amino acid sequence of the Ss protein, 30th, 32nd, 64th, 66th, 68th, 94th, and 129th. It is an antibody that recognizes amino acids at positions 185, 213, 214, and 258.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-D1), the light chain variable region is the above (L-D1), and the antibody of this combination is hereinafter referred to as "clone". Also called "2582". As described later, the clone 2582 has the amino acid sequences of the Ss protein at positions 30, 32, 64, 66, 68, 94, 129, 185, 213, 214, and 258. It is an antibody that recognizes the amino acid at the position.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibody (E) contains a polypeptide containing or consisting of the following (H-E) amino acid sequence as a heavy chain variable region. In addition, the antibody (E) contains a polypeptide containing or consisting of the following (L-E) amino acid sequence as a light chain variable region.

(HE) below (H-E1), (H -E2) or (H-E3) the amino acid sequence of (H-E1) of SEQ ID NO: 106 amino acid sequence SEQ ID NO 106: QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA DISYDGSEKYYADSVKG RFTIYRDNSKNTLYLQMNSLRAEDTAVYYCAK DFGGDNTAMVEYFFDF WGQGTLVTVSS
(H-E2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 106 (H-E3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 106. , Inserted and / or added amino acid sequence

(LE) following (L-E1), (L -E2) or (L-E3) of the amino acid sequence (L-E1) of SEQ ID NO: 107 amino acid sequence SEQ ID NO 107: DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY KASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYSPT FGQGTKVEIK
(L-E2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 107 (L-E3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 107. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-E1) is, for example, a sequence containing the amino acid sequences of (H1-E1) of HCDR1, (H2-E1) of HCDR2, and (H3-E1) of HCDR3. The amino acid sequence of (H-E2) includes, for example, the amino acid sequences of (H1-E1) of HCDR1, (H2-E1) of HCDR2, and HCDR3 (H3-E1), and the amino acid sequence of SEQ ID NO: 106. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-E3) includes, for example, the amino acid sequences of (H1-E1) of HCDR1, (H2-E1) of HCDR2, and HCDR3 (H3-E1), and the amino acid sequence of SEQ ID NO: 106. In, one or several amino acids may be deleted, substituted, inserted and / or added amino acid sequences.

The amino acid sequence of (L-E1) is, for example, a sequence containing the amino acid sequences of (L1-E1) of LCDR1, (L2-E1) of LCDR2, and (L3-E1) of LCDR3. The amino acid sequence of (L-E2) includes, for example, the amino acid sequences of (L1-E1) of LCDR1, (L2-E1) of LCDR2, and (L3-E1) of LCDR3, and the amino acid of SEQ ID NO: 107. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-E3) includes, for example, the amino acid sequences of (L1-E1) of LCDR1, (L2-E1) of LCDR2, and (L3-E1) of LCDR3, and the amino acid of SEQ ID NO: 107. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (E) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably, 30th, 32nd, 64th, 66th, 187th, 213th, and 214th in the amino acid sequence of the Ss protein. It is an antibody that recognizes amino acids at positions 216 and 217.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-E1), the light chain variable region is the above (L-E1), and the antibody of this combination is hereinafter referred to as "clone". Also called "2369". As will be described later, the clone 2369 recognizes the amino acids at positions 30, 32, 64, 66, 187, 213, 214, 216, and 217 in the amino acid sequence of the Ss protein. It is an antibody.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibody (F) contains a polypeptide containing or consisting of the following (H-F) amino acid sequence as a heavy chain variable region. In addition, the antibody (F) contains a polypeptide containing or consisting of the following (L-F) amino acid sequence as a light chain variable region.

(HF) below (H-F1), (H -F2) or (H-F3) the amino acid sequence (H-F1) of SEQ ID NO: 108 amino acid sequence SEQ ID NO 108: EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYDMH WVRQATGKGLEWVS AIGTAGDTYYPGSVKG RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR ADPYQLLGQHYYYGMDV WGQGTTVTVSS
(H-F2) Amino acid sequence having 70% or more identity with respect to the amino acid sequence of SEQ ID NO: 108 (H-F3) One or several amino acids are deleted or substituted in the amino acid sequence of SEQ ID NO: 108. , Inserted and / or added amino acid sequence

(LF) below (L-F1), (L -F2) or (L-F3) the amino acid sequence (L-F1) of SEQ ID NO: 109 amino acid sequence SEQ ID NO 109: EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLIT FGQGTRLEIK
Amino acid sequence having 70% or more identity with respect to the amino acid sequence of (L-F2) SEQ ID NO: 109 One or several amino acids are deleted or substituted in the amino acid sequence of (L-F3) SEQ ID NO: 109. , Inserted and / or added amino acid sequence

The amino acid sequence of (H-F1) is, for example, a sequence containing the amino acid sequences of (H1-F1) of HCDR1, (H2-F1) of HCDR2, and (H3-F1) of HCDR3. The amino acid sequence of (H-F2) includes, for example, the amino acid sequences of (H1-F1) of HCDR1, (H2-F1) of HCDR2, and HCDR3 (H3-F1), and the amino acid sequence of SEQ ID NO: 108. However, it may be an amino acid sequence having 70% or more identity. The amino acid sequence of (H-F3) includes, for example, the amino acid sequences of (H1-F1) of HCDR1, (H2-F1) of HCDR2, and HCDR3 (H3-F1), and the amino acid sequence of SEQ ID NO: 108. In, one or several amino acids may be deleted, substituted, inserted and / or added amino acid sequences.

The amino acid sequence of (L-F1) is, for example, a sequence containing the amino acid sequences of (L1-F1) of LCDR1, (L2-F1) of LCDR2, and (L3-F1) of LCDR3. The amino acid sequence of (L-F2) includes, for example, the amino acid sequences of (L1-F1) of LCDR1, (L2-F1) of LCDR2, and (L3-F1) of LCDR3, and the amino acid of SEQ ID NO: 109. It may be an amino acid sequence having 70% or more identity with respect to the sequence. The amino acid sequence of (L-F3) includes, for example, the amino acid sequences of (L1-F1) of LCDR1, (L2-F1) of LCDR2, and (L3-F1) of LCDR3, and the amino acid of SEQ ID NO: 109. The sequence may be an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.

The antibody (F) is, for example, an antibody that recognizes the Ss protein, and is preferably at positions 64, 66, 187, 213, and 214, or 64 in the amino acid sequence of the Ss protein. , 66th, 213, and 214th amino acids, more preferably in the amino acid sequence of the Ss protein, 30th, 32nd, 64th, 66th, 129th, 212th, 213. It is an antibody that recognizes amino acids at positions 214, 215, 216, 217, and 237.

In the antibody of the present invention, for example, the heavy chain variable region is the above (H-F1), the light chain variable region is the above (L-F1), and the antibody of this combination is hereinafter referred to as "clone". Also called "2660". As will be described later, the clone 2660 has positions 30, 32, 64, 66, 129, 212, 213, 214, 215, 216, and 217 in the amino acid sequence of the Ss protein. , And an antibody that recognizes the amino acid at position 237.

In the heavy chain variable region polypeptide and the light chain variable region polypeptide, the "identity" is, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, respectively. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

In the polypeptide of the heavy chain variable region and the polypeptide of the light chain variable region, "one or several" related to substitutions and the like are, for example, 1 to 30, 1 to 25, and 1 to 20, respectively. 1 to 17, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1-3 pieces, 1 or 2 pieces, 1 piece.

The antibodies (1) to (6) and (A) to (F) are, for example, amino acid sequences derived from antibodies isolated from humans.

The antibodies (1) to (6) and (A) to (F) are, for example, after cloning the gene encoding each antibody into an expression vector, and then host cells in the same manner as the expression vector in the production method of the present invention described later. By culturing the obtained transformant, the desired antibody can be obtained from the culture supernatant.

The reference antibody may be prepared, for example, by immunizing a mammal using the Sw protein as an immunogen and isolating the monoclonal antibody. In this case, in the preparation of the reference antibody, antibody-producing cells are isolated from the mammal to which the immunogen has been administered, and a hybridoma that produces a monoclonal antibody against the Sw protein is prepared. Then, the reference antibody can be prepared by identifying an antibody that recognizes an amino acid at the predetermined position in the Sw protein with respect to the antibody obtained from the hybridoma. The preparation of the monoclonal antibody can be carried out using, for example, a well-known method, and Reference 5 below can be referred to. Further, for the preparation of the hybridoma, for example, reference 6 below can be referred to. The antibody that recognizes the amino acid at the predetermined position can be detected, for example, by using the first to fourth detection methods of the present invention described later.
Reference 5: Ed Harlow et.al., “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory (1988)
Reference 6: G. KOHLER, et.al., “Continuous cultures of fused cells secreting antibody of predefined specificity”, Nature, 1975, volume 256, pages 495-497

The monoclonal antibody of the reference antibody may be obtained by, for example, a phage display method. In this case, in the phage display method, first, the phage antibody library is screened using the Sw protein and the mutant protein in the same manner as in the first to fourth detection methods of the present invention described later, and the Sw protein is screened. Phages that have binding to the amino acid at a predetermined position are selected. Next, in the phage display method, the antibody-corresponding sequence contained in the selected phage is isolated or sequenced, and the nucleic acid molecule encoding the antibody or antigen-binding domain is based on the isolated sequence or the determined sequence information. An expression vector containing the above is constructed. Then, the obtained expression vector is introduced into a host cell in the same manner as the expression vector in the production method of the present invention described later, and the obtained transformant is cultured to obtain the desired antibody from the culture supernatant. can.

In the present invention, the reference antibody may be a human antibody, a chimeric antibody, or a humanized antibody. Examples of the chimeric antibody include antibodies in which the variable region is a variable region derived from human immunoglobulin and the constant region is a constant region derived from a non-human animal (mouse, rat, hamster, chicken, etc.). In this case, the chimeric antibody can be produced, for example, by cutting out a variable region from the genes encoding the antibodies (1) to (6) and binding to a constant region derived from the non-human animal. The constant region derived from the non-human animal immunoglobulin may be any isotype such as IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA1, IgA2 and the like; IgD; IgE; and the like, but preferably. It is a constant region of mouse IgG.

The chimeric antibody is, for example, a chimeric antibody in which the variable region is a variable region derived from immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is a constant region derived from human immunoglobulin. You may. In this case, the chimeric antibody can be prepared, for example, as follows. Specifically, the Sw protein can be produced by immunizing a non-human animal such as a mouse, cutting out a variable region that binds to an antigen from the gene of the mouse monoclonal antibody, and binding to a human-derived constant region. The constant region derived from human immunoglobulin may be any isotype such as IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA1, IgA2 and the like; IgD; IgE; etc., but the constant region of human IgG is preferable. It is an area.

For the humanized antibody, for example, with reference to Japanese Patent Application Laid-Open No. 04-506458, Japanese Patent Application Laid-Open No. 62-296890, etc., CDRs of heavy chain variable region and light chain variable region were isolated, and human-derived immunoglobulins were isolated. It can be produced by a genetic engineering method of transplanting into a gene.

The human antibody, chimeric antibody, and humanized antibody are cloned into an expression vector, and then introduced into a host cell in the same manner as the expression vector in the production method of the present invention described later, and the obtained transformant is cultured. The desired antibody can be obtained from the culture supernatant.

According to the mutant protein of the present invention, when used as an active ingredient of a vaccine, it is possible to reduce the inducing ability of an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein. Since ACE2 is important for SARS2 to establish infection to a subject via S protein, it is considered that the ACE2 binding enhancing antibody contributes to ADE as a so-called infection enhancing antibody. Therefore, it is presumed that the mutant protein of the present invention can reduce the possibility of ADE occurring in vaccine recipients when used as an active ingredient of a vaccine. Therefore, the mutant protein of the present invention can be suitably used as an active ingredient of the pharmaceutical composition described later.

<Mutant polypeptide>
The present invention provides a mutant polypeptide capable of reducing the inducing ability of an antibody that enhances the ability of SARS2 to bind S protein to ACE2. The mutant polypeptide of the invention comprises a partial polypeptide having a partial sequence of a variant of the Spike protein of the invention, said partial polypeptide comprising at least one mutant amino acid in said variant. The mutant polypeptide of the present invention is characterized by containing a partial polypeptide having a partial sequence of the mutant protein of the present invention and containing the mutant amino acid, and other configurations and conditions are not particularly limited. The mutant polypeptide of the present invention can reduce the inducing ability of an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein when used as an active ingredient of a vaccine. The mutant polypeptide of the present invention can be referred to the description of the mutant protein of the present invention.

The mutant polypeptide contains a partial sequence of the mutant protein, that is, a partial amino acid sequence of the mutant protein, and the mutant amino acid in the mutant protein, that is, the 30th, 32nd, and 64th positions in the Ss protein. , 65, 66, 186, 187, 211, 213, 214, 216, and 217, including at least one or more amino acid residues into which the mutation has been introduced. good. The mutant protein replaces the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and / or 217th positions in the Ss protein. Or in addition, if the mutant polypeptide has mutations at positions 68, 94, 97, 129, 185, and / or 215, the mutant polypeptide is, for example, at least one of these into which the mutation has been introduced. It may include one or more amino acid residues.

Since the mutant polypeptide can, for example, induce a neutralizing antibody against the S protein and reduce the inducing ability of an antibody that enhances the binding ability of SARS2 to the ACE2 protein of the S protein, the S protein can be reduced. It is preferable to include the domain of the S protein as a partial sequence. Examples of the domain include an NTD region, an RBD region, and an S2 region in the Ss protein. Since the mutant polypeptide can effectively induce the neutralizing antibody and reduce the inducing ability of the antibody to enhance the binding ability of SARS2 S protein to ACE2 protein, for example, the NTD region and the NTD region and It is preferable to include the RBD region.

According to the mutant polypeptide of the present invention, when used as an active ingredient of a vaccine, the ability to induce an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein can be reduced. Therefore, the mutant polypeptide of the present invention can be suitably used as a component of the pharmaceutical composition described later.

<Nucleic acid>
The present invention provides a nucleic acid encoding the mutant protein of the present invention and / or the mutant polypeptide of the present invention. The nucleic acid of the present invention includes a nucleic acid molecule encoding a variant of the Spike protein of the present invention and / or a nucleic acid molecule encoding the mutant polypeptide of the present invention. The nucleic acid of the present invention is characterized by comprising a nucleic acid molecule encoding a variant of the Spike protein of the present invention and / or a nucleic acid molecule encoding the mutant polypeptide of the present invention, other configurations and conditions. There are no particular restrictions. Since the nucleic acid of the present invention contains a nucleic acid molecule encoding the mutant protein and / or the mutant polypeptide, an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein when used as an active ingredient of a vaccine. Inducibility can be reduced. For the nucleic acid of the present invention, the description of the mutant protein and mutant polypeptide of the present invention can be incorporated.

The nucleic acid of the present invention may contain a region other than the S protein in the genomic RNA of SARS2, or may contain a whole genomic RNA. For the whole genomic RNA, for example, the base sequence registered in Accession No .: NC_045512.2 in Genbank can be referred to.

In the present invention, the nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both. Further, the nucleic acid may be composed of a natural nucleic acid residue, a non-natural nucleic acid residue, or both. As a specific example, the nucleic acid includes, for example, DNA, RNA, and / or DNA / RNA composed of natural and / or non-natural nucleic acid residues. Examples of the unnatural nucleic acid residue include a modified nucleotide residue or a modified ribonucleotide residue in which a base, a sugar residue, or a sugar phosphate skeleton is modified in a nucleotide residue. When the sugar residue is modified, the non-natural nucleic acid residue is, for example, cEt (constrained ethyl bicyclic nucleic acid, manufactured by Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), ENA (registered). Trademarks, 2'-O, 4'-C-Ethylenebridged Nucleic Acid), etc. The nucleic acid may have, for example, a 5'cap at the 5'end.

In the present invention, the nucleic acid may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.

In the nucleic acid of the present invention, the nucleic acid molecule encoding the mutant protein of the present invention and / or the nucleic acid molecule encoding the mutant polypeptide corresponds, for example, based on the amino acid sequence of the mutant protein and the amino acid sequence of the mutant polypeptide. It can be designed by replacing it with a codon. The base sequence in the nucleic acid molecule may be codon-optimized.

The nucleic acid of the present invention can be synthesized, for example, by a genetic engineering method or an organic synthetic method. In this case, the nucleic acid of the present invention can also be referred to as synthetic DNA, synthetic RNA, or synthetic DNA / RNA.

Since the nucleic acid of the present invention contains a nucleic acid molecule encoding the mutant protein and / or the mutant polypeptide, an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein when used as an active ingredient of a vaccine. Inducibility can be reduced. Therefore, the nucleic acid of the present invention can be suitably used as a component of the pharmaceutical composition described later.

<Expression vector>
The present invention provides an expression vector encoding the mutant protein of the present invention and / or the mutant polypeptide of the present invention. The expression vector of the present invention contains the nucleic acid of the present invention. For the expression vector of the present invention, the description of the mutant protein, mutant polypeptide, and nucleic acid of the present invention can be incorporated. The expression vector of the present invention is useful for the synthesis (production) of the mutant protein and the mutant polypeptide of the present invention by a genetic engineering technique.

The expression vector can also be referred to as, for example, recombinant DNA.

The expression vector of the present invention is also an expression vector containing the mutant protein and / or the mutant polypeptide of the present invention. Therefore, the mutant protein of the present invention and / or the polynucleotide encoding the mutant polypeptide of the present invention is inserted into, for example, an expression vector. Hereinafter, an example in which a polynucleotide encoding the mutant protein (mutant protein polynucleotide) is inserted into the expression vector will be described, but the same applies to the description of the mutant polypeptide, and the mutant poly is the same. The description of the peptide can be incorporated by replacing "mutant protein" with "mutant polypeptide".

The expression vector of the mutant protein may contain, for example, the polynucleotide of the mutant protein so that the polynucleotide of the mutant protein can be expressed, and its composition is not particularly limited.

The expression vector of the mutant protein can be prepared, for example, by inserting the polynucleotide of the mutant protein into a vector serving as a skeleton (hereinafter, also referred to as “basic vector”). The type of the vector is not particularly limited, and can be appropriately determined, for example, according to the type of host cell (host).

Examples of the host include non-human hosts such as microorganisms, animal cells, plant cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, avian cells, mammalian cells, and the like. Examples of the prokaryote include bacteria of the genus Escherichia such as Escherichia coli, the genus Pseudomonas such as Pseudomonas putida, and bacteria such as mycobacteria. Examples of the eukaryote include yeasts such as Saccharomyces cerevisiae. The animal cells include, for example, COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells, and African green monkey cells, CV1 cells, HeLa cells, MDCK. Examples include cells, Vero cells, Hep-2 cells, etc., and the insect cells include, for example, Spodoptera frugiperda (Sf) cells (for example, Sf9, Sf21), Trichoplusia ni cells ( For example, High Five cells) and S2 cells of the genus Drosophila can be mentioned.

Examples of the vector (basic vector) include non-viral vectors and viral vectors. The virus vector may be, for example, baculovirus; poxvirus such as vaccinia virus, avipoxvirus, canary pox virus, sardine virus, raigmapoxvirus, pig pox virus; adenovirus such as canine adenovirus; adeno-associated virus; herpes. Examples include virus vectors such as virus; retrovirus; When transforming a host by a heat shock method as the introduction method, examples of the vector include a binary vector and the like. The vectors include, for example, pQE70, pQE60, pQE-9, pBluescript, Phasescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, pETDuet-1, pQE-80L, pUCP26Km. And so on. When transforming bacteria such as Escherichia coli, examples of the vector include pETDuet-1 vector (Novagen), for example, pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8 and the like. When transforming the yeast, examples of the vector include pFastBac1, pWINEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, pSVL, pYepSec1, pMFa, pYES2 and the like. When transforming the insect cells, examples of the vector include pAc, pVL and the like. When transforming the mammalian cells, examples of the vector include pCDM8, pMT2PC and the like.

The expression vector of the mutant protein preferably has, for example, a regulatory sequence that regulates the expression of the polynucleotide of the mutant protein and the expression of the mutant protein of the present invention encoded by the polynucleotide of the mutant protein. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, an origin of replication sequence (ori), and the like. In the expression vector of the mutant protein, the arrangement of the regulatory sequence is not particularly limited. In the expression vector of the mutant protein, the regulatory sequence may be arranged so as to be able to functionally regulate, for example, the expression of the polynucleotide of the mutant protein and the expression of the mutant protein encoded by the mutant protein, and is known. Can be placed according to the method. As the regulatory sequence, for example, a sequence provided in advance by the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, or a regulatory sequence provided by the basic vector may be used. It may be replaced with the regulatory sequence of.

The expression vector of the mutant protein may further have, for example, a coding sequence of a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, a cell surface receptor marker and the like.

Insertion of DNA, insertion of the regulatory sequence, and / or insertion of the coding sequence of the selectable marker into the vector may be carried out by, for example, a method using a restriction enzyme and a ligase, or a commercially available kit or the like. May be used.

<Transformant>
The present invention provides a transformant containing the nucleic acid of the present invention. The transformant of the present invention contains the nucleic acid of the present invention. The transformants of the present invention can incorporate the description of the mutant proteins, mutant polypeptides, and nucleic acids of the present invention. The transformant of the present invention is useful for the synthesis (production) of the mutant protein and / or mutant polypeptide.

In the transformant of the present invention, the nucleic acid of the present invention exists as an exogenous molecule. Therefore, the transformant of the present invention can be produced, for example, by introducing the nucleic acid of the present invention into the host.

The method for introducing the nucleic acid is not particularly limited, and a known method can be used. The above may be introduced, for example, by the expression vector. The introduction method can be appropriately set, for example, according to the type of the host. The introduction method includes, for example, an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a microglass tube, or the like. The direct injection method, the hydrodynamic method, the cationic liposome method, the method using an introduction aid, the method via Agrobacterium, and the like can be mentioned. Examples of the liposome include lipofectamine and cationic liposomes, and examples of the introduction aid include atelocollagen, nanoparticles and polymers. When the host is a microorganism, for example, E.I. colli or Ps. The method via putida or the like is preferable. The polynucleotide of the mutant protein and / or mutant polypeptide of the present invention may be introduced into the host by, for example, an expression vector of the mutant protein and / or mutant polypeptide of the present invention.

<Manufacturing method>
The present invention provides a method for producing the mutant protein and / or mutant polypeptide of the present invention. The method for producing the mutant protein and / or the mutant polypeptide of the present invention is not particularly limited, and examples thereof include production by a genetic engineering method. In this case, the production supplement of the present invention comprises an expression step of expressing the mutant protein and / or the polynucleotide encoding the mutant polypeptide. The production method of the present invention can be referred to the description of the mutant protein, mutant polypeptide, nucleic acid, expression vector, and transformant of the present invention.

Hereinafter, in the production method of the present invention, an example of expressing the mutant protein will be described, but the same applies to the description of the mutant polypeptide, and the description of the mutant polypeptide is described in that the “mutant protein” is “mutated”. It can be used by replacing it with "polypeptide".

Expression of the polynucleotide of the mutant protein may be carried out using, for example, the expression vector of the present invention. The method for expressing the polynucleotide of the mutant protein is not particularly limited, and a known method can be adopted. For example, a host may be used, or a cell-free protein synthesis system may be used.

In the former case, for example, it is preferable to use the host into which the polynucleotide of the mutant protein or an expression vector containing the same has been introduced, and to express the polynucleotide of the mutant protein in the host by culturing the host. As described above, for example, by introducing a polynucleotide of the mutant protein or an expression vector containing the same into a host, a transformant synthesizing the mutant protein of the present invention can be produced, and by culturing the transformant. , Can synthesize mutant proteins.

The method of culturing the host is not particularly limited and can be appropriately set according to the type of the host. The medium used for culturing is not particularly limited and can be appropriately determined according to the type of the host.

In the latter case, it is preferable to express the polynucleotide of the mutant protein in a cell-free protein synthesis system. In this case, an expression vector may be used for the expression of the polynucleotide of the mutant protein. The cell-free protein synthesis system can be carried out by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which a polynucleotide of the mutant protein has been introduced. Commercially available reagent kits can be used.

The second production method of the present invention may include, for example, a recovery step of recovering the mutant protein. In the recovery step, for example, a known method for purifying a protein or the like can be used. The purification method is not particularly limited, and examples thereof include salting out; density gradient centrifugation using cesium chloride, sucrose, iodixanol and the like; various column chromatography such as an ion exchange column and a gel filtration column. The solvent used in the various purification steps is not particularly limited, and for example, water, a buffer solution, or the like can be used. The pH of the solvent is, for example, 6-8.

The mutant protein obtained by the production method of the present invention may be used as it is as a crude protein (unpurified protein), may be used as a partially purified partially purified protein, or may be used as a single protein. It may be used as a purified protein purified in.

Further, in the production method of the present invention, the obtained mutant protein may be pulverized by, for example, freeze-drying, vacuum-drying, or spray-drying. In this case, in the production method of the present invention, for example, the mutant protein is previously prepared with an acetate buffer, a phosphate buffer, a triethanolamine buffer, a Tris hydrochloride buffer, a GOOD buffer (for example, PIPES, MES, MOPS, etc.) and the like. It may be dissolved in a buffer solution.

<Mutant SARS-CoV-2>
The present invention provides a mutant virus capable of reducing the inducing ability of an antibody that enhances the ability of SARS2 to bind S protein to ACE2. The SARS-CoV-2 mutant virus of the present invention contains a variant of the Spike protein of the present invention. The mutant virus of the present invention is characterized by containing the mutant protein, and other configurations and conditions are not particularly limited. Since the mutant virus of the present invention contains a mutant protein as the S protein, when used as an active ingredient of a vaccine, the ability to induce an antibody that enhances the binding ability of SARS2 to the ACE2 protein can be reduced. ..

In the present invention, the mutant virus may be an attenuated virus or an inactivated virus. The attenuation can be carried out, for example, by repeating subculture of the mutant virus, for example, in vitro. The inactivation can be carried out, for example, by irradiating the previrus with ultraviolet rays, radiation or the like.

In the mutant virus, the nucleotide sequence encoding the S protein in the wild-type SARS2 virus is changed (replaced) with the nucleotide sequence encoding the mutant protein, and the obtained polynucleotide encoding the SARS2 virus is obtained in the host cell. It can be prepared by expressing it. For the method of introducing the mutation and the method of preparation using a host cell, for example, reference 7 below can be referred to. Specifically, a cDNA containing the entire length of the genome is synthesized from the genomic RNA of SARS2 and cloned into an expression vector. Next, in the vector, a mutation is introduced into the base sequence encoding the S protein in the wild-type SARS2 virus, and an expression vector containing the base sequence encoding the mutant protein is prepared. Then, it can be prepared by transcribing the full-length RNA of the genome from the expression vector and introducing the obtained RNA into a host cell. As the host cell, for example, a host cell described later can be used, and a Vero cell such as a Vero E6 cell is preferable.
Reference 7: Yixuan J. Hou et.al., “SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo”, Science, 2020, eabe8499

Since the mutant virus of the present invention contains the mutant protein, when used as an active ingredient of a vaccine, it can reduce the inducing ability of an antibody that enhances the binding ability of SARS2 S protein to ACE2 protein. Therefore, the mutant virus of the present invention can be suitably used as a component of the pharmaceutical composition described later.

<VLP>
The present invention provides virus-like particles capable of reducing the inducing ability of an antibody that enhances the ability of SARS2 to bind S protein to ACE2. The virus-like particles of the present invention include variants of the Spike protein of the present invention and / or mutant polypeptides of the present invention. The VLP of the present invention is characterized by containing the mutant protein and / or the mutant polypeptide, and other configurations and conditions are not particularly limited. Since the VLP of the present invention contains the mutant protein and / or the mutant polypeptide as the S protein or a partial polypeptide thereof, the ability of SARS2 to bind to the ACE2 protein when used as an active ingredient of a vaccine. It is possible to reduce the inducing ability of an antibody that enhances the protein.

In the present invention, the VLP can be produced, for example, using an expression system or a host cell capable of forming a VLP. The VLP can be produced, for example, by expressing the S protein in a host cell together with a component of a virus particle used for preparing the VLP. As a method for producing the VLP, for example, Reference 8 below can be referred to.
Reference 8: L. Huhti et.al., “A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles”, Arch. Virol., 2010, vol. 155, No. 11, pages 1855 -1858

Since the VLP of the present invention contains the mutant protein and / or the mutant polypeptide, when used as an active ingredient of a vaccine, it reduces the inducing ability of an antibody that enhances the binding ability of SARS2 to S protein to ACE2 protein. sell. Therefore, the VLP of the present invention can be suitably used as a component of the pharmaceutical composition described later.

<Pharmaceutical composition>
The present invention provides a pharmaceutical composition that can be used as a vaccine. The pharmaceutical composition of the present invention comprises a variant of the Spike protein of the present invention, a mutant polypeptide, a mutant virus, a virus-like particle, a nucleic acid, and / or an expression vector, and a pharmaceutically acceptable carrier. It is a composition. The pharmaceutical composition of the present invention contains, as an active ingredient, a mutant of Peplomer protein, a mutant polypeptide, a mutant virus, a virus-like particle, a nucleic acid, and / or an expression vector (hereinafter, also referred to as “active ingredient”). The feature is that other configurations and conditions are not particularly limited. Since the pharmaceutical composition of the present invention contains, as an active ingredient, a polypeptide containing the mutant protein or a partial sequence thereof, or a nucleic acid encoding these, when used as an active ingredient of a vaccine, ACE2 of the S protein of SARS2 It can reduce the ability to induce antibodies that enhance their ability to bind to proteins.

The pharmaceutical composition of the present invention can induce an antibody against a Peplomer protein or a polypeptide having a partial sequence thereof, for example, by administering it to an administration subject. Therefore, the pharmaceutical composition of the present invention can also be referred to as a vaccine, a vaccine composition, or a vaccine preparation.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. The carrier is a suspending agent, a solubilizing agent, a stabilizer, an tonicity agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, a medium, a pH adjuster, and painlessness for administering the active ingredient. Examples thereof include agents, buffers, sulfur-containing reducing agents, antioxidants, etc., which can be appropriately added as long as the effects of the present invention are not impaired.

The suspending agent is not particularly limited, and examples thereof include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragant powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate and the like.

The solution auxiliary agent is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester.

The stabilizer is not particularly limited, and examples thereof include dextran 40, methyl cellulose, gelatin, sodium sulfite, and sodium metasulfite.

The tonicity agent is not particularly limited, and examples thereof include D-mannitol and sorbitol.

The preservative is not particularly limited, and examples thereof include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

The anti-adsorption agent is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hardened castor oil, and polyethylene glycol.

The sulfur-containing reducing agent is not particularly limited, and for example, N-acetylcysteine, N-acetylhomocysteine, thiochitoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, thio. Examples thereof include those having a sorbitol group such as sodium sulfate, glutathione, and thioalkanoic acid having 1 to 7 carbon atoms.

The antioxidant is not particularly limited, and is, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salt, L-ascorbic acid palmitate, L-ascorbic acid steer. Examples thereof include rate, sodium hydrogen sulfite, sodium sulfite, triamyl ascorbic acid, propyl ascorbic acid or a chelating agent such as ethylenediaminetetraacetic acid sodium (EDTA), sodium pyrophosphate, sodium metaphosphate and the like.

The pharmaceutical composition of the present invention further comprises an inorganic salt such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium hydrogencarbonate; an organic salt such as sodium citrate, potassium citrate, sodium acetate; Generally added components such as sugars such as glucose; and the like may be appropriately added.

The pharmaceutical composition of the present invention may further contain an immunostimulant. The immunostimulator is, for example, a Toll-like receptor stimulant such as aluminum hydroxide, aluminum phosphate, aluminum chloride, complete Freund's adjuvant, incomplete Freund's adjuvant, CpG oligonucleotide, Poly I: C, lipopolypolysaccharide (LPS). , Cytokines, lymphokines, chemokines and the like. The cytokines and lymphocains are, for example, interferons such as interferon (IFN-γ): inflammatory cytokines such as TNF-α; IL-1, IL-2, IL-3, IL-4, IL-12, IL-13. Interleukin such as; growth factors such as granulocyte macrophage (GM) colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF); Flt3 ligand; B7-1; B7-2; and the like. ..

The pharmaceutical composition of the present invention may be used , for example, in vitro or in vivo . The pharmaceutical composition of the present invention can be used, for example, as a research reagent or as a pharmaceutical product. In the former case, the pharmaceutical composition of the present invention can also be referred to as a test reagent or a test kit.

