JPWO2020130138A1 - Anti-preS1 antibody and its uses - Google Patents

Abstract

The present disclosure discloses an antibody against or a combination thereof against a preS1 antigen, a method for measuring a preS1 antigen using the antibody or a combination thereof, a composition and a kit for the measurement thereof, a method for evaluating HBV infection and a composition and a kit for the same, and the antibody or a combination thereof. Provided is a pharmaceutical composition containing a combination.

Description

This application claims priority with respect to Japanese Patent Application No. 2018-239719, which is incorporated herein by reference in its entirety.
The present disclosure relates to antibodies against the preS1 antigen of hepatitis B virus (HBV) and their uses.

HBV is a widespread worldwide virus that infects approximately 300 million people worldwide and persistently infects more than 200 million people. More than 1 million people die each year from cirrhosis and liver cancer derived from this virus, causing a serious infectious disease. Most of the viruses become persistent infections due to infection in early childhood, but there are cases in which the virus becomes chronic even when infected in adults. Some cases of persistent infection progress from chronic hepatitis to cirrhosis, and some develop hepatocellular carcinoma. In this way, persistent infection of hepatitis B virus is a serious health problem, and its treatment method, infection prevention method, pathological condition evaluation method, etc. are urgent issues.

HBV is a virus that has a partially double-stranded circular DNA of 3,200 bases as its genome. HBV particles consist of core particles with the HBV gene inside and an outer shell (envelope) that encloses them. Currently, HBs antigens present in the outer shell, HBc-related antigens present in core particles, HBe antigens released into the blood, antibodies against these antigens, and the amount of HBV DNA are used as markers for the diagnosis of HBV infection. , These are combined to determine the presence or absence of infection, the pathological condition, or the therapeutic effect. When HBV proliferates in hepatocytes, HBsAg is overproduced and released into the blood as small spherical or rod-shaped particles, resulting in complete HBV particles and incomplete HBV particles in the blood of HBV-infected patients. There are three types of virus particles, rod-shaped particles and small spherical particles. Antibodies to the HBsAg currently in use recognize all three types of virus particles. Since the blood of HBV-infected patients contains small spherical particles 10,000 to 100,000 times larger than complete HBV particles, most of the detected HBs antigens are derived from the small spherical particles.

The HBs antigen has three domains, S, preS1 and preS2, and lacks the L protein and preS1 domain composed of these three domains, and the M protein and preS1 domain composed of the S domain and preS2 domain. There is an S protein that lacks the preS2 domain and is composed only of the S domain. The preS1 domain binds to receptors on hepatocytes and plays an important role in HBV infection. HBsAg containing the preS1 domain (also called preS1 antigen) is present only in infectious complete HBV particles and rod-shaped particles, not in small spherical particles.

An object of the present disclosure is to provide an antibody against the preS1 antigen and its uses.

The present disclosure provides, in certain embodiments, an anti-preS1 antibody that binds to an epitope contained in an amino acid sequence selected from SEQ ID NOs: 2, 3 and 4.

The present disclosure, in a further embodiment, provides an anti-preS1 antibody that is selected from the following (a)-(i):
(A) CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 25 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 26 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(B) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 37 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 37, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(C) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(D) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 43 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(E) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 47 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(F) CDR1, which contains the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 52 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 55 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 56 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(G) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(H) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 63 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising; and (i) CDR1 comprising an amino acid sequence of SEQ ID NO: 65 or an amino acid sequence in which 1-3 amino acid residues have been deleted, substituted, or added.
CDR2 containing the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 67 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 69 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 70, and 1-3 in the amino acid sequence of SEQ ID NO: 71 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising.

The present disclosure provides, in a further embodiment, a combination of two or more anti-preS1 antibodies independently selected from said antibody.

The present disclosure, in a further embodiment, is a method of measuring the preS1 antigen, comprising contacting a sample obtained from a subject with the antibody and detecting a complex of the preS1 antigen and the antibody, as well as said. A composition and a kit for measuring a preS1 antigen containing an antibody or combination are provided.

The present disclosure, in a further aspect, comprises a method of assessing HBV infection, comprising measuring the preS1 antigen by the measurement method and comparing the measurement with a reference value, as well as the antibody or combination. A composition and a kit for evaluating HBV infection are provided.

The present disclosure, in a further embodiment, provides a pharmaceutical composition comprising said antibody or combination.

According to the present disclosure, an antibody against or a combination thereof against a preS1 antigen, a method for measuring a preS1 antigen using the antibody or a combination thereof, a composition and a kit for the measurement thereof, a method for evaluating HBV infection and a composition and a kit for the same, and the antibody or a combination thereof. A pharmaceutical composition comprising a combination is provided.

FIG. 1 shows the partial sequence of the preS1 domain of HBV of each genotype and the binding site of the antibody of the example. FIG. 2A shows the sequences of the light chain variable regions of antibodies 7, 10, 13, 39, 49, 55, 63, 41, and 1A. FIG. 2B shows the sequences of the heavy chain variable regions of antibodies 7, 10, 13, 39, 49, 55, 63, 41, and 1A. FIG. 3 shows the HBV infection blocking effect of antibodies 7, 10, 13, 15, 25, 27, 39, 41, 49, 51, 55 and 63. Culture supernatants of antibody-producing hybridomas were used. FIG. 4 shows the HBV infection inhibitory effect of antibodies 7, 10, 25, 41 and 55. Antibodies purified from the supernatant after the expanded culture of hybridomas were used. FIG. 5 shows the HBV infection inhibitory effect of antibodies 1A, 1B, 1C, 5A, 5B and 5C. Antibodies purified from the supernatant after the expanded culture of hybridomas were used. FIG. 6 shows the HBV infection inhibitory effect of the combination of antibody 41 and antibody 7 or 10. Hybridoma culture supernatants (0, 0.03, or 0.1 μg / ml) producing antibody 41 and hybridoma culture supernatants (0.003 or 0.01 μg / ml) producing antibody 7 or 10 were used. FIG. 7 shows the HBV infection inhibitory effect of the combination of antibody 41 and antibody 10 or 55. Antibody 41 (0, 0.03, or 0.1 μg / ml) and antibody 10 or 55 (0.01 or 0.03 μg / ml) purified from the supernatant after expanded culture of hybridomas were used. FIG. 8 shows the HBV infection inhibitory effect of the combination of antibody 49 and antibody 55. Antibody 49 (0, 0.01, or 0.03 μg / ml) and antibody 55 (0.01 or 0.03 μg / ml) purified from the supernatant after expanded culture of hybridomas were used. FIG. 9 shows the HBV infection inhibitory effect of the combination of antibody 824 having an epitope in the S domain and antibody 10 or 55. Antibody 824 (0 or 0.03 μg / ml) and antibody 10 or 55 (0.01 or 0.03 μg / ml) purified from the supernatant after expanded culture of hybridomas were used. FIG. 10 shows the preS1 antigen and background absorbance of the antibody used for solid phase formation. As the solid phase antibody, antibody 41 or antibody 5124A having an epitope in the S domain was used. The results are shown for each group in which the subject serum was divided by the measured values of HBe antigen and HBV-DNA. FIG. 11 shows a calibration curve of preS1 antigen by positive control serum. FIG. 12 shows a comparison of the measured values of the preS1 antigen of the immobilization antibody (antibody 5124A or 824). Subject No. and serum dilution rate are shown in the figure. FIG. 13 shows a comparison of the measured values of the preS1 antigen of the immobilization antibody (antibody 5124A or 824) in the serum of the subject different from that of FIG. Subject No. and serum dilution rate are shown in the figure. FIG. 14 shows the measured values of serum preS1 antigen when antibodies 10, 13, 39 or 55 were used as the detection antibody. Subject No. and serum dilution rate are shown in the figure. FIG. 15 shows the HBV infection blocking effect of antibodies 41, 1A, and 10. Antibodies (0.01, 0.1, or 1 μg / ml) purified from the supernatant after expanded culture of hybridomas were used. The measurement result of HBs antigen is shown. FIG. 16 shows the HBV infection blocking effect of antibodies 41, 1A, and 10. Antibodies (0.01, 0.1, or 1 μg / ml) purified from the supernatant after expanded culture of hybridomas were used. The measurement result of HBe antigen is shown. FIG. 17 shows the HBV infection inhibitory effect of the combination of antibody 824 and antibody 41, 1A, or 10. Antibody 824 (0.01 μg / ml) purified from the supernatant after the expanded culture of the hybridoma and antibody 41, 1A, or 10 (0.01 or 0.1 μg / ml) purified from the supernatant after the expanded culture of the hybridoma were used. The measurement result of HBs antigen is shown. FIG. 18 shows the HBV infection inhibitory effect of the combination of antibody 824 and antibody 41, 1A, or 10. Antibody 824 (0.01 μg / ml) purified from the supernatant after the expanded culture of the hybridoma and antibody 41, 1A, or 10 (0.01 or 0.1 μg / ml) purified from the supernatant after the expanded culture of the hybridoma were used. The measurement result of HBe antigen is shown. FIG. 19 shows the HBV infection inhibitory effect of a combination of two of antibodies 41, 1A, and 10. Antibodies (0.01 or 0.1 μg / ml) purified from the supernatant after the expanded culture of hybridomas were used. The measurement result of HBs antigen is shown. FIG. 20 shows the HBV infection inhibitory effect of a combination of two of antibodies 41, 1A, and 10. Antibodies (0.01 or 0.1 μg / ml) purified from the supernatant after the expanded culture of hybridomas were used. The measurement result of HBe antigen is shown.

In the present specification, amino acid residues are represented by the following abbreviations.
Ala or A: Alanine
Arg or R: Arginine
Asn or N: Asparagine
Asp or D: aspartic acid
Cys or C: Cysteine
Gln or Q: Glutamine
Glu or E: Glutamic acid
Gly or G: Glycine
His or H: Histidine
Ile or I: isoleucine
Leu or L: Leucine
Lys or K: Lysine
Met or M: Methionine
Phe or F: Phenylalanine
Pro or P: Proline
Ser or S: Serine
Thr or T: Threonine
Trp or W: Tryptophan
Tyr or Y: Tyrosine
Val or V: Valine

Hepatitis B virus (HBV) is divided into at least nine genotypes, A, B, C, D, E, F, G, H, and J. HBV in the present disclosure includes HBV of any genotype.

As used herein, the preS1 antigen means an HBs antigen (Hepatitis B surface antigen, HBsAg) containing a preS1 domain. The HBsAg in the present disclosure may be a protein of any genotype. The representative amino acid sequence of HBsAg (Genotype C) (accession No. MH887433) is shown in SEQ ID NO: 1, but the HBsAg of the present disclosure is not limited to those containing the amino acid sequence of SEQ ID NO: 1.

MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFGSGFTPPHGGLLGWSPQAQGILTTVPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPNMENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYNILSPFLPLLPIFFCLWVYI (SEQ ID NO: 1)

The preS1 domain, preS2 domain, and S domain in SEQ ID NO: 1 are the 1st to 119th, 120th to 174th, and 175th to 400th regions, respectively. One of ordinary skill in the art can easily obtain the amino acid sequence of HBsAg of each genotype from a database such as GenBank.

