Anti-hepatitis B virus antibody and application thereof

Abstract

The present invention relates to an antibody against hepatitis B surface antigen (HBsAg), a nucleic acid molecule encoding the antibody, and a pharmaceutical composition comprising the antibody. The anti-HBsAg antibody disclosed by the invention can be combined with HBsAg in a pH-dependent manner, and has enhanced capability of combining with FcRn and Fc gamma R, remarkably enhanced antigen removal effect and prolonged half-life period.

Description

Technical Field

[0001] The present invention relates to antibodies against hepatitis B surface antigen (HBsAg), nucleic acid molecules encoding said antibodies, and pharmaceutical compositions comprising said antibodies. The anti-HBsAg antibodies of the present invention can bind to HBsAg in a pH-dependent manner, and have enhanced abilities to bind to FcRn and FcγR, significantly enhanced antigen clearance effects and extended half-lives. Background Art

[0002] Hepatitis B virus infection, especially chronic HBV infection, is one of the most important public health problems globally. Chronic HBV infection can lead to a series of liver diseases such as chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Currently, the therapeutic drugs for chronic HBV infection can be mainly divided into interferons (IFNs) and nucleoside / nucleotide analogs. For HBV-infected individuals (such as CHB patients), the above drugs, either alone or in combination, can effectively inhibit virus replication in vivo and significantly reduce the HBV DNA level. However, neither the single nor the combined treatment with the above drugs can completely clear the HBV virus in infected individuals, and the response rate of HBsAg seroconversion or HBsAg serological conversion (a sign of complete clearance of HBV virus in infected individuals) is usually less than 5%.

[0003] Developing new drugs for the treatment of chronic HBV infection based on immunological means is one of the important research directions in this field. Currently, polyclonal Anti-HBs antibodies purified from the sera / plasmas of prophylactic hepatitis B vaccine responders or HBV-infected recoverers, namely high-titer hepatitis B immunoglobulin (HBIG), have been widely used to block mother-to-child vertical transmission of HBV, prevent HBV reinfection after liver transplantation in chronic HBV-infected individuals, and prevent infection in individuals accidentally exposed to HBV. However, directly using HBIG for the treatment of HBV-infected individuals (such as CHB patients) has no obvious curative effect, and it has many limitations such as a relatively small source of high-titer plasma, high price, unstable properties, and potential safety problems.

[0004] Therefore, it is urgent and necessary to develop innovative treatment methods and drugs that can more effectively clear the HBV virus, especially HBsAg, for HBV-infected individuals. Summary of the Invention

[0005] The inventors previously developed an anti-HBsAg antibody with pH-dependent antigen-binding ability, which enables the recycling of the antibody. On this basis, the inventors unexpectedly found that by introducing mutations that enhance the binding to FcRn and FcγR simultaneously in the Fc region, the clearance efficiency can be significantly improved and the half-life can be extended, thus obtaining a trifunctional hepatitis B therapeutic antibody with pH-dependent antigen binding / enhanced Fc-hFcγR interaction / enhanced Fc-hFcRn interaction. Accordingly, the following aspects are provided.

[0006] Antibodies of the present invention

[0007] In one aspect, the present invention provides an antibody capable of specifically binding to HBsAg, which comprises an antigen-binding domain that binds to HBsAg in a pH-dependent manner and an Fc domain, wherein the Fc domain comprises a first mutation that enhances the binding to FcRn (such as hFcRn) and a second mutation that enhances the binding to FcγR (such as hFcγR). <x

[0008] pH-dependent antigen binding

[0009] As used herein, the expression "binding in a pH-dependent manner" or an equivalent expression "pH-dependent binding" means binding with a higher affinity at neutral pH than at acidic pH, that is, the KD value or EC50 value of binding to HBsAg at acidic pH is higher than its KD value or EC50 value of binding to HBsAg at neutral pH.

[0010] In certain embodiments, the antibody binds to HBsAg with a higher affinity at neutral pH than at acidic pH. In certain embodiments, the neutral pH is pH 6.7 - pH 7.5, such as pH 7.4. In certain embodiments, the acidic pH is pH 4.0 - pH 6.5, such as pH 6.0.

[0011] In certain embodiments, the K of the antibody binding to HBsAg at acidic pH (such as pH 6.0) D compared with its K of binding to HBsAg at neutral pH (such as pH 7.4) D ratio (i.e., the value of K D (acidic pH) / K D (neutral pH)) is greater than 1. The K D can be measured by techniques well-known in the art, such as by SPR technology (such as Biacore).

[0012] In certain embodiments, the K of the antibody binding to HBsAg at pH 6.0 D compared with its K of binding to HBsAg at pH 7.4 DThe ratio is greater than 1, for example, not less than 1.5, or not less than 2. In certain embodiments, the KD value of the antibody of the present invention at neutral pH can be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less. In certain embodiments, the KD value of the antibody of the present invention at acidic pH can be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or more.

[0013] In certain embodiments, the ratio of the EC50 of the antibody binding to HBsAg at acidic pH (e.g., pH 6.0) to the EC50 of the antibody binding to HBsAg at neutral pH (e.g., pH 7.4) (i.e., the value of EC50(acidic pH) / EC50(neutral pH)) is greater than 1. In certain embodiments, the EC50 is measured by ELISA method, for example, calculated by the regression analysis of the dose-response curve generated by ELISA method.

[0014] In certain embodiments, the ratio of the EC50 of the antibody binding to HBsAg at pH 6.0 to the EC50 of the antibody binding to HBsAg at pH 7.4 is greater than 1, for example, not less than 1.5, or not less than 2.

[0015] In certain embodiments, the antibody contains at least one residue replaced by histidine in its CDR region.

[0016] In certain embodiments, the antigen-binding domain that binds to HBsAg in a pH-dependent manner is derived from the pH-dependent anti-HBsAg antibodies C26, C27, C32, D3, D4 or D5 described in Chinese Patent Application CN111978392A. In certain embodiments, the antigen-binding domain that binds to HBsAg in a pH-dependent manner is derived from the pH-dependent anti-HBsAg antibody D3 described in Chinese Patent Application CN111978392A.

[0017] In certain embodiments, the antigen-binding domain that binds to HBsAg in a pH-dependent manner comprises: heavy chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 3, 4 and 5, and light chain CDR1, CDR2 and CDR3 respectively comprising SEQ ID NOs: 5, 6 and 7.

[0018] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:1 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:2 or a sequence having at least 80% identity thereto. In certain embodiments, the antigen-binding domain comprises a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:1, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:2.

[0019] Enhanced binding to FcγR

[0020] The Fc domain of the antibody of the present invention comprises a first mutation that enhances binding to FcγR (such as hFcγR). The enhancement is compared to a reference Fc domain sequence that does not comprise the first mutation, such as a native Fc domain sequence.

[0021] Crosslinking of the Fc domain of a native IgG antibody to a receptor (FcγR) triggers a variety of effector functions, including phagocytosis, antibody-dependent cell cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In humans, three classes of FcγR have been characterized to date: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed (especially on leukocytes), is considered a core participant in antibody-mediated immunity, and can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, which play different functions in the immune system but bind IgG-Fc with similar low affinity, and the extracellular domains of these receptors are highly homologous; and (iii) FcγRIII (CD16), which binds IgG with medium to low affinity, and two forms of which have been found: FcγRIIIA, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is considered to mediate ADCC; and FcγRIIIB, which is highly expressed on neutrophils.

