Antibody compositions and methods for treating hepatitis b virus infection

Abstract

To provide a method for treating hepatitis B virus (HBV) infection in a subject.SOLUTION: Provided is a method comprising administering to the subject a single-dose pharmaceutical composition comprising an antibody having a specific sequence that binds to HBsAg.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] Sequence Listing Description The sequence listing associated with this application is provided in text format in lieu of hard copy and is hereby incorporated by reference herein. The name of the text file containing the sequence listing is 930285_412WO_SEQUENCE_LISTING.txt. The text file is 109 KB, was created on August 25, 2020, and is submitted electronically via EFS-Web.

[0002] The present disclosure relates to pharmaceutical antibody compositions and methods for the prevention and treatment of hepatitis B virus infection. [Background technology]

[0003] HBV consists of (i) an envelope containing three associated surface proteins (hepatitis B surface antigen, HBsAg) and lipids, and (ii) an icosahedral nucleocapsid enclosing the viral DNA genome and DNA polymerase. The HBV capsid is formed in the cytosol of infected cells during packaging of the RNA pregenome replication complex and acquires the ability to budding during synthesis of the viral DNA genome by reverse transcription of the pregenome in the particle lumen. The three HBV envelope proteins, S-HBsAg, M-HBsAg, and L-HBsAg, form complex transmembrane folds in the endoplasmic reticulum and form disulfide-linked homo- and heterodimers. During budding at intracellular membranes, a short linear domain of the cytosolic preS region interacts with binding sites on the capsid surface. Virions are then secreted into the bloodstream. Furthermore, surface proteins can bud in the absence of capsids to form secreted subviral particles (SVPs) that are also secreted in 3-4 log excess over virions. High levels of HBsAg can exhaust HBsAg-specific T cell responses and have been proposed as an important factor in viral immune tolerance in patients with chronic hepatitis B (CHB) (Chisari FV, Isogawa M, Wieland SF, Pathologie Biologie, 2010;58:258-66).

[0004] Hepatitis B virus can cause fatal acute and chronic liver infections. Acute hepatitis B is characterized by asymptomatic or silent viremia, with the risk of developing fulminant hepatitis (Liang TJ, Block TM, McMahon BJ, Ghany MG, Guo JT, Locarnini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of B hepatitis: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002 / hep.28025. [Epub ahead of print]). Although an effective vaccine against hepatitis B has been available since 1982, the WHO reports that 240 million people are chronically infected with hepatitis B, and more than 780,000 people die from hepatitis B complications each year. Approximately one-third of patients with chronic hepatitis B (CHB) develop cirrhosis, liver failure, and hepatocellular carcinoma, accounting for 600,000 deaths per year (Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo JT, Locamini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of hepatitis B: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002 / hep.28025. [Epub ahead of print]).

[0005] In patients infected with HBV, severe complications can develop as a result of coinfection or superinfection with HDV. According to the WHO, approximately 15 million people worldwide are infected with hepatitis D. HDV is considered a subviral satellite because it can only propagate in the presence of HBV. HDV is one of the smallest known animal viruses (40 nm), with a genome of only 1.6 kb encoding the S and L HDAg. All other proteins required for HDV genome replication, including RNA polymerase, are provided by the host cell, and the HDV envelope is provided by HBV. Upon introduction into permissive cells, the HDV RNA genome replicates and associates with multiple copies of HDV-encoded proteins to assemble ribonucleoprotein (RNP) complexes. RNPs are exported from the cell by HBV envelope proteins, which can also assemble lipoprotein vesicles that bud into the lumen of the pre-Golgi compartment before being secreted. Furthermore, HBV envelope proteins also provide a mechanism for targeting HDV to uninfected cells, thereby ensuring the spread of HDV. Summary of the Invention [Problem to be solved by the invention]

[0006] Complications due to HDV include an increased likelihood of experiencing liver failure and rapid progression to cirrhosis in acute infection and an increased likelihood of developing liver cancer in chronic infection. Hepatitis D, in combination with hepatitis B virus, has the highest case-fatality rate of all hepatitis infections, at 20% (Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. Gut. 2000 Mar;46(3):420-6). The only approved therapy for chronic HDV infection is interferon-alpha. However, treatment of HDV with interferon-alpha is relatively ineffective and poorly tolerated. Interferon-alpha treatment results in a sustained virologic response 6 months after treatment in one-quarter of patients. Nucleos(id)ide analogs (NAs) have also been widely tested in hepatitis delta, but they appear to be ineffective. Combination therapy with NAs and interferon has also proven disappointing (Zaigham Abbas, Minaam Abbas, Management of hepatitis delta: Need for novel therapeutic options. World J Gastroenterol 2015 August 28; 21(32): 9461-9465). Therefore, new treatment options are needed. [Means for solving the problem]

[0007] The figures provided herein are intended to explain the subject matter covered by this disclosure in more detail. The figures are not intended to limit the disclosure in any way. Throughout this disclosure, the exemplary antibody HBC34v35 (with or without Fc mutations such as MLNS and GAALIE) is also referred to as HBC34-v35 and HBC34-V35. Therefore, it is understood that HBC34v35, HBC34-v35, and HBC34-V35 have the same meaning. Similarly, the exemplary antibody HBC34v34 is also referred to as HBC34-v34 and HBC34-V34, and the exemplary antibody HBC34v7 is also referred to as HBC34-v7 and HBC34-V7. Furthermore, it will be understood that "MLNS-GAALIE" has the same meaning as "MLNS_GAALIE" (i.e., M428L+N434S+G236A+A330L+I332E mutations (EU numbering) in the Fc part). [Brief explanation of the drawings]

[0008] [Figure 1A] 1A-1B show the binding of HBC34-v7 and two modified antibodies of the present disclosure ("HBC34-v34"; "HBC34-v35") to HBsAg adw (1A) and HBsAg adr (1B) at the indicated concentrations, as determined by an antigen-based direct ELISA assay. All antibodies were produced as IgG1 (g1m17, 1 allotype). [Figure 1B] Same as above. [Figure 2A] Figures 2A-2K show the binding of HBC34-v7, HBC34-v34, and HBC34-v35 to all known HBsAg genotypes ((A)-(J)), respectively) and a mock control (K). Genotype-representative sequences showing the outer loops of the HBsAg antigen shown in Example 5 of PCT Publication No. WO 2017 / 060504 were used. Staining was performed by FACS. Antibody concentrations were as indicated on the y-axis of the graphs. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 3A] Figures 3A and 3B show the binding of HBC34-v7 and HBC34-v35 containing wild-type or mutant Fc regions to HBsAg adw in an antigen-based direct ELISA assay (two experiments; data from "Experiment 1" is shown in Figure 3A, and data from "Experiment 2" is shown in Figure 3B). Antigen binding curves are shown in the top panel of each figure. EC50 values ​​(determined by fitting the curves using Graphpad Prism) are shown in the middle panel of each figure. Binding to uncoated plates (control) is shown in the bottom panel of each figure. Fc regions: "HBC34v7" and "HBC34-v35" = wild-type; "HBC34-v35-MLNS" = M428L / N434S; "HBC34-v35-MLNS-GAALIE" = M428L / N434S / G236A / A330L / I332E. Three lots of HBC34-v35 were tested. Two lots of HBC34-v35-MLNS and two lots of HBC34-v35-MLNS-GAALIE were tested. One lot of HBC34-v7 was used. [Figure 3B] Same as above. [Figure 4]Figures 4-7 show the effect of HBC34-v35 on serum HBAg levels in an in vivo mouse model of HBV infection. AAV / HBV-infected SCID mice were transplanted with primary human hepatocytes and administered 1, 5, or 15 mg / kg of HBC34-v35 or PBS (control) as described in Example 5. Figure 4 shows serum HBV DNA concentrations before and after treatment. Figure 5 shows serum HBsAg concentrations before and after treatment. Figure 6 shows serum HBeAg concentrations before and after treatment. Figure 7 shows serum HBcrAg concentrations before and after treatment. [Figure 5] Same as above. [Figure 6] Same as above. [Figure 7] Same as above. [Figure 8A] Figures 8A-8E show the binding of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE to human FcγRs assessed by biolayer interferometry (BLI). 2 μg / ml of His-tagged human FcγRs ((A) FcγRIIa allele H131; (B) FcγRIIa allele R131; (C) FcγRIIIa allele F158; (D) FcγRIIIa allele V158; (E) FcγRIIb) were captured with an anti-pentaHis sensor for 6 min. The FcγR-loaded sensors were then exposed to a kinetics buffer solution (pH 7.1) containing 2 μg / ml of each mAb in the presence of 1 μg / ml of affiniPure F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific (crosslinking human mAbs through the Fab fragments) for 5 min (left part of plot), followed by a dissociation step in the same buffer for an additional 4 min (right part of plot). Binding and dissociation profiles were measured in real time as changes in the interference pattern using an Octet RED96 (ForteBio). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 9]Figure 9 shows the binding of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE to human C1q, as measured by Octet. Using an anti-human Fab (CH1) sensor, intact IgG1 of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE mAbs was captured via the Fab fragments at 10 μg / ml for 10 minutes. The IgG-loaded sensor was then exposed to a kinetics buffer solution (pH 7.1) containing 3 μg / ml of purified human C1q for 4 minutes (left part of the plot), followed by a dissociation step in the same buffer for another 4 minutes (right part of the plot). The binding and dissociation profiles were measured in real time as changes in the interference pattern using an Octet RED96 (ForteBio). [Figure 10A] Figures 10A and 10B show in vitro activation of human FcγRIIIa using receptor-binding activation of an NFAT-mediated luciferase reporter in engineered Jurkat cells. FcγRIIIa activation was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34v35-MLNS and HBC34-v35-MLNS-GAALIE and control (Ctr) mAbs were incubated with 0.2 μg / ml HBsAg for 25 minutes at 37°C. Jurkat effector cells (Promega) expressing either the low-affinity FcγRIIIa allele F158 (A) or the high-affinity FcγRIIIa allele V158 (B) were resuspended in assay buffer and then added to the assay plate. After 24 hours of incubation at 37°C, Bio-Glo-™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a luminometer (Bio-Tek). [Figure 10B] Same as above. [Figure 11A]Figures 11A and 11B show in vitro activation of human FcγRIIa using receptor-binding activation of an NFAT-mediated luciferase reporter in engineered Jurkat cells. Human FcγRIIa activation was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and a control mAb (Ctr) were incubated with HBsAg at 2 (A) or 0.2 μg / ml (B) for 25 minutes at 37°C. Jurkat effector cells expressing the FcγRIIa high-affinity allele H131 (Promega) were resuspended in assay buffer and then added to the assay plate. After 23 hours of incubation at 37°C, Bio-Glo-™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a luminometer (Bio-Tek). [Figure 11B] Same as above. [Figure 12] Figure 12 shows in vitro activation of human FcγRIIb using receptor-binding activation of an NFAT-mediated luciferase reporter in engineered Jurkat cells. Human FcγRIIb activation was tested using a validated commercially available bioreporter assay using recombinant HBsAg (Engerix B) as the target antigen. Serial dilutions of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE and a control mAb (Ctr) were incubated with 1 μg / ml HBsAg for 15 minutes at 37°C. Jurkat effector cells (Promega) expressing FcγRIIb were resuspended in assay buffer and then added to the assay plate. After 20 hours of incubation at 37°C, Bio-Glo™ Luciferase Assay Reagent (Promega) was added, and luminescence was quantified using a luminometer (Bio-Tek). [Figure 13A]Figures 13A and 13B show in vitro killing of PLC / PRF / 5 human hepatocellular carcinoma cells by human primary NK cells in the presence of HBC34-v35-MLNS and HBC34-v35-MLNS-GAALIE. (A) ADCC was tested using freshly isolated NK cells from a single donor previously genotyped for expression of heterozygous high (V158) and low (F158) affinity FcγRIIIa (F / V). Serial dilutions of HBC34-v35, HBC34-v35-MLNS, HBC34-v35-MLNS-GAALIE, 17.1.41, and control mAbs were added to the HBsAg-secreting hepatocellular carcinoma cell line PLC / PRF / 5 (also known as Alexander cells). PLC / PRF / 5 cells were incubated with the antibodies for 10 minutes at room temperature. NK cells were added to the assay plate (10:1 effector cell to target cell ratio) and incubated at 37°C for 4 hours. Cell death was determined by measuring lactate dehydrogenase (LDH) release. (B) Staining of PLC / PRF / 5 human hepatocellular carcinoma cells with HBC34v35 and 17.1.41 mAbs assessed by flow cytometry. Cells were extensively washed and fixed with formaldehyde (4%) or fixed and permeabilized (saponin 0.5%) before staining with various concentrations of HBC34-v35 and 17.1.41 mAbs. Binding of these human mAbs was detected by flow cytometry using Alexa Fluor® 647 AffiniPure F(ab')2 fragment goat anti-human IgG, an Fcγ fragment-specific antibody. [Figure 13B] Same as above. [Figure 14A]Figures 14A and 14B show the in vitro activation of primary human NK cells in the presence of HBC34v35-MLNS and HBC34-v35-MLNS-GAALIE and HBsAg. NK cell activation was tested using freshly isolated cells from two donors previously genotyped for expression of (A) homozygous high (V158) or (B) low (F158) affinity FcγRIIIa. Serial dilutions of HBC34-V35, HBC34-v35-MLNS-GAALIE, and HBC34-v35-LALA mAbs were incubated with NK cells for 4 hours. NK cell activation was measured by flow cytometry by staining NK cells with anti-CD107a mAb as a functional marker to identify NK cell activity. CD107a, also known as LAMP-1, is a marker for NK cell degranulation. [Figure 14B] Same as above. [Figure 15A] Figures 15A-15C show the evaluation schedule for healthy adult subjects in an exemplary single ascending dose (SAD) clinical trial of an exemplary pharmaceutical composition comprising the antibody HBC34-v35-MLNS-GAALIE, as described in Example 9. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16A] 16A-16E show the evaluation schedule for subjects with chronic HBV infection without cirrhosis and on nucleoside reverse transcriptase inhibitor (NRTI) therapy in an exemplary clinical trial described in Example 9. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 17A] 17A-17C show the time points of blood draws for pharmacokinetic measurements for subjects from an exemplary clinical trial described in Example 9. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 18]FIG. 18 shows the dosing schedule from an exemplary clinical trial described in Example 9. [Figure 19] FIG. 19 shows clinical laboratory evaluations from the exemplary clinical trial described in Example 9. [Figure 20] Figure 20 shows the upregulation of activation and costimulatory markers in monocyte-derived dendritic cells (moDCs) stimulated via immune complexes of HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in Example 10. [Figure 21] FIG. 21 shows cytokine secretion by moDCs stimulated via immune complexes of HBC34-v35-MLNS+HBsAg; or HBC34-v35-MLNS-GAALIE+HBsAg, as described in Example 10. [Figure 22A] 22A and 22B show the release of IFN-γ in whole blood cultures stimulated with immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. (A) IFN-γ concentration (log10); (B) IFN-γ fold change (log10), as described in Example 10. [Figure 22B] Same as above. [Figure 23A] 23A and 23B show IL-2 release in whole blood cultures stimulated with immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. (A) IL-2 concentration (loglO); (B) IL-2 fold change (loglO), normalized as described in Example 10. [Figure 23B] Same as above. [Figure 24A]Figures 24A and 24B show IFN-γ and IL-2 in whole blood cultures stimulated via immune complexes containing HBC34-v35-MLNS and HBsAg; or HBC34-v35-MLNS-GAALIE and HBsAg, as described in Example 10. (A) IFN-γ; 100 μg / ml mAb; (B) IL-2; IL-2 μg / ml mAb. [Figure 24B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure provides pharmaceutical compositions comprising antibodies that neutralize hepatitis B virus (HBV) infection, and methods of using those compositions. In certain embodiments, the antibodies bind HBsAg of a genotype selected from A, B, C, D, E, F, G, H, I, and J, or any combination thereof. In certain embodiments, the antibodies comprise heavy chain mutations that extend the in vivo half-life of the antibody (e.g., in humans) and heavy chain mutations that improve binding affinity to FcγR (e.g., human FcγRIIa, human FcγRIIIa, or both).

[0010] In some embodiments, the antibodies and pharmaceutical compositions are well tolerated by the subject when administered in a therapeutically effective amount. In some embodiments, the methods described herein comprise administering to a subject infected with HBV an antibody or pharmaceutical composition described herein.

[0011] Although antibodies that neutralize HBV, pharmaceutical compositions containing those antibodies, and methods for using such pharmaceutical compositions are described in detail below, it should be understood that the disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein does not limit the scope of the disclosure.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0013] Below, aspects of the present disclosure are described. Although certain embodiments are provided, it should be understood that the embodiments of the present disclosure can be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be interpreted as limiting the present disclosure to only the explicitly described embodiments. The present description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any disclosed subject matter. Furthermore, any permutation and combination of all described subject matter in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0014] Throughout this disclosure, unless the context otherwise requires, the term "comprise," and variations thereof, such as "comprises," and "comprising," are used synonymously with, for example, "having," "has," "including," "includes," etc., and will be understood to imply the inclusion of stated components, ratios, integers (including fractions thereof, where appropriate; e.g., tenths and hundredths of integers), concentrations, or steps, but not the exclusion of any other unrecited components, ratios, integers, concentrations, or steps. The term "consisting essentially of," which is not equivalent to "comprising," refers to the specified materials or steps of a claim, or to materials or steps that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain) or protein "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal or inter-domain amino acids) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., do not reduce activity by more than 50%, e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less) the activity of the domain(s), region(s), module(s), or protein (e.g., target binding affinity of a binding protein).

[0015] The term "consist of" is a specific embodiment of the term "comprise," excluding any other unrecited components, integers, or steps. In the context of the present disclosure, the term "comprising" encompasses the term "consisting of." The term "comprising" therefore encompasses "including" and "consisting." For example, a composition "comprising" X may consist exclusively of X, or it may include something additional, e.g., X+Y.

[0016] Furthermore, it should be understood that each individual compound or group of compounds resulting from the various combinations of structures and substituents described herein is disclosed by this application to the same extent as if each compound or group of compounds were individually described. Thus, selection of a particular structure or particular substituents is within the scope of this disclosure.

[0017] The terms "a," "an," and "the" and similar references used in the context of describing this disclosure (including the context of the claims) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of alternative words (e.g., "or") should be understood to mean either one, both, or any combination of the alternative words. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the disclosure as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the subject matter disclosed herein.

[0018] The word "substantially" does not exclude "completely"; for example, a composition that is "substantially free" of Y may be completely free of Y. In certain embodiments, "substantially" refers to a given amount, effect, or activity of a composition, method, or use of the present disclosure compared to a given amount, effect, or activity of a reference composition, method, or use, and represents a reduction in the amount, effect, or activity of 50% or less, e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less, of the amount, effect, or activity of the reference composition, method, or use.

[0019] The term "about" in reference to a numerical value x means x±10%, e.g., x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%. For example, in certain embodiments, "about" means ±20% of the indicated range, value, or structure.

[0020] "Optional" or "may, optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances when the element, component, event, or circumstance occurs and instances when it does not occur.

[0021] The term "disease," as used herein, is intended to be generally synonymous with, and used interchangeably with, the terms "disorder" and "condition" (such as medical condition), in that all of them reflect an abnormal condition of the human or animal body or one of its parts that impairs normal function, is typically manifested by characteristic signs and symptoms, and results in a reduction in the lifespan or quality of life of the afflicted human or animal.

[0022] As used herein, the term "therapeutically effective" refers to a quality or amount of a pharmaceutical composition or antibody described herein sufficient to provide a benefit to a subject. In the context of this disclosure, the benefit provided to the subject is treatment of hepatitis B virus infection. As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, palliative care, amelioration, and therapy. Benefits of treatment include improved clinical outcome; reduction or alleviation of symptoms associated with the disease; reduced onset of symptoms; improved quality of life; longer disease-free state; reduced extent of disease; stabilization of the disease state; delayed disease progression; remission; survival; extended survival; or any combination thereof. The terms "subject" and "patient" are used interchangeably herein to refer to a human who is susceptible to infection with HBV or who is already infected with HBV.

[0023] Doses are often expressed relative to body weight (i.e., of the subject). Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) may refer to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight" even if the term "body weight" is not explicitly stated.

[0024] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in the same way as naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and 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 chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in the same way as a naturally occurring amino acid.

[0025] As used herein, the terms "peptide," "polypeptide," and "protein," as well as variations of these terms, refer to molecules comprising at least two amino acids linked together by peptide bonds (either conventional or modified). For example, a peptide, polypeptide, or protein may be composed of multiple amino acids selected from the 20 amino acids defined by the genetic code, each linked to at least one other amino acid by a peptide bond. A peptide, polypeptide, or protein may be composed of L-amino acids and / or D-amino acids. The terms "peptide," "polypeptide," and "protein" also include "peptidomimetics," which are defined as peptide analogs containing non-peptide structural elements, allowing the peptide to mimic or antagonize the biological action of a natural parent peptide. In certain embodiments, peptidomimetics lack characteristics such as peptide bonds that are susceptible to enzymatic cleavage.

[0026] A peptide, polypeptide, or protein may contain or be composed of amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids. In certain embodiments, a peptide, polypeptide, or protein in the context of the present disclosure may contain amino acids that are modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), including those known in the art and described herein. Such modifications may occur anywhere in the polypeptide, for example, in the peptide backbone, the amino acid chain, or the carboxy- or amino-terminus. A peptide or polypeptide may be branched, such as after ubiquitination, or may be cyclic, with or without branching. The terms "peptide," "polypeptide," and "protein" also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of a nucleotide or nucleotide derivative, covalent immobilization of a lipid or lipid derivative, covalent immobilization of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, ceneroylation, sulfation, amino acid additions such as arginylation or ubiquitination.Such modifications are described in the literature (see, for example, Proteins Structure and Molecular Properties (1993) 2nd Ed., T.E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B.C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Thus, the terms "peptide," "polypeptide," and "protein" can include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like. Variants of the proteins, peptides, and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined or reference amino acid sequence described herein.

[0027] As used herein, "(poly)peptide" and "protein" may be used interchangeably in reference to a polymer of amino acid residues, such as multiple amino acid monomers linked by peptide bonds.

[0028] A "nucleic acid molecule" or "polynucleotide" or "nucleic acid" refers to a polymeric compound containing covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and the like.

[0029] Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA) (including cDNA, genomic DNA, and synthetic DNA), either of which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (antisense) strand. Polynucleotides (including oligonucleotides), and fragments thereof, may be generated, for example, by polymerase chain reaction (PCR) or in vitro translation, or may be generated by ligation, cleavage, endonuclease action, or exonuclease action.

[0030] A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequence may also contain introns, to the extent that introns can be removed through co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy of the genetic code, or by splicing, or both.

[0031] Variants of the nucleic acid molecules of the present disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid molecules of the defined or reference polynucleotides described herein, or hybridize to the polynucleotides under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the ability to encode fusion proteins or binding domains thereof having the functionality described herein, such as specific binding to a target molecule.

[0032] As used herein, the term "sequence variant" refers to any sequence having one or more changes compared to a reference sequence, where the reference sequence is any published sequence and / or a sequence listed in the "Table of Sequences and SEQ ID NOs" (Sequence Listing), i.e., SEQ ID NOs: 1-120. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments of sequence variants in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, whereas in certain embodiments of sequence variants in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. A "sequence variant," as used herein, may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence.

[0033] "Percent sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. Methods for determining sequence identity can be designed to give the best match between the sequences being compared. For example, sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison purposes. Percent sequence identity referred to herein is calculated over the length of the reference sequence unless otherwise specified. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using BLAST programs (e.g., BLAST2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of the present disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program, which refers to any set of values ​​or parameters originally loaded into the software when it is first initialized.

[0034] A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has a sequence change in which one or more nucleotides of the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by their standard single-letter designations (A, C, G, or T). Due to the degeneracy of the genetic code, a "sequence variant" of a nucleotide sequence may or may not result in a change in the respective reference amino acid sequence, i.e., an amino acid "sequence variant." In certain embodiments, a nucleotide sequence variant does not result in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucleotide sequence variant that results in a "non-silent" mutation is contemplated. In some embodiments, a nucleotide sequence variant of the present disclosure encodes an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. The nucleotide and amino acid sequences disclosed herein also refer to codon-optimized versions of a reference or wild-type nucleotide or amino acid sequence. In any of the embodiments described herein, the polynucleotides of the disclosure may be codon-optimized for a host cell containing the polynucleotide (see, e.g., Scholten et al., Clin. Immunol. 119: 135-145 (2006)).

[0035] A "sequence variant," in the context of an amino acid sequence, has a sequence change in which one or more amino acids are deleted, substituted, or inserted compared to a reference amino acid sequence. As a result of the changes, such a sequence variant has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having 10 or fewer changes, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence is "at least 90% identical" to the reference sequence.

[0036] "Conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding characteristics of a particular protein. Generally, a conservative substitution is one in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be classified into conservative substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic classification can include, for substitution purposes, Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0037] Amino acid sequence insertions can include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the N- or C-terminal fusion of an amino acid sequence to a reporter molecule or an enzyme.

[0038] Generally, the changes in the sequence variants do not eliminate or significantly reduce the desired functionality of the respective reference sequences. For example, it is preferred that the variant sequences disclosed herein do not significantly reduce or completely suppress the functionality of the sequence of an antibody or antigen-binding fragment thereof that binds to the same epitope and / or sufficiently neutralizes HBV and HDV infection, compared to the antibody or antigen-binding fragment having (or encoded by) the reference sequence. Guidance on determining which nucleotides and amino acid residues can be substituted, inserted, or deleted, respectively, without eliminating the desired structure or functionality can be found using known computer programs.

[0039] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence derived from a particular sequence may have essentially the same amino acid sequence as the sequence or portion thereof from which the nucleic acid or amino acid sequence is derived, thereby including sequence variants as defined above. A nucleic acid sequence or amino acid sequence derived from a particular peptide or protein may be derived from a corresponding domain of the particular peptide or protein. In this context, "corresponding" refers to possessing the same desired functionality or property. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another "transmembrane domain" (of another protein). "Corresponding" portions of peptides, proteins, and nucleic acids are therefore readily identifiable to those of skill in the art. Similarly, a sequence "derived from" another (e.g., "source") sequence can be identified by those of skill in the art as having its origin in the source sequence.

[0040] A nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived. However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations compared to the starting nucleic acid, peptide, polypeptide, or protein from which it is derived; in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant of the starting nucleic acid, peptide, polypeptide, or protein from which it is derived, as described above. For example, in a peptide / protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may occur.