As used herein, anti-preS1 antibody means an antibody that binds to an epitope in the preS1 domain of HBsAg. As used herein, the anti-S antibody means an antibody that binds to an epitope in the S domain of HBsAg, and the anti-preS2 antibody means an antibody that binds to an epitope in the preS2 domain of HBsAg. The term anti-HBs antibody means an antibody against the HBs antigen and is used to include an antibody that binds to an epitope in any of the S domain, preS1 domain and preS2 domain.

The term "antibody" as used herein is used to include various antibody structures such as polyclonal antibodies, monoclonal antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments and the like. Monoclonal antibodies include chimeric antibodies, humanized antibodies, and human antibodies. An antibody fragment means a molecule that comprises a portion of an antibody as a component, eg, but not limited to, the heavy and light chain variable regions ( _VH and _VL ), F (ab') ₂ , Fab', of the antibody. Examples include Fab, Fv, disulphide-linked FV (sdFv), Single-Chain FV (scFV) and polymers thereof. The type of antibody is not particularly limited, and examples thereof include antibodies derived from mice, rats, rabbits, goats, and humans.

The immunoglobulin class of an antibody is determined based on the heavy chain constant region. Immunoglobulin classes include IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chains are called α-chain, δ-chain, ε-chain, γ-chain, and μ-chain, respectively. The immunoglobulin class can be further subdivided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The immunoglobulin class of the antibody herein is not particularly limited. In certain embodiments, the antibody is IgG. In a further embodiment, the antibody is IgG1 or IgG4. The light chain of an antibody can be divided into a κ chain and a λ chain based on its constant region, but the antibody herein may have either a κ chain or a λ chain.

The variable regions of an antibody are usually composed of three complementarity determining regions (also referred to as CDRs) sandwiched between four framework regions (also referred to as FR). In the present specification, the variable region and CDR of an antibody are referred to as abYsis (Swindells, MB, Porter, CT, Couch, M., Hurst, J., Abhinandan, KR) of a web-based antibody search system, unless otherwise specified. , Nielsen, JH, Macindoe, G., Hetherington, J. and Martin, AC, 2017. abYsis: integrated antibody sequence and structure-management, analysis, and prediction. Journal of molecular biology, 429 (3), pp.356- Specified by Chothia Numbering Scheme in 364.).

The binding and binding affinity of the antibody to the antigen can be determined by enzyme immunoassay (EIA) (including ELISA), radioimmunoassay (RIA), chemoluminescence immunoassay (CIA), fluorescent immunoassay (FIA), etc. It can be confirmed by the immunoassay method of BIACORE (registered trademark) surface plasmon resonance assay. In certain embodiments, the anti-preS1 antibodies, 10 ^-8 M or less, for example ^{10 -8 M~10 -13 M, 10 -8} M~10 -12 M, 10 -9 M~10 -12 M or 10, ^{- It has} a dissociation constant (Kd) of 9 M to 10 ^{-11 M.} In certain embodiments, binding affinity is measured by the BIACORE® surface plasmon resonance assay.

The antibodies of the present disclosure use or use as an immunogen a peptide containing the amino acid sequence of PLGFFPDHQLDPAFG (SEQ ID NO: 2), NSNNPDWDFNPN (SEQ ID NO: 3), or DPAFGANSNN (SEQ ID NO: 4), or a peptide consisting of these amino acid sequences. Obtaining by a general method using a peptide containing the amino acid sequence of NNPDWDFNP (SEQ ID NO: 17), NNPDWDFN (SEQ ID NO: 21), or GFFPDHQLD (SEQ ID NO: 80), or a peptide consisting of these amino acid sequences as an immunogen. Can be done. Immunogens can be made by conventional peptide synthesis methods, for example by genetic engineering techniques or chemical synthesis. Alternatively, the antibody of the present disclosure can also be obtained by preparing an expression vector containing an antibody gene using a genetic engineering technique and expressing it in cells.

Polyclonal antibodies can be produced by the general method described in "Antibodies: A Laboratory Manual, Lane, HD et al. Eds., Cold Spring Harbor Laboratory Press, New York, 1989" and the like. Specifically, it can be produced by immunizing mammals such as rats, mice, rabbits, goats, and horses with the above-mentioned immunogen.

Monoclonal antibodies can be obtained by known methods such as a method for producing a hybridoma that produces an antibody, a method for producing an expression vector containing an antibody gene using a genetic engineering technique, and a method for expressing the antibody in cells.

Hybridomas that secrete monoclonal antibodies can be made according to the method described in Kohler et al., Nature 256: 495, 1975. First, the immunogen is mixed with a suitable substance for enhancing antigenicity (eg, keyhole limpet hemocyanin, bovine serum albumin, etc.) and, if necessary, an immunostimulant (such as Freund's complete or incomplete adjuvant). It immunizes non-human mammals such as rats, mice, rabbits, goats and horses. Usually, immunized animals are immunized multiple times at intervals of 3 to 10 days, and 1 to 100 μg of the immunogen peptide is administered. Then, immunocompetent cells (cells capable of producing antibodies in immune animals) are recovered from immune animals that have undergone multiple immunizations, and myeloma cells that are not capable of producing autoantibodies (eg, mice, rats, guinea pigs, hamsters, rabbits, or rabbits). Cell fusion with cells derived from mammals such as humans). For cell fusion, a polyethylene glycol method, an electric fusion method, or the like is used. Furthermore, cells that have succeeded in cell fusion are selected based on the selection marker possessed by the fused cells, and the reactivity of the antibody produced by the selected cells with respect to the immune source is confirmed by the above-mentioned immunoassay method or the like. A hybridoma that produces a monoclonal antibody is obtained. The monoclonal antibody can be isolated from the culture supernatant obtained by culturing the obtained hybridoma in vitro. It can also be cultured in vivo in ascites such as mice, rats, guinea pigs, hamsters or rabbits and isolated from ascites.

Monoclonal antibodies can also be obtained by preparing an expression vector containing an antibody gene and expressing it in a host cell (PJ Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY; P. Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; JW Goding., Monoclonal Antibodies: principals and practice., 1993 ACADEMIC PRESS). Furthermore, using a transgenic animal production technique, a transgenic animal (for example, bovine, goat, sheep or pig) in which the gene of the antibody of interest is integrated into an endogenous gene is produced, and the transgenic animal is contained in milk. It is also possible to obtain a monoclonal antibody derived from the antibody gene from.

The obtained monoclonal antibody is purified by appropriately combining methods well known in the art, for example, chromatography using a protein A column, ion exchange chromatography, hydrophobic chromatography, salting out method, gel filtration, affinity chromatography and the like. can do.

A chimeric antibody is an antibody containing different sequences derived from each other, and is, for example, an antibody in which different variable regions derived from each other and a constant region are linked. In certain embodiments, the chimeric antibody is composed of a variable region of an antibody derived from a non-human mammal and a constant region derived from a human antibody. For a chimeric antibody, for example, a polynucleotide encoding a variable region of an antibody derived from a non-human animal and a polynucleotide encoding a constant region of a human antibody are ligated, incorporated into an expression vector, and the expression vector is used as a host. It can be obtained by introducing and expressing it.

The CDR is a region that substantially determines the binding specificity of an antibody, and its amino acid sequence is rich in variety. On the other hand, the amino acid sequences constituting FR show high homology even among antibodies having different binding specificities. Therefore, the binding specificity of one antibody can be transplanted to another antibody by transplanting CDR.

Humanized antibodies are generally composed of CDRs of antibodies derived from non-human animals, FR derived from human antibodies and constant regions derived from human antibodies. Humanized antibodies can be obtained by transplanting CDRs of antibodies derived from non-human animals into human antibodies. Humanized antibodies can be produced by a variety of methods, one example being Overlap Extension PCR (Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). In this method, PCR is performed using an oligonucleotide having a portion that overlaps the end of the CDR of an antibody derived from a non-human animal (for example, a mouse antibody) and the FR of a human antibody as a primer, and is derived from a non-human animal. A polynucleotide in which the CDR of an antibody and the FR of a human antibody are linked is synthesized. Next, the obtained polynucleotide can be ligated with a polynucleotide encoding a constant region of a human antibody, incorporated into an expression vector, and the expression vector can be introduced into a host for expression to obtain a humanized antibody. can.

Methods for selecting FRs suitable for producing humanized antibodies are known, for example, FRs selected by the best fit method (Sims et al. J. Immunol. 151: 2296 (1993)) and light chains of human antibodies. Or FR (Carter et al. Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Presta et al. J. Immunol. 151: 2623 ( 1993)) can be used.

Human antibodies can be obtained, for example, by sensitizing human lymphocytes in vitro with the desired antigen and then fusing the sensitized lymphocytes with human myeloma cells (Japanese Patent Publication No. 1-59878). For human myeloma cells, which are fusion partners, for example, U266 can be used. Human antibodies can also be obtained by immunizing transgenic animals with the entire repertoire of human antibody genes with the desired antigen (Lonberg, Nat. Biotech. 23: 1117-1125, 2005). Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known (Antibody Phage Display: Methods and Protocols, Methods in Molecular Biology 178, 2001). For example, the variable region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage by the phage display method, a phage that binds to the antigen is selected, and the gene of the selected phage is analyzed to obtain an antigen. The DNA sequence encoding the variable region of the bound human antibody can be determined. The human antibody can then be obtained by in-frame ligation of this variable region sequence with the sequence of the human antibody constant region, inserting it into an appropriate expression vector, and introducing this expression vector into the host for expression. can.

Multispecific antibodies are antibodies that bind to at least two different sites. Examples of the multispecific antibody include bispecific antibody and trispecific antibody. In certain embodiments, the multispecific antibody binds the preS1 antigen to one or more other antigens. Multispecific antibodies can be produced, for example, by genetic engineering techniques or by binding two or more antibodies with different recognition antigens.

The antibody fragment can be obtained by digesting the antibody with a protease such as papain or pepsin. Alternatively, it can be obtained by introducing and expressing an expression vector containing a polynucleotide encoding an antibody fragment into a host cell (eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137; Hudson et al., Nat. Med., (2003) 9, 129-134).

As described above, an antibody can be obtained by introducing an expression vector containing a polynucleotide encoding the same into a cell and expressing it. Specifically, an expression vector is constructed so that the sequence encoding the antibody is expressed under an expression control region such as an enhancer or a promoter, and the host cell is transformed with this expression vector to express the antibody.

That is, the present disclosure also provides an expression vector containing a polynucleotide encoding an anti-preS1 antibody, and a transformed cell containing a polynucleotide capable of expressing the antibody.

As the host cell, for example, eukaryotic cells such as animal cells, plant cells, and fungal cells can be used. Animal cells include mammalian cells (eg, CHO, COS, NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero), amphibian cells (eg, Xenopus laevis), or insect cells (eg, Sf9, Sf21). , Tn5). Fungal cells include yeast (eg, Saccharomyces genus, eg, Saccharomyces cerevisiae), filamentous fungi (eg, Aspergillus genus, eg, Aspergillus niger), and the like. Will be. In addition, prokaryotic cells such as E. coli (for example, JM109, DH5α, HB101, etc.) and Bacillus subtilis can also be used as host cells. The vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, an electroporation method, a lipofection method, or the like.

