WO2024117057A1 - Antibody or antigen-binding fragment thereof, and immunological measurement device using same - Google Patents

Abstract

Provided are a novel anti-HSV monoclonal antibody, etc. The anti-HSV monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the envelope glycoprotein of herpes simplex virus (HSV), wherein: (i) the heavy chain variable region contains (a) the amino acid sequence of heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 2, (b) the amino acid sequence of heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 3, and (c) the amino acid sequence of heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 4, and (ii) the light chain variable region contains (a) the amino acid sequence of light chain CDR1 containing the amino acid sequence of SEQ ID NO: 6, (b) the amino acid sequence of light chain CDR2 containing the amino acid sequence of SEQ ID NO: 7, and (c) the amino acid sequence of light chain CDR3 containing the amino acid sequence of SEQ ID NO: 8; or (i) the heavy chain variable region contains (d) the amino acid sequence of heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 12, (e) the amino acid sequence of heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 13, and (f) the amino acid sequence of heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 14, and (ii) the light chain variable region contains (d) the amino acid sequence of light chain CDR1 containing the amino acid sequence of SEQ ID NO: 16, (e) the amino acid sequence of light chain CDR2 containing the amino acid sequence of SEQ ID NO: 17, and (f) the amino acid sequence of light chain CDR3 containing the amino acid sequence of SEQ ID NO: 18.

Description

Antibody or antigen-binding fragment thereof, and immunoassay device using same

The present invention relates to an anti-HSV monoclonal antibody or an antigen-binding fragment thereof, and an immunoassay device using the same.

There are two types of herpes simplex virus (HSV): herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).

Herpes simplex virus type 1 (HSV-1) tends to infect the lips and surrounding areas, primarily causing oral herpes. On the other hand, herpes simplex virus type 2 (HSV-2) tends to infect the genitals, primarily causing genital herpes. However, HSV-2 can also be detected in oral herpes, and HSV-1 can also be detected in genital herpes.

Herpes simplex viruses are DNA viruses that have an icosahedral capsid containing double-stranded nucleic acid, an envelope composed of a lipid bilayer, and a tegument filled between the capsid and the envelope. Herpes simplex viruses bind to infection receptors on the surface of the cell membrane, and the envelope fuses with the cytoplasmic membrane to allow the capsid to enter the cytoplasm. The viral genome then migrates from the capsid into the nucleus through nuclear pores and proliferates within the nucleus.

Herpes simplex virus infection may cause mild symptoms, painful blisters, or ulcers at the site of infection. If the virus infects the cornea, it can cause blindness, and if it infects the brain, it can cause herpes encephalitis, which can be fatal.
After initial infection, herpes simplex virus usually goes into a dormant (latent) state in the ganglia. The dormant (latent) herpes simplex virus periodically reactivates and grows, returning from the ganglia to the skin via nerve fibers, and may cause blisters or ulcers in the same area as the initial infection (recurrent episodes). It is known that such recurrences of herpes simplex can be triggered by stimuli such as colds, stress, mental stress, old age, anti-cancer drug treatment, and exposure to sunlight.
The incubation period for herpes simplex varies from person to person. For example, some people may experience a relapse after several years, while others may experience a relapse after only a few months.

Herpes simplex virus is highly contagious and cannot be eliminated by the immune system, so it remains dormant in nerve ganglia for the patient's entire life.
Herpes simplex is usually treated with antiviral drugs (e.g., acyclovir, famciclovir, valacyclovir, etc.) Since these antiviral drugs suppress the proliferation of the virus, they are preferably taken at an early stage after the onset of the disease.
From this perspective, there is a need for a method that can accurately detect the onset of herpes simplex early on in order to provide appropriate treatment.

For example, oral herpes caused by herpes simplex virus causes blisters and ulcers on the lips or around the lips, and may also cause itching, discomfort, and pain in the skin, which can be psychologically distressing for the patient.
From this viewpoint, there is a demand for prophylactic drugs having a preventive effect and therapeutic drugs having a therapeutic effect after the onset of herpes simplex for patients who frequently experience recurrence of herpes simplex.

For example, Patent Document 1 describes an invention relating to an anti-HSV gB monoclonal antibody or a binding fragment thereof. Patent Document 1 describes that an anti-HSV gB monoclonal antibody that specifically binds to the envelope glycoprotein B (gB), which is involved in the adsorption of herpes viruses, exhibits strong virus neutralizing activity and cell-to-cell transmission inhibitory activity.

International Publication No. 2019/044926

In these circumstances, there is a demand for new anti-HSV monoclonal antibodies, etc.

The present invention provides, for example, the following anti-HSV monoclonal antibodies, etc.

[1] An anti-herpes simplex virus (HSV) monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to an envelope glycoprotein of HSV,
(i) the heavy chain variable region is
(a) an amino acid sequence of a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2;
(b) an amino acid sequence of a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3; and (c) an amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4;
Contains
(ii) the light chain variable region is
(a) an amino acid sequence of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:6;
(b) an amino acid sequence of a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:7; and (c) an amino acid sequence of a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:8;
or (i) the heavy chain variable region comprises:
(d) an amino acid sequence of a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12;
(e) an amino acid sequence of a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) an amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;
Contains
(ii) the light chain variable region is
(d) the amino acid sequence of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16;
(e) an amino acid sequence of a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and (f) an amino acid sequence of a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18;
An antibody or an antigen-binding fragment thereof comprising:
[2] A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1, and
A light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:5; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:11, and
The antibody or antigen-binding fragment thereof according to [1] above, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15.
[3] The antibody or antigen-binding fragment thereof according to [1] or [2] above, which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 10; or a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 20.
[4] The antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, which specifically binds to envelope glycoprotein D (gD).
[5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4] above, wherein the herpes simplex virus (HSV) is HSV-1 and/or HSV-2.
[5-1] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, which has neutralizing activity against herpes simplex virus (HSV).
[6] An isolated nucleic acid molecule encoding the amino acid sequence of the antibody or antigen-binding fragment thereof according to any one of [1] to [5] and [5-1] above, or an isolated nucleic acid molecule which hybridizes under highly stringent conditions with any one of these nucleic acid molecules.
[7] An isolated nucleic acid molecule having a nucleotide sequence of any one of SEQ ID NOs: 21 to 24, or a nucleotide sequence having 90% or more identity to any one of SEQ ID NOs: 21 to 24.
[8] A recombinant expression vector incorporating the isolated nucleic acid molecule according to [7] above.
[9] An isolated host cell into which the recombinant expression vector according to [8] above has been introduced.
[10] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [5] and [5-1] above.
[11] An immunological measurement device for detecting herpes simplex virus (HSV), comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [5] and [5-1] above.
[12] An immunoassay device for detecting herpes simplex virus (HSV), comprising a sample dropping section, a labeling substance holding section, a detection section, and an absorption section,
An immunoassay device, wherein at least one of the labeling substance holding unit and the detection unit comprises the antibody or antigen-binding fragment thereof according to any one of [1] to [5] and [5-1] above.
[13] The immunoassay device according to [12] above, wherein both the labeling substance holding unit and the detection unit contain an antibody or an antigen-binding fragment thereof according to any one of [1] to [5] and [5-1] above.

The present invention provides novel anti-HSV monoclonal antibodies, etc. The anti-HSV monoclonal antibodies, etc. of the present invention have high sensitivity to, for example, herpes simplex virus (HSV). Therefore, for example, the presence or absence of herpes simplex onset can be accurately tested. In addition, it is possible to obtain prophylactic drugs having a preventive effect against herpes simplex, therapeutic drugs having a therapeutic effect against herpes simplex, etc.

1 is a schematic diagram of an immunological measurement device according to one embodiment of the present invention. 13 shows the results of reactivity evaluation of the immunological measurement device prepared in Example 4. These are the results of reactivity evaluation of a commercially available immunoassay device.

The following provides a detailed explanation of how to implement the present invention.

1. Explanation of Terms Scientific and technical terms used herein in connection with the present invention have the meanings commonly understood by those skilled in the art unless otherwise specified. Furthermore, unless otherwise required by context, singular terms include plurals and plural terms include the singular. In general, the nomenclature used in connection with the techniques of cell culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein is that which is well known and commonly used in the art.

The term "herpes simplex virus (HSV)" as used herein is a DNA virus that harbors a double-stranded DNA of approximately 150 kb. Recently, it has been classified as a virus of the Herpesviridae family, the Alphaherpesvirinae subfamily, and the Simplexvirus genus. When virus particles are examined under an electron microscope, they have a structure containing DNA in the center called the core, which is surrounded by a regular icosahedral capsid, and the outer layer is further wrapped in an envelope derived from the nuclear membrane. (Latest Skin Science System, Vol. 15, pp. 8-19, Yasumoto Shinichiro, 2003). There are two types of HSV: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).

As used in this specification, "herpes simplex" refers to an infection caused by herpes simplex virus (HSV), and includes herpes labialis, genital herpes, herpes encephalitis, herpetic gingivostomatitis, facial herpes, corneal herpes, buttock herpes, herpetic whitlow, Kaposi's varicelliform eruption, and the like. "Herpes simplex" is also sometimes called "herpes simplex virus infection."