[0022] In certain embodiments, the enhancement of the binding is determined by a K D value measurement, such as by SPR technology (such as Biacore).

[0023] In certain embodiments, the first mutation enhances binding to hFcγRIIIb at least.

[0024] In certain embodiments, the first mutation also enhances binding to hFcγRI, hFcγRIIa, and / or hFcγRIIIa.

[0025] In certain embodiments, the first mutation enhances antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the ADCP is mediated by neutrophils and / or DC cells.

[0026] In certain embodiments, the first mutation is selected from the following mutations according to EU numbering: G236A / S239D / A330L / I332E (ADLE), S239D / A330L / I332E (DLE), G236A / S239D / I332E (ADE), S239D / I332E (DE).

[0027] In certain embodiments, the first mutation is the following mutation according to EU numbering: S239D / A330L / I332E (DLE).

[0028] Enhanced binding to FcRn

[0029] The Fc domain of the antibody of the present invention contains a second mutation that enhances binding to FcRn (such as hFcRn). The enhancement is compared to a reference Fc domain sequence that does not contain the second mutation, such as a native Fc domain sequence.

[0030] In certain embodiments, the second mutation enhances binding to hFcRn at acidic pH (such as pH 6.0).

[0031] In certain embodiments, the enhancement of the binding is determined by the K D value measurement, for example, measured by SPR technology (such as Biacore). In certain embodiments, the enhancement of the binding is determined by the method in Example 2.3.2.

[0032] In certain embodiments, the second mutation is selected from the following mutations according to EU numbering: T256D / H285D / T307R / Q311V / A378V (D3-DLEddrvv), M428L / N434S (D3-DLEls), T307Q / N434S (D3-DLEqs), V259I / N315D / N434Y (D3-DLEidy).

[0033] In certain embodiments, the second mutation is the following mutation according to EU numbering: T307Q / N434S.

[0034] In certain embodiments, the first mutation is S239D / A330L / I332E and the second mutation is T307Q / N434S.

[0035] In certain embodiments, the Fc domain described in any of the above embodiments is IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0036] In certain embodiments, the Fc domain described in any of the above embodiments is human IgG, such as human IgG1, human IgG2, human IgG3, or human IgG4. In certain embodiments, the Fc domain is human IgG1.

[0037] Full-length antibody

[0038] In certain embodiments, the antibody of the present invention is a full-length antibody.

[0039] In certain embodiments, the full-length antibody comprises a heavy chain and a light chain.

[0040] In certain embodiments, the heavy chain comprises a heavy chain constant region (CH) shown in any one of SEQ ID NOs: 9-12. In certain embodiments, the heavy chain comprises the heavy chain constant region (CH) shown in SEQ ID NO: 10.

[0041] In certain embodiments, the heavy chain comprises a heavy chain constant region (CH) shown in any one of SEQ ID NOs: 14-17. In certain embodiments, the heavy chain comprises the heavy chain constant region (CH) shown in SEQ ID NO: 16.

[0042] In certain embodiments, the light chain comprises the light chain constant region (CL) shown in SEQ ID NO: 13.

[0043] In certain embodiments, the full-length antibody comprises: a heavy chain comprising VH shown in SEQ ID NO: 1 and CH shown in any one of SEQ ID NOs: 9-12, and a light chain comprising VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 13.

[0044] In certain embodiments, the full-length antibody comprises: a heavy chain comprising VH shown in SEQ ID NO: 1 and CH shown in SEQ ID NO: 10, and a light chain comprising VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 13.

[0045] In certain embodiments, the full-length antibody comprises: a heavy chain including VH shown in SEQ ID NO: 1 and CH shown in any one of SEQ ID NOs: 14-17, and a light chain including VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 13.

[0046] In certain embodiments, the full-length antibody comprises: a heavy chain including VH shown in SEQ ID NO: 1 and CH shown in SEQ ID NO: 16, and a light chain including VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 13.

[0047] scFv-Fc

[0048] In certain embodiments, the antibody of the present invention is scFv-Fc. ScFv-Fc refers to an scFv linked to an Fc domain.

[0049] In certain embodiments, the antibody comprises an scFv and an Fc domain from the N-terminus to the C-terminus.

[0050] In certain embodiments, the antibody comprises an Fc domain and an scFv from the N-terminus to the C-terminus.

[0051] In certain embodiments, the antigen-binding domain and the Fc domain are optionally linked by a peptide linker.

[0052] In certain embodiments, the Fc domain comprises at least CH2 and CH3. In certain embodiments, the Fc domain comprises a hinge region, CH2, and CH3.

[0053] In certain embodiments, the Fc domain comprises the Fc region sequence in the heavy chain constant region (CH) shown in any one of SEQ ID NOs: 9-12.

[0054] In certain embodiments, the Fc domain comprises the Fc region sequence in the heavy chain constant region (CH) shown in SEQ ID NO: 10.

[0055] In certain embodiments, the Fc domain comprises the Fc region sequence in the heavy chain constant region (CH) shown in any one of SEQ ID NOs: 14-17.

[0056] In certain embodiments, the Fc domain comprises the Fc region sequence in the heavy chain constant region (CH) shown in SEQ ID NO: 16.

[0057] In some embodiments, the scFv has a structure represented by VH-[L]-VL or VL-[L]-VH, where [L] is a peptide linker. In some embodiments, the peptide linker is selected from peptide linkers comprising one or more glycines (G) and / or serines (S). In some embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4.

[0058] In some embodiments, the scFv-Fc exists as a monomer. In some embodiments, the scFv-Fc exists as a dimer.

[0059] Antibody derivative

[0060] The antibodies of the present invention can be derivatized, for example, by being linked to another molecule (such as another polypeptide or protein). Generally, the derivatization of the antibody (such as, labeling) does not adversely affect its binding to HBsAg. Thus, the antibodies of the present invention are also intended to include such derivatized forms. For example, the antibodies of the present invention can be functionally linked (by chemical conjugation, gene fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (such as, to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody to another molecule (such as, avidin or a polyhistidine tag).

[0061] In another aspect, the present invention provides a multispecific antibody comprising the antibody of the present invention.

[0062] In some embodiments, the multispecific antibody specifically binds HBsAg and additionally specifically binds to other targets. In some embodiments, the multispecific antibody further comprises at least one second antigen-binding domain having a second binding specificity for a second target.

[0063] In some embodiments, the multispecific antibody comprises an antigen-binding domain that binds HBsAg in a pH-dependent manner as described herein, at least one second antigen-binding domain having a second binding specificity for a second target, and an Fc domain comprising a first mutation that enhances binding to FcRn (such as hFcRn) and a second mutation that enhances binding to FcγR (such as hFcγR).

[0064] In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody.

[0065] In another aspect, the present invention provides a conjugate comprising the antibody or multispecific antibody of the present invention and a conjugate moiety.

[0066] In certain embodiments, the conjugate moiety is selected from therapeutic agents. In certain embodiments, the conjugate is an antibody-drug conjugate (ADC). In certain embodiments, the therapeutic agent is an immunomodulator. In certain embodiments, the immunomodulator is a STING agonist. In certain embodiments, the immunomodulator is a TLR agonist, such as a TLR7 / 8 agonist.

[0067] In certain embodiments, the conjugate moiety is selected from detectable labels. A detectable label can be any substance detectable by means such as fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, or chemistry. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes, chemiluminescent substances (such as acridinium ester compounds), or biotin.

