JP2023053786A - Development of antibodies to prevent hepatitis B virus infection - Google Patents

Abstract

To provide novel HBs antigen-binding antibodies.SOLUTION: In one aspect, the present disclosure provides HBs antigen-binding antibodies or antigen-binding fragments thereof having the CDR sequences described herein. In one embodiment, the HBs antigen binding antibody is humanized. In one embodiment, compositions are provided for treating or preventing hepatitis B virus infection comprising the HBs antigen-binding antibodies or antigen-binding fragments thereof of the present disclosure. In one embodiment, a composition for treating an organ for transplantation is provided comprising an HBs antigen-binding antibody or antigen-binding fragment thereof of the present disclosure. A detection agent comprising an HBs antigen-binding antibody or antigen-binding fragment thereof of the disclosure is also provided.SELECTED DRAWING: None

Description

The present disclosure provides antibodies or antigen-binding fragments thereof that bind to the HBs antigen of hepatitis B virus. In particular, the present disclosure provides for hepatitis B virus inhibition and detection using these antibodies or antigen-binding fragments thereof.

Hepatitis B virus infection is known to cause hepatitis B and subsequent cirrhosis and liver cancer. Hepatitis B virus can be transmitted from person to person through blood. Drugs have been developed for the prevention and treatment of hepatitis B, such as the vaccine disclosed in US Pat.

In some cases, such as liver transplantation, it is necessary to directly inhibit hepatitis B virus infection, and human plasma-derived hepatitis B immunoglobulin (HBIG) preparations are commonly used.

Existing HBIG is manufactured by isolating antibodies from human blood carrying antibodies to hepatitis B and using viral inactivation techniques to remove potential sources of contamination. However, it is difficult to secure plasma, which is the raw material, and there is a problem with the supply of HBIG.

WO2019/123252

The present inventors have developed a novel antibody against the HBs antigen of hepatitis B virus. The produced antibodies included antibodies with high binding properties to HBs antigens and antibodies with binding properties also to HBs antigens having escape mutations. On this basis, the present disclosure provides HBs antigen-binding antibodies or antigen-binding fragments thereof, as well as inhibition and detection of hepatitis B virus using the same.

Accordingly, the present invention provides:
(Item 1)
HBs antigen binding comprising a heavy chain having a heavy chain variable region amino acid sequence comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences and a light chain having a light chain variable region amino acid sequence comprising light chain CDR1, CDR2 and CDR3 amino acid sequences An antibody or antigen-binding fragment thereof,
(1)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 1-5;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOS: 6-10;
the heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOs: 11-15;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 16-20;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 21-25;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 26-30, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences, or (2)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 31-35;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 36-40;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 41-45;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 46-50;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS: 51-55;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 56-60, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences;
(3)
said heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 61-65;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 66-70;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 71-75;
said light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 76-80;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS:81-85;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOs: 86-90, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences;
HBs antigen-binding antibody or antigen-binding fragment thereof.
(Item 2)
The heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences, respectively,
SEQ ID NOs: 1, 6, 11, 16, 21 and 26,
SEQ ID NOs: 2, 7, 12, 17, 22 and 27,
SEQ ID NOs: 3, 8, 13, 18, 23 and 28,
SEQ ID NOS: 4, 9, 14, 19, 24 and 29,
SEQ ID NOs: 31, 36, 41, 46, 51 and 56,
SEQ ID NOS: 32, 37, 42, 47, 52 and 57,
SEQ ID NOs: 33, 38, 43, 48, 53 and 58,
SEQ ID NOS: 34, 39, 44, 49, 54 and 59
SEQ ID NOs: 61, 66, 71, 76, 81 and 86,
SEQ ID NOs: 62, 67, 72, 77, 82 and 87,
SEQ ID NOs: 63, 68, 73, 78, 83 and 88, or SEQ ID NOs: 64, 69, 74, 79, 84 and 89
or contains a total of 1-6 amino acid mutations in the six CDR amino acid sequences;
The HBs antigen-binding antibody or antigen-binding fragment thereof according to item 1.
(Item 3)
3. The HBs antigen-binding antibody or antigen-binding fragment thereof according to item 1 or 2, wherein said amino acid mutation is independently an amino acid substitution, amino acid deletion or amino acid insertion.
(Item 4)
3. The HBs antigen-binding antibody or antigen-binding fragment thereof according to item 1 or 2, wherein said amino acid mutations are independently conservative amino acid substitutions.
(Item 5)
(1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:91 or at least 80% identical thereto, and a light chain variable region having an amino acid sequence of SEQ ID NO:92 or at least 80% identical thereto;
(2) a heavy chain variable region having an amino acid sequence of SEQ ID NO:93 or at least 80% identical thereto, and a light chain variable region having an amino acid sequence of SEQ ID NO:94 or at least 80% identical thereto, or (3) a SEQ ID NO: 5. Any one of items 1-4, having a heavy chain variable region having an amino acid sequence of 95 or at least 80% identical to SEQ ID NO: 96 or a light chain variable region having an amino acid sequence of at least 80% identical to SEQ ID NO:96 3. The HBs antigen-binding antibody or antigen-binding fragment thereof according to .
(Item 6)
The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 5, which binds to at least one of K141E mutant HBs, G145R mutant HBs and I152A mutant HBs.
(Item 7)
7. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 6, which binds to at least one of hepatitis B virus genotype A, B, C or D HBs.
(Item 8)
8. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 7, which has a KD value of less than 10 nM when the affinity for HBs is measured by biolayer interferometry.
(Item 9)
The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 8, which is humanized.
(Item 10)
A composition for treating or preventing hepatitis B virus infection, comprising the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 9.
(Item 11)
11. A composition according to item 10 for treating an organ for transplantation.
(Item 12)
A hepatitis B virus detection agent comprising the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 9.
(Item 13)
A nucleic acid encoding the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

It is contemplated in the present invention that one or more of the features described above may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the present invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present disclosure provides novel HBs antigen-binding antibodies. This novel antibody is useful in inhibiting hepatitis B virus and may replace existing HBIG.