The term "antibody" as used herein means an immunoglobulin molecule consisting of four polypeptide chains, i.e., two heavy chains (H chains or HC) and two light chains (L chains or LC), which are interconnected by disulfide bonds. The monoclonal antibodies of the present invention are also composed of immunoglobulin molecules each including two heavy chains (H chains) and two light chains (L chains). Each H chain consists of an H chain variable region (sometimes referred to as "HCVR" or "VH") and an H chain constant region (the H chain constant region consists of three domains, also called "CH1", "CH2", and "CH3" (collectively: CH)). Each L chain consists of an L chain variable region (sometimes referred to as "LCVR" or "VL") and an L chain constant region (the L chain constant region consists of one domain, also called "CL"), and the part before the start of each constant region is called the variable region. The constant region is also called the "constant region" or "constant part region". The variable region is also called the "variable part region".

VH and VL are particularly important in that they are involved in the binding specificity of the antibody. Because antibodies interact with target antigens primarily through amino acid residues in VH and VL, the amino acid sequences in the variable regions vary more between individual antibodies than sequences outside the variable regions. Furthermore, VH and VL can be further subdivided into regions called framework regions (FRs) that are more constant among various antibodies, and hypervariable regions called complementarity determining regions (CDRs). VH and VL each consist of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Based on the amino acid sequences representing the variable regions and the amino acid sequences of the CDRs, human monoclonal antibodies (fully human antibodies), monoclonal antibodies (including chimeric antibodies and humanized antibodies), or antigen-binding fragments thereof that can specifically bind to a target antigen can be obtained by techniques well known in the art. These antibodies are within the scope of the present invention.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") as used herein means one or more fragments of an antibody (e.g., VH) that have the ability to bind to an antigen. The fragment also includes a peptide having a minimal amino acid sequence that binds to an antigen. Examples of binding moieties included within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, (ii) an F(ab') ₂ fragment, (iii) an Fd fragment consisting of a VH and CH1 domain, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward ES. et al., Nature, 341:544-546, 1989), (vi) an isolated complementarity determining region (CDR) having sufficient framework for binding, (vii) a bispecific antibody, and (viii) a multispecific antibody. In this specification, when the term "antibody" is used without any particular distinction, it includes not only a full-length antibody but also such an "antigen-binding fragment".

Depending on the heavy chain constant region, antibodies are divided into gamma, mu, alpha, delta and epsilon chains, and these differences result in the formation of five immunoglobulin classes (isotypes): IgG, IgM, IgA, IgD and IgE. Furthermore, in humans, there are four subclasses of IgG: IgG1 to IgG4. On the other hand, antibodies are divided into kappa and lambda chains depending on the light chain constant region. The class (subclass) of the first antibody described below is IgG1 (lambda), and the class (subclass) of the second antibody is IgG1 (kappa).

2. Anti-HSV Monoclonal Antibody or Antigen-Binding Fragment thereof The anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention specifically binds to the envelope glycoprotein of herpes simplex virus (HSV). In this case, the HSV is HSV-1 and/or HSV-2, preferably HSV-1. In this specification, the term "specifically binds" means that one antibody recognizes a certain antigen.

Five envelope glycoproteins, gB, gC, gD, gH, and gL, are involved in the cell invasion of HSV (see, for example, Seikagaku, Vol. 84, No. 5, pp. 343-351, 2012). Of these, the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention preferably specifically binds to envelope glycoprotein D (gD). In this case, the anti-HSV monoclonal antibody or antigen-binding fragment thereof can specifically bind to the envelope glycoprotein with high sensitivity.
Thereby, the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention can neutralize the "biological activity" (or "viral activity" or "infectivity") (these terms are used interchangeably herein) of herpes simplex virus (HSV), or at least suppress infection. As a result, a pharmaceutical composition or the like comprising the anti-HSV monoclonal antibody or antigen-binding fragment thereof can be used as a prophylactic drug having a prophylactic effect against herpes simplex, a therapeutic drug having a therapeutic effect against herpes simplex, or the like.
Furthermore, the immunological measurement device containing the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention can accurately test for the presence or absence of herpes simplex onset.

An anti-HSV monoclonal antibody according to one embodiment of the present invention specifically binds to an envelope glycoprotein of herpes simplex virus (HSV), and (i) the heavy chain variable region contains (a) an amino acid sequence of heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, (b) an amino acid sequence of heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, and (c) an amino acid sequence of heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, and (ii) the light chain variable region contains (a) an amino acid sequence of light chain CDR1 comprising the amino acid sequence of SEQ ID NO:6, (b) an amino acid sequence of light chain CDR2 comprising the amino acid sequence of SEQ ID NO:7, and (c) an amino acid sequence of light chain CDR3 comprising the amino acid sequence of SEQ ID NO:8 (hereinafter, the above anti-HSV monoclonal antibody is also referred to as the "first antibody").

In a preferred embodiment, the first antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:5.

In a preferred embodiment, the first antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

In one embodiment, the heavy chain variable region (HCVR), the three complementarity determining regions of the heavy chain variable region (HCDR1, HCDR2, HCDR3), the light chain variable region (LCVR), the three complementarity determining regions of the light chain variable region (LCDR1, LCDR2, LCDR3), the heavy chain (HC), and the light chain (LC) of the first antibody are as shown in Table 1 below.

An anti-HSV monoclonal antibody according to one embodiment of the present invention specifically binds to the envelope glycoprotein of herpes simplex virus (HSV), and (i) the heavy chain variable region contains (d) an amino acid sequence of heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (e) an amino acid sequence of heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (f) an amino acid sequence of heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and (ii) the light chain variable region contains (d) an amino acid sequence of light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (e) an amino acid sequence of light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) an amino acid sequence of light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18 (hereinafter, the above anti-HSV monoclonal antibody is also referred to as the "second antibody").

In a preferred embodiment, the second antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:15.

In a preferred embodiment, the second antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 19 and a light chain having the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the heavy chain variable region (HCVR), the three complementarity determining regions of the heavy chain variable region (HCDR1, HCDR2, HCDR3), the light chain variable region (LCVR), the three complementarity determining regions of the light chain variable region (LCDR1, LCDR2, LCDR3), the heavy chain (HC), and the light chain (LC) of the second antibody are as shown in Table 2 below.

The amino acid sequence of SEQ ID NO:1 (heavy chain variable region: HCVR), the amino acid sequence of SEQ ID NO:5 (light chain variable region: LCVR), the amino acid sequence of SEQ ID NO:9 (heavy chain: HC), and the amino acid sequence of SEQ ID NO:10 (light chain: LC) contained in the first antibody may be substantially identical to these amino acid sequences. However, the substantially identical amino acid sequences include the three complementarity determining regions (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of SEQ ID NO:2 to 4 and the three complementarity determining regions (LCDR1, LCDR2, LCDR3) of the light chain variable region of SEQ ID NO:6 to 8.

Furthermore, the amino acid sequence of SEQ ID NO: 11 (heavy chain variable region: HCVR), the amino acid sequence of SEQ ID NO: 15 (light chain variable region: LCVR), the amino acid sequence of SEQ ID NO: 19 (heavy chain: HC), and the amino acid sequence of SEQ ID NO: 20 (light chain: LC) contained in the second antibody may be substantially identical to these amino acid sequences. However, the substantially identical amino acid sequences include the three complementarity determining regions (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of SEQ ID NO: 12 to 14 and the three complementarity determining regions (LCDR1, LCDR2, LCDR3) of the light chain variable region of SEQ ID NO: 16 to 18.

The term "substantially identical" as used herein means the presence of one or more deletions, substitutions, insertions, or additions of amino acid residues in the amino acid sequence of the antibody of the present invention, or, when any two or more of these are combined, means that there is a deletion, substitution, insertion, or addition of one or more amino acid residues at any one or more positions in the same sequence, and two or more of the deletions, substitutions, insertions, and additions may occur simultaneously.

Typically, an amino acid sequence that is substantially identical to the amino acid sequence of a specific heavy or light chain, its variable region, etc., shown in each of SEQ ID NOs: 1, 5, 9, 10, 11, 15, 19, and 20 includes an amino acid sequence that has at least, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or more, identity (or sequence identity) to each of SEQ ID NOs: 1, 5, 9, 10, 11, 15, 19, and 20.

For example, even monoclonal antibodies having an amino acid sequence in which approximately 10% or less (or 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.1% or less) of the number of amino acid residues in the amino acid sequence of the first antibody or the second antibody have been deleted, substituted, inserted, or added, or any combination of two or more of these mutations, can be included in the scope of the antibodies of the present invention, so long as they have an effect equivalent to the activity of the non-limiting monoclonal antibodies described above (i.e., specifically binding to the envelope glycoprotein of herpes simplex virus (HSV)).

The amino acids that make up natural proteins can be divided into groups based on the properties of their side chains, e.g., groups of amino acids with similar biochemical properties include aromatic amino acids (tyrosine, phenylalanine, tryptophan), basic amino acids (lysine, arginine, histidine), acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (serine, threonine, asparagine, glutamine), amino acids with hydrocarbon chains (alanine, valine, leucine, isoleucine, proline), and others (glycine, methionine, cysteine). Substitution of amino acid residues with side chains that have similar biochemical properties can be made while retaining the biological activity of the original protein.

For example, amino acid residues (including non-natural amino acids) in the same group as shown below may be mutually substitutable (while retaining the biological activity of the original protein). Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine, tryptophan.

The identity of amino acid sequences and nucleotide sequences described below can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S and Altschul S F, PNAS, 87:2264-2268, 1990; PNAS, 90:5873-5877, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul S F. et al., J Mol Biol, 215:403-410, 1990). When analyzing nucleotide sequences using BLASTN, the parameters are, for example, score = 100 and word length = 12. When analyzing amino acid sequences using BLASTX, the parameters are, for example, score = 50 and word length = 3. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Alternatively, when determining the identity of protein amino acid sequences, the amino acid sequences of the two proteins being compared are aligned and the number of identical amino acid residues is counted, giving the formula "(number of identical amino acid residues/number of amino acid residues in the entire protein) x 100 (%)".