[0068] In certain embodiments, the conjugate moiety is linked to the antibody or multispecific antibody of the present invention via a linker.

[0069] Preparation of antibodies

[0070] The antibody or multispecific antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chains of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions to express the antibody of the present invention.

[0071] In another aspect, the present invention provides an isolated nucleic acid molecule comprising: (i) a nucleotide sequence encoding the antibody of the present invention, or its heavy and light chains; or (ii) a nucleotide sequence encoding the multispecific antibody of the present invention or at least one polypeptide chain thereof.

[0072] In certain embodiments, the isolated nucleic acid molecule encodes the antibody of the present invention, or its heavy and light chains. In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain of the antibody of the present invention, and a second nucleotide sequence encoding the light chain of the antibody of the present invention. The first nucleotide sequence and the second nucleotide sequence are on the same or different isolated nucleic acid molecules.

[0073] In certain embodiments, the isolated nucleic acid molecule encodes a multispecific antibody of the invention, or at least one polypeptide chain thereof. In certain embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding each polypeptide chain of the multispecific antibody of the invention. The nucleotide sequences encoding each polypeptide chain are on the same or different isolated nucleic acid molecules.

[0074] In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the invention. In certain embodiments, the vector of the invention is, for example, a plasmid, a cosmid, a phage, etc.

[0075] In certain embodiments, the vector comprises nucleotide sequences encoding the antibody of the invention, or its heavy and light chains. In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain of the antibody of the invention, and a second nucleotide sequence encoding the light chain of the antibody of the invention. The first nucleotide sequence and the second nucleotide sequence are on the same or different vectors.

[0076] In certain embodiments, the vector comprises nucleotide sequences encoding the multispecific antibody of the invention, or at least one polypeptide chain thereof. In certain embodiments, the vector comprises nucleotide sequences encoding each polypeptide chain of the multispecific antibody of the invention. The nucleotide sequences encoding each polypeptide chain are on the same or different vectors.

[0077] In another aspect, the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, e.g., mouse cells, human cells, etc.). In certain embodiments, the host cell of the invention is a mammalian cell.

[0078] In another aspect, provided is a method for preparing the antibody or multispecific antibody of the invention, which comprises culturing the host cell of the invention under conditions that allow the expression of the antibody or multispecific antibody, and recovering the antibody or multispecific antibody from the cultured host cell culture.

[0079] Pharmaceutical composition

[0080] In another aspect, the invention provides a pharmaceutical composition comprising the antibody, multispecific antibody or conjugate of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0081] In certain embodiments, the pharmaceutical composition comprises an effective amount of the antibody.

[0082] In certain embodiments, the pharmaceutical composition comprises an effective amount of the multispecific antibody.

[0083] In certain embodiments, the pharmaceutical composition comprises an effective amount of the conjugate.

[0084] In certain embodiments, the pharmaceutical composition of the present invention may further comprise an additional pharmaceutically active agent. In certain embodiments, the additional pharmaceutically active agent is a drug for preventing or treating HBV infection or diseases associated with HBV infection (such as hepatitis B), such as interferon drugs, such as interferon or pegylated interferon. In certain embodiments, the additional pharmaceutically active agent is an immunomodulator, such as a STING agonist or a TLR agonist (such as a TLR7 / 8 agonist).

[0085] The pharmaceutical composition of the present invention can be formulated into a dosage form compatible with its intended route of administration. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the antibody described herein in a suitable solvent, and optionally, simultaneously incorporating other desired components (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use.

[0086] The pharmaceutical composition of the present invention can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration refers to a mode of administration that is typically by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, it can be administered via non-parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical.

[0087] Treatment

[0088] In another aspect, there is provided the use of the antibody, multispecific antibody, conjugate or pharmaceutical composition of the present invention in the preparation of a drug for preventing and / or treating HBV infection or diseases associated with HBV infection (such as hepatitis B) in a subject (such as a human), and / or for reducing the serum levels of HBV DNA and / or HBsAg in a subject (such as a human).

[0089] In another aspect, the present invention provides a method for preventing or treating HBV infection or diseases associated with HBV infection (such as hepatitis B) in a subject (such as a human), and / or for reducing the serum levels of HBV DNA and / or HBsAg in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of an antibody, multispecific antibody, conjugate or pharmaceutical composition according to the present invention.

[0090] In certain embodiments, the treatment comprises administering an effective amount of an antibody of the present invention or a pharmaceutical composition comprising the same.

[0091] In certain embodiments, the treatment comprises administering an effective amount of a multispecific antibody of the present invention or a pharmaceutical composition comprising the same.

[0092] In certain embodiments, the treatment comprises administering an effective amount of a conjugate of the present invention or a pharmaceutical composition comprising the same.

[0093] The antibody, multispecific antibody, conjugate or pharmaceutical composition provided by the present invention can be used alone or in combination with another pharmaceutically active agent. The other pharmaceutically active agent can be other antiviral reagents, such as interferon drugs, such as interferon or polyethylene glycol interferon; or immunomodulators, such as STING agonists or TLR agonists (such as TLR7 / 8 agonists).

[0094] The antibodies, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. The antibodies, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the active ingredient in a suitable solvent, and optionally, other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) are incorporated simultaneously, followed by filtration sterilization. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze-drying) for ease of storage and use. Such a sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (for example, 0.9% (w / v) NaCl), glucose solution (for example, 5% glucose), a solution containing a surfactant (for example, 0.01% polysorbate 20), a pH buffer solution (for example, phosphate buffer solution), Ringer's solution, and any combination thereof.

[0095] The antibodies, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, local (such as powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (such as intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route of administration and / or mode will vary according to the intended purpose. In certain embodiments, the antibodies, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus injection.

[0096] The antibodies, multispecific antibodies, conjugates or pharmaceutical compositions of the present invention can be formulated in dosage unit form for ease of administration. A dosage unit form refers to a physically discrete unit suitable as a single dose for the subject to be treated; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0097] The subjects described herein can be mammals, such as humans. In certain embodiments, the subject is an HBV-infected individual, such as a chronic HBV-infected individual or a chronic hepatitis B patient. In certain embodiments, the subject has been exposed to HBV or is at risk of exposure to HBV.

[0098] Term definition

[0099] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the operating steps such as cell culture, biochemistry, nucleic acid chemistry, immunology laboratory, etc. used herein are all conventional steps widely used in the corresponding fields. At the same time, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0100] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include various antibody structures as long as they exhibit the required antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0101] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the variable regions of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of ordinary skill in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those of ordinary skill in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0102] In the present invention, the CDRs contained in the antibodies of the present invention can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, or IMGT numbering systems. In certain embodiments, the CDRs contained in the antibodies of the present invention are preferably determined by the Kabat numbering system.

[0103] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues defined as above.

[0104] The term "antibody" is not limited by any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0105] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Herein, a "full-length heavy chain" refers to a polypeptide chain that, in the N-terminal to C-terminal direction, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of the IgE isotype, optionally further includes a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be from a single species, such as human; or can be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention contain two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize / bind the same antigen.

[0106] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, wherein the VL and VH are linked by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. In some cases, there may also be a disulfide bond between VH and VL of the scFv.

[0107] As used herein, the term "scFv-Fc" refers to an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus or N-terminus of the scFv. The Fc domain can follow VH or VL, depending on the orientation of the variable domains of the scFv (i.e., VH-VL or VL-VH).