[0041] As used herein, the term "mutation" refers to a change in a nucleic acid sequence and / or amino acid sequence compared to a reference sequence, e.g., a corresponding genomic sequence, a wild-type sequence, or a reference sequence. For example, compared to a reference genomic sequence, a mutation may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation (e.g., induced by enzymes, chemicals, or radiation), or a mutation obtained by site-directed mutagenesis (a molecular biological method for making specific and deliberate changes in nucleic acid and / or amino acid sequences). Thus, the term "mutation" or "mutating" should be understood to include physically creating mutations, for example, in a nucleic acid sequence or amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, and inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, mutations may be introduced into a nucleotide sequence encoding the amino acid sequence to express a (recombinant) mutant polypeptide. Mutation can be achieved, for example, by changing a codon in a nucleic acid molecule that encodes an amino acid (e.g., by changing one, two, or three nucleotide bases therein) (e.g., by site-directed mutagenesis) to provide a codon that encodes a different amino acid or that encodes the same amino acid, or by synthesizing a sequence variant.

[0042] The term "introduction" in the context of inserting a nucleic acid molecule into a cell means "transfection," or "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0043] As used herein, the term "recombinant" (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (antibody, protein, nucleic acid, etc.) that is prepared, expressed, created, or isolated by recombinant means and that does not exist in nature. "Recombinant" can be used synonymously with "modified" or "non-naturally occurring" and may refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic change or has been modified by the introduction of an exogenous nucleic acid molecule, where such change or modification is introduced by genetic engineering (i.e., human intervention). Genetic engineering includes, for example, modifications that introduce expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of other nucleic acid molecules in the genetic material of a cell. Additional modifications include, for example, non-coding regulatory regions, where the modifications alter the expression of a polynucleotide, gene, or operon.

[0044] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to an altered host cell or subject. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered so that the structure, activity, or both differ between the native and altered gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterologous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule (e.g., receptor, ligand, etc.) may not be endogenous to a host cell or subject; instead, a nucleic acid encoding such a gene, protein, or nucleic acid molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc.; the added nucleic acid molecule may be integrated into the host cell genome or may exist as extraneous genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and may encode a homologous polypeptide or activity, although the polynucleotide or polypeptide may be altered in structure, sequence, expression level, or any combination thereof. The non-endogenous polynucleotides or genes, and the encoded polypeptides or activities, can be from the same species, different species, or a combination thereof.

[0045] As used herein, the terms "endogenous" or "native" refer to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

[0046] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, it will be clear from the context.

[0047] The present disclosure is based in part on the design of antibodies and antigen-binding fragments capable of neutralizing hepatitis B and hepatitis delta viruses. Antibody and antigen-binding fragment embodiments according to the present description can be used in methods for preventing, treating, or attenuating HBV and HDV. In certain embodiments, the antibodies and antigen-binding fragments described herein bind to two or more different genotypes of hepatitis B virus surface antigen and two or more different infectious mutants of hepatitis B virus surface antigen. In certain embodiments, the antibodies and antigen-binding fragments described herein bind to all currently known genotypes of hepatitis B virus surface antigen and all currently known infectious mutants of hepatitis B virus surface antigen.

[0048] Antibodies and antigen-binding fragments thereof In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to an antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus for use in the pharmaceutical compositions and methods disclosed herein.

[0049] As used herein, and unless the context clearly dictates otherwise, "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (although it is understood that heavy-chain antibodies lacking light chains are also encompassed by the term "antibody"), as well as any antigen-binding portion or fragment of an intact antibody, e.g., an scFv, Fab, or F(ab')2 fragment, that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody. Thus, the term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, as well as heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise stated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass thereof, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0050] Thus, antibodies of the present disclosure may be of any isotype (e.g., IgA, IgG, IgM, referred to as α, γ, and μ heavy chains, respectively). For example, in certain embodiments, the antibody is of the IgG type. Within the IgG isotype, the antibody may be of the IgG1, IgG2, IgG3, or IgG4 subclass, e.g., IgG1. In some embodiments, antibodies include antibodies comprising constant regions comprising amino acid sequences from two different isotypes (e.g., by exchanging the amino acid sequences of the constant domains), e.g., an amino acid sequence from an IgA antibody and an amino acid sequence from an IgG antibody. Antibodies of the present disclosure may comprise a κ or λ light chain. In some embodiments, the antibody is of the IgG1 type and comprises a κ light chain.

[0051] As used herein, the terms "antigen-binding fragment," "fragment," and "antibody fragment" are used interchangeably and refer to any fragment of an antibody of the present disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv, or scFv. Furthermore, as used herein, the term "antibody" includes both antibodies and antigen-binding fragments thereof. Antibodies and antigen-binding fragments are further discussed herein.

[0052] Human antibodies are known (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies, or a selection thereof, in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigenic challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for preparing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).Human monoclonal antibodies may be prepared using improved EBV-B cell immortalization, as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. As used herein, the term "human antibody" also includes antibodies that have been modified, for example, in the variable region, to produce the properties of the antibodies and antibody fragments of the present disclosure. As used herein, the term "variable region" refers to the variable region of the light chain (V). L ), the variable region of the heavy chain (V H )] refers to each of the light and heavy chain pairs that are directly involved in binding of the antibody to the antigen.

[0053] As used herein, the term "variable region" [e.g., the variable region of a light chain (V L ), the variable region of the heavy chain (V H ) refers to the variable region of an antibody light chain or antibody heavy chain that is directly involved in binding the antibody to an antigen. L " or "VL" and "V H " or "VH" refer to the variable binding region from an antibody light chain and antibody heavy chain, respectively.

[0054] The variable binding region is composed of distinct, distinct subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to generally refer to noncontiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each variable region of an antibody has three CDRs. The VH and VL regions contain a total of six CDRs: HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3, also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively. The CDRs on the heavy and / or light chain may be separated in the primary amino acid sequence by framework regions, such that the framework regions (FRs) are regions in the variable domain that are less variable than the CDRs [i.e., from antibody to antibody (e.g., from antibody to antibody encoded by the same allele)]. For example, a chain (or each chain, respectively) may be composed of four framework regions separated by three CDRs. In certain embodiments, an antibody VH comprises four FRs and three CDRs arranged as follows: FR1-CDRH1-FR2-CDHR2-FR3-CDHR3-FR4, and an antibody VL comprises four FRs and three CDRs as follows: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4. Generally, the VH and VL together form an antigen-binding site through their respective CDRs, although it will be understood that in some cases the binding site can be formed by or include one, two, three, four, or five CDRs.

[0055] As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having up to one to three amino acid substitutions, deletions, or a combination thereof. Immunoglobulin sequences can be aligned to a numbering scheme (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), which allows equivalent residue positions to be noted and compared for different molecules using the Antigen Receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). It will be understood that in certain embodiments, an antibody or antigen-binding fragment of the present disclosure can include all or a portion of a heavy chain (HC), a light chain (LC), or both. For example, a full-length, intact IgG antibody monomer typically includes a VH, a CH1, a CH2, a CH3, a VL, and a CL. The Fc component is further described herein.

[0056] In the present disclosure, the positions of the CDR amino acids are defined according to the IMGT numbering system (IMGT: www.imgt.org / ; see Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

[0057] Table 1: Shows the amino acid sequences of the heavy chain variable region (VH), light chain variable region (VL), CDRs, heavy chain (HC), and light chain (LC) of certain exemplary antibodies according to the present disclosure. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0058] Fragments of the antibodies described herein can be obtained from the antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be obtained by cloning and expression of portions of the heavy or light chain sequences. Antibody "fragments" include Fab, Fab', F(ab'), and Fv fragments. The present disclosure also encompasses single-chain Fv fragments (scFvs) derived from the heavy and light chains of the antibodies described herein, including, for example, scFvs containing CDRs from an antibody according to the present disclosure; heavy or light chain monomers and dimers; single-domain heavy chain antibodies; single-domain light chain antibodies; and single-chain antibodies in which the heavy and light chain variable domains are linked by a peptide linker.

[0059] In certain embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a purified antibody, a single chain antibody, a Fab, a Fab', a F(ab')2, an Fv, or an scFv.

[0060] The antibodies and antigen-binding fragments of the present disclosure may be multispecific (i.e., bispecific, trispecific, tetraspecific, etc.) in embodiments and may be provided in any multispecific format as disclosed herein. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are multispecific antibodies, e.g., bispecific or trispecific antibodies. Bispecific antibody formats are described, for example, in Spiess et al., Mol. Immunol. 67(2):95 (2015), and Brinkmann and Kontermann, mAbs 9(2):182-212. (2017), the bispecific formats and methods for making the same are incorporated herein by reference, and include, for example, bispecific T cell engagers (BiTEs), DART, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triplebodies, TriBi minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, intrabodies, cross-Mab, These include Dual Action Fab (DAF) (2-in-1 or 4-in-1), DutaMabs, DT-IgG, Charge-Pairs, Fab-Arm Exchange, SEED bodies, Triomabs, LUZ-Y assembly, Fcab, κλ bodies, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (4-in-1). Bispecific or multispecific antibodies may comprise an HBV- and / or HDV-specific binding domain of the present disclosure in combination with another HBV- and / or HDV-specific binding domain of the present disclosure, or in combination with a different binding domain that specifically binds to HBV and / or HDV (e.g., at the same or a different epitope) or a binding domain that specifically binds to a different antigen.

[0061] Antibody fragments of the present disclosure may form monovalent or polyvalent interactions and may be included in various structures as described above. For example, scFv molecules can be synthesized to create trivalent "triabodies" or tetravalent "tetrabodies." scFv molecules may include a domain in the Fc region, resulting in a bivalent minibody. Furthermore, sequences of the present disclosure may be components of multispecific molecules in which the sequences of the present disclosure target epitopes of the present disclosure and other regions of the molecule that bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2s, trispecific Fab3s, bispecific scFvs, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

[0062] In some embodiments, the antibody may be present in a pharmaceutical composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the pharmaceutical composition is made up of other polypeptides, usually less than 60%, and more usually less than 50%.

[0063] Antibodies according to the present disclosure may be immunogenic in humans and / or non-human (or xenogeneic) hosts, such as mice. For example, the antibodies may have an idiotope that is immunogenic in a non-human host but not in a human host. Antibodies of the present disclosure for human use include antibodies that are not typically isolated from mice, goats, rabbits, rats, non-primate mammals, or other hosts, and in some cases are not obtained by humanization or from xenogeneic mice. In certain embodiments, antibodies according to the present disclosure are non-immunogenic or substantially non-immunogenic in humans.

[0064] Variant forms of the disclosed antibodies, engineered to reduce known or potential immunogenicity and / or other possible disadvantages, are also contemplated herein.

[0065] As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e., prevent, inhibit, reduce, interfere with, or interfere with, the ability of a pathogen to initiate and / or perpetuate infection in a host (e.g., a host organism or host cell). The terms "neutralizing antibody," "neutralizing antibody," or "neutralizing antibodies" are used interchangeably herein. These antibodies can be used alone or in combination (e.g., combining two or more of the antibodies of the present disclosure, or combining an antibody of the present disclosure with another agent, which may or may not be an antibody agent, including an antibody capable of neutralizing HBV B and / or D infection) as a prophylactic or therapeutic agent in an appropriate formulation in conjunction with active vaccination.

[0066] As used herein, "specifically binds" or "specific for" means 5 M -1 The affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction, with units of 1 / M) (which is the on-rate of this association reaction [K on ] and off-rate [K off [Ratio of β-Affinity to β-Affinity] refers to the association of a binding protein (e.g., an antibody or antigen-binding fragment thereof) or binding domain to a target molecule, which has a specific affinity to the target molecule (e.g., a ratio of β-Affinity to β-Affinity) but does not significantly associate or associate with any other molecules or components in the sample. Antibodies or binding domains can be classified as "high affinity" or "low affinity" binding proteins or domains. A "high affinity" binding protein or domain has a specific affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1A "low affinity" binding protein or binding domain refers to a binding protein or binding domain with a Ka of 10 7 M -1 Up to 10 6 M -1 up to or 10 5 M -1 Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, with units of M (e.g., 10 -5 M~10 -13 M). The terms "binding" and "specifically binding" and similar references do not encompass non-specific adhesion.

[0067] In certain embodiments, antibodies according to the present disclosure can bind to the antigenic loop region of HBsAg. The hepatitis B virus envelope generally comprises three "HBV envelope proteins" (also known as "HBsAg" or "hepatitis B surface antigen"): the S protein (for "small," also referred to as S-HBsAg), the M protein (for "middle," also referred to as M-HBsAg), and the L protein (for "large," also referred to as L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg share the same C-terminal extremity (226 amino acids, also referred to as the "S domain"), which corresponds to the S protein (S-HBsAg) and is important for viral assembly and infectivity. S-HBsAg, M-HBsAg, and L-HBsAg are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles through the Golgi apparatus. The S domain contains four predicted transmembrane (TM) domains, exposing both the N- and C-termini of the S domain to the lumen. Transmembrane domains TM1 and TM2 are thought to be required for cotranslational protein integration into the ER membrane, while transmembrane domains TM3 and TM4 are located at the C-terminal third of the S domain. The "antigenic loop region" of HBsAg is located between the predicted TM3 and TM4 transmembrane domains of the S domain of HBsAg, thereby comprising amino acids 101-172 of the S domain, a total of 226 amino acids (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). The infectivity determinants are present in the antigenic loop region of the HBV envelope protein. In particular, residues 119 to 125 of HBsAg contain a CXXC motif, which is thought to be important for the infectivity of HBV and HDV (Jaoude GA, Sureau C, Journal of Virology, 2005;79:10460-6).

[0068] When a position in the amino acid sequence of the S domain of HBsAg is referred to herein, such position is made with reference to the amino acid sequence set forth in SEQ ID NO: 3 (shown below) or a natural or artificial sequence variant thereof. MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 3, amino acids 101-172 are underlined)

[0069] For example, the phrase "amino acids 101-172 of the S domain" refers to amino acid residues 101-172 of the polypeptide according to SEQ ID NO: 3. However, those skilled in the art will understand that mutations or changes (including, but not limited to, substitutions, deletions, and / or additions, e.g., of different genotypes of HBsAg or different HBsAg mutants described herein) can occur naturally or be artificially introduced into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Thus, as used herein, the term "S domain of HBsAg" encompasses all such polypeptides, including, for example, the polypeptide according to SEQ ID NO: 3 and natural or artificial mutants thereof. Furthermore, when a sequence fragment of the S domain of HBsAg (e.g., amino acids 101-172 or amino acids 120-130 of the S domain of HBsAg) is described herein, this includes not only the corresponding sequence fragment of SEQ ID NO: 3, but also the corresponding sequence fragment of a natural or artificial mutant thereof. For example, the phrase "amino acid residues 101-172 of the S domain of HBsAg" encompasses amino acid residues 101-172 of SEQ ID NO: 3 and the corresponding fragment of mutants (natural or artificial mutants) thereof. As used herein, the phrases "corresponding sequence fragment" and "corresponding fragment" refer to a fragment that is located at the same position in a sequence when the sequences are subjected to optimized alignment, i.e., when the sequences are aligned to obtain the maximum percentage identity.

[0070] The M protein (M-HBsAg) corresponds to the S protein extended by a 55-amino acid N-terminal domain called "pre-S2." The L protein (L-HBsAg) corresponds to the M protein extended by a 108-amino acid N-terminal domain called "pre-S1" (genotype D). The pre-S1 and pre-S2 domains of the L protein are thought to play an important role in viral assembly on the inner surface of the virion (the cytoplasmic side of the ER) or in the outer surface (the luminal side of the ER) for interaction with target cells, which are important for viral infectivity. Furthermore, the HBV surface protein (HbsAg) is not only incorporated into the virion envelope but can also spontaneously bud from the membrane of the ER-Golgi intermediate compartment to form empty "subviral particles" (SVPs) that are released from cells by secretion.

[0071] In some embodiments, the antibody or antigen-binding fragment binds to the antigenic loop region of HBsAg and is capable of binding to all of S-HBsAg, M-HBsAg, and L-HBsAg.

[0072] In some embodiments, the antibody or antigen-binding fragment neutralizes infection by hepatitis B virus and hepatitis delta virus. In some embodiments, the antibody or antigen-binding fragment reduces viral infectivity of hepatitis B virus and hepatitis delta virus.

[0073] Standard "neutralization assays" may be utilized to study and quantify viral infectivity (or "neutralization") in the laboratory. For neutralization assays, animal viruses are typically propagated in cells and / or cell lines. Neutralization assays may be used in which cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen-binding fragment) to be tested. In such assays, readouts are obtained using the level of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant and / or by assessing HBcAg staining. For example, for HDV, delta antigen immunofluorescence staining may be assessed.

[0074] In a specific embodiment of an HBV neutralization assay, cultured cells, e.g., HepaRG cells, such as differentiated HepaRG cells, are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested. In such an embodiment, incubation is performed, for example, at 37°C for 16 hours. Incubation may be performed in culture medium (e.g., supplemented with 4% PEG 8000). After incubation, the cells are washed and further cultured. To measure viral infectivity, the levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted into the culture supernatant, for example, from days 7 to 11 postinfection, may be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBcAg staining may be assessed in an immunofluorescence assay. In an embodiment of an HDV neutralization assay, essentially the same assay as for HBV may be used, except that serum from an HDV carrier may be used (instead of HBV) as the inoculum for HDV infection of differentiated HepaRG cells. For detection, delta antigen immunofluorescence staining may be used as a readout.

[0075] Antibody embodiments of the present disclosure have high neutralizing capacity (e.g., in vitro). For example, in certain embodiments, the concentration of an antibody described herein required for 50% neutralization of hepatitis B virus (HBV) or hepatitis delta virus (HDV) is, for example, about 10 μg / ml or less. In other embodiments, the concentration of an antibody described herein required for 50% neutralization of HBV and HDV is about 5 μg / ml. In other embodiments, the concentration of an antibody described herein required for 50% neutralization of HBV and HDV is about 1 μg / ml. In yet other embodiments, the concentration of an antibody described herein required for 50% neutralization of HBV and HDV is about 750 ng / ml. In further embodiments, the concentration of an antibody described herein required for 50% neutralization of HBV and HDV (e.g., in vitro) is 500 ng / ml or less. In such embodiments, the concentration of antibodies described herein required for 50% neutralization of HBV and HDV may be selected from 450 ng / ml or less, 400 ng / ml or less, 350 ng / ml or less, 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, 175 ng / ml or less, 150 ng / ml or less, 125 ng / ml or less, 100 ng / ml or less, 90 ng / ml or less, 80 ng / ml or less, 70 ng / ml or less, 60 ng / ml or less, or 50 ng / ml or less.

[0076] Antibodies or antigen-binding fragments according to the present disclosure that are capable of neutralizing both HBV and HDV are useful for the prevention and treatment of hepatitis B and hepatitis D. Infection with HDV typically occurs simultaneously with or subsequent to infection with HBV (e.g., because HDV requires HBV support for its replication, inoculation of HDV in the absence of HBV does not cause hepatitis D), and hepatitis D is typically observed in chronic HBV carriers.

[0077] Embodiments of the disclosed antibodies promote clearance of HBsAg and HBV. In certain embodiments, the antibodies promote clearance of both HBV and hepatitis B virus subviral particles (SVPs). Clearance of HBsAg or subviral particles can be assessed by measuring the level of HBsAg, e.g., in a blood sample, e.g., of a hepatitis B patient. Similarly, clearance of HBV can be assessed by measuring the level of HBV, e.g., in a blood sample, e.g., of a hepatitis B patient.

[0078] In addition to infectious particles (HBV), sera from HBV-infected patients contain an excess (typically 1,000- to 100,000-fold excess) of empty subviral particles (SVPs), typically consisting only of the HBV envelope protein (HBsAg) in the form of relatively small spheres and fibers of variable length. Subviral particles have been shown to strongly enhance intracellular viral replication and HBV gene expression (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also relevant in the context of infectivity of HBV-containing serum, since infectivity depends not only on the number of viruses but also on the number of SVPs (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). Furthermore, excess subviral particles may act as decoys by absorbing neutralizing antibodies, thus delaying clearance of the infection. Achieving loss of hepatitis B surface antigen (HBsAg) is considered in some cases the endpoint of treatment and the closest outcome to curing chronic hepatitis B (CHB).

[0079] Antibody embodiments of the present disclosure may promote clearance of HBsAg. In certain embodiments, the antibodies promote clearance of subviral particles of hepatitis B virus. In some embodiments, the antibodies (e.g., in pharmaceutical compositions of the present disclosure) may be used to treat chronic hepatitis B.

[0080] In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment binds to HBsAg of a genotype selected from HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

[0081] In certain embodiments, an antibody or antigen-binding fragment of the present disclosure binds to one, two, three, four, five, six, seven, eight, nine, or ten of the following genotypes of HBsAg: A, B, C, D, E, F, G, H, I, and J. Examples of various HBsAg genotypes include the following: GenBank Accession No. J02203 (HBV-D, ayw3); GenBank Accession No. FJ899792.1 (HBV-D, adw2); GenBank Accession No. AM282986 (HBV-A); GenBank Accession No. D23678 (HBV-B1 Japan); GenBank Accession No. AB117758 (HBV-C1 Cambodia); GenBank Accession No. AB205192 (HBV-E Ghana); GenBank Accession No. X69798 (HBV-F4 Brazil); GenBank Accession No. AF160501 (HBV-G USA); GenBank Accession No. AY090454 (HBV-H Nicaragua); GenBank Accession No. AF241409 (HBV-I Vietnam); and GenBank Accession No. AB486012 (HBV-J Borneo). Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of various genotypes are provided herein (eg, SEQ ID NOS: 5-15).

[0082] In some embodiments, the antibody or antigen-binding fragment binds to at least six of the ten HBsAg genotypes: A, B, C, D, E, F, G, H, I, and J. In certain embodiments, the antibody or antigen-binding fragment binds to at least eight of the ten HBsAg genotypes: A, B, C, D, E, F, G, H, I, and J. In some embodiments, the antibody or antigen-binding fragment binds to all ten of the ten HBsAg genotypes: A, B, C, D, E, F, G, H, I, and J. HBV is differentiated into several genotypes according to its genome sequence. To date, eight known genotypes (A to H) of the HBV genome have been defined. In addition, two other genotypes, I and J, have also been identified (Sunbul M., 2014, World J Gastroenterol 20(18): 5427-5434). Genotype is known to influence disease progression, and differences between genotypes in response to antiviral treatment have been determined.

[0083] In some embodiments, an antibody or antigen-binding fragment according to the present disclosure binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of HBsAg mutants having mutations in the antigenic loop region, such mutants being HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg The mutants are selected from one or more of HBsAg D144A, HBsAg G145R, and HBsAg N146A. These mutants are naturally occurring mutants based on the S domain (SEQ ID NO: 4) of HBsAg genotype D, GenBank accession number FJ899792. The mutated amino acid residue in each of the mutants noted herein is indicated by its name.

[0084] SEQ ID NO:4 MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDY QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW LSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI (The antigenic loop region, i.e., amino acids 101 to 172, is underlined.)

[0085] The amino acid sequences of the antigenic loop regions of the S domains of various mutant HBsAg are shown in SEQ ID NOs: 16 to 33.

[0086] In certain embodiments, the antibody or antigen-binding fragment binds to at least 12 infectious HBsAg mutants selected from HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. In some such embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure binds to at least 15 infectious HBsAg mutants selected from HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. In some embodiments, the antibody or antigen-binding fragment binds to each of the following infectious HBsAg mutants: HBsAg Y100C / P120T; HBsAg P120T; HBsAg P120T / S143L; HBsAg C121S; HBsAg R122D; HBsAg R122I; HBsAg T123N; HBsAg Q129H; HBsAg Q129L; HBsAg M133H; HBsAg M133L; HBsAg M133T; HBsAg K141E; HBsAg P142S; HBsAg S143K; HBsAg D144A; HBsAg G145R; and HBsAg N146A.

[0087] In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces the serum concentration of HBV DNA in a mammal with HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces the serum concentration of HBsAg in a mammal with HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces the serum concentration of HBeAg in a mammal with HBV infection. In certain embodiments, the antibody or pharmaceutical composition comprising the same reduces the serum concentration of HBcrAg in a mammal with HBV infection.

[0088] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a related binding molecule, such as an immunoglobulin, chimeric antigen receptor, or other binding molecule, domain, or protein. Epitopic determinants generally comprise chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0089] In some embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least one, at least two, at least three, or at least four amino acids in the antigenic loop region of HBsAg. In certain embodiments, the antibody or antigen-binding fragment binds to at least two amino acids selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. In certain embodiments, the antibody or antigen-binding fragment binds to at least three amino acids selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. In some embodiments, the antibody or antigen-binding fragment binds to at least four amino acids selected from amino acids 115-133 of the S domain of HBsAg, amino acids 120-133 of the S domain of HBsAg, or amino acids 120-130 of the S domain of HBsAg. As used herein, the amino acid positions (e.g., 115-133, 120-133, 120-130) refer to the above-mentioned S domain of HBsAg present in all three HBV envelope proteins, S-HBsAg, M-HBsAg, and L-HBsAg, whereby S-HBsAg typically corresponds to the S domain of HBsAg.

[0090] As used herein in the context of an epitope, the term "formed by" means that the epitope to which an antibody or antigen-binding fragment thereof binds can be linear (continuous) or conformational (discontinuous). A linear or sequential epitope is one that is recognized by an antibody according to its linear sequence or primary structure of amino acids. A conformational epitope is recognized according to the three-dimensional shape and structure of a protein. Thus, if an epitope is linear and includes two or more amino acids located at positions selected from amino acids 115-133 or amino acids 120-133 of the S domain of HBsAg, the amino acids included in the epitope can be located adjacently in the primary structure (e.g., consecutive amino acids in the amino acid sequence). In the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as an epitope, and therefore the amino acids forming the epitope may or may not be located adjacently in the primary structure (i.e., they may or may not be consecutive amino acids in the amino acid sequence).

[0091] In certain embodiments, the epitope to which the antibody or antigen-binding fragment binds is a conformational epitope. In some embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least two amino acids in the antigenic loop region of HBsAg, wherein the at least two amino acids are selected from amino acids 120-133 or 120-130 of the S domain of HBsAg, and the at least two amino acids are not located adjacently (in the primary structure). In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least three amino acids in the antigenic loop region of HBsAg, wherein the at least three amino acids are selected from amino acids 120-133 or 120-130 of the S domain of HBsAg, and at least two of the three amino acids are not located adjacently (in the primary structure). In some embodiments, the binding protein binds to an epitope comprising at least four amino acids of an antigenic loop region of HBsAg, wherein the at least four amino acids are selected from amino acids 120-133 or amino acids 120-130 of the S domain of HBsAg, and at least two of the four amino acids are not located adjacently (in the primary structure).

[0092] Amino acids that are not adjacent in the primary structure and to which an antibody or antigen-binding fragment of the present disclosure binds (i.e., amino acids that form an epitope) are in some cases separated by one or more amino acids that are not bound by the antibody or antigen-binding fragment. In some embodiments, at least one, at least two, at least three, at least four, or at least five amino acids may be located between any two non-adjacent amino acids in the epitope.

[0093] In certain embodiments, the antibody or antigen-binding fragment binds to an epitope comprising at least amino acids P120, C121, R122, and C124 of the S domain of HBsAg. In other embodiments, the antibody or antigen-binding fragment of the present disclosure binds to SEQ ID NO: 88: PCRXC wherein X is any amino acid or no amino acid, X is any amino acid, or X is T, Y, R, S, or F, or X is T, Y, or R, or X is T or R.