In a preferred embodiment, the first antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:5.

In a preferred embodiment, the first antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:9, and a light chain comprising the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:10.

In a preferred embodiment, the second antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:15.

In a preferred embodiment, the second antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:19, and a light chain comprising the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:20.

The first or second antibody described above may be a human monoclonal antibody (fully human antibody), a chimeric antibody, or a humanized antibody, but is preferably a human monoclonal antibody. Note that a human monoclonal antibody (fully human antibody) has an antibody whose amino acid sequence is entirely the same as that of a human, a chimeric antibody is an antibody in which the variable region of a non-human antibody (e.g., a mouse antibody) is linked to the constant region of a human antibody, and a humanized antibody is an antibody in which the three complementarity determining regions of a non-human antibody (e.g., a mouse antibody) are transplanted into a human antibody.

The first or second antibody may be an antigen-binding fragment thereof.

In one embodiment, the antigen-binding fragment of the first antibody has a heavy chain variable region that contains (a) an amino acid sequence of heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, (b) an amino acid sequence of heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, and (c) an amino acid sequence of heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, and (ii) a light chain variable region that contains (a) an amino acid sequence of light chain CDR1 comprising the amino acid sequence of SEQ ID NO:6, (b) an amino acid sequence of light chain CDR2 comprising the amino acid sequence of SEQ ID NO:7, and (c) an amino acid sequence of light chain CDR3 comprising the amino acid sequence of SEQ ID NO:8, and specifically binds to an envelope glycoprotein of herpes simplex virus (HSV).

In one embodiment, the antigen-binding fragment of the first antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:5, and specifically binds to the envelope glycoprotein of herpes simplex virus (HSV).

In one embodiment, the antigen-binding fragment of the second antibody has a heavy chain variable region that contains (i) an amino acid sequence of a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (e) an amino acid sequence of a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and (f) an amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and (ii) a light chain variable region that contains (d) an amino acid sequence of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (e) an amino acid sequence of a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) an amino acid sequence of a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18, and specifically binds to an envelope glycoprotein of herpes simplex virus (HSV).

In one embodiment, the antigen-binding fragment of the second antibody contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:15, and specifically binds to the envelope glycoprotein of herpes simplex virus (HSV).

The antigen-binding fragment of the first or second antibody is preferably an F(ab) fragment or an F(ab') ₂ fragment, more preferably an F(ab') ₂ fragment.

3. Method for Producing Anti-HSV Monoclonal Antibody or Antigen-Binding Fragment thereof The above-mentioned anti-HSV monoclonal antibody or antigen-binding fragment thereof can be produced by known methods (for example, Riechmann L. et al., Nature, 332:323-327, 1988).

For example, the above-mentioned anti-HSV monoclonal antibody or antigen-binding fragment thereof can be obtained by isolating a cell clone that produces the antibody from the blood of a healthy individual (who may have a history of HSV infection) or an individual who has recovered from HSV infection (an individual in which HSV is in a dormant state after initial infection) through various steps, cloning the DNA that encodes the target antibody, and then carrying out gene expression and antibody production. Specific methods include, for example, the methods described in the Examples.

The antibody of the present invention can also be produced as a recombinant human antibody using known methods (see, for example, Boulianne GL et al., Nature, 312:643-646, 1984; Jones PT et al., Nature, 321:522-525, 1986). For example, the antibody of the present invention can be produced by culturing a host cell into which the vector of the present invention has been introduced and purifying the produced antibody from the culture supernatant or the like. More specifically, the antibody can be produced by constructing a human antibody expression vector by inserting the cDNA encoding VH and VL into an expression vector for animal cells containing genes encoding a human antibody CH and/or a human antibody CL prepared from the same cell or a different human cell, and introducing the human antibody expression vector into an animal cell for expression.

Furthermore, antigen-binding fragments of anti-HSV monoclonal antibodies, such as Fab, Fab', F(ab') ₂ , fragments of active antibody fragments linked via linkers or the like (e.g., single chain Fv (scFv) and disulfide stabilized Fv (dsFv)), and peptides containing active antibody fragments (e.g., peptides containing CDRs) can be produced by known methods, for example, by treating prepared antibodies with appropriate proteases or by methods using gene recombination techniques.

In addition, specific antibodies can be obtained by using phage display antibody technology, which utilizes genetic engineering technology to express recombinant antibodies on the surface of phages, to artificially shuffle VH and VL genes to diversify and express single chain fragment of variable region (scFv) antibodies as phage fusion proteins. This technology can avoid immunity and is highly regarded as a humanized antibody production technology that can replace cell fusion methods. Specific antibodies or antigen-binding fragments thereof produced using this technology with reference to the amino acid sequences in this specification, for example, SEQ ID NOs: 2 to 4 and SEQ ID NOs: 6 to 8, or SEQ ID NOs: 12 to 14 and SEQ ID NOs: 16 to 18, are also within the scope of the present invention.

Furthermore, antibodies obtained by applying Potellegent technology, which significantly improves the ADCC activity of an antibody by modifying the glycan portion of the antibody, to the antibody of the present invention (see Niwa R et al., Clin. Cancer Res., 10:6248-6255, 2004), and antibodies obtained by applying Complegnent technology, which improves CDC activity, to the antibody of the present invention (see Kanda Y. et al., Glycobiology, 17:104-118, 2007) are also within the scope of the present invention. Similarly, antibodies obtained by modifying the amino acid sequence of the antibody constant region to alter ADCC activity (WO 2007/039682 and WO 2007/100083) or CDC activity (WO 2007/011041 and WO 2011/091078) are also within the scope of the present invention.

Furthermore, the scope of the present invention also includes antibodies or antigen-binding fragments thereof obtained by applying a technique for partially replacing the Fc region (see WO 2006/071877) to confer protease resistance to the antibody and enable oral administration.

4. Isolated Nucleic Acid Molecules According to one aspect of the present invention, there is provided an isolated nucleic acid molecule, wherein the nucleic acid molecule is DNA or RNA, and typically is DNA.

In one embodiment, the isolated nucleic acid molecule is a nucleic acid molecule encoding the amino acid sequence of the above-mentioned antibody (anti-HSV monoclonal antibody) or an antigen-binding fragment thereof.

In another embodiment, the isolated nucleic acid molecule is a nucleic acid molecule that hybridizes under high stringency conditions with any of the nucleic acid molecules encoding the amino acid sequence of the above-mentioned antibody or antigen-binding fragment thereof. This makes it possible to obtain an isolated nucleic acid molecule that has a high identity with the nucleic acid molecule encoding the amino acid sequence of the above-mentioned antibody or antigen-binding fragment thereof (i.e., an isolated nucleic acid molecule selected from nucleic acids that hybridize with the nucleic acid molecule under high stringency conditions). Here, "having a high identity" means a degree of sequence identity that can hybridize to a specific nucleic acid sequence under high stringency conditions, for example, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or more than that. The identity of base sequences can be determined using the identity search algorithms mentioned above (Karlin S and Altschul SF, PNAS, 87:2264-2268, 1990; PNAS, 90:5873-5877, 1993).

Here, "highly stringent conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C (see, for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989), especially section 11.45 "Conditions for Hybridization of Oligonucleotide Probes"). Under these conditions, it is expected that polynucleotides (e.g., DNA) with higher identity can be obtained more efficiently as the temperature is increased. However, several factors, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, are thought to affect the stringency of hybridization, and a person skilled in the art can achieve similar stringency by appropriately selecting these factors.

In another embodiment, the isolated nucleic acid molecule has a base sequence of SEQ ID NO:21 to 24, or a base sequence having 90% or more identity to a base sequence of SEQ ID NO:21 to 24. Here, SEQ ID NO:21 is a base sequence (DNA) encoding the heavy chain (HC) of a first antibody, SEQ ID NO:22 is a base sequence (DNA) encoding the light chain (LC) of the first antibody, SEQ ID NO:23 is a base sequence (DNA) encoding the heavy chain (HC) of a second antibody, and SEQ ID NO:24 is a base sequence (DNA) encoding the light chain (LC) of the second antibody. The base sequences of SEQ ID NO:21 to 24 are specifically as follows:

SEQ ID NO:21:

SEQ ID NO:22:

SEQ ID NO:23:

SEQ ID NO:24

5. Recombinant Expression Vector According to one aspect of the present invention, there is provided a recombinant expression vector incorporating the above-mentioned isolated nucleic acid molecule.

In one embodiment, the recombinant expression vector is a recombinant expression vector incorporating at least one of an isolated nucleic acid molecule encoding the amino acid sequence of an anti-HSV monoclonal antibody or an antigen-binding fragment thereof, and an isolated nucleic acid molecule that hybridizes with any of these nucleic acid molecules under highly stringent conditions.

In another embodiment, the recombinant expression vector is a recombinant expression vector incorporating at least one of the isolated nucleic acid molecules having a base sequence of SEQ ID NO:21 to 24 and a base sequence having 90% or more identity to the base sequence of SEQ ID NO:21 to 24.

　The vector into which the isolated nucleic acid of the present invention is incorporated is not particularly limited, but may be a vector or a high expression vector that is generally used for the expression of protein genes and is particularly suitable for the expression of antibody genes. Non-limiting examples include vectors containing an FE promoter and/or a CMV enhancer.