[0108] As used herein, the terms "Fc domain", "Fc region", or "Fc portion" have the meaning commonly understood by those of ordinary skill in the art and are used interchangeably, and refer to a portion of the heavy chain constant region that includes CH2 and CH3. The Fc region of an antibody has a variety of different functions but is not involved in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as the B cell receptor); and B cell activation, among others. In some embodiments, the Fc region includes a hinge, CH2, and CH3. When the Fc region includes a hinge, the hinge regulates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4. Amino acid positions within the Fc domain can be numbered according to the Kabat EU numbering system, see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0109] The Fc domain can include either a native Fc region or a variant Fc region. A native Fc region contains an amino acid sequence that is identical to the amino acid sequence of the Fc region found in nature. For example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region contains an amino acid sequence that is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region can have altered effector functions (such as Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function) compared to the native Fc region. In some embodiments, a variant Fc region can have a modification that promotes dimerization. As used herein, a "monomer" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain that is capable of stabilizing its own association.

[0110] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) of that interaction. In the present invention, the term "K D" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding property between two molecules can be measured using methods well-known in the art, such as measuring using surface plasmon resonance (SPR) in a BIACORE instrument.

[0111] As used herein, the expression "binds in a pH-dependent manner" or an equivalent expression "pH-dependent binding" means that the KD value or EC50 value of the antibody of the present invention binding to HBsAg at acidic pH is higher than its KD value or EC50 value binding to HBsAg at neutral pH. The KD can be measured by techniques well-known in the art, such as by SPR technology (such as Biacore). In the present invention, the term "EC50" refers to the antibody-antigen half-maximal effective concentration, that is, the antibody concentration required for a specific antibody-antigen to reach 50% of the maximum binding effect, which is used to describe the binding ability between an antibody and an antigen. The smaller the EC50, the higher the binding ability between the antibody and the antigen. The antibody-antigen half-maximal effective concentration (EC50) can be measured using methods well-known in the art, such as by using an enzyme-linked immunosorbent assay (ELISA) in which the antigen binds to a solid-phase carrier and the antibody specifically binds to the antigen.

[0112] As used herein, a "neutralizing antibody" refers to an antibody that can significantly reduce or completely inhibit the virulence of a target virus (e.g., the ability to infect cells). Generally speaking, a neutralizing antibody can recognize and bind to a target virus and prevent the target virus from entering / infecting the cells of a subject. The antibody of the present invention is a neutralizing antibody.

[0113] However, it should be understood that in this application, the ability of an antibody to neutralize a virus is not directly equivalent to the ability of the antibody to clear the virus. As used herein, "neutralize a virus" means to neutralize the virulence of a target virus (i.e., significantly reduce or completely inhibit the virulence of the target virus) by inhibiting the process of the target virus entering / infecting the cells of a subject. As used herein, "clear the virus" means that the target virus in the body (whether it has infected cells or not) is eliminated from the body, so that the body changes towards the state before being infected by the virus (e.g., the serological test result of the virus turns negative). Therefore, generally speaking, a neutralizing antibody does not necessarily have the ability to clear the virus. However, in this application, the inventors unexpectedly found that the antibody of the present invention not only has the ability to neutralize HBV, but also has the ability to clear the virus (i.e., can clear HBV DNA and / or HBsAg in the body, clear HBV and HBV-infected cells in the body), thus having great clinical value.

[0114] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0115] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0116] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of a total of 6 positions match). Typically, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be accomplished by using, e.g., the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). Also, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), can be used with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0117] The notations for the twenty conventional amino acids referred to herein follow conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in the present invention, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0118] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, preservatives. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include but are not limited to sugars, NaCl and the like. Absorption delaying agents include but are not limited to monostearate and gelatin.

[0119] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom (e.g., HBV infection or a disease associated with HBV infection) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer deteriorating) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging the survival period compared to the expected survival period (if not treated). In the present application, the antibodies of the present invention have the ability to neutralize HBV, and thus can be used to prevent / deter an uninfected subject or its cells from being infected with HBV. In addition, the antibodies of the present invention have the ability to clear HBV (i.e., can clear HBV DNA and / or HBsAg in the body, clear HBV and HBV-infected cells in the body), and thus can be used to treat HBV infection or a disease associated with HBV infection in an infected subject.

[0120] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0121] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (such as HBV infection or a disease associated with HBV infection) refers to an amount sufficient to prevent, arrest, or delay the occurrence of the disease (such as HBV infection or a disease associated with HBV infection); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0122] Advantages of the invention

[0123] The present invention provides a trifunctional hepatitis B therapeutic antibody with pH-dependent antigen binding / enhanced Fc-hFcγR interaction / enhanced Fc-hFcRn interaction. The antibody of the present invention can reduce the serum levels of HBV DNA and / or HBsAg in a subject, and can effectively clear HBV and HBV-infected cells in the body. In particular, the antibody of the present invention has a significantly enhanced antigen clearance effect and antigen inhibition time compared with the prior art antibodies, and while improving the clinical cure rate, it can also reduce the dosing frequency and dose, having great clinical value.

[0124] The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and not to limit the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief description of the drawings

[0125] Figure 1 . Detection of the phagocytosis efficiency of phagocytes on HBsAg mediated by the Fc mutant antibody in human peripheral blood.

[0126] Figure 2 . Phagocytosis efficiency of phagocytes on HBsAg mediated by the Fc double mutant antibody in human peripheral blood.

[0127] Figure 3 . Detection results of the binding of the antibody to membrane-expressed hFcRn at pH 6.0.

[0128] Figure 4 . Schematic diagram of the construction of the engineered hepatitis B therapeutic antibody D3-DLEqs.

[0129] Figure 5. Detection results of the binding of D3-DLEqs and HBsAg at pH 7.4 and pH 6.0.

[0130] Figure 6 . Detection results of the binding of the antibody to membrane-expressed hFcRn at pH 6.0.

[0131] Figure 7 . Detection of the phagocytosis efficiency of the antibody-mediated different phagocytes on HBsAg in human peripheral blood.

[0132] Figure 8 . Detection of the serum antibody concentration of the antibody in hFcRn-Tg mice through the intravenous administration route.

[0133] Figure 9 . Detection of the serum antibody concentration of the antibody in hFcRn-Tg mice through the subcutaneous administration route.

[0134] Figure 10 . Detection of the serum HBsAg titer after subcutaneous administration of the antibody in the AAV-HBV-infected hFcγR-Tg mouse model. Detailed implementation mode

[0135] The present invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit the invention).

[0136] Those skilled in the art know that the examples describe the present invention by way of example and are not intended to limit the scope claimed in this application. The experimental methods in the examples are all conventional methods unless otherwise specified. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0137] Example 1. Screening of Fc point mutations for enhancing the phagocytosis of hepatitis B virus antigen by antibody-mediated human phagocytes

[0138] 1.1 Variable region sequence of D3 antibody

[0139] Using the pH-dependent anti-HBsAg antibody D3 developed in the early stage of the laboratory (described in detail in Chinese Patent Application CN111978392A) as the parental antibody, its heavy chain constant region was engineered by Fc engineering to maintain its variable region sequence and the corresponding pH-dependent HBsAg binding characteristics. Antibodies with pH-dependent HBsAg binding characteristics can maintain antigen binding activity under neutral conditions while significantly reducing antigen binding activity under acidic conditions, so that they can dissociate from HBsAg in acidic endosomes, circulate to the plasma and bind to the antigen again, achieving the effect that one molecule of antibody can repeatedly bind / neutralize multiple molecules of antigen. Its variable region sequence is shown in Table 1, and the CDR sequences determined according to the Kabat numbering system are shown in Table 2.