A comparison of HBV RNA levels when HBV-infected cells were treated with each monoclonal antibody clone or PreS1 peptide. The horizontal axis indicates the conditions of each treatment. NTC is drug-free. The vertical axis shows the amount of HBV RNA for each drug treatment compared to no drug addition. Fig. 2 shows changes in HBV RNA levels when HBV-infected cells were treated with various concentrations of each monoclonal antibody clone or HBIG. Each graph shows results for a different monoclonal antibody clone or HBIG, respectively. In each graph, the horizontal axis indicates the antibody concentration (ng/mL) added, and the vertical axis indicates the amount of HBV RNA for treatment at each concentration compared to no drug addition. The calculated IC50 value in each graph is also shown. Fig. 3 shows the results of testing the binding properties of various concentrations of monoclonal antibody clones to HBs antigen using the Octet system. Each graph shows results for different monoclonal antibody clones. FIG. 1 shows a schematic of an antibody-based infectivity inhibition test using hepatitis D virus (HDV) particles with the desired HBV-derived envelope. Figure 3 shows the effect of monoclonal antibody clones HB351 and HB1170 on infection with HDV with genotype A, B, C or D HBV envelopes. The horizontal axis indicates each treatment condition, and the vertical axis indicates the amount of HBV RNA compared to the case without drug addition. Above is a schematic of the experiment of Example 5. Below are the results of the experiment of Example 5. In the graph on the left, the vertical axis indicates the amount of HBV DNA in serum, and the horizontal axis indicates the date of blood sampling. In the graph on the right, the vertical axis indicates the amount of HBs antigen in serum, and the horizontal axis indicates the date of blood collection. Fig. 10 is an image obtained by examining the ability of HBs antigen-binding antibodies to recognize various alanine-substituted HBs antigens. HBs antigens having mutations C147A, T148A, C149A, I150A, P151A, and I142A are shown in order from the left. HB351 clone, HB1170 clone, and HBs antigen-binding antibody mix are shown in order from the top. Effect of each clone on HDV infection with HBV envelope of HBs antigen containing escape mutation G145R is shown. The horizontal axis indicates each treatment condition, and the vertical axis indicates the amount of HBV RNA compared to the case without drug addition. The left column shows the results of testing the binding properties of each humanized monoclonal antibody clone at various concentrations to the HBs antigen using the Octet system. The right column shows changes in HBV RNA levels when HBV-infected cells were treated with various concentrations of each humanized monoclonal antibody clone, together with calculated IC50 values, and the horizontal axis indicates the antibody concentration added (ng/mL). The vertical axis shows the amount of HBV RNA for treatment at each concentration compared to no drug addition. Each graph shows the results of humanized HB351 or humanized HB1170.

Hereinafter, the present disclosure will be described while showing the best mode. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Thus, articles in the singular (eg, “a,” “an,” “the,” etc. in the English language) should be understood to include their plural forms as well, unless otherwise stated. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

Definitions of terms and/or basic technical content particularly used in the present specification will be described below as appropriate.

As used herein, "inhibiting" a virus means reducing the infection of a cell by the virus, slowing the growth rate of the virus, reducing the virus and/or inactivating the virus. Suppression of virus can be detected by a reduction in the amount of viral protein or nucleic acid in a cell or sample.

As used herein, "hepatitis B virus (HBV)" refers to a DNA virus belonging to the genus Orthohepadnavirus in the family Hepadnaviridae. HBV genotypes include genotype A (including Aa/1 (Asian/African type), Ae/2 (European type), Ac/3 (Cameroon)), genotype B (Bj/1 (Japanese type), Ba/2 (Asian type), including B3, B4, and B5), genotype C (including Ce/1 (East Asian type), Cs/2 (South East type), C3, and C4), genotype D (including D1, D2, D3, D4, D5), genotype E, genotype F (including F1, F2, F3, F4), genotype G, genotype H, genotype J, etc. are known.

As used herein, "HBs" or "HBs antigen" refers generally to surface proteins of the hepatitis B virus. Surface proteins of hepatitis B virus include L protein having PreS1 region + PreS2 region + HBs region, M protein having PreS2 region + HBs region and S protein having HBs region. can also refer to proteins of For example, the amino acid sequence of the L protein of genotype A HBV with the entry name "A0A679DNY4_HBV" in the UniProt database is as follows. The underlined portion of 226 amino acids corresponds to the S protein, and the bolded portion (positions 124-147 in the S protein 226 amino acids) is called the 'a' determinant.

In the present specification, expressions such as HBs antigen-binding antibody and HBs-binding ability are used at any position or region (epitope) of any kind of protein (including mutants) belonging to HBs, unless otherwise specified. can be an object.

In this specification, unless otherwise specified, references to a molecule (e.g., protein, nucleic acid) refer to a molecule that performs a function similar to, but not to the same extent as, the molecule's biological function. It is understood that reference is also made to variants (eg, variants having modifications in the amino acid sequence). For example, the genotype A HBV L protein includes not only a protein having the amino acid sequence represented by SEQ ID NO: 107, but also a conjugate of this protein with a labeling molecule (such as a fluorescent label), replacing the endogenous L protein. Variants that allow HBV type A to grow in the host may also be included. Such variants include fragments of the original molecule, sequences of the original molecule aligned across amino acid or nucleic acid sequences of the same size, or aligned by computer homology programs known in the art. Molecules that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identical when compared to . Variants can include molecules with altered amino acids, such as alterations due to disulfide bond formation, glycosylation, lipidation, acetylation or phosphorylation.

As used herein, "amino acid" is used in the ordinary sense in the technical field and refers to a compound having a carboxyl group and an amino group in one molecule. Typically, the amino acids are alpha-amino acids, but may also be amino acids herein with more distant carboxyl and amino groups, such as beta- and gamma-amino acids. Furthermore, in this specification, unless otherwise indicated, an amino acid represents an L-isomer, and when it is a D-isomer, it is preceded by D (or d) (for example, D-Ala ( Alternatively, it is indicated by d-Ala)) or lower case letters (eg, a). The following are representative examples of amino acids, but other amino acids are also contemplated herein.

As used herein, the amino acid or nucleotide at the "corresponding position" has the same effect in a certain polypeptide molecule or polynucleotide molecule as the amino acid or nucleotide at a given position in the polypeptide or polynucleotide used as a reference for comparison. or an amino acid or nucleotide predicted to have. For example, one of ordinary skill in the art can align between the original molecule and its variants and set appropriate thresholds to identify corresponding positions. For example, insertion of a single amino acid may increase by one each corresponding position of the amino acid residue following the point of insertion.

When expressing an amino acid at a specific position, a combination of the number representing the specific position and the one-letter code or three-letter code of the amino acid can be used as appropriate. For example, the alanines in RIDPANGDTKYDPNFQG (SEQ ID NO:6) are present at positions 5 and 5 and can be represented as 5A, 5Ala, etc. in SEQ ID NO:6. The same amino acid may be represented at different positions when the referenced SEQ ID NOs are different.

For modification of amino acids in the amino acid sequence of a polypeptide, site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)), overlap extension PCR, etc. A known method can be employed as appropriate. In addition, a method of modifying an amino acid in which an amino acid other than a natural amino acid is substituted can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. US A. (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which an unnatural amino acid is bound to an amber suppressor tRNA complementary to a UAG codon (amber codon), which is one of stop codons, is preferably used. Used.

As used herein, amino acid "mutation" or "modification" refers to amino acid insertion, substitution or deletion. A single mutation or modification of an amino acid refers to an insertion of one amino acid, a substitution of one amino acid, or a deletion of one amino acid. Amino acid insertions also include amino acid additions to the ends of an amino acid sequence.

In the present specification, as an expression representing amino acid substitution, an expression in which the number representing a specific position is preceded and followed by the one-letter code or three-letter code of the amino acid before and after substitution can be used as appropriate. For example, the substitution G6V or Gly6Val used with reference to RIDPANGDTKYDPNFQG (SEQ ID NO: 6) represents a glycine to valine substitution at position 6.

