Hepatitis b antibodies

Abstract

The invention relates to an anti-HBsAg antibody, an antibody fragment and application of the anti-HBsAg antibody and the antibody fragment to prevention and treatment of hepatitis B virus infection and related diseases.

Description

[0001] This application is a divisional application of PCT application PCT / IB2019 / 054498, filed on May 30, 2019, with the invention name “Hepatitis B Antibody”. The date on which the PCT application entered the Chinese national phase is November 25, 2020, and the application number is 201980035357.4.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Serial No. 62 / 678,756, filed May 31, 2018, the contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to anti-hepatitis B surface antigen antibodies, antibody fragments and their use in reducing the likelihood of or treating hepatitis B virus infection. Background Art

[0005] Hepatitis B virus (HBV) is an enveloped hepatotropic virus that infects the liver and can cause chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma (HCC). Although there is a safe vaccine against HBV, at least 600,000 people die from HBV-related disorders worldwide each year. Disease progression is affected by viral load, genotype and specific viral mutations (Biswas et al., Med. Virol. [Medical Virology] 2013; 85: 1340-1347). Based on the antigenic determinants found in the HBV surface antigen (HBsAg), HBV is divided into ten genotypes or four serotypes (asw, adr, ayw and ayr).

[0006] HBV is a member of the Hepadnaviridae family and can only infect humans and primates. The virus particle consists of a small 3.2 kb partially double-stranded circular DNA surrounded by an envelope that interacts with hepatocytes. HBV first binds with low affinity to heparan sulfate proteoglycans on hepatocytes. Subsequently, the pre-S1 lipopeptide of the large envelope protein binds to its higher affinity receptor on hepatocytes, the bile acid transporter NCTP (sodium taurocholate co-transporting polypeptide). The virus then enters the cytoplasm by endocytosis.

[0007] HBV clearance and pathogenesis are largely mediated by the adaptive immune response in HBV infection (Guidotti et al., Annu Rev Pathol. [Annual Review of Pathology] 2006; 1: 23-61). In order for HBV to persist, it must not induce a response, or must escape or overwhelm the response. Interestingly, HBV "evades" the innate immune response simply by not inducing a response (Wieland et al., Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences of the United States] 2004; 101(17): 6669-74). On the other hand, viral persistence is characterized by a relatively low-responsive state of HBV-specific T cells (Chisari Annu Rev Immunol. [Annual Review of Immunology] 1995; 13: 29-60). Several viral proteins have been shown to modulate the adaptive immune response to HBV, suggesting that HBV can adopt an active evasion strategy that targets the adaptive immune response (Thimme et al., J Virol. [Journal of Virology] 2003; 77(1): 68-76). It has been previously reported that antiviral therapy can overcome CD8+ T cell hyporesponsiveness in chronic HBV infection, suggesting that T cells are present in these subjects but are exhausted (Boni et al., Hepatology. 2001; 33(4): 963-71). Induction of an effective HBV-specific CD8+ T cell response may depend on early CD4+ T cell priming, which is regulated by the size of the viral inoculum (Asabe J Virol. 2009; 83(19): 9652-62).

[0008] Currently approved antiviral therapies are two alpha-interferon (IFN-α) formulations and five nucleoside analogs. Although nucleosides inhibit HBV DNA polymerase activity with varying potency and barriers to resistance, this therapy does not eliminate the virus and patients are on this therapy for life. Therefore, better therapies are needed to suppress hepatitis B infection. Summary of the invention

[0009] The present disclosure relates to neutralizing antibodies and / or fragments thereof against hepatitis B, and antibodies that reduce the amount of hepatitis B surface antigen (HBsAg).

[0010] An antibody, wherein the antibody or antigen-binding fragment thereof specifically binds to HBsAg.

[0011] Antibodies, wherein the antibody or antigen binding fragment thereof specifically binds to HBsAg. In one embodiment, the antibody or antigen binding fragment thereof binds to HBsAg and mutations thereof.

[0012] Antibodies, wherein the antibody or antigen binding fragment specifically binds to and neutralizes hepatitis B. In one embodiment, the antibody or antigen binding fragment thereof neutralizes hepatitis B or hepatitis B containing mutations in HBsAg. In another embodiment, the antibody or antigen binding fragment thereof reduces the amount of HBsAg. In another embodiment, the antibody or antigen binding fragment thereof reduces the amount of HBsAg circulating in the blood.

[0013] An isolated 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;

[0014] (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;

[0015] (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;

[0016] (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;

[0017] (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;

[0018] (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;

[0019] (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;

[0020] (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;

[0021] (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;

[0022] (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;

[0023] (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;

[0024] (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;

[0025] (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;

[0026] (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;

[0027] (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

[0028] (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.

[0029] Antibodies in which one or two amino acids within a CDR have been modified, deleted or substituted.

[0030] Antibodies that retain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the heavy chain variable region or the light chain variable region.

[0031] 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.

[0032] An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

[0033] (i) a heavy chain variable region (vH) comprising SEQ ID NO: 18, and a light chain variable region (vL) comprising SEQ ID NO: 34;

[0034] (ii) a heavy chain variable region (vH) comprising SEQ ID NO: 50, and a light chain variable region (vL) comprising SEQ ID NO: 66;

[0035] (iii) a heavy chain variable region (vH) comprising SEQ ID NO: 82, and a light chain variable region (vL) comprising SEQ ID NO: 98;

[0036] (iv) a heavy chain variable region (vH) comprising SEQ ID NO: 114, and a light chain variable region (vL) comprising SEQ ID NO: 130;

[0037] (v) a heavy chain variable region (vH) comprising SEQ ID NO: 146, and a light chain variable region (vL) comprising SEQ ID NO: 162;

[0038] (vi) a heavy chain variable region (vH) comprising SEQ ID NO: 178, and a light chain variable region (vL) comprising SEQ ID NO: 194;

[0039] (vii) a heavy chain variable region (vH) comprising SEQ ID NO: 210, and a light chain variable region (vL) comprising SEQ ID NO: 226;

[0040] (viii) a heavy chain variable region (vH) comprising SEQ ID NO: 242, and a light chain variable region (vL) comprising SEQ ID NO: 258;

[0041] (ix) a heavy chain variable region (vH) comprising SEQ ID NO: 274, and a light chain variable region (vL) comprising SEQ ID NO: 290;

[0042] (x) a heavy chain variable region (vH) comprising SEQ ID NO: 306, and a light chain variable region (vL) comprising SEQ ID NO: 322;

[0043] (xi) a heavy chain variable region (vH) comprising SEQ ID NO: 338, and a light chain variable region (vL) comprising SEQ ID NO: 354;

[0044] (xii) a heavy chain variable region (vH) comprising SEQ ID NO: 370, and a light chain variable region (vL) comprising SEQ ID NO: 386;

[0045] (xiii) a heavy chain variable region (vH) comprising SEQ ID NO: 402, and a light chain variable region (vL) comprising SEQ ID NO: 418;

[0046] (xiv) a heavy chain variable region (vH) comprising SEQ ID NO: 434, and a light chain variable region (vL) comprising SEQ ID NO: 450;

[0047] (xv) a heavy chain variable region (vH) comprising SEQ ID NO: 466, and a light chain variable region (vL) comprising SEQ ID NO: 482; or

[0048] (xvi) a heavy chain variable region (vH) comprising SEQ ID NO: 498, and a light chain variable region (vL) comprising SEQ ID NO: 514.

[0049] Antibodies or fragments thereof that retain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the light chain variable region or the heavy chain variable region.

[0050] An antibody in which one, two, three, four or five but less than 10 amino acids in the light chain variable region or the heavy chain variable region have been modified, deleted or substituted.

[0051] 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.

[0052] The antibody or fragment thereof has reduced glycosylation or no glycosylation or hypofucosylation.

[0053] A pharmaceutical composition comprising an antibody or a fragment thereof, further comprising a pharmaceutically acceptable carrier.

[0054] The pharmaceutical composition, wherein the pharmaceutically acceptable carrier contains histidine or a sugar.

[0055] The pharmaceutical composition, wherein the sugar is sucrose.

[0056] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments, wherein at least 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 5%, or more, or more, of the antibodies in the composition have α2,3-linked sialic acid residues.

[0057] A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments, wherein none of the antibodies comprises a bisecting GlcNAc.

[0058] A pharmaceutical composition comprising an antibody or fragment thereof, wherein the composition is prepared as a lyophilisate.

[0059] A method of neutralizing hepatitis B virus infection, the method comprising administering an effective amount of an antibody to a patient in need thereof via injection or infusion.

[0060] The method, wherein the patient in need thereof is diagnosed with hepatitis B viruria or hepatitis B viremia.

[0061] The method, wherein the patient in need thereof is diagnosed with the presence of hepatitis B surface antigen (HBsAg) in the blood or serum.

[0062] A method of treating or reducing the likelihood of a hepatitis B virus-related disorder, the method comprising administering an effective amount of an antibody to a patient in need thereof via injection or infusion, and wherein the disorder is: liver failure, cirrhosis, or hepatocellular carcinoma.

[0063] The method, wherein the antibody or composition is reconstituted prior to injection or infusion.

[0064] The method wherein the antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[0065] The method, wherein the therapeutic agent is an antiviral agent.

[0066] The method wherein the antiviral agent is: lamivudine, entecavir and tenofovir or alpha-interferon.

[0067] The method wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0068] The method, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0069] The method, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0070] The method wherein the therapeutic agent is an additional anti-HBsAg antibody.

[0071] Antibodies or fragments thereof, for use as medicaments.

[0072] Antibodies or fragments thereof for neutralizing hepatitis B virus infection.

[0073] An antibody or fragment thereof for use in treating or reducing the likelihood of: liver failure, liver cirrhosis, and / or hepatocellular carcinoma.

[0074] The use as described in any one of the above, which is administered in combination with another therapeutic agent.

[0075] The use as described in any one of the above, wherein the therapeutic agent is an antiviral agent.

[0076] The use as described in any one of the above, wherein the antiviral agent is: lamivudine, entecavir and tenofovir or α-interferon.

[0077] The use as described in any one of the above, wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0078] The use as described in any of the above, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0079] The use as described above, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0080] The use as described in any one of the above, wherein the therapeutic agent is another anti-HBsAg antibody.

[0081] A nucleic acid encoding the antibody or antigen-binding fragment.

[0082] A vector comprising the nucleic acid.

[0083] A host cell comprising the vector.

[0084] A diagnostic reagent comprising the labeled antibody or antigen-binding fragment thereof.

[0085] The diagnostic agent, wherein the label is selected from the group consisting of radiolabels, fluorophores, chromophores, imaging agents and metal ions.

[0086] definition

[0087] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0088] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalently, reversibly and specifically binding to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer 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, namely CH1, 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, namely CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0089] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the disclosure). Antibodies may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0090] "Complementarity determining domain" or "complementarity determining region" ("CDR") refers interchangeably to the hypervariable regions of VL and VH. CDRs are target protein binding sites of antibody chains that are specific for such target proteins. There are three CDRs (CDR1-3, numbered sequentially from the N-terminus) in each human VL or VH, which together constitute approximately 15%-20% of the variable domain. CDRs can be referred to by their region and order. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of 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 remaining VL or VH segments (the so-called framework regions) show less amino acid sequence variation (Kuby, Immunology, 4th ed., Chapter 4, WH Freeman & Co., New York, 2000).