The binding of the obtained antibody to the antigen can be confirmed by immunoassays such as EIA (including ELISA), RIA, CIA, and FIA, and BIACORE (registered trademark) surface plasmon resonance assay.

Binding of the antibody to the antigen can also be confirmed by a competitive assay. For example, the antibody is any of the antibodies (a) to (i) described herein, any of the antibodies (a') to (i'), (a'') to (i''). Any antibody of, any antibody of (a''') to (i'''), or any antibody of (a'''') to (i''''), and preS1 antigen. It can be confirmed by examining whether or not there is a conflict in binding to.

In certain embodiments, the antibodies disclosed herein are any of (a)-(i) described herein, any of (a')-(i'), (a''). ~ (I''), any antibody (a'''') ~ (i''''), or (a'''') ~ (i'''') It is an antibody that competes with the antibody of the above in binding to the preS1 antigen. As used herein, an antibody that competes with a given antibody (ie, a reference antibody) means an antibody that significantly reduces the binding of the reference antibody to the preS1 antigen. Competitive experiments can be performed as follows. First, the test antibody and the reference antibody are mixed in various molar ratios. The test antibody is usually used in excess of the reference antibody (eg, 1x, 2x, 5x, 10x, 20x, 50x, or 100x). The reference antibody may be labeled with a suitable label (eg, biotin). The mixture is then added to the plate on which the preS1 antigen has been immobilized and reacted, then the plate is washed and labeled with a detection reagent (eg, horseradish peroxidase (HRP)) or biotinylated. When the test antibody was used, HRP-streptavidin) was added and the amount of labeled reference antibody bound to the preS1 antigen was measured. In certain embodiments, the antibodies of the present disclosure bind the reference antibody to the preS1 antigen by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95. Reduce by% or more.

In a further embodiment, the antibody of the present disclosure is any of the antibodies (a') to (i') described herein, any of the antibodies (a''') to (i'''). , Or an antibody that competes with any of the antibodies (a'''') to (i'''') in binding to the preS1 antigen; An antibody that competes with any antibody or any of the antibodies (a''') to (i''') in binding to the preS1 antigen; or (a''') to described herein. An antibody that competes with any of the antibodies (i''') in binding to the preS1 antigen.

In certain embodiments, the antibodies of the present disclosure bind to an epitope contained in an amino acid sequence selected from PLGFFPDHQLDPAFG (SEQ ID NO: 2), NSNNPDWDFNPN (SEQ ID NO: 3), or DPAFGANSNN (SEQ ID NO: 4). In certain embodiments, the antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 2 is an antibody that binds to the epitope contained in the amino acid sequence of GFFPDHQLD (SEQ ID NO: 80). In another embodiment, the antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 3 is an antibody that binds to the epitope contained in the amino acid sequence of NNPDWDFNP (SEQ ID NO: 17) or NNPDWDFN (SEQ ID NO: 21). Whether or not it binds to an epitope contained in a predetermined amino acid sequence can be confirmed by examining whether or not it binds to a peptide consisting of the amino acid sequence, as described in Examples.

In another embodiment, the antibodies of the present disclosure are selected from the following (a)-(i):
(A) CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 25 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 26 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(B) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 37 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 37, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(C) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(D) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 43 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(E) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 47 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(F) CDR1, which contains the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 52 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 55 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 56 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(G) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(H) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 63 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising; and (i) CDR1 comprising an amino acid sequence of SEQ ID NO: 65 or an amino acid sequence in which 1-3 amino acid residues have been deleted, substituted, or added.
CDR2 containing the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 67 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 69 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 70, and 1-3 in the amino acid sequence of SEQ ID NO: 71 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising.

In a further embodiment, the antibodies of the present disclosure are selected from the following (a')-(i'):
(A') CDR1, comprising the amino acid sequence of SEQ ID NO: 24,
CDR2 containing the amino acid sequence of SEQ ID NO: 25 and CDR3 containing the amino acid sequence of SEQ ID NO: 26
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 28,
CDR2 containing the amino acid sequence of SEQ ID NO: 29, and CDR3 containing the amino acid sequence of SEQ ID NO: 30
Antibodies containing heavy chain variable regions containing
(B') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(C') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(D') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 43.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 45
Antibodies containing heavy chain variable regions containing
(E') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 47.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(F') CDR1, comprising the amino acid sequence of SEQ ID NO: 50,
CDR2 containing the amino acid sequence of SEQ ID NO: 51 and CDR3 containing the amino acid sequence of SEQ ID NO: 52
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 54,
CDR2 containing the amino acid sequence of SEQ ID NO: 55, and CDR3 containing the amino acid sequence of SEQ ID NO: 56.
Antibodies containing heavy chain variable regions containing
(G') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 60 and CDR3 containing the amino acid sequence of SEQ ID NO: 61
Antibodies containing heavy chain variable regions containing
(H') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 61.
An antibody comprising a heavy chain variable region comprising; and (i') CDR1 comprising the amino acid sequence of SEQ ID NO: 65,
CDR2 containing the amino acid sequence of SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 67.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 69,
CDR2 containing the amino acid sequence of SEQ ID NO: 70, and CDR3 containing the amino acid sequence of SEQ ID NO: 71.

In a further embodiment, the antibodies of the present disclosure are selected from the following (a'') to (i''):
(A'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 23, or in the amino acid sequence of SEQ ID NO: 23 or the amino acid sequence of SEQ ID NO: 23. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 27. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 27 or the amino acid sequence of SEQ ID NO: 27, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(B'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 35. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(C'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 39. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 39 or the amino acid sequence of SEQ ID NO: 39. An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(D'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 41, or in the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence of SEQ ID NO: 41. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 44. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(E'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 46, or in the amino acid sequence of SEQ ID NO: 46 or the amino acid sequence of SEQ ID NO: 46. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 48. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 48 or the amino acid sequence of SEQ ID NO: 48, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(F'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 49, or in the amino acid sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 49. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 53. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO: 53, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(G'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 59. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 59 or the amino acid sequence of SEQ ID NO: 59, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(H'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 62. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of SEQ ID NO: 62, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added; and (i'') the amino acid sequence of SEQ ID NO: 64 and 80%. , 85%, 90%, or 95% or more of amino acid sequences, or 1 to 20, 1 to 10, 1 to 5 in the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of SEQ ID NO: 64. , Or a light chain variable region containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added, and 80%, 85%, 90%, or 95% or more of the amino acid sequence of SEQ ID NO: 68. In the amino acid sequence having the same sequence identity, or in the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of SEQ ID NO: 68, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are present. An antibody containing a heavy chain variable region containing an amino acid sequence that has been deleted, substituted, or added.

In a further embodiment, the antibodies of the present disclosure are selected from the following (a''') to (i'''):
(A''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27;
(B''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35;
(C''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39;
(D''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44;
(E''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48;
(F''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53;
(G''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
(H''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62; and (i''') the amino acid sequence of SEQ ID NO: 64. An antibody comprising a light chain variable region comprising, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68.

In certain embodiments, the antibodies of the present disclosure are selected from the following (a'''') to (i''''):
(A'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 23, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27. , And an antibody containing a heavy chain variable region containing CDR3;
(B'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35. , And an antibody containing a heavy chain variable region containing CDR3;
(C'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39. , And an antibody containing a heavy chain variable region containing CDR3;
(D'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 41, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 44. , And an antibody containing a heavy chain variable region containing CDR3;
(E'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 46, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 48. , And an antibody containing a heavy chain variable region containing CDR3;
(F'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 49, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 53. , And an antibody containing a heavy chain variable region containing CDR3;
(G'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59. , And an antibody containing a heavy chain variable region containing CDR3;
(H'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 62. , And an antibody comprising a heavy chain variable region comprising CDR3; and (i'''') a light chain variable region comprising CDR1, CDR2, and CDR3 in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64, and SEQ ID NO: An antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 in a heavy chain variable region comprising 68 amino acid sequences.
In this embodiment, the method of specifying the CDR is arbitrary. Preferably, the CDRs are Kabat's definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. 1991), Chothia's definition (Chothia et al., J. Mol.). Biol., 1987; 196: 901-917), Definition of AdM (Martin et al., Proc.Natl.Acad.Sci.USA, 1989; 86: 9268-9272), Definition of Contact (MacCallum et al., J) . Mol. Biol. 1996; 262: 732-745), and specified by either the definition of IMGT (Lefranc et al., Dev Comp Immunol. 2003; 27 (1): 55-77).

In the present specification, the amino acid sequence containing a predetermined amino acid sequence includes an amino acid sequence in which one or more amino acid residues are added to the predetermined amino acid sequence, and a sequence consisting of the predetermined amino acids.

As used herein, a heavy chain variable region or a light chain containing an amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of a predetermined heavy chain variable region or light chain variable region. 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted or substituted in the amino acid sequence of the variable region and the predetermined heavy chain variable region or light chain variable region. Alternatively, the heavy chain variable region or light chain variable region containing the added amino acid sequence includes an antibody in which the CDR in the amino acid sequence of the predetermined heavy chain variable region or light chain variable region is not modified.

As used herein, "sequence identity" with respect to an amino acid sequence refers to amino acid residues that match between two sequences that are optimally aligned (to maximize matching) over the entire region of the sequence to be compared. Means the proportion of. Here, the sequence to be compared may have additions or deletions (eg, gaps, etc.) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (eg, DDBJ (http://www.ddbj.nig.ac.jp)). Alternatively, it can be obtained using commercially available sequence analysis software (for example, Vector NTI (registered trademark) software, GENETYX (registered trademark) ver. 12).

Various methods are known as methods for modifying an amino acid sequence to obtain an antibody having desired properties. For example, variants with improved binding affinity can be obtained by methods based on phage display. In this method, for example, amino acid residues that affect the interaction between the antibody and the antigen are identified by the alanine scanning mutagenesis method, or the crystal structure of the antigen-antibody complex is analyzed and the contact point between the antibody and the antigen is analyzed. To determine the site of mutagenesis, such as by identifying. A mutant having a desired characteristic can be obtained by preparing a mutant in which the amino acid at this site is modified by error-prone PCR, site-specific mutation introduction, or the like, and screening the library of the obtained mutant. ..

The antibody may have a subclass of immunoglobulin selected for the regulation of antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cellular cytotoxicity (CDC) activity or pharmacokinetics, and the amino acid sequence or sugar in the Fc region. The chain may be modified. For example, IgG4 may be selected for the purpose of reducing complement activation capacity. In addition, modifications that reduce or enhance the binding to the Fc receptor or C1q, modifications that increase the binding affinity to FcRn to prolong the half-life in blood, and the like can be performed.

The antibody also binds to a polymer such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or a copolymer of polyethylene glycol and polypropylene glycol, for example to prolong the blood half-life of the antibody or improve stability. You may be doing it. Antibodies may also be bound to chemotherapeutic agents, toxic peptides, radiochemicals and the like.

Two or more antibodies of the present disclosure can also be used in combination. In certain embodiments, the antibody combinations of the present disclosure are combinations of antibodies that bind to different epitopes. Further, the antibody of the present disclosure may be combined with an anti-HBs antibody that binds to an epitope different from that of the anti-preS1 antibody of the present disclosure. In certain embodiments, the anti-preS1 antibodies of the present disclosure are used in combination with anti-S antibodies or anti-preS2 antibodies.