As used herein, "conservative amino acid substitution" refers to replacing an amino acid in a peptide with another amino acid having similar properties, and it can be expected that the properties of the peptide are similar before and after the conservative substitution. Examples of specific conservative amino acid substitutions are provided elsewhere herein.

As used herein, "antibody" is used in the broadest sense and encompasses various antibody structures, including monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).

As used herein, the term “humanized antibody” refers to an antibody having a structure in which the CDR regions of an antibody produced in a species other than human are incorporated into a human antibody. A “chimeric antibody” refers to an antibody having a structure in which the variable region of an antibody produced in a species other than human is incorporated into a human antibody.

As used herein, "antigen-binding fragment" refers to a partial structure of an antibody that has the ability to bind to an antigen of interest, and typically includes at least the heavy and light chain variable regions of an antibody. Antigen-binding fragments include, for example, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies and single chain variable region fragments (scFv).

As used herein, "variable region" or "variable domain" refers to the heavy or light chain domains of an antibody. The heavy and light chain variable domains (VH and VL, respectively) of antibodies each typically contain four conserved framework regions (FR) and three complementarity determining regions (CDR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)).

As used herein, "complementarity determining region" or "CDR" refers to the regions in the variable domain of an antibody that contribute specifically to antigen-binding. Antibodies typically contain six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several methods exist for determining CDR sequences, including the Kabat numbering system, the IMGT numbering system, the Paratome numbering system and the Chothia numbering system (e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., Public Health). Ｓｅｒｖｉｃｅ、Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ、Ｂｅｔｈｅｓｄａ、Ｍｄ．（Ｋａｂａｔナンバリングシステム）、Ｋｕｎｉｋ、Ａｓｈｋｅｎａｚｉ ａｎｄ Ｏｆｒａｎ、２０１２、「Ｐａｒａｔｏｍｅ：ａｎ ｏｎｌｉｎｅ ｔｏｏｌ ｆｏｒ ｓｙｓｔｅｍａｔｉｃ ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ｏｆ ａｎｔｉｇｅｎ－ｂｉｎｄｉｎｇ ｒｅｇｉｏｎｓ ｉｎ ａｎｔｉｂｏｄｉｅｓ ｂａｓｅｄ ｏｎ ｓｅｑｕｅｎｃｅ ｏｒ ｓｔｒｕｃｔｕｒｅ' Ｎｕｃｌ．Ａｃｉｄｓ Res., 40:W521-W524 (Paratome numbering system), Lefranc et al., 2003, Dev Comparat Immunol 27:55-77 (IMGT numbering system), Al-Lazikani et al., 1997, J. Mol. (See Chothia Numbering System).

"Framework" or "FR" refers to regions other than the complementarity determining regions (CDRs) in variable domains. The FRs of variable domains usually consist of four FR domains: FR1, FR2, FR3 and FR4. CDR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

As used herein, "constant region" or "constant domain" refers to the portion of an antibody other than the variable region. For example, IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded, with Thus, each heavy chain has a variable region (VH) and a heavy chain constant region (CH) comprising the CH1, hinge, CH2 and CH3 domains. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL) and a constant light chain (CL) domain. Two types of constant domains, called kappa (κ) and lambda (λ), are known in the light chains of naturally occurring antibodies.

As used herein, "Fc region" refers to a region comprising at least a portion of the CH3, CH2, and hinge regions of the constant domains of an antibody. Optionally, the Fc region can include a CH4 domain present in several antibody classes. The Fc region can include the entire hinge region of the constant domains of an antibody. The Fc region is recognized by T cells and the like, and is associated with effector functions and the like. For human IgG1, the heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present (numbering of amino acid residues in the Fc region is according to Kabat et al., (according to the EU numbering system (EU index) as described in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991).

A "class" of antibody is determined by the type of constant domain contained in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM. And some of these can be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

As used herein, "antigen" refers to a target molecule or portion thereof to which an antibody binds. An antigenic determinant present in an antigen is called an epitope, and antibodies bind to the epitope portion of the antigen. Epitopes include linear epitopes and conformational epitopes. A linear epitope in a protein antigen is an epitope whose primary amino acid sequence is recognized by an antibody. A linear epitope in a protein antigen is typically composed of at least 3, at least 5, eg 8-10, 6-20 amino acids. Conformal epitopes in protein antigens are epitopes that cannot be defined by the primary sequence of amino acids alone, in contrast to linear epitopes. For recognition of conformational epitopes, antibodies recognize the three-dimensional structure of proteins. Methods of determining epitope conformation include, but are not limited to, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance spectroscopy, site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), vol. 66, Morris (ed.).

As used herein, a comparison of the identity of amino acid sequences or base sequences is calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI BLAST 2.7.1 (issued on October 19, 2017). The value of "identity" in the present specification usually refers to the value when aligned under the default conditions using the above BLAST. "Identity" is calculated according to the known method as described above for the percentage of homologous amino acids or nucleotides in two or more amino acid or nucleotide sequences. Specifically, prior to calculating the percentage, the amino acid or base sequences of the amino acid or base sequences to be compared are aligned and amino acid or base sequences are added if necessary to maximize the percentage of identical amino acids or bases. Introduce gaps in part of the array. Alignment methods, ratio calculation methods, comparison methods, and computer programs related thereto are conventionally well known in the art (eg, BLAST, etc., as described above). Blastp can be used with default settings as an algorithm when comparing amino acid or base sequences with BLAST.

As used herein, the term "pharmaceutical ingredient" means any ingredient that can constitute a drug, for example, an active ingredient (which itself exhibits medicinal efficacy), an additive ingredient (which itself is not expected to have medicinal efficacy), However, components expected to play a certain role (for example, excipients, lubricants, surfactants, etc.) when included as a pharmaceutical can be exemplified. A pharmaceutical ingredient may be a single substance or a combination of multiple substances or agents. Any combination, such as a combination of an active ingredient and an additive ingredient, a combination of an adjuvant and an active ingredient, etc., may also be included.

As used herein, the term "active ingredient" refers to an ingredient that exerts an intended medicinal effect, and may be a single ingredient or multiple ingredients.

As used herein, the term "additive ingredient" refers to any ingredient that is not expected to have medicinal efficacy but plays a certain role when included as a medicine. Examples include pharmaceutically acceptable carriers, stabilizers, ( Auxiliary) Adjuvants, solubility improvers, solubilizers, diluents, excipients, buffers, binders, diluents, flavoring agents, and lubricants.

As used herein, "drug", "agent" or "agent" (both equivalent to agents in English) are broadly used interchangeably and are used to describe any agent capable of achieving its intended purpose. It may be matter or other elements (eg, energy such as light, radiation, heat, electricity, etc.). Such substances include, for example, antibodies, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including DNA such as cDNA and genomic DNA, and RNA such as mRNA). , polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.) etc.), these complex molecules and mixtures thereof.

