Anti-HBsAg antibody for treating chronic hepatitis delta

Abstract

The invention relates to an anti-HBsAg antibody, an antibody fragment and application thereof in preventing and treating hepatitis D virus infection and related diseases. The present invention provides a method of neutralizing hepatitis delta virus infection comprising administering an effective amount of the antibody or antigen-binding fragment thereof to a patient in need thereof by injection or infusion.

Description

Technical Field

[0001] This invention relates to anti-hepatitis B surface antigen antibody (anti-HBsAg), antibody fragments, and their use in reducing the likelihood of hepatitis D virus infection or treating hepatitis D infection. HBsAg is a key viral protein that causes hepatitis D virus infection.

[0002] Anti-HBsAg antibodies and their fragments are potent, selective, fully humanized immunoglobulin G1 (IgG1)-neutralizing monoclonal antibodies that target the antigenic loop in all forms of HBsAg protein (L- / M- / S-HBsAg). HBsAg is a key viral protein responsible for recognizing, binding to, and facilitating the entry of hepatitis D virus (HDV) into hepatocytes. Therefore, they can bind to HBsAg on the surface of HDV, neutralize and clear the virus, thereby exerting anti-HDV efficacy. Background Art Hepatitis D virus (HDV) is the pathogen of chronic hepatitis D (CHD), the most severe type of viral hepatitis. HDV infection can occur concurrently with hepatitis B virus (HBV), or more commonly, in patients already infected with chronic hepatitis B virus. Co-infection is associated with more severe liver disease, leading to accelerated progression of cirrhosis, hepatocellular carcinoma, and liver failure.

[0003] Although nucleoside inhibitors are effective in suppressing HBV replication, they have no effect on HDV replication. Antiviral therapy is urgently needed to prevent the progression of diseases such as cirrhosis, end-stage liver disease, and HCC. Despite its side effects, low response rate, and high relapse rate, pegylated interferon (PegIFN) is still used as an off-label treatment for hepatitis D (HDV) (Bahcecioglu, Ispiroglu et al., 2015; Rizzetto and Smedile, 2015). Among new drugs for CHD treatment, bulevirtide, a myristyl peptide administered daily via subcutaneous injection, blocks HBV and HDV entry by targeting the host NTCP receptor and has received conditional marketing authorization in Europe (Masetti and Aghemo, 2021; Lampertico, Roulot et al., 2022). Lonafarnib, a farnesyltransferase inhibitor, can therefore inhibit HDV release and is currently undergoing late-stage clinical evaluation (Yurdaydin, Keskin et al., 2022).

[0004] The currently approved antiviral drug bulevirtide works by binding to and blocking the receptors (targets) that allow hepatitis D virus (HDV) and hepatitis B virus to enter liver cells. By preventing the virus from entering cells, bulevirtide can limit the replication capacity of HDV and its effects in the body, thereby alleviating disease symptoms. Therefore, we need more effective therapies to suppress hepatitis D infection. Summary of the Invention

[0005] This invention relates to neutralizing antibodies against hepatitis D virus and / or fragments thereof, as well as antibodies that reduce the amount of hepatitis B surface antigen (HBsAg).

[0006] This invention relates to an antibody, or an antigen-binding fragment thereof, that specifically binds to HBsAg, as disclosed in US21210221871A1 and WO2019229699.

[0007] The antibody or its antigen-binding fragment specifically binds to HBsAg. In one embodiment, the antibody or its antigen-binding fragment binds to HBsAg and its mutants.

[0008] This invention relates to an antibody, or an antigen-binding fragment thereof, that specifically binds to and neutralizes hepatitis D virus. In one embodiment, the antibody or its antigen-binding fragment neutralizes hepatitis D virus and hepatitis D virus containing an HBsAg mutation. In another embodiment, the antibody or its antigen-binding fragment reduces the level of HBsAg. In yet another embodiment, the antibody or its antigen-binding fragment reduces the level of circulating HBsAg in the blood.

[0009] This invention relates to an isolated antibody, said antibody or its antigen-binding fragment comprising: (i) The heavy chain variable region comprising: (a) HCDR1 (CDR-complementary determinant region) of SEQ ID NO: 9, (b) HCDR2 of SEQ ID NO: 10, and (c) HCDR3 of SEQ ID NO: 11; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 25, (e) LCDR2 of SEQ ID NO: 26, and (f) LCDR3 of SEQ ID NO: 27; (ii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 41, (b) HCDR2 of SEQ ID NO: 42, (c) HCDR3 of SEQ ID NO: 43; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 57, (e) LCDR2 of SEQ ID NO: 58, and (f) LCDR3 of SEQ ID NO: 59; (iii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 73, (b) HCDR2 of SEQ ID NO: 74, (c) HCDR3 of SEQ ID NO: 75; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 89, (e) LCDR2 of SEQ ID NO: 90, and (f) LCDR3 of SEQ ID NO: 91; (iv) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 105, (b) HCDR2 of SEQ ID NO: 106, (c) HCDR3 of SEQ ID NO: 107; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 121, (e) LCDR2 of SEQ ID NO: 122, and (f) LCDR3 of SEQ ID NO: 123; (v) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 137, (b) HCDR2 of SEQ ID NO: 138, and (c) HCDR3 of SEQ ID NO: 139; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 153, (e) LCDR2 of SEQ ID NO: 154, and (f) LCDR3 of SEQ ID NO: 155; (vi) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 169, (b) HCDR2 of SEQ ID NO: 170, (c) HCDR3 of SEQ ID NO: 171; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 185, (e) LCDR2 of SEQ ID NO: 186, and (f) LCDR3 of SEQ ID NO: 187; (vii) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 201, (b) HCDR2 of SEQ ID NO: 202, (c) HCDR3 of SEQ ID NO: 203; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 217, (e) LCDR2 of SEQ ID NO: 218, and (f) LCDR3 of SEQ ID NO: 219; (viii) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 233, (b) HCDR2 of SEQ ID NO: 234, (c) HCDR3 of SEQ ID NO: 235; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 249, (e) LCDR2 of SEQ ID NO: 250, and (f) LCDR3 of SEQ ID NO: 251; (ix) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 265, (b) HCDR2 of SEQ ID NO: 266, and (c) HCDR3 of SEQ ID NO: 267; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 281, (e) LCDR2 of SEQ ID NO: 282, and (f) LCDR3 of SEQ ID NO: 283; (x) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 297, (b) HCDR2 of SEQ ID NO: 298, and (c) HCDR3 of SEQ ID NO: 299; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 313, (e) LCDR2 of SEQ ID NO: 314, and (f) LCDR3 of SEQ ID NO: 315; (xi) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 329, (b) HCDR2 of SEQ ID NO: 330, and (c) HCDR3 of SEQ ID NO: 331; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 345, (e) LCDR2 of SEQ ID NO: 346, and (f) LCDR3 of SEQ ID NO: 347; (xii) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 361, (b) HCDR2 of SEQ ID NO: 362, (c) HCDR3 of SEQ ID NO: 363; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 377, (e) LCDR2 of SEQ ID NO: 378, and (f) LCDR3 of SEQ ID NO: 379; (xiii) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 393, (b) HCDR2 of SEQ ID NO: 394, (c) HCDR3 of SEQ ID NO: 395; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 409, (e) LCDR2 of SEQ ID NO: 410, and (f) LCDR3 of SEQ ID NO: 411; (xiv) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 425, (b) HCDR2 of SEQ ID NO: 426, and (c) HCDR3 of SEQ ID NO: 427; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 441, (e) LCDR2 of SEQ ID NO: 442, and (f) LCDR3 of SEQ ID NO: 443; (xv) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 457, (b) HCDR2 of SEQ ID NO: 458, and (c) HCDR3 of SEQ ID NO: 459; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 473, (e) LCDR2 of SEQ ID NO: 474, and (f) LCDR3 of SEQ ID NO: 475; or (xvi) The heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 489, (b) HCDR2 of SEQ ID NO: 490, (c) HCDR3 of SEQ ID NO: 491; and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 505, (e) LCDR2 of SEQ ID NO: 506, and (f) LCDR3 of SEQ ID NO: 507.

[0010] One or two amino acids in the CDR of the antibody are modified, deleted, or replaced.

[0011] The antibodies of the present invention retain at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity in the variable heavy chain region or the variable light chain region.

[0012] Antibody y is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv), or antibody fragment.

[0013] This invention relates to an isolated antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: (i) Contains the heavy chain variable region (vH) of SEQ ID NO: 18 and the light chain variable region (vL) of SEQ ID NO: 34; (ii) Containing the heavy chain variable region (vH) of SEQ ID NO: 50 and the light chain variable region (vL) of SEQ ID NO: 66; (iii) The heavy chain variable region (vH) comprising SEQ ID NO: 82 and the light chain variable region (vL) comprising SEQ ID NO: 98; (iv) The heavy chain variable region (vH) comprising SEQ ID NO: 114 and the light chain variable region (vL) comprising SEQ ID NO: 130; (v) Contains the heavy chain variable region (vH) of SEQ ID NO: 146 and the light chain variable region (vL) of SEQ ID NO: 162; (vi) Contains the heavy chain variable region (vH) of SEQ ID NO: 178 and the light chain variable region (vL) of SEQ ID NO: 194; (vii) Contains the heavy chain variable region (vH) of SEQ ID NO: 210 and the light chain variable region (vL) of SEQ ID NO: 226; (viii) Contains the heavy chain variable region (vH) of SEQ ID NO: 242 and the light chain variable region (vL) of SEQ ID NO: 258; (ix) includes the heavy chain variable region (vH) of SEQ ID NO: 274 and the light chain variable region (vL) of SEQ ID NO: 290; (x) Contains the heavy chain variable region (vH) of SEQ ID NO: 306 and the light chain variable region (vL) of SEQ ID NO: 322; (xi) includes the heavy chain variable region (vH) of SEQ ID NO: 338 and the light chain variable region (vL) of SEQ ID NO: 354; (xii) Contains the heavy chain variable region (vH) of SEQ ID NO: 370 and the light chain variable region (vL) of SEQ ID NO: 386; (xiii) The heavy chain variable region (vH) comprising SEQ ID NO: 402 and the light chain variable region (vL) comprising SEQ ID NO: 418; (xiv) contains the heavy chain variable region (vH) of SEQ ID NO: 434 and the light chain variable region (vL) of SEQ ID NO: 450; (xv) Contains the heavy chain variable region (vH) of SEQ ID NO: 466 and the light chain variable region (vL) of SEQ ID NO: 482; or (xvi) includes the heavy chain variable region (vH) of SEQ ID NO: 498 and the light chain variable region (vL) of SEQ ID NO: 514.

[0014] The antibody or its fragment retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity in the variable light chain or variable heavy chain region.

[0015] One, two, three, four, or five, but less than 10, amino acids in the variable light chain or variable heavy chain region of the antibody are modified, deleted, or replaced.

[0016] The antibody is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, human antibody, single-chain antibody (scFv), or antibody fragment.

[0017] The antibody or its fragment has a reduced level of glycosylation, or no glycosylation, or low fucosylation.