In addition, the isolated nucleic acid molecule required for constructing an anti-HSV monoclonal antibody may be incorporated into one vector, or into two or more vectors. For example, the base sequence of SEQ ID NO:21 encoding the heavy chain (HC) of the first antibody and the base sequence of SEQ ID NO:22 encoding the light chain (LC) of the first antibody may both be incorporated into one vector, or the base sequence of SEQ ID NO:21 may be incorporated into a vector and the base sequence of SEQ ID NO:22 may be incorporated into another vector.

6. Isolated Host Cell According to one aspect of the present invention, there is provided an isolated host cell into which the above-described recombinant expression vector has been introduced.

Host cells into which expression vectors are introduced include, but are not limited to, cells commonly used for the expression of protein genes, and particularly cells suitable for the expression of antibody genes. Examples include bacteria (such as E. coli), actinomycetes, yeast, insect cells (such as SF9), and mammalian cells (COS-1, CHO, myeloma cells, etc.). To industrially produce recombinant antibodies, recombinant animal cell lines that stably produce high levels of the antibody, such as CHO cell lines, are generally used. Such recombinant cell lines can be created, cloned, and gene-amplified and screened for high expression using known methods (see, for example, Omasa T., J. Biosci. Bioeng., 94:600-605, 2002).

When the isolated nucleic acid molecule required for constructing an anti-HSV monoclonal antibody is incorporated into two or more vectors, all of them are generally introduced into a host cell. For example, when there is a first vector incorporating the base sequence of SEQ ID NO:21, and a second vector incorporating the base sequence of SEQ ID NO:22, both the first vector and the second vector are generally introduced into a host cell.

Anti-HSV monoclonal antibodies can be produced by culturing the isolated host cells of the present invention. Note that anti-HSV monoclonal antibodies are usually produced ex vivo.

The method for purifying the antibody is not particularly limited, and can be performed using known purification methods such as salting out, gel filtration, ion exchange chromatography, or affinity chromatography.

According to one embodiment of the present invention , there is provided a pharmaceutical composition comprising the above-mentioned antibody (anti-HSV monoclonal antibody) or an antigen-binding fragment thereof. In this case, the pharmaceutical composition may be used to prevent (prophylaxis) the recurrence of herpes simplex, or may be used to treat herpes simplex (for example, to improve blisters, ulcers, itchiness, discomfort, pain, etc.).

The content of the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention is preferably 0.0001 to 90% by mass based on the total mass of the pharmaceutical composition.
When the pharmaceutical composition is a liquid preparation, the content of the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention is preferably 0.001 to 1000 mg/mL, more preferably 0.1 to 200 mg/mL, even more preferably 0.1 to 100 mg/mL, and particularly preferably 1 to 50 mg/mL.

The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents.

Examples of pharma- ceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof. When used as an injection or the like, the pharmaceutical composition may contain pH adjusting agents and isotonicity agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride. The pharma-ceutically acceptable carrier may further contain minor amounts of auxiliary substances that enhance the shelf life or effectiveness of the antibody or antibody portion, such as wetting agents, emulsifiers, preservatives, buffers, stabilizers, surfactants, excipients, and antioxidants.

The amount of these pharma- ceutically acceptable carriers added is preferably 0.001 to 1000 times, and more preferably 0.01 to 100 times, the mass of the above-mentioned antibody (anti-HSV monoclonal antibody) or its antigen-binding fragment. The appropriate composition of the pharmaceutical composition in the formulation can be appropriately determined by those skilled in the art depending on the applicable disease, applicable administration route, etc.

The pharmaceutical compositions of the present invention can be in a variety of dosage forms. The dosage forms of the pharmaceutical compositions include liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injectable and infusible solutions), dispersions, suspensions, tablets, capsules, troches, pills, powders, liposomes, and suppositories. The dosage form can vary depending on the intended mode of administration and therapeutic application. For example, it can include injectable or infusible solutions, such as pharmaceutical compositions similar to those commonly used to passively immunize humans with other antibodies. For example, the mode of administration can be parenteral (e.g., intravenous, subcutaneous, transdermal, intraperitoneal, intramuscular, pulmonary, nasal). Alternatively, the pharmaceutical compositions can be administered by intravenous infusion or injection. Alternatively, the pharmaceutical compositions can be administered by intramuscular, subcutaneous, or intradermal injection. Alternatively, the pharmaceutical compositions can be administered by inhalation or nasal administration.

The effective amount of the pharmaceutical composition of the present invention is preferably 0.0001 to 100 mg/kg, more preferably 0.001 to 100 mg/kg, even more preferably 0.01 to 10 mg/kg, and particularly preferably 0.1 to 1 mg/kg. In this specification, the term "effective amount" refers to the amount of the anti-HSV monoclonal antibody or antigen-binding fragment thereof of the present invention that is required to be administered in order to exert an effect in the body.

The pharmaceutical composition of the present invention can be administered three times a day, twice a day, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 7 days (1 week), once every 10 days, once every 14 days (2 weeks), or once every 21 days (3 weeks).

The pharmaceutical composition according to the present invention can be produced by a known method. For example, in the case of an injection, an injectable pharmaceutical composition can be produced by mixing the anti-HSV monoclonal antibody or antigen-binding fragment thereof according to the present invention with a pharma- ceutical acceptable carrier. In this case, the injectable pharmaceutical composition can be dissolved in an injection solution at the time of use and administered, for example, intravenously.

8. Method for Treating Herpes Simplex According to one embodiment of the present invention, there is provided a method for treating herpes simplex, comprising administering the above-mentioned antibody (anti-HSV monoclonal antibody) or antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition to a patient suffering from herpes simplex. The method for treating herpes simplex includes prevention of recurrence (prophylaxis) of herpes simplex, treatment of herpes simplex (for example, improvement of blisters, ulcers, itchiness, discomfort, pain, etc.), etc.

Herpes simplex is not particularly limited, and may be oral herpes, genital herpes, herpes encephalitis, or other types of herpes simplex.

The causative virus of herpes simplex may be HSV-1 or HSV-2.

The administration form of the above-mentioned antibody (anti-HSV monoclonal antibody) or antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition may be, for example, parenteral (e.g., intravenous, subcutaneous, transdermal, intraperitoneal, intramuscular, pulmonary or nasal). Alternatively, the antibody, etc. may be administered by intravenous infusion or intravenous injection. Alternatively, the antibody, etc. may be administered by intramuscular, subcutaneous or intradermal injection. Alternatively, the antibody, etc. may be administered by inhalation or nasal administration.

9. Method for detecting herpes simplex virus (HSV) According to one aspect of the present invention, a method for detecting herpes simplex virus (HSV) is provided.

In one embodiment, the method for detecting herpes simplex virus (HSV) includes a reaction step of reacting HSV with an anti-HSV monoclonal antibody or a binding fragment thereof, or a derivative thereof, and a detection step of detecting HSV.

The reaction step involves reacting HSV with an anti-HSV monoclonal antibody or a binding fragment thereof, or a derivative thereof. The anti-HSV monoclonal antibody or a binding fragment thereof, or a derivative thereof, specifically binds to the envelope glycoprotein (preferably envelope glycoprotein D (gD)) of herpes simplex virus (HSV) of HSV, thereby forming a complex such as an HSV-anti-HSV monoclonal antibody.

In one embodiment, the derivative of the anti-HSV monoclonal antibody or its binding fragment is a labeled anti-HSV monoclonal antibody or its binding fragment. In this case, the label may be, but is not limited to, a fluorescent label, a luminescent label, a radioactive label, an enzyme label, biotin, magnetic beads, agarose beads, magnetic agarose beads, colored cellulose particles, gold colloid particles, etc.

The detection step is a step of detecting HSV-anti-HSV monoclonal antibodies. The detection method is not particularly limited, but includes ELISA (enzyme-linked immunosorbent assay), CLEIA (chemiluminescent enzyme immunoassay), dot blot, immunochromatography, and immunostaining.

For example, specific methods of ELISA include the direct sandwich method, the indirect sandwich method, the direct adsorption method, the indirect adsorption method, and the competitive method. Specific methods of immunostaining include the direct method, the indirect method, and the sensitization method.

The herpes simplex virus (HSV) detection method can detect HSV in a sample.

10. Immunoassay device According to one aspect of the present invention, there is provided an immunoassay device for detecting herpes simplex virus (HSV).

In one embodiment, the immunological measurement device includes the above-mentioned antibody (anti-HSV monoclonal antibody) or an antigen-binding fragment thereof.

In another embodiment, the immunological measurement device includes a sample dropping section, a labeling substance holding section, a detection section, and an absorption section, and at least one of the labeling substance holding section and the detection section includes the above-mentioned antibody (anti-HSV monoclonal antibody) or an antigen-binding fragment thereof.

The above-mentioned antibody (anti-HSV monoclonal antibody) or its antigen-binding fragment has high sensitivity to, for example, herpes simplex virus (HSV), and can therefore be used to accurately test for the onset of herpes simplex.

The immunological measurement device according to the present invention will be described below with reference to the drawings. Note that the drawings may be exaggerated for the purpose of explanation and may differ from the actual dimensions.

1 is a schematic diagram of an immunoassay device according to one embodiment of the present invention. The immunoassay device 1 has a sample dropping section 2, a labeled substance holding section 3, a detection section 4, and an absorption section 7. These are formed on a backing sheet 8. The labeled substance holding section 3 holds an anti-HSV antibody (labeled anti-HSV antibody) 3a labeled with colored cellulose particles. The detection section 4 has a test line 5 and a control line 6. The test line 5 holds (immobilizes) an anti-HSV antibody 5a, and the control line 6 holds (immobilizes) a capture antibody 6a that binds to the labeled antibody 3a. In a preferred embodiment, the labeled anti-HSV antibody 3a is a labeled second antibody, and the anti-HSV antibody 5a is a first antibody. In another preferred embodiment, the labeled anti-HSV antibody 3a is a labeled first antibody, and the anti-HSV antibody 5a is an antibody of the second antibody.