2. 2. Measurement of preS1 antigen The antibody of the present disclosure can be used for measurement of preS1 antigen. The methods of the present disclosure include contacting a sample obtained from a subject with an antibody of the present disclosure and detecting a complex of preS1 antigen and antibody.

Subjects include those who are infected with HBV and those who are suspected of being infected with HBV. The sample obtained from the subject may be any as long as it contains the preS1 antigen, and examples thereof include blood, plasma, serum, urine, sweat, milk, first milk, tears, saliva, semen, and vaginal discharge. The term "sample obtained from a subject" as used herein refers not only to the sample obtained from the subject itself, but also to the treatment necessary for forming a complex of the preS1 antigen and the anti-preS1 antibody in the sample. It is used to include the later sample.

The complex of preS1 antigen and antibody may be measured by any method known in the art. Examples of the measurement method include immunoassay methods such as EIA (including ELISA), RIA, CIA, FIA, and a method based on a latex agglutination reaction.

The antibody may be modified according to the measurement method. For example, enzymes (eg, horseradish peroxidase, alkaline phosphatase, luciferase, β-galactosidase), fluorescent substances (eg, fluorescein, rhodamine), radioisotopes (eg, ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I). ) Or the like, or may be modified with biotin in order to bind the label bound to avidin.

Immunoassay methods include direct method, indirect method, competitive method, sandwich method and the like. Each method is usually carried out as follows. In the direct method, the immobilized antigen is contacted with a labeled antigen-specific antibody. In the indirect method, the immobilized antigen is contacted with an antigen-specific primary antibody, followed by reaction with a labeled secondary antibody specific for the primary antibody. In the sandwich method, a immobilized antigen-specific antibody was contacted with the antigen, followed by binding of another labeled (or modified for binding to the label) antigen-specific antibody to the immobilized antibody. Contact with antigen. In the competitive method, the immobilized antigen-specific antibody is brought into contact with the antigen to be measured and the labeled antigen at a known concentration at the same time. After washing, the antigen-antibody complex can be measured based on the label.

In certain embodiments, the complex of preS1 antigen and antibody is measured by ELISA. In a further embodiment, the complex of preS1 antigen and antibody is measured by sandwich ELISA. In one embodiment of sandwich ELISA, an anti-HBs antibody that binds to an epitope different from the anti-preS1 antibody used is immobilized on a solid phase such as a microplate, and a sample obtained from the subject is added. After washing, the anti-preS1 antibody is added to the solid phase, and the complex of the preS1 antigen and the anti-preS1 antibody is measured based on the label. Alternatively, the complex of the antigen and the anti-preS1 antibody may be measured by immobilizing the anti-preS1 antibody on the solid phase and using an anti-HBs antibody that binds to a different epitope.

In certain embodiments, the methods of the present disclosure use a combination of the anti-preS1 antibody of the present disclosure with an anti-S antibody or an anti-preS2 antibody. In a further embodiment, the methods of the present disclosure use a combination of the anti-preS1 antibody and the anti-S antibody of the present disclosure. For example, the anti-preS1 antibody of the present disclosure is used as a detection antibody and the anti-S antibody is used as a immobilized antibody, or the anti-preS1 antibody of the present disclosure is used as a immobilized antibody and the anti-S antibody is used as a detection antibody. be able to. By using an antibody that recognizes an epitope at a site distant from the epitope of the antibody of the present disclosure, accurate measurement can be performed. Commercially available antibodies such as Hyb-824 and Hyb-5124A can be used as the anti-S antibody, and Hyb-5520 (both are Special Immunology Research Institute Co., Ltd.) can be used as the anti-preS2 antibody. Further, the anti-S antibody and the anti-preS2 antibody can be prepared according to the description of the present disclosure using a polypeptide having an amino acid sequence of the whole or a part of the S domain or the preS2 domain as an immunogen.

HBV infection can be assessed by measuring the preS1 antigen. For HBV infection, the preS1 antigen is measured by a method using an anti-HBs antibody that cannot distinguish between infectious HBV particles and small spherical particles, or by measuring the amount of HBe antigen or HBcr antigen or an antibody against them, or HBV DNA. It is possible to give new information that is different from the method. The evaluation of HBV infection includes determination of the presence or absence of infection, determination of the pathophysiology of HBV infection, determination of therapeutic effect on HBV infection, determination of prognosis of HBV infection target, and the like. The evaluation results of HBV infection may provide useful information for starting, discontinuing, or discontinuing treatment for HBV infection.

The evaluation of HBV infection is performed by comparing the measured value of the preS1 antigen of the subject with the reference value. The reference value is appropriately set according to the purpose of evaluation. The reference value may be a measured value of another sample measured at the same time as the sample obtained from the target, or may be a value set by a prior measurement. Standard statistical methods can be used for comparison.

In certain embodiments, the reference value is a measurement of a sample (negative control) that does not contain the preS1 antigen. In another embodiment, the reference value is a value (eg, an average value) set from the measured values of a plurality of subjects not infected with HBV. If the measured value of the subject is higher than the reference value (that is, when the preS1 antigen is detected), it can be determined that the subject is infected with HBV. In addition, the higher the measured value of the subject, the more severe the HBV infection can be determined. For example, the higher the measured value, the more infectious HBV particles in the subject and the larger the amount of HBV cccDNA (covalently closed circular DNA) in the liver, and the higher the possibility of re-exacerbation after discontinuation of treatment. Can be determined. Alternatively, it can be determined that the higher the measured value, the lower the probability of disappearance of HBsAg or persistent infection.

In a further embodiment, the reference value is a previous measurement of the same subject. If the measured value of the subject is higher than the reference value, it is determined that the HBV infection is progressing, and if it is lower than the reference value, it is determined that the HBV infection is improved.

In a further embodiment, the reference value is a value set from measurements of a plurality of subjects of a given pathology or prognosis of HBV infection. For example, if the measured value of the subject is higher or lower than the reference value, it is determined that the subject has the predetermined pathological condition or prognosis.

The antibodies of the present disclosure may be included in a composition or kit for measuring preS1 antigen and a composition or kit for evaluating HBV infection. In addition to the antibodies of the present disclosure, the composition for measurement may contain water, glycerol, preservatives, antioxidants, chelating agents, buffers and the like, and the form thereof is not limited, and for example, it may be a liquid or a solid. (For example, freeze-dried powder) may be used. In certain embodiments, the kit comprises an anti-S antibody or an anti-preS2 antibody in addition to the antibodies of the present disclosure. In a further embodiment, the kit comprises an anti-S antibody in addition to the antibodies of the present disclosure. For example, the anti-preS1 antibody of the present disclosure is used as a detection antibody and the anti-S antibody is used as a immobilized antibody, or the anti-preS1 antibody of the present disclosure is used as a immobilized antibody and the anti-S antibody is contained as a detection antibody. Can be done. The kit may further include equipment and / or reagents depending on the measurement method. For example, the kit may include a solid phase such as a microplate, a preS1 antigen as a standard antigen, a reaction and / or dilution buffer, a color reagent, a reaction stop solution, instructions for use, and the like. The antibody or anti-S antibody or anti-preS2 antibody of the present disclosure may be bound to the solid phase in the kit.

3. 3. Pharmaceutical Composition The antibody of the present disclosure can be used as an active ingredient of a pharmaceutical composition. In certain embodiments, the antibodies of the present disclosure are used for the treatment or prevention of HBV infection. Treatment of HBV infections includes treatment of symptoms or diseases associated with HBV infections, such as treatment of chronic or acute hepatitis B and cirrhosis. Prevention of HBV infection includes prevention of infection after a needle receiving accident of HBV contaminated needles, and prevention of infection after blood transfusion or organ transplantation.

The antibodies of the present disclosure are administered to a subject in an amount (referred to herein as an effective amount) capable of exerting the desired effect (eg, treatment or prevention of HBV infection). The dose of the antibody is appropriately selected depending on the administration method, the age, body weight, health condition and the like of the administration target. For example, 10 μg / kg to 100 mg / kg, 100 μg / kg to 10 mg / kg, or 1 mg / kg to 10 mg / kg per day for adults every day, or days, weeks or weeks. It can be administered once, but is not limited to this. The method of administering the antibody is also appropriately selected depending on the age, body weight, health condition and the like of the administration target. The administration method may be oral administration or parenteral administration, but parenteral administration is preferable. Examples of parenteral administration include subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration and the like, and intravenous administration is preferable.

The pharmaceutical composition can be formulated by a conventional method. Pharmaceutical compositions are pharmaceutically acceptable, such as sterile water, saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc., in addition to antibodies. Can include carriers or additives.

In certain embodiments, the pharmaceutical composition comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 2; or (a), (a'), (a''), (a''') or ( Contains antibodies of a''''). In another embodiment, the pharmaceutical composition comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3; or (b)-(h), (b')-(h'), (b''). )-(H''), (b''')-(h''') or (b'''')-(h''''). In another embodiment, the pharmaceutical composition comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 4; or (i), (i'), (i''), (i''') or Contains the antibody of (i'''').

Two or more antibodies of the present disclosure may be used in combination as the active ingredient of the pharmaceutical composition. In certain embodiments, the two or more antibodies of the present disclosure include antibodies that bind to different epitopes. In a further embodiment, the two or more antibodies of the present disclosure bind to one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and 1 that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3. Antibodies of more than one species; or antibodies of (a), (a'), (a''), (a''') or (a'''') and (b)-(h), (b). From') ~ (h'), (b'') ~ (h''), (b''') ~ (h''') or (b'''') ~ (h'''') Includes one or more antibodies selected. In a further embodiment, the two or more antibodies of the present disclosure bind to one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and 1 that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 4. Antibodies of more than one species; or antibodies of (a), (a'), (a''), (a''') or (a'''') and (i), (i'), ( Includes antibodies of i''), (i''') or (i''''). In a further embodiment, the two or more antibodies of the present disclosure bind to one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and 1 that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 4. Antibodies of more than one species; or (b)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') Or one or more antibodies selected from (b'''') to (h'''') and (i), (i'), (i''), (i''') or ( i'''') including antibodies. The antibody of the present disclosure may be used in combination with an anti-HBs antibody different from the antibody of the present disclosure, or may be used in combination with another therapeutic agent for HBV infection. Other therapeutic agents include interferon (eg, peginterferon α-2a) and nucleic acid analogs (eg, lamivudine, adefovir, entecavir, tenofovir). In the present specification, when two or more kinds of active ingredients are used in combination, all or some of the active ingredients may be contained in the same composition, and all the agents are contained in different compositions. May be good. In addition, the administration schedules of the two or more active ingredients may be the same or different.

Exemplary embodiments of the present disclosure are described below.