[0018] This invention relates to a pharmaceutical composition comprising the antibody or a fragment thereof, and further comprising a pharmaceutically acceptable carrier.

[0019] The pharmaceutical composition includes a pharmaceutically acceptable carrier containing histidine or sugar.

[0020] The sugar in the pharmaceutical composition is sucrose.

[0021] This invention relates to a pharmaceutical composition comprising multiple antibodies or antigen-binding fragments, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5% or more of the antibodies in the composition have α2,3-linked sialic acid residues.

[0022] This invention relates to a pharmaceutical composition comprising multiple antibodies or antigen-binding fragments, wherein the antibody does not comprise bipartite GlcNAc.

[0023] The pharmaceutical composition comprises an antibody or a fragment thereof, wherein the composition is prepared as a lyophilized product.

[0024] A method of neutralizing hepatitis D virus infection includes administering the composition or pharmaceutical composition to a patient who requires an effective amount of antibodies by injection or infusion.

[0025] The method includes diagnosing patients in need of hepatitis D viremia or hepatitis D viremia.

[0026] The method includes diagnosing hepatitis D surface antigen (HBsAg) in the blood or serum of patients in need.

[0027] This invention relates to a method for treating or reducing the likelihood of hepatitis D virus-related diseases, comprising administering an effective amount of antibodies to a patient in need by injection or infusion, wherein the disease is: liver failure, cirrhosis, or hepatocellular carcinoma.

[0028] The antibody or composition is further reconstituted prior to injection or infusion.

[0029] The antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[0030] The therapeutic agent is an antiviral agent.

[0031] The antiviral drugs include lamivudine, entecavir, and tenofovir or alpha-interferon.

[0032] The therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0033] The antagonist of the immune checkpoint inhibitor may be further selected from: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0034] The antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0035] The therapeutic agent is an additional anti-HBsAg antibody.

[0036] The antibody or its fragments will be used as a drug.

[0037] The antibody or a fragment thereof will be used to neutralize hepatitis D virus infection.

[0038] The antibody or fragment thereof will be used to treat or reduce the likelihood of liver failure, cirrhosis, and / or hepatocellular carcinoma.

[0039] Administered in combination with another therapeutic agent according to any of the above uses.

[0040] According to any of the above uses, including therapeutic agents that are antiviral agents, including siRNA, antisense, and gene editing methods targeting hepatitis B or hepatitis D.

[0041] According to any of the above uses, the antiviral agents include: lamivudine, entecavir, and tenofovir or alpha-interferon.

[0042] According to any of the above uses, including therapeutic agents that are antagonists of immune checkpoint inhibitors.

[0043] According to any of the above uses, the antagonists of immune checkpoint inhibitors are selected from: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0044] According to any of the above uses, the therapeutic agent is an agonist of an immunomodulator, wherein the agonist includes, but is not limited to, TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, or combinations thereof.

[0045] According to the above-described uses, the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0046] According to any of the above uses, including therapeutic agents, additional anti-HBsAg antibodies are included.

[0047] The present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment, a vector containing the nucleic acid, and a host cell containing the vector.

[0048] This invention relates to a diagnostic reagent comprising a labeled antibody or its antigen-binding fragment, wherein the label is selected from radiolabels, fluorophores, chromophores, imaging agents, and metal ions. Definition Unless otherwise stated, the following terms and phrases used in this invention shall have the following meanings: As used in this invention, the term "antibody" refers to a polypeptide of the immunoglobulin family that binds to a corresponding antigen in a non-covalent, reversible, and specific manner. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CHI, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs) and interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.

[0049] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the present invention). Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0050] The term "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refers to the hypervariable regions of the VL and VH. A CDR is a target protein binding site on the antibody chain and is specific to that target protein. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human VL or VH, comprising approximately 15-20% of the variable domain. CDRs can be referenced by their region and sequence. For example, "VHCDR1" or "HCDR1" refers to the first CDR in the heavy chain variable region. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for binding specificity. The remainder of the VL or VH, the so-called framework region, exhibits minor variations in its amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000).

[0051] The location of the CDR and the frame region can be determined using various well-known definitions in the art, such as those of Kabat, Chothia, IMGT, and AbM (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). The definition of the antigen-binding site is also described below: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); MacCallum et al., J.Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg M. JE (ed.), ProteinStructure Prediction, Oxford University Press, Oxford, 141-172 (1996). In the combined Kabat and Chothia numbering schemes, in some embodiments, the CDR corresponds to an amino acid residue that is part of the Kabat CDR, Chothia CDR, or both. For example, in some embodiments, CDR corresponds to amino acid residues 26-35 (HC CDR1), 50-65 (HC CDR2), and 95-102 (HC CDR3) in VH (e.g., mammalian VH, such as human VH); and amino acid residues 24-34 (LC CDR1), 50-56 (LC CDR2), and 89-97 (LC CDR3) in VL (e.g., mammalian VL, such as human VL).Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (according to "Kabat" numbering). Under IMGT, the CDR regions of the antibody can be determined using the IMGT / Domain Gap Align procedure.

[0052] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it will be recognized that the variable domains of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. Conventionally, the numbering of constant domains increases with their distance from the antigen-binding site or N-terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl-terminal domains of the heavy and light chains, respectively.

[0053] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen epitope (e.g., through binding, steric hindrance, stability / instability, spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab') fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; an F(ab)2 fragment, a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments (Ward et al., Nature 341:544-546, 1989) consisting of a VH domain; and isolated complementarity-determining regions (CDRs) or other epitope-binding fragments of the antibody. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via synthetic linkers using recombination methods, enabling the fabrication of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (“scFv”); see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single-chain antibodies are also intended to be included in the term “antigen-binding fragment.” These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art and are screened for their practicality in the same manner as intact antibodies.

[0054] Antigen-binding fragments can also be integrated into single-domain antibodies, large antibodies, micro antibodies, nanobodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can be grafted into peptide-based scaffolds, such as fibronectin type III (Fn3) (see US Patent 6,703,199 describing fibronectin peptide monomers). Antigen-binding fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which, together with a complementary light chain peptide, form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and US Patent 5,641,870).

[0055] As used in this invention, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a polypeptide, including antibody-antigen binding fragments having substantially the same amino acid sequence or originating from the same genetic source. The term also includes antibody molecular formulations consisting of a single molecule. Monoclonal antibody compositions exhibit single-specific binding specificity and affinity for a particular epitope.

[0056] As used herein, the term "human antibody" includes antibodies having a variable region, wherein both the frame and CDR regions are derived from human sequences. Furthermore, if an antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant versions of human germline sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0057] The human antibodies of this invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or somatic mutations in vivo, or conserved substitutions to promote stability or manufacture). As used herein, the term "recognition" refers to the discovery and interaction (e.g., binding) of an antibody or its antigen-binding fragment with an epitope, whether the epitope is linear or conformational. The term "epitaxy" refers to an antigenic site to which an antibody or antigen-binding fragment of this invention specifically binds. Epitopes may be formed from consecutive amino acids or from discontinuous amino acids arranged side-by-side through protein ternary folding. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids and have a unique spatial conformation. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, the epitope mapping protocol in *Methods in Molecular Biology*, Vol. 66, edited by GEMorris (1996)), or electron microscopy. A "complementary site" is the portion of an antibody that recognizes an antigenic epitope.

[0058] The terms “specific binding” or “selective binding,” when used to describe the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binder, refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological agents, such as in a biological sample, for example, in a blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or binder with specific binding specificity binds to a specific antigen at least twice the background and substantially not binds to other antigens present in the sample. In one aspect, under specified immunoassay conditions, an antibody or binder with specific binding specificity binds to a specific antigen at least ten (10) times the background and substantially not binds to other antigens present in the sample. Specific binding to an antibody or binder under such conditions may require selection of the antibody or reagent to determine its specificity for a specific protein. This selection may be achieved, as needed or where appropriate, by subtracting antibodies that cross-react with molecules of other species (e.g., mice or rats) or other subtypes. Alternatively, in some aspects, antibodies or antibody fragments that cross-react with certain desired molecules are selected.

[0059] The term "affinity" used in this invention refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable region of the antibody "arm" interacts with antigens at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0060] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. However, an isolated antibody that specifically binds to one antigen may be cross-reactive with other antigens. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0061] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known or inferred variable region amino acid sequences encoded by the human germline immunoglobulin variable region sequence. The corresponding human germline sequence can also be a human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with the reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be a frame region only, a complementarity-determining region only, a frame and a complementarity-determining region, a variable fragment (as defined above), or other combinations of sequences or subsequences containing a variable region. Sequence identity can be determined using the methods described in this invention, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence.

[0062] A variety of immunoassays can be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA is commonly used to select antibodies that specifically react with a protein (see, for example, Harlow & Lane, Using Antibodies, Laboratory Manual (1998), for describing immunoassays and conditions that can be used to determine specific immunoreactivity). Typically, the signal intensity produced by a specific or selective binding reaction is at least twice that of the background signal, and more typically, at least 10 to 100 times that of the background.

[0063] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (KD, time⁻¹) divided by the association rate constant (ka, time⁻¹, M⁻¹). The equilibrium dissociation constant can be measured using any method known in the art. The antibodies of the present invention typically have a value less than about 10. -7 Or 10 -8 The equilibrium dissociation constant of M is, for example, less than about 10. -9 M or 10 -10 M, in some respects, is less than approximately 10. -11 M, 10 -12 M or 10 -13 M. The term "bioavailability" refers to the systemic utilization (i.e., blood / plasma level) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates the time (rate) and total amount (degree) of a drug from its dosage form into systemic circulation.

[0064] As used herein, the phrase "consistently composed of..." refers to the genus or species of the active pharmaceutical ingredient included in the method or composition, and any excipients inactive for the intended purpose of the method or composition. In some aspects, the phrase "consistently composed of..." expressly excludes one or more additional active agents besides the anti-HBsAg antibody of the present invention.

[0065] The term "amino acid" refers to naturally occurring, synthetic, and non-natural amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon atom bonded to a hydrogen, carboxyl, amino, and R group, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methyl sulfone. These analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0066] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. For a specific nucleic acid sequence, a conservatively modified variant refers to a nucleic acid encoding the same or substantially the same amino acid sequence, or a nucleic acid encoding no amino acid sequence but whose sequences are substantially identical. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position designated by the codon for alanine, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. This nucleic acid variation is a "silent variant," a type of conservedly modified variant. Each nucleic acid sequence encoding a polypeptide in this invention also describes all possible silent variants of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variant of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0067] For polypeptide sequences, “conserved variants” include individual substitutions, deletions, or additions to the polypeptide sequence that result in the substitution of chemically similar amino acids. Conserved substitutions that provide functionally similar amino acids are well known in the art. Such conserved variants are complementary to polymorphic variants, interspecific homologs, and alleles, and are not excluded. The following eight groups contain amino acids that are conservedly substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some respects, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing amino acid sequences.