[0140] Table 1. Amino acid sequences of the heavy and light chain variable regions of antibody D3

[0141]

[0142] Table 2. Amino acid sequences of the CDRs of the heavy and light chain variable regions of antibody D3

[0143]

[0144]

[0145] 1.2 Fc point mutation design for enhancing antibody-mediated phagocytosis of hepatitis B virus antigen by human phagocytes

[0146] Antibodies can interact with FcγR on the surface of phagocytes through Fc to mediate antibody-dependent cellular phagocytosis (ADCP) to achieve specific clearance of viral antigens. In order to enhance the phagocytosis of hepatitis B virus antigen mediated by antibody D3 on human phagocytes and enhance the affinity with human FcγR (hFcγR), different point mutation combinations were introduced into its Fc domain, including G236A / S239D / A330L / I332E (ADLE), S239D / A330L / I332E (DLE), G236A / S239D / I332E (ADE), S239D / I332E (DE). The constant region sequences of each mutant antibody are shown in Table 3.

[0147] Table 3. Heavy and light chain constant region sequences of Fc mutant antibodies against hFcγR

[0148]

[0149]

[0150] 1.3 Preparation of Fc Mutant Antibodies with Enhanced hFcγR Binding

[0151] 1.3.1 Construction of Antibody Expression Vectors

[0152] The gene sequence encoding the heavy-chain variable region of the antibody was constructed into the PTT5-H vector (restriction enzyme sites AgeI / SalI) containing the heavy-chain constant region sequence, and the gene sequence of the light-chain variable region was constructed into the PTT5-K vector (restriction enzyme sites AgeI / BsiWI) containing the light-chain constant region sequence. The construction of the recombinant expression vector was commissioned to General Biosystems (Anhui) Co., Ltd. The recombinant vector was transformed into DH5α competent cells (Shenzhen Kangti). After growing on an ampicillin-resistant LB plate for 12 h, monoclonal colonies were picked and sent for sequencing (Shanghai Sangon). The correctly sequenced recombinant plasmid was extracted in large quantities using an endotoxin-free plasmid large-scale extraction kit (TianGen, DP117).

[0153] 1.3.2 Eukaryotic Expression and Purification of Antibodies

[0154] The antibody was expressed by transient transfection of Expi-293F cells with two plasmids. Expi-293F cells with a viability higher than 95% were prepared and inoculated into a 1 L cell culture flask at a density of 4×10 6 at 200 mL. Take 0.5 mg of the heavy-chain recombinant plasmid and 0.5 mg of the light-chain recombinant plasmid, mix them with 2 mg of PEI, shake vigorously for 8 s and then let stand for 8 min, and add the mixture to 200 mL of cells. After 4 h, 200 mL of Freestyle medium was supplemented, and the cells were placed in a 5% CO2 incubator at 37 °C for 7 days. The cell supernatant was collected, centrifuged at 10,000 rpm for 30 min, and the supernatant was taken for subsequent purification.

[0155] Filter the above cell supernatant with a 0.22 μm filter; turn on the AKTA instrument, first rinse the A pipeline and B pipeline with solution A (200 mM disodium hydrogen phosphate dodecahydrate) and solution B (100 mM citric acid monohydrate) respectively, and install the protein A column; balance the protein A column with solution A at a flow rate of 8 mL / min for more than 15 min. After the UV value, pH value and conductivity detected by the instrument are stable, proceed to the next step; load the sample at a flow rate of 6 - 10 mL / min. Subsequently, the UV value will increase, and this peak is the breakthrough peak. Continue to wash the column with solution A while collecting the breakthrough peak sample for detection. After the pH value no longer changes, introduce solution B at a flow rate of 6 - 10 mL / min. Subsequently, the pH value will decrease and the UV value will increase, and this peak is the elution peak. The antibody mainly exists in the elution peak, and collect the elution peak sample for detection; balance the column with solution A, then fill the pipeline and protein A column with 20% ethanol, remove the column, and store it at 4 °C. Dialyze the purified monoclonal antibody with 20 mM PBS buffer overnight, and measure the concentration by ultraviolet spectrophotometry or BCA and aliquot it into 1.5 mL tubes, and store it at -20 °C for later use.

[0156] 1.4 Screening of Fc mutant antibodies with enhanced hFcγR binding

[0157] 1.4.1 To evaluate the ADCP effect mediated by antibodies in human phagocytes, freshly isolated human peripheral blood was selected as the phagocytosis evaluation system, which contains various phagocytes such as monocytes, macrophages, neutrophils and dendritic (DC) cells.

[0158] Mix 1600 ng / mL Dylight 488-HBsAg with the antibody to be tested at 10 μg / mL and 0.5 μg / mL respectively in RPMI-1640 buffer in equal volumes (150 μL:150 μL), and incubate at 37 °C for 1 h. Take 250 μL of the mixed solution and add it to 1 mL of freshly isolated human peripheral blood (the peripheral blood is collected in an anticoagulant tube containing EDTA; from 4 donors), and incubate at 37 °C for 2 h. Treat the peripheral blood with 3 - 4 mL of red blood cell lysate (Solarbio Science & Technology Co., Ltd., Beijing) at room temperature for 15 - 30 min until there is no obvious red precipitate. Centrifuge at 1800 rpm for 10 min, and wash the cell pellet twice with PBS. Stain the cells with fluorescent antibodies against human CD45, F4 / 80, CD11c, CD14, and Ly-6G labeled with different fluorophores (other than Dylight 488) at 4 °C for 30 min. Wash the cells once with PBS, pass them through a cell sieve with a pore size of 70 μm, and perform flow cytometry analysis. Determine the subpopulations of human immune cells (Leukocyte), neutrophils (Neutrophil), and DC cells through flow cytometry gating strategy. Count the proportion of Dylight 488-positive cells and the mean fluorescence intensity in different phagocyte subpopulations, and analyze the phagocytosis efficiency of antibody-mediated HBsAg.

[0159] The results are as Figure 1 shown. Compared with the parental antibody D3, all 4 Fc mutant antibodies can mediate enhanced phagocytosis efficiency of viral antigens in the total population of immune cells in human peripheral blood (manifested as significantly increased antigen mean fluorescence intensity and positive cell proportion), among which D3-DLE mediates the highest phagocytosis efficiency. In addition, in neutrophils and DC cells, D3-DLE can also cause the strongest ADCP effect. Among them, neutrophils are the phagocyte population with the highest frequency in peripheral blood, which may be the reason why D3-DLE mediates the highest phagocytosis efficiency in the total population of immune cells.