As used herein, the term "label" refers to an entity (eg, substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When a plurality of target proteins or factors or means for capturing them are labeled by a fluorescence method, they are labeled with fluorescent substances having mutually different fluorescence emission maximum wavelengths. The difference in fluorescence emission maximum wavelength is preferably 10 nm or more. Fluorescent agents include Alexa ^™ Fluor, although any label that does not affect function can be used. Alexa ^™ Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that supports a wide range of fluorescent wavelengths. stable, bright, and insensitive to pH. Combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa ^™ 555 and Alexa ^™ 633, a combination of Alexa ^™ 488 and Alexa ^™ 555, and the like. Other fluorescent labels include cyanine dyes (eg, Cy3, Cy5, etc. of the CyDye ^TM series), rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF), AAIF (an iodine derivative of AAF), and the like. In the present disclosure, such labels can be utilized to modify the subject of interest so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods as appropriate depending on the label and the intended subject.

As used herein, the term "kit" refers to a unit provided with parts to be provided (eg, antibodies, instructions, etc.), usually divided into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed form for reasons such as stability, and is preferably used in a mixed form immediately before use. Such kits are advantageously provided with instructions or instructions, preferably describing how to use the parts provided or how to handle the reagents. In the present specification, when the kit is used as a reagent kit, the kit usually includes instructions describing how to use the antibody and the like.

As used herein, "instructions" are written instructions to a physician or other user how to use the present disclosure. The instructions contain language that instructs administration of the drug and the like of the present disclosure. The instructions may also include language that indicates the mode of administration. This instruction is prepared in accordance with the format prescribed by the regulatory authority of the country in which this disclosure is implemented (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.) and is approved by the regulatory authority. It is stated that it has received Instructions are so-called package inserts, which are usually provided in paper form, but are not limited thereto, for example, electronic medium (e.g., homepage provided on the Internet, e-mail) can also be provided.

The term "about" refers to plus or minus 10% of the indicated value. "About" when used for temperature refers to the indicated temperature plus or minus 5°C and "about" when used for pH refers to the indicated pH plus or minus 0.5.

(preferred embodiment)
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the disclosure, and it is understood that the scope of the disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used singly or in combination.

In one aspect, the present disclosure provides HBs antigen-binding antibodies. In one aspect, the present disclosure provides inhibition and detection of HBV using HBs antigen-binding antibodies. Any means for accomplishing this are contemplated within the scope of this disclosure. For example, even without explicit description, a description of a method of using an HBs antigen-binding antibody or an antigen-binding fragment thereof includes compositions comprising the antibody or antigen-binding fragment, uses of the antibody or antigen-binding fragment, and methods of the same. Embodiments reflecting other means, such as the same antibody or antigen-binding fragment for use, are also contemplated. Although the present disclosure is primarily described herein in terms of composition embodiments, a description of a composition containing a component for a use, a method for the same use of the component, and a description of the composition for the same use. Embodiments reflecting other means, such as the same use, are also contemplated.

(antibody performance)
The present invention provides HBs antigen-binding antibodies or antigen-binding fragments thereof capable of binding to HBs antigens. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof of the present invention includes genotype A (Aa/1 (Asian/African type), Ae/2 (European type), Ac/3 (Cameroon) ), genotype B (including Bj/1 (Japanese type), Ba/2 (Asian type), B3, B4, and B5), genotype C (Ce/1 (East Asian type), Cs/2 (South East type), C3, C4), genotype D (including D1, D2, D3, D4, D5), genotype E, genotype F (including F1, F2, F3, F4), genotype G, It can bind to HBs antigens of one or more of the HBV genotypes of genotype H and genotype J. In a preferred embodiment, the HBs antigen-binding antibodies or antigen-binding fragments thereof of the present invention are capable of binding HBs antigens of genotypes A, B, C and D HBV genotypes.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof of the present invention is less than 1000 nM, less than 300 nM, less than 300 nM, KD values less than 100 nM, less than 75 nM, less than 50 nM, less than 30 nM, or less than 20 nM, preferably less than 10 nM, less than 5 nM, less than 3 nM, or less than 2 nM, more preferably less than 1 nM, less than 0.3 nM, or less than 0.1 nM indicates Many antibody drugs show a KD value of less than 1 nM, but there is no specific reference value. Virus-neutralizing antibodies are commercially available with KD values of approximately 1-50 nM, and this level of affinity is considered sufficiently high. The HBs antigen-binding antibody or antigen-binding fragment thereof of the present invention can be useful as long as it can bind to any HBs antigen, and may or may not have selectivity and specificity for a particular HBs antigen. good. The HBs antigen-binding antibody or antigen-binding fragment thereof of the present invention may exhibit the above affinity as determined by surface plasmon resonance. (Biacore, etc.) and injected with different concentrations of purified HBs antigen-binding antibodies for 3 minutes, followed by washing with buffer for 5 minutes, the off-rate (kd) determined from the wash phase, and the on-rate (ka ) can be determined to calculate the affinity (KD).

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof of the present invention can also bind to HBs antigens having mutations. Escape mutants of HBs antigen that are resistant to treatment with HBIG are known, and the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein are also effective against such escape mutants. Therefore, it is expected that a wide range of HBV infections can be suppressed. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof can bind to HBs having the K141E, G145R or I152A mutation. In a preferred embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is capable of binding HBs with K141E or G145R mutations. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof can bind HBs having the K141E, G145R or I152A mutation with a KD value of less than 10 nM. In a preferred embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is capable of binding HBs with K141E or G145R mutations with a KD value of less than 10 nM.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof can recognize a conformational epitope of HBs antigen. Therefore, the binding properties to denatured HBs antigen and non-denatured HBs antigen are different, and it may be possible to distinguish between them. In one embodiment, the HBs antigen epitope recognized by the HBs antigen-binding antibody or antigen-binding fragment thereof may include isoleucine at position 152 (corresponding to position 326 of SEQ ID NO: 107).

(HBs antigen binding antibody)
In one aspect, the present disclosure provides specific HBs antigen-binding antibodies or antigen-binding fragments thereof. Clones HB351, HB1170, and HB1215 were prepared as monoclonal HBs antigen-binding antibodies as shown in Examples. The CDR sequences of these clones are as follows.

When an antibody or portion thereof (such as a variable region) is described herein as comprising a CDR or an amino acid sequence of a SEQ ID NO referring to a CDR, the antibody or portion thereof has that CDR amino acid sequence in the corresponding CDR region. and the extent of the CDR regions are defined. For example, when the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 91 other than DTYMY (positions 50-54 in SEQ ID NO: 91) is determined not to be the CDR1 region. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences, and a light chain CDR1, CDR2 and CDR3 amino acid sequence. A light chain having a light chain variable region amino acid sequence comprising the six CDR sequences specified in any of the following (1)-(3):
(1)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 1-5;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 6-10;
the heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 11-15;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 16-20;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 21-25;
the light chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 26-30;
(2)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 31-35;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 36-40;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 41-45;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 46-50;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS: 51-55;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOs: 56-60, or (3)
said heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 61-65;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 66-70;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 71-75;
said light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 76-80;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS:81-85;
Said light chain CDR3 amino acid sequence comprises any of SEQ ID NOs:86-90.