[0068] The term "optimized" as used in this invention refers to an altered nucleotide sequence that uses codons preferred in the production cells or organisms to encode the amino acid sequence. These production cells or organisms are typically eukaryotic cells, such as yeast cells, Pichia pastoris cells, fungal cells, Trichoderma cells, Chinese hamster ovary cells (CHO), or human cells. The optimized nucleotide sequence is engineered to fully or as much as possible retain the amino acid sequence originally encoded by the starting nucleotide sequence; this is also referred to as the "parental" sequence.

[0069] In the case of two or more nucleic acid or polypeptide sequences, the terms "percentage identical" or "percentage identity" refer to the degree to which two or more sequences or subsequences are identical. Two sequences are "identical" if they have the same amino acid or nucleotide sequence in the compared region. When performing maximum correspondence comparisons and alignments on a comparison window or specified region, two sequences are "substantially identical" if they have the same specified percentage of amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity, in the specified region, or, if not specified, throughout the entire sequence) when one of the following sequence comparison algorithms is used or measured by manual alignment and visual inspection. Optionally, identity is present in regions of at least about 30 nucleotides (or 10 amino acids) in length, or more preferably in regions of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0070] For sequence comparisons, one sequence typically serves as a reference sequence, which is then compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters.

[0071] As used in this invention, the "comparison window" includes segments of any consecutive positions selected from a group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, wherein, after optimal alignment of the two sequences, the sequence can be compared with a reference sequence of the same number of consecutive positions. Sequence alignment methods for comparison are well known in the art. The best sequence alignment for comparison can be achieved, for example, through local homology algorithms in Smith and Waterman in Adv. Appl. Math. 2:482c (1970), homology alignment algorithms in Needleman and Wunsch in J. Mol. Biol. 48:443 (1970), similarity search methods in Pearson and Lipman in Proc. Natl. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or through manual alignment and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, 2003).

[0072] Two examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST and BLAST2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). The algorithm first identifies high-scoring sequence pairs (HSPs) in the query sequence by recognizing short words of length W that match or satisfy a positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood word matches serve as the basis for initiating a search to find longer HSPs containing them. Word matching extends bidirectionally along each sequence until the cumulative alignment score increases. For nucleotide sequences, a cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word matching will stop extending in each direction when: the cumulative alignment score decreases by X numbers from its maximum value; the cumulative score drops to zero or lower due to the accumulation of one or more negative score residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and compares two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) with an alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and a comparison of the two strands.

[0073] The BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0074] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17, 1988), which has been integrated into the ALIGN program (version 2.0) using a PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970), which has been integrated into the GAP program of the GCG software package (available from the University of South Florida) using a BLOSUM62 or PAM250 matrix with GAP weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0075] Besides the percentage of sequence identity mentioned above, another indicator that two nucleic acid sequences or peptides are substantially identical is that the peptide encoded by the first nucleic acid exhibits immune cross-reactivity with antibodies generated against the peptide encoded by the second nucleic acid, as described below. Therefore, one peptide is usually substantially identical to the second peptide, for example, when the two peptides differ only in conserved substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complementary sequences hybridize under stringent conditions, as described below. Yet another indicator that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0076] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this invention, referring to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which may be synthetic, naturally occurring, or unnatural, have similar binding properties to the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise stated, specific nucleic acid sequences also implicitly encompass their conserved modified variants (e.g., degenerate codon substitutions) and their complementary sequences, as well as explicitly stated sequences. Specifically, as described below, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98). In the context of nucleic acids, the term "operable link" refers to a functional relationship between two or more segments of polynucleotides (e.g., DNA). Typically, it refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter or enhancer sequence is operably linked to the coding sequence. Typically, the promoter transcriptional regulatory sequence operably linked to the transcriptional sequence is physically adjacent to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not necessarily have to be physically adjacent to or close to the coding sequences that they enhance transcription.

[0077] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as to both naturally occurring and non-natural amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0078] The term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably in this document.

[0079] The terms "hepatitis," "hepatitis B virus," or "HBV" refer to members of the family Hepatoviridae and the genus Orthohepataviviridae. HBV is a double-stranded DNA virus.

[0080] HBsAgHBsAg "Hepatitis D," "Hepatitis D virus," or "HDV" refers to a virus belonging to the genus Orthohepadnavirus in the family Hepadnaviridae. It is a viral pathogen that requires the presence of hepatitis B virus (HBV) to replicate and cause infection. HDV is considered a defective virus because it cannot produce its own envelope proteins and therefore relies on HBV envelope proteins to form complete viral particles. It is primarily transmitted through blood contact or sexual contact. Compared to simple HBV infection, HDV infection can lead to more severe liver disease. HDV is a rare virus with a single-stranded circular RNA genome. This RNA genome is negative-stranded, meaning it can serve as a template for generating complementary positive-strand RNA.

[0081] "EC50" (half-maximal effective concentration) refers to the concentration at which a specific antibody induces a response that falls between the baseline control and the maximum possible effect after a specific exposure or treatment period. For example, EC50 refers to the antibody concentration at which 50% of a viral infection is neutralized.

[0082] "Neutralization" refers to the inhibition of viral infection of host cells, manifested as the loss of viral gene expression. Not bound by any single theory, the neutralization mechanism of a particular antibody may include blocking the interaction between viral capsid proteins and cell surface receptors, or disrupting any stage of viral entry and transport before the viral genome enters the host cell nucleus.

[0083] As used herein, the term "treatment" or "therapeutic method" refers to any disease or condition, in two senses: firstly, improving the disease or condition (i.e., slowing, halting, or alleviating the development of the disease or at least one of its clinical symptoms); secondly, alleviating or improving at least one bodily parameter, including parameters that the patient may not be aware of; and thirdly, regulating the disease or condition, whether physical (e.g., stabilizing perceptible symptoms), physiological (e.g., stabilizing bodily parameters), or both.

[0084] The term "reducing likelihood" refers to delaying the onset, development, or progression of a disease, infection, or symptom.

[0085] The terms "therapeutic acceptable dose" and "therapeutic effective dose" are used interchangeably to refer to a dose sufficient to achieve the desired effect (i.e., shrinking tumor size, inhibiting tumor growth, preventing metastasis, inhibiting or preventing viral, bacterial, fungal, or parasitic infections). In some respects, the therapeutic acceptable dose will not induce or cause adverse side effects. The therapeutic acceptable dose can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved. The "preventive effective dose" and "therapeutic effective dose" of the molecules of this invention can respectively prevent the occurrence of disease symptoms (including symptoms associated with polyomavirus infection) or reduce their severity.

[0086] "Combined administration" refers to the presence of two active agents in an individual's bloodstream simultaneously. The active agents in combined administration can be administered concurrently or sequentially. Attached Figure Description

[0087] Figure 1A The change of Log10 HDV RNA over time in 10 subjects in the F1 cohort, who were treated with 300 mg of HBsAgBJ once a week.

[0088] Figure 1B Changes in HDV RNA relative to baseline in 10 subjects in the F1 cohort at different treatment weeks.

[0089] Figure 2A Changes in ALT over time in 10 subjects in the F1 cohort who received 300 mg HBsAgBJ once a week.

[0090] Figure 2B Changes in ALT relative to baseline in 10 subjects in the F1 cohort at different treatment weeks. Detailed Implementation

[0091] This invention provides antibodies that bind to and neutralize hepatitis D, and antibody fragments (e.g., antigen-binding fragments). Furthermore, this invention provides antibodies with desirable pharmacokinetic characteristics and other desirable properties, thus enabling them to be used to treat or reduce the likelihood of hepatitis D-related liver failure, cirrhosis, or hepatocellular carcinoma. This invention also provides pharmaceutical compositions comprising said antibodies, and methods for preparing and using such pharmaceutical compositions for the prevention and treatment of hepatitis D infection and related diseases.

[0092] Anti-HBsAg antibody This invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg. The antibodies or antibody fragments (e.g., antigen-binding fragments) of this invention include, but are not limited to, the isolated human monoclonal antibodies or fragments thereof described in the following examples.

[0093] This invention provides, in some aspects, antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having an amino acid sequence of SEQ ID NO: 18, 50, 82, 114, 146, 178, 210, 242, 274, 306, 338, 370, 402, 434, 466, or 498 (Table 1). This invention also provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VHCDR having an amino acid sequence having any one of the VH CDRs listed in Table 1. In a particular aspect, this invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies comprise (or optionally constitute) one, two, three, or more VH CDRs having an amino acid sequence having any one of the VH CDRs listed in Table 1.

[0094] This invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VL domain having an amino acid sequence of SEQ ID NO: 34, 66, 98, 130, 162, 194, 226, 258, 290, 322, 354, 386, 418, 450, 482, or 514 (Table 1). This invention also provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 1. Specifically, this invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to HBsAg, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise (or optionally constitute) one, two, three, or more VL CDRs having an amino acid sequence of any one of the VL CDRs listed in Table 1.

[0095] Other antibodies or antibody fragments of the present invention (e.g., antigen-binding fragments) include mutated amino acids, but have at least 60%, 70%, 80%, 90%, or 95% identity in the CDR region with the CDR region shown in the sequences described in Table 1. In some aspects, it includes mutated amino acid sequences wherein the number of mutated amino acids in the CDR region does not exceed 1, 2, 3, 4, or 5 compared to the CDR region shown in the sequences described in Table 1.

[0096] This invention also provides nucleic acid sequences for the VH, VL, full-length heavy chain, and full-length light chain encoding antibodies that specifically bind to HBsAg. These nucleic acid sequences can be optimized for expression in mammalian cells. Table 1 Anti-HBV Antibodies Other antibodies included in this invention comprise antibodies in which the amino acids or nucleic acids encoding the amino acids have been mutated; but possess at least 60%, 70%, 80%, 90%, or 95% identity with the sequences described in Table 1. In some aspects, it comprises mutated amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids are mutated in the variable regions compared to those shown in the sequences described in Table 1, while retaining substantially the same therapeutic activity.

[0097] Because these antibodies can bind to HBsAg, VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding those amino acids) can be "mixed" to generate other HBsAg-binding antibodies. Such "mixed" HBsAg-binding antibodies can be detected using binding detection methods known in the art, such as ELISA and other detection methods described in the Examples section. When these chains are mixed, the VH sequence in a specific VH / VL pair should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence in a specific full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Likewise, the VL sequence in a specific VH / VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence in a specific full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence. Therefore, in one aspect, the present invention provides an isolated monoclonal antibody or its antigen-binding region, comprising: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 18, 50, 82, 114, 146, 178, 210, 242, 274, 306, 338, 370, 402, 434, 466 or 498 (Table 1); and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 34, 66, 98, 130, 162, 194, 226, 258, 290, 322, 354, 386, 418, 450, 482 or 514 (Table 1); wherein the antibody specifically binds to HBsAg.

[0098] In another aspect, the present invention provides (i) an isolated monoclonal antibody having: a full-length heavy chain comprising an amino acid sequence selected from SEQ ID NO: 20, 52, 84, 116, 148, 180, 212, 244, 276, 308, 340, 372, 404, 436, 468 or 500 (Table 1) optimized for expression in mammalian cells, and a full-length light chain comprising an amino acid sequence selected from SEQ ID NO: 36, 68, 100, 132, 164, 196, 228, 260, 292, 324, 356, 388, 420, 452, 484 or 516 (Table 1) optimized for expression in mammalian cells, or (ii) a functional protein comprising its antigen-binding portion.