[0091] The positions of CDRs and framework regions can be determined using a variety of definitions well known in the art, e.g., Kabat, Chothia, IMGT, and AbM (see, e.g., 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 ... Res. [Nucleic Acids Research], 29: 207-209 (2001); Al-Lazikani et al., J. Mol. Biol. [Journal of Molecular Biology], 273: 927-748 (1997)). The definition of antigen combining sites is also described in the following literature: 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 MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In the combined Kabat and Josiah numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Josiah CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HC CDR1), 50-65 (HC CDR2), and 95-102 (HC CDR3) in a VH (e.g., a mammalian VH, such as a human VH); and amino acid residues 24-34 (LC CDR1), 50-56 (LC CDR2), and 89-97 (LC CDR3) in a VL (e.g., a mammalian VL, such as a human VL).According to IMGT, the CDR amino acid residues in VH are numbered as approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered as approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (according to "Kabat" numbering). According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0092] Both the light chain and the heavy chain are divided into regions of structural homology and regions of functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and the heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the farther the constant region domain is from the antigen binding site or amino terminus of the antibody, the larger its number. 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 chain and light chain, respectively.

[0093] 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 epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, 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, a monovalent fragment consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementary determining regions (CDRs), or other epitope-binding fragments of antibodies.

[0094] In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to connect the two domains through a synthetic linker that enables them to form a single protein chain, wherein the VL region and the VH region pair to form a monovalent molecule (referred to as a single-chain Fv ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). The term "antigen-binding fragment" is also intended to encompass such single-chain antibodies. The antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0095] Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, in vivo antibodies, diabodies, triabodies, tetrabodies, v-NARs, and bi-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as type III fibronectin (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monomers).

[0096] The antigen-binding fragment can be incorporated into a single-chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641,870).

[0097] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to polypeptides, including antibodies and antigen-binding fragments, that have substantially identical amino acid sequences or are derived from the same genetic source. The term also includes preparations of antibody molecules having a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0098] As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework region and the CDR region are derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant forms of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, such as, for example, as described in Knappik et al., J. Mol. Biol. [Molecular Biology] 296: 57-86, 2000).

[0099] The human antibodies of the disclosure may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or by conservative substitution to improve stability or production).

[0100] As used herein, the term "recognition" refers to an antibody or antigen-binding fragment thereof that finds and interacts with its epitope (e.g., binds), whether or not the epitope is linear or conformational. The term "epitope" refers to a site on an antigen that specifically binds to an antibody or antigen-binding fragment of the present disclosure. An epitope can be formed by continuous amino acids or non-continuous amino acids juxtaposed due to the tertiary folding of a protein. An epitope formed by continuous amino acids is generally retained when exposed to a denaturing solvent, while an epitope formed by tertiary folding is generally lost when treated with a denaturing solvent. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include techniques in the art, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology [epitope mapping scheme in the method in molecular biology], Vol. 66, GE Morris ed. (1996)) or electron microscopy. The "paratope" is the part of an antibody that recognizes an antigen epitope.

[0101] The phrases "specific binding" or "selective binding" when used in the context of describing an interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or binding agent derived from an antibody refer to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological agents, for example, in a biological sample, such as blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or binding agent with a particular binding specificity binds to a particular antigen at least twice as much as background and does not substantially bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or binding agent with a particular binding specificity binds to a particular antigen at least ten (10) times as much as background and does not substantially bind to other antigens present in the sample in significant amounts. Specific binding to an antibody or binding agent under such conditions may require selection of the antibody or agent for its specificity for a particular protein. If desired or appropriate, such selection may be achieved by subtracting antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some aspects, antibodies or antibody fragments are selected that cross-react with certain desired molecules.

[0102] The term "affinity" as used herein 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 the antigen at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0103] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. However, an isolated antibody that specifically binds to one antigen may have cross-reactivity to other antigens. In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0104] 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 to a reference variable region amino acid sequence or subsequence compared to all other known or inferred variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A corresponding human germline sequence may be only framework regions, only complementary determining regions, framework regions and complementary determining regions, variable segments (as defined above), or other combinations of sequences or subsequences comprising variable regions. Sequence identity may be determined using the methods described herein, 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 to the reference variable region nucleic acid or amino acid sequence.

[0105] A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual (1998)). Typically, a specific or selective binding reaction will produce a signal that is at least 2 times higher than the background signal and more typically at least 10 to 100 times higher than the background signal.

[0106] 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 disclosed herein will generally have a dissociation constant of less than about 10 -7 or 10 -8 M, for example, is less than about 10 -9 M or 10 -10 M, in some aspects, is less than about 10 -11 M, 10 -12 M or 10 -13 The equilibrium dissociation constant of M.

[0107] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability is an absolute term that indicates a measure of the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.

[0108] As used herein, the phrase "consisting essentially of refers to the genus or species of active agents included in the method or composition, as well as any excipients that are inactive for the intended purpose of the method or composition. In some aspects, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the anti-HBsAg antibodies of the present disclosure. In some aspects, the phrase "consisting essentially of" explicitly excludes the inclusion of one or more additional active agents other than the anti-HBsAg antibodies of the present disclosure and the second co-administered agent.

[0109] The term "amino acid" refers to naturally occurring, synthetic and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an α-carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids.

[0110] The term "conservatively modified variant" is applicable to both amino acids and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids encoding the same or substantially the same amino acid sequence, or, in the case where the nucleic acid does not encode an amino acid sequence, to substantially the same sequence. 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 where a codon specifies alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled person will recognize that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of the nucleic acid encoding the polypeptide is implied in each of the described sequences.

[0111] For polypeptide sequences, "conservatively modified variants" include single substitutions, deletions or additions to a polypeptide sequence, whereby an amino acid is substituted with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs and alleles. The following eight groups contain amino acids that are conservative substitutions 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, e.g., Creighton, Proteins (1984). In some aspects, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of antibodies containing the amino acid sequence.

[0112] As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to use a preferred codon encoding an amino acid sequence in a production cell or organism, typically a eukaryotic cell, such as a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO), or a human cell. The optimized nucleotide sequence is engineered to retain, completely or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, which is also referred to as the "parent" sequence.

[0113] In the case of two or more nucleic acids or polypeptide sequences, the term "identical percentage" or "identity percentage" refers to the degree to which two or more sequences or subsequences are identical. If two sequences have the same amino acid sequence or nucleotide sequence in the region being compared, they are "identical". When comparing and aligning in a comparison window or a specified region to obtain maximum correspondence when using one of the following sequence comparison algorithms or by manual alignment and visual inspection measurement, if two sequences have a specified percentage of identical amino acid residues or nucleotides (that is, in a specified region or in the whole sequence when there is no provision, 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity), the two sequences are "substantially identical". Optionally, identity is present in a region of at least about 30 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0114] For sequence comparison, typically a sequence serves as a reference sequence, and the test sequence is compared with the reference sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm will calculate the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0115] As used herein, a "comparison window" includes a segment referring to any one of a plurality of contiguous positions selected from the group consisting of: 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein two sequences can be compared after optimal alignment of the sequence with a reference sequence of the same number of contiguous positions. Methods of sequence alignment for comparison are well known in the art. For example, by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the study of similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, 1996). Molecular Biology [Molecular Biology Experiment Manual], 2003), which can be used to optimally align sequences for comparison.

[0116] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. [Nucleic Acids Research] 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. [Molecular Biology] 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as the basis for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the 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. The expansion of the word hits in each direction terminates when the following situations occur: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score moves toward zero or lower due to the accumulation of one or more negative scoring residue alignments; or one end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and two chain comparisons as default values. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3 and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both strands.

[0117] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0118] The percent 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 incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available from the University of South Florida), using a BLOSUM 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0119] In addition to the above sequence identity percentages, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies produced for the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0120] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0121] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), and complementary sequences as well as explicitly indicated sequences. Specifically, as described in detail below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed 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).

[0122] In the case of nucleic acids, the term "operably connected" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcription regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, the promoter or enhancer sequence is operably connected to the coding sequence. Typically, the promoter transcription regulatory sequence operably connected to the transcribed sequence is physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcription regulatory sequences such as enhancers do not need to be physically adjacent or located very close to the position of the coding sequence that these transcription regulatory sequences enhance their transcription.

[0123] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, and to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0124] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

[0125] The terms "hepatitis B", "hepatitis B virus" or "HBV" refer to a member of the Hepadnaviridae family, genus Orthohepadnavirus. HBV is a double-stranded DNA virus. Based on HBsAg, the virus is divided into four major serotypes (adr, adw, ayr, ayw).

[0126] The terms "hepatitis B surface antigen," "HBsAg," or "HBVsAg" refer to a protein produced by the hepatitis B virus.

[0127] Table 1

[0128]

[0129]

[0130] "IC50" (half maximal inhibitory concentration) refers to the concentration of a particular antibody that induces a signal halfway (50%) between the baseline control and the maximum possible signal.

[0131] "EC50" (half maximal effective concentration) refers to the concentration of a particular antibody that induces a response halfway (50%) between baseline control and the maximum possible effect after a specific exposure or treatment period. For example, EC50 is the antibody concentration at which viral infection is neutralized by 50%.

[0132] "EC90" refers to the concentration of a particular antibody that induces a response corresponding to 90% of the maximal possible effect after a specific exposure or treatment period. For example, EC90 is the antibody concentration at which viral infection is neutralized by 90%.

[0133] "Neutralization" refers to inhibition of infection of host cells by a virus, as indicated by lack of viral gene expression. Without being bound by any one theory, the neutralization mechanism of a particular antibody may include blocking the interaction of viral capsid proteins with cell surface receptors, or disrupting any stage of the entry and trafficking process prior to delivery of the viral genome to the nucleus of the host cell.

[0134] As used herein, the terms "treat, treating or treatment" of any disease or disorder refer in one aspect to alleviating the disease or disorder (i.e., slowing or preventing or reducing the development of the disease or at least one clinical symptom thereof). In another aspect, "treat, treating or treatment" refers to alleviating or reducing at least one physical parameter, including those that cannot be discerned by the patient. In yet another aspect, "treat, treating or treatment" refers to regulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both.

[0135] The phrase "reduce the likelihood" means to delay the onset or development or progression of a disease, infection or disorder.

[0136] The terms "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to achieve the desired result (i.e., reduction in tumor size, inhibition of tumor growth, prevention of metastasis, inhibition or prevention of viral, bacterial, fungal or parasitic infection). In some aspects, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose and then incrementally increasing the dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of the molecules of the present disclosure can prevent the onset of disease symptoms or reduce the severity of disease symptoms, respectively, including symptoms associated with polyomavirus infection.