In one embodiment, the heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences in the HBs antigen-binding antibody or antigen-binding fragment thereof are, respectively,
(1)
SEQ ID NOs: 1, 6, 11, 16, 21 and 26 (Kabat numbering),
SEQ ID NOs: 2, 7, 12, 17, 22 and 27 (IMGT numbering),
SEQ ID NOs: 3, 8, 13, 18, 23 and 28 (Paratome numbering), or SEQ ID NOs: 4, 9, 14, 19, 24 and 29 (Chothia numbering),
(2)
SEQ ID NOs: 31, 36, 41, 46, 51 and 56 (Kabat numbering),
SEQ ID NOS: 32, 37, 42, 47, 52 and 57 (IMGT numbering),
SEQ ID NOs: 33, 38, 43, 48, 53 and 58 (Paratome numbering), or SEQ ID NOs: 34, 39, 44, 49, 54 and 59 (Chothia numbering)
(3)
SEQ ID NOs: 61, 66, 71, 76, 81 and 86 (Kabat numbering),
SEQ ID NOs: 62, 67, 72, 77, 82 and 87 (IMGT numbering),
SEQ ID NOs: 63, 68, 73, 78, 83 and 88 (Paratome numbering), or SEQ ID NOs: 64, 69, 74, 79, 84 and 89 (Chothia numbering)
is represented by a combination of

In one embodiment, the heavy chain CDR1-3 amino acid sequences in the HBs antigen-binding antibody or antigen-binding fragment thereof are, respectively,
(1)
SEQ ID NOs: 1, 6 and 11,
SEQ ID NOs: 2, 7 and 12,
SEQ ID NOs: 3, 8 and 13, or SEQ ID NOs: 4, 9 and 14,
(2)
SEQ ID NOS: 31, 36 and 41,
SEQ ID NOS: 32, 37 and 42,
SEQ ID NOs: 33, 38 and 43, or SEQ ID NOs: 34, 39 and 44,
(3)
SEQ ID NOs: 61, 66 and 71,
SEQ ID NOs: 62, 67 and 72,
SEQ ID NOs: 63, 68 and 73, or SEQ ID NOs: 64, 69 and 74
is represented by a combination of

In one embodiment, the light chain CDR1-3 amino acid sequences in the HBs antigen-binding antibody or antigen-binding fragment thereof are, respectively,
(1)
SEQ ID NOS: 16, 21 and 26,
SEQ ID NOS: 17, 22 and 27,
SEQ ID NOs: 18, 23 and 28, or SEQ ID NOs: 19, 24 and 29,
(2)
SEQ ID NOS: 46, 51 and 56,
SEQ ID NOS: 47, 52 and 57,
SEQ ID NOs: 48, 53 and 58, or SEQ ID NOs: 49, 54 and 59
(3)
SEQ ID NOS: 76, 81 and 86,
SEQ ID NOS: 77, 82 and 87,
SEQ ID NOs: 78, 83 and 88, or SEQ ID NOs: 79, 84 and 89
is represented by a combination of

In one embodiment, a total of 1, 2, 3, 4, 5 or 6 amino acid mutations may be included in the 6 CDR amino acid sequences identified above. In one embodiment, a total of 1 or 2 amino acid mutations may be included in the 6 CDR amino acid sequences identified above. In one embodiment, the amino acid mutations are SEQ ID NOs:5, 10, 15, 20, 25 and 30, SEQ ID NOs:35, 40, 45, 50, 55 and 60, or SEQ ID NOs:65, 70, 75, 80, 85 and at positions other than the 90 consensus CDR sequences. In one embodiment, the amino acid mutation is in the group of SEQ ID NOs: 1-4, the group of SEQ ID NOs: 6-9, the group of SEQ ID NOs: 11-14, the group of SEQ ID NOs: 16-19, the group of SEQ ID NOs: 21-24, Group of SEQ ID NOs: 26-29, Group of SEQ ID NOs: 31-34, Group of SEQ ID NOs: 36-39, Group of SEQ ID NOs: 41-44, Group of SEQ ID NOs: 46-49, Group of SEQ ID NOs: 51-54, SEQ ID NO: 56-59 group, SEQ ID NOS: 61-64 group, SEQ ID NOS: 66-69 group, SEQ ID NOS: 71-74 group, SEQ ID NOS: 76-79 group, SEQ ID NOS: 81-84 group or SEQ ID NO: 86- Occurs at an amino acid that appears only in the sequence of any one SEQ ID NO in each group of 89 groups.

In one embodiment, amino acid mutations in the CDR amino acid sequences are independently amino acid substitutions, amino acid deletions or amino acid insertions. In one embodiment, the amino acid mutations in the CDR amino acid sequences are independently conservative amino acid substitutions. In one embodiment, a conservative amino acid substitution is the replacement of an amino acid in the following group of amino acids with another amino acid within the same group.
Group 1: glycine, alanine, valine, leucine and isoleucine Group 2: serine and threonine Group 3: phenylalanine, tyrosine, tryptophan and tryptophan Group 4: lysine, hydroxylysine, arginine and histidine Group 5: cysteine, methionine , methionine sulfoxide and homocysteine Group 6: proline and hydroxyproline Group 7: glutamic acid and aspartic acid Group 8: glutamine and asparagine

The sequences of the heavy and light chain variable regions of clones HB351, HB1170 and HB1215 obtained in Examples and their humanized sequences are as follows.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises
(1) SEQ ID NO: 91 or 97 or a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity thereto; (2) SEQ ID NO: 93 or 99 or same; a heavy chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 95 or at least 80%, 85%, 90% thereof , 95%, 98%, or 99% identical amino acid sequences.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises
(1) a light chain variable region having SEQ ID NO: 92 or 98 or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical thereto;
(2) a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 94 or 100, or (3) SEQ ID NO: 96 or A light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to .

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises
(1) a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 91 or 97, or SEQ ID NO: 92 or 98, or a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity;
(2) a heavy chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 93 or 99, or SEQ ID NO: 94 or 100, or a light chain variable region having at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity; or (3) SEQ ID NO: 95 or at least 80%, 85%, 90% thereof; a heavy chain variable region having 95%, 98% or 99% amino acid sequence identity and SEQ ID NO: 96 or an amino acid sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto A light chain variable region having a

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is a humanized antibody or chimeric antibody or antigen-binding fragment thereof. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is a monoclonal antibody, polyclonal antibody, multispecific antibody (eg, bispecific antibody) or antigen-binding fragment thereof. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is isolated.

In one embodiment, the HBs antigen binding antibody is a full length antibody. In one embodiment, the antigen-binding fragment of the HBs antigen-binding antibody is Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies or single chain variable region fragments (scFv). In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is a monoclonal antibody, polyclonal antibody, multispecific antibody (eg, bispecific antibody) or antigen-binding fragment thereof. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof is isolated.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises one or more of a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and a CH4 domain. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises a CL domain. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof comprises an Fc region.