The administration target of the pharmaceutical composition of the present invention is not particularly limited. When the pharmaceutical composition of the present invention is used in vivo , the administration subject can, for example, refer to the above-mentioned example. When the pharmaceutical composition of the present invention is used in vitro , the administration target includes, for example, cells, tissues, organs, etc., and the cells include, for example, cells collected from a living body, cultured cells, and the like. Examples of the tissue or organ include a tissue (living tissue) or organ collected from a living body. Examples of the cells include iPS cells (induced pluripotent stem cells) and stem cells.

When the pharmaceutical composition of the present invention is used in vivo , the administration target may be a healthy person who is not infected with SARS2, a person who may be infected with SARS2, or a person who is infected with SARS2. It may be.

The conditions of use (administration conditions) of the pharmaceutical composition of the present invention are not particularly limited, and may be, for example, the type of active ingredient (protein, peptide, VLP, virus, nucleic acid, etc.) in the pharmaceutical composition, the type of administration target, and the like. Depending on the situation, the administration form, administration time, dose and the like can be appropriately set.

Examples of the method for administering the pharmaceutical composition of the present invention include intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intravenous administration and the like. Intramuscular administration or subcutaneous administration is preferable because it can be administered stably.

The dose of the pharmaceutical composition of the present invention is an amount capable of inducing an antibody against SARS2 when administered to a subject as compared with a non-administered subject, that is, an effective dose. The dose can be appropriately determined depending on, for example, the age, body weight, symptom, etc. of the administration subject. As a specific example, examples of the dose of the pharmaceutical composition include the following examples.
Mutant protein: 1 μg to 10 mg / time Mutant peptide: 1 μg to 100 mg / time Nucleic acid (mRNA): 1 μg to 10 mg / time Nucleic acid (DNA): 1 μg to 1000 mg / time Mutant virus: 1 × 10 ⁶ to 1 × 10 ¹³ viral particles / 1 time VLP: 1 μg 10 mg / time

The number of administrations of the pharmaceutical composition of the present invention is one or more. The plurality of times is, for example, two times, three times, four times, five times or more. The number of administrations may be appropriately determined while confirming the therapeutic effect of the elephant to be administered. In the case of multiple administrations, the administration interval can be appropriately determined while confirming the therapeutic effect of the administration target. For example, once a day, once a week, once every two weeks, once a month, once every three months 1 Times, once every 6 months, etc.

The pharmaceutical composition of the present invention can prevent or alleviate at least one symptom caused by SARS2 infection in an administered subject. Symptoms of SARS2 infection include, for example, rhinorrhea, sore throat, headache, hoarseness, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia and the like. The pharmaceutical compositions of the present invention may prevent or alleviate at least one symptom associated with SARS2 infection. The relief of the symptom can be evaluated subjectively or objectively, for example, self-evaluation by the administration subject; evaluation by a doctor; evaluation of QOL (Quality of Life); delay in progression of symptom of SARS2 infection or SARS2 infection. , Or evaluation of reduction of the severity of symptoms of SARS2 infection. The objective evaluation may be an evaluation by an animal or an evaluation by a human.

<Treatment method>
The present invention provides a method capable of imparting protective immunity to SARS2 to a subject and suppressing the induction of an ACE2 binding enhancing antibody. The treatment method of the present invention is a treatment method of a subject, and the subject is subjected to a mutant, a mutant polypeptide, a mutant virus, a virus-like particle, a nucleic acid, an expression vector, and / or a pharmaceutical composition of the Spike protein of the present invention. Use things. The treatment method of the present invention uses the active ingredient, a mutant of Peplomer protein, a mutant polypeptide, a mutant virus, a virus-like particle, a nucleic acid, and / or an expression vector (hereinafter, also referred to as “active ingredient”). The other steps and conditions are not particularly limited. Since the treatment method of the present invention contains, as an active ingredient, a polypeptide containing the mutant protein or a partial sequence thereof, or a nucleic acid encoding these, when the subject is treated, the S protein of SARS2 is converted to the ACE2 protein. It is possible to reduce the inducing ability of an antibody that enhances the binding ability of.

Since the treatment method of the present invention can impart protective immunity to SARS2, for example, it can be said to be, for example, a method of vaccination against SARS2, a method of inducing protective immunity, a method of stimulating an immune response, or a method of inducing an immune response. .. Further, since the treatment method of the present invention can induce an antibody against the S protein of SARS2 or a partial polypeptide thereof, it can also be said to be a method for inducing an antibody against the S protein of SARS2 or a partial polypeptide thereof. Furthermore, the treatment method of the present invention can induce an antibody against the S protein of SARS2 or a partial polypeptide thereof, for example, in which the induction of an antibody that enhances the binding activity of S protein of SARS2 to ACE2 is reduced. It can also be said that it is a method for inducing an antibody against the S protein of SARS2 or a partial polypeptide thereof, in which the induction of an antibody that enhances the binding activity of the S protein to ACE2 is reduced.

In the present invention, the active ingredient and the pharmaceutical composition can be administered to a subject to elicit protective immunity against SARS by inducing an immune response in the subject. Therefore, the treatment method of the present invention includes, for example, a step of administering the active ingredient and / or the pharmaceutical composition to the subject. Thereby, in the treatment method of the present invention, for example, in the subject, an immune response against the mutant protein and / or its partial polypeptide is induced, and a body fluid such as production of an antibody against the mutant protein and / or its partial polypeptide is induced. A cell-mediated immune response and / or a cell-mediated immune response by T cells or the like to SARS2 expressing the S protein occurs. The mutant protein has no mutation introduced into an amino acid other than the binding site of the ACE2 binding enhancing antibody, and the SARS2 neutralizing antibody is an antibody against the RBD region as shown in Examples described later. .. Therefore, in the treatment method of the present invention, for example, it is presumed that the inducing ability of the neutralizing antibody is induced in the same manner as when Sw protein is used as an active ingredient. Further, in the treatment method of the present invention, since the mutant protein and / or a partial polypeptide thereof is used, at least one mutation is introduced into the binding site of the ACE2 binding enhancing antibody, so that the epitope of the ACE2 binding enhancing antibody is introduced. Some or all of them are missing. Therefore, in the administration step, for example, the neutralizing antibody is induced, while the induction of the ACE2 binding enhancing antibody is reduced.

In the treatment method of the present invention, the administration step is carried out once or multiple times. As the administration conditions in the administration step, the description of the pharmaceutical composition of the present invention can be incorporated.

<Method for producing mutants of Peplomer protein>
The present invention provides a method for producing the mutant protein. The method of the present invention (hereinafter, also referred to as "method for producing a mutant") has reduced the ability to induce an antibody that enhances the ability of SARS-CoV-2 to bind Spike protein to angiotensin-converting enzyme 2 (ACE2). A method for producing a variant of the Spike protein, wherein in the amino acid sequence of the Spike protein (St protein) of the target SARS-CoV-2, 30 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. At least one amino acid residue selected from the group consisting of amino acids at positions 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. Includes a mutation step that introduces a mutation into the corresponding amino acid. The method for producing a mutant of the present invention is characterized in that a mutation is introduced into an amino acid at at least a predetermined position in the mutation step, and other steps and conditions are not particularly limited. According to the present invention, it is possible to produce a mutant of S protein having a reduced inducing ability of the ACE2 binding enhancing antibody.

The mutant protein obtained by the method for producing a variant of the present invention can reduce the inducing ability of an antibody that enhances the binding ability of SARS-CoV-2 Spike protein to angiotensin converting enzyme 2 (ACE2), and thus is an infection-enhancing antibody. It is believed that the occurrence of ADE due to induction or enhancement of infection can be reduced. Therefore, the method for producing a mutant of the present invention is a method for producing a mutant of Spike protein having a reduced ability to induce an infection-enhancing antibody or a method for producing a mutant of a Peplomer protein having a reduced ability to induce an ADE. You can also.

In the mutation step, a mutation is introduced into the amino acid at the predetermined position. The mutation step can be carried out, for example, by introducing a mutation into the base sequence (polynucleotide) encoding the St protein.

In the mutation step, the amino acids into which the mutation is introduced (amino acids to be mutated) are the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, and 214th positions in the Ss protein. One of the amino acid residues at positions 216 and 217 and the corresponding amino acid, or a plurality (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). ), For example, the latter because it can further suppress the inducing ability of the ACE2 binding enhancing antibody. In the mutation step, the amino acid residues at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein are used. It is presumed that the ability to induce the ACE2-binding enhancing antibody can be suppressed more efficiently by increasing the number of amino acids to be mutated in the corresponding amino acids.

When a mutation is introduced into one of the amino acids to be mutated in the mutation step, the positions of the amino acids to be mutated are, for example, positions 30, 32, 64, 65, 66, etc. in the Ss protein. Amino acids corresponding to amino acid residues at positions 186, 187, 211, 213, 214, 216, or 217, or positions 30, 32, 64, 66, 186, 187, 211. Amino acid residues corresponding to the amino acid residues at positions 213, 214, 216, or 217, preferably amino acid residues at positions 64, 65, 66, 187, 213, or 214. And the corresponding amino acid, the amino acid corresponding to the amino acid residue at position 64, 66, 187, 213, or 214, or the amino acid corresponding to the amino acid residue at position 64, 66, 213, or 214. Is.

When a mutation is introduced into a plurality of the amino acids to be mutated in the mutation step, the positions of the amino acids to be mutated are, for example, positions 30, 32, 64, 65, 66, and 186 in the Ss protein. Amino acids corresponding to any two or more amino acid residues in the amino acids at positions 187, 211, 213, 214, 216, and 217, or at positions 30, 32, 64, 66, It is an amino acid corresponding to any two or more amino acid residues in the amino acids at positions 186, 187, 211, 213, 214, 216, and 217, and is preferably at positions 64, 65, and 66. Amino acids corresponding to any two or more amino acid residues at positions, 187, 213, and 214, and residual amino acids at any two or more positions at positions 64, 66, 187, 213, and 214. The amino acid corresponding to the group, or the amino acid corresponding to any two or more amino acid residues at positions 64, 66, 213, and 214, more preferably (a) positions 64, 65, and. / Or corresponds to amino acid residues at positions 66, (b) 187, 213, and / or 214, or (c) 64, 65, 66, 187, 213, and / or 214. Amino acids, (d) amino acids corresponding to amino acid residues at positions 64, 66, 187, 213, and / or 214, or (e) positions 64, 66, 213, and / or 214. The amino acids corresponding to the amino acid residues of, more preferably (1) the amino acids corresponding to the amino acid residues at positions 64, 65, 66, 187, 213, and 214, and (2) 64. , Amino acids corresponding to the amino acid residues at positions 66, 187, 213, and 214, or (3) amino acids corresponding to the amino acid residues at positions 64, 66, 213, and 214.

In the mutation step, the amino acid to be mutated is, for example, 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, etc. in the Ss protein. And / or in addition to, or in addition to, amino acids corresponding to the amino acid residues at positions 68, 94, 97, 129, 185, and / or 215 may be used.

In the mutation step, the position of the amino acid to be mutated can further reduce, for example, the ability to induce an antibody that enhances the ability of SARS2 to bind to the ACE2 protein. Amino acids corresponding to one or more amino acid residues in the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein, or N30, F32, W64. , H66, F186, K187, N211, V213, R214, L216, and P217, which are amino acids corresponding to one or more amino acid residues, more preferably W64, F65, H66, K187, V213, and An amino acid corresponding to one or more amino acid residues in the amino acids of R214, W64, H66, K187, V213, and R214, or W64, H66, V213, and R214. The position of the amino acid to be mutated in the St protein can further reduce, for example, the ability to induce an antibody that enhances the ability of SARS2 to bind to the ACE2 protein. Therefore, the Ss in the St protein is more preferable. Amino acids corresponding to the amino acid residues of W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, or W64, H66, V213, and R214 in proteins.

In the mutation step, the amino acid to be mutated is, for example, in place of or in addition to N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and / or P217 in the Ss protein. , I68, S94, K97, K129, N185, and / or may be the amino acid corresponding to the amino acid residue of D215.

The type of amino acid mutation introduced in the mutation introduction step may be a range in which the function of the St protein is modified by the amino acid mutation, and the binding of the St protein after the mutation to the ACE2 protein by the amino acid mutation. It is preferable that the range is such that the induction of an antibody that enhances the ability can be suppressed. Therefore, the "mutation" can be, for example, a modification. Types of said mutations include, for example, amino acid deletions, substitutions, insertions and / or additions. The type of mutation introduced into the mutant protein may be one type or a plurality of types.

It is assumed that the type of mutation is, for example, easy to maintain the immunogenicity of a mutant protein having a mutant amino acid, and suppresses the influence on the induction of production of a neutralizing antibody crossing the mutant protein and the St protein. Therefore, replacement is preferable. When the mutation is a substitution, the substitution is preferably not the above-mentioned conservative substitution, and in particular, a substitution in which the charge of the side chain of the amino acid is changed is preferable. By introducing such a substitution as a mutation, the ability to induce an antibody that enhances the ability of SARS2 to bind to the ACE2 protein can be further reduced. Specific examples of the substitution include substitution with alanine (alanine substitution) and substitution with a sugar chain modification motif (NX [S / T]).

When the mutation is an alanine substitution, in the mutation step, the mutation of the amino acid to be mutated is in the St protein, in the Ss protein, N30, F32, W64, F65, H66, F186, K187, N211, V213, One of the amino acids corresponding to one or more amino acid residues in the amino acids of R214, L216, and P217, or one of the amino acids of N30, F32, W64, H66, F186, K187, N211, V213, R214, L216, and P217. Alternatively, it is an alanine substitution of an amino acid corresponding to a plurality of amino acid residues, more preferably W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, or W64, H66, V213. , And an alanine substitution of the amino acid corresponding to one or more amino acid residues in the amino acid of R214, more preferably W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and. R214, or the alanine substitution of the amino acid corresponding to the amino acid residue of W64, H66, V213, and R214.

When the mutation is a substitution with a sugar chain modification motif, in the mutation step, the mutation of the amino acid to be mutated is a substitution of N (asparagine) of the amino acid at position 64 in the Ss protein in the St protein, and 66. Substitution of the amino acid at the position with S (serine) or T (threonine) can be mentioned.

The substitution may be a combination of an alanine substitution and a substitution with a sugar chain modification motif. In the mutation step, the mutation of the mutation target amino acid is, for example, the same mutation except that W64 and H66 in the Ss protein are changed to W64N and H66S or W64N and H66T in the above-mentioned example of alanine substitution. be.

In the mutation step, for example, the mutation may be introduced only into the amino acid at the predetermined position by a site-specific mutation introduction method, or the mutation may be introduced only into the amino acid at an arbitrary position by a random mutation introduction method. You may.

The mutation step is a production step of introducing a mutation into the amino acid sequence of the St protein to generate an amino acid sequence of a candidate protein into which the mutation has been introduced into the amino acid sequence of the St protein, and a production step of the candidate protein in the Ss protein. , At least one amino acid selected from the group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. It is preferable to include a selection step of selecting a candidate protein having a mutation in the amino acid corresponding to the residue as a variant of the Picke protein having a reduced ability to induce an antibody that enhances the binding ability to ACE2.

The production step can be carried out, for example, by introducing a mutation into the base sequence (polynucleotide) encoding the St protein. The mutation introduction method may be a site-specific mutation introduction method or a random mutation introduction method. The number of mutations introduced in the production step is not particularly limited and may be any number, but preferably 70%, 75%, 80%, 85%, etc., with respect to the amino acid sequence of the St protein. Mutations are introduced to have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity.

The selection step can be carried out, for example, by decoding the base sequence of polynucleothio encoding the St protein (candidate protein) after introduction of the mutation, converting the obtained base sequence into an amino acid sequence, and analyzing the base sequence. Then, in the selection step, the amino acid sequence of the obtained St protein after introduction of the mutation is compared with the amino acid sequence of the Ss protein, and as a mutant in which the desired mutation is introduced into the amino acid to be mutated in the mutation step. Select.

In the selection step, the St protein after introduction of the mutation may be further selected based on the binding property with the reference antibody. In this case, in the selection step, a candidate protein having a mutation that reduces binding to the reference antibody is selected as the mutant from the St protein after the mutation is introduced. Specifically, in the selection step, for example, the binding property of the St protein after the mutation introduction and the reference antibody is significantly lower than the binding property of the St protein before the mutation introduction and the reference antibody. If so, the St protein after the introduction of the mutation may be selected. The binding property between the St protein after the introduction of the mutation and the reference antibody and the binding property between the St protein before the introduction of the mutation and the reference antibody can be evaluated, for example, in vitro. It can be evaluated in the same way as 5.

<Screening method for Spike protein>
The present invention provides a method for screening the mutant protein. The method of the present invention (hereinafter, also referred to as “screening method”) is a method of a Spike protein having a reduced ability to induce an antibody that enhances the ability of SARS-CoV-2 to bind to angiotensin converting enzyme 2 (ACE2). A method for screening variants, in the amino acid sequence of the Spike protein (Sc protein) of candidate SARS-CoV-2, at position 30 and 32 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. Corresponds to at least one amino acid residue selected from the group consisting of amino acids at positions 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. A selection step of selecting a Sc protein having a mutation in an amino acid as the mutant is included. The screening method of the present invention is characterized in that, in the selection step, a candidate protein having a mutation in an amino acid at at least a predetermined position is selected, and other steps and conditions are not particularly limited. According to the present invention, a mutant of the S protein having a reduced inducing ability of the ACE2 binding enhancing antibody can be screened.

Since the mutant protein obtained by the screening method of the present invention can reduce the inducing ability of an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Spike protein, it is possible to induce an infection-enhancing antibody or to induce an infection-enhancing antibody. It is believed that the occurrence of ADE due to infection-enhancing antibodies can be reduced. Therefore, the screening method of the present invention can be said to be a method for screening a variant of Spike protein having a reduced ability to induce an infection-enhancing antibody or a method for screening a variant of a Spike protein having a reduced ability to induce an ADE.

The selection step can be carried out in the same manner as the selection step of the method for producing a mutant of the present invention.

<Marker>
The present invention provides markers that can be indicators of enhanced SARS-CoV-2 infection. The marker of the present invention is an antibody (ACE2 binding enhancing antibody) that enhances the binding of wild SARS-CoV-2 Spike protein (Sw protein) to ACE2 protein in an Fc receptor-independent manner. The marker of the present invention is characterized by being the ACE2-binding enhancing antibody, and other configurations and conditions are not particularly limited. According to the marker of the present invention, the possibility of morbidity with SARS2 and the like can be tested. Specific examples of the marker of the present invention include the first to fourth markers of the present invention described later.

<First marker>
The present invention provides markers that can be indicators of enhanced SARS-CoV-2 infection. The SARS-CoV-2 infection-enhancing marker of the present invention is 30 in the wild-type SARS-CoV-2 Spike protein (Sw protein) and in the reference SARS-CoV-2 Spike protein (Ss protein, SEQ ID NO: 1). At least one amino acid residue selected from the group consisting of amino acids at positions 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. It is an antibody that recognizes the corresponding amino acid. The first marker of the present invention is characterized in that an antibody that recognizes an amino acid corresponding to an amino acid residue at a predetermined position in the Ss protein is used as a marker for enhancement of infection, and other configurations and conditions are particularly high. Not limited. According to the first marker of the present invention, the possibility of morbidity with SARS2 and the like can be tested.

In the first marker of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the first marker of the present invention can be, for example, a marker for enhancing the binding of the Spike protein of SARS-CoV-2 to ACE2 or a marker for enhancing the binding of SARS-CoV-2 to ACE2. Further, as described later, it is presumed that the infection-enhancing antibody is involved in the aggravation of SARS2 via ADE. Therefore, the first marker of the present invention can be, for example, a predictive marker or aggravation marker of ADE.

In the first marker of the present invention, the origin of the antibody is not particularly limited and can be appropriately set depending on, for example, the type of subject. For the origin of the antibody, for example, the above-mentioned example of the administration subject can be incorporated. The antibody is preferably derived from humans, for example.

In the first marker of the present invention, the antibody is an antibody that recognizes an amino acid corresponding to an amino acid residue at a predetermined position in the Ss protein in the Sw protein. The Sw protein may be the Ss protein.

The amino acids recognized by the antibody are the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, and 216th positions of the Ss protein in the Sw protein. And one of the mutant amino acids at position 217, or a plurality (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

When one amino acid is recognized by the antibody, the positions of the amino acids are, for example, in the Sw protein, in the Ss protein, at the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions. Amino acids corresponding to amino acid residues at positions 211, 213, 214, 216, or 217, or amino acids at positions 30, 32, 64, 66, 186, 187, 211, 213, 214. Amino acid corresponding to the amino acid residue at position 216 or 217, preferably the amino acid corresponding to the amino acid residue at position 64, 65, 66, 187, 213 or 214, 64. It is an amino acid corresponding to an amino acid residue at position 66, 187, 213, or 214, or an amino acid corresponding to an amino acid residue at position 64, 66, 213, or 214.

When there are a plurality of amino acids recognized by the antibody, the positions of the amino acids are, for example, in the Sw protein, in the Ss protein, at the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions. Amino acids corresponding to any two or more amino acid residues in the amino acids at positions 211, 213, 214, 216, and 217, or at positions 30, 32, 64, 66, 186, and 187. , 211, 213, 214, 216, and 217, which correspond to any two or more amino acid residues, preferably at positions 64, 65, 66, and 187. Amino acids corresponding to any two or more amino acid residues at positions 213 and 214, amino acids corresponding to any two or more amino acid residues at positions 64, 66, 187, and 214. , Or an amino acid corresponding to any two or more amino acid residues at positions 64, 66, 213, and 214, more preferably (a) positions 64, 65, and / or 66. (B) Amino acids corresponding to the amino acid residues at positions 187, 213, and / or 214, or (c) at positions 64, 65, 66, 187, 213, and / or 214, (d). ) With amino acids corresponding to the amino acid residues at positions 64, 66, 187, 213, and / or 214, or (e) with amino acid residues at positions 64, 66, 213, and / or 214. Corresponding amino acids, more preferably (1) amino acids corresponding to amino acid residues at positions 64, 65, 66, 187, 213, and 214, (2) amino acids at positions 64, 66, 187. Amino acids corresponding to the amino acid residues at positions 213 and 214, or (3) amino acids corresponding to the amino acid residues at positions 64, 66, 213, and 214.

The amino acids recognized by the antibody are the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, and 216th positions of the Ss protein in the Sw protein. And / or in addition to or in addition to, it may be an amino acid corresponding to the amino acid residues at positions 68, 94, 97, 129, 185, and / or 215.

The position of the amino acid recognized by the antibody is preferably, in the Sw protein, in the Ss protein, because, for example, an antibody that enhances the binding ability of SARS2 to the ACE2 protein can be detected more accurately. Amino acids corresponding to one or more amino acid residues in the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217, or N30, F32, W64, H66, F186. , K187, N211, V213, R214, L216, and P217, which are amino acids corresponding to one or more amino acid residues, more preferably W64, F65, H66, K187, V213, and R214, W64, An amino acid corresponding to one or more amino acid residues in the amino acids of H66, K187, V213, and R214, or W64, H66, V213, and R214. The position of the amino acid recognized by the antibody is more preferably in the Sw protein, in the Ss protein, because, for example, an antibody that enhances the binding ability of SARS2 to the ACE2 protein can be detected more accurately. , W64, F65, H66, K187, V213, and R214, W64, H66, K187, V213, and R214, or amino acids corresponding to the amino acid residues of W64, H66, V213, and R214.

The amino acids recognized by the antibody are, for example, in the Sw protein, in place of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and / or P217 in the Ss protein, or In addition, it may be an amino acid corresponding to the amino acid residue of I68, S94, K97, K129, N185, and / or D215.

The antibody is preferably an antibody that enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein, for example. The enhancement of the binding activity may be measured, for example, by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the antibody, or the antibody may be measured. , It may be measured indirectly by measuring whether or not there is an antibody that recognizes a predetermined position in the Ss protein. In the former case, the measurement can be carried out in the same manner as in Example 1 described later, for example. In the latter case, the measurement can be performed, for example, in the same manner as in Example 4 described later.

In the first marker of the present invention, the antibody may be one type or two or more types, that is, an antibody having the same epitope or an antibody having a different epitope.

The antibody may be a protein or mRNA transcribed from the gene of the protein of the antibody, but is preferably a protein.

<Second marker>
The present invention provides markers that can be indicators of enhanced SARS-CoV-2 infection. The SARS-CoV-2 infection enhancing marker of the present invention is a competitive antibody that competes with the reference antibody for binding to the Spike protein (Sw protein) of wild SARS-CoV-2, and the reference antibody is the Sw. In terms of protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213rd, and 214th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 216 and 217. The second marker of the present invention is characterized in that a competing antibody competing with the reference antibody is used as a marker for enhancing infection, and other configurations and conditions are not particularly limited. According to the second marker of the present invention, the possibility of morbidity with SARS2 and the like can be tested.

In the second marker of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the second marker of the present invention can be, for example, a marker for enhancing the binding of the Spike protein of SARS-CoV-2 to ACE2 or a marker for enhancing the binding of SARS-CoV-2 to ACE2. Further, as described later, it is presumed that the infection-enhancing antibody is involved in the aggravation of SARS2 via ADE. Therefore, the second marker of the present invention can be, for example, a predictive marker or aggravation marker of ADE.

In the second marker of the present invention, the origin of the antibody is not particularly limited and can be appropriately set depending on the type of the subject, for example. For the origin of the antibody, for example, the above-mentioned example of the administration subject can be incorporated. The antibody is preferably derived from humans, for example.

In the second marker of the present invention, the antibody is an antibody that competes with a reference antibody for binding to the Sw protein, and the reference antibody is an amino acid residue at a predetermined position in the Ss protein in the Sw protein. An antibody that recognizes the amino acid corresponding to the group. Competition with the reference antibody can be measured, for example, by measuring whether the binding of the Sw protein to one antibody suppresses the binding of the other antibody. Specifically, the Sw protein is brought into contact with the antibody. The resulting mixture is contacted with the reference antibody and the competition is based on whether the binding of the reference antibody to the Sw protein is suppressed (reduced or reduced) as compared to the absence of the antibody. Evaluate if it is occurring. When the binding between the reference antibody and the Sw protein is suppressed, it can be evaluated that the reference antibody and the antibody are in competition. On the other hand, when the binding between the reference antibody and the Sw protein is not suppressed, it can be evaluated that the reference antibody and the antibody do not compete with each other. In addition, although the example in which the Sw protein is sequentially brought into contact with the antibody and the reference antibody has been described, the measurement may be carried out by bringing the three parties into contact with each other at the same time. In this case, when the binding between the reference antibody and the Sw protein is suppressed as compared with the absence of the antibody, it can be evaluated that the reference antibody and the antibody are in competition. On the other hand, when the binding between the reference antibody and the Sw protein is not suppressed as compared with the absence of the antibody, it can be evaluated that the reference antibody and the antibody do not compete with each other.

In the second marker of the present invention, the antibody may be one type or two or more types, that is, an antibody having the same epitope or an antibody having a different epitope.

The antibody may be a protein or mRNA transcribed from the gene of the protein of the antibody, but is preferably a protein.

<Third marker>
The present invention provides markers that can be indicators of enhanced SARS-CoV-2 infection. The SARS-CoV-2 infection enhancing marker of the present invention binds to the Spike protein (Sw protein) of wild SARS-CoV-2, and in the Sw protein, the Spike protein (Ss protein, of the reference SARS-CoV-2). Selected from the group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in SEQ ID NO: 1). It is an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue. The third marker of the present invention is characterized in that an antibody that binds to the Sw protein and does not recognize the mutant protein is used as a marker for enhancing infection, and other configurations and conditions are not particularly limited. According to the third marker of the present invention, the possibility of morbidity with SARS2 and the like can be tested.

In the third marker of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the third marker of the present invention can be, for example, a marker for enhancing the binding of the Spike protein of SARS-CoV-2 to ACE2 or a marker for enhancing the binding of SARS-CoV-2 to ACE2. Further, as described later, it is presumed that the infection-enhancing antibody is involved in the aggravation of SARS2 via ADE. Therefore, the third marker of the present invention can be, for example, a predictive marker or aggravation marker of ADE.

In the third marker of the present invention, the origin of the antibody is not particularly limited and can be appropriately set depending on the type of the subject, for example. For the origin of the antibody, for example, the above-mentioned example of the administration subject can be incorporated. The antibody is preferably derived from humans, for example.

In the third marker of the present invention, in the Sw protein, in the Ss protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions, It is an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 214, 216, and 217, that is, the antibody is the Sw protein. In the Ss protein, selected from the group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. It can also be said to be an antibody that recognizes an amino acid corresponding to at least one amino acid residue. The antibody is at least one amino acid selected from the group consisting of amino acids at positions 30, 32, 64, 66, 186, 187, 211, 213, 214, 216, and 217. It may be an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the residue. In this case, the antibody is the Sw protein at positions 30, 32, 64, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. It can also be said that it is an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids of.

For the position of the mutated amino acid in the mutated protein, for example, the description of the amino acid sequence of (Sm1) in the mutated protein of the present invention can be incorporated.

In the third marker of the present invention, when the antibody does not recognize the mutant protein, the amount of the antibody binding to the mutant protein is significantly reduced (decreased) as compared with the amount of the antibody binding to the Sw protein. Or decreased), specifically, 10%, 20%, 30%, 40%, 50%, 60%, 70% as compared with the amount of the antibody bound to the Sw protein. , 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. The amount of the antibody bound to the mutant protein and the amount of the antibody bound to the Sw protein can be measured, for example, in the same manner as in Example 5 described later, and can be evaluated based on, for example, MFI (Mean Fluorescence Intensity).

In the third marker of the present invention, the antibody may be one type or two or more types, that is, an antibody having the same epitope or an antibody having a different epitope.

The antibody may be a protein or mRNA transcribed from the gene of the protein of the antibody, but is preferably a protein.

<Fourth marker>
The present invention provides markers that can be indicators of enhanced SARS-CoV-2 infection. The SARS-CoV-2 infection enhancing marker of the present invention is an antibody that competes with a reference antibody for binding to the Spike protein (Sw protein) of wild SARS-CoV-2, and the reference antibody is the Sw protein. In the Sw protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, and 211th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It is an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 213, 214, 216, and 217. The fourth marker of the present invention is characterized in that a competing antibody competing with the reference antibody is used as a marker for enhancing infection, and other configurations and conditions are not particularly limited. According to the fourth marker of the present invention, the possibility of morbidity with SARS2 and the like can be tested.

In the fourth marker of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the fourth marker of the present invention can be, for example, a marker for enhancing the binding of the Spike protein of SARS-CoV-2 to ACE2 or a marker for enhancing the binding of SARS-CoV-2 to ACE2. Further, as described later, it is presumed that the infection-enhancing antibody is involved in the aggravation of SARS2 via ADE. Therefore, the fourth marker of the present invention can be, for example, a predictive marker or aggravation marker of ADE.

In the fourth marker of the present invention, the origin of the antibody is not particularly limited and can be appropriately set depending on the type of the subject, for example. For the origin of the antibody, for example, the above-mentioned example of the administration subject can be incorporated. The antibody is preferably derived from humans, for example.

In the fourth marker of the present invention, the antibody is an antibody that competes with the reference antibody for binding to the Sw protein, the reference antibody recognizes the Sw protein, and in the Sw protein, the Ss protein. At least one amino acid residue selected from the group consisting of 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th. It is an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the group. Competition with the reference antibody can be evaluated, for example, in the same manner as described above for the second marker of the present invention. Whether or not the antibody recognizes a mutant protein can be evaluated, for example, in the same manner as in the description of the third marker of the present invention. The reference antibody is a residue of at least one amino acid selected from the group consisting of positions 30, 32, 64, 66, 186, 187, 211, 213, 214, 216, and 217. It may be an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the group. The mutant protein can be referred to the description of the mutant protein of the present invention.

In the fourth marker of the present invention, the antibody may be one type or two or more types, that is, an antibody having the same epitope or an antibody having a different epitope.

The antibody may be a protein or mRNA transcribed from the gene of the protein of the antibody, but is preferably a protein.

<Detection method>
The present invention provides a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2. The method for detecting the SARS-CoV-2 infection-enhancing antibody of the present invention is the ACE2 protein of the Spike protein (Sw protein) of wild SARS-CoV-2 in a biological sample of a subject in an Fc receptor-independent manner. It comprises a detection step of detecting an antibody that enhances binding to. The detection method of the present invention is characterized in that the ACE2-binding enhancing antibody is detected in the detection step, and other configurations and conditions are not particularly limited. According to the detection method of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected. Specific examples of the detection method of the present invention include the first to fourth detection methods of the present invention described later.