[0137] The term "co-administration" refers to the simultaneous presence of two active agents in the blood of an individual. Co-administered active agents may be delivered concurrently or sequentially. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 A / B-16A / B is a surface plasmon resonance (SPR / Biacore) measurement of the antibody showing the Kd for AD and AYHBV serotypes.

[0139] Figure 17-33 The antibodies were demonstrated to neutralize HBV infection.

[0140] Figure 34-50 The IC values ​​of antibodies against four genotypes (AD) are shown 50 .

[0141] Figure 51-66 The ICs of antibodies against four different clinical mutations are shown 50 .

[0142] Fig.67 The efficacy of the antibody in reducing the amount of HBsAg in the FRGN mouse model was shown. DETAILED DESCRIPTION

[0143] The disclosure provides antibodies, antibody fragments (e.g., antigen binding fragments) that bind and neutralize hepatitis B. In addition, the disclosure provides antibodies with desired pharmacokinetic characteristics and other desired properties, and thus can be used to reduce the likelihood of or treat hepatitis B-related liver failure, cirrhosis, or hepatocellular carcinoma. The disclosure also provides pharmaceutical compositions comprising antibodies, and methods for preparing and using such pharmaceutical compositions to prevent and treat hepatitis B infection and related disorders.

[0144] Anti-HBsAg antibodies

[0145] The present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HBsAg. Antibodies or antibody fragments (e.g., antigen binding fragments) of the present disclosure include, but are not limited to, human monoclonal antibodies or fragments thereof isolated as described in the following examples.

[0146] The present disclosure provides, in certain aspects, antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HBsAg, the antibodies or antibody fragments (e.g., antigen binding fragments) comprising 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 2). The present disclosure also 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) comprising a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 2. In a specific aspect, the present disclosure provides antibodies or antibody fragments (e.g., antigen binding fragments) that specifically bind to HBsAg, the antibodies comprising (or optionally consisting of) one, two, three, or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 2.

[0147] The present disclosure 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) comprising 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 2). The present disclosure also 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) comprising a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 2. In particular, the present disclosure 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 alternatively consist of): one, two, three or more VL CDRs having the amino acid sequence of any one of the VL CDRs listed in Table 2.

[0148] Other antibodies or antibody fragments (e.g., antigen-binding fragments) disclosed herein comprise amino acids that have been mutated but have at least 60%, 70%, 80%, 90% or 95% identity in the CDR regions to the CDR regions depicted in the sequences set forth in Table 2. In some aspects, it comprises a mutant amino acid sequence wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated within the CDR regions when compared to the CDR regions depicted in the sequences set forth in Table 2.

[0149] The present disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chain and full-length light chain of antibodies that specifically bind to HBsAg. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0150] Table 2: Anti-HBV Antibodies

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] Other antibodies of the disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been mutated; but have at least 60%, 70%, 80%, 90% or 95% identity to the sequences set forth in Table 2. In some aspects, it includes mutant amino acid sequences in which no more than 1, 2, 3, 4 or 5 amino acids in the variable region have been mutated when compared to the variable region depicted in the sequence set forth in Table 2, while retaining substantially the same therapeutic activity.

[0199] Since these antibodies can all bind to HBsAg, VH, VL, full-length light chain and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding amino acid sequences) can be "mixed and matched" to produce other HBsAg binding antibodies. Such "mixed and matched" HBsAg binding antibodies can be tested using binding assays known in the art (e.g., other assays described in ELISA and Examples section). When these chains are mixed and matched, the VH sequence from a specific VH / VL pairing should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a specific full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a specific VH / VL pairing should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a specific full-length heavy chain / full-length light chain pairing should be replaced with a structurally similar full-length light chain sequence. Thus, in one aspect, the disclosure provides an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 50, 82, 114, 146, 178, 210, 242, 274, 306, 338, 370, 402, 434, 466, or 498 (Table 2); and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 66, 98, 130, 162, 194, 226, 258, 290, 322, 354, 386, 418, 450, 482, or 514 (Table 2); wherein the antibody specifically binds to HBsAg.

[0200] In another aspect, the disclosure provides (i) an isolated monoclonal antibody having: a full-length heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 52, 84, 116, 148, 180, 212, 244, 276, 308, 340, 372, 404, 436, 468, or 500 that has been optimized for expression in mammalian cells (Table 2) and a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 68, 100, 132, 164, 196, 228, 260, 292, 324, 356, 388, 420, 452, 484, or 516 that has been optimized for expression in mammalian cells (Table 2) or (ii) a functional protein comprising an antigen binding portion thereof.

[0201] In another aspect, the disclosure provides HBsAg binding antibodies comprising heavy and light chain CDR1, CDR2 and CDR3, or a combination thereof, as described in Table 2. The amino acid sequence of the 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 the 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 the VH CDR3 of the antibody is 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 the VL CDR1 of the antibody is 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 the VL CDR2 of the antibody is 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 VL CDR3 of the antibody is shown in SEQ ID NO: 27, 59, 91, 123, 155, 187, 219, 251, 283, 315, 347, 379, 411, 443, 475 or 507.

[0202] In view of the fact that each of these antibodies can bind to HBsAg and the antigen binding specificity is mainly provided by CDR1, 2 and 3 regions, VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched), but each antibody must contain VH CDR1, 2 and 3 and VL CDR1, 2 and 3 to produce other HBsAg binding molecules. Such "mixed and matched" HBsAg binding antibodies can be tested using binding assays known in the art and those described in the examples (e.g., ELISA). When mixing and matching VH CDR sequences, CDR1, CDR2 and / or CDR3 sequences from specific VH sequences should be replaced with one or more structurally similar CDR sequences. Similarly, when mixing and matching VL CDR sequences, CDR1, CDR2 and / or CDR3 sequences from specific VL sequences should be replaced with one or more structurally similar CDR sequences. It will be readily apparent to those of ordinary skill 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 from the CDR sequences set forth herein for the monoclonal antibodies of the disclosure.

[0203] Thus, the present disclosure provides an isolated monoclonal antibody or antigen-binding region thereof, comprising: a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 41, 73, 105, 137, 169, 201, 233, 265, 297, 329, 361, 393, 425, 457, or 489; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 42, 74, 106, 138, 170, 202, 234, 266, 298, 330, 362, 394, 426, 458, or 490; a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ID NO: 11, 43, 75, 107, 139, 171, 203, 235, 267, 299, 331, 363, 395, 427, 459 or 491; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 57, 89, 121, 153, 185, 217, 249, 281, 313, 345, 377, 409, 441, 473 or 505; a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: NO:26, 58, 90, 122, 154, 186, 218, 250, 282, 314, 346, 378, 410, 442, 474 or 506; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:27, 59, 91, 123, 155, 187, 219, 251, 283, 315, 347, 379, 411, 443, 475 or 507; wherein the antibody specifically binds HBsAg.

[0204] In certain aspects, the antibody that specifically binds HBsAg is an antibody or antibody fragment (eg, antigen binding fragment) described in Table 2.

[0205] 1. Identification of Antibodies

[0206] The present disclosure provides antibodies and antibody fragments (eg, antigen binding fragments) that bind to HBsAg. In certain aspects, the antibodies and antibody fragments can bind to the same epitope within all four hepatitis B serotypes.

[0207] The present disclosure also provides antibodies and antibody fragments (e.g., antigen binding fragments) that bind to the same epitope as the HBsAg antibodies described in Table 2. Therefore, additional antibodies and antibody fragments (e.g., antigen binding fragments) can be identified based on their ability to cross-compete with other antibodies in a binding assay (e.g., competitively inhibit binding in a statistically significant manner). The ability of the test antibody to inhibit the binding of the antibodies and antibody fragments (e.g., antigen binding fragments) disclosed herein to HBsAg indicates that the test antibody can compete with such an antibody or antibody fragment (e.g., antigen binding fragment) for binding to HBsAg; according to non-limiting theory, such an antibody can bind to an epitope on HBsAg that is the same or related (e.g., structurally similar or spatially close) to the antibody or antibody fragment (e.g., antigen binding fragment) that competes with the antibody. In a certain aspect, the antibody that binds to the same epitope as the antibody or antibody fragment (e.g., antigen binding fragment) disclosed herein on HBsAg is a human monoclonal antibody or a humanized monoclonal antibody. Such human monoclonal antibodies or humanized monoclonal antibodies can be prepared and separated as described herein.

[0208] 2. Further changes to the Fc region framework

[0209] The present disclosure discloses specific HBsAg antibodies. These antibodies include modified antibodies or their antigen-binding fragments, and the modified antibodies or their antigen-binding fragments further include modifications to framework residues in VH and / or VL, such as to improve the characteristics of the antibody. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "revert" one or more framework residues to corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequences from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, somatic mutations can be "reverted" to germline sequences, such as by site-directed mutagenesis. Such "reverted" antibodies are also intended to be encompassed.

[0210] Another type of framework modification includes mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 2003 / 0153043 by Carr et al.

[0211] In addition to or as an alternative to modifications made in the framework or CDR regions, antibodies can be engineered to include modifications in the Fc region, typically altering one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular toxicity. In addition, the antibody can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, thereby again altering one or more functional properties of the antibody. Each of these aspects is described in further detail below.

[0212] In one aspect, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. Such methods are further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, e.g., to facilitate assembly of light and heavy chains or to increase or decrease the stability of the antibody.

[0213] In another aspect, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has impaired staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. The method is further described in detail in U.S. Pat. No. 6,165,745 to Ward et al.

[0214] In yet other aspects, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of complement. The method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260 of Winter et al.

[0215] In another aspect, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered CIq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). Such methods are described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0216] In another aspect, one or more amino acid residues are changed to change the ability of the antibody to fix complement. The method is described in, for example, PCT Publication WO 94 / 29351 of Bodmer et al. In a specific aspect, for IgG1 subclass and κ isotype, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced with one or more allotype amino acid residues. Allotype amino acid residues also include but are not limited to the constant region of the heavy chain of IgG1, IgG2 and IgG3 subclasses and the constant region of the light chain of κ isotype, as described by Jefferis et al., MAbs. [monoclonal antibodies] 1: 332-338 (2009).

[0217] In yet another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, by Presta in PCT Publication WO00 / 42072. In addition, the binding sites for FcγR1, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. [Journal of Biological Chemistry] 276: 6591-6604, 2001).

[0218] In yet another aspect, the glycosylation of the modified antibody. For example, an antibody without glycosylation can be prepared (i.e., the antibody lacks glycosylation). Glycosylation can be changed, for example, to increase the affinity of the antibody to the "antigen". Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, which eliminate one or more variable region framework glycosylation sites, thereby eliminating the glycosylation of the site. This kind of aglycosylation can increase the affinity of the antibody for the antigen. This method is described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 of Co et al.

[0219] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies are expressed, thereby producing antibodies with altered glycosylation. For example, 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 such a cell line exhibit low fucosylation. Presta described in PCT Publication WO 03 / 035835 a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., 2002, J. Biol. Chem. 277:26733-26740). Umana et al. described in PCT Publication WO 99 / 54342 a cell line engineered to express a glycoprotein modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0220] In another aspect, the antibody is modified to increase its biological half-life. A variety of methods can be used. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in Ward's U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain a salvage receptor binding epitope of two loops of the CH2 domain of the Fc region of IgG, as described in Presta et al. U.S. Patent Nos. 5,869,046 and 6,121,022.