When testing a sample containing HSV collected from a subject (detecting HSV in the sample), the sample is first dripped into the sample drip section 2. The dripped sample permeates from the sample drip section 2 into the labeled substance holding section 3. Here, the labeled anti-HSV antibody 3a contained in the labeled substance holding section 3 specifically binds to the envelope glycoprotein (preferably envelope glycoprotein D (gD)) of HSV contained in the sample, forming a labeled anti-HSV antibody-HSV complex.

The sample (containing the labeled anti-HSV antibody-HSV complex and the labeled anti-HSV antibody) permeates the detection section 4 and moves through the detection section 4 by capillary action (moving from left to right in Figure 1). As the sample passes through the test line 5, the anti-HSV antibody 5a specifically binds to the labeled anti-HSV antibody-HSV complex in the sample (more specifically, the HSV envelope glycoprotein in the complex) to form a labeled anti-HSV antibody-HSV-anti-HSV antibody complex. Because the anti-HSV antibody 5a is immobilized on the test line 5, the labeled anti-HSV antibody-HSV-anti-HSV antibody complex is retained on the test line 5 without moving with the sample.

As the sample (containing the labeled anti-HSV antibody-HSV complex and the labeled anti-HSV antibody) moves further through the detection section and passes through the control line 6, the capture antibody 6a binds to the labeled anti-HSV antibody-HSV complex (more specifically, the labeled anti-HSV antibody in the complex) and/or the labeled anti-HSV antibody in the sample to form a capture antibody-labeled anti-HSV antibody-HSV complex and/or a capture antibody-labeled anti-HSV antibody complex. Because the capture antibody 6a is immobilized on the control line 6, the capture antibody-labeled anti-HSV antibody-HSV complex and/or the capture antibody-labeled anti-HSV antibody complex are retained on the control line 6 without moving with the sample.

The sample (containing the labeled anti-HSV antibody-HSV complex and the labeled anti-HSV antibody) moves further through the detection section 4, where the sample is absorbed in the absorption section 7 to prevent backflow.

If the sample contains HSV, the HSV will form a complex with the labeled anti-HSV antibody 3a in the labeled substance holding section 3, and then form a complex with the anti-HSV antibody 5a in the test line 5, causing the test line 5 to develop a color. In this way, HSV in the sample can be detected. Note that if the sample does not contain HSV, no such complex is formed, and the test line 5 will not develop a color.

On the other hand, the capture antibody 6a in the control line 6 forms a complex with the labeled anti-HSV antibody 3a in the labeled substance holding section 3, so the control line 6 turns colored regardless of the presence or absence of HSV in the sample.

The components of the immunoassay device are explained below.

[Sample drop section]
The sample dropping section is preferably made of a material that absorbs the sample and easily transfers the sample to the labeling substance holding section. Examples of materials used for the sample dropping section include nonwoven fabrics using cellulose fiber, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, etc., cotton cloth, porous materials, agarose gel, paper, plastics, etc. These materials may be used alone or in combination of two or more.

[Labeled substance holding section]
The labeled substance holding portion includes, for example, a conjugate pad and a labeled anti-HSV antibody.

The conjugate pad may be made of, for example, paper, cellulose, nitrocellulose, polyester, acrylonitrile copolymer, glass fiber, rayon, or other nonwoven fibers.

The labeled anti-HSV antibody can be produced by labeling an anti-HSV antibody or its binding fragment with a labeling substance.

The anti-HSV antibody or binding fragment thereof is not particularly limited, but includes the anti-HSV monoclonal antibody or antigen-binding fragment thereof of the present invention, and known anti-HSV monoclonal antibodies or antigen-binding fragments thereof. Of these, at least one of a first antibody and its antigen-binding fragment, and a second antibody and its antigen-binding fragment is preferable, and at least one of a first antibody and a second antibody is more preferable. The above-mentioned anti-HSV antibody or binding fragment thereof may be used alone or in combination of two or more types.

The labeling substance is not particularly limited, but examples thereof include gold colloid particles, silver colloid particles, platinum-gold colloid particles, iron oxide colloid particles, sulfur particles, colored latex particles, colored cellulose particles, magnetic particles, etc. Among these, the labeling substance is preferably gold colloid particles, silver colloid particles, platinum-gold colloid particles, or colored cellulose particles, more preferably gold colloid particles or colored cellulose particles, and even more preferably colored cellulose particles. The above-mentioned labeling substances may be used alone or in combination of two or more kinds.

The average particle size of the labeled substance is not particularly limited, but is preferably 10 to 500 nm, more preferably 20 to 450 nm, even more preferably 30 to 400 nm, and particularly preferably 100 to 400 nm. In this specification, "particle size" refers to the maximum distance between two points on the contour line of the labeled substance. Also, "average particle size" refers to the average particle size of 200 labeled substances randomly selected from among objects observed in one field of view of a transmission electron microscope (TEM).

The content of the labeled anti-HSV antibody or binding fragment thereof in the labeled substance holding portion is preferably 0.05 to 1 μg/cm ² , more preferably 0.1 to 0.8 μg/cm ² , and even more preferably 0.15 to 0.7 μg/cm ² .

It is preferable that the labeled anti-HSV antibody or its binding fragment is impregnated into a conjugate pad. The labeled anti-HSV antibody can also be added directly to the sample.

[Detection unit]
The detection portion includes a membrane having a test line and a control line.

The membrane is not particularly limited, but examples include porous membranes made of nitrocellulose, cellulose acetate, cellulose, glass, nylon, polyethylene, polyethylene terephthalate, etc. Of these, it is preferable that the membrane is a nitrocellulose membrane.

The membrane may contain hydrophilic compounds such as sugars, amino acids, amino acid derivatives, surfactants, and alcohols. By containing a hydrophilic compound in the membrane, the surface tension on the membrane surface can be reduced, promoting capillary action. This can shorten the test (detection) time.

The test line is formed by retaining anti-HSV antibodies or binding fragments thereof in a fixed arrangement on the membrane.

The test line is usually placed upstream of the control line described below.
The test line is usually linear (line-like), but may also be circular, numeric, symbolic, or other shape.

The anti-HSV antibody or binding fragment thereof is not particularly limited, and examples thereof include the anti-HSV monoclonal antibody or antigen-binding fragment thereof of the present invention, and known anti-HSV monoclonal antibodies or antigen-binding fragments thereof. Of these, the anti-HSV antibody or binding fragment thereof is preferably at least one of a first antibody and its antigen-binding fragment, and a second antibody and its antigen-binding fragment, and more preferably at least one of a first antibody and a second antibody. The above-mentioned anti-HSV antibodies or binding fragments thereof may be used alone or in combination of two or more types.

The anti-HSV antibody in the test line may be the same as or different from the anti-HSV antibody that constitutes the labeled anti-HSV antibody in the label holding portion, but it is preferable that they are different.

In one embodiment, it is preferable that at least one of the labeled substance holding section and the detection section contains the anti-HSV monoclonal antibody or its antigen-binding fragment according to the present invention, and it is more preferable that both the labeled substance holding section and the detection section contain the anti-HSV monoclonal antibody or its antigen-binding fragment according to the present invention. The anti-HSV monoclonal antibody or its antigen-binding fragment according to the present invention may be contained as the labeled anti-HSV antibody in the labeled substance holding section, and as the anti-HSV antibody of the test line in the detection section.

In a preferred embodiment, the anti-HSV antibody contained in the labeled substance holding section (the anti-HSV antibody constituting the labeled anti-HSV antibody) is a first antibody or an antigen-binding fragment thereof, and the anti-HSV antibody contained in the detection section (the anti-HSV antibody of the test line) is a second antibody or an antigen-binding fragment thereof. In a more preferred embodiment, the anti-HSV antibody contained in the labeled substance holding section (the anti-HSV antibody constituting the labeled anti-HSV antibody) is the first antibody, and the anti-HSV antibody contained in the detection section (the anti-HSV antibody of the test line) is the second antibody.

In another preferred embodiment, the anti-HSV antibody contained in the labeled substance holding section (the anti-HSV antibody constituting the labeled anti-HSV antibody) is a second antibody or an antigen-binding fragment thereof, and the anti-HSV antibody contained in the detection section (the anti-HSV antibody of the test line) is a first antibody or an antigen-binding fragment thereof. In a more preferred embodiment, the anti-HSV antibody contained in the labeled substance holding section (the anti-HSV antibody constituting the labeled anti-HSV antibody) is a second antibody, and the anti-HSV antibody contained in the detection section (the anti-HSV antibody of the test line) is a first antibody.

In a preferred embodiment, the ratio of the content (mass%) of the labeled anti-HSV antibody contained in the labeled substance holding section to the content (mass%) of the anti-HSV antibody (anti-HSV antibody of the test line) contained in the detection section (content of anti-HSV antibody contained in the detection section/labeled anti-HSV antibody contained in the labeled substance holding section) is preferably 1 to 10, more preferably 3 to 7, and even more preferably 4 to 5. It is preferable that the content ratio is within the above range because it allows for high sensitivity detection. In a more preferred embodiment, the anti-HSV antibody constituting the labeled anti-HSV antibody is a first antibody, and the anti-HSV antibody contained in the detection section is a second antibody. In another embodiment, the anti-HSV antibody constituting the labeled anti-HSV antibody is a second antibody, and the anti-HSV antibody contained in the detection section is a first antibody.