<First detection method>
The present invention provides a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2. The method for detecting the SARS-CoV-2 infection-enhancing antibody of the present invention is to use the wild-type SARS-CoV-2 Spike protein (Sw protein) as a reference SARS-CoV-2 Spike protein in a biological sample of a subject. Consists of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in (Ss protein, SEQ ID NO: 1). It comprises a detection step of detecting an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group. In the first detection method of the present invention, in the detection step, an antibody that recognizes an amino acid corresponding to an amino acid residue at a predetermined position in the Ss protein is detected as an infection-enhancing antibody, that is, the first detection method. It is characterized by using a marker as an index, and other steps and conditions are not particularly limited. According to the first detection method of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

In the first detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the first detection method of the present invention is, for example, a method for detecting a binding-enhancing antibody for the Spike protein of SARS-CoV-2 to ACE2 or a method for detecting a binding-enhancing antibody for SARS-CoV-2 to ACE2. You can also do it.

According to the first detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody can be detected.

In the first detection method of the present invention, for example, the presence or absence of the antibody may be detected, or the amount of the antibody may be detected. In the former case, the first detection method of the present invention can be, for example, a qualitative analysis method or a qualitative measurement method. Further, in the latter case, the first detection method of the present invention can be, for example, a quantitative analysis method or a quantitative measurement method. The detection can also be, for example, inspection or detection.

In the present invention, the type of the biological sample is not particularly limited, and examples thereof include body fluids, body fluid-derived cells, organelles, tissues, and cells separated from the living body. Examples of the body fluid include blood samples, and specific examples thereof include whole blood, serum, and plasma. Examples of the body fluid-derived cells include blood-derived cells, and specific examples thereof include blood cell cells such as blood cells, white blood cells, and lymphocytes. The biological sample is preferably whole blood, serum, or plasma, more preferably serum or plasma, because it can reduce the burden on the patient or doctor, for example. When the biological sample is a liquid sample, the biological sample may be diluted with a solvent such as water or a buffer solution. When the biological sample is a solid sample, it is preferable to disperse the biological sample in the solvent.

In the first detection method of the present invention, the detection target in the detection step is an antibody that recognizes an amino acid corresponding to an amino acid at a predetermined position in the Ss protein in the Sw protein. Can be used. In the detection step, the detection target antibody may be one type or two or more types, that is, only antibodies having the same epitope may be detected, or antibodies having different epitopes may be detected together. good.

In the detection step, an antibody that recognizes an amino acid at a predetermined position in the Ss protein is detected in the Sw protein of the biological sample of the subject. The detection step can be carried out using, for example, the Sw protein and a mutant protein in which a mutation is introduced into the amino acid at the predetermined position in the Sw protein. For the mutant protein, the description of the amino acid sequence of (Sm1) in the mutant protein of the present invention can be incorporated. As a specific example, in the detection step, the biological sample is brought into contact with the Sw protein, and the binding or binding amount (also referred to as “amount of antibody”) between the antibody contained in the biological sample and the Sw protein is detected. (First detection step). Further, in the detection step, the biological sample is brought into contact with the mutant protein, and the binding or the amount of binding between the antibody contained in the biological sample and the mutant protein is detected (second detection step). The binding between the antibody contained in the biological sample and the Sw protein or the mutant protein can be measured by, for example, an ELISA method using an antigen-antibody reaction, flow cytometry, or the like.

Next, in the detection step, by comparing (evaluating) the results obtained in the first detection step and the second detection step, the antibody binding to the Sw protein is obtained in the Sw protein. It is possible to evaluate whether or not the amino acid at a predetermined position in the Ss protein is recognized (comparison step or evaluation step). Specifically, when the binding is used as an index, in the detection step, the antibody contained in the biological sample is bound to the Sw protein, and the antibody contained in the biological sample and the mutant protein are associated with each other. If no binding is found, the antibody contained in the biological sample can be evaluated to recognize the amino acid at a predetermined position in the Ss protein in the Sw protein, that is, the biological sample contains an infection-enhancing antibody. On the other hand, in the detection step, when the antibody contained in the biological sample does not bind to the Sw protein, or when the antibody contained in the biological sample binds to the Sw protein, the biological sample is bound. When the antibody contained in the above is bound to the mutant protein, the antibody contained in the biological sample does not recognize the amino acid at a predetermined position in the Ss protein in the Sw protein, that is, the biological sample is infected. It can be evaluated that it does not contain the enhancing antibody. When the binding amount is used as an index, in the detection step, the binding amount of the antibody contained in the biological sample and the mutant protein is compared with the binding amount of the antibody contained in the biological sample and the Sw protein. Then, when it is significantly decreased (for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94). %, 95%, 96%, 97%, 98%, or 99% or more), the antibody contained in the biological sample recognizes the amino acid at a predetermined position in the Ss protein in the Sw protein. That is, it can be evaluated that the biological sample contains an infection-enhancing antibody. When the binding amount of the antibody contained in the biological sample and the mutant protein is not significantly different from the binding amount of the antibody contained in the biological sample and the Sw protein, the antibody contained in the biological sample is said to be said. It can be evaluated that the Sw protein does not recognize the amino acid at a predetermined position in the Ss protein, that is, the biological sample does not contain the infection-enhancing antibody.

In the detection step, the N-terminal domain (NTD) may be used instead of the Sw protein. Further, in the detection step, the position of the amino acid recognized by the antibody can be changed by changing the position of the mutant amino acid in the mutant protein, whereby the epitope can be specified.

In the first detection method of the present invention, for example, it may be further evaluated whether the antibody in the biological sample is an antibody that enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein, for example. good. The enhancement of the binding activity can be measured, for example, by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the biological sample. The measurement can be performed, for example, in the same manner as in Example 1 described later.

<Second detection method>
The present invention provides a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2. The method for detecting the infection-enhancing antibody of SARS-CoV-2 of the present invention competes with the reference antibody for the binding of wild-type SARS-CoV-2 to the Spike protein (Sw protein) in the biological sample of the subject. The reference antibody comprises a detection step of detecting an antibody, wherein the reference antibody is located at positions 30, 32, 64, 65, etc. in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 66, 186, 187, 211, 213, 214, 216, and 217. The second detection method of the present invention is characterized in that, in the detection step, a competitive antibody that competes with the reference antibody is detected as an infection-enhancing antibody, that is, the second marker is used as an index. Other steps and conditions are not particularly limited. According to the first detection method of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

In the second detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the second detection method of the present invention is, for example, a method for detecting a binding-enhancing antibody for the Spike protein of SARS-CoV-2 to ACE2 or a method for detecting a binding-enhancing antibody for SARS-CoV-2 to ACE2. You can also do it.

According to the second detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody can be detected.

In the second detection method of the present invention, for example, the presence or absence of the antibody may be detected, or the amount of the antibody (the amount of the ACE2 binding enhancing antibody and / or the infection enhancing antibody) may be detected. .. In the former case, the second detection method of the present invention can be, for example, a qualitative analysis method or a qualitative measurement method. Further, in the latter case, the second detection method of the present invention can be, for example, a quantitative analysis method or a quantitative measurement method. The detection can also be, for example, inspection or detection.

In the second detection method of the present invention, the detection target in the detection step is an antibody that competes with the reference antibody for binding to the Sw protein, and the reference antibody is the Sw protein in the Ss protein. It is an antibody that recognizes an amino acid corresponding to an amino acid residue at a predetermined position, and the above description of the reference antibody can be incorporated. In the detection step, the detection target antibody may be one type or two or more types, that is, only antibodies having the same epitope may be detected, or antibodies having different epitopes may be detected together. good.

The detection step includes a detection step of detecting a competing antibody that competes with a reference antibody for binding to the Sw protein in a biological sample of the subject. Specifically, in the detection step, the Sw protein is brought into contact with the biological sample, and the binding or binding amount of the Sw protein to the antibody in the biological sample is detected (first detection step). Next, in the detection step, the Sw protein and the biological sample are brought into contact with each other in the presence of the reference antibody, and the binding or binding amount of the Sw protein to the antibody in the biological sample is detected (second). Detection process). The binding between the antibody contained in the biological sample and the Sw protein can be measured by, for example, an ELISA method using an antigen-antibody reaction, flow cytometry, or the like.

Next, in the detection step, by comparing (evaluating) the results obtained in the first detection step and the second detection step, the antibody contained in the biological sample binds to the Sw protein. Can be evaluated as to whether it is an antibody that competes with the reference antibody (comparison step or evaluation step). Specifically, when the binding is used as an index, in the detection step, the antibody contained in the biological sample is bound to the Sw protein, and in the presence of the reference antibody, the antibody is contained in the biological sample. If no binding between the antibody and the Sw protein is observed, the antibody contained in the biological sample is an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample is an infection-enhancing antibody. Can be evaluated as including. On the other hand, in the detection step, when the antibody contained in the biological sample is not bound to the Sw protein, or the antibody contained in the biological sample is bound to the Sw protein, and the reference antibody is found. If the antibody contained in the biological sample binds to the Sw protein even in the presence of, the antibody contained in the biological sample is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, , It can be evaluated that the biological sample does not contain the infection-enhancing antibody. When the binding amount is used as an index, in the detection step, the antibody contained in the biological sample and the Sw protein in the presence of the reference antibody are bound to the antibody contained in the biological sample and the Sw protein. (For example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%) when compared with the binding amount of , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more), the antibody contained in the biological sample is the reference antibody for binding to the Sw protein. It can be evaluated that it is a competing antibody, that is, the biological sample contains an infection-enhancing antibody. On the other hand, when the amount of binding between the antibody contained in the biological sample and the Sw protein in the presence of the reference antibody is not significantly different from the amount of binding between the antibody contained in the biological sample and the Sw protein, the biological sample It can be evaluated that the antibody contained in is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample does not contain an infection-enhancing antibody.

In the detection step, the biological sample may be brought into contact with the Sw protein, and then the obtained mixture may be brought into contact with the reference antibody to detect whether the reference antibody binds to the Sw protein. In this case, when the binding of the reference antibody to the Sw protein is not detected in the detection step, or when the binding of the reference antibody to the Sw protein is reduced as compared with the absence of the biological sample, the above. It can be evaluated that the antibody in the biological sample is an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample contains an infection-enhancing antibody. On the other hand, in the detection step, when the binding of the reference antibody to the Sw protein is detected, the antibody in the biological sample is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, the said. It can be evaluated that the biological sample does not contain the infection-enhancing antibody.

In the detection step, the N-terminal domain (NTD) may be used instead of the Sw protein.

In the second detection method of the present invention, for example, it may be further evaluated whether the antibody in the biological sample is an antibody that enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein, for example. good. The enhancement of the binding activity can be measured, for example, by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the biological sample. The measurement can be performed, for example, in the same manner as in Example 1 described later.

<Third detection method>
The present invention provides a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2. In the method for detecting a SARS-CoV-2 infection-enhancing antibody of the present invention, a biological sample of a subject is bound to the Spike protein (Sw protein) of wild-type SARS-CoV-2, and the reference SARS is used in the Sw protein. -In the Spike protein (Ss protein, SEQ ID NO: 1) of CoV-2, 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, And a detection step of detecting an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acid at position 217. The third detection method of the present invention is to detect an antibody that binds to the Sw protein and does not recognize the mutant protein as an infection-enhancing antibody in the detection step, that is, using the third marker as an index. It is characterized by use, and other steps and conditions are not particularly limited. According to the third detection method of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

In the third detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the third detection method of the present invention is, for example, a method for detecting a binding-enhancing antibody for the Spike protein of SARS-CoV-2 to ACE2 or a method for detecting a binding-enhancing antibody for SARS-CoV-2 to ACE2. You can also do it.

According to the third detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody can be detected.

In the third detection method of the present invention, for example, the presence or absence of the antibody may be detected, or the amount of the antibody may be detected. In the former case, the third detection method of the present invention can be, for example, a qualitative analysis method or a qualitative measurement method. Further, in the latter case, the second detection method of the present invention can be, for example, a quantitative analysis method or a quantitative measurement method. The detection can also be, for example, inspection or detection.

In the third detection method of the present invention, the detection target in the detection step binds to the Sw protein, and in the Sw protein, the 30th, 32nd, 64th, 65th, and 66th positions of the Ss protein, An antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. Is. For the mutant protein, the description of the amino acid sequence of (Sm1) in the mutant protein of the present invention can be incorporated. In the detection step, the detection target antibody may be one type or two or more types, that is, only antibodies having the same epitope may be detected, or antibodies having different epitopes may be detected together. good.

In the detection step, the biological sample is brought into contact with the Sw protein, and the binding or the amount of binding between the antibody contained in the biological sample and the Sw protein is detected (first detection step). Further, in the detection step, the biological sample is brought into contact with the mutant protein, and the binding or the amount of binding between the antibody contained in the biological sample and the mutant protein is detected (second detection step). The binding between the antibody contained in the biological sample and the Sw protein or the mutant protein can be measured by, for example, an ELISA method using an antigen-antibody reaction, flow cytometry, or the like.

Next, in the detection step, by comparing (evaluating) the results obtained in the first detection step and the second detection step, the antibody binding to the Sw protein does not recognize the mutant protein. Can be evaluated (comparison process or evaluation process). Specifically, when the binding is used as an index, in the detection step, the antibody contained in the biological sample is bound to the Sw protein, and the antibody contained in the biological sample and the mutant protein are associated with each other. If no binding is found, the antibody contained in the biological sample does not recognize the mutant protein, that is, the biological sample can be evaluated as containing an infection-enhancing antibody. On the other hand, in the detection step, when the antibody contained in the biological sample does not bind to the Sw protein, or when the antibody contained in the biological sample binds to the Sw protein, the biological sample is bound. When the antibody contained in the above is bound to the mutant protein, it can be evaluated that the antibody contained in the biological sample recognizes the mutant protein, that is, the biological sample does not contain the infection-enhancing antibody. When the binding amount is used as an index, in the detection step, the binding amount of the antibody contained in the biological sample and the mutant protein is compared with the binding amount of the antibody contained in the biological sample and the Sw protein. Then, when it is significantly decreased (for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94). %, 95%, 96%, 97%, 98%, or 99% or more), the antibody contained in the biological sample does not recognize the mutant protein, that is, the biological sample is an infection-enhancing antibody. Can be evaluated as including. When the binding amount of the antibody contained in the biological sample and the mutant protein is not significantly different from the binding amount of the antibody contained in the biological sample and the Sw protein, the antibody contained in the biological sample is said to be said. It can be evaluated that the mutant protein is recognized, that is, the biological sample does not contain the infection-enhancing antibody.

In the detection step, the N-terminal domain (NTD) may be used instead of the Sw protein. Further, in the detection step, the position of the amino acid recognized by the antibody can be changed by changing the position of the mutant amino acid in the mutant protein, whereby the epitope can be specified.

In the third detection method of the present invention, for example, it may be further evaluated whether the antibody in the biological sample is an antibody that enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein, for example. good. The enhancement of the binding activity can be measured, for example, by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the biological sample. The measurement can be performed, for example, in the same manner as in Example 1 described later.

<Fourth detection method>
The present invention provides a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2. The method for detecting the infection-enhancing antibody of SARS-CoV-2 of the present invention competes with the reference antibody for the binding of wild-type SARS-CoV-2 to the Spike protein (Sw protein) in the biological sample of the subject. Including a detection step of detecting an antibody, the reference antibody recognizes the Sw protein, and in the Sw protein, the 30th and 32nd positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. , 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217, the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids. Does not recognize mutant proteins that have mutations in. The fourth detection method of the present invention is characterized in that, in the detection step, a competitive antibody that competes with the reference antibody is detected as an infection-enhancing antibody, that is, the fourth marker is used as an index, and the like. The process and conditions of the above are not particularly limited. According to the fourth detection method of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

In the fourth detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody is used as an index. In the infection-enhancing antibody, it is considered that the antibody exhibits the function of enhancing the binding ability of SARS-CoV-2's Spike protein to ACE2, thereby producing the functionality of enhancing infection. Therefore, the fourth detection method of the present invention is, for example, a method for detecting a binding-enhancing antibody for the Spike protein of SARS-CoV-2 to ACE2 or a method for detecting a binding-enhancing antibody for SARS-CoV-2 to ACE2. You can also do it.

According to the fourth detection method of the present invention, the ACE2 binding enhancing antibody and / or the infection enhancing antibody can be detected.

In the fourth detection method of the present invention, for example, the presence or absence of the antibody may be detected, or the amount of the antibody may be detected. In the former case, the fourth detection method of the present invention can be, for example, a qualitative analysis method or a qualitative measurement method. Further, in the latter case, the fourth detection method of the present invention can be, for example, a quantitative analysis method or a quantitative measurement method. The detection can also be, for example, inspection or detection.

In the fourth detection method of the present invention, the detection target in the detection step is an antibody that competes with the reference antibody for binding to the Sw protein, and the reference antibody is an antibody that does not recognize the mutant protein. For example, the above description of the reference antibody can be incorporated. In the detection step, the detection target antibody may be one type or two or more types, that is, only antibodies having the same epitope may be detected, or antibodies having different epitopes may be detected together. good.

The detection step includes a detection step of detecting a competing antibody that competes with a reference antibody for binding to the Sw protein in a biological sample of the subject. Specifically, in the detection step, the Sw protein is brought into contact with the biological sample, and the binding or binding amount of the Sw protein to the antibody in the biological sample is detected (first detection step). Next, in the detection step, the Sw protein and the biological sample are brought into contact with each other in the presence of the reference antibody, and the binding or binding amount of the Sw protein to the antibody in the biological sample is detected (second). Detection process). The binding between the antibody contained in the biological sample and the Sw protein can be measured by, for example, an ELISA method using an antigen-antibody reaction, flow cytometry, or the like.

Next, in the detection step, by comparing (evaluating) the results obtained in the first detection step and the second detection step, the antibody contained in the biological sample binds to the Sw protein. Can be evaluated as to whether it is an antibody that competes with the reference antibody (comparison step or evaluation step). Specifically, when the binding is used as an index, in the detection step, the antibody contained in the biological sample is bound to the Sw protein, and in the presence of the reference antibody, the antibody is contained in the biological sample. If no binding between the antibody and the Sw protein is observed, the antibody contained in the biological sample is an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample is an infection-enhancing antibody. Can be evaluated as including. On the other hand, in the detection step, when the antibody contained in the biological sample is not bound to the Sw protein, or the antibody contained in the biological sample is bound to the Sw protein, and the reference antibody is found. If the antibody contained in the biological sample binds to the Sw protein even in the presence of, the antibody contained in the biological sample is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, , It can be evaluated that the biological sample does not contain the infection-enhancing antibody. When the binding amount is used as an index, in the detection step, the antibody contained in the biological sample and the Sw protein in the presence of the reference antibody are bound to the antibody contained in the biological sample and the Sw protein. (For example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%) when compared with the binding amount of , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more), the antibody contained in the biological sample is the reference antibody for binding to the Sw protein. It can be evaluated that it is a competing antibody, that is, the biological sample contains an infection-enhancing antibody. On the other hand, when the amount of binding between the antibody contained in the biological sample and the Sw protein in the presence of the reference antibody is not significantly different from the amount of binding between the antibody contained in the biological sample and the Sw protein, the biological sample It can be evaluated that the antibody contained in is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample does not contain an infection-enhancing antibody.

In the detection step, the biological sample may be brought into contact with the Sw protein, and then the obtained mixture may be brought into contact with the reference antibody to detect whether the reference antibody binds to the Sw protein. In this case, when the binding of the reference antibody to the Sw protein is not detected in the detection step, or when the binding of the reference antibody to the Sw protein is reduced as compared with the absence of the biological sample, the above. It can be evaluated that the antibody in the biological sample is an antibody that competes with the reference antibody for binding to the Sw protein, that is, the biological sample contains an infection-enhancing antibody. On the other hand, in the detection step, when the binding of the reference antibody to the Sw protein is detected, the antibody in the biological sample is not an antibody that competes with the reference antibody for binding to the Sw protein, that is, the said. It can be evaluated that the biological sample does not contain the infection-enhancing antibody.

In the detection step, the N-terminal domain (NTD) may be used instead of the Sw protein.

In the fourth detection method of the present invention, for example, it may be further evaluated whether the antibody in the biological sample is an antibody that enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein, for example. good. The enhancement of the binding activity can be measured, for example, by detecting the binding of the Sw protein to the ACE2 protein in the coexistence of the Sw protein, the ACE2 protein, and the biological sample. The measurement can be performed, for example, in the same manner as in Example 1 described later.

<Detection kit>
The present invention provides an antibody detection kit that can be an index for enhancing infection with SARS-CoV-2. The SARS-CoV-2 infection-enhancing antibody detection kit of the present invention is an antibody that enhances the binding of wild-type SARS-CoV-2 Spike protein (Sw protein) to ACE2 protein in an Fc receptor-independent manner. Includes detection reagents for. The detection kit of the present invention is characterized by including a detection reagent for the ACE2 binding enhancing antibody, and other configurations and conditions are not particularly limited. According to the kit of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected. Specific examples of the detection kit of the present invention include the first and second detection kits described later. When the detection kit of the present invention has one component, the detection kit of the present invention can be referred to as, for example, a detection reagent. The detection kit of the present invention can also be referred to as a test kit or a diagnostic kit.

<First detection kit>
The present invention provides an antibody detection kit that can be an index for enhancing infection with SARS-CoV-2. The SARS-CoV-2 infection-enhancing antibody detection kit of the present invention contains the Spike protein (Sw protein) of wild-type SARS-CoV-2 or its N-terminal domain, and the reference SARS-CoV-2 in the Sw protein. Consists of 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th positions in the Spike protein (Ss protein, SEQ ID NO: 1). Includes a mutant protein in which a mutation has been introduced into an amino acid corresponding to at least one amino acid residue selected from the group or its N-terminal domain. The first detection kit of the present invention is characterized by containing the Sw protein and the mutant protein, and other configurations and conditions are not particularly limited. According to the first kit of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

The first detection kit of the present invention can be suitably used, for example, for carrying out the first detection method and the third detection method of the present invention.

The first kit of the present invention comprises the Sw protein or its NTD and the mutant protein or its NTD. The position of the mutant protein or the mutant amino acid in the NTD thereof can be appropriately set according to, for example, the epitope of the antibody to be detected in the first kit of the present invention. The position of the mutated amino acid in the mutated protein or its NTD can be referred to, for example, the description of the amino acid sequence of (Sm1) in the mutated protein of the present invention. As the mutant protein, the mutant protein of the present invention may be used. In the mutant protein or its NTD, the position of the mutant amino acid and / the type of mutation may be one or more. By having a plurality of positions and / types of mutations of the mutant amino acid, the first kit of the present invention can simultaneously detect, for example, an antibody that recognizes different epitopes.

In the first kit of the present invention, the Sw protein or its NTD may be an isolated Sw protein or its NTD, a host cell expressing the Sw protein or its NTD, or the Sw protein or its NTD. It may be an Fc fusion protein in which NTD is fused with the Fc region of an antibody. When the isolated Sw protein or its NTD is used, the Sw protein or its NTD may be immobilized on a carrier. Fixation to the carrier is, for example, direct or indirect fixation. The direct fixation is a covalent fixation of the Sw protein or its NTD to the carrier. In the indirect fixation, the Sw protein or its NTD and a carrier are bound via, for example, a molecule capable of binding to the Sw protein or its NTD immobilized on the carrier (for example, an antibody or an aptamer). Means the state of being. The Sw protein or a molecule capable of binding to the NTD thereof binds to, for example, a site that does not compete with the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody in the binding to the Sw protein or its NTD, and preferably said. Sw protein binds to domains other than NTD, namely RBD and / or S2. When the carrier and the Sw protein or its NTD are indirectly immobilized, the carrier and the Sw protein or its NTD are, for example, simultaneously or simultaneously or simultaneously prior to mixing either one with the measurement target. Contact after mixing. The carrier can incorporate the above description.

In the first kit of the present invention, the mutant protein or its NTD may be an isolated mutant protein or its NTD, a host cell expressing the mutant protein or its NTD, or the mutant protein or its NTD. It may be an Fc fusion protein in which NTD is fused with the Fc region of an antibody. When the isolated mutant protein or NTD thereof is used, the mutant protein or NTD thereof may be immobilized on a carrier. The carrier can incorporate the above description.

In the first kit of the present invention, the Sw protein or its NTD and / or the mutant protein or its NTD may be labeled, for example. In this case, the label may be, for example, an enzyme such as alkaline phosphatase (ALP) or luciferase; a particle; a fluorescent substance such as a fluorescent protein or a fluorescent dye; a dye such as a dye substance; Luminescent substances; electron donors; enzyme color-developing substrates; haptens such as DNP (dinitrophenol) and TNP (trinitrophenol), vitamins such as biotin; and the like. When the label is an enzyme, the first kit of the present invention preferably further comprises a substrate for the enzyme. The label is preferably directly or indirectly bound to the Sw protein or its NTD and / or the mutant protein or its NTD.

The first kit of the present invention may contain, for example, a reagent for detecting the Sw protein or its NTD and / or an antibody bound to the mutant protein or its NTD. In this case, examples of the reagent include an antibody that recognizes the antibody, an aptamer, and the like (secondary antibody and the like). It is preferable that the secondary antibody or the like recognizes, for example, a constant region or an Fc region of the antibody. Further, when the Sw protein or its NTD and / or the mutant protein or its NTD is not labeled, the secondary antibody or the like is preferably labeled. Thereby, the first kit of the present invention can easily detect the binding of the Sw protein or its NTD to the antibody and / or the binding of the mutant protein or its NTD to the antibody.

The first kit of the present invention may further include, for example, other components. Examples of the component include the carrier, the substrate of the enzyme, a buffer solution, a washing solution, an instruction manual and the like. Each reagent in the first kit of the present invention may be contained in a separate container, or may be contained in the same container mixed or unmixed. In the latter case, the first kit of the present invention can also be said to be the first detection reagent. When the first kit of the present invention contains the substrate, the substrate is contained, for example, in a container separate from the Sw protein or its NTD and / or the mutant protein or its NTD.

The first kit of the present invention preferably comprises, for example, a carrier on which an antibody capable of binding to the RBD or S2 region of the Sw protein is immobilized, and the Sw protein. Further, the first kit of the present invention preferably further includes, for example, a detection reagent capable of detecting an antibody bound to a Sw protein derived from a measurement target (for example, a biological sample). Examples of the detection reagent include an antibody against the antibody, that is, a secondary antibody. The secondary antibody is preferably labeled.

The first kit of the present invention may contain an ACE2 protein in place of the mutant protein. In this case, the first kit of the present invention uses the binding of the ACE2 binding enhancing antibody contained in the sample to the Sw protein or its NTD, and the binding of the Sw protein or its NTD to the ACE2 protein. To detect. The ACE2 protein is preferably bound directly or indirectly to the label.

<Second detection kit>
The present invention provides an antibody detection kit that can be an index for enhancing infection with SARS-CoV-2. The SARS-CoV-2 infection-enhancing antibody detection kit of the present invention contains a wild-type SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain, and a reference antibody, wherein the reference antibody is described above. In the Sw protein, in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions, An antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 214, 216, and 217. The second detection kit of the present invention is characterized by containing the Sw protein and the reference antibody, and other configurations and conditions are not particularly limited. According to the second kit of the present invention, an antibody capable of enhancing the infection of SARS2 can be detected.

The second detection kit of the present invention can be suitably used, for example, for carrying out the second detection method and the fourth detection method of the present invention.

In the second kit of the present invention, the Sw protein or its NTD may be an isolated Sw protein or its NTD, a host cell expressing the Sw protein or its NTD, or the Sw protein or its NTD. It may be an Fc fusion protein in which NTD is fused with the Fc region of an antibody. When the isolated Sw protein or its NTD is used, the Sw protein or its NTD may be immobilized on a carrier. Fixation to the carrier is, for example, direct or indirect fixation. The direct fixation is a covalent fixation of the Sw protein or its NTD to the carrier. In the indirect fixation, the Sw protein or its NTD and a carrier are bound via, for example, a molecule capable of binding to the Sw protein or its NTD immobilized on the carrier (for example, an antibody or an aptamer). Means the state of being. The Sw protein or a molecule capable of binding to the NTD thereof binds to, for example, a site that does not compete with the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody in the binding to the Sw protein or its NTD, and preferably said. Sw protein binds to domains other than NTD, namely RBD and / or S2. The carrier can incorporate the above description.

In the second kit of the present invention, the position of the amino acid recognized by the reference antibody can be referred to, for example, the description of the reference antibody in the mutant protein of the present invention. In the reference antibody, the position of the amino acid to be recognized may be one or a plurality. By setting a plurality of positions of the amino acids to be recognized, the second kit of the present invention can detect, for example, antibodies recognizing different epitopes at the same time.

In the second kit of the present invention, the reference antibody may be an isolated reference antibody or a host cell expressing the reference antibody. When the reference antibody is used, the reference antibody may be immobilized on a carrier. The carrier can incorporate the above description. As the reference antibody, the antigen-binding fragment may be used.

In the second kit of the present invention, the Sw protein or its NTD and / or the reference antibody may be labeled, for example. In this case, the label may be, for example, an enzyme such as alkaline phosphatase (ALP) or luciferase; a particle; a fluorescent substance such as a fluorescent protein or a fluorescent dye; a dye such as a dye substance; Luminescent substances; electron donors; enzyme color-developing substrates; haptens such as DNP (dinitrophenol) and TNP (trinitrophenol), vitamins such as biotin; and the like. When the label is an enzyme, the second kit of the present invention preferably further comprises a substrate for the enzyme. The label is preferably directly or indirectly bound to the Sw protein or its NTD and / or the reference antibody.

The second kit of the present invention may contain, for example, an antibody bound to the Sw protein or its NTD, and / or a reagent for detecting the reference antibody. In this case, examples of the reagent include an antibody that recognizes the antibody, an aptamer, and the like (secondary antibody and the like). It is preferable that the secondary antibody or the like recognizes, for example, the constant region or Fc region of the antibody or the reference antibody. When the Sw protein or its NTD and / or the reference antibody is not labeled, the secondary antibody or the like is preferably labeled. Thereby, the second kit of the present invention binds the Sw protein or its NTD to the antibody and / or binds the Sw protein or its NTD to the reference antibody, or competes with the antibody and the reference antibody. Can be easily detected.

The second kit of the present invention may further include, for example, other components. Examples of the component include the carrier, the substrate of the enzyme, a buffer solution, a washing solution, an instruction manual and the like. Each reagent in the second kit of the present invention may be contained in a separate container, or may be contained in the same container mixed or unmixed. In the latter case, the first kit of the present invention can also be referred to as a second detection reagent. When the second kit of the present invention contains the substrate, the substrate is contained, for example, in a container separate from the Sw protein or its NTD and / or the mutant protein or its NTD.

The second kit of the present invention preferably comprises, for example, a carrier on which an antibody capable of binding to RBD or S2 of the Sw protein is immobilized, the Sw protein, and the reference antibody. Further, it is preferable that the second kit of the present invention further includes, for example, a detection reagent capable of detecting an antibody bound to a Sw protein derived from a measurement target (for example, a biological sample). Examples of the detection reagent include an antibody against the antibody, that is, a secondary antibody. The secondary antibody is preferably labeled.

The second kit of the present invention may not include, for example, the Sw protein or its NTD. In this case, the first kit of the present invention can also be said to be the reference antibody for use in a method for detecting an antibody that can be an index for enhancing infection with SARS-CoV-2.

The second kit of the present invention may contain an ACE2 protein in place of the reference antibody. In this case, the second kit of the present invention uses the binding of the ACE2 binding enhancing antibody contained in the sample to the Sw protein or its NTD, and the binding of the Sw protein or its NTD to the ACE2 protein. To detect. The ACE2 protein is preferably bound directly or indirectly to the label.

<Test method>
The present invention provides a method for testing the susceptibility (potential for morbidity) of SARS-CoV-2. The method of the present invention is a method for testing the susceptibility to SARS-CoV-2, and is a Fc receptor-independent Fc receptor-independent wild-type SARS-CoV-2 Spike protein in a biological sample of a subject. A measurement step of measuring an antibody that enhances the binding of (Sw protein) to the ACE2 protein is included. The test method of the present invention is characterized in that the ACE2-binding enhancing antibody is measured in the measurement step, and other configurations and conditions are not particularly limited. According to the test method of the present invention, the susceptibility to SARS2 infection can be tested. Specific examples of the test method of the present invention include the first to fourth test methods of the present invention described later.

<First test method>
The present invention provides a method for testing the susceptibility (potential for morbidity) of SARS-CoV-2. The method of the present invention is a test method for susceptibility to SARS-CoV-2, and is a reference SARS in the Spike protein (Sw protein) of wild-type SARS-CoV-2 in a biological sample of a subject. -In the Spike protein (Ss protein, SEQ ID NO: 1) of CoV-2, 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, And a measurement step of measuring an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acid at position 217. The first test method of the present invention is characterized in that, in the measurement step, an antibody that recognizes an amino acid corresponding to an amino acid residue at a predetermined position in the Ss protein is measured as an infection-enhancing antibody, and other The process and conditions are not particularly limited. According to the first test method of the present invention, the susceptibility to infection of SARS2 can be tested.