[1]
An anti-preS1 antibody that binds to an epitope contained in an amino acid sequence selected from SEQ ID NOs: 2, 3 and 4.
[2]
The antibody according to 1 above, which binds to an epitope contained in the amino acid sequence of SEQ ID NO: 2.
[3]
The antibody according to 2 above, wherein the antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 2 is an antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 80.
[4]
The antibody according to 1 above, which binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[5]
The antibody according to 4 above, wherein the antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 3 is an antibody that binds to the epitope contained in the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 21.
[6]
The antibody according to any one of 1 to 5 selected from the following (a) to (i):
(A) CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 25 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 26 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(B) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 37 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 37, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(C) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(D) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 43 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(E) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 47 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(F) CDR1, which contains the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 52 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 55 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 56 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(G) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(H) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 63 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising; and (i) CDR1 comprising an amino acid sequence of SEQ ID NO: 65 or an amino acid sequence in which 1-3 amino acid residues have been deleted, substituted, or added.
CDR2 containing the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 67 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 69 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 70, and 1-3 in the amino acid sequence of SEQ ID NO: 71 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising.
[7]
An anti-preS1 antibody selected from the following (a) to (i): antibody:
(A) CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 25 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 26 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(B) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 37 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 37, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(C) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(D) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 43 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(E) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 47 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(F) CDR1, which contains the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 52 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 55 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 56 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(G) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(H) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 63 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising; and (i) CDR1 comprising an amino acid sequence of SEQ ID NO: 65 or an amino acid sequence in which 1-3 amino acid residues have been deleted, substituted, or added.
CDR2 containing the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 67 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 69 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 70, and 1-3 in the amino acid sequence of SEQ ID NO: 71 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising.
[8]
The antibody according to any one of 1 to 7 above, wherein the antibody is any of the following (a') to (i'):
(A') CDR1, comprising the amino acid sequence of SEQ ID NO: 24,
CDR2 containing the amino acid sequence of SEQ ID NO: 25 and CDR3 containing the amino acid sequence of SEQ ID NO: 26
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 28,
CDR2 containing the amino acid sequence of SEQ ID NO: 29, and CDR3 containing the amino acid sequence of SEQ ID NO: 30
Antibodies containing heavy chain variable regions containing
(B') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(C') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(D') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 43.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 45
Antibodies containing heavy chain variable regions containing
(E') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 47.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(F') CDR1, comprising the amino acid sequence of SEQ ID NO: 50,
CDR2 containing the amino acid sequence of SEQ ID NO: 51 and CDR3 containing the amino acid sequence of SEQ ID NO: 52
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 54,
CDR2 containing the amino acid sequence of SEQ ID NO: 55, and CDR3 containing the amino acid sequence of SEQ ID NO: 56.
Antibodies containing heavy chain variable regions containing
(G') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 60 and CDR3 containing the amino acid sequence of SEQ ID NO: 61
Antibodies containing heavy chain variable regions containing
(H') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 61.
An antibody comprising a heavy chain variable region comprising; and (i') CDR1 comprising the amino acid sequence of SEQ ID NO: 65,
CDR2 containing the amino acid sequence of SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 67.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 69,
CDR2 containing the amino acid sequence of SEQ ID NO: 70, and CDR3 containing the amino acid sequence of SEQ ID NO: 71.
[9]
The antibody according to any one of 1 to 8 above, wherein the antibody is any of the following (a'') to (i''):
(A'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 23, or in the amino acid sequence of SEQ ID NO: 23 or the amino acid sequence of SEQ ID NO: 23. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 27. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 27 or the amino acid sequence of SEQ ID NO: 27, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(B'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 35. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(C'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 39. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 39 or the amino acid sequence of SEQ ID NO: 39. An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(D'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 41, or in the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence of SEQ ID NO: 41. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 44. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(E'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 46, or in the amino acid sequence of SEQ ID NO: 46 or the amino acid sequence of SEQ ID NO: 46. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 48. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 48 or the amino acid sequence of SEQ ID NO: 48, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(F'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 49, or in the amino acid sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 49. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 53. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO: 53, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(G'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 59. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 59 or the amino acid sequence of SEQ ID NO: 59, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(H'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 62. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of SEQ ID NO: 62, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added; and (i'') the amino acid sequence of SEQ ID NO: 64 and 80%. , 85%, 90%, or 95% or more of amino acid sequences, or 1 to 20, 1 to 10, 1 to 5 in the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of SEQ ID NO: 64. , Or a light chain variable region containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added, and 80%, 85%, 90%, or 95% or more of the amino acid sequence of SEQ ID NO: 68. In the amino acid sequence having the same sequence identity, or in the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of SEQ ID NO: 68, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are present. An antibody containing a heavy chain variable region containing an amino acid sequence that has been deleted, substituted, or added.
[10]
The antibody according to any one of 1 to 9 above, wherein the antibody is any of the following (a''') to (i'''):
(A''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27;
(B''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35;
(C''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39;
(D''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44;
(E''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48;
(F''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53;
(G''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
(H''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62; and (i''') the amino acid sequence of SEQ ID NO: 64. An antibody comprising a light chain variable region comprising, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68.
[11]
The antibody according to any one of 1 to 10 above, wherein the antibody is any of the following (a'''') to (i''''):
(A'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 23, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27. , And an antibody containing a heavy chain variable region containing CDR3;
(B'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35. , And an antibody containing a heavy chain variable region containing CDR3;
(C'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39. , And an antibody containing a heavy chain variable region containing CDR3;
(D'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 41, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 44. , And an antibody containing a heavy chain variable region containing CDR3;
(E'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 46, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 48. , And an antibody containing a heavy chain variable region containing CDR3;
(F'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 49, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 53. , And an antibody containing a heavy chain variable region containing CDR3;
(G'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59. , And an antibody containing a heavy chain variable region containing CDR3;
(H'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 62. , And an antibody comprising a heavy chain variable region comprising CDR3; and (i'''') a light chain variable region comprising CDR1, CDR2, and CDR3 in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64, and SEQ ID NO: An antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 in a heavy chain variable region comprising 68 amino acid sequences.
[12]
An anti-preS1 antibody that competes with the antibody according to any one of 7 to 11 above in binding to the preS1 antigen.
[13]
The anti-preS1 antibody according to 12 above, which competes with the antibody according to 8, 10, or 11 in binding to the preS1 antigen.
[14]
The anti-preS1 antibody according to 12 above, which competes with the antibody according to 8 or 10 in binding to the preS1 antigen.
[15]
The anti-preS1 antibody according to 12 above, which competes with the antibody according to 10 in binding to the preS1 antigen.

[16]
A combination of two or more anti-preS1 antibodies, wherein the two or more anti-preS1 antibodies are independently selected from the antibody according to any one of 1 to 15.
[17]
Two or more anti-preS1 antibodies
One or more antibodies that bind to the epitope contained in the amino acid sequence of SEQ ID NO: 2 and one or more antibodies that bind to the epitope contained in the amino acid sequence of SEQ ID NO: 3;
One or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 4; or to an epitope contained in the amino acid sequence of SEQ ID NO: 3. One or more antibodies that bind and one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 4.
The combination according to 16 above.
[18]
Two or more anti-preS1 antibodies
The combination according to 17 above, comprising one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[19]
Two or more anti-preS1 antibodies
The combination according to 17 above, comprising one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 4.
[20]
Two or more anti-preS1 antibodies
(A), (a'), (a''), (a''') or (a'''') antibody, and (b) to (h), (b') to (h'). ), (B'') to (h''), (b''') to (h''') or (b'''') to (h'''') Antibodies;
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H'''') and one or more antibodies, and (i), (i'), (i''), (i''') or (i''''). Antibodies; or (a), (a'), (a''), (a''') or (a'''') antibodies, and (i), (i'), (i'). '), The combination according to 17 above, comprising the antibody of (i''') or (i'''').
[21]
Two or more anti-preS1 antibodies
(A), (a'), (a''), (a''') or (a'''') antibody, and (b) to (h), (b') to (h'). ), (B'') to (h''), (b''') to (h''') or (b'''') to (h'''') The combination according to 18 above, comprising the antibody of.
[22]
Two or more anti-preS1 antibodies
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H'''') and one or more antibodies, and (i), (i'), (i''), (i''') or (i''''). The combination according to 19 above, comprising the antibody of.
[23]
Two or more anti-preS1 antibodies
(A), (a'), (a''), (a''') or (a'''') antibody and (b), (b'), (b''), (b''') Or (b'''') antibody;
(A), (a'), (a''), (a''') or (a'''') antibody and (c), (c'), (c''), (c''') Or (c'''') antibody;
(A), (a'), (a''), (a''') or (a'''') antibody and (g), (g'), (g''), (g''') Or (g'''') antibody;
(A), (a'), (a''), (a''') or (a'''') antibody and (i), (i'), (i''), (i''') Or (i'''') antibody; or (c), (c'), (c''), (c''') or (c'''') antibody and (i) , (I'), (i''), (i''') or (i'''') comprising the combination according to 20 above.

[24]
It is a method for measuring preS1 antigen.
Contacting the sample obtained from the subject with the antibody according to any one of 1 to 15 above, and
A method comprising detecting a complex of a preS1 antigen and the antibody.
[25]
24. The method of 24 above, wherein the antibody is an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[26]
Antibodies are (b) to (h), (b') to (h'), (b'') to (h''), (b''') to (h'''') or (b'). ''')-(H''''). The method according to 24 above, which is an antibody selected from the above.
[27]
The method according to any one of 24 to 26 above, wherein the complex of the preS1 antigen and the antibody is detected by ELISA.
[28]
The method according to any one of 24 to 27, wherein the antibody according to any one of 1 to 15 and an anti-HBs antibody that binds to a different epitope are used.
[29]
28. The method of 28 above, wherein the anti-HBs antibody is an anti-S antibody or an anti-preS2 antibody.

[30]
Hepatitis B virus infection evaluation method
A method comprising measuring the preS1 antigen by the method according to any one of 24-29 and comparing the measured value with a reference value.
[31]
30. The method of 30 above, further comprising administering to the subject an effective amount of the antibody or combination according to any of 1-23.

[32]
A composition for measuring a preS1 antigen, which comprises the antibody or combination according to any one of 1 to 23 above.
[33]
A composition for evaluation of hepatitis B virus infection, which comprises the antibody or combination according to any one of 1 to 23 above.
[34]
32. The composition of 32 or 33 above, comprising an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[35]
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H''''). The composition according to 32 or 33 above, which comprises an antibody selected from (h'''').

[36]
A kit for measuring preS1 antigen, which comprises the antibody or combination according to any one of 1 to 23 above.
[37]
A kit for evaluating hepatitis B virus infection, which comprises the antibody or combination according to any one of 1 to 23 above.
[38]
The kit according to 36 or 37 above, which comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[39]
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H''''). The kit according to 36 or 37 above, which comprises an antibody selected from (h'''').
[40]
The kit according to any one of 36 to 39, further comprising an anti-HBs antibody that binds to an epitope different from the antibody according to any one of 1 to 15.
[41]
The kit according to 40 above, wherein the anti-HBs antibody is an anti-S antibody or an anti-preS2 antibody.