In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof may have a structure classified as either IgA, IgD, IgE, IgG or IgM. In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof may have a structure classified as either IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. The HBs antigen-binding antibodies or antigen-binding fragments thereof described herein are contemplated to include any known modifications to antibodies, such as PEGylation.

In one aspect, the present disclosure provides nucleic acids encoding HBs antigen-binding antibodies or antigen-binding fragments thereof. Nucleic acids can encode any HBs antigen-binding antibody or antigen-binding fragment thereof described herein. The HBs antigen-binding antibody or antigen-binding fragment thereof may be encoded by a single nucleic acid molecule, or may be encoded by a plurality of divided nucleic acid molecules.

In Examples, the nucleotide sequences of the variable regions of clones HB351, HB1170 and HB1215 were determined as follows.

In one embodiment, the nucleic acid is
(1) SEQ ID NO: 101 or a base sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and/or SEQ ID NO: 102 or at least 80%, 85%, 90% thereof; %, 95%, 98%, or 99% identical base sequences,
(2) SEQ ID NO: 103 or a base sequence at least 80%, 85%, 90%, 95%, 98% or 99% identical thereto, and/or SEQ ID NO: 104 or at least 80%, 85%, 90% thereof; %, 95%, 98%, or 99% sequence identity, or (3) SEQ ID NO: 105 or a sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto; and/or SEQ ID NO: 106 or a base sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical thereto.

A person skilled in the art can use any system to produce the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein.

The produced HBs antigen-binding antibody or antigen-binding fragment thereof can be purified or isolated by any purification method. For example, immunoglobulin purification such as fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, protein A/G affinity chromatography, etc. law. In particular, when the monoclonal antibody is mouse IgG, it can be efficiently purified by affinity chromatography using a protein A binding carrier or an anti-mouse immunoglobulin binding carrier.

(Application/use)
In one aspect, the disclosure provides treatment or prevention of hepatitis B virus infection or detection of hepatitis B virus using the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein. In one embodiment, the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein can be used to treat or prevent hepatitis B virus infection. In a preferred embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof described herein can reduce hepatitis B virus infectivity as a neutralizing antibody against hepatitis B virus. It can be used in subjects already infected with hepatitis B virus, or it can be used to prevent new infections with hepatitis B virus. It can also be used to treat or prevent hepatitis D virus infection associated with hepatitis B virus infection. In one embodiment, the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein are used to treat organs for transplantation (eg, liver). In one embodiment, the HBs antigen-binding antibody or antigen-binding fragment thereof described herein is administered to a subject infected with hepatitis B virus or at risk for hepatitis B virus infection.

Since the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein can bind to HBs antigens having different escape mutations, different types of HBs antigen-binding antibodies or antigen-binding fragments thereof can be used simultaneously to achieve a wide range of escape. It may be advantageous to suppress mutant hepatitis B virus.

In one embodiment, the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein can be used to detect hepatitis B virus. Since the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein can exhibit different binding properties to mutated HBs antigens, they can also be used to determine hepatitis B virus mutations based on this. .

(Composition)
In one aspect, the disclosure provides compositions comprising the HBs antigen-binding antibodies or antigen-binding fragments thereof described herein. In one embodiment, compositions are provided for treating or preventing hepatitis B virus infection. In one embodiment, compositions are provided for detecting hepatitis B virus.

(dosage form, etc.)
Compositions described herein may be provided in various forms. The form of the composition may be, for example, injections, capsules, tablets, granules and the like. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. Aqueous solutions for injection may also contain physiological saline, sugar (eg, trehalose), NaCl, NaOH, or the like.

In one embodiment, compositions of the disclosure comprise a pharmaceutically acceptable carrier or excipient. Such carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals. Oil, sesame oil, etc. are included. Water is a preferred carrier when the drug is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable excipients include light silicic anhydride, microcrystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride. Sodium, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers include E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). In addition to these, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity Accelerators, flavoring agents and the like may also be included. In one embodiment, the pH of any liquid composition of the present disclosure is about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, It can be about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11 or ranges between any two of these values.

Any component of the compositions of the present disclosure can be provided as pharmaceutically acceptable salts, formed with free carboxyl groups, e.g., derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, and the like. salts formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and sodium, potassium, ammonium, calcium, and ferric hydroxides. It can be a salt formed with

In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for injectable administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form, for example a lyophilized powder or water-free concentrate, in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. can be supplied as goods. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

When applying the composition of the present disclosure to a subject, the subject is not particularly limited, and mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, monkeys, humans, etc.), It can be birds, reptiles, amphibians, arthropods, fish, and the like.

Amounts of the compositions and/or active ingredients of the present disclosure may vary with the nature of the disorder or condition to be treated or prevented, and can be determined by one of ordinary skill in the art using standard clinical techniques based on the description herein. In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulations can also vary with route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the attending physician and each patient's circumstances. The dosage of the composition and/or active ingredient of the present disclosure is not particularly limited, but for example, 0.00001, 0.0001, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg per dose It may be body weight or within any two of those values. The administration interval is not particularly limited. good too. The dose, administration interval and administration method may be appropriately selected depending on the patient's age, body weight, symptoms, target organ and the like.

The routes of administration of the compositions described herein are preferably those effective in treating or preventing viral infections, such as intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, Epidural or oral administration may be used. In one embodiment, the compositions of this disclosure can be used together with various delivery systems. Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules; use of receptor-mediated endocytosis; construction of therapeutic nucleic acids as part of retroviral vectors or other vectors; be. It is possible, and necessary, to administer the medicament by any suitable route, such as by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral, rectal and intestinal mucosa). An inhaler or nebulizer may be used, with an aerosolizing agent as appropriate, and may be co-administered with other agents. Administration can be systemic or local.

Compositions of the disclosure can be provided as kits. In one embodiment, the disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients that can be added to the compositions of the disclosure. Manufacture, use or sale for human administration by a governmental agency, optionally associated with such container, as prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products. It is also possible to indicate information indicating the authorization of

Procedures for formulating the compositions of the present disclosure as pharmaceuticals and the like are known in the art and described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, other countries' Pharmacopoeias, and the like. Accordingly, those of ordinary skill in the art, given the description herein, will be able to determine embodiments such as amounts to be used without undue experimentation.

In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "or". When we say "within a range" of "two values" herein, the range includes the two values themselves.

All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

The present disclosure has been described above showing preferred embodiments for ease of understanding. While the present disclosure will now be described based on the examples, the foregoing description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but only by the claims.

The reagents specifically used the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical, Nacalai, R&D Systems, USCN Life Science INC, etc.) can be substituted.

(Example 1: Production of HBs antigen-binding antibody)
HBs antigen-binding antibodies were obtained by immunizing mice with HBs antigen and preparing hybridomas using the mouse cells.