[0094] In other embodiments, the antibody or antigen-binding fragment of the disclosure has the sequence of SEQ ID NO: 80: TGPCRTC or an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:80.

[0095] In other embodiments, the antibody or antigen-binding fragment of the disclosure has the sequence of SEQ ID NO: 85: STTSTGPCRTC or an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:85.

[0096] In certain embodiments, the antibody or antigen-binding fragment of the present disclosure comprises at least amino acids 145-151 of the S domain of HBsAg. GNCTCIP (SEQ ID NO: 81) The antibody binds to an epitope comprising an amino acid sequence comprising:

[0097] In still other embodiments, the antibody or antigen-binding fragment of the present disclosure binds to an epitope comprising the amino acid sequence according to SEQ ID NO:80 and the amino acid sequence according to SEQ ID NO:81.

[0098] In other embodiments, the antibody or antigen-binding fragment of the present disclosure binds to an epitope comprising the amino acid sequence according to SEQ ID NO:85 and / or the amino acid sequence according to SEQ ID NO:87.

[0099] As noted above, the epitopes to which the antibodies or antigen-binding fragments of the present disclosure bind can be linear (continuous) or conformational (discontinuous). In some embodiments, the antibodies or antigen-binding fragments of the present disclosure bind to conformational epitopes, and in certain such embodiments, the conformational epitopes are only present under non-reducing conditions.

[0100] In certain embodiments, an antibody or antigen-binding fragment of the present disclosure binds to a linear epitope. In certain such embodiments, the linear epitope is present under both non-reducing and reducing conditions.

[0101] In certain embodiments, the antibody or antigen-binding fragment of the disclosure has the sequence identified as SEQ ID NO:1: X1X2X3TCX4X5X6AX7G wherein X1, X2, X3, X4, X5, X6, and X7 can be any amino acid (SEQ ID NO: 1).

[0102] In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are conservatively substituted amino acids compared to amino acids 120-130 of SEQ ID NO: 3. In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are conservatively substituted amino acids compared to amino acids 20-30 of any of SEQ ID NOs: 5-33.

[0103] In certain embodiments, the amino acid X1 of SEQ ID NO: 1 is a small amino acid. As used herein, a "small" amino acid refers to any amino acid selected from the group consisting of alanine, aspartic acid, asparagine, cysteine, glycine, proline, serine, threonine, and valine. In certain such embodiments, X1 is proline, serine, or threonine.

[0104] In certain embodiments, X2 of SEQ ID NO: 1 is a small amino acid. In certain embodiments, X2 may be selected from cysteine ​​or threonine.

[0105] In some embodiments, X3 in SEQ ID NO: 1 is a charged amino acid or an aliphatic amino acid. As used herein, a "charged" amino acid refers to any amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, and histidine. As used herein, an "aliphatic" amino acid refers to any amino acid selected from the group consisting of alanine, glycine, isoleucine, leucine, and valine. In certain embodiments, X3 is selected from arginine, lysine, aspartic acid, or isoleucine.

[0106] In some embodiments, X4 in SEQ ID NO: 1 is a small amino acid and / or a hydrophobic amino acid. As used herein, a "hydrophobic" amino acid refers to any amino acid selected from the group consisting of alanine, isoleucine, leucine, phenylalanine, valine, tryptophan, tyrosine, methionine, proline, and glycine. In certain embodiments, X4 is selected from methionine or threonine.

[0107] In some embodiments, X5 of SEQ ID NO: 1 is a small and / or hydrophobic amino acid. In certain embodiments, X5 is selected from threonine, alanine, or isoleucine.

[0108] In some embodiments, X6 of SEQ ID NO: 1 is a small and / or hydrophobic amino acid. In certain embodiments, X6 is selected from threonine, proline, or leucine.

[0109] In some embodiments, X7 in SEQ ID NO: 1 is a polar amino acid or an aliphatic amino acid. As used herein, "polar" amino acid refers to any amino acid selected from the group consisting of aspartic acid, asparagine, arginine, glutamic acid, histidine, lysine, glutamine, tryptophan, tyrosine, serine, and threonine. In certain embodiments, X7 is glutamine, histidine, or leucine.

[0110] In some embodiments, the binding protein according to the present disclosure has the sequence of SEQ ID NO:2: X1X2X3TCX4X5X6AX7G wherein the antigenic loop of HBsAg is formed by an amino acid sequence represented by X1 is P, T, or S, X2 is C or S, X3 is R, K, D, or I, X4 is M or T, X5 is T, A, or I, X6 is T, P, or L, and X7 is Q, H, or L. (SEQ ID NO: 2)

[0111] It is noted that the term "formed by," as used herein with respect to epitopes formed by the amino acid sequence according to SEQ ID NO: 1 or 2, does not imply that the disclosed binding proteins bind to necessarily every amino acid in SEQ ID NO: 1 or 2. In particular, the binding proteins may bind to only some of the amino acids in SEQ ID NO: 1 or 2, with other amino acid residues acting as "spacers."

[0112] In certain embodiments, an antibody or antigen-binding fragment according to the present disclosure binds to an epitope in an antigenic loop of HBsAg formed by one or more, two or more, three or more, or four or more amino acids of an amino acid sequence selected from SEQ ID NOs: 5-33 shown in Table 3 below.

[0113] In some embodiments, an antibody or antigen-binding fragment according to the present disclosure binds to an antigenic loop region of HBsAg having an amino acid sequence according to any one or more of SEQ ID NOs: 5-33, or sequence variants thereof, as set forth in Table 3 below. In certain embodiments, an antibody or antigen-binding fragment according to the present disclosure binds to all of the antigenic loop variants of HBsAg having an amino acid sequence according to any of SEQ ID NOs: 5-33, as set forth in Table 3 below.

[0114] Table 3: Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg of various genotypes and mutants used herein (residues 101-172 of the S domain of HBsAg, excluding SEQ ID NO: 16, which refers to residues 100-172 of the S domain of HBsAg to include relevant mutations). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0115] Thus, in certain aspects, the present disclosure provides a method for detecting a heavy chain variable region (V) comprising at least 90% identity to an amino acid sequence according to SEQ ID NO: 41 or 67. H ) and (ii) a light chain variable region (V) comprising at least 90% identity to an amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, or 110-120. L ), wherein the amino acid at position 40 of the VL according to the IMGT numbering is not a cysteine, and wherein the antibody or antigen-binding fragment thereof binds to an antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus.

[0116] In a further embodiment, (i) V H comprises at least 95% identity with an amino acid sequence according to SEQ ID NO: 41 or 67, and / or (ii) V L comprises at least 95% identity with an amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, or 110-120.

[0117] In certain embodiments, V L In another embodiment, the amino acid at position 40 of V is alanine. L In yet another embodiment, the amino acid at position 40 of V is serine. L The amino acid at position 40 is glycine.

[0118] In any of the embodiments disclosed herein, antibodies or antigen-binding fragments suitable for use in the pharmaceutical compositions and methods of the disclosure may comprise CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences according to SEQ ID NOs: (i) 34-36, 37, 38, and 40, respectively; (ii) 34, 66, 36, 37, 38, and 40, respectively; (iii) 34-36, 37, 39, and 40, respectively; (iv) 34, 66, 36, 37, 39, and 40, respectively; (v) 34-36, 37, 38, and 58, respectively; (vi) 34, 66, 36, 37, 38, and 58, respectively; (vii) 34-36, 37, 39, and 58, respectively; or (vii) 34, 66, 36, 37, 39, and 58, respectively.

[0119] In some embodiments, the V of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of the present disclosure L comprises or consists of an amino acid sequence according to SEQ ID NO: 89. In some embodiments, the V of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of the present disclosure L comprises or consists of an amino acid sequence according to SEQ ID NO: 90. In other embodiments, the V of an antibody or antigen-binding fragment suitable for use in the pharmaceutical compositions and methods of the disclosure Lcomprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 109 to 120. In certain embodiments, V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41. H comprises or consists of the amino acid sequence according to SEQ ID NO: 67.

[0120] In certain embodiments, V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41, and V L comprises or consists of the amino acid sequence according to SEQ ID NO: 89. In another embodiment, V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41, and V L comprises or consists of an amino acid sequence according to SEQ ID NO: 90. In certain embodiments, V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41, and V L comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 109 to 120. In another embodiment, V H comprises or consists of the amino acid sequence according to SEQ ID NO: 67, and V L comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 109 to 120.

[0121] In another aspect, the present disclosure provides a method for the production of a mAb comprising: (i) a heavy chain variable region (V) comprising at least 90% identity to an amino acid sequence according to SEQ ID NO: 95; H ) and (ii) a light chain variable region (V) comprising at least 90% identity with the amino acid sequence according to SEQ ID NO: 96. L ), wherein the antibody or antigen-binding fragment thereof binds to an antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus or hepatitis delta virus.

[0122] In further embodiments, (i) the VH comprises at least 95% identity to the amino acid sequence according to SEQ ID NO: 95, and / or (ii) the VL comprises at least 95% identity to the amino acid sequence according to SEQ ID NO: 96. In certain embodiments, the antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences according to SEQ ID NOs: 97-102, respectively.

[0123] In a particular embodiment, the VH comprises or consists of an amino acid sequence according to SEQ ID NO:95 and the VL comprises or consists of an amino acid sequence according to SEQ ID NO:96.

[0124] Fc part In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion. In certain embodiments, the Fc portion is derived from human origin, e.g., may be derived from human IgG1, IgG2, IgG3, and / or IgG4. In certain embodiments, the antibody or antigen-binding fragment may comprise an Fc portion derived from human IgG1.

[0125] As used herein, the term "Fc portion" refers to a sequence comprising or derived from a portion of an immunoglobulin heavy chain beginning with the hinge region immediately upstream of the papain cleavage site (e.g., residue 216 of native IgG, with 114 being the first residue of the heavy chain constant region) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, an Fc portion can be a complete Fc portion, or a portion thereof (e.g., a domain). In certain embodiments, a complete Fc portion comprises the hinge domain, the CH2 domain, and the CH3 domain (e.g., EU amino acids 216-446). An additional lysine residue (K) may be present at the very C-terminus of the Fc portion but is often cleaved from mature antibodies.

[0126] Amino acid positions in the Fc portion herein are numbered according to the EU numbering system of Kabat. See, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, 1983 and 1987. Amino acid positions in the Fc portion can also be numbered according to the IMGT numbering system (including the unique numbering of the C domain and exon numbering) and the Kabat numbering system.

[0127] In some embodiments, the Fc portion comprises at least one of a portion of the hinge (e.g., upper, middle, and / or lower hinge region), CH2 domain, CH3 domain, or variant, or fragment thereof. In some embodiments, the Fc portion comprises at least the hinge domain, CH2 domain, or CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. The amino acid sequence of an exemplary Fc portion of the human IgG1 isotype is provided in SEQ ID NO: 137. The Fc portion may comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc portion. For example, at least one or a portion of the hinge domain, CH2 domain, or CH3 domain may be deleted. For example, the Fc portion may comprise or consist of (i) a hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), (ii) a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iv) a hinge domain (or portion thereof), (v) a CH2 domain (or portion thereof), or (vi) a CH3 domain or portion thereof.

[0128] The Fc portion of the present disclosure can be modified so that it varies in amino acid sequence from the complete Fc portion of a naturally occurring immunoglobulin molecule while retaining or enhancing at least one desirable function imparted by the naturally occurring Fc portion. Such functions include, for example, Fc receptor (FcR) binding, modulation of antibody half-life (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement fixation. Some of the naturally occurring Fc portions involved in such functions have been described in the art.

[0129] For example, when an immunoglobulin molecule binds to an antigenic target to activate the complement cascade, a complex of C1q protein can bind to at least two IgG molecules or one IgM molecule (Ward, ES, and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, DR described that the heavy chain region containing amino acid residues 318 to 337 is involved in complement fixation (Mol. Immunol. 22 (1985) 161-206). Duncan, AR, and Winter, G. (Nature 332 (1988) 738-740) reported by site-directed mutagenesis that Glu318, Lys320, and Lys322 form the binding site for C1q. The role of residues Glu318, Lys320, and Lys322 in binding C1q was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated cell lysis.

[0130] For example, FcR binding can be mediated by the interaction of the Fc portion (of an antibody) with Fc receptors (FcRs), specialized cell surface receptors on cells, including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC; Van de Winkel, JG, and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are designated FcγR, those for IgE are designated FcεR, those for IgA are designated FcαR, and neonatal Fc receptors are designated FcRn. Fc receptor binding is described, for example, in Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0131] Cross-linking of receptors (FcγRs) by the Fc domain of natural IgG antibodies triggers a wide range of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as clearance of immune complexes and control of antibody production. Fc moieties that provide cross-linking of receptors (e.g., FcγRs) are contemplated herein. Three classes of Fcγ receptors have been characterized in humans to date: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds complexed IgG with moderate to low affinity, is widely expressed, particularly on leukocytes, and is thought to play a central role in antibody-mediated immunity. It can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, and binds IgG-Fc with similar low affinity, although the extracellular domains of these receptors are highly homologous. Finally, (iii) FcγRIII (CD16), which binds IgG with moderate to low affinity and is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is thought to mediate ADCC. It is found in two forms: FcγRIIIA and FcγRIIIB, which are highly expressed on neutrophils.

[0132] FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, as well as mast cells and eosinophils. Importantly, 75% of all FcγRIIB has been shown to be found in the liver (Ganesan, LP et al., 2012: "FcγRIIb on liver sinusoidal endothelium clears small immune complexes," Journal of Immunology 189: 4981-4988). FcγRIIB is abundantly expressed on liver sinusoidal endothelium, called LSECs, and on hepatic Kupffer cells, which are the primary site of clearance of small immune complexes (Ganesan, LP et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

[0133] In some embodiments, the antibodies and antigen-binding fragments thereof disclosed herein comprise an Fc portion, particularly an Fc region, for binding to FcγRIIb, e.g., an IgG-type antibody. Furthermore, as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933, the Fc portion can be engineered to introduce the mutations S267E and L328F to enhance FcγRIIB binding. This can enhance the clearance of immune complexes (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises an engineered Fc portion having the mutations S267E and L328F, particularly as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.

[0134] In B cells, FcγRIIB appears to function to suppress further immunoglobulin production and isotype switching, for example to IgE class. In macrophages, FcγRIIB appears to inhibit phagocytosis, as mediated by FcγRIIA. In eosinophils and mast cells, the b form may help suppress activation of these cells through binding of IgE to its separate receptor.

[0135] With respect to FcγRI binding, alteration of at least one of E233 to G236, P238, D265, N297, A327, and P329 of native IgG reduces FcγRI binding. Substitution of IgG2 residues at positions 233 to 236 with the corresponding positions in IgG1 and IgG4 reduces FcγRI binding of IgG1 and IgG4 by 10. 3 It reduced the antibody titer by 1-fold and abolished the response of human monocytes to antibody-sensitized red blood cells (Armour, KL, et al. Eur. J. Immunol. 29 (1999) 2613-2624).

[0136] With respect to FcγRII binding, for example, reduced binding to FcγRIIA is observed for at least one IgG mutation of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0137] The two allelic forms of human FcγRIIA are the "H131" variant, which binds IgG1 Fc with high affinity, and the "R131" variant, which binds IgG1 Fc with low affinity. See, e.g., Bruhns et al., Blood 113:3716-3725 (2009).

[0138] With regard to FcγRIII binding, for example, reduced binding to FcγRIIIA was observed for at least one mutation of E233 to G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376. Mapping of the binding site on human IgG1 for Fc receptors, the above-mentioned mutation sites, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0139] The two allelic forms of human FcγRIIIA are the "F158" variant, which binds IgG1 Fc with low affinity, and the "V158" variant, which binds IgG1 Fc with high affinity. See, e.g., Bruhns et al., Blood 113:3716-3725 (2009).

[0140] With regard to binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG: (i) the lower hinge region of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, specifically the loop and strand of the upper CH2 domain adjacent to the lower hinge region, e.g., the region of P331 (Wines, BD, et al., J. Immunol. 2000; 164: 5313-5318). Furthermore, while FcγRI appears to bind to the same site on IgG Fc, FcRn and Protein A bind to different sites on IgG Fc, which appear to be the CH2-CH3 interface (Wines, BD, et al., J. Immunol. 2000; 164: 5313-5318).

[0141] In some embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion comprising mutations that increase the binding affinity of the Fc portion to (i.e., one or more) Fcγ receptors, e.g., human FcγRIIa, human FcγRIIIa, or both (compared to a reference Fc portion or to an antibody comprising an Fc portion that does not comprise the mutations). See, e.g., Delillo and Ravetch, Cell 161(5):1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016). The Fc mutations and techniques described therein are incorporated herein by reference. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion that includes mutations selected from G236A; S239D; A330L; and I332E; or a combination comprising these, such as S239D / I332E; S239D / A330L / I332E; G236A / S239D / I332E; G236A / A330L / I332E (also referred to herein as "GAALIE"); or G236A / S239D / A330L / I332E.

[0142] In certain embodiments, the Fc portion may comprise or consist of at least a portion of the Fc portion involved in binding to FcRn (e.g., to human FcRn). In certain embodiments, the Fc portion comprises one or more amino acid modifications that improve binding affinity to FcRn, and in some embodiments, thereby extending the in vivo half-life of a molecule comprising the Fc portion (e.g., compared to a reference Fc portion or antibody that does not contain the modifications). In certain embodiments, the Fc portion comprises or is derived from an IgG Fc, and the half-life-extending mutations include any one or more of M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, E380A (EU numbering). In certain embodiments, the half-life-enhancing mutations include M428L / N434S (also referred to herein as "MLNS"). In certain embodiments, the half-life-enhancing mutations include M252Y / S254T / T256E. In certain embodiments, the half-life-enhancing mutations include T250Q / M428L. In certain embodiments, the half-life-enhancing mutations include P257I / Q311I. In certain embodiments, the half-life-enhancing mutations include P257I / N434H. In certain embodiments, the half-life-enhancing mutations include D376V / N434H. In certain embodiments, the half-life-enhancing mutations include T307A / E380A / N434A.

[0143] In some embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising the substitution mutations M428L / N434S. In some embodiments, the binding protein comprises an Fc portion comprising the substitution mutations G236A / A330L / I332E. In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising the G236A, A330L, and I332L mutations (GAALIE) and no S239D mutation. In some embodiments, the Fc portion comprises Ser at position 239. In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising the substitution mutations M428L / N434S and G236A / A330L / I332E. In certain embodiments, antibodies or antigen-binding fragments thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprise an Fc portion comprising the substitution mutations M428L / N434S and G236A / S239D / A330L / I332E. In certain further embodiments, the Fc portion does not comprise any substitution mutations other than M428L / N434S and G236A / S239D / A330L / I332E.

[0144] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises the CDRs and / or variable domains and / or heavy and / or light chains (antibodies HBC34, HBC34v7, HBC34v23, HBC34v31, HBC34v32, HBC34v33, HBC34v34, HBC34v35 (including variants of the HBC antibodies disclosed herein comprising a substitution mutation at position 40 of the light chain (e.g., replacement of the native cysteine ​​with alanine, serine, etc.))) according to any one of the exemplary anti-HBV antibodies disclosed herein and / or in PCT Publication No. WO2017 / 060504), and an Fc portion comprising the G236A, A330L, and I332E (GAALIE) mutations, and the Fc portion may further comprise the M428L / N434S (MLNS) mutations. In certain embodiments, the Fc portion does not include S239D.

[0145] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises a CDRH1 amino acid sequence according to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, a CDRH3 amino acid sequence according to SEQ ID NO: 36, a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 amino acid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequence according to SEQ ID NO: 58 or 40, and an Fc portion comprising a GAALIE mutation. In certain embodiments, the Fc portion further comprises a MLNS mutation.

[0146] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises a heavy chain variable domain (VH) amino acid sequence according to any one of SEQ ID NOs: 41 or 67 and a light chain variable domain (VL) amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, and 111-120, and an Fc portion comprising a GAALIE mutation. In certain embodiments, the Fc portion further comprises an MLNS mutation.

[0147] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises a heavy chain amino acid sequence according to SEQ ID NO: 138 or 91.

[0148] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises a CDRH1 amino acid sequence according to SEQ ID NO: 97, a CDRH2 amino acid sequence according to SEQ ID NO: 98, a CDRH3 amino acid sequence according to SEQ ID NO: 99, a CDRL1 amino acid sequence according to SEQ ID NO: 100, a CDRL2 amino acid sequence according to SEQ ID NO: 100, and a CDRL3 amino acid sequence according to SEQ ID NO: 102, and an Fc portion comprising a GAALIE mutation. In certain embodiments, the Fc portion further comprises a MLNS mutation.

[0149] In any of the embodiments of the present disclosure, a binding protein of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has enhanced binding to human FcγRIIa and / or human FcγRIIIa compared to a reference polypeptide (i.e., a polypeptide which may be a binding protein comprising an Fc portion that does not comprise a GAALIE mutation).

[0150] In certain embodiments, the reference polypeptide comprises an Fc portion that is a wild-type Fc portion, or an Fc portion that comprises one or more substitution mutations (or insertions or deletions), provided that the substitution mutations are not GAALIE. In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises the HBC34v35 antibody comprising GAALIE and MLNS mutations, and the reference polypeptide is HBC34v35 (comprising a wild-type Fc portion of the same isotype as the Fc portion of the antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure). In certain embodiments, the reference polypeptide does not comprise substitution mutations known or believed to affect binding to human FcγRIIa and / or human FcγRIIIa.

[0151] Binding between polypeptides, for example, binding between an Fc portion (or a binding protein comprising the same) and a human Fcγ receptor, for example, human FcγRIIA, human FcγRIIIA, or human Fc FcγRIIB, or a complement protein such as C1q, can be determined or detected by methods known in the art. For example, a biolayer interferometry (BLI) assay can be performed using an Octet® RED96 (ForteBio, Fremont, California, USA) instrument according to the manufacturer's instructions to determine real-time association and dissociation between a first polypeptide of interest (e.g., HBC34v35 containing a GAALIE mutation) and a second polypeptide of interest (e.g., FcγRIIA(H131), FcγRIIA(R131), FcγRIIIA(F158), FcγRIIIA(V158), or FcγRIIb) captured on a sensor substrate.

[0152] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), human FcγRIIIA(V158), or any combination thereof, compared to a reference polypeptide comprising an Fc portion that does not comprise a GAALIE mutation. In certain embodiments, enhanced binding is determined by an increased signal shift (e.g., one or more of a higher peak signal, a larger association rate, a slower dissociation rate, or a larger area under the curve) relative to a reference binding protein in a BLI assay. In certain embodiments, the BLI assay comprises use of an Octet® RED96 (ForteBio, Fremont, California, USA) instrument. In further embodiments, the BLI assay comprises a tagged human FcγR that is captured on an anti-pentatag sensor and exposed to the binding protein. In some embodiments, the binding protein comprises an IgG Fab and the BLI assay further comprises exposing the captured human FcγR to an antibody or antigen-binding fragment in the presence of an anti-IgG Fab binding fragment that crosslinks the binding protein through the Fab fragment.

[0153] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), and / or human FcγRIIIA(V158) compared to a reference polypeptide, wherein the enhanced binding comprises a signal shift (nanometers) in a BLI assay of 1.5-fold, 2-fold, 2.5-fold, 3-fold, or greater than the signal shift observed using the reference antibody.

[0154] In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), and human FcγRIIIA(V158) compared to a reference polypeptide.

[0155] In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has reduced binding to human FcγRIIB compared to a reference polypeptide. In certain embodiments, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and does not bind to human FcγRIIB, as determined, for example, by the absence of a statistically significant signal shift relative to baseline in a BLI assay.

[0156] In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and has reduced binding to human C1q (a complement protein) compared to a reference polypeptide. In certain embodiments, the binding protein comprises an Fc portion comprising a GAALIE mutation and does not bind to human C1q, as determined by the absence of a statistically significant signal shift relative to baseline in a BLI assay.

[0157] In any of the embodiments of the present disclosure, an antibody or antigen-binding fragment thereof suitable for use in the pharmaceutical compositions and methods of the present disclosure comprises an Fc portion comprising a GAALIE mutation and activates human FcγRIIA, human FcγRIIIA, or both to a greater extent than a reference polypeptide (i.e., a polypeptide which may be an antibody or antigen-binding fragment thereof comprising an Fc portion not comprising a GAALIE mutation). In certain embodiments, the reference polypeptide is a wild-type Fc portion or comprises an Fc portion comprising one or more substitution mutations, provided that the substitution mutation is not GAALIE. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HBC34v35 antibody having a GAALIE mutation (and other substitution mutations which may be, for example, MLNS), and the reference polypeptide is HBC34v35 having a wild-type Fc portion.

[0158] Activation of human FcγR can be determined or detected using methods known in the art. For example, a well-validated, commercially available bioreporter assay involves incubating an HBsAg-specific binding protein with recombinant HBsAg (Engelix B, GlaxoSmithKline) in the presence of Jurkat effector cells (Promega, Cat. no. G9798) stably expressing (i) the FcγR of interest and (ii) a firefly luciferase reporter under the control of an NFAT response element. Binding of Fc to the cell surface-expressed FcγR drives NFAT-mediated expression of the luciferase reporter gene. Bio-Glo™ Luciferase Assay Reagent (Promega) is then used to measure luminescence using a luminometer (e.g., Bio-Tek) according to the manufacturer's instructions. Activation is expressed as the average relative luminescence units (RLU) over background by applying the following formula: [RLU at [x] concentration of binding protein (eg, mAb) - RLU of background].

[0159] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an Fc portion comprising a GAALIE mutation and activates human FcγRIIA(H131), human FcγRIIIA(F158), and / or human FcγRIIIA(V158) to a greater extent than a reference polypeptide. In certain embodiments, a greater degree of activation refers to a higher peak emission and / or a larger area under the emission curve as determined using a luminescence bioreporter assay described herein. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an Fc portion comprising a GAALIE mutation and activates human FcγRIIA(H131), human FcγRIIA(R131), and human FcγRIIIA(F158) to a greater extent than a reference polypeptide, and a greater degree of activation can be represented by a peak RLU that is 1.5-fold, 2-fold, 2.5-fold, 3-fold, or more than the peak RLU observed using the reference polypeptide.

[0160] In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises an Fc portion comprising a GAALIE mutation and does not activate human FcγRIIB as determined by the absence of statistically significant and / or measurable RLU in the luminescent bioreporter assay described above.

[0161] In any of the embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises an Fc portion comprising a GAALIE mutation and activates human natural killer (NK) cells to a greater extent than a reference polypeptide in the presence of HBsAg. In certain embodiments, NK cell activation is determined by expression of CD107a (e.g., by flow cytometry). In certain embodiments, the NK cells comprise cells comprising a V158 / V158 homozygous, F158 / F158 homozygous, or V158 / F158 heterozygous FcγRIIIa genotype.