[42]
A pharmaceutical composition comprising the antibody or combination according to any one of 1 to 23 above.
[43]
42. The pharmaceutical composition according to 42 above, which comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 2.
[44]
42. The pharmaceutical composition according to 42 above, which comprises the antibody of (a), (a'), (a''), (a''') or (a'''').
[45]
42. The pharmaceutical composition according to 42 above, which comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
[46]
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H''''). The pharmaceutical composition according to 42 above, which comprises one or more antibodies selected from the above.
[47]
42. The pharmaceutical composition according to 42 above, which comprises an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO: 4.
[48]
42. The pharmaceutical composition according to 42 above, which comprises the antibody of (i), (i'), (i''), (i''') or (i'''').
[49]
Containing one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2, one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and / or included in the amino acid sequence of SEQ ID NO: 4. Combined with one or more antibodies that bind to an epitope that binds to one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and / or to an epitope contained in the amino acid sequence of SEQ ID NO: 4. Further comprising one or more antibodies;
Containing one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3, one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and / or included in the amino acid sequence of SEQ ID NO: 4. Used in combination with one or more antibodies that bind to an epitope that binds to one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and / or to an epitope contained in the amino acid sequence of SEQ ID NO: 4. It further comprises one or more antibodies; or contains one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 4, and one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2. / Or one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and / or one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2. Further comprises one or more antibodies that bind to an epitope contained in the amino acid sequence of
42. The pharmaceutical composition according to the above 42.
[50]
Contains one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2 and is used in combination with one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 or the amino acid of SEQ ID NO: 3. It further comprises one or more antibodies that bind to the epitope contained in the sequence; or contains one or more antibodies that bind to the epitope contained in the amino acid sequence of SEQ ID NO: 3 and is included in the epitope contained in the amino acid sequence of SEQ ID NO: 2. Combined with one or more antibodies that bind, or further comprising one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 2.
The pharmaceutical composition according to 49.
[51]
Contains one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3 and is used in combination with one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 4 or the amino acid of SEQ ID NO: 4. It further comprises one or more antibodies that bind to the epitope contained in the sequence; or contains one or more antibodies that bind to the epitope contained in the amino acid sequence of SEQ ID NO: 4 and is included in the epitope contained in the amino acid sequence of SEQ ID NO: 3. Combined with one or more antibodies that bind, or further comprising one or more antibodies that bind to an epitope contained in the amino acid sequence of SEQ ID NO: 3.
The pharmaceutical composition according to 49.
[52]
(A), (a'), (a''), (a''') or (a'''') containing antibodies, (b)-(h), (b')-(h'). ), (B'') to (h''), (b''') to (h''') or (b'''') to (h'''') And / or in combination with (i), (i'), (i''), (i''') or (i'''') antibodies or (b)-(h), (B') ~ (h'), (b'') ~ (h''), (b''') ~ (h''') or (b'''') ~ (h'''' ) And / or (i), (i'), (i''), (i''') or (i'''') antibody;
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H''''), including one or more antibodies selected from (a), (a'), (a''), (a''') or (a''''). And / or in combination with (i), (i'), (i''), (i''') or (i'''') antibody or (a), (a') , (A''), (a'''') or (a'''') antibody and / or (i), (i'), (i''), (i'''') or (i) '''') Further contains antibodies; or
(I), (i'), (i''), (i''') or (i'''') containing antibodies, (a), (a'), (a''), ( Antibodies of a''') or (a'''') and / or (b) ~ (h), (b') ~ (h'), (b'') ~ (h''), (b) Used in combination with one or more antibodies selected from'''') to (h'''') or (b'''') to (h''''), or (a), (a') , (A''), (a''') or (a'''') and / or (b)-(h), (b')-(h'), (b'')- Further comprising one or more antibodies selected from (h''), (b''') to (h''') or (b'''') to (h'''').
42. The pharmaceutical composition according to the above 42.
[53]
(A), (a'), (a''), (a''') or (a'''') containing antibodies, (b)-(h), (b')-(h'). ), (B'') to (h''), (b''') to (h''') or (b'''') to (h'''') (B) ~ (h), (b') ~ (h'), (b'') ~ (h''), (b'''') ~ (h'''' ) Or (b'''') to (h''''); or (b) to (h), (b') to (h'), ( One or more antibodies selected from b'') to (h''), (b''') to (h''') or (b'''') to (h'''') Containing and being used in combination with the antibody of (a), (a'), (a''), (a''') or (a''''), or (a), (a'), (a'). ''), Further comprising (a'''') or (a'''') antibodies,
52. The pharmaceutical composition according to 52.
[54]
(B)-(h), (b')-(h'), (b'')-(h''), (b''')-(h''') or (b'''')-(H''''), including one or more antibodies selected from (i), (i'), (i''), (i''') or (i''''). Combined with or further comprising (i), (i'), (i''), (i''') or (i'''') antibodies;
(I), (i'), (i''), (i''') or (i'''') containing antibodies, (b)-(h), (b')-(h'). ), (B'') to (h''), (b''') to (h''') or (b'''') to (h'''') (B) ~ (h), (b') ~ (h'), (b'') ~ (h''), (b'''') ~ (h'''' ) Or (b'''') to (h''''), further comprising one or more antibodies.
52. The pharmaceutical composition according to 52.
[55]
(A), (a'), (a''), (a''') or (a'''') comprising antibodies, (b), (b'), (b''), ( b''') or (b'''') antibody, (c), (c'), (c''), (c''') or (c'''') antibody, (g) ), (G'), (g''), (g''') or (g'''') antibody, or (i), (i'), (i''), (i'') ') Or (i'''') antibody, or (b), (b'), (b''), (b''') or (b'''') antibody, (C), (c'), (c''), (c''') or (c'''') antibody, (g), (g'), (g''), (g''') Or (g'''') antibody, or further comprising (i), (i'), (i''), (i''') or (i'''') antibody;
(B), (b'), (b''), (b''') or (b'''') containing antibodies, (a), (a'), (a''), ( Used in combination with an antibody of a''') or (a''''), or (a), (a'), (a''), (a''') or (a'''') Further contains antibodies;
(C), (c'), (c''), (c''') or contains (c'''') antibodies, (a), (a'), (a''), ( Used in combination with an antibody of a''') or (a'''') or an antibody of (i), (i'), (i''), (i''') or (i'''') Ruka, (a), (a'), (a''), (a''') or (a'''') antibody or (i), (i'), (i''), Further comprising (i''') or (i'''') antibodies;
Contains antibodies of (g), (g'), (g''), (g''') or (g''''), (a), (a'), (a''), ( Used in combination with an antibody of a''') or (a''''), or (a), (a'), (a''), (a''') or (a'''') And further comprises the antibody of (i), (i'), (i''), (i''') or (i''''), (a), (a'),. (A''), (a'''') or (a'''') antibody or (c), (c'), (c''), (c'''') or (c''''') In combination with the antibody of (a), (a'), (a''), (a''') or (a'''') or (c), (c') , (C''), (c''') or (c'''') further comprising,
52. The pharmaceutical composition according to 52.
[56]
The pharmaceutical composition according to any one of 42 to 55, which is used in combination with an anti-HBs antibody that binds to an epitope different from the antibody according to any one of 1 to 15, or further comprises the anti-HBs antibody.
[57]
56. The pharmaceutical composition according to 56 above, wherein the anti-HBs antibody is an anti-S antibody or an anti-preS2 antibody.
[58]
The pharmaceutical composition according to any one of 42 to 57 above, for treating or preventing hepatitis B virus infection.
[59]
The pharmaceutical composition according to any one of 42 to 58 above, for the treatment of chronic hepatitis B.

[60]
A method for treating or preventing hepatitis B virus infection, which comprises administering an effective amount of the antibody or combination according to any one of the above 1 to 23 to a subject in need of the treatment.
[61]
60. The method of 60, further comprising assessing the subject's hepatitis B virus infection by the method of 30.
[62]
The method according to 60 or 61 above for the treatment of chronic hepatitis B.

[63]
The antibody or combination according to any one of 1 to 23 above, for use in the treatment or prevention of hepatitis B virus infection.
[64]
The antibody or combination according to 63 above, for use in the treatment of chronic hepatitis B.

[65]
Use of any of the antibodies or combinations according to any of 1-23 above for the manufacture of a pharmaceutical for treating or preventing hepatitis B virus infection.
[66]
65. The use according to 65 above, for the manufacture of a pharmaceutical for the treatment of chronic hepatitis B.

株式会社アイティーエムにおいて、ペプチド21-35、34-48、および37-55を混合してマウスに免疫し、腸骨リンパ節法によりハイブリドーマを取得した。また、株式会社スクラムにおいて、ペプチドPep1およびPep2を混合してマウスに免疫し、ハイブリドーマを取得した。1. 1. Preparation of antibody
Antibodies were prepared using the following peptides derived from the preS1 domain of HBsAg.

At ITM Co., Ltd., peptides 21-35, 34-48, and 37-55 were mixed to immunize mice, and hybridomas were obtained by the iliac lymph node method. In addition, at Scrum Co., Ltd., peptides Pep1 and Pep2 were mixed and immunized with mice to obtain hybridomas.

ELISA was performed using the obtained antibody produced by the hybridoma and the peptide used for immunization. Each peptide was immobilized on a 96-well polystyrene plate, and a hybridoma culture supernatant was added and incubated. After washing, an anti-mouse rabbit antibody labeled with horseradish peroxidase (HRP) was added, and the supernatant showing a positive reaction was made positive, and a hybridoma that reacts with the peptide was selected. From the results in Table 2, from the hybridomas obtained using 21-35, 34-48, and 37-55, antibodies that react with 21-35 (15, 25, 41, and 51) and 34-48 and It was found that antibodies that responded to both 37-55 (7, 10, 13, 27, 39, 49, 55, and 63) were obtained (Table 2).

Antibodies that react with both 34-48 and 37-55 were considered to have epitopes at the intersection of both peptides (NSNNPDWDFNPN, SEQ ID NO: 3). To determine the epitope, antibodies 7, 10, and 55 were fine-mapped using the peptides shown in Table 3. An amino group-introduced plate (Sumitomo Bakelite) was used as the ELISA plate. 200 μl of 2% glutaraldehyde solution diluted with bicarbonate buffer (pH 9.6) was added to each well and incubated at room temperature for 2 hours to activate the amino group. Immediately after washing each well twice with 200 μl of sterile ultrapure water, 100 μl of each peptide solution for solid phase diluted with 0.2 M phosphate buffer (pH 6.0) to a concentration of 100 μM. It was added to each well and incubated overnight at 4 ° C to solidify. After solid phase, the antibody solution in the well was discarded and washed 3 times with 200 μl phosphate buffered saline (PBS, pH 7.4). 200 μl of blocking solution (PBS (pH 7.4) containing 5% skim milk and 0.05% Tween 20) was added to each well after washing, and the cells were incubated at 37 ° C. for 1 hour for blocking. The blocking solution in the wells was then discarded and washed 3 times with 200 μl PBST (PBS with 0.2% Tween 20, pH 7.4). The antibody purified from the hybridoma supernatant was diluted with a blocking buffer to a concentration of 1 μg / ml, 100 μl was added to each well, and the mixture was incubated at 37 ° C. for 1 hour. After incubation, the solution in each well was discarded and washed 3 times with 300 μl PBST. After washing, HRP-labeled commercially available antibodies, Goat anti-mouse IgG (H + L) and HRP conjugate (Proteintech Group, Inc), diluted 1: 2000 (0.5 μg / ml) with blocking buffer were used. 100 μl each was added to the wells and incubated at 37 ° C. for 1 hour. The solution in each well was then removed and washed 3 times with 300 μl PBST. Next, 100 μl of TMB substrate solution was added and reacted at 37 ° C. for 15 minutes, 100 μl of 2 M sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured.