In the first test method of the present invention, the susceptibility to SARS2 infection is tested (for example, diagnosis, evaluation, determination, prediction, etc.) by using the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody as an index. Or judge). Therefore, the first test method of the present invention can be, for example, a test, diagnosis, evaluation, determination, prediction, or determination method for the possibility of SARS2 morbidity. In addition, it is presumed that the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody also contributes to the aggravation of SARS2-infected persons. Therefore, the first test method of the present invention is, for example, a test, diagnosis, evaluation, judgment, prediction, or judgment method for aggravation or possibility of aggravation of SARS2, or aggravation at the time of SARS2 infection or in an infected person. Alternatively, it can be a test, diagnosis, evaluation, judgment, prediction, or judgment method for the possibility of aggravation. The first test method of the present invention excludes, for example, a doctor's judgment step.

In the first test method of the present invention, the susceptibility to infection is, for example, the possibility of infection, the risk of infection, the probability of infection, the susceptibility to morbidity, the possibility of morbidity, the risk of morbidity, and the like. It can also be called the probability of illness.

In the first test method of the present invention, the measurement step can be carried out in the same manner as the detection step in the first detection method of the present invention. For example, "detection" is read as "measurement" and the description thereof is incorporated. can.

In the first test method of the present invention, it is preferable to measure the amount of the antibody as the antibody in the measurement step. In this case, the first test method of the present invention further tests the possibility (possibility of morbidity) of the subject to be infected with SARS2 by comparing the amount of the antibody with the first threshold value. The first test step to be performed may be included. The first threshold value is a threshold value set based on the amount of the antibody in the biological sample of a healthy person and / or the amount of the antibody in the biological sample of a person (infected person) affected by SARS2. The first threshold value may be, for example, the amount of the antibody after treatment (for example, immediately after treatment) of the same SARS2-infected person.

The first threshold value can be obtained, for example, by using a biological sample isolated from a healthy person and / or a SARS2-infected person (hereinafter, also referred to as "reference biological sample"). Further, in the case of evaluation of prognosis, for example, a reference biological sample isolated after treatment from the same subject may be used. The first threshold value may be measured at the same time as the test biological sample of the subject, or may be measured in advance, for example. In the latter case, for example, it is not necessary to obtain the first threshold value every time the test biological sample of the subject is measured, which is preferable. It is preferable that the test biological sample of the subject and the reference biological sample are collected under the same conditions, and the amount of the antibody is measured under the same conditions.

In the first test step, the method for evaluating the susceptibility of a subject to SARS2 infection is not particularly limited, and can be appropriately determined depending on the type of the first threshold value. As a specific example, when the amount of the antibody in the test biological sample of the subject is significantly higher than the amount of the antibody in the reference biological sample of the healthy subject, the antibody in the reference biological sample of the SARS2-infected person. If the amount is the same (if there is no significant difference) and / or if it is significantly higher than the amount of the antibody in the reference biological sample of the SARS2-infected person, the subject may be infected with SARS2. It can be evaluated that there is or is likely to be infected. When the amount of the antibody in the test biological sample of the subject is the same as the amount of the antibody in the reference biological sample of the healthy subject (when there is no significant difference), the said in the reference biological sample of the healthy subject. If the amount of antibody is significantly lower and / or significantly lower than the amount of said antibody in the reference biological sample of the SARS2-infected person, the subject is unlikely or infected with SARS2. It can be evaluated that the possibility of doing so is low. Although the evaluation method in the first test step has been described by taking the possibility of infection as an example, the evaluation method may be carried out as a method for evaluating the possibility of aggravation. In this case, the possibility of infection can be said to be the possibility of aggravation. Further, in the first test step, by comparing the amount of the antibody in the test biological sample of the subject with the amount of the antibody in the reference biological sample of the SARS2-infected person for each severity, SARS2 The severity of the disease can be evaluated. Specifically, when the test biological sample of the subject has an expression level similar to that of the reference biological sample of any severity (when there is no significant difference), the subject , It can be evaluated that there is a possibility of the severity.

When evaluating the prognosis state in the first test step, for example, the evaluation may be made in the same manner as described above, or as the first threshold value, the antibody in the reference biological sample after treatment of the same subject. It can also be evaluated using the amount of. As a specific example, when the amount of the antibody in the test biological sample of the subject is significantly higher than the first threshold value, the subject may relapse, worsen, or become severe after the treatment. It can be evaluated that there is a possibility. Further, when the amount of the antibody in the test biological sample of the subject is the same as the first threshold value (when there is no significant difference) and / or when it is significantly lower than the first threshold value. The subject can be evaluated as having no or low possibility of recurrence, exacerbation or aggravation after the treatment.

In the present invention, for example, a biological sample of the same subject may be collected over time and the amount of the antibody in the biological sample may be compared. As a result, in the first test step, for example, if the amount of the antibody increases with time, it is possible to determine that the possibility of being infected with SARS2 has increased, the disease has become more severe, and the like. If the amount of the antibody decreases, it is possible to determine that the possibility of being infected with SARS2 has decreased or the disease has become milder.

The first test method of the present invention may further include a third measurement step of measuring the neutralizing antibody of the Sw protein with respect to the biological sample of the subject. The neutralizing antibody is, for example, an antibody that binds to the receptor binding domain (RBD) of the Sw protein. In the third measurement step, it is preferable to measure the amount of the neutralizing antibody as the neutralizing antibody. The neutralizing antibody can be measured using, for example, an isolated RBD or a host cell expressing RBD, and can be carried out in the same manner as in Example 1 described later.

When the first test method of the present invention includes the third measurement step, the first test method of the present invention measures the test by comparing the amount of the neutralizing antibody with the second threshold value. It is preferable to include a second test step of testing the possibility of a person being infected with SARS-CoV-2. In this case, the second test step is preferably carried out in combination with the first test step. The second threshold is the amount of the neutralizing antibody in the biological sample of the person affected by SARS2 (infected person) and / or the above-mentioned in the biological sample of the person who recovered after the disease (infection) of SARS2 (recovered person of SARS2). It is a threshold set based on the amount of neutralizing antibody. The person who has recovered from SARS2 is preferably a person who has recovered from infection with SARS2 within 1, 2, 3, or 6 months.

In the second test step, the method for evaluating the susceptibility of the subject to SARS2 infection is not particularly limited, and can be appropriately determined depending on the type of the second threshold value. As a specific example, when the amount of the antibody in the test biological sample of the subject is significantly higher than the amount of the antibody in the reference biological sample of the SARS2-infected person, the said in the reference biological sample of the SARS2 recoverer. If the amount of antibody is the same (if there is no significant difference) and / or if it is significantly higher than the amount of the antibody in the reference biological sample of the SARS2 recoverer, the subject may be infected with SARS2. It can be evaluated that there is no or the possibility of infection is low. When the amount of the antibody in the test biological sample of the subject is the same as the amount of the antibody in the reference biological sample of the SARS2-infected person (when there is no significant difference), the reference biological sample of the SARS2-infected person. The subject may be infected with SARS2 if it is significantly lower than the amount of said antibody in and / or if it is significantly lower than the amount of said antibody in the reference biological sample of the SARS2 recoverer. Or it can be evaluated that there is a high possibility of infection. Therefore, it is preferable to carry out the evaluation in the above-mentioned first test step for the subject who is evaluated to be or highly likely to be infected with SARS2. Although the evaluation method in the second test step has been described by taking the possibility of infection as an example, the evaluation method may be carried out as a method for evaluating the possibility of aggravation. In this case, the possibility of infection can be said to be the possibility of aggravation.

When the first test method of the present invention includes the third measurement step, the first test method of the present invention is obtained by the amount of the antibody obtained in the measurement step and the third measurement step. The evaluation may be performed based on the ratio with the amount of the neutralizing antibody. In this case, the first test method of the present invention is an antibody amount ratio (S / N) of the amount of antibody (S) in the measurement step and the amount of neutralizing antibody (N) in the third measurement step. A third test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the antibody amount ratio with the third threshold value is included. The third threshold was recovered, for example, after the antibody amount ratio in the biological sample of a healthy subject, the antibody amount ratio in the biological sample of a person suffering from SARS-CoV-2, and / or the morbidity of SARS-CoV-2. It is a threshold value set based on the antibody amount ratio in a biological sample of a person. The person who has recovered from SARS2 is preferably a person who has recovered from infection with SARS2 within 1, 2, 3, or 6 months.

In the third test step, the method for evaluating the susceptibility of the subject to SARS2 infection is not particularly limited, and can be appropriately determined depending on the type of the third threshold value. As a specific example, when the antibody amount ratio (S / N) in the test biological sample of the subject is significantly lower than the antibody amount ratio (S / N) in the reference biological sample of the SARS2-infected person. When it is the same as the antibody amount ratio (S / N) in the reference biological sample of the SARS2 recoverer (when there is no significant difference) and / or, the antibody amount ratio (S / N) in the reference biological sample of the SARS2 recoverer. ), It can be evaluated that the subject is unlikely to be infected with SARS2 or is unlikely to be infected with SARS2. Further, when the antibody amount ratio (S / N) in the test biological sample of the subject is significantly higher than the antibody amount ratio (S / N) in the reference biological sample of the healthy person, the healthy person When it is the same as the antibody amount ratio (S / N) in the reference biological sample of the above (when there is no significant difference) and / or, it is more significant than the antibody amount ratio (S / N) in the reference biological sample of the SARS2-infected person. If it is high, the subject can be evaluated as having a possibility of being infected with SARS2 or having a high possibility of being infected with SARS2. Although the evaluation method in the third test step has been described by taking the possibility of infection as an example, the evaluation method may be carried out as a method for evaluating the possibility of aggravation. In this case, the possibility of infection can be said to be the possibility of aggravation.

According to the first test method of the present invention, for example, in the first to third test steps, a subject who is evaluated to have a possibility or a high possibility of being infected with SARS2 may become severely ill. A step (administration step) of administering a therapeutic agent for SARS2 to a subject who is evaluated as having or is likely to be present may be further included. In this case, the test method of the present invention can also be referred to as a SARS2 test and treatment method. The therapeutic agent is, for example, remdecibir ((2S) -2-{(2R, 3S, 4R, 5R)-[5- (4-Aminopyrrolo [2,1-f] [1,2,4] triazin-7]. -yl) -5-cyano-3,4-dihydroxy-tetrahydro-furan-2-ylmethoxy] phenoxy- (S) -phosphorylamino} propionic acid 2-ethyl-butyl ester), favipiravir (6-fluoro-3-hydroxypyrazine-) 2-carboxamide), dexamethasone, anti-SARS2 antibodies such as REGN-COV2, protease inhibitors such as nafamostat and nelfinavir. For the therapeutic agent for SARS2, refer to, for example, the 3rd edition (Ministry of Health, Labor and Welfare) (https://www.mhlw.go.jp/content/000668291.pdf) of the guideline for the treatment of new coronavirus infection (COVID-19). can. For the administration conditions of the therapeutic agent in the administration step, for example, the usual administration conditions of each therapeutic agent can be referred to.

<Second test method>
The present invention provides a method for testing the susceptibility (potential for morbidity) of SARS-CoV-2. The method of the present invention is a method for testing the susceptibility to infection with SARS-CoV-2, and refers to the binding of wild-type SARS-CoV-2 to the Spike protein (Sw protein) of a biological sample of a subject. The reference antibody comprises a measurement step of measuring a competing antibody that competes with the reference antibody, wherein the reference antibody is located at positions 30 and 32 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. Amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. It is an antibody that recognizes. The second test method of the present invention is characterized in that, in the measurement step, a competitive antibody that competes with the reference antibody is measured as an infection-enhancing antibody, and other steps and conditions are not particularly limited. According to the second test method of the present invention, the susceptibility to infection with SARS2 can be tested.

In the second test method of the present invention, the susceptibility to SARS2 infection is tested (for example, diagnosis, evaluation, determination, prediction, etc.) by using the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody as an index. Or judge). Therefore, the second test method of the present invention can be, for example, a test, diagnosis, evaluation, determination, prediction, or determination method for the possibility of SARS2 morbidity. In addition, it is presumed that the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody also contributes to the aggravation of SARS2-infected persons. Therefore, the second test method of the present invention is, for example, a test, diagnosis, evaluation, judgment, prediction, or judgment method for aggravation or possibility of aggravation of SARS2, or aggravation at the time of SARS2 infection or in an infected person. Alternatively, it can be a test, diagnosis, evaluation, judgment, prediction, or judgment method for the possibility of aggravation. The second test method of the present invention excludes, for example, a doctor's judgment step.

In the second test method of the present invention, the measurement step can be carried out in the same manner as the detection step in the second detection method of the present invention. For example, "detection" is read as "measurement" and the description thereof is incorporated. can.

In the second test method of the present invention, the evaluation of the subject using the results obtained in the measurement step can be carried out in the same manner as in the first test method of the present invention, and the description thereof can be incorporated. ..

The second test method of the present invention, for example, in the first to third test steps, a subject who is evaluated to have a possibility or a high possibility of being infected with SARS2, has a possibility of becoming severe. A step (administration step) of administering a therapeutic agent for SARS2 to a subject who is evaluated as having or is likely to be present may be further included. In this case, the test method of the present invention can also be referred to as a SARS2 test and treatment method.

<Third test method>
The present invention provides a method for testing the susceptibility (potential for morbidity) of SARS-CoV-2. The method of the present invention is a test method for susceptibility to SARS-CoV-2.
The biological sample of the subject binds to the Spike protein (Sw protein) of wild-type SARS-CoV-2, and in the Sw protein, the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. At least one amino acid residue selected from the group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. A measurement step of measuring an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the group is included. The third test method of the present invention is characterized in that an antibody that binds to the Sw protein and does not recognize the mutant protein is measured as an infection-enhancing antibody in the measurement step, and the other steps and conditions are as follows. , There are no particular restrictions. According to the third test method of the present invention, the susceptibility to infection with SARS2 can be tested.

In the third test method of the present invention, the susceptibility to SARS2 infection is tested (for example, diagnosis, evaluation, determination, prediction, etc.) by using the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody as an index. Or judge). Therefore, the third test method of the present invention can be, for example, a test, diagnosis, evaluation, determination, prediction, or determination method for the possibility of SARS2 morbidity. In addition, it is presumed that the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody also contributes to the aggravation of SARS2-infected persons. Therefore, the third test method of the present invention is, for example, a test, diagnosis, evaluation, judgment, prediction, or judgment method for aggravation or possibility of aggravation of SARS2, or aggravation at the time of SARS2 infection or in an infected person. Alternatively, it can be a test, diagnosis, evaluation, judgment, prediction, or judgment method for the possibility of aggravation. The third test method of the present invention excludes, for example, a doctor's judgment step.

In the third test method of the present invention, the measurement step can be carried out in the same manner as the detection step in the third detection method of the present invention. For example, "detection" is read as "measurement" and the description thereof is incorporated. can.

In the third test method of the present invention, the evaluation of the subject using the results obtained in the measurement step can be carried out in the same manner as in the first test method of the present invention, and the description thereof can be incorporated. ..

The third test method of the present invention may, for example, in the first to third test steps, a subject evaluated to be or highly likely to be infected with SARS2, to be severely ill. A step (administration step) of administering a therapeutic agent for SARS2 to a subject who is evaluated as having or is likely to be present may be further included. In this case, the test method of the present invention can also be referred to as a SARS2 test and treatment method.

<Fourth test method>
The present invention provides a method for testing the susceptibility (potential for morbidity) of SARS-CoV-2. The method of the present invention is a method for testing the susceptibility to infection with SARS-CoV-2, and refers to the binding of wild-type SARS-CoV-2 to the Spike protein (Sw protein) of a biological sample of a subject. The reference antibody recognizes the Sw protein, and in the Sw protein, the Spike protein of the reference SARS-CoV-2 (Ss protein, SEQ ID NO: 1) includes a measurement step of measuring a competing antibody that competes with the reference antibody. At least one selected from the group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. It does not recognize mutant proteins that have mutations in the amino acid corresponding to the amino acid residue. The fourth test method of the present invention is characterized in that, in the measurement step, a competitive antibody that competes with the reference antibody is measured as an infection-enhancing antibody, and other steps and conditions are not particularly limited. According to the fourth test method of the present invention, the susceptibility to infection with SARS2 can be tested.

In the fourth test method of the present invention, the susceptibility to SARS2 infection is tested (for example, diagnosis, evaluation, determination, prediction, etc.) by using the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody as an index. Or judge). Therefore, the fourth test method of the present invention can be, for example, a test, diagnosis, evaluation, determination, prediction, or determination method for the possibility of SARS2 morbidity. In addition, it is presumed that the ACE2 binding enhancing antibody and / or the antibody infection enhancing antibody also contributes to the aggravation of SARS2-infected persons. Therefore, the fourth test method of the present invention is, for example, a test, diagnosis, evaluation, judgment, prediction, or judgment method for aggravation or possibility of aggravation of SARS2, or aggravation at the time of SARS2 infection or in an infected person. Alternatively, it can be a test, diagnosis, evaluation, judgment, prediction, or judgment method for the possibility of aggravation. The fourth test method of the present invention excludes, for example, a doctor's judgment step.

In the fourth test method of the present invention, the measurement step can be carried out in the same manner as the detection step in the fourth detection method of the present invention. For example, "detection" is read as "measurement" and the description thereof is incorporated. can.

In the fourth test method of the present invention, the evaluation of the subject using the results obtained in the measurement step can be carried out in the same manner as in the first test method of the present invention, and the description thereof can be incorporated. ..

The fourth test method of the present invention, for example, in the first to third test steps, a subject who is evaluated to have a possibility of being infected with SARS2 or has a high possibility of being infected with SARS2, has a possibility of becoming severe. A step (administration step) of administering a therapeutic agent for SARS2 to a subject who is evaluated as having or is likely to be present may be further included. In this case, the test method of the present invention can also be referred to as a SARS2 test and treatment method.

<Method of suppressing the aggravation of SARS-CoV-2>
The present invention provides a method capable of suppressing the aggravation of SARS-CoV-2. The method for suppressing the aggravation of SARS-CoV-2 of the present invention (hereinafter, also referred to as "suppression method") has the possibility of enhancing the infection of SARS-CoV-2 of the subject from the biological sample of the subject. The test step includes a test step of testing and an administration step of administering a therapeutic agent for SARS-CoV-2 to a subject evaluated as having a possibility of enhancing infection in the test step. , It is carried out by the first to fourth test methods of the present invention. The suppression method of the present invention is characterized in that the test step is carried out by the first to fourth test methods of the present invention, and other steps and conditions are not particularly limited. According to the suppression method of the present invention, it is possible to suppress the aggravation of SARS2-infected persons.

The suppression method of the present invention is, for example, an evaluation and treatment method for the aggravation of SARS-CoV-2 because it is possible to evaluate whether the subject is a SARS-infected person and treat the subject. can.

In the present invention, "severe" means, for example, a condition in which a respirator needs to be worn.

In the suppression method of the present invention, the test step can be carried out in the same manner as the first to fourth test methods of the present invention. In the administration step, the evaluation that there is a possibility of enhancing the infection can be carried out in the same manner as the test step in the first to fourth test methods, for example.

In the administration step, the therapeutic agent for SARS2 is, for example, remdecibir ((2S) -2-{(2R, 3S, 4R, 5R)-[5- (4-Aminopyrrolo [2,1-f] [1,2]. , 4] triazin-7-yl) -5-cyano-3,4-dihydroxy-tetrahydro-furan-2-ylmethoxy] phenoxy- (S) -phosphorylamino} propionic acid 2-ethyl-butyl ester), favipiravir (6- Fluoro-3-hydroxypyrazine-2-carboxamide), dexamethasone, anti-SARS2 antibodies such as R EGN-COV2, protease inhibitors such as nafamostat and nelfinavir. For the therapeutic agent for SARS2, refer to, for example, the 3rd edition (Ministry of Health, Labor and Welfare) (https://www.mhlw.go.jp/content/000668291.pdf) of the guideline for the treatment of new coronavirus infection (COVID-19). can. For the administration conditions of the therapeutic agent in the administration step, for example, the usual administration conditions of each therapeutic agent can be referred to.

<Reducing agent for infection-enhancing antibody>
The present invention provides a reducing agent capable of reducing an infection-enhancing antibody. The present invention is an antibody reducing agent (hereinafter, also referred to as "reducing agent") that enhances the binding ability of SARS-CoV-2's Spike protein to angiotensin converting enzyme 2 (ACE2), and is a wild-type Spike protein (hereinafter, also referred to as "reducing agent"). Sw protein) or its N-terminal domain. The reducing agent of the present invention is characterized by containing the Sw protein or its N-terminal domain, and other configurations and conditions are not particularly limited. According to the reducing agent of the present invention, for example, the infection-enhancing antibody in blood can be reduced.

As described above, it is presumed that the infection-enhancing antibody causes ADE by enhancing the binding to ACE2. Therefore, the infection enhancing antibody reducing agent of the present invention can be said to be, for example, an ACE2 binding enhancing antibody reducing agent.

In the reducing agent of the present invention, the Sw protein and its N-terminal domain (NTD) may be an isolated protein or polypeptide, or may coexist with other substances.

In the reducing agent of the present invention, the Sw protein and NTD are retained or immobilized on a carrier, for example, because they can be easily separated from the target after being contacted with the target for reducing the infection-enhancing antibody. It is preferable to have. The carrier is preferably a carrier formed of a material that is insoluble or substantially insoluble in an aqueous solvent such as water or a buffer solution. Examples of the carrier include beads made of agarose, resin and the like; hollow fiber membranes, microfiltration membranes, ultrafiltration membranes, reverse osmosis membranes, filters such as non-woven fabrics; porous bodies; and the like. The Sw protein or NTD can be retained or immobilized on the carrier, for example, by attaching an N-terminal amino group or a C-terminal carboxyl group to the linker and attaching the other end of the linker to the carrier. When the carrier has a functional group, the Sw protein or NTD may be immobilized by reacting an N-terminal amino group or a C-terminal carboxyl group with the functional group.

The object is not particularly limited, and is, for example, a biological sample, preferably blood, and more preferably blood derived from a SARS2-infected person.

Since the reducing agent of the present invention contains the Sw protein or its NTD, it contains the binding site of the infection-enhancing antibody. Therefore, according to the reducing agent of the present invention, the infection-enhancing antibody in the object can be reduced by causing an antigen-antibody reaction with the infection-enhancing antibody.

<Method of reducing infection-enhancing antibody>
The present invention provides a method capable of reducing an infection-enhancing antibody. The method of the present invention is a method for reducing an antibody that enhances the binding ability of SARS-CoV-2's Spike protein to angiotensin converting enzyme 2 (ACE2) (hereinafter referred to as "reduction method"), and is a method for reducing an antibody, which comprises a biological sample and a biological sample. It comprises a reduction step of reducing the antibody in the biological sample by contacting it with the reducing agent of the present invention. The reduction method of the present invention is characterized in that the antibody in the biological sample is reduced by contacting the biological sample with the reducing agent containing the Sw protein or its NTD, and other steps and conditions are described. , There are no particular restrictions. According to the reduction method of the present invention, the infection-enhancing antibody in the biological sample can be reduced.

The reduction method of the present invention may include an acquisition step of acquiring a biological sample from a target prior to carrying out the reduction step. The acquisition step may be a step of acquiring a biological sample isolated from the subject. The method for obtaining the biological sample from the target can be appropriately determined according to the type of the biological sample. When the biological sample is blood, the acquisition step can be carried out using, for example, a blood collection device such as a syringe, a blood collection device, or the like.

In the reduction step, the contact between the biological sample and the reducing agent can be carried out by coexisting the biological sample and the reducing agent, and specifically, the biological sample and the reducing agent are mixed. Can be carried out by. When the biological sample is a solid sample, the biological sample and the solvent are contacted in advance prior to the reduction step, or in the reduction step, the solvent is also brought into contact with the biological sample and the reducing agent. Is preferable. Examples of the solvent include aqueous solvents such as water and a buffer solution. In the reduction step, the infection-enhancing antibody contained in the biological sample binds to the Sw protein or NTD in the reducing agent by the contact, so that the infection-enhancing antibody in the biological sample can be reduced.

The contact conditions in the reduction step are not particularly limited, and examples thereof include temperatures and conditions under which the biological sample is not denatured. As a specific example, the temperature at the time of contact is, for example, 0 to 37 ° C. The contact time is, for example, 1 to 2 hours.

In the reduction step, the amount ratio of the biological sample to the reducing agent is not particularly limited, and can be appropriately determined, for example, according to the amount of reduction of the target infection-enhancing antibody. In the reduction step, when the reduction amount of the infection enhancing antibody is increased, the amount ratio (D / S) of the reducing agent (D) to the biological sample (S) is set to be relatively large. On the other hand, when the reduction amount of the infection enhancing antibody is reduced in the reduction step, the amount ratio (D / S) of the reducing agent to the biological sample is set to be relatively small.

The reduction method of the present invention may include a separation step of separating the biological sample and the reducing agent after the reduction step. The separation method can be appropriately determined, for example, depending on the form of the reducing agent. As a specific example, when the reducing agent is held on a carrier, the separation method can be carried out by a known solid-liquid separation method using a filter, a porous body or the like.

The reduction method of the present invention may include an administration step of administering the biological sample after the reduction step to the subject from which the biological sample has been obtained. In this case, in the reduction method of the present invention, it is preferable that the separation step is carried out after the reduction step, and the obtained liquid fraction is administered to the subject as the biological sample.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the embodiments described in the Examples. Unless otherwise specified, commercially available reagents and kits were used according to the protocol.

[Example 1]
It was confirmed that the anti-NTD antibody of SARS-CoV-2 enhances the binding of SARS2 to ACE2 of S protein.

(1) Preparation of antibody sample The amino acid sequences of the heavy chain variable region and the light chain variable region of 100 kinds of antibodies that bind to the S protein of SARS2 described in References 9 and 10 below for the antibody that binds to SARS2. An antibody sample was prepared based on the above. Specifically, the gene encoding the heavy chain variable region and the light chain variable region of each antibody is a partial sequence (sequence) of the secretory human IgG1 constant region (Genbank Accession No .: ARA90394 (protein), KY432415 (mRNA)). The gene encoding the gene (SEQ ID NO: 110) or the gene encoding the partial sequence (SEQ ID NO: 112) of the human kappa constant region (Genbank Accession No .: CAC20459 (protein), AJ294735 (mRNA)) (SEQ ID NO: 113) ) And cloned into a plasmid vector (pCAGGS). As a result, expression vectors of 100 types of antibody samples that bind to the spike protein of SARS-CoV-2 were prepared. The clone name of each antibody is given according to the description in References 9 and 10 below.
Reference 9: Chi X. et.al., “A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2.” Science, 2020, vol. 369, pages 650-655, doi 10.1126 / science.abc6952
Reference 10: SJ, Zost. Et al. “Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein.” Nat. Med., 2020, vol. 26, No. 9, pages 1422-1427, doi: 10.1038 / s41591-020-0998-x.

Human IgG1 constant region (SEQ ID NO: 110)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Gene encoding the human IgG1 constant region (SEQ ID NO: 111)
5'--3'

Human κ chain constant region (SEQ ID NO: 112)
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKVYACEVTHQGLSSPVTKSFNRGEC

Gene encoding the human kappa chain constant region (SEQ ID NO: 113)
5'-GCtGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG-3 '

As the host cell of the expression vector, 293T cells (obtained from RIKEN BioResource Center) were used. First, the 24-well plates, the 293T cells were seeded such that the 2 × 10 ⁵ cells / 500μl / well. Next, for the introduction of the expression vector into the 293T cells, a PEI max solution in which a transfection reagent (PEI max, manufactured by Polyscience) was dissolved in purified water so as to be 2 mg / ml was used. Specifically, a mixed solution of 2 μl PEI max solution and 50 μl of serum-free medium (OPTI-MEM®, manufactured by GIBCO), 0.8 μg of each antibody sample expression vector, and 50 μl of no serum. After addition to the serum medium, the mixture was mixed to prepare a transfection reagent. Then, the transfection reagent was added to each well, and the expression vector was introduced into 293T cells. After the introduction, the 293T cells were seeded in DMEM medium supplemented with 10% FBS (manufactured by Biological Industries), penicillin (final concentration 100 U / ml) and streptomycin (final concentration 100 μg / ml), and seeded at 37 ° C., 5%. The cells were cultured for 2 days under the condition of _{CO 2.} After the culture, the culture supernatant was collected.

(2) Purification of antibody samples Each antibody sample in the collected culture supernatant was purified using protein A. Specifically, a sample obtained by diluting 100 μl of the culture supernatant with phosphate buffered saline (PBS) 10-fold was purified by affinity chromatograph using Profinia (manufactured by Bio Rad). In the affinity chromatograph, a column containing protein A (Bil-Scale mini UNO Sphere SUPrA Cartridege, manufactured by Bio Rad) was used. Then, the sample was put on the column, and each antibody in the collected culture supernatant was reacted with protein A in the column. The column after the reaction was washed once with PBS. Then, 0.1 mol / l Glycine-HCl (pH 2.8) was added, and the antibody binding to the S protein of SARS2 was recovered from the column.

(3) Preparation of SARS2 S protein-expressing cell Codon optimal for the gene (SEQ ID NO: 2) encoding the full-length sequence of SARS2 S protein (Ss protein (SEQ ID NO: 1)) instead of the gene encoding each antibody. The gene encoding the encoded Ss protein (SEQ ID NO: 114) and the NTD region of the Ss protein of SARS2 (GenBank Accession No .: NC_045512.2 amino acid sequence 14 to 333 (SEQ ID NO: 3)) (SEQ ID NO: 3). 115), the gene encoding the RBD region of the Ss protein of SARS2 (GenBank Accession No .: 335-587th amino acid sequence of NC_045512.2 (SEQ ID NO: 4)) (SEQ ID NO: 116), or S2 of the Ss protein of SARS2. Example 1 above, except that a gene (SEQ ID NO: 117) encoding a region (GenBank Accession No .: NC_045512.2 amino acid sequence 588-1219 (SEQ ID NO: 5)) was used and pME18s was used as a plasmid vector. A SARS2 Ss protein expression vector (full length, NTD region, RBD region, or S2 region) was prepared in the same manner as in (1). Each of the above expression vectors was transfected into 293T cells to express full-length SARS2 Ss protein-expressing cells (full-length S protein-expressing cells), SARS2 Ss protein NTD region-expressing cells (SARS2-NTD-expressing cells), and SARS2 Ss protein RBD region expression. Cells (SARS2-RBD expressing cells) and SARS2 Ss protein S2 region expressing cells (SARS2-S2-expressing cells) were prepared. It is known that none of the 293T cells, HEK293 cells, and HEK293T cells used in the following experiments express the Fc receptor. Therefore, the following findings can be said to be a phenomenon that occurs by an Fc receptor-independent mechanism. A signal sequence, a Flag tag, and (MRAWIFFLLCLAGRALAASDYKDDDDKLE (SEQ ID NO: 118)) were added to the N-terminal of each protein. In the following examples, the mutant protein encodes an S protein (Ss protein) having a similar sequence. The Ss protein (Sw protein) used in the following examples was the amino acid sequence of the Ss protein.

Codon-optimized Ss protein coding gene (SEQ ID NO: 114)
5'-[AAGTTCGACGAAGACGATTCAGAACCAGTACTCAAGGGGGTCAAATTGCATTATACTtag] -3'

Gene encoding the NTD region (SEQ ID NO: 115)
5'--3'

Gene encoding the RBD region (SEQ ID NO: 116)
5'--3'

Gene encoding the S2 region (SEQ ID NO: 117)
5'--3'

(4) Preparation of biotinylated ACE2 mouse IgG2a Fc fusion protein An ACE2-Fc fusion protein expression vector encoding the hACE2-mouse IgG2a Fc fusion protein was prepared according to the methods described in References 11 and 12 below. ACE2-Fc fusion protein expressing cells were prepared in the same manner as in Example 1 (1) except that the ACE2-Fc fusion protein expression vector was used instead of the expression vector of each antibody, and the culture supernatant was collected. bottom. Then, the ACE2-Fc fusion protein was purified in the same manner as in Example 1 (2) except that the culture supernatant of the ACE2-Fc fusion protein-expressing cells was used instead of the culture supernatant of the antibody sample. The purified ACE2-Fc fusion protein was biotinylated using sulfo-NHS-LC-biotin (manufactured by Thermo Fisher Scientific) to prepare a biotinylated ACE2-Fc fusion protein.
Reference 11: Hirayasu K et.al. “Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.”, Nat. Microbiol. 2016, Vol. 1, Article number: 16054
Reference 12: Satoh T et.al., “PILRα is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein”, B. Cell., 2008, vol. 132, pages 935-944

(5) Confirmation of domain and ACE2-binding property The full-length Ss protein-expressing cell, SARS2-NTD-expressing cell, SARS2-RBD-expressing cell, or SARS2-S2-expressing cell of Example 1 (3) was applied to the above-mentioned Example 1 (1). ), A sample of each antibody (final concentration 3 μg / ml) was added in an amount of 10 μl. Then, the obtained reaction solution was incubated at 4 ° C. for 30 minutes to react. After the reaction, the obtained reaction solution was stained by adding an APC-labeled anti-mouse IgG antibody, and the obtained cells were analyzed using a flow cytometer (Atune ™, Thermo Fisher Scientific). As a result, the recognition site of each antibody in the Ss protein was confirmed. Controls were measured in the same manner except that the antibody sample was not added. Then, the measured value (MFI) in each antibody sample was corrected based on the measured value (MFI) in the control. FlowJo version 10.7 (manufactured by BD Biosciences) was used for the analysis of flow cytometry data (the same applies hereinafter).