[0099] In another aspect, the present invention provides an HBsAg-binding antibody comprising heavy and light chain CDR1, CDR2, and CDR3, or combinations thereof, as described in Table 1. The amino acid sequence of VH CDR1 of the antibody is shown in SEQ ID NO: 9, 41, 73, 105, 137, 169, 201, 233, 265, 297, 329, 361, 393, 425, 457, or 489. The amino acid sequence of VH CDR2 of the antibody is shown in SEQ ID NO: 10, 42, 74, 106, 138, 170, 202, 234, 266, 298, 330, 362, 394, 426, 458, or 490. The amino acid sequence of VH CDR3 of the antibody is as shown in SEQ ID NO: 11, 43, 75, 107, 139, 171, 203, 235, 267, 299, 331, 363, 395, 427, 459, or 491. The amino acid sequence of VL CDR1 of the antibody is as shown in SEQ ID NO: 25, 57, 89, 121, 153, 185, 217, 249, 281, 313, 345, 377, 409, 441, 473, or 505. The amino acid sequence of VL CDR2 of the antibody is as shown in SEQ ID NO: 26, 58, 90, 122, 154, 186, 218, 250, 282, 314, 346, 378, 410, 442, 474, or 506. The amino acid sequence of the antibody's VL CDR3 is shown in SEQ ID NO: 27, 59, 91, 123, 155, 187, 219, 251, 283, 315, 347, 379, 411, 443, 475 or 507.

[0100] Given that each of these antibodies can bind to HBsAg, and that antigen-binding specificity is primarily provided by CDR1, 2, and 3 regions, VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences can be “mixed” (i.e., CDRs from different antibodies can be mixed, but each antibody must contain both VH CDR1, 2, and 3 and VL CDR1, 2, and 3 to produce other HBsAg-binding molecules). Such “mixed” HBsAg-binding antibodies can be tested using binding assays known in the art and those described in the examples (e.g., ELISA). When VHCDR sequences are mixed, the CDR1, CDR2, and / or CDR3 sequences of a specific VH sequence should be substituted for structurally similar CDR sequences. Similarly, when VL CDR... When mixing sequences, the CDR3 sequence of specific VL sequences of CDR1 and CDR2 should be replaced with a structurally similar CDR sequence. It will be apparent to those skilled in the art that new VH and VL sequences can be generated by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences in the CDR sequences of the monoclonal antibodies shown herein.

[0101] Therefore, the present invention provides an isolated monoclonal antibody or its antigen-binding region, comprising a heavy chain CDR1 containing an amino acid sequence selected from SEQ ID NO: 9, 41, 73, 105, 137, 169, 201, 233, 265, 297, 329, 361, 393, 425, 457 or 489; a heavy chain CDR2 containing an amino acid sequence selected from SEQ ID NO: 10, 42, 74, 106, 138, 170, 202, 234, 266, 298, 330, 362, 394, 426, 458 or 490; and containing an amino acid sequence selected from SEQ ID NO: 11, 43, 75, 107, 139, 171, 203, 235, 267, 299, 331, 363, 395, 427, 459 or 491. The heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, 57, 89, 121, 153, 185, 217, 249, 281, 313, 345, 377, 409, 441, 473, or 505; the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, 58, 90, 122, 154, 186, 218, 250, 282, 314, 346, 378, 410, 442, 474, or 506; and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27, 59, 91, 123, 155, 187, 219, 251, 283, 315, 347, 379, 411, 443, 475, or 507. The amino acid sequence; wherein the antibody specifically binds to HBsAg and neutralizes HDV infection by blocking entry into and consuming HBsAg-containing particles.

[0102] In some respects, antibodies that specifically bind to HBsAg are the antibodies or antibody fragments (e.g., antigen-binding fragments) described in Table 1.

[0103] 1. Antibody identification This invention provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to HBsAg and neutralize HDV infection by blocking entry into and consuming HBsAg-containing particles. In some respects, the antibodies and antibody fragments can bind to the same epitopes in all four hepatitis serotypes.

[0104] This invention also provides antibodies and antibody fragments (e.g., antigen-binding fragments) that bind to the same epitopes as the HBsAg antibodies described in Table 1. Therefore, other antibodies and antibody fragments (e.g., antigen-binding fragments) can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibiting binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of the antibodies and antibody fragments (e.g., antigen-binding fragments) of this invention to HBsAg indicates that the test antibody can compete with the antibody or antibody fragment (e.g., antigen-binding fragment) for binding to HBsAg; according to a non-limiting theory, such antibodies can bind to the same or related (e.g., structurally similar or spatially proximate) epitopes on HBsAg with their competing antibodies or antibody fragments (e.g., antigen-binding fragments). In one aspect, antibodies that bind to the same epitopes on HBsAg as the antibodies or antibody fragments (e.g., antigen-binding fragments) of this invention are human or humanized monoclonal antibodies. These human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0105] 2. Further changes to the Fc region framework This invention discloses specific HBsAg antibodies. These antibodies include modified antibodies or antigen-binding fragments thereof, which further comprise modifications to framework residues in the VH and / or VL regions, for example, to improve antibody properties. Typically, such framework modifications are intended to reduce the immunogenicity of the antibody. For example, one approach is to “reverse mutation” one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody originated. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody originated. To restore the framework region sequence to its germline conformation, the somatic mutation can be “reverse mutation” to the germline sequence, for example, through site-directed mutagenesis. Such “reverse mutation” antibodies are also included.

[0106] Another type of framework modification involves mutating one or more residues within a framework region, or even within one or more CDR regions, to remove a T-cell epitope, thereby reducing the potential immunogenicity of the antibody. This approach is also known as “deimmunization,” and is described in more detail by Carr et al. in U.S. Patent Publication No. US 2003 / 0153043.

[0107] In addition to modifications within the frame region or CDR region, or as an alternative, antibodies can be engineered to modify the Fc region, typically to alter one or more functional properties, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, antibodies can be chemically modified (e.g., by attaching one or more chemical moieties to the antibody) or modified to alter their glycosylation, thereby again changing one or more functional properties. These aspects will be described in further detail below. On the one hand, the CH1 hinge region can be modified to alter the number of cysteine ​​residues, for example, by increasing or decreasing them. Bodmer et al. further described this method in US Patent No. 5,677,425. Changing the number of cysteine ​​residues in the CH1 hinge region can, for example, promote the assembly of light and heavy chains, or increase or decrease antibody stability.

[0108] On the other hand, mutations are made in the Fc hinge region of the antibody to reduce its biological half-life. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment, resulting in a weakening of the antibody's binding to staphylococcal protein A (SpA) relative to the binding of SpA in the native Fc hinge region. Ward et al. further described this method in detail in US Patent No. 6,165,745.

[0109] In other respects, the effector function of an antibody can be altered by changing the Fc region through the substitution of at least one amino acid residue for a different amino acid residue. For example, one or more amino acids can be substituted with different amino acid residues to change the antibody's affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in U.S. Patent Nos. US 5,624,821 and US 5,648,260, both proposed by Winter et al.

[0110] On the other hand, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues, thereby altering the C1q binding of the antibody and / or reducing or eliminating complement-dependent cytotoxicity (CDC). This method is described, for example, in U.S. Patent No. US6,194,551 to Idusogie et al.

[0111] In another aspect, one or more amino acid residues are altered to change the antibody's ability to fix complement. This method is described, for example, in PCT disclosure WO 94 / 29351 by Bodmer et al. In one specific aspect, one or more amino acids of the antibody or its antigen-binding fragment of the present invention are replaced by one or more isotype amino acid residues for IgG1 subclass and κ isotype. The isotype amino acid residues also include, but are not limited to, the heavy chain constant regions of IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the κ isotype, as described by Jefferis et al. in MAbs. 1:332-338 (2009).

[0112] In another aspect, the Fc region is modified by modifying one or more amino acids to enhance the ability of antibodies to induce antibody-dependent cytotoxicity (ADCC) and / or increase antibody affinity for the Fcγ receptor. This method is described, for example, in Presta's PCT publication WO 00 / 42072. Furthermore, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been located, and variants with improved binding affinity have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0113] In another aspect, the glycosylation of antibodies is modified. For example, glycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered, for example, to increase the antibody's affinity for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, thereby eliminating one or more variable region framework glycosylation sites, thus eliminating glycosylation at that site. Such glycosylation can increase the antibody's affinity for the antigen. This approach is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0114] Alternatively or additionally, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. This carbohydrate modification can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art, which can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, European patent application EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation. Presta's PCT publication WO03 / 035835 describes a variant CHO cell line, Lec13, which exhibits reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in this host cell line (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO 99 / 54342 describes a cell line engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), resulting in antibodies expressed in the engineered cell line exhibiting increased bifurcated GlcNac structure, thereby increasing ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17: 176-180, 1999).

[0115] On the other hand, antibodies can be modified to extend their biological half-life. Several methods are available to achieve this. For example, one or more mutations such as T252L, T254S, and T256F can be introduced, as described in Ward's authorized U.S. Patent 6,277,375. Alternatively, to extend the biological half-life, the antibody can be modified within the CH1 or CL region to include a salvage receptor-binding epitope derived from two loops of the CH2 domain of the IgG Fc region, as described by Presta et al. in U.S. Patents 5,869,046 and 6,121,022.

[0116] To minimize ADCC activity, specific mutations in the Fc region can produce "Fc-silencing" antibodies, which exhibit minimal interaction with effector cells. Typically, the "IgG Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including both the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as containing amino acid residues from position C226 or P230 to the C-terminus of the IgG antibody. Residues in the Fc region are numbered using the Kabat EU index. The C-terminal lysine residue (residue K447) in the Fc region may be removed during antibody production or purification.

[0117] Silent effector functions can be acquired through mutations in the antibody Fc region, as described in the art: LALA and N297A mutations (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); and D265A mutations (Baudino et al., 2008, J. Immunol. 181: 6664-69), see also Heusser et al. WO2012065950. Examples of Fc-silenced IgG1 antibodies include the LALA mutant containing L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silencing IgG1 antibody is the DAPA (D265A, P329A) mutation (US Patent 6,737,056). Another type of silenced IgG1 antibody contains the N297A mutation, resulting in a glycosylated / non-glycosylated antibody.

[0118] Fc silencing antibodies have no or low ADCC activity, meaning that Fc silencing antibodies exhibit ADCC activity of less than 50% specific cell lysis (low ADCC activity) or less than 1% specific cell lysis (no ADCC activity).

[0119] 3. Antibody production Anti-HBsAg antibodies and their antibody fragments (e.g., antigen-binding fragments) can be prepared by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers. Full-length monoclonal antibodies can be obtained, for example, through hybridoma or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0120] The present invention further provides polynucleotides encoding the antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or segments containing the complementarity-determining regions described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide selected from SEQ ID NO: 19, 51, 83, 115, 147, 179, 211, 243, 275, 307, 339, 371, 403, 435, 467, or 499. In some respects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide selected from SEQ ID NO: 35, 67, 99, 131, 163, 195, 227, 259, 291, 323, 355, 387, 419, 451, 483, or 515.