[0162] It is recognized that any antibody or antigen-binding fragment thereof comprising an Fc portion comprising a GAALIE mutation according to the present disclosure can exhibit or process any one or more of the characteristics described herein, such as enhanced binding to human FcγRIIA and / or human FcγRIIIA compared to a reference polypeptide, reduced binding to human FcγRIIB (and / or no binding to human FcγRIIB) compared to a reference polypeptide, reduced binding to human C1q (and / or no binding to human C1q) compared to a reference polypeptide, activating FcγRIIA, human FcγRIIIA, or both to a greater extent than a reference polypeptide, not activating human FcγRIIB, and / or activating human natural killer (NK) cells in the presence of HBsAg to a greater extent than a reference polypeptide (e.g., an antibody comprising an Fc portion specific for HBsAg and not comprising a GAALIE mutation).

[0163] Alternatively or additionally, the Fc portion of an antibody or antigen-binding fragment thereof of the present disclosure may comprise at least a portion known in the art of an Fc molecule required for binding Protein A, and / or the Fc portion of an antibody of the present disclosure comprises at least a portion known in the art of an Fc molecule required for binding Protein G. In some embodiments, the retained function includes clearance of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion comprises at least a portion known in the art of an FcγR required for binding. Thus, as outlined above, the Fc portion may comprise at least (i) the lower hinge region of native IgG Fc, in particular amino acid residues L, L, G, G (234-237, EU numbering), and (ii) an adjacent region of the CH2 domain of native IgG Fc, in particular the loop and strand of the upper CH2 domain adjacent to the lower hinge region, e.g., the region of P331, e.g., a region of at least 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of the upper CH2 domain of native IgG Fc surrounding P331, e.g., between amino acids 320 and 340 (EU numbering) of native IgG Fc.

[0164] In some embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure comprises an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc portions of antibody heavy chains. For example, an Fc region may be a monomeric or "single-chain" Fc region (i.e., scFc region). A single-chain Fc region consists of Fc portions linked in a single polypeptide chain (e.g., encoded by a single contiguous nucleic acid sequence). An exemplary scFc region is described in WO2008 / 143954A2, incorporated herein by reference. An Fc region may be or include a dimeric Fc region. A "dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc moieties (e.g., Fc regions of naturally occurring immunoglobulins) or a heterodimer of two non-identical Fc moieties (e.g., one Fc monomer of the dimeric Fc region contains at least one amino acid modification (e.g., a substitution, deletion, insertion, or chemical modification) that is not present in the other Fc monomer, or one Fc monomer may be truncated relative to the other).

[0165] Specific embodiments include antibodies and antigen-binding fragments having a heavy chain (e.g., VH-hinge-CH1-CH2-CH3) according to SEQ ID NO:91 or SEQ ID NO:92, and an antibody or antigen-binding fragment having a light chain (i.e., VL-CL) according to SEQ ID NO:93 or SEQ ID NO:94. In certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:91 and a light chain according to SEQ ID NO:93. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:92 and a light chain according to SEQ ID NO:94. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:91 and a light chain according to SEQ ID NO:94. In other embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:92 and a light chain according to SEQ ID NO:93. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:129. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain according to SEQ ID NO:138. These sequences are provided in the Sequence Listing.

[0166] The Fc moieties of the present disclosure may comprise Fc sequences or regions of the same or different classes and / or subclasses. For example, the Fc moieties may be derived from immunoglobulins (e.g., human immunoglobulins) of the IgG1, IgG2, IgG3, or IgG4 subclass, or any combination thereof. In certain embodiments, the Fc moieties of an Fc region are of the same class or subclass. However, an Fc region (or one or more Fc moieties of an Fc region) may be chimeric, such that a chimeric Fc region comprises Fc moieties derived from different immunoglobulin classes and / or subclasses. For example, at least two of the Fc moieties of a dimer or single-chain Fc region may be from different immunoglobulin classes and / or subclasses. In certain embodiments, a dimeric Fc region may comprise sequences from two or more different isotypes or subclasses, e.g., SEED bodies ("strand-exchange engineered domains"). See Davis et al., Protein Eng. Des. Sel. 23(4):195 (2010).

[0167] Additionally or alternatively, a chimeric Fc region can have one or more chimeric Fc moieties. For example, a chimeric Fc region or moiety can include one or more moieties derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or moiety is of a different subclass. For example, an Fc region or moiety of an Fc polypeptide can include a CH2 and / or CH3 domain derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2, or IgG4 subclass) and a hinge region derived from an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, an Fc region or moiety can include a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and a CH3 domain derived from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region can comprise an Fc portion (e.g., a complete Fc portion) derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and an Fc portion derived from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, the Fc region or portion can comprise a CH2 domain derived from an IgG4 immunoglobulin and a CH3 domain derived from an IgG1 immunoglobulin. For example, the Fc region or portion can comprise the CH1 and CH2 domains derived from an IgG4 molecule and the CH3 domain derived from an IgG1 molecule. For example, the Fc region or portion can comprise a portion of the CH2 domain derived from a particular antibody subclass, e.g., amino acids 292-340 of the CH2 domain. For example, the Fc region or portion can comprise amino acids 292-340 of the CH2 derived from the IgG4 portion and the remainder of the CH2 derived from the IgG1 portion (alternatively, CH2 292-340 can be derived from the IgG1 portion and the remainder of the CH2 derived from the IgG4 portion).

[0168] It is also recognized that any antibody, antigen-binding portion, or Fc region or portion of the present disclosure can be of any allotype and / or haplotype. For example, human immunoglobulin G allotypes include those disclosed in Jefferis and LeFranc, mAbs 1(4):1-7 (2009), including allotypes [G1m (1(a); 2(x); 3(f); and 17(z)]; G2m [23(n)]; G3m [21(g1); 28(g5); 11(b0); 5(b2); 13(b3); 14(b4); 10(b5); 15(s); 16(t); 6(c3); 24(c5); 26(u); and 27(v)]; A2m (1 and 2); and Km (1; 2; and 3) and haplotypes, and resulting amino acid sequences, and combinations thereof, are incorporated herein by reference. In certain embodiments, an antibody, antigen-binding fragment, or Fc region or portion of the present disclosure comprises the IgG1 allotype g1m17,k1.

[0169] Further, the Fc region or portion may (additionally or alternatively) comprise, for example, a chimeric hinge region. For example, the chimeric hinge may be derived, for example, partially from an IgG1, IgG2, or IgG4 molecule (e.g., the upper and lower middle hinge sequences) and partially from an IgG3 molecule (e.g., the middle hinge sequence). In another example, the Fc region or portion may comprise a chimeric hinge derived partially from an IgG1 molecule and partially from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge may be generated, for example, by introducing a proline substitution (Ser228Pro) at position EU228 of the middle hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may comprise amino acids at EU233-EU236 and / or a Ser228Pro mutation from an IgG2 antibody, with the remaining amino acids of the hinge being from an IgG4 antibody (e.g., a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP). Additional chimeric hinges that may be used in the Fc portion of antibodies according to the present disclosure are described in US2005 / 0163783A1.

[0170] In some embodiments of the antibodies or antigen-binding fragments thereof disclosed herein, the Fc portion or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from the Fc region or Fc portion of a human IgG molecule). However, the polypeptide may also comprise one or more amino acids from another mammalian species. For example, a primate Fc portion or primate binding site may be included in the subject polypeptide. Alternatively, one or more murine amino acids may be present in the Fc portion or Fc region.

[0171] nucleic acid molecule In another aspect, the present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof according to the present disclosure.

[0172] Table 4: Exemplary V according to the present disclosure H , V L The nucleotide sequences encoding CH, CL, HC, and LC are shown. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12]

[0173] Due to redundancy in the genetic code, the present disclosure also includes sequence variants of these nucleic acid sequences, particularly those sequence variants that encode the same amino acid sequence.

[0174] In certain embodiments, the polynucleotide or nucleic acid molecule comprises a nucleotide sequence that shares at least 80% identity with a nucleotide sequence according to any one of SEQ ID NOs: 103-110 and 130-136, and the nucleotide sequence is codon-optimized for expression by a host cell.

[0175] In certain embodiments, a nucleic acid molecule according to the present disclosure comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 103-110 and 130-136.

[0176] In certain embodiments, the polynucleotide is H The nucleotide sequence according to SEQ ID NO: 103 encoding V L In another embodiment, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 105, which encodes V H The nucleotide sequence according to SEQ ID NO: 103 encoding V L In another embodiment, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 104, which encodes V H The nucleotide sequence according to SEQ ID NO: 108 encoding V L The nucleotide sequence according to SEQ ID NO: 109 encodes:

[0177] VH The nucleotide sequence according to SEQ ID NO: 103 encoding V L Also provided herein is a polynucleotide encoding an antibody or antigen-binding fragment comprising or consisting of a nucleotide sequence according to SEQ ID NO: 110, which encodes: wherein the encoded antibody or antigen-binding fragment binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus or hepatitis delta virus.

[0178] In any of the embodiments of the present disclosure, the polynucleotide may comprise a nucleotide sequence according to SEQ ID NO: 130 encoding CH1-hinge-CH2-CH3 and / or comprises a nucleotide sequence according to SEQ ID NO: 131 encoding HC(VH-CH1-hinge-CH3-CH3). In some embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 132 encoding CL and / or comprises a nucleotide sequence according to SEQ ID NO: 133 encoding LC(VL-CL). In other embodiments, the polynucleotide comprises a nucleotide sequence according to SEQ ID NO: 134 encoding CL and / or a nucleotide sequence according to SEQ ID NO: 135 or SEQ ID NO: 136 encoding LC(VL-CL).

[0179] vector Further included within the scope of the present disclosure are vectors, e.g., expression vectors, that comprise nucleic acid molecules according to the present disclosure.

[0180] The term "vector" refers to a construct containing a nucleic acid molecule. A vector in the context of the present disclosure is suitable for incorporating or containing a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that allows for convenient storage of a nucleic acid molecule. That is, the vector may contain a sequence corresponding to a desired antibody or antibody fragment thereof according to the present disclosure.

[0181] As used herein, "expression vector" refers to a DNA construct comprising a nucleic acid molecule operably linked to suitable control sequences capable of effecting expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter (e.g., a heterologous promoter) to effect transcription, an appropriate operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. Any of the elements of an expression vector that contribute to the transcription of a nucleic acid molecule of interest may be heterologous to the vector. A vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector replicates and functions independently of the host genome, or, in some cases, is integrated into the genome itself. As used herein, the terms "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

[0182] A cloning vector is typically a vector that contains a cloning site, which is used to incorporate a nucleic acid sequence into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector.

[0183] A transfer vector may be a vector suitable for transferring a nucleic acid molecule into a cell or organism, such as a viral vector. A vector in the context of the present disclosure may be, for example, an RNA vector or a DNA vector. A vector may be a DNA molecule. For example, a vector in the context of the present application comprises a sequence suitable for replication of the vector, such as a cloning site, a selection marker such as an antibiotic resistance factor, and an origin of replication. In some embodiments, a vector in the context of the present application is a plasmid vector. In certain such embodiments, the vector comprises a lentiviral vector or a retroviral vector.

[0184] cell In a further aspect, the present disclosure also provides cells (also referred to as "host cells") that express an antibody, antigen-binding fragment, or fusion protein according to the present disclosure, or that contain a vector or polynucleotide according to the present disclosure.

[0185] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, insect cells, plant cells, and prokaryotic cells, including E. coli. In some embodiments, the cell is a mammalian cell. In certain such embodiments, the cells are mammalian cell lines, such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, YO cells, Sp2 / 0 cells, NS0 cells, human hepatocytes such as Hepa RG cells, myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells (e.g., TM4 cells), SV40-transformed monkey kidney CV1 line (COS-7), baby hamster kidney cells (BHK), African green monkey kidney cells (VERO-76), monkey kidney cells (CV1), human cervical carcinoma cells (HELA), human lung cells (W138), human hepatocytes (Hep G2), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), mouse mammary tumor (MMT) cells, and the like. 060562), TRI cells, MRC5 cells, and FS4 cells. Suitable mammalian host cell lines for antibody production also include those described, for example, in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0186] In certain embodiments, the host cell is a prokaryotic cell such as E. coli. Expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio / Technology 9:545-551 (1991)). For example, antibodies are produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523.

[0187] Useful insect cells for expressing the antibodies or antigen-binding fragments thereof of the present disclosure are known in the art and include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWT01 "Mimic™" cells. See, e.g., Palmberger et al., J. Biotechnol. 153(3-4):160-166 (2011). Numerous baculovirus strains have been identified for use with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0188] Eukaryotic microbes, such as filamentous fungi or yeast, are also suitable hosts for cloning or expressing protein-encoding vectors, including fungal and yeast strains that have "humanized" glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

[0189] Plant cells can also be used as hosts for expressing the antibodies or antigen-binding fragments thereof of the present disclosure. For example, PLANTIBODIES™ technology (described, for example, in U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429) employs transgenic plants to produce antibodies.

[0190] Any protein expression system compatible with the present disclosure may be used to produce the disclosed antibodies or antigen-binding fragments thereof. Suitable expression systems include transgenic animals as described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0191] In certain embodiments, cells may be transfected with a vector according to the present specification, including an expression vector. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, e.g., a eukaryotic cell. In the present context, the term "transfection" encompasses any method known to those skilled in the art for the introduction of a nucleic acid molecule into a cell, e.g., a eukaryotic cell, including a mammalian cell. Such methods include, for example, electroporation, lipofection based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection, such as DEAE-dextran or polyethyleneimine. In certain embodiments, the introduction is non-viral.

[0192] Additionally, cells of the present disclosure can be stably or transiently transfected with a vector according to the present disclosure, for example, to express an antibody or antigen-binding fragment thereof according to the present disclosure. In such embodiments, the cells are stably transfected with a vector described herein encoding a binding protein. Alternatively, the cells can be transiently transfected with a vector according to the present disclosure encoding a binding protein according to the present disclosure. In any of the embodiments of the present disclosure, the polynucleotide can be heterologous to the host cell.

[0193] In a related aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell of the disclosure under conditions and for a time sufficient to produce the antibody or antigen-binding fragment thereof.

[0194] Accordingly, the present disclosure also provides recombinant host cells that heterologously express an antibody or antigen-binding fragment thereof of the present disclosure. For example, the cell can be of a species different from the species from which the antibody was derived, wholly or partially (e.g., a CHO cell expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not naturally express the antibody or antigen-binding fragment. Furthermore, the host cell may impart post-translational modifications (PTMs, e.g., glycosylation or fucosylation) to the antibody or antigen-binding fragment that are not present in the native state of the antibody or antigen-binding fragment (or in the native state of the parent antibody from which the antibody or antigen-binding fragment is engineered or derived). Such PTMs may result in functional differences (e.g., reduced immunogenicity). Thus, an antibody or antigen-binding fragment of the disclosure produced by the host cells disclosed herein may contain one or more post-translational modifications that distinguish it from the antibody in its native state (or parent antibody) (e.g., a human antibody produced by CHO cells may contain more post-translational modifications that distinguish it from the antibody when isolated from a human and / or when produced by a natural human B cell or plasma cell).

[0195] Optional Additional Characteristics of the Antibody or Antigen-Binding Fragment The antibodies and antigen-binding fragments of the present disclosure may be linked to drugs, for example, for delivery to a treatment site, or to a detectable label to facilitate imaging of a site containing cells of interest. Methods for linking antibodies to drugs and detectable labels are known in the art, as are methods for imaging using detectable labels. Labeled antibodies can be employed in a wide variety of assays employing a wide range of labels. Detection of the formation of an antibody-antigen complex between an antibody (or antigen-binding fragment or fusion protein) of the present disclosure and an epitope of interest on HBsAg, particularly in the antigenic loop region of HBsAg, can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include the use of radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, dyes, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and other labels. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent material is luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeled agents can be used in a variety of known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISAs, fluorescent immunoassays, and the like. Thus, labeled antibodies and antigen-binding fragments according to the present disclosure can be used in assays such as those described in, for example, US Pat. Nos. 3,766,162, 3,791,932, 3,817,837, and 4,233,402.

[0196] The antibody or antigen-binding fragment thereof according to the present disclosure may be conjugated to a therapeutic moiety, such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. Such antibody conjugates can be used to modify a given biological response. The drug moiety should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

[0197] Techniques for conjugating such therapeutic moieties to antibodies are known. For example, Arnon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al. (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985); "Analysis," Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy,” in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.

[0198] Alternatively, an antibody or antigen-binding fragment thereof can be conjugated to a second antibody or antibody fragment thereof (or a second fusion protein) to form a heteroconjugate, as described in U.S. Pat. No. 4,676,980. Furthermore, a linker can be used between the label and the antibody herein, as described, for example, in U.S. Pat. No. 4,831,175. Antibodies, antigen-binding fragments, and fusion proteins can be directly labeled with radioactive iodine, indium, yttrium, or other radioactive particles known in the art, as described, for example, in U.S. Pat. No. 5,595,721. Treatment can also consist of a combination of treatments with conjugated or unconjugated antibodies and / or antigen-binding fragments administered simultaneously or sequentially, as described, for example, in WO 00 / 52031 and WO 00 / 52473.

[0199] The antibodies and antigen-binding fragments described herein may be attached to a solid support. Furthermore, the antibodies or functional antibody fragments thereof of the present disclosure can be chemically modified by covalently conjugating them to a polymer, for example, to increase their circulating half-life. Examples of polymers and methods for attaching them to peptides are provided in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. In some embodiments, the polymer may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula R(O-CH2-CH2). nThe PEG has an OR, where R can be hydrogen or a protecting group such as an alkyl or alkanol group. In certain embodiments, the protecting group can have 1 to 8 carbons. For example, the protecting group can be methyl. The symbol n is a positive integer. In one embodiment, n is 1 to 1,000. In another embodiment, n is 2 to 500. In some embodiments, the PEG has an average molecular weight selected from 1,000 to 40,000, 2,000 to 20,000, and 3,000 to 12,000. Furthermore, the PEG can have at least one hydroxy group, e.g., a terminal hydroxy group. For example, this is a terminal hydroxy group that is activated to react with a free amino group on the inhibitor. However, it is understood that the type and amount of reactive groups can be varied to achieve the covalently conjugated PEG / antibody herein.

[0200] In the context of the antibodies and antigen-binding fragments described herein, water-soluble polyoxyethylated polyols may also be utilized. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and others. In one embodiment, POG is used. While not wishing to be bound by any theory, the glycerol backbone of polyoxyethylated glycerol is the same backbone as mono-, di-, and triglycerides naturally occurring in, for example, animals and humans, so this branching would not necessarily be considered foreign in vivo. POG may have a molecular weight in the same range as PEG. Another drug delivery system that can be used to increase circulatory half-life is liposomes. Methods for preparing liposomal delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described, for example, in Poznansky et al. (1980) and Poznansky (1984), cited above.

[0201] Typically, the antibody or antigen-binding fragment is present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is made up of other polypeptides, usually less than 60%, more usually less than 50%.

[0202] The antibodies or antigen-binding fragments of the present disclosure may be immunogenic in a non-human (or xenogeneic) host, such as a mouse. In particular, the antibody, antigen-binding fragment, or fusion protein may have an idiotope that is immunogenic in a non-human host but not in a human host. In particular, such molecules of the present disclosure for human use include molecules that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained by humanization or from xenogeneic mice.

[0203] Production of antibodies, antigen-binding fragments, and fusion proteins Antibodies and antigen-binding fragments according to the present disclosure can be produced by any method known in the art. For example, the general methodology for producing monoclonal antibodies using hybridoma technology is known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In one embodiment, the alternative EBV immortalization method described in WO2004 / 076677 is used.

[0204] In one embodiment, antibodies are produced using the methods described in WO2004 / 076677. In such methods, antibody-producing B cells are transformed with EBV and a polyclonal B cell activator. To further enhance efficiency, additional stimulators of cell proliferation and differentiation may be added during the transformation step, as appropriate. These stimulators may be cytokines such as IL-2 and IL-15. In one embodiment, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, although its use is not essential. Immortalized B cells produced using these methods can be cultured and antibodies isolated therefrom using methods known in the art.

[0205] Another method for producing antibodies is described in WO2010 / 046775. In such a method, plasma cells are cultured in limited numbers or as single plasma cells in microwell culture plates. Antibodies can be isolated from the plasma cell culture. Furthermore, RNA can be extracted from the plasma cell culture and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, and cloned into an expression vector, which can then be transfected into HEK293T cells or other host cells. Cloning the nucleic acid into the expression vector, transfecting the host cells, culturing the transfected host cells, and isolating the produced antibody can be performed using any method known to those skilled in the art.

[0206] The antibodies may be further purified, if necessary, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Methods for the purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are known in the art.

[0207] Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies, antibody fragments, or fusion proteins of the present invention. The desired DNA sequence may be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as needed.

[0208] Any suitable host cell / vector system may be used for expression of DNA sequences encoding the antibody or fusion protein molecules of the present disclosure or fragments thereof. Bacterial, e.g., Escherichia coli and other microbial systems may be used in part for the expression of antibody fragments, such as Fab and F(ab')2 fragments, and particularly Fv fragments and single-chain antibody fragments, e.g., single-chain Fv. For the production of larger antibody molecules, including intact antibody molecules, eukaryotic, e.g., mammalian, host cell expression systems may be used. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma, or hybridoma cells.

[0209] The present disclosure also provides a process for producing an antibody or antigen-binding fragment according to the present disclosure, comprising culturing a host cell containing a vector encoding a nucleic acid of the present disclosure under conditions suitable for expression of protein from DNA encoding the antibody molecule of the present disclosure, and isolating the antibody molecule.

[0210] An antibody molecule or antibody fragment may contain only heavy or light chain polypeptides, in which case only sequences encoding the heavy or light chain polypeptides need be used to transfect the host cell. To produce a product containing both heavy and light chains, a cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both light and heavy chain polypeptides can be used.

[0211] Alternatively, antibodies and antigen-binding fragments according to the present disclosure may be produced by (i) expressing a nucleic acid sequence according to the present disclosure in a host cell, for example, by using a vector according to the present disclosure, and (ii) isolating the desired expressed product. Further, the method may include (iii) purifying the isolated antibody or antigen-binding fragment. Transformed B cells and cultured plasma cells may be screened for cells that produce antibodies and antigen-binding fragments with the desired specificity or function.

[0212] Screening may be performed by any immunoassay, such as ELISA, by staining of tissues or cells (including transfected cells), by neutralization assays, or by one of several other methods known in the art for identifying the desired specificity or function. The assay may select based on simple recognition of one or more antigens, or may additionally select based on the desired function, e.g., selecting neutralizing antibodies over merely antigen-binding antibodies, and selecting antibodies that can alter characteristics of target cells, such as their signaling cascades, their shape, their growth rate, their ability to affect other cells, their response to influence by other cells or other agents or changes in conditions, their differentiation state, etc.

[0213] Individual transformed B cell clones can then be produced from the culture of positive transformed B cells. The cloning step to separate individual clones from the mixture of positive cells may be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or other methods known in the art.

[0214] Nucleic acid from the cultured plasma cells can be isolated, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

[0215] The immortalized B cell clones or transfected host cells described herein can be used in a variety of ways for study, e.g., as a source of monoclonal antibodies and as a source of nucleic acid (DNA or mRNA) encoding the monoclonal antibody of interest.

[0216] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising an antibody that neutralizes hepatitis B virus and a pharmaceutically acceptable aqueous medium. A medium is typically understood to be a material suitable for storing, transporting, formulating, and / or administering a pharmaceutically active compound, particularly a compound such as an antibody according to the present disclosure. For example, the medium may be a physiologically acceptable liquid suitable for storing, transporting, and / or administering a pharmaceutically active compound, particularly an antibody according to the present disclosure.

[0217] The pharmaceutical compositions described herein are prepared for injection or infusion into a patient. In some embodiments, the compositions may be prepared for intravenous ("IV" or "iv"), intraarterial, or intraventricular infusion. In other embodiments, the compositions may be prepared for intravenous, intraarterial, intraventricular, intramedullary, intraperitoneal, intrathecal, or intraventricular injection. In certain embodiments, the compositions are prepared for subcutaneous ("SC" or "sc") injection. In certain embodiments, the compositions described herein are pharmaceutically acceptable sterile aqueous solutions that exhibit suitable pH, isotonicity, and stability for administration to a human subject. Suitable aqueous vehicles for formulating the compositions described herein include water (e.g., sterile water, USP Water for Injection) and isotonic vehicles such as sodium chloride for injection, Ringer's injection, lactated Ringer's injection, etc.

[0218] The pharmaceutical compositions herein comprise an antibody selected from the HBV-neutralizing antibodies herein. For example, in some embodiments, the pharmaceutical compositions herein comprise an (isolated) antibody comprising: (i) a heavy chain variable region (VH) comprising at least 90% identity to the amino acid sequence of SEQ ID NO: 41; and (ii) a light chain variable region (VL) comprising at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 59, 89, or 90, with the proviso that the amino acid at position 40 of the VL according to the IMGT numbering is not a cysteine, and wherein the antibody or antigen-binding fragment thereof binds to the antigenic loop region of HBsAg to neutralize infection by hepatitis B virus and hepatitis delta virus.

[0219] In certain embodiments, (i) the VH comprises at least 95% identity to an amino acid sequence according to SEQ ID NO: 41, and / or (ii) the VL comprises at least 95% identity to an amino acid sequence according to any one of SEQ ID NOs: 59, 89, or 90.

[0220] In certain embodiments, the amino acid at position 40 of the VL is alanine. In certain embodiments, the amino acid at position 40 of the VL is serine. In certain embodiments, the amino acid at position 40 of the VL is glycine.

[0221] In certain embodiments, the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences according to SEQ ID NOs: (i) 34-36, 37, 38, and 40, respectively; (ii) 34, 66, 36, 37, 38, and 40, respectively; (iii) 34-36, 37, 39, and 40, respectively; (iv) 34, 66, 36, 37, 39, and 40, respectively; (v) 34-36, 37, 38, and 58, respectively; (vi) 34, 66, 36, 37, 38, and 58, respectively; (vii) 34-36, 37, 39, and 58, respectively; or (viii) 34, 66, 36, 37, 39, and 58, respectively.

[0222] In certain embodiments, the VL comprises or consists of an amino acid sequence according to SEQ ID NO:89.

[0223] In certain embodiments, the VL comprises or consists of an amino acid sequence according to SEQ ID NO:90.

[0224] In certain embodiments, the VH comprises or consists of an amino acid sequence according to SEQ ID NO:41.

[0225] In certain embodiments, the VH comprises or consists of an amino acid sequence according to SEQ ID NO:41, and the VL comprises or consists of an amino acid sequence according to SEQ ID NO:89.

[0226] In certain embodiments, the VH comprises or consists of an amino acid sequence according to SEQ ID NO:41 and the VL comprises or consists of an amino acid sequence according to SEQ ID NO:90.

[0227] In certain embodiments, the antibody comprises a human antibody and / or a monoclonal antibody.

[0228] In certain embodiments, the antibody is a multispecific antibody, hi certain embodiments, the antibody is a bispecific antibody.