Further, after adding 10 μl of a sample (final concentration of 3 μg / ml) of each antibody of Example 1 (1) to the full-length Ss protein-expressing cells, the biotinylated ACE2- obtained in Example 1 (4) was added. 10 μl of Fc fusion protein (final concentration 2.7 μg / ml) was added. Then, the obtained reaction solution was incubated at 4 ° C. for 30 minutes to react. To 15 μl of the reaction solution after the reaction, 10 μl of APC-labeled streptavidin (final concentration 2 μg / ml) was added and stained, and the obtained cells were analyzed using the flow cytometer. As a result, the binding of the full-length Ss protein-expressing cells to ACE2 in the presence of each antibody was confirmed. Controls were measured in the same manner except that the antibody sample was not added. Then, the measured value in each antibody sample was corrected based on the measured value in the control. These results are shown in FIG.

FIG. 1 is a graph showing the binding of full-length Ss protein-expressing cells and ACE2 in the presence of each antibody recognition site in Ss protein and each antibody sample. In FIG. 1, the horizontal axis represents the type of antibody sample, and the vertical axis represents the relative values of the Ss protein's binding to the domain and the Ss protein's ACE2 binding. Although not shown, all clones were confirmed to bind to full-length Ss protein-expressing cells. As shown in FIG. 1, among each antibody clone, in the presence of 6 clones of anti-NTD antibody (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, clone 8D2) that bind to NTD of S protein of SARS2. It was found that the binding between the full-length Ss protein-expressing cells and ACE2 was enhanced. That is, it was found that the presence of these antibodies enhances the binding of the Ss protein to ACE2. Among the antibodies that bind to the RBD region and the S2 region, there is no antibody that enhances the binding ability of the Ss protein to ACE2, and some of the antibodies that bind to the NTD region have the ability to bind the Ss protein to ACE2. The enhancement suggests that the antibody that binds to a certain region of the antibody against the NTD region has a function of enhancing the binding ability of the Ss protein to ACE2. On the other hand, it was found that the presence of an antibody that binds to the RBD region reduces the binding of the Ss protein to ACE2. From these facts, it was found that there is an antibody that enhances the binding of Ss protein to ACE2 in a person infected with SARS-CoV-2. In addition, since ACE2 is important for establishing infection with SARS2, it was suggested that detection of these antibodies in subjects could be used to carry out tests for susceptibility to SARS2 and aggravation of SARS2. Furthermore, it was found that the anti-RBD antibody of the peaplomer of SARS2 can be used as a neutralizing antibody. As described above, 293T cells and the like do not express the Fc receptor essential for Fc receptor-dependent ADE. Therefore, it was found that the ACE2-binding enhancing antibody in the SARS2-infected person enhances the binding of the Ss protein to the ACE2 protein by an Fc receptor-independent mechanism.

Amino acid sequences were identified for 6 clones (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, clone 8D2) of an antibody (anti-NTD antibody) that enhances the binding of the Ss protein to ACE2. The amino acid sequence of each antibody is shown below. In each amino acid sequence, the underlined amino acid sequence corresponds to the amino acid sequences of CDR1, CDR2, and CDR3 from the N-terminus to the C-terminus, respectively.

Clone 2210 Heavy Chain Variable Region (SEQ ID NO: 98)
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRVEDTAVYYCAR DQEWFRELFLFDY WGQGTLVTVSS

Clone 2210 Light chain variable region (SEQ ID NO: 99)
DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKLLIY DASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPPT FGPGTKVDIK

Clone 2490 Heavy Chain Variable Region (SEQ ID NO: 100)
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMN WVRQAPGKGLEWVA NINQDGGEKYYVDSVRG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DPYDLYGDYGGTFDY WGQGTLVTVSS

Clone 2490 light chain variable region (SEQ ID NO: 101)
EIVLTQSPATLSVSPGERATLSC RASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTDFTLTISSLQSEDFALYYC QQYNNWWRT FGQGTKVEIN

Clone 8D double chain variable region (SEQ ID NO: 102)
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA NINQDGSEKYYVDSVKG RFTISRDNAKNSLYLQVNSLRAEDTAVYYCAR DWDYDILTGSWFGAFDI WGQGTTVTVSS

Clone 8D2 light chain variable region (SEQ ID NO: 103)
DIQMTQSPSSLSASVGDRVTITC RASQGIRNDLG WYQQKPGKAPKRLIY AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPLT FGGGTKVEIK

Clone 2582 Heavy Chain Variable Region (SEQ ID NO: 104)
QVQLVQSGSELKKPGASVKVSCKASGYTFT TYAMN WVRQAPGQGLEWMG WINTNTGNPTYAQGFTG RFVFSLDTSVNTAFLHIGSLKAEDTAVYYCAR DQDSGYPTYYYYYMDV WGKGTTVTVSS

Clone 2582 light chain variable region (SEQ ID NO: 105)
DIVMTQTPLSLSVTPGQPASISC KSSQSLLHSDGK TYLYWYLQKPGQSPQLLIY EVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQSIQPPLT FGGGTKVEIK

Clone 2369 Heavy Chain Variable Region (SEQ ID NO: 106)
QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA DISYDGSEKYYADSVKG RFTIYRDNSKNTLYLQMNSLRAEDTAVYYCAK DFGGDNTAMVEYFFDF WGQGTLVTVSS

Clone 2369 light chain variable region (SEQ ID NO: 107)
DIQMTQSPSTLSASVGDRVTITC RASQSISSWLA WYQQKPGKAPKLLIY KASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYSPT FGQGTKVEIK

Clone 2660 Heavy Chain Variable Region (SEQ ID NO: 108)
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYDMH WVRQATGKGLEWVS AIGTAGDTYYPGSVKG RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR ADPYQLLGQHYYYGMDV WGQGTTVTVSS

Clone 2660 light chain variable region (SEQ ID NO: 109)
EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLIT FGQGTRLEIK

(6) Confirmation of ACE2 binding property Next, the predetermined concentrations (0, 0.1, 0) of each of the above clones and the clone 2016 and the clone 4A8, which bind to the NTD region but have a weak ability to enhance the binding ability to ACE2. The binding to ACE2 was confirmed in the same manner as in Example 1 (5), except that each antibody was added so as to be (3, 1, 3 or 10 μg / ml). Clone 2016 and clone 4A8 were prepared in the same manner as in Examples 1 (1) and (2) based on the amino acid sequences described in References 9 and 10. Hereinafter, other antibody clones are also prepared based on the amino acid sequences described in References 9 and 10 in the same manner unless otherwise specified. These results are shown in FIG.

FIG. 2 is a graph showing the binding of full-length Ss protein-expressing cells and ACE2 in the presence of each antibody sample. In FIG. 2, the horizontal axis represents the concentration of the antibody sample, and the vertical axis represents the relative value of the ACE2 binding property of the Ss protein. As shown in FIG. 2, in clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, and clone 8D2, the binding property of Ss protein to ACE2 was enhanced in a concentration-dependent manner. On the other hand, in clone 2016 and clone 4A8, no enhancement of the binding property of Ss protein to ACE2 was observed at any concentration.

(7) Confirmation of effect of neutralizing antibody on ACE2 binding property Next, 5 clones (clone 2369, clone 2490, clone 2582, clone 8D2) and clone 4A8 having a weak enhancing activity for ACE2 binding ability were obtained. Each antibody was added so as to be 3 μg / ml, and clone C144 (anti-RBD antibody), which is a neutralizing antibody, was added to a predetermined concentration (0, 0.1, 0.3, 1, 3 or The binding to ACE2 was confirmed in the same manner as in Example 1 (6) except that the antibody was added so as to be 10 μg / ml). These results are shown in FIG.

FIG. 3 is a graph showing the binding of full-length Ss protein-expressing cells and ACE2 in the presence of each antibody sample. In FIG. 3, the horizontal axis represents the concentration of the C144 antibody, and the vertical axis represents the ACE binding property. As shown in FIG. 3, in the absence of C144, the binding property of the Ss protein to ACE2 was enhanced in a concentration-dependent manner, as in Example 1 (6). On the other hand, in the presence of C144, the binding of Ss protein to ACE2 was enhanced in a concentration-dependent manner, but the binding to ACE2 was suppressed as compared with the presence of C144. From these results, it was found that in SARS2-infected persons, there is an antibody that enhances the binding of Ss protein to ACE2, and that the enhancement of binding by the antibody can be suppressed by the neutralizing antibody.

[Example 2]
It was confirmed that the antibody that enhances the binding of S protein to ACE2 exhibits the infection enhancing effect of SARS2.

(1) Preparation of serum-derived anti-NTD antibody of SARS2-infected person Clone 2369, clone 2490, clone antibody 2582, and clone 8D2 were prepared in the same manner as in Example 1 (1).

(2) Preparation of SARS2 S Protein Expression Pseudotype Virus A gene expressing SARS2 full length Ss protein was cloned into a pME18s plasmid to obtain a SARS2 full length Ss protein expression vector. Then, the full-length Ss protein expression vector of SARS2 was transiently transfected into 293T cells to prepare full-length Ss protein expression cells of SARS2. Twenty-four hours after the transfection, the medium was replaced with a new medium, VSV-G-deficient vesicular stomatitis virus (VSV) incorporating the GFP reporter gene was added, and the cells were incubated for 2 hours to incubate the virus. Was infected. Next, the medium of the cells was washed with DMEM medium containing no FBS. Then, 10% FBS-added DMEM was added, and the cells were _{cultured at 37 ° C. and 5% CO 2} for 48 hours. After the culture, the supernatant containing the pseudotyped virus containing the full-length Ss protein of SARS2 was collected, dispensed, and stored at −80 ° C. until use.

(3) Preparation of constitutively expressing cells of ACE2 Encodes the coding region (GenBank Accession No .: NM_001371415.1 base sequence 50 to 2467) of human ACE2 (GenBank Accession No .: NP_001358344.1 (SEQ ID NO: 6)). The polynucleotide (SEQ ID NO: 7) was cloned into a pMXs vector to create a human ACE2 expression vector. Using polyethyleneimine (PEI; manufactured by Polysciences), the human ACE2 expression vector and the envelope protein expression vector of amphotropic virus were transfected into PlatE cells and cultured for 48 hours to express the retrovirus. After the culture, the culture supernatant containing the retrovirus was collected. After premixing the culture supernatant with a DOTAP liposome transfection reagent (manufactured by Roche), HEK293T cells were spin-transfected under the conditions of 2400 rpm, 32 ° C., and 2 hours, and then cultured for 3 days. After the culture, the collected cells were stained with a fluorescently labeled anti-ACE2 antibody (manufactured by R & D Systems), and the stained cells were selected for ACE2-positive cells with an SH800S cell sorter (manufactured by Sony), and the cells were ACE2 constitutive. Expressed cells were obtained.

(4) Infection test The ACE2 constitutive expression cells were seeded on a 96-well plate at a rate of 20000 cell / well, and 10 μl of a pseudo-type virus containing the full-length Ss protein of SARS2 was added. Next, the anti-NTD antibody (clone 2369, clone 2490, clone antibody 2582, clone 8D2, or clone 4A2) prepared in Example 2 (1) was added to a predetermined concentration (0, 0.03, 0.01, 0). .1, 1, or 10 μg / ml was added and incubated at 37 ° C. for 24 hours, thereby infecting the cells with pseudotyped virus. After the incubation, BD FACSVerse ™. Infection with pseudotyped virus was detected by detecting the GFP signal with a flow cytometer. A reference example is an anti-NTD antibody (clone 4A2) that does not have the ability to enhance the binding of Ss protein to ACE2 instead of the anti-NTD antibody. ) Was used. Further, the control was carried out in the same manner except that the anti-NTD antibody was not added. Then, each antibody was based on the infection amount or the infection rate of the pseudotype virus in the control. Relative values of the proportion of infected cells in the above were calculated. These results are shown in FIG.

FIG. 4 is a graph showing the infection rate of pseudotyped virus. In FIG. 4, the horizontal axis represents the concentration of the antibody sample, and the vertical axis represents the infection rate of the pseudotyped virus. As shown in FIG. 4, the infection rate of the pseudotype virus expressing the Ss protein of SARS2 in the presence of clone 2369, clone 2490, clone antibody 2582, and clone 8D2 as compared with the antibody of the reference example (clone 4A2). Was found to increase in a concentration-dependent manner. Although not shown, it has been confirmed that the infection rate also increases in a concentration-dependent manner when clone 2210 is used. From these results, it was found that an antibody that enhances the binding of Ss protein to ACE2 exhibits an infection enhancing effect of SARS2.

(5) SARS2 infection experiment Next, it was confirmed whether the antibody that enhances the binding of Ss protein to ACE2 has the SARS2 infection enhancing ability. First, as SARS2, KNG19-020 strain of SARS2 acquired at Kanagawa Prefectural Institute of Public Health was used. Specifically, the virus stock of SARS2 was amplified using VeroE6 cells expressing TMPRSS2. The resulting virus stock (6.25 log TCID50) was diluted 100-fold with 2% FBS-containing DMEM medium were mixed with 1 × 10 ⁵ cells / ml of infected target cells. At the time of the mixing, the clones were added so that the final concentration of the clone 2490 was 1 μg / ml. As the cells to be infected, HEK293 cells, the above-mentioned HEK293 cells expressing human ACE2, and Huh7 cells expressing human ACE were used. After the mixing, the cells were cultured at 37 ° C., 5% CO ₂ , and under wet conditions for 3 hours. After the culture, the virus was removed by washing the cells with DMEM. After removing the virus, the cells were further cultured for 2 days under the above culture conditions. Then, the culture supernatant after culturing was collected, and the virus RNA was extracted from the culture supernatant using a virus RNA extraction kit (manufactured by Qiagen). Then, using the obtained RNA and the following N2 primer set, the number of copies of the virus in the culture supernatant was measured by performing quantitative PCR. These results are shown in FIG.

-N2 primer set Forward primer (SEQ ID NO: 119)
5'-AGCCTCTTCTCGTTCCTCATCAC-3'
Reverse primer (SEQ ID NO: 120)
5'-CCGCCATTGCCAGCCATTC-3'

FIG. 5 is a graph showing the infection result of SARS2. In FIG. 5, (A) is the result of using HEK293 cells, (B) is the result of using HEK293 cells expressing ACE2, and (C) is the result of using Huh7. In FIGS. 5A to 5C, the horizontal axis indicates the presence or absence of an antibody, and the vertical axis indicates the amount of virus. As shown in FIG. 4 (A), in the cells not expressing ACE2 to which the Ss protein binds, the amount of SARS2 infection did not change depending on the presence or absence of the antibody. On the other hand, as shown in FIGS. 5 (B) and 5 (C), in ACE2-expressing cells, the presence of the antibody enhanced the amount of SARS2 infection. From these results, it was found that the antibody that enhances the binding of the Ss protein to ACE2 enhances the infection of ACE2-expressing cells by enhancing the binding of SARS2 to ACE2.

[Example 3]
It was confirmed that the antibody that enhances the binding of the S protein to ACE2 competes in the binding to the S protein.

(1) Preparation of anti-NTD antibody Clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, and clone 8D2 were prepared in the same manner as in Example 1 (1) (competitive antibody). Further, for the constant region, an antibody in which the constant region of each clone was mIgG2a was prepared in the same manner except that mouse IgG2a was used instead of human IgG1 (binder antibody). Further, as a control competing antibody, a clone 2147 antibody that binds to the S2 region was prepared in the same manner.

(2) Confirmation of competitiveness of anti-NTD antibody The six types of anti-NTD antibody (competitive antibody) of Example 3 (1) were introduced into the cells into which the full-length Ss protein of SARS2 and the gene of GFP of Example 1 (3) were introduced. ) Or a control antibody was added, and six types of anti-NTD antibodies (binder antibodies) prepared in Example 3 (1) above, each of which had a constant region of mouse IgG2a, were added. The concentration of each antibody was 10 μg / ml. After the addition, the mixture was incubated at 4 ° C. for 30 minutes. After the incubation, each cell was stained with APC-labeled anti-mouse IgG antibody. The control was added in place of the binder antibody at 2.7 μg / ml of the biotinylated ACE2-Fc fusion protein obtained in Example 1 (4), and then stained with APC-labeled streptomycin. , And carried out in the same manner (ACE2). Then, the fluorescence intensity (MFI) when the control competing antibody (combination of clone 2147 and ACE2) in the control was added was set to about 100, and the fluorescence intensity was calculated as a relative value. These results are shown in FIG.

FIG. 6 is a graph showing relative values of fluorescence intensity. As shown in FIG. 6, the relative values of the fluorescence intensities of the six types of anti-NTD antibodies (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, and clone 8D2) decreased in any combination. It turned out to compete with each other. In addition, it was reconfirmed that both antibodies enhance the binding of S protein to ACE2. From these facts, the antibody that enhances the binding property of S protein to ACE2 (infection enhancing antibody) is such that some or all of the binding sites of SARS2 in S protein overlap with each other, or the binding between S protein and antibody. It was found that they were bound to the extent that steric damage caused by the disease occurred. Further, it was found that an antibody that enhances the binding property of S protein to ACE2 can be detected by using the above 6 types of anti-NTD antibodies as reference antibodies and using the competitiveness of binding to S protein as an index.

[Example 4]
Epitope mapping of the antibody that enhances the binding of the S protein to ACE2 was performed, and it was confirmed that the same region in the S protein was recognized.

Same as Example 1 (3) above, except that an expression vector containing a gene encoding various mutant NTD-PILR-TM in which one amino acid is replaced with alanine in order from the N-terminal side was used for NTD of the Ss protein of SARS2. To prepare various mutant NTD-expressing cells. The mutant NTD-expressing cells were reacted with antibodies that enhance the binding of Ss protein to ACE2 (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, clone 8D2 (infection enhancing antibody)). After the reaction, the mutant NTD-expressing cells were stained with an APC-labeled anti-human IgG antibody (manufactured by Jackson ImmunoResearch). Further, the same procedure was carried out except that an antibody (clone 4A8) that did not enhance the binding property of the Ss protein to ACE2 or an antibody against Flag added to the N-terminal of NTD was used instead of the antibody. Clone 4A8 was prepared in the same manner as each antibody of Example 1 (1). The control was the same except that NTD-expressing cells of the Ss protein of wild-type SARS2 were used, and the fluorescence intensities of various mutant NTD-expressing cells and wild-type NTD-expressing cells (SARS-NTD-expressing cells) were compared. The fluorescence intensity of each sample was corrected by the fluorescence intensity when the anti-Flag antibody was used. These results are shown in FIGS. 7 and 8.

FIG. 7 is a graph showing changes in fluorescence intensity of infection-enhancing antibodies (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, clone 8D2). In FIG. 7, the horizontal axis represents the position of the mutant amino acid of NTD, and the vertical axis represents the relative value (logarithm) of the fluorescence intensity. Note that FIG. 7 shows the data of amino acids before and after the change in the relative value of the fluorescence intensity. FIG. 8 shows the positions of NTD amino acids commonly recognized by infection-enhancing antibodies. As shown in FIG. 7, each infection-enhancing antibody has different epitopes, but the 64th, 66th, 187th, 213rd, and 214th positions of the Ss protein, particularly the 64th and 66th positions. The amino acids at positions 2, 213, and 214 were recognized by a plurality of infection-enhancing antibodies, and it was presumed that the binding ability was reduced in the case of a mutant protein in which a mutation was introduced into these amino acids. The amino acid positions of the epitopes of each antibody were in agreement with the above-mentioned amino acids. Further, when the recognition sites of each antibody are compared as shown in the region surrounded by the white line in FIG. 8 (A), as shown in (B), the infection-enhancing antibody is a circle in the three-dimensional structure of the S protein of SARS2. It was found that the regions indicated by the shaped spots were commonly recognized. Moreover, since the recognition regions are similar in the three-dimensional structure in this way, it is presumed that competition among infection-enhancing antibodies has occurred. Further, in each infection-enhancing antibody, 8D2 well recognized the common site of the site recognized by other infection-enhancing antibodies, and was found to be suitable as a reference antibody when testing using competitiveness as an index.

[Example 5]
A mutant protein in which the recognition site of the infection-enhancing antibody was mutated was prepared, and it was confirmed that the infection-enhancing antibody did not bind.

Using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Inc.), according to the attached protocol, the amino acids at positions 64, 65, 66, 187, 213, and 214 in the Ss protein of full length SARS2. An expression vector encoding a mutant protein in which one or more of them was replaced with alanine was prepared. A cell expressing the mutant protein was prepared in the same manner as in Example 1 (3) except that the expression vector encoding the mutant protein was used instead of the expression vector of the full-length Ss protein of SARS2. Against cells expressing the mutant protein and RBD of infection-enhancing antibody (clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, clone 8D2), non-infection-enhancing NTD antibody (clone 4A8, clone 2016), or SARS2. A mouse antibody (clone 5-1-1) was allowed to coexist and incubated at 4 ° C. for 30 minutes. After the incubation, the cells were stained with the addition of APC-labeled anti-human IgG antibody or APC-labeled anti-mouse IgG antibody and analyzed with a flow cytometer. Control was carried out in the same manner except that the full-length Ss protein-expressing cells were used or the antibody was not used. These results are shown in FIG. Since the amino acid (F) at position 65 of the Ss protein is located inside the amino acids at positions 64 (W) and 66 (H) (inside the Ss protein), it is exposed on the surface of the Ss protein. It is not a recognition site for infection-enhancing antibodies. However, it has been separately confirmed that when the amino acid (F) at position 65 is combined with the alanine substitution, the binding of the infection-enhancing antibody is further reduced. It is presumed that this is because the positions of the amino acids at positions 64 and 66 are changed by the substitution with the amino acid at position 65.

FIG. 9 is a histogram showing the binding amount of each antibody. In FIG. 9A, the results of clone 2210, clone 2369, clone 2490, clone 2582, clone 2660, and clone 8D2 are shown from the top to the bottom. In FIG. 9A, the upper part of the histogram shows the amino acid to be mutated with alanine substitution. Further, in FIGS. 9 (B) and 9 (C), the upper figures show the results using wild-type spike protein-expressing cells, and the lower figures show the results using mutant protein-expressing cells. .. In FIGS. 9B and 9C, the clone names used at the top of each histogram or at the top of the histogram are shown. In each histogram of FIG. 9, the horizontal axis represents the amount of antibody bound, and the horizontal axis represents the number of cell counts. Further, in each histogram of FIG. 9A, the solid histogram shows the result using the cells expressing the mutant protein, and the shaded histogram shows the result (control) using the cell expressing the full-length Ss protein. .. In each of the histograms of FIGS. 9B and 9C, the solid histogram shows the result of adding each antibody, and the shaded histogram shows the result (control) of not adding each antibody. As shown in FIG. 9 (A), the 64-position and the 66-position and the 213 and 214-positions of the infection-enhancing antibody are obtained by substituting a plurality of the antibodies with alanine as compared with the case where one of them is replaced with alanine. The binding could be effectively suppressed. Further, as shown in FIGS. 9A to 9C, mutant proteins in which the amino acids at positions 64, 66, 213, and 214 of the Ss protein of SARS2 were replaced with alanine, positions 64, 66, and 187. Infection-enhancing antibodies are used for the mutant proteins in which the amino acids at positions 213 and 214 are substituted with alanine, and the mutant proteins in which the amino acids at positions 64, 65, 66, 187, 213, and 214 are substituted with alanine. Did not bind, only non-infection enhancing antibody and neutralizing antibody bound. Therefore, it was found that the mutant protein in which the recognition site of the infection-enhancing antibody was mutated did not bind to the infection-enhancing antibody, and the mutation was introduced at positions 64, 66, 213, and 214 of the Ss protein. It was found that this effectively suppresses the binding. In addition, in order for the infection-enhancing antibody to be induced in vivo, the S protein must have an epitope of the infection-enhancing antibody. Therefore, the SARS2 mutant spike protein in which the recognition site of the infection-enhancing antibody is mutated suppresses the induction of the antibody that enhances the binding of the S protein to ACE2 and the infection-enhancing antibody, and is an immunogen of the SARS2 vaccine. It was estimated that it could be used as.

[Example 6]
It was confirmed that the infection-enhancing antibody was present in the serum of the SARS2-infected person.

Serum was collected from critically ill patients diagnosed as infected with SARS2 by PCR (with a ventilator (hereinafter, the same applies), n = 3). All the sera of SARS2-infected persons used in this example were treated with 2% CHAPS for 5 minutes to inactivate SARS2 contained in the sera (hereinafter, the same applies). The serum was diluted 100-fold with HANKS buffer containing 0.1% BSA and 0.05% NaN _{3 to obtain diluted serum.} Next, 10 μl of diluted serum was added to the full-length Ss protein of SARS2 and GFP-expressing 293T cells of Example 1 (3) and reacted. To the reaction solution after the reaction, 10 μl of a reference antibody (8D2 of the binder antibody of Example 2) was added so as to be 35 ng / ml, and the reaction was further carried out. Then, it was stained with APC-labeled anti-mouse IgG antibody, and the fluorescence intensity of APC in GFP-positive cells was analyzed by the flow cytometer. As a comparative example, the same analysis was performed except that the serum of a healthy person (n = 2) was used instead of the serum of a SARS2-infected person. In addition, the control was tested in the same manner except that the reference antibody was not added. Regarding the collection and use of human serum, after obtaining approval from Osaka University, the collection and use were performed with the written consent of the provider (hereinafter, the same applies). These results are shown in FIG.

FIG. 10 is a histogram showing the amount of antibody bound. In FIG. 10, the horizontal axis represents the amount of antibody bound to the serum, and the horizontal axis represents the count number of cells. Further, in FIG. 10, each histogram shows the type of serum sample, the shaded histogram shows the result of the sample without the reference antibody added (control), and the solid histogram shows the sample with the reference antibody added. The result of is shown. As shown in FIG. 10, in the serum of healthy subjects, there was no change in the binding amount of the antibody derived from the serum due to competition with the reference antibody as compared with the control. That is, it was found that the infection-enhancing antibody was not contained. In contrast, in the serum of SARS2-infected individuals, the amount of binding of the antibody derived from the serum was reduced due to competition with the reference antibody as compared with the control. That is, it was found that the sera of SARS2-infected persons contained an infection-enhancing antibody. In addition, since infection-enhancing antibody was generated in critically ill patients infected with SARS2, it was confirmed that the infection-enhancing antibody could be used as a severity marker.

[Example 7]
The anti-NTD antibody titer, anti-RBD antibody titer, ACE2-binding property, and infection-enhancing antibody titer were evaluated in SARS2-infected person sera, and it was confirmed whether they correlate with each other.

(1) Measurement of anti-NTD antibody titer From the serum collected over time from the time of admission to the time of discharge of a SARS2 patient (n = 54, total number of samples 88), diluted 10 μl in the same manner as in Example 6 above. Serum was prepared. The diluted serum was added to the SARS2-NTD expressing cells of Example 1 (3) and reacted. Then, it was stained with APC-labeled anti-human IgG antibody, the fluorescence intensity of APC in GFP-positive cells was analyzed by the flow cytometer, and the anti-NTD antibody titer in the serum of a SARS2-infected person was measured.

(2) Measurement of anti-RBD antibody (neutralizing antibody) titer With the above-mentioned Example 7 (1) except that the SARS2-RBD-expressing cells of Example 1 (3) were used instead of the SARS2-NTD-expressing cells. Similarly, the anti-RBD antibody titer in the serum of SARS2-infected persons was measured.

(3) Measurement of Infection-Enhanced Antibody Tittle The serum was diluted 30-fold with a HANKS buffer solution containing _{0.1% BSA and 0.05% NaN 3 to obtain a 30-fold diluted serum.} To the full-length Ss protein of SARS2 and GFP-expressing 293T cells obtained in Example 1 (3), 10 μl of the 30-fold diluted serum was added and reacted. After the reaction, 10 μl of a reference antibody (8D2 of the binder antibody of Example 2) was added to the obtained reaction solution so as to have a concentration of 35 ng / ml, and the reaction was further carried out. Then, it was stained with APC-labeled anti-mouse IgG antibody, the fluorescence intensity of APC in GFP-positive cells was analyzed by the flow cytometer, and the infection-enhancing antibody titer was measured.

(4) Measurement of ACE2 binding property In the same manner as in Example 1 (5) except that the diluted serum was used instead of the antibody obtained in Example 1 (1), in the presence of the diluted serum. The binding property of ACE2 to the full-length Ss protein-expressing cell was confirmed. These results are shown in FIG.

FIG. 11 is a graph showing the relationship between the anti-NTD antibody titer, the anti-RBD antibody titer, the ACE2 binding property, and the infection-enhancing antibody titer in the serum of a SARS-CoV-2 infected person. In FIG. 11, (A) shows an outline of the measurement system used, (B) shows the relationship between ACE2 binding property and anti-RBD antibody titer, and (C) shows anti-NTD antibody titer and anti-RBD antibody titer. The valence relationship is shown, (D) shows the relationship between the infection-enhancing antibody titer and the anti-RBD antibody titer, and (E) shows the group with low anti-RBD antibody titer (low RBD antibody titer group) and the anti-RBD antibody titer. The relationship between the ACE2 binding property and the infection-enhancing antibody titer in the medium group (medium RBD antibody titer group) and the high anti-RBD antibody titer group (high RBD antibody titer group) is shown. In FIG. 11B, the horizontal axis represents the anti-RBD antibody titer and the vertical axis represents the ACE2 binding property. In FIG. 11C, the horizontal axis represents the anti-RBD antibody titer and the vertical axis represents the anti-NTD antibody titer. In FIG. 11 (D), the horizontal axis represents the anti-RBD antibody titer, and the vertical axis represents the infection-enhancing antibody titer. In FIG. 11 (E), the upper graph is the same as the graph of FIG. 11 (B), the horizontal axis shows the anti-RBD antibody titer, and the vertical axis shows the ACE2 binding property. The lower graph of FIG. 11 (E) shows the relationship between ACE2 binding and infection-enhancing antibody titer, and shows the results of the low RBD antibody titer group, the medium RBD antibody titer group, and the high RBD antibody titer group from the left. Further, in the lower graph of FIG. 11 (E), the horizontal axis shows the infection-enhancing antibody titer, and the vertical axis shows the ACE2 binding property.

As shown in FIG. 11 (B), it was confirmed that the anti-RBD antibody functions as a neutralizing antibody for SARS2 because the binding property between the full-length spike-expressing cells and ACE2 decreases as the anti-RBD antibody titer increases. .. Further, as shown in FIG. 11C, it was found that the increase in the anti-RBD antibody titer correlates with the increase in the anti-NTD antibody titer. That is, it was found that infection with SARS2 simultaneously induced antibodies against various sites of S protein. In addition, as shown in FIGS. 11B and 11D, ACE2-binding and infection were observed in the group of patients with low anti-RBD antibody titers (the shaded group in FIGS. 11B and 11D). Although there were variations in the enhanced antibody titer, the ACE2 binding property was higher than that in the group of patients with high anti-RBD antibody titers. Then, as shown in FIG. 11 (E), when the low RBD antibody titer group, the medium RBD antibody titer group, and the high RBD antibody titer group are compared, the infection-enhancing antibody titer also increases as the anti-RBD antibody titer increases. However, the ACE2 binding property was reduced. From these facts, in the early stage of SARS infection in which the increase in anti-RBD antibody titer is insufficient, if the infection-enhancing antibody titer is high, the binding of SARS2 to ACE2 is enhanced, and as a result, infection enhancement occurs. It was presumed that it would cause aggravation. Therefore, it was suggested that the infection-enhancing antibody titer is an indicator of infectivity and aggravation.