[0160] 1.4.2 Determine the affinity of the four Fc mutant antibodies for various hFcγRs by surface plasmon resonance (SPR) technology. The SPR experiment was carried out on a BIAcore 8K (GE Healthcare, USA) at 25 °C, and the buffer was HBS-EP +(Cytiva, USA). Human FcγRs were purchased from Sino Biological Inc. (Beijing, China), and the C-terminus of hFcγRs was fused with a 6×His tag. Briefly, antibodies were immobilized on a Protein A sensor chip (GE Healthcare, USA) at a density of 300 response units (RU). Recombinant hFcγRs at gradient dilutions were injected into the flow cell at a rate of 30 μL / min, with a concentration range of 400 to 3.125 nM (1:2 serial dilution). The binding time was 90 s, followed by a 120-s dissociation step. At the end of each cycle, the sensor surface was regenerated with glycine hydrochloride buffer (10 mM, pH 1.7; 50 μL / min, 30 s). The K D value was calculated using the 1:1 Langmuir binding model with BIAcore 8K evaluation software (GE Healthcare, USA).

[0161] As shown in Table 4, after introducing point mutations at the Fc region, D3-DLE showed enhanced affinity for various human FcγRs to varying degrees compared to the antibody D3 with wild-type Fc, especially the most significant improvement in affinity for hFcγRIIIb among the four Fc mutant antibodies. hFcγRIIIb is a type of FcγR mainly expressed on the surface of neutrophils, which explains why D3-DLE can mediate the strongest neutrophil phagocytosis.

[0162] Table 4. Affinity of antibodies for different human FcγRs determined by SPR.

[0163]

[0164]

[0165] Example 2. Screening of Fc double mutant combinations for simultaneously enhancing ADCP effect and prolonging antibody half-life

[0166] 2.1 Design of Fc double mutant combinations for simultaneously enhancing ADCP effect and prolonging antibody half-life

[0167] To further prolong the half-life of the D3-DLE antibody, point mutations T256D / H285D / T307R / Q311V / A378V (D3-DLEddrvv), M428L / N434S (D3-DLEls), T307Q / N434S (D3-DLEqs), and V259I / N315D / N434Y (D3-DLEidy) that can enhance binding to human FcRn (hFcRn) at acidic pH were introduced into its Fc domain. The constant region sequences of the four Fc double mutant antibodies are shown in Table 5.

[0168] Table 5. Heavy and light chain constant region sequences of Fc double mutant antibodies

[0169]

[0170]

[0171] Preparation of 2.2 Fc double mutant antibody

[0172] Using the same method as in Example 1.3, eukaryotic expression and purification of the Fc double mutant antibody were carried out.

[0173] 2.3 Screening of Fc double mutant antibody

[0174] 2.3.1 Verification of ADCP effect mediated by Fc double mutant antibody in human phagocytes

[0175] Using the same method as in Example 1.4.1, it was evaluated whether the antibody introducing the Fc double mutant combination could still maintain the enhanced ADCP effect in freshly isolated human peripheral blood.

[0176] The results are as Figure 2 shown. Compared with antibody D3-DLE, the ADCP efficiency mediated by most antibodies introducing Fc point mutations with enhanced affinity for hFcRn decreased, including D3-DLEddrvv, D3-DLEls and D3-DLEidy. However, antibody D3-DLEqs introducing the T307Q / N434S point mutation maintained an ADCP efficiency comparable to that of D3-DLE and could also mediate the strongest ADCP effect in neutrophils and DC cells.

[0177] 2.3.2 Verification of the affinity between Fc double mutant antibody and hFcRn under acidic pH conditions

[0178] Using the previously constructed MDCK cell line that can stably display hFcRn on the membrane (Kang, C., L. Xia, Y. Chen, T. Zhang, Y. Wang, B. Zhou, M. You, Q. Yuan, C. M. Tzeng, Z. An, W. Luo, N. Xia, Protein Cell 2018.9(1): p. 130-134.), the binding activity between the antibody D3-DLEqs with the strongest ADCP effect in Example 2.3.1 and hFcRn was evaluated.

[0179] After trypsin digestion of hFcRn-MDCK stable transfected cells, count and aliquot into a sterile 96-well cell plate, with 5×10 cells per well 5Individuals. Dilute the antibody with PBS containing 1% BSA and pH 6.0. The first well is 1 μg / μL, and perform 4-fold serial dilution using a 96-well microplate. Centrifuge the plate with the dispensed cells, aspirate the supernatant, then centrifuge and wash twice with PBS (1% BSA) at pH 6.0. Then add 100 μL of the diluted antibody and 1 μg of Dylight 650-human IgG, resuspend and mix well, incubate for 1 h at 4°C in the dark, and finally centrifuge and wash twice with PBS (1% BSA) at pH 6.0 and resuspend for flow cytometry analysis. Use empty cells resuspended with PBS at pH 6.0 as the negative control, and cells with only Dylight 650-human IgG (SIGMA-ALDRICH, USA) added as the positive control. Flow cytometry counts the mean fluorescence intensity (gMFI) of Dylight 650 on the cell surface. The blocking rate is calculated according to the following formula: (gMFI of the positive control group - gMFI of the experimental group) / gMFI of the positive control group × 100%.

[0180] The results are as Figure 3 shown. The ability of the antibody D3-DLE that does not introduce point mutations against FcRn to block the binding of human IgG to hFcRn is comparable to that of the parental antibody D3 with wild-type Fc, and the IC50 values are 150.9 μg / mL and 171.1 μg / mL respectively; while the efficiency of the Fc double mutant antibody D3-DLEqs in blocking the binding of human IgG to hFcRn is significantly improved, and the IC50 is 6.16 μg / mL, suggesting that the binding activity of D3-DLEqs to hFcRn at acidic pH is significantly enhanced.

[0181] So far, the three-functional optimized hepatitis B therapeutic antibody D3-DLEqs with pH-dependent antigen binding / enhanced Fc-hFcγR interaction / enhanced Fc-hFcRn interaction has been designed, and its schematic diagram is as Figure 4 shown.

[0182] Example 3. Evaluation of the in vitro HBsAg binding ability of D3-DLEqs

[0183] 3.1 Detect the pH-dependent binding ability of the antibody to HBsAg by ELISA.

[0184] First, use a BCA protein quantification kit to determine the concentration of the purified antibody, and uniformly dilute the antibody concentration to 10,000 ng / mL. Subsequently, perform a 3-fold serial dilution of the antibody concentration with 20% NBS, for a total of 11 concentration gradients. Then, add the diluted antibody to a commercial HBsAg plate (purchased from Beijing Wantai) and incubate at 37 °C for 1 h (in triplicate). Subsequently, wash the ELISA plate 5 times with PBST and centrifuge to dry. Then, add 100 μL of PBS at pH 7.4 and pH 6.0 to each well and incubate at 37 °C for 30 min. Wash 5 times with PBST of the corresponding pH and centrifuge to dry. Subsequently, add the GAH-HRP enzyme-labeled secondary antibody, incubate for 30 min, wash the plate 5 times with PBST and centrifuge to dry. And add the substrate TMB solution. After 15 min of color development, terminate the color reaction with H2SO4 and measure the reading at OD 450 / 630 value.

[0185] 3.2 The results are as Figure 5 shown. D3-DLEqs has good pH-dependent HBsAg binding activity, strongly binds to HBsAg at pH 7.4, with an EC50 of 51 ng / mL; however, the binding activity significantly decreases under acidic conditions at pH 6.0, with an EC50 of 1150 ng / mL.

[0186] Example 4. Determination of the affinity of D3-DLEqs for hFcγRs

[0187] Using the same method as in Example 1.4.2, determine the affinity of D3-DLEqs and its parental antibodies D3 and 162 for various hFcγRs by surface plasmon resonance (SPR) technology.