[0121] In some respects, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotides of SEQ ID NO:21, 53, 85, 117, 149, 181, 213, 245, 277, 309, 341, 373, 405, 437, 469, or 501. In some respects, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotides of SEQ ID NO:37, 69, 101, 133, 165, 197, 229, 261, 293, 325, 357, 389, 421, 453, 485, or 517.

[0122] The polynucleotides of this invention can encode only the variable region sequence of an anti-HBsAg antibody. They can also encode both the variable and constant regions of the antibody simultaneously. Some polynucleotide sequences encode polypeptides containing the heavy and light chain variable regions of an example anti-HBsAg antibody. Other polynucleotides encode two polypeptide fragments that are substantially identical to the heavy and light chain variable regions of the antibody, respectively.

[0123] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding anti-HBsAg antibodies or their binding fragments. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method proposed by Narang et al. in Meth. Enzymol. 68:90, 1979; the phosphodiester method proposed by Brown et al. in Meth. Enzymol. 68:109, 1979; the diethylphosphite method proposed by Beaucage et al. in Tetra. Lett. 22:1859, 1981; and the solid-phase support method in US Patent 4,458,066. Introducing mutations into polynucleotide sequences via PCR can be performed as described in the following literature, for example: PCR Techniques: Principles and Applications of DNA Amplification, HAErlich (ed.), Freeman Press, NY, NY, 1992; PCR Procedures: Methods and Applications, Innis et al. (ed.), Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0124] This invention also provides expression vectors and host cells for generating the aforementioned anti-HBsAg antibodies. Various expression vectors can be used to express polynucleotides encoding anti-HBsAg antibody chains or binding fragments. Both viral and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, free vectors (typically carrying expression cassettes for protein or RNA expression), and human artificial chromosomes (see, for example, Harrington et al., Nat Genet, 15:345, 1997). For example, non-viral vectors for expressing anti-HBsAg polynucleotides and peptides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include those based on retroviruses, adenoviruses, adeno-associated viruses, and herpesviruses, as well as those based on SV40, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semleeki Forest Virus (SFV). See Brent et al., ibid.; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0125] The choice of expression vector depends on the host cell to which the vector is intended to be expressed. Typically, expression vectors contain promoters and other regulatory sequences (e.g., enhancers) that are operatively linked to a polynucleotide encoding an anti-HBsAg antibody chain or fragment. In some respects, inducible promoters are used to prevent the expression of the inserted sequence under inducible conditions. Inducible promoters include, for example, arabinose promoters, lacZ promoters, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be amplified under non-inducible conditions without biasing the population towards sequences encoding the host cell that are more tolerant of the product being expressed. In addition to promoters, other regulatory elements may be needed or desired for efficient expression of the anti-HBsAg antibody chain or fragment. These elements typically include an ATG start codon and an adjacent ribosome-binding site or other sequences. Furthermore, expression efficiency can be enhanced by incorporating enhancers appropriate for the cell system used (see, for example, Scharf et al., ResultsProbl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol. 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to enhance expression in mammalian host cells.

[0126] Expression vectors can also provide a secretion signal sequence location to form a fusion protein with a polypeptide encoded by an inserted anti-HBsAg antibody sequence. More commonly, the inserted anti-HBsAg antibody sequence is linked to the signal sequence prior to insertion into the vector. Vectors used to receive sequences encoding variable domains of the light and heavy chains of anti-HBsAg antibodies sometimes also encode a constant region or a portion thereof. The vector allows the variable and constant regions to be expressed as a fusion protein, thereby producing the complete antibody or a fragment thereof. Typically, the constant region is human.

[0127] The host cells used to carry and express the anti-HBsAg antibody chain can be prokaryotic or eukaryotic. *Escherichia coli* is a prokaryotic host suitable for cloning and expressing the polynucleotides of this invention. Other suitable microbial hosts include *Bacillus*, such as *Bacillus subtilis*, and other Enterobacteriaceae, such as *Salmonella*, *Serratia marcescens*, and various *Pseudomonas* species. Expression vectors can also be constructed in these prokaryotic hosts, typically containing expression control sequences (e.g., origin of replication) compatible with the host cell. Furthermore, various known promoters can be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or promoter systems derived from *λ* phage. Promoters typically control expression, optionally containing an operon sequence and having ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-HBsAg antibodies. Combinations of insect cells with baculovirus vectors can also be used.

[0128] In other respects, mammalian host cells are used to express and produce the anti-HBsAg antibodies of the present invention. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., the myeloma hybridoma clones described in the examples) or mammalian cell lines carrying exogenous expression vectors. These cell lines include any normal or abnormal or immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. A general discussion of the expression of peptides using mammalian tissue cell cultures has been described, for example, in Winnacker's book *From Genes to Clones* (VCH Publishers, NY, 1987). Expression vectors for mammalian host cells may contain expression control sequences, such as origin of replication, promoters, and enhancers (see Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive promoters, cell type-specific promoters, stage-specific promoters, and / or regulated promoters. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-induced MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-induced CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0129] The methods for introducing expression vectors containing the target polynucleotide sequence vary depending on the cell host type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (see Sambrook et al., ibid.). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, shock transfection, virions, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced DNA uptake, and in vitro transduction. Stable expression is usually required for long-term, high-yield production of recombinant proteins. For example, expression vectors containing viral origin of replication or endogenous expression elements and selection marker genes can be used to prepare cell lines that stably express anti-HBsAg antibody chains or binding fragments. After vector introduction, cells can be grown in enrichment medium for 1–2 days before being transferred to selection medium. The purpose of selection markers is to confer resistance to selection; their presence allows cells that have successfully expressed the introduced sequence to grow in the selection medium. Stable transfected cells with resistance can be proliferated using tissue culture techniques appropriate to the cell type.

[0130] Therapeutic and diagnostic uses The antibodies and antibody fragments (e.g., antigen-binding fragments) of this invention can be used for a variety of purposes, including but not limited to hepatitis D virus infection and disease. In some aspects, the antibodies and antibody fragments (e.g., antigen-binding fragments) can be used to neutralize hepatitis D infection and to prevent or treat cirrhosis or liver cancer. The methods described can be in vitro, ex vivo, or in vivo.

[0131] On the one hand, antibodies and antibody fragments (e.g., antigen-binding fragments) can be used to detect the presence of HBsAg in biological samples. The term "detection" as used herein encompasses both quantitative and qualitative detection. In some respects, biological samples contain cells or tissues. In some respects, said tissues include normal and / or cancerous tissues with higher HBsAg expression levels than other tissues.

[0132] On one hand, the present invention provides a method for detecting the presence of HBsAg or hepatitis D virus in a biological sample. In some aspects, the method includes contacting the biological sample with an anti-HBsAg antibody under conditions allowing antibody-antigen binding, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, urine or blood samples.

[0133] The present invention also includes a method for diagnosing diseases associated with HBsAg expression. In some aspects, the method includes contacting test cells with an anti-HBsAg antibody; determining the expression level of HBsAg in the test cells (quantitative or qualitative) by detecting the binding of the antibody to HBsAg; and comparing the infection level in the test cells with the hepatitis D virus infection level in control cells (e.g., normal cells of the same tissue origin as the test cells or cells not infected with the virus), wherein a higher level of HBsAg in the test cells indicates the presence of a disease associated with hepatitis D infection. In some aspects, the test cells are taken from an individual suspected of being infected with hepatitis D virus.

[0134] In some aspects, the diagnostic or detection methods (such as those described above) include detecting the binding of HBsAg antibodies to hepatitis D virus-infected cells. An exemplary detection method for detecting the binding of anti-HBsAg antibodies to hepatitis D virus-infected cells is the "FACS" assay.

[0135] Several other methods can also be used to detect the binding of anti-HBsAg antibodies. These methods include, but are not limited to, antigen binding assays well known in the art, such as Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, and immunohistochemistry (IHC).

[0136] In some respects, anti-HBsAg antibodies are labeled. Labeling includes, but is not limited to, directly detectable markers or portions (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detectable portions (e.g., through enzymatic reactions or molecular interactions) such as enzymes or ligands.

[0137] In some respects, anti-HBsAg antibodies are immobilized on an insoluble matrix. Immobilization requires the separation of the anti-HBsAg antibody from free hepatitis D protein in solution. This is typically achieved by: rendering the anti-HBsAg antibody insoluble prior to the detection procedure, for example by adsorption onto a water-insoluble matrix or surface (Bennich et al., US Patent 3,720,760); or by covalent coupling (e.g., cross-linking with glutaraldehyde); or by rendering the anti-HBsAg antibody insoluble after it has formed a complex with the HBsAg protein, for example by immunoprecipitation.

[0138] Any of the above-mentioned diagnostic or testing procedures can be performed using the anti-HBsAg antibody described in this invention, in place of or with the addition of another anti-HBsAg antibody.

[0139] On one hand, the present invention provides a method for treating a disease, reducing the likelihood of developing a disease, or improving a disease, comprising administering the antibody or antibody fragment (e.g., an antigen-binding fragment) to a patient to treat the disease. In some aspects, the disease treated with the antibody or antibody fragment (e.g., an antigen-binding fragment) is hepatitis D virus infection. Examples of hepatitis D diseases that can be treated and / or prevented include, but are not limited to, liver failure, cirrhosis, and hepatocellular carcinoma. In some aspects, the infection is characterized by anti-HBsAg antibodies or antibody fragments (e.g., antigen-binding fragments) that can specifically bind to cells expressing HBsAg.

[0140] This invention provides a method for treating hepatitis D virus infection and liver failure, cirrhosis, and / or hepatocellular carcinoma, comprising administering a therapeutically effective amount of an antibody or antibody fragment (e.g., an antigen-binding fragment). In some respects, the subject is a human.

[0141] In some respects, methods for reducing hepatitis D virus infection include administering a therapeutically effective amount of antibody or antibody fragment (e.g., antigen-binding fragment) to the subject. In some respects, the subject is human. In some respects, the subject is immunosuppressed, immunocompromised, or has reduced immune function. For immunosuppressed subjects, the immunosuppressive dose may be increased or decreased due to the therapeutic effect of anti-HBsAg antibodies.

[0142] For the treatment of hepatitis D virus infection, the appropriate dose of HBsAg antibodies or antibody fragments (e.g., antigen-binding fragments) depends on a variety of factors, such as the type of infection being treated, the severity and duration of the infection, the responsiveness of the infection, the development of viral resistance to treatment, previous treatments, the patient's clinical history, and so on. Antibodies can be administered as a single dose or over a series of treatments lasting from several days to several months until a cure or reduction in infection (e.g., reduced viral urination or viral damage to the liver). The optimal dosing regimen can be calculated by measuring the accumulation of the drug in the patient and varies depending on the relative potency of individual antibodies or antibody fragments (e.g., antigen-binding fragments). In some aspects, the dosage is from 0.01 mg to 100 mg per kilogram of body weight (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg), and may be administered once or more daily, weekly, monthly, or annually. In some aspects, the antibody or antibody fragment of the present invention (e.g., antigen-binding fragment) is administered once every two weeks or once every three weeks. The attending physician can estimate the repetition rate of administration based on the measured half-life and the concentration of the antibody in body fluids or tissues.