[Example 8]
Changes in infection-enhancing antibody titer, anti-RBD antibody titer, and ACE2 binding after SARS2 infection were confirmed.

Infection in each patient's serum in the same manner as in Example 7 above, except that sera collected regularly after admission from 3 patients (severely ill patients) diagnosed as infected with SARS2 by PCR test were used. Changes in enhanced antibody titer, anti-RBD antibody titer, and ACE2 binding were confirmed. Patients with patient number 30 collect serum on days 1, 6, 8 and 13 after admission, and patients with patient number 74 collect serum on days 1, 8, 16, 19, 22 and 27 after admission. However, the patient with patient number 68 collected serum on days 1, 7, 13, 18, and 23 after admission. These results are shown in FIG.

FIG. 12 is a graph showing changes in infection-enhancing antibody titer, anti-RBD antibody titer, or ACE2-binding property in each infected person's serum. In FIG. 12, the horizontal axis indicates the elapsed time after admission, and the vertical axis indicates the anti-RBD antibody titer or ACE2-binding property. In FIG. 12, the upper three figures are graphs showing changes in infection-enhancing antibody titers, the middle three figures are graphs showing changes in anti-RBD antibody titers, and the lower three figures show ACE2 binding. It is a graph which shows. In each figure of FIG. 12, the results of patient number 30, patient number 74, and patient number 68 are shown from the left. As shown in FIG. 8, it was found that the infection-enhancing antibody titer was higher than the anti-RBD antibody titer in any of the patients. It was also found that in the early stage of infection in which the anti-RBD antibody titer is low (anti-RBD antibody is not produced), the ACE2 binding property increases as the infection-enhancing antibody titer increases. Therefore, it was confirmed that when the infection-enhancing antibody titer was high in the early stage of infection, the binding property of SARS2 to ACE2 was enhanced, and as a result, the infection was enhanced and the severity was caused. Therefore, it was found that the infection-enhancing antibody titer is an index of infectivity and aggravation.

[Example 9]
The anti-NTD antibody titer in the serum of non-infected persons with SARS-CoV-2 was confirmed.

Example 7 (purchased from GeorgeKingBio-Medical) of healthy Americans (n = 48) collected before June 2019 and not infected with SARS-CoV-2 (purchased from GeorgeKingBio-Medical). The anti-NTD antibody titer was measured in the same manner as in 1). In addition, the control was measured in the same manner except that the serum was not added. These results are shown in FIG.

FIG. 13 is a histogram showing the amount of anti-NTD antibody bound to each serum. In each histogram of FIG. 13, the horizontal axis is a graph showing the amount of anti-NTD antibody bound to the serum, and the vertical axis is the count of the number of cells. In FIG. 13, the shaded histogram shows the results of the control (no serum added), and the solid histogram shows the results of each serum sample. As shown in FIG. 13, it was confirmed that the anti-NTD antibody titer was present from 25 samples in the serum derived from a healthy subject. It has been suggested that Westerners have a higher infection rate and severity rate of infection with SARS2 than Asians. Since the anti-NTD antibody is an index containing an infection-enhancing antibody, it is presumed that a healthy person in which the anti-NTD antibody is found possesses the infection-enhancing antibody to a certain extent. For this reason, it was estimated that Westerners had a higher infection rate and aggravation rate than Asians because a certain percentage of people carry infection-enhancing antibodies. In addition, administration of a vaccine containing the full-length spike protein of SARS-CoV-2 as an active ingredient to a person who has an anti-NTD antibody in the serum may induce enhanced production of the infection-enhancing antibody. It was suggested that the mutant protein of the present invention could be used as an active ingredient of a vaccine to suppress the induction and enhancement of the production of an infection-enhancing antibody.

[Example 10]
We confirmed the mechanism by which the binding of the S protein to ACE2 is enhanced by the binding of the infection-enhancing antibody.

The mechanism by which the infection-enhancing antibody enhances the binding of S protein to ACE2 was investigated based on the data of crystal structure analysis of S protein. As the crystal structure of the S protein, the data of the crystal structure analysis of the S protein registered with the following PDB numbers in the Protein Data bank was used. Since the S protein is a trimer of A chain, B chain and C chain, the PDB number of each chain is shown. The data include data for crystal structure analysis in the structure in which the S protein has binding to ACE2 (RBD-Up) and crystal structure in the structure in which the S protein does not have binding to ACE2 (RBD-Down). Includes analysis data. Therefore, the data having the RBD-Up structure and the data having the RBD-Down structure were classified, and the data of each classification was superposed to create three-dimensional data. Then, in the three-dimensional data, changes in the positions of amino acids at the binding site of the infection-enhancing antibody were examined. The result is shown in FIG.

(Data used for RBD-down analysis)
7jjj-A, 7jjj-D, 7jji--A, 7jji-B, 7jji-C, 7jjj-B, 7jjj-E, 7jjj-C, 7jjj-F, 6xr8-A, 6xr8-B, 6xr8-C, 6zge -A, 6zge-B, 6zge-C, 6zgi-A, 6zgi-B, 6zgi-C, 6zgh-C, 7a94-B, 7a94-C, 6zgh-B, 6zoz-A, 6zoz-B, 6zoz-C , 7a93-C, 6zgg-A, 6zgg-C, 7a95-B, 7a97-C, 7a96-C, 6xlu-B, 6xlu-C, 6zp2-A, 6zp2-B, 6zp2-C, 6xlu-A, 6xM5 -A, 6xM5-B, 7c21-B, 6zxn-B, 6zxn-B, 6zxn-C, 7c21-C, 6xM4-C, 6xM3-C, 6xM3-A, 6xM0-A, 6xM4-A, 7cn9-C , 6xm5-C, 6xey-A, 6xey-B, 6xm0-C, 6xey-C, 6zp0-A, 6zp0-B, 6zp0-C, 7cai-C, 6zoy-A, 6zoy-B, 6zoy-C, 6x6p -A, 6x6p-B, 6x6p-C, 6zox-A, 6zox-B, 6zox-C, 6zp1-A, 6zp1-B, 6zp1-C, 7cn9-B, 6xf5-A, 6xf5-B, 6xf5-C , 7chh-C, 7byr-B, 6z97-A, 7chh-B, 6xf6-A, 6z43-B, 6zhd-B, 6xf6-C, 6z43-C, 6z97-C, 6zhd-C, 6zwv-A, 6zwv -B, 6zwv-C, 7jzl-A, 7jzn-A, 6xkl-B, 6xkl-C, 6vsb-B, 6vsb-C, 7byr-C, 6vxx-A, 6vxx-B, 6vxx-C, 6x29-A , 6x29-B, 6x29-C, 6x2c-A, 6x2c-B, 6x2c-C, 6vyb-A, 6x2a-A, 6x2b-A, 6xcM-A, 6x2a-C, 6vyb-C, 6wps-A, 6wps -B, 6wps-E, 6wpt-C, 6x79-A, 6x79-B, 6x79-C, 6wpt-A, 6zow-C, 6zp5-B, 6zp7-B, 6zow-B, 6zp5-C, 6zp7-C , 6zgh-A

(Data used for RBD-up analysis)
7a94-A, 7a93-A, 7a93-B, 6zgg-B, 7a95-C, 7a95-A, 7a97-B, 7a98-A, 7a98-B, 7a98-C, 7a96-A, 7a96-B, 7a97- A, 6zxn-A, 6xm3-B, 6xm4-B, 6xm0-B, 7c21-A, 7cak-A, 7cak-B, 7cak-C, 7cai-A, 7cai-B, 7cn9-A, 7chh-A, 6wpt-B, 6zow-A, 6xs6-C, 6xs6-A, 6xs6-B, 6zdh-A, 6zdh-B, 6zdh-C, 6z97-B, 6zp5-A, 6zp7-A, 6z43-A, 6zhd- A, 7jzn-C, 6xcm-B, 6xcm-C, 6xcn-A, 6xcn-C, 6xcn-E, 7jzl-C, 7jzn-B, 7jzl-B, 6xkl-A, 6vsb-A, 7byr-A, 6vyb-B, 6x2b-B, 6x2b-C, 6x2a-B, 6xf6-B

FIG. 14 is a diagram showing the structure of S protein. In FIG. 14, (A) shows the structure of RBD-Up, and (B) shows the structure of RBD-Down. As shown in FIGS. 14A and 14B, in the structure of RBD-down, the amino acid corresponding to the amino acid residue at position 64 in the Ss protein (amino acid at position 64 (W)) is inside the NTD region. There were many buried parts, and the exposure of the NTD region to the surface was small. On the other hand, in the structure of RBD-Up, most of the amino acids at position 64 were exposed on the surface of the NTD region. Further, as shown in FIGS. 14 (A) and 14 (B), in the structure of RBD-down, the amino acids at positions 64, 66, 187, 213, and 214 in the S protein are distributed broadly. On the other hand, in the structure of RBD-up, they were arranged in one place, that is, more proximal to each other. Further, when the infection-enhancing antibody binds to the S protein, the binding property of the S protein to ACE2 is enhanced. Therefore, when the infection-enhancing antibody binds to the S protein, the S protein has an RBD-Up structure. It is estimated that it has become. From these facts, it is presumed that the infection-enhancing antibody has enhanced binding of S protein to ACE2 by the following mechanism. When the infection-enhancing antibody binds to the S protein, the position in the S protein, particularly the amino acid position recognized by the infection-enhancing antibody, moves to the surface side of the S protein, and the recognition target amino acid is located proximally. And the binding of the S protein to the infection-enhancing antibody occurs. With the movement of the recognized amino acid, the positions of other amino acids of the S protein also change, and the structure of the S protein changes from the RBD-down structure to the RBD-Up structure. As a result, it is presumed that the RBD region of the S protein has a structure capable of binding to ACE2, and the binding property of the S protein to ACE2 is enhanced. That is, it was presumed that the infection-enhancing antibody changed the conformation of the S protein from the RBD-down structure to the RBD-up structure by binding to the S protein. In addition, it was presumed that it is capable of inducing Fc receptor-independent ADE in order to enhance the binding to ACE2 by such a mechanism. The present invention is not limited to the above estimation.

[Example 11]
It was confirmed that by introducing a mutation into the recognition site of the infection-enhancing antibody, the inducing ability of the infection-enhancing antibody was suppressed when administered to the subject.

As an antigen sample, cells in which the full-length Ss protein or the NTD region of the Ss protein was expressed in B16G10 cells were prepared. Specifically, first, the gene encoding the NTD region of the Ss protein of SARS2 was cloned into the pME18S expression vector to obtain the NTD expression vector of the Ss protein of SARS2. Using the PEI transfection reagent, the expression vector was transiently transfected into mouse B16G10 cells and cultured for 48 hours. After the culture, cells were collected, -30 ° C., after freezing for 30 minutes, the resulting frozen processed product (per mouse 3 × 10 ⁶ cells / 100 [mu] l) an equal volume of complete Freund's adjuvant ( Completely emulsified with Sigma-Aldrich) to prepare an antigen sample. The antigen sample was injected into the footpad and the base of the tail of a 7-week-old BALB / c mouse to immunize, and mouse serum was collected 14 days after the immunization. Further, except that a mutant protein or a mutant peptide (NTD region of the mutant protein) into which amino acid mutations of W64A, F65A, H66A, K187A, V213A, and R214A were introduced was used in the Ss protein or the NTD region of the Ss protein. , Mouse serum was collected in the same manner.

With respect to the obtained serum, the anti-RBD antibody titer, the anti-NTD antibody titer, and the infection-enhancing antibody titer were measured in the same manner as in Examples 7 (1) to (3). For the Ss protein and the mutant protein, the anti-RBD antibody titer and the infection-enhancing antibody titer were measured in order to examine changes in the inducing ability of the neutralizing antibody and the infection-enhancing antibody. Regarding the NTD region of the Ss protein and the mutant polypeptide, the anti-NTD antibody titer and the infection-enhancing antibody titer were measured in order to examine the antibody-inducing ability against NTD by mutation. Furthermore, regarding the NTD region of the Ss protein and the mutant polypeptide, in order to examine the antibody titer of the mutant protein against NTD, mutations were made using cells in which the same mutation as the mutant polypeptide was introduced into the SARS-NTD expressing cells. The antibody titer against the polypeptide was examined. These results are shown in FIG.

FIG. 15 is a graph showing the antibody titer in serum. In FIG. 15, (A) shows the outline of the immunization method, (B) and (C) show the result using the NTD region or the mutant polypeptide of the Ss protein, and (D) shows the result of using the immunization method. The outline is shown, and (E) and (F) show the result using the S protein or the mutant protein. In FIGS. 15 (B), (C) and (E), the horizontal axis represents an immunogen and the vertical axis represents the antibody titer of each antibody. Further, in FIG. 15 (F), the horizontal axis represents an immunogen, and the vertical axis represents the ratio of the infection-enhancing antibody titer to the anti-RBD antibody titer. As shown in FIG. 15B, when the NTD region of the Ss protein was used as an immunogen, no difference was observed in the inducing ability of the antibody against the NTD region of the Ss protein and the mutant polypeptide. On the other hand, when the mutant polypeptide was used as an immunogen, the ability of the Ss protein to induce an antibody against the NTD region and the mutant polypeptide was reduced. As shown in FIG. 15 (C), when the mutant polypeptide was used as an immunogen, the inducing ability of the infection-enhancing antibody was significantly reduced as compared with the case where the NTD region of the S protein was used as an immunogen. Further, as shown in FIG. 15 (E), when the Ss protein and the mutant protein were used as immunogens, it was possible to induce the same degree of anti-RBD antibody, that is, a neutralizing antibody. On the other hand, as shown in FIG. 15 (F), when the mutant protein was used as an immunogen, the ability to induce an infection-enhancing antibody against a neutralizing antibody was significantly reduced as compared with the case where the Ss protein was used as an immunogen. Was there. From the above, it was found that the ability to induce an infection-enhancing antibody can be suppressed when administered to a subject by introducing a mutation into the recognition site of the infection-enhancing antibody. Therefore, it was presumed that the mutant protein of the present invention can suppress the induction of the infection-enhancing antibody while maintaining the inducing ability of the neutralizing antibody, thus reducing the risk of ADE in the vaccinated person.

[Example 12]
It was confirmed that the amino acid at the recognition site of the infection-enhancing antibody in the S protein suppresses the binding of the S protein to ACE2.

(1) Preparation of mutant protein-expressing cells Except for expressing a mutant protein in which any one of the amino acids (64W, 66H, 187K, 213V, 214R) at the recognition site of the infection-enhancing antibody in the Ss protein was substituted with alanine. , Mutant protein-expressing cells were prepared in the same manner as in Example 1 (2).

(2) Binding to ACE2 The obtained mutant protein-expressing cells were used, and the binding to ACE2 was measured in the same manner as in Example 1 (5) except that no antibody was added.

(2) Preparation of SARS2 S protein expression pseudotyped virus Expresses a mutant protein in which any one of the amino acids (64W, 66H, 187K, 213V, 214R) at the recognition site of the infection-enhancing antibody in the Ss protein is replaced with alanine. A pseudotyped virus expressing a mutant protein was prepared in the same manner as in Example 2 (2) above, except that the virus was allowed to express the mutant protein. In addition, as a control, a pseudotyped virus expressing the Ss protein was prepared.

(4) Infection test Next, an infection experiment was performed on ACE2 constitutively expressing cells in the same manner as in Example 2 (4) except that the pseudotype virus prepared in Example 12 (3) was used. The proportion of infected cells was measured. These results are shown in FIG.

FIG. 16 is a graph showing the binding property to ACE2 and the infection rate of pseudotyped virus. In FIG. 16, (A) is a result showing the binding property of the mutant protein to ACE2, and (B) is a graph showing the result of the infection rate of the mutant protein-expressing pseudotyped virus. As shown in FIG. 16 (A), when a mutation is introduced into the amino acid corresponding to the amino acid residue at position 64, 187, or 213 in the Ss protein, the binding to ACE2 is not suppressed, but rather the binding is not suppressed. It was enhanced. On the other hand, when a mutation was introduced into the amino acid corresponding to the amino acid residue at the 65th, 66th, or 214th position of the Ss protein, the binding to ACE2 was suppressed. Further, as shown in FIG. 16 (B), when a mutation is introduced into the amino acid corresponding to the amino acid residue at the 64-position, 66-position, 187-position, 213-position, or 214-position in the Ss protein, the ACE2-expressing cell is introduced. The infection was not suppressed, but rather the infection was enhanced. On the other hand, when a mutation was introduced into the amino acid corresponding to the amino acid residue at position 65, 66, or 214 of the Ss protein, infection to ACE2-expressing cells was suppressed. These results suggest that the amino acid at the recognition site of the infection-enhancing antibody in the S protein suppresses the binding of the S protein to ACE2 and the infection mediated by the amino acid.

[Example 13]
It was confirmed that when the infection-enhancing antibody binds to the S protein, the conformation of the S protein is changed and the binding property of ACE2 is enhanced.

(1) Preparation of S protein-expressing cells The SR / PP / x1 mutant described in Reference 13 below is fixed in a closed (RBD-down) RBD region because a disulfide bond is formed in the S protein. It is known that it does not become an open (RBD-up) state. Therefore, using the SR / PP / x1 mutant, it was confirmed that the infection-enhancing antibody enhances the binding property of ACE2 by changing the conformation of the S protein. Specifically, first, the wild type was used in the same manner as in Example 1 (3) except that the polynucleotide (SEQ ID NO: 122) encoding the wild type S protein consisting of the amino acid sequence of SEQ ID NO: 121 below was used. Cells expressing S protein were prepared. In addition, in the wild-type S protein-expressing cells, cells expressing mutant proteins into which amino acid mutations of S383C, D985C, K986P, and V987P were introduced were prepared.
Reference 13: Xiaoli Xiong et.al., “A thermostable, closed SARS-CoV-2 spike protein trimer”, Nature Structural & Molecular Biology, vol. 27, pages 934-941

Wild-type S protein (SEQ ID NO: 121)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVCPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLCPPEAEVQIDRLITGR LQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

A polynucleotide encoding a wild-type S protein (SEQ ID NO: 122)
5'--3'

(2) Confirmation of ACE2 binding property After adding 10 μl of the clone 2490 or clone 8D2 (final concentration 3 μg / ml) to the wild-type S protein-expressing cell or mutant S protein-expressing cell of Example 13 (1), 10 μl of the biotinylated ACE2-Fc fusion protein (final concentration 2.7 μg / ml) obtained in Example 1 (4) was added. Then, the obtained reaction solution was incubated at 4 ° C. for 30 minutes to react. To 15 μl of the reaction solution after the reaction, 10 μl of APC-labeled streptavidin (final concentration 2 μg / ml) was added and stained, and the obtained cells were analyzed using the flow cytometer. As a result, the binding of the full-length Ss protein-expressing cells to ACE2 in the presence of each antibody was confirmed. Controls were measured in the same manner except that the antibody sample was not added. These results are shown in FIG.

FIG. 17 is a graph showing the binding of wild-type S protein-expressing cells or mutant S protein-expressing cells to ACE2 in the presence of each antibody sample. In FIG. 17, the horizontal axis represents the type of S protein, and the vertical axis represents ACE binding (MFI). As shown in FIG. 17, when wild-type S protein-expressing cells were used, the binding property of S protein to ACE2 was enhanced in the presence of clone 2490 and clone 8D2. On the other hand, when mutant S protein-expressing cells were used, the binding property of S protein to ACE2 did not change even in the presence of clone 2490 and clone 8D2. As described above, in the S-R / PP / x1 mutant expressed by the mutant S protein-expressing cell, the RBD region is fixed in the closed (RBD-down) and not open (RBD-up). Therefore, the infection-enhancing antibody changes the conformation of the S protein from RBD-down to RBD-up by binding to the above-mentioned predetermined position, thereby making the S protein bind to ACE2. It turned out to be increasing.

Although the present invention has been described above with reference to the embodiments and examples, the present invention 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 structure and details of the present invention within the scope of the present invention.