[0229] In certain embodiments, the antibody comprises an Fc portion.

[0230] In certain embodiments, the Fc portion comprises a mutation that enhances binding to (e.g., human) FcRn compared to a reference Fc portion that does not contain the mutation.

[0231] In certain embodiments, the Fc portion comprises a mutation that enhances binding to a (e.g., human) FcγR (e.g., FcγRIIa, FcγRIIIa, or both) relative to a reference Fc portion that does not contain the mutation.

[0232] In certain embodiments, the Fc portion is of or is derived from an IgG isotype.

[0233] In certain embodiments, mutations that enhance binding to FcRn include M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, E380A, or any combination thereof.

[0234] In certain embodiments, mutations that enhance binding to FcRn include (i) M428L / N434S, (ii) M252Y / S254T / T256E, (iii) T250Q / M428L, (iv) P257I / Q311I, (v) P257I / N434H, (vi) D376V / N434H, (vii) T307A / E380A / N434A, or (viii) any combination of (i)-(vii).

[0235] In certain embodiments, the mutation that enhances binding to FcRn comprises M428L / N434S.

[0236] In certain embodiments, the mutation that enhances binding to an FcγR comprises S239D, I332E, A330L, G236A, or any combination thereof.

[0237] In certain embodiments, the mutations that enhance binding to FcγR include (i) S239D / I332E, (ii) S239D / A330L / I332E, (iii) G236A / S239D / I332E, or (iv) G236A / A330L / I332E.

[0238] In certain embodiments, the mutations that enhance binding to FcγR comprise or consist of G236A / A330L / I332E. In some embodiments, the mutations that enhance binding to FcγR do not comprise S239D. In some embodiments, the Fc moiety comprises a native Ser (S) at position 239.

[0239] In certain embodiments, the Fc portion comprises the following amino acid substitution mutations: M428L, N434S, G236A, A330L, and I332E. In certain further embodiments, the Fc portion does not comprise any additional mutations.

[0240] In certain embodiments, the antibody comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO:91.

[0241] In certain embodiments, the antibody comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO:92.

[0242] In certain embodiments, the antibody comprises a light chain (LC) amino acid sequence according to SEQ ID NO:93.

[0243] In certain embodiments, the antibody comprises a light chain (LC) amino acid sequence according to SEQ ID NO:94.

[0244] In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:91 and an LC amino acid sequence according to SEQ ID NO:93.

[0245] In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:92 and an LC amino acid sequence according to SEQ ID NO:94.

[0246] In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:91 and an LC amino acid sequence according to SEQ ID NO:94.

[0247] In certain embodiments, the antibody comprises an HC amino acid sequence according to SEQ ID NO:92 and an LC amino acid sequence according to SEQ ID NO:93.

[0248] In some embodiments, a pharmaceutical composition herein comprises an (isolated) antibody comprising (i) a heavy chain (HC) comprising an amino acid sequence according to SEQ ID NO: 91, and (ii) a light chain (LC) comprising an amino acid sequence according to SEQ ID NO: 93, wherein the antibody binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus.

[0249] In some embodiments, the antibody binds to HBsAg of a genotype selected from HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

[0250] In some embodiments, the antibody or pharmaceutical composition reduces the serum concentration of HBV DNA in a mammal with HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces the serum concentration of HBsAg in a mammal with HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces the serum concentration of HBeAg in a mammal with HBV infection. In some embodiments, the antibody or pharmaceutical composition reduces the serum concentration of HBcrAg in a mammal with HBV infection.

[0251] In some embodiments, the pharmaceutical compositions herein comprise a heavy chain variable region (V) comprising a CDRH1 amino acid sequence according to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, and a CDRH3 amino acid sequence according to SEQ ID NO: 36. H ), and a light chain variable region (V) comprising a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 amino acid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequence according to SEQ ID NO: 58 or 40. L ), as well as antibodies comprising an Fc portion, wherein the Fc portion comprises G236A / A330L / I332E.

[0252] In certain embodiments, the Fc portion does not comprise S239D. In certain embodiments, the Fc portion comprises Ser (S) at position 239.

[0253] In certain embodiments, the Fc portion further comprises M428L / N434S.

[0254] In certain embodiments, V H comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 41 or 67, and V Lcomprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, and 111-120.

[0255] In other embodiments, the CDRH1 heavy chain variable region (V) comprises: (i) a CDRH1 amino acid sequence according to SEQ ID NO: 97, a CDRH2 amino acid sequence according to SEQ ID NO: 98, and a CDRH3 amino acid sequence according to SEQ ID NO: 99; H ), (ii) a light chain variable region (V) comprising a CDRL1 amino acid sequence according to SEQ ID NO: 100, a CDRL2 amino acid sequence according to SEQ ID NO: 100, and a CDRL3 amino acid sequence according to SEQ ID NO: 102 L ), and (iii) a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof comprising an Fc portion, wherein the Fc portion comprises G236A / A330L / I332E.

[0256] In certain such embodiments of the antibody of the pharmaceutical composition, V H comprises or consists of an amino acid sequence according to SEQ ID NO: 95, and V L comprises or consists of the amino acid sequence according to SEQ ID NO: 96.

[0257] In certain embodiments, the Fc portion does not comprise S239D, hi certain embodiments, the Fc portion further comprises M428L / N434S.

[0258] In some embodiments, the antibody of the pharmaceutical composition has enhanced binding to human FcγRIIA, human FcγRIIIA, or both relative to a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E (wherein human FcγRIIA can be H131 or R131 and / or human FcγRIIIA can be F158 or V158), has reduced binding to human FcγRIIB relative to a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E, and does not bind to human FcγRIIB. the polypeptide has reduced binding to human C1q compared to the polypeptide, does not bind human C1q, activates FcγRIIA, human FcγRIIIA, or both to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E (wherein human FcγRIIA can be H131 or R131, and / or human FcγRIIIA can be F158 or V158), does not activate human FcγRIIB, and / or activates human natural killer (NK) cells in the presence of HBsAg to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E.

[0259] The pharmaceutical composition comprises sufficient antibody material to facilitate administration of a therapeutically effective amount of the antibody to a patient. In some embodiments, the antibody is present at a concentration selected from 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, and 200 mg / mL. In other embodiments, the antibody is present in the composition at a concentration selected from greater than 50 mg / mL, greater than 75 mg / mL, greater than 100 mg / mL, greater than 125 mg / mL, greater than 150 mg / mL, greater than 175 mg / mL, greater than 200 mg / mL, greater than 225 mg / mL, and greater than 250 mg / mL. In other embodiments, the composition comprises the antibody at a concentration selected from the range of 50 mg / mL to 200 mg / mL, the range of 75 mg / mL to 225 mg / mL, and the range of 100 mg / mL to 200 mg / mL. In some embodiments, the composition comprises the antibody at a concentration ranging from 125 mg / ml to 150 mg / ml, hi yet other embodiments, the composition comprises the antibody at a concentration of 150 mg / mL.

[0260] Compositions herein may include one or more of a buffer, a surfactant or triblock copolymer, a salt (e.g., sodium chloride), and a stabilizer (a sugar alcohol, a disaccharide, or a polysaccharide stabilizer, and / or a stabilizing amino acid (e.g., arginine and / or glycine)). Additionally, if needed or desired, the compositions described herein may be formulated to further include one or more antioxidants (e.g., ascorbic acid, methionine, ethylenediaminetetraacetic acid (EDTA)).

[0261] The pharmaceutical compositions of the present disclosure maintain antibody viability and exhibit and maintain a pH suitable for injection or infusion. The compositions described herein generally have a pH in the range of about 5.5 to about 8.5. In certain embodiments, the pharmaceutical composition has a pH in the range of about 5.5 to about 6.5, e.g., 5.5 to 6.5. In certain embodiments, the pharmaceutical composition has a pH in the range of 5.8 to 6.2, e.g., about 6.0. In certain embodiments, the pH may be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the composition has a pH in the range of 6 to 8, e.g., about 7. In certain such embodiments, the pH may be about 6, e.g., 6.

[0262] The compositions may include a buffer to achieve and maintain a desired pH. Suitable buffers for use in the compositions described herein include, for example, acetate, citrate, histidine, succinate, phosphate, and hydroxymethylaminomethane (Tris) buffers. In certain embodiments, the compositions include a buffer selected from a histidine buffer and a phosphate buffer. In certain embodiments, the compositions have a pH of 6 and include a histidine buffer. In such embodiments, histidine may be included in the compositions at a concentration ranging from 10 mM to 40 mM (e.g., 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM). For example, in certain embodiments, the compositions herein have a pH of 6 and include histidine at a concentration selected from 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 40 mM.

[0263] The pharmaceutical compositions described herein may include a surfactant or triblock copolymer. Surfactants, sometimes referred to as "detergents," can serve one or more functions. For example, in aqueous antibody solutions, surfactants preserve antibody functionality, aid in dissolving the antibody or other excipients, and / or act to control microbial growth. Surfactants used in the compositions described herein include, for example, polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20). Additionally or alternatively, triblock copolymers such as poloxamer 188 may also be used. In some embodiments, the composition includes a surfactant at a concentration ranging from 0.01% to 0.05% (w / v). In such embodiments, the surfactant may be selected from polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. In certain embodiments, the pharmaceutical compositions herein include polysorbate 80 (Tween 80) at a concentration ranging from 0.01% to 0.05% (w / v). In other embodiments, the pharmaceutical compositions herein comprise polysorbate 80 (Tween 80) at a concentration of 0.02% (w / v).

[0264] When a composition according to the present disclosure includes a sugar alcohol, disaccharide, or polysaccharide stabilizer, the stabilizer may be selected from, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In certain embodiments, the stabilizer is a disaccharide. In certain embodiments, the pharmaceutical composition includes a disaccharide at a concentration selected from 4.0% to 10% (w / v). In certain such embodiments, the disaccharide is sucrose. In other embodiments, the pharmaceutical composition may be at 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11.0%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12.0%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.0%, 13.1%, 13.1%, 13 , 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, or 10.0% (w / v), or within a range bounded by and including any two of these values. In yet other embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7%, e.g., 7% (w / v).

[0265] In some embodiments, the composition is adapted for administration to a mammal, such as a human. In such embodiments, the composition is specially formulated to be sterile and pyrogen-free. Furthermore, the composition may be isotonic with respect to humans.

[0266] The compositions described herein may be prepared for direct administration to a subject (i.e., without a reconstitution or mixing step), or may be prepared as a lyophilized material that is reconstituted in an aqueous medium before injection or infusion into a patient. For direct administration to a subject, a pharmaceutical composition according to the present disclosure may be provided in, for example, a prefilled syringe or a vial, such as a glass vial. In some embodiments, the pharmaceutical composition of the present disclosure is supplied in an airtightly sealed container. In some embodiments, the composition may be in the form of a kit designed to reconstitute the combined composition immediately before administration to a subject. For example, a lyophilized antibody may be provided in the form of a kit together with sterile water or a sterile buffer.

[0267] Administration of a pharmaceutical composition according to the present disclosure in the methods and uses according to the present disclosure can be performed alone or in combination with a co-agent (also referred to herein as an "additional active ingredient"), which is useful for preventing and / or treating hepatitis B virus infection.

[0268] The present disclosure encompasses the administration of a pharmaceutical composition according to the present disclosure to a subject prior to, concurrently with, or after a co-drug or another therapeutic regimen useful for treating and / or preventing hepatitis B virus infection. The pharmaceutical composition administered in combination with the co-drug can be administered in the same or different compositions and by the same or different routes of administration. As used herein, the terms "combination therapy," "combined administration," "administered in combination," and the like refer to the combined action of drugs (administered "in combination"). For this purpose, the combined drugs are typically present at the site of action at the same time and / or within an overlapping time window. It is also possible that the effects due to one of the drugs are still continuing (even if the drug itself is no longer present at detectable levels) while the other drug is administered, thereby allowing the effects of both drugs to interact. However, a drug that was administered long before another drug (e.g., 1, 2, 3 months or more, or even a year) and is no longer present at detectable levels (or its effect is no longer ongoing) by the time the other drug is administered is typically not considered to have been administered "in combination."

[0269] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a PD-1 inhibitor, for example, a PD-1-specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any combination thereof.

[0270] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a PD-L1-specific antibody or binding fragment thereof, such as BMS-936559, duvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.

[0271] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0272] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of CTLA4. In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a CTLA4-specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

[0273] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a B7-H3-specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. The B7-H3 antibody binding fragment may be an scFv or fusion protein thereof, for example, as described in Dangaj et al., Cancer Res. 73:4820, 2013, and U.S. Patent No. 9,574,000 and PCT Patent Publications WO / 201640724A1 and WO2013 / 025779A1.

[0274] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of CD244.

[0275] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication WO2010 / 084158.

[0276] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of TIM3.

[0277] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of Gal9.

[0278] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0279] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of A2aR.

[0280] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of KIR, such as lirilumab (BMS-986015).

[0281] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0282] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an IDO inhibitor such as levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, or any combination thereof.

[0283] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0284] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0285] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of TIGIT, such as COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as COM701 (Compugen), or both.

[0286] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication WO2017 / 021526.

[0287] In certain embodiments, the compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0288] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0289] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with agents that increase the activity of (i.e., are agonists of) stimulatory immune checkpoint molecules. For example, compositions of the disclosure can be used with or without a CD137 (4-1BB) agonist (e.g., urelumab), a CD134 (OX-40) agonist (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (e.g., CDX-1127), a CD28 agonist (e.g., TGN1412, CD80, or CD86), a CD40 agonist (e.g., CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (e.g., IL-2), an agonist of GITR (e.g., the humanized monoclonal antibody described in PCT Patent Publication WO 2016 / 054638), an agonist of ICOS (CD278) (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof. In any of the embodiments disclosed herein, the method may comprise administering a pharmaceutical composition of the present disclosure in combination with one or more agonists of stimulatory immune checkpoint molecules, including any of the above, alone or in any combination.

[0290] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), an interferon (e.g., IFNα, IFNβ, or both), or any combination thereof. In some embodiments, the NRTI comprises one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil.

[0291] Medical Treatment and Use In a further aspect, the present disclosure provides for the use of a pharmaceutical composition according to the present disclosure in the treatment of infection by hepatitis B virus. In certain embodiments, the present disclosure provides a method for treating infection by hepatitis B virus, comprising a method comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure to a subject in need thereof.

[0292] In a therapeutic setting, the subject is infected with, diagnosed with, and / or exhibits symptoms of Hepatitis B virus infection. Notably, the terms "treatment" and "treatment / therapeutic" of Hepatitis B virus include (complete) cure as well as attenuation / reduction of Hepatitis B virus infection and / or associated symptoms (e.g., attenuation / reduction of the severity of the infection and / or symptoms, the number of symptoms, the duration of the infection and / or symptoms, or any combination thereof).

[0293] In certain embodiments, the subject is an adult. In certain embodiments, the subject is between 18 and 65 years old. In certain embodiments, the subject weighs between 40 kg and 125 kg.

[0294] In certain embodiments, the subject to whom the pharmaceutical composition of the present disclosure is administered has chronic HBV infection, as defined, for example, by positive serology for HBsAg, HBV DNA, and / or HBeAg on two occasions at least six months apart.

[0295] In certain embodiments, the subject of the pharmaceutical composition of the present disclosure is not having cirrhosis.The absence of cirrhosis is determined by fibroscan assessment (for example, within 6 months before the administration of a single dose of pharmaceutical composition) or liver biopsy (for example, within 12 months before the administration of a single dose of pharmaceutical composition), and preferably the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis.

[0296] In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), optionally within 120 days, and optionally within 60 days, prior to receiving a single dose of the pharmaceutical composition, in other words, the subject has already received an NRTI within 120 days, 60 days, etc., prior to receiving the pharmaceutical composition.

[0297] In certain embodiments, the NRTIs include one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil.

[0298] In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure has a serum HBV DNA concentration of less than 100 IU / mL (e.g., 99, 98, 97, 96, 95, 90, 80, 70, 60, etc.) for a period not exceeding 28 days prior to receiving the single dose.

[0299] In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure has a serum HBsAg concentration of less than 1,000 IU / mL before the single dose is administered.

[0300] In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure has a serum HBs surface antigen (HBsAg) concentration of 1,000 IU / mL or greater for a period not exceeding 28 days prior to receiving the single dose. HBsAg concentration can be determined, for example, using the Abbott ARCHITECT assay.

[0301] In certain embodiments, the subject receiving the pharmaceutical composition of the present disclosure was HBe antigen (HBeAg) negative for no more than 28 days prior to receiving the single dose.

[0302] In certain embodiments, the subject has tested negative for anti-HBs antibodies for no more than 28 days prior to administration of the single dose.

[0303] In certain embodiments, the subject to be administered the pharmaceutical composition of the present disclosure: (i) no fibrosis and / or no cirrhosis; and / or (ii) have a (serum) alanine aminotransferase (ALT) lower than twice the upper limit of normal (ULN);

[0304] In certain embodiments, the methods comprise administering a single dose of a pharmaceutical composition of the present disclosure.

[0305] In some embodiments, a single dose of the pharmaceutical composition comprises an antibody in the range of 2 to 18 mg / kg (of the subject's body weight), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 mg / kg.

[0306] In certain embodiments, a single dose of the pharmaceutical composition comprises up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of antibody. In certain embodiments, a single dose of the pharmaceutical composition comprises about 10, about 25, about 50, about 75, about 90, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, or about 3000 mg of antibody.

[0307] In certain embodiments, a single dose of the pharmaceutical composition comprises about 75 mg of antibody. In other embodiments, a single dose of the pharmaceutical composition comprises about 90 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition comprises up to 300 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition comprises up to 900 mg of antibody. In yet other embodiments, a single dose of the pharmaceutical composition comprises up to 3,000 mg of antibody.

[0308] In certain embodiments, a single dose of the pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, e.g., 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

[0309] In any of the methods for treating infection with hepatitis B virus described herein, the pharmaceutical composition can be administered by injection or infusion. When administered by infusion, the pharmaceutical composition may be administered, for example, by intravenous, intraarterial, or intraventricular infusion. When administered by injection, the pharmaceutical composition may be administered, for example, by intravenous, intraarterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular, or subcutaneous injection. In certain embodiments of the methods described herein, the pharmaceutical composition is administered by subcutaneous ("SC") injection or intravenous ("IV") injection.

[0310] Even if multiple injections or infusions are required to administer a defined dose, the dose is referred to as a "single dose" and the administration is considered a "single administration." Generally, when multiple injections or infusions are required to administer a defined single dose, the multiple injections or infusions are administered over a period of about 5 minutes or less, about 15 minutes or less, about 30 minutes or less, about 1 hour or less, about 2 hours or less, about 4 hours or less, about 6 hours or less, about 1 day or less, about 1 week or less, or about 1 month or less.

[0311] In certain embodiments, at about day 56 after administration of the single dose, the subject has a less than two-fold decrease in serum HBsAg (e.g., serum HBsAg concentration determined using an Abbott ARCHITECT assay) compared to the subject's serum HBsAg from day 0 to day 28 prior to administration of the single dose.

[0312] In certain embodiments, after administration of a single dose of the pharmaceutical composition (e.g., 56 days after administration of the single dose), the subject (i) has reduced or less severe intrahepatic spread of HBV compared to a reference subject (e.g., a subject of similar severity of HBV infection as the subject receiving the pharmaceutical composition and of the same sex, age, weight, and / or general health condition) who received a placebo or no treatment for HBV over the same period of time, and / or (ii) comprises an adaptive immune response to HBV, including, for example, an HBV-specific T cell response.

[0313] The present disclosure also includes the following exemplary embodiments.