[0188] The results are shown in Table 6. After introducing point mutations at the Fc site, D3-DLEqs enhanced the affinity for various human FcγRs to varying degrees compared to the two parental antibodies 162 and D3 with wild-type Fc; among them, the affinity for hFcγRIIIa and hFcγRIIIb increased most significantly, by 17.74-fold and 16.47-fold, respectively. These two types of FcγRs are widely distributed on the surface of various human phagocytic cells, including monocytes, macrophages, and neutrophils, and mediate the ADCP effect.

[0189] Table 6. SPR determination of the affinity of antibodies for different human FcγRs.

[0190]

[0191] Example 5. Determination of the binding activity of D3-DLEqs to hFcRn under acidic pH conditions

[0192] Using the same method as in Example 2.3.2, with parental antibody 162 and D3 and clinical antibody Vir-3434 as controls, the binding activity of D3-DLEqs to hFcRn was evaluated.

[0193] The results are as Figure 6 shown. The parental antibodies 162 and D3 with wild-type Fc had comparable abilities to block the binding of human IgG to hFcRn, with IC50 values of 200.7 μg / mL and 252.7 μg / mL, respectively; while the clinically engineered antibody Vir-3434 had a stronger ability to block the binding of human IgG to hFcRn, with an IC50 of 23.52 μg / mL. In contrast, D3-DLEqs had the strongest ability to block the binding of human IgG to hFcRn, with an IC50 of 9.9 μg / mL, suggesting that D3-DLEqs had a stronger binding activity to hFcRn at acidic pH than the clinical antibody Vir-3434.

[0194] Example 6. Phagocytosis of HBsAg mediated by D3-DLEqs in human peripheral blood

[0195] Using the same method as in Example 1.4.1, with parental antibody D3 and clinical antibody Vir-3434 as controls, the ADCP effect mediated by antibody D3-DLEqs in human phagocytes was evaluated.

[0196] The results are as Figure 7 shown. Compared with the parental antibody D3, both the clinically engineered antibody Vir-3434 and the antibody D3-DLEqs in this study could mediate a significantly enhanced phagocytosis efficiency of viral antigens in the complex system of human peripheral blood containing various immune cells, and the ADCP effect mediated by D3-DLEqs was stronger than that of Vir-3434. More importantly, an enhanced ADCP effect caused by D3-DLEqs was observed in multiple phagocyte subsets, including neutrophils, monocytes, and DC cells. The above results suggest that the engineered antibody D3-DLEqs has the potential to mediate enhanced clearance of HBV antigens in the human immune system.

[0197] Example 7. Evaluation of the antibody half-life of D3-DLEqs in hFcRn transgenic mice

[0198] 7.1 Based on the enhanced binding activity of D3-DLEqs to hFcRn at acidic pH observed in Examples 2 and 5, the half-life of the antibody in serum was further verified in hFcRn transgenic (hFcRn-Tg) mice.

[0199] In this experiment, 4-week-old hFcRn-Tg mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. 162, D3, and D3-DLEqs at a dose of 5 mg / kg were injected into the mice via the tail vein (i.v.) or subcutaneous administration (s.c.) (4-5 mice per group). Orbital venous plexus blood collection was performed on the mice on the 1st, 2nd, 4th, 6th, 8th, 11th, 13th, 16th, 22nd, 25th, 27th, and 29th days after antibody administration, and serum was collected by centrifugation at 12,000 rpm. By detecting the antibody concentration in the serum, the half-life of the antibody in vivo was analyzed.

[0200] 7.2 Quantitative detection of antibody concentration

[0201] (1) Preparation of reaction plates: The goat anti-human Fab domain-specific antibody (I5260-1ML; SIGMA-ALDRICH, USA) was diluted to 2 μg / mL with 20 mM PB buffer (Na2HPO4 / NaH2PO4 buffer, pH 7.4). 100 μL of the coating solution was added to each well in the ELISA plate and coated at 2-8 °C for 16-24 h, followed by coating at 37 °C for 2 h. The plate was washed once with PBST washing solution and spun dry. After washing, 200 μL of the blocking solution was added to each well and blocked at 37 °C for 2 h. Subsequently, the blocking solution was discarded, the plate was placed in a drying room to dry, and stored at 2-8 °C for later use.

[0202] (2) Sample dilution: The collected mouse serum was diluted with PBS solution into two gradients of 1:200 and 1:1000 for subsequent quantitative detection. The standard product was commercially available human IgG (IgG from human serum, I4506-10MG) diluted in gradients. The first well was 5 μg / mL, diluted in a 2-fold gradient, for a total of 12 gradients.

[0203] (3) Sample reaction: The diluted samples and standards were added to the blocked ELISA plate at 100 μL / well and reacted at 37 °C for 1 h. Subsequently, the reaction plate was washed 5 times with PBST and spun dry.

[0204] (4) Enzyme-labeled reagent reaction: To the ELISA plate, 100 μL / well of the goat anti-human Fc antibody dilution solution diluted with ED-11 at 1:5000 was added and reacted at 37 °C for 1 h. Subsequently, the plate was washed 5 times with PBST and spun dry.

[0205] (5) Luminescence reaction and measurement: Luminescence solution (100 μL / well) was added to the ELISA plate, and the termination solution (50 μL / well) was added after 15 minutes, and the light intensity was detected.

[0206] (6) Calculation of antibody concentration in mouse serum samples: A standard curve was drawn based on the results of the standard sample assay. The light intensity measurements of the mouse serum samples were then substituted into the standard curve to calculate the antibody concentration in the serum sample to be tested.

[0207] 7.3 The results are as follows Figure 8 、 Figure 9 As shown, whether administered intravenously or subcutaneously, the half-life of D3-DLEqs was significantly longer than that of the parental antibodies 162 and D3, compared with antibody concentrations in serum. More importantly, the half-life of D3-DLEqs was slightly longer than that of the clinical antibody Vir-3434. Comparing the two routes of administration, the half-life of D3-DLEqs was longer subcutaneously than intravenously, suggesting that D3-DLEqs may have better pharmacokinetic and pharmacodynamic performance when administered subcutaneously. It is important to note that the mice in this experiment did not have HBV virus or HBsAg in their bodies. Therefore, the half-life extension caused by D3-DLEqs is not dependent on the pH-dependent antigen-binding properties of the variable region, but rather on the increased affinity for hFcRn brought about by Fc engineering. These in vivo pharmacokinetic data are consistent with the in vitro hFcRn affinity data observed in Examples 2 and 5.

[0208] Example 8. Pharmacodynamic evaluation of D3-DLEqs in hFcγR transgenic mice

[0209] 8.1 In this example, to evaluate the in vivo therapeutic effect of D3-DLEqs in an HBV-infected mouse model and verify the Fc engineering effect of hFcγR affinity enhancement, 4-week-old hFcγR transgenic mice (hFcγR-Tg, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were used to culture 1×10 11 vg / mouse virus dose rAAV-HBV adeno-associated virus (adr serotype, purchased from Guangzhou Paizhen Biotechnology Co., Ltd.) was injected into the tail vein, and blood was collected for four consecutive weeks to monitor the virus titer of the mice. After the virus titer stabilized, 162 and D3-DLEqs at a dose of 10 mg / kg were injected subcutaneously into the mice (4 per group). Blood was collected from the orbital venous plexus of the mice 2 days before antibody administration, on the day of administration, and on days 1, 3, 5, 7, 10, 13, 17, 23, 29, and 35 after administration, and serum was collected by centrifugation at 12000 rpm. HBsAg in serum was uniformly tested after the experiment.