[0221] In order to minimize the ADCC activity of antibody, the specific mutation in Fc district has produced " Fc silence " antibody with minimum interaction with effector cells.In general, " IgG Fc district " is used to define the C-terminal region of immunoglobulin heavy chain, including native sequence Fc district and variant Fc district.Human IgG heavy chain Fc district is generally defined as the amino acid residue from position C226 or from position P230 to carboxyl terminal comprising IgG antibody.The numbering of residue in Fc district is the numbering of EU index of Kabat.For example, the C-terminal lysine (residue K447) in Fc district can be removed during the generation or purification of antibody.

[0222] Silent effector function can be obtained by mutations in the Fc region of antibodies and has been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. [Current Biotechnology Views] Vol. 20 (6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. [Journal of Immunology] 181: 6664-69) See also Heusser et al., WO 2012065950. An example of a silent Fc IgG1 antibody is the LALA mutant, which contains L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (US 6,737,056). Another silent IgG1 antibody contains the N297A mutation, which produces an aglycosylated / non-glycosylated antibody.

[0223] Fc-silent antibodies produce no or low ADCC activity, which means that the Fc-silent antibodies exhibit ADCC activity less than 50% specific cell lysis (low ADCC activity), or less than 1% specific cell lysis (no ADCC activity).

[0224] 3. Antibody Production

[0225] Anti-HBsAg antibodies and antibody fragments thereof (e.g., antigen binding fragments) can be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0226] The disclosure further provides polynucleotides encoding antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising complementary determining regions as described herein. In some aspects, the polynucleotides encoding the heavy chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 19, 51, 83, 115, 147, 179, 211, 243, 275, 307, 339, 371, 403, 435, 467 or 499. In some aspects, 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 to a polynucleotide selected from the group consisting of SEQ ID NO:35, 67, 99, 131, 163, 195, 227, 259, 291, 323, 355, 387, 419, 451, 483 or 515.

[0227] In some aspects, 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 to the polynucleotide of SEQ ID NO:21, 53, 85, 117, 149, 181, 213, 245, 277, 309, 341, 373, 405, 437, 469 or 501. In some aspects, 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 to the polynucleotide of SEQ ID NO:37, 69, 101, 133, 165, 197, 229, 261, 293, 325, 357, 389, 421, 453, 485, or 517.

[0228] The polynucleotides disclosed herein can encode only the variable region sequences of anti-HBsAg antibodies. They can also encode the variable regions and constant regions of antibodies. Some polynucleotide sequence encodings comprise a polypeptide of the variable regions of the heavy chain and light chain of an exemplary anti-HBsAg antibody. Some other polynucleotide encodings are two polypeptide segments substantially identical to the variable regions of the heavy chain and light chain of an antibody, respectively.

[0229] The polynucleotide sequence can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding an anti-HBsAg antibody or its binding fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. [Methods in Enzymology] 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. [Methods in Enzymology] 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. [Tetrahedron Letters], 22:1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Introduction of mutations into a polynucleotide sequence by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (ed.), Freeman Press, New York, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al., (eds.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0230] The disclosure also provides expression vectors and host cells for producing the anti-HBsAg antibodies described above. A variety of 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 produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Natural Genetics] 15: 345, 1997). For example, non-viral vectors that can be used to express anti-HBsAg polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, California), MPSV vectors and many other vectors known in the art for expressing other proteins. Useful viral vectors include retrovirus-, adenovirus-, adeno-associated virus-, herpes virus-based vectors, SV40-, papillomavirus-, HBP Epstein Barr virus-based vectors, vaccinia virus vectors, and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. [Annual Review of Microbiology] 49:807, 1995; and Rosenfeld et al., Cell [Cell] 68:143, 1992.

[0231] The selection of expression vector depends on the expected host cell to express the vector. Typically, expression vector contains promoters and other regulatory sequences (e.g., enhancers) operably connected to the polynucleotides encoding anti-HBsAg antibody chains or fragments. In some respects, inducible promoters are used to prevent the inserted sequence from being expressed under conditions outside the inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The cultivation of transformed organisms can be amplified under non-inducing conditions without causing the colony to be biased toward the coding sequence that its expression product is better tolerated by the host cell. In addition to promoters, other regulatory elements may also be needed or expected to efficiently express anti-HBsAg antibody chains or fragments. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be increased by including enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol. 153:516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0232] Expression vector can also provide secretion signal sequence position, to form fusion protein with the polypeptide encoded by the anti-HBsAg antibody sequence inserted.More commonly, the inserted anti-HBsAg antibody sequence is connected with the signal sequence before being included in the vector.The vector for receiving the sequence of the anti-HBsAg antibody light chain and heavy chain variable domain of coding also encodes constant region or its part sometimes.Such vector allows variable region to be expressed as fusion protein with constant region, thereby produces complete antibody or its fragment.Typically, such constant region is human constant region.

[0233] The host cell for carrying and expressing anti-HBsAg antibody chains can be prokaryotic or eukaryotic. Escherichia coli is a prokaryotic host that can be used for cloning and expressing the polynucleotides disclosed herein. Other microbial hosts suitable for use include bacillus, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, and the expression vectors typically contain expression control sequences (for example, origins of replication) compatible with host cells. In addition, there will be any number of known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems or promoter systems from bacteriophage lambda. Promoters typically optionally control expression together with operator sequences, and have ribosome binding site sequences, etc., for starting and completing transcription and translation. Other microorganisms such as yeast can also be used to express anti-HBsAg antibodies. Insect cells combined with baculovirus vectors can also be used.

[0234] In other aspects, mammalian host cells are used to express and produce the anti-HBsAg antibodies disclosed herein. For example, they can be hybridoma cell lines (for example, myeloma hybridoma clones described in the examples) expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normal non-immortal or normal or abnormal immortal animals or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. Utilizing mammalian tissue cell culture to express polypeptides, for example, Winnacker, From Genes to Clones [from genes to clones], VCH Publishers [VCH Press], NY, NY [New York City, New York], 1987, generally discussed. Expression vectors for mammalian host cells may include expression control sequences, such as replication origins, promoters and enhancers (see, e.g., Queen et al., Immunol. Rev. [Immunology Review] 89: 49-68, 1986), and 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 from mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific and / or regulatable or adjustable. Available promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone (dexamethasone) inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters and promoter-enhancer combinations known in the art.

[0235] The method for introducing the expression vector containing the target polynucleotide sequence varies according to the type of cell host.For example, calcium chloride transfection is generally used for prokaryotic cells, and calcium phosphate treatment or electroporation can be used for other cell hosts (generally referring to Sambrook et al., the same).Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated conversion, injection and microinjection, bombardment, virion, immunoliposome, polycation: nucleic acid conjugate, naked DNA, artificial virion, fusion (Elliot and O'Hare, Cell [cell] 88:223,1997) with herpes virus structural protein VP22, DNA uptake and in vitro transduction enhanced by medicament.For the long-term high yield production of recombinant protein, stable expression is generally desired.For example, the expression vector containing viral replication origin or endogenous expression element and selective marker gene can be used to prepare the cell line of stably expressing anti-HBsAg antibody chain or binding fragment.After introducing the vector, cells can be grown in enriched medium for 1 day-2 days, and then they are converted to selective medium. The purpose of the selectable marker is to confer resistance to the selection, and its presence allows growth of cells that successfully express the introduced sequences in a selective medium.Resistant, stably transfected cells can be propagated using tissue culture techniques appropriate to the cell type.

[0236] Therapeutic and diagnostic uses

[0237] The antibodies and antibody fragments (e.g., Fabs) disclosed herein can be used in a variety of applications, including but not limited to hepatitis B virus infection and disease. In certain aspects, the antibodies and antibody fragments (e.g., Fabs) can be used to neutralize hepatitis B infection and prevent or treat cirrhosis or liver cancer. The method of use can be in vitro, ex vivo, or in vivo.

[0238] In one aspect, antibodies, antibody fragments (e.g., Fabs) can be used to detect the presence of HBsAg in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In some aspects, the biological sample comprises cells or tissues. In some aspects, such tissues include normal tissues and / or cancerous tissues that express HBsAg at higher levels relative to other tissues.

[0239] In one aspect, the present disclosure provides a method for detecting the presence of HBsAg or hepatitis B in a biological sample. In some aspects, the method includes contacting the biological sample with an anti-HBsAg antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine or blood sample.

[0240] Also included is a method for diagnosing an obstacle associated with HBsAg expression. In some aspects, the method includes contacting the test cell with an anti-HBsAg antibody; determining the expression level of HBsAg in the test cell (quantitatively or qualitatively) by detecting the combination of the antibody and HBsAg; and comparing the infection level in the test cell with the hepatitis B virus infection level in the control cell (e.g., a normal cell or non-virus-infected cell having the same tissue source as the test cell), wherein the presence of a higher level of HBsAg in the test cell indicates the presence of an obstacle associated with hepatitis B infection compared to the control cell. In some aspects, the test cell is obtained from an individual suspected of having hepatitis B virus infection.

[0241] In certain aspects, a diagnostic or detection method such as the above method comprises detecting binding of HBsAg antibodies to hepatitis B virus infected cells. An exemplary assay for detecting binding of anti-HBsAg antibodies to hepatitis B infected cells is a "FACS" assay.

[0242] Certain other methods can be used to detect the combination of anti-HBsAg antibodies. Such methods include but are not limited to antigen binding assays known in the art, such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay, protein A immunoassay and immunohistochemistry (IHC).

[0243] In certain aspects, the anti-HBsAg antibody is labeled. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly detected (e.g., by enzymatic reactions or molecular interactions) moieties, such as enzymes or ligands.

[0244] In some aspects, anti-HBsAg antibodies are fixed on an insoluble matrix. Immobilization can separate anti-HBsAg antibodies from any hepatitis B protein that remains free in solution. Routinely, this is by making anti-HBsAg antibodies insoluble before the assay procedure, such as by being adsorbed to a water-insoluble matrix or surface (Bennich et al., U.S. Patent number 3,720,760) or by covalent coupling (e.g., cross-linked using glutaraldehyde), or by making anti-HBsAg antibodies insoluble (e.g., by immunoprecipitation) after forming a complex between anti-HBsAg antibodies and HBsAg proteins to complete.

[0245] Any of the above aspects of diagnosis or detection may be performed using an anti-HBsAg antibody of the present disclosure in place of or in addition to another anti-HBsAg antibody.

[0246] In one aspect, the present disclosure provides a method of treating, reducing the likelihood or ameliorating a disease, the method comprising administering an antibody, antibody fragment (e.g., an antigen binding fragment) to a patient, thereby treating the disease. In some aspects, the disease treated with an antibody, antibody fragment (e.g., an antigen binding fragment) is a hepatitis B virus infection. Examples of hepatitis B 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 cells expressing HBsAg, and anti-HBsAg antibodies, antibody fragments (e.g., antigen binding fragments) can specifically bind to these cells.