[0314] Embodiment 1. (i) A heavy chain variable region (V) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO: 41. H ), and (ii) a light chain variable region (V) comprising at least 90% identity to an amino acid sequence according to any one of SEQ ID NOs: 59, 89, or 90. L ), but V according to IMGT numbering L An isolated antibody or antigen-binding fragment thereof, wherein the amino acid at position 40 of is not cysteine ​​and binds to the antigenic loop region of HBsAg to neutralize infection by hepatitis B virus and hepatitis delta virus. Embodiment 2.(i)V H comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 41, and / or (ii) V L 2. The antibody or antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment has at least 95% identity to an amino acid sequence according to any one of SEQ ID NOs: 59, 89, or 90. Embodiment 3.V L 3. The antibody or antigen-binding fragment of embodiment 1 or 2, wherein the amino acid at position 40 of Embodiment 4.V L 3. The antibody or antigen-binding fragment of embodiment 1 or 2, wherein the amino acid at position 40 of is serine. Embodiment 5.V L 3. The antibody or antigen-binding fragment of embodiment 1 or 2, wherein the amino acid at position 40 of is glycine. Embodiment 6. The antibody or antigen-binding fragment of any one of embodiments 1 to 5, comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences according to SEQ ID NOs: (i) 34 to 36, 37, 38, and 40, respectively; (ii) 34, 66, 36, 37, 38, and 40, respectively; (iii) 34 to 36, 37, 39, and 40, respectively; (iv) 34, 66, 36, 37, 39, and 40, respectively; (v) 34 to 36, 37, 38, and 58, respectively; (vi) 34, 66, 36, 37, 38, and 58, respectively; (vii) 34 to 36, 37, 39, and 58, respectively; or (viii) 34, 66, 36, 37, 39, and 58, respectively. Embodiment 7.V L 7. The antibody or antigen-binding fragment of any one of embodiments 1 to 3 or 6, comprising or consisting of an amino acid sequence according to SEQ ID NO: 89. Embodiment 8.V L 7. The antibody or antigen-binding fragment of any one of embodiments 1, 2, 4, or 6, wherein said antibody or antigen-binding fragment comprises or consists of an amino acid sequence according to SEQ ID NO: 90. Embodiment 9.V H 9. The isolated antibody of any one of embodiments 1 to 8, wherein said antibody comprises or consists of an amino acid sequence according to SEQ ID NO: 41. Embodiment 10.V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41, and V L 10. The isolated antibody of any one of embodiments 1 to 3, 6, 7, or 9, wherein said antibody comprises or consists of an amino acid sequence according to SEQ ID NO: 89. Embodiment 11.V H comprises or consists of the amino acid sequence according to SEQ ID NO: 41, and V L 10. The isolated antibody of any one of embodiments 1, 2, 4, 6, 8, or 9, wherein said antibody comprises or consists of an amino acid sequence according to SEQ ID NO: 90. Embodiment 12. (i) A heavy chain variable region (V) comprising at least 90% identity to an amino acid sequence according to SEQ ID NO: 95. H ) and (ii) a light chain variable region (V) comprising at least 90% identity to the amino acid sequence according to SEQ ID NO: 96; L ), An isolated antibody or antigen-binding fragment thereof that binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus or hepatitis delta virus. Embodiment 13.(i)V H comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 95, and / or (ii)V L comprises at least 95% identity with the amino acid sequence according to SEQ ID NO: 96, 13. The antibody or antigen-binding fragment of embodiment 12. Embodiment 14. The antibody or antigen-binding fragment of embodiment 12 or 13, comprising the sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 according to SEQ ID NOs: 97-102, respectively. Embodiment 15. The antibody or antigen-binding fragment of any one of embodiments 1 to 14, comprising a human antibody, a monoclonal antibody, a purified antibody, a single-chain antibody, a Fab, a Fab', a F(ab')2, an Fv, or an scFv. Embodiment 16. The antibody or antigen-binding fragment of any one of embodiments 1 to 15, which is a multispecific antibody or antigen-binding fragment. Embodiment 17. The antibody or antigen-binding fragment of embodiment 16, which is a bispecific antibody or antigen-binding fragment. Embodiment 18. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17, comprising an Fc portion. Embodiment 19. The antibody or antigen-binding fragment of embodiment 18, wherein the Fc portion comprises a mutation that enhances binding to (e.g., human) FcRn compared to a reference Fc portion that does not contain the mutation. Embodiment 20. The antibody or antigen-binding fragment of embodiment 18 or 19, wherein the Fc portion comprises a mutation that enhances binding to a (e.g., human) FcγR compared to a reference Fc portion that does not contain the mutation. Embodiment 21. The antibody or antigen-binding fragment of any one of embodiments 18 to 20, wherein the Fc portion is of or is derived from an IgG isotype. Embodiment 22. The antibody or antigen-binding fragment of embodiment 21, wherein the mutations that enhance binding to FcRn comprise M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, E380A, or any combination thereof. Embodiment 23. The antibody or antigen-binding fragment of embodiment 21 or 22, wherein the mutations that enhance binding to FcRn include: (i) M428L / N434S, (ii) M252Y / S254T / T256E, (iii) T250Q / M428L, (iv) P257I / Q311I, (v) P257I / N434H, (vi) D376V / N434H, (vii) T307A / E380A / N434A, or (viii) any combination of (i) to (vii). Embodiment 24. The antibody or antigen-binding fragment of embodiment 23, wherein the mutations that enhance binding to FcRn comprise M428L / N434S. Embodiment 25. The antibody or antigen-binding fragment of any one of embodiments 20 to 24, wherein the mutations that enhance binding to FcγR comprise S239D, I332E, A330L, G236A, or any combination thereof. Embodiment 26. The antibody or antigen-binding fragment of embodiment 25, wherein the mutations that enhance binding to FcγR comprise: (i) S239D / I332E, (ii) S239D / A330L / I332E, (iii) G236A / S239D / I332E, or (iv) G236A / A330L / I332E. Embodiment 27. The antibody or antigen-binding fragment of embodiment 25 or 26, wherein the mutations that enhance binding to FcγR comprise or consist of G236A / A330L / I332E. Embodiment 28. The antibody or antigen-binding fragment of any one of embodiments 18 to 27, wherein the Fc portion comprises the following amino acid substitution mutations: M428L, N434S, G236A, A330L, and I332E. Embodiment 29. (i) A heavy chain variable region (V) comprising an amino acid sequence according to SEQ ID NO: 41. H ), and (ii) a light chain variable region (V) comprising the amino acid sequence according to SEQ ID NO: 89 L ), An isolated antibody or antigen-binding fragment thereof that binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus. Embodiment 30. The isolated antibody or antigen-binding fragment of embodiment 29, further comprising an Fc portion. Embodiment 31. The isolated antibody or antigen-binding fragment of embodiment 30, wherein the Fc portion is derived from an IgG isotype and comprises the following substitution mutations: M428L and N434S. Embodiment 32. The isolated antibody or antigen-binding fragment of embodiment 30 or 31, wherein the Fc portion is derived from an IgG isotype and comprises the following substitution mutations: G236A, A330L, and I332E. Embodiment 33. The isolated antibody or antigen-binding fragment of embodiment 32, wherein the Fc portion comprises the following substitution mutations: M428L, N434S, G236A, A330L, and I332E. Embodiment 34. The antibody or antigen-binding fragment of any one of embodiments 1 to 10, 15 to 33, comprising a heavy chain (HC) amino acid sequence according to SEQ ID NO: 91. Embodiment 35. The antibody or antigen-binding fragment of any one of embodiments 1 to 10 and 15 to 32, comprising a heavy chain (HC) amino acid sequence according to SEQ ID NO: 92. Embodiment 36. The antibody or antigen-binding fragment of any one of embodiments 1 to 3, 6, 7, 9, 10, and 15 to 35, comprising a light chain (LC) amino acid sequence according to SEQ ID NO: 93. Embodiment 37. The antibody or antigen-binding fragment of any one of embodiments 1, 2, 4, 6, 8, 11, and 15 to 28, comprising a light chain (LC) amino acid sequence according to SEQ ID NO: 94. Embodiment 38. The antibody or antigen-binding fragment of embodiment 34 or 36, comprising an HC amino acid sequence according to SEQ ID NO: 91 and an LC amino acid sequence according to SEQ ID NO: 93. Embodiment 39. The antibody or antigen-binding fragment of embodiment 35 or 37, comprising an HC amino acid sequence according to SEQ ID NO: 92 and an LC amino acid sequence according to SEQ ID NO: 94. Embodiment 40. The antibody or antigen-binding fragment of embodiment 34 or 37, comprising an HC amino acid sequence according to SEQ ID NO: 91 and an LC amino acid sequence according to SEQ ID NO: 94. Embodiment 41. The antibody or antigen-binding fragment of embodiment 35 or 36, comprising an HC amino acid sequence according to SEQ ID NO: 92 and an LC amino acid sequence according to SEQ ID NO: 93. Embodiment 42. (i) a heavy chain (HC) comprising an amino acid sequence according to SEQ ID NO: 91, and (ii) a light chain (LC) comprising an amino acid sequence according to SEQ ID NO: 93; An isolated antibody or antigen-binding fragment thereof that binds to the antigenic loop region of HBsAg and neutralizes infection by hepatitis B virus and hepatitis delta virus. Embodiment 43. The antibody or antigen-binding fragment of any one of embodiments 1 to 42, which binds to HBsAg of a genotype selected from HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof. Embodiment 44. The antibody or antigen-binding fragment of any one of embodiments 1 to 43, which reduces the serum concentration of HBV DNA in a mammal having an HBV infection. Embodiment 45. The antibody or antigen-binding fragment of any one of embodiments 1 to 44, which reduces the serum concentration of HBsAg in a mammal having an HBV infection. Embodiment 46 The antibody or antigen-binding fragment of any one of embodiments 1 to 45, which reduces the serum concentration of HBeAg in a mammal having an HBV infection. Embodiment 47 The antibody or antigen-binding fragment of any one of embodiments 1 to 46, which reduces the serum concentration of HBcrAg in a mammal having an HBV infection. Embodiment 48. (i) an antibody or antigen-binding fragment according to any one of embodiments 1 to 47, and (ii) instructions for using the composition to prevent, treat, attenuate, and / or diagnose hepatitis B infection and / or hepatitis D infection; Includes a kit. Embodiment 49. (i) a polymerase inhibitor, which may include lamivudine, adefovir, entecavir, telbivudine, tenofovir, or any combination thereof; (ii) interferons, which may include IFN beta and / or IFN alpha; (iii) a checkpoint inhibitor, which may include an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, and / or an anti-CTLA4 antibody or antigen-binding fragment thereof; (iv) an agonist of a stimulatory immune checkpoint molecule; or (v) Any combination of (viii) to (xii) 49. The kit of embodiment 48, further comprising: Embodiment 50. The kit of embodiment 49, wherein the polymerase inhibitor comprises lamivudine. Embodiment 51. Use of an antibody or antigen-binding fragment of any one of embodiments 1 to 47 in the manufacture of a medicament for preventing, treating, attenuating, and / or diagnosing hepatitis B infection and / or hepatitis D infection in a subject. Embodiment 52. A method of treating, preventing, and / or attenuating hepatitis B and / or hepatitis D infection in a subject, comprising administering to the subject an effective amount of (i) an antibody or antigen-binding fragment of any one of embodiments 1 to 47. Embodiment 53. The method of embodiment 52, further comprising administering to the subject one or more of: a polymerase inhibitor which may include lamivudine, adefovir, entecavir, telbivudine, tenofovir, or any combination thereof; an interferon which may include IFN-beta and / or IFN-alpha; a checkpoint inhibitor which may include an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof, and / or an anti-CTLA4 antibody or antigen-binding fragment thereof; an agonist of a stimulatory immune checkpoint molecule; or any combination thereof. Embodiment 54. The method of embodiment 52 or 53, wherein the hepatitis B infection is a chronic hepatitis B infection. Embodiment 55 The method of any one of embodiments 52 to 54, wherein the subject has undergone a liver transplant. Embodiment 56 The method of any one of embodiments 52 to 55, wherein the subject has not been immunized against hepatitis B. Embodiment 57. The method of any one of embodiments 52 to 56, wherein the subject is a newborn. Embodiment 58 The method of any one of embodiments 52 to 57, wherein the subject is undergoing or has undergone hemodialysis. Embodiment 59. A heavy chain variable region (V) comprising a CDRH1 amino acid sequence according to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, and a CDRH3 amino acid sequence according to SEQ ID NO: 36. H ), and a light chain variable region (V) comprising a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 amino acid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequence according to SEQ ID NO: 58 or 40; L ), and an Fc portion, wherein the Fc portion comprises G236A / A330L / I332E. Embodiment 60. The antibody or antigen-binding fragment of embodiment 59, wherein the Fc portion does not comprise S239D. Embodiment 61. The antibody or antigen-binding fragment of embodiment 59 or 60, wherein the Fc portion further comprises M428L / N434S. Embodiment 62.V H comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 41 or 67, and V L 62. The antibody or antigen-binding fragment of any one of embodiments 59 to 61, comprising or consisting of an amino acid sequence according to any one of SEQ ID NOs: 42, 59, 65, 89, 90, and 111-120. Embodiment 63. (i) A heavy chain variable region (V) comprising a CDRH1 amino acid sequence according to SEQ ID NO: 97, a CDRH2 amino acid sequence according to SEQ ID NO: 98, and a CDRH3 amino acid sequence according to SEQ ID NO: 99. H ), (ii) a light chain variable region (V) comprising a CDRL1 amino acid sequence according to SEQ ID NO: 100, a CDRL2 amino acid sequence according to SEQ ID NO: 100, and a CDRL3 amino acid sequence according to SEQ ID NO: 102; L ), and (iii) comprises an Fc portion; An isolated antibody or antigen-binding fragment thereof, wherein the Fc portion comprises G236A / A330L / I332E. Embodiment 64. The antibody or antigen-binding fragment of embodiment 63, wherein the Fc portion does not comprise S239D. Embodiment 65. The antibody or antigen-binding fragment of embodiment 63 or 64, wherein the Fc portion further comprises M428L / N434S. Embodiment 66.V H comprises or consists of an amino acid sequence according to SEQ ID NO: 95, and V L 66. The antibody or antigen-binding fragment of any one of embodiments 63 to 65, comprising or consisting of an amino acid sequence according to SEQ ID NO: 96. Embodiment 67. (i) has enhanced binding to human FcγRIIA, human FcγRIIIA, or both relative to a reference polypeptide comprising an Fc portion that does not contain G236A / A330L / I332E (wherein human FcγRIIA can be H131 or R131, and / or human FcγRIIIA can be F158 or V158); (ii) has reduced binding to human FcγRIIB relative to a reference polypeptide comprising an Fc portion that does not contain G236A / A330L / I332E; and (iii) does not bind to human FcγRIIB and to human C1q relative to a reference polypeptide comprising an Fc portion that does not contain G236A / A330L / I332E. 67. The antibody or antigen-binding fragment of any one of embodiments 63 to 66, having reduced binding to human C1q, does not bind to human C1q, activates FcγRIIA, human FcγRIIIA, or both to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E (wherein human FcγRIIA can be H131 or R131, and / or human FcγRIIIA can be F158 or V158), does not activate human FcγRIIB, and / or activates human natural killer (NK) cells in the presence of HBsAg to a greater extent than a reference polypeptide comprising an Fc portion that does not comprise G236A / A330L / I332E. Embodiment 68. A method of treating a hepatitis B virus infection in a subject, comprising administering to the subject a single dose of a composition comprising the antibody or antigen-binding fragment of any one of embodiments 1 to 47 or 59 to 67, at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 mg / kg or more of the antibody or antigen-binding fragment, or at a dose of up to 75 mg (i.e., including any integer or non-integer dose up to 75 mg), up to 300 mg, or up to 900 mg of the antibody or antigen-binding fragment. Embodiment 69. The method of embodiment 68, wherein the antibody or antigen-binding fragment comprises a heavy chain (HC) amino acid sequence according to SEQ ID NO: 91 and a light chain (LC) amino acid sequence according to SEQ ID NO: 93. Embodiment 70. The method of embodiment 68 or 69, wherein prior to said administering, the composition comprises the antibody or antigen-binding fragment at 150 mg / mL, optionally in sterile water, and further comprises 20 mM histidine, 7% sucrose, and 0.02% PS80 at pH 6. Embodiment 71. The subject (i) is 18 to 65 years of age, or older; (ii) weighs 40 kg or more and 125 kg or less; (iii) has chronic HBV infection as defined by positive serology for HBsAg, HBV DNA, or HBeAg on two occasions at least 6 months apart, based on previous or current laboratory records (or positive results based on any combination of these tests performed at least 6 months apart); (iv) does not have cirrhosis; (v) has received nucleoside reverse transcriptase inhibitor (NRTI) therapy, which may include tenofovir disoproxil / tenofovir alafenamide, entecavir, lamivudine, or adefovir / adefovir dipivoxil, for at least 4 months (120 days) before the single dose is administered; and (vi) has an HBV level of less than 100 IU / mL for a period not exceeding 4 weeks (28 days) before the single dose is administered. (ix) an HBsAg concentration equal to or greater than 1000 IU / mL for not more than four weeks (28 days) prior to administration of the single dose; (x) is HBeAg negative for not more than four weeks (28 days) prior to administration of the single dose; (xi) is negative for anti-hepatitis B antibodies for not more than four weeks (28 days) prior to administration of the single dose; (xii) has an alanine aminotransferase less than two times the ULN; or (xiii) any combination of (i)-(xii). Embodiment 72 The method of any one of embodiments 68 to 71, wherein the administering comprises subcutaneous injection. Embodiment 73. The method of any one of embodiments 68 to 72, wherein 8 weeks after administration of the single dose, the subject has a less than two-fold decrease in HBsAg compared to from 0 days to 4 weeks (28 days) prior to administration. Embodiment 74. A method of treating hepatitis B virus (HBV) infection in a subject, comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 47 or 59 to 67, wherein the antibody optionally comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93. Embodiment 75. The method of embodiment 74, wherein the single dose of the pharmaceutical composition comprises the antibody in the range of 2 to 18 mg / kg (body weight of the subject). Embodiment 76. The method of embodiment 74 or 75, wherein the single dose of the pharmaceutical composition comprises up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody. Embodiment 77. The method of any one of embodiments 74 to 76, wherein the single dose pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL. Embodiment 78. The method of any one of embodiments 74 to 77, wherein the single dose of pharmaceutical composition comprises about 75 mg of the antibody. Embodiment 79. The method of any one of embodiments 74 to 78, wherein the single dose of pharmaceutical composition comprises about 90 mg of the antibody. Embodiment 80. The method of any one of embodiments 74 to 78, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody. Embodiment 81. The method of any one of embodiments 74 to 78, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody. Embodiment 82. The method of any one of embodiments 74 to 78, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody. Embodiment 83 The method of any one of embodiments 74 to 82, comprising administering the single dose by subcutaneous injection. Embodiment 84 The method of any one of embodiments 74 to 83, comprising administering the single dose by intravenous injection. Embodiment 85. The method of any one of embodiments 74 to 84, wherein the pharmaceutical composition further comprises water, optionally water. Embodiment 86. The method of any one of embodiments 74 to 85, wherein the pharmaceutical composition further comprises histidine, suitably at a concentration in the range of 10 mM to 40 mM, for example 20 mM, in the pharmaceutical composition. Embodiment 87. The method of any one of embodiments 74 to 86, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w / v). Embodiment 88. The method of any one of embodiments 74 to 87, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), and wherein the polysorbate or poloxamer 188 is optionally present in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v). Embodiment 89. The method of any one of embodiments 74 to 88, wherein the pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.2. Embodiment 90. The pharmaceutical composition comprises: (i) 150 mg / mL of the antibody; (ii) USP Water; (iii) 20 mM histidine; (iv) 7% sucrose, and (v) 0.02% PS80 and wherein the pH is 6. Embodiment 91 The method of any one of embodiments 74 to 90, wherein the subject is an adult. Embodiment 92 The method of any one of embodiments 74 to 91, wherein the subject is between 18 and 65 years of age. Embodiment 93. The method of any one of embodiments 74 to 92, wherein the subject weighs between 40 kg and 125 kg. Embodiment 94. The method of any one of embodiments 74 to 93, wherein the subject has chronic HBV infection, e.g., as defined by positive serology for HBsAg, HBV DNA, and / or HBeAg on two occasions, the two occasions being at least 6 months apart. Embodiment 95 The method of any one of embodiments 74 to 94, wherein the subject does not have cirrhosis. Embodiment 96. The method of embodiment 95, wherein the absence of cirrhosis is determined by fibroscan assessment (e.g., within 6 months prior to administration of the single dose of the pharmaceutical composition) or liver biopsy (e.g., within 12 months prior to administration of the single dose of the pharmaceutical composition), preferably wherein the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis. Embodiment 97. The method of any one of embodiments 74 to 96, wherein the subject has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI) within 120 days, or even within 60 days, prior to administering the single dose. Embodiment 98. The method of embodiment 97, wherein the NRTIs comprise one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil. Embodiment 99. The method of any one of embodiments 74 to 98, wherein the subject has a serum HBV DNA concentration of less than 100 IU / mL for a period not exceeding 28 days prior to administration of the single dose. Embodiment 100. The method of any one of embodiments 74 to 99, wherein the subject has a serum HBsAg concentration of less than 1,000 IU / mL before the single dose is administered. Embodiment 101. The method of any one of embodiments 74 to 99, wherein the subject has a serum HBsAg concentration of 1,000 IU / mL or greater for a period not exceeding 28 days prior to administration of the single dose. Embodiment 102. The method of any one of embodiments 74 to 101, wherein the subject was HBe antigen (HBeAg) negative for no more than 28 days prior to administration of the single dose. Embodiment 103. The method of any one of embodiments 74 to 102, wherein the subject has been negative for anti-HBs antibodies for no more than 28 days prior to administration of the single dose. Embodiment 105. The method of any one of embodiments 74 to 103, wherein the subject (i) does not have fibrosis and / or does not have cirrhosis, and / or (ii) has an alanine aminotransferase (ALT) level that is less than two times the upper limit of normal (ULN) before the single dose is administered. Embodiment 106. The method of any one of embodiments 74 to 105, wherein, 56 days after administration of the single dose, the subject has a less than two-fold decrease in serum HBsAg (e.g., serum HBsAg concentration determined using an Abbott ARCHITECT assay) compared to the subject's serum HBsAg from days 0 to 28 prior to administration of the single dose. Embodiment 107. The method of any one of embodiments 74 to 106, wherein after administration of the single dose (e.g., 56 days after administration of the single dose), the subject (i) has reduced or less severe intrahepatic spread of HBV compared to a reference subject, and / or (ii) comprises an adaptive immune response to HBV. Embodiment 108 The method of any one of embodiments 74 to 107, wherein the subject is male. Embodiment 109. The method of any one of embodiments 74 to 107, wherein the subject is a female. Embodiment 110. A pharmaceutical composition comprising an antibody, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93, and the pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, for example, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL. Embodiment 111. The pharmaceutical composition of embodiment 110, comprising up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody. Embodiment 112. The pharmaceutical composition of embodiment 110 or 111, comprising about 75 mg of the antibody. Embodiment 113. The pharmaceutical composition of embodiment 110 or 111, comprising about 90 mg of the antibody. Embodiment 114. The pharmaceutical composition of embodiment 110 or 111, comprising about 300 mg of the antibody. Embodiment 115. The pharmaceutical composition of embodiment 110 or 111, comprising about 900 mg of the antibody. Embodiment 116. The pharmaceutical composition of embodiment 110 or 111, comprising about 3,000 mg of the antibody. Embodiment 117. A pharmaceutical composition according to any one of embodiments 110 to 116, further comprising water, optionally USP water. Embodiment 118. A pharmaceutical composition according to any one of embodiments 110 to 117, further comprising histidine in the pharmaceutical composition, optionally at a concentration of 10 mM to 40 mM, for example 20 mM. Embodiment 119.5%. The pharmaceutical composition of any one of embodiments 110 to 118, further comprising a disaccharide, such as sucrose, which may be 6%, 7%, 8%, or 9%, preferably about 7% (w / v). Embodiment 120. The pharmaceutical composition of any one of embodiments 110 to 119, further comprising a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS80), optionally present in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v). Embodiment 121. The pharmaceutical composition of any one of embodiments 110 to 120, having a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or of 5.8, 5.9, 6.0, 6.1, or 6.2. Embodiment 122. (i) 150 mg / mL of the antibody. (ii) USP Water; (iii) 20 mM histidine; (iv) 7% sucrose, and (v) 0.02% PS80 and having a pH of 6. [Example]

[0315] Below, specific examples are provided to illustrate embodiments and aspects of the present disclosure. However, the present disclosure should not be limited in scope by the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and practice the present disclosure. However, the present disclosure is not limited in scope by the exemplified embodiments. Indeed, various modifications of the present disclosure will be readily apparent to those skilled in the art from the foregoing description, accompanying drawings, and the following examples, in addition to those described herein. All such modifications are within the scope of the appended claims. [Example 1]

[0316] Generation and testing of engineered antibodies Analysis of several HBC34 antibody variants from PCT Publication No. WO 2017 / 060504 revealed an unpaired cysteine ​​amino acid at position 40 (IMGT numbering) in the light chain variable region, indicating potential liability. Without wishing to be bound by theory, the unpaired cysteine ​​residue may be potentially reactive and could trigger aggregation through intramolecular scrambling or intermolecular disulfide formation. The HBC34-V7 (WO 2017 / 060504) variant was modified to replace the cysteine ​​amino acid at position 40 with serine (thereby generating "HBC34-V34") or alanine (thereby generating "HBC34-V35"). The nucleotide sequences encoding these additional antibody variants were codon-optimized, and the antibodies were expressed as IgG1 (g1m17, 1 allotype) in ExpiCHO™ cells (ThermoFisher). The codon-optimized nucleotide sequences encoding the VH and VL domains of HBC34-V35 are shown in SEQ ID NOs: 103 and 104, respectively.

[0317] The ability of HBC34-V34 and HBC34-V35 to bind antigens was investigated using a direct antigen-binding ELISA. HBC34-V7 was used as a comparator. As shown in Figure 1, both HBC34-V34 and HBC34-V35 effectively bound two recombinant HBsAg antigens ("adw," upper panel; "adr," lower panel), and HBC34-V35 showed binding very similar to that of the parent HBC34-V7.

[0318] Mutant antibodies were tested for binding to all known HBsAg genotypes [(A)–(J)]. Briefly, human epithelial cells (Hep2 cells) were transfected with plasmids expressing HBsAg from each of the 10 HBV genotypes: A, B, C, D, E, F, G, H, I, and J. All antibodies were tested at multiple concentrations for staining of transiently transfected, permeabilized cells. Two days after transfection, Hep2 cells were harvested, fixed, and permeabilized with saponin for immunostaining with HBC34 and five selected variants. HBC34-V7 was included as a comparator. Antibody binding to transfected cells was analyzed using a Becton Dickinson FACSCanto2 (BD Biosciences) with FlowJo software (TreeStar). As shown in Figures 2A to 2J, HBC34-V34 and HBC34-V35 recognized all 10 HBV HBsAg genotypes, with HBC34-V35 showing slightly stronger staining than HBC34-V34.

[0319] These data indicate that the antibody variants HBC34-V34 and HBC34-V35 broadly recognize HBsAG and bind to HBsAG at levels comparable to those of HBC34-V7. [Example 2]

[0320] HBC antibodies with modified Fc regions bind antigens efficiently Modifications to the Fc region can provide benefits to therapeutic antibodies. HBC34-V35 was expressed as an IgG1 with a wild-type Fc, an Fc containing the "MLNS" mutation (M428L / N434S), or an Fc containing MLNS in combination with "GAALIE" (G239A / A330L / I332E). Each construct was tested in two separate antigen-binding ELISA experiments for binding to recombinant HBsAg (adw). Three (3) lots of HBC34-v35 (wild-type Fc) were tested. Two (2) lots of HBC34-V35-MLNS and two (2) lots of HBC34-V35-MLNS-GAALIE were tested. One (1) lot of HBC34v7 was tested as a comparator.

[0321] As shown in Figures 3A and 3B, the introduced Fc mutations did not affect the antigen-binding activity of HBC34-V35. The EC50 values ​​were generally low, although there was some variability between the various constructs and between the two experiments. [Example 3]

[0322] Additional Functional Tests HBC34-V35, HBC34-V35-MLNS, and HBC34-V35-MLNS-GAALIE were used to perform in vitro and in vivo neutralization studies. In one study, the antibodies were tested for neutralization activity using HBV-infected mouse PXB cells. In another study, the antibodies were tested using human hepatocytes infected with genotype C HBV.

[0323] Both tests use hebsubrin (human hepatitis B immunoglobulin) as a positive control. The following data will be captured at multiple time points: HBV DNA quantification; HBsAg quantification; HBeAg quantification; and hAlb quantification. [Example 4]

[0324] Identification and characterization of human monoclonal antibody HBC24 The human monoclonal antibody was isolated from a human patient in a manner similar to that described in Traggiai E. et al., 2004, Nat Med 10(8): 871-5. The antibody was characterized by determining the nucleotide and amino acid sequences of its variable region and the complementarity-determining regions (CDRs) therein, and was designated "HBC24." Thus, HBC24 has the CDRs, V and CDRs as shown in Table 3 above. H and V L It is a fully human IgG1 monoclonal antibody with the sequence HBC24. H and V L Exemplary nucleotide sequences encoding the following are shown in Table 4: [Example 5]

[0325] Clearance of HB antigen and inhibition of viral entry in a mouse model The effectiveness of the anti-HBV antibodies of the present disclosure in eliminating HBsAg was tested using immunodeficient mice transplanted with human hepatocytes. Briefly, primary human hepatocytes were transplanted into SCID mice in which mouse hepatocytes had been pre-destroyed by enzymes. The mice were T cell- and B cell-deficient. This model is useful for studying HBV infection, including invasion, spread, cccDNA regulation, hepatocyte-intrinsic immune responses, and viral integration into the host genome.

[0326] On day −28, mice were injected with rAAV8-1.3HBV strain ayw, type D at 1.0 × 10 per mouse. 7 Mice were inoculated with the viral genome via tail vein injection. Treatment was performed on day 0. AAV / HBV-infected mice (n = 4 per treatment group) were administered PBS (control) or HBC34-v35 (1, 5, or 15 mg / kg ip, 2x / week). The antibody was murine, excluding the antigen-binding Fab region.

[0327] Plasma and serum samples were collected periodically throughout the study to measure viral load, HBV DNA (by PCR), and HBV antigens (HBsAg, HBeAg, and HBcrAg). Mice were sacrificed at week 6. As shown in Figures 4–7, treatment with the highest dose of HBC34-v35 reduced viral load and viral entry into hepatocytes. [Example 6]

[0328] Generation and functional testing of germlined variants of HBC24 HBC24 is analyzed for the presence of somatic mutations in the variable region compared to the germline sequence. Identified somatic mutations are reverted to the germline sequence to generate HBC24 variants. HBC24 and variants are tested for binding (in vitro) and neutralization (in vitro; in vivo) of HBV and HBD serotypes using the assays described in Examples 1 and 3. [Example 7]

[0329] Introduction of Fc modifications into HBC24 and variants Additional HBC24 variants were generated containing MLNS and GAALIE mutations in both Fc monomers. The HC amino acid sequences of selected variants are shown in SEQ ID NOs: 120 and 121. The variants were tested for (1) in vitro binding to antigen; and (2) in vitro neutralization of HBV serotypes using the assays described in Examples 1 and 3. [Example 8]

[0330] In vitro effector function assays In vitro studies were performed to assess the ability of the Fc-modified HBC34 antibody to (1) bind and complement human FcγR; (2) activate FcγRIIa, FcγRIIb, and FcγRIIIa; and (3) promote ADCC and activate human natural killer (NK) cells. The test substances, cell lines, and reagents used were as described in Tables 5-7 below. The following abbreviations are used in this example: GLP = Good Laboratory Practice; ADCC = antibody-dependent cellular cytotoxicity; ADCP = antibody-dependent cellular phagocytosis; Fc = crystalline fragment; HBsAg = hepatitis B surface antigen; mAb = monoclonal antibody; PBS = phosphate-buffered saline; UHPL-SEC = ultra-high performance liquid size-exclusion chromatography; ATCC = American Type Culture Collection; FcγR = Fc gamma receptor; CHO cells = Chinese hamster ovary cells; RLU = relative light units; BLI = biolayer interferometry.

[0331] [Table 4-1] [Table 4-2]

[0332] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0333] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0334] Experimental procedure Measurement of binding to human Fcγ receptors Binding of HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE to human FcγRs was measured using an Octet instrument (BLI, Biolayer Interferometry). Briefly, 2 μg / ml of His-tagged human FcγRs (FcγRIIa allele H131, FcγRIIa allele R131, FcγRIIAa allele F158, FcγRIIIa allele V158, and FcγRIIb) were captured with an anti-pentaHis sensor for 6 min. The FcγR-loaded sensors were then exposed to a kinetics buffer solution (pH 7.1) containing 2 μg / ml of each mAb in the presence of 1 μg / ml of affiniPure F(ab')2 fragment goat anti-human IgG, F(ab')2 fragment specific (crosslinking human mAbs through the Fab fragments) for 4 min, followed by a dissociation step in the same buffer for another 4 min (right part of the plot). Association and dissociation profiles were measured in real time as changes in the interference pattern using an Octet RED96 (ForteBio).

[0335] Measurement of binding to human complement protein C1q The binding of HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE to human complement was measured using an Octet instrument (BLI, biolayer interferometry). Briefly, an anti-human Fab (CH1-specific) sensor was used to capture the intact IgG1 of HBC34v35 MLNS and HBC34-V35-MLNS-GAALIE mAbs via the Fab fragments at 10 μg / ml for 10 min. The IgG-loaded sensor was then exposed to a kinetics buffer solution (pH 7.1) containing 3 μg / ml of purified human C1q for 4 min (left part of the plot), followed by a dissociation step in the same buffer for another 4 min (right part of the plot). The binding and dissociation profiles were measured in real time as changes in the interference pattern using an Octet RED96 (ForteBio).