The results in Table 3 indicate that NNPDWDFN (SEQ ID NO: 21) is an epitope for antibodies 7 and 10 and NNPDWDFNP (SEQ ID NO: 17) is an epitope for antibody 55, or that the epitope may be included in this sequence. ..

Six types of antibodies (1A, 1B, 1C, 5A, 5B, 5C) were obtained from hybridomas obtained using the peptides Pep1 and Pep2. Since these antibodies reacted with both Pep1 and Pep2 peptides, it was considered that the epitope was present in the intersection of both peptides (DPAFGANSNN, SEQ ID NO: 4).

These epitopes had high sequence identity in HBV of each genotype (Fig. 1), and these antibodies were considered to recognize the preS1 antigen of each genotype.

The antibody sequence analysis tool abYsis (http://www.abysis.org/) was used to obtain the light chain and heavy chain variable regions (VR) of the obtained antibody, as well as the sequences of CDR1, 2 and 3 thereof. Identified (FIGS. 2A and B and Tables 4-1 to 4-9). Variable regions and CDRs were identified by the Chothia Numbering Scheme.

In the same manner as above, antibody 41 was fine-mapped using the peptides shown in Table 5. The results in Table 5 indicate that GFFPDHQLD (SEQ ID NO: 80) is an epitope for antibody 41, or that the epitope may be contained in this sequence.

2. 2. Prevention of HBV infection by antibody Antibodies obtained using human primary cultured hepatocytes prepared from the liver of human hepatocyte chimeric mouse (Tateno C, et al., PLoS One. 2015 Nov 4; 10 (11): e0142145) The effect of preventing HBV infection was examined. Human primary hepatocytes (10 ⁵ cells), HBV (about 20 virus particles / cell), were added to the culture supernatant of antibody-producing hybridoma or purified antibodies, after 13 days of culture, HBs antigen in culture supernatant Was measured by the CLIA method using an HBsAg detection kit (Sysmex Co., Ltd.). The purified antibody was obtained from the culture supernatant of the expanded antibody-producing hybridoma using an antibody purification column (Protenova Co., Ltd.). The antibody concentration in the purified antibody and the culture supernatant was quantified using the Mouse IgG1 ELISA kit. The results are shown as the average (IU / mL) of the measured values for each well. Compared to the control phosphate buffered saline (PBS), each antibody markedly suppressed the production of HBs antigen after culture and was shown to block HBV infection (FIGS. 3-5). ). In particular, antibodies 7, 10, 13, 39, 49, 55 and 63 showed a high infection-blocking effect (Fig. 3).

Similarly, the HBV infection inhibitory effect of the combination of antibody 41 and antibody 7 or 10 was investigated in human primary cultured hepatocytes. It was shown that the infection-preventing effect was enhanced by combining the culture supernatant of the hybridoma producing antibody 41 and the culture supernatant of the hybridoma producing antibody 7 or 10 (FIG. 6). A synergistic enhancement of the infection-suppressing effect was also observed by the combination of the purified antibody, antibody 41 and antibody 10 or 55 (Fig. 7). When the antibody 49 and the antibody 55 were combined, the virus growth inhibitory effect was enhanced in a dose-dependent manner by the combination as compared with the antibody 49 alone (FIG. 8). In addition, the combination of an IgG3 antibody (also referred to as antibody 824 in the present specification) of Hyb-824 (Special Immunology Research Institute Co., Ltd.) having an epitope in the S domain and antibody 10 or 55 also enhances the infection-preventing effect. Was observed (Fig. 9).

3. 3. Measurement of preS1 antigen (1) Examination of antibody used for solid phase formation (i)
An amino group-introduced plate (Sumitomo Bakelite) was used as the ELISA plate. 200 μl of 2% glutaraldehyde solution diluted with bicarbonate buffer (pH 9.6) was added to each well and incubated at room temperature for 2 hours to activate the amino group. Immediately after washing each well twice with 200 μl of sterile ultrapure water, 100 μl of a solution of immobilization antibody diluted to a concentration of 0.5 μg / ml with 0.2 M phosphate buffer (pH 6.0). Was added to each well and incubated overnight at 4 ° C to solidify. As the antibody for immobilization, an IgM antibody (also referred to as antibody 5124A in the present specification) of hybridoma clone 5124A having an epitope in the antibody 41 or S domain was used. In addition, in order to observe non-specific adsorption of HBV or enzyme-labeled antibody in serum, wells to which no antibody was added were prepared, and only 0.2 M phosphate buffer was added during the solid phase of the antibody. After solid phase, the antibody solution in the well was discarded and washed twice with 200 μl phosphate buffered saline (PBS, pH 7.4). After washing, 200 μl of blocking solution (PBS containing 3% BSA (pH 7.4)) was added to each well, and the mixture was incubated at room temperature for 2 hours for blocking. Then, the blocking solution in the wells was discarded, 100 μl of HBV-positive subject serum diluted 10-fold with the blocking solution was added to each well, and the mixture was incubated at 37 ° C. for 1 hour. No serum was added to the negative control (NTC), only the blocking solution was added. After incubation, the solution in each well was discarded and washed 3 times with 300 μl PBST (PBS containing 0.2% Tween 20, pH 7.4). After washing, 100 μl of HRP-labeled antibody 55 diluted with a blocking solution to a ratio of 1: 1000 (3 μg / ml) was added to each well, and incubation was performed at 37 ° C. for 1 hour. The solution in each well was then removed and washed 3 times with 300 μl PBST. Next, 100 μl of TMB substrate solution was added and reacted at room temperature for 15 minutes, 100 μl of 2 M sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured.

Both of the two types of antibodies compared had the ability to collect HBs antigen, but the antibody 5124A showed higher detection ability (Fig. 10). The absorbance due to non-specific adsorption of HBV or enzyme-labeled antibody was 0.128 ± 0.005 (0.087 to 0.313).

(2) Measurement of preS1 antigen in the serum of subjects Using antibody 5124A as the immobilized antibody, measurement of preS1 antigen in the sera of 10 HBV-negative subjects and 50 HBV-positive subjects by the same method as in (1) above. Was done. Table 6 shows the measured absorbance of the preS1 antigen in the serum of the subject and the measured value of the HBV marker by the existing method. The HBV-DNA value is determined by the real-time PCR method, and the HBs antigen and HBe antigen are determined by the CLIA method using the HBsAg detection kit and the HBeAg detection kit, respectively, using the fully automatic immunoassay device HISCL (Sysmex Corporation). It was measured. The HBc-related antigen was measured by the CLIA method using the hepatitis B virus core-related antigen kit Lumipulse HBcrAg (Fujirebio Corporation). The HBe antibody was measured by the CLIA method using Architect i2000SR (Abbott Japan Co., Ltd.) and Architect HBe antibody reagent. .. The absorbance of HBV-positive subjects ranged from 0.603 to 3.141. In the absorptiometry, the practical measurement range of absorbance is said to be 0.05 to 1.5, but when the HBsAg value (IU / ML) exceeds 1,000, there are many samples with an absorbance of over 1.5, and further dilution is required. It was considered. Absorbance of preS1 antigen Correlation with other HBV markers was analyzed using regression analysis, but no correlation was found with any of the markers (data not presented).

(3) Examination of serum dilution rate and antibody used for solid phase (ii)
An amino group-introduced plate (Sumitomo Bakelite) was used as the ELISA plate. 200 μl of 2% glutaraldehyde solution diluted with bicarbonate buffer (pH 9.6) was added to each well and incubated at room temperature for 2 hours to activate the amino group. Immediately after washing each well twice with 200 μl of sterile ultrapure water, 100 μl of a solution of immobilization antibody diluted to a concentration of 0.5 μg / ml with 0.2 M phosphate buffer (pH 6.0). Was added to each well and incubated overnight at 4 ° C. to solidify. As the solid phase antibody, antibody 5124A or antibody 824 was used. After solid phase, the antibody solution in the well was discarded and washed 3 times with 200 μl phosphate buffered saline (PBS, pH 7.4). To each well after washing, 100 μl of ChonBlock ™ Blocking / Sample Dilution Buffer (Chondrex) was added as a blocking solution, and the mixture was incubated at room temperature for 1 hour for blocking. The blocking solution in the wells was then discarded and washed 3 times with 200 μl PBST (PBS containing 0.2% Tween 20, pH 7.4). Using HBV patient serum with HBs antigen of 90,000 IU / ml and HBV-DNA of 9.1 Log copies / ml as positive control (PTC) serum, prepare a 50-fold diluted solution using a blocking solution, and further prepare a 3-fold diluted series. Then, 50 μl of each of the above antibodies was added to each of the immobilized wells. In addition, for the sera of HBV positive subjects and negative subjects, a 10-fold dilution series was prepared using a blocking solution, and 50 μl was also added to each well. Incubation was carried out at 37 ° C. for 1 hour, and after incubation, the solution in each well was discarded and washed 3 times with 200 μl PBST. After washing, 100 μl of HRP-labeled antibody 55 diluted with ChonBlock ™ Detection Antibody Dilution Buffer (Chondrex) to 1: 1000 (3 μg / ml) was added to each well, 37. Incubation was performed at ° C for 1 hour. The solution in each well was then removed and washed 3 times with 200 μl PBST. Next, 100 μl of TMB substrate solution was added and reacted at room temperature for 15 minutes, 100 μl of 2 M sulfuric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured.

When a calibration curve of preS1 antigen was prepared using the diluted series of PTC serum, the absorbance of antibody 5124A used for immobilization was 2.4, that of antibody 824 was up to around 2.3, and ^{an approximate curve of R 2} = 0.999 could be created. It was possible (Table 7, Figure 11). Table 8 shows the absorbance of the preS1 antigen in each dilution series of HBV-positive or negative subject sera and the measured values of HBs antigen measured by the existing method. The absorbance increased as the HBsAg value increased. The serum dilution ratio was considered to be 1:10 at 10,000 IU / ml or less, 1: 100 at around 10,000 IU / ml, and 1: 500 to 1: 1000 at around 100,000 IU / ml. When the absorbances of antibody 5124A and antibody 824 were compared at the antibody dilution ratio suitable for each serum, antibody 824 showed higher detectability than antibody 5124A (FIG. 12). The absorbance of HBV-negative subjects was as low as 0.056 (5124A) and 0.069 (824) even when serum was added at a concentration of 1:10, compared to the case of using PBS containing 3% BSA in the blocking solution and serum diluent. The absorbance of the background decreased.

(4) Examination of antibodies used for solid phase formation (iii)
Using the same method as in (3) above, the antibodies used for solid phase were compared in duplicate using the sera of other subjects. The antibody concentration during solid phase was 2 μg / ml. The average value of the absorbance at a wavelength of 450 nm is shown in FIG. Some subject sera (HBV (+) 64 and 65) had higher absorbance of antibody 5124A, while others had higher absorbance of antibody 824. Since it was suggested that antibody 824 has higher detection ability for more subject sera than antibody 5124A, we decided to use antibody 824 as the immobilized antibody in the subsequent tests.