The content of the anti-HSV antibody or binding fragment thereof in the test line is preferably 0.05 to 1 μg/cm ² , more preferably 0.075 to 0.75 μg/cm ² , and even more preferably 0.1 to 0.6 μg/cm ² .

The control line is formed by holding the capture antibody in a consistent position on the membrane.

The control line is usually located downstream from the test line.
The control line is usually linear (line-like), but may also be circular, numeric, symbolic, or other shape.
Additionally, the control line can be omitted.

The capture antibody is not particularly limited as long as it binds to the labeling substance of the labeled anti-HSV antibody or the labeling substance of the labeled anti-HSV antibody-HSV complex, and any known capture antibody can be used. The capture antibody is usually an anti-IgG antibody (e.g., a gold colloid-bound anti-IgG antibody).

The content of the capture antibody in the control line is preferably 0.05 to 1 μg/ ^cm2 , more preferably 0.075 to 0.75 μg/ ^cm2 , and even more preferably 0.1 to 0.5 μg/ ^cm2 .

[Absorption part]
The absorption section absorbs the sample that has passed through the detection section, thereby preventing liquid backflow.

The absorbent section includes an absorbent pad. Examples of the absorbent pad include, but are not limited to, cellulose fiber, glass fiber, cotton using pulp, nonwoven fabric, and filter paper.

[Backing sheet]
The backing sheet serves as a base material to support the immunoassay device. In general, the immunoassay device has a detection section disposed on the backing sheet, a label holding section and an absorption section disposed on the detection section, and a sample dropping section disposed on the label holding section.
The backing sheet is not particularly limited, but examples thereof include polystyrene, polyester, polypropylene, and vinyl chloride.

The present invention will be explained in detail below with reference to examples, but the present invention is not limited to these.

[Example 1: Preparation of first and second antibodies]
The first and second antibodies were prepared according to the following procedure.
(1) B cells (B lymphocytes) were isolated from peripheral blood mononuclear cells (PBMCs) collected from healthy individuals (possibly with a history of HSV infection), and the B cells were infected with Epstein-Barr virus (EBV). The infected B cells were seeded onto a 96-well plate and cultured for about three weeks to obtain an immortalized (transformed) B cell line (a human B cell line that has acquired the ability to proliferate).

(2) Anti-HSV antibodies in the culture supernatant produced by human B cells that had acquired the ability to proliferate were examined by immunostaining (indirect method) to confirm whether they bound to HSV-1 and HSV-2 gD glycoprotein (HSV gD), and wells producing anti-HSV antibodies were identified.

The immunostaining method (indirect method) was performed according to the following (a) to (d).

(a) Preparation of anti-HSV gD antibody screening plate An expression vector encoding HSV-1 gD or HSV-2 gD was transfected into CHO-K1 cells using a transfection reagent (Lipofectamine LTX, Invitrogen), and the cells were harvested after culturing for 16 hours (37°C, 5% _CO2 ).
Cells expressing HSV-1 gD or HSV-2 gD were suspended in 10% FCS-containing DMEM medium and seeded on a 96-well microwell plate (Greiner). After 24 hours of culture, the cells were fixed with 4% paraformaldehyde, washed, and then 0.2% Triton X-100 was added to perform membrane permeabilization treatment to prepare an anti-HSV gD antibody screening plate.

(b) Primary Antibody Reaction Anti-HSV antibody (1 μg/mL) was added to the anti-HSV gD antibody screening plate, and the plate was incubated at room temperature (25° C.) for 1 hour, and then washed.

(c) Secondary Antibody Reaction A fluorescently labeled anti-human IgG antibody (Anti-hIgG(γchain)-Alexa488, Invitrogen) was used as a secondary antibody, and reacted with the antibody bound to the antigen in the primary antibody reaction.

(d) Color development and evaluation After DAPI staining, stained images were taken with an imaging cytometer (In Cell Analyzer 2000, GE Healthcare) and analyzed using analysis software (In Cell Developer Toolbox 1.9.2, GE Healthcare).

(3) The culture supernatants from the identified anti-HSV antibody producing wells were used to confirm the isotypes of the anti-HSV antibodies by immunostaining (indirect method). The immunostaining (indirect method) was performed in the same manner as described in (2) above. However, as the secondary antibodies, antibodies specific to each isotype and subclass were used instead of the fluorescently labeled anti-human IgG antibody.
(4) From the identified anti-HSV antibody-producing wells, cell clones producing the target antibody were obtained using limiting dilution or the like.

(5) Total RNA from antibody-producing cells was reverse transcribed using an oligo-dT primer, and the resulting cDNA was used as a template to amplify an anti-HSV antibody gene by PCR. The primers used in PCR were designed based on a database of cDNAs encoding human IgG antibody H chains and L chains. In order to amplify full-length H chain cDNA and L chain cDNA, the 5'-end primer had a translation initiation point, and the 3'-end primer had a translation termination point.
(6) The obtained H-chain and L-chain genes were inserted into expression vectors, and these were simultaneously transfected into CHO-K1 cells using a transfection reagent (Lipofectamine LTX, Invitrogen). After two days, the culture supernatant was collected, and it was confirmed that the anti-HSV antibody (human IgG) in the supernatant bound to HSV-gD using an anti-HSV gD antibody screening plate.
(7) The base sequences of the H and L chains of the obtained anti-HSV antibody were confirmed by an ABI sequencer (ThermoFisher). From the obtained base sequences, the signal sequence of the antibody, the amino acid sequences of the H and L chains, the amino acid sequences of the variable regions, and the amino acid sequences of the complementarity determining regions (CDRs) were determined. The sequence numbers of the amino acid sequences of the H and L chains, the amino acid sequences of the variable regions, and the amino acid sequences of the complementarity determining regions (CDRs) of the first antibody are shown in Table 1 above, and the sequence numbers of the amino acid sequences of the H and L chains, the amino acid sequences of the variable regions, and the amino acid sequences of the complementarity determining regions (CDRs) of the second antibody are shown in Table 2 above. Kabat (www.bioinf.org.uk: Dr.Andrew CR Martin's Group, Antibodies: General Information) was used to analyze the CDRs. The isotype and subclass were also confirmed.
(8) The obtained H-chain and L-chain genes were inserted into an expression vector and introduced into CHO-K1 cells by electroporation. After culturing for 5 to 7 days, the culture supernatant was collected. This culture supernatant was subjected to affinity purification using a Protein A column (Cytiva) to obtain a purified antibody (anti-HSV antibody). After purification, the binding of the antibody to HSV gD was confirmed using an anti-HSV gD antibody screening plate. In addition, the antibody H-chain (approximately 50 kDa) and antibody L-chain (approximately 25 kDa) were confirmed by SDS-PAGE.

The signal sequence of the heavy chain of the first antibody is SEQ ID NO:25, and the signal sequence of the light chain is SEQ ID NO:26.
Furthermore, the signal sequence of the heavy chain of the second antibody is SEQ ID NO:27, and the signal sequence of the light chain is SEQ ID NO:28.
SEQ ID NOs:25 to 28 are as follows.

SEQ ID NO:25:
MKHLWFFLLLVAAPRWVLS
SEQ ID NO:26:
MAWTVLLLGLLSHCTASVT
SEQ ID NO:27:
MDWTWRVFCLLAVAPGAHS
SEQ ID NO:28:
MEAPAQLLFLLLLWLPDTTA

Example 2: Reactivity scores of first and second antibodies
The reactivity scores of the first and second antibodies were evaluated by the immunostaining method (indirect method) described in Example 1 (2). In this case, the first or second antibody was used as the primary antibody, and the scores calculated using analysis software (In Cell Developer Toolbox 1.9.2, GE Healthcare) were evaluated according to the following criteria. The results obtained for the first and second antibodies are shown in Table 3 below.

+++: Reactivity score is 2.0 or more ++: Reactivity score is 1.5 or more and less than 2.0 +: Reactivity score is 1.3 or more and less than 1.5 -: Reactivity score is less than 1.3

The results in Table 3 show that both the first and second antibodies show antigen reactivity to the envelope glycoprotein D (gD) of HSV-1 and HSV-2.

[Example 3: Inactivated antigen reactivity test of the first antibody and the second antibody]
The reactivity of the first and second antibodies with inactivated antigens was confirmed by ELISA (indirect method). At this time, HSV-1, HSV-2, and varicella zoster virus (VZV) were used as inactivated antigens.

The first or second antibody (1 μg/mL) was added to a 96-well microwell plate (Nunc) on which the inactivated antigen was immobilized, and ELISA was performed using an enzyme-labeled anti-human IgG antibody (MBL) as the secondary antibody. A substrate was added, and the color reaction (OD450) caused by the enzyme was quantified. The results are shown in Table 4 below.

The results in Table 4 show that the first and second antibodies show antigenic reactivity to HSV-1 and HSV-2, but not to VZV.

[Example 4: Evaluation of neutralizing activity using the first antibody]
As a first evaluation of the efficacy of the antibody, neutralizing activity was confirmed.
The neutralizing activity was evaluated based on the inhibition rate of HSV infection in Vero cells.
The viruses used were HSV-1 strain F (ATCC, VR-733) and HSV-2 strain G (ATCC, VR-734).

(1) Preparation of virus: A virus solution diluted with DMEM/4% FCS was added to Vero cells, and the cells were cultured (37°C, 5% _CO2 ) until the cytopathic effect (CPE) reached 95-100%. The cells and culture supernatant were centrifuged (3000 rpm, 4°C, 5 min) to collect the virus solution, which was then frozen and stored at -80°C.