[0336] Preparation of human NK cells from whole blood NK cells were freshly isolated from EDTA whole blood using the MACSxpress® NK Isolation Kit according to the manufacturer's instructions. Briefly, anticoagulated blood was mixed with 15 ml of NK isolation cocktail in a 50 ml tube and incubated at room temperature for 5 minutes using a rotator at approximately 12 revolutions per minute. The tube was then placed in the magnetic field of the MACSxpress® Separator for 15 minutes. Magnetically labeled cells adhere to the tube wall, while aggregated red blood cells settle to the bottom. Targeted NK cells were then collected from the supernatant while the tube was still inside the MACSxpress® Separator. NK cells were centrifuged, treated with distilled water to remove residual red blood cells, centrifuged again, and finally resuspended in AIM-V medium.

[0337] Determination of antibody-dependent NK cell killing MAbs were serially diluted 10-fold in AIM-V medium from 100 μg / ml to 0.001 μg / ml. Target cells (PLC / PRF / 5; MacNab et al., British Journal of Cancer, 34(5), 1976) were plated in round-bottom 384-well plates at 7.5 × 10 cells per 23 μl. 3100 cells / well were added, then serially diluted antibodies were added to each well (23 μl per well), and the antibody / cell mixture was incubated at room temperature for 10 minutes. After incubation, 7.5 × 10 human NK cells were added in 23 μl. 4 Cells were added at a cell density of 1000 / well, resulting in an effector-to-target ratio of 10:1. Control wells used to measure maximum lysis (target cells with 23 μl of 3% Triton x-100) and spontaneous lysis (target cells and effector cells without antibody) were also included. Plates were incubated at 37°C for 4 hours with 5% CO2. Cell death was determined by measuring lactate dehydrogenase (LDH) release using an LDH detection kit according to the manufacturer's instructions. Briefly, plates were centrifuged for 4 minutes at 400 × g, and 35 μl of the supernatant was transferred to a flat-bottom 384-well plate. LDH reagent was prepared, and 35 μl was added to each well. Using a kinetic protocol, absorbance at 490 nm and 650 nm was measured once every 2 minutes for 8 minutes. Percent specific lysis was determined using the following formula: (specific release - spontaneous release) / (maximum release - spontaneous release) × 100.

[0338] Determination of antibody-dependent NK cell activation Activation of primary NK cells was tested using freshly isolated cells from two donors previously genotyped for expression of homozygous high (V158 allele) or low (F158 allele) affinity FcγRIIIa. Serial dilutions of mAb (10-fold serial dilutions from 100 μg / ml to 0.0001 μg / ml in AIM-V medium) were incubated with NK cells for 4 hours. NK cell activation was measured by flow cytometry by staining NK cells with anti-CD107a mAb (anti-CD107 PE, BioLegend, diluted 1 / 35) as a functional marker of NK cell activity.

[0339] Determining antibody-dependent activation of human FcγRIIIa HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 4-fold in ADCC assay buffer from 5 μg / ml to 0.076 μg / ml. Target antigen (HBsAg from Engerix B, GlaxoSmithKline) was added to a white flat-bottom 96-well plate at 0.6 μg / ml in 25 μl, and then serially diluted antibodies were added to each well (25 μl per well), and the antibody / cell mixture was incubated at room temperature for 10 minutes. Effector cells for the ADCC bioassay were thawed and diluted to 7.5 × 10 in 25 μl. 4 Cells were added at a cell density of 1000 / well (final HBsAg concentration was 0.2 μg / ml). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous plate luminescence (wells containing only ADCP assay buffer) were also included. Plates were incubated at 37°C with 5% CO2 for 24 hours. Activation of human FcγRIIIa (V158 or F158 variant) in this bioassay results in NFAT-mediated expression of a luciferase reporter gene. Luminescence was measured in a luminometer using Bio-Glo™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (i.e., specific FcγRIIIa activation) are expressed as the mean relative light units (RLU) compared to background using the following formula: (RLU at mAb concentration x - RLU of background).

[0340] Determining antibody-dependent activation of human FcγRIIa HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold in ADCP assay buffer from 50 μg / ml to 0.00013 μg / ml. Target antigen (HBsAg from Engerix B) was added to a white flat-bottom 96-well plate at 0.6 or 6 μg / ml in 25 μl, and then serially diluted antibody was added to each well (25 μl per well), and the antigen / antibody mixture was incubated at room temperature for 25 minutes. Effector cells for the FcγRIIa activation bioassay were thawed and diluted to 50.0 × 10 in 25 μl. 4Cells were added at a cell density of 1000 μg / well (final HBsAg concentrations were 0.2 or 2 μg / ml, respectively). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous plate luminescence (wells containing only ADCP assay buffer) were also included. Plates were incubated at 37°C with 5% CO for 23 hours. Activation of human FcγRIIa (H131 variant) in this bioassay results in NFAT-mediated expression of a luciferase reporter gene. Luminescence was measured in a luminometer using Bio-Glo™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (i.e., specific FcγRIIa activation) are expressed as the mean relative light units (RLU) compared to background using the following formula: (RLU at concentration of mAb [x] - RLU of background).

[0341] Determining antibody-dependent activation of human FcγRIIb HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were serially diluted 5-fold in ADCP assay buffer from 100 μg / ml to 0.00026 μg / ml. Target antigen (HBsAg from Engerix B) was added to a white flat-bottom 96-well plate at 3 μg / ml in 25 μl, and then serially diluted antibody was added to each well (25 μl per well), and the antigen / antibody mixture was incubated at room temperature for 15 minutes. Effector cells for the FcγRIIb activation bioassay were thawed and diluted to 75.0 × 10 in 25 μl. 4Cells were added at a cell density of 1000 / well (final HBsAg concentration was 1 μg / ml). Control wells used to measure antibody-independent activation (containing HBsAg and effector cells but no antibody) and spontaneous plate luminescence (wells containing only ADCP assay buffer) were also included. Plates were incubated at 37°C with 5% CO2 for 20 hours. Activation of human FcγRIIb in this bioassay results in NFAT-mediated expression of a luciferase reporter gene. Luminescence was measured in a luminometer using Bio-Glo™ Luciferase Assay Reagent according to the manufacturer's instructions. Data (i.e., specific FcγRIIb activation) are expressed as the mean relative light units (RLU) compared to background using the following formula: (RLU at concentration of mAb [x] - RLU of background).

[0342] Determination of antibody binding to the human hepatocellular carcinoma cell line PLC / PRF / 5 PLC / PRF / 5 cells were trypsinized for 5 minutes at 37°C, transferred to 7 ml growth medium, centrifuged at 400 × g for 4 minutes at 4°C, and washed extensively in PBS at 4°C. Some cells were fixed with 4% formaldehyde (20 minutes at 4°C); others were fixed and then permeabilized with permeabilization buffer (20 minutes at 4°C). The cell pellet was resuspended in 2.64 ml of wash buffer (fixed cells) or permeabilization buffer (fixed and permeabilized cells) (Table 7) and dispensed into a 96-well round-bottom plate at 200 μl / well (equivalent to 100,000 cells / well). The plate was centrifuged at 400 g for 4 minutes at 4°C. Serial 1:5 5-point dilutions of the test antibody, starting from a final concentration of 10 μg / ml, were added to the cell-containing wells and incubated on ice for 30 minutes. After washing twice with wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells) at 400 x g for 4 minutes at 4°C, 50 μl / well of Alexa Fluor® 647-conjugated secondary antibody (Table 7) was added to the cells and incubated on ice for 20 minutes. Cells were washed twice more with wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells) and resuspended in 200 μl / well of wash buffer (fixed cells) or permeabilization buffer (fixed & permeabilized cells), and the signal (MFI, mean fluorescence intensity) was quantified using a cytofluorometer (BD FACSCanto II).

[0343] result Direct antiviral mechanisms are important for neutralizing HBV in vivo. Indirect Fc-dependent mechanisms of action mediated by the interaction of the Fc region with Fc gamma receptors (FcγRs) on immune cells may also contribute significantly to efficacy in vivo and mediation of endogenous immune responses. FcγR-dependent mechanisms can be assessed in vitro by measuring binding to FcγRs and antibody-dependent activation of human FcγRs (Hsieh, Y.-T., et al., Journal of Immunological Methods, 441(C), 56-66. doi.org / 10.1016 / j.jim.2016.12.002).

[0344] In this study, HBC34v35-MLNS and HBC34-V35-MLNS-GAALIE were compared for their ability to bind to the full set of human FcγRs (FcγRIIIa V158 and F158 alleles, FcγRIIa H131 and R131 alleles, and FcγRIIb) using biolayer interferometry (BLI Octet System, ForteBio). As shown in Figures 8A-8E, Fc carrying the MLNS-GAALIE mutations exhibit altered interactions with FcγRs; specifically, Fc carrying these mutations exhibit increased binding to FcγRIIIa and FcγRIIa and decreased binding to FcγRIIb. Not surprisingly, the binding of HBC34-V35-MLNS-GAALIE to C1q was abolished as measured by biolayer interferometry (Figure 9).

[0345] HBC34-V35-MLNS and HBC34-V35-MLNS-GAALIE were also tested for their ability to activate human FcγRIIIa and FcγRIIa using cell-based reporter bioassays. These assays utilize Jurkat cells engineered with an NFAT-mediated luciferase reporter to reflect human FcγR activation. HBC34v35-MLNS poorly or did not activate human FcγRIIIa and FcγRIIa in the presence of HBsAg, whereas HBC34-V35-MLNS-GAALIE demonstrated dose-dependent activation of all tested FcγRs (Figures 10A, 10B, 11A, and 11B). Conversely, HBC34-V35-MLNS-GAALIE did not activate FcγRIIb, even when tested at 100 μg / ml (Figure 12).

[0346] ADCC activity was also measured using natural killer (NK) cells isolated from human peripheral blood mononuclear cells of a single donor previously genotyped for expression of heterozygous high- (V158) and low- (F158) affinity FcγRIIIa (F / V). Killing of the hepatocellular carcinoma cell line PLC / PR / 5 was measured using the isolated NK cells upon exposure to HBC34v35; HBC34v35-MLNS; HBC34-V35-MLNS-GAALIE; or another mAb (17.1.41, targeting a different epitope on the antigenic loop of HBsAg; see Eren, R., et al., Hepatology, doi.org / 10.1053 / jhep.2000.9632; Galun, E., et al., Hepatology, doi.org / 10.1053 / jhep.2002.31867). No killing was observed in the presence of HBsAg-specific mAbs HBC34v35, HBC34v35-MLNS, HBC34-V35-MLNS-GAALIE, and 17.1.41 (Fig. 13A). The observed lack of antibody-dependent killing of PLC / PR / 5 cells may be related to the poor expression of HBsAg on the cell surface (Fig. 13B), which, without wishing to be bound by theory, may not be sufficient to trigger killing by NK cells. In contrast, when PLC / PR / 5 cells were fixed and permeabilized, high levels of HBsAg were detected in HBC34v35 and 17.1.41, indicating that most of the HBsAg was found either intracellularly or in a secreted form (i.e., subviral particles) (Fig. 13B).

[0347] Activation of primary human NK cells (V / F) in the presence of HBC34v35-MLNS or HBC34-V35-MLNS-GAALIE and HBsAg was also examined using anti-CD107a mAb. Data are shown in Figures 14A and 14B.

[0348] These in vitro data demonstrate that the HBV-specific binding proteins of the present disclosure, which have a GAALIE Fc mutation, bind to and activate low-affinity activating FcγRIIa and FcγRIIIa more effectively than non-GAALIE Fc parent antibodies. The GAALIE-containing binding proteins also did not bind to or activate low-affinity inhibitory FcγRIIb. The GAALIE-containing binding proteins also did not bind to C1q. Furthermore, the GAALIE-containing binding proteins did not promote ADCC in hepatocellular carcinoma cells, but activated human NK cells in the presence of soluble HBsAg. [Example 9]

[0349] Phase 1 clinical trial of HBC34-v35-MLNS-GAALIE A multicenter, phase 1, randomized, placebo-controlled study will be conducted to evaluate the safety, tolerability, pharmacokinetics, and antiviral activity of HBC34-v35-MLNS-GAALIE (comprising a heavy chain amino acid sequence set forth in SEQ ID NO: 91 and a light chain amino acid sequence set forth in SEQ ID NO: 93). The study sites are as follows: Part A (single center) and Part B / C (multicenter).

[0350] In Part A (maximum 40 patients), the primary objective is to evaluate the safety and tolerability of HBC34-v35-MLNS-GAALIE in healthy adult subjects. Secondary objectives are to characterize the serum pharmacokinetics (PK) of HBC34-v35-MLNS-GAALIE in healthy adult subjects and to evaluate the immunogenicity (induction of anti-drug antibodies (ADA)) of HBC34-v35-MLNS-GAALIE in healthy adult subjects.

[0351] In Parts B (maximum 56 patients) and C (maximum 24 patients), the primary objective is to evaluate the safety and tolerability of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. Secondary objectives are to characterize the serum PK of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis, evaluate the antiviral activity of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis, and evaluate the immunogenicity (induction of ADA) of HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis. Exploratory objectives include: assessing the effect of HBC34-v35-MLNS-GAALIE on additional viral parameters; assessing the effect of HBC34-v35-MLNS-GAALIE on immune responses (or exploratory biomarkers) in adult subjects with chronic HBV infection without cirrhosis; and assessing the impact of host genetic polymorphisms (or exploratory biomarkers) on response to HBC34-v35-MLNS-GAALIE in adult subjects with chronic HBV infection without cirrhosis.

[0352] Evaluation criteria details For Part A, the primary endpoints of the study are: Incidence of treatment-emergent adverse events (TEAEs) Clinical evaluation, including but not limited to laboratory test results Secondary endpoints of this study include: HBC34-v35-MLNS-GAALIE serum-free PK parameters, e.g.: C max ,Clast,T. max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z (IV only), CL (IV only), V z / F (SC only), and CL / F (SC only) Incidence and titer of ADA against HBC34v-35-MLNS-GAALIE (if applicable) For Parts B / C, the primary endpoints of this study are: Incidence of TEAEs Clinical evaluation, including but not limited to laboratory test results Secondary endpoints of this study include: HBC34-v35-MLNS-GAALIE serum-free and complete PK parameters, e.g.:C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λz, V z / F, and CL / F. Incidence and titer of ADA against HBC34-v35-MLNS-GAALIE (if applicable) Maximum reduction in serum HBsAg from baseline (pre-dose on Day 1) Exploratory endpoints in this study include: Assessment of additional viral parameters (e.g., HBV RNA and HBcrAg) Analysis of host immune responses Analysis of host factors determined by RNA sequencing Fc gamma receptor (FcγR) polymorphisms determined by genotyping IgG allotypes determined by genotyping may be included.

[0353] Planned number of subjects Part A: Up to 40 healthy adult subjects Part B: Up to 56 adult subjects with chronic HBV infection without cirrhosis who are HBeAg negative and have HBsAg <1000 IU / mL and are receiving nucleoside(t)ide reverse transcriptase inhibitor (NRTI) therapy Part C: Up to 24 adult subjects with chronic HBV infection without cirrhosis, on NRTI therapy, with HBsAg ≥ 1000 IU / mL

[0354] Diagnosis and main inclusion criteria Part A inclusion criteria include: Healthy adult subjects aged 18 years (or legal consent age, whichever is older) to 55 years, weighing ≥ 40 kg to ≤ 125 kg, were enrolled. Patients were in good health as determined by medical history (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety, and depression must be well controlled) and had no clinically significant findings from physical examination, 12-lead ECG, vital signs, and laboratory tests. Female subjects must have a negative pregnancy test or confirmation of postmenopausal status. Postmenopausal status is defined as 12 months of amenorrhea without an alternative medical cause. Women of childbearing potential (WOCBP) must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, cannot be breastfeeding, and be willing to use a highly effective method of contraception as disclosed herein from 14 days prior to study drug administration through 40 weeks after study drug administration.

[0355] Male subjects with a female partner of childbearing potential must agree to meet one of the following contraceptive requirements from the time of study drug administration until 40 weeks after study drug administration: vasectomy with documentation of azoospermia, or male condom use plus the partner's frequent use of highly effective contraception. Male subjects must also agree not to donate sperm from the time of study drug administration until 40 weeks after study drug administration. Patients agree not to donate blood during the course of the study.

[0356] Patients must be willing to comply with the study requirements and be able to provide written informed consent.

[0357] Part B / C inclusion criteria include: 1. 18 years old (or the age of legal consent, whichever is older) to 65 years old 2. Body weight ≥ 40 kg to ≤ 125 kg, with chronic HBV infection [defined by two positive serum HBsAg, HBV DNA, or HBeAg tests at least 6 months apart based on previous or current laboratory evidence (any combination of these tests performed 6 months apart is acceptable)]. 3. No cirrhosis 4. At least 2 months of NRTI therapy at screening and HBeAg negative. Examples of NRTI therapy include, but are not limited to, tenofovir disoproxil / tenofovir alafenamide; entecavir; lamivudine; adefovir / adefovir dipivoxil. 5. HBV DNA <100IU / mL at screening 6. HBsAg > detection limit 7. HBsAg <1000 IU / mL at screening (Part B only) 8. HBsAg ≥ 1000 IU / mL at screening (Part C only) 9. HBeAg negative at screening (Part B only) 10. Negative anti-HBs at screening 11. Except for chronic HBV infection, patients must be in good health as determined by medical history (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, asthma, anxiety, and depression must be well controlled), and there must be no clinically significant findings from physical examination, 12-lead ECG, vital signs, and laboratory tests. 12. Female subjects must have a negative pregnancy test or confirmation of postmenopausal status. Postmenopausal status is defined as 12 months of amenorrhea without an alternative medical cause. Women of childbearing potential must have a negative blood pregnancy test at screening and a negative urine pregnancy test on Day 1, cannot be breastfeeding, and must be willing to use highly effective contraception from 14 days prior to study drug administration through 40 weeks after study drug administration. 13. Male subjects with a female partner of childbearing potential must agree to meet one of the following contraceptive requirements from the time of study drug administration until 40 weeks after study drug administration: vasectomy with documentation of azoospermia, or male condom use plus one of the contraceptive options listed for contraception for WOCBP in the partner (see herein). Male subjects must also agree not to donate sperm from the time of the first study drug administration until 40 weeks after study drug administration. 14. Willing to comply with the requirements of the clinical trial and able to provide written informed consent.

[0358] Possibly effective birth control methods include: Established combined (estrogen- and progesterone-containing) use of oral, intravaginal, or transdermal hormonal contraceptive methods with ovulation inhibition, or established progesterone-only use of oral, injectable, or implantable hormonal contraceptive methods with ovulation inhibition. It is currently unknown whether HBC34-v35-MLNS_GAALIE affects the effectiveness of hormonal contraceptive methods; therefore, the use of an additional form of contraception (i.e., a barrier method) is recommended throughout the study and for 40 weeks after study drug administration. Placement of an intrauterine device Placement of an intrauterine hormone-releasing system Sterilization of the male partner (by providing adequate post-vasectomy documentation of the absence of sperm in the semen; for female subjects in the clinical trial, the vasectomized male partner should be the subject's only partner) True abstinence from contact with the opposite sex, consistent with the subject's preferred and usual lifestyle. Periodic abstinence (e.g., calendar, ovulation method, symptomatic-thermal method, postovulation method) and pull-out are not acceptable methods of contraception. Abstinent subjects must agree to use one of the above methods of contraception if they initiate sexual intercourse during the study and for up to 40 weeks after study drug administration, or while the subject is being followed in the study, whichever is longer. Barrier methods in combination with hormonal contraception as described above Postmenopausal status is defined as 12 months of amenorrhea without another medical cause. Male subjects with a female partner of childbearing potential must agree to meet one of the following contraceptive requirements from the time of study treatment administration until 40 weeks after study drug administration: Vasectomy with documented evidence of azoospermia Male condom use plus use of one of the contraceptive options listed above for WOCBP contraception (hormonal contraception, intrauterine device) Male subjects must also agree not to donate sperm for 40 weeks after their last dose of study drug.

[0359] Clinical trial participation period Part A: The duration of investigational drug treatment is a single dose. The estimated total time of the study, including screening and follow-up for each subject, is up to 28 weeks. Part B / C: The duration of investigational drug treatment is a single dose. The estimated total time of the study, including screening and follow-up for each subject, is up to 44 weeks.

[0360] Follow-up period Part A: All subjects will be followed for 24 weeks after receiving the study drug. Part B / C: All subjects will be followed for 8 weeks after study drug administration. Subjects with a >2-fold HBsAg reduction at week 8 will undergo extended follow-up for a total of up to 40 weeks or until HBsAg reduction is <2-fold compared to baseline on two consecutive samples, whichever comes first. Extended follow-up may be discontinued based on emerging data.

[0361] Clinical trial design The Safety Review Committee (SRC) will conduct an ongoing review of safety, tolerability, and antiviral activity data (Parts B and C only) at specified time points based on available data collected throughout the study. Primary data reviewed by the SRC for dose escalation and appropriate cohort enrollment are listed throughout the protocol, but additional relevant data from other cohorts will also be reviewed by the SRC, as indicated, to inform decisions. The trial will be conducted in three parts: Part A: A randomized, double-blind, placebo-controlled, single-ascending dose (SAD) study of HBC34-v35-MLNS-GAALIE administered by subcutaneous (SC) injection or intravenous (IV) infusion to healthy adult subjects. Part B: A randomized, double-blind, placebo-controlled, SAD study of HBC34-v35-MLNS-GAALIE administered by SC injection to adult subjects with chronic HBV infection without cirrhosis who are HBeAg negative and HBsAg <1000 IU / mL while on NRTI therapy. Part C: An optional, randomized, double-blind, placebo-controlled, SAD study of HBC34v35-MLNS-GAALIE administered by SC injection to adult subjects with chronic HBV infection without cirrhosis who are on NRTI therapy and have HBsAg ≥ 1000 IU / mL.

[0362] Overall risk / benefit assessment The potential risk to healthy adult subjects is based on the general safety risks observed with mAb class therapeutics and is not specific to HBC34-v35-MLNS-GAALIE: anaphylaxis and other severe allergic and injection / infusion-related reactions. The risk of developing such conditions specifically following administration of HBC34v35-MLNS-GAALIE is unknown. Part A of the trial will collect information on the safety and tolerability of HBC34v35-MLNS-GAALIE, as well as relevant data on its PK profile and anti-drug antibody (ADA) development. HBC34-v35-MLNS-GAALIE is not expected to provide benefit to healthy subjects enrolled in Part A of the study. Subjects will be monitored for important potential risks, and routine pharmacovigilance and risk minimization activities will be conducted. Compared to current standard of care, the potential benefits of HBC34-v35-MLNS-GAALIE in subjects with chronic HBV infection include: Reduction of serum HBsAg, inhibition of intrahepatic spread of HBV, elimination of infected hepatocytes, and stimulation of adaptive immune responses against HBV A well-tolerated, finite-duration, SC-administered pan-genotypic therapy for HBV infection In addition to anaphylaxis, other serious allergic reactions, and injection / infusion-related reactions, potential risks associated with administration of HBC34-v35-MLNS-GAALIE to subjects with chronic HBV infection include immune complex disease and hepatotoxicity due to ADCC / ADCP and / or cytotoxic T cell-mediated elimination of infected hepatocytes induced by the vaccine effect. The Part B / C study design includes several elements to mitigate these risks: Part B will enroll subjects with serum HBsAg <1000 IU / mL to reduce the risk of immune complex disease and hepatotoxicity. Additionally, Part B safety data will be reviewed by the SRC before subjects with potentially higher baseline HBsAg levels are enrolled in the appropriate Part C of the study. Parts B and C will enroll subjects who are on NRTIs, have HBV DNA <100 IU / mL at screening, and have good hepatic reserve and low levels of liver inflammation at baseline as determined by the following characteristics: ALT or AST ≤2 x ULN, no history of hepatic decompensation, and no significant fibrosis or cirrhosis. Two sentinel subjects will be randomized 1:1 to receive either HBC34-v35-MLNS-GAALIE or placebo. These sentinel subjects will be monitored for at least 72 hours after dosing, and if the investigator(s) have no safety concerns, the remaining six subjects in the same cohort will be dosed (five active and one placebo). Dose escalation will occur after SRC review of available safety data up to 4 weeks after dose administration to account for potential immune complex disease and the expected timing of hepatotoxicity due to ADCC / ADCP and / or vaccine effect-induced cytotoxic T cell-mediated clearance of infected hepatocytes. Design safety monitoring, including liver function tests, urinalysis, renal function, vital signs, and physical examination findings, to detect evidence of HBC34-v35-MLNS-GAALIE-related immune adverse events.

[0363] Part A Three sequential cohorts in Part A will evaluate 90 mg, up to 300 mg, and up to 900 mg administered by SC injection. The SRC will review available clinical and laboratory safety data for up to 2 weeks post-dose for all available subjects in the cohorts prior to dose escalation. Two additional optional cohorts in Part A may be added, evaluating up to 900 mg and 3000 mg administered by IV infusion. Enrollment for these optional cohorts may occur following SRC review of available week 2 data from all available subjects in Cohort 3a (up to 900 mg SC). All SC cohorts in Part A (Cohorts 1a, 2a, and 3a) will be enrolled consecutively, although additional cohort(s) may be enrolled concurrently if they investigate dose levels at or below those previously found to have an acceptable safety and tolerability profile in preceding cohorts in Part A. Within each cohort, two sentinel subjects will be randomized 1:1 to receive either HBC34-v35-MLNS-GAALIE or placebo. These subjects will be dosed and monitored in an inpatient setting for at least 24 hours; if the investigator has no safety concerns, the remaining subjects in the same cohort will be dosed. The remaining subjects will be randomized 5:1 to receive either HBC34-v35-MLNS-GAALIE or placebo. The maximum dose escalation factor for Part A will not exceed 5-fold.

[0364] Part B The first cohort of Part B (Cohort 1b) will be enrolled after SRC review of available Week 2 data from all available subjects in Cohort 1a (90 mg SC).

[0365] For Part B, five cohorts are planned to evaluate doses of 6 mg (Cohort 1b), 18 mg (Cohort 2b), up to 75 mg (Cohort 3b), up to 300 mg (Cohort 4b), and up to 900 mg (Cohort 5b) administered by SC injection. The SRC will review available clinical and laboratory safety and antiviral activity data for up to 4 weeks post-dose for all available subjects in the lead-up cohorts before dose escalation. Part B may include two additional optional cohorts following the same dosing schedule. The optional cohorts may be dosed at lower, equivalent, or intermediate dose levels compared to the dose levels investigated in the planned Part B cohorts, or may be dosed after Cohort 5b at a dose level not exceeding 900 mg. The maximum dose level for any optional cohort in Part B will not exceed the highest single dose found to have an acceptable safety and tolerability profile in Part A. Optional cohorts may be enrolled at any time within the planned Part B cohorts based on SRC approval.

[0366] All cohorts in Part B should be enrolled consecutively, but additional cohort(s) may be enrolled concurrently if they investigate dose levels at or below those previously found to have acceptable safety and tolerability profiles in prior cohorts in Parts A and B.

[0367] Within each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo via SC injection. These subjects will be dosed and monitored for at least 72 hours after dosing (including at least the first 24 hours of inpatient monitoring); if the investigator(s) have no safety concerns, the remaining subjects in the same cohort will be dosed. The remaining subjects will be randomized 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo via SC injection.