[0247] The present disclosure provides methods for treating hepatitis B virus infection and liver failure, cirrhosis and / or hepatocellular carcinoma, the methods comprising administering a therapeutically effective amount of an antibody, antibody fragment (eg, antigen binding fragment). In certain aspects, the subject is a human.

[0248] In some aspects, the method for reducing hepatitis B virus infection comprises administering a therapeutically effective amount of an antibody or antibody fragment (e.g., Fab) to a subject. In some aspects, the subject is a human. In some aspects, the subject is immunosuppressed, immunocompromised, or immunocompromised. For immunosuppressed subjects, due to the therapeutic effect of anti-HBsAg antibodies, the immunosuppressive amount can be increased or decreased.

[0249] In order to treat hepatitis B virus infection, the appropriate dosage of HBsAg antibody or antibody fragment (e.g., Fab) depends on multiple factors, such as the type of infection to be treated, the severity and course of infection, the responsiveness of infection, virus resistance to treatment, previous therapy, patient clinical history, etc. The antibody can be used once or in a series of treatments lasting several days to several months, or until a cure is achieved or the alleviation of infection is achieved (e.g., reducing viruria or viral damage to the liver). The optimal dosage regimen can be calculated by measuring the cumulative amount of the drug in the patient and will vary according to the relative effectiveness of a single antibody or antibody fragment (e.g., Fab). In some aspects, the dosage is 0.01 mg to 100 mg (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) / kg body weight, and can be administered once or multiple times daily, weekly, monthly, or annually. In some aspects, the antibody or antibody fragment (e.g., Fab) of the present disclosure is administered once every two weeks or once every three weeks. The treating physician can estimate the administration repetition rate based on the measured half-life and concentration of the antibody in body fluids or tissues.

[0250] Combination therapy

[0251] In some cases, the antibodies or antibody fragments (e.g., antigen-binding fragments) of the disclosure are combined with other therapeutic agents, such as other antiviral agents, antiallergic agents, antinausea agents (or antiemetic agents), analgesics, cytoprotective agents, immunosuppressants, and combinations thereof.

[0252] The term "pharmaceutical combination" as used herein refers to a fixed combination in the form of one dosage unit, or a non-fixed combination or a kit of parts for combined administration, wherein two or more therapeutic agents can be administered independently at the same time or separately within time intervals, especially when these time intervals allow the combination partners to exhibit a synergistic (e.g., synergistic) effect.

[0253] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic conditions or infections described in the present disclosure. Such administration encompasses the co-administration of these therapeutic agents in a substantially simultaneous manner, such as administration in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration encompasses the co-administration of each active ingredient in multiple or separate containers (e.g., capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also encompasses the use of each type of therapeutic agent at approximately the same time or in different time sequences. In either case, the treatment regimen will provide a beneficial effect of the drug combination in the treatment of conditions or disorders described herein.

[0254] Combination therapy may provide and prove to be "synergistic," i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the individual components when used alone. Synergy may be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered in alternation or in parallel in separate formulations; or (3) delivered by some other regimen. When delivered in alternation therapy, synergy may be obtained when the individual components are administered or delivered sequentially (e.g., by different injections in separate syringes). Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., sequentially, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0255] In one aspect, the present disclosure provides a method for treating hepatitis B infection, the method being applied to a subject in need thereof by administering an anti-HBsAg antibody together with an immunosuppressant therapy. The anti-HBsAg antibody will reduce the amount of HBsAg in the circulation and cause the immune system to respond to hepatitis B virus infection produced by immunosuppressant therapy before or after administration. Examples of immunosuppressant therapy include, but are not limited to, monophosphate dehydrogenase inhibitors, purine synthesis inhibitors, calcineurin inhibitors, or mTOR inhibitors. Specific examples of immunosuppressant therapy include, but are not limited to, mycophenolate mofetil (MMF), mycophenolate sodium, azathioprine, tacrolimus, sirolimus, and cyclosporine.

[0256] In one embodiment, the anti-HBsAg antibody is used in combination with a PD-1 inhibitor, for example, as described in WO 2015 / 026684 or WO 2016 / 057846. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab, Pembrolizumab, or Pidilizumab.

[0257] 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 Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8,008,449 and WO 2006 / 121168. In one embodiment, the inhibitor of PD-1 is nivolumab and has a sequence disclosed therein (or a sequence substantially identical or similar thereto, for example, a sequence at least 85%, 90%, 95% identical or higher to a specified sequence).

[0258] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as lambrolizumab, MK-3475, MK03475, SCH-900475 or Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134–44, US 8,354,509, and WO 2009 / 114335.

[0259] In one embodiment, the inhibitor of PD-1 is pembrolizumab disclosed in, for example, US 8,354,509 and WO 2009 / 114335, and has a sequence disclosed therein (or a sequence substantially identical or similar thereto, such as a sequence that is at least 85%, 90%, 95% identical or higher to the specified sequence.

[0260] In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2009 / 101611. Other anti-PD1 antibodies include AMP 514 (Amplymni), especially anti-PD1 antibodies disclosed in, for example, US 8,609,089, US2010028330 and / or US20120114649.

[0261] In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; disclosed, e.g., in WO 2010 / 027827 and WO 2011 / 066342), a PD-L2 Fc-fused soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0262] In one embodiment, the anti-HBsAg antibody is used in combination with an anti-PD-L1 antibody, for example, as described in WO 2016 / 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 WO 2010 / 077634. ​​In some embodiments, the anti-PD-L1 antibody is durvalumab, disclosed in WO 2011 / 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.

[0263] Pharmaceutical composition

[0264] To prepare a pharmaceutical or sterile composition comprising an anti-HBsAg antibody, the antibody of the disclosure is mixed with a pharmaceutically acceptable carrier or excipient. The composition may additionally contain one or more other therapeutic agents useful for neutralizing hepatitis B infection.

[0265] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY, 2000; Avis et al. (eds.), Pharmaceutical Dosage Forms: parenteral Medications, Marcel Dekker, NY, 1993; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekke, NY, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0266] In a specific aspect, the anti-HBsAg antibody is a lyophilisate in a vial containing the antibody. The lyophilisate can be reconstituted with water or a pharmaceutical carrier suitable for injection. For subsequent intravenous administration, the obtained solution is generally further diluted in a carrier solution.

[0267] The antibodies disclosed herein can be used to neutralize hepatitis B in patients with liver failure, cirrhosis and / or hepatocellular carcinoma and thus can be used as previously described in patients receiving A pharmaceutical carrier of sucrose and human albumin used in bone marrow transplant patients (DeRienzo et al. Pharmacotherapy 2000; 20: 1175-8). Alternatively, as described in WO 2003 / 105894, anti-HBsAg antibodies may be delivered via a cytotoxic agent such as directed against another antiviral antibody. The drug carrier is introduced into a transplant patient. In the present disclosure, the drug carrier is composed of histidine and / or glycine, sugar (eg sucrose) and polyol (eg polysorbate).

[0268] Selecting an administration regimen for a therapeutic agent depends on several factors, including the severity of infection, symptom level, and the accessibility of target cells in a biological matrix. In some aspects, an administration regimen maximizes the amount of a therapeutic agent delivered to a patient consistent with an acceptable level of side effects. Therefore, the amount of the biologic delivered depends in part on the severity of a specific entity and the illness being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available (see, e.g., 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 Antibodies 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).

[0269] The appropriate dose is determined by the clinician, for example, using parameters or factors known in the art or suspected to affect treatment or expected to affect treatment. Typically, the dose is started with an amount slightly less than the optimal dose and increased thereafter in small increments until the desired or optimal effect is achieved relative to any adverse side effects. Important diagnostic measures include, for example, diagnostic measures for infusion reaction symptoms.

[0270] The actual dosage level of the active ingredient in the pharmaceutical composition with anti-HBsAg antibodies can be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the neutralizing activity of the antibody, the route of administration, the time of administration, the half-life of the antibody in the patient, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors known in the medical field.

[0271] Compositions comprising antibodies or fragments thereof can be provided by continuous infusion, or in doses, for example, at intervals of one day, one week, or 1-7 times per week. Doses can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. A specific dosage regimen is one that involves a maximum dose or frequency of administration that avoids significant undesirable side effects.

[0272] For the antibodies described herein, the dosage administered to a patient can be 0.0001 mg / kg to 100 mg / kg of patient weight. The dosage can be between 0.0001 mg / kg and 20 mg / kg of patient weight, between 0.0001 mg / kg and 10 mg / kg of patient weight, between 0.0001 mg / kg and 5 mg / kg of patient weight, between 0.0001 mg / kg and 2 mg / kg of patient weight, between 0.0001 and 1 mg / kg of patient weight, between 0.0001 and 0.75 ... The dosage of the antibody or fragment thereof can be calculated by multiplying the patient's weight in kilograms (kg) by the dose to be administered in mg / kg.

[0273] The dosage of the antibody may then be repeated and each administration may be separated by 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.

[0274] The effective amount for a particular patient may vary depending on factors such as the condition being treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects (see, e.g., 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, England, 2001).

[0275] Administration can be by, for example, topical or cutaneous application, by injection or infusion performed intravenously, intraperitoneally, intracerebrally, intramuscularly, intraocularly, intraarterially, intracerebrospinal, intralesionally, or by sustained release systems or implants (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981). er, Chem. Tech. [Chemical Technology] 12: 98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 82: 3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 77: 4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). If necessary, the composition may also include a solubilizing agent or a local anesthetic such as lidocaine for relieving pain at the injection site, or both. In addition, pulmonary administration may also be employed, for example by using an inhaler or nebulizer and a formulation containing an atomizing agent. See, e.g., U.S. Patent Nos. 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 Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0276] The compositions of the present disclosure can also be administered using one or more of the various methods known in the art by one or more routes of administration. As will be appreciated by those skilled in the art, the administration route and / or mode will vary according to the desired result. The selected route of administration of the antibody includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion. Parenteral administration can represent the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered by non-parenteral routes, such as topical, epidermal or transmucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical. In one aspect, the antibodies of the present disclosure are administered by infusion. In another aspect, the antibodies are administered subcutaneously.

[0277] If the antibodies of the disclosure are administered in a controlled or sustained release system, a pump can be used to achieve controlled or sustained release (see Langer, supra; 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). Polymeric materials can be used to achieve controlled or sustained release of antibody therapy (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance [Controlled Drug Bioavailability, Drug Product Design and Performance], Smolen and Ball (eds.), Wiley [Wiley Publishing Company], New York [New York City], 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. [Journal of Polymer Science, Review of Polymer Chemistry] 23:61, 1983; see also Levy et al., Science [Science] 228:190, 1985; During et al., Ann. Neurol. [Neurological Record] 25:351, 1989; Howard et al., J. Neurosurg. [Journal of Neurosurgery] 7 1:105, 1989; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one aspect, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.Controlled or sustained release systems can be placed in proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in: Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138, 1984).