(2) Evaluation of neutralizing activity The virus solution and the purified antibody (first antibody) were mixed at 1:1 (v/v), and the virus was set to a final concentration of 5000 FFU/mL (500 FFU/well), and the antibody was set to a final concentration of 10 μg/mL and then diluted 4-fold in six stages.
The virus-antibody mixture was incubated at 37°C under 5% _CO2 for 1 hour, then added to Vero cells that had been seeded on a 96-well plate the previous day to reach 100% confluence, and incubated for another 1 hour. After washing, the cells were cultured in DMEM/2% FCS for 12 hours (35°C, 5% _CO2 ).
The cells were fixed with 4% paraformaldehyde and washed, and then 0.2% Triton X-100 was added to perform membrane permeabilization treatment.
Mouse anti-HSV-1/2-gD antibody (mixture of mouse anti-HSV-1-gD antibody and mouse anti-HSV-2-gD antibody, 1 μg/mL, ab6507, Abcam) was added to the fixed cells, reacted with HSV antigen on the infected Vero cells at room temperature (25° C.) for 1 hour, and then washed. After DAPI staining was performed using a fluorescently labeled anti-mouse IgG antibody (Anti-mouse IgG-Alexa488, A11001, Invitrogen) as a secondary antibody, images were taken with an imaging cytometer (In Cell Analyzer 2000, GE Healthcare) and the number of infected cells was counted. The lower the number of infected cells, the more the virus in the virus-antibody mixture added to the Vero cells has been neutralized, i.e., the higher the neutralizing activity of the added antibody.

A human monoclonal antibody (human IgG) having no specificity for HSV was used as a negative control in the evaluation of neutralizing activity, and the antibody mAb E317-103 against HSV-gD described in WO 2010/07851 was used as a positive control.
The average number of infected cells in all concentration ranges of the negative control was taken as the infection rate of 100%, and IC50 and IC90 were calculated from the infection rate at each concentration of the sample antibody. GraphPad Prism 6 was used for the calculation. The obtained results are shown in Table 5 below.

The results in Table 5 show that the first antibody has a neutralizing activity against HSV-1 strain F and HSV2 strain G that is greater than that of the positive control antibody mAb E317-103.

[Example 5: Evaluation of cell-to-cell infection blocking activity using the first antibody]
To evaluate the effectiveness of the first antibody, its activity of inhibiting cell-to-cell infection was evaluated by the following method.

The virus solution (150 PFU/well) was added to Vero cells that had been seeded on a 6-well plate the previous day to reach 100% confluence, and the cells were incubated at 37°C under 5% _CO2 for 1 hour. After washing, DMEM/2% FCS was added and the cells were incubated for another 1 hour.
The first antibody was diluted with DMEM/2% FCS containing 1% agarose (Seakem GTG Agarose, Lonza) and added at 2 mL/well. The antibody was diluted 4-fold in six steps from a final concentration of 40 μg/mL.
The cells were cultured (37°C, 5% _CO2 ) for 55 hours until plaques expanded. The cells were fixed with 4% paraformaldehyde, washed, and then stained with 0.1% Crystal violet (Fujifilm Wako Pure Chemicals). Stained images were photographed, and the area of the plaques was calculated using ImageJ. Note that the smaller the area of the plaques, the more the added antibody prevented the spread of infection from HSV-infected Vero cells to non-infected Vero cells, i.e., the higher the intercellular infection inhibitory activity of the added antibody.

As negative and positive controls in the cell-to-cell infection inhibitory activity evaluation, the same antibodies as those used in the neutralizing activity evaluation were used.
The average plaque area per well for all concentrations of the negative control was taken as 100% infection spread rate, and IC50 was calculated from the infection spread rate at each antibody concentration. Specifically, IC50 was calculated from the antibody concentrations and infection spread rates at two points that sandwiched an infection spread rate of 50%. The results are shown in Table 6 below.

The results in Table 6 show that the first antibody exhibits cell-to-cell infection inhibitory activity against HSV-1 strain F and HSV-2 strain G equivalent to that of the positive control antibody mAb E317-103.

[Example 6: Preparation of an immunoassay device using a first antibody and a second antibody and evaluation of the reactivity]
An immunoassay device using the first and second antibodies was prepared according to the following procedure: In Example 6, colored cellulose particles were used to label the labeled anti-HSV antibodies.

(1) Preparation of a solution of labeled anti-HSV antibody The first antibody was labeled with colored cellulose particles to prepare a solution of labeled anti-HSV antibody.
Specifically, 0.9 mL of 10 mM borate buffer (pH 9) was added to 0.1 mL of a 1% suspension of colored cellulose nanoparticles (Asahi Kasei Corporation, dark red), and then 0.1 mg of the first antibody solution was mixed and allowed to stand at 37 ° C. for 120 minutes. Then, 12 mL of borate buffer (pH 8.5) containing 1% casein was added and thoroughly stirred, and then allowed to stand at 37 ° C. for 60 minutes. Then, centrifugation was performed at 13,000 × g for 15 minutes, and the precipitate after removing the supernatant was redispersed in 12 mL of 50 mM borate buffer (pH 10). Centrifugation was performed at 13,000 × g for 15 minutes, and the precipitate after removing the supernatant was redispersed in 66 mM borate buffer (pH 9.2) containing 0.2% casein and 15% sucrose, and the final liquid volume was adjusted to 8 mL to obtain a solution of labeled anti-HSV antibody.

(2) Preparation of Labeled Substance Retaining Part A labeled substance retaining part was prepared using the solution of labeled anti-HSV antibody prepared in (1) above.
More specifically, 1.5 mL of the labeled anti-HSV antibody solution prepared in (1) above was added uniformly to a 10 mm x 300 mm glass fiber nonwoven fabric, and then dried at 37°C for 30 minutes to prepare a labeled substance retention portion.

(3) Preparation of Detection Section A detection section was prepared using the second antibody.
More specifically, a detection portion was prepared by applying a 1 mm-wide line of a second antibody at a concentration of 1.0 mg/mL at a position 10 mm parallel to one end of the long axis of a 25 mm x 300 mm nitrocellulose membrane (with a backing sheet) and a 0.5 mg/mL line of a goat anti-human IgG polyclonal antibody at a position 15 mm away, and then drying the line overnight at room temperature (25°C).
The area where the second antibody was applied was the test line, and the area where the goat anti-human IgG polyclonal antibody was applied was the control line.

(4) Preparation of Immunoassay Device An immunoassay device including a sample dropping section, a labeling substance holding section, a detection section, and an absorption section was prepared.
More specifically, the labeling substance holding part and the absorbing part (filter paper) prepared in (2) above were attached to the detection part prepared on the backing sheet in (3) above in this order from the upstream end where the sample flows. Next, the sample dropping part (cellulose nonwoven fabric) was attached to the labeling substance holding part. At this time, the detection part was attached in such a way that the test line (the part where the second antibody was applied) was on the upstream side (the side where the sample was dropped).
The plate was cut with a cutter to a width of 5 mm in a direction intersecting with each component, and assembled into a plastic housing case to prepare an immunoassay device.

The reactivity of the immunoassay device prepared above was evaluated.

A sample containing inactivated HSV-1 and a sample containing inactivated HSV-2 were prepared.
More specifically, an inactivated HSV-1 suspension or an inactivated HSV-2 suspension (Meridian) was diluted with TBS buffer containing 1% polyoxyethylene (10) octylphenyl ether to prepare samples containing inactivated HSV-1 and inactivated HSV-2. The concentrations of the samples were 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, or 500 ng/mL.

120 μL of a sample containing inactivated HSV-1 or a sample containing inactivated HSV-2 was dropped onto the sample drop section of the immunoassay device prepared above and allowed to develop. The color intensity of the test line in the detection section was measured 5, 10, and 15 minutes after dropping the sample using an immunochromato reader (Hamamatsu Photonics C10066-10). The results are shown in Figure 2.

For comparison with the immunological measurement device prepared above, evaluation was performed using a commercially available herpes simplex virus kit, Prime Check (registered trademark) HSV (manufactured by Alfresa Pharma Corporation).
In this case, the specimen extract included in the kit was used as a diluent to prepare the samples. Specifically, an inactivated HSV-1 suspension or an inactivated HSV-2 suspension (manufactured by Meridian) was diluted with the specimen extract included in the kit to prepare a sample containing inactivated HSV-1 and a sample containing inactivated HSV-2. The concentrations of the samples were 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL, or 2000 ng/mL.
The test was carried out for a sample containing inactivated HSV-1 or a sample containing inactivated HSV-2 according to the attached instructions, and the color intensity of the test line at the detection area was measured by an immunochromato reader 5 minutes, 10 minutes, and 15 minutes after the sample was dropped. The results are shown in Figure 3.

Referring to Figures 2 and 3, it can be seen that when a sample containing inactivated HSV-1 is used, the immunological measurement device of this embodiment has a sensitivity 10 times or more higher than that of a commercially available herpes simplex virus kit.

Furthermore, referring to Figures 2 and 3, when a sample containing inactivated HSV-2 is used, the immunological measurement device according to this embodiment has more than twice the sensitivity of a commercially available herpes simplex virus kit.

[Example 7: Preparation of half strips using first and second antibodies and evaluation of reactivity]
A half strip (an immunoassay device consisting of a detection section and an absorption section, but not having a sample dropping section or a labeling substance holding section) was prepared using the first and second antibodies according to the following procedure. In Example 7, gold colloid particles were used to label the labeled anti-HSV antibody.