<Additional notes>
Some or all of the above embodiments and examples may be described as, but not limited to, the following appendices.
<Mutant of Peplomer protein>
(Appendix 1)
A variant of the Spike protein of SARS-CoV-2, which is a protein consisting of the following (Sm) amino acid sequence:
(Sm) The amino acid sequence of the following (Sm1), (Sm2), or (Sm3):
(Sm1) In the amino acid sequence of the Spike protein (Sw protein) of wild SARS-CoV-2, the 30th, 32nd, and 64th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, It has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 65, 66, 186, 187, 211, 213, 214, 216, and 217. Amino acid sequence;
In the amino acid sequence of (Sm2) (Sm1), an amino acid sequence having 1 to 127 insertions, additions, substitutions and / or deletions and in which the mutated amino acid of (Sm1) is maintained;
(Sm3) An amino acid sequence having 90% or more identity with respect to the amino acid sequence of (Sm1) and maintaining the mutated amino acid of (Sm1).
(Appendix 2)
The amino acid sequence of (Sm1) is an amino acid sequence having a mutation in the amino acid corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein in the Sw protein. Variant.
(Appendix 3)
The amino acid sequence of (Sm1) is an amino acid sequence having a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein in the Sw protein, Appendix 1 or 2 The variant described.
(Appendix 4)
The amino acid sequence of (Sm1) is at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 of the Ss protein in the Sw protein. The variant according to any one of Supplementary note 1 to 3, which is an amino acid sequence having a mutation in the amino acid corresponding to the above.
(Appendix 5)
The amino acid sequence of (Sm1) is, in the Sw protein, in the Ss protein, at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64. The variant according to any one of Supplementary notes 1 to 4, which is an amino acid sequence having a mutation in the amino acid corresponding to the amino acid residue at the position, 65th, 66th, 187th, 213rd, and 214th positions.
(Appendix 6)
The mutation is a group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 in the Ss protein. The variant according to any of Supplementary notes 1 to 5, which reduces binding to an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from.
(Appendix 7)
The variant according to Appendix 6, wherein the antibody is an antibody containing the following (H) heavy dispersion variable region and the following (L) light chain variable region:
(H) Heavy chain variable region:
Amino acid sequence of SEQ ID NO: 8 (X ₁ X ₂ YX ₃ X ₄ X ₅ ), The amino acid sequence of heavy chain complementarity determining regions (HCDR) 1, SEQ ID NO: 9 (X). ₆ IX ₇ X ₈ X ₉ X ₁₀ GX ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ HCDR2 containing or consisting of), and the amino acid sequence of SEQ ID NO: 10 (X). ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ X ₃₂ X ₃₃ X ₃₄ X ₃₅ X ₃₆ X ₃₇ X ₃₈ X ₃₉ DX ₄₀ ), Or a heavy chain variable region comprising HCDR3 consisting of.
X ₁ Is S or T,
X ₂ Does not exist or is N,
X ₃ Is A, D, or W,
X ₄ Is M or W
X ₅ Is G, H, N, or S,
X ₆ Is A, D, N, T, V, or W,
X ₇ Is G, H, N, or S,
X ₈ Is Q, T, or Y,
X ₉ Is A, D, S, or N,
X ₁₀ Does not exist or is T,
X ₁₁ Is D, G, I, N, or S,
X ₁₂ Is E, N, P, S or T,
X ₁₃ Does not exist or is K,
X ₁₄ Is T or Y,
X ₁₅ Is A, N, P, or V,
X ₁₆ Is D, G, P, or Q,
X ₁₇ Is G or S
X ₁₈ Is F, L, or V,
X ₁₉ Is K, R, or T,
X ₂₀ Is G or S
X ₂₁ Does not exist or is A,
X ₂₂ Is D or R
X ₂₃ Is F, P, Q, T, or W,
X ₂₄ Does not exist or is D, E, G, or Y,
X ₂₅ Is G, S, Q, W, or Y,
X ₂₆ Is D, F, G, or L,
X ₂₇ Is I, L, N, R, or Y,
X ₂₈ Does not exist or is G, L, P, T, W, or Y,
X ₂₉ Does not exist or is A, G, Q, S, or T,
X ₃₀ Does not exist or is A, D, G, H, or M,
X ₃₁ Does not exist or is S, V, or Y,
X ₃₂ Does not exist or is E or S,
X ₃₃ Does not exist or is L, S, W, or Y,
X ₃₄ Does not exist or is F, G, T, or Y,
X ₃₅ Does not exist or is A, G, L, or Y,
X ₃₆ Does not exist or is A, T, or Y,
X ₃₇ Is F or Y,
X ₃₈ Does not exist or is C or G,
X ₃₉ Does not exist or is M,
X ₄₀ Is F, I, or V;
(L) Light chain variable region:
Amino acid sequence of SEQ ID NO: 11 (X ₄₁ X ₄₂ SX ₄₃ X ₄₄ X ₄₅ X ₄₆ X ₄₇ X ₄₈ X ₄₉ X ₅₀ X ₅₁ ), The amino acid sequence (X) of light chain complementarity determining regions (LCDR) 1, SEQ ID NO: 12. ₅₂ X ₅₃ SX ₅₄ X ₅₅ X ₅₆ X ₅₇ The amino acid sequence (X) of LCDR2 containing or consisting of, and SEQ ID NO: 13. ₅₈ QX ₅₉ X ₆₀ X ₆₁ X ₆₂ X ₆₃ X ₆₄ X ₆₅ X ₆₆ A light chain variable region comprising or comprising LCDR3 comprising or consisting of T).
X ₄₁ Is K, Q, or R,
X ₄₂ Does not exist or is A or S,
X ₄₃ Does not exist or is Q or V,
X ₄₄ Is G or S
X ₄₅ Does not exist or is I, L, or V,
X ₄₆ Does not exist or is L, R, or S,
X ₄₇ Does not exist or is H,
X ₄₈ Does not exist or is N or S,
X ₄₉ Does not exist or is D, N, Y, or W,
X ₅₀ Does not exist or is G or L
X ₅₁ Does not exist or is A, G, or K,
X ₅₂ Is A, D, E, G, or K,
X ₅₃ Is A or V,
X ₅₄ Is N, S, or T,
X ₅₅ Is L or R
X ₅₆ Is A, E, F, or Q,
X ₅₇ Is S or T,
X ₅₈ Is L, M, or Q,
X ₅₉ Is A, H, L, S, or Y,
X ₆₀ Is G, I, or N,
X ₆₁ Is N, S, or Q,
X ₆₂ Does not exist or is F, W, or Y,
X ₆₃ Does not exist or is P, S, or W,
X ₆₄ Is P or R
X ₆₅ Does not exist or is L,
X ₆₆ Does not exist or is I.
(Appendix 8)
The variant according to Appendix 6 or 7, wherein the antibody is at least one antibody selected from the group consisting of the following (1) to (5) and (6).
(1) Consists of heavy chain complementarity determining regions (HCDR) consisting of the amino acid sequence of SEQ ID NO: 14 (SYAMH), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 15 (VISYDGSNKYYADSVKG), and amino acid sequence of SEQ ID NO: 16 (DQEWFRELFLFDY). Heavy chain variable regions containing HCDR3, light chain complementarity determining regions (LCDRs) consisting of the amino acid sequence of SEQ ID NO: 17 (RASQGISSWLA), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 18 (DASSLQS), and amino acids of SEQ ID NO: 19. An amino acid comprising a light chain variable region comprising LCDR3 consisting of sequences (QQANSFPPT);
(2) A heavy chain variable region containing HCDR1 consisting of the amino acid sequence of SEQ ID NO: 20 (SYWMN), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 21 (NINQDGGEKYYVDSVRG), and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 22 (DPYDLYGDYGGTFDY). An antibody comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 23 (RASQSVSSNLA), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 24 (GASTRAT), and a light chain variable region containing LCDR3 consisting of the amino acid sequence of SEQ ID NO: 25 (QQYNNWWRT). ;
(3) A heavy chain variable region containing HCDR1 consisting of the amino acid sequence of SEQ ID NO: 26 (SYWMS), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 27 (NINQDGSEKYYVDSVKG), and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 28 (DWDYDILTGSWFGAFDI). An antibody comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 29 (RASQGIRNDLG), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 30 (AASSLQS), and a light chain variable region containing LCDR3 consisting of the amino acid sequence of SEQ ID NO: 31 (LQHNSYPLT). ;
(4) A heavy chain variable region containing HCDR1 consisting of the amino acid sequence of SEQ ID NO: 32 (TYAMN), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 33 (WINTNTGNPTYAQGFTG), and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 34 (DQDSGYPTYYYYYMDV). An antibody comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 35 (KSSQSLLHSDGK), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 36 (EVSNRFS), and a light chain variable region containing LCDR3 consisting of the amino acid sequence of SEQ ID NO: 37 (MQSIQPPLT). ;
(5) A heavy chain variable region containing HCDR1 consisting of the amino acid sequence of SEQ ID NO: 38 (SYAMH), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (DISYDGSEKYYADSVKG), and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 40 (DFGGDNTAMVEYFFDF). An antibody comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 41 (RASQSISSWLA), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 42 (KASSLES), and a light chain variable region containing LCDR3 consisting of the amino acid sequence of SEQ ID NO: 43 (QQYNSYSPT). ;
(6) A heavy chain variable region containing HCDR1 consisting of the amino acid sequence of SEQ ID NO: 44 (SYDMH), HCDR2 consisting of the amino acid sequence of SEQ ID NO: 45 (AIGTAGDTYYPGSVKG), and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 46 (ADPYQLLGQHYYYGMDV). An antibody comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 47 (QSVSSSYLA), LCDR2 consisting of the amino acid sequence of SEQ ID NO: 48 (GASSRAT), and a light chain variable region containing LCDR3 consisting of the amino acid sequence of SEQ ID NO: 49 (QQYGSSPLIT). ..
(Appendix 9)
The antibody is at least one selected from the group consisting of the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. Variant according to any one of Appendix 6 to 8, which is one antibody:
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109.
(Appendix 10)
The amino acid sequence of (Sm1) is selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Sw protein. The variant according to any one of Supplementary notes 1 to 9, which is an amino acid sequence having a mutation in the amino acid corresponding to at least one amino acid residue.
(Appendix 11)
The amino acid sequence of (Sm1) is selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Sw protein. The variant according to any one of Appendix 1 to 10, which is an amino acid sequence having a mutation in the amino acid corresponding to at least one amino acid residue.
(Appendix 12)
The amino acid sequence of (Sm1) is, in the Sw protein, in the Ss protein, W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, And the variant according to any one of Appendix 1 to 11, which is an amino acid sequence having a mutation in the amino acid corresponding to the amino acid residue of R214.
(Appendix 13)
The variant according to any of Supplementary notes 1 to 12, wherein the mutation is a substitution.
(Appendix 14)
The mutant according to any one of Supplementary notes 1 to 13, wherein the mutation is an alanine substitution.
(Appendix 15)
The amino acid sequence of (Sm1) is selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Sw protein. The variant according to any of Supplementary notes 1 to 14, which is an amino acid sequence having an alanine substitution in the amino acid corresponding to at least one amino acid residue.
(Appendix 16)
The amino acid sequence of (Sm1) corresponds to at least one amino acid residue selected from the group consisting of amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein in the Sw protein. The variant according to any of Supplementary notes 1 to 15, which is an amino acid sequence having an alanine substitution in.
(Appendix 17)
The amino acid sequence of (Sm1) is, in the Sw protein, in the Ss protein, W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, And the variant according to any of Appendix 1-16, which is an amino acid sequence having an alanine substitution in the amino acid corresponding to the amino acid residue of R214.
(Appendix 18)
The amino acid sequence of (Sm1) is the amino acid sequence of SEQ ID NO: 1, at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, The variant according to any of Supplementary notes 1 to 17, which is an amino acid sequence having a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acid at position 217.
<Mutant polypeptide>
(Appendix 19)
Containing a partial polypeptide having a partial sequence of a variant of the Peplomer protein according to any of Supplementary notes 1-18.
The partial polypeptide is a mutated polypeptide comprising at least one mutated amino acid in the variant.
(Appendix 20)
The mutant polypeptide according to Appendix 19, which comprises the N-terminal domain (NTD) and / or receptor binding domain (RBD) of the variant of the Peplomer protein according to any of Appendix 1-18.
<SARS-CoV-2>
(Appendix 21)
A mutant virus of SARS-CoV-2, which comprises a variant of the Peplomer protein according to any of Supplementary notes 1 to 18.
(Appendix 22)
The mutant virus according to Appendix 21, which is an attenuated virus or an inactivated virus.
<VLP>
(Appendix 23)
A virus-like particle comprising a variant of the Peplomer protein according to any one of Supplements 1 to 18 and / or a mutant polypeptide according to Supplement 19 or 20.
<Nucleic acid>
(Appendix 24)
A nucleic acid comprising a nucleic acid molecule encoding a variant of the Peplomer protein according to any of supplements 1 to 18 and / or a nucleic acid molecule encoding the mutant polypeptide according to supplement 19 or 20.
<Expression vector>
(Appendix 25)
An expression vector containing the nucleic acid according to Appendix 24.
(Appendix 26)
The expression vector according to Appendix 25, wherein the expression vector is a viral vector.
<Pharmaceutical composition>
(Appendix 27)
A variant of the Peplomer protein according to any one of Appendix 1 to 18, a mutant polypeptide according to Appendix 19 or 20, a mutant virus according to Appendix 21 or 22, a virus-like particle according to Appendix 23, a nucleic acid according to Appendix 24, and / Or a pharmaceutical composition comprising the expression vector according to Appendix 25 or 26 and a pharmaceutically acceptable carrier.
(Appendix 28)
27. The pharmaceutical composition according to Appendix 27, which comprises an immunostimulatory agent.
<Method for producing mutants of Peplomer protein>
(Appendix 29)
A method for producing a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (St protein) of the target SARS-CoV-2, the 30th, 32nd, 64th, 65th, and 66th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A mutation step of introducing a mutation into an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. Including, method.
(Appendix 30)
The method according to Appendix 29, wherein in the mutation step, a mutation is introduced into the amino acid corresponding to the amino acid residue at positions 64, 65, and / or 66 in the Ss protein in the St protein.
(Appendix 31)
The method according to Appendix 29 or 30, wherein in the mutation step, a mutation is introduced into the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the St protein.
(Appendix 32)
In the mutation step, in the St protein, the amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein. The method according to any of Supplementary note 29 to 31, wherein the mutation is introduced into.
(Appendix 33)
In the mutation step, in the St protein, the 64th, 66th, 213rd, and 214th, 64th, 66th, 187th, 213rd, and 214th, or 64th, 65th positions of the Ss protein are used. , 66, 187, 213, and 214, wherein mutations are introduced into the amino acids corresponding to the amino acid residues, according to any of Appendix 29-32.
(Appendix 34)
In the mutation step, in the St protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th positions of the Ss protein are used. 28. The method of any of Appendix 29-33, wherein a mutation is introduced that reduces binding to an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at the position.
(Appendix 35)
34. The method of Appendix 34, wherein the antibody is an antibody comprising the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 36)
The variant according to Appendix 34 or 35, wherein the antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 37)
The antibody is at least one selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. Variants according to any of Appendix 34-36, which are two antibodies:
(Appendix 38)
In the mutation step, in the St protein, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. 28. The method of any of Appendix 29-37, wherein a mutation is introduced into one amino acid residue and the corresponding amino acid.
(Appendix 39)
In the mutation step, the mutation is introduced into the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids W64, F65, H66, K187, V213, and R214 in the St protein. The method according to any one of Appendix 29 to 38.
(Appendix 40)
In the mutation step, in the St protein, the amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein. 28. The method of any of Appendix 29-39, wherein a mutation is introduced into the amino acid corresponding to the residue.
(Appendix 41)
The method according to any of Appendix 29-40, wherein the mutation is a substitution.
(Appendix 42)
The method according to any of Appendix 29-41, wherein the mutation is an alanine substitution.
(Appendix 43)
In the mutation step, in the St protein, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The method according to any of Appendix 29-42, wherein the amino acid corresponding to one amino acid residue is replaced with alanine.
(Appendix 44)
In the mutation step, in the St protein, the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein is replaced with alanine. The method according to any one of Appendix 29 to 43.
(Appendix 45)
In the mutation step, in the St protein, amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any of Appendix 29-44, wherein the amino acid corresponding to the residue is replaced with alanine.
(Appendix 46)
The mutation step
A production step of introducing a mutation into the amino acid sequence of the St protein to generate an amino acid sequence of a candidate protein into which the mutation has been introduced into the amino acid sequence of the St protein.
The candidate protein comprises amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. A selection step of selecting a candidate protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group as a variant of the Spike protein having a reduced ability to induce an antibody that enhances the binding ability to ACE2. ,
The method according to any of Supplementary note 29 to 45, comprising.
(Appendix 47)
In the production step, a mutation is introduced into the polynucleotide encoding the amino acid sequence of the St protein, and a polynucleotide encoding the amino acid sequence of the candidate protein in which the mutation is introduced into the amino acid sequence of the St protein is generated. 46. The method of Appendix 46, which produces the candidate protein.
(Appendix 48)
The method according to Supplementary note 46 or 47, wherein in the production step, a mutation is introduced so that the amino acid sequence of the candidate protein has 90% or more identity with respect to the amino acid sequence of the St protein.
(Appendix 49)
In the selection step, a candidate protein having a mutation in the amino acid corresponding to the amino acid residue at positions 64, 65, and / or 66 in the Ss protein is selected as the variant from the candidate protein. The method according to any of 46 to 48.
(Appendix 50)
In the selection step, a candidate protein having a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein is selected as the mutant from the candidate protein. The method according to any one of 46 to 49.
(Appendix 51)
In the selection step, the amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein from the candidate protein. The method according to any one of Appendix 46 to 50, wherein a candidate protein having a mutation in is selected as the mutant.
(Appendix 52)
In the selection step, from the candidate proteins, the 64th, 66th, 213rd, and 214th, 64th, 66th, 187th, 213th, and 214th, or 64th, 65th positions of the Ss protein are used. , 66, 187, 213, and 214. The method according to any one of Appendix 46 to 51, wherein a candidate protein having a mutation in the amino acid corresponding to the amino acid residue is selected as the variant.
(Appendix 53)
In the selection step, from the candidate proteins, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th positions of the Ss protein are selected. Any of Appendix 46 to 52, wherein a candidate protein having a mutation that reduces binding to an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at the position is selected as the variant. The method described in Crab.
(Appendix 54)
The method according to Supplementary note 53, wherein the antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 55)
The method according to Appendix 53 or 54, wherein the antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 56)
The antibody is at least one selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The method according to any of Appendix 53-55, which is one antibody.
(Appendix 57)
In the selection step, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein from the candidate proteins. The method according to any one of Appendix 46 to 56, wherein a candidate protein having a mutation in an amino acid corresponding to one amino acid residue is selected as the mutant.
(Appendix 58)
In the selection step, the candidate protein has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any one of Appendix 46 to 57, wherein the candidate protein is selected as the mutant.
(Appendix 59)
In the selection step, from the candidate proteins, amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any one of Appendix 46 to 58, wherein a candidate protein having a mutation in the amino acid corresponding to the residue is selected as the mutant.
(Appendix 60)
The method according to any of Appendix 46-59, wherein the mutation is a substitution.
(Appendix 61)
The variant according to any of Supplementary notes 46 to 60, wherein the mutation is an alanine substitution.
(Appendix 62)
In the selection step, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein from the candidate proteins. The method according to any one of Appendix 46 to 61, wherein a candidate protein having an alanine substitution in the amino acid corresponding to one amino acid residue is selected as the variant.
(Appendix 63)
In the selection step, alanine substitution is performed on the amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein from the candidate protein. The method according to any one of Appendix 46 to 62, wherein the candidate protein to have is selected as the variant.
(Appendix 64)
In the selection step, from the candidate proteins, amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any one of Appendix 46 to 63, wherein a candidate protein having an alanine substitution in the amino acid corresponding to the residue is selected as the variant.
(Appendix 65)
The method according to any one of Appendix 46 to 64, wherein the St protein is a protein consisting of the amino acid sequence of SEQ ID NO: 1.
<Screening method for Spike protein>
(Appendix 66)
A method for screening a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (Sc protein) of the candidate SARS-CoV-2, the 30th, 32nd, 64th, 65th, and 66th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A Sc protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. A method comprising a selection step of selecting as the variant.
(Appendix 67)
In the selection step, a Sc protein having a mutation in an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein is selected as the variant from the Sc protein. 66 The method according to the description.
(Appendix 68)
In the selection step, a Sc protein having a mutation in an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Ss protein is selected as the mutant from the Sc protein. 66 or 67.
(Appendix 69)
In the selection step, the amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein from the Sc protein. The method according to any of Supplementary note 66 to 68, wherein the Sc protein having a mutation in is selected as the mutant.
(Appendix 70)
In the selection step, from the Sc protein, the 64th, 66th, 213rd, and 214th, 64th, 66th, 187th, 213rd, and 214th, or 64th, 65th positions of the Ss protein are used. , 66, 187, 213, and 214. The method according to any one of Supplementary note 66 to 69, wherein a Sc protein having a mutation in an amino acid corresponding to the amino acid residue is selected as the mutant.
(Appendix 71)
In the selection step, from the Sc protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, 213th, 214th, 216th, and 217th positions of the Ss protein are used. Any of Appendix 66 to 70, wherein a Sc protein having a mutation that reduces binding to an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at the position is selected as the variant. The method described in Crab.
(Appendix 72)
The method according to Supplementary note 71, wherein the antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 73)
The method according to Appendix 71 or 72, wherein the antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 74)
The antibody is at least one selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The method according to any of Appendix 71-73, which is one antibody.
(Appendix 75)
In the selection step, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein from the Sc protein. The method according to any one of Appendix 66 to 74, wherein a Sc protein having a mutation in an amino acid corresponding to one amino acid residue is selected as the mutant.
(Appendix 76)
In the selection step, the Sc protein has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any of Supplementary note 66 to 75, wherein the Sc protein is selected as the mutant.
(Appendix 77)
In the selection step, the amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein from the Sc protein. The method according to any one of Appendix 66 to 77, wherein a Sc protein having a mutation in the amino acid corresponding to the residue is selected as the mutant.
(Appendix 78)
The method according to any of Supplementary note 66-77, wherein the mutation is a substitution.
(Appendix 79)
The variant according to any of Appendix 66-78, wherein the mutation is an alanine substitution.
(Appendix 80)
In the selection step, at least one selected from the group consisting of the amino acids N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein from the Sc protein. The method according to any of Supplementary note 66 to 79, wherein a Sc protein having an alanine substitution in the amino acid corresponding to one amino acid residue is selected as the variant.
(Appendix 81)
In the selection step, alanine substitution is performed from the Sc protein to the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids W64, F65, H66, K187, V213, and R214 in the Ss protein. The method according to any one of Appendix 66 to 80, wherein the Sc protein having is selected as the mutant.
(Appendix 82)
In the selection step, the amino acids of W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, and R214 in the Ss protein from the Sc protein. The method according to any one of Appendix 66 to 81, wherein a Sc protein having an alanine substitution in the amino acid corresponding to the residue is selected as the variant.
<Treatment method>
(Appendix 83)
The treatment method of a subject, wherein the subject includes a mutant of the Peplomer protein according to any one of Supplementary note 1 to 18, a mutant polypeptide according to Supplementary note 19 or 20, a mutant virus according to Supplementary note 21 or 22, and Supplementary note 23. The method of using the virus-like particles of the above, the nucleic acid of Appendix 24, the expression vector of Appendix 25 or 26, and / or the pharmaceutical composition of Appendix 27 or 28.
(Appendix 84)
The subject includes a variant of the Peplomer protein according to any one of Supplements 1 to 18, a mutant polypeptide according to Supplement 19 or 20, a mutant virus according to Supplement 21 or 22, a virus-like particle according to Supplement 23, and a description 24. 83. The method of Addendum 83, comprising the step of administering the nucleic acid of, the expression vector of Addendum 25 or 26, and / or the pharmaceutical composition of Addendum 27 or 28.
(Appendix 85)
The method according to Appendix 83 or 84, wherein the subject is a human.
(Appendix 86)
The method according to any of Appendix 83-85, wherein the procedure is vaccination.
(Appendix 87)
The method according to any of Appendix 83-86, wherein the treatment is the induction of an antibody against the SARS-CoV-2 Spike protein or a partial polypeptide thereof.
(Appendix 88)
The treatment reduced the induction of antibodies that enhance the binding activity of SARS-CoV-2's Spike protein to angiotensin converting enzyme 2 (ACE2), an antibody against SARS-CoV-2's Spike protein or a partial polypeptide thereof. The method according to any one of Appendix 83 to 87, which is the induction of the above.
<First infection enhancement marker>
(Appendix 89)
In the wild type SARS-CoV-2 Spike protein (Sw protein), the 30th, 32nd, 64th, 65th, and 66th positions in the standard SARS-CoV-2 Spike protein (Ss protein, SEQ ID NO: 1), SARS-CoV, an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 186, 187, 211, 213, 214, 216, and 217. -2 infection-enhancing marker.
(Appendix 90)
The infection-enhancing marker according to Appendix 89, wherein the antibody recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein.
(Appendix 91)
The infection-enhancing marker according to Appendix 89 or 90, wherein the antibody recognizes an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Ss protein.
(Appendix 92)
The antibody recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein, Appendix 89. To the infection enhancing marker according to any one of 91.
(Appendix 93)
The antibody is located at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65, 66, and 187 of the Ss protein. The infection enhancing marker according to any one of Supplementary note 89 to 92, which recognizes an amino acid corresponding to an amino acid residue at positions 2, 213, and 214.
(Appendix 94)
The antibody corresponds to an amino acid corresponding to at least one amino acid residue selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The infection-enhancing marker according to any one of Appendix 89 to 93, which recognizes.
(Appendix 95)
The infection-enhancing marker according to any one of Supplementary note 89 to 94, wherein the antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 96)
The infection-enhancing marker according to any one of Supplementary note 89 to 95, wherein the Sw protein is the Ss protein.
<Second infection enhancement marker>
(Appendix 97)
It is a competitive antibody that competes with the reference antibody for binding of wild-type SARS-CoV-2 to the Spike protein (Sw protein).
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. A marker for enhancing infection of SARS-CoV-2, which is an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, and 217. ..
(Appendix 98)
The infection enhancing marker according to Appendix 97, wherein the antibody recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein in the Sw protein.
(Appendix 99)
The infection-enhancing marker according to Appendix 97 or 98, wherein the antibody recognizes an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Sw protein.
(Appendix 100)
The antibody comprises an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. Recognize, the infection-enhancing marker according to any of Appendix 97-99.
(Appendix 101)
The antibody comprises the 64th, 66th, 213rd, and 214th, 64th, 66th, 187th, 213rd, and 214th, or 64th, 65th positions of the Ss protein in the Sw protein. The enhancement marker according to any one of Appendix 97 to 100, which recognizes amino acids corresponding to amino acid residues at positions 66, 187, 213, and 214.
(Appendix 102)
The antibody is the residue of at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Sw protein. The infection-enhancing marker according to any one of Appendix 97 to 101, which recognizes the amino acid corresponding to the group.
(Appendix 103)
The infection enhancing marker according to any one of Supplementary note 97 to 102, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 104)
The infection enhancing marker according to any one of Supplementary note 97 to 103, wherein the reference antibody is at least one antibody selected from the group consisting of the following (1) to (5) and (6).
(Appendix 105)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The infection-enhancing marker according to any one of Appendix 97 to 104, which is one antibody.
(Appendix 106)
The infection-enhancing marker according to any one of Supplementary note 97 to 105, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 107)
The infection-enhancing marker according to any one of Supplementary note 97 to 106, wherein the competing antibody enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein.
(Appendix 108)
The infection-enhancing marker according to any one of Supplementary note 97 to 107, wherein the Sw protein is the Ss protein.
<Third infection enhancement marker>
(Appendix 109)
It binds to the Spike protein (Sw protein) of wild-type SARS-CoV-2 and
In the Sw protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. SARS-CoV-2, an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 214, 216, and 217. marker.
(Appendix 110)
The infection-enhancing marker according to Appendix 109, wherein the mutant protein has a mutation in the amino acid corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein.
(Appendix 111)
The infection-enhancing marker according to Appendix 109 or 110, wherein the mutant protein has a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein.
(Appendix 112)
The mutant protein has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein. The infection enhancing marker according to any one of Supplementary notes 109 to 111.
(Appendix 113)
The mutant protein is the 64-position, 66-position, 213-position, and 214-position, 64-position, 66-position, 187-position, 213-position, and 214-position, or 64-position, 65-position, 66-position, 187-position in the Ss protein. The infection-enhancing marker according to any one of Appendix 109 to 112, which has a mutation in the amino acid corresponding to the amino acid residue at positions 213 and 214.
(Appendix 114)
The mutant protein corresponds to at least one amino acid residue selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The infection-enhancing marker according to any one of Appendix 109 to 113, which recognizes an amino acid.
(Appendix 115)
The infection-enhancing marker according to any one of Supplementary note 109 to 114, wherein the antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 116)
The infection-enhancing marker according to any one of Supplementary notes 109 to 115, wherein the Sw protein is the Ss protein.
<Fourth infection enhancement marker>
(Appendix 117)
It is an antibody that competes with the reference antibody for binding to the Spike protein (Sw protein) of wild-type SARS-CoV-2.
The reference antibody is
Recognize the Sw protein
In the Sw protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. A marker for enhancing infection of SARS-CoV-2, which is an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 214, 216, and 217.
(Appendix 118)
The infection-enhancing marker according to Appendix 117, wherein the mutant protein has a mutation in the amino acid corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein.
(Appendix 119)
The infection-enhancing marker according to Appendix 117 or 118, wherein the mutant protein has a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein.
(Appendix 120)
The mutant protein has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Ss protein. The infection enhancing marker according to any one of Supplementary notes 117 to 119.
(Appendix 121)
The mutant protein is the 64-position, 66-position, 213-position, and 214-position, 64-position, 66-position, 187-position, 213-position, and 214-position, or 64-position, 65-position, 66-position, 187-position in the Ss protein. The infection-enhancing marker according to any one of Appendix 117 to 120, which has a mutation in the amino acid corresponding to the amino acid residue at positions 213 and 214.
(Appendix 122)
The mutant protein corresponds to at least one amino acid residue selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The infection-enhancing marker according to any one of Appendix 117 to 121, which recognizes an amino acid.
(Appendix 123)
The infection-enhancing marker according to any one of Supplementary note 117 to 122, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 124)
The infection-enhancing marker according to any one of Supplementary note 117 to 123, wherein the competing antibody enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein.
(Appendix 125)
The infection-enhancing marker according to any one of Supplementary notes 117 to 124, wherein the Sw protein is the Ss protein.
<First method for detecting an infection-enhancing antibody>
(Appendix 126)
Regarding the biological sample of the subject, in the Spike protein (Sw protein) of wild-type SARS-CoV-2, the 30th, 32nd, and 64th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. Recognizes amino acids corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 65, 66, 186, 187, 211, 213, 214, 216, and 217. A method for detecting an infection-enhancing antibody of SARS-CoV-2, which comprises a detection step for detecting an antibody to be used.
(Appendix 127)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
Sw protein, a group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. The mutant protein or its N-terminal domain in which a mutation has been introduced into an amino acid corresponding to at least one amino acid residue selected from the above is brought into contact with the biological sample, and the mutant protein or its N-terminal domain and the biological sample are brought into contact with each other. 126. The detection method according to Appendix 126, which comprises a second detection step of detecting binding to the antibody in.
(Appendix 128)
The detection step is
By comparing the detection result of the antibody against the Sw protein or its N-terminal domain in the first detection step with the detection result of the antibody against the mutant protein or its N-terminal domain in the second detection step, the antibody can be obtained. The detection method according to Appendix 127, which comprises a comparison step for detection.
(Appendix 129)
In the detection step, in the Sw protein, an antibody that recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein is detected, according to any one of Supplementary note 126 to 128. The detection method described.
(Appendix 130)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the first mutant protein in which mutations have been introduced into the amino acids corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein or the N-terminal domain thereof, and the biological sample. 129. The detection method according to Appendix 129, which comprises a second detection step of detecting the binding of the first mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 131)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the first mutant protein or the N-terminal domain thereof in the second detection step. The detection method according to Appendix 130, which includes.
(Appendix 132)
In the detection step, in the Sw protein, an antibody that recognizes an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Ss protein is detected, according to any one of Supplementary formulas 126 to 128. The detection method described.
(Appendix 133)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the second mutant protein in which the mutation was introduced into the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein or the N-terminal domain thereof, and the biological sample. 132. The detection method according to Supplementary note 132, which comprises a second detection step of detecting the binding of the second mutant protein or its N-terminal domain to the antibody in the biological sample.
(Appendix 134)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the second mutant protein or the N-terminal domain thereof in the second detection step. Included, the detection method according to Appendix 133.
(Appendix 135)
In the detection step, in the Sw protein, in the Ss protein, at positions 64, 66, 213, and / or 214, 64, 66, 187, 213, and / or 214, or 64. The detection method according to any of Supplementary note 126 to 134, which detects an antibody that recognizes an amino acid corresponding to an amino acid residue at the position, 65, 66, 187, 213, and / or 214.
(Appendix 136)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the third mutant protein in which mutations are introduced into the amino acids at positions 64, 65, 66, 187, 213, and / or 214 of the Ss protein or the N-terminal domain thereof, and the N-terminal domain thereof. The detection method according to Appendix 135, which comprises a second detection step of contacting a biological sample to detect the binding of the third mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 137)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the third mutant protein or the N-terminal domain thereof in the second detection step. Included, the detection method according to Appendix 136.
(Appendix 138)
Addendums 126 to 137, which include an evaluation step of evaluating whether the antibody detected in the detection step enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein. The detection method described in any of the above.
(Appendix 139)
In the detection step, in the Sw protein, the amino acid corresponding to the amino acid residue of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and / or P217 in the Ss protein was added. The detection method according to any one of Appendix 126 to 138, which detects an antibody to be recognized.
(Appendix 140)
The detection method according to any one of Supplementary notes 126 to 139, wherein the Sw protein is the Ss protein.
<Detection method of second infection-enhancing antibody>
(Appendix 141)
A detection step of detecting a competing antibody that competes with a reference antibody for binding of wild-type SARS-CoV-2 to a Spike protein (Sw protein) in a biological sample of a subject is included.
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. SARS-CoV-2 infection-enhancing antibody, which is an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, and 217. Detection method.
(Appendix 142)
The detection method according to Appendix 141, wherein the reference antibody recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein in the Sw protein.
(Appendix 143)
The detection method according to Appendix 141 or 142, wherein the reference antibody recognizes an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Sw protein.
(Appendix 144)
The reference antibody is an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. The detection method according to any one of Appendix 141 to 143, which recognizes.
(Appendix 145)
The reference antibody is the Sw protein at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65 in the Ss protein. , 66th, 187th, 213rd, and 214th positions, the detection method according to any one of Appendix 141 to 144, which recognizes the amino acid corresponding to the amino acid residue.
(Appendix 146)
In the Sw protein, the reference antibody is at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The detection method according to any one of Appendix 141 to 145, which recognizes an amino acid corresponding to a residue.
(Appendix 147)
The detection method according to any one of Supplementary note 141 to 145, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 148)
The detection method according to any one of Supplementary note 141 to 147, wherein the reference antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 149)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The detection method according to any one of Appendix 141 to 148, which is one antibody.
(Appendix 150)
The detection method according to any one of Supplementary note 141 to 149, wherein the reference antibody enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein.
(Appendix 151)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
A second detection step of contacting the Sw protein or its N-terminal domain with the biological sample in the presence of the reference antibody to detect the binding of the Sw protein or its N-terminal domain to the antibody in the biological sample. The detection method according to any one of Appendix 141 to 150, which comprises.
(Appendix 152)
The detection step is
Competing with the reference antibody by comparing the detection result of the antibody against the Sw protein or its N-terminal domain in the first detection step with the detection result of the antibody against the Sw protein or its N-terminal domain in the second detection step. 151. The detection method according to Appendix 151, which comprises a comparative step of detecting a competing antibody.
(Appendix 153)
Addendum 141 to 152, which comprises an evaluation step of evaluating whether the antibody detected in the detection step enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein. The detection method described in any of the above.
(Appendix 154)
The detection method according to any one of Supplementary notes 141 to 153, wherein the Sw protein is the Ss protein.
<Third infection-enhancing antibody detection method>
(Appendix 155)
For the biological sample of the subject, it binds to the Spike protein (Sw protein) of wild-type SARS-CoV-2 and
In the Sw protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. SARS-CoV comprising a detection step of detecting an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 214, 216, and 217. -2 Method for detecting infection-enhancing antibody.
(Appendix 156)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
Sw protein, a group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. The mutant protein or its N-terminal domain in which a mutation has been introduced into an amino acid corresponding to at least one amino acid residue selected from the above is brought into contact with the biological sample, and the mutant protein or its N-terminal domain and the biological sample are brought into contact with each other. 155. The detection method according to Appendix 155, which comprises a second detection step of detecting binding to the antibody in.
(Appendix 157)
The detection step is
By comparing the detection result of the antibody against the Sw protein or its N-terminal domain in the first detection step with the detection result of the antibody against the mutant protein or its N-terminal domain in the second detection step, the antibody can be obtained. 156. The detection method according to Appendix 156, which comprises a comparison step for detection.
(Appendix 158)
In the detection step, an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 64, 65, and / or 66 in the Sw protein is detected in the Sw protein, Appendix 155. The detection method according to any one of 157 to 157.
(Appendix 159)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the first mutant protein in which mutations have been introduced into the amino acids corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein or the N-terminal domain thereof, and the biological sample. 158. The detection method according to Appendix 158, which comprises a second detection step of detecting the binding of the first mutant protein or its N-terminal domain to the antibody in the biological sample.
(Appendix 160)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the first mutant protein or the N-terminal domain thereof in the second detection step. 159. The detection method according to Supplementary note 159.
(Appendix 161)
In the detection step, an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Sw protein is detected in the Sw protein, Appendix 155. The detection method according to any one of 157 to 157.
(Appendix 162)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the second mutant protein in which the mutation was introduced into the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein or the N-terminal domain thereof, and the biological sample. 161 according to Appendix 161. The detection method comprises the second detection step of detecting the binding of the second mutant protein or its N-terminal domain to the antibody in the biological sample.
(Appendix 163)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the second mutant protein or the N-terminal domain thereof in the second detection step. Included, the detection method according to Appendix 162.
(Appendix 164)
In the detection step, in the Sw protein, a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at the 64th, 65th, 66th, 187th, 213rd, and / or 214th positions in the Ss protein. The detection method according to any one of Supplementary note 155 to 163, which detects an unrecognized antibody.
(Appendix 165)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
In the Sw protein, the third mutant protein in which mutations are introduced into the amino acids at positions 64, 65, 66, 187, 213, and / or 214 of the Ss protein or the N-terminal domain thereof, and the N-terminal domain thereof. 164. The detection method according to Appendix 164, which comprises a second detection step of contacting a biological sample to detect the binding of the third mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 166)
The detection step is
A comparative step of comparing the detection result of the Sw protein or the antibody against the N-terminal domain thereof in the first detection step with the detection result of the antibody against the third mutant protein or the N-terminal domain thereof in the second detection step. 165. The detection method according to Supplementary note 165.
(Appendix 167)
In the detection step, the Sw protein recognizes the amino acids corresponding to the amino acid residues of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The detection method according to any one of Supplementary note 155 to 166, which detects an antibody.
(Appendix 168)
Addendums 155 to 167, comprising an evaluation step of evaluating whether the antibody detected in the detection step enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein. The detection method described in any of the above.
(Appendix 169)
The detection method according to any one of Supplementary notes 155 to 168, wherein the Sw protein is the Ss protein.
<Fourth method for detecting infection-enhancing antibody>
(Appendix 170)
A detection step of detecting a competing antibody that competes with a reference antibody for binding of wild-type SARS-CoV-2 to a Spike protein (Sw protein) in a biological sample of a subject is included.
The reference antibody is
Recognize the Sw protein
In the Sw protein, in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213th positions. , 214-position, 216-position, and 217-position, detection of SARS-CoV-2 infection-enhancing antibody, which does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group. Method.
(Appendix 171)
The detection method according to Appendix 170, wherein the reference antibody does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 64, 65, and / or 66 in the Sw protein. ..
(Appendix 172)
The reference antibody does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Sw protein, according to Appendix 170 or 171. Detection method.
(Appendix 173)
The reference antibody is an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. The detection method according to any one of Appendix 170 to 172, which does not recognize a mutant protein having a mutation in.
(Appendix 174)
The reference antibody is the Sw protein at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65 in the Ss protein. , 66th, 187th, 213rd, and 214th positions, the detection method according to any one of Appendix 170 to 173, which does not recognize a mutant protein having a mutation in an amino acid corresponding to the amino acid residue.
(Appendix 175)
In the Sw protein, the reference antibody is at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The detection method according to any one of Appendix 170 to 174, which does not recognize a mutant protein having a mutation in an amino acid corresponding to a residue.
(Appendix 176)
The detection method according to any one of Supplementary note 170 to 175, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 177)
The detection method according to any one of Appendix 170 to 176, wherein the reference antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 178)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The detection method according to any one of Appendix 170 to 177, which is one antibody.
(Appendix 179)
The detection method according to any one of Supplementary note 170 to 178, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 180)
The detection step is
A first detection step of contacting the Sw protein or its N-terminal domain with the biological sample to detect binding of the Sw protein or its N-terminal domain to an antibody in the biological sample.
A second detection step of contacting the Sw protein or its N-terminal domain with the biological sample in the presence of the reference antibody to detect the binding of the Sw protein or its N-terminal domain to the antibody in the biological sample. The detection method according to any one of Appendix 170 to 179, including.
(Appendix 181)
The detection step is
Competing with the reference antibody by comparing the detection result of the antibody against the Sw protein or its N-terminal domain in the first detection step with the detection result of the antibody against the Sw protein or its N-terminal domain in the second detection step. 180. The detection method according to Appendix 180, which comprises a comparative step of detecting a competing antibody.
(Appendix 182)
The detection according to any one of Supplementary note 170 to 181, which comprises an evaluation step of evaluating whether the antibody detected in the detection step enhances the binding activity of the Sw protein to ACE2 by binding to the Sw protein. Method.
(Appendix 183)
The detection method according to any one of Appendix 170 to 182, wherein the Sw protein is the Ss protein.
<Detection kit for first and third infection-enhancing antibodies>
(Appendix 184)
The 30th and 32nd positions of the wild SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain and the reference SARS-CoV-2 Spike protein (Ss protein, SEQ ID NO: 1) in the Sw protein. Amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217. A kit for detecting an infection-enhancing antibody of SARS-CoV-2, which comprises a mutant protein in which a mutation has been introduced into the protein or an N-terminal domain thereof.
(Appendix 185)
The mutant protein is the first mutant protein in which a mutation is introduced into the amino acid corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein in the Sw protein, Appendix 184. Described detection kit.
(Appendix 186)
The mutant protein is a second mutant protein in which a mutation is introduced into the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Sw protein. Or the kit according to 185.
(Appendix 187)
The mutant protein is the Sw protein in which mutations are introduced into the amino acids corresponding to the amino acid residues at positions 64, 65, 66, 187, 213, and / or 214 in the Ss protein. The detection kit according to any one of Appendix 184 to 186, which is a mutant protein of 3.
(Appendix 188)
The detection kit according to any one of Supplements 184 to 187, which is used for the method for detecting an infection-enhancing antibody according to any one of Supplements 126 to 140 and 155 to 169.
<Detection kit for second and fourth infection-enhancing antibodies>
(Appendix 189)
It contains the Spike protein (Sw protein) of wild-type SARS-CoV-2 or its N-terminal domain and a reference antibody.
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. SARS-CoV-2 infection-enhancing antibody, which is an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, and 217. Detection kit.
(Appendix 190)
189. The detection kit according to Appendix 189, wherein the reference antibody recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein in the Sw protein.
(Appendix 191)
The detection kit according to Appendix 189 or 190, wherein the reference antibody recognizes in the Sw protein the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein.
(Appendix 192)
The reference antibody is an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. The detection kit according to any one of Appendix 189 to 191.
(Appendix 193)
The reference antibody is the Sw protein at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65 in the Ss protein. , 66, 187, 213, and 214, the detection kit according to any of Supplementary note 189 to 192, which recognizes the amino acid corresponding to the amino acid residue.
(Appendix 194)
In the Sw protein, the reference antibody is at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The detection kit according to any of Supplementary note 189 to 193, which recognizes the amino acid corresponding to the residue.
(Appendix 195)
The detection kit according to any one of Supplementary note 189 to 194, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 196)
The detection kit according to any one of Appendix 189 to 195, wherein the reference antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 197)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The detection kit according to any one of Supplementary notes 189 to 196, which is one antibody.
(Appendix 198)
The detection kit according to any one of Appendix 189 to 197, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 199)
The detection kit according to any one of Annex 189 to 198, which is used for the method for detecting an infection-enhancing antibody according to any one of Supplements 141 to 154 and 170 to 183.
<Test method for the first possibility of infection>
(Appendix 200)
This is a test method for susceptibility to SARS-CoV-2.
Regarding the biological sample of the subject, in the Spike protein (Sw protein) of wild-type SARS-CoV-2, the 30th, 32nd, and 64th positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. Recognizes amino acids corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 65, 66, 186, 187, 211, 213, 214, 216, and 217. A test method comprising a measurement step of measuring an amino acid to be used.
(Appendix 201)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
Sw protein, a group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. The mutant protein or its N-terminal domain in which a mutation has been introduced into an amino acid corresponding to at least one amino acid residue selected from the above is brought into contact with the biological sample, and the mutant protein or its N-terminal domain and the biological sample are brought into contact with each other. 200. The test method according to Appendix 200, which comprises a second measuring step of measuring the binding to the antibody in.
(Appendix 202)
The measurement step is
By comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the mutant protein or its N-terminal domain in the second measurement step, the antibody can be obtained. The test method according to Appendix 201, which comprises a comparative step of measurement.
(Appendix 203)
In the measurement step, in the Sw protein, an antibody that recognizes an amino acid corresponding to an amino acid residue at the 64-position, 65-position, and / or 66-position in the Ss protein is measured, according to any one of Appendix 200 to 202. The test method described.
(Appendix 204)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the first mutant protein in which mutations have been introduced into the amino acids corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein or the N-terminal domain thereof, and the biological sample. 203. The test method according to Appendix 203, which comprises a second measuring step of measuring the binding of the first mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 205)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the first mutant protein or its N-terminal domain in the second measurement step. Included, the test method according to Appendix 204.
(Appendix 206)
In the measurement step, in the Sw protein, an antibody that recognizes an amino acid corresponding to an amino acid residue at the 187th, 213th, and / or 214th positions in the Ss protein is measured, according to any of Supplementary note 200 to 205. The test method described.
(Appendix 207)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the second mutant protein in which the mutation was introduced into the amino acid corresponding to the amino acid residues at positions 187, 213, and / or 214 in the Ss protein or the N-terminal domain thereof, and the biological sample. 206. The test method according to Appendix 206, which comprises a second measuring step of measuring the binding of the second mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 208)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the second mutant protein or its N-terminal domain in the second measurement step. 207. The test method according to Appendix 207.
(Appendix 209)
In the measurement step, in the Sw protein, in the Ss protein, at positions 64, 66, 213, and / or 214, 64, 66, 187, 213, and / or 214, or 64. The test method according to any of Supplementary note 200 to 208, which measures an antibody that recognizes an amino acid corresponding to an amino acid residue at positions 65, 66, 187, 213, and / or 214.
(Appendix 210)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the third mutant protein in which mutations are introduced into the amino acids at positions 64, 65, 66, 187, 213, and / or 214 of the Ss protein or the N-terminal domain thereof, and the above-mentioned 209. The test method according to Appendix 209, which comprises contacting a biological sample and measuring the binding of the third mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 211)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the third mutant protein or its N-terminal domain in the second measurement step. Included, the test method according to Appendix 210.
(Appendix 212)
The test method according to any one of Supplementary note 200 to 211, wherein the amount of the antibody is measured as the antibody in the measurement step.
(Appendix 213)
The test method according to Appendix 212, comprising a first test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the antibody with a first threshold.
(Appendix 214)
The test according to Appendix 213, wherein the first threshold value is a threshold value set based on the amount of the antibody in the biological sample of a healthy person and / or the amount of the antibody in the biological sample of a person suffering from SARS-CoV-2. Method.
(Appendix 215)
In the first test step, when the amount of antibody in the biological sample of the subject is higher than the first threshold value, the subject is likely to be affected by SARS-CoV-2. , Appendix 214.
(Appendix 216)
The test method according to any one of Supplementary note 200 to 215, which comprises a third measuring step of measuring the neutralizing antibody of the Sw protein with respect to the biological sample of the subject.
(Appendix 217)
216. The test method according to Appendix 216, wherein the neutralizing antibody is an antibody that binds to the receptor binding domain of the Sw protein.
(Appendix 218)
The test method according to Appendix 216 or 217, wherein the amount of the neutralizing antibody is measured as the neutralizing antibody in the third measurement step.
(Appendix 219)
218. The test method according to Appendix 218, which comprises a second test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the neutralizing antibody with a second threshold value. ..
(Appendix 220)
The second threshold is the amount of the neutralizing antibody in the biological sample of the person affected by SARS-CoV-2 and / or the amount of the neutralizing antibody in the biological sample of the person who recovered after the onset of SARS-CoV-2. 219. The test method according to Appendix 219, which is a threshold value set based on the above.
(Appendix 221)
In the second test step, when the amount of antibody in the biological sample of the subject is higher than the second threshold value, the subject is unlikely to suffer from SARS-CoV-2. , Appendix 220.
(Appendix 222)
In the measurement step, the amount of the antibody as the antibody is measured.
A third measuring step of measuring the amount of the Neutralizing antibody of Sw protein with respect to the biological sample of the subject, and
A calculation step of calculating an antibody amount ratio (S / N) between the amount of antibody (S) in the measurement step and the amount of neutralizing antibody (N) in the third measurement step.
Any of Appendix 200-221 comprising a third test step of testing the subject's potential for infection with SARS-CoV-2 by comparing the antibody amount ratio to a third threshold. The test method described.
(Appendix 223)
The third threshold is the antibody amount ratio in the biological sample of a healthy person, the antibody amount ratio in the biological sample of a person suffering from SARS-CoV-2, and / or a person who has recovered after being affected by SARS-CoV-2. The test method according to Appendix 222, which is a threshold value set based on the antibody amount ratio in the biological sample of.
(Appendix 224)
In the third test step, when the antibody amount ratio (S / N) in the biological sample of the subject is higher than the third threshold value, the subject suffers from SARS-CoV-2. The test method according to Appendix 223, which is highly likely.
(Appendix 225)
In the measurement step, in the Sw protein, the amino acids corresponding to the amino acid residues of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein are recognized. The test method according to any one of Appendix 200 to 224, wherein the antibody is measured.
(Appendix 226)
The test method according to any one of Appendix 200 to 225, wherein the Sw protein is the Ss protein.
<Test method for the possibility of second infection>
(Appendix 227)
This is a test method for susceptibility to SARS-CoV-2.
A measurement step of measuring a competing antibody that competes with a reference antibody for binding of wild-type SARS-CoV-2 to a Spike protein (Sw protein) in a biological sample of a subject is included.
The reference antibody is the 30th, 32nd, 64th, 65th, 66th, 186th, and 187th positions of the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2 in the Sw protein. A test method, which is an antibody that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids at positions 211, 213, 214, 216, and 217.
(Appendix 228)
227. The test method according to Appendix 227, wherein the reference antibody recognizes an amino acid corresponding to an amino acid residue at positions 64, 65, and / or 66 in the Ss protein in the Sw protein.
(Appendix 229)
227 or 228 of the test method, wherein the reference antibody recognizes an amino acid corresponding to an amino acid residue at positions 187, 213, and / or 214 in the Sw protein.
(Appendix 230)
The reference antibody is an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. 227. The test method according to any one of Supplementary note 227 to 229.
(Appendix 231)
The reference antibody is the Sw protein at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65 in the Ss protein. , 66, 187, 213, and 214, the test method according to any of Supplementary note 227 to 230, which recognizes the amino acid corresponding to the amino acid residue.
(Appendix 232)
In the Sw protein, the reference antibody is at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The test method according to any of Supplementary note 227 to 231, which recognizes an amino acid corresponding to a residue.
(Appendix 233)
The test method according to any one of Supplementary note 227 to 232, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 234)
The test method according to any one of Supplementary note 227 to 233, wherein the reference antibody is at least one antibody selected from the group consisting of the following (1) to (5) and (6).
(Appendix 235)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The test method according to any of Supplementary note 227 to 234, which is one antibody.
(Appendix 236)
The test method according to any one of Supplementary note 227 to 235, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 237)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
A second measurement step in which the Sw protein or its N-terminal domain is brought into contact with the biological sample in the presence of the reference antibody, and the binding of the Sw protein or its N-terminal domain to the antibody in the biological sample is measured. 227. The test method according to any one of Supplementary note 227 to 236.
(Appendix 238)
The measurement step is
Competing with the reference antibody by comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the Sw protein or its N-terminal domain in the second measurement step. 237. The test method according to Appendix 237, which comprises a comparative step of measuring competing antibodies.
(Appendix 239)
The test method according to any one of Supplementary note 227 to 238, wherein the amount of the antibody is measured as the antibody in the measurement step.
(Appendix 240)
239. The test method according to Appendix 239, comprising a first test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the antibody with a first threshold.
(Appendix 241)
The test according to Appendix 240, wherein the first threshold value is a threshold value set based on the amount of the antibody in the biological sample of a healthy subject and / or the amount of the antibody in the biological sample of a person suffering from SARS-CoV-2. Method.
(Appendix 242)
In the first test step, when the amount of antibody in the biological sample of the subject is higher than the first threshold value, the subject is likely to be affected by SARS-CoV-2. , The test method according to Appendix 241.
(Appendix 243)
The test method according to any one of Supplementary note 227 to 242, which comprises a third measuring step of measuring a neutralizing antibody of Sw protein with respect to the biological sample of the subject.
(Appendix 244)
243. The test method according to Appendix 243, wherein the neutralizing antibody is an antibody that binds to the receptor binding domain of the Sw protein.
(Appendix 245)
The test method according to Appendix 243 or 244, wherein the amount of the neutralizing antibody is measured as the neutralizing antibody in the third measurement step.
(Appendix 246)
245. The test method according to Appendix 245, which comprises a second test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the neutralizing antibody with a second threshold value. ..
(Appendix 247)
The second threshold is the amount of the neutralizing antibody in the biological sample of the person affected by SARS-CoV-2 and / or the amount of the neutralizing antibody in the biological sample of the person who recovered after the onset of SARS-CoV-2. The test method according to Appendix 246, which is a threshold set based on the above.
(Appendix 248)
In the second test step, when the amount of antibody in the biological sample of the subject is higher than the second threshold value, the subject is unlikely to suffer from SARS-CoV-2. , Appendix 247.
(Appendix 249)
In the measurement step, the amount of the antibody as the antibody is measured.
A third measuring step of measuring the amount of the Neutralizing antibody of Sw protein with respect to the biological sample of the subject, and
A calculation step of calculating an antibody amount ratio (S / N) between the amount of antibody (S) in the measurement step and the amount of neutralizing antibody (N) in the third measurement step.
Any of Appendix 237-238, comprising a third test step of testing the subject's potential for infection with SARS-CoV-2 by comparing the antibody dose ratio to a third threshold. The test method described.
(Appendix 250)
The third threshold is the antibody amount ratio in the biological sample of a healthy person, the antibody amount ratio in the biological sample of a person suffering from SARS-CoV-2, and / or a person who has recovered after being affected by SARS-CoV-2. 249. The test method according to Appendix 249, which is a threshold value set based on the antibody amount ratio in the biological sample of.
(Appendix 251)
In the third test step, when the antibody amount ratio (S / N) in the biological sample of the subject is higher than the third threshold value, the subject suffers from SARS-CoV-2. The test method according to Appendix 250, which is highly likely.
<Test method for third infection-enhancing antibody>
(Appendix 252)
This is a test method for susceptibility to SARS-CoV-2.
For the biological sample of the subject, it binds to the Spike protein (Sw protein) of wild-type SARS-CoV-2 and
In the Sw protein, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213rd positions in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. , 214, 216, and 217. A test method comprising a measurement step of measuring an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids.
(Appendix 253)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
Sw protein, a group consisting of amino acids at positions 30, 32, 64, 65, 66, 186, 187, 211, 213, 214, 216, and 217 of the Ss protein. The mutant protein or its N-terminal domain in which a mutation has been introduced into an amino acid corresponding to at least one amino acid residue selected from the above is brought into contact with the biological sample, and the mutant protein or its N-terminal domain and the biological sample are brought into contact with each other. 252. The test method according to Appendix 252, which comprises a second measuring step of measuring the binding to the antibody in.
(Appendix 254)
The measurement step is
By comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the mutant protein or its N-terminal domain in the second measurement step, the antibody can be obtained. 253. The test method according to Appendix 253, which comprises a comparative step of measurement.
(Appendix 255)
In the measurement step, in the Sw protein, an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at the 64th, 65th, and / or 66th positions in the Ss protein is measured, Appendix 252. The test method according to any one of 254 to 254.
(Appendix 256)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the first mutant protein in which mutations have been introduced into the amino acids corresponding to the amino acid residues at positions 64, 65, and / or 66 in the Ss protein or the N-terminal domain thereof, and the biological sample. 255. The test method according to Appendix 255, which comprises a second measuring step of measuring the binding of the first mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 257)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the first mutant protein or its N-terminal domain in the second measurement step. Included, the test method according to Appendix 256.
(Appendix 258)
In the measurement step, in the Sw protein, an antibody that does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at the 187th, 213th, and / or 214th positions in the Ss protein is measured, Appendix 252. 257. The test method according to any one of.
(Appendix 259)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the second mutant protein in which the mutation was introduced into the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Ss protein or the N-terminal domain thereof, and the biological sample. 258. The test method according to Appendix 258, which comprises a second measuring step of measuring the binding of the second mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 260)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the second mutant protein or its N-terminal domain in the second measurement step. 259. The test method according to Appendix 259.
(Appendix 261)
In the measurement step, in the Sw protein, in the Ss protein, the 64th, 66th, 213rd, and 214th, 64th, 66th, 187th, 213rd, and 214th, or 64th, 65th positions. , 66, 187, 213, and 214. The test method according to any of Supplementary note 252 to 260, which measures an antibody that does not recognize a mutant protein having a mutation in an amino acid corresponding to an amino acid residue.
(Appendix 262)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
In the Sw protein, the third mutant protein in which mutations are introduced into the amino acids at positions 64, 65, 66, 187, 213, and / or 214 of the Ss protein or the N-terminal domain thereof, and the above-mentioned 261. The test method according to Appendix 261, comprising contacting a biological sample and measuring the binding of the third mutant protein or its N-terminal domain to an antibody in the biological sample.
(Appendix 263)
The measurement step is
A comparative step of comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the third mutant protein or its N-terminal domain in the second measurement step. 262, the test method according to Appendix 262.
(Appendix 264)
In the measurement step, in the Sw protein, the amino acids corresponding to the amino acid residues of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein are recognized. The test method according to any of Supplementary note 252 to 263, wherein the antibody is measured.
(Appendix 265)
The test method according to any one of Supplementary note 252 to 264, wherein the amount of the antibody is measured as the antibody in the measurement step.
(Appendix 266)
265. The test method according to Appendix 265, which comprises a first test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the antibody with a first threshold.
(Appendix 267)
The test according to Appendix 266, wherein the first threshold is a threshold set based on the amount of the antibody in a biological sample of a healthy subject and / or the amount of the antibody in a biological sample of a person suffering from SARS-CoV-2. Method.
(Appendix 268)
In the first test step, when the amount of antibody in the biological sample of the subject is higher than the first threshold value, the subject is likely to be affected by SARS-CoV-2. , Appendix 267.
(Appendix 269)
The test method according to any one of Supplementary note 252 to 268, which comprises a third measuring step of measuring the neutralizing antibody of the Sw protein with respect to the biological sample of the subject.
(Appendix 270)
269. The test method according to Appendix 269, wherein the neutralizing antibody is an antibody that binds to the receptor binding domain of the Sw protein.
(Appendix 271)
The test method according to Appendix 269 or 270, wherein the amount of the neutralizing antibody is measured as the neutralizing antibody in the third measurement step.
(Appendix 272)
271. The test method according to Appendix 271, which comprises a second test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the neutralizing antibody with a second threshold value. ..
(Appendix 273)
The second threshold is the amount of the neutralizing antibody in the biological sample of the person affected by SARS-CoV-2 and / or the amount of the neutralizing antibody in the biological sample of the person who recovered after the onset of SARS-CoV-2. 272. The test method according to Appendix 272, which is a threshold set based on the above.
Neutralizing antibody
(Appendix 274)
In the second test step, when the amount of antibody in the biological sample of the subject is higher than the second threshold value, the subject is unlikely to suffer from SARS-CoV-2. , Appendix 273.
(Appendix 275)
In the measurement step, the amount of the antibody as the antibody is measured.
A third measuring step of measuring the amount of the Neutralizing antibody of Sw protein with respect to the biological sample of the subject, and
A calculation step of calculating an antibody amount ratio (S / N) between the amount of antibody (S) in the measurement step and the amount of neutralizing antibody (N) in the third measurement step.
Any of Appendix 252-264, comprising a third test step of testing the subject's potential for infection with SARS-CoV-2 by comparing the antibody dose ratio to a third threshold. The test method described.
(Appendix 276)
The third threshold is the antibody amount ratio in the biological sample of a healthy person, the antibody amount ratio in the biological sample of a person suffering from SARS-CoV-2, and / or a person who has recovered after being affected by SARS-CoV-2. 275. The test method according to Appendix 275, which is a threshold value set based on the antibody amount ratio in the biological sample of.
(Appendix 277)
In the third test step, when the antibody amount ratio (S / N) in the biological sample of the subject is higher than the third threshold value, the subject suffers from SARS-CoV-2. The test method according to Appendix 276, which is highly likely.
(Appendix 278)
In the measurement step, in the Sw protein, the amino acids corresponding to the amino acid residues of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein are recognized. The test method according to any of Supplementary note 252 to 277, which measures an antibody.
(Appendix 279)
The test method according to any one of Appendix 252 to 278, wherein the Sw protein is the Ss protein.
<Test method for fourth infection-enhancing antibody>
(Appendix 280)
This is a test method for susceptibility to SARS-CoV-2.
A measurement step of measuring a competing antibody that competes with a reference antibody for binding of wild-type SARS-CoV-2 to a Spike protein (Sw protein) in a biological sample of a subject is included.
The reference antibody is
Recognize the Sw protein
In the Sw protein, in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2, the 30th, 32nd, 64th, 65th, 66th, 186th, 187th, 211th, and 213th positions. , 214, 216, and 217. A test method that does not recognize a mutant protein having a mutation in an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids.
(Appendix 281)
The test method according to Appendix 280, wherein the reference antibody does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 64, 65, and / or 66 in the Sw protein. ..
(Appendix 282)
The reference antibody does not recognize a mutant protein having a mutation in the amino acid corresponding to the amino acid residue at positions 187, 213, and / or 214 in the Sw protein, according to Appendix 280 or 281. Test method.
(Appendix 283)
The reference antibody is an amino acid corresponding to at least one amino acid residue selected from the group consisting of positions 64, 65, 66, 187, 213, and 214 in the Sw protein. The test method according to any of Supplementary note 280 to 282, which does not recognize a mutant protein having a mutation in.
(Appendix 284)
The reference antibody is the Sw protein at positions 64, 66, 213, and 214, 64, 66, 187, 213, and 214, or 64, 65 in the Ss protein. , 66, 187, 213, and 214, the test method according to any of Supplementary note 280 to 283, which does not recognize a mutant protein having a mutation in the corresponding amino acid.
(Appendix 285)
In the Sw protein, the reference antibody is at least one amino acid selected from the group consisting of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein. The test method according to any of Supplementary note 280 to 284, which does not recognize a mutant protein having a mutation in the amino acid corresponding to the residue.
(Appendix 286)
The test method according to any one of Supplementary note 280 to 285, wherein the reference antibody is an antibody containing the multiple variable region of (H) and the light chain variable region of (L).
(Appendix 287)
The test method according to any one of Appendix 280 to 286, wherein the reference antibody is at least one antibody selected from the group consisting of (1) to (5) and (6).
(Appendix 288)
The reference antibody is at least selected from the group consisting of (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and (F) 2660 antibody. The test method according to any one of Appendix 280 to 287, which is one antibody.
(Appendix 289)
The test method according to any one of Supplementary note 280 to 288, wherein the reference antibody enhances the binding activity of the Sw protein to angiotensin converting enzyme 2 (ACE2) by binding to the Sw protein.
(Appendix 290)
The measurement step is
The first measurement step of contacting the Sw protein or its N-terminal domain with the biological sample and measuring the binding of the Sw protein or its N-terminal domain with the antibody in the biological sample.
A second measurement step in which the Sw protein or its N-terminal domain is brought into contact with the biological sample in the presence of the reference antibody, and the binding of the Sw protein or its N-terminal domain to the antibody in the biological sample is measured. The test method according to any of Supplementary note 280 to 289, which comprises.
(Appendix 291)
The measurement step is
Competing with the reference antibody by comparing the measurement result of the antibody against the Sw protein or its N-terminal domain in the first measurement step with the measurement result of the antibody against the Sw protein or its N-terminal domain in the second measurement step. 290. The test method according to Appendix 290, which comprises a comparative step of measuring a competing antibody.
(Appendix 292)
The test method according to any one of Appendix 280 to 291 in which the amount of the antibody is measured as the antibody in the measurement step.
(Appendix 293)
292. The test method according to Appendix 292, comprising a first test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the antibody with a first threshold.
(Appendix 294)
The test according to Appendix 293, wherein the first threshold is a threshold set based on the amount of the antibody in a biological sample of a healthy subject and / or the amount of the antibody in a biological sample of a person suffering from SARS-CoV-2. Method.
(Appendix 295)
In the first test step, when the amount of antibody in the biological sample of the subject is higher than the first threshold value, the subject is likely to be affected by SARS-CoV-2. , Appendix 294.
(Appendix 296)
The test method according to any one of Supplementary note 280 to 295, which comprises a third measuring step of measuring the neutralizing antibody of the Sw protein with respect to the biological sample of the subject.
(Appendix 297)
The test method according to Appendix 296, wherein the neutralizing antibody is an antibody that binds to the receptor binding domain of the Sw protein.
(Appendix 298)
The test method according to Appendix 296 or 297, wherein the amount of the neutralizing antibody is measured as the neutralizing antibody in the third measurement step.
(Appendix 299)
298. The test method according to Appendix 298, which comprises a second test step of testing the possibility of the subject being infected with SARS-CoV-2 by comparing the amount of the neutralizing antibody with a second threshold value. ..
(Appendix 300)
The second threshold is the amount of the neutralizing antibody in the biological sample of the person affected by SARS-CoV-2 and / or the amount of the neutralizing antibody in the biological sample of the person who recovered after the onset of SARS-CoV-2. 299. The test method according to Appendix 299, which is a threshold set based on the above.
Neutralizing antibody
(Appendix 301)
In the second test step, when the amount of antibody in the biological sample of the subject is higher than the second threshold value, the subject is unlikely to suffer from SARS-CoV-2. , The test method according to Appendix 300.
(Appendix 302)
In the measurement step, the amount of the antibody as the antibody is measured.
A third measuring step of measuring the amount of the Neutralizing antibody of Sw protein with respect to the biological sample of the subject, and
A calculation step of calculating an antibody amount ratio (S / N) between the amount of antibody (S) in the measurement step and the amount of neutralizing antibody (N) in the third measurement step.
Any of Appendix 280-301, comprising a third test step of testing the subject's potential for infection with SARS-CoV-2 by comparing the antibody amount ratio to a third threshold. The test method described.
(Appendix 303)
The third threshold is the antibody amount ratio in the biological sample of a healthy person, the antibody amount ratio in the biological sample of a person suffering from SARS-CoV-2, and / or a person who has recovered after being affected by SARS-CoV-2. The test method according to Appendix 302, which is a threshold value set based on the antibody amount ratio in the biological sample of.
(Appendix 304)
In the third test step, when the antibody amount ratio (S / N) in the biological sample of the subject is higher than the third threshold value, the subject suffers from SARS-CoV-2. The test method according to Appendix 303, which is highly likely.
(Appendix 305)
In the measurement step, in the Sw protein, the amino acids corresponding to the amino acid residues of N30, F32, W64, F65, H66, F186, K187, N211, V213, R214, L216, and P217 in the Ss protein are recognized. The test method according to any of Supplementary note 280 to 304, wherein the antibody is measured.
(Appendix 306)
The test method according to any one of Appendix 280 to 305, wherein the Sw protein is the Ss protein.
<Method of suppressing the aggravation of SARS-CoV-2>
(Appendix 307)
A test step for testing the possibility of enhancing the infection of SARS-CoV-2 of the subject from a biological sample of the subject, and a test step.
The test step includes an administration step of administering a therapeutic agent for SARS-CoV-2 to a subject evaluated as having a possibility of enhancing infection.
The test step is a method for suppressing the aggravation of SARS-CoV-2, which is carried out by the test method according to any one of Appendix 200 to 306.
<Reducing agent for infection-enhancing antibody>
(Appendix 308)
It is an antibody reducing agent that enhances the binding ability of SARS-CoV-2's Spike protein to angiotensin converting enzyme 2 (ACE2).
A reducing agent comprising a wild-type Spike protein (Sw protein) or its N-terminal domain.
(Appendix 309)
Contains carriers
The reducing agent according to Appendix 308, wherein the Sw protein is retained on a carrier.
(Appendix 310)
309. The reducing agent according to Appendix 309, wherein the carrier is beads or a filter.
<Method of reducing infection-enhancing antibody>
(Appendix 311)
A method for reducing an antibody that enhances the ability of SARS-CoV-2 to bind the Spike protein to angiotensin converting enzyme 2 (ACE2).
A reduction method comprising a reduction step of reducing the antibody in the biological sample by contacting the biological sample with the reducing agent according to any one of Appendix 308 to 310.
(Appendix 312)
The reduction method according to Appendix 311, which comprises an acquisition step of acquiring the biological sample from the target prior to the reduction step.
(Appendix 313)
The reduction method according to Appendix 311 or 312, which comprises an administration step of administering the biological sample to a subject from which the biological sample has been obtained after the reduction step.