A purified recombinant protein adr-type HBs antigen (produced by PROSPEC, SEQ ID NO: 108) produced in CHO cells was administered to mice for initial immunization, followed by booster immunization. Thereafter, splenocytes were isolated and fused with mouse myeloma cells by PEG method to produce hybridoma cells. Hybridoma cells were cultured on the plate, and when colonies appeared, hybridomas producing monoclonal antibodies were screened.

Specifically, a solution of adr-type HBs antigen was dispensed into each well of the plate and incubated to immobilize the adr-type HBs antigen on the plate, followed by washing and blocking. A hybridoma culture supernatant was added to each well, incubated, and then washed. Thereafter, a horseradish peroxidase-labeled anti-mouse Ig polyclonal antibody was added to each well, allowed to react, washed, and positive wells were detected using an OPD (o-phenylenediamine) substrate solution. Of the hybridoma cells producing monoclonal antibodies in positive wells, hybridoma cells highly reactive to HBs antigen were cloned by limiting dilution to obtain single clones.

Mouse immunoglobulin subclasses of monoclonal antibodies produced by hybridoma cells obtained as single clones by cloning were determined. To identify the mouse immunoglobulin subclass, the hybridoma cell culture supernatant was used, and polyclonal antibodies for isotyping (anti-mouse IgG1 antibody, anti-mouse IgG2a antibody, anti-mouse IgG2b antibody, anti-mouse IgG3 antibody, anti-mouse κ antibody and Anti-mouse λ antibody: Bethyl Laboratory) was used for identification by a conventional ELISA method.

To see if the antibodies obtained are effective against a broad spectrum of HBV. The binding of each antibody to HBV genotypes A, B, C and D HBs antigens was tested. As a result, some antibodies showed binding only to specific genotypes of HBs antigens, but clones HB351, HB1170, HB1215, etc. showed binding to all genotypes of HBs antigens. It was tested whether each antibody could be used to detect the denatured HBs antigen of genotype C developed by gel electrophoresis by Western blotting, but none of the antibodies could be detected. From this, it is considered that the obtained antibody recognizes the stereoscopic epitope of the HBs antigen.

(Example 2: Ability of monoclonal antibody to suppress HBV)
Each monoclonal antibody obtained was tested for its ability to inhibit HBV.

HepG2-NTCP C4 cells were incubated for 24 hours in medium containing HBV and 25 μg/mL monoclonal antibody or 2.5 μM PreS1 peptide (control). After that, the medium was changed and incubated for 10 days. The amount of HBV RNA contained in these cultures was assessed by real-time qPCR.

The results are shown in FIG. It was confirmed that the obtained HBs antigen-binding antibodies such as clone HB351 contain antibodies capable of suppressing HBV.

Similar experiments were performed with different amounts of monoclonal antibody added to quantify the amount of HBV RNA contained in the cultures. For comparison, a commercially available HBIG was also tested. An HBV inhibition curve was generated by plotting the results at each concentration and the IC50 was calculated.

The results are shown in FIG. Clone 351 was found to be able to inhibit HBV at ng/mL levels of concentration. These clones are considered to be able to suppress HBV more efficiently than HBIG.

(Example 3: HBs antigen-binding ability of monoclonal antibody)
Clones HB351, HB1170, HB1215, etc. were evaluated for affinity with HBs antigen using the Octet system, and the KD value was measured.

Specifically, HBs antigen (Prosec Tany Technology Ltd. HBS 875) was biotinylated using Ez-Link (trademark) NHS-PEG4-biotin (ThermoFisher Scientific), and this was analyzed by Octet, an intermolecular interaction analyzer. Immobilization was performed using RED96e (Sartorius) at a concentration of 10 μg/mL over a streptavidin (SA) biosensor (Sartorius). After that, reacting with antibodies such as clones HB351, HB1170, HB1215 diluted 3-fold from 10 μM, K _on (association rate constant) and K _dis (dissociation rate constant) were calculated, and the equilibrium dissociation constant (KD) was calculated as K _dis /K _on .

The results are shown in Figure 3 and the table below. All monoclonal antibodies were confirmed to bind to biotinylated HBs antigen with high affinity. The calculated KD values were approximately 300 pM (clone HB351, HB1215) and approximately 3 nM (clone HB1170), respectively.

(Example 4: Suppression of viruses with HBV envelope proteins of each genotype)
We tested whether monoclonal antibodies could suppress infection with different genotypes of HBV. An overview is shown in FIG.

A system was constructed to generate hepatitis D virus (HDV) particles with the desired HBV-derived envelope. HDV is known as a satellite virus of HBV, the surface of the virus particle is covered by the envelope components of HBV, and the virus particle has delta antigen and gene RNA inside. Since HDV, like HBV, enters host cells via NTCP, this model is considered to be able to evaluate the steps of HBV infection. HDV particles covered by the HBV envelope of each genotype were obtained by transfecting Huh7 cells with plasmids encoding large HBs antigens of genotypes A, B, C or D and plasmids encoding the HDV genome. bottom. These HDV particles were then added to HepG2-18C cells simultaneously with HB351 or HB1170 monoclonal antibody (25 μg/mL), incubated for 10 days, and the amount of intracellular HDV RNA was measured.

The results are shown in FIG. Both HB351 and HB1170 were found to be able to suppress the infection of HDV with HBV envelopes of genotypes A, B, C and D.

(Example 5: Monoclonal antibody treatment in HBV producing mouse model)
Next, we tested the effects of HB351 and HB1170 in a mouse model of HBV production.

A summary of the experiment is shown at the top of FIG. Mice were dosed with HBV and HB351 or HB1170 and bled daily. HBs antigen and HBV DNA in the obtained serum were quantified.

The results are shown at the bottom of FIG. Also in mice, HB351 and HB1170 were found to be able to suppress HBV.

(Example 6: Recognition site of monoclonal antibody)
It was examined which site of the HBs antigen the monoclonal antibody recognizes.

Amino acids at positions 147 to 152 of the HBs antigen (corresponding to positions 321 to 326 of SEQ ID NO: 107 and positions 147 to 152 of SEQ ID NO: 108) were each replaced with alanine to examine whether the binding of the antibody was maintained. . The results are shown in FIG. Neither HB351 nor HB1170 was observed to bind to I152A mutated HBs. Isoleucine at position 152 was suggested to be an important amino acid residue in HBs recognition of HB351 and HB1170. Isoleucine at position 152 is conserved in almost all HBVs, and mutations at position 152 have been found to inhibit HBV particle formation. Therefore, HB351 and HB1170 are expected to be able to comprehensively suppress HBV. It has been observed that the HB1215 clone maintains binding properties even to HBs into which the I152A mutation has been introduced.

(Example 7: Neutralizing activity against HBIG escape mutants)
Escape mutants of HBs antigen that are resistant to treatment with HBIG are known. We examined whether monoclonal antibodies against these escape mutants are effective.