[0210] 8.2 Quantitative Detection of HBsAg

[0211] (1) Preparation of reaction plate: HBsAg quantitative detection kit was purchased from Beijing Wantai Biopharmaceutical Co., Ltd., and the chemiluminescence reaction plate was used.

[0212] (2) Sample dilution: Dilute the collected mouse serum with ED-11 solution at a ratio of 1:50 for subsequent quantitative detection. Dilute the HBsAg standard with ED-11 solution to 45 IU / mL, and then perform 5-fold serial dilutions for a total of 6 gradients;

[0213] (3) Sample reaction: Add the diluted samples and standards to the chemiluminescence plate at 100 μL / well and react at 37 °C for 1 h.

[0214] (4) Enzyme-labeled antibody reaction: Add the enzyme-labeled secondary antibody diluent in the kit to the chemiluminescence plate at 50 μL / well and react at 37 °C for 1 h. Subsequently, wash the plate 5 times with PBST and spin dry.

[0215] (5) Luminescence reaction and measurement: Add the luminescence solution (100 μL / well) to the chemiluminescence plate and perform light intensity detection.

[0216] (6) Calculation of HBsAg concentration in mouse serum samples: Plot a standard curve based on the measurement results of the standards. Then, substitute the light intensity measurement value of the mouse serum sample into the standard curve to calculate the HBsAg concentration in the serum sample to be tested.

[0217] 8.3 Results As Figure 10 shown, in the hFcγR-Tg mouse model with an initial average HBsAg titer of 10048.8 IU / mL, single subcutaneous injection of the parental antibody 162 at 10 mg / kg only decreased HBsAg to 2454.8 IU / mL, the clinical antibody Vir-3434 decreased HBsAg to 1394.1 IU / mL, while injection of D3-DLEqs rapidly decreased serum HBsAg to 539.2 IU / mL. Meanwhile, the serum HBsAg in the 162 treatment group rebounded to the baseline level within 2 weeks after dosing, and the serum HBsAg in the Vir-3434 treatment group rebounded to the baseline level around 3 weeks after dosing. In contrast, D3-DLEqs treatment effectively inhibited the HBsAg level for up to 35 days. In this treatment model, D3-DLEqs demonstrated stronger serum antigen clearance effects than the parental antibody 162 and the clinical antibody Vir-3434 under a high HBsAg background, and this significantly enhanced efficacy reflected the effects of variable region pH-dependent antigen binding modification and Fc domain modification to enhance hFcγR affinity.

[0218] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings that have been disclosed, and these changes are within the scope of protection of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody capable of specifically binding to HBsAg, comprising an antigen-binding domain that binds to HBsAg in a pH-dependent manner and an Fc domain, wherein, The Fc domain contains a first mutation that enhances binding to FcγR (such as hFcγR) and a second mutation that enhances binding to FcRn (such as hFcRn).

2. The antibody according to claim 1, wherein, The first mutation at least enhances binding to hFcγRIIIb; Preferably, the first mutation also enhances binding to hFcγRI, hFcγRIIa, and / or hFcγRIIIa; Preferably, the first mutation is selected from the following mutations according to EU numbering: G236A / S239D / A330L / I332E, S239D / A330L / I332E, G236A / S239D / I332E, S239D / I332E.

3. The antibody according to any one of claims 1-2, wherein, The first mutation is S239D / A330L / I332E.

4. The antibody according to any one of claims 1-3, wherein, The second mutation is selected from the following mutations according to EU numbering: T256D / H285D / T307R / Q311V / A378V, M428L / N434S, T307Q / N434S, V259I / N315D / N434Y; Preferably, the second mutation is T307Q / N434S.

5. The antibody according to any one of claims 1-4, wherein The first mutation is S239D / A330L / I332E, and the second mutation is T307Q / N434S.

6. The antibody according to any one of claims 1-5, wherein, The Fc domain is IgG, such as IgG1, IgG2, IgG3, or IgG4; Preferably, the Fc domain is human IgG1.

7. The antibody according to any one of claims 1-6, wherein, The antibody is a full-length antibody; Preferably, the antibody comprises a heavy chain and a light chain; Preferably, the heavy chain comprises a heavy chain constant region (CH) shown in any one of SEQ ID NOs: 14-17; Preferably, the heavy chain comprises the heavy chain constant region (CH) shown in SEQ ID NO: 16; Preferably, the light chain comprises the light chain constant region (CL) shown in SEQ ID NO:

13.

8. The antibody according to any one of claims 1-6, wherein, The antibody is scFv-Fc; Preferably, the antibody comprises scFv and an Fc domain from the N-terminus to the C-terminus; or, comprises an Fc domain and scFv from the N-terminus to the C-terminus.

9. The antibody according to any one of claims 1-8, wherein, The antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 3, 4, and 5, and light chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 5, 6, and 7.

10. The antibody according to claim 9, wherein, The antigen-binding domain comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 2 or a sequence having at least 80% identity thereto.

11. The antibody according to any one of claims 1-10, which is a full-length antibody and comprises: a heavy chain including VH shown in SEQ ID NO: 1 and CH shown in any one of SEQ ID NOs: 14-17, and, a light chain including VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO: 13; Preferably, the antibody comprises: a heavy chain including VH shown in SEQ ID NO: 1 and CH shown in SEQ ID NO: 16, and a light chain including VL shown in SEQ ID NO: 2 and CL shown in SEQ ID NO:

13.

12. A multispecific antibody, which comprises the antibody according to any one of claims 1-11; Preferably, the multispecific antibody specifically binds to HBsAg and additionally specifically binds to other targets; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody.

13. A conjugate, which comprises the antibody according to any one of claims 1-11 or the multispecific antibody according to claim 12, and a conjugate moiety linked to the antibody or the multispecific antibody; Preferably, the conjugate moiety is selected from detectable labels or therapeutic agents; Preferably, the conjugate moiety is selected from immunomodulators, such as STING agonists or TLR agonists (such as TLR7 / 8 agonists).

14. An isolated nucleic acid molecule, which encodes (i) the antibody according to any one of claims 1-11 or its heavy and light chains, or (ii) the multispecific antibody according to claim 12.

15. A vector, which comprises the nucleic acid molecule according to claim 14; preferably, the vector is a cloning vector or an expression vector.

16. A host cell, which comprises the nucleic acid molecule according to claim 14 or the vector according to claim 15.

17. A method for preparing the antibody according to any one of claims 1-11 or the multispecific antibody according to claim 12, which comprises culturing the host cell according to claim 16 under conditions allowing the expression of the antibody or the multispecific antibody, and recovering the antibody or the multispecific antibody from the cultured host cell culture.

18. A pharmaceutical composition, which contains the antibody according to any one of claims 1-11, the multispecific antibody according to claim 12 or the conjugate according to claim 13, and a pharmaceutically acceptable carrier and / or excipient.

19. Use of the antibody according to any one of claims 1-11, the multispecific antibody according to claim 12, the conjugate according to claim 13 or the pharmaceutical composition according to claim 18 in the preparation of a drug for preventing and / or treating HBV infection or a disease associated with HBV infection (such as hepatitis B) in a subject (such as a human), and / or for reducing the serum levels of HBV DNA and / or HBsAg in a subject (such as a human).

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