[0368] The maximum dose escalation factor for Part B will not exceed 5-fold.

[0369] Part C Part C may be conducted as appropriate based on the acceptable safety and tolerability profile of HBC34-v35-MLNS-GAALIE in HBeAg-negative subjects with HBsAg levels <1000 IU / mL in Part B. The first cohort of Part C will be enrolled after the SRC reviews available data through the Week 4 visit for all subjects in Parts A and B for the cohort of subjects in Part B receiving a dose that is commensurate with or higher than the proposed starting dose level in Part C.

[0370] Three optional cohorts may be enrolled in Part C. Each cohort may evaluate up to 900 mg administered by SC injection, with the dose utilized in the Part C cohort not exceeding the highest dose level in Part B found by the SRC to have an acceptable safety and tolerability profile. The cohorts may be enrolled simultaneously.

[0371] Within each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo via SC injection. These subjects will be dosed and monitored for at least 72 hours after dosing (including at least the first 24 hours of inpatient monitoring); if the investigator(s) have no safety concerns, the remaining subjects in the same cohort will be dosed. The remaining subjects will be randomized 5:1 to receive HBC34-v35-MLNS-GAALIE or placebo via SC injection.

[0372] Test Procedure Part A screening Healthy adult subjects will be enrolled in one of five cohorts (three planned cohorts, two ad hoc cohorts) in Part A. Screening will occur no more than four weeks prior to the Day 1 visit and will include written informed consent, determination of eligibility, collection of demographic and medical history according to the Schedule of Assessments (SoA), physical examination, vital signs, laboratory tests, 12-lead electrocardiogram (ECG), and other assessments. Eligible subjects will be admitted to the investigational site on Day -1 or Day 1. Eligibility criteria related to vital signs, pregnancy test, substance abuse, blood donation, the presence of any clinically significant acute conditions, and use of prescription, OTC, herbal, or investigational medications will be assessed on Day 1 to ensure eligibility to continue in the study. Any changes to medical history will also be assessed and recorded. Eligible subjects in each cohort will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to study drug administration. Subjects will receive a single dose of study drug (HBC34-v35-MLNS-GAALIE or placebo) on Day 1. Adverse events (AEs) related to screening activities will be collected from the time of consent onwards; any other events occurring during the screening period will be reported as medical history. All serious adverse events (SAEs) will be collected from the time of consent onwards. Screening viral serological parameters include: active infection with HIV, HCV, and HBV Day of administration (Day 1) Eligible subjects will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to administration of study drug on Day 1. Eligible subjects will receive a single dose of study drug on Day 1 and will undergo applicable evaluations. At the beginning of each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34v-35-MLNS-GAALIE or placebo. These subjects will be dosed and monitored in an inpatient setting for at least 24 hours. Vital signs, ECG, symptom-based physical examination(s), and AEs will be reviewed by the investigator; if the investigator has no safety concerns, the remaining subjects in the same cohort will be dosed. The remaining subjects in the cohort will be randomized 5:1 to receive a single dose of HBC34-v35-MLNS-GAALIE or placebo. All subjects will be closely monitored after dose administration. Follow-up period Subjects will be discharged after all study assessments on Day 2. All subsequent study visits will be outpatient. Subjects will return to the investigational site through Week 24 for in-person evaluations per the SoA, including but not limited to physical examination, vital signs, clinical laboratories, PK assessment, and review of AEs and concomitant medications.

[0373] Part B / C screening Screening will occur no more than 4 weeks prior to the Day 1 visit and will include written informed consent as per the SoA, determination of eligibility, collection of demographic and medical history, physical examination, vital signs, laboratory tests, 12-lead ECG, and other assessments. Adverse events related to screening activities will be collected from the time of consent onward; any other events occurring during the screening period will be reported in the medical history. All SAEs will be collected from the time of consent onward. Adult subjects with HBeAg-negative chronic HBV infection without cirrhosis and HBsAg <1000 IU / mL on NRTI therapy for ≥2 months will be enrolled in one of seven cohorts in Part B (5 planned cohorts, 2 ad hoc cohorts). Subject screening will occur ≤4 weeks prior to the Day 1 visit. Subjects will be admitted to the investigational site on Day -1 or Day 1. Eligibility criteria related to NRTI adherence, vital signs, pregnancy test, presence of any clinically significant acute condition, hepatic decompensation, and use of prescription, over-the-counter, herbal, or investigational medications will be assessed on Day 1 to ensure eligibility to continue in the study. Any changes to medical history will also be assessed and recorded. Eligible subjects in each cohort will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to administration of study drug on Day 1. Parts B and C subjects will undergo a Fibroscan evaluation to rule out the presence of cirrhosis. This is not necessary if the subject has had a Fibroscan in the 6 months prior to screening or a liver biopsy in the year prior to screening that confirms the absence of Metavir F3 fibrosis or F4 cirrhosis. Screening viral serology parameters are: active infection with HIV, HCV, and hepatitis delta virus. Subjects with a positive HCV serology result may undergo HCV-RT PCR reflex testing to determine eligibility. Chronic HBV infection is determined at screening and is defined as: two positive serum HBsAg, HBV DNA, or HBeAg tests at least six months apart based on previous or current laboratory documentation (any combination of these tests six months apart is acceptable). Day of administration (Day 1) Eligible subjects will be randomized to receive HBC34-v35-MLNS-GAALIE or placebo within 48 hours prior to administration of study drug on Day 1. Subjects will be admitted to the investigational site on Day 1. Eligible subjects will receive a single dose of study drug on Day 1 and will undergo applicable evaluations. At the beginning of each cohort, two sentinel subjects will be randomized 1:1 to receive HBC34-v35-MLNS-GAALIE or placebo. These subjects will be dosed and monitored for at least 72 hours post-dose (including hospital monitoring for at least the first 24 hours); if the investigator(s) have no safety concerns, the remaining subjects in the same cohort will be dosed. Vital signs, symptom-based physical examination(s), and AEs will be reviewed by the investigator(s) before dosing any additional subjects. The remaining subjects in the cohort will be randomized 5:1 to receive a single dose of the antibody composition or placebo. All subjects will be closely monitored after dose administration. Follow-up period Subjects will be discharged after all study assessments on Day 2. All subsequent study visits will be outpatient. Subjects will return to the investigational site by Week 8 for evaluations as per SoA, including but not limited to physical examination, vital signs, clinical laboratories, PK assessments, efficacy assessments, and review of AEs and concomitant medications. Extended follow-up period Subjects with a >2-fold HBsAg reduction at Week 8 will return to the study site for in-person evaluation as per the SoA until Week 40 or until the HBsAg reduction is <2-fold compared to baseline on two consecutive samples, whichever comes first. Extended follow-up may be discontinued based on emerging data.

[0374] Products, dosage, and method of administration HBC34v35-MLNS-GAALIE is supplied as a lyophilized solid, reconstituted with Sterile Water for Injection (USP) at a concentration of 150 mg / mL, and administered as a SC injection or IV infusion. The unit dose is based on volume and method of administration. When reconstituted to 150 mg / mL with Sterile Water for Injection, USP, the formulation, as administered, contains 20 mM histidine, 7% sucrose, 0.02% PS80, pH 6. The placebo is a 0.9% solution in sterile, preservative-free saline for IV infusion or SC injection. Cohort 1a: HBC34v35-MLNS-GAALIE, a single dose of 90 mg administered by SC injection Cohort 2a: HBC34v35-MLNS-GAALIE, a single dose of up to 300 mg administered by SC injection Cohort 3a: HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 4a (as appropriate): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by IV infusion Cohort 5a (as appropriate): HBC34v35-MLNS-GAALIE, a single dose of up to 3000 mg administered by IV infusion Cohort 1b: HBC34v35-MLNS-GAALIE, a single dose of 6 mg administered by SC injection Cohort 2b: HBC34v35-MLNS-GAALIE, a single dose of 18 mg administered by SC injection Cohort 3b: HBC34v35-MLNS-GAALIE, a single dose of up to 75 mg administered by SC injection Cohort 4b: HBC34v35-MLNS-GAALIE, a single dose of up to 300 mg administered by SC injection Cohort 5b: HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 6b (as needed): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 7b (as needed): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 1c (as appropriate): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 2c (as appropriate): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection Cohort 3c (as appropriate): HBC34v35-MLNS-GAALIE, a single dose of up to 900 mg administered by SC injection

[0375] [Table 7] IV = intravenous; SC = subcutaneous

[0376] Part B: A single-ascending dose study in subjects with chronic HBV infection In Part B, subjects with chronic HBV infection will receive a single dose of the investigational drug. The presence of HBsAg, the therapeutic target of HBC34-v35-MLNS-GAALIE, in subjects with chronic HBV infection alters the potential risks of HBC34-v35-MLNS-GAALIE administration. Potential risks include immune complex disease due to the formation of antigen-antibody complexes and hepatotoxicity due to elimination of infected hepatocytes via ADCC / ADCP and / or the "vaccine effect." To minimize risks to subjects, Part B will be conducted in subjects on NRTIs, with HBV DNA <100 IU / mL at screening, and with good hepatic reserve and low levels of liver inflammation, as determined by the absence of fibrosis / cirrhosis and ALT <2 × ULN. Five dose-level cohorts will be used in Part B. Dose escalation will occur approximately 3-4 times up to the planned maximum dose of 900 mg administered by SC injection: Two cohorts will be enrolled up to a maximum dose of 900 mg administered by SC injection. Cohort 7b may be enrolled for the purpose of collecting and evaluating immune response samples and liver fine needle aspiration samples, if available at select sites, including, but not limited to, cohort 7b. These dose levels are based on preclinical animal models and translational PK / PD modeling predicting significant HBsAg reduction for doses ranging from 2 to 15 mg / kg. Details regarding the dose escalation plan for Part B can be found in Table 9.

[0377] [Table 8] SC=subcutaneous

[0378] Optional Part C: Single-Ascending Dose Study in Subjects with Chronic HBV Infection To evaluate the safety, tolerability, and antiviral activity of HBC34-v35-MLNS-GAALIE in subjects with baseline HBsAg levels ≥1000 IU / mL, an optional Part C will be conducted after the safety, tolerability, and antiviral activity of HBC34-v35-MLNS-GAALIE has been established in HBeAg-negative subjects with HBsAg levels <1000 IU / mL in Part B. Part C will consist of three optional dose-level cohorts, each evaluating a dose of up to 900 mg administered by SC injection (Table 10). One or more optional cohorts in Part C may be enrolled for the purpose of collecting and evaluating immune response samples and liver fine needle aspiration samples at select sites, if applicable, including, but not limited to, those listed.

[0379] [Table 9]

[0380] Reference therapy, dosage, and method of administration: Subjects randomized to placebo will receive a sterile, preservative-free 0.9% saline solution by SC injection (Parts A, B, and C) or IV infusion (Part A only).

[0381] Local tolerability For all study parts, local tolerability assessments will be performed as per the assessment schedule (appearance of subjects receiving study drug by SC injection. The injection site(s) should be marked, located, and documented for subsequent observation. The injection site(s) should be monitored for pain / tenderness, swelling, redness, bruising, and pruritus. The timing of local tolerability assessments in Part A is shown in Figures 15A-15C. The timing of local tolerability assessments in Parts B / C is shown in Figures 16A-16E. Unscheduled visits will be allowed as needed to follow up any unresolved local tolerability symptoms at the investigator's discretion.

[0382] Substance abuse screening Urine for drug abuse screening will be collected for Parts A, B, and C of the trial. The panel will include amphetamines, cocaine, methadone, and opiates.

[0383] Pharmacokinetic evaluation Blood samples are collected to assess HBC34-v35-MLNS-GAALIE concentrations. The time points at which samples are collected for HBC34-v35-MLNS-GAALIE PK analysis in Part A of the clinical trial are indicated herein. The time points at which samples are collected for HBC34-v35-MLNS-GAALIE PK analysis in Parts B and C of the clinical trial are indicated herein.

[0384] Pharmacokinetic analysis Part A Free PK parameters of HBC34-v35-MLNS-GAALIE are computed using standard non-compartmental methods. Parameters include, but are not limited to, serum:C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z (IV only), CL (IV only), V z / F (SC only), and CL / F (SC only). Other parameters are calculated as needed. Part B / C Free and complete PK parameters of HBC34-v35-MLNS-GAALIE are computed using standard non-compartmental methods. Parameters include, but are not limited to, serum:C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z / F, and CL / F. Other parameters are calculated as needed. PK / pharmacodynamic analyses will be performed to investigate exposure-response relationships between PK parameters and selected antiviral variables.

[0385] Antiviral activity assay For Parts B and C, selected data related to the antiviral activity of HBC34-v35-MLNS-GAALIE, including HBsAg, anti-HBs, HBeAg, anti-HBe, HBV RNA, HBcrAg, and HBV DNA levels, are summarized by cohort and study visit with corresponding changes from baseline (n, mean, SD, median, Q1, Q3, min, and max). A summary (number and percentage of subjects) of HBsAg decline (defined as undetectable HBsAg on two consecutive separate measurements at least 2 weeks apart) is shown by cohort and study visit.

[0386] immunogenicity Collect blood samples for analysis of immunogenic responses to determine the presence / absence and titer of anti-drug antibodies (ADAs), if applicable, according to the time points defined in the evaluation schedule (Figures 15A-16E). If necessary, characterize samples for the neutralizing potential (NAb) of HBC34-v35-MLNS-GAALIE.

[0387] Evaluation of screening viral parameters, antiviral activity, and resistance surveillance During parts B and C, assessment of screening viral parameters includes HBsAg, anti-HBs, HBeAg (qualitative), and HBV DNA. Antiviral activity assessments performed after screening include HBsAg, anti-HBs, HBeAg (qualitative; should be collected only for Part C subjects who are HBeAg qualitatively positive at screening), HBeAg (quantitative; should be collected only for Part C subjects who are HBeAg qualitatively positive at screening), anti-HBe, HBV RNA, hepatitis B core-related antigen (HBcrAg), and HBV DNA. Resistance surveillance to monitor for the potential emergence of resistance to NRTIs or HBC34-v35-MLNS-GAALIE will be performed on all subjects receiving study drug. HBV genome sequencing will be attempted in subjects with confirmed HBV DNA breakthrough, defined by HBV DNA ≥ 500 IU / mL measured at two consecutive study visits, or in subjects who discontinue the study early with HBV DNA ≥ 500 IU / mL. Because it is not known at the time of the visit whether a subject has had a virologic breakthrough, resistance surveillance samples will be collected at all study visits noted in the SOA. Samples collected for resistance surveillance may be used to perform additional viral analysis, including viral sequencing.

[0388] Assessment of immune response To examine potential biomarkers of host immune response and infection, subjects may consent to optional substudies in which peripheral immune samples, with or without liver immune samples (by fine needle aspiration), will be collected at the time points outlined in Figures 15A-16E. These optional substudies and associated assessments will be performed when available at select facilities.

[0389] Fc gamma receptor (FcγR) genotyping and immunoglobulin allotyping Blood samples for FcγR genotyping and immunoglobulin allotyping will be collected at baseline for all subjects in Parts B and C to evaluate potential associations between Fc-gamma receptor gene polymorphisms or immunoglobulin allotypes and the antiviral activity of HBC34-v35-MLNS-GAALIE.

[0390] statistical methods Statistical analyses will be primarily descriptive. All study data will be presented in subject data tables. For all study parts, summary tables will present results by cohort for HBC34-v35-MLNS-GAALIE and placebo, with placebo subjects pooled across dose cohorts by route of administration for each part. This trial will be conducted in accordance with ethical principles that have their origins in the Declaration of Helsinki and are consistent with Good Clinical Practice (GCP) and applicable regulatory requirements, including the retention of essential documentation.

[0391] List of definitions of abbreviations and terms used in this example ADA anti-drug antibodies AE Adverse Event ALT alanine aminotransferase ANC absolute neutrophil count AP alkaline phosphatase AST aspartate aminotransferase Area under the AUC curve BLQ below limit of quantification BMI Body Mass Index BUN Blood urea nitrogen CLcr Creatinine clearance CRF Case Report Form CTCAE Common Terminology Criteria for Adverse Events DNA deoxyribonucleic acid ECG electrocardiogram eCRF Electronic Case Report Form End of EF follow-up End of ET treatment FDA Food and Drug Administration GCP Clinical Trial Implementation Standards GGT gamma glutamyltransferase GLP Good Laboratory Practice GNA Glycol Nucleic Acid HBcrAg hepatitis B core-related antigen HBeAg hepatitis B e antigen HBIG Hepatitis B immunoglobulin HBsAg Hepatitis B surface antigen HBV Hepatitis B virus HCC hepatocellular carcinoma HED Human Equivalent Dose Hgb hemoglobin ICF Informed Consent Document ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IgG immunoglobulin G IgM immunoglobulin M IEC Independent Ethics Committee INR International Normalized Ratio IRB Institutional Review Board IV (intravenous) IWRS Interactive Web Response System LDH lactate dehydrogenase LLN lower limit of reference LLOQ Lower limit of quantification LLT lower word mAb Monoclonal antibody MedDRA Drug Regulatory Glossary Nab neutralizing antibody NOAEL No Adverse Effect Level OTC OTC drugs PK Pharmacokinetics PT basic term Q1 1st quartile Q3 3rd quartile RBC red blood cells (number) RNA ribonucleic acid SAD Single Ascending Dose SAE serious adverse event SC subcutaneous SD standard deviation SoA Evaluation Schedule SOC major classification by organ SRC Safety Review Committee SUSAR Suspected Unknown Serious Adverse Reaction TCR tissue cross-reactivity TEAE Treatment-emergent adverse events US ULN upper limit of normal WBC white blood cells (number) WHO World Health Organization WOCBP Women of childbearing potential [Example 10]

[0392] Activation of dendritic cells by HBsAg:HBC34-v35 antibody immune complexes The activation of monocyte-derived (mo)DCs was examined in the presence of immune complexes (ICs) formed by HBC34-V35-MLNS_GAALIE (HC SEQ ID NO: 91, LC SEQ ID NO: 93) or HBC34-V35_MLNS (HC SEQ ID NO: 92, LC SEQ ID NO: 93) and HBsAg (provider: BioIVT) in the serum of HBV+ patients. Materials and Methods: CD14+ monocytes were isolated from human PBMCs from healthy donors (n=2) and cultured for 6 days in RPMI 1640 medium containing 10% FBS (Hyclone), 1% non-essential amino acids, 1% glutamine, 1% Pen / Strep, 1% sodium pyruvate, 50 μM β-mercaptoethanol, 50 ng / mL GM-CSF (Miltenyi), and 1000 U / mL IL-4 (R&D). Differentiated immature monocyte-derived DCs (moDCs) were then stimulated for 22 hours with HBsAg alone (sera from two patients at 1890 and 4460 IU / mL diluted to a final concentration of 250 IU / mL), HBsAg and HBC34-v35-MLNS or HBC34-v35-MLNS_GAALIE (mAb at 20–100 μg / mL), or mAb alone. Reagents were tested to be endotoxin-free. Surface expression of costimulatory markers CD83 and CD86 and HLA-DR was measured by flow cytometry. Levels of 10 (10) human proinflammatory cytokines (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFα) were measured using the Meso Scale Diagnostics (MSD) V-PLEX Proinflammatory Panel 1 Human Kit. Culture medium was used as a negative control. LPS (Sigma, 100 ng / ml) served as a positive control. The data are shown in Figures 20-24B. Immune complexes (ICs) of HBsAg and HBC34-v35-MLNS-GAALIE induced upregulation of costimulatory markers CD83 and CD86, as well as HLA-DR, on the surface of moDCs. Furthermore, ICs of HBsAg and HBC34-v35-MLNS-GAALIE induced moDCs to secrete cytokines TNFα, IL-6, and IL-10.

[0393] Table of Sequences and SEQ ID NOs (Sequence Listing): [Table 10-1] [Table 10-2] Table 10-3 Table 10-4 Table 10-5 Table 10-6 Table 10-7 Table 10-8 Table 10-9 Table 10-10 Table 10-11 Table 10-12 Table 10-13 Table 10-14 Table 10-15 Table 10-16 Table 10-17 Table 10-18 Table 10-19

[0394] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification or in the accompanying Application Data Sheets are hereby incorporated by reference in their entirety to the extent not inconsistent herewith. U.S. Provisional Application No. 62 / 893,742, filed August 29, 2019, is incorporated herein by reference in its entirety. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. 1. A method of treating hepatitis B virus (HBV) infection in a subject, the method comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO:

93.

2. 10. The method of claim 1, wherein the single dose of the pharmaceutical composition comprises the antibody in the range of 2 to 18 mg / kg (subject body weight).

3. 3. The method of claim 1 or 2, wherein the single dose of the pharmaceutical composition comprises up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody.

4. 4. The method of any one of claims 1 to 3, wherein the single dose of pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

5. 5. The method of any one of claims 1 to 4, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody.

6. 5. The method of any one of claims 1 to 4, wherein the single dose of the pharmaceutical composition comprises about 90 mg of the antibody.

7. 5. The method of any one of claims 1 to 4, wherein the single dose of the pharmaceutical composition comprises up to 300 mg of the antibody.

8. 5. The method of any one of claims 1 to 4, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody.

9. 5. The method of any one of claims 1 to 4, wherein the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody.

10. 10. The method of any one of claims 1 to 9, comprising administering the single dose by subcutaneous injection.

11. 10. The method of any one of claims 1 to 9, comprising administering the single dose by intravenous injection.

12. 12. The method of any one of claims 1 to 11, wherein the pharmaceutical composition further comprises water, optionally water.

13. 13. The method of any one of claims 1 to 12, wherein the pharmaceutical composition comprises histidine, suitably at a concentration in the range of 10 mM to 40 mM, such as 20 mM in the pharmaceutical composition.

14. 14. The method of any one of claims 1 to 13, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, at 5%, 6%, 7%, 8%, or 9%, as appropriate, preferably about 7% (w / v).

15. 15. The method of any one of claims 1 to 14, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), said polysorbate or poloxamer 188 being optionally present in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v).

16. 16. The method of any one of claims 1 to 15, wherein the pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.

2.

17. The pharmaceutical composition comprises: (i) 150 mg / mL of the antibody; (ii) USP Water; (iii) 20 mM histidine; (iv) 7% sucrose, and (v) 0.02% PS80 and the pH is 6.

18. 18. The method of any one of claims 1 to 17, wherein the subject is an adult.

19. 20. The method of claim 18, wherein the subject is between 18 and 65 years of age.

20. 20. The method of any one of claims 1 to 19, wherein the subject weighs between 40 kg and 125 kg.

21. 21. The method of any one of claims 1 to 20, wherein the subject has chronic HBV infection, e.g., as defined by positive serology for HBsAg, HBV DNA, and / or HBeAg on two occasions, the two occasions being at least 6 months apart.

22. 22. The method of any one of claims 1 to 21, wherein the subject does not have cirrhosis.

23. The absence of cirrhosis a fibroscan assessment (e.g., within 6 months prior to administration of the single dose of the pharmaceutical composition); or Liver biopsy (e.g., within 12 months prior to administration of the single dose of the pharmaceutical composition) 23. The method of claim 22, wherein the absence of cirrhosis is determined by the absence of Metavir F3 fibrosis or the absence of F4 cirrhosis.

24. 24. The method of any one of claims 1 to 23, wherein the subject has received a nucleoside (nucleotide) reverse transcriptase inhibitor (NRTI), optionally within 120 days, and optionally within 60 days, prior to administration of the single dose.

25. 25. The method of claim 24, wherein the NRTI comprises one or more of tenofovir, tenofovir disoproxil (e.g., tenofovir disoproxil fumarate), tenofovir alafenamide, entecavir, lamivudine, adefovir, and adefovir dipivoxil.

26. 26. The method of any one of claims 1 to 25, wherein the subject has a serum HBV DNA concentration of less than 100 IU / mL for no more than 28 days prior to administration of the single dose.

27. 27. The method of any one of claims 1 to 26, wherein the subject has a serum HBsAg concentration of less than 1,000 IU / mL before the single dose is administered.

28. 27. The method of any one of claims 1 to 26, wherein the subject has a serum HBsAg concentration of 1,000 IU / mL or greater for a period not exceeding 28 days prior to administration of the single dose.

29. 29. The method of any one of claims 1 to 28, wherein the subject was HBe antigen (HBeAg) negative for no more than 28 days prior to administration of the single dose.

30. 30. The method of any one of claims 1 to 29, wherein the subject has been negative for anti-HBs antibodies for no more than 28 days prior to administration of the single dose.

31. the subject, before administering the single dose, (i) does not have fibrosis and / or does not have cirrhosis, and / or (ii) have an alanine aminotransferase (ALT) level less than two times the upper limit of normal (ULN); 31. The method of any one of claims 1 to 30.

32. 32. The method of any one of claims 1 to 31, wherein, 56 days after administration of the single dose, the subject has a less than two-fold decrease in serum HBsAg (e.g., the concentration of serum HBsAg determined using an Abbott ARCHITECT assay) compared to the subject's serum HBsAg from days 0 to 28 prior to administration of the single dose.

33. After administration of the single dose (e.g., on day 56 after administration of the single dose), the subject: (i) have reduced or less severe intrahepatic spread of HBV compared to a reference subject; and / or (ii) comprising an adaptive immune response to HBV; 33. The method of any one of claims 1 to 32.

34. 34. The method of any one of claims 1 to 33, wherein the subject is male.

35. 34. The method of any one of claims 1 to 33, wherein the subject is female.

36. 1. A pharmaceutical composition comprising an antibody, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 91 and a light chain amino acid sequence of SEQ ID NO: 93, and the pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, for example 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

37. 37. The pharmaceutical composition of claim 36, comprising up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody.

38. 38. The pharmaceutical composition of claim 36 or 37, comprising about 75 mg of the antibody.

39. 38. The pharmaceutical composition of claim 36 or 37, comprising about 90 mg of the antibody.

40. 38. The pharmaceutical composition of claim 36 or 37, comprising about 300 mg of the antibody.

41. 38. The pharmaceutical composition of claim 36 or 37, comprising about 900 mg of the antibody.

42. 38. The pharmaceutical composition of claim 36 or 37, comprising about 3,000 mg of the antibody.

43. 43. The pharmaceutical composition of any one of claims 36 to 42, further comprising water, optionally USP water.

44. 44. A pharmaceutical composition according to any one of claims 36 to 43, further comprising histidine, suitably in a concentration of 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.

45. 45. A pharmaceutical composition according to any one of claims 36 to 44, further comprising a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8% or 9%, preferably about 7% (w / v).

46. 46. ​​A pharmaceutical composition according to any one of claims 36 to 45, further comprising a surfactant which may be a polysorbate, preferably polysorbate 80 (SP80), said polysorbate being present in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v).

47. 47. The pharmaceutical composition of any one of claims 36 to 46, having a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.

2.

48. (i) 150 mg / mL of the antibody; (ii) USP Water; (iii) 20 mM histidine; (iv) 7% sucrose, and (v) 0.02% PS80 and having a pH of 6.

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