[0143] combination therapy In some cases, the antibodies or antibody fragments of the present invention (e.g., antigen-binding fragments) are combined with other therapeutic agents, such as other antiviral agents, anti-allergy agents, antiemetics (or antiemetic drugs), analgesics, cytoprotective agents, immunosuppressants, and combinations thereof.

[0144] As used herein, the term "drug combination" refers to a fixed or non-fixed combination in the form of a dose unit, or a kit for combination administration, wherein two or more therapeutic agents may be administered simultaneously or separately at time intervals, particularly when such time intervals allow the combination partners to exhibit synergistic effects.

[0145] The term "combination therapy" refers to the use of two or more therapeutic agents to treat the disease or infection described herein. The administration includes co-administration in a substantially simultaneous manner, such as administration in a single capsule containing a fixed proportion of the active ingredients. Alternatively, the administration includes co-administration in multiple containers (e.g., capsules, powders, and liquids), or administration of each active ingredient in a separate container. Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, the administration also includes sequential administration of each type of therapeutic agent, approximately simultaneously or at different times. In either case, the treatment regimen will exert the beneficial effect of the combination of drugs in treating the disease or condition described herein.

[0146] Combination therapy can produce a “synergistic” or “synergistic effect”, meaning that the combined effect of the active ingredients is greater than the sum of the effects of using each ingredient alone. Synergistic effects can be achieved when the active ingredients are: (1) simultaneously formulated and administered or delivered in combination unit dosage forms; (2) delivered alternately or in parallel as individual formulations; or (3) using other methods. In alternating therapy, synergistic effects can be achieved when the ingredients are administered or delivered sequentially, for example, by using different syringes for different injections. Typically, in alternating therapy, the effective doses of each active ingredient are administered sequentially (i.e., consecutively); while in combination therapy, the effective doses of two or more active ingredients are administered together.

[0147] On one hand, the present invention provides a method for treating hepatitis D infection by administering anti-HBsAg antibodies and immunosuppressive therapy in combination to a subject in need. The anti-HBsAg antibody reduces the circulating HBsAg level and induces an immune response against hepatitis D virus infection induced by immunosuppressive therapy, either before or after administration of the immunosuppressive therapy. Examples of immunosuppressive therapies include, but are not limited to, monophosphate dehydrogenase inhibitors, purine synthesis inhibitors, calcineurin inhibitors, or mTOR inhibitors. Specific examples of immunosuppressive therapies include, but are not limited to, mycophenolate mofetil (MMF), sodium mycophenolate mofetil, azathioprine, tacrolimus, sirolimus, and cyclosporine.

[0148] In one embodiment, an anti-HBsAg antibody combination is used in combination with another anti-HBsAg antibody for the combined treatment of HBV and HDV.

[0149] In one embodiment, the anti-HBsAg antibody combination is used in conjunction with another antiviral agent, antagonist, or agonist for combination therapy of HBV and HDV.

[0150] According to any of the above uses, the therapeutic agent is an antiviral agent, including siRNA, antisense, and gene editing methods targeting hepatitis B or hepatitis D.

[0151] According to any of the above uses, the antiviral agent is: lamivudine, entecavir, and tenofovir or alpha-interferon.

[0152] According to any of the above uses, the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0153] According to any of the above uses, the antagonist of the immune checkpoint inhibitor is selected from: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0154] According to any of the foregoing uses, the therapeutic agent is an agonist of an immunomodulator, wherein the agonist includes, but is not limited to, TLR2, TLR3, TLR4, TLR7, TLR8, TLR9, or combinations thereof.

[0155] In another embodiment, the anti-HBsAg antibody combination is used in conjunction with a vaccine for both preventative and therapeutic purposes. When used for prevention, the vaccine composition of the present invention is administered before the appearance of any symptoms or clinical signs of pathogen infection. Prophylactic administration of the composition is intended to prevent or mitigate any subsequent infection. When used for prevention, a composition of antibodies or combinations thereof of the present invention is administered before the appearance of any symptoms or clinical signs of disease. Prophylactic administration of the composition is intended to prevent or mitigate one or more symptoms or clinical signs associated with the disease.

[0156] When used for treatment, vaccines are administered after symptoms or clinical signs of actual infection have been detected. When used for treatment, the antibodies or combinations thereof of the present invention are administered after symptoms or clinical signs of disease have been detected. Therapeutic administration of the compounds is intended to alleviate the symptoms or clinical signs of the disease.

[0157] In one embodiment, the anti-HBsAg antibody is used in combination with a PD-1 inhibitor, such as those described in WO2015 / 026684 or WO2016 / 057846. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab, Pembrolizumab, or Pidilizumab.

[0158] In some embodiments, the anti-PD-1 antibody is Nivolumab. Alternative names for Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is Nivolumab (CAS Registry No.: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone number 5C4) and other human monoclonal antibodies that specifically bind to PD1 have been disclosed in U.S. Patents US 8,008,449 and WO2006 / 121168. In one embodiment, the PD-1 inhibitor is Nivolumab and has the sequence disclosed therein (or a sequence substantially the same as or similar to it, e.g., a sequence with at least 85%, 90%, 95%, or higher identity to the specified sequence).

[0159] In some embodiments, the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab (also known as Lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA.RTM; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies have been disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, US Patent 8,354,509, and WO2009 / 114335.

[0160] In one embodiment, the PD-1 inhibitor is Pembrolizumab, which is disclosed, for example, in U.S. Patents US 8,354,509 and WO 2009 / 114335, and has the sequence disclosed therein (or a sequence that is substantially the same as or similar to it, such as a sequence that is at least 85%, 90%, 95% or higher identical to the specified sequence).

[0161] In some embodiments, the anti-PD-1 antibody is Pidilizumab. Pidilizumab (CT-011; CureTech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), such as the anti-PD-1 antibodies disclosed in US Patents US 8,609,089, US 2010028330, and / or US 20120114649.

[0162] In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion or a PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010 / 027827 and WO2011 / 066342), a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0163] In one embodiment, the anti-HBsAg antibody is used in combination with an anti-PD-L1 antibody, such as that described in WO2016 / 061142. In some embodiments, the anti-PD-L1 antibody is Atezolizumab, Avelumab, or Durvalumab. In some embodiments, the anti-PD-L1 antibody is Atezolizumab disclosed in WO2010 / 077634. ​​In some embodiments, the anti-PD-L1 antibody is Durvalumab disclosed in WO2011 / 066389. Other anti-PD-L1 antibodies include BMS-936559 (also known as MDX-1105) disclosed in WO2007 / 005874 and AMP-224 (also known as GSK2661380) disclosed in WO2010 / 027423.

[0164] Pharmaceutical Composition To prepare a pharmaceutical composition or sterile composition comprising anti-HBsAg antibodies, the antibodies described in this invention are mixed with a pharmaceutically acceptable carrier or excipient. The composition may also contain one or more other therapeutic agents suitable for neutralizing hepatitis D infection.

[0165] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in forms such as lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, Hardman et al., Pharmacological Basis of Goodman-Gilman Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmaceuticals, Lippincott, Williams, and Wilkins, New York, NY, 2000; Avis et al. (eds.), Drug Formulation: Parenteral Administration, Marcel Dekker, NY, 1993; Lieberman et al. (eds.), Drug Formulation: Tablets, Marcel Dekker, NY, 1990; Lieberman et al. (eds.), Drug Formulation: Dispersion Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0166] In one specific aspect, anti-HBsAg antibodies are lyophilized products packaged in vials. These lyophilized products can be reconstituted with water or a pharmaceutically suitable carrier for injection. To facilitate subsequent intravenous administration, the resulting solution is typically further diluted into a carrier solution.

[0167] The antibodies disclosed herein can be used to neutralize hepatitis D in patients with liver failure, cirrhosis, and / or hepatocellular carcinoma, and therefore can be used in sucrose and human serum albumin drug carriers previously used in bone marrow transplant patients treated with CytoGam.RTM (DeRienzo et al., Pharmacotherapy 2000; 20:1175-8). Alternatively, anti-HBsAg antibodies can be introduced into transplant patients via drug carriers, as described in WO2003 / 105894 for another antiviral antibody, Synagis.RTM. In that literature, the drug carrier consists of histidine and / or glycine, sugars (e.g., sucrose), and polyols (e.g., polysorbate).

[0168] The choice of treatment regimen depends on a number of factors, including the severity of the infection, the severity of symptoms, and the accessibility of target cells in the biological matrix. In some respects, the dosing regimen should maximize the therapeutic dose delivered to the patient while maintaining acceptable levels of side effects. Therefore, the dosage of the delivered biologic is partly dependent on the specific entity and the severity of the disease being treated. Guidance on selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, for example, Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines, and Arthritis, Marcel Dekker, New York, NY, 1991; Bach (ed.), Monoclonal Antibody and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY, 1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al., New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med. 344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619, 2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New Engl. J. Med. 343:1594-1602, 2000).

[0169] The appropriate dosage is determined by the clinician, for example, by using parameters or factors known or suspected in the art to affect or predicted to affect treatment. Typically, the dosage begins slightly below the optimal level and is then increased in small increments until the expected or optimal effect is achieved relative to any adverse side effects. Important diagnostic indicators include the diagnosis of symptoms such as infusion reactions.

[0170] The actual dose level of the active ingredient in an anti-HBsAg antibody pharmaceutical composition can be adjusted to obtain an amount of active ingredient that is effective in achieving the desired therapeutic effect for a specific patient, a specific composition, and a route of administration without causing toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the neutralizing activity of the antibody, route of administration, time of administration, half-life of the antibody in the patient, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition, the patient's age, sex, weight, physical condition, general health status, and medical history, as well as other factors known in the medical field.

[0171] Compositions containing antibodies or fragments thereof may be administered via continuous infusion or at intervals (e.g., daily, weekly, or weekly 1-7 times). Routes of administration include intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebral, or inhalation. A specific dosing regimen refers to the maximum dose or frequency of administration to avoid significant adverse side effects.

[0172] For the antibodies described in this article, the dosage administered to patients can be calculated from 0.0001 mg / kg to 50 mg / kg based on the patient's body weight. The dosage can be between 0.0001 mg / kg and 40 mg / kg, between 0.0001 mg / kg and 30 mg / kg, between 0.0001 mg / kg and 20 mg / kg, between 0.0001 mg / kg and 10 mg / kg, between 0.0001 mg / kg and 5 mg / kg, between 0.0001 and 2 mg / kg, between 0.0001 and 1 mg / kg, between 0.0001 mg / kg and 0.75 mg / kg, between 0.0001 mg / kg and 0.5 mg / kg, between 0.0001 mg / kg and 0.25 mg / kg, between 0.0001 and 0.15 mg / kg, between 0.0001 and 0.10 mg / kg, between 0.001 and 0.5 mg / kg, between 0.01 and 0.25 mg / kg, or between 0.01 and 0.10 mg / kg. The dosage of an antibody or its fragment can be calculated by multiplying the patient's weight (kg) by the required dose (mg / kg).