(5) Comparison of detection antibodies for preS1 antigen detection Using the same method as in (3) above, HRP-labeled antibody 10, 13, 39 or 55 was used as the detection antibody, and the detection ability was compared by 2. I went in a row. The solid-phase antibody concentration was 0.5 μg / ml, the detection antibody concentration was 1: 1000 as before, and 70 μl was added to each well. The average value of the absorbance at a wavelength of 450 nm is shown in FIG. The detectability of the four antibodies showed similar tendencies in all sera, indicating that any antibody can be used to measure the preS1 antigen. The measured values were highest for antibody 10, followed by antibodies 55, 39, and 13.

In the same manner as in Example 1, the effects of antibody 41, antibody 1A, and antibody 10 on HBV infection inhibition were examined using human primary cultured hepatocytes. The amounts of HBsAg and HBeAg in the culture supernatant were measured by the CLIA method using the HBsAg detection kit and the HBeAg detection kit (Sysmex Corporation). As a control, the group to which only HBV is added, and IgG1 (OY-TES-1 (G-5), santacruz: sc-390594) or IgG3 (Mouse IgG3 (isotype control), MBL: M078-3) instead of the antibody. ) Was added. Each antibody markedly suppressed the production of HBsAg and HBeAg after culture, and was shown to block HBV infection (FIGS. 15 and 16).

Similarly, the HBV infection inhibitory effect of the combination of antibody 824 with antibody 41, antibody 1A, or antibody 10 was examined. By combining antibody 824 having an epitope in the S domain with antibody 41, antibody 10 or antibody 1A, the effect of suppressing the production of HBsAg and HBeAg is enhanced in a dose-dependent manner, and a synergistic infection-preventing effect is observed. (Figs. 17 and 18). A synergistic infection-preventing effect was also observed by combining antibody 41, antibody 10 and antibody 1A, respectively (FIGS. 19 and 20).

Claims (22)

An anti-preS1 antibody selected from the following (a) to (i): antibody:
(A) CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 25 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 26 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 28 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 29 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 30 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(B) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 37 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 37, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(C) CDR1, which contains the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 34 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(D) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 43 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 45 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(E) CDR1, which contains the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 47 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 38 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(F) CDR1, which contains the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 51 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 52 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 54 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 55 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 56 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(G) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Antibodies containing heavy chain variable regions containing
(H) CDR1, which comprises the amino acid sequence of SEQ ID NO: 42 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 33 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 58 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 63 or the amino acid sequence, and 1-3 in the amino acid sequence of SEQ ID NO: 61 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising; and (i) CDR1 comprising an amino acid sequence of SEQ ID NO: 65 or an amino acid sequence in which 1-3 amino acid residues have been deleted, substituted, or added.
CDR2 containing the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 67 or 1 to 3 in the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
Light chain variable region, including
CDR1, which comprises the amino acid sequence of SEQ ID NO: 69 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted or added in the amino acid sequence.
CDR2 containing the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 70, and 1-3 in the amino acid sequence of SEQ ID NO: 71 or the amino acid sequence. CDR3 containing an amino acid sequence in which an amino acid residue has been deleted, substituted, or added.
An antibody comprising a heavy chain variable region comprising. The antibody according to claim 1, wherein the antibody is any of the following (a') to (i'):
(A') CDR1, comprising the amino acid sequence of SEQ ID NO: 24,
CDR2 containing the amino acid sequence of SEQ ID NO: 25 and CDR3 containing the amino acid sequence of SEQ ID NO: 26
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 28,
CDR2 containing the amino acid sequence of SEQ ID NO: 29, and CDR3 containing the amino acid sequence of SEQ ID NO: 30
Antibodies containing heavy chain variable regions containing
(B') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(C') CDR1, comprising the amino acid sequence of SEQ ID NO: 32,
CDR2 containing the amino acid sequence of SEQ ID NO: 33 and CDR3 containing the amino acid sequence of SEQ ID NO: 34
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(D') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 43.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 45
Antibodies containing heavy chain variable regions containing
(E') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 47.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 40 and CDR3 containing the amino acid sequence of SEQ ID NO: 38
Antibodies containing heavy chain variable regions containing
(F') CDR1, comprising the amino acid sequence of SEQ ID NO: 50,
CDR2 containing the amino acid sequence of SEQ ID NO: 51 and CDR3 containing the amino acid sequence of SEQ ID NO: 52
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 54,
CDR2 containing the amino acid sequence of SEQ ID NO: 55, and CDR3 containing the amino acid sequence of SEQ ID NO: 56.
Antibodies containing heavy chain variable regions containing
(G') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 60 and CDR3 containing the amino acid sequence of SEQ ID NO: 61
Antibodies containing heavy chain variable regions containing
(H') CDR1, comprising the amino acid sequence of SEQ ID NO: 42,
CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 58.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 36,
CDR2 containing the amino acid sequence of SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 61.
An antibody comprising a heavy chain variable region comprising; and (i') CDR1 comprising the amino acid sequence of SEQ ID NO: 65,
CDR2 containing the amino acid sequence of SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 67.
Light chain variable region, including
CDR1, comprising the amino acid sequence of SEQ ID NO: 69,
CDR2 containing the amino acid sequence of SEQ ID NO: 70, and CDR3 containing the amino acid sequence of SEQ ID NO: 71. The antibody according to claim 1 or 2, wherein the antibody is any of the following (a'') to (i''):
(A'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 23, or in the amino acid sequence of SEQ ID NO: 23 or the amino acid sequence of SEQ ID NO: 23. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 27. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 27 or the amino acid sequence of SEQ ID NO: 27, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(B'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 35. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(C'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 31, or in the amino acid sequence of SEQ ID NO: 31 or the amino acid sequence of SEQ ID NO: 31. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 39. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 39 or the amino acid sequence of SEQ ID NO: 39. An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(D'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 41, or in the amino acid sequence of SEQ ID NO: 41 or the amino acid sequence of SEQ ID NO: 41. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 44. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 44 or the amino acid sequence of SEQ ID NO: 44, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(E'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 46, or in the amino acid sequence of SEQ ID NO: 46 or the amino acid sequence of SEQ ID NO: 46. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 48. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 48 or the amino acid sequence of SEQ ID NO: 48, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(F'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 49, or in the amino acid sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 49. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 53. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO: 53, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(G'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 59. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 59 or the amino acid sequence of SEQ ID NO: 59, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added;
(H'') In the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 57, or in the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO: 57. A light chain variable region containing an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added, and the amino acid sequence of SEQ ID NO: 62. 1 to 20, 1 to 10 in the amino acid sequence having 80%, 85%, 90%, or 95% or more sequence identity, or the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of SEQ ID NO: 62, 1 An antibody containing a heavy chain variable region containing an amino acid sequence in which ~ 5 or 1-3 amino acid residues are deleted, substituted, or added; and (i'') the amino acid sequence of SEQ ID NO: 64 and 80%. , 85%, 90%, or 95% or more of amino acid sequences, or 1 to 20, 1 to 10, 1 to 5 in the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of SEQ ID NO: 64. , Or a light chain variable region containing an amino acid sequence in which 1-3 amino acid residues are deleted, substituted, or added, and 80%, 85%, 90%, or 95% or more of the amino acid sequence of SEQ ID NO: 68. In the amino acid sequence having the same sequence identity, or in the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of SEQ ID NO: 68, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are present. An antibody containing a heavy chain variable region containing an amino acid sequence that has been deleted, substituted, or added. The antibody according to any one of claims 1 to 3, wherein the antibody is any of the following (a''') to (i'''):
(A''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27;
(B''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35;
(C''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39;
(D''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44;
(E''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 48;
(F''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53;
(G''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
(H''') An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62; and (i''') the amino acid sequence of SEQ ID NO: 64. An antibody comprising a light chain variable region comprising, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68. The antibody according to any one of claims 1 to 4, wherein the antibody is any of the following (a'''') to (i''''):
(A'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 23, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 27. , And an antibody containing a heavy chain variable region containing CDR3;
(B'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35. , And an antibody containing a heavy chain variable region containing CDR3;
(C'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 31, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 39. , And an antibody containing a heavy chain variable region containing CDR3;
(D'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 41, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 44. , And an antibody containing a heavy chain variable region containing CDR3;
(E'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 46, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 48. , And an antibody containing a heavy chain variable region containing CDR3;
(F'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 49, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 53. , And an antibody containing a heavy chain variable region containing CDR3;
(G'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59. , And an antibody containing a heavy chain variable region containing CDR3;
(H'''') The light chain variable region containing the amino acid sequence of SEQ ID NO: 57, CDR1, CDR2, and CDR3, and the CDR1, CDR2 in the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 62. , And an antibody comprising a heavy chain variable region comprising CDR3; and (i'''') a light chain variable region comprising CDR1, CDR2, and CDR3 in a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64, and SEQ ID NO: An antibody comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 in a heavy chain variable region comprising 68 amino acid sequences. An anti-preS1 antibody that competes with the antibody according to any one of claims 1 to 5 in binding to the preS1 antigen. An anti-preS1 antibody that binds to an epitope contained in an amino acid sequence selected from SEQ ID NOs: 2, 3 and 4. The antibody according to claim 7, which binds to an epitope contained in the amino acid sequence of SEQ ID NO: 2. A combination of two or more anti-preS1 antibodies, wherein the two or more anti-preS1 antibodies are independently selected from the antibodies according to any one of claims 1-8. It is a method for measuring preS1 antigen.
Contacting the sample obtained from the subject with the antibody according to any one of claims 1 to 8, and contacting the sample with the antibody according to any one of claims 1 to 8.
A method comprising detecting a complex of a preS1 antigen and the antibody. The method according to claim 10, wherein the antibody according to any one of claims 1 to 8 and an anti-HBs antibody that binds to an epitope different from the antibody are used. 11. The method of claim 11, wherein the anti-HBs antibody is an anti-S antibody or an anti-preS2 antibody. Hepatitis B virus infection evaluation method
A method comprising measuring the preS1 antigen by the method according to any one of claims 10 to 12 and comparing the measured value with a reference value. A composition for measuring a preS1 antigen, which comprises the antibody or combination according to any one of claims 1 to 9. A composition for evaluating hepatitis B virus infection, which comprises the antibody or combination according to any one of claims 1 to 9. A kit for measuring a preS1 antigen, which comprises the antibody or combination according to any one of claims 1 to 9. A kit for evaluating hepatitis B virus infection, which comprises the antibody or combination according to any one of claims 1 to 9. The kit of claim 16 or 17, further comprising an anti-HBs antibody that binds to an epitope different from that of any of claims 1-9. The kit according to claim 18, wherein the anti-HBs antibody is an anti-S antibody or an anti-preS2 antibody. A pharmaceutical composition comprising the antibody or combination according to any one of claims 1-9. The pharmaceutical composition according to claim 20, for the treatment or prevention of hepatitis B virus infection. The pharmaceutical composition according to claim 20 or 21, for the treatment of chronic hepatitis B.

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