Using the same system using HDV as in Example 4, hepatitis D virus (HDV) particles with an HBV envelope expressing HBs antigen containing escape mutations (K141E, G145R) were produced. After that, these HDV particles were added to host cells simultaneously with monoclonal antibodies such as HB351, HB1170 and HB1215 in the same manner as in Example 4, and after incubation, the amount of intracellular HDV RNA was measured.

Results for escape mutation G145R are shown in FIG. It was found that HB351 can also successfully suppress HDV infection with an HBV envelope of HBs antigen containing the escape mutation G145R. For the escape mutation K141E, only HB1215 among the clones tested showed good inhibition of infection. The clones produced are also effective against various escape mutants of HBs antigens, and are therefore expected to be able to suppress a wide range of HBV infections.

(Example 8: Humanization of monoclonal antibody)
Humanized antibodies were generated based on clones HB351 and HB1170.

First, the coding nucleotide and amino acid sequences of these clones were determined. Each determined sequence is shown below.

CDR sequences were determined according to the Kabat, IMGT, Paratome and Chothia numbering system. Sequences determined as CDRs (cover sequences) in at least one of these four numbering systems were extracted, and humanized antibodies were produced while maintaining the cover sequences.

In the humanized antibody based on clone HB351, the humanization rate of the heavy chain variable region was 90.8% and the humanization rate of the light chain variable region was 85.5%. In the humanized antibody based on clone HB1170, the humanization rate of the heavy chain variable region was 90.2% and the humanization rate of the light chain variable region was 85.5%.

Based on the amino acid sequences of these humanized heavy chain variable regions and light chain variable regions, humanized antibodies were generated. Codons were optimized for CHO cells using GeneArt GeneOptimizer, an artificial gene was synthesized using Eurofin, and this artificial gene was seamlessly inserted into an antibody expression vector encoding a constant region to prepare a nucleic acid construct. A humanized antibody was produced using this nucleic acid construct, and it was confirmed that the desired humanized antibody was obtained.

(Example 9: Performance evaluation of humanized monoclonal antibody)
Humanized monoclonal antibodies HB351 and HB1170 were evaluated for affinity with HBs antigen and HBV inhibitory ability. The HBV inhibitory ability was evaluated in the same manner as in Example 2, and the affinity with HBs antigen was evaluated in the same manner as in Example 3.

The results are shown in Figure 9 and the table below. It can be said that all humanized antibodies have high affinity for HBs antigen, although their affinity for HBs antigen is slightly decreased by humanization. The calculated Kd values were approximately 300 pM for humanized HB351 and approximately 3 nM for humanized HB1170. Also, both humanized HB351 and humanized HB1170 were found to be able to suppress HBV at concentrations at the ng/mL level.

(Note)
While the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present disclosure is to be construed in scope only by the claims. It is understood that the patents, patent applications and publications cited herein are hereby incorporated by reference for their contents as if the contents themselves were specifically set forth herein. understood.

The present disclosure provides novel HBs antigen-binding antibodies that are useful in inhibiting hepatitis B virus and can replace existing HBIG.

Claims (13)

HBs antigen binding comprising a heavy chain having a heavy chain variable region amino acid sequence comprising heavy chain CDR1, CDR2 and CDR3 amino acid sequences and a light chain having a light chain variable region amino acid sequence comprising light chain CDR1, CDR2 and CDR3 amino acid sequences An antibody or antigen-binding fragment thereof,
(1)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 1-5;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOS: 6-10;
the heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOs: 11-15;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 16-20;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 21-25;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 26-30, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences, or (2)
the heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 31-35;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 36-40;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 41-45;
the light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 46-50;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS: 51-55;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 56-60, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences;
(3)
said heavy chain CDR1 amino acid sequence comprises any of SEQ ID NOs: 61-65;
the heavy chain CDR2 amino acid sequence comprises any of SEQ ID NOs: 66-70;
said heavy chain CDR3 amino acid sequence comprises any of SEQ ID NOS: 71-75;
said light chain CDR1 amino acid sequence comprises any of SEQ ID NOS: 76-80;
the light chain CDR2 amino acid sequence comprises any of SEQ ID NOS:81-85;
said light chain CDR3 amino acid sequence comprises any of SEQ ID NOs: 86-90, or said heavy and light chains have amino acid sequences comprising a total of 1-6 amino acid mutations in said 6 CDR amino acid sequences;
HBs antigen-binding antibody or antigen-binding fragment thereof. The heavy chain CDR1-3 and light chain CDR1-3 amino acid sequences, respectively,
SEQ ID NOs: 1, 6, 11, 16, 21 and 26,
SEQ ID NOs: 2, 7, 12, 17, 22 and 27,
SEQ ID NOs: 3, 8, 13, 18, 23 and 28,
SEQ ID NOS: 4, 9, 14, 19, 24 and 29,
SEQ ID NOs: 31, 36, 41, 46, 51 and 56,
SEQ ID NOS: 32, 37, 42, 47, 52 and 57,
SEQ ID NOs: 33, 38, 43, 48, 53 and 58,
SEQ ID NOS: 34, 39, 44, 49, 54 and 59
SEQ ID NOs: 61, 66, 71, 76, 81 and 86,
SEQ ID NOs: 62, 67, 72, 77, 82 and 87,
SEQ ID NOs: 63, 68, 73, 78, 83 and 88, or SEQ ID NOs: 64, 69, 74, 79, 84 and 89
or contains a total of 1-6 amino acid mutations in the six CDR amino acid sequences;
The HBs antigen-binding antibody or antigen-binding fragment thereof according to claim 1. 3. The HBs antigen-binding antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said amino acid mutations are independently amino acid substitutions, amino acid deletions or amino acid insertions. 3. The HBs antigen-binding antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said amino acid mutations are independently conservative amino acid substitutions. (1) a heavy chain variable region having an amino acid sequence of SEQ ID NO:91 or at least 80% identical thereto, and a light chain variable region having an amino acid sequence of SEQ ID NO:92 or at least 80% identical thereto;
(2) a heavy chain variable region having an amino acid sequence of SEQ ID NO:93 or at least 80% identical thereto, and a light chain variable region having an amino acid sequence of SEQ ID NO:94 or at least 80% identical thereto, or (3) a SEQ ID NO: 5. Any one of claims 1 to 4, having a heavy chain variable region having an amino acid sequence of 95 or at least 80% identical thereto, and a light chain variable region having an amino acid sequence of SEQ ID NO:96 or at least 80% identical thereto The HBs antigen-binding antibody or the antigen-binding fragment thereof according to the item. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to at least one of K141E mutant HBs, G145R mutant HBs and I152A mutant HBs. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to at least one of hepatitis B virus genotype A, B, C or D HBs. 8. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which has a KD value of less than 10 nM when the affinity with HBs is measured by biolayer interferometry. The HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is humanized. A composition for treating or preventing hepatitis B virus infection, comprising the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. 11. The composition of claim 10 for treating organs for transplantation. A detection agent for hepatitis B virus, comprising the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. A nucleic acid encoding the HBs antigen-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

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