[0173] Antibody doses can be repeated, and administration can be spaced at intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0174] For a given patient, the effective dose may vary depending on a number of factors, such as the condition being treated, the patient’s overall health, the method of administration, route and dosage, and the severity of side effects (see, for example, Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

[0175] The route of administration can be, for example, by local or skin application, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal fluid, intralesional injection or infusion, or by continuous release system or implant (see, for example, Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; US Patents US 6,350,466 and 6,316,024). If necessary, the composition may also contain a solubilizer or a local anesthetic (e.g., lidocaine) to relieve pain at the injection site, or both. Additionally, pulmonary administration may be used, for example by using an inhaler or nebulizer, and in combination with an aerosol. See, for example, U.S. Patents US 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO99 / 66903, the entire contents of which are incorporated herein by reference.

[0176] The compositions of the present invention can also be administered via one or more routes of administration, using one or more methods known in the art. Those skilled in the art will understand that the route and / or mode of administration will vary depending on the desired outcome. Routes of administration for antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. Parenteral administration can represent routes of administration other than enteral and local administration (typically by injection), including but not limited to intravenous, intramuscular, intra-articular, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of the present invention can be administered via non-parenteral routes, such as local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration. On one hand, the antibodies of the present invention are administered by infusion. On the other hand, the antibodies are administered by subcutaneous injection.

[0177] If the antibody of the present invention is administered in a controlled-release or sustained-release system, a pump can be used to achieve controlled-release or sustained-release (see Langer, ibid.; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymer materials can be used to achieve controlled or sustained release of antibody therapies (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Langer and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; also see Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 71:105, 1989; US Patent US). 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253.

[0178] Polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-vinyl acetate copolymer), poly(methacrylic acid), polyglycolic acid lactide (PLG), polyanhydride, poly(N-vinylpyrrolidone), polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactic acid (PLA), polylactic acid-glycolic acid lactide copolymer (PLGA), and polyorthoesters. On the one hand, the polymers used in sustained-release formulations are inert, free of leaching impurities, storage-stable, sterile, and biodegradable. Controlled-release or sustained-release systems can be placed near the preventative or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

[0179] Langer's review discusses controlled-release systems, Science 249:1527-1533, 1990. Any technique known to those skilled in the art can be used to prepare sustained-release formulations comprising one or more antibodies of the present invention. See, for example, U.S. Patent 4,526,938, PCT Publication WO 91 / 05548, PCT Publication WO 96 / 20698, Ning et al., Radiology & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rd. Bioact. Mater. 24:853-854, 1997; and Lam et al., Proc. Int'l. Symp. Control Rd. Bioact. Mater. 24:759-760, 1997, the entire contents of which are incorporated herein by reference.

[0180] If the antibody of the present invention is for topical application, it can be formulated as an ointment, cream, transdermal patch, lotion, gel, spray, aerosol, solution, emulsion, or other forms well known to those skilled in the art. See, for example, *Introduction to Remington Pharmaceutical Science and Pharmaceutical Dosage Forms*, 19th edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous to semi-solid or solid form is typically used, containing a carrier or one or more excipients compatible with topical application, and in some cases, its dynamic viscosity is greater than that of water. Suitable dosage forms include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, etc., which may be sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) as needed to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include aerosol sprays, in which the active ingredient (in some cases) is combined with a solid or liquid inert carrier, packaged in combination with pressurized volatiles (e.g., gaseous propellants such as Freon), or contained in squeeze bottles. Humectants or moisturizers may also be added to the pharmaceutical composition and dosage form if desired. Examples of such additives are well known in the art.

[0181] If the antibody-containing composition is administered nasally, it can be formulated as an aerosol, spray, mist, or drops. Specifically, the preventive or therapeutic agents used according to the invention can be conveniently delivered from a pressurized package or nebulizer in the form of an aerosol spray, using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). For pressurized aerosols, the dosage unit can be determined by providing a valve to quantitatively deliver the drug. Capsules and cartridges for inhalers or blowpipes (e.g., made of gelatin) can be formulated as a powder mixture containing the compound and a suitable powder matrix (e.g., lactose or starch).

[0182] Co-administration or treatment with a second therapeutic agent, such as an immunosuppressant, cytokine, steroid, chemotherapeutic agent, antibiotic, or radiotherapy, is known in the art (see, for example, Hardman et al., (eds.) (2001), Pharmacological Basis of Goodman-Gilman Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Advanced Practice Pharmacotherapy: Practical Approaches, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective dose of treatment may reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40%, or at least 50%.

[0183] Other therapies that can be administered in combination with anti-HBsAg antibodies (such as prophylactic or therapeutic agents) can be given at intervals of less than 5 minutes, less than 30 minutes, 1 hour, approximately 1 hour, approximately 1 to 2 hours, approximately 2 to 3 hours, approximately 3 to 4 hours, approximately 4 to 5 hours, approximately 5 to 6 hours, approximately 6 to 7 hours, approximately 7 to 8 hours, approximately 8 to 9 hours, approximately 9 to 10 hours, approximately 10 to 11 hours, approximately 11 to 12 hours, approximately 12 to 18 hours, approximately 18 to 24 hours, approximately 24 to 36 hours, approximately 36 to 48 hours, 48 ​​to 52 hours, 52 to 60 hours, 60 to 72 hours, 72 to 84 hours, and 84 to 96 hours. The interval may be 96 to 120 hours between the administration of the anti-HBsAg antibody described in this invention and the administration of two or more therapies during the same patient visit.

[0184] In some respects, anti-HBsAg antibodies can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​blocks many highly hydrophilic compounds. To ensure that anti-HBsAg antibodies can cross the BBB (if needed), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents US4,522,811, 5,374,548, and 5,399,331. Liposomes may contain one or more portions that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting components include folic acid or biotin (see, for example, US Patent 5,416,016 by Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptors (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al., (1994) J. Biol. Chem. 269:9090), see also K. Keinanen; MLLaukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0185] This invention provides a protocol for administering an antibody-containing pharmaceutical composition, alone or in combination with other therapies, to a subject in need. The combination therapy (e.g., a prophylactic or therapeutic agent) can be administered to the subject simultaneously or sequentially. Therapies within a combination therapy (e.g., a prophylactic or therapeutic agent) can also be cyclically administered. Cyclic therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administering a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration (i.e., cyclical administration) to reduce the development of resistance to one of the therapies (e.g., the agent), thereby avoiding or reducing the side effects of one of the therapies (e.g., the agent), and / or improving the efficacy of the therapy.

[0186] The therapies (e.g., prophylactic or therapeutic agents) in the combination therapies of this invention can be administered simultaneously to the subject. The term "simultaneously" is not limited to administering the therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather refers to administering a pharmaceutical composition containing an antibody or a fragment thereof to the subject in a sequence and at intervals such that the antibody can work synergistically with the other therapies to provide a greater benefit than when administered alone. For example, each therapy can be administered to the subject simultaneously or sequentially at different time points in any order; however, if not simultaneously, it should be administered at sufficiently close times to provide the desired therapeutic or preventative effect. Each therapy can be administered to the subject separately in any suitable form and via any appropriate route. In each respect, the therapy (e.g., a preventative or therapeutic agent) is administered to the subject at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 5 hours, about 5 hours to about 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, about 11 hours to about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 1 week. In other respects, two or more therapies (e.g., preventative or therapeutic agents) are administered within the same patient visit.

[0187] The preventive or therapeutic drugs in a combination therapy may be administered to the subject in the same pharmaceutical composition. Alternatively, the preventive or therapeutic drugs in a combination therapy may also be administered to the subject simultaneously in different pharmaceutical compositions. The preventive or therapeutic drugs may be administered to the subject via the same or different routes of administration.

[0188] Working Example Example 1. Production of test molecules HBsAgBJ is an HBsAg antibody produced by the Novartis Institutes for BioMedical Research Biologics Center. Myrcludex B (bulvertide) is provided by WuXi AppTec. Details are as follows: Compound information HDV and HBV The HDV inoculum used in the experiment was provided by Wuxi, which was produced by co-transfecting Huh7 cells with plasmid pAAV-1.2×HDV genotype 1-ITR141 and pAAV-HBsAg-141 ITR. The HBV inoculum was provided by WuXi AppTec, which was obtained by precipitating HepG2.2.15 cell culture supernatant using PEG8000.

[0189] Virus information Primary human hepatocytes (PHHs) The single cryopreserved primary human hepatocytes were provided by Wuxi.

[0190] Other test substances are as follows: The following are listed in US20210221871A1 and WO2019229699: NOV3832, NOV3833, NOV3831, NOV3540, NOV3357, NOV3834, NOV3835, NOV3836, NOV3837, NOV3838, NOV3839, NOV3840, NOV3841, NOV3842, NOV2603, and NOV3212.

[0191] Example 2. Neutralizing efficacy of HBsAgBJ against hepatitis B virus and hepatitis D virus. The neutralizing ability of HBsAgBJ against HBV and HDV infection was evaluated by de novo infection of primary human hepatocytes. In the de novo infection assay, HBsAgBJ showed potent neutralization against infectious HBV, with half-maximal effective concentrations (EC50) of 0.09 nM for HBsAg and 0.11 nM for HBeAg (Table 3). HBsAgBJ also showed potent neutralization against infectious HDV, with an EC50 of 0.01 nM (Table 4). Conversely, the entry inhibitor bulevirtide prevented HBV and HDV infection, with EC50 values ​​ranging from 0.77 to 1.45 nM.

[0192] Example 3. Pharmacological evaluation of HBsAgBJ in a mouse model of HBV / HDV co-infection. The efficacy of HBsAgBJ has been studied in a humanized mouse model of chronic HBV / HDV infection (1). A dose of 20 mg / kg of HBsAg can rapidly clear HBsAg and HDV RNA levels in the serum. In addition, HBsAgBJ can maintain the clearance of HBsAg and HDV RNA for more than 3 days. In contrast, the IgG control showed minimal inhibitory effect on HBsAg and HDV RNA levels in this model. (Marc Lütgehetmann et al., Humanicontroleric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation. Hepatology; 2012 Mar;55(3):685-94).

[0193] Example 4. Formulation The anti-HBsAg virus antibody described in this article is a monoclonal antibody, an IgG1 isotype, with a light chain of κ or λ, and can be lyophilized. For subsequent intravenous administration, the resulting solution is usually further diluted into a carrier solution to prepare a ready-to-use antibody solution for infusion. Important stability assays for selecting the most stable formulation include: size exclusion chromatography (for determining aggregation levels), subvisible particulate matter detection, and potency testing.