[0278] Controlled release systems are discussed in the review by Langer, Science 249:1527-1533, 1990. Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more antibodies of the disclosure. See, e.g., U.S. Pat. No. 4,526,938, PCT Publication WO 91 / 05548, PCT Publication WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Sciences. Science & Technology [PDA Journal of Pharmaceutical Science and Technology] 50: 372-397, 1995; Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. [Proceedings of the International Symposium on Controlled Release of Bioactive Materials] 24: 853-854, 1997; and Lam et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. [Proceedings of the International Symposium on Controlled Release of Bioactive Materials] 24: 759-760, 1997, each of which is incorporated herein by reference in its entirety.

[0279] If the antibodies of the present disclosure are applied topically, they can be formulated in the form of ointments, creams, transdermal patches, lotions, gels, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous to semisolid or solid forms are generally used, which contain a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some cases, greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments, etc., which, if desired, can be sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect a variety of properties, such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations, in which the active ingredient is packaged in combination with a solid or liquid inert carrier in some cases in a mixture with a pressurized volatile substance (e.g., a gas propellant such as freon) or in a squeeze bottle. If desired, moisturizers or wetting agents may also be added to the pharmaceutical composition and dosage form. Examples of such additional ingredients are well known in the art.

[0280] If the composition comprising the antibody is administered intranasally, it may be formulated in the form of an aerosol, spray, mist or in the form of drops. In particular, the prophylactic or therapeutic agent used in accordance with the present disclosure may be conveniently delivered in an aerosol spray presentation form from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, for example, gelatin) used in an inhaler or insufflator may be formulated as a powder mixture containing the compound and a suitable powder base such as lactose or starch.

[0281] Methods of co-administration or treatment with a second therapeutic agent (e.g., an immunosuppressant, cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation) are known in the art (see, e.g., Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, PA; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Philadelphia, Pa.). An effective amount of the therapeutic agent can reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40% or at least 50%.

[0282] Other therapeutic agents (e.g., prophylactic or therapeutic agents) that can be administered in combination with the anti-HBsAg antibodies of the present disclosure can be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 hour apart, about 1 hour apart to about 2 hours apart, about 2 hours apart to about 3 hours apart, about 3 hours apart to about 4 hours apart, about 4 hours apart to about 5 hours apart, about 5 hours apart to about 6 hours apart, about 6 hours apart to about 7 hours apart, about 7 hours apart to about 8 hours apart, about 8 hours apart to about 15 hours apart, about 10 hours apart to about 15 hours apart, about 15 hours apart to about 16 hours apart, about 17 hours apart to about 18 hours apart, about 19 hours apart to about 20 hours apart, about 21 hours apart to about 25 hours apart, about 22 hours apart to about 26 hours apart, about 24 hours apart to about 27 hours apart, about 25 hours apart to about 28 hours apart, about 26 hours apart to about 29 hours apart, about 30 hours apart to about 31 hours apart, about 30 hours apart to about 32 hours apart, about 30 hours apart to about 33 hours apart, about 30 hours apart to about 34 hours apart, about 30 hours apart to about 35 hours apart, about 30 hours apart to about 36 hours apart, about 30 hours apart to about 37 hours apart, about 30 hours apart to about 38 hours apart, about 30 hours apart to about 39 hours apart, about 30 hours apart to about 39 hours apart, about The two or more therapeutic agents may be administered at about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. Two or more therapeutic agents may be administered during the same visit of a patient.

[0283] In some aspects, anti-HBsAg antibodies can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. In order to ensure that anti-HBsAg antibodies cross the BBB (if necessary), they can be formulated in, for example, liposomes. For methods of preparing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can include one or more parts that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. [Journal of Clinical Pharmacology] 29: 685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to 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 receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al., (1994), J. Biol. Chem. [Journal of Biological Chemistry] 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. [Letter of the Federation of European Biochemical Societies] 346:123; JJ Killion; IJ Fidler (1994) Immunomethods [Immunomethods] 4:273.

[0284] The present disclosure provides a scheme for administering a pharmaceutical composition comprising an antibody to a subject in need thereof, either alone or in combination with other therapies. Combination therapy (e.g., a prophylactic or therapeutic agent) can be administered to a subject simultaneously or sequentially. The treatment (e.g., a prophylactic or therapeutic agent) of a combination therapy can also be administered in cycles. Cyclic therapy involves administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating the sequential administration, i.e., the cycle, to reduce the generation of drug resistance to a therapy (e.g., a medicament), to avoid or reduce the side effects of a therapy (e.g., a medicament) and / or to improve the efficacy of a therapy.

[0285] The therapy (e.g., preventive or therapeutic agent) of the combination therapy disclosed herein can be administered to the subject in parallel. The term "concurrently" is not limited to administering therapy (e.g., preventive or therapeutic agent) at exactly the same time, but means that a pharmaceutical composition comprising an antibody or its fragment is administered to the subject in a certain order and within a certain time interval, so that the antibody can work together with one or more other therapies to provide an increased benefit compared to administration in other ways. For example, each therapy can be administered to the subject at the same time or in any order at different time points in sequence; however, if not administered at the same time, the therapy should be administered sufficiently close in time to provide the desired treatment or preventive effect. Each therapy can be administered to the subject in any appropriate form and by any suitable route, respectively. In various aspects, therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 hour apart, about 2 hours apart, about 3 hours apart, about 4 hours apart, about 4 hours apart, about 5 hours apart, about 6 hours apart, about 7 hours apart, about 8 hours apart, about 9 hours apart, about 10 hours apart, about 11 hours apart, about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other aspects, two or more therapies (e.g., prophylactic or therapeutic agents) are administered to a patient at the same visit of the patient.

[0286] The prophylactic or therapeutic agent of the combination therapy can be administered to the subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent of the combination therapy can be administered to the subject in parallel in a separate pharmaceutical composition. The prophylactic or therapeutic agent can be administered to the subject by the same or different administration routes.

[0287] Examples

[0288] Example 1: Generation of anti-HBsAg viral antibodies

[0289] Human memory B cells from donors inoculated with HBV were expanded in vitro and selected for their ability to secrete IgG antibodies against HBsAg. Specific B cells were lysed and VH (heavy) and VL (light) chains were amplified by RT-PCR and subsequently sequenced and analyzed to identify key post-translational modification (PTM) sites. The plasmids of the VH and VL chains were then transfected into an IgG1 backbone vector in a CHO mammalian cell line to express a complete IgG1 antibody.

[0290] Methods for generating monoclonal antibodies using phage display technology are known in the art (Antibody Methods and Protocols, Methods in Molecular Biology, Vol. 901, 2012, Chapter 3:33). Briefly, solution panning was performed using streptavidin-coupled magnetic beads complexed with biotinylated recombinant HBsAg (TRINA Bioreactives AG, Catalog No. C028-3001994774 (AD serotype), C028-3001994774 (AY serotype) or Biorbyte, Catalog No. orb82536 (AD isotype)), and anti-HBsAg antibodies in a synthetic human germline framework antibody library in the form of Fab randomized in CDR-H3 were screened with increasing stringency. The isolate was first expressed as Fab and screened for binding to HBsAg serotypes AD and AY by ELISA. The selected isolates were then cloned and expressed as IgG1, reanalyzed by ELISA for binding to HBsAg (serotypes AD and AY) and functional activity in neutralization assays, and finally transfected into CHO mammalian cell lines to express complete IgG1 antibodies. Anti-HBsAg antibody affinity maturation was achieved by CDR-directed mutagenesis. Beneficial mutations have been identified by deep sequencing to compare the enrichment of the initial mutagenesis library after two rounds of phage display. Beneficial mutations selected individually and in combination were then cloned and expressed as IgG1, reanalyzed by ELISA for binding to HBsAg (serotypes AD and AY) and functional activity in neutralization assays, and finally transfected into CHO mammalian cell lines to express complete IgG1.

[0291] The changes in anti-HBsAg antibodies are provided in Table 3 .

[0292] Table 3

[0293]

[0294]

[0295] Example 2: Binding of anti-HBsAg antibodies to HBsAg

[0296] The binding affinity interactions (K) of anti-HBsAg antibodies to two major serotypes of HBsAg (AY and AD) were determined using surface plasmon resonance (SPR) technology. DHBsAg particles were immobilized on an S-series CM5 sensor chip at approximately 800 RU, and anti-HBsAg antibodies were flowed in 2-fold serial dilutions starting from 128 nM to assess binding using a Biacore T200 instrument (GE Heathcare, catalog number 28975001, Pittsburgh, PA). K was determined by fitting the curves with a 1:1 fitting model. D (O'Shannessy et al. Anal. Biochem [Analytical Biochemistry] 1993; 212: 457-468; Karlsson, J. Immunol. Methods. 1997; 200: 121-133).

[0297] K of anti-HBsAg antibodies measured by Biacore D The values ​​ranged from 110 pM to 40 nM and were comparable between the two major serotypes (AD and AY) for each antibody tested. The Biacore affinity data for anti-HBsAg antibodies are summarized in Table 4 and the SPR curves are shown in Figure 1 A / B-16A / B.

[0298] Table 4

[0299]

[0300]

[0301] Example 3: Neutralization of Hepatitis B Virus Infection by Anti-HBsAg Antibodies

[0302] Infectious HBV virus was purified from genotype D, serotype ayw cell culture derived HBV as described (Meier et al., J. Virol Hepat. [Journal of Viral Hepatitis] 2017; 24: 662-671). Anti-HBsAg antibodies were pre-incubated with the virus at 37 ° C for 1 hour to allow binding and neutralization. HepG2-hNTCP1 cells, which were generated in-house as described (Tropberger et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 2015; 112: E5715-E5724), were then exposed to the virus antibody mixture for 24 hours, replaced with fresh medium, and incubated for another 6 days to allow viral entry, cccDNA establishment and viral protein expression. The supernatant was recovered and analyzed for HBeAg levels by a custom in-house eAg AlphaScreen assay (Perkin Elmer, Bridgeville, Pennsylvania). Data were analyzed using an Envision plate reader (PerkinElmer, Catalog No. 2105-0010, Bridgeville, PA) and expressed as percent infection relative to untreated control wells.

[0303] All antibodies were able to neutralize HBV infection, EC 50 The values ​​ranged from 17 pM to 740 pM and this is shown in Table 5 and graphically in Figure 17-33 middle.

[0304] Table 5

[0305]

[0306] Example 4: Hepatitis B genotype binding

[0307] Anti-HBsAg antibodies were analyzed for binding to the four major genotypes (AD) of HBV by sandwich ELISA. Briefly, ELISA plates (Thermo Scientific, catalog number 15031) were coated with 5 μg / ml horse polyclonal HBsAg capture antibody (MyBiosource, catalog number MBS315002) for 2 hours at 37°C and then blocked with 5% milk overnight at 4°C. Supernatants collected from genotypes derived from HBV cell cultures in the pcDNA3.1 backbone (genotype A: AY934772, genotype B: AF121245, genotype C: DQ087960, genotype D: DQ219811.1) were combined with antibody-coated plates for 1 hour. The plates were washed (α diagnostic wash buffer, catalog number 80080) and incubated at room temperature for 1 hour with serial dilutions of anti-HBsAg antibodies in LowCross buffer (Candor, catalog number 100500). After incubation with anti-HBsAg antibodies, the plates were washed and incubated at room temperature for 1 hour with a secondary antibody (HRP-conjugated goat anti-human IgG Fab fragment, Jackson ImmunoResearch Inc, catalog number 109-035-097) diluted 1:2000 in dilution buffer (LowCross buffer, Candor, catalog number 100500). The plates were washed and reacted using tetramethylbenzidine (TMB) microwell peroxidase substrate (α diagnostic, catalog number 80091).