(1-1) Preparation of Detection Part A sheet made of nitrocellulose (manufactured by Advantec, IAB135, 300 mm x 25 mm) was cut to a width of 5 mm to prepare a sheet of 5 mm x 25 mm as a membrane.
Furthermore, a phosphate buffer (containing 0.2% by mass of bovine serum albumin (BSA) and 2% by mass of sucrose) was added to the first antibody or the second antibody to prepare an antibody solution with a concentration of 0.5 to 1.2 mg/mL.

The antibody solution prepared above was applied in a line 1 mm wide at a position 10 mm parallel to one end of the sheet, and then dried overnight at room temperature (25°C) to form a test line on the sheet.

(1-2) Preparation of Half Strip A detection part and an absorption part (filter paper) were attached on a backing sheet in this order from the upstream end through which the sample flows, to prepare a half strip with a three-layer structure.

The reactivity of the half strips prepared above was evaluated using the following procedure.

(2-1) Preparation of a solution of labeled anti-HSV antibody The first antibody or the second antibody was labeled with gold colloid particles to prepare a solution of labeled anti-HSV antibody.
Specifically, 100 mM borate buffer (pH 9.0) was added to the first antibody or the second antibody to prepare an antibody solution with a concentration of 0.05 to 0.12 mg/mL. Next, 0.1 mL of the antibody solution was added to 0.9 mL of a gold colloid particle solution (EMBC50 manufactured by BBI) with a diameter of 50 nm, and the mixture was mixed by inversion at room temperature (25°C) for 30 minutes. Next, 0.05 mL of a 2% aqueous sodium caseinate solution was added, and the mixture was mixed by inversion at room temperature for 5 minutes. After that, the mixture was centrifuged at 8000 x g for 5 minutes, and the precipitate after removing the supernatant was redispersed in Tris buffer (pH 8.5) and the final liquid volume was adjusted to 0.5 mL to obtain a solution of labeled anti-HSV antibody.

(2-2) Preparation of Samples 5 μL of each sample of inactivated HSV-1, inactivated HSV-2, and phosphate buffer solution (PBS) was added to a 96-well plate, and then 20 μL of the labeled anti-HSV antibody solution prepared in (2-1) above and 20 μL of 100 mM Tris buffer (pH 8.5) containing 2% by mass of nonionic surfactant (Triton X-100, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to prepare samples.

(2-3) Evaluation of reactivity The half strip prepared for the above reactivity evaluation was inserted into the sample prepared in (2-2) above, and after 15 minutes, the presence or absence of coloring of the line at the detection area (test line) was visually confirmed and evaluated according to the following criteria.

A: Strong coloring (positive)
B: Colored (positive)
C: Weak coloring (positive)
D: Very weak coloring is observed, but it is difficult to judge (negative)
E: No coloring (negative)

Here, the combinations of the anti-HSV antibodies contained in the test line of the half strip and the labeled anti-HSV antibodies added to the sample are as shown in Table 5 below, and are referred to as Examples 7-1 and 7-2, respectively. The combinations and evaluation results of Examples 7-1 and 7-2 are shown in Table 7 below.

The results in Table 7 show that high reactivity is observed even when gold colloid particles are used to label the labeled anti-HSV antibody.

[Example 8: Preparation of an immunoassay device using a first antibody and a second antibody and evaluation of the reactivity]
An immunoassay device using the first and second antibodies was prepared according to the following procedure: In Example 8, gold colloid particles were used to label the labeled anti-HSV antibody.

(1) Preparation of a solution of labeled anti-HSV antibody The first antibody or the second antibody was labeled with colloidal gold particles (average particle size: 40 nm, optical density (OD): 1.0) prepared by the citrate reduction method to prepare a solution of labeled anti-HSV antibody.
Specifically, 10 mL of a gold colloid particle solution prepared by the citrate reduction method was adjusted to an optimal pH, and a solution of the first or second antibody in an amount equivalent to the minimum coating amount was mixed and stirred for 10 minutes at room temperature (25° C.). At this time, the optimal pH and minimum coating amount of the first or second antibody were previously determined by a known method (such as the method described in Immunoassay Methods: From Basics to Advanced Technologies, edited by Biochemical Measurement Research Group, published by Kodansha in 2014).
Next, 1 mL of 1% sodium caseinate aqueous solution was added to the mixed solution and stirred for 15 minutes at room temperature (25° C.). The mixture was centrifuged at 8000×g for 10 minutes, and the supernatant was removed. The precipitate was redispersed in 100 mM Tris buffer containing 1% bovine serum albumin (BSA) and 2% sucrose, and the final volume was adjusted to 2.5 mL to obtain a solution of labeled anti-HSV antibody.

(2) Preparation of Labeled Substance Retaining Part A labeled substance retaining part was prepared using the solution of labeled anti-HSV antibody prepared in (1) above.
More specifically, 1.5 mL of the labeled anti-HSV antibody solution prepared in (1) above was added uniformly to a 10 mm x 300 mm glass fiber nonwoven fabric, and then dried at 37°C for 30 minutes to prepare a labeled substance retention portion.

(3) Preparation of detection area A 1.0 mg/mL concentration of the first antibody or the second antibody was applied in a line shape with a width of 1 mm to a position 10 mm away from one end of the long axis side of a 25 mm × 300 mm nitrocellulose membrane (with a backing sheet), and dried overnight at room temperature (25°C) to prepare a detection area.

(4) Preparation of Immunoassay Device An immunoassay device including a sample dropping section, a labeling substance holding section, a detection section, and an absorption section was prepared.

More specifically, the labeled substance holding part and the absorption part (filter paper) prepared in (2) above were attached in that order from the upstream end where the sample flows onto the detection part prepared on the backing sheet in (3) above. Next, the sample drip part (cellulose nonwoven fabric) was attached onto the labeled substance holding part. At this time, the detection part was attached in the direction that the test line was on the upstream side (the side where the sample drip part was attached).

The immunoassay device was created by cutting each component to a width of 5 mm in the direction crossing each component using a cutting machine.

The combinations of the antibodies contained in the labeling substance holding section and the antibodies contained in the detection section are as shown in Table 8 below, and are referred to as Example 8-1 and Example 8-2, respectively.

The reactivity of the immunoassay device prepared above was evaluated.

A sample containing inactivated HSV-1 and a sample containing inactivated HSV-2 were prepared.
More specifically, a sample containing inactivated HSV-1 and a sample containing inactivated HSV-2 were prepared by diluting an inactivated HSV-1 suspension or an inactivated HSV-2 suspension (manufactured by Meridian) with a TBS buffer containing 1% polyoxyethylene (10) octylphenyl ether. The concentrations of the samples were 0 ng/mL, 200 ng/mL, or 500 ng/mL.

120 μL of a sample containing inactivated HSV-1 or a sample containing inactivated HSV-2 was dropped onto the sample dropping section of the immunological measurement device prepared above and allowed to spread. Five and ten minutes after dropping the sample, the presence or absence of coloring of the test line in the detection section was visually confirmed and evaluated according to the following criteria. The results obtained are shown in Table 9 below.

A: Strong coloring (positive)
B: Colored (positive)
C: Weak coloring (positive)
D: Very weak coloring is observed, but it is difficult to judge (negative)
E: No coloring (negative)

The results in Table 9 show that high reactivity is observed even when gold colloid is used to label the labeled anti-HSV antibody.

REFERENCE SIGNS LIST 1 Immunological measurement device 2 Sample dropping section 3 Labeled substance holding section 3a Labeled anti-HSV antibody 4 Detection section 5 Test line 5a Anti-HSV antibody 6 Control line 6a Capture antibody 7 Absorption section 8 Backing sheet

Claims (13)

An anti-herpes simplex virus (HSV) monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to an envelope glycoprotein of HSV,
(i) the heavy chain variable region is
(a) an amino acid sequence of a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2;
(b) an amino acid sequence of a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:3; and (c) an amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4;
Contains
(ii) the light chain variable region is
(a) an amino acid sequence of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:6;
(b) an amino acid sequence of a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:7; and (c) an amino acid sequence of a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:8;
or (i) the heavy chain variable region comprises:
(d) the amino acid sequence of a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12;
(e) an amino acid sequence of a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) an amino acid sequence of a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14;
Contains
(ii) the light chain variable region is
(d) the amino acid sequence of a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 16;
(e) an amino acid sequence of a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and (f) an amino acid sequence of a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 18;
An antibody or an antigen-binding fragment thereof comprising: A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1; and
A light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:5; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:11, and
The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:9, and a light chain comprising the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:10; or comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:19 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:19, and a light chain comprising the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:20. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to envelope glycoprotein D (gD). The antibody or antigen-binding fragment thereof according to claim 1, wherein the herpes simplex virus (HSV) is HSV-1 and/or HSV-2. An isolated nucleic acid molecule encoding the amino acid sequence of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or an isolated nucleic acid molecule that hybridizes under highly stringent conditions with any of these nucleic acid molecules. An isolated nucleic acid molecule having a base sequence of SEQ ID NO:21 to 24, or a base sequence having 90% or more identity to a base sequence of SEQ ID NO:21 to 24. A recombinant expression vector incorporating the isolated nucleic acid molecule of claim 7. An isolated host cell into which the recombinant expression vector according to claim 8 has been introduced. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5. An immunoassay device for detecting herpes simplex virus (HSV), comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. An immunoassay device for detecting herpes simplex virus (HSV), comprising a sample dropping section, a labeling substance holding section, a detection section, and an absorption section,
An immunoassay device, wherein at least one of the labeling substance holding section and the detection section comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. The immunoassay device according to claim 12, wherein both the labeling substance holding section and the detection section contain the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.