[0194] Example 5. Preliminary efficacy of HBsAgBJ in clinical trials of patients with chronic hepatitis D. exist HBsAgBJ-001In this study, 10 participants with chronic hepatitis D (HDV) received weekly subcutaneous injections of 300 mg HBsAgBJ for up to 48 weeks. HDV RNA data were available for all 10 participants up to week 20 of treatment, with data available for 8 participants up to week 28. At weeks 12 and 24, the mean reduction in HDV RNA relative to baseline was 2.7 log10 IU / mL and 3.6 log10 IU / mL, respectively. To date, all 10 (100%) participants have achieved a virological response (≥2 log reductions or HDV RNA below the limit of quantitation (BLQ, 10 IU / ml in the assay, Victoria Infectious Disease Reference Laboratory, Melbourne, Australia). Six (60%) have achieved the HDV RNA BLQ. Of the nine participants with abnormal ALT at baseline, six (67%) have normalized ALT, while one had normal ALT at baseline and remained normal after treatment. The upper limit of normal ALT in the study was 40 for men and 30 for women, according to the central laboratory reference range. Six of the nine participants (67%) met the combined endpoint of virological response and ALT normalization, which is the endpoint for approval for hepatitis D. Data are shown in Figures 1A-1B and 2A-2B.

[0195] It should be understood that the examples and aspects described herein are for illustrative purposes only, and various modifications or alterations can be made by those skilled in the art based on them, and all such modifications or alterations should be covered within the spirit and scope of the invention and the appended claims. All references listed in the specification are incorporated herein in their entirety.

Claims

1. A method of neutralizing hepatitis B virus infection comprising administering to a patient in need thereof an effective amount of an antibody, wherein the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising: (a) HCDR1 (CDR-complementarity determining region) of SEQ ID NO: 9, (b) HCDR2 of SEQ ID NO: 10, (c) HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 25, (e) LCDR2 of SEQ ID NO: 26, and (f) LCDR3 of SEQ ID NO: 27; (ii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 41, (b) HCDR2 of SEQ ID NO: 42, (c) HCDR3 of SEQ ID NO: 43; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 57, (e) LCDR2 of SEQ ID NO: 58, and (f) LCDR3 of SEQ ID NO: 59; (iii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 73, (b) HCDR2 of SEQ ID NO: 74, (c) HCDR3 of SEQ ID NO: 75; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 89, (e) LCDR2 of SEQ ID NO: 90, and (f) LCDR3 of SEQ ID NO: 91; (iv) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 105, (b) HCDR2 of SEQ ID NO: 106, (c) HCDR3 of SEQ ID NO: 107; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 121, (e) LCDR2 of SEQ ID NO: 122, and (f) LCDR3 of SEQ ID NO: 123; (v) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 137, (b) HCDR2 of SEQ ID NO: 138, (c) HCDR3 of SEQ ID NO: 139; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 153, (e) LCDR2 of SEQ ID NO: 154, and (f) LCDR3 of SEQ ID NO: 155; by injection or infusion.(vi) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 169, (b) HCDR2 of SEQ ID NO: 170, (c) HCDR3 of SEQ ID NO: 171; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 185, (e) LCDR2 of SEQ ID NO: 186, and (f) LCDR3 of SEQ ID NO: 187; (vii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 201, (b) HCDR2 of SEQ ID NO: 202, (c) HCDR3 of SEQ ID NO: 203; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 217, (e) LCDR2 of SEQ ID NO: 218, and (f) LCDR3 of SEQ ID NO: 219; (viii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 233, (b) HCDR2 of SEQ ID NO: 234, (c) HCDR3 of SEQ ID NO: 235; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 249, (e) LCDR2 of SEQ ID NO: 250, and (f) LCDR3 of SEQ ID NO: 251; (ix) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 265, (b) HCDR2 of SEQ ID NO: 266, (c) HCDR3 of SEQ ID NO: 267; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 281, (e) LCDR2 of SEQ ID NO: 282, and (f) LCDR3 of SEQ ID NO: 283; (x) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 297, (b) HCDR2 of SEQ ID NO: 298, (c) HCDR3 of SEQ ID NO: 299; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 313, (e) LCDR2 of SEQ ID NO: 314, and (f) LCDR3 of SEQ ID NO: 315;(xi) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 329, (b) HCDR2 of SEQ ID NO: 330, (c) HCDR3 of SEQ ID NO: 331; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 345, (e) LCDR2 of SEQ ID NO: 346, and (f) LCDR3 of SEQ ID NO: 347; (xii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 361, (b) HCDR2 of SEQ ID NO: 362, (c) HCDR3 of SEQ ID NO: 363; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 377, (e) LCDR2 of SEQ ID NO: 378, and (f) LCDR3 of SEQ ID NO: 379; (xiii) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 393, (b) HCDR2 of SEQ ID NO: 394, (c) HCDR3 of SEQ ID NO: 395; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 409, (e) LCDR2 of SEQ ID NO: 410, and (f) LCDR3 of SEQ ID NO: 411; (xiv) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 425, (b) HCDR2 of SEQ ID NO: 426, (c) HCDR3 of SEQ ID NO: 427; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 441, (e) LCDR2 of SEQ ID NO: 442, and (f) LCDR3 of SEQ ID NO: 443; (xv) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 457, (b) HCDR2 of SEQ ID NO: 458, (c) HCDR3 of SEQ ID NO: 459; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 473, (e) LCDR2 of SEQ ID NO: 474, and (f) LCDR3 of SEQ ID NO: 475;or (xvi) a heavy chain variable region comprising: (a) HCDR1 of SEQ ID NO: 489, (b) HCDR2 of SEQ ID NO: 490, (c) HCDR3 of SEQ ID NO: 491; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 505, (e) LCDR2 of SEQ ID NO: 506, and (f) LCDR3 of SEQ ID NO: 507; or an antigen binding site thereof.

2. The method of claim 1, wherein, one or two amino acids of the CDRs of the antibody are modified, deleted, or replaced.

3. The method of claim 1, wherein, The antibody retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity in the variable heavy chain region or the variable light chain region.

4. The method of claim 1, wherein, The antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single chain antibody (scFv), or an antibody fragment.

5. A method of neutralizing hepatitis B virus infection comprising administering to a patient in need thereof an effective amount of an antibody, wherein the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region (vH) comprising SEQ ID NO: 18, and a light chain variable region (vL) comprising SEQ ID NO: 34; (ii) a heavy chain variable region (vH) comprising SEQ ID NO: 50, and a light chain variable region (vL) comprising SEQ ID NO: 66; (iii) a heavy chain variable region (vH) comprising SEQ ID NO: 82, and a light chain variable region (vL) comprising SEQ ID NO: 98; (iv) a heavy chain variable region (vH) comprising SEQ ID NO: 114, and a light chain variable region (vL) comprising SEQ ID NO: 130; (v) a heavy chain variable region (vH) comprising SEQ ID NO: 146, and a light chain variable region (vL) comprising SEQ ID NO: 162; (vi) a heavy chain variable region (vH) comprising SEQ ID NO: 178, and a light chain variable region (vL) comprising SEQ ID NO: 194; (vii) a heavy chain variable region (vH) comprising SEQ ID NO: 210, and a light chain variable region (vL) comprising SEQ ID NO: 226; (viii) a heavy chain variable region (vH) comprising SEQ ID NO: 242, and a light chain variable region (vL) comprising SEQ ID NO: 258; (ix) a heavy chain variable region (vH) comprising SEQ ID NO: 274, and a light chain variable region (vL) comprising SEQ ID NO: 290; (x) a heavy chain variable region (vH) comprising SEQ ID NO: 306, and a light chain variable region (vL) comprising SEQ ID NO: 322; (xi) a heavy chain variable region (vH) comprising SEQ ID NO: 338, and a light chain variable region (vL) comprising SEQ ID NO: 354; (xii) a heavy chain variable region (vH) comprising SEQ ID NO: 370, and a light chain variable region (vL) comprising SEQ ID NO: 386; (xiii) a heavy chain variable region (vH) comprising SEQ ID NO: 402, and a light chain variable region (vL) comprising SEQ ID NO: 418; (xiv) a heavy chain variable region (vH) comprising SEQ ID NO: 434, and a light chain variable region (vL) comprising SEQ ID NO: 450; (xv) a heavy chain variable region (vH) comprising SEQ ID NO: 466, and a light chain variable region (vL) comprising SEQ ID NO: 482; by injection or infusion.or, (xvi) a heavy chain variable region (vH) comprising SEQ ID NO: 498, and a light chain variable region (vL) comprising SEQ ID NO:

514.

6. The method of claim 5, wherein, The antibody or fragment thereof retains at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity in the variable light chain or variable heavy chain region.

7. The method of claim 5, wherein, The variable light chain or variable heavy chain region of the antibody has one, two, three, four, or five but less than 10 amino acids modified, deleted, or replaced.

8. The method of claim 5, wherein, The antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single chain antibody (scFv), or an antibody fragment.

9. The method of claim 1, wherein, The antibody or fragment thereof has reduced glycosylation level or is aglycosylated or is hypofucosylated.

10. The method of claim 1 or 5, wherein, The pharmaceutical composition comprises the antibody or fragment thereof of claim 1, further comprising a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising a plurality of antibodies or antigen binding fragments of claim 1, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5%, or more of the antibodies in the composition have an a2,3-linked sialic acid residue.

12. A pharmaceutical composition comprising a plurality of antibodies or antigen binding fragments of claim 1, wherein none of the antibodies comprise bisecting GlcNAc.

13. A pharmaceutical composition comprising the antibody or fragment thereof of claim 1, wherein, The pharmaceutical composition is prepared as a lyophilisate.

14. A method of treating or reducing the likelihood of a hepatitis D virus related disease, comprising administering to a patient in need thereof an effective amount of the antibody of claim 1 by injection or infusion, wherein the disease is: liver failure, liver cirrhosis, or hepatocellular carcinoma.

15. A diagnostic reagent comprising a labeled antibody or antigen binding fragment thereof of claim 1.

16. A pharmaceutical composition comprising a plurality of antibodies or antigen binding fragments of claim 5, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5%, or more of the antibodies in the composition have an a2,3-linked sialic acid residue.

17. A pharmaceutical composition comprising a plurality of antibodies or antigen binding fragments of claim 5, wherein none of the antibodies comprise bisecting GlcNAc.

18. A pharmaceutical composition comprising the antibody or fragment thereof of claim 5, wherein, The pharmaceutical composition is prepared as a lyophilisate.

19. A method of treating or reducing the likelihood of a hepatitis D virus related disease, comprising administering to a patient in need thereof an effective amount of the antibody of claim 5 by injection or infusion, wherein the disease is: liver failure, liver cirrhosis, or hepatocellular carcinoma.

20. A diagnostic reagent comprising a labeled antibody or antigen binding fragment thereof of claim 5.

21. A method of treating or reducing the likelihood of a hepatitis D virus associated disease comprising administering to a patient in need thereof an effective amount of an antibody by injection or infusion, wherein, The antibody or antigen binding fragment thereof comprises the sequences in Table 1.

22. A method of treating or reducing the likelihood of a hepatitis D virus associated disease comprising administering to a patient in need thereof by injection or infusion an effective amount of an antibody as listed in Table 1, wherein the disease is: liver failure, cirrhosis, or hepatocellular carcinoma.

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