[0308] Anti-HBsAg antibody NOV3834-36 against genotype A (IC 50 range from 2nM to 3.3nM), genotype B (IC 50 ranged from 1.2 nM to 1.7 nM) and genotype D (IC 50 ranged from 1.3 nM to 8.2 nM) showed similar binding but not for genotype C (IC 50 In contrast, the remaining anti-HBsAg antibodies were in the 4 major genotypes (IC 50 The IC values ​​of NOV3832 for genotype A ranged from 0.023 nM to 2.3 nM, genotype B ranged from 0.017 nM to 1.5 nM, genotype C ranged from 0.015 nM to 3.6 nM, and genotype D ranged from 0.026 nM to 1.3 nM. 50 The IC values ​​for all antibodies in Table 6 were 0.05 nM for genotype A, 0.02 nM for genotype B, 0.015 nM for genotype C and 0.043 nM for genotype D. 50Graphically shown in Figure 34-50 middle.

[0309] Table 6

[0310]

[0311]

[0312] Example 5: Identification of clinical mutations of hepatitis B by anti-HBsAg

[0313] Anti-HBsAg antibodies were analyzed for binding to 4 well-characterized vaccines and / or HBsAg clinical mutations (G145R, D144A, T126S, M133L) of HBsAg by sandwich ELISA. Mutations were generated by site-directed mutagenesis (New England Biolabs, catalog number E0554S) along Q5 of HBV genotype D (ayw serotype) and HBsAg sequences were cloned into pCl-neo vector (Promega, catalog number E1841) to generate stable cell lines. Briefly, ELISA plates (Thermo Fisher Scientific, catalog number 15031) were coated with 5 μg / ml horse polyclonal HBsAg capture antibody (McBio Resources, catalog number MBS315002) for 2 hours at 37°C and then blocked overnight with 5% milk at 4°C. The supernatant collected from the clinical mutation stable strain (G145R, D144A, T126S, M133L) derived from HBsAg cell culture was combined with the antibody coated plate for 1 hour. The plate was washed (α diagnostic wash buffer, catalog number 80080) and incubated at room temperature for 1 hour with the serial dilutions of anti-HBsAg antibody in LowCross buffer (Candor (Candor company), catalog number 100500). After the anti-HBsAg antibody was incubated, the plate was washed and incubated at room temperature for 1 hour with the second antibody (HRP-conjugated goat anti-human IgG Fab fragment, Jackson ImmunoResearch Inc (Jackson ImmunoResearch Inc), catalog number 109-035-097) diluted at 1:2000 in dilution buffer (LowCross buffer, Candor company, catalog number 100500) . The plates were washed and the reaction was carried out using tetramethylbenzidine (TMB) microwell peroxidase substrate (Alpha Diagnostics, Cat. No. 80091).

[0314] As shown in Table 7, anti-HBsAg antibodies NOV2603, NOV3212, NOV3357 and NOV3540 were able to bind to all four clinical mutations (G145RIC 50Values ​​ranged from 0.033 nM to 5.5 nM, D144A ranged from 0.079 nM to 2.1 nM, T126S ranged from 0.06 nM to 0.22 nM, and M133L ranged from 0.012 nM to 0.67 nM). Antibodies NOV3831, NOV3832, NOV3833, NOV3838, NOV3839, NOV3841, and NOV3842 showed loss of binding to the G145R mutation, but retained binding to the other 3 clinical mutations (D144A IC 50 In contrast, anti-HBsAg antibodies NOV3834-36 showed no binding to any of the four mutations (NOV3835, NOV3836) or had very reduced binding to only two of the clinical mutation panels (T126S, M133L) (NOV3835) (T126S IC 50 The value is 7.4nM, and the M133L IC 50 The results shown in Table 7 are also Figure 51-66 Represented graphically in .

[0315] Table 7

[0316]

[0317]

[0318] Example 6: Anti-HBsAg Antibodies Show Efficacy in the FRGN Mouse Model

[0319] In the FRGN mouse model of HBV infection, the in vivo efficacy of anti-HBsAg antibodies was determined by monitoring the loss of HBsAg after antibody administration. FRGN mice with human livers were purchased from Yecuris (FRGN KO on NOD, catalog number 10-0013) and were treated with 1.7×10 7HBV (genotype C) infection with 100 copies of HBV. After stable infection, mice were treated with 20 mg / ml anti-HBsAg antibody (n=2 / group) on days 0 and 21, and serum was collected at 0, 0.02, 0.3, 1, 3, 7, 14, 21, 24 and 28 days after initial antibody treatment. Free HBsAg levels were monitored by the protocol from Zhang et al., Gut [Intestinal] 2016; (4) 65: 658-671. In brief, serum was diluted 1:5 in sample diluent (provided by Alpha Diagnostic International, catalog number 4110), 0.5 volumes of lysis buffer (15% sodium dodecyl sulfate in 20 mM TrisHCL buffer-pH 8.0) were added, and the samples were incubated at 37 ° C for 1 hour. The samples were neutralized by adding 5 volumes of 4% CHAPS dissolved in 20 mM Tris HCL buffer-pH 8.0 and HBsAg levels were monitored using a HBsAg commercial ELISA kit (Alpha Diagnostics International, Cat. No. 4110) following the manufacturer's protocol. HBsAg levels were plotted as log fold change of pre-bleeding levels collected at time point 0. The maximum log fold change for this model was -3.9.

[0320] Anti-HBsAg antibodies NOV3212, NOV3357, NOV3540, NOV3831, NOV3832, NOV3834, NOV3838, NOV3841, and NOV3842 all significantly reduced HBsAg levels, with maximum log reductions ranging from -3.9 to -2.8, as shown in Table 8. In contrast, NOV2603 showed only a moderate log reduction (-1.1) in HBsAg. Fig.67 In the study, NOV3832 significantly reduced HBsAg at the 0.3 day time point and kept HBsAg levels down for 7 days until HBsAg levels slowly increased on day 14. After the second administration of NOV3832 on day 21, HBsAg levels decreased again and remained decreased until the end of the study on day 28. Therefore, this study demonstrates that the anti-HBsAg antibodies disclosed herein can significantly reduce HBsAg in serum in vivo.

[0321] Table 8

[0322]

[0323]

[0324] Example 7: Anti-HBsAg Cytotoxicity

[0325] The cytotoxicity of anti-HBsAg antibodies to de novo HBV-infected and uninfected HepG2-hNTCP1 cells was measured using CellTiter-Glo (Promega, catalog number G7570) (Tropberger et al. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 2015; 112: E5715-E5724), which monitors the metabolic activity of the cells by ATP concentration. Briefly, HepG2-hNTCP1 cells were infected with purified virus from genotype D (serotype ayw) cell culture-derived HBV or mock virus as described (Meier et al. J. Virol Hepat. [Journal of Viral Hepatitis] 2017; 24: 662-671). After infection, the cells were cultured for 4 days to allow viral entry, cccDNA establishment and viral protein expression, and the culture medium was removed and replaced with fresh culture medium containing serial dilutions of anti-HBsAg antibodies starting at 3.3 μM. Cells were incubated with the antibody mixture for an additional 4 days and then assayed with CellTiter-Glo reagent according to the manufacturer's protocol. Luminescence readings were performed using a PHERAstar microplate reader (BMG Labtech, Cary, NC) and given as a percentage of viability relative to a negative (no antibody) control.

[0326] All anti-HBsAg antibodies disclosed herein have no effect on cell viability in the presence or absence of virus. In short, the anti-HBsAg antibodies disclosed herein have no toxicity. This is in contrast to two disclosed antibodies CR8097 and HB48-59, which show cytotoxicity (CC50 range of uninfected HepG2-NTCP1 is from 2.24uM to 2.86uM, and HepG2-NTCP1 range of HBV infection is from 2.09uM to 2.11uM). Since CR8097 and HB48-59 antibodies show toxicity to uninfected cells at the same level as infected cells, this indicates that these antibodies are combined with normal cell proteins that are off-target.

[0327] Table 9

[0328]

[0329]

[0330] Example 8: Epitope binning of anti-HBsAg antibodies by SPR

[0331] Surface plasmon resonance (SPR) technology is used to carry out the epitope binning of anti-HBsAg antibodies and two major serotypes AY and AD of HBsAg. Antigens HBsAg AD and AY (TRINA Bioreactives AG, catalog number 0824 [AD serotype], #0823 [AY serotype], Naenikon, Switzerland) are fixed on the individual cells on the surface of CM5 chip with about 800RU by amine coupling. By subsequently applying a kind of antibody as the first antibody (saturation), then applying another antibody as the second antibody (competition), each pair of antibodies is tested to block the binding of each other to the epitope on its antigen, and vice versa. Saturated antibodies are applied for 120 seconds with 500nM. Competitive antibodies utilize the same conditions, but the first antibody of 500nM is supplemented to maintain saturation. The binding signal is corrected by subtracting the reference cell signal. The specific binding of the competing antibody in the presence of saturated antibodies is calculated by subtracting the saturated antibody plus buffer reference signal. All signals are normalized to the fixed ligand of 100RU. The specific binding signal obtained from the use as the competition antibody is expressed as the percentage of the binding signal obtained from the use of the antibody identical with the saturated (first) antibody, and its signal is considered as 100% combination. The combination value is less than 40% to show that the first antibody and the second antibody cover the same region, and the value is greater than 60% to represent different epi-positions. The results show that NOV3540, NOV3832, NOV3841 and NOV3842 do not compete with CR8087 or HBC34 antibodies. Therefore, NOV3540, NOV3832, NOV3841 and NOV3842 are combined with the epi-positions different from CR8087 or HBC34. The results are shown in the following table 10.

[0332] Table 10

[0333]

[0334]

[0335] Example 9: Preparation

[0336] The anti-HBsAg virus antibodies described herein are monoclonal antibodies, IgG1 isotypes with kappa or lambda light chains, and can be lyophilized. For subsequent intravenous administration, the obtained solution is usually further diluted into a carrier solution to prepare an antibody solution that is readily available for infusion. The important stability-indicating analytical methods for selecting the most stable formulations particularly encompass size exclusion chromatography, subvisible particle testing, and efficacy testing for determining aggregation levels.

[0337] It should be understood that the examples and aspects described herein are for illustrative purposes only and that various modifications or changes therefrom will be apparent to those skilled in the art and are included within the spirit and scope of this application and the scope of the appended claims.