As described above, according to the present invention, the ability to induce an antibody that enhances the ability of SARS2 to bind to the ACE2 protein can be reduced. Therefore, according to the mutant protein of the present invention, for example, the risk of ADE in a vaccinated person can be reduced. Therefore, the present invention is extremely useful in, for example, the pharmaceutical field.

Claims (16)

A variant of the Spike protein of SARS-CoV-2, which is a protein consisting of the following (Sm) amino acid sequence:
(Sm) The amino acid sequence of the following (Sm1), (Sm2), or (Sm3):
(Sm1) In the amino acid sequence of the Spike protein (Sw protein) of wild SARS-CoV-2, W64, H66, K187, V213, and in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It has a mutation in the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids of R214, and the mutations result in the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, ( D) Amino acid sequence with reduced binding to 2582 antibody, (E) 2369 antibody, and / or (F) 2660 antibody;
In the amino acid sequence of (Sm2) (Sm1), the mutated amino acid of (Sm1) having 1 to 127 insertions, additions, substitutions and / or deletions was maintained, and the mutation resulted in the following ( Amino acid sequence with reduced binding to A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) 2660 antibody;
The amino acid sequence of (Sm3) and (Sm1) has 90% or more identity, and the mutated amino acid of (Sm1) is maintained. ) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or amino acid sequence with reduced binding to (F) 2660 antibody;
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. The amino acid sequence of (Sm1) is that of the Sw protein, W64, H66, V213, and R214, W64, H66, K187, V213, and R214, or W64, F65, H66, K187, V213, in the Ss protein. The variant according to claim 1, which is an amino acid sequence having a mutation in the amino acid corresponding to the amino acid residue of R214. A mutant polypeptide comprising the N-terminal domain of a variant of the Spike protein according to claim 1 or 2. A mutant virus of SARS-CoV-2, which comprises a variant of the Spike protein according to claim 1 or 2. A virus-like particle comprising a variant of the Peplomer protein according to claim 1 or 2 and / or a mutant polypeptide according to claim 3. A nucleic acid comprising a nucleic acid molecule encoding a variant of the Spike protein according to claim 1 or 2 and / or a nucleic acid molecule encoding the mutant polypeptide according to claim 3. An expression vector containing the nucleic acid according to claim 6. A variant of the Spike protein according to claim 1 or 2, a mutant polypeptide according to claim 3, a mutant virus according to claim 4, a virus-like particle according to claim 5, a nucleic acid according to claim 6, and / or A pharmaceutical composition comprising the expression vector according to claim 7 and a pharmaceutically acceptable carrier. A method for producing a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (St protein) of the target SARS-CoV-2, from the amino acids of W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. at least one amino acid residue selected from the group consisting of and as product engineering mutations into the corresponding amino acid to generate a candidate proteins introduced,
The St protein after introduction of the mutation includes a selection step of selecting a candidate protein having a mutation whose binding to the reference antibody is reduced as the mutant.
The reference antibody is the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) 2660 antibody.
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. A method for screening a variant of a Peplomer protein in which the ability to induce an antibody that enhances the binding ability of SARS-CoV-2 to the angiotensin converting enzyme 2 (ACE2) of the Peplomer protein is reduced.
In the amino acid sequence of the Spike protein (Sc protein) of the candidate SARS-CoV-2, from the amino acids of W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, which have a mutation in the amino acid corresponding to at least one amino acid residue selected from the group. , (E) 2369 antibody and / or Sc protein whose binding to (F) 2660 antibody is reduced is selected as a variant of the Spike protein.
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. Regarding the biological sample of the subject, in the wild type SARS-CoV-2 Spike protein (Sw protein), in the reference SARS-CoV-2 Spike protein (Ss protein, SEQ ID NO: 1), W64, H66, K187, V213. , And a method for detecting an infection-enhancing antibody of SARS-CoV-2, which comprises a detection step of detecting an amino acid that recognizes an amino acid corresponding to at least one amino acid residue selected from the group consisting of amino acids of R214. A kit used to detect SARS-CoV-2 infection-enhancing antibody.
Wild-type SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain,
In the Sw protein, with at least one amino acid residue selected from the group consisting of the amino acids W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It has a mutation in the corresponding amino acid, and due to the mutation, the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) Detection kit containing a mutant protein with reduced binding to the 2660 antibody or its N-terminal domain:
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. A reagent used to detect SARS-CoV-2 infection-enhancing antibody.
Wild-type SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain,
In the Sw protein, with at least one amino acid residue selected from the group consisting of the amino acids W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It has a mutation in the corresponding amino acid, and due to the mutation, the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) Detection reagent containing a mutant protein with reduced binding to the 2660 antibody or its N-terminal domain:
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. It is a test method for the possibility of aggravation in SARS-CoV-2 infected persons.
Regarding the biological sample of the subject, in the wild type SARS-CoV-2 Spike protein (Sw protein), in the reference SARS-CoV-2 Spike protein (Ss protein, SEQ ID NO: 1), W64, H66, K187, V213. , And a measurement step of measuring the amount of antibody that recognizes the amino acid corresponding to at least one amino acid residue selected from the group consisting of the amino acids of R214.
Including a first test step of testing the possibility of aggravation of the subject by comparing the amount of the antibody with the first threshold.
The first threshold is the amount of the antibody in a biological sample isolated from a healthy person and / or a SARS-CoV-2 infected person.
In the first test step,
When the amount of the antibody in the biological sample of the subject is higher than the amount of the antibody in the biological sample of the healthy person, when it is the same as the amount of the antibody in the biological sample of the infected person, and / or the above. If the amount of the antibody is higher than the amount of the antibody in the biological sample of the infected person, the subject is likely to become severely ill or is likely to become severely ill.
When the amount of the antibody in the biological sample of the subject is the same as the amount of the antibody in the biological sample of the healthy person, when it is lower than the amount of the antibody in the biological sample of the healthy person, and / or the above. A test method in which if the amount of the antibody is lower than the amount of the antibody in the biological sample of the infected person, the subject is unlikely to become severe or is unlikely to become severe . A kit used to test the possibility of aggravation in SARS-CoV-2 infected individuals.
Wild-type SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain,
In the Sw protein, with at least one amino acid residue selected from the group consisting of the amino acids W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It has a mutation in the corresponding amino acid, and due to the mutation, the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) A test kit containing a mutant protein with reduced binding to the 2660 antibody or its N-terminal domain:
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109. A reagent used to test the possibility of aggravation in SARS-CoV-2 infected individuals.
Wild-type SARS-CoV-2 Spike protein (Sw protein) or its N-terminal domain,
In the Sw protein, with at least one amino acid residue selected from the group consisting of the amino acids W64, H66, K187, V213, and R214 in the Spike protein (Ss protein, SEQ ID NO: 1) of the reference SARS-CoV-2. It has a mutation in the corresponding amino acid, and due to the mutation, the following (A) 2210 antibody, (B) 2490 antibody, (C) 8D2 antibody, (D) 2582 antibody, (E) 2369 antibody, and / or (F) Test Reagents Containing Mutant Proteins with Reduced Binding to 2660 Antibodies or their N-Terminal Domains:
(A) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 98 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 99;
(B) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 100 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 101;
(C) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 102 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 103;
(D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 104 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 105;
(E) An antibody containing a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 106 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 107;
(F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 108 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 109.

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