[0338] In one aspect of the present invention, it relates to the following specific embodiments:

[0339] 1. An isolated 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;

[0340] (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;

[0341] (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;

[0342] (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;

[0343] (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;

[0344] (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;

[0345] (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;

[0346] (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;

[0347] (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;

[0348] (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;

[0349] (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;

[0350] (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;

[0351] (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;

[0352] (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;

[0353] (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

[0354] (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.

[0355] 2. The antibody of embodiment 1, wherein one or two amino acids within the CDR have been modified, deleted or substituted.

[0356] 3. The antibody of embodiment 1, which retains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the heavy chain variable region or the light chain variable region.

[0357] 4. The antibody of embodiment 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.

[0358] 5. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

[0359] (i) a heavy chain variable region (vH) comprising SEQ ID NO: 18, and a light chain variable region (vL) comprising SEQ ID NO: 34;

[0360] (ii) a heavy chain variable region (vH) comprising SEQ ID NO: 50, and a light chain variable region (vL) comprising SEQ ID NO: 66;

[0361] (iii) a heavy chain variable region (vH) comprising SEQ ID NO: 82, and a light chain variable region (vL) comprising SEQ ID NO: 98;

[0362] (iv) a heavy chain variable region (vH) comprising SEQ ID NO: 114, and a light chain variable region (vL) comprising SEQ ID NO: 130;

[0363] (v) a heavy chain variable region (vH) comprising SEQ ID NO: 146, and a light chain variable region (vL) comprising SEQ ID NO: 162;

[0364] (vi) a heavy chain variable region (vH) comprising SEQ ID NO: 178, and a light chain variable region (vL) comprising SEQ ID NO: 194;

[0365] (vii) a heavy chain variable region (vH) comprising SEQ ID NO: 210, and a light chain variable region (vL) comprising SEQ ID NO: 226;

[0366] (viii) a heavy chain variable region (vH) comprising SEQ ID NO: 242, and a light chain variable region (vL) comprising SEQ ID NO: 258;

[0367] (ix) a heavy chain variable region (vH) comprising SEQ ID NO: 274, and a light chain variable region (vL) comprising SEQ ID NO: 290;

[0368] (x) a heavy chain variable region (vH) comprising SEQ ID NO: 306, and a light chain variable region (vL) comprising SEQ ID NO: 322;

[0369] (xi) a heavy chain variable region (vH) comprising SEQ ID NO: 338, and a light chain variable region (vL) comprising SEQ ID NO: 354;

[0370] (xii) a heavy chain variable region (vH) comprising SEQ ID NO: 370, and a light chain variable region (vL) comprising SEQ ID NO: 386;

[0371] (xiii) a heavy chain variable region (vH) comprising SEQ ID NO: 402, and a light chain variable region (vL) comprising SEQ ID NO: 418;

[0372] (xiv) a heavy chain variable region (vH) comprising SEQ ID NO: 434, and a light chain variable region (vL) comprising SEQ ID NO: 450;

[0373] (xv) a heavy chain variable region (vH) comprising SEQ ID NO: 466, and a light chain variable region (vL) comprising SEQ ID NO: 482; or

[0374] (xvi) a heavy chain variable region (vH) comprising SEQ ID NO: 498, and a light chain variable region (vL) comprising SEQ ID NO: 514.

[0375] 6. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof retains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the light chain variable region or the heavy chain variable region.

[0376] 7. An antibody as described in embodiment 5, wherein one, two, three, four or five but less than 10 amino acids within the light chain variable region or the heavy chain variable region have been modified, deleted or substituted.

[0377] 8. The antibody of embodiment 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.

[0378] 9. The antibody of embodiment 1, wherein the antibody or fragment thereof has reduced glycosylation or no glycosylation or low fucosylation.

[0379] 10. A pharmaceutical composition comprising the antibody or fragment thereof according to embodiment 1, further comprising a pharmaceutically acceptable carrier.

[0380] 11. The pharmaceutical composition according to embodiment 10, wherein the pharmaceutically acceptable carrier contains histidine or a sugar.

[0381] 12. The pharmaceutical composition of embodiment 11, wherein the sugar is sucrose.

[0382] 13. A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments as described in embodiment 1, 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.

[0383] 14. A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments as described in embodiment 1, wherein none of the antibodies comprises a bisected GlcNAc.

[0384] 15. A pharmaceutical composition comprising the antibody or fragment thereof according to embodiment 1, wherein the composition is prepared as a lyophilisate.

[0385] 16. A method for neutralizing hepatitis B virus infection, the method comprising administering an effective amount of the antibody of embodiment 1 to a patient in need thereof via injection or infusion.

[0386] 17. The method of embodiment 16, wherein the patient in need thereof is diagnosed with hepatitis B viruria or hepatitis B viremia.

[0387] 18. The method of embodiment 16, wherein the patient in need thereof is diagnosed with the presence of hepatitis B surface antigen (HBsAg) in the blood or serum.

[0388] 19. A method of treating or reducing the likelihood of a hepatitis B virus-related disorder, the method comprising administering an effective amount of the antibody of embodiment 1 to a patient in need thereof via injection or infusion, and wherein the disorder is: liver failure, cirrhosis, or hepatocellular carcinoma.

[0389] 20. The method of embodiment 19, wherein the antibody or composition is reconstituted prior to injection or infusion.

[0390] 21. The method of embodiment 20, wherein the antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

[0391] 22. The method of embodiment 21, wherein the therapeutic agent is an antiviral agent.

[0392] 23. The method of embodiment 22, wherein the antiviral agent is: lamivudine, entecavir and tenofovir or alpha-interferon.

[0393] 24. A method as described in embodiment 23, wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0394] 25. A method as described in embodiment 24, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0395] 26. The method of embodiment 25, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0396] 27. The method of embodiment 21, wherein the therapeutic agent is an additional anti-HBsAg antibody.

[0397] 28. The antibody or fragment thereof according to embodiment 1, for use as a medicament.

[0398] 29. The antibody or fragment thereof as described in embodiment 1, for neutralizing hepatitis B virus infection.

[0399] 30. The antibody or fragment thereof of embodiment 1, for use in treating or reducing the likelihood of: liver failure, cirrhosis, and / or hepatocellular carcinoma.

[0400] 31. The use according to embodiment 30, which is administered in combination with another therapeutic agent.

[0401] 32. The use according to embodiment 31, wherein the therapeutic agent is an antiviral agent.

[0402] 33. The use according to embodiment 32, wherein the antiviral agent is: lamivudine, entecavir and tenofovir or alpha-interferon.

[0403] 34. The use as described in embodiment 31, wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

[0404] 35. The use as described in embodiment 34, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0405] 36. The use according to embodiment 35, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0406] 37. The use according to embodiment 31, wherein the therapeutic agent is another anti-HBsAg antibody.

[0407] 38. A nucleic acid encoding the antibody or antigen-binding fragment of embodiment 1.

[0408] 39. A vector comprising the nucleic acid of embodiment 38.

[0409] 40. A host cell comprising the vector of embodiment 39.

[0410] 41. A diagnostic reagent, comprising a labeled antibody or antigen-binding fragment thereof according to embodiment 1.

[0411] 42. The diagnostic reagent of embodiment 41, wherein the label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion.

Claims

1. An isolated anti-hepatitis B surface antigen antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: 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; The antigen binding fragment is selected from single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragment, F(ab)' fragment, F(ab)2 fragment, or Fv fragment consisting of the VL and VH domains of a single antibody arm.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a human engineered antibody.

5. An isolated anti-hepatitis B surface antigen antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (vH) comprising SEQ ID NO: 18, and a light chain variable region (vL) comprising SEQ ID NO: 34; The antigen binding fragment is selected from single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragment, F(ab)' fragment, F(ab)2 fragment, or Fv fragment consisting of the VL and VH domains of a single antibody arm. The antibody or antigen-binding fragment thereof according to claim 5 , wherein the antibody is a monoclonal antibody.

7. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

8. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody is a human engineered antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is aglycosylated or is hypofucosylated. 10 . A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 , and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable carrier contains histidine or a sugar.

12. The pharmaceutical composition of claim 11, wherein the sugar is sucrose.

13. A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 9, 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.

14. A pharmaceutical composition comprising a plurality of antibodies or antigen-binding fragments thereof according to any one of claims 1 to 9, wherein none of the antibodies comprises a bisected GlcNAc.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the composition is prepared as a lyophilisate.

16. Use of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 9 or a pharmaceutical composition as claimed in any one of claims 10 to 15 in the manufacture of a medicament for a method of neutralizing hepatitis B virus infection, wherein the method comprises administering an effective amount of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 9 or a pharmaceutical composition as claimed in any one of claims 10 to 15 to a patient in need thereof by injection or infusion.

17. The use according to claim 16, wherein the patient in need thereof is diagnosed with hepatitis B viruria or hepatitis B viremia.

18. The use of claim 16, wherein the patient in need thereof is diagnosed with the presence of hepatitis B surface antigen (HBsAg) in blood or serum.

19. Use of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9 or a pharmaceutical composition as described in any one of claims 10 to 15 in the manufacture of a medicament for treating or reducing the likelihood of a hepatitis B virus-related disorder, wherein the treatment or reduction comprises administering an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 9 or a pharmaceutical composition as described in any one of claims 10 to 15 to a patient in need thereof via injection or infusion, and wherein the disorder is: liver failure, cirrhosis or hepatocellular carcinoma.

20. The use of claim 19, wherein the antibody or antigen-binding fragment thereof or composition is reconstituted prior to injection or infusion.

21. The use of claim 20, wherein the antibody or antigen-binding fragment thereof or pharmaceutical composition is administered in combination with another therapeutic agent.

22. The use of claim 21, wherein the therapeutic agent is an antiviral agent.

23. The use according to claim 22, wherein the antiviral agent is: lamivudine, entecavir and tenofovir or alpha-interferon.

24. The use of claim 21, wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

25. The use of claim 24, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

26. The use according to claim 25, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

27. The use of claim 21, wherein the therapeutic agent is another anti-HBsAg antibody.

28. Use of the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 9 in the manufacture of a medicament for treating hepatitis B virus infection.

29. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 in the manufacture of a medicament for treating or reducing the likelihood of liver failure, cirrhosis, and / or hepatocellular carcinoma.

30. The use of claim 29, wherein the medicament is administered in combination with another therapeutic agent.

31. The use of claim 30, wherein the therapeutic agent is an antiviral agent.

32. The use according to claim 31, wherein the antiviral agent is: lamivudine, entecavir and tenofovir or alpha-interferon.

33. The use of claim 30, wherein the therapeutic agent is an antagonist of an immune checkpoint inhibitor.

34. The use of claim 33, wherein the antagonist of the immune checkpoint inhibitor is selected from the group consisting of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

35. The use according to claim 34, wherein the antagonist of the immune checkpoint inhibitor is an anti-PD-L1 antibody.

36. The use of claim 30, wherein the therapeutic agent is another anti-HBsAg antibody.

37. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

38. A vector comprising the nucleic acid of claim 37.

39. A host cell comprising the vector of claim 38.

40. A diagnostic reagent comprising a labeled antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

41. The diagnostic reagent of claim 40, wherein the label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion.

Images